AU2016259423B2 - Compositions and methods for treatment of microbial disorders - Google Patents

Abstract

The present invention relates to compositions and methods for treatment of microbial disorder by modulation of the host immune response. More particularly, the present invention relates to compositions that mediate an anti-microbial immune response, and methods of treating a microbial 5 infection using such compositions. 8422884_1 (GHMatters) P84050.AU.2

Description

The present invention relates to compositions and methods for treatment of microbial disorder by modulation of the host immune response. More particularly, the present invention relates to compositions that mediate an anti-microbial immune response, and methods of treating a microbial infection using such compositions.

8422884_1 (GHMatters) P84050.AU.2

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- 1 COMPOSITIONS AND METHODS FOR TREATMENT OF MICROBIAL DISORDERS

The entire disclosure in the complete specification of our Australian Patent Application No. 2014259514 is by this cross-reference incorporated into the present specification.

FIELD OF THE INVENTION

The present invention relates generally to the treatment of microbial disorders by modulation of the host immune response.

BACKGROUND

Infection by microbial pathogens represents a major cause of death worldwide and continues to pose a serious threat to global health (WHO, The World Health Report (2004)). For example, Attaching and effacing (A/E) bacterial pathogens, such as enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) are among the bacteria that cause diarrhea, morbidity and mortality, especially among infants and children in the developing world (2). E. coli O157:H7, one of the EHEC strains, caused many people to be hospitalized and 3 mortalities last year in the United States (MMWR Morb Mortal Wkly Rep 55, 1045 (Sep 29, 2006)). It is also believed that more than 90% of all cases of post-diarrhea hemolytic uremic syndrome (HUS) in industrialized countries were caused by E. coli O157:H7 infection (R. L. Siegler, Pediatr Clin North Am 42, 1505 (Dec,

1995)). Other EPEC strains such as E. coli O55:H7 also cause intestinal illness among infants world wide (T. S. Whittam et al., Infect Immun 61, 1619 (May, 1993)). Much of our knowledge on how hosts control the infection of A/E pathogens comes from the study of infection by Citrobacter rodentium, a natural pathogen in mice (L. Eckmann, Ann NY Acad Sci 1072, 28 (August 1, 2006)). Similar to the pathogenesis of EHEC or EHPC in human, intimately attaching of C. rodentium to murine colonic epithelial cells results in effacement of brush border microvilli, termed as attaching and effacing (A/E) lesion, and colonic hyperplasia (D. B. Schauer, S. Falkow, Infect Immun 61, 2486 (Jun, 1993)).

Both intestinal epithelial and immune cells play critical roles in host defense against A/E pathogens. The tight junctions of intestinal epithelial cells present the first barrier to prevent microbes leaving the intestinal lumen (T. T. MacDonald, G. Monteleone, Science 307, 1920 (March 25, 2005)). Additionally, epithelial cells secrete anti-microbial peptides to control pathogens in the gastrointestinal (Gl) tract (A. Takahashi et al., FEES Lett 508, 484 (Nov 23, 2001)). Studies with immune deficient mouse strains during C. rodentium infection established that CD4⁺ T cells, B cells, and anti-C. rodentium specific antibody responses are all essential components of the adaptive immunity to contain and eradicate infection (L. Bry, Μ. B. Brenner, J Immunol 172, 433 (January 1, 2004)). Many cytokines produced by lymphocytes during infection can enhance the innate immune

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-22016259423 17 Oct 2018 responses of epithelial cells. The specific functions of these cytokines, however, remain unclear during A/E pathogen infection.

IL-22, an IL-10 family cytokine, is produced by lymphocytes, particularly Thl7 cells (Y. Zheng et al., Nature 445, 648 (Leb 8, 2007)). Thl7 cells belong to a recently discovered CD4⁺ T helper subset that also produces IL-17. IL-17 has important functions in the control of extracellular bacterial infections (K. 1. Happel et al., J. Exp. Med. 202, 761 (September 19, 2005)). The role of IL-22, however, in host defense is still largely unknown. Tumor Necrosis Lactor (TNL)-related proteins are recognized in the art as a large family of proteins having a variety of activities ranging from host defense to immune regulation to apoptosis. TNL was first identified as a serum-derived factor that was cytotoxic for several transformed cell lines in vitro and caused necrosis of certain tumors in vivo. A similar factor in the superfamily was identified and referred to as lymphotoxin (LT). Due to observed similarities between TNL and LT in the early 1980's, it was proposed that TNL and LT be referred to as TNF-α and TNL-β, respectively. Scientific literature thus makes reference to both nomenclatures. As used in the present application, the term TNL refers to TNF-α. Later research revealed two forms of lymphotoxin, referred to as LTa and υΓβ. US 2005-0129614 describes another polypeptide member of the TNL ligand super-family based on structural and biological similarities, designated TL-5. Members of the TNL family of proteins exist in membrane-bound forms that act locally through cell-cell contact, or as secreted proteins. A family of TNL-related receptors react with these proteins and trigger a variety of signalling pathways including cell death or apoptosis, cell proliferation, tissue differentiation, and proinflammatory responses. TNF-α by itself has been implicated in inflammatory diseases, autoimmune diseases, viral, bacterial, and parasitic infections, malignancies, and/or neurodegenerative diseases and is a useful target for specific biological therapy in diseases such as RA and Crohn's disease.

It is to be understood that if any prior art publication is referred to herein, such reference does not constitute an admission that the publication forms a part of the common general knowledge in the art in Australia or any other country.

SUMMARY OF THE INVENTION

The present invention provides compositions and methods for treatment of microbial disorders by modulation of the host immune response. For example, an anti-microbial immune response in a host can be enhanced or inhibited by increasing or decreasing an activity of one or more antimicrobial polypeptides (AMPs) that mediate the anti-microbial immune response.

More particularly, the present invention provides AMPs, modulators thereof, and methods of using such compositions for treatment of microbial disorders. Such microbial disorders include, but are not limited to, infectious diseases, for example, EHEC- and EPEC-caused diarrhea, Inflammatory Bowel Disease (1BD) and, more particularly, Ulcerative Colitis (UC) and Crohn’s Disease (CD).

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-3AMPs of the present invention are polypeptides that mediate an anti-microbial immune response, and include, but are not limited to, LT, IL-6, IL-18, IL-22, IL-23 (including e.g., IL-23 p 19 or IL-23 p40), and Reg or Reg-related proteins encoded by the genes of the Reg super family. The Reg super family includes Reg and Reg-related genes from human, rat, and mouse and are grouped into four subclasses, types I, II, III, and IV. For example, type I includes human REG la, human REG 1β, rat Regl, and mouse Regl', type II includes mouse ReglR type III includes human REG III, human H1P/PAP (gene expressed in hepatocellular carcinoma-intestine-pancreas/gene encoding pancreatitisassociated protein), rat PAP/Peptide23, rat Reglll/PAPll, rat PAP 111, mouse Regllla, Regllip,

Regllly, mouse Regllld, and hamster INGAP (islet neogenesis-associated protein). Type IV contains human REG IV. In one aspect, the REG protein is encoded by a member of the human REG gene family which includes, but is not limited to, REG Ια, REG Ιβ, HIP/PAP, REG III, REG IV, and Regrelated sequence (RS).

In some aspects, the amino acid sequence of an AMP of the present invention comprises an amino acid sequence selected from the following group: SEQ ID NO: 2, SEQ ID NO: 4 , SEQ ID

NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40,

SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO:

52, SEQ ID NO: 54, and SEQ ID NO: 56.

In other aspects, the nucleic acid sequence encoding an AMP of the present invention comprises a nucleic acid sequence selected from the following group: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO:

27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID

NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, and SEQ ID NO: 55.

An activity of an AMP of the present invention can be increased or decreased and/or differentially regulated relative to the activity of another AMP or the same AMP. Examples of an activity of an AMP of the present invention, includes, but is not limited to, AMP expression, binding to a binding partner, signal transduction, anti-microbial activity, or other biological or immunological activity thereof.

In one aspect, an increase in the activity of one or more AMPs of the present invention results in an enhanced anti-microbial immune response in a subject.

In one aspect, AMPs of the present invention include, but are not limited to, polypeptides that 35 directly or indirectly interact with IL-22, e.g., polypeptides that are upstream or downstream of an IL22 signal transduction pathway that mediates host resistance to infection by a microbial pathogen

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-42016259423 17 Oct 2018 (e.g., a bacteria or virus). Examples of such AMPs include, but are not limited to, LT, IL-6, IL-18, and IL-23 (including e.g., IL-23 p 19 or IL-23 p40).

Modulators of the present invention include, but are not limited to, polypeptides and nucleic acid molecules (e.g., a DNA molecule or RNA molecule) that directly or indirectly modulate an activity of an AMP. Examples of such modulation include, but are not limited to, an increase, decrease, induction or activation, inhibition, or regulation (e.g., up or down regulation) of an activity of an AMP of the present invention.

In one aspect, the modulator indirectly modulates IL-22 activity by decreasing or inhibiting IL22 Binding Protein (BP) activity and thereby, increasing IL-22 activity. In a particular aspect, the modulator decreases or inhibits binding of IL-22 BP to IL-22 and thereby, increases IL-22 activity.

In some aspects, the modulator is a polypeptide e.g., a polypeptide that binds to or otherwise interacts with an AMP to increase, induce, or regulate an activity of an AMP. In one aspect, the modulator is a lusion polypeptide that modulates an activity of an AMP.

In one aspect, the modulator is an antibody that binds to an AMP. In a particular aspect, the 15 antibody is a monoclonal antibody. In another aspect, the antibody is an antibody fragment selected from a Fab, Fab’-SH, Fv, scFv, or (Fab’fi fragment. In another aspect, the antibody is a fusion polypeptide (e.g., an Fc lusion polypeptide). In another aspect, the antibody is a chimeric antibody.

In a particular aspect, the antibody is humanized. In another aspect, the antibody is a human antibody. In another aspect, the antibody binds to the same epitope as an antibody selected from a human, non-human primate, or other mammal (e.g., pig, sheep, rabbit, marmot, rat, or mouse). In a particular aspect, the antibody is an AMP agonist.

In another particular aspect, the modulator is a recombinant AMP or nucleic acid molecule encoding an AMP (e.g., a DNA or RNA molecule).

The present invention further provides methods of treating a microbial disorder by modulating 25 an anti-microbial immune response. In one aspect, the present invention provides a method of treating a microbial disorder, in a subject, comprising administering to the subject an effective amount of pharmaceutical composition comprising an AMP or modulator of the AMP, wherein the AMP is selected from a group consisting of: LT, IL-6, IL-18, IL-22, IL-23, REG Ια, REG Ιβ, HIP/PAP, REG III, REG IV and Reg-related sequence (RS). In one aspect the disorder is an infectious disease, for example, EHEC- or EPEC-caused diarrhea, Inflammatory Bowel Disease (IBD) or, more particularly, Ulcerative Colitis (UC) or Crohn’s Disease (CD).

In particular aspects, the present invention provides methods of modulating an anti-microbial immune response by stimulating or inhibiting an AMP-mediated signaling pathway and/or Thi_L-i7 cell lunction. Such methods are uselul for treatment of microbial disorders. For example, in one aspect, the present invention provides a method of enhancing an anti-microbial immune response by stimulating an AMP-mediated signaling pathway, e.g., and IL-22 and/or IL-23 mediated signaling

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- 5 pathway. In another aspect, the present invention provides methods of modulating an anti-microbial immune response by stimulating or inhibiting a cytokine-mediated signaling pathway. For example, in one aspect, the present invention provides methods of enhancing an anti-microbial immune response by stimulating a cytokine-mediated signaling pathway, e.g., an IL-22 and/or IL-23 signaling pathway. Moreover, the present invention provides methods of modulating an anti-microbial immune response by stimulating or inhibiting a ThiL-17 cell function.

In one aspect, the present invention provides a method of stimulating an AMP-mediated signaling pathway in a biological system, the method comprising providing an AMP agonist to the biological system. Examples of such a biological system include, but are not limited to, mammalian cells in an in vitro cell culture system or in an organism in vivo. In another aspect, the present invention provides a method of inhibiting an AMP-mediated signaling pathway in a biological system, the method comprising providing an AMP antagonist to the biological system.

In a particular aspect, the present invention provides a method of enhancing an anti-microbial immune response in a biological system by stimulating an IL-23 and/or IL-22 mediated signaling pathway in a biological system, the method comprising providing an IL-22 or IL-22 agonist to the biological system. In one aspect, an IL-22 agonist is IL-22. In another aspect, the IL-22 agonist is an antibody that binds to IL-22.

In another aspect, a method of inhibiting an IL-23-mediated signaling pathway in a biological system is provided, the method comprising providing an IL-22 antagonist to the biological system. In one aspect, the antagonist of IL-22 is an antibody, e.g., a neutralizing anti-IL-22 antibody and/or a neutralizing anti-IL-22R antibody.

In another aspect, the present invention provides a method of stimulating a ThiL-17 cell function, the method comprising exposing a ThiL-17 cell to an agonist of an AMP that mediates the IL-23 mediated signaling pathway (e.g., IL-23, IL-6, or IL-22). Such methods are useful for treating a microbial disorder. In one aspect, an IL-22 agonist is IL-22. In another aspect, the IL-22 agonist is an antibody that binds to IL-22.

In another aspect, a method of inhibiting a Thin? cell function is provided, the method comprising exposing a ThiL-17 cell to an antagonist of an AMP that mediates the IL-23 mediated signaling pathway (e.g., IL-23, IL-6, or IL-22). In one aspect the antagonist is an anti-IL-22 antibody,

e.g., a neutralizing anti-IL-22 antibody.

Exemplary ThiL-17 cell functions include, but are not limited to, stimulation of cell-mediated immunity (delayed-type hypersensitivity); recruitment of innate immune cells, such as myeloid cells (e.g., monocytes and neutrophils) to sites of inflammation; and stimulation of inflammatory cell infdtration into tissues. In one aspect, a Thin? cell function is mediated by IL-23 and/or IL-22.

In a further aspect, the present invention provides a method of treating an infection by a microbial pathogen (e.g., a bacteria or virus), in a subject, comprising administering to the subject an

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-62016259423 17 Oct 2018 effective amount of pharmaceutical composition comprising an AMP or modulator of the AMP, wherein the AMP is selected from a group consisting of: LT, IL-6, IL-18,, IL-22, IL-23, REG Ια, REG Ιβ, HIP/PAP, REG III, REG IV and Reg-related sequence (RS).

In another aspect, the present invention provides a method of treating a microbial disorder, in a 5 subject, comprising contacting cells of the subject with a nucleic acid molecule (e.g., a DNA or RNA molecule) encoding an AMP or modulator of the AMP, wherein the AMP is selected from a group consisting of: LT, IL-6, IL-18, IL-22, IL-23, REG Ια, REG Ιβ, HIP/PAP, REG III, REG IV and Regrelated sequence (RS). In one aspect the disorder is an infectious disease, for example, EHEC- or EPEC-caused diarrhea, Inflammatory Bowel Disease (IBD) or, more particularly, Ulcerative Colitis (UC) or Crohn’s Disease (CD).

In another aspect, the present invention provides a method of modulating the activity of an AMP in cells of a subject infected with a microbial pathogen (e.g., a bacteria or virus), comprising contacting the cells with a nucleic acid molecule (e.g., a DNA or RNA molecule) encoding an AMP or modulator of the AMP, wherein the AMP is selected from a group consisting of: LT, IL-6, IL-18,

IL-22, IL-23, REG Ια, REG Ιβ, HIP/PAP, REG III (e.g., REG ΙΙΙβ or REGIIIy), REG IV, and Regrelated sequence (RS).

Examples of a microbial pathogen include, but are not limited to, a bacteria or virus. In one aspect, the microbial pathogen is a bacteria e.g., a gram-negative or gram-positive bacteria. In a particular aspect, the bacteria is a gram-negative bacteria. In another aspect, the bacteria is an attaching or effacing (A/E) bacteria and, more particularly, an enterohemorrhagic Escherichia coli (EHEC) or enteropathogenic E. Coli (EPEC). In one aspect, the bacteria is enteropathogenic E. coli (EHEC) is E. coli 0157:H7 or E. coli 055:H7.

In another aspect, the present invention provides polynucleotides encoding an AMP of the present invention, or modulator thereof. In another aspect, the invention provides a vector comprising the polynucleotide. In another aspect, the invention provides a host cell comprising the vector. In one aspect, the host cell is a eukaryotic cell. In another aspect, the host cell is a CHO cell, yeast cell, or bacterial cell (e.g., E. coli).

In one aspect, the present invention provides a method of making an antibody that binds to an AMP of the present invention, wherein the method comprises culturing the host cell under conditions suitable for expression of the polynucleotide encoding the antibody, and isolating the antibody. In a particular aspect, the invention provides a method of making an antibody that is an agonist of an AMP of the present invention.

In one aspect, the present invention provides a method of detecting the presence of an AMP in a biological sample, comprising contacting the biological sample with an antibody to the AMP, under conditions permissive for binding of the antibody to the AMP, and detecting whether a complex is formed between the antibody and AMP.

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-7In another aspect, the present invention provides a kit comprising one or more AMPs of the present invention and/or modulators thereof. In another aspect, the present invention provides a kit comprising one or more one or more pharmaceutical compositions each comprising an AMP of the present invention or modulator thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 depicts data demonstrating host defense against C. rodentium infection. Fig. 1(A) depicts the results of a real-time RT-PCR analysis on receptor subunits for IL-22 in uninfected wildtype mouse GI track; Fig. l(B-F) depicts a real-time RT-PCR analysis on various cytokine expressions in wildtype mouse colons upon C. rodentium infection; Fig. 1(G) depicts survival of C57B1/6 (n=5), IL-23p40 (n=5), and IL-6 (n=5) mice after C. rodentium infection; and Fig. 1(H) depicts a time course real-time RT-PCR analysis on IL-22 and IL-17 expressions in C57B1/6,1L23p40'', and IL-6 mouse colons upon C. rodentium infection. For C. rodentium infection, the mice were orally inoculated with 2xl0⁹ CFU of bacteria. All of the above data are representative of two independent experiments.

Fig. 2 depicts data demonstrating that IL-22 deficiency renders mice susceptible to C. rodentium infection. 6-7 weeks old IL-22'⁷' (Fig. 2(A-C)), IL-17RC'⁷' (D), IL-20RP'⁷' mice (Fig. 2(F)) or wildtype mice (Fig. 2(A-C, E)) were orally inoculated with 2xl0⁹ CFU of C. rodentium and weighed at indicated time points. Histologic analysis of colons from IL-22'⁷' and wildtype mice 8 days post inoculation using hematoxylin-and-eosin (H&E) staining (Fig. 2(B and C)). Arrows indicate submucosal inflammation (Fig. 2(B)), and bacterial invasion into mucosal glands (Fig. 2(C)). Representative data are shown (bars = 100 pm for Fig. 2(B) and bars = 25 pm for Fig. 2(C)).

Wildtype C57B1/6 mice received 150 pg of anti-IL-22 mAb or isotype control IgGl mAb intraperitoneally, every other day, starting on day 0 or day 8 post inoculation (Fig. 2(E)). * p < 0.05, ** p < 0.01, *** p < 0.001. All data are representative of two independent experiments.

Fig. 3 depicts data demonstrating the effect of IL-22 deficiency in mice during C. rodentium infection. C57BF6 mice (Fig. 3(A, B, and F)), IL-22'⁷' and wildtype mice (Fig. 3(C-E, and G)) were orally inoculated with 2xl0⁹ CFU of C. rodentium. Mice also received 150 pg of anti-IL-22 mAb or isotype control IgGl mAb intraperitoneally every other day starting from the same day as C.

rodentium inoculation (Fig. 3(A and B)). On day 10, colons were photographed and individual colon length was measured (Fig. 3(A)). Histologic analysis of colons was performed using hematoxylinand-eosin (H&E) staining (Fig. 3(B)). Histologic analysis of colons and livers (day 8) from infected IL-22'⁷' and wildtype mice was performed using H&E staining (Fig. 3(C and E)). Arrows in Fig. 3(C) indicate colonic transmural inflammation and ulceration. Fig. 3(E) depicts a hepatic septic microabscess in the IL-22'⁷' mouse. Representative data are shown in Fig. 3(C), where the bars = 500 pm for the upper panels and bars = 100 pm for the lower panels. In Fig.3(E), the bars = 25 pm. Fig.

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- 8 3(D) depicts the logio CFU of C. rodentium in colon, liver, spleen, and mesenteric lymph node. Fig. 3(F-G) depicts the serum anti-C. rodentium IgG levels by ELISA. * p < 0.05. All of the above data are representative of two independent experiments.

Fig. 4 depicts data demonstrating that IL-22 induces anti-microbial Regill family protein 5 expression upon C. rodentium infection. In vitro culture of C57BF6 mouse colons were treated with pg of IL-22 for 24 hours, RNA were isolated and used for microarray analysis (Fig.4(A)) and realtime RT-PCR analysis (Fig. 4(B)). In Fig. 4(C), IL-22'⁷' mice and wildtype littermates were orally inoculated with 2xl0⁹ CFU of C. rodentium, and real-time RT-PCR was performed on RNA isolated from individual mouse colon collected on indicated time points. All data are representative of two independent experiments.

Fig. 5 depicts data demonstrating the targeted disruption of the murine 1L-17RC gene. Fig.

5(A) depicts the strategy for generation of 1L-17RC knockout mice. Exons 1-5 (open boxes) encompassing the 1L-17RC coding sequence was replaced with a neomycin resistance cassette. Fig. 5(B) depicts the genotyping of offspring from wildtype (WT) and knockout (KO) mice using the indicated primer sets (Pl, P2 and P3). Tail tip fibroblasts from WT and KO mice were generated and stimulated with various concentrations of 1L-17A and 1L-17F in vitro for 24 hours, and culture supernatant were collected for IL-6 ELISA (Fig. 5(C)).

Fig. 6 depicts data of a real-time RT-PCR analysis on IL-19, IL-20 and IL-24 expression in wildtype mouse colons upon C. rodentium infection, over time. C57B1/6 mice were orally inoculated with 2x10⁹ CFU of C. rodentium. Colons were collected at indicated time points and isolated RNAs were used for real-time RT-PCR analysis.

Fig. 7 depicts data demonstrating lL-20Ra and IL-20P|f expression in the G1 tract. Real-time RT-PCR analysis on receptor subunits for IL-19, IL-20 and IL-24 in uninfected wildtype mouse G1 tract.

Fig. 8 depicts data demonstrating targeted disruption of the murine IL-20Rf gene. Fig. 8(A) depicts the strategy for generation of IL-20 R β knockout mice. Exon 1 (open boxes) was replaced with a neomycin resistance cassette. Fig. 8(B) depicts the phenotyping of offspring from wildtype (WT), heterozygous (HET) and knockout (KO) mice using the indicated primer sets (pi, p2 and p3). Fig. 8(C) WT and KO mouse ears were injected intradermally with 500 ng recombinant IL-20 in 20 μΐ

PBS or with 20 μΐ PBS alone. 24 hours later, mouse ears were collected for RNA isolation. Isolated RNAs were used for real-time RT-PCR analysis for genes known to be upregulated upon IL-20 signaling.

Fig. 9 depicts data of a histologic analysis of mouse colons from anti-lL-22 mAh treated wildtype mice inoculated with C. rodentium. C57B1/6 mice were orally inoculated with 2xl0⁹ CFU of C. rodentium. Mice also received 150 pg of anti-lL-22 mAh or isotype control IgGl mAh intraperitoneally every other day starting from the same day as C. rodentium inoculation. On day 10,

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-92016259423 17 Oct 2018 routine histologic analysis of colons was performed using hematoxylin-and-eosin (H&E) staining. Arrows indicate mucosal ulceration with transmural inflammation. Representative images are shown, bars = 500 pm for the upper panels and bars = 250 pm for the lower panels.

Fig. 10 depicts data demonstrating serum lg levels in IL-22'⁷' mice and wildtype littermates 5 during C. rodentium infection. IL-22'⁷' and wild type littermates mice were orally inoculated with

2xl0⁹ CFU of C. rodentium. On indicated time points, mouse blood were collected. Levels of total serum IgM and IgG (Fig. 10(A)) and serum anti-C. rodentium IgG2a, IgG2b, IgG2c and IgG3 (Fig. 10(B)) were determined by ELISA. All data are representative of two independent experiments.

Fig. 11 depicts data demonstrating an ex vivo colon culture ELISA of IL-22 (Fig. 11(A)) and

IL-17 (Fig. 11(B)) expression in C57B1/6, IL-23pl9'⁷', and IL-6'⁷' mouse colons after C. rodentium infection. For C. rodentium infection, mice were orally inoculated with 2xl0⁹ CFU of bacteria. All data are representative of at least two independent experiments.

Fig. 12 depicts a FACS analysis of IL-22R expression on isolated mouse IEL, LPMCs and colonic epithelial cells (Fig. 12 (A)), and a FACS analysis of IL-22R expression on primary human colonic epithelial cells (Fig. 12 (B)). All data are representative of at least two independent experiments.

Fig. 13 depicts data demonstrating that IL-22, produced by dendritic cells (DCs), is critical for innate immune responses against C. rodentium infection. In Fig. 13(A), Rag2'⁷' and wildtype Balb/c mice were orally inoculated with 2xl0⁹ CFU of C. rodentium. In Fig. 13(B and C), the mice also received 150 pg of isotype control IgGl mAh or anti-IL-22 mAh intraperitoneally every other day starting at the same day as bacteria inoculation and were weighed at the indicated time points. Fig. 13(B) depicts a time course real-time RT-PCR analysis, and Fig. (13(C) depicts an ex vivo colon culture ELISA of IL-22 and IL-17 expression in colons of wildtype Balb/c and Rag2'⁷' mice following C. rodentium infection. Fig. 13(D) depicts the immunohistochemical staining for IL-22, CD 11c, and

DAPI in day 4 colons from C. rodentium infected Rag2'⁷' miceMagnification: 400x. Fig. 13(E) depicts data demonstrating that IL-23 directly induces IL-22 production, as measured by ELISA, from isolated murine CD1 lc⁺ DCs in vitro. All data are representative of two independent experiments.

Fig. 14 depicts data demonstrating that IL-22 can induce STAT3 activation in human colon cells lines. In Fig. 14(A), IL-22'⁷' mice and wildtype littermates were orally inoculated with 2xl0⁹

CFU of C. rodentium. One group of IL-22'⁷' mice also received mPegl I Ιγ-lg fusion protein. Animals were weighed and monitored at the indicated time points. * p < 0.05, ** p < 0.01. In Fig. 14(B), IL23 directly induces IL-22 production from isolated human DCs, measured by ELISA. Fig. 14(C) depicts IL-22R expression by FACS on human colon cell lines. Fig. 14(D) depicts a Western blotting showing that IL-22 can induce STAT3 activation in human colon cell lines. Fig. 14(E) depicts a real35 time RT-PCR analysis for Regllip and Regllfy expression in human colonic epithelial cell lines treated with IL-22. All data are representative of two independent experiments.

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Fig. 15 depicts the characterization of anti-lL-22 mAh for immunohistochemisty. Fig. 15(A) depicts colon sections from day4 C. rodentium infected IL-22-/- and wildtype mice or uninfected wildtype mice, stained with Alexa555 conjugated anti-lL-22 mAh (8E11) or isotype control. Fig 15(B) depicts cell pellets of lL-22-expressing 293 cells stained with Alexa555 conjugated anti-lL-22 mAh (8E11) or isotype control. The magnification is at 200x.

Fig. 16 depicts a time-course analysis on Regllly and Regllip expression in C57BE6 and lL-23p 19-/- mouse colons following C. rodentium infection. C57B1/6 and !L-23pl9-/- mice were orally inoculated with 2x109 CFU of C. rodentium. At the indicated time points, mouse colons were collected for RNA extraction and subsequently real-time

RT-PCR analysis on mouse Regllly and Regllip expression.

Fig. 17 depicts a time-course analysis on other Reg family members expressions in IL-22-/and wildtype mouse colons following C. rodentium infection. IL-22-/- and wild type littermates mice were orally inoculated with 2x109 CFU of C. rodentium. At the indicated time points, mouse colons were collected for RNA extraction and subsequently real-time RT-PCR analysis.

Fig. 18 depicts data demonstrating that recombinant human Reglll_ fusion protein can partially protect IL-22-/- following C. rodentium infection. IL-22-/- mice and wildtype littermates were orally inoculated with 2x109 CFU of C. rodentium. One group of IL-22-/- mice also received human RegHI -cFlag fusion proteins. Animals were weighed and monitored at the indicated time points. * p < 0.05.

Fig. 19 A-C depicts 161 genes differentially expressed in colon, from IL-22 treatment.

Fig. 20 depicts the 2D hierarchical clustering of 161 genes differentially expressed in colon from IL-22 treatment, where selected genes were clustered by iterative agglomeration of vectors most highly linked by Pearson correlation coefficient, with data for agglomerated vectors summarized by average linkage.

Fig. 21 depicts data demonstrating LTbRFc and anti-IL-22 mAh both lead to mortality after

C. rodentium infection.

Fig. 22 depicts data demonstrating LT pathway regulation of multiple upstream aspects involved in IL-22 production.

Fig. 23 depicts data demonstrating IL-22 partially rescues the defects seen in LTbR treated mice.

Fig. 24 depicts data demonstrating anti-IL-22 mAh treatment leads to reduced colon follicles, compromised B/T organization, and reduced DC, T cell and B cell numbers in the colon.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compositions and methods for treatment of microbial disorders by modulation of the host immune response.

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The present inventors discovered a novel cytokine pathway that mediates immune response and resistance of mammals to infectious microbial pathogens. In particular, the present inventors discovered that IL-22 is one of the key cytokines that bridges adaptive immune response and innate epithelial defense during early infection of an attaching or effacing (A/E) bacterial pathogen.

As shown herein, cytokines such as IL-22 that are produced by immune cells during the early stages of infection are necessary for intestinal epithelial cells to elicit a full-anti-microbial response and wound-healing response in order to prevent systemic invasion of pathogenic microbes into the host. The studies herein show that IL-22 protects the integrity of the intestinal epithelial barrier and prevents bacterial invasion with systemic spread. Further, the studies herein indicate that IF-22 is involved in the elicitation of the early anti-bacterial IgG responses, and is indespensable for the induction of anti-microbial lectins, such as Regill// and Regl 1 Ιγ, from colonic epithelial cells during bacterial infection. The lack of either or both of these mechanisms may contribute to the compromised host defense response with increased systemic spread and mortality in IL-22 mice during C. rodentium infection.

As shown herein, the induction of Regl 11 β and Regl I Ιγ indicates that IL-22 may have broader functions in controlling various bacterial infections. The studies herein further support the role of ThiL-17 cells and their effector cytokines in infectious disorders and autoimmune disorders. Further, the studies herein indicate that 1F-22 and its downstream products, such as Regl 11 β and Regl I Ιγ, may be beneficial for the treatment of infectious disorders.

Therefore, the present invention provides methods of treating such microbial disorders by modulation of the host immune response. For example, an anti-microbial immune response in a subject can be enhanced or inhibited by increasing or decreasing an activity of one or more antimicrobial polypeptides (AMPs) that mediate the anti-microbial immune response.

More particularly, the present invention provides AMPs, modulators thereof, and methods of using such compositions for treatment of microbial disorders. Such microbial disorders include, but are not limited to, infectious diseases, for example, EHEC- and EPEC-caused diarrhea, Inflammatory Bowel Disease (1BD) and, more particularly, Ulcerative Colitis (UC) and Crohn’s Disease (CD).

All references, including patents, applications, and scientific literature, cited herein are hereby incorporated by reference, in their entirety.

GENERAL TECHNIQUES

The techniques and procedures described or referenced herein are generally well understood and commonly employed using conventional methodology by those skilled in the art, such as, for example, the widely utilized methodologies described in Sambrook et al., Molecular Cloning: A

Laboratory Manual 3rd. edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Current Protocols in Molecular Biology (F. M. Ausubel, et al. eds., (2003)); the series Methods

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- 12 2016259423 17 Oct 2018 in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (M. J. MacPherson, B. D.

Hames and G. R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Animal Cell Culture (R. 1. Freshney, ed. (1987)); Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J.

E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R. 1. Freshney), ed., 1987); Introduction to Cell and Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B. Griffiths, andD. G. Newell, eds., 1993-8) J. Wiley and Sons; Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M. Miller and Μ. P. Calos, eds., 1987); PCR: The

Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (J. E.

Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999);

Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (D. Catty., ed., 1RL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using Antibodies: A

Laboratory Manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (V. T. DeVita et al., eds., J.B. Lippincott Company, 1993).

I. DEFINITIONS

In the claims which follow and in the description of the invention, except where the context requires otherwise due to express language or necessary implication, the word “comprise” or variations such as “comprises” or “comprising” is used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.

For purposes of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition set forth below conflicts with any document incorporated herein by reference, the definition set forth below shall control.

An anti-microbial polypeptide” or “AMP” is a polypeptide that mediates, or otherwise effects, an anti-microbial immune response to a microbial pathogen, and encompasses encompasses a fragment, variant, analog, derivative or mimetic thereof that retains an AMP activity, e.g., an antimicrobial activity, or activity for modulating an anti-microbial immune response. These methods can be used to treat subjects that are infected with or at risk for infection with an infectious microbial pathogen, e.g., a virus or bacterium. The activity of the AMP can be modulated or differentially regulated (e.g., up or down regulated) relative to another AMP or the same AMP.

An AMP of the present invention encompasses a native AMP and variant forms thereof (which are further defined herein), and may be isolated from a variety of sources, such as from human tissue

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- 13 2016259423 17 Oct 2018 or from another source, or prepared by recombinant or synthetic methods. A native AMP may be from any species, e.g., murine or human. AMPs of the present invention include, but are not limited to, LT, IL-6, IL-18, IL-22, IL-23 (including e.g., IL-23 p 19 or IL-23 p40), and Reg or Reg-related proteins encoded by the genes of the Reg super family. The Reg super family includes Reg and Reg5 related genes from human, rat, and mouse and are grouped into four subclasses, types 1,11, 111, and IV. For example, type 1 includes human REG la, human REG Ιβ, rat Regl, and mouse RegR, type 11 includes mouse ReglR, type 111 includes human REG 111, human H1P/PAP (gene expressed in hepatocellular carcinoma-intestine-pancreas/gene encoding pancreatitis-associated protein), rat PAP/Peptide23, rat Reglll/PAPll, rat PAP 111, mouse Regllla, Regl 11β, Regllly, mouse Regl 11δ. and hamster INGAP (islet neogenesis-associated protein). Type IV contains human REG IV.

Additionally, human Reg-related Sequence (RS) is reportedly a pseudogene. In one embodiment, the REG protein is encoded by a member of the human REG gene family which includes, but is not limited to, REG la, REG Ιβ, H1P/PAP, REG 111, REG IV, and Reg-related sequence (RS).

Lymphotoxin (LT) is a trimeric cytokine in the tumor necrosis family; expressed by activated

T, B, and NK cells; and involved in inflammatory response signaling and secondary lymphoid organ architecture. “Lymphotoxin-“ or “LT” is defined herein as a biologically active polypeptide having the amino acid sequence shown in FIG. 2A of US Pat. No. 5,824,509. “LT” is defined to specifically exclude human TNFa or its natural animal analogues (Pennica et al., Nature 312:20/27: 724-729 (1984) and Aggarwal et al., J. Biol. Chem. 260: 2345-2354 (1985)). As used herein, “LT” refers to one or more LT subunits as described herein.

“Lymphotoxin-α” or “LTa” is defined to specifically exclude human LT|! as defined, for example, in US 5,661,004. “Lymphotoxina-3 trimer” or “LTa3” refers to a homotrimer of LTa monomers. This homotrimer is anchored to the cell surface by the ΕΤβ, transmembrane and cytoplasmic domains.

“Lymphotoxin-αβ” or “ΕΤαβ” or ‘ΈΤαβ complex” refers to a heterotrimer of LTa with ΕΤβ.

These heterotrimers contain either two subunits of LTa and one subunit of ΕΤβ (ΕΤα2β 1), or one subunit of LTa and two of ΕΤβ (ΕΤα1β2). The term “ΕΤαβ” or “LTab” as used herein refers to a heterotrimer made up of one subunit of LTa and two of ΕΤβ (ΕΤα1β2).

“Tumor necrosis factor receptor-1” or “TNFR1” and “tumor necrosis factor receptor-Π” or “TNFRII” refer to cell-surface TNF receptors for the LTa3 homotrimer, also known as p55 and p75, respectively.

“Lymphotoxin-β receptor” or “ΕΤβ-R” refers to the receptor to which the ΕΤαβ heterotrimers bind.

In some embodiments, the amino acid sequence of an AMP of the present invention comprises an amino acid sequence selected from the following group: SEQ ID NO: 2 (human IL-6), SEQ ID

NO: 4 (human 1L-12B), SEQ ID NO: 6 (human IL-18), SEQ ID NO: 8 (human IL-22), SEQ ID NO:

(human IL-23 p 19 or 1L-23A), SEQ ID NO: 12 (human REGIA), SEQ ID NO: 14 (human

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REGIB), SEQ ID NO: 16 (human REG3A, variant 1), SEQ ID NO: 18 (human REG3A, variant 2), SEQ ID NO: 20 (human REG3A, variant 3), SEQ ID NO: 22 (human REG3G, variant 2), SEQ ID NO: 24 (human REG3G, variant 1), SEQ ID NO: 26 (human REG4), SEQ ID NO: 28 (murine IL-6), SEQ ID NO: 30 (murine IL-12B), SEQ ID NO: 32 (murine IL-18), SEQ ID NO: 34 (murine IL-22),

SEQ ID NO: 36 (murine IL-23 p 19 or IL-23A), SEQ ID NO: 38 (murine PAP), SEQ ID NO: 40 (murine REG1), SEQ ID NO: 42 (murine REG2), SEQ ID NO: 44 (murine REG3A), SEQ ID NO: 46 (murine REG3D), SEQ ID NO: 48 (murine REG4), SEQ ID NO: 50 (human LTa), SEQ ID NO: 52 (human LTP), SEQ ID NO: 54 (murine LTa), and SEQ ID NO: 56 (murine LTP).

In other embodiments, the nucleic acid sequence encoding an AMP of the present invention 10 comprises a nucleic acid sequence selected from the following group: SEQ ID NO: 1 (human IL-6),

SEQ ID NO: 3 (human IL-12B), SEQ ID NO: 5 (human IL-18), SEQ ID NO: 7 (human IL-22), SEQ ID NO: 9 (human IL-23 p 19 or IL-23A), SEQ ID NO: 11 (human REGIA), SEQ ID NO: 13 (human REGIB), SEQ ID NO: 15 (human REG3A, variant 1), SEQ ID NO: 17 (human REG3A, variant 2), SEQ ID NO: 19 (human REG3A, variant 3), SEQ ID NO: 21 (human REG3G, variant 2), SEQ ID

NO: 23 (human REG3G, variant 1), SEQ ID NO: 25 (human REG4), SEQ ID NO: 27 (murine IL-6), SEQ ID NO: 29 (murine IL-12B), SEQ ID NO: 31 (murine IL-18), SEQ ID NO: 33 (murine IL-22), SEQ ID NO: 35 (murine IL-23 p 19 or IL-23A), SEQ ID NO: 37 (murine PAP), SEQ ID NO: 39 (murine REG1), SEQ ID NO: 41 (murine REG2), SEQ ID NO: 43 (murine REG3A), SEQ ID NO: 45 (murine REG3D), SEQ ID NO: 47 (murine REG4), SEQ ID NO: 49 (human LTa), SEQ ID NO: 51 (human LTP), SEQ ID NO: 53 (murine LTa), and SEQ ID NO: 55 (murine LTP).

A native sequence AMP polypeptide” or a “native sequence AMP polypeptide refers to a polypeptide comprising the same amino acid sequence as a corresponding AMP polypeptide derived from nature. Such native sequence AMP polypeptides can be isolated from nature or can be produced by recombinant or synthetic means. The terms specifically encompass naturally-occurring truncated or secreted forms of the specific AMP polypeptide (e.g., an IL-22 lacking its associated signal peptide), naturally-occurring variant forms (e.g., alternatively spliced forms), and naturally-occurring allelic variants of the polypeptide. In various embodiments of the invention, the native sequence AMP polypeptides disclosed herein are mature or full-length native sequence polypeptides.

A “variant ” polypeptide, refers to an active polypeptide having at least about 80% amino acid sequence identity with a full-length native polypeptide sequence. Ordinarily, a variant polypeptide will have at least about 80% amino acid sequence identity, alternatively at least about 81% amino acid sequence identity, alternatively at least about 82% amino acid sequence identity, alternatively at least about 83% amino acid sequence identity, alternatively at least about 84% amino acid sequence identity, alternatively at least about 85% amino acid sequence identity, alternatively at least about

86% amino acid sequence identity, alternatively at least about 87% amino acid sequence identity, alternatively at least about 88% amino acid sequence identity, alternatively at least about 89% amino

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- 15 2016259423 17 Oct 2018 acid sequence identity, alternatively at least about 90% amino acid sequence identity, alternatively at least about 91 % amino acid sequence identity, alternatively at least about 92% amino acid sequence identity, alternatively at least about 93% amino acid sequence identity, alternatively at least about 94% amino acid sequence identity, alternatively at least about 95% amino acid sequence identity, alternatively at least about 96% amino acid sequence identity, alternatively at least about 97% amino acid sequence identity, alternatively at least about 98% amino acid sequence identity, and alternatively at least about 99% amino acid sequence identity to a full-length or mature native polypeptide sequence.

Percent (%) amino acid sequence identity, is defined as the percentage of amino acid residues 10 in a candidate sequence that are identical with the amino acid residues in a specific or reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For amino acid sequence comparisons, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows:

100 times the fraction X/Y where X is the number of amino acid residues scored as identical matches by the sequence alignment program in that program’s alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. As examples of % amino acid sequence identity calculations using this method, Tables 1 and 2 below demonstrate how to calculate the % amino acid sequence identity of the amino acid sequence designated “Reference Protein” to the amino acid sequence designated “IL-22”, wherein IL-22 represents the amino acid sequence of an IL-22 polypeptide of interest, Reference Protein represents the amino acid sequence of a polypeptide against which the IL-22 polypeptide of interest is being compared, and X, Y and Z each represent different amino acid residues.

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Table 1

IL-22 XXXXXXXXXXXXXXX (Length = 15 amino acids)

Reference Protein XXXXXYYYYYYY (Length = 12 amino acids) % amino acid sequence identity = (the number of identically matching amino acid residues between the two polypeptide sequences) divided by (the total number of amino acid residues of the IL-22 polypeptide) = divided by 15 = 33.3%

Table 2

IL-22 XXXXXXXXXX (Length = 10 amino acids)

Reference Protein XXXXXYYYYYYZZYZ (Length = 15 amino acids) % amino acid sequence identity = (the number of identically matching amino acid residues between the two polypeptide sequences) divided by (the total number of amino acid residues of the IL-22 polypeptide) = divided by 10 = 50%

An isolated biological molecule, such as the various polypeptides, polynucleotides, and antibodiesdisclosed herein, refers to a biological molecule that has been identified and separated and/or recovered from at least one component of its natural environment.

Active or activity, with reference to a polypeptide, refers to a biological and/or an 25 immunological activity of a native polypeptide, wherein “biological” activity refers to a biological function of a native polypeptide other than the ability to induce the production of an antibody against an antigenic epitope possessed by the native polypeptide. An “immunological” activity refers to the ability to induce the production of an antibody against an antigenic epitope possessed by a native polypeptide.

The term antagonist is used in the broadest sense, and includes any molecule that partially or fully blocks, inhibits, or neutralizes a biological activity of a polypeptide. Also encompassed by “antagonist” are molecules that fully or partially inhibit the transcription or translation of mRNA encoding the polypeptide. Suitable antagonist molecules include, e.g., antagonist antibodies or antibody fragments; fragments or amino acid sequence variants of a native polypeptide; peptides;

antisense oligonucleotides; small organic molecules; and nucleic acids that encode polypeptide

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- 17 2016259423 17 Oct 2018 antagonists or antagonist antibodies.. Reference to “an” antagonist encompasses a single antagonist or a combination of two or more different antagonists.

The term agonist is used in the broadest sense and includes any molecule that partially or fully mimics a biological activity of a polypeptide, e.g., a native AMP. Also encompassed by “agonist” are molecules that stimulate the transcription or translation of mRNA encoding the polypeptide. Suitable agonist molecules include, e.g., agonist antibodies or antibody fragments; a native polypeptide; fragments or amino acid sequence variants of a native polypeptide; peptides; antisense oligonucleotides; small organic molecules; and nucleic acids that encode polypeptides agonists or antibodies. Reference to “an” agonist encompasses a single agonist or a combination of two or more different agonists.

An “anti-microbial immune response” includes, but is not limited to, resistance or defense to infection by a microbial pathogen. Such resistance or defense can result in an inhibition or decrease in microbial infectivity, replication, proliferation or other activity of a microbial pathogen. In particular, treatment resulting in an anti-microbial immune response can result in the alleviation of a microbial disorder or symptom of a microbial disorder.

Alleviation, “alleviating” or equivalents thereof, refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to ameliorate, prevent, slow down (lessen), decrease or inhibit the targeted microbial disorder or symptom thereof. Those in need of treatment include those already with the disorder as well as those prone to having the disorder or those in whom the disorder is to be prevented.

With reference to treating a microbial disorder, “treatment”, “treating”, or equivalents thereof, refers to alleviating a microbial disorder or a symptom of a microbial disorder, in a subject having the disorder.

Chronic administration refers to administration of an agent(s) in a continuous mode as 25 opposed to an acute mode, so as to maintain the initial therapeutic effect for an extended period of time. “Intermittent” administration is treatment that is not consecutively done without interruption, but rather is cyclic in nature.

Mammal for purposes of treatment refers to any animal classified as a mammal, including humans, rodents (e.g., mice and rats), and monkeys; domestic and farm animals; and zoo, sports, laboratory, or pet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc. In some embodiments, the mammal is selected from a human, rodent, or monkey. Similarly, “subject” for the purposes of treatment, refers to a mammalian subject, and includes both human and veterinary subjects.

Administration in combination with one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order.

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Carriers as used herein include pharmaceutically acceptable carriers, excipients, or stabilizers which are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Often the physiologically acceptable carrier is an aqueous pH buffered solution.

Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN™, polyethylene glycol (PEG), and PLURON1CS™.

Antibodies (Abs) and immunoglobulins (Igs) are glycoproteins having similar structural characteristics. While antibodies exhibit binding specificity to a specific antigen, immunoglobulins include both antibodies and other antibody-like molecules which generally lack antigen specificity.

Polypeptides of the latter kind are, for example, produced at low levels by the lymph system and at increased levels by myelomas.

The terms antibody and “immunoglobulin” are used interchangeably in the broadest sense and include monoclonal antibodies (e.g., full length or intact monoclonal antibodies), polyclonal antibodies, monovalent antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies so long as they exhibit the desired biological activity) and may also include certain antibody fragments (as described in greater detail herein). An antibody can be chimeric, human, humanized and/or affinity matured.

An antibody that specifically binds to a particular antigen refers to an antibody that is capable of binding the antigen with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting the antigen. Preferably, the extent of binding of such an antibody to a non-target polypeptide is less than about 10% of the binding of the antibody to the target antigen as measured, e.g., by a radioimmunoassay (R1A). In certain embodiments, an antibody that binds to a target antigen has a dissociation constant (Kd) of < ΙμΜ, < 100 nM, < 10 nM, < 1 nM, or < 0.1 nM.

The “variable region” or “variable domain” of an antibody refers to the amino-terminal domains of the heavy or light chain of the antibody. The variable domain of the heavy chain may be referred to as “VH.” The variable domain of the light chain may be referred to as “VL.” These domains are generally the most variable parts of an antibody and contain the antigen-binding sites.

The term “variable” refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called

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-19 2016259423 17 Oct 2018 complementarity-determining regions (CDRs) or hypervariable regions (HVRs) both in the lightchain and the heavy-chain variable domains. The more highly conserved portions of variable domains are called the framework regions (FR). The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a beta-sheet configuration, connected by three

CDRs, which form loops connecting, and in some cases forming part of, the beta-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health,

Bethesda, MD (1991)). The constant domains are not involved directly in the binding of an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibodydependent cellular toxicity.

The “light chains” of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (k) and lambda (λ), based on the amino acid sequences of their constant domains.

Depending on the amino acid sequences of the constant domains of their heavy chains, antibodies (immunoglobulins) can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGi, lgG+, IgG3, IgG4, IgAi, and lgA+. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known and described generally in, for example, Abbas et al. Cellular and Mol. Immunology, 4th ed. (2000). An antibody may be part of a larger fusion molecule, formed by covalent or noncovalent association of the antibody with one or more other proteins or peptides.

The terms “full length antibody,” “intact antibody” and “whole antibody” are used herein interchangeably to refer to an antibody in its substantially intact form, not antibody fragments as defined below. The terms particularly refer to an antibody with heavy chains that contain the Fc region.

Antibody fragments” comprise only a portion of an intact antibody, wherein the portion retains at least one, and as many as most or all, of the functions normally associated with that portion when present in an intact antibody. In one embodiment, an antibody fragment comprises an antigen binding site of the intact antibody and thus retains the ability to bind antigen. In another embodiment, an antibody fragment, for example one that comprises the Fc region, retains at least one of the biological functions normally associated with the Fc region when present in an intact antibody, such as FcRn binding, antibody half life modulation, ADCC function and complement binding. In one embodiment, an antibody fragment is a monovalent antibody that has an in vivo half life substantially

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-20 2016259423 17 Oct 2018 similar to an intact antibody. For example, such an antibody fragment may comprise on antigen binding arm linked to an Fc sequence capable of conferring in vivo stability to the fragment.

Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with a single antigen-binding site, and a residual “Fc” fragment, whose name reflects its ability to crystallize readily. Pepsin treatment yields an F(ab’)2 fragment that has two antigencombining sites and is still capable of cross-linking antigen.

“Fv” is the minimum antibody fragment which contains a complete antigen-binding site. In one embodiment, a two-chain Fv species consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. In a single-chain Fv (scFv) species, one heavy- and one light-chain variable domain can be covalently linked by a flexible peptide linker such that the light and heavy chains can associate in a “dimeric” structure analogous to that in a two-chain Fv species. It is in this configuration that the three CDRs of each variable domain interact to define an antigenbinding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

The Fab fragment contains the heavy- and light-chain variable domains and also contains the constant domain of the light chain and the first constant domain (CHI) of the heavy chain. Fab’ fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CHI domain including one or more cysteines from the antibody hinge region. Fab’-SH is the designation herein for Fab’ in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab’)2 antibody fragments originally were produced as pairs of Fab’ fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

“Single-chain Fv” or “scFv” antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding. For a review of scFv see Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag,

New York, pp. 269-315 (1994).

The term “diabodies” refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies may be bivalent or bispecific. Diabodies are described more fully in, for example, EP 404,097; WO93/1161; Hudson et

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-21 2016259423 17 Oct 2018 al. (2003) Nat. Med. 9:129-134; and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al. (2003) Nat. Med. 9:129-134.

The term monoclonal antibody as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible mutations, e.g., naturally occurring mutations, that may be present in minor amounts. Thus, the modifier “monoclonal” indicates the character of the antibody as not being a mixture of discrete antibodies. In certain embodiments, such a monoclonal antibody typically includes an antibody comprising a polypeptide sequence that binds a target, wherein the target-binding polypeptide sequence was obtained by a process that includes the selection of a single target binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection process can be the selection of a unique clone from a plurality of clones, such as a pool of hybridoma clones, phage clones, or recombinant DNA clones. It should be understood that a selected target binding sequence can be further altered, for example, to improve affinity for the target, to humanize the target binding sequence, to improve its production in cell culture, to reduce its immunogenicity in vivo, to create a multispecific antibody, etc., and that an antibody comprising the altered target binding sequence is also a monoclonal antibody of this invention. In contrast to polyclonal antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. In addition to their specificity, monoclonal antibody preparations are advantageous in that they are typically uncontaminated by other immunoglobulins.

The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by a variety of techniques, including, for example, the hybridoma method (e.g., Kohler et al., Nature, 256: 495 (1975); Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2^nd ed. 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981)), recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567), phage display technologies (see,

e.g., Clackson et al., Nature, 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992);

Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132(2004), and technologies for producing human or human-like antibodies in animals that have parts or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (see, e.g., WO98/24893; WO96/34096; WO96/33735; WO91/10741;

Jakobovits et al., Proc. Natl. Acad. Sci. USA 90: 2551 (1993); Jakobovits et al., Nature 362: 255-258

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-22 2016259423 17 Oct 2018 (1993); Bruggemann et al., Year in Immunol. 7:33 (1993); U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016; Marks et al., Bio. Technology 10: 779-783 (1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature 368: 812-813 (1994); Fishwild et al., Nature Biotechnol. 14: 845-851 (1996); Neuberger, Nature Biotechnol. 14: 826 (1996) and Lonberg and Huszar, Intern. Rev. Immunol. 13: 65-93 (1995).

The monoclonal antibodies herein specifically include chimeric antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).

Humanized forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In one embodiment, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit, or nonhuman primate having the desired specificity, affinity, and/or capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications may be made to further refine antibody performance. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a nonhuman immunoglobulin, and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also the following review articles and references cited therein: Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998);

Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428433 (1994).

A “human antibody” is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human and/or has been made using any of the techniques for making human antibodies as disclosed herein. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.

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Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGl, lgG2, lgG3, lgG4, IgA, and lgA2.

An “affinity matured” antibody is one with one or more alterations in one or more HVRs thereof which result in an improvement in the affinity of the antibody for antigen, compared to a parent antibody which does not possess those alteration(s). In one embodiment, an affinity matured antibody has nanomolar or even picomolar affinities for the target antigen. Affinity matured antibodies may be produced by procedures known in the art. Marks et al. Bio/Technology 10:77910 783 (1992) describes affinity maturation by VH and VL domain shuffling. Random mutagenesis of

HVR and/or framework residues is described by: Barbas et al. Proc Nat. Acad. Sci. USA 91:38093813 (1994); Schier et al. Gene 169:147-155 (1995); Yeltoneia/. J. Immunol. 155:1994-2004 (1995); Jackson et al., J. Immunol. 154(7):3310-9 (1995); and Hawkins et al, J. Mol. Biol. 226:889-896 (1992).

A “blocking” antibody, “neutralizing” antibody, or “antagonist” antibody is one which inhibits or reduces a biological activity of the antigen it binds. Such antibodies may substantially or completely inhibit the biological activity of the antigen.

An “agonist antibody,” as used herein, is an antibody which partially or fully mimics a biological activity of a polypeptide of interest.

Antibody “effector functions” refer to those biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody, and vary with the antibody isotype. Examples of antibody effector functions include: Cl q binding and complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g. B cell receptor); and B cell activation.

“Binding affinity” generally refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally bind antigen slowly and tend to dissociate readily, whereas high-affinity antibodies generally bind antigen faster and tend to remain bound longer. A variety of methods of measuring binding affinity are known in the art, any of which can be used for purposes of the present invention. Specific illustrative embodiments are described in the following.

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In one embodiment, the “Kd” or “Kd value” according to this invention is measured by a radiolabeled antigen binding assay (R1A) performed with the Fab version of an antibody of interest and its antigen as described by the following assay. Solution binding affinity of Fabs for antigen is measured by equilibrating Fab with a minimal concentration of (1 ^^I)-l_abeled antigen in the presence of a titration series of unlabeled antigen, then capturing bound antigen with an anti-Fab antibodycoated plate (Chen, et al., (1999) J. Mol. Biol. 293:865-881). To establish conditions for the assay, microtiter plates (Dynex) are coated overnight with 5 pg/ml of a capturing anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), and subsequently blocked with 2% (w/v) bovine serum albumin in PBS for two to five hours at room temperature (approximately 23°C). In a non-adsorbent plate (Nunc #269620), 100 pM or 26 pM [ 125j]._antjg_{en are} mixed with serial dilutions of a Fab of interest (e.g., consistent with assessment of the anti-VEGF antibody, Fab-12, in Presta et al., (1997) Cancer Res. 57:4593-4599). The Fab of interest is then incubated overnight; however, the incubation may continue for a longer period (e.g., about 65 hours) to ensure that equilibrium is reached. Thereafter, the mixtures are transferred to the capture plate for incubation at room temperature (e.g., for one hour). The solution is then removed and the plate washed eight times with 0.1% Tween-20 in PBS. When the plates have dried, 150 μΐ/well of scintillant (MicroScint-20; Packard) is added, and the plates are counted on a Topcount gamma counter (Packard) for ten minutes. Concentrations of each Fab that give less than or equal to 20% of maximal binding are chosen for use in competitive binding assays.

According to another embodiment, the Kd or Kd value is measured by surface plasmon resonance assays using a BIAcoi'c ' ^-2000 or a BIAcoi'c ' ^-3000 (BlAcore, Inc., Piscataway, NJ) at 25°C with immobilized antigen CM5 chips at ~10 response units (RU). Briefly, carboxymethylated dextran biosensor chips (CM5, BlAcore Inc.) are activated with TV-ethyl-TV (3dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and TV-hydroxysuccinimide (NHS) according to the supplier’s instructions. Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 pg/ml (~0.2 μΜ) before injection at a flow rate of 5 μΐ/minute to achieve approximately 10 response units (RU) of coupled protein. Following the injection of antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetics measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with 0.05% Tween 20 (PBST) at 25°C at a flow rate of approximately 25 μΐ/min. Association rates (k_on) and dissociation rates (k_off) are calculated using a simple one-to-one

Langmuir binding model (BlAcore Evaluation Software version 3.2) by simultaneously fitting the association and dissociation sensorgrams. The equilibrium dissociation constant (Kd) is calculated as the ratio k_off/k_on See, e.g., Chen, Y., et al., (1999) J. Mol. Biol. 293:865-881. If the on-rate exceeds

10^ M’l s‘l by the surface plasmon resonance assay above, then the on-rate can be determined by using a fluorescent quenching technique that measures the increase or decrease in fluorescence

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-25 2016259423 17 Oct 2018 emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm band-pass) at 25°C of a 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in the presence of increasing concentrations of antigen as measured in a spectrometer, such as a stop-flow equipped spectrophometer (Aviv Instruments) or a 8000-series SLM-Aminco spectrophotometer (ThermoSpectronic) with a stirred cuvette.

An “on-rate,” “rate of association,” “association rate,” or “ko_n” according to this invention can also be determined as described above using a BIAcore'^-2000 or a BIAcot'e ' M-3000 system (BIAcore, Inc., Piscataway, NJ).

An isolated antibody is one which has been identified and separated and/or recovered from a 10 component of its natural environment. Contaminant components of its natural environment are materials which would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In preferred embodiments, the antibody will be purified (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.

The word label when used herein refers to a detectable compound or composition which is conjugated directly or indirectly to a molecule (such as a nucleic acid, polypeptide, or antibody) so as to generate a labeled molecule. The label may be detectable by itself (e.g. radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition, resulting in a detectable product.

By solid phase is meant a non-aqueous matrix to which a molecule (such as a nucleic acid, polypeptide, or antibody) can adhere. Examples of solid phases encompassed herein include those formed partially or entirely of glass (e.g., controlled pore glass), polysaccharides (e.g., agarose), polyacrylamides, polystyrene, polyvinyl alcohol and silicones. In certain embodiments, depending on the context, the solid phase can comprise the well of an assay plate; in others it is a purification column (e.g., an affinity chromatography column). This term also includes a discontinuous solid phase of discrete particles, such as those described in U.S. Patent No. 4,275,149.

A liposome is a small vesicle composed of various types of lipids, phospholipids and/or surfactant which is useful for delivery of a drug (such as a nucleic acid, polypeptide, antibody, agonist or antagonist) to a mammal. The components of the liposome are commonly arranged in a bilayer formation, similar to the lipid arrangement of biological membranes.

A “small molecule” or “small organic molecule” is defined herein as an organic molecule

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-26 2016259423 17 Oct 2018 having a molecular weight below about 500 Daltons.

An “oligopeptide” that binds to a target polypeptide is an oligopeptide that is capable of binding the target polypeptide with sufficient affinity such that the oligopeptide is useful as a diagnostic and/or therapeutic agent in targeting the polypeptide. In certain embodiments, the extent of binding of an oligopeptide to an unrelated, non-target polypeptide is less than about 10% of the binding of the oligopeptide to the target polypeptide as measured, e.g., by a surface plasmon resonance assay. In certain embodiments, an oligopeptide bnds to a target polypeptide with a dissociation constant (Kd) of < ΙμΜ, < 100 nM, < 10 nM, < 1 nM, or < 0.1 nM.

An “organic molecule” that binds to a target polypeptide is an organic molecule other than an 10 oligopeptide or antibody as defined herein that is capable of binding a target polypeptide with sufficient affinity such that the organic molecule is useful as a diagnostic and/or therapeutic agent in targeting the polypeptide. In certain embodiments, the extent of binding of an organic molecule to an unrelated, non-target polypeptide is less than about 10% of the binding of the organic molecule to the target polypeptide as measured, e.g., by a surface plasmon resonance assay. In certain embodiments, an organic molecule binds to a target polypeptide with a dissociation constant (Kd) of < 1 μΜ, <100 nM, < 10 nM, < 1 nM, or < 0.1 nM.

A “biological system” is an in vitro, ex vivo, or in vivo system comprising mammalian cells that share a common signaling pathway.

“Microbial disorder” refers to a disease or condition wherein a microbial pathogen causes, mediates, or otherwise contributes to a morbidity of the disease or condition. Also included are diseases in which stimulation or intervention of an anti-microbial response has an ameliorative effect on progression of the disease. Included within this term are infectious diseases or conditions, and opportunistic diseases resulting from primary infection by a microbial pathogen. Examples of such infectious disease, include, but are not limited to, EHEC- and EPEC-caused diarrhea, Inflammatory

Bowel Disease (IBD) and, more particularly, Ulcerative Colitis (UC) and Crohn’s Disease (CD).

The term T cell mediated disease” means a disease in which T cells directly or indirectly mediate or otherwise contribute to a morbidity in a mammal. The T cell mediated disease may be associated with cell mediated effects, lymphokine mediated effects, etc., and even effects associated with B cells if the B cells are stimulated, for example, by the lymphokines secreted by T cells.

An “autoimmune disorder” or “autoimmunity” refers to any condition in which a humoral or cell-mediated immune response is mounted against a body’s own tissue. An “IL-23 mediated autoimmune disorder” is any autoimmune disorder that is caused by, maintained, or exacerbated by IL-23 activity.

“Inflammation” refers to the accumulation of leukocytes and the dilation of blood vessels at a site of injury or infection, typically causing pain, swelling, and redness,

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-27 2016259423 17 Oct 2018 “Chronic inflammation” refers to inflammation in which the cause of the inflammation persists and is difficult or impossible to remove.

“Autoimmune inflammation” refers to inflammation associated with an autoimmune disorder. “Arthritic inflammation” refers to inflammation associated with arthritis.

“Inflammatory bowel disease” or “IBD” refers to a chronic disorder characterized by inflammation of the gastrointestinal tract. IBD encompasses ulcerative colitis, which affects the large intestine and/or rectum, and Crohn’s disease, which may affect the entire gastrointestinal system but more commonly affects the small intestine (ileum) and possibly the large intestine.

The term effective amount is a concentration or amount of a molecule (e.g., a nucleic acid, polypeptide, agonist, or antagonist) that results in achieving a particular stated purpose. An “effective amount” may be determined empirically. A therapeutically effective amount is a concentration or amount of a molecule which is effective for achieving a stated therapeutic effect. This amount may also be determined empirically.

The term cytotoxic agent as used herein refers to a substance that inhibits or prevents the 15 lunction of cells and/or causes destruction of cells. The term is intended to include radioactive isotopes (e.g., I¹³¹,1¹²⁵, Y⁹⁰ and Re¹⁸⁶), chemotherapeutic agents, and toxins such as enzymatically active toxins of bacterial, lungal, plant or animal origin, or fragments thereof.

A growth inhibitory agent when used herein refers to a compound or composition which inhibits growth of a cell, especially a cell overexpressing any of a gene, either in vitro or in vivo.

Thus, a growth inhibitory agent is one which significantly reduces the percentage of cells overexpressing such genes in S phase. Examples of growth inhibitory agents include agents that block cell cycle progression (at a place other than S phase), such as agents that induce G1 arrest and M-phase arrest. Classical M-phase blockers include the vincas (vincristine and vinblastine), taxol, and topo II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Those agents that arrest G1 also spill over into S-phase arrest, for example, DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C. Further information can be found in The Molecular Basis of Cancer, Mendelsohn and Israel, eds., Chapter 1, entitled Cell cycle regulation, oncogens, and antineoplastic drugs by Murakami et al. (WB Saunders: Philadelphia, 1995), especially p. 13.

The term cytokine is a generic term for proteins released by one cell population which act on another cell population as intercellular mediators. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones. Included among the cytokines are growth hormone such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; fibroblast growth factor; prolactin; placental

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-28 2016259423 17 Oct 2018 lactogen; tumor necrosis factor-α and -β; lymphotoxin-α and -β, mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF-β; platelet-growth factor; transforming growth factors (TGFs) such as TGF-α and TGF-β; insulin-like growth factor-I and -II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon-α, -β, and -γ; colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs) such as IL-1, IL-Ια, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-6, IL-17, IL-18, IL-22, IL-23; a tumor necrosis factor such as TNF-α or TNF-β; and other polypeptide factors including L1F and kit ligand (KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of the native sequence cytokines.

As used herein, the term “inflammatory cells” designates cells that enhance the inflammatory response such as mononuclear cells, eosinophils, macrophages, and polymorphonuclear neutrophils (PMN).

II. Compositions and Methods of the Invention

A. Anti-Microbial Polypeptides (AMP) and Modulators Thereof

Anti-microbial polypeptides (AMPs) of the present invention are polypeptides that mediate, or otherwise effect, an anti-microbial immune response to a microbial pathogen. AMPs of the present invention include, but are not limited to, LT, IL-6, IL-22, IL-23 (including e.g., IL-23 p 19 or IL-23 p40), and Reg or Reg-related proteins encoded by the genes of the Reg super family. The Reg super family includes Reg and Reg-related genes from human, rat, and mouse and are grouped into four subclasses, types 1,11, 111, and IV. For example, type 1 includes human REG la, human REG 1β, rat Regl, and mouse RegR type 11 includes mouse ReglR type 111 includes human REG 111, human

H1P/PAP (gene expressed in hepatocellular carcinoma-intestine-pancreas/gene encoding pancreatitis associated protein), rat PAP/Peptide23, rat Reglll/PAPll, rat PAP 111, mouse Regllla, Regllip, Regllly, mouse Regllld, and hamster INGAP (islet neogenesis-associated protein). Type IV contains human REG IV. Additionally, human Reg-related Sequence (RS) is reportedly a pseudogene. In one embodiment, the REG protein is encoded by a member of the human REG gene family which includes, but is not limited to, REG la, REG Ιβ, H1P/PAP, REG 111, REG IV, and Reg-related sequence (RS).

In some aspects, the amino acid sequence of an AMP of the present invention comprises an amino acid sequence selected from the following group: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO:

18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID

NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ

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ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52,

SEQ ID NO: 54, and SEQ ID NO: 56.

In other aspects, the nucleic acid sequence encoding an AMP of the present invention comprises a nucleic acid sequence selected from the following group: SEQ ID NO: 1, SEQ ID NO: 3,

SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO:

27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID

NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ

ID NO: 51, SEQ ID NO: 53, and SEQ ID NO: 55.

An activity of an AMP of the present invention can be increased or decreased and/or differentially regulated relative to the activity of another AMP or the same AMP. Examples of an activity of an AMP of the present invention, includes, but is not limited to, AMP expression, signal transduction, binding to a binding partner, anti-microbial response, or other biological or immunological activity thereof.

In one embodiment, an increase in the activity of one or more AMPs of the present invention results in an enhanced or induced anti-microbial immune response in a subject.

In one embodiment, AMPs of the present invention include, but are not limited to, polypeptides that directly or indirectly interact with IL-22, e.g., polypeptides that are upstream or downstream of an IL-22 signal transduction pathway that mediates host resistance to infection by a microbial pathogen (e.g., a bacteria or virus). Examples of such AMPs include, but are not limited to, LT, IL-6, IL-18, and IL-23 (including e.g., IL-23 p 19 or IL-23 p40).

Modulators of the present invention include, but are not limited to, polypeptides and nucleic acid molecules (e.g., a DNA molecule or RNA molecule) that directly or indirectly modulate an activity of an AMP. Examples of such modulation include, but are not limited to, an increase, decrease, induction or activation, inhibition, or regulation (e.g., up or down regulation) of an activity of an AMP of the present invention.

In a particular embodiment, the modulator indirectly modulates IL-22 activity by decreasing or inhibiting IL-22 Binding Protein (BP) activity and thereby, increasing IL-22 activity. In a further embodiment, the modulator decreases or inhibits binding of IL-22 BP to IL-22 and thereby, increases

IL-22 activity.

In some embodiments, the modulator is a polypeptide e.g., a polypeptide that binds to or otherwise interacts with an AMP to increase, induce, or regulate an activity of an AMP. In one embodiment, the modulator is a fusion polypeptide that modulates an activity of an AMP.

In one embodiment, the modulator is an antibody that binds to an AMP. In a particular 35 embodiment, the antibody is a monoclonal antibody. In another embodiment, the antibody is an antibody fragment selected from a Fab, Fab’-SH, Fv, scFv, or (Fab’fi fragment. In another

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-30 2016259423 17 Oct 2018 embodiment, the antibody is a fusion polypeptide (e.g., an Fc fusion polypeptide). In another embodiment, the antibody is a chimeric antibody. In a particular embodiment, the antibody is humanized. In another embodiment, the antibody is a human antibody. In another embodiment, the antibody binds to the same epitope as an antibody selected from a human, non-human primate, or other mammal (e.g., pig, sheep, rabbit, marmot, rat, or mouse). In a particular embodiment, the antibody is an AMP agonist.

In a particular embodiment, the modulator is a recombinant AMP or nucleic acid molecule encoding an AMP (e.g., a DNA or RNA molecule).

In another particular embodiment, the modulator is a recombinant AMP or nucleic acid molecule encoding an AMP (e.g., a DNA or RNA molecule) that can be expressed in a cell.

AMPs of the present invention encompass native full-length or mature AMPs as well as variants thereof. AMP variants can be prepared by introducing appropriate nucleotide changes into the DNA encoding an AMP, and/or by synthesis of the desired anti-microbial polypeptide. Those skilled in the art will appreciate that amino acid changes may alter post-translational processing of a polypeptide of the present invention, such as changing the number or position of glycosylation sites or altering the membrane anchoring characteristics.

Variations in native AMP or in various domains of the AMP, as described herein, can be made, for example, using any of the techniques and guidelines for conservative and non-conservative mutations set forth, for instance, in U.S. Patent No. 5,364,934. Variations may be a substitution, deletion or insertion of one or more codons encoding the AMP that results in a change in the amino acid sequence of the AMP as compared with a native sequence AMP. Optionally, the variation is by substitution of at least one amino acid with any other amino acid in one or more domains of the AMP. Guidance in determining which amino acid residue may be inserted, substituted or deleted without adversely affecting the desired activity may be found by comparing the sequence of the AMP with that of homologous known protein molecules and minimizing the number of amino acid sequence changes made in regions of high homology. Amino acid substitutions can be the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, such as the replacement of a leucine with a serine, i.e., conservative amino acid replacements. Insertions or deletions may optionally be in the range of about 1 to 5 amino acids. The variation allowed may be determined by systematically making insertions, deletions or substitutions of amino acids in the sequence and testing the resulting variants for activity exhibited by the full-length or mature native sequence.

In particular embodiments, conservative substitutions of interest are shown in Table 3 under the heading of preferred substitutions. If such substitutions result in a change in biological activity, then more substantial changes, denominated exemplary substitutions in Table 6, or as further described below in reference to amino acid classes, are introduced and the products screened.

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Table 3

5	Original Residue	Exemplary Substitutions	Preferred Substitutions
	Ala (A)	val; leu; ile	val
	Arg (R)	lys; gin; asn	lys
	Asn (N)	gin; his; lys; arg	gin
	Asp (D)	glu	glu
10	Cys (C)	ser	ser
	Gin (Q)	asn	asn
	Glu (E)	asp	asp
	Gly (G)	pro; ala	ala
	His (H)	asn; gin; lys; arg	arg
15	He (1)	leu; val; met; ala; phe;
		norleucine	leu
	Leu (L)	norleucine; ile; val;
		met; ala; phe	ile
	Lys (K)	arg; gin; asn	arg
20	Met (M)	leu; phe; ile	leu
	Phe (F)	leu; val; ile; ala; tyr	leu
	Pro (P)	ala	ala
	Ser (S)	thr	thr
	Thr (T)	ser	ser
25	Trp (W)	tyr; phe	tyr
	Tyr(Y)	trp; phe; thr; ser	phe
	Val (V)	ile; leu; met; phe; ala; norleucine	leu

Substantial modifications in function or immunological identity of the AMP polypeptide are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Naturally occurring residues are divided into groups based on common side-chain properties:

(1) hydrophobic: norleucine, met, ala, val, leu, ile;

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-32 2016259423 17 Oct 2018 (2) neutral hydrophilic: cys, ser, thr;

(3) acidic: asp, glu;

(4) basic: asn, gin, his, lys, arg;

(5) residues that influence chain orientation: gly, pro; and (6) aromatic: trp, tyr, phe.

Non-conservative substitutions will entail exchanging a member of one of these classes for another class. Such substituted residues also may be introduced into the conservative substitution sites or, more preferably, into the remaining (non-conserved) sites.

The variations can be made using methods known in the art such as oligonucleotide-mediated 10 (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed mutagenesis [Carter et al., Nucl. Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487 (1987)], cassette mutagenesis [Wells et al., Gene, 34:315 (1985)], restriction selection mutagenesis [Wells et al., Philos. Trans. R. Soc. London SerA, 317:415 (1986)] or other known techniques can be performed on cloned DNA to produce a DNA encoding a variant AMP.

Fragments of an AMP or other polypeptides of the present invention are also provided herein.

Such fragments may be truncated at the N-terminus or C-terminus, or may lack internal residues, for example, when compared with a full length native protein. Certain fragments lack amino acid residues that are not essential for a desired biological activity of an AMP or polypeptide of the present invention. Accordingly, in certain embodiments, a fragment of an AMP or other polypeptide of the present invention, is biologically active. In certain embodiments, a fragment of full length AMP lacks the N-terminal signal peptide sequence. In certain embodiments, a fragment of full-length AMP is a soluble form of a membrane-bound AMP. For example, a soluble form of AMP may lack all or a substantial portion of the transmembrane domain.

Covalent modifications of AMPs or other polypeptides of the present invention are included within the scope of this invention. One type of covalent modification includes reacting targeted amino acid residues of a polypeptide of the present invention with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C-terminal residues of the polypeptide. Derivatization with bifunctional agents is useful, for instance, for crosslinking the polypeptide to a water-insoluble support matrix or surface for use in the method for purifying antibodies to the polypeptide, and vice-versa. Commonly used crosslinking agents include, e.g., l,l-bis(diazoacetyl)2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3'dithiobis(succinimidylpropionate), bifunctional maleimides such as bis-N-maleimido-l,8-octane and agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate.

Other modifications include deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, respectively, hydroxylation of proline and lysine,

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-33 2016259423 17 Oct 2018 phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the α-amino groups of lysine, arginine, and histidine side chains [T.E. Creighton, Proteins: Structure and Molecular Properties, W.EI. Freeman & Co., San Francisco, pp. 79-86 (1983)], acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.

Another type of covalent modification of a polypeptide of the present invention included within the scope of this invention comprises altering the native glycosylation pattern of the polypeptide. Altering the native glycosylation pattern is intended for purposes herein to mean deleting one or more carbohydrate moieties found in the native sequence of a polypeptide of the present invention (either by removing the underlying glycosylation site or by deleting the glycosylation by chemical and/or enzymatic means), and/or adding one or more glycosylation sites that are not present in the native sequence of the polypeptide. In addition, the phrase includes qualitative changes in the glycosylation of the native proteins, involving a change in the nature and proportions of the various carbohydrate moieties present.

A polypeptide of the present invention may also be modified in a way to form a chimeric molecule comprising the polypeptide fused to another, heterologous polypeptide or amino acid sequence. In one embodiment, a chimeric molecule comprises a fusion of the polypeptide with a tag polypeptide which provides an epitope to which an anti-tag antibody can selectively bind. The epitope tag is generally placed at the amino- or carboxyl- terminus of the polypeptide. The presence of such epitope-tagged forms of the polypeptide can be detected using an antibody against the tagged polypeptide. Also, provision of the epitope tag enables the AMP to be readily purified by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag. Various tag polypeptides and their respective antibodies are well known in the art. Examples include poly-histidine (poly-his) or poly-histidine-glycine (poly-his-gly) tags; the flu EIA tag polypeptide and its antibody 12CA5 [Field et al., Mol. Cell. Biol., 8:2159-2165 (1988)]; the c-myc tag and the 8F9,

3C7, 6E10, G4, B7 and 9E10 antibodies thereto [Evan et al., Molecular and Cellular Biology, 5:36103616 (1985)]; and the Elerpes Simplex virus glycoprotein D (gD) tag and its antibody [Paborsky et al., Protein Engineering, 3(6):547-553 (1990)]. Other tag polypeptides include the Flag-peptide [EIopp et al., BioTechnology, 6:1204-1210 (1988)]; the KT3 epitope peptide [Martin et al., Science, 255:192194 (1992)]; an alpha-tubulin epitope peptide [Skinner et al., J. Biol. Chem., 266:15163-15166 (1991)]; and the T7 gene 10 protein peptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA,

87:6393-6397 (1990)].

In another embodiment, a chimeric molecule may comprise a fusion of a polypeptide of the present invention with an immunoglobulin or a particular region of an immunoglobulin. For a bivalent form of the chimeric molecule (also referred to as an “immunoadhesin”), such a fusion could be to the Fc region of an IgG molecule. The Ig fusions preferably include the substitution of a soluble form of a polypeptide of the present invention (e.g., an AMP or polypeptide modulator thereof) in

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-34 2016259423 17 Oct 2018 place of at least one variable region within an Ig molecule. In a particularly preferred embodiment, the immunoglobulin fusion includes the hinge, CH2 and CH3, or the hinge, CHI, CH2 and CH3 regions of an IgGl molecule. For the production of immunoglobulin fusions see also US Patent No. 5,428,130 issued June 27, 1995.

1. Preparation of Polypeptides

Polypeptides of the present invention may be prepared by routine recombinant methods, e.g., culturing cells transformed or transfected with a vector containing a nucleic acid encoding an AMP or polypeptide modulator thereof. Host cells comprising any such vector are also provided. By way of example, host cells may be CHO cells, E. coli, or yeast. A process for producing any of the herein described polypeptides is further provided and comprises culturing host cells under conditions suitable for expression of the desired polypeptide and recovering the desired polypeptide from the cell culture.

In other embodiments, the invention provides chimeric molecules comprising any of the herein described polypeptides fused to a heterologous polypeptide or amino acid sequence. Examples of such chimeric molecules include, but are not limited to, any of the herein described polypeptides fused to an epitope tag sequence or an Fc region of an immunoglobulin.

Alternative methods, which are well known in the art, may be employed to prepare a polypeptide of the present invention. For example, a sequence encoding a polypeptide or portion thereof, may be produced by direct peptide synthesis using solid-phase techniques [see, e.g., Stewart et al., Solid-Phase Peptide Synthesis, W.H. Freeman Co., San Francisco, CA (1969); Merrifield, L Am. Chem. Soc., 85:2149-2154 (1963)]. In vitro protein synthesis may be performed using manual techniques or by automation. Automated synthesis may be accomplished, for instance, using an Applied Biosystems Peptide Synthesizer (Foster City, CA) using manufacturer's instructions. Various portions of a polypeptide of the present invention or portion thereof may be chemically synthesized separately and combined using chemical or enzymatic methods to produce the full-length polypeptide or portion thereof.

Recombinantly expressed polypeptides of the present invention may be recovered from culture medium or from host cell lysates. The following procedures are exemplary of suitable purification procedures: by fractionation on an ion-exchange column; ethanol precipitation; reverse phase HPLC;

chromatography on silica or on a cation-exchange resin such as DEAE; chromatofocusing; SDSPAGE; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75; protein A Sepharose columns to remove contaminants such as IgG; and metalchelating columns to bind epitopetagged forms of a polypeptide of the present invention. Various methods of protein purification may be employed and such methods are known in the art and described for example in Deutscher, Methods in Enzymology, 182 (1990); Scopes, Protein Purification: Principles and Practice, Springer-Verlag, New York (1982). The purification step(s) selected will depend, for example, on the nature of the

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-35 2016259423 17 Oct 2018 production process used and the particular polypeptide produced. LT polypeptides may be purified by expressing a tagged LT polypeptide such as, for example, an LTa-tagged polypeptide (SEQ ID

NO:61).

2. Detection of Gene Expression

Expression of a gene encoding a polypeptide of the present invention can be detected by various methods in the art, e.g, by detecting expression of mRNA encoding the polypeptide. As used herein, the term “detecting” encompasses quantitative or qualitative detection. By detecting gene expression of a polypeptide of the present invention, one can identify, e.g., those tissues that express this gene. Gene expression may be measured using certain methods known to those skilled in the art,

e.g., Northern blotting, (Thomas, Proc. Natl. Acad. Sci. USA, 77:5201-5205 [1980]); quantitative

PCR; or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein. Alternatively, gene expression may be measured by immunological methods, such as immunohistochemical staining of tissue sections and assay of cell culture or body fluids, to quantitate directly the expression of gene product. Antibodies useful for immunohistochemical staining and/or assay of sample fluids encompass any of the antibodies provided herein. Conveniently, the antibodies may be prepared against a native sequence encoding e.g., an AMP of the present invention; against a synthetic peptide comprising a fragment of the AMP sequence; or against an exogenous sequence fused to AMP polypeptide or fragment thereof (including a synthetic peptide).

B. Antibodies

Antibodies that bind to any of the above- or below- described polypeptides are provided. In one embodiment, an isolated antibody that binds to an AMP of the present invention and thereby modulates AMP activity, e.g., increasing an activity of the AMP. Exemplary antibodies include polyclonal, monoclonal, humanized, human, bispecific, and heteroconjugate antibodies. An antibody may be an antibody fragment, e.g., a Fab, Fab’-SH, Fv, scFv, or (Fab’)2 fragment. In one embodiment, an isolated antibody that binds to an IL-22 is provided. In one such embodiment, an antibody partially or completely increases the activity of an AMP of the present invention.

Exemplary monoclonal antibodies that bind an AMP of the present invention are described herein. These antibodies include the anti-IL-22 antibodies designated 3F11.3 (“3F11”), 11H4.4 (“11H4”), and 8E11.9 (“8E11”), and the anti-lL-22R antibodies designated 7E9.10.8 (“7E9”),

8A12.32 (“8A12”), 8H11.32.28 (“8H11”), and 12H5. In one embodiment, a hybridoma that produces any of those antibodies is provided. In one embodiment, monoclonal antibodies that compete with 3F11.3, 11H4.4, or 8E11.9 for binding to IL-22 are provided. In another embodiment, monoclonal antibodies that bind to the same epitope as 3F11.3, 11H4.4, or 8E11.9 are provided. In another embodiment, monoclonal antibodies that compete with 7E9, 8A12, 8H11, or 12H5 for binding to IL-22R are provided. In one embodiment, monoclonal antibodies that bind to the same

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-36 2016259423 17 Oct 2018 epitope as 7E9, 8A12, 8H11, or 12H5 are provided. Various embodiments of antibodies are provided below:

1. Polyclonal Antibodies

Antibodies may comprise polyclonal antibodies. Methods of preparing polyclonal antibodies 5 are known to the skilled artisan. Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant. Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections. The immunizing agent may include the polypeptide of interest or a fusion protein thereof. It may be useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized. Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. Examples of adjuvants which may be employed include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate). The immunization protocol may be selected by one skilled in the art without undue experimentation.

2. Monoclonal Antibodies

Antibodies may, alternatively, be monoclonal antibodies. Monoclonal antibodies may be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes may be immunized in vitro.

The immunizing agent will typically include the polypeptide of interest or a fusion protein thereof. Generally, either peripheral blood lymphocytes (PBLs) are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell [Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103]. Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed. The hybridoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such

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-37 2016259423 17 Oct 2018 as HAT medium. More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection, Manassas, Virginia. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies [Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63].

The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies that bind to the polypeptide of interest. Preferably, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (R1A) or enzymelinked immunoabsorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem, 107:220 (1980).

After the desired hybridoma cells are identified, the clones may be subcloned by limiting dilution procedures and grown by standard methods [Goding, supra]. Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPM1-1640 medium. Alternatively, the hybridoma cells may be grown in vivo as ascites in a mammal.

The monoclonal antibodies secreted by the subclones may be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

Monoclonal antibodies can be made by using combinatorial libraries to screen for antibodies with the desired activity or activities. For example, a variety of methods are known in the art for generating phage display libraries and screening such libraries for antibodies possessing the desired binding characteristics. Such methods are described generally in Hoogenboom et al. (2001) in Methods in Molecular Biology 178:1-37 (O’Brien et al., ed., Human Press, Totowa, NJ), and in certain embodiments, in Lee et al. (2004) J. Mol. Biol. 340:1073-1093.

In principle, synthetic antibody clones are selected by screening phage libraries containing phage that display various fragments of antibody variable region (Fv) fused to phage coat protein.

Such phage libraries are panned by affinity chromatography against the desired antigen. Clones expressing Fv fragments capable of binding to the desired antigen are adsorbed to the antigen and thus separated from the non-binding clones in the library. The binding clones are then eluted from the antigen, and can be further enriched by additional cycles of antigen adsorption/elution. Any of the antibodies of the invention can be obtained by designing a suitable antigen screening procedure to select for the phage clone of interest followed by construction of a full length antibody clone using the Fv sequences from the phage clone of interest and suitable constant region (Fc) sequences described

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-38 2016259423 17 Oct 2018 in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, N1H Publication 913242, Bethesda MD (1991), vols. 1-3.

The monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567. DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells of the invention serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. The DNA also may be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences [U.S. Patent No. 4,816,567; Morrison et al., supral or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. Such a non15 immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody.

3. Monovalent Antibodies

Monovalent antibodies are also provided. Methods for preparing monovalent antibodies are well known in the art. For example, one method involves recombinant expression of immunoglobulin light chain and modified heavy chain. The heavy chain is truncated generally at any point in the Fc region so as to prevent heavy chain crosslinking. Alternatively, the relevant cysteine residues are substituted with another amino acid residue or are deleted so as to prevent crosslinking.

In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly, Fab fragments, can be accomplished using routine techniques known in the art.

4. Antibody Fragments

Antibody fragments are also provided. Antibody fragments may be generated by traditional means, such as enzymatic digestion, or by recombinant techniques. In certain circumstances there are advantages of using antibody fragments, rather than whole antibodies. The smaller size of the fragments allows for rapid clearance, and may lead to improved access to solid tumors. For a review of certain antibody fragments, see Hudson et al. (2003) Nat. Med. 9:129-134.

Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived via proteolytic digestion of intact antibodies (see, e.g.,

Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992); and Brennan et al., Science, 229:81 (1985)). However, these fragments can now be produced directly by recombinant

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-39 2016259423 17 Oct 2018 host cells. Fab, Fv and ScFv antibody fragments can all be expressed in and secreted from E. coli, thus allowing the facile production of large amounts of these fragments. Antibody fragments can be isolated from the antibody phage libraries discussed above. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab')2 fragments (Carter et al.,

Bio/Technology 10:163-167 (1992)). According to another approach, F(ab')2 fragments can be isolated directly from recombinant host cell culture. Fab and F(ab')2 fragment with increased in vivo half-life comprising salvage receptor binding epitope residues are described in U.S. Pat. No.

5,869,046. Other techniques for the production of antibody fragments will be apparent to the skilled practitioner. In certain embodiments, an antibody is a single chain Fv fragment (scFv). See WO

93/16185; U.S. Pat. Nos. 5,571,894; and 5,587,458. Fv and scFv are the only species with intact combining sites that are devoid of constant regions; thus, they may be suitable for reduced nonspecific binding during in vivo use. scFv fusion proteins may be constructed to yield fusion of an effector protein at either the amino or the carboxy terminus of an scFv. See Antibody Engineering, ed. Borrebaeck, supra. The antibody fragment may also be a linear antibody, e.g., as described in U.S.

Pat. No. 5,641,870, for example. Such linear antibodies may be monospecific or bispecific.

5. Humanized Antibodies

Humanized antibodies are also provided. Various methods for humanizing non-human antibodies are known in the art. For example, a humanized antibody can have one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as import residues, which are typically taken from an import variable domain. Humanization can be essentially performed following the method of Winter and co-workers (Jones et al. (1936) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-327; Verhoeyen et al.

(1988) Science 239:1534-1536), by substituting hypervariable region sequences for the corresponding sequences of a human antibody. Accordingly, such humanized antibodies are chimeric antibodies (U.S. Patent No. 4,816,567) wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some hypervariable region residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be used in making the humanized antibodies can be important to reduce antigenicity. According to the so-called best-fit method, the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable-domain sequences. The human sequence which is closest to that of the rodent is then accepted as the human framework for the humanized antibody (Sims et al. (1993) J. Immunol. 151:2296; Chothia et al. (193Τ) J. Mol. Biol. 196:901. Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of

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-40 2016259423 17 Oct 2018 light or heavy chains. The same framework may be used for several different humanized antibodies (Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285; Prestaeia/. (1993) J. Immunol., 151:2623.

It is further generally desirable that antibodies be humanized with retention of high affinity for the antigen and other favorable biological properties. To achieve this goal, according to one method, humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable threedimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved. In general, the hypervariable region residues are directly and most substantially involved in influencing antigen binding.

6. Human Antibodies

Human antibodies are also provided. Human antibodies can be constructed by combining Fv clone variable domain sequence(s) selected from human-derived phage display libraries with known human constant domain sequences(s) as described above. Alternatively, human monoclonal antibodies of the invention can be made by the hybridoma method. Human myeloma and mousehuman heteromyeloma cell lines for the production of human monoclonal antibodies have been described, for example, byKozbor J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York,

1987); and Boemer et a/., J. Immunol., 147: 86 (1991).

It is now possible to produce transgenic animals (e.g. mice) that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been described that the homozygous deletion of the antibody heavy-chain joining region (JH) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germ-line immunoglobulin gene array in such germ-line mutant mice will result in the production of human antibodies upon antigen challenge. See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci USA, 90: 2551 (1993); Jakobovits et al., Nature, 362: 255 (1993); Bruggermann et al., Year in Immunol., 7: 33 (1993).

Gene shuffling can also be used to derive human antibodies from non-human, e.g. rodent, antibodies, where the human antibody has similar affinities and specificities to the starting non-human antibody. According to this method, which is also called epitope imprinting, either the heavy or

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-41 2016259423 17 Oct 2018 light chain variable region of a non-human antibody fragment obtained by phage display techniques as described herein is replaced with a repertoire of human V domain genes, creating a population of non-human chain/human chain scFv or Fab chimeras. Selection with antigen results in isolation of a non-human chain/human chain chimeric scFv or Fab wherein the human chain restores the antigen binding site destroyed upon removal of the corresponding non-human chain in the primary phage display clone, i.e. the epitope governs (imprints) the choice of the human chain partner. When the process is repeated in order to replace the remaining non-human chain, a human antibody is obtained (see PCT WO 93/06213 published April 1, 1993). Unlike traditional humanization of non-human antibodies by CDR grafting, this technique provides completely human antibodies, which have no FR or CDR residues of non-human origin.

7. Bispecific Antibodies

Bispecific antibodies are also provided. Bispecific antibodies are monoclonal antibodies that have binding specificities for at least two different antigens. In certain embodiments, bispecific antibodies are human or humanized antibodies. In certain embodiments, one of the binding specificities is for a polypeptide of interest and the other is for any other antigen. In certain embodiments, bispecific antibodies may bind to two different epitopes of a polypeptide of interest. Bispecific antibodies may also be used to localize cytotoxic agents to cells which express a polypeptide of interest, such a cell surface polypeptide. These antibodies possess a TAT226-binding arm and an arm which binds a cytotoxic agent, such as, e.g., saporin, anti-interferon-α, vinca alkaloid, ricin A chain, methotrexate or radioactive isotope hapten. Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g. F(ab')2bispecific antibodies).

Methods for making bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two heavy chains have different specificities (Milstein and Cuello,

Nature, 305: 537 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule, which is usually done by affinity chromatography steps, is rather cumbersome, and the product yields are low. Similar procedures are disclosed in WO 93/08829 published May 13, 1993, and in Traunecker et al., EMBOJ., 10: 3655 (1991).

According to a different approach, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. The fusion, for example, is with an immunoglobulin heavy chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. In certain embodiments, the first heavy35 chain constant region (CHI), containing the site necessary for light chain binding, is present in at least one of the fusions. DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the

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-42 2016259423 17 Oct 2018 immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism. This provides for great flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments when unequal ratios of the three polypeptide chains used in the construction provide the optimum yields. It is, however, possible to insert the coding sequences for two or all three polypeptide chains in one expression vector when the expression of at least two polypeptide chains in equal ratios results in high yields or when the ratios are of no particular significance.

In one embodiment of this approach, the bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. It was found that this asymmetric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, as the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides for a facile way of separation. This approach is disclosed in WO 94/04690. For further details of generating bispecific antibodies see, for example,

Suresh et al., Methods in Enzymology, 121:210 (1986).

According to another approach, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture. The interface comprises at least a part of the Ch3 domain of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g. tyrosine or tryptophan). Compensatory cavities of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.

Bispecific antibodies include cross-linked or heteroconjugate antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (US Patent No. 4,676,980), and for treatment of HIV infection (WO 91/00360, WO 92/00373, and EP 03089). Heteroconjugate antibodies may be made using any convenient cross-linking method. Suitable cross30 linking agents are well known in the art, and are disclosed in US Patent No. 4,676,980, along with a number of cross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragments have also been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science, 229: 81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab')2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular

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-43 2016259423 17 Oct 2018 disulfide formation. The Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.

Recent progress has facilitated the direct recovery of Fab'-SH fragments from E. coli, which can be chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med., 175: 217-225 (1992) describe the production of a fully humanized bispecific antibody F(ab')2 molecule. Each Fab' fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody. The bispecific antibody thus formed was able to bind to cells overexpressing the HER2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.

Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol., 148(5):1547-1553 (1992). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers. The diabody technology described by Hollinger et al.,

Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See Gruber et al., J. Immunol., 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tutt et al. J. Immunol. 147: 60 (1991).

8. Multivalent Antibodies

Multivalent antibodies are also provided. A multivalent antibody may be internalized (and/or catabolized) faster than a bivalent antibody by a cell expressing an antigen to which the antibodies bind. The antibodies of the present invention can be multivalent antibodies (which are other than of the IgM class) with three or more antigen binding sites (e.g. tetravalent antibodies), which can be readily produced by recombinant expression of nucleic acid encoding the polypeptide chains of the antibody. The multivalent antibody can comprise a dimerization domain and three or more antigen binding sites. In certain embodiments, the dimerization domain comprises (or consists of) an Fc

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-44 2016259423 17 Oct 2018 region or a hinge region. In this scenario, the antibody will comprise an Fc region and three or more antigen binding sites amino-terminal to the Fc region. In certain embodiments, a multivalent antibody comprises (or consists of) three to about eight antigen binding sites. In one such embodiment, a multivalent antibody comprises (or consists of) four antigen binding sites. The multivalent antibody comprises at least one polypeptide chain (for example, two polypeptide chains), wherein the polypeptide chain(s) comprise two or more variable domains. For instance, the polypeptide chain(s) may comprise VDl-(Xl)n -VD2-(X2)n -Fc, wherein VD1 is a first variable domain, VD2 is a second variable domain, Fc is one polypeptide chain of an Fc region, XI and X2 represent an amino acid or polypeptide, and n is 0 or 1. For instance, the polypeptide chain(s) may comprise: VH-CH1-flexible linker-VH-CHl -Fc region chain; or VH-CH1-VH-CH1 -Fc region chain. The multivalent antibody herein may further comprise at least two (for example, four) light chain variable domain polypeptides. The multivalent antibody herein may, for instance, comprise from about two to about eight light chain variable domain polypeptides. The light chain variable domain polypeptides contemplated here comprise a light chain variable domain and, optionally, further comprise a CL domain.

9. Single-Domain Antibodies

Single-domain antibodies are also provided. A single-domain antibody is a single polyeptide chain comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 BI). In one embodiment, a single-domain antibody consists of all or a portion of the heavy chain variable domain of an antibody.

10. Antibody Variants

In some embodiments, amino acid sequence modification(s) of the antibodies described herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody. Amino acid sequence variants of the antibody may be prepared by introducing appropriate changes into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of, residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics. The amino acid alterations may be introduced in the subject antibody amino acid sequence at the time that sequence is made.

A uselul method for identification of certain residues or regions of the antibody that are preferred locations for mutagenesis is called alanine scanning mutagenesis as described by Cunningham and Wells (1989) Science, 244:1081-1085. Here, a residue or group of target residues are identified (e.g., charged residues such as arg, asp, his, lys, and glu) and replaced by a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to affect the interaction of the amino

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-45 2016259423 17 Oct 2018 acids with antigen. Those amino acid locations demonstrating functional sensitivity to the substitutions then are refined by introducing further or other variants at, or for, the sites of substitution. Thus, while the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation per se need not be predetermined. For example, to analyze the performance of a mutation at a given site, ala scanning or random mutagenesis is conducted at the target codon or region and the expressed immunoglobulins are screened for the desired activity.

Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g. for ADEPT) or a polypeptide which increases the serum half-life of the antibody.

In certain embodiments, an antibody of the invention is altered to increase or decrease the extent to which the antibody is glycosylated. Glycosylation of polypeptides is typically either N15 linked or O-linked. N-linked refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.

Addition or deletion of glycosylation sites to the antibody is conveniently accomplished by altering the amino acid sequence such that one or more of the above-described tripeptide sequences (for N-linked glycosylation sites) is created or removed. The alteration may also be made by the addition, deletion, or substitution of one or more serine or threonine residues to the sequence of the original antibody (for O-linked glycosylation sites).

Where the antibody comprises an Fc region, the carbohydrate attached thereto may be altered. For example, antibodies with a mature carbohydrate structure that lacks fucose attached to an Fc region of the antibody are described in US Pat Appl No US 2003/0157108 (Presta, L.). See also US

2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Antibodies with a bisecting N-acetylghicosamine (GlcNAc) in the carbohydrate attached to an Fc region of the antibody are referenced in WO 2003/011878, Jean-Mairet et al. and US Patent No. 6,602,684, Umana et al. Antibodies with at least one galactose residue in the oligosaccharide attached to an Fc region of the antibody are reported in WO 1997/30087, Patel et al. See, also, WO 1998/58964 (Raju, S.) and WO 1999/22764 (Raju, S.) concerning antibodies with altered carbohydrate attached to the Fc region thereof. See also US 2005/0123546 (Umana et al.) on antigen-binding molecules with modified glycosylation.

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In certain embodiments, a glycosylation variant comprises an Fc region, wherein a carbohydrate structure attached to the Fc region lacks fucose. Such variants have improved ADCC function. Optionally, the Fc region further comprises one or more amino acid substitutions therein which further improve ADCC, for example, substitutions at positions 298, 333, and/or 334 of the Fc region (Eu numbering of residues). Examples of publications related to “defucosylated” or “fucosedeficient” antibodies include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; W02005/053742; Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004). Examples of cell lines producing defucosylated antibodies include Lee 13 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US Pat Appl No US 2003/0157108 Al, Presta, L; and WO 2004/056312 Al, Adams et al., especially at Example 11), and knockout cell lines, such as alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004)).

Another type of variant is an amino acid substitution variant. These variants have at least one amino acid residue in the antibody molecule replaced by a different residue. Sites of interest for substitutional mutagenesis include the hypervariable regions, but FR alterations are also contemplated. Conservative substitutions are shown in Table 3 above under the heading of preferred substitutions. If such substitutions result in a desirable change in biological activity, then more substantial changes, denominated exemplary substitutions in Table 3, or as further described above in reference to amino acid classes, may be introduced and the resulting antibodies screened for the desired binding propeties.

One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g. a humanized or human antibody). Generally, the resulting variant(s) selected for further development will have modified (e.g., improved) biological properties relative to the parent antibody from which they are generated. A convenient way for generating such substitutional variants involves affinity maturation using phage display. Briefly, several hypervariable region sites (e.g. 6-7 sites) are mutated to generate all possible amino acid substitutions at each site. The antibodies thus generated are displayed from filamentous phage particles as fusions to at least part of a phage coat protein (e.g., the gene 111 product of M13) packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g. binding affinity). In order to identify candidate hypervariable region sites for modification, scanning mutagenesis (e.g., alanine scanning) can be performed to identify hypervariable region residues contributing significantly to antigen binding. Alternatively, or additionally, it may be beneficial to analyze a crystal structure of the antigen-antibody complex to identify contact points between the antibody and antigen. Such contact residues and neighboring residues are candidates for substitution

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-47 2016259423 17 Oct 2018 according to techniques known in the art, including those elaborated herein. Once such variants are generated, the panel of variants is subjected to screening using techniques known in the art, including those described herein, and antibodies with superior properties in one or more relevant assays may be selected for further development.

Nucleic acid molecules encoding amino acid sequence variants of the antibody are prepared by a variety of methods known in the art. These methods include, but are not limited to, isolation from a natural source (in the case of naturally occurring amino acid sequence variants) or preparation by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared variant or a non-variant version of the antibody.

It may be desirable to introduce one or more amino acid modifications in an Fc region of antibodies of the invention, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgGl, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g. a substitution) at one or more amino acid positions including that of a hinge cysteine.

In accordance with this description and the teachings of the art, it is contemplated that in some embodiments, an antibody of the invention may comprise one or more alterations as compared to the wild type counterpart antibody, e.g. in the Fc region. These antibodies would nonetheless retain substantially the same characteristics required for therapeutic utility as compared to their wild type counterpart. For example, it is thought that certain alterations can be made in the Fc region that would result in altered (i.e., either improved or diminished) Clq binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as described in WO99/51642. See also Duncan & Winter Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO94/29351 concerning other examples of Fc region variants. WO00/42072 (Presta) and WO 2004/056312 (Lowman) describe antibody variants with improved or diminished binding to FcRs. The content of these patent publications are specifically incorporated herein by reference. See, also, Shields et al. J. Biol. Chem. 9(2): 6591-6604 (2001). Antibodies with increased half lives and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), are described in US2005/0014934A1 (Hinton et al.). These antibodies comprise an Fc region with one or more substitutions therein which improve binding of the Fc region to FcRn. Polypeptide variants with altered Fc region amino acid sequences and increased or decreased Clq binding capability are described in US patent No. 6,194,55 IB 1, WO99/51642. The contents of those patent publications are specifically incorporated herein by reference. See, also, Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

In one embodiment, the invention provides antibodies comprising modifications in the interface of Fc polypeptides comprising the Fc region, wherein the modifications facilitate and/or promote

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-48 2016259423 17 Oct 2018 heterodimerization. These modifications comprise introduction of a protuberance into a first Fc polypeptide and a cavity into a second Fc polypeptide, wherein the protuberance is positionable in the cavity so as to promote complexing of the first and second Fc polypeptides. Methods of generating antibodies with these modifications are known in the art, e.g., as described in U.S. Pat. No. 5,731,168.

11. Antibody Derivatives

Antibodies can be further modified to contain additional nonproteinaceous moieties that are known in the art and readily available. Preferably, the moieties suitable for derivatization of the antibody are water soluble polymers. Non-limiting examples of water soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3-dioxolane, poly1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, propropylene glycol homopolymers, prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer may be of any molecular weight, and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer are attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative will be used in a therapy under defined conditions, etc.

In another embodiment, conjugates of an antibody and nonproteinaceous moiety that may be selectively heated by exposure to radiation are provided. In one embodiment, the nonproteinaceous moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. 102: 11600-11605 (2005)). The radiation may be of any wavelength, and includes, but is not limited to, wavelengths that do not harm ordinary cells, but which heat the nonproteinaceous moiety to a temperature at which cells proximal to the antibody-nonproteinaceous moiety are killed.

In certain embodiments, an antibody may be labeled and/or may be immobilized on a solid support. In a further embodiment, an antibody is an anti-idiotypic antibody.

12. Heteroconjugate Antibodies

Heteroconjugate antibodies are also provided. Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells [U.S. Patent No. 4,676,980], and for treatment of HIV infection [WO 91/00360; WO 92/200373; EP 03089]. It is contemplated that the antibodies may be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents.

For example, immunotoxins may be constructed using a disulfide exchange reaction or by forming a

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-49 2016259423 17 Oct 2018 thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4mercaptobutyrimidate and those disclosed, for example, in U.S. Patent No. 4,676,980.

13. Effector Function Engineering

It may be desirable to modify an antibody with respect to effector function, so as to enhance,

e.g., the effectiveness of the antibody in treating a microbial disorder. For example, cysteine residue(s) may be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. Homodimeric antibodies with enhanced anti-anti-microbial activity may also be prepared using heterobifunctional cross-linkers. Alternatively, an antibody can be engineered that has dual Fc regions and may thereby have enhanced activity.

14. Vectors, Host Cells, and Recombinant Methods

For recombinant production of an antibody, in one embodiment, the nucleic acid encoding it is isolated and inserted into a replicable vector for further cloning (amplification of the DNA) or for expression. DNA encoding the antibody is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody). Many vectors are available. The choice of vector depends in part on the host cell to be used. Generally, host cells are of either prokaryotic or eukaryotic (generally mammalian) origin. It will be appreciated that constant regions of any isotype can be used for this purpose, including IgG, IgM, IgA, IgD, and IgE constant regions, and that such constant regions can be obtained from any human or animal species.

a)Generating antibodies using prokaryotic host cells:

(1) Vector Construction

Polynucleotide sequences encoding polypeptide components of an antibody can be obtained using standard recombinant techniques. Desired polynucleotide sequences may be isolated and sequenced from antibody producing cells such as hybridoma cells. Alternatively, polynucleotides can be synthesized using nucleotide synthesizer or PCR techniques. Once obtained, sequences encoding the polypeptides are inserted into a recombinant vector capable of replicating and expressing heterologous polynucleotides in prokaryotic hosts. Many vectors that are available and known in the art can be used for the purpose of the present invention. Selection of an appropriate vector will depend mainly on the size of the nucleic acids to be inserted into the vector and the particular host cell to be transformed with the vector. Each vector contains various components, depending on its function (amplification or expression of heterologous polynucleotide, or both) and its compatibility with the particular host cell in which it resides. The vector components generally include, but are not limited to: an origin of replication, a selection marker gene, a promoter, a ribosome binding site (RBS), a signal sequence, the heterologous nucleic acid insert and a transcription termination sequence.

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In general, plasmid vectors containing replicon and control sequences which are derived from species compatible with the host cell may be used in connection with these hosts. The vector ordinarily carries a replication site, as well as marking sequences which are capable of providing phenotypic selection in transformed cells. For example, E. coli is typically transformed using pBR322, a plasmid derived from an E. coli species. pBR322 contains genes encoding ampicillin (Amp) and tetracycline (Tet) resistance and thus provides easy means for identifying transformed cells. pBR322, its derivatives, or other microbial plasmids or bacteriophage may also contain, or be modified to contain, promoters which can be used by the microbial organism for expression of endogenous proteins. Examples of pBR322 derivatives used for expression of particular antibodies are described in detail in Carter et al., U.S. Patent No. 5,648,237.

In addition, phage vectors containing replicon and control sequences that are compatible with the host microorganism can be used as transforming vectors in connection with these hosts. For example, bacteriophage such as XGEM.TM.-ll may be utilized in making a recombinant vector which can be used to transform susceptible host cells such as E. coli FE392.

An expression vector of the invention may comprise two or more promoter-cistron pairs, encoding each of the polypeptide components. A promoter is an untranslated regulatory sequence located upstream (5') to a cistron that modulates its expression. Prokaryotic promoters typically fall into two classes, inducible and constitutive. Inducible promoter is a promoter that initiates increased levels of transcription of the cistron under its control in response to changes in the culture condition,

e.g. the presence or absence of a nutrient or a change in temperature.

A large number of promoters recognized by a variety of potential host cells are well known.

The selected promoter can be operably linked to cistron DNA encoding the light or heavy chain by removing the promoter from the source DNA via restriction enzyme digestion and inserting the isolated promoter sequence into the vector of the invention. Both the native promoter sequence and many heterologous promoters may be used to direct amplification and/or expression of the target genes. In some embodiments, heterologous promoters are utilized, as they generally permit greater transcription and higher yields of expressed target gene as compared to the native target polypeptide promoter.

Promoters suitable for use with prokaryotic hosts include the PhoA promoter, the β30 galactamase and lactose promoter systems, a tryptophan (trp) promoter system and hybrid promoters such as the tac or the trc promoter. However, other promoters that are functional in bacteria (such as other known bacterial or phage promoters) are suitable as well. Their nucleotide sequences have been published, thereby enabling a skilled worker operably to ligate them to cistrons encoding the target light and heavy chains (Siebenlist et al. (1980) Cell 20: 269) using linkers or adaptors to supply any required restriction sites.

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In one embodiment of the invention, each cistron within the recombinant vector comprises a secretion signal sequence component that directs translocation of the expressed polypeptides across a membrane. In general, the signal sequence may be a component of the vector, or it may be a part of the target polypeptide DNA that is inserted into the vector. The signal sequence selected for the purpose of this invention should be one that is recognized and processed (i.e. cleaved by a signal peptidase) by the host cell. For prokaryotic host cells that do not recognize and process the signal sequences native to the heterologous polypeptides, the signal sequence is substituted by a prokaryotic signal sequence selected, for example, from the group consisting of the alkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II (STII) leaders, LamB, PhoE, PelB, OmpA and MBP.

In one embodiment of the invention, the signal sequences used in both cistrons of the expression system are STII signal sequences or variants thereof.

In another embodiment, the production of the immunoglobulins according to the invention can occur in the cytoplasm of the host cell, and therefore does not require the presence of secretion signal sequences within each cistron. In that regard, immunoglobulin light and heavy chains are expressed, folded and assembled to form functional immunoglobulins within the cytoplasm. Certain host strains (e.g., the E. coli trxB- strains) provide cytoplasm conditions that are favorable for disulfide bond formation, thereby permitting proper folding and assembly of expressed protein subunits. Proba and Pluckthun Gene, 159:203 (1995).

Antibodies of the invention can also be produced by using an expression system in which the quantitative ratio of expressed polypeptide components can be modulated in order to maximize the yield of secreted and properly assembled antibodies of the invention. Such modulation is accomplished at least in part by simultaneously modulating translational strengths for the polypeptide components.

One technique for modulating translational strength is disclosed in Simmons et al., U.S. Pat.

No. 5,840,523. It utilizes variants of the translational initiation region (TIR) within a cistron. For a given TIR, a series of amino acid or nucleic acid sequence variants can be created with a range of translational strengths, thereby providing a convenient means by which to adjust this factor for the desired expression level of the specific chain. TIR variants can be generated by conventional mutagenesis techniques that result in codon changes which can alter the amino acid sequence. In certain embodiments, changes in the nucleotide sequence are silent. Alterations in the TIR can include, for example, alterations in the number or spacing of Shine-Dalgamo sequences, along with alterations in the signal sequence. One method for generating mutant signal sequences is the generation of a codon bank at the beginning of a coding sequence that does not change the amino acid sequence of the signal sequence (i.e., the changes are silent). This can be accomplished by changing the third nucleotide position of each codon; additionally, some amino acids, such as leucine, serine, and arginine, have multiple first and second positions that can add complexity in making the

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-52 2016259423 17 Oct 2018 bank. This method of mutagenesis is described in detail in Yansura et al. (1992) METHODS: A Companion to Methods in Enzymol. 4:151-158.

In one embodiment, a set of vectors is generated with a range of T1R strengths for each cistron therein. This limited set provides a comparison of expression levels of each chain as well as the yield of the desired antibody products under various T1R strength combinations. T1R strengths can be determined by quantifying the expression level of a reporter gene as described in detail in Simmons et al. U.S. Pat. No. 5, 840,523. Based on the translational strength comparison, the desired individual TIRs are selected to be combined in the expression vector constructs of the invention.

Prokaryotic host cells suitable for expressing antibodies of the invention include Archaebacteria 10 and Eubacteria, such as Gram-negative or Gram-positive organisms. Examples of useful bacteria include Escherichia (e.g., E. coli), Bacilli (e.g., B. subtilis), Enterobacteria, Pseudomonas species (e.g., P. aeruginosa), Salmonella typhimurium, Serratia marcescans, Klebsiella, Proteus, Shigella, Rhizobia, Vitreoscilla, or Paracoccus. In one embodiment, gram-negative cells are used. In one embodiment, E. coli cells are used as hosts for the invention. Examples of E. coli strains include strain W3110 (Bachmann, Cellular and Molecular Biology, vol. 2 (Washington, D.C.: American Society for Microbiology, 1987), pp. 1190-1219; ATCC Deposit No. 27,325) and derivatives thereof, including strain 33D3 having genotype W3110 AfhuA (AtonA) ptr3 lac Iq lacL8 AompTA(nmpcfepE) degP41 kanR (U.S. Pat. No. 5,639,635). Other strains and derivatives thereof, such as E. coli 294 (ATCC 31,446), E. coli B, E. coliX 1776 (ATCC 31,537) and E. coli RV308(ATCC 31,608) are also suitable. These examples are illustrative rather than limiting. Methods for constructing derivatives of any of the above-mentioned bacteria having defined genotypes are known in the art and described in, for example, Bass et al., Proteins, 8:309-314 (1990). It is generally necessary to select the appropriate bacteria taking into consideration replicability of the replicon in the cells of a bacterium. For example, E. coli, Serratia, or Salmonella species can be suitably used as the host when well known plasmids such as pBR322, pBR325, pACYC177, or pKN410 are used to supply the replicon. Typically the host cell should secrete minimal amounts of proteolytic enzymes, and additional protease inhibitors may desirably be incorporated in the cell culture.

(2) Antibody Production

Host cells are transformed with the above-described expression vectors and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.

Transformation means introducing DNA into the prokaryotic host so that the DNA is replicable, either as an extrachromosomal element or by chromosomal integrant. Depending on the host cell used, transformation is done using standard techniques appropriate to such cells. The calcium treatment employing calcium chloride is generally used for bacterial cells that contain substantial cell10751098_1 (GHMatters) P84050.AU.2 17-Oct-18

-53 2016259423 17 Oct 2018 wall barriers. Another method for transformation employs polyethylene glycol/DMSO. Yet another technique used is electroporation.

Prokaryotic cells used to produce the polypeptides of the invention are grown in media known in the art and suitable for culture of the selected host cells. Examples of suitable media include luria broth (LB) plus necessary nutrient supplements. In some embodiments, the media also contains a selection agent, chosen based on the construction of the expression vector, to selectively permit growth of prokaryotic cells containing the expression vector. For example, ampicillin is added to media for growth of cells expressing ampicillin resistant gene.

Any necessary supplements besides carbon, nitrogen, and inorganic phosphate sources may also 10 be included at appropriate concentrations introduced alone or as a mixture with another supplement or medium such as a complex nitrogen source. Optionally the culture medium may contain one or more reducing agents selected from the group consisting of glutathione, cysteine, cystamine, thioglycollate, dithioerythritol and dithiothreitol.

The prokaryotic host cells are cultured at suitable temperatures. In certain embodiments, for E. 15 coli growth, growth temperatures range from about 20° C to about 39°C; from about 25° C to about 37°C; or about 30°C. The pH of the medium may be any pH ranging from about 5 to about 9, depending mainly on the host organism. In certain embodiments, for E. coli, the pH is from about 6.8 to about 7.4, or about 7.0.

If an inducible promoter is used in the expression vector of the invention, protein expression is 20 induced under conditions suitable for the activation of the promoter. In one embodiment of the invention, PhoA promoters are used for controlling transcription of the polypeptides. Accordingly, the transformed host cells are cultured in a phosphate-limiting medium for induction. In certain embodiments, the phosphate-limiting medium is the C.R.A.P. medium (see, e.g., Simmons et al., J. Immunol. Methods (2002), 263:133-147). A variety of other inducers may be used, according to the vector construct employed, as is known in the art.

In one embodiment, the expressed polypeptides of the present invention are secreted into and recovered from the periplasm of the host cells. Protein recovery typically involves disrupting the microorganism, generally by such means as osmotic shock, sonication or lysis. Once cells are disrupted, cell debris or whole cells may be removed by centrifugation or filtration. The proteins may be further purified, for example, by affinity resin chromatography. Alternatively, proteins can be transported into the culture media and isolated therein. Cells may be removed from the culture and the culture supernatant being filtered and concentrated for further purification of the proteins produced. The expressed polypeptides can be further isolated and identified using commonly known methods such as polyacrylamide gel electrophoresis (PAGE) and Western blot assay.

In one embodiment of the invention, antibody production is conducted in large quantity by a fermentation process. Various large-scale fed-batch fermentation procedures are available for

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-54 2016259423 17 Oct 2018 production of recombinant proteins. Large-scale fermentations have at least 1000 liters of capacity, and in certain embodiments, about 1,000 to 100,000 liters of capacity. These fermentors use agitator impellers to distribute oxygen and nutrients, especially glucose (the preferred carbon/energy source). Small scale fermentation refers generally to fermentation in a fermentor that is no more than approximately 100 liters in volumetric capacity, and can range from about 1 liter to about 100 liters.

In a fermentation process, induction of protein expression is typically initiated after the cells have been grown under suitable conditions to a desired density, e.g., an OD550 of about 180-220, at which stage the cells are in the early stationary phase. A variety of inducers may be used, according to the vector construct employed, as is known in the art and described above. Cells may be grown for shorter periods prior to induction. Cells are usually induced for about 12-50 hours, although longer or shorter induction time may be used.

To improve the production yield and quality of the polypeptides of the invention, various fermentation conditions can be modified. For example, to improve the proper assembly and folding of the secreted antibody polypeptides, additional vectors overexpressing chaperone proteins, such as

Dsb proteins (DsbA, DsbB, DsbC, DsbD and or DsbG) or FkpA (a peptidylprolyl cis,trans-isomerase with chaperone activity) can be used to co-transform the host prokaryotic cells. The chaperone proteins have been demonstrated to facilitate the proper folding and solubility of heterologous proteins produced in bacterial host cells. Chen et al. (1999) J. Biol. Chem. 274:19601-19605; Georgiou et al., U.S. Patent No. 6,083,715; Georgiou et al., U.S. Patent No. 6,027,888; Bothmann and

Pluckthun (2000) J. Biol. Chem. 275:17100-17105; Ramm and Pluckthun (2000) J. Biol. Chem. 275:17106-17113; Arie et al. (2001) Mol. Microbiol. 39:199-210.

To minimize proteolysis of expressed heterologous proteins (especially those that are proteolytically sensitive), certain host strains deficient for proteolytic enzymes can be used for the present invention. For example, host cell strains may be modified to effect genetic mutation(s) in the genes encoding known bacterial proteases such as Protease 111, OmpT, DegP, Tsp, Protease 1,

Protease Mi, Protease V, Protease VI and combinations thereof. Some E. coli protease-deficient strains are available and described in, for example, Joly et al. (1998), supra', Georgiou et al., U.S. Patent No. 5,264,365; Georgiou et al., U.S. Patent No. 5,508,192; Hara et al., Microbial Drug Resistance, 2:63-72 (1996).

In one embodiment, E. coli strains deficient for proteolytic enzymes and transformed with plasmids overexpressing one or more chaperone proteins are used as host cells in the expression system of the invention.

(3) Antibody Purification

In one embodiment, an antibody produced herein is further purified to obtain preparations that are substantially homogeneous for further assays and uses. Standard protein purification methods known in the art can be employed. The following procedures are exemplary of suitable purification

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-55 2016259423 17 Oct 2018 procedures: fractionation on immunoaffinity or ion-exchange columns, ethanol precipitation, reverse phase HPLC, chromatography on silica or on a cation-exchange resin such as DEAE, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, and gel filtration using, for example, Sephadex G-75.

In one embodiment, Protein A immobilized on a solid phase is used for immunoaffinity purification of the antibody products of the invention. Protein A is a 41kD cell wall protein from Staphylococcus aureas which binds with a high affinity to the Fc region of antibodies. Lindmark et al (1983) J. Immunol. Meth. 62:1-13. The solid phase to which Protein A is immobilized can be a column comprising a glass or silica surface, or a controlled pore glass column or a silicic acid column.

In some applications, the column is coated with a reagent, such as glycerol, to possibly prevent nonspecific adherence of contaminants.

As the first step of purification, a preparation derived from the cell culture as described above can be applied onto a Protein A immobilized solid phase to allow specific binding of the antibody of interest to Protein A. The solid phase would then be washed to remove contaminants non-specifically bound to the solid phase. Finally the antibody of interest is recovered from the solid phase by elution, b) Generating antibodies using eukaryotic host cells:

A vector for use in a eukaryotic host cell generally includes one or more of the following nonlimiting components: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

(1) Signal sequence component

A vector for use in a eukaryotic host cell may also contain a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide of interest. The heterologous signal sequence selected may be one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. In mammalian cell expression, mammalian signal sequences as well as viral secretory leaders, for example, the herpes simplex gD signal, are available. The DNA for such a precursor region is ligated in reading frame to DNA encoding the antibody.

(2) Origin of replication

Generally, an origin of replication component is not needed for mammalian expression vectors. For example, the SV40 origin may typically be used only because it contains the early promoter.

(3) Selection gene component

Expression and cloning vectors may contain a selection gene, also termed a selectable marker. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, where relevant, or (c) supply critical nutrients not available from complex media.

One example of a selection scheme utilizes a drug to arrest growth of a host cell. Those cells that are successfully transformed with a heterologous gene produce a protein conferring drug

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-56 2016259423 17 Oct 2018 resistance and thus survive the selection regimen. Examples of such dominant selection use the drugs neomycin, mycophenolic acid and hygromycin.

Another example of suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up the antibody nucleic acid, such as DHFR, thymidine kinase, metallothionein-1 and -11, preferably primate metallothionein genes, adenosine deaminase, ornithine decarboxylase, etc.

For example, in some embodiments, cells transformed with the DHFR selection gene are first identified by culturing all of the transformants in a culture medium that contains methotrexate (Mtx), a competitive antagonist of DHFR. In some embodiments, an appropriate host cell when wild-type

DHFR is employed is the Chinese hamster ovary (CHO) cell line deficient in DHFR activity (e.g., ATCC CRL-9096).

Alternatively, host cells (particularly wild-type hosts that contain endogenous DHFR) transformed or co-transformed with DNA sequences encoding an antibody, wild-type DHFR protein, and another selectable marker such as aminoglycoside 3'-phosphotransferase (APH) can be selected by cell growth in medium containing a selection agent for the selectable marker such as an aminoglycosidic antibiotic, e.g., kanamycin, neomycin, or G418. See U.S. Patent No. 4,965,199.

(4) Promoter component

Expression and cloning vectors usually contain a promoter that is recognized by the host organism and is operably linked to nucleic acid encoding a polypeptide of interest (e.g., an antibody).

Promoter sequences are known for eukaryotes. For example, virtually all eukaryotic genes have an AT-rich region located approximately 25 to 30 bases upstream from the site where transcription is initiated. Another sequence found 70 to 80 bases upstream from the start of transcription of many genes is a CNCAAT region where N may be any nucleotide. At the 3' end of most eukaryotic genes is an AATAAA sequence that may be the signal for addition of the poly A tail to the 3' end of the coding sequence. In certain embodiments, any or all of these sequences may be suitably inserted into eukaryotic expression vectors.

Transcription from vectors in mammalian host cells is controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis30 B virus and Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g., the actin promoter or an immunoglobulin promoter, from heat-shock promoters, provided such promoters are compatible with the host cell systems.

The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication. The immediate early promoter of the human cytomegalovirus is conveniently obtained as a Hindlll E restriction fragment. A system for expressing DNA in mammalian hosts using the bovine papilloma virus as a vector is

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-57 2016259423 17 Oct 2018 disclosed in U.S. Patent No. 4,419,446. A modification of this system is described in U.S. Patent No. 4,601,978. See also Reyes et al., Nature 297:598-601 (1982), describing expression of human βinterferon cDNA in mouse cells under the control of a thymidine kinase promoter from herpes simplex virus. Alternatively, the Rous Sarcoma Virus long terminal repeat can be used as the promoter.

(5) Enhancer element component

Transcription of DNA encoding an antibody of this invention by higher eukaryotes is often increased by inserting an enhancer sequence into the vector. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin). Typically, however, one will use an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. See also Yaniv, Nature 297:17-18 (1982) describing enhancer elements for activation of eukaryotic promoters. The enhancer may be spliced into the vector at a position 5' or 3' to the antibody polypeptide15 encoding sequence, but is generally located at a site 5' from the promoter.

(6) Transcription termination component

Expression vectors used in eukaryotic host cells may also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5' and, occasionally 3', untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding an antibody. One useful transcription termination component is the bovine growth hormone polyadenylation region. See WO94/11026 and the expression vector disclosed therein.

(7) Selection and transformation of host cells

Suitable host cells for cloning or expressing the DNA in the vectors herein include higher eukaryote cells described herein, including vertebrate host cells. Propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines are monkey kidney CV1 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., J. Gen Virol.

36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)) ; mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 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

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-58 2016259423 17 Oct 2018 mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N. Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).

Host cells are transformed with the above-described expression or cloning vectors for antibody production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.

(8) Culturing the host cells

The host cells used to produce an antibody of this invention may be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPM1-1640 (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., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Patent Re. 30,985 may be used as culture media for the host cells. 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 GENTAMYC1N™ 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 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 those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.

(9) Purification of antibody

When using recombinant techniques, the antibody can be produced intracellularly, or directly secreted into the medium. If the antibody is produced intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, may be removed, for example, by centrifugation or ultrafiltration. Where the antibody is secreted into the medium, supernatants from such expression systems may be first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of adventitious contaminants.

The antibody composition prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being a convenient technique. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the antibody. Protein A can be used to purify antibodies that are based on human γ 1, γ2, or γ4 heavy chains (Findmark et al., J. Immunol. Methods 62:1-13 (1983)). Protein G is recommended for all

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-59 2016259423 17 Oct 2018 mouse isotypes and for human γ3 (Guss et al., EMBO J. 5:15671575 (1986)). The matrix to which the affinity ligand is attached may be agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. Where the antibody comprises a CH3 domain, the Bakerbond ABX™resin (J. T. Baker, Phillipsburg, NJ) is useful for purification. Other techniques for protein purification such as fractionation on an ion-exchange column, ethanol precipitation, Reverse Phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE™ chromatography on an anion or cation exchange resin (such as a polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also available depending on the antibody to be recovered.

Following any preliminary purification step(s), the mixture comprising the antibody of interest and contaminants may be subjected to further purification, for example, by low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5-4.5, preferably performed at low salt concentrations (e.g., from about 0-0.25M salt).

In general, various methodologies for preparing antibodies for use in research, testing, and clinical use are well-established in the art, consistent with the above-described methodologies and/or as deemed appropriate by one skilled in the art for a particular antibody of interest.

C. Agonists and Antagonists

Agonists and antagonists of an AMP of the present inventions are provided. Such AMP modulators are encompassed in the present invention and useful for treating a microbial disorder as provided herein.

In one embodiment, an agonist or antagonist of an AMP of the present invention is an antibody, e.g., and IL-22 antibody or an anti-IL-22R antibody. In certain embodiments, an anti-IL-22 antibody is an agonistic antibody that promotes the interaction of IL-22 with IL-22R. In another embodiment, an anti-IL-22 antibody is an antagonistic antibody that fully or partially blocks the interaction of IL22 with IL-22R. In certain embodiments, an anti-IL-22R antibody binds to the extracellular ligand binding domain of an IL-22R. For example, an anti-IL-22R antibody may bind to the extracellular ligand binding domain of human IL-22R, which is found in SEQ ID NO:3 from about amino acids 18-228.

In a particular embodiment, an IL-22 agonist is an antibody that binds IL-22BP and blocks or inhibits binding of IL-22BP to IL-22, and thereby induces or increases an IL-22 activity (e.g., binding to IL-22R).

In another embodiment, an agonist or antagonist of an AMP of the present invention is an oligopeptide that binds to the AMP. In one embodiment, an oligopeptide binds to the extracellular ligand binding domain of IL-22R. Oligopeptides may be chemically synthesized using known oligopeptide synthesis methodology or may be prepared and purified using recombinant technology.

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-60 2016259423 17 Oct 2018

Such oligopeptides are usually at least about 5 amino acids in length, alternatively at least about 6, 7,

8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21,22, 23,24, 25, 26, 27, 28, 29,30,31,32,33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,

90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 amino acids in length. Such oligopeptides may be identified without undue experimentation using well known techniques. In this regard, it is noted that techniques for screening oligopeptide libraries for oligopeptides that are capable of specifically binding to a polypeptide target are well known in the art (see, e.g., U.S. Patent Nos. 5,556,762, 5,750,373, 4,708,871, 4,833,092, 5,223,409, 5,403,484, 5,571,689, 5,663,143; PCT Publication Nos. WO 84/03506 and WO84/03564; Geysen et al., Proc. Natl. Acad. Sci. U.S.A., 81:3998-4002 (1984); Geysen et al., Proc. Natl. Acad. Sci. USA, 82:178-182 (1985); Geysen et al., in Synthetic Peptides as Antigens, 130-149 (1986); Geysen et al., J. Immunol. Meth., 102:259-274 (1987); Schoofs et al., T Immunol., 140:611-616 (1988), Cwirla, S. E. et al. (1990) Proc. Natl. Acad. Sci. USA, 87:6378; Lowman, H.B. et al. (1991) Biochemistry, 30:10832; Clackson, T. et al. (1991) Nature, 352: 624; Marks, J. D. et al. (1991), J. Mol. Biol., 222:581; Kang, A.S. et al. (1991) Proc. Natl. Acad. Sci. USA, 88:8363, and Smith, G. P. (1991) Current Opin. Biotechnol., 2:668).

In yet another embodiment, an agonist or antagoist of an AMP of the present invention is an organic molecule that binds to the AMP, other than an oligopeptide or antibody as described herein. An organic molecule may be, for example, a small molecule. In one embodiment, an organic molecule binds to the extracellular domain of an 1L-22R. An organic molecule that binds to an AMP of the present invention may be identified and chemically synthesized using known methodology (see, e.g., PCT Publication Nos. WO00/00823 and WOOO/39585). Such organic molecules are usually less than about 2000 daltons in size, alternatively less than about 1500, 750, 500, 250 or 200 daltons in size, wherein such organic molecules that are capable of binding to an AMP of the present invention may be identified without undue experimentation using well known techniques. In this regard, it is noted that techniques for screening organic molecule libraries for molecules that are capable of binding to a polypeptide target are well known in the art (see, e.g., PCT Publication Nos. WO00/00823 and WOOO/39585).

In a particular embodiment, an IL-22 agonist is an organic molecule that binds 1L-22BP and blocks or inhibits binding of 1L-22BP to IL-22, and thereby induces or increases an IL-22 activity (e.g., binding to 1L-22R).

In a particular embodiment, an IL-22 antagonist is a soluble IL-22 receptor, e.g., a form of 1L22R that is not membrane bound. Such soluble forms of 1L-22R may compete with membrane-bound 1L-22R for binding to IL-22. In certain embodiments, a soluble form of 1L-22R may comprise all or a ligand-binding portion of an extracellular domain of 1L-22R, e.g., all or a ligand-binding portion of a polypeptide comprising amino acids 18-228 of SEQ ID NOG. In certain embodiments, a soluble

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-61 2016259423 17 Oct 2018 form of 1L-22R lacks a transmembrane domain. For example, a soluble form of human IL-22R may lack all or a substantial portion of the transmembrane domain from about amino acids 229-251 of SEQ ID NO:3.

A naturally occurring, soluble receptor for IL-22 has been reported. See Dumoutier L. et al.,

Cloning and characterization of IL-22 binding protein, a natural antagonist of IL-10-related T cellderived inducible factor/IL-22, J. Immunol. 166:7090-7095 (2001); and Xu W. et al., A soluble class 11 cytokine receptor, 1L-22RA2, is a naturally occurring IL-22 antagonist, Proc. Natl. Acad. Sci. U.S.A. 98:9511-9516 (2001). That receptor is variously designated “1L-22BP” or “1L-22RA2” in the art. The sequence of a human 1L-22BP is shown in Figure 4. The term “1L-22BP” or “IL-22 binding protein” as used herein refers to any native 1L-22BP from any vertebrate source, including mammals such as primates (e.g. humans and monkeys) and rodents (e.g., mice and rats), unless otherwise indicated.

In yet another embodiment, an antagonist of IL-22 is an antisense nucleic acid that decreases expression of the IL-22 or 1L-22R gene (i.e., that decreases transcription of the IL-22 or 1L-22R gene and/or translation of IL-22 or 1L-22R mRNA). In certain embodiments, an antisense nucleic acid binds to a nucleic acid (DNA or RNA) encoding IL-22 or 1L-22R. In certain embodiments, an antisense nucleic acid is an oligonucleotide of about 10-30 nucleotides in length (including all points between those endpoints). In certain embodiments, an antisense oligonucleotide comprises a modified sugar-phosphodiester backbones (or other sugar linkages, including phosphorothioate linkages and linkages as described in WO 91/06629), wherein such modified sugar-phosphodiester backbones are resistant to endogenous nucleases. In one embodiment, an antisense nucleic acid is an oligodeoxyribonucleotide, which results in the degradation and/or reduced transcription or translation of mRNA encoding IL-22 or 1L-22R. In certain embodiments, an antisense nucleic acid is an RNA that reduces expression of a target nucleic acid by “RNA interference” (“RNAi”). For review of

RNAi, see, e.g., Novina et al. (2004) Nature 430:161-164. Such RNAs are derived from, for example, short interfering RNAs (siRNAs) and microRNAs. siRNAs, e.g., may be synthesized as double stranded oligoribonucleotides of about 18-26 nucleotides in length. Id.

In yet another embodiment, agonists of IL-22 are provided. Exemplary agonists include, but are not limited to, native IL-22 or 1L-22R; fragments, variants, or modified forms of IL-22 or 1L-22R that retain at least one activity of the native polypeptide; agents that are able to bind to and activate 1L-22R; and agents that induce overexpression of IL-22 or 1L-22R or nucleic acids encoding IL-22 or 1L-22R.

D. Pharmaceutical Formulations

The invention provides pharmaceutical formulations. In one embodiment, a pharmaceutical formulation comprises 1) an active agent, e.g., any of the above-described polypeptides, antibodies,

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-62 2016259423 17 Oct 2018 agonists, or antagonists; and 2) a pharmaceutically acceptable carrier. In a further embodiment, a pharmaceutical formulation lurther comprises at least one additional therapeutic agent.

Pharmaceutical formulations are prepared for storage by mixing an agent having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. [1980]), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Znprotein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

Lipofections or liposomes can also be used to deliver an agent into a cell. Where the agent is an antibody fragment, the smallest inhibitory fragment which specifically binds to the target protein is preferred. For example, based upon the variable region sequences of an antibody, peptide molecules can be designed which retain the ability to bind the target protein sequence. Such peptides can be synthesized chemically and/or produced by recombinant DNA technology (see, e.g., Marasco et al., Proc. Natl. Acad. Sci. USA 90, 7889-7893 [1993]). Antibodies disclosed herein may also be formulated as immunoliposomes. Liposomes containing an antibody are prepared by methods known in the art, such as described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl Acad. Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Patent No. 5,013,556. Particularly uselul liposomes can be generated by the reverse-phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEGPE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter. Fab' fragments of an antibody of the present invention can be conjugated to liposomes as described in Martin et al., J. Biol. Chem., 257: 286-288 (1982) via a disulfide-interchange reaction.

A chemotherapeutic agent (such as doxorubicin) is optionally contained within the liposome. See

Gabizon et al., J. National Cancer Inst., 81( 19): 1484(1989).

An agent may also be entrapped in microcapsules prepared, for example, by coacervation

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-63 2016259423 17 Oct 2018 techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatinmicrocapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical

Sciences 16th edition, Osol, A. Ed. (1980).

Sustained-release preparations of an agent may be prepared. Suitable examples of sustainedrelease preparations include semipermeable matrices of solid hydrophobic polymers containing the agent, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl10 methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of Lglutamic acid and γ-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acidglycolic acid copolymers such as the LUPRON DEPOTTM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods. When encapsulated antibodies remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 37°C, resulting in a loss of biological activity and possible changes in immunogenicity. Rational strategies can be devised for stabilization depending on the mechanism involved. For example, if the aggregation mechanism is discovered to be intermolecular S-S bond formation through thio-disulfide interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions.

A pharmaceutical formulation herein may also contain more than one active compound as necessary for the particular indication being treated. For example, in one embodiment, a pharmaceutical formulation containing more than one active compound comprises 1) at least one agonist of IL-22, e.g., an antibody that binds to IL-22 and/or an antibody that binds to IL-22R; and 2) at least one antibody that binds to IL-6 or IL-23 (wherein any number of the antibodies listed in 2) may be selected in any combination). In another embodiment, a pharmaceutical formulation contains two or more active compounds having complementary activities.

E. Methods of Treatment

The present invention further provides methods of treating a microbial disorder. In another embodiment, the present invention provides a method of treating a microbial disorder, in a subject, comprising administering to the subject an effective amount of pharmaceutical composition comprising an AMP or modulator of the AMP, wherein the AMP is selected from a group consisting of: LT, IL-6, IL-18, IL-22, IL-23, REG Ια, REG Ιβ, HIP/PAP, REG III, REG IV and Reg-related

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-64 2016259423 17 Oct 2018 sequence (RS). In one embodiment the disorder is EHEC- or EPEC-caused diarrhea, Inflammatory Bowel Disease (1BD) or, more particularly, Ulcerative Colitis (UC) and Crohn’s Disease (CD).

In one embodiment, the present invention provides a method of treating an infection by a microbial pathogen (e.g., a bacteria or virus), in a subject, comprising administering to the subject an effective amount of pharmaceutical composition comprising an AMP or modulator of the AMP, wherein the AMP is selected from a group consisting of: LT, IL-6, IL-18, IL-22, IL-23, REG la,

REG Ιβ, HIP/PAP, REG 111, REG IV and Reg-related sequence (RS).

In another embodiment, the present invention provides a method of modulating the activity of an AMP in cells of a subject infected with a microbial pathogen (e.g., a bacteria or virus), comprising contacting the cells with an AMP or modulator of the AMP, wherein the AMP is selected from a group consisting of: LT, IL-6, IL-18, IL-22, IL-23, REG Ια, REG Ιβ, HIP/PAP, REG III (e.g., REG ΙΙΙβ or REGIIIy), REG IV, and Reg-related sequence (RS).

In another embodiment, the present invention provides a method of treating a microbial disorder, in a subject, comprising contacting cells of the subject with a nucleic acid molecule (e.g., a

DNA or RNA molecule) encoding an AMP or modulator of the AMP, wherein the AMP is selected from a group consisting of: LT, IL-6, IL-18, IL-22, IL-23, REG Ια, REG Ιβ, HIP/PAP, REG III, REG IV and Reg-related sequence (RS). In one embodiment the disorder is EHEC- or EPEC-caused diarrhea, Inflammatory Bowel Disease (IBD) or, more particularly, Ulcerative Colitis (UC) or Crohn’s Disease (CD).

In another embodiment, the present invention provides a method of modulating the activity of an AMP in cells of a subject infected with a microbial pathogen (e.g., a bacteria or virus), comprising contacting the cells with a nucleic acid molecule (e.g., a DNA or RNA molecule) encoding an AMP or modulator of the AMP, wherein the AMP is selected from a group consisting of: LT, IL-6, IL-18, IL-22, IL-23, REG Ια, REG Ιβ, HIP/PAP, REG III (e.g., REG ΙΙΙβ or REGIIIy), REG IV, and Reg25 related sequence (RS).

Examples of a microbial pathogen include, but are not limited to, a bacteria or virus. In one embodiment, the microbial pathogen is a bacteria e.g., a gram-negative or gram-positive bacteria. In a particular embodiment, the bacteria is a gram-negative bacteria. In another embodiment, the bacteria is an attaching or effacing (A/E) bacteria and, more particularly, an enterohemorrhagic Escherichia coli (EHEC) or enteropathogenic E. Coli (EPEC). In one embodiment, the bacteria is enteropathogenic E. coli (EHEC) is E. coli 0157:H7 or E. coli 055:H7.

The therapeutic methods of the present invention comprise one or more compositions or pharmaceutical formulations of the present invention. Such methods include in vitro, ex vivo, and in vivo therapeutic methods, unless otherwise indicated.

In various embodiments, the present invention provides methods of modulating an antimicrobial immune response by stimulating or inhibiting an AMP-mediated signaling pathway and/or

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ThiL-17 cell function. Such methods are useful for treatment of microbial disorders. For example, in one embodiment, the present invention provides a method of enhancing an anti-microbial immune response by stimulating an AMP-mediated signaling pathway, e.g., and IF-22 and/or IF-23 mediated signaling pathway. In another embodiment, the present invention provides methods of modulating an anti-microbial immune response by stimulating or inhibiting a cytokine-mediated signaling pathway. For example, in one embodiment, the present invention provides methods of enhancing an antimicrobial immune response by stimulating a cytokine-mediated signaling pathway, e.g., an IF-22 and/or IF-23 signaling pathway. Moreover, the present invention provides methods of modulating an anti-microbial immune response by stimulating or inhibiting a Thi_L-i7 cell function.

In one embodiment, the present invention provides a method of stimulating an AMP-mediated signaling pathway in a biological system, the method comprising providing an AMP agonist to the biological system. Examples of such a biological system include, but are not limited to, mammalian cells in an in vitro cell culture system or in an organism in vivo. In another embodiment, the present invention provides a method of inhibiting an AMP-mediated signaling pathway in a biological system, the method comprising providing an AMP antagonist to the biological system.

In a particular embodiment, the present invention provides a method of enhancing an antimicrobial immune response in a biological system by stimulating an IF-23 and/or IF-22 mediated signaling pathway in a biological system, the method comprising providing an IF-22 or IF-22 agonist to the biological system. In one embodiment, an IF-22 agonist is IF-22. In another embodiment, the

IF-22 agonist is an antibody that binds to IF-22.

In another embodiment, a method of inhibiting an IF-23-mediated signaling pathway in a biological system is provided, the method comprising providing an IF-22 antagonist to the biological system. In one embodiment, the antagonist of IF-22 is an antibody, e.g., a neutralizing anti-lF-22 antibody and/or a neutralizing anti-lF-22R antibody.

In another embodiment, the present invention provides a method of stimulating a ThiL-17 cell function, the method comprising exposing a ThiL-17 cell to an agonist of an AMP that mediates the IF23 mediated signaling pathway (e.g., IF-23,1F-6, or IF-22). Such methods are useful for treating a microbial disorder. In one embodiment, an IF-22 agonist is IF-22. In another embodiment, the IF-22 agonist is an antibody that binds to IF-22.

In another embodiment, a method of inhibiting a Thin? cell function is provided, the method comprising exposing a ThiL-17 cell to an antagonist of an AMP that mediates the IF-23 mediated signaling pathway (e.g., IF-23,1F-6, or IF-22). In one embodiment the antagonist is an anti-lF-22 antibody, e.g., a neutralizing anti-lF-22 antibody.

Exemplary ThiL-17 cell functions include, but are not limited to, stimulation of cell-mediated immunity (delayed-type hypersensitivity); recruitment of innate immune cells, such as myeloid cells (e.g., monocytes and neutrophils) to sites of inflammation; and stimulation of inflammatory cell

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-66 2016259423 17 Oct 2018 infiltration into tissues. In one embodiment, a ThiL-17 cell function is mediated by IL-23 and/or IL-22. Compositions of the present invention are administered to a mammal, preferably a human, in accord with known methods, such as intravenous administration as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intra5 articular, intrasynovial, intrathecal, oral, topical, or inhalation (intranasal, intrapulmonary) routes. Intravenous or inhaled administration of polypeptides and antibodies is preferred.

For the treatment or reduction in the severity of a microbial disorder, the appropriate dosage of a composition of the invention will depend on the type of disorder to be treated, as defined above, the severity and course of the disorder, whether the agent is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the compound, and the discretion of the attending physician. The compound is suitably administered to the patient at one time or over a series of treatments.

For example, depending on the type and severity of a disorder, about 1 pg/kg to 15 mg/kg (e.g., 0.1-20 mg/kg) of a polypeptide or antibody is an initial candidate dosage for administration to a patient, whether, for example, by one or more separate administrations, or by continuous infusion. A typical daily dosage might range from about 1 pg/kg to 100 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

F. Diagnostic Methods and Methods of Detection

In one embodiment, the present invention provides a method of detecting the presence of an AMP in a biological sample, comprising contacting the biological sample with an antibody to the AMP, under conditions permissive for binding of the antibody to the AMP, and detecting whether a complex is formed between the antibody and AMP.

In one embodiment, the present invention provides a method of monitoring treatment of a microbial disorder in a subject, wherein the method comprises detecting the level of expression of a gene encoding an AMP in a test sample of tissue cells obtained from the subject in need of treatment, and the expression level in the test sample is detected. The detection may be qualitative or quantitative. In one embodiment, the test sample comprises blood or serum. In one embodiment, detecting the level of expression of a gene encoding an AMP comprises (a) contacting an anti-AMP antibody with a test sample obtained from the mammal, and (b) detecting the formation of a complex between the antibody and an AMP in the test sample. The antibody may be linked to a detectable label. Complex formation can be monitored, for example, by light microscopy, flow cytometry, fluorimetry, or other techniques known in the art. The test sample may be obtained from an individual suspected of having a microbial disorder.

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In one embodiment, detecting the level of expression of a gene encoding an AMP polypeptide comprises detecting the level of mRNA transcription from the gene. Levels of mRNA transcription may be detected, either quantitatively or qualitatively, by various methods known to those skilled in the art. Levels of mRNA transcription may also be detected directly or indirectly by detecting levels of cDNA generated from the mRNA. Exemplary methods for detecting levels of mRNA transcription include, but are not limited to, real-time quantitative RT-PCR and hybridization-based assays, including microarray-based assays and fdter-based assays such as Northern blots.

In another embodiment, the present invention provides a method of detecting the presence of an AMP in a biological sample, comprising contacting the biological sample with an antibody to the

AMP, under conditions permissive for binding of the antibody to the AMP, and detecting whether a complex is formed between the antibody and AMP.

In another embodiment, the present invention concerns a diagnostic kit containing an anti-AMP in suitable packaging. The kit preferably contains instructions for using the antibody to detect an AMP. In one embodiment, the diagnostic kit is for diagnosing a microbial disorder. In one embodiment, the diagnostic kit is for diagnosing a microbial infection.

In another embodiment, the present invention provides a kit comprising one or more AMPs of the present invention and/or modulators thereof. In another embodiment, the present invention provides a kit comprising one or more one or more pharmaceutical compositions each comprising an AMP of the present invention or modulator thereof.

G. Assays

1. Cell-Based Assays and Animal Models

Cell-based assays and animal models for immune diseases are useful in practicing certain embodiments of the invention. Certain cell-based assays provided in the Examples below are useful, e.g., for testing the efficacy of IL-22 antagonists or agonists.

In vivo animal models are also useful in practicing certain embodiments of the invention.

Exemplary animal models are also described in the Examples below. The in vivo nature of such models makes them predictive of responses in human patients. Animal models of immune related diseases include both non-recombinant and recombinant (transgenic) animals. Non-recombinant animal models include, for example, rodent, e.g., murine models. Such models can be generated by introducing cells into syngeneic mice using standard techniques, e.g., subcutaneous injection, tail vein injection, spleen implantation, intraperitoneal implantation, implantation under the renal capsule, etc.

Graft-versus-host disease models provide a means of assessing T cell reactivity against MHC antigens and minor transplant antigens. Graft-versus-host disease occurs when immunocompetent cells are transplanted into immunosuppressed or tolerant patients. The donor cells recognize and respond to host antigens. The response can vary from life threatening severe inflammation to mild cases of diarrhea and weight loss. A suitable procedure for assessing graft-versus-host disease is

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-68 2016259423 17 Oct 2018 described in detail in Current Protocols in Immunology, above, unit 4.3.

An animal model for skin allograft rejection is a means of testing the ability of T cells to mediate in vivo tissue destruction and a measure of their role in transplant rejection. The most common and accepted models use murine tail-skin grafts. Repeated experiments have shown that skin allograft rejection is mediated by T cells, helper T cells and killer-effector T cells, and not antibodies. Auchincloss, H. Jr. and Sachs, D. H., Fundamental Immunology, 2nd ed., W. E. Paul ed., Raven Press, NY, 1989, 889-992. A suitable procedure is described in detail in Current Protocols in Immunology, above, unit 4.4. Other transplant rejection models which can be used to test the compounds of the invention are the allogeneic heart transplant models described by Tanabe, M. et al,

Transplantation (1994) 58:23 and Tinubu, S. A. et al, J. Immunol. (1994) 4330-4338.

Contact hypersensitivity is a simple in vivo assay for cell mediated immune function (delayed type hypersensitivity). In this procedure, cutaneous exposure to exogenous haptens which gives rise to a delayed type hypersensitivity reaction which is measured and quantitated. Contact sensitivity involves an initial sensitizing phase followed by an elicitation phase. The elicitation phase occurs when the T lymphocytes encounter an antigen to which they have had previous contact. Swelling and inflammation occur, making this an excellent model of human allergic contact dermatitis. A suitable procedure is described in detail in Current Protocols in Immunology, Eds. J. E. Cologan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach and W. Strober, John Wiley & Sons, Inc., 1994, unit 4.2. See also Grabbe, S. and Schwarz, T, Immun. Today 19 (1): 37-44 (1998) .

Additionally, the compositions of the invention can be tested on animal models for psoriasislike diseases. For example, compositions of the invention can be tested in the scid/scid mouse model described by Schon, Μ. P. et al, Nat. Med. (1997) 3:183, in which the mice demonstrate histopathologic skin lesions resembling psoriasis. Another suitable model is the human skin/scid mouse chimera prepared as described by Nickoloff, B. J. et al, Am. J. Path. (1995) 146:580. Another suitable model is described in Boyman et al., J Exp Med. (2004) 199(5):731-6, in which human prepsoriatic skin is grafted onto AGR129 mice, leading to the development of psoriatic skin lesions.

Knock out animals can be constructed which have a defective or altered gene encoding a polypeptide identified herein, as a result of homologous recombination between the endogenous gene encoding the polypeptide and a DNA molecule in which that gene has been altered. For example, cDNA encoding a particular polypeptide can be used to clone genomic DNA encoding that polypeptide in accordance with established techniques. A portion of the genomic DNA encoding a particular polypeptide can be deleted or replaced with another gene, such as a gene encoding a selectable marker which can be used to monitor integration. Typically, several kilobases of unaltered flanking DNA (both at the 5' and 3' ends) are included in the vector [see e.g., Thomas and Capecchi,

Cell, 51:503 (1987) for a description of homologous recombination vectors]. The vector is introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced DNA has

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-69 2016259423 17 Oct 2018 homologously recombined with the endogenous DNA are selected [see e.g., Li et al., Cell, 69:915 (1992)]. The selected cells are then injected into a blastocyst of an animal (e.g., a mouse or rat) to form aggregation chimeras [see e.g., Bradley, in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (1RL, Oxford, 1987), pp. 113-152], A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term to create a knock out animal. Progeny harboring the homologously recombined DNA in their germ cells can be identified by standard techniques and used to breed animals in which all cells of the animal contain the homologously recombined DNA. Knockout animals can be characterized for instance, for their ability to defend against certain pathological conditions and for their development of pathological conditions due to absence of the polypeptide.

2. Screening Assays for Drug Candidates

Screening assays for drug candidates are designed to identify compounds that bind to or complex with a polypeptide identified herein or a biologically active fragment thereof, or otherwise interfere with the interaction of a polypeptide with other cellular proteins. Such screening assays will include assays amenable to high-throughput screening of chemical libraries, making them particularly suitable for identifying small molecule drug candidates. Small molecules contemplated include synthetic organic or inorganic compounds, including peptides, preferably soluble peptides, (poly)peptide-immunoglobulin fusions, and, in particular, antibodies including, without limitation, poly- and monoclonal antibodies and antibody fragments, single-chain antibodies, anti-idiotypic antibodies, and chimeric or humanized versions of such antibodies or fragments, as well as human antibodies and antibody fragments. The assays can be performed in a variety of formats, including protein-protein binding assays, biochemical screening assays, immunoassays and cell based assays, which are well characterized in the art. All assays are common in that they call for contacting a test compound with a polypeptide identified herein under conditions and for a time sufficient to allow the polypeptide to interact with the test compound.

In binding assays, the interaction is binding and the complex formed can be isolated or detected in the reaction mixture. In a particular embodiment, a polypeptide or the test compound is immobilized on a solid phase, e.g., on a microtiter plate, by covalent or non-covalent attachments. Non-covalent attachment generally is accomplished by coating the solid surface with a solution of the polypeptide or test compound and drying. Alternatively, an immobilized antibody, e.g., a monoclonal antibody specific for a polypeptide to be immobilized, can be used to anchor the polypeptide to a solid surface. The assay is performed by adding the non-immobilized component, which may be labeled by a detectable label, to the immobilized component, e.g., the coated surface containing the anchored component. When the reaction is complete, the non-reacted components are removed, e.g., by washing, and complexes anchored on the solid surface are detected. When the originally nonimmobilized component carries a detectable label, the detection of label immobilized on the surface

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-70 2016259423 17 Oct 2018 indicates that complexing occurred. Where the originally non-immobilized component does not carry a label, complexing can be detected, for example, by using a labelled antibody specifically binding the immobilized complex.

If the test compound interacts with but does not bind to a particular polypeptide identified 5 herein, its interaction with that protein can be assayed by methods well known for detecting proteinprotein interactions. Such assays include traditional approaches, such as, cross-linking, coimmunoprecipitation, and co-purification through gradients or chromatographic columns. In addition, protein-protein interactions can be monitored by using a yeast-based genetic system described by Fields and co-workers [Fields and Song, Nature (London) 340, 245-246 (1989); Chien et al., Proc.

Natl. Acad. Sci. USA 88, 9578-9582 (1991)] as disclosed by Chevray and Nathans, Proc. Natl. Acad. Sci. USA 89, 5789-5793 (1991). Many transcriptional activators, such as yeast GAL4, consist of two physically discrete modular domains, one acting as the DNA-binding domain, while the other one functioning as the transcription activation domain. The yeast expression system described in the foregoing publications (generally referred to as the two-hybrid system) takes advantage of this property, and employs two hybrid proteins, one in which the target protein is fused to the DNAbinding domain of GAL4, and another, in which candidate activating proteins are fused to the activation domain. The expression of a GALl-/acZ reporter gene under control of a GAL4-activated promoter depends on reconstitution of GAL4 activity via protein-protein interaction. Colonies containing interacting polypeptides are detected with a chromogenic substrate for β -galactosidase. A complete kit (MATCHMAKER™) for identifying protein-protein interactions between two specific proteins using the two-hybrid technique is commercially available from Clontech. This system can also be extended to map protein domains involved in specific protein interactions as well as to pinpoint amino acid residues that are crucial for these interactions.

To identify compounds that interfere with the interaction of a polypeptide identified herein and other intra- or extracellular component(s), a reaction mixture may be prepared containing the polypeptide and the component under conditions allowing for the interaction of the polypeptide with the component. To test the ability of a test compound to inhibit the interaction, the reaction mixture is prepared in the absence and in the presence of the test compound. If there is a decrease in the interaction of the polypeptide with the component in the presence of the test compound, then the test compound is said to inhibit the interaction of the polypeptide with the component.

In certain embodiments, methods for identifying agonists or antagonists of an AMP comprise contacting an AMP with a candidate agonist or antagonist molecule and measuring a detectable change in one or more biological activities normally associated with the AMP. Such activities include, but are not limited to, those described in the Examples below.

In one embodiment, the present invention provides methods for identifying agonists of an IL-22 polypeptide comprise contacting an IL-22 polypeptide with a candidate agonist molecule and

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-71 2016259423 17 Oct 2018 measuring a detectable change in one or more biological activities normally associated with the IL-22 polypeptide. Such activities include, but are not limited to, those described in the Examples below.

3. Antibody binding assays

Antibody binding studies may be carried out in any known assay method, such as competitive 5 binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. Zola,

Monoclonal Antibodies: A Manual of Techniques, pp.147-158 (CRC Press, Inc., 1987).

Competitive binding assays rely on the ability of a labeled standard to compete with the test sample analyte for binding with a limited amount of antibody. The amount of target protein in the test sample is inversely proportional to the amount of standard that becomes bound to the antibodies. To facilitate determining the amount of standard that becomes bound, the antibodies preferably are insolubilized before or after the competition, so that the standard and analyte that are bound to the antibodies may conveniently be separated from the standard and analyte which remain unbound.

Sandwich assays involve the use of two antibodies, each capable of binding to a different immunogenic portion, or epitope, of the protein to be detected. In a sandwich assay, the test sample analyte is bound by a first antibody which is immobilized on a solid support, and thereafter a second antibody binds to the analyte, thus forming an insoluble three-part complex. See, e.g., US Pat No. 4,376,110. The second antibody may itself be labeled with a detectable moiety (direct sandwich assays) or may be measured using an anti-immunoglobulin antibody that is labeled with a detectable moiety (indirect sandwich assay). For example, one type of sandwich assay is an ELISA assay, in which case the detectable moiety is an enzyme.

Immunhistochemistry may also be used to determine the cellular location of an antigen to which an antibody binds. For immunohistochemistry, the tissue sample may be fresh or frozen or may be embedded in paraffin and fixed with a preservative such as formalin, for example. Articles of Manufacture

In another embodiment, the present invention provides an article of manufacture comprising compositions useful for the diagnosis or treatment of the microbial disorders described herein. The article of manufacture comprises a container and an instruction. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is effective for diagnosing or treating the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The active agent in the composition is usually a polypeptide, an antibody, an agonist, or an antagonist of the invention. An instruction or label on, or associated with, the container indicates that the composition is used for diagnosing or treating the condition of choice. The article of manufacture may further comprise a second container comprising a pharmaceutically-acceptable buffer, such as phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other

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-72 2016259423 17 Oct 2018 materials desirable from a commercial and user standpoint, including other buffers, diluents, fdters, needles, syringes, and package inserts with instructions for use.

In one embodiment, the invention provides an article of manufacture, comprising:

(a) a composition of matter comprising an AMP or modulator thereof (e.g., an IL-22 agonist);

(b) a container containing said composition; and (c) a label affixed to said container, or a package insert included in said container, referring to the use of said agonist in the treatment of an microbial disorder. The composition may comprise an effective amount of the agonist.

EXAMPLES

The following are examples of methods and compositions of the invention, and are provided herein for illustrative purposes, and are not intended to limit the scope of the present invention. It is understood that various other embodiments may be practiced, given the general description provided herein.

Commercially available reagents referred to in the examples were used according to manufacturer's instructions unless otherwise indicated. The source of those cells identified in the following examples, and throughout the specification, by ATCC accession numbers is the American Type Culture Collection, Manassas, VA.

The data presented herein demonstrate for the first time that IL-22 is one of the key cytokines that bridges adaptive immune response and innate epithelial defense during early infection of an A/E bacterial pathogen. As shown herein, the induction of RegI I Ιβ and RegIIΙγ also indicates that IL-22 may have broader functions in controlling various bacterial infections. The data further supports the role of Thl7 cells and their effector cytokines in infectious diseases and autoimmune diseases. Finally, the present studies indicate that IL-22 and its downstream products, such as Rcgl 11 β and

RegI I Ιγ, may be beneficial for the treatment of certain infectious diseases.

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Example 1: IL-23 is essential for IL-22 regulation during an infectious disease process.

The data herein demonstrate that IL-23 is essential for IL-22 regulation during an infectious disease process.

Both IL-22 receptor pairs, 1L-22R and IL-10RQ chains, are expressed in the Gl tract of wildtype 5 C57B1/6 mice (Fig. 1 A). Their expression in the duodenum, jejunum, ileum, and colon are higher than they are in the skin, a tissue where IL-22 has been shown to induce hyperplasia. Consistently both colonic epithelial cells and subepithelial myofibroblasts have been reported to respond to IL-22. During C. rodentium infection, IL-22 was induced in the colon of wildtype mice (Fig. IB), as were cytokines that promote Thl7 cell differentiation, including the p 19 and p40 subunit of IL-23 (Fig. 1C10 D), and IL-6 (Fig. IE). All of these cytokines were rapidly induced, with peak expression around day post inoculation. In contrast, IL-17 induction had slower kinetics and reached its maximum level at day 12 post inoculation (Fig. IF).

Since either IL-23 or IL-6 promotes IL-22 production from T cells in vitro, the present inventors sought to first define their role in regulation of IL-22 production during C. rodentium infection. When comparing the survival rate of wildtype, p 19, p40, and IL-6 mice after C.

rodentium infection, we consistently found all the mice from either the p40'^z group (Fig. 1G) or the p 19^z group (data not shown) died 10 days post inoculation. Interestingly, 60% mortality was also observed in the 1L-6'^Z group around day 12 (Fig. 1G), indicating that IL-6 is required, to a certain extent, for a total control of C. rodentium infection. Next we examined IL-22 and IL-17 expression in both pl9'^z and 1L-6'^Z mice (Fig. IH). While, IL-17 expression was not altered in pl9'^z mice (15), induction of IL-22 was diminished in pl9'^z mice compared to wildtype mice. In 1L-6'^Z mice, however, while the peak level of IL-22 was comparable to that of wildtype mice, its induction was significantly delayed (Fig IH). Furthermore, in 1L-6'^Z mice, the induction of IL-17 was significantly reduced, consistent with an essential role of IL-6 for IL-17 production.

Directly measuring 1L-22/1L-17 proteins by ELISA in ex vivo culture of colons from infected mice confirmed the kinetics of 1L-22/1L-17 production and the absence of IL-22 induction from pl9'^z mice during C. rodentium infection (Fig. 11). These data for the first time demonstrate that IL-23 is essential for IL-22 regulation during an infectious disease process.

These data for the first time demonstrate that IL-23 is essential for IL-22 regulation during an infectious disease process.

Example 2: IL-22 is a key downstream effector cytokine that contributes to the biology of IL-23 in controlling microbial infection.

The altered regulation of IL-22 in both IL-23 deficient and 1L-6'^Z mice indicated that IL-22 may play a critical role in the host defense against C. rodentium infection. To further examine the role of IL-22, IL-22' mice were inoculated with C. rodentium. While wildtype littermates transiently lost

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-74 2016259423 17 Oct 2018 weight but were able to fully recover after day 6, IL-22'⁷' mice continued losing weight following C. rodentium infection (Fig. 2A). About 80% of IL-22'⁷' mice became moribund or died 12 days post C. rodentium inoculation (Fig. 2A). Histologic analysis of the colons from day 8 infected IL-22'⁷' mice demonstrated increased mucosal thickness when compared with that of WT mice (Fig. 2B).

Coincidently, there was also increased submucosal inflammation (Arrow, Fig. 2B). Furthermore, while in control mice, C. rodentium infection was predominantly superficial, large numbers of bacteria penetrated deeply into colonic crypts in IL-22'⁷' mice (Arrows, Fig. 2C). FACS analysis with an anti-IL-22R antibody (Fig. 12) revealed that IL-22R was expressed by E-cadherin positive primary murine colonic epithelial cells, but not by CD45⁺ intra-epithelial lymphocytes (IEL) or lamina propria mononuclear cells (LPMCs) (Fig. 12A). Similarly, primary human colonic epithelial cells also expressed IL-22R (Fig. 12B). These data suggest that colonic epithelial cells were directly targeted by IL-22.

These data support the importance of IL-22 in host defense against C. rodentium infection, and indicate that IL-22 may be one of the key downstream effector cytokines that contribute to the biology of IL-23 in controlling microbial infections.

Example 3: IL-17A and IL-17F pathways are not required for host defense against C. rodentium infection.

The partial impairment of host defense in IL-6'⁷' mice against C. rodentium could also be explained by the delayed induction of IL-22 in these mice (Fig. IH, left panel). However, it is also possible that lethality in C. rodentium infected IL-6'⁷' mice may have been due to their inability to upregulate IL-17 (Fig. IH, right panel). The IL-17 pathway is crucial for the control of many extracellular bacterial infections, such as Klebsiella pneumoniae. IL-17 signals through IL-17R and IL-17RC (D. Toy et al., J Immunol 177, 36 (July 1, 2006)), and induces proinflammatory responses from many cell types, including epithelial cells (J. Witowski, K. Ksiazek, A. Jorres, Cell Mol Life Sci 61, 567 (Mar, 2004)). To analyze the role of the IL-17 pathway during C. rodentium infection, IL17RC'⁷' mice were generated (Fig. 5). Compared to wildtype littermates, there was no obvious defect in IL-17RC'⁷' mice in terms of development or composition of T cells, B cells and other immune cells (data not shown). However, fibroblasts generated from the tail tip (Fig. 5C) or lung tissue (data not shown) of IL-17RC'⁷' mice were completely incapable of producing IL-6 when stimulated with either IL-17A or IL-17F, indicating that IL-17RC is an essential receptor for both IL-17A and IL-17F mediated functions. Following C. rodentium inoculation, both IL-17RC'⁷' mice and wildtype littermates survived the course of infection without any significant loss of weight (Fig. 2D) or any histologic differences in the colon (data not shown).

These results indicate that IL-17A and IL-17F pathways were not required for host defense against C. rodentium infection, directly excluding the possibility that defective IL-17 production is the

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-75 2016259423 17 Oct 2018 major cause of observed mortality in IL-6'⁷' mice. Thus, the delayed induction of IL-22 observed in IL-6'⁷' mice might be the reason that these mice were incapable of surviving the infection. Other factors downstream of IL-6, however, may also be important. The results from IL-6'⁷' mice imply that the early induction of IL-22 might be critical for the host to mount a sufficient response against C.

rodentium infection in order to prevent lethality.

Example 3: IL-22 plays a critical role in the early stage of bacterial infection.

To determine whether early induction of IL-22 is critical for the host to mount a sufficient response against C. rodentium infection in order to prevent lethality, anti-IL-22 neutralizing antibody was administrated every other day starting either at day 0 or at day 8 post inoculation of C. rodentium. As expected, mice that received anti-IL-22 mAh at the same time as the bacterial inoculation continued to lose weight, and all became moribund or died 12 days post inoculation. In contrast, all isotype control antibody treated animals survived (Fig. 2E). Mice that received anti-IL-22 mAh starting 8 days post inoculation had a similar outcome as did isotype mAh treated mice, with full recovery from infection.

Therefore, these data indicate that IL-22 plays a critical role in the early stage of C. rodentium infection, but plays no role during the later phase of host defense when bacteria are being eradicated.

Example 4: IL-19, IL-20, and IL-24 are dispensable for host defense against bacterial infection.

Other IL-10 family cytokines, IL-19, IL-20, and IL-24, all induce similar biological functions as those induced by IL-22 in human epidermal keratinocytes (S. M. Sa et al., J Immunol 178, 2229 (February 15, 2007).). IL-19, IL-20, and IL-24 were all upregulated in wildtype mouse colon during C. rodentium infection (Fig. 6). They may, therefore, play similar role as does IL-22 during C.

rodentium infection. IL-19 signals through IL-20Ra and IL-20R|3 chains. IL-20 and IL-24 can signal through two different receptor pairs, IL-20Ra/IL-20RP and IL-22R/IL-20RP (J.-C. Renauld, Nature Reviews Immunology 3, 667 (2003)). Therefore, IL-20 R β is the common receptor chain for these three cytokines. In the GI tract, expression of IL-20Ra and IL-20R3 chains was significantly lower than the expression of these chains in skin (Fig. 7).

To critically address the role of these three cytokines during C. rodentium infection, IL-20R|3 mice were generated (Fig. 8). These mice exhibited normal development with similar lymphocyte composition and development in all major lymphoid organs when compared to wildtype mice (data not shown). The ear skin from these mice failed to upregulate SI00 family proteins when treated with recombinant IL-20, indicating a defect in IL-20 signaling in vivo (Fig. 8C).

IL-20RP'⁷' mice survived C. rodentium infection as well as wildtype mice did (Fig. 2F), demonstrating that IL-19, IL-20, and IL-24 are dispensable for host defense against C. rodentium.

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Example 5: IL-22 deficiency may compromise epithelial integrity during the early stage of C. rodentium infection.

The present inventors examined the downstream mechanisms of IL-22 during C. rodentium 5 infection. Both IL-22'⁷' mice and wildtype mice treated with anti-IL-22 mAh on day 0 developed more severe bloody diarrhea and an increased incidence of rectal prolapse compared to control mice 8 days post inoculation of C. rodentium (data not shown). Colons from IL-22'⁷' mice (data not shown) or day 0 anti-IL-22 mAh treated mice were thickened and shortened 10 days post inoculation (Lig. 3A), as well as having a smaller cecum, compared to control mice. Histologic analysis further revealed increased inflammation in colons lacking IL-22 signaling (Lig. 3B). There were also marked multifocal mucosal ulceration and multiple foci of transmural inflammation in both IL-22'⁷' and antiIL-22 mAh treated mice (Lig. 3C, and Lig. 9). Lurthermore, the bacterial burdens in mesenteric lymph node, spleen, and liver of IL-22'⁷' mice were significantly increased compared to those of wildtype mice. Interestingly, the difference in bacterial burdens in colons of wildtype mice and IL15 22'⁷' mice was negligible (Lig. 3D). Consistent with these results, there was also evidence of systemic bacterial spread, particularly in the livers of IL-22'⁷' mice, where multifocal hepatocellular necrosis with embolic microabscessation was evident (Lig. 3E).

In conclusion, these data indicate that the epithelial integrity is compromised in IL-22'⁷' mice during the early stage of C. rodentium infection.

Example 6: IL-22 deficiency leads to a reduction in anti-bacterial IgG titers

Previous studies established the essential role of anti-C. rodentium antibodies in the clearance of bacteria. Transferring serum from wildtype mice post-infection fully rescued CD4'⁷' mice from death following C. rodentium challenge (Bry et al. J Immunol. 172, 433-441 (2004)). In our studies,

IL-22 deficient mice became moribund or died starting around day 8, when antibody responses were not fully developed in wildtype mice (Lig. 3L). On day 8, anti-C. rodentium antibody titers were 50 fold less than those on day 16 in wildtype mice. However, when we compared the titers of anti-C rodentium antibodies on day 8 from wildtype and IL-22'⁷' mice, there was an unexpected significant reduction in the anti-C rodentium IgG titer in IL-22'⁷' mice compared to that in wildtype mice (Lig.

3G). In contrast, there was no decrease in total IgG, IgM, IgA or anti-C rodentium IgM and IgA titers in IL-22'⁷' mice (Lig. 10A and data not shown). Lurther IgG subtype analysis revealed that while there was no anti-C rodentium IgGl in either wildtype or IL-22'⁷' mice 8 days post inoculation, other anti-C rodentium IgG subtypes, including IgG2a, IgG2b, IgG2c and IgG3, were all significantly reduced in IL-22'⁷' mice (Lig. 10B). It was unlikely that differences in anti-bacterial specific IgG contributed to clearance of C. rodentium from the colon at this time point, especially since IgG is not targeted to the colonic mucosal lumen, and colonic bacterial burdens in both wildtype

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-77 2016259423 17 Oct 2018 and IL-22'⁷' mice were similar (Fig. 3D). It is possible, though that circulating anti-C. rodentium IgG may be important in controlling penetration of C. rodentium through the intestinal epithelial barrier, and preventing systemic spread, since a recent study demonstrated that circulating IgG, but not secretory IgA or IgM, was required for systemic clearance of C. rodentium (C. Maaser et al., Infect.

Immun. 72, 3315 (June 1, 2004)). How IL-22 deficiency leads to a reduction in anti-bacterial IgG titers is unclear. It is unlikely that IL-22 directly acts on B cells, since the expression of IL-22R is not detectable onB cells (S. Lecart et al., Int. Immunol. 14, 1351 (November 1, 2002)). Nonetheless, reduced anti-C. rodentium IgG might be one of the factors that contribute to the defective host defense response in IL-22'⁷' mice during C. rodentium infection.

Example 7: IL-22 was indispensable for the induction of anti-microbial lectins, such as RegHip and RegHIy, from colonic epithelial cells during bacterial infection.

IL-22 treatment of colon tissues from uninfected wildtype mice ex vivo upregulated many antimicrobial proteins, including S100A8, S100A9, RegHip, RegHIy, haptoglobin, SAA3, and lactotransferrin by microarray analysis (Fig.s 4A, 19, and 20). The induction of these proteins was confirmed by real-time RT-PCR (Fig. 4B and data not shown). During C. rodentium infection, however, only S100A8, S100A9, RegHip and RegHIy were differentially expressed in IL-22'⁷' mice compared to wildtype mice (Fig. 4C). All other genes were either not induced or were not different in colons of wildtype vs. IL-22'⁷' mice (data not shown). Expression of both S100A8 and S100A9 was slightly higher in the colons of IL-22'⁷' mice than it was in wildtype colon on day 4 and day 6, suggesting that differential expression of these proteins was most likely not responsible for the increased mortality observed in IL-22'⁷' mice during C. rodentium infection. Differences were not found in the expression of defensins, proteins that are important in host defense of infected epithelium (T. Ganz, Science 286, 420 (October 15, 1999)), between wildtype and IL-22'⁷' mice (data not shown).

Interestingly, the upregulation of RegHip and RegIIΙγ observed in wild type mice was completely abolished in IL-22'⁷' mice post C. rodentium inoculation (Fig. 4C), indicating that these two proteins had potential functions in controlling C. rodentium infection. Both Regl 11 β and RegHIy belong to a family of secreted C-type lectin proteins (H. L. Cash, C. V. Whitham, L. V. Hooper, Protein Expression and Purification 48, 151 (2006)). RegHip and RegHIy expression levels increase dramatically in response to bacterial colonization as well as following other inflammatory stimuli in mice (S. A. Keilbaugh et al., Gut 54, 623 (May 1, 2005) H. Ogawa et al., Inflammatory Bowel Diseases 9, 162 (2003) H. Ogawa, K. Fukushima, I. Sasaki, S. Matsuno, Am J Physiol Gastrointest Liver Physiol 279, G492 (Sep, 2000)).

RegHip or ReglIΙγ may prevent the invasion of C. rodentium deep into the colonic crypts, as we saw no differences in bacterial burdens from the colons of IL-22'⁷' vs. wildtype mice (Fig. 3D). Alternatively, RegHip or RegHIy proteins may act as autocrine growth factors that play a role in

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-78 2016259423 17 Oct 2018 epithelial repair and/or protection in the setting of intestinal inflammation (H. Ogawa et al., Inflammatory Bowel Diseases 9, 162 (2003); S. L. Pull, J. M. Doherty, J. C. Mills, J. 1. Gordon, T. S. Stappenbeck, PNAS 102, 99 (January 4, 2005); V. Moucadel et al., EurJ Cell Biol 80, 156 (Feb, 2001)).

Example 8: Adaptive immunity is not essential for IL-22 mediated early host defense against C. rodentium infection

The above data suggested roles of IL-22 in both innate immunity and adaptive immunity. Therefore, we used recombination activating gene 2 deficient (Rag2'^/_) mice to critically examine the function of IL-22 in innate vs. adaptive immunity during C. rodentium infection. Rag2 mice gradually lost weight and eventually became moribund or died around day 30, due to their lack of B and T cells, and their consequent inability to mount anti-C. rodentium antibody responses (Fig. 13A). In contrast to p 19'^/_ or IL-22 mice, none of the Rag2 mice lost more than 10% of their body weight or died during the first two weeks of infection. Furthermore, Rag2 mice treated with anti-lL-22 mAh lost weight very rapidly (Fig. 13A), similar to WT mice treated with anti-lL-22 mAh (Fig. 2E). All Rag2 mice treated with anti-lL-22 mAh became moribund or died around day 10 (Fig. 13). These data suggest that the IL-22 pathway is still active in Rag2 mice, and that IL-22 is essential to protect mice from death during the early phase of C. rodentium infection in the absence of adaptive immunity. These data also indicate that reduction in anti-C. rodentium IgG titers was insufficient cause for the morbidity and mortality observed in IL-22 mice following C. rodentium infection, as lack of antibody production in Rag2 mice alone did not cause rapid weight loss and early death following infection.

IL-22 production in Rag2 mice was comparable with that of WT mice following C. rodentium infection (Fig. 13B). In contrast, induction of 1L-17A was significantly reduced in Rag2 mice (Fig.

13B and C). T cells and B cells, therefore, were not the sources of IL-22 in this model.

Immunohistochemical staining with an anti-lL-22 mAh (Fig. 15) detected IL-22 positive cells in the colon of WT mice infected with C. rodentium, but not in uninfected colon or colon from infected IL22'⁷' mice. IL-22 positive cells primarily co-localized with CD1 lc⁺ cell clusters in the colon of Rag2'^/_ mice (Fig. 13D), but not with F4/80, Gr-1, or DX5 positive cells (data not shown). In addition, IL-23 induced IL-22 production directly from CDllc⁺ DCs in vitro (Fig. 13E). Taken together, our data demonstrate that DCs are one of the major sources of IL-22 production during C. rodentium infection, and that IL-23 can directly promote IL-22 production from DCs.

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Example 9: RegHI plays an important role during bacterial infection.

Interestingly, the upregulation of Regl I Ιβ and Regl I Ιγ observed in wild type mice was completely abolished in IL-22'⁷' mice (Fig. 4C), as well as in pl9'⁷'mice, (Fig. 16 ) post C. rodentium inoculation. Regllip and Regllly belong to a family of secreted C-type lectin proteins. We found that other family members, including Regl, Regll, Regllla, and Regllld (Fig. 17), but not ReglV (data not shown), were also upregulated in C. rodentium infected colons, and that their induction was completely abolished in IL-22'⁷' mice. Exogenous mouse Regllly fusion protein (rmRegllly) significantly protected IL-22⁷ mice from the weight loss induced by the C. rodentium infection, and approximately 50% of rmRegllly fusion protein treated animals survived the infection, whereas 100% of control treated IL-22'⁷' mice became moribund or died (Fig. 14A). These data support the hypothesis that Reg family proteins, such as Regllly, mediate essential functions in controlling C. rodentium infection downstream of IL-22.

Finally, the presence of the IL-23/IL-22/Reg axis was also validated in a human system. Human IL-23 induces hIL-22 production from human DCs (Fig. 14B). Primary human colonic epithelial cells (Fig. 12B) and the human colonic epithelial cell lines, HT29 and HCT15, express IL-22R (Fig. 14C). In vitro, primary human colonic epithelial cells grew slowly, and gradually lost their expression of IL22R during expansion (data not shown). Therefore, we used colonic epithelial cell lines to test their response to human IL-22. IL-22 induced STAT3 activation in these colonic epithelial cell lines (Fig. 14D), and both Regllip and Regllly were significantly induced by IL-22 (Fig. 14E). Importantly, human Regllly fusion protein (rhRegllly), like rmRegllly D'usion protein, also reduced the mortality of IL-22'⁷' mice, to 40% following C. rodentium infection, versus 100% mortality in control treated IL-22'⁷' mice (Fig. 18). In conclusion, our data imply that the IL-22 pathway may play an essential role in controlling bacterial infections, particularly A/E bacterial infections, in the human GI tract.

Summary

The present inventors demonstrate herein that IL-22 plays an indispensable role in early host defense against attaching and effacing (A/E) bacterial pathogens.

The data herein indicate that IL-22 protects the integrity of the intestinal epithelial barrier and prevents bacterial invasion with systemic spread through two mechanisms. First, IL-22 is involved in the elicitation of the early anti-bacterial IgG responses. Second, IL-22 is indispensable for the induction of anti-microbial lectins, such as Regllip and ReglIΙγ, from colonic epithelial cells during bacterial infection. The lack of either or both of these mechanisms may contribute to the compromised host defense response with increased systemic spread and mortality in IL-22'⁷' mice during C. rodentium infection.

While adaptive immune responses are essential for clearance of these pathogens (L. Bry, Μ. B.

Brenner, J Immunol 172, 433 (January 1, 2004)), cytokines such as IL-22 that are produced by

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-802016259423 17 Oct 2018 immune cells during the early stages of infection are also necessary for intestinal epithelial cells to elicit a full anti-microbial response and wound healing response in order to prevent systemic invasion of pathogenic bacteria into the host. As shown herein, the induction of Pcgl I Ιβ and Regllly also indicates that IL-22 may have broader functions in controlling various bacterial infections. The data further supports the role of Thl7 cells and their effector cytokines in infectious diseases and autoimmune diseases. Finally, the present studies indicate that IL-22 and its downstream products, such as RegIΠβ and Regllly, may be beneficial for the treatment of certain infectious diseases.

Materials and Methods 10 Mice

C57B1/6, IL-12p40'^z, and IL-6 ' mice were purchased from the Jackson Laboratory. IL-22' mice and IL-12pl9^z were generated as described before (11, Fig.5). IL-17RC'^z'and Ιί-20Κβ 'mice were generated by Lexicon Pharmaceuticals (The Woodlands, TX) by using strategies as described (Fig.5 and Fig.8). Briefly, knockout mice were made by standard homologous recombination using depicted targeting vectors. Targeting vectors are electroporated into 129 strain ES cells and targeted clones are identified. Targeted clones are microinjected into host blastocysts to produce chimeras. Chimeras are bred with C57B1/6 animals to produce FI heterozygotes. Heterozygotes are intercrossed to produce F2 wild type, heterozygote and homozygote cohorts. Mice used in these studies were genotyped by tail DNA via PCR using a pool of three primers. The primers used for wild-type allele amplification of IL-20Rβ-^z- mice were 5’-GTG GAA GCT ACT TGA TGA GTA GGG-3’ (pi) and 5’-AGA TGC GAA AAT GGA GAT TAA AAG-3’ (p2), which yielded a 595 bp product. The primers used for mutant allele amplification of IL-20Rβ'^z mice were 5’-CTA CCC GTG ATA TTG CTG AAG AG-3’ (p3) and p2, which yielded a 351 bp product. The primers used for wild-type allele amplification of IL-17RC'^Z' mice were 5’-GAG CCT GAA GAA GCT GGA AA-3’ (P3) and 5’-CAA

GTG TTG GCA GAG ATG GA-3’ (P2), which yielded a 534 bp product. The primers used for mutant allele amplification of IL-17RC'^Z- mice were 5’-TCG CCT TCT TGA CGA GTT CT-3’ (Pl) and P2, which yielded a 404 bp product.

Bacteria strain and infection of mice

6-8 weeks old mice were fasted for 8h before oral inoculation with 2x10⁹ C. rodentium strain

DBS 100 (ATCC 51459; American Type Culture Collection) in a total volume of 200 pi per mouse. While fasting, animals had access to water. Inoculation and all subsequent manipulations were conducted in BL-2 biosafety cabinets. Animals were allowed access to food after inoculation. Bacteria were prepared by incubation with shaking at 37°C overnight in LB broth. The relative concentration of bacteria was assessed by measuring absorbance at OD600 and each inoculation culture was serially diluted and plated to confirm CFU administered.

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Tissue collection, histology and CFU counts

Control or infected mice were inoculated as described. Samples of whole blood, spleen, liver, mesenteric lymph node, and colon were removed under aseptic conditions. The colon was dissected to the anal canal, and the terminal 0.5-cm piece was used for CFU analysis. Proximal segments were fixed in 10% neutral buffered formalin. Sections were stained with H&E to evaluate tissue pathology. Spleen, liver, mesenteric lymph node, and colon were weighed and homogenized. Homogenates were serially diluted and plated in triplicates to MacConkey agar (Remel). C. rodentium colonies were identified as pink colonies. Colonies were counted after 24 h of incubation at 37°C to determine the logio CFU per gram of tissues.

RNA isolation and real-time RT-PCR

Cell and tissue RNA were isolated by RNeasy Mini Kit (Qiagen) according to the manufacture’s directions. Real-time RT-PCR was conducted on an ABI 7500 Real-Time PCR system (Applied Biosystems) with primers and probes using TaqMan one-step RT-PCR master mix reagents (Applied Biosystems). The sequences for primers and probes were as follows: mIL-22, forward, 5’TCC GAG GAG TCA GTG CTA AA-3’, reverse, 5’-AGA ACG TCT TCC AGG GTG AA-3’, and probe, 5’-TGA GCA CCT GCT TCA TCA GGT AGC A-3’ (FAM, TAMRA); mIL-17A, forward, 5’GCT CCA GAA GGC CCT CAG A-3’, reverse, 5’-CTT TCC CTC CGC ATT GAC A-3’, and probe,

5’- ACC TCA ACC GTT CCA CGT CAC-3’ (FAM, TAMRA); mouse ribosomal housekeeping gene

RPL-19, forward, 5’-GCA TCC TCA TGG AGC ACA T-3’, reverse, 5’-CTG GTC AGC CAG GAG CTT-3’, and probe, 5’-CTT GCG GGC CTT GTC TGC CTT-3’ (FAM, TAMRA); mIL-19, forward,

5’-AGC CTG GAT TGA CAG GAA TC-3’, reverse, 5’-GAT AAT CAG ACG AGG CGT TTC-3’, and probe, 5’-TCT GGA AAC TCC TGC AGC CTG ACA C-3’ (FAM, TAMRA); mIL-20, forward,

5 ’ -TTT GGG AGA ACT AGG CAT TCT T-3 ’, reverse, 5 ’ -TCT TGG ACA GGA GTG TTC TCA3’, and probe, 5’-CAG CCT CTC CAC TTT CAT CTA TAG CAT CTC C-3’ (FAM,TAMRA); mlL24, forward, 5’-GCT CTC CAT GCC ATT TCA A-3’, reverse, 5’-TGG CCA AGG GTC TGA AGT3’, and probe, 5’-TGT ACA TCC CTG CTG TCC TCA AGG C-3’ (FAM, TAMRA); mIL-6, forward, 5’-TCC AAT GCT CTC CTA ACA GAT AAG-3’, reverse, 5’-CAA GAT GAA TTG GAT

GGT CTT G-3 ’, and probe, 5 ’ -TCC TTA GCC ACT CCT TCT GTG ACT CCA-3 ’ (FAM,

TAMRA); mS100A8, forward, 5’-TGT CCT CAG TTT GTG CAG AAT ATA AA-3’, reverse, 5’TCA CCA TCG CAA GGA ACT CC-3’, and probe 5’-CGA AAA CTT GTT CAG AGA ATT GGA CAT CAA TAG TGA-3’ (FAM, TAMRA); mS100A9, forward, 5’-GGT GGA AGC ACA GTT GGC A-3’, reverse, 5’-GTG TCC AGG TCC TCC ATG ATG-3’, and probe, 5’-TGA AGA AAG

AGA AGA GAA ATG AAG CCC TCA TAA ATG-3 ’ (FAM, TAMRA); mRegllly, forward, 5’-ATG

GCT CCT ATT GCT ATG CC-3’, reverse, 5’-GAT GTC CTG AGG GCC TCT T-3’, and probe, 5’10751098_1 (GHMatters) P84050.AU.2 17-Oct-18

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TGG CAG GCC ATA TCT GCA TCA TAC C-3’ (FAM, TAMRA); mPAP/HIP/Reglllp, forward,

5’- ATG GCT CCT ACT GCT ATG CC-3’, reverse, 5’- GTG TCC TCC AGG CCT CTT T-3’, and probe, 5’-TGA TGC AGA ACT GGC CTG CCA-3’ (FAM, TAMRA); mIL-12p40, forward, 5’-ACA TCT ACC GAA GTC CAA TGC A-3’, reverse, 5’-GGA ATT GTA ATA GCG ATC CTG AGC-3’, and probe, 5’-TGC ACG CAG ACA TTC CCG CCT-3’ (FAM, TAMRA); mIL-23pl9, forward, 5’GGT GGC TCA GGG AAA TGT-3’, reverse, 5’-GAC AGA GCA GGC AGG TAC AG-3’, and probe, 5’-CAG ATG CAC AGT ACT CCA GAC AGC AGC-3’ (FAM, TAMRA); mlL-20Rp, forward, 5’-CAG GTG CTT CCA GTC CGT CT-3’, reverse, 5’-CTC TCC TGG AAT CCC CAA AGT-3’, and probe, 5’-CAG CAC AGA TGC CAA CGG CCT CAT-3’ (FAM, TAMRA); mlL10 20Ra, forward, 5’-CTG GCC GCT TCG GGA CGC-3’, reverse, 5’-AAC CAC AGA AGA CAC

AAG GAA CTG-3’, and probe, 5’-TCT GCT GCT GGC CGC TTC GG-3’ (FAM, TAMRA); mlL22R, forward, 5’-GCT GGA CTC CCT TGT GTG T-3’, reverse, 5’-CAC ATG GCC TCA GTC TCA A-3’, and probe, 5’-CGC GGG ACC CTC ATC CTT TG-3’ (FAM, TAMRA); mlL-lORp, forward,

5’-TCC ACA GCA CCT GAA GGA GTT-3’, reverse, 5’-GGA GGG AAG GAG AAC AGC AGA15 3’, and probe, 5’-TGG GCC ACC CCC ATC ACA GC-3’ (FAM, TAMRA). Reactions were run in duplicates and samples were normalized to the control housekeeping gene RPL-19 and reported according to the AACt method: AACt = JCt_sampie - MCtreference.

Ig ELISA

Analyses were performed on serum from collected whole blood as previously described (10).

Briefly, ELISA plates (Nunc) were coated with heat-killed C. rodentium or with a goat anti-mouse lg capture Ab diluted 1/1000 in PBS (SouthemBiotech). Coated plates were washed in PBS plus 0.05% Tween20, blocked for 1 h with 300 μΐ of blocking buffer (PBS + 0.5 % BSA + 10 PPM Proclin), and washed before addition of serially diluted standards (mouse monoclonal IgA, IgG, lgG3, and IgM from SouthemBiotech; IgGl, lgG2a, and lgG2b isotypes from Sigma-Aldrich; mouse lgG2c obtained from Bethyl Laboratories) or unknowns. Samples were incubated for 4 hours at room temperature. Plates were washed five times and the lg isotypes were detected with goat anti-mouse IgA, IgM, IgG, IgGl, lgG2a, lgG2b, lgG2c, and lgG3 (SouthemBiotech) conjugated to horseradish peroxidase (HRP), diluted 1/4,000 in assay diluent (PBS + 0.5 % BSA + 0.05 % Tween 20+10 PPM Proclin, pH

7.4), and incubated for 1 hour at room temperature. After washing, TMB peroxidase substrate was added to each well and allowed to develop for 15 minutes, then stop solution (1 M Phosphoric acid) were added to each well. Absorbance was read at 450 nm in a Molecular Devices (Sunnyvale, CA) plate reader atOD450.

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In vitro colon culture

Colons were removed from C57B1/6 mice. After cleaning with cold PBS, colons were cut longitudinally. Colons were placed in a 100mm Petri dish with 10ml HBSS (Mediatech) buffer containing 2.5 pg/ml of Fungizone-Amphotericin B, 10 pg/ml Gentamicin, 100 U/ml Penicillin and

100 pg/ml Streptomycin (all from G1BCO, Invitrogen). Colons were gently scraped to remove mucus at the edge of the Petri dish and were transferred to a new Petri dish with fresh HBSS buffer. Colons were cut into 1-2 mm pieces and transferred to a 24-well plate with 50mg colons/lml/well in RPM1 buffer containing 10% heat inactivated FCS (HyClone), 2.5 pg/ml of Fungizone-Amphotericin B, 10 pg/ml Gentamicin, 2 mM L-Glutamine, 100 U/ml Penicillin and 100 pg/ml Streptomycin. 10 pg/ml of IL-22 (R & D systems) were added to the culture and incubated in 37 °C for 24 hours.

Microarray analysis

Quantity and quality of total RNA samples was determined using an ND-1000 spectrophotometer (Nanodrop Technologies) and Bioanalyzer 2100 (Agilent Technologies), respectively. The method for preparation of Cy-dye labeled cRNA and array hybridization was provided by Agilent Technologies. Briefly, total RNA sample was converted to double-stranded cDNA and then to Cy-dye labeled cRNA using Agilent’s Low RNA Input Fluorescent Linear Amplification Kit. The labeled cRNA was purified using RNeasy mini kit (Qiagen). cRNA yield and Cy-dye incorporation was determined using ND-1000 spectrophotometer. 750 ng of the labeled cRNA was fragmented and hybridized to the Agilent’s Whole Mouse Genome array as described in manufacturer’s In situ Hybridization kit-plus. All samples were labeled with Cy5 and hybridized against Cy3 labeled universal mouse reference (Stratagene). Following hybridization, the arrays were washed, dried and scanned on Agilent’s DNA microarray scanner. Agilent’s Feature Extraction software 8.5 was used to analyze acquired array images. For microarray data clustering (Fig. 20), expression data was processed to Agilent log-ratio data by standard methods. Selected genes were clustered by iterative agglomeration of vectors most highly linked by Pearson correlation coefficient, with data for agglomerated vectors summarized by average linkage.

In vitro mouse tail tip fibroblast culture and stimulation

To establish tail tip fibroblasts (TTFs), the tails from 1L-17RC'' adult mice and wild type littermates were peeled, minced into 1 cm pieces, placed on culture dishes, and incubated in high glucose DMEM (containing 10% FCS, 2 nm glutamine, 100 U/ml Penicillin and 100 pg/ml Streptomycin) for 5 days. Cells that migrated out of the graft pieces were transferred to new plates (passage 2) and maintained in the same media. We used TTFs at passage 3 for stimulation experiments. TTFs were seeded into 24-well plate at a density of 1.2xl0⁵ per well. Twenty four hours after seeding, recombinant murine 1L-17A and 1L-17F (R&D Systems) were added to the culture

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-842016259423 17 Oct 2018 medium at various concentrations. Cell culture supernatant was harvested 24 hours after addition of cytokines and levels of murine IL-6 was measured by enzyme linked immunosorbent assay (ELISA) by mouse IL-6 ELISA set (BD Biosciences) following manufacturer’s instructions.

Blockade of murine IL-22 in vivo

Blocking anti-mouse IL-22 (Clone 8E11, isotype mouse IgGl) mAh (//) was intraperitoneally injected before (Day 0) or 8 days after (Day 8) C. rodentium infection at a dose of 150 pg/mouse every other day. Certain control group also received isotype control IgGl mAh.

Statistics

Statistical significance was calculated by one -way or two-way ANOVA using Prism software (GraphPad). Allp values < 0.05 are considered significant, and are indicated in the text. Unless otherwise specified, all studies for which data are presented are representative of at least two independent experiments.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the descriptions and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literatures cited herein are expressly incorporated in their entirety by reference.

Example 10: The LT pathway is mediated by IL-22 during Citrobacer rodentium infection.

To help determine if IL-22 is important for the mortality caused by LT blockade, we performed a rescue experiment in which we expressed IL-22 in the mouse at the same time of LTbR-Fc treatment. The method we used for IL-22 expression was hydrodynamic tail vein delivery of plasmid DNA encoding mouse IL-22. Human LTbR-Ig was constructed as follows: human LTbR encompassing the extracellular domain (position 1 through position 224; SEQ ID NO:57) was cloned into a modified pRK5 expression vector encoding the human IgGl Fc region (SEQ ID NO:58) downstream of the LTbR sequence. Proteins were overexpressed in CHO cells and purified by protein A affinity chromatography. Murine LTbR.Ig was constructed as follows: murine LTbR encompassing the extracellular domain (position 1 through position 222; SEQ ID NO:59) was cloned into a modified pRK5 expression vector encoding the murine IgG2a Fc region (SEQ ID NO:60) downstream of the LTbR sequence.

In Figure 21, we find that LTbR-Fc produces a similar weight loss curve (Figure 21 right panel) and death curve (Figure 21 left panel) to IL-22 blockade which led us to examine the relationship between LT and IL-22. C. rodetium infection leads to early expression of IL-22 in the colon.

Fig. 21 shows the percent survival of mice inoculated with Citrobacter rodentium. 6-8 week old Balb/c mice were fasted for 8h before oral inoculation with 2x109 C. rodentium strain DBS 100

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-85 2016259423 17 Oct 2018 (ATCC 51459; American Type Culture Collection) in a total volume of 200 μΐ per mouse. While fasting, animals had access to water. Inoculation and all subsequent manipulations were conducted in BL-2 biosafety cabinets. Animals were allowed access to food after inoculation. Bacteria were prepared by incubation with shaking at 37°C overnight in LB broth. The relative concentration of bacteria was assessed by measuring absorbance at OD600 and each inoculation culture was serially diluted and plated to confirm CFU administered. On the day of inoculation, mice were also injected with 150ug of anti-gpl20 mAh, anti-lL-22 8E11 mAh, or LTbR-Fc 3 times per week.

LT pathway regulate multiple upstream aspects that important for IL-22 production. Figure 22 provides data on the LT pathway after infection with C. rodentium. A, C, E. Colons were harvested at different timepoints after infection with C. rodentium. Mice were injected with 150ug anti-gpl20 or LTbR-Fc every other day. RNA was extracted using Qiagen RNeasy Kit. Taqman analysis was performed to determine expression of IL-22, Regllg, p 19, or p40 relative to the day 0 timepoint. B. On day 4 after infection, colons were collected. After cleaning with cold PBS, colons were cut longitudinally. Colons were placed in a 100 mm Petri dish with 10 ml HBSS (Mediatech) buffer containing 2.5 pg/ml of Fungizone-Amphotericin B, 10 pg/ml Gentamicin, 100 U/ml Penicillin and 100 pg/ml Streptomycin (all from G1BCO, Invitrogen). Colons were gently scraped to remove mucus at the edge of the Petri dish and were transferred to a new Petri dish with fresh HBSS buffer. Colons were cut into 1-2 mm pieces and transferred to a 24-well plate with 50 mg colons/1 ml/well in RPM1 buffer containing 10 % heat inactivated FCS (HyClone), 2.5 pg/ml of Fungizone-Amphotericin

B, 10 pg/ml Gentamicin, 2 mM L-Glutamine, 100 U/ml Penicillin and 100 pg/ml Streptomycin. 10 ng/ml of rmlL-22 (R & D systems) were added to the culture and incubated in 37°C for 24 hours. Supernatants were collected for an IL-22 ELISA. D. Day 6 colon lamina propria cells. DC determined by CD1 lc+ and MHC II+. Colons were harvested and flushed with HBSS without calcium and magnesium (Invitrogen) with 2% FBS and lOmM HEPES. Colons were cut longitudinally, and then sectioned into 2-4 cm pieces, and the pieces were transferred to a 10 cm dish with HBSS without calcium and magnesium, 2% FBS, ImM EDTA, lOmM HEPES, and ImM DTT (Sigma-Aldrich). IEL fractions were collected and discarded after a 45 minute incubation at 37°C while shaking at 200 rpm. For LPMCs isolation, the remaining epithelial layer was peeled off and the colon pieces were diced and placed into RPMI containing 10 % FCS, 20mM HEPES, and 0.5 mg/ml collagenase/dispase (Roche Diagnostics). Colon pieces were incubated for one hour at 37°C while shaking. Isolated epithelial cells were washed and used for FACS analysis.

In Figure 22, we find that LTbR-Fc blocked the induction of IL-22 as well as Reglllg which has been shown to be induced by IL-22 (Figure 22A-C). Dendritic cells have previously been shown to produce IL-22 and we find a slight reduction of DC numbers in the lamina propria of the colon 6 days after infection (Figure 22D). The decrease in IL-22 caused by LTbR-Fc is most likely due the loss of

10751098_1 (GHMatters) P84050.AU.2 17-Oct-18

-862016259423 17 Oct 2018

1L-23, since both p 19 and p40 expression is inhibited after LTbR-Fc treatment during infection (Figure 22E).

IL-22 partially rescues the defects seen in LTbR treated mice. Figure 23 provides data concerning the effect of IL-22 on LTbR treated mice. A. Test of expression of IL-22 in serum and

Reglllg in colon after tail vein injection of IL-22 plasmid. B. Rescue of LTbREc effects with IL-22 plasmid.

On day-1, animals were weighed and grouped, extra mice were euthanized. After weighing, all animals were fasted 14 h. The next morning (day 0), all mice were orally inoculated with 2-4 X 10e9 CFU of C. rodentium in 200 ul PBS. 150ug control mAh or Fc fusion protein was injected i.p. in

200ul PBS three times per week for two weeks starting on the same day as bacteria inoculation. Food was replaced back by investigators after inoculation. Six hours later plasmid DNA was injected by tail vein. Tail vein injection experiments: 1) DNA construct (pRK vector or pRK-mIL-22) was diluted in Ringer's to a concentration to yield a final dose of 10 micrograms/mouse/injection. 2) Each mouse was injected intravenously in the tail vein with approximately 1.6ml of the solution containing DNA in Ringer's. 3) Doses were administered as a bolus intravenous injection (tail vein) over a period of 45 seconds (8 seconds maximum) for maximum DNA uptake. Mice were restrained without anesthesia in a conical acrylic restrainer with a heating element to increase body temperature and dilate blood vessels. 4) Disposable sterile syringes were used for each animal. Animals were continuously monitored until they are clinically normal. 5) Animals were observed for any adverse clinical signs for at least 20 minutes post dose. If animals were not clinically normal by 1 hour post dose, they were euthanized or they were monitored until they were clinically normal. Moribund animals were euthanized. All manipulations were performed in BL-2 biosafety cabinets. During infection, moribund animals or those showing unalleviated distress or rectal prolapse were euthanized.

The mice were monitored for 4 weeks everyday. Between day 5 to day 17 when LTbR-Fc treated mice might become moribund, the mice were monitored twice per day including weekends. Fecal pellets were collected every week to measure CFU of C. rodentium. Mice were weighed once per week during the study. If mice exhibited a weight loss of 15% or more, they were weighed daily. If the weight loss exceeds 20%, the mice were euthanized. At the end of the study, all mice were euthanized and spleen, and colon were collected for histology, RNA or FACS analysis.

As shown in Figure 23 A, we can detect expression of IL-22 in the mouse serum beginning at 2 hours post-injection, with expression declining at 72 hours. We can also detect expression of Reglllg in the colon, suggesting that active IL-22 can act on the colon when expressed in this manner. IL-22 could partially rescue mortality and weight loss induced by LTb-Fc treatment during infection (Figure 23B).

Treatment of mice with IL-22 mAh (8E11). Figure 24 shows data demonstrating that treatment with IL-22 mAh 8E11 leads to reduced colon follicles, compromised B/T cell organization,

10751098_1 (GHMatters) P84050.AU.2 17-Oct-18

-872016259423 17 Oct 2018 and reduced DC, T cell, and B cell numbers in the colon. A. Six days after infection, colons were harvested and cut longitudinally. After a 30 minute incubation in HBSS without calcium and magnesium, 2% FBS, ImM EDTA, lOmM HEPES, and ImM DTT, colons were gently scraped to remove epithelial cells. Follicles were identified as white, round masses. There were five mice per group and each colon was counted and plotted as total follicles found or total follicles greater than 1mm found. B. Six days after infection, colons were flushed with cold PBS and quick frozen in OCT. Six micron sections were cut, dried, then fixed in acetone. Sections were blocked with 10% serum, the incubated with anti-CD5 FITC and anti-B220 APC at lOug/ml each. Images were capture on a NIKON BX61 microscope. C. Six days after infection colon lamina propria cells were isolated at described above. FAC analysis was performed to determine the number of dendritic cells, CD3 T cells, and B cells after 8E11 treatment.

As shown in Figure 24, we treated mice with either IL-22 blocking antibody or LTbR-Fc and counted lymphoid follicles in the colon after six days post infection in order to determine if IL-22 could have a role in formation of colon lymphoid structures. We found a decrease in follicles greater than 1mm, suggesting IL-22 and LT could be important for the increase in follicle size after infection (Figure 24A). Histological analysis shows that blocking IL-22 disrupted the T and B cell zones of the follicle while LT blockade had a similar effect (Figure 24B). We next determined whether blockade of IL-22 leads to a change in cell numbers in the colon lamina propria. We found that IL-22 blockade led to decreases in DC, T cell, and B cell numbers during infection. In conclusion, IL-22 appears to be important for lymphoid follicle formation and may be an important downstream component of the lymphotoxin pathway in the colon.

10751098_1 (GHMatters) P84050.AU.2 17-Oct-18

2016259423 17 Oct 2018

Claims (28)

1. THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

   1. Use of an anti-microbial polypeptide (AMP) comprising REG III in the manufacture of a medicament for treating a microbial disorder in a subject, wherein said microbial disorder is

   5 inflammatory bowel disease (IBD), and wherein the REG III is human REG ΙΙΙβ and/or human REG ΙΙΙγ.
2. 2. A method of treating a microbial disorder in a subject, comprising administering to said subject an effective amount of an anti-microbial polypeptide (AMP) comprising REG III, wherein said

   10 microbial disorder is inflammatory bowel disease (IBD), and wherein the REG 111 is human REG ΙΙΙβ and/or human REG ΙΙΙγ.
3. 3. A use or method according to claim 1 or 2, wherein said IBD is Crohn’s disease or ulcerative colitis (UC).
4. 4. A use or method according to any one of claims 1 to 3, wherein said AMP comprises a sequence selected from a group of amino acid sequences consisting of: SEQ ID NO: 22, SEQ ID NO: 24, or the mature form of the AMP thereof.

   10751098_1 (GHMatters) P84050.AU.2 17-Oct-18

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   2016259423 18 Nov 2016

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    Probeset:

    Fifl IQA A„51_P256827 HJ· A_52_P700056

    A_51_P402943

    A_51_P235945

    A_51_P169671

    A_52_P472324

    A_52_P329207

    A_52_P295432

    A_52_P321831

    A_51_P279437

    A_51_P246166

    A_52_P230938

    A_51_P384894

    A_51_P156955

    A_51_P303160

    A_51_P461703

    A_51_P374476

    A_51_P481958

    A_51_P464703

    A_51_P132185

    A_51„P496795

    A„51„P2961O9

    A_51_P228768

    A_51_P466535

    A_51_P387239

    A_51_P337308

    A_51_P346938

    A_51_P504546

    A_51_P386870

    A_51„P216456

    A_51_P212491

    A_52_P48681

    A_52_P424959

    A_52_P232813

    A_51_P181891

    A_51_P249989

    A_52_P487686

    A_52_P168962

    A_51_P485312

    A_52_P99353

    A_52_P252931

    A_52_P278538

    A_52_P85132

    A_52„P407198

    A_52_P638459

    A_51„P335559

    A_51_P240801

    A_52_P627816

    A_52_P65686O

    A_51J>180314

    A_51_P328870

    A_51_P482908

    A_51_P257951

    A_51_P441426

    P-value Log2 FC ID

    0 5.674 NM_013650

    0.001 3.722 BC089618

    0.001 3.637 NM_009114

    0.007 2.668 NM_Q17370

    0.042 2.523 NM_011036

    0.012 2.351 NM„011414

    0.006 2.262 NM_007969

    0.015 2.111 NM_009141

    0.007 2.062 XM_894811

    0.013 2.032 NM_029662

    0.01 1.983 NM_007969

    0.008 1.966 NM_010741

    0.019 1.837 NMJ301001332

    0.045 1.837 NM_013459

    0.03 1.821 NM_007482

    0.034 1.804 NM_031188

    0.019 1.742 NM_008220

    0.012 1.735 NM_016674

    0.019 1.727 NM_021443

    0.031 1.724 NM_001001332

    0.019 1,669 NM_145555

    0.01 1.63 NM_028618

    0.043 1.591 NM_011409

    0.014 1.571 AK172918

    0.04 1.569 NM_021792

    0.03 1.547 NMJ311315

    0.031 1.541 NM_029796

    0.047 1.536 NM_027406

    0.026 1.53 NM_011472

    0.024 1.522 NM„009311

    0.025 1.517 NM_133232

    0.018 1.514 NM_016674

    0.038 1.498 NM .153457

    0.047 1.484 NM_203320

    0.011 1,458 NM_028113

    0.014 1.455 NM_145133

    0.042 1.405 NM 001001332

    0.023 1.387 NM_007646

    0.026 1.372 NM_013653

    0.019 1.37 AK009488

    0.037 1.358 NM_013505

    0.039 1.349 NM_008218

    0.034 1.331 NM_172440

    0.023 1.322 NM_183153

    0.03 1.317 BC033508

    0,019 1.311 AK034993

    0,018 1.308 BC046640

    0.032 1,292 NM_019984

    0.01 1.273 NM_026232

    0.045 1.258 AK011413

    0.011 1.249 D86599

    0.041 1.237 NM_025549

    0.041 1.235 NM_020509

    0.029 1.234 NM_O19932

    Symbol

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    Aldhlll

    Sprr2f

    Tael

    Pfkfb3

    Cidnl

    Rtnl

    Gml960

    2600011E07R.|k

    T2bp

    Stfal

    Cd38

    Ccl5

    Dsc2

    Hba-al

    Stxbp5l

    A430060F13Rik

    CcIS

    2610307008Rik

    Tgml

    Slc25a30

    Mupl

    Oxtr

    Arrdc4

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    Cxcl4
22. 22/28

    2016259423 18 Nov 2016

    Fig. 19B

    Probeset P-value Log2 FC ID Symbol A_51„P128463 0.049 1.232 XM_485455 Grrpl A_51_P239750 0.008 1.196 NM_008380 Inhba A_51_P413866 0.044 1.188 NM_008198 Cfb A_51_P462192 0.037 1.18 NM_138648 Olrl A_51_P291417 0.026 1.172 NM_009378 Thbd A_51_P459176 0.013 1.162 XM_899837 1700095J03Rik A„51_P122321 0.012 1.161 NM_133775 9230117N10Rik A_51_P110301 0.023 1.158 NM_009778 C3 A_51_P294807 0.039 1.154 NM_013515 Stom A_51_P504991 0.027 1.151 NM„181542 SlfnlO A_51_P440743 0.041 1.148 NM_009886 Celsrl A_51_P141012 0.019 1.137 AK173296 Slitrkl A_51_P445726 0.043 1.128 NM_011095 Lilrb3 A_51_P397968 0.03 1.087 AK087113 A_51_P464408 0.034 1.078 NM_177314 5330439B14Rik A_52_P597634 0.041 1.077 NM_021457 Fzdl A_51_P149714 0.038 1.066 NM_026835 Ms4a6d A_52_P409833 0,027 1.04 NM_008872 Plat A_51_P172853 0.03 1.04 NM_009841 Cdl4 A_52_P2616l 0.026 1.039 NM_008987 Ptx3 A_52_.P164821 0.034 1,038 XM_905096 LOC631323 A_52_P987411 0.036 1.038 AK032717 A_51_P370552 0.037 1.037 NM_030061 9230117E20Rik A_52_P616392 0.017 1.032 NM_183426 Stno A_51_P181899 0.027 1.021 NM_028113 2600011E07Rik A_52_P104075 0.037 1.021 AK052629 Freml A_52_P621991 0.048 1.019 NM_009684 Apafl A_52_P616949 0.036 1.014 NM_027893 Pvrl4 A_51_P5O4988 0.022 1.01 NM_172796 Slfn9 A_52_P87713 0.031 1.008 NM„011593 Timpl A_52_P1012710 0.034 1.005 AK075639 A„52_P491718 0.049 -1.002 AK047841 Mbtps2 A_51_P191331 0.015 -1.003 NM.011379 Sipal A_52_P680941 0.028 -1.003 NM 010284 Ghr A_52_P1027837 0.038 -1.003 AK034309 9330175M20Rik A„52_P787453 0.021 -1.019 AK045835 A_52_P484194 0.025 -1.023 NM_001025602 Illrll A_52_P524426 0.03 -1.023 NM_001003815 Epb4,lll A_52_P828218 0.027 -1.027 NM_001030294 Olfm4 A_52_P674087 0.011 -1,028 AK036172 Prtg A_52_P826055 0,032 -1.028 AK134458 2310020J12Rik A_52_P563505 0.022 -1.033 AK007074 170009 5A21RIk A_52_P44454 0.015 -1.034 P052591-1 A„51_P398037 0.018 -1.039 AK048407 Scn3a A„52_P1132297 0.017 -1.058 AK052006 A_52_P35598 0.034 -1.064 AK051891 D230015P20Rik A_51_P151983 0.009 -1.066 NM_019744 Ncoa4 A_51_P130719 0.014 -1.066 NM_023047 Dpysl5 A_52_P218833 0.018 -1.066 ENSMUSTOOOOOO' A_52_P1084199 0.022 -1.078 AK036139 9630039A02Rik A_51_P138496 0,021 -1.08 AK046037 A_52_P1124261 0.046 -1.086 AK081465 A_52„P158438 0.023 -1.094 AK006669 1700041C23RIk A_52_P187046 0.022 -1.098 NM_022724 Suv39h2
23. 23/28

    2016259423 18 Nov 2016

    Fig. 19C

    Probeset P-value Log2 FC ID Symbol A_52_P344098 0.027 -1.098 AK040876 Pde7b A_51_P314669 0.034 -1.1 NM_011128 Pnliprp2 A 52 P475356 0.02 -1.107 NM_011445 Sox6 A 52.P177454 0.043 -1.114 AK045474 Rab34 A.52 P972196 0.015 -1.12 AK084612 A_52_P1140731 0.024 -1.122 AK089673 A_51_P249302 0.017 -1.125 NM_011994 Abed 2 A_52_P866925 0.031 -1.133 AK006067 1700017I07Rik A_52„P956057 0.026 -1.17 AK081935 A_52_P939063 0.012 -1.171 AK032576 A_51„P243750 0.037 -1.175 NM_012025 Racgapl A_52_P205710 0.047 -1.189 NM_021611 Mylc2pl A_52_P1188576 0.017 -1.197 AK082439 A_51_P282523 0.032 -1.201 NM_054044 Gprl24 A_52„P642801 0.009 -1,202 XM_618738 Latsl A_51_P312615 0.038 -1.207 NM_010377 Histlhlt A_52_P214828 0.022 -1.209 NM_011253 Rbmylal A„52_P158660 0.024 -1.231 XM_001002308 4833417AllRik A_51_P104255 0.012 -1.237 NM_001039226 LOC625026 A_52_P811599 0.035 -1.244 AK084144 A_52_P668855 0.016 -1.246 AK042778 9330154J02Rik A_51...P399974 0.035 -1.246 NM_146997 Olfrl78 A__52_P355709 0.007 -1.247 XM„973329 LOC664837 A_52 .P207356 0.048 -1.249 AK052515 Aspm A_52_P1188835 0.034 -1.256 NM_001002780 F830005K03Rik A_51_P407915 0.013 -1.263 AK040806 A530026G17 A_51_P352743 0.045 -1.285 NM_0010l3616 Trim6 A_51_P191139 0.012 -1.293 AK050699 D030005H02Rik A_52_P1139473 0.007 -1.294 AK018751 0610037L18Rik A_52_P69506 0.042 -1.294 NM_177566 ArhgeflS A_52_P134003 0.042 -1.327 NM_001033289 Slc9a2 A_52_P519943 0.004 -1.337 BC044804 Fads6 A_51_P366413 0.009 -1.394 AK033346 8030498J20Rlk A_52_P988314 0.029 -1.401 AK083509 A_52_P778486 0.029 -1.404 AK016521 4932412D23Rik A_52_P162486 0.009 -1.409 NM_007542 Bgn A_51_P242952 0,032 -1.411 AK079868 A_52_P267243 0.039 -1.413 NM_010200 Fgfl3 A_52_P328714 0.018 -1.422 AK133758 Fbxw2 A~51_P238040 0.018 -1.424 AK030263 A_51_P327166 0.006 -1.426 AK015801 A_51_P201035 0.027 -1.443 NM_145924 Cenpi A_52_P169595 0.034 -1.455 NM_177669 A630098G03Rik A_52_P507877 0.04 -1.463 NM_007729 Colllal A_52_P224818 0.012 -1,468 NM_172483 2fpl80 A_51_P270050 0.041 -1.485 AK078370 6530421E24Rik A_51_P309099 0.02 -1.489 AK050657 A 51_P103261 0.034 -1.533 NM_009198 Slcl7al A_52_P535430 0.021 -1.561 NM_207130 Abcal6 A_52_P1O83387 0.015 -1.585 AK005856 1700011B04Rik A_51_P152873 0.014 -1.71 NM_207670 Gripapl A_52_P569873 0.012 -1,778 XM_127913 4930455B06Rik A_51_P396119 0.008 -2.489 AK047973 Ttll5
24. 24/28

    2016259423 18 Nov 2016

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    2016259423 18 Nov 2016

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    2016259423 18 Nov 2016
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    Fig. 24

    Isotype 8E11

    2016259423 18 Nov 2016

    2265275_1 Sequence Listing <110> Genentech, Inc.

    Abbas, Alexander R.

    Danilenko, Dimitry M. de Sauvage, Frederic J.

    Ghilardi, Nico P.

    Modrusan, Zora Ouyang, Wenjun Valdez, Patricia A.

    Zheng, Yan <120> Compositions and Methods for Treatment of Microbial Disorders <130> GNE-0303PCT <140> Not yet Assigned <141> Herewith <150> 60/986,170 <151> 2007-11-07 <150> 61/013,620 <151> 2007-12-13 <150> 61/015,620 <151> 2007-12-20 <160> 61 <210> 1 <211> 1131 <212> DNA <213> Homo sapiens <400> 1

    cattctgccc tcgagcccac cgggaacgaa agagaagctc tatctcccct 50 ccaggagccc agctatgaac tccttctcca caagcgcctt cggtccagtt 100 gccttctccc tggggctgct cctggtgttg cctgctgcct tccctgcccc 150 agtaccccca ggagaagatt ccaaagatgt agccgcccca cacagacagc 200 cactcacctc ttcagaacga attgacaaac aaattcggta catcctcgac 250 ggcatctcag ccctgagaaa ggagacatgt aacaagagta acatgtgtga 300 aagcagcaaa gaggcactgg cagaaaacaa cctgaacctt ccaaagatgg 350 ctgaaaaaga tggatgcttc caatctggat tcaatgagga gacttgcctg 400 gtgaaaatca tcactggtct tttggagttt gaggtatacc tagagtacct 450 ccagaacaga tttgagagta gtgaggaaca agccagagct gtgcagatga 500 gtacaaaagt cctgatccag ttcctgcaga aaaaggcaaa gaatctagat 550 gcaataacca cccctgaccc aaccacaaat gccagcctgc tgacgaagct 600 gcaggcacag aaccagtggc tgcaggacat gacaactcat ctcattctgc 650 gcagctttaa ggagttcctg cagtccagcc tgagggctct tcggcaaatg 700 tagcatgggc acctcagatt gttgttgtta atgggcattc cttcttctgg 750 tcagaaacct gtccactggg cacagaactt atgttgttct ctatggagaa 800

    Page 1

    2265275_1

    2016259423 18 Nov 2016

    ctaaaagtat gagcgttagg acactatttt aattattttt aatttattaa 850 tatttaaata tgtgaagctg agttaattta tgtaagtcat atttatattt 900 ttaagaagta ccacttgaaa cattttatgt attagttttg aaataataat 950 ggaaagtggc tatgcagttt gaatatcctt tgtttcagag ccagatcatt 1000 tcttggaaag tgtaggctta cctcaaataa atggctaact tatacatatt 1050 tttaaagaaa tatttatatt gtatttatat aatgtataaa tggtttttat 1100 accaataaat ggcattttaa aaaattcagc a 1131

    <210> 2 <211> 212 <212> PRT <213> Homo sapiens <400> 2

    Met Asn Ser 1 Phe Ser Thr Ser Ala Phe 5 Gly 10 Pro Val Ala Phe Ser 15 Leu Gly Leu Leu Leu Val Leu Pro Al a Al a Phe Pro Ala Pro Val 20 25 30 Pro Pro Gly Glu Asp Ser Lys Asp Val Al a Al a Pro His Arg Gin 35 40 45 Pro Leu Thr Ser Ser Glu Arg lie Asp Lys Gin lie Arg Tyr lie 50 55 60 Leu Asp Gly lie Ser Al a Leu Arg Lys Glu Thr cys Asn Lys Ser 65 70 75 Asn Met cys Glu Ser Ser Lys Glu Al a Leu Al a Glu Asn Asn Leu 80 85 90 Asn Leu Pro Lys Met Al a Glu Lys Asp Gly cys Phe Gin Ser Gly 95 100 105 Phe Asn Glu Glu Thr cys Leu Val Lys lie lie Thr Gly Leu Leu 110 115 120 Glu Phe Glu Val Tyr Leu Glu Tyr Leu Gin Asn Arg Phe Glu Ser 125 130 135 Ser Glu Glu Gin Al a Arg Al a Val Gin Met Ser Thr Lys Val Leu 140 145 150 lie Gin Phe Leu Gin Lys Lys Al a Lys Asn Leu Asp Ala lie Thr 155 160 165 Thr Pro Asp Pro Thr Thr Asn Al a Ser Leu Leu Thr Lys Leu Gin 170 175 180 Al a Gin Asn Gin T rp Leu Gin Asp Met Thr Thr Hi s Leu lie Leu 185 190 195 Arg Ser Phe Lys Glu Phe Leu Gin Ser Ser Leu Arg Ala Leu Arg 200 205 210

    Gin Met <210> 3 <211> 2347

    Page 2

    2265275_1

    2016259423 18 Nov 2016 <212> DNA <213> Homo sapiens <400> 3

    ctgtttcagg gccattggac tctccgtcct gcccagagca agatgtgtca 50 ccagcagttg gtcatctctt ggttttccct ggtttttctg gcatctcccc 100 tcgtggccat atgggaactg aagaaagatg tttatgtcgt agaattggat 150 tggtatccgg atgcccctgg agaaatggtg gtcctcacct gtgacacccc 200 tgaagaagat ggtatcacct ggaccttgga ccagagcagt gaggtcttag 250 gctctggcaa aaccctgacc atccaagtca aagagtttgg agatgctggc 300 cagtacacct gtcacaaagg aggcgaggtt ctaagccatt cgctcctgct 350 gcttcacaaa aaggaagatg gaatttggtc cactgatatt ttaaaggacc 400 agaaagaacc caaaaataag acctttctaa gatgcgaggc caagaattat 450 tctggacgtt tcacctgctg gtggctgacg acaatcagta ctgatttgac 500 attcagtgtc aaaagcagca gaggctcttc tgacccccaa ggggtgacgt 550 gcggagctgc tacactctct gcagagagag tcagagggga caacaaggag 600 tatgagtact cagtggagtg ccaggaggac agtgcctgcc cagctgctga 650 ggagagtctg cccattgagg tcatggtgga tgccgttcac aagctcaagt 700 atgaaaacta caccagcagc ttcttcatca gggacatcat caaacctgac 750 ccacccaaga acttgcagct gaagccatta aagaattctc ggcaggtgga 800 ggtcagctgg gagtaccctg acacctggag tactccacat tcctacttct 850 ccctgacatt ctgcgttcag gtccagggca agagcaagag agaaaagaaa 900 gatagagtct tcacggacaa gacctcagcc acggtcatct gccgcaaaaa 950 tgccagcatt agcgtgcggg cccaggaccg ctactatagc tcatcttgga 1000 gcgaatgggc atctgtgccc tgcagttagg ttctgatcca ggatgaaaat 1050 ttggaggaaa agtggaagat attaagcaaa atgtttaaag acacaacgga 1100 atagacccaa aaagataatt tctatctgat ttgctttaaa acgttttttt 1150 aggatcacaa tgatatcttt gctgtatttg tatagttaga tgctaaatgc 1200 tcattgaaac aatcagctaa tttatgtata gattttccag ctctcaagtt 1250 gccatgggcc ttcatgctat ttaaatattt aagtaattta tgtatttatt 1300 agtatattac tgttatttaa cgtttgtctg ccaggatgta tggaatgttt 1350 catactctta tgacctgatc catcaggatc agtccctatt atgcaaaatg 1400 tgaatttaat tttatttgta ctgacaactt ttcaagcaag gctgcaagta 1450 catcagtttt atgacaatca ggaagaatgc agtgttctga taccagtgcc 1500 atcatacact tgtgatggat gggaacgcaa gagatactta catggaaacc 1550 tgacaatgca aacctgttga gaagatccag gagaacaaga tgctagttcc 1600

    Page 3

    2265275_1

    2016259423 18 Nov 2016

    catgtctgtg aagacttcct ggagatggtg ttgataaagc aatttagggc 1650 cacttacact tctaagcaag tttaatcttt ggatgcctga attttaaaag 1700 ggctagaaaa aaatgattga ccagcctggg aaacataaca agaccccgtc 1750 tctacaaaaa aaatttaaaa ttagccaggc gtggtggctc atgcttgtgg 1800 tcccagctgt tcaggaggat gaggcaggag gatctcttga gcccaggagg 1850 tcaaggctat ggtgagccgt gattgtgcca ctgcatacca gcctaggtga 1900 cagaatgaga ccctgtctca aaaaaaaaaa tgattgaaat taaaattcag 1950 ctttagcttc catggcagtc ctcaccccca cctctctaaa agacacagga 2000 ggatgacaca gaaacaccgt aagtgtctgg aaggcaaaaa gatcttaaga 2050 ttcaagagag aggacaagta gttatggcta aggacatgaa attgtcagaa 2100 tggcaggtgg cttcttaaca gccctgtgag aagcagacag atgcaaagaa 2150 aatctggaat ccctttctca ttagcatgaa tgaacctgat acacaattat 2200 gaccagaaaa tatggctcca tgaaggtgct acttttaagt aatgtatgtg 2250 cgctctgtaa agtgattaca tttgtttcct gtttgtttat ttatttattt 2300 atttttgcat tctgaggctg aactaataaa aactcttctt tgtaatc 2347

    <210> 4 <211> 328 <212> PRT <213> Homo sapiens <400> 4

    Met Cys 1 Hi s Gln Gln 5 Leu Val Ile Ser T rp 10 Phe Ser Leu Val Phe 15 Leu Al a Ser Pro Leu Val Al a Ile T rp Glu Leu Lys Lys Asp Val 20 25 30 Tyr Val Val Glu Leu Asp T rp Tyr Pro Asp Al a Pro Gly Glu Met 35 40 45 Val Val Leu Thr cys Asp Thr Pro Glu Glu Asp Gly Ile Thr T rp 50 55 60 Thr Leu Asp Gln Ser Ser Glu Val Leu Gly Ser Gly Lys Thr Leu 65 70 75 Thr Ile Gln Val Lys Glu Phe Gly Asp Al a Gly Gln Tyr Thr cys 80 85 90 Hi s Lys Gly Gly Glu Val Leu Ser Hi s Ser Leu Leu Leu Leu Hi s 95 100 105 Lys Lys Glu Asp Gly Ile T rp Ser Thr Asp Ile Leu Lys Asp Gln 110 115 120 Lys Glu Pro Lys Asn Lys Thr Phe Leu Arg cys Glu Ala Lys Asn 125 130 135 Tyr Ser Gly Arg Phe Thr cys T rp T rp Leu Thr Thr Ile Ser Thr 140 145 150 Asp Leu Thr Phe Ser Val Lys Ser Ser Arg Gly Ser Ser Asp Pro 155 160 165

    Page 4

    2265275_1

    2016259423 18 Nov 2016

    Gin Gly Val Thr Cys Gly Ala Ala 170 Thr Leu 175 Ser Al a Glu Arg Val 180 Arg Gly Asp Asn Lys Glu Tyr Glu Tyr Ser Val Glu cys Gin Glu 185 190 195 Asp Ser Al a cys Pro Al a Al a Glu Glu Ser Leu Pro lie Glu Val 200 205 210 Met Val Asp Al a Val Hi s Lys Leu Lys Tyr Glu Asn Tyr Thr Ser 215 220 225 Ser Phe Phe lie Arg Asp lie lie Lys Pro Asp Pro Pro Lys Asn 230 235 240 Leu Gin Leu Lys Pro Leu Lys Asn Ser Arg Gin Val Glu Val Ser 245 250 255 T rp Glu Tyr Pro Asp Thr T rp Ser Thr Pro Hi s Ser Tyr Phe Ser 260 265 270 Leu Thr Phe cys Val Gin Val Gin Gly Lys Ser Lys Arg Glu Lys 275 280 285 Lys Asp Arg Val Phe Thr Asp Lys Thr Ser Al a Thr Val lie cys 290 295 300 Arg Lys Asn Al a Ser lie Ser Val Arg Al a Gin Asp Arg Tyr Tyr 305 310 315 Ser Ser Ser T rp Ser Glu T rp Al a Ser Val Pro cys Ser 320 325

    <210> 5 <211> 1145 <212> DNA <213> Homo sapiens <400> 5

    attctctccc cagcttgctg agccctttgc tcccctggcg actgcctgga 50 cagtcagcaa ggaattgtct cccagtgcat tttgccctcc tggctgccaa 100 ctctggctgc taaagcggct gccacctgct gcagtctaca cagcttcggg 150 aagaggaaag gaacctcaga ccttccagat cgcttcctct cgcaacaaac 200 tatttgtcgc aggaataaag atggctgctg aaccagtaga agacaattgc 250 atcaactttg tggcaatgaa atttattgac aatacgcttt actttatagc 300 tgaagatgat gaaaacctgg aatcagatta ctttggcaag cttgaatcta 350 aattatcagt cataagaaat ttgaatgacc aagttctctt cattgaccaa 400 ggaaatcggc ctctatttga agatatgact gattctgact gtagagataa 450 tgcaccccgg accatattta ttataagtat gtataaagat agccagccta 500 gaggtatggc tgtaactatc tctgtgaagt gtgagaaaat ttcaactctc 550 tcctgtgaga acaaaattat ttcctttaag gaaatgaatc ctcctgataa 600 catcaaggat acaaaaagtg acatcatatt ctttcagaga agtgtcccag 650 gacatgataa taagatgcaa tttgaatctt catcatacga aggatacttt 700

    Page 5

    2265275_1

    2016259423 18 Nov 2016

    ctagcttgtg aaaaagagag agaccttttt aaactcattt tgaaaaaaga 750 ggatgaattg ggggatagat ctataatgtt cactgttcaa aacgaagact 800 agctattaaa atttcatgcc gggcgcagtg gctcacgcct gtaatcccag 850 ccctttggga ggctgaggcg ggcagatcac cagaggtcag gtgttcaaga 900 ccagcctgac caacatggtg aaacctcatc tctactaaaa atacaaaaaa 950 ttagctgagt gtagtgacgc atgccctcaa tcccagctac tcaagaggct 1000 gaggcaggag aatcacttgc actccggagg tagaggttgt ggtgagccga 1050 gattgcacca ttgcgctcta gcctgggcaa caacagcaaa actccatctc 1100 aaaaaataaa ataaataaat aaacaaataa aaaattcata atgtg 1145

    <210> 6 <211> 193 <212> PRT <213> Homo sapiens <400> 6

    Met 1 Al a Al a Glu Pro 5 Val Glu Asp Asn Cys 10 Ile Asn Phe Val Al a 15 Met Lys Phe Ile Asp Asn Thr Leu Tyr Phe Ile Al a Glu Asp Asp 20 25 30 Glu Asn Leu Glu Ser Asp Tyr Phe Gly Lys Leu Glu Ser Lys Leu 35 40 45 Ser Val Ile Arg Asn Leu Asn Asp Gin Val Leu Phe Ile Asp Gin 50 55 60 Gly Asn Arg Pro Leu Phe Glu Asp Met Thr Asp Ser Asp cys Arg 65 70 75 Asp Asn Al a Pro Arg Thr Ile Phe Ile Ile Ser Met Tyr Lys Asp 80 85 90 Ser Gin Pro Arg Gly Met Al a Val Thr Ile Ser Val Lys cys Glu 95 100 105 Lys Ile Ser Thr Leu Ser cys Glu Asn Lys Ile Ile Ser Phe Lys 110 115 120 Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys Ser Asp Ile 125 130 135 Ile Phe Phe Gin Arg Ser Val Pro Gly Hi s Asp Asn Lys Met Gin 140 145 150 Phe Glu Ser Ser Ser Tyr Glu Gly Tyr Phe Leu Al a cys Glu Lys 155 160 165 Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu 170 175 180 Gly Asp Arg Ser Ile Met Phe Thr Val Gin Asn Glu Asp 185 190

    <210> 7 <211> 1147 <212> DNA <213> Homo sapiens

    Page 6

    2265275_1

    2016259423 18 Nov 2016 <400> 7

    cgaccaggtt ctccttcccc agtcaccagt tgctcgagtt agaattgtct 50 gcaatggccg ccctgcagaa atctgtgagc tctttcctta tggggaccct 100 ggccaccagc tgcctccttc tcttggccct cttggtacag ggaggagcag 150 ctgcgcccat cagctcccac tgcaggcttg acaagtccaa cttccagcag 200 ccctatatca ccaaccgcac cttcatgctg gctaaggagg ctagcttggc 250 tgataacaac acagacgttc gtctcattgg ggagaaactg ttccacggag 300 tcagtatgag tgagcgctgc tatctgatga agcaggtgct gaacttcacc 350 cttgaagaag tgctgttccc tcaatctgat aggttccagc cttatatgca 400 ggaggtggtg cccttcctgg ccaggctcag caacaggcta agcacatgtc 450 atattgaagg tgatgacctg catatccaga ggaatgtgca aaagctgaag 500 gacacagtga aaaagcttgg agagagtgga gagatcaaag caattggaga 550 actggatttg ctgtttatgt ctctgagaaa tgcctgcatt tgaccagagc 600 aaagctgaaa aatgaataac taaccccctt tccctgctag aaataacaat 650 tagatgcccc aaagcgattt tttttaacca aaaggaagat gggaagccaa 700 actccatcat gatgggtgga ttccaaatga acccctgcgt tagttacaaa 750 ggaaaccaat gccacttttg tttataagac cagaaggtag actttctaag 800 catagatatt tattgataac atttcattgt aactggtgtt ctatacacag 850 aaaacaattt attttttaaa taattgtctt tttccataaa aaagattact 900 ttccattcct ttaggggaaa aaacccctaa atagcttcat gtttccataa 950 tcagtacttt atatttataa atgtatttat tattattata agactgcatt 1000 ttatttatat cattttatta atatggattt atttatagaa acatcattcg 1050 atattgctac ttgagtgtaa ggctaatatt gatatttatg acaataatta 1100 tagagctata acatgtttat ttgacctcaa taaacacttg gatatcc 1147

    <210> 8 <211> 179 <212> PRT <213> Homo sapiens <400> 8

    Met Al a Al a Leu Gin Lys Ser Val Ser Ser Phe Leu Met Gly Thr 1 5 10 15 Leu Al a Th r Ser Cys Leu Leu Leu Leu Al a Leu Leu Val Gin Gly 20 25 30 Gly Al a Al a Al a Pro lie Ser Ser Hi s cys Arg Leu Asp Lys Ser 35 40 45 Asn Phe Gl n Gin Pro Tyr lie Thr Asn Arg Thr Phe Met Leu Al a 50 55 60 Lys Glu Al a Ser Leu Al a Asp Asn Asn Thr Asp Val Arg Leu lie 65 70 75

    Page 7

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    Gly Glu Lys Leu Phe 80 Hi s Gly Val Ser Met 85 Ser Glu Arg cys Tyr 90 Leu Met Lys Gin Val Leu Asn Phe Thr Leu Glu Glu Val Leu Phe 95 100 105 Pro Gin Ser Asp Arg Phe Gin Pro Tyr Met Gin Glu Val Val Pro 110 115 120 Phe Leu Al a Arg Leu Ser Asn Arg Leu Ser Thr cys His lie Glu 125 130 135 Gly Asp Asp Leu Hi s lie Gin Arg Asn Val Gin Lys Leu Lys Asp 140 145 150 Thr Val Lys Lys Leu Gly Glu Ser Gly Glu lie Lys Ala lie Gly 155 160 165 Glu Leu Asp Leu Leu Phe Met Ser Leu Arg Asn Al a cys lie 170 175

    <210> 9 <211> 1049 <212> DNA <213> Homo sapiens <400> 9

    aaaacaacag gaagcagctt acaaactcgg tgaacaactg agggaaccaa 50 accagagacg cgctgaacag agagaatcag gctcaaagca agtggaagtg 100 ggcagagatt ccaccaggac tggtgcaagg cgcagagcca gccagatttg 150 agaagaaggc aaaaagatgc tggggagcag agctgtaatg ctgctgttgc 200 tgctgccctg gacagctcag ggcagagctg tgcctggggg cagcagccct 250 gcctggactc agtgccagca gctttcacag aagctctgca cactggcctg 300 gagtgcacat ccactagtgg gacacatgga tctaagagaa gagggagatg 350 aagagactac aaatgatgtt ccccatatcc agtgtggaga tggctgtgac 400 ccccaaggac tcagggacaa cagtcagttc tgcttgcaaa ggatccacca 450 gggtctgatt ttttatgaga agctgctagg atcggatatt ttcacagggg 500 agccttctct gctccctgat agccctgtgg gccagcttca tgcctcccta 550 ctgggcctca gccaactcct gcagcctgag ggtcaccact gggagactca 600 gcagattcca agcctcagtc ccagccagcc atggcagcgt ctccttctcc 650 gcttcaaaat ccttcgcagc ctccaggcct ttgtggctgt agccgcccgg 700 gtctttgccc atggagcagc aaccctgagt ccctaaaggc agcagctcaa 750 ggatggcact cagatctcca tggcccagca aggccaagat aaatctacca 800 ccccaggcac ctgtgagcca acaggttaat tagtccatta attttagtgg 850 gacctgcata tgttgaaaat taccaatact gactgacatg tgatgctgac 900 ctatgataag gttgagtatt tattagatgg gaagggaaat ttggggatta 950 tttatcctcc tggggacagt ttggggagga ttatttattg tatttatatt 1000 gaattatgta cttttttcaa taaagtctta tttttgtggc taaaaaaaa 1049

    Page 8

    2265275_1

    2016259423 18 Nov 2016 <210> 10 <211> 189 <212> PRT <213> Homo sapiens <400> 10

    Met 1 Leu Gly Ser Arg 5 Ala Val Met Leu Leu 10 Leu Leu Leu Pro T rp 15 Thr Al a Gin Gly Arg Al a Val Pro Gly Gly Ser Ser Pro Al a T rp 20 25 30 Thr Gin cys Gin Gin Leu Ser Gin Lys Leu cys Thr Leu Al a T rp 35 40 45 Ser Al a Hi s Pro Leu Val Gly Hi s Met Asp Leu Arg Glu Glu Gly 50 55 60 Asp Glu Glu Thr Thr Asn Asp Val Pro Hi s lie Gin cys Gly Asp 65 70 75 Gly cys Asp Pro Gin Gly Leu Arg Asp Asn Ser Gin Phe cys Leu 80 85 90 Gin Arg lie Hi s Gin Gly Leu lie Phe Tyr Glu Lys Leu Leu Gly 95 100 105 Ser Asp lie Phe Thr Gly Glu Pro Ser Leu Leu Pro Asp Ser Pro 110 115 120 Val Gly Gin Leu Hi s Al a Ser Leu Leu Gly Leu Ser Gin Leu Leu 125 130 135 Gin Pro Glu Gly Hi s Hi s T rp Glu Thr Gin Gin lie Pro Ser Leu 140 145 150 Ser Pro Ser Gin Pro T rp Gin Arg Leu Leu Leu Arg Phe Lys lie 155 160 165 Leu Arg Ser Leu Gin Al a Phe Val Al a Val Al a Al a Arg Val Phe 170 175 180 Al a Hi s Gly Al a Al a Thr Leu Ser Pro 185

    <210> 11 <211> 808 <212> DNA <213> Homo sapiens <400> 11 gatataaagc tcctacagct acctggcctg agaagccaac tcagactcag 50 ccaacagaga ttgttgattt gcctcttaag caagagattc attgcagctc 100 agcatggctc agaccagctc atacttcatg ctgatctcct gcctgatgtt 150 tctgtctcag agccaaggcc aagaggccca gacagagttg ccccaggccc 200 ggatcagctg cccagaaggc accaatgcct atcgctccta ctgctactac 250 tttaatgaag accgtgagac ctgggttgat gcagatctct attgccagaa 300 catgaattcg ggcaacctgg tgtctgtgct cacccaggcc gagggtgcct 350 ttgtggcctc actgattaag gagagtggca ctgatgactt caatgtctgg 400

    Page 9

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    attggcctcc atgaccccaa aaagaaccgc cgctggcact ggagcagtgg 450 gtccctggtc tcctacaagt cctggggcat tggagcccca agcagtgtta 500 atcctggcta ctgtgtgagc ctgacctcaa gcacaggatt ccagaaatgg 550 aaggatgtgc cttgtgaaga caagttctcc tttgtctgca agttcaaaaa 600 ctagaggcaa ctggaaaata catgtctaga actgatccag caattacaac 650 ggagtcaaaa attaaaccgg accatctctc caactcaact caacctggac 700 actctcttct ctgctgagtt tgccttgtta atcttcaata gttttaccta 750 ccccagtctt tggaacccta aataataaaa ataaacatgt ttccactatt 800 gtgctgtc 808

    <210> 12 <211> 166 <212> PRT <213> Homo sapiens

    <400> 12 Met 1 Al a Gln Thr Ser 5 Ser Tyr Phe Met Leu 10 lie Ser cys Leu Met 15 Phe Leu Ser Gin Ser Gin Gly Gin Glu Al a Gin Thr Glu Leu Pro 20 25 30 Gin Al a Arg lie Ser cys Pro Glu Gly Thr Asn Al a Tyr Arg Ser 35 40 45 Tyr cys Tyr Tyr Phe Asn Glu Asp Arg Glu Thr T rp Val Asp Al a 50 55 60 Asp Leu Tyr cys Gin Asn Met Asn Ser Gly Asn Leu Val Ser Val 65 70 75 Leu Thr Gin Al a Glu Gly Al a Phe Val Al a Ser Leu lie Lys Glu 80 85 90 Ser Gly Thr Asp Asp Phe Asn Val T rp lie Gly Leu His Asp Pro 95 100 105 Lys Lys Asn Arg Arg T rp Hi s T rp Ser Ser Gly Ser Leu Val Ser 110 115 120 Tyr Lys Ser T rp Gly lie Gly Al a Pro Ser Ser Val Asn Pro Gly 125 130 135 Tyr cys Val Ser Leu Thr Ser Ser Thr Gly Phe Gin Lys T rp Lys 140 145 150 Asp Val Pro cys Glu Asp Lys Phe Ser Phe Val cys Lys Phe Lys 155 160 165

    Asn <210> 13 <211> 773 <212> DNA <213> Homo sapiens <400> 13 aagccacctc aagtggacaa ggcacttacc aacagagatt gctgatttgc 50 Page 10

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    tccttaagca agagattcac tgccgctaag catggctcag accaactcgt 100 tcttcatgct gatctcctcc ctgatgttcc tgtctctgag ccaaggccag 150 gagtcccaga cagagctgcc taatccccga atcagctgcc cagaaggcac 200 caatgcctat cgctcctact gctactactt taatgaagac cctgagacct 250 gggttgatgc agatctctat tgccagaaca tgaattcagg caacctggtg 300 tctgtgctca cccaggcgga gggtgccttc gtggcctcac tgattaagga 350 gagtagcact gatgacagca atgtctggat tggcctccat gacccaaaaa 400 agaaccgccg ctggcactgg agtagtgggt ccctggtctc ctacaagtcc 450 tgggacactg gatccccgag cagtgctaat gctggctact gtgcaagcct 500 gacttcatgc tcaggattca agaaatggaa ggatgaatct tgtgagaaga 550 agttctcctt tgtttgcaag ttcaaaaact agaggaagct gaaaaatgga 600 tgtctagaac tggtcctgca attactatga agtcaaaaat taaactagac 650 tatgtctcca actcagttca gaccatctcc tccctaatga gtttgcatcg 700 ctgatcttca gtaccttcac ctgtctcagt ctctagagcc ctgaaaaata 750 aaaacaaact tatttttatc cag 773

    <210> 14 <211> 166 <212> PRT <213> Homo sapiens

    <400> 14 Thr Asn 5 Ser Phe Phe Met Leu 10 lie Ser Ser Leu Met 15 Met 1 Al a Gln Phe Leu Ser Leu Ser Gin Gly Gin Glu Ser Gin Thr Glu Leu Pro 20 25 30 Asn Pro Arg lie Ser cys Pro Glu Gly Thr Asn Al a Tyr Arg Ser 35 40 45 Tyr cys Tyr Tyr Phe Asn Glu Asp Pro Glu Thr T rp Val Asp Al a 50 55 60 Asp Leu Tyr cys Gin Asn Met Asn Ser Gly Asn Leu Val Ser Val 65 70 75 Leu Thr Gin Al a Glu Gly Al a Phe Val Al a Ser Leu lie Lys Glu 80 85 90 Ser Ser Thr Asp Asp Ser Asn Val T rp lie Gly Leu His Asp Pro 95 100 105 Lys Lys Asn Arg Arg T rp Hi s T rp Ser Ser Gly Ser Leu Val Ser 110 115 120 Tyr Lys Ser T rp Asp Thr Gly Ser Pro Ser Ser Al a Asn Al a Gly 125 130 135 Tyr cys Al a Ser Leu Thr Ser cys Ser Gly Phe Lys Lys T rp Lys 140 145 150 Asp Glu Ser cys Glu Lys Lys Phe Ser Phe Val cys Lys Phe Lys 155 160 165

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    Asn <210> 15 <211> 807 <212> DNA <213> Homo sapiens <400> 15

    aaaccatacc atatcccacc agagagtgac tcctgattgc ctcctcaagt 50 cgcagacact atgctgcctc ccatggccct gcccagtgta tcttggatgc 100 tgctttcctg cctcatgctg ctgtctcagg ttcaaggtga agaaccccag 150 agggaactgc cctctgcacg gatccgctgt cccaaaggct ccaaggccta 200 tggctcccac tgctatgcct tgtttttgtc accaaaatcc tggacagatg 250 cagatctggc ctgccagaag cggccctctg gaaacctggt gtctgtgctc 300 agtggggctg agggatcctt cgtgtcctcc ctggtgaaga gcattggtaa 350 cagctactca tacgtctgga ttgggctcca tgaccccaca cagggcaccg 400 agcccaatgg agaaggttgg gagtggagta gcagtgatgt gatgaattac 450 tttgcatggg agagaaatcc ctccaccatc tcaagccccg gccactgtgc 500 gagcctgtcg agaagcacag catttctgag gtggaaagat tataactgta 550 atgtgaggtt accctatgtc tgcaagttca ctgactagtg caggagggaa 600 gtcagcagcc tgtgtttggt gtgcaactca tcatgggcat gagaccagtg 650 tgaggactca ccctggaaga gaatattcgc ttaattcccc caacctgacc 700 acctcattct tatctttctt ctgtttcttc ctccccgctg tcatttcagt 750 ctcttcattt tgtcatacgg cctaaggctt taaagagcaa taaaattttt 800

    agtctgc 807 <210> 16 <211> 175 <212> PRT <213> Homo sapiens

    <400> 16 Al a Leu Pro Ser Val 10 Ser T rp Met Leu Leu 15 Met 1 Leu Pro Pro Met 5 Ser cys Leu Met Leu Leu Ser Gin Val Gin Gly Glu Glu Pro Gin 20 25 30 Arg Glu Leu Pro Ser Al a Arg lie Arg cys Pro Lys Gly Ser Lys 35 40 45 Al a Tyr Gly Ser Hi s cys Tyr Al a Leu Phe Leu Ser Pro Lys Ser 50 55 60 T rp Thr Asp Al a Asp Leu Al a cys Gin Lys Arg Pro Ser Gly Asn 65 70 75 Leu Val Ser Val Leu Ser Gly Al a Glu Gly Ser Phe Val Ser Ser 80 85 90

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    Leu Val Lys Ser lie 95 Gly Asn Ser Tyr Ser Tyr Val 100 T rp lie Gly 105 Leu Hi s Asp Pro Thr Gin Gly Thr Glu Pro Asn Gly Glu Gly T rp 110 115 120 Glu T rp Ser Ser Ser Asp Val Met Asn Tyr Phe Al a T rp Glu Arg 125 130 135 Asn Pro Ser Thr lie Ser Ser Pro Gly Hi s cys Al a Ser Leu Ser 140 145 150 Arg Ser Thr Al a Phe Leu Arg T rp Lys Asp Tyr Asn cys Asn Val 155 160 165 Arg Leu Pro Tyr Val cys Lys Phe Thr Asp 170 175

    <210> 17 <211> 784 <212> DNA <213> Homo sapiens <400> 17

    aaaccatacc atatcccacc agagagtcgc agacactatg ctgcctccca 50 tggccctgcc cagtgtatct tggatgctgc tttcctgcct catgctgctg 100 tctcaggttc aaggtgaaga accccagagg gaactgccct ctgcacggat 150 ccgctgtccc aaaggctcca aggcctatgg ctcccactgc tatgccttgt 200 ttttgtcacc aaaatcctgg acagatgcag atctggcctg ccagaagcgg 250 ccctctggaa acctggtgtc tgtgctcagt ggggctgagg gatccttcgt 300 gtcctccctg gtgaagagca ttggtaacag ctactcatac gtctggattg 350 ggctccatga ccccacacag ggcaccgagc ccaatggaga aggttgggag 400 tggagtagca gtgatgtgat gaattacttt gcatgggaga gaaatccctc 450 caccatctca agccccggcc actgtgcgag cctgtcgaga agcacagcat 500 ttctgaggtg gaaagattat aactgtaatg tgaggttacc ctatgtctgc 550 aagttcactg actagtgcag gagggaagtc agcagcctgt gtttggtgtg 600 caactcatca tgggcatgag accagtgtga ggactcaccc tggaagagaa 650 tattcgctta attcccccaa cctgaccacc tcattcttat ctttcttctg 700 tttcttcctc cccgctgtca tttcagtctc ttcattttgt catacggcct 750 aaggctttaa agagcaataa aatttttagt ctgc 784

    <210> 18 <211> 175 <212> PRT <213> Homo sapiens <400> Met 18 Leu Pro Pro Met Ala Leu Pro Ser Val Ser Trp Met Leu Leu 1 5 10 15

    Ser Cys Leu Met Leu Leu Ser Gin Val Gin Gly Glu Glu Pro Gin 20 25 30

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    Arg Glu Leu Pro Ser Ala 35 Arg lie Arg cys 40 Pro Lys Gly Ser Lys 45 Al a Tyr Gly Ser Hi s cys Tyr Al a Leu Phe Leu Ser Pro Lys Ser 50 55 60 T rp Thr Asp Al a Asp Leu Al a cys Gin Lys Arg Pro Ser Gly Asn 65 70 75 Leu Val Ser Val Leu Ser Gly Al a Glu Gly Ser Phe Val Ser Ser 80 85 90 Leu Val Lys Ser lie Gly Asn Ser Tyr Ser Tyr Val T rp lie Gly 95 100 105 Leu Hi s Asp Pro Thr Gin Gly Thr Glu Pro Asn Gly Glu Gly T rp 110 115 120 Glu T rp Ser Ser Ser Asp Val Met Asn Tyr Phe Al a T rp Glu Arg 125 130 135 Asn Pro Ser Thr lie Ser Ser Pro Gly Hi s cys Al a Ser Leu Ser 140 145 150 Arg Ser Thr Al a Phe Leu Arg T rp Lys Asp Tyr Asn cys Asn Val 155 160 165 Arg Leu Pro Tyr Val cys Lys Phe Thr Asp 170 175

    <210> 19 <211> 1002 <212> DNA <213> Homo sapiens <400> 19

    gggagggtcc cttcctcagg gagcacagga actctgagac tcagcaaggg 50 tgtcctggga gggctcgggg atgggagagt acacagattc acaactcatt 100 cagaactgta gaagatgatg gatgtgacca agatcacttt agtcctaggg 150 gactagagaa ggaaaatgac atgaggcagt ggggtatctg tgtgttctcc 200 cactgaccac gctttcttta gtgactcctg attgcctcct caagtcgcag 250 acactatgct gcctcccatg gccctgccca gtgtatcttg gatgctgctt 300 tcctgcctca tgctgctgtc tcaggttcaa ggtgaagaac cccagaggga 350 actgccctct gcacggatcc gctgtcccaa aggctccaag gcctatggct 400 cccactgcta tgccttgttt ttgtcaccaa aatcctggac agatgcagat 450 ctggcctgcc agaagcggcc ctctggaaac ctggtgtctg tgctcagtgg 500 ggctgaggga tccttcgtgt cctccctggt gaagagcatt ggtaacagct 550 actcatacgt ctggattggg ctccatgacc ccacacaggg caccgagccc 600 aatggagaag gttgggagtg gagtagcagt gatgtgatga attactttgc 650 atgggagaga aatccctcca ccatctcaag ccccggccac tgtgcgagcc 700 tgtcgagaag cacagcattt ctgaggtgga aagattataa ctgtaatgtg 750 aggttaccct atgtctgcaa gttcactgac tagtgcagga gggaagtcag 800

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    2265275_1 cagcctgtgt ttggtgtgca actcatcatg ggcatgagac cagtgtgagg actcaccctg gaagagaata ttcgcttaat tcccccaacc tgaccacctc attcttatct ttcttctgtt tcttcctccc cgctgtcatt tcagtctctt cattttgtca tacggcctaa ggctttaaag agcaataaaa tttttagtct gc 1002 <210> 20 <211> 175 <212> PRT <213> Homo sapiens <400> 20

    850

    900

    950

    1000

    Met 1 Leu Pro Pro Met Ala 5 Leu Pro Ser Val 10 Ser T rp Met Leu Leu 15 Ser cys Leu Met Leu Leu Ser Gin Val Gin Gly Glu Glu Pro Gin 20 25 30 Arg Glu Leu Pro Ser Al a Arg lie Arg cys Pro Lys Gly Ser Lys 35 40 45 Al a Tyr Gly Ser Hi s cys Tyr Al a Leu Phe Leu Ser Pro Lys Ser 50 55 60 T rp Thr Asp Al a Asp Leu Al a cys Gin Lys Arg Pro Ser Gly Asn 65 70 75 Leu Val Ser Val Leu Ser Gly Al a Glu Gly Ser Phe Val Ser Ser 80 85 90 Leu Val Lys Ser lie Gly Asn Ser Tyr Ser Tyr Val T rp lie Gly 95 100 105 Leu Hi s Asp Pro Thr Gin Gly Thr Glu Pro Asn Gly Glu Gly T rp 110 115 120 Glu T rp Ser Ser Ser Asp Val Met Asn Tyr Phe Al a T rp Glu Arg 125 130 135 Asn Pro Ser Thr lie Ser Ser Pro Gly Hi s cys Al a Ser Leu Ser 140 145 150 Arg Ser Thr Al a Phe Leu Arg T rp Lys Asp Tyr Asn cys Asn Val 155 160 165 Arg Leu Pro Tyr Val cys Lys Phe Thr Asp 170 175

    <210> 21 <211> 847 <212> DNA <213> Homo sapiens <400> 21 ccatccctga gatcttttta taaaaaaccc agtctttgct gaccagacaa 50 agcataccag atctcaccag agagtcgcag acactatgct gcctcccatg 100 gccctgccca gtgtgtcctg gatgctgctt tcctgcctca ttctcctgtg 150 tcaggttcaa ggtgaagaaa cccagaagga actgccctct ccacggatca 200 gctgtcccaa aggctccaag gcctatggct ccccctgcta tgccttgttt 250

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    ttgtcaccaa aatcctggat ggatgcagat ctggcttgcc agaagcggcc 300 ctctggaaaa ctggtgtctg tgctcagtgg ggctgaggga tccttcgtgt 350 cctccctggt gaggagcatt agtaacagct attcatacat ctggattggg 400 ctccatgacc ccacacaggg ctctgagcct gatggagatg gatgggagtg 450 gagtagcact gatgtgatga attactttgc atgggagaaa aatccctcca 500 ccatcttaaa ccctggccac tgtgggagcc tgtcaagaag cacaggattt 550 ctgaagtgga aagattataa ctgtgatgca aagttaccct atgtctgcaa 600 gttcaaggac tagggcaggt gggaagtcag cagcctgagc ttggcgtgca 650 gctcatcatg gacatgagac cagtgtgaag actcaccctg gaagagaata 700 ttctccccaa actgccctac ctgactacct tgtcatgatc ctccttcttt 750 ttcctttttc ttcaccttca tttcaggctt ttctctgtct tccatgtctt 800 gagatctcag agaataataa taaaaatgtt actttatact taaaaaa 847

    <210> 22 <211> 175 <212> PRT <213> Homo sapiens <400> 22

    Met 1 Leu Pro Pro Met Ala 5 Leu Pro Ser Val 10 Ser T rp Met Leu Leu 15 Ser cys Leu lie Leu Leu cys Gin Val Gin Gly Glu Glu Thr Gin 20 25 30 Lys Glu Leu Pro Ser Pro Arg lie Ser cys Pro Lys Gly Ser Lys 35 40 45 Al a Tyr Gly Ser Pro cys Tyr Al a Leu Phe Leu Ser Pro Lys Ser 50 55 60 T rp Met Asp Al a Asp Leu Al a cys Gin Lys Arg Pro Ser Gly Lys 65 70 75 Leu Val Ser Val Leu Ser Gly Al a Glu Gly Ser Phe Val Ser Ser 80 85 90 Leu Val Arg Ser lie Ser Asn Ser Tyr Ser Tyr lie T rp lie Gly 95 100 105 Leu Hi s Asp Pro Thr Gin Gly Ser Glu Pro Asp Gly Asp Gly T rp 110 115 120 Glu T rp Ser Ser Thr Asp Val Met Asn Tyr Phe Al a T rp Glu Lys 125 130 135 Asn Pro Ser Thr lie Leu Asn Pro Gly Hi s cys Gly Ser Leu Ser 140 145 150 Arg Ser Thr Gly Phe Leu Lys T rp Lys Asp Tyr Asn cys Asp Al a 155 160 165 Lys Leu Pro Tyr Val cys Lys Phe Lys Asp 170 175

    <210> 23 <211> 947

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    2016259423 18 Nov 2016 <212> DNA <213> Homo sapiens <400> 23

    ccatccctga gatcttttta taaaaaaccc agtctttgct gaccagacaa 50 agcataccag atctcaccag agagtcctag gggactacag aaggaaaaag 100 acaagaggca gtaggatatc tgtgtgtcct cccgctgacc acacttcctt 150 tagtgacccg attgcctcct caagtcgcag acactatgct gcctcccatg 200 gccctgccca gtgtgtcctg gatgctgctt tcctgcctca ttctcctgtg 250 tcaggttcaa ggtgaagaaa cccagaagga actgccctct ccacggatca 300 gctgtcccaa aggctccaag gcctatggct ccccctgcta tgccttgttt 350 ttgtcaccaa aatcctggat ggatgcagat ctggcttgcc agaagcggcc 400 ctctggaaaa ctggtgtctg tgctcagtgg ggctgaggga tccttcgtgt 450 cctccctggt gaggagcatt agtaacagct attcatacat ctggattggg 500 ctccatgacc ccacacaggg ctctgagcct gatggagatg gatgggagtg 550 gagtagcact gatgtgatga attactttgc atgggagaaa aatccctcca 600 ccatcttaaa ccctggccac tgtgggagcc tgtcaagaag cacaggattt 650 ctgaagtgga aagattataa ctgtgatgca aagttaccct atgtctgcaa 700 gttcaaggac tagggcaggt gggaagtcag cagcctgagc ttggcgtgca 750 gctcatcatg gacatgagac cagtgtgaag actcaccctg gaagagaata 800 ttctccccaa actgccctac ctgactacct tgtcatgatc ctccttcttt 850 ttcctttttc ttcaccttca tttcaggctt ttctctgtct tccatgtctt 900 gagatctcag agaataataa taaaaatgtt actttatacg taaaaaa 947

    <210> 24 <211> 175 <212> PRT <213> Homo sapiens

    <400> 24 Pro Met 5 Al a Leu Pro Ser Val 10 Ser Trp Met Leu Leu 15 Met 1 Leu Pro Ser cys Leu lie Leu Leu cys Gin Val Gin Gly Glu Glu Thr Gin 20 25 30 Lys Glu Leu Pro Ser Pro Arg lie Ser cys Pro Lys Gly Ser Lys 35 40 45 Al a Tyr Gly Ser Pro cys Tyr Al a Leu Phe Leu Ser Pro Lys Ser 50 55 60 T rp Met Asp Al a Asp Leu Al a cys Gin Lys Arg Pro Ser Gly Lys 65 70 75 Leu Val Ser Val Leu Ser Gly Al a Glu Gly Ser Phe Val Ser Ser 80 85 90 Leu Val Arg Ser lie Ser Asn Ser Tyr Ser Tyr lie T rp lie Gly 95 100 105 Page 17

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    Leu Hi s Asp Pro Thr Gln Gly Ser Glu Pro Asp Gly Asp Gly T rp 110 115 120 Glu T rp Ser Ser Thr Asp Val Met Asn Tyr Phe Al a T rp Glu Lys 125 130 135 Asn Pro Ser Thr Ile Leu Asn Pro Gly Hi s cys Gly Ser Leu Ser 140 145 150 Arg Ser Thr Gly Phe Leu Lys T rp Lys Asp Tyr Asn cys Asp Al a 155 160 165 Lys Leu Pro Tyr Val cys Lys Phe Lys Asp 170 175

    <210> 25 <211> 1285 <212> DNA <213> Homo sapiens <400> 25

    ataagacttt tatggatgga ttgtttttct caaataatat tatcgctttg 50 tgactaaagt aaagattatt aattcctgag gcaagaagat ataaaagctc 100 cagaaacgtt gactgggacc actggagaca ctgaagaagg caggggccct 150 tagagtcttg gttgccaaac agatttgcag atcaaggaga acccaggagt 200 ttcaaagaag cgctagtaag gtctctgaga tccttgcact agctacatcc 250 tcagggtagg aggaagatgg cttccagaag catgcggctg ctcctattgc 300 tgagctgcct ggccaaaaca ggagtcctgg gtgatatcat catgagaccc 350 agctgtgctc ctggatggtt ttaccacaag tccaattgct atggttactt 400 caggaagctg aggaactggt ctgatgccga gctcgagtgt cagtcttacg 450 gaaacggagc ccacctggca tctatcctga gtttaaagga agccagcacc 500 atagcagagt acataagtgg ctatcagaga agccagccga tatggattgg 550 cctgcacgac ccacagaaga ggcagcagtg gcagtggatt gatggggcca 600 tgtatctgta cagatcctgg tctggcaagt ccatgggtgg gaacaagcac 650 tgtgctgaga tgagctccaa taacaacttt ttaacttgga gcagcaacga 700 atgcaacaag cgccaacact tcctgtgcaa gtaccgacca tagagcaaga 750 atcaagattc tgctaactcc tgcacagccc cgtcctcttc ctttctgcta 800 gcctggctaa atctgctcat tatttcagag gggaaaccta gcaaactaag 850 agtgataagg gccctactac actggctttt ttaggcttag agacagaaac 900 tttagcattg gcccagtagt ggcttctagc tctaaatgtt tgccccgcca 950 tccctttcca cagtatcctt cttccctcct cccctgtctc tggctgtctc 1000 gagcagtcta gaagagtgca tctccagcct atgaaacagc tgggtctttg 1050 gccataagaa gtaaagattt gaagacagaa ggaagaaact caggagtaag 1100 cttctagacc ccttcagctt ctacaccctt ctgccctctc tccattgcct 1150

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    2265275_1 gcaccccacc ccagccactc aactcctgct tgtttttcct ttggccatag 1200 gaaggtttac cagtagaatc cttgctaggt tgatgtgggc catacattcc 1250 tttaataaac cattgtgtac ataagaaaaa aaaaa 1285 <210> 26 <211> 158 <212> PRT <213> Homo sapiens <400> 26

    Met 1 Al a Ser Arg Ser 5 Met Arg Al a Lys Thr Gly Val 20 Leu Gly Al a Pro Gly T rp Phe 35 Tyr Hi s Arg Lys Leu Arg Asn 50 T rp Ser Tyr Gly Asn Gly Al a 65 Hi s Leu Al a Ser Thr lie Al a 80 Glu Tyr Pro lie T rp lie Gly 95 Leu Hi s Gin T rp lie Asp Gly 110 Al a Met Lys Ser Met Gly Gly 125 Asn Lys Asn Asn Phe Leu Thr 140 T rp Ser Hi s Phe Leu cys Lys 155 Tyr Arg

    Leu Leu Leu 10 Leu Leu Ser cys Leu 15 Asp lie lie 25 Met Arg Pro Ser cys 30 Lys Ser Asn 40 cys Tyr Gly Tyr Phe 45 Asp Al a Glu 55 Leu Glu cys Gin Ser 60 Al a Ser lie 70 Leu Ser Leu Lys Glu 75 lie Ser Gly 85 Tyr Gin Arg Ser Gin 90 Asp Pro Gin 100 Lys Arg Gin Gin T rp 105 Tyr Leu Tyr 115 Arg Ser T rp Ser Gly 120 Hi s cys Al a 130 Glu Met Ser Ser Asn 135 Ser Pro Asn Glu 145 cys Asn Lys Arg Gin 150

    <210> 27 <211> 1087 <212> DNA <213> Mus musculus <400> 27

    ccaagaacga tagtcaattc cagaaaccgc tatgaagttc ctctctgcaa 50 gagacttcca tccagttgcc ttcttgggac tgatgctggt gacaaccacg 100 gccttcccta cttcacaagt ccggagagga gacttcacag aggataccac 150 tcccaacaga cctgtctata ccacttcaca agtcggaggc ttaattacac 200 atgttctctg ggaaatcgtg gaaatgagaa aagagttgtg caatggcaat 250 tctgattgta tgaacaacga tgatgcactt gcagaaaaca atctgaaact 300 tccagagata caaagaaatg atggatgcta ccaaactgga tataatcagg 350 aaatttgcct attgaaaatt tcctctggtc ttctggagta ccatagctac 400 ctggagtaca tgaagaacaa cttaaaagat aacaagaaag Page 19 acaaagccag 450

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    agtccttcag agagatacag aaactctaat tcatatcttc aaccaagagg 500 taaaagattt acataaaata gtccttccta ccccaatttc caatgctctc 550 ctaacagata agctggagtc acagaaggag tggctaagga ccaagaccat 600 ccaattcatc ttgaaatcac ttgaagaatt tctaaaagtc actttgagat 650 ctactcggca aacctagtgc gttatgccta agcatatcag tttgtggaca 700 ttcctcactg tggtcagaaa atatatcctg ttgtcaggta tctgacttat 750 gttgttctct acgaagaact gacaatatga atgttgggac actattttaa 800 ttatttttaa tttattgata atttaaataa gtaaacttta agttaattta 850 tgattgatat ttattatttt tatgaagtgt cacttgaaat gttatatgtt 900 atagttttga aatgataacc taaaaatcta tttgatataa atattctgtt 950 acctagccag atggtttctt ggaatgtata agtttacctc aatgaattgc 1000 taatttaaat atgtttttaa agaaatcttt gtgatgtatt tttataatgt 1050 ttagactgtc ttcaaacaaa taaattatat tatattt 1087

    <210> 28 <211> 211 <212> PRT <213> Mus musculus <400> 28

    Met 1 Lys Phe Leu Ser 5 Ala Arg Asp Phe Hi s 10 Pro Val Ala Phe Leu 15 Gly Leu Met Leu Val Thr Thr Thr Al a Phe Pro Thr Ser Gin Val 20 25 30 Arg Arg Gly Asp Phe Thr Glu Asp Thr Thr Pro Asn Arg Pro Val 35 40 45 Tyr Thr Thr Ser Gin Val Gly Gly Leu lie Thr Hi s Val Leu T rp 50 55 60 Glu lie Val Glu Met Arg Lys Glu Leu cys Asn Gly Asn Ser Asp 65 70 75 cys Met Asn Asn Asp Asp Al a Leu Al a Glu Asn Asn Leu Lys Leu 80 85 90 Pro Glu lie Gin Arg Asn Asp Gly cys Tyr Gin Thr Gly Tyr Asn 95 100 105 Gin Glu lie cys Leu Leu Lys lie Ser Ser Gly Leu Leu Glu Tyr 110 115 120 Hi s Ser Tyr Leu Glu Tyr Met Lys Asn Asn Leu Lys Asp Asn Lys 125 130 135 Lys Asp Lys Al a Arg Val Leu Gin Arg Asp Thr Glu Thr Leu lie 140 145 150 Hi s lie Phe Asn Gin Glu Val Lys Asp Leu Hi s Lys lie Val Leu 155 160 165 Pro Thr Pro lie Ser Asn Al a Leu Leu Thr Asp Lys Leu Glu Ser 170 175 180 Page 20

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    Gin Lys Glu Trp Leu Arg Thr Lys Thr Ile Gin Phe Ile Leu Lys 185 190 195

    Ser Leu Glu Glu Phe Leu Lys Val Thr Leu Arg Ser Thr Arg Gin 200 205 210

    Thr <210> 29 <211> 1951 <212> DNA <213> Mus musculus <400> 29

    agaaggaaca gtgggtgtcc aggcacatca gaccaggcag ctcgcagcaa 50 agcaaggtaa gttctctcct cttccctgtc gctaactccc tgcatctaga 100 ggctgtccag attcagactc caggggacag gctacccctg aaccaggcag 150 cgtgggagtg ggatgtgtcc tcagaagcta accatctcct ggtttgccat 200 cgttttgctg gtgtctccac tcatggccat gtgggagctg gagaaagacg 250 tttatgttgt agaggtggac tggactcccg atgcccctgg agaaacagtg 300 aacctcacct gtgacacgcc tgaagaagat gacatcacct ggacctcaga 350 ccagagacat ggagtcatag gctctggaaa gaccctgacc atcactgtca 400 aagagtttct agatgctggc cagtacacct gccacaaagg aggcgagact 450 ctgagccact cacatctgct gctccacaag aaggaaaatg gaatttggtc 500 cactgaaatt ttaaaaaatt tcaaaaacaa gactttcctg aagtgtgaag 550 caccaaatta ctccggacgg ttcacgtgct catggctggt gcaaagaaac 600 atggacttga agttcaacat caagagcagt agcagttccc ctgactctcg 650 ggcagtgaca tgtggaatgg cgtctctgtc tgcagagaag gtcacactgg 700 accaaaggga ctatgagaag tattcagtgt cctgccagga ggatgtcacc 750 tgcccaactg ccgaggagac cctgcccatt gaactggcgt tggaagcacg 800 gcagcagaat aaatatgaga actacagcac cagcttcttc atcagggaca 850 tcatcaaacc agacccgccc aagaacttgc agatgaagcc tttgaagaac 900 tcacaggtgg aggtcagctg ggagtaccct gactcctgga gcactcccca 950 ttcctacttc tccctcaagt tctttgttcg aatccagcgc aagaaagaaa 1000 agatgaagga gacagaggag gggtgtaacc agaaaggtgc gttcctcgta 1050 gagaagacat ctaccgaagt ccaatgcaaa ggcgggaatg tctgcgtgca 1100 agctcaggat cgctattaca attcctcatg cagcaagtgg gcatgtgttc 1150 cctgcagggt ccgatcctag gatgcaacgt tggaaaggaa agaaaagtgg 1200 aagacattaa ggaagaaaaa tttaaactca ggatggaaga gtcccccaaa 1250 agctgtcttc tgcttggttg gctttttcca gttttcctaa gttcatcatg 1300

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    acacctttgc tgatttctac atgtaaatgt taaatgcccg cagagccagg 1350 gagctaatgt atgcatagat attctagcat tccacttggc cttatgctgt 1400 tgaaatattt aagtaattta tgtatttatt aatttatttc tgcatttcac 1450 atttgtatac caagatgtat tgaatatttc atgtgctcgt ggcctgatcc 1500 actgggacca ggccctatta tgcaaattgt gagcttgtta tcttcttcaa 1550 cagctcttca atcagggctg cgtaggtaca ttagcttttg tgacaaccaa 1600 taagaacata atattctgac acaagcagtg ttacatattt gtgaccagta 1650 aagacatagg tggtatttgg agacatgaag aagctgtaaa gttgactctg 1700 aagagtttag cactagtttc aacaccaaga aagacttttt agaagtgata 1750 ttgataagaa accagggcct tctttagaag ggtacctaaa tttaaaagaa 1800 ttttgaaagg ctgggtatcg gtggtatatg cttttaattc cagcactcag 1850 gagaccaagg caggcagatc tctgtgagtt tgaggacagc ctggtgtaca 1900 gagggagttc cagcacagcc agtgccacac agaaattctg tctcaaaaac 1950 a 1951

    <210> 30 <211> 335 <212> PRT <213> Mus musculus

    <400> 30 Τ rp 10 Phe Al a lie Val Leu 15 Met 1 cys Pro Gin Lys 5 Leu Thr lie Ser Leu Val Ser Pro Leu Met Al a Met Τ rp Glu Leu Glu Lys Asp Val 20 25 30 Tyr Val Val Glu Val Asp Τ rp Thr Pro Asp Al a Pro Gly Glu Thr 35 40 45 Val Asn Leu Thr cys Asp Thr Pro Glu Glu Asp Asp lie Thr Τ rp 50 55 60 Thr Ser Asp Gin Arg Hi s Gly Val lie Gly Ser Gly Lys Thr Leu 65 70 75 Thr lie Thr Val Lys Glu Phe Leu Asp Al a Gly Gin Tyr Thr cys 80 85 90 Hi s Lys Gly Gly Glu Thr Leu Ser Hi s Ser Hi s Leu Leu Leu Hi s 95 100 105 Lys Lys Glu Asn Gly lie Τ rp Ser Thr Glu lie Leu Lys Asn Phe 110 115 120 Lys Asn Lys Thr Phe Leu Lys cys Glu Al a Pro Asn Tyr Ser Gly 125 130 135 Arg Phe Thr cys Ser Τ rp Leu Val Gin Arg Asn Met Asp Leu Lys 140 145 150 Phe Asn lie Lys Ser Ser Ser Ser Ser Pro Asp Ser Arg Al a Val 155 160 165 Thr cys Gly Met Al a Ser Leu Ser Al a Glu Lys Val Thr Leu Asp

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    2265275_1

    170 175 180

    Gin Arg Asp Tyr Glu 185 Lys Tyr Ser Val Ser 190 cys Gin Glu Asp Val 195 Thr cys Pro Thr Al a Glu Glu Thr Leu Pro lie Glu Leu Al a Leu 200 205 210 Glu Al a Arg Gin Gin Asn Lys Tyr Glu Asn Tyr Ser Thr Ser Phe 215 220 225 Phe lie Arg Asp lie lie Lys Pro Asp Pro Pro Lys Asn Leu Gin 230 235 240 Met Lys Pro Leu Lys Asn Ser Gin Val Glu Val Ser T rp Glu Tyr 245 250 255 Pro Asp Ser T rp Ser Thr Pro Hi s Ser Tyr Phe Ser Leu Lys Phe 260 265 270 Phe Val Arg lie Gin Arg Lys Lys Glu Lys Met Lys Glu Thr Glu 275 280 285 Glu Gly cys Asn Gin Lys Gly Al a Phe Leu Val Glu Lys Thr Ser 290 295 300 Thr Glu Val Gin cys Lys Gly Gly Asn Val cys Val Gin Al a Gin 305 310 315 Asp Arg Tyr Tyr Asn Ser Ser cys Ser Lys T rp Al a cys Val Pro

    320 325 330

    Cys Arg Val Arg Ser 335 <210> 31 <211> 866 <212> DNA <213> Mus musculus <400> 31

    ggcacagctg gacctggtgg gggttctctg tggttccatg ctttctggac 50 tcctgcctgc tggctggagc tgctgacagg cctgacatct tctgcaacct 100 ccagcatcag gacaaagaaa gccgcctcaa accttccaaa tcacttcctc 150 ttggcccagg aacaatggct gccatgtcag aagactcttg cgtcaacttc 200 aaggaaatga tgtttattga caacacgctt tactttatac ctgaagaaaa 250 tggagacctg gaatcagaca actttggccg acttcactgt acaaccgcag 300 taatacggaa tataaatgac caagttctct tcgttgacaa aagacagcct 350 gtgttcgagg atatgactga tattgatcaa agtgccagtg aaccccagac 400 cagactgata atatacatgt acaaagacag tgaagtaaga ggactggctg 450 tgaccctctc tgtgaaggat agtaaaatgt ctaccctctc ctgtaagaac 500 aagatcattt cctttgagga aatggatcca cctgaaaata ttgatgatat 550 acaaagtgat ctcatattct ttcagaaacg tgttccagga cacaacaaga 600 tggagtttga atcttcactg tatgaaggac actttcttgc ttgccaaaag 650 gaagatgatg ctttcaaact cattctgaaa aaaaaggatg Page 23 aaaatgggga 700

    2265275_1

    2016259423 18 Nov 2016 taaatctgta atgttcactc tcactaactt acatcaaagt taggtgggga 750 gggtttgtgt tccagaaaga tgattagcac acatgcgcct tgtgatgacc 800 tcgcctgtat ttccataaca gaatacccga ggctgcatga tttatagagt 850 aaacacgttt atttgt 866 <210> 32 <211> 192 <212> PRT <213> Mus musculus <400> 32

    Met Ala 1 Al a Met Ser 5 Glu Asp Ser Cys Val 10 Asn Phe Lys Glu Met 15 Met Phe lie Asp Asn Thr Leu Tyr Phe lie Pro Glu Glu Asn Gly 20 25 30 Asp Leu Glu Ser Asp Asn Phe Gly Arg Leu Hi s cys Thr Thr Al a 35 40 45 Val lie Arg Asn lie Asn Asp Gin Val Leu Phe Val Asp Lys Arg 50 55 60 Gin Pro Val Phe Glu Asp Met Thr Asp lie Asp Gin Ser Al a Ser 65 70 75 Glu Pro Gin Thr Arg Leu lie lie Tyr Met Tyr Lys Asp Ser Glu 80 85 90 Val Arg Gly Leu Al a Val Thr Leu Ser Val Lys Asp Ser Lys Met 95 100 105 Ser Thr Leu Ser cys Lys Asn Lys lie lie Ser Phe Glu Glu Met 110 115 120 Asp Pro Pro Glu Asn lie Asp Asp lie Gin Ser Asp Leu lie Phe 125 130 135 Phe Gin Lys Arg Val Pro Gly Hi s Asn Lys Met Glu Phe Glu Ser 140 145 150 Ser Leu Tyr Glu Gly Hi s Phe Leu Al a cys Gin Lys Glu Asp Asp 155 160 165 Al a Phe Lys Leu lie Leu Lys Lys Lys Asp Glu Asn Gly Asp Lys 170 175 180 Ser Val Met Phe Thr Leu Thr Asn Leu Hi s Gin Ser 185 190

    <210> 33 <211> 1121 <212> DNA <213> Mus musculus <400> 33 cctaaacagg ctctcctctc acttatcaac tgttgacact tgtgcgatct 50 ctgatggctg tcctgcagaa atctatgagt ttttccctta tggggacttt 100 ggccgccagc tgcctgcttc tcattgccct gtgggcccag gaggcaaatg 150 cgctgcccgt caacacccgg tgcaagcttg aggtgtccaa cttccagcag 200

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    ccgtacatcg tcaaccgcac ctttatgctg gccaaggagg ccagccttgc 250 agataacaac acagacgtcc ggctcatcgg ggagaaactg ttccgaggag 300 tcagtgctaa agatcagtgc tacctgatga agcaggtgct caacttcacc 350 ctggaagacg ttctgctccc ccagtcagac aggttccagc cctacatgca 400 ggaggtggta cctttcctga ccaaactcag caatcagctc agctcctgtc 450 acatcagcgg tgacgaccag aacatccaga agaatgtcag aaggctgaag 500 gagacagtga aaaagcttgg agagagtgga gagatcaagg cgattgggga 550 actggacctg ctgtttatgt ctctgagaaa tgcttgcgtc tgagcgagaa 600 gaagctagaa aacgaagaac tgctccttcc tgccttctaa aaagaacaat 650 aagatccctg aatggacttt tttactaaag gaaagtgaga agctaacgtc 700 catcatcatt agaagatttc acatgaaacc tggctcagtt gaaaaagaaa 750 atagtgtcaa gttgtccatg agaccagagg tagacttgat aaccacaaag 800 attcattgac aatattttat tgtcactgat gatacaacag aaaaataatg 850 tactttaaaa aattgtttga aaggaggtta cctctcattc ctttagaaaa 900 aaagcttatg taacttcatt tccatatcca atattttata tatgtaagtt 950 tatttattat aagtatacat tttatttatg tcagtttatt aatatggatt 1000 tatttataga aacattatct gctattgata tttagtataa ggcaaataat 1050 atttatgaca ataactatgg aaacaagata tcttaggctt taataaacac 1100 atggatatca taaaaaaaaa a 1121

    <210> 34 <211> 179 <212> PRT <213> Mus musculus

    <400> 34 Leu Gin 5 Lys Ser Met Ser Phe 10 Ser Leu Met Gly Thr 15 Met 1 Ala Val Leu Al a Al a Ser cys Leu Leu Leu lie Al a Leu T rp Ala Gin Glu 20 25 30 Al a Asn Al a Leu Pro Val Asn Thr Arg cys Lys Leu Glu Val Ser 35 40 45 Asn Phe Gin Gin Pro Tyr lie Val Asn Arg Thr Phe Met Leu Al a 50 55 60 Lys Glu Al a Ser Leu Al a Asp Asn Asn Thr Asp Val Arg Leu lie 65 70 75 Gly Glu Lys Leu Phe Arg Gly Val Ser Al a Lys Asp Gin cys Tyr 80 85 90 Leu Met Lys Gin Val Leu Asn Phe Thr Leu Glu Asp Val Leu Leu 95 100 105 Pro Gin Ser Asp Arg Phe Gin Pro Tyr Met Gin Glu Val Val Pro 110 115 120

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    Phe Leu Thr Lys Leu Ser Asn Gin Leu Ser Ser cys His lie Ser 125 130 135 Gly Asp Asp Gin Asn lie Gin Lys Asn Val Arg Arg Leu Lys Glu 140 145 150 Thr Val Lys Lys Leu Gly Glu Ser Gly Glu lie Lys Ala lie Gly 155 160 165 Glu Leu Asp Leu Leu Phe Met Ser Leu Arg Asn Al a cys Val 170 175

    <210> 35 <211> 1359 <212> DNA <213> Mus musculus <400> 35

    cgcttagaag tcggactaca gagttagact cagaaccaaa ggaggtggat 50 agggggtcca caggcctggt gcagatcaca gagccagcca gatctgagaa 100 gcagggaaca agatgctgga ttgcagagca gtaataatgc tatggctgtt 150 gccctgggtc actcagggcc tggctgtgcc taggagtagc agtcctgact 200 gggctcagtg ccagcagctc tctcggaatc tctgcatgct agcctggaac 250 gcacatgcac cagcgggaca tatgaatcta ctaagagaag aagaggatga 300 agagactaaa aataatgtgc cccgtatcca gtgtgaagat ggttgtgacc 350 cacaaggact caaggacaac agccagttct gcttgcaaag gatccgccaa 400 ggtctggctt tttataagca cctgcttgac tctgacatct tcaaagggga 450 gcctgctcta ctccctgata gccccatgga gcaacttcac acctccctac 500 taggactcag ccaactcctc cagccagagg atcacccccg ggagacccaa 550 cagatgccca gcctgagttc tagtcagcag tggcagcgcc cccttctccg 600 ttccaagatc cttcgaagcc tccaggcctt tttggccata gctgcccggg 650 tctttgccca cggagcagca actctgactg agcccttagt gccaacagct 700 taaggatgcc caggttccca tggctaccat gataagacta atctatcagc 750 ccagacatct accagttaat taacccatta ggacttgtgc tgttcttgtt 800 ttgtttgttt tgcgtgaagg gcaaggacac cattattaaa gagaaaagaa 850 acaaacccca gagcaggcag ctggctagag aaaggagctg gagaagaaga 900 ataaagtctc gagcccttgg ccttggaagc gggcaagcag ctgcgtggcc 950 tgaggggaag ggggcggtgg catcgagaaa ctgtgagaaa acccagagca 1000 tcagaaaaag tgagcccagg ctttggccat tatctgtaag aaaaacaaga 1050 aaaggggaac attatacttt cctgggtggc tcagggaaat gtgcagatgc 1100 acagtactcc agacagcagc tctgtacctg cctgctctgt ccctcagttc 1150 taacagaatc tagtcactaa gaactaacag gactaccaat acgaactgac 1200 aaatactacc actatgacct gtgacaaagc tgcatattta ttaagtggga 1250

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    2265275_1 agggaacttt tgatattatt tatccttgta acagtataga tgatggttat 1300 ttattctatt tataaggaat tatgtatttt ttttttcaat aaagatttat 1350 ttatgtggc 1359 <210> 36 <211> 196 <212> PRT <213> Mus musculus

    <400> 36 Arg Ala 5 Val lie Met Leu 10 T rp Leu Leu Pro T rp 15 Met 1 Leu Asp cys Val Thr Gin Gly Leu Al a Val Pro Arg Ser Ser Ser Pro Asp T rp 20 25 30 Al a Gin cys Gin Gin Leu Ser Arg Asn Leu cys Met Leu Al a T rp 35 40 45 Asn Al a Hi s Al a Pro Al a Gly Hi s Met Asn Leu Leu Arg Glu Glu 50 55 60 Glu Asp Glu Glu Thr Lys Asn Asn Val Pro Arg lie Gin cys Glu 65 70 75 Asp Gly cys Asp Pro Gin Gly Leu Lys Asp Asn Ser Gin Phe cys 80 85 90 Leu Gin Arg lie Arg Gin Gly Leu Al a Phe Tyr Lys His Leu Leu 95 100 105 Asp Ser Asp lie Phe Lys Gly Glu Pro Al a Leu Leu Pro Asp Ser 110 115 120 Pro Met Glu Gin Leu Hi s Thr Ser Leu Leu Gly Leu Ser Gin Leu 125 130 135 Leu Gin Pro Glu Asp Hi s Pro Arg Glu Thr Gin Gin Met Pro Ser 140 145 150 Leu Ser Ser Ser Gin Gin T rp Gin Arg Pro Leu Leu Arg Ser Lys 155 160 165 lie Leu Arg Ser Leu Gin Al a Phe Leu Al a lie Al a Ala Arg Val 170 175 180 Phe Al a Hi s Gly Al a Al a Thr Leu Thr Glu Pro Leu Val Pro Thr 185 190 195

    Al a <210> 37 <211> 759 <212> DNA <213> Mus musculus <400> 37 acaccatcca gatctctgga agacagacaa gatgctgcct ccaacagcct 50 gctccgtcat gtcctggatg ctgctctcct gcctgatgct cttatctcag 100 gttcaaggtg aagactccct gaagaatata ccctccgcac gcattagttg 150 ccccaagggc tcccaggctt atggctccta ctgctatgcc ttgtttcaga 200

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    taccacagac ctggtttgat gcagaactgg cctgccaaaa gaggcctgga 250 ggacacctcg tatctgtgct caatagcgct gaggcttcat tcttgtcctc 300 catggtgaag agaacaggaa acagctacca atacacttgg attgggctcc 350 atgaccccac tctgggtgca gaacccaatg gaggtggatg ggaatggagt 400 aacaatgacg tgatgaatta ctttaactgg gagaggaacc catctactgc 450 cttagaccgt gctttctgtg gcagcttgtc aagagcttct ggatttctaa 500 aatggagaga tatgacatgt gaggtgaagt tgccctatgt ctgcaaattt 550 actggttaaa cttatcagac agcaaacatc ccgaatttgt cttgaagagc 600 atcatggaca agggacaaaa tgtgaagact cacctagaaa aagcattttc 650 tatctacagt ccacattaga gccttaatct gctctttcca tatctgtctt 700 tagtcctttt ggtataagtt tgggctcaat tctaaaataa aaataagctt 750

    tctgtcaca 759 <210> 38 <211> 175 <212> PRT <213> Mus musculus

    <400> 38 Met 1 Leu Pro Pro Thr Ala 5 cys Ser Val Met 10 Ser T rp Met Leu Leu 15 Ser cys Leu Met Leu Leu Ser Gin Val Gin Gly Glu Asp Ser Leu 20 25 30 Lys Asn lie Pro Ser Al a Arg lie Ser cys Pro Lys Gly Ser Gin 35 40 45 Al a Tyr Gly Ser Tyr cys Tyr Al a Leu Phe Gin lie Pro Gin Thr 50 55 60 T rp Phe Asp Al a Glu Leu Al a cys Gin Lys Arg Pro Gly Gly Hi s 65 70 75 Leu Val Ser Val Leu Asn Ser Al a Glu Al a Ser Phe Leu Ser Ser 80 85 90 Met Val Lys Arg Thr Gly Asn Ser Tyr Gin Tyr Thr T rp lie Gly 95 100 105 Leu Hi s Asp Pro Thr Leu Gly Al a Glu Pro Asn Gly Gly Gly T rp 110 115 120 Glu T rp Ser Asn Asn Asp Val Met Asn Tyr Phe Asn T rp Glu Arg 125 130 135 Asn Pro Ser Thr Al a Leu Asp Arg Al a Phe cys Gly Ser Leu Ser 140 145 150 Arg Al a Ser Gly Phe Leu Lys T rp Arg Asp Met Thr cys Glu Val 155 160 165 Lys Leu Pro Tyr Val cys Lys Phe Thr Gly 170 175

    <210> 39 <211> 769

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    2016259423 18 Nov 2016 <212> DNA <213> Mus musculus <400> 39

    aagacacctt ggtctcagcc tgcagagatc gttgagttgc atcctaagca 50 aaagactgtc tgctgctcag catggctagg aacgcctact tcatcctgct 100 ctcatgcctg atcgtcctgt ctccaagcca aggccaggaa gctgaagaag 150 acctgccatc tgccaggatc agttgcccag aaggttccaa tgcctacagc 200 tcctattgtt actacttcac tgaagaccgt ttaacttggg ctgatgcaga 250 tcttttttgc cagaacatga attcaggcta cctggtgtca gttctcagtc 300 aggctgaggg caactttgtg gcctctctga ttaaggagag tggcactaca 350 gacgccaatg tctggactgg actccatgat cccaaaagga atcgtcgctg 400 gcactggagc agtgggtctc tgtttctcta caaatcctgg gcaactgggt 450 ctcctaacag ttccaatcgt ggctactgtg tatctctgac ttcaaacaca 500 ggatacaaga aatggaagga tgacaactgt gatgcccaat actcatttgt 550 ctgcaagttc aaaggctgaa gtcacctgaa aaaaaatagt catataaagc 600 aaaattgaaa ttactatagt gtcagaaatt aaattggacc atctatcaaa 650 agcaaattag atcctgtctt cctggagaga cattcttgct tcactgtcct 700 atggtacctg tatctccatt attttctgga aatttgcaca actgaaataa 750 aaacaccttt acaatgttg 769

    <210> 40 <211> 165 <212> PRT <213> Mus musculus

    <400> 40 Asn Al a 5 Tyr Phe lie Leu Leu 10 Ser cys Leu lie Val 15 Met 1 Al a Arg Leu Ser Pro Ser Gin Gly Gin Glu Al a Glu Glu Asp Leu Pro Ser 20 25 30 Al a Arg lie Ser cys Pro Glu Gly Ser Asn Al a Tyr Ser Ser Tyr 35 40 45 cys Tyr Tyr Phe Thr Glu Asp Arg Leu Thr T rp Al a Asp Al a Asp 50 55 60 Leu Phe cys Gin Asn Met Asn Ser Gly Tyr Leu Val Ser Val Leu 65 70 75 Ser Gin Al a Glu Gly Asn Phe Val Al a Ser Leu lie Lys Glu Ser 80 85 90 Gly Thr Thr Asp Al a Asn Val T rp Thr Gly Leu Hi s Asp Pro Lys 95 100 105 Arg Asn Arg Arg T rp Hi s T rp Ser Ser Gly Ser Leu Phe Leu Tyr 110 115 120 Lys Ser T rp Al a Thr Gly Ser Pro Asn Ser Ser Asn Arg Gly Tyr 125 130 135

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    Cys Val Ser Leu Thr Ser Asn Thr Gly Tyr Lys Lys Trp Lys Asp 140 145 150

    Asp Asn Cys Asp Ala Gln Tyr Ser Phe Val Cys Lys Phe Lys Gly 155 160 165 <210> 41 <211> 692 <212> DNA <213> Mus musculus <400> 41

    agtattcatt attcctagct gacagaaatt attgatttag aatttaaatc 50 gaagactgtc tgctgatcag catggctcag aacaatgtat accttatcct 100 gttcttatgc ctgatgttcc tgtcatacag ccaaggccag gtagctgaag 150 aagacttccc cttggctgaa aaagaccttc cttctgccaa aatcaactgc 200 ccagagggtg ccaacgccta tggttcctac tgttattatc taattgaaga 250 ccgtttgacc tggggggagg ctgatctctt ttgccagaac atgaatgcag 300 gtcacctggt gtcaatactc agccaggctg agagcaactt tgtggcctcg 350 ctggttaagg agagtggtac tacagcttcc aatgtctgga ctggacttca 400 tgaccctaaa agtaaccgtc gttggcactg gagcagtggc tccctatttc 450 tcttcaagtc atgggccact ggagctccaa gcactgccaa ccgtggttat 500 tgtgtatcgc tgacttcaaa cacagcatac aaaaaatgga aggacgaaaa 550 ctgtgaggca cagtactcct ttgtctgcaa gttcagagcc taaagtcacc 600 tgaagaacag atgtgcagaa ctctgttact atatattata aatgaaagaa 650 gaccatctat ctgcaaaata aatactcttt cctgtaataa ct 692

    <210> 42 <211> 173 <212> PRT <213> Mus musculus

    <400> 42 Met Ala 1 Gln Asn Asn 5 Val Tyr Leu Ile Leu 10 Phe Leu cys Leu Met 15 Phe Leu Ser Tyr Ser Gln Gly Gln Val Al a Glu Glu Asp Phe Pro 20 25 30 Leu Al a Glu Lys Asp Leu Pro Ser Al a Lys Ile Asn cys Pro Glu 35 40 45 Gly Al a Asn Al a Tyr Gly Ser Tyr cys Tyr Tyr Leu lie Glu Asp 50 55 60 Arg Leu Thr T rp Gly Glu Al a Asp Leu Phe cys Gln Asn Met Asn 65 70 75 Al a Gly Hi s Leu Val Ser Ile Leu Ser Gln Al a Glu Ser Asn Phe 80 85 90 Val Al a Ser Leu Val Lys Glu Ser Gly Thr Thr Al a Ser Asn Val 95 100 105

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    T rp Thr Gly Leu Hi s Asp Pro Lys Ser Asn Arg Arg T rp Hi s T rp 110 115 120 Ser Ser Gly Ser Leu Phe Leu Phe Lys Ser T rp Al a Thr Gly Al a 125 130 135 Pro Ser Thr Al a Asn Arg Gly Tyr cys Val Ser Leu Thr Ser Asn 140 145 150 Thr Al a Tyr Lys Lys T rp Lys Asp Glu Asn cys Glu Ala Gin Tyr 155 160 165

    Ser Phe Val Cys Lys Phe Arg Ala 170 <210> 43 <211> 837 <212> DNA <213> Mus musculus <400> 43

    gcccttcctt caaatcctat cataaagcag tcacctttgt cctgacaaac 50 catctcagat ctctacaaga gagacaagat gctgcctcac ctggtcctca 100 acagtatttc ctggatgctg ctctcctgcc tgttgtttgt atttcaggta 150 caaggtgaag acttccagaa ggaagtgccc tctccacgta ccagctgccc 200 catgggttac aaggcttatc gctcccactg ctatgcctta gttatgacac 250 ctaaatcctg gtttcaagca gatctggtct gccagaagag accctcagga 300 catctcgtgt ctattcttag tggaggtgag gcttcctttg tgtcctcctt 350 ggtgaacggc agagtggaca actaccaaga catctggatt gggctccatg 400 atccaacaat gggtcaacaa cccaatggag gtggatggga gtggagtaac 450 tccgatgtgc tgaattatct taactgggat ggggatcctt cctctactgt 500 caaccgtggt cactgtggga gtctgacagc aagttcaggg tttctgaagt 550 ggggagacta ttactgcgat gggacattac catttgtctg caagttcaag 600 cagtagacaa gcagcatcca gcatttatca tgaagctccc catgacaagg 650 gatgaaatac aagaattcac ccggcaaggc tgtacttgct ttacagttgt 700 gcatcagact tattctggtt ttctgtcctc tttcatccat ctccttcccc 750 ttacttcagg cttttcaata tagttcctgc tttgcaatct tgcagataaa 800 taataaatac aacattttgg ttttactttt gtgtttt 837

    <210> 44 <211> 175 <212> PRT <213> Mus musculus <400> 44

    Met 1 Leu Pro Hi s Leu 5 Val Leu Asn Ser lie 10 Ser Trp Met Leu Leu 15 Ser cys Leu Leu Phe Val Phe Gin Val Gin Gly Glu Asp Phe Gin 20 25 30 Lys Glu Val Pro Ser Pro Arg Thr Ser cys Pro Met Gly Tyr Lys

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    35 40 45 Al a Tyr Arg Ser Hi s cys Tyr Al a Leu Val Met Thr Pro Lys Ser 50 55 60 T rp Phe Gin Al a Asp Leu Val cys Gin Lys Arg Pro Ser Gly Hi s 65 70 75 Leu Val Ser lie Leu Ser Gly Gly Glu Al a Ser Phe Val Ser Ser 80 85 90 Leu Val Asn Gly Arg Val Asp Asn Tyr Gin Asp lie T rp lie Gly 95 100 105 Leu Hi s Asp Pro Thr Met Gly Gin Gin Pro Asn Gly Gly Gly T rp 110 115 120 Glu T rp Ser Asn Ser Asp Val Leu Asn Tyr Leu Asn T rp Asp Gly 125 130 135 Asp Pro Ser Ser Thr Val Asn Arg Gly Hi s cys Gly Ser Leu Thr 140 145 150 Al a Ser Ser Gly Phe Leu Lys T rp Gly Asp Tyr Tyr cys Asp Gly 155 160 165 Thr Leu Pro Phe Val cys Lys Phe Lys Gin

    170 175 <210> 45 <211> 788 <212> DNA <213> Mus musculus

    <400> 45 cccggggcga aatcacctct gagctgtcaa agcattgcag acctctgtat 50 agacagatat accatggtgt ctcacaagac ccttcatagc atgtcctgga 100 tgctactgtg ttgcctgatg tccctttctt gggtacaagg ggaacaatcc 150 cagaaaaaac tgtcttctcc acgcatcagc tgtccccaag aagcccaagc 200 ttatggctcc tattgctatt tactgattct ggaaccacag acctgggcta 250 atgcagagat ccactgccag aagcatttct caggacacct ggcatttctg 300 ctcacttatg gggaaattat ctttgtgtcc tctctggtga aaaacagttt 350 gaccacattc ccatacatct ggattggact ccatgatctg tcacttggga 400 gtttgcccaa tgaaaatgga tggaagtgga gcagctctga ccctctgacc 450 ttctataact gggagatccc accctccatg tctgcacacc acggttactg 500 cgcagctttg tctcaggcct caggttatca gaagtggaga gattattatt 550 gtgacccaac atttccctat gtctgcaaat tcaagggtta ggccagttct 600 gatttcaact gcctgaaagt atcctgaaga tcacatagac aaaggagcga 650 gcatgatggc tcaccaagaa agtccttctc acaccccgac accgaattcc 700 tcatctcatc tctgctgttc ttccataagt gtattctctg gggactctgg 750 cctaaggatt cggagaacta taataaaatt tagtcaat 788

    <210> 46

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    2265275_1

    2016259423 18 Nov 2016 <211> 175 <212> PRT <213> Mus musculus <400> 46

    Met 1 Val Ser Hi s Lys 5 Thr Leu His Ser Met 10 Ser Trp Met Leu Leu 15 cys cys Leu Met Ser Leu Ser T rp Val Gin Gly Glu Gin Ser Gin 20 25 30 Lys Lys Leu Ser Ser Pro Arg Ile Ser cys Pro Gin Glu Al a Gin 35 40 45 Al a Tyr Gly Ser Tyr cys Tyr Leu Leu Ile Leu Glu Pro Gin Thr 50 55 60 T rp Al a Asn Al a Glu Ile Hi s cys Gin Lys Hi s Phe Ser Gly Hi s 65 70 75 Leu Al a Phe Leu Leu Thr Tyr Gly Glu Ile Ile Phe Val Ser Ser 80 85 90 Leu Val Lys Asn Ser Leu Thr Thr Phe Pro Tyr Ile T rp Ile Gly 95 100 105 Leu Hi s Asp Leu Ser Leu Gly Ser Leu Pro Asn Glu Asn Gly T rp 110 115 120 Lys T rp Ser Ser Ser Asp Pro Leu Thr Phe Tyr Asn T rp Glu Ile 125 130 135 Pro Pro Ser Met Ser Al a Hi s Hi s Gly Tyr cys Al a Ala Leu Ser 140 145 150 Gin Al a Ser Gly Tyr Gin Lys T rp Arg Asp Tyr Tyr cys Asp Pro 155 160 165 Thr Phe Pro Tyr Val cys Lys Phe Lys Gly 170 175

    <210> 47 <211> 1024 <212> DNA <213> Mus musculus <400> 47

    ccgactcaag ctgaaaaagg cagggttctc ggagtgttgc ttccaatcag 50 attgcaggtc taggagaatc cacaaaaaag agaagaaaag aaaagaaaaa 100 aactggaacg ggctctgagg gccttgaaat caaagcattg aagtatcatc 150 tccatcgaaa gaggaagatg gcttacaaag gcgtgcggct actcttactg 200 ctgagctggg tagctggccc cgaagtcctg agcgatatct tgagacccag 250 ctgtgcccca ggatggtttt actataggtc ccactgctat ggatacttcc 300 ggaagctaag aaactggtct catgctgagc tggagtgtca gtcatatgga 350 aatggatccc atctggcatc tgtcttgaat caaaaggaag ccagtgtcat 400 atcaaagtac ataactggct atcagagaaa cctgcctgtg tggattggcc 450 tgcatgaccc acaaaagaag caattatggc agtggactga tgggtctaca 500 aacctgtaca gacgctggaa tcccaggaca aagagtgaag Page 33 ccaggcattg 550

    2265275_1

    2016259423 18 Nov 2016

    cgctgagatg aaccccaagg ataaattctt aacttggaac aaaaatggat 600 gtgccaaccg ccaacacttc ctgtgcaagt ataagacata gagcaaaaat 650 caagcgtcta ccagccttgc acaaactctt cccacttccc tctcacctgg 700 tggctgatct aatcattatc ccagagtaaa cactgtagca aacattgagg 750 aggcctccag ggcactggct atcaagccct gcttagcatg gtgggacagt 800 ggcttccggt ctcagagttt agcatggtgg gacggtgact tccggtctca 850 gagattagca tggtgggaca agggcttctg gtctccgtgt tcactctaca 900 atcctttctg gtactcccct tccctctcat tgtcttaaac ageaatgett 950 aacaagctag aaatgtgctt tcttgactac tgcgtctctg tcaaaccagt 1000 aaagttttgg agccaagaaa cage 1024

    <210> 48 <211> 157 <212> PRT <213> Mus musculus <400> 48

    Met 1 Ala Tyr Lys Gly 5 Val Arg Leu Leu Leu 10 Leu Leu Ser T rp Val 15 Al a Gly Pro Glu Val Leu Ser Asp lie Leu Arg Pro Ser cys Al a 20 25 30 Pro Gly T rp Phe Tyr Tyr Arg Ser Hi s cys Tyr Gly Tyr Phe Arg 35 40 45 Lys Leu Arg Asn T rp Ser Hi s Al a Glu Leu Glu cys Gin Ser Tyr 50 55 60 Gly Asn Gly Ser Hi s Leu Al a Ser Val Leu Asn Gin Lys Glu Al a 65 70 75 Ser Val lie Ser Lys Tyr lie Thr Gly Tyr Gin Arg Asn Leu Pro 80 85 90 Val T rp lie Gly Leu Hi s Asp Pro Gin Lys Lys Gin Leu T rp Gin 95 100 105 T rp Thr Asp Gly Ser Thr Asn Leu Tyr Arg Arg T rp Asn Pro Arg 110 115 120 Thr Lys Ser Glu Al a Arg Hi s cys Al a Glu Met Asn Pro Lys Asp 125 130 135 Lys Phe Leu Thr T rp Asn Lys Asn Gly cys Al a Asn Arg Gin Hi s 140 145 150 Phe Leu cys Lys Tyr Lys Thr

    155 <210> 49 <211>

    <212> DNA <213> Homo sapiens <400> 49

    1 atgacaccac ctgaacgtct cttcctccca agggtgtgtg gcaccaccct acacctcctc Page 34

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    2265275_1

    61 cttctggggc tgctgctggt tctgctgcct ggggcccagg ggctccctgg tgttggcctc

    121 acaccttcag ctgcccagac tgcccgtcag caccccaaga tgcatcttgc ccacagcacc

    181 ctcaaacctg ctgctcacct cattggagac cccagcaagc agaactcact gctctggaga

    241 gcaaacacgg accgtgcctt cctccaggat ggtttctcct tgagcaacaa ttctctcctg

    301 gtccccacca gtggcatcta cttcgtctac tcccaggtgg tcttctctgg gaaagcctac

    361 tctcccaagg ccacctcctc cccactctac ctggcccatg aggtccagct cttctcctcc

    421 cagtacccct tccatgtgcc tctcctcagc tcccagaaga tggtgtatcc agggctgcag

    481 gaaccctggc tgcactcgat gtaccacggg gctgcgttcc agctcaccca gggagaccag

    541 ctatccaccc acacagatgg catcccccac ctagtcctca gccctagtac tgtcttcttt

    601 ggagccttcg ctctgtag <210> 50 <211>

    <212> PRT <213> Homo sapiens <400> 50

    MTPPERLFLPRVCGTTLHLLLLGLLLVLLPGAQGLPGVGLTPSAAQTARQHPKMHLAHSTLKPAAHLIGDPSK

    QNSLLWRANTDRAFLQDGFSLSNNSLLVPTSGIYFVYSQWFSGKAYSPKATSSPLYLAHEVQLFSSQYPFHV

    PLLSSQKMVYPGLQEPWLHSMYHGAAFQLTQGDQLSTHTDGIPHLVLSPSTVFFGAFAL <210> 51 <211>

    <212> DNA <213> Homo sapiens <400> 51

    1 atgggggcac tggggctgga gggcaggggt gggaggctcc aggggagggg ttccctcctg

    61 ctagctgtgg caggagccac ttctctggtg accttgttgc tggcggtgcc tatcactgtc

    121 ctggctgtgc tggccttagt gccccaggat cagggaggac tggtaacgga gacggccgac

    181 cccggggcac aggcccagca aggactgggg tttcagaagc tgccagagga ggagccagaa

    241 acagatctca gccccgggct cccagctgcc cacctcatag gcgctccgct gaaggggcag

    301 gggctaggct gggagacgac gaaggaacag gcgtttctga cgagcgggac gcagttctcg

    361 gacgccgagg ggctggcgct cccgcaggac ggcctctatt acctctactg tctcgtcggc

    421 taccggggcc gggcgccccc tggcggcggg gacccccagg gccgctcggt cacgctgcgc

    481 agctctctgt accgggcggg gggcgcctac gggccgggca ctcccgagct gctgctcgag

    541 ggcgccgaga cggtgactcc agtgctggac ccggccagga gacaagggta cgggcctctc

    601 tggtacacga gcgtggggtt cggcggcctg gtgcagctcc ggaggggcga gagggtgtac

    661 gtcaacatca gtcaccccga tatggtggac ttcgcgagag ggaagacctt ctttggggcc

    721 gtgatggtgg ggtga <210> 52 <211>

    <212> PRT <213> Homo sapiens <400> 52

    MGALGLEGRGGRLQGRGSLLLAVAGATSLVTLLLAVPITVLAVLALVPQDQGGLVTETADPGAQAQQGLGFQKLPEEEPE

    TDL

    SPGLPAAHLIGAPLKGQGLGWETTKEQAFLTSGTQFSDAEGLALPQDGLYYLYCLVGYRGRAPPGGGDPQGRSVTLRSSL

    YRA

    GGAYGPGTPELLLEGAETVTPVLDPARRQGYGPLWYTSVGFGGLVQLRRGERVYVNISHPDMVDFARGKTFFGAVMVG <210> 53 <211>

    <212> DNA <213> Mus musculus <400> 53

    1 atgacactgc tcggccgtct ccacctcttg agggtgcttg gcacccctcc tgtcttcctc

    61 ctggggctgc tgctggccct gcctctaggg gcccagggac tctctggtgt ccgcttctcc

    121 gctgccagga cagcccatcc actccctcag aagcacttga cccatggcat cctgaaacct

    181 gctgctcacc ttgttgggta ccccagcaag cagaactcac tgctctggag agcaagcacg

    241 gatcgtgcct ttctccgaca tggcttctct ttgagcaaca actccctcct gatccccacc

    301 agtggcctct actttgtcta ctcccaggtg gttttctctg gagaaagctg ctcccccagg

    361 gccattccca ctcccatcta cctggcacac gaggtccagc tcttttcctc ccaatacccc

    Page 35

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    421 ttccatgtgc ctctcctcag tgcgcagaag tctgtgtatc cgggacttca aggaccgtgg 481 gtgcgctcaa tgtaccaggg ggctgtgttc ctgctcagta agggagacca gctgtccacc 541 cacaccgacg gcatctccca tctacacttc agccccagca gtgtattctt tggagccttt 601 gcactgtag <210> 54 <211>

    <212> PRT <213> Mus musculus <400> 54

    MTLLGRLHLL RVLGTPPVFL LGLLLALPLG AQGLSGVRFS AARTAHPLPQ KHLTHGILKPAAHLVGYPSK QNSLLWRAST DRAFLRHGFS LSNNSLLIPT SGLYFVYSQV VFSGESCSPRAIPTPIYLAH EVQLFSSQYP FHVPLLSAQK SVYPGLQGPW VRSMYQGAVF LLSKGDQLSTHTDGISHLHF SPSSVFFGAFAL <210> 55 <211>

    <212> DNA <213> Mus musculus <400> 55

    1 atgacactgc tcggccgtct ccacctcttg agggtgcttg gcacccctcc tgtcttcctc

    61 ctggggctgc tgctggccct gcctctaggg gcccagggac tctctggtgt ccgcttctcc

    121 gctgccagga cagcccatcc actccctcag aagcacttga cccatggcat cctgaaacct

    181 gctgctcacc ttgttgggta ccccagcaag cagaactcac tgctctggag agcaagcacg

    241 gatcgtgcct ttctccgaca tggcttctct ttgagcaaca actccctcct gatccccacc

    301 agtggcctct actttgtcta ctcccaggtg gttttctctg gagaaagctg ctcccccagg

    361 gccattccca ctcccatcta cctggcacac gaggtccagc tcttttcctc ccaatacccc

    421 ttccatgtgc ctctcctcag tgcgcagaag tctgtgtatc cgggacttca aggaccgtgg

    481 gtgcgctcaa tgtaccaggg ggctgtgttc ctgctcagta agggagacca gctgtccacc

    541 cacaccgacg gcatctccca tctacacttc agccccagca gtgtattctt tggagccttt

    601 gcactgtag <210> 56 <211>

    <212> PRT <213> Mus musculus <400> 56

    MGTRGLQGLG GRPQGRGCLL LAVAGATSLV TLLLAVPITV LAVLALVPQD QGRRVEKIIGSGAQAQKRLD DSKPSCILPS PSSLSETPDP RLHPQRSNAS RNLASTSQGP VAQSSREASAWMTILSPAAD STPDPGVQQL PKGEPETDLN PELPAAHLIG AWMSGQGLSW EASQEEAFLRSGAQFSPTHG LALPQDGVYY LYCHVGYRGR TPPAGRSRAR SLTLRSALYR AGGAYGRGSPELLLEGAETV TPWDPIGYG SLWYTSVGFG GLAQLRSGER VYVNISHPDM VDYRRGKTFF GAVMVG <210> 57 <211>

    <212> DNA <213> Homo sapiens <400> 57

    ATGCTCCTGCCTTGGGCCACCTCTGCCCCCGGCCTGGCCTGGGGGCCTCTGGTGCTGGGCCTCTTCGGGCTCCTGGCAGC

    ATCGCAGCCCCAGGCGGTGCCTCCATATGCGTCGGAGAACCAGACCTGCAGGGACCAGGAAAAGGAATACTATGAGCCCC

    AGCACCGCATCTGCTGCTCCCGCTGCCCGCCAGGCACCTATGTCTCAGCTAAATGTAGCCGCATCCGGGACACAGTTTGT

    GCCACATGTGCCGAGAATTCCTACAACGAGCACTGGAACTACCTGACCATCTGCCAGCTGTGCCGCCCCTGTGACCCAGT

    GATGGGCCTCGAGGAGATTGCCCCCTGCACAAGCAAACGGAAGACCCAGTGCCGCTGCCAGCCGGGAATGTTCTGTGCTG

    CCTGGGCCCTCGAGTGTACACACTGCGAGCTACTTTCTGACTGCCCGCCTGGCACTGAAGCCGAGCTCAAAGATGAAGTT

    GGGAAGGGTAACAACCACTGCGTCCCCTGCAAGGCAGGGCACTTCCAGAATACCTCCTCCCCCAGCGCCCGCTGCCAGCC

    CCACACCAGGTGTGAGAACCAAGGTCTGGTGGAGGCAGCTCCAGGCACTGCCCAGTCCGACACAACCTGCAAAAATCCAT

    TAGAGCCACTGCCCCCAGAGATGTCAGGAGGG <210> 58 <211>

    Page 36

    2016259423 18 Nov 2016

    2265275_1 <212> DNA <213> Homo sapiens <400> 58

    CGCGCCCAGGTCACCGACAAAGCTGCGCACTATACTCTGTGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTC

    AGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACG

    TGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGG

    GAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTA

    CAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAAC

    CACAGGTGTACACCCTGCCCCCATCCCGGGAAGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTC

    TATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGA

    CTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCT

    CCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAAT <210> 59 <211>

    <212> DNA <213> Mus musculus <400> 59

    ATGCGCCTGCCCCGGGCCTCCTCTCCCTGCGGCCTGGCCTGGGGGCCACTCCTGCTGGGACTCAGCGGGCTTCTGGTGGC

    CTCTCAGCCCCAGCTGGTGCCCCCTTATCGCATAGAAAACCAGACTTGCTGGGACCAGGACAAGGAATACTACGAGCCCA

    TGCACGACGTCTGCTGCTCCCGCTGTCCCCCAGGCGAGTTTGTCTTTGCGGTATGCAGCCGCAGCCAAGACACGGTTTGC

    AAGACTTGCCCCCATAATTCCTATAATGAACACTGGAACCATCTCTCCACCTGCCAGCTGTGCCGCCCCTGTGACATTGT

    GCTGGGCTTTGAGGAGGTTGCCCCTTGCACCAGCGATCGGAAAGCCGAGTGCCGCTGTCAGCCGGGGATGTCCTGTGTGT

    ATCTGGACAATGAGTGTGTGCACTGTGAGGAGGAGCGGCTTGTACTCTGCCAGCCTGGCACAGAAGCCGAGGTCACAGAT

    GAAATTATGGATACTGACGTCAACTGTGTCCCCTGTAAGCCGGGACACTTCCAGAACACTTCCTCCCCTCGAGCCCGCTG

    TCAACCCCATACCAGATGTGAGATCCAGGGCCTGGTGGAGGCAGCTCCAGGTACCTCCTACTCGGATACCATCTGTAAAA <210> 60 <211>

    <212> DNA <213> Mus musculus <400> 60

    CTCGAGGACCCACAATCAAGCCCTGTCCTCCATGCAAATGCCCAGCACCTAACCTCTTGGGTGGACCATCCGTCTTCATC

    TTCCCTCCAAAGATCAAGGATGTACTCATGATCTCCCTGAGCCCCATAGTCACATGTGTGGTGGTGGATGTGAGCGAGGA

    TGACCCAGATGTCCAGATCAGCTGGTTTGTGAACAACGTGGAAGTACACACAGCTCAGACACAAACCCATAGAGAGGATT

    ACGACAGTACTCTACGCGTGGTCAGTGCCCTCCCCATCCAGCACCAGGACTGGATGAGTGGCAAGGAGTTCAAATGCAAG

    GTCAACAACAAAGACCTCCCAGCGCCCATCGAGAGAACCATCTCAAAACCCAAAGGGTCAGTAAGAGCTCCACAGGTATA

    TGTCTTGCCTCCACCAGAAGAAGAGATGACTAAGAAACAGGTCACTCTGACCTGCATGGTCACAGACTTCATGCCTGAAG

    ACATTTACGTGGAGTGGACCAACAACGGGAAAACAGAGCTAAACTACAAGAACACTGAACCAGTCCTGGACTCTGATGGT

    TCTTACTTCATGTACAGCAAGCTGAGAGTGGAAAAGAAGAACTGGGTGGAAAGAAATAGCTACTCCTGTTCAGTGGTCCA

    CGAGGGTCTGCACAATCACCACACGACTAAGAGCTTCTCCCGGACTCCGGGTAAATGAAAGCTTGGCCGCCATGGCCC <210> 61 <211>

    <212> PRT <213> Homo sapiens <400> 61

    MKHQHQHQHQHQHQMHQAQTARQHPKMHLAHSTLKPAAHLIGDPSKQNSLLWRANTDRAFLQDGFSLSNNSLLVPTSGIYFVYSQWFSGKAYSPKATSSPLYLAH

    E

    VQLFSSQYPFHVPLLSSQKMVYPGLQEPWLHSMYHGAAFQLTQGDQLSTHTDGIPHLVLSPSTVFFGAFAL

    Page 37