Antibodies to kynurenine

Abstract

The present invention relates to a monoclonal antibody or an antigen-binding fragment thereof, or a synthetic product and a biotechnological derivative thereof, which specifically binds to kynurenine and acts as a detection marker for inflammation in an individual. Furthermore, the present invention provides a reliable method and a test kit for detecting inflammation by measuring kynurenine levels in a body fluid, preferably saliva. By using the monoclonal antibody of the present invention, the inventors provide a tool for measuring the level of KYN easily and quickly without the need for trained personnel when saliva is used as a sample. This is also beneficial in pediatric care and monitoring various diseases.

Description

[Technical field]

[0001] The present invention relates to antibodies against kynurenines and methods of use thereof. [Background technology]

[0002] Kynurenine (KYN) is an aromatic non-proteinogenic amino acid and a metabolic product of tryptophan (TRP). TRP is an essential amino acid required not only for protein synthesis but also for the biosynthesis of neurotransmitters such as serotonin and melatonin. Most of the TRP ingested from the diet is catabolized to kynurenine via the kynurenine pathway (KP) by the enzymes tryptophan-2,3-dioxygenase (TDO) produced in the liver and indoleamine-2,3-dioxygenase (IDO-1), produced in many tissues in response to immune activation. Kynurenine is involved in several physiological processes, including neurotransmission and immune responses. KP contains neurotoxic and neuroprotective metabolites, the delicate balance of which has been demonstrated in several pathological processes.

[0003] During inflammation, which is the first line of defense of the innate immune system against dangerous proteins, the enzymes IDO-1 and TDO are activated by inflammatory cytokines such as IFN-α, IFN-β, IFN-γ, TNFα, and IL-6, and therefore KYN levels are increased by activation of the innate immune system.

[0004] Alterations in the kynurenine pathway are associated with many diseases, including viral and bacterial infections, cancer, and diabetes. Imbalances in the kynurenine pathway have also been shown to be associated with a variety of diseases that result in cognitive decline. Furthermore, alterations in kynurenine metabolites have been shown to correlate with infarct volume, mortality in stroke patients, and cognitive impairment after stroke.

[0005] Because KYN has been implicated in a variety of disease states, there is a need in the art to be able to quickly and easily measure levels of KYN, which preferably can be performed by the patient themselves, without the need for a physician or other trained personnel.

[0006] The currently preferred method for measuring KYN levels is HPLC (High Performance Liquid Chromatography), which uses adsorption and flow rate to separate, identify and quantify different components in a mixture. Another method used in the prior art is to measure the level of IDO-1 and extrapolate it to the level of KYN. Antibodies that bind to different components of the kynurenine pathway have also been described for use in enzyme-linked immunosorbent assays (ELISA), immunofluorescence, immunochemistry and western blotting of serum, plasma and / or dried blood samples.

[0007] The methods described in the prior art are not suitable for determining the amount of kynurenine in a sample quickly, easily and advantageously without the involvement of a physician or other trained person.Therefore, there is a need for antibodies against KYN to detect said amino acid not only in blood samples but also in other body samples, such as saliva, which can preferably be taken non-invasively by the patient himself.This is particularly important in pediatric care and in the monitoring of various diseases.

[0008] It is therefore an object of the present invention to provide an antibody that specifically binds to kynurenine. The antibody provided by the present invention is preferably capable of detecting KYN in body fluids, preferably saliva, where the levels of KYN are low, and is preferably used in an assay that can be performed by an individual himself. Summary of the Invention

[0009] The subject matter of the invention is defined in the independent claims. Preferred embodiments of the invention are defined in the respective dependent claims.

[0010] In accordance with the present invention, there are provided monoclonal antibodies, or antigen-binding fragments thereof, or synthetic and biotechnological derivatives thereof, that specifically bind kynurenine and act as markers for detecting inflammation in an individual.

[0011] According to a preferred embodiment, there is provided a monoclonal antibody, or an antigen-binding fragment thereof, or synthetic and biotechnological derivatives thereof, that binds to kynurenine in a body fluid. According to another preferred embodiment, the body fluid is serum, saliva and / or cerebrospinal fluid (CSF). In a particularly preferred embodiment, the body fluid is saliva.

[0012] According to another preferred embodiment, the monoclonal antibody, or antigen-binding fragment thereof, or synthetic and biotechnological derivatives thereof, is defined by one or more complementarity determining regions (CDRs) and / or by a variable heavy chain (VH) and optionally a variable light chain (VL) region consisting of said CDRs or related sequences.

[0013] Furthermore, according to a preferred embodiment, the monoclonal antibody, or antigen-binding fragment thereof, or synthetic and biotechnological derivatives thereof, comprises a variable region of the heavy chain (VH) having the sequence set forth in SEQ ID NO:1.

[0014] According to another preferred embodiment, the monoclonal antibody, or antigen-binding fragment thereof, or synthetic and biotechnological derivatives thereof, comprises a variable region of the light chain (VL) having the sequence set forth in SEQ ID NO:2.

[0015] According to yet another preferred embodiment, the monoclonal antibody, or antigen-binding fragment thereof, or synthetic and biotechnological derivatives thereof, comprises one or more complementarity determining regions, CDRs, and / or a variable heavy chain (VH) region consisting of an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:1, and a variable light chain (VL) region consisting of an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:2.

[0016] According to yet another preferred embodiment, the monoclonal antibody, or antigen-binding fragment thereof, or synthetic and biotechnological derivatives thereof, is characterized by a VH region and optionally a VL region comprising three CDRs, designated CDRH1, CDRH2, CDRH3 and CDRL1, CDRL2, CDRL3, respectively, which define the binding specificity of the antibody, or antigen-binding fragment thereof, or synthetic and biotechnological derivatives thereof.

[0017] According to the present invention, a VH region or its CDRs alone may constitute a complete antigen-binding site. In a particular embodiment, an antibody comprises a VH region or its CDRs alone as defined herein. In a particular embodiment, an antibody comprises a VH region or its CDRs, in particular as defined herein, together with a VL region or its CDRs.

[0018] In yet another preferred embodiment, the present invention relates to a monoclonal antibody, or an antigen-binding fragment thereof, or synthetic and biotechnological derivatives thereof, wherein at least one CDR is selected from the heavy chain CDRs defined by SEQ ID NO:3; SEQ ID NO:4; and SEQ ID NO:5; and the light chain CDRs defined by SEQ ID NO:6; SEQ ID NO:7; and SEQ ID NO:8.

[0019] Furthermore, according to a preferred embodiment, the monoclonal antibody, or antigen-binding fragment thereof, or synthetic and biotechnological derivatives thereof, comprises A heavy chain CDR1 (CDRH1) domain having the amino acid sequence set forth in SEQ ID NO:3; A heavy chain CDR2 (CDRH2) domain having the amino acid sequence set forth in SEQ ID NO:4; A heavy chain CDR3 (CDRH3) domain having the amino acid sequence set forth in SEQ ID NO:5; and A light chain CDR1 (CDRL1) domain having the amino acid sequence set forth in SEQ ID NO:6; A light chain CDR2 (CDRL2) domain having the amino acid sequence set forth in SEQ ID NO: 7; A light chain CDR3 (CDRL3) domain having the amino acid sequence set forth in SEQ ID NO:8; Includes.

[0020] According to yet another preferred embodiment, the monoclonal antibody, or antigen-binding fragment thereof, or synthetic and biotechnological derivatives thereof, comprises a VH region consisting of CDRH1 of SEQ ID NO:3, CDRH2 of SEQ ID NO:4, and CDRH3 of SEQ ID NO:5, and optionally a VL region consisting of CDRL1 of SEQ ID NO:6, CDRL2 of SEQ ID NO:7, and CDRL3 of SEQ ID NO:8.

[0021] In another preferred embodiment, up to 1, 2 or 3 amino acid residues are substituted in CDRH1. In another preferred embodiment, up to 1, 2 or 3 amino acid residues are substituted in CDRH2. In yet another preferred embodiment, up to 1, 2 or 3 amino acid residues are substituted in CDRH3. In yet another preferred embodiment, up to 1, 2 or 3 amino acid residues are substituted in CDRL1. In yet another preferred embodiment, up to 1, 2 or 3 amino acid residues are substituted in CDRL2. In yet another preferred embodiment, up to 1, 2 or 3 amino acid residues are substituted in CDRL3.

[0022] In a further preferred embodiment, up to 1, 2 or 3 amino acid residues are deleted in CDRH1. In another preferred embodiment, up to 1, 2 or 3 amino acid residues are deleted in CDRH2. In yet another preferred embodiment, up to 1, 2 or 3 amino acid residues are deleted in CDRH3. In another preferred embodiment, up to 1, 2 or 3 amino acid residues are deleted in CDRL1. In yet another preferred embodiment, up to 1, 2 or 3 amino acid residues are deleted in CDRL2. In yet another preferred embodiment, up to 1, 2 or 3 amino acid residues are deleted in CDRL3.

[0023] According to another preferred embodiment, amino acids can be exchanged by conservative amino acid substitutions in other parts of the antibody, its antigen-binding fragment, or its synthetic and biotechnological derivatives, without altering the specificity of the antibody for KYN.

[0024] According to a preferred embodiment, the present invention provides a monoclonal antibody, or an antigen-binding fragment thereof, or synthetic and biotechnological derivatives thereof. In a preferred embodiment, the isolated antigen-binding protein is a Fab fragment, a Fab' fragment, a F(ab')2 fragment, an Fv fragment, a diabody, a nanobody or a single chain antibody molecule. In another preferred embodiment, the isolated antigen-binding protein is of the IgG1-, IgG2-, IgG3- or IgG4 type. In yet another preferred embodiment, the antibody may be of the IgA-, IgD-, IgE- or IgM type.

[0025] According to another preferred embodiment, the present invention provides a monoclonal antibody of the IgG1 type. In yet another preferred embodiment, the present invention provides an scFv fragment.

[0026] According to yet another preferred embodiment, the monoclonal antibody, or antigen-binding fragment thereof, or synthetic and biotechnological derivatives thereof, is administered at a concentration of 1×10 -5 M to 1×10 -8 The affinity between M (K D In another preferred embodiment, the monoclonal antibody, or antigen-binding fragment thereof, or synthetic and biotechnological derivatives thereof, can bind to kynurenine at a concentration of 1×10 -5 Less than M, preferably 1×10 -6 Less than M, more preferably less than 1×10 -7 Affinity (K D In yet another preferred embodiment, the antibody of the invention can bind to kynurenine at a concentration of 1×10 -5 M or less, preferably 1×10 -6 M or less, preferably 1×10 -7 K below M D Combine with.

[0027] According to yet another preferred embodiment, the monoclonal antibodies, or antigen-binding fragments thereof, or synthetic and biotechnological derivatives thereof, are human.

[0028] In accordance with the present invention, there are provided isolated nucleic acid molecules encoding the monoclonal antibodies of the invention, or antigen-binding fragments thereof, or synthetic and biotechnological derivatives thereof.

[0029] According to the present invention, there is provided an expression vector comprising the nucleic acid molecule.

[0030] According to the present invention there is provided a method for producing a monoclonal antibody, or an antigen-binding fragment thereof, or synthetic and biotechnological derivatives thereof, said method comprising the steps of: - introducing said expression vector into an isolated host cell, - growing the cells under conditions that allow the production of the antibody, or an antigen-binding fragment thereof, or synthetic and biotechnological derivatives thereof; - recovering the antibodies, fragments or derivatives thus produced.

[0031] According to the present invention there is provided an in vitro method for detecting a disease or condition in an individual, wherein the level of kynurenine in a body fluid is determined using a monoclonal antibody of the invention, or an antigen-binding fragment thereof, or synthetic and biotechnological derivatives thereof, and the value of kynurenine measured in the individual to be diagnosed is compared with a mean value obtained from a comparable cohort of healthy individuals, whereby the value of kynurenine in the patient is increased.

[0032] According to another preferred embodiment, the measurement of kynurenine in body fluids is used in the management of a patient's treatment.

[0033] According to yet another preferred embodiment, the measurement of KYN in body fluids is used to monitor the health status of an individual. According to yet another preferred embodiment, the present invention relates to diagnostic uses of antibodies or antigen-binding fragments thereof, or synthetic and biotechnological derivatives thereof. Applications include, but are not limited to, detection of inflammation, detection of various diseases and / or biological markers in samples, preferably saliva.

[0034] According to yet another preferred embodiment, the body fluid is serum, saliva or cerebrospinal fluid (CSF). In a particularly preferred embodiment, the body fluid is saliva.

[0035] Moreover, according to a preferred embodiment, the level of kynurenine in the patient is at least 2 times higher than that of a healthy control group. More preferably, the level of kynurenine in the patient is at least 3 μM in serum, at least 1 μM in saliva, and at least 1 μM in CSF. More preferably, the level of KYN in the patient is 4 μM to 25 μM in serum, more preferably 5 μM to 17 μM, more preferably 5.5 μM to 12 μM, and more preferably 6 μM to 9 μM. More preferably, the level of KYN in the patient is 1.5 μM to 4 μM in saliva. More preferably, the level of KYN in the patient is 1.5 μM to 4 μM in CSF.

[0036] In yet another preferred embodiment, the disease or condition detected in the present invention is selected from at least one of neurodegenerative diseases, transplantation, infectious diseases, dialysis, dental diseases, sports medicine, gynecology, gastroenterology, cancer, diabetes, multiple sclerosis, asthma, and concussion.

[0037] More preferably, the neurodegenerative disease includes, but is not limited to, Alzheimer's disease, Parkinson's disease, vascular dementia, Huntington's disease, and post-operative cognitive impairment.

[0038] More preferably, the transplant includes, but is not limited to, solid organ transplant, stem cell transplant, corneal transplant, bone transplant, graft versus host disease.

[0039] More preferably, the diseases include, but are not limited to, infection with severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), sepsis, tuberculosis, and infection with cytomegalovirus (CMV).

[0040] More preferably, sports medicine includes, but is not limited to, overtraining syndrome.

[0041] More preferably, gynecology includes, but is not limited to, eclampsia.

[0042] More preferably, the gastroenterology includes, but is not limited to, Morbus clonal.

[0043] More preferably, the dental disease includes, but is not limited to, periodontitis.

[0044] Additionally, the methods of the present invention are useful for monitoring the physical fitness of an individual.

[0045] According to another preferred embodiment, the in vitro method is an ELISA test, or a lateral flow immunochromatographic method (LFA), or a microfluidic test.

[0046] Thus, the in vitro method disclosed herein can be based on different principles, which are explained in more detail in the figure legends. One preferred principle is known as Lateral Flow Immunochromatographic Assay (LFA). Such an LFA test can be easily performed by the patient without the help of a doctor or other specialist, when saliva is used as sample.

[0047] In another preferred embodiment, a single component of the lateral flow assay is adapted to indicate the presence of kynurenine only if kynurenine above a certain threshold value is present in the sample.

[0048] In another preferred embodiment, the in vitro method of the invention is carried out as an ELISA. Different configurations of ELISA tests are known. ELISA types are direct ELISA, sandwich ELISA, competitive ELISA and / or reverse ELISA.

[0049] In a further preferred embodiment, the in vitro test method is a microfluidic test. The test can be used to perform ELISA or LFA tests on paper. The test consists of two parts: a sliding strip containing an active sensing area and a structure surrounding the sliding strip that holds stored reagents such as buffers, antibodies, enzyme substrates, and distributes fluids. To perform the test, samples of body fluids and water are added, the sliding strip is moved at a scheduled time, and the resulting color of the sensing area is analyzed visually or by a flatbed scanner.

[0050] According to the present invention, a test kit is provided for measuring kynurenine levels in body fluids using the antibodies of the present invention. Such a test kit comprises suitable means for carrying out the methods of the present invention, which may work on different principles. The kit comprises at least one antibody according to the present invention that specifically binds to kynurenine.

[0051] In another preferred embodiment, the kit may also include a second antibody. When using two antibodies, such antibodies do not bind to the same epitope, in order to allow the formation of a sandwich by a first antibody and a second antibody that specifically binds to kynurenine or its derivatives. Preferably, the second antibody is an enzyme-linked secondary antibody that is applied as a detection antibody.

[0052] According to a preferred embodiment, the test kit is an ELISA test kit, an LFA test kit, or a microfluidic test kit.

[0053] According to yet another preferred embodiment, the body fluid used in the test kit is serum, saliva, or CSF. In a particularly preferred embodiment, the body fluid is saliva.

[0054] A preferred test kit would consist of the following components: 1. Sample Pad – an absorbent pad onto which the test sample is applied; 2. Conjugate or reagent pad - antibody specific for the target analyte bound to colored particles (usually colloidal gold particles or latex microspheres); 3. Reaction membrane - usually a hydrophobic nitrocellulose or cellulose acetate membrane on which anti-target analyte antibodies are immobilized in a line across the membrane as a capture zone or test line (there may also be a control zone containing an antibody specific for the conjugated antibody); 2. Wick or waste reservoir - a further absorbent pad designed to draw and collect the sample across the reaction membrane by capillary action.

[0055] The strip components are typically fixed to an inert backing and can display capture and control zones in a simple dipstick format, or in a plastic casing with a sample port and reaction window.

[0056] The term "antibody" as used herein is intended to refer to any antigen-binding molecule, for example, in some embodiments, an immunoglobulin molecule composed of four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (composed of domains CH1, CH2, and CH3). Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL).

[0057] The term "variable region" or "variable domain" as used herein refers to a domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable regions of the heavy and light chains (VH and VL, respectively) of native antibodies generally have a similar structure. They are further divided into regions of hypervariability called complementarity determining regions (CDRs) and highly conserved regions called framework regions (FRs). The framework regions serve to position and align the CDRs in three-dimensional space. The CDRs contain residues that contact the antigen and are primarily involved in binding to the antigen. Generally, each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Unless otherwise indicated, CDR residues and other residues in the antibodies of the invention are numbered according to Kabat,l et al. (Sequences of Proteins of Immunoogical Interest, 5th Ed, Public Health Service, National Institutes of Health, Bethesda, MD, 1991). The CDR amino acid sequences of the antibodies of the invention are set forth in SEQ ID NOs:3 to 8, and the nucleic acid sequences of the CDRs of the antibodies of the invention are set forth in SEQ ID NOs:11 to 16. The amino acid sequence of the VH region of the antibodies of the invention is set forth in SEQ ID NO:1, the nucleic acid sequence of the VH region is set forth in SEQ ID NO:9, the amino acid sequence of the VL region of the antibodies of the invention is set forth in SEQ ID NO:2, and the nucleic acid sequence of the VL region is set forth in SEQ ID NO:10. Silent mutations in the nucleic acid sequences, i.e., exchanges of bases that do not change the amino acid sequence, are also encompassed by the invention and do not affect the properties of the antibodies of the invention. Substitution of up to 1, 2, or 3 amino acid residues per CDR, substitution of up to 1, 2, or 3 amino acid residues in all three CDRs of one light or heavy chain, or deletion of one or more CDRs are also possible. It has been described in the scientific literature that antibodies can be missing one or two CDRs for binding. When a CDR or its amino acid residue is deleted, it is usually replaced with an amino acid that occupies the corresponding position in another human antibody sequence or a consensus of such sequences.The substitution positions within the CDRs and the amino acids to be substituted can also be selected empirically. Empirical substitutions include conservative and non-conservative substitutions.

[0058] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies. In general, there are two types of antibodies: polyclonal and monoclonal. Polyclonal antibodies are a mixture of antibodies obtained from the serum of immunized animals (usually goats, rabbits, mice, and rats). By adsorbing to the antigen by affinity chromatography, antibodies against different epitopes of the antigen can be purified together, even though the antibodies are produced by different B cells. Polyclonal antibodies, due to the method of their production, show a wide range of specificities, and as a result, their effects are difficult to predict.

[0059] In contrast, monoclonal antibodies are antibodies that are generated by cloning a unique B cell. All subsequent antibodies thus generated can be traced back to a unique parent cell. Monoclonal antibodies may have monospecific activity, binding only to the same epitope (the part of the antigen that the antibody recognizes). However, bispecific monoclonal antibodies can also be generated by expanding the target of one monoclonal antibody to two epitopes. Monoclonal antibodies exhibit advantageous binding profiles compared to polyclonal antibodies. Furthermore, the individual monoclonal antibodies that make up the population are identical and / or bind to the same epitope, with the exception of variant antibodies that may, for example, include naturally occurring mutations or arise during the manufacture of the monoclonal antibody preparation, which are generally present in trace amounts. Polyclonal antibody preparations usually contain different antibodies against different epitopes, whereas each monoclonal antibody in a monoclonal antibody preparation is directed against a single epitope on the antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.

[0060] The term "human antibody," as used herein, comprises antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human monoclonal antibodies of the invention can include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), e.g., in the CDRs.

[0061] As used herein, the term "antibody fragment" refers to a molecule other than a full-length antibody, which constitutes a part of a full-length antibody and binds to the same antigen as the full-length antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv), and fragments containing CDRs or isolated CDRs. The antibody fragment of the present invention refers to one or more fragments of an antibody that retains the ability to specifically bind to kynurenine.

[0062] As used herein, the term "antigen-binding site" refers to the site of an antibody that recognizes and binds to an antigen. The antigen-binding site is formed by multiple amino acid residues from the heavy and light chain variable domains of an antibody and is arranged in close spatial proximity in the tertiary structure of the Fv region.

[0063] The term "scFv" as used herein refers to an antibody fragment format consisting of the variable regions of the heavy (VH) and light (VL) chains of an antibody, or two copies of the VH or VL chains of an antibody, linked by a short, flexible peptide linker that enables the scFv to form the desired structure for antigen binding. scFvs are fusion proteins of the variable regions of the heavy (VH) and light (VL) chains of immunoglobulins, and can be readily expressed in a functional form in E. coli and other host cells.

[0064] In the present specification, the term "synthetic and biotechnological derivatives" refers to artificial fragments, synthesized by chemical or recombinant techniques, that have the desired antigen binding specificity of the antibody of the invention and that retain the functional properties of the antibody. For example, these derivatives can have modifications in regions that are important / not important for antigen binding. The antibody derivatives can be obtained from the antibody according to the invention, for example, by exchange of one or more constant regions and / or linkages with other molecules. Thus, an exchange of constant regions for isotype can be performed, for example, converting an antibody of class IgM into an antibody of class IgG while maintaining its antigen specificity. This isotype switching can be performed by cell biological or molecular biological methods well known in the art. Another possibility for providing derivatives of the antibody according to the invention is the exchange of amino acids in other parts of the antibody by conservative amino acid substitutions.

[0065] A "conservative amino acid substitution" is one in which an amino acid residue is replaced with another amino acid residue having a side chain (R group) with similar chemical properties (e.g., similar charge or hydrophobicity). In general, conservative amino acid substitutions do not substantially change the functional properties of a protein. When two or more amino acid sequences differ from each other by conservative substitutions, the percentage of similarity can be adjusted upwards to correct for the conservative nature of the substitutions. Means for making this adjustment are well known to those of skill in the art.

[0066] Examples of amino acids that have similar chemical side chains and can be substituted for one another to form conservative substitutions include the following: 1) Aliphatic side chains: glycine, alanine, valine, leucine, isoleucine 2) Aliphatic hydroxyl side chains: serine and threonine 3) Amide-containing side chains: Asparagine and Histidine 4) Aromatic side chains: phenylalanine, tyrosine, tryptophan 5) Basic side chains: lysine, arginine, histidine 6) Acidic side chains: aspartic acid and glutamic acid, and 7) Sulfur-containing side chains: cysteine ​​and methionine.

[0067] Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic-aspartic, and asparagine-glutamine.

[0068] Polypeptide sequence similarity is usually measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, GCG software includes programs such as GAP and BESTFIT, which can be used with default parameters to determine sequence homology and sequence identity between closely related polypeptides, such as homologous polypeptides from different species and wild-type and mutant proteins. Polypeptide sequences can also be compared using FASTA with default or recommended parameters. The program GCG version 6.1 FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of the best overlapping regions between the query and search sequences. Another preferred algorithm for comparing the sequences of the present invention to a database containing a large number of sequences from different organisms is the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters.

[0069] The term "sequence identity" when referring to a nucleic acid or antigen-binding fragment thereof indicates that when optimally aligned with another nucleic acid (or its complementary strand), with appropriate nucleotide insertions or deletions, there is at least about 90% identity of the nucleotide sequence, more preferably at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% of the nucleotide bases, as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST or GAP as described above.

[0070] The term "sequence identity" as applied to polypeptides refers to two peptide sequences that share at least 90%, more preferably at least 95%, at least 98% or at least 99% sequence identity when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights. Preferably, residue positions that are not identical differ by conservative amino acid substitutions.

[0071] As used herein, the term "affinity" means that an antibody, or an antigen-binding fragment thereof, or synthetic and biotechnological derivatives thereof, forms a complex with an antigen that is relatively stable under physiological conditions. Affinity is determined by the equilibrium dissociation constant (K D ) is 1×10 -5 Less than M, preferably 1×10 -6 M or less, more preferably 1×10 -7 M (K D (The smaller the value, the stronger the binding.) Antibody affinity is a measure of the strength of the bond between an antibody and an antigen, with low affinity antibodies binding weakly and high affinity antibodies binding strongly.

[0072] Terms such as "specifically bind" or "specifically binds" mean that an antibody, an antigen-binding fragment thereof, or synthetic and biotechnological derivatives thereof specifically and selectively recognizes its antigen. In comparison with affinity, the specificity of an antibody is a measure of the compatibility of the antibody with its antigen, with low specificity antibodies exhibiting cross-reactivity with other proteins and high specificity antibodies not exhibiting cross-reactivity with other proteins.

[0073] As used herein, the term "K D " is intended to refer to the equilibrium dissociation constant of a particular ligand-light source interaction. Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, e.g., BIACORE®, etc.

[0074] As used herein, the term "high affinity antibody" refers to an antibody that has a binding affinity for kynurenine, K, as measured by surface plasmon resonance (BIACORE®). D 10 -5 Less than M, preferably 10 -6 Less than M, more preferably 10 -7 Less than M (K D This refers to a monoclonal antibody (the smaller the value, the stronger the binding).

[0075] The term "antigen," as used herein, refers to a molecule or portion of a molecule capable of binding to a selective binding agent, such as an antigen binding protein (including, for example, an antibody or an immunologically functional fragment thereof). An antigen can have one or more epitopes capable of interacting with different antigen binding proteins, such as antibodies.

[0076] As used herein, the term "surface plasmon resonance" refers to an optical phenomenon that allows the analysis of real-time biospecific interactions by detecting changes in protein concentration within a biosensor matrix, for example using a BIACORE® system.

[0077] The term "competition", when used in the context of an antibody, its antigen-binding fragment, or its synthetic and biotechnological derivatives, means the competition between a reference antibody and another antigen-binding molecule in specifically binding to a target protein (also called "antigen", which in the specific case of the present invention is KYN). Such antigen-binding molecules can be selected from the list of natural ligands for KYN, other antibodies or their antigen-binding fragments, diabodies, nanobodies, anticalins, single-chain antibody variable regions, etc. This competition can be determined by an assay in which the reference antibody blocks or inhibits the specific binding of another antigen-binding molecule, for example a test antibody, to KYN.

[0078] For example, solid-phase direct or indirect radioimmunoassays (RIA), solid-phase direct or indirect enzyme immunoassays (EIA), sandwich assays, competitive assays, or solid-phase direct labeling assays.

[0079] The term "kynurenine(s)" as used herein refers to the amino acid itself as well as its esters and salts. Thus, kynurenine and / or its esters and salts are included in the term.

[0080] The present invention provides a monoclonal antibody, or an antigen-binding fragment thereof, or synthetic and biotechnological derivatives thereof, that specifically binds to kynurenine and acts as a detection marker for inflammation in an individual. The present invention also provides a method for detecting a disease or condition in an individual using the monoclonal antibody, or an antigen-binding fragment thereof, or synthetic and biotechnological derivatives thereof, by measuring KYN in a body fluid. Furthermore, means in the form of a test kit for carrying out the method of the present invention are provided. This test can be easily carried out by an individual or the patient himself, when saliva is used as the sample, at any time when such a measurement seems appropriate. Thus, the present invention provides a reliable method for the rapid and easy detection of various diseases and conditions. Furthermore, the present invention provides a reliable method for the early measurement and prediction of the development of inflammation. This is beneficial for the treatment of patients. The method disclosed herein should be used in conjunction with clinical parameters. The relative value of kynurenine can preferably be interpreted in conjunction with other clinical parameters. The present invention substantially contributes to the prognostic value of the diagnosis. The method of the present invention is improved by comparing the value of kynurenine measured in the patient to be diagnosed with the average value obtained from a comparable cohort of people not affected by the disease.

[0081] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by those skilled in the art. Furthermore, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. In general, the nomenclature and techniques used in connection with biochemistry, enzymology, molecular biology, cell biology, microbiology, genetics, protein and nucleic acid chemistry and hybridization described herein are well known and commonly used in the art. It is also noted that aspects of the present invention have been described with reference to different subject matter. In particular, some aspects have been described with reference to method type claims, while other aspects have been described with reference to use type claims and / or product type claims, respectively. However, a person skilled in the art will appreciate from the above and following description that, unless otherwise stated, any combination between features belonging to one type of subject matter, as well as any combination related to different types of subject matter, is considered to be disclosed herein. In particular, combinations of features related to method type claims and features related to use type claims and / or product type claims are considered to be disclosed. Furthermore, it is noted that the term "consisting of" does not exclude other elements or steps. Furthermore, elements described in association with different embodiments may be combined.

[0082] The invention and its embodiments will now be explained in more detail with reference to the drawings. [Brief description of the drawings]

[0083] [Figure 1] FIG. 1 shows a schematic of the kynurenine pathway. [Diagram 2] FIG. 2 shows the chemical structure of kynurenine. [Figure 3a] Figure 3a shows the correlation between serum and saliva kynurenine concentrations in healthy subjects. [Figure 3b] FIG. 3b shows the levels of KYN in serum and saliva. [Figure 4]Figure 4 shows the difference in KYN levels between healthy and diagnosed patients. [Figure 5a] 5a and 5b respectively show the results of a titration ELISA of the antibody of the invention YU552-A01. [Figure 5b] 5a and 5b respectively show the results of a titration ELISA of the antibody of the invention YU552-A01. [Figure 6] FIG. 6 shows the results of competitive ELISA for the antibody of the invention, YU552-A01. [Figure 7] FIG. 7 shows the affinity measurement of the antibody of the present invention using surface plasmon resonance. [Figure 8] FIG. 8 shows affinity measurements of prior art anti-KYN antibodies using surface plasmon resonance. [Figure 9] FIG. 9 shows a schematic diagram of the phage display technology. [Figure 10] FIG. 10 shows a table of measurements of KYN levels using prior art antibodies against KYN. [Figure 11] FIG. 11 shows the results of ELISA testing of serum samples with the antibody of the invention YU552-A01. [Figure 12] FIG. 12 shows the results of an LFA test of saliva samples using the antibody of the present invention YU552-A01.

[0084] In Figure 1, the kynurenine pathway (KP), the major pathway for tryptophan degradation in higher eukaryotes, is depicted. The enzymes are shown in italics. Tryptophan is an essential amino acid and is metabolized by various pathways, but the major pathway is the KP. The first enzyme in this pathway, indoleamine-2,3-dioxygenase, IDO-1, is strongly stimulated by inflammatory molecules, especially interferon-γ. Thus, when the immune response is activated, the KP is often systematically upregulated. The biological significance is that tryptophan depletion and kynurenine production play an important modular role in the immune response. The inventors have surprisingly found that inflammation in an individual can be detected by measuring kynurenine concentrations in body fluids.

[0085] The central intermediate of KP is L-kynurenine (L-KYN), and the metabolic pathway splits into two distinct branches. L-KYN is converted via kynurenine aminotransferase (KAT) to the neuroprotective kynurenic acid (KYNA) or the neurotoxic 3-hydroxy-kynurenine (3-HK). 3-HK is further metabolized in a series of enzymatic steps, ultimately converting it to NAD. + is obtained.

[0086] Increased kynurenine is important in inflammatory metabolism to produce anti-inflammatory substances in the body. Kynurenine can cross the blood-brain barrier and enter the brain. Therefore, high conversion of TRP to kynurenine also increases the risk of neuroinflammatory or neurotoxic disorders. Figure 2 shows the chemical structure of the amino acid KYN. KYN is a very small molecule (molecular weight 209) and is synthesized by the enzymes TDO and IDO-1 in the kynurenine pathway (KP) mentioned above. The chemical formula of KYN is C 10 H 12 It is N2O3.

[0087] Figure 3a shows the results of measuring KYN concentrations in serum and saliva samples. The inventors of the present invention were able to determine a reliable relationship between kynurenine concentrations in serum and saliva using a color reaction. There is a linear correlation (R2=0,902) between the measurements of kynurenine in serum and saliva. The relationship is 3.8 (serum):1 (saliva), which is not true for many other proteins.

[0088] With regard to the color reaction, before the content of kynurenine is measured, components that may adversely affect a correct and accurate test result must be removed. Said undesirable components of the sample that may interfere with a correct test result are preferably removed by precipitation. Such precipitation is preferably carried out using trichloric acid. However, it is also possible to use methods other than using trichloric acid for deproteinization of the components of the sample. After removing the disturbing components of the sample by precipitation, it may be necessary to separate the phases by centrifugation. The supernatant is preferably reacted with Ehrlich's reagent. After color development, the sample is measured by measuring the absorbance at an appropriate wavelength. Preferably, the test is performed quantitatively or semi-quantitatively. The test method uses a calibration curve or a certain threshold value is set in the test kit to avoid false positives. As already mentioned, the inventors found that the concentration of KYN measured using a color reaction in saliva (x-axis) or serum (y-axis) correlates in μM. This was surprising, since not all components that can be detected in serum can also be detected in saliva. The method of the invention is based in particular on the inventors' surprising discovery that kynurenine levels in saliva are highly proportional to kynurenine levels in serum samples, and therefore the method of the invention is preferably carried out with saliva.

[0089] The quantification of kynurenine is preferably performed quantitatively or semi-quantitatively, since it is important to detect changes in kynurenine levels outside the normal range. When saliva is used as the sample, it is particularly advantageous that the in vitro method can be performed without a physician or other trained person.

[0090] Saliva is a complex body fluid that is clear and slightly acidic. Like blood, saliva contains various enzymes, hormones, antibodies, antibacterial components, growth factors, etc. Many of these components pass from blood to saliva through the intercellular spaces. Thus, most compounds found in blood are also present in saliva. Thus, saliva is functionally equivalent to serum in reflecting the physiological state of the body, including emotional, hormonal, nutritional, and metabolic changes. However, although saliva contains a variety of components with diagnostic properties, there are several major limitations that have prevented those skilled in the art from fully realizing the potential of this body fluid for disease detection. For example, the low concentration of components compared to the levels in blood, the lack of conclusive biomarkers for specific diseases, the lack of simple and inexpensive sampling methods with minimal discomfort, and the lack of an accurate, easy-to-use, portable platform to facilitate (early) disease detection. Furthermore, another problem with using saliva as a sample is the reproducibility of saliva testing.

[0091] This shows that saliva cannot be used to measure all parameters. For example, salivary measurement of glucose levels in diabetic patients has so far been unsuccessful. The same goes for the detection of various viruses in saliva. This was also evident during the corona pandemic, when various saliva tests were unable to detect SARS-CoV-2, the causative agent of corona. As a result, saliva cannot be simply used and measurements in saliva are not self-evident. This applies not only to the choice of matrix (serum, plasma, saliva), but also to the methodology and the technology finally chosen.

[0092] Figure 3b shows the results of KYN measurement in serum and saliva of 304 healthy control subjects. The measurement was performed using a color reaction. As can be seen from the graph, the relationship between KYN in serum and KYN in saliva is 3.8:1. Therefore, the KYN level in saliva is significantly lower than that in serum. As already mentioned above, the currently preferred method for detecting KYN is HPLC. This is especially because KYN is a very small molecule, and therefore the production of antibodies that recognize said molecule is difficult in itself. Moreover, KYN has immunological activity, and therefore inhibits antibody production. Furthermore, the inventors have been able to show that the level of KYN in saliva is significantly lower than in serum, and that it is difficult to produce antibodies that can detect KYN even in body fluids with low KYN levels, such as saliva. Due to the low occurrence of KYN in body fluids such as saliva, it is very important that the antibody against KYN has high specificity and sensitivity to the antigen in order to detect this amino acid in body fluids such as saliva. Therefore, the antibody against KYN in particular must have high specificity for its antigen.

[0093] Figure 4 shows the results of investigating KYN levels in patients with various diseases. The study included 304 normal controls (healthy subjects), 314 patients in a stable state after kidney transplantation (stable Tx), 96 patients with acute rejection after kidney transplantation (AR), 274 patients with infection, and 48 patients with sepsis. Measurements were performed using serum samples using a colorimetric reaction.

[0094] As can be seen, KYN levels are only slightly elevated in stable transplant recipients compared to healthy controls. This is due to sufficient immunosuppression in stable transplant recipients, which leads to a concomitant reduction in the innate immune response. Thus, inflammation is minimal in this group. In contrast, KYN levels are significantly elevated in patients with an activated immune system, such as transplant recipients with acute rejection, infections, and sepsis, compared to KYN levels in healthy controls.

[0095] Figures 5a and 5b show a titration ELISA, respectively. This assay is commonly used to test the specific binding of an antibody to its antigen. In a titration ELISA, a fixed concentration of antigen is titrated with different concentrations of added antibody. The antigen is immobilized on a solid surface and after binding to the antigen, the free antibody is removed. The bound antibody remains with the antigen, which is immobilized on the surface of a microtiter well. The amount of bound antigen is quantified with a detection antibody, resulting in a detectable signal.

[0096] The antibody YU552-A01 of the present invention showed specific binding to the biotinylated KYN (Strep+Kynurenin-eBio) used in antibody selection. No binding was detected to other coat antigens (Figure 5a) or to the conjugate of streptavidin and bovine serum albumin (BSA) (Figure 5b). Thus, the antibody of the present invention is highly specific to the antigen KYN and does not show cross-reactivity.

[0097] In FIG. 6 the result of a competitive ELISA test is shown. In general, different configurations of ELISA tests are known. The types of ELISA are direct ELISA, sandwich ELISA, competitive ELISA and / or reverse ELISA. Usually so-called sandwich ELISA is carried out. In such ELISA tests, a compound that specifically binds to the analyte is fixed to a solid surface (e.g. the bottom of a microtiter well). To avoid non-specific binding, the non-specific binding sites are saturated (e.g. with skim milk powder). Usually several microtiter wells are coated with this component so that the sample can be easily diluted to measure the content of the analyte. The binding of the analyte to the corresponding well is usually detected with another antibody that binds to another region of the target molecule to avoid negative interference of the binding. Such an antibody is usually coupled to a signal generating means, which is an enzyme such as horseradish peroxidase (HRP). The presence of the analyte to be detected can be confirmed by adding a precursor molecule that is converted by the signal generating molecule into another molecule with different properties. For example, if kynurenine is present in one well, the antibody binds to this molecule and the activity of the signal generating means (e.g. HRP) generates a color signal whose intensity is proportional to the amount of bound target molecule. The reaction is measured quantitatively, allowing for an accurate determination of the amount of analyte to be detected in the body fluid.

[0098] Another type of ELISA is the competitive ELISA, in which a compound that binds to the analyte is immobilized on a solid surface and an analyte sample containing the analyte (KYN in the present invention) is added to bind to the compound, which is the antibody of the present invention YU552-A01. The less analyte there is in the sample, the more free binding sites there are on the compound. These free binding sites bind to the synthetically labeled analyte and are visualized by a color reaction. Thus, the less analyte there is in the sample, the more synthetically labeled analyte is bound to the compound and the stronger the signal. Conversely, the more KYN there is in the sample and the more it binds to the antibody of the present invention, the less detectable the signal will be. The detection of the analyte and the measurement of its concentration are possible indirectly by detection of the label and, if necessary, quantitative determination.

[0099] Competitive inhibition is therefore measured by determining the amount of label in the presence of a compound, which is usually present in excess.

[0100] Typically, a competing compound, when present in excess, inhibits specific binding of a reference antigen binding protein to a common antigen by at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% or more. In some embodiments, binding is inhibited by at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% or more.

[0101] As can be seen from FIG. 6, the antibody of the present invention YU552-A01 showed a decrease in ELISA binding signal in the presence of the bound target antigen KYN-ebio compared to the negative control phosphate buffered saline (PBS). The unconjugated free target antigen L-KYN showed an even decreased ELISA signal, which means that more unconjugated free KYN binds to the antibody of the present invention. As can be seen from FIG. 6, this blocking effect is better detected at lower antibody concentrations. Thus, the antibody of the present invention binds to the antigen KYN itself and does not show cross-reactivity with the conjugate biotin. These results indicate that the antibody of the present invention recognizes KYN itself and does not recognize the biotin of the linker. This result also supports the high specificity of the antibody of the present invention for KYN.

[0102] FIG. 7 shows the results of surface plasmon resonance measurements performed using the antibody YU552-A01 of the present invention. Surface plasmon resonance spectroscopy (SPR spectroscopy) is a spectroscopic method that measures the refractive index of an analyte with very high resolution. The advantage of this method is that it allows tracking of binding kinetics in real time without affecting the analyte. To measure protein interactions, a gold film capable of adsorbing biomolecules is prepared on a prism. By covering the film with molecules, the refractive index of the film changes, allowing this method to measure with very high sensitivity.

[0103] For SPR, different concentrations of antibodies were used, as shown in Figure 7. As is clear from the graph, binding of antibody YU552-A01 to free KYN but not to immobilized KYN was detected.

[0104] FIG. 8 shows the results of SPR spectroscopy using a prior art KYN antibody. As the prior art antibody, the antibody sc-69890 from Santa Cruz, which is still commercially available, was used. This antibody is a mouse monoclonal antibody against N-formyl-KYN. As can be seen from the graph, the prior art antibody cannot bind to free KYN. The antibody of the present invention and the prior art antibody were produced by different manufacturing processes. As a result, the two have different properties and structures. Therefore, not all antibodies raised against KYN show the same properties in terms of specificity and affinity for the antigen KYN. This also shows the need for antibodies that reliably recognize KYN in different body fluids, preferably saliva.

[0105] Figure 9 illustrates the phage display method. In general, complete isolation of human or humanized monoclonal antibodies can be achieved in a variety of ways. One method uses mice in which the immunoglobulin genes have been replaced with human genes. The mice mount an antibody response using human antibody sequences. Another method uses a bacteriophage display library of human variable region sequences and selects for antigen binding in vitro. The selected genes are combined with human constant region genes to reconstitute a complete IgG antibody. A third approach utilizes B cells to produce antibodies. As outlined in more detail in the Examples, suspension panning can be used for high throughput antibody identification.

[0106] The antibodies of the present invention were generated using the phage display method. In principle, phage display is an experimental technique for studying protein-protein, protein-peptide and protein-DNA interactions, which uses bacteriophages (viruses that infect bacteria) to link proteins with the genetic information that codes for them. In this technique, a gene that codes for a protein of interest is fused to the coat protein gene of a phage, which "displays" the protein on the outside of the phage and contains the gene for the protein on the inside, thus linking genotype and phenotype. Such displaying phages can be screened against other proteins, peptides and DNA sequences to detect interactions of the displayed protein with other molecules. In this way, large libraries of proteins can be screened and amplified in a process called in vitro selection, which is similar to natural selection. There are three categories of phage display: 1. Phage display of antibody libraries 2. Phage display of peptide libraries; 3. Phage display of cDNA libraries.

[0107] The scheme of antibody phage display is shown in Figure 9. In antibody phage particles, the antibody genes and the function they code for (antigen binding) are physically linked. This allows affinity selection of monoclonal human antibodies in a test tube (this process is called "panning"). Panning can be performed in the presence of soluble competitors to eliminate cross-reacting antibodies. Panning under defined biochemical conditions selects only those antibodies that function under those conditions. Sequential panning against two different, but homologous, antigens is also possible, allowing functional selection of antibodies that bind to structural features common to the two proteins. Panning is followed by screening of monoclonal antibodies against the antigens used, followed by production of the antibodies.

[0108] Antibodies developed by phage display can be produced in high yields in E. coli and other cell systems, a major advantage over antibodies produced in animals.

[0109] Figure 10 shows the results of measuring various prior art antibodies against KYN in serum and / or saliva samples from nine post-renal transplant patients and one healthy control using an ELISA method to detect KYN.

[0110] The prior art antibodies used in the measurements were as follows: IDK ELISA K7728: Polyclonal antibody against L-KYN included in the ELISA kit.

[0111] ABIN 6560056: Mouse monoclonal antibody against L-KYN. St.Cruz 69890: Mouse monoclonal antibody against N-Formyl-KYN.

[0112] So far, due to the difficulty in generating antibodies that bind to KYN, only a few antibodies against KYN have been reported, not all of which are commercially available. None of these antibodies were generated by phage display, but in different animals. Moreover, none of these antibodies were developed for LFA testing. As is well known, one cannot simply switch to a testing system for which an antibody has not been developed. Therefore, it was not possible to test the prior art antibodies in another testing system, such as LFA. However, in order to be able to test the specificity of the prior art antibodies, ELISA tests were performed with serum and / or saliva samples from patients after kidney transplantation, either stably engrafted (patient 176) or with acute rejection (patients 139, 180, 1433, 1286, 1365, 1288, 1274), and from healthy controls.

[0113] As already mentioned, KYN levels in patients are at least two times higher than in healthy controls. This relationship could not be reliably detected in samples tested with prior art antibodies, not in serum samples, and even more so in saliva samples. One antibody was unable to detect KYN at all in saliva.

[0114] This result shows the difficulty of generating antibodies that reliably recognize KYN even in body fluids with low KYN levels, such as saliva. Figure 11 shows the results of a competitive ELISA test of serum samples using the antibody YU552-A01 of the present invention. As can be seen from the graph, the antibody of the present invention is able to detect KYN in serum samples. The grey bars show the results of the colorimetric measurement depicted in Example 4 and also referred to in Figure 4. In comparison, the black dotted bars show the results of the ELISA test using the antibody YU552-A01 of the present invention. The inventors have been able to show that the antibody of the present invention is as reliable as the colorimetric measurement in detecting KYN in serum samples. As can be seen from the graph, the antibody of the present invention is able to detect KYN in different patient samples.

[0115] Abbreviations / Terminology in Figure 11: Ktx rejection = samples from patients after kidney transplantation who showed acute rejection Ktx = stable condition, sample from patient after kidney transplant Long-COVID = Samples from patients infected with the SARS-CoV-2 virus that causes corona infection and who suffer from Long-COVID syndrome n = number of samples included in the measurement, i.e. number of patients ns=Not Specified Monoclonal Ab = Monoclonal antibody Colorimetric measurement = colorimetric measurement

[0116] Figure 12 is a photograph of an LFA test of a saliva sample using the antibody of the present invention. LFA tests are simple devices aimed at detecting the presence (or absence) of a target analyte without the need for specialized and expensive equipment, but there are also many lab-based applications supported by a reading device. Generally, these tests are used for medical diagnostics, either at home, point-of-care, or laboratory use. A widespread and well-known application is the home pregnancy test, as well as the recently widely used "corona test". The technology is based on a series of capillary beds, such as porous paper strips or sintered polymers. Each of these elements has the ability to spontaneously transport a liquid (e.g., saliva). The first element (sample pad) acts as a sponge, retaining excess sample liquid. Once soaked, the fluid moves to the second element (conjugate pad) where the manufac- turer stores what they call the conjugate, which is a dried version of bioactive particles in a matrix of salts and sugars, containing everything that ensures an optimized chemical reaction between the target molecule and the chemical partner immobilized on the particle surface. While the sample liquid dissolves the salt-sugar matrix, the particles also dissolve, and the sample and conjugate mix while flowing through the porous structure. In this way, the analyte, while bound to the particles, travels further through the third capillary bed. This material has one or more areas (often called stripes) where a third molecule has been immobilized by the manufacturer. By the time the sample-conjugate mix reaches these stripes, the analyte is bound on the particle and a third "capture" molecule binds the complex. After a while, as more liquid passes through the strip, the particles accumulate and the stripe area changes color. There are usually at least two stripes: one (the control) captures any particle, thereby indicating that the reaction conditions and technique were successful; the second contains a specific capture molecule and captures only the particles on which the analyte molecule is immobilized. After passing through these reaction zones, the liquid enters the last porous material, the wick. Lateral flow tests work as competitive or sandwich assays.

[0117] In principle, particles of any color can be used. However, latex (blue) and nanometer-sized gold particles (red) are the most commonly used. Gold particles are red due to localized surface plasmon resonance. Fluorescent or magnetically labeled particles can also be used. However, these require the use of an electronic reader to evaluate the test results. The sample is first contacted with colored particles labeled with antibodies against the target analyte. The test line also contains antibodies against the same target, but may bind to a different epitope on the analyte. In a positive sample, the test line is indicated by a colored band. Although not strictly necessary, it is desirable for most test kits to incorporate a second line containing antibodies that pick up free latex / gold to ensure that the test has been performed correctly.

[0118] The LFA test shown in FIG. 12 is a competitive assay. As already explained, in a competitive assay, an antibody specific for an antigen is bound (immobilized) to a solid phase. A sample solution containing the analyte and a second solution (tracer solution) containing the antigen to be detected in a known concentration and labeled with a detection system are added. During the reaction time (incubation time), the unknown concentration of unlabeled antigen present in the sample solution and the known concentration of labeled antigen compete for binding sites on the immobilized antibody. As a result, the higher the antigen concentration in the sample, the less the labeled antigen binds to the antibody. The detection of the antigen and the measurement of its concentration are possible indirectly by the detection of the label and, if necessary, the quantitative determination of the label. Thus, the less the signal, the more unlabeled KYN in the sample binds to the antibody of the invention YU552-A01.

[0119] In the depicted LFA test, a commercially available saliva sample (Invent Human Specimen) was used. Different concentrations of KYN (0 μM, 1.25 μM, 2.5 μM, 5 μM, 10 μM, 20 μM) were added to ensure there was enough KYN for the antibodies of the present invention to bind and for the assay to function.

[0120] As is evident from the photographs of different LFAs showing various KYN concentrations, antibody YU552-A01 is able to detect KYN in saliva samples. As the concentration of KYN increases, the signal decreases, since unlabeled KYN competes with the labeled antigen to bind to the antibody of the present invention. The signal of the sample without KYN is due to the fact that KYN is always present in the saliva of human samples.

[0121] Thus, the present invention provides a reliable method and test kit for detecting inflammation by measuring kynurenine levels in body fluids, preferably saliva. By using the monoclonal antibody of the present invention, the present inventors provide a tool for measuring KYN levels easily and quickly without the need for trained personnel when saliva is used as a sample. This is also beneficial for pediatric care and monitoring various diseases. The present invention is further illustrated by the following examples, which should not be construed as limiting the scope of the invention.

[0122] Example 1: Discovery of monoclonal antibodies As already mentioned, the phage display method was applied to identify the antibodies of the present invention. As the library, a naive scFv library was used. As the antigens, L-kynurenine and 3-hydroxy-DL-kynurenine from Sigma Aldrich were used. Biotinylated kynurenine, kynurenine alone, streptavidin + biotinylated kynurenine and kynurenine-BSA were used as immobilized targets. As counter selection, streptavidin, IVIG (intravenous immunoglobulin) and N protein standard (Siemens Healthcare) were used.

[0123] In total, 384 clones were isolated from the lambda and kappa selections. 29 sequence clusters with binding antibodies were identified. Based on a rational approach, the number of clones was reduced for IgG conversion.

[0124] 1.1 Antibody selection using phage display Antibody selection was performed as follows: In the panning method, the antigen was immobilized on a Costar High binding 96-well plate (Corning). 1 μg of the antigen was added to phosphate-buffered saline (PBS, 8 g / l NaCl, 0.2 g / l KCl, 1.8 g / l Na2HPO4 * 2H2O, 0.24 g / l KH2PO4) and coated onto wells overnight at 4° C. Next, wells were blocked with 350 μl of BSA PBST (2% (w / v) bovine serum albumin, BSA in PBS; 0.05% Tween 20) for 1 h at RT (room temperature) and washed three times with PBST (PBS; 0.05% Tween 20).

[0125] For immobilization of biotinylated antigen, 2 μg of streptavidin in PBS was immobilized as described above. After blocking of the wells, 1 μg of biotinylated antigen in 200 μl PBS was immobilized for 1 h and washed three times with PBST.

[0126] Before adding the library to the coated wells, dilute the library (5 × 10 10 The phage particles were preincubated with 2% BSA-PBST (and 5 μg streptavidin for panning on biotinylated antigens) on blocked wells for 1 h at RT. The library was transferred to the coated wells, incubated for 2 h at RT, and washed 10 times. Bound phages were eluted with 200 μl trypsin (10 μg / ml) for 30 min at 37°C and used for the next panning round.

[0127] The eluted phage solution was transferred to a 96 deep-well plate (Greiner Bio-One, Frickenhausen, Germany) and incubated with 190 μl of E. coli TG1 (OD 600=0.5) at 37°C for 30 min, then incubated at 37°C and 650 rpm for 30 min to infect the phage particles. Four ml of 2xYT-GA (1.6% (w / v) tryptone; 1% (w / v) yeast extract; 0.5% (w / v) NaCl (pH 7.0), 100 mM D-glucose, 100 μg / ml ampicillin) was added and incubated at 37°C and 650 rpm for 1 h, after which 5 × 10 10 cfu (colony forming units) of M13KO7 helper phage were added, and the infected bacteria were then incubated at 37°C for 30 min, 650 rpm for 30 min, and then centrifuged at 3220xg for 10 min.

[0128] The supernatant was discarded and the pellet was resuspended in fresh 2xYT-AK (1.6% (w / v) Tryptone; 1% (w / v) Yeast extract; 0.5% (w / v) NaCl (pH 7.0), 100 μg / ml ampicillin, 50 μg / ml kanamycin). Phage antibodies were amplified overnight at 30°C and 650 rpm and used for the next panning round. A total of three panning rounds were performed. In each round, the stringency of the washing procedure was increased (20-fold for panning round 2 and 30-fold for panning round 3). In the third round, XL1Blue cells were used for infection as in the case of TG1. After the third panning round, monoclonal antibody clones for screening ELISA were selected using titer plates.

[0129] 1.2 Screening of monoclonal recombinant binders using E. coli scFv supernatant Soluble antibody fragments (scFv) were produced in 96-well polypropylene microtiter plates (MTPs) (IJ96PP, Greiner BioOne). 150 μl of 2xYT-GA was inoculated with bacteria carrying scFv-expressing phagemids. The MTPs were incubated overnight in an MTP shaker (SOK3190, Axon) at 37°C, 300 rpm, and 70% humidity. 10 μl of the overnight culture was inoculated into 140 μl of 2xYT-GA in the MTP wells and incubated at 37°C, 300 rpm, and 70% humidity until the bacteria reached an OD600 of 0.5. Bacteria were harvested by centrifugation at 3220xg for 10 min, and the supernatant was discarded. To induce expression of the antibody genes, the pellet was suspended in 150 μl of 2xYT supplemented with 100 μg / ml ampicillin and 50 μM isopropyl-βD-thiogalactopyranoside (IPTG) and cultured overnight at 30° C. and 300 rpm.

[0130] Bacteria were pelleted by centrifugation at 3220×g for 10 min at 4° C. The supernatant containing the scFv was transferred to a fresh MTP and stored at 4° C. prior to ELISA analysis.

[0131] For ELISA, 20 ng of antigen or 40 ng of streptavidin, followed by 20 ng of biotinylated antigen after blocking, were coated onto 384-well microtiter plates (Greiner PS, F-bottom) in PBS (pH 7.4) overnight at 4°C. After coating, wells were washed three times with PBST and blocked with 1% BSA-PBST for 1 h at RT, followed by three washes with PBST. Supernatants containing monoclonal scFv were mixed with 1% BSA-PBST (1:2) and incubated in the antigen-coated plates for 1.5 h (RT), followed by three repeated PBST washes. Bound scFv were detected using anti-c-myc antibody (diluted in 2% BSA-PBST) and horseradish peroxidase (HRP)-conjugated goat anti-mouse serum (A0168, Sigma) (diluted 1:50.000 in 2% BSA-PBST).

[0132] Bound antibodies were visualized with tetramethylbenzidine (TMB) substrate (20 parts of TMB solution A (30 mM 50% potassium citrate; 1% (w / v) citric acid (pH 4.1)) mixed with one part of TMB solution B (10 mM TMB; 10% (v / v) acetone; 90% (v / v) ethanol; 80 mM H2O2 (30%))). After stopping the reaction by adding 1NH2SO4, the absorbance was measured at 450 nm with a reference at 620 nm using an ELISA plate reader (Epoch, BioTek). The base sequences of the monoclonal conjugates were determined and analyzed.

[0133] 1.3 Characterization of IgG in Titrimetric ELISA For titration ELISA, antibodies were converted to mouse IgG2a format, expressed in HEK293 cells, and purified. Purified IgG was titrated from 10 μg / ml to 0.01 μg / ml on streptavidin + biotinylated KYN, streptavidin, KYN, and KYN-BSA. IgG was detected using goat-anti-mouse IgG(Fc)-HRP (A0168, Sigma).

[0134] Example 2: Binding and competition assays Specific binding was examined by titration ELISA. Thus, 200 ng of streptavidin in PBS was coated onto wells of a 96-well plate (Costar 96-well plate, Corning #9018). Alternatively, 200 ng of L-KYN, 3-hydroxykynurenine, and L-KYN-BSA conjugates diluted in PBS were directly coated for 1 h at room temperature (RT). After washing three times with H2O containing 0.05% (v / v) Tween 20 (H2O-T), the wells were blocked with 300 μl / well 1% BSA-PBST for 10 min and washed three times with H2O-T. 200 ng of biotinylated KYN or 3-hydroxykynurenin was loaded onto the streptavidin-coated plate for 1 h at RT, and unbound antigen was removed by washing three times with H2O-T. Antibodies were diluted starting at 10 μg / ml in 1X BSA-PBST in 1 / 10 steps. After washing (3xH2O-T), 100 μl / well of detection antibody was added for 1 h at RT (Goat anti-mouse IgG (Fc specific), HRP coupled, Sigma #A0168). Unbound detection antibody was washed out with an additional 3x H2O-T and 100 μl of TMB substrate (9.5 parts + 0.5 parts TMB A (9.73 g / L potassium citrate, adjusted to pH 4.1 with citric acid) + TMB B (90% (v / v) absolute EtOH, 10% (v / v) acetone, 2.4 g / L tetramethylbenzidine, 0.907% (v / v) 30% H2O2) was added. The reaction was stopped by the addition of 1N H2SO4 and absorbance was measured at 450 nm with a reference at 620 nm. Antibody YU552-A01 showed specific binding to biotinylated L-KYN, but no binding was observed to other coated antigens.

[0135] For the competition assay, biotinylated KYN was immobilized via streptavidin on 96-well plates (Costar 96-well plates, Corning #9018) and washed similarly to the binding assay. Antibody YU552-A01 was pre-diluted to 10 μg / ml in KYN, biotinylated KYN, and PBS without KYN, respectively. Serial 1 / 10 dilutions of antibody and antibody-KYN mixture were prepared in PBS and incubated for 15 min at RT. After washing the coated plates 3xH2O-T, 100 μl of antibody solution was transferred to the coated wells and incubated for 1 h at RT. Unbound antibody was removed by washing 3 times with 300 μl H2O-T and 100 μl / well of detection antibody (goat anti-mouse IgG (Fc specific), HRP coupled, Sigma #A0168) was added for 1 h at RT. Unbound detection antibody was washed off with an additional 3x H2O-T and 100 μl of TMB substrate (9.5 parts + 0.5 parts TMB A (9.73 g / L potassium citrate, adjusted to pH 4.1 with citric acid) + TMB B (90% (v / v) pure EtOH, 10% (v / v) acetone, 2.4 g / L tetramethylbenzidine, 0.907% (v / v) 30% H2O2) was added. The reaction was stopped by the addition of 1N H2SO4 and the absorbance was measured at 450 nm with a reference at 620 nm.

[0136] Measurements were normalized to the ELISA signal obtained without the addition of competing antigen (PBS). Generally, a lower ELISA signal indicates greater antigen binding.

[0137] Example 3: Affinity study For affinity measurements by SPR spectroscopy, the antibodies of the invention were immobilized on a CM7 chip and incubated with increasing concentrations of kynurenine and biotin-kynurenine. Measurements were performed in a buffered aqueous system. For both analytes, 1:1 binding to the antibody was determined. The affinity value for kynurenine was K D :2.09×10 -5 M for biotin-kynurenine and K for D 1.27×10 -5The K was 1.2 M. The experiments were repeated using a CM5 chip. In these experiments, biotin-kynurenine was immobilized and incubated with increasing concentrations of the antibody of the invention. In this experimental setup, the affinity was not determined, only the avidity. D The value is 3.75×10 -7 It's M.

[0138] Affinity measurements showed that the antibody of the present invention strongly binds to the antigen KYN, and further showed that the antibody has no cross-reactivity and is highly specific to the antigen KYN (FIGS. 5 and 6).

[0139] In contrast, the prior art antibody sc-69890 from St. Cruz was also tested for affinity to KYN, and this antibody was unable to bind to non-immobilized biotin-KYN (Figure 8).

[0140] These results indicate that antibodies produced by different methods have different properties in binding to their antigens and therefore cannot be used interchangeably.

[0141] Example 4: Color response 4.1 General approach Kynurenine-mediated tryptophan metabolism can be quantified in biological fluids by color reactions that have been known for decades. In general, detection by the formation of a colored reaction product can be performed by standard methods.

[0142] A microplate reader is a laboratory device designed to detect biological, chemical or physical events in samples in microtiter plates. It is widely used in research, drug discovery, bioassay validation, quality control, and manufacturing processes in the pharmaceutical and biotechnology industries and academic institutions. Sample reactions can be assayed in microtiter plates in 6-1536 well formats. The most commonly used microplate format in academic and clinical diagnostic laboratories is 96 wells (8x12 matrix), with reaction volumes of 100 to 200 μL per well being typical.

[0143] Common detection modes in microplate assays are absorbance, fluorescence intensity, luminescence, time-resolved fluorescence, and fluorescence polarization. Absorbance detection has been available in microplate readers for over 30 years and is used in assays such as ELISA assays, protein and nucleic acid quantification, and enzyme activity assays. A light source illuminates the sample with a specific wavelength (selected with optical filters or a monochromator), and a photodetector on the other side of the well measures how much of the initial (100%) light is transmitted through the sample.

[0144] 4.2 Test Contents This test was developed as a correction. A color reagent was prepared and dilutions of a standard solution of kynurenine were also prepared. The color reaction is carried out in the so-called "Ehrlich-Reagenz" and produces a yellow color. A solution of 100% by weight of dimethylaminobenzaldehyde in 20% HCl is called "Ehrlich-Reagenz". This color reagent is also useful for the detection of primary amino groups, pyrrole and indole derivatives. The colorimetric determination of the concentration is carried out in monochromatic light. A standard solution of kynurenine was prepared with L-kynurenine sulfate.

[0145] An equal amount of sample was thoroughly mixed with 100 μl of trichloroacetic acid (30%). After centrifugation, the supernatant was measured. The absorbance of each sample at 492 nm was compared with the absorbance of the same sample at 650 nm or 690 nm. The absorbance of the control (average of 5 wells) was then subtracted from the absorbance of each well. By constructing a standard curve, the kynurenine concentration in each sample could be determined.

[0146] Example 5: ELISA testing of serum samples with antibodies of the invention The binding of KYN to the antibody YU552-A01 of the present invention was tested using competitive ELISA. Serum samples from patients with different diseases and healthy individuals were pretreated with TCA according to the colorimetric assay shown in Example 4 above. 50 μl / well of serum samples were added to the wells of a microtiter plate. YU552-A01 was added at a concentration of 0.25 μg / ml, 50 μl / well, and then the plate was shaken at 170 rpm on a shaking platform for 1 h at RT. The plate was then washed three times with PBS-T, 250 μl / well, followed by another wash with PBS (250 μl / well, 1x). Then, 100 μl / well of HRP was added and incubated for 1 h at RT. Again, the plate was washed three times with PBS-T, 250 μl / well, and once with PBS, 250 μl / well. 1-Step Ultra TMB (100 μl / well) was added and incubated for approximately 15 to 20 minutes, after which the reaction was stopped with 2M H2SO4 (100 μl / well). OD was measured at 450 nm.

[0147] Between each incubation step, the plates or the corresponding rows were provided with a sealing foil to avoid contamination.

Claims

1. A monoclonal antibody or antigen-binding fragment thereof, or synthetic and biotechnological derivatives thereof, that specifically binds to kynurenine and acts as a detectable marker for inflammation in an individual.

2. binds to kynurenine in body fluids, preferably serum, saliva and / or cerebrospinal fluid (CSF), more preferably saliva; 2. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, or its synthetic and biotechnological derivatives.

3. The monoclonal antibody or antigen-binding fragment thereof, or synthetic and biotechnological derivatives thereof, comprises one or more complementarity determining regions (CDRs) and / or a variable heavy chain (VH) region consisting of an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:1; and a variable light chain (VL) region consisting of an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:

2.

2. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, or its synthetic and biotechnological derivatives.

4. At least one CDR Heavy chain CDRs defined by SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, and Light chain CDRs as defined in SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:

8. and / or The monoclonal antibody or antigen-binding fragment thereof, or synthetic and biotechnological derivatives thereof, A heavy chain CDR1 (CDRH1) domain having the amino acid sequence set forth in SEQ ID NO: 3; A heavy chain CDR2 (CDRH2) domain having the amino acid sequence set forth in SEQ ID NO: 4; A heavy chain CDR3 (CDRH3) domain having the amino acid sequence set forth in SEQ ID NO:5; and A light chain CDR1 (CDRL1) domain having the amino acid sequence set forth in SEQ ID NO:6; A light chain CDR2 (CDRL2) domain having the amino acid sequence set forth in SEQ ID NO: 7; A light chain CDR3 (CDRL3) domain having the amino acid sequence set forth in SEQ ID NO:

8. A monoclonal antibody or an antigen-binding fragment thereof according to any one of claims 1 to 3, or synthetic and biotechnological derivatives thereof.

5. Kynurenine and 10 －５ M, preferably 10 －６ Less than M, more preferably 10 －７ Affinity (K Ｄ ) can specifically bind to A monoclonal antibody or an antigen-binding fragment thereof according to any one of claims 1 to 4, or synthetic and biotechnological derivatives thereof.

6. An isolated nucleic acid molecule encoding the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, or its synthetic and biotechnological derivatives.

7. An expression vector comprising the nucleic acid molecule of claim 6.

8. A step of introducing the expression vector of claim 7 into an isolated host cell; Growing the cells under conditions that allow the production of the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, or synthetic and biotechnological derivatives thereof; recovering the antibody or fragment or derivative thus produced; The method includes the steps of: A monoclonal antibody or an antigen-binding fragment thereof according to any one of claims 1 to 5, or synthetic and biotechnological derivatives thereof.

9. 1. An in vitro method for detecting a disease or condition in an individual, comprising: A method, wherein the level of kynurenine in a body fluid is measured using a monoclonal antibody or its antigen-binding fragment, or synthetic and biotechnological derivatives thereof, according to any one of claims 1 to 5, and the value of kynurenine measured in the individual to be diagnosed is compared with the mean value obtained from a comparable cohort of healthy individuals, whereby the value of kynurenine in the patient is increased.

10. The measurement of kynurenine in said body fluids is used in the management of the patient's treatment. and / or Used to monitor personal health conditions, 10. The in vitro method of claim 9.

11. The body fluid is serum, saliva and / or cerebrospinal fluid (CSF); An in vitro method according to any one of claims 9 to 10.

12. Kynurenine levels in patients are at least two-fold higher than in healthy controls; An in vitro method according to any one of claims 9 to 11.

13. The disease or condition is selected from at least one of neurodegenerative diseases, transplantation, infectious diseases, dialysis, dental diseases, sports medicine, gynecology, gastroenterology, cancer, diabetes, multiple sclerosis, asthma, and concussion. An in vitro method according to any one of claims 9 to 12.

14. ELISA test, lateral flow immunochromatography or microfluidic test; An in vitro method according to any one of claims 9 to 13.

15. A test kit for determining the concentration of kynurenine in a body fluid using the monoclonal antibody or antigen-binding fragment thereof, or synthetic and biotechnological derivatives thereof, according to any one of claims 1 to 5.

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