Anti-il-27 antibodies and uses thereof

Abstract

This disclosure relates to anti-IL-27 antibodies and their antigen-binding portions. This disclosure also relates to methods for treating or improving one or more symptoms of diseases such as cancer by administering such antibodies or their antigen-binding portions. This disclosure also relates to methods for detecting IL-27, for example, in an individual or sample.

Description

Technical Field

[0001] The present disclosure generally relates to compositions and methods for modulating IL-27 signaling. More specifically, the present disclosure relates to immunogenic compositions (e.g., antibodies, antibody fragments, and analogs thereof) that bind to IL-27 and modulate IL-27 signaling. Prior Art

[0002] In recent years, increasing evidence has shown that the immune system is an important obstacle to tumor formation and development. The existence of natural T cells with anti-tumor potential or activity in cancer patients has rationalized the development of immunotherapy in oncology. Immune cells, such as T cells, macrophages and natural killer cells, can exhibit anti-tumor activity and effectively control the occurrence and growth of malignant tumors. Tumor-specific antigens or tumor-associated antigens can induce immune cells to recognize and eliminate malignant tumors (Chen & Mellman, (2013) Immunity 39 (1): 1-10). Despite the existence of tumor-specific immune responses, malignant tumors often escape or avoid immune attacks through various immune regulatory mechanisms, resulting in uncontrolled tumor occurrence and development (Motz & Coukos, (2013) Immunity 39 (1): 61-730). In fact, an emerging feature of cancer is the use of these immune regulatory mechanisms and the suppression of anti-tumor immune responses, resulting in tumor evasion and escape from immune killing (Hanahan and Weinberg (2011) Cell 144 (5): 646-674).

[0003] IL-27 is a heterodimeric cytokine composed of two subunits (EBI3 and IL-27p28). 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 IL-27Rα (WSX1) and the gp130 chain, which primarily mediates signaling through STAT1 and STAT3. Initial reports characterized IL-27 as an immunopotentiating cytokine that often acts synergistically with IL-12 to support CD4+ T cell proliferation, T helper (Th) 1 cell differentiation, and IFN-γ production. Subsequent studies have demonstrated that IL-27 has complex immunomodulatory functions that can result in either pro-inflammatory or anti-inflammatory effects depending on the biological context and the experimental model used. IL-27 may drive the expression of different immunomodulatory molecules in human cancer cells, which may support local derangement 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).

[0004] Despite significant advances in cancer treatment and management, there remains a need for new and effective therapies for the treatment and management of cancer. Summary of the invention

[0005] Disclosed herein are antibodies, or antigen-binding portions thereof, that antagonize IL-27 and specifically bind to an antigenic determinant comprising one or more of the following amino acids: (i) amino acids 37 to 56 corresponding to SEQ ID NO: 2 (IL-27p28), (ii) amino acids 142 to 164 corresponding to SEQ ID NO: 2 (IL-27p28), or (iii) both (i) and (ii). In some aspects, the antibody, or antigen-binding portion thereof, specifically binds to an antigenic determinant comprising one or more amino acids 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, or Glu164 of SEQ ID NO: 2 (IL-27p28).

[0006] In some aspects, the antibodies or antigen-binding portions thereof of the present disclosure specifically bind to an antigenic determinant comprising Asp146, Arg149, and / or Phe153 of SEQ ID NO: 2 (IL-27p28). In some aspects, the antigenic determinant further comprises His150 and / or Leu156 of SEQ ID NO: 2 (IL-27p28). In some aspects, the antigenic determinant further comprises Gln37, Leu38, Glu42, Leu142, and / or Glu164 of SEQ ID NO: 2 (IL-27p28). In some aspects, the antigenic determinant further comprises Glu46, Val49, Ser50, and / or Leu162 of SEQ ID NO: 2 (IL-27p28). In some aspects, the antigenic determinant further comprises one or more amino acids selected from Leu53, Lys56, Asp143, Arg145, Leu147, Arg152, Ala157, Gly159, Phe160, Asn161, or Pro163 of SEQ ID NO: 2 (IL-27p28).

[0007] In some aspects, an antibody of the present disclosure, or an antigen-binding portion thereof, specifically binds to an antigenic determinant consisting of or consisting essentially of Gln37, Leu38, Glu42, Glu46, Val49, Ser50, Leu142, Asp146, Arg149, His150, Phe153, Leu156, Leu162, and Glu164 of SEQ ID NO: 2 (IL-27p28).

[0008] In some aspects, an antibody or antigen-binding portion thereof of the present disclosure specifically binds to an antigenic determinant consisting of 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 of SEQ ID NO: 2 (IL-27p28).

[0009] In other aspects, the antibody or antigen-binding portion thereof that specifically binds to an antigenic determinant comprising one or more amino acids 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 of SEQ ID NO: 2 (IL-27p28) does not comprise a heavy and light chain CDR selected from the group consisting of: (i) the heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 9, 10, and 11, respectively, and (ii) the heavy chain CDR2, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 11, 12, and 13, respectively. NO:17, 18 and 19; (ii) the heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO:31, 32 and 33, respectively, and the light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO:39, 40 and 41, respectively; (iii) the heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO:53, 54 and 55, respectively, and the light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO:61, 62 and 63, respectively; (iv) the heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO:75, 76 and 77, respectively, and the light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO:83, 84 and 85, respectively; (v) the heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: or (vi) the heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 119, 120 and 121, respectively, and the light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 127, 128 and 129, respectively.

[0010] In other aspects, the antibody or antigen-binding portion thereof that specifically binds to an antigenic determinant comprising one or more amino acids 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 of SEQ ID NO: 2 does not comprise a heavy and light chain CDR selected from the group consisting of: (i) the heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 12, 13, and 14, respectively, and (ii) the heavy chain CDR2, CDR3 sequences set forth in SEQ ID NOs: 14, 15, and 16, respectively. NO:20, 21 and 22; (ii) the heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO:34, 35 and 36, respectively, and the light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO:42, 43 and 44, respectively; (iii) the heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO:56, 57 and 58, respectively, and the light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO:64, 65 and 66, respectively; (iv) the heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO:78, 79 and 80, respectively, and the light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO:86, 87 and 88, respectively; (v) the heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: or (vi) the heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 122, 123 and 124, respectively, and the light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 130, 131 and 132, respectively.

[0011] In some aspects, the heavy chain CDR1 of the antibody, 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, the heavy chain CDR1 of the antibody, or antigen-binding portion thereof, does not consist of N-FTF[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- Composed of [G / S]ISSS[S / G][S / A]YI[L / Y]YADSVKG-C (SEQ ID NO: 149).

[0012] In other aspects, the antibody or antigen-binding portion thereof that specifically binds to an antigenic determinant comprising one or more amino acids 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 of SEQ ID NO: 2 (IL-27p28) 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 consisting of SEQ ID NO: 148; NO: 121; and the light chain CDR1, CDR2 and CDR3 sequences set forth 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 a heavy chain CDR3 sequence set forth in SEQ ID NO: 124; and the light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 130, 131 and 132, respectively.

[0013] In some aspects, the antibody or antigen-binding portion thereof that specifically binds to an antigenic determinant comprising one or more amino acids 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 of 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 consisting of N-AR[X] The heavy chain CDR3 sequence consists of n=6-15DX-C (SEQ ID NO: 153); and the light chain CDR1 consists of N-QS[X] n=1-3SS[X] n=0-4Y-C (SEQ ID NO: 154), the light chain CDR2 consists of N-XXS-C (SEQ ID NO: 155) and the light chain CDR3 sequence consists of N-QQXXXXP[X] n=0-1T-C (SEQ ID NO: 156).

[0014] Disclosed herein are antibodies or antigen-binding portions thereof that exhibit at least one or more of the following properties: (i) binding to human IL-27 with an equilibrium dissociation constant (KD) of 15 nM or less; (ii) blocking IL-27 binding to the IL-27 receptor; (iii) inhibiting or reducing STAT1 and / or STAT3 phosphorylation in cells; (iv) inhibiting or reducing IL-27-mediated inhibition of CD161 expression in cells; (v) inhibiting or reducing IL-27-mediated PD-L1 and / or TIM-3 expression in cells; and (vi) inducing or enhancing PD-1-mediated secretion of one or more cytokines from cells.

[0015] In some aspects, the isolated antibody, or antigen-binding portion thereof, binds to human IL-27 with an equilibrium dissociation constant (KD) of 15 nM or less.

[0016] In other aspects, the isolated antibody or its antigen-binding portion inhibits or reduces STAT1 and / or STAT3 phosphorylation in cells. In some aspects, the isolated antibody or its antigen-binding portion reduces STAT1 and / or STAT3 phosphorylation in immune cells or cancer cells.

[0017] In some aspects, the isolated antibody or antigen binding portion thereof inhibits or reduces the inhibition of CD161 expression in cells. In some aspects, the isolated antibody or antigen binding portion thereof inhibits or reduces the inhibition of CD161 expression in immune cells.

[0018] In other aspects, the isolated antibody or its antigen binding portion inhibits or reduces the expression of PD-L1 and / or TIM-3 in cells. In some aspects, the isolated antibody or its antigen binding portion inhibits or reduces the expression of PD-L1 and / or TIM-3 in immune cells or cancer cells. In some aspects, the isolated antibody or its antigen binding portion inhibits or reduces the expression of PD-L1 in cancer cells.

[0019] In some aspects, the isolated antibody or its antigen-binding portion induces or enhances PD1-mediated secretion of one or more cytokines from cells. In some aspects, the one or more cytokines are IFNg (IFNγ), IL-17, TNFa (TNFα) or IL-6. In some aspects, the antibody or its antigen-binding portion is selected from the group consisting of: IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD and IgE antibodies. In other aspects, the antibody or its antigen-binding portion is an IgG1 antibody or an IgG4 antibody. In some aspects, the antibody or its antigen-binding portion comprises an Fc domain containing at least one mutation. A pharmaceutical composition is also disclosed herein, which comprises any of the described isolated antibodies or their antigen-binding portions, and a pharmaceutically acceptable carrier. A nucleic acid comprising a nucleotide sequence encoding a light chain, a heavy chain, or both a light and heavy chain of an isolated antibody or its antigen-binding portion is also disclosed. An expression vector comprising the nucleic acid is disclosed herein. Cells transformed with the expression vector are further disclosed.

[0020] The present disclosure also provides a method for producing an antibody or an antigen-binding portion thereof that specifically binds to human IL-27, comprising maintaining a cell transformed with an expression vector under conditions that allow expression of the antibody or an antigen-binding portion thereof. In some aspects, the method further comprises obtaining the antibody or an antigen-binding portion thereof.

[0021] Disclosed herein are methods for inhibiting or reducing STAT1 and / or STAT3 phosphorylation in a cell, comprising contacting the cell with an antibody or an antigen-binding portion thereof, wherein the antibody or the antigen-binding portion thereof inhibits or reduces STAT1 and / or STAT3 phosphorylation in the cell.

[0022] Further disclosed is a method of inhibiting or reducing the inhibition of CD161 expression in a cell, comprising contacting the cell with an antibody or an antigen-binding portion thereof, wherein the antibody or the antigen-binding portion thereof inhibits or reduces the inhibition of CD161 expression in the cell.

[0023] Also disclosed are methods of inhibiting or reducing the expression of PD-L1 and / or TIM-3 in a cell, comprising contacting the cell with an antibody or an antigen-binding portion thereof, wherein the antibody or the antigen-binding portion thereof inhibits the expression of PD-L1 and / or TIM-3 in the cell.

[0024] Also disclosed are methods of inducing or enhancing the secretion of one or more cytokines from a cell, comprising contacting the cell with an antibody or an antigen-binding portion thereof, wherein the antibody or the antigen-binding portion thereof induces or enhances PD-1-mediated secretion of one or more cytokines from the cell.

[0025] Further disclosed are methods of stimulating an immune response in a subject, comprising administering to the subject an effective amount of the disclosed isolated antibody or antigen-binding fragment or the disclosed pharmaceutical composition.

[0026] Further disclosed are methods of treating cancer in a subject, comprising administering to the subject an effective amount of the disclosed isolated antibody or antigen-binding fragment or the disclosed pharmaceutical composition.

[0027] Disclosed herein are methods for stimulating an immune response in a subject or treating cancer in a subject. The methods include administering to a subject an effective amount of a disclosed isolated antibody or antigen-binding portion thereof or a disclosed pharmaceutical composition, wherein the antibody, antigen-binding portion thereof or pharmaceutical composition inhibits or reduces STAT1 and / or STAT3 phosphorylation in a cell, thereby stimulating an immune response or treating cancer.

[0028] Further disclosed are methods for stimulating an immune response in a subject or treating cancer in a subject. The methods include administering to a subject an effective amount of the disclosed isolated antibody or antigen-binding portion thereof or the disclosed pharmaceutical composition, wherein the antibody, antigen-binding portion thereof or pharmaceutical composition inhibits or reduces the inhibition of CD161 expression in cells, thereby stimulating an immune response or treating cancer.

[0029] Further disclosed are methods for stimulating an immune response in a subject or treating cancer in a subject. The method comprises administering to a subject an effective amount of the disclosed isolated antibody or antigen-binding portion thereof or the disclosed pharmaceutical composition, wherein the antibody, antigen-binding portion thereof or pharmaceutical composition inhibits or reduces the expression of PD-L1 and / or TIM-3 on cells, thereby stimulating an immune response or treating cancer.

[0030] Further disclosed are methods of stimulating an immune response in a subject or treating cancer in a subject. The methods include administering to a subject an effective amount of the disclosed isolated antibody or antigen-binding portion thereof or the disclosed pharmaceutical composition, wherein the antibody, antigen-binding portion thereof or pharmaceutical composition induces or enhances PD-1-mediated secretion of one or more cytokines from cells, thereby stimulating an immune response or treating cancer.

[0031] In some aspects, the cancer 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), melanoma, head and neck cancer (e.g., squamous head and neck cancer), colorectal cancer, bladder cancer, endometrial cancer, prostate cancer, thyroid cancer, hepatocellular carcinoma, gastric cancer, brain cancer, lymphoma (e.g., DL-BCL), leukemia (e.g., AML), or kidney cancer (e.g., renal cell carcinoma, e.g., clear cell renal carcinoma).

[0032] Disclosed herein are methods for enhancing one or more activities of an anti-PD-1 antibody (e.g., enhancing PD-1-mediated cytokine secretion; enhancing anti-PD-1-mediated TNFα secretion; enhancing anti-PD-1-mediated IL-6 secretion from cells exposed to anti-PD-1 antibodies). The method comprises exposing cells to the disclosed antibody or antigen-binding portion thereof simultaneously or sequentially with the anti-PD-1 antibody, thereby enhancing one or more activities of the anti-PD-1 antibody.

[0033] Further disclosed is a pharmaceutical composition comprising an anti-PD-1 antibody, the disclosed antibody or an antigen-binding portion thereof, and a pharmaceutically acceptable carrier.

[0034] Also disclosed are kits comprising an anti-PD-1 antibody and a disclosed antibody or antigen-binding portion thereof for simultaneous or sequential administration, and instructions for use thereof.

[0035] Disclosed herein are any of the disclosed methods for stimulating an immune response or treating cancer, wherein the disclosed separated antibody or its antigen binding portion is administered in combination with one or more additional therapeutic agents or procedures. The second therapeutic agent or procedure is selected from a group consisting of chemotherapy, targeted anticancer therapy, oncolytic drugs, cytotoxic agents, immune-based therapies, cytokines, surgical procedures, radiation procedures, activators of costimulatory molecules, inhibitors of inhibitory molecules, vaccines or cellular immunotherapy, or a combination thereof. In some aspects, one or more additional therapeutic agents are PD-1 antagonists, PD-L1 inhibitors, TIM-3 inhibitors, LAG-3 inhibitors, TIGIT inhibitors, CD112R inhibitors, TAM inhibitors, STING agonists, 4-1BB agonists, tyrosine kinase inhibitors, agents targeting adenosine axis (e.g., CD39 antagonists, CD73 antagonists or A2AR, A2BR or dual A2AR / A2BR antagonists), CCR8 antagonists, CTLA4 antagonists, VEG-F inhibitors or a combination thereof. In other aspects, one or more additional therapeutic agents are PD-1 antagonists. In some aspects, the 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, the PD-L1 inhibitor is selected from the group consisting of: FAZ053, Atezolizumab, Avelumab, Durvalumab, and BMS-936559. In other aspects, 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 (CD-122 biased agonist), Tivozanib (FOTIVDA®), abexinostat, Ipilimumab (YERVOY®), Trelimumab (tremelimu mab), Pazopanib (VOTRIENT®), Sorafenib (NEXAVAR®), Temsirolimus (TORISEL®), Ramucirumab (CYRAMZA®), Niraparib, Savolitinib, Vorolanib (X-82), Regorafenib (STIVARGO®), Donafenib (multikinase inhibitor), Camrelizumab (SHR-1210), Pexastimogen devacirepvec (JX-594), Cyramza®, apatinib (YN968D1), encapsulated doxorubicin (THERMODOX®), tivantinib (ARQ197), ADI-PEG 20, binimetinib, apatinib mesylate (mesylate), nintedanib, lirilumab, nivolumab (OPDIVO®), pembrolizumab (KEYTRUDA®), atezolizumab (TECENTRIQ®), avelumab (BAVENCIO®), durvalumab (IMFIMZI®), cemiplimab-rwlc (LIBTAYO®), tislelizumab and spartalizumab. In some aspects, one or more additional therapeutic agents are TIM-3 inhibitors, optionally wherein the TIM-3 inhibitor is MGB453 or TSR-022. In some aspects, one or more additional therapeutic agents are LAG-3 inhibitors, optionally wherein the LAG-3 inhibitor is selected from the group consisting of: LAG525, BMS-986016, and TSR-033. In some aspects, one or more additional therapeutic agents are TIGIT inhibitors. In other aspects, one or more additional therapeutic agents are CD112R inhibitors. In some aspects, one or more additional therapeutic agents are TAM (Axl, Mer, Tyro) inhibitors. In some aspects, one or more additional therapeutic agents are 4-1BB agonists. In other aspects, one or more additional therapeutic agents are tyrosine kinase inhibitors (TKIs). In some aspects, TKIs are selected from imatinib, dasatinib, nilotinib, bosutinib, or ponatinib. In some aspects, one or more additional agents are agents targeting the adenosine axis. In some aspects, the agent targeting the adenosine axis is selected from a CD39 antagonist, a CD73 antagonist, an A2AR antagonist, an A2BR antagonist, or a dual A2AR / A2BR antagonist. In some aspects, one or more additional therapeutic agents are CD39 antagonists. Examples of CD39 antagonists include those described in US2019 / 0284295 (Surface Oncology, Inc.), which is incorporated herein by reference. In some aspects, one or more additional therapeutic agents are CD73 antagonists. Examples of CD73 antagonists include small molecule CD73 inhibitors such as AB421 (Arcus), CD73 antibodies or antigen-binding portions thereof that bind to CD73 such as MEDI9447 (Medimmune), BMS-986179 (Bristol MeyersSquibb), or as described in US2018 / 0009899 (Corvus), which is incorporated herein by reference in its entirety. In some aspects, one or more additional therapeutic agents are A2AR antagonists, A2BR antagonists, or dual A2AR / A2BR antagonists. Examples of A2AR, A2BR and dual A2AR / A2BR antagonists include Preladenant / SCH420814 (Merck / Schering, CAS Reg. No. 377727-87-2), described in Hodgson et al. (2009) J Pharmacol Exp Ther 330(1):294-303 and incorporated herein by reference in its entirety; ST-4206 (Leadiant Biosciences), described in U.S. Pat. No. 9,133,197 and incorporated herein by reference in its entirety; KW-6356 (Kyowa Hakko Kogyo), Tozadenant / SYN-115 (Acorda), Istradefylline / KW-6002 (Kyowa Hakko Kogyo, CAS Reg. No.: 155270-99-8), which is described in LeWitt et al., (2008) Ann Neurol 63(3):295-302 and is incorporated herein by reference in its entirety; theophylline (CAS Reg. No.: 58-55-9), NIR178 (Novartis); AB928 (Arcus Biosciences), GBV-2034 (Globavir), Vibaene (Vipadenant;Redox / Juno),AZD4635 / HTL-1071 (AstraZeneca / Heptares), which is described in WO2011 / 095625 and is incorporated herein by reference in its entirety; CPI-444 / V81444 (Corvus / PBF509 (Palobiofarma / Novartis), which is described in WO2009 / 156737 and is incorporated herein by reference in its entirety; US 8,796,284 and WO 2017 / 025918 are described and incorporated herein by reference in their entirety; A2AR antagonists, which are described in US8114845, US9029393, US20170015758 or US20160129108, all of which are incorporated herein by reference in their entirety; 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, one or more additional therapeutic agents are CCR8 antagonists. In some aspects, the CCR8 antagonist is selected from a small molecule and an antibody. In some aspects, one or more additional therapeutic agents are CTLA4 antagonists. In some aspects, the CTLA4 antagonist is selected from the group consisting of: Yervoy® (ipilimumab or antibody 10D1, which is described in PCT Publication WO 01 / 14424), tralimumab (formerly ticilimumab, CP-675,206), a monoclonal or anti-CTLA-4 antibody described in any of the following publications: WO 98 / 42752; WO 00 / 37504; U.S. Pat. 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): antibodies tract No. 2505 (antibody CP-675206); and Mokyr et al. (1998) Cancer Cell Biology 23(14): 1154-116. Res. 58:5301-5304. Any anti-CTLA-4 antibody disclosed in WO2013 / 173223 may also be used. In some aspects, one or more additional therapeutic agents are VEG-F inhibitors. In some aspects, the VEG-F inhibitor is selected from cabozantinib, pazopanib, bevacizumab, sunitinib, axitinib, lenvatinib, sorafenib, regorafenib, ponatinib, cabozantinib, vandetanib, ramucirumab or bevacizumab.

[0036] Disclosed herein are uses of the disclosed antibodies or antigen-binding portions thereof or the disclosed pharmaceutical compositions for stimulating an immune response in a subject or for treating cancer in a subject, optionally in combination with one or more additional therapeutic agents or procedures.

[0037] Further disclosed are kits comprising the disclosed antibodies or antigen-binding portions thereof or the disclosed pharmaceutical compositions and instructions for use to stimulate an immune response in a subject or to treat cancer in a subject, optionally in combination with one or more additional therapeutic agents or procedures.

[0038] Also disclosed are kits comprising the disclosed antibodies, or antigen-binding portions thereof, and instructions for use in detecting IL-27 in a sample from a subject, optionally with instructions for use in detecting an IL-27-associated cancer in the subject. [definition] [] []

[0039] Unless otherwise specified, the terms used in the claims and the specification are defined as set forth below.

[0040] It should be noted that as used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural references unless the context clearly dictates otherwise.

[0041] As used herein, "about" is understood by one of ordinary skill in the art and varies to some extent depending on the context in which it is used. If one of ordinary skill in the art is unsure of the use of the term given the context in which the term is used, "about" means up to plus or minus 10% of the specified value.

[0042] As used herein, the term "agonist" refers to any molecule that partially or completely promotes, induces, increases and / or activates the biological activity of the natural polypeptide disclosed herein. Suitable agonist molecules include, among others, agonist antibodies or antibody fragments, fragments of natural polypeptides, peptides or proteins, or amino acid sequence variants. In some aspects, activation in the presence of an agonist is observed in a dose-dependent manner. In some aspects, under comparable conditions, the measured signal (e.g., 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%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% higher than the signal measured using a negative control. Also disclosed herein are methods for identifying agonists suitable for use in the methods of the present disclosure. For example, such methods include, but are not limited to, binding assays such as enzyme-linked immunosorbent assays (ELISA), FORTE BIO® systems, and radioimmunoassays (RIA). Such assays measure the ability of an agonist to bind to a polypeptide of interest (e.g., a receptor or ligand), and thus indicate the ability of the agonist to promote, increase, or activate the activity of the polypeptide. The efficacy of an agonist can also be determined using a functional assay, such as the ability of the agonist to activate or promote the function of a polypeptide. For example, a functional assay may include contacting a polypeptide with a candidate agonist molecule and measuring detectable changes in one or more biological activities typically associated with the polypeptide. The potency of an agonist is typically defined by its EC 50 value (the concentration required to activate 50% of the agonist response). The lower the EC 50 value, the greater the potency of the agonist and the lower the concentration required to activate the maximum biological response.

[0043] As used herein, the term "alanine scanning" refers to a technique for determining the contribution of a particular wild-type residue to the stability or function (e.g., binding affinity) of a given protein or polypeptide. The technique involves substituting alanine residues for wild-type residues in a polypeptide, followed by assessing the stability or function (e.g., binding affinity) of the alanine-substituted derivative or mutant polypeptide and comparing it to the wild-type polypeptide. Techniques for substituting alanine for wild-type residues in a polypeptide are known in the art.

[0044] The term "amelioration" refers to any therapeutically beneficial result in the treatment of a disease state (eg, cancer), including prevention, lessening of severity or progression, remission, or cure.

[0045] As used herein, the term "amino acid" refers to naturally occurring and synthetic amino acids, and to amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those amino acids encoded by the genetic code, as well as those amino acids that are later modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as naturally occurring amino acids (i.e., alpha carbon bound to hydrogen, carboxyl group, amine group, and R group), such as homoserine, norleucine, methionine sulfoxide, methionine methylsulfoxide. These analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of amino acids but function in a manner similar to naturally occurring amino acids.

[0046] Amino acids are referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Likewise, nucleotides may be referred to by their commonly recognized single-letter codes.

[0047] As used herein, "amino acid substitution" refers to the replacement of at least one existing amino acid residue in a predetermined amino acid sequence (the amino acid sequence of the starting polypeptide) with a second, different "replacement" amino acid residue. "Amino acid insertion" refers to the incorporation of at least one other amino acid into a predetermined amino acid sequence. Although insertions typically consist of the insertion of one or two amino acid residues, larger "peptide insertions" may be made, such as insertions of about three to about five or even up to about ten, fifteen or twenty amino acid residues. As disclosed above, one or more of the inserted residues may be naturally occurring or non-naturally occurring. "Amino acid deletion" refers to the removal of at least one amino acid residue from a predetermined amino acid sequence.

[0048] As used herein, the terms "amount" or "level" are used in the broadest sense and refer to the amount, concentration, or abundance of a substance (e.g., a metabolite, a small molecule, a protein, an mRNA, a marker). When referring to a metabolite or a small molecule (e.g., a drug), the terms "amount", "level", and "concentration" are often used interchangeably and generally refer to a detectable amount in a biological sample. "Elevated level" or "increased level" refers to an increase in the amount, concentration, or abundance of a substance in a sample, such as a control sample or an internal control from one or more individuals not suffering from a disease or disorder (e.g., cancer). In some aspects, an increased level of a substance (e.g., a drug) in a sample refers to an increase in the amount of the substance by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% relative to the amount of the substance in a control sample, as determined by techniques known in the art (e.g., HPLC). "Decreased levels" refers to a decrease in the amount, concentration, or abundance of a substance (e.g., a drug) in a subject, such as a control or internal control from one or more subjects not suffering from a disease or disorder (e.g., cancer). In some aspects, a reduced level is an almost undetectable amount, concentration, or abundance. In some aspects, a reduction in the level of a substance (e.g., a drug) in a sample refers to a decrease in the amount of the substance by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% relative to the amount of the substance in a control sample as determined by techniques known in the art (e.g., HPLC).

[0049] When referring to a protein, mRNA or marker, such as those described herein, the terms "level of expression" or "expression level" are often used interchangeably and generally refer to the detectable amount of a protein, mRNA or marker in a biological sample. In some aspects, the detectable amount or detectable level of a protein, mRNA or marker is correlated with the likelihood of a response to an agent, such as those described herein. "Expression" generally refers to the process by which the information contained within a gene is converted into a structure (e.g., a protein marker, such as PD-L1) that is present and functioning in a cell. Thus, as used herein, "expression" may refer to transcription into polynucleotides, translation into polypeptides, or even polynucleotide and / or polypeptide modifications (e.g., post-translational modifications of polypeptides). Fragments of transcribed polynucleotides, translated polypeptides, or modifications of polynucleotides and / or polypeptides (e.g., post-translational modifications of polypeptides) should also be considered expressed, whether they arise from transcripts generated by alternative splicing or from degraded transcripts, or from post-translational processing of polypeptides (e.g., by proteolysis). "Expressed genes" include genes that are transcribed into polynucleotides as mRNA and then translated into polypeptides, and also include genes that are transcribed into RNA but not translated into polypeptides (e.g., transfer RNA and ribosomal RNA). "Increased expression", "increased expression level" or "increased level" refers to an increase in the expression or level of a substance in a sample relative to a control sample, such as one or more individuals not suffering from a disease or disorder (e.g., cancer) or an internal control. In some aspects, the increased expression of a substance (e.g., a protein marker, such as PD-L1) in a sample refers to an increase in the amount of the substance by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% relative to the amount of the substance in a control sample, as determined by techniques known in the art (e.g., FACS). "Decreased expression," "reduced expression level," or "reduced level" refers to a decrease in the expression or level of a substance (e.g., a protein marker) in a subject relative to a control, such as one or more subjects not suffering from a disease or disorder (e.g., cancer) or an internal control. In some aspects, the decreased expression is almost no expression. In some aspects, reduced expression of a substance (e.g., a protein marker) in a sample refers to a decrease in the amount of the substance relative to the amount of the substance in a control sample by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%, as determined by techniques known in the art (e.g., FACS).

[0050] As used herein, the term "angiogenesis" or "neovascularization" refers to the process by which new blood vessels develop from pre-existing blood 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). In response to growth factor or hormonal signals or hypoxic or ischemic conditions, 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) become activated to migrate, proliferate, and differentiate into structures with luminal structures, forming new blood vessels. During ischemia, such as occurs in cancer, the need for increased oxygenation and delivery of nutrients apparently induces the secretion of angiogenic factors by the diseased tissue; these factors stimulate new blood vessel formation. Several additional terms are associated with angiogenesis.

[0051] As used herein, the term "antagonist" refers to an inhibitor of a target molecule and can be used synonymously with the term "inhibitor" herein. As used herein, the term "antagonist" refers to any molecule that partially or completely blocks, inhibits or neutralizes the biological activity of a natural polypeptide disclosed herein. Suitable antagonist molecules include, among others, antagonistic antibodies or antibody fragments, fragments of natural polypeptides, peptides or proteins, or amino acid sequence variants. In some aspects, inhibition in the presence of an antagonist is observed in a dose-dependent manner. In some aspects, under comparable conditions, the measured signal (e.g., 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%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% lower than the signal measured using a negative control. Also disclosed herein are methods of identifying antagonists suitable for use in the methods of the disclosure. For example, such methods include, but are not limited to, binding assays such as enzyme-linked immunosorbent assays (ELISA), ForteBio® systems, radioimmunoassays (RIA), Meso Scale Discovery assays (e.g., Meso Scale Discovery electrochemiluminescence (MSD-ECL), and bead-based Luminex® assays. These assays measure the ability of an antagonist to bind to a polypeptide of interest (e.g., a receptor or ligand) and are therefore indicative of the ability of the antagonist to inhibit, neutralize, or block the activity of the polypeptide. The efficacy of an antagonist can also be determined using functional assays, such as the ability of an antagonist to inhibit the function of a polypeptide or agonist. For example, a functional assay can comprise contacting a polypeptide with a candidate antagonist molecule and measuring a detectable change in one or more biological activities typically associated with the polypeptide. The potency of an antagonist is typically defined by its IC 50 value (the concentration required to inhibit the agonist response by 50%). The lower the IC 50 value, the greater the potency of the antagonist and the lower the concentration required to inhibit the maximal biological response.

[0052] As used herein, the phrase "antibodies or antigen-binding portions thereof that antagonize human IL-27" refers to antibodies that antagonize at least one art-recognized activity of human IL-27 (e.g., IL-27 biological activity and / or downstream pathways mediated by IL-27 signaling or other IL-27-mediated functions), e.g., associated with a reduction (or decrease) in human IL-27 activity of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more. Additional examples of IL-27 biological activities and / or downstream pathways mediated by IL-27 signaling or other IL-27-mediated functions are described in further detail below and elsewhere herein.

[0053] As used herein, the term "anti-IL-27 antagonist antibody" (interchangeably referred to as "anti-IL-27 antibody") refers to an antibody that specifically binds to IL-27 and inhibits IL-27 biological activity and / or downstream pathways mediated by IL-27 signaling or other IL-27-mediated functions. Anti-IL-27 antagonist antibodies encompass antibodies that block, antagonize, suppress, inhibit or reduce IL-27 biological activity (e.g., ligand binding, enzymatic activity), including downstream pathways mediated by IL-27 signaling or function, such as receptor binding and / or eliciting a cellular response to IL-27 or its metabolites. In some aspects, the anti-IL-27 antagonist antibodies provided by the present disclosure bind to human IL-27 and prevent, block or inhibit human IL-27 from binding to its cognate or normal receptor (e.g., IL-27 receptor) or one or more receptor subunits (e.g., gp130 and / or IL-27Rα (also known as WSX1 / TCCR)). In some aspects, the anti-IL-27 antagonist antibody prevents, blocks or inhibits human IL-27 binding to gp130. In some aspects, the anti-IL-27 antagonist antibody prevents, blocks or inhibits human IL-27 binding to IL-27Rα. In some aspects, the anti-IL-27 antagonist antibody prevents, blocks or inhibits dimerization of IL-27 monomers. In some aspects, the anti-IL-27 antibody does not specifically bind to EBI3 monomers. In some aspects, the anti-IL-27 antibody specifically binds to IL-27p28 monomers. In some embodiments, the anti-IL-27 antibody specifically binds to a non-adjacent antigenic determinant comprising p28, but does not bind to an EBI3 monomer. In some aspects, the anti-IL-27 antibody inhibits or reduces STAT1 and / or STAT3 phosphorylation in cells. In some aspects, anti-IL-27 antibodies inhibit or reduce the inhibition of CD161 expression in cells (e.g., improve or reduce the inhibition of CD161 expression in cells mediated by IL-27). In some aspects, anti-IL-27 antibodies inhibit or reduce PD-L1 and / or TIM-3 expression in cells. In some aspects, anti-IL-27 induces or enhances PD-1-mediated secretion of one or more cytokines from cells. In some aspects, anti-IL-27 antagonist antibodies bind to human IL-27 and stimulate or enhance anti-tumor responses. In some aspects, anti-IL-27 antagonist antibodies bind to human IL-27 with an affinity of 15nM or less. In some aspects, anti-IL-27 antagonist antibodies bind to human IL-27 and include a wild-type or mutant IgG1 heavy chain constant region or a wild-type or mutant IgG4 heavy chain constant region. Examples of anti-IL-27 antagonist antibodies are provided herein.

[0054] As used herein, the term "antibody" refers to a complete antibody comprising two light chain polypeptides and two heavy chain polypeptides. Complete antibodies include different antibody isotypes, including IgM, IgG, IgA, IgD and IgE antibodies. The term "antibody" includes polyclonal antibodies, monoclonal antibodies, chimeric or chimeric antibodies, humanized antibodies, primatized antibodies, deimmunized antibodies and fully human antibodies. Antibodies can be produced or obtained in any of a variety of species, for example, mammals such as humans, non-human primates (e.g., gorillas, baboons or chimpanzees), horses, cattle, pigs, sheep, goats, dogs, cats, rabbits, guinea pigs, gerbils, hamsters, rats and mice. The antibody can be a purified or recombinant antibody. As used herein, the term "antibody fragment", "antigen binding fragment" or similar terms refer to an antibody fragment that retains the ability to bind to a target antigen (e.g., IL-27) and inhibits the activity of the target antigen. Such fragments include, for example, single-chain antibodies, single-chain Fv fragments (scFv), Fd fragments, Fab fragments, Fab' fragments, or F(ab')2 fragments. A scFv fragment is a single polypeptide chain comprising both the heavy and light chain variable regions of the antibody that produces the scFv. In addition, intrabodies, microbodies, trifunctional antibodies, and bifunctional antibodies are also included in the definition of antibodies 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 disclosures of which are each incorporated herein by reference in their entirety.

[0055] As used herein, the term "antibody fragment" also includes, for example, single domain antibodies such as camelized 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 Application Publication Nos. WO 94 / 04678 and WO 94 / 25591; and U.S. Pat. No. 6,005,079, all of which are incorporated herein by reference in their entirety. In some aspects, the present disclosure provides single domain antibodies comprising two VH domains with modifications to form a single domain antibody.

[0056] In some aspects, the antigen binding fragment comprises the variable region of the heavy chain polypeptide and the variable region of the light chain polypeptide. In some aspects, the antigen binding fragment described herein comprises the CDRs of the light chain and heavy chain polypeptide of an antibody.

[0057] The term "antigen presenting cell" or "APC" is a cell that displays foreign antigens complexed with MHC on its surface. T cells recognize this complex using 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 by phagocytosis or receptor-mediated endocytosis.

[0058] The term "antigen presentation" refers to the process by which an APC captures an antigen and makes it available for recognition by T cells, for example as a component of an MHC-I and / or MHC-II complex.

[0059] As used herein, the term "apoptosis" refers to the process of planned cell death in multicellular organisms, such as humans. The highly regulated biochemical and molecular events leading to apoptosis produce observable and characteristic morphological changes in cells, including membrane blebbing, cell volume shrinkage, chromosomal DNA condensation and fragmentation, and mRNA decay. A common method for identifying cells undergoing apoptosis, including T cells, is to expose the cells to a fluorophore-binding protein (Annexin V). Annexin V is commonly used to detect apoptotic cells by its ability to bind to phosphatidylserine on the outer leaflet of the plasma membrane, an early indicator that cells are undergoing the apoptotic process.

[0060] As used herein, the term "B cell" (or "B lymphocyte") refers to a type of white blood cell of the lymphocyte subtype. B cells play a role in the humoral immunity component of the adaptive immune system by secreting antibodies. B cells also present antigens and secrete cytokines. Unlike the other two classes of lymphocytes, T cells and natural killer cells, B cells express B cell receptors (BCRs) on their cell membranes. The BCR allows B cells to bind to specific antigens against which they elicit an antibody response.

[0061] As used herein, the term "binds to immobilized IL-27" refers to the ability of the antibodies of the present disclosure to bind to IL-27, for example, expressed on the surface of a cell or attached to a solid support.

[0062] As used herein, the term "bispecific" or "bifunctional antibody" refers to an artificial hybrid antibody with two different heavy / light chain pairs and two different binding sites. Bispecific antibodies can be produced by a variety of methods, including fusion of hybridomas or connection of Fab' fragments. See, e.g. Songsivilai & Lachmann, (1990) Clin. Exp. Immunol. 79:315-321; Kostelny et al., (1992) J. Immunol. 148:1547-1553.

[0063] Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain / light chain pairs, where the two heavy chain / light chain pairs have different specificities (Milstein and Cuello, (1983) Nature 305: 537-539). The antibody variable domain with the desired binding specificity (antibody-antigen combining site) can be fused to the immunoglobulin constant domain sequence. The heavy chain variable region is preferably fused to the immunoglobulin heavy chain constant domain including at least a portion of the hinge, CH2 and CH3 regions. For further details of exemplary currently known methods for producing bispecific antibodies, see, e.g., Suresh et al., (1986) Methods Enzymol. 121:210; PCT 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 also include cross-linked or heteroconjugate antibodies. Heteroconjugate antibodies can be prepared using any convenient cross-linking method. Suitable cross-linking agents are well known in the art and are disclosed in U.S. Pat. No. 4,676,980, along with a number of cross-linking techniques.

[0064] A variety of techniques have also been described for making and isolating bispecific antibody fragments directly from recombinant cell culture. For example, bispecific antibodies are produced using a leucine zipper. See, e.g., Kostelny et al. (1992) J Immunol 148(5):1547-1553. By gene fusion, leucine zipper peptides from the Fos and Jun proteins can be linked to the Fab' portions of two different antibodies. 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 making bispecific antibody fragments. The fragments comprise a heavy chain variable domain (VH) linked to a light chain variable domain (VL) by a linker that is too short to allow pairing of the two domains on the same chain. Thus, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of the other fragment, thereby forming two antigen binding sites. Another strategy for making bispecific antibody fragments by using single-chain Fv (scFv) dimers has also been reported. See, e.g., Gruber et al. (1994) J Immunol 152:5368. Alternatively, the antibody may be a "linear antibody" as described, e.g., in 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 regions. Linear antibodies may be bispecific or monospecific.

[0065] Antibodies with more than two valencies (eg, trispecific antibodies) are contemplated and are described, for example, in Tutt et al. (1991) J Immunol 147:60.

[0066] The present disclosure 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 molecule is designed such that two different light chain variable domains (VL) from two different parent antibodies are linked in tandem by recombinant DNA technology directly or via a short linker, followed by a light chain constant domain. Similarly, the heavy chain comprises two different heavy chain variable domains (VH) linked in tandem, followed by a constant domain CH1 and an Fc region. Methods for making 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, the bispecific antibody is a tandem Fab immunoglobulin in which the light chain variable region with a second specificity is fused to the heavy chain variable region of an intact antibody. Such antibodies are described, for example, in International Patent Application Publication No. WO 2015 / 103072.

[0067] As used herein, "cancer antigen" or "tumor antigen" refers to (i) tumor-specific antigens, (ii) tumor-associated antigens, (iii) cells expressing tumor-specific antigens, (iv) cells expressing 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 or antigen-presenting cell or material associated with cancer.

[0068] As used herein, the term "cancer-specific immune response" refers to an immune response induced by the presence of a tumor, cancer cell, or cancer antigen. In certain aspects, the response comprises proliferation of cancer antigen-specific lymphocytes. In certain aspects, the response comprises expression and upregulation of antibodies and T-cell receptors and the formation and release of lymphokines, chemokines, and cytokines. The innate and acquired immune systems interact to elicit an antigenic response against a tumor, cancer cell, or cancer antigen. In certain aspects, the cancer-specific immune response is a T cell response.

[0069] The term "cancer" is recognized in the art and refers to malignant tumors of epithelial or endocrine tissue, including respiratory cancer, gastrointestinal cancer, genitourinary cancer, testicular cancer, breast cancer, prostate cancer, endocrine cancer, and melanoma. The anti-IL-27 antibodies described herein can be used to treat patients who have any type of cancer, including renal cancer or melanoma or any viral disease, are suspected of having such a cancer or disease, or may be at high risk for developing such a cancer or disease. Exemplary cancers include cancers formed from tissues of the cervix, lung, prostate, breast, head and neck, colon, and ovary. The term also encompasses carcinosarcoma, including malignant tumors composed of cancerous and sarcomatous tissue. "Adenocarcinoma" refers to a cancer derived from glandular tissue or a cancer in which the tumor cells form a recognizable glandular structure.

[0070] As used herein, the term "CD112R" refers to a member of the poliovirus receptor-like protein and is a co-inhibitory receptor for human T cells. CD112R is an inhibitory receptor expressed primarily by T cells and NK cells and competes with the activating receptor CD226 for CD112 binding. The interaction of CD112 with CD112R has a higher affinity than the interaction with CD226, and thus effectively regulates CD226-mediated cell activation. Anti-CD112R antagonists that block the interaction with CD112 limit inhibitory signaling directly downstream of CD112R, while at the same time promoting greater immune cell activation by increasing the interaction of CD226 with CD112. As used herein, the term "CD112R inhibitor" refers to an agent that interferes with, blocks or inhibits the biological function or activity of CD112R.

[0071] As used herein, the term "CD137" (or "4-1BB") refers to a member of the tumor necrosis factor (TNF) receptor superfamily. 4-1BB is a co-stimulatory immune checkpoint molecule primarily used for activated T cells. Cross-linking of CD137 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 increases one or more functions of 4-1BB. An exemplary 4-1BB agonist is Utomitumab (PF-05082566), which is a fully human IgG2 monoclonal antibody that targets this 4-1BB in order to stimulate T cells.

[0072] As used herein, the term "CD161" (alternatively referred to as killer cell lectin-like receptor subfamily B, member 1 (KLRB1); NK1.1, or NKR-P1A) refers to a member of the C-type lectin superfamily. CD161 is a marker for T cells and CD161 expression is associated with T cell infiltration into the tumor microenvironment of many different cancer types. CD161 is further described in Fergusson et al., (2014) Cell Reports 9(3):1075-1088, which is incorporated herein by reference in its entirety.

[0073] As used herein, the term "IL-27" or "interleukin 27" refers to the IL-27 cytokine. IL-27 is related to the IL-6 / IL-12 cytokine family and is a heterodimeric cytokine that includes a first subunit called Epstein-Barr virus-induced gene 3 (EBI3; also known as IL-27 subunit β and IL-27B) and a second subunit called IL-27p28 (also known as IL30, IL-27 subunit α and IL-27A). IL-27 is primarily 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 pro-inflammatory effects, many studies suggest that IL-27 plays an important role as an immunosuppressant (Shimizu et al. (2006) J. Immunol. 176:7317-7324, Hisada et al. (2004) Cancer Res. 64:1152-1156, Diakowski (2013) supra). Although IL-27 was originally described as a factor that promotes the initiation of Th1 responses, it was later found to play a significant T-cell suppressive function by limiting Th1 responses, inhibiting Th2 and Th17 cell differentiation, and regulating the development of Tr1 and other T regulatory cell populations (Dietrich et al. (2014) J. Immunol. 192:5382-5389). In addition to its role as an immunomodulator, IL-27 also regulates angiogenesis, erythropoiesis, and osteoclastogenesis (supra).

[0074] IL-27 signals through a heterodimeric type I cytokine receptor (IL-27 receptor or IL-27R), which comprises a first subunit called WSX1 (also known as IL-27 receptor subunit α, IL-27RA, T-cell cytokine receptor type 1 (TCCR), and cytokine receptor-like 1 (CRL1)) and a second subunit called gp130 (also known as interleukin-6 signal transducer (IL6ST), interleukin-6 receptor subunit β (IL-6RB), and oncostatin M-type receptor). gp130 is also a receptor subunit for IL-6 family cytokines (Liu et al. (2008) Scan. J. Immunol. 68:22-299, Diakowski (2013) supra). IL-27 signaling through IL-27R activates multiple signaling cascades, including the JAK-STAT and p38MAPK pathways.

[0075] It is also believed that EBI3 has biological functions that are independent of p28 or IL-27 heterodimers. For example, EBI3 also interacts with p35 to form the heterodimeric cytokine IL-35 (Yoshida et al. (2015) Annu. Rev Immunol. 33:417-43) 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):1217-28).

[0076] The amino acid sequence of an exemplary human EBI3 protein is provided in SEQ ID NO: 1 (NCBI Reference Sequence: NP_005746.2; N-MTPQLLL ALVLWASCPPCSGRKGPPAALTLPRVQCRASRYPIAVDCSWTLPPAPNSTSPVSFIATYRLGMAARGHSWPCLQQTPTSTSCTITDVQLFSMAPYVLNVTAVHPWGSSSSFVPFITEHIIKPDPPEGVRLSPLAERQLQVQWEPPGSWPFPEIFSLKYWIRYKRQGAARFHRVGPIEATSFILRAVRPRARYYVQVAAQDLTDYGELSDWSLPATATMSLGK-C). The amino acid sequence of an exemplary human p28 protein is provided in SEQ ID NO: 2 (NCBI Reference Sequence: NP_663634.2; N-MGQTAGDLGWRLSLLLLPLLLVQAGVWG FPRPPGRPQLSLQELRREFTVSLHLARKLLSEVRGQAHRFAESHLPGVNLYLLPLGEQLPDVSLTFQAWRRLSDPERLCFISTTLQPFHALLGGLGTQGRWTNMERMQLWAMRLDLRDLQRHLRFQVLAAGFNLPEEEEEEEEEEEEERKGLLPGALGSALQGPAQVSWPQLLSTYRLLHSLELVLSRAVRELLLLSKAGHSVWPLGFPTLSPQP-C) is provided in SEQ ID NO: 3 (NCBI reference sequence: NP_004834.1; N-MRGGRGAPFWLWPLPKLALLPLLWVLFQRTRPQGSAGPLQCYGVGPLGDLNCSWEPLGDLGAPSELHLQSQKYRSNKTQTVAVAAGRSWVAIPREQLTMSDKLLVWGTKAGQPLWPPVFVNLETQMKPNAPRLGPDVDFSEDDPLEATVHWAPPTWPSHKVLICQFHYRRCQEAAWTLLEPELKTIPLTPVEIQDLELATGYKVYGRCRMEKEEDLWGEWSPILSFQTPPSAPKDVWVSGNLCGTPGGEEPLLLWKAPGPCVQVSYKVWFWVGGRELSPEGITCCCSLIPSGAEWARVSAVNATSWEPLTNLSLVCLDSASAPRSVAVSSIAGSTELLVTWQPGPGEPLEHVVDWARDGDPLEKLNWVRLPPGNLSALLPGNFTVGVPYRITVTAVSASGLASASSVWGFREELAPLVGPTLWRLQDAPPGTPAIAWGEVPRHQLRGHLTHYTLCAQSGTSPSVCMNVSGNTQSVTLPDLPWGPCELWVTASTIAGQGPPGPILRLHLPDNTLRWKVLPGILFLWGLFLLGCGLSLATSGRCYHLRHKVLPRWVWEKVPDPANSSSGQPHMEQVPEAQPLGDLPILEVEEMEPPPVMESSQPAQATAPLDSGYEKHFLPTPEELGLLGPPRPQVLA-C).The amino acid sequence of an exemplary human gp130 protein is provided in SEQ ID NO: 4 (NCBI reference sequence: NP_002175.2; N-MLTLQTWLVQALFIFLT TESTGELLDPCGYISPESPVVQLHSNFTAVCVLKEKCMDYFHVNANYIVWKTNHFTIPKEQYTIINRTASSVTFTDIASLNIQLTCNILTFGQLEQNVYGITIISGLPPEKPKNLSCIVNEGKKMRCEWDGGRETHLETNFTLKSEWATHKFADCKAKRDTPTSCTVDYSTVYFVNIEVWVEAENALGKVTSDHINFDPVYKVKPNPPHNLSVINSEELSSILKLTWTNPSIKSVIILKYNIQYRTKDASTWSQIPPEDTASTRSSFTVQDLKPFTEYVFRIRCMKEDGKGYWSDWSEEASGITYEDRPSKAPSFWYKIDPSHTQGYRTVQLVWKTLPPFEANGKILDYEVTLTRWKSHLQNYTVNATKLTVNLTNDRYLATLTVRNLVGKSDAAVLTIPACDFQATHPVMDLKAFPKDNMLWVEWTTPRESVKKYILEWCVLSDKAPCITDWQQEDGTVHRTYLRGNLAESKCYLITVTPVYADGPGSPESIKAYLKQAPPSKGPTVRTKKVGKNEAVLEWDQLPVDVQNGFIRNYTIFYRTIIGNETAVNVDSSHTEYTLSSLTSDTLYMVRMAAYTDEGGKDGPEFTFTTPKFAQGEIEAIVVPVCLAFLLTTLLGVLFCFNKRDLIKKHIWPNVPDPSKSHIAQWSPHTPPRHNFNSKDQMYSDGNFTDVSVVEIEANDKKPFPEDLKSLDLFKKEKINTEGHSSGIGGSSCMSSSRPSISSSDENESSQNTSSTVQYSTVVHSGYRHQVPSVQVFSRSESTQPLLDSEERPEDLQLVDHVDGGDGILPRQQYFKQNCSQHESSPDISHFERSKQVSSVNEEDFVRLKQQISDHISQSCGSGQMKMFQEVSAADAFGPGTEGQVERFETVGMEAATDEGMPKSYLPQTVRQGGYMPQ-C).

[0077] As used herein, the term "competition" when used in the context of antigen binding proteins (e.g., immunoglobulins, antibodies, or antigen binding fragments thereof) competing for binding to the same antigenic determinant refers to the interaction between antigen binding proteins as determined by an assay (e.g., competitive binding assay; cross-blocking assay) in which a test antigen binding protein (e.g., test antibody) inhibits (e.g., reduces or blocks) specific binding of a reference antigen binding protein (e.g., reference antibody) to a common antigen (e.g., IL-27 or a fragment thereof).

[0078] A polypeptide or amino acid sequence "derived from" a specified polypeptide or protein relates to the origin of the polypeptide. Preferably, a polypeptide or amino acid sequence derived from a specific sequence has an amino acid sequence that is substantially identical to that sequence or a portion thereof, wherein the portion consists of at least 10 to 20 amino acids, preferably at least 20 to 30 amino acids, more preferably at least 30 to 50 amino acids, or the sequence origin of the polypeptide or amino acid sequence can be identified in other ways by a person of ordinary skill in the art. A polypeptide derived from another peptide may have one or more mutations relative to the starting polypeptide, such as having been substituted by another amino acid residue or having one or more amino acid residues inserted or deleted.

[0079] Polypeptide can comprise non-natural amino acid sequence.Such variants necessarily have less than 100% sequence identity or similarity with the starting molecule.In some aspects, the amino acid sequence that variant has will have about 75% to less than 100% amino acid sequence identity or similarity with the amino acid sequence of the starting polypeptide, more preferably about 80% to less than 100%, more preferably about 85% to less than 100%, more preferably about 90% to less than 100% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) and more preferably about 95% to less than 100%, for example, within the length range of the variant molecule.

[0080] In certain aspects, the antibodies of the present disclosure are encoded by nucleotide sequences. The nucleotide sequences of the present invention can be used in many applications, including: cloning, gene therapy, protein expression and purification, mutation introduction, DNA vaccination of hosts in need, antibody production for passive immunization, PCR, primer and probe production, etc.

[0081] It is also understood by those of ordinary skill that antibodies suitable for use in the methods disclosed herein can be altered to vary the sequence of the naturally occurring or native sequence from which it is derived while retaining the desired activity of the native sequence. For example, nucleotide or amino acid substitutions can be made to produce conservative substitutions or changes at "non-essential" amino acid residues. Mutations can be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis.

[0082] Antibodies suitable for use in the methods disclosed herein may include conservative amino acid substitutions at one or more amino acid residues, for example, at essential or non-essential amino acid residues. A "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art and include 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 non-essential amino acid residue in a binding polypeptide is preferably replaced with another amino acid residue from the same side chain family. In certain aspects, a string of amino acids can be replaced with a structurally similar string that differs in the order and / or composition of the side chain family members. Alternatively, in certain aspects, mutations can be introduced randomly along all or a portion of the coding sequence, such as by saturation mutagenesis, and the resulting mutants can be incorporated into the binding polypeptides of the invention and screened for their ability to bind to a desired target.

[0083] As used herein, the term antigen "cross-presentation" refers to the presentation of foreign protein antigens to T cells via MHC class I and class II molecules on APCs.

[0084] As used herein, the term "cross-reactivity" refers to the ability of an antibody of the disclosure to bind to IL-27 from a different species. For example, an antibody of the disclosure that binds to human IL-27 may also bind to IL-27 from another species. As used herein, cross-reactivity is measured by detecting specific reactivity with purified antigen in a binding assay (e.g., SPR, ELISA) or binding to cells that physiologically express IL-27, or otherwise functionally interacting with such cells. Methods for determining cross-reactivity include standard binding assays as described herein, for example, by Biacore™ surface plasmon resonance (SPR) analysis using a Biacore™ 2000 SPR instrument (Biacore AB, Uppsala, Sweden), or flow cytometry.

[0085] As used herein, the term "cytotoxic T lymphocyte (CTL) response" refers to an immune response induced by cytotoxic T cells. CTL responses are mainly mediated by CD8+ T cells.

[0086] As used herein, the term "dendritic cell" or "DC" refers to a type of antigen presenting cell that is a bone marrow (BM) derived white blood cell and is the most efficient type of antigen presenting cell. DCs capture and process antigens, converting proteins into peptides that are presented on major histocompatibility complex (MHC) molecules recognized by T cells. DCs are heterogeneous, such as myeloid and plasmacytoid DCs; while all DCs are capable of taking up antigen, processing and presenting to naive T cells, DC subtypes have different markers and differ in location, migration pathways, detailed immune functions, and dependence on infectious or inflammatory stimuli for the generation of these DC subtypes. During the development of adaptive immune responses, the phenotype and function of DCs play a role in eliciting tolerance, memory, and polarized T-helper 1 (Th1), Th2, and Th17 differentiation.

[0087] As used herein, the term "dendritic cell activation" refers to the transition from immature to mature dendritic cells; and activated dendritic cells include mature dendritic cells and dendritic cells in the transition process, in which the expression of CD80 and CD86 that induce costimulatory signals is increased due to activation stimulation. Mature human dendritic cells are cells that are positive for the expression of CD40, CD80, CD86 and HLA-II class (e.g., HLA-DR). Immature dendritic cells can be distinguished from mature dendritic cells, for example, based on markers selected from the group consisting of CD80 and CD86. For these markers, immature dendritic cells are weakly positive and preferably negative, while mature dendritic cells are positive. The distinction between mature dendritic cells is routinely performed by those skilled in the art, and the corresponding markers and methods for measuring their expression as described above are also well known to those skilled in the art.

[0088] As used herein, the term "EC50" refers to the concentration of an antibody, or antigen-binding portion thereof, that induces a response in an in vitro or in vivo assay that is 50% of the maximal response, ie, halfway between the maximal response and the baseline.

[0089] As used herein, 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 an amount sufficient to cure or at least partially stop the disease and its complications in a patient already suffering from the disease. The amount effective for this use will depend on the severity of the condition being treated and the general state of the patient's own immune system.

[0090] As used herein, the term "antigenic determinant" or "antigenic determinant" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds. The term "antigenic determinant mapping" refers to a process or method for identifying a binding site or antigenic determinant of an antibody or its antigen-binding fragment on its target protein antigen. Provided herein are methods and techniques for antigenic determinant mapping. Antigenic determinants can be formed by contiguous amino acids or non-contiguous amino acids juxtaposed by the tertiary folding of proteins. Antigenic determinants formed by contiguous amino acids are generally retained upon exposure to denaturing solvents, while antigenic determinants formed by tertiary folding are generally lost upon treatment with denaturing solvents. Antigenic determinants typically include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in a unique spatial configuration. Methods for determining which antigenic determinants are bound by a given antibody (i.e., antigenic determinant localization) 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 reactivity with a given anti-IL-27 antibody. Methods for determining the spatial configuration of an antigenic determinant include techniques in the art and those described herein, e.g., x-ray crystallography and 2-dimensional nuclear magnetic resonance (see, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GE Morris, ed. (1996)).

[0091] The present disclosure also encompasses antibodies that bind to an antigenic determinant on IL-27 that comprises all or part of the antigenic determinant (e.g., the same or overlapping region or regions between or across regions) recognized by a specific antibody described herein.

[0092] The present disclosure also encompasses antibodies that bind to the same antigenic determinant as the antibodies described herein and / or antibodies that compete with the antibodies described herein for binding to human IL-27. Antibodies that recognize the same antigenic determinant or compete for binding can be identified using conventional techniques. Such techniques include, for example, immunoassays that show the ability of one antibody to block the binding of another antibody to a target antigen, i.e., competitive binding assays. Competitive binding is determined in an assay in which the immunoglobulin being tested inhibits specific binding of a reference antibody to a common antigen, such as IL-27. Many types of competitive binding assays are known, for example: solid phase direct or indirect radioimmunoassays (RIA), solid phase direct or indirect enzyme immunoassays (EIA), sandwich competition assays (see Stahli et al., Methods in Enzymology 9:242 (1983)); solid phase direct biotin-avidin EIA (see Kirkland et al., J. Immunol. 137:3614 (1986)); solid phase direct labeled assays, solid phase direct labeled sandwich assays (see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988)); solid phase direct labeled RIA using an I-125 label (see Morel et al., Mol. Immunol. 25(1):7 (1988)); solid phase direct biotin-avidin EIA (Cheung et al., Virology 176:546 (1990)); and direct labeled RIA. (Moldenhauer et al., Scand. J. Immunol. 32:77 (1990)). Typically, the assay involves the use of purified antigen bound to a solid surface, or cells bearing either of the unlabeled test immunoglobulins and labeled reference immunoglobulins. Competitive inhibition is measured by determining the amount of label bound to the solid surface or cells in the presence of the test immunoglobulin. Typically, the test immunoglobulin is present in excess. Typically, when the competing antibody is present in excess, it will inhibit specific binding of the reference antibody to the common antigen by at least 50-55%, 55-60%, 60-65%, 65-70%, 70-75% or more.

[0093] Other techniques include, for example, methods for antigenic determinant mapping, such as x-ray analysis of crystals of antigen:antibody complexes that provide atomic resolution of antigenic determinants and mass spectrometry combined with hydrogen / deuterium (H / D) exchange to study the conformation and dynamics of antigen:antibody interactions. Other methods monitor the binding of antibodies to antigenic fragments or mutant variations of antigens, where loss of binding due to modification of amino acid residues within the antigenic sequence is often considered indicative of antigenic determinant composition. In addition, computational combinatorial methods for antigenic determinant mapping can also be used. These methods rely on the ability of related antibodies to affinity isolate specific short peptides from combinatorial phage-displayed peptide libraries. These peptides are then considered as leader peptides for defining the antigenic determinant corresponding to the antibody used to screen the peptide library. For antigenic determinant mapping, computational algorithms have also been developed that show the mapping of conformationally discontinuous epitopes.

[0094] As used herein, the term "Fc-mediated effector function" or "Fc effector function" refers to the biological activity of an antibody other than the primary function and use of the antibody. For example, the effector function of a therapeutic agonist antibody is a biological activity 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 receptors (e.g., B cell receptor); lack of activation of platelets expressing Fc receptors; and B cell activation. Many effector functions begin with Fc binding to Fcγ receptors. In some aspects, a tumor antigen-targeted antibody has an effector function, e.g., ADCC activity. In some aspects, a tumor antigen-targeted antibody described herein comprises a variant constant region that has an increased effector function (e.g., increased ability to mediate ADCC) relative to an unmodified form of the constant region.

[0095] As used herein, the term "Fc receptor" refers to a polypeptide present on the surface of immune effector cells that is bound by the Fc region of an antibody. In some aspects, the Fc receptor is an Fcγ receptor. There are three subclasses of Fcγ receptors, namely FcγRI (CD64), FcγRII (CD32), and FγcRIII (CD16). All four IgG isotypes (IgG1, IgG2, IgG3, and IgG4) bind to and activate the Fc receptors FcγRI, FcγRIIA, and FcγRIIIA. FcγRIIB is an inhibitory receptor, and therefore antibodies bound to this receptor do not activate complement and cellular responses. FcγRI is a high affinity receptor that binds to IgG in monomeric form, while FcγRIIA and FcγRIIA are low affinity receptors that bind to IgG only in multimeric form and with slightly lower affinity. Antibody binding to Fc receptors and / or C1q is controlled by specific residues or domains within the Fc region. Binding also depends on residues located in the hinge region and in the CH2 portion of the antibody. In some aspects, the agonist and / or therapeutic activity of the antibodies described herein depends on binding of the Fc region to an Fc receptor (e.g., FcγR). In some aspects, the agonist and / or therapeutic activity of the antibodies described herein is enhanced due to binding of the Fc region to an Fc receptor (e.g., FcγR).

[0096] A list of certain Fc receptor sequences used in the present disclosure is set forth in Table 13 below.

[0097] As used herein, the term "glycosylation pattern" is defined as the pattern of carbohydrate units covalently attached to a protein, more specifically an immunoglobulin. The glycosylation pattern of a heterologous antibody can be characterized as being substantially similar to the glycosylation pattern that occurs naturally on antibodies produced by a non-human transgenic animal species when the skilled artisan recognizes that the glycosylation pattern of a heterologous antibody is more similar to that in a non-human transgenic animal species than the species from which the CH genes of the transgene originate.

[0098] As used herein, the term "human antibody" includes antibodies having variable and constant regions (if present) of human germline immunoglobulin sequences. The human antibodies of the present disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutagenesis 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. NY Acad. Sci. 764: 536-546). However, the term "human antibody" does not include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences (ie, humanized antibodies).

[0099] As used herein, the term "heterologous antibody" is defined with respect to the transgenic non-human organism that produces such an antibody. This term refers to an antibody having an amino acid sequence or encoding nucleic acid sequence corresponding to that found in an organism not composed of a transgenic non-human animal, and generally from a species other than a transgenic non-human animal.

[0100] The terms "induce an immune response" and "enhance an immune response" are used interchangeably and refer to the stimulation of an immune response (ie, passive or adaptive) to a specific antigen. The term "induce" as used with respect to the induction of CDC or ADCC refers to the stimulation of a specific direct cell killing mechanism.

[0101] As used herein, the term "immunogenic cell death" (alternatively referred to as "immunogenic apoptosis") refers to a cell death mode associated with activation of one or more signaling pathways that induces pro-death expression and release of damage-associated molecular pattern (DAMP) molecules (e.g., adenosine triphosphate, ATP) from tumor cells, leading to increased immunogenicity of tumor cells and death of tumor cells in an immunogenic manner (e.g., by phagocytosis). As used herein, the term "immunogenic cell death inducer" refers to a chemical, biological or pharmacological agent that induces an immunogenic cell death process, pathway or mode. [] []

[0102] As used herein, the terms "inhibit", "reduce" or "block" (e.g., involving inhibition or reduction of human IL-27-mediated phosphorylation of STAT1 and / or STAT3 in cells) are used interchangeably and include both partial and complete inhibition / blocking. Inhibition / blocking of IL-27 reduces or alters the normal level or type of activity that occurs in the absence of inhibition or blocking. Inhibition and blocking are also intended to include any measurable decrease in the binding affinity of IL-27 when contacted with an anti-IL-27 antibody as compared to IL-27 not contacted with the anti-IL-27 antibody, for example, inhibiting the binding of IL-27 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%.

[0103] As used herein, the terms "inhibit angiogenesis," "reduce angiogenesis," and "reduce angiogenesis" refer to reducing the level of angiogenesis in a tissue to at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or less of the amount in a corresponding control tissue, and preferably to the same level observed in a control tissue.

[0104] As used herein, the term "inhibit growth" (e.g., involving cells) is intended to include any measurable reduction in cell growth, e.g., inhibiting cell growth by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100%.

[0105] As used herein, a subject "in need of prevention," "in need of treatment," or "in need thereof" refers to a subject who, according to the judgment of an appropriate medical practitioner (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 (such as treatment with a composition comprising an anti-IL-27 antibody).

[0106] The term "in vivo" refers to processes that occur in living organisms.

[0107] As used herein, the term "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds to human IL-27 is substantially free of antibodies that specifically bind to antigens other than IL-27). However, an isolated antibody that specifically binds to an antigenic determinant may have cross-reactivity with other IL-27 proteins from different species. However, in a specific binding assay as described herein, the antibody continues to exhibit specific binding to human IL-27. In addition, the isolated antibody is typically substantially free of other cellular material and / or chemicals. In some aspects, a combination of "isolated" antibodies having different IL-27 specificities are combined in a well-defined composition.

[0108] As used herein, the term "isolated nucleic acid molecule" refers to a nucleic acid encoding an antibody or antibody portion (e.g., VH, VL, CDR3) that binds to IL-27, and is intended to refer to a nucleic acid molecule in which the nucleotide sequence encoding the antibody or antibody portion does not contain other nucleotide sequences encoding antibodies or antibody portions that bind to antigens other than IL-27, which other sequences may naturally flank the nucleic acid in human genomic DNA. For example, a sequence selected from the sequences set forth in Table 12 corresponds to a nucleotide sequence comprising the heavy chain (VH) and light chain (VL) variable regions of the anti-IL-27 antibody monoclonal antibody described herein.

[0109] As used herein, "isotype" refers to the class of antibodies encoded by the heavy chain constant region gene (e.g., IgM or IgG1). In some aspects, the human monoclonal antibodies of the present disclosure are of the IgG1 isotype. In some aspects, the human monoclonal antibodies of the present disclosure are of the IgG2 isotype. In some aspects, the human monoclonal antibodies of the present disclosure are of the IgG3 isotype. In some aspects, the human monoclonal antibodies of the present disclosure are of the IgG4 isotype. As will be apparent to one 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 alignments with known antibodies, published Fc variant sequences, and conserved sequences.

[0110] As used herein, the term "isotype switching" refers to the phenomenon whereby the class or isotype of an antibody changes from one Ig class to one of the other Ig classes.

[0111] As used herein, the term "KD" or "KD" refers to the equilibrium dissociation constant of the binding reaction between an antibody and an antigen. The value of KD is a numerical representation of the ratio of the antibody dissociation rate constant (kd) to the antibody association rate constant (ka). The value of KD is inversely related to the binding affinity of the antibody to the antigen. The smaller the KD value, the greater the affinity of the antibody for its antigen. Affinity is the strength of the binding of a single molecule to its ligand and is usually measured and reported by the equilibrium dissociation constant (KD), which is used to evaluate and rank the strength of bimolecular interactions.

[0112] As used herein, the term "kd" or "kd" (or "k dissociation" or "k dissociation") refers to the dissociation rate constant for the dissociation of an antibody from an antibody / antigen complex. The value of kd is a numerical representation of the fraction of the complex that dissociates or dissociates per second and is expressed in units of sec-1.

[0113] As used herein, the term "ka" or "ka" (or "k association" or "k association") refers to the association rate constant of the association of an antibody with an antigen. The value of ka is a numerical representation of the number of antibody / antigen complexes formed per second in a 1 molar (1 M) solution of the antibody and antigen, and is expressed in units of M-1sec-1.

[0114] As used herein, the term "leukocyte" refers to the type of white blood cells involved in defending the body against infectious organisms and foreign substances. Leukocytes are produced in the bone marrow. There are 5 major types of leukocytes, which are subdivided into 2 major groups: polymorphonuclear leukocytes (neutrophils, eosinophils, basophils) and mononuclear leukocytes (monocytes and lymphocytes).

[0115] As used herein, the term "lymphocyte" refers to a type of white blood cell or leukocyte that is involved in the body's immune defense. There are two main types of lymphocytes: B-cells and T-cells.

[0116] As used herein, the terms "linked", "fused" or "fusion" are used interchangeably. These terms refer to the joining together of two or more elements or components or domains by whatever means, including chemical conjugation or recombinant means. Methods of chemical conjugation (e.g., using heterobifunctional cross-linking agents) are known in the art.

[0117] As used herein, "local administration" or "local delivery" refers to delivery that does not rely on transport of a composition or agent to its intended target tissue or site 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 a target tissue or site, the composition or agent, or one or more components thereof, can diffuse to the intended target tissue or site.

[0118] As used herein, "MHC molecules" refers to two types of molecules, namely MHC class I and MHC class II. MHC class I molecules present antigens to specific CD8+ T cells and MHC class II molecules present antigens to specific CD4+ T cells. Antigens delivered exogenously to APCs are primarily processed to associate with MHC class II. In contrast, antigens delivered endogenously to APCs are primarily processed to associate with MHC class I.

[0119] As used herein, the term "monoclonal antibody" refers to an antibody that displays a single binding specificity and affinity for a particular antigenic determinant. Thus, the term "human monoclonal antibody" refers to an antibody that displays a single binding specificity and has a variable region and, optionally, a constant region derived from human germline immunoglobulin sequences. In some aspects, human monoclonal antibodies are produced by a fusion tumor that includes a B cell obtained from a transgenic non-human animal (e.g., a transgenic mouse) that has a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell.

[0120] As used herein, the term "monocyte" refers to a type of white blood cell and can differentiate into macrophages and dendritic cells in order to carry out an immune response.

[0121] As used herein, the term "natural killer (NK) cells" refers to a type of cytotoxic lymphocyte. These cells are large, usually granular, non-T, non-B lymphocytes that kill certain tumor cells and play an important role in innate immunity to viruses and other intracellular pathogens, as well as in antibody-dependent cell-mediated cytotoxicity (ADCC).

[0122] As used herein, the term "naturally occurring" as applied to an object refers to the fact that an object can exist in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and has not been intentionally modified by man in the laboratory is naturally occurring.

[0123] As used herein, the term "non-switched isotype" refers to the isotype class of heavy chains produced when no isotype switching occurs; the CH gene encoding the non-switched isotype is usually the first CH gene immediately downstream of the functionally rearranged VDJ gene. Isotype switching is classified as either traditional or non-traditional isotype switching. Traditional isotype switching occurs by a recombination event involving at least one switch sequence region in a transgene. Non-traditional isotype switching can occur, for example, by homologous recombination between human σμ and human Σμ (delta-associated deletion). Alternative non-traditional switching mechanisms, such as inter-transgene and / or inter-chromosomal recombination, can occur and achieve isotype switching.

[0124] As used herein, the term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in single-stranded or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with 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, modifications at the second base can also be conservative. The term nucleic acid can be used interchangeably with gene, cDNA, and mRNA encoded by a gene.

[0125] As used herein, a polynucleotide may be composed of any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. For example, a polynucleotide may be composed 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, and RNA that is a mixture of single-stranded and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or more commonly double-stranded or a mixture of single-stranded and double-stranded regions. In addition, a polynucleotide may be composed of triple-stranded regions comprising RNA or DNA or both RNA and DNA. A polynucleotide may also contain one or more modified bases or a DNA or RNA backbone that has been modified for stability or other reasons. "Modified" bases include, for example, tritylated bases and unusual bases such as inosine. Various modifications may be made to RNA and DNA; thus, "polynucleotide" includes chemically, enzymatically, or metabolically modified forms.

[0126] 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 it affects the transcription of the sequence. For transcriptional regulatory sequences, operably linked means that the linked DNA sequences are contiguous and, where necessary to join two protein coding regions, contiguous and in reading frame. For switch sequences, operably linked means that the sequences are capable of effecting switch recombination.

[0127] As used herein, "parenteral administration" administration) and parenteral administration The terms “intravenous,” “intraocular,” “intraocular,” “intramuscular,” “intraarterial,” “intrathecal,” “intraorbital,” “intracardiac,” “intradermal,” “intraperitoneal,” “transtracheal,” “subcutaneous,” “subcutaneous,” “intraarticular,” “subcapsular,” “subarachnoid,” “intraspinal,” “epidural,” “intracerebral,” “incranial,” “intracarotid,” and “intrasternal” injection and infusion are intended to include, but are not limited to, intravenous, intranasal, intraocular, intramuscular, intraarticular, intraarticular, intracarotid, and intrasternal injection and infusion.

[0128] As used herein, the term "patient" includes human and other mammalian subjects receiving prophylactic or therapeutic treatment.

[0129] As used herein, the term "PD-1 antagonist" refers to any 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, a PD-1 antagonist blocks the binding of PD-L1 to PD-1 and / or the binding of PD-L2 to PD-1. In some aspects, a PD-1 antagonist specifically binds to PD-1. In some aspects, a PD-1 antagonist specifically binds to PD-L1.

[0130] In the context of two or more nucleic acid or polypeptide sequences, the term "percent identity" refers to two or more sequences or subsequences that have a specified percentage of identical nucleotides or amino acid residues 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 over a region of the sequences being compared, e.g., a functional domain, or alternatively, over the entire length of the two sequences being compared. For sequence comparison, typically one sequence serves as a reference sequence to which a test sequence is compared. When using a sequence comparison algorithm, both the test sequence and the reference sequence are entered into a computer, subsequence coordinates are set if necessary, and then the sequence algorithm program parameters are set. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the set program parameters.

[0131] Optimal alignment of sequences for comparison can be performed, 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 search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of such algorithms (GAP, BestFit, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally Ausubel et al., below).

[0132] An 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 through the National Center for Biotechnology Information website.

[0133] As generally used herein, "pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms that are suitable, within the scope of sound medical judgment, for use in contact with the tissues, organs and / or body fluids of humans and animals without excessive toxicity, irritation, allergic response or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0134] As used herein, "pharmaceutically acceptable carrier" means and includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. The composition may include a pharmaceutically acceptable salt, for example, an acid addition salt or a base addition salt (see, for example, Berge et al. (1977) J Pharm Sci 66: 1-19).

[0135] As used herein, the terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to polymers 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 naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.

[0136] As used herein, the term "preventing" when used with respect to a condition refers to the administration of a composition that reduces the frequency of symptoms of, or delays the onset of, a medical condition in a subject relative to a subject not receiving the composition.

[0137] As used herein, the term "purified" or "isolated" as applied to any protein (antibody or fragment) described herein refers to a polypeptide that is separated or purified from components (e.g., proteins or other naturally occurring biological or organic molecules) that naturally accompany the polypeptide in the prokaryotic organism that expresses the protein, such as other proteins, lipids, and nucleic acids. 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.

[0138] As used herein, the term "planned cell death protein 1" or "PD-1" refers to the planned cell death protein 1 polypeptide, which belongs to the CD28 family of immunoinhibitory receptors and is encoded by the PDCD1 gene in humans. Alternative names or synonyms of PD-1 include: PDCD1, PD1, CD279, and SLEB2. PD-1 is primarily expressed on previously 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 (hPD-1), variants, isoforms, and species homologs of hPD-1, and analogs that share at least one common antigenic determinant with hPD-1. The complete hPD-1 sequence can be found under GenBank accession number AAC51773.

[0139] As used herein, the term "programmed death ligand-1" or "PD-L1" refers to one of the two cell surface glycoprotein ligands for PD-1 (the other being PD-L2) that downregulates T-cell activation and cytokine secretion after binding to PD-1. Alternative names and synonyms for PD-L1 include: PDCD1L1, PDL1, B7H1, B7-4, CD274, and B7-H. As used herein, the term "PD-L1" includes human PD-L1 (hPD-L1), variants, isoforms, and species homologs of hPD-L1, and analogs that share at least one common antigenic determinant with hPD-L1. The complete hPD-L1 sequence can be found under GenBank accession number Q9NZQ7.

[0140] PD-1 is known as an immunosuppressive 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 can act as an immune checkpoint, which can lead to a decrease in 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). Immunosuppression can be reversed by inhibiting the local interaction of PD-1 with PD-L1 or PD-L2; the effect is additive when 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).

[0141] For many cancers, tumor survival and proliferation are maintained by tumor-mediated immune checkpoint regulation. This regulation can lead to the disruption of anti-cancer immune system function. For example, recent studies have shown that immune checkpoint receptor ligands such as PD-L1 or PD-L2 can downregulate immune system activity in the tumor microenvironment and promote cancer immune evasion, particularly by suppressing T cells, through expression by tumor cells. PD-L1 is abundantly expressed by various human cancers (Dong et al., (2002) Nat Med 8:787-789). The receptor for PD-L1, PD-1, is expressed on lymphocytes (e.g., activated T cells) and is generally involved in downregulating the immune system and promoting self-tolerance, particularly by suppressing T cells. However, when the PD-1 receptor expressed on T cells binds to the cognate PD-L1 ligand on tumor cells, the resulting T cell suppression results in impaired immune responses against the tumor (e.g., a decrease in tumor-infiltrating lymphocytes or the establishment of immune evasion of cancer cells).

[0142] In larger sample groups such as ovarian, renal, colorectal, pancreatic, liver cancers and melanoma, PD-L1 expression has been shown to be associated with a poor prognosis and reduced overall survival, regardless 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 Res 11:2947-2953). 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, 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 expression on tumor lymphocytes was found to mark dysfunctional T cells. PD-1 antagonists, such as those that affect 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 PD-L2, have been developed and represent a novel class of anti-tumor inhibitors that act by modulating immune cell-tumor cell interactions.

[0143] As used herein, the term "rearrangement" refers to the configuration of a heavy or light chain immunoglobulin locus in which the V segment is positioned adjacent to the DJ or J segment in a configuration encoding a substantially complete VH or VL domain, respectively. Rearranged immunoglobulin gene loci can be identified by comparison to germline DNA; rearranged loci will have at least one recombined heptamer / nonamer homology element.

[0144] As used herein, the term "recombinant host cell" (or simply "host cell") is intended to refer to a cell into which a recombinant expression vector has been introduced. It should be understood that these terms are intended to refer not only to a particular individual cell, but also to the progeny of that cell. Because certain modifications may occur in the progeny due to mutation or environmental influences, such progeny may not in fact be identical to the parent cell, but are still included in the scope of the term "host cell" as used herein.

[0145] As used herein, the term "recombinant human antibody" includes all human antibodies prepared, expressed, generated or isolated by recombinant means, such as (a) antibodies isolated from animals (e.g., mice) genetically or chromosomally transfected with human immunoglobulin genes or hybridomas prepared therefrom, (b) antibodies isolated from host cells transformed to express antibodies (e.g., from transfectomas), (c) antibodies isolated from recombinant, combinatorial human antibody libraries, and (d) antibodies prepared, expressed, generated or isolated by any other means that involve splicing human immunoglobulin gene sequences to other DNA sequences. These recombinant human antibodies contain variable and constant regions that utilize specific human germline immunoglobulin sequences encoded by germline genes, but include subsequent rearrangements and mutations that occur, for example, during antibody maturation. As is known in the art (see, e.g., Lonberg (2005) Nature Biotech. 23(9): 1117-1125), the variable region contains the antigen binding domain, which is encoded by various genes that are rearranged to form antibodies specific for foreign antigens. In addition to rearrangement, the variable region can be further modified by a variety of single amino acid changes (called somatic mutations or hypermutations) to increase the affinity of the antibody for foreign antigens. The constant region changes in further response to the antigen (i.e., isotype switching). Thus, the rearranged and somatically mutated nucleic acid molecules encoding light and heavy chain immunoglobulin polypeptides in response to an antigen may not have sequence identity to the original nucleic acid molecule, but instead may be substantially identical or similar (i.e., have at least 80% identity).

[0146] As used herein, the term "reference antibody" (interchangeably used with "reference mAb") or "reference antigen binding protein" refers to an antibody or antigen binding fragment thereof that binds to a specific antigenic determinant on IL-27 and is used to establish a relationship between itself and one or more different antibodies, wherein the relationship is binding of the reference antibody and one or more different antibodies to the same antigenic determinant on IL-27. As used herein, the term implies that an anti-IL-27 antibody is suitable as a competitor in a test or assay, such as a test or assay described herein (e.g., a competitive binding assay), wherein the assay is suitable for discovering, identifying or developing one or more different antibodies that bind to the same antigenic determinant.

[0147] As used herein, the terms "specific binding", "selective binding", "selectively binds" and "specifically binds" refer to the binding of an antibody to an antigenic determinant on a predetermined antigen. Typically, when measured by surface plasmon resonance (SPR) technology in a BIACORE 2000 instrument using recombinant human IL-27 as an analyte and using the antibody as a ligand, the antibody binds with an equilibrium dissociation constant (KD) of about less than 10-6M, such as about less than 10-7M, 10-8M, 10-9M or 10-10M or even lower, and the affinity of binding to the predetermined antigen is at least two times greater than its affinity for binding to a nonspecific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely related antigen. In certain aspects, when measured by surface plasmon resonance (SPR) technology in a BIACORE 2000 instrument using recombinant human IL-27 as an analyte and the antibody as a ligand, the antibody binds with an equilibrium dissociation constant (KD) of about less than 100 nM (10-7M), optionally about less than 50 nM (5 x 10-8M), optionally about less than 15 nM (1.5 x 10-8M), optionally about less than 10 nM (10-8M), optionally about less than 5 nM (5 x 10-9M), optionally about less than 1 nM (10-9M), optionally about less than 0.1 nM (10-10M), optionally about less than 0.01 nM (10-11M), and binds to the predetermined antigen with an affinity that is at least two times greater than its affinity for binding to a nonspecific antigen other than the predetermined antigen or a closely related antigen (e.g., BSA, casein). The phrases "an antibody that recognizes an antigen" and "an antibody specific for an antigen" are used interchangeably herein with the term "an antibody that specifically binds to an antigen."

[0148] As used herein, the term "STAT1 phosphorylation" refers to the phosphorylation of the signal transducer and activator of transcription 1 (STAT1) polypeptide, i.e., the transcription factor encoded by the STAT1 gene in humans. STAT molecules are phosphorylated by receptor-associated kinases, resulting in activation and dimerization by forming homo- or heterodimers that translocate to the nucleus to act 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 results in phosphorylation of STAT1 (pSTAT1). STAT1 plays a key role in the expression of genes involved in cell survival, viability, or pathogen response. Methods for determining STAT1 phosphorylation due to 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).

[0149] As used herein, the term "STAT3 phosphorylation" refers to the phosphorylation of the signal transducer and activator of transcription 3 (STAT3) polypeptide, i.e., the transcription factor encoded by the STAT3 gene in humans. STAT3 mediates the expression of various genes in response to cellular stimulation and thus plays a key role in many cellular processes such as cell growth and apoptosis. Methods for determining STAT3 phosphorylation due to 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).

[0150] As used herein, the term "switch sequence" refers to those DNA sequences that are responsible for switch recombination. The "switch donor" sequence, usually the μ switch region, is located 5' (i.e., upstream) of the region of the construct to be deleted during switch recombination. The "switch acceptor" region is located between the region of the construct to be deleted and the replacement constant region (e.g., γ, ε, etc.). Because there is no specific site where recombination always occurs, the final gene sequence is usually not predictable from the construct.

[0151] 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 individuals with immune disorders. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cows, chickens, amphibians, reptiles, etc.

[0152] For nucleic acids, the term "substantial homology" indicates that two nucleic acids, or designated sequences thereof, when optimally aligned and compared, are identical in at least about 80% of the nucleotides, usually at least about 90% to 95%, and more preferably at least about 98% to 99.5% of the nucleotides, with appropriate nucleotide insertions or deletions. Alternatively, substantial homology exists when a segment will hybridize under selective hybridization conditions to the complement of that strand.

[0153] The percent identity between the two sequences varies with the number of identical positions shared by the sequences (i.e., % homology = number of identical positions / total number of positions x 100), taking into account the number of gaps that need to be introduced to achieve optimal alignment of the two sequences and the length of each gap. Comparison of sequences and determination of the percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the following non-limiting examples.

[0154] The percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software suite (available at http: / / www.gcg.com) using the NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight 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)) incorporated into the ALIGN program (version 2.0) using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. (48): 444-453 (1970)) algorithm incorporated into the GAP program in the GCG software suite (available at http: / / www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0155] The nucleic acid and protein sequences of the present disclosure can further be used as "query sequences" to perform searches against public databases, e.g., to identify related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed using the NBLAST program, score = 100, wordlength = 12, to obtain nucleotide sequences homologous to the nucleic acid molecules of the present invention. BLAST protein searches can be performed using the XBLAST program, score = 50, wordlength = 3, to obtain amino acid sequences homologous to the protein molecules of the present invention. To obtain gapped alignments for comparison purposes, gapped BLAST as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402 can be used. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (eg, XBLAST and NBLAST) can be used. See http: / / www.ncbi.nlm.nih.gov.

[0156] Nucleic acids can be present in whole cells, cell lysates, or partially purified or substantially purified forms. Nucleic acids are "isolated" or "made substantially pure" when purified from other cellular components or other contaminants (e.g., other cellular nucleic acids or proteins) by standard techniques, including alkaline / SDS treatment, CsCl gradient centrifugation (CsCl banding), 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).

[0157] Although the nucleic acid compositions of the present disclosure are often in natural sequence (except for modified restriction sites and the like), mutations may be generated from cDNA, genome, or mixtures thereof to provide gene sequences according to standard techniques. For coding sequences, such mutations may affect the amino acid sequence as desired. Specifically, DNA sequences substantially homologous to or derived from natural V, D, J, constant, switch, and other such sequences described herein are contemplated (where "derived" indicates that a sequence is identical to or modified from another sequence).

[0158] As used herein, the term "STING" (or TMEM173) refers to a stimulator of interferon genes, i.e., a protein that acts as both a direct cytoplasmic DNA sensor and an adaptor protein. In humans, STING is encoded by the TMEM173 gene. STING plays an important role in innate immunity. When cells are infected with intracellular pathogens, such as viruses, mycobacteria, and intracellular parasites, STING induces the production of type I interferons. Type I interferons mediated by STING protect infected cells and neighboring cells from local infection by binding to the same cells and neighboring cells that secrete the interferon. An exemplary amino acid sequence of STING is provided in the NCBI Genbank database under accession number NP_001288667.

[0159] 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 the cell surface. There are multiple T cell subsets, including, but not limited to, T helper cells (also known as TH cells or CD4+T cells) and subtypes, including TH1, TH2, TH3, TH17, TH9 and TFH cells, cytotoxic T cells (also known as TC cells, CD8+T cells, cytotoxic T lymphocytes, T-killer cells, killer T cells), memory T cells and 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 T reg cells or suppressor T cells) and subtypes, including CD4+FOXP3+T reg cells, CD4+FOXP3-T reg cells, Tr1 cells, Th3 cells and T reg17 cells, natural killer T cells (also known as NKT cells), mucosal-associated invariant T cells (MAIT) and gamma delta T cells (γδ T cells), including Vγ9 / Vδ2 T cells. Any one or more of the aforementioned or unmentioned T cells can be the target cell type for the methods of use of the present invention.

[0160] As used herein, 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 proliferation.

[0161] As used herein, the term "therapeutically effective amount" or "therapeutically effective dose" or similar terms used herein are intended to refer to the amount of an agent (e.g., an anti-IL-27 antibody or antigen-binding fragment thereof) that elicits a desired biological or medical response (e.g., improvement of one or more symptoms of cancer).

[0162] As used herein, the term "TAM receptor" refers to TAM receptor protein tyrosine kinases (TYRO3, AXL and MER). TAM receptors are involved in regulating immune system homeostasis. In the case of cancer, TAM receptors have a dual regulatory role, controlling the initiation and progression of tumor development and simultaneously controlling the relevant anti-tumor responses of different immune cells. Further description of TAM receptors is found in Paolino and Penninger (2016) Cancers 8(97): doi:10.3390 / cancers8100097). As used herein, the term "TAM receptor inhibitor" or "TAM inhibitor" refers to an agent that inhibits, blocks or reduces the function or activity of TAM receptors.

[0163] Unless otherwise indicated, the term "TIGIT" or "T cell immunoreceptor with Ig and ITIM domains" as used herein refers to any native TIGIT from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). TIGIT is also known in the art as DKFZp667A205, FLJ39873, protein 9 containing V-set and immunoglobulin domains, protein 3 containing V-set and transmembrane domains, VSIG9, VSTM3, and WUCAM. The term also encompasses naturally occurring variants of TIGIT, such as splice variants or allelic variants. The amino acid sequence of an exemplary human TIGIT can be found under UniProt accession number Q495A1.

[0164] As used herein, the term "treat", "treating" or "treatment" refers to therapeutic or preventive measures as described herein. "Treatment" methods employ administration of the human antibodies of the present invention to a subject in need of such treatment, e.g., a subject in need of enhanced immune response to a particular antigen or a subject who will eventually acquire the disorder, to prevent, cure, delay the onset of the disorder or recurrence of the disorder, reduce its severity or ameliorate one or more symptoms thereof, or to prolong the survival of the subject beyond the expected survival without such treatment.

[0165] As used herein, the term "tumor microenvironment" (alternatively "cancer microenvironment"; abbreviated as "TME") refers to the cellular environment or background in which a tumor or neoplasm exists, including surrounding blood vessels and non-cancerous cells (including but not limited to immune cells, fibroblasts, bone marrow-derived inflammatory cells and lymphocytes). Signaling molecules and extracellular matrix also constitute the TME. Tumors are closely related to and always interact with the surrounding microenvironment. Tumors can affect the microenvironment by releasing extracellular signals, thereby promoting tumor angiogenesis and inducing peripheral immune tolerance, and immune cells in the microenvironment can affect the growth and evolution of tumor cells.

[0166] As used herein, the term "unrearranged" or "germline configuration" refers to a configuration in which a V segment is not recombined so as to be immediately adjacent to a D or J segment.

[0167] As used herein, the term "vector" is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plastid," which refers to a circular double-stranded DNA loop into which other DNA segments can be joined. Another type of vector is a viral vector, in which other DNA segments can be joined to the viral genome. Certain vectors are capable of self-replication in the host cell into which they are introduced (e.g., bacterial vectors with bacterial replication origins, and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of the host cell when introduced into the host cell, thereby replicating along with the host genome. In addition, certain vectors are capable of directing the expression of genes to which they are operably linked. These vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, the utility of expression vectors in recombinant DNA technology is often in the form of plastids. In this specification, "plastid" and "vector" can be used interchangeably because plastids are the most commonly used form of vectors. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

[0168] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Preferred methods and materials are described below, but methods and materials similar or equivalent to those described herein can also be used to practice or test the methods and compositions disclosed herein. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. Simple diagram description

[0169] [ [picture] [1]] is a table providing affinity data for anti-IL-27 antibodies that are capable of binding to an antigenic determinant comprising one or more amino acids of Gln37, Leu38, Glu42, Glu46, Val49, Ser50, Leu142, Asp146, Arg149, His150, Phe153, Leu156, Leu162 and Glu164 of SEQ ID NO: 2 (IL-27p28). Affinity measurements were performed using ForteBio and Meso Scale Discovery methods. [ [picture] [2A]] is a graph depicting that anti-IL-27 antibodies inhibit IL-27-mediated STAT1 phosphorylation in human PBMCs as measured by flow cytometry, as indicated. [picture] [2B]] is a graph depicting that anti-IL-27 antibodies inhibit IL-27-mediated STAT1 phosphorylation in U937 cells as measured by flow cytometry, as indicated. [picture] [2C]] is a graph depicting that anti-IL-27 antibodies inhibited IL-27-mediated STAT1 phosphorylation in HUT-78 cells as measured by flow cytometry as indicated. [ [picture] [3]] is a graph showing that the anti-IL-27 antibody of the present disclosure ("anti-IL-27 Ab1") inhibits IL-27-mediated pSTAT1 in human whole blood T cells. [ [picture] [4]] is a graph depicting that a range of concentrations of anti-IL-27 antibodies reversed IL-27-mediated inhibition of CD161 expression in T cells as indicated. CD161 expression was determined using flow cytometry. [ [picture] [5A]] is a graph depicting the extent to which anti-IL-27 antibodies enhance PD-1-mediated TNFα secretion in human PBMCs as measured by ELISA. [picture] [5B]] is a graph depicting the extent to which anti-IL-27 antibodies enhance PD-1-mediated IL-6 secretion in human PBMCs as measured by ELISA. [picture] [5C-5D]] show that after PD-1 blockade, IL-27 inhibited cytokine production (IL-17A, Figure 5C; and IFNγ, Figure 5D) and was restored in combination with anti-IL-27 Ab1 (Abbreviations: Ctrl = control, ns = not significant, PBMC = peripheral blood mononuclear cells, rhIL-27 = recombinant human IL-27). [ [picture] [5E-5H]] summarizes the observed cytokine induction of TNFα (Figure 5E), IFNγ (Figure 5F), IL-6 (Figure 5G), and IL-17A (Figure 5H) in activated PBMC cultures from several individual donors including healthy controls and patients with RCC, HCC, and ovarian cancer, where these cells were contacted with anti-IL-27 Ab1 antibody, αPD-1 antibody, or a combination of anti-IL-27 Ab1 and αPD-1 antibody. [] [] [[] [picture] [6A]] is a graph depicting that treatment of human monocytes with anti-IL-27 antibodies inhibits IL-27-mediated PD-L1 expression as measured by flow cytometry. [picture] [6B]] is a graph depicting that treatment of human monocytes with anti-IL-27 antibodies inhibits IL-27-mediated TIM3 expression as determined by flow cytometry. [picture] [6C]] is a graph depicting that treatment of resting human T cells with anti-IL-27 antibody inhibits IL-27-mediated PD-L1 expression as determined by flow cytometry. [ [picture] [7A]] is a dot plot depicting the number of surface lung B16F10 metastatic nodules (lung nodules) from B16F10 tumor-bearing mice treated with anti-IL27 antibody (anti-IL-27 Ab1), isotype control antibody, αWSX-1 antibody, or combined αPD-1 and αCTLA-4 antibodies as indicated, as determined by visual counting of nodules in lungs isolated from mice. [ [picture] [7B]] provides graphs depicting the growth kinetics of bioluminescent B16-Luc tumors in mice treated with anti-IL-27 antibody (anti-IL-27 Ab1) or isotype control antibody as determined by bioluminescent imaging analysis. [picture] [7C-7F]] shows a series of images of fixed, sectioned lung tissue stained with hematoxylin and eosin isolated from B16F10 tumor-bearing mice treated with anti-IL27 antibody (anti-IL-27 Ab1) (Figure 7D), isotype control antibody (Figure 7C), αWSX-1 antibody (Figure 7E), or combined αPD-1 and αCTLA-4 antibodies (Figure 7F), as indicated. [ [picture] [7G]] is a dot plot depicting total tumor area as a percentage of total tissue area of ​​fixed, sectioned lung tissue B16F10 tumor tissue isolated from B16F10 tumor-bearing mice treated with anti-IL27 antibody (anti-IL-27 Ab1), isotype control antibody, αWSX-1 antibody, or combination αPD-1 and αCTLA-4 antibodies, as indicated, as determined by image analysis software. Similar reductions in the number of superficial lung metastases and total tumor area were observed for IL-27RA (WSX-1)-mediated antibody blockade and for anti-PD-1 + anti-CTLA-4 combination therapy. [ [picture] [8A]] provides a volcano plot of microarray data depicting genes with >1.0 log 2 fold change in expression (black dots) in splenocytes isolated from mice overexpressing IL-27 following treatment with IL-27 minicircles. The x-axis shows the log2 fold change in gene expression (IL-27 minicircle treatment versus control). The y-axis is the t-test p-value showing the probability of the fold change for each gene. [ [picture] [8B]] provides a depiction as indicated, such as [picture] [8A] Graph showing expression levels of selected immune regulatory genes in splenocytes. [picture] [8C-8F]] shows that ectopic expression of human IL-27 induces inhibitory receptor expression on murine T cells in vivo (by flow cytometry analysis) and anti-IL-27 Ab1 reduces inhibitory receptor expression on T cells after IL-27 minicircle treatment in vivo. Six-week-old female Balb / c mice were injected with empty vector (control) or hIL-27 minicircle (Figures 8C and 8D). Five days after transfection, PBMCs and (Figures 8E and 8F) total splenocytes were collected and the cells were stained and analyzed by flow cytometry. The expression of the indicated markers was analyzed on CD4+ T cells (Figures 8C and 8E) and CD8+ T cells (Figures 8D and 8F). Analyses were performed using FlowJo software. [ [picture] [8G]] shows that anti-IL-27 Ab1 inhibits the detection of minicircle-derived human IL-27 in murine plasma. [ [picture] [9]] is the crystal ribbon structure of the IL-27-anti-IL-27 Ab1 complex determined 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: 1022-25). The heavy chain, light chain, p28 and EBI-3 are colored in yellow, red, gray and green, respectively. [picture] [9] showed that anti-IL-27 Ab1 binds to the p28 molecule of IL-27. [ [picture] [10A-10B]] shows the interaction of human IL-27 heterodimers with WSX-1 ( [picture] [10A]) and gp130( [picture] [10B]) Graph of binding affinity. [ [picture]

[11] ] is a ribbon diagram of p28, showing the residues where anti-IL-27 Ab1 binds to p28. LC = light chain of anti-IL-27 Ab1; HC = heavy chain of anti-IL-27 Ab1 [ [picture]

[12] ] is a ribbon diagram of the structural alignment of IL-27 / anti-IL-27 Ab1 Fab and IL-23 / IL-23R (PDB ID: 5MZV). The alignment was performed in 3D space using a superposition of the complexes of p28 and p19. [ [picture]

[13] ] is a ribbon diagram of the structural alignment of IL-27 / anti-IL-27 Ab1 Fab and IL-6 / IL-6Ra / gp130. The alignment was performed in 3D space using a superposition of the complexes of p28 and IL-6. [ [picture] [14A-14B]] is a ribbon diagram of the binding interface between p28 and EBI3, where [picture] [14B] shows [picture] [14A] Magnification to illustrate the location of the salt bridge interaction and aromatic / hydrophobic interactions between p28 and EBI3. [] [] [[] [picture] [15A-15B]] is p28 ( [picture] [15A]) and EBI3( [picture] [15B]) of the sequence alignment. As indicated, arrows point to conserved salt-bridging amino acids and conserved hydrophobic amino acids. [ [picture] [16A]] is a ribbon diagram showing the structural alignment of the IL-27 heterodimer with IL-6 / IL-6Ra. [picture] [16B-16C]] is the sequence alignment of IL-27 and IL-6 / IL-6Ra. Arrows point to conserved salt-bridging amino acids and conserved hydrophobic amino acids. [picture] [16D]] is a ribbon diagram showing several p28 and EBI3 interactions that are conserved for IL-6Ra. [ [picture]

[17] ] is a table presenting the binding affinity data of human IL-27 and gp130, WSX-1 and anti-p28 antibodies. [ [picture] [18A]] is a sequence alignment of mouse and human p28 amino acid sequences. [picture] [18B]] is a ribbon diagram centered on residue 162 (Leu in the human sequence and Cys in the mouse sequence). [ [picture] [19A]] shows the electrostatic surface potential of human IL-27. [picture] [19B]] shows the primary sequence of human IL-27, showing the αA, αB, αC, αD helices, and the unresolved CD loop with the poly-Glu sequence. [ [picture] [20A]] is a graphical representation showing the differential expression of EBI3, IL-27p28, and IL-27RA in RCC tumors (1) and normal kidney tissue (2). [picture] [20B-20D]] is in accordance with EBI3( [picture] [20B]), IL-27p28 ( [picture] [20C]) and IL-27RA ( [picture] Kaplan-Meier curves (percentage of death-free survival in days) of RCC patients stratified by high (1) or low (2) expression of HER2 [20D]). Data were generated using 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). [ [picture] [21A-21B]] show the IL-27-induced gene expression signature in activated human CD4+ T cells. [picture] [21A] is a scatter plot of the fold change of IL-27-treated CD4+ T cells compared to untreated controls for two separate donors. [picture] [21B] Shows the top 31 genes in the IL-27 signature in CD4+ T cells. Fifteen of the 31 genes (marked with stars) were associated with poor outcome. Data were generated using TCGA. [ [picture] [22A-22B]] is for the RCC ( [picture] [22A]) and BRCA ( [picture] [22B]) Graphical representation of genome-wide risk ratios associated with expression of IL-27 signature genes in tumor samples. Data were generated using TCGA. [ [picture] [23A]] is a graphical representation of EBI3 plasma levels in RCC patients as compared to healthy donor serum and serum from pregnant females (positive control) as measured using an EBI3-specific antibody pair. [picture] [23B]] shows EBI3 levels in separate RCC patient groups grouped by tumor stage. [picture] [23C]] shows the overall survival in RCC patients stratified by serum EBI3 levels and [ [picture] [23D]] shows disease-free survival. [ [picture] [24A-24B]] are graphical representations of the effects of anti-IL-27 Ab1 on tumor growth and lung metastasis in the orthotopic Renca model. [picture] [24A] [and] [24B] shows the net primary tumor weight (kidney) and number of lung metastases in control and anti-IL-27 Ab1 treated Renca mice. (*P < 0.05; unpaired t test) [ [picture] [25A-25B]] show the orthotopic Hepa1-6-luc tumor model ( [picture] [25A]), the effect of anti-IL-27 Ab1 as a single agent on the mean in situ Hepa1-6 tumor flux over time as compared to isotype control ( [picture] [25B]). Error bars indicate standard error. [ [picture] [26A-26F]] show dose-dependent inhibition of orthotopic Hepa1-6 tumor growth after continuous administration of anti-IL-27 Ab1 ( [picture] [26A]). [picture] [26B] shows the average bioluminescence imaging ("BLI", photons / second) at 5, 8, 13 and 16 days after implantation for administration of control and anti-IL-27 Ab1 (5 mg / kg, 25 mg / kg and 50 mg / kg). [picture] [26C-26F] showed that in the control ( [picture] [26C]) and anti-IL-27 Ab1 5 mg / kg ( [picture] [26D]), 25 mg / kg( [picture] [26E]), and 50 mg / kg group ( [picture] [26F]), BLI (photons / second) of individual animals at 5, 8, 13, and 16 days after implantation. [ [picture] [27A-27C]] show the regulation of gene expression in Hepa1-6 liver after administration of anti-IL-27 Ab1 ( [picture] [27A] [and] [27B]). [picture] [27C] is a volcano plot of genes regulated by administration of anti-IL-27 Ab 1. The following Tables 11A-11B provide a list of up-regulated and down-regulated genes shown in Figure 27B. [ [picture] [28A-28E]] shows the expression of various IL-27 component genes ( [picture] [28A]); CD274, TIGIT, LAG3, HAVCR2 and PDCD1 ( [picture] [28B]);TGFA and TGFB1( [picture] [28C]);AFP( [picture] [28D]); and TNFRSF10B, TNFRSF1A and PDGFA ( [picture] [28E]) is a graphical representation of the performance. [ [picture] [29A-29B]] are graphical representations of the relative expression of various macrophage and NK transcript marker genes in the tumor microenvironment (TME) after administration of anti-IL-27 Ab1. [ [picture]

[30] ] is a graphical representation of the expression of NK-associated receptors after administration of anti-IL-27 Ab1 or isotype control. [ [picture]

[31] ] shows the relative expression of various cell surface markers after administration of anti-IL-27 Ab1 as compared to IgG isotype control. Ratios were obtained by normalizing target marker transcript levels to PTPRC levels. Directionality is expressed as the difference between the anti-IL-27 Ab1 ratio and the IgG ratio. [ [picture] [32A-32D]] shows the expression of IL17A ( [picture] [32A]), IFNg (IFNγ) [picture] [32B]), TNFa (TNFα) [picture] [32C]) and IL-10 ( [picture] [32D]) is a bar graph showing the performance of [ [picture] [33A-33D]] shows the expression of IL17A ( [picture] [33A]), IFNg (IFNγ) [picture] [33B]), TNFa (TNFα) [picture] [33C]) and IL-10 ( [picture] [33D]) scatter plot. [ [picture]

[34] ] is a volcano plot representing the log 2 fold change in gene expression after IL-27 inhibition compared to control (x-axis) compared to the significance (p-value) of the change in gene expression after treatment with anti-IL-27 Ab1 compared to control (y-axis). [ [picture]

[35] ] is a scatter plot showing the expression of TNFSF15 in activated PBMCs after incubation in anti-IL-27 Ab1 or isotype control (IgG). [ [picture] [36A-36B]] shows the activation of cells cultured in the presence of two different batches of anti-IL-27 Ab1 (1 µg / mL) or isotype control ( [picture] [36A]) and stationary ( [picture] [36B]) Bar graph of TNFSF15 expression in PBMCs. [ [picture]

[37] ] is a bar graph showing the fold change of TNFSF15 transcript after IL-27 inhibition with anti-IL-27 Ab1 compared to isotype control in various cell types as indicated. [ [picture]

[38] ] is a bar graph showing the fold expression of TNFSF15 transcripts after treatment with anti-IL-27 Ab1, anti-CD39 antibody and two anti-CD112R antibodies as indicated. [ [picture] [39A-39B]] shows that TNFSF15 transcripts ( [picture] [39A]) and secreted TNFSF15 protein ( [picture] [39B]) with delayed kinetics. Implementation

[0170] [via] [EFS-WEB] [Citation of a sequence listing submitted electronically] The contents of the Sequence Listing (Name: _____; Size: _____ bytes; and Creation Date: ______) submitted electronically with this application as an ASCII text file are incorporated herein by reference in their entirety. [Cross-reference to related applications] [] []

[0171] This PCT application claims the benefit of priority to U.S. Provisional Application No. 62 / 906,008, filed on September 25, 2019, and No. 63 / 081,705, filed on September 22, 2020; each of which is incorporated herein by reference in its entirety.

[0172] The present disclosure provides, at least in part, antibody molecules that bind with high affinity and specificity to specific antigenic determinants on human IL-27p28. As used herein, the terms "IL-27" and "IL27" interchangeably refer to the heterodimeric cytokine IL-27 composed of two different subunits, which are encoded by two different genes: Epstein-Barr virus-induced gene 3 (EBI3) and IL-27p28. IL-27 has both pro-inflammatory and anti-inflammatory properties, with different effects on hematopoietic and non-hematopoietic cells.

[0173] Thus, in one aspect, the present disclosure provides an isolated antibody or antigen-binding portion thereof that specifically binds to and antagonizes human IL-27, wherein the antibody or antigen-binding portion thereof specifically binds to an antigenic determinant disclosed herein and exhibits at least one or more of the following properties: (i) binds to human IL-27 with an equilibrium dissociation constant (KD) of 15 nM or less; (ii) blocking IL-27 binding to IL-27 receptor; (iii) inhibiting or reducing STAT1 and / or STAT3 phosphorylation in cells; (iv) inhibiting or reducing IL-27-mediated inhibition of CD161 expression in cells; (v) inhibiting or reducing IL-27-mediated PD-L1 and / or TIM-3 expression in cells; (vi) inducing or enhancing PD-1-mediated secretion of one or more cytokines from cells; and (vii) A combination of (i) to (vi).

[0174] Additional aspects of the invention include nucleic acid molecules encoding antibody molecules, expression vectors, host cells, and methods of preparing antibody molecules. Immunoconjugates, multi- or bispecific molecules, and pharmaceutical compositions comprising the antibody molecules are also provided. The anti-IL-27 antibody molecules disclosed herein can be used to treat, prevent, and / or diagnose cancerous or malignant conditions, e.g., solid and liquid tumors (e.g., leukemias, e.g., lymphomas, e.g., AML), lung cancers (e.g., non-small cell lung cancer), pancreatic cancer, breast cancers (e.g., triple-negative breast cancer), melanomas, testicular cancers, sarcomas, head and neck cancers (e.g., squamous head and neck cancer), liver cancers (e.g., hepatocellular carcinoma (HCC)), colorectal cancers, ovarian cancers, brain cancers (e.g., glioblastoma multiforme), or kidney cancers (e.g., renal cell carcinomas, e.g., clear cell renal carcinomas). [anti] [IL-27] [Antibodies and antigen-binding fragments thereof] [] []

[0175] The present disclosure provides antibodies and antigen-binding portions thereof that specifically bind to IL-27p28 and antagonize IL-27, particularly human IL-27.

[0176] The present disclosure relates to isolated antibodies or antigen-binding portions thereof that antagonize human IL-27, wherein the antibodies or antigen-binding portions thereof specifically bind to an antigenic determinant comprising one or more of the following amino acids: (i) amino acids 37 to 56 corresponding to SEQ ID NO: 2 (IL-27p28), (ii) amino acids 142 to 164 corresponding to SEQ ID NO: 2 (IL-27p28), or (iii) both (i) and (ii). In some aspects, an isolated antibody of the present disclosure that antagonizes human IL-27, or an antigen-binding portion thereof, specifically binds to an antigenic determinant comprising one or more amino acids 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, or Glu164 of SEQ ID NO: 2 (IL-27p28).

[0177] In some aspects, the antibodies or antigen-binding portions thereof of the present disclosure specifically bind to an antigenic determinant comprising Asp146, Arg149 and / or Phe153 of SEQ ID NO: 2 (IL-27p28). In some aspects, the antibodies or antigen-binding portions thereof of the present disclosure specifically bind to an antigenic determinant comprising Asp146, Arg149 and Phe153 of SEQ ID NO: 2 (IL-27p28). In some aspects, the antigenic determinant comprises Asp146, Arg149, His150 and Phe153 of SEQ ID NO: 2 (IL-27p28). In some aspects, the antigenic determinant comprises Asp146, Arg149, Phe153 and Leu156 of SEQ ID NO: 2 (IL-27p28). In some aspects, the antigenic determinant comprises Asp146, Arg149, His150, Phe153, and Leu156 of SEQ ID NO: 2 (IL-27p28).

[0178] In some aspects, the antibodies or antigen-binding portions thereof of the present disclosure specifically bind to an antigenic determinant comprising Leu142, Asp146, Arg149, His150, Phe153, Leu156, and Glu164 of SEQ ID NO: 2 (IL-27p28). In some aspects, the antigenic determinant comprises Gln37, Leu38, Glu42, Asp146, Arg149, His150, Phe153, and Leu156 of SEQ ID NO: 2 (IL-27p28). In some aspects, the antibodies of the present disclosure, or antigen-binding portions thereof, specifically bind to an antigenic determinant comprising Gln37, Leu38, Glu42, Leu142, Asp146, Arg149, His150, Phe153, Leu156, and Glu164 of SEQ ID NO: 2 (IL-27p28).

[0179] In some aspects, the antibodies or antigen-binding portions thereof of the present disclosure specifically bind to an antigenic determinant comprising Leu142, Asp146, Arg149, His150, Phe153, Leu156, Leu162, and Glu164 of SEQ ID NO: 2 (IL-27p28). In some aspects, the antibodies or antigen-binding portions thereof of the present disclosure specifically bind to an antigenic determinant comprising Glu46, Val49, Ser50, Leu142, Asp146, Arg149, His150, Phe153, Leu156, and Glu164 of SEQ ID NO: 2 (IL-27p28). In some aspects, the antibodies of the present disclosure, or antigen-binding portions thereof, specifically bind to an antigenic determinant comprising Gln37, Leu38, Glu42, Glu46, Val49, Ser50, Leu142, Asp146, Arg149, His150, Phe153, Leu156, Leu162, and Glu164 of SEQ ID NO: 2 (IL-27p28).

[0180] In some aspects, an antibody of the present disclosure, or an antigen-binding portion thereof, specifically binds to an antigenic determinant consisting of or consisting essentially of Gln37, Leu38, Glu42, Glu46, Val49, Ser50, Leu142, Asp146, Arg149, His150, Phe153, Leu156, Leu162, and Glu164 of SEQ ID NO: 2 (IL-27p28).

[0181] In some aspects, the antibodies of the present disclosure, or antigen-binding portions thereof, specifically bind to an antigenic determinant comprising Gln37, Leu38, Glu42, Glu46, Val49, Ser50, Leu142, Asp146, Arg149, His150, Phe153, Leu156, Leu162, and Glu164 of 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 of SEQ ID NO: 2 (IL-27p28).

[0182] In some aspects, the antibodies of the present disclosure, or antigen-binding portions thereof, specifically bind to an antigenic determinant comprising Gln37, Leu38, Glu42, Glu46, Val49, Ser50, Leu142, Asp146, Arg149, His150, Phe153, Leu156, Leu162, and Glu164 of 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 of SEQ ID NO: 2 (IL-27p28).

[0183] In some aspects, an antibody of the present disclosure, or an antigen-binding portion thereof, specifically binds to an antigenic determinant consisting of, 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 of SEQ ID NO: 2 (IL-27p28).

[0184] In some aspects, an antibody or antigen-binding portion thereof of the present disclosure specifically binds to an antigenic determinant consisting of 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 of SEQ ID NO: 2 (IL-27p28).

[0185] In some aspects, the disclosure provides an isolated antibody or antigen-binding portion thereof that specifically binds to an antigenic determinant 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 of SEQ ID NO: 2 (IL-27p28) and antagonizes human IL-27, wherein the antibody or antigen-binding portion thereof exhibits at least one or more of the following properties: (i) (i) inhibiting or reducing STAT1 and / or STAT3 phosphorylation in cells; (ii) inhibiting or reducing the inhibition of CD161 expression in cells; (iii) inhibiting or reducing the expression of PD-L1 and / or TIM-3 in cells; (iv) inducing or enhancing PD-1-mediated secretion of one or more cytokines from cells; and (vii) a combination of (i)-(vi).

[0186] In some aspects, the isolated antibody, or antigen-binding portion thereof, binds to an antigenic determinant of one or more amino acids at Gln37, Leu38, Glu42, Glu46, Val49, Ser50, Leu53, Lys56, Leu142, Aspl43, Arg145, Aspl46, Leu147, Arg149, His150, Arg152, Phe153, Leu156, Ala157, Gly159, Phe160, Asn161, Leu162, Pro163, and Glu164 of SEQ ID NO: 2 (human IL-27p28) with an equilibrium dissociation constant (KD) of 15 nM or less.

[0187] In some aspects, the isolated antibody or antigen-binding portion thereof binds to recombinant human IL-27p28 or murine IL-27p28.

[0188] In some aspects, the isolated antibody or its antigen-binding portion inhibits or reduces STAT1 and / or STAT3 phosphorylation in a cell. In some aspects, the cell is an immune cell. In some aspects, the cell is a cancer cell.

[0189] In some aspects, the isolated antibody or antigen-binding portion thereof inhibits or reduces the inhibition of CD161 expression in a cell (eg, improves or alleviates the inhibition of CD161 expression in a cell). In some aspects, the cell is an immune cell.

[0190] In some aspects, the isolated antibody or antigen-binding portion thereof inhibits or reduces PD-L1 and / or TIM-3 expression in a cell. In some aspects, PD-L1 expression is inhibited or reduced. In some aspects, TIM-3 expression is inhibited or reduced. In some aspects, both PD-L1 expression and TIM-3 expression are reduced. In some aspects, the cell is an immune cell. In some aspects, the antibody is a monoclonal antibody.

[0191] In some aspects, the 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, the one or more cytokines are TNFα. In some aspects, the one or more cytokines are IL-6. In some aspects, the one or more cytokines are TNFα and IL-6. In some aspects, the cell is an immune cell.

[0192] In some aspects, the isolated antibody or its antigen-binding portion is selected from the group consisting of: IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD and IgE antibodies. In some aspects, the antibody is an IgG1 antibody or an IgG4 antibody. In some aspects, the antibody comprises a wild-type IgG1 heavy chain constant region. In some aspects, the antibody comprises a wild-type IgG4 heavy chain constant region. In some aspects, the antibody comprises an Fc domain containing at least one mutation. In some aspects, the antibody comprises a mutant IgG1 heavy chain constant region. In some aspects, the antibody comprises a mutant IgG4 heavy chain constant region. In some aspects, the mutant IgG4 heavy chain constant region comprises any one of the substitutions S228P, L235E, L235A according to EU numbering, or a combination thereof.

[0193] In some aspects, the disclosure provides an isolated antibody, or antigen-binding portion thereof, that binds to substantially the same antigenic determinant on IL-27 as the antibody, or antigen-binding portion thereof, according to any of the preceding aspects.

[0194] In some aspects, the disclosure provides an isolated antibody, or antigen-binding portion thereof, that binds to at least one of the amino acid residues 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 of SEQ ID NO: 2 (IL-27p28) to which the antibody, or antigen-binding portion thereof, according to any of the preceding aspects binds.

[0195] In some aspects, the disclosure provides an isolated antibody, or antigen-binding portion thereof, wherein a mutation of an antigenic determinant (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 of SEQ ID NO: 2 (IL-27p28)) bound by the antibody, or antigen-binding portion thereof, inhibits, reduces, or blocks binding to both the antibody, or antigen-binding portion thereof, and the antibody, or antigen-binding portion thereof according to any of the preceding aspects.

[0196] In some aspects, the antibody or its antigen-binding portion 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 the light chain CDR1 consists of N-XXXXXXLFSSNXKXYXX-C. In some aspects, the antibody or its antigen-binding portion 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 the light chain CDR3 consists of N-XXXASAXXX-C. In some aspects, the antibody or its antigen-binding portion 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 the heavy chain CDR2 consists of N-XXSSSXSYXYXXXXXX XC. In some aspects, the 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 the heavy chain CDR3 consists of N-XXXXGRTSYTATXHNXXXX-C, wherein X is any amino acid.

[0197] In some aspects, the antibody or its antigen-binding portion 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 the light chain CDR1 consists of N-XXXXXXLFSSNXKXYXX-C and the light chain CDR3 consists of N-XXXASAXXX-C. In some aspects, the antibody or its antigen-binding portion 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 the heavy chain CDR2 consists of N-XXSSSXSYXYXXXXXXX-C and the heavy chain CDR3 consists of N-XXXXGRTSYTATXHNXXXX-C, wherein X is any amino acid.

[0198] In some aspects, the 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 the light chain CDR1 consists of N-XXXXXXLFSSNXKXYXX-C, the light chain CDR3 consists of N-XXXASAXXX-C, the heavy chain CDR2 consists of N-XXSSSXSYXYXXXXXXX-C, and the heavy chain CDR3 consists of N-XXXXGRTSYTATXHNXXXX-C, wherein X is any amino acid.

[0199] In some aspects, the disclosure provides an isolated antibody, or antigen-binding portion thereof, that specifically binds to an antigenic determinant comprising one or more amino acids at Gln37, Leu38, Glu42, Glu46, Val49, Ser50, Leu53, Lys56, Leu142, Aspl43, Arg145, Aspl46, Leu147, Arg149, His150, Arg152, Phe153, Leu156, Ala157, Gly159, Phe160, Asn161, Leu162, Pro163, and Glu164 of SEQ ID NO: 2 (IL-27p28), wherein the antibody, or antigen-binding portion thereof, does not comprise heavy and light chain CDRs selected from the group consisting of: (i) the heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 9, 10 and 11, respectively, and the light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 17, 18 and 19, respectively; (ii) the heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 31, 32 and 33, respectively, and the light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 39, 40 and 41, respectively; (iii) the heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 53, 54 and 55, respectively, and the light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 61, 62 and 63, respectively; (iv) the heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 75, 76 and 77, respectively, and the light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 83, 84 and 85, respectively; (v) the heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 97, 98 and 99, respectively, and the light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 105, 106 and 107, respectively; or (vi) the heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 119, 120 and 121, respectively, and the light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 127, 128 and 129, respectively.

[0200] In some aspects, the disclosure provides an isolated antibody, or antigen-binding portion thereof, that specifically binds to an antigenic determinant comprising or consisting of Gln37, Leu38, Glu42, Glu46, Val49, Ser50, Leu142, Asp146, Arg149, His150, Phe153, Leu156, Leu162, and Glu164 of SEQ ID NO: 2 (IL-27p28), wherein the antibody, or antigen-binding portion thereof, does not comprise heavy and light chain CDRs selected from the group consisting of: (i) the heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 9, 10 and 11, respectively, and the light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 17, 18 and 19, respectively; (ii) the heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 31, 32 and 33, respectively, and the light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 39, 40 and 41, respectively; (iii) the heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 53, 54 and 55, respectively, and the light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 61, 62 and 63, respectively; (iv) the heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 75, 76 and 77, respectively, and the light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 83, 84 and 85, respectively; (v) the heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 97, 98 and 99, respectively, and the light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 105, 106 and 107, respectively; or (vi) the heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 119, 120 and 121, respectively, and the light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 127, 128 and 129, respectively.

[0201] In some aspects, the disclosure provides an isolated antibody, or antigen-binding portion thereof, that specifically binds to an antigenic determinant comprising or consisting of Gln37, Leu38, Glu42, Glu46, Val49, Ser50, Leu53, Lys56, Leu142, Aspl43, Arg145, Aspl46, Leu147, Arg149, His150, Arg152, Phe153, Leu156, Ala157, Gly159, Phe160, Asn161, Leu162, Pro163, and Glu164 of SEQ ID NO: 2 (IL-27p28), wherein the antibody, or antigen-binding portion thereof, does not comprise heavy and light chain CDRs selected from the group consisting of: (i) the heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 9, 10 and 11, respectively, and the light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 17, 18 and 19, respectively; (ii) the heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 31, 32 and 33, respectively, and the light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 39, 40 and 41, respectively; (iii) the heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 53, 54 and 55, respectively, and the light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 61, 62 and 63, respectively; (iv) the heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 75, 76 and 77, respectively, and the light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 83, 84 and 85, respectively; (v) the heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 97, 98 and 99, respectively, and the light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 105, 106 and 107, respectively; or (vi) the heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 119, 120 and 121, respectively, and the light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 127, 128 and 129, respectively.

[0202] In some aspects, the disclosure provides an isolated antibody, or antigen-binding portion thereof, that specifically binds to an antigenic determinant comprising one or more amino acids at Gln37, Leu38, Glu42, Glu46, Val49, Ser50, Leu53, Lys56, Leu142, Aspl43, Arg145, Aspl46, Leu147, Arg149, His150, Arg152, Phe153, Leu156, Ala157, Gly159, Phe160, Asn161, Leu162, Pro163, and Glu164 of SEQ ID NO: 2 (IL-27p28), wherein the antibody, or antigen-binding portion thereof, does not comprise heavy and light chain CDRs selected from the group consisting of: (i) the heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 12, 13 and 14, respectively, and the light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 20, 21 and 22, respectively; (ii) the heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 34, 35 and 36, respectively, and the light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 42, 43 and 44, respectively; (iii) the heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 56, 57 and 58, respectively, and the light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 64, 65 and 66, respectively; (iv) the heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 78, 79 and 80, respectively, and the light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 86, 88 and 89, respectively; (v) the heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 100, 101 and 102, respectively, and the light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 108, 109 and 110, respectively; or (vi) the heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 122, 123 and 124, respectively, and the light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 130, 131 and 132, respectively.

[0203] In some aspects, the disclosure provides an isolated antibody, or antigen-binding portion thereof, that specifically binds to an antigenic determinant comprising or consisting of Gln37, Leu38, Glu42, Glu46, Val49, Ser50, Leu142, Asp146, Arg149, His150, Phe153, Leu156, Leu162, and Glu164 of SEQ ID NO: 2 (IL-27p28), wherein the antibody, or antigen-binding portion thereof, does not comprise heavy and light chain CDRs selected from the group consisting of: (i) the heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 12, 13 and 14, respectively, and the light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 20, 21 and 22, respectively; (ii) the heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 34, 35 and 36, respectively, and the light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 42, 43 and 44, respectively; (iii) the heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 56, 57 and 58, respectively, and the light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 64, 65 and 66, respectively; (iv) the heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 78, 79 and 80, respectively, and the light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 86, 88 and 89, respectively; (v) the heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 100, 101 and 102, respectively, and the light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 108, 109 and 110, respectively; or (vi) the heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 122, 123 and 124, respectively, and the light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 130, 131 and 132, respectively.

[0204] In some aspects, the disclosure provides an isolated antibody, or antigen-binding portion thereof, that specifically binds to an antigenic determinant comprising or consisting of Gln37, Leu38, Glu42, Glu46, Val49, Ser50, Leu53, Lys56, Leu142, Aspl43, Arg145, Aspl46, Leu147, Arg149, His150, Arg152, Phe153, Leu156, Ala157, Gly159, Phe160, Asn161, Leu162, Pro163, and Glu164 of SEQ ID NO: 2 (IL-27p28), wherein the antibody, or antigen-binding portion thereof, does not comprise heavy and light chain CDRs selected from the group consisting of: (i) the heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 12, 13 and 14, respectively, and the light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 20, 21 and 22, respectively; (ii) the heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 34, 35 and 36, respectively, and the light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 42, 43 and 44, respectively; (iii) the heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 56, 57 and 58, respectively, and the light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 64, 65 and 66, respectively; (iv) the heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 78, 79 and 80, respectively, and the light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 86, 88 and 89, respectively; (v) the heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 100, 101 and 102, respectively, and the light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 108, 109 and 110, respectively; or (vi) the heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 122, 123 and 124, respectively, and the light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 130, 131 and 132, respectively.

[0205] In some aspects, the disclosure provides a polypeptide that specifically binds to Gln37, Leu38, Glu42, Glu46, Val49, Ser50, Leu53, Lys56, Leu142, Asp143, Arg145, Asp146, Leu147, Arg149, His150, Arg152, Phe153, Leu156, Ala157, Gly159, Phe160, Asp161, Asp162, Asp163, Asp164, Asp165, Asp166, Leu167, Arg168, His169, Asp170, Asp171, Asp172, Asp173, Asp174, Asp175, Asp176, Leu168, Arg177, Asp178, Asp179, Asp180, Asp181, Asp182, Asp183, Asp184, Asp185, Asp186, An isolated antibody or antigen-binding portion thereof that contains an antigenic determinant of one or more amino acids n161, Leu162, Pro163 and Glu164, wherein the 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 the 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 the heavy chain CDR2 does not consist of N-ISSS[S / G][S / A]YI-C (SEQ ID NO: 146).

[0206] In some aspects, the disclosure provides an isolated antibody or antigen-binding portion thereof that specifically binds to an antigenic determinant comprising or consisting of Gln37, Leu38, Glu42, Glu46, Val49, Ser50, Leu142, Asp146, Arg149, His150, Phe153, Leu156, Leu162, and Glu164 of SEQ ID NO: 2 (IL-27p28), wherein the 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 the 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 the heavy chain CDR2 does not consist of N-ISSS[S / G][S / A]YI-C (SEQ ID NO: 146) composition.

[0207] In some aspects, the disclosure provides an isolated antibody or antigen-binding portion thereof that specifically binds to an antigenic determinant 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 of SEQ ID NO: 2 (IL-27p28), wherein the 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 the heavy chain CDR1 is not composed 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).

[0208] In some aspects, the disclosure provides a polypeptide that specifically binds to Gln37, Leu38, Glu42, Glu46, Val49, Ser50, Leu53, Lys56, Leu142, Asp143, Arg145, Asp146, Leu147, Arg149, His150, Arg152, Phe153, Leu156, Ala157, Gly159, Phe160, Asn161, An isolated antibody or antigen-binding portion thereof that contains an antigenic determinant of one or more amino acids 161, Leu162, Pro163 and Glu164, wherein the 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 the heavy chain CDR1 does not contain N-FTF[S / A / R][S / R][T / Y][G / S]MN-C (SEQ ID NO: 148) and / or the heavy chain CDR2 does not contain N-[G / S]ISSS[S / G][S / A]YI[L / Y] YADSVKG-C (SEQ ID NO: 149).

[0209] In some aspects, the disclosure provides an isolated antibody or antigen-binding portion thereof that specifically binds to an antigenic determinant comprising or consisting of Gln37, Leu38, Glu42, Glu46, Val49, Ser50, Leu142, Asp146, Arg149, His150, Phe153, Leu156, Leu162, and Glu164 of SEQ ID NO: 2 (IL-27p28), wherein the 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 the heavy chain CDR1 does not comprise N-FTF[S / A / R][S / R][T / Y][G / S]MN-C (SEQ ID NO: 148) and / or the heavy chain CDR2 does not comprise N-[G / S]ISSS[S / G][S / A] YI[L / Y]YADSVKG-C (SEQ ID NO: 149).

[0210] In some aspects, the disclosure provides an isolated antibody or antigen-binding portion thereof that specifically binds to an antigenic determinant 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 of SEQ ID NO: 2 (IL-27p28), wherein the 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 the heavy chain CDR1 does not comprise N-FTF. [S / A / R][S / R][T / Y][G / S]MN-C (SEQ ID NO: 148) and / or the heavy chain CDR2 does not contain N-[G / S]ISSS[S / G][S / A]YI[L / Y]YADSVKG-C (SEQ ID NO: 149).

[0211] In some aspects, the disclosure provides an isolated antibody or antigen-binding portion thereof that specifically binds to an antigenic determinant comprising one or more amino acids 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 of SEQ ID NO: 2 (IL-27p28), wherein the 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 set forth in SEQ ID NO: 121; and light chain CDR1, CDR2, and CDR3 sequences set forth 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 a heavy chain CDR3 sequence set forth in SEQ ID NO: 124; and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 130, 131, and 132, respectively.

[0212] In some aspects, the disclosure provides an isolated antibody, or antigen-binding portion thereof, that specifically binds to an antigenic determinant comprising or consisting of Gln37, Leu38, Glu42, Glu46, Val49, Ser50, Leu142, Asp146, Arg149, His150, Phe153, Leu156, Leu162, and Glu164 of SEQ ID NO: 2 (IL-27p28), wherein the 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 set forth in SEQ ID NO: 121; and light chain CDR1, CDR2, and CDR3 sequences set forth 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 a heavy chain CDR3 sequence set forth in SEQ ID NO: 124; and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 130, 131, and 132, respectively.

[0213] In some aspects, the disclosure provides an isolated antibody, or antigen-binding portion thereof, that specifically binds to an antigenic determinant 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 of SEQ ID NO: 2 (IL-27p28), wherein the 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 set forth in SEQ ID NO: 121; and light chain CDR1, CDR2, and CDR3 sequences set forth 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 a heavy chain CDR3 sequence set forth in SEQ ID NO: 124; and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 130, 131, and 132, respectively.

[0214] In some aspects, the disclosure provides an isolated antibody or antigen-binding portion thereof that specifically binds to an antigenic determinant comprising one or more amino acids 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 of SEQ ID NO: 2 (IL-27p28), wherein the antibody or antigen-binding portion thereof 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: 146), a heavy chain CDR3 consisting of N-IXXXXXXX-C (SEQ ID NO: 147), a heavy chain CDR4 consisting of N-IXXXXXXX-C (SEQ ID NO: 148), a heavy chain CDR5 consisting of N- 152) and a heavy chain CDR2 composed of N-AR[X] n=6-15DX-C (SEQ ID NO: 153); and a light chain CDR1 composed of N-QS[X] n=1-3SS[X] n=0-4Y-C (SEQ ID NO: 154), a light chain CDR2 composed of N-XXS-C (SEQ ID NO: 155) and a light chain CDR3 sequence composed of N-QQXXXXP[X] n=0-1T-C (SEQ ID NO: 156).

[0215] In some aspects, the disclosure provides an isolated antibody or antigen-binding portion thereof that specifically binds to an antigenic determinant comprising or consisting of Gln37, Leu38, Glu42, Glu46, Val49, Ser50, Leu142, Asp146, Arg149, His150, Phe153, Leu156, Leu162, and Glu164 of SEQ ID NO: 2 (IL-27p28), wherein the antibody or antigen-binding portion thereof 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=6-15DX-C (SEQ ID NO: 153); and a heavy chain CDR4 consisting of N-QS[X] n=1-3SS[X] n=0-4Y-C (SEQ ID NO: 154). The light chain CDR1 is composed of N-XXS-C (SEQ ID NO: 154), the light chain CDR2 is composed of N-XXS-C (SEQ ID NO: 155), and the light chain CDR3 sequence is composed of N-QQXXXXP[X] n=0-1T-C (SEQ ID NO: 156).

[0216] In some aspects, the disclosure provides an isolated antibody or antigen-binding portion thereof that specifically binds to an antigenic determinant 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 of SEQ ID NO: 2 (IL-27p28), wherein the antibody or antigen-binding portion thereof 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: 146), a heavy chain CDR3 consisting of N-IXXXXXXX-C (SEQ ID NO: 147), a heavy chain CDR4 consisting of N-IXXXXXXX-C (SEQ ID NO: 148), a heavy chain CDR5 consisting of N- 152) and a heavy chain CDR2 composed of N-AR[X] n=6-15DX-C (SEQ ID NO: 153); and a light chain CDR1 composed of N-QS[X] n=1-3SS[X] n=0-4Y-C (SEQ ID NO: 154), a light chain CDR2 composed of N-XXS-C (SEQ ID NO: 155) and a light chain CDR3 sequence composed of N-QQXXXXP[X] n=0-1T-C (SEQ ID NO: 156).

[0217] In some aspects, the disclosure provides an isolated antibody or antigen-binding portion thereof that antagonizes IL-27 and specifically binds to an antigenic determinant comprising one or more amino acids 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 of SEQ ID NO: 2 (IL-27p28), wherein the antibody or antigen-binding portion thereof comprises heavy and light chain variable regions, wherein the heavy chain variable region does not comprise a sequence selected from the group consisting of SEQ ID NO: The invention relates to an amino acid sequence of the group consisting of SEQ ID NOs: 15, 37, 59, 81, 103 and 125; and wherein the light chain variable region does not comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 23, 45, 67, 89, 111 and 133.

[0218] In some aspects, the disclosure provides an isolated antibody or antigen-binding portion thereof that antagonizes IL-27 and specifically binds to an antigenic determinant comprising one or more amino acids 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 of SEQ ID NO: 2 (IL-27p28), wherein the antibody or antigen-binding portion thereof comprises heavy and light chain variable regions, wherein the heavy chain variable region and the light chain variable region are not an amino acid sequence 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 NOs: 59 and 67, respectively; (iv) SEQ ID NOs: 81 and 89, respectively; (v) SEQ ID NOs: 103 and 111, respectively; and (vi) SEQ ID NOs: 125 and 133, respectively.

[0219] In some aspects, the disclosure provides an isolated antibody or antigen-binding portion thereof that antagonizes IL-27 and specifically binds to an antigenic determinant 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 of SEQ ID NO: 2 (IL-27p28), wherein the antibody or antigen-binding portion thereof comprises heavy and light chain variable regions, wherein the heavy chain variable region does not comprise an antigen-binding region selected from the group consisting of SEQ ID NO: The invention relates to a light chain variable region comprising an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 37, 59, 81, 103 and 125; and wherein the light chain variable region does not comprise an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 23, 45, 67, 89, 111 and 133.

[0220] In some aspects, the disclosure provides an isolated antibody or antigen-binding portion thereof that antagonizes IL-27 and specifically binds to an antigenic determinant comprising one or more amino acids 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 of SEQ ID NO: 2 (IL-27p28), wherein the antibody or antigen-binding portion thereof comprises heavy and light chain variable regions, wherein the heavy chain variable region and the light chain variable region do not comprise an amino acid sequence that is at least 90% identical to an amino acid sequence 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 NOs: 59 and 67, respectively; (iv) SEQ ID NOs: 81 and 89, respectively; (v) SEQ ID NOs: 103 and 111, respectively; and (vi) SEQ ID NOs: 125 and 133, respectively.

[0221] In some aspects, the disclosure provides an isolated antibody or antigen-binding portion thereof that antagonizes IL-27 and specifically binds to an antigenic determinant comprising one or more amino acids 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 of SEQ ID NO: 2 (IL-27p28), wherein the antibody or antigen-binding portion thereof comprises a heavy chain and a light chain, wherein the heavy chain does not comprise a sequence selected from the group consisting of SEQ ID NO: The light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 20, 42, 71, 93 and 1115.

[0222] In some aspects, the disclosure provides an isolated antibody or antigen-binding portion thereof that antagonizes IL-27 and specifically binds to an antigenic determinant 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 of SEQ ID NO: 2 (IL-27p28), wherein the antibody or antigen-binding portion thereof comprises heavy and light chains, wherein the heavy chain does not comprise an antigen-binding residue selected from the group consisting of SEQ ID NO: The invention relates to a light chain comprising an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 20, 42, 71, 93 and 115; and wherein the light chain does not comprise an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 20, 42, 71, 93 and 115.

[0223] In some aspects, the disclosure provides an isolated antibody or antigen-binding portion thereof that antagonizes IL-27 and specifically binds to an antigenic determinant comprising one or more amino acids 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 of SEQ ID NO: 2 (IL-27p28), wherein the antibody or antigen-binding portion thereof comprises a heavy chain and a light chain, wherein the heavy chain does not comprise a sequence selected from the group consisting of SEQ ID NO: The light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 71, 49, 71, 93, 115 and 137.

[0224] In some aspects, the disclosure provides an isolated antibody or antigen-binding portion thereof that antagonizes IL-27 and specifically binds to an antigenic determinant 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 of SEQ ID NO: 2 (IL-27p28), wherein the antibody or antigen-binding portion thereof comprises heavy and light chains, wherein the heavy chain does not comprise an antigen-binding residue selected from the group consisting of SEQ ID NO: The invention relates to a light chain comprising an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 29, 51, 73, 95, 117 and 139; and wherein the light chain does not comprise an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 71, 49, 71, 93, 115 and 137.

[0225] In some aspects, the disclosure provides an isolated antibody or antigen-binding portion thereof that antagonizes IL-27 and specifically binds to an antigenic determinant comprising one or more amino acids 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 of SEQ ID NO: 2 (IL-27p28), wherein the antibody or antigen-binding portion thereof comprises a heavy chain and a light chain, and wherein the heavy chain and the light chain do not comprise an amino acid sequence selected from the group consisting of: (i) SEQ ID NOs: 25 and 27, respectively; (ii) SEQ ID NOs: 47 and 49, respectively; (iii) SEQ ID NOs: 69 and 71, respectively; (iv) SEQ ID NOs: 91 and 93, respectively; (v) SEQ ID NOs: 113 and 115, respectively; and (vi) SEQ ID NOs: 135 and 137, respectively.

[0226] In some aspects, the disclosure provides an isolated antibody or antigen-binding portion thereof that antagonizes IL-27 and specifically binds to an antigenic determinant comprising one or more amino acids 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 of SEQ ID NO: 2 (IL-27p28), wherein the antibody or antigen-binding portion thereof comprises a heavy chain and a light chain, and wherein the heavy chain and the light chain do not comprise an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of: (i) SEQ ID NOs: 25 and 27, respectively; (ii) SEQ ID NOs: 47 and 49, respectively; (iii) SEQ ID NOs: 69 and 71, respectively; (iv) SEQ ID NOs: 91 and 93, respectively; (v) SEQ ID NOs: 113 and 115, respectively; and (vi) SEQ ID NOs: 135 and 137, respectively.

[0227] In some aspects, the disclosure provides an isolated antibody or antigen-binding portion thereof that antagonizes IL-27 and specifically binds to an antigenic determinant comprising one or more amino acids 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 of SEQ ID NO: 2 (IL-27p28), wherein the antibody or antigen-binding portion thereof comprises heavy and light chains, and wherein the heavy and light chains do not comprise an amino acid sequence selected from the group consisting of: (i) SEQ ID NOs: 29 and 27, respectively; (ii) SEQ ID NO: 51 and 49, respectively; (iii) SEQ ID NOs: 73 and 72, respectively; (iv) SEQ ID NO: 95 and 93, respectively; (v) SEQ ID NOs: 117 and 115, respectively; and (vi) SEQ ID NOs: 139 and 137, respectively.

[0228] In some aspects, the disclosure provides an isolated antibody or antigen-binding portion thereof that antagonizes IL-27 and specifically binds to an antigenic determinant comprising one or more amino acids of Gln37, Leu38, Glu42, Glu46, Val49, Ser50, Leu53, Lys56, Leu142, Aspl43, Arg145, Aspl46, Leu147, Arg149, His150, Arg152, Phe153, Leu156, Ala157, Gly159, Phe160, Asn161, Leu162, Pro163, and Glu164 of SEQ ID NO: 2 (IL27-p28), wherein the antibody or antigen-binding portion thereof comprises heavy and light chains, and wherein the heavy and light chains do not comprise an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of: (i) SEQ ID NOs: 29 and 27, respectively; (ii) SEQ ID NO: 51 and 49, respectively; (iii) SEQ ID NOs: 73 and 72, respectively; (iv) SEQ ID NO: 95 and 93, respectively; (v) SEQ ID NOs: 117 and 115, respectively; and (vi) SEQ ID NOs: 139 and 137, respectively. [Produce resistance] [IL-27] [Methods of Antibodies and Antigen-binding Fragments thereof] [] []

[0229] The present disclosure also provides methods of producing any anti-IL-27 antibody or antigen-binding fragment thereof described herein. In some aspects, the method of preparing an antibody described herein may include immunizing a subject (e.g., a non-human mammal) with a suitable immunogen. Suitable immunogens for producing any of the antibodies described herein are set forth herein. For example, to produce an antibody that binds to IL-27p28, one skilled in the art may 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 IL-27. In some aspects, a full-length human IL-27p28 monomer polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2 is used as an immunogen.

[0230] A suitable individual (e.g., a non-human mammal) can be immunized with an appropriate antigen, along with multiple subsequent booster immunizations sufficient to elicit antibody production in the mammal. The immunogen can be administered to the individual (e.g., a non-human mammal) together with an adjuvant. Adjuvants suitable for producing antibodies in an individual 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 skeletons, trehalose dimycolate, monophosphatidyl lipid A, methanol extractable residue (MER) of tuberculosis, complete or incomplete Freund's adjuvant; viral adjuvants; chemical adjuvants, such as aluminum hydroxide and iodoacetate and cholesterol 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, e.g., 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.

[0231] In some aspects, the methods include preparing 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., B cells of the spleen) of the immunized mammal can be isolated two to four days after at least one booster immunization with the immunogen and then grown briefly in culture, and then fused with cells of a suitable myeloma cell line. The cells can be fused in the presence of a fusion promoter such as vaccinia virus or polyethylene glycol. The hybrid cells obtained in the fusion are cloned, and cell clones that secrete the desired antibody are selected. For example, spleen cells of a Balb / c mouse immunized with a suitable immunogen can be fused with cells of the myeloma cell line PAI or the myeloma cell line Sp2 / 0-Ag 14. After fusion, the cells are expanded at regular intervals in a suitable culture medium supplemented with a selection medium (e.g., HAT medium) to prevent normal myeloma cells from growing out of the desired hybridoma cells. The resulting hybrid cells are then screened for secretion of the desired antibody, e.g., an antibody that binds to human IL-27 and in some aspects, one skilled in the art can identify anti-IL-27 antibodies from a non-immune biased library, as described, e.g., in U.S. Pat. No. 6,300,064 (Knappik et al.; Morphosys AG) and Schoonbroodt et al. (2005) Nucleic Acids Res 33(9):e81.

[0232] In some aspects, the methods described herein may involve, for example, phage display technology, bacterial display, yeast surface display, eukaryotic virus display, mammalian cell display, and cell-free (e.g., ribosome display) antibody screening technology (see, e.g., 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. (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) or used in combination therewith.

[0233] Methods for identifying antibodies using a variety of phage display methods are known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles carrying polynucleotide sequences encoding them. Such phages can be used to display antigen binding domains of antibodies expressed from a pedigree or combinatorial antibody library (e.g., human or murine), such as Fab, Fv, or disulfide-stabilized Fv antibody fragments. Phages used in these methods are typically filamentous phages, such as fd and M13. Antigen binding domains are expressed as proteins 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 prepare the immunoglobulins or fragments thereof described herein include those described 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 PCT Publication Nos. WO 90 / 02809, WO 91 / 10737, WO 92 / 01047, WO 92 / 18619, WO 93 / 11236, WO 95 / 15982, and WO Suitable methods are also described in, for example, U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 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.

[0234] In some aspects, the phage display antibody libraries can be generated using mRNA collected from B cells from immunized mammals. For example, a spleen cell sample containing B cells can be isolated from a mouse 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), supra; Benny KC Lo (2004), supra; and Borrebaek (1995), supra. Phage display libraries are constructed using cDNA encoding the variable regions of the heavy and light chain polypeptides of immunoglobulins. Methods for generating such libraries 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.

[0235] In some aspects, a combination of selection and screening can be used to identify antibodies of interest from, for example, a population of fusion tumor-derived 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), supra; Ames et al. (1995), supra; Kettleborough et al. (1994), supra; Persic et al. (1997), supra; and Burton et al. (1994), supra. For example, a plurality of phagemid vectors each encoding a fusion protein of a phage coat protein (e.g., pIII, pVIII, or pIX of M13 phage) and a different antigen combination region are generated using standard molecular biology techniques and then introduced into a bacterial (e.g., E. coli) population. In some aspects, phage expression in bacteria may require the use of helper phages. In some aspects, helper phage is not required (see, e.g., Chasteen et al., (2006) Nucleic Acids Res 34(21): e145). Phage produced from bacteria are recovered and then contacted with target antigen, e.g., bound to a solid support (immobilized). Phage can also be contacted with antigen in solution, and the complex then bound to a solid support.

[0236] Subpopulations of antibodies screened using the above methods can be characterized for their specificity and binding affinity for 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 assays, SPR assays, immunoprecipitation assays, affinity chromatography, and equilibrium dialysis as described above. Immunoassays that can be used to analyze the immunospecific binding and cross-reactivity of antibodies include, but are not limited to, competitive and non-competitive assay systems using techniques such as Western blotting, RIA, enzyme-linked immunosorbent assays (ELISA), "sandwich" immunoassays, immunoprecipitation assays, immunodiffusion assays, agglutination assays, complement fixation assays, immunoradiometric assays, fluorescent immunoassays, and protein A immunoassays. These assays are routine and well known in the art.

[0237] In aspects where the CDR amino acid sequences are selected to be short (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 U.S. Pat. No. 6,995,259. With respect to a given nucleic acid sequence encoding an acceptor antibody, the region encoding the CDRs in the nucleic acid sequence can be replaced with a chemically synthesized nucleic acid using standard molecular biology techniques. The 5' and 3' ends of the chemically synthesized nucleic acids can be synthesized to include sticky-terminal restriction enzyme sites for cloning the nucleic acids into nucleic acids encoding the variable regions of the donor antibody.

[0238] In some aspects, the anti-IL-27 antibodies described herein comprise an altered heavy chain constant region that has reduced (or no) effector function relative to its corresponding unaltered constant region. Effector functions involving the constant region of the anti-IL-27 antibody can be modulated by altering the properties of the constant or Fc region. The altered effector function includes, for example, modulation of one or more of the following activities: 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 effector function activity exhibited by the antibody of the invention containing the altered constant region, as compared to the activity of the unaltered form of the constant region. In a specific aspect, modulation includes situations in which the activity is eliminated or completely absent.

[0239] In one aspect, the anti-IL-27 antibodies described herein comprise an IgG4 heavy chain constant region. In one aspect, the IgG4 heavy chain constant region is a wild-type IgG4 heavy chain constant region. In another aspect, the IgG4 constant region comprises a mutation, such as S228P and one or both of L235E or L235A, such as according to EU numbering (Kabat, EA et al., supra). In one aspect, the anti-IL-27 antibodies described herein comprise an IgG1 constant region. In one aspect, the IgG1 heavy chain constant region is a wild-type IgG1 heavy chain constant region. In another aspect, the IgG1 heavy chain constant region comprises a mutation.

[0240] An altered constant region with altered FcR binding affinity and / or ADCC activity and / or altered CDC activity is a polypeptide with enhanced or diminished FcR binding activity and / or ADCC activity and / or CDC activity compared to the unaltered form of the constant region. An altered constant region exhibiting increased FcR binding binds to at least one FcR with an affinity greater than that of the unaltered polypeptide. An altered constant region exhibiting decreased FcR binding binds to at least one FcR with an affinity less than that of the unaltered form of the constant region. Such variants that exhibit reduced FcR binding may have little or no appreciable FcR binding, e.g., 9-50% (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, 7, 6, 5, 4, 8, 3, 3, 2, or 1%) of FcR binding as compared to the level of binding of a native sequence immunoglobulin constant or Fc region to FcR. Similarly, an altered constant region that exhibits modulated ADCC and / or CDC activity may exhibit increased or decreased ADCC and / or CDC activity compared to the unaltered constant region. For example, in some aspects, an anti-IL-27 antibody comprising an altered constant region can exhibit about 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 the ADCC and / or CDC activity of the unaltered form of the constant region. The anti-IL-27 antibodies described herein that comprise an altered constant region that exhibits reduced ADCC and / or CDC may exhibit reduced or no ADCC and / or CDC activity.

[0241] In some aspects, the anti-IL-27 antibodies described herein exhibit reduced or no effector function. In some aspects, the anti-IL-27 antibodies comprise a hybrid constant region or a portion thereof, such as a G2 / G4 hybrid 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). See above.

[0242] In some aspects, the anti-IL-27 antibody may contain an altered constant region that exhibits enhanced or reduced complement-dependent cytotoxicity (CDC). Modulated CDC activity may be achieved by introducing one or more amino acid substitutions, insertions or deletions into the Fc region of the antibody. See, e.g., U.S. Pat. No. 6,194,551. Alternatively or additionally, cysteine ​​residues may be introduced into the Fc region, thereby allowing interchain disulfide bonds to form in this region. The homodimeric antibody thus produced may have improved or reduced internalization capacity and / or increased or reduced complement-mediated cell killing. See, e.g., Caron et al. (1992) J Exp Med 176:1191-1195 and Shopes (1992) Immunol 148:2918-2922; PCT Publication Nos. WO 99 / 51642 and WO 94 / 29351; Duncan and Winter (1988) Nature 322:738-40; and U.S. Pat. No. 5,648,260 and No. 5,624,821. [Recombinant Antibody Expression and Purification] [] []

[0243] The antibodies or antigen-binding fragments thereof described herein can be produced using a variety of molecular biology and protein chemistry techniques known in the art. For example, nucleic acids encoding one or both of the heavy and light chain polypeptides of the antibody can be inserted into an expression vector containing transcriptional and translational regulatory sequences, including, 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. Such regulatory sequences include promoters 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, such as in mammalian or insect cells for expression and in prokaryotic hosts for cloning and amplification.

[0244] Several possible vector systems can be used for the expression of heavy and light chain polypeptides cloned from nucleic acids in mammalian cells. One type of vector relies on the integration of the desired gene sequence into the host cell genome. Cells that stably integrate DNA can be selected by simultaneously introducing drug resistance genes such as Escherichia 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 be connected to the DNA gene sequence to be expressed, or introduced into the same cell by co-transfection (Wigler et al. (1979) Cell 16: 77). The second type of vector utilizes DNA elements that confer autonomous replication ability to the extrachromosomal plastid. Such vectors may be derived from animal viruses such as bovine papilloma virus (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).

[0245] Such expression vectors can be introduced into cells in a manner suitable for subsequent expression of the nucleic acid. The method of introduction is primarily indicated by the target cell type discussed below. Exemplary methods include CaPO precipitation, liposome fusion, cationic liposomes, electroporation, viral infection, dextran-mediated transfection, polybrene-mediated transfection, protoplast fusion, and direct microinjection.

[0246] Suitable host cells for expression of antibodies or antigen-binding fragments thereof include yeast, bacteria, insect, plant and mammalian cells. 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.

[0247] In some aspects, the antibody or fragment thereof can be expressed in and purified from a transgenic animal (e.g., a transgenic mammal). For example, the antibody can be produced in a transgenic non-human 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.

[0248] Antibodies and fragments thereof can be produced from cells by culturing host cells transformed with an expression vector containing nucleic acid encoding the antibody or fragment under conditions sufficient to allow expression of the protein and for an amount of time sufficient to allow expression of the protein. Such conditions for protein expression vary with the choice of expression vector and host cell and are readily determined by one skilled in the art through routine experimentation. For example, antibodies expressed in E. coli can be refolded from inclusion bodies (see, e.g., Hou et al. (1998) Cytokine 10:319-30). Bacterial expression systems and methods of using them are known in the art (see Current Protocols in Molecular Biology, Wiley & Sons, and Molecular Cloning -- A Laboratory Manual -- 3rd Ed., Cold Spring Harbor Laboratory Press, New York (2001)). The choice of codons, suitable expression vectors and suitable host cells varies depending on a variety of factors and can be easily optimized when necessary. The antibodies (or fragments thereof) described herein can be expressed in mammalian cells or other expression systems including, but not limited to, yeast, baculovirus and in vitro expression systems (see, e.g., Kaszubska et al. (2000) Protein Expression and Purification 18: 213-220).

[0249] After expression, the antibodies and fragments thereof can be isolated. The antibodies or fragments thereof can be isolated or purified in a variety of ways known to those skilled in the art, depending on what other components are present in the sample. Standard purification methods include electrophoretic, molecular, immunological and chromatographic techniques, including ion exchange, hydrophobic, affinity and reverse phase HPLC chromatography. For example, the antibodies 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 are also useful. See, e.g., Scopes (1994) "Protein Purification, 3rd edition," Springer-Verlag, New York City, New York. The degree of purification necessary varies depending on the desired use. In some cases, purification of the expressed antibody or fragment thereof is not necessary.

[0250] Methods for determining the yield or purity of the purified antibody or fragment thereof are known in the art and include, for example, Bradford analysis, UV spectroscopy, biuret protein analysis, Lowry protein analysis, amidomelanoprotein analysis, high pressure liquid chromatography (HPLC), mass spectrometry (MS), and gel electrophoresis methods (e.g., using protein stains such as Coomassie blue or colloidal silver staining). [Modified Antibody or Antigen-binding Fragment thereof] [] []

[0251] The antibody or its antigen-binding fragment can be modified after its expression and purification. Such modifications can be covalent or non-covalent modifications. Such modifications can be introduced into the antibody or fragment by, for example, reacting the targeted amino acid residues of the polypeptide with an organic derivatizing agent capable of reacting with the selected side chain or terminal residue. 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 antibody or fragment.

[0252] In some aspects, the antibody or antigen-binding fragment thereof can be conjugated to a heterologous moiety. The heterologous moiety can 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 radiolabel, an enzyme label, a fluorescent label, a heavy metal label, a luminescent label, or an affinity tag (such as biotin or streptavidin). Suitable heterologous polypeptides include, for example, antigenic tags (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)), which are used to purify antibodies or fragments. Heterologous polypeptides also include polypeptides suitable for use as diagnostic or detectable markers (e.g., enzymes), such as luciferase, fluorescent proteins (e.g., green fluorescent protein (GFP)) or chloramphenicol acetyltransferase (CAT). Suitable radiolabels include, for example, 32P, 33P, 14C, 125I, 131I, 35S and 3H. Suitable fluorescent labels include, but are not limited to, luciferin, fluorescein isothiocyanate (FITC), green fluorescent protein (GFP), DyLight™ 488, phycoerythrin (PE), propidium iodide (PI), PerCP, PE-Alexa Fluor® 700, Cy5, isophycocyanin, and Cy7. Luminescent labels include, for example, any of a variety of luminescent lanthanide (e.g., europium or zirconium) chelates. For example, suitable europium chelates include europium chelates of diethylenetriaminepentaacetic acid (DTPA) or tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). Enzyme labels include, for example, alkaline phosphatase, CAT, luciferase, and horseradish peroxidase.

[0253] Two proteins (e.g., an antibody and a heterologous moiety) can be cross-linked using any of a variety of known chemical cross-linking agents. Examples of such cross-linking agents are those that link amino acid residues via bonds including "hindered" disulfide bonds. In such bonds, the disulfide bonds within the cross-linking unit are protected (by hindering groups on either side of the disulfide bond) from reduction by, for example, reduced glutathione or the action of the enzyme disulfide reductase. One suitable reagent, 4-succinimidyloxycarbonyl-α-methyl-α(2-pyridyldisulfide)toluene (SMPT), forms such bonds between two proteins using a terminal lysine on one of the proteins and a terminal cysteine ​​on the other. Heterobifunctional reagents that cross-link via different coupling moieties on each protein can also be used. Other suitable cross-linking agents include, but are not limited to, agents that link two amine groups (e.g., N-5-azido-2-nitrobenzyloxybutane), two sulfhydryl groups (e.g., 1,4-bis-maleimidobutane), an amine group and a sulfhydryl group (e.g., m-maleimidobenzyl-N-hydroxybutanediamide ester), an amine group and a carboxyl group (e.g., 4-[p-azidosalicylamido]butylamine), and an amine group and a guanidinium group present in the side chain of arginine (e.g., p-azidophenylglyoxal monohydrate).

[0254] In some aspects, the radiolabel can be directly attached to the amino acid backbone of the antibody. Alternatively, the radiolabel can be included as part of a larger molecule (e.g., m-[125I]iodophenyl-N-hydroxysuccinimide (125I in [125I]mIPNHS), which is attached to a free amine group to form a m-iodophenyl (mIP) derivative of the protein of interest (see, e.g., Rogers et al. (1997) J Nucl Med 38:1221-1229) or to a chelate (e.g., to DOTA or DTPA), which is in turn attached to the protein backbone. Methods for attaching such radiolabels or larger molecules / chelates containing them to the antibodies or antigen-binding fragments described herein are known in the art. Such methods involve incubating the protein with the radiolabel under conditions (e.g., pH, salt concentration, and / or temperature) that promote binding of the radiolabel or chelate to the protein (see, e.g., U.S. Pat. No. 6,001,329).

[0255] Methods for conjugating fluorescent labels (sometimes referred to as fluorophores) to proteins (e.g., antibodies) are known in the art of protein chemistry. For example, fluorophores can be conjugated to free amine groups (e.g., from lysine) or sulfhydryl groups (e.g., from cysteine) of proteins using succinimidyl (NHS) ester or tetrafluorophenyl (TFP) ester moieties attached to the fluorophores. In some aspects, the fluorophores can be conjugated to a heterobifunctional cross-linker moiety, such as sulfo-SMCC. Suitable conjugation methods involve incubating the antibody protein or fragment thereof with the fluorophore under conditions that promote binding of the fluorophore to the protein. See, e.g., Welch and Redvanly (2003) "Handbook of Radiopharmaceuticals: Radiochemistry and Applications,” John Wiley and Sons (ISBN 0471495603).

[0256] In some aspects, antibodies or fragments can be modified, for example, with a moiety that improves the stability and / or retention of the antibodies in circulation (e.g., in blood, serum, or other tissues). For example, antibodies or fragments can be pegylated or hesylated (Fresenius Kabi, Germany; see, e.g., Pavisić et al. (2010) Int J Pharm 387(1-2):110-119) as described, for example, by 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). The stabilizing moiety can improve the stability or retention of the antibody (or fragment) by at least 1.5 (e.g., at least 2, 5, 10, 15, 20, 25, 30, 40, or 50 or more) times.

[0257] In some aspects, the antibodies or antigen-binding fragments thereof described herein may be glycosylated. In some aspects, the antibodies or antigen-binding fragments thereof described herein may be subjected to enzymatic or chemical treatment, or produced from cells, such that the antibody or fragment has reduced or no glycosylation. Methods for producing antibodies with reduced glycosylation are known in the art and are described, for example, in U.S. Pat. No. 6,933,368; Wright et al. (1991) EMBO J 10(10):2717-2723; and Co et al. (1993) Mol Immunol 30:1361. [Medical compositions and formulations] [] []

[0258] In certain aspects, the present invention provides a pharmaceutical composition comprising an anti-IL-27 antibody and a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and / or adjuvant.

[0259] In some aspects, the acceptable formulation materials are preferably non-toxic to the recipient at the dosages and concentrations used. In some aspects, the formulation material(s) are for sc and / or IV administration. In some aspects, the pharmaceutical composition may contain formulation materials for modifying, maintaining or preserving, for example, pH, osmotic concentration, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption or penetration of the composition. In certain aspects, suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium bisulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrate, phosphate or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, β-cyclodextrin or hydroxypropyl β-cyclodextrin); fillers; monosaccharides, disaccharides and other carbohydrates (such as glucose, mannose or dextrin); proteins (such as serum albumin, gelatin or immunoglobulins); coloring, flavoring 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, phenethyl 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; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancers (such as sucrose or sorbitol); tonicity enhancers (such as alkali metal halides, preferably sodium chloride or potassium chloride; mannitol sorbitol); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants. (Remington's Pharmaceutical Sciences, 18th Edition, AR Gennaro, ed., Mack Publishing Company (1995). In certain aspects, the formulation comprises PBS; 20 mM NaOAC (pH 5.2), 50 mM NaCl; and / or 10 mM NAOAC (pH 5.2), 9% sucrose. In certain aspects, the optimal pharmaceutical composition will be determined by one skilled in the art depending on, for example, the intended route of administration, delivery form, and desired dosage.See, e.g., Remington's Pharmaceutical Sciences, supra. In certain aspects, such compositions may affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the anti-IL-27 antibody.

[0260] In certain aspects, the primary vehicle or carrier in the pharmaceutical composition may be aqueous or non-aqueous in nature. For example, in certain aspects, a suitable vehicle or carrier may be water for injection, saline solution, or artificial cerebrospinal fluid, possibly supplemented with other materials commonly used in compositions for parenteral administration. In certain aspects, the saline solution comprises isotonic phosphate-buffered saline. In certain aspects, neutral buffered saline or saline mixed with serum albumin is a further exemplary vehicle. In certain aspects, the pharmaceutical composition comprises a Tris buffer of about pH 7.0-8.5, or an acetate buffer of about pH 4.0-5.5, which may further include sorbitol or a suitable substitute thereof. In certain aspects, a composition comprising an anti-IL-27 antibody may be prepared in the form of a lyophilized cake or aqueous solution for storage by mixing a selected composition having a desired purity with a formulation agent (Remington's Pharmaceutical Sciences, above) selected as appropriate. Furthermore, in certain aspects, the compositions comprising the anti-IL-27 antibody can be formulated as a lyophilizate using a suitable excipient such as sucrose.

[0261] In some aspects, the pharmaceutical composition may be selected for parenteral delivery. In some aspects, the compositions may be selected for inhalation or for delivery via the digestive tract, such as orally. The preparation of such pharmaceutically acceptable compositions is within the capabilities of those skilled in the art.

[0262] In certain aspects, the formulation components are present at concentrations acceptable to the site of administration. In certain aspects, a buffer is used to maintain the composition at physiological pH or slightly lower pH, typically in the pH range of about 5 to about 8.

[0263] In certain aspects, when parenteral administration is contemplated, the therapeutic composition may be in the form of a pyrogen-free, parenterally acceptable aqueous solution comprising an anti-IL-27 antibody in a pharmaceutically acceptable vehicle. In certain aspects, the vehicle for parenteral injection is sterile distilled water, wherein the anti-IL-27 antibody is formulated as a sterile, isotonic solution and appropriately preserved. In certain aspects, the preparation may involve formulating the desired molecule with an agent that can provide controlled or sustained release of the product, such as injectable microspheres, biocorrodible ions, polymeric compounds (such as polylactic acid or polyglycolic acid), beads, or liposomes, which can then be delivered via reservoir injection. In certain aspects, hyaluronic acid may also be used, and it may have the effect of promoting duration in circulation. In certain aspects, an implantable drug delivery device may be used to introduce the desired molecule.

[0264] In certain aspects, the pharmaceutical composition can be formulated for inhalation. In certain aspects, the anti-IL-27 antibody can be formulated as a dry powder for inhalation. In certain aspects, an inhalation solution comprising the anti-IL-27 antibody can be formulated with a propellant for aerosol delivery. In certain aspects, the solution can be atomized. Pulmonary administration is further described in PCT Application No. PCT / US94 / 001875, which describes pulmonary delivery of chemically modified proteins.

[0265] In certain aspects, it is contemplated that the formulation may be administered orally. In certain aspects, the anti-IL-27 antibody administered in this manner may be formulated with or without carriers conventionally used to compound solid dosage forms such as tablets and capsules. In certain aspects, the capsule may be designed to release the active portion of the formulation while in the gastrointestinal tract, when bioavailability is maximized and pre-systemic degradation is minimized. In certain aspects, at least one additional agent may be included to promote absorption of the anti-IL-27 antibody. In certain aspects, diluents, flavoring agents, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrants, and binders may also be used.

[0266] In certain aspects, the pharmaceutical composition may involve an effective amount of an anti-IL-27 antibody mixed with a non-toxic excipient suitable for tablet manufacture. In certain aspects, the solution can be prepared in unit dosage form by dissolving the tablet in sterile water or another appropriate vehicle. In certain aspects, suitable excipients include, but are not limited to, inert diluents such as calcium carbonate, sodium carbonate or sodium bicarbonate, lactose or calcium phosphate; or binders such as starch, gelatin or acacia; or lubricants such as magnesium stearate, stearic acid or talc.

[0267] Other pharmaceutical compositions will be apparent to those skilled in the art, including formulations involving anti-IL-27 antibodies in the form of sustained or controlled delivery formulations. In certain aspects, techniques for formulating a variety of other sustained or controlled delivery means, such as liposome carriers, bioerodible microparticles or porous beads, and reservoir injections, are also known to those skilled in the art. See, for example, PCT Application No. PCT / US93 / 00829, which describes controlled release of porous polymeric microparticles for delivery of pharmaceutical compositions. In certain aspects, sustained release formulations may include a semipermeable polymer matrix in the form of a shaped article, such as a film or microcapsule. Sustained release matrices may include polyesters, hydrogels, polylactic acid (U.S. Pat. No. 3,773,919 and EP 058,481), copolymers of L-glutamic acid and γ-ethyl-L-glutamic acid ester (Sidman et al., Biopolymers, 22: 547-556 (1983)), poly (2-hydroxyethyl-methacrylate) (Langer et al., J. Biomed. Mater. Res., 15: 167-277 (1981) and Langer, Chem. Tech., 12: 98-105 (1982)), ethylene-vinyl acetate (Langer et al. supra) or poly-D-(-)-3-hydroxybutyric acid (EP 133,988). In certain aspects, the sustained release composition may also include liposomes which may be prepared by any of several methods known in the art. See, e.g., Eppstein et al., Proc. Natl. Acad. Sci. USA, 82:3688-3692 (1985); EP 036,676; EP 088,046 and EP 143,949.

[0268] Pharmaceutical compositions intended for in vivo administration are typically sterile. In certain aspects, this can be accomplished by filtering through a sterile filtration membrane. In certain aspects in which the composition is lyophilized, sterilization using this method can be performed before or after lyophilization and reconstitution. In certain aspects, compositions for parenteral administration can be stored in lyophilized form or in solution. In certain aspects, parenteral compositions are generally placed in a container with a sterile access port (e.g., an intravenous solution bag or a vial with a stopper pierceable by a hypodermic needle).

[0269] In certain aspects, once the pharmaceutical composition has been formulated, it can be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder. In certain aspects, the formulations can be stored in a ready-to-use form or in a form that is reconstituted prior to administration (e.g., lyophilized).

[0270] In certain aspects, kits are provided for producing single-dose administration units. In certain aspects, the kit may contain a first container with a dried protein and a second container with an aqueous formulation. In certain aspects, kits containing single and multi-chamber prefilled syringes (e.g., liquid syringes and lyophilized formulation syringes) are included.

[0271] In certain aspects, the effective amount of a pharmaceutical composition comprising an anti-IL-27 antibody to be used therapeutically depends, for example, on the therapeutic context and goal. Those skilled in the art will appreciate that, according to certain aspects, the appropriate dosage level for treatment varies in part depending on the molecule delivered, the indication for which the anti-IL-27 antibody is used, the route of administration, and the patient's size (weight, body surface or organ size) and / or condition (age and general health). In certain aspects, the clinician can titrate the dose and change the route of administration to obtain the optimal therapeutic effect.

[0272] In some aspects, the frequency of dosing takes into account the pharmacokinetic parameters of the anti-IL-27 antibody in the formulation used. In some aspects, the clinician administers the composition until a dose is reached to achieve the desired effect. In some aspects, the composition can therefore be administered in a single dose, or in two or more doses over time (which may or may not contain equal amounts of the desired molecule), or in a continuous infusion via, for example, an implanted device or catheter. Further refinements of appropriate doses are routinely performed by persons of ordinary skill and within the scope of the tasks routinely performed by them. In some aspects, appropriate doses can be determined using appropriate dose-response data.

[0273] In certain aspects, the route of administration of the pharmaceutical composition is according to known methods, such as oral; by injection by intravenous, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, subcutaneous, intraocular, intraarterial, intraportal or intralesional routes; by sustained release system or by implant device. In certain aspects, the compositions may be administered by bolus or continuously by infusion, or by implant device. In certain aspects, the individual elements of the combination therapy may be administered by different routes.

[0274] In certain aspects, the composition may also be administered locally via implantation of a membrane, sponge, or another appropriate material into which the desired molecule has been absorbed or encapsulated. In certain aspects in which an implant device is used, the device may be implanted into any suitable tissue or organ, and delivery of the desired molecule may be via diffusion, timed release bolus, or continuous administration. In certain aspects, it may be desirable to use a pharmaceutical composition comprising an anti-IL-27 antibody ex vivo. In such cases, cells, tissues, and / or organs that have been removed from a patient are exposed to a pharmaceutical composition comprising an anti-IL-27 antibody, after which the cells, tissues, and / or organs are subsequently implanted back into the patient.

[0275] In certain aspects, anti-IL-27 antibodies can be delivered by implanting certain cells that have been genetically engineered to express and secrete the polypeptides using methods such as those described herein. In certain aspects, the cells can be animal or human cells and can be autologous, heterologous or xenogeneic. In certain aspects, the cells can be immortalized. In certain aspects, in order to reduce the chance of an immune response, the cells can be encapsulated to avoid infiltration of surrounding tissues. In certain aspects, the encapsulation material is typically a biocompatible, semipermeable polymer cover or membrane that allows release of the protein product but prevents destruction of the cells by the patient's immune system or by other harmful factors from surrounding tissues. [application] [] []

[0276] The compositions described herein can be used for multiple diagnostic and therapeutic applications. For example, detectably labeled antigen binding molecules can be used in assays to detect the presence or amount of a target antigen 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, such as where the composition binds to and inhibits complement proteins, the composition can be used as a positive control in assays designed to identify additional novel compounds that inhibit complement activity or can be used to treat complement-related disorders. For example, an IL-27 inhibitory composition can be used as a positive control in assays to identify additional compounds (e.g., small molecules, aptamers, or antibodies) that reduce or eliminate IL-27 production. The composition can also be used in treatment methods as described below.

[0277] In some aspects, the disclosure provides methods for detecting IL-27 in a biological sample or subject, comprising (i) contacting the sample or subject (and, optionally, a reference sample or subject) with any of the antibodies described herein under conditions that allow interaction of the antibody molecule with IL-27 to occur, and (ii) detecting formation of a complex between the antibody molecule and the sample or subject (and, optionally, a reference sample or subject). [Set] [] []

[0278] The kit may include an anti-IL-27 antibody as disclosed herein, and instructions for use. The kit may include an anti-IL-27 antibody in a suitable container, one or more controls, and various buffers, reagents, enzymes, and other standard components well known in the art. In some aspects, the disclosure provides a kit comprising an anti-IL-27 antibody or antigen-binding portion as disclosed herein, and instructions for use to stimulate an immune response in a subject, or to treat cancer in a subject, optionally with instructions for use in combination with one or more additional therapeutic agents or procedures as disclosed herein.

[0279] The container may include at least one vial, well, test tube, flask, bottle, syringe or other container component in which the anti-IL-27 antibody can be placed and, in some cases, appropriately aliquoted. When additional components are provided, the kit may contain additional containers in which such components can be placed. The kit may also include components containing the anti-IL-27 antibody and any other reagent containers in a tightly sealed container for commercial sale. Such containers may include injection or blow molded plastic containers in which the desired vials are retained. The container and / or kit may include a label with instructions for use and / or warnings. [How to use] [] []

[0280] The compositions of the present invention have a number of in vitro and in vivo utilities involving the detection and / or quantification of IL-27 and / or antagonism of IL-27 function.

[0281] In some aspects, the present disclosure provides a method for inhibiting or reducing STAT1 and / or STAT3 phosphorylation in a cell, the method comprising contacting the cell with an isolated antibody or antigen-binding fragment provided by the present disclosure, wherein the antibody or antigen-binding portion thereof inhibits or reduces STAT1 and / or STAT3 phosphorylation in the cell.

[0282] In some aspects, the disclosure provides a method for inhibiting or reducing the inhibition of CD161 expression in a cell, the method comprising contacting the cell with an isolated antibody or antigen-binding fragment provided by the disclosure, wherein the antibody or antigen-binding portion thereof inhibits or reduces the inhibition of CD161 expression in the cell.

[0283] In some aspects, the present disclosure provides a method for inhibiting or reducing the expression of PD-L1 and / or TIM-3 in a cell, the method comprising contacting the cell with an isolated antibody or antigen-binding fragment provided by the present disclosure, wherein the antibody or antigen-binding portion thereof inhibits or reduces the expression of PD-L1 and / or TIM-3 in the cell.

[0284] In some aspects, the present disclosure provides a method for inducing or enhancing the secretion of one or more cytokines from a cell, the method comprising contacting the cell with an isolated antibody or antigen-binding fragment provided by the present disclosure, wherein the antibody or antigen-binding portion thereof induces or enhances PD-1-mediated secretion of one or more cytokines from the cell.

[0285] In some aspects, the present disclosure provides a method of stimulating an immune response in a subject, the method comprising administering to the subject an effective amount of an isolated antibody or antigen-binding portion thereof that specifically binds to and antagonizes IL-27 provided by the present disclosure, or a pharmaceutical composition comprising the antibody or antigen-binding portion thereof and a pharmaceutically acceptable carrier.

[0286] In some aspects, the present disclosure provides a method of treating cancer in a subject, comprising administering to the subject an effective amount of an isolated antibody or antigen-binding portion thereof that specifically binds to and antagonizes IL-27 provided by the present disclosure, or a pharmaceutical composition comprising the antibody or antigen-binding portion thereof and a pharmaceutically acceptable carrier.

[0287] In some aspects, the present disclosure provides a method for stimulating an immune response in a subject or treating cancer in a subject, the method comprising administering to the subject an effective amount of an isolated antibody or antigen-binding fragment provided by the present disclosure, or a pharmaceutical composition comprising the antibody or its antigen-binding portion and a pharmaceutically acceptable carrier, wherein the antibody or its antigen-binding portion or the pharmaceutical composition inhibits or reduces STAT1 and / or STAT3 phosphorylation in cells, thereby stimulating an immune response or treating cancer.

[0288] In some aspects, the present disclosure provides a method for stimulating an immune response in a subject or treating cancer in a subject, the method comprising administering to the subject an effective amount of an isolated antibody or antigen-binding fragment provided by the present disclosure, or a pharmaceutical composition comprising an antibody or antigen-binding portion thereof and a pharmaceutically acceptable carrier, wherein the antibody or antigen-binding portion thereof or the pharmaceutical composition inhibits or reduces the inhibition of CD161 expression in cells, thereby stimulating an immune response or treating cancer.

[0289] In some aspects, the present disclosure provides a method for stimulating an immune response in a subject or treating cancer in a subject, the method comprising administering to the subject an effective amount of an isolated antibody or antigen-binding fragment provided by the present disclosure, or a pharmaceutical composition comprising an antibody or its antigen-binding portion and a pharmaceutically acceptable carrier, wherein the antibody or its antigen-binding portion or the pharmaceutical composition inhibits or reduces the expression of PD-L1 and / or TIM-3 in cells, thereby stimulating an immune response or treating cancer.

[0290] In some aspects, the present disclosure provides a method for stimulating an immune response in a subject or treating cancer in a subject, the method comprising administering to the subject an effective amount of an isolated antibody or antigen-binding fragment provided by the present disclosure, or a pharmaceutical composition comprising the antibody or its antigen-binding portion and a pharmaceutically acceptable carrier, wherein the antibody or its antigen-binding portion or the pharmaceutical composition induces or enhances PD-1-mediated secretion of one or more cytokines from cells, thereby stimulating an immune response or treating cancer.

[0291] In some aspects, the cancer 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 head and neck cancer), colorectal cancer, bladder cancer, endometrial cancer, prostate cancer, thyroid cancer, hepatocellular carcinoma, gastric cancer, brain cancer, lymphoma (e.g., DL-BCL), leukemia (e.g., AML), or kidney cancer (e.g., renal cell carcinoma, e.g., clear cell renal carcinoma).

[0292] The above compositions are particularly useful in methods of treating or preventing various cancers in a subject. The compositions can be administered to a subject (e.g., a human subject) using a variety of methods that depend in part on the route of administration. The route can be, for example, intravenous injection or infusion (IV), subcutaneous injection (SC), intraperitoneal (IP) injection, intramuscular injection (IM), or intrathecal injection (IT). The injection can be in the form of a bolus or continuous infusion.

[0293] Administration can be achieved, for example, by local infusion, injection, or by means of an implant. The implant can have a porous, non-porous, or gel-like material, including a membrane (such as a silicone rubber membrane) or a fiber. The implant can be configured for sustained or periodic release of the composition to the subject. See, for example, U.S. Patent Application Publication No. 20080241223; U.S. Patent Nos. 5,501,856; 4,863,457; and 3,710,795; EP 488401; and EP 430539, the disclosures of which are each incorporated herein by reference in their entirety. The composition can be delivered to the subject by means of an implantable device based on, for example, a diffusive, corrosive, or convective system, such as an osmotic pump, a biodegradable implant, an electrodiffusion system, an electroosmotic system, a vapor pressure pump, an electrolytic pump, an effervescent pump, a piezoelectric pump, a corrosion-based system, or an electromechanical system.

[0294] In some aspects, the anti-IL-27 antibody or antigen-binding fragment thereof is therapeutically delivered to a subject via local administration.

[0295] The appropriate dosage of an antibody or fragment thereof described herein (which dosage is capable of treating or preventing cancer in an individual) may depend on various factors including, for example, the age, sex, and weight of the individual to be treated and the specific inhibitor compound used. For example, a different dosage of a complete anti-IL-27 antibody may be required to treat an individual with cancer, as compared to the dosage of an IL-27 binding Fab' antibody fragment required to treat the same individual. Other factors that affect the dosage administered to an individual include, for example, the type or severity of the cancer. For example, an individual with metastatic melanoma may require administration of an anti-IL-27 antibody at a different dosage than an individual with glioblastoma. Other factors may include, for example, other medical conditions that affect the individual concurrently or previously, the individual's overall health, diet, time of administration, excretion rate, drug combinations, and any other additional therapeutic agents administered to the individual. It should also be understood that the specific dosage and treatment regimen for any particular individual also depends on the judgment of the treating medical practitioner (e.g., a doctor or nurse). Suitable dosages are described herein.

[0296] The 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 readily determined by one of ordinary skill in the art based in part on the effect of the administered antibody, or if more than one agent is used, based on the combined effect of the antibody and one or more additional active agents. The therapeutically effective amount of an antibody or fragment thereof described herein may also vary depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody (and one or more additional active agents) to elicit a desired response in the individual, e.g., reduction of tumor growth. For example, a therapeutically effective amount of an anti-IL-27 antibody may inhibit a particular disorder, and / or any one of the symptoms of a particular disorder known in the art or described herein (reducing its severity or eliminating its occurrence) and / or preventing the above. A therapeutically effective amount is also an amount in which any toxic or deleterious effects of the composition are outweighed by the therapeutically beneficial effects.

[0297] Suitable human doses of any of the antibodies or fragments thereof described herein can be further evaluated in, for example, a Phase I dose escalation study. 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 Agents and Chemotherapy 50(10):3499-3500.

[0298] In some aspects, the composition contains any antibody or antigen-binding fragment 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 the composition as a whole is therapeutically effective. For example, the composition may contain an anti-IL-27 antibody described herein and an alkylating agent, wherein the antibody and the agent are each at a concentration that, when combined, is therapeutically effective to treat or prevent cancer (e.g., melanoma) in a subject.

[0299] The toxicity and therapeutic efficacy of the compositions can be determined by known medical procedures in cell culture or experimental animals (e.g., animal models of any of the cancers described herein). Such procedures can be used, for example, to determine the LD 50 (the dose that is lethal to 50% of the population) and the ED 50 (the dose that is therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, and it can be expressed as the ratio LD 50 / ED 50. Antibodies or antigen-binding fragments thereof that exhibit high therapeutic indices are preferred. Although compositions that exhibit toxic side effects can be used, care should be taken to design delivery systems that target the compositions to sites of affected tissues and minimize potential damage to normal cells and thereby reduce side effects.

[0300] Data obtained from cell culture assays and animal studies can be used to elucidate dosage ranges for use in humans. The dosage of such antibodies or antigen-binding fragments thereof is generally within a range of circulating concentrations of the antibody or fragment that include the ED 50 with little toxicity. The dosage may vary within this range depending on the dosage form used and the route of administration used. With respect to the anti-IL-27 antibodies described herein, therapeutically effective doses may be initially estimated from cell culture assays. The dosage may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC 50 (i.e., the antibody concentration that achieves half-maximal inhibition of symptoms) as determined in cell culture. This information can be used to more accurately determine the appropriate dosage in humans. The levels in plasma can be measured, for example, by high performance liquid chromatography. In some aspects, for example, where local administration (e.g., to the eye or joint) is desired, cell culture or animal models may be used to determine the dosage required to achieve a therapeutically effective concentration in the local site.

[0301] In some aspects, the methods can be performed in combination with other cancer therapies. For example, the composition can be administered to an individual simultaneously with, before, or after radiation, surgery, targeted or cytotoxic chemotherapy, chemoradiation therapy, hormone therapy, immunotherapy, gene therapy, cell transplantation therapy, precision medicine, genome editing therapy, or other drug therapy.

[0302] As described above, the compositions described herein (e.g., anti-IL-27 compositions) can be used to treat a variety of cancers, such as, but not limited to: Kaposi's sarcoma, leukemia, acute lymphocytic leukemia, acute myeloid leukemia, myeloid blasts, promyelocytes, myelomonocytic monocytic leukemia, 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 lymphoma, King's disease, non-Hodgkin's lymphoma, multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, solid tumors, sarcomas and carcinomas, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, osteosarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovial tumor, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon sarcoma, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary Adenocarcinoma, Cystic adenocarcinoma, Medullary carcinoma, Bronchogenic carcinoma, Renal cell carcinoma, Hepatocellular carcinoma (HCC), Liver cancer, Cholangiocarcinoma, Choriocarcinoma, Seminoma, Embryonal carcinoma, Wilm's tumor, Cervical cancer, Uterine cancer, Testicular tumor, Lung cancer, Small cell lung cancer, Non-small cell lung cancer, Bladder cancer, Epithelial cancer, Glioma, Astrocytoma, Medulloblastoma, Craniopharyngioma, Ependymoma, Pineal tumor, Hemangioblastoma, Acoustic neuroma, Oligodendroglioma, Hemangioma, Melanoma, Neuroblastoma, Retinoblastoma, Nasopharyngeal carcinoma, Esophageal cancer, Basal cell carcinoma, Biliary tract cancer, bladder cancer, bone cancer, brain and central nervous system (CNS) cancer, cervical cancer, choriocarcinoma, colorectal cancer, connective tissue cancer, digestive system cancer, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, stomach cancer, intraepithelial neoplasia, kidney cancer, laryngeal cancer, liver cancer, lung cancer (small cell, large cell), melanoma, neuroblastoma; oral cancer (e.g. lips, tongue, oral cavity and pharynx), ovarian cancer, pancreatic cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer; respiratory system cancer, sarcoma, skin cancer, stomach cancer, testicular cancer, thyroid cancer, uterine cancer and urinary system cancer. [Combination therapy] [] []

[0303] In some aspects, the anti-IL-27 antibodies or antigen-binding portions thereof provided by the present disclosure can be combined with one or more additional therapeutic agents or therapies, e.g., another therapeutic agent or therapy for cancer. For example, an 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 the combination provides a therapeutic benefit to a subject having or at risk of developing cancer.

[0304] In some aspects, the 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, the anti-IL-27 antibody, or antigen binding portion thereof, is administered first and the one or more additional therapeutic agents are administered subsequently (e.g., sequentially). In some aspects, the one or more additional therapeutic agents are administered first and the anti-IL-27 antibody is administered subsequently.

[0305] The anti-IL-27 antibodies or antigen-binding fragments thereof described herein can replace or enhance a previously or currently administered therapy. For example, during treatment with an anti-IL-27 antibody or antigen-binding fragment thereof, the administration of one or more additional therapeutic agents can be stopped or attenuated, such as administered at a lower level. In some aspects, the administration of the previous therapy can be maintained. In some aspects, the previous therapy is maintained until the level of the anti-IL-27 antibody reaches a level sufficient to provide a therapeutic effect.

[0306] In some aspects, the present disclosure provides a method of treating cancer in a subject, the method comprising administering to the subject an effective amount of an isolated antibody or antigen-binding portion thereof that specifically binds to and antagonizes IL-27 provided by the present disclosure, and one or more additional therapeutic agents or procedures, wherein the second therapeutic agent or procedure is selected from the group consisting of chemotherapy, targeted anti-cancer therapy, oncolytic drugs, cytotoxic agents, immune-based therapies, cytokines, surgical procedures, radiation procedures, activators of co-stimulatory molecules, inhibitors of inhibitory molecules, vaccines or cellular immunotherapy, or a combination thereof.

[0307] In some aspects, one or more additional therapeutic agents are PD-1 antagonists, TIM-3 inhibitors, LAG-3 inhibitors, TIGIT inhibitors, CD112R inhibitors, TAM inhibitors, STING agonists, 4-1BB agonists, or combinations thereof. In some aspects, one or more additional therapeutic agents are CD39 antagonists, CD73 antagonists, CCR8 antagonists, or combinations thereof. In some aspects, anti-CD73 is, for example, any anti-CD73 antibody disclosed in U.S. Publication No. 2019 / 0031766 A1, which is incorporated herein by reference in its entirety. In some aspects, anti-CD39 is, for example, any anti-CD39 antibody disclosed in International Publication No. WO2019 / 178269 A2, which is incorporated herein by reference in its entirety.

[0308] In some aspects, one or more additional therapeutic agents are PD-1 antagonists. In some aspects, the PD-1 antagonists are selected from the group consisting of: PDR001, nivolumab, pembrolizumab, pilizumab, MEDI0680, REGN2810, TSR-042, PF-06801591, and AMP-224. In certain aspects, one or more additional therapeutic agents are PD-L1 inhibitors. In some aspects, the PD-L1 inhibitors are selected from the group consisting of: FAZ053, atezolizumab, avelumab, durvalumab, and BMS-936559. In some aspects, the present disclosure provides a method for enhancing one or more activities of an anti-PD-1 antibody (e.g., enhancing PD-1-mediated cytokine secretion; enhancing anti-PD-1-mediated TNFα secretion; enhancing anti-PD-1-mediated IL-6 secretion from cells exposed to anti-PD-1 antibodies), the method comprising exposing the cells to an antibody or an antigen-binding portion thereof provided by the present disclosure simultaneously or sequentially with the anti-PD-1 antibody, thereby enhancing one or more activities of the anti-PD-1 antibody.

[0309] In some aspects, the one or more additional therapeutic agents are sunitinib (Sutent®), cabozantinib (CABOMETYX®), axitinib (INLYTA ®), lenvatinib (LENVIMA ®), everolimus (AFINITOR ®), bevacizumab (AVASTIN ®), icadolstat, NKTR-214 (CD-122 biased agonist), tivozanib (FOTIVDA ®), abexinostat, ipilimumab (YERVOY ®), trolimusab, pazopanib (VOTRIENT ®), sorafenib (NEXAVAR ®), temsirolimus (TORISEL ®), ramucirumab (CYRAMZA ®), niraparib, savolitinib, voronimumab (X-82), regorafenib (STIVARGO ®), donafenib (multikinase inhibitor), camizumab (SHR-1210), pexastimogene devacirepvec (JX-594), ramucirumab (CYRAMZA ®), apatinib (YN968D1), encapsulated doxorubicin (THERMODOX ®), tivantinib (ARQ197), ADI-PEG 20, binitinib, apatinib mesylate, nintedanib, rilulumab, nivolumab (OPDIVO ®), pembrolizumab (KEYTRUDA ®), atezolizumab (TECENTRIQ ®), avelumab (BAVENCIO ®), durvalumab (IMFIMZI ®), cemiplizumab-rwlc (LIBTAYO ®), tislelizumab, and spartalizumab.

[0310] In some aspects, the one or more additional therapeutic agents is a TIM-3 inhibitor, optionally wherein the TIM-3 inhibitor is MGB453 or TSR-022.

[0311] In some aspects, the one or more additional therapeutic agents is a LAG-3 inhibitor, optionally wherein the LAG-3 inhibitor is selected from the group consisting of LAG525, BMS-986016, and TSR-033.

[0312] In some aspects, one or more additional therapeutic agents are TIGIT inhibitors. In some aspects, one or more additional therapeutic agents are CD112R inhibitors. In some aspects, one or more additional therapeutic agents are TAM (Axl, Mer, Tyro) inhibitors. In some aspects, one or more additional therapeutic agents are STING agonists. In some aspects, one or more additional therapeutic agents are 4-1BB agonists.

[0313] In some aspects, the one or more additional therapeutic agents are tyrosine kinase inhibitors, agents targeting the adenosine axis (e.g., CD39 antagonists, CD73 antagonists, or A2AR, A2BR, or dual A2AR / A2BR antagonists), CCR8 antagonists, CTLA4 antagonists, VEG-F inhibitors, or combinations thereof. [Combination with chemotherapeutic agents]

[0314] Chemotherapeutic agents suitable for administration and / or co-administration with the compositions of the present invention include, for example, paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunomycin, (daunorubicin), dihydroxyanthraquinone, mitoxantrone mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol and puromycin and its analogs or homologues. Other agents include, for example, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil dacarbazine), alkylating agents (e.g., dichloromethyl diethylamine, thiotepa (thioTEPA), chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, cis-dichlorodiamine platinum (II) (DDP), procarbazine, hexamethylmelamine, cisplatin, carboplatin, oxaliplatin The present invention relates to the treatment of leukemia and leukemia with the following: oxaliplatin, nedaplatin, satraplatin or triplatinium tetranitrate), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomcin (formerly actinomycin), bleomycin, mithramycin and anthramycin (AMC)), and antimitotic agents (e.g., vincristine and vinblastine), and temozolomide. [] [] [and] [PD-1 / PD-L1] [Combination of antagonists]

[0315] In some aspects, an anti-IL-27 antibody or antigen-binding portion thereof provided by the present disclosure is combined (e.g., administered in combination) with one or more PD-1 antagonists that specifically bind to human PD-1 or PD-L1 and inhibit PD-1 / PD-L1 biological activity and / or downstream pathways and / or cellular processes mediated by human PD-1 / PD-L1 signaling or other human PD-1 / PD-L1-mediated functions.

[0316] Therefore, PD-1 antagonists are provided herein, which directly or allosterically block, antagonize, suppress, inhibit or reduce the biological activity of PD-1 / PD-L1, including downstream pathways and / or cellular processes mediated by PD-1 / PD-L1 signaling, such as receptor binding and / or induced cellular responses to PD-1 / PD-L1. PD-1 antagonists that reduce the number or amount of human PD-1 or PD-L1 produced by cells or individuals are also provided herein.

[0317] In some aspects, the disclosure provides PD-1 antagonists that bind to human PD-1 and prevent, inhibit or reduce the binding of PD-L1 to PD-1. In some aspects, the PD-1 antagonists bind to mRNA encoding PD-1 or PD-L1 and prevent translation. In some aspects, the PD-1 antagonists bind to mRNA encoding PD-1 or PD-L1 and cause degradation and / or conversion.

[0318] In some aspects, a PD-1 antagonist inhibits PD-1 signaling or function. In some aspects, a PD-1 antagonist blocks PD-1 from binding to PD-L1, PD-L2, or both. In some aspects, a PD-1 antagonist blocks PD-1 from binding to PD-L1. In some aspects, a PD-1 antagonist blocks PD-1 from binding to PD-L2. In some aspects, a PD-1 antagonist blocks PD-1 from binding to PD-L1 and PD-L2. In some aspects, a PD-1 antagonist specifically binds to PD-1. In some aspects, a PD-1 antagonist specifically binds to PD-L1. In some aspects, a PD-1 antagonist specifically binds to PD-L2.

[0319] In some aspects, a PD-1 antagonist inhibits the binding of PD-1 to its cognate ligand. In some aspects, a PD-1 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, a PD-1 antagonist does not inhibit the binding of PD-1 to its cognate ligand.

[0320] In some aspects, a 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, a PD-1 antagonist is an antibody or an antigen-binding fragment thereof that specifically binds to human PD-1. In some aspects, a PD-1 antagonist is an antibody or an antigen-binding fragment thereof that specifically binds to human PD-L1. In some aspects, a PD-1 antagonist is an antibody or an antigen-binding fragment that binds to human PD-L1 and inhibits PD-L1 from binding to PD-1. In some aspects, a PD-1 antagonist is an antibody or an antigen-binding fragment that binds to human PD-1 and inhibits PD-L1 from binding to PD-1.

[0321] Several immune checkpoint antagonists that inhibit or interfere with the interaction between PD-1 and either or both of its ligands PD-L1 and PD-L2 are in clinical development or are currently available to clinicians for the treatment of cancer.

[0322] 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 by the present disclosure include, but are not limited to: KEYTRUDA® (pembrolizumab, MK-3475, h409A11; see US8952136, US8354509, US8900587, and EP2170959, all of which are incorporated herein by reference in their entirety; Merck), OPDIVO® (nivolumab, BMS-936558, MDX-1106, ONO-4538; see US7595048, US8728474, US9073994, US9067999, EP1537878, US8008449, US8779105, and EP2161336, all of which are incorporated herein by reference in their entirety; Bristol Myers Squibb), MEDI0680 (AMP-514), BGB-A317 and BGB-108 (BeiGene), 244C8 and 388D4 (see WO2016106159, which is incorporated herein by reference in its entirety; Enumeral Biomedical), PDR001 (Novartis), and REGN2810 (Regeneron). Thus, in some aspects, the PD-1 antagonist is pembrolizumab. In some aspects, the PD-1 antagonist is nivolumab. [] []

[0323] 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 by the present disclosure include, but are not limited to: BAVENCIO® (avelumab, MSB0010718C, see WO2013 / 79174, which is incorporated herein by reference in its entirety; Merck / Pfizer), IMFINZI® (durvalumab, In some embodiments, the PD-1 antagonist is avelumab. In some embodiments, the PD-1 antagonist is durvalumab. In some embodiments, the PD-1 antagonist is atezolizumab.

[0324] In some aspects, the PD-1 antagonist is an immunoadhesin that specifically binds to human PD-1 or human PD-L1, for example, a fusion protein comprising an 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 immunoadhesin molecules that specifically bind to PD-1 are described in WO2010 / 027827 and WO2011 / 066342, both of which are incorporated herein by reference in their entirety. In some aspects, the 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.

[0325] One of ordinary skill in the art understands that any PD-1 antagonist that binds to PD-1 or PD-L1 and interferes with the PD-1 / PD-L1 signaling pathway is suitable for the compositions, methods, and uses disclosed herein.

[0326] In some aspects, the PD-1 / PD-L1 antagonist is a small molecule, a nucleic acid, a peptide, a peptide mimetic, a protein, a carbohydrate, a carbohydrate derivative, or a glycopolymer. Exemplary small molecule PD-1 inhibitors are described in Zhan et al., (2016) Drug Discov Today 21(6):1027-1036. [and] [TIM-3] [Combination of inhibitors] [] []

[0327] In some aspects, an anti-IL-27 antibody or antigen binding portion thereof provided by the present disclosure is combined with a TIM-3 inhibitor (e.g., administered in combination). The TIM-3 inhibitor can be an antibody, an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide. In some aspects, the TIM-3 inhibitor is selected from MGB453 (Novartis), TSR-022 (Tesaro), or LY3321367 (Eli Lilly). In some aspects, an anti-IL-27 antibody or antigen binding portion thereof is administered in combination with MGB453. In some aspects, an anti-IL-27 antibody or antigen binding portion thereof is administered in combination with TSR-022. [and] [LAG-3] [Combination of inhibitors] [] []

[0328] In some aspects, an anti-IL-27 antibody or antigen-binding portion thereof provided by the present disclosure is combined with a LAG-3 inhibitor (e.g., administered in combination). The LAG-3 inhibitor can be an antibody, an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide. In some aspects, 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] [] []

[0329] In some aspects, the anti-IL-27 antibodies or antigen-binding portions thereof provided by the present disclosure are combined with TIGIT inhibitors, kinase inhibitors (e.g., tyrosine kinase inhibitors (TKI)), CD112R inhibitors, TAM receptor inhibitors, STING agonists and / or 4-1BB agonists, or combinations thereof (e.g., administered in combination). In some aspects, the anti-IL-27 antibodies or antigen-binding portions thereof provided by the present disclosure are combined with tyrosine kinase inhibitors, agents targeting the adenosine axis (e.g., CD39 antagonists, CD73 antagonists, or A2AR, A2BR, or dual A2AR / A2BR antagonists), CCR8 antagonists, CTLA4 antagonists, VEG-F inhibitors, or combinations thereof (e.g., administered in combination) [Detection method] [] []

[0330] In some embodiments, the anti-IL-27 antibodies or antigen-binding fragments thereof described herein can be used in methods for detecting and / or quantifying human IL-27 in a biological sample. Thus, the anti-IL-27 antibodies or antigen-binding fragments thereof as described herein can be used to diagnose, predict and / or determine the progression of a disease (e.g., cancer) in a patient.

[0331] Monitoring a subject (e.g., a human patient) for improvement of a cancer as defined herein means assessing the subject for a change in a disease parameter, such as a decrease in tumor growth. In some embodiments, the assessment is performed at least one (1) hour after administration, such as at least 2, 4, 6, 8, 12, 24, or 48 hours, or at least 1, 2, 4, 10, 13, 20, or more days, or at least 1, 2, 4, 10, 13, 20, or more weeks. The subject may be assessed in one or more of the following periods: before treatment begins; during treatment; or after one or more elements of the treatment have been administered. The assessment may include assessing the need for further treatment, such as assessing whether the dosage, frequency of administration, or duration of treatment should be changed. It may also include assessing the need to add or abandon a selected treatment modality, such as adding or abandoning any of the treatments for the cancer described herein.

[0332] In some embodiments, the present disclosure provides a method for detecting IL-27 in a sample from a subject, the method comprising (a) contacting a sample from a subject with a detection antibody under conditions that allow the detection antibody to form a detection antibody-IL-27 complex if IL-27 is present in the sample, wherein the detection antibody is an antibody or an antigen-binding fragment thereof provided by the present disclosure; and (b) detecting the presence of the complex (if any) produced in step (a).

[0333] In some embodiments, the present disclosure provides a method for detecting IL-27-related cancer in a subject, the method comprising the steps of: (a) contacting a sample from a subject suspected of having an IL-27-related cancer with a detection antibody under conditions that allow the detection antibody to form a detection antibody-IL-27 complex if IL-27 is present in the sample, wherein the detection antibody is an antibody or an antigen-binding fragment thereof provided by the present disclosure; and (b) detecting the presence of the complex (if any) produced in step (a). In some embodiments, the detection antibody is coupled to a detectable label. In some embodiments, the method further comprises contacting the sample with a capture antibody if IL-27 is present in the sample so as to produce a complex comprising IL-27 and the capture antibody, wherein the capture antibody is an antibody or an antigen-binding portion thereof provided by the present disclosure.

[0334] In some embodiments, the capture antibody is immobilized on a solid support. In some embodiments, the sample is contacted with the capture antibody prior to the detection antibody. In some embodiments, the sample is a body fluid sample. In some embodiments, the liquid sample is blood, serum, plasma, cell lysate, or tissue lysate.

[0335] In some embodiments, the cancer is selected from renal cell carcinoma (RCC), hepatocellular carcinoma, lung cancer, gastroesophageal cancer, ovarian cancer, endometrial cancer, melanoma, leukemia, and lymphoma. In some embodiments, the cancer is renal cell carcinoma (RCC). In other embodiments, the cancer is hepatocellular carcinoma (HCC). In some embodiments, the cancer is selected from leukemia and lymphoma. In some embodiments, the cancer is acute myeloid leukemia (AML). [Example] [] []

[0336] Although the present invention has been described with reference to specific aspects of the present invention, it will be appreciated by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the present invention. In addition, many modifications may be made to adapt specific circumstances, materials, compositions of matter, methods, and method steps to the purpose, spirit, and scope of the present disclosure. All such modifications are intended to be within the scope of the present invention. [Example] [1] [:Generated in yeast to specifically bind to humans] [IL-27] [Of] [P28] [Subunit Antibody] [IL-27] [Antibody] [] []

[0337] Anti-IL-27 antibodies representing multiple antigenic determinant bins were selected from eight natural human synthetic yeast libraries using the methods described below. Materials and Methods

[0338] Eight natural human synthetic yeast libraries, each with a diversity of ~10 9, were propagated as previously described (see, e.g., Xu et al., (2013) Protein Eng Des Sel 26(10):663-670; WO2009036379; WO2010105256; and WO2012009568, all of which are incorporated herein by reference in their entirety). For the first two rounds of selection, magnetic bead sorting techniques were performed using the Miltenyi MACS system as previously described (see, e.g., Siegel et al. (2004) J Immunol Methods 286(1-2):141-153, incorporated herein by reference in its entirety).

[0339] Briefly, yeast cells (~10 10 cells / library) were incubated with 3 mL of 100 nM biotinylated antigen (recombinant human IL-27; R&D Systems) in wash buffer (phosphate buffered saline (PBS) / 0.1% bovine serum albumin (BSA)) at 30°C for 30 min. After washing once with 40 mL of ice-cold wash buffer, the cell pellet was resuspended in 20 mL of wash buffer, and streptavidin MicroBeads (500 μL) were added to the yeast and incubated at 4°C for 15 min. Subsequently, the yeast cells were pelleted, resuspended in 20 mL of wash buffer, and loaded onto a Miltenyi LS column. After loading 20 mL, the column was washed 3 times with 3 mL of wash buffer. The column was then removed from the magnetic field, and the yeast cells were eluted with 5 mL of growth medium and then grown overnight. The following selection rounds were performed using flow cytometry. Approximately 2×10 7 yeast cells were pelleted, washed three times with wash buffer, and incubated at 30°C under equilibrium conditions with decreasing concentrations of biotinylated antigen (100 to 1 nM), either with 30 nM of biotinylated antigen of different species to obtain species cross-reactivity or with polyspecific depletion reagent (PSR) to remove nonspecific antibodies from selection. For PSR depletion, the library was incubated with a 1:10 dilution of the biotinylated PSR reagent.

[0340] The yeast cells were then washed twice with wash buffer and stained with LC-FITC (1:100 dilution) and SA-633 (1:500 dilution) or EAPE (1:50 dilution) secondary reagents for 15 min at 4°C. After washing twice with wash buffer, the cell pellet was resuspended in 0.3 mL wash buffer and transferred to a filter-capped sorting tube. Sorting was performed using a FACS ARIA sorter (BD Biosciences) and the sorting gate was set to select antibodies with the desired properties. Selection rounds were repeated until a population with all the desired properties was obtained. After the final sorting round, the yeast cells were plated and individual colonies were harvested for characterization. Light chain diversification

[0341] A light chain diversification protocol was used during the primary discovery phase to further discover and improve antibodies.

[0342] Light chain batch diversification protocol: Heavy chain plasmids from natural selection output were extracted from yeast via disruption and grabbing, propagated in E. coli and subsequently purified, and transformed into a light chain library with a diversity of 5 x 10 6. Selections were performed using one MACS round and four FACS rounds using the same conditions as natural discovery. Antibody Optimization

[0343] Antibody optimization was performed by introducing diversity into the heavy and light chain variable regions as described below.

[0344] CDRH1 and CDRH2 selection: CDRH3 of a single antibody was recombined into a pre-made library with CDRH1 and CDRH2 variants of 1 x 108 diversity and selection was performed using one MACS round and four FACS rounds as described in Natural Discovery. In different FACS rounds, the library was interrogated for PSR binding, species cross-reactivity and affinity pressure by titration or parental Fab pre-complexation, and sorting was performed in order to obtain populations with desired properties. Antibody production and purification

[0345] Yeast clones were grown to saturation and then induced at 30°C with shaking for 48 h. After induction, yeast cells were pelleted and the supernatant was harvested for purification. IgG was purified using a protein A column and eluted with acetic acid, pH 2.0. Fab fragments were generated by papain digestion and purified via KappaSelect (GE Healthcare LifeSciences). ForteBio KD Measurement

[0346] ForteBio affinity measurements were generally performed on Octet RED384 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), incorporated herein by reference in its entirety). Briefly, ForteBio affinity measurements were performed by loading IgG inline onto an AHQ sensor. The sensor was equilibrated offline in assay buffer for 30 min and then monitored inline for 60 s to establish a baseline. The IgG-loaded sensor was exposed to 100 nM antigen for 3 min and then transferred to assay buffer for 3 min for dissociation rate measurements. All kinetics were analyzed using a 1:1 binding model. Recombinant human IL-27 protein (R&D Systems Cat: 2526-IL) was used as antigen. Affinity measurements of anti-IL-27 antibodies are shown in [picture] [1] Chinese. ForteBio Antigenic Determinant Binning / Ligand Blocking

[0347] Epitope binning / ligand blocking was performed using a standard sandwich format cross-blocking assay. A control anti-target IgG was loaded onto the AHQ sensor and unoccupied Fc binding sites on the sensor were blocked with an irrelevant human IgG1 antibody. The sensor was then exposed to 100 nM target antigen followed by a second anti-target antibody or ligand. Additional binding of the second antibody or ligand after antigen association indicates unoccupied epitopes (non-competitors), while no binding indicates epitope blocking (competitor or ligand blocking). MSD-SET Kinetic Assay

[0348] Equilibrium affinity measurements were performed as described previously (Estep et al., 2013). Solution equilibrium titrations (SET) were performed in PBS + 0.1% IgG-free BSA (PBSF), with antigen held constant at 10-100 pM and incubated with 3- to 5-fold serial dilutions of antibody starting at 5-100 nM (experimental conditions were sample dependent). Antibodies (20 nM in PBS) were coated onto standard binding MSD-ECL plates overnight at 4°C or for 30 min at room temperature. The plates were then blocked for 30 min at 700 rpm with shaking, followed by three washes with wash buffer (PBSF + 0.05% Tween 20). SET samples were applied to the plates and incubated for 150 s at 700 rpm with shaking, followed by one wash. Antigen captured on the plates was detected with 250 ng / mL sulfotag-labeled streptavidin in PBSF by incubation on the plates for 3 min. The plates were washed three times with wash buffer and then read on an MSD Sector Imager 2400 instrument using 1x Read Buffer T with surfactant. In Prism, the percentage of free antigen was plotted as a function of titrated antibody and fitted to a quadratic equation to extract the KD. To improve throughput, a liquid handling robot was used throughout the MSD-SET experiment, including SET sample preparation. [Example] [2] [:anti] [IL-27] [Antibodies and Recombinant Human] [IL-27] [Combination] [] []

[0349] The ability of the anti-IL-27 antibodies described in Example 1 to bind to recombinant human IL-27 was assessed by ELISA. Briefly, Nunc MaxiSorp ELISA plates (Affymetrix #44-2404-21) were coated with 100 μL / well recombinant human IL-27 (R&D Systems #2526-IL / CF) (0.5 μg / mL diluted in PBS), sealed, and incubated overnight at 4°C. The plates were washed 3 times with 100 μL / well of wash buffer (PBS + 0.01% Tween). The plates were then blocked with 200 μL / well of blocking buffer (PBS + 0.1% BSA + 0.01% Tween) for 1 hour at room temperature (RT) with shaking. The blocking buffer was poured off and 100 μL of dilution control and anti-IL-27 antibody were added per well as indicated. A 10-point serial dilution of each antibody was generated by diluting the antibody 1:10 starting from the highest concentration of 1 μg / mL. The plate was incubated for 1-2 hours at RT with shaking. The plate was washed 3 times with 100 μL / well of wash buffer. 100 μL / well of anti-human IgG secondary antibody (SouthernBiotech; Cat. # 2014-05) (1:5000 dilution in blocking buffer) was added. The plate was then incubated for 1 hour at RT with shaking. After the 1 hour incubation, the plate was washed 3 times with 100 μL / well of wash buffer. To develop the plate, 100 μL / well of TMB buffer (Life Technologies #00-2023) was added. Blue development in the wells of the standard curve was observed and once the highest concentration of the diluted control antibody reached a dark blue color (5-10 minutes), 50 μL / well of STOP solution (Thermo Fisher #SS04) was added (the color changed to yellow). Within 30 minutes of stopping the reaction, read the developed plates at 450 nm (minus 570 nm for wavelength correction).

[0350] For example, biochemical affinity and specificity studies showed that the anti-IL-27 antibody Anti-IL-27 Ab1 binds to the p28 subunit (but not to the EBI3 subunit) of the heterodimeric cytokine IL-27. Anti-IL-27 Ab1 binds to human, non-human primate, and rodent recombinant IL-27, and the extent of binding varies between species. The binding specificity of Anti-IL 27 Ab1 for IL-27 was confirmed by testing against a panel of approximately 4500 cell surface and soluble molecules, and no off-target binding was observed. The binding specificity of IL-27 for its receptor IL-27RA (WSX-1) was also confirmed; no other cell surface receptors bind human IL-27. The ability of Anti-IL-27 Ab1 to block the interaction between human IL-27 and IL-27RA (WSX-1) was confirmed by surface plasmon resonance.

[0351] The binding of the antibodies disclosed herein is evaluated in several model systems. Since human IL-27 is biologically active on mouse cells, the systemic overexpression of human IL-27 delivered using DNA minicircles in mice is used to analyze the in vivo effects mediated by IL-27 by whole genome microarray analysis, flow cytometry, and serum cytokine analysis. Many markers regulated by IL-27 in vivo are consistent with the findings in human cell-based assays. Anti-IL-27 Ab3 is also evaluated in a disseminated B16 tumor model. In this case, treatment with anti-IL-27 Ab3 shows consistent results with the phenotypes observed in mice lacking various components of IL-27 ligands (IL-27p28, EBI3) or receptors (IL-27RA).

[0352] In summary, these studies demonstrate that anti-IL-27 Ab1 (and its class-one anti-IL-27 Ab3) can phenocopy IL 27 deficiency in mice, bind specifically and with high affinity to IL-27, and can inhibit its immunosuppressive effects alone or in combination with PD-L1 blockade. [Example] [3] [:anti] [IL27] [Antibody inhibition in vitro] [STAT1] [Phosphorylation] [] []

[0353] IL-27 signaling through the IL-27 receptor (IL-27R) leads to phosphorylation of the signal transducer and activator of transcription 1 (STAT1) polypeptide (pSTAT1). The anti-IL-27 antibodies described in Example 1 were tested by flow cytometry 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.

[0354] Test the ability of anti-IL-27 antibodies to inhibit STAT1 phosphorylation in IL-27-mediated human whole blood. Briefly, EDTA anticoagulated human whole blood stored at room temperature was used for this assay. 45 μL of blood was distributed to each well of a deep well, round bottom plate (Phenix #850356) and heated at 37°C on a plate warmer (EchoTherm IC20) or in a 37°C incubator for 30 minutes. In a polypropylene V-bottom plate (Corning #3363), anti-IL-27 antibodies were diluted to 10x the highest concentration in endotoxin-free PBS (Teknova #P0300). Anti-IL-27 antibodies were serially diluted as needed in endotoxin-free PBS. PBS alone was added to the wells to obtain unstimulated and stimulated controls. 5 μL of each dilution was added to the wells of 45 μL of blood and mixed on a plate shaker for 15 seconds at 1000 RPM (Eppendorf Mix Mate). Incubate the plate at 37°C on a plate warmer or in a 37°C incubator for 60 minutes.

[0355] A 10 μg vial of recombinant human IL-27 (R&D Systems # 2526-IL) was reconstituted to 100 μg / mL by adding 100 μL PBS + 0.1% BSA (made from 10% BSA Sigma #A1595). A working stock of recombinant hIL-27 (rhIL-27) was prepared by diluting to 200 ng / mL in endotoxin-free PBS. After 60 minutes incubation, 5 μL of 200 ng / mL rhIL-27 was added to each well of stimulated blood. 5 μL PBS was added to the unstimulated control wells. The plate was shaken on a plate shaker at 1000 RPM for 15 seconds. The plate was incubated at 37°C for 30 minutes.

[0356] After 30 minutes incubation, cells were fixed. Lysis / Fix Reagent (BD #558049) was diluted 1:5 in sterile water (Hyclone #SH3052902) and warmed to 37°C in a water bath. 500 μL of Lysis / Fix Reagent was added to each well of the deep well plate and the plate was mixed on a plate shaker at 1000 RPM for 15 seconds. The plate was incubated at 37°C for 15 min.

[0357] After 15 minutes incubation, the plate was centrifuged at 1500 RPM for 5 minutes at room temperature (Eppendorf centrifuge 5810R) and the supernatant was discarded by flicking. 1 mL of endotoxin-free PBS was added to each well and the plate was shaken on a plate shaker at 1000 RPM for 15 seconds. The plate was centrifuged at 1500 RPM for 5 minutes at room temperature (Eppendorf centrifuge 5810R) and the supernatant was discarded by flicking. The cell pellet remained in the plate.

[0358] The cell pellet was resuspended in 50 μL 1:200 CD14-Pacific Blue (Biolegend #325616) in FACS buffer (PBS, Gibco #14190-144 / 2% FBS, Sigma #F8317 / 1 mM EDTA, Fisher #BP2482) and transferred to a U-bottom 96-well plate (Costar #3799). The plate was sealed with a plate sealer (VWR #89134-432) and incubated at room temperature in the dark for 30 minutes.

[0359] After 30 minutes incubation, 150 μL of FACS buffer was added to each well and the plate was centrifuged at 1500 RPM for 5 minutes at room temperature. Then, with pipetting, the cell pellet was resuspended in 100 μL of Perm III (stored at -20°C) (BD #558050) and the plate was sealed with a plate sealer and lid. The plate was incubated at -20°C overnight or at 4°C for 15 minutes.

[0360] After incubation, 150 μL of PBS was added and the plate was centrifuged at 1500 RPM for 5 minutes at room temperature. The supernatant was discarded from the plate by flicking and the plate was resuspended in PBS as described below. [surface] In 50 μL of the staining mixture prepared as described in [6]:

[0361] The plate was incubated at room temperature in the dark for 1 hour. After the 1 hour 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 discarded from the plate by flicking and the plate was resuspended in 100 μL of FACS buffer for analysis by flow cytometry.

[0362] The ability of the anti-IL-27 antibodies described in Example 1 to inhibit IL-27-mediated STAT1 phosphorylation in pooled human PBMCs was tested by flow cytometry. Briefly, cryovials of human PBMCs (peripheral blood mononuclear cells) obtained 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 conical falcon tube. 2-3 mL of complete RPMI-1640 (Gibco, 61870-036) was slowly added to the thawed cells and the cells were gently vortexed or flicked for suspension. With complete RPMI-1640, the conical tube was filled to 10 mL and the tube was inverted for mixing. The conical tube was centrifuged at 1400 RPM for 8 minutes at room temperature.

[0363] PBMC cells were resuspended in warm, serum-free RPMI-1640 at a density of 4 million cells / mL and plated at a density of 200,000 cells / well (50 μL) in a round-bottom 96-well plate (Costar, 3799). In the first column of a 96-well polypropylene plate, anti-IL-27 antibody was diluted in serum-free RPMI-1640 to a top concentration of 40 μg / mL (final 10 μg / mL). Serial dilutions (1:2, 1:3, etc.) were performed as needed in the remainder of the first 10 columns of the plate. In a round-bottom plate, fifty microliters (μL) of antibody stock solution (4x) were added to the first 10 columns of the PBMC cell plate. In columns 11 and 12, 50 μL of serum-free RPMI-1640 cell culture medium was added. The plate was then incubated at 37°C for 2 hours.

[0364] After the 2 hour incubation, 100 μL of 50 ng / ml recombinant human IL-27 (R&D Systems, 2526-IL) diluted in serum-free RPMI-1640 cell culture medium was added to each well (except for control wells containing serum-free medium alone or antibody alone) to a final concentration of 25 ng / ml. 100 μL of serum-free RPMI-1640 cell culture medium was added to control wells or wells with antibody alone. The plate was incubated at 37°C for 20 minutes.

[0365] After the 20 minute incubation, 50 μL of 4% PFA (Pierce, 28906) in DI water was added directly to each well and the plate was incubated at 37°C for 5 minutes to fix the cells. The plate was centrifuged at 2000 RPM for 5 minutes. The medium was discarded by flicking and the plate was washed with 150 μL DPBS. The wash step was repeated 2 more times. Using a 12-channel pipette, 50 μL of ice-cold 90% methanol (MeOH) (Sigma, 439193) diluted in H 2O was quickly added to each well. When adding MeOH, be particularly careful to mix the wells. The plate was incubated at 20°C for at least 15 minutes. 100 μL DPBS was added to the top of the 90% methanol in each well and the plate was centrifuged at 2000 RPM for 5 minutes. The plate contents were discarded by flicking and the plate was washed 3 times as previously described. After the last wash, the cell pellet remained in the well of the plate.

[0366] Pelleted PBMCs were stained with 1:100 pSTAT1 PE (BD Phosflow, 526069) in FACS buffer (2% FBS, 2 mM EDTA in DPBS) for 45 minutes at room temperature in the dark. Take special care to mix the wells with a 12-channel pipette when adding the stain. After the 45-minute incubation, 100 μL of FACS buffer was added to each well and the plate was centrifuged at 2000 RPM for 5 minutes. The supernatant was discarded by flicking and the plate was washed twice as previously described. The cells were resuspended in 100 μL of FACS buffer and analyzed by flow cytometry.

[0367] like [picture] [2A] shows that anti-IL-27 antibodies inhibit phosphorylation of STAT1 in human pooled PBMCs. Anti-IL-27 antibody anti-IL-27 Ab3 inhibits phosphorylation of STAT1 in pooled human PBMCs with an average IC 50 of 140.5 ng / mL (n=2). Anti-IL-27 antibody anti-IL-27 Ab1 inhibits phosphorylation of STAT1 in pooled human PBMCs with an average IC 50 of 58.3 ng / mL (n=3).

[0368] By basically [picture] The 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 as described in [2A]. [picture] [2B] shows that, as indicated, anti-IL-27 antibodies inhibited phosphorylation of STAT1 in U-937 cells. Anti-IL-27 Ab3 inhibited phosphorylation of STAT1 in U937 cells with an average IC 50 of 81 ng / mL (n=2). Antibody anti-IL-27 Ab1 inhibited phosphorylation of STAT1 in U937 cells with an average IC 50 of 96 ng / mL (n=2).

[0369] By basically [picture] [2A] described flow cytometry to test the ability of anti-IL-27 antibodies to inhibit IL-27-mediated STAT1 phosphorylation in the cutaneous T-cell lymphoma cell line HUT-78, which does not express cell surface Fc receptors. [picture] [2C] shows that anti-IL-27 antibodies inhibited the phosphorylation of STAT1 in HUT-78 cells. Anti-IL-27 Ab3 inhibited the phosphorylation of STAT1 in HUT-78 cells with an average IC50 of 80 ng / mL (n=1). Antibody anti-IL-27 Ab1 inhibited the phosphorylation of STAT1 in HUT-78 cells with an average IC50 of 95 ng / mL (n=1).

[0370] The present disclosure also evaluates IL-27 inhibition of anti-IL-27 Ab1 in a whole blood assay across species. To characterize anti-IL-27 Ab1 activity across species, recombinant IL-27 from humans, cynomolgus monkeys, rats, and mice were tested for stimulation of pSTAT1 signaling in T lymphocytes obtained from whole blood samples of these species (data not shown).

[0371] Briefly, whole blood was warmed to 37°C, then pre-incubated with anti-IL-27 Ab1 for 60 minutes, and 20 ng / mL of human IL-27 was added. The samples were incubated for an additional 30 minutes. Leukocytes were fixed, and erythrocytes were lysed. After washing, fixed cells were permeabilized and stained with anti-CD3 and anti-phospho-STAT1 (Y701). After 1 hour 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.

[0372] Representative data of anti-IL-27 Ab1 signaling inhibition in human T cells are shown in [picture] [3]. Consistent with the observations regarding the affinity of anti-IL-27 Ab1 to different species, the potency of anti-IL-27 Ab1 in inhibiting IL-27 signaling was strongest in humans, followed by cynomolgus monkeys, rats, and mice (see, e.g., [surface] [7]). Abbreviations: IC 50 = half maximal inhibitory concentration, N / A = not applicable [Example] [4] [:anti] [IL-27] [Antibody reduction] [IL-27] [Mediation] [CD161] [Suppression] [] []

[0373] The C-type lectin CD161 is a marker for T cells, whose expression is inhibited by IL-27. The ability of the anti-IL-27 antibodies described in Example 1 to reverse IL-27-mediated inhibition of CD161 in pooled human PBMC cells was tested by flow cytometry. Briefly, cryovials of human PBMCs (peripheral blood mononuclear cells) obtained 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 conical falcon tube. 2-3 mL of complete RPMI-1640 (Gibco, 61870-036) was slowly added to the thawed cells and the cells were gently vortexed or flicked for suspension. With complete RPMI-1640, the conical tube was filled to 10 mL and the tube was inverted for mixing. The conical tube was centrifuged at 1400 RPM for 8 minutes at room temperature.

[0374] During the 5-day assay, avoid using the outer wall to minimize evaporation effects. The outer wells were filled with 200 μL / well of DPBS (Gibco, 14190-144). PBMC cells were resuspended in warm, complete RPMI-1640 at a density of 2 million cells / mL. Purified human anti-CD3 antibody (Biolegend, UCTH1, #300402) was added at a concentration of 0.5 μg / mL (this is 2X final concentration). 100 μL / well of this cell mixture (200,000 cells / well) was plated in a round-bottom 96-well plate (Costar, 3799).

[0375] In the first column of a 96-well polypropylene plate, dilute anti-IL-27 antibody in complete RPMI-1640 to a top concentration of 40 μg / mL (final 10 ug / mL). Perform serial dilutions (1:2, 1:3, etc.) as needed in the remainder of the first 10 columns of the plate. In a round-bottom plate, add 50 μL of antibody stock (4x) to the first 10 columns of the PBMC cell plate. In columns 11 and 12, add 50 μL of complete RPMI-1640.

[0376] After adding the anti-IL-27 antibody, 50 μL of 100 ng / mL recombinant human IL-27 (R&D Systems, #2526-IL) diluted in complete RPMI-1640 was added to each well (except for control wells containing serum-free medium or antibody alone) to a final concentration of 25 ng / mL. Fifty μL of complete RPMI-1640 was added to the control wells. The plates were incubated at 37° C. in a tissue culture incubator with minimal disturbance for 5 days.

[0377] After 5 days of incubation, the plates were removed from the incubator and agitated on a plate shaker at 600 RPM for 30 seconds. The plates were centrifuged at 1800 RPM for 5 minutes. The culture medium was removed and set aside for additional assays and the plates were washed with 150 μL DPBS (Gibco, #14190-144). The wash step was repeated 2 more times. The cell pellet was washed with 50 μL / well of the following [surface] [8] Dyeing with the dye mixture described:

[0378] The plate was agitated on a plate shaker at 600 RPM for 30 seconds and the plate was incubated at room temperature in the dark for 30 minutes.

[0379] After the 30 minute incubation, the plates were centrifuged and the supernatant discarded by flicking. The plates were washed 2 times as previously described. After the final wash, the cell pellets were fixed by adding 50 μL of 4% PFA (Pierce, 28906) in DI water for 10 minutes at room temperature. 100 μL of FASC buffer was added to each well and the plates were centrifuged at 1800 RPM for 5 minutes. The cells were resuspended in 100 μL of FACS buffer and read by flow cytometry.

[0380] like [picture] It was shown in [4] that, as indicated, anti-IL-27 antibodies reduced IL-27-mediated inhibition of CD161. [Example] [5] [:anti] [IL-27] [Antibody Enhancement] [PD-1] [Mediation] [TNFα] [、] [IL-6] [and other cytokines secretion, including anti- [IL-27] [Additional in vitro characterization of antibodies] [] []

[0381] The ability of anti-IL-27 antibodies to enhance PD-1-mediated secretion of TNFα and IL-6 in human PBMC cells from cancer patients was tested. Human PBMC cells from cancer patients were cultured essentially as described in Example 4, with the addition of wells receiving 1 μg / mL of anti-PD-1 antibodies as indicated. Supernat...

Claims

1. A method for detecting IL-27 in a sample, the method comprising (a) contacting the sample with a detection antibody under conditions that allow the detection antibody to form a detection antibody-IL-27 complex if IL-27 is present in the sample; and (b) detecting the presence of the complex produced in step (a), wherein the detection antibody comprises a heavy chain CDR and a light chain CDR, wherein: (i) the heavy chain CDR1, CDR2 and CDR3 sequences are shown in SEQ ID NOs: 9, 10 and 11, respectively, and the light chain CDR1, CDR2 and CDR3 sequences are shown in SEQ ID NOs: 17, 18 and 19, respectively; (ii) the heavy chain CDR1, CDR2 and CDR3 sequences are shown in SEQ ID NOs: 31, 32 and 33, respectively, and the light chain CDR1, CDR2 and CDR3 sequences are shown in SEQ ID NOs: 39, 40 and 41, respectively; (iii) the heavy chain CDR1, CDR2 and CDR3 sequences are shown in SEQ ID NOs: 53, 54 and 55, respectively, and the light chain CDR1, CDR2 and CDR3 sequences are shown in SEQ ID NOs: 61, 62 and 63, respectively; (iv) the heavy chain CDR1, CDR2 and CDR3 sequences are shown in SEQ ID NOs: (v) the heavy chain CDR1, CDR2 and CDR3 sequences are shown in SEQ ID NOs: 97, 98 and 99, respectively, and the light chain CDR1, CDR2 and CDR3 sequences are shown in SEQ ID NOs: 105, 106 and 107, respectively; or (vi) the heavy chain CDR1, CDR2 and CDR3 sequences are shown in SEQ ID NOs: 119, 120 and 121, respectively, and the light chain CDR1, CDR2 and CDR3 sequences are shown in SEQ ID NOs: 127, 128 and 129, respectively.

2. A method for detecting IL-27-related cancer, the method comprising the steps of: (a) contacting a sample obtained from an individual suspected of having an IL-27-related cancer with a detection antibody under conditions that allow the detection antibody to form a detection antibody-IL-27 complex if IL-27 is present in the sample; and (b) detecting the presence of the complex if produced in step (a), wherein the detection antibody comprises a heavy chain CDR and a light chain CDR, wherein: (i) the heavy chain CDR1, CDR2 and CDR3 sequences are shown in SEQ ID NOs: 9, 10 and 11, respectively, and the light chain CDR1, CDR2 and CDR3 sequences are shown in SEQ ID NOs: 17, 18 and 19, respectively; (ii) the heavy chain CDR1, CDR2 and CDR3 sequences are shown in SEQ ID NOs: 31, 32 and 33, respectively, and the light chain CDR1, CDR2 and CDR3 sequences are shown in SEQ ID NOs: 39, 40 and 41, respectively; (iii) the heavy chain CDR1, CDR2 and CDR3 sequences are shown in SEQ ID NOs: 53, 54 and 55, respectively, and the light chain CDR1, CDR2 and CDR3 sequences are shown in SEQ ID NOs: 61, 62 and 63, respectively; (iv) the heavy chain CDR1, CDR2 and CDR3 sequences are shown in SEQ ID NOs: (v) the heavy chain CDR1, CDR2 and CDR3 sequences are shown in SEQ ID NOs: 97, 98 and 99, respectively, and the light chain CDR1, CDR2 and CDR3 sequences are shown in SEQ ID NOs: 105, 106 and 107, respectively; or (vi) the heavy chain CDR1, CDR2 and CDR3 sequences are shown in SEQ ID NOs: 119, 120 and 121, respectively, and the light chain CDR1, CDR2 and CDR3 sequences are shown in SEQ ID NOs: 127, 128 and 129, respectively.

3. The method of claim 1 or 2, wherein the detection antibody is coupled to a detectable label.

4. The method of any one of claims 1 to 3, wherein the method further comprises: if IL-27 is present in the sample, contacting the sample with a capture antibody to produce a complex comprising IL-27 and the capture antibody, wherein the capture antibody comprises a heavy chain CDR and a light chain CDR, wherein: (i) the heavy chain CDR1, CDR2 and CDR3 sequences are shown in SEQ ID NOs: 9, 10 and 11, respectively, and the light chain CDR1, CDR2 and CDR3 sequences are shown in SEQ ID NOs: 17, 18 and 19, respectively; (ii) the heavy chain CDR1, CDR2 and CDR3 sequences are shown in SEQ ID NOs: 31, 32 and 33, respectively, and the light chain CDR1, CDR2 and CDR3 sequences are shown in SEQ ID NOs: 39, 40 and 41, respectively; (iii) the heavy chain CDR1, CDR2 and CDR3 sequences are shown in SEQ ID NOs: 53, 54 and 55, respectively, and the light chain CDR1, CDR2 and CDR3 sequences are shown in SEQ ID NOs: 61, 62 and 63, respectively; (iv) the heavy chain CDR1, CDR2 and CDR3 sequences are shown in SEQ ID NOs: (v) the heavy chain CDR1, CDR2 and CDR3 sequences are shown in SEQ ID NOs: 97, 98 and 99, respectively, and the light chain CDR1, CDR2 and CDR3 sequences are shown in SEQ ID NOs: 105, 106 and 107, respectively; or (vi) the heavy chain CDR1, CDR2 and CDR3 sequences are shown in SEQ ID NOs: 119, 120 and 121, respectively, and the light chain CDR1, CDR2 and CDR3 sequences are shown in SEQ ID NOs: 127, 128 and 129, respectively.

5. The method of claim 4, wherein the capture antibody is immobilized on a solid support.

6. The method of claim 4 or 5, wherein the sample is contacted with the capture antibody before the detection antibody.

7. The method of any one of claims 1 to 6, wherein the sample is a body fluid sample.

8. The method of claim 7, wherein the body fluid sample is blood, serum, plasma, cell lysate or tissue lysate.

9. The method of any one of claims 2 to 8, wherein the cancer is selected from renal cell carcinoma (RCC), hepatocellular carcinoma (HCC), lung cancer, gastroesophageal cancer, ovarian cancer, endometrial cancer, melanoma, leukemia and lymphoma.

10. The method of claim 9, wherein the cancer is renal cell carcinoma (RCC).

11. The method of claim 9, wherein the cancer is hepatocellular carcinoma (HCC).

12. The method of claim 9, wherein the cancer is selected from leukemia and lymphoma.

13. The method of claim 9, wherein the cancer is ovarian cancer.

14. The method of claim 9, wherein the cancer is gastric cancer.

15. The method of claim 9, wherein the cancer is gastroesophageal cancer.

16. The method of claim 9, wherein the cancer is non-small cell lung cancer.

17. The method of any one of claims 1 to 16, wherein the detection antibody comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region and the light chain variable region respectively comprise an amino acid sequence selected from the group consisting of: (i) SEQ ID NOs: 15 and 23; (ii) SEQ ID NOs: 37 and 45; (iii) SEQ ID NOs: 59 and 67; (iv) SEQ ID NOs: 81 and 89; (v) SEQ ID NOs: 103 and 111; and (vi) SEQ ID NOs: 125 and 133.

18. The method of any one of claims 1 to 17, wherein the detection antibody comprises a heavy chain and a light chain, and the heavy chain and the light chain respectively comprise an amino acid sequence selected from the group consisting of: (i) SEQ ID NOs: 25 and 27; (ii) SEQ ID NOs: 29 and 27; (iii) SEQ ID NOs: 47 and 49; (iv) SEQ ID NOs: 51 and 49; (v) SEQ ID NOs: 69 and 71; (vi) SEQ ID NOs: 73 and 71; (vii) SEQ ID NOs: 91 and 93; (viii) SEQ ID NOs: 95 and 93; (ix) SEQ ID NOs: 113 and 115; (x) SEQ ID NOs: 117 and 115; (xi) SEQ ID NOs: 135 and 137; and (xii) SEQ ID NOs: 139 and 137.

19. The method of any one of claims 1 to 18, wherein the capture antibody comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region and the light chain variable region respectively comprise an amino acid sequence selected from the group consisting of: (i) SEQ ID NOs: 15 and 23; (ii) SEQ ID NOs: 37 and 45; (iii) SEQ ID NOs: 59 and 67; (iv) SEQ ID NOs: 81 and 89; (v) SEQ ID NOs: 103 and 111; and (vi) SEQ ID NOs: 125 and 133.

20. The method of any one of claims 1 to 19, wherein the capture antibody comprises a heavy chain and a light chain, and the heavy chain and the light chain respectively comprise an amino acid sequence selected from the group consisting of: (i) SEQ ID NOs: 25 and 27; (ii) SEQ ID NOs: 29 and 27; (iii) SEQ ID NOs: 47 and 49; (iv) SEQ ID NOs: 51 and 49; (v) SEQ ID NOs: 69 and 71; (vi) SEQ ID NOs: 73 and 71; (vii) SEQ ID NOs: 91 and 93; (viii) SEQ ID NOs: 95 and 93; (ix) SEQ ID NOs: 113 and 115; (x) SEQ ID NOs: 117 and 115; (xi) SEQ ID NOs: 135 and 137; and (xii) SEQ ID NOs: 139 and 137.

21. An isolated antibody or antigen-binding portion thereof that antagonizes human IL-27, wherein the antibody or antigen-binding portion thereof comprises: a) heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 9, 10 and 11, respectively, and light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 17, 18 and 19, respectively; b) heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 12, 13 and 14, respectively, and light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 20, 21 and 22, respectively; c) heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 31, 32 and 33, respectively, and light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 39, 40 and 41, respectively; d) heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 41, 42 and 43, respectively. 34, 35 and 36, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 42, 43 and 44, respectively; e) 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; f) 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; g) 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: h) 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; i) 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 j) 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.

22. The antibody or antigen-binding portion thereof of claim 21, wherein the antibody or antigen-binding portion thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region respectively comprise an amino acid sequence selected from the group consisting of: (i) SEQ ID NOs: 15 and 23; (ii) SEQ ID NOs: 37 and 45; (iii) SEQ ID NOs: 59 and 67; (iv) SEQ ID NOs: 81 and 89; and (v) SEQ ID NOs: 103 and 111.

23. The antibody or antigen-binding portion thereof of claim 21, wherein the antibody or antigen-binding portion thereof comprises a heavy chain and a light chain, wherein the heavy chain and the light chain respectively comprise an amino acid sequence selected from the group consisting of: (i) SEQ ID NOs: 25 and 27; (ii) SEQ ID NOs: 29 and 27; (iii) SEQ ID NOs: 47 and 49; (iv) SEQ ID NOs: 51 and 49; (v) SEQ ID NOs: 69 and 71; (vi) SEQ ID NOs: 73 and 71; (vii) SEQ ID NOs: 91 and 93; (viii) SEQ ID NOs: 95 and 93; (ix) SEQ ID NOs: 113 and 115; and (x) SEQ ID NOs: 117 and 115.

24. The antibody or antigen-binding portion thereof of claim 21, wherein the antibody or antigen-binding portion thereof specifically binds an antigenic determinant comprising one or more of the following amino acids of SEQ ID NO: 2 (IL-27p28): 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.

25. The antibody or antigen-binding portion thereof of any one of claims 21 to 23, wherein the antibody or antigen-binding portion thereof exhibits at least one or more of the following properties: (i) binds to human IL-27 with an equilibrium dissociation constant (KD) of 15 nM or less; (ii) blocks IL-27 binding to IL-27 receptor; (iii) inhibits or reduces STAT1 and / or STAT3 phosphorylation in cells; (iv) inhibits or reduces IL-27-mediated inhibition of CD161 expression in cells; (v) inhibits or reduces IL-27-mediated PD-L1 and / or TIM-3 expression in cells; and (vi) induces or enhances PD-1-mediated secretion of one or more cytokines from cells.

26. The antibody or antigen-binding portion thereof of any one of claims 21 to 23, wherein the antibody is selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD and IgE antibodies.

27. A pharmaceutical composition comprising the isolated antibody or antigen-binding portion thereof according to any one of claims 21 to 26 and a pharmaceutically acceptable carrier.

28. A nucleic acid molecule or a group of nucleic acid molecules encoding the isolated antibody or antigen-binding portion thereof of any one of claims 21 to 26.

29. An expression vector comprising the nucleic acid molecule of claim 28.

30. A cell comprising the expression vector of claim 29.

31. A method for producing an antibody or an antigen-binding portion thereof that specifically binds to human IL-27, the method comprising maintaining the cell of claim 30 under conditions that allow expression of the antibody or the antigen-binding portion thereof.

32. The method of claim 31, further comprising obtaining the antibody or antigen-binding portion thereof.

33. A method for inhibiting or reducing STAT1 and / or STAT3 phosphorylation in a cell in vitro, the method comprising contacting the cell with the isolated antibody or antigen-binding portion thereof of any one of claims 21 to 26, wherein the antibody or antigen-binding portion thereof inhibits or reduces STAT1 and / or STAT3 phosphorylation in the cell.

34. A method for inhibiting or reducing the inhibition of CD161 expression in a cell in vitro, the method comprising contacting the cell with the isolated antibody or antigen-binding portion thereof of any one of claims 21 to 26, wherein the antibody or antigen-binding portion thereof inhibits or reduces the inhibition of CD161 expression in the cell.

35. A method for inhibiting or reducing the expression of PD-L1 and / or TIM-3 in a cell in vitro, the method comprising contacting the cell with the isolated antibody or antigen-binding portion thereof of any one of claims 21 to 26, wherein the antibody or antigen-binding portion thereof inhibits or reduces the expression of PD-L1 and / or TIM-3 in the cell.

36. A method for inducing or enhancing the secretion of one or more cytokines from a cell in vitro, the method comprising contacting the cell with the isolated antibody or antigen-binding portion thereof of any one of claims 21 to 26, wherein the antibody or antigen-binding portion thereof induces or enhances PD-1-mediated secretion of one or more cytokines from the cell.

37. Use of the antibody or antigen-binding portion thereof of any one of claims 21 to 23 or the pharmaceutical composition of claim 27 in the manufacture of a medicament for stimulating an immune response in a subject.

38. Use of the antibody or antigen-binding portion thereof of any one of claims 21 to 23 or the pharmaceutical composition of claim 27 in the manufacture of a medicament for treating cancer in a subject.

39. Use of the antibody or antigen-binding portion thereof of any one of claims 21 to 23 or the pharmaceutical composition of claim 27 in the manufacture of a medicament for enhancing one or more activities of an anti-PD-1 antibody in a subject, comprising enhancing PD-1-mediated cytokine secretion, enhancing anti-PD-1-mediated TNFα secretion, and enhancing anti-PD-1-mediated IL-6 secretion from cells exposed to the anti-PD-1 antibody, wherein the antibody or antigen-binding portion thereof is administered simultaneously or sequentially with the anti-PD-1 antibody to enhance one or more activities of the anti-PD-1 antibody.

40. The use of claim 38, wherein the antibody or antigen-binding portion thereof is administered in combination with one or more additional therapeutic agents or procedures, wherein the additional therapeutic agent or procedure is selected from the group consisting of chemotherapy, targeted anti-cancer therapy, oncolytic drugs, cytotoxic agents, immune-based therapies, cytokines, surgical procedures, radiation procedures, activators of co-stimulatory molecules, inhibitors of inhibitory molecules, vaccines, cellular immunotherapy, and combinations thereof.

41. The use of claim 40, wherein the one or more additional therapeutic agents are PD-1 antagonists, PD-L1 inhibitors, TIM-3 inhibitors, LAG-3 inhibitors, TIGIT inhibitors, CD112R inhibitors, TAM inhibitors, STING agonists, 4-1BB agonists, or combinations thereof.

42. The use of claim 41, wherein the one or more additional therapeutic agents are PD-1 antagonists.

43. The use of claim 42, wherein the PD-1 antagonist is selected from the group consisting of: PDR001, nivolumab, pembrolizumab, pidilizumab, MEDI0680, REGN2810, TSR-042, PF-06801591 and AMP-224.

44. The use of claim 41, wherein the PD-L1 inhibitor is selected from the group consisting of: FAZ053, Atezolizumab, Avelumab, Durvalumab and BMS-936559.

45. The use of claim 40, wherein the one or more additional therapeutic agents are: (i) selected from the group consisting of sunitinib (SUTENT®), cabozantinib (CABOMETYX®), axitinib (INLYTA®), lenvatinib (LENVIMA®), everolimus (AFINITOR®), bevacizumab (AVASTIN®), epacadostat, NKTR-214 (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, OPDIVO®, KEYTRUDA®, TECENTRIQ®, BAVENCIO®, IMFIMZI®, LIBTAYO®, tislelizumab, and spartalizumab;(ii) a TIM-3 inhibitor selected from MGB453 or TSR-022; (iii) a LAG-3 inhibitor selected from the group consisting of LAG525, BMS-986016 and TSR-033; (iv) a TIGIT inhibitor; (v) a CD112R inhibitor; (vi) a TAM (Axl, Mer, Tyro) inhibitor; (vii) a 4-1BB agonist; or (viii) a tyrosine kinase inhibitor (TKI). ; 46. ​​A kit comprising the isolated antibody or antigen-binding portion thereof of any one of claims 21 to 26 or the pharmaceutical composition of claim 27, and instructions for use for stimulating an immune response in a subject or treating cancer in a subject.