Anti-IL-27 antibodies and uses thereof

Abstract

To provide an anti IL-27 antibody and use thereof.SOLUTION: The present disclosure relates to an anti IL-27 antibody and an antigen-binding portion thereof. The present disclosure also relates to a method for treating or improving one or more symptoms of a disease such as cancer by administering the antibody and an antigen-binding portion thereof. The present disclosure also relates to a method for detecting IL-27 in a subject or a sample, for example. The present specification discloses an antibody that specifically binds to human IL-27 (interleukin 27) and antagonizes it at high affinity and specificity, or an antigen-binding portion thereof.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62 / 779,341, filed December 13, 2018, the entire contents of which are incorporated herein by reference.

[0002] The present disclosure relates generally to compositions and methods for modulating IL-27 signaling. More specifically, the present disclosure relates to immunogenic compositions (e.g., antibodies, antibody fragments, etc.) that bind to IL-27 and modulate IL-27 signaling. [Background technology]

[0003] In recent years, a growing body of evidence has suggested that the immune system acts as a significant obstacle to tumor formation and progression. The principle that naturally occurring T cells with anti-tumor capacity or activity exist in patients with cancer has rationalized the development of immunotherapeutic approaches in oncology. Immune cells, such as T cells, macrophages, and natural killer cells, can exhibit anti-tumor activity and effectively control the development and growth of malignant tumors. Tumor-specific or tumor-associated antigens can induce immune cells to recognize and eliminate malignant tumors (Chen et al., 2013). & Mellman, (2013) Immunity 39(1):1-10). Despite the presence of tumor-specific immune responses, malignant tumors often escape or evade immune attack through various immunoregulatory mechanisms, resulting in failure to control tumor development and progression (Motz & Coukos, (2013) Immunity 39(1):61-730). Indeed, an emerging hallmark of cancer is the exploitation of these immunoregulatory mechanisms and the neutralization of antitumor immune responses, resulting in tumor evasion and escape from immune killing (Hanahan and Weinberg (2011) Cell 144(5):646-674).

[0004] 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 signal transduction through a heterodimeric receptor composed of IL-27Rα (WSX1) and gp130 chains, which mediate signal transduction through STAT1 and STAT3. Initial reports characterized IL-27 as an immune-enhancing cytokine that supports CD4+ T cell proliferation, T helper (Th)1 cell differentiation, and IFN-γ production, often acting in concert with IL-12. Subsequent studies have demonstrated that IL-27 exhibits complex immunomodulatory functions, resulting in either pro- or anti-inflammatory effects depending on the biological context and experimental model used. IL-27 can drive the expression of different immunoregulatory molecules in human cancer cells, which may assist in localized disruption of immune responses in vivo (Fabbi et al., (2017) Mediators Inflamm 3958069, published online February 1, 2017, doi:10.1155 / 2017 / 3958069, and references contained therein). Despite the significant advances that have been made in the treatment and management of cancer, there remains a continuing need for new and effective therapies for the treatment and management of cancer. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Chen & Mellman,(2013) Immunity 39(1):1-10 [Non-patent document 2] Motz & Coukos,(2013) Immunity 39(1):61-730 [Non-patent document 3] Hanahan and Weinberg (2011) Cell 144(5):646-674 Summary of the Invention [Means for solving the problem]

[0006] Disclosed herein are antibodies, or antigen-binding portions thereof, that specifically bind to and antagonize human IL-27 (interleukin-27) with high affinity and specificity. Nucleic acid molecules encoding the antibody molecules, expression vectors, host cells, and methods for making the antibody molecules are also provided. Pharmaceutical compositions comprising the antibody molecules are also provided. The anti-IL-27 antibodies, or antigen-binding portions thereof, disclosed herein can be used (alone or in combination with other therapeutic agents or procedures) to treat, prevent, and / or diagnose disorders, including immune deficiencies and cancer. Accordingly, disclosed herein are compositions and methods for treating and / or diagnosing various disorders, including cancer and immune deficiencies, using anti-IL-27 antibody molecules.

[0007] In one aspect, the disclosure provides an isolated monoclonal antibody or antigen-binding portion thereof, wherein the antibody or antigen-binding portion thereof comprises heavy and light chain CDRs selected from the group consisting of: (i) heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 706, 707, and 708, respectively, and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 714, 715, and 716, respectively; (ii) heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 728, 729, and 730, respectively, and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 736, 737, and 738, respectively; (iii) heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 750, 751, and 752, respectively, and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 758, 759, and 760, respectively; and (iv) heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 772, 773, and 774, respectively, and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 780, 781, and 782, respectively.

[0008] In one aspect, the disclosure provides an isolated monoclonal antibody or antigen-binding portion thereof, wherein the antibody or antigen-binding portion thereof comprises heavy and light chain CDRs selected from the group consisting of: (i) heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 709, 710, and 711, respectively, and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 717, 718, and 719, respectively; (ii) heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 731, 732, and 733, respectively, and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 739, 740, and 741, respectively; (iii) heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 753, 754, and 755, respectively, and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 761, 762, and 763, respectively; and (iv) heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 775, 776, and 777, respectively, and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 783, 784, and 785, respectively.

[0009] In one aspect, the disclosure provides an isolated monoclonal antibody or antigen-binding portion thereof, wherein the antibody or antigen-binding portion thereof comprises heavy chain CDRs and light chain CDRs, wherein the heavy chain CDR1, CDR2, and CDR3 sequences are set forth in SEQ ID NOs: 706, 707, and 708, respectively, and the light chain CDR1, CDR2, and CDR3 sequences are set forth in SEQ ID NOs: 714, 715, and 716, respectively.

[0010] In one aspect, the disclosure provides an isolated monoclonal antibody or antigen-binding portion thereof, wherein the antibody or antigen-binding portion thereof comprises heavy chain CDRs and light chain CDRs, wherein the heavy chain CDR1, CDR2, and CDR3 sequences are set forth in SEQ ID NOs: 728, 729, and 730, respectively, and the light chain CDR1, CDR2, and CDR3 sequences are set forth in SEQ ID NOs: 736, 737, and 738, respectively.

[0011] In one aspect, the disclosure provides an isolated monoclonal antibody or antigen-binding portion thereof, wherein the antibody or antigen-binding portion thereof comprises heavy chain CDRs and light chain CDRs, wherein the heavy chain CDR1, CDR2, and CDR3 sequences are set forth in SEQ ID NOs: 750, 751, and 752, respectively, and the light chain CDR1, CDR2, and CDR3 sequences are set forth in SEQ ID NOs: 758, 759, and 760, respectively.

[0012] In one aspect, the disclosure provides an isolated monoclonal antibody or antigen-binding portion thereof, wherein the antibody or antigen-binding portion thereof comprises heavy chain CDRs and light chain CDRs, wherein the heavy chain CDR1, CDR2, and CDR3 sequences are set forth in SEQ ID NOs: 772, 773, and 774, respectively, and the light chain CDR1, CDR2, and CDR3 sequences are set forth in SEQ ID NOs: 780, 781, and 782, respectively.

[0013] In one aspect, the disclosure provides an isolated monoclonal antibody or antigen-binding portion thereof, 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 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 712, 734, 756, and 778, and the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 720, 742, 764, and 786.

[0014] In one aspect, the disclosure provides an isolated monoclonal antibody or antigen-binding portion thereof, wherein the antibody or antigen-binding portion thereof comprises a heavy chain variable region and a light chain variable region comprising an amino acid sequence selected from the group consisting of: (i) SEQ ID NOs: 712 and 720, respectively; (ii) SEQ ID NOs: 734 and 742, respectively; (iii) SEQ ID NOs: 756 and 764, respectively; and (iv) SEQ ID NOs: 71775 and 786, respectively.

[0015] In one aspect, the disclosure provides an isolated monoclonal antibody or antigen-binding portion thereof, 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 comprises an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 712, 734, 756, and 778, and the light chain variable region comprises an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 720, 742, 764, and 786.

[0016] In one aspect, the disclosure provides an isolated monoclonal antibody or antigen-binding portion thereof, wherein the antibody or antigen-binding portion thereof comprises a heavy chain variable region and a light chain variable region comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of: (i) SEQ ID NOs: 712 and 720, respectively; (ii) SEQ ID NOs: 734 and 742, respectively; (iii) SEQ ID NOs: 756 and 764, respectively; and (iv) SEQ ID NOs: 778 and 786, respectively.

[0017] In one aspect, the disclosure provides an isolated monoclonal antibody or antigen-binding portion thereof, wherein the antibody or antigen-binding portion thereof comprises a heavy chain variable region and a light chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 712 and 720, respectively.

[0018] In one aspect, the disclosure provides an isolated monoclonal antibody or antigen-binding portion thereof, wherein the antibody or antigen-binding portion thereof comprises a heavy chain variable region and a light chain variable region comprising amino acid sequences at least 90% identical to the amino acid sequences set forth in SEQ ID NOs: 712 and 720, respectively.

[0019] In one aspect, the disclosure provides an isolated monoclonal antibody or antigen-binding portion thereof, wherein the antibody or antigen-binding portion thereof comprises a heavy chain variable region and a light chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 734 and 742, respectively.

[0020] In one aspect, the disclosure provides an isolated monoclonal antibody or antigen-binding portion thereof, wherein the antibody or antigen-binding portion thereof comprises a heavy chain variable region and a light chain variable region comprising amino acid sequences at least 90% identical to the amino acid sequences set forth in SEQ ID NOs: 734 and 742, respectively.

[0021] In one aspect, the disclosure provides an isolated monoclonal antibody or antigen-binding portion thereof, wherein the antibody or antigen-binding portion thereof comprises a heavy chain variable region and a light chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 756 and 764, respectively.

[0022] In one aspect, the disclosure provides an isolated monoclonal antibody or antigen-binding portion thereof, wherein the antibody or antigen-binding portion thereof comprises a heavy chain variable region and a light chain variable region comprising amino acid sequences at least 90% identical to the amino acid sequences set forth in SEQ ID NOs: 756 and 764, respectively.

[0023] In one aspect, the disclosure provides an isolated monoclonal antibody or antigen-binding portion thereof, wherein the antibody or antigen-binding portion thereof comprises a heavy chain variable region and a light chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 778 and 786, respectively.

[0024] In one aspect, the disclosure provides an isolated monoclonal antibody or antigen-binding portion thereof, wherein the antibody or antigen-binding portion thereof comprises a heavy chain variable region and a light chain variable region comprising amino acid sequences at least 90% identical to the amino acid sequences set forth in SEQ ID NOs: 778 and 786, respectively.

[0025] In one aspect, the disclosure provides an isolated monoclonal antibody or antigen-binding portion thereof, wherein the antibody or antigen-binding portion thereof comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 722, 744, 766, and 788, and the light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 724, 746, 768, and 790.

[0026] In one aspect, the disclosure provides an isolated monoclonal antibody or antigen-binding portion thereof, wherein the antibody or antigen-binding portion thereof comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 722, 744, 766, and 788, and the light chain comprises an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 724, 746, 768, and 790.

[0027] In one aspect, the disclosure provides an isolated monoclonal antibody or antigen-binding portion thereof, wherein the antibody or antigen-binding portion thereof comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 726, 748, 770, and 792, and the light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 724, 746, 768, and 790.

[0028] In one aspect, the disclosure provides an isolated monoclonal antibody or antigen-binding portion thereof, wherein the antibody or antigen-binding portion thereof comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 726, 748, 770, and 792, and the light chain comprises an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 724, 746, 768, and 790.

[0029] In one aspect, the disclosure provides an isolated monoclonal antibody or antigen-binding portion thereof, wherein the antibody or antigen-binding portion thereof comprises a heavy chain and a light chain comprising an amino acid sequence selected from the group consisting of: (i) SEQ ID NOs: 722 and 724, respectively; (ii) SEQ ID NOs: 744 and 746, respectively; (iii) SEQ ID NOs: 766 and 768, respectively; and (iv) SEQ ID NOs: 788 and 790, respectively.

[0030] In one aspect, the disclosure provides an isolated monoclonal antibody or antigen-binding portion thereof, wherein the antibody or antigen-binding portion thereof comprises a heavy chain and a light chain comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of: (i) SEQ ID NOs: 722 and 724, respectively; (ii) SEQ ID NOs: 744 and 746, respectively; (iii) SEQ ID NOs: 766 and 768, respectively; and (iv) SEQ ID NOs: 788 and 790, respectively.

[0031] In one aspect, the disclosure provides an isolated monoclonal antibody or antigen-binding portion thereof, wherein the antibody or antigen-binding portion thereof comprises a light chain and a light chain comprising an amino acid sequence selected from the group consisting of: (i) SEQ ID NOs: 726 and 724, respectively; (ii) SEQ ID NOs: 748 and 746, respectively; (iii) SEQ ID NOs: 770 and 768, respectively; and (iv) SEQ ID NOs: 792 and 790, respectively.

[0032] In one aspect, the disclosure provides an isolated monoclonal antibody or antigen-binding portion thereof, wherein the antibody or antigen-binding portion thereof comprises a light chain and a light chain comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of: (i) SEQ ID NOs: 726 and 724, respectively; (ii) SEQ ID NOs: 748 and 746, respectively; (iii) SEQ ID NOs: 770 and 768, respectively; and (iv) SEQ ID NOs: 792 and 790, respectively.

[0033] In one aspect, the disclosure provides an isolated monoclonal antibody or antigen-binding portion thereof, wherein the antibody or antigen-binding portion thereof comprises a heavy chain and a light chain comprising the amino acid sequences set forth in SEQ ID NOs: 722 and 724, respectively.

[0034] In one aspect, the disclosure provides an isolated monoclonal antibody or antigen-binding portion thereof, wherein the antibody or antigen-binding portion thereof comprises a heavy chain and a light chain comprising amino acid sequences at least 90% identical to the amino acid sequences set forth in SEQ ID NOs: 722 and 724, respectively.

[0035] In one aspect, the disclosure provides an isolated monoclonal antibody or antigen-binding portion thereof, wherein the antibody or antigen-binding portion thereof comprises a heavy chain and a light chain comprising the amino acid sequences set forth in SEQ ID NOs: 744 and 746, respectively.

[0036] In one aspect, the disclosure provides an isolated monoclonal antibody or antigen-binding portion thereof, wherein the antibody or antigen-binding portion thereof comprises a heavy chain and a light chain comprising amino acid sequences at least 90% identical to the amino acid sequences set forth in SEQ ID NOs: 744 and 746, respectively.

[0037] In one aspect, the disclosure provides an isolated monoclonal antibody or antigen-binding portion thereof, wherein the antibody or antigen-binding portion thereof comprises a heavy chain and a light chain comprising the amino acid sequences set forth in SEQ ID NOs: 766 and 768, respectively.

[0038] In one aspect, the disclosure provides an isolated monoclonal antibody or antigen-binding portion thereof, wherein the antibody or antigen-binding portion thereof comprises a heavy chain and a light chain comprising amino acid sequences at least 90% identical to the amino acid sequences set forth in SEQ ID NOs: 766 and 768, respectively.

[0039] In one aspect, the disclosure provides an isolated monoclonal antibody or antigen-binding portion thereof, wherein the antibody or antigen-binding portion thereof comprises a heavy chain and a light chain comprising the amino acid sequences set forth in SEQ ID NOs: 788 and 790, respectively.

[0040] In one aspect, the disclosure provides an isolated monoclonal antibody or antigen-binding portion thereof, wherein the antibody or antigen-binding portion thereof comprises a heavy chain and a light chain comprising amino acid sequences at least 90% identical to the amino acid sequences set forth in SEQ ID NOs: 788 and 790, respectively.

[0041] In one aspect, the disclosure provides an isolated monoclonal antibody or antigen-binding portion thereof, wherein the antibody or antigen-binding portion thereof comprises a heavy chain and a light chain comprising the amino acid sequences set forth in SEQ ID NOs: 726 and 724, respectively.

[0042] In one aspect, the disclosure provides an isolated monoclonal antibody or antigen-binding portion thereof, wherein the antibody or antigen-binding portion thereof comprises a heavy chain and a light chain comprising amino acid sequences at least 90% identical to the amino acid sequences set forth in SEQ ID NOs: 726 and 724, respectively.

[0043] In one aspect, the disclosure provides an isolated monoclonal antibody or antigen-binding portion thereof, wherein the antibody or antigen-binding portion thereof comprises a heavy chain and a light chain comprising the amino acid sequences set forth in SEQ ID NOs: 748 and 746, respectively.

[0044] In one aspect, the disclosure provides an isolated monoclonal antibody or antigen-binding portion thereof, wherein the antibody or antigen-binding portion thereof comprises a heavy chain and a light chain comprising amino acid sequences at least 90% identical to the amino acid sequences set forth in SEQ ID NOs: 748 and 746, respectively.

[0045] In one aspect, the disclosure provides an isolated monoclonal antibody or antigen-binding portion thereof, wherein the antibody or antigen-binding portion thereof comprises a heavy chain and a light chain comprising the amino acid sequences set forth in SEQ ID NOs: 770 and 768, respectively.

[0046] In one aspect, the disclosure provides an isolated monoclonal antibody or antigen-binding portion thereof, wherein the antibody or antigen-binding portion thereof comprises a heavy chain and a light chain comprising amino acid sequences at least 90% identical to the amino acid sequences set forth in SEQ ID NOs: 770 and 768, respectively.

[0047] In one aspect, the disclosure provides an isolated monoclonal antibody or antigen-binding portion thereof, wherein the antibody or antigen-binding portion thereof comprises a heavy chain and a light chain comprising the amino acid sequences set forth in SEQ ID NOs: 792 and 790, respectively.

[0048] In one aspect, the disclosure provides an isolated monoclonal antibody or antigen-binding portion thereof, wherein the antibody or antigen-binding portion thereof comprises a heavy chain and a light chain comprising amino acid sequences at least 90% identical to the amino acid sequences set forth in SEQ ID NOs: 792 and 790, respectively.

[0049] In some embodiments, the present disclosure provides a pharmaceutical composition comprising the isolated monoclonal antibody or antigen-binding portion thereof of the above aspect and a pharmaceutically acceptable carrier.

[0050] In some embodiments, the present disclosure provides a nucleic acid comprising a nucleotide sequence encoding the light chain, the heavy chain, or both the light and heavy chains of the isolated monoclonal antibody, or antigen-binding portion thereof, of any of the above aspects.

[0051] In some embodiments, the disclosure provides an expression vector comprising the nucleic acid.

[0052] In some embodiments, the disclosure provides a cell transformed with the expression vector.

[0053] In some embodiments, the present disclosure provides that the isolated monoclonal antibody or antigen-binding fragment thereof is capable of specifically binding to and / or specifically antagonizing human IL-27.

[0054] In some embodiments, the disclosure provides a method for making a monoclonal antibody or antigen-binding portion thereof that specifically binds human IL-27, the method comprising maintaining the cell under conditions that allow expression of the monoclonal antibody or antigen-binding portion thereof. In some embodiments, the method further comprises obtaining the monoclonal antibody or antigen-binding portion thereof.

[0055] In some embodiments, 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 any of the above-described isolated monoclonal antibodies or antigen-binding fragments, wherein the antibody or antigen-binding portion thereof inhibits or reduces STAT1 and / or STAT3 phosphorylation in the cell.

[0056] In some embodiments, the present disclosure provides a method for inhibiting or reducing inhibition of CD161 expression in a cell, the method comprising contacting the cell with any of the above-described isolated monoclonal antibodies or antigen-binding fragments, wherein the antibody or antigen-binding portion thereof inhibits or reduces inhibition of CD161 expression in the cell.

[0057] In some embodiments, the disclosure provides methods for inhibiting or reducing PD-L1 and / or TIM-3 expression in a cell, the method comprising contacting the cell with any of the above-described isolated monoclonal antibodies or antigen-binding fragments, wherein the antibody or antigen-binding portion thereof inhibits or reduces PD-L1 and / or TIM-3 expression in the cell.

[0058] In some embodiments, the present disclosure provides methods for inducing or enhancing secretion of one or more cytokines from a cell, the method comprising contacting the cell with any of the above-described isolated monoclonal antibodies or antigen-binding fragments, wherein the antibody or antigen-binding portion thereof induces or enhances PD-1-mediated secretion of one or more cytokines from the cell.

[0059] In some embodiments, 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 any of the above-described isolated monoclonal antibodies or antigen-binding fragments or the above-described pharmaceutical compositions.

[0060] In some embodiments, the present disclosure provides a method of treating cancer in a subject, the method comprising administering to the subject an effective amount of any of the above-described isolated monoclonal antibodies or antigen-binding fragments or the above-described pharmaceutical compositions.

[0061] In some embodiments, the present disclosure provides methods of stimulating an immune response or treating cancer in a subject, the method comprising administering to the subject an effective amount of any of the above-described isolated monoclonal antibodies or antigen-binding fragments or above-described pharmaceutical compositions, wherein the antibody or antigen-binding portion thereof or the pharmaceutical composition inhibits or reduces STAT1 and / or STAT3 phosphorylation in cells, thereby stimulating the immune response or treating the cancer.

[0062] In some embodiments, the present disclosure provides a method of stimulating an immune response or treating cancer in a subject, the method comprising administering to the subject an effective amount of any of the above-described isolated monoclonal antibodies or antigen-binding fragments or above-described pharmaceutical compositions, wherein the antibody or antigen-binding portion thereof or pharmaceutical composition inhibits or reduces inhibition of CD161 expression in cells, thereby stimulating the immune response or treating the cancer.

[0063] In some embodiments, the present disclosure provides methods of stimulating an immune response or treating cancer in a subject, the method comprising administering to the subject an effective amount of any of the above-described isolated monoclonal antibodies or antigen-binding fragments or above-described pharmaceutical compositions, wherein the antibody or antigen-binding portion thereof, or pharmaceutical composition inhibits or reduces PD-L1 and / or TIM-3 expression on cells, thereby stimulating the immune response or treating the cancer.

[0064] In some embodiments, the present disclosure provides methods of stimulating an immune response or treating cancer in a subject, the method comprising administering to the subject an effective amount of any of the above-described isolated monoclonal antibodies or antigen-binding fragments or above-described pharmaceutical compositions, wherein the antibody or antigen-binding portion thereof, or pharmaceutical composition induces or enhances PD-1-mediated secretion of one or more cytokines from cells, thereby stimulating the immune response or treating the cancer.

[0065] In some embodiments, 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., head and neck squamous cell carcinoma), 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., renal clear cell carcinoma)).

[0066] In some embodiments, the present disclosure provides 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 the anti-PD-1 antibody), comprising exposing a cell to any of the antibodies or antigen-binding portions thereof, either simultaneously or sequentially with the anti-PD-1 antibody, thereby enhancing one or more activities of the anti-PD-1 antibody.

[0067] In some embodiments, the disclosure provides anti-PD-1 antibodies, and pharmaceutical compositions comprising any of the above antibodies or antigen-binding portions thereof, and a pharmaceutically acceptable carrier.

[0068] In some embodiments, the disclosure provides kits comprising an anti-PD-1 antibody for simultaneous or sequential administration, and any of the antibodies or antigen-binding portions thereof, and instructions for their use.

[0069] In some embodiments, the present disclosure provides that any of the above-described isolated monoclonal antibodies or antigen-binding portions thereof is administered in combination with 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 drug, cytotoxic agent, immunotherapy, cytokine, surgery, radiation therapy, activator of costimulatory molecules, inhibitor of inhibitory molecules, vaccine, or cellular immunotherapy, or a combination thereof.

[0070] In some embodiments, the disclosure provides that the one or more additional therapeutic agents is a PD-1 antagonist, a PD-L1 inhibitor, a TIM-3 inhibitor, a LAG-3 inhibitor, a TIGIT inhibitor, a CD112R inhibitor, a TAM inhibitor, a STING agonist, a 4-1BB agonist, a CTLA-4 inhibitor, a CD73 inhibitor, a CD39 inhibitor, an A2AR inhibitor, an IDO inhibitor, peg-IL-2, peg IL-10, a CD40 agonist, or a combination thereof.

[0071] In some embodiments, the disclosure provides that the one or more additional therapeutic agents is a PD-1 antagonist.

[0072] In some embodiments, the present disclosure provides that the PD-1 antagonist is selected from the group consisting of: PDR001, nivolumab, pembrolizumab, pidilizumab, MEDI0680, REGN2810, TSR-042, PF-06801591, AMP-224, AB122, and JTX-4014.

[0073] In some embodiments, the present disclosure provides that the PD-L1 inhibitor is selected from the group consisting of: FAZ053, atezolizumab, avelumab, durvalumab, and BMS-936559.

[0074] In some embodiments, the present disclosure provides methods for treating rheumatoid arthritis, wherein the one or more additional therapeutic agents are sunitinib (Sutent®), cabozantinib (Cabometyx®), axitinib (Inlyta®), lenvatinib (Lenvima®), everolimus (Afinitor®), bevacizumab (Avastin®), epacadostat, NKTR-214 (CD-122 bias), or rituximab (rituximab). type agonist), tivozanib (Fotivda®), abexinostat, ipilimumab (Yervoy®), tremelimumab, pazopanib (Votrient®), sorafenib (Nexavar®), temsirolimus (Torisel®), ramucirumab (Cyramza®), niraparib, savolitinib, boranib (X-82), regorafenib ( Stivargo®), donafenib (multi-targeted kinase inhibitor), camrelizumab (SHR-1210), pexastimodine devasilepvec (JX-594), ramucirumab (Cyramza®), apatinib (YN968D1), encapsulated doxorubicin (Thermodox®), tivantinib (ARQ197), ADI-PEG20, binimetinib, apatinib mesylate, nintedanib The present invention provides that the anti-cancer agent is selected from the group consisting of danib, lirilumab, nivolumab (Opdivo®), pembrolizumab (Keytruda®), atezolizumab (Tecentriq®), avelumab (Bavencio®), durvalumab (Imfimzi®), cemiplimab-rwlc (Libtayo®), tislelizumab, and spartalizumab.

[0075] In some embodiments, the present disclosure provides that the one or more additional therapeutic agents is a TIM-3 inhibitor, optionally wherein the TIM-3 inhibitor is MGB453 or TSR-022.

[0076] In some embodiments, the present disclosure provides that 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.

[0077] In some embodiments, the present disclosure provides that the one or more additional therapeutic agents is a TIGIT inhibitor.

[0078] In some embodiments, the present disclosure provides that the one or more additional therapeutic agents is a CD112R inhibitor.

[0079] In some embodiments, the present disclosure provides that the one or more additional therapeutic agents is a TAM (Axl, Mer, Tyro) inhibitor.

[0080] In some embodiments, the present disclosure provides that the one or more additional therapeutic agents is a 4-1BB agonist.

[0081] In some embodiments, the present disclosure provides that the one or more additional therapeutic agents is a tyrosine kinase inhibitor (TKI).

[0082] In certain aspects, the disclosure provides an isolated SRF388 monoclonal antibody, or antigen-binding portion thereof. In some embodiments, the disclosure provides that the isolated SRF388 monoclonal antibody, or antigen-binding portion thereof, is administered in further combination with one or more additional therapeutic agents or procedures selected from the group consisting of chemotherapy, targeted anti-cancer therapy, oncolytic drug, cytotoxic agent, immunotherapy, cytokine, surgery, radiation therapy, activator of costimulatory molecules, inhibitor of inhibitory molecules, vaccine, or cellular immunotherapy, or a combination thereof. In some embodiments, the one or more additional therapeutic agents are a PD-1 antagonist, a PD-L1 inhibitor, a TIM-3 inhibitor, a LAG-3 inhibitor, a TIGIT inhibitor, a CD112R inhibitor, a TAM inhibitor, a STING agonist, a 4-1BB agonist, a CTLA-4 inhibitor, a CD73 inhibitor, a CD39 inhibitor, an A2AR inhibitor, an IDO inhibitor, a NEKTAR, peg-IL-2, peg-IL-10, a CD40 agonist, or a combination thereof. In some embodiments, the disclosure provides that the PD-1 antagonist is selected from the group consisting of PDR001, nivolumab, pembrolizumab, pidilizumab, MEDI0680, REGN2810, TSR-042, PF-06801591, AMP-224, AB122, and JTX-4014.

[0083] In one aspect, the disclosure provides a method for detecting IL-27 or EBI3 or p28 in a sample from a subject, the method comprising the steps of: (a) contacting a sample from the subject with a detection antibody under conditions that allow the detection antibody to form a detection antibody-EBI3 complex or a detection antibody-p28 complex if IL-27 or EBI3 or p28 is present in the sample, wherein the detection antibody is any of the antibodies or antigen-binding fragments thereof, or any antibody described in Table 12; and (b) detecting the presence of the complex formed in step (a), if present.

[0084] In one aspect, the disclosure provides a method of detecting an IL-27-associated cancer in a subject, the method comprising: (a) contacting a sample from a subject suspected of having an IL-27-associated cancer with a detector antibody under conditions that allow the detector antibody to form a detector antibody-EBI3 complex or a detector antibody-p28 complex if IL-27 or EBI3 or p28 is present in the sample, wherein the detector antibody is any of the antibodies or antigen-binding portions thereof, or any antibody described in Table 12; and (b) detecting the presence of the complex formed in step (a), if present.

[0085] In some embodiments, the detection antibody is conjugated to a detectable label.

[0086] In some embodiments, the method further comprises contacting the sample with a capture antibody to form a complex comprising IL-27 or EBI3 and the capture antibody if IL-27 or EBI3 is present in the sample, wherein the capture antibody is any of the above antibodies or antigen-binding portions thereof.

[0087] In some embodiments, the capture antibody has one or more CDRs of Ab7, and optionally is Ab7.

[0088] In some embodiments, the capture antibody is immobilized on a solid support.

[0089] In some embodiments, the sample is contacted with the capture antibody before the detection antibody.

[0090] In some embodiments, the sample is a bodily fluid sample.

[0091] In some embodiments, the liquid sample is blood, serum, plasma, a cell lysate, or a tissue lysate.

[0092] In some embodiments, the cancer is selected from renal cell carcinoma (RCC), hepatocellular carcinoma (HCC), lung cancer, gastroesophageal cancer, ovarian cancer, endometrial cancer, melanoma, leukemia, and lymphoma.

[0093] In some embodiments, the cancer is renal cell carcinoma (RCC) or the cancer is hepatocellular carcinoma (HCC).

[0094] In some embodiments, the cancer is selected from leukemia and lymphoma, or the cancer is ovarian cancer.

[0095] In some embodiments, the present disclosure provides the use of any of the above-described isolated monoclonal antibodies or antigen-binding portions thereof, or the above-described pharmaceutical compositions, optionally used in combination with one or more additional therapeutic agents or procedures, to stimulate an immune response in a subject or to treat cancer in a subject.

[0096] In some embodiments, the present disclosure provides kits comprising any of the above-described isolated monoclonal antibodies or antigen-binding portions thereof or the above-described pharmaceutical compositions and instructions for use in stimulating an immune response in a subject or treating cancer in a subject, optionally containing instructions for use in combination with one or more additional therapeutic agents or procedures.

[0097] In some embodiments, the present disclosure provides a kit comprising any of the above-described isolated monoclonal antibodies or antigen-binding portions thereof and instructions for use in detecting IL-27 in a sample from a subject, and optionally containing instructions for use in detecting an IL-27-associated cancer in a subject.

[0098] In some embodiments, the present disclosure provides that the isolated monoclonal antibody or antigen-binding portion thereof includes an antibody or antigen-binding portion thereof that antagonizes IL-27.

[0099] In some embodiments, the present disclosure provides that the isolated monoclonal antibody or antigen-binding portion thereof includes an antibody or antigen-binding portion thereof that inhibits or reduces STAT1 and / or STAT3 phosphorylation in a cell, wherein the cell is an immune cell or the cell is a cancer cell.

[0100] In some embodiments, the present disclosure provides that any of the isolated monoclonal antibodies or antigen-binding portions thereof includes an antibody or antigen-binding portion thereof that inhibits or reduces inhibition of CD161 expression in a cell. In some embodiments, the present disclosure provides that the cell is an immune cell.

[0101] In some embodiments, the disclosure provides that the isolated monoclonal antibody or antigen-binding portion thereof includes an antibody or antigen-binding portion thereof that inhibits or reduces PD-L1 and / or TIM-3 expression in a cell. In some embodiments, the disclosure provides that the cell is an immune cell or a cancer cell. In some embodiments, when the cell is a cancer cell, the antibody or antigen-binding portion thereof inhibits or reduces PD-L1 expression in the cancer cell.

[0102] In some embodiments, the present disclosure provides that the isolated monoclonal antibody or antigen-binding portion thereof includes an antibody or antigen-binding portion thereof that induces or enhances PD-1-mediated secretion of one or more cytokines from a cell. In some embodiments, the one or more cytokines are IFNg, TNFα, or IL-6. In some embodiments, the one or more cytokines are IFNg, IL-17, TNFα, or IL-6. In some embodiments, the one or more cytokines are TNFα. In some embodiments, the cell is an immune cell.

[0103] In some embodiments, the present disclosure provides that the isolated monoclonal antibody or antigen-binding portion thereof includes an antibody selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE antibodies. In some embodiments, the antibody is an IgG1 antibody or an IgG4 antibody. In some embodiments, the antibody comprises a wild-type IgG1 heavy chain constant region. In some embodiments, the antibody comprises a wild-type IgG4 heavy chain constant region.

[0104] In some embodiments, the present disclosure provides that the isolated monoclonal antibody or antigen-binding portion thereof comprises an Fc domain comprising at least one mutation. In some embodiments, the antibody comprises a mutated IgG1 heavy chain constant region. In some embodiments, the antibody comprises a mutated IgG4 heavy chain constant region. In some embodiments, the mutated IgG4 heavy chain constant region comprises either an S228P substitution, an L235E substitution, an L235A substitution, or a combination thereof, according to EU numbering.

[0105] In certain aspects, the present disclosure provides an isolated monoclonal antibody, or antigen-binding portion thereof, that binds to substantially the same epitope as any of the above-described antibodies, or antigen-binding portions thereof.

[0106] In certain aspects, the present disclosure provides an isolated monoclonal antibody, or antigen-binding portion thereof, that binds to at least one of the amino acid residues bound by any of the above-described antibodies, or antigen-binding portions thereof.

[0107] In one aspect, the disclosure provides an isolated monoclonal antibody or antigen-binding portion thereof, wherein mutation of the epitope bound by the antibody or antigen-binding portion thereof inhibits, reduces, or blocks both binding to the antibody or antigen-binding portion thereof and binding to an antibody or antigen-binding portion thereof corresponding to any of the foregoing antibodies or antigen-binding fragments thereof.

[0108] In one aspect, the disclosure provides an isolated monoclonal antibody or antigen-binding portion thereof that binds to an epitope on IL-27, wherein the epitope is identical to or similar to an epitope bound by an antibody molecule described in Table 12.

[0109] In one aspect, the present disclosure provides an isolated monoclonal antibody or antigen-binding portion thereof that specifically binds to and antagonizes human WSX-1, wherein the antibody or antigen-binding portion thereof comprises heavy chain CDRs and light chain CDRs selected from the group consisting of heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 802, 803, and 804, respectively, and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 805, 806, and 807, respectively.

[0110] In one aspect, the disclosure provides an isolated monoclonal antibody or antigen-binding portion thereof that specifically binds to human WSX-1, wherein the antibody or antigen-binding portion thereof comprises a heavy chain variable region and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 794 and 796, respectively.

[0111] In one aspect, the present disclosure provides an isolated monoclonal antibody or antigen-binding portion thereof that specifically binds to human WSX-1 and comprises a light chain constant region, wherein the light chain constant region comprises the amino acid sequence set forth in SEQ ID NO:798.

[0112] In one aspect, the disclosure provides an isolated monoclonal antibody or antigen-binding portion thereof that specifically binds to human WSX-1, wherein the antibody or antigen-binding portion thereof comprises heavy chain CDRs and light chain CDRs that are at least 90% identical to the group consisting of the heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 802, 803, and 804, respectively, and heavy chain CDRs and light chain CDRs that are at least 90% identical to the group consisting of the light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 805, 806, and 807, respectively.

[0113] In one aspect, the present disclosure provides an isolated monoclonal antibody or antigen-binding portion thereof that specifically binds to human WSX-1, wherein the antibody or antigen-binding portion thereof comprises a heavy chain variable region and a light chain variable region that are at least 90% identical to the group consisting of SEQ ID NOs: 794 and 796, respectively.

[0114] In one aspect, the present disclosure provides an isolated monoclonal antibody or antigen-binding portion thereof that specifically binds to human WSX-1 and comprises a light chain constant region, wherein the light chain constant region is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:798.

[0115] In some embodiments, the present disclosure provides a pharmaceutical composition comprising an isolated monoclonal antibody, or antigen-binding portion thereof, that is any of the WSX-1 binding antibodies described above, and a pharmaceutically acceptable carrier.

[0116] In some embodiments, the present disclosure provides a nucleic acid comprising a nucleotide sequence encoding the light chain, the heavy chain, or both the light and heavy chains of an isolated monoclonal antibody, or antigen-binding portion thereof, that is any of the WSX-1-binding antibodies described above. In some embodiments, the present disclosure provides an expression vector comprising the nucleic acid described above. In some embodiments, the present disclosure provides a cell transformed with the expression vector described above. In some embodiments, the present disclosure provides a method for producing a monoclonal antibody, or antigen-binding portion thereof, that specifically binds to human WSX-1, comprising maintaining the cell described above under conditions that permit expression of the monoclonal antibody, or antigen-binding portion thereof. In some embodiments, the present disclosure provides a method for detecting WSX-1 in a sample from a subject, the method comprising: (a) contacting the sample from the subject with a detection antibody under conditions that allow the detection antibody to form a detection antibody-WSX-1 complex if WSX-1 is present in the sample, wherein the detection antibody is an antibody or antigen-binding fragment thereof that corresponds to any of the WSX-1-binding antibodies described above; and (b) detecting the presence of the complex formed in step (a), if present.

[0117] In some embodiments, the present disclosure provides a method for detecting a WSX-1-associated cancer in a subject, the method comprising: (a) contacting a sample from a subject suspected of having a WSX-1-associated cancer with a detection antibody under conditions that allow the detection antibody to form a detection antibody-WSX-1 complex if WSX-1 is present in the sample, wherein the detection antibody is an antibody or antigen-binding portion thereof that corresponds to any of the WSX-1-binding antibodies described above; and (b) detecting the presence of the complex formed in step (a), if present.

[0118] In some embodiments, the present disclosure provides the use of an isolated monoclonal antibody or antigen-binding portion thereof, which is any of the above-described WSX-1 binding antibodies, or the above-described pharmaceutical composition, optionally used in combination with one or more additional therapeutic agents or procedures, to stimulate an immune response in a subject or treat cancer in a subject.

[0119] In some embodiments, the present disclosure provides a kit comprising an isolated monoclonal antibody or antigen-binding portion thereof that is any of the WSX-1 binding antibodies described above, or the pharmaceutical composition described above, and instructions for use to stimulate an immune response in a subject or treat cancer in a subject, optionally containing instructions for use in combination with one or more additional therapeutic agents or procedures.

[0120] definition Terms used in the claims and this specification are defined as follows, unless otherwise specified.

[0121] It must 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.

[0122] As used herein, "about" will be understood by one of ordinary skill in the art and will vary to some extent depending on the context in which it is used. If the use of the term is not clear to a person of ordinary skill in the art given the context in which it is used, "about" will mean up to ±10% of the particular value.

[0123] As used herein, the term "agonist" refers to any molecule that partially or fully promotes, induces, increases, and / or activates the biological activity of a native polypeptide disclosed herein. Suitable agonist molecules include, inter alia, agonist antibodies or antibody fragments, fragments or amino acid sequence variants of native polypeptides, peptides, or proteins. In some embodiments, activation in the presence of an agonist is observed in a dose-dependent manner. In some embodiments, 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 under comparable conditions. Methods for identifying agonists suitable for use in the disclosed methods are also disclosed herein. For example, these methods include, but are not limited to, binding assays such as enzyme-linked immunosorbent assays (ELISAs), Forte Bio© systems, and radioimmunoassays (RIAs). These assays determine 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 potency of an agonist, such as the ability of an agonist to activate or promote the function of a polypeptide, can also be determined using a functional assay. For example, a functional assay can include contacting a polypeptide with a candidate agonist molecule and measuring a detectable change in one or more biological activities typically associated with the polypeptide. The potency of an agonist is typically determined by its EC 50 The EC value is defined as the concentration required to activate the agonist response by 50%. 50The lower the value, the more potent the agonist and the lower the concentration required to maximally activate a biological response.

[0124] As used herein, the term "alanine scanning" refers to a technique used to determine the contribution of a particular wild-type residue to the stability or function(s) (e.g., binding affinity) of a given protein or polypeptide. This technique involves substituting an alanine residue for the wild-type residue of the polypeptide, followed by evaluation of the stability or function(s) (e.g., binding affinity) of the alanine-substituted derivative or mutant polypeptide and comparison with the wild-type polypeptide. Techniques for substituting alanine for wild-type residues of polypeptides are known in the art.

[0125] The term "amelioration" refers to any beneficial outcome of therapy in the treatment of a condition, e.g., cancer, including prevention, reduction in severity or progression, remission, or cure of the condition.

[0126] As used herein, the term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those 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 a naturally occurring amino acid (i.e., a carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group), such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refer to compounds that have a structure that is different from the general chemical structure of an amino acid, but that function in a manner similar to a naturally occurring amino acid.

[0127] As used herein, amino acids may be referred to by either the commonly known amino acid three-letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Similarly, nucleotides may be referred to by their commonly accepted single-letter codes.

[0128] As used herein, an "amino acid substitution" refers to the replacement of at least one amino acid residue present in a predetermined amino acid sequence (the amino acid sequence of the starting polypeptide) with another, different, "substitution" amino acid residue. An "amino acid insertion" refers to the incorporation of at least one additional amino acid into a predetermined amino acid sequence. Insertions usually consist of the insertion of one or two amino acid residues, although larger "peptide insertions," e.g., insertions of about 3 to about 5 amino acid residues, or even up to about 10, 15, or 20 amino acid residues, can also be made. The inserted residue(s) can be naturally occurring or non-naturally occurring, as disclosed above. An "amino acid deletion" refers to the removal of at least one amino acid residue from a predetermined amino acid sequence.

[0129] 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, small molecule, protein, mRNA, marker). When referring to a metabolite or small molecule (e.g., a drug), the terms "amount," "level," and "concentration" are generally 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 compared to a control sample, e.g., from an individual or individuals not afflicted with a disease or disorder (e.g., cancer), or an internal standard. In some embodiments, an increase in the level of a substance (e.g., a drug) in a sample refers to an increase of 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% in the amount of the substance compared to the amount of the substance in a control sample, as determined by techniques known in the art (e.g., HPLC). A "reduced level" refers to a decrease in the amount, concentration, or abundance of a substance (e.g., a drug) in an individual compared to a control, e.g., an individual or individuals not afflicted with a disease or disorder (e.g., cancer), or an internal standard. In some embodiments, a reduced level is an amount, concentration, or abundance that is barely or completely undetectable. In some embodiments, a reduction in the level of a substance (e.g., a drug) in a sample refers to about a 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% decrease in the amount of the substance compared to the amount of the substance in a control sample, as determined by techniques known in the art (e.g., HPLC).

[0130] When referring to a protein, mRNA, or marker such as those described herein, the terms "level of expression" or "expression level" are generally used interchangeably and typically 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 associated with the likelihood of response to an agent such as those described herein. "Expression" typically refers to the process by which information contained within a gene is converted into a structure (e.g., a protein marker such as PD-L1) that is present and functional within a cell. Thus, as used herein, "expression" can refer to transcription into a polynucleotide, translation into a polypeptide, or even modification of a polynucleotide and / or polypeptide (e.g., post-translational modification of a polypeptide). Fragments of a transcribed polynucleotide, a translated polypeptide, or modified (e.g., post-translational modification of a polypeptide) polynucleotide and / or polypeptide are also considered expressed, regardless of whether they are derived from a transcript generated by alternative splicing or a degraded transcript, or from post-translational processing of a polypeptide, e.g., by proteolysis. "Expressed genes" include those that are transcribed into polynucleotides as mRNA and then translated into polypeptides, as well as those that are further transcribed into RNA but are not translated into polypeptides (e.g., transfer RNA and ribosomal RNA). "Increased expression," "elevated expression levels," or "elevated levels" refer to increased expression or increased levels of a substance in a sample compared to a control sample, e.g., an individual or individuals not afflicted with a disease or disorder (e.g., cancer), or an internal standard. In some embodiments, increased expression of a substance (e.g., a protein marker such as PD-L1) in a sample refers to an increase of 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% in the amount of the substance compared to the amount of the substance in a control sample, as determined by techniques known in the art (e.g., FACS)."Decreased expression," "decreased expression level," or "decreased level" refers to a decrease in expression or level of a substance (e.g., a protein marker) in an individual compared to a control, e.g., an individual or individuals not afflicted with a disease or disorder (e.g., cancer), or an internal standard. In some embodiments, decreased expression is little or no expression. In some embodiments, decreased expression of a substance (e.g., a protein marker) in a sample refers to about a 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% decrease in the amount of the substance compared to the amount of the substance in a control sample, as determined by techniques known in the art (e.g., FACS).

[0131] As used herein, the term "angiogenesis" or "neovascularization" refers to the process of new blood vessels developing from pre-existing vessels (Varner et al., (1999) Angiogen. 3:53-60; Mousa et al., (2000) Angiogen. Stim. Inhib. 35:42-44; Kim et al., (2000) Amer. J. Path. 156:1345-1362; Kim et al., (2000) J. Biol. Chem. 275:33920-33928; Kumar et al. (2000) Angiogenesis: From Molecular to Integrative Pharm. 169-180). Endothelial cells derived from pre-existing blood vessels or circulating endothelial stem cells (Takahashi et al., (1995) Nat. Med. 5:434-438; Isner et al., (1999) J. Clin. Invest. 103:1231-1236) are activated to migrate, proliferate, and differentiate into lumen-bearing structures to form new blood vessels in response to growth factor or hormonal cues or hypoxic or ischemic conditions. During ischemia, such as occurs in cancer, the need for increased oxygen load and nutrient delivery apparently induces the secretion of angiogenic factors by the affected tissue, which stimulate new blood vessel formation. Several additional terms are associated with angiogenesis.

[0132] As used herein, the term "antagonist" refers to any molecule that partially or completely blocks, inhibits, or neutralizes the biological activity of the native polypeptide disclosed herein.Suitable antagonist molecules include, in particular, antagonist antibodies or antibody fragments, fragments or amino acid sequence variants of native polypeptides, peptides, or proteins.In some embodiments, inhibition in the presence of an antagonist is observed in a dose-dependent manner. In some embodiments, 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 under comparable conditions. Also disclosed herein are methods for identifying antagonists suitable for use in the disclosed methods. For example, these methods include, but are not limited to, binding assays such as enzyme-linked immunosorbent assays (ELISAs), Forte Bio© systems, radioimmunoassays (RIAs), Meso Scale Discovery assays (e.g., Meso Scale Discovery electrochemiluminescence (MSD-ECL)), and Luminex® bead-based assays. These assays determine the ability of an antagonist to bind to a polypeptide of interest (e.g., a receptor or ligand), and thus indicate the ability of the antagonist to inhibit, neutralize, or block the activity of the polypeptide. The potency of an antagonist, such as the ability of an antagonist to inhibit the function of a polypeptide or agonist, can also be determined using functional assays. For example, a functional assay can include 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 measured by its IC 50It is defined by the IC value (the concentration required to inhibit the agonist response by 50%). 50 The lower the value, the more potent the antagonist and the lower the concentration required to maximally inhibit the biological response.

[0133] As used herein, the phrase "antibody or antigen-binding portion thereof that antagonizes human IL-27" refers to an antibody that antagonizes at least one art-recognized activity of human IL-27 (e.g., IL-27 biological activity and / or downstream pathway(s) mediated by IL-27 signaling, or other function mediated by IL-27), e.g., associated with a decrease (or reduction) of human IL-27 activity by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more. Additional examples of IL-27 biological activity and / or downstream pathway(s) mediated by IL-27 signaling, or other function mediated by IL-27, are described in more detail below and elsewhere herein.

[0134] 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 pathway(s) mediated by IL-27 signaling, or other function mediated by IL-27. Anti-IL-27 antagonist antibodies include antibodies that block, antagonize, suppress, inhibit, or reduce IL-27 biological activity (e.g., ligand binding, enzymatic activity), including downstream pathways or functions mediated by IL-27 signaling, e.g., receptor binding and / or eliciting cellular responses to IL-27 or its metabolites. In some embodiments, anti-IL-27 antagonist antibodies provided by the present disclosure bind to human IL-27 and prevent, block, or inhibit binding of human IL-27 to its cognate or canonical 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 embodiments, anti-IL-27 antagonist antibodies prevent, block, or inhibit binding of human IL-27 to gp130. In some embodiments, anti-IL-27 antagonist antibodies prevent, block, or inhibit binding of human IL-27 to IL-27Rα. In some embodiments, anti-IL-27 antagonist antibodies prevent, block, or inhibit dimerization of IL-27 monomers. In some embodiments, anti-IL-27 antibodies specifically bind to EBI3 monomers. In some embodiments, the anti-IL-27 antibody specifically binds to the IL-27p28 monomer. In some embodiments, the anti-IL-27 antibody specifically binds to both IL-27 monomers. In some embodiments, the anti-IL-27 antibody specifically binds to a discontinuous epitope that includes both EBI3 and p28. In some embodiments, the anti-IL-27 antibody inhibits or reduces STAT1 and / or STAT3 phosphorylation in a cell. In some embodiments, the anti-IL-27 antibody inhibits or reduces inhibition of CD161 expression in a cell (e.g., ameliorate or alleviate IL-27-mediated inhibition of CD161 expression in a cell).In some embodiments, the anti-IL-27 antibody inhibits or reduces PD-L1 and / or TIM-3 expression in cells. In some embodiments, the anti-IL-27 antibody induces or enhances PD-1-mediated secretion of one or more cytokines from cells. In some embodiments, the anti-IL-27 antagonist antibody binds to human IL-27 and stimulates or enhances an anti-tumor response. In some embodiments, the anti-IL-27 antagonist antibody binds to human IL-27 with an affinity of 15 nM or less. In some embodiments, the anti-IL-27 antagonist antibody binds to human IL-27 and comprises a wild-type or mutant IgG1 heavy chain constant region or a wild-type or mutant IgG4 heavy chain constant region. Examples of anti-IL-27 antagonist antibodies are provided herein.

[0135] As used herein, the term "antibody" refers to a full-length antibody comprising two light polypeptide chains and two heavy polypeptide chains. Full-length antibodies include different antibody isotypes, including IgM, IgG, IgA, IgD, and IgE antibodies. The term "antibody" includes polyclonal, monoclonal, chimeric, humanized, primatized, deimmunized, and fully human antibodies. Antibodies can be made in or derived from any of a variety of mammalian species, including humans, non-human primates (e.g., orangutans, baboons, or chimpanzees), horses, cows, pigs, sheep, goats, dogs, cats, rabbits, guinea pigs, gerbils, hamsters, rats, and mice. Antibodies can be purified or recombinant. As used herein, the terms "antibody fragment," "antigen-binding fragment," or similar terms refer to fragments of antibodies that retain the ability to bind to a target antigen (e.g., IL-27) and inhibit 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. An scFv fragment is a single polypeptide chain that contains both the heavy and light chain variable regions of the antibody from which the scFv is derived. Additionally, intrabodies, minibodies, triabodies, and diabodies are also included within the definition of antibody and are adaptable for use in the methods described herein. See, e.g., 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 each of which are incorporated herein by reference in their entirety).

[0136] As used herein, the term "antibody fragment" also includes single domain antibodies, such as, for example, camelized single domain antibodies. See, e.g., 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 Publications WO 94 / 04678 and WO 94 / 25591; and U.S. Patent No. 6,005,079, all of which are incorporated herein by reference in their entireties. In some embodiments, the present disclosure provides single domain antibodies comprising two VH domains that have been engineered to form a single domain antibody.

[0137] In some embodiments, the antigen-binding fragment comprises a variable region of a heavy chain polypeptide and a variable region of a light chain polypeptide. In some embodiments, the antigen-binding fragment described herein comprises the CDRs of the light and heavy chain polypeptides of the antibody.

[0138] The term "antigen-presenting cell" or "APC" refers to a cell that presents 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 (e.g., THP-1), B lymphoblastoid cells (e.g., C1R.A2, 1518 B-LCL), and monocyte-derived dendritic cells (DCs). Some APCs internalize antigens by phagocytosis or receptor-mediated endocytosis.

[0139] The term "antigen presentation" refers to the process by which APCs capture antigens and enable their recognition by T cells, for example as components of MHC-I and / or MHC-II complexes.

[0140] As used herein, the term "apoptosis" refers to the process of programmed cell death that occurs in multicellular organisms (e.g., humans). The highly regulated biochemical and molecular events that lead to apoptosis can cause cells to undergo observable and characteristic morphological changes, including membrane blebbing, cell volume shrinkage, chromatin DNA condensation and fragmentation, and mRNA degradation. A common method for identifying cells, including T cells, undergoing apoptosis is to expose the cells to a fluorophore-conjugated protein (annexin V). Annexin V is commonly used to detect apoptotic cells due to its ability to bind to phosphatidylserine in the outer leaflet of the cell membrane, an early indicator that the cell is undergoing the apoptotic process.

[0141] As used herein, the term "B cell" (or "B lymphocyte") refers to a lymphocyte subtype of white blood cell. B cells function in the humoral immune component of the adaptive immune system by secreting antibodies. B cells also present antigens and secrete cytokines. B cells differ from the other two classes of lymphocytes, T cells and natural killer cells, in that they express a B cell receptor (BCR) on their cell membrane. The BCR enables B cells to bind to specific antigens, thereby initiating an antibody response against those antigens.

[0142] As used herein, the term "binds to immobilized IL-27" refers to the ability of a human antibody of the disclosure to bind to IL-27 that is expressed, for example, on a cell surface or that is bound to a solid support.

[0143] As used herein, the term "bispecific" or "bifunctional antibody" refers to an artificial hybrid antibody having two different heavy / light chain pairs and thus two different binding sites. Bispecific antibodies can be produced by a variety of methods, including hybridoma fusion or linking of Fab' fragments. See, e.g., Songsivilai & Lachmann, (1990) Clin. Exp. Immunol. 79:315-321; Kostelny et al., (1992) J. Immunol. 148:1547-1553.

[0144] Originally, recombinant production of bispecific antibodies was based on the coexpression of two immunoglobulin heavy / light chain pairs, where the two heavy / light chain pairs have different specificities (Milstein and Cuello, (1983) Nature 305:537-539). Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. Fusion of the heavy chain variable region to an immunoglobulin heavy chain constant domain comprising at least part of the hinge, CH2, and CH3 regions is preferred. For further details regarding exemplary methods currently known 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) See J. Immunol. 147:60. Bispecific antibodies also include cross-linked or heteroconjugate antibodies. Heteroconjugate antibodies may be made 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.

[0145] Various techniques for producing and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. See, e.g., Kostelny et al. (1992) J Immunol 148(5):1547-1553. The leucine zipper peptides from the Fos and Jun proteins can be linked to the Fab' portions of two different antibodies by gene fusion. Antibody homodimers can be reduced at the hinge region to form monomers and then reoxidized to form antibody heterodimers. This method can also be used to produce homodimeric antibodies. The "diabody" technology described by Hollinger et al. (1993) Proc Natl Acad Sci USA 90:6444-6448 provides an alternative mechanism for producing bispecific antibody fragments. These fragments comprise a heavy chain variable domain (VH) connected by a linker to a light chain variable domain (VL). The linker is too short to allow pairing between 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 another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of 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, for example, 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), which form a pair of antigen-binding regions. Linear antibodies may be bispecific or monospecific.

[0146] Antibodies with more than two valencies (e.g., trispecific antibodies) are also contemplated, see, e.g., Tutt et al. (1991) J Immunol 147:60.

[0147] 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. DVD-Ig molecules are engineered such that two different light chain variable domains (VL) from two different parent antibodies are linked in tandem by recombinant DNA techniques, either directly or via a short linker, and then linked to a light chain constant domain. Similarly, the heavy chain contains two different heavy chain variable domains (VH) linked in tandem, followed by a constant domain CH1 and an Fc region. Methods for generating DVD-Ig molecules from two parent antibodies are further described, for example, in PCT Publication Nos. WO08 / 024188 and WO07 / 024715. In some embodiments, bispecific antibodies are tandem Fab (Fabs-in-Tandem) immunoglobulins in which a light chain variable region with a second specificity is fused to a heavy chain variable region of a whole antibody. Such antibodies are described, for example, in International Patent Application Publication No. WO2015 / 103072.

[0148] As used herein, "cancer antigen" refers to (i) tumor-specific antigens, (ii) tumor-associated antigens, (iii) cells expressing tumor-specific antigens, (iv) cells expressing tumor-associated antigens, (v) fetal 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 substance associated with cancer.

[0149] As used herein, the term "cancer-specific immune response" refers to an immune response elicited by the presence of a tumor, cancer cells, or cancer antigen. In certain embodiments, this response includes proliferation of cancer antigen-specific lymphocytes. In certain embodiments, this response includes expression and upregulation of antibodies and T cell receptors, and the formation and release of lymphokines, chemokines, and cytokines. Both the innate and adaptive immune systems interact to mount an antigen response against a tumor, cancer cells, or cancer antigen. In certain embodiments, the cancer-specific immune response is a T cell response.

[0150] The term "carcinoma" is art-recognized and refers to malignant tumors of epithelial and endocrine tissues, including carcinomas of the respiratory system, gastrointestinal system, genitourinary system, testicular cancer, breast cancer, prostate cancer, endocrine system, and melanoma. The anti-IL-27 antibodies described herein can be used to treat patients who have, are suspected of having, or may be at high risk of developing any type of cancer, including kidney cancer or melanoma, or any viral disease. Exemplary carcinomas include those formed from tissue of the cervix, lung, prostate, breast, head and neck, colon, and ovary. The term also includes carcinosarcomas, which include malignant tumors composed of carcinomatous and sarcomatous tissue. "Adenocarcinoma" refers to a carcinoma derived from glandular tissue or in which tumor cells form recognizable glandular structures.

[0151] As used herein, the term "CD112R" refers to a member of the poliovirus receptor-like protein family and is a co-inhibitory receptor for human T cells. CD112R is preferentially expressed on T cells and inhibits signals mediated by the T cell receptor. CD112, which is widely expressed on antigen-presenting cells and tumor cells, is the ligand for CD112R. CD112R binds to CD112 in competition with CD226. Disruption of the CD112R-CD112 interaction enhances human T cell responses. CD112R is a novel checkpoint for human T cells through its interaction with CD112. As used herein, the term "CD112R inhibitor" refers to an agent that destroys, blocks, or inhibits the biological function or activity of CD112R.

[0152] 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 costimulatory immune checkpoint molecule primarily for activated T cells. Cross-linking of CD137 enhances T cell proliferation, IL-2 secretion, survival, and cytolytic responses. As used herein, the term "4-1BB agonist" refers to an agent that stimulates, induces, or enhances one or more functions of 4-1BB. An exemplary 4-1BB agonist is utomilumab (PF-05082566), a fully human IgG2 monoclonal antibody that targets 4-1BB and stimulates T cells.

[0153] As used herein, the term "CD161" (alternatively known 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 of T cells, and CD161 expression has been 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.

[0154] 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 containing a first subunit known as Epstein-Barr virus-induced gene 3 (EBI3; also known as IL-27 subunit β and IL-27B) and a second subunit known as 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) Arch Immunol. Ther. Exp. 58:417-425). Adv. Clin. Exp. Med. (2013) 22(5):683-691). Although IL-27 can have proinflammatory effects, numerous studies suggest an important role for IL-27 as an immunosuppressant (Shimizu et al. (2006) J. Immunol. 176:7317-7324, Hisada et al. (2004) Cancer Res. 64:1152-1156, Diakowski (2013) (ibid.)). IL-27 was initially described as a factor promoting the initiation of Th1 responses, but it was later found to fulfill a primary T cell suppressive function by limiting Th1 responses, inhibiting Th2 and Th17 cell differentiation, and regulating the development of Tr1 and other regulatory T cell populations (Dietrich et al. (2014) J. Immunol. 192:5382-5389). In addition to its role as an immunomodulator, IL-27 also regulates angiogenesis, hematopoiesis, and osteoclastogenesis (Id.).

[0155] IL-27 signals through a heterodimeric type I cytokine receptor (IL-27 receptor or IL-27R) that contains a first subunit known as WSX1 (also known as IL-27 receptor subunit alpha, IL-27RA, T-cell cytokine receptor type 1 (TCCR), and cytokine receptor-like 1 (CRL1)) and a second subunit known as gp130 (also known as interleukin-6 signal transducer (IL6ST), interleukin-6 receptor subunit beta (IL-6RB), and oncostatin M 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 p38 MAPK pathways.

[0156] EBI3 is also thought to have biological functions 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).

[0157] The amino acid sequence of an exemplary human EBI3 protein is provided in SEQ ID NO: 698 (NCBI Reference Sequence: NP_005746.2; N- [ka] The amino acid sequence of an exemplary human p28 protein is provided in SEQ ID NO: 699 (NCBI Reference Sequence: NP_663634.2; N- [ka] The amino acid sequence of an exemplary human WSX1 protein is provided in SEQ ID NO: 700 (NCBI Reference Sequence: NP_004834.1; N- [ka] The amino acid sequence of an exemplary human gp130 protein is provided in SEQ ID NO: 701 (NCBI Reference Sequence: NP_002175.2; N- [ka]

[0158] As used herein, the term "compete," when used in the context of antigen binding proteins (e.g., immunoglobulins, antibodies, or antigen-binding fragments thereof) that compete for binding to the same epitope, refers to an interaction between antigen binding proteins as determined by an assay (e.g., competitive binding assay, cross-blocking assay, etc.) in which a test antigen binding protein (e.g., a test antibody) inhibits (e.g., reduces or blocks) the specific binding of a reference antigen binding protein (e.g., a reference antibody) to a common antigen (e.g., IL-27 or a fragment thereof).

[0159] A polypeptide or amino acid sequence "derived from" a specified polypeptide or protein refers to the origin of the polypeptide. Preferably, a polypeptide or amino acid sequence derived from a particular sequence is an amino acid sequence essentially identical to that sequence or a portion thereof, the portion comprising at least 10-20 amino acids, preferably at least 20-30 amino acids, more preferably at least 30-50 amino acids, of that amino acid sequence or an amino acid sequence otherwise identifiable to one of skill in the art as having its origin in that sequence. A polypeptide derived from another peptide may have one or more mutations compared to the starting polypeptide, e.g., one or more amino acid residues substituted for another amino acid residue, or one or more amino acid residues inserted or deleted.

[0160] Polypeptides may contain non-naturally occurring amino acid sequences. Such variants necessarily have less than 100% sequence identity or similarity with the starting molecule. In certain embodiments, variants have less than about 75% to 100%, more preferably less than about 80% to 100%, more preferably less than about 85% to 100%, more preferably less than about 90% to 100%, (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%), and most preferably less than about 95% to 100% amino acid sequence identity or similarity with the amino acid sequence of the starting polypeptide, e.g., over the entire length of the variant molecule.

[0161] In certain embodiments, there is one amino acid difference between the starting polypeptide sequence and the sequence derived therefrom. Identity or similarity to this sequence is defined herein as the percentage of amino acid residues in the candidate sequence that are identical (i.e., the same residues) to the starting amino acid residues after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. In certain embodiments, a polypeptide consists of, consists essentially of, or comprises an amino acid sequence selected from the sequences set forth in Table 12. In certain embodiments, a polypeptide comprises an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from the sequences set forth in Table 12. In certain embodiments, a polypeptide comprises a contiguous amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a contiguous amino acid sequence selected from the sequences set forth in Table 12. In certain embodiments, a polypeptide comprises an amino acid sequence having at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, or 500 (or any integer within these numerical ranges) contiguous amino acids of an amino acid sequence selected from the sequences set forth in Table 12.

[0162] In certain embodiments, the antibodies of the present disclosure are encoded by a nucleotide sequence. The nucleotide sequences of the present invention may be useful for a number of applications, including cloning, gene therapy, protein expression and purification, mutagenesis, DNA vaccination of a host in need thereof, antibody generation for, e.g., passive immunization, PCR, primer and probe generation, and the like. In certain embodiments, the nucleotide sequences of the present invention consist of, consist essentially of, or comprise a nucleotide sequence selected from the sequences set forth in Table 12. In certain embodiments, the nucleotide sequence comprises a nucleotide sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleotide sequence selected from the sequences set forth in Table 12. In certain embodiments, the nucleotide sequence comprises a contiguous nucleotide sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a contiguous nucleotide sequence selected from the sequences set forth in Table 12. In certain embodiments, the polypeptide comprises a nucleotide sequence having at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, or 500 (or any integer within these numerical ranges) contiguous nucleotides of a nucleotide sequence selected from the sequences set forth in Table 12.

[0163] It will also be understood by those skilled in the art that antibodies suitable for use in the methods disclosed herein can be modified so as to differ in sequence from the naturally occurring or native sequence from which they are derived, but retain the desired activity of the native sequence. For example, nucleotide or amino acid substitutions that result in conservative substitutions or changes of "non-essential" amino acid residues can be made. Mutations can be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis.

[0164] Antibodies suitable for use in the methods disclosed herein may contain 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 with similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar 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 embodiments, a consecutive sequence of amino acids can be replaced with a structurally similar consecutive sequence that differs in the order and / or composition of the side chain family members. Alternatively, in certain embodiments, mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resulting variants can be incorporated into binding polypeptides of the invention and screened for their ability to bind to a desired target.

[0165] 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.

[0166] As used herein, the term "cross-reactivity" refers to the ability of an antibody of the present disclosure to bind to IL-27 from a different species. For example, an antibody of the present 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 by detecting binding to or otherwise functional interaction with cells that physiologically express IL-27. Methods for determining cross-reactivity include standard binding assays described herein, for example, by Biacore™ surface plasmon resonance (SPR) analysis using a Biacore™ 2000 SPR instrument (Biacore AB, Uppsala, Sweden) or flow cytometry techniques.

[0167] As used herein, the term "cytotoxic T lymphocyte (CTL) response" refers to an immune response elicited by cytotoxic T cells. CTL responses are primarily CD8 + It is mediated by T cells.

[0168] As used herein, the term "dendritic cell" or "DC" refers to a type of antigen-presenting cell, a white blood cell derived from bone marrow (BM) that is the most potent type of antigen-presenting cell. DCs capture and process antigens, converting proteins into peptides displayed on major histocompatibility complex (MHC) molecules that are recognized by T cells. DCs are heterogeneous, e.g., myeloid DCs and plasmacytoid DCs. Although all DCs can take up, process, and present antigens to naive T cells, DC subtypes possess distinct markers and differ in their localization, migration pathways, detailed immunological functions, and dependence on infectious or inflammatory stimuli for their development. During the development of adaptive immune responses, DC phenotype and function play a role in initiating immune tolerance, memory, and differentiation into polarized T helper 1 (Th1), Th2, and Th17 subtypes.

[0169] As used herein, the term "dendritic cell activation" refers to the transition from immature dendritic cells to mature dendritic cells. Activated dendritic cells encompass both mature dendritic cells and dendritic cells in the transitional stage in which the expression of CD80 and CD86, which induce costimulatory signals upon activation, is elevated. Mature human dendritic cells are cells positive for the expression of CD40, CD80, CD86, and HLA class II (e.g., HLA-DR). Immature dendritic cells can be distinguished from mature dendritic cells based on markers selected from the group consisting of CD80 and CD86. Immature dendritic cells are weakly positive, or preferably negative, for these markers, whereas mature dendritic cells are positive. The distinction between mature dendritic cells is routinely performed by those skilled in the art, and the above-mentioned markers and methods for measuring their expression are also known to those skilled in the art.

[0170] As used herein, the term "EC 50 " refers to the concentration of an antibody or antigen-binding portion thereof that induces 50% of the maximal response, i.e., a response halfway between the maximal response and baseline, in either an in vitro or in vivo assay.

[0171] As used herein, the term "effective dose" or "effective administration amount" 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 treat or at least partially arrest the progression of a disease and its complications in a patient already suffering from the disease. Amounts effective for this use will depend on the severity of the disorder being treated and the general state of the patient's own immune system.

[0172] As used herein, the term "epitope" or "antigenic determinant" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds. As used herein, the term "epitope mapping" refers to a process or method for identifying the binding site or epitope of an antibody or antigen-binding fragment thereof on its target protein antigen. Epitope mapping methods and techniques are provided herein. Epitopes can be formed both from contiguous amino acids or non-contiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed by contiguous amino acids are usually retained upon exposure to denaturing solvents, while epitopes formed by tertiary folding are usually lost upon treatment with denaturing solvents. An epitope typically contains 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 epitope is bound by a given antibody (i.e., epitope mapping) are well known in the art and include, for example, immunoblotting and immunoprecipitation assays, in which overlapping or consecutive peptides derived from IL-27 are tested for reactivity with a given anti-IL-27 antibody. Methods for determining the spatial configuration of epitopes include techniques in the art and those described herein, such as x-ray crystallography and two-dimensional nuclear magnetic resonance (e.g., Epitope Mapping). See Protocols in Methods in Molecular Biology, Vol. 66, GE Morris, Ed. (1996).

[0173] Antibodies that bind to epitopes on IL-27 (e.g., the same or overlapping regions, or regions between or spanning regions), including all or part of the epitopes recognized by the specific antibodies described herein, are also encompassed by the present disclosure.

[0174] Antibodies that bind the same epitope and / or that compete with the antibodies described herein for binding to human IL-27 are also encompassed by the present disclosure. Antibodies that recognize the same epitope or compete for binding can be identified using routine 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 measured in an assay in which the immunoglobulin being tested inhibits the specific binding of a reference antibody to a common antigen, such as IL-27. Many types of competitive binding assays are known, such as solid-phase direct or indirect radioimmunoassays (RIAs), solid-phase direct or indirect enzyme immunoassays (EIAs), sandwich competition assays (see Stahl et al., Methods in Enzymology 9:242 (1983)); solid-phase direct biotin-avidin EIAs (see Kirkland et al., J. Immunol. 137:3614 (1986)); solid-phase direct label assays, solid-phase direct label sandwich assays (see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988)); solid-phase direct label RIAs using I-125 labels (see Morel et al., Mol. Immunol. 25(1):7 (1988)); solid-phase direct biotin-avidin EIAs (see Cheung et al., Virology 176:546 (1990); and direct-labeled RIA (Moldenhauer et al., Scand. J. Immunol. 32:77 (1990)). Such assays typically involve the use of purified antigen bound to a solid surface or cells bearing either of these, an unlabeled test immunoglobulin, and a labeled reference immunoglobulin. Competitive inhibition is measured by determining the amount of label bound to the solid surface or cells in the presence of the test immunoglobulin. The test immunoglobulin is usually present in excess. Typically, when a competing antibody is present in excess, it inhibits the specific binding of the reference antibody to a common antigen by at least 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, or more.

[0175] Other techniques include epitope mapping methods such as X-ray analysis of crystals of antigen:antibody complexes, which provide atomic resolution of epitopes, and mass spectrometry combined with hydrogen / deuterium (H / D) exchange, which study the conformation and dynamics of antigen:antibody interactions. Other methods monitor the binding of antibody fragments or mutant forms of the antigen, where loss of binding due to alterations of amino acid residues within the antigen sequence is often considered an indication of epitope content. Additionally, combinatorial computational methods for epitope mapping can also be used. These methods rely on the ability of an antibody of interest to affinity isolate specific short peptides from a combination of phage-displayed peptide libraries. The peptides are then used as clues to define the epitope corresponding to the antibody, which is used to screen the peptide library. Computational algorithms for epitope mapping have also been developed that have been shown to map non-contiguous conformational epitopes.

[0176] As used herein, the term "Fc-mediated effector function" or "Fc effector function" refers to a biological activity of an antibody other than the primary function and target 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 cellular cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); lack of activation of platelets expressing Fc receptors; and B cell activation. Many effector functions are initiated by binding of Fc to Fcγ receptors. In some embodiments, antibodies targeting tumor antigens have effector functions, such as ADCC activity. In some embodiments, the antibodies targeting tumor antigens described herein comprise a variant constant region that has enhanced effector function (e.g., enhanced ability to mediate ADCC) compared to an unmodified version of the constant region.

[0177] As used herein, the term "Fc receptor" refers to a polypeptide found on the surface of immune effector cells that is bound by the Fc region of an antibody. In some embodiments, the Fc receptor is an Fcγ receptor. There are three subclasses of Fcγ receptors: FcγRI (CD64), FcγRII (CD32), and FcγRIII (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; therefore, antibody binding to this receptor does not activate complement and cellular responses. FcγRI is a high-affinity receptor that binds monomeric IgG, while FcγRIIA and FcγRIIA are low-affinity receptors that bind only multimeric IgG. Binding of antibodies to Fc receptors and / or C1q is directed by specific residues or domains within the Fc region. Binding also depends on residues present in the hinge region and CH2 portion of the antibody. In some embodiments, the agonistic 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 embodiments, the agonistic and / or therapeutic activity of the antibodies described herein is enhanced by binding of the Fc region to an Fc receptor (e.g., FcγR).

[0178] A list of specific Fc receptor sequences used in this disclosure is provided below as Table 13.

[0179] As used herein, the term "glycosylation pattern" is defined as the pattern of carbohydrate units covalently attached to a protein, more specifically, an immunoglobulin protein. If one skilled in the art recognizes that the glycosylation pattern of a heterologous antibody is more similar to the glycosylation pattern in a non-human transgenic animal species than to the glycosylation pattern in the species from which the transgenic CH gene is derived, the glycosylation pattern of the heterologous antibody can be characterized as substantially similar to the glycosylation pattern naturally present on antibodies produced by the non-human transgenic animal species.

[0180] As used herein, the term "human antibody" includes antibodies having variable and, if present, constant regions derived from 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 mutation in vivo) (see, e.g., Lonberg et al., (1994) Nature 368(6474):856-859; Lonberg, (1994) Handbook of Experimental Pharmacology 113:49-101; Lonberg & Huszar, (1995) Intern. Rev. Immunol. 13:65-93, and Harding & Lonberg, (1995) Ann. 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 (i.e., humanized antibodies).

[0181] As used herein, the term "heterologous antibody" is defined in relation to the transgenic non-human organism producing such an antibody. This term refers to an antibody found in an organism not consisting of the transgenic non-human animal, and generally has an amino acid sequence or its corresponding coding nucleic acid sequence derived from a species other than that of the transgenic non-human animal.

[0182] The terms "eliciting an immune response" and "enhancing an immune response" are used interchangeably and refer to the stimulation of an immune response (i.e., a passive or adaptive immune response) against a particular antigen. The term "eliciting" when used in reference to eliciting CDC or ADCC refers to the stimulation of a specific direct cell-killing mechanism.

[0183] As used herein, the term "immunogenic cell death" (alternatively known as "immunogenic apoptosis") refers to a mode of cell death associated with the activation of one or more signaling pathways that cause the expression and release of damage-associated molecular pattern (DAMP) molecules (e.g., adenosine triphosphate (ATP)) from tumor cells prior to cell death, thereby resulting in increased immunogenicity of the tumor cells and tumor cell death (e.g., by phagocytosis) in an immunogenic manner. As used herein, the term "immunogenic cell death-inducing agent" refers to a chemical, biological, or pharmacological agent that induces an immunogenic cell death process, pathway, or mode.

[0184] As used herein, the terms "inhibit," "reduce," or "block" (e.g., with respect to inhibiting or reducing human IL-27-mediated phosphorylation of STAT1 and / or STAT3 in a cell) are used interchangeably and include partial and complete inhibition / blocking. Inhibition / blocking of IL-27 reduces or alters the normal level or type of activity that occurs without inhibition or blockage. 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 compared to IL-27 not contacted with an anti-IL-27 antibody, e.g., inhibiting IL-27 binding 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%.

[0185] As used herein, the terms "inhibit angiogenesis," "reduce angiogenesis," and "reducing angiogenesis" refer to reducing the level of angiogenesis in a tissue by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or less compared to the amount in a corresponding control tissue, and most preferably to the same level observed in the control tissue.

[0186] As used herein, the term "inhibit growth" (e.g., with respect to a cell) is intended to include any measurable decrease in cell growth, e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100% inhibition of cell growth.

[0187] As used herein, a subject "in need of prevention," "in need of treatment," or "in need of" refers to one who, according to the judgment of an appropriate medical practitioner (e.g., a physician, nurse, or clinical nurse in the case of a human, or a veterinarian in the case of a non-human mammal), would reasonably benefit from a given treatment (e.g., treatment with a composition comprising an anti-IL-27 antibody).

[0188] The term "in vivo" refers to a process that occurs in a living organism.

[0189] As used herein, the term "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds 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 epitope may have cross-reactivity to other IL-27 proteins from different species. However, the antibody will continue to exhibit specific binding to human IL-27 in the specific binding assays described herein. In addition, an isolated antibody is typically substantially free of other cellular material and / or chemicals. In some embodiments, a combination of "isolated" antibodies with different IL-27 specificities is combined in a defined composition.

[0190] As used herein, the term "isolated nucleic acid molecule" refers to an antibody or antibody portion that binds IL-27 (e.g., V H , V L , CDR3) of the heavy chain variable region (V) of the anti-IL-27 monoclonal antibody described herein. The term "antibody" refers to a nucleic acid molecule encoding the heavy chain variable region (V) of the anti-IL-27 monoclonal antibody described herein, and is intended to refer to a nucleic acid molecule in which the nucleotide sequence encoding an antibody or antibody portion does not contain other nucleotide sequences encoding antibodies or antibody portions that bind to antigens other than IL-27, and such 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 may be selected from the heavy chain variable region (V) of the anti-IL-27 monoclonal antibody described herein. H ) and the light chain variable region (V L ) corresponds to a nucleotide sequence containing

[0191] As used herein, "isotype" refers to the antibody class (e.g., IgM or IgG1) encoded by the heavy chain constant region genes. In some embodiments, human monoclonal antibodies of the present disclosure are of the IgG1 isotype. In some embodiments, human monoclonal antibodies of the present disclosure are of the IgG2 isotype. In some embodiments, human monoclonal antibodies of the present disclosure are of the IgG3 isotype. In some embodiments, human monoclonal antibodies of the present disclosure are of the IgG4 isotype. As will be apparent to one of skill in the art, identifying antibody isotypes (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA1 IgA2, IgD, and IgE) is routine in the art and generally requires a combination of sequence alignment with known antibodies, published Fc variant sequences, and conserved sequences.

[0192] As used herein, the term "isotype switching" refers to the phenomenon in which the class or isotype of an antibody changes from one Ig class to another Ig class isotype.

[0193] As used herein, the terms "KD" or "K D " refers to the equilibrium dissociation constant of the binding reaction between an antibody and an antigen. D The value is a numerical expression of the ratio of the antibody's off-rate constant (kd) to the antibody's on-rate constant (ka). D The K value is inversely correlated with the binding affinity of the antibody for the antigen. D The smaller the value, the higher the affinity of the antibody for its antigen. Affinity is the strength of binding of a single molecule to its ligand and is typically measured as the equilibrium dissociation constant (K D ), which is used to assess and rank the strength of bimolecular interactions.

[0194] As used herein, the terms "Kd" or "k d (or "koff" or "k off") is intended to refer to the off-rate constant for dissociation of an antibody from an antibody / antigen complex. The kd value is a numerical expression of the rate at which the complex disintegrates or dissociates per second, sec -1 It is expressed in units of:

[0195] As used herein, the terms "ka" or "k a (or "kon" or "k on ") is intended to refer to the on-rate constant for the association of an antibody with an antigen. The ka value is a numerical expression of the number of antibody / antigen complexes formed per second in a 1 molar (1M) solution of antibody and antigen, and M -1 sec -1 It is expressed in units of .

[0196] As used herein, the term "leukocyte" refers to a type of white blood cell that is involved in defending the body against infectious organisms and foreign substances. Leukocytes are produced in the bone marrow. There are five main types of white blood cells, which are subdivided into two main groups: polymorphonuclear leukocytes (neutrophils, eosinophils, basophils) and mononuclear cells (monocytes and lymphocytes).

[0197] As used herein, the term "lymphocyte" refers to a type of white blood cell or leukocyte that is involved in the body's immune defenses. There are two main types of lymphocytes: B cells and T cells.

[0198] As used herein, the terms "linked," "fused," or "fusion" are used interchangeably. These terms refer to the joining of two or more elements or components or domains together by whatever means, including chemical conjugation or recombinant means. Methods of chemical conjugation (e.g., using heterobifunctional crosslinkers) are known in the art.

[0199] As used herein, " local administration " or " local delivery " refers to the delivery of composition or drug to intended target tissue or site without relying on transport through the vascular system.For example, composition can be delivered by injection or implantation of composition or drug, or by injection or implantation of device containing composition or drug.After local administration near target tissue or site, composition or drug or one or more components thereof can diffuse to intended target tissue or site.

[0200] As used herein, "MHC molecule" refers to two types of molecules, 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 processed to bind primarily to MHC class II. In contrast, antigens delivered endogenously to APCs are processed to bind primarily to MHC class I.

[0201] As used herein, the term "monoclonal antibody" refers to an antibody that exhibits binding specificity and affinity only for a particular epitope. Accordingly, the term "human monoclonal antibody" refers to an antibody that displays a single binding specificity and has variable and optionally constant regions derived from human germline immunoglobulin sequences. In some embodiments, human monoclonal antibodies are obtained from a transgenic non-human animal, e.g., a transgenic mouse, whose genome includes human heavy chain and light chain transgenes, and are produced by a hybridoma comprising a B cell fused with an immortalized cell.

[0202] As used herein, the term "monocyte" refers to a type of white blood cell that can differentiate into macrophages and dendritic cells to influence the immune response.

[0203] As used herein, the term "natural killer (NK) cells" refers to a type of cytotoxic lymphocyte. These 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 cellular cytotoxicity (ADCC).

[0204] As used herein, the term "naturally occurring" refers to the fact that the object can be found in nature when applied to an object.For example, a polypeptide or polynucleotide sequence that exists in an organism (including viruses) that can be isolated from a natural source and has not been intentionally modified by laboratory personnel is naturally occurring.

[0205] As used herein, the term "nonswitched isotype" refers to the class of heavy chain isotypes produced when isotype switching has not occurred. The CH gene encoding a nonswitched isotype is usually the first CH gene immediately downstream of the functionally rearranged VDJ gene. Isotype switching is classified as classical isotype switching or nonclassical isotype switching. Classical isotype switching occurs by recombination events involving at least one switch sequence region in the transgene. Nonclassical isotype switching is mediated by, for example, human σ μ and human Σ μ Alternative non-classical switching mechanisms, such as inter-transgene and / or inter-chromosomal recombination, can specifically initiate and activate isotype switching.

[0206] As used herein, the term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- 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 creating 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 is used interchangeably with gene, cDNA, and mRNA encoded by a gene.

[0207] As used herein, a polynucleotide can be composed of any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA, or modified RNA or DNA. For example, a polynucleotide can 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, or hybrid molecules containing DNA and RNA that may be single-stranded or, more typically, double-stranded, or that are a mixture of single-stranded and double-stranded regions. In addition, a polynucleotide can be composed of RNA, or DNA, or triple-stranded regions containing both RNA and DNA. A polynucleotide can also contain one or more modified bases or modified DNA or RNA backbones for stability or other reasons. "Modified" bases include, for example, tritylated bases and unusual bases such as inosine. Various modifications can be made to DNA and RNA. Thus, "polynucleotide" encompasses chemically, enzymatically, or metabolically modified forms.

[0208] A nucleic acid is "operably linked" when it is placed into 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. With respect to transcriptional regulatory sequences, operably linked means that the DNA sequences being linked are contiguous and, where necessary to join two protein-coding regions, contiguous and in reading frame. With respect to switch sequences, operably linked indicates that the sequences are capable of effecting switch recombination.

[0209] As used herein, "parenteral administration," "parenterally administered," and other grammatically equivalent phrases refer to modes of administration other than enteral and topical administration, typically by injection, including, but not limited to, intravenous, intranasal, intraocular, intramuscular, intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intrathecal, intrathecal, epidural, intracerebral, intracranial, intracarotid, and intrasternal injection and infusion.

[0210] As used herein, the term "patient" includes human and other mammalian subjects receiving either prophylactic or therapeutic treatment.

[0211] As used herein, the term "PD-1 antagonist" refers to any compound or biomolecule that inhibits the PD-1 signaling pathway in a cell (e.g., an immune cell) or otherwise inhibits the function of PD-1. In some embodiments, the PD-1 antagonist blocks the binding of PD-L1 to PD-1 and / or the binding of PD-L2 to PD-1. In some embodiments, the PD-1 antagonist specifically binds to PD-1. In some embodiments, the PD-1 antagonist specifically binds to PD-L1.

[0212] 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 nucleotides or amino acid residues that are identical when compared and aligned for maximum correspondence, as determined using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to those of skill in the art) or by visual inspection. Depending on the application, the "percent identity" can be over a region of the sequences being compared, such as 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 test sequences are compared. When using a sequence comparison algorithm, the test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence(s) relative to the reference sequence, based on the designated program parameters.

[0213] Optimal sequence alignment 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 computer implementations of these 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., infra).

[0214] One example of an algorithm that is 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.

[0215] As generally used herein, "pharmaceutically acceptable" refers to compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues, organs, and / or body fluids of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0216] As used herein, "pharmaceutically acceptable carrier" refers to and includes any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. The compositions may include pharmaceutically acceptable salts, such as acid addition salts or base addition salts (see, e.g., Berge et al. (1977) J Pharm Sci 66:1-19).

[0217] As used herein, the terms "polypeptide," "peptide," and "protein" are used interchangeably to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of a corresponding naturally occurring amino acid, as well as to naturally occurring and non-naturally occurring amino acid polymers.

[0218] As used herein, the term "prevention," when used in reference to a condition, refers to the administration of a composition that reduces the frequency of or delays the onset of symptoms of a medical condition in a subject compared to a subject not administered the composition.

[0219] As used herein, the terms "purified" or "isolated" when applied to any of the proteins (antibodies or fragments) described herein refer to a polypeptide that has been separated or purified from components that naturally accompany it (e.g., proteins or other naturally occurring biomolecules or organic molecules), e.g., other proteins, lipids, and nucleic acids in prokaryotes that express the protein. Typically, a polypeptide is purified if it constitutes at least 60% by weight (e.g., at least 65, 70, 75, 80, 85, 90, 92, 95, 97, or 99% by weight) of the total protein in a sample.

[0220] As used herein, the term "programmed cell death protein 1" or "PD-1" refers to the programmed cell death protein 1 polypeptide, an immunosuppressive receptor belonging to the CD28 family, which in humans is encoded by the PDCD1 gene. Alternative names or synonyms of PD-1 include: PDCD1, PD1, CD279, and SLEB2. PD-1 is primarily expressed in vivo on preactivated T cells, B cells, and myeloid cells 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 other species homologs of hPD-1, as well as analogs that share at least one shared epitope with hPD-1. The full-length hPD-1 sequence can be found under GenBank accession number AAC51773.

[0221] As used herein, the term "programmed death-ligand-1" or "PD-L1" refers to one of two cell surface glycoprotein ligands for PD-1 (the other being PD-L2) that downregulates T cell activation and cytokine secretion upon 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 other species homologs of hPD-L1, and analogs that share at least one shared epitope with hPD-L1. The full-length hPD-L1 sequence can be found under GenBank accession number Q9NZQ7.

[0222] 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 may act as an immune checkpoint, which may result in a reduction 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). Immune suppression can be reversed by inhibiting the local interaction of PD-1 with PD-L1 or PD-L2, and this 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).

[0223] For some cancers, tumor survival and growth are maintained by tumor-mediated immune checkpoint modulation. This modulation can lead to the disruption of the immune system's anti-cancer functions. For example, recent studies have shown that the expression of immune checkpoint receptor ligands, such as PD-L1 or PD-L2, by tumor cells can downregulate the activity of the immune system in the tumor microenvironment, particularly by suppressing T cells, thereby promoting cancer immune evasion. PD-L1 is abundantly expressed by various human cancers (Dong et al., (2002) Nat Med 8:787-789). PD-1, the receptor for PD-L1, is expressed on lymphocytes (e.g., activated T cells) and is normally involved in downregulating the immune system and promoting self-tolerance, particularly by suppressing T cells. However, when PD-1 receptors expressed on T cells bind to their cognate PD-L1 ligands on tumor cells, the resulting T-cell suppression contributes to impaired immune responses to tumors (e.g., reduction of tumor-infiltrating lymphocytes or the occurrence of immune evasion by cancer cells).

[0224] For example, in large sample sets of ovarian, renal, colorectal, pancreatic, liver, and melanoma cancers, PD-L1 expression has been shown to correlate with poor prognosis and reduce overall survival regardless of subsequent treatment (e.g., Dong et al., (2002) Nat Med 8(8):793-800; Yang et al., (2008) Invest Ophthalmol Vis Sci 49(6):2518-2525; Ghebeh et al., (2006) Neoplasia 8:190-198; Hamanishi et al., (2007) Proc Nat Acad Sci USA 104:3360-3365;Thompson et al.,(2006) Clin Genitourin Cancer 5:206-211;Nomi et al.,(2005) Clin Cancer Res 11:2947-2953;Inman et al.,(2007) Cancer 109:1499-1505;Shimauchi et al. al.,(2007) Int J Cancer 121:2585-2590; Gao et al., (2009) Clin Cancer Res 15:971-979; Nakanishi et al., (2007) Cancer Immunol Immunother 56:1173-1182; Hino et al., (2010) Cancer 116(7):1757-1766). Similarly, PD-1 expression on tumor lymphocytes has been shown to characterize dysfunctional T cells in breast cancer (Kitano et al., (2017) ESMO Open 2(2):e000150) and melanoma (Kleffel et al., (2015) Cell 162(6):1242-1256). For example, 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 suppressors that function by modulating immune cell-tumor cell interactions.

[0225] As used herein, the term "rearranged" refers to a V segment that is a nearly complete V H or V L This refers to the configuration of a heavy or light chain immunoglobulin locus in which the DJ or J segments, respectively, are located immediately adjacent to the DJ or J segments in an arrangement that encodes the domains. Rearranged immunoglobulin loci can be identified by comparison with germline DNA. Rearranged loci have at least one recombined heptamer / nonamer homology element.

[0226] 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 such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in subsequent generations due to either mutation or environmental influences, such progeny may not actually be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein.

[0227] 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) transgenic or transchromosomal for human immunoglobulin genes or hybridomas prepared from such animals, (b) antibodies isolated from host cells, e.g., transfectomas, transformed to express the antibody, (c) antibodies isolated from the combination of recombinant human antibody libraries, and (d) antibodies prepared, expressed, generated, or isolated by any other means, including splicing human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies contain variable and constant regions that utilize specific human germline immunoglobulin sequences encoded by germline genes, but also 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), variable regions contain antigen-binding domains, which are encoded by different genes that undergo rearrangement to form antibodies specific to foreign antigens. In addition to rearrangement, variable regions can be further modified by multiple single amino acid changes (termed somatic mutation or hypermutation) to increase the affinity of the antibody for the foreign antigen. The constant regions change in further response to antigen (i.e., isotype switching). Thus, nucleic acid molecules rearranged and somatically mutated in response to antigen that encode light and heavy chain immunoglobulin polypeptides need not share sequence identity with the original nucleic acid molecules, but instead are substantially identical or similar (i.e., have at least 80% identity).

[0228] As used herein, the term "reference antibody" (used interchangeably with "reference mAb") or "reference antigen-binding protein" refers to an antibody or antigen-binding fragment thereof that binds to a specific epitope on human IL-27 and is used to establish a relationship between itself and one or more different antibodies, where the relationship is the binding of the reference antibody and the one or more different antibodies to the same epitope on IL-27. As used herein, the term also refers to an anti-IL-27 antibody that is useful as a competitor in tests or assays such as those described herein (e.g., competitive binding assays), where the assays are useful for discovering, identifying, or developing one or more different antibodies that bind to the same epitope.

[0229] As used herein, the terms "specific binding," "selective binding," "selectively binds," and "specifically binds" refer to an antibody that binds to an epitope on a predetermined antigen. Typically, the antibody binds to an epitope of about 10 ng / mL as determined by surface plasmon resonance (SPR) technology on a BIACORE 2000 instrument using recombinant human IL-27 as the analyte and the antibody as the ligand. -6 Less than m, e.g., about 10 -7 Under M, 10 -8 Under M, 10 -9 Less than M or 10 -10 The equilibrium dissociation constant (K D ) or even lower, and binds to a predetermined antigen with an affinity that is at least two-fold higher than the affinity of binding to a nonspecific antigen other than the predetermined antigen or a closely related antigen (e.g., BSA, casein). In certain embodiments, an antibody that specifically binds to IL-27 has an affinity of about 100 nM (10) as determined by surface plasmon resonance (SPR) technology on a BIACORE 2000 instrument using recombinant human IL-27 as the analyte and the antibody as the ligand. -7 M), optionally less than about 50 nM (5 × 10 -8 M), optionally less than about 15 nM (1.5 × 10 -8 M), optionally less than about 10 nM (10 -8 M), optionally less than about 5 nM (5 × 10 -9 M), optionally less than about 1 nM (10-9 M), optionally less than about 0.1 nM (10 -10 M), optionally less than about 0.01 nM (10 -11 M) D ), or an even lower equilibrium dissociation constant, where binding to a given antigen occurs with an affinity that is at least two-fold higher than the affinity of the antibody for binding to a nonspecific antigen other than the given antigen or a closely related antigen (e.g., BSA, casein). The phrases "antibody that recognizes an antigen" and "antibody specific for an antigen" are used interchangeably herein with the term "antibody that specifically binds to an antigen."

[0230] As used herein, the term "STAT1 phosphorylation" refers to the phosphorylation of the signal transducer and activator of transcription 1 (STAT1) polypeptide, a transcription factor encoded by the STAT1 gene in humans. STAT molecules are phosphorylated by receptor-associated kinases, which cause activation and dimerization by forming homo- or heterodimers, which translocate to the nucleus and function as transcription factors. STAT1 can be activated (i.e., phosphorylated) in response to signal transduction via several ligands, including IL-27. IL-27 signaling via IL-27R results in the phosphorylation of STAT1 (pSTAT1). STAT1 plays an important role in the expression of genes involved in cell survival, viability, or pathogen response. Methods for measuring STAT1 phosphorylation as a result of IL-27 signaling include, but are not limited to, flow cytometry analysis of cells labeled with an antibody that specifically recognizes phosphorylated STAT1 (see, e.g., Tochizawa et al., (2006) J Immunol Methods 313(1-2):29-37).

[0231] As used herein, the term "STAT3 phosphorylation" refers to the phosphorylation of the signal transducer and activator of transcription 3 (STAT3) polypeptide, a transcription factor encoded by the STAT3 gene in humans. STAT3 mediates the expression of various genes in response to cellular stimuli and thus plays an important role in numerous cellular processes, such as cell proliferation and apoptosis. Methods for measuring STAT3 phosphorylation as a result of IL-27 signaling include, but are not limited to, flow cytometry analysis of cells or cell extracts labeled with an antibody that specifically recognizes phosphorylated STAT3 (see, e.g., Fursov et al., (2011) Assay Drug Dev Technol 9(4):420-429).

[0232] As used herein, the term "switch sequence" refers to a DNA sequence that is responsible for switch recombination. A "switch donor" sequence, typically a μ switch region, is located 5' (i.e., upstream) of the construct region that is removed during switch recombination. A "switch acceptor" region is located between the construct region that is removed and the replacement constant region (e.g., γ, ε, etc.). Because there are no specific sites where recombination will always occur, the final gene sequence is usually not predictable from the construct.

[0233] 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 subjects with immune deficiencies. 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.

[0234] With respect to nucleic acids, the term "substantial homology" indicates that two nucleic acids, or their designated sequences, when optimally aligned and compared, are identical in at least about 80% of the nucleotides, usually at least about 90%-95% of the nucleotides, and more preferably at least about 98%-99.5% of the nucleotides, with appropriate nucleotide insertions or deletions. Alternatively, substantial homology exists when the segments will hybridize under selective hybridization conditions to the complement of the strand.

[0235] The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology = # of identical positions / total # of positions x 100), and takes into account the number of gaps and the length of each gap that need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of the percent identity between two sequences can be performed using a mathematical algorithm, such as that described in the non-limiting examples below.

[0236] The percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package (available at http: / / www.gcg.com) using the NWSgapdna.CMP matrix and gap weights of 40, 50, 60, 70, or 80, and length weights 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)) as 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. Furthermore, the percent identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) incorporated into the GAP program of the GCG software package (available at http: / / www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0237] Furthermore, the nucleic acid and protein sequences of the present disclosure can be used as "query sequences" to perform searches against public databases, for example, 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. To obtain nucleotide sequences homologous to the nucleic acid molecules of the present invention, BLAST nucleotide searches can be performed with the NBLAST program, score = 100, word length = 12. To obtain amino acid sequences homologous to the protein molecules of the present invention, BLAST protein searches can be performed with the XBLAST program, score = 50, word length = 3. To obtain gapped alignments for comparison purposes, Gapped BLAST can be performed with the NBLAST program, score = 100, word length = 12. Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of each program (e.g., XBLAST and NBLAST) can be used. See http: / / www.ncbi.nlm.nih.gov.

[0238] Nucleic acids may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. Nucleic acids are "isolated" or "substantially pure" when purified from other cellular components or other contaminants, such as other cellular nucleic acids or proteins, by standard techniques, including alkaline / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and others well known in the art. See F. Ausubel, et al., eds., Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987).

[0239] The nucleic acid compositions of the present disclosure are often naturally occurring sequences (excluding modified restriction sites, etc.) derived from cDNA, genomic, or mixtures thereof, but can be mutated by standard techniques to obtain the gene sequence. With respect to coding sequences, these mutations can have a desired effect on the amino acid sequence. Specifically contemplated are DNA sequences that are substantially homologous to or derived from naturally occurring V, D, J, constant, and switch sequences, as well as other such sequences described herein (where "derived" indicates that the sequence is identical to or modified from another sequence).

[0240] As used herein, the term "STING" (or TMEM173) refers to stimulator of interferon genes, a protein that functions 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. STING induces type I interferon production when cells are infected with intracellular pathogens, such as viruses, mycobacteria, and intracellular parasites. STING-mediated type I interferon protects infected cells and nearby cells from local infection by binding to the same cell that secretes it and nearby cells. An exemplary amino acid sequence of STING is provided in the NCBI Genbank database under accession number NP_001288667.

[0241] 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 T cell receptors on the cell surface. There are several subsets of T cells, including but not limited to T helper cells (T H cells or CD4 + T cells) and T H 1. T H 2. T H 3. T H 17, T H 9 and T FH The subtype of T cells includes cytotoxic T cells (T C cells, CD8+ T cells, also known as cytotoxic T lymphocytes, T killer cells, killer T cells), memory T cells and central memory T cells (T CM cells), effector-memory T cells (T EM and T EMRA cells) and resident memory T cells (T RM cells), including its subtype, regulatory T cells (T reg T cells (also known as suppressor T cells) and CD4 + FOXP3 + T reg cells, CD4 + FOXP3 - T reg cells, Tr1 cells, Th3 cells and T reg 17 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 above-mentioned T cells or T cells not mentioned may be the target cell type for the methods of use of the present invention.

[0242] As used herein, the term "T cell-mediated response" includes, but is not limited to, effector T cells (e.g., CD8 + cells) and helper T cells (e.g., CD4 + The term "T cell-mediated response" refers to any response mediated by T cells, including T cells. T cell-mediated responses include, for example, cytotoxicity and proliferation of T cells.

[0243] As used herein, the term "therapeutically effective amount" or "therapeutically effective dose" or similar terms used herein is intended to mean an 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., amelioration of one or more symptoms of cancer).

[0244] 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 cancer, TAM receptors have a dual regulatory role: controlling the initiation and progression of tumor development and, concomitantly, the associated anti-tumor response by various immune cells. Further description of TAM receptors is provided 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 a TAM receptor.

[0245] As used herein, the term "TIGIT" or "T cell immunoreceptor having Ig and ITIM domains" refers to any native TIGIT from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise specified. TIGIT is also known in the art as DKFZp667A205, FLJ39873, V-set and immunoglobulin domain-containing protein 9, V-set and transmembrane domain-containing protein 3, VSIG9, VSTM3, and WUCAM. 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.

[0246] As used herein, the terms "treat," "treating," and "treatment" refer to therapeutic or prophylactic measures as described herein. "Treatment" methods employ administration of a human antibody of the present disclosure to a subject in need of such treatment, e.g., a subject in need of an enhanced immune response to a particular antigen, or a subject likely to eventually acquire such a disorder, to prevent, cure, delay, reduce the severity of, or ameliorate one or more symptoms of a disorder or a recurrent disorder, or to extend the subject's survival beyond that expected in the absence of such treatment.

[0247] As used herein, the term "tumor microenvironment" (or "cancer microenvironment"; abbreviated as TME) refers to the cellular environment or surroundings in which a tumor or neoplasm resides, 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 the extracellular matrix also comprise the TME. Tumors and the surrounding microenvironment are closely associated and constantly interact. While tumors can influence the microenvironment by releasing extracellular signals, promoting tumor angiogenesis, and inducing peripheral immune tolerance, immune cells in the microenvironment can influence the growth and evolution of tumor cells.

[0248] As used herein, the term "unrearranged" or "germline configuration" refers to a configuration in which a V segment has not recombined directly adjacent to a D or J segment.

[0249] 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 "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, into which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" or simply "expression vectors." In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" can be used interchangeably, as the plasmid is the most commonly used form of vector. However, the invention is intended to include other forms of expression vectors that serve equivalent functions, such as viral vectors (e.g., replication defective retroviruses, adenoviruses, and adeno-associated viruses).

[0250] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the methods and compositions of this disclosure, preferred methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. [Brief explanation of the drawings]

[0251] [Figure 1]

[0023] Figure 1 shows a table providing affinity data for the indicated anti-IL-27 antibodies. Affinity measurements were performed using ForteBio and Meso Scale Discovery methods. [Figure 2] Binding of the indicated anti-IL-27 antibodies to plate-bound recombinant IL-27 as measured by ELISA is shown. [Figure 3A] 1 shows a bar graph depicting inhibition of IL-27-mediated phosphorylation of STAT1 in human whole blood by the indicated anti-IL-27 antibodies as measured by flow cytometry. [Figure 3B] 1 shows a graph depicting inhibition of IL-27-mediated phosphorylation of STAT1 in human PBMCs by the indicated anti-IL-27 antibodies as measured by flow cytometry. [Figure 3C] 1 shows a graph depicting inhibition of IL-27-mediated phosphorylation of STAT1 in U937 cells by the indicated anti-IL-27 antibodies, as measured by flow cytometry. [Figure 3D] 1 shows a bar graph depicting inhibition of IL-27-mediated phosphorylation of STAT1 in HUT-78 cells by the indicated anti-IL-27 antibodies as measured by flow cytometry. [Figure 3E] 1 shows a graph demonstrating that SRF388 inhibits IL-27-mediated pSTAT1 in human whole blood T cells. [Figure 4] 1 shows reversal of IL-27-mediated inhibition of CD161 expression in T cells by various concentrations of the indicated anti-IL-27 antibodies. CD161 expression was determined using flow cytometry. [Figure 5A] Figure 1 shows the extent to which anti-IL-27 antibodies enhance PD-1-mediated TNFα secretion in human PBMCs, as measured by ELISA. [Figure 5B] 1 shows the extent to which anti-IL-27 antibodies enhance PD-1-mediated IL-6 secretion in human PBMCs, as measured by ELISA. [Figure 5C]FIG. 1 shows dot plots demonstrating that SRF388 in combination with PD-1 blockade results in increased cytokine production in PBMCs from healthy donors and patients with RCC (abbreviations: CBA = cytometry bead array, IFNγ = interferon gamma, MSD = Meso Scale Discovery, PBMC = peripheral blood mononuclear cells, PD-1 = programmed death receptor 1, RCC = renal cell carcinoma, TNFα = tumor necrosis factor alpha). [Figure 5D] Figure 1 shows that IL-27 inhibits cytokine production after PD-1 blockade and that in combination with SRF388 cytokine production is restored (Abbreviations: Ctrl = control, ns = not significant, PBMC = peripheral blood mononuclear cells, rhIL-27 = recombinant human IL-27). [Figure 5E-1] For various cell types as indicated, the observed cytokine induction (specifically, TNFα, IFNγ, IL-6, and IL-17A) in PBMC cultures is shown when such cells are contacted with SRF388 antibody, αPD-1 antibody, or a combination of SRF388 and αPD-1 antibodies. [Figure 5E-2] Same as above. [Figure 5F] The effect of different concentrations of the indicated individual antibodies (SRF405, SRF410, SRF411, SRF414, SRF416, SRF536, SRF543, SRF529, SRF381, SRF388, and Ab7) on pSTAT1 signaling in U937 (lymphoma) cells is shown. [Figure 5G] The effect of different concentrations of these individual antibodies on pSTAT1 signaling in PBMCs (peripheral blood mononuclear cells) is shown. [Figure 5H] The effect of different concentrations of these individual antibodies on CD161 signaling in PBMCs (peripheral blood mononuclear cells) is shown. [Figure 5I] The effect of different concentrations of these individual antibodies (except SRF414) on PD-L1 signaling in CD4 T lymphocytes (CD4 cells) is shown. [Figure 5J]The effect of different concentrations of these individual antibodies (except SRF529) on PD-L1 signaling in monocytes is shown. [Figure 5K] The effect of different concentrations of each of these antibodies (except SRF529) on TIM-3 signaling in monocytes is shown. [Figure 6A] Figure 1 shows inhibition of IL-27-mediated PD-L1 expression by treatment of human monocytes with anti-IL-27 antibodies, as measured by flow cytometry. [Figure 6B] Figure 1 shows dose-dependent inhibition of IL-27-mediated PD-L1 expression by treatment of human monocytes with various concentrations of an anti-IL-27 antibody that specifically binds to EBI3 monomer, as measured by flow cytometry. [Figure 6C] Figure 1 shows the inhibition of IL-27-mediated TIM3 expression by treatment of human monocytes with anti-IL-27 antibody, as measured by flow cytometry. [Figure 6D] Figure 1 shows inhibition of IL-27-mediated PD-L1 expression by treatment of human resting T cells with anti-IL-27 antibodies, as measured by flow cytometry. [Figure 7A] 1 shows dot plots depicting the number of lung surface B16F10 metastatic nodules (pulmonary nodules) in B16F10 tumor-bearing mice treated with the indicated anti-IL27 antibody (SRF388), isotype control antibody, αWSX-1 antibody, or a combination of αPD-1 and αCTLA-4 antibodies, as determined by visual counting of lung nodules isolated from mice. [Figure 7B] Figure 1 shows the growth kinetics of bioluminescent B16-Luc tumors in mice treated with anti-IL-27 antibody (SRF388) or isotype control antibody, as measured by bioluminescence imaging analysis. [Figure 7C] Shown is a series of images of lung tissue isolated, fixed, sectioned, and stained with hematoxylin and eosin from B16F10 tumor-bearing mice treated with the indicated anti-IL27 antibody (SRF388), an isotype control antibody, αWSX-1 antibody, or a combination of αPD-1 and αCTLA-4 antibodies. [Figure 7D]Dot plots showing total tumor area as a percentage of total tissue area, as measured by image analysis software, are shown for B16F10 tumor tissue isolated, fixed, sectioned, and stained with hematoxylin and eosin from mice bearing B16F10 tumors treated with the indicated anti-IL-27 antibody (SRF388), isotype control antibody, αWSX-1 antibody, or a combination of αPD-1 and αCTLA-4 antibodies. Similar reductions in the number of lung surface metastases and total tumor area were observed with anti-IL-27RA (WSX-1) antibody-mediated blockade and with anti-PD-1 plus anti-CTLA-4 combination therapy. [Figure 8A] A scatter plot showing microarray data of genes with a log2 fold change in expression (black circles) >1.0 in splenocytes isolated from mice overexpressing IL-27 after treatment with IL-27 minicycles is shown. [Figure 8B] Expression levels of selected immunoregulatory genes as indicated in splenocytes as in Figure 8A are shown. [Figure 8C] These figures show that ectopic expression of human IL-27 induces inhibitory receptor expression on mouse T cells in vivo, and that SRF388 reduces inhibitory receptor expression on T cells in vivo after IL-27 minicycle treatment. Six-week-old female Balb / c mice were injected with empty vector (control) or hIL-27 minicycles. PBMCs (upper left and right panels) and total splenocytes (lower left and right panels) were harvested 5 days after transfection, and the cells were stained and analyzed by flow cytometry. Expression of the indicated markers was analyzed for CD4+ T cells (upper left and lower left panels) and CD8+ T cells (upper right and lower right panels). Analysis was performed using FlowJo software. [Figure 8D] Figure 1 shows that SRF388 inhibits the detection of minicycle-derived human IL-27 in mouse plasma. [Figure 9-1] A tabular summary of the properties of selected monoclonal antibodies is provided. [Figure 9-2] Same as above. [Figure 10A-1]A table of antibody sequences is shown with sequence divisions reflecting NT numbering. In both Figures 10A and 10B, highlighted amino acids in the CDR sequences indicate variations from the germline-encoded sequence. As will be apparent to one of skill in the art, antibody numbering, including determination of CDR sequences, framework sequences, etc., can be performed in a number of art-recognized ways, including by the NT and IMGT numbering systems shown in Figures 10A and 10B and used elsewhere herein. [Figure 10A-2] Same as above. [Figure 10A-3] Same as above. [Figure 10A-4] Same as above. [Figure 10B-1] A table of antibody sequences (corresponding to the sequence listing in Figure 10A) is shown, with sequence divisions reflecting ImMunoGeneTics (IMGT) numbering. In both Figures 10A and 10B, highlighted amino acids in the CDR sequences indicate variations from the germline-encoded sequence. As will be apparent to one of skill in the art, antibody numbering, including determination of CDR sequences, framework sequences, and the like, can be performed in a number of art-recognized ways, including by the NT and IMGT numbering systems shown in Figures 10A and 10B and used elsewhere herein. [Figure 10B-2] Same as above. [Figure 10B-3] Same as above. [Figure 10B-4] Same as above. [Figure 11](A) Dot plots showing serum levels of EBI3 or IL-27 in patients with cancer or healthy controls. Serum samples were tested for EBI3 (A) or IL-27 (B) levels using the antibody pair described in Example 15. Values ​​were extrapolated using a standard curve using recombinant human IL-27. The mean + 2SD of healthy donors (normal controls) was used as an arbitrary cutoff value for serum samples to be considered positive. The total number of positive samples is shown above each cancer classification. (B) Dot plots showing that, using the same parameters as described for (A), several ovarian cancer samples showed detectable levels of IL-27 in serum, which was also found in serum from three patients with B-cell lymphoma and one patient with endometrial cancer. [Figure 12A] 1 shows a graph demonstrating that the combination of anti-p28 capture antibody SRF381 and anti-EBI3 antibody Ab7 detects recombinant human IL-27 by MSD immunoassay in an IL-27 concentration-dependent manner. [Figure 12B] 1 shows a graph demonstrating that the combination of anti-EBI3 capture antibody SRF557 and anti-EBI3 antibody Ab7 detects recombinant human IL-27 by MSD immunoassay in an IL-27 concentration-dependent manner. [Figure 12C] 1 shows a graph showing the concentration of EBI3 in 174 serum samples from cancer patients (renal cell carcinoma (RCC), ovarian cancer, hepatocellular carcinoma (HCC), acute myeloid leukemia (AML), diffuse large B-cell lymphoma (DLBCL), sarcoma, melanoma, head and neck squamous cell carcinoma (HNSCC), Hodgkin's lymphoma, gastric cancer, endometrial cancer) and healthy (normal) patients as measured by the Meso Scale Discovery (MSD) assay. [Figure 12D]1 shows a graph showing the concentration of IL-27 in 174 serum samples from cancer patients (renal cell carcinoma (RCC), ovarian cancer, hepatocellular carcinoma (HCC), acute myeloid leukemia (AML), diffuse large B-cell lymphoma (DLBCL), sarcoma, melanoma, head and neck squamous cell carcinoma (HNSCC), Hodgkin's lymphoma, gastric cancer, endometrial cancer) and healthy (normal) patients as measured by the Meso Scale Discovery (MSD) assay. [Figure 12E] 1 shows a bar graph demonstrating that anti-IL-27 antibodies can be combined with SRF381 to detect recombinant human IL-27 by MSD immunoassay. [Figure 12F] 1 shows a bar graph demonstrating that anti-IL-27 antibodies can be combined with Ab7 to detect recombinant human IL-27 by ELISA. DETAILED DESCRIPTION OF THE INVENTION

[0252] The present disclosure provides, at least in part, antibody molecules that bind to human IL-27 with high affinity and high specificity. In one embodiment, human antibodies that bind to IL-27 are disclosed herein. As used herein, the terms "IL-27" and "IL27" refer interchangeably to IL-27, a heterodimeric cytokine composed of two distinct subunits encoded by two distinct genes: Epstein-Barr virus-induced gene 3 (EBI3) and IL-27p28. IL-27 has both pro- and anti-inflammatory properties and diverse effects on hematopoietic and non-hematopoietic cells.

[0253] Accordingly, in one aspect, the present disclosure provides a monoclonal antibody or antigen-binding portion thereof that specifically binds to and antagonizes human IL-27, wherein the antibody or antigen-binding portion thereof exhibits at least one or more of the following properties: (i) an equilibrium dissociation constant (K) of 15 nM or less D ) binding to human IL-27; (ii) blocking the binding of IL-27 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; (vi) inducing or enhancing PD-1-mediated secretion of one or more cytokines from the cell; and (vii) A combination of (i) to (vi).

[0254] In another aspect, the disclosure provides a monoclonal antibody or antigen-binding portion thereof that specifically binds to human IL-27 and inhibits or reduces IL-27 biological activity or IL-27 signaling.

[0255] Additional aspects of the present invention include nucleic acid molecules encoding the antibody molecules, expression vectors, host cells, and methods for producing the 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 cancer or malignant diseases, such as solid tumors and liquid tumors (e.g., leukemia, e.g., lymphoma, e.g., AML), lung cancer (e.g., non-small cell lung cancer), pancreatic cancer, breast cancer (e.g., triple-negative breast cancer), melanoma, testicular cancer, sarcoma, head and neck cancer (e.g., head and neck squamous cell carcinoma), liver cancer (e.g., hepatocellular carcinoma (HCC)), colorectal cancer, ovarian cancer, brain cancer (e.g., glioblastoma multiforme), or kidney cancer (e.g., renal cell carcinoma (e.g., renal clear cell carcinoma)).

[0256] Anti-IL-27 antibodies and antigen-binding fragments thereof The present disclosure provides antibodies and antigen-binding portions thereof that specifically bind to and antagonize IL-27, particularly human IL-27. Provided herein are isolated monoclonal antibodies, or antigen-binding portions thereof, that specifically bind to human IL-27, comprising the heavy and light chain CDRs and variable sequences set forth in Table 12.

[0257] In some embodiments, the present disclosure provides isolated monoclonal antibodies or antigen-binding portions thereof that specifically bind to and antagonize human IL-27, wherein the antibodies or antigen-binding portions thereof exhibit at least one or more of the following properties: (i) an equilibrium dissociation constant (K) of 15 nM or less. D (ii) blocking the binding of IL-27 to the IL-27 receptor; (iii) inhibiting or reducing STAT1 and / or STAT3 phosphorylation in the cell; (iv) inhibiting or reducing the inhibition of CD161 expression in the cell; (v) inhibiting or reducing PD-L1 and / or TIM-3 expression in the cell; (vi) inducing or enhancing PD-1-mediated secretion of one or more cytokines from the cell; and (vii) a combination of (i)-(vi).

[0258] In some embodiments, the isolated monoclonal antibody or antigen-binding portion thereof has an equilibrium dissociation constant (K D ) binds to human IL-27.

[0259] In some embodiments, the isolated monoclonal antibody or antigen-binding portion thereof binds to recombinant human IL-27 or to murine IL-27.

[0260] In some embodiments, the isolated monoclonal antibody or antigen-binding portion thereof inhibits or reduces STAT1 and / or STAT3 phosphorylation in a cell. In some embodiments, the cell is an immune cell. In some embodiments, the cell is a cancer cell.

[0261] In some embodiments, the isolated monoclonal antibody or antigen-binding portion thereof inhibits or reduces inhibition of CD161 expression in the cell (e.g., ameliorates or alleviates inhibition of CD161 expression in the cell). In some embodiments, the cell is an immune cell.

[0262] In some embodiments, the isolated monoclonal antibody, or antigen-binding portion thereof, inhibits or reduces PD-L1 and / or TIM-3 expression in a cell. In some embodiments, PD-L1 expression is inhibited or reduced. In some embodiments, TIM-3 expression is inhibited or reduced. In some embodiments, both PD-L1 expression and TIM-3 expression are reduced. In some embodiments, the cell is an immune cell.

[0263] In some embodiments, the isolated monoclonal antibody, or antigen-binding portion thereof, induces or enhances PD-1-mediated secretion of one or more cytokines from a cell. In some embodiments, the one or more cytokines are TNFα. In some embodiments, the one or more cytokines are IL-6. In some embodiments, the one or more cytokines are TNFα and IL-6. In some embodiments, the cell is an immune cell.

[0264] In some embodiments, the isolated monoclonal antibody, or antigen-binding portion thereof, is selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE antibodies. In some embodiments, the antibody is an IgG1 antibody or an IgG4 antibody. In some embodiments, the antibody comprises a wild-type IgG1 heavy chain constant region. In some embodiments, the antibody comprises a wild-type IgG4 heavy chain constant region. In some embodiments, the antibody comprises an Fc domain comprising at least one mutation. In some embodiments, the antibody comprises a mutated IgG1 heavy chain constant region. In some embodiments, the antibody comprises a mutated IgG4 heavy chain constant region. In some embodiments, the mutated IgG4 heavy chain constant region comprises either a S228P substitution, a L235E substitution, a L235A substitution, or a combination thereof, according to EU numbering.

[0265] In some embodiments, the present disclosure provides an isolated monoclonal antibody, or antigen-binding portion thereof, that binds to substantially the same epitope on IL-27 as the antibody, or antigen-binding portion thereof, of any one of the preceding embodiments.

[0266] In some embodiments, the present disclosure provides an isolated monoclonal antibody, or antigen-binding portion thereof, that binds to at least one of the amino acid residues comprising IL-27, which is bound by the antibody, or antigen-binding portion thereof, of any one of the preceding embodiments.

[0267] In some embodiments, the present disclosure provides an isolated monoclonal antibody or antigen-binding portion thereof, wherein a mutation in the epitope on IL-27 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 of any one of the preceding embodiments.

[0268] In some embodiments, the present disclosure provides isolated monoclonal antibodies or antigen-binding portions thereof that bind to an epitope on IL-27, wherein the epitope is the same as or similar to an epitope bound by an antibody molecule described in Table 12.

[0269] In some embodiments, the present disclosure provides an isolated monoclonal antibody or antigen-binding portion thereof that specifically binds to and antagonizes human IL-27, wherein the antibody or antigen-binding portion thereof comprises heavy and light chain CDRs selected from the group consisting of: (i) heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 706, 707, and 708, respectively, and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 714, 715, and 716, respectively; (ii) heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 728, 729, and 730, respectively, and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 736, 737, and 738, respectively; (iii) heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 750, 751, and 752, respectively, and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 758, 759, and 760, respectively; and (iv) heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 750, 751, and 752, respectively, and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 758, 759, and 760, respectively.

[0270] In some embodiments, the present disclosure provides an isolated monoclonal antibody or antigen-binding portion thereof that specifically binds to and antagonizes human IL-27, wherein the antibody or antigen-binding portion thereof comprises heavy chain CDRs and light chain CDRs, wherein the heavy chain CDR1, CDR2, and CDR3 sequences are set forth in SEQ ID NOs: 161, 162, and 163, respectively, and the light chain CDR1, CDR2, and CDR3 sequences are set forth in SEQ ID NOs: 169, 170, and 171, respectively.

[0271] In some embodiments, the present disclosure provides an isolated monoclonal antibody or antigen-binding portion thereof that specifically binds to and antagonizes human IL-27, wherein the antibody or antigen-binding portion thereof comprises heavy and light chain CDRs selected from the group consisting of: (i) heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 709, 710, and 711, respectively, and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 717, 718, and 719, respectively; (ii) heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 731, 732, and 733, respectively, and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 739, 740, and 741, respectively; (iii) heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 753, 754, and 755, respectively, and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 761, 762, and 763, respectively; and (iv) heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 775, 776, and 777, respectively, and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 783, 784, and 785, respectively.

[0272] In some embodiments, the present disclosure provides an isolated monoclonal antibody or antigen-binding portion thereof that specifically binds to and antagonizes human IL-27, wherein the antibody or antigen-binding portion thereof comprises heavy chain CDRs and light chain CDRs, wherein the heavy chain CDR1, CDR2, and CDR3 sequences are set forth in SEQ ID NOs: 164, 165, and 166, respectively, and the light chain CDR1, CDR2, and CDR3 sequences are set forth in SEQ ID NOs: 172, 173, and 174, respectively.

[0273] In some embodiments, the present disclosure provides an isolated monoclonal antibody or antigen-binding portion thereof that specifically binds to and antagonizes human IL-27, wherein the antibody or antigen-binding portion thereof comprises a heavy chain CDR and a light chain CDR, wherein the amino acid sequences of heavy chain CDR1, CDR2, and CDR3 are SYSMS (SEQ ID NO: 23), YISYDGGSAYYPDTVKG (SEQ ID NO: 24), and HGDYDDDDAMDY (SEQ ID NO: 25), respectively, and the amino acid sequences of light chain CDR1, CDR2, and CDR3 are RASENIYSYLA (SEQ ID NO: 26), NAETLTE (SEQ ID NO: 27), and QHHYGTPLT (SEQ ID NO: 28), respectively.

[0274] In some embodiments, the disclosure provides an isolated monoclonal antibody or antigen-binding portion thereof that specifically binds to and antagonizes human IL-27, 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 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 712, 734, 756, and 778, and the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 720, 742, 764, and 786.

[0275] In some embodiments, the disclosure provides an isolated monoclonal antibody or antigen-binding portion thereof that specifically binds to and antagonizes human IL-27, wherein the antibody or antigen-binding portion thereof comprises a heavy chain variable region and a light chain variable region comprising an amino acid sequence selected from the group consisting of: (i) SEQ ID NOs: 712 and 720, respectively; (ii) SEQ ID NOs: 734 and 742, respectively; (iii) SEQ ID NOs: 756 and 764, respectively; and (iv) SEQ ID NOs: 778 and 786, respectively.

[0276] In some embodiments, the disclosure provides an isolated monoclonal antibody or antigen-binding portion thereof that specifically binds to and antagonizes human IL-27, 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 comprises an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 712, 734, 756, and 778, and the light chain variable region comprises an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 720, 742, 764, and 786.

[0277] In some embodiments, the disclosure provides an isolated monoclonal antibody or antigen-binding portion thereof that specifically binds to and antagonizes human IL-27, wherein the antibody or antigen-binding portion thereof comprises a heavy chain variable region and a light chain variable region comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of: (i) SEQ ID NOs: 712 and 720, respectively; (ii) SEQ ID NOs: 734 and 742, respectively; (iii) SEQ ID NOs: 756 and 764, respectively; and (iv) SEQ ID NOs: 778 and 786, respectively.

[0278] In some embodiments, the present disclosure provides an isolated monoclonal antibody or antigen-binding portion thereof that specifically binds to and antagonizes human IL-27, wherein the antibody or antigen-binding portion thereof comprises a heavy chain variable region and a light chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 167 and 175, respectively.

[0279] In some embodiments, the present disclosure provides an isolated monoclonal antibody or antigen-binding portion thereof that specifically binds to and antagonizes human IL-27, wherein the antibody or antigen-binding portion thereof comprises a heavy chain variable region and a light chain variable region comprising amino acid sequences at least 90% identical to the amino acid sequences set forth in SEQ ID NOs: 167 and 175, respectively.

[0280] In some embodiments, the disclosure provides an isolated monoclonal antibody or antigen-binding portion thereof that specifically binds to and antagonizes human IL-27, wherein the antibody or antigen-binding portion thereof comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 722, 744, 766, and 788, and the light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 724, 746, 768, and 790.

[0281] In some embodiments, the disclosure provides an isolated monoclonal antibody or antigen-binding portion thereof that specifically binds to and antagonizes human IL-27, wherein the antibody or antigen-binding portion thereof comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 722, 744, 766, and 788, and the light chain comprises an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 724, 746, 768, and 790.

[0282] In some embodiments, the disclosure provides an isolated monoclonal antibody or antigen-binding portion thereof that specifically binds to and antagonizes human IL-27, wherein the antibody or antigen-binding portion thereof comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 726, 748, 770, and 792, and the light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 724, 746, 768, and 790.

[0283] In some embodiments, the disclosure provides an isolated monoclonal antibody or antigen-binding portion thereof that specifically binds to and antagonizes human IL-27, wherein the antibody or antigen-binding portion thereof comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 726, 748, 770, and 792, and the light chain comprises an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 724, 746, 768, and 790.

[0284] In some embodiments, the present disclosure provides an isolated monoclonal antibody or antigen-binding portion thereof that specifically binds to and antagonizes human IL-27, wherein the antibody or antigen-binding portion thereof comprises a heavy chain and a light chain comprising an amino acid sequence selected from the group consisting of: (i) SEQ ID NOs: 722 and 724, respectively; (ii) SEQ ID NOs: 744 and 746, respectively; (iii) SEQ ID NOs: 766 and 768, respectively; and (iv) SEQ ID NOs: 788 and 790, respectively.

[0285] In some embodiments, the disclosure provides an isolated monoclonal antibody or antigen-binding portion thereof that specifically binds to and antagonizes human IL-27, wherein the antibody or antigen-binding portion thereof comprises a heavy chain and a light chain comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of: (i) SEQ ID NOs: 722 and 724, respectively; (ii) SEQ ID NOs: 744 and 746, respectively; (iii) SEQ ID NOs: 766 and 768, respectively; and (iv) SEQ ID NOs: 788 and 790, respectively.

[0286] In some embodiments, the present disclosure provides an isolated monoclonal antibody or antigen-binding portion thereof that specifically binds to and antagonizes human IL-27, wherein the antibody or antigen-binding portion thereof comprises a heavy chain and a light chain comprising an amino acid sequence selected from the group consisting of: (i) SEQ ID NOs: 726 and 724, respectively; (ii) SEQ ID NOs: 748 and 746, respectively; (iii) SEQ ID NOs: 770 and 768, respectively; and (iv) SEQ ID NOs: 792 and 790, respectively.

[0287] In some embodiments, the disclosure provides an isolated monoclonal antibody or antigen-binding portion thereof that specifically binds to and antagonizes human IL-27, wherein the antibody or antigen-binding portion thereof comprises a heavy chain and a light chain comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of: (i) SEQ ID NOs: 726 and 724, respectively; (ii) SEQ ID NOs: 748 and 746, respectively; (iii) SEQ ID NOs: 770 and 768, respectively; and (iv) SEQ ID NOs: 792 and 790, respectively.

[0288] In some embodiments, the present disclosure provides an isolated monoclonal antibody or antigen-binding portion thereof that specifically binds to and antagonizes human IL-27, wherein the antibody or antigen-binding portion thereof comprises a heavy chain and a light chain comprising the amino acid sequences set forth in SEQ ID NOs: 177 and 179, respectively.

[0289] In some embodiments, the present disclosure provides an isolated monoclonal antibody or antigen-binding portion thereof that specifically binds to and antagonizes human IL-27, wherein the antibody or antigen-binding portion thereof comprises a heavy chain and a light chain comprising amino acid sequences at least 90% identical to the amino acid sequences set forth in SEQ ID NOs: 177 and 179, respectively.

[0290] In some embodiments, the present disclosure provides an isolated monoclonal antibody or antigen-binding portion thereof that specifically binds to and antagonizes human IL-27, wherein the antibody or antigen-binding portion thereof comprises a heavy chain and a light chain comprising the amino acid sequences set forth in SEQ ID NOs: 181 and 179, respectively.

[0291] In some embodiments, the present disclosure provides an isolated monoclonal antibody or antigen-binding portion thereof that specifically binds to and antagonizes human IL-27, wherein the antibody or antigen-binding portion thereof comprises a heavy chain and a light chain comprising amino acid sequences at least 90% identical to the amino acid sequences set forth in SEQ ID NOs: 181 and 179, respectively.

[0292] Anti-IL-27 receptor (WSX-1) antibody and its antigen-binding fragment WSX-1 is a class I cytokine receptor that is homologous in both sequence and structure to the β2 chain of the IL-12R. This receptor binds to resting / naive CD4 + T cells and CD8 + It is highly expressed by T cells. Recent studies have identified IL-27, a heterodimeric cytokine composed of EBI3 and IL-27p28 subunits, as a ligand for WSX-1. EBI3, a member of the class I cytokine receptor family, shares significant structural homology with IL-12p40, while IL-27p28 is closely related to IL-12p35. In addition to the structural similarity between the ligand / receptor pairs IL-12 / IL-12R and IL-27 / WSX-1, some reports have also demonstrated functional similarities. While IL-12R plays a crucial role in the development of Th1-type responses, WSX-1-deficient cells have been reported to have impaired IFN-γ production during the early stages of Th1 differentiation. Furthermore, recombinant IL-27, like IL-12, can enhance Th1 differentiation in highly purified naive helper T cells. As a result of these studies, there was early agreement that IL-27 / WSX-1, like the IL-12 / IL-12R interaction, is a key factor in the early differentiation of Th1 responses.

[0293] Thus, in some embodiments, the present disclosure provides an isolated monoclonal antibody or antigen-binding portion thereof that specifically binds to and antagonizes human WSX-1, wherein the antibody or antigen-binding portion thereof comprises a heavy chain CDR and a light chain CDR, wherein the heavy chain CDR1, CDR2, and CDR3 amino acid sequences are SNNAAWN (SEQ ID NO: 802), RTYYRSKWYNDYALSVKS (SEQ ID NO: 803), and GLPMVPFDS (SEQ ID NO: 804), respectively, and the light chain CDR1, CDR2, and CDR3 sequences are RASQSISSWLA (SEQ ID NO: 805), KASSLES (SEQ ID NO: 806), and QQYDSFSMYT (SEQ ID NO: 807), respectively.

[0294] In some embodiments, the present disclosure provides an isolated monoclonal antibody or antigen-binding portion thereof that specifically binds to and antagonizes human WSX-1, 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 comprises the amino acid sequence set forth in SEQ ID NO:794 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:796.

[0295] In some embodiments, the antibody or antigen-binding portion thereof that specifically binds to and antagonizes human WSX-1 comprises a light chain constant region, wherein the light chain constant region comprises the amino acid sequence set forth in SEQ ID NO:798.

[0296] In some embodiments, the antibody or antigen-binding portion thereof that specifically binds to and antagonizes human WSX-1 comprises a heavy chain constant region, wherein the heavy chain constant region comprises the amino acid sequence set forth in SEQ ID NO:799.

[0297] Methods for producing anti-IL-27 antibodies and antigen-binding fragments thereof The present disclosure also features methods for producing any of the anti-IL-27 antibodies or antigen-binding fragments thereof described herein. In some embodiments, methods for preparing the antibodies described herein can 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 described herein. For example, to produce an antibody that binds to IL-27, one skilled in the art can immunize a suitable subject with IL-27 (e.g., a non-human mammal such as a rat, mouse, gerbil, hamster, dog, cat, pig, goat, horse, or non-human primate). In some embodiments, a full-length human IL-27 antibody comprising the amino acid sequence set forth in SEQ ID NO:97 is used. An EBI3 monomer polypeptide is used as an immunogen. In some embodiments, a full-length human IL-27p28 monomer polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 98 is used as an immunogen.

[0298] A suitable subject (e.g., a non-human mammal) can be immunized with an appropriate antigen, with subsequent booster immunizations, sufficient times to induce the production of antibodies by the mammal. The immunogen can be administered to the subject (e.g., a non-human mammal) along with an adjuvant. Adjuvants useful for generating antibodies in a subject include, but are not limited to, protein adjuvants; bacterial adjuvants, such as whole bacteria (BCG, Corynebacterium parvum, or Salmonella minnesota) and bacterial components, including cell wall skeleton, trehalosedimycolic acid, monophosphoryl lipid A, methanol extract residue of Mycobacterium tuberculosis (MER), and complete or incomplete Freund's adjuvant; viral adjuvants; and chemical adjuvants, such as aluminum hydroxide, iodoacetic acid, and cholesteryl hemisuccinate. Other adjuvants that can be used in methods to induce an immune response include, for example, cholera toxin and parapoxvirus proteins. See also Bieg et al. (1999) Autoimmunity 31(1):15-24. See, e.g., Lodmell et al. (2000) Vaccine 18:1059-1066; Johnson et al. (1999) See also J Med Chem 42:4640-4649; Baldridge et al. (1999) Methods 19:103-107; and Gupta et al. (1995) Vaccine 13(14):1263-1276.

[0299] In some embodiments, the method involves preparing a hybridoma cell line secreting a monoclonal antibody that binds to the immunogen. For example, a suitable mammal, such as a house mouse, is immunized with an IL-27 polypeptide as described above. Antibody-producing cells (e.g., splenic B cells) from the immunized mammal are isolated 2-4 days after at least one boost of the immunogen and then briefly grown in culture before fusion with cells of a suitable myeloma cell line. The cells may be fused, for example, in the presence of a fusion promoter, such as vaccinia virus or polyethylene glycol. The hybrid cells resulting from the fusion are cloned, and cell clones secreting the desired antibody are selected. For example, spleen cells from a Balb / c mouse immunized with a suitable immunogen are transfected with the myeloma cell line PAI or the myeloma cell line Sp2 / 0-Ag. The hybridoma cells may be fused with human IL-27 cells. After fusion, the cells are grown at regular intervals in a suitable culture medium supplemented with a selective medium, such as HAT medium, to prevent normal myeloma cells from overgrowing the desired hybridoma cells. The resulting hybrid cells are then screened for secretion of a desired antibody, e.g., an antibody that binds to human IL-27. In some embodiments, one skilled in the art can identify anti-IL-27 antibodies from a non-immune biased library, as described, for example, in U.S. Pat. No. 6,300,064 (Knappik et al.; Morphosys AG) and Schoonbroodt et al. (2005) Nucleic Acids Res 33(9):e81.

[0300] In some embodiments, the methods described herein are directed to, for example, phage display technology, bacterial display, yeast surface display, eukaryotic viral display, mammalian cell display, and cell-free (e.g., ribosome display) antibody screening technologies (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).

[0301] Methods for identifying antibodies using various phage display methods are known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles that carry the polynucleotide sequences encoding them. Such phage can be used to display antigen-binding domains of antibodies expressed from a repertoire or combinatorial antibody library (e.g., human or murine), such as Fab, Fv, or disulfide-bond-stabilized Fv antibody fragments. The phage used in these methods are typically filamentous phage such as fd and M13. The antigen-binding domains are expressed as recombinant fusion proteins with either the phage coat protein 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 generate 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 Methods 184:177-186; Immunol 24:952-958; Persic et al. (1997) Gene 187:9-18; Burton et al. (1994) Advances in Immunology 57:191-280; and those disclosed in PCT Publication Nos. WO90 / 02809, WO91 / 10737, WO92 / 01047, WO92 / 18619, WO93 / 11236, WO95 / 15982, and WO95 / 20401. Suitable methods are also described, for example, in U.S. Patent Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743, and 5,969,108.

[0302] In some embodiments, phage display antibody libraries can be generated using mRNA collected from B cells from an immunized mammal. For example, a splenocyte 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 can be constructed using cDNAs encoding the variable regions of immunoglobulin heavy and light chain polypeptides. 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.

[0303] In some embodiments, a combination of selection and screening can be used to identify antibodies of interest, for example, from a population of hybridoma-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. al. (1995) (supra); Kettleborough et al. (1994) (supra); Persic et al. (1997) (supra); and Burton et al. (1994) (supra). For example, multiple phagemid vectors, each encoding a fusion protein of a bacteriophage coat protein (e.g., pIII, pVIII, or pIX of M13 phage) and a different antigen-binding domain, are generated using standard molecular biology techniques and then introduced into a population of bacteria (e.g., E. coli). In some embodiments, expression of bacteriophage in bacteria may require the use of a helper phage. In some embodiments, a helper phage is not required (see, e.g., Chasteen et al., (2006) Nucleic Acids Res 34(21):e145). The phage produced from the bacteria are recovered and then contacted with the target antigen, for example, bound (immobilized) to a solid support. The phage may be contacted with the antigen in solution, after which the complex is bound to the solid support.

[0304] Antibody subpopulations screened using the above methods can be characterized for their specificity and binding affinity to a particular antigen (e.g., human IL-27) using any immunological or biochemical method known in the art. For example, the specific binding of an antibody to IL-27 can be determined using, for example, immunological or biochemical methods, including, 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 blots, RIAs, ELISAs (enzyme-linked immunosorbent assays), "sandwich" immunoassays, immunoprecipitation assays, immunodiffusion assays, agglutination assays, complement fixation assays, immunoradiometry, fluorescence immunoassays, and protein A immunoassays. Such assays are routine and known in the art.

[0305] It will be appreciated that the above methods may also be used, for example, to determine whether an anti-IL-27 antibody does not bind to full-length human IL-27 and / or IL-27 protein.

[0306] In embodiments where the selected CDR amino acid sequences are short sequences (e.g., 10-15 amino acids or less in length), the nucleic acids encoding the CDRs can be prepared using methods such as those described in, for example, Shiraishi et al. (2007) Nucleic Acids Symposium Series 51(1):129-130 and U.S. Patent No. 6,995,259. For a given nucleic acid sequence encoding an acceptor antibody, the region of the nucleic acid sequence encoding the CDRs can be replaced with chemically synthesized nucleic acids using standard molecular biology techniques. The 5' and 3' ends of the chemically synthesized nucleic acids can be synthesized to contain cohesive end restriction enzyme sites for use in cloning the nucleic acid into the nucleic acid encoding the variable region of the donor antibody.

[0307] In some embodiments, the anti-IL-27 antibodies described herein comprise an altered heavy chain constant region that has reduced effector function (or no effector function) compared to the corresponding unaltered constant region. Effector functions associated with the constant region of an anti-IL-27 antibody can be modulated by altering the properties of the constant region or Fc region. Altered effector functions include, 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 proinflammatory responses. Modulation refers to an increase, decrease, or elimination of an effector function activity exhibited by a subject antibody comprising an altered constant region compared to the activity of the unaltered constant region. In certain embodiments, modulation includes a situation in which the activity is abolished or completely absent.

[0308] In one embodiment, an anti-IL-27 antibody described herein comprises an IgG4 heavy chain constant region. In one embodiment, the IgG4 heavy chain constant region is a wild-type IgG4 heavy chain constant region. In another embodiment, the IgG4 constant region comprises mutations, e.g., S228P and one or both of L235E or L235A, according to EU numbering (Kabat, EA, et al., supra). Exemplary sequences of wild-type and mutant IgG4 constant regions for use in the antibodies of the present disclosure are set forth in Table 12. In one embodiment, an anti-IL-27 antibody described herein comprises an IgG1 constant region. In one embodiment, the IgG1 heavy chain constant region is a wild-type IgG1 heavy chain constant region. In another embodiment, the IgG1 heavy chain constant region comprises mutations. Exemplary sequences of wild-type and mutant IgG4 constant regions for use in the antibodies of the present disclosure are set forth in Table 12.

[0309] An altered constant region with altered FcR binding affinity and / or altered ADCC activity and / or altered CDC activity is a polypeptide that has enhanced or diminished FcR binding activity and / or ADCC activity and / or CDC activity compared to the unmodified constant region. An altered constant region that exhibits increased FcR binding binds to at least one FcR with higher affinity than the unmodified polypeptide. An altered constant region that exhibits decreased FcR binding binds to at least one FcR with lower affinity than the unmodified constant region. Such variants that exhibit reduced binding to FcR may have little or no measurable binding to FcR (e.g., have 0-50% (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%) binding to FcR compared to the binding level of a native sequence immunoglobulin constant region 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 embodiments, an anti-IL-27 antibody comprising an altered constant region may exhibit about 0-50% (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 constant region. The anti-IL-27 antibodies described herein that comprise an altered constant region that exhibits reduced ADCC and / or CDC activity may exhibit reduced ADCC and / or CDC activity, or may not exhibit ADCC and / or CDC activity.

[0310] In some embodiments, the anti-IL-27 antibodies described herein exhibit reduced or no effector function. In some embodiments, the anti-IL-27 antibodies comprise a hybrid constant region or portion thereof, e.g., 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 supra.

[0311] In some embodiments, anti-IL-27 antibodies can comprise modified constant regions that exhibit enhanced or reduced complement-dependent cytotoxicity (CDC). Modulated CDC activity can be achieved by introducing one or more amino acid substitutions, insertions, or deletions into the Fc region of the antibody. See U.S. Patent No. 6,194,551. Alternatively or additionally, cysteine ​​residue(s) can be introduced into the Fc region, allowing interchain disulfide bond formation in this region. Homodimeric antibodies thus generated can have improved or reduced internalization capability and / or increased or decreased 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. Nos. 5,648,260 and 5,624,821.

[0312] Recombinant antibody expression and purification The antibodies or antigen-binding fragments thereof described herein can be produced using a variety of techniques known in the fields of molecular biology and protein chemistry. For example, a nucleic acid encoding one or both of the antibody heavy and light chain polypeptides can be inserted into an expression vector containing transcriptional and translational control sequences, including, for example, promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, transcriptional termination signals, polyadenylation signals, and enhancer or activator sequences. Control sequences include promoters and transcriptional start and stop sequences. In addition, an expression vector can contain two or more replication systems so that it can be maintained in two different organisms, such as mammalian or insect cells for expression and a prokaryotic host for cloning and amplification.

[0313] Several vector systems are available for the expression of cloned heavy and light chain polypeptides from nucleic acids in mammalian cells. Some vectors rely on integration of the desired gene sequences into the host cell genome. Cells with stably integrated DNA can be selected by co-introducing a drug resistance gene, such as E. coli gpt (Mulligan and Berg (1981) Proc Natl Acad Sci USA 78:2072) or Tn5 neo (Southern and Berg (1982) Mol Appl Genet 1:327). The selectable marker gene can either be linked to the DNA gene sequences to be expressed or introduced into the same cell by co-transfection (Wigler et al. (1979) Cell 16:77). A second type of vector utilizes a DNA element that confers autonomous replication capability on an extrachromosomal plasmid. These vectors can be derived from animal viruses such as bovine papillomavirus (Sarver et al. (1982) Proc Natl Acad Sci USA, 79:7147), cytomegalovirus, polyomavirus (Deans et al. (1984) Proc Natl Acad Sci USA 81:1292), or SV40 virus (Lusky and Botchan (1981) Nature 293:79).

[0314] Expression vectors can be introduced into cells by any method suitable for subsequent expression of the nucleic acid. The method of introduction is determined primarily by the targeted 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.

[0315] Suitable host cells for expression of antibodies or antigen-binding fragments thereof include yeast, bacterial, 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.

[0316] In some embodiments, antibodies or fragments thereof can be expressed in transgenic animals (e.g., transgenic mammals) and purified from the transgenic animals. For example, antibodies can be produced in transgenic non-human mammals (e.g., rodents) and isolated from their 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.

[0317] Antibodies and fragments thereof can be produced from host cells transformed with an expression vector containing nucleic acid encoding the antibody or fragment by culturing the cells under conditions and for a period of time sufficient to allow expression of the protein. Such conditions for protein expression will vary with the choice of expression vector and host cell, and will be easily ascertained by one of ordinary skill 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 for their use 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 will vary depending on many factors and can be easily optimized as needed. 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).

[0318] After expression, antibodies and fragments thereof can be isolated. Depending on other components present in the sample, antibodies or fragments thereof can be isolated or purified by a variety of methods known to those skilled in the art. Standard purification methods include electrophoretic, molecular, immunological, and chromatographic techniques, including ion-exchange, hydrophobic, affinity, and reverse-phase HPLC chromatography. For example, antibodies can be purified using a standard anti-antibody column (e.g., a Protein A or Protein G column). Ultrafiltration and diafiltration techniques, in conjunction with protein concentration, are also useful. See, e.g., Scopes (1994) "Protein Purification, 3 rd edition,” Springer-Verlag, New York City, New York. The degree of purification required will vary depending on the desired use. In some cases, purification of the expressed antibody or fragment thereof is not required.

[0319] Methods for determining the yield or purity of a purified antibody or fragment thereof are known in the art and include, for example, Bradford assay, ultraviolet spectroscopy, Biuret protein assay, Lowry protein assay, amido black protein assay, high performance liquid chromatography (HPLC), mass spectrometry (MS), and gel electrophoresis (e.g., using a protein stain such as Coomassie Blue or colloidal silver stain).

[0320] Modification of antibodies or antigen-binding fragments thereof Antibodies or antigen-binding fragments thereof can be modified after their expression and purification. Modifications can be covalent or non-covalent. Such modifications can be introduced into antibodies or fragments, for example, by reacting targeted amino acid residues of the polypeptide with organic derivatizing agents capable of reacting with selected side chains or terminal residues. Suitable sites for modification can be selected using any of a variety of criteria, including, for example, structural or amino acid sequence analysis of the antibody or fragment.

[0321] In some embodiments, an antibody or antigen-binding fragment thereof may be conjugated to a heterologous moiety. The heterologous moiety can be, for example, a heterologous polypeptide, a therapeutic agent (e.g., a toxin or drug), or a detectable label, including, but not limited to, a radioactive label, an enzymatic 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: 405)), polyhistidine (6-His; HHHHHH (SEQ ID NO: 406)), hemagglutinin (HA; YPYDVPDYA (SEQ ID NO: 407)), glutathione-S-transferase (GST), or maltose-binding protein (MBP)) for use in purifying the antibody or fragment. Heterologous polypeptides also include polypeptides useful 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, 32 P, 33 P, 14 C. 125 I, 131 I, 35 S, and 3H. Suitable fluorescent labels include, but are not limited to, fluorescein, fluorescein isothiocyanate (FITC), green fluorescent protein (GFP), DyLight™ 488, phycoerythrin (PE), propidium iodide (PI), PerCP, PE-Alexa Fluor® 700, Cy5, allophycocyanin, and Cy7. Luminescent labels include, for example, any of a variety of luminescent lanthanide (e.g., europium or terbium) chelates. For example, suitable europium chelates include the 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.

[0322] Two proteins (e.g., an antibody and a heterologous moiety) can be crosslinked using any of a number of known chemical crosslinkers. Examples of such crosslinkers are those that link two amino acid residues through a bond containing a "hindered" disulfide bond. In these bonds, the disulfide bond within the crosslinking unit is protected from reduction (by shielding groups on either side of the disulfide bond), for example, by the action of reduced glutathione or the enzyme disulfide reductase. One suitable reagent, 4-succinimidyloxycarbonyl-α-methyl-α(2-pyridyldithio)toluene (SMPT), forms a bond between two proteins, utilizing a terminal lysine on one protein and a terminal cysteine ​​on the other. Heterobifunctional reagents that crosslink via different binding moieties on each protein can also be used. Other useful cross-linking agents include, but are not limited to, reagents that link two amino groups (e.g., N-5-azido-2-nitrobenzoyloxysuccinimide), two sulfhydryl groups (e.g., 1,4-bis-maleimidobutane), an amino group and a sulfhydryl group (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester), an amino group and a carboxyl group (e.g., 4-[p-azidosalicylamido]butylamine), and an amino group and a guanidinium group present in the side chain of arginine (e.g., p-azidophenylglyoxal monohydrate).

[0323] In some embodiments, 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., a meta-[iota]cyl ester bonded to a free amino group to form a meta-iodophenyl (mIP) derivative of the protein of interest). 125 Iodophenyl-N-hydroxysuccinimide ([ 125 I]mIPNHS) 125 I (e.g. Rogers et al. (1997) J Nucl Med 38:1221-1229)) or as part of a chelate (e.g., DOTA or DTPA), which is then attached to the protein backbone. Methods for conjugating a radiolabel or a larger molecule / chelate containing a radiolabel to the antibodies or antigen-binding fragments thereof described herein are known in the art. Such methods include 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).

[0324] 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 amino groups (e.g., lysine) or sulfhydryl groups (e.g., cysteine) of proteins using succinimidyl (NHS) ester or tetrafluorophenyl (TFP) ester moieties attached to the fluorophore. In some embodiments, fluorophores can be conjugated to heterobifunctional crosslinking moieties such as sulfo-SMCC. A suitable conjugation method involves incubating an antibody protein, or a fragment thereof, with the fluorophore under conditions that promote binding of the fluorophore to the protein. See, for example, Welch and Redvanly (2003) “Handbook of Radiopharmaceuticals: Radiochemistry and Applications,” John Wiley and Sons (ISBN 0471495603).

[0325] In some embodiments, the antibody or fragment can be modified with a moiety that improves stabilization and / or retention of the circulating antibody, for example, in blood, serum, or other tissues. For example, the antibody or fragment can be modified with a moiety that improves stabilization and / or retention of the circulating antibody in blood, serum, or other tissues, for example, as described in, e.g., Lee et al. (1999) Bioconjug Chem 10(6):973-8; Kinstler et al. (2002) Advanced Drug Deliveries Reviews 54:477-485; and Roberts et al. (2002) Advanced Drug Delivery Reviews 54:459-476, or may be PEGylated (Fresenius Kabi, Germany; see, e.g., Pavisic et al. (2010) Int J Pharm 387(1-2):110-119). The stabilizing moiety may improve the stability or retention of the antibody (or fragment) by at least 1.5-fold (e.g., at least 2, 5, 10, 15, 20, 25, 30, 40, or 50-fold or more).

[0326] In some embodiments, the antibodies or antigen-binding fragments thereof described herein may be glycosylated. In some embodiments, the antibodies or antigen-binding fragments thereof described herein may be enzymatically or chemically treated or produced from cells so that the antibodies or fragments have reduced or no glycosylation. Methods for producing antibodies with reduced glycosylation are known in the art and are described, for example, in U.S. Patent No. 6,933,368; Wright et al. (1991) EMBO J 10(10):2717-2723; and Co et al. (1993) Mol Immunol 30:1361.

[0327] Pharmaceutical Compositions and Formulations In certain embodiments, the present invention provides pharmaceutical compositions comprising an anti-IL-27 antibody together with a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and / or adjuvant.

[0328] In certain embodiments, acceptable formulation materials are preferably nontoxic to recipients at the dosages and concentrations employed. In certain embodiments, the formulation material(s) are for subcutaneous and / or intravenous administration. In certain embodiments, pharmaceutical compositions may contain formulation materials to modify, maintain, or preserve, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, absorption, or penetration of the composition.In certain embodiments, suitable formulation materials include, but are not limited to, amino acids (e.g., glycine, glutamine, asparagine, arginine, or lysine); antimicrobial agents; antioxidants (e.g., ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (e.g., borate, bicarbonate, Tris-HCl, citrate, phosphate, or other organic acids); bulking agents (e.g., mannitol or glycine); chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)); complexing agents (e.g., caffeine, polyvinylpyrrolidone, β-cyclodextrin, or hydroxypropyl-β-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (e.g., glucose, mannose, or dextrin); proteins (e.g., serum albumin, gelatin, or immunoglobulins); colorants, flavorings, and diluents; emulsifiers; hydrophilic polymers (e.g., polyvinylpyrrolidone); low molecular weight polypeptides. peptides; salt-forming counterions (e.g., sodium); preservatives (e.g., benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide); solvents (e.g., glycerin, propylene glycol, or polyethylene glycol); sugar alcohols (e.g., mannitol or sorbitol); suspending agents; surfactants or wetting agents (e.g., Pluronic ( These may include, for example, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, Triton, tromethamine, lecithin, cholesterol, tyloxapol; stability enhancers (e.g., sucrose or sorbitol); tonicity enhancers (e.g., 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, A.R. Gennaro, ed., Mack Publishing Company (1995)).In certain embodiments, the formulation contains PBS; 20 mM NaOAC (pH 5.2), 50 mM NaCl; and / or 10 mM NaOAC (pH 5.2), 9% sucrose. In certain embodiments, the optimal pharmaceutical composition will be determined by one of skill in the art depending on, for example, the intended route of administration, delivery format, and desired dosage. See, e.g., Remington's Pharmaceutical Sciences, supra. In certain embodiments, such compositions may affect the physical state, stability, rate of in vivo release, and / or rate of in vivo clearance of the anti-IL-27 antibody.

[0329] In certain embodiments, the primary vehicle or carrier in a pharmaceutical composition can be either aqueous or non-aqueous in nature. For example, in certain embodiments, a suitable vehicle or carrier can be water for injection, saline, or artificial cerebrospinal fluid, optionally supplemented with other ingredients common in compositions for parenteral administration. In certain embodiments, saline comprises isotonic phosphate-buffered saline. In certain embodiments, further exemplary vehicles are neutral buffered saline or saline mixed with serum albumin. In certain embodiments, the pharmaceutical composition comprises Tris buffer of about pH 7.0-8.5 or acetate buffer of about pH 4.0-5.5, which may therefore further comprise sorbitol or a suitable substitute. In certain embodiments, compositions comprising anti-IL-27 antibodies can be prepared by adding any compounding agent (e.g., Remington's Pharmaceuticals, Inc.) to the selected composition having the desired purity. The anti-IL-27 antibody composition may be prepared for storage in the form of a lyophilized cake or aqueous solution by mixing with an appropriate excipient such as sucrose.

[0330] In some embodiments, pharmaceutical compositions may be selected for parenteral delivery. In certain embodiments, compositions may be selected for inhalation or 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.

[0331] In certain embodiments, formulation components are present in concentrations that are acceptable to the site of administration. In certain embodiments, a buffer is used to maintain the composition at physiological pH or slightly lower, typically within a pH range of about 5 to about 8.

[0332] In certain embodiments, when intended for parenteral administration, the therapeutic composition may be in the form of a pyrogen-free, parenterally acceptable aqueous solution containing an anti-IL-27 antibody in a pharmaceutically acceptable vehicle. In certain embodiments, the parenteral injection vehicle is sterile distilled water in which the anti-IL-27 antibody is formulated as a sterile, isotonic solution and appropriately maintained. In certain embodiments, the preparation may involve formulating the desired molecule with an agent capable of providing controlled or sustained release of the product, such as injectable microspheres, bioerodible particles, polymeric compounds (e.g., polylactic acid or polyglycolic acid), beads, or liposomes, and the product may then be delivered by depot injection. In certain embodiments, hyaluronic acid may also be used and may have the effect of enhancing the duration of circulation. In certain embodiments, the desired molecule may be introduced using an implantable drug delivery device.

[0333] In certain embodiments, pharmaceutical compositions can be formulated for inhalation. In certain embodiments, anti-IL-27 antibodies can be formulated as dry powders for inhalation. In certain embodiments, inhalation solutions comprising anti-IL-27 antibodies can be formulated with a propellant for aerosol delivery. In certain embodiments, the solution can be nebulized. Pulmonary administration is further described in PCT Application No. PCT / US94 / 001875, which describes pulmonary delivery of chemically modified proteins.

[0334] In certain embodiments, it is contemplated that the formulation may be administered orally. In certain embodiments, the anti-IL-27 antibody administered in this manner may be formulated with or without carriers customarily used in formulating solid dosage forms such as tablets and capsules. In certain embodiments, the formulation may be designed to release the active portion at a point in the gastrointestinal tract where bioavailability is maximized and pre-systemic degradation is minimized. In certain embodiments, at least one additive may be included to promote absorption of the anti-IL-27 antibody. In certain embodiments, diluents, flavorings, low-melting waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders may also be used.

[0335] In certain embodiments, pharmaceutical compositions may contain an effective amount of anti-IL-27 antibody in a mixture with non-toxic excipients suitable for tablet manufacture. In certain embodiments, a unit-dose solution may be prepared by dissolving tablets in sterile water or another suitable vehicle. In certain embodiments, suitable excipients include, but are not limited to, inert diluents such as calcium carbonate, sodium carbonate or bicarbonate, lactose, or calcium phosphate; or binders such as starch, gelatin, or acacia; or lubricants such as magnesium stearate, stearic acid, or talc.

[0336] Additional pharmaceutical compositions will be apparent to those skilled in the art, including formulations comprising anti-IL-27 antibodies in sustained- or controlled-delivery formulations. In certain embodiments, techniques for constructing various other sustained- or controlled-delivery means, such as liposome carriers, bioerodible microparticles or porous beads, and depot injections, are also known to those skilled in the art. See, for example, PCT Application No. PCT / US93 / 00829, which describes controlled release via porous polymer microparticles for delivery of pharmaceutical compositions. In certain embodiments, sustained-release preparations may comprise semipermeable polymer matrices in the form of shaped articles, e.g., films or microcapsules. Sustained-release matrices include polyesters, hydrogels, polylactic acid (U.S. Pat. No. 3,773,919 and EP 058481), copolymers of L-glutamic acid and gamma-ethyl-L-glutamic acid (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-hydroxybutyrate (EP 133,988). In certain embodiments, sustained-release compositions may also include liposomes, which can be prepared by any of several methods known in the art. See, for example, Eppstein et al., Proc. Natl. Acad. Sci. USA, 82:3688-3692 (1985); EP 036,676; EP 088046 and EP 143,949.

[0337] Pharmaceutical compositions used for in vivo administration are often sterile. In certain embodiments, sterility can be achieved by filtration through sterile filtration membranes. In certain embodiments in which the composition is lyophilized, sterilization using this method can be performed either before or after lyophilization and reconstitution. In certain embodiments, compositions for parenteral administration can be stored in lyophilized form or in a solution. In certain embodiments, parenteral compositions are typically placed into a container having a sterile access port, for example, an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle.

[0338] In certain embodiments, once a pharmaceutical composition has been formulated, it may be stored in a sterile vial as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder. In certain embodiments, such formulations may be stored either in a ready-to-use form or in a form that is reconstituted (e.g., lyophilized) prior to administration.

[0339] In certain embodiments, kits for preparing single-dose administration units are provided. In certain embodiments, the kits can include both a first container with a dried protein and a second container with an aqueous formulation. In certain embodiments, kits include kits containing single- or multi-chamber pre-filled syringes (e.g., liquid syringes and lyosyringes).

[0340] In certain embodiments, the effective amount of a pharmaceutical composition comprising an anti-IL-27 antibody used for treatment will depend, for example, on the therapeutic situation and purpose. Accordingly, one of skill in the art will understand that appropriate dosage levels for treatment according to certain embodiments will vary, in part, depending on the molecule being delivered, the indication for which the anti-IL-27 antibody is being used, the route of administration, and the size (weight, body surface area, or organ size) and / or condition (age and health) of the patient. In certain embodiments, a clinician may titrate the dosage and modify the route of administration to obtain optimal therapeutic effect.

[0341] In certain embodiments, the frequency of administration takes into account the pharmacokinetic parameters of the anti-IL-27 antibody in the formulation used. In certain embodiments, the clinician administers the composition until a dosage is reached that achieves the desired effect. Thus, in certain embodiments, the composition can be administered as a single dose, or as two or more doses over time (which may or may not contain the same amount of the desired molecule), or as a continuous infusion via an implanted device or catheter. Further refinement of the appropriate dosage is routinely performed by those of skill in the art and is within the scope of tasks routinely performed by those skilled in the art. In certain embodiments, the appropriate dosage can be ascertained through the use of appropriate dose-response data.

[0342] In certain embodiments, the route of administration of the pharmaceutical composition is according to known methods, for example, by oral, intravenous, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, subcutaneous, intraocular, intraarterial, intraportal, or intralesional injection, or by sustained release system, or by implantation device. In certain embodiments, the composition may be administered by bolus injection, or continuously by infusion, or by implantation device. In certain embodiments, the individual components of the combination therapy may be administered by different routes.

[0343] In certain embodiments, compositions may be administered locally by implantation of a membrane, sponge, or another suitable material into which the desired molecule has been absorbed or encapsulated. In certain embodiments in which an implantable device is used, the device may be implanted into any suitable tissue or organ, and delivery of the desired molecule may be by diffusion, timed bolus release, or continuous administration. In certain embodiments, it may be desirable to use a pharmaceutical composition comprising an anti-IL-27 antibody in an ex vivo manner. In such instances, cells, tissues, and / or organs removed from a patient are exposed to a pharmaceutical composition comprising an anti-IL-27 antibody, and then the cells, tissues, and / or organs are transplanted back into the patient.

[0344] In certain embodiments, anti-IL-27 antibodies may be delivered by implanting specific cells genetically engineered to express and secrete the polypeptide, such as using the methods described herein. In certain embodiments, such cells may be animal or human cells and may be autologous, non-autologous, or xenogeneic. In certain embodiments, the cells may be immortalized. In certain embodiments, to reduce the likelihood of an immune response, the cells may be encapsulated to avoid infiltration of surrounding tissues. In certain embodiments, the encapsulating material is typically a biocompatible, semipermeable polymeric enclosure or membrane that allows release of the protein product(s) but prevents destruction of the cells by the patient's immune system or by other harmful factors from surrounding tissues.

[0345] Purpose The compositions described herein can be used in numerous 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 inhibition of target antigen function. For example, in some embodiments 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 are otherwise useful for treating complement-related diseases. For example, an IL-27-inhibiting composition can be used as a positive control in assays to identify additional compounds (e.g., small molecules, aptamers, or antibodies) that reduce or suppress IL-27 production. The compositions can also be used in therapeutic methods, as described in more detail below.

[0346] In some embodiments, the present disclosure provides methods for detecting IL-27 in a biological sample or a subject, the method comprising: (i) contacting the sample or the subject (and optionally a reference sample or subject) with any of the antibodies described herein under conditions that allow interaction of the antibody molecule and IL-27 to occur; and (ii) detecting the formation of a complex between the antibody molecule and the sample or the subject (and optionally the reference sample or subject).

[0347] kit In some embodiments, a kit can include an anti-IL-27 antibody disclosed herein and instructions for use. The kit can include, in suitable containers, an anti-IL-27 antibody, one or more controls, as well as various buffers, reagents, enzymes, and other standard components known in the art. In some aspects, the disclosure provides kits comprising an anti-IL-27 antibody or antigen-binding portion disclosed herein and instructions for use in stimulating an immune response in a subject or treating cancer in a subject, optionally containing instructions for use in combination with one or more additional therapeutic agents or procedures disclosed herein.

[0348] The container may include at least one vial, well, test tube, flask, bottle, syringe, or other container means into which the anti-IL-27 antibody is placed, and in some instances, appropriately aliquoted. If additional components are provided, the kit may include additional containers into which such components may be placed. The kit may also include a means for storing the anti-IL-27 antibody and any other reagent containers under close control for commercial sale. Such containers may include injection- or blow-molded plastic containers into which the desired vials are retained. The container and / or kit may include a label with instructions and / or warnings.

[0349] How to use The compositions of the present invention have many in vitro and in vivo utilities, including the detection and / or quantification of IL-27 and / or antagonism of the function of IL-27.

[0350] In some embodiments, 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 monoclonal 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.

[0351] In some embodiments, the present disclosure provides a method for inhibiting or reducing inhibition of CD161 expression in a cell, the method comprising contacting the cell with an isolated monoclonal antibody or antigen-binding fragment provided by the present disclosure, wherein the antibody or antigen-binding portion thereof inhibits or reduces inhibition of CD161 expression in the cell.

[0352] In some embodiments, the present disclosure provides methods for inhibiting or reducing PD-L1 and / or TIM-3 expression in a cell, the method comprising contacting the cell with an isolated monoclonal antibody or antigen-binding fragment provided by the disclosure, wherein the antibody or antigen-binding portion thereof inhibits or reduces PD-L1 and / or TIM-3 expression in the cell.

[0353] In some embodiments, the present disclosure provides methods for inducing or enhancing secretion of one or more cytokines from a cell, the method comprising contacting the cell with an isolated monoclonal 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.

[0354] In some embodiments, the present disclosure provides methods of stimulating an immune response in a subject, the method comprising administering to the subject an effective amount of an isolated monoclonal antibody or antigen-binding portion thereof that specifically binds to and antagonizes IL-27, as provided by the present disclosure, or a pharmaceutical composition comprising the antibody or antigen-binding portion thereof and a pharmaceutically acceptable carrier.

[0355] In some embodiments, the present disclosure provides methods of treating cancer in a subject, the method comprising administering to the subject an effective amount of an isolated monoclonal antibody or antigen-binding portion thereof that specifically binds to and antagonizes IL-27, as provided by the present disclosure, or a pharmaceutical composition comprising the antibody or antigen-binding portion thereof and a pharmaceutically acceptable carrier.

[0356] In some embodiments, the present disclosure provides methods of stimulating an immune response or treating cancer in a subject, the method comprising administering to the subject an effective amount of an isolated monoclonal antibody or antigen-binding portion thereof provided by the present disclosure, or a pharmaceutical composition comprising the 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 STAT1 and / or STAT3 phosphorylation in a cell, thereby stimulating the immune response or treating the cancer.

[0357] In some embodiments, the present disclosure provides methods of stimulating an immune response or treating cancer in a subject, the method comprising administering to the subject an effective amount of an isolated monoclonal antibody or antigen-binding portion thereof provided by the present disclosure, or a pharmaceutical composition comprising the antibody or antigen-binding portion thereof and a pharmaceutically acceptable carrier, wherein the antibody or antigen-binding portion thereof or pharmaceutical composition inhibits or reduces inhibition of CD161 expression in cells, thereby stimulating the immune response or treating the cancer.

[0358] In some embodiments, the present disclosure provides methods of stimulating an immune response or treating cancer in a subject, the method comprising administering to the subject an effective amount of an isolated monoclonal antibody or antigen-binding portion thereof provided by the present disclosure, or a pharmaceutical composition comprising the antibody or antigen-binding portion thereof and a pharmaceutically acceptable carrier, wherein the antibody or antigen-binding portion thereof or pharmaceutical composition inhibits or reduces PD-L1 and / or TIM-3 expression in cells, thereby stimulating the immune response or treating the cancer.

[0359] In some embodiments, the present disclosure provides methods of stimulating an immune response or treating cancer in a subject, the method comprising administering to the subject an effective amount of an isolated monoclonal antibody or antigen-binding portion thereof provided by the present disclosure, or a pharmaceutical composition comprising the antibody or antigen-binding portion thereof and a pharmaceutically acceptable carrier, wherein the antibody or antigen-binding portion thereof or pharmaceutical composition induces or enhances PD-1-mediated secretion of one or more cytokines from cells, thereby stimulating the immune response or treating the cancer.

[0360] In some embodiments, 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., head and neck squamous cell carcinoma), 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., renal clear cell carcinoma)).

[0361] The above-described compositions are particularly useful in methods for 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, depending in part on the route of administration. The route can be, for example, intravenous injection or infusion (IV), subcutaneous injection (SC), intraperitoneal injection (IP), intramuscular injection (IM), or intrathecal injection (IT). The injection can be a bolus injection or continuous infusion.

[0362] Administration can be performed, for example, by local infusion, injection, or implant. Implants can be of porous, non-porous, or gelatinous materials, including, for example, membranes such as silastic membranes or fibers. Implants can be configured for sustained or periodic release of the composition into a subject. See, for example, U.S. Patent Application No. 20080241223; U.S. Patent Nos. 5,501,856; 4,863,457; and 3,710,795; EP 488401; and EP 430539 (the disclosures of each of which are incorporated herein by reference in their entirety). The composition can be delivered to a subject by, for example, an implantable device based on a diffusive, erodible, 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, an erosion-based system, or an electromechanical system.

[0363] In some embodiments, the anti-IL-27 antibody or antigen-binding fragment thereof is therapeutically delivered to a subject by local administration.

[0364] The appropriate dose of an antibody or fragment thereof described herein, capable of treating or preventing cancer in a subject, can depend on various factors, including, for example, the age, sex, and weight of the subject being treated, as well as the particular inhibitory compound used. For example, the dose of whole anti-IL-27 antibody required to treat a subject with cancer may be different compared to the dose of an IL-27-binding Fab' antibody fragment required to treat the same subject. Other factors that affect the dose administered to a subject include, for example, the type or severity of the cancer. For example, a subject with metastatic melanoma may require a different dose of anti-IL-27 antibody than a patient with glioblastoma. Other factors include, for example, other medical conditions the subject has or has previously had, the subject's health condition, the subject's genetic predisposition, diet, time of administration, excretion rate, drug combinations, and any other additional treatments administered to the subject. It should also be understood that the specific dosage and treatment regimen for any particular subject will depend on the judgment of the treating medical professional (e.g., a doctor or nurse). Suitable dosages are described herein.

[0365] A pharmaceutical composition may comprise a therapeutically effective amount of an anti-IL-27 antibody or antigen-binding fragment thereof described herein. Such an effective amount can be readily determined by one of skill in the art based, in part, on the efficacy of the administered antibody or the combination of the antibody and one or more additional active agents when two or more agents are used. A therapeutically effective amount of an antibody or fragment thereof described herein may vary depending on factors such as the individual's condition, age, sex, and weight, as well as the ability of the antibody (and one or more additional active agents) to elicit a desired response in the individual, e.g., reduced tumor growth. For example, a therapeutically effective amount of an anti-IL-27 antibody may inhibit (reduce the severity of or eliminate the occurrence of) and / or prevent a particular disorder and / or any of the symptoms of a particular disorder known in the art or described herein. A therapeutically effective amount is also one in which any toxic or adverse effects of the composition are outweighed by the therapeutically beneficial effects.

[0366] Suitable human doses of any of the antibodies or fragments thereof described herein can be further evaluated, for example, in a Phase I dose escalation study. See, e.g., 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.

[0367] In some embodiments, a composition contains any of the antibodies or antigen-binding fragments thereof described herein and one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or 10 or more) additional therapeutic agents such that the composition as a whole is therapeutically effective. For example, a composition can include an anti-IL-27 antibody described herein and an alkylating agent, wherein the antibody and agent are each at concentrations that, when combined, are therapeutically effective to treat or prevent cancer (e.g., melanoma) in a subject.

[0368] The toxicity and therapeutic efficacy of such compositions can be determined by known pharmaceutical procedures in cell culture or experimental animals (e.g., animal models of any of the cancers described herein). These procedures can be performed, for example, using LD 50 (lethal dose for 50% of the population) and ED 50 The dose ratio between toxic and therapeutic effects is the therapeutic index, and the LD 50 / ED 50 It can be expressed as a ratio. Antibodies or antigen-binding fragments thereof that exhibit high therapeutic indices are preferred. When compositions that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of the affected tissue, minimizing the potential for damage to normal cells, thereby reducing side effects.

[0369] The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such antibodies or antigen-binding fragments thereof is generally within the range of ED 50 The therapeutically effective dose of an anti-IL-27 antibody described herein may be estimated initially by cell culture assays. The dose may be determined based on the IC50 determined in cell culture. 50 The antibody may be formulated in animal models to achieve a circulating plasma concentration range that includes the antibody concentration range (i.e., the antibody concentration that achieves a half-maximal inhibition of symptoms). Such information can be used to more accurately determine useful dosages in humans. Plasma levels can be measured, for example, by high performance liquid chromatography. In some embodiments, for example, when local administration (e.g., to the eye or joint) is desired, cell culture or animal models can be used to determine the dose required to achieve a therapeutically effective concentration within the local site.

[0370] In some embodiments, the methods of the present invention can be performed in combination with other cancer treatments. For example, the compositions can be administered to a subject simultaneously with, prior to, or after radiation therapy, surgery, targeted or cytotoxic chemotherapy, chemoradiotherapy, hormone therapy, immunotherapy, gene therapy, cell transplantation therapy, precision medicine, genome editing therapy, or other drug therapy.

[0371] As previously mentioned, the compositions described herein (e.g., anti-IL-27 compositions) can be used to treat a variety of cancers, including, but not limited to, Kaposi's sarcoma, leukemia, acute lymphocytic leukemia, acute myeloid leukemia, myeloblastic promyelocytic myelomonocytic monocytic erythroleukemia, chronic leukemia, chronic myeloid (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, and the like. Lymphoma), Hodgkin's disease, Non-Hodgkin's disease, Multiple myeloma, Waldenstrom's macroglobulinemia, Heavy chain disease, Solid tumors, Sarcoma and carcinoma, Fibrosarcoma, Myxosarcoma, Liposarcoma, Chondrosarcoma, Osteogenic sarcoma, Osteosarcoma, Chordoma, Angiosarcoma, Endosarcoma, Lymphangiosarcoma, Lymphangioendothelial sarcoma, Synovioma, Mesothelioma, Ewing's tumor, Leiomyosarcoma, Rhabdominisarcoma Myosarcoma, 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, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma (HCC), liver cancer, cholangiocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicular tumor, lung cancer, small cell lung cancer, non-small cell Lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, nasopharyngeal cancer, esophageal cancer, basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, cancer of the brain and central nervous system (CNS), cervical cancer, choriocarcinoma, colon cancer, connective tissue cancer, and cancer of the gastrointestinal system cancer of the endometrium, esophagus, eye, head and neck, stomach, intraepithelial neoplasia, kidney, pharyngeal, liver, lung (small cell, large cell), melanoma, neuroblastoma, oral cancer (e.g., lip, tongue, mouth, and pharynx), ovarian, pancreatic, retinoblastoma, rhabdomyosarcoma, rectal cancer; cancer of the respiratory system, sarcoma, skin, stomach, testicular, thyroid, uterine, and urinary system.

[0372] Combination Therapy In some embodiments, the anti-IL-27 antibodies, or antigen-binding portions thereof, provided by the present disclosure may be combined with one or more additional therapeutic agents or treatments, e.g., another therapeutic agent or treatment for cancer. For example, an anti-IL-27 antibody, or antigen-binding portion thereof, may 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 the subject having or at risk of developing cancer.

[0373] In some embodiments, the anti-IL-27 antibody, or antigen-binding portion thereof, and the one or more additional therapeutic agents are administered simultaneously (e.g., concurrently). In other embodiments, the anti-IL-27 antibody, or antigen-binding portion thereof, is administered first, and the one or more additional therapeutic agents are administered second (e.g., sequentially). In some embodiments, the one or more additional therapeutic agents are administered first, and the anti-IL-27 antibody is administered second.

[0374] The anti-IL-27 antibodies or antigen-binding fragments thereof described herein can replace or augment previously or currently administered therapies. For example, upon treatment with an anti-IL-27 antibody or antigen-binding fragment thereof, the administration of one or more additional therapeutic agents can be terminated or reduced (e.g., administered at a lower level). In some embodiments, the administration of the previous therapy can be maintained. In some embodiments, the previous therapy can be maintained until the level of the anti-IL-27 antibody reaches a level sufficient to provide a therapeutic effect.

[0375] In some embodiments, 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 monoclonal antibody, or antigen-binding portion thereof, that specifically binds to and antagonizes IL-27, provided by the present disclosure, in combination with one or more additional therapeutic agents or treatments, wherein the second therapeutic agent or treatment is selected from the group consisting of chemotherapy, targeted anti-cancer therapy, oncolytic drug, cytotoxic agent, immunotherapy, cytokine, surgery, radiation therapy, activator of costimulatory molecules, inhibitor of inhibitory molecules, vaccine, or cellular immunotherapy, or a combination thereof.

[0376] In some embodiments, the one or more additional therapeutic agents is a PD-1 antagonist, a TIM-3 inhibitor, a LAG-3 inhibitor, a TIGIT inhibitor, a CD112R inhibitor, a TAM inhibitor, a STING agonist, a 4-1BB agonist, or a combination thereof.

[0377] In some embodiments, the one or more additional therapeutic agents are PD-1 antagonists. In some embodiments, the PD-1 antagonists are selected from the group consisting of: PDR001, nivolumab, pembrolizumab, pidilizumab, MEDI0680, REGN2810, TSR-042, PF-06801591, AMP-224, AB122, and JTX-4014. In certain embodiments, the one or more additional therapeutic agents are PD-L1 inhibitors. In some embodiments, the PD-L1 inhibitors are selected from the group consisting of: FAZ053, atezolizumab, avelumab, durvalumab, and BMS-936559. In some embodiments, the present disclosure provides 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 the anti-PD-1 antibody), the method comprising exposing a cell to an antibody, or 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.

[0378] In some embodiments, the one or more additional therapeutic agents are sunitinib (Sutent®), cabozantinib (Cabometyx®), axitinib (Inlyta®), lenvatinib (Lenvima®), everolimus (Afinitor®), bevacizumab (Avastin®), epacadostat, NKTR-214 (CD-122 bias), or rituximab (rituximab). type agonists), tivozanib (Fotivda®), abexinostat, ipilimumab (Yervoy®), tremelimumab, pazopanib (Votrient®), sorafenib (Nexavar®), temsirolimus (Torisel®), ramucirumab (Cyramza®), niraparib, savolitinib, borolinib (X-82), regorafenib phenytoin (Stivargo®), donafenib (multi-targeted kinase inhibitor), camrelizumab (SHR-1210), pexastimodine devasilepvec (JX-594), ramucirumab (Cyramza®), apatinib (YN968D1), encapsulated doxorubicin (Thermodox®), tivantinib (ARQ197), ADI-PEG20, binimetinib, apatinib silate, nintedanib, lirilumab, nivolumab (Opdivo®), pembrolizumab (Keytruda®), atezolizumab (Tecentriq®), avelumab (Bavencio®), durvalumab (Imfimzi®), cemiplimab-rwlc (Libtayo®), tislelizumab, and / or spartalizumab.

[0379] In some embodiments, the one or more additional therapeutic agents is a TIM-3 inhibitor, optionally wherein the TIM-3 inhibitor is MGB453 or TSR-022.

[0380] In some embodiments, 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.

[0381] In some embodiments, the one or more additional therapeutic agents are TIGIT inhibitors. In some embodiments, the one or more additional therapeutic agents are CD112R inhibitors. In some embodiments, the one or more additional therapeutic agents are TAM (Axl, Mer, Tyro) inhibitors. In some embodiments, the one or more additional therapeutic agents are STING agonists. In some embodiments, the one or more additional therapeutic agents are 4-1BB agonists.

[0382] Combination with chemotherapy drugs Suitable chemotherapeutic agents for combination and / or co-administration with the compositions of the invention include, for example, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracenedione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, and analogs or homologs thereof. Additional agents include, for example, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, dacarbazine), alkylating agents (e.g., mechlorethamine, thioTEPA, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, cis-dichlorodiamineplatinum(II) (DDP), procarbazine, Anti-mitotic drugs include cyclosporine, cyclosporine, cyclosporine tetranitrate ...

[0383] Combination with PD-1 / PD-L1 antagonists In some embodiments, the anti-IL-27 antibodies, or antigen-binding portions thereof, provided by the present disclosure are 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 pathway(s) and / or cellular processes mediated by human PD-1 / PD-L1 signaling or other human PD-1 / PD-L1-mediated functions.

[0384] Thus, provided herein are PD-1 antagonists that directly or allosterically block, antagonize, inhibit, inhibit, or reduce downstream pathways and / or cellular processes mediated by PD-1 / PD-L1 signaling, e.g., PD-1 / PD-L1 biological activities, including receptor binding and / or eliciting a cellular response to PD-1 / PD-L1. Also provided herein are PD-1 antagonists that reduce the content or amount of human PD-1 or PD-L1 produced by a cell or a subject.

[0385] In some embodiments, the disclosure provides PD-1 antagonists that bind to human PD-1 and prevent, inhibit, or reduce binding of PD-L1 to PD-1. In some aspects, the PD-1 antagonist binds to the mRNA encoding PD-1 or PD-L1 and prevents translation. In some embodiments, the PD-1 antagonist binds to the mRNA encoding PD-1 or PD-L1 and causes its degradation and / or turnover.

[0386] In some embodiments, the PD-1 antagonist inhibits PD-1 signaling or PD-1 function. In some embodiments, the PD-1 antagonist blocks the binding of PD-1 to PD-L1, PD-L2, or both PD-L1 and PD-L2. In some embodiments, the PD-1 antagonist blocks the binding of PD-1 to PD-L1. In some embodiments, the PD-1 antagonist blocks the binding of PD-1 to PD-L2. In some embodiments, the PD-1 antagonist blocks the binding of PD-1 to PD-L1 and PD-L2. In some embodiments, the PD-1 antagonist specifically binds to PD-1. In some embodiments, the PD-1 antagonist specifically binds to PD-L1. In some embodiments, the PD-1 antagonist specifically binds to PD-L2.

[0387] In some embodiments, the PD-1 antagonist inhibits the binding of PD-1 to its cognate ligand. In some embodiments, the PD-1 antagonist inhibits the binding of PD-1 to PD-L1, the binding of PD-1 to PD-L2, or the binding of PD-1 to both PD-L1 and PD-L2. In some embodiments, the PD-1 antagonist does not inhibit the binding of PD-1 to its cognate ligand.

[0388] In some embodiments, the PD-1 antagonist is an isolated monoclonal antibody (mAb) or antigen-binding fragment thereof that specifically binds to PD-1 or PD-L1. In some embodiments, the PD-1 antagonist is an antibody or antigen-binding fragment thereof that specifically binds to human PD-1. In some embodiments, the PD-1 antagonist is an antibody or antigen-binding fragment thereof that specifically binds to human PD-L1. In some embodiments, the PD-1 antagonist is an antibody or antigen-binding fragment that binds to human PD-L1 and inhibits binding of PD-L1 to PD-1. In some embodiments, the PD-1 antagonist is an antibody or antigen-binding fragment that binds to human PD-1 and inhibits binding of PD-L1 to PD-1.

[0389] Several immune checkpoint antagonists that inhibit or disrupt the interaction between PD-1 and one or both of its ligands, PD-L1 and PD-L2, are in clinical development or are currently available to clinicians to treat cancer.

[0390] Examples of anti-human PD-1 monoclonal antibodies or antigen-binding fragments thereof that may be included in the PD-1 antagonist in any of the compositions, methods, and uses provided by the present disclosure include, but are not limited to, the following: KEYTRUDA® (pembrolizumab, MK-3475, h409A11); US8952136, US8354509, US8900587, and EP2170959 (all of which are incorporated by reference in their entirety). See Merck), OPDIVO® (nivolumab, BMS-936558, MDX-1106, ONO-4538; see US 7595048, US 8728474, US 9073994, US 9067999, EP 1537878, US 8008449, US 8779105, and EP 2161336 (all of which are incorporated 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 by reference herein in its entirety; Enumeral Biomedical), PDR001 (Novartis), and REGN2810 (Regeneron). Thus, in some embodiments, the PD-1 antagonist is pembrolizumab. In some embodiments, the PD-1 antagonist is nivolumab.

[0391] Examples of anti-human PD-L1 monoclonal antibodies or antigen-binding fragments thereof that may be included in the 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, ME DI4736), TECENTRIQ® (atezolizumab, MPDL3280A, RG7446; see WO 2010 / 077634, which is incorporated herein by reference in its entirety; Roche), MDX-1105 (BMS-936559, 12A4; see US 7943743 and WO 2013 / 173223, both of which are incorporated herein by reference in their entirety; Medarex / BMS), and FAZ053 (Novartis). Thus, 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.

[0392] In some embodiments, the PD-1 antagonist is an immunoadhesin that specifically binds to human PD-1 or human PD-L1, e.g., a fusion protein comprising the extracellular portion or PD-1-binding portion of PD-L1 or PD-L2 fused to a constant region, such as the Fc region, of an immunoglobulin molecule. Examples of immunoadhesion molecules that specifically bind to PD-1 are described in WO2010 / 027827 and WO2011 / 066342, both of which are incorporated by reference in their entireties. In some embodiments, the PD-1 antagonist is AMP-224 (also known as B7-DCIg), a PD-L2-FC fusion protein that specifically binds to human PD-1.

[0393] It will be appreciated by those skilled in the art that any PD-1 antagonist that binds to PD-1 or PD-L1 and disrupts the PD-1 / PD-L1 signaling pathway is suitable for the compositions, methods, and uses disclosed herein.

[0394] In some embodiments, the PD-1 / PD-L1 antagonist is a small molecule, nucleic acid, peptide, peptidomimetic, protein, carbohydrate, carbohydrate derivative, or glycopolymer. Exemplary small molecule PD-1 inhibitors are described in Zhan et al., (2016) Drug Discov Today 21(6):1027-1036.

[0395] Combination with TIM-3 inhibitors In some embodiments, an anti-IL-27 antibody, or antigen-binding portion thereof, provided by the present disclosure is combined (e.g., administered in combination) with a TIM-3 inhibitor. The TIM-3 inhibitor can be an antibody, antigen-binding fragment thereof, immunoadhesin, fusion protein, or oligopeptide. In some embodiments, the TIM-3 inhibitor is selected from MGB453 (Novartis), TSR-022 (Tesaro), or LY3321367 (Eli Lilly). In some embodiments, the anti-IL-27 antibody, or antigen-binding portion thereof, is administered in combination with MGB453. In some embodiments, the anti-IL-27 antibody, or antigen-binding portion thereof, is administered in combination with TSR-022.

[0396] Combination with LAG-3 inhibitors In some embodiments, an anti-IL-27 antibody or antigen-binding portion thereof provided by the present disclosure is combined (e.g., administered in combination) with a LAG-3 inhibitor. The LAG-3 inhibitor can be an antibody, antigen-binding fragment thereof, immunoadhesin, fusion protein, or oligopeptide. In some embodiments, the LAG-3 inhibitor is selected from LAG525 (Novartis), BMS-986016 (Bristol-Myers Squibb), TSR-033 (Tesaro), MK-4280 (Merck & Co.), or REGN3767 (Regeneron).

[0397] Other combinations In some embodiments, an anti-IL-27 antibody, or antigen-binding portion thereof, provided by the present disclosure is combined with (e.g., administered in combination with) a TIGIT inhibitor, a kinase inhibitor (e.g., a tyrosine kinase inhibitor (TKI)), a CD112R inhibitor, a TAM receptor inhibitor, a STING agonist and / or a 4-1BB agonist, a CTLA-4 inhibitor, a CD73 inhibitor, a CD39 inhibitor, an A2AR inhibitor, an IDO inhibitor, a NEKTAR, peg-IL-2, peg-IL-10, a CD40 agonist, or a combination thereof.

[0398] Detection Method 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, are useful for diagnosing, predicting, and / or determining the progression of a disease (e.g., cancer) in a patient.

[0399] As defined herein, monitoring a subject (e.g., a human patient) for improvement of cancer means evaluating the subject for changes in disease parameters, e.g., a decrease in tumor growth. In some embodiments, the evaluation is performed at least one (1) hour after administration, e.g., 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 evaluated at one or more of the following time periods: before initiating treatment; during treatment; or after one or more components of treatment have been administered. Evaluation may include assessing the need for further treatment, e.g., whether the dosage, frequency of administration, or duration of treatment should be changed. Evaluation may also include assessing the need to add or discontinue a selected therapy, e.g., add or discontinue any of the cancer treatments described herein.

[0400] In some embodiments, the present disclosure provides methods for detecting IL-27 in a sample from a subject, the method comprising: (a) contacting the sample from the subject with a detector antibody under conditions that allow the detector antibody to form a detector antibody-IL-27 complex if IL-27 is present in the sample, wherein the detector antibody is an antibody or antigen-binding fragment thereof provided by the present disclosure; and (b) detecting the presence of the complex formed in step (a), if present.

[0401] In some embodiments, the present disclosure provides a method for detecting an IL-27-associated cancer in a subject, the method comprising: (a) contacting a sample from a subject suspected of having an IL-27-associated cancer with a detector antibody under conditions that allow the detector antibody to form a detector antibody-IL-27 complex if IL-27 is present in the sample, wherein the detector antibody is an antibody or antigen-binding portion thereof provided by the present disclosure; and (b) detecting the presence of the complex formed in step (a), if present. In some embodiments, the detector antibody is conjugated to a detectable label. In some embodiments, the method further comprises contacting the sample with a capture antibody to form a complex comprising IL-27 and the capture antibody if IL-27 is present in the sample, wherein the capture antibody is an antibody or antigen-binding portion thereof provided by the present disclosure.

[0402] In some embodiments, the capture antibody is immobilized on a solid support. In some embodiments, the sample is contacted with the capture antibody before the detection antibody. In some embodiments, the sample is a bodily fluid sample. In some embodiments, the liquid sample is blood, serum, plasma, cell lysate, or tissue lysate.

[0403] 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]

[0404] While the present disclosure has been described with reference to specific embodiments thereof, it should be understood 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 disclosure. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps to the objective, spirit, and scope of the present disclosure. All such modifications are intended to be within the scope of the present disclosure.

[0405] Example 1: Generation and characterization of anti-IL-27 antibodies that specifically bind to the IL-27 EBI3 monomer This example describes the generation of anti-IL-27 antibodies that specifically bind to the EBI3 subunit of human IL-27. Briefly, BALB / c mice were immunized with a human EBI3 immunization vector (Aldevron) and used to generate and isolate hybridomas expressing anti-EBI3 monoclonal antibodies. The isolated hybridomas included hybridomas expressing anti-IL-27 antibody molecules designated herein as Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, and Ab8. Hybridoma supernatants were analyzed by flow cytometry of mammalian cells expressing surface-targeted human EBI3. All hybridoma supernatants tested bound to EBI3-expressing cells (data not shown).

[0406] An exemplary isolated anti-EBI3 antibody, Ab7 (hereinafter referred to as "Ab7"; hereinafter referred to as "Ab7-V"). H0 " and the immunoglobulin heavy chain variable region hereinafter referred to as "Ab7-V L0 The immunoglobulin light chain variable region (containing the immunoglobulin light chain variable region designated as "") was sequenced and further characterized below (the amino-terminal signal peptide sequence is not shown).

[0407] The heavy chain variable region of the isolated Ab7 antibody (Ab7-V H0 ) has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-constant region): [ka]

[0408] Heavy chain variable region Ab7-V H0 The nucleic acid sequence encoding has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4): [ka]

[0409] The light chain variable region of the isolated Ab7 antibody (Ab7-V L0 ) has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-constant region): [ka]

[0410] Isolated Ab7 antibody heavy chain (Ab7-V H0 -mIgG2a) has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-constant region): [ka]

[0411] Light chain variable region Ab7-V L0 The nucleic acid sequence encoding has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4): [ka]

[0412] Isolated Ab7 antibody (Ab7-V L0 The light chain of IL-16-mκ has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-constant region): [ka]

[0413] Humanized Ab7 antibodies were designed using methods known in the art. Briefly, a series of fully humanized antibodies was constructed using the V region gene sequence encoding the murine monoclonal Ab7 antibody. The variable region genes were cloned into vectors encoding the human IgG1 heavy chain constant domain and the human kappa light chain constant domain. Chimeric and humanized antibodies were transiently expressed in mammalian cells. Humanization of the isolated Ab7 heavy chain variable region resulted in five humanized heavy chain variable region variants (hereinafter referred to as "Ab7-V"). H1 ", "Ab7-V H2 ", "Ab7-V H3 ", "Ab7-V H4 " and "Ab7-V H5 Humanization of the isolated Ab7 light chain variable region resulted in four humanized light chain variable region variants (hereinafter referred to as "Ab7-V"). L1 ", "Ab7-V L2 ", "Ab7-V L3 " and "Ab7-V L4 ").

[0414] The protein sequences defining the humanized Ab7 variant variable regions and the nucleotide sequences encoding the humanized Ab7 variant variable regions are summarized below (amino-terminal signal peptide sequences are not shown).

[0415] Heavy chain variable region Ab7-V H1 has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4): [ka]

[0416] Heavy chain variable region Ab7-V H1 The nucleic acid sequence encoding has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4): [ka]

[0417] Heavy chain variable region Ab7-V H2 has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4): [ka]

[0418] Heavy chain variable region Ab7-V H2 The nucleic acid sequence encoding has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4): [ka]

[0419] Heavy chain variable region Ab7-V H3 has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4): [ka]

[0420] Heavy chain variable region Ab7-V H3 The nucleic acid sequence encoding has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4): [ka]

[0421] Heavy chain variable region Ab7-V H4 has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4): [ka]

[0422] Heavy chain variable region Ab7-V H4 The nucleic acid sequence encoding has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4): [ka]

[0423] Heavy chain variable region Ab7-V H5 has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4): [ka]

[0424] Heavy chain variable region Ab7-V H5 The nucleic acid sequence encoding has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4): [ka]

[0425] Light chain variable region Ab7-V L1 has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4): [ka]

[0426] Light chain variable region Ab7-V L1 The nucleic acid sequence encoding has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4): [ka]

[0427] Light chain variable region Ab7-V L2 has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4): [ka]

[0428] Light chain variable region Ab7-VL2 The nucleic acid sequence encoding has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4): [ka]

[0429] Light chain variable region Ab7-V L3 has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4): [ka]

[0430] Light chain variable region Ab7-V L3 The nucleic acid sequence encoding has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4): [ka]

[0431] Light chain variable region Ab7-V L4 has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4): [ka]

[0432] Light chain variable region Ab7-V L4 The nucleic acid sequence encoding has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4): [ka]

[0433] The heavy and light chain CDR amino acid sequences (Kabat definition) of the original Ab7 antibody isolated are shown in Table 1. [Table 1-1] [Table 1-2]

[0434] To generate the entire heavy or light chain sequences of the chimeric and humanized antibodies, each of the heavy chain variable regions described above was combined with a human IgG1 constant region, and each of the light chain variable regions described above was combined with a human kappa constant region.

[0435] The full protein sequences defining the heavy and light chains of the chimeric and humanized Ab7 variants are summarized below (amino-terminal signal peptide sequences not shown).

[0436] Heavy chain Ab7-V H0 - IgG1 has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-constant region): [ka]

[0437] Heavy chain Ab7-V H0 - the nucleic acid sequence encoding IgG1 has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-constant region): [ka] [ka]

[0438] Heavy chain Ab7-V H1 - IgG1 has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-constant region): [ka]

[0439] Heavy chain Ab7-V H1- the nucleic acid sequence encoding IgG1 has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-constant region): [ka] [ka]

[0440] Heavy chain Ab7-V H2 - IgG1 has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-constant region): [ka]

[0441] Heavy chain Ab7-V H2 - the nucleic acid sequence encoding IgG1 has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-constant region): [ka] [ka]

[0442] Heavy chain Ab7-V H3 - IgG1 has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-constant region): [ka]

[0443] Heavy chain Ab7-V H3 - the nucleic acid sequence encoding IgG1 has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-constant region): [ka]

[0444] Heavy chain Ab7-V H4 - IgG1 has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-constant region): [ka]

[0445] Heavy chain Ab7-V H4 - the nucleic acid sequence encoding IgG1 has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-constant region): [ka]

[0446] Heavy chain Ab7-V H5 - IgG1 has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-constant region): [ka]

[0447] Heavy chain Ab7-V H5 - the nucleic acid sequence encoding IgG1 has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-constant region): [ka]

[0448] Light chain Ab7-V L0 -κ has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-constant region): [ka]

[0449] Light chain Ab7-V L0The nucleic acid sequence encoding CDR-κ has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-constant region): [ka]

[0450] Light chain Ab7-V L1 -κ has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-constant region): [ka]

[0451] Light chain Ab7-V L1 The nucleic acid sequence encoding CDR-κ has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-constant region): [ka]

[0452] Light chain Ab7-V L2 -κ has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-constant region): [ka]

[0453] Light chain Ab7-V L2 The nucleic acid sequence encoding CDR-κ has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-constant region): [ka]

[0454] Light chain Ab7-V L3 -κ has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-constant region): [ka]

[0455] Light chain Ab7-V L3 The nucleic acid sequence encoding CDR-κ has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-constant region): [ka]

[0456] Light chain Ab7-V L4 -κ has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-constant region): [ka]

[0457] Light chain Ab7-V L4 The nucleic acid sequence encoding CDR-κ has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-constant region): [ka]

[0458] It is also contemplated that the variable region sequences can be fused to constant region sequences of other antibodies to create full-length immunoglobulin heavy and light chains. For example, Ab7-V H0 , Ab7-V H1 , Ab7-V H2 , Ab7-V H3 , Ab7-V H4 , or Ab7-V H5 The heavy chain variable region can be combined with a human IgG2, IgG3, IgG4, or IgG4 (e.g., IgG4mt or IgG4mt2) containing one or more amino acid substitutions in the constant region. L0 , Ab7-V L1 , Ab7-V L2 , Ab7-V L3 , or Ab7-V L4The light chain variable region can be combined with a human lambda constant region.

[0459] DNA fragments encoding the heavy and light chain variable regions of Ab7 and the above-mentioned humanized Ab7 variants, with flanking restriction enzyme sites, were synthesized for cloning into the pANT expression vector (Antitope) system for IgG1 heavy chains and κ light chains. All constructs were verified by sequencing. The heavy and light chain combinations shown in Table 2 were transiently transfected into HEK293 EBNA adherent cells (LGC Standards, Teddington, UK) using the PEI transfection method and incubated for 7 days after transfection. [Table 2-1] [Table 2-2]

[0460] Antibodies were purified from cell culture supernatants on a Protein A-conjugated Sepharose column (GE Healthcare, Little Chalfont, UK), buffer exchanged into 1xDPBS (pH 7.2), and analyzed using extinction coefficients (Ec) based on the predicted amino acid sequence. (0.1%) ) to OD 280nm 1 μg of each antibody was analyzed by SDS-PAGE, and bands consistent with the typical antibody profile were observed (data not shown).

[0461] In vitro characterization of anti-EBI3 antibodies The Ab7 antibodies and Ab7 antibody variants generated as described in Table 2 were tested in a series of in vitro assays to confirm their biological characteristics and activities.

[0462] A binding competition ELISA was established to evaluate the binding of humanized Ab7 antibody variants compared to the chimeric Ab7.1 antibody. Assay plates were coated with 1 μg / mL human IL-27 (hIL-27) (R&D Systems, Abingdon, UK) diluted in 1x DPBS (pH 7.2) and incubated overnight at 4°C. All antibodies were diluted to 25 μg / mL in 2% BSA / DPBS and serially diluted threefold down the plate to generate eight-point binding curves. Antibody dilutions were premixed with biotinylated Ab7.1 antibody at a constant final concentration of 0.08 μg / mL. The antibody mixture was then transferred onto the coated assay plate and incubated at room temperature for 1 hour. Binding of biotinylated Ab7.1 antibody was detected using streptavidin-peroxidase conjugate (Sigma-Aldrich, Gillingham, UK) and TMB substrate (ThermoFisher, Loughborough, UK). The reaction was stopped with 1 M HCl, and the absorbance was read at 450 nm on a Dynex Technologies MRX TC II plate reader.

[0463] IC of tested antibodies 50 Absolute values ​​were determined using a four-parameter logistic curve. IC of the chimeric antibody on each plate 50 IC normalized to 50 The results are summarized in Table 3. L4 With the exception of variants containing the light chain, all humanized Ab7 variants generated have binding profiles comparable to that of the chimeric Ab7.1 antibody, with most variants matching the chimeric Ab7.1 antibody within 2-fold. [Table 3]

[0464] The antibody was further characterized by surface plasmon resonance (SPR). Kinetic experiments were performed on a Biacore T200 (GE Healthcare, Uppsala, Sweden). All experiments were performed using HBS-P+ running buffer (pH 7.4) (GE Healthcare, Little Chalfont, UK) with hIL-27 (R&D Systems, Abingdon, UK) at 25°C. Human IL-27 (hIL-27) antigen was captured to approximately 16 RU on a CM5 chip. Immobilization was performed at a protein concentration of 1 μg / mL in 10 mM acetate buffer (pH 5.0). Antibody was used in a three-point, 3-fold dilution range from 3.3 nM to 30 nM in HBS-P+ buffer, without regeneration between concentrations. For three injections of increasing antibody concentrations, the association phase was monitored for 75 s each time, and the dissociation phase was measured once for 250 s after the last antibody injection. Regeneration of the hIL-27 surface was performed for 120 s using a single injection of 2 M MgCl2. To confirm the stability of the surface and analyte during kinetic cycling, multiple replicates (n = 4) with chimeric antibodies were performed throughout the assay.

[0465] Due to the bivalent nature of the antibodies, the data were analyzed using a 1:1 binding model and a bivalent analyte model. The results of the 1:1 binding model analysis are summarized in Table 4, and the results from the bivalent analyte model are summarized in Table 5. [Table 4-1] [Table 4-2] [Table 5-1] [Table 5-2]

[0466] Single cycle kinetics using the 1:1 model (Table 4) were consistent with results from the bivalent analyte model (Table 5) and competitive ELISA for Ab7-V. L4It was demonstrated that the humanized variants did not bind to hIL-27, and that the remaining variants bound within 2-fold of the chimeric antibody.

[0467] In summary, the binding affinity of the Ab7.1 chimeric antibody was 1 nM as determined by single-cycle kinetics using a 1:1 binding model. L4 All humanized variants had a KD of 1.5 nM or less, except for the variant containing: These results demonstrate that the Ab7 antibody and Ab7 antibody variants bind with high affinity to the EBI3 subunit of IL-27.

[0468] Example 2: Generation of anti-IL-27 antibodies that specifically bind to the EBI3 and / or p28 subunits of human IL-27 in yeast Additional anti-IL-27 monoclonal antibodies representing multiple epitope bins were selected from eight naive human synthetic yeast libraries using the methods described below.

[0469] Materials and Methods Each is about 10 9 Eight naive human synthetic yeast libraries representing a diversity of 100 were grown 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 by reference in their entirety). For the first two rounds of selection, magnetic bead sorting using the Miltenyi MACS system was performed as previously described (see, e.g., Siegel et al. (2004) J Immunol Methods 286(1-2):141-153, which is incorporated by reference in its entirety).

[0470] In summary, yeast cells (approximately 10 10The 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)) for 30 min at 30°C. After one wash 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. The yeast cells were then pelleted, resuspended in 20 ml of wash buffer, and loaded onto a Miltenyi LS column. After loading 20 ml, the column was washed three times with 3 ml of wash buffer. The column was then removed from the magnetic field, and the yeast cells were eluted with 5 ml of growth medium and then grown overnight. The next round of selection was performed using flow cytometry. Approximately 2 × 10 7 Yeast cells were pelleted, washed three times with wash buffer, and incubated at 30°C with either decreasing concentrations of biotinylated antigen (100–1 nM) under equilibrium conditions, 30 nM biotinylated antigen from different species to obtain cross-species cross-reactivity, or polyspecific removal reagent (PSR) to remove nonspecific antibodies from the selection. For PSR removal, the library was incubated with a 1:10 dilution of biotinylated PSR reagent.

[0471] The yeast cells were then washed twice with wash buffer and stained with LC-FITC (1:100 dilution) and either SA-633 (1:500 dilution) or EAPE (1:50 dilution) secondary reagents for 15 minutes at 4°C. After washing twice with wash buffer, the cell pellet was resuspended in 0.3 mL of wash buffer and transferred to a strainer-capped sort tube. Sorting was performed using a FACS ARIA sorter (BD Biosciences) to determine sort gates for selecting antibodies with the desired properties. Selection rounds were repeated until a population possessing all of the desired properties was obtained. After the final round of sorting, the yeast cells were plated, and individual colonies were harvested for characterization.

[0472] Light chain diversification To further discover and improve the antibody, a light chain diversification protocol was used during the main discovery phase.

[0473] Light chain batch diversification protocol: Heavy chain plasmids resulting from naive library selection were extracted from yeast by disruption and excision, propagated in E. coli, and subsequently purified from E. coli, yielding 5 x 10 6 This was converted into a light chain library with a diversity of 1. Selection was performed by one round of MACS and four rounds of FACS using the same conditions as for the discovery of the naive library.

[0474] Antibody Optimization Antibody optimization was carried out by introducing diversity into the heavy and light chain variable regions as described below.

[0475] CDRH1 and CDRH2 selection: CDRH3 of a single antibody was selected from 1 × 10 8 The resulting CDRH1 and CDRH2 variants were recombined into a pre-generated library containing a diversity of CDRH1 and CDRH2 variants, and selection was performed by one round of MACS and four rounds of FACS as described for the discovery of the naive library. In different FACS rounds, the library was examined for PSR binding, species cross-reactivity, and affinity selection pressure by dilution or pre-complexing with the original Fab, and selection was performed to obtain populations with the desired properties.

[0476] Antibody production and purification Yeast clones were grown to saturation and then induced for 48 hours with shaking at 30°C. After induction, yeast cells were pelleted and the supernatant was collected 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 by KappaSelect (GE Healthcare LifeSciences).

[0477] ForteBio K D measurement ForteBio affinity measurements were performed on an Octet RED384 generally as previously described (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 onto an AHQ sensor online. The sensor was equilibrated offline in assay buffer for 30 minutes and then monitored online for 60 seconds to establish a baseline. The IgG-loaded sensor was exposed to 100 nM antigen for 3 minutes and then transferred to assay buffer for 3 minutes for off-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 the antigen. The affinity measurements of anti-IL-27 antibodies are shown in Figure 1.

[0478] ForteBio Epitope Binning / Ligand Blocking 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 the available Fc binding sites on the sensor were blocked with an irrelevant human IgG1 antibody. The sensor was then exposed to 100 nM of target antigen, followed by a second anti-target antibody or ligand. Additional binding by the second antibody or ligand after antigen binding indicates an available epitope (non-competitor), whereas no binding indicates epitope blocking (competitor or ligand blocking).

[0479] MSD-SET kinetics assay Equilibrium affinity measurements were performed as previously described (Estep et al., 2013). Solution equilibrium titration (SET) was performed with antigen held constant at 10–100 pM in PBS + 0.1% IgG-free BSA (PBSF) and incubated with 3–5-fold serial dilutions of antibody starting at 5–100 nM (experimental conditions were sample-dependent). Antibody (20 nM in PBS) was coated onto a standard MSD-ECL binding plate overnight at 4°C or for 30 minutes at room temperature. The plate was then blocked for 30 minutes with shaking at 700 rpm, followed by three washes with wash buffer (PBSF + 0.05% Tween® 20). SET samples were applied and incubated on the plate for 150 seconds with shaking at 700 rpm, followed by one wash. Antigen captured on the plate was detected by incubating the plate with 250 ng / mL Sulfotag-labeled streptavidin in PBSF for 3 minutes. The plate was washed three times with wash buffer and then read on an MSD Sector Imager 2400 instrument using 1x Read Buffer T containing detergent. Percent free antigen was plotted in Prism as a function of diluted antibody and fit to a quadratic equation to obtain K D To improve throughput, a liquid handling robot was used throughout the MSD-SET experiments, including SET sample preparation.

[0480] Example 3: Binding of anti-IL-27 antibodies to recombinant human IL-27 The ability of anti-IL-27 antibodies described in Example 2 to bind recombinant human IL-27 was assessed by ELISA. Briefly, Nunc MaxiSorp ELISA plates (Affymetrix #44-2404-21) were coated with 100 μL / well of 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 three 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 decanted, and 100 μL of the indicated diluted control and anti-IL-27 antibodies were added per well. A 10-point serial dilution was made for each antibody by diluting the antibody 1:10, starting with a top concentration of 1 μg / mL. The plates were incubated for 1-2 hours with shaking at room temperature. The plates were washed three times with 100 μL / well of wash buffer. 100 μL / well of anti-human IgG secondary antibody (SouthernBiotech; catalog number 2014-05) (diluted 1:5000 in blocking buffer) was added. The plates were then incubated for 1 hour with shaking at room temperature. After the 1-hour incubation, the plates were washed three times with 100 μL / well of wash buffer. To settle the plates, 100 μL / well of TMB buffer (Life Technologies #00-2023) was added. Blue color development was observed in the standard curve wells. As soon as the highest concentration of diluted control antibody reached a deep blue color (5-10 minutes), 50 μL / well of stop solution (Thermo Fisher #SS04) was added (the color changed to yellow). The developed plate was read at 450 nm within 30 minutes of stopping the reaction (the reading at 570 nm was subtracted for wavelength correction).

[0481] The anti-IL-27 antibodies bind to recombinant human IL-27, as shown in Figure 2. An IgG isotype control antibody (IgG control) was used as a comparative control.

[0482] For example, biochemical affinity and specificity studies showed that the anti-IL-27 antibody SRF388 binds to the p28 subunit (but not the EBI3 subunit) of the heterodimeric cytokine IL-27. SRF388 bound to recombinant IL-27 from human, nonhuman primates, and rodents, with the extent of binding varying between species. The binding specificity of SRF388 to IL-27 was confirmed by testing against a panel of approximately 4,500 cell surface and soluble molecules, and no off-target binding was observed. The binding specificity of IL-27 to its receptor, IL-27RA (WSX-1), was also confirmed; no other cell surface receptors bound human IL-27. The ability of SRF388 to block the interaction between human IL-27 and IL-27RA (WSX-1) was confirmed by surface plasmon resonance.

[0483] The binding of the antibodies disclosed herein was evaluated in several model systems. Because human IL-27 is biologically active in mouse cells, we utilized systemic overexpression of human IL-27 in mice using minicircle DNA delivery and analyzed IL-27-mediated in vivo effects by whole-genome microarray analysis, flow cytometry, and serum cytokine analysis. Many of the markers regulated by IL-27 in vivo were consistent with findings in human cell-based assays. SRF381 was also evaluated in a disseminated B16 tumor model. In this setting, treatment with SRF381 demonstrated phenotypes consistent with those observed in mice lacking various components of the IL-27 ligand (IL-27 p28, EBI3) or receptor (IL-27RA).

[0484] Collectively, these studies demonstrate that SRF388 (and its sister, SRF381) can mimic the phenotype of IL-27 deficiency in mice, bind IL-27 specifically and with high affinity, and inhibit its immunosuppressive effects alone or in combination with PD-L1 blockade.

[0485] Example 4: Anti-IL27 antibodies inhibit STAT1 phosphorylation in vitro IL-27 signaling through the IL-27 receptor (IL-27R) results in phosphorylation of the signal transducer and activator of transcription 1 (STAT1) polypeptide (pSTAT1). The anti-IL-27 antibodies described in Example 2 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.

[0486] Anti-IL-27 antibodies were tested for their ability to inhibit IL-27-mediated STAT1 phosphorylation in human whole blood. Briefly, EDTA-anticoagulated human whole blood stored at room temperature was used in this assay. 45 μL of blood was dispensed into each well of a round-bottom, deep-well plate (Phenix #850356) and warmed at 37°C on a plate warmer (EchoTherm IC20) or in a 37°C incubator for 30 minutes. Anti-IL-27 antibodies were diluted to 10x the highest concentration in endotoxin-free PBS (Teknova #P0300) in a polypropylene V-bottom plate (Corning #3363). Anti-IL-27 antibodies were serially diluted in endotoxin-free PBS as desired. For unstimulated and stimulated controls, PBS alone was added to the wells. 5 μL of each dilution was added to a well containing 45 μL of blood and mixed by shaking for 15 seconds at 1000 RPM on an Eppendorf MixMate plate shaker. Plates were incubated for 60 minutes at 37°C in a plate warmer or in a 37°C incubator.

[0487] 10 μg / vial of recombinant human IL-27 (R&D Systems #2526-IL) was reconstituted to 100 μg / mL by adding 100 μL of 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 of incubation, 5 μL of 200 ng / mL rhIL-27 was added to each well of stimulated blood. 5 μL of PBS was added to unstimulated control wells. The plates were shaken at 1000 RPM for 15 seconds on a plate shaker. The plates were incubated at 37°C for 30 minutes.

[0488] After a 30-minute incubation, cells were fixed. Lysis / Fixation 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 / Fixation Reagent was added to each well of the deep-well plate, and the plate was mixed for 15 seconds at 1000 RPM on a plate shaker. The plate was incubated at 37°C for 15 minutes.

[0489] After 15 minutes of incubation, the plates were centrifuged at 1500 RPM for 5 minutes at room temperature (Eppendorf centrifuge 5810R), and the supernatant was discarded by flicking. One mL of endotoxin-free PBS was added per well, and the plates were shaken at 1000 RPM for 15 seconds on a plate shaker. The plates were 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.

[0490] The cell pellet was resuspended in 50 μL of CD14-Pacific Blue (Biolegend #325616) at 1:200 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 in the dark at room temperature for 30 minutes.

[0491] After 30 minutes of incubation, 150 μL of FACS buffer was added to each well, and the plates were centrifuged at 1500 RPM for 5 minutes at room temperature. The cell pellet was then resuspended in 100 μL of Perm III (stored at -20°C) (BD #558050) by pipetting, and the plates were sealed with plate sealers and lids. The plates were then incubated at -20°C overnight or at 4°C for 15 minutes.

[0492] 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 50 μL of staining cocktail prepared as described in Table 6 below. [Table 6]

[0493] The plate was incubated in the dark at room temperature 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 by flicking, and the plate was resuspended in 100 μL of FACS buffer for analysis by flow cytometry.

[0494] As shown in Figure 3A, anti-IL-27 antibodies inhibit STAT1 phosphorylation in human whole blood. Anti-IL-27 antibody Ab14 had an IC of 24.7 ng / mL in human whole blood. 50inhibited STAT1 phosphorylation.

[0495] The anti-IL-27 antibodies described in Example 2 were further tested by flow cytometry for their ability to inhibit IL-27-mediated STAT1 phosphorylation in pooled human PBMCs. Briefly, cryovials of human PBMCs (peripheral blood mononuclear cells) obtained from buffy coats were removed from liquid nitrogen storage and quickly thawed in a 37°C water bath. The contents of each cryovial were removed with a P1000 pipette and transferred to a 15 mL Falcon conical tube. 2–3 mL of complete RPMI-1640 (Gibco, 61870-036) was slowly added to the thawed cells, and the cells were suspended by gentle swirling or flicking. The conical tube was filled to 10 mL with complete RPMI-1640 and mixed by inversion. The conical tube was centrifuged at 1400 RPM for 8 minutes at room temperature.

[0496] PBMC cells were resuspended in warm serum-free RPMI-1640 at a density of 4,000,000 cells per mL and plated in a round-bottom 96-well plate (Costar, 3799) at a density of 200,000 cells per well (50 μL). Anti-IL-27 antibody was diluted in serum-free RPMI-1640 to a maximum concentration of 40 μg / mL in column 1 of the 96-well polypropylene plate (resulting in a final concentration of 10 μg / mL). Serial dilutions were per...

Claims

1. 1. A composition comprising a detector antibody for use in a method for detecting IL-27, EBI3, or p28 in a sample from a subject, the method comprising: (a) contacting a sample from the subject with the detector antibody under conditions that allow the detector antibody to form a detector antibody-EBI3 complex or a detector antibody-p28 complex if IL-27, EBI3, or p28 is present in the sample; and (b) detecting the presence of the complex formed in step (a), if any, wherein the detector antibody comprises a monoclonal antibody, or antigen-binding portion thereof, that specifically binds to human IL-27, wherein the antibody or antigen-binding portion thereof does not inhibit STAT1 phosphorylation, and wherein the antibody or antigen-binding portion thereof: (i) heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 706, 707, and 708, respectively, and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 714, 715, and 716, respectively; (ii) heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 750, 751, and 752, respectively, and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 758, 759, and 760, respectively; (iii) heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 772, 773, and 774, respectively, and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 780, 781, and 782, respectively; (iv) heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 709, 710, and 711, respectively, and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 717, 718, and 719, respectively; (v) heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 753, 754, and 755, respectively, and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 761, 762, and 763, respectively; (vi) the heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 775, 776, and 777, respectively, and the light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 783, 784, and 785, respectively; A composition comprising heavy and light chain CDRs selected from the group consisting of:

2. 1. A composition comprising a detector antibody for use in a method for detecting an IL-27-associated cancer in a subject, the method comprising: (a) contacting a sample from a subject with the detector antibody under conditions that allow the detector antibody to form a detector antibody-EBI3 complex or a detector antibody-p28 complex if IL-27, EBI3, or p28 is present in the sample; and (b) detecting the presence of the complex formed in step (a), if any, wherein the detector antibody comprises a monoclonal antibody, or antigen-binding portion thereof, that specifically binds to human IL-27, and wherein the antibody or antigen-binding portion thereof does not inhibit STAT1 phosphorylation, and wherein the antibody or antigen-binding portion thereof: (i) heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 706, 707, and 708, respectively, and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 714, 715, and 716, respectively; (ii) heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 750, 751, and 752, respectively, and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 758, 759, and 760, respectively; (iii) heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 772, 773, and 774, respectively, and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 780, 781, and 782, respectively; (iv) heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 709, 710, and 711, respectively, and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 717, 718, and 719, respectively; (v) heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 753, 754, and 755, respectively, and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 761, 762, and 763, respectively; (vi) the heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 775, 776, and 777, respectively, and the light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 783, 784, and 785, respectively; A composition comprising heavy and light chain CDRs selected from the group consisting of:

3. 3. The composition of claim 1, wherein the method further comprises contacting the sample with a capture antibody to generate a complex comprising IL-27 or EBI3 and the capture antibody if IL-27 or EBI3 is present in the sample.

4. The capture antibody (i) a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 23; (ii) a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 24; (iii) a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 25; (iv) a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 26; (v) a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 27; and (vi) a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 28 The composition of claim 3 comprising:

5. (a) the capture antibody comprises a heavy chain variable region comprising an amino acid sequence selected from the amino acid sequences set forth in SEQ ID NOs: 1, 5, 7, 9, 11, and 13, and a light chain variable region comprising an amino acid sequence selected from the amino acid sequences set forth in SEQ ID NOs: 3, 17, 19, and 21; (b) the detection antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 712, and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 720; or (c) both (a) and (b); The composition according to claim 3 or 4.

6. The composition of any one of claims 1 to 5, wherein the detection antibody is conjugated to a detectable label.

7. The composition of any one of claims 3 to 5, wherein the capture antibody is immobilized on a solid support.

8. The composition of any one of claims 3 to 5 or 7, wherein the sample is contacted with the capture antibody before the detection antibody.

9. The composition according to any one of claims 1 to 8, wherein the sample is a body fluid sample.

10. 10. The composition of claim 9, wherein the body fluid sample is blood, serum, plasma, a cell lysate, or a tissue lysate.

11. 11. The composition of any one of claims 2 to 10, 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.

12. A monoclonal antibody, or antigen-binding portion thereof, that specifically binds to human IL-27, wherein the antibody or antigen-binding portion thereof does not inhibit STAT1 phosphorylation, and the antibody or antigen-binding portion thereof: (i) heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 706, 707, and 708, respectively, and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 714, 715, and 716, respectively; (ii) heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 750, 751, and 752, respectively, and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 758, 759, and 760, respectively; (iii) heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 772, 773, and 774, respectively, and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 780, 781, and 782, respectively; (iv) heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 709, 710, and 711, respectively, and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 717, 718, and 719, respectively; (v) heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 753, 754, and 755, respectively, and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 761, 762, and 763, respectively; (vi) the heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 775, 776, and 777, respectively, and the light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 783, 784, and 785, respectively; A monoclonal antibody or antigen-binding portion thereof, comprising heavy and light chain CDRs selected from the group consisting of:

13. the antibody or antigen-binding portion thereof, (i) SEQ ID NOs: 712 and 720, respectively; (ii) SEQ ID NOs: 756 and 764, respectively; and (iii) SEQ ID NOs: 778 and 786, respectively The composition of any one of claims 1 to 11, or the monoclonal antibody or antigen-binding portion thereof of claim 12, comprising a heavy chain variable region and 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:

14. the antibody or antigen-binding portion thereof, (i) SEQ ID NOs: 722 and 724, respectively; (ii) SEQ ID NOs: 766 and 768, respectively; (iii) SEQ ID NOs: 788 and 790, respectively; (iv) SEQ ID NOs: 726 and 724, respectively; (v) SEQ ID NOs: 770 and 768, respectively; and (vi) SEQ ID NOs: 792 and 790, respectively 14. The composition of any one of claims 1 to 11 or 13, or the monoclonal antibody or antigen-binding portion thereof of claim 12 or 13, comprising a heavy chain and 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:

15. The composition of any one of claims 1 to 11, 13, or 14, or the monoclonal antibody or antigen-binding portion thereof of claims 12 to 14, wherein the antibody or antigen-binding portion thereof comprises a heavy chain variable region and a light chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 712 and 720, respectively.

16. The composition of any one of claims 1 to 11 or 13 to 15, or the monoclonal antibody or antigen-binding portion thereof of any one of claims 12 to 15, wherein the antibody or antigen-binding portion thereof antagonizes IL-27.

17. 17. The composition of any one of claims 1 to 11 or 13 to 16, or the monoclonal antibody or antigen-binding portion thereof of any one of claims 12 to 16, wherein the antibody is selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE antibodies.

18. The composition of any one of claims 1 to 11 or 13 to 17, or the monoclonal antibody, or antigen-binding portion thereof, of any one of claims 12 to 17, wherein the antibody is an IgG1 antibody or an IgG4 antibody.

19. 19. The composition of any one of claims 1 to 11 or 13 to 18, or the monoclonal antibody or antigen-binding portion thereof of any one of claims 12 to 18, wherein the antibody comprises a wild-type IgG1 heavy chain constant region or a wild-type IgG4 heavy chain constant region.

20. A composition according to any one of claims 1 to 11 or 13 to 19, or a monoclonal antibody or antigen-binding portion thereof according to any one of claims 12 to 19, wherein the antibody comprises an Fc domain comprising at least one mutation.

21. 21. The composition of claim 20, or a monoclonal antibody or antigen-binding portion thereof, comprising a variant IgG1 heavy chain constant region or a variant IgG4 heavy chain constant region.

22. 22. The composition of claim 21, or the monoclonal antibody or antigen-binding portion thereof, wherein the variant IgG4 heavy chain constant region comprises a substitution selected from S228P, L235E, L235A, and combinations thereof, according to EU numbering.

23. A pharmaceutical composition comprising the monoclonal antibody or antigen-binding portion thereof according to any one of claims 12 to 22 and a pharmaceutically acceptable carrier.

24. A nucleic acid comprising a nucleotide sequence encoding the light and heavy chains of the monoclonal antibody or antigen-binding portion thereof according to any one of claims 12 to 22.

25. 25. An expression vector comprising the nucleic acid of claim 24.

26. A cell transformed with the expression vector of claim 25.

27. 27. A method for producing a monoclonal antibody or antigen-binding portion thereof that specifically binds to human IL-27, comprising maintaining the cell of claim 26 under conditions that allow expression of the monoclonal antibody or antigen-binding portion thereof.

28. 28. The method of claim 27, further comprising obtaining the monoclonal antibody or antigen-binding portion thereof.

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