WO2025115009A1

WO2025115009A1 - Peptides for urinary diagnostics and antibodies for their detection

Info

Publication number: WO2025115009A1
Application number: PCT/IL2024/051120
Authority: WO
Inventors: Shani SHENHAR-TSARFATY; Igal RIKLIN; Keren LEVINSTEIN HALLAK; Peleg ITAY; Yael SCHNEIDMAN
Original assignee: Accurine Ltd
Current assignee: Accurine Ltd
Priority date: 2023-11-28
Filing date: 2024-11-27
Publication date: 2025-06-05
Anticipated expiration: 2026-05-28

Abstract

The present invention provides methods 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, by detecting in a urine sample of the subject a level of at least one protein fragment; determining for the detected protein fragments whether the level is above or below a respective threshold; and predicting whether the infection is bacterial and/or viral based on the determination. The present invention further provides methods of predicting inflammation, methods of treatment based on the above pre dictions, and suitable diagnostic kits. Furthermore, the present invention provides specific antibodies for detecting protein fragments in urine.

Description

PEPTIDES FOR URINARY DIAGNOSTICS AND ANTIBODIES FOR THEIR

DETECTION

FIELD OF THE INVENTION

The present disclosure is generally directed to the diagnostics of infectious diseases. Specifically, 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.

BACKGROUND OF THE INVENTION

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%.

Typically, 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. However, 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.

Accordingly, fast, simple, and easy to use non-invasive diagnostic tests for infections could decrease morbidity and mortality by increasing early administration of antibiotics to patients with bacterial infections, and reducing unnecessary administration of antibiotics to patients without bacterial infections, such as those having a viral infection.

SUMMARY OF INVENTION

The following embodiments are described and illustrated in conjunction with compositions and methods which are meant to be exemplary and illustrative, not limiting in scope. In various embodiments, one or more of the above-described problems have been reduced or eliminated, while other embodiments are directed to other advantages or improvements.

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.

In some embodiments, there is provided 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, the method 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).

In some embodiments, 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, sequences at least 90%, 95%, 98%, or 99% identical to the peptide sequences, sequences including them, and intervening protein sequences.

In some embodiments, the at least one protein is selected from CRP, BTLA, EGF, FGA, LGALS9, SAA, and combinations thereof.

In some embodiments, 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. In some embodiments, 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.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, the at least one protein is at least 6 proteins including CRP, BTLA, EGF, FGA, LGALS9, and SAA.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, 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. In some embodiments, the one or more binding agents include at least one antibody specific to at least one of the protein fragments. In some embodiments, the one or more binding agents includes at least one monoclonal antibody defined in Table 2. In some embodiments, 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.

In some embodiments, 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.

In some embodiments, for each protein fragment 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.

In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

In some embodiments, the infection is an upper respiratory tract infection (URTI). In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

In some embodiments, 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.

In some embodiments, there is provided 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.

In some embodiments, 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.

In some embodiments, there is provided a method for treating a viral infection in a subject afflicted with an infection, the method 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.

In some embodiments, 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.

In some embodiments, there is provided a method for predicting efficacy of a treatment in a subject afflicted with a bacterial and/or a viral infection, the method 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. 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).

In some embodiments, there is provided a kit for predicting in a urine sample whether an infection is a bacterial infection or a viral infection, the kit 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. 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.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, 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. In some embodiments, the one or more binding agent includes at least one monoclonal antibody defined in Table 2. In some embodiments, 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.

In some embodiments, there is provided a method for predicting an elevated blood CRP level by a urine test, the method 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).

In some embodiments, there is provided a kit for predicting an elevated blood CRP level by a urine test, the kit 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.

In some embodiments, there is provided a method for treating an inflammation in a subject, the method 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.

In some embodiments, there is provided a method for predicting efficacy of a treatment of inflammation in a subject, the method 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.

In some embodiments, there is provided 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).

In some embodiments, there is provided an isolated polypeptide capable of specifically binding a CRP-derived protein fragment or peptide, 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. 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.

In some embodiments, there is provided 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. 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.

In some embodiments, the VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3, include sequences substantially identical to sequences set forth in:

SEQ ID Nos. 183, 184, 185, 186, 187, and 188, respectively;

SEQ ID Nos. 203, 204, 205, 206, 207, and 207, respectively;

SEQ ID Nos. 223, 224, 225, 226, 227, and 228, respectively;

SEQ ID Nos. 243, 244, 245, 246, 247, and 248, respectively;

SEQ ID Nos. 263, 264, 265, 266, 267, and 268, respectively; or

SEQ ID Nos. 283, 284, 285, 286, 287, and 288, respectively.

In some embodiments, the VH and the VL regions include sequences substantially identical to 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.

In some embodiments, 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).

In some embodiments, the binding is under urinary conditions.

In some embodiments, 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.

In some embodiments, there is provided a nucleic acid encoding the isolated polypeptide disclosed herein. In some embodiments, there is provided a vector including the nucleic acid disclosed herein. In some embodiments, there is provided a cell including the isolated polypeptide disclosed herein.

In some embodiments, there is provided the isolated polypeptide disclosed herein or the monoclonal antibody disclosed herein for use in the methods disclosed herein.

In addition to the exemplary embodiments described above, further embodiments will become apparent by reference to the figures and by study of the following detailed descriptions. BRIEF DESCRIPTION OF DRAWINGS

The invention will now be described in relation to certain examples and embodiments with reference to the following illustrative figures.

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. Left histograms - Classifier cohort; right histograms - Validation cohort 1A. YCANRPHVTWCK (BTLA); IB. RLFWTDTGINPR (EGF); 1C. RAPDLQDLPWQVK (PROZ); ID. VHLIVQVSPK (NTM); IE. RQDNEILIFWSK (CRP); IF. VFHLTVAEPHAEPPPR (MXRA8); 1G. FEDGGYVVCNTR (LGALS9); 1H. IALVITDGR (COL6A1); II. GLYDVVSVLR (ICOSLG); 1J. ENFVLTTAK (PROZ).

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. Left histograms - Classifier cohort; right histograms - Validation cohort 2A. GLLSGWAR (PROZ); 2B. TDGCQHFCLPGQESYTCSCAQGYR (PROZ); 2C. EANYIGSDK (SAA); 2D. MNLYGFHGGQR (TNXB); 2E. AAAATGTIFTFR (SERPINA5); 2F. YMHLFSTIK (CNTFR); 2G. GSESGIFTNTK (FGA); 2H. QCVPHDQCACGVLTSEK (PROZ); 21. CQCPAGAALQADGR (THBD); 2J LAGLGLQQLDEGLFSR (VASN).

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).

DETAILED DESCRIPTION OF THE INVENTION

In the following description, various aspects of the disclosure will be described. For the purpose of explanation, specific configurations and details are set forth in order to provide a thorough understanding of the different aspects of the disclosure. However, it will also be apparent to one skilled in the art that the disclosure may be practiced without specific details being presented herein. Furthermore, well-known features may be omitted or simplified in order not to obscure the disclosure.

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.

These most indicative peptides were identified by statistical methods generally based on an analysis involving searching for peptides present above a certain level in urine, for which the entropy gain from separating the urine samples into two populations (bacterial infections and viral infections) based on an intensity cutoff for that peptide in the LC-MS analysis, was the highest, thereby finding peptides which could best separate the bacterial and the viral infections. 80 peptides in which the entropy gain was highest were selected first (out of above 50,000 peptides originating from 5,838 proteins), and the best 20 peptides were selected based on additional statistical testing. Finally, the best six protein fragments derived from six different proteins were selected based on an additional analysis of the levels of each peptide, as described in the methods (see Figs. 1 and 2). Combinations of the six most indicative peptides were also assessed, and best combinations are offered based on this analysis, as detailed below.

It should be noted that 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). This may be attributed to kidney processes such as glomerular filtration and tubular absorption, responsible for restricting the release of most plasma proteins into the blood.

Accordingly, the presence and levels of certain proteins, protein fragments, or peptides in urine cannot a priori be assumed, even if their blood levels are known.

A method of diagnosis by detecting protein fragments from specific proteins in urine

In some embodiments, there is provided 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, the method 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).

In some embodiments, 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. In some embodiments, the at least one protein is selected from CRP, BTLA, EGF, FGA, LGALS9, SAA, and combinations thereof. In some embodiments, the at least one protein is selected from BTLA, EGF, FGA, LGALS9, SAA, and combinations thereof.

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 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.

The terms “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. In some embodiments, the peptide or protein fragment has a length of about 3-500 amino acids. In some embodiments, 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. In some embodiments, the peptide or protein fragment has a length of at least about 5, 10, or 20 amino acids. In some embodiments, 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.

In some embodiments, “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. In some embodiments, the high probability is a probability of at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%. In some embodiments, “predicting” means increasing a probability of predicting a desired parameter.

In some embodiments, predicting that the infection is bacterial means that the infection has a high probability of being a bacterial infection. In some embodiments, 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.

In some embodiments, 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.

In step (a) of the method, a urine sample from the subject is analyzed by determining the level of certain protein fragments (or peptides). As explained above and in the experimental section below, 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.

The term “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.

In some embodiments, 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.

TDGCQHFCLPGQESYTCSCAQGYR, EANYIGSDK, FFGHGAEDSLADQAANEWGR, GPGGVWAAEAISDAR, RGPGGVWAAEAISDAR, DPNHFRPAGLPEK, MNLYGFHGGQR, AAAATGTIFTFR, YMHLFSTIK, GSESGIFTNTK, TVIGPDGHK, GDSTFESK, TVIGPDGHKEVTK, VTSGSTTTTR, QCVPHDQCACGVLTSEK, CQCPAGAALQADGR, LAGLGLQQLDEGLFSR, sequences at least 90%, 95%, 98%, or 99% identical to the peptide sequences, sequences including them, and combinations thereof.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, 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, EANYIGSDK, FFGHGAEDSLADQAANEWGR, GPGGVWAAEAISDAR, RGPGGVWAAEAISDAR, DPNHFRPAGLPEK, MNLYGFHGGQR, AAAATGTIFTFR, YMHLFSTIK, GSESGIFTNTK, TVIGPDGHK, GDSTFESK, TVIGPDGHKEVTK, VTSGSTTTTR,

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.

The term “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.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, the detection method is a method for identifying protein fragments or peptides, such as MS or by high-performance liquid chromatography (HPLC).

In some embodiments, 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.

Accordingly, in some embodiments, 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.

It is appreciated that “binding” (and “no 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. In this way, 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. However, it should be clear from the detection method and any corresponding instructions of the method whether the result indicates binding or no binding. In some embodiments, the term “binding” as used herein means “specifically binding” as defined below.

In some embodiments, 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. Nonlimiting examples for 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.

In some embodiments, the one or more binding agents include antibodies. In some embodiments, the one or more binding agents include monoclonal antibodies.

In some embodiments, the one or more binding agents include at least one antibody specific to at least one of the protein fragments.

In some embodiments, 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.

In step (b) the method involves determining for the at least one protein fragment whether the level is above or below a respective threshold.

The term “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.

The term “respective threshold” refers to a threshold appropriate for a specific protein fragment detected by the specific method. It is noted that the respective threshold may be different between different protein fragments. In some embodiments, each protein fragment independently has a respective threshold, which may be the same or different from respective thresholds of other protein fragments. In some embodiments, 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.

In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, the threshold concentration is a reference level of the protein fragment which has been determined by calculation, such as a statistical analysis.

Accordingly, in some embodiments, the respective threshold is a reference level of the protein fragment. In some embodiments, the respective threshold is a reference level retrieved from a database. In some embodiments, 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. In some embodiments, the respective threshold is a reference level of the protein fragment representing a normal urinary level of the protein fragment. In some embodiments, 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. In such embodiments where the respective threshold is explicit (a reference level), the detecting in step (a) includes measuring the level of the protein fragment, and the determining in step (b) includes comparing the measured level of the protein fragment to the respective threshold.

In some embodiments, 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. For example, in order to detect a protein fragment by any detection method, there is a threshold level of the protein fragment under which level it would not be detected by the detection method.

Accordingly, in some embodiments, 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.

In some embodiments, 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.

In such embodiments, where the threshold is implicit, 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.

In some embodiments, 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.

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. In some embodiments, 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.

In some embodiments, 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 nonlimiting example may be the appearance of a color band representing the tested protein fragment in a lateral flow assay.

In some embodiments, the predicting in step (c) requires further processing, computation, or calculation. In some embodiments, 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. In some embodiments, the determination in step (b) is performed by a processor, as noted above, and the determination is further processed in step (c). In some embodiments, 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.

In some embodiments, 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. In a particular embodiment, the algorithm is selected from the group consisting of gradient boosted trees, random forest, regularized regression, and combinations thereof.

In some embodiments, the at least one protein fragment is a single protein fragment.

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 above the respective threshold, then 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.

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 above the respective threshold, then 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.

The terms “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. Examples for bacterial infection- related protein fragments include peptides derived from the proteins CRP, FGA, and/or SAA. Examples for viral infection-related protein fragments include peptides derived from the proteins BTLA, EGF, and LGALS9.

In some embodiments, the at least one protein fragment is more than one protein fragment, such as 2, 3, 4, 5, or 6 protein fragments.

In some embodiments, 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.

Accordingly, in some embodiments, 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.

In some embodiments, 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.

When the combination of protein fragments includes only bacterial infection-related protein fragments, the level of at least one protein fragment being above the respective threshold predicts that the infection is a bacterial infection. When the combination of protein fragments includes only viral infection-related protein fragments, the level of at least one protein fragment being above the respective threshold predicts that the infection is a viral infection.

When 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; and 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.

When 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).

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, 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; and FGA, LGALS9, and SAA.

In some embodiments, 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.

In some embodiments, 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 SAA; CRP, EGF, LGALS9, and SAA; CRP, FGA, LGALS9, and SAA; and EGF, FGA, LGALS9, and SAA.

In some embodiments, 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 FGA; and BTLA, CRP, FGA, and LGALS9.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, the at least one protein does not include TRAIL.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, 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.

As explained above, in some embodiments, in each of the above embodiments, 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.

As also explained above, since the peptide sequences presented herein were determined following trypsinization of longer peptides in the urine samples, the sequences presented here are most likely parts of longer sequences. Accordingly, in some embodiments, the protein fragments of the invention include the presented sequences. As a consequence, it is appreciated that overlapping peptides (i.e., peptide sequences which overlap with sequences presented herein), that are derived from the longer sequences (of the same protein), or 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. Accordingly, 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.

The term “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.

Finally, since polymorphisms and small variations may exist for almost any protein, 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.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, 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, FAVNFQTGFSGNDIAFHFNPR, QNGSWGPEER, GMPFDECFEVQSSDFK, VMVNGIEFVQYFHR, and THMPFQK; and the SAA-derived peptide sequences EANYIGSDK, FFGHGAEDSEADQAANEWGR, GPGGVWAAEAISDAR, GNYDAAKRGPGGVW, RGPGGVWAAEAISDAR, and DPNHFRPAGEPEK, sequences at least 90%, 95%, 98%, or 99% identical to the peptide sequences, and sequences including them.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, the subject is a child. In some embodiments, the subject is an adult. In some embodiments, the subject is an elderly adult.

It should be clarified that 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.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, the infection is a URTI.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, 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 method of diagnosis by detecting protein fragments in urine by antibodies

In some embodiments, there is provided 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, the method 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).

In general, definitions and embodiments mentioned above, and which may be relevant to the embodiments related to detecting protein fragments by antibodies, also apply here, and vice versa. Some particularly relevant embodiments may be pointed out or explicitly repeated. For terms used herein, unless stated otherwise, their definition and embodiments are intended to be the same as above (mutatis mutandis).

In some embodiments, the at least one protein is selected from BTLA, C0L6A1, CNTFR, EGF, FGA, ICOSLG, LGALS9, MXRA8, NTM, PROZ, SAA, SERPINA5, THBD, TNXB, and VASN. In some embodiments, 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. In some embodiments, 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.

In some embodiments, 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.

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. As also explained above, 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). Accordingly, 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.

It is appreciated that 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. As also explained above, 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. Alternatively, 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.

In some embodiments, 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.

Accordingly, in some embodiments, 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.

In some embodiments, 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.

In some embodiments, in the case of the infection being a viral infection and not a bacterial infection, it is possible that no treatment is needed, and 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 of treating a bacterial infection following detection

In some embodiments, there is provided 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.

In general, definitions and embodiments mentioned above, and which may be relevant to the treatment embodiments also apply here, and vice versa. Some particularly relevant embodiments may be pointed out or explicitly repeated. For terms used herein, unless stated otherwise, their definition and embodiments are intended to be the same as above (mutatis mutandis).

The term “afflicted with an infection” means that the subject is suffering from an infection but the infection causative agent is not known. In some embodiments, 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 with symptoms which are typical to a bacterial or to a viral infection. In some embodiments, the subject presents with symptoms which are typical to a specific infection. However, in order to provide the appropriate treatment (e.g. to not administer antibiotics unnecessarily), it needs to be verified whether the infection is caused by a bacterial agent or by a viral agent.

The term “treating” or “treatment”, as used herein, 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.

In some embodiments, the at least one protein is selected from CRP, BTLA, C0L6A1, CNTFR, EGF, FGA, ICOSLG, LGALS9, MXRA8, NTM, PROZ, SAA, SERPINA5, THBD, TNXB, VASN.

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 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 of treating a viral infection following detection

In some embodiments, there is provided 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.

In general, definitions and embodiments mentioned above and which may be relevant to the treatment embodiments also apply here, and vice versa. Some particularly relevant embodiments may be pointed out or explicitly repeated. For terms used herein, unless stated otherwise, their definition and embodiments are intended to be the same as above (mutatis mutandis).

In some embodiments, 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.

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 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 of predicting efficacy of a treatment

In some embodiments, there is provided a method for predicting efficacy of a treatment in a subject afflicted with a bacterial and/or a viral infection, the method 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. 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).

In general, definitions and embodiments mentioned above and which may be relevant to the efficacy prediction embodiments also apply here, and vice versa. Some particularly relevant embodiments may be pointed out or explicitly repeated. For terms used herein, unless stated otherwise, their definition and embodiments are intended to be the same as above (mutatis mutandis).

In some embodiments, 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.

As explained above with reference to detecting, in some embodiments, 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.

When the detection in step (a) includes measuring levels of the protein fragments in the urine sample, then determining in step (d) involves comparing the first level with the second level.

When the detection in step (a) includes detection of protein fragments, such as by binding agents (e.g. by antibodies), then the determining in 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.

The interpretation of the determination in step (d) to provide a prediction in step (e) depends on the infection and on the peptide(s). 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.

A kit for diagnosing bacterial vs. viral infection

In some embodiments, there is provided a kit for predicting in a urine sample whether an infection is a bacterial infection or a viral infection, the kit 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. 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.

In general, definitions and embodiments mentioned above, and which may be relevant to the kit embodiments also apply here, and vice versa. Some particularly relevant embodiments may be pointed out or explicitly repeated. For terms used herein, unless stated otherwise, their definition and embodiments are intended to be the same as above (mill al is mutandis).

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, 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. In some embodiments, 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.

In some embodiments, 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. Nonlimiting examples for 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.

In some embodiments, 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.

In some embodiments, 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.

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.

As shown in Example 3, 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.

In some embodiments, there is provided a method for predicting an elevated blood CRP level by a urine test, the method 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).

In general, definitions and embodiments mentioned above and which may be relevant to the inflammation prediction embodiments, also apply to the present embodiments, and vice versa. Some particularly relevant embodiments may be pointed out or explicitly repeated. For terms used herein, unless stated otherwise, their definition and embodiments are intended to be the same as above (mutatis mutandis).

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 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.

In some embodiments, predicting an elevated blood CRP level means that blood CRP level has a high probability of being elevated.

In some embodiments, 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).

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, 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 sequences, and sequences including them

A kit for predicting inflammation (predicting blood CRP levels

In some embodiments, there is provided a kit for predicting an elevated blood CRP level by a urine test, the kit 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.

In general, definitions and embodiments mentioned above and which may be relevant to the kit embodiments, also apply here, and vice versa. Some particularly relevant embodiments may be pointed out or explicitly repeated. For terms used herein, unless stated otherwise, their definition and embodiments are intended to be the same as above (mill al is mutandis).

A method of treating inflammation

In some embodiments, there is provided a method for treating an inflammation in a subject, the method 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.

In general, definitions and embodiments mentioned above and which may be relevant to the inflammation prediction embodiments, also apply here, and vice versa. Some particularly relevant embodiments may be pointed out or explicitly repeated. For terms used herein, unless stated otherwise, their definition and embodiments are intended to be the same as above (mutatis mutandis).

In some embodiments, the anti-inflammatory agent is selected from anti-inflammatory drugs, immunosuppressants, and corticosteroids. For example, 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 the efficacy of treating inflammation

In general, definitions and embodiments mentioned above and which may be relevant to the efficacy prediction embodiments also apply here, and vice versa. Some particularly relevant embodiments may be pointed out or explicitly repeated.

In some embodiments, 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. Similarly, in some embodiments, 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.

Isolated polypeptides and antibodies against urinary protein fragments

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.

In order to facilitate detection of the protein fragments, antibodies were developed against peptides disclosed herein, as detailed in Example 4. Examples for the binding characteristics of the antibodies produced are presented in Table 12 and Figs. 3A-3K. It is noted that the screening for successful binding was done under conditions which simulate urine conditions (e.g. salts, urea, and pH similar to human urine, e.g., about 6), so that the selected antibodies are capable of binding to their respective antigen in urine. In this respect it should be noted that since the conditions in urine are unique and rather harsh, it cannot be assumed that any antibody capable of binding a specific peptide or a protein fragment under any conditions, such as in other bodily fluids (e.g. blood) may be able to bind it under urinary conditions.

The term “urinary conditions”, as used herein, 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.

In some embodiments, 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.

Below are provided specific isolated polypeptides which are capable of binding protein fragments derived from a protein selected from CRP, BTLA, LGALS9, EGF, FGA, and SAA.

It is appreciated that 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. In other words, the isolated polypeptides (e.g. antibodies) 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.

Several standard methods exist for determining CDRs in a variable region sequence. These methods include the non-limiting examples of Kabat, Chothia, IMGT (International ImMunoGeneTics information system), and AbM.

Accordingly, in some embodiments, 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.

Alternatively, in some embodiments, the six CDRs are defined by their sequence. When defined by sequences, 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.

In some embodiments, 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).

In some embodiments, the amino acid sequences of the isolated polypeptide are defined by the nucleotide sequences encoding them.

Definitions and embodiments mentioned above and which may be relevant to the isolated polypeptides and antibodies embodiments, also apply here, and vice versa. Some particularly relevant embodiments may be pointed out or explicitly repeated. For terms used herein, unless stated otherwise, their definition and embodiments are intended to be the same as above (mutatis mutandis).

The above definitions and embodiments apply to all of the isolated polypeptides defined below.

Anti-CRP antibodies

In some embodiments, there is provided 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.

In some embodiments, 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. 183, 184, 185, 186, 187, and 188, respectively;

SEQ ID Nos. 203, 204, 205, 206, 207, and 207, respectively;

SEQ ID Nos. 223, 224, 225, 226, 227, and 228, respectively;

SEQ ID Nos. 243, 244, 245, 246, 247, and 248, respectively;

SEQ ID Nos. 263, 264, 265, 266, 267, and 268, respectively; or

SEQ ID Nos. 283, 284, 285, 286, 287, and 288, respectively.

In some embodiments, the VH and the VL regions include, or consist of, sequences identical or substantially identical to 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.

In some embodiments, there is provided 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, 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.

In some embodiments, 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. 193, 194, 195, 196, 197, and 198, respectively;

SEQ ID Nos. 213, 214, 215, 216, 217, and 218, respectively;

SEQ ID Nos. 233, 234, 235, 236, 237, and 238, respectively;

SEQ ID Nos. 253, 254, 255, 256, 257, and 258, respectively;

SEQ ID Nos. 273, 274, 275, 276, 277, and 278, respectively; or

SEQ ID Nos. 293, 294, 295, 296, 297, and 298, respectively.

In some embodiments, 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. 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.

In some embodiments, 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). In some embodiments, the CRP-derived protein fragment or peptide includes, or consists of, a sequence set forth in SEQ ID No: 861. In some embodiments, the CRP-derived protein fragment or peptide includes, or consists of, a sequence set forth in SEQ ID No: 862.

In some embodiments, there is provided 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. In some embodiments, there is provided 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: 861 (CRP-P01), under urinary conditions. In some embodiments, there is provided 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.

Anti-BTLA antibodies

In some embodiments, there is provided 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. 61 and 62, respectively; 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.

SEQ ID Nos. 03, 04, 05, 06, 07, and 08, respectively;

SEQ ID Nos. 23, 24, 25, 26, 27, and 28, respectively;

SEQ ID Nos. 43, 44, 45, 46, 47, and 48, respectively;

SEQ ID Nos. 63, 64, 65, 66, 67, and 68, respectively;

SEQ ID Nos. 83, 84, 85, 86, 87, and 88, respectively;

SEQ ID Nos. 103, 104, 105, 106, 107, and 108, respectively;

SEQ ID Nos. 123, 124, 125, 126, 127, and 128, respectively; SEQ ID Nos. 143, 144, 145, 146, 147, and 148, respectively; or

SEQ ID Nos. 163, 164, 165, 166, 167, and 168, respectively.

SEQ ID Nos. 01 and 02, respectively;

SEQ ID Nos. 21 and 22, respectively;

SEQ ID Nos. 41 and 42, respectively;

SEQ ID Nos. 61 and 62, respectively;

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.

In some embodiments, there is provided 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. 51 and 52, respectively; 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.

SEQ ID Nos. 13, 14, 15, 16, 17, and 18, respectively;

SEQ ID Nos. 33, 34, 35, 36, 37, and 38, respectively;

SEQ ID Nos. 53, 54, 55, 56, 57, and 58, respectively;

SEQ ID Nos. 73, 74, 75, 76, 77, and 78, respectively;

SEQ ID Nos. 93, 94, 95, 96, 97, and 98, respectively;

SEQ ID Nos. 113, 114, 115, 116, 117, and 118, respectively;

SEQ ID Nos. 133, 134, 135, 136, 137, and 138, respectively;

SEQ ID Nos. 153, 154, 155, 156, 157, and 158, respectively; or SEQ ID Nos. 173, 174, 175, 176, 177, and 178, respectively.

SEQ ID Nos. 11 and 12, respectively;

SEQ ID Nos. 31 and 32, respectively;

SEQ ID Nos. 51 and 52, respectively;

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.

In some embodiments, 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). In some embodiments, the BTLA-derived protein fragment or peptide includes, or consists of, a sequence set forth in SEQ ID No: 867. In some embodiments, the BTLA-derived protein fragment or peptide includes, or consists of, a sequence set forth in SEQ ID No: 868.

In some embodiments, there is provided 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. In some embodiments, there is provided 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: 867, under urinary conditions. In some embodiments, there is provided 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.

Anti-LGALS9 antibodies

In some embodiments, there is provided an isolated polypeptide capable of specifically binding a LGALS9-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. 661 and 662, respectively. In some embodiments, 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.

In some embodiments, 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.

In some embodiments, there is provided an isolated polypeptide capable of specifically binding a LGALS9-derived protein fragment or peptide, wherein 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), and 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.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, 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). In some embodiments, the LGALS9-derived protein fragment or peptide includes, or consists of, a sequence set forth in SEQ ID No: 882. In some embodiments, the LGALS9-derived protein fragment or peptide includes, or consists of, a sequence set forth in SEQ ID No: 883.

In some embodiments, there is provided 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. In some embodiments, there is provided 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. In some embodiments, there is provided 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

In some embodiments, there is provided 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. 741 and 742, respectively; SEQ ID Nos. 761 and 762, respectively; 781 and 782, respectively; SEQ ID Nos. 801 and 802, respectively; SEQ ID Nos. 821 and 822, respectively; SEQ ID Nos. or SEQ ID Nos. 841 and 842, respectively.

SEQ ID Nos. 683, 684, 685, 686, 687, and 688, respectively;

SEQ ID Nos. 703, 704, 705, 706, 707, and 708, respectively;

SEQ ID Nos. 723, 724, 725, 726, 727, and 728, respectively;

SEQ ID Nos. 743, 744, 745, 746, 747, and 748, respectively;

SEQ ID Nos. 763, 764, 765, 766, 767, and 768, respectively;

SEQ ID Nos. 783, 784, 785, 786, 787, and 788, respectively;

SEQ ID Nos. 803, 804, 805, 806, 807, and 808, respectively;

SEQ ID Nos. 823, 824, 825, 826, 827, and 828, respectively; or

SEQ ID Nos. 843, 844, 845, 846, 847, and 848, respectively.

SEQ ID Nos. 681 and 682, respectively;

SEQ ID Nos. 701 and 702, respectively;

SEQ ID Nos. 721 and 722, respectively;

SEQ ID Nos. 741 and 742, respectively;

SEQ ID Nos. 761 and 762, respectively;

SEQ ID Nos. 781 and 782, respectively;

SEQ ID Nos. 801 and 802, respectively;

SEQ ID Nos. 821 and 822, respectively; or

SEQ ID Nos. 841 and 842, respectively.

In some embodiments, there is provided 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. 731 and 732, respectively; SEQ ID Nos. 751 and 752, respectively; SEQ ID Nos. 771 and 772, respectively; SEQ ID Nos. 791 and 792, respectively; SEQ ID Nos. 811 and 812, respectively; SEQ ID Nos. 831 and 832, respectively; or SEQ ID Nos. 851 and 852, respectively.

SEQ ID Nos. 693, 694, 695, 696, 697, and 698, respectively;

SEQ ID Nos. 713, 714, 715, 716, 717, and 718, respectively;

SEQ ID Nos. 733, 734, 735, 736, 737, and 738, respectively;

SEQ ID Nos. 753, 754, 755, 756, 757, and 758, respectively;

SEQ ID Nos. 773, 774, 775, 776, 777, and 778, respectively;

SEQ ID Nos. 793, 794, 795, 796, 797, and 798, respectively;

SEQ ID Nos. 813, 814, 815, 816, 817, and 818, respectively;

SEQ ID Nos. 833, 834, 835, 836, 837, and 838, respectively; or

SEQ ID Nos. 853, 854, 855, 856, 857, and 858, respectively.

In some embodiments, 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;

SEQ ID Nos. 711 and 712, respectively;

SEQ ID Nos. 731 and 732, respectively;

SEQ ID Nos. 751 and 752, respectively;

SEQ ID Nos. 771 and 772, respectively;

SEQ ID Nos. 791 and 792, respectively;

SEQ ID Nos. 811 and 812, respectively;

SEQ ID Nos. 831 and 832, respectively; or

SEQ ID Nos. 851 and 852, respectively.

In some embodiments, 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

GNYDAAKRGPGGAWAAEVISNAR (SEQ ID No: 892, SAA-P04). In some embodiments, 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.

In some embodiments, there is provided 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. In some embodiments, there is provided 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. In some embodiments, there is provided 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: 890, under urinary conditions. In some embodiments, there is provided 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: 891, under urinary conditions. In some embodiments, there is provided 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: 892, under urinary conditions.

Anti-EGF antibodies

In some embodiments, there is provided 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. 361 and 362, respectively; SEQ ID Nos. 381 and 382, respectively; SEQ ID Nos. 401 and 402, respectively; SEQ ID Nos. 421 and 422, respectively; SEQ ID Nos. 441 and 442, respectively; or SEQ ID Nos. 461 and 462, respectively. In some embodiments, 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. 303, 304, 305, 306, 307, and 308, respectively;

SEQ ID Nos. 323, 324, 325, 326, 327, and 328, respectively;

SEQ ID Nos. 343, 344, 345, 346, 347, and 348, respectively;

SEQ ID Nos. 363, 364, 365, 366, 367, and 368, respectively;

SEQ ID Nos. 383, 384, 385, 386, 387, and 388, respectively;

SEQ ID Nos. 403, 404, 405, 406, 407, and 408, respectively;

SEQ ID Nos. 423, 424, 425, 426, 427, and 428, respectively;

SEQ ID Nos. 443, 444, 445, 446, 447, and 448, respectively; or

SEQ ID Nos. 463, 464, 465, 466, 467, and 468, respectively.

SEQ ID Nos. 301 and 302, respectively;

SEQ ID Nos. 321 and 322, respectively;

SEQ ID Nos. 341 and 342, respectively;

SEQ ID Nos. 361 and 362, respectively;

SEQ ID Nos. 381 and 382, respectively;

SEQ ID Nos. 401 and 402, respectively;

SEQ ID Nos. 421 and 422, respectively;

SEQ ID Nos. 441 and 442, respectively; or

SEQ ID Nos. 461 and 462, respectively.

In some embodiments, there is provided 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, 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. 351 and 352, respectively; SEQ ID Nos. 371 and 372, respectively; SEQ ID Nos. 391 and 392, respectively; SEQ ID Nos. 411 and 412, respectively; SEQ ID Nos. 431 and 432, respectively; SEQ ID Nos. 451 and 452, respectively; or SEQ ID Nos. 471 and 472, respectively.

SEQ ID Nos. 313, 314, 315, 316, 317, and 318, respectively;

SEQ ID Nos. 333, 334, 335, 336, 337, and 338, respectively;

SEQ ID Nos. 353, 354, 355, 356, 357, and 358, respectively;

SEQ ID Nos. 373, 374, 375, 376, 377, and 378, respectively;

SEQ ID Nos. 393, 394, 395, 396, 397, and 398, respectively;

SEQ ID Nos. 413, 414, 415, 416, 417, and 418, respectively;

SEQ ID Nos. 433, 434, 435, 436, 437, and 438, respectively;

SEQ ID Nos. 453, 454, 455, 456, 457, and 458, respectively; or

SEQ ID Nos. 473, 474, 475, 476, 477, and 478, respectively.

SEQ ID Nos. 311 and 312, respectively;

SEQ ID Nos. 331 and 332, respectively;

SEQ ID Nos. 351 and 352, respectively;

SEQ ID Nos. 371 and 372, respectively;

SEQ ID Nos. 391 and 392, respectively;

SEQ ID Nos. 411 and 412, respectively;

SEQ ID Nos. 431 and 432, respectively;

SEQ ID Nos. 451 and 452, respectively; or

SEQ ID Nos. 471 and 472, respectively.

In some embodiments, 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). In some embodiments, the EGF-derived protein fragment or peptide includes, or consists of, a sequence set forth in SEQ ID No: 870. In some embodiments, the EGF- derived protein fragment or peptide includes, or consists of, a sequence set forth in SEQ ID No: 871.

In some embodiments, there is provided 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. In some embodiments, there is provided an antibody capable of specifically binding to an EGF-derived protein fragment or peptide including, or consisting of, a sequence set forth in SEQ ID No: 870, under urinary conditions. In some embodiments, there is provided an antibody capable of specifically binding to an EGF-derived protein fragment or peptide including, or consisting of, a sequence set forth in SEQ ID No: 871, under urinary conditions.

Anti-FGA antibodies

In some embodiments, there is provided 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. 521 and 522, respectively; SEQ ID Nos. 541 and 542, respectively; SEQ ID Nos. 561 and 562, respectively; SEQ ID Nos. 581 and 582, respectively; SEQ ID Nos. 601 and 602, respectively; SEQ ID Nos. 621 and 622, respectively; or SEQ ID Nos. 641 and 642, respectively.

SEQ ID Nos. 483, 484, 485, 486, 487, and 488, respectively;

SEQ ID Nos. 503, 504, 505, 506, 507, and 508, respectively;

SEQ ID Nos. 523, 524, 525, 526, 527, and 528, respectively;

SEQ ID Nos. 543, 544, 545, 546, 547, and 548, respectively;

SEQ ID Nos. 563, 564, 565, 566, 567, and 568, respectively;

SEQ ID Nos. 583, 584, 585, 586, 587, and 588, respectively;

SEQ ID Nos. 603, 604, 605, 606, 607, and 608, respectively;

SEQ ID Nos. 623, 624, 625, 626, 627, and 628, respectively; or

SEQ ID Nos. 643, 644, 645, 646, 647, and 648, respectively.

SEQ ID Nos. 481 and 482, respectively;

SEQ ID Nos. 501 and 502, respectively;

SEQ ID Nos. 521 and 522, respectively;

SEQ ID Nos. 541 and 542, respectively;

SEQ ID Nos. 561 and 562, respectively;

SEQ ID Nos. 581 and 582, respectively; SEQ ID Nos. 601 and 602, respectively;

SEQ ID Nos. 621 and 622, respectively; or

SEQ ID Nos. 641 and 642, respectively.

In some embodiments, there is provided 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. 531 and 532, respectively; SEQ ID Nos. 551 and 552, respectively; SEQ ID Nos. 571 and 572, respectively; SEQ ID Nos. 591 and 592, respectively; SEQ ID Nos. 611 and 612, respectively; SEQ ID Nos. 631 and 632, respectively; or SEQ ID Nos. 651 and 652, respectively.

SEQ ID Nos. 493, 494, 495, 496,497, and 498, respectively;

SEQ ID Nos. 513, 514, 515,516, 517, and 518, respectively;

SEQ ID Nos. 533, 534,535, 536,537, and 538, respectively;

SEQ ID Nos. 553, 554, 555, 556, 557, and 558, respectively;

SEQ ID Nos. 573, 574, 575, 576, 577, and 578, respectively;

SEQ ID Nos. 593, 594, 595,596, 597, and 598, respectively;

SEQ ID Nos. 613, 614, 615, 616,617, and 618, respectively;

SEQ ID Nos. 633, 634, 635, 636, 637, and 638, respectively; or

SEQ ID Nos. 653, 654, 655,656, 657, and 658, respectively.

SEQ ID Nos. 491 and 492, respectively;

SEQ ID Nos. 511 and 512, respectively;

SEQ ID Nos. 531 and 532, respectively;

SEQ ID Nos. 551 and 552, respectively;

SEQ ID Nos. 571 and 572, respectively;

SEQ ID Nos. 591 and 592, respectively;

SEQ ID Nos. 611 and 612, respectively; SEQ ID Nos. 631 and 632, respectively; or

SEQ ID Nos. 651 and 652, respectively.

In some embodiments, 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). In some embodiments, the FGA-derived protein fragment or peptide includes, or consists of, a sequence set forth in SEQ ID No: 876. In some embodiments, the FGA-derived protein fragment or peptide includes, or consists of, a sequence set forth in SEQ ID No: 877.

In some embodiments, there is provided 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. In some embodiments, there is provided an antibody capable of specifically binding to an FGA-derived protein fragment or peptide including, or consisting of, a sequence set forth in SEQ ID No: 876, under urinary conditions. In some embodiments, there is provided an antibody capable of specifically binding to an FGA-derived protein fragment or peptide including, or consisting of, a sequence set forth in SEQ ID No: 877, under urinary conditions.

General features of the isolated polypeptides

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.

Accordingly, the isolated polypeptide may be in any form suitable for detection of the respective protein fragment or peptide. Non-limiting examples of 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.

The term “antibody”, as used herein 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.

The term “Fv” (fragment variable), as used herein, relates to an antibody variable domain, including heavy and light chain variable regions.

The term “Fab” (fragment antigen binding), as used herein, relates to the heavy and light chain variable regions and further includes the 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.

The term “F(ab’)2”, as used herein 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.

The term “scFv”, as used herein 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.

The term “chimeric”, as used herein relates to an antibody or fragment thereof including sequences from more than one species.

The term “humanized”, as used herein relates to an antibody or fragment thereof which is produced in a non-human species (such as a mouse) but includes human sequences. For example, the framework sequences which separate the CDRs in the variable region may be replaced, or partly replaced, with human sequences.

The term “CAR-B”, as used herein, 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.

In some embodiments, 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 an antibody.

In some embodiments, the antibody is a monoclonal antibody.

In some embodiments, 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.

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.

The term “specifically binding”, as used herein, 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. In some embodiments, 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. In some embodiments, specific binding is defined as binding with an EC50 of less than about 10. In some embodiments, specific binding is defined as binding with an EC50 of less than about 1. In some embodiments, specific binding is defined as binding with an EC50 of less than about 0.5. In some embodiments, 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.

In some embodiments, the isolated polypeptide is further conjugated to a functional agent for using in various applications. Nonlimiting examples for 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. horseradish peroxidase (HRP) or alkaline phosphatase (AP), for 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.; oligonucleotides for immobilization and nucleic acid-based detection methods; polymers such as polyethylene glycol (PEG), for improving solubility and stability; and small molecules and haptens such as drugs, toxins, for targeted detection and quantification.

In some embodiments, the isolated polypeptide is conjugated to gold nanoparticles.

Nucleic acids, vectors, host cells, and hybridomas

In some embodiments, there is provided a 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.

In some embodiments, there is provided a vector including the nucleic acid molecule disclosed herein.

In some embodiments, there is provided a host cell including the nucleic acid molecule disclosed herein, the vector disclosed herein, and/or the isolated polypeptide disclosed herein.

In some embodiments, there is provided a hybridoma which is capable of producing the isolated polypeptide disclosed herein.

Definitions and embodiments mentioned above and which may be relevant to the present chapter (nucleic acids, vectors, host cells, hybridoma) also apply here, and vice versa. Some particularly relevant embodiments may be pointed out or explicitly repeated.

For terms used herein, unless stated otherwise, their definition and embodiments are intended to be the same as above (mutatis mutandis).

Combinations including the isolated polypeptide

For certain applications, a combination of more than one antibody against the same protein is needed. For example, 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.

In some embodiments, there is provided 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).

Definitions and embodiments mentioned above and which may be relevant to the present chapter (combinations) also apply here, and vice versa. Some particularly relevant embodiments may be pointed out or explicitly repeated.

In some embodiments, 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). In other words, in some embodiments, the isolated polypeptide and the additional polypeptide bind to the same protein-derived- protein fragment or peptide.

In some embodiments, the additional polypeptide does not specifically bind the protein- derived protein fragment or peptide bound by the isolated polypeptide. In other words, in some embodiments, the isolated polypeptide and the additional polypeptide bind to different protein- derived fragments or peptides of the same protein.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, 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.

Diagnostic methods and uses of the isolated polypeptide

In some embodiments, there is provided 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.

In some embodiments, there is provided 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.

In some embodiments, 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.

In some embodiments, the diagnostic method is a diagnostic method disclosed herein. In some embodiments, the sample is a urine sample. In some embodiments, 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.

In some embodiments, 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.

Definitions and embodiments mentioned above and which may be relevant to the diagnostic methods and use embodiments also apply here, and vice versa. Some particularly relevant embodiments may be pointed out or explicitly repeated. For terms used herein, unless stated otherwise, their definition and embodiments are intended to be the same as above (mutatis mutandis).

Methods of treatment by using the isolated polypeptide for diagnosis

In some embodiments, there is provided 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.

For example, if the diagnostic step detects a bacterial infection, the subject may be treated with suitable antibiotics. If a systemic inflammation is diagnosed, then the subject may be treated with anti-inflammatory agents.

In some embodiments, the method of treating is a method of treating disclosed herein.

Definitions and embodiments mentioned above and which may be relevant to the treatment methods embodiments, also apply here, and vice versa. Some particularly relevant embodiments may be pointed out or explicitly repeated. For terms used herein, unless stated otherwise, their definition and embodiments are intended to be the same as above (mulalis mutandis).

Kits and devices for using the isolated polypeptide

In some embodiments, there is provided a diagnostic kit including the isolated polypeptide disclosed herein or the combinations disclosed herein, and instructions for use.

Definitions and embodiments mentioned above and which may be relevant to the kits embodiments, also apply here, and vice versa. Some particularly relevant embodiments may be pointed out or explicitly repeated. For terms used herein, unless stated otherwise, their definition and embodiments are intended to be the same as above (initial is mutandis).

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.

In some embodiments, the kit further includes an additional polypeptide, as described above.

In some embodiments, 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.

In some embodiments, 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. In some embodiments, 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. In some embodiments, the functional agent is a fluorescent label or nanoparticle.

In some embodiments, 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.

In some embodiments, the diagnostic kit is a diagnostic kit disclosed herein.

Table 2: Protein SEQ ID Nos. for antibodies

SEQ ID No.

A An FtiKbo ady name V VWH V VLI ^V _C ^H _DR1 ^V _C ^H _DR2 ^V _CD ^H _R3 ^V _C ^L _DR1 ^V _C ^L _DR2 ^V _C ^L _DR3

BTLA-POl-mAbOl 1 2 3 4 5 6 7 8

BTLA-P01-mAb02 21 22 23 24 25 26 27 28

BTLA-P01-mAb03 41 42 43 44 45 46 47 48

BTLA-P01-mAb04 61 62 63 64 65 66 67 68

BTLA-P01-mAb05 81 82 83 84 85 86 87 88

BTLA-P01-mAb06 101 102 103 104 105 106 107 108

BTLA-P01-mAb07 121 122 123 124 125 126 127 128

BTLA-P01-mAb08 141 142 143 144 145 146 147 148

BTLA-P01-mAb09 161 162 163 164 165 166 167 168

CRP-POl-mAblO 181 182 183 184 185 186 187 188

CRP-POl-mAbl l 201 202 203 204 205 206 207 208

CRP-P01-mAbl2 221 222 223 224 225 226 227 228 CRP-P01-mAbl3 241 242 243 244 245 246 247 248

CRP-P01-mAbl4 261 262 263 264 265 266 267 268

CRP-P01-mAbl5 281 282 283 284 285 286 287 288

EGF-P01-mAbl6 301 302 303 304 305 306 307 308

EGF-P01-mAbl7 321 322 323 324 325 326 327 328

EGF-P01-mAbl8 341 342 343 344 345 346 347 348

EGF-P01-mAbl9 361 362 363 364 365 366 367 368

EGF-P01-mAb20 381 382 383 384 385 386 387 388

EGF-P01-mAb22 421 422 423 424 425 426 427 428

EGF-P01-mAb23 441 442 443 444 445 446 447 448

EGF-P02-mAb21 401 402 403 404 405 406 407 408

EGF-P02-mAb24 461 462 463 464 465 466 467 468

FGA-P01-mAb30 581 582 583 584 585 586 587 588

FGA-P01-mAb31 601 602 603 604 605 606 607 608

FGA-P01-mAb32 621 622 623 624 625 626 627 628

FGA-P02-mAb25 481 482 483 484 485 486 487 488

FGA-P02-mAb26 501 502 503 504 505 506 507 508

FGA-P02-mAb27 521 522 523 524 525 526 527 528

FGA-P02-mAb28 541 542 543 544 545 546 547 548

FGA-P02-mAb29 561 562 563 564 565 566 567 568

FGA-P02-mAb33 641 642 643 644 645 646 647 648

T GAT SQ-PO1 -

A?ri 661 662 663 664 665 666 667 668 mAb34

SAA-P01-mAb35 681 682 683 684 685 686 687 688

SAA-P01-mAb36 701 702 703 704 705 706 707 708

SAA-P01-mAb38 741 742 743 744 745 746 747 748

SAA-P01-mAb41 801 802 803 804 805 806 807 808

SAA-P01-mAb42 821 822 823 824 825 826 827 828

SAA-P02-mAb37 721 722 723 724 725 726 727 728

SAA-P02-mAb39 761 762 763 764 765 766 767 768

SAA-P02-mAb40 781 782 783 784 785 786 787 788

SAA-P02-mAb43 841 842 843 844 845 846 847 848

Table 3: Nucleic acid SEQ ID Nos. for antibodies

SEQ ID No.

VH VH VH VL VL VL

Ab name VH V _{CDR1 CDR2 CDR3 CDR1 CDR2 CDR3}

BTLA-POl-mAbOl 11 12 13 14 15 16 17 18

BTLA-P01-mAb02 31 32 33 34 35 36 37 38

BTLA-P01-mAb03 51 52 53 54 55 56 57 58

BTLA-P01-mAb04 71 72 73 74 75 76 77 78

BTLA-P01-mAb05 91 92 93 94 95 96 97 98

BTLA-P01-mAb06 111 112 113 114 115 116 117 118

BTLA-P01-mAb07 131 132 133 134 135 136 137 138

BTLA-P01-mAb08 151 152 153 154 155 156 157 158

BTLA-P01-mAb09 171 172 173 174 175 176 177 178 CRP-POl-mAblO 191 192 193 194 195 196 197 198

CRP-POl-mAbl l 211 212 213 214 215 216 217 218

CRP-P01-mAbl2 231 232 233 234 235 236 237 238

CRP-P01-mAbl3 251 252 253 254 255 256 257 258

CRP-P01-mAbl4 271 272 273 274 275 276 277 278

CRP-P01-mAbl5 291 292 293 294 295 296 297 298

EGF-P01-mAbl6 311 312 313 314 315 316 317 318

EGF-P01-mAbl7 331 332 333 334 335 336 337 338

EGF-P01-mAbl8 351 352 353 354 355 356 357 358

EGF-P01-mAbl9 371 372 373 374 375 376 377 378

EGF-P01-mAb20 391 392 393 394 395 396 397 398

EGF-P01-mAb22 431 432 433 434 435 436 437 438

EGF-P01-mAb23 451 452 453 454 455 456 457 458

EGF-P02-mAb21 411 412 413 414 415 416 417 418

EGF-P02-mAb24 471 472 473 474 475 476 477 478

FGA-P01-mAb30 591 592 593 594 595 596 597 598

FGA-P01-mAb31 611 612 613 614 615 616 617 618

FGA-P01-mAb32 631 632 633 634 635 636 637 638

FGA-P02-mAb25 491 492 493 494 495 496 497 498

FGA-P02-mAb26 511 512 513 514 515 516 517 518

FGA-P02-mAb27 531 532 533 534 535 536 537 538

FGA-P02-mAb28 551 552 553 554 555 556 557 558

FGA-P02-mAb29 571 572 573 574 575 576 577 578

FGA-P02-mAb33 651 652 653 654 655 656 657 658

T GAT SO-POT - 671 672 673 674 675 676 677 678 mAb34

SAA-P01-mAb35 691 692 693 694 695 696 697 698

SAA-P01-mAb36 711 712 713 714 715 716 717 718

SAA-P01-mAb38 751 752 753 754 755 756 757 758

SAA-P01-mAb41 811 812 813 814 815 816 817 818

SAA-P01-mAb42 831 832 833 834 835 836 837 838

SAA-P02-mAb37 731 732 733 734 735 736 737 738

SAA-P02-mAb39 771 772 773 774 775 776 777 778

SAA-P02-mAb40 791 792 793 794 795 796 797 798

SAA-P02-mAb43 851 852 853 854 855 856 857 858

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.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains.

The term "a" and "an" refers to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

The term "about" when referring to a measurable value such as an amount, a ratio, and the like, is meant to encompass variations of ±10% of the indicated value, as such variations are also suitable to perform the disclosed invention. Any numerical values appearing in the application are intended to be construed as if preceded by “about”, unless indicated otherwise.

The term “substantially identical”, as used herein relates to a sequence identity of at least about 95%, 96% 97%, 98%, or 99%.

The term “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. In some embodiments, a nucleic acid molecule is a nucleic acid vector (usually a DNA vector) which includes elements such as genes, promoters, linkers, etc.

The term “bp”, as used herein, means base pair, or base pairs.

The term “aa”, as used herein, means amino acid or amino acids.

While certain embodiments of the invention have been illustrated and described, it will be clear that the invention is not limited to the embodiments described herein. Numerous modifications, changes, variations, substitutions, and equivalents will be apparent to those skilled in the art without departing from the spirit and scope of the present invention as described by the claims, which follow.

The following examples are presented in order to more fully illustrate some embodiments of the invention. They should in no way be construed, however, as limiting the broad scope of the invention. One skilled in the art can readily devise many variations and modifications of the principles disclosed herein without departing from the scope of the invention.

EXAMPLES

Methods

Patients

The study was based on three separate cohorts of patients, as detailed in Table 4.

Table 4: Patient’s cohorts

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.

Sample preparation

Blood and urine samples were collected from patients upon hospitalization. Routine chemistry was analyzed immediately, and aliquots of serum and urine were frozen in -80°c. For proteomics analysis, the samples were concentrated using 3 kDa molecular weight cutoff filters and the Classifier and Validation cohorts were subjected to in-solution tryptic digestion (resulting in cleavage at the carboxyl side of a Lysine (K) or Arginine (R)), followed by a desalting step. The trypsinization step was required in order to facilitate analysis by a mass spectrometer (MS). Handling the endogenous peptides samples further involved concentration of low molecular weight cut off spin columns, followed by Sephadex G-25 gel filtration columns and Elution steps.

Liquid chromatography mass spectrometry (LC-MS)

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.

Data processing

Raw data were processed with MaxQuant vl.6.6.0 or DIANN vl.8.1. Quantification was based on the label-free quantification (LFQ) method, based on unique peptides.

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 for a fraction of samples is calculated according to the following function: W(p) = — p log₂ (p) — (1 — p) log₂ (1 — p)- If P represents the fraction of bacterial samples out of the group of total samples identified by an examined peptide, then entropy is calculated for the bacterial fraction, and for each threshold, also for the subgroups of the bacterial fraction which are above (pA) and below (pB) the threshold. 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).

It is noted that 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.

Example 1. Identifying peptides indicative of bacterial vs. viral upper respiratory tract infections (URTI)

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.

Following validation of the top 80 peptides in the Validation cohort, 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

SEQ ID Nos for all sequences are provided in Table 1 above.

Cross validation of the type Leave One Out (LOO) was further used to reduce bias. Briefly, one patient is removed at each iteration, and entropy calculations are repeated each time to find the highest entropy threshold and to train a logistic regression model. The patient removed is used as the test for the model. Following establishing predictions for all patients, the accuracy, precision, and additional parameters are calculated for the bacterial infection-related as the positive assumption. The results are presented in Table 6 (for the same peptides presented in Table 5). B and V represent average log intensities for the bacterial and viral subgroups, respectively.

Table 6: Top 20 peptides after LOO, sorted by the mean entropy gain

SEQ ID Nos for all sequences are provided in Table 1 above.

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.

Example 2. Identifying peptides combinations indicative of bacterial vs. viral upper respiratory tract infections (URTI)

The best 6 peptides were selected based on the histograms in Figs. 1 and 2, and combinations of 2, 3, and 4 peptides were tested for predicting bacterial or viral infection. Tables 7-9 show performance metrics of peptide combinations for distinguishing bacterial from non-bacterial infections using Leave-One-Out Cross Validation (LOOCV). For each combination, 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).

Table 7: Combinations of 2 peptides

Table 8: Combinations of 3 peptides

Table 9: Combinations of 4 peptides

It appears that all combinations of more than one peptide increase the probability of detecting bacterial from viral infections. Additionally, it appears that a combination of peptides from 3 proteins provides a sufficiently good prediction, and that the addition of proteins beyond 3 may not be as meaningful.

Example 3. Correlation of urinary peptides with blood CRP

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.

Table 10: Correlation to blood CRP of the top peptides, sorted by Spearman correlation

SEQ ID Nos for all sequences are provided in Table 1 above.

From above table, the best predictors for blood CRP levels were CRP, SAA, and LRG1 peptides.

Example 4: Preparing monoclonal antibodies (mAbs) against urine peptides

The peptides presented in Table 11, which were identified by methods disclosed above (and most of which appear in tables presented above or include sequences flanking these peptides), were used for preparing mAbs, as detailed below.

Table 11: Target human peptides

FGA-P01 GSESGIFTNTK

FGA P02671

FGA-P02 QFTSSTSYNR

SEQ ID Nos for all sequences are provided in Table 1 above.

Animal immunization

For immunization, the peptides were conjugated at the N-terminus to keyhole limpet hemocyanin (KLH).

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. For primary immunization, total 40 pg of peptides with Titermax® adjuvants were intraperitoneally injected into each mouse as planned. After 3 days, total 40 pg of peptides with CpG plus Alum adjuvants were intraperitoneally injected into each mouse in order to enhance the immune response, and subsequent boosts were administered 3-4 days apart. Blood samples from each mouse were collected 3 days after boost 4. The antibody titers in sera (test bleed (TB) 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.

Test blood (TB) test by ELISA

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. Following 4 washes with PBST (1%BSA and 0.05% Tween®-20 in PBS) using Biotek (Elx 405), Primary bleed (PB) and Test Bleed (TB) sera were diluted by 1:100 and were 10-fold serially diluted (6 points, including 0 point) by assay buffer or urine buffer (1%BSA and 0.05% Tween-20 in simulated urine solution) respectively. 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. 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). Data Analysis was performed using Graphpad prism 6 software.

Spleen lymphocyte/splenocyte harvest and culture

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. 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.

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 xlO⁷ SP2/0 cells were re-suspended in 20 mL fusion medium.

Electrofusion

5xl0⁷ SP2/0 cells were added to IxlO⁸ 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. After electrofusion, the fused cells were placed in the slot for an additional 10 min. Cells were resuspended in DMEM with 20% FBS + hypoxanthine and thymidine (HT, Corning-cellgro, 25- 047-C1), adjusted to 0.5xl0⁶ cells/mL, and seeded 100 pL/well in a 96-well plate. Plates were transferred carefully to a 37°C, 5 % CO2 incubator. 100 pL DMEM, 20%FBS, 2xHAT (medium containing hypoxanthine, aminopterin, and thymidine, Sigma, H0262) was then added 24 hrs post- electro-fusion. Cell growth condition and potential contamination were monitored daily. When cell colonies reached 1-2 mm in diameter (usuallylO-14 days post fusion), hybridoma supernatant was collected for screening.

Elybridoma supernatant screening

For screening, 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 cm² flasks. Hybridoma cells were then frozen down.

For the hybridoma ELISA screening, typically, 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).

Results

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.

Table 12: EC50 values of mAbs

Ab Name EC50 P01 EC50 P02

BTLA-POl-mAbOl 0.05318 / ll'l'I.A-P()l inAb02 04)4417 7

BTLA-P01-mAb03 0.1529 / BTLA-P01-mAb04 0.0372 0.2875

BTLA-P01-mAb05 0.1524 /

“7”: EC50 could not be determined (the graph did not reach a plateau) Table 13: Amino add sequences for mAbs

QIQLVQSGPELKKPGETVKISCKAS

GYTFTDYSVHWVKQTPGKGLKWI BTLA-P01-mAb08 VH GWINTETGEPTYADDFKGRFAFSLE 151

TSASTAYLQINNLKNEDTATYFCTS TADVWGAGTTVTVSS _

DVVMTQTPLSLPVSLGDQASISCRS

SQSLVHSNGNTYLHWYLQKPGQSP BTEA-P01-mAb08 VL KLLIYKVSNRFSGVPDRFSGSGSGT 152

DFTLKISRVEAEDLGVYFCSQSTHIP

WTFGGGTKEEIE

QVQLQQSGAELVRPGASVKLSCKA

SVYTFTGQWIEWVKQRPGHGLEWI EGF-P01-mAbl7 VH AEILPGSGDTHYNEKFKGKATFTA 331

DTSSYTAYMQLSSLTTEDSAIYYCV RHYGEHYWGQGTTLTVSS _

DVLLTQTPLSLPVSLGDQVSISCRSS

QTIVHSNGNTYLEWFLQKPGQSPK EGF-P01-mAbl7 VL LLIYKVSNRFSGVPARFRGSGSGTD 332

FTLKISRVEAEDLGVYYCFQGSHVP

YTFGGGTKLEIK

EFQLQQSGPELVKPGASVKISCKAS

GFSFTDYNMNWMKQSNGKSLEWI EGF-P01-mAb23 VH GLINPKYGTTTYNQKFKGKATLTV 451

DQSSSIAYMQLNSLTSEDSAVYYCL YDGYYDYWGQGTTLTVSS _

DILMTQSPSSMSVSLGDTVSITCHA

SQVISNNIGWLQQKPGKSFKGLIYL EGF-P01-mAb23 VL GTNLEDGVPSRFSGSGSGADYSLTI 452

SSLESEDFADYYCVQYAQFPYTFG GGTKLEIK

EVQLQQSGAELVRSGASVKLSCTA

SGFNIKVYYLHWVKQKPDQGLEWI FGA-P02-mAb26 VH GWIDPENGDTEYVPNLQGKATMT 511

ADISSNTAYLQLSSLTSEDTAVYYC NVYGNYDHLMDYWGQGTSVTVSS

DVVMTQTPLTLSVTIGQPASISCKS

GQSLLDSDGKTYLNWLLQRPGQSP FGA-P02-mAb26 VL KRLIYLVSKLDSGVPDRFTGSGSGT 512

DFTLKISRVEAEDLGVYYCWQGTH

FPWTFGGGTKLEIK

QVQLHQSGTELMKPGASVKISCKA

TGYKFSSYWIEWIKVRPGHGLEWI SAA-P01-mAb38 VH GEILPRNGSAYYIEKFKGKATFTAD 751

TSSNTAYMQLSSLTSDDSAVYYCA RFGQNYFDYWGQGTTLTVSS _

DVEMTQTPLSLPVSLGDQASISCRS

SQSIVHSNGNTYLEWYLQKPGQSP SAA-P01-mAb38 VL KLLIYKVSNRFSGVPDRFSGSGSGT 752

DFTLKISRVEAEDLGVYYCFQGSH

VPYTFGGGTKLEIK

EVQLQQSGPELVKPGASVKISCKAS

GYTFTDYYINWVKQSHGKSLEWIG SAA-P01-mAb41 VH DINPNNGDTSHNQKFKGKATLTVD 811

KSSSTAYMEVRSLTSEDSAIYYCAT GDWYFDVWGTGTTVTVSS _

DVVMTQTPLTLSVTTGQPASISCKS

SQSLLDSDGKTYLNWLLQRPGQSP SAA-P01-mAb41 VL KRLIYRVSKLDSGVPDRFTGSGSGT 812

DFTLKISRVEAEDLGVYYCWQATH

FPRTFGGGTKLEIK

Ab name: Protein-Peptide-antibody number; SID: SEQ ID No.

Claims

1. A monoclonal antibody capable of specifically binding a CRP-derived protein fragment or peptide comprising a sequence set forth in SEQ ID NO: 861 and/or SEQ ID NO: 862.

2. An isolated polypeptide capable of specifically binding a CRP-derived protein fragment or peptide comprising 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. 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.

3. The isolated polypeptide of claim 2, wherein the VH-CDR1, VH-CDR2, VH-CDR3, VL- CDR1, VL-CDR2, and VL-CDR3, comprise sequences substantially identical to sequences set forth in:

SEQ ID Nos. 183, 184, 185, 186, 187, and 188, respectively;

SEQ ID Nos. 203, 204, 205, 206, 207, and 207, respectively;

SEQ ID Nos. 223, 224, 225, 226, 227, and 228, respectively;

SEQ ID Nos. 243, 244, 245, 246, 247, and 248, respectively;

SEQ ID Nos. 263, 264, 265, 266, 267, and 268, respectively; or

SEQ ID Nos. 283, 284, 285, 286, 287, and 288, respectively.

4. The isolated polypeptide of claim 2 or 3, wherein the VH and the VL regions comprise sequences substantially identical to 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.

5. The isolated polypeptide of any one of claims 2-4, or the monoclonal antibody of claim 1, wherein the binding is under urinary conditions.

6. The isolated polypeptide of any one of claims 2-5, wherein 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.

7. The isolated polypeptide of claim 6, wherein the antibody is a monoclonal antibody.

8. A nucleic acid encoding the monoclonal antibody of claim 1 or the isolated polypeptide of any one of claims 2-7.

9. A vector comprising the nucleic acid of claim 8.

10. A cell comprising the monoclonal antibody of claim 1, the isolated polypeptide of any one of claims 2-7, the nucleic acid of claim 8, or the vector of claim 9.

11. 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, the method comprising: 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, COL6A1, 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).

12. The method of 11, wherein the at least one protein fragment comprises 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, sequences at least 90%, 95%, 98%, or 99% identical to the peptide sequences, sequences comprising them, and intervening protein sequences.

13. The method of 11 or 12, wherein the at least one protein is selected from CRP, BTLA, EGF, FGA, LGALS9, SAA, and combinations thereof.

14. The method of claim 13, wherein: the level of at least one protein fragment derived from CRP, FGA, and/or SAA being above the respective threshold predicts a bacterial infection; and/or the level of at least one protein fragment derived from BTEA, EGF, and/or EGAES9 being above the respective threshold predicts a viral infection.

15. The method of claim 13 or 14, wherein the at least one protein is at least 2 proteins comprising BTEA and CRP; BTLA and FGA; BTLA and SAA; CRP and SAA; CRP and FGA; CRP and EGF; or EGF and FGA.

16. The method of claim 13 or 14, wherein the at least one protein is at least 3 proteins comprising 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.

17. The method of claim 13 or 14, wherein the at least one protein is at least 4 proteins comprising 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.

18. The method of claim 13 or 14, wherein the at least one protein is at least 5 proteins comprising 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.

19. The method of claim 13 or 14, wherein the at least one protein is at least 6 proteins comprising CRP, BTLA, EGF, FGA, LGALS9, and SAA.

20. The method of any one of claims 13-19, wherein the at least one protein fragment comprises 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 comprising them, and intervening protein sequences.

21. The method of any one of claims 13-20, wherein the at least one protein fragment comprises 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 comprising them, and intervening protein sequences.

22. The method of any one of claims 11-21, wherein the detecting in step (a) comprises 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.

23. The method of claim 22, wherein the one or more binding agents are selected from antibodies, functional fragments of antibodies, aptamers; and/or any agents capable of specifically binding to the protein fragment.

24. The method of claim 23, wherein the one or more binding agents comprise at least one antibody specific to at least one of the protein fragments.

25. The method of claims 23 or 24, wherein the one or more binding agents comprises at least one monoclonal antibody defined in Table 2, or comprises 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.

26. The method of any one of claims 22-25, wherein the detecting the binding is conducted by an assay selected from a lateral flow assay (LFA), a fluorescence activated cell sorting (FACS), enzyme-linked immunosorbent assay (ELISA), a dipstick, a dot-blot, an antibody chip, and magnetic beads.

27. The method of any one of claims 22-26, wherein for each protein fragment the respective threshold is a threshold of detection of the protein fragment by the one or more binding agents.

28. The method of any one of claims 11-26, wherein 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.

29. The method of any one of claims 11-26, wherein 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.

30. The method of any one of claims 11-29, wherein the subject is a mammal.

31. The method of claim 30, wherein the subject is a human.

32. The method of any one of claims 11-31, wherein the infection is an upper respiratory tract infection (URTI).

33. The method of claim 32, wherein 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.

34. The method of claim 32 or 33, wherein the URTI is caused by a bacterial agent selected from: group A streptococcus, Staphylococcus aureus, Moraxella catarrhalis, Haemophilus influenzae, Streptococcus pneumoniae, and combinations thereof.

35. The method of claim 32 or 33, wherein the URTI is caused by a viral agent selected from: rhinovirus, coronavirus, adenovirus, influenza virus, human parainfluenza virus, and combinations thereof.

36. The method of any one of claims 11-35, further comprising a step of treating the subject with an antibiotic treatment when the infection is predicted to be bacterial, and/or treating the subject with an antiviral treatment when the infection is predicted to be viral.

37. A method for treating a bacterial infection in a subject afflicted with an infection, comprising: 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.

38. The method of claim 37, wherein 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.

39. A method for treating a viral infection in a subject afflicted with an infection, the method comprising: 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.

40. The method of claim 39, wherein 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.

41. A method for predicting efficacy of a treatment in a subject afflicted with a bacterial and/or a viral infection, the method comprising: 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 comprising an antibiotic and/or an antiviral agent; 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; and e. predicting whether the treatment is effective based on the determination in step (d).

42. A kit for predicting in a urine sample whether an infection isa bacterial infection or a viral infection, the kit comprising: 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. 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.

43. The kit of claim 42, wherein 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 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 a viral infection.

44. The kit of claim 42 or 43, wherein the kit further comprises reagents for use with an assay based on a lateral flow assay (LFA), a fluorescence activated cell sorting (FACS), enzyme- linked immunosorbent assay (ELISA), a dipstick, a dot-blot, an antibody chip, and magnetic beads.

45. The kit of any one of claims 42-44, wherein the one or more binding agent are selected from antibodies, functional fragments of antibodies, aptamers, and/or any agents capable of specifically binding to a protein fragment.

46. The kit of any one of claims 42-45, wherein the one or more binding agent comprises at least one monoclonal antibody defined in Table 2, or comprises at least one monoclonal antibody defined by a variable heavy chain (VH) region and a variable light chain (VL) region and/or complementarity determining region (CDR) sequences presented in Table 13.

47. A method for predicting an elevated blood CRP level by a urine test, the method comprising: 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 comprising 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).

48. A kit for predicting an elevated blood CRP level by a urine test, the kit comprising: a. one or more binding agents each capable of binding to at least one protein fragment comprising a sequence selected from peptide sequences presented in Table 10, sequences at least 90%, 95%, 98%, or 99% identical to the peptide sequences, sequences comprising 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.

49. A method for treating an inflammation in a subject, the method comprising: a. detecting in a urine sample of the subject a level of at least one protein fragment comprising a sequence selected from peptide sequences presented in Table 10, sequences at least 90%, 95%, 98%, or 99% identical to the peptide sequences, sequences comprising 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.

50. A method for predicting efficacy of a treatment of inflammation in a subject, the method comprising: a. detecting in a first urine sample of the subject a first level of at least one protein fragment comprising a sequence selected from peptide sequences presented in Table 10, sequences at least 90%, 95%, 98%, or 99% identical to the peptide sequences, sequences comprising 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.

51. The isolated polypeptide of any one of claims 2-7 or the monoclonal antibody of claim 1, for use in the method of any one of claims 11, 37, 39, 41, 47, 49, or 50.