WO2017172509A1

WO2017172509A1 - Il-21 antibodies and uses thereof

Info

Publication number: WO2017172509A1
Application number: PCT/US2017/023946
Authority: WO
Inventors: Stuart Willis Bright; Karen Leigh Cox; Julian Davies; Angus John MACDONALD; Andrea Paula MARTIN; Joshua Dade POORBAUGH; Oliver SCHROEDER; Sean Edward SISSONS; Xiao-fen WANG
Original assignee: Eli Lilly And Company
Priority date: 2016-03-31
Filing date: 2017-03-24
Publication date: 2017-10-05
Also published as: CA3018792C; CA3018792A1; JP2019514349A; JP7010833B2; US11267882B2; CN109069600A; CN109069600B; EP3436060A1; US20190233508A1; EP3436060A4

Abstract

Provided are antibodies, or an antigen-binding fragment thereof, that binds human IL-21, These antibodies are useful in immunoassays of IL-21 levels, and/or in vivo, ex vivo or in vitro immunochemical and other imaging methods for determining the presence of IL-21 and/or quantifying IL-21 levels, and for diagnostic, prognostic and predictive purpose, and or optimizing therapeutic regimens in patients in which IL-21 signaling is implicated in pathogenesis.

Description

IL-21 ANTIBODIES AND USES THEREOF The present invention relates to the field of medicine. More specifically, the present invention relates to antibodies that bind human interleukin-21 (IL-21) to form a detectable IL-21/anti-IL-21 antibody complex useful in determination of IL-21 levels found in human biological matrices with high sensitivity (sub picogram per milliliter (pg/ml)). In particular, the present invention relates to determination of femtogram per milliliter levels of IL-21 in vitro assays.

IL-21 is an important cytokine involved in the pathogenesis of inflammatory diseases, including allergic diseases, cancer, and autoimmune diseases, particularly, psoriasis, systemic lupus erythematosus (SLE), , chronic inflammatory bowel disease and Sjögren’s syndrome. Elevated serum levels of IL-21 are reportedly associated with disease severity in patients with autoimmune diseases such as psoriasis, SLE or

Sjögren’s syndrome. An enzyme-linked immunosorbent assay (ELISA) kit for detecting human IL-21 is commercially available, but it has a low limit of detection of 16 pg/mL (Weir et al. Cytokine 60 (2012) 220-225). The kit is, however, unable to detect the true level of IL-21 in patient samples.

There is, therefore, a need for anti-IL-21 antibodies that possess higher binding affinity and selectivity to human IL-21 resulting in enhanced sensitivity in IL-21 determinations, or, when used in ELISA assays, provide minimal interference and broad dilutional linearity. Preferably, the antibodies are monoclonal antibodies, and include, for example, two or more distinct antibodies that recognize two or more distinct epitopes on IL-21 so that a pair of antibodies can bind simultaneously to IL-21 in an assay.

There is a need for anti-IL-21 antibodies that bind IL-21, which permits diagnostic assessment of IL-21 levels, before, during, and/or after treatment of the patient, with minimal plasma protein interference and with higher sensitivity than commercially available assays. Accordingly, the present invention seeks to provide alternative anti-IL- 21 antibodies that specifically bind to human IL-21. The present invention further seeks to provide a rapid and convenient method for quantifying human IL-21 in vitro at femtogram per milliliter (fg/ml) levels. Accordingly, a first aspect of the present invention provides an antibody, or antigen-binding fragment thereof, that binds human IL-21, comprising a light chain variable region (LCVR) and a heavy chain variable region (HCVR), wherein said LCVR comprises three light chain complementarity determining regions (LCDRs) and said HCVR comprises three heavy chain complementarity determining regions (HCDRs), wherein the amino acid sequences of said three LCDRs and said three HCDRs are selected from the group consisting of:

a) RASQDISNYLN (SEQ ID NO: 1), YTSRLHS (SEQ ID NO: 2), QQFHTLRTF (SEQ ID NO: 3), GYTFTDYWMH (SEQ ID NO: 4), LIDTSDSYTIYNQKFKG (SEQ ID NO: 5), and YGPLAMDY (SEQ ID NO: 6);

b) RASKSIEKYIA (SEQ ID NO: 7), AGGTLQS (SEQ ID NO: 8), QQHEEYPLT (SEQ ID NO: 9), GYDFTGYTMN (SEQ ID NO: 10), LINPYNGGTAYSPKFKG (SEQ ID NO: 11), and THYYGSEYTGMDY (SEQ ID NO: 12); and

c) KSSQSLLDVDGKTYLN (SEQ ID NO: 13), LVSKLDS (SEQ ID NO: 14), WQGTHFPYT (SEQ ID NO: 15), GYFFTLYMMH (SEQ ID NO: 16),

YINPSSGYTEYNQKFKD (SEQ ID NO: 17), and DFDY (SEQ ID NO: 18). In a further embodiment, the present invention provides an antibody, or antigen- binding fragment thereof, that binds human IL-21, wherein said antibody or antigen- binding fragment thereof comprises a LCVR and a HCVR, wherein the amino acid sequences of said LCVR and said HCVR are selected from the group consisting of:

a) the amino sequence of SEQ ID NO: 19 and the amino sequence of SEQ ID NO:

20;

b) the amino sequence of SEQ ID NO: 21 and the amino sequence of SEQ ID NO:

22; and

c) the amino sequence of SEQ ID NO: 23 and the amino sequence of SEQ ID NO:

24.

In another embodiment, the present invention provides an antibody that binds human IL-21, wherein said antibody comprises a light chain and a heavy chain, wherein the amino acid sequences of said light chain and said heavy chain are selected from the group consisting of:

a) the amino sequence of SEQ ID NO:25 and the amino sequence of SEQ ID NO:26; b) the amino sequence of SEQ ID NO:27 and the amino sequence of SEQ ID NO:28; and

c) the amino sequence of SEQ ID NO:29 and the amino sequence of SEQ ID NO:30.

In another embodiment, the present invention provides an antibody that binds human IL-21, wherein said antibody comprises two light chains and two heavy chains, wherein the amino acid sequences of each of said light chains and each of said heavy chains are selected from the group consisting of:

c) the amino sequence of SEQ ID NO:29 and the amino sequence of SEQ ID NO:30.

The present invention also provides a polynucleotide comprising a nucleotide sequence encoding the LCVR and/or the HCVR, or the light chain and/or the heavy chain of the antibodies of the present invention, wherein said polynucleotide has the nucleotide sequence as shown in SEQ ID NOs: 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42.

The present invention also provides a recombinant expression vector comprising a polynucleotide encoding the LCVR and/or the HCVR, or the light chain and/or the heavy chain of the antibodies of the present invention.

In another embodiment, the present invention provides a host cell which has been transformed by an expression vector comprising a polynucleotide encoding the LCVR and/or the HCVR, or the light chain and/or the heavy chain of the antibodies of the present invention.

In another embodiment, the present invention further provides an antibody or antigen-binding fragment comprising a detectable label, wherein said detectable label is selected from the group consisting of a chromophore, a chromogen, a dye, a fluorescent agent, a fluorogenic agent, a phosphorescent agent, a chemiluminescent agent, a bioluminescent agent, a radionuclide, a positron emission tomography-imageable agent, and a magnetic resonance-imageable agent.

In another embodiment, the present invention provides a composition comprising an antibody or antigen-binding fragment thereof of the present invention, and an acceptable carrier, diluent, or excipient. In another embodiment, the present invention provides an in vitro method of detecting or quantifying human IL-21 in a sample of tissue or body fluid comprising: contacting said sample with said antibody or antigen-binding fragment thereof of the present invention; optionally, removing any non-specifically bound antibody or antigen- binding fragment thereof; and detecting or quantifying the amount of the antibody or antigen-binding fragment thereof, which is specifically bound to human IL-21 in said sample quantitatively, semi-quantitatively or qualitatively.

In another aspect, the present invention provides an antibody or antigen-binding fragment thereof of the present invention for use in diagnostic, prognostic, and/or patient monitoring procedure in vitro.

In another aspect, the present invention provides a kit for use in in vitro detecting or quantifying human IL-21 in a sample of tissue or body fluid, comprising

a) a first reagent, wherein said first reagent is an antibody or antigen-binding

fragment thereof, comprising a LCVR having the amino sequence of SEQ ID NO: 21 and a HCVR having the amino sequence of SEQ ID NO: 22; and

b) a second reagent, wherein said second reagent is an antibody or antigen-binding fragment comprising a LCVR having the amino sequence of SEQ ID NO: 19 and a HCVR having the amino sequence of SEQ ID NO: 20.

Preferably, the sample of tissue or body fluid is a plasma sample or a serum sample.

According to yet another aspect of the present invention, there is provided an antibody according to the present invention for use in in vitro measurement of the amount of IL-21 in a human sample.

As used herein, the term“IL-21” (also known as interleukin-21) means a type I cytokine that exerts pleiotropic effects on both innate and adaptive immune responses. IL- 21 is produced by activated CD4 positive T cells including, follicular T helper and Th17 cells. The amino acid and cDNA sequences of human IL-21 are listed as SEQ ID NOs:43 and 45, respectively.

An antibody or a full-length antibody is an immunoglobulin molecule comprising two heavy chains and two light chains interconnected by disulfide bonds. The amino terminal portion of each chain includes a variable region of about 100-110 amino acids primarily responsible for antigen recognition via the complementarity determining regions (CDRs) contained therein. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. The antibodies of the present invention are monoclonal antibodies (“mAbs”). Monoclonal antibodies can be produced, for example, by hybridoma technologies, e.g. CDR-grafting, or combinations of such or other technologies known in the art. In another embodiment of the present invention, there is provided an antibody, or the nucleic acid encoding the same, in isolated form. As used herein, the term“isolated” refers to a protein, peptide or nucleic acid that is not found in nature and is free or substantially free from other macromolecular species found in a cellular environment.“Substantially free”, as used herein, means the protein, peptide or nucleic acid of interest comprises more than 80% (on a molar basis) of the

macromolecular species present, preferably more than 90% and more preferably more than 95%.

“Antigen-binding fragment”, as used herein, refers to antigen-binding fragments of antibodies, i.e. antibody fragments that retain the ability to bind specifically to the antigen bound by the full-length antibody. Examples of antigen-binding fragment include, but are not limited to, Fab fragments, Fab’ fragments, F(ab’)₂ fragments, and single chain Fv fragments. Preferably, the antibody fragment is a Fab fragment.

The CDRs are interspersed with regions that are more conserved, termed framework regions (“FR”). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) is composed of 3 CDRs and 4 FRs, arranged from amino- terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The 3 CDRs of the light chain are referred to as“LCDR1, LCDR2, and LCDR3” and the 3 CDRs of the heavy chain are referred to as“HCDR1, HCDR2, and HCDR3”. The CDRs contain most of the residues which form specific interactions with the antigen. Three systems of CDR assignments for antibodies are commonly used for sequence delineation. The Kabat CDR definition (Kabat et al.,“Sequences of Proteins of Immunological Interest,” National Institutes of Health, Bethesda, Md. (1991)) is based upon antibody sequence variability. The Chothia CDR definition (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)) is based on three-dimensional structures of antibodies and topologies of the CDR loops. The Chothia CDR definitions are identical to the Kabat CDR definitions with the exception of HCDR1 and HCDR2. The North CDR definition (North et al.,“A New Clustering of Antibody CDR Loop Conformations”, Journal of Molecular Biology, 406, 228-256 (2011)) is based on affinity propagation clustering with a large number of crystal structures. The Kabat CDR definition is used in the present invention except HCDR1, which is defined with Kabat and Chothia.

Another aspect of the present invention pertains to isolated nucleic acid molecules encoding any of the aforementioned anti-IL-21 antibodies, expression vectors comprising the nucleic acid molecules, and host cells comprising the nucleic acid molecules.

Additionally, the present invention provides expression vectors containing the polynucleotide sequences previously described operably linked to a control sequence such as an expression sequence, a promoter and/or an enhancer sequence. A variety of expression vectors for the efficient synthesis of antibody polypeptide in prokaryotic systems, such as bacteria and eukaryotic systems, including but not limited to, yeast and mammalian cell culture systems have been developed. The vectors of the present invention can comprise segments of chromosomal, non-chromosomal and synthetic DNA sequences.

Methods for producing and purifying antibodies and antigen-binding fragments are 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, New York, chapters 5-8 and 15, ISBN 0-87969-314-2. Antigen-binding fragments can also be prepared by conventional methods. The present invention also provides recombinant host cells containing the recombinant vectors previously described. Cell lines of particular preference are selected based on high levels of expression, constitutive expression of protein of interest and minimal contamination from host proteins. Mammalian cell lines available as hosts for expression are well known in the art and include many immortalized cell lines, such as but not limited to, COS-7 cells, Chinese Hamster Ovary (CHO) cells, Baby Hamster Kidney (BHK) cells and many others including cell lines of lymphoid origin such as lymphoma, myeloma, or hybridoma cells. Preferred host cells for transformation of vectors and expression of the antibodies of the present invention are mammalian cells, e.g., NSO cells (non-secreting (0) mouse myeloma cells), Human embryonic kidney (HEK) 293, SP20 and Chinese hamster ovary (CHO) cells and other cell lines of lymphoid origin such as lymphoma, myeloma, or hybridoma cells. Antibodies of the present invention can be expressed in cell lines other than in hybridomas. Other eukaryotic hosts, such as yeasts, can be alternatively used. The antibodies and more specifically the antigen binding fragments thereof can also be produced from prokaryotic cells such as Escherichia coli. Nucleic acids, which comprise a sequence encoding an antibody according to the present invention, can be used for transformation of a suitable mammalian host cell.

The present invention further provides methods of purifying any of the aforementioned anti-IL-21 antibodies. The engineered antibodies or antigen binding fragments of the present invention may be prepared and purified using known methods.

The anti-IL-21 antibodies disclosed herein are useful for diagnostic, prognostic, and/or patient monitoring procedures, by detecting the level of IL-21 present in or on cells, tissues, or organs, whether in vivo and/or in various forms of ex vivo preparations, and in bodily fluids. The term“body fluid” refers to any fluid or fluid-like material derived from the body of a normal or diseased subject, such as blood, serum, plasma, lymph, bone marrow, urine, saliva, tears, cerebrospinal fluid, milk, amniotic fluid, bile, urine, bronchial fluid, ascites fluid, pus, and any other biological fluid product. Also included within the meaning of fluid-like materials are organ or tissue extracts, and culture media in which cells or tissue preparation from a subject have been incubated. An anti-IL-21 antibody described herein can be conjugated to an enzyme and used in an enzyme-linked immunosorbent assay (ELISA). Such assays are described in detail in, for example, Butler (1994)“ELISA” (Chapter 29), In: van Oss, C. J. et al., eds.,

Immunochemistry, Marcel Dekker, Inc., New York, pp.759-803. The present anti-IL-21 antibodies can also be used in radioimmunoassay and fluorescence-activated cell sorting (FACS) analysis of IL-21 expression.

As used herein, the term“contacting” refers to bringing an antibody or antigen- binding fragment thereof and an antigen or a target protein, e.g., IL-21, together in such a manner that form detectable antigen/antibody complex useful in detecting or quantifying the antigen or target protein in sample of tissue or body fluid in vitro assays. Such contacting can be accomplished in vitro, e.g., in a test tube, a microplate or the like. Alternately,“contacting” refers to mixing together an antibody or antigen-binding fragment thereof with a liquid such as serum, or plasma in vitro assays. Antibody Compositions and Methods

There are well-known methods in the art that a skilled artisan may use to form stable, detectable antigen-antibody complexes (see, e.g., Antibodies, A Laboratory Manual by Harlow and Lane (current edition), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, for conditions permitting formation of detectable antigen/antibody complexes).

The anti-IL-21 antibodies or antigen-binding fragment thereof of the present invention or the IL-21/anti-IL-21 antibody complexes described herein can be detectably labeled using any art-known means (see, e.g., Antibody Engineering Volume 2,

Kontermann, Roland; Dubel, Stefan (Eds.)). Labels can be, for example, without limitation, light-emitting or light-absorbing agents, chromophores, chromogens, magnetic or iron particles, dyes, fluorescents, fluorophores, phosphorescents, chemiluminescents, bioluminescents agent, radionuclides, enzymes, positron emission tomographic- imageable agents, magnetic micro-beads, ferrofluid nanoparticles, secondary antibodies, and magnetic resonance-imageable agents.

The term“detectably labeled” means that the anti-IL-21 antibody, or antigen- binding fragment thereof of the present invention, or a complex of IL-21/anti-IL-21 antibody has attached to it, either covalently or non-covalently, a useful detectable label. In direct conjugate-labeled antibody methods, many different useful labels can be employed including, for example, prosthetic group complexes, chromophores, chromogens (color-producing substrates), dyes, fluorescent compounds, fluorogenic compounds, radioactive isotopes, paramagnetic isotopes, and compounds that can be imaged by positron emission tomography (PET) and magnetic resonance imaging (MRI). Useful radiolabels, which are detected simply by gamma counter, scintillation counter, PET scanning, or autoradiography, include ³H, ¹²⁴I, ¹²⁵I, ¹³¹I, ³⁵S, and ¹⁴C. For in vivo diagnosis, radionuclides can be bound to an antibody or antigen-binding fragments either directly or indirectly using a chelating agent such as DTPA and EDTA. Examples of such radionuclides include ⁹⁹Tc, ¹²³I, ¹²⁵I, ¹³¹I, ¹¹¹In, ⁹⁷Ru, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁷²As, ⁸⁹Zr, ⁹⁰Y and ²⁰¹Tl. Other suitable labels are art-known or can be determined by routine

experimentation. In indirect methods, a secondary antibody can be conjugated with, for example but not restricted to an enzyme or fluorescent labels. Binding of the secondary antibody to the primary antibody, which is bound to the target antigen, can then be detected by reaction with a chromogenic substrate of the enzyme under appropriate conditions to yield a detectable signal.

Colorimetric detection can be used, employing chromogenic compounds that have, or result in, chromophores with high extinction coefficients, and which are therefore easily detectable. When later exposed to its substrate under appropriate reaction conditions, the enzyme will react with the substrate to produce a chemical label that can be detected, for example, by spectrophotometric, fluorometric, or visual means.

Enzymes commonly used for this purpose include horseradish peroxidase, alkaline phosphatase, glucose-6-phosphate dehydrogenase, malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase, α - glycerophosphate dehydrogenase, triose phosphate isomerase, asparaginase, glucose oxidase, β-galactosidase, ribonuclease, urease, catalase, glucoamylase, and

acetylcholinesterase. Examples of suitable prosthetic group complexes include, e.g., without limit, streptavidin/biotin and avidin/biotin. Use of chromogens is preferred because assays employing them can be easily performed in clinical diagnostic laboratories and reviewed by a pathologist with equipment commonly available in these laboratories. Commonly used chromogens include diaminobenzidine (DAB); DAB with enhancement; 3-amino-9-ethyl carbazole (AEC); 4-chloro-1-naphthol (4-CN); Hanker- Yates reagent; alpha-naphthol pyronin; 3,3’,5,5’-tetramethylbenzidine (TMB); Fast Blue BB; Fast Red TR; new fuchsin; BCIP-NBT; tetrazolium; tetranitoblue tetrazolium (TNBT); and immunogold with silver enhancement.

Useful fluorescent labels include umbelliferone, fluorescein, fluorescein isothiocyanate, dichlorotriazinylamine fluorescein, rhodamine, a dansyl group, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, fluorescamine, and Cy5 (Haugland ((1996) Handbook of Fluorescent Probes and Research Chemicals, Sixth Ed., Molecular Probes, Eugene, Ore.).

The anti-IL-21 antibodies, or antigen-binding fragments thereof, or IL-21/anti-IL- 21 antibody complexes of the present invention can also be detectably labeled using fluorescence-emitting metals such as ¹⁵²Eu⁺, or other members of the lanthanide series, by attaching them using such metal chelating groups as diethylenetriaminepentaacetic acid (DTPA) or ethylenediamine-tetraacetic acid (EDTA).

The anti-IL-21 antibody, or antigen-binding fragment thereof, or IL-21/anti-IL-21 antibody complexes of the present invention can also be detectably labeled by coupling them to a phosphorescent or chemiluminescent compound that can then be detected by the phosphorescence or luminescence that arises during the course of a chemical reaction. Examples of useful chemiluminescent compounds include luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt, and oxalate ester. Likewise, a bioluminescent compound such as luciferin, luciferase, or aequorin can be used to label an antibody or antigen-binding fragment thereof of the present invention. The presence of a bioluminescent protein is determined by detecting the presence of luminescence.

An antibody, or antigen-binding fragment thereof, of the present invention can also be attached to solid supports, which are particularly useful for immunoassays or purification of a target antigen. Such solid supports include, e.g., without limitation, beads, e.g., microscopic paramagnetic beads, glass, cellulose, poly-acrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene. Use of the Antibodies of the Invention in Immunoassays

A particular protein such as IL-21 can be measured by a variety of immunoassay methods including, e.g., without limitation, competitive and non-competitive assay systems using techniques such as, e.g., without limitation, Western blots,

radioimmunoassays, ELISA (enzyme linked immunosorbent assay),“sandwich” immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, and protein A immunoassays. For a review of immunological and immunoassay procedures in general, see for example Stites and Terr (eds.) (1991) Basic and Clinical Immunology (7th ed.). Moreover, the immunoassays of the present invention can be performed in many configurations as is known in the art (See for example Maggio (ed.) (1980) Enzyme Immunoassay CRC Press, Boca Raton, Fla.; Gosling J P 2000 Immunoassays: A Practical Approach

(Practical Approach Series) Oxford Univ Press; Diamandis & Christopoulus, 1996 Immunoassay Academic Press, San Diego, Calif.).

Immunoassays for quantitation can be performed by a variety of art-known methods. In brief, immunoassays to measure IL-21 can be either competitive or noncompetitive binding assays. In competitive binding assays, the sample to be analyzed competes with a labeled analyte for specific binding sites on a capture agent bound to a solid surface. Preferably, the capture agent is an antibody of the present invention, such as Ab2-1, which specifically binds to IL-21. The concentration of labeled analyte bound to the capture agent is inversely proportional to the amount of free analyte present in the sample.

In some embodiments, human IL-21 in a body fluid, e.g., serum or plasma, can be quantified using Quanterix’s SIMOA™ technology, which can enable protein

quantification at fg/ml levels. SIMOA™ technology (named for single molecule array) is based upon the isolation of individual immunocomplexes on paramagnetic beads using standard ELISA reagents. The main difference between Simoa and conventional immunoassays lies in the ability to trap single molecules in femtoliter-sized wells, allowing for a“digital” readout of each individual bead to determine if it is bound to the target analyte or not. The digital nature of the technique allows an average of 1000× sensitivity increase over conventional assays with CVs<10%. Commercially available SIMOA™ technology platforms offer multiplexing options up to a 10-plex on a variety of analyte panels, and assays can be automated. Multiplexing experiments can generate large amounts of data. Therefore, in some embodiments, a computer system is utilized to automate and control data collection settings, organization, and interpretation.

In a further embodiment, samples from human normal control and from patients with different diseases (in autoimmune diseases such as psoriasis, systemic lupus erythematosus (SLE), , chronic inflammatory bowel disease and Sjogren’s Syndrome) in the IL-21 Quanterix SIMOA^TM assay can be analysed. An“elevated level” of IL-21 may be determined by comparing the diseased samples to the healthy control samples. The term“elevated level” refers to a“cut point” above which patients may preferentially respond to therapy by administration of a therapeutic antibody that binds to IL-21.

Preferably, the“cut point” is 2 standard deviations (SD) above the mean of the healthy controls and is, more preferably, 3 standard deviations (SD) above the mean of the healthy controls.

Any observed significant increases in plasma IL-21 in autoimmune diseases compared with healthy control subjects can be used in patient tailoring whereby a“cut- point” based on IL-21 measurements in a clinical trial is determined. In this regard, IL-21 levels can be used to identify subgroups of patients that preferentially respond to a therapy. This identification can be done with IL-21 levels alone or in combination with other baseline patient characteristics or biomarkers, for example, CRP.

A variety of approaches may be employed to identify IL-21 cut points that define a responding patient subgroup in each disease state or indication of interest (see

Lipkovich I, Dmitrienko A, D’Agostino BR. Tutorial in biostatistics: data-driven subgroup identification and analysis in clinical trials. Statistics in medicine.2017;36(1): doi:10.1002/sim.7064; Foster JC, Taylor JMG, Ruberg SJ. Subgroup identification from randomized clinical trial data. Statistics in medicine.2011;30(24):10.1002/sim.4322. doi:10.1002/sim.4322; Ruberg SJ, Chen L, Wang Y. The mean does not mean as much anymore: finding sub-groups for tailored therapeutics. Clinical Trials.2010;7(5):

doi:10.1177/1740774510369350.

Accordingly, the present invention provides a method of selecting a patient population having an autoimmune disease such as psoriasis, systemic lupus

erythematosus (SLE), Crohn’s disease, chronic inflammatory bowel disease and

Sjogren’s Syndrome and having elevated IL-21 levels comprising assaying a plasma sample from a patient, determining levels of IL-21 present and administering an effective amount of a therapeutic IL-21 antibody when the plasma IL-21 levels are elevated.

Another embodiment of the present invention provides a therapeutic antibody that binds to human IL-21 for use in treating an autoimmune disease such as psoriasis, systemic lupus erythematosus (SLE), Crohn’s disease, chronic inflammatory bowel disease and Sjogren’s Syndrome) in a patient. An example of a therapeutic IL-21 antibody is one such those disclosed in WO 2015/142637 (Eli Lilly & Company). Such an antibody consists of two antibody heavy chains and two antibody light chains, in which each heavy chain comprises a heavy chain variable domain, the amino acid sequence of which is the sequence of SEQ ID NO:1 disclosed in WO 2015/142637, and in which each light chain comprises a light chain variable domain, the amino acid sequence of which is the sequence of SEQ ID No: 2 disclosed in WO 2015/142637.

The following examples are offered for illustrative purpose only, and are not intended to limit the scope of the present invention. Example 1

Antibody Expression and Purification

The polypeptides of the variable regions of the heavy chain and light chain, the complete heavy chain and light chain amino acid sequences of anti-human IL-21 antibodies, AbM2, Ab2-1 and Ab3-1, and the nucleotide sequences encoding the same, are listed below in the section entitled“SEQUENCE LISTING”. The amino acid sequences and the corresponding SEQ ID NOs of the CDRs of AbM2, Ab2-1 and Ab3-1 are shown below in Tables 1A and 1B; the SEQ ID NOs of the amino acid sequences as well as the encoding DNA sequences of variable regions and full-length of light and heavy chains of AbM2, Ab2-1 and Ab3-1 in Table 1C.

Table 1A

Table 1B

The anti-human IL-21 antibodies of the present invention, including, but not limited to, AbM2, Ab2-1 and Ab3, may be expressed transiently in HEK293 or CHO cells using vectors known in the art to be suitable for expression in HEK293 or CHO cells, following standard transfection procedures. Briefly, a recombinant vector or vectors comprising SEQ ID NO: 33 and SEQ ID NO: 34, or SEQ ID NO: 37 and SEQ ID NO: 38, or , SEQ ID NO: 41 and SEQ ID NO: 42 may be constructed and used to transiently transfect HEK293 EBNA cells. Transfected cells are cultured in standard serum-free medium 10 containing geneticin (G418) and tobramycin for 48 to 120 hours at 37 °C after transfection. The anti-human IL-21 antibody may be purified using Protein A MabSelect chromatrography resin (GE Healthcare, #17-5199-01) that is pre-equilibrated with PBS, pH7.2, or a HiLoad Superdex 20026/60 preparative grade size-exclusion chromatography column (GE Healthcare, #28-9893-36) that is pre-equilibrated with PBS, pH7.2. The bound protein is subsequently eluted with 10mM citrate, pH3 and the pooled fractions immediately neutralized with a 1:10 dilution of 1M Tris, pH8. The neutralized pool is concentrated using Amicon Ultra-15 concentrators (Millipore, #UFC903024). Example 2

IL-21 Antibody pairing analysis

Antibodies that may pair (or bind simultaneously) in an ELISA-based assay are determined using a surface plasmon resonance (SPR) assay on a Biacore 2000 instrument primed with HBS-P (GE Healthcare catalog BR-1003-68, 10 mM HEPES pH 7.4 + 150 mM NaCl + 0.0005% surfactant P20) running buffer and analysis temperature at 25 C. A CM4 chip containing immobilized goat anti-mouse IgG Fc specific antibody (Jackson ImmunoResearch catalog 115-005-008) is used to capture an antibody to IL-21. An IL- 21 antibody is captured on a test flow cell. Excess Mouse IgG isotype control antibody is injected to block remaining capacity to capture antibody. Human IL-21 is captured by the IL-21 antibody. A second antibody is injected to test for additive binding to the captured IL-21.

The antibodies of the present invention, AbM2, Ab2-1, and Ab3-1, are conjugated to beads (Quanterix Cat# 101360) at 0.5mg/mL and are biotinylated according to the manufacturer’s protocol at a ratio of 40 to 1 biotin to antibody. Nine combinations of the three antibody pairs are generated and analyzed against a recombinant IL-21 reference curve (100ng/mL– 1.0fg/mL). The beads are diluted in bead diluent (Quanterix, Cat #100458) and the detection antibodies are diluted in sample/detection buffer (Quanterix, Cat # 101359). Two pairs of antibodies are moved into further optimization due to the ability to discriminate IL-21 concentrations in the fg/mL range. The first pair is Ab2-1 and AbM2 which perform well in either orientation. The second pair is Ab3-21 as capture and Ab2-1 as detection.

Further optimization of the antibody pairs is performed to increase both sensitivity and percent recovery. The concentrations of both the capture and detection antibody are varied in a series of experiments to determine the optimal sensitivity. The capture antibodies are tested at three antibody concentrations (0.1, 0.5, and 1.0mg/ml). The detection antibodies are tested at three concentrations (0.5, 1.0, and 1.5mg/ml) using a 40X biotin to antibody ratio. The combinations yield 18 different pairs of antibody combinations. Recombinant human IL-21 protein is used as the standard over a range of 10,000 to 0.64 fg/ml. Antigen is diluted in assay diluent (PBS + 1% BSA) (Gibco Cat#20012-043 and Meso Scale Discovery Cat #R93BA-1 respectively). The capture antibodies are diluted in bead diluent (Quanterix, Cat #100458) and the detection antibodies are diluted in the sample/detection buffer (Quanterix, Cat # 101359). The optimal pair of antibodies and antibody concentrations is determined to be Ab 2-1 as capture antibody on the bead (1.0 mg/ml) and AbM2 biotinylated as detection (0.5 mg/ml).

Recombinant human IL-21 protein (25-155), as shown in SEQ ID NO: 44, can be expressed in Escherichia coli and found as an insoluble inclusion body. The inclusion body is isolated, solubilized in high-concentration urea buffer, and the solubilized material is purified by ion-exchange chromatography. The resulting main peak fractions are pooled and subjected to a sequential dialysis refolding process. The main peak fractions are then purified to homogeneity using reverse-phase chromatography. The main peak fractions are pooled, lyophilized by freeze-drying, resuspended in PBS, pH7.2 buffer and stored at -80C as working aliquots.

Example 3

Quanterix Simoa™ Assay

Anti-IL-21 antibody Ab2-1 is conjugated to carboxylated paramagnetic beads (Quanterix Cat# 100451) according to the standard Quanterix protocol at 1.0 mg/ml. Anti-IL-21 antibody AbM2 is biotinylated according to the standard Quanterix protocol (40:1 biotin ratio). For each run on the Quanterix, Ab2-1 beads (approximately 5 million beads/ml) are prepared in bead diluent (Quanterix Cat #100458) and biotinylated AbM2 antibody (0.5 μg/mL) is diluted in sample/detection buffer (Quanterix Cat # 101359) to appropriate volumes. Streptavidin-beta-galactosidase (SBG) (Quanterix Cat#100439) is prepared in SBG diluent (Quanterix Cat # 100376) at 150 pM. IL-21 recombinant protein or samples are diluted in assay buffer (600 mM NaCl, 0.5% Tween 20, 25% FBS, 2% BSA and 200 μg/ml HBR in PBS (Boston BioProducts Cat# BM-244; Thermo Scientific Cat #28320; Gibco Cat# 16010-159; Meso Scale Discovery Cat# R93BA-2; Scantibodies Cat# 3KC534-075 and Hyclone Cat# SH30258.01 respectively) at appropriate dilutions. Ab2-1 beads, biotinylated AbM2 antibody, calibrators, SBG, and supplied resorufin-beta- D galactopyranoside (RGP) (Quanterix Cat#10030) reagents are loaded into the instrument and run as a two-step Homebrew method according to the Simoa™ HD-1 Analyzer User Guide at room temperature. Binding data of Ab2-1 to recombinant human IL-21 protein is shown in Table 1 and Figure 1. To determine the spike and recovery in the Quanterix Simoa assay, different amounts of recombinant IL-21 are spiked into the human serum matrix. The percentage recovery is summarized in Table 2 and Figures 2 and 3. LLOQ in serum matrix was calculated as 30 fg/ml. Exploratory Validation has also been done in heparin plasma with comparable results for dilutional linearity, spike recovery and total error.

Table 1. IL-21 Quanterix Assay Binding Data

*AEB = average enzyme per bead

The data in Table 1 and Figure 1 demonstrate that the IL-21 Quanterix assay has a large dynamic range from 0.0003-5 pg/ml of IL-21 with a lower limit of Quantification of 0.03 pg/ml as calculated in serum matrix.

Table 2. IL-21 spike and recovery in human serum

The developed IL-21 Quanterix Simoa assay demonstrates an acceptable percent recovery for a given amount of IL-21 in serum. Example 4

Analysis of human normal control samples and diseased samples in the IL-21 Quanterix SIMOA™ assay

Human normal control samples and Sjögren’s and SLE patient samples, including 13 healthy, 11 Sjögren’s, and 14 SLE serum samples, are run in the IL-21 Quanterix SIMOA™ Homebrew assay. The samples are run in a Quanterix SIMOA™ Homebrew assay at a 1:2 dilution in the IL-21 Assay Buffer: NaCl (600mM, Boston BioProducts, Cat # BM-244), newborn calf serum (25%, Gibco, Cat #16010-159), tween 20 (0.5%, Thermo Scientific, Cat # 28320), BSA (2%, MSD, Cat # R93BA-2), and Heterophilic blocker (200ug/ml, Scantibodies, Cat # 3KC534-075) in PBS (1x, Hyclone, cat # SH30258.01) Ab 2.1 antibody is conjugated to beads for capture, and biotinylated AbM2 for detection. The results are presented in Figure 1. There is a significant difference in IL-21 levels between the normal healthy controls and both the Sjögren’s and SLE patients’ serum. In this regard, significant increases in plasma IL-21 in autoimmune diseases vs. healthy control subjects were observed.

Figure 1. Individual data points for the healthy controls and diseased samples.

SEQUENCE LISTING SEQ ID NO:1; PRT1; Artificial sequence RASQDISNYLN SEQ ID NO:2; PRT1; Artificial sequence YTSRLHS SEQ ID NO:3; PRT1; Artificial sequence QQFHTLRTF SEQ ID NO:4; PRT1; Artificial sequence GYTFTDYWMH SEQ ID NO:5; PRT1; Artificial sequence LIDTSDSYTIYNQKFKG SEQ ID NO:6; PRT1; Artificial sequence YGPLAMDY SEQ ID NO:7; PRT1; Artificial sequence RASKSIEKYIA SEQ ID NO:8; PRT1; Artificial sequence AGGTLQS SEQ ID NO:9; PRT1; Artificial sequence QQHEEYPLT SEQ ID NO:10; PRT1; Artificial sequence GYDFTGYTMN SEQ ID NO:11; PRT1; Artificial sequence LINPYNGGTAYSPKFKG SEQ ID NO:12; PRT1; Artificial sequence THYYGSEYTGMDY SEQ ID NO:13; PRT1; Artificial sequence KSSQSLLDVDGKTYLN SEQ ID NO:14; PRT1; Artificial sequence LVSKLDS SEQ ID NO:15; PRT1; Artificial sequence WQGTHFPYT SEQ ID NO:16; PRT1; Artificial sequence GYFFTLYMMH SEQ ID NO:17; PRT1; Artificial sequence YINPSSGYTEYNQKFKD SEQ ID NO:18; PRT1; Artificial sequence DFDY SEQ ID NO:19; PRT1; Artificial sequence

Claims

WE CLAIM: 1. An antibody, or antigen-binding fragment thereof, that binds human IL-21, comprising a light chain variable region (LCVR) and a heavy chain variable region (HCVR), wherein said LCVR comprises three light chain complementarity determining regions (LCDRs) and said HCVR comprises three heavy chain complementarity determining regions (HCDRs), wherein the amino acid sequences of said three LCDRs and said three HCDRs are selected from the group consisting of:

a) RASQDISNYLN (SEQ ID NO: 1), YTSRLHS (SEQ ID NO: 2),

QQFHTLRTF (SEQ ID NO: 3), GYTFTDYWMH (SEQ ID NO: 4), LIDTSDSYTIYNQKFKG (SEQ ID NO: 5), and YGPLAMDY (SEQ ID NO: 6);

b) RASKSIEKYIA (SEQ ID NO: 7), AGGTLQS (SEQ ID NO: 8),

QQHEEYPLT (SEQ ID NO: 9), GYDFTGYTMN (SEQ ID NO: 10), LINPYNGGTAYSPKFKG (SEQ ID NO: 11), and THYYGSEYTGMDY (SEQ ID NO: 12); and

c) KSSQSLLDVDGKTYLN (SEQ ID NO: 13), LVSKLDS (SEQ ID NO:

14), WQGTHFPYT (SEQ ID NO: 15), GYFFTLYMMH (SEQ ID NO: 16), YINPSSGYTEYNQKFKD (SEQ ID NO: 17), and DFDY (SEQ ID NO: 18).

2. The antibody, or antigen-binding fragment thereof of claim 1, wherein the amino acid sequences of said LCVR and said HCVR are selected from the group consisting of:

a) the amino sequence of SEQ ID NO: 19 and the amino sequence of SEQ ID NO: 20;

b) the amino sequence of SEQ ID NO: 21 and the amino sequence of SEQ ID NO: 22; and

c) the amino sequence of SEQ ID NO: 23 and the amino sequence of SEQ ID NO: 24.

3. The antibody of claim 1 or claim 2, wherein said antibody comprises a light chain and a heavy chain, wherein the amino acid sequences of said light chain and said heavy chain are selected from the group consisting of:

a) the amino sequence of SEQ ID NO:25 and the amino sequence of SEQ ID NO:26;

b) the amino sequence of SEQ ID NO:27 and the amino sequence of SEQ ID NO:28; and

c) the amino sequence of SEQ ID NO:29 and the amino sequence of SEQ ID NO:30.

4. The antibody of any one of claims 1 to 3, wherein said antibody comprises two light chains and two heavy chains, wherein the amino acid sequences of each of said light chains and each of said heavy chains are selected from the group consisting of: a) the amino sequence of SEQ ID NO:25 and the amino sequence of SEQ ID NO:26;

c) the amino sequence of SEQ ID NO:29 and the amino sequence of SEQ ID NO:30.

5. A polynucleotide comprising a nucleotide sequence encoding the LCVR and/or the HCVR, or the light chain and/or the heavy chain according to any one of claims 1 to 4.

6. The polynucleotide of claim 5, wherein said polynucleotide has the nucleotide sequence as shown in SEQ ID NOs: 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42.

7. A recombinant expression vector comprising a polynucleotide according to claim 5 or claim 6.

8. A host cell which has been transformed by the vector according to claim 7.

9. The antibody or antigen-binding fragment thereof of any one of claims 1 to 4, further comprising a detectable label.

10. The antibody, or antigen-binding fragment thereof of claim 9, wherein said detectable label is selected from the group consisting of a chromophore, a chromogen, a dye, a fluorescent agent, a fluorogenic agent, a phosphorescent agent, a chemiluminescent agent, a bioluminescent agent, a radionuclide, a positron emission tomography-imageable agent, and a magnetic resonance-imageable agent.

11. An antibody or antigen-binding fragment thereof of any one of claims 1 to 4 for use in diagnostic, prognostic, and/or patient monitoring procedure in vitro.

12. A composition comprising an antibody or antigen-binding fragment thereof, of any one of claims 1 to 4, and an acceptable carrier, diluent, or excipient.

13. An in vitro method of detecting or quantifying human IL-21 in a sample of tissue or body fluid comprising:

a) contacting said sample with the antibody or antigen-binding fragment thereof of any one of claims 1 to 4;

b) optionally, removing any non-specifically bound antibody or antigen- binding fragment thereof; and

c) detecting or quantifying the amount of the antibody or antigen-binding fragment thereof, which is specifically bound to human IL-21 in said sample.

14. A method of detecting or quantifying human IL-21 according to claim 13 wherein the method consists of an enzyme-linked immunosorbent assay (ELISA).

15. A kit for use in detecting or quantifying human IL-21 in vitro in a sample of tissue or body fluid, comprising:

a) a first reagent, wherein said first reagent is an antibody or antigen-binding fragment thereof, comprising a LCVR having the amino sequence of SEQ ID NO: 21 and a HCVR having the amino sequence of SEQ ID NO: 22; and

b) a second reagent, wherein said second reagent is an antibody or antigen- binding fragment comprising a LCVR having the amino sequence of SEQ ID NO: 19 and a HCVR having the amino sequence of SEQ ID NO: 20.

16. The method of claim 13 or the kit of claim 14, wherein the sample is a plasma sample or a serum sample.

17. An antibody according to any one of claims 1 to 10 for use in in vitro measurement of the amount of IL-21 in a human sample.