Compounds and methods targeting interleukin-19

Abstract

The present invention provides compounds and methods targeting human interleukin-19, including therapeutic antibodies, pharmaceutical compositions and diagnostic applications useful in the field of immune-mediated diseases including psoriasis, atopic dermatitis, psoriatic arthritis, bronchial asthma and diabetic nephropathy.

Claims

1. A method of detecting IL-19 in a patient sample comprising the steps of: contacting the patient sample with a first antibody; and detecting a binding of the first antibody to IL-19 in the patient sample, wherein, the first antibody comprises a light chain variable region (LCVR) and a heavy chain variable region (HCVR), wherein the LCVR comprises complementarity determining regions (CDRs) LCDR1 set forth in SEQ ID NO. 3, LCDR2 set forth in SEQ ID NO. 4, and LCDR3 set forth in SEQ ID NO. 5 and the HCVR comprises CDRs HCDR1 set forth in SEQ ID NO. 7, HCDR2 set forth in SEQ ID NO. 8 and HCDR3 set forth in SEQ ID NO. 9.

2. The method of claim 1 further comprising the step of: contacting the patient sample with a second antibody, wherein the second antibody comprises a light chain variable region (LCVR) and a heavy chain variable region (HCVR), wherein the LCVR comprises complementarity determining regions (CDRs) LCDR1 set forth in SEQ ID NO. 11, LCDR2 set forth in SEQ ID NO. 12, and LCDR3 set forth in SEQ ID NO. 13 and the HCVR comprises CDRs HCDR1 set forth in SEQ ID NO. 15, HCDR2 set forth in SEQ ID NO. 16 and HCDR3 set forth in SEQ ID NO. 17.

3. The method of claim 1, wherein the patient sample is plasma.

4. The method of claim 1, wherein the first antibody comprises an IgG1 heavy chain.

5. The method of claim 4, wherein the first antibody further comprises a kappa light chain.

6. The method of claim 2, wherein the second antibody comprises an IgG1 heavy chain.

7. The method of claim 6, wherein the second antibody further comprises a kappa light chain.

8. The method of claim 2, wherein one of the first antibody and the second antibody comprises a detectable label.

9. The method of claim 1, wherein the patient sample is blood.

10. The method of claim 1, wherein the patient sample is serum.

Description

EXAMPLES

(1) Expression of IL-19 Antibodies

(2) Murine-derived IL-19 antibodies of the present invention are generated employing hybridoma methodology (e.g., as as first described by Kohler et al, Nature, 256:495 (1975)). Briefly, the mouse is immunized with recombinant human IL-19 and lymphocytes capable of producing antibodies that hind human IL-19 are isolated and fused with a myeloma cell line using a suitable fusing agent for forming a hybridoma cell (Coding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Hybridomas are seeded and grown in a suitable culture medium (preferably containing one or more substances inhibiting survival of unfused myeloma cells). Binding specificity of monoclonal antibodies produced by hybridomas is then determined by by an in vitro binding assay (e.g., immunoprecipitation, radioimmunoassay (RIA), or enzyme-linked immunosorbent assay (ELISA)). Preferred hybridomas may be subcloned by limiting dilution procedures and grown by standard methods including in vivo as ascites tumors in an animal (Goding, Monoclonal Antibodies: principles and Practice, pp. 59-103 (Academic Press, 1986)). Monoclonal antibodies secreted by the hybridomas (and or subclones) are purified according to conventional procedures such as, for example, affinity chromatography (e.g., protein A or protein G-Sepharose) or ion-exchange chromatography, hydroxylapatite chromatography, gel electrophoresis, or the like. Affinity maturation of antibodies may be performed according to methods known in the field.

(3) cDNA encoding antibodies of the present invention is sequenced using conventional procedures. cDNA sequences encoding the heavy and light chains may be cloned and engineered into a GS (glutamine synthetase) expression vector. The engineered immunoglobulin expression vector may then be stably transfected into CHO cells. As one of skill in the art will appreciate, mammalian expression of antibodies will result in glycosylation, typically at highly conserved N-glycosylation sites in the Fc region. Stable clones may be verified for expression of an antibody specifically binding to human IL-19. Positive clones may be expanded into serum-free culture medium for antibody production in bioreactors. Media, into which an antibody has been secreted, may be purified by conventional techniques. For example, the medium may 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 nonspecific binding components. The bound antibody is eluted, for example, by pH gradient and antibody fractions are detected, such as by SDS-PAGE, and then pooled. The antibody may be concentrated and/or sterile filtered using common techniques. Soluble aggregate and multimers may be effectively removed by common techniques, including size exclusion, hydrophobic interaction, ion exchange, or hydroxyapatite chromatography. The product may be immediately frozen, for example at −70° C., or may be lyophilized. CDR sequences of exemplified embodiments of murine-derived IL-19 antibodies of the present invention, which have been affinity optimized as known in the art, are provided in Table 1.

(4) TABLE-US-00001 TABLE 1 Murine Immunization-Derived Antibody CDR Amino Acid Sequences Light Chain CDRs SEQ ID NOs. Heavy Chain CDRs SEQ ID NOs. Antibody LCDR1 LCDR2 LCDR3 HCDR1 HCDR2 HCDR3 M_1 3 4 5 7 8 9 M_2 11 12 13 15 16 17 M_3 19 20 21 23 24 25 M_4 27 28 29 31 32 33 M_5 303 304 305 307 308 309

(5) Rabbit-derived IL-19 antibodies of the present invention are generated after obtaining antibody gene sequences directly from B a rabbit is immunized with recombinant human IL-19 and mRNA is isolated from antigen-specific B cells enriched from PBMCs. Nucleic acid sequence encoding the heavy and light chain variable regions from this library are then cloned into a cell-based display system. Functional binding fragments are isolated from library, the individual gene sequences determined, cloned for recombinant IgG expression, and purified essentially as described above with regard to murine-derived IL-19 antibodies. CDR sequences of exemplified embodiments of rabbit-derived IL-19 antibodies of the present invention, which have been affinity optimized as known in the art, are provided in Table 2.

(6) TABLE-US-00002 TABLE 2 Rabbitt Immunization-Derived Antibody CDR Amino Acid Sequences. Light Chain CDRs SEQ ID NOs. Heavy Chain CDRs SEQ ID NOs. Antibody LCDR1 LCDR2 LCDR3 HCDR1 HCDR2 HCDR3 R_1 35 36 37 39 40 41 R_2 43 44 45 47 48 49 R_3 51 52 53 55 56 57 R_4 59 60 61 63 64 65 R_5 90 91 92 93 94 95 R_6 96 97 98 99 100 101 R_7 102 103 104 105 106 107 R_8 108 109 110 111 112 113 R_9 114 115 116 117 118 119 R_10 120 121 122 123 124 125 R_11 126 127 128 129 130 131 R_12 132 133 134 135 136 137 R_13 138 139 140 141 142 143 R_14 144 145 146 147 148 149 R_15 150 151 152 153 154 155 R_16 156 157 158 159 160 161 R_17 162 163 164 165 166 167 R_18 168 169 170 171 172 173 R_19 174 175 176 177 178 179 R_20 180 181 182 183 184 185 R_21 186 187 188 189 190 191 R_22 192 193 194 195 196 197 R_23 198 199 200 201 202 203 R_24 204 205 206 207 208 209 R_25 210 211 212 213 214 215 R_26 216 217 218 219 220 221 R_27 222 223 224 225 226 227 R_28 228 229 230 231 232 233 R_29 234 235 236 237 238 239 R_30 240 241 242 243 244 245 R_31 246 247 248 249 250 251 R_32 252 253 254 255 256 257 R_33 258 259 260 261 262 263 R_34 264 265 266 267 268 269 R_35 270 271 272 273 274 275 R_36 276 277 278 279 280 281 R_37 282 283 284 285 286 287 R_38 286 287 288 289 290 291 R_39 295 296 297 299 300 301

(7) Phage-derived IL-19 antibodies of the present invention are isolated from antibody phage libraries employing common techniques such as described above, as described in McCafferty et al., Nature, 348:552-554 (1990), Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991). cDNA sequences encoding the heavy and light chains of phage-derived antibodies of the present invention may be cloned and engineered into a GS (glutamine synthetase) expression vector for recombinant expression in a competent cell line, such as CHO cells. CDR sequences of exemplified embodiments of phage-derived IL-19 antibodies of the present invention are provided in Table 3.

(8) TABLE-US-00003 TABLE 3 Phage-Derived Antibody CDR Amino Acid Sequences. Light Chain CDRs SEQ ID NOs. Heavy Chain CDRs SEQ ID NOs. Antibody LCDR1 LCDR2 LCDR3 HCDR1 HCDR2 HCDR3 P_1 67 68 69 71 72 73 P_2 75 76 77 79 80 81 P_3 83 84 85 87 88 89
Binding Kinetics and Affinity

(9) Bio-layer interferometry (BLI) assay, measured with a Octet Red96® instrument available from ForteBio (using HBS-EP+ running buffer (GE Healthcare, 10 mM Hepes pH7.4+150 mM NaCl+3 mM EDTA+0.05% surfactant P20) at 25° C.), is used to measure binding of the exemplified IL-19 antibodies of the present invention to recombinant human IL-19 (having the amino acid sequence set for in SEQ ID NO: 1).

(10) Except as noted, all reagents and materials are from ForteBio (Freemont, Calif.). An AMQ biosensor is used to immobilize antibody of interest for analysis. Exemplified antibody samples of the present invention (R_1, R_2, R_3, R_4, R_39, M_1, M_2 and M_5) are prepared at 5 μg/mL by dilution into running buffer. Recombinant human IL-19 is prepared to concentrations of 270, 90, 30, 10, 3.33, 1.11, 0.370, and 0 (blank) nM by dilution into running buffer. Each analysis consists of: (1) capturing antibody samples on biosensors for 300 secs; (2) establishing a baseline by incubating antibody loaded biosensors with running buffer for 60 secs; (3) incubating antibody loaded biosensors with serially diluted recombinant human IL-19 for 300 secs to monitor association phase; (4) return of biosensor to running buffer to monitor dissociation phase.

(11) Binding data is processed using standard double-referencing and fit to a 1:1 binding model using Data Analysis v9.0 evaluation software, to determine the association rate (k.sub.on, M.sup.−1s.sup.−1 units), dissociation rate (k.sub.off, s.sup.−1 units), and R.sub.max (nm units). The equilibrium dissociation constant (KO) was calculated from the relationship K.sub.D=k.sub.off/k.sub.on, and is in molar units. Results are provided in Table 4.

(12) TABLE-US-00004 TABLE 4 SPR binding data to recombinant human IL-19. Exemplified k.sub.on k.sub.off K.sub.D* Antibody (M.sup.−1s.sup.−1 units) (M.sup.−1s.sup.−1 units) (M) R_1 3.33 × 10.sup.4   1.04 × 10.sup.−5 3.11 × 10.sup.−10 R_2 4.28 × 10.sup.5   4.42 × 10.sup.−5 1.03 × 10.sup.−10 R_3 3.91 × 10.sup.5 <1.00 × 10.sup.−7 <1.00 × 10.sup.−12   R_4 1.52 × 10.sup.5   1.30 × 10.sup.−3 8.52 × 10.sup.−9  R_39 2.06 × 10.sup.5   3.48 × 10.sup.−2 1.70 × 10.sup.−7  M_1 1.10 × 10.sup.6   6.32 × 10.sup.−5 5.76 × 10.sup.−11 M_2 6.37 × 10.sup.4   1.85 × 10.sup.−4 2.90 × 10.sup.−9  M_5 6.88 × 10.sup.5   3.58 × 10.sup.−5 5.21 × 10.sup.−11 *K.sub.D results are considered relative as the results are not normalized for influence of avidity.
Epitope Mapping

(13) PEPperCHIP® peptide microarray linear epitope mapping of exemplified antibody M_1 against human IL-19 is performed, according to manufacturer instructions, for high resolution linear epitope mapping. Briefly, exemplified antibody M-1 is incubated with a custom PEPperCHIP® peptide microarray comprising overlapping 12-mer peptide fragments of human IL-19. Scanning intensity is resolved using manufacturer software. An epitope, consisting of residues 95-102 (EPNPKILR) of SEQ ID NO. 1 is revealed, according to PEPperCHIP® analysis, for exemplified antibody M_1. Other murine-derived and rabbit-derived exemplified antibodies of the present invention do not yield a linear epitope, indicating human IL-19 conformational epitope binding.

(14) Hydrogen deuterium exchange coupled with mass spectrometry (HDX-MS) is performed to map epitope regions of human IL-19 recombinant protein for exemplified antibodies M_2, M_3, M_5 and R_39. Briefly, HDX-MS is performed on a Waters nanoACQUITY system with HDX technology, including a LEAP HDX robotic liquid handling system and mass analysis is performed on a Waters Xevo G2—Tof mass spectrometer. The complex of human IL-19 with exemplified antibodies M_2, M_3, M_5, and R_39 is prepared at the molar ratio of 1:1.2 in 10 mM sodium phosphate buffer, pH 7.4 containing 150 mM NaCl (1×PBS buffer). The deuterium exchange experiment is initiated adding 55 uL of D20 buffer containing 0.1×PBS to 5 ul of human IL-19 or the human IL-19/antibody complex at 15° C. for various amounts of time (0 s, 10 s, 1 min, 10 min, 60 min, 120/240 min). The reaction is quenched using equal volume of was 0.32M TCEP, 0.1M phosphate pH 2.5 for two minutes at 1° C. 50 μL of the quenched reaction is injected on to an on-line pepsin column (Waters BEH Enzymate) at 14° C., using 0.2% formic acid in water as the mobile phase at a flow rate of 100 μL/min for 4 min. The resulting peptic peptides are then separated on a C18 column (Waters, Acquity UPLC BEH C18, 1.7 μm, 1.0 mm×50 mm) fit with a Vanguard trap column using a 3 to 85% acetonitrile (containing 0.2% formic acid) gradient over 10 min at a flow rate of 50 μL/min. The separated peptides are directed into a Waters Xevo G2 time-of-flight (qTOF) mass spectrometer. The mass spectrometer is set to collect data in the MS.sup.E, ESI.sup.+ mode; in a mass acquisition range of m/z 255.00-1950.00; with a scan time of 0.5 s. The Xevo G2 is calibrated with Glu-fibrinopeptide prior to use. All acquired data is mass corrected using a 2 μg/ml solution of LeuEnk in 50% ACN, 50% H.sub.2O and 0.1% FA at a flowrate of 5 μl/min every 30 s (m/z of 556.2771). The peptides are initially identified by Waters Protein Lynx Global Server 3.02. The processing parameters are set to low energy threshold at 100.0 counts, an elevated energy threshold at 50.0 counts and an intensity threshold at 1500.0 counts. The resulting peptide list is imported to Waters DynamX 3.0 software, with threshold of 5 ppm mass error, 20% fragments ions per peptide based on peptide length. The relative deuterium incorporation for each peptide is determined by processing the MS data for deuterated samples along with the non-deuterated control in DynamX.

(15) Sequence coverage from 90.9 to 94.8% of human IL-19 protein, with HDX-MS as described, is observed. When in complex with the exemplified antibodies, decreased deuterium uptake is observed at the residues of SEQ ID NO. 1 as denoted: M_2 and M_3: residues 67-75 (QIIKPLDVC) and 125-136 (RQCHCRQEATNA); M_5 residues 90-100 (FKDHQEPNPKI); and R_39 residues 42-60 (QEIKRAIQAKDTFPNVTIL), 90-107 (FKDHQEPNPKILRKISSI), and 149-160 (VHAAAIKSLGEL).

(16) Binning Experiments

(17) Binning experiments involve competing monoclonal antibodies against one another in a pairwise and combinatorial fashion for binding to a specific antigen. A “bin” is a relative concept, based upon the epitope regions represented within the panel of monoclonal antibodies being tested. Two antibodies belong to the same bin if they cannot pair with one another and share the same blocking profile when tested against the other antibodies (or bins of antibodies) in the test panel. Binning of exemplified antibodies of the present invention may be performed by cross-competition binding assays using the Octet Red96®, available from ForteBio, according to manufacturer instruction. Briefly, to determine if two antibodies share overlapping epitope regions, an exemplified antibody is labeled with biotin and captured onto streptavidin sensor tip. The coated biosensor tip is then incubated with recombinant human IL-19 to saturate the capture antibody binding sites. The capture antibody-antigen complex is then incubated with a detection antibody. A change in wavelength is detected if the detection antibody is capable of binding. Antibodies with a same binding profile are grouped together into the same bin. Results are presented in Table 5.

(18) TABLE-US-00005 TABLE 5 Antibody Binning Groups Exemplified Binning Antibody Group M_1 1 M_2 2 M_3 2 M_4 1 M_5 1 R_1 3 R_2 3 R_3 3 R_4 4
Neutralization of IL-19 In Vitro

(19) Antibodies of the present invention are expected to neutralize IL-19. Neutralization of IL-19 activity by antibodies of the present invention may be assessed by one or more of the IL-19/IL-19 receptor binding assay formats, as well as IL-19 binding assays, for example, as described below.

(20) In an example, IL-19 is radiolabeled, for example, with iodine-125 or tritium. Cells (e.g., transfected with the IL-19 receptor, transformed keratinocytes that endogenously express the IL-19 receptor, or primary human cells such as keratinocytes that express the IL-19 receptor) expressing the IL-19 receptor such as IL-20R1 are used in the assay which may be conducted in buffered media, such as HBSS with calcium and magnesium and with whole cells. Accordingly, the cells may be incubated with the labeled IL-19 in the assay buffer at 4, 20 or 37° C. for 1 to 6 hours. A readout provides the amount of label bound to the cells after separation of unbound tracer, such as with filtration though a glass fiber filter. Alternatively, neutralization may be assessed by way of a proximity based assay, such as with SPA beads. Further, a neutralization assay utilizing non-radioactive label IL-19 protein may be used.

(21) Such neutralization assays involve pre-incubation of the antibody being assessed with the labeled IL-19 (for example, for 1 hour) before addition to the binding assay (as well as control samples in which no antibody targeting IL-19 is involved). Concentrations of labeled IL-19 near the 50% binding level (EC50) may be used, as well as varying concentrations (for example, in assessing a dose response of the antibody such as from about 100 micromolar down to about 1 picomolar). Antibody inhibition assessed for a range allows for determination of potency (IC50).

(22) According to another method for assessing neutralization of IL-19 by antibodies of the present invention, the IL-19 protein is labeled with a fluorescent dye for flow cytometry (e.g., Alexa-647) and used to label cells, such as human keratinocytes. The binding may then be measured using flow cytometry. Neutralization of IL-19 by the antibody is assessed by pre-incubating the antibody with the labeled IL-19 (for example, for 1 hour at 4° C.) before adding the mixture to the cells (with staining occurring for about 3 hours at 4° C.). Concentrations of fluorescently labeled IL-19 near its 50% binding level (EC50) may used, as well as varying concentrations (for example, in assessing a dose response of the antibody such as from about 100 micromolar down to about 1 picomolar). Antibody inhibition of binding of the labeled IL-19 to its receptor is reflected by measurement of loss of labeled cells, and a potency (IC50) for the antibody may be determined.

(23) Alternatively, a biophysical assay such as bio-layer interferometry (BLI) may be used for assessing neutralization of IL-19 by antibodies of the present invention. Binding between a ligand immobilized on the biosensor tip surface and an analyte in solution produces an increase in optical thickness at the biosensor tip, which results in a wavelength shift (expressed in nm). According to such assay, the IL-19 receptor (i.e., IL-20R1) is expressed in a membrane-free manner (such as with an Fc-fusion e.g. IL20Rbeta Fc chimera protein from R&D Systems catalog 1788-IR-050). AMQ or anti-rabbit conjugated biosensors (ForteBio) are used to immobilize anti-IL-19 antibody of interst (M_1, M_2, M_3, M_5 and R_39). The immobilized antibodies are then incubated with recombinant human IL-19 protein diluted to 100 nM using HBS-EP+ running buffer (GE Healthcare, 10 mM Hepes pH7.4+150 mM NaCl+3 mM EDTA+0.05% surfactant P20) for 240-300 secs. The human IL-19, after binding to the anti-IL-19 antibody, is assessed by incubation with human IL-20R beta Fc-fusion protein for 240-300 secs. The ability of the antibody to block or neutralize binding of the IL-19 ligand to the soluble receptor is observed as a minimal (<0.025 nm) increase in the wavelength during this step of the assay. Results are provided in Table 6.

(24) TABLE-US-00006 TABLE 6 In vitro neutralization. IL-20R beta Fc Exemplified protein binding Antibody response (nm) Neutralization M_1 −0.0075 Yes M_2 0.3614 No M_3 0.3539 No M_5 −0.0124 Yes R_39 −0.0421 Yes

(25) Another method of assessing neutralization of IL-19 by antibodies of the present invention includes addition of such antibody, pre-incubated with human IL-19, to human keratinocytes. Exogenous IL-19 to human keratinocytes induces expression of additional IL-19 and other inflammatory molecules such as IL-8, CCL20 and S100A7. After pre-incubation of the antibody and IL-19 (for example, for 1 hr at 4° C.), the antibody-IL-19 mixture is added to cultured human keratinocytes. The cells are then cultured for 1 to 48 hrs and one or more of IL-19, IL-8, CCL20 and S100A7 (or other molecule expressed downstream of IL-19) in the supernatant is measured, for example, by ELISA (alternatively, mRNA of the downstream molecule may be measured). Antibody inhibited IL-19 function will demonstrate a reduced expression of the downstream molecule by the cultured keratinocytes.

(26) PathHunter® eXpress IL20RA/IL20RB dimerization assay (DiscoverX product code 93-1027E3) is used to assess ability of exemplified antibodies to prevent binding of human IL-19 recombinant protein in a cell-based assay format. The assay detects ligand induced dimerization of two subunits of a receptor-dimer pair. The cells have been engineered to co-express one receptor subunit fused to enzyme donor and a second dimer partner fused to enzyme acceptor. Binding of an agonist to one receptor subunit induces it to interact with its dimer partner, forcing complementation of the two enzyme fragments resulting in the formation of a functional enzyme that hydrolizes a substrate to generate a chemiluminescent signal. Briefly, cells are plated at 2500 cells per well and cultured at 37° C./5% CO.sub.2 for 4 hours before addition of human IL-19 recombinant protein pre-mixed with various concentrations, ranging from 10 to 0.00001 μg/ml including a buffer only control, of exemplified antibodies (M_1 and M_5). Human IL-19 recombinant protein with and without exemplified antibodies mixture is then incubated with cells overnight at 37° C./5% CO.sub.2. Substrate buffer is added to cells and incubated at room temperature for 1 hour in the dark before luminescent detection. The concentration of exemplified antibody resulting in inhibition of 50% of signal (IC50) and the maximum signal inhibition percentage (% inhibition) is tabulated for eight experiments with standard error of the mean (SEM) denoted below. Results are provided in Table 7.

(27) TABLE-US-00007 TABLE 7 Cell-based neutralization. Exemplified IC50 +/− SEM Antibody (μg/ml) % inhibition +/− SEM M_1 0.48 +/− 0.06  98.59 +/− 0.42 M_5 0.64 +/− 0.20 105.03 +/− 1.38
IL-19 Assay

(28) Plaque type psoriasis is currently measured based on measures of overall body surface involvement (BSA) and/or assessments of degree of erythema, thickness and scale of psoriasis lesions (PAST). However, given subjective input required with these methods they may not be linear depending on severity of skin involvement. No single blood-derived marker has been identified which allows for assessing overall psoriasis activity. Therefore, a more objective and reproducible method to determine severity is desired. The present invention provides a highly sensitive and specific assay to measure IL-19 levels in patients samples such as blood, serum and plasma. As illustrated herein, the IL-19 assay of the present invention provides an accurate diagnostic tool for therapy responsiveness (i.e., a predictive biomarker), disease reoccurrence (i.e., a prognostic biomarker), disease onset, and disease severity in patients with moderate-to-severe Ps0.

(29) According to an exemplified embodiment, a sandwich ELISA assay for the sensitive detection of IL-19 is provided herein. The assay utilizes exemplified IL-19 antibodies of the present invention, for example, as set forth in Table 1, 2 or 3. According to an exemplified embodiment, a first IL-19 antibody (selected from Table 1, 2 or 3) is utilized as an IL-19 capture antibody and a second IL-19 antibody (selected from Table 1, 2 or 3) is utilized as an IL-19 reporter antibody. In some embodiments the first and second IL-19 antibodies are selected from separate epitope bins (for example, in specific embodiments, exemplified IL-19 antibody M_1 is paired with exemplified IL-19 antibody M_2). According to some embodiments, one milligram of the first IL-19 antibody (the capture antibody) is biotinylated using Pierce biotinylation kit (Cat #) and one milligram of the second IL-19 antibody (the reporter antibody) is labeled with ruthenium using MesoScale Discovery (MSD) kit for electrochemiluminescent (ECL) detection. According to such embodiment, labeled antibodies are evaluated using MALDI-TOF to ensure suitable labeling, and then diluted in 50% glycerol and stored at −20° C. prior to use.

(30) Streptavidin-coated 96-well MSD plates are washed three times with TBST (Tris buffered saline containing 10 mmol/L Tris pH 7.40, 150 mmol/L NaCl with 1 mL Tween 20/L) and then blocked with TBS-T plus 1% BSA for 1 hour at room temperature. Plates are again washed and wells are then incubated with biotinylated IL-19 capture antibody (1 mg/L) for 1 hour. Thereafter, plates are again washed prior to patient sample testing.

(31) During patient sample testing, a standard curve is generated using 50 μL of recombinant human IL-19 control standard (serially diluted IL-19 recombinant protein ranging from 100-0.0001 ng/L, and including a zero blank, in assay buffer of 50 mmol/L HEPES, pH 7.40, 150 mmol/L NaCl, 10 mL/L Triton X-100, 5 mmol/L EDTA, and 5 mmol/L EGTA). Data from ten separate standard curves, prepared as described herein, shows a dynamic range of 10.sup.−1 pg/mL to 10.sup.5 pg/mL of IL-19 (providing an sensitive and broad dynamic range in the therapeutic and diagnostic assays provided herein). Patient samples (which, according to the present invention, may include blood, serum or plasma) are diluted 1:4 in assay buffer and added to respective wells. The plate is incubated overnight at 4° C. Following incubation, wells are aspirated and washed 3 times with TB ST. Thereafter, 50 μL of ruthenium-labeled IL-19 detection antibody (0.5 mg/L) is added to the wells for a 1-hour incubation at room temperature. Following incubation, wells are aspirated and washed 3 times with TBST. Thereafter, 150 μl of 2×MSD read buffer is added. Ruthenium electrochemiluminescence in the wells is detected using a MSD Sector 6000 plate reader. Data is analyzed and IL-19 MSD immunoassay calibration curve fitting is performed using MesoScale Discovery software. SAS® software version 9.4 (PROC MIXED) is used for assessing treatment effects on IL-19 levels using a mixed effects model with an unstructured covariance matrix and log.sub.10 transformed IL-19 concentrations (SAS. Version 9.4 for UNIX; SAS Institute Inc.: Cary, N.C., 2016). Statistical analysis is generated with the ggplot and pROC packages using R version 3.3.3 statistical computing environment (www.R-project.org, Vienna, Austria, 2017).

(32) IL-19 Serum Concentrations in Ps0 Patients Versus Healthy Groups

(33) A study of IL-19 levels in serum of 125 Ps0 patients, pre-treatment, were compared to IL-19 serum levels of 36 healthy volunteer samples. Using an IL-19 assay essentially as described above, with a capture antibody selected from bin 1 (specifically, exemplified IL-19 antibody M_1) and a detection antibody selected from bin 2 (specifically, exemplified IL-19 antibody M_2), serum IL-19 concentrations (pg/ml) of each patient sample is measured. The geometric mean of serum IL-19 concentrations for healthy volunteers (n=36) is measured at 11 pg/mL (with a range of 4 to 51 pg/mL, and a 95% confidence level at less than 21 pg/mL) whereas the geometric mean of serum IL-19 concentrations for Ps0 patients (pre-treatment) (n=112) is measured at 87 pg/mL. Thus, the present invention provides an IL-19 blood-based assay allowing for the diagnosis of Ps0 patients.

(34) Ps0 Study in Anti-IL-17 Treatment Groups

(35) A study of IL-19 levels, in serum of 125 Ps0 patients treated with a therapeutic antibody targeting IL-17, ixekizumab, is performed. The study includes five treatment group doses of: 10 mg (n=24), 25 mg (n=23), 75 mg (n=26), or 150 mg (n=28) of ixekizumab or placebo (n=24). Administration of all treatment doses is subcutaneous, and doses are administered starting at week 0 and every 2 weeks thereafter up to week 16 (inclusive). Using an IL-19 assay as described above, with a capture antibody selected from bin 1 (specifically, exemplified IL-19 antibody M_1]) and a detection antibody selected from bin 5 (specifically, exemplified IL-19 antibody M_2), serum IL-19 concentrations (pg/ml) of each patient is measured at week 0 (pre-treatment dosing), week 2 and week 12. Serum IL-19 concentration levels, percent PASI change and PASI 75 response are provided in Tables 8-10.

(36) As shown, 36 out of 41 (87.8%) patients with a greater than or equal to 5-fold reduction in IL-19 from week 0 to week 2 achieved PASI 75 or greater by week 16 (whereas only 24 of 56 (42.9%) patients with less than a 5-fold reduction in IL19 from week 0 to week 2 achieved a PASI 75 response by week 16. Further, 37 out of 42 (88%) patients with a greater than or equal to 5-fold reduction in IL-19 from week 0 to week 9 achieved PASI 75 or greater by week 12 (whereas only 22 out of 53 (41.5%) patients with less than a 5-fold reduction in IL19 from week 0 to week 16 achieved a PASI 75 response by week 16 (data reflects a drop-out of 2 patients between weeks 2 and 12). For the placebo treatment group, no significant change in IL-19 concentrations were observed during the 16-week trial period.

(37) TABLE-US-00008 TABLE 8 IL-19 serum levels (geometric mean) per treatment group. Treatment Week 0 Week 2 Week 16 Group (baseline IL-19 pg/mL) (IL-19 pg/mL) (IL-19 pg/mL) 150  87.1 13.9 11.9 (n = 28) 75  86.7 11.6  9.4 (n = 26) 25  89.0 22.3 13.6 (n = 23) 10 111.6 45.3 57.3 (n = 24) Placebo  67.4 66.7 57.2 (n = 24)

(38) Table 8 provides data showing IL-19 measured in treatment groups over 16 weeks of treatment with placebo or various ixekizumab doses.

(39) TABLE-US-00009 TABLE 9 Patients achieving at least PASI 75 at week 16 per treatment group. Week 2 Treatment (number of patients achieving Group at least PASI 75) 150  22 (n = 24) 75 21 (n = 24) 25 16 (n = 20) 10 5 (n = 19) Placebo 1 (n = 20)

(40) Table 9 provides the the number of patients, per treatment group, achieveing PASI 75 by week 16.

(41) TABLE-US-00010 TABLE 10 Assessment of serum IL-19 concentration and PASI at week 16. % patients with % patients with serum IL-19 serum IL-19 % of patients concentration ≤ concentration ≤ with serum IL-19 21 pg/mL 21 pg/mL not Treatment concentration ≤ achieving at achieving at Group 21pg/mL least PASI 75 least PASI 75 150  92% (23/25) .sup. 95% (19/20)  5% (1/20) (n = 25) 75 91.7% (22/24).sup.  86.4% (19/22) 13.6% (3/22)  (n = 24) 25 70% (14/20) 92.9% (13/14)  7.1% (1/14) (n = 20) 10 42.1% (8/19)   62.5% (5/8)  37.5% (3/8).sup.  (n = 19) Placebo 25% (5/20)  20% (1/5)  80% (4/5) (n = 20)

(42) Table 10 presents correlations of IL-19 serum levels and PASI in psoriasis patients after 16 weeks of placebo or various ixekizumab treatments (21 ng/L indicates the upper limit of the normal range of IL-19 in healthy subjects). It was noted that PASI 100 improvements at 16 weeks were preceded by reduction of circulating IL-19 to near normal concentrations after 2 weeks of treatment.

(43) The data provided in Tables 8-10 demonstrate that the IL-19 assay of the present invention provides a valuable tool for diagnosis and therapeutic prognostication of Ps0 patients treated with IL-17 antibodies.

(44) Ps0 Study in Anti-TNFα Treatment Groups

(45) A study of IL-19 levels, in serum of 35 Ps0 patients that were complete responders when treated with the FDA approved TNFα antagonist, entanercept, is performed. Treatment groups of 50 mg of etanercept (n=35) administered biweekly or placebo are compared. Administration of both treatment groups is subcutaneous. Using an IL-19 assay as described above, with a capture antibody selected from bin 1 (specifically, exemplified IL-19 antibody M_1) and a detection antibody selected from bin 2 (specifically, exemplified IL-19 antibody M_2), serum IL-19 concentrations (pg/ml) of each patient is measured at week 0 (pre-treatment dosing), week 1, week 4 and week 12. IL-19 levels are assessed for prognostic value with PASI improvement at weeks 4 and 12. Serum IL-19 concentration levels are presented in Table 11; prognostic values at week 4 showing correlations of IL-19 serum levels and PASI in psoriasis patients (21 ng/L indicates the upper limit of the normal range of IL-19 in healthy subjects) is presented in Table 12; prognostic values at week 12 P showing correlations of IL-19 serum levels and PASI in psoriasis patients (21 ng/L indicates the upper limit of the normal range of IL-19 in healthy subjects) is presented in Table 13.

(46) TABLE-US-00011 TABLE 11 IL-19 (pg/mL) serum levels (geometric mean) per treatment group. Treatment Week 0 Group (baseline) Week 1 Week 4 Week 12 Etanercept 98.2 42.3 24.3 14.2 (n = 35)

(47) TABLE-US-00012 TABLE 12 Assessment of serum IL-19 concentration and PASI at week 4. % patients with % patients with serum IL-19 serum IL-19 % of patients concentration ≤ concentration ≤ with serum IL-19 21 pg/mL 21 pg/mL not Treatment concentration ≤ achieving at achieving at Group 21 pg/mL least PASI 75 least PASI 75 Etanercept 37.9% (44/116) 22.7% (10/44) 77.3% (34/44) (n = 160)

(48) TABLE-US-00013 TABLE 13 Assessment of serum IL-19 concentration and PASI at week 12. % patients with % patients with serum IL-19 serum IL-19 % of patients concentration ≤ concentration ≤ with serum IL-19 21 pg/mL 21 pg/mL not Treatment concentration ≤ achieving at achieving at Group 21 pg/mL least PASI 75 least PASI 75 Etanercept 42.9% (69/161) 49.3% (34/69) 50.7% (35/69) (n = 161)

(49) As shown above, on average TNF antagonist-treated patients experienced a median reduction in serum IL-19 of greater than 40 (pg/mL) after 1 week and a median reduction in serum IL-19 of grater than 70 (pg/mL) at week 4. The data provided in Tables 11-13 demonstrate that the IL-19 assay of the present invention provides a valuable tool for diagnosis and therapeutic prognostication of Ps0 patients treated with TNFα antibodies.

(50) Ps0 Study in Anti-IL-23 Treatment Groups

(51) A study of IL-19 levels, in serum of Ps0 patients treated with a therapeutic antibody targeting IL-23, mirikizumab, is performed. Eight treatment groups of: 5 mg, 20 mg, 60 mg, 120 mg, 200 mg, 350 mg, and 600 mg of mirikizumab, or placebo, are assessed. Administration of each treatment group, as a single subcutaneous dose, occurs at day 0. Using an IL-19 assay as described above, with a capture antibody selected from bin 1 (specifically, exemplified IL-19 antibody M_1) and a detection antibody selected from bin 2 (specifically, exemplified IL-19 antibody M_2), serum IL-19 concentrations (pg/ml) of each patient is measured between visits 2 and 12. IL-19 levels are also assessed for prognostic value with PASI improvement between visits 2 and 12. Serum IL-19 concentration levels are presented in Table 14; a comparative of serum levels at week 8 is presented in Table 15; and prognostic values at week 8 showing correlations of IL-19 serum levels and PASI in psoriasis patients (21 ng/L indicates the upper limit of the normal range of IL-19 in healthy subjects) are presented in Table 16. Decreases in serum IL-19 correlated with improvement in PASI score demonstrating the IL-19 assay of the present invention provides a valuable tool for diagnosis and therapeutic prognostication of Ps0 patients treated with IL-23 antibodies.

(52) TABLE-US-00014 TABLE 14 IL-19 (geometric mean) serum levels per treatment group. Treatment Day 1 Group (baseline) Day 15 Day 29 Day 57 Day 71  5 16.2 13.9 10.4 13.2 14.5 (n = 5)  20 37.2 27.2 25.4 33.5 28.4 (n = 5)  60 49.8 9.0 10.8 11.9 10.9 (n = 5) 120 52.4 26.6 25.2 22.2 18.2 (n = 5) 200 69.6 36.0 23.3 21.9 22.5 (n = 5) 350 58.4 35.2 22.4 23.7. 18.0 (n = 5) 600 57.3 30.1 21.8 14.4. 15.8 (n = 5) placebo 58.9 52.2 47.0 40.4. 47.1 (n = 7)

(53) TABLE-US-00015 TABLE 15 IL-19 (geometric mean) serum levels per treatment group. Treatment Week 0 Week 8 Group (Baseline) (End of induction) LY 300 mg 106.5 18.2 (n = 50) Placebo 158.0 154.9 (n = 52)

(54) TABLE-US-00016 TABLE 16 Assessment of serum IL-19 concentration and PASI at week 8. % patients with % patients with serum IL-19 serum IL-19 % of patients concentration ≤ concentration ≤ with serum IL-19 21 pg/mL 21 pg/mL not Treatment concentration ≤ achieving at achieving at Group 21 pg/mL least PASI 75 least PASI 75 LY 300 mg 58.3% (28/48) 71.4% (20/28) 28.6% (8/28) (n = 48) Placebo 13.7% (7/51)  28.6% (2/7)  71.4% (5/7)  (n = 51)

(55) Ps0 Study in JAK1 and JAK2 Kinase Inhibitor Treatment Groups

(56) A study of IL-19 levels, in serum of Ps0 patients treated with the therapeutic selective JAK1 and JAK2 inhibitor, baricitinib, is performed. Treatment groups of 2 mg, 4 mg, 8 mg, and 10 mg of baricitinib, or placebo, are assessed. Each treatment group is orally administered once daily. Using an IL-19 assay as described above, with a capture antibody selected from bin 1 (specifically, exemplified IL-19 antibody M_1) and a detection antibody selected from bin 2 (specifically, exemplified IL-19 antibody M_2), serum IL-19 concentrations (pg/ml) of each patient is measured at baseline and following each treatment. IL-19 levels are also assessed for prognostic value with PASI improvement. Serum IL-19 concentration levels are presented in Table 17. PASI prognostic data (at week 12) showing correlations of IL-19 serum levels and PASI in psoriasis patients (21 ng/L indicates the upper limit of the normal range of IL-19 in healthy subjects) are presented in Table 18. The data demonstrates a decreases in serum IL-19 correlates with improvement in PASI score demonstrating the IL-19 assay of the present invention provides a valuable tool for diagnosis and therapeutic prognostication of Ps0 patients treated with JAK1 and JAK2 inhibitors.

(57) TABLE-US-00017 TABLE 17 IL-19 serum levels (geometric mean) per treatment group. Treatment Week 0 Group (baseline) Week 2 Week 12 2 182.3 98.5 64.8 (n = 32) 4 134.9 70.2 60.1 (n = 72) 8 177.3 67.0 47.5 (n = 64) 10  110.1 39.7 25.7 (n = 69) placebo 116.9 131.8 95.3 (n = 34)

(58) TABLE-US-00018 TABLE 18 Assessment of serum IL-19 concentration and PASI at week 12. % patients with % patients with serum IL-19 serum IL-19 % of patients concentration ≤ concentration ≤ with serum IL-19 21 pg/mL 21 pg/mL not Treatment concentration ≤ achieving at achieving at Group 21 pg/mL least PASI 75 least PASI 75 2 13.8% (4/29)  50% (2/4)   50% (2/4) (n = 29) 4 31.8% (21/66) 47.6% (10/21)  52.4% (11/21) (n = 66) 8 38.9% (21/54) 85.7% (18/21) 14.3% (3/21) (n = 54) 10  46.6% (27/58) 77.8% (21/27) 22.2% (6/27) (n = 58) Placebo 14.8% (4/27)  100% (4/4).sup.  .sup. 0% (0/4) (n = 27)

(59) AD Study in JAK1 and JAK2 Kinase Inhibitor Treatment Groups

(60) A study of IL-19 levels, in serum of 123 patients with moderate-to-sever atopic dermatitis treated with the therapeutic selective JAK1 and JAK2 inhibitor, baricitinib, is performed. Treatment groups of 2 mg and 4 mg of baricitinib, or placebo, are compared. Each treatment group is orally administered once daily. Using an IL-19 assay as described above, with a capture antibody selected from bin 1 (specifically, exemplified IL-19 antibody M_1) and a detection antibody selected from bin 2 (specifically, exemplified IL-19 antibody M_2), serum IL-19 concentrations (pg/ml) of each patient is measured at baseline (pre-treatment) and at weeks 4 and 16. IL-19 levels are assessed for prognostic value with EASI score improvement. Serum IL-19 concentration levels are presented in Table 19; EASI prognostic values (at week 16) showing correlations of IL-19 serum levels in AD patients (21 ng/L indicates the upper limit of the normal range of IL-19 in healthy subjects) are presented in Table 20. The data demonstrates baseline IL-19 concentrations in AD patients were found to be elevated compared to normal (geometric mean of 34 pg/mL in AD patients). The data also demonstrates a decreases in serum IL-19 at weeks 4 and 16 correlates with improvement in EASI score at week 16 demonstrating the IL-19 assay of the present invention provides a valuable tool for diagnosis and therapeutic prognostication of Ps0 patients treated with JAK1 and JAK2 inhibitors.

(61) TABLE-US-00019 TABLE 19 IL-19 (geometric mean) serum levels per treatment group. Treatment Week 0 Group (baseline) Week 4 Week 16 2 27.8 18.8 25.1 (n = 37) 4 30.3 18.0 21.1 (n = 38) placebo 44.4 27.5 23.6 (n = 49)

(62) TABLE-US-00020 TABLE 20 Assessment of serum IL-19 concentration and EASI at week 16. % patients with % patients with serum IL-19 serum IL-19 % of patients concentration ≤ concentration ≤ with serum IL-19 21 pg/mL 21 pg/mLnot Treatment concentration ≤ achieving at achieving at Group 21 pg/mL least EASI 75 least EASI 75 2 51.9% (14/27) 64.3% (9/14) 35.7% (5/14) (n = 27) 4 63.0% (17/27)  58.8% (10/17) 41.2% (7/17) (n = 27) Placebo .sup. 50% (14/28) 64.3% (9/14) 35.7% (5/14) (n = 28)

(63) IL-19 Serum Concentrations in Renal Failure and Diabetes Patient Groups

(64) IL-19 levels are measured in healthy donors (n=20), renal failure patients (n=16), diabetes patients without renal failure (n=20), and diabetes patients with renal failure (n=21). Using an IL-19 assay as described above, with a capture antibody selected from bin 1 (specifically, exemplified IL-19 antibody M_1) and a detection antibody selected from bin 2 (specifically, exemplified IL-19 antibody M_2), baseline serum IL-19 concentrations (pg/ml) of each patient is measured. The data demonstrates markedly elevated IL-19 levels in renal failure patients (40±6 pg/mL), diabetes patients without renal failure (17±3 pg/mL), and diabetes patients with renal failure (46±9 pg/mL) as compared to healthy donors (8±1 pg/mL) IL-19 levels. Thus, the IL-19 assay of the present invention provides a valuable tool for diagnosis and therapeutic prognostication of diabetes and renal failure patients.

(65) PsA Study in Anti-IL-17 Treatment Groups

(66) A study of IL-19 levels, in serum of 309 PsA patients treated with a therapeutic antibody targeting IL-17, ixekizumab, is performed. The study includes three treatment groups: (i.) anti-IL-17 treatment group, administered ixekizumab 160 mg at baseline followed by an 80 mg dose administered every two weeks thereafter for 12 weeks (n=103); (ii.) anti-IL-17 treatment group, administered ixekizumab 160 mg at baseline followed by an 80 mg dose administered every four weeks thereafter for 12 weeks (n=107); or (iii.) placebo (n=105). Administration of all treatment doses is subcutaneous. Using an IL-19 assay as described above, with a capture antibody selected from bin 1 (specifically, exemplified IL-19 antibody M_1) and a detection antibody selected from bin 2 (specifically, exemplified IL-19 antibody M_2), serum IL-19 concentrations (pg/ml) of each patient is measured at week 0 (pre-treatment dosing), week 4 and week 12. Serum IL-19 concentration levels are presented in Table 21; week 4 and 12 PAST prognostic values showing correlations of IL-19 serum levels and PAST in patients (21 ng/L indicates the upper limit of the normal range of IL-19 in healthy subjects) are presented in Tables 22 and 23, respectively.

(67) TABLE-US-00021 TABLE 21 IL-19 (geometric mean) serum levels per treatment group. Treatment Week 0 Group (baseline) Week 4 Week 12 Ixe 23.0 6.0 6.1 Q2W (n = 103) Ixe 31.3 7.6 7.3 Q4W (n = 107) placebo 23.7 27.0 28.4 (n = 105)

(68) TABLE-US-00022 TABLE 22 Assessment of serum IL-19 concentration at week 4 and PASI at week 12. % patients with % patients with serum IL-19 serum IL-19 % of patients concentration ≤ concentration ≤ with serum IL-19 21 pg/mL 21 pg/mL not Treatment concentration ≤ achieving at achieving at Group 21 pg/mL least PASI 75 least PASI 75 Ixe 97.5% (79/81) 72.2% (57/79) 27.8% (22/79) Q2W (n = 81) Ixe 90.8% (79/87) 73.4% (58/79) 26.6% (21/79) Q4W (n = 87) Placebo 46.4% (39/84) 20.5% (8/39)  79.5% (31/39) (n = 84)

(69) TABLE-US-00023 TABLE 23 Assessment of serum IL-19 concentration at week 12 and PASI at week 12. % patients with % patients with serum IL-19 serum IL-19 % of patients concentration ≤ concentration ≤ with serum IL-19 21 pg/mL 21 pg/mL not Treatment concentration ≤ achieving at achieving at Group 21 pg/mL least PASI 75 least PASI 75 Ixe 96.7% (87/90) 73.1% (57/78) 26.9% (21/78) Q2W (n = 90) Ixe 93.5% (87/93) 74.1% (60/81) 25.9% (21/81) Q4W (n = 93) Placebo 43.0% (37/86) 24.3% (9/37)  75.7% (28/37) (n = 86)

(70) Table 21 shows baseline IL-19 levels in psoriatic arthritis patients are increased compared to the reference value of healthy volunteers (represented by the shaded grey region). Placebo treatment does not result in significant change in IL-19 over the 12-week time study. However, both ixekizumab treatment groups show lowering of IL-19 to near normal levels after 4 weeks; lowering which is sustained over the 12 week treatment. Tables 22 and 23 show the relationship between IL-19 levels in PsA patients after either 4 or 12 weeks of placebo or ixekizumab treatment groups and the PASI score at 12 weeks. PASI 100 improvements at 12 weeks were correlated with a reduction of circulating IL-19 concentrations to near normal levels, with the majority of the poor PASI responders being in the placebo group. The data provided in Tables 21-23 demonstrate that the IL-19 assay of the present invention provides a valuable tool for diagnosis and therapeutic prognostication of PsA patients treated with IL-17 antibodies.