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WO2025115009A1 - Peptides pour diagnostics urinaires et anticorps pour leur détection - Google Patents

Peptides pour diagnostics urinaires et anticorps pour leur détection Download PDF

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Publication number
WO2025115009A1
WO2025115009A1 PCT/IL2024/051120 IL2024051120W WO2025115009A1 WO 2025115009 A1 WO2025115009 A1 WO 2025115009A1 IL 2024051120 W IL2024051120 W IL 2024051120W WO 2025115009 A1 WO2025115009 A1 WO 2025115009A1
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Prior art keywords
seq
crp
protein
sequences
protein fragment
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Inventor
Shani SHENHAR-TSARFATY
Igal RIKLIN
Keren LEVINSTEIN HALLAK
Peleg ITAY
Yael SCHNEIDMAN
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Accurine Ltd
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Accurine Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • 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
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • 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
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4737—C-reactive protein
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
    • 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
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/493—Physical analysis of biological material of liquid biological material urine

Definitions

  • the present disclosure is generally directed to the diagnostics of infectious diseases.
  • the invention relates to urinary peptides and protein fragments used as biomarkers for distinguishing between bacterial and viral infections or for diagnosing or monitoring inflammation, and antibodies for their detection.
  • Antibiotic resistance has been largely attributed to the overuse and misuse of antibiotics. A major reason for this is incorrect diagnosis, often caused by the similar clinical features of infections caused by bacterial and by viral agents. While antibiotics is usually the treatment of choice to a bacterial infection, it is often at best useless when treating viral infections, and in worse scenarios may cause adverse effects and development of resistant bacteria. Nevertheless, when in doubt, prescribing antibiotics is usually the default, so as not to miss treating a bacterial infection. The rate of inappropriate antibiotic prescriptions in the hospital setting is estimated at 30 to 50%.
  • diagnostic tools today are based on specific microbiological diagnostic tests such as culture, serology and more recently nucleic acid-based tests usually directed at finding the causative agent.
  • these tests involve challenges such as cases where the infection site is not readily accessible or unknown and so cannot be sampled, long times and expertise needed for microbiological laboratory assays, entailing dependency on health care professionals, and more.
  • the present invention is based, inter alia, on the surprising finding that certain peptides and protein fragments, found in urine samples of individuals suffering from an infection, were present at different levels in patients with a bacterial infection and in patients with a viral infection, and could consequently be used as biomarkers for distinguishing between a bacterial infection and a viral infection.
  • a method for predicting in a urine sample of a subject suspected of having an infection, whether the infection is a bacterial infection and/or a viral infection including: a. detecting in a urine sample of the subject a level of at least one protein fragment derived from at least one protein selected from: CRP, BTLA, EGF, FGA, LGALS9, SAA, C0L6A1, CNTFR, ICOSLG, MXRA8, NTM, PROZ, SERPINA5, THBD, TNXB, VASN, and combinations thereof; b. determining for the at least one protein fragment whether the level is above or below a respective threshold; and c. predicting whether the infection is bacterial and/or viral based on the determination in step (b).
  • the at least one protein fragment includes a sequence selected from peptide sequences GYSIFSYATK, RQDNEILIFWSK, YCANRPHVTWCK, RQSEHSILAGDPFELECPVK, RLFWTDTGINPR, LCSDIDECEMGVPVCPPASSK, LFWIQYNR, LYWCDAK, CISEGEDATCQCLK, RAPDLQDLPWQVK, VHLIVQVSPK, APLTKPLK, YEVQGEVFTKPQLWP, VFHLTVAEPHAEPPPR, FEDGGYVVCNTR, FAVNFQTGFSGNDIAFHFNPR, QNGSWGPEER, VMVNGILFVQYFHR, THMPFQK, IALVITDGR, GLYDVVSVLR, ENFVLTTAK, GLLSGWAR,
  • TDGCQHFCLPGQESYTCSCAQGYR EANYIGSDK, FFGHGAEDSLADQAANEWGR
  • GPGGVWAAEAISDAR RGPGGVWAAEAISDAR
  • DPNHFRPAGLPEK MNLYGFHGGQR
  • AAAATGTIFTFR YMHLFSTIK
  • GSESGIFTNTK TVIGPDGHK
  • GDSTFESK TVIGPDGHKEVTK
  • VTSGSTTTTR QCVPHDQCACGVLTSEK
  • CQCPAGAALQADGR LAGLGLQQLDEGLFSR
  • the at least one protein is selected from CRP, BTLA, EGF, FGA, LGALS9, SAA, and combinations thereof.
  • the level of at least one protein fragment derived from CRP, FGA, and/or SAA being above the respective threshold indicates that the infection is predicted to be a bacterial infection; and/or the level of at least one protein fragment derived from BTLA, EGF, and/or LGALS9 being above the respective threshold indicates that the infection is predicted to be a viral infection.
  • the at least one protein is at least 2 proteins including BTLA and CRP; BTLA and FGA; BTLA and SAA; CRP and SAA; CRP and FGA; CRP and EGF; or EGF and FGA.
  • the at least one protein is at least 3 proteins including CRP, BTLA, and LGALS9; CRP, SAA, and BTLA; CRP, SAA, and FGA; CRP, SAA, and EGF; CRP, SAA, and LGALS9; BTLA, FGA, and LGALS9; CRP, LGALS9, and SAA; EGF, FGA, and LGALS9; or FGA, LGALS9, and SAA.
  • the at least one protein is at least 4 proteins including CRP, BTLA, FGA, and SAA; BTLA, EGF, FGA, and LGALS9; BTLA, FGA, LGALS9, and SAA; CRP, EGF, FGA, and LGALS9; or CRP, FGA, LGALS9, and SAA.
  • the at least one protein is at least 5 proteins including CRP, BTLA, EGF, FGA, and LGALS9; CRP, BTLA, EGF, LGALS9, and SAA; CRP, BTLA, FGA, LGALS9, and SAA; BTLA, EGF, FGA, LGALS9, and SAA; or CRP, EGF, FGA, LGALS9, and SAA.
  • the at least one protein is at least 6 proteins including CRP, BTLA, EGF, FGA, LGALS9, and SAA.
  • the at least one protein fragment includes a sequence selected from the CRP-derived peptide sequences GYSIFSYATK, RQDNEILIFWSK, APLTKPLK, and YEVQGEVFTKPQLWP; the BTLA-derived peptide sequences YCANRPHVTWCK and RQSEHSILAGDPFELECPVK; the EGF-derived peptide sequences RLFWTDTGINPR, LCSDIDECEMGVPVCPPASSK, LFWIQYNR, LYWCDAK, and CISEGEDATCQCLK; the FGA-derived peptide sequences GSESGIFTNTK, TVIGPDGHK, GDSTFESK, TVIGPDGHKEVTK, and VTSGSTTTTR; the LGALS9-derived peptide sequences FEDGGYVVCNTR, FAVNFQTGFSGNDIAFHFNPR, QNGSWGPEER,
  • VMVNGILFVQYFHR, and THMPFQK the SAA-derived peptide sequences EANYIGSDK, FFGHGAEDSLADQAANEWGR, GPGGVWAAEAISDAR, RGPGGVWAAEAISDAR, and DPNHFRPAGLPEK; sequences at least 90%, 95%, 98%, or 99% identical to the peptide sequences, sequences including them, and intervening protein sequences.
  • the at least one protein fragment includes a sequence selected from peptide sequences GYSIFSYATK (CRP), RQDNEILIFWSK (CRP), YCANRPHVTWCK (BTLA), RLFWTDTGINPR (EGF), GSESGIFTNTK (FGA), FEDGGYVVCNTR (LGALS9), EANYIGSDK (SAA), sequences at least 90%, 95%, 98%, or 99% identical to the peptide sequences, sequences including them, and intervening protein sequences.
  • CRP GYSIFSYATK
  • RQDNEILIFWSK CPP
  • BTLA YCANRPHVTWCK
  • BTLA RLFWTDTGINPR
  • GSESGIFTNTK FGA
  • FEDGGYVVCNTR LGALS9
  • EANYIGSDK SAA
  • the detecting in step (a) includes contacting the urine sample with one or more binding agents capable of specifically binding to the at least one protein fragment; and detecting the binding of the one or more binding agents to the at least one protein fragment.
  • the one or more binding agents are selected from antibodies, including monoclonal antibodies, polyclonal antibodies, recombinant antibodies, rodent antibodies humanized antibodies, and chimeric antibodies; functional fragments of antibodies including scFv, Fv, Fab, Fab’ and F(ab')2 fragments; aptamers; and/or any agents capable of specifically binding to a protein fragment.
  • the one or more binding agents include at least one antibody specific to at least one of the protein fragments.
  • the one or more binding agents includes at least one monoclonal antibody defined in Table 2.
  • the one or more binding agents includes at least one monoclonal antibody defined by variable heavy chain (VH) regions and variable light chain (VL) regions and/or complementarity determining regions (CDR)s presented in Table 13.
  • the detecting the binding is conducted by an assay selected from a lateral flow assays (LFA), a fluorescence activated cell sorting (FACS), enzyme-linked immunosorbent assay (ELISA), a dot-blot, a dipstick, an antibody chip, and magnetic beads.
  • LFA lateral flow assays
  • FACS fluorescence activated cell sorting
  • ELISA enzyme-linked immunosorbent assay
  • a dot-blot a dipstick, an antibody chip, and magnetic beads.
  • the respective threshold is a threshold of detection of the protein fragment by the one or more binding agents. In some embodiments, for each protein fragment the respective threshold is a reference level of the protein fragment representing a level of the protein fragment in a urine sample from a reference subject afflicted with a viral infection or a bacterial infection. In some embodiments, for each protein fragment of the at least one protein fragment, the respective threshold is a reference level of the protein fragment representing a level of the protein fragment in a urine sample from the subject at a previous time point.
  • the subject is a mammal. In some embodiments, the subject is a human.
  • the infection is an upper respiratory tract infection (URTI).
  • the URTI is selected from streptococcal pharyngitis (strep throat), bacterial tracheitis, sinusitis, epiglottitis, and viral URTIs caused by rhinovirus, coronavirus, adenovirus, influenza virus, and/or human parainfluenza virus.
  • the URTI is caused by a bacterial agent selected from: group A streptococcus (including Streptococcus pyogenes), Staphylococcus aureus, Moraxella catarrhalis, Haemophilus influenzae, Streptococcus pneumoniae, and combinations thereof.
  • the URTI is caused by a viral agent selected from: rhinovirus, coronavirus, adenovirus, influenza virus (including influenza virus type A, B, and/or C), human parainfluenza virus, and combinations thereof.
  • the method further includes a step of treating the subject with an antibiotic treatment when the infection is predicted to be a bacterial infection, and/or treating the subject with an antiviral treatment when the infection is predicted to be a viral infection.
  • a method for treating a bacterial infection in a subject afflicted with an infection including: a. detecting in a urine sample of the subject a level of at least one protein fragment derived from at least one protein selected from: CRP, BTLA, EGF, FGA, LGALS9, SAA, C0L6A1, CNTFR, ICOSLG, MXRA8, NTM, PROZ, SERPINA5, THBD, TNXB, VASN, and combinations thereof; b. determining for the at least one protein fragment whether the level is above or below a respective threshold; c. predicting that the infection is bacterial based on the determination in step (b); and d. treating the subject with antibiotics.
  • predicting that the infection is bacterial is based on the level of at least one protein fragment derived from CRP, FGA, and/or SAA being above the respective threshold.
  • a method for treating a viral infection in a subject afflicted with an infection including: a. detecting in a urine sample of the subject a level of at least one protein fragment derived from at least one protein selected from: CRP, BTLA, EGF, FGA, LGALS9, SAA, C0L6A1, CNTFR, ICOSLG, MXRA8, NTM, PROZ, SERPINA5, THBD, TNXB, VASN, and combinations thereof; b. determining for the at least one protein fragment whether the level is above or below a respective threshold; c. predicting that the infection is viral based on the determination in step (b); and d. treating the subject with an antiviral agent.
  • predicting that the infection is viral is based on the level of at least one protein fragment derived from BTLA, EGF, and/or LGALS9 being above the respective threshold.
  • a method for predicting efficacy of a treatment in a subject afflicted with a bacterial and/or a viral infection including: a. detecting in a first urine sample of the subject a first level of at least one protein fragment derived from at least one protein selected from: CRP, BTLA, EGF, FGA, LGALS9, SAA, C0L6A1, CNTFR, ICOSLG, MXRA8, NTM, PROZ, SERPINA5, THBD, TNXB, VASN, and combinations thereof; b. administering to the subject a treatment including an antibiotic and/or an antiviral agent; c.
  • step (d) detecting in a second urine sample of the subject a second level of the at least one protein fragment; d. determining for the at least one protein fragment whether the second level is above or below the first level; and e. predicting whether the treatment is effective based on the determination in step (d).
  • kits for predicting in a urine sample whether an infection is a bacterial infection or a viral infection including: a. one or more binding agents each capable of binding to at least one protein fragment derived from at least one protein selected from CRP, BTLA, EGF, FGA, LGALS9, SAA, C0L6A1, CNTFR, ICOSLG, MXRA8, NTM, PROZ, SERPINA5, THBD, TNXB, VASN; b.
  • the instructions for use indicate that the level of at least one protein fragment derived from CRP, FGA, and/or SAA being above the respective threshold predicts that the infection is a bacterial infection; and/or the level of at least one protein fragment derived from BTLA, EGF, and/or LGALS9 being above the respective threshold predicts that the infection is a viral infection.
  • the kit further includes reagents for use with an assay based on LFA, FACS, ELISA, a dot blot, a dipstick, an antibody chip, a multiplex bead immunoassay.
  • the one or more binding agent are selected from antibodies, including monoclonal antibodies, polyclonal antibodies, recombinant antibodies, rodent antibodies humanized antibodies, and chimeric antibodies; functional fragments of antibodies including scFv, Fv, Fab, Fab’ and F(ab')2 fragments; aptamers; and/or any agents capable of specifically binding to the protein fragment.
  • the one or more binding agent includes at least one monoclonal antibody defined in Table 2.
  • the one or more binding agent includes at least one monoclonal antibody defined by variable heavy chain (VH) regions and variable light chain (VL) regions and/or complementarity determining regions (CDR)s presented in Table 13.
  • a method for predicting an elevated blood CRP level by a urine test including: a. detecting in a urine sample of the subject a level of at least one protein fragment having a sequence selected from sequences presented in Table 10, sequences including them, and intervening protein sequences; b. determining for the at least one protein fragment whether the level is above or below a respective threshold; and c. predicting whether blood CRP level is elevated, based on the determination in step (b).
  • kits for predicting an elevated blood CRP level by a urine test including: a. one or more binding agents each capable of binding to at least one protein fragment including a sequence selected from peptide sequences presented in Table 10, sequences at least 90%, 95%, 98%, or 99% identical to the peptide sequences, sequences including them, and intervening protein sequences b. at least one reagent for detecting the binding of the one or more binding agents to the at least one protein fragment in a urine sample, thereby indicating that the level of the at least one protein fragment bound by the one or more binding agents in the urine sample is above a respective threshold; and c. instructions for use.
  • a method for treating an inflammation in a subject including: a. detecting in a urine sample of the subject a level of at least one protein fragment including a sequence selected from peptide sequences presented in Table 10, sequences at least 90%, 95%, 98%, or 99% identical to the peptide sequences, sequences including them, and intervening protein sequences; b. determining for the at least one protein fragment whether the level is above or below a respective threshold; c. predicting that blood CRP level is elevated, based on the determination in step (b); and d. treating the subject with antibiotics or with an anti-inflammatory agent.
  • a method for predicting efficacy of a treatment of inflammation in a subject including: a. detecting in a first urine sample of the subject a first level of at least one protein fragment including a sequence selected from peptide sequences presented in Table 10, sequences at least 90%, 95%, 98%, or 99% identical to the peptide sequences, sequences including them, and intervening protein sequences; b. administering to the subject an antibiotic or an anti-inflammatory treatment; c. detecting in a second urine sample of the subject a second level of the at least one protein fragment; d. determining for the at least one protein fragment whether the second level is above or below the first level, thereby predicting increased or a decreased inflammation, respectively; and e. predicting whether the treatment is effective based on whether the determination in step (d) indicates decreased inflammation.
  • a monoclonal antibody capable of specifically binding a CRP-derived protein fragment or peptide including a sequence set for the in SEQ ID NO: 861 (GYSIFSYATKRQDNEILIFWSK) and/or SEQ ID NO: 862 (RQDNEILIFWSK).
  • an isolated polypeptide capable of specifically binding a CRP-derived protein fragment or peptide
  • the isolated polypeptide includes a variable heavy chain (VH) region including three complementarity determining regions (CDRs) (VH-CDR1, VH-CDR2, VH-CDR3) and a variable light chain (VL) region including three CDRs (VL-CDR1, VL-CDR2, and VL-CDR3)
  • VH-CDRs and three VL-CDRs are defined by a standard method selected from Kabat, Chothia, IMGT, and AbM, based on VH and VL regions sequences set forth in: SEQ ID Nos. 181 and 182, respectively; SEQ ID Nos. 201 and 202, respectively; SEQ ID Nos. 221 and 222, respectively; SEQ ID Nos. 241 and 242, respectively; SEQ ID Nos. 261 and 262, respectively; or SEQ ID Nos. 281 and 282; respectively.
  • an isolated polypeptide capable of specifically binding a CRP-derived protein fragment or peptide including a sequence set forth in SEQ ID NO: 862, wherein the isolated polypeptide includes a variable heavy chain (VH) region including three complementarity determining regions (CDRs) (VH-CDR1, VH-CDR2, VH-CDR3) and a variable light chain (VL) region including three CDRs (VL-CDR1, VL-CDR2, and VL-CDR3), and the three VH-CDRs and three VL-CDRs are defined by a standard method selected from Kabat, Chothia, IMGT, and AbM, based on VH and VL regions sequences set forth in: SEQ ID Nos.
  • VH variable heavy chain
  • CDRs variable light chain
  • SEQ ID Nos. 201 and 202 respectively; SEQ ID Nos. 221 and 222, respectively; SEQ ID Nos. 241 and 242, respectively; SEQ ID Nos. 261 and 262, respectively; or SEQ ID Nos. 281 and 282; respectively.
  • the VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3, include sequences substantially identical to sequences set forth in:
  • the VH and the VL regions include sequences substantially identical to sequences set forth in:
  • the CRP-derived protein fragment or peptide includes a sequence set forth in SEQ ID NO: 861 (GYSIFSYATKRQDNEILIFWSK) and/or SEQ ID NO: 862 (RQDNEILIFWSK).
  • the binding is under urinary conditions.
  • the isolated polypeptide is selected from an antibody, an Fv fragment, an Fab fragment, an F(ab’)2 fragment, an scFv, a chimeric or a humanized antibody or antibody fragment, and a CAR-B. In some embodiments, the isolated polypeptide is a monoclonal antibody.
  • nucleic acid encoding the isolated polypeptide disclosed herein.
  • a vector including the nucleic acid disclosed herein.
  • a cell including the isolated polypeptide disclosed herein.
  • Figs. 1A-1J show histograms for each peptide of the top 10, presenting the count of patients categorized as bacterial or as viral vs. a logarithm of the intensity of the peptide in the LC-MS analysis.
  • Figs. 2A-2J show histograms for each peptide of the top 11-20, presenting the count of patients categorized as bacterial or as viral vs. a logarithm of the intensity of the peptide in the LC- MS analysis.
  • Figs. 3A-3K show ELISA binding curves of purified monoclonal antibodies against CRP- P01 and CRP-P02 (Figs. 3A and 3B, respectively), BTLA-P01 and BTLA-P02 (Figs. 3C and 3D, respectively), EGF-P01 and EGF-P02 (Figs. 3E and 3F, respectively), FGA-P01 and FGA-P02 (Figs. 3G and 3H, respectively), SAA-P01 and SAA-P02 (Figs. 31 and 3 J, respectively), and LGALS9-P01 (Fig. 3K).
  • the present invention is based on the finding that certain peptides, which are derived by protease cleavage from certain proteins found in urine (and therefore representing these proteins), were found at different levels in urine samples of subjects carrying bacterial infections and in urine samples of subjects carrying viral infections. Following a statistical analysis described in more detail below and in the experimental section, the most discriminative (indicative) peptides were selected, that could be used to distinguish bacterially-infected subjects from virally -infected subjects.
  • the peptides mentioned hereinbelow were identified by the inventors following trypsinization of the urine samples in order to allow analysis by mass spectrometry (MS). Accordingly, the identified peptides represent larger peptides (protein fragments or proteins) which are present in the urine of the respective subjects.
  • the findings of the present invention are especially surprising since urinary levels of proteins in general do not correlate well with their blood levels.
  • This also relates to proteins proposed as blood biomarkers for various conditions, including biomarkers for distinguishing between bacterial and viral infection, e.g., tumor necrosis factor-related apoptosis-inducing ligand (TRAIL).
  • TRAIL tumor necrosis factor-related apoptosis-inducing ligand
  • a method for predicting in a urine sample of a subject suspected of having an infection, whether the infection is a bacterial infection and/or a viral infection including: a. detecting in a urine sample of the subject a level of at least one protein fragment derived from at least one protein selected from: CRP, BTLA, COL6A1, CNTFR, EGF, FGA, ICOSLG, LGALS9, MXRA8, NTM, PROZ, SAA, SERPINA5, THBD, TNXB, VASN, and combinations thereof; b. determining for the at least one protein fragment whether the level is above or below a respective threshold; and c. predicting whether the infection is bacterial and/or viral based on the determination in step (b).
  • the at least one protein is selected from CRP, BTLA, C0L6A1, CNTFR, EGF, FGA, ICOSLG, LGALS9, MXRA8, NTM, PROZ, SAA, SERPINA5, THBD, TNXB, VASN, and combinations thereof.
  • the at least one protein is selected from CRP, BTLA, EGF, FGA, LGALS9, SAA, and combinations thereof.
  • the at least one protein is selected from BTLA, EGF, FGA, LGALS9, SAA, and combinations thereof.
  • the method is a method for predicting in a urine sample of a subject suspected of having an infection, whether the infection is a bacterial infection, i.e. an infection caused by a bacterial agent. In some embodiments, the method is a method for predicting in a urine sample of a subject suspected of having an infection, whether the infection is a viral infection, i.e. an infection caused by a viral agent.
  • peptide or protein fragment are used herein interchangeably and relate to an amino acid sequence that is a part of a protein which is found in urine.
  • the peptide or protein fragment has a length of about 3-500 amino acids.
  • the peptide or protein fragment has a length of about 5-200, 5-100, 5-90, 5-80, 5-70, 5-60, 5-50, 5-40, 5-30, 5-25, or 7-80 amino acids.
  • the peptide or protein fragment has a length of at least about 5, 10, or 20 amino acids.
  • the peptide or protein fragment does not include the complete protein.
  • the term “suspected of having an infection” with reference to a subject means that the subject presents with symptoms typical of an infection in general, such as fever, pain, feeling weak and/or tired, signs of inflammation, etc. In some embodiments, the subject presents symptoms which are typical to a bacterial or to a viral infection. In some embodiments, the subject presents symptoms which are typical to a specific infection.
  • “predicting” means predicting a desired parameter with a high probability. This is meant to reflect the fact that the predicting in step (c) most likely does not indicate a 100% certainty. However, the prediction is expected to provide a reasonable basis for determining the desired parameter, and treating the subject with a suitable agent, if needed.
  • the desired parameter may be, e.g., an infection being bacterial and/or viral, or blood CRP level being elevated.
  • the high probability is a probability of at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%.
  • “predicting” means increasing a probability of predicting a desired parameter.
  • predicting that the infection is bacterial means that the infection has a high probability of being a bacterial infection.
  • predicting that the infection is viral means that that the infection has a high probability of being a viral infection.
  • the term “increasing the probability of predicting”, reflects the fact that the prediction according to the methods of the invention may be done in addition to other parameters such as physical features of the subject, e.g., that are typical to a bacterial or to a viral infection (e.g., fever, presence of additional symptoms such as rash, puss, immune system activity parameters such as types of cells involved in the immune response, etc.). Accordingly, the methods of the invention may be used as an additional test, which increases the probability of a correct prediction. However, in some embodiments, the methods of the invention are used without additional parameters. In some embodiments, the methods of the invention are suitable for use by the subject without a need for a health care professional. In some embodiments, the methods of the invention are suitable for use in the home of the subject. The advantage is that it allows the subject to know, for example, whether or not there is a need to reach a physician in order to receive antibiotics.
  • the high probability is higher than a reference probability, where the methods of the invention are not used. In some embodiments, the high probability is higher than a reference probability by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%. In some embodiments, the increasing the probability is by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%, compared to a reference where the methods of the invention are not used.
  • step (a) of the method a urine sample from the subject is analyzed by determining the level of certain protein fragments (or peptides).
  • the peptides disclosed herein were selected based on a statistical analysis which predicted the most informative peptides for differentiating between a bacterial and a viral infection.
  • urine sample may refer to any form of urine that may be tested, including, for example, a urine sample provided in a container such as a urine cup; urine used directly during urination, such as urinating on a device, such as a stick, which is capable of detecting the presence of protein fragments or peptides in the urine; and urine taken from a urine output device such as a urine bag, a diaper, or a catheter.
  • a urine sample provided in a container such as a urine cup
  • urine used directly during urination such as urinating on a device, such as a stick, which is capable of detecting the presence of protein fragments or peptides in the urine
  • urine taken from a urine output device such as a urine bag, a diaper, or a catheter.
  • the at least one protein fragment is derived from at least one protein selected from: CRP: C-reactive protein; BLTA: B- and T-lymphocyte attenuator; COL6A1: collagen VI, Alpha- 1 chain; CNTFR: ciliary neurotrophic factor receptor; EGF: epidermal growth factor; FGA: fibrinogen alpha chain; ICOSLG: inducible T-cell co-stimulator (ICOS) ligand, CD275; LGALS9: galectin 9; MXRA8: matrix remodeling associated 8; NTM: neurotrimin; PROZ: Protein Z, vitamin K dependent plasma glycoprotein; SAA: serum amyloid A; SERPINA5: serpin family A member 5; THBD: thrombomodulin; TNXB: tenascin XB protein; and VASN: vasorin.
  • CRP C-reactive protein
  • BLTA B- and T-lymphocyte attenuator
  • COL6A1 collagen VI, Alpha- 1 chain
  • the at least one protein fragment includes a sequence selected from peptide sequences GYSIFSYATK, RQDNEILIFWSK, YCANRPHVTWCK, RQSEHSILAGDPFELECPVK, RLFWTDTGINPR, LCSDIDECEMGVPVCPPASSK, LFWIQYNR, LYWCDAK, CISEGEDATCQCLK, RAPDLQDLPWQVK, VHLIVQVSPK, APLTKPLK, YEVQGEVFTKPQLWP, VFHLTVAEPHAEPPPR, FEDGGYVVCNTR, FAVNFQTGFSGNDIAFHFNPR, QNGSWGPEER, VMVNGILFVQYFHR, THMPFQK, IALVITDGR, GLYDVVSVLR, ENFVLTTAK, GLLSGWAR,
  • TDGCQHFCLPGQESYTCSCAQGYR EANYIGSDK, FFGHGAEDSLADQAANEWGR
  • GPGGVWAAEAISDAR RGPGGVWAAEAISDAR
  • DPNHFRPAGLPEK MNLYGFHGGQR
  • AAAATGTIFTFR YMHLFSTIK
  • GSESGIFTNTK TVIGPDGHK
  • GDSTFESK TVIGPDGHKEVTK
  • VTSGSTTTTR QCVPHDQCACGVLTSEK
  • CQCPAGAALQADGR LAGLGLQQLDEGLFSR
  • the at least one protein is at least 2, 3, 4, 5, or 6 proteins selected from CRP, BTLA, COL6A1, CNTFR, EGF, FGA, ICOSLG, LGALS9, MXRA8, NTM, PROZ, SAA, SERPINA5, THBD, TNXB, VASN, and combinations thereof.
  • the at least one protein is at least 2, 3, 4, 5, or 6 proteins selected from CRP, BTLA, EGF, FGA, LGALS9, SAA, and combinations thereof. In some embodiments, the at least one protein is at least 2, 3, 4, 5, or 6 proteins selected from BTLA, EGF, FGA, LGALS9, SAA, and combinations thereof.
  • the at least one protein fragment is at least 2, 3, 4, 5, or 6 protein fragments, each including at least one sequence selected from peptide sequences GYSIFSYATK, RQDNEILIFWSK, YCANRPHVTWCK, RQSEHSILAGDPFELECPVK, RLFWTDTGINPR, LCSDIDECEMGVPVCPPASSK, LFWIQYNR, LYWCDAK, CISEGEDATCQCLK, RAPDLQDLPWQVK, VHLIVQVSPK, APLTKPLK, YEVQGEVFTKPQLWP, VFHLTVAEPHAEPPPR, FEDGGYVVCNTR, FAVNFQTGFSGNDIAFHFNPR, QNGSWGPEER, VMVNGILFVQYFHR, THMPFQK, IALVITDGR, GLYDVVSVLR, ENFVLTTAK, GLLSGWAR, TDGCQHFCLPGQESYTCSCAQGYR, EAN
  • QCVPHDQCACGVLTSEK CQCPAGAALQADGR, LAGLGLQQLDEGLFSR, sequences at least 90%, 95%, 98%, or 99% identical to the peptide sequences, sequences including them, and combinations thereof.
  • the detecting of the levels of protein fragments according to step (a) may be conducted by any suitable method known in the art for detecting or for measuring levels of specific protein fragments in a urine sample.
  • level refers to an indication for the amount of the protein fragment present in the urine sample, and is not necessarily an exact value or number.
  • a level may be, e.g., a concentration, or a value corresponding to a concentration by any method of measurement.
  • a level may also be an amount of the protein fragment that is sufficient for detection by any method, such as an amount of the protein fragment sufficient for binding by a specific binding agent.
  • the level obtained by the detecting step is quantitative, i.e., in some embodiments, the detecting includes measuring a level of the protein fragments in the urine sample.
  • the level is not quantitative, e.g., when the detecting provides an indication of whether the detected protein fragment is present (e.g., detected) or absent (e.g., not detected), or whether the level of the detected protein fragment is above a certain threshold, such as the threshold of detection by a certain assay or by a certain binding agent, but without an explicit measurement of the level (e.g., the concentration in urine) of the detected protein fragment.
  • a certain threshold such as the threshold of detection by a certain assay or by a certain binding agent, but without an explicit measurement of the level (e.g., the concentration in urine) of the detected protein fragment.
  • the detection method is a method for identifying protein fragments or peptides, such as MS or by high-performance liquid chromatography (HPLC).
  • the protein fragments are detected by a specific binding agent, which specifically binds to the detected protein fragment, and the binding is further detected by a suitable assay.
  • the detecting in step (a) includes contacting the urine sample with one or more binding agents capable of specifically binding to the at least one protein fragment; and detecting the binding of the one or more binding agents to the at least one protein fragment.
  • binding may not necessarily be in absolute terms, but may be used to indicate binding of the isolated polypeptide to the relevant peptide, as detected (binding) or not detected (no binding) by a certain method used.
  • the binding threshold depends on the method used, e.g. on an antibody binding constant or on the sensitivity or other agents used in the method.
  • binding means “specifically binding” as defined below.
  • the one or more binding agents include a binding agent capable of binding to the at least one protein fragment. In some embodiments, each of the one or more binding agents is capable of binding to a single protein fragment. In some embodiments, the one or more binding agents include at least one binding agent which is capable of binding to more than a single protein fragment, and at least one binding agent capable of binding to a single protein fragment.
  • Nonlimiting examples for suitable binding agents include antibodies, such as monoclonal antibodies, polyclonal antibodies, recombinant antibodies, rodent antibodies humanized antibodies, and chimeric antibodies; functional fragments of antibodies capable of specifically binding to protein fragments or peptides, such as scFv, Fv, Fab, Fab’, and F(ab')2 fragments; aptamers, i.e. short sequences of artificial DNA, RNA, XNA (xeno nucleic acid), or peptide, that are capable of binding to a specific target molecule; and/or any agent that is capable of specifically binding to a protein fragment.
  • agents capable of binding to more than a single protein fragment include antibodies, such as bispecific or trispecific antibodies or similar constructs which are designed to bind to more than a single entity.
  • the one or more binding agents include antibodies. In some embodiments, the one or more binding agents include monoclonal antibodies.
  • the one or more binding agents include at least one antibody specific to at least one of the protein fragments.
  • the one or more binding agents include at least one isolated polypeptide and/or antibody disclosed hereinbelow.
  • Nonlimiting examples for suitable assays include, for example, fluorescent assays, colorimetric assays, radioactive assays, magnetic beads assays, etc. More specific examples for suitable assays include a lateral flow assay (LFA), fluorescence activated cell sorting (FACS), enzyme-linked immunosorbent assay (ELISA), a dot blot, a dipstick, an antibody chip, a multiplex bead immunoassay.
  • LFA lateral flow assay
  • FACS fluorescence activated cell sorting
  • ELISA enzyme-linked immunosorbent assay
  • step (b) the method involves determining for the at least one protein fragment whether the level is above or below a respective threshold.
  • threshold generally refers to a reference level used for comparing to a level of a detected protein fragment in order to determine whether the level of the protein fragment is above or below the threshold.
  • each protein fragment independently has a respective threshold, which may be the same or different from respective thresholds of other protein fragments.
  • a protein fragment may have different respective thresholds in different situations. For example, the respective threshold for a certain protein fragment may vary based on the means of detection. Additionally, the respective threshold may vary based on whether the method is intended for detection of a bacterial infection, a viral infection, or either a bacterial or a viral infection. Further, the respective threshold for a protein fragment may be different depending on whether the protein fragment is used alone or in combination with additional protein fragments.
  • the threshold or reference level is explicit, e.g., the threshold corresponds to a certain concentration of the protein fragment, above which the detected protein fragment is determined in step (b) to be above the threshold, and below which the detected protein fragment is determined in step (b) to be below the threshold.
  • the threshold concentration is a urinary concentration of the detected protein fragment, typical to a bacterial infection or to a viral infection in reference individuals.
  • the threshold concentration is a typical urinary concentration of the detected protein fragment found in bacterially infected or virally infected individuals and retrieved from a database.
  • the threshold concentration is an earlier- measured concentration of the protein fragment in the subject, for follow-up purposes, such as follow-up of the patient’s recovery following treatment, as described in more detail below.
  • the threshold concentration is a reference level of the protein fragment which has been determined by calculation, such as a statistical analysis.
  • the respective threshold is a reference level of the protein fragment.
  • the respective threshold is a reference level retrieved from a database.
  • the respective threshold is a reference level of the protein fragment representing a level of the protein fragment in a urine sample from a reference subject afflicted with a viral infection or with a bacterial infection.
  • the respective threshold is a reference level of the protein fragment representing a normal urinary level of the protein fragment.
  • the respective threshold is a reference level of the protein fragment representing a level of the protein fragment in a urine sample from the subject at a previous time point.
  • the detecting in step (a) includes measuring the level of the protein fragment
  • the determining in step (b) includes comparing the measured level of the protein fragment to the respective threshold.
  • the threshold is implicit, i.e., the threshold value is not explicitly used, but is rather manifested in the ability to detect the protein fragment.
  • the threshold level of the protein fragment under which level it would not be detected by the detection method.
  • the respective threshold is a level of the protein fragment that corresponds to a threshold level of detection of the protein fragment by the detection method used.
  • the respective threshold is a level of the protein fragment that corresponds to a threshold level of detection of the protein fragment by the one or more binding agents.
  • the detecting in step (a) results in determining for each of the detected protein fragments whether it is present at a level above or below a respective threshold, in step (b), based on whether or not the protein fragment was detected by the detecting method used.
  • the protein fragment level detected in step (a) is sent to a processing unit which determines in step (b) whether the level is above or below the respective threshold, e.g., by comparing it to the respective threshold.
  • step (b) The determinations made in step (b) with respect to whether protein fragment levels are above or below their respective thresholds are used in step (c) in order to predict whether the infection is bacterial and/or viral.
  • the determinations of protein fragments being at levels above or below their respective thresholds are used in order to predict that the infection has a lower probability of being a bacterial infection and/or has a lower probability of being a viral infection, in other words, to rule out a bacterial or a viral infection.
  • the predicting in step (c) may simply involve identifying the appearance of a positive result, such as by viewing, imaging, or otherwise detecting a positive outcome of a color, fluorescent, or radioactive reaction, which indicates the presence of the tested protein fragment at a level above the threshold of detection.
  • a positive result such as by viewing, imaging, or otherwise detecting a positive outcome of a color, fluorescent, or radioactive reaction, which indicates the presence of the tested protein fragment at a level above the threshold of detection.
  • a nonlimiting example may be the appearance of a color band representing the tested protein fragment in a lateral flow assay.
  • the predicting in step (c) requires further processing, computation, or calculation.
  • the predicting is conducted by a processor, which computes, based on the determinations in step (b), whether it predicts that the infection is a bacterial infection and/or a viral infection.
  • the determination in step (b) is performed by a processor, as noted above, and the determination is further processed in step (c).
  • the determination in step (b) may be based on images, such as by a camera, a cell phone, or another appropriate imaging device, and the image may be viewed or analyzed by a processor.
  • the predicting in step (c) includes processing by a computer analysis, e.g., by using machine learning algorithms such as learning and pattern recognition algorithms, clustering algorithms, supervised classification algorithms including, but not limited to, gradient boosted trees, random forest, regularized regression, multiple linear regression (MLR), principal component regression (PCR), partial least squares (PLS), discriminant function analysis (DFA) including linear discriminant analysis (LDA), nearest neighbor, artificial neural networks, multilayer perceptrons (MLP), generalized regression neural network (GRNN), and combinations thereof, or non-supervised clustering algorithms, including, but not limited to, K-means, spectral clustering, hierarchical clustering, gaussian mixture models, and combinations thereof.
  • the algorithm is selected from the group consisting of gradient boosted trees, random forest, regularized regression, and combinations thereof.
  • the at least one protein fragment is a single protein fragment.
  • the infection is predicted to be a bacterial infection. In some embodiments, if the single protein fragment is a bacterial infection-related protein fragment and the determination that the level of the protein fragment is below the respective threshold, then the infection is predicted to be a viral infection.
  • the infection is predicted to be a viral infection. In some embodiments, if the single protein fragment is a viral infection-related protein fragment and the determination that the level of the protein fragment is below the respective threshold, then the infection is predicted to be a bacterial infection.
  • bacterial infection-related protein fragments and “viral infection-related protein fragments”, as used here, relate to protein fragments, or peptides, found to be increased in urine of bacterially- or virally -infected patients, respectively.
  • bacterial infection-related protein fragments include peptides derived from the proteins CRP, FGA, and/or SAA.
  • viral infection-related protein fragments include peptides derived from the proteins BTLA, EGF, and LGALS9.
  • the at least one protein fragment is more than one protein fragment, such as 2, 3, 4, 5, or 6 protein fragments.
  • the at least one protein fragment includes a combination of protein fragments. In some embodiments, the combination of protein fragments includes only bacterial infection-related protein fragments. In some embodiments, the combination of protein fragments includes only viral infection-related protein fragments. In some embodiments, the combination of protein fragments includes fragments derived from both groups.
  • the level of all bacterial infection-related protein fragments being above the respective threshold predicts that the infection is a bacterial infection. In some embodiments, the level of all bacterial infection-related protein fragments being below the respective threshold predicts that the infection is a viral infection.
  • the level of all viral infection-related protein fragments being above the respective threshold predicts that the infection is a viral infection. In some embodiments, the level of all viral infection-related protein fragments being below the respective threshold predicts that the infection is a bacterial infection.
  • the level of at least one protein fragment being above the respective threshold predicts that the infection is a bacterial infection.
  • the level of at least one protein fragment being above the respective threshold predicts that the infection is a viral infection.
  • the combination of protein fragments includes protein fragments of both the bacterial- related and the viral-related groups
  • the levels of all bacterial-related fragments being above the respective threshold and the levels of all viral-related protein fragments being below the respective threshold predicts that the infection is a bacterial infection
  • the levels of all viral infection- related protein fragments being above the respective threshold and the levels of all bacterial infection-related protein fragments being below the respective threshold predicts that the infection is a viral infection.
  • the at least one peptide includes a combination of protein fragments including both bacterial infection-related protein fragments and viral infection-related protein fragments and levels of bacterial infection-related protein fragments and viral infection-related protein fragments are both above their respective thresholds or both below their respective thresholds, further processing is required in order for the prediction in step (c).
  • the bacterial infection-related protein fragments include at least one protein fragment derived from CRP, FGA, and/or SAA. In some embodiments, the viral infection- related protein fragments include at least one protein fragment derived from BTLA, EGF, and/or LGALS9.
  • the at least one protein is selected from CRP, BTLA, EGF, FGA, LGALS9, and SAA. In some embodiments, the at least one protein is selected from BTLA, EGF, FGA, LGALS9, and SAA.
  • the at least one protein includes at least 1, 2, 3, 4, 5, or 6 proteins selected from CRP, BTLA, EGF, FGA, LGALS9, and SAA. In some embodiments, the at least one protein includes at least 1, 2, 3, 4, 5, or 6 proteins selected from BTLA, EGF, FGA, LGALS9, and SAA.
  • the at least one protein includes at least two proteins selected from BTLA and CRP; BTLA and EGF; BTLA and FGA; BTLA and LGALS9; BTLA and SAA; CRP and EGF; CRP and FGA; CRP and LGALS9; CRP and SAA; EGF and FGA; EGF and LGALS9; EGR and SAA; FGA and LGALS9; FGA and SAA; or LGSLS9 and SAA.
  • the at least one protein includes at least two proteins selected from CRP and EGF; EGF and FGA; BTLA and CRP; BTLA and FGA; BTLA and SAA; CRP and FGA; CRP and SAA; EGF and SAA; EGF and LGALS9; or FGA and SAA.
  • the at least one protein includes at least three proteins selected from BTLA, CRP, and EGF; BTLA, CRP, and FGA; BTLA, CRP, and LGALS9; BTLA, CRP, and SAA; BTLA, EGF, and FGA; BTLA, EGF, and LGALS9; BTLA, EGF, and SAA; BTLA, FGA, and LGALS9; BTLA, FGA, and SAA; BTLA, LGALS9, and SAA; CRP, EGF, and FGA; CRP, EGF, and LGALS9; CRP, EGF, and SAA; CRP, FGA, and LGALS9; CRP, FGA, LGALS9, and SAA; CRP, LGALS9, and SAA; EGF, FGA, and LGALS9; EGF, FGA, and SAA; EGF, LGALS9, and SAA; EGF, FGA, and SAA; EGF, FGA, and SAA; EGF, FGA, and LGALS
  • the at least one protein includes at least three proteins selected from FGA, LGALS9, and SAA; BTLA, FGA, and LGALS9; EGF, FGA, and LGALS9; BTLA, CRP, and LGALS9; CRP, LGALS9, and SAA; CRP, EGF, and LGALS9; BTLA, CRP, and FGA; BTLA, CRP, and EGF; CRP EGF, and FGA; and BTLA, FGA, and SAA.
  • the at least one protein includes at least four proteins selected from BTLA, CRP, EGF, and FGA; BTLA, CRP, EGF, and LGALS9; BTLA, CRP, EGF, and SAA; BTLA, CRP, FGA, and LGALS9; BTLA, CRP, FGA, and SAA; BTLA, CRP, LGALS9, and SAA; BTLA, EGF, FGA, and LGALS9; BTLA, EGF, FGA, and SAA; BTLA, EGF, LGALS9, and SAA; BTLA, FGA, LGALS9, and SAA; CRP, EGF, FGA, and LGALS9; CRP, EGF, FGA, and LGALS9; CRP, EGF, FGA, and SAA; CRP, EGF, LGALS9, and SAA; CRP, FGA, LGALS9, and SAA; CRP, FGA, LGALS9, and SAA; CRP, EGF, FGA, and SAA;
  • the at least one protein includes at least four proteins selected from CRP, FGA, LGALS9, and SAA; BTLA, FGA, LGALS9, and SAA; CRP, EGF, FGA, and LGALS9; BTLA, CRP, FGA, and SAA; BTLA, EGF, FGA, and LGALS9; EGF, FGA, LGALS9, and SAA; BTLA, CRP, EGF, and LGALS9; CRP, EGF, FGA, and SAA; BTLA, CRP, EGF, and SAA; BTLA, CRP, EGF, and FGA; and BTLA, CRP, FGA, and LGALS9.
  • the at least one protein includes at least five proteins selected from BTLA, EGF, FGA, LGALS9, and SAA; BTLA, CRP, FGA, LGALS9, and SAA; CRP, EGF, FGA, LGALS9, and SAA; BTLA, CRP, EGF, FGA, and LGALS9; BTLA, CRP, EGF, LGALS9, and SAA; and BTLA, CRP, EGF, FGA, and SAA.
  • the at least one protein includes CRP, BTLA, EGF, FGA, LGALS9, and SAA. In some embodiments, the at least one protein includes BTLA, EGF, FGA, LGALS9, and SAA.
  • the at least one protein does not include TRAIL.
  • the level of at least one protein fragment derived from CRP, FGA, and/or SAA being above the respective threshold predicts that the infection is a bacterial infection.
  • the level of each of at least two protein fragments derived from CRP, FGA, and/or SAA being above the respective threshold predicts that the infection is a bacterial infection.
  • the level of each of at least three protein fragments derived from CRP, FGA, and SAA being above the respective threshold predicts that the infection is a bacterial infection.
  • the level of at least one protein fragment derived from BTLA, EGF, and/or LGALS9 being above the respective threshold predicts that the infection is a viral infection.
  • the level of each of at least two protein fragments derived from BTLA, EGF, and/or LGALS9 being above the respective threshold predicts that the infection is a viral infection.
  • the level of each of at least three protein fragments derived from BTLA, EGF, and LGALS9 being above the respective threshold predicts that the infection is a viral infection.
  • the prediction when the level of bacterial infection related protein fragments (derived from CRP, FGA, and/or SAA) is below the respective threshold, the prediction is for a viral infection, and when the level of viral infection related protein fragments (derived from BTLA, EGF, and/or LGALS9) is below the respective threshold, the prediction is for a bacterial infection.
  • Urine is produced predominantly from plasma that is filtered by the kidneys. As a result, as indicated above, most of the proteins and protein fragments present in blood are not present in urine. It is therefore not productive to analyze blood-derived biomarkers by urinalysis, and it is not possible to predict which biomarkers may appear in urine, based on blood biomarkers. Except for TRAIL, already noted above, another example is CRP, which is known to be elevated in blood during inflammation, and is routinely tested in blood. However, none of the tests for detecting CRP in blood could be used to detect CRP in urine, and till this day urine CRP is not tested in correlation with inflammation. Nevertheless, since urine analysis is not invasive and does not require a medical specialist, urine may be more readily used basing a prediction.
  • the protein fragments of the invention include the presented sequences.
  • overlapping peptides i.e., peptide sequences which overlap with sequences presented herein
  • protein fragments derived from protein sequences that are between two protein fragments derived from the same protein are expected to function in the same way as the protein fragments of the invention.
  • the protein fragments of the invention encompass sequences which include, are included in, or overlap with, the sequences presented herein. It is further noted that several sequences may be parts of a single protein.
  • intervening protein sequences relates to sequences of the protein from which at least two presented peptide sequences are derived, that which are located between at least two peptide sequences.
  • the protein fragments found in a sample from a particular subject may not be identical to the protein fragments of the invention. Accordingly, in some embodiments, the protein fragments of the invention also encompass peptides having sequences at least 90%, 95%, 98%, or 99% identical to the indicated peptide sequences. In some embodiments, the protein fragments of the invention also encompass protein fragments having sequences with 1, 2, or 3 amino acid differences from the indicated peptide sequences.
  • the at least one protein fragment includes any protein fragments derived from CRP, BTLA, EGF, FGA, LGALS9, and/or SAA. In some embodiments, the at least one protein fragment includes any protein fragments derived from BTLA, EGF, FGA, LGALS9, and/or SAA.
  • the BTLA-derived protein fragments include sequences selected from peptide sequences YCANRPHVTWCK, YVTDVKSASERP, RQSEHSILAGDPFELECPVK, sequences at least 90%, 95%, 98%, or 99% identical to the peptide sequences, sequences including them, and intervening protein sequences.
  • the CRP-derived protein fragments include sequences selected from peptide sequences GYSIFSYATK, RQDNEILIFWSK, APLTKPLK, ESDTSYVSLK, YEVQGEVFTKPQLWP, sequences at least 90%, 95%, 98%, or 99% identical to the peptide sequences, sequences including them, and intervening protein sequences.
  • the EGF-derived protein fragments include sequences selected from peptide sequences RLFWTDTGINPR, LCSDIDECEMGVPVCPPASSK, LFWIQYNR, RIYWVDLER, LYWCDAK, CISEGEDATCQCLK, sequences at least 90%, 95%, 98%, or 99% identical to the peptide sequences, sequences including them, and intervening protein sequences.
  • the FGA-derived protein fragments include sequences selected from peptide sequences GSESGIFTNTK, TVIGPDGHK, GDSTFESK, QFTSSTSYNR, TVIGPDGHKEVTK, VTSGSTTTTR, sequences at least 90%, 95%, 98%, or 99% identical to the peptide sequences, sequences including them, and intervening protein sequences.
  • the LGALS9-derived protein fragments include sequences selected from peptide sequences FEDGGYVVCNTR, FAVNFQTGFSGNDIAFHFNPR, QNGSWGPEER, GMPFDLCFLVQSSDFK, VMVNGILFVQYFHR, THMPFQK, sequences at least 90%, 95%, 98%, or 99% identical to the peptide sequences, sequences including them, and intervening protein sequences.
  • the SAA-derived protein fragments include sequences selected from peptide sequences EANYIGSDK, FFGHGAEDSLADQAANEWGR, GPGGVWAAEAISDAR, RGPGGVWAAEAISDAR, GNYDAAKRGPGGVW, DPNHFRPAGLPEK, sequences at least 90%, 95%, 98%, or 99% identical to the peptide sequences, sequences including them, and intervening protein sequences.
  • the at least one protein fragment includes a sequence selected from the CRP-derived peptide sequences GYSIFSYATK, RQDNEILIFWSK, APLTKPLK, ESDTSYVSLK, and YEVQGEVFTKPQLWP; the BTLA-derived peptide sequences YCANRPHVTWCK, YVTDVKSASERP, and RQSEHSILAGDPFELECPVK; the EGF-derived peptide sequences RLFWTDTGINPR, LCSDIDECEMGVPVCPPASSK, LFWIQYNR, RIYWVDLER, LYWCDAK, and CISEGEDATCQCLK; the FGA-derived peptide sequences GSESGIFTNTK, TVIGPDGHK, GDSTFESK, QFTSSTSYNR, TVIGPDGHKEVTK, and VTSGSTTTTR; the LGALS 9 -derived peptide sequences FEDGGYVVCNTR, FAVNFQTGFSGNDIAFHF
  • the at least one protein fragment includes a sequence selected from peptide sequences GYSIFSYATK, RQDNEIEIFWSK, YCANRPHVTWCK, RQSEHSIEAGDPFEEECPVK, APETKPEK, YEVQGEVFTKPQEWP, REFWTDTGINPR, ECSDIDECEMGVPVCPPASSK, EFWIQYNR, EYWCDAK, CISEGEDATCQCEK, GSESGIFTNTK, TVIGPDGHK, GDSTFESK, TVIGPDGHKEVTK, VTSGSTTTTR, FEDGGYVVCNTR, FAVNFQTGFSGNDIAFHFNPR, QNGSWGPEER,
  • VMVNGIEFVQYFHR THMPFQK
  • EANYIGSDK FFGHGAEDSEADQAANEWGR
  • GPGGVWAAEAISDAR RGPGGVWAAEAISDAR
  • DPNHFRPAGLPEK sequences at least 90%, 95%, 98%, or 99% identical to the peptide sequences, and sequences including them.
  • the at least one protein fragment includes a sequence selected from peptide sequences GYSIFSYATK (CRP), RQDNEILIFWSK (CRP), YCANRPHVTWCK (BTLA), RLFWTDTGINPR (EGF), GSESGIFTNTK (FGA), FEDGGYVVCNTR (LGALS9), and EANYIGSDK (SAA), sequences at least 90%, 95%, 98%, or 99% identical to the peptide sequences, and sequences including them.
  • the subject may be any subject, including a mammal such as a human or an animal subject. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human subject. It is noted that the present invention is suitable for veterinary applications.
  • the subject is a child. In some embodiments, the subject is an adult. In some embodiments, the subject is an elderly adult.
  • the presence of a bacterial infection in the subject does not preclude the presence of a viral infection at the same time, and vice versa. Accordingly, the subject may be suffering from both a bacterial and a viral infection.
  • the infection is chronic. In some embodiments, the infection is acute. In some embodiments, the infection is systemic. In some embodiments, the infection is local.
  • the infection is selected from: abscess, bacteremia, bronchitis, cellulitis, cholangitis, cholecystitis, colitis, cytomegalovirus (CMV) infection, dengue infection, dental infection, diverticulitis, empyema, endocarditis, Epstein-Barr virus (EBV) infection, folliculitis, herpes zoster infection, influenza, lower respiratory tract infection, measles, meningitis, mononucleosis, myositis, osteomyelitis, parainfluenza bronchitis, parotitis, peritonitis, pharyngitis, pneumonia, rickettsia infection, stemitis, upper respiratory tract infection (URTI), varicella- zoster virus (VZV) infection, and combinations thereof.
  • CMV cytomegalovirus
  • EBV Epstein-Barr virus
  • folliculitis herpes zoster infection
  • influenza lower respiratory tract
  • the infection is a URTI.
  • the URTI is selected from: bacterial tracheitis, bronchitis, cytomegalovirus (CMV) infection, epiglottitis, measles, meningitis, mumps, pneumonia, sinusitis, streptococcal pharyngitis (strep throat), and viral URTIs, such as those caused by adenovirus, coronavirus, influenza, parainfluenza virus respiratory syncytial virus (RSV), rhinovirus, and/or varicella zoster virus.
  • CMV cytomegalovirus
  • the URTI is caused by gram positive bacteria. In some embodiments, the URTI is caused by gram negative bacteria. In some embodiments, the URTI is caused by a bacterial agent selected from: Bordetella pertussis, Burkholderia pseudomallei, Chlamydophila pneumoniae, Corynebacterium diphtheriae, Haemophilus influenzae, Moraxella catarrhalis, Mycoplasma pneumoniae, Staphylococcus aureus, Streptococcus pyogenes (Group A Streptococcus), Streptococcus pneumoniae (Pneumococcus), and combinations thereof.
  • Bordetella pertussis Burkholderia pseudomallei
  • Chlamydophila pneumoniae Corynebacterium diphtheriae
  • Haemophilus influenzae Haemophilus influenzae
  • Moraxella catarrhalis Mycoplasma pneumoniae
  • Staphylococcus aureus Streptococcus
  • the URTI is caused by a viral agent selected from: adenoviruses, CMV, coronaviruses (e.g. SARS-CoV-2; COVID- 19), human metapneumovirus, influenza virus (including influenza virus type A, B, and/or C), measles virus, mumps virus, parainfluenza virus, respiratory syncytial virus, rhinoviruses, RSV, varicella zoster virus, and combinations thereof.
  • a viral agent selected from: adenoviruses, CMV, coronaviruses (e.g. SARS-CoV-2; COVID- 19), human metapneumovirus, influenza virus (including influenza virus type A, B, and/or C), measles virus, mumps virus, parainfluenza virus, respiratory syncytial virus, rhinoviruses, RSV, varicella zoster virus, and combinations thereof.
  • a method for predicting in a urine sample of a subject suspected of having an infection, whether the infection is a bacterial infection and/or a viral infection including; a. contacting a urine sample of the subject with one or more antibodies capable of specifically binding to at least one protein fragment derived from at least one protein selected from: CRP, BTLA, C0L6A1, CNTFR, EGF, FGA, ICOSLG, LGALS9, MXRA8, NTM, PROZ, SAA, SERPINA5, THBD, TNXB, VASN, and combinations thereof; b. determining binding of the one or more antibodies to the at least one peptide; and c. predicting whether the infection is bacterial and/or viral based on the determination in step (b).
  • the at least one protein is selected from BTLA, C0L6A1, CNTFR, EGF, FGA, ICOSLG, LGALS9, MXRA8, NTM, PROZ, SAA, SERPINA5, THBD, TNXB, and VASN.
  • the at least one protein is selected from CRP, BTLA, EGF, FGA, LGALS9, and SAA.
  • the at least one protein is selected from BTLA, EGF, FGA, LGALS9, and SAA.
  • the at least one protein fragment includes a sequence selected from peptide sequences GYSIFSYATK (CRP), RQDNEILIFWSK (CRP), YCANRPHVTWCK (BTLA), RLFWTDTGINPR (EGF), GSESGIFTNTK (FGA), FEDGGYVVCNTR (LGALS9), EANYIGSDK (SAA), sequences at least 90%, 95%, 98%, or 99% identical to the peptide sequences, sequences including them, and combinations thereof.
  • CRP GYSIFSYATK
  • RQDNEILIFWSK CPP
  • BTLA YCANRPHVTWCK
  • BTLA RLFWTDTGINPR
  • GSESGIFTNTK FGA
  • FEDGGYVVCNTR LGALS9
  • EANYIGSDK SAA
  • At least one of the antibodies capable of specifically binding to at least one protein fragment is an isolated polypeptide and/or an antibody disclosed hereinbelow.
  • step (a) Contacting the urine sample in step (a) is done at suitable conditions, which facilitate binding of antibodies to the protein fragments or peptides they are specific for.
  • antibodies may have a single specificity, i.e., capable of specifically binding to a single peptide, or may have multiple specificities (such as bispecific or trispecific antibodies, etc.), which are capable of binding to more than one peptide.
  • Detecting the antibodies binding to the protein fragments step (b) may be conducted by any suitable method, as also mentioned above.
  • the detecting may be done by detecting antibody- peptide complexes, by methods based, for example, on fluorescence, colorimetric assays, and magnetic beads, or on binding partners (such as biotin and avidin).
  • the antibodies may be labelled, such as fluorescently-labelled, radioactively labelled, or by any other label that may be later detected.
  • Methods for detecting antibody binding may include LFA, FACS, ELISA, a dot blot assay, a dipstick, an antibody chip, a multiplex bead immunoassay, etc.
  • protein fragments detected by antibodies in step (b) are considered to be above their respective threshold, which in this case is the threshold of detection of the protein fragment by the respective antibody under conditions used.
  • the predicting in step (c) may be done by methods known in the art as explained hereinabove, such as by visual detection of a color reaction, by imaging, by computer processing, etc.
  • the interpretation of the protein fragments detected in step (b) for the predicting in step (c) may be straightforward, such as when bacterial infection-related protein fragments are detected, the prediction is for a bacterial infection; and when viral infection-related protein fragments are detected, the prediction is for a viral infection.
  • the interpretation of the protein fragments detected in step (b) for the predicting in step (c) may not be straightforward and may require further processing, such as by a computation device.
  • the method further includes a treatment step. Since the method disclosed above allows to predict whether the infection is bacterial or viral, it allows to determine which treatment is suitable, such as an antibiotic (or antibacterial) treatment, or an antiviral treatment. It is also understood that the method facilitates avoiding unnecessarily taking antibiotics, when no bacterial infection is diagnosed by the methods of the invention, e.g., when no bacterial infection-related protein fragment is found to be present at a level above the respective threshold.
  • a treatment step Since the method disclosed above allows to predict whether the infection is bacterial or viral, it allows to determine which treatment is suitable, such as an antibiotic (or antibacterial) treatment, or an antiviral treatment. It is also understood that the method facilitates avoiding unnecessarily taking antibiotics, when no bacterial infection is diagnosed by the methods of the invention, e.g., when no bacterial infection-related protein fragment is found to be present at a level above the respective threshold.
  • the method further includes a step of treating the subject with an antibiotic when the infection is predicted to be a bacterial infection, or treating the subject with an antiviral treatment when the infection is predicted to be a viral infection.
  • the antibiotic or antiviral treatment may be adapted to the specific case, based on additional symptoms.
  • suitable antibiotics include broad- spectrum gram-positive antibiotics, such as vancomycin and linezolid; broad- spectrum gram-negative antibiotics, such as penicillin (e.g., piperacillin and tazobactam), cephalosporin (e.g., cefoperazone, cefotaxime, cefepime and cefpirome); imipenem (e.g., imipenem monohydrate); and aminoglycosides (e.g., gentamicin, tobramycin, amikacin, plazomicin, streptomycin, neomycin, and paromomycin); and combinations thereof.
  • penicillin e.g., piperacillin and tazobactam
  • cephalosporin e.g., cefoperazone, cefotaxime, cefepime and cefpirome
  • imipenem e.g., imipenem monohydrate
  • aminoglycosides e.g., gentamicin, tobramycin
  • the infection being a viral infection and not a bacterial infection
  • the benefit is in avoiding unnecessary antibiotics.
  • Suitable antiviral treatments may include broad spectrum anti-viral drugs, or drugs specific to certain viruses, based on symptoms or on further analysis.
  • Administration regimens and doses are generally known in the art and may be calculated based on specific features relevant to a specific case.
  • a method for treating a bacterial infection in a subject afflicted with an infection including: a. detecting in a urine sample of the subject a level of at least one protein fragment derived from at least one protein selected from: CRP, BTLA, C0L6A1, CNTFR, EGF, FGA, ICOSLG, LGALS9, MXRA8, NTM, PROZ, SAA, SERPINA5, THBD, TNXB, VASN, and combinations thereof; b. determining for the at least one protein fragment whether the level is above or below a respective threshold; c. predicting that the infection is bacterial based on the determination in step (b); and d. treating the subject with an antibacterial agent or antibiotics.
  • the term “afflicted with an infection” means that the subject is suffering from an infection but the infection causative agent is not known.
  • the subject presents with symptoms typical of an infection in general, such as fever, pain, feeling weak and/or tired, signs of inflammation, etc.
  • the subject presents with symptoms which are typical to a bacterial or to a viral infection.
  • the subject presents with symptoms which are typical to a specific infection.
  • it needs to be verified whether the infection is caused by a bacterial agent or by a viral agent.
  • treating refers to means of obtaining a desired physiological effect.
  • the effect may be therapeutic in terms of partially or completely curing a disease and/or symptoms attributed to the disease.
  • the term includes inhibiting the disease, i.e. arresting its development; or ameliorating the disease, i.e. causing regression of the disease, e.g., by eliminating or ameliorating its symptoms.
  • the at least one protein is selected from CRP, BTLA, C0L6A1, CNTFR, EGF, FGA, ICOSLG, LGALS9, MXRA8, NTM, PROZ, SAA, SERPINA5, THBD, TNXB, VASN.
  • the at least one protein is selected from CRP, BTLA, EGF, FGA, LGALS9, and SAA. In some embodiments, the at least one protein is selected from BTLA, EGF, FGA, LGALS9, and SAA.
  • predicting that the infection is bacterial is based on at least one protein fragment derived from CRP, FGA, and/or SAA being present at a level above the respective threshold. In some embodiments, predicting that the infection is bacterial is based on at least one protein fragment derived from CRP, FGA, and/or SAA being present at a level above the respective threshold. In some embodiments, predicting that the infection is bacterial is based on at least three protein fragments derived from CRP, FGA, and/or SAA being present at a level above the respective threshold.
  • a method for treating a viral infection in a subject afflicted with an infection including: a. detecting in a urine sample of the subject a level of at least one protein fragment derived from at least one protein selected from: CRP, BTLA, COL6A1, CNTFR, EGF, FGA, ICOSLG, LGALS9, MXRA8, NTM, PROZ, SAA, SERPINA5, THBD, TNXB, VASN, and combinations thereof; b. determining for the at least one protein fragment whether the level is above or below a respective threshold; c. predicting that the infection is viral based on the determination in step (b); and d. treating the subject with an antiviral agent.
  • the at least one protein is selected from CRP, BTLA, COL6A1, CNTFR, EGF, FGA, ICOSLG, LGALS9, MXRA8, NTM, PROZ, SAA, SERPINA5, THBD, TNXB, and VASN.
  • the at least one protein is selected from CRP, BTLA, EGF, FGA, LGALS9, and SAA. In some embodiments, the at least one protein is selected from BTLA, EGF, FGA, LGALS9, and SAA.
  • predicting that the infection is viral is based on at least one protein fragment derived from BTLA, EGF, and/or LGALS9 being present at a level above the respective threshold. In some embodiments, predicting that the infection is viral is based on at least one protein fragment derived from BTLA, EGF, and/or LGALS9 being present at a level above the respective threshold. In some embodiments, predicting that the infection is viral is based on at least three protein fragments derived from BTLA, EGF, and/or LGALS9 being present at a level above the respective threshold.
  • a method for predicting efficacy of a treatment in a subject afflicted with a bacterial and/or a viral infection including: a. detecting in a first urine sample of the subject a first level of at least one protein fragment derived from at least one protein selected from: CRP, BTLA, C0L6A1, CNTFR, EGF, FGA, ICOSLG, LGALS9, MXRA8, NTM, PROZ, SAA, SERPINA5, THBD, TNXB, VASN, and combinations thereof; b. administering to the subject a treatment including an antibiotic and/or an antiviral agent; c.
  • step (d) detecting in a second urine sample of the subject a second level of the at least one peptide; d. determining for the at least one protein fragment whether the second level is above or below the first level; and e. predicting whether the treatment is effective based on the determination in step (d).
  • the at least one protein is selected from CRP, BTLA, C0L6A1, CNTFR, EGF, FGA, ICOSLG, LGALS9, MXRA8, NTM, PROZ, SAA, SERPINA5, THBD, TNXB, VASN.
  • the at least one protein is selected from CRP, BTLA, EGF, FGA, LGALS9, and SAA. In some embodiments, the at least one protein is selected from BTLA, EGF, FGA, LGALS9, and SAA.
  • the first urine sample is taken prior to start of treatment. In some embodiments, the first urine sample is taken after treatment has already started.
  • the first level of the protein fragment represents the level of the protein fragment prior to the treatment session which is evaluated, and the second level of the protein fragments represents the level of the protein fragment after the evaluated treatment session.
  • the detecting in steps (a) and (c) includes measuring levels of the protein fragments. In some embodiments, the detecting in steps (a) and (c) does not include measuring levels, but rather detecting the presence, such as by using a specific binding agent, as explained above.
  • step (d) involves comparing the first level with the second level.
  • step (d) involves comparing the results between step (a) and step (c), i.e., if a protein fragment was detected in step (a) and not in step (c) then it is determined that the second level of the protein fragment is below the first level, and if a protein fragment was detected in step (c) and not in step (a) then it is determined that the second level of the protein fragment is above the first level.
  • step (d) determines whether the infection is a bacterial infection and the protein fragment is a bacterial infection-related peptide. For example, if the infection is a bacterial infection and the protein fragment is a bacterial infection-related peptide, then the second level being below the first level indicates that the treatment is effective. Similarly, if the infection is a viral infection and the protein fragment is a viral infection-related peptide, then the second level being below the first level indicates that the treatment is effective.
  • kits for predicting in a urine sample whether an infection is a bacterial infection or a viral infection including: a. one or more binding agents each capable of binding to at least one protein fragment derived from at least two proteins selected from CRP, BTLA, C0L6A1, CNTFR, EGF, FGA, ICOSLG, LGALS9, MXRA8, NTM, PROZ, SAA, SERPINA5, THBD, TNXB, VASN; b.
  • the at least one protein is selected from CRP, BTLA, C0L6A1, CNTFR, EGF, FGA, ICOSLG, LGALS9, MXRA8, NTM, PROZ, SAA, SERPINA5, THBD, TNXB, VASN.
  • the at least one protein is selected from CRP, BTLA, EGF, FGA, LGALS9, and SAA. In some embodiments, the at least one protein is selected from BTLA, EGF, FGA, LGALS9, and SAA.
  • the instructions for use indicate that the detection of at least one bacterial infection-related protein fragment indicating levels above the respective threshold, predicts a bacterial infection; and/or the detection of at least one viral infection-related protein fragment indicating levels above the respective threshold, predicts a viral infection.
  • the instructions for use indicate that the detection of at least one protein fragment derived from CRP, FGA, and/or SAA, indicating levels above the respective threshold, predicts a bacterial infection; and/or the detection of at least one protein fragment derived from BTLA, EGF, and/or LGALS9, indicating levels above the respective threshold, predicts a viral infection.
  • the kit further includes reagents for use with an assay based on a fluorescent assay, a colorimetric assay, a radioactive assay, a magnetic beads assay, etc.
  • the kit further includes reagents for use with LFA, FACS, ELISA, a dipstick, an antibody chip, a multiplex bead immunoassay, or a dot blot, all assays known in the art.
  • the one or more binding agents include antibodies, such as monoclonal antibodies, polyclonal antibodies, recombinant antibodies, rodent antibodies humanized antibodies, and chimeric antibodies; functional fragments of antibodies capable of specifically binding to protein fragments, such as scFv, Fv, Fab, Fab’, and F(ab')2 fragments; aptamers, i.e. short sequences of artificial DNA, RNA, XNA (xeno nucleic acid), or peptide, that are capable of binding to a specific target molecule; and/or any agent that is capable of specifically binding to a peptide.
  • agents capable of binding to more than a single protein fragment include antibodies, such as bispecific or trispecific antibodies or similar constructs which are designed to bind to more than a single entity.
  • the one or more binding agents are antibodies. In some embodiments, the one or more binding agents are monoclonal antibodies. In some embodiments, the one or more binding agents are aptamers.
  • the one or more binding agents include at least one isolated polypeptide and/or antibody disclosed hereinbelow.
  • the kit further includes reagents and/or a device for performing a lateral flow assay.
  • Lateral flow assays are known in the art, and generally include a series of pads including capillary beds, along which a tested urine (fluid) sample moves. Some of the pads include one or more immobilized binding agents (such as antibodies) and necessary reagents, such that when a protein fragment in the urine sample binds to an immobilized binding agent, a color reaction is produces, by principles similar to the affinity chromatography principles on which an ELISA test is based. This color reaction provides a positive detection of the bound peptide.
  • immobilized binding agents such as antibodies
  • a method for predicting inflammation (predicting blood CRP levels)
  • Blood CRP is a known inflammation-related protein, and its level in blood is often used to determine the level of inflammation. Nevertheless, the ability to predict an elevated blood CRP level by a urine test would facilitate fast and simple testing by any subject suspected of suffering from inflammation without the need for medical personnel. The result would help to determine whether to seek medical treatment. It is noted that although CRP has been tested in blood for many years, it is not being tested in urine and tests which are used for measuring CRP levels in blood are not suitable for testing in urine samples.
  • the urinary levels of several protein fragments could be correlated with the blood levels of CRP, and could therefore serve as predictive markers for elevated blood CRP levels and therefore for inflammation.
  • a method for predicting an elevated blood CRP level by a urine test including: a. detecting in a urine sample of the subject a level of at least one protein fragment including a sequence selected from peptide sequences presented in Table 10, sequences at least 90%, 95%, 98%, or 99% identical to the peptide sequences, sequences including them, and intervening protein sequences; b. determining for the at least one protein fragment whether the level is above or below a respective threshold; and c. predicting whether blood CRP level is elevated, based on the determination in step (b).
  • the subject is a mammal. In some embodiments, the subject is a human subject. It is noted that the present invention is suitable for veterinary applications.
  • the term “elevated” with reference to blood CRP relates to the prediction of a blood CRP levels that is higher than normal and medically related to inflammation, or an increase in blood CRP levels.
  • the level of the indicative protein fragments in the urine does not necessarily predict a specific level of blood CRP, but generally predicts that blood CRP is elevated, thereby predicting the presence of inflammation.
  • predicting an elevated blood CRP level means that blood CRP level has a high probability of being elevated.
  • the at least one protein fragment is selected from protein fragments derived from CRP, SAA, LRG1 (Leucine -rich repeat-containing G protein-coupled receptor 1), and/or LBP (lipopolysaccharide binding protein).
  • the CRP-derived protein fragments include sequences selected from peptide sequences GYSIFSYATK, RQDNEILIFWSK, ESDTSYVSLK, APLTKPLK, YEVQGEVFTKPQLWP, sequences at least 90%, 95%, 98%, or 99% identical to the peptide sequences, sequences including them, and intervening protein sequences.
  • the SAA-derived protein fragments include sequences selected from peptide sequences FFGHGAEDSLADQAANEWGR, EANYIGSDK, GPLQLER, GPGGAWAAEVISNAR sequences at least 90%, 95%, 98%, or 99% identical to the peptide sequences, sequences including them, and intervening protein sequences.
  • the LRG1 -derived protein fragments include sequence selected from peptide sequences GPLQLER, YLFLNGNK, VAAGAFQGLR, LHLEGNK, WLQAQKDK, GQTLLAVAK, TLDLGENQLETLPPDLLR, ALGHLDLSGNR, ENQLEVLEVSWLHGLK, DLLLPQPDLR, LQELHLSSNGLESLSPEFLR, CAGPEAVKGQTLLAVAK, YLFLNGNKLAR, sequences at least 90%, 95%, 98%, or 99% identical to the peptide sequences, sequences including them, and intervening protein sequences.
  • the LBP-derived protein fragments include sequences selected from peptide sequences LAEGFPLPLLKR, LAEGFPLPLLK, ATAQMLEVMFK, ITLPDFTGDLR, ITGFLKPGK, sequences at least 90%, 95%, 98%, or 99% identical to the peptide sequences, sequences including them, and intervening protein sequences.
  • the at least one protein fragment includes a sequence selected from peptide sequences GYSIFSYATK, RQDNEILIFWSK, ESDTSYVSLK, APLTKPLK, YEVQGEVFTKPQLWP, FFGHGAEDSLADQAANEWGR, EANYIGSDK, GPLQLER, YLFLNGNK, VAAGAFQGLR, LHLEGNK, WLQAQKDK, GQTLLAVAK, TLDLGENQLETLPPDLLR, ALGHLDLSGNR, ENQLEVLEVSWLHGLK, DLLLPQPDLR, LQELHLSSNGLESLSPEFLR, CAGPEAVKGQTLLAVAK, YLFLNGNKLAR, GPGGAWAAEVISNAR, LAEGFPLPLLKR, LAEGFPLPLLK, ATAQMLEVMFK, ITLPDFTGDLR, ITGFLKPGK, sequences at least 90%, 95%, 98%, or 99% identical to the peptide
  • a kit for predicting inflammation (predicting blood CRP levels
  • kits for predicting an elevated blood CRP level by a urine test including: a. one or more binding agents each capable of binding to at least one protein fragment having a sequence selected from peptide sequences presented in Table 10, sequences at least 90%, 95%, 98%, or 99% identical to the peptide sequences, sequences including them, and intervening protein sequences; b. at least one reagent for detecting the binding of the one or more binding agents to the at least one protein fragment in a urine sample, thereby indicating that the level of the at least one protein fragment bound by the one or more binding agents in the urine sample is above a respective threshold; and c. instructions for use.
  • a method for treating an inflammation in a subject including: a. detecting in a urine sample of the subject a level of at least one protein fragment having a sequence selected from peptide sequences presented in Table 10, sequences at least 90%, 95%, 98%, or 99% identical to the peptide sequences, sequences including them, and intervening protein sequences; b. determining for the at least one protein fragment whether the level is above or below a respective threshold; c. predicting that blood CRP level is elevated, based on the determination in step (b); and d. treating the subject with an anti-inflammatory agent.
  • the anti-inflammatory agent is selected from anti-inflammatory drugs, immunosuppressants, and corticosteroids.
  • anti-inflammatory drugs include, without limitation, non-steroidal anti-inflammatory drugs (NSAIDs) and anti-cytokine agents such as anti-IL6 mAb (e.g. Actemra), anti-IL-1 mAb (e.g. Anakinra) and anti-TNF mAb (e.g. Remicade);
  • immunosuppressants or immunosuppressive agents have negative immunoregulatory activities and include e.g. cyclosporine and methotrexate;
  • corticosteroids have both antiinflammatory and immunoregulatory activity and include e.g. prednisone, dexamethasone, and hydrocortisone.
  • a method for predicting efficacy of a treatment of inflammation in a subject including: a. detecting in a first urine sample of the subject a first level of at least one protein fragment including a sequence selected from peptide sequences presented in Table 10, sequences at least 90%, 95%, 98%, or 99% identical to the peptide sequences, sequences including them, and intervening protein sequences; b. administering to the subject an antibiotic or an anti-inflammatory treatment; c. detecting in a second urine sample of the subject a second level of the at least one protein fragment; d. determining for the at least one protein fragment whether the second level is above or below the first level, thereby predicting increased or a decreased inflammation, respectively; and e. predicting whether the treatment is effective based on whether the determination in step (d) indicates decreased inflammation.
  • the invention also provides methods, as described above, based on combinations of proteins and/or protein fragments disclosed herein with respect to bacterial and/or viral infections and proteins and/or protein fragments disclosed herein with respect to predicting an elevated blood CRP level.
  • the invention also provides kits, as described above, based on combinations of proteins and/or protein fragments disclosed herein with respect to bacterial and/or viral infections and proteins and/or protein fragments disclosed herein with respect to predicting elevated blood CRP levels.
  • Methods of administration include, but are not limited to, parenteral, e.g., intravenous, intraperitoneal, intramuscular, subcutaneous; mucosal (e.g., oral, sublingual, intranasal, buccal, vaginal, rectal, intraocular), intrathecal, topical, and intradermal routes. Administration can be systemic or local.
  • Table 1 List of peptides mentioned in the application with SEQ ID Nos.
  • the present invention further relates to the development of polypeptides, such as antibodies, which specifically bind to protein fragments found in urine which are useful for urinary diagnosis of inflammation and distinguishing bacterial from viral infections.
  • the antibodies of the invention may be used for detecting the relevant protein fragments in urine of patient, thereby facilitating diagnosis of the relevant conditions.
  • urinary conditions means conditions similar to the urine environment, having a similar pH (such as about 6) and/or some similar ingredients, such as urea, and certain typical salts.
  • Examples for solutions which simulate urinary conditions are artificial or synthetic (or simulated) urine products such as Biochemazone artificial urine products.
  • the urinary conditions mean having a pH of about 4.6-8, about 5-7, about 5.5-6.5, or about 6. In some embodiments, the urinary conditions mean including at least one of urea, creatinine, uric acid, and ammonia. In some embodiments, urinary conditions further means including certain salts or proteins.
  • any one of the protein-derived fragments may be an isolated peptide or a peptide which is part of polypeptide or a protein, such as the protein from which the protein fragment peptide is derived, or a part thereof.
  • the isolated polypeptides e.g. antibodies
  • the isolated polypeptides may also be capable of binding larger peptides or polypeptides including the protein- derived fragments or peptides, as well as the proteins from which the peptides are derived.
  • Each of the isolated polypeptides includes two variable regions: a heavy chain variable (VH) region and a light chain variable (VE) region, and six complementarity-determining regions (CDRs), including VH region CDRs: VH-CDR1, VH-CDR2, and VH-CDR3; and VL region CDRs: VL-CDR1, VL-CDR2, and VL-CDR3.
  • VH heavy chain variable
  • VE light chain variable
  • CDRs complementarity-determining regions
  • the six CDRs are defined by any standard method known in the art based on sequences of a VH and a VL regions. In some embodiments, the six CDRs are defined by the Kabat method.
  • the six CDRs are defined by their sequence.
  • the method of defining the six CDRs may be any method acceptable in the art, including any of the methods mentioned herein (Kabat, Chothia, IM GT, and AbM), for example the Kabat method.
  • the isolated polypeptide binds the protein fragment of peptide under conditions similar to urine environment, such as including certain salts, urea, and a similar pH (e.g. about 6).
  • amino acid sequences of the isolated polypeptide are defined by the nucleotide sequences encoding them.
  • an isolated polypeptide capable of specifically binding a CRP-derived protein fragment or peptide, wherein the isolated polypeptide includes a VH region including three CDRs (VH-CDR1, VH-CDR2, VH-CDR3) and a VL region including three CDRs (VL-CDR1, VL-CDR2, and VL-CDR3), and the six CDRs are defined by a standard method selected from Kabat, Chothia, IMGT, and AbM, based on VH and VL regions sequences set forth in: SEQ ID Nos. 181 and 182, respectively; SEQ ID Nos. 201 and 202, respectively; SEQ ID Nos. 221 and 222, respectively; SEQ ID Nos. 241 and 242, respectively; SEQ ID Nos. 261 and 262, respectively; or SEQ ID Nos. 281 and 282; respectively.
  • the VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3, include, or consist of, sequences identical or substantially identical to sequences set forth in:
  • the VH and the VL regions include, or consist of, sequences identical or substantially identical to sequences set forth in:
  • an isolated polypeptide capable of specifically binding a CRP-derived protein fragment or peptide
  • the isolated polypeptide includes a VH region including three CDRs (VH-CDR1, VH-CDR2, VH-CDR3) and a VL region including three CDRs (VL-CDR1, VL-CDR2, and VL-CDR3)
  • the six CDRs are defined by a standard method selected from Kabat, Chothia, IM GT, and AbM, based on VH and VL regions encoded by nucleotide sequences set forth in: 191 and 192, respectively; SEQ ID Nos. 211 and 212, respectively; SEQ ID Nos. 231 and 232, respectively; SEQ ID Nos. 251 and 252, respectively; SEQ ID Nos. 271 and 272, respectively; or SEQ ID Nos. 291 and 292, respectively.
  • the VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3, are encoded by nucleotide sequences including, or consisting of, sequences identical or substantially identical to sequences set forth in:
  • the VH and the VL regions are encoded by nucleotide sequences including, or consisting of, sequences identical or substantially identical to sequences set forth in:
  • the CRP-derived protein fragment or peptide includes, or consists of, a sequence selected from GYSIFSYATKRQDNEILIFWSK (SEQ ID No: 861, CRP-P01) and RQDNEILIFWSK (SEQ ID No: 862, CRP-P02).
  • the CRP-derived protein fragment or peptide includes, or consists of, a sequence set forth in SEQ ID No: 861.
  • the CRP-derived protein fragment or peptide includes, or consists of, a sequence set forth in SEQ ID No: 862.
  • a monoclonal antibody capable of specifically binding to a CRP-derived protein fragment or peptide including, or consisting of, a sequence selected from SEQ ID No: 861 (CRP-P01) and SEQ ID No: 862 (CRP-P02), under urinary conditions.
  • a monoclonal antibody capable of specifically binding to a CRP-derived protein fragment or peptide including, or consisting of, a sequence set forth in SEQ ID No: 862 (CRP-P02), under urinary conditions.
  • an isolated polypeptide capable of specifically binding a BTLA-derived protein fragment or peptide, wherein the isolated polypeptide includes a VH region including three CDRs (VH-CDR1, VH-CDR2, VH-CDR3) and a VL region including three CDRs (VL-CDR1, VL-CDR2, and VL-CDR3), and the six CDRs are defined by a standard method selected from Kabat, Chothia, IMGT, and AbM, based on VH and VL regions sequences set forth in: SEQ ID Nos. 01 and 02, respectively; SEQ ID Nos. 21 and 22, respectively; SEQ ID Nos. 41 and 42, respectively; SEQ ID Nos.
  • SEQ ID Nos. 81 and 82 respectively; SEQ ID Nos. 101 and 102, respectively; SEQ ID Nos. 121 and 122, respectively; SEQ ID Nos. 141 and 142, respectively; or SEQ ID Nos. 161 and 162, respectively.
  • the VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3, include, or consist of, sequences identical or substantially identical to sequences set forth in:
  • the VH and the VL regions include, or consist of, sequences identical or substantially identical to sequences set forth in:
  • an isolated polypeptide capable of specifically binding a BTLA-derived protein fragment or peptide, wherein the isolated polypeptide includes a VH region including three CDRs (VH-CDR1, VH-CDR2, VH-CDR3) and a VL region including three CDRs (VL-CDR1, VL-CDR2, and VL-CDR3), and the six CDRs are defined by a standard method selected from Kabat, Chothia, IM GT, and AbM, based on VH and VL regions encoded by nucleotide sequences set forth in: SEQ ID Nos. 11 and 12, respectively; SEQ ID Nos. 31 and 32, respectively; SEQ ID Nos.
  • SEQ ID Nos. 71 and 72 respectively; SEQ ID Nos. 91 and 92, respectively; SEQ ID Nos. Ill and 112, respectively; SEQ ID Nos. 131 and 132, respectively; SEQ ID Nos. 151 and 152, respectively; or SEQ ID Nos. 171 and 172, respectively.
  • the VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3, are encoded by nucleotide sequences including, or consisting of, sequences identical or substantially identical to sequences set forth in:
  • the VH and the VL regions are encoded by nucleotide sequences including, or consisting of, sequences identical or substantially identical to sequences set forth in:
  • the BTLA-derived protein fragment or peptide includes, or consists of, a sequence selected from RQSEHSILAGDPFELECPVKYCANRPHVTWCK (SEQ ID No: 867, BTLA-P01) and YCANRPHVTWCK (SEQ ID No: 868, BTLA-P02).
  • the BTLA-derived protein fragment or peptide includes, or consists of, a sequence set forth in SEQ ID No: 867.
  • the BTLA-derived protein fragment or peptide includes, or consists of, a sequence set forth in SEQ ID No: 868.
  • an antibody capable of specifically binding to a BTLA-derived protein fragment or peptide including, or consisting of, a sequence selected from SEQ ID No: 867 (BTLA-P01) and SEQ ID No: 868 (BTLA-P02), under urinary conditions.
  • an antibody capable of specifically binding to a BTLA-derived protein fragment or peptide including, or consisting of, a sequence set forth in SEQ ID No: 868, under urinary conditions.
  • an isolated polypeptide capable of specifically binding a LGALS9-derived protein fragment or peptide
  • the isolated polypeptide includes a VH region including three CDRs (VH-CDR1, VH-CDR2, VH-CDR3) and a VL region including three CDRs (VL-CDR1, VL-CDR2, and VL-CDR3)
  • the six CDRs are defined by a standard method selected from Kabat, Chothia, IMGT, and AbM, based on VH and VL regions sequences set forth in: SEQ ID Nos. 661 and 662, respectively.
  • the VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3, include, or consist of, sequences identical or substantially identical to sequences set forth in SEQ ID Nos. 663, 664, 665, 666, 667, and 668, respectively.
  • the VH and the VL include, or consist of, sequences identical or substantially identical to sequences set forth in SEQ ID Nos. 661 and 662, respectively.
  • an isolated polypeptide capable of specifically binding a LGALS9-derived protein fragment or peptide
  • the isolated polypeptide includes a VH including three CDRs (VH-CDR1, VH-CDR2, VH-CDR3) and a VL including three CDRs (VL-CDR1, VL-CDR2, and VL-CDR3)
  • the six CDRs are defined by a standard method selected from Kabat, Chothia, IMGT, and AbM, based on VH and VL regions encoded by nucleotide sequences set forth in: SEQ ID Nos. 671 and 672, respectively.
  • the VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3, are encoded by nucleotide sequences including, or consisting of, sequences identical or substantially identical to sequences set forth in SEQ ID Nos. 673, 674, 675, 676, 677, and 678, respectively.
  • the VH and the VL regions are encoded by nucleotide sequences including, or consisting of, sequences identical or substantially identical to sequences set forth in SEQ ID Nos. 671 and 672, respectively.
  • the LGALS9-derived protein fragment or peptide includes, or consists of, a sequence selected from FEDGGYVVCNTRQNGSWGPEER (SEQ ID No: 882, LGAL-P01) and FEDGGYVVCNT (SEQ ID No: 883, LGAL-P02).
  • the LGALS9-derived protein fragment or peptide includes, or consists of, a sequence set forth in SEQ ID No: 882.
  • the LGALS9-derived protein fragment or peptide includes, or consists of, a sequence set forth in SEQ ID No: 883.
  • an antibody capable of specifically binding to an LGALS9-derived protein fragment or peptide including, or consisting of, a sequence selected from SEQ ID No: 882 (LGAL-P01) and SEQ ID No: 883 (LGAL-P02), under urinary conditions.
  • an antibody capable of specifically binding to an LGALS9- derived protein fragment or peptide including, or consisting of, a sequence set forth in SEQ ID No: 882, under urinary conditions.
  • an antibody capable of specifically binding to an LGALS9-derived protein fragment or peptide including, or consisting of, a sequence set forth in SEQ ID No: 883, under urinary conditions.
  • Anti-SAA antibodies including, or consisting of, a sequence selected from SEQ ID No: 882 (LGAL-P01) and SEQ ID No: 883 (LGAL-P02), under urinary conditions.
  • an antibody capable of specifically binding to an LGALS9-derived protein fragment or peptide including, or consisting of, a sequence set forth in SEQ ID No: 883
  • an isolated polypeptide capable of specifically binding a SAA-derived protein fragment or peptide, wherein the isolated polypeptide includes a VH region including three CDRs (VH-CDR1, VH-CDR2, VH-CDR3) and a VL region including three CDRs (VL-CDR1, VL-CDR2, and VL-CDR3), and the six CDRs are defined by a standard method selected from Kabat, Chothia, IMGT, and AbM, based on VH and VL regions sequences set forth in: SEQ ID Nos. 681 and 682, respectively; SEQ ID Nos. 701 and 702, respectively; SEQ ID Nos. 721 and 722, respectively; SEQ ID Nos.
  • the VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3, include, or consist of, sequences identical or substantially identical to sequences set forth in:
  • the VH and the VL regions include, or consist of, sequences identical or substantially identical to sequences set forth in:
  • an isolated polypeptide capable of specifically binding a SAA-derived protein fragment or peptide, wherein the isolated polypeptide includes a VH region including three CDRs (VH-CDR1, VH-CDR2, VH-CDR3) and a VL region including three CDRs (VL-CDR1, VL-CDR2, and VL-CDR3), and the six CDRs are defined by a standard method selected from Kabat, Chothia, IM GT, and AbM, based on VH and VL regions encoded by nucleotide sequences set forth in: SEQ ID Nos. 691 and 692, respectively; SEQ ID Nos. 711 and 712, respectively; SEQ ID Nos.
  • the VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3, are encoded by nucleotide sequences including, or consisting of, sequences identical or substantially identical to sequences set forth in:
  • the VH and the VL regions are encoded by nucleotide sequences including, or consisting of, sequences identical or substantially identical to sequences set forth in: SEQ ID Nos. 691 and 692, respectively;
  • the SAA-derived protein fragment or peptide includes, or consists of, a sequence selected from GAEDSLADQAANKWGR (SEQ ID No: 889, SAA-P01), GNYDAAKRGPGGAWAAEVITDAR (SEQ ID No: 890, SAA-P02),
  • GAEDSLADQAANEWGR (SEQ ID No: 891, SAA-P03), and
  • the SAA-derived protein fragment or peptide includes, or consists of, a sequence set forth in SEQ ID No: 889. In some embodiments, the SAA-derived protein fragment or peptide includes, or consists of, a sequence set forth in SEQ ID No: 890. In some embodiments, the SAA-derived protein fragment or peptide includes, or consists of, a sequence set forth in SEQ ID No: 891. In some embodiments, the SAA-derived protein fragment or peptide includes, or consists of, a sequence set forth in SEQ ID No: 892.
  • an antibody capable of specifically binding to an SAA-derived protein fragment or peptide including, or consisting of, a sequence selected from SEQ ID No: 889 (SAA-P01), SEQ ID No: 890 (SAA-P02), SEQ ID No: 891 (SAA-P03), and SEQ ID No: 892 (SAA-P04), under urinary conditions.
  • an antibody capable of specifically binding to an SAA-derived protein fragment or peptide including, or consisting of, a sequence set forth in SEQ ID No: 889, under urinary conditions.
  • an isolated polypeptide capable of specifically binding a EGF-derived protein fragment or peptide, wherein the isolated polypeptide includes a VH region including three CDRs (VH-CDR1, VH-CDR2, VH-CDR3) and a VL region including three CDRs (VL-CDR1, VL-CDR2, and VL-CDR3), and the six CDRs are defined by a standard method selected from Kabat, Chothia, IMGT, and AbM, based on VH and VL regions sequences set forth in: SEQ ID Nos. 301 and 302, respectively; SEQ ID Nos. 321 and 322, respectively; SEQ ID Nos. 341 and 342, respectively; SEQ ID Nos.
  • VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3, include, or consist of, sequences identical or substantially identical to sequences set forth in:
  • the VH and the VL regions include, or consist of, sequences identical or substantially identical to sequences set forth in:
  • an isolated polypeptide capable of specifically binding a EGF-derived protein fragment or peptide
  • the isolated polypeptide includes a VH region including three CDRs (VH-CDR1, VH-CDR2, VH-CDR3) and a VL region including three CDRs (VL-CDR1, VL-CDR2, and VL-CDR3)
  • the six CDRs are defined by a standard method selected from Kabat, Chothia, IM GT, and AbM, based on VH and VL regions encoded by nucleotide sequences set forth in: SEQ ID Nos. 311 and 312, respectively; SEQ ID Nos. 331 and 332, respectively; SEQ ID Nos.
  • the VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3, are encoded by nucleotide sequences including, or consisting of, sequences identical or substantially identical to sequences set forth in:
  • the VH and the VL regions are encoded by nucleotide sequences including, or consisting of, sequences identical or substantially identical to sequences set forth in:
  • the EGF-derived protein fragment or peptide includes, or consists of, a sequence selected from RIYWVDLER (SEQ ID No: 870, EGF-P01) and RLFWTDTGINPR (SEQ ID No: 871, EGF-P02).
  • the EGF-derived protein fragment or peptide includes, or consists of, a sequence set forth in SEQ ID No: 870.
  • the EGF- derived protein fragment or peptide includes, or consists of, a sequence set forth in SEQ ID No: 871.
  • an antibody capable of specifically binding to an EGF-derived protein fragment or peptide including, or consisting of, a sequence selected from SEQ ID No: 870 (EGF-P01) and SEQ ID No: 871 (EGF-P02), under urinary conditions.
  • an isolated polypeptide capable of specifically binding a FGA-derived protein fragment or protein fragment or peptide, wherein the isolated polypeptide includes a VH region including three CDRs (VH-CDR1, VH-CDR2, VH-CDR3) and a VL region including three CDRs (VL-CDR1, VL-CDR2, and VL-CDR3), and the six CDRs are defined by a standard method selected from Kabat, Chothia, IMGT, and AbM, based on VH and VL regions sequences set forth in: SEQ ID Nos. 481 and 482, respectively; SEQ ID Nos. 501 and 502, respectively; SEQ ID Nos.
  • the VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3, include, or consist of, sequences identical or substantially identical to sequences set forth in:
  • the VH and the VL regions include, or consist of, sequences identical or substantially identical to sequences set forth in:
  • an isolated polypeptide capable of specifically binding a FGA-derived protein fragment or peptide, wherein the isolated polypeptide includes a VH region including three CDRs (VH-CDR1, VH-CDR2, VH-CDR3) and a VL region including three CDRs (VL-CDR1, VL-CDR2, and VL-CDR3), and the six CDRs are defined by a standard method selected from Kabat, Chothia, IM GT, and AbM, based on VH and VL regions encoded by nucleotide sequences set forth in: SEQ ID Nos. 491 and 492, respectively; SEQ ID Nos. 511 and 512, respectively; SEQ ID Nos.
  • the VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3, are encoded by nucleotide sequences including, or consisting of, sequences identical or substantially identical to sequences set forth in:
  • the VH and the VL regions are encoded by nucleotide sequences including, or consisting of, sequences identical or substantially identical to sequences set forth in:
  • the FGA-derived protein fragment or peptide includes, or consists of, a sequence selected from GSESGIFTNTK (SEQ ID No: 876, FGA-P01) and QFTSSTSYNR (SEQ ID No: 877, FGA-P02).
  • the FGA-derived protein fragment or peptide includes, or consists of, a sequence set forth in SEQ ID No: 876.
  • the FGA-derived protein fragment or peptide includes, or consists of, a sequence set forth in SEQ ID No: 877.
  • an antibody capable of specifically binding to an FGA-derived protein fragment or peptide including, or consisting of, a sequence selected from SEQ ID No: 876 (FGA-P01) and SEQ ID No: 877 (FGA-P02), under urinary conditions.
  • the CDRs define the binding specificity of the antibody and are therefore sufficient to define the antibody.
  • the CDRs may be engrafted in any polypeptide sequence suitable to provide the general 3D structure of the antibody in order to carry out the specific binding. Accordingly, the CDRs do not necessarily have to be linked by any specific antibody sequences, as long as they are connected by sequences which provide the general paratope 3D structure. It is also appreciated that any carrier structure that is capable of assuming or mimicking a structure of an antibody paratope, may be engrafted with the specific CDRs of the invention to provide the isolated polypeptide of the invention with the same specificity, and is intended to be encompassed by the present invention.
  • the isolated polypeptide may be in any form suitable for detection of the respective protein fragment or peptide.
  • suitable forms include: an antibody or an antigen-binding fragment thereof, a single-chain variable fragment (scFv), a chimeric or a humanized antibody or antigen-binding fragment thereof, and a chimeric antigen receptor (CAR)-B.
  • antibody is an immunoglobulin having two heavy chains and two light chains, each chain having a variable region and a constant region, and wherein the variable regions of the heavy and light chains form antigen binding regions.
  • Antigen-binding fragments of an antibody include an Fv fragment, an Fab fragment, and an F(ab’)2 fragment.
  • Fv fragment variable
  • Fab fragment antigen binding
  • CHI region of the heavy and light chains. It may be obtained by a papain digestion above the hinge region, such that the hinge region is not included.
  • F(ab’)2 relates to two Fab regions linked by a disulfide bond of the hinge region. It may be obtained by a pepsin digestion below the hinge region, such that the hinge region is included and connects the two Fab fragments.
  • scFv relates to a fusion protein which includes the variable regions of the heavy and light chains connected by a short linker to make a single polypeptide chain.
  • chimeric as used herein relates to an antibody or fragment thereof including sequences from more than one species.
  • humanized relates to an antibody or fragment thereof which is produced in a non-human species (such as a mouse) but includes human sequences.
  • the framework sequences which separate the CDRs in the variable region may be replaced, or partly replaced, with human sequences.
  • CAR-B relates to a B-cell antigen receptor in which the signaling domains have been fused to an antigen recognition domain specific to a certain antigen, such as from a monoclonal antibody.
  • the isolated polypeptide is selected from an antibody, an Fv fragment, an Fab fragment, an F(ab’)2 fragment, an scFv, a chimeric or a humanized antibody or antibody fragment, and a CAR-B .
  • the isolated polypeptide is an antibody.
  • the antibody is a monoclonal antibody.
  • the Fv fragment, Fab fragment, F(ab’)2 fragment, scFv, chimeric or humanized antibody or single domain, or antigen-binding fragment thereof, and/or CAR-B are derived from a monoclonal antibody, or based on a sequence derived from a monoclonal antibody.
  • the antibody when the antibody includes a C-region, it may be derived from any isotype suitable for the desired use, including the common isotypes IgG, IgM, IgD, IgA, or IgE, and a combination thereof. Additionally, the light chain may be kappa or lambda, or a combination thereof. In some embodiments, the heavy chain C-region is derived from an IgG. In some embodiments, the light chain is kappa. In some embodiments, the light chain is lambda.
  • specific binding relates to binding that is sufficiently specific to detect the respective protein or protein fragment or peptide, without significant background of other proteins, protein fragments, or peptides.
  • specific binding is defined as binding with an EC50 (half maximal effective concentration) of less than about 10, 8, 5, 2, 1, 0.8, 0.5, 0.4, 0.3, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, or 0.04 nM.
  • specific binding is defined as binding with an EC50 of less than about 10.
  • specific binding is defined as binding with an EC50 of less than about 1.
  • specific binding is defined as binding with an EC50 of less than about 0.5.
  • specific binding is defined as binding with an association rate constant (Ka) of at least about 10’ 9 , 10’ 8 , or 10’ 7 M 1 .
  • the isolated polypeptide is further conjugated to a functional agent for using in various applications.
  • functional agents may include fluorophores (fluorescent dyes) such as Fluorescein isothiocyanate (FITC), Phycoerythrin (PE), Allophycocyanin (APC), and Alexa Fluor dyes, for fluorescence detection (such as in flow cytometry); enzymes, e.g.
  • HRP horseradish peroxidase
  • AP alkaline phosphatase
  • assays such as enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, etc.
  • biotin for binding to avidin during ELISA, western blotting, immunoprecipitation
  • radioisotopes e.g., Iodine-CRP-p01125, Iodine-131, Indium-I l l for radio-immunoassays (RIA) and nuclear imaging
  • chemotherapeutic agents such as monomethyl auristatin E (MMAE), doxorubicin, and calicheamicin in case of therapy
  • toxins such as Pseudomonas exotoxin, Ricin A-chain, e.g., for cancer therapy
  • nanoparticles and other particles such as latex beads, gold nanoparticles, iron oxide nanoparticles, quantum dots for drug delivery, imaging, etc.
  • the isolated polypeptide is conjugated to gold nanoparticles.
  • nucleic acid molecule including a sequence encoding the isolated polypeptide disclosed herein.
  • the nucleic acid molecule may include a promoter, terminator, and further transcription and/or regulation-related elements, for expression of the isolated polypeptide in a suitable cell, e.g. for producing the isolated polypeptide.
  • a vector including the nucleic acid molecule disclosed herein.
  • a host cell including the nucleic acid molecule disclosed herein, the vector disclosed herein, and/or the isolated polypeptide disclosed herein.
  • hybridoma which is capable of producing the isolated polypeptide disclosed herein.
  • nucleic acids, vectors, host cells, hybridoma also apply here, and vice versa. Some particularly relevant embodiments may be pointed out or explicitly repeated.
  • a combination of more than one antibody against the same protein is needed.
  • a lateral flow test requires one antibody (capture antibody) which binds to the analyte, such as the protein or peptide target in urine, and is immobilized at the test line (e.g., to a nitrocellulose membrane or substrate), and another antibody (detection antibody) binding to the same analyte and conjugated to a detectable label such as gold nanoparticles, colored latex beads, or fluorescent dyes, for detecting the bound analyte.
  • capture antibody capture antibody
  • detection antibody binding to the same analyte and conjugated to a detectable label
  • a detectable label such as gold nanoparticles, colored latex beads, or fluorescent dyes
  • a polypeptide combination including an isolated polypeptide as disclosed herein which specifically binds to a protein or to a protein-derived fragment or peptide (e.g. to be used as the capture antibody or as the detection antibody), and an additional polypeptide which specifically binds to the same protein from which the protein-derived fragment or peptide is derived, but does not interfere with the binding of the isolated polypeptide to the protein (e.g. to be used as the second antibody needed for the test - the detection antibody or the capture antibody, respectively).
  • an isolated polypeptide as disclosed herein which specifically binds to a protein or to a protein-derived fragment or peptide (e.g. to be used as the capture antibody or as the detection antibody)
  • an additional polypeptide which specifically binds to the same protein from which the protein-derived fragment or peptide is derived, but does not interfere with the binding of the isolated polypeptide to the protein (e.g. to be used as the second antibody needed for the test - the detection antibody or the capture antibody, respectively).
  • the protein fragment or peptide to which the additional polypeptide binds is the same as the protein-derived protein fragment or peptide (to which the isolated polypeptide binds).
  • the isolated polypeptide and the additional polypeptide bind to the same protein-derived- protein fragment or peptide.
  • the additional polypeptide does not specifically bind the protein- derived protein fragment or peptide bound by the isolated polypeptide.
  • the isolated polypeptide and the additional polypeptide bind to different protein- derived fragments or peptides of the same protein.
  • the additional polypeptide is also an isolated polypeptide as disclosed herein. In some embodiments, the additional polypeptide is not an isolated polypeptide as disclosed herein, and may be, e.g. an antibody, such as a commercially-available antibody against the protein from which the protein fragment or peptide is derived.
  • the additional polypeptide binds to the protein-derived fragments or peptides under urinary conditions. In some embodiments, the isolated polypeptide binds to the protein-derived fragments or peptides under urinary conditions. In some embodiments, both the isolated polypeptide and the additional polypeptide bind to the protein-derived fragments or peptides under urinary conditions.
  • the isolated polypeptide and the additional polypeptide do not compete with each other in a competition assay for binding the protein.
  • the lack of competition indicates that the isolated polypeptide and the additional polypeptide will not interfere with each other in a test, such as a lateral flow test.
  • a diagnostic method including detecting a protein- derived fragment (or peptide) in a sample from a subject in need of a diagnosis by contacting the sample with the isolated polypeptide disclosed herein or with the combination disclosed herein.
  • the isolated polypeptide disclosed herein or the combination disclosed herein for use in a diagnostic method including detecting a protein-derived fragment (or peptide) by using the isolated polypeptide disclosed herein or the combination disclosed herein.
  • the diagnostic method is a method for diagnosis of whether an infection is a bacterial or a viral infection. In some embodiments, the diagnostic method is a method for diagnosis or detection of infection, systemic infection, systemic inflammation, cancer, autoimmune condition, kidney disease, infectious disease, urinary tract infection (URTI), cardiovascular disease, cardiac infection, a rheumatic condition, and/or a metabolic disorder.
  • URTI urinary tract infection
  • the diagnostic method is a diagnostic method disclosed herein.
  • the sample is a urine sample.
  • the detecting is done by the specific binding of the isolated polypeptide disclosed herein or the combination disclosed herein to a urinary biomarker in the sample. In some embodiments, the binding is under urinary conditions.
  • the method includes using more than one different isolated polypeptide. In some embodiments, at least one of the isolated polypeptides is conjugated to a functional molecule. In some embodiments, each of the isolated polypeptides is conjugated to a different functional molecule.
  • a method of treating a subject in need thereof including a step of detecting a protein-derived fragment (or peptide) in a sample from the subject by contacting the sample with the isolated polypeptide disclosed herein or with the combination disclosed herein; and a step of treating the subject based on the results of the diagnostic step.
  • the subject may be treated with suitable antibiotics. If a systemic inflammation is diagnosed, then the subject may be treated with anti-inflammatory agents.
  • the method of treating is a method of treating disclosed herein.
  • a diagnostic kit including the isolated polypeptide disclosed herein or the combinations disclosed herein, and instructions for use.
  • the diagnostic kit may be used with assays such as LFA, ELISA, western blot, immunohistochemistry (IHC), flow cytometry, chemiluminescent immunoassay (CLIA), radioimmunoassay (RIA), magnetic bead-based immunoassay, ELISPOT (enzyme-linked immunoSpot), immunoprecipitation, dot blot, and biotin-streptavidin immunoassay.
  • assays such as LFA, ELISA, western blot, immunohistochemistry (IHC), flow cytometry, chemiluminescent immunoassay (CLIA), radioimmunoassay (RIA), magnetic bead-based immunoassay, ELISPOT (enzyme-linked immunoSpot), immunoprecipitation, dot blot, and biotin-streptavidin immunoassay.
  • the kit further includes an additional polypeptide, as described above.
  • the isolated polypeptide and/or the additional polypeptide is an antibody. In some embodiments, the isolated polypeptide and/or the additional polypeptide is conjugated to a functional agent.
  • the diagnostic kit includes the combination disclosed herein, wherein the isolated polypeptide is conjugated to a functional agent, and the additional polypeptide is conjugated to a different functional agent.
  • the functional agent may be any agent suitable for the method of detection the kit is used for.
  • the isolated or the additional polypeptide, whichever functions as a detection antibody in a test such as a lateral flow assay, is conjugated to gold nanoparticles, for use in a colloidal gold (colorimetric) detection method.
  • the functional agent is a fluorescent label or nanoparticle.
  • the kit further includes a detection device, such as a lateral flow device and/or an immunoassay device-based reader designed to rapidly and accurately measure specific protein levels in patient samples. It may utilize technologies like fluorescence, chemiluminescence, or electrochemical detection for precise measurement.
  • a detection device such as a lateral flow device and/or an immunoassay device-based reader designed to rapidly and accurately measure specific protein levels in patient samples. It may utilize technologies like fluorescence, chemiluminescence, or electrochemical detection for precise measurement.
  • the diagnostic kit is a diagnostic kit disclosed herein.
  • EGF-P01-mAbl8 351 352 353 354 355 356 357 358
  • EGF-P01-mAbl9 371 372 373 374 375 376 377 378
  • EGF-P02-mAb24 471 472 473 474 475 476 477 478
  • the antibody name includes the protein it targets, a peptide it binds to, and a serial antibody number. It is noted that many BLTA, CRP, and LGALS9 antibodies bind both P01 and P02 of the same protein (these peptides overlap in sequence, as seen from Table 11), however at different affinities, as can be seen from Fig. 3. Additionally, SAA antibodies binding P01 also bind to SAA- P03, and SAA antibodies binding P02 also bind to SAA-P04. Finally, as may be seen from Fig. 3, some antibodies also bind to some extent two peptides from the same protein, although the peptides do not overlap in sequence.
  • an element means one element or more than one element.
  • substantially identical relates to a sequence identity of at least about 95%, 96% 97%, 98%, or 99%.
  • nucleic acid molecule is a molecule including at least one nucleotide sequence.
  • a nucleic acid molecule may be linear, circular, or branched, and the nucleotides may be modified or unmodified.
  • a nucleic acid molecule is a nucleic acid vector (usually a DNA vector) which includes elements such as genes, promoters, linkers, etc.
  • base means base pair, or base pairs.
  • amino acid means amino acid or amino acids.
  • the Classifier cohort was used for classifying the peptides into bacterial infection-related or viral infection-related.
  • the Validation cohort was used for validating the classification. While the peptides for the Classifier and the Validation cohorts were retrieved following tryptic digestion, the endogenous peptides group was not treated with trypsin.
  • the bacterial infections included Bacterial pneumonia, and the viral infections included CMV, Influenza, parainfluenza, Meningitis aseptic, Varicella zoster virus (VZV), bronchitis, Respiratory Syncytial Virus (RSV), Pneumonia, measles.
  • the resulting peptides were analyzed using nanoflow liquid chromatography (nanoAcquity) coupled to high resolution, high mass accuracy mass spectrometry (Fusion Lumos). Each sample was analyzed separately in a random order in a discovery mode. Intensity was used for data processing.
  • the classification of peptides as bacterial infection-related or viral infection-related was based, inter alia, on calculating entropy gain based on MS intensity thresholds for one or more peptide per protein, and selecting a threshold for which the entropy gain is highest.
  • Entropy gain is the difference between the weighted average of the entropies H(pA) and H(pB) (i.e., the sum of H(pA) and H(pB) each multiplied by the ratio of subfraction/total bacterial fraction) and the entropy of the bacterial fraction H(p).
  • the entropy function is symmetrical in the sense that it returns the same value regardless of whether p is the fraction of bacterial samples or of viral samples.
  • 80 peptides out of >50,000 peptides were initially identified in urine samples of the classier cohort patients by LC-MS as having the highest entropy gains. 80 peptides initially identified were selected originating from 5,838 proteins, and provide a short list of peptides that can distinguish bacterial from viral infections.
  • the top 20 peptides were selected by having the highest mean entropy gain across both cohorts. These peptides are presented in Table 5.
  • Table 5 Top 20 peptides identified, sorted by the mean entropy gain
  • Table 6 Top 20 peptides after LOO, sorted by the mean entropy gain
  • Histograms presenting the number of samples (count) diagnosed as bacterial vs. viral infection for each peptide, on a log scale of the intensity threshold is presented in Figs. 1A-1J for peptides 1-10 in Tables 5/6, and in Figs. 2A-2J for peptides 11-20 in Tables 5/6.
  • test performance metrics include overall accuracy (proportion of correct classifications across all samples), sensitivity (proportion of non-bacterial cases correctly identified), specificity (proportion of bacterial cases correctly identified), and bacterial misclassification rate (proportion of bacterial samples incorrectly classified).
  • Bacterial infection is characterized by elevated blood levels of CRP (Brian Clyne, Jonathan S Olshaker, The C-reactive protein, The Journal of Emergency Medicine, Volume 17, Issue 6, 1999, Pages 1019-1025). Accordingly, the inventors tested which peptides were best correlated with blood CRP concentrations in the Classifier cohort. A log scale was used for both the blood CRP levels and peptides intensities.
  • KLH keyhole limpet hemocyanin
  • mice 6-8 weeks old SJL mice and Balb/c mice (Shanghai SLAC Laboratory Animal Center) were immunized with peptides mixture. Mice were housed under Specific Pathogen Free (SPF) conditions.
  • SPF Specific Pathogen Free
  • the antibody titers in sera were subjected to analysis by enzyme-linked immunosorbent assay (ELISA). Mice with a strong immune response as determined by serum titer were selected and used for hybridoma generation. All the treatment of the animals were strictly followed the ethical committee guidelines.
  • Peptides were conjugated to bovine serum albumin (BSA) and diluted to proper final concentrations into lx phosphate-buffered saline (PBS), coated 100 pL/well on ELISA plate (cat: 9018, Coming). Following overnight incubation at 4°C, plates were blocked with 250 pL assay buffer (1% BSA and 0.05% Tween-20 in PBS) for 1 hr at 37°C.
  • BSA bovine serum albumin
  • PBS lx phosphate-buffered saline
  • PBST Primary bleed
  • TB Test Bleed
  • 100 pL/well of the diluted serum solution were added to the plate, incubated for 1 hr at 37°C and wash 4 times with PBST.
  • 100 pL/well secondary antibody anti-mouse-Fc-HRP (Sigma, A0168, 1:5000) were added and incubated for 0.5 hr at 37°C.
  • mice selected for fusion were given a final intraperitoneal boost with total 40pg peptide mixture for each target without adjuvant. Three days later the mice were euthanized by carbon dioxide asphyxiation following an approved Institutional Animal Care and Use Committee (IACUC) protocol, and a blood sample, lymphocytes and splenocytes were collected. Serum was generated and used as a positive control (designated as final bleed (FB)) at the hybridoma screening stage. Lymphocytes /splenocytes were centrifuged at 400 g (or 1000 rpm) for 5 min and the supernatant was discarded.
  • IACUC Institutional Animal Care and Use Committee
  • Lymphocyte/splenocytes were re-suspended in 5 mL red cell lysis buffer, incubated for 5 min at 4°C, the reaction was stopped by the addition of Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) to 50 mL. Lymphocyte/splenocytes were then centrifuged at 400 g for 5 min, and re-suspended in DMEM.
  • DMEM Dulbecco's Modified Eagle Medium
  • FBS fetal bovine serum
  • SP2/0 mouse spleen cells were collected and centrifuged at 400 g for 5 min at room temperature. Culture medium was aspirated and the cells were re-suspended in 20 mL fusion media. Typically, 2.5 xlO 7 SP2/0 cells were re-suspended in 20 mL fusion medium.
  • 5xl0 7 SP2/0 cells were added to IxlO 8 lymphocyte/splenocytes (7:3) to give a final ratio of lymphocytes/splenocytes: SP2/0 of 2:1.
  • the cells were centrifuged at 400 g for 5 minutes, and the medium was discarded.
  • the cell pellet was washed twice with 25ml electrofusion (EF) solution (QIWEN BITECH Cat#CEB005) by centrifuging for 5 minutes at 1500 rpm. and re-suspended in EF solution.
  • the mixture of cells was placed in a fusion slot of a BTX ECM2001 electrofusion system and the fusion was effectuated by a cell fusion generator using an optimized program.
  • the peptides were conjugated to biotin at both the N and the C termini.
  • Fusion plates were monitored for growth and fed weekly. Wells with cell growth were screened by primary screening assays in 10-14 days with ELISA assay. After 3 days, secondary screening by confirmatory ELISA was performed.
  • Hybridoma clones that exhibited binding to target peptides by ELISA were expanded into 24- well plates. Hybridoma supernatant was collected from 24- well cultures and tested confirmatory ELISA binding assay. Clones that specifically bound target peptides in assay buffer and in urine solution were selected for subcloning.
  • Subcloning was performed by limiting dilution for the desired positive parental clones. 96- well plates containing subcloned cells were incubated in a CO2 incubator and the cells were expanded for 7 days. ELISA with target peptides was performed for the subcloning plates. Based on results for each subcloning cell line, subcloned wells (single clones) with a strong and specific positive signal were expanded into a 24-well plate, and the resultant hybridoma supernatants were evaluated by confirmatory ELISA binding assay. Subclones with the specific binding to target peptides in assay buffer and in urine solution were expanded to T-75 cm 2 flasks. Hybridoma cells were then frozen down.
  • BSA conjugated peptide were diluted to proper final concentrations into IxPBS, coated 100 pL/well on ELISA plate (cat: 9018, Coming). Following overnight incubation at 4°C, plates were blocked with 250 pL assay buffer (1%BSA and 0.05% Tween-20 in PBS) for 1 hr at 37°C. Following 4 washes with PBST using Biotek (Elx 405), hybridoma supernatant (or 1:1 diluted by simulated urine solution) were added to the plate, incubated for 1 hr at 37°C and wash 4 times with PBST.
  • 100 pL/well secondary antibody (anti- mouse-Fc-HRP (Sigma, A0168, 1:5000) were added and incubated for 0.5 hr at 37°C. Following 4 washes with PBST, 100 pL/well of TMB substrate was added and incubated at room temperature for 5 min. 100 pL/well of IN HC1 were then added to terminate reaction. Plates were read using ELISA plate reader at 450nm wavelength (instrument SpectraMax M5e).
  • Hybridomas were screened for antibodies against proteins including the peptides presented in Table 11. Sequences of the most specific antibodies are presented in Table 13 below.
  • Figs. SASK show ELISA binding of mAbs against the corresponding peptides (Figs. 3A-3B: mAbs against CRP P01 and P02, respectively; Figs. 3C-3D: mAbs against BTLA P01 and P02, respectively; Figs. 3E-3F: mAbs against EGF P01 and P02, respectively; Figs. 3G-3H: mAbs against FGA P01 and P02, respectively; Figs. 3I-JB: mAbs against SAA P01 and P02, respectively; Fig. 3K: mAb against LAGLS9 P01). EC50 values for the same antibodies are presented in Table 12.

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Abstract

La présente invention concerne des méthodes permettant de prédire dans un échantillon d'urine d'un sujet suspecté d'avoir une infection, si l'infection est une infection bactérienne et/ou une infection virale, par détection dans un échantillon d'urine du sujet d'un niveau d'au moins un fragment de protéine ; de déterminer pour les fragments de protéine détectés si le niveau est supérieur ou inférieur à un seuil respectif ; et de prédire si l'infection est bactérienne et/ou virale sur la base de la détermination. La présente invention concerne en outre des méthodes de prédiction d'une inflammation, des méthodes de traitement basées sur les prédictions ci-dessus, et des kits de diagnostic appropriés. En outre, la présente invention concerne des anticorps spécifiques pour détecter des fragments de protéine dans l'urine.
PCT/IL2024/051120 2023-11-28 2024-11-27 Peptides pour diagnostics urinaires et anticorps pour leur détection Pending WO2025115009A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009107170A1 (fr) * 2008-02-29 2009-09-03 学校法人日本大学 Anticorps anti-crp et son utilisation
WO2010115749A2 (fr) * 2009-03-30 2010-10-14 Nordic Bioscience A/S Dosage d'un biomarqueur de fibrose
WO2021117044A1 (fr) * 2019-12-11 2021-06-17 Ichilov Tech Ltd. Essai non invasif pour différencier des infections bactériennes et virales
WO2021204984A1 (fr) * 2020-04-09 2021-10-14 B.R.A.H.M.S Gmbh Biomarqueurs pour le diagnostic d'infections des voies respiratoires

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009107170A1 (fr) * 2008-02-29 2009-09-03 学校法人日本大学 Anticorps anti-crp et son utilisation
WO2010115749A2 (fr) * 2009-03-30 2010-10-14 Nordic Bioscience A/S Dosage d'un biomarqueur de fibrose
WO2021117044A1 (fr) * 2019-12-11 2021-06-17 Ichilov Tech Ltd. Essai non invasif pour différencier des infections bactériennes et virales
WO2021204984A1 (fr) * 2020-04-09 2021-10-14 B.R.A.H.M.S Gmbh Biomarqueurs pour le diagnostic d'infections des voies respiratoires

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