WO2017085693A1

WO2017085693A1 - Reporter gene assay methods for identifying and analyzing multi-specific binding proteins

Info

Publication number: WO2017085693A1
Application number: PCT/IB2016/056982
Authority: WO
Inventors: Matthias NAUMER; Thore SCHMEDT; Renate KRON
Original assignee: AbbVie Deutschland GmbH & Co. KG
Priority date: 2015-11-19
Filing date: 2016-11-18
Publication date: 2017-05-26

Abstract

Novel assays, methods, cells and kits for determining therapeutic efficacy of multi- specific binding proteins are provided.

Description

REPORTER GENE ASSAY METHODS FOR IDENTIFYING AND

ANALYZING MULTI- SPECIFIC BINDING PROTEINS

Field

[0001] Methods, assays, cells and kits for determining the efficacy of multivalent and multi-specific binding proteins (e.g., bi-specific therapeutics), as well as methods and assays for screening therapeutics, are provided.

Background of the Invention

[0002] Multi-specific binding proteins can simultaneously target two or more mediators of disease by a single pharmaceutical entity. For example, dual variable domain immunoglobulin (DVD-Ig) binding protein technology provides distinct technological, scientific and drug development advantages compared to conventional mAbs and to previous efforts to create multi-specific antibodies known in the art. The approach is compatible with any antibody, including humanized mAbs and fully-human mAbs, and can be extended beyond antibodies to receptor proteins and other, similar binding molecules. Multi-specific, e.g., bispecific, binding proteins can provide improved efficacy because they target multiple disease-causing molecules and pathways, and can address redundant disease processes in which two or more different molecules have the same disease-causing effect.

[0003] A need exists for rapidly assaying and screening the in vitro efficacy of multi- specific binding proteins such as, e.g., DVD-Ig binding proteins.

SUMMARY

[0004] The subject invention is based on the development of a novel combinatorial cell- based reporter assay that can be used to determine the efficacy of multi-specific protein therapeutics for binding one or more target molecules or for reducing ligand-receptor binding.

[0005] An aspect of the invention provides a method for assessing the efficacy of a multi- specific therapeutic for binding to one or more ligands. The method includes the steps of contacting a reporter cell expressing a promoter response element responsive to ligand- receptor binding operably linked to a nucleic acid sequence encoding a detectable moiety with one or more ligands, and allowing the one or more ligands to bind one or more receptors and mediate cell signaling; producing a detectable moiety, and contacting the cell with a multi-specific therapeutic, wherein a decrease in detectable moiety correlates with efficacy of the multi-specific therapeutic to bind one or more ligands.

[0006] In certain exemplary embodiments, the one or more ligands are cytokines, e.g., one or more of interleukin-17 (IL-17), tumor necrosis factor a (T Fa), interleukin-1 alpha (IL-la), and interleukin-1 beta (IL-Ιβ). In a particular embodiment, the one or more ligands are IL-17 and TNFa or IL-l and IL-Ιβ.

[0007] An aspect of the invention provides a method for assessing the efficacy of a multi- specific therapeutic for binding to each of two ligands. The method includes the steps of contacting a reporter cell expressing a promoter response element responsive to ligand- receptor binding operably linked to a nucleic acid sequence encoding a detectable moiety with two ligands, and allowing each of the ligands to bind its receptor and mediate cell signaling, producing a detectable moiety, and contacting the cell with a multi-specific therapeutic, wherein a decrease in detectable moiety production correlates with the efficacy of the multi-specific therapeutic for binding one or both of the ligands.

[0008] In certain exemplary embodiments, the two ligands are cytokines, e.g., two of interleukin-17 (IL-17), tumor necrosis factor a (TNFa), interleukin-1 alpha (IL-la), and interleukin-1 beta (IL-Ιβ). In a particular embodiment, the two ligands are IL-17 and TNFa or IL-la and IL-Ιβ.

[0009] In certain exemplary embodiments, the multi-specific therapeutic is a bi-specific therapeutic binding protein, e.g., a dual variable domain immunoglobulin (DVD-Ig). In other exemplary embodiments, the detectable moiety is visibly detectable, e.g., luciferase.

[0010] In certain exemplary embodiments, the promoter response element is a lipocalin

(LCN) 2 promoter response element, responsive to IL-17 and TNFa receptor binding, or an IL-8 promoter response element, responsive to IL-la and IL-Ιβ receptor binding. In certain exemplary embodiments, the cell is a cervical cancer (HeLa) cell, an

adenocarcinomic human alveolar basal epithelial (A549) cell, a human embryonic kidney HEK293 cell, or a lymphoma U937 cell. In certain exemplary embodiments, the bispecific binding protein comprises anti-TNFa and anti-IL-17 or anti-IL-la and anti-IL- 1β binding protein sequences. [0011] An aspect of the invention provides a method for assessing the efficacy of a multi- specific therapeutic binding protein (e.g., a DVD-Ig) to bind one or both of IL-17 and TNFa. The method includes the steps of contacting a cell with IL-17 and allowing it to bind its receptor, wherein IL-17-receptor binding mediates expression of a detectable moiety via an LCN 2 promoter response element, contacting the cell with TNFa and allowing it to bind its receptor, wherein TNFa-receptor binding mediates expression of a detectable moiety via the LCN 2 promoter response element, and contacting the cell with a multi-specific therapeutic, wherein a decrease in detectable moiety correlates with efficacy of the multi-specific therapeutic for binding one or both of IL-17 and TNFa.

[0012] An aspect of the invention provides a method for assessing the efficacy of a multi- specific therapeutic (e.g., a DVD-Ig) to bind one or both of IL-la and IL-Ιβ. The method includes the steps of contacting a cell with IL-la and allowing it to bind its receptor, wherein IL- la-receptor binding mediates expression of a detectable moiety via an IL-8 promoter response element; contacting the cell with IL-Ιβ and allowing it to bind its receptor, wherein IL-i -receptor binding mediates expression of a detectable moiety via the IL-8 promoter response element, and contacting the cell with a multi-specific therapeutic, wherein a decrease in detectable moiety correlates with efficacy of the multi- specific therapeutic for binding one or both of IL-la and IL-Ιβ.

[0013] An aspect of the invention provides a reporter cell responsive to DVD-Ig binding to one or both of IL-17 and TNFa. The reporter cell includes an LCN 2 promoter response element operably linked to a nucleic acid sequence encoding a detectable moiety, and one or more receptors that mediate signaling in the reporter cell upon one or both of IL-17- and TNFa-receptor binding via the LCN 2 promoter response element, wherein the signaling in the reporter cell promotes expression of the detectable moiety, and wherein DVD-Ig binding to one or both of IL-17 and TNFa decreases expression of the detectable moiety. In certain exemplary embodiments, a kit comprising the reporter cell and optional instructions for use are provided.

[0014] An aspect of the invention provides a reporter cell responsive to DVD-Ig binding to one or both of IL-la and IL-Ιβ. The reporter cell includes an IL-8 promoter response element operably linked to a nucleic acid sequence encoding a detectable moiety, and one or more receptors that mediate signaling in the reporter cell upon one or both of IL-la- and IL-i -receptor binding via the IL-8 promoter response element, wherein the signaling in the reporter cell promotes expression of the detectable moiety, and wherein DVD-Ig binding of one or both of IL-la and IL-Ιβ decreases expression of the detectable moiety. In certain exemplary embodiments, a kit comprising the reporter cell and optional instructions for use are provided.

BRIEF DESCRIPTION OF THE FIGURES

[0015] The foregoing and other features and advantages of the present invention will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings. The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0016] Figure 1 schematically depicts a circular plasmid map of the

pGL4[/wc2i7hIL8/Hygro] vector.

[0017] Figure 2 depicts luminescence data obtained for A549 cells transfected with

pGL4.32[/wc2i7NF-KB-RE/Hygro] vector. A549 cells were transfected using FuGENE^® HD Reagent for 24 hours, then treated with the indicated concentrations of IL-la (top row) or IL-Ιβ (bottom row) for 6 (gray line) or 24 hours (black line) at 37°C.

Luminescence data was collected using ONE-Glo™ Reagent and plotted as Relative Light Units (RLUs) or treated/untreated RLU ratio (Fold Response).

[0018] Figure 3 depicts luminescence data obtained for A549 cells transfected with

pGL4 [Zwc2i7hIL8/Hygro] vector. A549 cells were transfected using FuGENE^® HD Reagent for 24 hours, then treated with the indicated concentrations of IL-la (top row) or IL-Ιβ (bottom row) for 6 (gray line) or 24 hours (black line) at 37°C. Luminescence data was collected and plotted as RLUs or Fold Response.

[0019] Figure 4 depicts luminescence data obtained for A549 cells transfected with

pGL4.27[/wc2iVminP/Hygro] control vector. A549 cells were transfected using

FuGENE^® HD Reagent for 24 hours, then treated with the indicated concentrations of IL- la (left graph) or IL-Ιβ (right graph) for 6 (gray line) or 24 (black line) hours at 37°C. Luminescence data was collected using O E-Glo™ Reagent and plotted as RLUs or Fold Response.

[0020] Figures 5A and 5B depict luminescence data for NF-KB-RE-/WC2,P HEK293 cells transfected with IL1R1 (Figure 5 A) or pGL4.27[/wc2i7minP/Hygro] (Figure 5B).

GloResponse™ NF-KB-RE-/WC2,P HEK293 cells were transfected with IL1R1 or pGL4.27[/wc2i7minP/Hygro] (negative control) using FuGE E^® HD Reagent for 24 hours, then treated with the indicated concentrations of IL-l (top row in Figure 5 A; left graph in Figure 5B) or IL-Ιβ (bottom row in Figure 5 A; right graph in Figure 5B) for 6 hours (gray line) or 24 hours (black line) at 37°C. Luminescence data was collected using ONE-Glo™ Reagent and plotted as RLUs or Fold Response.

[0021] Figures 6A-6C depict bar graphs showing primary screen results. Raw data

depicting RLUs (Figure 6A), fold-response results (Figure 6B), and CELLTITER-GLO^® Luminescent Cell Viability Assay results (Figure 6C) are provided. A549 cells were transfected with pGL4[IL8-luc2P/Hygro] Vector using FuGENE^® HD Reagent for 48 hours and then kept under selection drug Hygromycin B for 2 weeks. From the stable pool, single clones were isolated by limited dilution and expanded. Each bar represents a cell population from a single clone. Cells were stimulated with Π.1β at 0.6pg/ml (EC50) or 0.3ng/ml (EClOO) for 6 hours at 37°C or left untreated (ECO). Luminescence data was collected using ONE-Glo™ Reagent and plotted as RLUs (Figure 6A) or Fold Response (Figure 6B). To monitor well to well cell number differences, another copy of the cell plate was tested using CellTiter-Glo^® Luminescent Cell Viability Assay (Figure 6C).

[0022] Figures 7A and 7B depict secondary screen results. ONE-Glo™ assay results

(Figure 7A) and fold-response results (Figure 7B) are provided. Selected A549 cell clones, stably transfected with pGL4[IL8-luc2P/Hygro], were stimulated with the indicated 1 :4 serial dilutions of ILi (lng/ml as top concentration) for 6 hours at 37°C. Luminescence data was collected using ONE-Glo™ Reagent and plotted as RLUs (Figure 7A) or Fold Response (Figure 7B).

[0023] Figures 8A-8F depict response curves for clones 1C-B3 and 0.3B-C1 in response to IL-la or IL-Ιβ. Figures 8A-8D provide the results for Clone 1C-B3 RLU, Clone 1C- B3 fold-response, Clone 0.3B-C1 RLU, and Clone 0.3B-C1 fold-response, respectively. The final two candidate clones were stimulated with the indicated 1 :4 serial dilutions of ILla (lng/ml as top concentration) (gray line) or ILip (top concentration at lng/ml) (black line) for 6 hours at 37°C. Luminescence data was collected using O E-Glo Reagent as described above. Figure 8E provides the data for the dose response of clone 1C-B3 in the presence of increasing concentrations of IL-la (gray line) or IL-Ιβ (black line). GloResponse™ IL8-luc2P/A549 clone 1C-B3 cells were plated at 20,000 cells/well for 24 hours and then incubated for an additional 6 hours with the indicated

concentrations of IL-la or IL-Ιβ. Luminescence data was collected using ONE-Glo™ reagent and plotted as RLUs. Figure 8F provides the data for the dose response of clone 1C-B3 in the presence of 40 pg/mL pre-mixed IL-la/IL-Ιβ, and increasing amounts of anti-IL-la/IL-Ιβ DVD-Ig. GloResponse™ IL8-luc2P/A549 clone 1C-B3 cells were plated at 30,000 cells/well for 24 hours. IL-la/IL-Ιβ and the indicated concentrations of anti-IL-la/IL-Ιβ DVD-Ig reference standard (black line) or drug substance sample (gray line) were pre-incubated for one hour and added to the cells. Luminescence data was collected after 4 hours of incubation using ONE-Glo™ reagent and plotted as RLUs.

[0024] Figure 9 depicts a circular plasmid map of the pGL4[lwc2_JP/LCN2/Hygro] vector.

[0025] Figures 1 OA- IOC depict results from a synergy study of IL-17 (IL-17 A) and

TNFa in HeLa cells (Figure 10A), A549 cells (Figure 10B), and U937 cells (Figure IOC). Cells were transfected with either pGL4[lwc2i7LCN2/Hygro], pGL4.32[/wc2i7NF-KB- RE/Hygro], pGL4.44[/wc2i7APl RE/Hygro], or pGL4[/wc2i7hIL8/Hygro] under optimized conditions. After 24 hours, cells were treated with IL-17 and TNFa at various concentrations for either 6 (left column) or 24 hours (right column). Luminescence data was collected using ONE-Glo™ Reagent and plotted as RLUs.

[0026] Figures 11A and 11B depict dose response curves showing the synergistic effect of TNF-a (Figure 11A) and IL-17 (IL-17A) (Figure 1 IB) using the LCN2 promoter vector in HeLa cells. Cells were transfected with pGL4[lwc2_JP/LCN2/Hygro] and then treated for 6 hours with a dose-response of IL-17 in the presence or absence of lng/mL TNF-a (Figure 11A) or a dose-response of TNF-a in the presence or absence of

200ng/mL IL-17 (Figure 1 IB). Luminescence data was collected using ONE-Glo™ Reagent and plotted as RLUs.

[0027] Figures 12A-12C depict bar graphs showing initial screen results. Figures 12A and 12B provide data for RLU in log scale and for RLU in linear scale, respectively. Figure 12C provides the fold-response over untreated cells. HeLa cells were transfected with pGL4[lwc2i⁵/LCN2/Hygro] vector and then kept under the selection drug Hygromycin B. Single cell clones were isolated and expanded from the stable cell pool by limited dilution. Bar graphs show the response of individual cell clones after treatment with 50ng/mL IL-17 (IL-17A) and 4ng/mL T F-α (stimulated) for 6 hours, or left untreated (unstimulated). Luminescence data was collected using ONE-Glo™

Reagent and plotted as indicated.

[0028] Figures 13A and 13B depict fold-responses over untreated cells for various

clones. Figure 13A provides data for HeLa cell clones 1A7, 1C9, 1D9, 1F5, 1G1, and 1G3. Figure 13B provides data for HeLa cell clones 1H3, 2H1, 2H3, 2H7, and 3F1.

Background RLU values are indicated in the upper left corner of each graph. The selected HeLa cell clones, stably transfected with pGL4[lwc2_JP/LCN2/Hygro], were stimulated for 6 hours at 37°C with various TNFa concentrations as indicated on the graphs. Luminescence data was collected using ONE-Glo™ Reagent and plotted as Fold Response.

[0029] Figures 14A and 14B depict fold-responses of clones 1D9 (Figure 14A) and 1F5

(Figure 14B) seeded at different cell densities in the presence of various concentrations of TNFa. Fold Response over background values are indicated in the upper left corner of each graph. Cells were seeded at 5000, 10,000 or 20,000 cells per well and stimulated for 6 hours with various cytokine concentrations in a matrix-type format as indicated on the graphs. Luminescence data was collected using ONE-Glo™ Reagent and plotted as RLUs.

[0030] Figures 15A and 15B depict fold-responses of clones 1D9 (Figure 15B) and 1F5

(Figure 15 A) plated at 20,000, 40,000 or 60,000 cells/well and stimulated for 6 hours by the indicated dose-response of IL-17 in the presence or absence of 20 ng/mL TNFa. Luminescence data was collected using ONE-Glo™ Reagent and plotted as Fold

Response.

[0031] Figure 16 depicts a synergistic TNFa dose response of clone 1F5. Absence of

IL-17 (inverted triangle); presence of 5 ng/mL IL-17 (circle); presence of 25 ng/mL IL-17 (square); presence of 200 ng/mL IL-17 (triangle). HeLa_LCN2-luc2P clone 1F5 were plated for 24 hours and incubated with various concentrations of IL-17 (IL-17 A) and TNFa. Luminescence data was collected using ONE-Glo™ Reagent and plotted as RLUs.

[0032] Figure 17 depicts the dose response of clone 1F5 in the presence of 3 ng/mL

TNFa, 50 ng/mL IL-17, and increasing amounts of TNFa/IL-17 DVD-Ig. HeLa_LCN2- luc2P clone 1F5 were plated for 24 hours and incubated with various concentrations of TNFa/IL-17 DVD-Ig. Luminescence data was collected using O E-Glo™ Reagent and plotted as RLUs.

DETAILED DESCRIPTION

[0033] Reporter assays, methods, kits, and cells responsive to multivalent and/or multi- specific binding proteins capable of binding immune cell receptors and/or ligands are provided.

I. Definitions

[0034] Unless otherwise defined herein, scientific and technical terms used herein have the meanings that are commonly understood by those of ordinary skill in the art. In the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The use of "or" means "and/or" unless stated otherwise. The use of the term "including," as well as other forms, such as "includes" and "included," is not limiting.

[0035] Generally, nomenclatures used in connection with cell and tissue culture,

molecular biology, immunology, microbiology, analytical chemistry, synthetic organic chemistry, medicinal chemistry, pharmaceutical chemistry, genetics and protein and nucleic acid chemistry as well as hybridization and laboratory procedures and techniques described herein are those well-known and commonly used in the art. The methods and techniques provided herein are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

Enzymatic reactions and purification techniques are performed according to

manufacturer's specifications, as commonly accomplished in the art or as described herein.

[0036] That the disclosure may be more readily understood, select terms are defined

below. [0037] The term "multi-specific binding protein" means a polypeptide having two, three, four or more distinct antigen binding sites, such that it can simultaneously bind to at least two, three, four or more targets and have specificity for two, three, four or more different targets.

[0038] The term "bispecific binding protein" means a polypeptide having at least two distinct antigen binding sites, such that it can simultaneously bind to at least two targets and have specificity for two different targets, i.e., either two different antigens or two different epitopes on the same antigen, with the proviso that the antigen binding sites of the bispecific binding protein are not antibody Fc regions. The two targets may be located on the same molecule, e.g., different epitopes on the same antigen, or may be located on separate (e.g., soluble) molecules, e.g., on two different cells, on a cell and a soluble antigen, on two soluble antigens, on two cytokines, etc. Bispecific binding proteins include bispecific antibodies but also include fusion proteins comprising known antibody components as well as a variety of other formats, including a DVD-Ig molecule, a BiTe^® molecule, a DART^® molecule, a DuoBody^™ molecule, a scFv/diabody-IgG molecule, a cross-over multi-specific {e.g., bispecific) molecule, a 2-in-l bispecific molecule, a knob-in-hole multi-specific {e.g., bispecific) molecule, a CovXBody molecule, an affibody molecule, a scFv/diabody-CH2/CH3 bispecific molecule, a IgG- non-Ig protein scaffold-based multi-specific {e.g., bispecific) molecule, and a

scFv/diabody linked to normal human protein like human serum albumin-bispecific molecule. Examples of different formats of bispecific binding proteins can be found in, e.g., US Patent Nos. 7,612,181; ,869,620; 5,864,019; 5,844,094; 5,990,275; 5,856,456; 6,476, 198; European Patent No. EP 0517024 Bl; US Patent Publication Nos.

20140155581; 20130058937; 20150018529; 20140051833; and 20140200331 each of which is incorporated herein by reference in its entirety for all purposes.

[0039] The term "ligand" means any substance capable of binding to, or of being bound by, another substance. Similarly, the term "antigen" means any substance to which an antibody may be generated. Although "antigen" is commonly used in reference to an antibody binding substrate, and "ligand" is often used when referring to receptor binding substrates, these terms encompass a wide range of overlapping chemical entities. For the avoidance of doubt, antigen and ligand are used interchangeably throughout herein. Antigens/ligands may be a peptide, a polypeptide, a protein, an aptamer, a polysaccharide, a sugar molecule, a carbohydrate, a lipid, an oligonucleotide, a polynucleotide, a synthetic molecule, an inorganic molecule, an organic molecule, and any combination thereof. In certain embodiments, a ligand is a cytokine.

[0040] The term "receptor" means a protein or polypeptide associated with a cell

membrane or wall having a high specific affinity for one or more ligands. Ligand binding may cause a conformational change in the receptor and may modify one or more activities in a cell. In certain embodiments, a receptor is a cytokine receptor.

[0041] The term "antibody" means an immunoglobulin (Ig) molecule, which is generally comprised of four polypeptide chains, two heavy chains (HC) and two light chains (LC), or a functional fragment, mutant, variant, or derivative thereof, that retains the epitope binding features of an Ig molecule. Such fragment, mutant, variant, or derivative antibody formats are known in the art. In an embodiment of a full-length antibody, each heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region (CH). The CH is comprised of three domains, CHI, CH2 and CH3. Each light chain is comprised of a light chain variable region (VL) and a light chain constant region (CL). The CL is comprised of a single CL domain. The VH and VL can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). Generally, each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy -terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2), or subclass.

[0042] The term "Fc region" defines the C-terminal region of an immunoglobulin heavy chain, which may be generated by papain digestion of an intact antibody. The Fc region may be a native sequence Fc region or a variant Fc region. The Fc region of an immunoglobulin generally comprises two constant domains, a CH2 domain and a CH3 domain, and optionally comprises a CH4 domain. Replacements of amino acid residues in the Fc portion to alter antibody effector function are known in the art (e.g., US Patent Nos. 5,648,260 and 5,624,821). The Fc region mediates several important effector functions, e.g., cytokine induction, antibody dependent cell mediated cytotoxicity

(ADCC), phagocytosis, complement dependent cytotoxicity (CDC), and half- life/clearance rate of antibody and antigen-antibody complexes. In some cases these effector functions are desirable for a therapeutic immunoglobulin but in other cases might be unnecessary or even deleterious, depending on the therapeutic objectives.

[0043] The term "antigen-binding portion" of a binding protein means one or more

fragments of a binding protein (e.g., an antibody or a receptor) that retain the ability to specifically bind to an antigen. The antigen-binding portion of a binding protein can be a fragment of a full-length antibody, as well as bispecific, dual specific, or multi-specific format; specifically binding to two or more different antigens. Examples of binding fragments encompassed within the term "antigen-binding portion" of a binding protein include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) an F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CHI domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment, which comprises a single variable domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv). Such single chain antibody fragments are also intended to be encompassed within the term "antigen-binding portion" of an antibody. Other forms of single chain antibodies, or fragments thereof, such as diabodies, are also encompassed. In addition, single chain antibodies also include "linear antibodies" comprising a pair of tandem Fv segments (VH-CH1-VH-CH1) which, together with complementary light chain polypeptides, form a pair of antigen binding regions.

[0044] The term "monovalent binding protein" means a binding protein comprising one antigen (ligand) binding site for each antigen. The term "multivalent binding protein" means a binding protein comprising two or more antigen (ligand) binding sites for the same antigen. In an embodiment, the multivalent binding protein is engineered to have three or more antigen binding sites, and is not a naturally occurring antibody. The term "multi-specific binding protein" refers to a binding protein capable of binding two or more related or unrelated targets. In an embodiment, a monovalent binding protein may be multi-specific in that it possesses one binding domain for each of the different target antigens.

[0045] The term "linker" means an amino acid residue or a polypeptide comprising two or more amino acid residues joined by peptide bonds that are used to link two

polypeptides (e.g., two VH or two VL domains). Such linker polypeptides are well known in the art (see, e.g., Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444- 6448; Poljak et a/. (1994) Structure 2: 1121-1123).

[0046] The term "epitope" means a region of an antigen that is bound by a binding

protein, e.g., a polypeptide and/or other determinant capable of specific binding to an immunoglobulin or cell receptor (e.g., T cell receptor). In certain embodiments, epitope determinants include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl, and, in certain embodiments, may have specific three dimensional structural characteristics, and/or specific charge characteristics. In an embodiment, an epitope comprises the amino acid residues of a region of an antigen known to bind to the complementary site on the specific binding partner. Binding proteins "bind to the same epitope" if the antibodies cross-compete (one prevents the binding or modulating effect of the other). Structural definitions of epitopes

(overlapping, similar, identical) are informative; and functional definitions encompass structural (binding) and functional (modulation, competition) parameters. Different regions of proteins may perform different functions such that specific epitopes on a cytokine may interact with its cytokine receptor to bring about receptor activation whereas other epitopes of the cytokine may be required for its stabilization. To abrogate the negative effects of cytokine signaling, the cytokine may be targeted with a binding protein that binds specifically to the receptor interacting epitope, thereby preventing the binding of its receptor. Alternatively, a binding protein may target the epitope

responsible for cytokine stabilization, thereby designating the protein for degradation. Methods of visualizing and modeling epitope recognition are known to one skilled in the art (US Patent No. 9,035,027).

[0047] An "affinity matured antibody" means an antibody with one or more alterations in one or more CDRs thereof that result in an improvement in the affinity of the antibody for its antigen, compared to a parent antibody that does not possess those alteration(s).

Exemplary affinity matured antibodies will have nanomolar or even picomolar affinities for their target antigen. Affinity matured antibodies are produced by procedures known in the art. Marks et al. (1992) BioTechnology 10:779-783 describes affinity maturation by VH and VL domain shuffling. Random mutagenesis of CDR and/or framework residues is described by Barbas et al. (1994) Proc. Nat. Acad. Sci. USA 91 :3809-3813; Schier et al. (1995) Gene 169: 147-155; Yelton et al. (1995) J. Immunol. 155: 1994-2004; Jackson et al. (1995) J. Immunol. 154(7):3310-9; and Hawkins et al. (1992) J. Mol. Biol. 226:889-896. Mutation at selective mutagenesis positions, contact or hypermutation positions with an activity enhancing amino acid residue is described, for example, in U.S. Patent No. 6,914,128.

[0048] The term "CDR-grafted antibody" means an antibody that comprises heavy and light chain variable region sequences in which the sequences of one or more of the CDR sequences of VH and/or VL are replaced with CDR sequences of another antibody. For example, the two antibodies can be from different species, such as antibodies having murine heavy and light chain variable regions in which one or more of the murine CDRs has been replaced with human CDR sequences.

[0049] The term "humanized antibody" means an antibody from a non-human species that has been altered to be more "human-like," i.e., more similar to human germline sequences. One type of humanized antibody is a CDR-grafted antibody, in which human CDR sequences are introduced into non-human VH and VL sequences to replace the corresponding non-human CDR sequences. A "humanized antibody" is also an antibody or a variant, derivative, analog or fragment thereof that comprises framework region (FR) sequences having substantially (e.g., at least 80%, at least 85%, at least 90%, at least 95%), at least 98%> or at least 99%> identity to) the amino acid sequence of a human antibody and at least one CDR having substantially the amino acid sequence of a non- human antibody. A humanized antibody may comprise substantially all of at least one, and typically two, variable domains (Fab, Fab', F(ab') 2, FabC, Fv) in which the sequence of all or substantially all of the CDRs correspond to those of a non-human immunoglobulin and the sequence of all or substantially all of the FR regions are those of a human immunoglobulin. The humanized antibody also may include the CHI, hinge, CH2, CH3, and CH4 regions of the human heavy chain. In an embodiment, a humanized antibody also comprises at least a portion of a human immunoglobulin Fc region. In some embodiments, a humanized antibody only contains a humanized light chain. In some embodiments, a humanized antibody only contains a humanized heavy chain. In some embodiments, a humanized antibody only contains a humanized variable domain of a light chain and/or humanized variable domain of a heavy chain. In some embodiments, a humanized antibody contains a light chain as well as at least the variable domain of a heavy chain. In some embodiments, a humanized antibody contains a heavy chain as well as at least the variable domain of a light chain.

[0050] The term "bispecific antibody" means an antibody that binds two different targets or two different regions of a target. In an embodiment, a bispecific antibody is an antibody that binds one antigens or epitopes on one of its two binding arms (one pair of HC/LC), and binds a different antigen or epitope on its second binding arm (a different pair of HC/LC). In an embodiment, a bispecific antibody has two distinct antigen binding arms (in both specificity and CDR sequences). In an embodiment, the bispecific antibody is monovalent for each antigen to which it binds. Bispecific antibodies have been produced using the quadroma technology (Milstein and Cuello (1983) Nature

305(5934):537-40) based on the somatic fusion of two different hybridoma cell lines expressing murine monoclonal antibodies with the desired specificities of the bispecific antibody. Because of the random pairing of two different Ig heavy and light chains within the resulting hybrid-hybridoma (or quadroma) cell line, up to ten different immunoglobulin species are generated, of which only one is the functional bispecific antibody. The presence of mispaired by-products, and significantly reduced production yields, means sophisticated purification procedures are required.

[0051] In an embodiment, bispecific antibodies can also be produced by chemical

conjugation of two different mAbs (Staerz et al. (19S5) Nature 314(6012):628-31) or smaller antibody fragments (Brennan et al. (1985) Science 229(4708):81-3). Bispecific antibodies and methods of making them are described in Glennie et al. (1987) J.

Immunol. 139(7):2367-75; Kriangkum et al. (2001) Biomol. Engin. 18(2):3140;

Economides et al. (2003) Nature Med. 9(l):47-52; Nakanishi et al. (2001) Ann. Rev. Immunol. 19:423-74; Grade et al. (1999) J. Clin. Invest. 104(10): 1393-401; Leung et al. (2000) J. Immunol. 164(12): 6495-502; Ito et al. (2003) J. Immunol. 170(9):4802-9; and Kami et al. (2002) J. Neuroimmunol. 125(1-2): 134-40.

[0052] In an embodiment, "bispecific diabodies (Dbs)" are produced from scFv

fragments by reducing the length of the linker connecting the VH and VL domain to approximately 5 residues (Peipp and Valerius (2002) Biochem. Soc. Trans. 30(4):507-l 1). Bispecific diabodies and methods of making them are described in Mack et al. (2005) Proc. Natl. Acad. Sci. USA 92(15):7021-5; and Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90(14):6444-8.18.

[0053] In an embodiment, "single-chain diabodies (scDb)" represent an alternative

strategy for improving the formation of bispecific diabody-like molecules (Holliger and Winter (1997) Cancer Immunol. Immunother. 45(3-4): 128-30; Wu et al. (1996)

Immunotechnol. 2(l):21-36). Bispecific single-chain diabodies and methods of making them are described in Holliger and Winter (1997) Cancer Immunol. Immunother.

45(34): 128-30; Wu et al. (1996) Immunotechnol. 2(l):21-36; Pluckthun and Pack (1997) Immunotechnol. 3(2):83-105; Ridgway et al. (1996) Protein Engin. 9(7):617-21); Lu et al. (2004) J. Biol. Chem. 279(4):2856-65; PCT Publication No. WO 0177342; and Miller et al. (2003) J. Immunol. 170(9):4854-61.

[0054] In an embodiment, the terms "dual variable domain binding protein" and "dual variable domain immunoglobulin" mean a binding protein that has two variable domains in each of its two binding arms {e.g., a pair of HC/LC), each of which is able to bind to an antigen. In certain embodiments, the dual variable domain binding proteins may be monospecific, i.e., capable of binding one antigen or epitope or multi-specific, i.e., capable of binding two or more antigens or epitopes. In certain embodiments, each variable domain binds the same antigen or epitope. In certain embodiments, at least one binding site comprises a ligand binding site (e.g., a receptor), capable of binding one or more receptor ligands (e.g., cytokines). In certain embodiments, a dual variable domain binding protein has two identical antigen binding arms, with identical specificity and identical CDR sequences, and is bivalent for each antigen to which it binds. Dual variable domain binding proteins comprising two heavy chain dual variable domain polypeptides and two light chain dual variable domain polypeptides are referred to as a DVD-Ig protein. In certain embodiments, each half of a four chain dual variable domain binding protein comprises a heavy chain dual variable domain polypeptide, and a light chain dual variable domain polypeptide, and two antigen binding sites. In certain embodiments, each binding site comprises a heavy chain variable domain and a light chain variable domain with a total of 6 CDRs involved in antigen binding per antigen binding site. [0055] The terms "parent antibody," "parent receptor," or "parent binding protein" mean a pre-existing binding protein from which a portion, e.g., a functional binding domain, is utilized to construct a novel binding protein, e.g., a bispecific binding protein.

[0056] The term "biological activity" means a biological property of a molecule (whether present naturally as found in vivo, or provided or enabled by recombinant means).

Biological properties include, but are not limited to, binding a receptor or receptor ligand, inducing cell proliferation, inhibiting cell growth, inducing the production or activation of other cytokines, inducing apoptosis, and enzymatic activity.

[0057] The term "neutralizing" means counteracting the biological activity of an

antigen/ligand (e.g., a cytokine) when a binding protein specifically binds to the antigen/ligand. In an embodiment, the neutralizing binding protein binds to an antigen/ligand and reduces its biologically activity by at least about 20%, 40%, 60%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more.

[0058] The term "specificity" means the ability of a binding protein to selectively bind an antigen/ligand.

[0059] The term "affinity" means the strength of the interaction between a binding

protein and an antigen/ligand, and is determined by the sequence and/or structure of the binding domain(s) of the binding protein as well as by the nature of the antigen/ligand, such as its size, shape, and/or charge. Binding proteins may be selected for affinities that provide desired therapeutic end-points while minimizing negative side-effects. Affinity may be measured using methods known to one skilled in the art (US Patent No.

9,035,027).

[0060] The term "potency" means the ability of a binding protein to achieve a desired effect, and is a measurement of its therapeutic efficacy. Potency may be assessed using methods known to one skilled in the art (US Patent No. 9,035,027).

[0061] The term "biological function" refers the specific in vitro or in vivo activity of a binding protein. Binding proteins may target several classes of antigens/ligands and achieve desired therapeutic outcomes through multiple mechanisms of action. Binding proteins may target soluble proteins, cell surface antigens, as well as extracellular protein deposits. Binding proteins may agonize, antagonize, and/or neutralize the activity of their targets. Binding proteins may assist in the clearance of the targets to which they bind, or may result in cytotoxicity when bound to cells. Portions of two or more antibodies may be incorporated into a multivalent format to achieve distinct functions in a single binding protein molecule. The in vitro assays and in vivo models used to assess biological function are known to one skilled in the art (US Patent No. 9,035,027).

[0062] The term "label," "detectable label," "detectable marker" or "detectable moiety" can be used interchangeably and refer to molecules, proteins, compounds or the like that produce a detectable signal, e.g., a signal that is detectable in one or more reporter assays and/or reporter cells described herein. Examples of detectable markers include various radioactive moieties, enzymes, prosthetic groups, fluorescent markers, luminescent markers, bioluminescent markers, metal particles, protein-protein binding pairs, protein- antibody binding pairs and the like. Examples of fluorescent proteins include, but are not limited to, yellow fluorescent protein (YFP), green fluorescence protein (GFP), cyan fluorescence protein (CFP), umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, phycoerythrin and the like. Examples of bioluminescent markers include, but are not limited to, luciferase (e.g., bacterial, firefly, click beetle and the like), luciferin, aequorin and the like. Examples of enzyme systems having visually detectable signals include, but are not limited to, galactosidases, glucorimidases, phosphatases, peroxidases, cholinesterases and the like.

125 131 35 14 3

Identifiable markers also include radioactive compounds such as I, I, S, C, H, 9⁰Y, ⁹⁹Tc, ^U1ln, ¹⁷⁷Lu, ¹⁶⁶Ho, and ¹⁵³Sm. Identifiable markers are commercially available from a variety of sources.

[0063] Fluorescent labels are described in many reviews, including Haugland, Handbook of Fluorescent Probes and Research Chemicals, Ninth Edition (Molecular Probes, Inc., Eugene, 2002); Keller and Manak, DNA Probes, 2nd Edition (Stockton Press, New York, 1993); Eckstein, editor, Oligonucleotides and Analogues: A Practical Approach (IRL Press, Oxford, 1991); and Wetmur (1991) Crit. Rev. Biochem. Mol. Biol. 26:227-259. Particular methodologies applicable to the invention are disclosed in US Patent Nos. 4,757, 141; 5, 151,507; and 5,091,519. As used herein, the term "fluorescent label" means a signaling moiety that conveys information through the fluorescent absorption and/or emission properties of one or more molecules. Such fluorescent properties include fluorescence intensity, fluorescence lifetime, emission spectrum characteristics, energy transfer, and the like. [0064] Other useful fluorophores include, but are not limited to, ALEXA FLUOR 350,

ALEXA FLUOR™ 532, ALEXA FLUOR™ 546, ALEXA FLUOR™, 568, ALEXA FLUOR™ 594, ALEXA FLUOR™ 647, BODIPY 493/503, BODIPY FL, BODIPY R6G, BODIPY 530/550, BODIPY TMR, BODIPY 558/568, BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, BODIPY 630/650, BODIPY 650/665, Cascade Blue, Cascade Yellow, Dansyl, lissamine rhodamine B, Marina Blue, Oregon Green 488, Oregon Green 514, Pacific Blue, rhodamine 6G, rhodamine green, rhodamine red, tetramethyl rhodamine, Texas Red (available from Molecular Probes, Inc., Eugene, OR), Cy2, Cy3.5, Cy5.5, Cy7 (Amersham Biosciences, Piscataway, N.J.) and the like. FRET tandem fluorophores may also be used, including, but not limited to, PerCP-Cy5.5, PE-Cy5, PE-Cy5.5, PE-Cy7, PE-Texas Red, APC-Cy7, PE-Alexa dyes (610, 647, 680), APC-Alexa dyes and the like.

[0065] Metallic silver or gold particles may be used to enhance signal of fluorescent labels (Lakowicz et al. (2003) Bio Techniques 34:62).

[0066] Biotin, or a derivative thereof, may also be used as a detectable label using

subsequent binding by a detectably labeled avidin/streptavidin derivative (e.g., phycoerythrin-conjugated streptavidin), or a detectably labeled anti-biotin antibody. Digoxigenin may be subsequently bound by a detectably labeled anti-digoxigenin antibody (e.g., fluoresceinated anti-digoxigenin).

[0067] In one embodiment the following hapten/antibody pairs are used for detection, in which each of the antibodies is derivatized with a detectable label: biotin/a-biotin, digoxigenin/a-digoxigenin, dinitrophenol (DNP)/a-DNP, 5-Carboxyfluorescein

(FAM)/a-FAM.

[0068] Favorable properties of fluorescent labeling agents to be used in the practice of the invention include high molar absorption coefficient, high fluorescence quantum yield, and photostability. In certain embodiments, labeling fluorophores desirably exhibit absorption and emission wavelengths in the visible (i.e., between 400 and 750 nm) rather than in the ultraviolet range of the spectrum (i.e., lower than 400 nm). Other desirable properties of the fluorescent moiety may include cell permeability and low toxicity, for example if labeling of the nucleic acid polymer is to be performed in a cell or an organism. [0069] In certain embodiments, response elements are provided for use in the assays and methods described herein. In certain embodiments, a response element is present within a promoter sequence and regulates (i.e., activates) transcription of a nucleic acid sequence operably linked to the promoter sequence. In certain embodiments, one or more intracellular signal transduction components bind to the response element to activate transcription. In certain embodiments, a response element controls expression of a nucleic acid sequence encoding one or more detectable moieties. In certain embodiments, a response element may consist of multiple and sometimes overlapping binding sites for DNA binding factors and is referred to as a multiresponse element (MRE). In certain embodiments, a response element is a cytokine response element that contains one or more binding sites for cytokine-specific nuclear transcription factors, e.g., IL-6 response element (IL-6 RE), IFN-stimulated response elements (ISREs), and the like.

[0070] Response elements are well-known in the art and include, but are not limited to, antigen receptor response element (ARRE), conserved lymphokine element (CLE), cyclic AMP-responsive element (CRE), CREB response element, estrogen receptor response element (ERE), glucocorticoid receptor response element (GRE), heat shock factor response element (HSE), serum response factor element (SRE), glucocorticoid response element (GRE), IL-6 responsive element (IL-6RE), interferon response sequence (IRS), serum response element (SRE), TGF-β-Ι inhibitory element (TIE), TP A response element (TRE), serum lipocalin-2 response element (LCNRE), Rev responsive element (RRE), and the like. A database of suitable response elements can be found at the Transcriptional Regulatory Element Database (TRED) website, included by reference in its entirety. In particular embodiments, the lipocalin (LCN) 2 promoter RE and/or the IL-8 responsive element (IL-8RE), is provided for use in the methods described herein. Other useful response elements include, but are not limited to, ARE, SRE, p53-RE, NFkB-RE, NFAT- RE, STAT-RE, SBE, IL-8-RE, BRE, ISRE, EGR1 promoter, Myc-RE, cAMP-RE, MTFl-RE, MTF2-RE, SRF-RE and the like. Suitable response elements are described in the art.

[0071] The term "vector" means a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid," is a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Other vectors include RNA vectors. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" may be used interchangeably as the plasmid is the most commonly used form of vector. However, other forms of expression vectors are also included, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions. A group of pHybE vectors (US Patent No. 8, 187,836) were used for parental binding protein and monovalent binding protein cloning.

[0072] The terms "recombinant host cell" or "host cell" refer to a cell into which

exogenous DNA has been introduced. In an embodiment, host cells include prokaryotic and eukaryotic cells. In an embodiment, eukaryotic cells include protist, fungal, plant and animal cells. In another embodiment, host cells include but are not limited to the prokaryotic cell hne E. coli; mammalian cell lines HeLa, CHO, HEK293, COS, NSO, SP2 and PER.C6; the insect cell line Sf9; and the yeast cell Saccharomyces cerevisiae.

[0073] The term "transfection" encompasses a variety of techniques commonly used for the introduction of exogenous nucleic acid (e.g., DNA) into a host cell, e.g.,

electroporation, calcium-phosphate precipitation, lipofection, DEAE-dextran transfection and the like.

[0074] The term "cytokine" refers to a protein released by one cell population that acts on another cell population as an intercellular mediator. The term "cytokine" includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of the native sequence cytokines.

[0075] The term "control" or "calibrator" refers to a composition known to not contain analyte ("negative control") or to contain analyte ("positive control"). A positive control can comprise a known concentration of analyte. A "positive control" can be used to establish assay performance characteristics and is a useful indicator of the integrity of reagents (e.g., analytes).

[0076] The term "specific binding partner" means a member of a specific binding pair. A specific binding pair comprises two different molecules that specifically bind to each other through chemical or physical means. Therefore, in addition to antigen and antibody specific binding, other specific binding pairs can include biotin and avidin (or streptavidin), carbohydrates and lectins, complementary nucleotide sequences, effector and receptor molecules, cofactors and enzymes, enzyme inhibitors and enzymes, and the like. Furthermore, specific binding pairs can include members that are analogs of the original specific binding members, for example, an analyte-analog. Immunoreactive specific binding members include antigens, antigen fragments, and antibodies, including monoclonal and polyclonal antibodies as well as complexes, fragments, and variants (including fragments of variants) thereof, whether isolated or recombinantly produced.

[0077] The terms "IL- 17" and "human IL- 17" ("ML- 17") include a homodimeric protein comprising two 15 kD IL-17A proteins (hIL-17A/A) and a heterodimeric protein comprising a 15 kD IL-17A protein and a 15 kD IL-17F protein ("hIL-17A/F"). The amino acid sequences of ML-17A and ML-17F are shown in Table 1. The term "hIL-17" includes recombinant hIL-17 (rhIL-17), which can be prepared by standard recombinant expression methods.

Table 1. Sequence of Human IL-17A and Human IL-17F

Sequence Sequence

Identifier

Protein

123456789012345678901234567901234567890

Human IL-17A SEQ ID NO. : 1 GI IPRNPGCPNSEDKNFPRTVMVNLNIHNRNTNTNPK

RSSDYYNRSTSPWNLHRNEDPERYPSVIWEAKCRHLGC INADGNVDYHMNSVPIQQEILVLRREPPHCPNSFRLEK ILVSVGCTCVTPIVHHVA

Human IL-17F SEQ ID NO. : 2 RKIPKVGH FFQKPESCPPVPGGSMKLDIGI INENQRV

SMSRNIESRSTSPWNYTVTWDPNRYPSEVVQAQCRNLG CINAQGKEDISMNSVPIQQETLVVRRKHQGCSVSFQLE KVLVTVGCTCVTPVIHHVQ [0078] The term "human T F-α" ("hTNF-a," or simply "hTNF") means a 17 kD secreted form and a 26 kD membrane associated form of a human cytokine, the biologically active form of which is composed of a trimer of noncovalently bound 17 kD molecules. The structure of hTNF-α is described further in, for example, Pennica et al. (1984) Nature 312:724-729; Davis et al. (1987) Biochem. 26: 1322-1326; and Jones et al. (1989) Nature 338:225-228. The term hTNF-a includes recombinant human TNF-a ("rhTNF-a"). The amino acid sequence of hTNF-a is shown in Table 2.

Table 2. Sequence Of Human TNF-a

[0079] The phrase "IL-17/TNF-a binding protein" means a bispecific binding protein

(e.g., DVD-Ig protein) that binds IL-17 and TNF-a. The relative positions of the TNF-a binding region and IL-17 binding region within the bispecific binding protein are not fixed (e.g., VD1 or VD2 of the DVD-Ig protein) unless specifically specified herein.

[0080] The term "human IL-Ιβ" (abbreviated herein as hIL-Ιβ, or herein simply IL-Ιβ) includes a pleiotropic cytokine involved in various immune responses, inflammatory processes, and hematopoiesis. The term human IL-Ιβ includes recombinant human IL-Ιβ (rh IL-Ιβ) that can be prepared by standard recombinant expression methods.

[0081] The term "human IL-la" (abbreviated herein as hIL-Ια, or herein simply IL-la) includes a cytokine that stimulates thymocyte proliferation by inducing IL-2 release, B- cell maturation and proliferation, and fibroblast growth factor activity. IL-la proteins are involved in the inflammatory response, being identified as endogenous pyrogens, and are reported to stimulate the release of prostaglandin and collagenase from synovial cells. The term human IL-l includes recombinant human IL-l (rh IL-l ) that can be prepared by standard recombinant expression methods.

[0082] The amino acid sequences of human IL-la and IL-Ιβ are shown in Table 3.

Table 3. Sequence Of Human IL- 1 a And Human IL- 1 β

[0083] Cells, compositions and methods for determining efficacy of multi-specific

therapeutic binding proteins and methods of making the same are provided. The multi- specific therapeutic binding proteins can be generated using various techniques.

Expression vectors, host cells and methods of generating the multi-specific therapeutic binding proteins are provided in this disclosure.

[0084] The antigen-binding variable domains of the binding proteins of this invention can be obtained from parent binding proteins, including polyclonal Abs, monoclonal Abs, and / or receptors capable of binding antigens of interest. These parent binding proteins may be naturally occurring or may be generated by recombinant technology. The person of ordinary skill in the art is well familiar with many methods for producing isolated antibodies and/or receptors, including, but not limited to, hybridoma techniques, selected lymphocyte antibody method (SLAM), a phage, yeast, or RNA-protein fusion display or other library, immunizing a non-human animal comprising at least some of the human immunoglobulin locus, and preparation of chimeric, CDR-grafted, and humanized antibodies. See, e.g., US Patent No. 9,035,027. Variable domains may also be prepared using affinity maturation techniques. The binding variable domains of the binding proteins can also be obtained from isolated receptor molecules obtained by extraction procedures known in the art (e.g., using solvents, detergents, and/or affinity

purifications), or determined by biophysical methods known in the art (e.g., X-ray crystallography, MR, interferometry, and/or computer modeling).

[0085] An embodiment is provided comprising selecting parent binding proteins with at least one or more properties desired in the binding protein molecule. In an embodiment, the desired property is one or more of those used to characterize antibody parameters, such as, for example, antigen specificity, affinity to antigen, potency, biological function, epitope recognition, stability, solubility, production efficiency, immunogenicity, pharmacokinetics, bioavailability, tissue cross reactivity, or orthologous antigen binding.

[0086] The linker sequence may be a single amino acid or a polypeptide sequence. In an embodiment, the choice of linker sequences is based on crystal structure analysis of several Fab molecules. There is a natural flexible linkage between the variable domain and the CH1/CL constant domain in Fab or antibody molecular structure. This natural linkage may contain approximately 10-12 amino acid residues, contributed by 4-6 residues from the C-terminus of a V domain and 4-6 residues from the N-terminus of a CL/CHl domain. The binding proteins may be generated using N-terminal 5-6 amino acid residues, or 1 1-12 amino acid residues, of CL or CHI as a linker in the light chain and heavy chains, respectively. The N-terminal residues of CL or CHI domains, particularly the first 5-6 amino acid residues, can adopt a loop conformation without strong secondary structures, and therefore can act as flexible linkers between the two variable domains. The N-terminal residues of CL or CHI domains are natural extension of the variable domains, as they are part of the Ig sequences, and therefore their use may minimize to a large extent any immunogenicity potentially arising from the linkers and junctions.

[0087] Other linker sequences may include any sequence of any length of a CL/CHl domain but not all residues of a CL/CHl domain; for example the first 5-12 amino acid residues of a CL/CHl domain; the light chain linkers can be from CK or C ; and the heavy chain linkers can be derived from CHI of any isotype, including Cyl, Cyl, Cy3, Cy4, Cal, Ca2, C5, Ce, and Cμ. Linker sequences may also be derived from other proteins such as Ig-like proteins (e.g., TCR, FcR, KIR); G/S based sequences (e.g., G4S repeats); hinge region-derived sequences; and other natural sequences from other proteins.

[0088] In an embodiment, one or more constant domains are linked to the variable

domains using recombinant DNA techniques. In an embodiment, a sequence comprising one or more heavy chain variable domains is linked to a heavy chain constant domain and a sequence comprising one or more light chain variable domains is linked to a light chain constant domain. In an embodiment, the constant domains are human heavy chain constant domains and human light chain constant domains, respectively. In an embodiment, the heavy chain is further linked to an Fc region. The Fc region may be a native sequence Fc region or a variant Fc region. In another embodiment, the Fc region is a human Fc region. In another embodiment, the Fc region includes Fc region from IgGl, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD.

[0089] Detailed description of specific binding proteins capable of binding specific

targets, and methods of making the same, is provided in the Examples section below.

II. Cell-Based Assays

Ligands

[0090] In certain embodiments of the invention, ligands are provided that bind to one or more cellular receptors for use in the assays and methods described herein. In certain embodiments, ligands bind to one or more receptors that mediate cell signaling.

Exemplary ligands include, but are not limited to cytokines, such as: Type I cytokines, e.g., interleukin-2 (IL-2), IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12 (IL- 12A, IL-12B), IL-13, IL-14, IL-15, IL-16, IL-17 (IL-17A, IL-17F), IL-19, IL-20, IL-21, IL-22, IL-23 (IL-23 A, IL-23B), IL-24, IL-25 (IL-17E), IL-26, IL-27 (IL-27B), IL-28, IL- 29, IL-30, IL-31, IL-32, IL-35, IL-36, stem cell factor (c-Kit ligand), granulocyte- monocyte colony-stimulating factor (GM-CSF), monocyte CSF (M-CSF, CSF1), granulocyte CSF (G-CSF, CSF3) and the like; Type II cytokines, e.g., interferon a (IFN- a), IFN-β, IFN-γ, interleukin-10 (IL-10), IL-19, IL-22, IL-26, interferon- s (type III interferons), leukemia inhibitory factor (LIF), oncostatin M, and the like; tumor necrosis factor (TNF) superfamily cytokines, e.g., TNF (T FSF1), lymphotoxin-a (LTa,

TNFSF1), lymphotoxin-αβ (LTa ), BAFF (CD257, TNFSF13B), APRIL (CD256, TNFSF13), osteoprotegrin (OPG, TNFRSFl IB), CD40L, CD27L, CD30L, FASL, 4- 1BBL, and TRAIL and the like; interleukin 1 (IL-1) family cytokines, e.g., IL-la, IL-Ιβ, IL-1 receptor antagonist (IL-IRa), IL-18, IL-33 and the like; and other cytokines such as, e.g., transforming growth factor-β (TGF-β). In particular embodiments, ligands including one or any combination of T Fa, IL-17, IL-la, and/or IL-Ιβ are provided for use in the assays and methods described herein.

[0091] In exemplary embodiments, cells of the invention can bind two or more distinct ligands to mediate cell signaling and subsequent expression of one or more detectable labels. In certain embodiments, cells of the invention can bind a ligand pair to mediate cell signaling and subsequent expression of one or more detectable labels. Ligand pairs can bind to the same or to different receptors. In exemplary embodiments, ligand pairs include, but are not limited to, TNFa / IL-17, IL-la / IL-Ιβ, and IL-1 β / IL-17.

Receptors

[0092] In certain embodiments of the invention, receptors are provided for use in the assays and methods described herein. In certain embodiments, receptors of the invention mediate cell signaling (i.e., signal transduction). In other embodiments, receptors of the invention are endogenous and present at the surface of a cell used in a cell-based assay described herein. In other embodiments, receptors of the invention are exogenous and are expressed at the surface of a cell that has been engineered to express one or more exogenous receptors using routine methods of molecular biology. In certain

embodiments of the invention, one or more endogenous receptors and/or one or more exogenous receptors are expressed at the surface of the same cell. In certain

embodiments, receptors of the invention bind one or more ligands, e.g., one or more cytokines. In other embodiments, receptors of the invention bind ligand pairs, e.g., cytokine pairs.

[0093] Exemplary receptors include, but are not limited to, cytokine receptors (e.g., interleukin receptors (ILRs), tumor necrosis factor receptors (TNFRs)), growth factor receptors (e.g., TGF receptors, epidermal growth factor (EGF) receptors), extracellular matrix receptors, chemokine receptors, hormone receptors, transmitter receptors, survival factor receptors, and the like. In particular embodiments, one or more ILRs and/or one or more TNFRs are provided for use in the assays and methods described herein. [0094] Interleukin receptors include, but are not limited to, CD121a/ILlRl,

CD121b/ILlR2, CD25/IL2RA, CD122/IL2RB, CD132/IL2RG, CD123/IL3RA,

CD131/IL3RB, CD124/IL4R, CD132/IL2RG, CD125/IL5RA, CD131/IL3RB,

CD126/IL6RA, CD130/IR6RB, CD127/IL7RA, CD132/IL2RG, CXCR1/IL8RA, CXCR2/IL8RB/CD128, CD129/IL9R, CD210/IL10RA, CDW210B/IL10RB, ILl lRA, CD212/IL12RB1, IR12RB2, IL13R, IL15RA, CD4, CDw217/IL17RA, IL17RB,

CDw218a/IL18Rl, IL20R, IL21R, IL22R, IL23R, LY6E, IL20R1, IL27RA, IL28R, and IL31RA.

[0095] TNFRs include, but are limited to: 4- 1BB/T FRSF9/CD 137, lymphotoxin

R/TNFRSF3, BAFF R/TNFRSF13C NGF R/T FRSF16, BCMA/TNFRSF 17

Osteoprotegerin/TNFRSFl lB, CD27/TNFRSF7 OX40/T FRSF4, CD30/T FRSF8 RA K/T FRSF 11 A, CD40/T FRSF5 RELT/TNFRSF 19L, DcR3/T FRSF6B

TACI/T FRSF13B, DcTRAIL R1/TNFRSF23 TNFRH3/T FRSF26, DcTRAIL

R2/TNFRSF22 TNF RI/TNFRSF1A, DR3/T FRSF25 TNF RII/TNFRSF1B,

DR6/T FRSF21 TRAIL Rl/TNFRSFIOA, EDA2R/TNFRSF27/XEDAR TRAIL

R2/T FRSF10B, EDAR TRAIL R3/T FRSF10C, Fas/T FRSF6/CD95 TRAIL

R4/T FRSF 10D, GITR/T FRSF 18 TRO Y/T FRSF 19, HVEM/T FRSF 14 and

TWEAK R/TNFRSF12.

[0096] In certain exemplary embodiments, a cell is provided that expresses at its surface two, three, four or more receptors. In certain embodiments, a cell expresses receptor pairs that can bind ligand pairs, e.g., cytokine ligand pairs. In exemplary embodiments, receptor pairs include, but are not limited to, CD 120a or CD 120b / CD217, CD 12 la or CD121b / CD217, CD121a / CD121b, CD121a / CD121a or CD121b.

Vectors

[0097] Examples of expression vectors suitable for expression in prokaryotic cells such as E. coli include, for example, plasmids of the types: pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids and pUC- derived plasmids; expression vectors suitable for expression in yeast include, for example, YEP24, YIP5, YEP51, YEP52, pYES2, and YRP17; and expression vectors suitable for expression in mammalian cells include, for example, pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectors. [0098] Vectors can be targeted for delivery to host cells via conventional transformation or transfection techniques. In certain aspects, the introduced vector and the target cells together are capable of targeted recombination. As used herein, the terms

"transformation" and "transfection" are intended to refer to a variety of art-recognized techniques for introducing an exogenous nucleic acid sequence (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran- mediated transfection, lipofection, electroporation, optoporation, injection and the like. Suitable methods for transforming or transfecting cells can be found in Sambrook et al, Current Protocols in Molecular Biology. John Wiley & Sons, Inc., 1998; Methods in Enzymol. (Vols. 68, 100, 101, 118, and 152-155) (1979, 1983, 1986 and 1987); and DNA Cloning, D. M. Clover, Ed., IRL Press, Oxford (1985), and other laboratory manuals.

Cells

[0099] In certain embodiments, cells are provided that can be used to assess the ability of a multi-specific therapeutic to reduce binding of one or more ligands to one or more receptors. In certain embodiments, the cells provided herein produce a detectable signal when contacted with one or more ligands, which signal is decreased when ligand-receptor binding is reduced or inhibited by a molecule such, e.g., as a multi-specific binding protein. The level of detectable signal produced by cells in the presence of ligand and in the absence multi-specific binding protein can be detected and compared to the level of detected signal produced by cells in the presence of ligand and in the presence of the multi-specific binding protein to determine the efficacy of the multi-specific binding protein for inhibiting ligand-receptor binding. In certain embodiments, ligand-receptor binding is reduced by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91% about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% or more, or any percentage points or ranges between these percentages. In certain embodiments, ligand-receptor binding is reduced by between about 5% and about 95%, between about 10% and about 90%, between about 20% and about 90%, between about 30%) and about 90%, between about 35% and about 60%, between about 40% and about 65%), between about 50% and about 70%, between about 55% and about 75%, between about 60% and about 80%>, between about 65%> and about 85%>, between about 70% and about 90%), between about 75% and about 95%, or between about 80%> and about 99%, or any percentage points within these ranges.

[0100] A host cell can be any prokaryotic or eukaryotic cell. For example, host cells can be cells such as yeast, insect cells, plant cells, reptilian cells, fish cells, amphibian cells (such as Xenopus cells), or mammalian cells. Suitable mammalian host cells for the assays and methods of the invention include Chinese Hamster Ovary (CHO cells) (including dhfir- CHO cells, described in Urlaub and Chasin (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g., as described in Kaufman and Sharp (1982) o/. Biol. 159:601-621), NSO myeloma cells, COS cells and SP2 cells. Other examples of useful mammalian host cell lines are monkey kidney CVl line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al. (1997) J. Gen Virol. 36:59); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/- DHFR (CHO, Urlaub et al. (1980) Proc. Natl. Acad. Sci. USA 77:4216); mouse Sertoli cells (TM4, Mather (1980) Biol. Reprod. 23:243-251); monkey kidney cells (CVl ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al. (1982) Annals NY Acad. Sci. 383 :44- 68); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).

[0101] Other suitable target cells are known to those skilled in the art. Both cultured and explanted cells may be used according to the invention. The present invention is also adaptable for in vivo use using viral vectors including, but not limited to, replication defective retroviruses, adenoviruses, adeno-associated viruses and the like. For in vivo use, marker selection can be applied to the entire organism such as, for example, by using an automated worm sorter (Union Biometrica, Zurich Switzerland).

[0102] Host cells useful in the present invention include human cells including, but not limited to, embryonic cells, fetal cells, and adult stem cells. Human stem cells may be obtained, for example, from a variety of sources including embryos obtained through in vitro fertilization, from umbilical cord blood, from bone marrow and the like. In one aspect of the invention, target human cells are useful as donor-compatible cells for transplantation, e.g., via alteration of surface antigens of non-compatible third-party donor cells, or through the correction of genetic defect in cells obtained from the intended recipient patient. In another aspect of the invention, target cells, including without limitation human cells, are useful for the production of therapeutic proteins, peptides, antibodies and the like.

[0103] The host cells of the invention may be cultured in a variety of media.

Commercially available media such as Ham's F10™ (Sigma), Minimal Essential Medium™ ((MEM), Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium™ ((DMEM), Sigma) are suitable for culturing the host cells. In addition, any of the media described in Ham et al. (1979) Meth. Enz. 58:44; Barnes et al. (1980) Anal. Biochem. 102:255; US Patent Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or US Patent No. Re. 30,985 may be used as culture media for the host cells, the entire teachings of which are incorporated herein by reference. Any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as gentamycin drug), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art. The culture conditions, such as temperature, pH, and the like, are generally those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.

Multi-Specific Therapeutic Proteins

[0104] In certain embodiments, the present invention is directed to methods of screening and/or determining efficacy of multi-specific therapeutic proteins. In one embodiment, a cell expressing one or more target receptors which, upon ligand binding, express a detectable label, can be used to determine efficacy of one or more multi-specific therapeutic proteins to bind to one or more target receptors and/or inhibit ligand binding to one or more target receptors. Target receptors may be endogenously expressed by the cell, and/or exogenously expressed, e.g., by an expression vector. The ability of the multi-specific therapeutic protein to alter ligand binding to one or more target receptors can be determined by observing an increase or a decrease of a detectable label under transcriptional control of one or more response elements that are responsive to ligand binding to the one or more target receptors.

[0105] In certain embodiments, the assay is a cell-based assay comprising contacting a cell expressing one or more target receptors and one or more detectable labels that are expressed upon binding of one or more ligands to the one or more target receptors.

Determining the ability of a multi-specific therapeutic protein to modulate receptor-ligand binding can be accomplished, for example, by determining the level of detectable label expressed.

[0106] In certain embodiments, a multi-specific therapeutic protein of the invention is a bi-specific therapeutic selected from the group consisting of a TNFa / IL-17 binding protein, IL-la / IL-Ιβ binding protein, or IL-Ιβ / IL-17 binding protein.

[0107] It will be readily apparent to those skilled in the art that other suitable

modifications and adaptations of the compositions and methods described herein may be made using suitable equivalents without departing from the scope of the embodiments disclosed herein.

Exemplification

[0108] Having now described certain embodiments in detail, the same will be more

clearly understood by reference to the following examples, which are included for purposes of illustration only and are not intended to be limiting.

Example 1. Anti-TNF/Anti-IL-17 DVD-Ig Binding Protein Construct

[0109] A human anti -human TNF/anti -human IL-17 dual variable domain

immunoglobulin (DVD-Ig) protein was constructed (Table 4). Table 4. Variable and Constant Domain Sequences of Anti-IL-17/TNF DVD-Ig Binding Protein

EVQLVESGGGLVQPGRSLR LSCAASGFTFDDYAMHWVR QAPGKGLEWVSAITWNSGH

DVD HEAVY SEQ ID NO.:4 IDYADSVEGRFTISRDNAK VARIABLE NSLYLQMNSLRAEDTAVYY

D2E7-GS10-B6-17 CAKVSYLSTASSLDYWGQG DVD-Ig Protein TLVTVSSGGGGSGGGGSEV

QLVQSGAEVKKPGSSVKVS CKASGGSFGGYGIGWVRQA PGQGLEWMGGITPFFGFAD YAQKFQGRVTITADESTTT AYMELSGLTSDDTAVYYCA RDPNEFWNGYYSTHDFDSW GQGTTVTVSS

D2E7 VH SEQ ID NO.:5 EVQLVESGGGLVQPGRSLR

LSCAASGFTFDDYAMHWVR QAPGKGLEWVSAITWNSGH IDYADSVEGRFTISRDNAK NSLYLQMNSLRAEDTAVYY CAKVSYLSTASSLDYWGQG TLVTVSS

LINKER SEQ ID NO.:6 GGGGSGGGGS

B6-17 VH SEQ ID NO.:7 EVQLVQSGAEVKKPGSSVK

VSCKASGGSFGGYGIGWVR QAPGQGLEWMGGITPFFGF ADYAQKFQGRVTITADEST TTAYMELSGLTSDDTAVYY CARDPNEFWNGYYSTHDFD SWGQGTTVTVSS

CH SEQ ID NO.:8 ASTKGPSVFPLAPSSKSTS

GGTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFPAVL QSSGLYSLSSWTVPSSSL GTQTYICNVNHKPSNTKVD KKVEPKSCDKTHTCPPCPA PELLGGPSVFLFPPKPKDT LMISRTPEVTCVWDVSHE DPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRWSVLT VLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPRE PQVYTLPPSRDELTKNQVS LTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDG SFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKS LSLSPGK DVD LIGHT SEQ ID NO.:9 DIQMTQSPSSLSASVGDRV

TITCRASQGIRNYLAWYQQ VARIABLE KPGKAPKLLIYAASTLQSG

D2E7-GS10-B6-17 VPSRFSGSGSGTDFTLTIS

SLQPEDVATYYCQRYNRAP DVD-Ig Protein YTFGQGTKVEIKRGGSGGG

GSGEIVLTQSPDFQSVTPK EKVTITCRASQDIGSELHW YQQKPDQPPKLLIKYASHS TSGVPSRFSGSGSGTDFTL TINGLEAEDAGTYYCHQTD SLPYTFGPGTKVDIKR

D2E7 VL SEQ ID NO.: 10 DIQMTQSPSSLSASVGDRV

TITCRASQGIRNYLAWYQQ KPGKAPKLLIYAASTLQSG VPSRFSGSGSGTDFTLTIS SLQPEDVATYYCQRYNRAP YTFGQGTKVEIKR

LINKER SEQ ID NO.: l l GGSGGGGSG*

B6-17 VL SEQ ID NO.: 12 EIVLTQSPDFQSVTPKEKV

TITCRASQDIGSELHWYQQ KPDQPPKLLIKYASHSTSG VPSRFSGSGSGTDFTLTIN GLEAEDAGTYYCHQTDSLP YTFGPGTKVDIKR

CL SEQ ID NO.: 13 TVAAPSVFI FPPSDEQLKS

GTASWCLLNNFYPREAKV QWKVDNALQSGNSQESVTE QDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSS PVTKSFNRGEC

Example 2. Anti-IL-la/Anti-IL-Ιβ DVD-Ig Binding Protein Constructs A human anti-human IL-la / Anti-human IL-Ιβ DVD-Ig binding protein was constructed (Table 5). Table 5. Sequence Identifier Amino Acid Sequence Variable and

Constant

Domain

Sequences of

Anti-IL-loi /

Anti-IL-Ιβ

DVD-Ig Binding

Proteins

12345678901234567890

E26.13-SS-X3 SEQ ID NO: 17 EVQLVESGGGVVQPGRSLRL

SCSASGFIFSRYDMSWVRQA

DVD-Ig HEAVY

PGKGLEWVAYISHGGAGTYY VARIABLE PDSVKGRFTISRDNSKNTLF LQMDSLRPEDTGVYFCARGG VTKGYFDVWGQGTPVTVSSA STKGPQVQLVESGGGVVQPG RSLRLSCTASGFTFSMFGVH WVRQAPGKGLEWVAAVSYDG SNKYYAESVKGRFTISRDNS KNILFLQMDSLRLEDTAVYY CARGRPKVVIPAPLAHWGQG TLVTFSS

Table 5. Sequence Identifier Amino Acid Sequence Variable and

Constant

Domain

Sequences of

Anti-IL-loi /

Anti-IL-Ιβ

DVD-Ig Binding

Proteins

12345678901234567890

E26.13 VH SEQ ID NO: 18 EVQLVESGGGVVQPGRSLRL

SCSASGFIFSRYDMSWVRQA PGKGLEWVAYISHGGAGTYY PDSVKGRFTISRDNSKNTLF LQMDSLRPEDTGVYFCARGG VTKGYFDVWGQGTPVTVSS

LINKER SEQ ID NO: 19 ASTKGP

X3 VH SEQ ID NO: 20 QVQLVESGGGVVQPGRSLRL

SCTASGFTFSMFGVHWVRQA PGKGLEWVAAVSYDGSNKYY AESVKGRFTISRDNSKNILF LQMDSLRLEDTAVYYCARGR PKVVIPAPLAHWGQGTLVTF SS

CH SEQ ID NO: 21 ASTKGPSVFPLAPSSKSTSG

GTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPEAAGG PSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPGK

E26.13-SS-X3 SEQ ID NO: 22 DIQMTQSPSSLSASVGDRVT

ITCRASGNIHNYLTWYQQTP

DVD-Ig LIGHT

GKAPKLLIYNAKTLADGVPS VARIABLE RFSGSGSGTDYTFTISSLQP EDIATYYCQHFWSIPYTFGQ GTKLQITRTVAAPDIQMTQS Table 5. Sequence Identifier Amino Acid Sequence Variable and

Constant

Domain

Sequences of

Anti-IL-loi /

Anti-IL-Ιβ

DVD-Ig Binding

Proteins

12345678901234567890

PSSVSASVGDRVTITCRASQ GISSWLAWYQQKPGKAPKLL IYEASNLETGVPSRFSGSGS GSDFTLTISSLQPEDFATYY CQQTSSFLLSFGGGTKVEHK R

E26.13 VL SEQ ID NO: 23 DIQMTQSPSSLSASVGDRVT

ITCRASGNIHNYLTWYQQTP GKAPKLLIYNAKTLADGVPS RFSGSGSGTDYTFTISSLQP EDIATYYCQHFWSIPYTFGQ GTKLQITR

Linker SEQ ID NO: 24 TVAAP

X3 VL SEQ ID NO: 25 DIQMTQSPSSVSASVGDRVT

ITCRASQGISSWLAWYQQKP GKAPKLLIYEASNLETGVPS RFSGSGSGSDFTLTISSLQP EDFATYYCQQTSSFLLSFGG GTKVEHKR

CL SEQ ID NO:26 TVAAPSVFIFPPSDEQLKSG

TASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKS FNRGEC

E26.13-LL-X3 SEQ ID NO: 27 EVQLVESGGGVVQPGRSLRL

SCSASGFIFSRYDMSWVRQA

DVD-Ig HEAVY

PGKGLEWVAYISHGGAGTYY VARIABLE PDSVKGRFTISRDNSKNTLF LQMDSLRPEDTGVYFCARGG VTKGYFDVWGQGTPVTVSSA STKGPSVFPLAPQVQLVESG GGVVQPGRSLRLSCTASGFT FSMFGVHWVRQAPGKGLEWV AAVSYDGSNKYYAESVKGRF TISRDNSKNILFLQMDSLRL Table 5. Sequence Identifier Amino Acid Sequence Variable and

Constant

Domain

Sequences of

Anti-IL-loi /

Anti-IL-Ιβ

DVD-Ig Binding

Proteins

12345678901234567890

EDTAVYYCARGRPKVVIPAP LAHWGQGTLVTFSS

E26.13 VH SEQ ID NO: 28 EVQLVESGGGVVQPGRSLRL

Linker SEQ ID NO:29 ASTKGPSVFPLAP

X3 VH SEQ ID NO: 30 QVQLVESGGGVVQPGRSLRL

CH SEQ ID NO: 31 ASTKGPSVFPLAPSSKSTSG

E26.13-LL-X3 SEQ ID NO: 32 DIQMTQSPSSLSASVGDRVT

ITCRASGNIHNYLTWYQQTP DVD-Ig LIGHT

GKAPKLLIYNAKTLADGVPS Table 5. Sequence Identifier Amino Acid Sequence Variable and

Constant

Domain

Sequences of

Anti-IL-loi /

Anti-IL-Ιβ

DVD-Ig Binding

Proteins

12345678901234567890

VARIABLE RFSGSGSGTDYTFTISSLQP EDIATYYCQHFWSIPYTFGQ GTKLQITRTVAAPSVFIFPP DIQMTQSPSSVSASVGDRVT ITCRASQGISSWLAWYQQKP GKAPKLLIYEASNLETGVPS RFSGSGSGSDFTLTISSLQP EDFATYYCQQTSSFLLSFGG GTKVEHKR

E26.13 VL SEQ ID NO: 33 DIQMTQSPSSLSASVGDRVT

Linker SEQ ID NO: 34 TVAAPSVFIFPP

X3 VL SEQ ID NO: 35 DIQMTQSPSSVSASVGDRVT

CL SEQ ID NO: 36 TVAAPSVFIFPPSDEQLKSG

X3-SS- E26.13 SEQ ID NO: 37 QVQLVESGGGVVQPGRSLRL

SCTASGFTFSMFGVHWVRQA

DVD-Ig HEAVY

PGKGLEWVAAVSYDGSNKYY VARIABLE AESVKGRFTISRDNSKNILF LQMDSLRLEDTAVYYCARGR PKVVIPAPLAHWGQGTLVTF SSASTKGPEVQLVESGGGVV QPGRSLRLSCSASGFIFSRY Table 5. Sequence Identifier Amino Acid Sequence

Variable and

Constant

Domain

Sequences of

Anti-IL-loi /

Anti-IL-Ιβ

DVD-Ig Binding

Proteins

12345678901234567890

DMSWVRQAPGKGLEWVAYIS HGGAGTYYPDSVKGRFTISR DNSKNTLFLQMDSLRPEDTG VYFCARGGVTKGYFDVWGQG TPVTVSS

X3 VH SEQ ID NO: 38 QVQLVESGGGVVQPGRSLRL

Linker SEQ ID NO: 39 ASTKGP

E26.13 VH SEQ ID NO: 0 EVQLVESGGGVVQPGRSLRL

CH SEQ ID NO: 1 ASTKGPSVFPLAPSSKSTSG

GTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPEAAGG PSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPGK Table 5. Sequence Identifier Amino Acid Sequence Variable and

Constant

Domain

Sequences of

Anti-IL-loi /

Anti-IL-Ιβ

DVD-Ig Binding

Proteins

12345678901234567890

X3-SS- E26.13 SEQ ID NO: 2 DIQMTQSPSSVSASVGDRVT

ITCRASQGISSWLAWYQQKP

DVD-Ig LIGHT

GKAPKLLIYEASNLETGVPS VARIABLE RFSGSGSGSDFTLTISSLQP EDFATYYCQQTSSFLLSFGG GTKVEHKRTVAAPDIQMTQS PSSLSASVGDRVTITCRASG NIHNYLTWYQQTPGKAPKLL

lYNAKTLADGVPSRFSGSGS GTDYTFTISSLQPEDIATYY CQHFWSIPYTFGQGTKLQIT R

X3 VL SEQ ID NO: 3 DIQMTQSPSSVSASVGDRVT

LINKER SEQ ID NO: 44 TVAAP

E26.13 VL SEQ ID NO: 45 DIQMTQSPSSLSASVGDRVT

CL SEQ ID NO: 46 TVAAPSVFIFPPSDEQLKSG

X3-LL- E26.13 SEQ ID NO: 47 QVQLVESGGGVVQPGRSLRL

SCTASGFTFSMFGVHWVRQA

DVD-Ig HEAVY

PGKGLEWVAAVSYDGSNKYY VARIABLE AESVKGRFTISRDNSKNILF LQMDSLRLEDTAVYYCARGR Table 5. Sequence Identifier Amino Acid Sequence

Variable and

Constant

Domain

Sequences of

Anti-IL-loi /

Anti-IL-Ιβ

DVD-Ig Binding

Proteins

12345678901234567890

PKVVIPAPLAHWGQGTLVTF SSASTKGPSVFPLAPEVQLV ESGGGVVQPGRSLRLSCSAS GFI FSRYDMSWVRQAPGKGL EWVAYISHGGAGTYYPDSVK GRFTISRDNSKNTLFLQMDS LRPEDTGVYFCARGGVTKGY FDVWGQGTPVTVSS

X3 VH SEQ ID NO: 8 QVQLVESGGGVVQPGRSLRL

LINKER SEQ ID NO: 9 ASTKGPSVFPLAP

E26.13 VH SEQ ID NO: 50 EVQLVESGGGVVQPGRSLRL

CH SEQ ID NO: 51 ASTKGPSVFPLAPSSKSTSG

GTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPEAAGG PSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRW Table 5. Sequence Identifier Amino Acid Sequence Variable and

Constant

Domain

Sequences of

Anti-IL-loi /

Anti-IL-Ιβ

DVD-Ig Binding

Proteins

12345678901234567890

QQGNVFSCSVMHEALHNHYT QKSLSLSPGK

X3-LL- E26.13 SEQ ID NO: 52 DIQMTQSPSSVSASVGDRVT

ITCRASQGISSWLAWYQQKP

DVD-Ig LIGHT

GKAPKLLIYEASNLETGVPS VARIABLE RFSGSGSGSDFTLTISSLQP EDFATYYCQQTSSFLLSFGG GTKVEHKRTVAAPSVFIFPP DIQMTQSPSSLSASVGDRVT ITCRASGNIHNYLTWYQQTP GKAPKLLIYNAKTLADGVPS RFSGSGSGTDYTFTISSLQP EDIATYYCQHFWSIPYTFGQ GTKLQITR

X3 VL SEQ ID NO: 53 DIQMTQSPSSVSASVGDRVT

LINKER SEQ ID NO: 54 TVAAPSVFIFPP

E26.13 VL SEQ ID NO: 55 DIQMTQSPSSLSASVGDRVT

CL SEQ ID NO:56 TVAAPSVFIFPPSDEQLKSG

TASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKS FNRGEC Table 5. Sequence Identifier Amino Acid Sequence Variable and

Constant

Domain

Sequences of

Anti-IL-loi /

Anti-IL-Ιβ

DVD-Ig Binding

Proteins

12345678901234567890

E26.35-SS-X3 SEQ ID NO: 57 EVQLVESGGGVVQPGRSLRL JM SCSASGFIFSRYDMSWVRQA PGKGLEWVAYISHGGAGTYY

DVD-Ig HEAVY

PDSVKGRFTISRDNSKNTLF VARIABLE LQMDSLRAEDTAVYYCARGG VYKGYFDVWGQGTPVTVSSA STKGPQVQLVESGGGVVQPG RSLRLSCTASGFTFSMFGVH WVRQAPGKGLEWVAAVSYDG SNKYYAESVKGRFTISRDNS KNILFLQMDSLRLEDTAVYY CARGRPKVVIPAPLAHWGQG TLVTVSS

E26.35 VH SEQ ID NO: 58 EVQLVESGGGVVQPGRSLRL

SCSASGFIFSRYDMSWVRQA PGKGLEWVAYISHGGAGTYY PDSVKGRFTISRDNSKNTLF LQMDSLRAEDTAVYYCARGG VYKGYFDVWGQGTPVTVSS

LINKER SEQ ID NO:59 ASTKGP

X3 JM VH SEQ ID NO: 60 QVQLVESGGGVVQPGRSLRL

SCTASGFTFSMFGVHWVRQA PGKGLEWVAAVSYDGSNKYY AESVKGRFTISRDNSKNILF LQMDSLRLEDTAVYYCARGR PKVVIPAPLAHWGQGTLVTV SS

CH SEQ ID NO: 61 ASTKGPSVFPLAPSSKSTSG

GTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPEAAGG PSVFLFPPKPKDTLMISRTP Table 5. Sequence Identifier Amino Acid Sequence Variable and

Constant

Domain

Sequences of

Anti-IL-loi /

Anti-IL-Ιβ

DVD-Ig Binding

Proteins

12345678901234567890

EVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPGK

E26.35-SS-X3 SEQ ID NO: 62 DIQMTQSPSSLSASVGDRVT JM ITCRASGNIHNYLTWYQQTP GKAPKLLIYNAKTLADGVPS

DVD-Ig LIGHT

RFSGSGSGTDYTFTISSLQP VARIABLE EDIATYYCQHFWSIPYTFGQ GTKLQITRTVAAPDIQMTQS PSSVSASVGDRVTITCRASQ GISSWLAWYQQKPGKAPKLL

lYEASNLETGVPSRFSGSGS GSDFTLTISSLQPEDFATYY CQQTSSFLLSFGGGTKVEIK R

E26.35 VL SEQ ID NO: 63 DIQMTQSPSSLSASVGDRVT

LINKER SEQ ID NO: 64 TVAAP

X3 JM VL SEQ ID NO: 65 DIQMTQSPSSVSASVGDRVT

ITCRASQGISSWLAWYQQKP GKAPKLLIYEASNLETGVPS RFSGSGSGSDFTLTISSLQP EDFATYYCQQTSSFLLSFGG GTKVEIKR

CL SEQ ID NO: 66 TVAAPSVFIFPPSDEQLKSG Table 5. Sequence Identifier Amino Acid Sequence Variable and

Constant

Domain

Sequences of

Anti-IL-loi /

Anti-IL-Ιβ

DVD-Ig Binding

Proteins

12345678901234567890

E26.13 JM-SS- SEQ ID NO: 67 EVQLVESGGGVVQPGRSLRL

X3 SCSASGFIFSRYDMSWVRQA

PGKGLEWVAYISHGGAGTYY

DVD-Ig HEAVY

PDSVKGRFTISRDNSKNTLF VARIABLE LQMDSLRPEDTGVYFCARGG VTKGYFDVWGQGTTVTVSSA STKGPQVQLVESGGGVVQPG RSLRLSCTASGFTFSMFGVH WVRQAPGKGLEWVAAVSYDG SNKYYAESVKGRFTISRDNS KNILFLQMDSLRLEDTAVYY CARGRPKVVIPAPLAHWGQG TLVTFSS

E26.13 JM VH SEQ ID NO: 68 EVQLVESGGGVVQPGRSLRL

SCSASGFIFSRYDMSWVRQA PGKGLEWVAYISHGGAGTYY PDSVKGRFTISRDNSKNTLF LQMDSLRPEDTGVYFCARGG VTKGYFDVWGQGTTVTVSS

LINKER SEQ ID NO: 69 ASTKGP

X3 VH SEQ ID NO: 70 QVQLVESGGGVVQPGRSLRL

CH SEQ ID NO: 71 ASTKGPSVFPLAPSSKSTSG

GTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQT Table 5. Sequence Identifier Amino Acid Sequence Variable and

Constant

Domain

Sequences of

Anti-IL-loi /

Anti-IL-Ιβ

DVD-Ig Binding

Proteins

12345678901234567890

YICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPEAAGG PSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPGK

E26.13 JM-SS- SEQ ID NO: 72 DIQMTQSPSSLSASVGDRVT

X3 ITCRASGNIHNYLTWYQQTP

GKAPKLLIYNAKTLADGVPS

DVD-Ig LIGHT

RFSGSGSGTDYTFTISSLQP VARIABLE EDIATYYCQHFWSIPYTFGQ GTKLEIKRTVAAPDIQMTQS PSSVSASVGDRVTITCRASQ GISSWLAWYQQKPGKAPKLL

lYEASNLETGVPSRFSGSGS GSDFTLTISSLQPEDFATYY CQQTSSFLLSFGGGTKVEHK R

E26.13 JM VL SEQ ID NO: 73 DIQMTQSPSSLSASVGDRVT

ITCRASGNIHNYLTWYQQTP GKAPKLLIYNAKTLADGVPS RFSGSGSGTDYTFTISSLQP EDIATYYCQHFWSIPYTFGQ GTKLEIKR

LINKER SEQ ID NO: 74 TVAAP

X3 VL SEQ ID NO: 75 DIQMTQSPSSVSASVGDRVT

ITCRASQGISSWLAWYQQKP GKAPKLLIYEASNLETGVPS RFSGSGSGSDFTLTISSLQP EDFATYYCQQTSSFLLSFGG Table 5. Sequence Identifier Amino Acid Sequence Variable and

Constant

Domain

Sequences of

Anti-IL-loi /

Anti-IL-Ιβ

DVD-Ig Binding

Proteins

12345678901234567890

GTKVEHKR

CL SEQ ID NO:76 TVAAPSVFIFPPSDEQLKSG

E26.35-SS-X3 SEQ ID NO: 77 EVQLVESGGGVVQPGRSLRL

SCSASGFIFSRYDMSWVRQA

DVD-Ig HEAVY

PGKGLEWVAYISHGGAGTYY VARIABLE PDSVKGRFTISRDNSKNTLF LQMDSLRAEDTAVYYCARGG VYKGYFDVWGQGTPVTVSSA STKGPQVQLVESGGGVVQPG RSLRLSCTASGFTFSMFGVH WVRQAPGKGLEWVAAVSYDG SNKYYAESVKGRFTISRDNS KNILFLQMDSLRLEDTAVYY CARGRPKVVIPAPLAHWGQG TLVTFSS

E26.35 VH SEQ ID NO: 78 EVQLVESGGGVVQPGRSLRL

LINKER SEQ ID NO:79 ASTKGP

X3 VH SEQ ID NO: 80 QVQLVESGGGVVQPGRSLRL

CH SEQ ID NO: 81 ASTKGPSVFPLAPSSKSTSG Table 5. Sequence Identifier Amino Acid Sequence Variable and

Constant

Domain

Sequences of

Anti-IL-loi /

Anti-IL-Ιβ

DVD-Ig Binding

Proteins

12345678901234567890

E26.35-SS-X3 SEQ ID NO: 82 DIQMTQSPSSLSASVGDRVT

ITCRASGNIHNYLTWYQQTP

DVD-Ig LIGHT

GKAPKLLIYNAKTLADGVPS VARIABLE RFSGSGSGTDYTFTISSLQP EDIATYYCQHFWSIPYTFGQ GTKLQITRTVAAPDIQMTQS PSSVSASVGDRVTITCRASQ GISSWLAWYQQKPGKAPKLL

lYEASNLETGVPSRFSGSGS GSDFTLTISSLQPEDFATYY CQQTSSFLLSFGGGTKVEHK R

E26.35 VL SEQ ID NO: 83 DIQMTQSPSSLSASVGDRVT

LINKER SEQ ID NO: 84 TVAAP

X3 VL SEQ ID NO: 85 DIQMTQSPSSVSASVGDRVT

ITCRASQGISSWLAWYQQKP Table 5. Sequence Identifier Amino Acid Sequence Variable and

Constant

Domain

Sequences of

Anti-IL-loi /

Anti-IL-Ιβ

DVD-Ig Binding

Proteins

12345678901234567890

GKAPKLLIYEASNLETGVPS RFSGSGSGSDFTLTISSLQP EDFATYYCQQTSSFLLSFGG GTKVEHKR

CL SEQ ID NO: 86 TVAAPSVFIFPPSDEQLKSG

E26.13-SS-X3 SEQ ID NO: 87 EVQLVESGGGVVQPGRSLRL JM SCSASGFIFSRYDMSWVRQA PGKGLEWVAYISHGGAGTYY

DVD-Ig HEAVY

PDSVKGRFTISRDNSKNTLF VARIABLE LQMDSLRPEDTGVYFCARGG VTKGYFDVWGQGTPVTVSSA STKGPQVQLVESGGGVVQPG RSLRLSCTASGFTFSMFGVH WVRQAPGKGLEWVAAVSYDG SNKYYAESVKGRFTISRDNS KNILFLQMDSLRLEDTAVYY CARGRPKVVIPAPLAHWGQG TLVTVSS

E26.13 VH SEQ ID NO: 88 EVQLVESGGGVVQPGRSLRL

LINKER SEQ ID NO: 89 ASTKGP

X3 JM VH SEQ ID NO: 90 QVQLVESGGGVVQPGRSLRL

SCTASGFTFSMFGVHWVRQA PGKGLEWVAAVSYDGSNKYY AESVKGRFTISRDNSKNILF LQMDSLRLEDTAVYYCARGR PKVVIPAPLAHWGQGTLVTV Table 5. Sequence Identifier Amino Acid Sequence Variable and

Constant

Domain

Sequences of

Anti-IL-loi /

Anti-IL-Ιβ

DVD-Ig Binding

Proteins

12345678901234567890

SS

CH SEQ ID NO: 91 ASTKGPSVFPLAPSSKSTSG

E26.13-SS-X3 SEQ ID NO: 92 DIQMTQSPSSLSASVGDRVT JM ITCRASGNIHNYLTWYQQTP GKAPKLLIYNAKTLADGVPS

Anti-IL- RFSGSGSGTDYTFTISSLQP

lalpha/beta

EDIATYYCQHFWSIPYTFGQ DVD-Ig LIGHT

GTKLQITRTVAAPDIQMTQS VARIABLE PSSVSASVGDRVTITCRASQ GISSWLAWYQQKPGKAPKLL

lYEASNLETGVPSRFSGSGS GSDFTLTISSLQPEDFATYY CQQTSSFLLSFGGGTKVEIK R

E26.13 VL SEQ ID NO: 93 DIQMTQSPSSLSASVGDRVT

ITCRASGNIHNYLTWYQQTP GKAPKLLIYNAKTLADGVPS RFSGSGSGTDYTFTISSLQP EDIATYYCQHFWSIPYTFGQ GTKLQITR Table 5. Sequence Identifier Amino Acid Sequence Variable and

Constant

Domain

Sequences of

Anti-IL-loi /

Anti-IL-Ιβ

DVD-Ig Binding

Proteins

12345678901234567890

LINKER SEQ ID NO: 94 TVAAP

X3 JM VL SEQ ID NO: 95 DIQMTQSPSSVSASVGDRVT

CL SEQ ID NO: 96 TVAAPSVFIFPPSDEQLKSG

E26.13 JM-LL- SEQ ID NO: 97 EVQLVESGGGVVQPGRSLRL

X3 SCSASGFIFSRYDMSWVRQA

PGKGLEWVAYISHGGAGTYY

DVD-Ig HEAVY

PDSVKGRFTISRDNSKNTLF VARIABLE LQMDSLRPEDTGVYFCARGG VTKGYFDVWGQGTTVTVSSA STKGPSVFPLAPQVQLVESG GGVVQPGRSLRLSCTASGFT FSMFGVHWVRQAPGKGLEWV AAVSYDGSNKYYAESVKGRF TISRDNSKNILFLQMDSLRL EDTAVYYCARGRPKVVIPAP LAHWGQGTLVTFSS

E26.13 JM VH SEQ ID NO: 98 EVQLVESGGGVVQPGRSLRL

LINKER SEQ ID NO: 99 ASTKGPSVFPLAP

X3 VH SEQ ID NO: 100 QVQLVESGGGVVQPGRSLRL

SCTASGFTFSMFGVHWVRQA Table 5. Sequence Identifier Amino Acid Sequence Variable and

Constant

Domain

Sequences of

Anti-IL-loi /

Anti-IL-Ιβ

DVD-Ig Binding

Proteins

12345678901234567890

PGKGLEWVAAVSYDGSNKYY AESVKGRFTISRDNSKNILF LQMDSLRLEDTAVYYCARGR PKVVIPAPLAHWGQGTLVTF SS

CH SEQ ID NO: 101 ASTKGPSVFPLAPSSKSTSG

E26.13 JM-LL- SEQ ID NO: 102 DIQMTQSPSSLSASVGDRVT

X3 ITCRASGNIHNYLTWYQQTP

GKAPKLLIYNAKTLADGVPS

DVD-Ig LIGHT

RFSGSGSGTDYTFTISSLQP VARIABLE EDIATYYCQHFWSIPYTFGQ GTKLEIKRTVAAPSVFIFPP DIQMTQSPSSVSASVGDRVT ITCRASQGISSWLAWYQQKP GKAPKLLIYEASNLETGVPS RFSGSGSGSDFTLTISSLQP EDFATYYCQQTSSFLLSFGG GTKVEHKR

E26.13 JM VL SEQ ID NO: 103 DIQMTQSPSSLSASVGDRVT

ITCRASGNIHNYLTWYQQTP GKAPKLLIYNAKTLADGVPS Table 5. Sequence Identifier Amino Acid Sequence

Variable and

Constant

Domain

Sequences of

Anti-IL-loi /

Anti-IL-Ιβ

DVD-Ig Binding

Proteins

12345678901234567890

RFSGSGSGTDYTFTISSLQP EDIATYYCQHFWSIPYTFGQ GTKLEIKR

LINKER SEQ ID NO: 104 TVAAPSVFIFPP

X3 VL SEQ ID NO: 105 DIQMTQSPSSVSASVGDRVT

CL SEQ ID NO: 106 TVAAPSVFIFPPSDEQLKSG

Example 3. Reporter-Based Bioassay To Measure IL-1 Potency

[0111] A reporter-based bioassay was developed to measure binding of IL-la and IL-

1β to their receptor. The bioassay is based on responsiveness of the IL-8 promoter to IL- la and IL-Ιβ stimulation.

Example 3.1. Molecular Biology Of The Bioassay

[0112] A nucleic acid encoding the human IL-8 promoter region (-273 to +45) was

created synthetically. The synthetic nucleic acid was transferred to a Flexi Vector pGL4.15 [/wc2i7Hygro] vector (Promega, Cat. #E6701) via a standard FLEXI® Vector reaction. The human IL-8 promoter region insert was sequence verified. A maxi-prep was prepared. Figure 1 depicts a circular plasmid map of the pGL4[/wc2_JP/hIL8/Hygro] vector.

Table 6. The pGL4[/wc2i7hIL8/Hygro Vector (Promega Catalogue No. PI 975)

Example 3.2. Feasibility Study In A549 Cells

[0113] The ability of A549 cells transfected with various promoter reporter construct to provide a read-out upon the binding of IL-l and IL-Ιβ to their endogenously expressed receptor was assessed.

Cell Thaw and Culture

[0114] Human lung carcinoma A549 cells were obtained from ATCC (CCL-185),

thawed, cultured in F-12K media (ATCC #30-2004) containing 10% fetal bovine serum (FBS) (ATCC #30-2020) in a 5% C0₂, 37 °C, humidified incubator and expanded for assays.

Transient Transfection in A549 Cells

[0115] A549 cells were harvested by Trypsin/EDTA (Gibco #25200) detachment,

counted and plated at 2xl0⁴ cells per well in ΙΟΟμΙ F-12K medium + 10% FBS in white 96-well tissue culture treated plates (Costar #3917) and cultured overnight in a 5% C0₂, 37 °C, humidified incubator. Transfection complexes were assembled in 12x75 mm polystyrene tubes (Fisher #14-956-3D) and incubated 10-15 minutes at RT using

FuGENE® HD Transfection Reagent (Promega, Cat. #E2311), C0₂ independent (C0₂-) medium (Gibco #18045) and the vectors pGL4.32[/Hc2i7NF-KB-RE/Hygro],

pGL4[/wc2iVhIL8/Hygro] or pGL4.27[/wc2i7minP/Hygro] as described in Table 7. The three vectors contained different response elements to measure activation of the NF-KB (DNA 1) and IL-8 (DNA 2) signaling pathway, or to serve as a negative control (DNA 3), determining background activity of the pGL4 vector construct with a minimal promoter sequence (minP), respectively.

Table 7. Transient Transfection Setup

[0116] A549 cells were transfected by the addition of 5μ1 of FuGENE® HD/DNA

complex per well. Plates were gently mixed briefly on a plate shaker and incubated 24 hours in a 5% C0₂, 37 °C, humidified incubator. After 24 hours, the medium was removed and replaced with 90μ1Λνε11 of fresh F-12K+ 10% FBS. Human Interleukin-la (hIL-1 a, Cell Signaling, Cat. #5236sc), human Interleukin-ΐβ (hIL-1 β, Cell Signaling, Cat. #8900sc), or PBS as vehicle control were prepared in F-12K+0.5% cFBS (HyClone #SH30068.02) at lOx concentration. Cells were treated with ΙΟμΙ/well of lOx compound for 6 hours or 24 hours at 37 °C. ONE-Glo™ Luciferase Assay System was reconstituted by thawing and combining ONE-Glo™ Substrate and Buffer). The plate was removed from the incubator and equilibrated at RT for 10-15 minutes. Equal volume ONE-Glo™ Reagent (ΙΟΟμΙ/well) was added to cells. The plate was incubated for 10-15 minutes at RT. Luminescence was measured in a GLOMax® Multi+ Plate Reader for a 0.5 second integration time at RT. Data was analyzed as Relative Light Units (RLUs) or

treated/untreated RLU ratio (Fold Response).

[0117] Figure 2 depicts A549 cells transfected with the luc2P/NF -κΒ -RE/Hy gro

construct. A549 cells plated at 2 x 10⁴ per well overnight were transfected with pGL4.32[/wc2i7NF-kB-RE/Hygro] vector using FuGENE® HD Reagent for 24 hours, then treated with hIL-Ια or hIL-Ιβ for 6 or 24 hours at 37 °C. Luminescence data was collected for 0.5 seconds using a GLOMax® Multi+ Plate Reader and plotted as RLUs or Fold Response. [0118] Figure 3 depicts A549 cells transfected with the /wc2_JP/hIL8/Hygro construct.

A549 cells plated at 2xl0⁴ per well overnight were transfected with

pGL4[/wc2i7hIL8/Hygro] vector using FUGENE® HD Reagent for 24 hours, then treated with hIL-Ι or hIL-Ι β for 6 or 24 hours at 37 °C. Luminescence data was collected and plotted as in Figure 2.

[0119] Figure 4 depicts A549 cells transfected with the /wc2_JP/minP/Hygro construct.

A549 cells plated at 2xl0⁴ per well overnight were transfected with

pGL4.27[/wc2i7minP/Hygro] control DNA using FUGENE® HD Reagent for 24 hours, then treated with hIL-Ια or hIL-Ι β for 6 or 24 hours at 37 °C. Luminescence data was collected and plotted as in Figure 2.

[0120] Reporter genes pGL4.32[/wc2 VNF-KB-RE/Hygro] vector and pGL4 [Zwc2i7hIL8/

Hygro] vector tested in A549 cells showed dose dependent response to both hIL-la and hIL-Ι β at 6 hours or 24 hours. However, /MC2_jP/NF-KB-RE showed higher fold-induction compared to the response from luc2PI\s5L%. Up to 50-fold induction was observed for both hIL-la and hIL-Ι β in A549 cells using IUC2P/NF-KB-KE as a reporter gene at 6 hours, with only 5- to 7-fold response at 24 hours induction. Using luc2P/ JL$ as a reporter gene in A549 cells, 10- to 15-fold induction for both hIL-la and hIL-Ι β was shown at 6 hours, and up to a 5- to 6-fold response was shown at 24 hours. Calculated EC50 values of hIL-1 a and hIL-Ι β were comparable. The pGL4.27[/wc2i⁵/minP/Hygro] vector served as a negative control determining background activity of the pGL4 vector construct with a minimal promoter sequence (minP) that does not get activated upon IL- la/IL-Ιβ stimulation.

Example 3.3. Feasibility Study in NF-KB-RE-/WC2,P HEK293 Cell Line

[0121] The ability of a commercially available NF-KB-RE-/WC2_jP HEK293 reporter cell line to provide a read-out upon IL-la and IL-Ι β binding after transient transfection of the cells with the respective human IL1-R1 receptor was assessed as an alternative to the experiments using A549 cells endogenously expressing the IL1-R1 receptor.

Cell Thaw and Culture

[0122] A GloResponse™ NF-KB-RE-/WC2,P HEK293 cell line (Promega, Cat. #E8520) was thawed, cultured in DMEM medium (Gibco #1 1995) containing 10% FBS (ATCC #30-2020) in a 5% C0₂, 37 °C, humidified incubator and expanded for assays. Homo sapiens interleukin 1 receptor, type 1 was obtained from OriGene Technologies Inc. (SCI 19624). Transfection quality plasmid DNA was generated by Aldevron.

Transient Transfection in NF-KB-RE-/WC2,P HEK293 Cells

[0123] GloResponse™ NF-KB-RE-/WC2,P HEK293 cells were harvested by

Trypsin/EDTA (Gibco #25200) detachment method, counted and plated at 2xl0⁴ cells per well in 100 μΐ of DMEM + 10% FBS in white 96-well tissue culture treated plates (Costar #3917) and cultured overnight in a 5% C0₂, 37 °C, humidified incubator. Transfection complexes were assembled in 12 x 75mm polystyrene tubes using FUGE E^® FID

Reagent and an IL-1 receptor expression plasmid (ILl-Rl) or the

pGL4.27[/wc2i7minP/Hygro] vector, which served as a negative control, in C0₂- medium and incubated 10-15 minutes at RT.

Table 8. Transient Transfection Setup

[0124] GloResponse™ NF-KB-RE-/WC2,P HEK293 cells were transfected by addition of

5μ1 of FuGENE^® HD/DNA complex per well. Plates were gently mixed briefly on a plate shaker and incubated 24 hours in a 5% C0₂, 37 °C humidified incubator. After 24 hours, the existing medium was removed and replaced with 90 μΐ/well fresh DMEM + 10% FBS medium. Human Interleukin- la (hIL-l , Cell Signaling, Cat. #5236sc), human

Interleukin- 1β (hIL-Ιβ, Cell Signaling, Cat. #8900sc) or PBS as vehicle control were prepared in DMEM+0.5% cFBS (HyClone #SH30068.02) at a lOx concentration. Cells were treated with ΙΟμΙ/well of lOx compound for 6 or 24 hours at 37 °C. ONE-Glo™ Reagent was reconstituted as described above. Plate was removed from the incubator and equilibrated at RT for 10-15 minutes. An equal volume ONE-Glo™ Reagent (100 μΐ/well) was added to the cells. The plate was incubated for 10-15 minutes at RT. Luminescence was measured in a GloMax Multi+ Plate Reader for a 0.5 second integration time at RT. Data was analyzed as RLUs or Fold Response.

[0125] Figures 5A and 5B depict luminescence data for the NF-KB-KE-IUC2P HEK293

Cell Line. Cells plated at 2xl0⁴ per well overnight were transfected with ILIRI or pGL4.27[/wc2i⁵/minP/Hygro] as negative control using FUGE E^® FID Reagent for 24 hours, then treated with hIL-l or hIL-Ιβ for 6 hours or 24 hours at 37 °C.

Luminescence data was collected as described above.

[0126] An IL1R1 -dependent hIL-la and hIL-Ιβ dose response in NF-KB-KE-IUC2P

HEK293 cells was demonstrated. Fold-induction for both hIL-la and hIL-Ιβ in IL1R1 transfected F -KB-BE-IUC2P HEK293 cells were relatively low: 1.5- to 2-fold at 6 hours or 24 hours of induction, potentially due to low or lack of key components in the pathway. Calculated EC50 values of hIL-la and hIL-Ιβ were comparable. This experiment demonstrated the feasibility of introducing receptors in commercially available reporter cell lines to broaden their applicability. However, in this case the fold- response was too low to proceed with this option.

Example 3.4. Stable Cell Line Generation

[0127] Based on the results of the above feasibility studies (Example 3.1-3.3) the

generation of a stable A549 cell line expressing the pGL4 [/wci VhILS/Hygro] plasmid was initiated. The IL-8 promoter was chosen as the optimal response element because it is efficiently activated by IL-la and IL-Ιβ and therefore closely reflects the presumed in vivo mode of action.

Example 3.4.1. Assay Materials and Reagents Required Materials

Table 9. Stable Cell Line

Cell Line Clone Passage # of Cells/Vial % Mycoplasma Visible Name # # Vials Viability Test Bacterial

Contamination

GloResponse™ 1C- 6 2 <2.0 x 94% Negative Negative

stable cell line

Reagents:

-White, tissue culture-treated, 96-well plates (Costar, Cat. #3917); miscellaneous tissue culture reagents and equipment

-Serum-free medium: Opti-MEM (Invitrogen, Cat. #11058)

-FuGE E^® HD Transfection Reagent (Promega, Cat. #E2311)

-Hygromycin B (Invitrogen, Cat. #10687-010)

-Trypsin (Invitrogen, Cat. #25200)

-O E-Glo™ Luciferase Assay System (Promega, Cat. #E6110)

-CellTiter-Glo^® Luminescent Cell Viability Assay (Promega, Cat. #G7570)

-hILl (Cell Signaling, Cat. #5236sc)

-hILI (Cell Signaling, Cat. #8900sc)

-Cell culture and assay medium (for growth and assay of GloResponse™ IL8- luc2P/A549 Cells):

90% (final concentration) F12-K (ATCC catalogue #30-2004)

10% (final concentration) FBS (ATCC catalogue #20-2020)

250 μg/mL hygromycin B (Invitrogen catalogue #10687-010)

-Freezing medium:

85% (final concentration) F12-K (ATCC catalogue #30-2004)

10% (final concentration) FBS (ATCC catalogue #20-2020)

5% (final concentration) DMSO (Sigma catalogue #D2650)

Example 3.4.2. Experimental Workflow

[0128] As the first step towards obtaining single cell clones, a pool of cells stably

expressing the plasmid was generated. Limited dilution of the stable cell pool were made to yield a single cell per well. Successful single cell clones were then tested for their ability to be stimulated by IL-Ιβ (primary screen). Select clones were chosen for further characterization. Stable Pool Generation

[0129] A549 cells were plated in a T75 flask at 6.7 x lOVcm² in 20 ml cell culture medium without antibiotics. The next day, cells were transfected with pGL4[IL8- liic2P/Hygro] vector using FUGENE^® FID at 3 : 1 ratio according to the protocol. Forty- eight (48) hours post-transfection, cells were dissociated with trypsin, counted, centrifuged (228 x g, 5 minutes at room temperature (RT)) and seeded into a T150 flask in complete growth medium plus selection drug Hygromycin B at 250 μg/ml. The same concentration of selection drug Hygromycin B was applied to non-transfected cells as negative control. Cells were kept under selection with Hygromycin B at 37 °C/5% C0₂ with medium change every other day for 2 weeks. From the stable pool, single clones were isolated by limited dilution and expanded. Each well represented a cell population from a single clone.

Stable Clone Selection by Limited Dilution Cloning Strategy

[0130] A stable pool of GloResponse™ TL%-luc2P/ A549 cells were counted and diluted to 1 x 10⁵ cells/ml in complete medium + Hygromycin (250μg/ml):

-Dilute 1 x 10⁵ cells/ml, 1 : 10 in complete medium = 1 x 10⁴ cells/ml (1ml + 9ml) -Dilute 1 x 10⁴ cells/ml, 1 : 10 in complete medium = 1 x 10³ cells/ml (1ml + 9ml) -Dilute 1 x 10³ cells/ml, 1 : 10 in selection medium to 100 cells/ml (3ml + 27ml) -Dilute 100 cells/ml, 1 :3 in selection medium = 33 cells/ml (10ml + 20ml)

-Dilute 100 cells/ml, 1 : 10 in selection medium = 10 cells/ml (3ml + 27ml)

-Dilute 33 cells/ml, 1 : 10 in selection medium = 3.3 cells/ml (5ml + 45ml)

The following volumes of each dilution in selection medium were prepared:

-30ml of 33 cells/ml

-30ml of 10 cells/ml

-50ml of 3.3 cells/ml

ΙΟΟμΙ of each dilution into 96-well tissue culture plates were plated:

-33 cells/ml = 3.3 cells/well (1 plate)

-10 cells/ml = 1 cell/well (4 plates)

-3.3 cells/ml = 0.33 cells/well (2 plates) Primary Screen of GloResponse IL8-/wc2i7A549 Clones

[0131] A primary screen was performed to characterize stable cell clones regarding their response to IL-Ιβ.

[0132] A total of 92 clones were isolated and expanded. During clone expansion, a

fraction of cells from each clone was plated in a 96-well plate (100 μΐ in F12-K + 10% FBS +250 μg/ml Hygromycin B). When cells became approximately 50% confluent, cells were stimulated with IL-Ιβ at 0 pg/ml, 0.6 pg/ml (EC₅₀) or 0.3ng/ml (EC₁₀₀) for 6 hours at 37 °C. All dilutions were made in F12-K + 10%FBS. A ONE-Glo™ Luciferase Assay System was reconstituted by thawing and combining ONE-Glo™ Substrate and Buffer. The plate was removed from the incubator and equilibrated at RT for 10-15 minutes. An equal volume of ONE-Glo™ Reagent (ΙΟΟμΙ/well) was added to cells. The plate was incubated for 10-15 minutes at RT. Luminescence was measured in a

GLOMax^® Multi + Plate Reader for a 0.5 second integration time at RT. Data was analyzed as RLUs or Fold Response. To monitor well-to-well cell number differences, another copy of the cell plate was tested using CellTiter-GLO^® Luminescent Cell Viability Assay.

Secondary Screen of GloResponse™ JL8-luc2P/ A549 Clones

[0133] A total of 15 clones with acceptable background and assay window were

expanded. Each selected clone was plated in a 96-well plate at 2.0 x 104 cells/well in 90 μΐ of in F12-K + 10% FBS + 250 μg/ml Hygromycin B and incubated overnight. The next day, each selected clone was stimulated with 1 :4 serial dilutions of Π.1β (lng/ml as top concentration) for 6 hours at 37 °C. All dilutions were made in F12-K + 10% FBS. A ONE-Glo™ Luciferase Assay System was reconstituted by thawing and combining ONE- Glo™ Substrate and Buffer. The plate was removed from the incubator and equilibrated at RT for 10-15 minutes. An equal volume of ONE-Glo™ Reagent (100 μΐ/well) was added to cells. The plate was incubated for 10-15 minutes at RT. Luminescence was measured in a GLOMax^® Multi + Plate Reader for 0.5 sec integration time at RT. Data was analyzed as RLUs, or Fold Response. Final Clone Selection Using ILl or ILi Dose Responses

[0134] The final two candidate GloResponse™ l S-luc2P/ A549 clones (clone 1C-B3 and clone 0.3B-C1) were plated in F12-K+105FBS+250 μg/ml Hygromycin B and incubated overnight. The next day cells were stimulated with 1 :4 serial dilutions of ILl (top concentration at 1 ng/ml) or ILi (top concentration at 1 ng/ml) for 6 hours at 37 °C. All dilutions were made in F12-K + 10% FBS. A O E-Glo™ Luciferase Assay System was reconstituted by thawing and combining ONE-Glo™ Substrate and Buffer. The plate was removed from the incubator and equilibrated at RT for 10-15 minutes. An equal volume of ONE-Glo™ Reagent (100 / well) was added to cells. The plate was incubated for 10-15 minutes at RT. Luminescence was measured in a GLOMax^® Multi + Plate Reader for a 0.5 second integration time at RT. Data was analyzed as RLUs, or Fold Response.

Results

[0135] A bar graph of each representative clone using raw data of RLUs is depicted in

Figure 6A. A bar graph of each representative clone using Fold Response is depicted in Figure 6B. A bar graph of each representative clone's cell viability data using CellTiter- Glo^® Luminescent Cell Viability Assay is depicted in Figure 6C. Several clones showed promising activation of the luciferase reporter gene upon stimulation with IL-Ι β and hence, were further evaluated.

[0136] Dose response curves (RLUs) of positive clones selected from the primary screen in response to IL-Ιβ are depicted in Figure 7 A. Dose response curves (Fold Responses) of positive clones selected from the primary screen in response to IL-Ιβ are depicted in Figure 7B. Based on RLU values and Fold-Response, two clones (0.3B-F10 and 1C-B3) were selected for further characterization (see Figures 8A to 8D) and assay development.

[0137] Clone 1C-B3 dose response curves screen in response to IL-la or IL-Ιβ using

RLU are depicted in Figure 8 A, and fold-responses are depicted in Figure 8B. Clone 0.3B-C1 dose response curves in response to IL-la or IL-Ιβ using RLU are depicted in Figure 8C, and fold-responses are depicted in Figure 8D. Clone 1C-B3 showed overall higher RLU values in the range of 10⁶ and a fold-response of 40 upon stimulation with IL-la or IL-Ιβ. Clone 0.3B-C1 showed overall lower RLU values in the range of 10⁴ and a fold response of 60 upon stimulation with IL-la or IL-Ιβ. Conclusions

[0138] A GloResponse™ IL8-luc2P/A549 stable cell line was generated. During the secondary screen, two groups of clones were identified: clones with a higher background (10⁵ RLU) but relatively lower fold-induction (20-40 fold) and clones with low background (10² -10³ RLU) but higher fold response (60-80 fold). One clone from each group was evaluated for a full-dose functional response to hILla or hILi . Calculated EC50 values of hIL-Ια and hIL-Ιβ were comparable for both clones. Clone 1C-B3 was selected as final clone and underwent further evaluation regarding the response upon hILla or hlLip stimulation alone (Figure 8E) or stimulation both cytokines in combination with increasing amounts of the anti-IL-la/IL-Ιβ DVD-Ig ABT-981 (Figure 8F).

Example 4. Reporter-Based Bioassay To Measure TNF-a/IL-17 Potency

[0139] A reporter-based bioassay was developed to measure binding of T F-a and IL-

17 to their respective receptors. The bioassay was based on responsiveness of the human LCN2 promoter to TNF-a and IL-17 stimulation. HeLa cells were identified as optimal signal transducers upon stimulation with TNF-a and IL-17.

Example 4.1. Molecular Biology

[0140] The nucleotide sequence encoding the human LCN2 promoter region was inserted into the pGL4.15 [luc2P/Hygro] vector (Promega catalogue #E6701) to generate the pGL4[luc2P/LCN2/Hygro] vector, and transfection-quality DNA was generated (20 μg, 3.1 μg/mL in JM109 cells (Promega catalogue #P1975)). A vector map for

pGL4[luc2P/LCN2/Hygro] is depicted in Figure 9.

[0141] The pGL4[luc2P/LCN2/Hygro] vector sequence is as follows:

GGCCTAA.CTGGCCGGTACCAGAGGTGCAGCACTCCGGGAA.TGTCCCTCACTCTCCCCGTCCCTCTGTCTTGCCCAA.TC CTGACCAGGTGCAGAAATCTTGCCAAGTGTTTCCGCAGGAGTTGCTGGCAATTGCCTCACATTCCTGGCCTTGGCAAA GAATGAATCAACCCACCCTAGATCCCATAAATAGGGCCACCCAGGTGAGCCTCTCACTCGCCACCTCCTCTTCCACCC CTGCCAAAGCTTGGCAATCCGGTACTGTTGGTAAAGCCACCATGGAAGATGCCAAAAACATTAAGAAGGGCCCAGCGC CATTCTACCCACTCGAAGACGGGACCGCCGGCGAGCAGCTGCACAAAGCCATGAAGCGCTACGCCCTGGTGCCCGGCA CCATCGCCTTTACCGACGCACATATCGAGGTGGACATTACCTACGCCGAGTACTTCGAGATGAGCGTTCGGCTGGCAG AAGCTATGAAGCGCTATGGGCTGAATACAAACCATCGGATCGTGGTGTGCAGCGAGAATAGCTTGCAGTTCTTCATGC CCGTGTTGGGTGCCCTGTTCATCGGTGTGGCTGTGGCCCCAGCTAA.CGACATCTACAA.CGAGCGCGAGCTGCTGAA.CA GCATGGGCATCAGCCAGCCCACCGTCGTATTCGTGAGCAAGAAAGGGCTGCAAAAGATCCTCAACGTGCAAAAGAAGC T AC C GAT C AT AC AAAAGAT CAT CAT CAT G GAT AGC AAGAC C GAC T AC C AG G G C T T C C AAAG CAT GT AC AC C T T C GT GA CTTCCCATTTGCCACCCGGCTTCAACGAGTACGACTTCGTGCCCGAGAGCTTCGACCGGGACAAAACCATCGCCCTGA TCATGAACAGTAGTGGCAGTACCGGATTGCCCAAGGGCGTAGCCCTACCGCACCGCACCGCTTGTGTCCGATTCAGTC ATGCCCGCGACCCCATCTTCGGCAACCAGATCATCCCCGACACCGCTATCCTCAGCGTGGTGCCATTTCACCACGGCT TCGGCATGTTCACCACGCTGGGCTACTTGATCTGCGGCTTTCGGGTCGTGCTCATGTACCGCTTCGAGGAGGAGCTAT TCTTGCGCAGCTTGCAAGACTATAAGATTCAATCTGCCCTGCTGGTGCCCACACTATTTAGCTTCTTCGCTAAGAGCA CTCTCATCGACAAGTACGACCTAAGCAACTTGCACGAGATCGCCAGCGGCGGGGCGCCGCTCAGCAAGGAGGTAGGTG AGGCCGTGGCCAAACGCTTCCACCTACCAGGCATCCGCCAGGGCTACGGCCTGACAGAAACAACCAGCGCCATTCTGA TCACCCCCGAAGGGGACGACAAGCCTGGCGCAGTAGGCAAGGTGGTGCCCTTCTTCGAGGCTAAGGTGGTGGACTTGG ACACCGGTAAGACACTGGGTGTGAACCAGCGCGGCGAGCTGTGCGTCCGTGGCCCCATGATCATGAGCGGCTACGTTA ACAACCCCGAGGCTACAAACGCTCTCATCGACAAGGACGGCTGGCTGCACAGCGGCGACATCGCCTACTGGGACGAGG ACGAGCACTTCTTCATCGTGGACCGGCTGAAGAGCCTGATCAAATACAAGGGCTACCAGGTAGCCCCAGCCGAACTGG AGAGCATCCTGCTGCAACACCCCAACATCTTCGACGCCGGGGTCGCCGGCCTGCCCGACGACGATGCCGGCGAGCTGC CCGCCGCAGTCGTCGTGCTGGAACACGGTAAAACCATGACCGAGAAGGAGATCGTGGACTATGTGGCCAGCCAGGTTA CAACCGCCAAGAAGCTGCGCGGTGGTGTTGTGTTCGTGGACGAGGTGCCTAAAGGACTGACCGGCAAGTTGGACGCCC GCAAGATCCGCGAGATTCTCATTAAGGCCAAGAAGGGCGGCAAGATCGCCGTGAATTCTCACGGCTTCCCTCCCGAGG TGGAGGAGCAGGCCGCCGGCACCCTGCCCATGAGCTGCGCCCAGGAGAGCGGCATGGATAGACACCCTGCTGCTTGCG CCAGCGCCAGGATCAACGTCTAAGGCCGCGACTCTAGAGTCGGGGCGGCCGGCCGCTTCGAGCAGACATGATAAGATA CATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGC T T TAT T T GT AAC CAT T AT AAG C T G C AAT AAAC AAGT T AAC AAC AAC AAT T G CAT T CAT T T TAT GT T T C AG GT T C AG G G GGAGGTGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTAAAATCGATAAGGATCCGTTTGCGTATTG GGCGCTCTTCCGCTGATCTGCGCAGCACCATGGCCTGAAATAACCTCTGAAAGAGGAACTTGGTTAGCTACCTTCTGA GGCGGAAAGAACCAGCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATG CAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGC ATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCC CATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCTGCCTCTGAGCTATTCCAG AAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTCGATTCTTCTGACACTAGCGCCACCATGAAG AAGCCCGAACTCACCGCTACCAGCGTTGAAAAATTTCTCATCGAGAAGTTCGACAGTGTGAGCGACCTGATGCAGTTG TCGGAGGGCGAAGAGAGCCGAGCCTTCAGCTTCGATGTCGGCGGACGCGGCTATGTACTGCGGGTGAATAGCTGCGCT GATGGCTTCTACAAAGACCGCTACGTGTACCGCCACTTCGCCAGCGCTGCACTACCCATCCCCGAAGTGTTGGACATC GGCGAGTTCAGCGAGAGCCTGACATACTGCATCAGTAGACGCGCCCAAGGCGTTACTCTCCAAGACCTCCCCGAAACA GAGCTGCCTGCTGTGTTACAGCCTGTCGCCGAAGCTATGGATGCTATTGCCGCCGCCGACCTCAGTCAAACCAGCGGC TTCGGCCCATTCGGGCCCCAAGGCATCGGCCAGTACACAACCTGGCGGGATTTCATTTGCGCCATTGCTGATCCCCAT GTCTACCACTGGCAGACCGTGATGGACGACACCGTGTCCGCCAGCGTAGCTCAAGCCCTGGACGAACTGATGCTGTGG GCCGAAGACTGTCCCGAGGTGCGCCACCTCGTCCATGCCGACTTCGGCAGCAACAACGTCCTGACCGACAACGGCCGC ATCACCGCCGTAATCGACTGGTCCGAAGCTATGTTCGGGGACAGTCAGTACGAGGTGGCCAACATCTTCTTCTGGCGG CCCTGGCTGGCTTGCATGGAGCAGCAGACTCGCTACTTCGAGCGCCGGCATCCCGAGCTGGCCGGCAGCCCTCGTCTG CGAGCCTACATGCTGCGCATCGGCCTGGATCAGCTCTACCAGAGCCTCGTGGACGGCAACTTCGACGATGCTGCCTGG GCTCAAGGCCGCTGCGATGCCATCGTCCGCAGCGGGGCCGGCACCGTCGGTCGCACACAAATCGCTCGCCGGAGCGCA GCCGTATGGACCGACGGCTGCGTCGAGGTGCTGGCCGACAGCGGCAACCGCCGGCCCAGTACACGACCGCGCGCTAAG GAGGTAGGTCGAGTTTAAACTCTAGAACCGGTCATGGCCGCAATAAAATATCTTTATTTTCATTACATCTGTGTGTTG GTTTTTTGTGTGTTCGAACTAGAT

GCTGTCGACCGATGCCCTTGAGAGCCTTCAACCCAGTCAGCTCCTTCCGGTGGGCGCGGGGCATGACTATCGTCGCCG CACTTATGACTGTCTTCTTTATCATGCAACTCGTAGGACAGGTGCCGGCAGCGCTCTTCCGCTTCCTCGCTCACTGAC TCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCA GGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCG TTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGA CTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATAC CTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTC GTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTT GAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTA GGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTG CTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTT TTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGAC GCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTA AAT T AAAAAT GAAGT T T T AAAT C AAT C T AAAGT AT AT AT GAGT AAAC T T G GT C T GAC AG C G G C C G C AAAT G C T AAAC C ACTGCAGTGGTTACCAGTGCTTGATCAGTGAGGCACCGATCTCAGCGATCTGCCTATTTCGTTCGTCCATAGTGGCCT GACTCCCCGTCGTGTAGATCACTACGATTCGTGAGGGCTTACCATCAGGCCCCAGCGCAGCAATGATGCCGCGAGAGC CGCGTTCACCGGCCCCCGATTTGTCAGCAATGAACCAGCCAGCAGGGAGGGCCGAGCGAAGAAGTGGTCCTGCTACTT TGTCCGCCTCCATCCAGTCTATGAGCTGCTGTCGTGATGCTAGAGTAAGAAGTTCGCCAGTGAGTAGTTTCCGAAGAG TTGTGGCCATTGCTACTGGCATCGTGGTATCACGCTCGTCGTTCGGTATGGCTTCGTTCAACTCTGGTTCCCAGCGGT CAAGCCGGGTCACATGATCACCCATATTATGAAGAAATGCAGTCAGCTCCTTAGGGCCTCCGATCGTTGTCAGAAGTA AGTTGGCCGCGGTGTTGTCGCTCATGGTAATGGCAGCACTACACAATTCTCTTACCGTCATGCCATCCGTAAGATGCT TTTCCGTGACCGGCGAGTACTCAACCAAGTCGTTTTGTGAGTAGTGTATACGGCGACCAAGCTGCTCTTGCCCGGCGT CTATACGGGACAACACCGCGCCACATAGCAGTACTTTGAAAGTGCTCATCATCGGGAATCGTTCTTCGGGGCGGAAAG ACTCAAGGATCTTGCCGCTATTGAGATCCAGTTCGATATAGCCCACTCTTGCACCCAGTTGATCTTCAGCATCTTTTA CTTTCACCAGCGTTTCGGGGTGTGCAAAAACAGGCAAGCAAAATGCCGCAAAGAAGGGAATGAGTGCGACACGAAAAT GTTGGATGCTCATACTCGTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTACTAGTACGTCTCTCAAGGATAAG TAAGTAATATTAAGGTACGGGAGGTATTGGACAGGCCGCAATAAAATATCTTTATTTTCATTACATCTGTGTGTTGGT TTTTTGTGT GAAT C GAT AGT AC T AAC AT AC G C T CT C CAT C AAAAC AAAAC GAAAC AAAAC AAAC TAG C AAAAT AG G C T GTCCCCAGTGCAAGTGCAGGTGCCAGAACATTTCTCT

(SEQ ID NO: 16)

Example 4.2. Initial Feasibility Study In HeLa, A549 and U937 Cells [0142] The ability of HeLa, A549 and U937 cells transfected with various promoter reporter constructs to provide a read-out upon the binding of TNF-a and IL-17 to their respective receptors was assessed.

Cell Thaw and Culture

[0143] HeLa, A549 and U937 cells were thawed and cultured under optimized conditions in the respective growth media (DMEM (Life Technologies #11995), F-12 (Life

Technologies #31765) or RPMI (Life Technologies #22400), respectively) containing 10% fetal bovine serum (FBS) (Hyclone #SH30070.02) in a 5% C0₂, 37 °C, humidified incubator and expanded for assays.

Transient Transfection of HeLa, A549 and U937 Cells

[0144] Cells were plated into 96 well assay plates (Costar Cat. #3917) in growth media and incubated overnight.

a. HeLa cells were plated at 10,000 cells/well in DMEM (Life Technologies Cat.

#11995) + 10% FBS (Hyclone Cat. #SH30070.02).

b. A549 cells were plated at 15,000 cells/well in F-12 (Life Technologies Cat. #31765) + 10% FBS.

c. U937 cells were plated at 10,000 cells/well in RPMI (Life Technologies Cat. #22400) + 10% FBS.

[0145] The next day, cells were transfected with either pGL4[luc2P/LCN2/Hygro],

pGL4.32[luc2P/NF-KB-RE/Hygro], pGL4.44[luc2P/APl RE/Hygro], or

pGL4[luc2P/hIL8/Hygro] vectors. The four vectors contained different response elements to measure activation of the LCN2, F-κΒ, API and IL-8 signaling pathways, respectively. Each cell line was transfected according to previously optimized conditions:

[0146] a. HeLa and A549 cells were transfected using Fugene HD (Promega Cat.

#E2311), using a 3 : 1 lipid:DNA ratio, and adding 1/lOth volume of transfection complex per well to a final concentration of O. ^g DNA and 0.3μΕ Fugene HD per well.

[0147] b. U937 cells were transfected using a new transfection reagent currently being developed by Promega, using a 6: 1 lipid:DNA ratio, and adding 1/lOth volume of transfection complex per well to a final concentration of O. ^g DNA and 0.6μΕ transfection reagent per well. [0148] Approximately 24 hours post-transfection, media was aspirated (for adherent cells only) and replaced with assay media (DMEM + 0.5% FBS for HeLa cells, or F-12 + 0.5% FBS for A549 cells). Since U937 cells are suspension cells, an aspiration or media change step was not required.

[0149] Cells were treated with IL- 17 (IL- 17A) (Life Technologies Cat. #PHC0174) and

TNFa (Promega Cat. #G5241) at various concentrations for either 6 or 24 hours (Figures 1 OA- IOC). Concentrations were arranged in a matrix-type format as indicated in Figures 1 OA- IOC in order to test all combinations of cytokine concentrations. Luminescence data was collected using O E-Glo™ Reagent and plotted as RLUs. Using the F-κΒ, API, and hIL8 reporter vectors, TNFa response alone could be measured; however a clear synergistic effect of both cytokines was not seen. The pGL4[luc2P/LCN2/Hygro] reporter vectors could measure the synergistic effect of TNFa and IL-17A in HeLa cells, while this effect was less obvious in A549 and U937 cells. HeLa cells were chosen to further investigate the synergetic effect of TNFa and IL-17A in order to establish a stable reporter cell line.

Repeat Study

[0150] The optimized conditions determined in the initial feasibility experiment were used with expanded concentrations (9 point dose-response of one agonist, holding the other one constant). HeLa cells were transfected with the LCN2 promoter vector in a flask (instead of an assay plate), and then plated in assay media, starved overnight and stimulated for 6 hours with an expanded dose-range of each cytokine in the presence or absence of the alternate cytokine at a single concentration.

[0151] 2.5 million HeLa cells were plated in a T75 flask in DMEM + 10% FBS and

incubated overnight. The next day, cells were transfected with

pGL4[/wc2_JP/LCN2/Hygro] using Fugene HD, using a 3 : 1 lipid:DNA ratio, and adding 1/lOth volume of transfection complex directly to the flask. 1.5ml Optimem was incubated with 15 μg DNA and 45 μΕ Fugene HD for 10 minutes at RT, then added to the existing 15 mL volume in the flask. Approximately 7-8 hours post-transfection, cells were harvested with trypsin and resuspended in assay media (DMEM + 0.5% charcoal - dextran stripped FBS (Life Technologies Cat. #12676). 20,000 cells/well were plated in a white 96-well assay plate and incubated overnight. The next day, cells were treated with a dose-response of either IL-17 or TNFa in the presence or absence of a single concentration of the alternate agonist. After a 6 hour stimulation time, O E-Glo™ Luciferase Assay was performed and plates were read on a GLOMax^® plate reader.

[0152] Figures 11 A and 1 IB depict the synergistic effect of both IL-17 and T Fa that was measured using the LCN2 promoter vector in HeLa cells. Cells were transfected and plated as described above, and then treated for 6 hours with a dose-response of IL-17 in the presence or absence of lng/mL TNFa (Figure 11A) or a dose-response of TNFa in the presence or absence of 200ng/mL IL-17 (Figure 1 IB). The LCN2 promoter reporter could measure the synergistic effect of both cytokines in HeLa cells. This experiment demonstrated the capability of the LCN2 promoter reporter to detect the synergistic effect of IL-17 and TNFa in HeLa cells transfected with pGL4[lwc2i7LCN2/Hygro].

Example 4.3. Stable Cell Line Development

[0153] Based on the results of the feasibility study, stable clones were developed with the

LCN2-Luc2P reporter in HeLa cells, using standard transfection, selection, and limiting dilution cloning methods. Approximately 100 clones were screened for a response to IL- 17A and TNFa. Responsive clones were expanded and re-tested to confirm response. Two final clones were chosen, and multiple cell seeding densities were evaluated to promote maximum response. Cryopreserved cells were tested for bacterial contamination (visual inspection) and mycoplasma (Lonza MycoAlert).

Example 4.3.1. Initial Clone Screen

Protocol

[0154] 102 separate clones were harvested and plated in 96 well assay plates (Costar Cat.

#3917) in growth media (DMEM (Life Technologies Cat. #11995) + 10% FBS (Hy clone Cat. #SH30070.02)) and incubated overnight at 37°C/5% C0₂ until the cells had reattached. Cell density was not determined. The next day, the media was aspirated and replaced with assay media (DMEM + 0.5% charcoal/dextran-stripped FBS (Life

Technologies Cat. #12676)). After an overnight incubation in assay media, cells were treated with 50 ng/ml IL-17A (eBioscience Cat. #14-8180) and 4 ng/ml TNFa (Promega Cat. #G5241) or left untreated. After a six hour stimulation time, ONE-Glo™ Luciferase Assay was performed and plates were read on GLOMax^® plate reader. Results

[0155] Of the 102 clones tested, 17 had a response greater than 5-fold. Of these 17

clones, 9 clones had a response greater than 10-fold, and 2 clones had a response greater than 20-fold. Responding clones were expanded for further testing. Figures 12A-12C depict initial clone screen results. Clones were stimulated for 6 hours in the presence or absence of 4ng/ml T Fa and 50ng/ml IL-17A, as described above. Figures 12A and B depict the raw RLUs, in both log and linear scale. Figure 12C depicts the fold response over untreated cells.

Example 4.3.2. Secondary Clone Screen

[0156] Eleven clones were expanded into T150 flasks, and tested with various

concentrations of IL-17A and TNFa for 6 hours. Concentrations were arranged in a matrix-type format so that all combinations of cytokine concentrations were tested.

Protocol

[0157] Clones were plated in 96 well assay plates at 20,000 cells per well in assay media and incubated overnight. Edge wells were not used. Cells were harvested with 0.05% Trypsin-EDTA (Life Technologies Cat. #23500. After the cells were detached, the trypsin was neutralized with growth media, the cells were centrifuged at 200 x g for 5 minutes, and the cell pellet was resuspended in assay media. Cells were then counted and the volume of assay media was adjusted for appropriate plating density. After an overnight incubation in assay media at 37°C/5% C0₂, cells were stimulated with a dose- response of IL-17A and a dose-response of TNFa in a matrix-type format. After a 6 hour stimulation time, a ONE-Glo™ Luciferase Assay was performed and the plates were read on GLOMax^® plate reader. Figures 13 A and 13B depict secondary clone screen results. Responsive clones were expanded, plated at 20,000 cells per well in assay media, incubated overnight at 37°C/5% C0₂, and then stimulated for 6 hours with various cytokine concentrations as indicated on the graphs described above. Graphs showing the fold response over untreated cells are shown for each clone. 8 of the 11 clones tested showed dose-response curves. Background RLU values are indicated in red font in the upper left corner of each graph for each of the 8 clones. The clones differed in the fold- response as well as in the observed background RLU. Conclusion

[0158] Clones 1D9 and 1F5 were chosen for further evaluation. These two clones were chosen for their large assay window as well as their differing background RLU values. Clone 1D9 had a very low background, with RLU values in the hundreds. Clone 1F5 had a higher background, with RLU values in the thousands. The rest of the clones were frozen down as back-ups, with the exception of the three that had poor response (1G1, 1G3, and 2H3).

Example 4.3.3. Cell Density Evaluation

[0159] Clones 1D9 and 1F5 were plated at various cell densities and tested with various concentrations of IL-17A and TNFa for 6 hours. Concentrations were arranged in a matrix-type format so that all combinations of cytokine concentrations were tested.

Protocol

[0160] Clones were plated in 96 well white assay plates at 5000, 10,000, or 20,000 cells per well in assay media and incubated overnight at 37°C/5% C0₂. Edge wells were not used. Cells were stimulated with a dose-response of IL-17A and a dose-response of TNFa in a matrix-type format. After a 6 hour stimulation time, a ONE-Glo™ Luciferase Assay was performed and the plates were read on GLOMax^® plate reader. Figures 14A and 14B depict the cell density evaluation. Clones 1D9 (Figure 14A) and 1F5 (Figure 14B) were plated at 5000, 10,000, or 20,000 cells per well in assay media, incubated overnight at 37°C/5% C0₂, and then stimulated for 6 hours with various cytokine concentrations as indicated on the graphs described above. Graphs showing the raw RLU values are shown for each clone. In red text in the upper left corner of each graph is the fold response over background.

Conclusion

[0161] Increasing cell density resulted in increasing assay window for each clone. Clone

1F5 has approximately 10 times more total RLUs than clone 1D9, and it has a lower fold response over background. Example 4.3.3.1 Additional Cell Density Evaluation

[0162] An experiment was performed to test even higher cell densities (20,000, 40,000 or

60,000 cells per well) since the response had not plateaued with the previous experiment. Also, the IL-17A concentrations were left-shifted slightly to capture the full dose- response curve. Instead of a matrix format, the IL-17A dose-response was tested in the presence or absence of 20 ng/ml T Fa.

Protocol

[0163] Clones were plated in 96 well assay plates at 20,000, 40,000 or 60,000 cells per well in assay media and incubated overnight. Edge wells were not used. After an overnight incubation at 37°C/5% C0₂ in assay media, cells were stimulated with a dose of IL-17A and a single concentration of TNFa. After a 6 hour stimulation time, a ONE- Glo™ Luciferase Assay was performed and plates were read on GLOMax^® plate reader. Figures 15A and 15B depict results of the repeat of cell density evaluation. Clones 1D9 (Figure 15B) and 1F5 (Figure 15 A) were plated at 20,000, 40,000 or 60,000 cells per well in assay media, incubated overnight, and then stimulated for 6 hours with a dose of IL- 17A in the presence or absence of 20ng/ml TNFa, as indicated on the graphs (n=3). The EC₅₀ for IL-17A is approximately 3-4 ng/ml in all of the graphs. The highest fold response for clone 1D9 was 100 (at 40,000 cells/well) and the highest fold response for clone 1F5 was 55 (at 60,000 cells/well).

Conclusion

[0164] Several responsive clones were isolated and frozen down. Clone 1D9

(CS159409A) and 1F5 (CS159409B) were chosen for further evaluation using multiple cell seeding densities, and were tested to be free of mycoplasma contamination. Cell density optimization: clone 1D9 at 40,000 cells/well provided highest fold response of 100; clone 1F5 at 60,000 cells/well provided highest fold response of 55.

Example 4.4. Summary and Conclusions

[0165] The LCN2 promoter reporter was capable of detecting the synergistic effect of treatment with IL-17A and TNFa together. The NF-κΒ, AP-1, and hIL-8 reporters only detected the effect of TNFa and the addition of IL-17A does not increase the response. Without intending to be bound by scientific theory, HeLa cells transfected in bulk, starved overnight, and treated with cytokines for 6 hours appeared be the optimal assay condition. The synergistic effect of both cytokines was clearly observed.

Example 5. Single Reporter Assay That Detects T Fa/IL-17 DVD-Ig-T Fa and

TNFa/IL-17 DVD-Ig -IL-17 Binding

[0166] A reporter line (i.e., HeLa_LCN2-luc2P, exemplified by clone 1F5) was generated as described above that was synergistically activated by TNFa and IL-17. As shown in Figure 16, a synergistic effect of clone 1F5 was observed at various concentrations of IL- 17 in the presence of increasing concentrations of TNFa. Figure 17 depicts the dose response of HeLa_LCN2-luc2P in the presence of 3 ng/mL TNFa, 50 ng/mL IL-17, and increasing amounts of TNFa/IL-17 DVD-Ig.

Example 6. Additive Dual Reporter Assay

[0167] A reporter line was generated in which a response element was independently activated by each of two ligands (such as, e.g., each of two cytokines). The titration of each ligand resulted in a complete dose response curve. In contrast to a synergistic reporter, the combined signal of an additive reporter was the addition of the two individual signals.

[0168] The reporter cell line generated, A549_IL8-luc2P, was additively activated by IL- la and IL-Ιβ. This reporter cell line was used to detect a DVD targeting IL-la and IL- 1β.

Incorporation by Reference

[0169] The contents of all cited references (including literature references, patents, patent applications, and websites) that maybe cited throughout this application are hereby expressly incorporated by reference in their entirety for any purpose, as are the references cited therein. The disclosure will employ, unless otherwise indicated, conventional techniques of immunology, molecular biology and cell biology, and pathology, which are well known in the art.

[0170] The present disclosure also incorporates by reference in their entirety techniques well known in the field of molecular biology and drug delivery. These techniques include, but are not limited to, techniques described in the following publications:

Ausubel et al. (eds.) (1993) CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NY; Ausubel et al. (eds.) (4th ed.; 1999) SHORT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NY; Bergman et al. (1998) J. Pharmacol. Exp. Ther.

284(1): 111-5; Braen et al. (2010) Int. J. Toxicol. 29(3):259-67; Smolen and Ball (eds.) (1984) CONTROLLED DRUG BIOAVAILABILITY, DRUG PRODUCT DESIGN AND

PERFORMANCE, John Wiley & Sons, NY; Garg and Balthasar (2009) AAPS J. 11(3):553- 7; Giege and Ducruix (2nd ed.; 2009) CRYSTALLIZATION OF NUCLEIC ACIDS AND

PROTEINS, a Practical Approach, pp. 201-16, Oxford University Press, NY; Goodson (1981) in: MEDICAL APPLICATIONS OF CONTROLLED RELEASE, 2: 115-138 (CRC Press, Florida); Hammerling et al. (1981) in: MONOCLONAL ANTIBODIES AND T-CELL

HYBRIDOMAS, pp. 563-681, Elsevier, NY; Harlow et al. (1988) ANTIBODIES: A

LABORATORY MANUAL, Cold Spring Harbor Laboratory Press, 2nd ed. (Cold Water Press, New York); Kabat et al. (1987 and 1991) SEQUENCES OF PROTEINS OF

IMMUNOLOGICAL INTEREST, National Institutes of Health, Bethesda, MD; Kabat et al. (5^th ed.; 1991) SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, U.S. Department of Health and Human Services, NIH Publication No. 91-3242; Kontermann and Dubel (eds.) (2001) ANTIBODY ENGINEERING, Springer- Verlag, NY (ISBN 3-540-41354-5);Kriegler (1990) Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY; Levites et al. (Epub 2006) FASEB J. 20(14):2576-8; Lu and Weiner (eds.) (2001) CLONING AND EXPRESSION VECTORS FOR GENE FUNCTION ANALYSIS, BioTechniques Press, MA; Langer and Wise (eds.) (1974) MEDICAL APPLICATIONS OF CONTROLLED RELEASE, CRC Press, FLA; Old and Primrose (3d ed.; 1985) PRINCIPLES OF GENE MANIPULATION: AN

INTRODUCTION TO GENETIC ENGINEERING Blackwell Scientific Publications, MA 2:409; Robinson (ed.) (1978) SUSTAINED AND CONTROLLED RELEASE DRUG DELIVERY SYSTEMS, Marcel Dekker, Inc., NY; Sambrook et al. (eds.) (2nd ed.; 1989) MOLECULAR CLONING: A LABORATORY MANUAL Cold Spring Harbor Laboratory Press, NY; Shen et al. (2004) Adv. Drug Deliv. Rev. 14:56(12): 1825-57; Skripuletz et al. (2008) Am. J. Phys.

172(4): 1053-61; Winnacker (1987) FROM GENES TO CLONES: INTRODUCTION TO GENE TECHNOLOGY, VCH Publishers, NY; EQUIVALENTS The disclosure may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting of the disclosure. Scope of the disclosure is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are therefore intended to be embraced herein.

Claims

WHAT IS CLAIMED IS:

1. A method for assessing efficacy of a multi-specific therapeutic for binding of each of two ligands, the method comprising the steps of:

contacting a reporter cell expressing a promoter response element operably linked to a nucleic acid sequence encoding a detectable moiety with two ligands, and allowing each of the ligands to bind its receptor and mediate cell signaling;

producing a detectable moiety; and

contacting the cell with a multi-specific therapeutic,

wherein a decrease in detectable moiety production correlates with efficacy of the multi-specific therapeutic to bind each of the two ligands.

2. The method of claim 1, wherein the multi-specific therapeutic is a bi-specific therapeutic.

3. The method of claim 2, wherein the bi-specific therapeutic is a dual variable domain immunoglobulin (DVD-Ig).

4. The method of any one of claims 1 to 3, wherein the detectable moiety is visibly

detectable.

5. The method of any one of claims 1 to 4, wherein the detectable moiety is luciferase.

6. A method for assessing efficacy of a DVD-Ig for binding a ligand, the method comprising the steps of:

contacting a reporter cell expressing a promoter response element operably linked to a nucleic acid sequence encoding a detectable moiety with one or more ligands, and allowing the one or more ligands to bind one or more receptors and mediate cell signaling;

producing a detectable moiety; and

contacting the cell with a DVD-Ig,

wherein a decrease in detectable moiety correlates with efficacy of the DVD-Ig to bind the ligand.

7. The method of any one of claims 1 to 6, wherein the two ligands or the one or more ligands are cytokines.

8. The method of claim 7, wherein the cytokines are selected from the group consisting of interleukin-17 (IL-17), interleukin-1 alpha (IL-la), interleukin-1 beta (IL-Ιβ), and tumor necrosis factor a (TNFa).

9. The method of claim 7 or 8, wherein the cytokines are IL-17 and TNFa or IL-la and IL- 1β.

10. The method of any one of claims 1 to 9, wherein the promoter response element is a

lipocalin (LCN) 2 promoter response element or an IL-8 promoter response element.

11. The method of any one of claims 1 to 10, wherein the cell is a cervical cancer (HeLa) cell or an adenocarcinomic human alveolar basal epithelial (A549) cell.

12. The method of any one of claims 3 to 11, wherein the DVD-Ig comprises anti-TNFa and anti-IL-17.

13. The method of any one of claims 3 to 11, wherein the DVD-Ig comprises anti-IL-la and anti-IL-Ιβ.

14. A method for assessing the efficacy of a multi-specific therapeutic to bind one or both of IL-17 and TNFa, comprising the steps of:

contacting a cell with IL-17 and allowing it to bind its receptor, wherein nonreceptor binding mediates expression of a detectable moiety via an LCN 2 promoter response element;

contacting the cell with TNFa and allowing it to bind its receptor, wherein TNFa- receptor binding mediates expression of a detectable moiety via the LCN 2 promoter response element; and contacting the cell with a multi-specific therapeutic, wherein a decrease in detectable moiety correlates with efficacy of the multi-specific therapeutic for binding one or both of IL-17 and TNFa.

15. The method of claim 14, wherein the multi-specific therapeutic is a DVD-Ig.

16. A method for assessing the efficacy of a multi-specific therapeutic to bind one or both of IL-la and IL-Ιβ, comprising the steps of:

contacting a cell with IL-la and allowing it to bind its receptor, wherein IL- la- receptor binding mediates expression of a detectable moiety via an IL-8 promoter response element;

contacting the cell with IL-Ιβ and allowing it to bind its receptor, wherein nonreceptor binding mediates expression of a detectable moiety via the IL-8 promoter response element; and

contacting the cell with a multi-specific therapeutic, wherein a decrease in detectable moiety correlates with efficacy of the multi-specific therapeutic for binding one or both of IL-la and IL-Ιβ.

17. The method of claim 16, wherein the multi-specific therapeutic is a DVD-Ig.

18. A reporter cell responsive to DVD-Ig binding to one or both of IL-17 and TNFa, wherein the reporter cell comprises:

an LCN 2 promoter response element operably linked to a nucleic acid sequence encoding a detectable moiety; and

one or more receptors that mediate signaling in the reporter cell upon one or both of IL-17- and TNFa-receptor binding via the LCN 2 promoter response element, wherein the signaling in the reporter cell promotes expression of the detectable moiety, and wherein DVD-Ig binding of one or both of IL-17 and TNFa decreases expression of the detectable moiety.

19. A kit comprising the reporter cell of claim 18, and optional instructions for use.

20. A reporter cell responsive to DVD-Ig binding to one or both of IL-la and IL-Ιβ, wherein the reporter cell comprises:

an IL-8 promoter response element operably linked to a nucleic acid sequence encoding a detectable moiety; and

one or more receptors that mediate signaling in the reporter cell upon one or both of IL-la and IL-i -receptor binding via the IL-8 promoter response element, wherein the signaling in the reporter cell promotes expression of the detectable moiety, and wherein DVD-Ig binding of one or both of IL-la and IL-Ιβ decreases expression of the detectable moiety.

21. A kit comprising the reporter cell of claim 20, and optional instructions for use.