Antibodies and methods targeting interleukin-19

Abstract

The present invention provides compounds and methods of targeting human interleukin-19, including therapeutic antibodies, pharmaceutical compositions, and methods of use thereof, which are useful in the field of immune-mediated diseases, including psoriasis, atopic dermatitis, asthma, psoriatic arthritis, rheumatoid arthritis, central axis type spondyloarthritis, inflammatory bowel disease, and colitis.

Description

[0001] The present invention belongs to the field of medicine. More specifically, the present invention relates to antibodies against human interleukin-19 (IL-19), pharmaceutical compositions comprising such antibodies, and methods of using such antibodies. It is expected that the antibodies and methods of the present invention can be used in the field of autoimmune and chronic inflammatory diseases (collectively referred to herein as immune-mediated diseases), particularly diseases such as psoriasis (PsO), atopic dermatitis (AD), asthma, psoriatic arthritis (PsA), rheumatoid arthritis (RA), axial spondyloarthritis (AxSpA), inflammatory bowel disease (IBD), colitis, including treatment thereof.

[0002] Interleukin-19 (IL-19) is a cytokine reported to belong to the interleukin-10 cytokine family (which includes IL-10, 20, 22, 24, and 26 as well as some virally encoded cytokines). IL-19 is reported to participate in the IL-20R complex signaling pathway and is expressed in resting monocytes, macrophages, B cells, and epithelial cells (including keratinocytes). In addition, studies have reported the involvement of IL-19 in immune-mediated diseases (e.g., Konrad et al., ScientificReports 9, article number 5211 (2019) and Steinert et al., J.Immunol 2017;199:2570-2584 (September 2017).

[0003] Autoimmune disease is a form of immune-mediated disease caused by the body's immune response to its own tissues. Autoimmune diseases are often chronic and can be debilitating or even life-threatening. PsO is a chronic autoimmune disease with systemic manifestations, including PsA, cardiovascular disease, metabolic syndrome, and affective disorders. AD and many other forms of chronic autoimmune diseases (such as asthma, PsO, PsA, RA, IBD, colitis, and AxSpA) affect the axial skeleton and / or the peripheral skeleton.

[0004] Current FDA-approved treatments for immune-mediated diseases include corticosteroids, which are typically used to treat acute inflammation, and biologics that target TNFα, interleukin-12, interleukin-17, and interleukin-23. Although these treatments have been shown to be effective in alleviating symptoms in subgroups of patients, a subset of patients do not respond or fail to respond to existing treatments. Therefore, there remains a need for additional therapies that target different human targets and pathways to treat immune-mediated diseases.

[0005] Although IL-19 antibodies are known in the art (WO 2019 / 143585), there are no approved IL-19 antibody therapeutics to date. In addition, nonspecific binding (e.g., binding to serum proteins) has been shown to be a challenge in the field of IL-19 antibodies. Therefore, there is still an unmet need for antibodies, pharmaceutical compositions and methods targeting human IL-19, which can be used to treat immune-mediated diseases such as AD, asthma, PsO, RA, IBD, colitis, AxSpA, PsA, etc. Such IL-19 antibodies should have good therapeutic properties, including being a potent neutralizer (i.e., antagonist) of human IL-19, having high binding affinity and low nonspecific binding to human IL-19, including nonspecific binding to serum proteins. Such IL-19 antibodies should also have therapeutically acceptable pharmacokinetic (Pk) characteristics and show low immunogenicity. Such IL-19 antibodies should also be suitable for commercial production, including high solubility levels and low aggregation levels. The IL-19 antibodies provided by the present disclosure can meet these needs for treating immune-mediated diseases.

[0006] Therefore, in certain embodiments, the present invention provides antibodies against human IL-19. According to some embodiments, the antibodies of the present invention have an antagonistic effect on human IL-19. The embodiments of the present invention provide antibodies comprising a light chain variable region (LCVR) and a heavy chain variable region (HCVR), wherein the LCVR comprises complementary determining regions (CDRs) LCDR1, LCDR2 and LCDR3, and the HCVR comprises CDRs HCDR1, HCDR2 and HCDR3, wherein the amino acid sequence of HCDR1 is SEQ ID NO.7, the amino acid sequence of HCDR2 is SEQ ID NO.8, the amino acid sequence of HCDR3 is SEQ ID NO.9 or SEQ ID NO.10, the amino acid sequence of LCDR1 is SEQ ID NO.2, the amino acid sequence of LCDR2 is SEQ ID NO.3, and the amino acid sequence of LCDR3 is SEQID NO.4. According to some embodiments, the antibodies of the present invention also include antibodies comprising CDRs, the amino acid sequences of these CDRs having at least 95% homology with the amino acid sequences of the CDRs herein.

[0007] According to some embodiments, the amino acid sequence of HCDR3 is SEQ ID NO. 9. According to some such embodiments, the amino acid sequence of HCVR is SEQ ID NO. 11 and the amino acid sequence of LCVR is SEQ ID NO. 5. According to some embodiments, the antibodies of the invention also include antibodies comprising LCVR and HCVR whose amino acid sequences are at least 95% homologous to the amino acid sequences of LCVR and HCVR herein.

[0008] According to some embodiments, the amino acid sequence of HCDR3 is SEQ ID NO. 10. According to some such embodiments, the amino acid sequence of HCVR is SEQ ID NO. 13 and the amino acid sequence of LCVR is SEQ ID NO. 5. According to some embodiments, the antibodies of the invention also include antibodies comprising LCVR and HCVR whose amino acid sequences are at least 95% homologous to the amino acid sequences of LCVR and HCVR herein.

[0009] According to some embodiments, the antibodies of the present invention comprise a heavy chain (HC) and a light chain (LC), wherein the amino acid sequence of the HC is SEQ ID NO. 12 or 14 and the amino acid sequence of the LC is SEQ ID NO. 6. In some such embodiments, the amino acid sequence of the HC is SEQ ID NO. 12. In some embodiments, the amino acid sequence of the HC is SEQ ID NO. 14. According to some embodiments, the antibodies of the present invention also include antibodies comprising HC and LC, the amino acid sequences of which are at least 95% homologous to the amino acid sequences of the HC and LC herein.

[0010] Further embodiments of the present invention include nucleic acids comprising sequences encoding SEQ ID NO:6, 12 or 14. Additional embodiments include vectors comprising a first nucleic acid sequence encoding SEQ ID NO:12 or 14 and a second nucleic acid sequence encoding SEQ ID NO:6. Additional embodiments also include a first vector comprising a nucleic acid sequence encoding SEQ ID NO:12 or 14 and a second vector comprising a nucleic acid sequence encoding SEQ ID NO:6. Still further embodiments include cells comprising one or more vectors of the present invention. In addition, the present invention provides a method of producing an antibody, the method comprising culturing a cell of the present disclosure under conditions such that the antibody is expressed and recovering the expressed antibody from the culture medium.

[0011] Embodiments of the invention also include antibodies that bind to murine IL-19, the antibodies comprising a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein the HCVR comprises complementarity determining regions (CDRs) HCDR1, HCDR2, and HCDR3, and the LCVR comprises CDRs LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of HCDR1 is SEQ ID NO.21, the amino acid sequence of HCDR2 is SEQ ID NO.22, the amino acid sequence of HCDR3 is SEQ ID NO.23, the amino acid sequence of LCDR1 is SEQ ID NO.16, the amino acid sequence of LCDR2 is SEQ ID NO.17, and the amino acid sequence of LCDR3 is SEQ ID NO.18. According to some embodiments, the amino acid sequence of HCVR is SEQ ID NO.24 and the amino acid sequence of LCVR is SEQ ID NO.19. According to some embodiments, the antibodies of the invention include antibodies comprising a heavy chain (HC) and a light chain (LC), wherein the amino acid sequence of HC is SEQ ID NO.25 and the amino acid sequence of LC is SEQ ID NO.20. According to some embodiments, the antibodies of the present invention also include antibodies whose amino acid sequences have at least 95% homology to the amino acid sequences herein.

[0012] Another embodiment of the invention includes an antibody that binds to murine IL-19, the antibody comprising a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein the HCVR comprises complementarity determining regions (CDRs) HCDR1, HCDR2 and HCDR3, and the LCVR comprises CDRs LCDR1, LCDR2 and LCDR3, wherein the amino acid sequence of HCDR1 is SEQ ID NO.33, the amino acid sequence of HCDR2 is SEQ ID NO.34, the amino acid sequence of HCDR3 is SEQ ID NO.35, the amino acid sequence of LCDR1 is SEQ ID NO.28, the amino acid sequence of LCDR2 is SEQ ID NO.29, and the amino acid sequence of LCDR3 is SEQ ID NO.30. According to some embodiments, the amino acid sequence of HCVR is SEQ ID NO.36 and the amino acid sequence of LCVR is SEQ ID NO.31. According to some embodiments, the antibodies of the invention include antibodies comprising a heavy chain (HC) and a light chain (LC), wherein the amino acid sequence of HC is SEQ ID NO.37 and the amino acid sequence of LC is SEQ ID NO.32. According to some embodiments, the antibodies of the present invention also include antibodies whose amino acid sequences have at least 95% homology with the amino acid sequences herein. In addition, according to some embodiments, the antibodies of the present invention that bind to murine IL-19 do not completely bind to murine IL-19.

[0013] Further embodiments of the invention include nucleic acid sequences encoding SEQ ID NO: 26 or 27. Additional embodiments include vectors comprising a first nucleic acid sequence encoding SEQ ID NO: 26 and a second nucleic acid sequence encoding SEQ ID NO: 27. Alternatively, embodiments of the invention include nucleic acid sequences encoding SEQ ID NO: 38 or 39. Additional embodiments include vectors comprising a first nucleic acid sequence encoding SEQ ID NO: 38 and a second nucleic acid sequence encoding SEQ ID NO: 39.

[0014] Further embodiments include cells comprising one or more vectors of the invention. In addition, the invention provides a method of producing an antibody, the method comprising culturing the cell of the invention under conditions such that the antibody is expressed and recovering the expressed antibody from the culture medium.

[0015] According to some embodiments, provided herein is a pharmaceutical composition comprising an antibody of the present invention and one or more pharmaceutically acceptable carriers, diluents or excipients.

[0016] In addition, provided herein are methods of treating AD, asthma, PsO, PsA, RA, AxSpA, IBD, colitis, or PsA, the methods comprising administering to a patient in need thereof an effective amount of an antibody of the invention or a pharmaceutical composition of the disclosure.

[0017] Embodiments of the invention include antibodies of the invention for use in therapy. According to some embodiments, antibodies of the invention are provided herein for use in treating AD, asthma, PsO, PsA, RA, AxSpA, IBD, colitis, or PsA. In addition, antibodies of the invention are provided herein for use in the production of a medicament for treating AD, asthma, PsO, PsA, RA, AxSpA, IBD, colitis, or PsA.

[0018] As used herein, the term "antibody" refers to an immunoglobulin molecule that binds an antigen. Embodiments of antibodies include monoclonal antibodies, polyclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, bispecific or multispecific antibodies, or conjugated antibodies. These antibodies may belong to any class (e.g., IgG, IgE, IgM, IgD, IgA) and any subclass (e.g., IgG1, IgG2, IgG3, IgG4).

[0019] Exemplary antibodies of the present disclosure are immunoglobulin G (IgG) type antibodies, which are composed of four polypeptide chains: two heavy chains (HC) and two light chains (LC) cross-linked by interchain disulfide bonds. The amino terminal portion of each of the four polypeptide chains contains a variable region of about 100 to 125 or more amino acids that is primarily responsible for antigen recognition. The carboxyl terminal portion of each of the four polypeptide chains contains a constant region that is primarily responsible for effector function. Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region. Each light chain is composed of a light chain variable region (VL) and a light chain constant region. IgG isotypes can be further divided into subclasses (e.g., IgG1, IgG2, IgG3, and IgG4).

[0020] VH and VL regions can be further subdivided into hypervariable regions known as complementary determining regions (CDRs), which are interspersed with more conservative regions known as framework regions (FRs). CDRs are exposed to the surface of the protein and are important regions of the antigen-binding specificity of antibodies. Each VH and VL is composed of three CDRs and four FRs, which are arranged in the following order from amino-terminal to carboxyl-terminal: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In this article, the three CDRs of the heavy chain are referred to as "HCDR1, HCDR2 and HCDR3", and the three CDRs of the light chain are referred to as "LCDR1, LCDR2 and LCDR3". These CDRs contain most of the residues that form specific interactions with the antigen. The assignment of amino acid residues to CDRs can be performed according to well-known schemes, including those described in Kabat (Kabat et al., "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1991)), Chothia (Chothia et al., "Canonical structures for the hypervariable regions of immunoglobulins," Journal of Molecular Biology, 196, 901-917 (1987); Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins," Journal of Molecular Biology, 273, 927-948 (1997)), North (North et al., "A New Clustering of Antibody CDR Loop Conformations," Journal of Molecular Biology, 274, 937-951 (1998)), and the like. Biology, 406, 228-256 (2011)) or IMGT (International Immunogenetics Database available at www.imgt.org; see Lefranc et al., Nucleic Acids Res. 1999; 27: 209-212).

[0021] Embodiments of the present disclosure also include antibody fragments or antigen-binding fragments, as used herein, which comprise at least a portion of an antibody that retains the ability to specifically interact with an antigen or antigenic epitope, such as Fab, Fab', F(ab')2, Fv fragments, scFv antibody fragments, scFab, disulfide-linked Fv (sdFv), Fd fragments.

[0022] The antibodies of the present invention are monoclonal antibodies. A monoclonal antibody is an antibody derived from a single copy or clone (including, for example, any eukaryotic, prokaryotic or phage clone), regardless of the method of its production. For example, a monoclonal antibody can be produced by hybridoma technology, recombinant technology, phage display technology, synthetic technology (e.g., CDR-grafting), or a combination of such or other technologies known in the art.

[0023] Methods for producing and purifying antibodies are well known in the art and can be found, for example, in Harlow and Lane (1988), Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring harbor, NY, Chapters 5-8 and 15, ISBN 0-87969-314-2. For example, mice or rabbits (including transgenic mice or rabbits known in the art) can be immunized with human IL-19 or a portion thereof, and the resulting antibodies can be recovered, screened, purified, and the amino acid sequence determined using conventional methods well known in the art.

[0024] In a specific embodiment of the invention, the antibody or nucleic acid encoding the antibody is provided in an isolated form. As used herein, the term "isolated" refers to a protein, peptide or nucleic acid that is free or substantially free of other macromolecular species found in the cellular environment.

[0025] The antibodies of the invention can be prepared and purified using known methods. For example, cDNA sequences encoding HC (e.g., the amino acid sequence given by SEQ ID NO.12 or 14) and LC (e.g., the amino acid sequence given by SEQ ID NO.6) can be cloned and engineered into a GS (glutamine synthetase) expression vector. The engineered immunoglobulin expression vector can then be stably transfected into CHO cells. As will be appreciated by those skilled in the art, mammalian expression of antibodies results in glycosylation, typically at the highly conserved N-glycosylation sites in the Fc region. Stable clones can be verified to express antibodies that specifically bind to human IL-19 (e.g., as represented by a recombinantly produced peptide comprising SEQ ID NO.1). Positive clones can be amplified in serum-free culture medium for use in the production of antibodies in a bioreactor. The culture medium in which the antibodies have been secreted can be purified by conventional techniques. For example, the culture medium can be conveniently applied to a protein A or G Sepharose FF column that has been equilibrated with a compatible buffer such as phosphate-buffered saline. The column is washed to remove non-specific binding components. For example, the bound antibody is eluted by a pH gradient, and the antibody fractions are detected, such as by SDS-PAGE, and then combined. Common techniques can be used to concentrate and / or sterile filter the antibody. Soluble aggregates and polymers can be effectively removed by common techniques, including size exclusion, hydrophobic interaction, ion exchange or hydroxyapatite chromatography. The product can be frozen immediately, for example, at -70°C, or can be lyophilized.

[0026] The antibodies of the present invention can be used to treat patients. More specifically, it is expected that the antibodies of the present invention can be used to treat immune-mediated diseases or disorders, including AD, asthma, PsO, PsA, RA, AxSpA, IBD, colitis and PsA. "Treatment" as used interchangeably herein is intended to refer to all processes in which there may be a slowing, interruption, prevention, control, cessation or reversal of the progression of the disorder described herein, but does not necessarily mean that all disorder symptoms are completely eliminated. Treatment includes administering an antibody of the present invention or a pharmaceutical composition thereof to treat a disease or condition in a human being who will benefit from reduced IL-19 activity, and includes: (a) inhibiting further progression of the disease, i.e., preventing its development; or (b) alleviating the disease, i.e., causing regression of the disease or disorder, alleviating its symptoms or complications, or reducing the "suddenness" of disease manifestations.

[0027] As used interchangeably herein, the terms "patient," "subject," and "individual" refer to a human. In certain embodiments, the patient is further characterized by having a disease, disorder, or condition (e.g., an immune-mediated disease) that would benefit from reduced IL-19 activity. In other embodiments, the patient is further characterized by being at risk for an immune-mediated disease, disorder, or condition that would benefit from reduced IL-19 activity.

[0028] As used herein, unless otherwise indicated, the term "binding" refers to the ability of a protein or molecule to form a chemical bond or attractive interaction with another protein or molecule that brings the two proteins or molecules into proximity as determined by common methods known in the art.

[0029] As referred to herein, the term "epitope" refers to the amino acid residues in an antigen that are bound by an antibody. An epitope can be a linear epitope, a conformational epitope, or a hybrid epitope.

[0030] The term "epitope" may be used to refer to a structural epitope. According to some embodiments, a structural epitope may be used to describe the region of an antigen that is covered by an antibody (e.g., the footprint of an antibody when bound to an antigen). In some embodiments, a structural epitope may describe the amino acid residues of an antigen that are within a specified proximity (e.g., within a specified number of angstroms) of the amino acid residues of an antibody.

[0031] The term "epitope" may also be used to refer to a functional epitope. According to some embodiments, a functional epitope may be used to describe the amino acid residues of an antigen that interact with the amino acid residues of an antibody in a manner that contributes to the binding energy between the antigen and the antibody.

[0032] Epitopes can be determined according to different experimental techniques (also referred to as "epitope mapping techniques"). It should be understood that the determination of epitopes can vary based on the different epitope mapping techniques used, and can also vary with the different experimental conditions used, such as due to conformational changes or cleavage of antigens induced by specific experimental conditions. Epitope mapping techniques are known in the art (e.g., Rockberg and Nilvebrant, Epitope Mapping Protocols: Methods in Molecular Biology, Humana Press, 3rd edition, 2018), including but not limited to X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, site-directed mutagenesis, species exchange mutagenesis, alanine scanning mutagenesis, hydrogen-deuterium exchange (HDX) and cross-blocking assays.

[0033] The antibodies of the invention can be incorporated into pharmaceutical compositions that can be prepared by methods well known in the art and include the antibodies of the invention and one or more pharmaceutically acceptable carriers and / or diluents (e.g., Remington, The Science and Practice of Pharmacy, 22nd edition, Loyd V. ed., Pharmaceutical Press, 2012, which provides a compilation of formulation techniques generally known to practitioners). Suitable carriers for pharmaceutical compositions include any material that retains molecular activity when combined with the antibodies of the invention and does not react with the patient's immune system.

[0034] Pharmaceutical compositions comprising antibodies of the invention may be administered to patients at risk of or exhibiting a disease or disorder as described herein by parenteral routes (e.g., subcutaneously, intravenously, intraperitoneally, intramuscularly, or transdermally). The pharmaceutical compositions of the invention contain an "effective" or "therapeutically effective" amount (as used interchangeably herein) of an antibody of the invention. An effective amount refers to the amount (dosage and time period and mode of administration) necessary to achieve the desired therapeutic outcome. The effective amount of an antibody may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody to elicit a desired response in the individual. An effective amount is also an amount in which the therapeutically beneficial effects of the antibodies of the invention exceed any toxic or deleterious effects thereof.

[0035] As used in the present disclosure, the homology percentage in the context of two or more amino acid sequences refers to two or more sequences of the same amino acid residues of a specified percentage when comparing and aligning to obtain maximum correspondence, as measured using a sequence comparison algorithm (e.g., BLASTP and BLASTN or other algorithms available to the technician) or by visual inspection. Depending on the application, the homology percentage may be present in the region of the sequence compared, such as in a functional domain, or alternatively, in the total length of two sequences to be compared. For example, the homology percentage of a sequence can be compared with a reference sequence. For example, when using a sequence comparison algorithm, a test sequence and a reference sequence can be input into a computer (and if necessary, subsequence coordinates and sequence algorithm program parameters can also be further specified). Then, based on the specified program parameters, the sequence comparison algorithm calculates the sequence identity or homology percentage of the test sequence relative to the reference sequence. Exemplary sequence alignment and / or homology algorithms can be obtained by Smith and Waterman, Adv. Appl. Math. 2: 482 (1981), Needleman and Wunsch, J. Mol. Biol. 48: 443 (1970), Pearson and Lipman, Proc. Nat'l. Acad. Sci. USA 85: 2444 (1988), GAP, BESTFIT, FASTA and TFASTA (in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.) or by visual inspection (see generally Ausubel et al., below). An example of an algorithm suitable for determining percentages of sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215: 403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov / ). BRIEF DESCRIPTION OF THE DRAWINGS

[0036] The features and advantages of the present invention will become more apparent to those skilled in the art after considering the following detailed description taken in conjunction with the accompanying drawings.

[0037] Figure 1 Shown are changes in total clinical scores over time comparing vehicle-treated mice, anti-inflammatory Ab-treated mice, and naive mice in a psoriasis-like mouse model for genetic analysis.

[0038] Figure 2A and Figure 2B represents the total clinical score over time for a single study evaluating the efficacy of anti-murine IL-19 Ab4 in a psoriasis-like mouse model ( Figure 2A ) and the area under the curve ( Figure 2B ).

[0039] Figure 3 demonstrated that in an atopic dermatitis-like mouse model used for genetic analysis, ear thickness was increased in isotype-treated mice compared with naive or anti-inflammatory treated mice.

[0040] Figure 4 Shown are IL-19 protein levels in the ears during MC903-induced inflammation compared to the naive group.

[0041] Figure 5 demonstrated the efficacy of anti-murine IL-19 Ab3 in an atopic dermatitis-like mouse model.

[0042] Fig. 6A and Figure 6B Shown is a reduction in IL-4 and IL-13 secretion from the ear in anti-murine IL-19 Ab3-treated mice compared to isotype-treated mice.

[0043] Figure 7 Changes in ear thickness were compared between isotype controls of naive mice and post-challenge mice treated with anti-inflammatory agents in a mouse model of contact dermatitis.

[0044] Figure 8 Shown are IL-19 protein levels in mice following FITC challenge when compared to naive mice.

[0045] Fig. 9 and Fig.10 Shown is a comparison of two separate studies using anti-murine IL-19 Ab4 in a mouse model of contact dermatitis.

[0046] Fig.11 Shown is the relationship between IL-19 expression and changes in ear thickness in an atopic dermatitis-like mouse model of skin inflammation.

[0047] Fig.12 Shown is the relationship between IL-19 expression and the area under the curve of the total clinical score in a psoriasis-like mouse model of skin inflammation.

[0048] Fig.13 IL-19 expression in an atopic dermatitis-like mouse model of skin inflammation (disease peak vs. remission) is shown. Example

[0049] Expression of Exemplary Anti-IL-19 Antibodies

[0050] Table 1 shows exemplary anti-IL-19 antibodies of the present invention. The cDNA sequences encoding the heavy and light chains of the exemplary anti-IL-19 antibodies of the present disclosure can be cloned and engineered into GS (glutamine synthetase) expression vectors for recombinant expression in competent cell lines (such as CHO cells). The relationship between the various regions of the exemplary anti-IL-19 antibodies is as follows (the numbering of amino acids uses linear numbering; the assignment of amino acids to variable domains is based on the International Immunogenetics Information System (International Immunogenetics Information System) found at www.imgt.org). ); the assignment of amino acids to CDR domains is based on the well-known Northern numbering convention, with the exception of HCDR2, which has its C-terminus based on the well-known Kabat numbering convention).

[0051] Table 1: SEQ ID NOs of exemplary antibody amino acid sequences .

[0052]

[0053] The exemplified antibodies of the present disclosure are identified as having high binding affinity and being chemically and physically stable, including aggregation and solubility consistent with therapeutic parenteral administration. The exemplified antibodies of the present disclosure are also identified as having low immunogenicity (including low nonspecific and / or serum binding) and having pharmacokinetic properties consistent with therapeutic parenteral administration for the treatment of immune-mediated diseases.

[0054] Binding affinity

[0055] use The Meso Scale Discovery Solution Equilibrium Titration (MSD-SET) assay measured by Imager 6000 (Meso Scale Diagnostics) was used to evaluate the binding of the exemplary IL-19 antibodies of the present invention to biotinylated human IL-19 (having the amino acid sequence set forth in SEQ ID NO: 1).

[0056] Unless otherwise stated, all reagents and materials are from Meso Scale Diagnostics (Rockville, Maryland). The recombinant human IL-19 reagent was biotinylated using the EZ-link sulfo-NHS-biotin kit (Thermo catalog number A39257). In brief, 20 pM biotinylated human IL-19 was mixed with a 3-fold dilution series of the exemplified antibody samples of the present invention (mAb 1 and mAb 2) in a 1:1 ratio to obtain the following final concentrations: 10 pM biotinylated human IL-19 and an 11-point 3-fold antibody gradient from 1 nM to 0.017 pM. The mixture was incubated at 37°C for 72 hours. MSD 96-well plates (multi-array 96-well plates, catalog number L15XA-3) were coated overnight at 4°C with 20 nM exemplified antibodies in phosphate-buffered saline (PBS) per well at 30 μl. The plate was then washed three times with 150 μL of wash buffer per well (0.05% Tween-20 in PBS, VWR catalog number 9005-64-5) and blocked with 150 μL of blocking buffer per well (PBS + 3% Blocker A buffer, catalog number R93BA-1) for 45 minutes at 37°C. After three wash cycles, 50 μL of the IL-19 and antibody mixture per sample was transferred to each well and incubated with shaking (700 rpm) at 37°C for 150 seconds. After handling the samples and three wash cycles, 100 μL of detection antibody (1 μg / mL MSD sulfonate tag streptavidin antibody, catalog number R32AD1) was added to each well and incubated with shaking at 37°C for 3 minutes. After washing three times with wash buffer, 150 μL of 1X Read Buffer T (Catalog Number R92TC-1) was added to the well and incubated 5 minutes after adding the buffer. The samples were analyzed on Imager 6000 (Meso Scale Diagnostics). The exemplified antibodies were tested in duplicate in each individual experiment, and three independent experiments were performed.

[0057] The equilibrium K was determined by fitting a sigmoidal curve to the electrochemiluminescence (ECL) response versus log(IL-19 concentration) plotted with normalized ECL values ​​using the Assay Development Kit. D . K D Determined as a “curve fit” at 10 pM immobilized IL-19 ligand. The reported average K D The values ​​were calculated from three independent experiments, and the data variability is expressed as the standard deviation (SD) of three independent runs.

[0058] Table 2: MSD affinity data for recombinant human IL-19 .

[0059] Exemplary antibodies <![CDATA[Steady-state K D (pM)]]> SD(pM) N Ab 1 13.1 ±1.8 3 Ab_2 6.8 ±0.7 3

[0060] *K D Results are reported as equilibrium binding of 10 pM immobilized biotinylated human IL19.

[0061] Table 2 demonstrates that the exemplified anti-IL-19 antibodies mAb1 and mAb2 have low picomolar binding affinity for human IL-19 in vitro.

[0062] In vitro neutralization of IL-19

[0063] It is expected that the antibodies of the invention can neutralize IL- 19. Neutralization of IL-19 activity by the antibodies of the invention can be assessed by an IL-19 / IL-19 receptor (IL20Ra / IL20Rb) dimerization assay format, for example as described below.

[0064] Briefly, U20S cells expressing IL20Ra and IL20Rb (DiscoVerx, catalog number 93-1027C3) were cultured in assay complete cell plating medium (DiscoVerx, catalog number 93-0563R5A). Subconfluent cells were removed with assay complete cell detachment reagent (DiscoVerx, catalog number 92-0009), centrifuged, washed, resuspended in assay complete cell plating reagent, and plated at 2,500 cells per well in 100 μl per well on white well clear bottom 96-well plates. Cells were cultured for 4 hours and then treated with 10 μl per well of a 11x mixture of antibody and IL-19. The corresponding antibodies were diluted in DiscoveRx protein dilution buffer (DiscoveRx, catalog number 92-0023M) at a starting concentration of 10 μg / ml (1x) and mixed 1:3 with 22x human (SEQ ID NO.1) or cynomolgus monkey (SEQ ID NO.15) IL-19 (1x equals 100 ng / ml) diluted in DiscoveRx protein dilution buffer for dose response titration. The antibody and ligand mixtures were pre-incubated for 20 minutes; assay medium was used for "untreated" controls, and isotype control antibodies were used as negative controls. The cells were then incubated overnight at 37°C. Luminescence readouts were counted at 0.1 to 1 seconds / well (Perkin Elmer Victor3) using PathHunter Rapid Detection Kit X (DiscoveRx, catalog number 93-0247) according to the manufacturer's instructions. Statistical analysis was performed using GraphPad Prism version 9. The percentage of inhibition was calculated for each run in triplicate, and the average was combined. Data are reported as IC50 and 95% confidence interval (95% CI, symmetric) of sigmoidal curve fit (four parameters). Inhibition of human and cynomolgus monkey IL-19-induced receptor dimerization signaling by exemplified anti-IL-19 antibodies is provided in Table 3.

[0065] Table 3: IL-19 neutralization in vitro .

[0066]

[0067] In vitro neutralization of IL-19-induced phosphorylated STAT3 signaling

[0068] IL-19 in vitro neutralization can be further evaluated essentially as described below. A431 dermoid epithelial cells (ATCC #CRL-1555) express the IL-19 receptor (IL20Ra / IL20Rb). The interaction of IL-19 with the IL-19 receptor induces phosphorylation of STAT3 in A431 dermoid epithelial cells. Briefly, A431 cells were grown in growth medium [HyClone #sh30284.01), 10% HI FBS (Gibco #10082147) and 1X PenStrep (Gibco #15140-122)]. Cells were dissociated in 0.05% trypsin-PBS and plated at 30,000 cells / 100 μl medium in a 96-well plate (Falcon #353072) in serum-free medium (Opti-MEM (Gibco #31985-070), 0.5% BSA (Gibco #15260-037) and 1x PenStrep). The cells were then incubated overnight at 37°C. Ab 1 or Ab 2 or isotype controls were prepared at a final starting concentration of 20 nM and diluted 2-fold for 8-point titration. The antibodies were pre-incubated with human (SEQ ID NO.1) or cynomolgus monkey IL-19 (SEQID NO.15) (100 ng / ml) for 20 minutes. The cells were then treated and stimulated with the antibody:IL-19 mixture for 15 minutes. Thereafter, the culture medium was removed and the phosphorylation of STAT3 was determined using a phosphorylated STAT3 (Tyr705) kit (Meso Scale Diagnostics, LLC #K150SVD) according to the manufacturer's protocol (Note: cells can be frozen after the culture medium is removed and before phosphorylation is determined). Phosphorylated STAT3 activation was determined by chemiluminescence induction and displayed as relative luminescence units (RLU) using MesoScale Diagnostics SECTOR Imager. Statistical analysis was performed using GraphPad Prism version 9. The percent inhibition of the runs performed in triplicate was calculated and the averages were combined. Data are reported as IC50 and 95% confidence intervals (95% CI, symmetric) of sigmoid curve fits (four parameters).

[0069] Table 4 shows that the exemplary anti-IL-19 antibodies reduced the phosphorylated STAT3 signal induced by human and cynomolgus monkey IL-19.

[0070] Table 4: IL-19 neutralization in vitro .

[0071]

[0072] Tables 3 and 4 show that exemplary anti-IL-19 antibodies Ab 1 and Ab 2 neutralize human and cynomolgus monkey IL-19 in vitro.

[0073] In vivo target engagement

[0074] In vivo target engagement can be assessed essentially as described below. In brief, total plasma IL-19 concentration (free IL-19 and IL-19 bound to the exemplified IL-19 antibody) is determined essentially as described herein. Biotinylated IL19 antibody is coated on a streptavidin plate. The exemplified Ab 2 sample is diluted 1:4 in dilution buffer to a concentration of 10 μg / mL. A ruthenium-labeled anti-IgG4 antibody bound to Ab 2 is used as a detection antibody.

[0075] Four cynomolgus monkeys were evaluated for baseline plasma IL-19 levels, and the measurement results were below the detection limit (about 20pg / mL). Thereafter, two cynomolgus monkeys were administered Ab 2 by IV, and two cynomolgus monkeys were administered Ab 2 subcutaneously. Thereafter, total plasma IL-19 concentrations were measured. Total plasma IL-19 concentrations showed an increase over time, reaching a peak at 336 hours (1651.0pg / mL and 4467.5pg / mL for IV; 1382.0pg / mL and 2127.6pg / mL for SC). These results demonstrate that the exemplified IL-19 antibodies of the present disclosure engage IL-19 in vivo and maintain target engagement over time.

[0076] In vivo pharmacokinetics of exemplary antibodies

[0077] The pharmacokinetic parameters of the exemplary IL-19 antibodies of the present disclosure can be evaluated essentially as described herein. Briefly, a single IV or SC dose of 5 mg / kg Ab1 or Ab2 in PBS (pH 7.4) was administered to male Sprague Dawley rats at a volume of 1 mL / kg. Blood was collected from each animal and processed into serum at 1, 6, 12, 24, 48, 96, 120, 168, 240, 336, 504, and 672 hours after the IV dose or at 3, 6, 12, 24, 48, 96, 120, 168, 240, 336, 504, and 672 hours after the SC dose.

[0078] Serum concentrations of Ab1 and Ab2 were determined by plate-based total human IgG ELISA. Goat anti-human IgG F(ab')2 antibody was coated on ELISA plates at 1 mg / mL as capture reagent. After incubation with serum standards, controls or samples, plate-bound Ab1 or Ab2 was detected using mouse anti-human IgG4 pFc horseradish peroxidase (diluted 1:10,000). Pharmacokinetic parameters for each animal (N=3) were calculated using non-compartmental analysis (NCA), and the parameters were summarized by mean and standard deviation (SD) when appropriate. NCA and summary statistics were calculated using Phoenix WinNonlin 8.1 or Excel. As shown in Table 5, both Ab1 and Ab2 exhibited extended pharmacokinetic characteristics.

[0079] Table 5: Plasma pharmacokinetic parameters of exemplary IL-19 antibodies

[0080]

[0081]

[0082] *N=1; parameters are reported as mean or mean (SD) when appropriate; Abbreviations: C0=maximum concentration extrapolated to time 0 hours after IV bolus dose, C max = maximum serum concentration after SC dose, T max =C max time, AUC 0-inf = area under the serum concentration-time curve from time 0 to infinity, CL = clearance after IV administration, CL / F = apparent CL after SC administration, T 1 / 2 = half-life, %F = bioavailability after SC administration, calculated as (mean AUC 0-inf .SC / Average AUC 0- inf .IV)*100%.

[0083] Immunogenicity analysis

[0084] Dendritic cell (DC) internalization

[0085] Dendritic cell (DC) internalization assay assesses the internalization of CD14+ monocyte-derived dendritic cells to molecules. In brief, CD14+ monocytes are separated from peripheral blood mononuclear cells (PBMC), and are cultured and differentiated into DC according to standard protocols. Using Ficoll (#17-1440-02, GE Healthcare) and Sepmate 50 (#15450, STEMCELL Technologies), PBMC is separated from LRS-WBC by density gradient centrifugation. CD14+ microbead kit (#130-050-201, Miltenyi Biotec) is used to separate CD14+ monocytes by positive selection according to the manufacturer's manual. Then in RPMI medium (complete RPMI medium or medium purchased from Life Technologies) containing L-glutamine and 25mM HEPES supplemented with 10% FBS, 1mM sodium pyruvate, 1x penicillin-streptomycin, 1x non-essential amino acids and 55μM 2-mercaptoethanol, the cells were cultured at 1 million / mL with 1000 units / mL GM-CSF and 600 units / mL IL-4 for 6 days to drive into immature dendritic cells (MDDC). The medium was changed on the 2nd and 5th days. On the 6th day, the cells were gently collected with a cell scraper and used for experiments. In order to obtain mature DC, cells were treated with 1μg / mL LPS for 4 hours.

[0086] The individual test molecules were normalized to 1 mg / mL with PBS and then further diluted to 8 μg / mL in complete RPMI medium. The detection probe Fab-TAMRA-QSY7 was diluted to 5.33 μg / mL in complete RPMI medium. The antibody and Fab-TAMRA-QSY7 were mixed in equal volumes and incubated in the dark at 4°C for 30 minutes to form a complex. MDDCs were resuspended in complete RPMI medium at 4 million / mL and seeded in 96-well round-bottom plates at 50 μL per well, to which 50 μL of antibody / probe complex was added. The cells were incubated at 37°C in a CO2 incubator for 24 hours. The cells were washed with 2% FBS PBS and resuspended in 100 μL 2% FBS PBS containing Cytox Green live / dead dye. Data were collected on a BD LSR Forttessa X-20 and analyzed in FlowJo. Live single cells were gated and the percentage of TAMRA fluorescence-positive cells was recorded as a readout. To allow comparison of molecules with data generated from different donors, a normalized internalization index was used. The internalization signal was normalized to the IgG1 isotype (normalized internalization index = 0) and the internal positive control PC (normalized internalization index = 100) using the following formula:

[0087]

[0088] Where X TAMRA , IgG1 isotype TAMRA and PC TAMRA are the percentages of the TAMRA-positive population for test molecule X, IgG1 isotype and PC, respectively.

[0089] Table 6: DC internalization assessment of exemplary IL-19 antibodies

[0090] Exemplary antibodies Internalization Index Ab1 9.5 Ab2 10.3

[0091] For the normalized internalization index, 0 to 15 was considered low, >15 to 30 was considered low to moderate, >30 to 60 was considered moderate, and >60 was considered high immunogenicity risk.

[0092] Table 6 demonstrates that the exemplified anti-IL-19 antibodies Abl and Ab2 have a low risk of DC internalization.

[0093] MHC-associated peptide proteomics

[0094] MHC-associated peptide proteomics (MAPP) profiles peptides presented by human leukocyte antigen class II (HLA-II) on human dendritic cells that were previously treated with test molecules to assess immunogenicity. Briefly, primary human dendritic cells from a panel of 10 normal human donors were prepared from buffy coats by isolating CD-14-positive cells and differentiated into immature dendritic cells by incubation for 3 days at 37°C and 5% CO2 in complete RPMI medium (Thermo Fisher Scientific, catalog number A2596101) containing 5% serum replacement with 20ng / ml IL-4 and 40ng / ml GM-CSF as described (Knierman et al., 2020). Approximately 5×10 6 3 micromolar concentrations of test antibodies were added to each cell, and fresh medium containing 5 μg / ml LPS was replaced after 5 hours of incubation to convert the cells into mature dendritic cells. The next day, the mature cells were lysed in 1 mL RIPA buffer containing protease inhibitors and DNase. The lysate was stored at -80°C until sample analysis.

[0095] HLA-II molecules were separated from thawed lysates using a biotinylated anti-pan-HLA class II antibody (clone Tu39) using an automated liquid handling system. The bound receptor-peptide complex was eluted with 5% acetic acid, 0.1% TFA. The eluted HLA-II peptides were passed through a pre-washed 10k MWCO filter to remove high molecular weight proteins. The separated HLA-II peptides were analyzed by nano LC / MS using a Thermo easy1200 nLC-HPLC system equipped with a Thermo LUMOS mass spectrometer. A 65-minute gradient separation was performed using a 75μm×7cm YMC-ODS C18 column with a flow rate of 250nL / min, 0.1% formic acid in water as the A solvent, and 80% acetonitrile containing 0.1% formic acid as the B solvent. Mass spectrometry was run in full scan mode at 240,000 resolution, followed by a 3-second data-dependent MS / MS cycle consisting of an ion trap rapid scan with HCD and EThcD fragmentation.

[0096] The identification of peptides was generated by an internal proteomics pipeline (Higgs et al., 2008) using multiple search algorithms (without enzyme search parameters) against a bovine / human database containing the test molecule sequences. Peptides identified from the test molecules were compared with the parental sequence. A summary was created for all test molecules, which annotated the donor percentage of peptides containing non-germline residues and the number of different regions in the test molecule that displayed peptides containing non-germline residues. An increase in the degree of display of non-germline peptides was associated with an increased risk of immunogenicity.

[0097] Table 7: MAPP results of exemplary IL-19 antibodies

[0098]

[0099] Table 7 demonstrates that the exemplary anti-IL-19 antibodies Abl and Ab2 have a low-risk MAPP signature.

[0100] T cell proliferation assay

[0101] T cell proliferation assay evaluates the ability of the exemplified antibodies to activate CD4+ T cells by inducing cell proliferation. Briefly, cryopreserved PBMCs from 10 healthy donors were used, CD8+ T cells were depleted from PBMCs, and labeled with 1 μM carboxyfluorescein diacetate succinimidyl ester (CFSE). PBMCs were incubated at 4×10 6 10 cells / ml / well were seeded in 5% CTS TMImmune cell SR (Gibco, catalog number A2596101) in AIM-V medium (Life Technologies, catalog number 12055-083) and tested in triplicate in 2.0 mL containing different exemplified antibodies, DMSO control, medium control, keyhole limpet hemocyanin (KLH; positive control). Cells were cultured and incubated for 7 days at 37°C and 5% CO2. On day 7, samples were stained with the following cell surface markers: anti-CD3, anti-CD4, anti-CD14, anti-CD19 and DAPI to use a BD LSRFortessa equipped with a high throughput sampler (HTS) TM Viability was measured by flow cytometry. Software (FlowJo, LLC, TreeStar) analyzes data and calculates cell division index (CDI). The CDI of every kind of exemplified antibody is calculated by dividing the percentage of proliferation CFSEdimCD4+T cells from antibody stimulation wells by the percentage of proliferation CFSEdimCD4+T cells in unstimulated wells. CDI of ≥2.5 is considered as representing positive response. The donor frequency percentage of all donors is assessed.

[0102] Table 8: T cell proliferation results of exemplary IL-19 antibodies

[0103]

[0104] Proliferation of <30% donors is considered low; 30% to 40% is considered moderate; and >40% is considered high immunogenicity risk.

[0105] Table 8 demonstrates that the exemplified anti-IL-19 antibodies Abl and Ab2 have low risk T cell proliferation immunogenicity characteristics.

[0106] Pre-existing reactivity assay

[0107] The pre-existing reactivity assay evaluates the presence of reactivity derived from pre-existing anti-drug antibodies (PEA) and potentially other cross-reactive proteins in untreated normal human serum. In short, diluted sera from a group of at least 50 untreated donors are captured overnight on a plate coated with biotinylated exemplified antibodies. The next day, the captured reactive proteins are eluted with acid and then neutralized in the presence of biotinylated and ruthenated exemplified antibodies. If anti-drug antibodies are present, a complex of the exemplified antibodies will be formed. The complex is captured by a streptavidin-coated Mesoscale plate, and the resulting signal is referred to as the first layer signal (expressed as electrochemiluminescence). The first layer signal is confirmed in the second layer by adding an excess of unlabeled exemplified antibodies in the detection step, which results in inhibition of the first layer signal. The presence of pre-existing anti-drug antibodies is expressed as the 90th percentile of the second layer inhibition.

[0108] Table 9: Pre-existing reactivity results for exemplary IL-19 antibodies

[0109] Exemplary antibodies 90th percentile of Tier 2 suppression Ab1 20.3 Ab2 26.1

[0110] Results <30% are low, 30% to 55% are moderate, and >55% are high immunogenicity risk.

[0111] Table 9 demonstrates that the exemplified anti-IL-19 antibodies Abl and Ab2 have a low risk of PEA reactivity.

[0112] Illustrated physical and chemical properties of antibodies

[0113] Exemplified antibodies Abl and Ab2 exhibit solubility, low aggregation, chemical stability, and physical stability characteristics necessary for parental therapeutic administration.

[0114] Solubility :

[0115] Sufficiently high solubility is required to enable convenient administration. For example, a 1 mg / kg dose administered to a 100 kg patient by 1.0 mL injection will require a solubility of 100 mg / ml. In addition, it is also desirable to maintain the monomer state of the antibody at high concentrations without high molecular weight (HMW) aggregation. The solubility of the exemplified antibody was analyzed by concentrating 15 mg of the exemplified antibody to a volume less than 100 μl using a 10 K molecular weight cutoff filter (Amicon UC filter, Millipore, catalog number UFC903024). The ultimate concentration of the sample was measured by the UV absorbance at A280 using Nanodrop 2000 (Thermo Scientific). According to the procedure substantially as described above, the exemplified antibody showed a solubility greater than 200 mg / ml (pH 7.4 in PBS buffer). In addition, only low levels of HMW (from about 3.8% to about 4.35%) were present at high concentrations, and phase separation was not observed.

[0116] Chemical and physical stability :

[0117] Chemical stability is beneficial for developing pharmaceutical formulations with sufficient shelf life. The chemical stability of the exemplified antibodies was evaluated by preparing the exemplified antibodies at a concentration of 100 mg / ml in a buffer solution of pH 6. In an accelerated degradation study, the prepared samples were incubated for four weeks at 4° C. and 35° C. CE-SDS and aSEC were used to evaluate antibody changes reflecting chemical changes according to standard procedures. Following the procedure essentially as described above, the exemplified antibodies exhibited the chemical stability results presented in Table 10.

[0118] Table 10. % change in the main peak and flux measured by CE-SDS over four weeks relative to samples incubated at 4°C. Summary of % change in HMW aggregates measured by aSEC .

[0119]

[0120] The results provided in Table 10 demonstrate that the exemplified antibodies had a percentage decrease in the main peak of about 0.6% to 1.9% after storage for 4 weeks at 35° C. In addition, mass spectrometry analysis demonstrated that only minimal degradation (about 0.2% CDR PTM changes in all CDR sequences) was observed after storage for 4 weeks at 35° C., indicating that the exemplified antibodies have sufficient chemical stability to facilitate the development of solution formulations with sufficient shelf life.

[0121] IL-19 expression and activity in a psoriasis-like mouse model

[0122] Eight-week-old female BALB / c mice (Envigo, Inc., Indianapolis, IN) were allowed free access to food and water. 30 mg of 3.75% imiquimod (IMQ) cream (Zyclara) (Bausch Health Companies Inc., Laval, Quebec, Canada) was applied daily to the shaved back of the mice (2×2 cm area). Mice were randomized into treatment groups based on body weight, with 6 mice in each group. Mice were subcutaneously administered isotype control / vehicle, anti-inflammatory positive control antibody, or anti-murine IL-19 Ab ("mAb4", comprising the HCVR of SEQ ID NO. 36 and the LCVR of SEQ ID NO. 31) one day before the application of IMQ. Clinical scores of 0 to 4 were given based on absence, mild, moderate, marked, or severe changes in erythema, thickness, and scaling, with 12 as the maximum total score. All mice were sacrificed on day 8.

[0123] Figure 1 Shown is the change in total clinical score over time comparing vehicle-treated mice, anti-inflammatory Ab-treated mice, and naive mice. This study was used for further genetic analysis to evaluate IL-19 mRNA levels in this model. Figure 2A and Figure 2B represents the total clinical score over time for a single study evaluating the efficacy of mAb4 in this model ( Figure 2A ) and the area under the curve ( Figure 2B ). The 10 mg / kg dose of mAb4 improved the total clinical score by 25% and reduced the last day clinical score by 46% when compared to the isotype control. However, mice in the high treatment group at 20 mg / kg did not improve in the total clinical score when compared to the isotype control.

[0124] This study demonstrates that anti-IL-19 antibodies have the potential to treat psoriasis-like skin diseases. In this regard, the results demonstrate that the antibodies of the present invention reduce inflammation in a murine inflammation model.

[0125] IL-19 expression and activity in an atopic dermatitis-like mouse model

[0126] Balb / cJ aged 6 to 8 weeks were anesthetized by inhalation of isoflurane (5%), and the thickness of both ears was measured using a digital caliper and recorded as baseline ear measurements. On day 1, 10 μL of MC903 (Tocris Bioscience) was applied to the dorsal and ventral sides of each ear (40 μL in total) with a pipette. After administration of MC903, the mice were kept under anesthesia until the treatment area was dry. Ear measurements were recorded twice or three times a week. The thickness of the left and right ears (inflammation) of each mouse was averaged every day, and the thickness changes between measurements were calculated and recorded. The standard protocol was stimulated four times in total on days 1, 4, 6 and 8. Mice were given 10 mg / kg of isotype control Ab, anti-inflammatory agents as positive controls, or anti-mouse IL-19 Ab ("mAb 3", comprising HCVR of SEQ ID NO.24 and LCVR of SEQ ID NO.19) on days 0 and 7. The ears were prepared for culture by manually separating the dermis with forceps and placing in RPMI medium. IL-19 levels in ear cultures were measured by an ELISA developed in-house using mAb3 and mAb4, which do not compete for binding with each other. IL-4 and IL-13 levels from ear cultures were assessed using a custom U-Plex biomarker MSD panel 1 according to the manufacturer's protocol (MesoScale Discovery). Data analysis and statistical significance analysis were performed using GraphPad Prism version 9.

[0127] Figure 3 The results compared the isotype control with normal unchallenged and untreated mice until the peak of inflammation on day 14. The isotype control had no effect on the inflammatory response measured by ear thickness with a precision electronic caliper. The untreated and unchallenged controls showed no inflammatory response. Treatment with a known anti-inflammatory agent at 10 mg / kg per week for two weeks (days 0 and 7) significantly reduced the inflammatory effects of MC903 stimulation and persisted until day 21 ( Figure 3 ). These studies were used for further genetic analysis to evaluate IL-19 mRNA levels in this model. Figure 4 It was demonstrated that IL-19 protein levels in the ears were significantly increased during MC903-induced inflammation compared to the naive group, confirming the elevation of IL-19 in this skin inflammation model.

[0128] Figure 5The efficacy of "mAb 3" in this model was confirmed, which contained the HCVR of SEQ ID NO. 24 and the LCVR of SEQ ID NO. 19. mAb 3 significantly reduced ear thickness at days 11 and 13 compared to mice treated with isotype controls. In addition, IL-4 and IL-13 secretion in the ears of isotype treated mice was elevated compared to initial untreated mice ( Fig. 6A and Figure 6B ). mAb3 reduced the secretion of IL-4 and IL-13 in ear culture compared to the isotype group. This confirms that mAb3 not only reduces ear inflammation but also reduces the production of Th2 cytokines in this AD-like mouse inflammation model. The results confirm that the antibodies of the present invention reduce inflammation in the mouse inflammation model.

[0129] IL-19 expression and activity in a mouse model of contact dermatitis

[0130] 6 to 8 week old C57BL / 6J were anesthetized by inhalation of isoflurane (5%), and the thickness of both ears was measured using a digital caliper and recorded as baseline ear measurements. 24 hours after challenge, or on day 7, the ears were measured again. On day 0, the ventral sides of all mice were shaved under inhalation of 5% isoflurane anesthesia. 100 μl of fluorescein isothiocyanate isomer I (FITC; Sigma) in DBP: acetone was applied to the shaved area. The mice were kept under anesthesia until the solution had dried. On day 1, the administration and application procedures of FITC in DBP: acetone were repeated. On day 5, mice were treated with isotype control Ab or anti-mouse antibody mAb4 (comprising HCVR of SEQ ID NO.36 and LCVR of SEQ ID NO.31). On day 6, after baseline measurements were taken, mice were challenged with 10 μL of FITC in DBP: acetone and applied to both sides of both ears, a total of 40 μL per mouse. Ears were prepared for culture by manually separating the dermis with forceps and placing in RPMI medium. IL-19 levels in ear cultures were measured by an ELISA developed in-house using mAb3 and another anti-murine IL-19 Ab that do not compete for binding with each other. Data analysis and statistical significance were performed using GraphPad Prism version 9

[0131] Figure 7 The results of the isotype control were compared with naive mice and challenged mice treated with anti-inflammatory agents until the peak of inflammation on day 7. The isotype control had no effect on the inflammatory response measured by ear thickness with a precision electronic caliper. The naive unchallenged control showed no inflammatory response. Treatment with anti-inflammatory agents at a dose of 10 mg / kg for 24 hours before challenge significantly reduced the inflammatory effects of FITC challenge. In addition, Figure 8It was demonstrated that IL-19 protein levels were elevated in FITC-challenged mice when compared to naive mice, confirming the elevation of IL-19 in this model of skin inflammation.

[0132] Fig. 9 and Fig.10 are the results of two separate studies comparing the use of mAb4 in this model. Fig. 9 In the replicate study ( Fig.10 ), mAb4 significantly reduced ear thickness at high doses (20% inhibition) compared to the isotype control group, but did not significantly reduce ear thickness at medium doses (10 mg / kg) and low doses (3 mg / kg). In summary, this demonstrates that mAb4 at high doses (30 mg / kg) reduced inflammation induced in this contact dermatitis model. The results demonstrate that the antibodies of the present invention reduce inflammation in a murine inflammation model.

[0133] IL-19 gene expression in an in vivo model of skin inflammation

[0134] To assess changes in IL19 gene expression in these mouse models of skin inflammation, tissue RNA levels were evaluated using qPCR and NanoString analysis. RNA was isolated from selected tissues using Qiagen RNeasy (Cat. No. 74181) and performed using a centrifugation protocol according to the manufacturer's recommended animal tissue protocol.

[0135] Gene expression levels were determined by two-step TaqMan RT-PCR using a customized 96 marker gene expression panel and Viia-7 platform (Applied Biosystems). First, RNA previously purified from mouse tissue was reverse transcribed into cDNA using the Qiagen QuantiTect reverse transcription kit according to the method recommended by the manufacturer. The resulting cDNA was diluted 1:10 for downstream applications. RT-PCR was performed according to the method recommended by the manufacturer for Applied Biosystems TaqMan custom plates. Gene expression levels were determined by NanoString using the "Mouse Cancer Immune Profiling Panel" according to the manufacturer's recommended protocol. Previously purified RNA was diluted to 20 ng / ul in RNase-free water, and a total of 100 ng (in 5ul) was used to prepare the NanoString column. According to the data processing method recommended by the manufacturer, nSolver advanced analysis software (version 2.0.115) was used to normalize the original mRNA counts. All mRNA counts were first scaled to within-sample binding density controls, and experimental genes were further normalized to the housekeeping gene index following a dynamic housekeeping gene selection across the entire dataset to minimize housekeeping gene differences. Normalized gene counts were then compared.

[0136] IL19 gene expression was assessed by qRT-PCR or NanoString and was found to be elevated in mouse models of skin inflammation representative of atopic dermatitis and psoriasis ( Figure 1 and Figure 3 Increased IL19 gene expression correlated strongly with measures of disease pathology in tissues, such as increased ear thickness in an atopic dermatitis-like model ( Fig.11 ) and total clinical score of psoriasis ( Fig.12 In addition, treatment significantly reduced IL19 gene expression, which was comparable to the histopathological changes observed in the same tissues ( Figure 1 and Figure 3 ). In the atopic dermatitis-like model, IL19 mRNA was the only transcript that was most elevated as observed by differential expression analysis of the genes evaluated in this study ( Fig.13 ). Taken together, these findings suggest that IL19 gene expression is strongly associated with tissue inflammation in skin diseases.

[0137] Preparation of anti-murine IL-19 antibodies (mAb 3 and mAb 4)

[0138] Rabbits were immunized with mouse IL-19. Splenocytes were obtained and a library derived from splenocytes was constructed by amplifying the heavy chain variable region (VH) and light chain variable region (VL) genes and combining them into single-chain Fabs to express using yeast cell surface display. mAb 3 and mAb 4 were obtained after panning the library with mouse IL-19. The cloned sequences were determined and used to construct expression plasmids for recombinant IgG expression.

[0139] mAb3 and mAb4 were expressed as recombinant rabbit IgG after co-transfection into CHO cells and purified using MabSelect (Protein A) resin.

[0140] Sequence Listing

[0141] SEQ ID NO: 1 (human IL-19)

[0142]

[0143] SEQ ID NO: 2 (LCDR1 of exemplified Abl and Ab2)

[0144] RASQDIRSDFG

[0145] SEQ ID NO: 3 (LCDR2 of exemplary Ab 1 and Ab 2)

[0146] YAASLS

[0147] SEQ ID NO: 4 (LCDR3 of exemplary Ab 1 and Ab 2)

[0148] LQDYNYPWT

[0149] SEQ ID NO: 5 (Exemplary LCVR of Ab1 and Ab2)

[0150]

[0151] SEQ ID NO: 6 (LC of exemplary Ab 1 and Ab 2)

[0152]

[0153] SEQ ID NO:7 (HCDR1 of exemplary Ab 1 and Ab 2)

[0154] KASGYTFTGYYLH

[0155] SEQ ID NO: 8 (HCDR2 of exemplary Ab 1 and Ab 2) WINPNSGGTNYAQKFQG

[0156] SEQ ID NO:9 (Exemplary HCDR3 of Ab 1)

[0157] ARDIVVLPPAIGFDY

[0158] SEQ ID NO: 10 (Exemplary Ab ≥ HCDR3)

[0159] ARDIVVLPPAIGFDL

[0160] SEQ ID NO: 11 (Exemplary HCVR of Ab 1)

[0161]

[0162] SEQ ID NO:12 (Exemplary Ab 1 HC)

[0163]

[0164] SEQ ID NO:13 (Exemplary Ab2 HCVR)

[0165]

[0166] SEQ ID NO:14 (Exemplary Ab 2 HC)

[0167]

[0168] SEQ ID NO:15(Food crab IL-19)

[0169]

[0170] SEQ ID NO:16 (Exemplary Ab 3 LCDR1)

[0171] QASESVYNKNWLS

[0172] SEQ ID NO:17 (Exemplary Ab 3 LCDR2)

[0173] DSSDLAS

[0174] SEQ ID NO:18 (Exemplary Ab 3 LCDR3)

[0175] GGSYTDTYV

[0176] SEQ ID NO:19 (Exemplary Ab 3 LCVR)

[0177]

[0178] SEQ ID NO:20 (Exemplary Ab 3 LC)

[0179]

[0180] SEQ ID NO:21 (Exemplary Ab 3 HCDR1)

[0181] GFDLSSTYYMS

[0182] SEQ ID NO:22 (Exemplary Ab3 HCDR2)

[0183] SIVTSGRSGASYYANWAKG

[0184] SEQ ID NO:23 (Exemplary Ab3 HCDR3)

[0185] DLPAPTDDRGL

[0186] SEQ ID NO:24 (Exemplary Ab 3 HCVR)

[0187]

[0188] SEQ ID NO:25 (Exemplary Ab 3 HC)

[0189]

[0190]

[0191] SEQ I D NO:26 (exemplary Ab 3 LC)

[0192]

[0193] SEQ ID NO:27 (Exemplary Ab 3 HC)

[0194]

[0195]

[0196] SEQ ID NO:28 (Exemplary Ab 4 LCDR1)

[0197] MAINTENANCE

[0198] SEQ ID NO:29 (Exemplary Ab 4 LCDR2)

[0199] GASVLTD

[0200] SEQ ID NO:30 (Exemplary Ab 4 LCDR3)

[0201] QSYNDGSSSGDAHV

[0202] SEQ ID NO:31 (Exemplary Ab4 LCVR)

[0203]

[0204] SEQ ID NO:32 (Exemplary Ab 4 LC)

[0205]

[0206] SEQ ID NO:33 (Exemplary Ab 4 HCDR1)

[0207] GFPLSSYGFS SEQ ID NO:34 (Exemplary Ab4 HCDR2)

[0208] YLDPVFGSTLSAHTVNG

[0209] SEQ ID NO:35 (Exemplary Ab 4 HCDR3)

[0210] GIGYVYYGYTYDL

[0211] SEQ ID NO:36 (Exemplary Ab4 HCVR)

[0212]

[0213] SEQ ID NO:37 (Exemplary Ab4 HC)

[0214]

[0215] SEQ ID NO:38 (Exemplary Ab 4 LC)

[0216]

[0217] SEQ ID NO:39 (Exemplary Ab 4 HC)

[0218]

[0219]

Claims

1. An antibody that binds to human IL-19, comprising a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein the HCVR comprises complementarity determining regions (CDRs) HCDR1, HCDR2 and HCDR3, and the LCVR comprises CDRs LCDR1, LCDR2 and LCDR3, wherein the amino acid sequence of HCDR1 is SEQ ID NO.7, the amino acid sequence of HCDR2 is SEQ ID NO.8, the amino acid sequence of HCDR3 is SEQ ID NO.9 or SEQ ID NO.10, the amino acid sequence of LCDR1 is SEQ ID NO.2, the amino acid sequence of LCDR2 is SEQ ID NO.3, and the amino acid sequence of LCDR3 is SEQ ID NO.

4.

2. The antibody according to claim 1, wherein HCDR3 is SEQ ID NO.

9.

3. The antibody according to claim 2, wherein the amino acid sequence of the HCVR is SEQ ID NO. 11 and the amino acid sequence of the LCVR is SEQ ID NO.

5. The antibody according to claim 1 , wherein HCDR3 is SEQ ID NO.

10.

5. The antibody of claim 4, wherein the amino acid sequence of the HCVR is SEQ ID NO. 13 and the amino acid sequence of the LCVR is SEQ ID NO.

5.

6. An antibody comprising a heavy chain (HC) and a light chain (LC), wherein the amino acid sequence of the HC is SEQ ID NO. 12 or 14 and the amino acid sequence of the LC is SEQ ID NO.

6.

7. The antibody according to claim 6, wherein the amino acid sequence of the HC is SEQ ID NO.

12. The antibody according to claim 6 , wherein the amino acid sequence of the HC is SEQ ID NO.

14.

9. A pharmaceutical composition comprising the antibody according to any one of claims 1 to 8 and one or more pharmaceutically acceptable carriers, diluents or excipients.

10. A method for treating AD, asthma, PsO, PsA, RA, AxSpA, IBD, colitis or PsA, comprising administering an effective amount of the antibody according to any one of claims 1 to 8 or the pharmaceutical composition according to claim 9 to a patient in need thereof.

11. The antibody according to any one of claims 1 to 8 for use in therapy.

12. The antibody according to any one of claims 1 to 8 for use in the treatment of AD, asthma, PsO, PsA, RA, AxSpA, IBD, colitis or PsA.

13. The antibody according to any one of claims 1 to 8, for use in the manufacture of a medicament for the treatment of AD, asthma, PsO, PsA, RA, AxSpA, IBD, colitis or PsA.

14. A nucleic acid comprising a sequence encoding SEQ ID NO: 6, 12 or 14.

15. A vector comprising a first nucleic acid sequence encoding SEQ ID NO: 12 or 14 and a second nucleic acid sequence encoding SEQ ID NO:

6.

16. A cell comprising the vector according to claim 15.

17. A composition comprising a first vector and a second vector, the first vector comprising a nucleic acid sequence encoding SEQ ID NO: 12 or 14 and the second vector comprising a nucleic acid sequence encoding SEQ ID NO:

6.

18. A cell comprising the first vector and the second vector according to claim 17.

19. A method of producing an antibody, comprising culturing the cell according to claim 16 or claim 18 under conditions such that the antibody is expressed and recovering the expressed antibody from the culture medium.

20. An antibody produced by culturing the cell according to claim 16 or claim 18 under conditions such that the antibody is expressed and recovering the expressed antibody from the culture medium.

21. An antibody that binds to human IL-19, comprising a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein the HCVR comprises complementarity determining regions (CDRs) HCDR1, HCDR2, and HCDR3, and the LCVR comprises CDRs LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of HCDR1 is SEQ ID NO.21, the amino acid sequence of HCDR2 is SEQ ID NO.22, the amino acid sequence of HCDR3 is SEQ ID NO.23, the amino acid sequence of LCDR1 is SEQ ID NO.16, the amino acid sequence of LCDR2 is SEQ ID NO.17, and the amino acid sequence of LCDR3 is SEQ ID NO.

18.

22. The antibody of claim 21, wherein the amino acid sequence of the HCVR is SEQ ID NO. 24 and the amino acid sequence of the LCVR is SEQ ID NO.

19.

23. An antibody comprising a heavy chain (HC) and a light chain (LC), wherein the amino acid sequence of the HC is SEQ ID NO. 25 and the amino acid sequence of the LC is SEQ ID NO.

20.

24. An antibody that binds to human IL-19, comprising a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein the HCVR comprises complementarity determining regions (CDRs) HCDR1, HCDR2, and HCDR3, and the LCVR comprises CDRs LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of HCDR1 is SEQ ID NO.33, the amino acid sequence of HCDR2 is SEQ ID NO.34, the amino acid sequence of HCDR3 is SEQ ID NO.35, the amino acid sequence of LCDR1 is SEQ ID NO.28, the amino acid sequence of LCDR2 is SEQ ID NO.29, and the amino acid sequence of LCDR3 is SEQ ID NO.

30.

25. The antibody of claim 24, wherein the amino acid sequence of the HCVR is SEQ ID NO. 36 and the amino acid sequence of the LCVR is SEQ ID NO.

31.

26. An antibody comprising a heavy chain (HC) and a light chain (LC), wherein the amino acid sequence of the HC is SEQ ID NO. 37 and the amino acid sequence of the LC is SEQ ID NO.

32.

27. The antibody of any one of claims 21 to 26, wherein the antibody is an anti-murine IL-19 antibody.

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