EA052811B1 - ANTIBODIES AGAINST IL-27 AND THEIR APPLICATION - Google Patents

Abstract

The invention relates to antibodies against IL-27 and antigen-binding portions thereof. This invention also relates to methods for treating or ameliorating one or more symptoms of a disease, such as cancer, by administering said antibodies or antigen-binding portions thereof. This invention also relates to methods for detecting IL-27, for example, in a subject or in a sample.

Description

The contents of the electronic sequence listing in the ASCII text file (named 4416_009PC02_Seqlisting_ST25.txt; size: 156863 bytes; created on September 25, 2020) filed with this application are incorporated herein by reference in their entirety.

Cross-reference to related applications

This invention claims priority to U.S. Provisional Patent Application No. 62/906,008, filed September 25, 2019, and U.S. Provisional Patent Application No. 63/081,705, filed September 22, 2020, the contents of which are incorporated herein by reference in their entirety.

Field of invention

This invention generally relates to compositions and methods for modulating IL-27-associated signaling. More specifically, this invention relates to immunogenic compositions (e.g., antibodies, antibody fragments, etc.) that bind to IL-27 and modulate IL-27 signaling.

State of the art of the invention

In recent years, mounting evidence has emerged that the immune system acts as a significant barrier preventing the formation and progression of tumors. The principle that cancer patients possess naturally occurring T cells with antitumor potential or activity has led to the development of immunotherapeutic approaches in oncology. Immune cells such as T cells, macrophages, and natural killer cells can exhibit antitumor activity and effectively control the occurrence and growth of malignant tumors. Tumor-specific or tumor-associated antigens can induce immune cells to recognize and destroy malignant tumors (Chen & Mellman, (2013) Immunity 39 (1): 1–10). Despite the presence of a tumor-specific immune response, malignant tumors often avoid or elude immune attack through various immunomodulatory mechanisms, resulting in an inability to control tumor initiation and progression (Motz & Coukos, (2013) Immunity 39 (1): 61–730). Indeed, an emerging hallmark of cancer is the use of such immunomodulatory mechanisms and the incapacitation of the antitumor immune response, resulting in tumor evasion from immunological killing (Hanahan and Weinberg (2011) Cell 144 (5): 646–674).

IL-27 is a heterodimeric cytokine composed of two subunits (EBI3 and IL27p28). IL-27 is structurally related to both the IL-12 and IL-6 cytokine families. IL-27 binds to and mediates signaling through a heterodimeric receptor composed of the IL-27Pα (WSX1) and gp130 chains, which mediate signaling primarily through STAT1 and STAT3. In early publications, IL-27 was characterized as an immunostimulatory cytokine that supports CD4+ T-cell proliferation, T helper (Th)1 differentiation, and IFN-γ production, often acting in concert with IL-12. Subsequent studies have shown that IL-27 exerts complex immunomodulatory functions, resulting in either pro- or anti-inflammatory effects depending on the biological context and experimental models used. IL-27 can drive the expression of various immunoregulatory molecules in human cancer cells, which may contribute to local impairment of the immune response in vivo (Fabbi et al. (2017) Mediators Inflamm 3958069; published online February 1, 2017, doi:10.1155/2017/3958069; and references therein).

Despite significant advances in treatment and supportive care for patients with cancer, there remains a continuing need for new and effective treatments and supportive care for cancer.

Brief description of the invention

Disclosed herein are antibodies or antigen-binding portions thereof that are IL-27 antagonists and specifically bind to an epitope comprising one or more of the following amino acids: (i) amino acids 37 to 56 of SEQ ID NO: 2 (IL-27p28); (ii) amino acids 142 to 164 of SEQ ID NO: 2 (IL-27p28); or (iii) both (i) and (ii). In some aspects of the invention, the antibody or antigen-binding portion thereof specifically binds to an epitope comprising one or more amino acids Gln37, Leu38, Glu42, Glu46, Val49, Ser50, Leu53, Lys56, Leu142, Asp143, Arg145, Asp146, Leu147, Arg149, His150, Arg152, Phe153, Leu156, Ala157, Gly159, Phe160, Asn161, Leu162, Pro163 or Glu164 according to SEQ ID NO: 2 (IL27p28).

In some aspects of the invention, an antibody of the invention or an antigen-binding portion of the antibody specifically binds to an epitope comprising Asp146, Arg149, and/or Phe153 according to SEQ ID NO: 2 (IL-27p28). In some aspects of the invention, the epitope further comprises His150 and/or Leu156 according to SEQ ID NO: 2 (IL-27p28). In some aspects of the invention, the epitope further comprises Gln37,

- 1 052811

Leu38, Glu42, Leu142 and/or Glu164 according to SEQ ID NO: 2 (IL-27p28). In some aspects of the invention, the epitope further comprises Glu46, Val49, Ser50 and/or Leu162 according to SEQ ID NO: 2 (IL-27p28). In some aspects of the invention, the epitope further comprises one or more amino acids Leu53, Lys56, Asp143, Arg145, Leu147, Arg152, Ala157, Gly159, Phe160, Asn161 or Pro163 according to SEQ ID NO: 2 (HH-27p28).

In some aspects of the invention, an antibody of the invention or an antigen-binding portion of said antibody specifically binds to an epitope consisting of or consisting essentially of Gln37, Leu38, Glu42, Glu46, Val49, Ser50, Leu142, Asp146, Arg149, His150, Phe153, Leu156, Leu162 and Glu164 according to SEQ ID NO: 2 (HH-27p28).

In some aspects of the invention, an antibody of the invention or an antigen-binding portion of the antibody specifically binds to an epitope consisting or consisting essentially of Gln37, Leu38, Glu42, Glu46, Val49, Ser50, Leu53, Lys56, Leu142, Asp143, Arg145, Asp146, Leu147, Arg149, His150, Arg152, Phe153, Leu156, Ala157, Gly159, Phe160, Asn161, Leu162, Pro163 and Glu164 according to SEQ ID NO: 2 (HH-27p28).

In other aspects of the invention, an antibody or antigen-binding portion thereof that specifically binds to an epitope comprising one or more of the amino acids Gln37, Leu38, Glu42, Glu46, Val49, Ser50, Leu53, Lys56, Leu142, Asp143, Arg145, Asp146, Leu147, Arg149, His150, Arg152, Phe153, Leu156, Ala157, Gly159, Phe160, Asn161, Leu162, Pro163 and Glu164 according to SEQ ID NO: 2 (HH-27p28) lacks heavy and light chain CDRs selected from the group consisting of: (i) the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 9, 10 and 11, respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 18 and 19, respectively; (ii) the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 31, 32 and 33, respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 39, 40 and 41, respectively; (iii) the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 53, 54 and 55, respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 61, 62 and 63, respectively; (iv) the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 75, 76 and 77, respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 83, 84 and 85, respectively; (v) the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 97, 98 and 99, respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 105, 106 and 107, respectively; or (vi) the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 119, 120 and 121, respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 127, 128 and 129, respectively.

In other aspects of the invention, an antibody or antigen-binding portion thereof that specifically binds to an epitope comprising one or more of the amino acids Gln37, Leu38, Glu42, Glu46, Val49, Ser50, Leu53, Lys56, Leu142, Asp143, Arg145, Asp146, Leu147, Arg149, His150, Arg152, Phe153, Leu156, Ala157, Gly159, Phe160, Asn161, Leu162, Pro163 and Glu164 according to SEQ ID NO: 2 (HH-27p28) lacks heavy and light chain CDRs selected from the group consisting of: (i) the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 12, 13 and 14, respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 20, 21 and 22, respectively; (ii) the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 34, 35 and 36, respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 42, 43 and 44, respectively; (iii) the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 56, 57 and 58, respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 64, 65 and 66, respectively; (iv) the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 78, 79 and 80, respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 86, 87 and 88, respectively; (v) the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 100, 101 and 102, respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 108, 109 and 110, respectively; or (vi) the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 122, 123 and 124, respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 130, 131 and 132, respectively.

In some aspects of the invention, the antibody heavy chain CDR1 or antigen-binding portion thereof does not consist of N-GFTF[S/A/R][S/R][T/Y][G/S]-C (SEQ ID NO: 144) and/or the heavy chain CDR2 does not consist of N-ISSS[S/G][S/A]YI-C (SEQ ID NO: 146). In some aspects of the invention, the antibody heavy chain CDR1 or antigen-binding portion thereof does not consist of NFTF[S/A/R][S/R][T/Y][G/S]MN-C (SEQ ID NO: 148) and/or the heavy chain CDR2 does not consist of N[G/S]ISSS[S/G][S/A]YI[L/Y]YADSVKG-C (SEQ ID NO: 149).

In other aspects of the invention, an antibody or antigen-binding portion thereof that specifically binds to an epitope comprising one or more amino acids of Gln37,

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Leu38, Glu42, Glu46, Val49, Ser50, Leu53, Lys56, Leu142, Asp143, Arg145, Asp146, Leu147, Arg149, His150, Arg152, Phe153, Leu156, Ala157, Gly159, Phe160, Asn161, Leu162, Pro163 and Glu164 according to SEQ ID NO: 2 (IL-27p28) do not contain: (i) a heavy chain CDR1 consisting of N-GFTFXXXX-C (SEQ ID NO: 145), a heavy chain CDR2 consisting of N-ISSSXXYI-C (SEQ ID NO: 147), and the heavy chain CDR3 sequence presented in SEQ ID NO: 121; and the light chain CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 127, 128, and 129, respectively; or (ii) a heavy chain CDR1 consisting of N-FTFXXXXMN-C (SEQ ID NO: 150), a heavy chain CDR2 consisting of NXISSSXXYIXYADSVKG-C (SEQ ID NO: 151), and a heavy chain CDR3 sequence shown in SEQ ID NO: 124; and the light chain CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 130, 131, and 132, respectively.

In other aspects of the invention, an antibody or antigen-binding portion thereof that specifically binds to an epitope comprising one or more of the amino acids Gln37, Leu38, Glu42, Glu46, Val49, Ser50, Leu53, Lys56, Leu142, Asp143, Arg145, Asp146, Leu147, Arg149, His150, Arg152, Phe153, Leu156, Ala157, Gly159, Phe160, Asn161, Leu162, Pro163 and Glu164 according to SEQ ID NO: 2 (IL-27p28) does not comprise: a heavy chain CDR1 consisting of N-GFTFXXXX-C (SEQ ID NO: 145), a heavy chain CDR2 consisting of N-IXXXXXXX-C (SEQ ID NO: 152), and a heavy chain CDR3 sequence consisting of N-AR[X] _n = ₆ _ ₁₅ DX-C (SEQ ID NO: 153); and a light chain CDR1 consisting of N-QS[X] _n = ₁ . ₃ SS[X] _n = ₀ . ₄ YC (SEQ ID NO: 154), a light chain CDR2 consisting of N-XXS-C (SEQ ID NO: 155), and a light chain CDR3 sequence consisting of N-QQXXXXP[X] _n=0-1 TC (SEQ ID NO: 156), respectively.

Disclosed herein are antibodies or antigen-binding portions thereof that exhibit at least one or more of the following properties: (i) bind to human IL-27 with an equilibrium dissociation constant (KD) of 15 nM or less; (ii) block the binding of IL-27 to the IL-27 receptor; (iii) inhibit or reduce the phosphorylation of STAT1 and/or STAT3 in a cell; (iv) inhibit or reduce IL-27-mediated inhibition of CD161 expression in a cell; (v) inhibit or reduce IL-27-mediated expression of PD-L1 and/or TIM-3 in a cell; and (vi) induce or enhance PD-1-mediated secretion of one or more cytokines from a cell.

In some aspects of the present invention, said isolated antibody or antigen-binding portion thereof binds to human IL-27 with an equilibrium dissociation constant (KD) of 15 nM or less.

In other aspects of the present invention, said isolated antibody or antigen-binding portion thereof inhibits or reduces phosphorylation of STAT1 and/or STAT3 in a cell. In some aspects of the present invention, said isolated antibody or antigen-binding portion thereof reduces phosphorylation of STAT1 and/or STAT3 in an immune cell or a cancer cell.

In some aspects of the present invention, said isolated antibody or antigen-binding portion thereof inhibits or reduces the inhibition of CD161 expression in a cell. In some aspects of the present invention, said isolated antibody or antigen-binding portion thereof inhibits or reduces the inhibition of CD161 expression in an immune cell.

In other aspects of the invention, said isolated antibody or antigen-binding portion thereof inhibits or reduces the expression of PD-L1 and/or TIM-3 in a cell. In some aspects of the invention, said isolated antibody or antigen-binding portion thereof inhibits or reduces the expression of PD-L1 and/or TIM-3 in an immune cell or a cancer cell. In some aspects of the invention, said isolated antibody or antigen-binding portion thereof inhibits or reduces the expression of PD-L1 in a cancer cell.

In some aspects of the invention, said isolated antibody or antigen-binding portion thereof induces or enhances PD1-mediated secretion of one or more cytokines from a cell. In some aspects of the invention, said one or more cytokines are IFN-gamma (IFN-γ), IL-17, TNF-alpha (TNF-α), or IL-6. In some aspects of the invention, the antibody or antigen-binding portion thereof is selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE antibodies. In other aspects of the invention, the antibody or antigen-binding portion thereof is an IgG1 antibody or an IgG4 antibody. In some aspects of the invention, the antibody or antigen-binding portion thereof comprises an Fc domain comprising at least one mutation. Also provided herein are pharmaceutical compositions comprising any of the described isolated antibodies or antigen-binding portions thereof and a pharmaceutically acceptable carrier. Also provided are nucleic acids comprising a nucleotide sequence encoding the light chain, heavy chain, or both light and heavy chains of said isolated antibody, or an antigen-binding portion thereof. Also provided herein is an expression vector comprising said nucleic acid. Also provided is a cell transformed with said expression vector.

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The present invention also provides methods for producing an antibody that specifically binds human IL-27, or an antigen-binding portion thereof, comprising culturing a cell transformed with said expression vector under conditions that allow expression of said antibody or antigen-binding portion thereof. In some aspects of the present invention, said method further comprises producing the antibody or antigen-binding portion thereof.

This document provides a method for inhibiting or reducing phosphorylation of STAT1 and/or STAT3 in a cell, comprising contacting said cell with said antibody or antigen-binding portion thereof, wherein said antibody or antigen-binding portion thereof inhibits or reduces phosphorylation of STAT1 and/or STAT3 in the cell.

Also provided is a method for inhibiting or reducing the inhibition of CD161 expression in a cell, comprising bringing said cell into contact with said antibody or antigen-binding portion thereof, wherein said antibody or antigen-binding portion thereof inhibits or reduces the inhibition of CD161 expression in the cell.

Also provided is a method for inhibiting or reducing the expression of PD-L1 and/or TIM-3 in a cell, comprising bringing said cell into contact with said antibody or antigen-binding portion thereof, wherein said antibody or antigen-binding portion thereof inhibits the expression of PD-L1 and/or TIM-3 in the cell.

Also provided is a method for inducing or enhancing the secretion of one or more cytokines from a cell, comprising bringing said cell into contact with said antibody or antigen-binding portion thereof, wherein said antibody or antigen-binding portion thereof induces or enhances PD-1-mediated secretion of one or more cytokines from the cell.

Also provided is a method for stimulating an immune response in a subject, comprising administering to said subject an effective amount of an isolated antibody as provided herein, or an antigen-binding fragment of said antibody, or a pharmaceutical composition as provided herein.

Also provided is a method for treating an oncological disease in a subject, comprising administering to said subject an effective amount of an isolated antibody as provided herein, or an antigen-binding fragment of said antibody, or a pharmaceutical composition as provided herein.

This document provides a method for stimulating an immune response or treating an oncological disease in a subject. This method comprises administering to said subject an effective amount of an isolated antibody as provided herein, or an antigen-binding portion of said antibody, or a pharmaceutical composition as provided herein, wherein said antibody, its antigen-binding portion, or pharmaceutical composition inhibits or reduces phosphorylation of STAT1 and/or STAT3 in a cell, thereby stimulating an immune response or treating an oncological disease.

A method for stimulating an immune response or treating an oncological disease in a subject is also provided. Said method comprises administering to said subject an effective amount of an isolated antibody as provided herein, or an antigen-binding portion of said antibody, or a pharmaceutical composition as provided herein, wherein said antibody, its antigen-binding portion, or pharmaceutical composition inhibits or reduces the inhibition of CD161 expression in a cell, thereby stimulating an immune response or treating an oncological disease.

A method for stimulating an immune response or treating an oncological disease in a subject is also provided. Said method comprises administering to said subject an effective amount of an isolated antibody as provided herein, or an antigen-binding portion of said antibody, or a pharmaceutical composition as provided herein, wherein said antibody, its antigen-binding portion, or pharmaceutical composition inhibits or reduces the expression of PD-L1 and/or TIM-3 on a cell, thereby stimulating an immune response or treating an oncological disease.

A method for stimulating an immune response or treating an oncological disease in a subject is also provided. Said method comprises administering to said subject an effective amount of an isolated antibody as provided herein, or an antigen-binding portion of said antibody, or a pharmaceutical composition as provided herein, wherein said antibody, its antigen-binding portion, or pharmaceutical composition induces or enhances PD-1-mediated secretion of one or more cytokines from a cell, thereby stimulating an immune response or treating an oncological disease.

In some aspects of the invention, the oncological disease treated by the method is selected from lung cancer (e.g., non-small cell lung cancer), sarcoma, testicular cancer, ovarian cancer, pancreatic cancer, breast cancer (e.g., triple-negative breast cancer),

- 4 052811 breast), melanoma, head and neck cancer (e.g., squamous cell carcinoma of the head and neck), colorectal cancer, bladder cancer, endometrial cancer, prostate cancer, thyroid cancer, hepatocellular carcinoma, stomach cancer, brain cancer, lymphoma (e.g., diffuse large B-cell lymphoma - DL-BCL), leukemia (e.g., acute myeloid leukemia - AML), or kidney cancer (e.g., renal cell carcinoma, e.g., clear cell renal cell carcinoma).

This document provides a method for enhancing one or more functions of an anti-PD-1 antibody (e.g., enhancing PD-1-mediated cytokine secretion; enhancing anti-PD-1 antibody-mediated TNF-α secretion; enhancing anti-PD-1 antibody-mediated IL-6 secretion from a cell exposed to anti-PD-1 antibodies). The method comprises exposing the cell to the antibody provided herein or an antigen-binding portion thereof simultaneously with or sequentially with the anti-PD-1 antibody, thereby enhancing said one or more functions of the anti-PD-1 antibodies.

Also provided is a pharmaceutical composition comprising an anti-PD-1 antibody, the antibody or antigen-binding portion thereof as provided herein, and a pharmaceutically acceptable carrier.

Also provided is a kit comprising an anti-PD-1 antibody and the antibody or antigen-binding portion thereof provided herein, for simultaneous or sequential administration, and instructions for use thereof.

This document describes all of the methods provided herein for stimulating an immune response or treating an oncological disease, wherein the isolated antibody provided herein, or an antigen-binding portion thereof, is administered in combination with one or more additional therapeutic agents or procedures. The second therapeutic agent or procedure is selected from the group consisting of: chemotherapy, targeted anticancer therapy, an oncolytic drug, a cytotoxic agent, immune therapy, a cytokine, a surgical procedure, a radiation procedure, an activator of a costimulatory molecule, an inhibitor of an inhibitory molecule, a vaccine, or cellular immunotherapy, or a combination thereof. In some aspects of the present invention, said one or more additional therapeutic agents are a PD-1 antagonist, a PD-L1 inhibitor, a TIM-3 inhibitor, a LAG-3 inhibitor, a TIGIT inhibitor, a CD112R inhibitor, a TAM inhibitor, a STING agonist, a 41BB agonist, a tyrosine kinase inhibitor, an agent targeting the adenosine axis (e.g., a CD39 antagonist, a CD73 antagonist, an A2AR antagonist or an A2BR antagonist, or a dual A2AR/A2BR antagonist), a CCR8 antagonist, a CTLA4 antagonist, a VEG-F inhibitor, or a combination thereof. In other aspects of the present invention, said one or more additional therapeutic agents are a PD-1 antagonist. In some aspects of the present invention, said PD-1 antagonist is selected from the group consisting of: PDR001, nivolumab, pembrolizumab, pidilizumab, MEDI0680, REGN2810, TSR-042, PF-06801591, and AMP-224. In some aspects of the present invention, said PDL1 inhibitor is selected from the group consisting of: FAZ053, atezolizumab, avelumab, durvalumab, and BMS-936559. In other aspects of the invention, said one or more additional therapeutic agents are selected from the group consisting of: sunitinib (SUTENT®), cabozantinib (CABOMETYX®), axitinib (INLYTA®), lenvatinib (LENVIMA®), everolimus (AFINITOR®), bevacizumab (AVASTIN®), epacadostat, NKTR-214 (a CD-122 biased agonist), tivozanib (FOTIVDA®), abexinostat, ipilimumab (YERVOY®), tremelimumab, pazopanib (VOTRIENT®), sorafenib (NEXAVAR®), temsirolimus (TORISEL®), ramucirumab (CYRAMZA®), niraparib, savolitinib, vorolanib (X-82), regorafenib (STIVARGO®), donafenib (multikinase inhibitor), camrelizumab (SHR-1210), pexastimogene devacirepvec (JX-594), ramucirumab (Cyramza®), apatinib (YN968D1), encapsulated doxorubicin (THERMODOX®), tivantinib (ARQ197), ADI-PEG 20, binimetinib, apatinib mesylate, nintedanib, lirilumab, nivolumab (OPDIVO®), pembrolizumab (KEYTRUDA®), atezolizumab (TECENTRIQ®), avelumab (BAVENCIO®), durvalumab (IMFIMZI®), cemiplimab-rwlc (LIBTAYO®), tislelizumab and spartalizumab. In some aspects of the invention, said one or more additional therapeutic agents is a TIM-3 inhibitor, optionally wherein said TIM-3 inhibitor is MGB453 or TSR-022. In some aspects of the invention, said one or more additional therapeutic agents is a LAG-3 inhibitor, optionally wherein said LAG-3 inhibitor is selected from the group consisting of LAG525, BMS-986016 and TSR-033. In some aspects of the invention, said one or more additional therapeutic agents is a TIGIT inhibitor. In other aspects of the invention, said one or more additional therapeutic agents is a CD112R inhibitor. In some aspects of the invention, said one or more additional therapeutic agents is a TAM (Axl, Mer, Tyro) inhibitor. In some aspects of the invention, said one or more additional therapeutic agents

- 5 052811 are a 4-1BB agonist. In other aspects of the invention, said one or more additional therapeutic agents are a tyrosine kinase inhibitor (TKI). In some aspects of the invention, the TKI is selected from imatinib, dasatinib, nilotinib, bosutinib, or ponatinib. In some aspects of the invention, said one or more additional agents are an adenosine axis targeting agent. In some aspects of the invention, said adenosine axis targeting agent is selected from a CD39 antagonist, a CD73 antagonist, an A2AR antagonist, an A2BR antagonist, or a dual A2AR/A2BR antagonist. In some aspects of the invention, said one or more additional therapeutic agents are a CD39 antagonist. Examples of CD39 antagonists include those described in U.S. Patent No. US2019/0284295 (Surface Oncology, Inc.), which is incorporated herein by reference. In some aspects of the invention, the one or more additional therapeutic agents are a CD73 antagonist. Examples of CD73 antagonists include small molecule CD73 inhibitors such as AB421 (Arcus), an anti-CD73 antibody or antigen-binding portion thereof that binds to CD73, such as MEDI9447 (Medimmune), BMS986179 (Bristol Meyers Squibb), or those described in U.S. Patent No. US2018/0009899 (Corvus), which is incorporated herein by reference in its entirety. In some aspects of the invention, the one or more additional therapeutic agents are an A2AR antagonist, an A2BR antagonist, or a dual A2AR/A2BR antagonist. Examples of A2AR, A2BR, and dual A2AR/A2BR antagonists include preladenant/SCH 420814 (Merck/Schering, CAS Registry Number: 377727-87-2), which is described in Hodgson et al. (2009) J Pharmacol Exp Ther 330 (1): 294-303, which is incorporated herein by reference in its entirety; ST-4206 (Leadiant Biosciences), which is described in U.S. Patent No. 9,133,197, which is incorporated herein by reference in its entirety; KW-6356 (Kyowa Hakko Kogyo), tozadenant/SYN-115 (Acorda), istradefylline/KW-6002 (Kyowa Hakko Kogyo, CAS Registry Number: 155270-99-8), which is described in LeWitt et al. (2008) Ann Neurol 63 (3): 295–302, which is incorporated herein by reference in its entirety; theophylline (CAS Registry Number: 58-55-9), NIR178 (Novartis); AB928 (Arcus Biosciences), GBV2034 (Globavir), vipadenant (Redox/Juno), AZD4635/HTL-1071 (AstraZeneca/Heptares), which is described in WO 2011/095625 and is incorporated herein by reference in its entirety; CPI-444/V81444 (Corvus/Genentech), which is described in WO 2009/156737 and is incorporated herein by reference in its entirety; PBF509 (Palobiofarma/Novartis), which is described in U.S. Patent No. US 8,796,284 and WO 2017/025918, which are incorporated herein by reference in their entireties; A2AR antagonists described in U.S. Patent Nos. US 8,114,845, US 9,029,393, US 20170015758, or US 20160129108, which are incorporated herein by reference in their entireties; and ATL-444, MSX-3, SCH-58261, SCH-412,348, SCH-442,416, VER-6623, VER-6947, VER-7835, CGS-15943, or ZM-241,385. In some aspects of the invention, the one or more additional therapeutic agents is a CCR8 antagonist. In some aspects of the invention, the CCR8 antagonist is selected from a small molecule and an antibody. In some aspects of the invention, the one or more additional therapeutic agents is a CTLA4 antagonist. In some aspects of the invention, the CTLA4 antagonist is selected from the group consisting of Yervoy® (ipilimumab or the 10D1 antibody described in PCT Patent Publication No. WO 01/14424), tremelimumab (formerly ticilimumab, CP-675,206), a monoclonal antibody, or an anti-CTLA-4 antibody described in any of the following publications: WO 98/42752; WO 00/37504; U.S. Patent No. 6,207,156; Hurwitz et al. (1998) Pro. Natl. Acad. Sci. USA 95 (17): 10067 10071; Camacho et al. (2004) J. Clin. Oncology 22 (145): antibody No. 2505 (antibody CP-675206); and Mokyr et al. (1998) Cancer Res. 58: 5301-5304. Any of the anti-CTLA-4 antibodies described in WO 2013/173223 can also be used. In some aspects of the invention, the one or more additional therapeutic agents are a VEG-F inhibitor. In some aspects of the invention, the VEG-F inhibitor is selected from cabozantinib, paopanib, bevacizumab, sunitinib, axitinib, lenvantinib, sorafenib, regorafenib, ponatinib, cabozantinib, vandetanib, ramucirumab, or bevacizumab.

Provided herein is the use of an antibody as provided herein, or an antigen-binding portion of said antibody, or a pharmaceutical composition as provided herein, for stimulating an immune response in a subject or for treating an oncological disease in a subject, optionally for use in combination with one or more additional therapeutic agents or procedures.

Also provided is a kit comprising an antibody as provided herein, or an antigen-binding portion of said antibody, or a pharmaceutical composition as provided herein, and instructions for use for stimulating an immune response in a subject or for

- 6 052811 treatment of an oncological disease in a subject, optionally with instructions for use in combination with one or more additional therapeutic agents or procedures.

Also provided is a kit comprising the antibody provided herein, or an antigen-binding portion of said antibody, and instructions for use for detecting IL-27 in a sample taken from a subject, optionally with instructions for use for detecting IL-27-associated cancer in a subject.

Definitions

The terms used in the claims of this invention and in the description of this invention are defined as set out below unless otherwise indicated.

It should be noted that when used in the context of the description of this appended claim of the present invention, the singular includes the plural unless the context clearly dictates otherwise.

When used in the context of this document, the term "about" will be understood by those skilled in the art and will vary to some extent depending on the context in which it is used. In cases where the term is used that are not understood by those skilled in the art in the context in which it is used, the term "about" will be understood to mean up to plus or minus 10% of the specific value.

As used herein, the term "agonist" refers to any molecule that partially or fully promotes, induces, increases, and/or activates the biological activity of a native polypeptide as described herein. Suitable agonist molecules include, but are not limited to, agonist antibodies or fragments of such antibodies, fragments, or amino acid sequence variants of native polypeptides, peptides, or proteins. In some aspects of the invention, activation in the presence of an agonist is dose-dependent. In some aspects of the invention, a measurable signal (e.g., an indicator of biological activity) is at least about 5%, at least about 10 ...

15%, 30%, 45%, 60%, 75%, 90%, by by by by by at least less less less less at least at least at least at least on on on on about about about about about

20%, 35%, 50%, 65%, 80%, by by by by by at least less less less less at least at least at least at least on on on on about about about about about

25%, 40%, 55%, 70%, 85%, by by by by at least about 95% or at least about 100% higher than the signal measured in a negative control sample under comparable conditions. Also provided herein are methods for identifying agonists suitable for use in the methods of the present invention. For example, such methods include, but are not limited to, binding assays such as enzyme-linked immunosorbent assay (ELISA), FORTE BIO® systems, and radioimmunoassay (RIA). Such assays determine the ability of an agonist to bind a polypeptide of interest (e.g., a receptor or ligand) and, therefore, indicate the ability of such agonist to stimulate, enhance, or activate the activity of the polypeptide. Agonist potency can also be determined using functional assays, such as assays for the ability of an agonist to activate or stimulate the function of a given polypeptide. For example, a functional assay may involve contacting a polypeptide with a potential agonist molecule and measuring a detectable change in one or more biological activities typically associated with the polypeptide. Agonist potency is typically determined by its _EC50 value (the concentration required to elicit 50% of the agonist response). The lower the _EC50 value, the greater the agonist potency and the lower the concentration required to elicit the maximal biological response.

As used herein, the term "alanine scanning" refers to a technique used to determine the contribution of a particular wild-type residue to the stability or function(s) (e.g., binding affinity) of a given protein or polypeptide. This technique involves replacing a wild-type residue in a polypeptide with an alanine residue, followed by assessing the stability or function(s) (e.g., binding affinity) of the alanine-substituted derivative or mutant polypeptide and comparing it to the wild-type polypeptide. Techniques for replacing a wild-type residue in a polypeptide with alanine are known in the art.

The term amelioration refers to any therapeutically beneficial outcome of treatment of a disease condition, such as cancer, including prevention, reduction in severity or slowing of progression, remission, or cure of the disease condition.

As used in this document, the term amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to naturally occurring amino acids. Naturally occurring amino acids are those amino acids that are encoded by

- 7 052811 genetic code, as well as amino acids that are subsequently modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. The term amino acid analogs refers to compounds that have a basic chemical structure identical to that of a naturally occurring amino acid, i.e., a carbon bonded to a hydrogen, a carboxyl group, an amine group, and an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones but retain the same basic chemical structure as the naturally occurring amino acid. The term amino acid mimetics refers to chemical compounds that have a structure that differs from the overall chemical structure of the amino acid but function similarly to the naturally occurring amino acid.

Amino acids may be designated in this document using either the generally accepted three-letter codes or the single-letter codes recommended by the IUPAC-IUB Commission on Biochemical Nomenclature. Similarly, nucleotides may be designated using the generally accepted single-letter codes.

As used herein, the term "amino acid substitution" refers to the replacement of at least one existing amino acid residue in a given amino acid sequence (the amino acid sequence of the original polypeptide) with a different, replacement amino acid residue. The term "amino acid insertion" refers to the inclusion of at least one additional amino acid in a given amino acid sequence. Although an insertion will typically consist of one or two amino acid residues, larger peptide insertions can also be made, such as insertions of about three to about five, or even up to about ten, fifteen, or twenty amino acid residues. The inserted residue may be naturally occurring or non-naturally occurring, as described above. The term "amino acid deletion" refers to the removal of at least one amino acid residue from a given amino acid sequence.

When used in the context of this document, the term "amount" or "level" is used in its broadest sense and refers to the amount, concentration, or content of a compound (e.g., a metabolite, small molecule, protein, mRNA, marker). When referring to a metabolite or small molecule (e.g., a drug), the terms "amount," "level," and "concentration" are typically used interchangeably and typically refer to the detectable amount in a biological sample. The terms "elevated levels" or "increased levels" refer to an increase in the amount, concentration, or content of a compound in a sample compared to a control sample, such as in an individual or individuals not suffering from a given disease or disorder (e.g., cancer), or compared to an internal control. In some aspects of the invention, an increased level of a compound (e.g., a drug) in a sample refers to an increase in the amount of said compound by about 5%, by about 10%, by about 15%, by about 20%, by about 25%, by about 30%, by about 35%, by about 40%, by about 45%, by about 50%, by about 55%, by about 60%, by about 65%, by about 70%, by about 75%, by about 80%, by about 85%, by about 90%, by about 95%, by about 96%, by about 97%, by about 98%, by about 99%, or by about 100% compared to the amount of said compound in a control sample, as determined by techniques known in the art (e.g., by high performance liquid chromatography - HPLC). The term "reduced level" refers to a decrease in the amount, concentration, or content of a compound (e.g., a drug) in an individual compared to a control, such as an individual or individuals not suffering from a given disease or disorder (e.g., cancer), or compared to an internal control. In some aspects of the invention, a reduced level is a small or undetectable amount, concentration, or content. In some aspects of the invention, a reduced level of a compound (e.g., a drug) in a sample refers to a decrease in the amount of said compound by about 5%, by about 10%, by about 15%, by about 20%, by about 25%, by about 30%, by about 35%, by about 40%, by about 45%, by about 50%, by about 55%, by about 60%, by about 65%, by about 70%, by about 75%, by about 80%, by about 85%, by about 90%, by about 95%, by about 96%, by about 97%, by about 98%, by about 99%, or by about 100% compared to the amount of said compound in a control sample, as determined by techniques known in the art (e.g., by HPLC).

When referring to a protein, mRNA, or marker, such as those described herein, the terms expression level or expression level are generally used interchangeably and generally refer to the detectable amount of the protein, mRNA, or marker in a biological sample. In some aspects of the invention, the detectable amount or detectable level of the protein, mRNA, or marker is related to the likelihood of a response to an agent, such as those described herein. The term expression generally refers to the process by which the information contained in a gene is converted into structures (e.g., a protein marker,

- 8 052811 (such as PD-L1) present and active in the cell in question. Therefore, when used in the context of this document, the term "expression" may refer to transcription into a polynucleotide, translation into a polypeptide, or even modifications of a polynucleotide and/or polypeptide (e.g., post-translational modification of a polypeptide). Fragments of a transcribed polynucleotide, translated polypeptide, or modification of a polynucleotide and/or polypeptide (e.g., post-translational modification of a polypeptide) should also be considered as expressed, regardless of whether they originate from a transcript obtained by alternative splicing, from a degraded transcript, or as a result of post-translational processing of the polypeptide, for example, by proteolysis. The term "expressed genes" includes those genes that are transcribed into a polynucleotide as mRNA and then translated into a polypeptide, as well as those genes that are transcribed into RNA but not translated into a polypeptide (e.g., transfer and ribosomal RNAs). The terms "increased expression," "increased expression levels," or "increased levels" refer to increased expression or increased levels of a compound in a sample compared to a control sample, such as in an individual or individuals not suffering from a given disease or disorder (e.g., cancer), or compared to an internal control. In some aspects of the invention, increased expression of a compound (e.g., a protein marker such as PD-L1) in a sample refers to an increase in the amount of said compound by about 5%, by about 10%, by about 15%, by about 20%, by about 25%, by about 30%, by about 35%, by about 40%, by about

45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about

80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about

99% or about 100% compared to the amount of the specified compound in a control sample, as determined by techniques known in the art (e.g., fluorescence-activated cell sorting - FACS). The terms "reduced expression," "reduced expression levels," or "reduced levels" refer to reduced expression or reduced levels of a compound (e.g., a protein marker) in an individual compared to a control, such as an individual or individuals not suffering from a given disease or disorder (e.g., cancer), or compared to an internal control. In some aspects of the invention, reduced expression is little or no expression. In some aspects of the invention, reduced expression of a compound (e.g., a protein marker) in a sample refers to a decrease in the amount of said compound by about 5%, by about 10%, by about 15%, by about 20%, by about 25%, by about 30%, by about 35%, by about 40%, by about 45%, by about 50%, by about 55%, by about 60%, by about 65%, by about 70%, by about 75%, by about 80%, by about 85%, by about 90%, by about 95%, by about 96%, by about 97%, by about 98%, by about 99%, or by about 100% compared to the amount of said compound in a control sample, as determined by techniques known in the art (e.g., by FACS).

As used in the context of this document, the term angiogenesis or neovascularization refers to the process by which new blood vessels develop from pre-existing vessels (Varner et al. (1999) Angiogen. 3: 53–60; Mousa et al. (2000) Angiogen. Stim. Inhib. 35: 42–44; Kim et al. (2000) Amer. J. Path. 156: 1345–1362; Kim et al. (2000) J. Biol. Chem. 275: 33920–33928; Kumar et al. (2000) Angiogenesis: From Molecular to Integrative Pharm. 169–180).

Endothelial cells from pre-existing blood vessels or from circulating endothelial stem cells (Takahashi et al. (1995) Nat. Med. 5: 434–438; Isner et al. (1999) J. Clin. Invest. 103: 1231–1236) are activated to migrate, proliferate, and differentiate into luminal structures, forming new blood vessels, in response to growth factor or hormonal signals, or under conditions of hypoxia or ischemia. During ischemia, such as that occurring in cancer, the need for increased oxygenation and nutrient delivery appears to trigger the secretion of angiogenic factors by the affected tissue; these factors stimulate the formation of new blood vessels. Several additional terms are associated with angiogenesis.

As used herein, the term "antagonist" refers to an inhibitor of a target molecule and may be used herein as a synonym for the term "inhibitor." As used herein, the term "antagonist" refers to any molecule that partially or completely blocks, inhibits, or neutralizes the biological activity of a native polypeptide as described herein. Suitable antagonist molecules include, but are not limited to, antagonist antibodies or fragments of such antibodies, fragments, or amino acid sequence variants of native polypeptides, peptides, or proteins. In some aspects of the invention, inhibition in the presence of an antagonist is dose-dependent. In some aspects of the invention, a measurable signal (e.g., an indicator of biological activity) is at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%,

- 9 052811 at least about 35%, at least about 50%, at least about 65%, at least about 80%, at least about 40%, at least about 55%, at least about 70%, at least about 85%, at least about 45%, at least about 60%, at least about 75%, at least about 90%, at least about 95%, or at least about 100% lower than the signal measured in a negative control sample under comparable conditions. Also provided herein are methods for identifying antagonists suitable for use in the methods of the present invention. For example, such methods include, but are not limited to, binding assays such as enzyme-linked immunosorbent assay (ELISA), ForteBio® systems, radioimmunoassay (RIA), the Meso Scale Discovery assay (e.g., the Meso Scale Discovery electrochemiluminescence assay (MSD-ECL)), and the Luminex® bead-based assay. Such assays determine the ability of an antagonist to bind a polypeptide of interest (e.g., a receptor or ligand) and, therefore, indicate the ability of such antagonist to inhibit, neutralize, or block the activity of the polypeptide. Antagonist potency can also be determined by functional assays, such as the ability of an antagonist to inhibit the function of a given polypeptide or agonist. For example, a functional assay may involve contacting a polypeptide with a potential antagonist molecule and measuring a detectable change in one or more biological activities typically associated with the polypeptide. Antagonist activity is typically determined by its IC50 value (the concentration required to inhibit 50% of the agonist response). The lower the _IC50 value, the higher the potency of the antagonist and the lower the concentration required to inhibit the maximum biological response.

When used in the context of this document, the phrase "an antibody that exhibits antagonism against human IL-27," or an antigen-binding portion thereof, refers to an antibody that exhibits antagonism against at least one activity of human IL-27 known in the art (e.g., an IL-27 biological activity and/or a downstream pathway(s) mediated by IL-27 signaling or another IL-27-mediated function), such as by reducing (or decreasing) human IL-27 activity by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more. Additional examples of biological activity of IL-27 and/or downstream pathway(s) mediated by IL-27 signaling or other IL-27-mediated function are described in more detail below and elsewhere in this document.

As used herein, the term "IL-27 antagonist antibody" (interchangeably referred to as "anti-IL-27 antibody") refers to an antibody that specifically binds to IL-27 and inhibits the biological activity of IL-27 and/or a downstream pathway(s) mediated by IL-27 signaling or another function mediated by IL-27. "IL-27 antagonist antibody" includes antibodies that block, antagonize, suppress, inhibit, or reduce the biological activity of IL-27 (e.g., ligand binding, enzymatic activity), including downstream pathways mediated by IL-27 signaling or function, such as receptor binding and/or the initiation of a cellular response to IL-27 or its metabolites. In some aspects of the invention, the antagonist IL-27 antibody provided herein binds to human IL-27 and prevents, blocks, or inhibits the binding of human IL-27 to its native or normal receptor (e.g., the IL-27 receptor), or to one or more subunits of the receptor (e.g., gp130 and/or IL-27Rα (also known as WSX1/TCCR)). In some aspects of the invention, the antagonist IL-27 antibody prevents, blocks, or inhibits the binding of human IL-27 to gp130. In some aspects of the invention, the antagonist IL-27 antibody prevents, blocks, or inhibits the binding of human IL-27 to IL-27Rα. In some aspects of the invention, said antagonist IL-27 antibody prevents, blocks, or inhibits the dimerization of IL-27 monomers. In some aspects of the invention, said anti-IL-27 antibody does not specifically bind to an EBI3 monomer. In some aspects of the invention, said anti-IL-27 antibody specifically binds to an IL-27p28 monomer. In some embodiments, said anti-IL-27 antibody specifically binds to a non-contiguous epitope comprising P28, but does not bind to an EBI3 monomer. In some aspects of the invention, said anti-IL-27 antibody inhibits or reduces phosphorylation of STAT1 and/or STAT3 in a cell. In some aspects of the invention, said anti-IL-27 antibody inhibits or reduces the inhibition of CD161 expression in a cell (e.g., attenuates or eliminates IL-27-mediated inhibition of CD161 expression in a cell). In some aspects of the invention, said anti-IL-27 antibody inhibits or reduces the expression of PD-L1 and/or TIM-3 in a cell. In some aspects of the invention, said anti-IL-27 antibody induces or enhances PD-1-mediated secretion of one or more

- 10 052811 number of cytokines from a cell. In some aspects of the invention, an antagonist IL-27 antibody binds to human IL-27 and stimulates or enhances an anti-tumor response. In some aspects of the invention, said antagonist IL-27 antibody binds to human IL-27 with an affinity of 15 nM or less. In some aspects of the invention, said antagonist IL-27 antibody binds to human IL-27 and comprises a wild-type or mutant IgG1 heavy chain constant region, or a wild-type or mutant IgG4 heavy chain constant region. Examples of antagonist IL-27 antibodies are provided herein.

As used herein, the term "antibody" refers to a whole antibody comprising two light chain polypeptides and two heavy chain polypeptides. Whole antibodies include various antibody isotypes, including IgM, IgG, IgA, IgD, and IgE antibodies. The term "antibody" includes a polyclonal antibody, a monoclonal antibody, a chimerized or chimeric antibody, a humanized antibody, a primatized antibody, a deimmunized antibody, and a fully human antibody. An antibody may be derived from or produced in any of a variety of species, such as mammals such as humans, non-human higher primates (e.g., orangutans, baboons, or chimpanzees), horses, cattle, pigs, sheep, goats, dogs, cats, rabbits, guinea pigs, gerbils, hamsters, rats, and mice. An antibody may be a recombinant antibody. As used herein, the terms "antibody fragment,""antigen-bindingfragment," or similar terms refer to a fragment of an antibody that retains the ability to bind to a target antigen (e.g., IL27) and inhibit the activity of said target antigen. Such fragments include, for example, a single-chain antibody, a single-chain Fv fragment (sFv), an Fd fragment, a Fab fragment, a Fab' fragment, or an F(ab') ₂ fragment. An sFv fragment is a single polypeptide chain that includes the variable regions of both the heavy and light chains of the antibody from which the sFv is derived. Additionally, intrabodies, minibodies, tribodies, and diabodies are also included within the definition of an antibody and are suitable for use in the methods described herein. See, for example, Todorovska et al. (2001) J. Immunol. Methods 248 (1): 47-66; Hudson and Kortt, (1999) J. Immunol. Methods 231 (1): 177–189; Poljak, (1994) Structure 2 (12): 1121–1123; Rondon and Marasco, (1997) Annu. Rev. Microbiol. 51: 257–283, the contents of each of which are incorporated herein by reference in their entirety.

As used herein, the term antibody fragment also includes, for example, single-domain antibodies, such as camel single-domain antibodies. See, for example, Muyldermans et al. (2001) Trends Biochem. Sci. 26: 230-235; Nuttall et al. (2000) Curr. Pharm. Biotech. 1: 253-263; Reichmann et al. (1999) J. Immunol. Meth. 231: 25-38; PCT Patent Publication Nos. WO 94/04678 and WO 94/25591; and U.S. Patent No. 6,005,079, which are incorporated herein by reference in their entireties. In some aspects of the present invention, single domain antibodies comprising two VH domains are provided, with modifications that result in the formation of single domain antibodies.

In some aspects of the invention, the antigen-binding fragment comprises the variable region of a heavy chain polypeptide and the variable region of a light chain polypeptide. In some aspects of the invention, the antigen-binding fragment described herein comprises the CDRs of a light chain polypeptide and a heavy chain polypeptide of an antibody.

The term antigen-presenting cell or APC refers to a cell that displays a foreign antigen complexed with MHC on its surface. T cells recognize this complex via the T cell receptor (TCR). Examples of APCs include, but are not limited to, B cells, dendritic cells (DCs), peripheral blood mononuclear cells (PBMCs), monocytes (such as THP-1), B lymphoblastoid cells (such as C1R.A2, 1518 B-LCL), and monocyte-derived dendritic cells (DCs). Some APCs internalize antigens either through phagocytosis or receptor-mediated endocytosis.

The term antigen presentation refers to the process by which APCs capture antigens and enable their recognition by T cells, such as as a component of an MHC-I and/or MHC-II conjugate.

As used in this document, the term apoptosis refers to the process of programmed cell death occurring in multicellular organisms (e.g., humans). The tightly regulated biochemical and molecular events that lead to apoptosis can result in observable and characteristic cellular morphological changes, including membrane blebbing, cell volume reduction, condensation and fragmentation of chromosomal DNA, and mRNA decay. A common method for identifying cells, including T cells, undergoing apoptosis is by exposing them to a fluorophore-conjugated protein (annexin V). Annexin V is widely used to detect apoptotic cells by its ability to bind to phosphatidylserine on the outer layer of the plasma membrane, which is an early indicator that a cell is undergoing apoptosis.

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As used in this document, the term B cell (alternatively B lymphocyte) refers to a type of white blood cell of the lymphocyte subtype. B cells function in the humoral immunity component of the adaptive immune system by secreting antibodies. B cells also present antigen and secrete cytokines. B cells, unlike the other two classes of lymphocytes, T cells and natural killer cells, express B cell receptors (BCRs) on their cell membrane. BCRs allow the B cell to bind to a specific antigen, against which it initiates antibody production.

When used in the context of this document, the term "binds to immobilized IL-27" refers to the ability of an antibody of the present invention to bind to IL-27, such as expressed on the surface of a cell or attached to a solid support.

As used herein, the term bispecific antibody or bifunctional antibody refers to an artificial hybrid antibody containing two different heavy/light chain pairs and two different binding sites. Bispecific antibodies can be produced by a variety of methods, including hybridoma fusion or Fab' fragment fusion. See, e.g., Songsivilai & Lachmann, (1990) Clin. Exp. Immunol. 79: 315-321; Kostelny et al. (1992) J. Immunol. 148: 1547-1553.

Traditionally, recombinant production of bispecific antibodies has been based on the coexpression of two immunoglobulin light chain/heavy chain pairs, wherein the two light chain/heavy chain pairs have different specificities (Milstein and Cuello, (1983) Nature 305: 537-539). The variable domains of antibodies with the desired binding specificities (the portions of the antibody that combine with the antigen) can be fused to immunoglobulin constant domain sequences. The fusion of the heavy chain variable region is preferably accomplished with an immunoglobulin heavy chain constant domain comprising at least a portion of the hinge, CH2, and CH3 regions. For further information on exemplary currently known methods for producing bispecific antibodies, see, for example, Suresh et al. (1986) Methods Enzymol. 121: 210; Patent Publication No. WO 96/27011; Brennan et al. (1985) Science 229: 81; Shalaby et al., J. Exp. Med. (1992) 175: 217–225; Kostelny et al. (1992) J. Immunol. 148 (5): 1547–1553; Hollinger et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444–6448; Gruber et al. (1994) J. Immunol. 152: 5368; and Tutt et al. (1991) J. Immunol. 147: 60. Bispecific antibodies include cross-linked or heteroconjugate antibodies. Heteroconjugate antibodies can be prepared using any suitable cross-linking methods. Suitable cross-linking agents are well known in the art and are described in U.S. Patent No. 4,676,980, along with a number of cross-linking techniques.

Various techniques for producing and isolating bispecific antibody fragments directly from recombinant cell culture have also been described in the literature. For example, bispecific antibodies have been produced using leucine zippers. See, e.g., Kostelny et al. (1992) J Immunol 148 (5): 1547–1553. Leucine zipper peptides from the Fos and Jun proteins can be linked to the Fab' portions of two different antibodies by gene fusion. Antibody homodimers can be reduced at the hinge region to form monomers and then reoxidized to form antibody heterodimers. This method can also be used to produce antibody homodimers. The diabody technology described by Hollinger et al. (1993) Proc Natl Acad Sci USA 90: 6444–6448 provides an alternative mechanism for producing bispecific antibody fragments. The fragments contain a heavy-chain variable domain (VH) linked to a light-chain variable domain (VL) by a linker that is too short to allow connection between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to join with the complementary VL and VH domains of the other fragment, thereby forming two antigen-binding sites. Another strategy for producing bispecific antibody fragments using single-chain Fv dimers (ssFv) has also been reported. See, e.g., Gruber et al. (1994) J Immunol 152: 5368. Alternatively, the antibodies can be linear antibodies, as described, e.g., by Zapata et al. (1995) Protein Eng. 8 (10): 1057–1062. Briefly, these antibodies comprise a pair of tandem Fd segments (VH–CH1–VH–CH1) that form a pair of antigen-binding sites. Linear antibodies can be bispecific or monospecific.

Antibodies with more than two valencies (e.g., trispecific antibodies) are discussed and described, for example, in Tutt et al. (1991) J Immunol 147: 60.

The present invention also encompasses variant forms of multispecific antibodies, such as the dual variable domain immunoglobulin (DVD-Ig) molecules described in Wu et al. (2007) Nat Biotechnol 25 (11): 1290-1297. The DVD-Ig molecules are designed such that two different light chain variable domains (VL) from two different parent antibodies are linked in tandem, either directly or through a short linker using recombinant DNA techniques, followed by the light chain constant domain. Similarly, the heavy chain comprises two different heavy chain variable domains (VH) linked in tandem, followed by

- 12 052811 CH1 constant domain and Fc region. Methods for producing DVD-Ig molecules from two parent antibodies are further described, for example, in PCT Publication Nos. WO 08/024188 and WO 07/024715. In some aspects of the invention, the bispecific antibody is a Fab-in-tandem immunoglobulin in which a light chain variable region with a second specificity is fused to a heavy chain variable region of the whole antibody. Such antibodies are described, for example, in International Patent Application Publication No. WO 2015/103072.

As used in the context of this document, the term cancer antigen refers to: (i) tumor-specific antigens; (ii) tumor-associated antigens; (iii) cells that express tumor-specific antigens; (iv) cells that express tumor-associated antigens; (v) embryonic antigens on tumors; (vi) autologous tumor cells; (vii) tumor-specific membrane antigens; (viii) tumor-associated membrane antigens; (ix) growth factor receptors; (x) growth factor ligands; and (xi) any other type of antigen, antigen-presenting cell, or material associated with cancer.

As used herein, the term "immune response to cancer" refers to an immune response induced by the presence of tumors, cancer cells, or cancer antigens. In some aspects of the invention, said response includes the proliferation of lymphocytes specific for the cancer antigen. In certain aspects of the invention, said response includes the expression and activation of antibodies and T-cell receptors, as well as the production and release of lymphokines, chemokines, and cytokines. Both the innate and adaptive immune systems interact to initiate antigenic responses against tumors, cancer cells, or cancer antigens. In some aspects of the invention, the immune response to cancer is a T-cell response.

The term "carcinoma" is recognized in the art and refers to malignant neoplasms of epithelial or endocrine tissues, including carcinomas of the respiratory system, carcinomas of the gastrointestinal tract, carcinomas of the genitourinary system, testicular carcinoma, breast carcinoma, prostate carcinoma, endocrine carcinoma, and melanoma. The anti-IL-27 antibodies described herein can be used to treat patients who have, are suspected of having, or who may be at high risk for developing any type of cancer, including renal cell carcinoma or melanoma, or any viral disease. Typical carcinomas include those arising from tissue of the cervix, lung, prostate, breast, head and neck, colon, and ovary. This term also includes carcinosarcomas, which include malignant tumors composed of carcinomatous and sarcomatous tissues. The term adenocarcinoma refers to a carcinoma originating from glandular tissue or in which tumor cells form recognizable glandular structures.

As used in this document, the term CD112R refers to one of the poliovirus receptor-like proteins and is a coinhibitory receptor for human T cells. CD112R is an inhibitory receptor expressed primarily on T cells and NK cells and competes with the activating receptor CD226 for binding to CD112. CD112 interacts with CD112R with higher affinity than with CD226 and thus effectively regulates CD226-mediated cell activation. Antagonists directed against CD112R, which block interaction with CD112, limit inhibitory signaling immediately downstream of CD112R while simultaneously promoting greater immune cell activation by increasing the interaction of CD226 with CD112. When used in the context of this document, the term "CD112R inhibitor" refers to an agent that disrupts, blocks, or inhibits the biological function or activity of CD112R.

As used herein, the term CD137 (alternatively 4-1BB) refers to a member of the tumor necrosis factor (TNF) receptor superfamily. 4-1BB is a costimulatory immune checkpoint molecule primarily for activated T cells. CD137 crosslinking enhances T cell proliferation, IL-2 secretion, survival, and cytolytic activity. As used herein, the term 4-1BB agonist refers to an agent that stimulates, induces, or enhances one or more functions of 4-1BB. An exemplary 4-1BB agonist is utomilumab (PF-05082566), a fully human IgG2 monoclonal antibody that targets 4-1BB to stimulate T cells.

As used in the context of this document, the term CD161 (alternatively known as killer cell lectin-like receptor B subfamily 1 (KLRB1); NK1.1 or NKR-P1A) refers to a member of the C-type lectin superfamily. CD161 is a T cell marker, and CD161 expression has been associated with T cell infiltration into the tumor microenvironment in a number of different types of cancer. CD161 is further described in Fergusson et al. (2014) Cell Reports 9 (3): 1075–1088, which is incorporated herein by reference in its entirety.

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When used in the context of this document, the term IL-27 or interleukin 27 refers to the cytokine IL-27. IL-27 is a cytokine related to the IL-6/IL-12 cytokine family and is a heterodimeric cytokine that contains a first subunit known as Epstein-Barr virus-induced gene 3 (EBI3; also known as the IL-27 β subunit and IL27B) and a second subunit known as IL-27p28 (also known as IL-30, IL-27 α subunit, and IL-27A). IL-27 is predominantly synthesized by activated antigen-presenting cells, including monocytes, endothelial cells, and dendritic cells (Jankowski et al. (2010) Arch Immunol. Ther. Exp. 58: 417–425, Diakowski et al. (2013) Adv. Clin. Exp. Med. (2013) 22 (5): 683–691). Although IL-27 can have proinflammatory effects, many studies suggest an important role for IL-27 as an immunosuppressant (Shimizu et al. (2006) J. Immunol. 176: 7317–7324, Hisada et al. (2004) Cancer Res. 64: 1152–1156, Diakowski (2013), see above). Although initially described as a factor that promotes the initiation of Th1 responses, IL-27 was later found to mediate a major suppressive function of T cells by limiting Th1 responses, inhibiting the differentiation of Th2 and Th17 cells, and regulating the development of Tr1 and other regulatory T cell populations (Dietrich et al. (2014) J. Immunol. 192: 5382–5389). In addition to its role as an immunoregulator, IL-27 also regulates angiogenesis, hematopoiesis, and osteocalcitogenesis (see the publication cited above).

IL-27 signals through the heterodimeric cytokine receptor type I (IL-27 receptor or IL-27R), which contains a first subunit known as WSX1 (also known as the IL-27 receptor α subunit, IL-27RA, T-cell cytokine receptor type 1 (TCCR), and cytokine receptor-like 1 (CRL1)) and a second subunit known as gp130 (also known as the interleukin-6 signal transducer (IL6ST), interleukin-6 receptor β subunit (IL-6RB), and oncostatin M receptor). gp130 is also a subunit of the IL-6 family cytokine receptor (Liu et al. (2008) Scan. J. Immunol. 68:22–299, Diakowski (2013), see above). IL-27 signaling through IL-27R activates multiple signaling cascades, including the JAK-STAT and p38 MAPK pathways.

The EBI3 subunit is also thought to have biological functions independent of p28 or the IL-27 heterodimer. For example, the EBI3 subunit also interacts with p35 to form the heterodimeric cytokine IL-35 (Yoshida et al. (2015) Annu. Rev Immunol. 33: 417–443) and has been shown to be selectively overexpressed in certain cell types without a corresponding increase in p28 or IL-27 (Larousserie et al. (2005) Am. J. Pathol. 166(4): 1217–1228).

The amino acid sequence of an exemplary human EBI3 protein is shown in SEQ ID NO: 1 (NCBI reference sequence: NP_005746.2;

Nmtpqlllalvlwascppcsgrkgppaaltlprvqcrasrypiavdcswtlppapnstspvsfiatyrlgmaarghswpclqqtptstsctitdvqlfsmapyvlnvtav hpwgssssfvpfitehiikpdppegvrlsplaerqlqvqweppgswpfpeifslkywirykrqgaarfhrvgpieatsfilravrpraryyvqvaaqdltdygelsd wslpatatmslgk-C). The amino acid sequence of an exemplary human p28 protein is shown in SEQ ID NO: 2 (NCBI Reference Sequence: NP 663634.2; Nmgqtagdlgwrlsllllplllvqagvwgfprppgrpqlslqelrreftvslhlarkllsevrgqahrfaeshlpgvnlyllplgeqlpdvsltfqawrrlsdperlcfisttlq pihallgglgtqgrwtamermqlwarnrldlrdlqrhlrfqvlaagfnlpeeeeeeeeeeeerkgllpgalgsalqgpaqvswpqllstyrllhslelvlsravre 1111s kaghsvwplgfptlspqp-C).

The amino acid sequence of an exemplary human WSX1 protein is shown in SEQ ID NO: 3 (NCBI reference sequence: NP_004834.1;

Nmrggrgapfwlwplpklallpllwvlfqrtrpqgsagplqcygvgplgdlncsweplgdlgapselhlqsqkyrsnktqtvavaagrswvaipreqltmsdkllvw gtkagqplwppvfvnletqmkpnaprlgpdvdfseddpleatvhwapptwpshkvlicqfhyrrcqeaawtllepelktipltpveiqdlelatgykvygrcrme keedlwgewspilsfqtppsapkdvwvsgnlcgtpggeeplllwkapgpcvqvsykvwfwvggrelspegitcccslipsgaewarvsavnatswepltnlslv cldsasaprsvavssiagstellvtwqpgpgeplehvvdwardgdpleklnwvrlppgnlsallpgnftvgvpyritvtavsasglasassvwgfreelaplvgptlw rlqdappgtpaiawgevprhqlrghlthytlcaqsgtspsvcmnvsgntqsvtlpdlpwgpcelwvtastiagqgppgpilrlhlpdntlrwkvlpgilflwglfllgc glslatsgrcyhlrhkvlprwvwekvpdpansssgqphmeqvpeaqplgdlpileveemepppvmessqpaqatapldsgyekhilptpeelgllgpprpqvla -C).

The amino acid sequence of an exemplary human gp130 protein is shown in SEQ ID NO: 4 (NCBI reference sequence: NP_002175.2;

- 14 052811

Nmltlqtwlvqalfiflttestgelldpcgyispespvvqlhsnftavcvlkekcmdyfhvnanyivwktnhftipkeqytiinrtassvtftdiaslniqltcniltfgqleq nvygitiisglppekpknlscivnegkkmrcewdggrethletnftlksewathkfadckakrdtptsctvdystvyfvnievwveaenalgkvtsdhinfdpvyk vkpnpphnlsvinseelssilkltwtnpsiksviilkyniqyrtkdastwsqippedtastrssftvqdlkpfteyvfrircmkedgkgywsdwseeasgityedrpsk apsfwykidpshtqgyrtvqlvwktlppfeangkildyevtltrwkshlqnytvnatkltvnltndrylatltvmlvgksdaavltipacdfqathpvmdlkafpkdn mlwvewttpresvkkyilewcvlsdkapcitdwqqedgtvhrtylrgnlaeskcylitvtpvyadgpgspesikaylkqappskgptvrtkkvgkneavlewdql pvdvqngfmiytifyrtiignetavnvdsshteytlssltsdtlymvrmaaytdeggkdgpeftfttpkfaqgeieaiwpvclafllttllgvlfcfiikrdlikkhiwpn vpdpskshiaqwsphtpprhnfnskdqmysdgnftdvsvveieandkkpfpedlksldlfkkekmteghssgiggsscmsssrpsisssdenessqntsstvqys tvvhsgyrhqvpsvqvfsrsestqplldseerpedlqlvdhvdggdgilprqqyfkqncsqhesspdishferskqvssvneedfvrlkqqisdhisqscgsgqmk mfqevsaadafgpgtegqverfetvgmeaatdegmpksylpqtvrqggympq-C).

As used in the context of this document, the term compete, when used in the context of antigen-binding proteins (e.g., immunoglobulins, antibodies, or antigen-binding fragments thereof) that compete for binding to the same epitope, refers to an interaction between the antigen-binding proteins, as determined by an assay (e.g., a competitive binding assay; a cross-blocking assay), wherein a test antigen-binding protein (e.g., a test antibody) inhibits (e.g., reduces or blocks) the specific binding of a reference antigen-binding protein (e.g., a reference antibody) to a common antigen (e.g., IL-27 or a fragment thereof).

A polypeptide or amino acid sequence derived from a designated protein or polypeptide refers to the origin of the polypeptide. Preferably, a polypeptide or amino acid sequence derived from a particular sequence has an amino acid sequence that is substantially identical to this sequence or a portion thereof, wherein said portion consists of at least 10-20 amino acids, preferably at least 20-30 amino acids, more preferably at least 30-50 amino acids, or which is otherwise identified by a person skilled in the art as being derived from said sequence. Polypeptides derived from another peptide may have one or more mutations relative to the original polypeptide, for example, one or more amino acid residues that have been replaced by another amino acid residue, or which have one or more insertions or deletions of amino acid residues.

The polypeptide may comprise an amino acid sequence that is not found in nature. Such variants are necessarily less than 100% identical or similar to the sequence of the parent molecule. In some aspects of the invention, said variant will have an amino acid sequence that is from about 75% to less than 100% identical or similar to the amino acid sequence of the parent polypeptide, more preferably from about 80% to less than 100%, more preferably from about 85% to less than 100%, more preferably from about 90% to less than 100% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) and most preferably from about 95% to less than 100% identical or similar to the amino acid sequence of the parent polypeptide, for example, along the entire length of said variant molecule.

In some aspects of the present invention, the antibodies of the present invention are encoded by a nucleotide sequence. The nucleotide sequences of the present invention can be used for a variety of applications, including: cloning, gene therapy, protein expression and purification, introducing mutations, DNA vaccination of a host in need thereof, antibody generation, for example, for passive immunization, PCR, primer and probe generation, and the like.

One skilled in the art will also appreciate that antibodies suitable for use in the methods provided herein can be altered so that their sequence differs from the naturally occurring or native sequences from which they were derived, while maintaining the desired activity inherent in said native sequences. For example, nucleotide or amino acid substitutions can be made that result in conservative substitutions or changes in nonessential amino acid residues. Mutations can be introduced using standard techniques known in the art, such as site-directed mutagenesis and polymerase chain reaction (PCR)-mediated mutagenesis.

Antibodies suitable for use in the methods provided herein may contain conservative amino acid substitutions at one or more amino acid residues, such as essential or nonessential amino acid residues. A conservative amino acid substitution is one in which one amino acid residue is replaced by another amino acid residue having a similar side chain. Amino acid families

- 15,052,811 residues having similar side chains have been identified in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a nonessential amino acid residue in a binding polypeptide is preferably replaced by another amino acid residue from the same side chain family. In another aspect, an amino acid sequence can be conservatively replaced with a structurally similar sequence that differs in the order and/or composition of the amino acids that are members of the side chain family. Alternatively, in some aspects of the present invention, mutations can be introduced randomly throughout or part of the coding sequence, for example, by saturation mutagenesis, and the resulting mutants can be incorporated into the binding polypeptides of the present invention and screened for their ability to bind to the desired target molecule.

When used in the context of this document, the term antigen cross-presentation refers to the presentation of exogenous protein antigens to T cells via MHC class I and class II molecules on APCs.

As used herein, the term "cross-reacts" refers to the ability of an antibody of the invention to bind to IL-27 from another species. For example, an antibody of the invention that binds human IL-27 may also bind IL-27 from other species. As used herein, cross-reactivity is measured by detecting specific reactivity with purified antigen in binding assays (e.g., surface plasmon resonance (SPR) or ELISA) or by binding to or otherwise functionally interacting with cells physiologically expressing IL-27. Methods for determining cross-reactivity include standard binding assays as described herein, such as by Biacore™ surface plasmon resonance (SPR) analysis using a Biacore™ 2000 SPR analyzer (Biacore AB, Uppsala, Sweden), or by flow cytometric techniques.

As used in this document, the term cytotoxic T lymphocyte (CTL) response refers to the immune response induced by cytotoxic T cells. CTL responses are mediated primarily by CD8+ T cells.

As used in this document, the term dendritic cell or DC refers to a type of antigen-presenting cell that is a white blood cell originating from the bone marrow (BM) and is the most active type of antigen-presenting cell. DCs capture and process antigens, converting proteins into peptides that are then presented on major histocompatibility complex (MHC) molecules recognized by T cells. DCs are a heterogeneous population, such as myeloid and plasmacytoid DCs; although all DCs are capable of taking up antigen, processing it, and presenting it to naive T cells, DC subtypes have distinct markers and differ in their location, migratory routes, detailed immunological function, and dependence on infectious or inflammatory stimuli for their formation. During the development of the adaptive immune response, DC phenotype and function play a role in the initiation of tolerance, memory, and differentiation of polarized T helper 1 (Th1), Th2, and Th17 cells.

As used in the context of this document, the term dendritic cell activation refers to the transition of dendritic cells from immature to mature; activated dendritic cells include mature dendritic cells and dendritic cells in transition, wherein the expression of CD80 and CD86, which induce costimulatory signals, is increased by activating stimuli. Mature human dendritic cells are cells that are positive for the expression of CD40, CD80, CD86, and HLA class II (e.g., HLA-DR). An immature dendritic cell can be distinguished from a mature dendritic cell, for example, based on markers selected from the group consisting of CD80 and CD86. An immature dendritic cell is weakly positive, and preferably negative, for these markers, while a mature dendritic cell is positive. Persons skilled in the art routinely separate mature dendritic cells, and the relevant markers described above and methods for measuring their expression are also well known to those skilled in the art.

When used in the context of this document, the term EC ₅₀ refers to that concentration of an antibody or antigen-binding portion thereof that induces a response in either an in vitro assay or an in vivo assay that is 50% of the maximal response, i.e., midway between the maximal response and the baseline.

As used in this document, the term effective dose or effective dosage is defined as an amount sufficient to achieve, or at least partially achieve, the desired effect. The term therapeutically effective dose is defined as

- 16 052811, a quantity sufficient to cure or at least partially interrupt the disease and its complications in a patient already suffering from the disease. Amounts effective for such use depend on the severity of the disease being treated and the overall health of the patient's immune system.

As used in this document, the term epitope or antigenic determinant refers to a site on an antigen to which an immunoglobulin or antibody specifically binds. The term epitope mapping refers to the process or method of identifying the binding site, or epitope, of an antibody or its antigen-binding fragment on its target protein antigen. This document presents methods and techniques for epitope mapping. Epitopes can be formed by either contiguous or non-contiguous amino acids located adjacent to each other as a result of protein folding into a tertiary structure. Epitopes formed from contiguous amino acids are generally preserved upon exposure to denaturing solvents, whereas epitopes formed as a result of folding into a tertiary structure are generally lost upon treatment with denaturing solvents. Typically, an epitope comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in a unique spatial conformation. Methods for determining which epitopes a given antibody binds (i.e., epitope mapping) are well known in the art and include, for example, immunoblotting and immunoprecipitation assays in which overlapping or adjacent peptides from IL-27 are tested for their reactivity with a given anti-IL-27 antibody. Methods for determining the spatial conformation of epitopes include methods known in the art and methods described herein, such as X-ray crystallography and two-dimensional nuclear magnetic resonance (see, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996)).

The present invention also encompasses antibodies that bind to an epitope on IL-27 that comprises all or a portion of the epitope recognized by the particular antibodies described herein (e.g., the same or an overlapping region, or a region within or encompassing such a region).

The present invention also encompasses antibodies that bind the same epitope and/or antibodies that compete with the antibodies described herein for binding to human IL-27. Antibodies that recognize the same epitope or compete for binding can be identified using routine techniques. Such techniques include, for example, an immunoassay that measures the ability of one antibody to block the binding of another antibody to a target antigen, i.e., a competitive binding assay. Competitive binding is determined in an assay in which the test immunoglobulin inhibits the specific binding of a reference antibody to a common antigen, such as IL-27. Many types of competitive binding assays are known, such as: solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme-linked immunosorbent assay (ELISA), sandwich competitive assay (see Stahli et al., Methods in Enzymology 9: 242 (1983)); solid-phase direct biotin-avidin ELISA (see Kirkland et al., J. Immunol. 137: 3614 (1986)); solid-phase direct labeling assay, solid-phase direct labeling sandwich assay (see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988)); solid-phase direct labeling RIA using I-125 label (see Morel et al., Mol. Immunol. 25 (1): 7 (1988)); Solid-phase direct biotin-avidin ELISA (see Cheung et al., Virology 176: 546 (1990)); and direct-labeling RIA (Moldenhauer et al., Scand. J. Immunol. 32: 77 (1990)). Typically, these assays involve the use of purified antigen bound to a solid surface or cells bearing either, unlabeled immunoglobulin of interest, and labeled reference immunoglobulin. Competitive inhibition is assessed by determining the amount of label bound to the solid surface or cells in the presence of the immunoglobulin of interest. Typically, the immunoglobulin of interest is present in excess. Typically, if a competing antibody is present in excess, it will inhibit the specific reference antibody with the common antigen by at least 50-55%, 55-60%, 60-65%, 65-70%, 70-75% or more.

Other techniques include, for example, epitope mapping methods such as X-ray crystallography of antigen-antibody complexes, which provides atomic resolution of the epitope, and hydrogen-deuterium (H/D) exchange mass spectrometry, which studies the conformation and dynamics of the antigen-antibody interaction. Other methods examine the binding of an antibody to antigen fragments or mutant variations of an antigen, with loss of binding due to modification of an amino acid residue in the sequence of a given antigen often considered an indication of the presence of an epitope component. In addition, computational combinatorial methods can be used for epitope mapping. These methods rely on the ability of the antibody of interest to affinity isolate specific short peptides from combinatorial phage display libraries. Such peptides are then used as the basis for determining the epitope corresponding to the antibody used to screen for a given

- 17 052811 peptide libraries. Computational algorithms have also been developed for epitope mapping, which have been shown to map conformational discontinuous epitopes.

As used herein, the term Fc-mediated effector functions or Fc effector functions refers to biological activities of an antibody other than the primary function and purpose of the antibody. For example, effector functions of a therapeutic agonist antibody are biological activities other than activation of a target protein or pathway. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptor expression (e.g., B cell receptor); lack of activation of Fc receptor-expressing platelets; and B cell activation. Many effector functions begin with Fc binding to the Fcy receptor. In some aspects of the invention, an antibody targeted to a tumor antigen has an effector function, such as ADCC activity. In some aspects of the invention, the tumor antigen-targeted antibody provided herein comprises a variant constant region having enhanced effector function (e.g., enhanced ability to mediate ADCC) compared to an unmodified form of the constant region.

As used herein, the term "Fc receptor" refers to a polypeptide found on the surface of immune effector cells that binds to the Fc region of an antibody. In some aspects of the invention, the Fc receptor is an Fc receptor. There are three subclasses of Fc receptors: FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16). All four IgG isotypes (IgG1, IgG2, IgG3, and IgG4) bind and activate Fc receptors—FcγRI, FcγRIIA, and FcγRIIIA. FcγRIIB is an inhibitory receptor; therefore, antibody binding to this receptor does not activate complement or cellular responses. FcγRI is a high-affinity receptor that binds IgG in monomeric form, while FcγRIIA and FcγRIIIA are low-affinity receptors that bind IgG only in multimeric form and have a slightly lower affinity. Binding of an antibody to the Fc receptor and/or C1q is regulated by specific residues or domains within the Fc regions. Binding also depends on residues located in the hinge region and in the CH2 portion of the antibody. In some aspects of the invention, the agonist and/or therapeutic activities of the antibodies provided herein depend on binding of the Fc region to the Fc receptor (e.g., FcγR). In some aspects of the invention, the agonist and/or therapeutic activities of the antibodies provided herein are enhanced by binding of the Fc region to the Fc receptor (e.g., FcγR).

A list of specific Fc receptor sequences useful in the present invention is provided in Table 13 below.

As used in the context of this document, the term "glycosylation profile" is defined as the pattern of arrangement and composition of carbohydrate units that are covalently attached to a protein, more particularly to an immunoglobulin protein. The glycosylation profile of a heterologous antibody may be characterized as substantially similar to the glycosylation profile that occurs naturally in antibodies produced by transgenic animal species other than humans when one of ordinary skill in the art would recognize the glycosylation profile of the heterologous antibody as being more similar to the glycosylation profile of the transgenic animal species other than humans than to the species from which the CH genes of the transgene are derived.

As used herein, the term "human antibody" includes antibodies having variable and constant regions (if any) derived from human germline immunoglobulin sequences. Human antibodies of the present invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro, or by somatic mutation in vivo) (see, e.g., Lonberg et al. (1994) Nature 368 (6474): 856-859); Lonberg, (1994) Handbook of Experimental Pharmacology 113: 49-101; Lonberg & Huszar, (1995) Intern. Rev. Immunol. 13: 65-93, and Harding & Lonberg, (1995) Ann. N.Y. Acad. Sci. 764: 536–546). However, when used in the context of this document, the term human antibody does not include antibodies in which CDR sequences derived from the germlines of other mammalian species, such as mice, have been inserted into human framework sequences (i.e., humanized antibodies).

As used in this document, the term "heterologous antibody" refers to a transgenic, non-human organism that produces such an antibody. This term refers to an antibody that has an amino acid sequence or nucleic acid coding sequence that corresponds to those found in the organism.

- 18 052811 which is not a transgenic non-human animal and is generally from a species other than the species of the transgenic non-human animal.

The terms "immune response induction" and "immune response enhancement" are used interchangeably and refer to the stimulation of an immune response (i.e., either passive or adaptive) to a specific antigen. The term "induce," when used in relation to the induction of CDC or ADCC, refers to the stimulation of specific mechanisms of direct cell killing.

As used herein, the term immunogenic cell death (also known as immunogenic apoptosis) refers to a mode of cell death associated with the activation of one or more signaling pathways that induce the pre-death expression and release of damage-associated molecular fragments (DAMPs) (e.g., adenosine triphosphate, ATP) from a tumor cell, resulting in increased immunogenicity of the tumor cell and death of the tumor cell in an immunogenic manner (e.g., by phagocytosis). As used herein, the term immunogenic cell death-inducing agent refers to a chemical, biological, or pharmacological agent that induces the process, pathway, or method of immunogenic cell death.

When used in the context of this document, the terms "inhibits," "reduces," or "blocks" (e.g., in relation to inhibiting or reducing cellular STAT1 and/or STAT3 phosphorylation mediated by human IL-27) are used interchangeably and encompass both partial and complete inhibition/blocking. Inhibition/blocking of IL-27 reduces or alters the normal level or type of activity that would occur in the absence of inhibition or blocking. Inhibition and blocking also include any measurable decrease in the binding affinity of IL-27 when in contact with an anti-IL-27 antibody compared to IL-27 not in contact with the anti-IL-27 antibody, such as IL-27 binding being inhibited by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%.

When used in the context of this document, the terms inhibits angiogenesis, reduces angiogenesis, and reduces angiogenesis refer to a reduction in the level of angiogenesis in a tissue to a value that is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or less than the amount in the corresponding control tissue, and most preferably is at the same level as observed in the control tissue.

When used in the context of this document, the term "inhibits growth" (e.g., with respect to cells) is intended to include any measurable reduction in cell growth, such as inhibiting cell growth by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100%.

When used in the context of this document, the term "in need of prophylaxis, in need of treatment, or in need thereof" refers to someone who, in the opinion of an appropriate healthcare professional (e.g., a physician, nurse, or nurse practitioner in the case of humans; a veterinarian in the case of non-human mammals), would appropriately benefit from a given treatment (e.g., treatment with a composition comprising an anti-IL-27 antibody).

The term in vivo refers to processes occurring within a living organism.

As used herein, the term "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds to human IL-27 is substantially free of antibodies that specifically bind antigens other than IL-27). However, an isolated antibody that specifically binds to an epitope may have cross-reactivity with other IL-27 proteins from different species. However, said antibody continues to exhibit specific binding to human IL-27 in a specific binding assay as described herein. In addition, an isolated antibody is typically substantially free of other cellular material and/or chemicals. In some aspects of the invention, a combination of isolated antibodies having different specificities for IL-27 are combined into a well-defined composition.

As used in the context of this document, the term "isolated nucleic acid molecule" refers to nucleic acids encoding antibodies or antibody portions (e.g., V _H , V L , CDR3) that bind to IL-27 and is intended to refer to a nucleic acid molecule in which the nucleotide sequences encoding said antibody or portion thereof do not contain other nucleotide sequences encoding antibodies or antibody portions that bind antigens other than IL-27, while other sequences may naturally flank the nucleic acid in human genomic DNA. For example, a sequence selected from the set of sequences presented in Table 12,

- 19 052811 corresponds to nucleotide sequences containing the variable regions of the heavy chain (V _H ) and light chain (V _L ) of the monoclonal antibodies against IL-27 described in this document.

As used herein, the term "isotype" refers to the class of antibodies (e.g., IgM or IgG1) encoded by the heavy chain constant region genes. In some aspects of the invention, a human monoclonal antibody of the invention is of the IgG1 isotype. In some aspects of the invention, a human monoclonal antibody of the invention is of the IgG2 isotype. In some aspects of the invention, a human monoclonal antibody of the invention is of the IgG3 isotype. In some aspects of the invention, a human monoclonal antibody of the invention is of the IgG4 isotype. As will be apparent to those skilled in the art, identification of antibody isotypes (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA1 IgA2, IgD, and IgE) is routine in the art and typically involves a combination of sequence alignment with known antibodies, published Fc variant sequences, and conserved sequences.

When used in the context of this document, the term isotype switching refers to the phenomenon by which the class or isotype of an antibody changes from one Ig class to one of the other Ig classes.

When used in this document, the term KD or _KD refers to the equilibrium dissociation constant in the binding reaction between an antibody and an antigen. The _KD value is a numerical expression of the ratio of the antibody dissociation rate constant (kD) to the antibody association rate constant ( _ka ). The _KD value is inversely proportional to the binding affinity of the antibody to the antigen. The lower the _KD value, the higher the affinity of the antibody for its antigen. Affinity represents the binding strength of an individual molecule to its ligand and is commonly measured and displayed using the equilibrium dissociation constant (KD), which is used to evaluate and rank the strength of bimolecular interactions.

When used in the context of this document, the term kd or kd (alternatively koff or k _off ) is intended to refer to the rate constant for the dissociation of an antibody from an antibody-antigen complex. The kd value is a numerical representation of the fraction of complexes that dissociate or dissociate per second and is expressed in units of s ^-1 .

When used in the context of this document, the term ka or k_a(alternatively kon or k_on) is used to denote the rate constant of antibody-antigen association. The ka value is a numerical representation of the number of antibody-antigen complexes formed per second in a 1-molar (1 M) solution of antibody and antigen and is expressed in units of M 1s^-1.

As used in this document, the term "leukocyte" refers to a type of white blood cell involved in defending the body against infectious organisms and foreign substances. Leukocytes are produced in the bone marrow. There are five main types of leukocytes, divided into two main groups: polymorphonuclear leukocytes (neutrophils, eosinophils, basophils) and mononuclear leukocytes (monocytes and lymphocytes).

As used in this document, the term lymphocyte refers to a type of white blood cell, or leukocyte, that plays a role in the body's immune defense. There are two main types of lymphocytes: B cells and T cells.

When used in the context of this document, the terms "linked," "fused," or "fusion" are used interchangeably. These terms refer to the joining of two or more elements, components, or domains together by any means, including chemical conjugation or recombinant methods. Chemical conjugation methods (e.g., using heterobifunctional cross-linkers) are known in the art.

As used in the context of this document, the term "local administration" or "local delivery" refers to delivery that is independent of transport of a composition or therapeutic agent to the intended target tissue or target area via the vascular system. For example, a composition can be delivered by injection or implantation of the composition or agent, or by injection or implantation of a device containing the composition or agent. Following local administration near the target tissue or target area, the composition, agent, or one or more components thereof can diffuse to the intended target tissue or target area.

As used in this document, the term MHC molecules refers to two types of molecules: MHC class I and MHC class II. MHC class I molecules present antigen to certain CD8+ T cells, while MHC class II molecules present antigen to certain CD4+ T cells. Antigens delivered exogenously to APCs are processed primarily for association with MHC class II. Conversely, antigens delivered endogenously to APCs are processed primarily for association with MHC class I.

When used in the context of this document, the term monoclonal antibody refers to an antibody that exhibits a single binding specificity and affinity for a particular

- 20 052811 epitope. Accordingly, the term "human monoclonal antibody" refers to an antibody that exhibits a single binding specificity and that has variable and optional constant regions derived from human germline immunoglobulin sequences. In some aspects of the invention, human monoclonal antibodies are produced by a hybridoma that includes a B cell derived from a transgenic non-human animal, such as a transgenic mouse, having a genome comprising a human heavy chain transgene and a human light chain transgene fused to an immortalized cell.

When used in the context of this document, the term monocyte refers to a type of white blood cell and can differentiate into macrophages and dendritic cells to carry out an immune response.

As used in this document, the term natural killer (NK) cells refers to a type of cytotoxic lymphocyte. These are large, typically granular, non-T, non-B lymphocytes that kill specific tumor cells and play a critical role in innate immunity to viruses and other intracellular pathogens, as well as in antibody-dependent cell-mediated cytotoxicity (ADCC).

When used in the context of this document, the term "naturally occurring" refers to the fact that an object can be found in nature. For example, polypeptide or polynucleotide sequences present in an organism (including viruses) that can be isolated from a natural source and that have not been intentionally altered by humans in a laboratory are naturally occurring.

As used in this document, the term non-switchable isotype refers to the isotypic class of the heavy chain that is produced when isotype switching does not occur; the CH gene encoding the non-switchable isotype is typically the first CH gene located immediately downstream of the functionally rearranged VDJ gene. Isotype switching is classified as classical or non-classical isotype switching. Classical isotype switching occurs as a result of recombination events involving at least one region of the switching sequence in a given transgene. Non-classical isotype switching can occur, for example, by homologous recombination between human σ _μ and human Σ _μ (δ-associated deletion). Alternative non-classical switching mechanisms, such as intertransgene and/or interchromosomal recombination, among others, can occur and cause isotype switching.

When used in the context of this document, the term nucleic acid refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form.

Unless otherwise specified, this term includes nucleic acids containing known analogs of natural nucleotides that exhibit binding properties similar to those of the reference nucleic acid and are metabolized in a manner similar to those of naturally occurring nucleotides. Unless otherwise specified, a specific nucleic acid sequence is also understood to include its conservatively modified variants (e.g., degenerate codon substitutions) and complementary sequences, as well as the explicitly stated sequence. In particular, degenerate codon substitutions can be obtained by creating sequences in which the third position of one or more selected (or all) codons is substituted with a mixed base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19: 5081, 1991; Ohtsuka et al., Biol. Chem. 260: 2605-2608, 1985; and Cassol et al., 1992; Rossolini et al., Mol. Cell. Probes 8: 91-98, 1994). For arginine and leucine, the second base modifications can also be conservative. The term nucleic acid is used interchangeably herein to refer to a gene, cDNA, and mRNA encoded by a gene.

The polynucleotides used in the present invention may consist of any polyribonucleotides or polydeoxyribonucleotides, which may be unmodified RNA or DNA, or modified RNA or DNA. For example, polynucleotides may consist of single-stranded and double-stranded DNA, DNA that is a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, as well as RNA that is a mixture of single-stranded and double-stranded regions, hybrid molecules containing DNA and RNA, which may be single-stranded or, more typically, double-stranded, or a mixture of single-stranded and double-stranded regions. In addition, a polynucleotide may consist of triple-stranded regions containing RNA or DNA, or both RNA and DNA. A polynucleotide may also contain one or more modified bases, or DNA or RNA backbones modified for stability or for other reasons. Modified bases include, for example, tritylated bases and unusual bases such as inosine. DNA and RNA can be modified in various ways; thus, a polynucleotide includes chemically, enzymatically, or metabolically modified forms.

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A nucleic acid is operably linked when it is in a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if they influence the transcription of said sequence. For transcription-regulating sequences, the term "operably linked" means that the linked DNA sequences are contiguous and, if necessary for joining two protein-coding regions, are adjacent and in reading frame. For switch sequences, the term "operably linked" indicates that the sequences are capable of mediating switch recombination.

When used in the context of this document, parenteral administration, administered parenterally, and other grammatically equivalent phrases refer to routes of administration other than enteral and local administration, typically accomplished by injection and including, without limitation, intravenous, intranasal, intraocular, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, intracerebral, intracranial, intracarotid, or intrasternal injections and infusions.

When used in the context of this document, the term patient includes humans and other mammalian subjects receiving prophylactic or therapeutic treatment.

As used herein, the term "PD-1 antagonist" refers to any chemical compound or biological molecule that inhibits the PD-1 signaling pathway or otherwise inhibits PD-1 function in a cell (e.g., an immune cell). In some aspects of the invention, a PD-1 antagonist blocks the binding of PD-L1 to PD-1 and/or PD-L2 to PD1. In some aspects of the invention, a PD-1 antagonist specifically binds PD-1. In some aspects of the invention, a PD-1 antagonist specifically binds PD-L1.

The term "percent identity," when used in the context of two or more nucleic acid sequences or polypeptide sequences, refers to two or more sequences or subsequences that have a specified percentage of nucleotides or amino acid residues that are the same when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to those skilled in the art) or by visual inspection. Depending on the application, the percent identity may exist within a region of the sequence being compared, such as a functional domain, or, alternatively, may exist over the entire length of the two sequences being compared. For sequence comparison, one sequence is typically considered the reference sequence to which the sequences being compared are compared. When using an algorithm for sequence comparison, the sequences being compared and the reference sequences are entered into a computer, the coordinates of the subsequences are designated if necessary, and the parameters of the program—the sequence comparison algorithm—are set. The sequence comparison algorithm then calculates the percent identity for the test sequence(s) relative to the reference sequence based on the specified program parameters.

Optimal alignment of sequences for comparison can be achieved, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2: 482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48: 443 (1970), by the similarity search method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI, USA), or by visual assessment (see generally Ausubel et al., below).

One example of an algorithm suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215: 403–410 (1990). Software for performing BLAST analyses is publicly available from the website of the National Center for Biotechnology Information.

When used in the context of this document, the term pharmaceutically acceptable is generally used to refer to those compounds, materials, compositions and/or drug formulations that are medically suitable for use in contact with the tissues, organs and/or bodily fluids of humans and animals, without undue toxicity, irritation, allergic reaction or other problems or complications, in proportion to a reasonable benefit/risk ratio.

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As used in the context of this document, the term "pharmaceutically acceptable carrier" means and includes all solvents, dispersion media, coating agents, antibacterial and antifungal agents, isotonic and absorption-delaying agents, etc., that are physiologically compatible. The composition may include a pharmaceutically acceptable salt, such as an acid addition salt or a base addition salt (see, for example, Berge et al. (1977) J Pharm Sci 66: 1-19).

When used in this document, the terms polypeptide, peptide, and protein are used interchangeably to refer to a polymer of amino acid residues. These terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of the corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.

As used in the context of this document, the term "prophylaxis," when used in relation to a pathological condition, refers to the administration of a composition that reduces the incidence or delays the onset of symptoms of the pathological condition in a subject, compared to a subject who does not receive the composition.

As used in the context of this document, the term "purified" or "isolated," when used with respect to any of the proteins (antibodies or fragments) described herein, refers to a polypeptide that has been separated or purified from components (e.g., proteins or other naturally occurring biological or organic molecules) that naturally accompany it, such as other proteins, lipids, and nucleic acids in prokaryotes expressing said proteins. Typically, a polypeptide is purified when it constitutes at least 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, or 99%) by weight of the total protein in a sample.

As used herein, the term programmed cell death protein 1, or PD-1, refers to the programmed cell death protein 1 polypeptide, an immunoinhibitory receptor belonging to the CD28 family and encoded by the PDCD1 gene in humans. Alternative names or synonyms for PD-1 include PDCD1, PD1, CD279, and SLEB2. PD-1 is expressed predominantly on pre-activated T cells, B cells, and myeloid cells in vivo and binds to two ligands, PD-L1 and PD-L2. As used herein, the term PD-1 includes human PD-1 (4PD-1), variants, isoforms, and species homologs of 4PD-1, as well as analogs that share at least one epitope with 4PD-1. The complete sequence of 4PD-1 can be found under GenBank accession number AAC51773.

As used in this document, the term programmed death ligand 1, or PD-L1, is one of two cell surface glycoprotein ligands for PD-1 (the other is PD-L2) that suppresses T cell activation and cytokine secretion when bound to PD-1. Alternative names and synonyms for PD-L1 include the following: PDCD1L1, PDL1, B7H1, B7-4, CD274, and B7-H. As used in this document, the term PD-L1 includes human PD-L1 (4PD-L1), variants, isoforms, and species homologs of 4PD-L1, as well as analogs that share at least one epitope with 4PD-L1. The complete sequence of 4PD-L1 can be found under GenBank accession number Q9NZQ7.

PD-1 is known as an immunoinhibitory protein that negatively regulates TCR signaling (Ishida, Y. et al. (1992) EMBO J. 11: 3887–3895; Blank, C. et al. (Epub 2006 Dec. 29) Immunol. Immunother. 56 (5): 739–745). The interaction between PD-1 and PD-L1 may act as an immune checkpoint, which may result in decreased T cell receptor-mediated proliferation (Dong et al. (2003) J. Mol. Med. 81: 281–7; Blank et al. (2005) Cancer Immunol. Immunother. 54: 307–314; Konishi et al. (2004) Clin. Cancer Res. 10: 5094–100). Immune suppression can be reversed by inhibiting the local interaction of PD-1 with PD-L1 or with PD-L2; This effect is additive if the interaction of PD-1 with PD-L2 is also blocked (Iwai et al. (2002) Proc. Nat'l. Acad. Sci. USA 99: 12293–7; Brown et al. (2003) J. Immunol. 170: 1257–66).

In some cancers, tumor survival and proliferation are maintained by tumor-mediated modulation of immune checkpoints. Such modulation may impair the anti-cancer functions of the immune system. For example, recent studies have shown that expression of immune checkpoint receptor ligands, such as PD-L1 or PD-L2, by tumor cells can suppress immune activity in the tumor microenvironment and promote cancer defense against the immune system, in particular by suppressing T cells. PD-L1 is highly expressed in a variety of human cancers (Dong et al. (2002) Nat Med 8: 787–789). PD-1, the receptor for PD-L1, is expressed on lymphocytes (e.g., activated T cells) and is normally involved in reducing immune reactivity and promoting self-tolerance, in particular by suppressing T cells. However, when PD-1 receptors expressed on T cells bind to their associated PD-L1 ligands on

- 23 052811 tumor cells, the resulting T-cell suppression contributes to the impairment of the immune response against the tumor (for example, a decrease in the level of tumor-infiltrating lymphocytes or persistent evasion of the immune response by cancer cells).

In large sets of samples, such as ovarian cancer, renal cell carcinoma, colorectal cancer, pancreatic cancer, liver cancer, and melanoma, PD-L1 expression has been shown to correlate with poor prognosis and reduce overall survival independent of subsequent treatment (see, e.g., Dong et al. (2002) Nat Med 8(8):793–800; Yang et al. (2008) Invest Ophthalmol Vis Sci 49(6):2518–2525; Ghebeh et al. (2006) Neoplasia 8:190–198; Hamanishi et al. (2007) Proc Nat Acad Sci USA 104:3360–3365; Thompson et al. (2006) Clin Genitourin Cancer 5:206–211; Nomi et al. (2005) Clin Cancer Res 11: 2947 - 2953; Inman et al. (2007) Cancer 109: 1499 - 1505; Shimauchi et al. (2007) Int J Cancer 121: 2585 - 2590; Gao et al. (2009) Clin Cancer Res 15: 971 - 979; Nakanishi et al. (2007) Cancer Immunol Immunother 56: 1173 -1182; Hino et al. (2010) Cancer 116 (7): 1757 - 1766). Similarly, PD-1 expression on tumor lymphocytes has been found to mark dysfunctional T cells in breast cancer (Kitano et al. (2017) ESMO Open 2(2):e000150) and melanoma (Kleffel et al. (2015) Cell 162(6):1242–1256). PD-1 antagonists that, for example, interfere with the function of the PD-1/PD-L1/PD-L2 signaling axis and/or disrupt the interaction between PD-1 and PD-L1 and/or PDL2 have been developed, representing a new class of antitumor inhibitors that function by modulating the interaction between immune cells and tumor cells.

As used in the context of this document, the term "rearranged" refers to a configuration of the immunoglobulin heavy chain or light chain locus in which the V segment is positioned immediately adjacent to the D-J or J segment in a conformation encoding substantially the complete VH or VL domain, respectively. A rearranged immunoglobulin gene locus can be identified by comparison with germline DNA; a rearranged locus will have at least one rearranged heptameric/nonamer homology element.

When used in the context of this document, the term "recombinant host cell" (or simply "host cell") refers to a cell into which a recombinant expression vector has been introduced. It should be understood that such terms are intended to refer not only to the specific cell identified but also to cells derived from it. Because some modifications may occur in subsequent generations due to mutations or environmental influences, such progeny may not be essentially identical to the parent cell, but are nonetheless included within the term "host cell" when used in the context of this document.

As used in the context of this document, the term recombinant human antibody includes all human antibodies that are produced, expressed, created, or isolated by recombinant methods, such as: (a) antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes, or a hybridoma derived therefrom; (b) antibodies isolated from a host cell transformed to express the antibody, such as from a transfectoma; (c) antibodies isolated from a combinatorial library of recombinant human antibodies; and (d) antibodies produced, expressed, created, or isolated by any other methods that involve splicing of human immunoglobulin gene sequences with other DNA sequences. Such recombinant human antibodies comprise variable and constant regions that utilize specific human germline immunoglobulin sequences encoded by genes of said germline, but incorporate subsequent rearrangements and mutations that occur, for example, during antibody maturation. As is known in the art (see, for example, Lonberg (2005) Nature Biotech. 23 (9): 1117–1125), the variable region comprises an antigen-binding domain that is encoded by different genes that rearrange to form an antibody specific for a foreign antigen. In addition to rearrangement, the variable region can be further modified by multiple substitutions of individual amino acids (called somatic mutation or hypermutation) to increase the affinity of the antibody for the foreign antigen. The constant region will change upon subsequent response to the antigen (i.e., upon isotype switching). Therefore, rearranged and somatically mutated nucleic acid molecules that encode immunoglobulin light and heavy chain polypeptides in response to the antigen may not have sequence identity with the original nucleic acid molecules but instead will be essentially identical or similar (i.e., at least 80% identical).

As used in the context of this document, the term reference antibody (used interchangeably with the term reference Ab), or reference antigen-binding protein, refers to an antibody or antigen-binding fragment thereof that binds to a specific epitope on IL-27 and is used to establish a relationship between it and one or more different antibodies, wherein said relationship is binding of said reference antibody and said one or more other antibodies to the same epitope on IL-27. As used in the context of this document, this term means an antibody

- 24 052811 against IL-27, which can be used in a test or assay, such as those described herein (e.g., a competitive binding assay), as a competing molecule, wherein said assay is suitable for detecting, identifying or developing one or more different antibodies that bind to the same epitope.

When used in the context of this document, the terms specific binding, selective binding, selectively binds, and specifically binds refer to the binding of an antibody to an epitope on a predetermined antigen. Typically, antibody binding is characterized by an equilibrium dissociation constant (K _D ) of less than about 10 ^-6 M, such as less than about 10 ^-7 , 10 ^-8 M, 10 ^-9 M, or 10 ^-10 M, or even less, when determined by surface plasmon resonance (SPR) technology on a BIACORE 2000 analyzer using recombinant human IL-27 as an analyte and the antibody as a ligand that binds to a predetermined antigen with an affinity that is at least twofold greater than its binding affinity to a non-specific antigen (e.g., bovine serum albumin (BSA), casein) other than the predetermined antigen or a closely related antigen. In certain aspects of the invention, an antibody that specifically binds to IL-27 is characterized by an equilibrium dissociation constant (KD) for the binding of less than about 100 nM (10 ^-7 M), optionally less than about 50 nM (5 * 10 ^-8 M), optionally less than about 15 nM (1.5 ^ 10 ^-8 M), optionally less than about 10 nM (10 ^-8 M), optionally less than about 5 nM (5 * 10 ^-9 M), optionally less than about 1 nM (10 ^-9 M), optionally less than about 0.1 nM (10 ^-10 M), optionally less than about 0.01 nM (10 ^-11 M), or even less, when determined using surface plasmon resonance (SPR) technology. The BIACORE 2000 analyzer uses recombinant human IL-27 as the analyte and the antibody as the ligand, wherein binding to the predetermined antigen occurs with an affinity that is at least twofold greater than the binding affinity of the antigen to a non-specific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely related antigen. The phrases "antibody recognizing an antigen" and "antigen-specific antibody" are used interchangeably herein with the term "antibody that specifically binds to an antigen."

As used in this document, the term STAT1 phosphorylation refers to the phosphorylation of signal transducer and activator of transcription polypeptide 1 (STAT1), a transcription factor encoded by the STAT1 gene in humans. STAT molecules are phosphorylated by receptor-associated kinases, which induce activation and dimerization by forming homodimers or heterodimers that translocate to the nucleus to function as transcription factors. STAT1 can be activated (i.e., phosphorylated) in response to signaling through several ligands, including IL-27. IL-27 signaling through IL-27R leads to STAT1 phosphorylation (pSTAT1). STAT1 plays a key role in the expression of genes involved in cell survival, viability, and pathogen response. Methods for detecting STAT1 phosphorylation as a result of IL-27 signaling include, but are not limited to, flow cytometric analysis of cells labeled with antibodies that specifically recognize phosphorylated STAT1 (see, e.g., Tochizawa et al. (2006) J Immunol Methods 313 (1-2): 29-37).

As used in this context, the term STAT3 phosphorylation refers to the phosphorylation of signal transducer and activator of transcription polypeptide 3 (STAT3), a transcription factor encoded by the STAT3 gene in humans. STAT3 mediates the expression of numerous genes in response to cellular stimuli and thus plays a key role in many cellular processes, such as cell growth and apoptosis. Methods for detecting STAT3 phosphorylation as a result of IL-27 signaling include, but are not limited to, analysis of cells or cell extracts labeled with antibodies that specifically recognize phosphorylated STAT3 (see, e.g., Fursov et al. (2011) Assay Drug Dev Technol 9 (4): 420–429).

As used in this document, the term switch sequence refers to those DNA sequences responsible for switch recombination. The switch donor sequence, typically the μ switch region, will be located 5' (i.e., upstream) of the construct region to be removed during switch recombination. The switch acceptor region will be located between the construct region to be removed and the replacement constant region (e.g., γ, ε, etc.). Because there is no specific site where recombination always occurs, the final gene sequence cannot usually be predicted from the construct.

As used herein, the term "subject" includes any human or non-human animal. For example, the methods and compositions of the present invention can be used to treat a subject with an immune disorder. The term "non-human animal" includes all vertebrate animals, such as mammals and non-human animals.

- 25 052811 mammals such as non-human primates, sheep, dogs, cows, chickens, amphibians, reptiles, etc.

For nucleic acids, the term substantial homology means that two nucleic acids or their specified sequences, when optimally aligned and compared, are identical, with appropriate nucleotide insertions or deletions, in at least about 80% of the nucleotides, typically at least about 90% to about 95%, and more preferably at least about 98% to about 99.5% of the nucleotides. Alternatively, substantial homology exists when segments will hybridize under selective hybridization conditions to a strand complementary to a given strand.

The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology = number of identical positions * 100), taking into account the number of gaps and the length of each gap that must be introduced for optimal alignment of the two sequences. Sequence comparison and determination of the percent identity between the two sequences can be achieved using a mathematical algorithm, as described in the non-limiting examples presented below.

The percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package (available at http://www.gcg.com) using the NWSgapdna.CMP matrix and a gap opening penalty of 40, 50, 60, 70, or 80 and a gap length penalty of 1, 2, 3, 4, 5, or 6. The percent identity between two nucleotide or amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4: 11–17 (1989)) included in the ALIGN program (version 2.0) using the PAM120 residue substitution weight table, a gap length penalty of 12, and a gap opening penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, J. Mol. Biol. (48): 444-453 (1970)), which is included in the GAP program in the GCG software package (available at http://www.gcg.com), using the Blossum 62 matrix or the PAM250 matrix, a gap opening penalty of 16, 14, 12, 10, 8, 6, or 4, and a gap length penalty of 1, 2, 3, 4, 5, or 6.

The nucleic acid and protein sequences of the present invention can be further used as a query sequence to search publicly available databases, for example, to identify related sequences. Such searches can be performed using the NBLAST and XBLAST (version 2.0) programs according to Altschul, et al. (1990) J. Mol. Biol. 215: 403-10. To obtain nucleotide sequences homologous to the nucleic acid molecules described herein, nucleotide sequence searches can be performed using BLAST with the NBLAST program, weight=100, wordlength=12. To obtain amino acid sequences homologous to the protein molecules described herein, BLAST searches can be performed for proteins with the XBLAST program, weight=50, wordlength=3. To obtain gapped alignments for comparison purposes, Gapped BLAST can be used as described in Altschul et al. (1997) Nucleic Acids Res. 25 (17): 3389–3402. When using BLAST and Gapped BLAST programs, the default parameters of the corresponding programs (e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.

Nucleic acids may be present in whole cells, in a cell lysate, or in partially purified or substantially pure form. Nucleic acid is isolated or substantially pure if it is purified from other cellular components or other contaminating compounds, such as other cellular nucleic acids or proteins, using standard techniques, including alkali/sodium dodecyl sulfate (SDS) treatment, CsCl separation, column chromatography, agarose gel electrophoresis, and other techniques well known in the art. See F. Ausubel, et al., ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987).

The nucleic acid compositions of the present invention, although often having a native sequence (except for modified restriction sites, etc.), can be mutated from either cDNA or genomic DNA, or mixtures thereof, according to standard techniques, to produce gene sequences. For coding sequences, such mutations may affect the amino acid sequence, if desired. In particular, DNA sequences are provided that are substantially homologous to or derived from the native V, D, J sequences, constant sequences, switch sequences, and other similar sequences described herein (whereby derived from means that a given sequence is identical to another sequence or modified from it).

When used in the context of this document, the term STING (alternatively TMEM173) refers to the stimulator of interferon genes, a protein that also acts as a direct sensor

- 26 052811 cytosolic DNA, and as an adapter protein. In humans, STING is encoded by the TMEM173 gene. STING plays a crucial role in innate immunity. STING induces the production of type I interferon when cells are infected with intracellular pathogens, such as viruses, mycobacteria, and intracellular parasites. STING-mediated type I interferon protects infected cells and nearby cells from local infection by binding to the same cell that secretes it and to nearby cells.

The illustrative amino acid sequence for STING is submitted to the NCBI Genbank database under the accession number NP_001288667.

The term T cell refers to a type of white blood cell that can be distinguished from other white blood cells by the presence of a T cell receptor on their cell surface. There are several subsets of T cells, including, but not limited to, helper T cells (also known as TH cells or CD4+ T cells) and their subtypes, including _TH1 , _TH2 , _TH3 , _TH17 , _TH9 , and _TFH cells, cytotoxic T cells (also known as _TC cells, CD8+ T cells, cytotoxic T lymphocytes, killer T cells, killer T cells), memory T cells and their subtypes, including central memory T cells ( _TCM cells), effector memory T cells ( _TEM and _TEMRA cells), and resident memory T cells ( _TRM cells), regulatory T cells (also known as Treg cells or suppressor T cells) and their subtypes, including CD4+ FOXP3+ Treg cells, CD4+ ^FOXP3 Treg cells, Tr1 cells, Th3 cells and _Treg 17 cells, natural killer T cells (also known as NKT cells), invariant mucosal-associated T cells (TACOs), and gamma-delta T cells (γδ T cells), including Vγ9/Vδ2 T cells. Any one or more of the aforementioned or unmentioned T cells may be the target cell type for the method of use according to the present invention.

As used in this document, the term "T-cell-mediated response" refers to any response mediated by T cells, including, but not limited to, effector T cells (e.g., CD8 ⁺ cells) and helper T cells (e.g., CD4 ⁺ cells). T-cell-mediated responses include, for example, T-cell cytotoxicity and T-cell proliferation.

When used in the context of this document, the terms therapeutically effective amount, or therapeutically effective dose, or similar terms as used herein are intended to refer to the amount of an agent (e.g., an anti-IL27 antibody or antigen-binding fragment thereof) that will produce a desired biological or medical response (e.g., improvement in one or more symptoms of an oncological disease).

As used in this document, the term TAM receptor refers to the TAM receptor protein tyrosine kinases (TYRO3, AXL, and MER). TAM receptors are involved in the regulation of immune homeostasis. In cancer, TAM receptors perform a dual regulatory role, controlling tumor initiation and progression and, at the same time, the associated antitumor responses of various immune cells. Further description of TAM receptors can be found in Paolino and Penninger (2016), Cancers 8 (97):

doi:10.3390/cancers8100097). When used in the context of this document, the term TAM receptor inhibitor or TAM inhibitor refers to an agent that inhibits, blocks, or reduces the function, or activity, of the TAM receptor.

As used herein, the term TIGIT, or T cell immunoreceptor with Ig and ITIM domains, refers to any native TIGIT from any vertebrate animal, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise noted. TIGIT is also known in the art as DKFZp667A205, FLJ39873, V-set and immunoglobulin domain-containing protein 9, V-set and transmembrane domain-containing protein 3, VSIG9, VSTM3, and WUCAM. This term also encompasses naturally occurring TIGIT variants, such as splice variants or allelic variants. The amino acid sequence of an exemplary human TIGIT can be found in UniProt under the accession number Q495A1.

The terms "treat," "treats," and "treatment," as used herein, refer to the therapeutic or prophylactic measures described herein. Methods of treatment include administering a human antibody of the invention to a subject in need of such treatment, for example, a subject in need of an enhanced immune response against a particular antigen or a subject who may eventually develop such a disorder, to prevent, cure, delay, reduce the severity, or alleviate one or more symptoms of said disorder or a recurrent disorder, or to prolong the survival of the subject beyond the period expected in the absence of such treatment.

As used in the context of this document, the term tumor microenvironment (TME; alternatively, cancer microenvironment) refers to the cellular environment or milieu in which a tumor or neoplasm exists, including surrounding blood vessels, as well as non-cancerous cells, including, but not limited to, immune system cells, fibroblasts,

- 27 052811 bone marrow inflammatory cells and lymphocytes. The MOR also includes signaling molecules and the extracellular matrix. Tumors and their surrounding microenvironment are closely linked and constantly interact. Tumors can influence the microenvironment by releasing extracellular signals, promoting tumor angiogenesis, and inducing peripheral immune tolerance, while immune cells in the microenvironment can influence tumor cell growth and development.

When used in the context of this document, the term unrearranged or germline configuration refers to a configuration in which the V segment is not recombined so as to be located directly adjacent to the D or J segment.

As used in this document, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid linked to it. One type of vector is a plasmid, a term that refers to a circular double-stranded loop of DNA into which additional DNA segments can be ligated. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication within the host cell into which they are introduced (e.g., bacterial vectors that have a bacterial origin of replication and mammalian episomal vectors). Other vectors (e.g., non-episomal mammalian vectors) can integrate into the host cell's genome upon introduction into that host cell and thus replicate along with the host genome. Furthermore, some vectors are capable of directing the expression of genes to which they are functionally linked. Such vectors are referred to herein as recombinant expression vectors (or simply expression vectors). Expression vectors used in recombinant DNA techniques are typically plasmids. In the present disclosure, plasmid and vector may be used interchangeably, as plasmids are the most commonly used form of vector. However, it is envisaged that the present invention includes other forms of expression vectors, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), which perform equivalent functions.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this invention. All publications, patent applications, patents, and other documents mentioned herein are hereby incorporated by reference in their entirety.

Brief description of graphic materials

Fig. 1 is a table showing the affinity data of anti-IL-27 antibodies that bind to an epitope containing one or more of the amino acids Gln37, Leu38, Glu42, Glu46, Val49, Ser50, Leu142, Asp146, Arg149, His150, Phe153, Leu156, Leu162, and Glu164 according to SEQ ID NO: 2 (IL-27p28). Affinity measurements were performed using the ForteBio and Meso Scale Discovery methods.

Fig. 2A is a graph showing the inhibition of IL-27-mediated STAT1 phosphorylation in human peripheral blood mononuclear cells (PBMCs) by anti-IL-27 antibodies, as indicated, as measured by flow cytometry. Fig. 2B is a graph showing the inhibition of IL-27-mediated STAT1 phosphorylation in U937 cells by anti-IL-27 antibodies, as indicated, as measured by flow cytometry. Fig. 2C is a graph showing the inhibition of IL-27-mediated STAT1 phosphorylation in HUT-78 cells by anti-IL-27 antibodies, as indicated, as measured by flow cytometry.

Fig. 3 is a graph showing that the anti-IL-27 antibody of the present invention (anti-IL-27 Ab1) inhibits IL-27-mediated pSTAT1 in human whole blood T cells.

Fig. 4 is a graph showing the change in IL-27-mediated inhibition of CD161 expression in T cells over the indicated concentration range of anti-IL-27 antibodies. CD161 expression was determined by flow cytometry.

Fig. 5A is a graph showing the extent to which anti-IL-27 antibodies enhance PD-1-mediated TNF-α secretion in human PBMCs, as measured by ELISA. Fig. 5B is a graph showing the extent to which anti-IL-27 antibodies enhance PD-1-mediated IL-6 secretion in human PBMCs, as measured by ELISA. Figs. 5C-5D show that IL-27 inhibits cytokine production (IL-17A, Fig. 5C; and IFN-γ, Fig. 5D) after PD-1 blockade and is restored in combination with anti-IL-27 Ab1 (abbreviations: contr.=control, n/s=not significant, PBMCs=peripheral blood mononuclear cells, rhIL-27=recombinant human IL-27). In Fig. 5E - 5H is presented in general terms

- 28 052811 observed induction of the cytokines TNF-α (Fig. 5E), IFN-γ (Fig. 5F), IL-6 (Fig. 5G), and IL-17A (Fig. 5H) in activated PBMC cultures from several individual donors, including healthy controls and patients with renal cell carcinoma (RCC), hepatocellular carcinoma (HCC), and ovarian cancer, when such cells were contacted with anti-IL-27 Ab1, αPD-1, or a combination of anti-IL-27 Ab1 and αPD-1.

Fig. 6A is a graph showing the inhibition of IL-27-mediated PD-L1 expression upon treatment of human monocytes with an anti-IL-27 antibody, as measured by flow cytometry. Fig. 6B is a graph showing the inhibition of IL-27-mediated TIM3 expression upon treatment of human monocytes with an anti-IL-27 antibody, as measured by flow cytometry. Fig. 6C is a graph showing the inhibition of IL-27-mediated PD-L1 expression upon treatment of resting human T cells with an anti-IL-27 antibody, as measured by flow cytometry.

Fig. 7A is a dot plot showing the number of B16F10 superficial metastatic nodules in the lungs (lung nodules) from mice bearing B16F10 tumors treated with anti-IL27 antibody (anti-IL-27 Ab1), isotype control antibody, αWSX-1 antibody, or a combination of αPD-1 antibody and αCTLA-4 antibody, as indicated, as measured by visual counting of nodules in lungs isolated from mice. Fig. 7B is a graph showing the growth time course of bioluminescent B16-Luc tumors in mice treated with anti-IL-27 antibody (anti-IL-27 Ab1) or isotype control antibody, as measured by bioluminescence imaging analysis. 7C-7F show a series of images of fixed hematoxylin and eosin-stained lung tissue sections isolated from mice bearing B16F10 tumors treated with anti-IL-27 antibody (anti-IL-27 Ab1) (Fig. 7D), isotype control antibody (Fig. 7C), αWSX-1 antibody (Fig. 7E), or a combination of αPD-1 antibody and αCTLA-4 antibody (Fig. 7F), as indicated. Fig. 7G is a dot plot showing total tumor area as a percentage of total fixed, hematoxylin and eosin-stained, dissected lung tissue from B16F10 cancer patients isolated from B16F10 tumor-bearing mice treated with anti-IL-27 antibody (anti-IL-27 Ab1), isotype control antibody, αWSX-1 antibody, or a combination of αPD-1 antibody and αCTLA-4 antibody, as indicated, as measured by computerized image analysis. Similar reductions in superficial lung metastases and total tumor area were observed with anti-IL-27RA (WSX-1) antibody-mediated blockade and with combination therapy with anti-PD-1 antibody and anti-CTLA-4 antibody.

Figure 8A is a scatterplot of the microarray data for genes whose expression changed >1.0 log2-fold (black dots) in splenocytes isolated from IL-27-overexpressing mice following IL-27 minicircle treatment. The x-axis shows the log2-fold change in gene expression (IL-27 minicircle treatment versus control). The y-axis shows the t-test p-value, which represents the probability of a fold change for each gene. Figure 8B is a graph showing the expression level of selected immunomodulatory genes, as indicated, in splenocytes as in Figure 8A. Figures 8C–8F show that ectopic expression of human IL-27 induces inhibitory receptor expression (as analyzed by flow cytometry) on murine T cells in vivo and that anti-IL-27 Ab1 reduces inhibitory receptor expression on T cells in vivo after treatment with IL-27 minicircles. Six-week-old female Balb/c mice were injected with empty vector (control) or hIL-27 minicircle (Figs. 8C and 8D). PBMCs and (Figs. 8E and 8F) total splenocytes were harvested 5 days after transfection, stained, and analyzed by flow cytometry. Expression of the indicated markers was analyzed on CD4+ T cells (Figs. 8C and 8E) and CD8+ T cells (Figs. 8D and 8F). Analysis was performed using FlowJo software. Fig. 8G shows that anti-IL-27 antibody Ab1 inhibits the detection of human minicircle-derived IL-27 in mouse plasma.

Fig. 9 is a ribbon diagram of the crystal structure of the IL-27 Ab1 antibody complex determined using Phaser molecular replacement software (McCoy et al. (2007) J. Appl. Cyrst. 40: 658–74) and Molrep (Vagin et al. (1997) J. Appl. Cyrst. 30: 1022–25). The heavy chain, light chain, p28, and EBI-3 are colored yellow, red, gray, and green, respectively. Fig. 9 shows that the anti-IL-27 Ab1 antibody is bound to the p28 molecule of IL-27.

Figs. 10A-10B are graphs showing the binding affinity of human IL-27 heterodimer to WSX-1 (Fig. 10A) and gp130 (Fig. 10B) in the presence (dark gray line) or absence (light gray line) of anti-IL-27 antibody Ab1, as measured by surface plasmon resonance.

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Fig. 11 is a ribbon diagram of p28 showing the residues at which anti-IL-27 Ab1 antibody binds to p28. LC=anti-IL-27 Ab1 light chain; TC=anti-IL-27 Ab1 heavy chain.

Fig. 12 is a ribbon diagram of the structural alignment of IL-27/Fab antibody Ab1 against IL-23/IL-23R (PDB ID: 5MZV). The superposition of complexes using p28 and p19 was aligned in 3-dimensional space.

Fig. 13 is a ribbon diagram of the structural alignment of IL-27/Fab antibody Ab1 against IL-6/IL-6Pa/gp130. The superposition of complexes using p28 and IL-6 was aligned in 3-dimensional space.

Figs. 14A-14B are ribbon diagrams of the binding surface of p28 and EBI3, and Fig. 14B shows an enlargement of Fig. 14A to illustrate the location of salt bridge interactions and aromatic/hydrophobic interactions between p28 and EBI3.

Fig. 15A-15B are sequence alignments of p28 (Fig. 15A) and EBI3 (Fig. 15B) in several animal species. Arrows point to conserved amino acids that form salt bridges and conserved hydrophobic amino acids, as indicated.

Fig. 16A is a ribbon diagram illustrating the structural alignment of the IL-27 heterodimer with IL-6/IL-6Ra. Figs. 16B-16C are sequence alignments of IL-27 and IL-6/IL-6Ra. Arrows point to conserved amino acids that form salt bridges and conserved hydrophobic amino acids. Fig. 16D is a ribbon diagram illustrating several interactions of p28 with EBI3 that are conserved by IL-6Ra.

Fig. 17 is a table showing the binding affinity data of human IL-27 and gp130, WSX-1, and anti-p28 antibody.

Fig. 18A is an alignment of the amino acid sequences of mouse and human p28. Fig. 18B is a ribbon diagram focusing on residue 162 (Leu in the human sequence and Cys in the mouse sequence).

Figure 19A shows the electrostatic surface potential of human IL-27. Figure 19B shows the primary structure of human IL-27, which shows the αA, αB, αC, αD helices and the unresolved CD loop with the poly-Glu sequence.

Fig. 20A is a graphical representation illustrating the differential expression of EBI3, IL-27p28, and IL-27RA in RCC tumor (1) and normal renal tissue (2). Figs. 20B-20D are Kaplan-Meier curves (percentage of death-free survival in days) for RCC patients stratified by high (1) or low (2) expression of EBI3 (Fig. 20B), IL-27p28 (Fig. 20C), and IL-27RA (Fig. 20D). Data were obtained using The Cancer Genome Atlas (TCGA) as described previously (see, e.g., Li et al., Cancer Research. 2017; 77(21):e108–e110; Li et al., Genome Biology 2016; 17(1):174).

Figures 21A–21B show IL-27-induced gene expression profiles in activated human CD4+ T cells. Figure 21A is a scatter plot of the fold change of IL-27-treated CD4+ T cells compared to the untreated control for two separate donors. Figure 21B shows the top 31 genes in the IL-27 profile in CD4+ T cells. Fifteen of these 31 genes (marked with an asterisk) were associated with poor outcome. The data were generated using ARGs.

Figs. 22A–22B are graphical representations of genome-wide hazard ratios associated with IL-27 profile gene expression in RCC (Fig. 22A) and BRCA (Fig. 22B) tumor samples. Data were generated using ARGs.

Fig. 23A is a graphical representation of plasma EBI3 levels in patients with RCC compared to healthy donor serum and pregnant woman serum (positive control), measured using a pair of EBI3-specific antibodies. Fig. 23B shows EBI3 levels in a separate cohort of patients with RCC grouped by tumor stage. Fig. 23C shows overall survival, and Fig. 23D shows disease-free survival in patients with RCC, stratified by serum EBI3 levels.

Figs. 24A-24B are graphical representations of the effect of anti-IL-27 Ab1 antibody on tumor growth and lung metastasis in an orthotopic renal cell carcinoma model (Renca model). Fig. 24A and Fig. 24B show the net primary tumor (kidney) weight and the number of lung metastases in control mice and anti-IL-27 Ab1 antibody-treated Renca mice. (*P < 0.05; unpaired t test).

Figures 25A–25B show the effect of single-agent anti-IL-27 Ab1 on the mean photon flux from Hepa1-6 orthotopic tumor over time compared to the isotype control (Figure 25B) in the Hepa1-6-luc orthotopic tumor model (Figure 25A). Error bars (whiskers) represent the standard error.

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Figures 26A-26F show the dose-dependent inhibition of Hepa1-6 orthotopic tumor growth after serial administration of anti-IL-27 Ab1 (Figure 26A). Figure 26B shows the mean bioluminescence imaging (BLI, photons/second) at 5, 8, 13, and 16 days post-implantation for control and anti-IL-27 Ab1 (at doses of 5 mg/kg, 25 mg/kg, and 50 mg/kg). Figure 26C - 26F show the mean BLV (photons/second) at 5, 8, 13, and 16 days post-implantation in individual animals for control (Fig. 26C) and anti-IL-27 Ab1 in the 5 mg/kg (Fig. 26D), 25 mg/kg (Fig. 26E), and 50 mg/kg (Fig. 26F) dose groups.

Figures 27A-27C show the modulation of gene expression in Hepa1-6 liver after administration of the anti-IL-27 Ab1 antibody (Figure 27A and Figure 27B). Figure 27C is a scatter plot of the data for genes modulated by administration of the anti-IL-27 Ab1 antibody. Tables 11A-11B below provide lists of the up- and down-regulated genes shown in Figure 27B.

Fig. 28A-28E are graphs illustrating the expression of various components of the IL-27 gene (Fig. 28A); CD274, TIGIT, LAG3, HAVCR2, and PDCD1 (Fig. 28B); TGF-alpha and TGF-beta-1 (Fig. 28C); AFP (Fig. 28D); and TNFRSF10B, TNFRSF1A, and PDGFA (Fig. 28E) after administration of anti-IL-27 antibody Ab1.

Fig. 29A - 29B are graphical representations of the relative expression of various macrophage marker genes and NK transcripts in the tumor microenvironment (TME) after administration of anti-IL-27 Ab1.

Fig. 30 is a graphical representation of NK-associated receptor expression following administration of either the anti-IL-27 Ab1 antibody or an isotype control.

Figure 31 shows the relative expression of various cell surface markers after administration of the anti-IL-27 Ab1 antibody compared to the isotype control IgG. Ratios were obtained by normalizing the target marker transcript level to the PTPRC gene transcript level. Directivity is expressed as the difference between the ratio obtained for the anti-IL-27 Ab1 antibody and the ratio obtained for IgG.

Fig. 32A-32D are bar graphs showing the expression of IL-17A (Fig. 32A), IFN-γ (IFN-gamma) (Fig. 32B), TNF-α (TNF-alpha) (Fig. 32C), and IL-10 (Fig. 32D) in cultured PBMCs stimulated with anti-CD3 antibody (0.25 μg/ml) for 3 days in the presence or absence of IL-27 (100 ng/ml).

Fig. 33A-33D are scatter plots showing the expression of IL-17A (Fig. 33A), IFN-γ (IFN-gamma) (Fig. 33B), TNF-α (TNF-alpha) (Fig. 33C), and IL-10 (Fig. 33D) in cultured PBMCs stimulated with anti-CD3 antibody (0.25 μg/ml) for 4 days in the presence or absence of anti-IL-27 Ab1 (1 μg/ml).

Fig. 34 is a scatter plot showing the log2-fold change in gene expression after IL-27 inhibition compared to control (x-axis) versus the significance (p-value) of gene expression changes after treatment with anti-IL-27 Ab1 compared to control (y-axis).

Fig. 35 is a scatter plot showing the expression of TNFSF15 in activated PBMCs after culture with anti-IL-27 Ab1 or isotype control (IgG).

Fig. 36A-36B are bar graphs showing the expression levels of TNFSF15 in activated (Fig. 36A) and quiescent (Fig. 36B) PBMCs cultured in the presence of two different lots of anti-IL-27 Ab1 (1 μg/ml) or with an isotype control.

Fig. 37 is a bar graph depicting the fold changes in TNFSF15 transcript levels following IL-27 inhibition with anti-IL-27 Ab1 compared to isotype control in various cell types as indicated.

Fig. 38 is a bar graph showing the fold changes in TNFSF15 transcript levels following treatment with anti-IL-27 Ab1, anti-CD39, and two anti-CD112R antibodies, as indicated.

Fig. 39A-39B are bar graphs showing the expression levels of TNFSF15 transcript (Fig. 39A) and secreted TNFSF15 protein (Fig. 39B) after blocking IL27 with anti-IL-27 Ab1 in activated PBMCs with delayed kinetics.

Detailed description of the invention

The present invention provides, at least in part, antibody molecules that bind to a specific epitope of human IL-27p28 with high affinity and specificity. As used herein, the terms IL-27 and IL27 interchangeably refer to the heterodimeric cytokine IL-27, which consists of two distinct subunits encoded by two different genes: Epstein-Barr virus-induced gene 3 (EBI3) and IL-27p28. IL-27 possesses both proinflammatory and anti-inflammatory properties with diverse effects on hematopoietic and non-hematopoietic cells.

Accordingly, in one aspect of the present invention, there is provided an isolated antibody that specifically binds to and antagonizes human IL-27, or

- 31 052811 an antigen-binding portion of said antibody, wherein said antibody or its antigen-binding portion specifically binds to the epitopes presented in this document and exhibits at least one or more of the following properties:

(i) bind to human IL-27 with an equilibrium dissociation constant (K _D ) of 15 nM or less;

(ii) block the binding of IL-27 to the IL-27 receptor;

(iii) inhibit or reduce phosphorylation of STAT1 and/or STAT3 in the cell;

(iv) inhibit or reduce IL-27-mediated inhibition of CD161 expression in the cell;

(v) inhibit or reduce IL-27-mediated expression of PD-L1 and/or TIM-3 in a cell;

(vi) induce or enhance PD-1-mediated secretion of one or more cytokines from a cell; and (vii) a combination of (i)-(vi).

Additional aspects of the present invention include nucleic acid molecules encoding antibody molecules, expression vectors, host cells, and methods for producing said antibody molecules. Immunoconjugates, multi- or bispecific molecules, and pharmaceutical compositions comprising said antibody molecules are also provided. The anti-IL-27 antibody molecules provided herein can be used to treat, prevent, and/or diagnose cancer or malignancy, such as solid and liquid tumors (e.g., leukemia, e.g., lymphoma, e.g., AML), lung cancer (e.g., non-small cell lung cancer), pancreatic cancer, breast cancer (e.g., triple negative breast cancer), melanoma, testicular cancer, sarcoma, head and neck cancer (e.g., squamous cell carcinoma of the head and neck), liver cancer (e.g., hepatocellular carcinoma (HCC)), colorectal cancer, ovarian cancer, brain cancer (e.g., glioblastoma multiforme), or kidney cancer (e.g., renal cell carcinoma, e.g., clear cell renal cell carcinoma).

Antibodies against IL-27 and their antigen-binding fragments.

The present invention relates to antibodies and antigen-binding portions thereof that specifically bind to IL-27p28 and exhibit antagonism towards IL-27, in particular towards human IL-27.

The present invention is directed to an isolated antibody that exhibits antagonism towards human IL-27, or to an antigen-binding portion of said antibody, wherein said antibody or antigen-binding portion thereof specifically binds to an epitope comprising one or more of the following amino acids: (i) amino acids 37 to 56 according to SEQ ID NO: 2 (IL-27p28); (ii) amino acids 142 to 164 according to SEQ ID NO: 2 (IL-27p28); or (iii) both (i) and (ii). In some aspects of the invention, an isolated antibody of the invention that exhibits antagonism towards human IL-27, or an antigen-binding portion of said antibody, specifically binds to an epitope comprising one or more of the following amino acids: Gln37, Leu38, Glu42, Glu46, Val49, Ser50, Leu53, Lys56, Leu142, Asp143, Arg145, Asp146, Leu147, Arg149, His150, Arg152, Phe153, Leu156, Ala157, Gly159, Phe160, Asn161, Leu162, Pro163 or Glu164 according to SEQ ID NO: 2 (IL-27p28).

In some aspects of the invention, an antibody of the invention or an antigen-binding portion of said antibody specifically binds to an epitope comprising Asp146, Arg149 and/or Phe153 according to SEQ ID NO: 2 (IL-27p28). In some aspects of the invention, an antibody of the invention or an antigen-binding portion of said antibody specifically binds to an epitope comprising Asp146, Arg149 and Phe153 according to SEQ ID NO: 2 (IL-27p28). In some aspects of the invention, said epitope comprises Asp146, Arg149, His150 and Phe153 according to SEQ ID NO: 2 (IL-27p28). In some aspects of the present invention, said epitope comprises Asp146, Arg149, Phe153 and Leu156 according to SEQ ID NO: 2 (IL-27p28). In some aspects of the present invention, said epitope comprises Asp146, Arg149, His150, Phe153 and Leu156 according to SEQ ID NO: 2 (IL-27p28).

In some aspects of the invention, an antibody of the invention or an antigen-binding portion of said antibody specifically binds to an epitope comprising Leu142, Asp146, Arg149, His150, Phe153, Leu156 and Glu164 according to SEQ ID NO: 2 (IL-27p28). In some aspects of the invention, said epitope comprises Gln37, Leu38, Glu42, Asp146, Arg149, His150, Phe153 and Leu156 according to SEQ ID NO: 2 (IL-27p28). In some aspects of the invention, an antibody of the invention or an antigen-binding portion of said antibody specifically binds to an epitope comprising Gln37, Leu38, Glu42, Leu142, Asp146, Arg149, His150, Phe153, Leu156 and Glu164 according to SEQ ID NO: 2 (IL-27p28).

In some aspects of the invention, an antibody of the invention or an antigen-binding portion of the antibody specifically binds to an epitope comprising Leu142, Asp146, Arg149, His150, Phe153, Leu156, Leu162 and Glu164 according to SEQ ID NO: 2 (IL27p28). In some aspects of the invention, an antibody of the invention or

- 32 052811 the antigen-binding portion of said antibody specifically binds to an epitope comprising Glu46, Val49, Ser50, Leu142, Asp146, Arg149, His150, Phe153, Leu156 and Glu164 according to SEQ ID NO: 2 (IL-27p28). In some aspects of the present invention, the antibody of the present invention or the antigen-binding portion of said antibody specifically binds to an epitope comprising Gln37, Leu38, Glu42, Glu46, Val49, Ser50, Leu142, Asp146, Arg149, His150, Phe153, Leu156, Leu162 and Glu164 according to SEQ ID NO: 2 (IL-27p28).

In some aspects of the invention, an antibody of the invention or an antigen-binding portion of said antibody specifically binds to an epitope consisting of or consisting essentially of Gln37, Leu38, Glu42, Glu46, Val49, Ser50, Leu142, Asp146, Arg149, His150, Phe153, Leu156, Leu162 and Glu164 according to SEQ ID NO: 2 (IL-27p28).

In some aspects of the invention, an antibody of the invention or an antigen-binding portion of said antibody specifically binds to an epitope comprising Gln37, Leu38, Glu42, Glu46, Val49, Ser50, Leu142, Asp146, Arg149, His150, Phe153, Leu156, Leu162 and Glu164 according to SEQ ID NO: 2 (IL-27p28), and at least one residue selected from the group consisting of: Leu53, Lys56, Asp143, Leu147, Arg152, Ala157, Gly159, Phe160 or Asn161 according to SEQ ID NO: 2 (IL-27p28).

In some aspects of the invention, an antibody of the invention or an antigen-binding portion of said antibody specifically binds to an epitope comprising Gln37, Leu38, Glu42, Glu46, Val49, Ser50, Leu142, Asp146, Arg149, His150, Phe153, Leu156, Leu162 and Glu164 according to SEQ ID NO: 2 (IL-27p28), and at least one residue selected from the group consisting of: Leu53, Lys56, Asp143, Arg145, Leu147, Arg152, Ala157, Gly159, Phe160, Asn161 or Pro163 according to SEQ ID NO: 2 (IL-27p28).

In some aspects of the invention, an antibody of the invention or an antigen-binding portion of the antibody specifically binds to an epitope consisting or consisting essentially of Gln37, Leu38, Glu42, Glu46, Val49, Ser50, Leu53, Lys56, Leu142, Asp143, Asp146, Leu147, Arg149, His150, Arg152, Phe153, Leu156, Ala157, Gly159, Phe160, Asn161, Leu162 and Glu164 according to SEQ ID NO: 2 (IL-27p28).

In some aspects of the invention, an antibody of the invention or an antigen-binding portion of the antibody specifically binds to an epitope consisting or consisting essentially of Gln37, Leu38, Glu42, Glu46, Val49, Ser50, Leu53, Lys56, Leu142, Asp143, Arg145, Asp146, Leu147, Arg149, His150, Arg152, Phe153, Leu156, Ala157, Gly159, Phe160, Asn161, Leu162, Pro163 and Glu164 according to SEQ ID NO: 2 (IL-27p28).

In some aspects of the present invention, there are provided an isolated antibody that specifically binds to an epitope comprising one or more of the amino acids Gln37, Leu38, Glu42, Glu46, Val49, Ser50, Leu53, Lys56, Leu142, Asp143, Arg145, Asp146, Leu147, Arg149, His150, Arg152, Phe153, Leu156, Ala157, Gly159, Phe160, Asn161, Leu162, Pro163 and Glu164 according to SEQ ID NO: 2 (IL-27p28) and exhibits antagonism towards human IL-27, or an antigen-binding portion of said antibody, wherein said antibody or antigen-binding portion thereof exhibits at least one or more of the following properties: (i) bind to human IL-27 with an equilibrium dissociation constant (K _D ) of 15 nM or less; (ii) block the binding of IL-27 to the IL-27 receptor; (iii) inhibit or reduce the phosphorylation of STAT1 and/or STAT3 in a cell; (iv) inhibit or reduce IL-27-mediated inhibition of CD161 expression in a cell; (v) inhibit or reduce the expression of PD-L1 and/or TIM-3 in a cell; (vi) induce or enhance PD-1-mediated secretion of one or more cytokines from a cell; and (vii) a combination of (i)(vi).

In some aspects of the present invention, said isolated antibody or antigen-binding portion thereof binds to an epitope comprising one or more amino acids Gln37, Leu38, Glu42, Glu46, Val49, Ser50, Leu53, Lys56, Leu142, Asp143, Arg145, Asp146, Leu147, Arg149, His150, Arg152, Phe153, Leu156, Ala157, Gly159, Phe160, Asn161, Leu162, Pro163 and Glu164 according to SEQ ID NO: 2 (human IL-27p28) with an equilibrium dissociation constant (K _D ) of 15 nM or less.

In some aspects of the present invention, said isolated antibody or antigen-binding portion thereof binds to recombinant human IL-27p28 or murine IL-27p28.

In some aspects of the invention, said isolated antibody or antigen-binding portion thereof inhibits or reduces phosphorylation of STAT1 and/or STAT3 in a cell. In some aspects of the invention, said cell is an immune cell. In some aspects of the invention, said cell is a cancer cell.

In some aspects of the present invention, said isolated antibody or antigen-binding portion thereof inhibits or reduces the inhibition of CD161 expression in a cell (e.g., attenuates or eliminates the inhibition of CD161 expression in a cell). In some aspects of the present invention, said cell is an immune system cell.

- 33 052811

In some aspects of the invention, said isolated antibody or antigen-binding portion thereof inhibits or reduces the expression of PD-L1 and/or TIM-3 in a cell. In some aspects of the invention, PD-L1 expression is inhibited or reduced. In some aspects of the invention, TIM-3 expression is inhibited or reduced. In some aspects of the invention, both PD-L1 expression and TIM-3 expression are reduced. In some aspects of the invention, said cell is an immune cell. In some aspects of the invention, said antibodies are monoclonal antibodies.

In some aspects of the invention, said isolated antibody or antigen-binding portion thereof induces or enhances PD-1-mediated secretion of one or more cytokines from a cell. In some aspects of the invention, said one or more cytokines are TNF-α. In some aspects of the invention, said one or more cytokines are IL-6. In some aspects of the invention, said one or more cytokines are TNF-α and IL-6. In some aspects of the invention, said cell is an immune system cell.

In some aspects of the present invention, said isolated antibody or antigen-binding portion thereof is selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE antibodies. In some aspects of the present invention, said antibody is an IgG1 antibody or an IgG4 antibody. In some aspects of the present invention, said antibody comprises a wild-type IgG1 heavy chain constant region. In some aspects of the present invention, said antibody comprises a wild-type IgG4 heavy chain constant region. In some aspects of the present invention, said antibody comprises an Fc domain comprising at least one mutation. In some aspects of the present invention, said antibody comprises a mutant IgG1 heavy chain constant region. In some aspects of the present invention, said antibody comprises a mutant IgG4 heavy chain constant region. In some aspects of the present invention, said mutant IgG4 heavy chain constant region comprises any of the substitutions S228P, L235E, L235A, or a combination thereof, according to the EU numbering system.

In some aspects of the present invention, there is provided an isolated antibody or antigen-binding portion thereof that binds to substantially the same epitope on IL-27 as an antibody or antigen-binding portion thereof according to any of the above aspects.

In some aspects of the present invention, there are provided an isolated antibody or antigen-binding portion thereof that binds to at least one amino acid residue selected from the group consisting of Gln37, Leu38, Glu42, Glu46, Val49, Ser50, Leu53, Lys56, Leu142, Asp143, Arg145, Asp146, Leu147, Arg149, His150, Arg152, Phe153, Leu156, Ala157, Gly159, Phe160, Asn161, Leu162, Pro163 and Glu164 according to SEQ ID NO: 2 (IL-27p28), bound by the antibody or antigen-binding portion thereof according to any of the above aspects.

In some aspects of the invention, an isolated antibody or antigen-binding portion thereof is provided, wherein a mutation in an epitope (Gln37, Leu38, Glu42, Glu46, Val49, Ser50, Leu53, Lys56, Leu142, Asp143, Arg145, Asp146, Leu147, Arg149, His150, Arg152, Phe153, Leu156, Ala157, Gly159, Phe160, Asn161, Leu162, Pro163, and Glu164 according to SEQ ID NO: 2 (IL-27p28)) bound by said antibody or antigen-binding portion thereof inhibits, reduces, or blocks binding to both said antibody or antigen-binding portion thereof and to an antibody or antigen-binding portion thereof according to any of said above aspects.

In some aspects of the present invention, said antibody or antigen-binding portion thereof comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2, and a light chain CDR3, wherein said light chain CDR1 consists of NXXXXXXLFSSNXKXYXX-C. In some aspects of the present invention, said antibody or antigen-binding portion thereof comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2, and a light chain CDR3, wherein said light chain CDR3 consists of N-XXXASAXXX-C. In some aspects of the present invention, said antibody or antigen-binding portion thereof comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2, and a light chain CDR3, wherein said heavy chain CDR2 consists of N-XXSSSXSYXYXXXXXXX-C. In some aspects of the present invention, said antibody or antigen-binding portion thereof comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2 and a light chain CDR3, wherein said heavy chain CDR3 consists of N-XXXXGRTSYTATXHNXXXX-C, where X represents any amino acid.

In some aspects of the present invention, said antibody or antigen-binding portion thereof comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2, and a light chain CDR3, wherein said light chain CDR1 consists of NXXXXXXLFSSNXKXYXX-C and said light chain CDR3 consists of N-XXXASAXXX-C. In some aspects of the present invention, said antibody or antigen-binding portion thereof

- 34 052811 contain heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2 and light chain CDR3, wherein said heavy chain CDR2 consists of NXXSSSXSYXYXXXXXXX-C and said heavy chain CDR3 consists of NXXXXGRTSYTATXHNXXXX-C, where X represents any amino acid.

In some aspects of the present invention, said antibody or antigen-binding portion thereof comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2 and a light chain CDR3, wherein said light chain CDR1 consists of NXXXXXXLFSSNXKXYXX-C, said light chain CDR3 consists of N-XXXASAXXX-C, said heavy chain CDR2 consists of N-XXSSSXSYXYXXXXXXX-C and said heavy chain CDR3 consists of N-XXXXGRTSYTATXHNXXXX-C, where X represents any amino acids.

In some aspects, the present invention provides an isolated antibody or antigen-binding portion thereof that specifically binds to an epitope comprising one or more of the amino acids Gln37, Leu38, Glu42, Glu46, Val49, Ser50, Leu53, Lys56, Leu142, Asp143, Arg145, Asp146, Leu147, Arg149, His150, Arg152, Phe153, Leu156, Ala157, Gly159, Phe160, Asn161, Leu162, Pro163, and Glu164 according to SEQ ID NO: 2 (IL-27p28), wherein said antibody or antigen-binding portion thereof lacks heavy and light chain CDRs selected from the group consisting of:

(i) the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 9, 10 and 11, respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 17, 18 and 19, respectively;

(ii) the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 31, 32 and 33, respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 39, 40 and 41, respectively;

(iii) the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 53, 54 and 55, respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 61, 62 and 63, respectively;

(iv) the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 75, 76 and 77, respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 83, 84 and 85, respectively;

(v) the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 97, 98 and 99, respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 105, 106 and 107, respectively; or (vi) the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 119, 120 and 121, respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 127, 128 and 129, respectively.

In some aspects of the present invention, there are provided an isolated antibody or antigen-binding portion thereof that specifically binds to an epitope comprising or consisting of Gln37, Leu38, Glu42, Glu46, Val49, Ser50, Leu142, Asp146, Arg149, His150, Phe153, Leu156, Leu162 and Glu164 according to SEQ ID NO: 2 (IL-27p28), wherein said antibody or antigen-binding portion thereof lacks heavy and light chain CDRs selected from the group consisting of:

(i) the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 9, 10 and 11, respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 17, 18 and 19, respectively;

(ii) the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 31, 32 and 33, respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 39, 40 and 41, respectively;

(iii) the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 53, 54 and 55, respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 61, 62 and 63, respectively;

(iv) the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 75, 76 and 77, respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 83, 84 and 85, respectively;

(v) the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 97, 98 and 99, respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 105, 106 and 107, respectively; or (vi) the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 119, 120 and 121, respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 127, 128 and 129, respectively.

In some aspects of the present invention, there is provided an isolated antibody or antigen-binding portion thereof that specifically binds to an epitope comprising or consisting of Gln37, Leu38, Glu42, Glu46, Val49, Ser50, Leu53, Lys56, Leu142, Asp143, Arg145, Asp146,

- 35 052811

Leu147, Arg149, His150, Arg152, Phe153, Leu156, Ala157, Gly159, Phe160, Asn161, Leu162, Pro163 and Glu164 according to SEQ ID NO: 2 (IL-27p28), wherein said antibody or antigen-binding portion thereof does not contain CDRs of the heavy and light chains, selected from the group consisting of:

(i) the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 9, 10 and 11, respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 17, 18 and 19, respectively;

(ii) the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 31, 32 and 33, respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 39, 40 and 41, respectively;

(iii) the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 53, 54 and 55, respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 61, 62 and 63, respectively;

(iv) the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 75, 76 and 77, respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 83, 84 and 85, respectively;

(v) the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 97, 98 and 99, respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 105, 106 and 107, respectively; or (vi) the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 119, 120 and 121, respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 127, 128 and 129, respectively.

In some aspects, the present invention provides an isolated antibody or antigen-binding portion thereof that specifically binds to an epitope comprising one or more of the amino acids Gln37, Leu38, Glu42, Glu46, Val49, Ser50, Leu53, Lys56, Leu142, Asp143, Arg145, Asp146, Leu147, Arg149, His150, Arg152, Phe153, Leu156, Ala157, Gly159, Phe160, Asn161, Leu162, Pro163, and Glu164 according to SEQ ID NO: 2 (IL-27p28), wherein said antibody or antigen-binding portion thereof lacks heavy and light chain CDRs selected from the group consisting of:

(i) the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 12, 13 and 14, respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 20, 21 and 22, respectively;

(ii) the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 34, 35 and 36, respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 42, 43 and 44, respectively;

(iii) the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 56, 57 and 58, respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 64, 65 and 66, respectively;

(iv) the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 78, 79 and 80, respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 86, 88 and 89, respectively;

(v) the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 100, 101 and 102, respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 108, 109 and 110, respectively; or (vi) the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 122, 123 and 124, respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 130, 131 and 132, respectively.

In some aspects of the present invention, there are provided an isolated antibody or antigen-binding portion thereof that specifically binds to an epitope comprising or consisting of Gln37, Leu38, Glu42, Glu46, Val49, Ser50, Leu142, Asp146, Arg149, His150, Phe153, Leu156, Leu162 and Glu164 according to SEQ ID NO: 2 (IL-27p28), wherein said antibody or antigen-binding portion thereof lacks heavy and light chain CDRs selected from the group consisting of:

(i) the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 12, 13 and 14, respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 20, 21 and 22, respectively;

(ii) the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 34, 35 and 36, respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 42, 43 and 44, respectively;

(iii) the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 56, 57 and 58, respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 64, 65 and 66, respectively;

- 36 052811 (iv) the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 78, 79 and 80, respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 86, 88 and 89, respectively;

(v) the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 100, 101 and 102, respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 108, 109 and 110, respectively; or (vi) the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 122, 123 and 124, respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 130, 131 and 132, respectively.

In some aspects, the present invention provides an isolated antibody or antigen-binding portion thereof that specifically binds to an epitope comprising or consisting of Gln37, Leu38, Glu42, Glu46, Val49, Ser50, Leu53, Lys56, Leu142, Asp143, Arg145, Asp146, Leu147, Arg149, His150, Arg152, Phe153, Leu156, Ala157, Gly159, Phe160, Asn161, Leu162, Pro163, and Glu164 according to SEQ ID NO: 2 (IL-27p28), wherein said antibody or antigen-binding portion thereof lacks heavy and light chain CDRs selected from the group consisting of:

(i) the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 12, 13 and 14, respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 20, 21 and 22, respectively;

(ii) the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 34, 35 and 36, respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 42, 43 and 44, respectively;

(iii) the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 56, 57 and 58, respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 64, 65 and 66, respectively;

(iv) the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 78, 79 and 80, respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 86, 88 and 89, respectively;

(v) the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 100, 101 and 102, respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 108, 109 and 110, respectively; or (vi) the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 122, 123 and 124, respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 130, 131 and 132, respectively.

In some aspects of the invention, there are provided an isolated antibody or antigen-binding portion thereof that specifically binds to an epitope comprising one or more of the amino acids Gln37, Leu38, Glu42, Glu46, Val49, Ser50, Leu53, Lys56, Leu142, Asp143, Arg145, Asp146, Leu147, Arg149, His150, Arg152, Phe153, Leu156, Ala157, Gly159, Phe160, Asn161, Leu162, Pro163, and Glu164 according to SEQ ID NO: 2 (IL-27p28), wherein said antibody or antigen-binding portion thereof comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2, and a light chain CDR3. chains, and wherein said heavy chain CDR1 does not consist of N-GFTF[S/A/R][S/R][T/Y][G/S]-C (SEQ ID NO: 144) and/or said heavy chain CDR2 does not consist of N-ISSS[S/G][S/A]YI-C (SEQ ID NO: 146).

In some aspects of the invention, there are provided an isolated antibody or antigen-binding portion thereof that specifically binds to an epitope comprising or consisting of Gln37, Leu38, Glu42, Glu46, Val49, Ser50, Leu142, Asp146, Arg149, His150, Phe153, Leu156, Leu162, and Glu164 according to SEQ ID NO: 2 (IL-27p28), wherein said antibody or antigen-binding portion thereof comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2, and a light chain CDR3, and wherein said heavy chain CDR1 does not consist of N-GFTF[S/A/R][S/R][T/Y][G/S]-C (SEQ ID NO: 144) and/or said heavy chain CDR2 chain does not consist of N-ISSS[S/G][S/A]YI-C (SEQ ID NO: 146).

In some aspects of the invention, there are provided an isolated antibody or antigen-binding portion thereof that specifically binds to an epitope comprising or consisting of Gln37, Leu38, Glu42, Glu46, Val49, Ser50, Leu53, Lys56, Leu142, Asp143, Arg145, Asp146, Leu147, Arg149, His150, Arg152, Phe153, Leu156, Ala157, Gly159, Phe160, Asn161, Leu162, Pro163, and Glu164 according to SEQ ID NO: 2 (IL-27p28), wherein said antibody or antigen-binding portion thereof comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2, and a light chain CDR3. chains, and wherein said heavy chain CDR1 does not consist of N-GFTF[S/A/R][S/R][T/Y][G/S]-C (SEQ ID NO: 144) and/or said heavy chain CDR2 does not consist of N-ISSS[S/G][S/A]YI-C (SEQ ID NO: 146).

In some aspects of the present invention, there is provided an isolated antibody or antigen-binding portion thereof that specifically binds to an epitope comprising one or

- 37 052811 a greater number of amino acids Gln37, Leu38, Glu42, Glu46, Val49, Ser50, Leu53, Lys56, Leu142, Asp143, Arg145, Asp146, Leu147, Arg149, His150, Arg152, Phe153, Leu156, Ala157, Gly159, Phe160, Asn161, Leu162, Pro163 and Glu164 according to SEQ ID NO: 2 (IL-27p28), wherein said antibody or antigen-binding portion thereof comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2 and a light chain CDR3, and wherein said heavy chain CDR1 does not consist of N-FTF[S/A/R][S/R][T/Y][G/S]MN-C (SEQ ID NO: 148) and/or said heavy chain CDR2 does not contain N-[G/S]ISSS[S/G][S/A]YI[L/Y]YADSVKG-C (SEQ ID NO: 149).

In some aspects, the invention provides an isolated antibody or antigen-binding portion thereof that specifically binds to an epitope comprising or consisting of Gln37, Leu38, Glu42, Glu46, Val49, Ser50, Leu142, Asp146, Arg149, His150, Phe153, Leu156, Leu162, and Glu164 according to SEQ ID NO: 2 (IL-27p28), wherein said antibody or antigen-binding portion thereof comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2, and a light chain CDR3, and wherein said heavy chain CDR1 does not consist of N-FTF[S/A/R][S/R][T/Y][G/S]MN-C (SEQ ID NO: 148) and/or said The heavy chain CDR2 does not contain N-[G/S]ISSS[S/G][S/A]YI[L/Y]YADSVKG-C (SEQ ID NO: 149).

In some aspects of the invention, there are provided an isolated antibody or antigen-binding portion thereof that specifically binds to an epitope comprising or consisting of Gln37, Leu38, Glu42, Glu46, Val49, Ser50, Leu53, Lys56, Leu142, Asp143, Arg145, Asp146, Leu147, Arg149, His150, Arg152, Phe153, Leu156, Ala157, Gly159, Phe160, Asn161, Leu162, Pro163, and Glu164 according to SEQ ID NO: 2 (IL-27p28), wherein said antibody or antigen-binding portion thereof comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2, and a light chain CDR3. chains, and wherein said heavy chain CDR1 does not consist of N-FTF[S/A/R][S/R][T/Y][G/S]MN-C (SEQ ID NO: 148) and/or said heavy chain CDR2 does not comprise N-[G/S]ISSS[S/G][S/A]YI[L/Y]YADSVKG-C (SEQ ID NO: 149).

In some aspects, the present invention provides an isolated antibody or antigen-binding portion thereof that specifically binds to an epitope comprising one or more of the amino acids Gln37, Leu38, Glu42, Glu46, Val49, Ser50, Leu53, Lys56, Leu142, Asp143, Arg145, Asp146, Leu147, Arg149, His150, Arg152, Phe153, Leu156, Ala157, Gly159, Phe160, Asn161, Leu162, Pro163, and Glu164 according to SEQ ID NO: 2 (IL-27p28), wherein said antibody or antigen-binding portion thereof does not comprise:

(i) a heavy chain CDR1 consisting of N-GFTFXXXX-C (SEQ ID NO: 145), a heavy chain CDR2 consisting of N-ISSSXXYI-C (SEQ ID NO: 147), and the heavy chain CDR3 sequence shown in SEQ ID NO: 121; and the light chain CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 127, 128, and 129, respectively; or (ii) a heavy chain CDR1 consisting of N-FTFXXXXMN-C (SEQ ID NO: 150), a heavy chain CDR2 consisting of N-XISSSXXYIXYADSVKG-C (SEQ ID NO: 151), and the heavy chain CDR3 sequence shown in SEQ ID NO: 124; and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 130, 131 and 132, respectively.

In some aspects of the present invention, there is provided an isolated antibody or antigen-binding portion thereof that specifically binds to an epitope comprising or consisting of Gln37, Leu38, Glu42, Glu46, Val49, Ser50, Leu142, Asp146, Arg149, His150, Phe153, Leu156, Leu162 and Glu164 according to SEQ ID NO: 2 (IL-27p28), wherein said antibody or antigen-binding portion thereof does not comprise:

(i) a heavy chain CDR1 consisting of N-GFTFXXXX-C (SEQ ID NO: 145), a heavy chain CDR2 consisting of N-ISSSXXYI-C (SEQ ID NO: 147), and the heavy chain CDR3 sequence shown in SEQ ID NO: 121; and the light chain CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 127, 128, and 129, respectively; or (ii) a heavy chain CDR1 consisting of N-FTFXXXXMN-C (SEQ ID NO: 150), a heavy chain CDR2 consisting of N-XISSSXXYIXYADSVKG-C (SEQ ID NO: 151), and the heavy chain CDR3 sequence shown in SEQ ID NO: 124; and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 130, 131 and 132, respectively.

In some aspects, the present invention provides an isolated antibody or antigen-binding portion thereof that specifically binds to an epitope comprising or consisting of Gln37, Leu38, Glu42, Glu46, Val49, Ser50, Leu53, Lys56, Leu142, Asp143, Arg145, Asp146, Leu147, Arg149, His150, Arg152, Phe153, Leu156, Ala157, Gly159, Phe160, Asn161, Leu162, Pro163, and Glu164 according to SEQ ID NO: 2 (IL-27p28), wherein said antibody or antigen-binding portion thereof does not comprise:

(i) a heavy chain CDR1 consisting of N-GFTFXXXX-C (SEQ ID NO: 145), a heavy chain CDR2 consisting of N-ISSSXXYI-C (SEQ ID NO: 147), and a heavy chain CDR3 sequence shown in SEQ ID NO: 121; and a light chain CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 127, 128, and 129, respectively; or

- 38 052811 (ii) a heavy chain CDR1 consisting of N-FTFXXXXMN-C (SEQ ID NO: 150), a heavy chain CDR2 consisting of N-XISSSXXYIXYADSVKG-C (SEQ ID NO: 151), and a heavy chain CDR3 sequence presented in SEQ ID NO: 124; and the light chain CDR1, CDR2, and CDR3 sequences presented in SEQ ID NOs: 130, 131, and 132, respectively.

In some aspects, the invention provides an isolated antibody or antigen-binding portion thereof that specifically binds to an epitope comprising one or more of the amino acids Gln37, Leu38, Glu42, Glu46, Val49, Ser50, Leu53, Lys56, Leu142, Asp143, Arg145, Asp146, Leu147, Arg149, His150, Arg152, Phe153, Leu156, Ala157, Gly159, Phe160, Asn161, Leu162, Pro163, and Glu164 according to SEQ ID NO: 2 (IL-27p28), wherein said antibody or antigen-binding portion thereof lacks a heavy chain CDR1 consisting of N-GFTFXXXX-C (SEQ ID NO: 145), a heavy chain CDR2 consisting of N-GFTFXXXX-C (SEQ ID NO: 146), a heavy chain CDR3 consisting of N-GFTFXXXX-C (SEQ ID NO: 147), a heavy chain CDR4 consisting of N-GFTFXXXX-C (SEQ ID NO: 148), a heavy chain CDR5 consisting of N-GFTFXXXX-C (SEQ ID NO: 149), a heavy chain CDR6 consisting of N-GFTFXXXX-C (SEQ ID NO: 149), a heavy chain CDR7 consisting of N-GFTFXXXX-C (SEQ ID NO: 149), a heavy chain CDR8 consisting of N-GFTFXXXX-C (SEQ ID NO: 149), a heavy chain CDR9 consisting of N-GFTFXXXX-C (SEQ ID NO: 149), a heavy chain CDR10 consisting of N-GFTFXXXX-C (SEQ ID NO: 149), a heavy chain CDR21 consisting of N-GFTFXXXX-C (SEQ ID NO: 149), a heavy chain CDR chain consisting of N-IXXXXXXX-C (SEQ ID NO: 152), heavy chain CDR3 consisting of N-AR[X] _n = ₆ _ ₁₅ DX-C (SEQ ID NO: 153); and light chain CDR1 consisting of NQS[X] _n = ₁ _ ₃ SS[X] _n = ₀ _ ₄ YC (SEQ ID NO: 154), light chain CDR2 consisting of N-XXS-C (SEQ ID NO: 155), light chain CDR3 consisting of N-QQXXXXP[X] _n = _0-1 TC (SEQ ID NO: 156), respectively.

In some aspects, the invention provides an isolated antibody or antigen-binding portion thereof that specifically binds to an epitope comprising or consisting of Gln37, Leu38, Glu42, Glu46, Val49, Ser50, Leu142, Asp146, Arg149, His150, Phe153, Leu156, Leu162, and Glu164 according to SEQ ID NO: 2 (IL-27p28), wherein said antibody or antigen-binding portion thereof lacks a heavy chain CDR1 consisting of N-GFTFXXXX-C (SEQ ID NO: 145), a heavy chain CDR2 consisting of N-IXXXXXXX-C (SEQ ID NO: 152), a heavy chain CDR3 consisting of N-AR[X] _n = ₆ _ ₁₅ DX-C (SEQ ID NO: 153); and light chain CDR1 consisting of N-QS[X] _n = _1.3 SS[X] _n = ₀ _ ₄ YC (SEQ ID NO: 154), light chain CDR2 consisting of N-XXS-C (SEQ ID NO: 155), light chain CDR3 consisting of N-QQXXXXP[X] _n=0-1 TC (SEQ ID NO: 156), respectively.

In some aspects, the invention provides an isolated antibody or antigen-binding portion thereof that specifically binds to an epitope comprising or consisting of Gln37, Leu38, Glu42, Glu46, Val49, Ser50, Leu53, Lys56, Leu142, Asp143, Arg145, Asp146, Leu147, Arg149, His150, Arg152, Phe153, Leu156, Ala157, Gly159, Phe160, Asn161, Leu162, Pro163, and Glu164 according to SEQ ID NO: 2 (IL-27p28), wherein said antibody or antigen-binding portion thereof lacks a heavy chain CDR1 consisting of N-GFTFXXXX-C (SEQ ID NO: 145), a heavy chain CDR2, consisting of N-IXXXXXXX-C (SEQ ID NO: 152), heavy chain CDR3 consisting of N-AR[X] _n = ₆ _ ₁₅ DX-C (SEQ ID NO: 153); and light chain CDR1 consisting of N-QS[X] _n = 1. ₃ SS[X] _n = ₀ _ ₄ YC (SEQ ID NO: 154), light chain CDR2 consisting of N-XXS-C (SEQ ID NO: 155), light chain CDR3 consisting of N-QQXXXXP[X] _{n = 0-1} TC (SEQ ID NO: 156), respectively.

In some aspects of the present invention, there are provided an isolated antibody or antigen-binding portion thereof that exhibits antagonism towards IL-27 and specifically binds to an epitope comprising one or more of the amino acids Gln37, Leu38, Glu42, Glu46, Val49, Ser50, Leu53, Lys56, Leu142, Asp143, Arg145, Asp146, Leu147, Arg149, His150, Arg152, Phe153, Leu156, Ala157, Gly159, Phe160, Asn161, Leu162, Pro163 and Glu164 according to SEQ ID NO: 2 (IL27p28), wherein said antibody or antigen-binding portion thereof comprises variable regions of the heavy and light chains, wherein said the variable region of the heavy chain does not comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 15, 37, 59, 81, 103 and 125; and wherein said variable region of the light chain does not comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 23, 45, 67, 89, 111 and 133.

In some aspects, the present invention provides an isolated antibody or antigen-binding portion thereof that exhibits antagonism towards IL-27 and specifically binds to an epitope comprising one or more of the amino acids Gln37, Leu38, Glu42, Glu46, Val49, Ser50, Leu53, Lys56, Leu142, Asp143, Arg145, Asp146, Leu147, Arg149, His150, Arg152, Phe153, Leu156, Ala157, Gly159, Phe160, Asn161, Leu162, Pro163 and Glu164 according to SEQ ID NO: 2 (IL-27p28), wherein said antibody or antigen-binding portion thereof comprises heavy and light chain variable regions, wherein said the variable region of the heavy chain and said variable region of the light chain do not represent amino acid sequences selected from the group consisting of:

(i) SEQ ID NO: 15 and 65, respectively;

(ii) SEQ ID NO: 37 and 45, respectively;

(iii) SEQ ID NO: 59 and 67, respectively;

(iv) SEQ ID NO: 81 and 89, respectively;

(v) SEQ ID NO: 103 and 111, respectively; and (vi) SEQ ID NO: 125 and 133, respectively.

In some aspects of the present invention, there is provided an isolated antibody or antigen-binding portion thereof that exhibits antagonism towards IL-27 and specifically binds to an epitope comprising one or more of the amino acids Gln37, Leu38, Glu42, Glu46, Val49, Ser50, Leu53, Lys56, Leu142, Asp143, Arg145, Asp146, Leu147, Arg149, His150, Arg152,

- 39 052811

Phe153, Leu156, Ala157, Gly159, Phe160, Asn161, Leu162, Pro163 and Glu164 according to SEQ ID NO: 2 (IL-27p28), wherein said antibody or antigen-binding portion thereof comprises variable regions of heavy and light chains, wherein said variable region of the heavy chain does not comprise amino acid sequences that are at least 90% identical to amino acid sequences selected from the group consisting of SEQ ID NO: 15, 37, 59, 81, 103 and 125; and wherein said variable region of the light chain does not contain amino acid sequences that are at least 90% identical to amino acid sequences selected from the group consisting of SEQ ID NO: 23, 45, 67, 89, 111 and 133.

In some aspects, the present invention provides an isolated antibody or antigen-binding portion thereof that exhibits antagonism towards IL-27 and specifically binds to an epitope comprising one or more of the amino acids Gln37, Leu38, Glu42, Glu46, Val49, Ser50, Leu53, Lys56, Leu142, Asp143, Arg145, Asp146, Leu147, Arg149, His150, Arg152, Phe153, Leu156, Ala157, Gly159, Phe160, Asn161, Leu162, Pro163 and Glu164 according to SEQ ID NO: 2 (IL-27p28), wherein said antibody or antigen-binding portion thereof comprises heavy and light chain variable regions, wherein said the variable region of the heavy chain and said variable region of the light chain do not comprise amino acid sequences that are at least 90% identical to amino acid sequences selected from the group consisting of:

(i) SEQ ID NO: 15 and 65, respectively;

(ii) SEQ ID NO: 37 and 45, respectively;

(iii) SEQ ID NO: 59 and 67, respectively;

(iv) SEQ ID NO: 81 and 89, respectively;

(v) SEQ ID NO: 103 and 111, respectively; and (vi) SEQ ID NO: 125 and 133, respectively.

In some aspects, the invention provides an isolated antibody or antigen-binding portion thereof that exhibits antagonism towards IL-27 and specifically binds to an epitope comprising one or more of the amino acids Gln37, Leu38, Glu42, Glu46, Val49, Ser50, Leu53, Lys56, Leu142, Asp143, Arg145, Asp146, Leu147, Arg149, His150, Arg152, Phe153, Leu156, Ala157, Gly159, Phe160, Asn161, Leu162, Pro163 and Glu164 according to SEQ ID NO: 2 (IL-27p28), wherein said antibody or antigen-binding portion thereof comprises a heavy chain and a light chain, wherein said heavy chain does not comprise amino acid sequences selected from the group consisting of SEQ ID NO: 25, 47, 69, 91, 113 and 135; and wherein said light chain does not comprise amino acid sequences selected from the group consisting of SEQ ID NO: 20, 42, 71, 93 and 115.

In some aspects, the invention provides an isolated antibody or antigen-binding portion thereof that exhibits antagonism towards IL-27 and specifically binds to an epitope comprising one or more of the amino acids Gln37, Leu38, Glu42, Glu46, Val49, Ser50, Leu53, Lys56, Leu142, Asp143, Arg145, Asp146, Leu147, Arg149, His150, Arg152, Phe153, Leu156, Ala157, Gly159, Phe160, Asn161, Leu162, Pro163 and Glu164 according to SEQ ID NO: 2 (IL-27p28), wherein said antibody or antigen-binding portion thereof comprises a heavy and a light chain, wherein said heavy chain does not comprise the amino acid sequences that are at least 90% identical to amino acid sequences selected from the group consisting of SEQ ID NOs: 25, 47, 69, 91, 113, and 135; and wherein said light chain does not contain amino acid sequences that are at least 90% identical to amino acid sequences selected from the group consisting of SEQ ID NOs: 20, 42, 71, 93, and 115.

In some aspects, the invention provides an isolated antibody or antigen-binding portion thereof that exhibits antagonism towards IL-27 and specifically binds to an epitope comprising one or more of the amino acids Gln37, Leu38, Glu42, Glu46, Val49, Ser50, Leu53, Lys56, Leu142, Asp143, Arg145, Asp146, Leu147, Arg149, His150, Arg152, Phe153, Leu156, Ala157, Gly159, Phe160, Asn161, Leu162, Pro163 and Glu164 according to SEQ ID NO: 2 (IL-27p28), wherein said antibody or antigen-binding portion thereof comprises a heavy chain and a light chain, wherein said heavy chain does not comprise amino acid sequences selected from the group consisting of SEQ ID NO: 29, 51, 73, 95, 117 and 139; and wherein said light chain does not comprise amino acid sequences selected from the group consisting of SEQ ID NO: 71, 49, 71, 93, 115 and 137.

In some aspects, the invention provides an isolated antibody or antigen-binding portion thereof that exhibits antagonism towards IL-27 and specifically binds to an epitope comprising one or more of the amino acids Gln37, Leu38, Glu42, Glu46, Val49, Ser50, Leu53, Lys56, Leu142, Asp143, Arg145, Asp146, Leu147, Arg149, His150, Arg152, Phe153, Leu156, Ala157, Gly159, Phe160, Asn161, Leu162, Pro163 and Glu164 according to SEQ ID NO: 2 (IL-27p28), wherein said antibody or antigen-binding portion thereof comprises a heavy and

- 40 052811 a light chain, wherein said heavy chain does not contain amino acid sequences that are at least 90% identical to amino acid sequences selected from the group consisting of SEQ ID NO: 29, 51, 73, 95, 117 and 139; and wherein said light chain does not contain amino acid sequences that are at least 90% identical to amino acid sequences selected from the group consisting of SEQ ID NO: 71, 49, 71, 93, 115 and 137.

In some aspects, the invention provides an isolated antibody or antigen-binding portion thereof that exhibits antagonism towards IL-27 and specifically binds to an epitope comprising one or more of the amino acids Gln37, Leu38, Glu42, Glu46, Val49, Ser50, Leu53, Lys56, Leu142, Asp143, Arg145, Asp146, Leu147, Arg149, His150, Arg152, Phe153, Leu156, Ala157, Gly159, Phe160, Asn161, Leu162, Pro163 and Glu164 according to SEQ ID NO: 2 (IL-27p28), wherein said antibody or antigen-binding portion thereof comprises a heavy chain and a light chain, and wherein said heavy chain and said light chain does not contain amino acid sequences selected from the group consisting of:

(i) SEQ ID NO: 25 and 27, respectively;

(ii) SEQ ID NO: 47 and 49, respectively;

(iii) SEQ ID NO: 69 and 71, respectively;

(iv) SEQ ID NO: 91 and 93, respectively;

(v) SEQ ID NO: 113 and 115, respectively; and (vi) SEQ ID NO: 135 and 137, respectively.

In some aspects, the invention provides an isolated antibody or antigen-binding portion thereof that exhibits antagonism towards IL-27 and specifically binds to an epitope comprising one or more of the amino acids Gln37, Leu38, Glu42, Glu46, Val49, Ser50, Leu53, Lys56, Leu142, Asp143, Arg145, Asp146, Leu147, Arg149, His150, Arg152, Phe153, Leu156, Ala157, Gly159, Phe160, Asn161, Leu162, Pro163 and Glu164 according to SEQ ID NO: 2 (IL-27p28), wherein said antibody or antigen-binding portion thereof comprises a heavy chain and a light chain, and wherein said heavy chain and said light chain does not contain amino acid sequences that are at least 90% identical to amino acid sequences selected from the group consisting of:

(i) SEQ ID NO: 25 and 27, respectively;

(ii) SEQ ID NO: 47 and 49, respectively;

(iii) SEQ ID NO: 69 and 71, respectively;

(iv) SEQ ID NO: 91 and 93, respectively;

(v) SEQ ID NO: 113 and 115, respectively; and (vi) SEQ ID NO: 135 and 137, respectively.

In some aspects, the present invention provides an isolated antibody or antigen-binding portion thereof that exhibits antagonism towards IL-27 and specifically binds to an epitope comprising one or more of the amino acids Gln37, Leu38, Glu42, Glu46, Val49, Ser50, Leu53, Lys56, Leu142, Asp143, Arg145, Asp146, Leu147, Arg149, His150, Arg152, Phe153, Leu156, Ala157, Gly159, Phe160, Asn161, Leu162, Pro163 and Glu164 according to SEQ ID NO: 2 (IL-27p28), wherein said antibody or antigen-binding portion thereof comprises a heavy chain and a light chain, and wherein said heavy chain and said light chain does not contain amino acid sequences selected from the group consisting of:

(i) SEQ ID NO: 29 and 27, respectively;

(ii) SEQ ID NO: 51 and 49, respectively;

(iii) SEQ ID NO: 73 and 72, respectively;

(iv) SEQ ID NO: 95 and 93, respectively;

(v) SEQ ID NO: 117 and 115, respectively; and (vi) SEQ ID NO: 139 and 137, respectively.

In some aspects, the present invention provides an isolated antibody or antigen-binding portion thereof that exhibits antagonism towards IL-27 and specifically binds to an epitope comprising one or more of the amino acids Gln37, Leu38, Glu42, Glu46, Val49, Ser50, Leu53, Lys56, Leu142, Asp143, Arg145, Asp146, Leu147, Arg149, His150, Arg152, Phe153, Leu156, Ala157, Gly159, Phe160, Asn161, Leu162, Pro163 and Glu164 according to SEQ ID NO: 2 (IL-27p28), wherein said antibody or antigen-binding portion thereof comprises a heavy chain and a light chain, and wherein said heavy chain and said light chain does not contain amino acid sequences that are at least 90% identical to amino acid sequences selected from the group consisting of:

(i) SEQ ID NO: 29 and 27, respectively;

(ii) SEQ ID NO: 51 and 49, respectively;

(iii) SEQ ID NO: 73 and 72, respectively;

(iv) SEQ ID NO: 95 and 93, respectively;

- 41 052811 (v) SEQ ID NO: 117 and 115, respectively; and (vi) SEQ ID NO: 139 and 137, respectively.

Methods for obtaining antibodies against IL-27 and their antigen-binding fragments.

The present invention also provides methods for producing any of the IL-27 antibodies or antigen-binding fragments of the antibodies described herein. In some aspects of the present invention, methods for producing an antibody provided herein may comprise immunizing a subject (e.g., a non-human mammal) with a suitable immunogen. Suitable immunogens for producing any of the antibodies described herein are provided herein. For example, to generate an antibody that binds to IL-27p28, one skilled in the art can immunize a suitable subject (e.g., a non-human mammal such as a rat, mouse, gerbil, hamster, dog, cat, pig, goat, horse, or non-human primate) with interleukin IL-27. In some aspects of the present invention, a full-length monomeric human IL-27p28 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2 is used as an immunogen.

A suitable subject (e.g., a non-human mammal) can be immunized with the appropriate antigen, followed by a number of booster immunizations sufficient to elicit antibody production in the mammal. The immunogen can be administered to the subject (e.g., a non-human mammal) together with an adjuvant. Adjuvants useful for eliciting antibody in a subject include, but are not limited to, protein adjuvants; bacterial adjuvants such as whole bacteria (BCG, Corynebacterium parvum, or Salmonella minnesota) and bacterial components including cell wall skeleton, trehalose dimycolate, monophosphoryl lipid A, methanol extractable residue (MER) of tubercle bacillus, complete or incomplete Freund's adjuvant; viral adjuvants; Chemical adjuvants such as aluminum hydroxide, iodoacetate, and cholesteryl hemisuccinate. Other adjuvants that can be used in methods for inducing an immune response include, for example, cholera toxin and parapoxvirus proteins. See also Bieg et al. (1999) Autoimmunity 31 (1): 15–24. See also, for example, Lodmell et al. (2000) Vaccine 18: 1059–1066; Johnson et al. (1999) J Med Chem 42: 4640–4649; Baldridge et al. (1999) Methods 19: 103–107; and Gupta et al. (1995) Vaccine 13 (14): 1263–1276.

In some aspects of the present invention, the methods include generating a hybridoma cell line that secretes a monoclonal antibody that binds to an immunogen. For example, a suitable mammal, such as a laboratory mouse, is immunized with an IL-27 polypeptide as described above. Antibody-producing cells (e.g., spleen B cells) from the immunized mammal can be isolated two to four days after at least one booster immunization with the immunogen and then briefly grown in culture before fusion with cells of a suitable myeloma cell line. Cell fusion can be performed in the presence of a fusion promoter, such as, for example, vaccinia virus or polyethylene glycol. The hybrid cells obtained from the fusion are cloned, and cell clones secreting the desired antibodies are selected. For example, spleen cells from a Balb/c mouse immunized with a suitable immunogen can be fused with cells of the PAI myeloma cell line or with cells of the Sp2/0-Ag 14 myeloma cell line. After fusion, the cells are expanded in a suitable culture medium, supplemented with a selection medium, such as hypoxanthine-aminopterin-thymidine medium (HAT medium), at regular intervals to prevent overgrowth of normal myeloma cells relative to the desired hybridoma cells. The resulting hybrid cells are then screened for secretion of desired antibodies, such as an antibody that binds to human IL-27, and in some aspects of the invention, one skilled in the art can identify an anti-IL-27 antibody from a non-immunologically targeted library, as described, for example, in U.S. Patent No. 6,300,064 (to Knappik et al.; Morphosys AG) and Schoonbroodt et al. (2005) Nucleic Acids Res 33 (9): e81.

In some aspects of the invention, the methods described herein can include or be used in combination with, for example, phage display, bacterial display, yeast surface display, eukaryotic viral display, mammalian cell display, and cell-free antibody screening techniques (e.g., ribosome display) (see, for example, Etz et al. (2001) J Bacteriol 183: 6924-6935; Cornelis (2000) Curr Opin Biotechnol 11: 450-454; Klemm et al. (2000) Microbiology 146: 3025-3032; Kieke et al. (1997) Protein Eng 10: 1303-1310; Yeung et al. (2002) Biotechnol Prog 18: 212-220; Boder et al. (2002) Biotechnol Prog 18: 212-220; al. (2000) Methods Enzymology 328: 430 - 444; Grabherr et al. (2001) Comb Chem High Throughput Screen 4: 185 - 192; Michael et al. (1995) Gene Ther 2: 660 - 668; Pereboev et al. (2001) J Virol 75: 7107 - 7113; Schaffitzel et al. (1999) J Immunol Methods 231: 119 - 135; and Hanes et al. (2000) Nat Biotechnol 18: 1287 - 1292).

Methods for identifying antibodies using various phage display methods are known in the art. In phage display methods, the functional domains of an antibody

- 42 052811 are displayed on the surface of phage particles that carry the polynucleotide sequences encoding them. Such phage can be used to display antigen-binding domains of antibodies, such as Fab, Fv, or disulfide-stabilized Fv fragments of antibodies, expressed from a repertoire or combinatorial library of antibodies (e.g., human or murine). Phages used in such methods are typically filamentous phages such as fd and M13. The antigen-binding domains are expressed as a protein recombinantly fused to any of the phage coat proteins pIII, pVIII, or pIX. See, e.g., Shi et al. (2010) JMB 397: 385–396. Examples of phage display methods that can be used to produce the immunoglobulins or fragments thereof described herein include those presented in Brinkman et al. (1995) J Immunol Methods 182: 41–50; Ames et al. (1995) J Immunol Methods 184: 177–186; Kettleborough et al. (1994) Eur J Immunol 24: 952–958; Persic et al. (1997) Gene 187: 9–18; Burton et al. (1994) Advances in Immunology 57: 191–280; and in International Patent Publication Nos. WO 90/02809, WO 91/10737, WO 92/01047, WO 92/18619, WO 93/11236, WO 95/15982, and WO 95/20401. Suitable methods are also shown, for example, in U.S. Patents No. 5,698,426; No. 5,223,409; No. 5,403,484; No. 5,580,717; No. 5,427,908; No. 5,750,753; No. 5,821,047; No. 5,571,698; No. 5,427,908; No. 5,516,637; No. 5,780,225; No. 5,658,727; No. 5,733,743; and No. 5,969,108.

In some aspects of the present invention, phage display antibody libraries can be generated using mRNA collected from B cells of immunized mammals. For example, a spleen cell sample containing B cells can be isolated from mice immunized with an IL-27 polypeptide as described above. mRNA can be isolated from the cells and converted to cDNA using standard molecular biology techniques. See, e.g., Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, ^2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Harlow and Lane (1988), as cited above; Benny KC Lo (2004), as cited above; and Borrebaek (1995), as cited above. cDNA encoding the variable regions of immunoglobulin heavy chain and light chain polypeptides is used to generate the phage display library. Methods for generating such a library are described, for example, in Merz et al. (1995) J Neurosci Methods 62 (1 - 2): 213 - 9; Di Niro et al. (2005) Biochem J 388 (Pt 3): 889 - 894; and Engberg et al. (1995) Methods Mol Biol 51: 355 - 376.

In some aspects of the present invention, a combination of selection and screening can be used to identify an antibody of interest, for example, from a population of hybridoma antibodies or a phage display antibody library. Suitable methods are known in the art and are described, for example, in Hoogenboom (1997) Trends in Biotechnology 15: 62-70; Brinkman et al. (1995), as cited above; Ames et al. (1995), as cited above; Kettleborough et al. (1994), as cited above; Persic et al. (1997), as cited above; and Burton et al. (1994), as cited above. For example, a plurality of phagemid vectors, each encoding a fusion protein of a bacteriophage coat protein (e.g., pIII, pVIII, or pIX of phage M13) and another antigen-binding region, are produced using standard molecular biology techniques and then introduced into a population of bacteria (e.g., E. coli). Expression of the bacteriophage in bacteria may, in some aspects of the invention, require the use of a helper phage. In some aspects of the invention, a helper phage is not required (see, e.g., Chasteen et al. (2006) Nucleic Acids Res 34 (21): e145). Phage produced by the bacteria are isolated and then contacted, for example, with a target antigen bound to a solid support (immobilized). The phage can also be contacted with the antigen in solution, after which the resulting complex is bound to a solid support.

A subpopulation of antibodies isolated by screening using the above methods can be characterized for their specificity and binding affinity to a particular antigen (e.g., human IL-27p28) using any immunological or biochemical method known in the art. For example, specific binding of an antibody to IL-27p28 can be determined, for example, using immunological or biochemical methods such as, but not limited to, ELISA, SPR, immunoprecipitation, affinity chromatography, and equilibrium dialysis, as described above. Immunoassays that can be used to analyze immune-specific binding and cross-reactivity of antibodies include, but are not limited to, competitive and non-competitive assay systems using methods such as Western blotting, RIA, ELISA (enzyme-linked immunosorbent assay), sandwich immunoassays, immunoprecipitation assays, immunodiffusion assays, agglutination assays, complement fixation assays, immunoradiometric assays, fluorescence immunoassays, and protein A immunoassays. Such assays are routine and well known in the art.

In aspects of the present invention in which the selected CDR amino acid sequences are short sequences (e.g., less than 10-15 amino acids in length), nucleic acids encoding the CDRs can be chemically synthesized as described, for example, in Shiraishi et al. (2007) Nucleic Acids Symposium Series 51(1):129-130 and in U.S. Pat.

- 43 052811

6995259. For a given nucleic acid sequence encoding an acceptor antibody, the region of the nucleic acid sequence encoding the CDR can be replaced with chemically synthesized nucleic acids using standard molecular biology techniques. The 5' and 3' ends of the chemically synthesized nucleic acids can be synthesized to contain cohesive-end restriction enzyme sites for use in cloning the nucleic acids into the nucleic acid encoding the variable region of the donor antibody.

In some aspects of the invention, the anti-IL-27 antibodies described herein comprise an altered heavy chain constant region that has reduced effector function (or no effector function) compared to the corresponding unaltered constant region. Effector functions associated with the constant region of an anti-IL-27 antibody can be modulated by altering the properties of the constant region or the Fc region. Altered effector functions include, for example, modulation of one or more of the following functions: antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), apoptosis, binding to one or more Fc receptors, and pro-inflammatory responses. Modulation refers to an increase, decrease, or elimination of the activity of an effector function exhibited by a subject antibody comprising an altered constant region compared to the activity of the unaltered form of the constant region. In certain aspects of the invention, modulation includes situations in which the activity is disabled or completely absent.

In one aspect of the invention, the anti-IL-27 antibodies described herein comprise an IgG4 heavy chain constant region. In one aspect of the invention, said IgG4 heavy chain constant region is a wild-type IgG4 heavy chain constant region. In another aspect of the invention, said IgG4 constant region comprises a mutation, such as one or both of S228P and L235E or L235A, such as according to the EU numbering system (Kabat, EA, et al., supra). In one aspect of the invention, the anti-IL27 antibodies described herein comprise an IgG1 constant region. In one aspect of the invention, said IgG1 heavy chain constant region is a wild-type IgG1 heavy chain constant region. In another aspect of the invention, said IgG1 heavy chain constant region comprises a mutation.

An altered constant region with altered FcR binding affinity and/or ADCC activity and/or altered CDC activity is a polypeptide that has either increased or decreased FcR binding activity and/or ADCC activity and/or CDC activity compared to an unaltered form of the constant region. An altered constant region exhibiting increased FcR binding binds at least one FcR with greater affinity than the unaltered polypeptide. An altered constant region exhibiting decreased FcR binding binds at least one FcR with lower affinity than the unaltered form of the constant region. Such variants that exhibit reduced FcR binding may exhibit little or no detectable FcR binding, e.g., exhibiting 0% to 50% (e.g., less than 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1%) of FcR binding compared to the FcR binding level of the native sequence immunoglobulin constant region or Fc region. Similarly, an altered constant region that exhibits modulated ADCC and/or CDC activity may exhibit either increased or decreased ADCC and/or CDC activity compared to an unaltered constant region. For example, in some aspects of the invention, an anti-IL-27 antibody comprising an altered constant region may exhibit from about 0% to about 50% (e.g., less than 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1%) ADCC activity and (or) CDC compared to the unmodified constant region. The anti-IL-27 antibody described herein, which contains an altered constant region exhibiting reduced ADCC and/or CDC, may exhibit reduced ADCC and/or CDC activity, or none at all.

In some aspects of the invention, the anti-IL-27 antibody described herein exhibits reduced or no effector function. In some aspects of the invention, the anti-IL-27 antibody comprises a hybrid constant region or portion thereof, such as a hybrid G2/G4 constant region (see, e.g., Burton et al. (1992) Adv Immun 51: 1-18; Canfield et al. (1991) J Exp Med 173: 1483-1491; and Mueller et al. (1997) Mol Immunol 34 (6): 441,452).

In some aspects of the present invention, an anti-IL-27 antibody may comprise an altered constant region exhibiting increased or decreased complement-dependent cytotoxicity (CDC). Modulation of CDC activity may be achieved by administering one or more of the following:

- 44 052811 more amino acid substitutions, insertions, or deletions in the Fc region of the antibody. See, e.g., U.S. Patent No. 6,194,551. Alternatively or in addition, cysteine residue(s) may be introduced into the Fc region, thereby providing for the formation of an interchain disulfide bond in this region. The homodimeric antibody thus produced may have improved or decreased internalization capacity and/or increased or decreased complement-mediated cell killing activity. See, e.g., Caron et al. (1992) J Exp Med 176: 1191-1195 and Shopes (1992) Immunol 148: 2918-2922; PCT International Patent Publication Nos. WO 99/51642 and WO 94/29351; Duncan and Winter (1988) Nature 322: 738–40; and U.S. Patent Nos. 5,648,260 and 5,624,821.

Expression and purification of recombinant antibodies.

The antibodies or antigen-binding fragments thereof described herein can be produced using a variety of techniques known in the fields of molecular biology and protein chemistry. For example, a nucleic acid encoding one or both heavy and light chain polypeptides of an antibody can be inserted into an expression vector that contains transcriptional and translational regulatory sequences, which include, for example, promoter sequences, ribosome binding sites, transcriptional start and stop sequences, translational start and stop sequences, transcriptional terminator signals, polyadenylation signals, and enhancer or activator sequences. Regulatory sequences include a promoter and transcriptional start and stop sequences. In addition, the expression vector can include more than one replication system, so that it can be maintained in two different organisms, for example, in mammalian or insect cells for expression and in a prokaryotic host for cloning and amplification.

Several possible vector systems are available for the expression of cloned heavy and light chain polypeptides from nucleic acids in mammalian cells. One class of vectors is based on the integration of the desired gene sequences into the host cell genome. Cells with stably integrated DNA can be selected by the simultaneous introduction of drug resistance genes, such as E. coli gpt (Mulligan and Berg (1981) Proc Natl Acad Sci USA 78: 2072) or Tn5 neo (Southern and Berg (1982) Mol Appl Genet 1: 327). The selectable marker gene can either be directly linked to the DNA sequences to be expressed or introduced into the same cell by cotransfection (Wigler et al. (1979) Cell 16: 77). A second class of vectors uses DNA elements that confer autonomous replication capability on an extrachromosomal plasmid. Such vectors can be derived from animal viruses such as bovine papillomavirus (Sarver et al. (1982) Proc Natl Acad Sci USA 79: 7147), cytomegalovirus, polyoma virus (Deans et al. (1984) Proc Natl Acad Sci USA 81: 1292), or SV40 virus (Lusky and Botchan (1981) Nature 293: 79).

Expression vectors can be introduced into cells using a method suitable for subsequent nucleic acid expression. The route of introduction is largely dictated by the target cell type, as discussed below. Illustrative methods include _CaPO4 precipitation, liposome fusion, cationic liposomes, electroporation, viral infection, dextran-mediated transfection, polybrene-mediated transfection, protoplast fusion, and direct microinjection.

Suitable host cells for expressing antibodies or their antigen-binding fragments include yeast, bacteria, insects, plants, and mammals. Of particular interest are bacteria such as E. coli, fungi such as Saccharomyces cerevisiae and Pichia pastoris, insect cells such as SF9, mammalian cell lines (e.g., human cell lines), and primary cell lines.

In some aspects of the invention, the antibody or fragment thereof can be expressed and purified from transgenic animals (e.g., transgenic mammals). For example, the antibody can be produced in a transgenic mammal (e.g., a rodent) and isolated from milk as described, for example, in Houdebine (2002) Curr Opin Biotechnol 13 (6): 625-629; van Kuik-Romeijn et al. (2000) Transgenic Res 9 (2): 155-159; and Pollock et al. (1999) J Immunol Methods 231 (1-2): 147-157.

Antibodies and fragments thereof can be produced from cells by culturing a host cell transformed with an expression vector containing a nucleic acid encoding the antibodies or fragments thereof under conditions and for a time sufficient to ensure expression of the proteins. Such conditions for protein expression will vary depending on the choice of expression vector and host cell and are readily determined by one skilled in the art using routine experimentation. For example, antibodies expressed in E. coli can be obtained by refolding from inclusion bodies (see, e.g., Hou et al. (1998) Cytokine 10: 319–30). Bacterial expression systems and methods for using them are well known in the art (see Current Protocols in Molecular Biology, Wiley & Sons, and Molecular Cloning—A Laboratory Manual— ^3rd Ed.,

- 45 052811

Cold Spring Harbor Laboratory Press, New York (2001)). The choice of codons, appropriate expression vectors, and suitable host cells will vary depending on a number of factors and can be easily optimized if necessary. The antibody (or fragment thereof) described herein can be expressed in mammalian cells or other expression systems, including, but not limited to, yeast, baculoviruses, and in vitro expression systems (see, e.g., Kaszubska et al. (2000) Protein Expression and Purification 18: 213–220).

After expression, antibodies and their fragments can be isolated. The antibody or fragment can be isolated or purified by a variety of methods known to those skilled in the art, depending on what other components are present in the sample. Standard purification methods include electrophoretic, molecular, immunoassay, and chromatographic techniques, including ion exchange, hydrophobic interaction, affinity chromatography, and reversed-phase HPLC. For example, an antibody can be purified using a standard anti-antibody column (e.g., a protein A or protein G column). Ultrafiltration and diafiltration techniques in combination with protein concentration can also be used. See, e.g., Scopes (1994) Protein Purification, ^3rd edition, Springer-Verlag, New York City, New York. The degree of purification will necessarily depend on the desired application. In some cases, there is no need to purify the expressed antibody or its fragments.

Methods for determining the yield or purity of a purified antibody or fragment thereof are known in the art and include, for example, the Bradford assay, UV spectroscopy, biuret protein assay, Lowry protein assay, amide black protein assay, high pressure liquid chromatography (HPLC), mass spectrometry (MS), and gel electrophoresis methods (e.g., using a protein dye such as Coomassie blue or colloidal silver).

Modification of antibodies or their antigen-binding fragments.

Antibodies or their antigen-binding fragments can be modified after expression and purification. Modifications can be covalent or non-covalent. Such modifications can be introduced into antibodies or their fragments, for example, by reacting target amino acid residues of a given polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues. Suitable sites for modification can be selected using any of a variety of criteria, including, for example, structural analysis or amino acid sequence analysis of the antibodies or their fragments.

In some aspects of the present invention, antibodies or antigen-binding fragments thereof may be conjugated to a heterologous moiety. Such a heterologous moiety may be, for example, a heterologous polypeptide, a therapeutic agent (e.g., a toxin or a drug), or a detectable label, such as, but not limited to, a radioactive label, an enzymatic label, a fluorescent label, a heavy metal label, a luminescent label, or an affinity label such as biotin or streptavidin. Suitable heterologous polypeptides include, for example, an antigen tag (FLAG (DYKDDDDK (SEQ ID NO: 141)), polyhistidine (6-His; HHHHHH (SEQ ID NO: 142), hemagglutinin (HA; YPYDVPDYA (SEQ ID NO: 143)), glutathione S-transferase (GST) or maltose binding protein (MBP).

MBP)), for use in the purification of antibodies or their fragments. Heterologous polypeptides also include polypeptides (e.g., enzymes) that can be used as diagnostic or detectable markers, such as luciferase, fluorescent protein (e.g., green fluorescent protein (GFP)) or chloramphenicol acetyltransferase (CAT). Suitable radioactive labels include, for example, ^32P , ^33P , ^14C , ^125I , ^131I , ^35S , and ^3H . Suitable fluorescent labels include, but are not limited to, fluorescein, fluorescein isothiocyanate (FITC), green fluorescent protein (GFP), DyLight™ 488, phycoerythrin (PE), propidium iodide (PI), PerCP, PE-Alexa Fluor® 700, Cy5, allophycocyanin, and Cy7. Luminescent labels include, for example, any of a variety of fluorescent lanthanide chelates (e.g., europium or terbium). For example, suitable europium chelates include europium chelate and diethylenetriaminepentaacetic acid (DTPA) or tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). Enzymatic labels include, for example, alkaline phosphatase, ChAT, luciferase, and horseradish peroxidase.

Two proteins (e.g., an antibody and a heterologous fragment) can be cross-linked using any of a number of known chemical cross-linking agents. Examples of such cross-linking agents are those that link two amino acid residues via a linkage involving a hindered disulfide bond. In such linkages, the disulfide bond in the cross-linking unit is protected (by sterically blocking groups on either side of the disulfide bond) from reduction by, for example, reduced glutathione or the enzyme disulfide reductase. One suitable reagent, 4-succinimidyloxycarbonyl-α-methyl-α(2-pyridyldithio)toluene (SMPT), forms such a linkage between two proteins using

- 46 052811 terminal lysine on one protein and terminal cysteine on the other. Heterobifunctional reagents that cross-link via different binding groups on each protein can also be used. Other suitable cross-linking agents include, but are not limited to, reagents that link two amino groups (e.g., N-5-azido-2-nitrobenzoyloxysuccinimide), two sulfhydryl groups (e.g., 1,4-bis-maleimidobutane), an amino group and a sulfhydryl group (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester), an amino group and a carboxyl group (e.g., 4-[p-azidosalicylamido]butylamine), and an amino group and a guanidinium group that are present in the side chain of arginine (e.g., p-azidophenylglyoxal monohydrate).

In some aspects of the present invention, the radioactive label may be directly conjugated to the amino acid backbone of the antibody. Alternatively, the radiolabel may be incorporated as part of a larger molecule (e.g., ¹²⁵ I in meta-[ ^125I ]iodophenyl-Nhydroxysuccinimide ([ ^125I ]mIPNHS) which couples to free amino groups to form meta- ^iodophenyl (mIP) derivatives of the corresponding proteins (see, e.g., Rogers et al. (1997) J NuclMed 38: 1221-1229) or as a chelate (e.g., with TCTC or DTPC) which is in turn linked to the protein backbone. Methods for conjugating radiolabels or larger molecules/chelates containing them to the antibodies described herein or to antigen-binding fragments thereof are known in the art. Such methods include involves incubating radioactively labeled proteins under specific conditions (e.g., pH, salt concentration, and/or temperature) that facilitate binding of the radioactive label or chelate to the protein (see, e.g., U.S. Patent No. 6,001,329).

Methods for conjugating a fluorescent label (sometimes referred to as a fluorophore) to a protein (e.g., an antibody) are known in the field of protein chemistry. For example, fluorophores can be conjugated to free amino groups (e.g., lysines) or sulfhydryl groups (e.g., cysteines) of proteins using succinimidyl ester (NHS) or tetrafluorophenyl ester (TFP) groups attached to the fluorophores. In some aspects of the present invention, fluorophores can be conjugated to a heterobifunctional cross-linking moiety, such as sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC). Suitable conjugation methods include incubating the antibody protein or fragment thereof with a fluorophore under conditions that facilitate binding of the fluorophore to the protein. See, for example, Welch and Redvanly (2003) Handbook of Radiopharmaceuticals: Radiochemistry and Applications, John Wiley and Sons (ISBN 0471495603).

In some aspects of the invention, antibodies or fragments thereof can be modified, e.g., with a moiety that improves stabilization and/or retention of the antibody in circulation, e.g., in blood, serum, or other tissues. For example, an antibody or fragment thereof can be PEGylated as described, e.g., in Lee et al. (1999) Bioconjug Chem 10 (6): 973-8; Kinstler et al. (2002) Advanced Drug Deliveries Reviews 54: 477-485; and Roberts et al. (2002) Advanced Drug Delivery Reviews 54: 459-476; or HES (using hydroxyethyl starch - HES) (Fresenius Kabi, Germany; see, e.g., Pavisic et al. (2010) Int J Pharm 387 (1-2): 110-119). The stabilizing moiety can improve the stability or retention of the antibody (or fragment thereof) by at least 1.5-fold (e.g., at least 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, or 50-fold or more).

In some aspects of the invention, the antibodies or antigen-binding fragments thereof described herein may be glycosylated. In some aspects of the invention, an antibody or antigen-binding fragment thereof described herein may be enzymatically or chemically treated, or obtained from a cell, such that the antibody or fragment thereof has a low degree of glycosylation or is not glycosylated at all. Methods for producing antibodies with a low degree of glycosylation are known in the art and are described, for example, in U.S. Patent No. 6,933,368; and in Wright et al. (1991) EMBO J 10 (10): 2717-2723; and Co et al. (1993) Mol Immunol 30: 1361.

Pharmaceutical compositions and formulations.

In some aspects of the present invention, a pharmaceutical composition is provided comprising an anti-IL-27 antibody together with pharmaceutically acceptable diluents, a carrier, a solubilizer, an emulsifier, a preservative, and/or an adjuvant.

In certain aspects of the invention, suitable formulation materials are preferably non-toxic to recipients at the dosages and concentrations employed. In some aspects of the invention, the formulation material(s) are intended for subcutaneous and/or intravenous administration. In certain aspects of the invention, the pharmaceutical composition may comprise formulation materials to modify, maintain, or preserve, for example, pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution rate, or release rate.

- 47 052811 adsorption or permeability of said composition. In certain aspects of the invention, suitable materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine, proline, or lysine); antimicrobial agents; antioxidants (such as ascorbic acid, sodium sulfite, or sodium hydrosulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates, or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-betacyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); colors, flavors and diluents; emulsifiers; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenylethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerol, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; Surface-active substances or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol, tyloxapol); stability-enhancing agents (such as sucrose or sorbitol); tonicity-enhancing agents (such as alkali metal halides, preferably sodium or potassium chloride, mannitol, sorbitol); carriers; diluents; excipients and/or pharmaceutical adjuvants. (Remington's Pharmaceutical Sciences, ^18th Edition, A.R. Gennaro, ed., Mack Publishing Company (1995). In some aspects of the invention, the dosage formulation comprises PBS; 20 mM sodium acetate, pH 5.2, 50 mM NaCl; and/or 10 mM sodium acetate, pH 5.2, 9% sucrose. In certain aspects of the invention, the optimal pharmaceutical composition is determined by one of skill in the art depending on, for example, the intended route of administration, the delivery form, and the desired dosage. See, for example, Remington's Pharmaceutical Sciences, as noted above. In certain aspects of the invention, such compositions can affect the physical state, stability, in vivo release rate, and/or in vivo clearance rate of the anti-IL-27 antibody.

In certain aspects of the present invention, the primary carrier medium or vehicle in the pharmaceutical composition may be either aqueous or non-aqueous in nature. For example, in certain aspects of the present invention, a suitable carrier medium or vehicle may be water for injection, physiological saline, or artificial cerebrospinal fluid, optionally supplemented with other materials frequently used in parenteral compositions. In certain aspects of the present invention, the physiological solution comprises isotonic phosphate-buffered saline. In certain aspects of the present invention, additional exemplary carriers include neutral buffered saline or saline mixed with serum albumin. In certain aspects of the present invention, the pharmaceutical compositions comprise a Tris buffer with a pH of about 7.0-8.5 or an acetate buffer with a pH of about 4.0-5.5, and may further comprise sorbitol or a suitable substitute thereof. In certain aspects of the present invention, compositions containing an anti-IL-27 antibody can be prepared for storage by mixing the selected composition, having the required degree of purity, with optimal formulation agents (see Remington's Pharmaceutical Sciences, as noted above) in the form of a lyophilized mass or an aqueous solution. Furthermore, in certain aspects of the present invention, a composition containing an anti-IL-27 antibody can be formulated as a lyophilisate using appropriate excipients, such as sucrose.

In certain aspects of the present invention, pharmaceutical compositions may be selected for parenteral delivery. In certain aspects of the present invention, compositions may be selected for inhalation or for delivery via the gastrointestinal tract, such as oral delivery.

The preparation of such pharmaceutically acceptable compositions is within the skill of the art.

In certain aspects of the present invention, the components of the drug formulation are preferably present in concentrations that are appropriate for the specific site of administration. In certain aspects of the present invention, buffers are used to maintain the composition at a physiological pH or slightly lower pH, typically within the pH range of about 5 to about 8.

In certain aspects of the present invention, if parenteral administration is contemplated, the therapeutic compositions may be in the form of a pyrogen-free, parenterally acceptable aqueous solution comprising an anti-IL-27 antibody and a pharmaceutically acceptable carrier. In certain aspects of the present invention, the parenteral injection carrier is

- 48 052811 sterile distilled water to which an anti-IL-27 antibody has been added, in the form of a sterile isotonic solution with an appropriate preservative. In certain aspects of the present invention, the preparation may include a finished drug formulation of the desired molecule with an agent such as injectable microspheres, biodegradable particles, polymeric compounds (such as polylactic acid or polyglycolic acid), granules, or liposomes that can provide controlled or sustained release of the product, which can be delivered by depot injection. In certain aspects of the present invention, hyaluronic acid can also be used, the effect of which is to provide an extended period of residence in the circulation. In certain aspects of the present invention, implantable drug delivery devices can be used to administer the desired molecule.

In certain aspects of the present invention, the pharmaceutical composition is formulated for inhalation. In certain aspects of the present invention, the anti-IL-27 antibody may be formulated as a dry powder for inhalation. In certain aspects of the present invention, an inhalation solution containing the anti-IL-27 antibody may be formulated with a propellant for aerosol delivery. In certain aspects of the present invention, the solutions may be nebulized. Inhalation administration is further described in International Patent Document No. PCT/US94/001875, which describes the inhalation administration of chemically modified proteins.

In certain aspects of the present invention, it is contemplated that the medicinal compositions can be administered orally. In certain aspects of the present invention, the anti-IL-27 antibody administered in this manner can be formulated with or without carriers commonly used in the formulation of solid dosage forms, such as tablets or capsules. In certain aspects of the present invention, the capsule can be designed so that the release of the active portion of the medicinal composition occurs at a point in the gastrointestinal tract where bioavailability is maximized and presystemic degradation is minimized. In certain aspects of the present invention, at least one additional agent can be added to facilitate absorption of the anti-IL-27 antibody. In certain aspects of the present invention, diluents, flavoring agents, refractory waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders can also be used.

In certain aspects of the present invention, a pharmaceutical composition may comprise an effective amount of an anti-IL-27 antibody in admixture with non-toxic excipients suitable for the manufacture of tablets. In certain aspects of the present invention, solutions in unit dose form can be prepared by dissolving tablets in sterile water or another suitable vehicle. In certain aspects of the present invention, suitable excipients include, but are not limited to, inert diluents such as calcium carbonate, sodium carbonate or bicarbonate, lactose or calcium phosphate; or binders such as starch, gelatin or acacia; or lubricating agents such as magnesium stearate, stearic acid or talc.

Additional pharmaceutical compositions will be apparent to those skilled in the art, including anti-IL-27 antibody formulations characterized by prolonged or controlled delivery. In certain aspects of the present invention, those skilled in the art are also familiar with techniques for formulating various other sustained or controlled delivery vehicles, such as liposomal carriers, biodegradable microparticles or porous microgranules, and depot injections. See, for example, PCT Application No. PCT/US93/00829, which describes the controlled release of porous polymeric microparticles for the delivery of pharmaceutical compositions. In certain aspects of the present invention, sustained-release preparations may comprise semipermeable polymer matrices in the form of shaped articles, such as films or microcapsules. Matrices that provide sustained release may include polyesters, hydrogels, polylactides (U.S. Patent No. 3,773,919 and European Patent Application No. EP 058481), copolymers of L-glutamic acid and gamma-ethyl-E-glutamate (Sidman et al., Biopolymers, 22: 547-556 (1983)), poly(2-hydroxyethyl methacrylate) (Eanger et al., J. Biomed. Mater. Res., 15: 167-277 (1981); and Eanger, Chem. Tech., 12: 98-105 (1982)), ethylene vinyl acetate (Eanger et al., supra), or poly-P(-)-3-hydroxybutyric acid (EP 133988). In certain aspects of the present invention, sustained release compositions may also include liposomes, which can be prepared by any of several methods known in the art. See, for example, Eppstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688-3692 (1985); European Patent Application Nos. EP 036676; EP 088046; and EP 143949.

A pharmaceutical composition for use in in vivo administration is generally sterile. In certain aspects of the invention, this can be achieved by filtration through sterile filter membranes. In certain aspects of the invention, if the composition is lyophilized, sterilization using this method can be performed before or after lyophilization and reconstitution. In certain aspects of the invention

- 49 052811 parenteral compositions can be stored in lyophilized form or in solution. In certain aspects of the present invention, parenteral compositions are generally placed in a container having a sterile entry port, such as an intravenous solution bag or a vial having a stopper pierced by a hypodermic injection needle.

In certain aspects of the present invention, after preparation of the pharmaceutical composition, it can be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder. In certain aspects of the present invention, such medicinal compositions can be stored in a ready-to-use form or in a form (e.g., lyophilized) that is reconstituted prior to administration.

Certain aspects of the present invention also provide kits for preparing a single dose for administration. In certain aspects of the present invention, said kit comprises a first container containing a dry protein and a second container containing an aqueous preparation. Certain aspects of the present invention also provide kits containing single- and multi-chamber prefilled syringes (e.g., liquid-containing syringes and lyophilisate-containing syringes).

In certain aspects of the present invention, the effective amount of a therapeutically useful pharmaceutical composition comprising an anti-IL-27 antibody will depend, for example, on the circumstances and goals of the treatment. One skilled in the art will appreciate that, according to certain aspects of the present invention, suitable dosage levels for treatment will vary depending, in part, on the molecule being delivered, the indication for which the anti-IL-27 antibody is used, the route of administration, and the size (body weight, body surface area, or organ size) and/or condition (age and general health) of the patient. In certain aspects of the present invention, the treating physician may titrate the dose and vary the route of administration to achieve the optimal therapeutic effect.

In certain aspects of the present invention, the frequency of dosing will take into account the pharmacokinetic parameters of the anti-IL-27 antibody in the formulation used. In certain aspects of the present invention, the treating physician will administer the composition until a dose that results in the desired effect is reached. Thus, in certain aspects of the present invention, the composition can be administered as a single dose, or as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion through an implantable device or catheter. Further improvement in the use of the appropriate dose is routinely performed by those of ordinary skill in the art and is within the scope of tasks routinely performed by them. In certain aspects of the present invention, appropriate doses can be determined based on relevant dose-effect data.

In certain aspects of the present invention, the route of administration of the pharmaceutical composition corresponds to known methods, for example, oral administration, administration by intravenous, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, subcutaneous, intraocular, intraarterial, intraportal or intralesional injections; using sustained release systems or implantable devices. In certain aspects of the present invention, the compositions can be administered by bolus injection or continuously by infusion, or using an implantable device. In certain aspects of the present invention, the individual elements of the combination therapy can be administered by different routes.

In certain aspects of the present invention, the composition can be administered locally by implanting a membrane, sponge, or other suitable material in which the desired molecule is absorbed or encapsulated. In certain aspects of the present invention, if an implantable device is used, said device can be implanted into any suitable tissue or organ, and delivery of the desired molecule can be accomplished by diffusion, a delayed-release bolus, or continuous administration. In certain aspects of the present invention, it may be desirable to administer a pharmaceutical composition containing an anti-IL-27 antibody ex vivo. In such cases, cells, tissues, and/or organs that have been removed from the patient's body are treated with a pharmaceutical composition containing an anti-IL-27 antibody, after which these cells, tissues, and/or organs are reimplanted into the patient's body.

In certain aspects of the invention, the anti-IL-27 antibody may be delivered by implantation of certain cells that have been genetically engineered by methods such as those described herein to express and secrete the polypeptides. In certain aspects of the invention, such cells may be animal or human cells and may be autologous, heterologous, or xenogeneic. In certain aspects of the invention, the cells may be immortalized. In certain aspects of the invention, to reduce the likelihood of an immunological response, the cells may be encapsulated to avoid

- 50 052811 infiltration of surrounding tissues. In certain aspects of the present invention, encapsulating materials typically comprise biocompatible, semipermeable polymeric shells or membranes that allow the release of the protein product(s) but prevent cell destruction by the patient's immune system or other harmful factors from surrounding tissues.

Application options.

The compositions described herein can be used in a variety of diagnostic and therapeutic applications. For example, detectably labeled antigen-binding molecules can be used in assays to detect the presence or amount of target antigens in a sample (e.g., a biological sample). The compositions can be used in in vitro assays to study the inhibition of target antigen function. In some aspects of the invention, for example, in which the compositions bind to and inhibit a complement protein, the compositions can be used as positive controls in assays designed to identify additional novel compounds that inhibit complement activity or are otherwise useful for treating a complement-related disorder. For example, a composition that inhibits IL-27 can be used as a positive control in an assay to identify additional compounds (e.g., small molecules, aptamers, or antibodies) that reduce or eliminate IL-27 production. The compositions can also be used in therapeutic methods, as described in detail below.

In some aspects of the present invention, there is provided a method for detecting IL-27 in a biological sample or in a subject, comprising: (i) contacting said sample or said subject (and, optionally, a reference sample or subject) with any antibody described herein under conditions that allow interaction between the antibody molecule and IL-27, and (ii) detecting the formation of a complex between the antibody molecule and the sample or subject (and, optionally, a reference sample or subject).

Sets.

A kit may include an anti-IL-27 antibody, as described herein, and instructions for use. Kits may include, in a suitable container, an anti-IL-27 antibody, one or more controls, and various buffers, reagents, enzymes, and other standard ingredients well known in the art. In some aspects, the present invention provides a kit comprising an anti-IL-27 antibody or an antigen-binding portion thereof, as described herein, and instructions for use to stimulate an immune response in a subject or to treat an oncological disease in a subject, optionally with instructions for use in combination with one or more additional therapeutic agents or procedures, as described herein.

The container may include at least one vial, well, test tube, flask, bottle, syringe, or other type of container into which the anti-IL-27 antibody can be placed and, in some cases, appropriately aliquoted. In cases where an additional component is provided, the kit contains one or more additional containers into which the component can be placed. These kits may also include means for holding containers of the anti-IL-27 antibody or any other reagent tightly packaged for commercial sale. Such containers may include plastic containers manufactured by injection or blow molding that contain the desired vials. The containers and/or kits may include labeling with instructions for use and/or warnings.

Methods of application.

The compositions of the present invention have numerous in vitro and in vivo uses, including the detection and/or quantification of IL-27 and/or antagonism of IL-27 function.

In some aspects of the present invention, a method is provided for inhibiting or reducing the phosphorylation of STAT1 and/or STAT3 in a cell, comprising contacting said cell with an isolated antibody or antigen-binding portion thereof provided herein, wherein said antibody or antigen-binding portion thereof inhibits or reduces the phosphorylation of STAT1 and/or STAT3 in the cell.

In some aspects of the present invention, a method is provided for inhibiting or reducing the inhibition of CD161 expression in a cell, comprising contacting said cell with an isolated antibody or antigen-binding portion thereof provided herein, wherein said antibody or antigen-binding portion thereof inhibits or reduces the inhibition of CD161 expression in the cell.

In some aspects of the present invention, there is provided a method of inhibiting or reducing the expression of PD-L1 and/or TIM-3 in a cell, comprising contacting said cell with an isolated antibody or antigen-binding portion thereof as provided herein,

- 51 052811 wherein the said antibody or its antigen-binding portion inhibits the expression of PD-L1 and/or TIM-3 in a cell.

In some aspects of the present invention, a method for inducing or enhancing the secretion of one or more cytokines from a cell is provided, comprising contacting said cell with an isolated antibody or antigen-binding portion thereof provided herein, wherein said antibody or antigen-binding portion thereof induces or enhances PD-1-mediated secretion of one or more cytokines from the cell.

In some aspects of the present invention, a method for stimulating an immune response in a subject is provided, comprising administering to said subject an effective amount of an isolated antibody or antigen-binding portion thereof that specifically binds to and antagonizes IL-27, as provided herein, or a pharmaceutical composition comprising said antibody or antigen-binding portion thereof and a pharmaceutically acceptable carrier.

In some aspects of the present invention, a method of treating an oncological disease in a subject is provided, comprising administering to said subject an effective amount of an isolated antibody or antigen-binding portion thereof that specifically binds to and antagonizes IL-27, as provided herein, or a pharmaceutical composition comprising said antibody or antigen-binding portion thereof and a pharmaceutically acceptable carrier.

In some aspects of the present invention, a method for stimulating an immune response or treating an oncological disease in a subject is provided, comprising administering to said subject an effective amount of an isolated antibody or antigen-binding fragment as provided herein, or a pharmaceutical composition comprising said antibody or antigen-binding portion thereof and a pharmaceutically acceptable carrier, wherein said antibody, antigen-binding portion thereof, or pharmaceutical composition inhibits or reduces phosphorylation of STAT1 and/or STAT3 in a cell, thereby stimulating an immune response or treating an oncological disease.

In some aspects of the present invention, a method for stimulating an immune response or treating an oncological disease in a subject is provided, comprising administering to said subject an effective amount of an isolated antibody or antigen-binding fragment as provided herein, or a pharmaceutical composition comprising said antibody or antigen-binding portion thereof and a pharmaceutically acceptable carrier, wherein said antibody, antigen-binding portion thereof, or pharmaceutical composition inhibits or reduces the inhibition of CD161 expression in a cell, thereby stimulating an immune response or treating an oncological disease.

In some aspects of the present invention, a method for stimulating an immune response or treating an oncological disease in a subject is provided, comprising administering to said subject an effective amount of an isolated antibody or antigen-binding fragment as provided herein, or a pharmaceutical composition comprising said antibody or antigen-binding portion thereof and a pharmaceutically acceptable carrier, wherein said antibody, antigen-binding portion thereof, or pharmaceutical composition inhibits or reduces the expression of PD-L1 and/or TIM-3 in a cell, thereby stimulating an immune response or treating an oncological disease.

In some aspects of the present invention, a method for stimulating an immune response or treating an oncological disease in a subject is provided, comprising administering to said subject an effective amount of an isolated antibody or antigen-binding fragment as provided herein, or a pharmaceutical composition comprising said antibody or antigen-binding portion thereof and a pharmaceutically acceptable carrier, wherein said antibody, antigen-binding portion thereof, or pharmaceutical composition induces or enhances PD-1-mediated secretion of one or more cytokines from a cell, thereby providing stimulation of an immune response or treatment of an oncological disease.

In some aspects of the invention, the oncological disease is selected from lung cancer (e.g., non-small cell lung cancer), sarcoma, testicular cancer, ovarian cancer, pancreatic cancer, breast cancer (e.g., triple-negative breast cancer), melanoma, head and neck cancer (e.g., squamous cell carcinoma of the head and neck), colorectal cancer, bladder cancer, endometrial cancer, prostate cancer, thyroid cancer, hepatocellular carcinoma, gastric cancer, brain cancer, lymphoma (e.g., DLBCL), leukemia (e.g., AML), or kidney cancer (e.g., renal cell carcinoma, e.g., clear cell renal cell carcinoma).

The above-described compositions are useful, among other things, in methods for treating or preventing various types of cancer in a subject. These compositions can be administered to a subject, such as a human subject, by a variety of routes depending, in part, on

- 52 052811 routes of administration. The route of administration may be, for example, intravenous (IV) injection or infusion, subcutaneous (SC) injection, intraperitoneal (IP) injection, intramuscular (IM) injection, or intrathecal (IT) injection. The injection may be a bolus injection or a continuous infusion.

Administration can be accomplished, for example, by local infusion, injection, or by means of an implant. The implant can be of a porous, nonporous, or gelatinous material, including membranes such as silicone membranes, or fibers. The implant can be configured to release the composition into the subject in a sustained or periodic manner. See, for example, U.S. Patent Application Publication No. 20080241223; U.S. Patent Nos. 5,501,856; No. 4,863,457; and No. 3,710,795; European Patent Publications EP 488401; and EP 430539; the disclosure of each of which is incorporated herein by reference in its entirety. The composition can be delivered to the subject's body using an implantable device based on, for example, diffusion, erosion or convective systems, such as osmotic pumps, biodegradable implants, electrodiffusion systems, electroosmotic systems, steam pressure pumps, electrolytic pumps, effervescent pumps, piezoelectric pumps, erosion-based systems or electromechanical systems.

In some aspects of the present invention, the anti-IL-27 antibody or antigen-binding fragment thereof is therapeutically delivered to a subject by local administration.

A suitable dose of the antibody or fragment thereof composition described herein that can treat or prevent cancer in a subject may depend on a variety of factors, including, for example, the age, gender, and body weight of the subject being treated, and the particular inhibitor used. For example, treating a subject with cancer may require a different dose of the whole anti-IL-27 antibody than the dose of the IL-27-binding Fab' fragment of the antibody required to treat the same subject. Other factors that influence the dose administered to a subject include, for example, the type or severity of the cancer. For example, a subject with metastatic melanoma may require a different dose of the anti-IL-27 antibody than a subject with glioblastoma. Other factors may include, for example, other medical conditions present or previously present in the subject, the subject's overall health, the subject's genetic predisposition, diet, timing of administration, rate of elimination, drug combinations, and any other additional therapeutic agents being administered to the subject. Furthermore, it should be understood that the specific dose and treatment regimen for any given subject may also be adjusted based on the judgment of the healthcare professional (e.g., the attending physician or nurse) treating the subject. Suitable doses are described in this document.

A pharmaceutical composition may include a therapeutically effective amount of an anti-IL-27 antibody or antigen-binding fragment thereof described herein. Such effective amounts can be determined by one of skill in the art, in part based on the effect of the administered antibody or the combinatorial effect of the antibody and one or more additional active agents, if more than one agent is used. A therapeutically effective amount of an antibody or antigen-binding fragment thereof described herein may vary depending on factors such as the stage of the disease, age, sex, and body weight of the individual, as well as the ability of the antibody (as well as one or more additional active agents) to elicit a desired response in the individual, such as a reduction in tumor growth. For example, a therapeutically effective amount of an anti-IL-27 antibody may inhibit (reduce the severity or eliminate the occurrence of) and/or prevent a particular disorder and/or any of the symptoms of a particular disorder known in the art or described herein. A therapeutically effective amount is also one in which the therapeutically beneficial effects outweigh any toxic or deleterious effects of the composition.

Suitable human doses of any of the antibodies or fragments thereof described herein can be further evaluated, for example, in phase I dose escalation studies. See, for example, van Gurp et al. (2008) Am J Transplantation 8 (8): 1711–1718; Hanouska et al. (2007) Clin Cancer Res 13 (2, part 1): 523–531; and Hetherington et al. (2006) Antimicrobial 5 Agents and Chemotherapy 0 (10): 3499–3500.

In some aspects of the invention, a composition comprises any of the antibodies or antigen-binding fragments thereof described herein and one or more (e.g., two, three, four, five, six, seven, eight, nine, 10, or 11, or more) additional therapeutic agents, such that said composition as a whole is therapeutically effective. For example, a composition may comprise an anti-IL-27 antibody described herein and an alkylating agent, wherein said antibody and agent are at a concentration such that,

- 53 052811 their combination is therapeutically effective for the treatment or prevention of an oncological disease (eg, melanoma) in a subject.

The toxicity and therapeutic efficacy of such compositions can be determined using known pharmaceutical procedures in cell cultures or in experimental animals (e.g., in animal models of any of the cancers described herein). Such procedures can be used, for example, to determine the LD ₅₀ (the dose lethal to 50% of the population) and ED ₅₀ (the dose therapeutically effective for 50% of the population). The dose ratio between the toxic and therapeutic effects is the therapeutic index and can be expressed as the LD ₅₀ /ED ₅₀ ratio. Preferably, the antibody or antigen-binding fragment thereof has a high therapeutic index. Although compositions that exhibit toxic side effects can be used, care should be taken to develop a delivery system that targets such compounds to the site of diseased tissue and minimizes potential damage to normal cells, thereby reducing side effects.

Results obtained from cell culture and animal studies can be used to determine a dose range for use in humans. The dose of such antibodies or their antigen-binding fragments is typically within the range of circulating concentrations of these antibodies or fragments—concentrations that include the ED ₅₀ with little or no toxicity. The dose may vary within this range depending on the dosage form and route of administration used. For the anti-IL-27 antibody described herein, the therapeutically effective dose can be initially estimated based on cell culture assays. The dose can be developed in animal models to achieve a range of circulating plasma concentrations that includes the IC ₅₀ (i.e., the concentration of the active ingredient that achieves half-maximal inhibition of symptoms) determined in cell culture. This information can be used to more accurately determine doses suitable for humans. Plasma concentrations can be measured, for example, by high-performance liquid chromatography. In some aspects of the present invention, for example, when local administration is desired (e.g., to the eye or to a joint), cell culture or animal modeling can be used to determine the dose required to achieve a therapeutically effective concentration at the local site.

In some aspects of the present invention, the methods can be performed in combination with other oncology treatments. For example, the composition can be administered to a subject concurrently with, before, or after radiation, surgery, targeted or cytotoxic chemotherapy, chemoradiation, hormonal therapy, immunotherapy, gene therapy, cell transplant therapy, precision medicine, genome editing therapy, or other pharmacotherapy.

As described above, the compositions described herein (e.g., anti-IL-27 antibody compositions) can be used to treat a variety of cancers, including, but not limited to, Kaposi's sarcoma, leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, myeloblastic, promyelocytic, myelomonocytic, monocytic erythroleukemia, chronic leukemia, chronic myelocytic (granulocytic) leukemia, chronic lymphocytic leukemia, mantle cell lymphoma, primary central nervous system lymphoma, Burkitt's lymphoma and marginal zone B-cell lymphoma, polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, solid tumors, sarcomas and carcinomas, fibrosarcoma, myxosarcoma, liposarcoma, chrondrosarcoma, osteosarcoma, osteosarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon sarcoma, colorectal carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, Hepatocellular carcinoma (HCC), hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, non-small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, retinoblastoma, nasopharyngeal carcinoma, esophageal carcinoma, basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain cancer and central nervous system (CNS), cervical cancer, choriocarcinoma, colorectal cancer, connective tissue cancer, gastrointestinal cancer, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer, intraepithelial

- 54 052811 neoplasm, kidney cancer, laryngeal cancer, liver cancer, lung cancer (small cell, large cell), melanoma, neuroblastoma; oral cancer (such as lip, tongue, mouth and pharynx cancer), ovarian cancer, pancreatic cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer; respiratory system cancer, sarcoma, skin cancer, stomach cancer, testicular cancer, thyroid cancer, uterine cancer and urinary tract cancer.

Combination therapy.

In some aspects of the present invention, the anti-IL-27 antibody or antigen-binding portion thereof provided herein can be combined with one or more additional therapeutic agents or treatment methods, such as another therapeutic agent or treatment method for an oncological disease. For example, the anti-IL-27 antibody or antigen-binding portion thereof can be administered to a subject (e.g., a human patient) in combination with one or more additional therapeutic agents, wherein such combination provides a therapeutic benefit to a subject who has or is at risk of developing an oncological disease.

In some aspects of the invention, an anti-IL-27 antibody, or antigen-binding portion thereof, and one or more additional therapeutic agents are administered at the same time (e.g., simultaneously). In other aspects of the invention, an anti-IL-27 antibody, or antigen-binding portion thereof, is administered first in time, and one or more additional therapeutic agents are administered second in time (e.g., sequentially). In some aspects of the invention, one or more additional therapeutic agents are administered first in time, and the anti-IL-27 antibody is administered second in time.

The anti-IL-27 antibody or antigen-binding fragment thereof described herein may replace or complement a previously or concurrently administered therapy. For example, during treatment with the anti-IL-27 antibody or antigen-binding fragment thereof, administration of one or more additional therapeutic agents may be discontinued or reduced, such as at lower doses. In some aspects of the invention, administration of the prior therapeutic agent may be maintained. In some aspects of the invention, administration of the prior therapeutic agent will continue until the level of the anti-IL-27 antibody reaches a level sufficient to provide a therapeutic effect.

In some aspects of the present invention, the present invention provides a method of treating an oncological disease in a subject, comprising administering to said subject an effective amount of an isolated antibody or antigen-binding portion thereof that specifically binds to IL-27 and has an antagonistic effect on IL-27, in accordance with the present invention, in combination with one or more additional therapeutic agents or procedures, wherein said second therapeutic agent or procedure is selected from the group consisting of: chemotherapy, targeted anticancer therapy, an oncolytic drug, a cytotoxic agent, immune therapy, a cytokine, a surgical procedure, a radiation procedure, an activator of a costimulatory molecule, an inhibitor of an inhibitory molecule, a vaccine or cellular immunotherapy, or a combination thereof.

In some aspects of the invention, the one or more additional therapeutic agents are a PD-1 antagonist, a TIM-3 inhibitor, a LAG-3 inhibitor, a TIGIT inhibitor, a CD112R inhibitor, a TAM inhibitor, a STING agonist, a 4-1BB agonist, or a combination thereof. In some aspects of the invention, the one or more additional therapeutic agents are a CD39 antagonist, a CD73 antagonist, a CCR8 antagonist, or a combination thereof. In some aspects of the invention, the anti-CD73 antibody is any anti-CD73 antibody disclosed, for example, in US Patent Publication 2019/0031766 A1, which is incorporated herein by reference in its entirety. In some aspects of the present invention, the anti-CD39 antibody is any anti-CD39 antibody as disclosed, for example, in International Patent Publication No. WO 2019/178269 A2, which is incorporated herein by reference in its entirety.

In some aspects of the present invention, said one or more additional therapeutic agents is a PD-1 antagonist. In some aspects of the present invention, said PD-1 antagonist is selected from the group consisting of: PDR001, nivolumab, pembrolizumab, pidilizumab, MEDI0680, REGN2810, TSR-042, PF-06801591, and AMP-224. In certain aspects of the present invention, said one or more additional therapeutic agents is a PD-L1 inhibitor. In some aspects of the present invention, said PD-L1 inhibitor is selected from the group consisting of: FAZ053, atezolizumab, avelumab, durvalumab, and BMS-936559. In some aspects of the present invention, there is provided a method for enhancing one or more functions of an anti-PD-1 antibody (e.g., enhancing PD-1-mediated cytokine secretion; enhancing anti-PD-1 antibody-mediated TNF-α secretion; enhancing anti-PD-1 antibody-mediated IL-6 secretion from a cell exposed to anti-PD-1 antibodies), the method comprising exposing the cell to the antibody or

- 55 052811 its antigen-binding portion, as presented in this document, simultaneously with or sequentially with an antibody against PD-1, thereby providing for enhancement of one or more functions of said antibody against PD-1.

In some aspects of the invention, the one or more additional therapeutic agents are sunitinib (Sutent®), cabozantinib (CABOMETYX®), axitinib (INLYTA®), lenvatinib (LENVIMA®), everolimus (AFINITOR®), bevacizumab (AVASTIN®), epacadostat, NKTR-214 (a CD-122 biased agonist), tivozanib (FOTIVDA®), abexinostat, ipilimumab (YERVOY®), tremelimumab, pazopanib (VOTRIENT®), sorafenib (NEXAVAR®), temsirolimus (TORISEL®), ramucirumab (CYRAMZA®), niraparib, savolitinib, vorolanib (X-82), regorafenib (STIVARGO®), donafenib (multikinase inhibitor), camrelizumab (SHR-1210), pexastimogene devacirepvec (JX-594), ramucirumab (CYRAMZA®), apatinib (YN968D1), encapsulated doxorubicin (THERMODOX®), tivantinib (ARQ197), ADI-PEG 20, binimetinib, apatinib mesylate, nintedanib, lirilumab, nivolumab (OPDIVO®), pembrolizumab (KEYTRUDA®), atezolizumab (TECENTRIQ®), avelumab (BAVENCIO®), durvalumab (IMFIMZI®), cemiplimab-rwlc (LIBTAYO®), tislelizumab and spartalizumab.

In some aspects of the invention, said one or more additional therapeutic agents is a TIM-3 inhibitor, optionally wherein said TIM-3 inhibitor is MGB453 or TSR-022.

In some aspects of the present invention, said one or more additional therapeutic agents is a LAG-3 inhibitor, optionally wherein said LAG-3 inhibitor is selected from the group consisting of LAG525, BMS-986016 and TSR-033.

In some aspects of the invention, said one or more additional therapeutic agents are a TIGIT inhibitor. In some aspects of the invention, said one or more additional therapeutic agents are a CD112R inhibitor. In some aspects of the invention, said one or more additional therapeutic agents are a TAM (Axl, Mer, Tyro) inhibitor. In some aspects of the invention, said one or more additional therapeutic agents are a STING agonist. In some aspects of the invention, said one or more additional therapeutic agents are a 4-1BB agonist.

In some aspects of the present invention, said one or more additional therapeutic agents are a tyrosine kinase inhibitor, an adenosine axis targeting agent (e.g., a CD39 antagonist, a CD73 antagonist, an A2AR antagonist or an A2BR antagonist, or a dual A2AR/A2BR antagonist), a CCR8 antagonist, a CTLA4 antagonist, a VEG-F inhibitor, or a combination thereof.

Combination with chemotherapeutic agents.

Chemotherapeutic agents suitable for combination and/or co-administration with the compositions of the present invention include, for example: taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoside, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracinatedione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin and analogs or homologues thereof. Other agents include, for example, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thiotepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, platinum(II) cisdichlorodiamine (DDP), procarbazine, altretamine, cisplatin, carboplatin, oxaliplatin, nedaplatin, satraplatin, or triplatin tetranitrate), anthracycline (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC), antimitotic agents (eg, vincristine and vinblastine), and temozolomide.

Combination with PD-1/PD-L1 antagonists.

In some aspects of the invention, the IL-27 antibodies or antigen-binding portions thereof provided herein are combined with (e.g., administered in combination with) one or more PD-1 antagonists that specifically bind to human PD-1 or PD-L1 and inhibit the biological activity of PD-1/PD-L1 and/or the pathway(s) downstream thereof, and/or the cellular processes mediated by the human PD-1/PD-L1 signaling pathway(s), or other functions mediated by human PD-1/PD-L1.

Accordingly, provided herein are PD-1 antagonists that directly or allosterically block, antagonize, suppress, inhibit, or reduce the biological activity of PD-1/PD-L1, including signaling pathways and/or cellular processes mediated by the PD-1/PD-L1 signaling pathway(s), such as receptor binding and/or initiation of a cellular response to PD-1/PD-L1. Also provided herein are PD- antagonists 56 052811

1, which reduce the amount or level of human PD-1 or human PD-L1 produced by a cell or subject.

In some aspects of the invention, a PD-1 antagonist is provided that binds human PD-1 and prevents, inhibits, or reduces the binding of PD-L1 to PD-1. In some aspects of the invention, said PD-1 antagonist binds to mRNA encoding PD-1 or PD-L1 and prevents translation. In some aspects of the invention, said PD-1 antagonist binds to mRNA encoding PD-1 or PD-L1 and causes their degradation and/or metabolism.

In some aspects of the invention, said PD-1 antagonist inhibits PD-1 signaling or PD-1 function. In some aspects of the invention, said PD-1 antagonist blocks PD-1 binding to PD-L1, PD-L2, or both PD-L1 and PD-L2. In some aspects of the invention, said PD-1 antagonist blocks PD-1 binding to PD-L1. In some aspects of the invention, said PD-1 antagonist blocks PD-1 binding to PD-L2. In some aspects of the invention, said PD-1 antagonist blocks PD-1 binding to PDL1 and/or PD-L2. In some aspects of the invention, said PD-1 antagonist specifically binds PD-1. In some aspects of the invention, said PD-1 antagonist specifically binds PD-L1. In some aspects of the present invention, said PD-1 antagonist specifically binds PD-L2.

In some aspects of the invention, said PD-1 antagonist inhibits the binding of PD1 to its ligand. In some aspects of the invention, said PD1 antagonist inhibits the binding of PD-1 to PD-L1, PD-1 to PD-L2, or PD-1 to both PD-L1 and PD-L2. In some aspects of the invention, said PD-1 antagonist does not inhibit the binding of PD-1 to its ligand.

In some aspects of the invention, said PD-1 antagonist is an isolated antibody (mAb) or an antigen-binding fragment thereof that specifically binds to PD-1 or PD-L1. In some aspects of the invention, said PD-1 antagonist is an antibody or an antigen-binding fragment thereof that specifically binds to human PD-1. In some aspects of the invention, said PD-1 antagonist is an antibody or an antigen-binding fragment thereof that specifically binds to human PD-L1. In some aspects of the invention, said PD-1 antagonist is an antibody or an antigen-binding fragment thereof that binds to human PD-L1 and inhibits the binding of PD-L1 to PD-1. In some aspects of the invention, said PD-1 antagonist is an antibody or an antigen-binding fragment thereof that specifically binds to human PD-1 and inhibits the binding of PD-L1 to PD-1.

Several immune checkpoint antagonists that inhibit or disrupt the interaction between PD-1 and one or both of its ligands, PD-L1 and PD-L2, are in clinical development or currently available to clinicians for the treatment of cancer.

Examples of anti-human PD-1 antibodies or antigen-binding fragments thereof that may comprise a PD-1 antagonist in any of the compositions, methods, and uses provided herein include, but are not limited to: KEYTRUDA® (pembrolizumab, MK-3475, h409A11, see US 8952136, US 8354509, US 8900587, and EP 2170959, each of which is incorporated herein by reference in its entirety; Merck), OPDIVO® (nivolumab, BMS-936558, MDX-1106, ONO-4538; see US 7595048, US 8728474, US 9073994, US 9067999, EP 1,537,878, US 8,008,449, US 8,779,105, and EP 2,161,336, each of which is incorporated herein by reference in its entirety; Bristol Myers Squibb), MEDI0680 (AMP-514), BGB-A317 and BGB-108 (BeiGene), 244C8 and 388D4 (see patent document WO 2016106159, which is incorporated herein by reference in its entirety; Enumeral Biomedical), PDR001 (Novartis), and REGN2810 (Regeneron). Accordingly, in some aspects of the invention, said PD-1 antagonist is pembrolizumab. In some aspects of the invention, said PD-1 antagonist is nivolumab.

Examples of anti-human PD-L1 antibodies or antigen-binding fragments thereof that may comprise a PD-1 antagonist in any of the compositions, methods, and uses provided herein include, but are not limited to, BAVENCIO® (avelumab, MSB0010718C, see WO 2013/79174, which is incorporated herein by reference in its entirety; Merck/Pfizer), IMFINZI® (durvalumab, MEDI4736), TECENTRIQ® (atezolizumab, MPDL3280A, RG7446; see WO 2010/077634, which is incorporated herein by reference in its entirety; Roche), MDX-1105 (BMS936559, 12A4; see US Patent 7943743 and WO 2013/173223, each of which is incorporated herein by reference in its entirety; Medarex/BMS), and FAZ053 (Novartis). Accordingly, in some aspects of the invention, said PD-1 antagonist is avelumab. In some aspects of the invention, said PD-1 antagonist is

- 57 052811 is durvalumab. In some aspects of this invention, said PD-1 antagonist is atezolizumab.

In some aspects of the invention, said PD-1 antagonist is an immunoadhesin that specifically binds to human PD-1 or human PD-L1, such as a fusion protein comprising the extracellular or PD-1-binding portion of PD-L1 or PD-L2 fused to a constant region, such as the Fc region of an immunoglobulin molecule. Examples of immunoadhesion molecules that specifically bind to PD-1 are described in patent documents WO 2010/027827 and WO 2011/06634, each of which is incorporated herein by reference in its entirety. In some aspects of the invention, said PD-1 antagonist is AMP-224 (also known as B7-DCIg), which is a PD-L2-FC fusion protein that specifically binds to human PD-1.

One of ordinary skill in the art will appreciate that any PD-1 antagonist that binds to PD-1 or PD-L1 and disrupts the PD-1/PD-L1 signaling pathway is suitable for the compositions, methods, and uses described herein.

In some aspects of the present invention, the PD-1/PD-L1 antagonist is a small molecule, nucleic acid, peptide, peptide mimetic, protein, carbohydrate, carbohydrate derivative, or glycopolymer. Illustrative small molecule PD-1 inhibitors are described in Zhan et al. (2016) Drug Discov Today 21 (6):1027–1036.

Combinations with TIM-3 inhibitors.

In some aspects of the invention, an anti-IL-27 antibody or antigen-binding portion thereof provided herein is combined (e.g., administered in combination) with a TIM-3 inhibitor. The TIM-3 inhibitor can be an antibody, an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide. In some aspects of the invention, the TIM-3 inhibitor is selected from MGB453 (Novartis), TSR-022 (Tesaro), or LY3321367 (Eli Lilly). In some aspects of the invention, an anti-IL-27 antibody or antigen-binding portion thereof is administered in combination with MGB453. In some aspects of the invention, an anti-IL-27 antibody or antigen-binding portion thereof is administered in combination with TSR-022.

Combinations with LAG-3 inhibitors.

In some aspects of the invention, an anti-IL-27 antibody or antigen-binding portion thereof provided herein is combined with (e.g., administered in combination with) a LAG-3 inhibitor. The LAG-3 inhibitor can be an antibody, an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide. In some aspects of the invention, the LAG-3 inhibitor is selected from LAG525 (Novartis), BMS-986016 (Bristol-Myers Squibb), TSR-033 (Tesaro), MK-4280 (Merck & Co), or REGN3767 (Regeneron).

Other combinations.

In some aspects of the invention, an anti-IL-27 antibody or antigen-binding portion thereof provided herein is combined with (e.g., administered in combination with) a TIGIT inhibitor, a kinase inhibitor (e.g., a tyrosine kinase inhibitor (TKI)), a CD112R inhibitor, a TAM receptor inhibitor, a STING agonist, and/or a 4-1BB agonist, or a combination thereof. In some aspects of the invention, an anti-IL-27 antibody or antigen-binding portion thereof provided herein is combined with (e.g., administered in combination with) a tyrosine kinase inhibitor, an adenosine axis-targeting agent (e.g., a CD39 antagonist, a CD73 antagonist, or an A2AR, A2BR, or dual A2AR/A2BR antagonist), a CCR8 antagonist, a CTLA4 antagonist, a VEG-F inhibitor, or a combination thereof.

Detection methods.

In some embodiments of the present invention, the anti-IL-27 antibody or antigen-binding fragment thereof described herein can be used in methods for detecting and/or quantifying human IL-27 in a biological sample. Accordingly, the anti-IL-27 antibody or antigen-binding fragment thereof described herein is useful for diagnosing, prognosing, and/or determining the progression of a disease (e.g., cancer) in a patient.

Monitoring a subject (e.g., a human patient) for improvement in an oncological disease, as defined herein, means assessing the subject for a change in a disease parameter, such as a decrease in tumor growth. In some embodiments, said assessment is conducted for at least one (1) hour, such as for at least 2, 4, 6, 8, 12, 24, or 48 hours, or for at least 1 day, 2 days, 4 days, 10 days, 13 days, 20 days, or more, or for at least 1 week, 2 weeks, 4 weeks, 10 weeks, 13 weeks, 20 weeks, or more after administration. Said assessment of the subject may be conducted at one or more of the following times: before treatment; during treatment; or after administration of one or more elements of treatment. The assessment may include assessing the need for further treatment, such as assessing the need for a change in the dose, frequency of administration, or duration of treatment. The assessment may also include

- 58 052811 assessment of the need to add or exclude a selected therapeutic intervention, such as adding or excluding any of the cancer treatments described in this document.

In some embodiments of the present invention, there is provided a method for detecting IL-27 in a sample taken from a subject, said method comprising: (a) contacting a sample taken from a subject with a detecting antibody under conditions that allow said detecting antibody to form a detecting antibody-IL-27 complex, if IL-27 is present in said sample, wherein said detecting antibody is an antibody or antigen-binding fragment thereof as provided herein; and (b) detecting the presence of said complex, if any, formed in step (a).

In some embodiments of the present invention, there is provided a method for detecting an IL-27-associated cancer in a subject, said method comprising the following steps: (a) contacting a sample from a subject suspected of having an IL-27-associated cancer with a detecting antibody under conditions that allow said detecting antibody to form a detecting antibody-IL-27 complex, if IL27 is present in said sample, wherein said detecting antibody is an antibody or antigen-binding portion thereof as provided herein; and (b) detecting the presence of said complex, if any, formed in step (a). In some embodiments of the present invention, the detecting antibody is conjugated to a detectable label. In some embodiments of the present invention, said method further comprises contacting said sample with a capture antibody to produce a complex comprising IL-27 and the capture antibody, if IL-27 is present in said sample, wherein said capture antibody is an antibody or antigen-binding portion thereof as provided herein.

In some embodiments of the invention, said capture antibody is immobilized on a solid support. In some embodiments, said sample is contacted with said capture antibody prior to contact with said detection antibody. In some embodiments, said sample is a sample of a biological body fluid. In some embodiments, said sample of a biological body fluid is blood, blood serum, blood plasma, cell lysates, or tissue lysates.

In some embodiments of the invention, the oncological disease is selected from renal cell carcinoma (RCC), hepatocellular carcinoma, lung cancer, gastroesophageal cancer, ovarian cancer, endometrial cancer, melanoma, leukemia, and lymphoma. In some embodiments of the invention, the oncological disease is renal cell carcinoma (RCC). In other embodiments of the invention, the oncological disease is hepatocellular carcinoma (HCC). In some embodiments of the invention, the oncological disease is leukemia and lymphoma. In some embodiments of the invention, the oncological disease is acute myeloid leukemia (AML).

Examples

While this invention has been described with reference to specific aspects, those skilled in the art will recognize that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of this invention. Furthermore, numerous modifications may be made to adapt a particular situation, material, composition, process, process step, or stage to the objective, spirit, and scope of this invention. All such modifications are included within the scope of this invention.

Example 1. Production of antibodies against IL-27 in yeast that specifically bind the P28 subunits of human IL-27.

Anti-IL-27 antibodies representing multiple epitope groups were selected from eight synthetic yeast human antibody libraries using the methods described below.

Materials and methods.

Eight synthetic yeast libraries of naive human antibodies, each ~ ¹⁰⁹ in size, were propagated as previously described (see, e.g., Xu et al. (2013) Protein Eng Des Sel 26 (10): 663–670; WO2009036379; WO2010105256; and WO2012009568, each of which is incorporated herein by reference in its entirety). For the first two rounds of selection, magnetic bead sorting was used using the Miltenyi MACS system, as previously described (see, e.g., Siegel et al. (2004) J Immunol Methods 286 (1–2): 141–153, which is incorporated herein by reference in its entirety).

- 59 052811

Briefly, yeast cells (~10 ¹⁰ cells per library) were incubated with 3 mL of biotinylated antigen (recombinant human IL-27; R & D Systems) at 100 nM for 30 min at 30°C in wash buffer (phosphate-buffered saline (PBS)/0.1% bovine serum albumin (BSA)). After washing once with 40 mL of ice-cold wash buffer, the cell pellet was resuspended in 20 mL of wash buffer, streptavidin microbeads (500 µL) were added to the yeast, and the cells were incubated for 15 min at 4°C. Yeast cells were then pelleted, resuspended in 20 mL of wash buffer, and loaded onto a Miltenyi LS column. After loading 20 ml, the column was washed three times with 3 ml of wash buffer. The column was then removed from the magnetic field, yeast cells were eluted with 5 ml of growth medium, and grown overnight. Subsequent rounds of selection were performed using flow cytometry. Approximately 2 x 10 ⁷ yeast cells were pelleted, washed three times with wash buffer, and incubated at 30°C either with decreasing concentrations of biotinylated antigen (from 100 to 1 nM) under equilibrium conditions, or with 30 nM biotinylated antigens of different species to obtain species cross-reactivity, or with polyspecificity reduction reagent (PSR) to remove nonspecific antibodies from selection. For PSR depletion, the libraries were incubated with a 1:10 dilution of biotinylated PSR reagent.

Yeast cells were then washed twice with wash buffer and stained with LCFITC (diluted 1:100) and secondary reagents—either SA-633 (diluted 1:500) or EAPE (diluted 1:50)—for 15 min at 4°C. After washing twice with wash buffer, cell pellets were resuspended in 0.3 ml of wash buffer and transferred to filter sorting tubes. Sorting was performed using a FACS ARIA sorter (BD Biosciences) with gating to select antibodies with the desired characteristics. Selection rounds were repeated until a population with all the desired characteristics was obtained. After the final round of sorting, yeast cells were plated, and individual colonies were picked for characterization.

Light chain differentiation.

The light chain differentiation protocol was used in the primary discovery phase for further antibody detection and refinement.

Light chain batch differentiation protocol: heavy chain plasmids from the naive selection source were extracted from yeast using the smash-and-grab method, propagated, and then purified from E. coli and transformed into a light chain library with 5*106 diversity. Selection was performed using one round of MACS and four rounds of FACS using the same conditions as for naive detection.

Antibody optimization.

Antibody optimization was performed by introducing diversity into the variable regions of the heavy chain and light chain as described below.

Selection of CDRH1 and CDRH2: CDRH3 from a single antibody was recombined into a pre-generated library with CDRH1 and CDRH2 variants with a diversity of 1 x 10 and selected using one round of MACS and four rounds of FACS, as described for naive detection. In various FACS rounds, the libraries were screened for binding to the RSP, species cross-reactivity, and affinity pressure by titration or pre-complexation of parental Fabs, and sorted to obtain a population with the desired characteristics.

Obtaining and purifying antibodies.

Yeast clones were propagated to saturation and then induced for 48 hours at 30°C with shaking. After induction, the yeast cells were pelleted, and the supernatants were collected for purification. IgG was purified using a protein A column and eluted with acetic acid, pH 2.0. Fab fragments were obtained by papain digestion and purified by passage through KappaSelect (GE Healthcare LifeSciences).

KD measurements using ForteBio technology.

ForteBio affinity measurements were performed on an Octet RED384 analyzer generally as described previously (see, e.g., Estep et al., High throughput solution-based measurement of antibody-antigen affinity and epitope binning. Mabs 5 (2), 270–278 (2013), which is incorporated by reference herein in its entirety). Briefly, ForteBio affinity measurements were performed by loading IgG in real time onto AHQ sensors. The sensors were equilibrated offline in assay buffer for 30 min and then monitored in real time for 60 s to establish a baseline. The IgG-loaded sensors were exposed to 100 nM antigen for 3 min and then transferred to assay buffer for 3 min to measure the off-rate. All kinetics were analyzed using a 1:1 binding model. Recombinant human IL-27 protein (R&D Systems, catalog no. 2526-IL) was used as the antigen. The results of anti-IL-27 antibody affinity measurements are shown in Fig. 1.

- 60 052811

Epitope-specific ligand sorting/blocking using ForteBio technology.

Epitope-specific ligand sorting/blocking was performed using a standard sandwich cross-blocking assay. Control anti-target IgG was applied to AHQ sensors, and unoccupied Fc-binding sites on the sensor were blocked with a human IgG1 antibody of no interest. The sensors were then exposed to the target antigen at a concentration of 100 nM, followed by a second anti-target antibody or ligand. Additional binding by the second antibody or ligand after association with the antigen indicates an unoccupied epitope (non-competing), while lack of binding indicates epitope blocking (competitor or ligand blocking).

Kinetic analysis using MSD-RTR technology.

Equilibrium affinity measurements were performed as described previously (Estep et al., 2013). Equilibrium titration assays (SETs) were performed in PBS + 0.1% BSA without IgG (PBSF) at a constant antigen concentration of 10–100 pM and incubated with 3–5-fold serial dilutions of antibodies starting from 5–100 nM (experimental conditions depended on the sample). Antibodies (20 nM in PBS) were coated onto the wells of standard MSD-ECL plates for binding overnight at 4°C or at room temperature for 30 min. The plates were then blocked for 30 min with shaking at 700 rpm, followed by three washes with wash buffer (BSABI) + 0.05% Tween 20). Samples for RTP were applied to the plates and incubated for 150 s with shaking at 700 rpm, followed by one wash. Antigen captured on the plate was detected with 250 ng/ml sulfamethoxazole-labeled streptavidin in BSABI by incubation on the plate for 3 min. The plates were washed three times with wash buffer and then read on an MSD Sector Imager 2400 using 1× Read Buffer T with surfactant. The percentage of free antigen was plotted as a function of titrated antibody in Prism software and fitted into a quadratic model to obtain the KD. To improve throughput, liquid handling robots were used in all MSD-RTP experiments, including sample preparation for RTP.

Example 2. Binding of anti-IL-27 antibodies to recombinant human IL-27.

The ability of the anti-IL-27 antibodies described in Example 1 to bind to recombinant human IL-27 was assessed using ELISA. Briefly, Nunc MaxiSorp ELISA plates (Affymetrix, Cat. No. 44-2404-21) were coated with recombinant human IL-27 (R & D Systems, Cat. No. 2526IL/CF) at a concentration of 100 μl/well (0.5 μg/ml diluted in PBS), sealed, and incubated overnight at 4°C. The plates were washed three times with wash buffer (PBS + 0.01% Tween), 100 μl/well. The plates were then blocked with blocking buffer (PBS + 0.1% BSA + 0.01% Tween), 200 µl/well, for 1 h at room temperature (RT) with shaking. The blocking buffer was decanted, and 100 µl per well of diluted control and anti-IL-27 antibodies were added as indicated. For each antibody, a 10-point serial dilution was performed by diluting the antibodies 1:10, starting with a maximum concentration of 1 µg/ml. The plates were incubated for 1-2 h at RT with shaking. The plates were washed three times with wash buffer, 100 µl/well. Anti-human IgG secondary antibody (SouthernBiotech; catalog #2014-05) (1:5000 diluted in blocking buffer) was added to the plates at 100 µl/well. The plates were then incubated for 1 h at RT. After the 1-h incubation, the plates were washed three times with wash buffer, 100 µl/well. For development of the plates, a solution of tetramethylbenzidine (TMB; Life Technologies, catalog #00-2023) in buffer was added at 100 µl/well. The development of a blue color in the wells of the standard curve was observed, and as soon as the highest concentration of diluted control antibodies reached a dark blue color (5-10 min), stop solution (Thermo Fisher, catalog no.: SS04) was added at 50 µl/well (the color changed to yellow). The developed plates were read at 450 nm (minus the value read at 570 nm for wavelength correction) for 30 min after stopping the reaction.

For example, biochemical affinity and specificity studies showed that anti-IL-27 Ab1 binds to the p28 subunit (but not the EBI3 subunit) of the heterodimeric cytokine IL-27. Anti-IL-27 Ab1 bound to recombinant IL-27 from humans, non-human primates, and rodents, and the extent of binding varied between species. The binding specificity of anti-IL-27 Ab1 was confirmed by testing against a panel of ~4,500 cell surface and soluble molecules, and no off-target binding was observed. The binding specificity of IL-27 to its receptor IL-27RA (WSX-1) was also confirmed; no other cell surface receptor bound human IL-27. The ability of the anti-IL-27 antibody Ab1 to block the interaction between human IL-27 and IL-27RA (WSX-1) was confirmed using surface plasmon resonance.

The binding of the antibodies described in this paper was assessed in several model systems. Because human IL-27 is biologically active when applied to mouse cells, systemic overexpression of human IL-27 in mice was used using DNA minicircle analysis.

- 61 052811 IL-27-mediated effects in vivo were analyzed using whole-genome multiplex analysis, flow cytometry, and serum cytokine analysis. The results of the study of many of the markers modulated by IL-27 in vivo are consistent with the results of analyses in human cells. Anti-IL-27 Ab3 was also evaluated in a disseminated B16 tumor model. Under these conditions, treatment with anti-IL-27 Ab3 yielded results consistent with phenotypes observed in mice deficient in various components of the IL-27 ligand (IL-27p28, EBI3) or receptor (IL-27RA).

Taken together, these studies demonstrate that anti-IL-27 Ab1 (and its related anti-IL-27 Ab3) can phenocopy IL-27 deficiency in mice, bind IL-27 specifically and with high affinity, and can inhibit its immunosuppressive effects, either alone or in combination with PD-L1 blockers.

Example 3. Anti-IL-27 antibodies inhibit STAT1 phosphorylation in vitro.

IL-27 signaling through the IL-27 receptor (IL-27R) leads to phosphorylation of the signal transducer and activator of transcription 1 (pSTAT1) polypeptide STAT1. The anti-IL-27 antibodies described in Example 1 were tested for their ability to inhibit IL-27-mediated STAT1 phosphorylation in human whole blood, human PBMCs, U937 myeloid cells (a histiocytic lymphoma cell line), and HUT-78 T-cell lymphoma cells using flow cytometry.

Anti-IL-27 antibodies were tested for their ability to inhibit IL-27-mediated STAT1 phosphorylation in human whole blood. Briefly, EDTA-anticoagulated human whole blood stored at room temperature was used in this assay. Forty-five microliters of blood were dispensed into each well of a deep-well round-bottom plate (Phenix, catalog #850356) and heated for 30 min at 37°C using a plate warmer (EchoTherm IC20) or in a 37°C incubator. Anti-IL-27 antibodies were diluted to 10-fold the maximum concentration in endotoxin-free PBS (Teknova, catalog # P0300) in a polypropylene V-bottom well plate (Corning, catalog # 3363). Anti-IL-27 antibodies were serially diluted to the desired concentration in endotoxin-free PBS. Wells for unstimulated and stimulated controls received PBS alone. 5 μl of each dilution was added to a well with 45 μl of blood and mixed by shaking on a plate shaker for 15 s at 1000 rpm (Eppendorf Mix Mate). The plate was incubated for 60 min at 37°C on a plate warmer or in an incubator set at 37°C.

A 10 μg vial of recombinant human IL-27 (R&D Systems, catalog #2526-IL) was reconstituted to 100 μg/mL by adding 100 μL of PBS + 0.1% BSA (made from 10% BSA, Sigma, catalog #A1595). A working solution of recombinant hIL-27 (rhIL-27) was prepared by diluting to 200 ng/mL in endotoxin-free PBS. After a 60-min incubation, 5 μL of rhIL-27 at a concentration of 200 ng/mL were added to each well containing stimulated blood. Unstimulated control wells received 5 μL of PBS. The plate was shaken on a shaker for 15 s at 1000 rpm and incubated for 30 min at 37°C.

After a 30-minute incubation, the cells were fixed. Lyse/Fix reagent (BD, catalog #558049) was diluted 1:5 in sterile water (Hyclone, catalog #SH3052902) and heated to 37°C in a water bath. 500 µl of Lyse/Fix reagent was added to each well of a deep-well plate, and the plate was shaken on a plate shaker for 15 s at 1000 rpm. The plate was incubated for 15 min at 37°C.

After a 15-minute incubation, the plate was centrifuged for 5 minutes at 1,500 rpm at room temperature (Eppendorf 5810R centrifuge), and the supernatant was removed by shaking. 1 ml of endotoxin-free PBS was added to each well, and the plate was shaken on a plate shaker for 15 seconds at 1,000 rpm. The plate was centrifuged for 5 minutes at 1,500 rpm at room temperature (Eppendorf 5810R centrifuge), and the supernatant was removed by shaking. The cell pellets remained in the plate.

Cell pellets were resuspended in 50 µl of 1:200 CD14-Pacific Blue reagent (Biolegend, Cat. No. 325616) in FACS buffer (PBS, Gibco, Cat. No. 14190-144/2% FBS, Sigma, Cat. No. F8317/1 mM EDTA, Fisher, Cat. No. BP2482) and transferred to a 96-well U-bottom plate (Costar, Cat. No. 3799). The plate was sealed with plate film (VWR, Cat. No. 89134-432) and incubated for 30 min at room temperature in the dark.

After a 30-minute incubation, 150 µl of FACS buffer was added to each well, and the plate was centrifuged at 1500 rpm for 5 min at room temperature. The cell pellets were then resuspended in 100 µl of Perm III reagent (stored at -20°C) (BD, catalog #558050) by pipetting, and the plate was sealed with plate sealing film and a lid. The plate was incubated overnight at -20°C or for 15 min at 4°C.

After incubation, 150 µl of PBS was added and the plate was centrifuged at 1500 rpm for 5 min at room temperature. The supernatant was removed from the plate by

- 62 052811 shaken out and the contents of the plate were resuspended in 50 µl of staining mixture prepared as described in Table 6 below.

Table 6

Catalog number BD Antibody Color Breeding 561807 CD3 FITC 1:10 562069 Y701 pSTATl RE 1:100 562071 Y705 pSTAT3 ARS 1:20

The plate was incubated for 1 hour at room temperature in the dark. After 1 hour of incubation, 100 µl of FACS buffer was added, and the plate was centrifuged at 1500 rpm for 5 minutes at room temperature. The supernatant was removed from the plate by shaking, and the plate contents were resuspended in 100 µl of FACS buffer for flow cytometric analysis.

The anti-IL-27 antibodies described in Example 1 were tested for their ability to inhibit IL-27-mediated STAT1 phosphorylation in pooled human PBMC samples using flow cytometry. Briefly, frozen cryovials of human PBMCs (peripheral blood mononuclear cells) derived from buffy coats were removed from liquid nitrogen storage and rapidly thawed in a 37°C water bath. The contents of each cryovial were removed with a P1000 pipette and transferred to a 15 mL Falcon conical tube. 2-3 mL of complete RPMI-1640 medium (Gibco, cat. #61870-036) was slowly added to the thawed cells, and they were resuspended by gentle vortexing or shaking. Conical tubes were filled to 10 ml with complete RPMI-1640 medium and inverted to mix the contents. The conical tubes were centrifuged at 1400 rpm at room temperature for 8 minutes.

PBMCs were resuspended at a density of 4 million cells/mL in warm serum-free RPMI-1640 medium and seeded at a density of 200,000 cells/well (50 μL) in a 96-well round-bottom plate (Costar, catalog #3799). Anti-IL-27 antibodies were diluted in serum-free RPMI-1640 medium in the first row of a 96-well polypropylene plate to a maximum concentration of 40 μg/mL (the final concentration will be 10 μg/mL). Serial dilutions to the desired concentrations (1:2, 1:3, etc.) were performed in the remaining wells of the first 10 rows of the plate. Fifty microliters (µl) of the antibody stock solution (4*) were added to the wells of the first 10 rows of a round-bottomed plate containing PBMC cells. Rows 11 and 12 were each supplemented with 50 µl of serum-free RPMI-1640 culture medium. The plate was then incubated at 37°C for 2 hours.

After a 2-hour incubation, 100 μl of recombinant human IL-27 (R&D Systems, catalog #2526-IL) diluted to a concentration of 50 ng/ml in serum-free RPMI-1640 culture medium was added to each well (except control wells, which contained only serum-free medium or antibody only) to a final concentration of 25 ng/ml. 100 μl of serum-free RPMI-1640 culture medium was added to control wells or wells containing antibody alone. The plate was incubated for 20 min at 37°C.

After a 20-minute incubation, 50 µl of 4% paraformaldehyde (PFA; Pierce, catalog #28906) in deionized water was added directly to each well, and the plate was incubated at 37°C for 5 min to fix the cells. The plate was centrifuged at 2000 rpm for 5 min. The medium was removed by shaking, and the plate was washed with 150 µl of Dulbecco's phosphate-buffered saline (DPBS). The washing steps were repeated two more times. Ice-cold 90% methanol (MeOH) (Sigma, cat. #439193) diluted in _H2O was quickly added to each well using a 12-channel pipette. Care was taken to mix the contents of each well when adding MeOH. The plate was incubated at 20°C for at least 15 min. 100 µL of PBS was added to each well on top of the 90% methanol, and the plate was centrifuged at 2000 rpm for 5 min. The plate contents were removed by shaking, and the plate was washed three times as described previously. After the final wash, cell pellets remained in the wells of the plate.

The precipitated PBMCs were stained with pSTAT1 PE reagent (BD Phosflow, Cat. No. 526069) 1:100 in FACS buffer (2% PBS, 2 mM EDTA in PBS) for 45 min at room temperature in the dark. When adding the dye, special care was taken to mix the contents of each well using a 12-channel pipette. After 45 min of incubation, 100 μl of FACS buffer were added to each well and the plate was centrifuged at 2000 rpm for 5 min. The supernatant

- 63 052811 The liquid was removed by shaking, and the plate was washed twice as described previously. Cells were resuspended in 100 µl of FACS buffer and analyzed by flow cytometry.

As shown in Fig. 2A, anti-IL-27 antibodies inhibited STAT1 phosphorylation in pooled human PBMC samples. Anti-IL-27 Ab3 inhibited STAT1 phosphorylation with a mean IC ₅₀ of 140.5 ng/mL (n = 2) in pooled human PBMC samples. Anti-IL-27 Ab1 inhibited STAT1 phosphorylation with a mean IC ₅₀ of 58.3 ng/mL (n = 3) in pooled human PBMC samples.

Anti-IL-27 antibodies were further tested for their ability to inhibit IL-27-mediated STAT1 phosphorylation in U937 cells, a cell line known to express Fc receptors, using flow cytometry, essentially as described for Fig. 2A. As shown in Fig. 2B, anti-IL-27 antibodies inhibited STAT1 phosphorylation in U-937 cells as indicated. Anti-IL-27 Ab3 inhibited STAT1 phosphorylation with a mean _IC50 of 81 ng/mL (n=2) in U937 cells. Anti-IL-27 Ab1 inhibited STAT1 phosphorylation with a mean IC50 of 96 ng/mL (n=2) in U937 cells.

Anti-IL-27 antibodies were further tested for their ability to inhibit IL-27-mediated STAT1 phosphorylation in the cutaneous T-cell lymphoma cell line HUT78, which does not express cell surface Fc receptors, by flow cytometry, essentially as described for Fig. 2A. As shown in Fig. 2C, anti-IL-27 antibodies inhibit STAT1 phosphorylation in HUT-78 cells. Anti-IL-27 Ab3 inhibited STAT1 phosphorylation with an _IC50 of 80 ng/mL (n=1) in HUT-78 cells. Anti-IL-27 Ab1 inhibited STAT1 phosphorylation with an _IC50 of 95 ng/mL (n=1) in HUT-78 cells.

In this study, we also assessed the inhibition of IL-27 by the anti-IL-27 Ab1 antibody in different species in a whole blood assay. To characterize the activity of the anti-IL-27 Ab1 antibody in different species, recombinant IL-27 from human, cynomolgus macaque, rat, and mouse was tested for its stimulation of pSTAT1 signaling in T cells from whole blood samples obtained from these species (data not shown).

Briefly, whole blood was warmed to 37°C, followed by a 60-min preincubation with anti-IL-27 Ab1, and human IL-27 was added at a concentration of 20 ng/mL. Samples were incubated for an additional 30 min. Leukocytes were fixed, and erythrocytes were lysed. After washing, fixed cells were permeabilized and stained with anti-CD3 and anti-PhosPho-STATI (Y701) antibodies. After a 1-h incubation, samples were washed and resuspended for flow cytometry. Percent inhibition was calculated using stimulated and unstimulated control wells, and _IC50 values were calculated using GraphPad Prism.

Representative data on the signaling inhibition induced by anti-IL-27 Ab1 in human T cells are shown in Fig. 3. Consistent with the observations made regarding the affinity of anti-IL-27 Ab1 in different species, the potency of inhibiting IL-27 signaling by anti-IL-27 Ab1 was highest in human, followed by the activity in cynomolgus monkey, rat, and mouse (see, e.g., Table 7).

Table 7

IC ₅₀ values of the At1 antibody against IL-27 in peripheral blood T cells of different types

Types Average ICso, ng/ml Standard deviation Quantity Human 78.4 35 7 Jovanese macaque 118.1 36.4 4 Rat 273.2 133.5 8 Mouse 1721 N/A 1 (pool of 10)

Abbreviations: IC ₅₀ = half-maximal inhibitory concentration, N/A = not applicable.

Example 4. Reduction of IL-27-mediated inhibition of CD161 by anti-IL-27 antibodies.

CD161, a C-type lectin, is a T-cell marker whose expression is downregulated by IL-27. The anti-IL-27 antibodies described in Example 1 were tested for their ability to reverse IL-27-mediated inhibition of CD161 in pooled human PBMC samples using flow cytometry. Briefly, frozen cryovials containing pooled human PBMC (peripheral blood mononuclear cells) samples derived from buffy coats were removed from liquid nitrogen storage and rapidly thawed in a water bath at

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37°C. The contents of each cryovial were removed with a P1000 pipette and transferred to a 15 ml Falcon conical tube. 2–3 ml of complete RPMI-1640 medium (Gibco, catalog #61870-036) were slowly added to the thawed cells and they were resuspended by gentle shaking or vortexing. Conical tubes were filled to 10 ml with complete RPMI-1640 medium and inverted to mix their contents. Conical tubes were centrifuged at 1400 rpm at room temperature for 8 min.

The use of outer wells was avoided to minimize the effects of evaporation during the 5-day study. The outer wells were filled with PBS (Gibco, catalog #14190-144) at 200 µL per well. PBMCs were resuspended at a density of 2 million cells/mL in warm complete RPMI-1640 medium. Purified human anti-CD3 antibody (Biolegend, UCTH1, catalog #300402) was added at a concentration of 0.5 µg/mL (2-fold higher than the final concentration). 100 µL per well of this cell mixture (200,000 cells/well) were seeded in a 96-well round-bottom plate (Costar, catalog #3799).

Anti-IL-27 antibodies were diluted in complete RPMI-1640 medium in the first row of a 96-well polypropylene plate to a maximum concentration of 40 μg/ml (the final concentration will be 10 μg/ml). Serial dilutions to the required concentrations (1:2, 1:3, etc.) were performed in the remaining wells of the first 10 rows of the plate. Fifty μl of the antibody stock solution (4*) was added to the first 10 rows of the plate—the wells containing PBMC cells in the round-bottomed plate. Fifty μl of complete RPMI-1640 medium was added to rows 11 and 12.

After addition of anti-IL-27 antibodies, 50 μl of recombinant human IL-27 (R&D Systems, catalog #2526-IL) diluted to 100 ng/ml in complete RPMI-1640 medium (except control wells, which contained serum-free medium or antibody only) were added to each well to obtain a final concentration of 25 ng/ml. Control wells were supplemented with 50 μl of complete RPMI-1640 medium. The plate was incubated for 5 days at 37°C in a cell culture incubator with minimal intervention.

After 5 days of incubation, the plate was removed from the incubator and shaken on a plate shaker for 30 sec at 600 rpm. The plate was centrifuged at 1800 rpm for 5 min. The medium was removed and saved for further analysis, and the plate was washed with 150 µl of PBS (Gibco, #14190-144). The wash was repeated two more times. The cell pellet was stained with 50 µl of staining mixture per well, as described in Table 8 below.

Table 8

Catalog number (Biolegend) Antibody target Color Breeding 300532 CD4 BV421 1:100 304204 CD45RO FITC 1:100 339910 CD161 AF647 1:100 353410 CCR6 RE 1:100

The plate was shaken on a shaker for 30 s at 600 rpm and incubated for 30 min at room temperature in the dark.

After a 30-minute incubation, the plate was centrifuged, and the supernatant was removed by shaking. The plate was washed twice as described previously. After the final wash, the cell pellet was fixed by adding 50 µl of 4% PFA (Pierce, catalog #28906) in deionized water at room temperature for 10 min. 100 µl of FACS buffer was added to each well, and the plate was centrifuged at 1800 rpm for 5 min. Cells were resuspended in 100 µl of FACS buffer and analyzed by flow cytometry.

As shown in Fig. 4, anti-IL-27 antibodies were shown to reduce IL-27-mediated inhibition of CD161.

Example 5. Enhancement of PD-1-mediated secretion of TNF-α, IL-6, and other cytokines by anti-IL-27 antibodies, including further characterization of anti-IL-27 antibodies in vitro.

Anti-IL-27 antibodies were tested for their ability to enhance PD-1-mediated TNF-α and IL-6 secretion in human cancer patient-derived PBMCs. Human cancer patient-derived PBMCs were cultured essentially as described in Example 4, with the addition of wells supplemented with anti-PD-1 antibody as indicated at a concentration of 1 μg/mL. Supernatants from this study were assayed for TNF-α and IL-6 using the CBA Human Cytokine Multiplex Th1/Th2/Th17 Kit (BD, Cat. # 560484). As shown in Figs. 5A and 5B, anti-IL-27 antibodies enhance PD-1-mediated TNF-α and IL-6 secretion in pooled human PBMC samples.

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The results of the studies described in this paper also demonstrate the cytokine-inducing activity of the anti-IL-27 Ab1 antibody as monotherapy and in combination with the anti-PD-1 antibody in human PBMCs. IL-27 is known to negatively regulate the expression of several inflammatory cytokines. To determine the effect of IL-27 blockade on cytokine production, human PBMCs from healthy donors, patients with RCC, and patients with ovarian cancer were activated with the anti-CD3 antibody in the presence or absence of the anti-IL-27 Ab1 antibody for several days and the levels of secreted cytokines, including IL-17, IFN-γ (IFN-gamma), TNF-α (TNF-alpha), and IL-6, were analyzed. Briefly, PBMCs isolated from fresh whole blood of 4 healthy donors, 5 patients with RCC, and 2 patients with ovarian cancer were activated with anti-CD3 antibody at 0.25 μg/mL in the absence or presence of anti-IL-27 Ab1 (1 μg/mL), anti-PD1 antibody (pembrolizumab, 1 μg/mL), or both. After 5 days, supernatants were collected and analyzed for TNF-α (A) or IFN-γ (B) levels by MSD or CBA technologies. Data shown represent fold changes in cytokine production compared to stimulation with anti-CD3 antibody alone. Statistical analysis was performed using paired t-tests (*p < 0.005).

In these assays, anti-PD-1 antibody was used as a control, and a combination of PD-1 and IL-27 blockade was also tested, as shown in Fig. 5C. Treatment with anti-IL-27 Ab1 resulted in increased TNF-α production in 6 of 11 PBMC samples tested (defined as a >2-fold increase), including 2 of 4 healthy donors, 3 of 5 RCC patients, and 1 of 2 ovarian cancer patients. When tested in a subset of donors, this activity was dose-dependent with anti-IL-27 Ab1 (data not shown). Treatment with the anti-PD-1 antibody (pembrolizumab) resulted in increased TNF-α levels in 2 of 11 donors studied (1 of 5 with RCC and 1 of 2 with ovarian cancer), while the combination of anti-IL-27 Ab1 and anti-PD-1 resulted in increases in 10 of 11 donors. The increase in TNF-α observed with the combination treatment was additive in 8 of 10 responding cell samples. An additive effect on IFN-γ production was observed in these cultures after treatment with anti-IL-27 Ab1 and anti-PD-1 (10 of 11 donors); however, responses to anti-PD-1 alone were more frequent (10 of 11 donors) compared with anti-IL-27 Ab1 (2 of 11 donors). Taken together, these data indicate that anti-IL-27 Ab1 increases TNF-α levels in activated PBMC cultures from healthy donors and cancer patients, and that the combination of anti-IL-27 Ab1 and anti-PD-1 results in higher TNF-α and IFN-γ levels compared to either treatment alone.

To further explore the role of IL-27 and PD-1 blockade, the same activated PBMC culture system was used to determine whether IL-27 could directly counteract the effect of PD-1 blockade-induced cytokine overproduction. Briefly, freshly isolated PBMCs from human whole blood were activated with anti-CD3 antibody at a concentration of 0.25 μg/ml. Cells were treated with either control IgG1 (1 μg/ml), αPD-1 antibody (pembrolizumab, 1 μg/ml) alone, rhIL-27 (25 ng/ml) plus αPD-1, or rhIL-27 plus αPD-1 with anti-IL-27 Ab1 (1 μg/ml) at 37°C for 5 days. Supernatants were collected for cytokine detection by CBA technology. As an example, cytokine (IL-17A and IFN-γ) results in samples from 4 healthy donors are shown as fold changes compared to controls. The mean and standard deviation are shown. Statistical analysis was performed using a paired t-test (* p < 0.05, ** p < 0.01). Similar results were also observed in PBMCs from patients with RCC. PD-1 blockade increased both IL-17 and IFN-γ in these cultures, and IL-27 could completely inhibit this activity, a response that was reversed in the presence of anti-IL-27 Ab1, as shown in Fig. 5D. These data indicate that IL-27 can attenuate the effects on cytokine production induced by anti-PD-1 antibody treatment.

Thus, IL-27 was shown to inhibit anti-PD-1 antibody-mediated proinflammatory cytokine production in activated human PBMCs, an effect that was blocked by anti-IL-27 Ab1. Furthermore, anti-IL-27 Ab1, in combination with PD-1 blockade, resulted in increased cytokine production in activated PBMCs from healthy donors and patients with RCC. Thus, blockade of IL-27 with anti-IL-27 Ab1 enhances immune cell activation by altering the expression of immunoregulatory receptors and increasing inflammatory cytokine production.

In addition to characterizing individual anti-IL-27 antibodies in the presence of anti-IL-27 antibody (herein anti-IL-27 Ab1), αPD-1 antibody, or a combination of anti-IL-27 antibody and αPD-1 antibody, further characterization of cytokine induction/secretion was performed (Fig. 5E-5H, specifically for TNF-α, IFN-γ, IL-6, and IL-17A).

Example 6. Inhibition of IL-27-mediated PD-L1 and TIM3 expression by anti-IL-27 antibodies.

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The anti-IL-27 antibodies described in Example 1 were tested for their ability to inhibit IL-27-mediated PD-L1 and TIM-3 expression in pooled human monocyte samples using flow cytometry.

Fresh monocytes were isolated from human buffy coat using ROSETTESEP™ Human Monocyte Enrichment Mix (Stemcell, Cat. No. 15068).

The use of outer wells was avoided to minimize the effects of evaporation during the 5-day study. The outer wells were filled with PBS, 200 µl per well (Gibco, catalog #14190-144).

Monocytes were resuspended at a density of 2 million cells/ml in warm complete RPMI1640 medium. 100 µl of this cell mixture per well (200,000 cells/well) were seeded into a 96-well round-bottom plate (Costar, catalog #3799).

Anti-IL-27 antibodies were diluted in complete RPMI-1640 medium in the first row of a 96-well polypropylene plate to a maximum concentration of 40 μg/ml (the final concentration will be 10 μg/ml). Serial dilutions to the desired concentrations (1:2, 1:3, etc.) were performed in the remaining wells of the first 10 rows of the plate. 50 μl of the antibody stock solution (4*) were added to the wells of the first 10 rows of the plate containing PBMC cells in a round-bottomed plate. 1250 μl of complete RPMI-1640 medium were added to rows 11 and 12.

Following addition of anti-IL-27 antibodies, 50 μl of recombinant human IL-27 (R&D Systems, catalog #2526-IL) diluted to a concentration of 80 ng/ml in complete RPMI-1640 medium (except control wells, which contained serum-free medium or antibody only) were added to each well to obtain a final concentration of 20 ng/ml. Control wells received 100 μl of serum-free RPMI-1640 medium. The plate was incubated for 3 days at 37°C with minimal intervention.

After 3 days of incubation, the plate was removed from the incubator and shaken on a plate shaker for 30 sec at 600 rpm. The plate was centrifuged at 1800 rpm for 5 min. The medium was removed by shaking, and the plate was washed with 150 µl of PBS (Gibco, catalog #14190-144). Washing was repeated twice. The cell pellet was stained with 50 µl of staining mixture per well, as described in Table 9 below:

Table 9

Catalog number (Biolegend) Antibody target Color Breeding 345006 TIM3 RE 1:100 301310 CDllb ARS 1:100 329714 PD-L1 BV421 1:100

The plate was shaken on a shaker for 30 s at 600 rpm and incubated for 30 min at 4°C in the dark.

After a 30-minute incubation, the plate was centrifuged, and the supernatant was removed by shaking. The plate was washed twice as described previously. After the final wash, the cell pellet was fixed by adding 50 µl of 4% PFA (Pierce, catalog #28906) in deionized (DI) water at room temperature for 10 min. 100 µl of FACS buffer was added to each well, and the plate was centrifuged at 1800 rpm for 5 min. Cells were resuspended in 100 µl of FACS buffer and analyzed by flow cytometry.

As shown in Fig. 6A and 6B, anti-IL-27 antibodies potently inhibited IL-27-mediated PD-L1 and TIM3 expression in pooled human monocyte samples.

Anti-IL-27 antibodies were further tested for their ability to inhibit IL-27-mediated PD-L1 expression in resting T cells (inactivated), essentially as described for Fig. 6A and 6B. Resting T cells were isolated from human buffy coat using ROSETTESEP™ Human T Cell Enrichment Mix (Stemcell, Cat. #15061).

In the final stage of the analysis, cell pellets were stained with 50 µl per well of the staining mixture as described in Table 10 below:

- 67 052811

Table 10

Catalog number (Biolegend) Antibody target Color Breeding 345006 TIM3 RE 1:100 555349 CD4 ARS 1:100 329714 PD-L1 BV421 1:100 555366 CD8 FITC 1:100

The plate was shaken on a plate shaker for 30 s at 600 rpm and incubated for 30 min at 4°C in the dark.

After a 30-min incubation, the plate was centrifuged and the supernatant was removed by shaking. The plate was washed twice as described previously. After the final wash, the cell pellet was fixed by adding 50 μl of 4% PFA (Pierce, cat. #28906) in DI water at room temperature for 10 min. 100 μl of FACS buffer was added to each well, and the plate was centrifuged at 1800 rpm for 5 min. Cells were resuspended in 100 μl of FACS buffer and analyzed by flow cytometry. As shown in Fig. 6C, anti-IL-27 antibodies potently inhibited IL-27-mediated PD-L1 expression in pooled human resting T cell samples.

Example 7. In vivo efficacy of anti-IL-27 antibody in the B16F10 disseminated melanoma model.

A melanoma lung metastasis model was used to evaluate the antitumor activity of IL-27 blockade using the clinical candidate antibody Ab1 against IL-27. The growth of disseminated B16F10 lung metastases is known to be significantly reduced in EBI3- and Il27ra-deficient (Wsx-1) mice (Sauer et al., J. Immunology 181: 6148–6157). Because lung nodule size and growth kinetics depend on the number of transferred B16F10 cells and can develop variably and rapidly, the combination of an anti-PD-1 antibody and an anti-CTLA-4 antibody was studied as a reference therapeutic activity. Pretreatment with the anti-IL-27 antibody Ab1 resulted in a significant reduction in overall tumor burden.

Briefly, six- to eight-week-old female C57BL/6 mice (n=10/group) were inoculated intravenously (i.v.) with either 2.5 × 105 B16F10 cells or 1 × 105 B16-Luc cells via the tail vein in 200 μL phosphate-buffered saline (PBS). Animals received 1 mg of anti-IL-27 Ab1 (Wuxi; lot #2108SD170316K01X01I01) or 1 mg of human polyclonal isotype control (Bioxcell; catalog #BE0092; lot #658417D1) by intraperitoneal injection (i.p.). Antibodies were administered once weekly, starting 7 days before tumor injection, for a total of four doses (days -7, 0, 7, and 14). For visual counting of lung metastases, mice bearing B16F10 tumors were euthanized by _CO2 asphyxiation 18 days after tumor cell injection, and the lungs were perfused with PBS via cardiac puncture, removed, and fixed in 10% neutral-buffered formalin for 24 h. The fixed lungs were then transferred to 70% ethanol, and superficial lung metastases were visually counted. For immunohistochemistry, formalin-fixed lungs (n=5/group) were embedded in paraffin, sectioned, and stained with hematoxylin and eosin to quantify the total tumor area as a percentage of the total tissue area in each section. To visualize lung tumor metastasis in vivo, animals bearing B16-Luc tumors were injected intravenously via the tail vein with 3 mg of VivoGlo D-luciferin in 200 μL of PBS (Promega) twice weekly. Five minutes after luciferin injection, animals were anesthetized, and bioluminescence imaging was performed using an IVIS Lumina LT Series III imaging system. Images were analyzed using Living Image software (version 4.5.5), and data were displayed as total photon flux measurements per second (photons/sec).

As shown in Figs. 7A–7G, treatment of B16F10 tumor-bearing mice with anti-IL-27 Ab1 resulted in a significant reduction in total tumor burden, as measured by both the total number of superficial lung metastases (pulmonary nodule count, Fig. 7A) and the reduction in tumor area in lung tissue sections by immunohistochemistry (IHC) analysis (Figs. 7C–7F and Fig. 7G). Blockade of p28 with anti-IL-27 Ab1 resulted in a 42% reduction in B16 pulmonary nodule count compared to isotype control treatment. Treatment with Ab1 antibody against IL-27 significantly inhibited (p=0.0079) the growth of B16F10 lung metastases compared with isotype control (21.6 ± 8.4 versus 37.6 ± 10.9 pulmonary nodules, respectively). Treatment with Ab1 antibody against IL-27 resulted in an 83% reduction in the total area of tumor metastases in the lungs, according to

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IHC (16.43 ± 1.39% in the isotype control group versus 2.83 ± 1.45% in the anti-IL-27 Ab1 group). Similarly, bioluminescence imaging showed that anti-IL-27 Ab1 significantly (p=0.0062) delayed the growth of B16-Luc lung metastases (Fig. 7B). Similar reductions in the number of superficial lung metastases and total tumor area were observed with anti-IL-27RA (WSX1) antibody-mediated blockade and with anti-PD-1 and anti-CTLA-4 combination therapy, as shown in Fig. 7G. These data were obtained from 2 independent experiments, in which the reference anti-PD-1 and anti-CTLA-4 combination demonstrated antitumor activity. B16F10 cells (2.5*105) were injected intravenously into C57BL/6 mice (n=10/group). Mice were injected intraperitoneally with 1 mg of either anti-IL-27 Ab1, anti-IL-27RA (WSX-1), or an isotype control human IgG antibody (days -7, 0, 7, 14). Some animals were injected intraperitoneally with anti-PD-1 and anti-CTLA-4 antibodies (days 0, 4, 7, and 11). Lungs were obtained from animals (n=5/group) with B16F10 lung metastases treated as described above, sectioned, and stained with hematoxylin and eosin. B16F10 tumor tissue was distinguished from normal lung tissue based on hematoxylin and eosin staining of lung sections from treated animals (Fig. 7A). Tumor area was calculated as a percentage of total lung area (Fig. 7G). Statistical analysis was performed using a paired t-test. Taken together, these data indicate that anti-IL-27 Ab1 can phenocopy IL-27RA (WSX-1) and EBI3 deficiency in a tumor model and exhibits activity similar to combined PD-1 and CTLA-4 blockade.

These data demonstrate that treatment with an anti-IL-27 antibody (anti-IL-27 Ab1) results in antitumor effects, reducing both tumor growth and metastasis to a greater extent than treatment with a control isotype antibody that does not bind IL-27.

Example 8. Determination of gene expression profiles in splenocytes obtained from mice hydrodynamically transfected with human IL-27 minicircles.

To examine the effects of IL-27 on T cell phenotype in vivo, DNA minicircles encoding IL-27 were used to overexpress IL-27 in mice, and T cell responses were assessed by RNA sequencing and flow cytometry. Human IL-27 is known to be a cross-reactive immunoglobulin and can induce pSTAT1 and PD L1 signaling in mouse splenocytes in vitro. This species-specific cross-reactivity was used to examine the effects of human IL-27 overexpression in mice and its inhibition by anti-IL-27 Ab1. For this purpose, DNA minicircles encoding human IL-27 (p28 linked to an EBI3 glycine serine linker) were administered to mice by hydrodynamic transfection as described below, resulting in high systemic levels of IL-27.

Hydrodynamic transfection of human IL-27 minicircles.

Six-week-old female BALB/c mice were injected with 20 μg of either empty vector or linked human IL-27 minicircle DNA (System Biosciences, Palo Alto, CA, USA) in 2 ml of 0.9% saline via the tail vein over 5 s. Following injection, animals were transferred to an empty cage with a heating pad to recover for 5 min. Whole blood was collected into plasma separation tubes containing K2-EDTA 24 h after minicircle injection, and plasma IL-27 levels were confirmed by ELISA. PBMCs and total splenocytes were collected 5 days posttransfection, and cells were stained and analyzed by flow cytometry. Expression of these markers was analyzed on CD4 ⁺ T cells and CD8 ⁺ T cells. Analysis was performed using FlowJo software.

Determination of gene expression profiles.

Mouse splenocytes were obtained by mechanical dissociation of whole spleens followed by erythrocyte lysis with sodium ammonium chloride buffer (SAB). Total RNA was extracted from splenocytes using the RNEASY® Mini Kit (Qiagen, catalog #74104) and adjusted to 20 ng/µl in nuclease-free water (Qiagen, catalog #19101). Gene expression profiling was performed on GENECHIP™ Mouse Gene 2.0 ST Arrays (Applied Biosystems, catalog #902118). RNA sample processing, hybridization, and array scanning were performed using standard Affymetrix GENECHIP™ protocols at the Boston University Microarray and Sequencing Resource (BUMSR). All CEL files were normalized using the Robust Multi-array Average (RMA) method (Irizarry et al., 2003), and gene expression data were preprocessed by removing unexpressed probes and discarding transcripts with high inter-repeat coefficients of variance. Subsequent analyses (mean expression, fold change, t-test) were performed in R (version 3.6.2).

Flow cytometric analysis.

Whole blood and spleen were obtained from mice five days after miniring injection. Splenocytes were obtained from IL-27-expressing mice five days after transfection. Single splenocyte suspensions were prepared by mechanical dissociation through a nylon cell culture bag.

- 69 052811 40 μm filter followed by RBC lysis in ACN buffer. Whole blood cells were stained directly followed by RBC lysis and fixation in BD Phosflow Lyse/Fix buffer according to the manufacturer's instructions (BD Biosciences, San Jose, CA, USA). FcγRIII/II was blocked by preincubating cells with rat anti-mouse CD16/CD32 monoclonal antibody (mAb) (1 μg per million cells; Biolegend, San Diego, CA, USA) in PBS with 2% FBS and 2 mM EDTA. Cells were stained with APC-, PE-, Brilliant Violet 510-, and Brilliant Violet 711-conjugated mAbs against mouse CD4 (clone GK1.5), CD8 (53-6.7), PD-L1 (10F.9G2), TIM3 (RMT3-23), LAG3 (C9B7W), and TIGIT (1G9) (Biolegend). Cell-associated fluorescence was measured using an LSRFortessa X-20 flow cytometer (BD Biosciences), and data analysis was performed using FlowJo software (Tree Star, Ashland, OR, USA).

Statistical analysis.

Statistical significance was determined using GraphPad Prism software using paired or unpaired Student's t-tests or Student's t-tests, as indicated. When using the t-test, zero values were subtracted by 0.1 to make them nonzero. P values less than 0.05 were considered significant.

IL-27 promotes the expression of inhibitory receptors by T cells in vivo.

The expression of more than 400 genes changed >Log2-fold in response to IL-27 treatment, as shown in Fig. 8A. A subset of these genes is shown in Tables 11A–11B. These genes included those encoding immunoinhibitory receptors that play a key role in the immune response. As shown in Fig. 8B, the expression of Ly6a (encoding Sca-1), Lag3, Tigit, and IL-10 was increased on splenocytes in response to IL-27. There was also a trend toward IL-27-mediated increase in the expression of Ctla4 and Cd274 (encoding PD-L1), representing less than 1-fold induction (data not shown). To verify these expression data, flow cytometry was used to assess the expression of PD-L1, LAG-3, TIGIT, and TIM-3 proteins on T cells from these mice. Administration of IL-27 minicircles increased the expression of PD-L1, LAG-3, and TIGIT in splenic and peripheral blood CD4 ⁺ T cells (PBMCs). In CD8+ T cells, IL-27 minicircles increased PD-L1, LAG-3, TIGIT, and TIM-3. As shown in Fig. 8C–8F, administration of IL-27 minicircles increased PD-L1, Lag-3, and Tigit in splenic and peripheral blood CD4 ⁺ T cells. In CD8 ⁺ T cells, IL-27 minicircles increased PD-L1, Lag-3, Tigit, and Tim-3. These data suggest that IL-27 may play a key role in directing the expression of immunoregulatory receptors in vivo.

To investigate the ability of Ab1 to block minicircle-derived human IL-27 in vivo, both target molecule interaction using enzyme-linked immunosorbent assay (ELISA) and immune receptor expression in splenocytes were examined. Five days after IL-27 transfection and exposure to anti-IL-27 Ab1 (50 mg/kg), plasma was collected from mice for analysis of IL-27 heterodimer and EBI3 levels using Meso Scale Discovery (MSD) technology. The IL-27 heterodimer assay utilized an anti-p28 capture antibody, which cross-blocks anti-IL-27 Ab1, and a human-specific anti-EBI3 detection antibody; therefore, if anti-IL-27 Ab1 binds to IL-27, its detection will be masked. The EBI3 assay utilized both capture and detection antibodies specific to 2 different epitopes of human EBI3, and since minicircle-derived IL-27 is a tethered heterodimer, the assay allows detection of total IL-27 independent of binding to the anti-IL-27 Ab1 antibody.

Briefly, 6-week-old female Balb/c mice were injected with empty vector (control) or human IL-27. Mice were exposed to 1 mg of either anti-IL-27 Ab1 or an isotype control IgG1 anti-DNP antibody 7 days before and on the day of minicircle transfection (days -7 and 0). Whole blood was collected, and plasma was analyzed for IL-27 (Figure 8G) using Meso Scale Discovery technology. Figure 8G shows that treatment with anti-IL-27 Ab1 completely inhibited IL-27 detection in plasma by MSD. Similar data were observed when anti-IL-27 Ab1 was tested at 25 mg/kg. These data indicate that anti-IL-27 Ab1 at 25 mg/kg or higher can completely saturate minicircle-derived IL-27 in vivo. This complete interaction with the target molecule was also confirmed in a functional assay of pSTAT1.

To evaluate the ability of anti-IL-27 Ab1 to block IL-27 activity in vivo, the expression of PD L1, Tim-3, Lag-3, and Tigit in mouse PBMCs and splenocytes was analyzed using flow cytometry. Anti-IL-27 Ab1 significantly blocked IL-27-induced PD-L1 and Lag 3 expression in CD4+ PBMCs and PD-L1, Tim-3, Lag-3, and Tigit expression in CD8+ PBMCs.

Treatment with anti-IL-27 Ab1 also blocked IL-27-induced expression of PD-L1, Lag-3, and TIGIT in CD4+ splenocytes and expression of PD-L1 and Lag 3 in CD8+ splenocytes. These data suggest that anti-IL-27 Ab1 can both interact with and block the activity of human IL-27 in vivo.

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These results demonstrate that ectopic expression of IL-27 in vivo leads to upregulation of multiple inhibitory receptors and several other molecules with immunomodulatory activity in splenocytes by T cells. These data suggest that antagonism of IL-27 (e.g., by treatment with an anti-IL-27 antibody) will reduce the expression of inhibitory receptors on T cells, thereby enhancing the immune response.

PA table

Gene activation in response to IL-27 administration

Gene designation Frequency of change p-value Gene identification Frequency of change p- zpachepie GM4841 3,824228667 0.012331653 SLC26A3 2,286 0.3487 LY6A 3,568709 0.000991642 DPCR1 2,285 0.0653 IIGP1 3,294783 0.000455248 GM 11844 2,279 0.1054 TUBB1 3,145617 0.002782618 CYP4A32 2,269 0.2133 MRO 3,112051333 0.026667968 MIR1928 2,267 0.0134 CTSG 2.954178333 0.018177934 GKN3 2,266 0.3840 RRVR 2.878417333 0.006623845 KRT4 2,264 0.1539 ELANE 2.845762333 0.024770656 PGC 2,262 0.2583 MT2 2.757525667 0.004548988 HIST1H2BA 2,260 0.0390 MUCH 2.696042667 0.018195326 SPRR2A3 2,254 0.1974 F83OO16BO8RIK 2.603319667 0.011330985 IGKV12-41 2,235 0.3548 GM4951 2.583066667 0.01529863 PARD3BOS3 2,227 0.1149 APOL11B 2.565688667 0.006885943 CLCA4B 2,225 0.4177 GZMB 2.515642667 0.004012308 GM23744 2,208 0.1217 PRTN3 2.450033 0.028785527 VMN2R-PS129 2,200 0.0021 CLCA3A1 2.345778333 0.018175529 GM23241 2,194 0.1644 GM11505 2.331628667 0.023451392 MYOZ2 2,191 0.2091 IL-10 2.324278 0.008857442 MIR192 2,191 0.0553 GBP 11 2,307386 0.005204674 MY111 2,188 0.1653 PF4 2.270045 0.016210658 5033403H07RIK 2,187 0.1231 IRG1 2,269440667 0.003861367 MIRLET7F-2 2,186 0.1617 CES2G 2.252403333 0.033921335 GM25009 2,185 0.1243 OASL2 2,243393 0.00470682 MUC13 2,182 0.4412 LAG3 2,203067 0.002539095 PPP1R3A 2,178 0.1968 II1ST1H2AG 2,197938 0.03499933 OLFR798 2,172 0.0036 OAS1G 2,184084667 0.007645573 SERPINA12 2,154 0.0309 MFSD2B 2,179382333 0.030664254 GM25864 2,152 0.0404 RI1AG 2,146212667 0.072130267 N-R5S155 2,151 0.0691

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T1G1T 2.145155 0.005421349 CYP2C65 2,142 0.1438 SLC6A4 2.121439 0.009945773 GM 14750 2,141 0.0525 SHCBP1 2,092208667 0.038685767 GM23026 2,134 0.0863 BC023105 2,079652 0.002582842 HAMP2 2,125 0.2675 PKLR 2.045597333 0.053459171 GM24527 2,125 0.0777 TFR2 2.037887333 0.033394313 OLFR891 2,125 0.0731 F13A1 2,010803333 0.010612157 OLFR68 2,125 0.1746 HIST1H2AB 2.002687333 0.025241 TRAV6-2 2,123 0.1684 SERPINA3F 1.991916 0.002220854 CYP4F40 2,119 0.1848 ERMAP 1,940590333 0.042680673 TRDN 2,119 0.2396 MCPT8 1,930212667 0.018921256 CFTR 2,117 0.3208 SLC26A1 1.926420667 0.025126602 XIRP2 2,109 0.2031 PRKAR2B 1.924056667 0.016881189 CYP8B1 2,107 0.0912 BIRC5 1.914826333 0.032959509 GM23629 2,105 0.0590 FADS2 1.91223 0.013830993 GM25076 2,104 0.0980 TOP2A 1.88339 0.034852729 1700080G11RIK 2,103 0.1621 NCAPG 1.881082333 0.036612626 N-R5S96 2,100 0.0493 A730089K16RIK 1,870258667 0.061491704 GM 11027 2,097 0.3009 MNS1 1,860366 0.014834618 SLC22A29 2,089 0.2663 GP9 1.859234333 0.002956835 GSTT2 2,082 0.2372 GF11B 1,852962 0.0291761 GM23911 2,081 0.1903 NUF2 1,850953 0.032872517 OLFR724 2,079 0.0925 CHIL3 1.848470667 0.001494932 GM23277 2,078 0.0912 KIF11 1.83252 0.040535377 A4GNT 2,076 0.2431 ALOX 12 1,823174 0.006931665 MUP21 2,074 0.1149 ADGRG7 1.822065 0.017542834 GM24147 2,074 0.2669 KLF1 1.820262333 0.060689421 MYBPC1 2,067 0.1392 E2F8 1.817455333 0.069143254 CYP3A59 2,063 0.2628 ATP1B2 1.811274333 0.024016123 ACTN2 2,063 0.1471 KIF2C 1.811223333 0.061246458 MIR29A 2,062 0.0340 FADS3 1.803859667 0.050197663 TFF1 2,061 0.0803 MS4A6D 1.801871667 0.01036878 PLA2G1B 2,060 0.4477 SLC25A21 1.801518667 0.048282826 GM23021 2,060 0.1111 HIST1H1B 1,800434 0.037242995 GM22607 2,045 0.1383 CKAP2L 1.783850333 0.061646634 RPL10L 2,044 0.0692 SAMD14 1.782388333 0.02384128 ACE2 2,038 0.3505 CARI 1.770098667 0.025511071 PRAMEL3 2,038 0.1490 DEPDC1A 1.765839667 0.03875749 SULT1B1 2,032 0.2495 CEN PE 1.765095667 0.039288425 ARL14 2,026 0.0952 ASPM 1.753558 0.054145246 GM11337 2,024 0.0270 CCNB2 1.751291667 0.036336189 CNN1 2,020 0.0217

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RY K 1.749673 0.035413093 LR1T2 2,019 0.0533 MMP14 1.747348667 0.010510984 ACOT4 2,015 0.2120 BUB1 1.738322667 0.02139336 LOC100125594 2,014 0.0208 MYOID 1.734508 0.006655486 GLB1L2 2,012 0.0396 PARVB 1.733820333 0.010799396 GM25605 2,008 0.1329 GM5593 1.728317 0.008578526 GM15384 2,002 0.0255 CCNA2 1.724975667 0.026630845 GM94 1,997 0.1095 PRR11 1.724352667 0.04875805 OLFR1113 1,997 0.1277 AQP1 1.719081667 0.064562051 G630018N14RIK 1,997 0.3017 CASP3 1.709594333 0.009087444 GM23232 1,994 0.0672 K1F15 1.708689667 0.026782535 GM16378 1,994 0.0357 ASNS 1.708074333 0.037928738 GM22838 1,994 0.0875 CPOX 1.706113 0.030298001 HSD3B5 1,992 0,2019 MT1 1.699002667 0.010159345 OLFR746 1,989 0.0331 CDC6 1.694586667 0.049970924 GM24254 1,988 0.1301 GBP2B 1.694018 0.000929009 GM24232 1,986 0.2212 GBP2 1.689558667 0.004961011 PRAP1 1,986 0.3720 HMMR 1.687253333 0.063769534 ALPI 1,986 0.4192 KIF20A 1.686718333 0.020639142 MIR3964 1,983 0.0301 GSTM5 1.681807333 0.04316743 GM22787 1,983 0.0183 REEP6 1.677204667 0.056071877 GM26081 1,982 0.1771 GM12250 1.675485 0.003251181 GM22242 1,980 0.3169 GBP 10 1.673458 0.009333312 IGHV1-77 1,980 0.4742 ATP7B 1.671614 0.029798535 OLFR740 1,980 0.2191 GM22973 1.663495667 0.004472758 OLFR1201 1,979 0.0339 CASC5 1.659578 0.044333895 GM22654 1,976 0.5101 ADD2 1.659553667 0.053938067 OLFR747 1,975 0.0719 CAMP 1.659111667 0.066745225 GM22521 1,973 0.1073 CLEC5A 1.654882667 0.00665055 VMM RI 72 1,972 0.0731 MUP-PS12 6,332 0.0021 GM 13773 1,971 0.2179 GM26184 5,617 0.0850 CLEC2H 1,970 0.2041 GKN2 4,822 0.0378 OLFR714 1,967 0.0816 MYH8 3,854 0.3190 GM22284 1,965 0.1376 MIR101B 3,695 0.0111 GM26342 1,962 0.0242 GKN1 3,485 0.0451 OLFR190 1,961 0.0184 SLN 3,473 0.1822 CCDC152 1,960 0.0375 MUC5AC 3,341 0.0628 GM26048 1,959 0.1807 2310057J18RIK 3,041 0.4100 GM24410 1,959 0.1844 PSCA 3,027 0.1219 LGALS4 1,957 0.0215 GM25623 2,948 0.0133 OLFR969 1,956 0.0946 FAM83B 2,905 0.0202 GM6222 1,956 0.0950

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GM23852 2,882 0.0763 OLFR913 1,953 0.3111 SPTSSB 2,873 0.0952 4930557A04RIK 1,953 0.0645 MIRLET7F-1 2,794 0.0447 GM24750 1,950 0.2385 MUP-PS16 2,778 0.0114 GM23043 1,948 0.0490 ANXA1O 2,689 0.1047 STFA1 1,946 0.0803 LGALS2 2,685 0.1337 SUCNR1 1,942 0.1078 SSHL1 2,672 0.4078 MYOM1 1,941 0.1753 AKP3 2,660 0.3665 KLK1B22 1,938 0.1263 SLC13A1 2,622 0.2936 GM23917 1,934 0.1949 GM766 2,621 0.4035 GM 15949 1,932 0.0450 GM24537 2,617 0.1649 OLFR709-PS1 1,930 0.0722 GM24138 2,606 0.0374 OBP1A 1,928 0.3421 SIS 2,585 0.3777 SULT2A1 1,927 0.3528 2210407C18RIK 2,584 0.0600 GM24786 1,923 0.2103 S100G 2,580 0.3877 SGK2 1,916 0.1567 GM26354 2,540 0.1097 MIRLET7C-1 1,911 0.0725 CKMT2 2,538 0.2680 VMN1R90 1,910 0.1018 GM26055 2,525 0.2157 GM23342 1,909 0.0298 GM5885 2,479 0.0305 REG3G 1,908 0.1599 AGR2 2,458 0.1845 GM24839 1,906 0.2631 VMN1R167 2,454 0.0932 N-R5S157 1,905 0.1167 GM25167 2,444 0.0264 GM25023 1,905 0.0253 ATP5C1-PS 2,437 0.0477 CES2B 1,902 0.1386 GM24470 2,434 0.1676 IGHV8-11 1,901 0.1555 MY 112 2,403 0.1632 GM25982 1,899 0.0542 MIR326 2,375 0.0059 ATP4A 1,899 0.3968 MIR122 2,375 0.0242 GM26162 1,898 0.0147 MCPT9 2,375 0.0623 PNPLA3 1,898 0.0031 PSG18 2,359 0.0115 AU015336 1,893 0.0794 MUC6 2,358 0.3108 GM24621 1,891 0.0840 VMN2R115 2,352 0.1111 CYP2C40 1,891 0.4000 GM25498 2,351 0.0086 GM25602 1,891 0.1527 2010106E10RIK 2,339 0.4607 ADGRG7 1,889 0.3205 ERBB4 2,328 0.0350 SLC30A10 1,888 0.1288 LYPD8 2,320 0.1374 GM25836 1,888 0.1934 CYP2A5 2,305 0.2330 GM25629 1,887 0.1192 BTNL5-PS 2,301 0.1684 GM24861 1,886 0.0396 GM24549 2,289 0.1138 GM21057 1,884 0.0850 GM24465 2,287 0.2356

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Table 11B

Gene suppression in response to IL-27 administration

Gene designation Frequency of change p-value Gene designation Frequency of change p- value DEFB44-PS 0.187 0.1696 1700049E17RIK2 0.380 0.0045 C1S2 0.187 0.0076 IGH-VJ558 0.382 0.4348 HIST1H1B 0.204 0.1347 PRRG4 0.382 0.1000 GM15114 0.205 0.1127 GM7391 0.383 0.0206 GM2005 0.205 0.0169 CD 109 0.384 0.0892 KHDC1C 0.211 0.0820 TMEM173 0.385 0.0912 GM2005 0.224 0.0023 CDH17 0.385 0.0338 SERPINB7 0.224 0.1389 KITL 0.385 0.0287 SERP1NB11 0.225 0.0594 SKA3 0.388 0.1991 CXCL3 0.230 0.1516 PRC1 0.390 0.0796 CDH10 0.230 0.0466 MVOAT1 0.390 0.0339 MIR1949 0.235 0.1307 TOR2A 0.394 0.0659 SLC7A11 0.236 0.0195 1700049E17RIK2 0.395 0.0084 LUZP4 0.249 0.0952 SPEER4D 0.396 0.0119 IGHV1-81 0.252 0.5116 HIST1H3I 0.396 0.3818 PTPRTOS 0.252 0.0338 TUBA1A 0.396 0.0139 RP1 0.254 0.1538 GM22265 0.396 0.0909 GM15127 0.254 0.0500 TRBJ1-7 0.397 0.1567 CRISP 1 0.255 0.0865 KIF20B 0.398 0.0922 GM15107 0.255 0.0591 SERPINB2 0.398 0.0259 SERP1NB5 0.256 0.0402 GM 14402 0.398 0.2496 GM7665 0.256 0.0034 PLK4 0.399 0.0728 TMEM252 0.259 0.0173 C330027C09R1K 0.400 0.1046 PNMA5 0.262 0.0922 GM15091 0.401 0.0763 CXCL15 0.263 0.1037 NDC80 0.401 0.0773 CLEC2G 0.265 0.0388 MYBL1 0.401 0.1659 MMRP13 0.267 0.1687 KIF2C 0.401 0.0703 GM15093 0.269 0.0741 GM 12603 0.401 0.0856 VMN1R53 0.270 0.0648 CYP11A1 0.403 0.2635 GM14409 0.276 0.1558 FANCI 0.403 0.1782 GM11884 0.278 0.1682 CCNA2 0.404 0.0598 SLC16A4 0.278 0.1159 RACGAP1 0.406 0.0734 MMRP12 0.280 0.0786 NCAPH 0.406 0.1125 KIF5C 0.281 0.1003 GM 16094 0.407 0.0317 APELA 0.283 0.0601 SP25P 0.407 0.3075 AFP (AFP) 0.283 0.0424 GM 15398 0.408 0.1043 1GKV1-122 0.286 0.0090 KNTC1 0.409 0.1335 A63OO95E13RIK 0.292 0.0905 BST1 0.409 0.0928 GM15109 0.297 0.0058 KIF11 0.412 0.0752

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OLFR111 0.299 0.2107 GM23576 0.412 0.2618 TNS4 0.301 0.0381 SHCBP1 0.412 0.1361 PLEKI1S1 0.301 0.0784 1GIIV1-42 0.413 0.4474 LNCENC1 0.305 0.0939 GPRC5A 0.415 0.0333 THBS1 0.309 0.0243 TNFRSF10B 0.416 0.1047 PLATR14 0.313 0.0812 IL23A 0.418 0.1414 RASSF9 0.314 0.1125 ERICH2 0.419 0.1444 ITGA2 0.319 0.0699 AN LN 0.420 0.0409 LOCI 02634388 0.320 0.0213 CASC5 0.420 0.1178 LOC102634388 0.320 0.0213 GM17689 0.421 0.2773 GM15093 0.320 0.0380 SOX4 0.422 0.0795 CEP55 0.321 0.1007 GM22069 0.422 0.0494 A630038E17R1K 0.321 0.1334 PRR11 0.423 0.0750 GM25552 0.323 0.1261 SEMA3C 0.423 0.0208 RPS26 0.327 0.1840 RETNLA 0.423 0.0429 STRA6 0.330 0.0498 FRMD7 0.424 0.0211 GM15093 0.331 0.0403 TNFRSF11B 0.425 0.0592 PSAT1 0.332 0.0926 RAD54L 0.426 0.1604 AKR1C18 0.332 0.0983 GM10488 0.426 0.0074 DEPDC1A 0.333 0.1814 PLAT 0.427 0.0647 GM10439 0.335 0.0694 GM13790 0.427 0.1161 GM24916 0.335 0.2031 DTL 0.427 0.1700 STC1 0.337 0.0829 SERPINB9B 0.429 0.1064 DPPA2 0.337 0.0887 ASNS 0.430 0.0471 E030011O05RIK 0.337 0.0486 GM15097 0.430 0.1722 GM20756 0.337 0.0218 TMED6 0.432 0.2648 GM22771 0.339 0.2897 SERPINE1 0.433 0.0345 IGKV6-32 0.340 0.4435 TPX2 0.433 0.0588 AIM2 0.340 0.0091 CENPK 0.433 0.1772 C920009B18RIK 0.341 0.0220 2810429I04RIK 0.434 0.2171 BUB1 0.349 0.0755 4930461G14RIK 0.434 0.1799 T1CRR 0.350 0.1772 111ST1H2AG 0.435 0.1874 MIS18BP1 0.353 0.0909 IER3 0.435 0.1414 MAGEA6 0.353 0.2067 CHRNB1 0.436 0.0500 CHIL3 0.354 0.0651 GM5431 0.437 0.1300 IGHV1-78 0.357 0.2850 GM13247 0.438 0.2035 STYLE 0.358 0.1413 AI506816 0.438 0.0773 GM26735 0.360 0.0654 GM7942 0.438 0.0432 REG2 0.360 0.4969 CCNB1 0.440 0.0362 SPRR1A 0.361 0.0056 ZFP345 0.440 0.2490 PARPBP 0.362 0.1471 TGHV8-8 0.440 0.4507

-76052811

PADI4 0.366 0.0975 ATAD5 0.442 0.1143 GM14139 0.367 0.0423 KDELR3 0.444 0.0873 GPC3 0.368 0.2139 CDC25C 0.445 0.0353 MS4A6D 0.370 0.0176 IGKV4-72 0.445 0.1803 ATR10A 0.371 0.0977 OLFR99 0.447 0.1622 KIF23 0.371 0.0432 GM25544 0.448 0.1385 GM20757 0.371 0.2248 MIR101C 0.448 0.0818 GM2318 0.372 0.0136 HYDIN 0.448 0.1499 GM2318 0.372 0.0136 IGHV5-16 0.449 0.3210 GM2318 0.372 0.0136 NOP58 0.449 0.1379 GM2318 0.372 0.0136 CEL 0.449 0.5062 IGHV1-9 0.372 0.3585 MS4A4A 0.449 0.1126 SRV1 0.374 0.5445 BCL2A1B 0.450 0.0035 TNFSF4 0.374 0.1420 IGKV4-59 0.450 0.4975 RNASE1 0.375 0.2935 CCL3 0.450 0.1070 T1MP1 0.376 0.0765 GM2933 0.451 0.1650 GM6020 0.379 0.4836 FAM102B 0.451 0.1330

Table 12

List of sequences

SEQ ID NO Description Subsequence Ab2-A antibody against IL-27 9 HCDR1 (IMGT) GFTFSSYS 10 HCDR2 (IMGT) ISSSSSSYI 11 HCDR3 (IMGT) ARDGGRTSYTATAHNWFDP 12 HCDR1 (NT) FTFSSYSMN 13 HCDR2 (NT) SISSSSSSYIYYADSVKG 14 HCDR3 (NT) ARDGGRTSYTATAHNWFDP 15 VH EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEW VSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYC ARDGGRTSYTATAHNWFDPWGQGTLVTVSS 16 VH DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCCTGGTCAAGCCTGGGG GGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGC TATAGCATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGT GGGTCTCATCCATTAGTAGTAGTAGTAGTTACATATACTACGCAGAC TCAGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACT CACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCGGT GTACTACTGCGCCAGAGATGTGGAAGAACGTCCTACACCGCCACA

-77 052811

GCCCACAATTGGTTCGACCCCTGGGGACAGGGTACATTGGTCACCGT STSSTSA 17 LCDR1 (IMGT) QSVLFSSNNKNY 18 LCDR2 (IMGT) WAS 19 LCDR3 (IMGT) QQHASAPPT 20 LCDR1 (NT) KSSQSVLFSSNNKNYLA 21 LCDR2 (NT) WASTRES 22 LCDR3 (NT) QQHASAPPT 23 VL DIVMTQSPDSLAVSLGERAT1NCKSSQSVLFSSNNKNYLAWYQQKPGQP PKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHA SAPPTFGGGTKVEIK 24 VL DNA GACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGG CGAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTATTCA GCTCCAACAATAAGAACTACTTAGCTTGGTACCAGCAGAAACCAGG ACAGCCTCCTAAGCTGCTCATTTACTGGGCATCTACCCGGGAATCCG GGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACT CTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTG TCAGCAGCACGCCAGTGCCCCTCCTACTTTTGGCGGAGGGACCAAGG TTGAGATCAAA 25 Heavy chain EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEW VSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYC ARDGGRTSYTATAIINWFDPWGQGTLVTVSSASTKGPSVFPLAPSSKST SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS VVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHN AKTKPREEQYN ST YR VVS VLTVLHQD WLNGKE YKCKV SNK ALP APIEKTTSK AKGQPR EPQ VYTLPPS RDELTKNQ VSLTCLVKGFYP SDIA V EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK 26 Heavy chain DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCCTGGTCAAGCCTGGGG GGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGC TATAGCATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGT GGGTCTCATCCATTAGTAGTAGTAGTAGTTACATATACTACGCAGAC TCAGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACT CACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCGGT GT ACTA CTGCGCC AG AG ATGGTGGA AG A ACGTC CTAC ACCGCCACA GCCCACAATTGGTTCGACCCCTGGGGACAGGGTACATTGGTCACCGT CTCCTCAGCGAGCACCAAAGGCCCGAGCGTGTTTCCGCTGGCGCCGA GCAGCAAAAGCACCAGCGGCGGCACCGCGGCGCTGGGCTGCCTGGT GAAAGATTATTTTCCGGAACCGGTGACCGTGAGCTGGAACAGCGGC GCGCTGACCAGCGGCGTGCATACCTTTCCGGCGGTGCTGCAGAGCA

-78052811

GCGGCCTGTATAGCCTGAGCAGCGTGGTGACCGTGCCGAGCAGCAG CCTGGGCACCCAGACCTATATTTGCAACGTGAACCATAAACCGAGC AACACCAAAGTGGATAAAAAAGTGGAACCGAAAAGCTGCGATAAA ACCCATACCTGCCCGCCGTGCCCGGCGCCGGAACTGCTGGGCGGCCC GAGCGTGTTTCTGTTTCCGCCGAAACCGAAAGATACCCTGATGATTA GCCGCACCCCGGAAGTGACCTGCGTGGTGGTGGATGTGAGCCATGA AGATCCGGAAGTGAAATTTAACTGGTATGTGGATGGCGTGGAAGTG CATAACGCGAAAACCAAACCGCGCGAAGAACAGTATAACAGCACCT ATCGCGTGGTGAGCGTGCTGACCGTGCTGCATCAGGATTGGCTGAAC GGCAAAGAATATAAATGCAAAGTGAGCAACAAAGCGCTGCCGGCGC CGATTGAAAAAACCATTAGCAAAGCGAAAGGCCAGCCGCGCGAACC GCAGGTGTATACCCTGCCGCCGAGCCGCGATGAACTGACCAAAAAC CAGGTGAGCCTGACCTGCCTGGTGAAAGGCTTTTATCCGAGCGATAT TGCGGTGGAATGGGAAAGCAACGGCCAGCCGGAAAACAACTATAAA ACCACCCCGCCGGTGCTGGATAGCGATGGCAGCTTTTTTCTGTATAG CAAACTGACCGTGGATAAAAGCCGCTGGCAGCAGGGCAACGTGTTT AGCTGCAGCGTGATGCATGAAGCGCTGCATAACCATTATACCCAGA AAAGCCTGAGCCTGAGCCCGGGCAAA 27 Light chain DIVMTQSPDSLAVSLGERATINCKSSQSVLFSSNNKNYLAWYQQKPGQP PKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHA SAPPTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPR EAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK VY ACEVTHQGLS SP VTK SFNRGEC 28 Light chain DNA GACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGG CGAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTATTCA GCTCCAACAATAAGAACTACTTAGCTTGGTACCAGCAGAAACCAGG ACAGCCTCCTAAGCTGCTCATTTACTGGGCATCTACCCGGGAATCCG GGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACT CTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTG TCAGCAGCACGCCAGTGCCCCTCCTACTTTTGGCGGAGGGACCAAGG TTGAGATCAAACGTACGGTGGCCGCTCCCTCCGTGTTCATCTTCCCA CCCTCCGACGAGCAGCTGAAGTCCGGCACCGCCTCCGTCGTGTGCCT GCTGAACAACTTCTACCCTGCGAGGCCAAAGTGCAGTGGAAAGTG GACAACGCCCCTGCAGTCCGGCAACTCCCAGGAATCCGTCACCGAGC AGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACCCTG TCCAAGGCCGACTACGAGAAGCACAAAGTGTACGCCTGCGAAGTGA CCCACCAGGGCCTGTCCAGCCCCGTGACCAAGTCCTTCAACCGGGGC GAGTGC Ab2-B antibody against IL-27 29 Heavy chain EVQL VESGGGLVKPGG SLRLSC A A SGFTF S SYSMN WVRQ APGKGLE W VSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYC ARDGGRTSYTATAHNWFDPWGQGTLVTVSSASTKGPSVFPLAPCSRST

-79052811

SESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS VVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEV EALHNHYTQKSLSLSLG 30 Heavy chain DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCCTGGTCAAGCCTGGGG GGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGC TATAGCATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGT GGGTCTCATCCATTAGTAGTAGTAGTAGTTACATATACTACGCAGAC TCAGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACT CACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCGGT GTACTACTGCGCCAGAGATGTGGAAGAACGTCCTACACCGCCACA GCCCACAATTGGTTCGACCCCTGGGGACAGGGTACATTGGTCACCGT CTCCTCAGCTTCCACCAAGGGCCCCTCCGTGTTCCCTCTGGCCCCTTG CTCCCGGTCCACCTCCGAGTCTACCGCCGCTCTGGCTGCCTCGTGA AGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACTCTGGCGCC CTGACCTCCGGCGTGCACACCTTCCCTGCCGTGCTGCAGTCCTCCGG CCTGTACTCCCTGTCCAGCGTCGTGACCGTGCCCTCCTCCAGCCTGG GCACCAAGACCTACACCTGTAACGTGGACCACAAGCCCTCCAACAC CAAAGTGGACAAGCGGGTGGAATCTAAGTACGGCCCTCCCTGCCCTT CCTGCCCTGCCCCTGAGTTCCTGGGCGGACCTTCCGTGTTCCTGTTCC CTCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCTGAAGTG ACCTGCGTGGTGGTGGACGTGTCCCAGGAAAGATCCCGAAGTCCAGTT CAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAG CCCAGAGAGAACAGTTCAACTCCACCTACCGGGTGGTGTCCGTGCT GACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGC AAAGTGTCCAACAAGGCCTGCCCTCCAGCATCGAAAAGACCATCT CCAAGGCCAAGGGCCAGCCCCGCGAGCCCCAAGTGTACACCCTGCC TCCCAGCCAGGAAGAGATGACCAAGAATCAAGTGTCCCTGACTTGT CTGGTCAAGGGCTTCTACCCCTCCGATATCGCCGTGGAGTGGGAGTC CAACGGCCAGCCCGAGAACAACTACAAGACCACCCCTCCCGTGCTG GACTCCGACGGCTCCTTCTTCCTGTACTCTCGGCTGACCGTGGACAA GTCCCGGTGGCAGGAAGGCAACGTCTTCTCCTGCTCCGTGATGCACG AGGCCCTGCACAACCACTACACCAGAAGTCCCTGTCCCTGTCTCTG GGC Antibody AL3-A against IL-27 31 HCDRl (IMGT) GFTFRSYG 32 HCDR2 (IMGT) ISSSSSSYI 33 HCDR3 (IMGT) ARDGGRTSYTATAHNWFDP

- 80 052811

34 HCDR1 (NT) FTFRSYGMN 35 HCDR2 (NT) SISSSSSSYIYYADSVKG 36 HCDR3 (NT) ARDGGRTSYTATAHNWFDP 37 VH EVQLVESGGGLVKPGGSLRLSCAASGFTFRSYGMNWVRQAPGKGLEW VSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYC ARDGGRTSYTATAHNWFDPWGQGTLVTVSS 38 VH DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCCTGGTCAAGCCTGGGG GGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCCGGAGC TATGGGATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGT GGGTCTCATCCATTAGTAGTAGTAGTAGTTACATATACTACGCAGAC TCAGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACT CACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCGGT GTACTACTGCGCCAGAGATGTGGAAGAACGTCCTACACCGCCACA GCCCACAATTGGTTCGACCCCTGGGGACAGGGTACATTGGTCACCGT CTCCTCA 39 LCDR1 (IMGT) QSVLFSSNNKNY 40 LCDR2 (IMGT) WAS 41 LCDR3 (IMGT) QQHASAPPT 42 LCDR1 (NT) KSSQSVLFSSNNKNYLA 43 LCDR2 (NT) WASTRES 44 LCDR3 (NT) QQHASAPPT 45 VL DIVMTQSPDSLAVSLGERATINCKSSQSVLFSSNNKNYLAWYQQKPGQP PKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHA SAPPTFGGGTKVEIK 46 VL DNA GACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGG CGAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTATTCA GCTCCAACAATAAGAACTACTTAGCTTGGTACCAGCAGAAACCAGG ACAGCCTCCTAAGCTGCTCATTTACTGGGCATCTACCCGGGAATCCG GGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACT CTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTG TCAGCAGCACGCCAGTGCCCCTCCTACTTTTGGCGGAGGGACCAAGG TTGAGATCAAA 47 Heavy chain EVQLVESGGGLVKPGGSLRLSCAASGFTFRSYGMNWVRQAPGKGLEW VSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYC ARDGGRTSYTATAHNWFDPWGQGTLVTVSSASTKGPSVFPLAPSSKST SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS VVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV

- 81 052811

EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK 48 Heavy chain DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCCTGGTCAAGCCTGGGG GGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCCGGAGC TATGGGATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGT GGGTCTCATCCATTAGTAGTAGTAGTAGTTACATATACTACGCAGAC TCAGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACT CACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCGGT GTACTACTGCGCCAGAGATGTGGAAGAACGTCCTACACCGCCACA GCCCACAATTGGTTCGACCCCTGGGGACAGGGTACATTGGTCACCGT CTCCTCAGCGAGCACCAAAGGCCCGAGCGTGTTTCCGCTGGCGCCGA GCAGCAAAAGCACCAGCGGCGGCACCGCGGCGCTGGGCTGCCTGGT GAAAGATTATTTTCCGGAACCGGTGACCGTGAGCTGGAACAGCGGC GCGCTGACCAGCGGCGTGCATACCTTTCCGGCGGTGCTGCAGAGCA GCGGCCTGTATAGCCTGAGCAGCGTGGTGACCGTGCCGAGCAGCAG CCTGGGCACCCAGACCTATATTTGCAACGTGAACCATAAACCGAGC AACACCAAAGTGGATAAAAAAGTGGAACCGAAAAGCTGCGATAAA ACCCATACCTGCCCGCCGTGCCCGGCGCCGGAACTGCTGGGCGGCCC GAGCGTGTTTCTGTTTCCGCCGAAACCGAAAGATACCCTGATGATTA GCCGCACCCCGGAAGTGACCTGCGTGGTGGTGGATGTGAGCCATGA AGATCCGGAAGTGAAATTTAACTGGTATGTGGATGGCGTGGAAGTG CATAACGCGAAAACCAAACCGCGCGAAGAACAGTATAACAGCACCT ATCGCGTGGTGAGCGTGCTGACCGTGCTGCATCAGGATTTGGCTGAAC GGCAAAGAATATAAATGCAAAGTGAGCAACAAAGCGCTGCCGGCGC CGATTGAAAAAACCATTAGCAAAGCGAAAGGCCAGCCGCGCGAACC GCAGGTGTATACCCTGCCGCCGAGCCGCGATGAACTGACCAAAAAC CAGGTGAGCCTGACCTGCCTGGTGAAAGGCTTTTATCCGAGCGATAT TGCGGTGGAATGGGAAAGCAACGGCCAGCCGGAAAACAACTATAAA ACCACCCCGCCGGTGCTGGATAGCGATGGCAGCTTTTTTCTGTATAG CAAACTGACCGTGGATAAAAGCCGCTGGCAGCAGGGCAACGTGTTT AGCTGCAGCGTGATGCATGAAGCGCTGCATAACCATTATACCCAGA AAAGCCTGAGCCTGAGCCCGGGCAAA 49 Light chain DIVMTQSPDSLAVSLGERATINCKSSQSVLFSSNNKNYLAWYQQKPGQP PKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHA SAPPTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPR EAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK VYACEVTHQGLSSPVTKSFNRGEC 50 Light chain DNA GACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGG CGAGAGGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTATTCCA GCTCCAACAATAAGAACTACTTAGCTTGGTACCAGCAGAAACCAGG ACAGCCTCCTAAGCTGCTCATTTACTGGGCATCTACCCGGGAATCCG GGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACT

- 82 052811

CTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTG TCAGCAGCACGCCAGTGCCCCTCCTACTTTTGGCGGAGGGACCAAGG TTGAGATCAAACGTACGGTGGCCGCTCCCTCCGTGTTCATCTTCCCA CCCTCCGACGAGCAGCTGAAGTCCGGCACCGCCTCCGTCGTGTGCCT GCTGAACAACTTCTACCCTGCGAGGCCAAAGTGCAGTGGAAAGTG GACAACGCCCCTGCAGTCCGGCAACTCCCAGGAATCCGTCACCGAGC AGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACCCTG TCCAAGGCCGACTACGAGAAGCACAAAGTGTACGCCTGCGAAGTGA CCCACCAGGGCCTGTCCAGCCCCGTGACCAAGTCCTTCAACCGGGGC GAGTGC Antibody A13-B against IL-27 51 Heavy chain EVQLVESGGGLVKPGGSLRLSCAASGFTFRSYGMNWVRQAPGKGLEW VSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYC ARDGGRTSYTATAHNWFDPWGQGTLVTVSSASTKGPSVFPLAPCSRST SESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS VVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI EKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMH EALHNHYTQKSLSLSLG 52 Heavy chain DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCCTGGTCAAGCCTGGGG GGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCCGGAGC TATGGGATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGT GGGTCTCATCCATTAGTAGTAGTAGTAGTTACATATACTACGCAGAC TCAGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACT CACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCGGT GTACTACTGCGCCAGAGATGTGGAAGAACGTCCTACACCGCCACA GCCCACAATTGGTTCGACCCCTGGGGACAGGGTACATTGGTCACCGT CTCCTCAGCTTCCACCAAGGGCCCCTCCGTGTTCCCTCTGGCCCCTTG CTCCCGGTCCACCTCCGAGTCTACCGCCGCTCTGGCTGCCTCGTGA AGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACTCTGGCGCC CTGACCTCCGGCGTGCACACCTTCCCTGCCGTGCTGCAGTCCTCCGG CCTGTACTCCCTGTCCAGCGTCGTGACCGTGCCCTCCTCCAGCCTGG GCACCAAGACCTACACCTGTAACGTGGACCACAAGCCCTCCAACAC CAAAGTGGACAAGCGGGTGGAATCTAAGTACGGCCCTCCCTGCCCTT CCTGCCCTGCCCCTGAGTTCCTGGGCGGACCTTCCGTGTTCCTGTTCC CTCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCTGAAGTG ACCTGCGTGGTGGTGGACGTGTCCCAGGAAAGATCCCGAAGTCCAGTT CAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAG CCCAGAGAGAACAGTTCAACTCCACCTACCGGGTGGTGTCCGTGCT GACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGC

- 83 052811

AAAGTGTCCAACAAGGGCCTGCCCTCCAGCATCGAAAAGACCATCT CCAAGGCCAAGGGCCAGCCCCGCGAGCCCCAAGTGTACACCCTGCC TCCCAGCCAGGAAGAGATGACCAAGAATCAAGTGTCCCTGACTTGT CTGGTCAAGGGCTTCTACCCCTCCGATATCGCCGTGGAGTGGGAGTC CAACGGCCAGCCCGAGAACAACTACAAGACCACCCCTCCCGTGCTG GACTCCGACGGCTCCTTCTTCCTGTACTCTCGGCTGACCGTGGACAA GTCCCGGTGGCAGGAAGGCAACGTCTTCTCCTGCTCCGTGATGCACG AGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGTCTCTG GGC Antibody Ab4-A against IL-27 53 HCDR1 (IMGT) GFTFSRTG 54 HCDR2 (IMGT) ISSSSSSYI 55 HCDR3 (IMGT) ARDGGRTSYTATAHNWFDP 56 HCDR1 (NT) FTFSRTGMN 57 HCDR2 (NT) SISSSSSSYIYYADSVKG 58 HCDR3 (NT) ARDGGRTSYTATAHNWFDP 59 VH EVQLVESGGGLVKPGGSLRLSCAASGFTFSRTGMNWVRQAPGKGLEW VSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYC ARDGGRTSYTATAHNWFDPWGQGTLVTVSS 60 VH DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCCTGGTCAAGCCTGGGG GGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGG ACTGGGATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAAT GGGTCTCATCCATTAGTAGTAGTAGTAGTTACATATACTACGCAGAC TCAGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACT CACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCGGT GTACTACTGCGCCAGAGATGTGGAAGAACGTCCTACACCGCCACA GCCCACAATTGGTTCGACCCCTGGGGACAGGGTACATTGGTCACCGT CTCCTCA 61 LCDR1 (IMGT) QSVLFSSNNKNY 62 LCDR2 (IMGT) WAS 63 LCDR3 (IMGT) QQHASAPPT 64 LCDR1 (NT) KSSQSVLFSSNNKNYLA 65 LCDR2 (NT) WASTRES 66 LCDR3 (NT) QQHASAPPT 67 VL DIVMTQSPDSLAVSLGERATINCKSSQSVLFSSNNKNYLAWYQQKPGQP PKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHA SAPPTFGGGTKVEIK 68 VL DNA GACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGG CGAGAGGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTATTCCA GCTCCAACAATAAGAACTACTTAGCTTGGTACCAGCAGAAACCAGG ACAGCCTCCTAAGCTGCTCATTTACTGGGCATCTACCCGGGAATCCG

- 84 052811

GGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACT CTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTG TCAGCAGCACGCCAGTGCCCCTCCTACTTTTGGCGGAGGGACCAAGG TTGAGATCAAA 69 Heavy chain EVQLVESGGGLVKPGGSLRLSCAASGFTFSRTGMNWVRQAPGKGLEW VSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYC ARDGGRTSYTATAHNWFDPWGQGTLVTVSSASTKGPSVFPLAPSSKST SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS VVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK 70 Heavy chain DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCCTGGTCAAGCCTGGGG GGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGG ACTGGGATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAAT GGGTCTCATCCATTAGTAGTAGTAGTAGTTACATATACTACGCAGAC TCAGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACT CACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCGGT GTACTACTGCGCCAGAGATGTGGAAGAACGTCCTACACCGCCACA GCCCACAATTGGTTCGACCCCTGGGGACAGGGTACATTGGTCACCGT CTCCTCAGCGAGCACCAAAGGCCCGAGCGTGTTTCCGCTGGCGCCGA GCAGCAAAAGCACCAGCGGCGGCACCGCGGCGCTGGGCTGCCTGGT GAAAGATTATTTTCCGGAACCGGTGACCGTGAGCTGGAACAGCGGC GCGCTGACCAGCGGCGTGCATACCTTTCCGGCGGTGCTGCAGAGCA GCGGCCTGTATAGCCTGAGCAGCGTGGTGACCGTGCCGAGCAGCAG CCTGGGCACCCAGACCTATATTTGCAACGTGAACCATAAACCGAGC AACACCAAAGTGGATAAAAAAGTGGAACCGAAAAGCTGCGATAAA ACCCATACCTGCCCGCCGTGCCCGGCGCCGGAACTGCTGGGCGGCCC GAGCGTGTTTCTGTTTCCGCCGAAACCGAAAGATACCCTGATGATTA GCCGCACCCCGGAAGTGACCTGCGTGGTGGTGGATGTGAGCCATGA AGATCCGGAAGTGAAATTTAACTGGTATGTGGATGGCGTGGAAGTG CATAACGCGAAAACCAAACCGCGCGAAGAACAGTATAACAGCACCT ATCGCGTGGTGAGCGTGCTGACCGTGCTGCATCAGGATTTGGCTGAAC GGCAAAGAATATAAATGCAAAGTGAGCAACAAAGCGCTGCCGGCGC CGATTGAAAAAACCATTAGCAAAGCGAAAGGCCAGCCGCGCGAACC GCAGGTGTATACCCTGCCGCCGAGCCGCGATGAACTGACCAAAAAC CAGGTGAGCCTGACCTGCCTGGTGAAAGGCTTTTATCCGAGCGATAT TGCGGTGGAATGGGAAAGCAACGGCCAGCCGGAAAACAACTATAAA ACCACCCCGCCGGTGCTGGATAGCGATGGCAGCTTTTTTCTGTATAG CAAACTGACCGTGGATAAAAGCCGCTGGCAGCAGGGCAACGTGTTT

- 85 052811

AGCTGCAGCGTGATGCATGAAGCGCTGCATAACCATTATACCCAGA AAAGCCTGAGCCTGAGCCCGGGCAAA 71 Light chain DIVMTQSPDSLAVSLGERATINCKSSQSVLFSSNNKNYLAWYQQKPGQP PKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHA SAPPTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPR EAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK VYACEVTHQGLSSPVTKSFNRGEC 72 Light chain DNA GACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGG CGAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTATTCA GCTCCAACAATAAGAACTACTTAGCTTGGTACCAGCAGAAACCAGG ACAGCCTCCTAAGCTGCTCATTTACTGGGCATCTACCCGGGAATCCG GGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACT CTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTG TCAGCAGCACGCCAGTGCCCCTCCTACTTTTGGCGGAGGGACCAAGG TTGAGATCAAACGTACGGTGGCCGCTCCCTCCGTGTTCATCTTCCCA CCCTCCGACGAGCAGCTGAAGTCCGGCACCGCCTCCGTCGTGTGCCT GCTGAACAACTTCTACCCTGCGAGGCCAAAGTGCAGTGGAAAGTG GACAACGCCCCTGCAGTCCGGCAACTCCCAGGAATCCGTCACCGAGC AGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACCCTG TCCAAGGCCGACTACGAGAAGCACAAAGTGTACGCCTGCGAAGTGA CCCACCAGGGCCTGTCCAGCCCCGTGACCAAGTCCTTCAACCGGGGC GAGTGC Antibody AЬ4-B against IL-27 73 Heavy chain EVQLVESGGGLVKPGGSLRLSCAASGFTFSRTGMNWVRQAPGKGLEW VSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYC ARDGGRTSYTATAHNWFDPWGQGTLVTVSSASTKGPSVFPLAPCSRST SESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS VVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI EKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMH EALHNHYTQKSLSLSLG 74 Heavy chain DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCCTGGTCAAGCCTGGGG GGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGG ACTGGGATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAAT GGGTCTCATCCATTAGTAGTAGTAGTAGTTACATATACTACGCAGAC TCAGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACT CACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCGGT GTACTACTGCGCCAGAGATGTGGAAGAACGTCCTACACCGCCACA GCCCACAATTGGTTCGACCCCTGGGGACAGGGTACATTGGTCACCGT CTCCTCAGCTTCCACCAAGGGCCCCTCCGTGTTCCCTCTGGCCCCTTG

- 86 052811

CTCCCGGTCCACCTCCGAGTCTACCGCCGCTCTGGCTGCCTCGTGA AGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACTCTGGCGCC CTGACCTCCGGCGTGCACACCTTCCCTGCCGTGCTGCAGTCCTCCGG CCTGTACTCCCTGTCCAGCGTCGTGACCGTGCCCTCCTCCAGCCTGG GCACCAAGACCTACACCTGTAACGTGGACCACAAGCCCTCCAACAC CAAAGTGGACAAGCGGGTGGAATCTAAGTACGGCCCTCCCTGCCCTT CCTGCCCTGCCCCTGAGTTCCTGGGCGGACCTTCCGTGTTCCTGTTCC CTCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCTGAAGTG ACCTGCGTGGTGGTGGACGTGTCCCAGGAAAGATCCCGAAGTCCAGTT CAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAG CCCAGAGAGAACAGTTCAACTCCACCTACCGGGTGGTGTCCGTGCT GACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGC AAAGTGTCCAACAAGGCCTGCCCTCCAGCATCGAAAAGACCATCT CCAAGGCCAAGGGCCAGCCCCGCGAGCCCCAAGTGTACACCCTGCC TCCCAGCCAGGAAGAGATGACCAAGAATCAAGTGTCCCTGACTTGT CTGGTCAAGGGCTTCTACCCCTCCGATATCGCCGTGGAGTGGGAGTC CAACGGCCAGCCCGAGAACAACTACAAGACCACCCCTCCCGTGCTG GACTCCGACGGCTCCTTCTTCCTGTACTCTCGGCTGACCGTGGACAA GTCCCGGTGGCAGGAAGGCAACGTCTTCTCCTGCTCCGTGATGCACG AGGCCCTGCACAACCACTACACCAGAAGTCCCTGTCCCTGTCTCTG GGC Antibody Ab5-A against IL-27 75 HCDR1 (IMGT) GFTFSRYG 76 HCDR2 (IMGT) ISSSSSAYI 77 HCDR3 (IMGT) ARDGGRTSYTATAHNWFDP 78 HCDR1 (NT) FTFSRYGMN 79 HCDR2 (NT) SISSSSSAYILYADSVKG 80 HCDR3 (NT) ARDGGRTSYTATAHNWFDP 81 VH EVQLVESGGGLVKPGGSLRLSCAASGTFFSRYGMNWVRQAPGKGLEW VSSISSSSAYILYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYC ARDGGRTSYTATAHNWFDPWGQGTLVTVSS 82 VH DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCCTGGTCAAGCCTGGGG GGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGG TATGGGATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGT GGGTCTCATCCATTAGTAGTAGTAGTGCTTACATACTGTACGCAGAC TCAGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACT CACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCGGT GTACTACTGCGCCAGAGATGTGGAAGAACGTCCTACACCGCCACA GCCCACAATTGGTTCGACCCCTGGGGACAGGGTACATTGGTCACCGT CTCCTCA 83 LCDR1 (IMGT) QSVLFSSNNKNY

- 87 052811

84 LCDR2 (IMGT) WAS 85 LCDR3 (IMGT) QQHASAPPT 86 LCDR1 (NT) KSSQSVLFSSNNKNYLA 87 LCDR2 (NT) WASTRES 88 LCDR3 (NT) QQHASAPPT 89 VL DIVMTQSPDSLAVSLGERATINCKSSQSVLFSSNNKNYLAWYQQKPGQP PKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHA SAPPTFGGGTKVEIK 90 VL DNA GACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGG CGAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTATTCA GCTCCAACAATAAGAACTACTTAGCTTGGTACCAGCAGAAACCAGG ACAGCCTCCTAAGCTGCTCATTTACTGGGCATCTACCCGGGAATCCG GGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACT CTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTG TCAGCAGCACGCCAGTGCCCCTCCTACTTTTGGCGGAGGGACCAAGG TTGAGATCAAA 91 Heavy chain EVQLVESGGGLVKPGGSLRLSCAASGTFFSRYGMNWVRQAPGKGLEW VSSISSSSAYILYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYC ARDGGRTSYTATAHNWFDPWGQGTLVTVSSASTKGPSVFPLAPSSKST SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS VVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK 92 Heavy chain DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCCTGGTCAAGCCTGGGG GGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGG TATGGGATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGT GGGTCTCATCCATTAGTAGTAGTAGTGCTTACATACTGTACGCAGAC TCAGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACT CACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCGGT GTACTACTGCGCCAGAGATGTGGAAGAACGTCCTACACCGCCACA GCCCACAATTGGTTCGACCCCTGGGGACAGGGTACATTGGTCACCGT CTCCTCAGCGAGCACCAAAGGCCCGAGCGTGTTTCCGCTGGCGCCGA GCAGCAAAAGCACCAGCGGCGGCACCGCGGCGCTGGGCTGCCTGGT GAAAGATTATTTTCCGGAACCGGTGACCGTGAGCTGGAACAGCGGC GCGCTGACCAGCGGCGTGCATACCTTTCCGGCGGTGCTGCAGAGCA GCGGCCTGTATAGCCTGAGCAGCGTGGTGACCGTGCCGAGCAGCAG CCTGGGCACCCAGACCTATATTTGCAACGTGAACCATAAACCGAGC AACACCAAAGTGGATAAAAAAGTGGAACCGAAAAGCTGCGATAAA

- 88 052811

ACCCATACCTGCCCGCCGTGCCCGGCGCCGGAACTGCTGGGCGGCCC GAGCGTGTTTCTGTTTCCGCCGAAACCGAAAGATACCCTGATGATTA GCCGCACCCCGGAAGTGACCTGCGTGGTGGTGGATGTGAGCCATGA AGATCCGGAAGTGAAATTTAACTGGTATGTGGATGGCGTGGAAGTG CATAACGCGAAAACCAAACCGCGCGAAGAACAGTATAACAGCACCT ATCGCGTGGTGAGCGTGCTGACCGTGCTGCATCAGGATTGGCTGAAC GGCAAAGAATATAAATGCAAAGTGAGCAACAAAGCGCTGCCGGCGC CGATTGAAAAAACCATTAGCAAAGCGAAAGGCCAGCCGCGCGAACC GCAGGTGTATACCCTGCCGCCGAGCCGCGATGAACTGACCAAAAAC CAGGTGAGCCTGACCTGCCTGGTGAAAGGCTTTTATCCGAGCGATAT TGCGGTGGAATGGGAAAGCAACGGCCAGCCGGAAAACAACTATAAA ACCACCCCGCCGGTGCTGGATAGCGATGGCAGCTTTTTTCTGTATAG CAAACTGACCGTGGATAAAAGCCGCTGGCAGCAGGGCAACGTGTTT AGCTGCAGCGTGATGCATGAAGCGCTGCATAACCATTATACCCAGA AAAGCCTGAGCCTGAGCCCGGGCAAA 93 Light chain DIVMTQSPDSLAVSLGERATINCKSSQSVLFSSNNKNYLAWYQQKPGQP PKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHA SAPPTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPR EAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK VYACEVTHQGLSSPVTKSFNRGEC 94 Light chain DNA GACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGG CGAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTATTCA GCTCCAACAATAAGAACTACTTAGCTTGGTACCAGCAGAAACCAGG ACAGCCTCCTAAGCTGCTCATTTACTGGGCATCTACCCGGGAATCCG GGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACT CTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTG TCAGCAGCACGCCAGTGCCCCTCCTACTTTTGGCGGAGGGACCAAGG TTGAGATCAAACGTACGGTGGCCGCTCCCTCCGTGTTCATCTTCCCA CCCTCCGACGAGCAGCTGAAGTCCGGCACCGCCTCCGTCGTGTGCCT GCTGAACAACTTCTACCCTGCGAGGCCAAAGTGCAGTGGAAAGTG GACAACGCCCCTGCAGTCCGGCAACTCCCAGGAATCCGTCACCGAGC AGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACCCTG TCCAAGGCCGACTACGAGAAGCACAAAGTGTACGCCTGCGAAGTGA CCCACCAGGGCCTGTCCAGCCCCGTGACCAAGTCCTTCAACCGGGGC GAGTGC Antibody A15-B against IL-27 95 Heavy chain EVQLVESGGGLVKPGGSLRLSCAASGTFFSRYGMNWVRQAPGKGLEW VSSISSSSAYILYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYC ARDGGRTSYTATAHNWFDPWGQGTLVTVSSASTKGPSVFPLAPCSRST SESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS VVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEV

- 89 052811

EALHNHYTQKSLSLSLG 96 Heavy chain DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCCTGGTCAAGCCTGGGG GGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGG TATGGGATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGT GGGTCTCATCCATTAGTAGTAGTAGTGCTTACATACTGTACGCAGAC TCAGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACT CACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCGGT GTACTACTGCGCCAGAGATGTGGAAGAACGTCCTACACCGCCACA GCCCACAATTGGTTCGACCCCTGGGGACAGGGTACATTGGTCACCGT CTCCTCAGCTTCCACCAAGGGCCCCTCCGTGTTCCCTCTGGCCCCTTG CTCCCGGTCCACCTCCGAGTCTACCGCCGCTCTGGCTGCCTCGTGA AGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACTCTGGCGCC CTGACCTCCGGCGTGCACACCTTCCCTGCCGTGCTGCAGTCCTCCGG CCTGTACTCCCTGTCCAGCGTCGTGACCGTGCCCTCCTCCAGCCTGG GCACCAAGACCTACACCTGTAACGTGGACCACAAGCCCTCCAACAC CAAAGTGGACAAGCGGGTGGAATCTAAGTACGGCCCTCCCTGCCCTT CCTGCCCTGCCCCTGAGTTCCTGGGCGGACCTTCCGTGTTCCTGTTCC CTCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCTGAAGTG ACCTGCGTGGTGGTGGACGTGTCCCAGGAAAGATCCCGAAGTCCAGTT CAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAG CCCAGAGAGAACAGTTCAACTCCACCTACCGGGTGGTGTCCGTGCT GACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGC AAAGTGTCCAACAAGGCCTGCCCTCCAGCATCGAAAAGACCATCT CCAAGGCCAAGGGCCAGCCCCGCGAGCCCCAAGTGTACACCCTGCC TCCCAGCCAGGAAGAGATGACCAAGAATCAAGTGTCCCTGACTTGT CTGGTCAAGGGCTTCTACCCCTCCGATATCGCCGTGGAGTGGGAGTC CAACGGCCAGCCCGAGAACAACTACAAGACCACCCCTCCCGTGCTG GACTCCGACGGCTCCTTCTTCCTGTACTCTCGGCTGACCGTGGACAA GTCCCGGTGGCAGGAAGGCAACGTCTTCTCCTGCTCCGTGATGCACG AGGCCCTGCACAACCACTACACCAGAAGTCCCTGTCCCTGTCTCTG GGC Antibody Ab6-A against IL-27 97 HCDRl (IMGT) GFTFASYG 98 HCDR2 (IMGT) ISSSSSSYI 99 HCDR3 (IMGT) ARDGGRTSYTATAHNWFDP 100 HCDRl (NT) FTFASYGMN 101 HCDR2 (NT) SISSSSSSYIYYADSVKG 102 HCDR3 (NT) ARDGGRTSYTATAHNWFDP

- 90 052811

103 VH EVQLVESGGGLVKPGGSLRLSCAASGFFASYGMNWVRQAPGKGLEW VSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYC ARDGGRTSYTATAHNWFDPWGQGTLVTVSS 104 VH DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCCTGGTCAAGCCTGGGG GGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCGCTAGCT ATGGGATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTG GGTCTCATCCATTAGTAGTTCTAGTAGTTACATATACTACGCAGACT CAGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTC ACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCGGTG TACTACTGCGCCAGAGATGTGGAAGAACGTCCTACACCGCCACAG CCCACAATTGGTTCGACCCCTGGGGACAGGGTACATTGGTCACCGTC TSCTSA 105 LCDR1 (IMGT) QSVLFSSNNKNY 106 LCDR2 (IMGT) WAS 107 LCDR3 (IMGT) QQHASAPPT 108 LCDR1 (NT) KSSQSVLFSSNNKNYLA 109 LCDR2 (NT) WASTRES BY LCDR3 (NT) QQHASAPPT 111 VL DIVMTQSPDSLAVSLGERATINCKSSQSVLFSSNNKNYLAWYQQKPGQP PKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHA SAPPTFGGGTKVEIK 112 VL DNA GACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGG CGAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTATTCA GCTCCAACAATAAGAACTACTTAGCTTGGTACCAGCAGAAACCAGG ACAGCCTCCTAAGCTGCTCATTTACTGGGCATCTACCCGGGAATCCG GGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACT CTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTG TCAGCAGCACGCCAGTGCCCCTCCTACTTTTGGCGGAGGGACCAAGG TTGAGATCAAA 113 Heavy chain EVQLVESGGGLVKPGGSLRLSCAASGFTFASYGMNWVRQAPGKGLEW VSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYC ARDGGRTSYTATAHNWFDPWGQGTLVTVSSASTKGPSVFPLAPSSKST SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS VVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK 114 Heavy chain DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCCTGGTCAAGCCTGGGG GGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCGCTAGCT

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ATGGGATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTG GGTCTCATCCATTAGTAGTTCTAGTAGTTACATATACTACGCAGACT CAGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTC ACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCGGTG TACTACTGCGCCAGAGATGGTGGAAGAACGTCCTACACCGCCACAG CCCACAATTGGTTCGACCCCTGGGGACAGGGTACATTGGTCACCGTC TCCTCAGCGAGCACCAAAGGCCCGAGCGTGTTTCCGCTGGCGCCGA GCAGCAAAAGCACCAGCGGCGGCACCGCGGCGCTGGGCTGCCTGGT GAAAGATTATTTTCCGGAACCGGTGACCGTGAGCTGGAACAGCGGC GCGCTGACCAGCGGCGTGCATACCTTTCCGGCGGTGCTGCAGAGCA GCGGCCTGTATAGCCTGAGCAGCGTGGTGACCGTGCCGAGCAGCAG CCTGGGCACCCAGACCTATATTTGCAACGTGAACCATAAACCGAGC AACACCAAAGTGGATAAAAAAGTGGAACCGAAAAGCTGCGATAAA ACCCATACCTGCCCGCCGTGCCCGGCGCCGGAACTGCTGGGCGGCCC GAGCGTGTTTCTGTTTCCGCCGAAACCGAAAGATACCCTGATGATTA GCCGCACCCCGGAAGTGACCTGCGTGGTGGTGGATGTGAGCCATGA AGATCCCGGAAGTGAAATTTAACTGGTATGTGGATGGCGTGGAAGTG CATAACGCGAAAACCAAACCGCGCGAAGAACAGTATAACAGCACCT ATCGCGTGGTGAGCGTGCTGACCGTGCTGCATCAGGATTTGGCTGAAC GGCAAAGAATATAAATGCAAAGTGAGCAACAAAGCGCTGCCGGCGC CGATTGAAAAAACCATTAGCAAAGCGAAAGGCCAGCCGCGCGAACC GCAGGTGTATACCCTGCCGCCGAGCCGCGATGAACTGACCAAAAAC CAGGTGAGCCTGACCTGCCTGGTGAAAGGCTTTTATCCGAGCGATAT TGCGGTGGAATGGGAAAGCAACGGCCAGCCGGAAAACAACTATAAA ACCACCCCGCCGGTGCTGGATAGCGATGGCAGCTTTTTTCTGTATAG CAAACTGACCGTGGATAAAAGCCGCTGGCAGCAGGGCAACGTGTTT AGCTGCAGCGTGATGCATGAAGCGCTGCATAACCATTATACCCAGA AAAGCCTGAGCCTGAGCCCGGGCAAA 115 Light chain DIVMTQSPDSLAVSLGERATINCKSSQSVLFSSNNKNYLAWYQQKPGQP PKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHA SAPPTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPR EAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK VYACEVTHQGLSSPVTKSFNRGEC 116 Light chain DNA GACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGG CGAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTATTCA GCTCCAACAATAAGAACTACTTAGCTTGGTACCAGCAGAAACCAGG ACAGCCTCCTAAGCTGCTCATTTACTGGGCATCTACCCGGGAATCCG GGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACT CTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTG TCAGCAGCACGCCAGTGCCCCTCCTACTTTTGGCGGAGGGACCAAGG TTGAGATCAAACGTACGGTGGCCGCTCCCTCCGTGTTCATCTTCCCA CCCTCCGACGAGCAGCTGAAGTCCGGCACCGCCTCCGTCGTGTGCCT

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GCTGAACAACTTCTACCCTGCGAGGCCAAAGTGCAGTGGAAAGTG GACAACGCCCCTGCAGTCCGGCAACTCCCAGGAATCCGTCACCGAGC AGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACCCTG TCCAAGGCCGACTACGAGAAGCACAAAGTGTACGCCTGCGAAGTGA CCCACCAGGGCCTGTCCAGCCCCGTGACCAAGTCCTTCAACCGGGGC GAGTGC Antibody A6-B against IL-27 117 Heavy chain EVQLVESGGGLVKPGGSLRLSCAASGFTFASYGMNWVRQAPGKGLEW VSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYC ARDGGRTSYTATAHNWFDPWGQGTLVTVSSASTKGPSVFPLAPCSRST SESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS VVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI EKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMH EALHNHYTQKSLSLSLG 118 Heavy chain DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCCTGGTCAAGCCTGGGG GGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCGCTAGCT ATGGGATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTG GGTCTCATCCATTAGTAGTTCTAGTAGTTACATATACTACGCAGACT CAGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTC ACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCGGTG TACTACTGCGCCAGAGATGTGGAAGAACGTCCTACACCGCCACAG CCCACAATTGGTTCGACCCCTGGGGACAGGGTACATTGGTCACCGTCTCCTCAGCTTCCACCAAGGGCCCCTCCGTGTTCCCTCTGGCCCCTTGC TCCCGGTCCACCTCCGAGTCTACCGCCGCTCTGGCTGCCTCGTGAA GGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACTCTGGCGCCC TGACCTCCGGCGTGCACACCTTCCCTGCCGTGCTGCAGTCCTCCGGC CTGTACTCCCTGTCCAGCGTCGTGACCGTGCCCTCCTCCAGCCTGGGG CACCAAGACCTACACCTGTAACGTGGACCACAAGCCCTCCAACACC AAAGTGGACAAGCGGGTGGAATCTAAGTACGGCCCTCCCTGCCCTTC CTGCCCTGCCCCTGAGTTCCTGGGCGGACCTTCCGTGTTCCTGTTCCC TCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCTGAAGTG ACCTGCGTGGTGGTGGACGTGTCCCAGGAAAGATCCCGAAGTCCAGTT CAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAG CCCAGAGAGAACAGTTCAACTCCACCTACCGGGTGGTGTCCGTGCT GACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGC AAAGTGTCCAACAAGGCCTGCCCTCCAGCATCGAAAAGACCATCT CCAAGGCCAAGGGCCAGCCCCGCGAGCCCCAAGTGTACACCCTGCC TCCCAGCCAGGAAGAGATGACCAAGAATCAAGTGTCCCTGACTTGT CTGGTCAAGGGCTTCTACCCCTCCGATATCGCCGTGGAGTGGGAGTC

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CAACGGCCAGCCCGAGAACAACTACAAGACCACCCCTCCCGTGCTG GACTCCGACGGCTCCTTCTTCCTGTACTCTCGGCTGACCGTGGACAA GTCCCGGTGGCAGGAAGGCAACGTCTTCTCCTGCTCCGTGATGCACG AGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGTCTCTG GGC Anti-IL-27 antibody Abl-A 119 HCDR1 (IMGT) GFTFRSYG 120 HCDR2 (IMGT) ISSSGSYI 121 HCDR3 (IMGT) ARDGGRTSYTATAHNWFDP 122 HCDR1 (NT) FTFRSYGMN 123 HCDR2 (NT) GISSSGSYIYYADSVKG 124 HCDR3 (NT) ARDGGRTSYTATAHNWFDP 125 VH EVQLVESGGGLVKPGGSLRLSCAASGFTFRSYGMNWVRQAPGKGLEW VSGISSSGSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYC ARDGGRTSYTATAHNWFDPWGQGTLVTVSS 126 DNKUN GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCCTGGTCAAGCCTGGGG GGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCCGTAGCT ATGGGATGAACTGGGTCCGCCAGGCTCCAGGAAGGGGCTGGAGTG GGTCTCAGGTATTAGTAGTAGTGGTAGTTACATATACTACGCAGACT CAGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTC ACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCGGTG TACTACTGCGCCAGAGATGTGGAAGAACGTCCTACACCGCCACAG CCCACAATTGGTTCGACCCCTGGGGACAGGGTACATTGGTCACCGTC TCCTCA 127 LCDR1 (IMGT) QSVLFSSNNKNY 128 LCDR2 (IMGT) WAS 129 LCDR3 (IMGT) QQHASAPPT 130 LCDR1 (NT) KSSQSVLFSSNNKNYLA 131 LCDR2 (NT) WASTRES 132 LCDR3 (NT) QQHASAPPT 133 VL DIVMTQSPDSLAVSLGERATINCKSSQSVLFSSNNKNYLAWYQQKPGQP PKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHA SAPPTFGGGTKVEIK 134 VL DNA GACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGG CGAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTATTCA GCTCCAACAATAAGAACTACTTAGCTTGGTACCAGCAGAAACCAGG ACAGCCTCCTAAGCTGCTCATTTACTGGGCATCTACCCGGGAATCCG GGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACT CTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTG TCAGCAGCACGCCAGTGCCCCTCCTACTTTTGGCGGAGGGACCAAGG TTGAGATCAAA

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135 Heavy chain EVQLVESGGGLVKPGGSLRLSCAASGFTFRSYGMNWVRQAPGKGLEW VSGISSSGSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYC ARDGGRTSYTATAHNWFDPWGQGTLVTVSSASTKGPSVFPLAPSSKST SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS VVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK 136 Heavy chain DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCCTGGTCAAGCCTGGGG GGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCCGTAGCT ATGGGATGAACTGGGTCCGCCAGGCTCCAGGAAGGGGCTGGAGTG GGTCTCAGGTATTAGTAGTAGTGGTAGTTACATATACTACGCAGACT CAGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTC ACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCGGTG TACTACTGCGCCAGAGATGGTGGAAGAACGTCCTACACCGCCACAG CCCACAATTGGTTCGACCCCTGGGGACAGGGTACATTGGTCACCGTC TCCTCAGCGAGCACCAAAGGCCCGAGCGTGTTTCCGCTGGCGCCGA GCAGCAAAAGCACCAGCGGCGGCACCGCGGCGCTGGGCTGCCTGGT GAAAGATTATTTTCCGGAACCGGTGACCGTGAGCTGGAACAGCGGC GCGCTGACCAGCGGCGTGCATACCTTTCCGGCGGTGCTGCAGAGCA GCGGCCTGTATAGCCTGAGCAGCGTGGTGACCGTGCCGAGCAGCAG CCTGGGCACCCAGACCTATATTTGCAACGTGAACCATAAACCGAGC AACACCAAAGTGGATAAAAAAGTGGAACCGAAAAGCTGCGATAAA ACCCATACCTGCCCGCCGTGCCCGGCGCCGGAACTGCTGGGCGGCCC GAGCGTGTTTCTGTTTCCGCCGAAACCGAAAGATACCCTGATGATTA GCCGCACCCCGGAAGTGACCTGCGTGGTGGTGGATGTGAGCCATGA AGATCCGGAAGTGAAATTTAACTGGTATGTGGATGGCGTGGAAGTG CATAACGCGAAAACCAAACCGCGCGAAGAACAGTATAACAGCACCT ATCGCGTGGTGAGCGTGCTGACCGTGCTGCATCAGGATTTGGCTGAAC GGCAAAGAATATAAATGCAAAGTGAGCAACAAAGCGCTGCCGGCGC CGATTGAAAAAACCATTAGCAAAGCGAAAGGCCAGCCGCGCGAACC GCAGGTGTATACCCTGCCGCCGAGCCGCGATGAACTGACCAAAAAC CAGGTGAGCCTGACCTGCCTGGTGAAAGGCTTTTATCCGAGCGATAT TGCGGTGGAATGGGAAAGCAACGGCCAGCCGGAAAACAACTATAAA ACCACCCCGCCGGTGCTGGATAGCGATGGCAGCTTTTTTCTGTATAG CAAACTGACCGTGGATAAAAGCCGCTGGCAGCAGGGCAACGTGTTT AGCTGCAGCGTGATGCATGAAGCGCTGCATAACCATTATACCCAGA AAAGCCTGAGCCTGAGCCCGGGCAAA 137 Light chain DIVMTQSPDSLAVSLGERATINCKSSQSVLFSSNNKNYLAWYQQKPGQP PKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHA

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SAPPTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPR EAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK VYACEVTHQGLSSPVTKSFNRGEC 138 Light chain DNA GACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGG CGAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTATTCA GCTCCAACAATAAGAACTACTTAGCTTGGTACCAGCAGAAACCAGG ACAGCCTCCTAAGCTGCTCATTTACTGGGCATCTACCCGGGAATCCG GGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACT CTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTG TCAGCAGCACGCCAGTGCCCCTCCTACTTTTGGCGGAGGGACCAAGG TTGAGATCAAACGTACGGTGGCCGCTCCCTCCGTGTTCATCTTCCCA CCCTCCGACGAGCAGCTGAAGTCCGGCACCGCCTCCGTCGTGTGCCT GCTGAACAACTTCTACCCTGCGAGGCCAAAGTGCAGTGGAAAGTG GACAACGCCCCTGCAGTCCGGCAACTCCCAGGAATCCGTCACCGAGC AGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACCCTG TCCAAGGCCGACTACGAGAAGCACAAAGTGTACGCCTGCGAAGTGA CCCACCAGGGCCTGTCCAGCCCCGTGACCAAGTCCTTCAACCGGGGC GAGTGC Antibody Abi-B against IL-27 139 Heavy chain EVQLVESGGGLVKPGGSLRLSCAASGFTFRSYGMNWVRQAPGKGLEW VSGISSSGSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYC ARDGGRTSYTATAHNWFDPWGQGTLVTVSSASTKGPSVFPLAPCSRST SESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS VVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI EKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMH EALHNHYTQKSLSLSLG 140 Heavy chain DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCCTGGTCAAGCCTGGGG GGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCCGTAGCT ATGGGATGAACTGGGTCCGCCAGGCTCCAGGAAGGGGCTGGAGTG GGTCTCAGGTATTAGTAGTAGTGGTAGTTACATATACTACGCAGACT CAGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTC ACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCGGTG TACTACTGCGCCAGAGATGTGGAAGAACGTCCTACACCGCCACAG CCCACAATTGGTTCGACCCCTGGGGACAGGGTACATTGGTCACCGTCTCCTCAGCTTCCACCAAGGGCCCCTCCGTGTTCCCTCTGGCCCCTTGC TCCCGGTCCACCTCCGAGTCTACCGCCGCTCTGGCTGCCTCGTGAA GGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACTCTGGCGCCC TGACCTCCGGCGTGCACACCTTCCCTGCCGTGCTGCAGTCCTCCGGC CTGTACTCCCTGTCCAGCGTCGTGACCGTGCCCTCCTCCAGCCTGGGG

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CACCAAGACCTACACCTGTAACGTGGACCACAAGCCCTCCAACACC AAAGTGGACAAGCGGGTGGAATCTAAGTACGGCCCTCCCTGCCCTTC CTGCCCTGCCCCTGAGTTCCTGGGCGGACCTTCCGTGTTCCTGTTCCC TCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCTGAAGTG ACCTGCGTGGTGGTGGACGTGTCCCAGGAAGATCCCGAAGTCCAGTT CAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAG CCCAGAGAGGGAACAGTTCAACTCCACCTACCGGGTGGTGTCCGTGCT GACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGC AAAGTGTCCAACAAGGGCCTGCCCCTCCAGCATCGAAAAGACCATCT CCAAGGCCAAGGGCCAGCCCCGCGAGCCCCAAGTGTACACCCTGCC TCCCAGCCAGGAAGAGATGACCAAGAATCAAGTGTCCCTGACTTGT CTGGTCAAGGGCTTCTACCCCTCCGATATCGCCGTGGAGTGGGAGTC CAACGGCCAGCCCGAGAACAACTACAAGACCACCCCTCCCGTGCTG GACTCCGACGGCTCCTTCTTCCTGTACTCTCGGCTGACCGTGGACAA GTCCCGGTGGCAGGAAGGCAACGTCTTCTCCTGCTCCGTGATGCACG AGGCCCTGCACAACCACTACACCAGAAGTCCCTGTCCCTGTCTCTG GGC 141 FLAG DYKDDDDK 142 6-HIS HHHHHH 143 HA YPYDVPDYA

Table 13

Fc sequences (=CH ₂ +CH ₃ )

Names e Pseudo him Amino acid sequence Human IgGl 1.0 EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 5) Human IgG4 4.0 ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDP EVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGN VFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 6) Human IgG4 (S228P) 4.1 ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDP EVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGN VFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 7)

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Human IgG4 (S228P/ L235E) 4.2 ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDP EVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGN VFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 8)

Example 9. Binding properties of anti-IL-27 antibody α1 and IL-27 receptor blockade.

The association and dissociation of recombinant human IL-27 was determined over a concentration range from 0 to 5.0 μg/ml with the anti-IL-27 antibody AB at a concentration of 1 μg/ml. The final kinetic binding parameters are presented in Table 14 along with the binding model fit parameters (R ² and χ ² ), which demonstrate a good agreement between the experimental data and the model.

Human IL-27 demonstrated the highest binding affinity for the anti-IL-27 antibody among all species tested in this study (3.86 pM). Rat and cynomolgus macaque recombinant IL-27 also exhibited high affinity for the anti-IL-27 antibody, with values of 80.9 pM and 37.4 pM, respectively, although slightly weaker than the corresponding human protein. Mouse recombinant IL-27 had the lowest affinity for the anti-IL-27 antibody compared to the corresponding human protein, with a value in the nanomolar range (4.43 nM), as indicated by its slower association rate and higher dissociation rate.

Table 14

Summary of IL-27 binding to IL-27 antibody α1 and interspecies cross-reactivity

Analyte Kd (M) ka (1/Ms) ka (1/s) Full χ ² Full R ² Human IL-27 3.86E-12 5D0E+05 1.97E-06 0.4055 0.9991 IL-27 mice 4.43E-09 5.50E+04 2.44E-04 0.6732 0.9963 IL-27 rats 8.09E-11 2.34E+06 1.89E-04 0.4685 0.9945 IL-27 Javan macaque 3.74E-11 ZD8E+05 1D9E-05 1,3431 0.9979

Abbreviations: IL-27=interleukin 27, k _a =association constant, kj = dissociation constant, K _D = binding affinity.

Note: ^R2 values > 0.95 and ^χ2 values < 3.0 indicate that the obtained data accurately match the mathematical model.

Example 10. Alignment of CDR sequences.

Several subgroups of anti-IL-27 antibodies according to the present invention share sequence homology in their CDR regions, providing a variety of CDR sequence variants that have been confirmed to retain their functionality. It is expressly contemplated herein that the following consensus CDR sequences are fully supported and therefore included within the scope of the present invention.

For the anti-IL-27 antibody AL1, anti-IL-27 antibody AL3, anti-IL-27 antibody AL4, anti-IL-27 antibody AL5, anti-IL-27 antibody AL6, and anti-IL-27 antibody AL7, alignment of the CDR sequences of each of these anti-IL-27 antibodies revealed extensive homology interspersed with variable residues. In particular, alignment of the heavy chain CDR1 revealed the following variable residues:

HCDR1 (IMGT).

CLUSTAL O (1.2.4) multiple sequence alignment program

1 GFTFRSYG 8 (SEQ ID NO: 119) 5 GFTFRSYG 8 (SEQ ID NO: 31) 4 GFTFASYG 8 (SEQ ID NO: 97) 2 GFTFSRTG 8 (SEQ ID NO: 53) 3 GFTFSRYG 8 (SEQ ID NO: 75) 6 GFTFSSYS 8 (SEQ ID NO: 9) ### *

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Thus, the heavy chain CDR1 (IMGT) consensus sequence for these homologous antibodies is N-GFTF[S/A/R][S/R][T/Y][G/S]-C (SEQ ID NO: 144) and, accordingly, the more generalized heavy chain CDR1 (IMGT) consensus sequence provided herein is N-GFTFXXXXC (SEQ ID NO: 145), where X is any amino acid residue.

Alignment of the CDR2 (IMGT) sequences of the heavy chain of anti-IL-27 antibody AL1, anti-IL-27 antibody AL3, anti-IL-27 antibody AL4, anti-IL-27 antibody AL5, anti-IL-27 antibody AL6, and anti-IL-27 antibody AL7 revealed the following:

HCDR2 (IMGT).

CLUSTAL O (1.2.4) multiple sequence alignment program

10 ISSSGSY1 8 (SEQ ID NO: 120) 11ISSSSSSYI 8 (SEQ ID NO: 98) 7ISSSSSYI 8 (SEQ ID NO: 32) 9 ISSSSSYI 8 (SEQ ID NO: 54) 8 ISSSSAYI 8 (SEQ ID NO: 76) 12 ISSSSSYI 8 (SEQ ID NO: 10)

Thus, the heavy chain CDR2 (IMGT) consensus sequence for these homologous antibodies is N-ISSS[S/G][S/A]YI-C (SEQ ID NO: 146) and, accordingly, the more generalized heavy chain CDR2 (IMGT) consensus sequence provided herein is N-ISSSXXYI-C (SEQ ID NO: 147), where X is any amino acid residue.

Alignment of human CDR1 (NT) and human CDR2 (NT) sequences also revealed the following:

HCDR1 (NT).

CLUSTAL O (1.2.4) multiple sequence alignment program

13 FTFRSYGMN 9 (SEQ ID NO: 34) 16FTFRSYGMN 9 (SEQ ID NO: 122) 17FTFASYGMN 9 (SEQ ID NO: 100) 14 FTFSRTGMN 9 (SEQ ID NO: 56) 15 FTFSRYGMN 9 (SEQ ID NO: 78) 18FTFSSYSMN 9 (SEQ ID NO: 12)

HCDR2 (NT).

CLUSTAL O (1.2.4) multiple sequence alignment program

23 GISSSGSYIYYADSVKG 17 (SEQ ID NO: 123) 19 SISSSSSSYIYYADSVKG 17 (SEQ ID NO: 35) 20 SISSSSSSYIYYADSVKG 17 (SEQ ID NO: 57) 22 SISSSSSSYIYYADSVKG 17 (SEQ ID NO: 101) 21 SISSSSSAYILYADSVKG 17 (SEQ ID NO: 79) 24 SISSSSSSYIYYADSVKG 17 (SEQ ID NO: 13)

Thus, the heavy chain CDR1 (NT) and CDR2 (NT) consensus sequences for these homologous antibodies are the sequence NFTF[S/A/R][S/R][T/Y][G/S]MN-C (SEQ ID NO: 148) and the sequence N[G/S]ISSS[S/G][S/A]YI[L/Y]YADSVKG-C (SEQ ID NO: 149), respectively. Taking into account these consensus sequences, the more general heavy chain CDR1 (NT) and CDR2 (NT) consensus sequences provided herein are the sequence N-FTFXXXXMN-C (SEQ ID NO: 150) and the sequence NXISSSXXYIXYADSVKG-C (SEQ ID NO: 151), respectively, where X is any amino acid residue.

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The heavy chain CDR3 region (IMGT or NT) and the light chain CDR regions CDR1 (IMGT or NT), CDR2 (IMGT or NT), and CDR3 (IMGT or NT) were completely conserved between the anti-IL-27 antibody AL1, anti-IL-27 antibody AL3, anti-IL-27 antibody AL4, anti-IL-27 antibody AL5, anti-IL-27 antibody AL6, and anti-IL-27 antibody AL7.

Example 11. Crystallization and epitope determination of the IL-27 complex - Fab antibody Abi against IL-27.

Initial crystallization studies were performed with the human IL-27-anti-IL-27 Fab antibody Abi complex at a concentration of 10.1 mg/ml in 25 mM Tris pH 7.5, approximately 30 mM sodium chloride, and 5% glycerol. Preliminary crystallization conditions were determined using the PACT screen (Newman et al. (2005) Acta Cryst. D 61: 1426).

Crystals were obtained under various conditions, including PACT A2 (0.1 M SPG (succinic acid, sodium dihydrogen phosphate, and glycine) pH 5 and 25% PEG 1500). These crystals were used to generate new seeding material and set up a microarray seeding experiment (MM3) using the JCSG+ screening (D'Arcy et al. (2007) Acta Cryst. D Biol Cryst. 63: 550 54).

Data sets were collected at 100 K on a 104 unit, Diamond Light Source, Didcot, UK (λ=0.9795 A) with an Eiger2 XE 16M detector. Data were processed using the autoPROC program (Kabash, (2010) Acta. Cryst. D. Biol. Cyrst. 66: 125–32; Vonrhein et al. (2011) Acta Cryst. Biol. Cryst. 67: 292–302) and anisotropically truncated using STARANISO software (Tickle et al, STRANISO. Cambridge, United Kingdom: Global Phasing Ltd. (2018)), also incorporated into the Aimless program (Evans et al. (2013) Acta Cryst. Biol. Cryst. 69: 1204–14).

The structure was solved using the molecular replacement software Phaser (McCoy et al. (2007) J. Appl. Cyrst. 40: 658–74) and Molrep (Vagin et al. (1997) J. Appl. Cyrst. 30: 102–225) (FIG. 9). As shown in Fig. 9, the Fab of the anti-IL-27 antibody Abi is bound to the p28 molecule of IL-27. The electron density for the entire epitope–paratope region is well defined. The interactions between the anti-IL-27 antibody Abi and p28 are shown in Table 15.

Table 15

Interaction map of anti-IL-27 antibody Abi with p28 (< 4.0 A)

IL-27 (p28) Fab antibodies Abi against IL-27 Distance (A) Type Remainder Atom Remainder Atom Loop CDR Gln37 o ^eI Ser32 N LI 3.0 11-connection c ^b Phe31 c ^dl LI 3.6 Hydrophobic Leu38 c® Phe31 c ^z LI 4.1 Hydrophobic Glu42 o ^el Ser99 o ^g L3 2.7 H-bond Glu46 o ^e2 Tyr59 o ^h H2 2.7 H-bond Val49 c ^sl Tyr57 c ^dl H2 3.5 Hydrophobic Ser50 o® Tyr57 o'* H2 2.7 H-bond Leul42 from ⁶² Ser54 c ^a H2 3.8 Hydrophobic Aspl46 _o dU Ser53 o ^g H2 3.7 H-bond o ^d2 Ser54 o ⁶ H2 2.7 H-bond o ^d2 Ser56 o ^g H2 4.0 11-connection Argl49 No. Thrl03 O 113 3.1 H-bond No. Thrl06 O H3 2.9 H-bond NH ² GlylOl O H3 3.0 H-bond Hisl50 No. ² Thrl08 o ^gI H3 2.7 H-bond

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N ^dl , c ^el Tyr57 c ^e2 , c ^d2 H2 3.5-3.7 R-stacking Phel53 c ^z HisllO N ^dl H3 3.6 Hydrophobic c ^d2 Ala 107 c ^b H3 3.7 Hydrophobic c ^el Tyr38 c ^e2 LI 3.8 Hydrophobic Leul56 O Ser33 o ⁸ LI 2.6 H-bond Tyrl05 C ^dl H3 3.9 Hydrophobic c ^dl Ser33 c ^b LI 3.9 Hydrophobic Leul62 O Tyrl05 o ^h H3 2.8 H-bond Glul64 o ^el Thrl03 o ⁸ H3 2.6 H-bond o ^e2 Lys36 N ^z LI 3.9 Ionic

Example 12. Further epitope mapping studies.

Further epitope mapping studies were performed using the crystal structure of the IL-27 - Fab complex of the anti-IL-27 Ab1 antibody. The interaction regions between the Fab molecule of the anti-IL-27 Ab1 antibody and IL-27 were investigated using the Qt-PISA and NCONT programs in the CCP4 software package (Winn, et al. (2011) Acta Cryst. D Biol. Cryst. 67: 235 - 42). The Coot program (Emsley, et al. (2010) Acta Cryst. D Biol. Cyrst. 66: 486 - 501) was used for data analysis.

Epitope residues were defined as IL-27 amino acids within 4 A of the Fab, based on NCONT estimation in CCP4. In addition to the previously identified residues listed in Table 15, additional epitope residues were identified in these studies. All interactions between IL-27p28 and the Fab of the anti-IL-27 Ab1 antibody within 4 A are presented in Table 16A below.

Table 16A

CDR Fab antibodies Abi against IL-27 IL-27 residue (p28) LC-CDR1 Leu 30 Gin 37 Phe 31 Gin 37, Leu 38, Phe 153, Ala 157 Ser 32 Gin 37 Ser 33 Gin 37, Leu 156, Gly 159, Phe 160, Asn 161 Asn 34 Leu 156 Lys 36 Glu 164 Tyr38 Phe 153 LC-CDR3 Ala 98 Leu 38 Ser 99 Leu 38, Glu 42 Ala 100 Glu 42 HC-CDR1 Ser 31 Arg 145* HC-CDR2 Ser 52 Asp 146, His 150

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Ser 53 Leu 142, Asp 146 Ser 54 Leu 142, Asp 143, Asp 146 Ser 56 Leu 53, Lys 56, Asp 146 Tug 57 Vai 49, Ser 50, Leu 53, Asp 146, Leu 147, His 150 Tug 59 Glu 46 Asn 74 Leu 142** HC-CDR3 Gly 101 Arg 149 Arg 102 Arg 149 Thr103 Arg 149, Glu 164 Ser 104 Arg 149, Arg 152, Phe 153, Leu 156 Tyr 105 Phe 153, Leu 156, Leu 162, Pro 163*, Glu 164 Thr106 Arg 149, Phe 153 Ala 107 Arg 149, His 150, Phe 153 Thr108 His 150 His 110 Glu 42, Glu 46, Phe 153 Asn 111 Arg 149

Binding and blocking studies were performed using PIR for both WSX-1 and gpl30 for the human IL-27 heterodimer. Human IL-27 bound to WSX-1 with high affinity, and anti-IL-27 antibody AA was able to completely inhibit binding (Fig. 10A). Human IL-27 bound to gpl30 with lower affinity, and anti-IL-27 antibody AA did not inhibit IL-27 binding to gpl30 (Fig. 10B).

The IL-27 antibody AB interacts with the aA and aC helices and the initial portion of the poly-Glu sequence (Fig. 11). CDRs 2 and 3 of the heavy chain have the longest contacts with p28 (Table 16B).

Table 16B

Contacts between IL27-p28 and the anti-IL-27 antibody AN

IL27 (p28) Fab antibodies against IL-27 Distance (A) Type Remainder Atom Remainder Atom Loop CDR Gln37 o ^el Ser32 O ^e LI 3.0 H-bond c ^b Phe31 c ^dl LI 3.6 Hydrophobic Leu38 c ^s Phe31 c ^z LI 3.8 Hydrophobic Glu42 o ^eI Ser99 o ^s L3 2.8 H-bond Glu46 o ^eI Tyr59 o ^h H2 2.8 H-bond Val49 c ^sl Tyr57 H2 3.6 Hydrophobic Ser50 o ^g Tyr57 o ^h H2 2.7 H-bond Leul42 c ^d2 Ser54 c ^a 112 3.9 Hydrophobic

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Aspl46 o ^dl Ser53 O ^s H2 2.7 H-bond Ser54 O ⁸ H2 2.7 H-bond Ser56 o ⁸ H2 2.9 H-bond Argl49 No. Thrl03 O H3 2.8 H-bond No. Thrl06 0 FROM 2.8 11-connection NH ² GlylOl 0 FROM 3.0 H-bond His 150 N ^e2 Thrl08 about ⁸¹ FROM 2.8 H-bond N ^dl , c ^el Tyr57 c ^eI , c ^dI H2 3.5-3.6 R-staking Phel53 from ⁷ HisllO FROM 3.8 Hydrophobic c ^d2 Ala 107 c ^b FROM 3.6 Hydrophobic c ^el Tyr38 c ^e2 L1 3.7 Hydrophobic Leul56 O Ser33 o ^s L1 2.7 H-bond c ^dl Tyrl05 c ^dl FROM 3.7 Hydrophobic c ^d2 Ser33 c ^b L1 3.8 Hydrophobic Leu 162 O Tyr105 o ^h FROM 3.1 H-bond Glul64 o ^el Thrl03 o ^s from 2.7 H-bond o ^e2 Lys36 N ^z L1 3.9 Ionic

Figure 12 shows a superposition of IL-27/anti-IL-27 antibody and IL-23/IL-23β complexes using p28 and IL-6 for three-dimensional alignment. The gpl30 binding site on IL-6 overlaps with the anti-IL-27 antibody binding site on p28. However, the IL-23β binding site on p19 does not overlap with the anti-IL-27 antibody binding site on p28.

Figure 13 shows a superposition of IL-27/anti-IL-27 antibody and IL-6/IL-6Pa/gp130 complexes using p28 and IL-6 for three-dimensional alignment. In this case, the gp130 binding site on IL-6 overlaps with the anti-IL-27 antibody binding site on p28. Additionally, IL-6Pa is aligned with EBI-3.

Sequence alignments from different animals show that the p28 residues involved in specific interactions with EBI3 are highly conserved, and the same is true for the EBI3 residues involved in specific interactions with p28 (Figs. 14A–15B). This includes several conserved salt-bridge amino acid residues and several conserved hydrophobic amino acid residues, indicated by arrows.

Structural alignment of the IL-27 heterodimer with IL-6/IL-6RA shows that the secondary structure, domains, and alpha-carbon backbone are well matched between both heterodimers (Fig. 16A, 16D), and several p28 interactions with EBI3 are potentially conserved between IL-6RA (Fig. 16D).

Binding affinity data for human IL-27 show that p28 had weak or no binding to either gpl30 or WSX-1 individually (Fig. 17). EBI3 did not bind gpl30 alone but had moderate affinity for WSX-1. High-affinity binding to human IL-27 was observed only in the presence of the assembled heterodimer. The affinity of EBI3 for p28 was 5 nM.

The amino acids in human p28 that interact with the anti-IL-27 antibody AN are largely conserved in the mouse sequence (Fig. 18A); however, the anti-IL-27 antibody interacts with both Gln37 and Leul62 for human p28, and Gln37 is absent in the mouse sequence, while Leul62 corresponds to Cys in the mouse sequence (Fig. 18B). Additional disulfide bonds in mouse p28 may also disrupt the local structural epitope at which the anti-IL-27 antibody AN binds.

There was also an unresolved CD loop with a poly-Glu sequence, including a large region of positive charges from the Arg residues in the aC helix (Fig. 19A - 19B).

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Example 13. Targeting IL-27 expression in renal cell carcinoma.

IL-27 expression is elevated in renal cell carcinoma (RCC), with elevated levels of EBI3, IL-27p28, and IL-27RA in RCC tumor tissue compared to the expression of each in normal kidney tissue (Fig. 20A). High expression of each of EBI3 (Fig. 20B), IL-27RA (Fig. 20C), and IL-27p28 (Fig. 20D) correlated with reduced survival in human subjects compared to low expression of each of these transcripts.

IL-27 induces a reproducible gene expression profile in activated human CD4+ T cells. Peripheral blood mononuclear cells (PBMCs) from individual donors were activated with anti-CD3 antibody ± recombinant human IL-27 (rhIL-27) for 3 days. CD4+ T cells were sorted by FACS, and gene expression was analyzed by microarray. T cells activated with CD3 and rhIL-27 showed upregulation or downregulation of numerous genes, including increased expression of PDCD1, HAVCR2, CD274, LGALS9, GBP5, LAMP3, RGS1, IL12RB2, RSAD2, IFIT3, and IFI44L, while GZMA and CD200 were downregulated (Fig. 21A). The 31 top genes in the IL-27 expression profile in CD4+ T cells are listed in Fig. 21B. Notably, 15 of the 31 genes were associated with adverse outcomes, namely, AIM2, ALPK1, APOL1, GBP5, IFI44, IRF1, LAMP3, LOC400696, PARP3, RGS1, SAMD9L, SOCS1, STAT1, TNFSF13B, and XAF1. Twelve of the top genes in the expression profile (including STAT1, GBP5, IFI44, XAF1, and SOCS1) were associated with adverse outcomes in RCC (Fig. 22A), but these same genes were not associated with adverse outcomes in breast cancer (BC) (Fig. 22B).

Furthermore, plasma EBI3 levels in patients with RCC may predict disease outcome. Serum samples were collected from patients with RCC during kidney removal surgery, and EBI3 levels were measured using a pair of EBI3-specific antibodies. Mean EBI3 levels were elevated in the sera of patients with RCC compared with those of healthy donors (Fig. 23A). Serum from pregnant donors was used as a positive control. EBI3 levels were highest in subjects with stage 4 RCC compared with stage 2 or stage 3 (Fig. 23B); overall survival (Fig. 23C) and disease-free survival (Fig. 23D) were higher in RCC patients with low serum EBI3 levels.

To test the efficacy of anti-IL-27 Ab1 treatment in vivo in a mouse RCC model, Renca cells were orthotopically implanted into the left kidney. Three days after implantation, mice were injected intraperitoneally with anti-IL-27 Ab1 or human IgG1 isotype control (50 mg/kg twice weekly) for 2 weeks. After 21 days, tissues were harvested, both kidneys were weighed to calculate net tumor weight, and lung metastases were visually counted. Although mean tumor weight remained largely constant (Fig. 24A), lung metastasis was significantly reduced in mice receiving anti-IL-27 Ab1 compared to mice receiving the isotype control (Fig. 24B).

These data demonstrate that elevated levels of IL-27p28, EBI3, and IL-27RA transcripts in tumors from patients with RCC are associated with an unfavorable prognosis. Anti-IL-27 Ab1 demonstrates monoactivity in an orthotopic RCC model in vivo, and IL-27 blockade with anti-IL-27 Ab1 represents a promising strategy for patients with RCC who have high circulating levels of EBI3.

Example 14. In vivo targeting of IL-27 using anti-IL-27 antibody Ab1 in a mouse model of orthotopic HCC Hepa1-6.

To study the in vivo efficacy of anti-IL-27 Ab1 in a liver cancer model, Hepa1-6-Luc tumor cells were injected into mouse livers, and on days 5, 8, 12, and 15 post-implantation, the animals were administered anti-IL-27 Ab1 at a dose of 50 mg/kg by i.p. injection (Fig. 25A). Total photon flux was reduced to near baseline in mice receiving anti-IL-27 Ab1 compared to mice receiving the isotype control hIgG1 (Fig. 25B).

The response to anti-IL-27 Ab1 in this mouse model was dose-dependent. Mice were treated with isotype control, 5 mg/kg anti-IL-27 Ab1, 25 mg/kg anti-IL-27 Ab1, or 50 mg/kg anti-IL-27 Ab1 on days 5, 8, 12, and 15 after implantation (Fig. 26A). Tumor growth was slowest, close to baseline, in mice treated with 25 or 50 mg/kg anti-IL-27 Ab1 (Figs. 26B–26F).

To determine whether the previously identified preclinical changes in gene expression induced by anti-IL-27 Ab1 are relevant in this model, the expression of a number of biomarker genes was analyzed following administration of anti-IL-27 Ab1 (Figs. 27A–27C). The top 200 genes with the greatest repression are shown in Table 17A, and the top 200 genes with the greatest induction are shown in Table 17B. The complete lists of up- and down-regulated genes are presented above in Tables 11A–11B.

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Table 17A

200 genes in the liver of Hepal-6 mice with the greatest repression after administration of the anti-IL-27 antibody AN

Gene Frequency of change meaning Gene Frequency of change meaning DEFB44-PS 0.18731 0.16963 GM7391 0.38252 0.02063 C1S2 0.18735 0.00760 GM7391 0.38252 0.02063 HIST1H1B 0.20429 0.13468 GM7391 0.38252 0.02063 GM15114 0.20467 0.11272 GM7391 0.38252 0.02063 G M2005 0.20501 0.01692 GM7391 0.38252 0.02063 KHDC1C 0.21082 0.08196 GM7391 0.38252 0.02063 GM2005 0.22434 0.00235 CD109 0.38422 0.08923 SERPINB7 0.22443 0.13886 TMEM173 0.38467 0.09117 SERP1NB11 0.22512 0.05938 CD1117 0.38479 0.03381

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CXCL3 0.22982 0.15165 KIEL 0.38506 0.02871 CDH10 0.23008 0.04656 SKA3 0.38794 0.19907 M1R1949 0.23543 0.13071 PRC1 0.38977 0.07960 SLC7A11 0.23650 0.01951 MBOAT1 0.39032 0.03388 LUZP4 0.24896 0.09520 TOP2A 0.39357 0.06590 IGHV1-81 0.25160 0.51163 1700049E17RIK2 0.39548 0.00842 PTPRTOS 0.25229 0.03379 SPEER4D 0.39557 0.01189 RP1 0.25355 0.15378 HTST1H3I 0.39572 0.38176 GM15127 0.25399 0.05002 TUBA 1A 0.39616 0.01385 CRISP 1 0.25472 0.08655 GM22265 0.39624 0.09085 GM15107 0.25489 0.05910 TRBJ1-7 0.39684 0.15669 SERPINB5 0.25593 0.04019 KTF20B 0.39753 0.09225 GM7665 0.25600 0.00338 SERP1NB2 0.39766 0.02587 TMEM252 0.25889 0.01729 GM14402 0.39768 0.24963 RNML5 0.26178 0.09220 PLK4 0.39883 0.07277 CXCL15 0.26260 0.10373 C330027C09RIK 0.40017 0.10461 CLEC2G 0.26543 0.03883 GM15091 0.40058 0.07631 MMP13 0.26736 0.16866 NDC80 0.40091 0.07729 GM 15093 0.26907 0.07413 MYBL1 0.40096 0.16592 VMN1R53 0.27031 0.06485 K1F2C 0.40131 0.07030 GM 14409 0.27619 0.15580 GM12603 0.40134 0.08558 GM11884 0.27767 0.16819 CYP11A1 0.40271 0.26346 SLC16A4 0.27777 0.11590 FANCI 0.40316 0.17817 MMP12 0.27973 0.07864 CCNA2 0.40376 0.05984 KIF5C 0.28118 0.10032 RACGAP1 0.40619 0.07336 APELA 0.28255 0.06005 NCAPII 0.40648 0.11248 AFP (ΛΦΠ) 0.28256 0.04236 GM16094 0.40657 0.03168 IGKV1-122 0.28592 0.00900 C11251I 0.40668 0.30753 A630095E13RIK 0.29162 0.09055 GM15398 0.40844 0.10428 GM15109 0.29701 0.00582 KNTC1 0.40858 0.13345 OLFR1U 0.29889 0.21068 BST1 0.40915 0.09282 TNS4 0.30067 0.03812 K1F11 0.41151 0.07520 PLEKHS1 0.30148 0.07842 GM23576 0.41226 0.26181 LNCENC1 0.30470 0.09388 SHCBP1 0.41247 0.13607 THBS1 0.30908 0.02429 1GHV1-42 0.41274 0.44744 PLATR14 0.31317 0.08123 GPRC5A 0.41537 0.03331 RASSF9 0.31383 0.11251 TNFRSF10B 0.41617 0.10473 ITGA2 0.31895 0.06993 IL23A 0.41843 0.14139 LOC102634388 0.32002 0.02127 ER1CH2 0.41910 0.14436 LOC102634388 0.32002 0.02127 ANLN 0.41967 0.04088 GM 15093 0.32036 0.03800 CASC5 0.41996 0.11775

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CEP55 0.32092 0.10073 GM17689 0.42053 0.27734 A630038E17RIK 0.32118 0.13340 SOX4 0.42211 0.07946 GM25552 0.32317 0.12614 GM22069 0.42215 0.04943 RPS26 0.32662 0.18403 PRR11 0.42277 0.07500 STRA6 0.33040 0.04984 SEMA3C 0.42285 0.02076 GM 15093 0.33144 0.04030 RETNLA 0.42307 0.04289 PSAT1 0.33163 0.09265 FRMD7 0.42356 0.02106 AKR1C18 0.33224 0.09827 TNFRSF11B 0.42500 0.05920 DEPDC1A 0.33251 0.18136 RAD54L 0.42591 0.16039 GM 1043 9 0.33495 0.06941 GM10488 0.42650 0.00744 GM24916 0.33509 0.20312 PLAT 0.42677 0.06470 STC1 0.33666 0.08291 GM13790 0.42685 0.11605 DPPA2 0.33711 0.08874 DTL 0.42697 0.16996 E030011O05RIK 0.33714 0.04863 SERPINB9B 0.42886 0.10643 GM20756 0.33749 0.02179 ASNS 0.42974 0.04714 GM22771 0.33862 0.28971 GM15097 0.42977 0.17217 1GKV6-32 0.33990 0.44350 TMED6 0.43183 0.26485 A1M2 0.34040 0.00912 SERP1NE1 0.43312 0.03450 C920009B18RIK 0.34064 0.02201 TPX2 0.43325 0.05879 BUB1 0.34878 0.07548 CENPK 0.43347 0.17724 TICRR 0.34960 0.17718 2810429I04RIK 0.43385 0.21706 M1S18BP1 0.35250 0.09094 4930461G14RIK 0.43432 0.17990 MAGEA6 0.35303 0.20666 HIST1H2AG 0.43516 0.18735 C1I1L3 0.35389 0.06510 1ER3 0.43524 0.14140 IGHV1-78 0.35720 0.28499 CHRNB1 0.43554 0.04999 ST1L 0.35848 0.14130 GM5431 0.43690 0.13001 GM26735 0.36001 0.06537 GM13247 0.43774 0.20349 REG 2 0.36041 0.49690 A1506816 0.43795 0.07730 SPRR1A 0.36134 0.00562 GM7942 0.43806 0.04319 PARPBP 0.36204 0.14708 CCNB1 0.44021 0.03617 PADI4 0.36592 0.09746 ZFP345 0.44040 0.24896 GM14139 0.36657 0.04225 1GI1V8-8 0.44047 0.45075 GPC3 0.36787 0.21392 ATAD5 0.44240 0.11428 MS4A6D 0.37021 0.01764 KDELR3 0.44372 0.08725 ATP10A 0.37100 0.09774 CDC25C 0.44462 0.03533 KIF23 0.37107 0.04316 IGKV4-72 0.44464 0.18026 GM20757 0.37125 0.22483 OLFR99 0.44744 0.16218 GM2318 0.37176 0.01361 GM25544 0.44759 0.13854 GM2318 0.37176 0.01361 M1R101C 0.44785 0.08179 GM2318 0.37176 0.01361 HYD1N 0.44832 0.14992 GM2318 0.37176 0.01361 1GHV5-16 0.44859 0.32098 IGHV1-9 0.37183 0.35845 NOP58 0.44866 0.13787 CPB1 0.37389 0.54454 CEL 0.44913 0.50615 TNFSF4 0.37410 0.14201 MS4A4A 0.44926 0.11256 RNASE 1 0.37521 0.29355 BCL2A1B 0.44968 0.00346 TIMP1 0.37633 0.07652 IGKV4-59 0.44969 0.49749 GM6020 0.37895 0.48362 CCL3 0.45007 0.10701 1700049E17RIK2 0.37958 0.00452 GM2933 0.45077 0.16505 IGH-VJ558 0.38192 0.43479 FAM102B 0.45149 0.13304 PRRG4 0.38250 0.10001

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Table 17B

200 genes in the liver of Hepa1-6 mice with the greatest induction after administration of the Ab1 antibody against IL-27

Gene Frequency of change meaning Gene Frequency of change meaning MUP-PS12 6.33152 0.00215 OLFR724 2,07902 0.09250 GM26184 5.61658 0.08504 GM23277 2,07764 0.09120 GKN2 4.82174 0.03776 A4GNT 2,07584 0.24306 MYH8 3.85386 0.31903 MUP21 2,07390 0.11490 MIR101B 3.69523 0.01108 GM24147 2,07383 0.26694 GKN1 3.48462 0.04511 MYBPC1 2,06732 0.13917 SLN 3.47316 0.18221 CYP3A59 2.06303 0.26277 MUC5AC 3.34061 0.06276 ACTN2 2.06253 0.14714 2310057J18RIK 3.04114 0.41002 MIR29A 2,06200 0.03405 PSCA 3,02691 0.12186 TFF1 2,06146 0.08032 GM25623 2.94838 0.01329 PLA2G1B 2,05956 0.44772 FAM83B 2,90452 0.02016 GM23021 2,05954 0.11112 GM23852 2.88192 0.07627 GM22607 2,04477 0.13834 SPTSSB 2.87259 0.09524 RPL10L 2,04430 0.06920 MIRLET7F-1 2.79416 0.04469 ACE2 2,03841 0.35049 MUP-PS16 2.77768 0.01138 PRAMEL3 2,03798 0.14902 ANXA10 2.68901 0.10472 SULT1B1 2.03237 0.24951 LGALS2 2.68519 0.13369 ARL14 2.02633 0.09522 CHIA1 2.67246 0.40779 GM11337 2.02394 0.02702 AKRZ 2,66042 0.36655 CNN1 2.02048 0.02173 SLC13A1 2.62180 0.29356 LRIT2 2.01905 0.05334 GM766 2.62086 0.40345 ASOT4 2.01479 0.21202 GM24537 2.61729 0.16494 LOC100125594 2.01433 0.02079 GM24138 2.60556 0.03739 GLB1L2 2.01156 0.03963 SIS 2.58535 0.37774 GM25605 2,00818 0.13287 2210407C18RIK 2.58374 0.06005 GM15384 2.00171 0.02548 S100G 2,58040 0.38773 GM94 1.99674 0.10946

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GM26354

2,54047

0.10968

OLFR1113

1.99669

0.12774

CKMT2 2.53798 0.26801 G630018N14RIK 1.99666 0.30168 GM26055 2.52487 0.21572 GM23232 1.99430 0.06721 GM5885 2.47858 0.03053 GM16378 1.99415 0.03571 AGR2 2.45816 0.18450 GM22838 1.99362 0.08752 VMN1R167 2.45428 0.09317 HSD3B5 1.99243 0.20188 GM25167 2.44373 0.02641 OLFR746 1.98892 0.03312 ATP5C1-PS 2.43743 0.04767 GM24254 1.98755 0.13006 GM24470 2.43383 0.16763 GM24232 1.98629 0.22120 MYH2 2.40309 0.16324 PRAP1 1.98616 0.37196 MIR326 2.37544 0.00590 ALPI 1.98615 0.41922 MIR122 2.37543 0.02420 MIR3964 1.98298 0.03007 MCPT9 2.37520 0.06227 GM22787 1.98276 0.01829 PSG18 2.35852 0.01151 GM26081 1.98214 0.17707 MUC6 2.35850 0.31084 GM22242 1.98012 0.31686 VMN2R115 2.35187 0.11114 IGHV1-77 1.97991 0.47423 GM25498 2.35108 0.00863 OLFR740 1.97966 0.21907 2010106E10RIK 2.33919 0.46065 OLFR1201 1.97856 0.03392 ERBB4 2.32759 0.03502 GM22654 1.97558 0.51008 LYPD8 2.31999 0.13737 OLFR747 1.97523 0.07186 CYP2A5 2,30476 0.23296 GM22521 1,97342 0.10732 BTNL5-PS 2.30131 0.16835 VMN1R172 1.97211 0.07313 GM24549 2,28941 0.11379 GM13773 1.97144 0.21792 GM24465 2.28686 0.23556 CLEC2H 1,97034 0.20412 SLC26A3 2,28613 0.34866 OLFR714 1,96697 0.08163 DPCR1 2.28459 0.06528 GM22284 1,96490 0.13765 GM 11844 2.27856 0.10541 GM26342 1.96171 0.02416 CYP4A32 2.26879 0.21331 OLFR190 1.96140 0.01845 MIR1928 2.26702 0.01338 CCDC152 1,96048 0.03753 GKN3 2,26609 0.38398 GM26048 1,95935 0.18068 KRT4 2.26351 0.15392 GM24410 1.95853 0.18441 PGC 2.26227 0.25834 LGALS4 1.95650 0.02146 HIST1H2BA 2,26012 0.03903 OLFR969 1,95620 0.09460 SPRR2A3 2.25406 0.19740 GM6222 1,95616 0.09501 IGKV12-41 2.23462 0.35478 OLFR913 1,95340 0.31106 PARD3BOS3 2.22662 0.11494 4930557A04RIK 1,95314 0.06455 CLCA4B 2.22470 0.41766 GM24750 1,95027 0.23848 GM23744 2.20755 0.12172 GM23043 1.94750 0.04897 VMN2R-PS129 2,20013 0.00212 STFA1 1.94574 0.08031 GM23241 2,19443 0.16439 SUCNR1 1.94177 0.10776 MYOZ2 2,19124 0.20907 MYOM1 1.94136 0.17534

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MIR192 2,19083 0.05530 KLK1B22 1,93841 0.12634 MYH1 2.18765 0.16532 GM23917 1.93358 0.19490 5033403H07RIK 2,18709 0.12308 GM15949 1.93187 0.04498 MIRLET7F-2 2,18612 0.16167 OLFR709-PS1 1,93037 0.07218 GM25009 2,18474 0.12429 OBP1A 1.92773 0.34212 MUC13 2,18238 0.44121 SULT2A1 1.92697 0.35277 PPP1R3A 2,17804 0.19680 GM24786 1,92300 0.21032 OLFR798 2.17172 0.00360 SGK2 1.91561 0.15674 SERPINA12 2,15429 0.03094 MIRLET7C-1 1,91086 0.07251 GM25864 2.15155 0.04041 VMN1R90 1,90960 0.10180 N-R5S155 2,15090 0.06906 GM23342 1,90947 0.02984 CYP2C65 2.14174 0.14376 REG3G 1,90771 0.15987 GM14750 2.14105 0.05245 GM24839 1,90586 0.26313 GM23026 2,13427 0.08632 N-R5S157 1,90509 0.11667 HAMP2 2,12517 0.26750 GM25023 1,90492 0.02529 GM24527 2,12509 0.07768 CES2B 1.90228 0.13857 OLFR891 2.12488 0.07314 IGHV8-11 1,90067 0.15551 OLFR68 2,12477 0.17461 GM25982 1.89862 0.05418 TRAV6-2 2.12335 0.16838 ATP4A 1.89851 0.39680 CYP4F40 2.11940 0.18482 GM26162 1.89809 0.01472 TRDN 2.11902 0.23956 PNPLA3 1.89778 0.00313 CFTR 2.11685 0.32080 AU015336 1.89289 0.07944 XIRP2 2.10948 0.20311 GM24621 1.89139 0.08404 CYP8B1 2,10691 0.09121 CYP2C40 1.89106 0.40000 GM23629 2,10532 0.05899 GM25602 1.89074 0.15275 GM25076 2.10385 0.09801 ADGRG7 1.88894 0.32051 1700080G11RIK 2.10309 0.16206 SLC30A10 1.88809 0.12880 N-R5S96 2.09968 0.04925 GM25836 1.88784 0.19345 GM 11027 2,09669 0.30087 GM25629 1.88723 0.11919 SLC22A29 2,08861 0.26634 GM24861 1.88583 0.03961 GSTT2 2,08187 0.23716 GM21057 1,88436 0.08496 GM23911 2.08111 0.19027

Notably, anti-IL-27 Ab1 was found to downregulate several key inhibitory genes, including PD-L1, TIGIT, and AFP expression (Figs. 28B–28D), with no significant changes in the expression of EBI3, IL-27, and IL-27RA (Fig. 28A). The TGF-β pathway was also found to be downregulated after anti-IL-27 Ab1 administration, with reduced expression of TNFRSF10B, TNFRSF1A, and PDGFA (Fig. 28C and Fig. 28E).

Furthermore, anti-IL-27 Ab1 treatment alters tumor cell immune infiltration. Anti-IL-27 Ab1 promotes upregulation of macrophage and NK cell transcript levels in the tumor microenvironment (TME; Fig. 29A). Specifically, anti-IL-27 Ab1 upregulates CD206 and CD163 (Fig. 29B), which are key macrophage-associated markers; and anti-IL-27 Ab1 was found to modulate specific NK-associated receptors (Fig. 3O). The heterogeneity of NK marker modulation suggests that anti-IL-27 Ab1 affects NK function and not just infiltration in HEPA1-6 tumors. In addition, various other cell surface markers showed modulated expression after treatment with anti-IL-27 Ab1 (Fig. 31).

Example 15. TNFSF15 as a biomarker of IL-27 inhibition.

TNFSF15 (soluble tumor necrosis factor 15), also known as TL1A (TNF-like ligand 1A) or VEGFI (vascular endothelial growth factor inhibitor), is a cytokine of the tumor necrosis factor family that is known to play a role in inhibiting angiogenesis (ref: VEGI, a novel cytokine of the tumor necrosis factor family, is an angiogenesis inhibitor that suppresses the growth of colon carcinomas in vivo. FASEB J. 1999 Human Genome Sciences, Inc.) and can act as a T cell costimulator by inducing the production of inflammatory cytokines (see Migone et al., Immunity 16 (3): 479–92 (March 2002); Jin et al., Mucosal Immunology 6: 886–99 (December

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19, 2012). TNFSF15 has been shown to be upregulated following IL-27 blockade following treatment with an IL-27 inhibitor, supporting its utility as a biomarker in assessing the efficacy of IL-27 inhibition following administration of a therapeutic agent designed to inhibit IL-27.

IL-27 inhibited the production of the cytokines IL-17A, IFN-γ (or IFN-gamma), and TNF-α (or TNF-alpha) in activated PBMCs. Pooled human PBMCs from 3 donors were stimulated with anti-CD3 antibody (0.25 μg/ml) for 3 days in the presence or absence of IL-27 (100 ng/ml). Supernatants were collected and analyzed for the effect of IL-27 on IL-17A, IFN-γ, TNF-α, and IL-10 by bead cytometry; IL-27 reduced the production of IL-17A, IFN-γ, and TNF-α and had no effect on IL10 production (Fig. 32A–32D).

Conversely, blocking IL-27 with anti-IL-27 Ab1 in activated PBMCs resulted in increased cytokine production. PBMCs from 3–4 individual donors were stimulated with anti-CD3 (0.25 μg/ml) for 4 days in the presence or absence of anti-IL-27 Ab1 (1 μg/ml). Supernatants were collected and analyzed for effects on IL-17A, IFN-γ, TNF-α, and IL-10 by bead cytometry; anti-IL-27 Ab1 resulted in increased IL-17A, IFN-γ, and TNF-α production (Fig. 33A–33D).

To identify other changes in gene expression that could be monitored as pharmacodynamic or biomarker indicators of anti-IL-27 Ab1 activity, gene expression profiling was performed by microarray analysis of activated PBMCs. PBMCs from three separate donors were stimulated with anti-CD3 antibody (0.25 μg/ml) in the presence or absence of anti-IL-27 Ab1 (1 μg/ml) for 24 h. RNA was isolated from each sample and processed for gene expression profiling by microarray analysis to determine the effect of IL-27 inhibition. Several genes were differentially expressed, as seen in scatter plots, following IL-27 inhibition by anti-IL-27 Ab1 compared to the isotype control, including MMP1, MMP10, and TNFSF15, as shown in Table 18. Fig. 34 is a scatter plot displaying the 1og2-fold change in gene expression after IL-27 inhibition compared to control (x-axis) versus the significance (p-value) of the gene expression changes after treatment with anti-IL-27 Ab1 compared to control (y-axis). TNFSF15 transcript levels were significantly increased after IL-27 blockade. TNFSF15 gene expression levels were reproducibly increased after treatment with anti-IL-27 Ab1 compared to isotype control in all 3 individual donors, as shown in Fig. 35.

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Table 18

Transcription profiles in PBMCs in vitro after IL-27 inhibition of the 200 genes with the highest induction caused by IL-27 inhibition

Gene designation Fold change: [antibody A1 against IL-27 / IgGl] p-value: IL-27 antibody α1 versus IgG1 LOC1053750 02 1.645118 0.000457 OR2AG1 1.633194 0.02143 SNORD115- 10 1.61366 0.042825 M1R548AK. 1.594121 0.047753 VTRNA2-1 1.539071 0.018135 LOC1053758 37 1.528625 0.042765 MIR4540 1.506249 0.014019 TNFSF15 1.48874 0.00196 LOC1053779 50 1.474449 0.017305 LOC1053752 91 1.455875 0.037183 LOC392232 1.442977 0.034733 IFNA6 1.435639 0.032471 MIG7 1.433659 0.011024 LOC1053694 73 1.428921 0.045657 GAGE1 1.427557 0.025117 LOC1005060 98 1.426037 0.008256 LOC1053776 43 1.416114 0.011246 LOC1053704 79 1.414996 0.016319 LOCI 019273 42 1.387702 0.001705

Gene designation Fold change [Antibody AI against IL-27 / IgGl] p-value of IL-27 antibody compared to IgGl MIR519B 0.542762 0.017631 MIR4272 0.633358 0.045813 MIR4712 0.662079 0.013235 MIR4738 0.666032 0.008113 MIR4668 0.676758 0.002044 MIR634 0.67895 0.010185 L1NCO1233 0.695199 0.03774 MIR19B2 0.706896 0.045961 LOC105378073 0.712528 0.010495 LOC105370770 0.716513 0.010176 KSSAT198 0.72041 0.040506 LOC101927552 0.721552 0.002026 MIR1302-1 0.72292 0.031298 ARON 0.729982 0.018512 OR56A3 0.731084 0.014148 SD1M1 0.73208 0.001115 LOC101928395 0.736856 0.03984 LCE1F 0.739268 0.025556 LOC100129476 0.740716 0.005034

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spoil 1.384138 0.019734 LOC1053741 46 1.372296 0.042536 MMP10 1.35563 0.035631 MIR513A1 1.349552 0.003188 LOC1053731 87 1.33789 0.011411 MIR302E 1.329896 0.049451 LOC1053747 15 1.328902 0.039526 CRYGC 1.326275 0.002843 NR0B1 1.322219 0.03641 LOC1053728 23 1.322197 0.008592 KRT74 1.321165 0.032883 LOC1053755 38 1.319595 0.019808 LOC1053782 20 1.319239 0.004044 SNORD116- 23 1.314349 0.035597 ROCK1P1 1.311881 0.009781 LOC1053757 34 1.310484 0.01621 MIR874 1.30894 0.00796 LOC1024672 16 1.306665 0.029327 MIR18B 1,305409 0.02179 LOC1053786 88 1.30501 0.005119 PIPSL 1.303637 0.013713 LOC1053744 84 1.302454 0.01914

C6orflO 0.741035 0.045023 DDX46 0.744975 0.031001 MIR544B 0.745497 0.000331 LOC105371152 0.745893 0.001107 HBZ 0.746633 0.032099 LINC00173 0.75377 0.046112 MIR95 0.754058 0.029894 LOC105369893 0.758578 0.042461 RNU6-58P 0.761545 0.022808 EPYC 0.763574 0.015155 WWC2-AS1 0.764054 2.67E-05 LOC105370359 0.766521 0.01068 MIR519A2 0.766808 0.045329 NSAP11 0.768519 0.020859 TDGF1 0.771131 0.010419 LOC646588 0.771593 0.022006 LOCI 01927281 0.773448 0.028332 LOC105374670 0.777386 0.028457 A2MP1 0.778456 0.001304 SLC7A11 0.779049 0.048778 PRH2 0.780025 0.029219 SNORD114-19 0.781894 0.040583

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LRRC46 1.299851 0.040688 SMG7-AS1 1.298713 0.039351 LOC1053787 49 1,295692 0.02812 CT55 1.295094 0.029095 MPDZ 1,288722 0.005299 H1ST1H4B 1,288471 0.02363 LOC1053792 23 1,287376 0.007854 POM121L4P 1.28655 0.006754 HIST1H2AH 1,285763 0.045671 OR6C74 1,284028 0.021357 KRT33A 1.28161 0.041615 PLCE1-AS1 1,280197 0.023448 ALX1 1.279203 0.042523 KCNN3 1,274928 0.01882 LOC1053771 24 1,274321 0.01119 BMP 15 1,272919 0.013839 RPLP0P2 1.271495 0.003828 LOC1053740 24 1.271237 0.040873 OR4C5 1,270368 0.008923 LOC1053762 34 1,269085 0.000332 LINC01591 1,268954 0.010567 LOC1019286 63 1,265242 0.003128 LOC1053758 90 1,264046 0.005132 IGHV3-16 1,2619 0.010831 TRIM55 1,260372 0.014182

LOC105373550 0.78246 0.003341 LOC101927413 0.785604 0.025946 MIR765 0.785901 0.027588 ALDH2 0.787067 0.03251 LOC105370607 0.787785 0.029023 ZNF717 0.788458 0.019536 MIR7-2 0.78937 0.024631 OR2Y1 0.791148 0.005362 FCGR1C 0.791398 0.036053 LOC102723879 0.792285 0.021851 LOC153910 0.793028 0.026202 M1R410 0.793302 0.007555 LOG 102725105 0.794407 0.019386 LOC105373461 0.798351 0.021561 LOC105373109 0.799694 0.035357 LOC105376899 0.8002 0.011314 LOC105375857 0.800606 0.049069 LOC105371883 0.802577 0.001618 M1R3121 0.804293 0.01562 KRTAP19-2 0.805488 0.007923 LOC105370057 0.805791 0.036764 LOC105375969 0.806373 0.002891 TMED3 0.807779 0.049844 LOC105371856 0.809355 0.000543 EYAI 0.809515 0.008405

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LOC1053710 18 1,259721 0.026344 LOC1053719 76 1,256944 0.030406 LOC1001293 45 1,256654 0.011358 LOC1053758 75 1.254562 0.047657 TM4SF1 1.253654 0.008913 LOC1053752 73 1.25346 0.035173 LOC1019282 79 1.252743 0.015819 MIR4493 1.251366 0.046562 LOC1053764 25 1,249945 0.029786 LOC1053762 43 1.248505 0.004647 OR4K17 1.24847 0.035361 LINC00865 1,248322 0.007719 CAV3 1,246213 0.024171 LOC1053698 30 1.244553 0.000338 RSI 1.244259 0.009886 LOC1019292 84 1,243628 0.009664 SPATAI 7- AS1 1,243442 0.036463 C9orfl29 1.241344 0.008005 TEX 12 1,239195 0.039282 OLIG2 1,235907 0.016016 SRGAP1 1.232365 0.0168

LOC105372147 0.810403 0.018231 SNORT) 115-20 0.81087 0.01113 LOC105378424 0.812013 0.025289 LOC105379104 0.812998 0.026458 LOC105378078 0.815137 0.043079 PCAT18 0.815152 0.012051 CRYBAI 0.815488 0.008079 DOCK4-AS1 0.816945 0.003652 PFN4 0.817389 0.017892 LOC105373456 0.817412 0.047871 LOC105376207 0.817593 0.03227 RUFY4 0.817735 0.047204 PGM5P2 0.820132 0.047774 FLJ20712 0.822198 0.042585 LOC105378741 0.822688 0.023776 KIR3DL2 0.822791 0.017144 IRS4 0.824149 0.020883 RNF175 0.824277 0.040132 MIR4473 0.824601 0.01576 C21orf62-ASl 0.825875 0.029134 TPI1P3 0.826368 0.005535

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LOC1053743 23 1.23136 0.006316 MIR1283-1 1,230642 0.005018 GJD4 1,229305 0.016043 LOC1053747 70 1,229204 0.023076 IGFL2 1,226273 0.0209 LOC1053769 44 1,225754 0.017134 ALG1L 1,224177 0.002154 PCDHB18P 1,223959 0.009905 RIMBP2 1,223519 0.039249 OR52B4 1.22324 0.024734 ADGRL2 1,223099 0.02921 SCG5 1,222368 0.013456 LOC1053765 70 1.221999 0.007463 LOC654841 1,221214 0.016472 HAS2-AS1 1,221198 0.047307 SPATA24 1,220361 0.009933 LINC01538 1.218128 0.007969 CCDC85C 1,217577 0.040344 LOC1053705 71 1,216124 0.013425 LOC1019274 99 1.214266 0.029007 TFPI2 1.213888 0.020451 TSSK3 1,212747 0.003353 LOC1053718 18 1.21189 0.040895 NPY1R 1,211641 0.033039 LOC1053765 30 1,211121 0.008511

OR6K2 0.82656 0.027951 LOC105375971 0.827067 0.028154 LOC105377841 0.827522 0.046396 LOC105370772 0.828585 0.019601 OR5H6 0.828925 0.033792 LOC400958 0.829718 0.027201 KCNAB1-AS2 0.830199 0.046563 ANXA8L1 0.830719 0.017418 LOC102724520 0.831113 0.019589 ALS2CR11 0.831379 0.005442 LINC01052 0.831661 0.038213 MIR492 0.8348 0.041657 LOC101928119 0.835502 0.004564 SYT4 0.836312 0.025009 LOC101928421 0.836388 0.04031 CASC23 0.838526 0.026392 LOC105375855 0.838859 0.027901 LINC00366 0.839834 0.009281 DEPDC7 0.841512 0.035067 LOCI 027243 30 0.842675 0.033285 PLA2G2D 0.843078 0.032033 LINC01166 0.844555 0.044378 RPE65 0.845494 0.005261 GLIS3 0.845755 0.047774 LINC01523 0.84612 0.021712

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EVX1-AS 1,208195 0.026851 NPAS3 1,207602 0.019453 OR51Q1 1,206806 0.037877 LOC1019274 18 1,206024 0.018913 SSTR2 1,20578 0.043155 LOC653653 1,205761 0.036703 LOC1053722 49 1,203904 0.010203 LOC1027236 39 1,203543 0.007388 LOC1053754 76 1,203038 0.015508 FKBP7 1,201988 0.042042 HRAT56 1,201942 0.020195 MIR4778 1,201939 0.017579 LOC1053781 80 1,201664 0.020417 SCARA3 1,200146 0.008278 LOC1019302 94 1,199177 0.022741 GUCY1A2 1,197934 0.000492 MGAM2 1,197312 0.042246 TEX 14 1,197177 0.037082 OR2L13 1,19687 0.022817 IGSF10 1,195837 0.02574 SFRP2 1,194806 0.008784 GALNT16 1,194743 0.022064 TM4SF19 1,194237 0.042563 LOC1019270 55 1,194215 0.029334 XIRP2-AS1 1,194173 0.045563 KCNJ16 1,194028 0.018938

LOC105374787 0.84724 0.001573 IGLV3-32 0.84882 0.010713 LOC105373595 0.849597 0.020159 LOC728040 0.849791 0.01671 LINC00597 0.849976 0.037685 MIR3591 0.850417 0.045077 KRTAP10-3 0.851393 0.03041 ZC3H12B 0.853207 0.026583 LOC105370690 0.853241 0.001048 ZIC3 0.853712 0.039829 FGF10-AS1 0.853729 0.035366 PMS2P5 0.854195 0.048446 SUGT1P1 0.854792 0.034214 LOC105369293 0.855263 0.044491 DEFBI19 0.85586 0.008256 LOC105369488 0.856419 0.018871 NRXN1 0.856769 0.047832 LVCAT8 0.856829 0.028297 KIZ-AS1 0.857975 0.033864 LOC105378283 0.858492 0.036091 MIR1290 0.858598 0.031938 GKAP1 0.858622 0.042287 LGR5 0.858793 0.01244 NUDT10 0.85898 0.015489 DSCAM-AS1 0.85983 0.003654 LOC105376640 0.86136 0.004118

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LOC1053705 61 1,194006 0.046886 ASIC2 1,193784 0.020987 LOC401191 1,19219 0.013408 INCAI 1,189251 0.007965 LOC1053761 61 1,188411 0.016143 ADAMTS3 1,188383 0.048302 LOC1019282 31 1,187861 0.031467 CMYA5 1,187321 0.024627 IGSF5 1,187268 0.046046 MIR3177 1,187017 0.041887 GPC4 1,186929 0.023815 OC90 1,186462 0.046711 ST8SIA6- AS1 1,185617 0.034159 SPDYE4 1,185393 0.007728 CHODL-AS1 1,185235 0.029759 USP17L17 1,184052 0.037856 LOC1053769 75 1,182382 0.011412 LOC1019271 09 1,180499 0.008781 CLCNKB 1.179758 0.033508 NR1I2 1.179102 0.020533 CASC17 1,179066 0.018346 FAM 19 A3 1,177994 0.002009 LOC1053755 86 1,177235 0.025561 SUMO1P3 1,176228 0.027566 FLJ44874 1,175661 0.037981 NACAP1 1,175123 0.02547

LOC105377693 0.862866 0.038576 SFTA3 0.863502 0.038347 CYYR1-AS1 0.864454 0.033992 C9orf3 0.864736 0.006764 LOC105373886 0.864772 0.012476 LOC105370060 0.865766 0.0341 CTBP2 0.866251 0.009633 LOC101927328 0.86679 0.033119 ZNF449 0.866794 0.036778 OΟΓΗ 0.86879 0.02991 MAP1LC3B2 0.869085 0.00058 ASPN 0.87022 0.049977 LOC105370018 0.873226 0.033161 LOC105376347 0.87418 0.033977 OR52D1 0.874677 0.037917 SFTA1P 0.87492 0.012883 LOC105376658 0.877883 0.038524 LINC00092 0.878605 0.03258 ERVK3-2 0.878835 0.001504 LOC105379385 0.878896 0.02955 ATP8B3 0.879183 0.00631 CTRB2 0.880249 0.025135 LINC01527 0.880518 0.040258 KRTAP19-7 0.880746 0.030687 LOC105376244 0.881151 0.036515 LOC105376554 0.881259 0.022378

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PTPN3 1,175009 0.028207 SSX2B 1,174369 0.003748 SSX2B 1,174369 0.003748 LOXHD1 1,173847 0.008937 LINC01104 1,172936 0.038443 SERPINI1 1,172725 0.00357 LOC1019283 87 1,172215 0.007858 CDH12 1,171839 0.00615 LPIN3 1.171832 0.027473 LOC1053695 75 1,171058 0.022101 SHC3 1,170409 0.014611 AIFM3 1,170074 0.001628 LOC1053705 64 1,16947 0.031193 KCNJ14 1,169446 0.03716 LOC1027233 23 1,16941 0.005804 LOC1019296 08 1,169147 0.005278 LINC01039 1,168287 0.028847 LOC1053749 80 1,168286 0.039283 GAL 1,167357 0.013476 MIR107 1,1655 0.014498 SCGB1A1 1,165175 0.007183 LOC1019272 77 1,164914 0.022585 LMOD3 1,164303 0.013973 MIR520G 1,163434 0.033228 LINC01618 1,162641 0.049271

ZNF30 0.8823 0.024368 LINC00894 0.88431 0.020883 MIR592 0.884366 0.023417 LOH12CR2 0.886427 0.036694 ZNF846 0.887147 0.019874 LINC01202 0.887201 0.036865 CACNG4 0.8877 0.031884 LOC642648 0.888364 0.019056 LOC105370526 0.888491 0.002669 TRIM45 0.889074 0.046329 AQP11 0.889428 0.005461 ANKEF1 0.889878 0.019532 PTGDR2 0.890525 0.042594 LOC100129869 0.891023 0.036875 RBM12B-AS1 0.892387 0.00632 LOC105378081 0.894442 0.029207 MIR365A 0.895999 0.04973 LINC00581 0.896004 0.048047 LYPD8 0.896025 0.042973 OR14I1 0.896295 0.007798 FAM217A 0.89866 0.019317 TMEM254-AS1 0.898681 0.049183 LARS2-AS1 0.898922 0.014049 CCDC150 0.899017 0.002703 GLI1 0.899604 0.00209

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LOC1019295 28 1.16255 0.024828 LOC1053757 10 1,162468 0.036178 LOC1027247 32 1,162206 0.015044 GEM 1.161606 0.005818 HLX-AS1 1,161435 0.03007 LOC441239 1,160728 0.018365 LOC1053738 23 1,157242 0.000638 TINAG 1,157194 0.039893 MFAP2 1,155841 0.026956 TUB-AS1 1,154582 0.031326

CD163L1 0.90077 0.019563 MAR2K2 0.901533 0.028712 DIRC3 0.901772 0.047553 IQCC 0.901863 0.009081 IRX6 0.901966 0.041501 TRIM63 0.903387 0.048605 SPATA18 0.90354 0.004953 LPEQ6126 0.903553 0.030286 RIPPLY3 0.905238 0.027728 ZNF192P1 0.905716 0.021822

In a subsequent experiment, pooled human PBMC samples were either stimulated with anti-CD3 antibody (0.25 μg/ml) for 24 h or left unstimulated in the presence of two different lots of anti-IL-27 Ab1 antibody (1 μg/ml) or an isotype control. RNA was collected, and the relative amount (RA) of TNFSF15 was determined by qPCR, measuring TNFSF15 transcripts using gene-specific Taqman probes. The results confirmed the increase in TNFSF15 expression following IL-27 inhibition and were found to be dependent on immune cell activation, as TNFSF15 expression was not increased following anti-IL-27 Ab1 treatment in resting PBMCs, as shown in Fig. 36A–36B.

The relative increase in TNFSF15 transcript levels was further enhanced by the addition of monocytes to PBMC cultures. Pooled human PBMC samples were either left quiescent (quiescent PBMCs), or monocytes isolated from PBMCs were added to PBMCs at a 1:2 ratio (quiescent PBMCs + monocytes), or monocytes were left quiescent (quiescent monocytes), or PBMCs were activated with anti-CD3 antibody (0.25 μg/ml) (activated PBMCs), or monocytes isolated from PBMCs were added to PBMCs at a 1:2 ratio and activated with anti-CD3 antibody (0.25 μg/ml) (activated PBMCs + monocytes). Cells were cultured for 24 h, then RNA was isolated to determine TNFSF15 levels by qPCR. The values shown in Fig. 37 represent the fold change in TNFSF15 transcript after IL-27 inhibition with anti-IL-27 Ab1 compared to the isotype control.

The effect of increasing TNFSF15 transcript levels was specific to IL-27 blockade and was not observed with other antibodies targeting CD39 or CD112R, as shown in Fig. 38. Here, pooled human PBMC samples were supplemented with monocyte-derived macrophages and stimulated with anti-CD3 antibody (0.25 μg/ml) for 24 h in the presence of control IgG1 antibody, anti-IL-27 antibody (anti-IL-27 Ab1 (1 μg/ml), anti-CD39 IgG4 antibody (1 μg/ml), anti-CD112R IgG1 antibody (1 μg/ml), or anti-CD112R IgG4 antibody (1 μg/ml). RNA was collected after 24 h; TNFSF15 transcript was measured in cell culture supernatants using the Human TL1A/TNFSF15 DuoSet ELISA kit (R & D Systems).

Furthermore, as shown in Fig. 39A and Fig. 39B, an increase in the secreted TNFSF15 protein level was also observed after blocking IL-27 with anti-IL-27 Ab1 in activated PBMCs, which showed delayed kinetics compared to the transcript (Figs. 39A-39B). Pooled human PBMC samples were supplemented with monocyte-derived macrophages and stimulated with anti-CD3 antibody (0.25 μg/ml) in the presence of control IgG1 or anti-IL-27 antibody (anti-IL-27 Ab1, 1 μg/ml). RNA was collected at days 1, 2, and 5, and the amount of TNFSF15 transcript (AT) was determined by qPCR at each time point. Culture supernatants were collected at different times; TNFSF15 protein was measured using sandwich ELISA.

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Claims (20)

CLAUSES OF THE INVENTION 1. An isolated antibody that exhibits antagonism against human IL-27, or an antigen-binding portion of said antibody, wherein said antibody or antigen-binding portion thereof specifically binds to an epitope comprising: (i) amino acids 37 to 56 of SEQ ID NO: 2 (IL-27p28); (ii) amino acids 142 to 164 of SEQ ID NO: 2 (IL-27p28); or (iii) both (i) and (ii), wherein said antibody or antigen-binding portion thereof comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2, and a light chain CDR3, wherein said light chain CDR1 consists of N-XXXXXXLFSSNXKXYXX-C, said light chain CDR3 consists of N-XXXASAXXX-C, said heavy chain CDR2 consists of NXXSSSXSYXYXXXXXXX-C, and said heavy chain CDR3 consists of NXXXXGRTSYTATXHNXXXX-C, wherein X is any amino acid. 2. An antibody or antigen-binding portion thereof according to claim 1, characterized in that said antibody or antigen-binding portion thereof does not contain CDRs of the heavy and light chains selected from the group consisting of: (i) the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 9, 10 and 11, respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 17, 18 and 19, respectively; (ii) the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 31, 32 and 33, respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 39, 40 and 41, respectively; (iii) the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 53, 54 and 55, respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 61, 62 and 63, respectively; (iv) the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 75, 76 and 77, respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 83, 84 and 85, respectively; (v) the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 97, 98 and 99, respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 105, 106 and 107, respectively; (vi) the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 119, 120 and 121, respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 127, 128 and 129, respectively; (vii) the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 12, 13 and 14, respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 20, 21 and 22, respectively; (viii) the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 34, 35 and 36, respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 42, 43 and 44, respectively; (ix) the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 56, 57 and 58, respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 64, 65 and 66, respectively; (x) the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 78, 79 and 80, respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 86, 87 and 88, respectively; (xi) the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 100, 101 and 102, respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 108, 109 and 110, respectively; and (xii) the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 122, 123 and 124, respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 130, 131 and 132, respectively. 3. An antibody or antigen-binding portion thereof according to claim 1 or 2, characterized in that said antibody or antigen-binding portion thereof exhibits at least one or more of the following properties: (i) bind to human IL-27 with an equilibrium dissociation constant (K _D ) of 15 nM or less; (ii) block the binding of IL-27 to the IL-27 receptor; (iii) inhibit or reduce phosphorylation of STAT1 and/or STAT3 in the cell; (iv) inhibit or reduce IL-27-mediated inhibition of CD161 expression in the cell; (v) inhibit or reduce IL-27-mediated expression of PD-L1 and/or TIM-3 in a cell; and - 121 052811 (vi) induce or enhance PD-1-mediated secretion of one or more cytokines from a cell. 4. An isolated antibody or antigen-binding portion thereof according to any one of claims 1 to 3, characterized in that said antibody is selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD and IgE antibodies. 5. A pharmaceutical composition comprising an isolated antibody or an antigen-binding portion thereof according to any one of claims 1 to 4 and a pharmaceutically acceptable carrier. 6. A method for inhibiting or reducing phosphorylation of STAT1 and/or STAT3 in a cell, comprising contacting said cell with an isolated antibody or antigen-binding portion thereof according to any one of claims 1-4, wherein said antibody or antigen-binding portion thereof inhibits or reduces phosphorylation of STAT1 and/or STAT3 in the cell. 7. A method for inhibiting or reducing the inhibition of CD161 expression in a cell, comprising contacting said cell with an isolated antibody or antigen-binding portion thereof according to any one of claims 1-4, wherein said antibody or antigen-binding portion thereof inhibits or reduces the inhibition of CD161 expression in the cell. 8. A method for inhibiting or reducing the expression of PD-L1 and/or TIM-3 in a cell, comprising contacting said cell with an isolated antibody or antigen-binding portion thereof according to any one of claims 1-4, wherein said antibody or antigen-binding portion thereof inhibits the expression of PD-L1 and/or TIM-3 in the cell. 9. A method for inducing or enhancing the secretion of one or more cytokines from a cell, comprising contacting said cell with an isolated antibody or antigen-binding portion thereof according to any one of claims 1-4, wherein said antibody or antigen-binding portion thereof induces or enhances PD-1-mediated secretion of one or more cytokines from the cell. 10. A method for stimulating an immune response in a subject, comprising administering to said subject an effective amount of an isolated antibody or an antigen-binding portion thereof according to any one of claims 1-4, or a pharmaceutical composition according to claim 5. 11. A method for treating IL-27-associated cancer in a subject, comprising administering to said subject an effective amount of an isolated antibody or antigen-binding portion thereof according to any one of claims 1-4, or a pharmaceutical composition according to claim 5. 12. The method according to claim 11, wherein the IL-27-associated oncological disease is selected from lung cancer, sarcoma, testicular cancer, ovarian cancer, pancreatic cancer, breast cancer, melanoma, head and neck cancer, colorectal cancer, bladder cancer, endometrial cancer, prostate cancer, thyroid cancer, hepatocellular carcinoma, gastric cancer, brain cancer, lymphoma, leukemia, or kidney cancer. 13. The method according to claim 11, wherein the IL-27-associated cancer is non-small cell lung cancer. 14. The method according to claim 11, wherein the IL-27-associated oncological disease is hepatocellular carcinoma. 15. The method according to claim 11, wherein the IL-27-associated cancer is renal cell carcinoma. 16. The method according to claim 11, wherein the IL-27-associated oncological disease is gastric cancer. 17. A method for enhancing one or more functions of an anti-PD-1 antibody, comprising exposing a cell to an antibody or antigen-binding portion thereof according to any one of claims 1-4, simultaneously with or sequentially with an anti-PD-1 antibody, thereby enhancing one or more functions of the anti-PD1 antibody. 18. The method according to claim 17, wherein said one or more functions of the anti-PD-1 antibody are selected from the group consisting of: PD-1-mediated cytokine secretion; anti-PD-1 antibody-mediated TNF-α secretion; anti-PD-1 antibody-mediated IL-6 secretion, and combinations thereof. 19. The method according to any one of claims 10-16, characterized in that said isolated antibody or antigen-binding portion thereof is administered in combination with one or more additional therapeutic agents or procedures, wherein said second therapeutic agent or procedure is selected from the group consisting of: chemotherapy, targeted anticancer therapy, an oncolytic drug, a cytotoxic agent, immune therapy, a cytokine, a surgical procedure, a radiation procedure, an activator of a costimulatory molecule, an inhibitor of an inhibitory molecule, a vaccine, or cellular immunotherapy, or a combination thereof. 20. The method according to claim 19, characterized in that said one or more additional therapeutic agents are a PD-1 antagonist, a PD-L1 inhibitor, a TIM-3 inhibitor, a LAG-3 inhibitor, a TIGIT inhibitor, a CD112R inhibitor, a TAM inhibitor, a STING agonist, a 41BB agonist, or a combination thereof. - 122 -