Antibodies against urinary crp

Abstract

The present invention provides isolated polypeptides including antibodies capable of specifically binding CRP or CRP -derived peptides under urinary conditions, methods of using such isolated polypeptides and antibodies in diagnostics of inflammation, and kits including them.

Description

[0001] ANTIBODIES AGAINST URINARY CRP

[0002] FIELD OF THE INVENTION

[0003] The present disclosure is generally directed to antibodies against urinary C-reactive protein (CRP) and their uses. More specifically, the invention relates to antibodies against CRP, which may be used for diagnosis and / or monitoring of inflammation by testing a urine sample, and to methods and kits containing such antibodies.

[0004] BACKGROUND OF THE INVENTION

[0005] Biomarkers are widely used in the diagnosis, prognosis, and monitoring of various diseases. As opposed to testing blood, which is usually a more direct test and thus frequently used, urine collection is non-invasive and easy to store, making it a natural candidate for screening, diagnosis, and monitoring of diseases for which biomarkers can be found in urine.

[0006] Urinary biomarkers are used in clinical applications including pregnancy testing and prenatal screening, cancer detection, and more. Published applications WO 2021 / 117045 and WO 2021 / 117044, also present urinary biomarkers for diagnosing systemic inflammation and for distinguishing between bacterial and viral infection, respectively.

[0007] C-reactive protein (CRP) is a blood protein which is known to be increased during inflammation and infection. Testing of blood CRP levels by a urine-based test will simplify diagnosis and facilitate at-home testing for inflammation without the need for a care provider.

[0008] Accordingly, developing reagents and assays for urinary testing to predict blood CRP levels is needed.

[0009] SUMMARY OF INVENTION

[0010] 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.

[0011] In some embodiments, there is provided an isolated polypeptide capable of specifically binding C-reactive protein (CRP) or a CRP-derived peptide under urinary conditions. In some embodiments, the isolated polypeptide is capable of specifically binding CRP. In some embodiments, the isolated polypeptide is capable of specifically binding a CRP-derive peptide.

[0012] In some embodiments, the isolated polypeptide includes a heavy chain variable (VH) region including three complementarity determining region (CDR) sequences (VH-CDR1, VH-CDR2, VH-CDR3) and a light chain variable (VL) region including three CDR sequences (VL-CDR1, VL-CDR2, and VL-CDR3), and the six CDR sequences are defined by a standard method selected from Kabat, Chothia, IMGT, and AbM, based on VH and VL regions sequences of an antibody selected from the antibodies CRP-PRT-mAbOl, 03, 04, 06, 07, 09, 10, 11, 15, 16, and 20, and CRP-POl-mAb 10-15.

[0013] In some embodiments, the isolated polypeptide includes a heavy chain variable (VH) region including three complementarity determining region (CDR) sequences and a light chain variable (VL) region including three CDR sequences, and the six CDR sequences are defined by a standard method selected from Kabat, Chothia, IMGT, and AbM, based on VH and VL regions sequences selected from: SEQ ID Nos. 01 and 02; SEQ ID Nos. 41 and 42; SEQ ID Nos. 61 and 62; SEQ ID Nos. 101 and 102; SEQ ID Nos. 121 and 122; SEQ ID Nos. 161 and 162; SEQ ID Nos. 181 and 182; SEQ ID Nos. 201 and 202; SEQ ID Nos. 281 and 282; SEQ ID Nos. 301 and 302; SEQ ID Nos. 341 and 342; SEQ ID Nos. 361 and 362; SEQ ID Nos. 381 and 382; SEQ ID Nos. 421 and 422; SEQ ID Nos. 441 and 442; SEQ ID Nos. 461 and 462; SEQ ID Nos. 481 and 482; SEQ ID Nos. 501 and 502; and SEQ ID Nos. 521 and 522.

[0014] In some embodiments, the six CDR sequences of the isolated polypeptide include sequences substantially identical to CDR sequences of an antibody selected from CRP-PRT-mAbOl, 03, 04, 06, 07, 09, 10, 11, 15, 16, and 20, and CRP-POl-mAb 10-15.

[0015] In some embodiments, the six CDR sequences of the isolated polypeptide include sequences substantially identical to six CDR sequences selected from: SEQ ID Nos. 03, 04, 05, 06, 07, and 08; SEQ ID Nos. 43, 44, 45, 46, 47, and 48; SEQ ID Nos. 63, 64, 65, 66, 67, and 68; SEQ ID Nos.

[0016] 103, 104, 105, 106, 107, and 108; SEQ ID Nos. 123, 124, 125, 126, 127, and 128; SEQ ID Nos.

[0017] 163, 164, 165, 166, 167, and 168; SEQ ID Nos. 183, 184, 185, 186, 187, and 188; SEQ ID Nos.

[0018] 203, 204, 205, 206, 207, and 208; SEQ ID Nos. 283, 284, 285, 286, 287, and 288; SEQ ID Nos.

[0019] 303, 304, 305, 306, 307, and 308; SEQ ID Nos. 383, 384, 385, 386, 387, and 388; SEQ ID Nos.

[0020] 423, 424, 425, 426, 427, and 428; SEQ ID Nos. 443, 444, 445, 446, 447, and 448; SEQ ID Nos.

[0021] 463, 464, 465, 466, 467, and 468; SEQ ID Nos. 483, 484, 485, 486, 487, and 488; SEQ ID Nos.

[0022] 503, 504, 505, 506, 507, and 508; and SEQ ID Nos. 523, 524, 525, 526, 527, and 528.

[0023] In some embodiments, the VH and VL region sequences of the isolated polypeptide include sequences substantially identical to VH and VL sequences of an antibody selected from CRP-PRT-mAbOl, 03, 04, 06, 07, 09, 10, 11, 15, 16, and 20, and CRP-POl-mAb 10-15.

[0024] In some embodiments, the VH and VL region sequences of the isolated polypeptide include sequences substantially identical to VH and VL sequences of selected from: SEQ ID Nos. 01 and 02; SEQ ID Nos. 41 and 42; SEQ ID Nos. 61 and 62; SEQ ID Nos. 101 and 102; SEQ ID Nos. 121 and 122; SEQ ID Nos. 161 and 162; SEQ ID Nos. 181 and 182; SEQ ID Nos. 281 and 282; SEQ ID Nos. 301 and 302; SEQ ID Nos. 381 and 382; SEQ ID Nos. 421 and 422; SEQ ID Nos.

[0025] 441 and 442; SEQ ID Nos. 461 and 462; SEQ ID Nos. 481 and 482; SEQ ID Nos. 501 and 502; and SEQ ID Nos. 521 and 522.

[0026] In some embodiments, the six CDR sequences of the isolated polypeptide include sequences substantially identical to CDR sequences encoded by nucleotide sequences of an antibody selected from CRP-PRT-mAbOl, 03, 04, 06, 07, 09, 10, 11, 15, 16, and 20, and CRP-P01-mAbl0-15.

[0027] In some embodiments, the six CDR sequences of the isolated polypeptide include sequences substantially identical to six CDR sequences encoded by nucleic acid sequences selected from: SEQ ID Nos. 13, 14, 15, 16, 17, and 18; SEQ ID Nos. 53, 54, 55, 56, 57, and 58; SEQ ID Nos. 73, 74, 75, 76, 77, and 78; SEQ ID Nos. 113, 114, 115, 116, 117, and 118; SEQ ID Nos. 133, 134, 135, 136, 137, and 138; SEQ ID Nos. 173, 174, 175, 176, 177, and 178; SEQ ID Nos. 193, 194, 195, 196, 197, and 198; SEQ ID Nos. 213, 214, 215, 216, 217, and 218; SEQ ID Nos. 293, 294, 295, 296, 297, and 298; SEQ ID Nos. 313, 314, 315, 316, 317, and 318; SEQ ID Nos. 393, 394, 395, 396, 397, and 398; SEQ ID Nos. 433, 434, 435, 436, 437, and 438; SEQ ID Nos. 453, 454, 455, 456, 457, and 458; SEQ ID Nos. 473, 474, 475, 476, 477, and 478; SEQ ID Nos. 493, 494, 495, 496, 497, and 498; SEQ ID Nos. 513, 514, 515, 516, 517, and 518; and SEQ ID Nos. 533, 534, 535, 536, 537, and 538.

[0028] In some embodiments, the VH and VL regions include sequences substantially identical to VH and VL sequences encoded by nucleotide sequences of an antibody selected from CRP-PRT-mAbOl, 03, 04, 06, 07, 09, 10, 11, 15, 16, and 20, and CRP-POl-mAb 10-15.

[0029] In some embodiments, the VH and VL regions of the isolated polypeptide include sequences substantially identical to VH and VL sequences encoded by nucleotide sequences selected from: SEQ ID Nos. 11 and 12; SEQ ID Nos. 51 and 52; SEQ ID Nos. 71 and 72; SEQ ID Nos. 111 and 112; SEQ ID Nos. 131 and 132; SEQ ID Nos. 171 and 172; SEQ ID Nos. 191 and 192; SEQ ID Nos. 211 and 212; SEQ ID Nos. 291 and 292; SEQ ID Nos. 311 and 312; SEQ ID Nos. 391 and 392; SEQ ID Nos. 431 and 432; SEQ ID Nos. 451 and 452; SEQ ID Nos. 471 and 472; SEQ ID Nos. 491 and 492; SEQ ID Nos. 511 and 512; and SEQ ID Nos. 531 and 532.

[0030] In some embodiments, the CRP-derived peptide includes a sequence selected from GYSIFSYATKRQDNEILIFWSK (SEQ ID NO: 541) and RQDNEILIFWSK (SEQ ID NO:

[0031] 542).

[0032] In some embodiments, the CRP is a human CRP. In some embodiments, the CRP is from a non-human mammal.

[0033] In some embodiments, the isolated polypeptide is capable of specifically binding C-reactive protein (CRP) or a CRP-derived peptide under urinary conditions.

[0034] In some embodiments, the isolated polypeptide is selected from an antibody or an antigenbinding 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.

[0035] In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody fragment is an Fv fragment, an Fab fragment, or an F(ab’)2 fragment.

[0036] In some embodiments, the isolated polypeptide is, or derived from, an IgG.

[0037] In some embodiments, the isolated polypeptide is conjugated to a functional moiety.

[0038] In some embodiments, the specific binding is characterized by an EC50 of less than about 10, l, or 0.1 nM.

[0039] In some embodiments, there if provided an isolated nucleic acid molecule including at least one sequence encoding the isolated polypeptide disclosed herein.

[0040] In some embodiments, there is provided a host cell including the isolated polypeptide disclosed herein, or the nucleic acid molecule disclosed herein. In some embodiments, the host cell is a hybridoma cell.

[0041] In some embodiments, there is provided a combination including a first isolated polypeptide which is an isolated polypeptide disclosed herein, and an additional polypeptide capable of specifically binding CRP or a CRP-derived peptide, wherein the additional polypeptide does not interfere with binding of the isolated polypeptide to the CRP or CRP-derived peptide. In some embodiments, the additional polypeptide is an isolated polypeptide as disclosed herein. In some embodiments, the additional polypeptide is different from the first isolated polypeptide.

[0042] In some embodiments, the isolated polypeptide has six CDR sequences substantially identical to six CDR sequences set forth in SEQ ID Nos. 43-48, and the additional polypeptide has six CDR sequences substantially identical to six CDR sequences selected from: SEQ ID Nos. 103-108 and SEQ ID Nos. 163-168.

[0043] In some embodiments, the isolated polypeptide has VH and VL sequences substantially identical to VH and VL sequences set forth in SEQ ID Nos. 41-42, and the additional polypeptide has VH and VL sequences substantially identical to VH and VL sequences selected from: 101-102 and SEQ ID Nos. 161-162.

[0044] In some embodiments, the isolated polypeptide has six CDR sequences substantially identical to six CDR sequences set forth in SEQ ID Nos. 203-208, and the additional polypeptide has six CDR sequences substantially identical to six CDR sequences selected from: SEQ ID Nos.

[0045] 103-108 and SEQ ID Nos. 163-168.

[0046] In some embodiments, the isolated polypeptide has VH and VL sequences substantially identical to VH and VL sequences set forth in SEQ ID Nos. 201-202, and the additional polypeptide has VH and VL sequences substantially identical to VH and VL sequences selected from: 101-102 and SEQ ID Nos. 161-162.

[0047] In some embodiments, the isolated polypeptide has six CDR sequences substantially identical to six CDR sequences set forth in SEQ ID Nos. 43-48, and the additional polypeptide has six CDR sequences substantially identical to six CDR sequences set forth in SEQ ID Nos. 3-8.

[0048] In some embodiments, the isolated polypeptide has VH and VL sequences substantially identical to VH and VL sequences set forth in SEQ ID Nos. 41-42, and the additional polypeptide has VH and VL sequences substantially identical to VH and VL sequences set forth in SEQ ID Nos. 1-2.

[0049] In some embodiments, there is provided a monoclonal antibody capable of specifically binding C-reactive protein (CRP) or a CRP -derived peptide under urinary conditions.

[0050] In some embodiments, there is provided a monoclonal antibody capable of specifically binding C-reactive protein (CRP)-derived peptide including the sequence GYSILSYATKRQDNEILIEWSK (CRP-P01, SEQ ID NO: 541) or RQDNEILIFWSK (CRP-P02, SEQ ID NO: 542), under urinary conditions.

[0051] In some embodiments, there is provided a method for predicting in a urine sample of a subject, whether the subject has elevated blood CRP levels, the method including:

[0052] a. contacting a urine sample of the subject with one or more isolated polypeptides disclosed herein;

[0053] b. detecting binding of the one or more isolated polypeptides to the CRP or CRP -derived peptide; and

[0054] c. predicting whether the subject has elevated blood CRP levels based on the detecting in step (b), thereby indicating that the subject suffers from an inflammation, wherein binding of the one or more isolated polypeptides to the CRP or CRP-derived peptide predicts that the subject has elevated blood CRP levels, thereby indicating that the subject suffers from an inflammation.

[0055] In some embodiments, the inflammation is systemic inflammation.

[0056] In some embodiments, the elevated blood CRP levels are at least 100 or 120 mg / L.

[0057] In some embodiments, the method further includes a step of treating the subject with an antiinflammatory agent when the subject is predicted to be suffering from inflammation.

[0058] In some embodiments, the detecting the binding is conducted by an assay selected from a lateral flow test, a fluorescence activated cell sorting (LACS), enzyme-linked immunosorbent assay (ELISA), a dipstick, an antibody chip, and magnetic beads.

[0059] In some embodiments, the subject is a human. In some embodiments, the subject is a nonhuman mammal.

[0060] In some embodiments, the urine sample is selected from a device containing urine, a urine cup, a urine bag, a urine diaper, or a urine catheter.

[0061] In some embodiments, there is provided a kit for predicting in a urine sample of a subject whether the subject has elevated blood CRP levels, thereby indicating that the subject suffers from inflammation, the kit including:

[0062] a. one or more one or more isolated polypeptides disclosed herein;

[0063] b. at least one reagent for detecting the binding of the one or more isolated polypeptides to CRP or a CRP-derived peptide in a urine sample; and

[0064] c. instructions for use.

[0065] In some embodiments, the kit further includes reagents for use with an assay based on a lateral flow test, FACS, ELISA, a dipstick, an antibody chip, or a multiplex bead immunoassay.

[0066] 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.

[0067] BRIEF DESCRIPTION OF DRAWINGS

[0068] The invention will now be described in relation to certain examples and embodiments with reference to the following illustrative figures.

[0069] Fig. 1 shows a correlation analysis between blood and urinary CRP levels in 99 paired samples. Due to inherently different measurement scales (blood CRP: mg / L; urinary CRP: mass spectrometry intensity values), the data are presented as percentile ranks (0-100%) on both axes to enable direct comparison. Each point represents a single sample, with its position determined by its relative rank in both measurements rather than absolute values. The analysis revealed a strong positive correlation (Spearman's p = 0.84, 95% confidence interval: [0.74, 0.90], p = 3.06 / 1027). The black solid line shows the fitted regression line (slope = 0.84), while the gray dashed line represents the theoretical perfect correlation (slope = 1). Blood CRP concentrations (mg / L) are displayed above the plot, with their corresponding percentile ranks shown below, providing both absolute and relative measurements for reference. This strong correlation demonstrates the potential utility of urinary CRP measurement as a non-invasive alternative to blood CRP testing.

[0070] Figs. 2A-2C show binding characteristics of the antibodies of the invention. Fig. 2A shows ELISA binding of purified monoclonal antibodies against CRP-His protein in assay buffer. EC80 values for CRP-PRT-mAbO3 and CRP-PRT-mAbll were 0.007529 pg / ml and 0.01284 pg / ml (respectively). Figs. 2B-2C show ELISA binding of purified monoclonal antibodies against CRP-His protein in simulated urine.

[0071] Figs. 3A-3B show sandwich ELISA binding to combinations of antibodies in simulated urine. Fig. 3A. Sandwich ELISA using CRP-PRT-mAbl5 as the capture antibody. Detection was performed with serial dilutions of biotinylated CRP-PRT-mAb03 or -mAb20. Dose-dependent binding to CRP-His is observed. mlgGl was used as a negative control. Fig. 3B. Sandwich ELISA using CRP-PRT-mAb03 as the capture antibody. Serial dilutions of biotinylated CRP-PRT-mAbOl, -mAbO4, -mAbO6, -mAbO9, -mAblO, -mAbl5, or -mAbl6 were used for detection, with mlgGl used as a negative control. Dose-dependent binding to CRP-His is observed.

[0072] Figs. 4A-4B show prediction of blood CRP levels by a urine-based ELISA. Fig.4A. presents a receiver Operating Characteristic (ROC) for prediction of blood CRP > 80 mg / L using the urine sandwich ELISA assay with CRP-PRT-mAb03 as capture antibody and CRP-PRT-mAbO9 as detection antibody, showing AUC = 0.886. sensitivity = 88.9%, and specificity = 80.0%. The solid line represents the true positive rate plotted against the false positive rate across discrimination thresholds, while the dashed diagonal line indicates random classification. Fig. 4B. Urinary ELISA signal (hybrid format using CRP-PRT-mAb03 of the invention with a commercial antibody (Polyclonal Rabbit- anti CRP-HRP) stratified by blood CRP categories (0-10, 10-50, 50-100, > 100 mg / L), illustrating increasing urine signal intensity with higher systemic CRP levels. Boxes represent the interquartile range (IQR); horizontal lines indicate medians; diamonds represent means; whiskers denote 1.5 x IQR; and circles indicate outliers.

[0073] DETAILED DESCRIPTION OF THE INVENTION

[0074] 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.

[0075] C-reactive protein (CRP) is a protein found in blood plasma, and which blood levels are known to be elevated in response to inflammation. Urine is produced predominantly from plasma that is filtered by the kidneys. As a result, most of the proteins and peptides present in blood are not present in urine. It is therefore not a priori productive to analyze blood-derived biomarkers by urinalysis, and it is not possible to predict which biomarkers will be present in urine, based on blood biomarkers. With respect to CRP, none of the tests for detecting CRP in blood could be used to detect CRP in urine. Nevertheless, since urine analysis is not invasive and does not require a medical specialist, urine may be more readily used for basing a prediction.

[0076] As it turns out, CRP levels in urine do correlate with blood CRP levels, as shown in Fig. 1.

[0077] This indicates that high urine CRP is predictive of high blood CRP, which correlates with inflammation or infection. Accordingly, testing CRP levels in urine may be used for predicting blood CRP levels, and the present invention is therefore directed to development of reagents for detection of CRP or peptides thereof in urine. Furthermore, PCT publication no. WO2025 / 115009 by the inventors shows prediction of blood CRP levels based on detection of CRP -derived peptides in urine samples from patients.

[0078] To this end, the present invention relates to the development of polypeptides, such as antibodies, which are capable of specifically binding to CRP or peptides derived from CRP in urine. Such polypeptides or antibodies are especially useful for diagnosis of inflammation by using a urine sample. The antibodies of the invention may be used for detecting the CRP protein or peptides in urine of patient, thereby facilitating diagnosis of the relevant conditions.

[0079] In the present application, antibodies were developed against CRP (e.g., human CRP of UniProt accession no. P02741) and peptides derived from CRP, as described below, and were screened for their ability to bind CRP in simulated urine. Examples for the binding characteristics of the antibodies produced are shown in Fig. 2 and Example 2. 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 specifically 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 under any conditions, such as in other bodily fluids (e.g. blood) may be able to bind it under urinary conditions.

[0080] Below are provided specific isolated polypeptides which are capable of specifically binding CRP and peptides derived therefrom, and methods and uses of such polypeptides.

[0081] Isolated polypeptides and antibodies against CRP

[0082] In some embodiments, there is provided an isolated polypeptide capable of specifically binding a CRP protein target or a peptide thereof.

[0083] The term “C-reactive protein (CRP)” is intended to include all forms of the CRP protein, including pentameric and monomeric forms.

[0084] In some embodiments, the isolated polypeptide is capable of specifically binding the CRP or CRP -derived peptide in a urine sample. In some embodiments, the isolated polypeptide is capable of specifically binding the CRP or CRP -derived peptide under conditions similar to urine environment, also termed herein “urinary conditions”.

[0085] The term “urinary conditions”, as used herein, means a urine environment, such as urine (e.g. a urine sample), or conditions similar to the urine environment, such as a solution 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 condition (also termed herein “simulated urine”) are artificial or synthetic (or simulated) urine products such as Biochemazone artificial urine products. In some embodiments, the term “urinary conditions” includes urine, such as a urine sample.

[0086] In some embodiments, the urinary conditions relate to a urine sample. In some embodiments, the urinary conditions mean a solution 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 a solution including at least one of urea, creatinine, uric acid, and ammonia. In some embodiments, urinary conditions further mean a solution including certain salts or proteins.

[0087] Accordingly, in some embodiments, there is provided an isolated polypeptide capable of specifically binding CRP or a CRP -derived peptide, in urine, or in a urine sample.

[0088] Accordingly, in some embodiments, there is provided an isolated polypeptide capable of specifically binding CRP or a CRP -derived peptide under urinary conditions.

[0089] The terms “peptide”, as used herein, relates to any peptide or protein fragment having a sequence included in a CRP protein, that can bind to an isolated protein or antibody disclosed herein.

[0090] It is appreciated that isolated polypeptides identified as capable of binding to a complete CRP protein were generated in the present invention by immunizing against the complete protein. They may therefore bind a sequence that overlaps with any of the peptides disclosed herein, or they may bind a different sequence of the complete protein. It is also appreciated that peptides of CRP are not limited to the peptides disclosed herein.

[0091] In some embodiments, the peptide has a length of about 3-500 amino acids. In some embodiments, the peptide 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 has a length of at least about 5, 10, or 20 amino acids. In some embodiments, the peptide does not include the complete protein.

[0092] In some embodiments, the CRP is a human CRP. In some embodiments, the CRP is from a non-human mammal.

[0093] Each of the isolated polypeptides includes two different variable regions: a heavy chain variable (VH) region and a light chain variable (VL) region, and six complementarity-determining regions (CDRs), including three VH region CDRs: VH-CDR1, VH-CDR2, and VH-CDR3; and three VL region CDRs: VL-CDR1, VL-CDR2, and VL-CDR3.

[0094] 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.

[0095] 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. In some embodiments, the six CDRs are defined by the Kabat method.

[0096] Alternatively, in some embodiments, the six CDRs are defined by their sequence. When defined by sequences, the method of defining the sequences of the six CDRs may be any method acceptable in the art, including any of the methods mentioned herein (Kabat, Chothia, IMGT, and AbM), for example the Kabat method.

[0097] In some embodiments, the amino acid sequences of the isolated polypeptide or parts thereof (such as the VH, VL, and any of the CDRs) are defined by nucleotide sequences encoding them.

[0098] The above definitions and embodiments apply to all of the isolated polypeptides defined below.

[0099] It is noted that each of the antibodies mentioned by their name in the application is defined by VH, VL, and six CDR sequences presented in Table 1 and Table 6 (and / or encoded by sequences presented in Table 2 and Table 6). Accordingly, the antibody names clearly and unambiguously define the antibodies even without using the SEQ ID Nos explicitly in each embodiment.

[0100] In some embodiments, CRP-derived peptide includes, or consists of, a sequence selected from GYSIFSYATKRQDNEILIFWSK (CRP-P01, SEQ ID NO: 541) and RQDNEILIFWSK (CRP-P02, SEQ ID NO: 542).

[0101] In some embodiments, there is provided a monoclonal antibody capable of specifically binding to CRP under urinary conditions. In some embodiments, there is provided a monoclonal antibody capable of specifically binding to a CRP-derived peptide including, or consisting of, a sequence selected from SEQ ID NO: 541 and SEQ ID NO: 542, under urinary conditions.

[0102] In some embodiments, there is provided an isolated polypeptide capable of specifically binding a CRP protein target or a CRP-derived 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 of an antibody selected from the antibodies CRP-PRT-mAbOl, 03, 04, 06, 07, 09, 10, 11, 15, 16, and 20, and CRP-P01-mAbl0-15.

[0103] The VH and VL region amino acid sequences corresponding to the antibodies CRP-PRT-mAbOl, 03, 04, 06, 07, 09, 10, 11, 15, 16, and 20, and CRP-P01-mAbl0-15 are set forth in: SEQ ID Nos. 01 and 02; SEQ ID Nos. 41 and 42; SEQ ID Nos. 61 and 62; SEQ ID Nos. 101 and 102; SEQ ID Nos. 121 and 122; SEQ ID Nos. 161 and 162; SEQ ID Nos. 181 and 182; SEQ ID Nos.

[0104] 201 and 202; SEQ ID Nos. 281 and 282; SEQ ID Nos. 301 and 302; SEQ ID Nos. 381 and 382; SEQ ID Nos. 421 and 422; SEQ ID Nos. 441 and 442; SEQ ID Nos. 461 and 462; SEQ ID Nos.

[0105] 481 and 482; SEQ ID Nos. 501 and 502; and SEQ ID Nos. 521 and 522, respectively.

[0106] The six CDR amino acid sequences of antibodies CRP-PRT-mAbOl, 03, 04, 06, 07, 09, 10, 11, 15, 16, and 20, and CRP-P01-mAbl0-15 are set forth in: SEQ ID Nos. 03, 04, 05, 06, 07, and 08; SEQ ID Nos. 43, 44, 45, 46, 47, and 48; SEQ ID Nos. 63, 64, 65, 66, 67, and 68; SEQ ID Nos.

[0107] 103, 104, 105, 106, 107, and 108; SEQ ID Nos. 123, 124, 125, 126, 127, and 128; SEQ ID Nos.

[0108] 163, 164, 165, 166, 167, and 168; SEQ ID Nos. 183, 184, 185, 186, 187, and 188; SEQ ID Nos.

[0109] 203, 204, 205, 206, 207, and 208; SEQ ID Nos. 283, 284, 285, 286, 287, and 288; SEQ ID Nos.

[0110] 303, 304, 305, 306, 307, and 308; SEQ ID Nos. 383, 384, 385, 386, 387, and 388; SEQ ID Nos.

[0111] 423, 424, 425, 426, 427, and 428; SEQ ID Nos. 443, 444, 445, 446, 447, and 448; SEQ ID Nos.

[0112] 463, 464, 465, 466, 467, and 468; SEQ ID Nos. 483, 484, 485, 486, 487, and 488; SEQ ID Nos.

[0113] 503, 504, 505, 506, 507, and 508; and SEQ ID Nos. 523, 524, 525, 526, 527, and 528, respectively.

[0114] In some embodiments, the six CDR sequences of the isolated polypeptide, include, or consist of, sequences identical or substantially identical to CDR sequences of an antibody selected from the antibodies CRP-PRT-mAbOl, 03, 04, 06, 07, 09, 10, 11, 15, 16, and 20, and CRP-POl-mAb 10-15.

[0115] In some embodiments, the VH and the VL regions of the isolated polypeptide include, or consist of, sequences identical or substantially identical to VH and VL sequences of an antibody selected from the antibodies CRP-PRT-mAbOl, 03, 04, 06, 07, 09, 10, 11, 15, 16, and 20, and CRP-POl-mAb 10-15.

[0116] The term “substantially identical”, as used herein relates to a sequence identity of at least about 95%, 96% 97%, 98%, or 99%. In some embodiments, when the compared sequences are VH or VL sequence, all differences between the sequences are not in CDR regions.

[0117] The VH and VL nucleotide sequences of antibodies CRP-PRT-mAbOl, 03, 04, 06, 07, 09, 10, 11, 15, 16, and 20, and CRP-POl-mAb 10- 15 are set forth in: SEQ ID Nos. 11 and 12; SEQ ID Nos. 51 and 52; SEQ ID Nos. 71 and 72; SEQ ID Nos. 111 and 112; SEQ ID Nos. 131 and 132; SEQ ID Nos. 171 and 172; SEQ ID Nos. 191 and 192; SEQ ID Nos. 211 and 212; SEQ ID Nos. 291 and 292; SEQ ID Nos. 311 and 312; SEQ ID Nos. 391 and 392; SEQ ID Nos. 431 and 432; SEQ ID Nos. 451 and 452; SEQ ID Nos. 471 and 472; SEQ ID Nos. 491 and 492; SEQ ID Nos.

[0118] 511 and 512; and SEQ ID Nos. 531 and 532, respectively.

[0119] The six CDR nucleotide sequences of antibodies CRP-PRT-mAbOl, 03, 04, 06, 07, 09, 10, 11, 15, 16, and 20, and CRP-P01-mAbl0-15 are set forth in: SEQ ID Nos. 13, 14, 15, 16, 17, and 18; SEQ ID Nos. 53, 54, 55, 56, 57, and 58; SEQ ID Nos. 73, 74, 75, 76, 77, and 78; SEQ ID Nos.

[0120] 113, 114, 115, 116, 117, and 118; SEQ ID Nos. 133, 134, 135, 136, 137, and 138; SEQ ID Nos.

[0121] 173, 174, 175, 176, 177, and 178; SEQ ID Nos. 193, 194, 195, 196, 197, and 198; SEQ ID Nos.

[0122] 213, 214, 215, 216, 217, and 218; SEQ ID Nos. 293, 294, 295, 296, 297, and 298; SEQ ID Nos.

[0123] 313, 314, 315, 316, 317, and 318; SEQ ID Nos. 393, 394, 395, 396, 397, and 398; SEQ ID Nos.

[0124] 433, 434, 435, 436, 437, and 438; SEQ ID Nos. 453, 454, 455, 456, 457, and 458; SEQ ID Nos.

[0125] 473, 474, 475, 476, 477, and 478; SEQ ID Nos. 493, 494, 495, 496, 497, and 498; SEQ ID Nos.

[0126] 513, 514, 515, 516, 517, and 518; andSEQ ID Nos. 533, 534, 535, 536, 537, and 538, respectively.

[0127] In some embodiments, the six CDR sequences of the isolated polypeptide are defined by a standard method selected from Kabat, Chothia, IMGT, and AbM, based on VH and VL regions sequences encoded by nucleotide sequences of an antibody selected from the antibodies CRP-PRT-mAbOl, 03, 04, 06, 07, 09, 10, 11, 15, 16, and 20, and CRP-POl-mAb 10-15.

[0128] In some embodiments, the six CDR of the isolated polypeptide, include, or consist of, sequences identical or substantially identical to CDR sequences encoded by nucleotide sequences of an antibody selected from the antibodies CRP-PRT-mAbOl, 03, 04, 06, 07, 09, 10, 11, 15, 16, and 20, and CRP-POl-mAb 10- 15.

[0129] In some embodiments, the VH and the VL regions of the isolated polypeptide include, or consist of, sequences identical or substantially identical to VH and VL sequences encoded by nucleotide sequences of an antibody selected from the antibodies CRP-PRT-mAbOl, 03, 04, 06, 07, 09, 10, 11, 15, 16, and 20, and CRP-POl-mAb 10-15.

[0130] In some embodiments, the isolated polypeptide is selected from the antibodies (the SEQ ID Nos. of the VH, VL, and six CDRs of each antibody are indicated in parentheses): CRP-PRT-mAbOl (SEQ ID Nos: 1-8), CRP-PRT-mAb03 (SEQ ID Nos: 41-48), CRP-PRT-mAbO4 (SEQ ID Nos: 61-68), CRP-PRT-mAbO6 (SEQ ID Nos: 101-108), CRP-PRT-mAbO7 (SEQ ID Nos: 121-128), CRP-PRT-mAbO9 (SEQ ID Nos: 161-168), CRP-PRT-mAblO (SEQ ID Nos: 181-188), CRP-PRT-mAbll (SEQ ID Nos: 201-208), CRP-PRT-mAbl5 (SEQ ID Nos: 281-288), CRP-PRT-mAb16 (SEQ ID Nos: 301-308), and CRP-PRT-mAb20 (SEQ ID Nos: 381-388).

[0131] In some embodiments, the isolated polypeptide is CRP-PRT-mAbO 1. In some embodiments, the isolated polypeptide is CRP-PRT-mAb03. In some embodiments, the isolated polypeptide is CRP-PRT-mAbO4. In some embodiments, the isolated polypeptide is CRP-PRT-mAbO6. In some embodiments, the isolated polypeptide is CRP-PRT-mAbO7. In some embodiments, the isolated polypeptide is CRP-PRT-mAbO9. In some embodiments, the isolated polypeptide is CRP-PRT-mAblO. In some embodiments, the isolated polypeptide is CRP-PRT-mAbll. In some embodiments, the isolated polypeptide is CRP-PRT-mAbl5. In some embodiments, the isolated polypeptide is CRP-PRT-mAbl6. In some embodiments, the isolated polypeptide is CRP-PRT-mAb20.

[0132] The CDRs disclosed herein 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.

[0133] The isolated polypeptide may be in any form suitable for detection of the CRP or CRP-derived peptide. Non-limiting examples of suitable forms include: an antibody or an antigenbinding 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.

[0134] The term “antibody”, as used herein is an immunoglobulin having two heavy chains and two light chains, each chain having a variable region (as described above) and a constant region, and wherein the variable regions of the heavy and light chains form antigen binding regions. It is noted that while classic antibodies are monospecific, i.e., having a single specificity targeting a single antigen or epitope, an antibody may also be multi-specific, i.e., capable of binding more than one distinct antigen or epitope.

[0135] Antigen-binding fragments of an antibody include an Fv fragment, an Fab fragment, and an F(ab’)2 fragment.

[0136] The term “Fv” (fragment variable), as used herein, relates to an antibody variable domain, including heavy and light chain variable regions.

[0137] 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.

[0138] 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.

[0139] 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.

[0140] The term “chimeric”, as used herein relates to an antibody or fragment thereof including sequences from more than one species.

[0141] 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, framework sequences, which are the less variable sequences separating the CDRs in the variable region, may be replaced, or partly replaced, with human sequences.

[0142] 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.

[0143] 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.

[0144] In some embodiments, the isolated polypeptide is an antibody. In some embodiments, the antibody is a monoclonal antibody.

[0145] In some embodiments, the isolated polypeptide is an antibody fragment. In some embodiments, the antibody fragment is selected from an Fv fragment, an Fab fragment, and an F(ab’)2 fragment.

[0146] In some embodiments, the Fv fragment, Fab fragment, F(ab’)2 fragment, scFv, chimeric or humanized antibody 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.

[0147] When the antibody includes a constant (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. Sequences of constant regions are well known in the art.

[0148] The term “specifically binding”, as used herein, relates to binding that is sufficiently specific so as to specifically detect the respective protein or peptide, without significant background of other proteins 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 EC80 (80% 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 10-7M-1.

[0149] It is further appreciated that specifically binding a protein target does not preclude binding to fragments of the protein target.

[0150] In some embodiments, the 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-125, Iodine-131, Indium-Ill 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.

[0151] In some embodiments, the polypeptide is further conjugated to gold nanoparticles.

[0152] Nucleic acids, vectors, host cells, and hybridomas for generation of antibodies of the invention

[0153] In some embodiments, there is provided at least one nucleic acid molecule including at least one sequence encoding the isolated polypeptide disclosed herein. In some embodiments, the nucleic acid molecule includes VH and VL, and / or six CDR sequences encoding an antibody selected from CRP-PRT-mAbOl, 03, 04, 06, 07, 09, 10, 11, 15, 16, and 20, and CRP-POl-mAblO- 15, as disclosed above with reference to the isolated polypeptides of the invention. In some embodiments, the nucleic acid molecule includes a set of VH and VL, and / or six CDR nucleotide sequences encoding a set of VH and VL, and / or six CDR sequences which together define an antibody presented in Table 1. In some embodiments, the nucleic acid molecule includes a set of VH and VL, and / or six CDR sequences which together define an antibody presented in Table 2.

[0154] Since an antibody is composed of several sequences corresponding, e.g., to the light and heavy chains, or to the six CD Rs, it is appreciated that the nucleic acid may be more than a single nucleic acid, and that sequence encoding the isolated polypeptide may include more than one sequence which together encode the isolated polypeptide. For example, the heavy chain and the light chain, or the six CDRs, of an antibody may be encoded on separate nucleic acid molecules, or may be encoded on the same nucleic acid molecules, but separated by a different sequence.

[0155] The nucleic acid molecule may further include sequences encoding additional antibody parts such as, e.g., heavy / light constant regions or parts thereof, and / or a hinge region. The nucleic acid molecule may further include a promoter, terminator, and further transcription and / or regulation elements, for expression of the polypeptide in a suitable cell, e.g. for producing the isolated polypeptide.

[0156] In some embodiments, there is provided a vector including the nucleic acid molecule disclosed herein. The vector may be any vector suitable for expression of the sequences encoding the isolated polypeptide in a desired cell, i.e., include a suitable promoter and terminator and any additional elements required for expression in the suitable cell. Nonlimiting examples for such vectors include plasmids, viral vectors, etc.

[0157] 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. The host cell may be any cell suitable for expression and / or production (or mass production) of the isolated polypeptides of the invention. In some embodiments, the host cell is an eukaryotic cell. In some embodiments, the host cell is a cell of a cell line or in a cell culture. In some embodiments, the host cell is a cell derived from an immune system cell. In some embodiments, the host cell is a human cell. In some embodiments, the host cell is a hybridoma cell.

[0158] In some embodiments, there is provided a hybridoma cell which is capable of producing the isolated polypeptide disclosed herein. In some embodiments, the hybridoma cell includes the nucleic acid molecule disclosed herein. In some embodiments, the hybridoma cell includes a nucleic acid molecule encoding the isolated polypeptide disclosed herein.

[0159] 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).

[0160] Combinations including the isolated polypeptide

[0161] 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.

[0162] In some embodiments, there is provided a polypeptide combination, including an isolated polypeptide as disclosed herein, and an additional polypeptide which is capable of specifically binding to the CRP without interfering with the binding of the isolated polypeptide to the protein target.

[0163] In some embodiments, the isolated polypeptide is used as capture antibody and the additional peptide is used as detection antibody. In some embodiments, the isolated polypeptide is used as detection antibody and the additional peptide is used as capture antibody.

[0164] 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. For terms used herein, unless stated otherwise, their definition and embodiments are intended to be the same as above (mutatis mutandis).

[0165] 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 which is capable of specifically recognizing CRP, e.g., a commercially-available anti-CRP antibody. Some examples for such antibodies include CRP Antibody (500-P242-100UG, PeproTech), HRP Anti-C Reactive Protein antibody (Abeam, abl9175), and anti-C-Reactive Protein antibody produced in goat (C8284, Sigma- Aldrich).

[0166] In some embodiments, the additional polypeptide is capable of specifically binding to CRP or CRP -derived peptide under urinary conditions (e.g., in urine, in a urine sample, or in a urine environment). In some embodiments, the isolated polypeptide is capable of specifically binding to CRP or CRP -derived peptide under urinary conditions. In some embodiments, both the isolated polypeptide and the additional polypeptide are capable of specifically binding to the CRP or CRP-derived peptide under urinary conditions. In some embodiments, both the isolated polypeptide and the additional polypeptide are capable of specifically binding to the same CRP-derived peptide.

[0167] 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 binding test, such as a lateral flow test.

[0168] Figs. 3A-3B demonstrate examples for sandwich assays including anti-CRP antibodies. Additionally, Example 3 (Table 5) present an analysis of interaction between different antibody pairs, highlighting combinations of antibodies which may be used together without interfering with each other’s binding.

[0169] In some embodiments, the isolated polypeptide is CRP-PRT-mAbO3 and the additional polypeptide is selected from CRP-PRT-mAbOl, CRP-PRT-mAbO4, CRP-PRT-mAbO6, CRP-PRT-mAbO7, CRP-PRT-mAbO9, CRP-PRT-mAblO, CRP-PRT-mAbl5, CRP-PRT-mAbl6, and CRP-PRT-mAb20.

[0170] In some embodiments, the isolated polypeptide has six CDR sequences substantially identical to six CDR sequences set forth in SEQ ID Nos. 43-48, and the additional polypeptide has six CDR sequences substantially identical to six CDR sequences selected from: SEQ ID Nos. 3-8; SEQ ID Nos. 63-68; SEQ ID Nos. 103-108; SEQ ID Nos. 123-128; SEQ ID Nos. 163-168; SEQ ID Nos. 183-188; SEQ ID Nos. 283-288; SEQ ID Nos. 303-308; and SEQ ID Nos. 383-388.

[0171] In some embodiments, the isolated polypeptide has VH and VL sequences substantially identical to VH and VL sequences set forth in SEQ ID Nos. 41-42, and the additional polypeptide has VH and VL sequences substantially identical to VH and VL sequences selected from: SEQ ID Nos. 1-2; SEQ ID Nos. 61-62; SEQ ID Nos. 101-102; SEQ ID Nos. 121-122; SEQ ID Nos. 161-162; SEQ ID Nos. 181-182; SEQ ID Nos. 281-282; SEQ ID Nos. 301-302; and SEQ ID Nos. 381-382.

[0172] In some embodiments, the isolated polypeptide is CRP-PRT-mAbll and the additional polypeptide is selected from CRP-PRT-mAbOl, CRP-PRT-mAbO4, CRP-PRT-mAbO6, CRP-PRT-mAbO7, CRP-PRT-mAbO9, CRP-PRT-mAblO, CRP-PRT-mAbl5, CRP-PRT-mAbl6, and CRP-PRT-mAb20.

[0173] In some embodiments, the isolated polypeptide has six CDR sequences substantially identical to six CDR sequences set forth in SEQ ID Nos. 203-208, and the additional polypeptide has six CDR sequences substantially identical to six CDR sequences selected from: SEQ ID Nos.

[0174] 3-8; SEQ ID Nos. 63-68; SEQ ID Nos. 103-108; SEQ ID Nos. 123-128; SEQ ID Nos. 163-168; SEQ ID Nos. 183-188; SEQ ID Nos. 283-288; SEQ ID Nos. 303-308; and SEQ ID Nos. 383-388. In some embodiments, the isolated polypeptide has VH and VL sequences substantially identical to VH and VL sequences set forth in SEQ ID Nos. 201-202, and the additional polypeptide has VH and VL sequences substantially identical to VH and VL sequences selected from: SEQ ID Nos. 1-2; SEQ ID Nos. 61-62; SEQ ID Nos. 101-102; SEQ ID Nos. 121-122; SEQ ID Nos. 161-162; SEQ ID Nos. 181-182; SEQ ID Nos. 281-282; SEQ ID Nos. 301-302; and SEQ ID Nos. 381-382.

[0175] In some embodiments, the isolated polypeptide is CRP-PRT-mAbl5 and the additional polypeptide is CRP-PRT-mAbl6.

[0176] In some embodiments, the isolated polypeptide has six CDR sequences substantially identical to six CDR sequences set forth in SEQ ID Nos. 283-288, and the additional polypeptide has six CDR sequences substantially identical to six CDR sequences set forth in SEQ ID Nos. 303-308.

[0177] In some embodiments, the isolated polypeptide has VH and VL sequences substantially identical to VH and VL sequences set forth in SEQ ID Nos. 281-282, and the additional polypeptide has VH and VL sequences substantially identical to VH and VL sequences set forth in SEQ ID Nos. 301-302.

[0178] In some embodiments, the isolated polypeptide is CRP-PRT-mAbl5 and the additional polypeptide is CRP-PRT-mAb03. In some embodiments, the isolated polypeptide is CRP-PRT-mAbl5 and the additional polypeptide is CRP-PRT-mAb20.

[0179] In some embodiments, the isolated polypeptide is CRP-PRT-mAbOl and the additional polypeptide is CRP-PRT-mAb03. In some embodiments, the isolated polypeptide is CRP-PRT-mAbOl and the additional polypeptide is CRP-PRT-mAb20. In some embodiments, the isolated polypeptide is CRP-PRT-mAb03 and the additional polypeptide is CRP-PRT-mAbO4. In some embodiments, the isolated polypeptide is CRP-PRT-mAb03 and the additional polypeptide is CRP-PRT-mAbO6. In some embodiments, the isolated polypeptide is CRP-PRT-mAb03 and the additional polypeptide is CRP-PRT-mAbO9. In some embodiments, the isolated polypeptide is CRP-PRT-mAb03 and the additional polypeptide is CRP-PRT-mAblO. In some embodiments, the isolated polypeptide is CRP-PRT-mAb03 and the additional polypeptide is CRP-PRT-mAbl5. In some embodiments, the isolated polypeptide is CRP-PRT-mAb03 and the additional polypeptide is CRP-PRT-mAbl6. In some embodiments, the isolated polypeptide is CRP-PRT-mAbO6 and the additional polypeptide is CRP-PRT-mAb20. In some embodiments, the isolated polypeptide is CRP-PRT-mAbO9 and the additional polypeptide is CRP-PRT-mAb20. In some embodiments, the isolated polypeptide is CRP-PRT-mAbl5 and the additional polypeptide is CRP-PRT-mAbl6. In some embodiments, the isolated polypeptide is CRP-PRT-mAbl5 and the additional polypeptide is CRP-PRT-mAb20. In some embodiments, the isolated polypeptide is CRP-PRT-mAbl6 and the additional polypeptide is CRP-PRT-mAb20.

[0180] In some embodiments, the isolated polypeptide is CRP-PRT-mAbO3 and the additional polypeptide is a commercial antibody. In some embodiments, the isolated polypeptide is CRP-PRT-mAb20 and the additional polypeptide is a commercial antibody.

[0181] In some embodiments, the combination of isolated polypeptide and additional polypeptide exhibits a wave shift of at least about 0.2 nanometer in an Octet binding assay with CRP as antigen.

[0182] General diagnostics using the antibodies of the invention

[0183] In some embodiments, there is provided a diagnostic method including detecting CRP or a CRP-derived 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.

[0184] In some embodiments, there is provided the isolated polypeptide disclosed herein or the combination disclosed herein, for use in a diagnostic method including detecting CRP or a CRP-derived peptide by using the isolated polypeptide disclosed herein or the combination disclosed herein.

[0185] Definitions and embodiments mentioned above and which may be relevant to the present chapter (diagnostics) 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).

[0186] In some embodiments, the diagnostic method is a method for diagnosis or detection of inflammation, infection, systemic infection or inflammation, cancer, autoimmune condition, kidney disease, infectious disease, urinary tract infection (URI), cardiovascular disease, cardiac infection, a rheumatic condition, and / or a metabolic disorder.

[0187] 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 CRP or a CRP-derived peptide in the urine sample. In some embodiments, the binding is under urinary conditions.

[0188] 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. Diagnosing inflammation

[0189] 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.

[0190] As shown in Fig. 1, CRP urinary levels correlate with CRP blood levels, and could therefore serve as predictive markers for elevated blood CRP levels and therefore for inflammation.

[0191] In some embodiments, there is provided a method for predicting in a urine sample of a subject, whether the subject has elevated blood CRP levels, the method including;

[0192] a. contacting a urine sample of the subject with one or more isolated polypeptides disclosed herein;

[0193] b. detecting binding of the one or more isolated polypeptides to CRP or a CRP-derived peptide; and

[0194] c. predicting whether the subject has elevated blood CRP levels based on the detecting in step (b), thereby indicating that the subject suffers from an inflammation, wherein detected binding of the one or more isolated polypeptides to CRP or a CRP-derived peptide predicts that the subject has elevated blood CRP levels, thereby indicating that the subject suffers from an inflammation.

[0195] 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).

[0196] 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 on a stick, which is capable of detecting the presence of peptides in the urine; and urine taken from a urine output device such as a device already containing urine (e.g. a stick after urination), a urine bag, a diaper, or a catheter. In some embodiments, the urine sample has previously been obtained from the subject.

[0197] Contacting the urine sample in step (a) is generally done under suitable conditions (such as pH, salt concentrations), which facilitate binding of isolated polypeptides to CRP or to a CRP- derived peptide. Such conditions may be empirically determined for each antibody.

[0198] In some embodiments, the CRP -derived peptide includes at least one sequence selected from sequences having at least 90%, 95%, 98%, or 99% identity to GYSIFSYATKRQDNEILIFWSK (CRP-P01, SEQ ID NO: 541) or to RQDNEILIFWSK (CRP-P02, SEQ ID NO: 542). In some embodiments, the CRP-derived peptide includes at least one sequence selected from SEQ ID NO: 541 and SEQ ID NO: 542

[0199] The term “elevated” with reference to blood CRP relates to a level of blood CRP that corresponds to inflammation. In some embodiments, an elevated blood CRP level is a blood CRP level which corresponds to severe inflammation. In some embodiments, an elevated blood CRP level is at least 10, 20, 50, 100, or 120 mg / L. In some embodiments, an elevated blood CRP level is at least 120 mg / L.

[0200] Detecting the isolated polypeptides binding to the proteins or peptides in step (b) may be conducted by any suitable method, such as fluorescent assays, colorimetric assays, radioactive assays, magnetic beads assays, methods based on binding partners (such as biotin and avidin), etc. More specific examples for suitable assays include fluorescence activated cell sorting (FACS), enzyme-linked immunosorbent assay (ELISA), a dot blot assay, a dipstick, an antibody chip, a multiplex bead immunoassay, and a lateral flow test. Accordingly, the isolated polypeptides may be labelled, such as fluorescently-labelled, radioactively labelled, or by any other label that may be later detected.

[0201] It is appreciated that the sensitivity of detection depends on the method used, e.g. on an antibody concentration and binding constant and / or on the concentrations and sensitivities of detection agents used in the method, as well as on additional parameters. Accordingly the sensitivity of detection may be calibrated such that detection of binding corresponds to a desired level of blood CRP, such as 100 mg / L or 120 mg / L.

[0202] The predicting in step (c) may be conducted by methods known in the art, such as by visual detection of a color reaction which directly indicates a positive result (i.e., elevated blood CRP level), by imaging, by computer processing, etc.

[0203] 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., blood CRP level being above a certain threshold as a sign of inflammation. In some embodiments, “predicting” means increasing a probability of predicting a desired parameter. In some embodiments, the high probability is a probability of at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%.

[0204] In some embodiments, the predicting in step (c) requires further processing, computation, or calculation. In some embodiments, the predicting is conducted by a processor, In some embodiments, the detection in step (b) is performed by a processor, as noted above, and the detection is further processed in step (c). In some embodiments, the detection 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.

[0205] 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.

[0206] In some embodiments, the inflammation is systemic inflammation.

[0207] 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.

[0208] 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.

[0209] In some embodiments, the subject has an immunodeficiency or is susceptible to infection due to treatments such as CAR-T therapy or chemotherapy.

[0210] In some embodiments, the subject may need to be monitored to evaluate treatment efficacy. In some embodiments, the method further includes a treatment step. In some embodiments, the treatment step includes treating the subject with an anti-inflammatory agent.

[0211] 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.

[0212] Administration regimens and doses are generally known in the art and may be calculated based on specific features relevant to a specific case.

[0213] Methods of treatment

[0214] In some embodiments, there is provided a method of treating a subject in need thereof, including a step of detecting CRP or a peptide thereof 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.

[0215] In some embodiments, there is provided a method for treating inflammation in a subject in need thereof, including:

[0216] a. contacting a urine sample of the subject with one or more isolated polypeptides disclosed herein;

[0217] b. detecting binding of the one or more isolated polypeptides to the CRP or CRP -derived peptide;

[0218] c. predicting that the subject suffers from inflammation based on the detection of binding of the one or more isolated polypeptides to the CRP or CRP-derived peptide; and d. treating the subject with an anti-inflammatory agent.

[0219] 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).

[0220] 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.

[0221] 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. Methods for predicting efficacy of treatment

[0222] In some embodiments, there is provided a method for predicting efficacy of a treatment in a subject suffering from severe inflammation, the method including:

[0223] a. administering to the subject a treatment including an anti-inflammatory agent; b. contacting a urine sample of the subject, taken after administration, with one or more isolated polypeptides disclosed herein;

[0224] c. detecting binding of the one or more isolated polypeptides to CRP or a CRP-related peptide; and

[0225] d. predicting whether the treatment is effective based on the detecting in step (c).

[0226] In some embodiments, if binding is detected in step (c) then treatment is not effective. In some embodiments, if binding is not detected in step (c) then treatment is effective.

[0227] 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 (mutatis mutandis).

[0228] Diagnostic kits and devices

[0229] In some embodiments, there is provided a diagnostic kit including the isolated polypeptide disclosed herein or the combinations disclosed herein, and instructions for use.

[0230] In some embodiments, there is provided a kit for predicting in a urine sample of a subject whether the subject has elevated blood CRP levels, the kit including:

[0231] a. one or more isolated polypeptides disclosed herein;

[0232] b. at least one reagent for detecting the binding of the one or more isolated polypeptides to CRP or a CRP-derived peptide in a urine sample; and

[0233] c. instructions for use.

[0234] 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 (mutatis mutandis).

[0235] In some embodiments, the reagent is suitable for use with an assay based on a fluorescent assay, a colorimetric assays, a radioactive assays, a magnetic beads assays, etc. In some embodiments, the reagent is suitable for use with lateral flow (LFA), ELISA, dipstick, antibody chip, 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.

[0236] In some embodiments, the kit is a single-strip kit. In some embodiments, the kit is a multiplestrip kit.

[0237] In some embodiments, the kit further includes reagents and / or a device for performing a lateral flow assay.

[0238] 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 peptide 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.

[0239] In some embodiments, the kit further includes an additional isolated polypeptide, as described above.

[0240] 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, such as described hereinabove.

[0241] 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 test, 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.

[0242] 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.

[0243] In some embodiments, there is provided an article of manufacture including the isolated polypeptide disclosed herein. In some embodiments, the article of manufacture is a lateral flow device. Table 1: protein SEQ ID Nos. for antibodies

[0244] SEQ ID No.

[0245] VH VH VH VL VL VL

[0246] Antibody name VH VL

[0247] CDR1 CDR2 CDR3 CDR1 CDR2 CDR3 CRP-PRT-mAb01 1 2 3 4 5 6 7 8 CRP-PRT-mAb03 41 42 43 44 45 46 47 48 CRP-PRT-mAb04 61 62 63 64 65 66 67 68 CRP-PRT-mAb06 101 102 103 104 105 106 107 108 CRP-PRT-mAb07 121 122 123 124 125 126 127 128 CRP-PRT-mAb09 161 162 163 164 165 166 167 168 CRP-PRT-mAb10 181 182 183 184 185 186 187 188 CRP-PRT-mAb11 201 202 203 204 205 206 207 208 CRP-PRT-mAb15 281 282 283 284 285 286 287 288 CRP-PRT-mAb16 301 302 303 304 305 306 307 308 CRP-PRT-mAb20 381 382 383 384 385 386 387 388 CRP-P01-mAb10 421 422 423 424 425 426 427 428 CRP-P01-mAb11 441 442 443 444 445 446 447 448 CRP-P01-mAb12 461 462 463 464 465 466 467 468 CRP-P01-mAb13 481 482 483 484 485 486 487 488 CRP-P01-mAb14 501 502 503 504 505 506 507 508

[0248]

[0249] CRP-P01-mAb15 521 522 523 524 525 526 527 528

[0250] Table 2: DNA SEQ ID Nos. for antibodies

[0251] SEQ ID No.

[0252] VH VH VH VL VL VL

[0253] Antibody name VH VL

[0254] CDR1 CDR2 CDR3 CDR1 CDR2 CDR3 CRP-PRT-mAb01 11 12 13 14 15 16 17 18 CRP-PRT-mAb03 51 52 53 54 55 56 57 58 CRP-PRT-mAb04 71 72 73 74 75 76 77 78 CRP-PRT-mAb06 111 112 113 114 115 116 117 118 CRP-PRT-mAb07 131 132 133 134 135 136 137 138 CRP-PRT-mAb09 171 172 173 174 175 176 177 178 CRP-PRT-mAb10 191 192 193 194 195 196 197 198 CRP-PRT-mAb11 211 212 213 214 215 216 217 218 CRP-PRT-mAb15 291 292 293 294 295 296 297 298 CRP-PRT-mAb16 311 312 313 314 315 316 317 318 CRP-PRT-mAb20 391 392 393 394 395 396 397 398 CRP-P01-mAb10 431 432 433 434 435 436 437 438 CRP-P01-mAb11 451 452 453 454 455 456 457 458 CRP-P01-mAb12 471 472 473 474 475 476 477 478 CRP-P01-mAb13 491 492 493 494 495 496 497 498 CRP-P01-mAb14 511 512 513 514 515 516 517 518

[0255]

[0256] CRP-P01-mAb15 531 532 533 534 535 536 537 538 The antibody name includes the protein it targets (CRP), the target it was raised against (PRT - a complete protein; P-01 - a specific peptide), and a serial antibody number. It is noted that CRP anti-POl antibodies were found to also bind P02 (since these peptides overlap in sequence), but at a different affinity.

[0257] 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.

[0258] 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.

[0259] 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.

[0260] 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.

[0261] The term “bp”, as used herein, means base pair, or base pairs.

[0262] The term “aa”, as used herein, means amino acid or amino acids.

[0263] 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.

[0264] 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.

[0265] EXAMPLES

[0266] Methods

[0267] Animal immunization

[0268] For immunization, CRP (UniProt Acc P02741) protein or peptides thereof, namely CRP- P01 (GYSIFSYATKRQDNEILIFWSK, SEQ ID NO: 541) and CRP-P02 (RQDNEILIFWSK, SEQ ID NO: 542) were conjugated at the N-terminus to keyhole limpet hemocyanin (KLH).

[0269] 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.

[0270] Test blood (TB) test by ELISA

[0271] Peptides were conjugated to bovine serum albumin (BSA) and diluted to proper final concentrations into 1× 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 such as Artificial Urine Solution (BIOCHEMAZONE, Cat No.:#BZ186) 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.

[0272] Spleen lymphocyte / splenocyte harvest and culture

[0273] 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.

[0274] 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 ×107SP2 / 0 cells were re-suspended in 20 mL fusion medium.

[0275] Electrofusion

[0276] 5×107SP2 / 0 cells were added to 1×108lymphocyte / 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 containing polyethylene glycol (PEG) (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 re-suspended in DMEM with 20% FBS + hypoxanthine and thymidine (HT, Coming-cellgro, 25-047-C1), adjusted to 0.5x106cells / 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 (usually 10- 14 days post fusion), hybridoma supernatant was collected for screening.

[0277] Hybridoma supernatant screening

[0278] 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.

[0279] 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 (e.g. Artificial Urine Solution (BIOCHEMAZONE, Cat No.:#BZ186)) were selected for subcloning

[0280] 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 cm2flasks. Hybridoma cells were then frozen down.

[0281] For the hybridoma ELISA screening, typically, BSA conjugated peptide were diluted to proper final concentrations into 1×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 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).

[0282] Purification of mouse antibodies

[0283] Hybridoma cells were seeded at (1-2) x 105 cells / mL in culture medium (DMEM with 10% FBS and 1% Pen / Strep) into T-75 flasks. Upon reaching 75% confluence or 5x105 cells / mL and over 85% cell viability, cells were expanded at 1: 1 ratio with fresh FBS-Free medium to allow the cells gradually adapted to the low-serum medium (7-10 days).

[0284] The cells were inoculated into a roller bottle pre-filled with 200 mL antibody production medium at the density of (0.25-0.5) ×105cells / mL. The inoculated roller bottles were incubated in roller culture incubator and cultured for 14-16 days. The cell suspension was centrifuged and filtered with a 0.45 pM filtration capsule to remove the cell debris. The antibodies were purified from the culture supernatants by Protein A affinity column (GE, Cat# 17- 1279-02). The collected antibodies were dialyzed against PBS, pH7.4, at 2-8°C overnight with 3 times buffer exchanges and sterile filtered with a 0.22 pM syringe filter. Antibody concentrations were determined by absorbance at 280nm, and purity was assessed by SDS-PAGE and SEC-HPLC. Endotoxin levels were determined with the Limulus Amebocyte Lysate (LAL) method (Charles River, Cat#R1708K).

[0285] Purified mAb characterization by ELISA

[0286] Recombinant CRP-his protein was diluted with coating buffer (PBS) to 1 pg / mL, 100 pL of diluted protein was coated onto 96-well ELISA plates and incubated at 4°C overnight. The plates were washed 3 times with washing buffer (PBST-PBS, 0.05% Tween-20, pH 7.4). 200 pL / well blocking buffer (1% BSA in PBST, pH 7.4) was added to each well and incubated for 1 hrs at 37°C. Thereafter, 100 pL of 21 purified mAbs (initial concentration at 66.6 nM, 1:5 dilution in blocking buffer or simulated urine solution (Artificial Urine Solution, BIOCHEMAZONE, Cat:#BZ186) containing 1% BSA and 0.05% Tween 20) were added to each well of the 96-well ELISA plate respectively, and incubated at 37°C for Ihrs. The plates were washed 3 times with washing buffer. 100 pL / well of anti-mouse IgG (Fey specific)-HRP (Sigma, A0168; 1:5000 in blocking buffer) were added to each well and incubated for 0.5 hr at 37°C, and the plates were washed 3 times with washing buffer. 100 pL / well of TMB substrate was added to each well and incubated at room temperature for 10 min. 50 pL / well of 1 N HC1 was added to terminate the reaction, and the absorbance at 450 nm was measured with a microplate reader.

[0287] Epitope binning

[0288] Epitope binning pretest to ensure the EC80 of CRP-his binding to 21 different mAbs: coated 100 pL / well 1 pg / mL 21 different mAbs (mlgG was coated as negative control) and serially diluted CRP-his (1:5 dilution for 8 dose from 1 pg / mL) were co-incubated at 37°C for 1 hrs, following by addition of secondary antibody (Rabbit anti-his-HRP, Abeam, Ab 1187; 1:5000 in blocking buffer) at 37°C for 1 hr. Detailed steps can refer to the above ELISA steps. According to the results, the coating concentrations of different mAbs were adjusted to get accurate EC80 of CRP-his binding to 21 different mAbs. Finally, Ipg / mL mAbs were coated to acquire accurate EC80 of CRP-his.

[0289] Epitope binning: Coated 100 pL / well 1 pg / mL purified mAbs. 50 pL competitive antibodies (40-fold concentration of coating capture antibodies) and 50 pL CRP-his (using the concentration of EC80x2) were added to each well of the 96-well ELISA plate and incubated at 37°C for 1.5 hrs, following by addition of secondary antibody dilution (Rabbit anti-his-HRP, Abeam, Ab 1187; 1:5000 in blocking buffer) at 37°C for 1 hr. Detailed steps could refer to the above ELISA steps. The inhibition rate of each mAb against the rest of mAbs was calculated using the following equation, OD450 (mIgG-mAb) / OD450 mlgG* 100%. The higher of inhibition rate, the more likely the antibody shared the closer bin with the test antibody. And then the inhibition rate of mAb pairs were plotted to a data matrix.

[0290] Biomolecular interaction analyses by Octet

[0291] The Octet RED384 system (ForteBio) was used to perform label-free biomolecular interaction analyses. The binding response in Octet systems is measured in nanometers (nm), which reflects the wavelength shift caused by the change in optical thickness at the biosensor's surface during binding events. This wavelength shift corresponds to the amount of material (antibody / antigen) bound to the surface, with a greater shift indicating more binding.

[0292] The experimental procedure began with a baseline measurement for sensor equilibration in PBS for 60 seconds, followed by the immobilization of the capture antibody (loaded 1st) onto the AMC biosensor for 240 seconds. After an additional baseline step (60 seconds), the biosensors were blocked to minimize nonspecific binding (300 seconds). Antigen loading was carried out for 240 seconds, followed by an association step of the detection antibody for 300 seconds. The dissociation phase, which is next, lasted 50 seconds, and the biosensors were regenerated with 10 mM Glycine -HC1 for 5 seconds.

[0293] The proteins and antibodies used included CRP-his (concentration: 0.62 mg / mL, molecular weight: 23.87 kDa, Cytiva) and mouse IgG subclasses (mIgG1, mIgG2a, mIgG2b, and mIgG2c) at concentrations ranging from 1.343 to 2.19 mg / mL. Data analyses were performed using the ForteBio Octet Data Analyses HT software V12.0.

[0294] Total RNA Isolation

[0295] According to NucleoZOL Reagent’s protocol. Generally, prepared ~1E7 hybridoma cells, removed cell culture medium and lysed cells by addition of at least 1 mL NucleoZOL (MACHEREY-NAGEL, 740404.200) to the culture disk (diameter 3.5 cm, 10 cm2). Next, complete lysis was ensured by repeated pipetting. Then 200 pL RNase-free water per 500 pL NucleoZOL were added to the lysate. The sample was shaken vigorously for 15s and incubated at room temperature for 5 min. Samples were then centrifuged for 15 min at 12,000 g at room temperature. 500 pL of supernatant was transferred to a fresh tube and a layer of the supernatant was left above the DNA / protein pellet. Then 500 pL of isopropanol per 500 pL supernatant was added in order to precipitate RNA. Samples were then incubated at room temperature for 10 min. Samples were centrifuged for 10 min at 12,000 g, after which supernatant was removed and discarded. 500 pL 75 % ethanol was used when precipitating in 1.5 mL tubes. For larger tubes, 500 pL 75 % ethanol per 1 mL supernatant was added. The pellets were centrifuged for 3 min at 8,000 g. Ethanol was removed from the pellets by pipetting. The 75 % ethanol washing step was repeated. The RNA pellets were later dissolved in RNase-free water to obtain an RNA concentration of 1μg / μL.

[0296] Example 1: Correlation between serum CRP concentrations and urinary proteomic CRP intensity

[0297] Spearman correlation analysis (r = 0.844, n = 99) shown in Fig. 1 demonstrated a strong positive association between CRP levels measured in serum using a wide-range immunoturbidimetric assay (Beckman Coulter, Brea, CA, USA) and CRP protein intensity quantified by the urine-based proteomic platform. Serum CRP concentrations were determined as part of routine hospital testing using the WR-CRP method (analytical range 0.2-480 mg / L) and served as the clinical gold standard for comparison. Urinary CRP proteomic intensity results are presented as percentile ranks.

[0298] Example 2: Preparing monoclonal antibodies (mAbs) against urinary peptides Hybridomas were screened for antibodies capable of binding to CRP (UniProt Acc P02741) and two peptides thereof, namely CRP-P01 (GYSIFSYATKRQDNEILIFWSK, SEQ ID NO: 541) and CRP-P02 (RQDNEILIFWSK, SEQ ID NO: 542) by dilution of 1:1 in simulated urine. Amino acid sequences of the most specific antibodies are presented in Table 6 below. Fig. 2A shows ELISA binding of mAbs to the corresponding target in simulated urine, as part of an EC80 binding pretest. Figs. 2B and 2C show ELISA binding of mAbs to the corresponding target in simulated urine. The respective EC50 values for the results in Fig. 2B, 2C for the mAbs binding to CRP or a peptide thereof, are presented in Table 3, as well as the results for CRP-POl-mAblO-mAbl5, which were obtained in simulated urine. Furthermore, results for binding of pairs of capture / detection antibodies to CRP by are presented in Table 4 and Figs. 3A-3B.

[0299] Table 3: EC50 values of mAbs (nM)

[0300] Ab Name EC50 - CRP EC50 - P01 EC50 - P02

[0301] CRP-PRT-mAbOl 0.09904

[0302] CRP-PRT-mAb03 0.09567

[0303] CRP-PRT-mAbO4 0.05697

[0304] CRP-PRT-mAbO6 0.117

[0305] CRP-PRT-mAbO7 0.06293

[0306] CRP-PRT-mAbO9 0.1246

[0307] CRP-PRT-mAblO 0.128

[0308] CRP-PRT-mAbll 0.1088

[0309] CRP-PRT-mAbl5 0.08949

[0310] CRP-PRT-mAbl6 0.1105 CRP-PRT-mAb20 0.131

[0311] CRP-POl-mAblO 0.1622 0.03585

[0312] CRP-POl-mAbll 0.0675 0.04601

[0313] CRP-P01-mAbl2 0.2676 0.1235

[0314] CRP-P01-mAbl3 0.1091 0.07181

[0315] CRP-P01-mAbl4 0.6598 0.04516

[0316] CRP-P01-mAbl5 0.3944 0.1516

[0317] Table 4: Octet results for CRP-His binding with different capture antibodies and with CRP-PRT-mAbO3 as the detection antibody

[0318] Binding Response

[0319] Capture antibody

[0320] (nm)

[0321] CRP-PRT-mAb03 0.0684

[0322] CRP-PRT-mAbOl 0.4808

[0323] CRP-PRT-mAbO6 0.4462

[0324] CRP-PRT-mAblO 0.4095

[0325] CRP-PRT-mAbl5 0.4845

[0326]

[0327] CRP-PRT-mAbl6 0.2541

[0328] Example 3: Epitope binning and sandwich ELISA validation of CRP monoclonal antibody pairs.

[0329] The assays were designed to identify compatible antibody pairs that recognize distinct, noncompeting epitopes on a CRP protein. Table 5A presents an epitope binning matrix determined by biolayer interferometry (Octet® RED384 (Sartorius, Cat. No. 18-5156)). Values represent binding response levels between antibody pairs. Bold highlights indicates non-competing antibodies (binding response > 0.2 nm) capable of simultaneous binding to CRP. Table 5B presents results of a sandwich ELISA validation confirming functional antibody pairing. Values represent optical density at 450 nm for each capture-detection combination. Bold highlights indicate high-performing sandwich pairs (OD > 0.8), with optimal combinations achieving 1.3-2.3 OD units. Control antibody (mlgG) and incompatible pairs showed baseline signals < 0.1 OD units. All data represent mean values of duplicate assays. As can be seen, most results overlap between the two methods.

[0330] Table 5A. Epitope binning matrix (Octet)

[0331] AB# mAbOl mAb03 mAbO6 mAbl5 mAbl6 mAb20 Control mAbOl 0.038 0.481 0.046 0.068 0.061 0.538 -0.017 mAb03 0.266 0.068 0.279 0.218 0.293 0.059 -0.012 mAbO6 0.037 0.446 0.051 0.068 0.058 0.474 -0.013 mAbO9 0.065 0.357 0.102 0.109 0.141 0.337 -0.010 mAbl5 0.052 0.484 0.072 0.047 0.305 0.563 -0.027 mAbl6 0.048 0.254 0.068 0.03 0.028 0.282 -0.044 mAb20 0.228 0.045 0.229 0.194 0.276 0.069 -0.024 Control 0.058 0.099 0.085 0.092 0.076 0.088 -0.002

[0332] Table 5B. Epitope binning matrix (ELISA)

[0333] AB# mAbOl mAb03 mAbO6 mAbl5 mAbl6 mAb20 Control mAbOl 0.063 1.317 0.066 0.059 0.052 1.463 0.051 mAb03 2.297 0.059 2.265 2.106 1.853 0.061 0.052 mAbO6 0.066 1.483 0.066 0.064 0.053 1.488 0.053 mAbl5 0.071 1.715 0.205 0.099 0.132 1.799 0.053 mAbl6 0.077 0.59 0.216 0.11 0.053 0.815 0.053 mAb20 1.698 0.343 1.767 1.706 1.455 0.06 0.054 Control 0.052 0.052 0.083 0.061 0.05 0.054 0.054

[0334] Example 4: Prediction of blood CRP levels by urine-based ELISA.

[0335] Fig. 4A shows as receiver operating characteristic (ROC) curve illustrating the performance of the urine CRP ELISA assay in identifying patients with systemic (mega-)inflammation defined by serum CRP > 80 mg / L (n = 38). The ELISA was performed using CRP-PRT-mAb03 as the capture CRP-PRT-mAbO9 as the detection antibody. The assay demonstrated high diagnostic accuracy with AUC = 0.886, overall accuracy = 84.2%, balanced accuracy = 84.4%, sensitivity = 88.9%, and specificity = 80.0%. Fig. 4B shows a box-and-whisker plots showing urinary CRP signal intensity (ELISA results) stratified by serum CRP concentration categories (0-10, 10-50, 50-100, and >100 mg / L), as determined by a wide-range immunoturbidimetric assay (Beckman Coulter, Brea, CA, USA). The urine ELISA was performed using CRP-PRT-mAb03 as the capture antibody and a commercial polyclonal rabbit anti-CRP antibody for detection. A significant positive trend was observed, with higher urinary CRP concentrations corresponding to higher serum CRP.

[0336] Example 5: Detection of CRP in urine

[0337] The monoclonal antibodies described above are used in a lateral flow assay for detecting the target proteins or peptides thereof in urine. For that purpose, according to some embodiments, a first monoclonal antibody targeting CRP or a peptide thereof is immobilized on a substrate, such as a nitrocellulose membrane, of a lateral flow device. A second monoclonal antibody targeting CRP or a peptide thereof is conjugated to a suitable agent for detection, such as gold nanoparticles, for use in a colorimetric detection method, and added to the lateral flow device, or to a kit for using with the lateral flow device. At least the first or the second antibody is selected from the monoclonal antibodies described herein, while the other antibody may either also be selected from antibodies described herein, or obtained from a different source (such a commercial antibody capable of recognizing its target in urine conditions). When the lateral flow device is contacted with urine which contains the target protein above a certain threshold, a colored line will appear.

[0338] Table 6: Amino acid sequences of mAbs

[0339] Prot. Ab name Ab part Protein Sequence DNA SID SID

[0340] 001 CRP-PRT-mAbOl VH QVQLQQTGTELVRPGTSVKVSCKA Oil SGYGIINYLIEWVKQRPGQGLEWIG VINPGSGVTNNNEI< FI< GI< ATLTAD KSSNTAYMQLSSLTSEDSAVYFCA RSPLYDYDRDWYFDVWGTGTTVT VSS

[0341] 002 CRP-PRT-mAbOl VL DIKMTQSPSSMYAFLGERVTITCKA 012 SQDINSYLSWFQQKPGKSPKTLIYR ANRLVDGVPSRFSGSGSGQDYSLTI SSLEYEDMGIYYCLQYDEFPVTFG AGTKLELQ

[0342] 003 CRP-PRT-mAbOl VH CDR1 NYLIE 013 004 CRP-PRT-mAbOl VH CDR2 VINPGSGVTNNNEKFKG 014 005 CRP-PRT-mAbOl VH CDR3 RSPLY 015 006 CRP-PRT-mAbOl VL CDR1 KASQDINSYLS 016 007 CRP-PRT-mAbOl VL CDR2 RANRLVD 017 008 CRP-PRT-mAbOl VL CDR3 LQYDEFPVT 018 0

[0343]

[0344] 041 CRP-PRT-mAb03 VH EVQLQQSGPELVKPGSSVKMSCKA 051 SGYTFTDYNMHWVKQSHGKALE WIGYIHPNNGGTTYNQKFKGKATL TVNKSSSTAYMELRSLTSEDSAVY YCAMDDRWGQGTLVTVSA

[0345] 042 CRP-PRT-mAb03 VL DIVMTQSPSSLAVSVGQKVSMSCK 052 SSQTLLSSSNQKNYLAWYQQKPGQ SPKLLVYLASTRESGVPDRFIGSGS GTDFTLTISSVQAEDLADYFCQQHF SIPVTFGAGTKLELK

[0346] 043 CRP-PRT-mAb03 VH CDR1 DYNMH 053 044 CRP-PRT-mAb03 VH CDR2 YIHPNNGGTTYNQKFKG 054 045 CRP-PRT-mAb03 VH CDR3 DDR 055 "046 CRP - RT -niAbO3 VL^CDRl KSSQ I I. LSSSNQKNX l^A 056 047 ( RP-PI<r-inAb()3 Vl ( DR2 LASTRES 057 048 CRP-PRT-mAb03 VL CDR3 QQHFSIPVT 058 061 CRP-PRT-mAbO4 VH EVQLQQSGPDLVKPGASVKISCKA 071 SGYTFTDLYMNWVKESHGKSLEWI GEINPTNGGTTYNQKFKGKATLTV DKS S STAYMELRSLTSED S A VYYC ARGFYVLFDYWGQGTTLTVS S

[0347] 062 CRP-PRT-mAb04 VL DVLMTQTPLSLPVSLGDQASISCRS 072

[0348] SQSIVHSNGNTYLEWYLQKPGQSP KLLIYKVSNRFSGVPDRFSGSGSGT DFTLKISRVEAEDLGVYYCFQGSH VPPTFGGGTKLEIK

[0349] 063 CRP-PRT-mAbO4 VH CDR1 DLYMN 073 064 CRP-PRT-mAbO4 VH CDR2 EINPTNGGTTYNQKFKG 074 065 CRP-PRT-mAbO4 VH CDR3 GFYVLFDY 075 066 CRP-PRT-mAb04 VL CDR1 RSSQSIVHSNGNTYLE 076 067 CRP-PRT-mAbO4 VL CDR2 KVSNRFS 077 0

[0350]

[0351] 68 CRP-PRT-mAbO4 VL CDR3 FQGSHVPPT 078 101 CRP-PRT-mAbO6 VH QVQLQQTGTELVRPGTSVKVSCKA 111 SGYAFTNYLIEWVKQRPGQGLEWI GVINPGSGVTNNNEKFKGKATLTT DRSSNTAYMQLSSLTSEDSAVYFC ARSPLFDYDRDWYFDVWGTGTTV TVSS 102 CRP-PRT-mAb06 VL DIKMTQSPSSMYAFLGERVTITCKA 112

[0352] SQDINSYLSWFQQKPGKSPKTLIYR ANRLVDGVPSRVSGSGSGQDYSLTI SSLEYEDVGIYYCLQYDKFPVTFGA GTKLELK

[0353] 103 CRP-PRT-mAbO6 VH CDR1 NYLIE 113 104 CRP-PRT-mAb06 VH CDR2 VINPGSGVTNNNEKFKG 114 105 CRP-PRT-mAb06 VH CDR3 RSPLF 115 106 CRP-PRT-mAbO6 VL CDR1 KASQDINSYLS 116 107 CRP-PRT-mAbO6 VL CDR2 RANRLVD 117 108 CRP-PRT-mAbO6 VL CDR3 LQYDKFPVT 118

[0354]

[0355] 121 CRP-PRT-mAbO7 VH EVQLQQSGPELVKPGASVKISCKAS 131 GYTFTDYYLNWVKQSHGKSLEWI GDMNSKNGGPTYNQKFKGKATLII DKSSNTAYMELRSLTSEDSAVYYC ARYHSYGSYYEAYWGQGTLVTVS

[0356] A

[0357] 122 CRP-PRT-mAbO7 VL DIQMTQTTSSLSASLGDRVTITCRA 132 SQDISNYLNWYQQKPDGTVKLLIY YTSRLHSGVPSRFSGSGSGTDYSLT ISNLEQEDLATYFCQQGNTLPLTFG GGTKLELK

[0358] 123 CRP-PRT-mAb07 VH CDR1 DYYLN 133 124 CRP-PRT-mAbO7 VH CDR2 DMNSKNGGPTYNQKFKG 134 125 CRP-PRT-mAbO7 VH CDR3 YHSYGSYYEAY 135 126 CRP-PRT-mAbO7 VL CDR1 RASQDISNYLN 136 127 CRP-PRT-mAbO7 VL CDR2 YTSRLHS 137 128 CRP-PRT-mAbO7 VL CDR3 QQGNTLPLT 138 1

[0359]

[0360] 61 CRP-PRT-mAbO9 VH EVQLQQSGPVLVKPGASVKMSCK 171 ASGYTFTDYYTNWVKQ SHGKSLE WIGVINPYNGRTSYNQNFKGKATL TFDKSSSTAFMELNSLTSEESAVYY CAREGYNSIYGASFDYWGQGTTLT

[0361] vss 162 CRP-PRT-mAbO9 VL QIVLTQSPAIMSASPGEKVTISCSAS 172 SSVSNMYWYQQKPGSSPKPWIYRT SNLASGVPGRFSGSGSGTIYSLTISS MEAEDAATYYCQQYNSYPPTFGG GTKLEIK

[0362] 163 CRP-PRT-mAbO9 VH CDR1 DYYTN 173 164 CRP-PRT-mAbO9 VH CDR2 VINPYNGRTSYNQNFKG 174 165 CRP-PRT-mAbO9 VH CDR3 EGYNSIYGASFDY 175 166 CRP-PRT-mAbO9 VL CDR1 SASSSVSNMY 176 167 CRP-PRT-mAbO9 VL CDR2 RTSNLAS 177 168 CRP-PRT-mAbO9 VL CDR3 QQYNSYPPT 178

[0363]

[0364] 181 CRP-PRT-mAb10 VH QVQLQQTGTELVRPGTSVKVSCKA 191 SGYGIINYLIEWVKQRPGQGLEWIG VINPGSGVSNNNEKFKGKATLTAD KSSNTAYMQLSSLTSEDSAVYFCA RSPLYDYDRDWYFDVWGTGTTVT

[0365] vss

[0366] 182 CRP-PRT-mAblO VL DIKMTQSPSSMYAFLGERVTITCKA 192 SQDINNYLSWFQQKPGKSPKTLIYR ANRLVDGVPSRFSGSGSGQDYSLTI SSLDYEDMGIYYCLQYDKFPVTFG AGTKLELK

[0367] 183 CRP-PRT-mAblO VH CDR1 NYLIE 193 184 CRP-PRT-mAblO VH CDR2 VINPGSGVSNNNEKFKG 194 185 CRP-PRT-mAblO VH CDR3 SPLYDYDRDWYFDV 195 186 CRP-PRT-mAblO VL CDR1 KASQDINNYLS 196 187 CRP-PRT-mAb10 VL CDR2 RANRLVD 197 188 ~;Rp:pR |:nibi()V|( DR3I QYDIxM’V r 198 201 CRP-PRT-mAb 11 VH EVQLQQSGPELVKPGSSVKMSCMA 211 SGYTFTDYNMHWVKQSHGKALE WIGYIHPNNGGTTYNQKFKGKATL TVNKSSSTAYMELRSLTSEDSAVY YCAMDDRWGQGTLVTVSA

[0368] 202 CRP-PRT-mAb11 VL DIVMTQSPSSLAVSVGQKVSMSCK 212

[0369]

[0370] SSQSLLSSSNQKNYLAWYQQKPGQ SPKLLVYLASTRESGVPDRFIGSGS GTDFTLTISSVQAEDLADYFCQQHF SIPVTFGTGTKLELK

[0371] 203 CRP-PRT-mAb 11 VH CDR1 DYNMH 213 204 CRP-PRT-mAb 11 VH CDR2 YIHPNNGGTTYNQKFKG 214 205 CRP-PRT-mAb11 VH CDR3 WGQGTLVTVSA 215 206 CRP-PRT-mAbll VL CDR1 KSSQSLLSSSNQKNYLA 216 207 CRP-PRT-mAbll VL CDR2 LASTRES 217 208 CRP-PRT-mAbll VL CDR3 QQHFSIPVT 218 281 CRP-PRT-mAb 15 VH EAQLQQSGAELVRSGASVKLSCTA 291 SGFNIKDYYMHWVKQRPEQGLEW IGWIDPENGDTDYAPKFQGKATMT TDTS SNTAYLQLS SLTSEDTA VYYC NAHGNVVDWGQGTLVTVSA CRP-PRT-mAbl5 VL DVLMTQTPLSLPVSLGDQASISCRS 292 SQSLVHSNGNTYLQWYLQKPGQSP KLLIYKVSDRFSGVPDRFSGSGSGT DFTLKISRVEAEDLGVYYCFQGSH VPWTFGGGTKLEIK CRP-PRT-mAbl5 VH CDR1 DYYMH 293 CRP-PRT-mAbl5 VH CDR2 WIDPENGDTDYAPKFQG 294 CRP-PRT-mAbl5 VH CDR3 HGNVV 295 CRP-PRT-mAbl5 VL CDR1 RSSQSLVHSNGNTYLQWYLQ 296 CRP-PRT-mAbl5 VL CDR2 KVSDRFS 297 CRP-PRT-mAbl5 VL CDR3 FQGSHVPWT 298

[0372]

[0373] (;R|) |)|<rmAb | 6 V| |lA’QlA IAGGGi W SGFTYNTYAMNWVRQAPGKGLEW VARIRNKSKNYATDYADSVKDRFT LSRDDSQSMVYLQMNNLKTEDTAI YYCVKGLLLYWGQGTLVTVSA CRP-PRT-mAbl6 VL DVVLTQTPLYVPVNIGAQASISCKC 312 SKSLLNSDGFIYLEWYLQKPGQSPQ LQIYLVCNRFSGVPDRFSGSGSGTD FTLNISRVEEEDLGVYSWNQSNYLP YTFGGGTKLEIK CRP-PRT-mAbl6 VH CDR1 TYAMN 313 CRP-PRT-mAbl6 VH CDR2 RIRNKSKNYATDYADSVKD 314 CRP-PRT-mAbl6 VH CDR3 GLLLY 315 306 CRP-PRT-mAb16 VL CDR1 KCSKSLLNSDGFIYLE 316 307 CRP-PRT-mAb16 VL CDR2 LVCNRFS 317 CRP-PRT-mAbl6 VL CDR3 NQSNYLPYT 318 CRP-PRT-mAb20 VH EVQLQQSGPELVKPGSSVKMSCMA 391

[0374] SGYTFTDYNMHWVKQSHGKALE WIGYIHPNNGGTTYNQKFKGKATL TVNKSSSTAYMELRSLTSEDSAVY YCAMDDRWGQGTLVSVSA 382 CRP-PRT-mAb20 VL DIVMTQSPSSLAVSVGQKVSMSCK 392

[0375] SSQRLLSSSNQKNYLAWYQQKPGQ SPKLLVYLASTRESGVPDRFIGSGS GTDFTLTISSVQAEDLADYFCQQHF SIPVTFGAGTKLELK CRP-PRT-mAb20 VH CDR1 DYNMH 393 ( I< I’44<rHnAb2() Vir YIHPXXGG i rYV^ 394 CRP-PRT-mAb20 VH CDR3 DDR 395 CRP-PRT-mAb20 VL CDR1 KSSQRLLSSSNQKNYLA 396 CRP-PRT-mAb20 VL CDR2 LASTRES 397 CRP-PRT-mAb20 VL CDR3 QQHFSIPVT 398 CRP-POl-mAblO VH QVQLQQSGAELARPGASVKLSCKA 431

[0376] SGYTFPKYWMQWVKQRPRQGLE WIGAIYPEDDDSRYAQKFRDKATL TADKSSSTAYMQLTSLTSEDSAVY YCARFRYDSSPYTLDYWGQGTSVT

[0377] vss

[0378]

[0379] 422 CRP-POl-mAblO VL DVVMTQTPLSLPVSLGDQASISCRS 432

[0380] SQSLVYSNGNTYLHWYLQKPGQSP KLLIYKVSNRFSGVPDRFSGSGSGT DFIFKISRVEAEDLGVYFCSQSTHV PLTFGAGTKLELK

[0381] 423 CRP-POl-mAblO VH CDR1 KYWMQ 433 424 CRP-POl-mAblO VH CDR2 AIYPEDDDSRYAQKFRD 434 425 CRP-POl-mAblO VH CDR3 FRYDSSPYTLDY 435 426 CRP-POl-mAblO VL CDR1 RSSQSLVYSNGNTYLH 436 "427 (•RP-P()l-inAbl()Vl" (DR2 KVSNRFS 437 428 CRP-POl-mAblO VL CDR3 SQSTHVPLT 438 441 CRP-POl-mAbll VH QVQLQQSGAELVRPGTSVKISCKA 451

[0382] SGYTFTNYWLGCIRQRPGHGLEWI GNIHPGGGYTNYNE1< FI< GI< ATLTA DTSSSTAYMQLSSLTSDDSAVYFC ASGNSGNPFAYWGQGTLVTVSA

[0383] 442 CRP-POl-mAbll VL DIRMTQSPSSLSASLGERVSLTCRA 452

[0384] SQEISAFLTWLQQKPDGTIKRLIYA ASTLDYGVPKRFSGSRSGSGYSLTI SSLESEDFADYYCLQYASYPFTFGS RTKLEMR

[0385] 443 CRP-POl-mAbll VH CDR1 NYWLG 453 444 CRP-POl-mAbll VH CDR2 NIHPGGGYTNYNEKFKG 454 445 CRP-POl-mAbll VH CDR3 GNSGNPFAY 455 446 CRP-POl-mAbll VL CDR1 RASQEISAFLT 456 "447 CRP-POlmiAbll VL CDR2 AASTLDY 457 448 CRP-POl-mAbll VL CDR3 LQYASYPFT 458 461 CRP-P01-mAbl2 VH QVQLQQSGTELVRPGTSVKISCKAS 471

[0386] GYTFTHYWLGWIKQRPGHGLEWI GNISPGGIYTNYNENFKGKATLTAD TSSSFAYMQLSSLTSEDSAVYFCAN GDYGNPFAYWGQGTLVTVSA

[0387] 462 CRP-P01-mAbl2 VL DIQMTQSPSSLSASLGERVSLTCRT 472

[0388] SQEISGYLSWLQQKPDGTIKRLIYA ASTLDSSVPKRFSGSRSGSDYSLTIS SLESEDFADYYCLQYASYPFTFGSG TKLEIR

[0389] 463 CRP-P01-mAb12 VH CDR1 HYWLG 473 464 CRP-P01-mAbl2 VH CDR2 NISPGGIYTNYNENFKG 474 465 CRP-P01-mAbl2 VH CDR3 GDYGNPFAY 475 466 CRP-P01-mAbl2 VL CDR1 RTSQEISGYLS 476 467 CRP-P01-mAb12 VL CDR2 AASTLDS 477 468 CRP-P01-mAbl2 VL CDR3 LQYASYPFT 478 481 CRP-POl-mAbl3 VH EVKLDETGGGLVQPGGPMKLSCV 491

[0390] ASGFTFSDYWMNWVRQSPEKGLE WVAQIRNKFYNYETYYSDSVRGRF TISRDDSKSSVYLQMNNLGTEDMG IYSCTSYRYGFAYWGQGTLVTVSA 482 CRP-P01-mAbl3 VL QAVVTQESALTTSPGETVTLTCRSS 492

[0391] TGAVTASNYVSWVQEKPDHLFTSL IGSTNNRRPGVPARFSGSLIGDKAA LTITGAQTEDEAIYFCALRYSNYFV FGGGTKLTVL

[0392] 483 CRP-P01-mAbl3 VH CDR1 DYWMN 493 484 CRP-P01-mAbl3 VH CDR2 QIRNKFYNYETYYSDSVRG 494 485 CRP-P01-mAbl3 VH CDR3 SYRYGFAY 495 486 CRP-P01-mAbl3 VL CDR1 RSSTGAVTASNYVS 496 487 CRP-P01-mAb13 VL CDR2 STNNRRP 497 488 CRP-P01-mAbl3 VL CDR3 ALRYSNYFV 498 501 CRP-P01-mAbl4 VH DVQLQESGPGLVKPSQSLSLTCSVT 511

[0393] GYSITSGYYWNWIRQFPGNKLEW MGYIRYDGSNKYTPSLKNRISITRD TSKNQFFLKLNSVTTEDTGTYFCA GVFTTVEIMDWYSDVWGTGTTVT

[0394] vss

[0395] 502 CRP-P01-mAbl4 VL DIVMTQAAFSNPVILGTSASISCRSS 512

[0396] KSLLYSNGITYLYWYLQRPGQSPQ LLIYQMSNLASGVPDRFSSSGSGTD FTLRISRVEAEDVGVYFCAQNLEL WTFGGGTKLEIK

[0397] 503 CRP-P01-mAb14 VH CDR1 SGYYWN 513 504 CRP-P01-mAbl4 VH CDR2 YIRYDGSNKYTPSLKN 514 505 CRP-P01-mAbl4 VH CDR3 FTTVEIMDWYSDV 515 506 CRP-P01-mAbl4 VL CDR1 RSSKSLLYSNGITYLY 516 507 CRP-P01-mAbl4 VL CDR2 QMSNLAS 517 508 CRP-P01-mAb14 VL CDR3 AQNLELWT 518 521 CRP-P01-mAbl5 VH QVQLQQSGTELVRPGTSVKMSCKA 531

[0398] SGYTFTNQWIGWTKQRPGHDLEWI GNIYPGGDYTNYNEKFKGKATLTA DRSSSTAYMQFSSLTSEDSAIYYCA RGYYGDPFASWGQGTLVTVSA

[0399] 522 CRP-P01-mAbl5 VL DIQMTQSPSSLSASLGERVSLTCRA 532

[0400] SQDIGS SLNWLQQGPDGTIKRLIYA TSSVDSGVPKRFSGSRSGSDYSLTIS SLESEDFVDYYCLQYASFPFTFGSG TKLEKK

[0401] 523 CRP-P01-mAbl5 VH CDR1 NQWIG 533 "524 (•RP-P()i-mAbl5 VH CDR2 NIYPGGDYTb^^ 534 525 CRP-P01-mAbl5 VH CDR3 GYYGDPFAS 535 526 CRP-P01-mAbl5 VL CDR1 RASQDIGSSLN 536 527 CRP-P01-mAbl5 VL CDR2 ATSSVDS 537 "528 (■RI’-POI-inAbl^ VI 7( DR3 I QYASI'I’I'I 538 Ab name: Protein-Peptide / complete protein-antibody number; SID: SEQ ID No.

[0402]

Claims

CLAIMS1. An isolated polypeptide capable of specifically binding C-reactive protein (CRP) or a CRP- derived peptide under urinary conditions.

2. The isolated polypeptide of claim 1, wherein the CRP-derived peptide comprises a sequence selected from GYSIFSYATKRQDNEILIFWSK (SEQ ID NO: 541) and RQDNEILIFWSK (SEQ ID NO: 542)3. The isolated polypeptide of claim 1 or 2, comprising a heavy chain variable (VH) region comprising three complementarity determining region (CDR) sequences and a light chain variable (VL) region comprising three CDR sequences, and the six CDR sequences are defined by a standard method selected from Kabat, Chothia, IMGT, and AbM, based on VH and VL regions sequences selected from: SEQ ID Nos. 41 and 42; SEQ ID Nos. 01 and 02; SEQ ID Nos. 61 and 62; SEQ ID Nos. 101 and 102; SEQ ID Nos. 121 and 122; SEQ ID Nos. 161 and 162; SEQ ID Nos. 181 and 182; SEQ ID Nos. 201 and 202; SEQ ID Nos. 281 and 282; SEQ ID Nos. 301 and 302; SEQ ID Nos. 381 and 382; SEQ ID Nos. 421 and 422; SEQ ID Nos. 441 and 442; SEQ ID Nos. 461 and 462; SEQ ID Nos. 481 and 482; SEQ ID Nos. 501 and 502; and SEQ ID Nos. 521 and 522.

4. The isolated polypeptide of claim 3, wherein the six CDR sequences comprise sequences substantially identical to six CDR sequences selected from: SEQ ID Nos. 43, 44, 45, 46, 47, and 48; SEQ ID Nos. 03, 04, 05, 06, 07, and 08; SEQ ID Nos. 63, 64, 65, 66, 67, and 68; SEQ ID Nos. 103, 104, 105, 106, 107, and 108; SEQ ID Nos. 123, 124, 125, 126, 127, and 128; SEQ ID Nos. 163, 164, 165, 166, 167, and 168; SEQ ID Nos. 183, 184, 185, 186, 187, and 188; SEQ ID Nos. 203, 204, 205, 206, 207, and 208; SEQ ID Nos. 283, 284, 285, 286, 287, and 288; SEQ ID Nos. 303, 304, 305, 306, 307, and 308; SEQ ID Nos. 383, 384, 385, 386, 387, and 388; SEQ ID Nos. 423, 424, 425, 426, 427, and 428; SEQ ID Nos. 443, 444, 445, 446, 447, and 448; SEQ ID Nos. 463, 464, 465, 466, 467, and 468; SEQ ID Nos. 483, 484, 485, 486, 487, and 488; SEQ ID Nos. 503, 504, 505, 506, 507, and 508; and SEQ ID Nos. 523, 524, 525, 526, 527, and 528.

5. The isolated polypeptide of claim 3 or 4, wherein the VH and VL region sequences comprise sequences substantially identical to VH and VL sequences selected from: SEQ ID Nos. 41 and 42; SEQ ID Nos. 01 and 02; SEQ ID Nos. 61 and 62; SEQ ID Nos. 101 and 102; SEQ ID Nos.121 and 122; SEQ ID Nos. 161 and 162; SEQ ID Nos. 181 and 182; SEQ ID Nos. 201 and202; SEQ ID Nos. 281 and 282; SEQ ID Nos. 301 and 302; SEQ ID Nos. 381 and 382; SEQ ID Nos. 421 and 422; SEQ ID Nos. 441 and 442; SEQ ID Nos. 461 and 462; SEQ ID Nos. 481 and 482; SEQ ID Nos. 501 and 502; and SEQ ID Nos. 521 and 522.

6. The isolated polypeptide of any one of claims 3-5, wherein the six CDR sequences comprise sequences substantially identical to six CDR sequences encoded by nucleic acid sequences selected from: SEQ ID Nos. 53, 54, 55, 56, 57, and 58; SEQ ID Nos. 13, 14, 15, 16, 17, and 18; SEQ ID Nos. 73, 74, 75, 76, 77, and 78; SEQ ID Nos. 113, 114, 115, 116, 117, and 118; SEQ ID Nos. 133, 134, 135, 136, 137, and 138; SEQ ID Nos. 173, 174, 175, 176, 177, and 178; SEQ ID Nos. 193, 194, 195, 196, 197, and 198; SEQ ID Nos. 213, 214, 215, 216, 217, and 218; SEQ ID Nos. 293, 294, 295, 296, 297, and 298; SEQ ID Nos. 313, 314, 315, 316, 317, and 318; SEQ ID Nos. 393, 394, 395, 396, 397, and 398; SEQ ID Nos. 433, 434, 435, 436, 437, and 438; SEQ ID Nos. 453, 454, 455, 456, 457, and 458; SEQ ID Nos. 473, 474, 475, 476, 477, and 478; SEQ ID Nos. 493, 494, 495, 496, 497, and 498; SEQ ID Nos. 513, 514, 515, 516, 517, and 518; and SEQ ID Nos. 533, 534, 535, 536, 537, and 538.

7. The isolated polypeptide of any one of claims 3-6, wherein the VH and VL region sequences comprise sequences substantially identical to VH and VL sequences encoded by nucleic acid sequences selected from: SEQ ID Nos. 51 and 52; SEQ ID Nos. 11 and 12; SEQ ID Nos. 71 and 72; SEQ ID Nos. 111 and 112; SEQ ID Nos. 131 and 132; SEQ ID Nos. 171 and 172; SEQ ID Nos. 191 and 192; SEQ ID Nos. 211 and 212; SEQ ID Nos. 291 and 292; SEQ ID Nos. 311 and 312; SEQ ID Nos. 391 and 392; SEQ ID Nos. 431 and 432; SEQ ID Nos. 451 and 452; SEQ ID Nos. 471 and 472; SEQ ID Nos. 491 and 492; SEQ ID Nos. 511 and 512; and SEQ ID Nos. 531 and 532.

8. The isolated polypeptide of any one of claims 1-7, wherein the CRP is human CRP.

9. The isolated polypeptide of any one of claims 1-8, wherein the isolated polypeptide is selected from 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.

10. The isolated polypeptide of claim 9, wherein the isolated polypeptide is a monoclonal antibody.

11. The isolated polypeptide of any one of claims 1-10, wherein the isolated polypeptide is conjugated to a functional moiety.

12. The isolated polypeptide of any one of claims 1-11, wherein the specific binding is characterized by an EC50 of less than about 10, 1, or 0.1 nM.

13. An isolated nucleic acid molecule comprising at least one sequence encoding the isolated polypeptide of any one of claims 1-12.

14. A host cell comprising the isolated polypeptide of any one of claims 1-12, or the nucleic acid molecule of claim 13.

15. The host cell of claim 14, wherein the host cell is a hybridoma cell.

16. A combination comprising the isolated polypeptide of any one of claims 1-12, and an additional polypeptide capable of specifically binding CRP or a CRP-derived peptide, wherein the additional polypeptide does not interfere with binding of the isolated polypeptide to the CRP or CRP-derived peptide.

17. The combination of claim 16, wherein the additional polypeptide is an isolated polypeptide of any one of claims 1-12, different from the isolated polypeptide of claim 16.

18. The combination of claim 17, wherein the isolated polypeptide has six CDR sequences substantially identical to six CDR sequences set forth in SEQ ID Nos. 43-48, and the additional polypeptide has six CDR sequences substantially identical to six CDR sequences selected from: SEQ ID Nos. 103-108 and SEQ ID Nos. 163-168.

19. The combination of claim 17, wherein the isolated polypeptide has VH and VL sequences substantially identical to VH and VL sequences set forth in SEQ ID Nos. 41-42, and the additional polypeptide has VH and VL sequences substantially identical to VH and VL sequences selected from: 101-102 and SEQ ID Nos. 161-162.

20. The combination of claim 17, wherein the isolated polypeptide has six CDR sequences substantially identical to six CDR sequences set forth in SEQ ID Nos. 43-48, and the additional polypeptide has six CDR sequences substantially identical to six CDR sequences set forth in SEQ ID Nos. 3-8.

21. The combination of claim 17, wherein the isolated polypeptide has VH and VL sequences substantially identical to VH and VL sequences set forth in SEQ ID Nos. 41-42, and the additional polypeptide has VH and VL sequences substantially identical to VH and VL sequences set forth in SEQ ID Nos. 1-2.

22. A method for predicting in a urine sample of a subject, whether the subject has elevated blood CRP levels, the method comprising:a. contacting a urine sample of the subject with one or more isolated polypeptides of any one of claims 1-12;b. detecting binding of the one or more isolated polypeptides to CRP or a CRP-derived peptide; andc. predicting whether the subject has elevated blood CRP levels based on the detecting in step (b), thereby indicating that the subject suffers from an inflammation, wherein binding of the one or more isolated polypeptides to the CRP or CRP-derived peptide predicts that the subject has elevated blood CRP levels, thereby indicating that the subject suffers from an inflammation.

23. The method of claim 22, wherein the inflammation is systemic inflammation.

24. The method of claim 22 or 23, wherein the elevated blood CRP levels are at least 100 or 120 mg / L.

25. The method of any one of claims 22-24, wherein the subject is a human.

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 test, a fluorescence activated cell sorting (FACS), enzyme- linked immunosorbent assay (ELISA), a dipstick, an antibody chip, and magnetic beads.

27. The method of any one of claims 22-26, further comprising a step of treating the subject with an anti-inflammatory agent when the subject is predicted to be suffering from inflammation.

28. A kit for predicting in a urine sample of a subject whether the subject has elevated blood CRP levels, the kit comprising:a. one or more one or more isolated polypeptides of any one of claims 1-12;b. at least one reagent for detecting the binding of the one or more isolated polypeptides to CRP or CRP-derived peptide in a urine sample; andc. instructions for use.

29. The kit of claim 28, 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.

30. The kit of claim 28 or 29, wherein the kit further comprises a urine collection device selected from a urine cup, a urine bag, a urine diaper, and a urine catheter.

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