Anti-IL-27 antibodies and uses thereof

Abstract

The present disclosure relates to anti-IL-27 antibodies and antigen-binding portions thereof. The present disclosure also relates to methods for treating or alleviating one or more symptoms of a disease such as cancer by administering the antibodies or antigen-binding portions thereof. The present disclosure also relates to methods for detecting, for example, IL-27 in a subject or sample.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 779,341, filed December 13, 2018, which is incorporated herein by reference in its entirety. Technical Field

[0003] The present disclosure generally relates to compositions and methods for modulating IL-27 signaling. More specifically, the present disclosure relates to immunogenic compositions (e.g., antibodies, antibody fragments, etc.) that bind to IL-27 and modulate IL-27 signaling. Background Art

[0004] In recent years, increasing evidence has shown that the immune system is a significant barrier to tumor formation and progression. The existence of naturally occurring T cells with anti-tumor potential or activity in cancer patients has rationalized the development of immunotherapy 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 and Mellman, (2013) Immunity 39(1):1-10). Despite the existence of tumor-specific immune responses, malignant tumors often evade or avoid immune attacks through various immune regulatory mechanisms, resulting in an inability to control tumor development and progression (Motz and Coukos, (2013) Immunity 39(1):61-730). In fact, an emerging hallmark of cancer is the exploitation of these immune regulatory mechanisms and the inability to function in anti-tumor immune responses, leading to tumor evasion and immune killing escape (Hanahan and Weinberg (2011) Cell 144(5):646-674).

[0005] 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 a heterodimeric receptor composed of IL-27Rα (WSX1) and gp130 chains and mediates signal transduction through the heterodimeric receptor, which is mainly mediated by 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, usually in synergy with IL-12. Subsequent studies have shown that IL-27 exhibits complex immunomodulatory functions, resulting in pro-inflammatory or anti-inflammatory effects depending on the biological environment and experimental model used. IL-27 can drive the expression of different immune regulatory molecules in human cancer cells, which may support local deregulation of the immune response in vivo (Fabbi et al. (2017) Mediators Inflamm 3958069. Published online February 1, 2017, doi:10.1155 / 2017 / 3958069, and references therein).

[0006] Despite significant advances in cancer treatment and management, there remains a continuing need for new and effective therapies for the treatment and management of cancer. Summary of the Invention

[0007] Disclosed herein are antibodies or antigen-binding portions thereof that bind to and antagonize human IL-27 (interleukin 27) with high affinity and specificity. Also provided are nucleic acid molecules encoding the antibody molecules, expression vectors, host cells, and methods for preparing the antibody molecules. Also provided are pharmaceutical compositions comprising the antibody molecules. 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 conditions (including immune conditions and cancer). Thus, disclosed herein are compositions and methods for treating and / or diagnosing various conditions (including cancer and immune conditions) using anti-IL-27 antibody molecules.

[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 a heavy chain CDR and a light chain CDR selected from the group consisting of:

[0009] (i) the heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 706, 707, and 708, respectively, and the light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 714, 715, and 716, respectively;

[0010] (ii) the heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 728, 729, and 730, respectively, and the light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 736, 737, and 738, respectively;

[0011] (iii) the heavy chain CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 750, 751, and 752, respectively, and the light chain CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 758, 759, and 760, respectively; and

[0012] (iv) the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 772, 773 and 774, respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown 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 CDR and a light chain CDR selected from the group consisting of:

[0014] (i) the heavy chain CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 709, 710, and 711, respectively, and the light chain CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 717, 718, and 719, respectively;

[0015] (ii) the heavy chain CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 731, 732, and 733, respectively, and the light chain CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 739, 740, and 741, respectively;

[0016] (iii) the heavy chain CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 753, 754, and 755, respectively, and the light chain CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 761, 762, and 763, respectively; and

[0017] (iv) the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 775, 776 and 777, respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 783, 784 and 785, respectively.

[0018] In one aspect, the present 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 shown in SEQ ID NOs: 706, 707, and 708, respectively, and the light chain CDR1, CDR2, and CDR3 sequences are shown in SEQ ID NOs: 714, 715, and 716, respectively.

[0019] In one aspect, the present 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 shown in SEQ ID NOs: 728, 729, and 730, respectively, and the light chain CDR1, CDR2, and CDR3 sequences are shown in SEQ ID NOs: 736, 737, and 738, respectively.

[0020] In one aspect, the present 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 shown in SEQ ID NOs: 750, 751, and 752, respectively, and the light chain CDR1, CDR2, and CDR3 sequences are shown in SEQ ID NOs: 758, 759, and 760, respectively.

[0021] In one aspect, the present 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 shown in SEQ ID NOs: 772, 773, and 774, respectively, and the light chain CDR1, CDR2, and CDR3 sequences are shown in SEQ ID NOs: 780, 781, and 782, respectively.

[0022] In one aspect, the present 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 wherein the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 720, 742, 764, and 786.

[0023] In one aspect, the present 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:

[0024] (i) SEQ ID NOs: 712 and 720, respectively;

[0025] (ii) SEQ ID NOs: 734 and 742, respectively;

[0026] (iii) SEQ ID NOs: 756 and 764, respectively; and

[0027] (iv) SEQ ID NOs: 71, 775 and 786, respectively.

[0028] In one aspect, the present 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 that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 712, 734, 756, and 778; and wherein the light chain variable region comprises an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 720, 742, 764, and 786.

[0029] In one aspect, the present 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 that is at least 90% identical to an amino acid sequence selected from the group consisting of:

[0030] (i) SEQ ID NOs: 712 and 720, respectively;

[0031] (ii) SEQ ID NOs: 734 and 742, respectively;

[0032] (iii) SEQ ID NOs: 756 and 764, respectively; and

[0033] (iv) SEQ ID NOs: 778 and 786, respectively.

[0034] In one aspect, the present 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 shown in SEQ ID NOs: 712 and 720, respectively.

[0035] In one aspect, the present 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 that is at least 90% identical to the amino acid sequence shown in SEQ ID NOs: 712 and 720, respectively.

[0036] In one aspect, the present 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 shown in SEQ ID NOs: 734 and 742, respectively.

[0037] In one aspect, the present 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 that is at least 90% identical to the amino acid sequence shown in SEQ ID NOs: 734 and 742, respectively.

[0038] In one aspect, the present 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 shown in SEQ ID NOs: 756 and 764, respectively.

[0039] In one aspect, the present 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 that is at least 90% identical to the amino acid sequence shown in SEQ ID NOs: 756 and 764, respectively.

[0040] In one aspect, the present 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 shown in SEQ ID NOs: 778 and 786, respectively.

[0041] In one aspect, the present 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 that is at least 90% identical to the amino acid sequence shown in SEQ ID NOs: 778 and 786, respectively.

[0042] In one aspect, the present 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 wherein the light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 724, 746, 768, and 790.

[0043] In one aspect, the present 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 that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 722, 744, 766, and 788; and wherein the light chain comprises an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 724, 746, 768, and 790.

[0044] In one aspect, the present 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 wherein the light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 724, 746, 768, and 790.

[0045] In one aspect, the present 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 that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 726, 748, 770, and 792; and wherein the light chain comprises an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 724, 746, 768, and 790.

[0046] In one aspect, the present 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:

[0047] (i) SEQ ID NOs: 722 and 724, respectively;

[0048] (ii) SEQ ID NOs: 744 and 746, respectively;

[0049] (iii) SEQ ID NOs: 766 and 768, respectively; and

[0050] (iv) SEQ ID NOs: 788 and 790, respectively.

[0051] In one aspect, the present 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 that is at least 90% identical to an amino acid sequence selected from the group consisting of:

[0052] (i) SEQ ID NOs: 722 and 724, respectively;

[0053] (ii) SEQ ID NOs: 744 and 746, respectively;

[0054] (iii) SEQ ID NOs: 766 and 768, respectively; and

[0055] (iv) SEQ ID NOs: 788 and 790, respectively.

[0056] In one aspect, the present 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:

[0057] (i) SEQ ID NOs: 726 and 724, respectively;

[0058] (ii) SEQ ID NOs: 748 and 746, respectively;

[0059] (iii) SEQ ID NOs: 770 and 768, respectively; and

[0060] (iv) SEQ ID NOs: 792 and 790, respectively.

[0061] In one aspect, the present 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 that is at least 90% identical to an amino acid sequence selected from the group consisting of:

[0062] (i) SEQ ID NOs: 726 and 724, respectively;

[0063] (ii) SEQ ID NOs: 748 and 746, respectively;

[0064] (iii) SEQ ID NOs: 770 and 768, respectively; and

[0065] (iv) SEQ ID NOs: 792 and 790, respectively.

[0066] 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 shown in SEQ ID NOs: 722 and 724, respectively.

[0067] In one aspect, the present 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 that is at least 90% identical to the amino acid sequence shown in SEQ ID NOs: 722 and 724, respectively.

[0068] 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 shown in SEQ ID NOs: 744 and 746, respectively.

[0069] In one aspect, the present 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 that is at least 90% identical to the amino acid sequence shown in SEQ ID NOs: 744 and 746, respectively.

[0070] 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 shown in SEQ ID NOs: 766 and 768, respectively.

[0071] In one aspect, the present 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 that is at least 90% identical to the amino acid sequence shown in SEQ ID NOs: 766 and 768, respectively.

[0072] 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 shown in SEQ ID NOs: 788 and 790, respectively.

[0073] In one aspect, the present 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 that is at least 90% identical to the amino acid sequence shown in SEQ ID NOs: 788 and 790, respectively.

[0074] 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 shown in SEQ ID NOs: 726 and 724, respectively.

[0075] In one aspect, the present 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 that is at least 90% identical to the amino acid sequence shown in SEQ ID NOs: 726 and 724, respectively.

[0076] 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 shown in SEQ ID NOs: 748 and 746, respectively.

[0077] In one aspect, the present 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 that is at least 90% identical to the amino acid sequence shown in SEQ ID NOs: 748 and 746, respectively.

[0078] 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 shown in SEQ ID NOs: 770 and 768, respectively.

[0079] In one aspect, the present 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 that is at least 90% identical to the amino acid sequence shown in SEQ ID NOs: 770 and 768, respectively.

[0080] 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 shown in SEQ ID NOs: 792 and 790, respectively.

[0081] In one aspect, the present 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 that is at least 90% identical to the amino acid sequence shown in SEQ ID NOs: 792 and 790, respectively.

[0082] In some embodiments, the present disclosure provides a pharmaceutical composition comprising the isolated monoclonal antibody or antigen-binding portion thereof according to the above aspects, and a pharmaceutically acceptable carrier.

[0083] In some embodiments, the present disclosure provides a nucleic acid comprising a nucleotide sequence encoding the light chain, heavy chain, or both the light chain and heavy chain of the isolated monoclonal antibody or antigen-binding portion thereof of the above aspects.

[0084] In some embodiments, the present disclosure provides an expression vector comprising the above-mentioned nucleic acid.

[0085] In some embodiments, the present disclosure provides a cell transformed with the above-mentioned expression vector.

[0086] In some embodiments, the present disclosure provides that the above-mentioned isolated monoclonal antibody or antigen-binding fragment thereof can specifically bind to human IL-27 and / or specifically antagonize human IL-27.

[0087] In some embodiments, the present disclosure provides a method for producing a monoclonal antibody or antigen-binding portion thereof that specifically binds to human IL-27, the method comprising maintaining the above-described cells under conditions that permit 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.

[0088] In some embodiments, the present disclosure provides a method of inhibiting or reducing STAT1 and / or STAT3 phosphorylation in a cell, the method comprising contacting the cell with any one of the isolated monoclonal antibodies or antigen-binding fragments described above, wherein the antibody or antigen-binding portion thereof inhibits or reduces STAT1 and / or STAT3 phosphorylation in the cell.

[0089] In some embodiments, the present disclosure provides a method of inhibiting or reducing the inhibition of CD161 expression in a cell, the method comprising contacting the cell with any one of the isolated monoclonal antibodies or antigen-binding fragments described above, wherein the antibody or antigen-binding portion thereof inhibits or reduces the inhibition of CD161 expression in the cell.

[0090] In some embodiments, the present disclosure provides a method of inhibiting or reducing PD-L1 and / or TIM-3 expression in a cell, the method comprising contacting the cell with any one of the isolated monoclonal antibodies or antigen-binding fragments described above, wherein the antibody or antigen-binding portion thereof inhibits or reduces PD-L1 and / or TIM-3 expression in the cell.

[0091] In some embodiments, the present disclosure provides a method of inducing or enhancing the secretion of one or more cytokines from a cell, the method comprising contacting the cell with any one of the isolated monoclonal antibodies or antigen-binding fragments described above, wherein the antibody or antigen-binding portion thereof induces or enhances PD-1-mediated secretion of one or more cytokines from the cell.

[0092] In some embodiments, the present disclosure provides a method of stimulating an immune response in a subject, 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.

[0093] In some embodiments, the present disclosure provides a method of treating cancer in a subject, comprising administering to the subject an effective amount of any one of the above-described isolated monoclonal antibodies or antigen-binding fragments or the above-described pharmaceutical compositions.

[0094] In some embodiments, the present disclosure provides a method of stimulating an immune response in a subject or treating cancer in a subject, the method comprising administering to the subject an effective amount of the above-described isolated monoclonal antibody or antigen-binding fragment or any one of the above-described pharmaceutical compositions, wherein the antibody or antigen-binding portion thereof or pharmaceutical composition inhibits or reduces STAT1 and / or STAT3 phosphorylation in cells, thereby stimulating the immune response or treating the cancer.

[0095] In some embodiments, the present disclosure provides a method of stimulating an immune response in a subject or treating cancer in a subject, the method comprising administering to the subject an effective amount of the isolated monoclonal antibody or antigen-binding fragment described above, or any one of the pharmaceutical compositions described above, wherein the antibody or antigen-binding portion thereof, or pharmaceutical composition inhibits or reduces the inhibition of CD161 expression in cells, thereby stimulating the immune response or treating the cancer.

[0096] In some embodiments, the present disclosure provides a method of stimulating an immune response in a subject or treating cancer in a subject, the method comprising administering to the subject an effective amount of the isolated monoclonal antibody or antigen-binding fragment described above, or any one of the pharmaceutical compositions described above, wherein the antibody or antigen-binding portion thereof, or pharmaceutical composition inhibits or reduces the expression of PD-L1 and / or TIM-3 on cells, thereby stimulating the immune response or treating the cancer.

[0097] In some embodiments, the present disclosure provides a method of stimulating an immune response in a subject or treating cancer in a subject, the method comprising administering to the subject an effective amount of the isolated monoclonal antibody or antigen-binding fragment described above, or any one of the pharmaceutical compositions described above, 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.

[0098] 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., squamous head and neck cancer), colorectal cancer, bladder cancer, endometrial cancer, prostate cancer, thyroid cancer, hepatocellular carcinoma, gastric cancer, brain cancer, lymphoma (e.g., DL-BCL), leukemia (e.g., AML), or kidney cancer (e.g., renal cell carcinoma, e.g., clear cell renal carcinoma).

[0099] In some embodiments, the present disclosure provides a method of 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 the cell to any one of the above-mentioned antibodies, or antigen-binding portions thereof, simultaneously or sequentially with the anti-PD-1 antibody, thereby enhancing one or more activities of the anti-PD-1 antibody.

[0100] In some embodiments, the present disclosure provides a pharmaceutical composition comprising an anti-PD-1 antibody and any one of the above antibodies or antigen-binding portions thereof, and a pharmaceutically acceptable carrier.

[0101] In some embodiments, the present disclosure provides a kit comprising an anti-PD-1 antibody and any one of the above antibodies or antigen-binding portions thereof for simultaneous or sequential administration, and instructions for use thereof.

[0102] In some embodiments, the present disclosure provides that any of the above-described isolated monoclonal antibodies, or antigen-binding portions thereof, are 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 drugs, cytotoxic agents, immune-based therapies, cytokines, surgical procedures, radiation procedures, activators of co-stimulatory molecules, inhibitors of inhibitory molecules, vaccines, or cellular immunotherapy, or a combination thereof.

[0103] In some embodiments, the present 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.

[0104] In some embodiments, the present disclosure provides that one or more additional therapeutic agents is a PD-1 antagonist.

[0105] 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.

[0106] 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.

[0107] In some embodiments, the present disclosure provides that the one or more additional therapeutic agents are selected from the group consisting of: sunitinib Cabozantinib Axitinib Lenvatinib Everolimus Bevacizumab Edoxetine, NKTR-214 (CD-122-biased agonist), Tivozanib Abetastat, ipilimumab Tremelimumab, pazopanib Sorafenib Sirolimus Ramucirumab Niraparib, savolitinib, vololanib (X-82), regorafenib Donafenib (multikinase inhibitor), camrelizumab (SHR-1210), pexastimogene devacirepvec (JX-594), ramucirumab Apatinib (YN968D1), encapsulated doxorubicin Tivantinib (ARQ197), ADI-PEG 20, bimetinib, apatinib mesylate, nintedanib, lirilumab, nivolumab Pembrolizumab Atezolizumab Avelumab Durvalumab Cemipril-rwlc Tislelizumab and spartalizumab.

[0108] In some embodiments, the present disclosure provides that one or more additional therapeutic agents is a TIM-3 inhibitor, optionally wherein the TIM-3 inhibitor is MGB453 or TSR-022.

[0109] 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.

[0110] In some embodiments, the present disclosure provides that the one or more additional therapeutic agents is a TIGIT inhibitor.

[0111] In some embodiments, the present disclosure provides that the one or more additional therapeutic agents is a CD112R inhibitor.

[0112] In some embodiments, the present disclosure provides that the one or more additional therapeutic agents is a TAM (Axl, Mer, Tyro) inhibitor.

[0113] In some embodiments, the present disclosure provides that the one or more additional therapeutic agents is a 4-1BB agonist.

[0114] In some embodiments, the present disclosure provides that the one or more additional therapeutic agents is a tyrosine kinase inhibitor (TKI).

[0115] In one aspect, the present disclosure provides an isolated SRF388 monoclonal antibody or an antigen-binding portion thereof. In some embodiments, the present disclosure provides that the isolated SRF388 monoclonal antibody or its antigen-binding portion is further administered in combination with one or more additional therapeutic agents or procedures, the additional therapeutic agents or procedures being selected from the group consisting of: chemotherapy, targeted anti-cancer therapy, oncolytic drugs, cytotoxic agents, immune-based therapies, cytokines, surgical procedures, radiation procedures, activators of costimulatory molecules, inhibitors of inhibitory molecules, vaccines or cellular immunotherapy or a combination thereof. In some embodiments, the present disclosure provides that one or more additional therapeutic agents are PD-1 antagonists, PD-L1 inhibitors, TIM-3 inhibitors, LAG-3 inhibitors, TIGIT inhibitors, CD112R inhibitors, TAM inhibitors, STING agonists, 4-1BB agonists, CTLA-4 inhibitors, CD73 inhibitors, CD39 inhibitors, A2AR inhibitors, IDO inhibitors, NEKTAR, peg-IL-2, pegIL-10, CD40 agonists or a combination thereof. 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.

[0116] In one aspect, the present disclosure provides a method for detecting IL-27 or EBI3 or p28 in a sample from a subject, the method comprising the following steps: (a) contacting the sample from the subject with a detection antibody under conditions allowing the detection antibody to form a detection antibody-EBI3 complex or a detection antibody-p28 complex in the presence of IL-27 or EBI3 or p28 in the sample, wherein the detection antibody is any one of the above-mentioned antibodies or antigen-binding fragments thereof or any antibody described in Table 12; and (b) detecting the presence of the complex (if any) produced in step (a).

[0117] In one aspect, the present disclosure provides a method for detecting IL-27-related cancer in a subject, the method comprising the steps of: (a) contacting a sample from a subject suspected of having an IL-27-related cancer with a detection antibody under conditions that allow the detection antibody to form a detection antibody-EBI3 complex or a detection antibody-p28 complex in the presence of IL-27 or EBI3 or p28 in the sample, wherein the detection antibody is any one of the above-mentioned antibodies or antigen-binding portions thereof or any antibody described in Table 12; and (b) detecting the presence of the complex (if any) produced in step (a).

[0118] In some embodiments, the detection antibody is conjugated to a detectable label.

[0119] In some embodiments, the method further comprises contacting the sample with a capture antibody to produce a complex comprising IL-27 or EBI3 and the capture antibody in the presence of IL-27 or EBI3 in the sample, wherein the capture antibody is any one of the above antibodies or antigen-binding portions thereof.

[0120] In some embodiments, the capture antibody has one or more CDRs of Ab7, optionally is Ab7.

[0121] In some embodiments, the capture antibody is immobilized on a solid support.

[0122] In some embodiments, the sample is contacted with the capture antibody prior to the detection antibody.

[0123] In some embodiments, the sample is a bodily fluid sample.

[0124] In some embodiments, the body fluid sample is blood, serum, plasma, cell lysate, or tissue lysate.

[0125] 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.

[0126] In some embodiments, the cancer is renal cell carcinoma (RCC), or the cancer is hepatocellular carcinoma (HCC).

[0127] In some embodiments, the cancer is selected from leukemia and lymphoma, or the cancer is ovarian cancer.

[0128] In some embodiments, the present disclosure provides use of any of the above-described isolated monoclonal antibodies, or antigen-binding portions thereof, or the above-described pharmaceutical compositions for stimulating an immune response in a subject or for treating cancer in a subject, optionally in combination with one or more additional therapeutic agents or procedures.

[0129] In some embodiments, the present disclosure provides a kit comprising any of the above-described isolated monoclonal antibodies or antigen-binding portions thereof or the above-described pharmaceutical compositions and instructions for use for stimulating an immune response in a subject or treating cancer in a subject, optionally with instructions for use in combination with one or more additional therapeutic agents or procedures.

[0130] 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 detecting IL-27 in a sample from a subject, optionally with instructions for detecting an IL-27-associated cancer in the subject.

[0131] In some embodiments, the present disclosure provides that the isolated monoclonal antibody, or antigen-binding portion thereof, described above comprises an antibody, or antigen-binding portion thereof, that antagonizes IL-27.

[0132] In some embodiments, the present disclosure provides that the above-mentioned isolated monoclonal antibody or its antigen-binding portion thereof comprises an antibody or its antigen-binding portion thereof that inhibits or reduces STAT1 and / or STAT3 phosphorylation in a cell. In some embodiments, the present disclosure provides that the above-mentioned cell is an immune cell, or the cell is a cancer cell.

[0133] In some embodiments, the present disclosure provides that any of the above-described isolated monoclonal antibodies or antigen-binding portions thereof include antibodies or antigen-binding portions thereof that inhibit or reduce inhibition of CD161 expression in cells. In some embodiments, the present disclosure provides that the cell is an immune cell.

[0134] In some embodiments, the present disclosure provides that the above-mentioned isolated monoclonal antibodies or antigen-binding portions thereof include antibodies or antigen-binding portions thereof that inhibit or reduce the expression of PD-L1 and / or TIM-3 in cells. In some embodiments, the present disclosure provides that the cells are immune cells or cancer cells. In some embodiments, when the cells are cancer cells, the antibodies or antigen-binding portions thereof inhibit or reduce the expression of PD-L1 in cancer cells.

[0135] In some embodiments, the present disclosure provides that the above-mentioned isolated monoclonal antibodies or antigen-binding portions thereof include antibodies or antigen-binding portions thereof that induce or enhance PD-1-mediated secretion of one or more cytokines from cells. 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 cells are immune cells.

[0136] In some embodiments, the present disclosure provides that the isolated monoclonal antibody or antigen-binding portion thereof comprises 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.

[0137] 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 mutant IgG1 heavy chain constant region. In some embodiments, the antibody comprises a mutant IgG4 heavy chain constant region. In some embodiments, according to EU numbering, the mutant IgG4 heavy chain constant region comprises any one of the substitutions S228P, L235E, L235A, or a combination thereof.

[0138] In one aspect, the disclosure provides an isolated monoclonal antibody, or antigen-binding portion thereof, that binds to substantially the same epitope as any of the above antibodies, or antigen-binding portions thereof.

[0139] In one aspect, the disclosure provides an isolated monoclonal antibody, or antigen-binding portion thereof, that binds to at least one amino acid residue to which any of the above antibodies, or antigen-binding portions thereof, binds.

[0140] In one aspect, the present disclosure provides an isolated monoclonal antibody, or antigen-binding portion thereof, wherein mutation of the epitope to which the antibody, or antigen-binding portion thereof, binds inhibits, reduces, or blocks binding to the antibody, or antigen-binding portion thereof, and binding to the antibody, or antigen-binding portion thereof, according to any of the above antibodies or antigen-binding fragments thereof.

[0141] 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 the same as or similar to the epitope bound by the antibody molecule described in Table 12.

[0142] 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 the heavy chain CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 802, 803, and 804, respectively, and the light chain CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 805, 806, and 807, respectively.

[0143] In one aspect, the present disclosure provides an isolated monoclonal antibody, or an antigen-binding portion thereof, that specifically binds 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.

[0144] In one aspect, the present disclosure provides an isolated monoclonal antibody, or antigen-binding portion thereof, that specifically binds human WSX-1, comprising a light chain constant region, wherein the light chain constant region comprises the amino acid sequence shown in SEQ ID NO: 798.

[0145] In one aspect, the present disclosure provides an isolated monoclonal antibody, or antigen-binding portion thereof, that specifically binds human WSX-1, wherein the antibody, or antigen-binding portion thereof, comprises heavy and light chain CDRs that are at least 90% identical to the group consisting of the heavy chain CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 802, 803, and 804, respectively, and at least 90% identical to the group consisting of the light chain CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 805, 806, and 807, respectively.

[0146] In one aspect, the present disclosure provides an isolated monoclonal antibody, or antigen-binding portion thereof, that specifically binds human WSX-1, wherein the antibody, or antigen-binding portion thereof, comprises heavy and light chain variable regions that are at least 90% identical to the group consisting of SEQ ID NOs: 794 and 796, respectively.

[0147] In one aspect, the present disclosure provides an isolated monoclonal antibody, or antigen-binding portion thereof, that specifically binds human WSX-1, comprising a light chain constant region, wherein the light chain constant region is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 798.

[0148] In some embodiments, the present disclosure provides a pharmaceutical composition comprising an isolated monoclonal antibody, or antigen-binding portion thereof, of any of the above-described WSX-1 binding antibodies, and a pharmaceutically acceptable carrier.

[0149] In some embodiments, the present disclosure provides a nucleic acid comprising a nucleotide sequence encoding the light chain, heavy chain, or both of the light and heavy chains of an isolated monoclonal antibody or antigen-binding portion thereof of any of the above-mentioned WSX-1 binding antibodies. In some embodiments, the present disclosure provides an expression vector comprising the aforementioned nucleic acid. In some embodiments, the present disclosure provides a cell transformed with the aforementioned expression vector. 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, the method comprising maintaining the aforementioned cell under conditions that allow 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 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-WSX-1 complex in the presence of WSX-1 in the sample, wherein the detection antibody is an antibody or antigen-binding fragment thereof according to any of the above-mentioned WSX-1 binding antibodies; and (b) detecting the presence of the complex (if any) produced in step (a).

[0150] In some embodiments, the present disclosure provides a method for detecting a WSX-1-associated cancer in a subject, the method comprising the steps of: (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 in the presence of WSX-1 in the sample, wherein the detection antibody is an antibody or antigen-binding portion thereof according to any of the above-described WSX-1 binding antibodies; and (b) detecting the presence of the complex (if any) produced in step (a).

[0151] In some embodiments, the present disclosure provides for use of an isolated monoclonal antibody, or antigen-binding portion thereof, of any of the aforementioned WSX-1 binding antibodies, or the aforementioned pharmaceutical compositions for stimulating an immune response in a subject or for treating cancer in a subject, optionally in combination with one or more additional therapeutic agents or procedures.

[0152] In some embodiments, the present disclosure provides a kit comprising an isolated monoclonal antibody or antigen-binding portion thereof of any of the above-described WSX-1 binding antibodies or a pharmaceutical composition described above and instructions for use for stimulating an immune response in a subject or treating cancer in a subject, optionally with instructions for use in combination with one or more additional therapeutic agents or procedures.

[0153] definition

[0154] Unless otherwise stated, the terms used in the claims and the specification are defined as follows.

[0155] It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0156] As used herein, "about" will be understood by persons of ordinary skill and will vary to some extent depending on the context in which it is used. If the use of the term is unclear to persons of ordinary skill, given the context in which it is used, "about" will mean up to plus or minus 10% of the specified value.

[0157] As used herein, the term "agonist" refers to any molecule that partially or completely promotes, induces, increases and / or activates the biological activity of the natural polypeptide disclosed herein. Suitable agonist molecules specifically include agonist antibodies or antibody fragments, fragments or amino acid sequence variants, peptides or proteins of natural polypeptides, etc. In some embodiments, activation in the presence of an agonist is observed in a dose-dependent manner. In some embodiments, the signal (for example, biological activity) measured 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. Also disclosed herein is a method for identifying an agonist suitable for the disclosed method. For example, these methods include, but are not limited to, binding assays such as enzyme-linked immunosorbent assay (ELISA), Forte Systems and radioimmunoassays (RIA). These assays determine the ability of an agonist to bind to a target polypeptide (e.g., a receptor or ligand) and are therefore indicative of the ability of the agonist to promote, increase, or activate the activity of the polypeptide. The efficacy of an agonist can also be determined using functional assays, such as the ability of an agonist to activate or promote the function of a polypeptide. For example, a functional assay may comprise 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 measured by its EC 50 EC value (the concentration required to activate 50% of the agonist response). 50 The lower the value, the more potent the agonist is and the lower the concentration required to activate a maximal biological response.

[0158] As used herein, the term "alanine scanning" refers to a technique for determining the contribution of a particular wild-type residue to the stability or one or more functions (e.g., binding affinity) of a given protein or polypeptide. The technique comprises replacing the wild-type residue in a polypeptide with an alanine residue, and then evaluating the stability or one or more functions (e.g., binding affinity) of the alanine-substituted derivative or mutant polypeptide and comparing it with the wild-type polypeptide. Techniques for replacing wild-type residues in polypeptides with alanine are known in the art.

[0159] The term "ameliorate" refers to any therapeutically beneficial result in the treatment of a disease state (eg, cancer), including prevention, lessening of severity or progression, remission, or cure thereof.

[0160] 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 the 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 naturally occurring amino acids, which is a carbon bound to a hydrogen, a carboxyl group, an amino group, and an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. The analogs have modified R groups (e.g., norleucine) or a modified peptide backbone, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimetics refer to compounds whose structure is different from the general chemical structure of an amino acid, but whose function is similar to that of a naturally occurring amino acid.

[0161] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Likewise, nucleotides may be referred to by their commonly accepted single-letter codes.

[0162] As used herein, "amino acid substitution" refers to the replacement of at least one existing amino acid residue in a predetermined amino acid sequence (the amino acid sequence of the starting polypeptide) with a second, different "replacement" amino acid residue. "Amino acid insertion" refers to the incorporation of at least one additional amino acid into a predetermined amino acid sequence. Although insertions typically consist of the insertion of one or two amino acid residues, larger "peptide insertions" can also be performed, such as insertions of about 3 to about 5 or even up to about 10, 15, or 20 amino acid residues. As disclosed above, the one or more inserted residues can be naturally occurring or non-naturally occurring. "Amino acid deletion" refers to the removal of at least one amino acid residue from a predetermined amino acid sequence.

[0163] As used herein, the terms "amount" or "level" are used in the broadest sense and refer to the amount, concentration or abundance of a substance (e.g., a metabolite, a small molecule, a protein, an mRNA, a marker). When referring to a metabolite or a small molecule (e.g., a drug), the terms "amount," "level," and "concentration" are often used interchangeably and generally refer to the detectable amount in a biological sample. "Elevated levels" or "increased levels" refer to an increase in the amount, concentration, or abundance of a substance in a sample relative to a control sample (such as from one or more individuals not suffering from a disease or disorder (e.g., cancer)) or an internal control. In some embodiments, the level of an increase in a substance (e.g., a drug) in a sample refers to an increase in the amount of the substance relative to the amount of the substance in a control sample by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, as determined by techniques known in the art (e.g., HPLC). A "reduced level" refers to a decrease in the quantity, concentration or abundance of a substance (e.g., a drug) in an individual relative to a control (such as one or more individuals from a patient not suffering from a disease or disorder (e.g., cancer)) or an internal control. In some embodiments, the reduced level is rarely or undetectable. In some embodiments, a decreased level of a substance (e.g., a drug) in a sample refers to a decrease in the amount of the substance by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% relative to the amount of the substance in a control sample as determined by techniques known in the art (e.g., HPLC).

[0164] 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 generally refer to the detectable amount of a protein, mRNA or marker in a biological sample. In some aspects, the detectable amount or detectable level of a protein, mRNA or marker is associated with the likelihood of response to an agent (such as those described herein). "Expression" generally refers to the process by which the information contained in a gene is converted into a structure (e.g., a protein marker such as PD-L1) that is present and functional in a cell. Thus, as used herein, "expression" may refer to transcription into a polynucleotide, translation into a polypeptide, or even polynucleotide and / or polypeptide modification (e.g., post-translational modification of a polypeptide). Fragments of transcribed polynucleotides, translated polypeptides, or polynucleotide and / or polypeptide modifications (e.g., post-translational modification of a polypeptide) should also be considered to be expressed, whether they are derived from transcripts produced by alternative splicing or degradation, or from post-translational processing of a polypeptide (e.g., by proteolysis). "Expressed genes" include genes that are transcribed into polynucleotides (as mRNA) and then translated into polypeptides, and also include those genes that are transcribed into ribonucleic acids but not translated into polypeptides (e.g., transfer RNA and ribosomal RNA). "Elevated expression," "elevated expression level," or "elevated level" refers to increased expression or elevated levels of a substance in a sample relative to a control sample (such as one or more individuals not suffering from a disease or disorder (e.g., cancer)) or an internal control. In some embodiments, the expression of an increase in a substance (e.g., a protein marker, such as PD-L1) in a sample refers to an increase in the amount of the substance relative to the amount of the substance in the control sample by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, as determined by techniques known in the art (e.g., FACS). "Reduced expression," "reduced expression level," or "reduced level" refers to the expression or reduced level of a substance (e.g., a protein marker) in an individual relative to a control (such as one or more individuals not suffering from a disease or disorder (e.g., cancer)) or an internal control. In some embodiments, reduced expression is little or no expression. In some embodiments, decreased expression of a substance (e.g., a protein marker) in a sample refers to a decrease in the amount of the substance by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% relative to the amount of the substance in a control sample as determined by techniques known in the art (e.g., FACS).

[0165] As used herein, the term "angiogenesis" or "neovascularization" refers to the process by which new blood vessels develop from pre-existing blood vessels (Varner et al., (1999) Angiogen. 3:53-60; Mousa et al., (2000) Angiogen. Stim. Inhib. 35:42-44; Kim et al., (2000) Amer. J. Path. 156:1345-1362; Kim et al., (2000) J. Biol. Chem. 275:33920-33928; Kumar et al. (2000) Angiogenesis: From Molecular to Integrative Pharm. 169-180). 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), become activated in response to growth factor or hormonal signals or hypoxic or ischemic conditions to migrate, proliferate, and differentiate into luminal structures, thereby forming new blood vessels. During ischemia, as occurs in cancer, the need for increased oxygenation and nutrient delivery apparently causes the affected tissue to secrete angiogenic factors; these factors stimulate the formation of new blood vessels. Several other terms are associated with angiogenesis.

[0166] As used herein, the term "antagonist" refers to any molecule that partially or completely blocks, inhibits or neutralizes the biological activity of a natural polypeptide disclosed herein. Suitable antagonist molecules specifically include antagonist antibodies or antibody fragments, fragments or amino acid sequence variants of natural polypeptides, peptides or proteins, etc. 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 is a method for identifying an antagonist suitable for the disclosed method. For example, these methods include, but are not limited to, binding assays such as enzyme-linked immunosorbent assay (ELISA), Forte systems, radioimmunoassays (RIA), Meso Scale Discovery assays (e.g., Meso Scale Discovery electrochemiluminescence (MSD-ECL), and bead-based Assays. These assays determine the ability of an antagonist to bind to a target polypeptide (e.g., a receptor or ligand) and are therefore indicative of the ability of the antagonist to inhibit, neutralize, or block the activity of the polypeptide. The efficacy of an antagonist antibody can also be determined using functional assays, such as the ability of the antagonist to inhibit the function of a polypeptide or agonist. For example, a functional assay can involve 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 50 The IC value is defined as the concentration required to inhibit the agonist response by 50%. 50 The lower the value, the more potent the antagonist is and the lower the concentration required to inhibit the maximal biological response.

[0167] 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 one or more downstream pathways mediated by IL-27 signaling or other IL-27-mediated functions), for example, involving a reduction (or decrease) in 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 one or more downstream pathways mediated by IL-27 signaling or other IL-27-mediated functions are described in more detail below and elsewhere herein.

[0168] 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 one or more downstream pathways mediated by IL-27 signaling or other IL-27-mediated functions. 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 mediated by IL-27 signaling or function, such as receptor binding and / or eliciting a cellular response to IL-27 or its metabolites). In some embodiments, the anti-IL-27 antagonist antibodies provided herein bind to human IL-27 and prevent, block or inhibit the binding of human IL-27 to its cognate or normal receptor (e.g., IL-27 receptor) or one or more receptor subunits (e.g., gp130 and / or IL-27Rα (also known as WSX1 / TCCR)). In some embodiments, the anti-IL-27 antagonist antibody prevents, blocks, or inhibits the binding of human IL-27 to gp130. In some embodiments, the anti-IL-27 antagonist antibody prevents, blocks, or inhibits the binding of human IL-27 to IL-27Rα. In some embodiments, the anti-IL-27 antagonist antibody prevents, blocks, or inhibits the dimerization of IL-27 monomers. In some embodiments, the anti-IL-27 antibody specifically binds to an EBI3 monomer. In some embodiments, the anti-IL-27 antibody specifically binds to an IL-27p28 monomer. In some embodiments, the anti-IL-27 antibody specifically binds to two IL-27 monomers. In some embodiments, the anti-IL-27 antibody specifically binds to a non-contiguous epitope comprising both EBI3 and p28. In some embodiments, the anti-IL-27 antibody inhibits or reduces STAT1 and / or STAT3 phosphorylation in cells. In some embodiments, anti-IL-27 antibodies inhibit or reduce the inhibition of CD161 expression in cells (e.g., improve or alleviate IL-27-mediated inhibition of CD161 expression in cells). In some embodiments, anti-IL-27 antibodies inhibit or reduce PD-L1 and / or TIM-3 expression in cells. In some embodiments, anti-IL-27 induces or enhances the secretion of one or more cytokines mediated by PD-1 from cells. In some embodiments, anti-IL-27 antagonist antibodies bind to human IL-27 and stimulate or enhance anti-tumor response. In some embodiments, anti-IL-27 antagonist antibodies bind to human IL-27 with an affinity of 15nM or less. In some embodiments, anti-IL-27 antagonist antibodies bind to human IL-27 and comprise 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.

[0169] As used herein, the term "antibody" refers to a complete antibody comprising two light chain polypeptides and two heavy chain polypeptides. Complete antibodies include different antibody isotypes, including IgM, IgG, IgA, IgD and IgE antibodies. The term "antibody" includes polyclonal antibodies, monoclonal antibodies, chimeric or chimeric antibodies, humanized antibodies, primatized antibodies, deimmunized antibodies and fully human antibodies. Antibodies can be prepared in any of a variety of species or derived from any of a variety of species, such as mammals such as humans, non-human primates (e.g., orangutans, baboons or chimpanzees), horses, cattle, pigs, sheep, goats, dogs, cats, rabbits, guinea pigs, gerbils, hamsters, rats and mice. Antibodies can be purified or recombinant antibodies. As used herein, the terms "antibody fragment," "antigen-binding fragment," or similar terms refer to antibody fragments 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. A scFv fragment is a single polypeptide chain that includes both the heavy and light chain variable regions of the antibody from which the scFv is derived. In addition, the definition of antibody also includes intrabodies, minibodies, triabodies, and diabodies, which are suitable 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.

[0170] As used herein, the term "antibody fragment" also includes, for example, single domain antibodies, such as camelized single domain antibodies. See, for example, Muyldermans et al., (2001) Trends Biochem. Sci. 26: 230-235; Nuttall et al., (2000) Curr. Pharm. Biotech. 1: 253-263; Reichmann et al., (1999) J. Immunol. Meth. 231: 25-38; PCT Application Publication Nos. WO 94 / 04678 and WO 94 / 25591 and U.S. Patent No. 6,005,079, all of which are incorporated herein by reference in their entirety. In some embodiments, the present disclosure provides single domain antibodies comprising two VH domains with modifications that enable the formation of single domain antibodies.

[0171] In some embodiments, the antigen binding fragment comprises the variable region of a heavy chain polypeptide and the variable region of a light chain polypeptide.In some embodiments, the antigen binding fragment described herein comprises the CDRs of the light chain and heavy chain polypeptides of an antibody.

[0172] The term "antigen presenting cell" or "APC" is a cell that displays a foreign antigen in complex with MHC on its surface. T cells recognize this complex via the T cell receptor (TCR). Examples of APCs include, but are not limited to, B cells, dendritic cells (DCs), peripheral blood mononuclear cells (PBMCs), monocytes (such as THP-1), B lymphoblastoid cells (such as C1R.A2, 1518B-LCL), and monocyte-derived dendritic cells (DCs). Some APCs internalize antigens by phagocytosis or receptor-mediated endocytosis.

[0173] The term "antigen presentation" refers to the process by which an APC captures an antigen and makes it available for recognition by T cells (eg, as components of MHC-I and / or MHC-II conjugates).

[0174] As used herein, the term "apoptosis" refers to a programmed cell death process that occurs in multicellular organisms (such as humans). The highly regulated biochemical and molecular events that cause apoptosis can result in observable and unique morphological changes to the cell, including blebbing, cell volume shrinkage, chromosomal DNA condensation and fracture, and mRNA decay. A common method for identifying cells (including T cells) undergoing apoptosis is to expose the cells to a protein (annexin V) conjugated with a fluorophore. Annexin V is typically used to detect apoptotic cells by its ability to bind to the phosphatidylserine on the plasma membrane outer leaflet, and binding to the phosphatidylserine on the plasma membrane outer leaflet is an early indicator that a cell is undergoing apoptosis.

[0175] As used herein, the term "B cell" (or "B lymphocyte") refers to a type of white blood cell of the lymphocyte subtype. B cells play a role in the humoral immunity component of the adaptive immune system by secreting antibodies. B cells also present antigens and secrete cytokines. B cells, unlike the other two types of lymphocytes (T cells and natural killer cells), express B cell receptors (BCRs) on their cell membranes. The BCR allows B cells to bind to specific antigens, which will initiate an antibody response against the antigen.

[0176] As used herein, the term "binds to immobilized IL-27" refers to the ability of an antibody of the present disclosure to bind to IL-27 (e.g., IL-27 expressed on the surface of a cell or attached to a solid support).

[0177] As used herein, the term "bispecific" or "bifunctional antibody" refers to an artificial hybrid antibody having two different heavy / light chain pairs and two different binding sites. Bispecific antibodies can be produced by a variety of methods, including fusion of hybridomas or linking of Fab' fragments. See, for example, Songsivilai & Lachmann, (1990) Clin. Exp. Immunol. 79:315-321; Kostelny et al., (1992) J. Immunol. 148:1547-1553.

[0178] Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain / light chain pairs, wherein the two heavy chain / light chain pairs have different specificities (Milstein and Cuello, (1983) Nature 305:537-539). The antibody variable domains with the desired binding specificity (antibody-antigen binding site) can be fused to the immunoglobulin constant domain sequence. Preferably, the heavy chain variable region is fused to the immunoglobulin heavy chain constant region (including at least a portion of the hinge region, CH2 region, and CH3 region). For further details of illustrative 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) J. Immunol. 147:60. Bispecific antibodies also include cross-linked or heteroconjugated antibodies. Heteroconjugated antibodies can be prepared using any convenient cross-linking method. Suitable cross-linking agents are well known in the art and are disclosed in U.S. Patent No. 4,676,980, along with many cross-linking techniques.

[0179] Various techniques for preparing 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, for example, Kostelny et al. (1992) J Immunol 148(5):1547-1553. Leucine zipper peptides from Fos and Jun proteins can be connected to the Fab' portions of two different antibodies by gene fusion. Antibody homodimers can be reduced to monomers at the hinge region and then oxidized to form antibody heterodimers. The method can also be used to produce antibody homodimers. The "double antibody" technology described by Hollinger et al. (1993) Proc Natl Acad Sci USA 90:6444-6448 provides an alternative mechanism for preparing bispecific antibody fragments. The fragment comprises a heavy chain variable domain (VH) connected to a light chain variable domain (VL) by a linker, and 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 preparing bispecific antibody fragments by using single-chain Fv (scFv) dimers has also been reported. See, for example, Gruber et al. (1994) J Immunol 152:5368. Alternatively, the antibody can be a "linear antibody" as described in Zapata et al. (1995) Protein Eng. 8 (10): 1057-1062. In brief, these antibodies comprise a pair of tandem Fd segments (VH-CH1-VH-CH1) that form a pair of antigen-binding regions. Linear antibodies can be bispecific or monospecific.

[0180] Antibodies with more than two valencies (eg, trispecific antibodies) are contemplated and described in, eg, Tutt et al. (1991) J Immunol 147:60.

[0181] The present disclosure also includes variant forms of multispecific antibodies, such as Wu et al. (2007) Nat Biotechnol 25 (11): Dual variable domain immunoglobulin (DVD-Ig) molecules described in 1290-1297. DVD-Ig molecules are designed so that two different light chain variable domains (VL) from two different parent antibodies are directly connected in series or connected via a short linker by recombinant DNA technology, followed by a light chain constant domain. Similarly, the heavy chain comprises two different heavy chain variable domains (VH) connected in series, followed by a constant region CH1 and an Fc region. Methods for preparing DVD-Ig molecules from two parent antibodies are further described in, for example, PCT Publication Nos. WO 08 / 024188 and WO 07 / 024715. In some embodiments, bispecific antibodies are Fab tandem immunoglobulins, in which the light chain variable region with a second specificity is fused to the heavy chain variable region of a complete antibody. Such antibodies are described in, for example, International Patent Application Publication No. WO 2015 / 103072.

[0182] As used herein, "cancer antigen" or "tumor antigen" refers to (i) a tumor-specific antigen, (ii) a tumor-associated antigen, (iii) a cell expressing a tumor-specific antigen, (iv) a cell expressing a tumor-associated antigen, (v) an embryonic antigen on a tumor, (vi) an autologous tumor cell, (vii) a tumor-specific membrane antigen, (viii) a tumor-associated membrane antigen, (ix) a growth factor receptor, (x) a growth factor ligand, and (xi) any other type of antigen or antigen-presenting cell or material associated with cancer.

[0183] As used herein, the term "cancer-specific immune response" refers to an immune response induced by the presence of a tumor, cancer cell, or cancer antigen. In certain embodiments, the response comprises proliferation of cancer antigen-specific lymphocytes. In certain embodiments, the response comprises expression and upregulation of antibodies and T cell receptors and the formation and release of lymphokines, chemokines, and cytokines. The innate and acquired immune systems interact to initiate an antigenic response against a tumor, cancer cell, or cancer antigen. In certain embodiments, the cancer-specific immune response is a T cell response.

[0184] The term "cancer" is recognized in the art and refers to a malignant tumor of epithelial or endocrine tissue, including respiratory cancer, gastrointestinal cancer, genitourinary cancer, testicular cancer, breast cancer, prostate cancer, endocrine cancer, and melanoma. The anti-IL-27 antibodies described herein can be used to treat patients who have, are suspected of having, or are at high risk of developing any type of cancer (including renal cancer or melanoma) or any viral disease. Exemplary cancers include those formed by tissues of the cervix, lung, prostate, breast, head and neck, colon, and ovary. The term also includes carcinosarcoma, which includes malignant tumors composed of carcinomatous tissue and sarcoma tissue. "Adenocarcinoma" refers to a cancer that originates from glandular tissue or in which tumor cells form a recognizable glandular structure.

[0185] As used herein, the term "CD112R" refers to a member of the poliovirus receptor-like protein and is a co-inhibitory receptor on human T cells. CD112R is preferentially expressed on T cells and inhibits T cell receptor-mediated signals. CD112, which is widely expressed on antigen-presenting cells and tumor cells, is a ligand for CD112R. CD112R competes with CD226 for binding to CD112. Disruption of the CD112R-CD112 interaction can enhance human T cell responses. CD112R acts as a new checkpoint on human T cells through 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.

[0186] As used herein, the term "CD137" (or "4-1BB") refers to a member of the tumor necrosis factor (TNF) receptor superfamily. 4-1BB is a co-stimulatory immune checkpoint molecule, primarily targeting activated T cells. Cross-linking of CD137 can enhance T cell proliferation, IL-2 secretion, survival, and cytolytic activity. As used herein, the term "4-1BB agonist" refers to an agent that stimulates, induces, or increases one or more functions of 4-1BB. An exemplary 4-1BB agonist is utolumab (PF-05082566), a fully human IgG2 monoclonal antibody that targets this 4-1BB to stimulate T cells.

[0187] 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 for T cells, and for many different cancer types, CD161 expression has been associated with T cell infiltration into the tumor microenvironment. CD161 is further described in Fergusson et al., (2014) Cell Reports 9(3): 1075-1088, which is incorporated herein by reference in its entirety.

[0188] 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 comprising a first subunit called Epstein-Barr virus-induced gene 3 (EBI3; also known as IL-27 subunit β and IL-27B) and a second subunit called IL-27p28 (also known as IL30, IL-27 subunit α and IL-27A). IL-27 is primarily synthesized by activated antigen-presenting cells (including monocytes, endothelial cells, and dendritic cells) (Jankowski et al. (2010) Arch Immunol. Ther. Exp. 58: 417-425; Diakowski et al. (2013) Adv. Clin. Exp. Med. (2013) 22 (5): 683-691). Although IL-27 can have pro-inflammatory effects, many studies have shown that IL-27 plays an important role as an immunosuppressant (Shimizu et al. (2006) J. Immunol. 176: 7317-7324; Hisada et al. (2004) Cancer Res. 64: 1152-1156; Diakowski (2013) supra). Although IL-27 was initially described as a factor that promotes the initiation of Th1 responses, it was later found that IL-27 plays a major T cell suppressive function by limiting Th1 responses, inhibiting Th2 and Th17 cell differentiation, and regulating the development of Tr1 and other T regulatory cell populations (Dietrich et al. (2014) J. Immunol. 192: 5382-5389). In addition to its role as an immunomodulator, IL-27 also regulates angiogenesis, hematopoiesis, and osteoclastogenesis (supra).

[0189] IL-27 signals through a heterodimeric type I cytokine receptor (IL-27 receptor or IL-27R), which comprises a first subunit called WSX1 (also known as IL-27 receptor subunit α, IL-27RA, T cell cytokine receptor type 1 (TCCR), and cytokine receptor-like 1 (CRL1)) and a second subunit called gp130 (also known as interleukin-6 signal transducer (IL6ST), interleukin-6 receptor subunit β (IL-6RB), and oncostatin M 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.

[0190] It is also believed that EBI3 has 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 expression (Larousserie et al. (2005) Am. J. Pathol. 166 (4): 1217-28).

[0191] The amino acid sequence of an exemplary human EBI3 protein is provided in SEQ ID NO: 698 (NCBI Reference Sequence: NP_005746.2; N-MTPQLLLALVLWASCPPCSGRKG PPAALTLPRVQCRASRYPIAVDCSWTLPPAPNSTSPVSFIATYRLGMAARGHSWPCLQQTPTSTSCTITDVQLFSMAPYVLNVTAVHPWGSSSSFVPFITEHIIKPDPPEGVRLSPLAERQLQVQWEPPGSWPFPEIFSLKYWIRYKRQGAARFHRVGPIEATSFILRAVRPRARYYVQVAAQDLTDYGELSDWSLPATATMSLGK-C). The amino acid sequence of an exemplary human p28 protein is provided in SEQ ID NO: 699 (NCBI Reference Sequence: NP_663634.2; N-MGQTAGDLGWRLSLLLLPLLLVQAGVWGFPRPPGRPQLSLQELRREFTVSLHLARKLLSEVRGQAHRFAESHLPGVNLYLLPLGEQLPDVSLTFQAWRRLSDPERLCFISTTLQPFHALLGGLGTQGRWTNMERMQLWAMRLDLRDLQRHLRFQVLAAGFNLPEEEEEEEEEEEEERKGLLPGALGSALQGPAQVSWPQLLSTYRLLHSLELVLSRAVRELLLLSKAGHSVWPLGFPTLSPQP-C).The amino acid sequence of an exemplary human WSX1 protein is provided in SEQ ID NO: 700 (NCBI reference sequence: NP_004834.1; N-MRGGRGAPFWLWPLPKLALLPLLWVLFQRTRPQGSAGPLQCYGVGPLGDLNCSWEPLGDLGAPSELHLQSQKYRSNKTQTVAVAAGRSWVAIPREQLTMSDKLLVWGTKAGQPLWPPVFVNLETQMKPNAPRLGPDVDFSEDDPLEATVHWAPPTWPSHKVLICQFHYRRCQEAAWTLLEPELKTIPLTPVEIQDLELATGYKVYGRCRMEKEEDLWGEWSPILSFQTPPSAPKDVWVSGNLCGTPGGEEPLLLWKAPGPCVQVSYKVWFWVGGRELSPEGITCCCSLIPSGAEWARVSAVNATSWEPLTNLSLVCLDSASAPRSVAVSSIAGSTELLVTWQPGPGEPLEHVVDWARDGDPLEKLNWVRLPPGNLSALLPGNFTVGVPYRITVTAVSASGLASASSVWGFREELAPLVGPTLWRLQDAPPGTPAIAWGEVPRHQLRGHLTHYTLCAQSGTSPSVCMNVSGNTQSVTLPDLPWGPCELWVTASTIAGQGPPGPILRLHLPDNTLRWKVLPGILFLWGLFLLGCGLSLATSGRCYHLRHKVLPRWVWEKVPDPANSSSGQPHMEQVPEAQPLGDLPILEVEEMEPPPVMESSQPAQATAPLDSGYEKHFLPTPEELGLLGPPRPQVLA-C).The amino acid sequence of an exemplary human gp130 protein is provided in SEQ ID NO: 701 (NCBI Reference Sequence: NP_002175.2; N-MLTLQTWLVQALFIFLTTESTGELLDPCGYISPESPVVQLHSNFTAVCVLKEKCMDYFHVNANYIVWKTNHFTIPKEQYTIINRTASSVTFTDIASLNIQLTCNILTFGQLEQNVYGITIISGLPPEKPKNLSCIVNEGKKMRCEWDGGRETHLETNFTLKSEWATHKFADCKAKRDTPTSCTVDYSTVYFVNIEVWVEAENALGKVTSDHINFDPVYKVKPNPPHNLSVINSEELSSILKLTWTNPSIKSVIILKYNIQYRTKDASTWSQIPPEDTASTRSSFTVQDLKPFTEYVFRIRCMKEDGKGYWSDWSEEASGITYEDRPSKAPSFWYKIDPSHTQGYRTVQLVWKTLPPFEANGKILDYEVTLTRWKSHLQNYTVNATKLTVNLTNDRYLATLTVRNLVGKSDAAVLTIPACDFQATHPVMDLKAFPKDNMLWVEWTTPRESVKKYILEWCVLSDKAPCITDWQQEDGTVHRTYLRGNLAESKCYLITVTPVYADGPGSPESIKAYLKQAPPSKGPTVRTKKVGKNEAVLEWDQLPVDVQNGFIRNYTIFYRTIIGNETAVNVDSSHTEYTLSSLTSDTLYMVRMAAYTDEGGKDGPEFTFTTPKFAQGEIEAIVVPVCLAFLLTTLLGVLFCFNKRDLIKKHIWPNVPDPSKSHIAQWSPHTPPRHNFNSKDQMYSDGNFTDVSVVEIEANDKKPFPEDLKSLDLFKKEKINTEGHSSGIGGSSCMSSSRPSISSSDENESSQNTSSTVQYSTVVHSGYRHQVPSVQVFSRSESTQPLLDSEERPEDLQLVDHVDGGDGILPRQQYFKQNCSQHESSPDISHFERSKQVSSVNEEDFVRLKQQISDHISQSCGSGQMKMFQEVSAADAFGPGTEGQVERFETVGMEAATDEGMPKSYLPQTVRQGGYMPQ-C).

[0192] As used herein, the term "competition," 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., a competitive binding assay; a cross-blocking assay) in which a test antigen binding protein (e.g., a test antibody) inhibits (e.g., reduces or blocks) 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).

[0193] A polypeptide or amino acid sequence "derived from" a specified polypeptide or protein refers to the source of the polypeptide. Preferably, a polypeptide or amino acid sequence derived from a particular sequence has an amino acid sequence that is substantially identical to that sequence or a portion thereof (wherein the portion consists of at least 10-20 amino acids, preferably at least 20-30 amino acids, more preferably at least 30-50 amino acids), or is otherwise recognizable by one of ordinary skill in the art as being derived from that sequence. A polypeptide derived from another peptide may have one or more mutations relative to the starting polypeptide, e.g., one or more amino acid residues that have been substituted with another amino acid residue or have one or more amino acid residue insertions or deletions.

[0194] Polypeptide can comprise non-naturally occurring amino acid sequence.Such variant must have less than 100% sequence identity or similarity with starting molecule.In certain embodiments, variant will have for example on the length of variant molecule with the amino acid sequence of starting polypeptide and have about 75% to less than 100%, more preferably about 80% to less than 100%, more preferably about 85% to less than 100%, more preferably about 90% to less than 100% (for example, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%), most preferably about 95% to less than 100% amino acid sequence identity or similarity amino acid sequence.

[0195] In certain embodiments, the starting polypeptide sequence has an amino acid difference with the sequence derived therefrom.Identity or similarity relative to the sequence are defined in this article as follows: after comparing sequences and introducing room (if necessary) to realize the sequence identity of maximum percentage, the percentage ratio of the amino acid residue identical with the initial sequence (that is, identical residues) in the candidate sequence. In certain embodiments, polypeptide is made up of the amino acid sequence of the sequence shown in Table 12, is substantially made up of or comprises the amino acid sequence. In certain embodiments, polypeptide comprises at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence with the amino acid sequence of the sequence shown in Table 12. In certain embodiments, the 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 sequence shown in Table 12. In certain embodiments, the 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 numbers) contiguous amino acids of an amino acid sequence selected from the sequence shown in Table 12.

[0196] In certain embodiments, the antibody of the present disclosure is encoded by a nucleotide sequence. The nucleotide sequence of the present invention can be used for a variety of applications, including cloning, gene therapy, protein expression and purification, mutation introduction, DNA vaccination of a host in need thereof, antibody generation (for example, passive immunity), PCR, primers and probes generation, etc. In certain embodiments, the nucleotide sequence of the present invention includes, is composed of, or is essentially composed of a nucleotide sequence selected from the sequence shown in Table 12. In certain embodiments, the nucleotide sequence includes a nucleotide sequence 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 the nucleotide sequence selected from the sequence shown 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 sequence shown in Table 12. In certain embodiments, the nucleotide sequence 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 numbers) contiguous nucleotides of a nucleotide sequence selected from the sequence shown in Table 12.

[0197] It will also be understood by those of ordinary skill in the art that antibodies suitable for use in the methods disclosed herein can be altered so that they differ in sequence from the naturally occurring or native sequences from which they are derived while retaining the desired activity of the native sequence. For example, nucleotide or amino acid substitutions that result in conservative substitutions or alterations at "non-essential" amino acid residues can be made. Mutations can be introduced by standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis.

[0198] The antibody that is suitable for method disclosed herein can comprise conservative amino acid replacement at one or more amino acid residues, for example, at essential or non-essential amino acid residues." conservative amino acid replacement " is the amino acid replacement in which amino acid residue is replaced by amino acid residues with similar side chains. Families of amino acid residues with similar side chains have been defined in the art, including basic side chains (for example, lysine, arginine, histidine), acidic side chains (for example, aspartic acid, glutamic acid), uncharged polar side chains (for example, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (for example, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (for example, threonine, valine, isoleucine) and aromatic side chains (for example, tyrosine, phenylalanine, tryptophan, histidine). Therefore, the non-essential amino acid residues in the binding polypeptide are preferably replaced by another amino acid residue from the same side chain family. In certain embodiments, a stretch of amino acids can be replaced by a structurally similar stretch that differs in sequence and / or in the composition of side chain family members. Alternatively, in certain embodiments, mutations can be introduced randomly along all or part of a coding sequence, for example, by saturation mutagenesis, and the resulting mutants can be incorporated into binding polypeptides of the invention and screened for their ability to bind to a desired target.

[0199] 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.

[0200] 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 cells that physiologically express IL-27 or otherwise functionally interacting with the cells. Methods for determining cross-reactivity include standard binding assays described herein, for example, by Biacore TM Surface plasmon resonance (SPR) analysis using Biacore TM 2000 SPR instrument (Biacore AB, Uppsala, Sweden) or flow cytometry technology.

[0201] As used herein, the term "cytotoxic T lymphocyte (CTL) response" refers to an immune response induced by cytotoxic T cells. CTL responses are primarily induced by CD8 + T cell mediated.

[0202] As used herein, the term "dendritic cell" or "DC" refers to a type of antigen-presenting cell that is a bone marrow (BM)-derived leukocyte and is the most potent antigen-presenting cell type. DCs capture and process antigens, converting proteins into peptides that are presented on major histocompatibility complex (MHC) molecules recognized by T cells. DCs are heterogeneous, such as myeloid and plasmacytoid DCs; although all DCs are able to take up antigens, process antigens, and present them to naive T cells, DC subtypes have different markers and differ in their localization, migration pathways, detailed immune functions, and dependence on infection or inflammatory stimuli (for their generation). During the development of the adaptive immune response, the phenotype and function of DCs play a role in initiating tolerance, memory, and polarized T-helper 1 (Th1), Th2, and Th17 differentiation.

[0203] As used herein, the term "dendritic cell activation" refers to the transition from immature dendritic cells to mature dendritic cells; and activated dendritic cells include mature dendritic cells and dendritic cells in the transition process, wherein the expression of CD80 and CD86 that induce costimulatory signals is activated and stimulated to increase. Mature human dendritic cells are cells that are positive for the expression of CD40, CD80, CD86, and HLA-II classes (e.g., HLA-DR). For example, based on the markers of the group consisting of CD80 and CD86, immature dendritic cells can be distinguished from mature dendritic cells. Immature dendritic cells are weakly positive for these markers, preferably negative, while mature dendritic cells are positive. The distinction between mature dendritic cells is routinely performed by those skilled in the art, and the above-mentioned respective markers and methods for measuring their expression are also well known to those skilled in the art.

[0204] As used herein, the term "EC 50 ” refers to the concentration of an antibody, or antigen-binding portion thereof, that induces a response that is 50% of the maximal response (ie, halfway between the maximal response and the baseline) in an in vitro or in vivo assay.

[0205] As used herein, the term "effective dose" or "effective dosage" is defined as an amount sufficient to achieve or at least partially achieve the desired effect. The term "therapeutically effective dose" is defined as an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. The effective amount for this use depends on the severity of the condition being treated and the general state of the patient's own immune system.

[0206] As used herein, the term "epitope" or "antigenic determinant" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds. The term "epitope mapping" refers to the process or method of identifying the binding site or epitope of an antibody or its antigen-binding fragment on its target protein antigen. Methods and techniques for epitope mapping are provided herein. An epitope can be formed by either contiguous amino acids or by non-contiguous amino acids brought into close proximity by the tertiary folding of a protein. Epitopes formed by contiguous amino acids are generally retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are generally lost upon treatment with denaturing solvents. An epitope generally comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in a unique spatial conformation. Methods for determining which epitopes a given antibody binds (i.e., epitope mapping) are well known in the art and include, for example, immunoblotting and immunoprecipitation assays, in which overlapping or contiguous peptides of IL-27 are tested for reactivity with a given anti-IL-27 antibody. Methods for determining the spatial conformation of epitopes include techniques in the art and those described herein, e.g., x-ray crystallography and two-dimensional nuclear magnetic resonance (see, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, ed. (1996)).

[0207] The present disclosure also includes antibodies that bind to an epitope on IL-27 that comprises all or a portion (e.g., the same or overlapping regions or regions between or across the regions) of an epitope recognized by a specific antibody described herein.

[0208] The present disclosure also includes antibodies that bind to the same epitope and / or antibodies that compete with the antibodies described herein for binding to human IL-27. Antibodies that recognize the same epitope or compete for binding can be identified using conventional techniques. Such techniques include, for example, immunoassays that show the ability of one antibody to block the binding of another antibody to a target antigen, i.e., competitive binding assays. Competitive binding is determined in an assay in which the immunoglobulin tested inhibits specific binding of a reference antibody to a common antigen (such as IL-27). Many types of competitive binding assays are known, for example: solid phase direct or indirect radioimmunoassays (RIAs), solid phase direct or indirect enzyme immunoassays (EIAs), sandwich competition assays (see Stahli et al., Methods in Enzymology 9:242 (1983)); solid phase direct biotin-avidin EIA (see Kirkland et al., J. Immunol. 137:3614 (1986)); solid phase direct labeled assays, solid phase direct labeled sandwich assays (see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988)); solid phase direct labeled RIA using an I-125 label (see Morel et al., Mol. Immunol. 25(1):7 (1988)); solid phase direct biotin-avidin EIA (Cheung et al., Virology 176:546 (1990)); and directly labeled RIA. (Moldenhauer et al., Scand. J. Immunol. 32:77 (1990)). Typically, such assays involve the use of purified antigen bound to a solid surface or cells bearing either, 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. Typically, the test immunoglobulin is present in excess. Typically, when the competing antibody is present in excess, it will inhibit specific binding of the reference antibody to the common antigen by at least 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, or more.

[0209] In some embodiments, the present invention relates to the epitope mapping method, for example, antigen: antibody complex crystal x-ray analysis, which provides the atomic resolution of epi-position, and the mass spectrometry analysis of hydrogen / deuterium (H / D) exchange combination, which studies the conformation and kinetics of antigen: antibody interactions. Other methods monitor the combination of antibody and antigen fragment or the mutant form of antigen, wherein the combination loss caused by the modification of amino acid residues in the antigen sequence is generally considered to be the indication of epitope components. In addition, the calculation combination method for epitope mapping can also be used. These methods rely on the ability of affinity separation of target antibody specific short peptides from combination phage display peptide library. Then peptide is considered as the leader corresponding to the epi-position definition of the antibody for screening peptide library. For epitope mapping, computational algorithms have also been developed, and described algorithms have been shown to map the discontinuous epitope of conformation.

[0210] As used herein, the term "Fc-mediated effector function" or "Fc effector function" refers to the biological activity of an antibody other than the primary function and purpose of the antibody. For example, the effector function of a therapeutic agnostic antibody is a biological activity other than the activation of a target protein or pathway. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); lack of activation of platelets expressing Fc receptors; and B cell activation. Many effector functions begin with the binding of Fc to Fcγ receptors. In some embodiments, tumor antigen-targeted antibodies have effector functions, such as ADCC activity. In some embodiments, the tumor antigen-targeted antibodies described herein comprise variant constant regions that have enhanced effector functions (e.g., enhanced ability to mediate ADCC) relative to the unmodified form of the constant region.

[0211] 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. Fcγ receptors have three subclasses: FcγRI (CD64), FcγRII (CD32), and FγcRIII (CD16). All four IgG isotypes (IgG1, IgG2, IgG3, and IgG4) bind to and activate Fc receptors FcγRI, FcγRIIA, and FcγRIIIA. FcγRIIB is an inhibitory receptor, so antibodies bound to this receptor do not activate complement and cellular responses. FcγRI is a high-affinity receptor that binds to IgG in monomeric form, while FcγRIIA and FcγRIIA are low-affinity receptors that only bind to IgG in multimeric form and have slightly lower affinity. The binding of antibodies to Fc receptors and / or C1q is controlled by specific residues or domains in the Fc region. Binding also depends on the residues located in the hinge region and in the CH2 portion of the antibody. In some embodiments, the agonist and / or therapeutic activity of the antibodies described herein is dependent on binding of the Fc region to an Fc receptor (e.g., FcγR). In some embodiments, the agonist and / or therapeutic activity of the antibodies described herein is enhanced by binding of the Fc region to an Fc receptor (e.g., FcγR).

[0212] A list of certain Fc receptor sequences employed in the present disclosure is shown in Table 13 below.

[0213] As used herein, the term "glycosylation pattern" is defined as the pattern of carbohydrate units covalently attached to a protein, more specifically an immunoglobulin. The glycosylation pattern of a heterologous antibody can be characterized as being substantially similar to the glycosylation pattern naturally occurring on antibodies produced by a non-human transgenic animal species when one of ordinary skill in the art recognizes the glycosylation pattern of the heterologous antibody as being more similar to the glycosylation pattern in the species from which the CH genes of the transgene are derived than to the glycosylation pattern in the non-human transgenic animal species.

[0214] As used herein, the term "human antibody" includes antibodies having the variable and constant regions of human germline immunoglobulin sequences, if present. 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 (ie, humanized antibodies).

[0215] As used herein, the term "heterologous antibody" is defined relative to the transgenic non-human organism that produces such antibody. The term refers to an antibody having an amino acid sequence or encoding nucleic acid sequence that corresponds to an amino acid sequence or encoding nucleic acid sequence found in an organism that does not consist of the transgenic non-human animal, and typically is from a species other than the transgenic non-human animal.

[0216] The terms "induce an immune response" and "enhance an immune response" are used interchangeably and refer to the stimulation of an immune response (ie, passive or adaptive) to a specific antigen. The term "induce" as used with respect to inducing CDC or ADCC refers to the stimulation of specific direct cell killing mechanisms.

[0217] As used herein, the term "immunogenic cell death" (or "immunogenic apoptosis") refers to a cell death mode associated with the activation of one or more signal transduction pathways that induce the pre-death expression and release of damage-associated molecular pattern (DAMP) molecules (e.g., adenosine triphosphate, ATP) from tumor cells, thereby leading to enhanced tumor cell immunogenicity and tumor cell death in an immunogenic manner (e.g., by phagocytosis). As used herein, the term "immunogenic cell death inducer" refers to a chemical, biological or pharmaceutical agent that induces an immunogenic cell death process, pathway or mode.

[0218] As used herein, the terms "inhibit," "reduce," or "block" (e.g., relating to inhibiting or reducing STAT1 and / or STAT3 phosphorylation mediated by human IL-27 in cells) are used interchangeably and encompass both partial and complete inhibition / blocking. Inhibition / blocking of IL-27 reduces or alters the normal level or type of activity that would occur in the absence of inhibition or blocking. Inhibition and blocking 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 the anti-IL-27 antibody, for example, inhibiting the binding of IL-27 by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%.

[0219] As used herein, the terms "inhibit angiogenesis," "diminishes angiogenesis," and "reduces angiogenesis" refer to reducing the level of angiogenesis in a tissue to at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or less of the amount in a corresponding control tissue, and most preferably to an amount that is the same as the level observed in the control tissue.

[0220] As used herein, the term "inhibit growth" (e.g., with respect to cells) is intended to include any measurable decrease in cell growth, such as inhibition of cell growth by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100%.

[0221] As used herein, a subject "in need of prevention," "in need of treatment," or "in need thereof" is one who, in the judgment of an appropriate medical practitioner (e.g., a physician, nurse, or paramedic in the case of a human; a veterinarian in the case of a non-human mammal), would reasonably benefit from a given treatment (such as treatment with a composition comprising an anti-IL-27 antibody).

[0222] The term "in vivo" refers to processes that occur in living organisms.

[0223] As used herein, the term "isolated antibody" refers to an antibody that is substantially free of other antibodies with different antigenic specificities (e.g., an isolated antibody that specifically binds to human IL-27 is substantially free of antibodies that specifically bind to antigens other than IL-27). However, an isolated antibody that specifically binds to an epitope may have cross-reactivity with other IL-27 proteins from different species. However, in a specific binding assay as described herein, the antibody continues to show specific binding to human IL-27. In addition, the isolated antibody is generally substantially free of other cellular material and / or chemicals. In some embodiments, a combination of "isolated" antibodies with different IL-27 specificities are combined in a well-defined composition.

[0224] As used herein, the term "isolated nucleic acid molecule" refers to a nucleic acid molecule encoding an antibody or antibody portion (e.g., V H 、V L , CDR3) nucleic acid, is intended to mean a nucleic acid molecule in which the nucleotide sequence encoding the antibody or antibody portion is free of other nucleotide sequences encoding antibodies or antibody portions that bind antigens other than IL-27, which other sequences may naturally flank the nucleic acid in human genomic DNA. For example, a sequence selected from the sequences shown in Table 12 corresponds to a heavy chain (V) comprising the anti-IL-27 antibody monoclonal antibody described herein. H ) and light chain (V L ) nucleotide sequence of the variable region.

[0225] As used herein, "isotype" refers to the antibody class (e.g., IgM or IgG1) encoded by the heavy chain constant region gene. In some embodiments, the human monoclonal antibodies of the present disclosure are IgG1 isotypes. In some embodiments, the human monoclonal antibodies of the present disclosure are IgG2 isotypes. In some embodiments, the human monoclonal antibodies of the present disclosure are IgG3 isotypes. In some embodiments, the human monoclonal antibodies of the present disclosure are IgG4 isotypes. It will be apparent to those skilled in the art that the identification of antibody isotypes (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE) is conventional in the art and generally involves a combination of sequence alignments with known antibodies, disclosed Fc variant sequences, and conserved sequences.

[0226] As used herein, the term "isotype switching" refers to a phenomenon in which the class or isotype of an antibody changes from one Ig class to another Ig class.

[0227] As used herein, the term "KD" or "K D " refers to the equilibrium dissociation constant of the binding reaction between antibody and antigen. K DThe value of K is the ratio of the antibody dissociation rate constant (kd) to the antibody association rate constant (ka). D The value of K is inversely proportional to the binding affinity of the antibody to the antigen. D The smaller the value, the greater the affinity of the antibody for its antigen. Affinity is the strength with which a single molecule binds to its ligand, usually expressed as the equilibrium dissociation constant (K D ) is measured and reported, and this constant is used to evaluate and rank the ordering strength of bimolecular interactions.

[0228] As used herein, the term "kd" or "k d ” (or “koff” or “k off ”) is intended to refer to the dissociation rate constant for the dissociation of an antibody from an antibody / antigen complex. The Kd value is expressed as the fraction of the complex that decays or dissociates per second, expressed in seconds -1 Indicated as a unit.

[0229] As used herein, the term "ka" or "k a ” (or “kon” or “k on ”) is intended to refer to the association rate constant for the association of an antibody with an antigen. The value of ka is the number of antibody / antigen complexes formed per second in a 1 molar (1 M) solution of antibody and antigen, expressed in M -1 sec -1 Indicated as a unit.

[0230] As used herein, the term "leukocyte" refers to a type of white blood cell that is involved in protecting the body from infectious organisms and foreign substances. Leukocytes are produced in the bone marrow. There are five main types of leukocytes, which are divided into two major categories: polymorphonuclear leukocytes (neutrophils, eosinophils, basophils) and mononuclear leukocytes (monocytes and lymphocytes).

[0231] As used herein, the term "lymphocyte" refers to a type of leukocyte or white blood cell that participates in the body's immune defense. There are two main types of lymphocytes: B cells and T cells.

[0232] As used herein, the terms "linked," "fused," or "fusion" are used interchangeably. These terms refer to the joining together of two or more elements, components, or domains by any means, including chemical conjugation or recombinant means. Methods of chemical conjugation (e.g., using heterobifunctional cross-linking agents) are known in the art.

[0233] As used herein, "local administration" or "local delivery" refers to delivery of a composition or agent that does not rely on transport of the composition or agent through the vascular system to its intended target tissue or site. For example, the composition or agent can be delivered by injection or implantation, or by injection or implantation of a device containing the composition or agent. Following local administration near the target tissue or site, the composition or agent, or one or more components thereof, can diffuse to the intended target tissue or site.

[0234] As used herein, "MHC molecules" refer 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 primarily processed for association with MHC class II. In contrast, antigens delivered endogenously to APCs are primarily processed for association with MHC class I.

[0235] As used herein, the term "monoclonal antibody" refers to an antibody that exhibits a single binding specificity and affinity for a particular epitope. Thus, the term "human monoclonal antibody" refers to an antibody that exhibits a single binding specificity and has a variable region and, optionally, a constant region, that are derived from human germline immunoglobulin sequences. In some embodiments, human monoclonal antibodies are produced by hybridomas comprising B cells obtained from a transgenic non-human animal (e.g., a transgenic mouse) fused to immortalized cells having a genome comprising a human heavy chain transgene and a light chain transgene.

[0236] As used herein, the term "monocyte" refers to a type of white blood cell that can differentiate into macrophages and dendritic cells to carry out an immune response.

[0237] 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 against viruses and other intracellular pathogens and in antibody-dependent cell-mediated cytotoxicity (ADCC).

[0238] As used herein, the term "naturally occurring" as applied to an object refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence present in an organism (including a virus) is naturally occurring if the polypeptide or polynucleotide sequence can be isolated from a source in nature and has not been intentionally modified by man in the laboratory.

[0239] As used herein, the term "non-switched isotype" refers to the isotype class of heavy chains produced when isotype switching has not occurred; the CH gene encoding the non-switched isotype is usually the first CH gene immediately downstream of the functionally rearranged VDJ gene. Isotype switching is divided into classical isotype switching and non-classical isotype switching. Classical isotype switching occurs through a recombination event involving at least one switch sequence region in a transgene. Non-classical isotype switching can be achieved by, for example, human σ μ With people μ Alternative non-classical switching mechanisms, such as intergenic and / or interchromosomal recombination, can occur and achieve isotype switching.

[0240] As used herein, the term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof that exist in single-stranded or double-stranded form. Unless otherwise specified, the term includes nucleic acids containing known natural nucleotide analogs that have similar binding properties to reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specified, a specific nucleic acid sequence also implicitly includes conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences as well as sequences explicitly specified. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed bases 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 may also be conservative.The term nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene.

[0241] As used herein, a polynucleotide can be composed of any polyribonucleotide or polydeoxyribonucleotide, which can 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 a hybrid molecule comprising DNA and RNA that can be single-stranded regions or more commonly double-stranded regions or a mixture of single-stranded and double-stranded regions. In addition, a polynucleotide can be composed of a triple-stranded region comprising RNA or DNA or RNA and DNA. A polynucleotide can also contain one or more modified bases or a DNA or RNA backbone modified for stability or other reasons. "Modified" bases include, for example, tritylated bases and rare bases, such as inosine. DNA and RNA can be subjected to various modifications; therefore, "polynucleotide" includes chemically, enzymatically, or metabolically modified forms.

[0242] A nucleic acid is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence. With respect to transcriptional regulatory sequences, operably linked means that the linked DNA sequences are contiguous and, where necessary, connect two protein coding regions (the two protein coding regions are contiguous and in reading frame). With respect to switch sequences, operably linked means that the sequence is capable of effecting switch recombination.

[0243] As used herein, "parenteral administration," "parenterally administered," and other grammatically equivalent phrases refer to modes of administration other than enteral and topical administration, usually by injection, including but not limited to intravenous, intranasal, intraocular, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intracerebral, intracranial, intracarotid, and intrasternal injection and infusion.

[0244] As used herein, the term "patient" includes humans and other mammalian subjects receiving prophylactic or therapeutic treatment.

[0245] As used herein, the term "PD-1 antagonist" refers to any compound or biological molecule that inhibits the PD-1 signaling pathway or otherwise inhibits PD-1 function in a cell (e.g., an immune cell). In some embodiments, the PD-1 antagonist blocks the binding of PD-L1 to PD-1 and / or 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.

[0246] In the context of two or more nucleic acids or peptide sequences, the term "percentage identity" refers to two or more sequences or subsequences of identical nucleotides or amino acids having a specific percentage as measured using one of the following sequence comparison algorithms (e.g., BLASTP and BLASTN or other algorithms available to those skilled in the art) or by visual estimation when comparing and aligning with regard to maximum consistency. Depending on the application, "percentage identity" may be present in the region of the compared sequences, for example, in a functional domain, or in the full length of two sequences to be compared. For sequence comparison, generally, a sequence serves as a reference sequence for comparison with a test sequence. When using a sequence comparison algorithm, test and reference sequence are input into a computer, and if necessary, subsequence coordinates are specified, and sequence algorithm program parameters are specified. Then, the sequence comparison algorithm calculates the sequence identity percentages of one or more test sequences relative to the reference sequence according to the specified program parameters.

[0247] Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally, Ausubel et al., infra).

[0248] An example of an algorithm suitable for determining percentage 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 analysis is publicly available through the National Center for Biotechnology Information website.

[0249] As generally used herein, "pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues, organs and / or body fluids of humans and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit / risk ratio.

[0250] As used herein, "pharmaceutically acceptable carrier" means and includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. that are physiologically compatible. The composition may include a pharmaceutically acceptable salt, such as an acid addition salt or a base addition salt (see, e.g., Berge et al. (1977) J Pharm Sci 66: 1-19).

[0251] 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 is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.

[0252] As used herein, the term "preventing," when used in connection with a disorder, refers to the administration of a composition that reduces the frequency or delays the onset of symptoms of the disorder in a subject relative to a subject that has not received the composition.

[0253] As used herein, the term "purified" or "isolated" for any protein (antibody or fragment) described herein refers to a polypeptide that is separated or purified from components (e.g., proteins or other naturally occurring biological or organic molecules) with which it naturally accompanies it (e.g., other proteins, lipids, and nucleic acids in the prokaryotic organism in which the protein is expressed). Typically, a polypeptide is purified when it constitutes at least 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, or 99%) of the total protein in a sample by weight.

[0254] As used herein, the term "programmed cell death protein 1" or "PD-1" refers to the programmed cell death protein 1 polypeptide, an immunoinhibitory receptor belonging to the CD28 family, and is encoded by the PDCD1 gene in humans. Alternative names or synonyms for PD-1 include: PDCD1, PD1, CD279, and SLEB2. PD-1 is primarily expressed in vivo on previously activated 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 species homologs of hPD-1, as well as analogs that have at least one common epitope with hPD-1. The complete hPD-1 sequence can be found under GenBank accession number AAC51773.

[0255] As used herein, the term "programmed death ligand-1" or "PD-L1" is one of the two cell surface glycoprotein ligands of PD-1 (the other is PD-L2), which downregulates T cell activation and cytokine secretion when bound to PD-1. Alternative names and synonyms for PD-L1 include: PDCD1L1, PDL1, B7H1, B7-4, CD274, and B7-H. The term "PD-L1" as used herein includes human PD-L1 (hPD-L1), variants, isoforms, and species homologs of hPD-L1, as well as analogs that have at least one common epitope with hPD-L1. The complete hPD-L1 sequence can be found under GenBank accession number Q9NZQ7.

[0256] PD-1 is known as an immunosuppressive protein that negatively regulates TCR signaling (Ishida, Y. et al. (1992) EMBO J. 11: 3887-3895; Blank, C. et al. (Epub 2006 Dec 29) Immunol. Immunother. 56(5): 739-745). The interaction between PD-1 and PD-L1 can act as an immune checkpoint, which can lead to a decrease in T cell receptor-mediated proliferation (Dong et al. (2003) J. Mol. Med. 81: 281-7; Blank et al. (2005) Cancer Immunol. Immunother. 54: 307-314; Konishi et al. (2004) Clin. Cancer Res. 10: 5094-100). Immunosuppression can be reversed by inhibiting the local interaction of PD-1 with PD-L1 or PD-L2; the effects are 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).

[0257] For several cancers, the survival and proliferation of tumors are maintained by tumor-mediated immune checkpoint regulation. This regulation can lead to the destruction of anti-cancer immune system function. For example, recent studies have shown that tumor cells express immune checkpoint receptor ligands, such as PD-L1 or PD-L2, which can specifically downregulate the immune system activity in the tumor microenvironment by suppressing T cells and promote cancer immune escape. PD-L1 is expressed in large quantities in a variety of human cancers (Dong et al., (2002) Nat Med 8:787-789). PD-L1 receptor PD-1 is expressed on lymphocytes (e.g., activated T cells) and is generally involved in downregulating (particularly by suppressing T cells) the immune system and promoting self-tolerance. However, when the PD-1 receptor expressed on T cells binds to the homologous PD-L1 ligand on tumor cells, the resulting T cell suppression leads to an impaired immune response against the tumor (e.g., tumor-infiltrating lymphocytes decrease or cancer cells establish immune escape).

[0258] In large cohorts of, for example, ovarian, renal, colorectal, pancreatic, liver, and melanoma cancers, PD-L1 expression has been shown to be associated with poor prognosis and decreased overall survival, regardless of subsequent treatment (see, e.g., Dong et al., (2002) Nat Med 8(8):793-800; Yang et al., (2008) Invest Ophthalmol Vis Sci 49(6):2518-2525; Ghebeh et al., (2006) Neoplasia 8:190-198; Hamanishi et al., (2007) Proc Nat Acad Sci USA 104:3360-3365; Thompson et al., (2006) Clin Genitourin Cancer 5:206-211; Nomi et al., (2005) Clin Cancer Res 11:2947-2953; Inman et al., (2007) Cancer 109:1499-1505; Shimauchi et al., (2007) Int J Cancer 121:2585-2590; Gao et al., (2009) Clin Cancer Res 15:971-979; Nakanishi et al., (2007) Cancer Immunol Immunother 56:1173-1182; Hino et al., (2010) Cancer 116(7):1757-1766). Similarly, PD-1 expression on tumor lymphocytes was found to signal dysfunctional T cells in breast cancer (Kitano et al. (2017) ESMO Open 2(2):e000150) and melanoma (Kleffel et al. (2015) Cell 162(6):1242-1256). PD-1 antagonists, 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 antitumor inhibitors that act by modulating immune cell-tumor cell interactions.

[0259] As used herein, the term "rearranged" refers to the configuration of a heavy or light chain immunoglobulin locus in which the V segments encode essentially the entire V domain, respectively. H or V L The conformation of the domain is immediately adjacent to the DJ or J segment. Rearranged immunoglobulin loci can be identified by comparison with germline DNA; rearranged loci will have at least one recombined heptamer / nonamer homology element.

[0260] 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 refer not only to the specific subject cell, but also to the progeny of such a cell. Because certain modifications may occur in subsequent generations due to mutations 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.

[0261] 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) or hybridomas prepared therefrom that are transgenic or transgenic for human immunoglobulin genes, (b) antibodies isolated from host cells transformed to express the antibodies, such as transfectomas, (c) antibodies isolated from recombinant combinatorial human antibody libraries, and (d) antibodies prepared, expressed, generated, or isolated by any other means involving splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies comprise variable and constant region sequences that utilize specific human germline immunoglobulin sequences, but include subsequent rearrangements and mutations that occur, for example, during antibody maturation. As is known in the art (see, e.g., Lonberg (2005) Nature Biotech. 23(9): 1117-1125), the variable region comprises an antigen binding domain, which is encoded by various genes that rearrange to form an antibody specific for a foreign antigen. In addition to rearrangement, the variable region can be further modified by multiple single amino acid changes (called somatic mutations or hypermutations) to increase the affinity of the antibody for the foreign antigen. The constant region will change with further response to the antigen (i.e., isotype switching). Therefore, nucleic acid molecules encoding light and heavy chain immunoglobulin polypeptides that rearrange and somatically mutate in response to an antigen may not have completely identical sequences to the original nucleic acid molecules, but rather have substantially identical or similar sequences (i.e., having at least 80% identity).

[0262] As used herein, the term "reference antibody" (interchangeably used with "reference mAb") or "reference antigen-binding protein" is an antibody or antigen-binding fragment thereof that binds to a specific epitope on IL-27 and is used to establish a relationship between itself and one or more different antibodies, wherein the relationship is that the reference antibody and the one or more different antibodies bind to the same epitope on IL-27. As used herein, the term refers to an anti-IL-27 antibody that can be used as a competitor in an assay or assay, such as those described herein (e.g., a competitive binding assay), wherein the assay can be used to discover, identify, or develop one or more different antibodies that bind to the same epitope.

[0263] As used herein, the terms "specific binding," "selective binding," "selectively binds," and "specifically binds" refer to the binding of an antibody to an epitope on a predetermined antigen. Typically, when recombinant human IL-27 is used as the analyte and the antibody is used as the ligand in a BIACORE 2000 instrument using surface plasmon resonance (SPR) technology, the antibody binds to an epitope with a specific binding affinity of less than 10 -6 M, such as approximately less than 10 -7 , 10 -8 M, 10 -9 M or 10 -10 M or even lower equilibrium dissociation constant (K D ) and its affinity for binding to the predetermined antigen is at least twice that of its affinity for binding to a non-specific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely related antigen. In certain embodiments, when measured by surface plasmon resonance (SPR) technology in a BIACORE 2000 instrument using recombinant human IL-27 as an analyte and the antibody as a ligand, the antibody specifically binds to IL-27 at an affinity of approximately less than 100 nM (10 -7 M), optionally less than about 50 nM (5 x 10 -8 M), optionally less than about 15 nM (1.5 x 10 -8 M), optionally less than about 10 nM (10 -8 M), optionally less than about 5 nM (5 x 10 -9 M), optionally less than about 1 nM (10 -9 M), optionally less than about 0.1 nM (10 -10 M), optionally less than about 0.01 nM (10 -11 M) or even lower equilibrium dissociation constants (K D ), wherein binding to the predetermined antigen occurs with an affinity that is at least two times greater than the affinity of the antibody for binding to a nonspecific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely related antigen. The phrases "an antibody that recognizes an antigen" and "an antibody that is specific for an antigen" are used interchangeably herein with the term "an antibody that specifically binds to an antigen."

[0264] As used herein, the term "STAT1 phosphorylation" refers to the phosphorylation of signal transducer and activator of transcription 1 (STAT1) polypeptide (which is 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 homodimers or heterodimers, which translocate to the nucleus to act as transcription factors. STAT1 can be activated (i.e., phosphorylated) in response to signal transduction via several ligands including IL-27. IL-27 signal transduction by IL-27R leads to STAT1 (pSTAT1) phosphorylation. STAT1 plays a key role in gene expression related to cell survival, viability, or pathogen response. The method for determining STAT1 phosphorylation due to IL-27 signal transduction includes, but is not limited to, flow cytometry analysis of cells labeled with antibodies specifically recognizing phosphorylated STAT1 (see, for example, Tochizawa et al., (2006) J Immunol Methods 313 (1-2): 29-37).

[0265] As used herein, the term "STAT3 phosphorylation" refers to the phosphorylation of the signal transducer and activator of transcription 3 (STAT3) polypeptide, which is a transcription factor encoded by the STAT3 gene in humans. STAT3 mediates the expression of a variety of genes in response to cell stimulation and therefore plays a key role in many cellular processes (such as cell growth and apoptosis). Methods for determining STAT3 phosphorylation due to IL-27 signal transduction include, but are not limited to, analysis of cells or cell extracts labeled with antibodies that specifically recognize phosphorylated STAT3 (see, for example, Fursov et al., (2011) Assay Drug Dev Technol 9(4): 420-429).

[0266] As used herein, the term "switch sequence" refers to those DNA sequences responsible for switch recombination. The μ "switch donor" sequence, typically the switch region, will be 5' (i.e., upstream) of the region of the construct that is deleted during switch recombination. The "switch acceptor" region will be located between the region of the construct to be deleted and the replacement constant region (e.g., γ, ε, etc.). Because there is no specific site where recombination always occurs, the final gene sequence cannot usually be predicted from the construct.

[0267] As used herein, the term "subject" includes any human or non-human animal. For example, the methods and compositions of the present invention can be used to treat a subject with an immune disorder. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cows, chickens, amphibians, reptiles, etc.

[0268] With respect to nucleic acids, the term "substantial homology" means that two nucleic acids, or designated sequences thereof, when optimally aligned and compared, are identical in at least about 80% of the nucleotides, typically at least about 90% to 95%, and more preferably at least about 98% to 99.5% of the nucleotides, with appropriate nucleotide insertions or deletions. Alternatively, substantial homology exists when segments will hybridize under selective hybridization conditions to the complement of a strand.

[0269] The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology = number of identical positions / total number of positions × 100), taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap. Comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm as described in the following non-limiting examples.

[0270] The percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package (available at http: / / www.gcg.com) using the NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. The percent identity between two nucleotide or amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4:11-17 (1989)) incorporated into the ALIGN program (version 2.0) using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Additionally, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. (48): 444-453 (1970)) algorithm incorporated into the GAP program in the GCG software package (available at http: / / www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix, a gap weight of 16, 14, 12, 10, 8, 6, or 4, and a length weight of 1, 2, 3, 4, 5, or 6.

[0271] The nucleic acid and protein sequences disclosed herein can be further 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. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, word length = 12, to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score = 50, word length = 3, to obtain amino acid sequences homologous to the protein molecules of the invention. To obtain gapped alignments for comparison purposes, gapped BLAST programs such as those described in Altschul et al. (1997) Nucleic Acids Res. 25(17): 3389-3402 can be utilized. When utilizing BLAST and gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http: / / www.ncbi.nlm.nih.gov.

[0272] Nucleic acids can exist in intact cells, cell lysates, or partially purified or substantially pure forms. A nucleic acid is "isolated" or "substantially purified" when it is purified from other cellular components or other contaminants (e.g., other cellular nucleic acids or proteins) by standard techniques, including alkaline / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and other techniques known in the art. See, F. Ausubel, et al., eds., Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987).

[0273] The nucleic acid compositions of the present disclosure, derived from cDNA, genomic or mixtures thereof, although typically native sequences (except for modified restriction sites, etc.), can be mutated according to standard techniques for providing gene sequences. For coding sequences, these mutations can affect the amino acid sequence as desired. In particular, DNA sequences that are substantially homologous to or derived from native V sequences, D sequences, J sequences, constant sequences, switch sequences, and other such sequences described herein are contemplated (where "derived from" means that the sequence is identical to or modified from another sequence).

[0274] As used herein, the term "STING" (or TMEM173) refers to the stimulator of interferon genes, which is a protein that acts as a direct cytosolic DNA sensor and adapter protein. In humans, STING is encoded by the TMEM173 gene. STING plays an important role in innate immunity. When cells are infected with intracellular pathogens (such as viruses, mycobacteria, and intracellular parasites), STING induces the production of type I interferon. Type I interferon mediated by STING protects infected cells and nearby cells from local infection by binding to the same cell and nearby cells that secrete it. An exemplary amino acid sequence of STING is provided by the NCBI Genbank database with accession number NP_001288667.

[0275] The term "T cell" refers to a type of white blood cell that is distinguished from other white blood cells by the presence of a T cell receptor on the cell surface. There are multiple subpopulations of T cells, including but not limited to T helper cells (also known as T cells). H cells or CD4 + T cells) and subtypes, including T H 1. T H 2. T H 3. T H 17. T H 9 and T FH cells; cytotoxic T cells (also known as T C cells, CD8 + T cells, cytotoxic T lymphocytes, T killer cells, killer T cells); memory T cells and subtypes, including 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); regulatory T cells (also known as T reg cells or suppressor T cells) and subtypes, including CD4 + FOXP3 + T reg cells, CD4 + FOXP3 - T reg cells, Tr1 cells, Th3 cells and T reg 17 cells; natural killer T cells (also known as NKT cells); mucosal-associated invariant T cells (MAIT) and γδT cells (γδT cells), including Vγ9 / Vδ2T cells. Any one or more of the aforementioned or unmentioned T cells can be a target cell type for the use method of the present invention.

[0276] As used herein, the term "T cell-mediated response" refers to a response mediated by T cells, including but not limited to effector T cells (e.g., CD8+ cells) and helper T cells (e.g., CD4 + T cell-mediated responses include, for example, T cell cytotoxicity and proliferation.

[0277] As used herein, the term "therapeutically effective amount" or "therapeutically effective dose" or similar terms used herein are intended to mean an amount of an agent (e.g., an anti-IL-27 antibody or antigen-binding fragment thereof) that will elicit a desired biological or medical response (e.g., improvement of one or more symptoms of cancer).

[0278] As used herein, the term "TAM receptor" refers to TAM receptor protein tyrosine kinases (TYRO3, AXL and MER). TAM receptors are involved in the regulation of immune system homeostasis. In the cancer environment, TAM receptors have a dual regulatory effect, thereby controlling the initiation and progression of tumor development, and simultaneously controlling the relevant anti-tumor responses of various immune cells. A further description of TAM receptors is found in Paolino and Penninger (2016) Cancers 8(97): doi: 10.3390 / cancers8100097). As used herein, the term "TAM receptor inhibitor" or "TAM inhibitor" refers to an agent that inhibits, blocks or reduces the function or activity of TAM receptors.

[0279] As used herein, unless otherwise indicated, 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). TIGIT is also known in the art as DKFZp667A205, FLJ39873, protein 9 containing V group and immunoglobulin domains, protein 3 containing V group and immunoglobulin domains, VSIG9, VSTM3, and WUCAM. The term also encompasses naturally occurring variants of TIGIT, such as splice variants or allelic variants. The amino acid sequence of an exemplary human TIGIT can be found under UniProt accession number Q495A1.

[0280] As used herein, the term "treatment" refers to treatment or preventive measures as described herein. The method of "treatment" employs administering 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 who may eventually acquire such a condition) to prevent, cure, delay, reduce the severity of the condition or recurring condition, or ameliorate one or more symptoms thereof, or to prolong the subject's survival beyond that which would be expected in the absence of such treatment.

[0281] As used herein, the term "tumor microenvironment" (or "cancer microenvironment"; abbreviated as TME) refers to the cellular environment or background in which a tumor or neoplasm exists, including surrounding blood vessels and non-cancerous cells, including but not limited to immune cells, fibroblasts, bone marrow-derived inflammatory cells and lymphocytes. Signaling molecules and extracellular matrix also constitute the TME. Tumors and the surrounding microenvironment are closely related and constantly interact with each other. Tumors can affect the microenvironment by releasing extracellular signals, promoting tumor angiogenesis and inducing peripheral immune tolerance, and immune cells in the microenvironment can affect the growth and evolution of tumor cells.

[0282] As used herein, the term "unrearranged" or "germline configuration" refers to the configuration of a V segment in which the V segments are not recombined so as to be in close proximity to a D or J segment.

[0283] As used herein, the term "vector" is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid connected thereto. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop into which another DNA segment can be connected. Another type of vector is a viral vector, in which another DNA segment can be connected to a viral genome. Some vectors can be autonomously replicated in the host cell into which they are introduced (for example, bacterial vectors and additional mammalian vectors with bacterial replication origins). Other vectors (for example, non-additional mammalian vectors) can be integrated into the genome of the host cell after being introduced into the host cell, and thus replicated together with the host genome. In addition, some vectors can guide the gene expression operably connected thereto. Such vectors are referred to as "recombinant expression vectors" (or simply "expression vectors") in this article. Typically, useful expression vectors in recombinant DNA technology exist in the form of plasmids. In this specification, "plasmid" and "vector" can be used interchangeably because plasmids are the most commonly used vector forms. However, the present invention is intended to include such other forms of expression vectors, such as viral vectors (for example, replication-defective retroviruses, adenoviruses and adeno-associated viruses), which have equivalent functions.

[0284] 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. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the presently disclosed methods and compositions. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. BRIEF DESCRIPTION OF THE DRAWINGS

[0285] Figure 1is a table providing affinity data for the indicated anti-IL-27 antibodies. Affinity measurements were performed using ForteBio and Meso Scale Discovery methods.

[0286] Figure 2 is a graph depicting binding of the indicated anti-IL-27 antibodies to plate-bound recombinant IL-27 as measured by ELISA.

[0287] Figures 3A-3E They are a bar chart, two graphs, a bar chart and a graph. Figure 3A is a graph depicting the inhibition of IL-27-mediated STAT1 phosphorylation in human whole blood by the indicated anti-IL-27 antibodies as measured by flow cytometry. Figure 3B is a graph depicting the inhibition of IL-27-mediated STAT1 phosphorylation in human PBMCs by the indicated anti-IL-27 antibodies as measured by flow cytometry. Figure 3C is a graph depicting the inhibition of IL-27-mediated STAT1 phosphorylation in U937 cells by the indicated anti-IL-27 antibodies as measured by flow cytometry.

[0288] Figure 3D is a graph depicting the inhibition of IL-27-mediated STAT1 phosphorylation in HUT-78 cells by the indicated anti-IL-27 antibodies as measured by flow cytometry. Figure 3E This figure shows that SRF388 inhibits IL-27-mediated pSTAT1 in human whole blood T cells.

[0289] Figure 4 is a graph depicting reversal of IL-27-mediated inhibition of CD161 expression in T cells by a range of concentrations of anti-IL-27 antibodies as indicated. CD161 expression was determined using flow cytometry.

[0290] Figure 5A is a graph depicting the extent to which anti-IL-27 antibodies enhance PD-1-mediated TNFα secretion in human PBMCs as measured by ELISA. Figure 5B is a graph depicting the extent to which anti-IL-27 antibodies enhance PD-1-mediated IL-6 secretion in human PBMCs as measured by ELISA. Figure 5C is a dot plot showing that the combination of SRF388 and PD-1 blockade results in increased cytokine production in PBMCs from healthy donors and RCC patients (Abbreviations: CBA = cytometric bead array, IFNγ = interferon gamma, MSD = MesoScale Discovery, PBMC = peripheral blood mononuclear cells, PD-1 = programmed death receptor-1, RCC = renal cell carcinoma, TNFα = tumor necrosis factor alpha). Figure 5DIL-27 is shown to inhibit cytokine production after PD-1 blockade and is restored in combination with SRF388 (Abbreviations: Ctrl = control, ns = not significant, PBMC = peripheral blood mononuclear cells, rhIL-27 = recombinant human IL-27). Figure 5E Summarizes the cytokine induction (particularly TNFα, IFNγ, IL-6, and IL-17A) observed for various indicated cell types in PBMC cultures when such cells were contacted with SRF388 antibody, αPD-1 antibody, or a combination of SRF388 and αPD-1 antibodies. Figure 5F Shown are the effects of varying concentrations of individual antibodies as indicated (SRF405, SRF410, SRF411, SRF414, SRF416, SRF536, SRF543, SRF529, SRF381, SRF388, and Ab7) on pSTAT1 signaling in U937 (lymphoma) cells. Figure 5G Shown are the effects of different concentrations of these individual antibodies on pSTAT1 signaling in PBMCs (peripheral blood mononuclear cells). Figure 5H Shown are the effects of different concentrations of these individual antibodies on CD161 signal in PBMCs (peripheral blood mononuclear cells). Figure 5I Shown are the effects of different concentrations of these individual antibodies (excluding SRF414) on PD-L1 signaling in CD4 T lymphocytes (CD4 cells). Figure 5J Shown are the effects of different concentrations of these individual antibodies (excluding SRF529) on PD-L1 signaling in monocytes. Figure 5K Shown are the effects of different concentrations of these individual antibodies (excluding SRF529) on TIM-3 signaling in monocytes.

[0291] Figure 6A is a graph depicting inhibition of IL-27-mediated PD-L1 expression by treatment of human monocytes with anti-IL-27 antibodies as determined by flow cytometry. Figure 6B is a graph depicting dose-dependent inhibition of IL-27-mediated PD-L1 expression by treating human monocytes with a range of concentrations of an anti-IL-27 antibody that specifically binds to EBI3 monomers, as determined by flow cytometry. Figure 6C is a graph depicting the inhibition of IL-27-mediated TIM3 expression by treatment of human monocytes with anti-IL-27 antibodies as determined by flow cytometry. Figure 6D is a graph depicting inhibition of IL-27-mediated PD-L1 expression by treatment of resting human T cells with anti-IL-27 antibodies as determined by flow cytometry.

[0292] Figure 7Aare dot plots depicting the number of surface lung B16F10 metastatic nodules (lung nodules) from B16F10 tumor-bearing mice treated with anti-IL27 antibody (SRF388), isotype control antibody, αWSX-1 antibody, or combined αPD-1 and αCTLA-4 antibodies as indicated, as determined by visual counts of nodules in lungs isolated from mice. Figure 7B Provided are graphs depicting the growth kinetics of bioluminescent B16-Luc tumors in mice treated with anti-IL-27 antibody (SRF388) or isotype control antibody as determined by bioluminescent imaging analysis. Figure 7C Shown are a series of images of fixed, sectioned lung tissue isolated from B16F10 tumor-bearing mice treated with anti-IL27 antibody (SRF388), isotype control antibody, αWSX-1 antibody, or combined αPD-1 and αCTLA-4 antibodies as indicated, stained with hematoxylin and eosin. Figure 7D Is a dot plot depicting total tumor area as a percentage of total tissue area of ​​fixed, sectioned lung tissue B16F10 tumor tissue isolated from B16F10 tumor-bearing mice treated with anti-IL27 antibody (SRF388), isotype control antibody, αWSX-1 antibody, or combined αPD-1 and αCTLA-4 antibodies as indicated, stained with hematoxylin and eosin. Similar reductions in the number of surface lung metastases and total tumor area were observed with IL-27RA (WSX-1)-mediated antibody blockade and with anti-PD-1 + anti-CTLA-4 combination therapy.

[0293] Figure 8A Provided are scatter plots depicting microarray data for genes whose expression changed by >1.0 log2 fold change (black dots) in splenocytes isolated from IL-27 overexpressing mice following treatment with IL-27 minicircles. Figure 8B Provide description as Figure 8A Figure 2. Expression levels of selected immunoregulatory genes in splenocytes, as indicated. Figure 8C Ectopic expression of human IL-27 is shown to induce inhibitory receptor expression on murine T cells in vivo, and SRF388 reduces inhibitory receptor expression on T cells in vivo after IL-27 minicircle treatment. Six-week-old female Balb / c mice were injected with empty vector (control) or hIL-27 minicircle. PBMCs (upper left and upper right panels) and total splenocytes (lower left and lower right panels) were collected 5 days after transfection, and the cells were stained and analyzed by flow cytometry. The expression of the indicated markers was analyzed on CD4+ T cells (upper left and lower left panels) and CD8+ T cells (upper right and lower right panels). FlowJo software was used for analysis. Figure 8D SRF388 was shown to inhibit the detection of minicircle-derived human IL-27 in murine plasma.

[0294] Figure 9 A tabular summary of the properties of selected monoclonal antibodies is provided.

[0295] Figure 10A Antibody sequence diagrams are presented, with sequence divisions reflecting NT numbering. Figure 10B Presents antibody sequence diagrams (corresponding to Figure 10A Sequence diagram of ), where sequence divisions reflect ImMunoGeneTics (IMGT) numbering. Figure 10A and Figure 10B In the , the highlighted amino acids in the CDR sequences represent mutations from the germline encoded sequence. It will be apparent to those skilled in the art that antibody numbering can be performed in a variety of art-recognized ways, including determining CDR sequences, framework sequences, etc., including by Figure 10A and Figure 10B The NT and IMGT numbering systems are presented in and used elsewhere in this paper.

[0296] Figure 11A Figures are 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 antibodies described in Example 15. Values ​​are extrapolated from a standard curve using recombinant human IL-27. The mean + 2SD of healthy donors (normal controls) was used as an arbitrary cutoff for serum samples to be considered positive. The total number of positive samples is indicated on the corresponding cancer classification. Figure 11B is a scatter plot showing that several ovarian cancer samples showed detectable levels of IL-27 in serum, using Figure 11A Regarding the same parameters, detectable levels of IL-27 were also observed in sera from three patients with B-cell lymphoma and one patient with endometrial cancer.

[0297] Figure 12A is a graph showing that anti-p28 capture antibody SRF381 is paired with anti-EBI3 antibody Ab7 to detect recombinant human IL-27 in an IL-27 concentration-dependent manner by MSD immunoassay. Figure 12B Graph showing that anti-EBI3 capture antibody SRF557 was paired with anti-EBI3 antibody Ab7 to detect recombinant human IL-27 in an IL-27 concentration-dependent manner by MSD immunoassay. Figures 12C-12DFigure 2 shows the expression of EBI3 in 174 serum samples from cancerous (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 lymphoma, gastric cancer, endometrial cancer) and healthy (normal) patients as determined by Meso Scale Discovery (MSD) assay. Figure 12C ) and IL-27( Figure 12D ) concentration. Figure 12E is a bar graph showing that anti-IL-27 antibodies can be paired with SRF381 to detect recombinant human IL-27 by MSD immunoassay. Figure 12F is a bar graph showing that anti-IL-27 antibodies can be paired with Ab7 to detect recombinant human IL-27 by ELISA. DETAILED DESCRIPTION

[0298] The present disclosure provides, at least in part, antibody molecules that bind to human IL-27 with high affinity and specificity. In one embodiment, disclosed herein are human antibodies that bind to IL-27. As used herein, the terms "IL-27" and "IL27" refer interchangeably to the heterodimeric cytokine IL-27, which is composed of two different subunits and encoded by two different genes: Epstein-Barr virus-induced gene 3 (EBI3) and IL-27p28. IL-27 has pro-inflammatory and anti-inflammatory properties and has different effects on hematopoietic and non-hematopoietic cells.

[0299] Thus, 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:

[0300] (i) with an equilibrium dissociation constant (K) of 15 nM or less D ) binds to human IL-27;

[0301] (ii) blocking the binding of IL-27 to the IL-27 receptor;

[0302] (iii) inhibiting or reducing STAT1 and / or STAT3 phosphorylation in cells;

[0303] (iv) inhibiting or reducing IL-27-mediated inhibition of CD161 expression in cells;

[0304] (v) inhibiting or reducing IL-27-mediated PD-L1 and / or TIM-3 expression in cells;

[0305] (vi) inducing or enhancing PD-1-mediated secretion of one or more cytokines from cells; and

[0306] (vii) A combination of (i)-(vi).

[0307] In other aspects, the present disclosure provides monoclonal antibodies, or antigen-binding portions thereof, that specifically bind to human IL-27 and inhibit or reduce IL-27 biological activity or IL-27 signaling.

[0308] Other aspects of the present invention include nucleic acid molecules encoding antibody molecules, expression vectors, host cells, and methods for preparing antibody molecules. Immunoconjugates, multi- or bispecific molecules, and pharmaceutical compositions comprising antibody molecules are also provided. The anti-IL-27 antibody molecules disclosed herein can be used to treat, prevent, and / or diagnose cancerous or malignant conditions, such as solid tumors and liquid tumors (e.g., leukemias, such as lymphomas, such as AML), lung cancer (e.g., non-small cell lung cancer), pancreatic cancer, breast cancer (e.g., triple-negative breast cancer), melanoma, testicular cancer, sarcoma, head and neck cancer (e.g., squamous head and neck cancer), 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, such as clear cell renal carcinoma).

[0309] Anti-IL-27 antibodies and antigen-binding fragments thereof

[0310] 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 shown in Table 12.

[0311] 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 ) binds to human IL-27; (ii) blocks the binding of IL-27 to the IL-27 receptor; (iii) inhibits or reduces STAT1 and / or STAT3 phosphorylation in cells; (iv) inhibits or reduces the inhibition of CD161 expression in cells; (v) inhibits or reduces PD-L1 and / or TIM-3 expression in cells; (vi) induces or enhances PD-1-mediated secretion of one or more cytokines from cells; and (vii) a combination of (i)-(vi).

[0312] In some embodiments, the isolated monoclonal antibody, or antigen-binding portion thereof, has an equilibrium dissociation constant (K D ) binds to human IL-27.

[0313] In some embodiments, the isolated monoclonal antibody, or antigen-binding portion thereof, binds to recombinant human IL-27 or murine IL-27.

[0314] In some embodiments, the isolated monoclonal antibody or its antigen-binding portion 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.

[0315] In some embodiments, the isolated monoclonal antibody or antigen-binding portion thereof inhibits or reduces the inhibition of CD161 expression in a cell (eg, improves or alleviates the inhibition of CD161 expression in a cell). In some embodiments, the cell is an immune cell.

[0316] 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.

[0317] In some embodiments, the isolated antibody or antigen-binding portion thereof induces or enhances PD-1-mediated secretion of one or more cytokines from a cell. In some 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.

[0318] 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 containing at least one mutation. In some embodiments, the antibody comprises a mutant IgG1 heavy chain constant region. In some embodiments, the antibody comprises a mutant IgG4 heavy chain constant region. In some embodiments, according to EU numbering, the mutant IgG4 heavy chain constant region comprises any one of the substitutions S228P, L235E, L235A, or a combination thereof.

[0319] In some embodiments, the present invention 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, according to any one of the preceding embodiments.

[0320] In some embodiments, the present invention provides an isolated monoclonal antibody, or antigen-binding portion thereof, that binds to at least one of the amino acid residues comprising IL-27 bound by the antibody, or antigen-binding portion thereof, according to any one of the preceding embodiments.

[0321] In some embodiments, the present disclosure provides an isolated monoclonal antibody, or antigen-binding portion thereof, wherein mutation of 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, according to any one of the preceding embodiments.

[0322] In some embodiments, the disclosure provides an isolated monoclonal antibody, or antigen-binding portion thereof, that binds to an epitope on IL-27, wherein the epitope is the same as or similar to the epitope bound by the antibody molecules described in Table 12.

[0323] 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 selected from the group consisting of:

[0324] (i) the heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 706, 707, and 708, respectively, and the light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 714, 715, and 716, respectively;

[0325] (ii) the heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 728, 729, and 730, respectively, and the light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 736, 737, and 738, respectively;

[0326] (iii) the heavy chain CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 750, 751, and 752, respectively, and the light chain CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 758, 759, and 760, respectively; and

[0327] (iv) the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 750, 751 and 752, respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 758, 759 and 760, respectively; and

[0328] 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.

[0329] 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 selected from the group consisting of:

[0330] (i) the heavy chain CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 709, 710, and 711, respectively, and the light chain CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 717, 718, and 719, respectively;

[0331] (ii) the heavy chain CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 731, 732, and 733, respectively, and the light chain CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 739, 740, and 741, respectively;

[0332] (iii) the heavy chain CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 753, 754, and 755, respectively, and the light chain CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 761, 762, and 763, respectively; and

[0333] (iv) the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 775, 776 and 777, respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 783, 784 and 785, respectively.

[0334] 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.

[0335] 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 amino acid sequences of the 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 wherein the amino acid sequences of the light chain CDR1, CDR2, and CDR3 are RASENIYSYLA (SEQ ID NO: 26), NAETLTE (SEQ ID NO: 27), and QHHYGTPLT (SEQ ID NO: 28), respectively.

[0336] 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, 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 wherein the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 720, 742, 764, and 786.

[0337] 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 an amino acid sequence selected from the group consisting of:

[0338] (i) SEQ ID NOs: 712 and 720, respectively;

[0339] (ii) SEQ ID NOs: 734 and 742, respectively;

[0340] (iii) SEQ ID NOs: 756 and 764, respectively; and

[0341] (iv) SEQ ID NOs: 778 and 786, respectively.

[0342] 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, wherein the heavy chain variable region comprises an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 712, 734, 756, and 778; and wherein the light chain variable region comprises an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 720, 742, 764, and 786.

[0343] 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 an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of:

[0344] (i) SEQ ID NOs: 712 and 720, respectively;

[0345] (ii) SEQ ID NOs: 734 and 742, respectively;

[0346] (iii) SEQ ID NOs: 756 and 764, respectively; and

[0347] (iv) SEQ ID NOs: 778 and 786, respectively.

[0348] 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 shown in SEQ ID NOs: 167 and 175, respectively.

[0349] 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 an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NOs: 167 and 175, respectively.

[0350] 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, wherein the heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 722, 744, 766, and 788; and wherein the light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 724, 746, 768, and 790.

[0351] 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, wherein the heavy chain comprises an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 722, 744, 766, and 788; and wherein the light chain comprises an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 724, 746, 768, and 790.

[0352] 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, wherein the heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 726, 748, 770, and 792; and wherein the light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 724, 746, 768, and 790.

[0353] 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, wherein the heavy chain comprises an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 726, 748, 770, and 792; and wherein the light chain comprises an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 724, 746, 768, and 790.

[0354] 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:

[0355] (i) SEQ ID NOs: 722 and 724, respectively;

[0356] (ii) SEQ ID NOs: 744 and 746, respectively;

[0357] (iii) SEQ ID NOs: 766 and 768, respectively; and

[0358] (iv) SEQ ID NOs: 788 and 790, respectively.

[0359] 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 that is at least 90% identical to an amino acid sequence selected from the group consisting of:

[0360] (i) SEQ ID NOs: 722 and 724, respectively;

[0361] (ii) SEQ ID NOs: 744 and 746, respectively;

[0362] (iii) SEQ ID NOs: 766 and 768, respectively; and

[0363] (iv) SEQ ID NOs: 788 and 790, respectively.

[0364] 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:

[0365] (i) SEQ ID NOs: 726 and 724, respectively;

[0366] (ii) SEQ ID NOs: 748 and 746, respectively;

[0367] (iii) SEQ ID NOs: 770 and 768, respectively; and

[0368] (iv) SEQ ID NOs: 792 and 790, respectively.

[0369] 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 that is at least 90% identical to an amino acid sequence selected from the group consisting of:

[0370] (i) SEQ ID NOs: 726 and 724, respectively;

[0371] (ii) SEQ ID NOs: 748 and 746, respectively;

[0372] (iii) SEQ ID NOs: 770 and 768, respectively; and

[0373] (iv) SEQ ID NOs: 792 and 790, respectively.

[0374] 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 shown in SEQ ID NOs: 177 and 179, respectively.

[0375] 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 that is at least 90% identical to the amino acid sequence shown in SEQ ID NOs: 177 and 179, respectively.

[0376] 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 shown in SEQ ID NOs: 181 and 179, respectively.

[0377] 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 that is at least 90% identical to the amino acid sequence shown in SEQ ID NOs: 181 and 179, respectively.

[0378] Anti-IL-27 receptor (WSX-1) antibodies and antigen-binding fragments thereof

[0379] WSX-1 is a class I cytokine receptor that is homologous in sequence and structure to the β2 chain of IL-12R. + T cells and CD8 + This receptor is highly expressed on T cells. Recent studies have identified the heterodimeric cytokine IL-27, composed of the subunits EBI3 and IL-27p28, as a ligand for WSX-1. EBI3, a member of the class I cytokine receptor family, shares significant structural homology with IL-12p40, and IL-27p28 is closely related to IL-12p35. In addition to the structural similarities between the IL-12 / IL-12R and IL-27 / WSX-1 ligand / receptor pairs, functional similarities have also been reported. Although the IL-12R plays a key role in the development of Th1-type responses, WSX-1-deficient cells have been reported to have impaired IFN-γ production during early Th1 differentiation. Furthermore, like IL-12, recombinant IL-27 is able to enhance Th1 differentiation in highly purified naive helper T cells. As a result of these studies, the early consensus is that, like the IL-12 / IL-12R interaction, IL-27 / WSX-1 is an important factor in the initial differentiation of Th1 responses.

[0380] Accordingly, 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 heavy and light chain CDRs, 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.

[0381] 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 shown in SEQ ID NO: 794; and wherein the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 796.

[0382] 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 shown in SEQ ID NO: 798.

[0383] 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 shown in SEQ ID NO: 799.

[0384] Methods for producing anti-IL-27 antibodies and antigen-binding fragments thereof

[0385] 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, the methods for preparing the antibodies described herein may include immunizing a subject (e.g., a non-human mammal) with a suitable immunogen. Suitable immunogens for producing any of the antibodies described herein are described herein. For example, to produce antibodies that bind to IL-27, one can immunize a suitable subject (e.g., a non-human mammal, such as a rat, mouse, gerbil, hamster, dog, cat, pig, goat, horse, or non-human primate) with IL-27. In some embodiments, the full-length human IL-27 EBI3 monomeric polypeptide comprising the amino acid sequence shown in SEQ ID NO: 97 is used as an immunogen. In some embodiments, the full-length human IL-27p28 monomeric polypeptide comprising the amino acid sequence shown in SEQ ID NO: 98 is used as an immunogen.

[0386] Suitable subjects (e.g., non-human mammals) can be immunized with a suitable antigen, followed by multiple booster immunizations sufficient to induce the production of antibodies in the mammal. The immunogen can be administered to the subject (e.g., non-human mammal) together with an adjuvant. Adjuvants for producing antibodies in the subject include, but are not limited to, protein adjuvants; bacterial adjuvants, such as whole bacteria (BCG, anaerobic Corynebacterium parvum, or Salmonella minnesota) and bacterial components, including cell wall skeletons, trehalose dimycolate, monophosphoryl lipid A, methanol extractable residue (MER) of tubercle bacillus, complete or incomplete Freund's adjuvant; viral adjuvants; chemical adjuvants, such as aluminum hydroxide and iodoacetate and cholesterol hemisuccinate. Other adjuvants that can be used in methods of inducing an immune response include, for example, cholera toxin and parapoxvirus proteins. See also Bieg et al. (1999) Autoimmunity 31(1):15-24. See also, e.g., Lodmell et al. (2000) Vaccine 18: 1059-1066; Johnson et al. (1999) J Med Chem 42: 4640-4649; Baldridge et al. (1999) Methods 19: 103-107; and Gupta et al. (1995) Vaccine 13(14): 1263-1276.

[0387] In some embodiments, the method comprises preparing a hybridoma cell line that secretes a monoclonal antibody that binds to an immunogen. For example, a suitable mammal, such as a laboratory mouse, is immunized with an IL-27 polypeptide as described above. Antibody-producing cells (e.g., splenic B cells) from the immunized mammal can be isolated 2 to 4 days after at least one booster immunization with the immunogen and then briefly grown in culture before being fused with cells from a suitable myeloma cell line. The cells can be fused in the presence of a fusion promoter (such as vaccinia virus or polyethylene glycol). The hybrid cells obtained in the fusion are cloned, and cell clones that secrete the desired antibody are selected. For example, spleen cells from Balb / c mice immunized with a suitable immunogen can be fused with cells from the myeloma cell line PAI or the myeloma cell line Sp2 / 0-Ag 14. After fusion, the cells are expanded in a suitable culture medium that is supplemented with a selective medium, such as HAT medium, at regular intervals to prevent normal myeloma cells from outgrowing the desired hybridoma cells. The resulting hybrid cells are then screened for secretion of the desired antibody (e.g., an antibody that binds to human IL-27), and in some embodiments, one can identify anti-IL-27 antibodies from the non-immune-biased libraries described in U.S. Patent No. 6,300,064 (to Knappik et al.; Morphosys AG) and Schoonbroodt et al. (2005) Nucleic Acids Res 33(9):e81.

[0388] In some embodiments, the methods described herein can involve or be used in combination with, for example, phage display technology, bacterial display, yeast surface display, eukaryotic virus display, mammalian cell display, and cell-free (e.g., ribosome display) antibody screening technology (see, e.g., Etz et al. (2001) J Bacteriol 183:6924-6935; Cornelis (2000) Curr Opin Biotechnol 11:450-454; Klemm et al. (2000) Microbiology 146:3025-3032; Kieke et al. (1997) Protein Eng 10:1303-1310; Yeung et al. (2002) Biotechnol Prog 18:212-220; Boder et al. (2000) Methods Enzymology 328:430-444; Grabherr et al. (2001) Comb Chem High Throughput Screen 4:185-192; Michael et al. (1995) Gene Ther 2:660-668; Pereboev et al. (2001) J Virol 75:7107-7113; Schaffitzel et al. (1999) J Immunol Methods 231:119-135; and Hanes et al. (2000) Nat Biotechnol 18:1287-1292).

[0389] 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 carrying polynucleotide sequences encoding them. Such phage can be used to display the antigen-binding domains of antibodies, such as Fab, Fv, or disulfide-stabilized Fv antibody fragments, expressed from a library or combinatorial antibody library (e.g., human or mouse). The phage used in these methods is typically a filamentous phage, such as fd and M13. The antigen-binding domain is expressed as a protein recombinantly fused to any one of the phage coat proteins pIII, pVIII, or pIX. See, for example, Shi et al. (2010) JMB 397 :385-396. Examples of phage display methods that can be used to prepare immunoglobulins or fragments thereof described herein include those disclosed in Brinkman et al. (1995) J Immunol Methods 182 :41-50; Ames et al. (1995) J Immunol Methods 184:177-186; Kettleborough et al. (1994) Eur J Immunol 24 :952-958; Persic et al. (1997) Gene 187 :9-18; Burton et al. (1994) Advances in Immunology 57 :191-280; and PCT Publication Nos. WO 90 / 02809, WO 91 / 10737, WO 92 / 01047, WO 92 / 18619, WO 93 / 11236, WO 95 / 15982 and WO 95 / 20401. Suitable methods are also described in, for example, 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.

[0390] In some embodiments, phage display antibody libraries can be generated using mRNA collected from B cells of an immunized mammal. For example, a spleen cell sample containing B cells can be isolated from a mouse immunized with an IL-27 polypeptide as described above. mRNA can be isolated from the cells and converted into cDNA using standard molecular biology techniques. See, for example, 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 K.C. Lo (2004), supra; and Borrebaek (1995), supra. cDNAs encoding the variable regions of immunoglobulin heavy and light chain polypeptides are used to construct phage display libraries. 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(Pt3) :889-894; and Engberg et al. (1995) Methods Mol Biol 51 :355-376.

[0391] In some embodiments, a combination of selection and screening can be used to identify an antibody of interest from, for example, a population of antibodies produced by hybridomas or a phage-displayed antibody library. Suitable methods are known in the art and are described, for example, in Hoogenboom (1997) Trends in Biotechnology. 15 :62-70; Brinkman et al. (1995), supra; Ames et al. (1995), supra; Kettleborough et al. (1994), supra; Persic et al. (1997), supra; and Burton et al. (1994), supra. For example, standard molecular biology techniques are used to produce a variety of phagemid vectors (each encoding a fusion protein of a phage coat protein (e.g., pIII, pVIII, or pIX of M13 phage) and a different antigen binding region), which are then introduced into a bacterial population (e.g., E. coli). In some embodiments, expression of phage 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 is recovered and then contacted with a (fixed) target antigen, for example, bound to a solid support. Phage can also be contacted with the antigen in solution and the complex subsequently bound to a solid support.

[0392] Any immunological or biochemical method known in the art can be used to characterize the specificity and binding affinity of the antibody subgroup screened using the above method to a specific antigen (e.g., human IL-27). For example, the specific binding of an antibody to IL-27 can be determined, for example, using an immunological or biochemical method, such as, but not limited to, ELISA assays, SPR assays, immunoprecipitation assays, affinity chromatography, and equilibrium dialysis as described above. Immunoassays that can be used to analyze the immunospecific binding and cross-reactivity of an antibody include, but are not limited to, competitive and non-competitive assay systems using techniques such as Western blotting, RIA, ELISA (enzyme-linked immunosorbent assay), "sandwich" immunoassays, immunoprecipitation assays, immunodiffusion assays, agglutination assays, complement fixation assays, immunoradiometric assays, fluorescent immunoassays, and protein A immunoassays. Such assays are conventional and well known in the art.

[0393] It will be appreciated that the above methods can also be used to determine, for example, whether an anti-IL-27 antibody does not bind to full-length human IL-27 and / or IL-27 protein.

[0394] In embodiments where the selected CDR amino acid sequence is a short sequence (e.g., less than 10-15 amino acids in length), the CDR may be selected as described, for example, in Shiraishi et al. (2007) Nucleic Acids Symposium Series 51(1) : 129-130 and U.S. Patent No. 6,995,259, chemical synthesis of nucleic acids encoding CDRs. For a given nucleic acid sequence encoding an acceptor antibody, the region of the nucleic acid sequence encoding the CDRs can be replaced using standard molecular biology techniques. The 5' and 3' ends of the chemically synthesized nucleic acid can be synthesized to include sticky end restriction enzyme sites for cloning the nucleic acid into the nucleic acid encoding the variable region of the donor antibody.

[0395] In some embodiments, the anti-IL-27 antibodies described herein comprise an altered heavy chain constant region that has reduced (or no) effector function relative to its corresponding unaltered constant region. The effector function of the constant region of the anti-IL-27 antibody can be adjusted by changing the properties of the constant region or Fc region. The altered effector function includes, for example, modulation of one or more of the following activities: antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), apoptosis, binding to one or more Fc receptors, and pro-inflammatory response. Modulation refers to the enhancement, reduction, or elimination of the effector function activity exhibited by the subject antibody comprising the altered constant region compared to the activity of the unaltered form of the constant region. In specific embodiments, modulation includes situations where the activity is eliminated or completely absent.

[0396] In one embodiment, the anti-IL-27 antibodies described herein comprise 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, for example, according to EU numbering (Kabat, EA et al., supra), the IgG4 constant region comprises a mutation, for example, one or both of S228P and L235E or L235A. Representative sequences of wild-type and mutant IgG4 constant regions for antibodies of the present disclosure are shown in Table 12. In one embodiment, the anti-IL-27 antibodies described herein comprise 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 a mutation. Representative sequences of wild-type and mutant IgG4 constant regions for antibodies of the present disclosure are shown in Table 12.

[0397] An altered constant region having altered FcR binding affinity and / or ADCC activity and / or altered CDC activity is a polypeptide that has increased or decreased FcR binding activity and / or ADCC activity and / or CDC activity compared to the unaltered form of the constant region. An altered constant region that exhibits enhanced binding to an FcR binds to at least one FcR with greater affinity than the unaltered polypeptide. An altered constant region that exhibits decreased binding to an FcR binds to at least one FcR with less affinity than the unaltered form of the constant region. Such variants that exhibit decreased binding to an FcR may have little or no significant binding to an FcR, e.g., 0 to 50% (e.g., less than 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 37%, 38%, 39 ... %, 5%, 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 binding to FcR. Similarly, the altered constant region that exhibits adjusted ADCC and / or CDC activity can exhibit enhanced 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 to 50% (e.g., less than 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%) of the ADCC and / or CDC activity of the unaltered form of the constant region. The anti-IL-27 antibodies described herein that exhibit reduced ADCC and / or CDC and comprise an altered constant region may exhibit reduced or no ADCC and / or CDC activity.

[0398] 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 a portion thereof, such as a G2 / G4 hybrid constant region (see, e.g., Burton et al. (1992) Adv Immun51 :1-18; Canfield et al. (1991) J Exp Med 173 :1483-1491; and Mueller et al. (1997) Mol Immunol 34(6) :441-452). See above.

[0399] In some embodiments, the anti-IL-27 antibody may comprise an altered constant region that exhibits enhanced or reduced complement-dependent cytotoxicity (CDC). CDC activity may be adjusted by introducing one or more amino acid substitutions, insertions, or deletions into the Fc region of the antibody. See, for example, U.S. Patent No. 6,194,551. Alternatively or additionally, one or more cysteine ​​residues may be introduced into the Fc region, thereby allowing interchain disulfide bonds to form in this region. The homodimeric antibody thus produced may have increased or decreased internalization capacity 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.

[0400] Expression and purification of recombinant antibodies

[0401] Antibodies as described herein or their antigen-binding fragments can be produced using various techniques known in the art of molecular biology and protein chemistry. For example, nucleic acids encoding one or both of the heavy and light chain polypeptides of the antibody can be inserted into expression vectors comprising transcriptional and translational regulatory sequences, including, for example, promoter sequences, ribosome binding sites, transcriptional initiation and termination sequences, translational initiation and termination sequences, transcriptional terminator signals, polyadenylation signals, and enhancer or activator sequences. Regulatory sequences include promoters and transcriptional initiation and termination sequences. In addition, expression vectors can include more than one replication system so that they can be maintained in two different organisms, such as by expression in mammalian or insect cells, and by cloning and amplification in prokaryotic hosts.

[0402] Several possible vector systems can be used to express heavy and light chain polypeptides cloned from nucleic acids in mammalian cells. One type of vector relies on integration of the desired gene sequence into the host cell genome. Cells with stably integrated DNA can be modified by simultaneously introducing a drug resistance gene such as E. coli gpt (Mulligan and Berg (1981) Proc Natl Acad Sci USA78 :2072) or Tn5 neo (Southern and Berg (1982) Mol Appl Genet 1 :327). The selectable marker gene can be linked to the DNA sequence to be expressed, or the selectable marker gene can be introduced into the same cell by co-transfection (Wigler et al. (1979) Cell 16 :77). The second type of vector utilizes DNA elements that confer autonomous replication capability to extrachromosomal plasmids. 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).

[0403] The expression vector can be introduced into the cell in a manner suitable for subsequent expression of the nucleic acid. As discussed below, the method of introduction is primarily determined by the target cell type. Exemplary methods include CaPO precipitation, liposome fusion, cationic liposomes, electroporation, viral infection, dextran-mediated transfection, polybrene-mediated transfection, protoplast fusion, and direct microinjection.

[0404] Suitable host cells for expressing antibodies or antigen-binding fragments thereof include yeast, bacteria, insects, plants and mammalian cells. Of particular interest are bacteria such as Escherichia 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.

[0405] In some embodiments, antibodies or fragments thereof can be expressed in and purified from transgenic animals (e.g., transgenic mammals). For example, as described in, for example, Houdebine (2002) Curr Opin Biotechnol 13(6) :625-629; van Kuik-Romeijn et al. (2000) Transgenic Res 9(2):155-159; and Pollock et al. (1999) J Immunol Methods 231(1-2):147-157, antibodies can be produced in transgenic non-human mammals (e.g., rodents) and isolated from milk.

[0406] Antibodies and fragments thereof can be produced from cells by culturing host cells transformed with an expression vector containing a nucleic acid encoding the antibody or fragment under conditions sufficient to allow expression of the protein for an amount of time. Such conditions for protein expression will vary with the choice of expression vector and host cell and will be readily determined by one skilled in the art through routine experimentation. For example, antibodies expressed in E. coli can be refolded from inclusion bodies (see, e.g., Hou et al. (1998) Cytokine 10 : 319-30). Bacterial expression systems and methods of using them are well known in the art (see Current Protocols in Molecular Biology, Wiley & Sons, and Molecular Cloning--A Laboratory Manual--3rd Edition, Cold Spring Harbor Laboratory Press, New York (2001)). The selection of codons, suitable expression vectors, and suitable host cells will vary according to 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, for example, Kaszubska et al. (2000) Protein Expression and Purification 18 :213-220).

[0407] After expression, antibodies and fragments thereof can be separated. Antibodies or their fragments can be separated or purified using a variety of methods known to those skilled in the art, depending on the other components present in the sample. Standard purification methods include electrophoretic techniques, molecular techniques, immunological techniques, and chromatographic techniques, including ion exchange chromatography, hydrophobic chromatography, affinity chromatography, and reversed-phase HPLC chromatography. For example, antibodies can be purified using standard anti-antibody columns (e.g., protein-A or protein-G columns). Ultrafiltration and diafiltration techniques combined with protein concentration are also useful. See, for example, Scopes (1994) "Protein Purification, 3rd Edition," Springer-Verlag, New York City, New York. The required degree of purification will vary depending on the desired use. In some cases, purification of the expressed antibody or its fragment is not necessary.

[0408] Methods for determining the yield or purity of purified antibodies or fragments thereof are known in the art and include, for example, the Bradford assay, ultraviolet spectroscopy, biuret protein assay, Lowry protein assay, amidoblack protein assay, high pressure liquid chromatography (HPLC), mass spectrometry (MS), and gel electrophoresis methods (e.g., using protein stains such as Coomassie blue or colloidal silver staining).

[0409] Modification of antibodies or antigen-binding fragments thereof

[0410] The antibody or its antigen-binding fragment can be modified after expression and purification. The modification can be covalent or non-covalent modification. Such modifications can be introduced into the antibody or fragment by, for example, reacting the targeted amino acid residues of the polypeptide with an organic derivatizing agent that can react with the selected side chain or terminal residue. Any one of a plurality of criteria (including, for example, structural analysis or amino acid sequence analysis of the antibody or fragment) can be used to select a suitable modification site.

[0411] In some embodiments, the antibody or antigen-binding fragment thereof can be conjugated to a heterologous moiety. The heterologous moiety can be, for example, a heterologous polypeptide, a therapeutic agent (e.g., a toxin or a drug), or a detectable label, such as, but not limited to, a radiolabel, an 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, antigen 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 purification of the antibody or fragment. Heterologous polypeptides also include polypeptides (e.g., enzymes) used as diagnostic or detectable markers, 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 3 H. Suitable fluorescent labels include, but are not limited to, fluorescein, fluorescein isothiocyanate (FITC), green fluorescent protein (GFP), DyLight TM 488, Phycoerythrin (PE), Propidium Iodide (PI), PerCP, PE-Alexa 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 diethylenetriaminepentaacetic acid (DTPA) or tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). Enzymatic labels include, for example, alkaline phosphatase, CAT, luciferase, and horseradish peroxidase.

[0412] Two proteins (e.g., an antibody and a heterologous moiety) can be cross-linked using any of a number of known chemical cross-linking agents. Examples of such cross-linking agents are those that link two amino acid residues via a linkage comprising a "hindered" disulfide bond. In these linkages, the disulfide bond within the cross-linking unit is protected (by hindering groups on either side of the disulfide bond) from reduction by, for example, the action of reduced glutathione or disulfide reductase. A suitable reagent, 4-succinimidyloxycarbonyl-α-methyl-α(2-pyridyldithio)toluene (SMPT), utilizes a terminal lysine on one protein and a terminal cysteine ​​on the other to form such a linkage between two proteins. Heterobifunctional reagents that cross-link via different coupling 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 the guanidinium group present in the side chain of arginine (e.g., p-azidophenylglyoxal monohydrate).

[0413] In some embodiments, the radiolabel can be conjugated directly to the amino acid backbone of the antibody. Alternatively, the radiolabel can be incorporated as part of a larger molecule (e.g., m-[ 125 I] Iodophenyl-N-hydroxysuccinimide ([ 125 I]mIPNHS) 125 I) comprises, which binds to a free amino group to form a meta-iodophenyl (mIP) derivative of the protein of interest (see, e.g., Rogers et al. (1997) J Nucl Med 38: 1221-1229) or chelates (e.g., chelates with DOTA or DTPA), which are then bound to the protein backbone. Methods for conjugating radiolabels or larger molecules / chelates containing them to the antibodies or antigen-binding fragments described herein are known in the art. Such methods involve incubating the protein with the radiolabel under conditions (e.g., pH, salt concentration, and / or temperature) that are favorable for binding of the radiolabel or chelator to the protein (e.g., see U.S. Patent No. 6,001,329).

[0414] Methods for conjugating fluorescent markers (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 succinimide (NHS) esters or tetrafluorophenyl (TFP) ester moieties connected to fluorophores. In some embodiments, fluorophores can be conjugated to heterobifunctional cross-linking agent moieties (such as sulfo-SMCC). Suitable conjugation methods involve incubating antibody proteins or fragments thereof with fluorophores under conditions that promote binding of fluorophores to proteins. See, for example, Welch and Redvanly (2003) "Handbook of Radiopharmaceuticals:Radiochemistry and Applications," John Wiley and Sons (ISBN 0471495603).

[0415] In some embodiments, the antibody or fragment may be modified, for example, with a moiety that improves the stability and / or retention of the antibody in circulation (e.g., in blood, serum, or other tissues). For example, the antibody or fragment may be modified as described, for example, in Lee et al. (1999) Bioconjug Chem 10(6) :973-8; Kinstler et al. (2002) Advanced Drug Deliveries Reviews 54 :477-485; and Roberts et al. (2002) Advanced Drug Delivery Reviews 54 The antibody or fragment may be pegylated as described in SEQ ID NO: 459-476, or may be HESylated (Fresenius Kabi, Germany; see e.g. et al. (2010) Int J Pharm 387(1-2)The stabilizing portion can increase the stability or retention of the antibody (or fragment) by at least 1.5 fold (e.g., at least 2 fold, 5 fold, 10 fold, 15 fold, 20 fold, 25 fold, 30 fold, 40 fold, or 50 fold or more).

[0416] In some embodiments, the antibodies or antigen-binding fragments thereof described herein can be glycosylated. In some embodiments, the antibodies or antigen-binding fragments thereof described herein can be enzymatically or chemically treated, or produced from cells, such that the antibodies or fragments have reduced or absent 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.

[0417] Pharmaceutical compositions and preparations

[0418] In certain embodiments, the present invention provides a pharmaceutical composition comprising an anti-IL-27 antibody and a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and / or adjuvant.

[0419] In certain embodiments, acceptable formulation materials are preferably non-toxic to the recipient at the dosages and concentrations employed. In certain embodiments, one or more formulation materials are for subcutaneous and / or intravenous administration. In certain embodiments, pharmaceutical compositions may include formulation materials that are used to alter, maintain, or preserve, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption, or penetration of the composition. In certain embodiments, suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine, or lysine); antimicrobial agents; antioxidants (such as ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (such as borates, bicarbonates, Tris-HCl, citrates, phosphates, or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, β-cyclodextrin, or hydroxypropyl-β-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose, or dextrin); proteins (such as serum albumin, gelatin, or immunoglobulins); colorants, flavorings, and diluents; emulsifiers; hydrophilic polymers (such as polysorbate 60, dextrin); pyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenylethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerol, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronic, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol, tyloxapol); stability enhancers (such as sucrose or sorbitol); tonicity enhancers (such as alkali metal halides, preferably sodium chloride or potassium chloride, mannitol sorbitol); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants. (Remington's Pharmaceutical Sciences, 18th ed., A.R. Gennaro, ed., Mack Publishing Company (1995). In certain embodiments, the formulation comprises PBS; 20 mM NaOAC (pH 5.2), 50 mM NaCl; and / or 10 mM NAOAC (pH 5.2), 9% sucrose. In certain embodiments, the optimal pharmaceutical composition will be determined by one skilled in the art based on, for example, the intended route of administration, delivery form, 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.

[0420] In certain embodiments, the primary vehicle or carrier in the pharmaceutical composition may be aqueous or non-aqueous in nature. For example, in certain embodiments, a suitable vehicle or carrier may be water for injection, physiological saline solution, or artificial cerebrospinal fluid, which may be supplemented with other materials commonly used in compositions for parenteral administration. In certain embodiments, the saline comprises isotonic phosphate-buffered saline. In certain embodiments, neutral buffered saline or saline mixed with serum albumin is a further exemplary vehicle. In certain embodiments, the pharmaceutical composition comprises a Tris buffer having a pH of about 7.0-8.5, or an acetate buffer having a pH of about 4.0-5.5, which may further comprise sorbitol or a suitable alternative thereof. In certain embodiments, a composition comprising an anti-IL-27 antibody can be prepared for storage by mixing a selected composition having the desired purity with an optional formulation (Remington's Pharmaceutical Sciences, supra) in the form of a lyophilized cake or aqueous solution. In addition, in certain embodiments, the composition comprising an anti-IL-27 antibody can be formulated as a lyophilizate using a suitable excipient such as sucrose.

[0421] In certain embodiments, pharmaceutical compositions can be selected for parenteral delivery. In certain embodiments, compositions can be selected for suction or delivered by the digestive tract (such as oral). The preparation of such pharmaceutically acceptable compositions is within the capabilities of those skilled in the art.

[0422] In certain embodiments, the formulation components are present in concentrations 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 the pH range of about 5 to about 8.

[0423] In certain embodiments, when envisioning parenteral administration, therapeutic compositions can be present in a pharmaceutically acceptable vehicle in the form of a pyrogen-free, parenteral acceptable aqueous solution, the aqueous solution comprising anti-IL-27 antibodies. In certain embodiments, the vehicle for parenteral injection is sterile distilled water, wherein the anti-IL-27 antibodies are formulated into sterile isotonic solutions, and are suitably preserved. In certain embodiments, the preparation can be directed to preparing the desired molecule together with an agent (such as injectable microspheres, bioerodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads or liposomes), the agent can provide controlled or sustained release of the product, and the product can then be delivered by reservoir injection. In certain embodiments, hyaluronic acid can also be used, the hyaluronic acid can have the effect of promoting the duration in the circulation. In certain embodiments, an implantable drug delivery device can be used to introduce the desired molecule.

[0424] In certain embodiments, the pharmaceutical composition can be formulated for inhalation. In certain embodiments, the anti-IL-27 antibody can be formulated as a dry powder for inhalation. In certain embodiments, an inhalation solution containing the anti-IL-27 antibody can be formulated with a propellant for aerosol delivery. In certain embodiments, the solution can be atomized. Pulmonary administration is further described in PCT Application No. PCT / US94 / 001875, which describes pulmonary delivery of chemically modified proteins.

[0425] In certain embodiments, it is envisioned that the formulation can be administered orally. In certain embodiments, the anti-IL-27 antibody administered in this manner may be formulated with or without carriers commonly used to formulate solid dosage forms such as tablets and capsules. In certain embodiments, the capsule may be designed to release the active portion of the formulation at a point in the gastrointestinal tract where bioavailability is maximized and pre-systemic degradation is minimized. In certain embodiments, at least one additional agent may be included to promote absorption of the anti-IL-27 antibody. In certain embodiments, diluents, flavorings, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrants, and binders may also be used.

[0426] In certain embodiments, pharmaceutical compositions may comprise an effective amount of an anti-IL-27 antibody in admixture with a nontoxic excipient suitable for tablet production. In certain embodiments, a solution can be prepared in unit dosage form by dissolving the tablet 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 sodium bicarbonate, lactose, or calcium phosphate; or binders such as starch, gelatin, or acacia; or lubricants such as magnesium stearate, stearic acid, or talc.

[0427] Other pharmaceutical compositions will be apparent to those skilled in the art, including formulations comprising anti-IL-27 antibodies in sustained or controlled release formulations. In certain embodiments, techniques for formulating a variety of other sustained or controlled delivery components (such as liposome carriers, bioerodible microparticles or porous beads, and reservoir injections) are also known to those skilled in the art. See, for example, PCT Application No. PCT / US93 / 00829 describing controlled release of porous polymer microparticles for delivery of pharmaceutical compositions. In certain embodiments, sustained release formulations may include a semipermeable polymer matrix in the form of a shaped article (e.g., a film or microcapsule). Sustained-release matrices may include polyesters, hydrogels, polylactides (U.S. Pat. No. 3,773,919 and EP 058,481), copolymers of L-glutamic acid and γ-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-hydroxybutyric acid (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 088,046 and EP 143,949.

[0428] The pharmaceutical composition for internal use is usually sterile. In certain embodiments, this can be achieved by filtering with a sterile filtration membrane. In certain embodiments, when freeze-dried compositions, sterilization using this method can be carried out before or after freeze-drying and reconstitution. In certain embodiments, compositions for parenteral administration can be stored in a lyophilized form or a solution form. In certain embodiments, parenteral compositions are usually placed in a container with a sterile entrance, for example, an intravenous solution bag or a bottle with a stopper that can be pierced by a hypodermic needle.

[0429] In certain embodiments, once the pharmaceutical composition is formulated, it can be stored in a sterile vial in the form of a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder. In certain embodiments, such formulations can be stored in a ready-to-use or administered reconstituted form (e.g., lyophilized).

[0430] In certain embodiments, kits for producing single-dose administration units are provided. In certain embodiments, the kit may comprise a first container containing a dried protein and a second container containing an aqueous formulation. In certain embodiments, kits comprising single-chamber and multi-chamber prefilled syringes (e.g., liquid syringes and lyophilizing syringes) are included.

[0431] In certain embodiments, the effective amount of a pharmaceutical composition comprising an anti-IL-27 antibody to be used therapeutically will depend, for example, on the therapeutic context and objectives. Those skilled in the art will appreciate that, according to certain embodiments, the appropriate dosage level for treatment will therefore vary depending, in part, on the molecule being delivered, the indication for which the anti-IL-27 antibody is being used, the route of administration, and the patient's size (weight, body surface area, or organ size) and / or condition (age and general health). In certain embodiments, the clinician can titrate the dosage and modify the route of administration to achieve the optimal therapeutic effect.

[0432] In certain embodiments, the frequency of administration will take into account the pharmacokinetic parameters of the anti-IL-27 antibody in the formulation used. In certain embodiments, the clinician will administer the composition until the dose that achieves the desired effect is reached. In certain embodiments, the composition can therefore be administered over time in a single dose or two or more doses (which may or may not contain the same amount of the desired molecule), or as a continuous infusion via an implant or catheter. Further refinement of the appropriate dosage is routinely performed by those of ordinary skill in the art and is within the scope of the tasks they routinely perform. In certain embodiments, the appropriate dosage can be determined by using appropriate dose-response data.

[0433] In certain embodiments, the route of administration of the pharmaceutical composition is according to known methods, such as oral administration, by injection via intravenous, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, subcutaneous, intraocular, intraarterial, intraportal or intralesional routes; by sustained release system or implant device. In certain embodiments, the composition can be administered by bolus injection or continuously administered by infusion, or administered by implant device. In certain embodiments, the individual components of the combination therapy can be administered by different routes.

[0434] In certain embodiments, the composition can be administered topically by implanting a membrane, sponge, or another suitable material onto which the desired molecule has been adsorbed or encapsulated. In certain embodiments, when an implant device is used, the device can be implanted in any suitable tissue or organ, and the delivery of the desired molecule can be carried out by diffusion, timed-release injection, 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 cases, 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 implanted back into the patient's body.

[0435] In certain embodiments, anti-IL-27 antibodies can be delivered by implanting certain genetically engineered cells to express and secrete polypeptides using methods such as those described herein. In certain embodiments, such cells can be animal or human cells and can be autologous, heterologous, or xenogeneic. In certain embodiments, the cells can be immortalized. In certain embodiments, in order to reduce the chance of an immune response, the cells can be encapsulated to avoid infiltration of surrounding tissues. In certain embodiments, the encapsulating material is typically a biocompatible, semipermeable polymer shell or membrane that allows the release of one or more protein products but prevents the cells from being destroyed by the patient's immune system or other harmful factors from surrounding tissues.

[0436] application

[0437] The compositions described herein can be used for a variety of diagnostic and therapeutic applications. For example, detectably labeled antigen binding molecules can be used to detect the presence or amount of target antigens in a sample (e.g., a biological sample). The compositions can be used for in vitro determinations of the inhibition of target antigen function. In some embodiments, such as where the compositions are combined with complement proteins and inhibit complement proteins, the compositions can be used as a positive control in determinations, and the determinations are designed to identify other novel compounds that inhibit complement activity or are otherwise used to treat complement-related disorders. For example, IL-27 inhibitory compositions can be used as a positive control in determinations to identify other compounds (e.g., small molecules, aptamers, or antibodies) that reduce or eliminate IL-27 production. The compositions can also be used for methods of treatment described in detail below.

[0438] In some embodiments, the present disclosure provides methods of detecting IL-27 in a biological sample or subject, comprising (i) contacting the sample or subject (and optionally a reference sample or subject) with any of the antibodies described herein under conditions permissive for interaction between the antibody molecule and IL-27, and (ii) detecting formation of a complex between the antibody molecule and the sample or subject (and optionally a reference sample or subject).

[0439] medicine box

[0440] The kit may include an anti-IL-27 antibody as disclosed herein and instructions for use. The kit may include the anti-IL-27 antibody, one or more controls, and various buffers, reagents, enzymes, and other standard components known in the art in suitable containers. In some aspects, the present disclosure provides a kit comprising an anti-IL-27 antibody or antigen-binding portion disclosed herein, and instructions for use for stimulating an immune response in a subject or treating cancer in a subject, optionally in combination with one or more additional therapeutic agents or procedures described herein.

[0441] The container may include at least one vial, well, test tube, flask, bottle, syringe, or other container device into which the anti-IL-27 antibody can be placed and, in some cases, into which the anti-IL-27 antibody can be appropriately aliquoted. If additional components are provided, the kit may contain additional containers into which such components can be placed. The kit may also include a device for sealingly containing the anti-IL-27 antibody and any other reagent containers for commercial sale. Such containers may include injection-molded or blow-molded plastic containers into which the desired vials are retained. The container and / or kit may include a label with instructions for use and / or warnings.

[0442] How to use

[0443] The compositions of the present invention have a number of in vitro and in vivo uses, including the detection and / or quantification of IL-27 and / or antagonism of IL-27 function.

[0444] 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.

[0445] In some embodiments, the present disclosure provides a method of inhibiting or reducing the 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 the inhibition of CD161 expression in the cell.

[0446] In some embodiments, the present disclosure provides a method of 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 present disclosure, wherein the antibody or antigen-binding portion thereof inhibits or reduces PD-L1 and / or TIM-3 expression in the cell.

[0447] In some embodiments, the present disclosure provides a method of inducing or enhancing the 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.

[0448] In some embodiments, the present disclosure provides a method for stimulating an immune response in a subject, comprising administering to the subject an effective amount of an isolated monoclonal antibody or antigen-binding portion thereof that specifically binds to and antagonizes human IL-27 provided by the present disclosure, or a pharmaceutical composition comprising the antibody or antigen-binding portion thereof and a pharmaceutically acceptable carrier.

[0449] In some embodiments, the present disclosure provides a method of treating cancer in a subject, 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, or a pharmaceutical composition comprising the antibody or antigen-binding portion thereof and a pharmaceutically acceptable carrier.

[0450] In some embodiments, the present disclosure provides a method for stimulating an immune response in a subject or treating cancer in a subject, the method comprising administering to the subject an effective amount of an isolated monoclonal antibody or antigen-binding fragment provided by the present disclosure, or a pharmaceutical composition comprising the antibody or its antigen-binding portion and a pharmaceutically acceptable carrier, wherein the antibody or its antigen-binding portion or pharmaceutical composition inhibits or reduces STAT1 and / or STAT3 phosphorylation in cells, thereby stimulating an immune response or treating cancer.

[0451] In some embodiments, the present disclosure provides a method for stimulating an immune response in a subject or treating cancer in a subject, the method comprising administering to the subject an effective amount of an isolated monoclonal antibody or antigen-binding fragment provided by the present disclosure, or a pharmaceutical composition comprising the antibody or its antigen-binding portion and a pharmaceutically acceptable carrier, wherein the antibody or its antigen-binding portion or pharmaceutical composition inhibits or reduces the inhibition of CD161 expression in cells, thereby stimulating the immune response or treating cancer.

[0452] In some embodiments, the present disclosure provides a method of stimulating an immune response in a subject or treating cancer in a subject, the method comprising administering to the subject an effective amount of an isolated monoclonal antibody or antigen-binding fragment provided by the present disclosure, or a pharmaceutical composition comprising the antibody or its antigen-binding portion and a pharmaceutically acceptable carrier, wherein the antibody or its antigen-binding portion or pharmaceutical composition inhibits or reduces PD-L1 and / or TIM-3 expression in cells, thereby stimulating an immune response or treating cancer.

[0453] In some embodiments, the present disclosure provides a method of stimulating an immune response in a subject or treating cancer in a subject, the method comprising administering to the subject an effective amount of an isolated monoclonal antibody or antigen-binding fragment provided by the present disclosure, or a pharmaceutical composition comprising the antibody or its antigen-binding portion and a pharmaceutically acceptable carrier, wherein the antibody or its antigen-binding portion or pharmaceutical composition induces or enhances PD-1-mediated secretion of one or more cytokines from cells, thereby stimulating an immune response or treating cancer.

[0454] 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., squamous head and neck cancer), colorectal cancer, bladder cancer, endometrial cancer, prostate cancer, thyroid cancer, hepatocellular carcinoma, gastric cancer, brain cancer, lymphoma (e.g., DL-BCL), leukemia (e.g., AML), or kidney cancer (e.g., renal cell carcinoma, e.g., clear cell renal carcinoma).

[0455] The above compositions are particularly useful for methods of treating or preventing a variety of cancers in a subject. 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 (IP) injection, intramuscular injection (IM), or intrathecal injection (IT). The injection can be a bolus or continuous infusion.

[0456] Administration can be achieved by, for example, local infusion, injection or by implantation. Implants can be porous, non-porous or gelatinous materials, including membranes, such as salivary elastic membranes or fibers. Implants can be configured to continuously or periodically release compositions to a subject. See, for example, U.S. Patent Application Publication No. 20080241223; U.S. Patent Nos. 5,501,856, 4,863,457 and 3,710,795; EP488401; and EP 430539, each of which is incorporated herein by reference in its entirety. Compositions can be delivered to a subject by an implantable device (e.g., osmotic pump, biodegradable implant, electrodiffusion system, electroosmotic system, vapor pressure pump, electrolytic pump, bubbling pump, piezoelectric pump, erosion-based system or electromechanical system) based on, for example, a diffusion system, an erodible system or a convection system.

[0457] In some embodiments, the anti-IL-27 antibody or antigen-binding fragment thereof is therapeutically delivered to a subject by local administration.

[0458] The appropriate dosage of an antibody or fragment thereof described herein (the dosage being capable of treating or preventing cancer in a subject) may depend on a variety of factors, including, for example, the age, sex, and weight of the subject to be treated and the specific inhibitor compound used. For example, a different dose of a whole anti-IL-27 antibody may be required to treat a subject with cancer than the dose of an IL-27-binding Fab' antibody fragment required to treat the same subject. Other factors that affect the dosage administered to a subject include, for example, the type or severity of the cancer. For example, a subject with metastatic melanoma may require administration of a different dose of an anti-IL-27 antibody than a subject with glioblastoma. Other factors may include, for example, other medical conditions that may affect the subject concurrently or previously, the subject's general health, the subject's genetic predisposition, diet, time of administration, excretion rate, drug combination, and any other additional therapeutic agents administered to the subject. It should also be understood that the specific dosage and treatment regimen for any particular subject will also depend on the judgment of the practitioner (e.g., a doctor or nurse). Suitable dosages are described herein.

[0459] The pharmaceutical composition may comprise a therapeutically effective amount of an anti-IL-27 antibody or antigen-binding fragment thereof as described herein. If more than one agent is used, one of ordinary skill in the art can readily determine such an effective amount based, in part, on the effect of the administered antibody or the combined effect of the antibody and one or more additional active agents. The therapeutically effective amount of an antibody or fragment thereof as described herein may also vary depending on factors such as the individual's disease state, age, sex, and weight, as well as the ability of the antibody (and one or more additional active agents) to elicit a desired response (e.g., reduction in tumor growth) in the individual. For example, a therapeutically effective amount of an anti-IL-27 antibody can inhibit (reduce the severity or eliminate the occurrence of) and / or prevent any symptom of a particular condition and / or a particular condition known in the art or described herein. A therapeutically effective amount is also an amount in which any toxic or deleterious effects of the composition are outweighed by the therapeutically beneficial effects.

[0460] Suitable human doses of any of the antibodies or fragments thereof described herein can be further evaluated in, for example, a Phase I dose escalation study. See, for example, van Gurp et al. (2008) Am J Transplantation 8(8):1711-1718; Hanouska et al. (2007) Clin Cancer Res 13(2, part 1):523-531; and Hetherington et al. (2006) Antimicrobial Agents and Chemotherapy 50(10):3499-3500.

[0461] In some embodiments, the composition comprises any of the antibodies or antigen-binding fragments thereof described herein and one or more (e.g., two, three, four, five, six, seven, eight, nine, 10, or 11 or more) additional therapeutic agents such that the composition as a whole is therapeutically effective. For example, the composition may comprise an anti-IL-27 antibody described herein and an alkylating agent, wherein the concentrations of each of the antibody and agent, when combined, are therapeutically effective for treating or preventing cancer (e.g., melanoma) in a subject.

[0462] The toxicity and therapeutic efficacy of such compositions can be determined by known pharmaceutical procedures in cell cultures or experimental animals (e.g., animal models of any of the cancers described herein). These procedures can be used, for example, to determine the LD 50 (lethal dose for 50% of the population) and ED 50 (the therapeutically effective dose in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD 50 / ED 50 Antibodies or antigen-binding fragments thereof that exhibit high therapeutic indices are preferred. Although compositions that exhibit toxic side effects may be used, care should be taken to design delivery systems that target such compounds to the site of affected tissue and minimize potential damage to normal cells, thereby reducing side effects.

[0463] The data obtained from cell culture assays and animal studies can be used to formulate a range of dosages for use in humans. Dosages of such antibodies or antigen-binding fragments thereof will generally range from about 100 mg / dL to about 100 mg / dL, including the ED 50 The dosage may vary within this range depending on the dosage form employed and the route of administration used. For the anti-IL-27 antibodies described herein, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve an IC value, including that determined in cell culture. 50 The range of circulating plasma concentrations of the antibody (i.e., the antibody concentration that achieves half-maximal inhibition of symptoms) can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography. In some embodiments, for example, where local administration is desired (e.g., to the eye or joints), cell culture or animal modeling can be used to determine the dose required to achieve a therapeutically effective concentration in the local area.

[0464] In some embodiments, the methods can be combined with other therapeutic agents for cancer. For example, the composition can be administered to a subject simultaneously with, before, or after radiation, surgery, targeted or cytotoxic chemotherapy, chemoradiotherapy, hormone therapy, immunotherapy, gene therapy, cell transplantation therapy, precision medicine, genome editing therapy, or other drug therapy.

[0465] As described above, the compositions described herein (e.g., anti-IL-27 compositions) can be used to treat a variety of cancers, such as, but not limited to, Kaposi's sarcoma, leukemias, acute lymphocytic leukemia, acute myeloid leukemia, myeloblasts promyelocyte myelomonocytic monocyticerythroleukemia, chronic leukemia, chronic myeloid (granulocytic) leukemia, chronic lymphocytic leukemia, mantle cell lymphoma, primary central nervous system lymphoma, Burkitt lymphoma and marginal zone B-cell lymphoma, polycythemia vera lymphoma, Hodgkin's disease, non-Hodgkin's disease, multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, solid tumors, sarcomas and carcinomas, fibrosarcomas, myxosarcoma, liposarcoma, Chondrosarcoma, osteogenic sarcoma, osteosarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon sarcoma, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma (HCC), hepatoma, bile duct carcinoma, choriocarcinoma Chorionic carcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicular tumors, lung cancer, small cell lung cancer, non-small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, nasopharyngeal cancer, esophageal cancer, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain and central nervous system cancer (CNS) cancer, cervical cancer, choriocarcinoma, colorectal cancer, connective tissue cancer, digestive system cancer, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, stomach cancer, intraepithelial neoplasia, kidney cancer, laryngeal cancer, liver cancer, lung cancer (small cell, large cell), melanoma, neuroblastoma; oral cancer (e.g., lip, tongue, mouth and pharynx), ovarian cancer, pancreatic cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer; respiratory system cancer, sarcoma, skin cancer, stomach cancer, testicular cancer, thyroid cancer, uterine cancer and urinary system cancer.

[0466] Combination therapy

[0467] In some embodiments, the anti-IL-27 antibodies, or antigen-binding portions thereof, provided herein can 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, can be administered to a subject (e.g., a human patient) in combination with one or more additional therapeutic agents, where the combination provides a therapeutic benefit to a subject having or at risk of developing cancer.

[0468] In some embodiments, the anti-IL-27 antibody, or antigen-binding portion thereof, and one or more additional therapeutic agents are administered at the same time (e.g., simultaneously). In other embodiments, the anti-IL-27 antibody, or antigen-binding portion thereof, is administered at a first time and the one or more additional therapeutic agents are administered at a second time (e.g., sequentially). In some embodiments, the one or more additional therapeutic agents are administered at a first time and the anti-IL-27 antibody is administered at a second time.

[0469] The anti-IL-27 antibodies or antigen-binding fragments thereof described herein can replace or enhance a previously or currently administered therapy. For example, after treatment with an anti-IL-27 antibody or antigen-binding fragment thereof, administration of one or more additional therapeutic agents can be stopped or reduced, for example, by administering one or more additional therapeutic agents at a lower level. In some embodiments, administration of the previous therapy can be maintained. In some embodiments, the previous therapy will be maintained until the level of the anti-IL-27 antibody reaches a level sufficient to provide a therapeutic effect.

[0470] In some embodiments, the present disclosure provides a method of treating cancer in a subject, 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 procedures, wherein the second therapeutic agent or procedure is selected from the group consisting of chemotherapy, targeted anti-cancer therapy, oncolytic drugs, cytotoxic agents, immune-based therapies, cytokines, surgical procedures, radiation procedures, activators of costimulatory molecules, inhibitors of inhibitory molecules, vaccines, or cellular immunotherapy, or a combination thereof.

[0471] 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.

[0472] In some embodiments, the one or more additional therapeutic agents are PD-1 antagonists. In some embodiments, 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. In certain embodiments, the one or more additional therapeutic agents are PD-L1 inhibitors. In some embodiments, the PD-L1 inhibitor is selected from the group consisting of: FAZ053, atezolizumab, avelumab, durvalumab, and BMS-936559. In some embodiments, the present disclosure provides a method of 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 the 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.

[0473] In some embodiments, the one or more additional therapeutic agents is sunitinib Cabozantinib Axitinib Lenvatinib Everolimus Bevacizumab Edoxetine, NKTR-214 (CD-122-biased agonist), Tivozanib Abetastat, ipilimumab Tremelimumab, pazopanib Sorafenib Sirolimus Ramucirumab Niraparib, savolitinib, vololanib (X-82), regorafenib Donafenib (multikinase inhibitor), Camrelizumab (SHR-1210), Dipesatimoquin (JX-594), Ramucirumab Apatinib (YN968D1), encapsulated doxorubicin Tivantinib (ARQ197), ADI-PEG 20, bimetinib, apatinib mesylate, nintedanib, lirilumab, nivolumab Pembrolizumab Atezolizumab Avelumab Durvalumab Cemipril-rwlc Tislelizumab and / or spartalizumab.

[0474] 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.

[0475] 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.

[0476] In some embodiments, one or more additional therapeutic agents are TIGIT inhibitors. In some embodiments, one or more additional therapeutic agents are CD112R inhibitors. In some embodiments, one or more additional therapeutic agents are TAM (Axl, Mer, Tyro) inhibitors. In some embodiments, one or more additional therapeutic agents are STING agonists. In some embodiments, one or more additional therapeutic agents are 4-1BB agonists.

[0477] Combination with chemotherapeutic agents

[0478] Suitable chemotherapeutic agents for use in combination and / or co-administration with the compositions of the present invention include, for example, paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenotoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthrancindione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, and analogs or homologs thereof. Other agents include, for example, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thiotepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, cis-dichlorodiamineplatinum (II) (DDP), procarbazine, hexamethylmelamine, cisplatin, carboplatin, oxaliplatin, nedaplatin, satraplatin, or triplatin tetranitrate), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and antimitotic agents (e.g., vincristine and vinblastine), and temozolomide.

[0479] Combination with PD-1 / PD-L1 antagonists

[0480] 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 one or more downstream pathways and / or cellular processes mediated by human PD-1 / PD-L1 signaling or other human PD-1 / PD-L1-mediated functions.

[0481] Thus, provided herein are PD-1 antagonists that directly or allosterically block, antagonize, suppress, inhibit, or reduce PD-1 / PD-L1 biological activity, including downstream pathways and / or cellular processes mediated by PD-1 / PD-L1 signaling, such as receptor binding and / or initiation of cellular responses to PD-1 / PD-L1. Also provided herein are PD-1 antagonists that reduce the number or amount of human PD-1 or PD-L1 produced by a cell or subject.

[0482] In some embodiments, the present disclosure provides PD-1 antagonists that bind to human PD-1 and prevent, inhibit, or reduce the binding of PD-L1 to PD-1. In some aspects, the PD-1 antagonists bind to mRNA encoding PD-1 or PD-L1 and prevent translation. In some embodiments, the PD-1 antagonists bind to mRNA encoding PD-1 or PD-L1 and cause degradation and / or turnover.

[0483] In some embodiments, a PD-1 antagonist inhibits PD-1 signaling or function. In some embodiments, a PD-1 antagonist blocks the binding of PD-1 to PD-L1, PD-L2, or PD-L1 and PD-L2. In some embodiments, a PD-1 antagonist blocks the binding of PD-1 to PD-L1. In some embodiments, a PD-1 antagonist blocks the binding of PD-1 to PD-L2. In some embodiments, a PD-1 antagonist blocks the binding of PD-1 to PD-L1 and PD-L2. In some embodiments, a PD-1 antagonist specifically binds to PD-1. In some embodiments, a PD-1 antagonist specifically binds to PD-L1. In some embodiments, a PD-1 antagonist specifically binds to PD-L2.

[0484] In some embodiments, a PD-1 antagonist inhibits the binding of PD-1 to its cognate ligand. In some embodiments, a PD-1 antagonist inhibits the binding of PD-1 to PD-L1, PD-1 to PD-L2, or PD-1 to PD-L1 and PD-L2. In some embodiments, a PD-1 antagonist does not inhibit the binding of PD-1 to its cognate ligand.

[0485] In some embodiments, the PD-1 antagonist is an isolated monoclonal antibody (mAb) or an antigen-binding fragment thereof that specifically binds to PD-1 or PD-L1. In some embodiments, the PD-1 antagonist is an antibody or an antigen-binding fragment thereof that specifically binds to human PD-1. In some embodiments, the PD-1 antagonist is an antibody or an antigen-binding fragment thereof that specifically binds to human PD-L1. In some embodiments, the PD-1 antagonist is an antibody or an antigen-binding fragment that binds to human PD-L1 and inhibits the binding of PD-L1 to PD-1. In some embodiments, the PD-1 antagonist is an antibody or an antigen-binding fragment that binds to human PD-1 and inhibits the binding of PD-L1 to PD-1.

[0486] 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 for the treatment of cancer.

[0487] Examples of anti-human PD-1 monoclonal antibodies or antigen-binding fragments thereof that may comprise a PD-1 antagonist in any of the compositions, methods, and uses provided by the present disclosure include, but are not limited to: (pembrolizumab, MK-3475, h409A11; see US8952136, US8354509, US8900587 and EP2170959, all of which are incorporated herein by reference in their entirety; Merck), (nivolumab, BMS-936558, MDX-1106, ONO-4538; see US7595048, US8728474, US9073994, US9067999, EP1537878, US8008449, US8779105 and EP2161336, all of which are herein incorporated by reference in their entirety; Bristol Myers Squibb), MEDI0680 (AMP-514), BGB-A317 and BGB-108 (BeiGene), 244C8 and 388D4 (see WO2016106159, which are herein incorporated by reference in their 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.

[0488] Examples of anti-human PD-L1 monoclonal antibodies or antigen-binding fragments thereof that can comprise a PD-1 antagonist in any of the compositions, methods, and uses provided by the present disclosure include, but are not limited to: (avelumab, MSB0010718C, see WO2013 / 79174, which is incorporated herein by reference in its entirety; Merck / Pfizer), (Durvalumab, MEDI4736), (atezolizumab, MPDL3280A, RG7446; see WO2010 / 077634, which is incorporated herein by reference in its entirety; Roche), MDX-1105 (BMS-936559, 12A4; see US7943743 and WO2013 / 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.

[0489] In some embodiments, the PD-1 antagonist is an immunoadhesin that specifically binds to human PD-1 or human PD-L1, for example, a fusion protein comprising the extracellular portion of PD-L1 or PD-L2 or a PD-1 binding portion fused to a constant region of an immunoglobulin molecule (such as an Fc region). Examples of immunoadhesins that specifically bind to PD-1 are described in WO2010 / 027827 and WO2011 / 066342, both of which are incorporated herein by reference in their entirety. In some embodiments, the PD-1 antagonist is AMP-224 (also known as B7-DCIg), which is a PD-L2-FC fusion protein that specifically binds to human PD-1.

[0490] One of ordinary skill in the art will understand 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.

[0491] In some embodiments, the PD-1 / PD-L1 antagonist is a small molecule, a nucleic acid, a peptide, a peptide mimetic, a protein, a carbohydrate, a carbohydrate derivative, or a glycopolymer. Exemplary small molecule PD-1 inhibitors are described in Zhan et al. (2016) Drug Discov Today 21(6): 1027-1036.

[0492] Combination with TIM-3 inhibitors

[0493] In some embodiments, an anti-IL-27 antibody or antigen-binding portion thereof provided by the present disclosure is combined with a TIM-3 inhibitor (e.g., administered in combination). The TIM-3 inhibitor can be an antibody, an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide. In some embodiments, the TIM-3 inhibitor is selected from MGB453 (Novartis), TSR-022 (Tesaro), or LY3321367 (Eli Lilly). In some embodiments, an anti-IL-27 antibody or antigen-binding portion thereof is administered in combination with MGB453. In some embodiments, an anti-IL-27 antibody or antigen-binding portion thereof is administered in combination with TSR-022.

[0494] Combination with LAG-3 inhibitors

[0495] In some embodiments, an anti-IL-27 antibody or antigen-binding portion thereof provided herein is combined with a LAG-3 inhibitor (e.g., administered in combination). The LAG-3 inhibitor can be an antibody, an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide. In some 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).

[0496] Other combinations

[0497] In some embodiments, an anti-IL-27 antibody, or antigen-binding portion thereof, provided herein is combined (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, NEKTAR, peg-IL-2, peg IL-10, a CD40 agonist, or a combination thereof.

[0498] Detection method

[0499] 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 described herein can be used to diagnose, prognose, and / or determine the progression of a disease (e.g., cancer) in a patient.

[0500] As defined herein, monitoring a subject (e.g., a human patient) for improvement of cancer means assessing a change in a subject's disease parameter, such as a decrease in tumor growth. In some embodiments, the assessment is performed at least one (1) hour after administration, e.g., at least 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, or 48 hours, or at least 1 day, 2 days, 4 days, 10 days, 13 days, 20 days, or longer, or at least 1 week, 2 weeks, 4 weeks, 10 weeks, 13 weeks, 20 weeks, or longer. The subject may be assessed during one or more of the following periods: before treatment begins; during treatment; or after one or more therapeutic components have been administered. The assessment may include assessing the need for further treatment, e.g., assessing whether the dose, frequency of administration, or duration of treatment should be changed. It may also include assessing the need to add or withdraw a selected treatment modality, e.g., adding or withdrawing any cancer treatment described herein.

[0501] In some embodiments, the present disclosure provides a method for detecting IL-27 in a sample from a subject, the method comprising: (a) contacting the sample from the subject with a detection antibody under conditions allowing the detection antibody to form a detection antibody-IL-27 complex in the presence of IL-27 in the sample, wherein the detection antibody is an antibody or antigen-binding fragment thereof provided by the present disclosure; and (b) detecting the presence of the complex (if any) produced in step (a).

[0502] In some embodiments, the present disclosure provides a method for detecting IL-27-related cancer in a subject, the method comprising the following steps: (a) contacting a sample from a subject suspected of having an IL-27-related cancer with a detection antibody under conditions that allow the detection antibody to form a detection antibody-IL-27 complex in the presence of IL-27 in the sample, wherein the detection antibody is an antibody or antigen-binding portion thereof provided by the present disclosure; and (b) detecting the presence of the complex (if any) produced in step (a). In some embodiments, the detection antibody is coupled to a detectable label. In some embodiments, the method further comprises contacting the sample with a capture antibody to produce a complex comprising IL-27 and the capture antibody in the presence of IL-27 in the sample, wherein the capture antibody is an antibody or antigen-binding portion thereof provided by the present disclosure.

[0503] In some embodiments, the capture antibody is immobilized on a solid support. In some embodiments, the sample is contacted with the capture antibody prior to the detection antibody. In some embodiments, the sample is a body fluid sample. In some embodiments, the body fluid sample is blood, serum, plasma, cell lysate, or tissue lysate.

[0504] 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).

[0505] Example

[0506] Although the present disclosure has been described with reference to specific embodiments thereof, it will 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 specific circumstances, materials, compositions of matter, processes, and process steps to the goals, spirit, and scope of the present disclosure. All such modifications are intended to be within the scope of the present disclosure.

[0507] Example 1: Generation and Characterization of Anti-IL-27 Antibodies That Specifically Bind to IL-27 EBI3 Monomers

[0508] This example describes the production 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 immune vector (Aldevron) and used to generate and isolate hybridomas expressing anti-EBI3 monoclonal antibodies. Isolated hybridomas included those expressing anti-IL-27 antibody molecules (referred to herein as Abl, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, and Ab8). Hybridoma supernatants were analyzed by flow cytometry on mammalian cells expressing surface-targeted human EBI3. All hybridoma clone supernatants tested bound to cells expressing EBI3 (data not shown).

[0509] For the exemplary isolated anti-EBI3 antibody Ab7 (hereinafter referred to as "Ab7", including the antibody hereinafter referred to as "Ab7-V H0 " and hereinafter referred to as "Ab7-V L0 " " immunoglobulin light chain variable region) were sequenced and further characterized below (the amino-terminal signal peptide sequence is not shown).

[0510] The isolated heavy chain variable region of Ab7 antibody (Ab7-V H0 ) has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-constant region):

[0511] EVKLVESGGGLVQPGGSLKLFCAASGFTFTSYSMSWVRQTPEKRLEWVAYISYDGGSAYYPDTVKGRFSISRDNAKKTLYLQMSSLKSEDTAMYYCARHGDYDDDDAMDYWGQGTSVTVSS (SEQ ID NO: 1).

[0512] Encoding the heavy chain variable region Ab7-V H0 The nucleic acid sequence has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4):

[0513] GAGGTGAAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAAACTCTTCTGTGCAGCCTCTGGATTCACCTTTACCAGCTATTCCATGAGCTGGGTCCGCCAGACTCCAGAGAAGAGGCTGGAGTGGGTCGCATACATTAGTTATGATGGTGGTAGCGCCTACTACCCT GACACTGTGAAGGGCCGGTTCTCCATCTCCAGAGACAATGCCAAGAAAACCCTGTATCTGCAAATGAGCAGCCTGAAGTCTGAGGACACGGCCATGTATTACTGTGCAAGACATGGAGACTATGACGACGACGACGCGATGGACTACTGGGGCCAAGGAACCTCAGTCACCGTCTCCTCA(SEQ IDNO:2).

[0514] The isolated light chain variable region of Ab7 antibody (Ab7-V L0 ) has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-constant region):

[0515] DIQMTQSPASSLSASVGETVTITCRASENIYSYLAWYQQKQ GKSPQLLVYNAETLTEGVPSRFSGSGSGTQFSLKINSLQPEDFG NYYCQHHYGTPLTFGAGTKLDLK (SEQ ID NO: 3).

[0516] The isolated heavy chain of Ab7 antibody (Ab7-V H0 -mIgG2a) has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-constant region):

[0517] EVKLVESGGGLVQPGGSLKLFCAASGFTFTSYSMSWVRQTPEKRLEWVAYISYDGGSAYYPDTVKGRFSISRDNAKKTLYLQMSSLKSEDTAMYYCARHGDYDDDDAMDYWGQGTSVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK(SEQ ID NO:183).

[0518] Nucleic acid sequence encoding the light chain variable region Ab7-V L0 has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4):

[0519] GACATCCAGATGACCCAGTCTCCAGCCTCCCTGTCTGCATCTGTAGGAGAAACTGTCACCATCACTTGCCGGGCAAGTGAGAACATTTACAGCTATTTAGCATGGTATCAGCAGAAACAGGGGAAATCTCCTCAGCTCCTGGTCTATAATGCAGAAACCTTGACAGAAGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACACAATTCTCTCTCAAGATCAACAGTCTGCAACCTGAAGATTTTGGGAATTACTACTGTCAACATCATTACGGTACCCCGCTCACATTCGGCGCTGGGACCAAGCTGGATCTGAAA(SEQ ID NO:4).

[0520] Light chain of the isolated Ab7 antibody (Ab7-V L0-mκ) has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-constant region):

[0521] DIQMTQSPASSLSASVGETVTITCRASENIYSYLAWYQQKQGKSPQLLVYNAETLTEGVPSRFSGSGSGTQFSLKINSLQPEDFGNYYCQHHYGTPLTFGAGTKLDLKA DAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC(SEQ IDNO:184).

[0522] Humanized Ab7 antibodies were designed using methods known in the art. Briefly, the V region gene sequences encoding the mouse monoclonal Ab7 antibody were used to construct a series of fully humanized antibodies. The variable region genes were cloned into vectors encoding human IgG1 heavy chain constant domains and human kappa light chain constant domains. Chimeric and humanized antibodies were transiently expressed in mammalian cells. Humanization of the isolated Ab7 heavy chain variable region produced 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 generated four humanized light chain variable region variants (hereinafter referred to as “Ab7-V L1 ”, “Ab7-V L2 ”, “Ab7-V L3 ” and “Ab7-V L4 ”).

[0523] The protein sequences defining the humanized Ab7 variant variable regions and the nucleotide sequences encoding the humanized Ab7 variant variable regions are summarized below (the amino-terminal signal peptide sequence is not shown).

[0524] Heavy chain variable region Ab7-V H1 Having the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4):

[0525] EVKLVESGGGLVQPGGSLRLSCAASGFTFTSYSMSWVRQAPGKGLEWVAYISYDGGSAYYPDTVKGRFTISRDNSKKTLYLQMSSLKSEDTAMYYCARHGDYDDDDAMDYWGQGTSVTVSS (SEQ ID NO: 5).

[0526] Encoding the heavy chain variable region Ab7-V H1 The nucleic acid sequence has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4):

[0527] GAGGTGAAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTACCAGCTATTCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCGCATACATTAGTTATGATGGTGGTAGCGCCTACTACCCT GACACTGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAAAACCCTGTATCTGCAAATGAGCAGCCTGAAGTCTGAGGACACGGCCATGTATTACTGTGCAAGACATGGAGACTATGACGACGACGACGCGATGGACTACTGGGGCCAAGGAACCTCAGTCACCGTCTCCTCA(SEQ IDNO:6).

[0528] Heavy chain variable region Ab7-V H2 Having the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4):

[0529] EVQLVESGGGLVQPGGSLRLSCAASGFTFTSYSMSWVRQAPGKGLEWVAYISYDGGSAYYPDTVKGRFTISRDNSKNTLYLQMSSLKSEDTAMYYCARHGDYDDDDAMDYWGQGTLVTVSS (SEQ ID NO: 7).

[0530] Encoding the heavy chain variable region Ab7-V H2 The nucleic acid sequence has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4):

[0531] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTACCAGCTATTCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCGCATACATTAGTTATGATGGTGGTAGCGCCTACTACCCT GACACTGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACCCTGTATCTGCAAATGAGCAGCCTGAAGTCTGAGGACACGGCCATGTATTACTGTGCAAGACATGGAGACTATGACGACGACGACGCGATGGACTACTGGGGCCAAGGAACCCTGGTCACCGTCTCCTCA(SEQ IDNO:8).

[0532] Heavy chain variable region Ab7-V H3 Having the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4):

[0533] EVKLVESGGGLVQPGGSLRLSCAASGFTFTSYSMSWVRQAPGKGLEWVAYISYDGGSAYYPDTVKGRFTISRDNSKKTLYLQMNSLRAEDTAVYYCARHGDYDDDDAMDYWGQGTLVTVSS (SEQ ID NO: 9).

[0534] Encoding the heavy chain variable region Ab7-V H3 The nucleic acid sequence has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4):

[0535] GAGGTGAAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTACCAGCTATTCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCGCATACATTAGTTATGATGGTGGTAGCGCCTACTATCCT GACACTGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAAAACCCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCAAGACATGGAGACTATGACGACGACGACGCGATGGACTACTGGGGCCAAGGAACCCTGGTCACCGTCTCCTCA(SEQ IDNO:10).

[0536] Heavy chain variable region Ab7-V H4 Having the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4):

[0537] EVQLVESGGGLVQPGGSLRLSCAASGFTFTSYSMSWVRQAPGKGLEWVAYISYDGGSAYYPDTVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHGDYDDDDAMDYWGQGTLVTVSS (SEQ ID NO: 11).

[0538] Encoding the heavy chain variable region Ab7-V H4 The nucleic acid sequence has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4):

[0539] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTACCAGCTATTCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCGCATACATTAGTTATGATGGTGGTAGCGCCTACTATCCT GACACTGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACCCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCAAGACATGGAGACTATGACGACGACGACGCGATGGACTACTGGGGCCAAGGAACCCTGGTCACCGTCTCCTCA(SEQ IDNO:12).

[0540] Heavy chain variable region Ab7-V H5 Having the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4):

[0541] EVQLVESGGGLVQPGGSLRLSCAASGFTFTSYSMSWVRQAPGKGLEWVSYISYDGGSAYYPDTVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHGDYDDDDAMDYWGQGTLVTVSS (SEQ ID NO: 13).

[0542] Encoding the heavy chain variable region Ab7-V H5 The nucleic acid sequence has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4):

[0543] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTACCAGCTATTCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGTCTTACATTAGTTATGATGGTGGTAGCGCCTACTATCCT GACACTGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACCCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCAAGACATGGAGACTATGACGACGACGACGCGATGGACTACTGGGGCCAAGGAACCCTGGTCACCGTCTCCTCA(SEQ IDNO:14).

[0544] Light chain variable region Ab7-V L1 Having the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4):

[0545] DIQMTQSPSSSLSASVGDRVTITCRASENIYSYLAWYQQKQ GKAPKLLVYNAETLTEGVPSRFSGSGSGTDFTLTISSLQPEDFA NYYCQHHYGTPLTFGQGTKLDIK (SEQ ID NO: 15).

[0546] Encoding the light chain variable region Ab7-V L1 The nucleic acid sequence has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4):

[0547] GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTGAGAACATTTACAGCTATTTAGCATGGTATCAGCAGAAACAGGGGAAAGCCCCTAAGCTCCTGGTCTATAATGCAGAAACCTTG ACAGAAGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAAATTACTACTGTCAACATCATTACGGTACCCCGCTCACATTCGGCCAAGGGACCAAGCTGGATATCAAA(SEQ ID NO:16).

[0548] Light chain variable region Ab7-V L2 Having the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4):

[0549] DIQMTQSPSSSLSASVGDRVTITCRASENIYSYLAWYQQKPGKAPKLLVYNAETLTEGVPSRFSGSGSGTDFTLTISSLQPEDFAN YYCQHHYGTPLTFGQGTKLEIK (SEQ ID NO: 17).

[0550] Encoding the light chain variable region Ab7-V L2 The nucleic acid sequence has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4):

[0551] GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTGAGAACATTTACAGCTATTTAGCATGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGGTCTATAATGCAGAAACCTTG ACAGAAGGGGTCCCATCAAGGTTCAGCGGCAGTGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAAATTACTACTGTCAACATCATTACGGTACCCCGCTCACATTCGGCCAAGGGACCAAGCTGGAAATCAAA(SEQ ID NO:18).

[0552] Light chain variable region Ab7-V L3 Having the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4):

[0553] DIQMTQSPSSSLSASVGDRVTITCRASENIYSYLAWYQQKPGKAPKLLVYNAETLTEGVPSRFSGSGSGTDFTLTISSLQPEDFAT YYCQHHYGTPLTFGQGTKLEIK (SEQ ID NO: 19).

[0554] Encoding the light chain variable region Ab7-V L3 The nucleic acid sequence has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4):

[0555] GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTGAGAACATTTACAGCTATTTAGCATGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGGTCTATAATGCAGAAACCTTG ACAGAAGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACATCATTACGGTACCCCGCTCACATTCGGCCAAGGGACCAAGCTGGAAATCAAA(SEQ ID NO:20).

[0556] Light chain variable region Ab7-V L4 Having the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4):

[0557] DIQMTQSPSSSLSASVGDRVTITCRASENIYSYLNWYQQKPG KAPKLLVYNAETLTEGVPSRFSGSGSGTDFTLTISSLQPEDFAT YYCQHHYGTPLTFGQGTKLEIK (SEQ ID NO: 21).

[0558] Encoding the light chain variable region Ab7-V L4 The nucleic acid sequence has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4):

[0559] GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTGAGAACATTTACAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGGTCTATAATGCAGAAACCTTG ACAGAAGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACATCATTACGGTACCCCGCTCACATTCGGCCAAGGGACCAAGCTGGAAATCAAA(SEQ ID NO:22).

[0560] The isolated heavy chain CDR and light chain CDR amino acid sequences of the parent Ab7 antibody are shown in Table 1 (Kabat definition).

[0561] Table 1

[0562]

[0563] To generate complete chimeric and humanized heavy or light chain antibody sequences, each of the above heavy chain variable regions was combined with a human IgG1 constant region, and each of the above light chain variable regions was combined with a human kappa constant region.

[0564] The protein sequences defining the complete heavy and light chains of the chimeric and humanized Ab7 variants are summarized below (the amino-terminal signal peptide sequence is not shown).

[0565] Heavy chain Ab7-V H0 - IgG1 has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-constant region):

[0566] EVKLVESGGGLVQPGGSLKLFCAASGFTFTSYSMSWVRQTPEKRLEWVAYISYDGGSAYYPDTVKGRFSISRDNAKKTLYLQMSSLKSEDTAMYYCARHGDYDDDDAMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:29).

[0567] Nucleic acid sequence encoding heavy chain Ab7-V H0 -IgG1 has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-constant region):

[0568]

[0569] Heavy chain Ab7-V H1 - IgG1 has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-constant region):

[0570] EVKLVESGGGLVQPGGSLRLSCAASGFTFTSYSMSWVRQAPGKGLEWVAYISYDGGSAYYPDTVKGRFTISRDNSKKTLYLQMSSLKSEDTAMYYCARHGDYDDDDAMDYWGQ GTSVTVSSASTKGPSVFPLAPSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:31).

[0571] Encoding heavy chain Ab7-V H1 - The nucleic acid sequence of IgG1 has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-constant region):

[0572]

[0573] Heavy chain Ab7-V H2 - IgG1 has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-constant region):

[0574] EVQLVESGGGLVQPGGSLRLSCAASGFTFTSYSMSWVRQAPGKGLEWVAYISYDGGSAYYPDTVKGRFTISRDNSKNTLYLQMSSLKSEDTAMYYCARHGDYDDDDAMDYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:33).

[0575] Encoding heavy chain Ab7-V H2 - The nucleic acid sequence of IgG1 has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-constant region):

[0576]

[0577] Heavy chain Ab7-V H3 - IgG1 has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-constant region):

[0578] EVKLVESGGGLVQPGGSLRLSCAASGFTFTSYSMSWVRQAPGKGLEWVAYISYDGGSAYYPDTVKGRFTISRDNSKKTLYLQMNSLRAEDTAVYYCARHGDYDDDDAMDYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:35).

[0579] Encoding heavy chain Ab7-V H3 - The nucleic acid sequence of IgG1 has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-constant region):

[0580]

[0581] Heavy chain Ab7-V H4 - IgG1 has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-constant region):

[0582] EVQLVESGGGLVQPGGSLRLSCAASGFTFTSYSMSWVRQAPGKGLEWVAYISYDGGSAYYPDTVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHGDYDDDDAMDYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:37).

[0583] Encoding heavy chain Ab7-V H4 - The nucleic acid sequence of IgG1 has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-constant region):

[0584]

[0585] Heavy chain Ab7-V H5 - IgG1 has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-constant region):

[0586] EVQLVESGGGLVQPGGSLRLSCAASGFTFTSYSMSWVRQAPGKGLEWVSYISYDGGSAYYPDTVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHGDYDDDDAMDYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:39).

[0587] Encoding heavy chain Ab7-V H5 - The nucleic acid sequence of IgG1 has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-constant region):

[0588]

[0589] Light chain Ab7-V L0 -Kappa has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR 3-FR4-constant region):

[0590] DIQMTQSPASSLSASVGETVTITCRASENIYSYLAWYQQKQGKSPQLLVYNAETLTEGVPSRFSGSGSGTQFSLKINSLQPEDFGNYYCQHHYGTPLTFGAGTKLDLKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:41).

[0591] Encoding light chain Ab7-V L0 -The nucleic acid sequence of κ has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-constant region):

[0592] GACATCCAGATGACCCAGTCTCCAGCCTCCCTGTCTGCATCTGTAGGAGAAACTGTCACCATCACTTGCCGGGCAAGTGAGAACATTTACAGCTATTTAGCATGGTATCAGCAGAAACAGGGGAAATCTCCTCAGCTCCTGGTCTATAATGCAGAAACCTTGACAGAAGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACACAATTCTCTCTCAAGATCAACAGTCTGCAACCTGAAGATTTTGGGAATTACTACTGTCAACATCATTACGGTACCCCGCTCACATTCGGCGCTGGGACCAAGCTGGATCTGAAACGAACGGTGGCCGCGCCGAGCGTCTTCATCTTCCCGCCTTCCGACGAGCAGCTCAAGTCCGGGACCGCCTCCGTAGTATGCCTCCTCAATAACTTCTACCCCCGGGAGGCGAAGGTCCAGTGGAAGGTCGACAACGCCCTCCAATCGGGCAACTCCCAGGAGTCGGTGACCGAGCAGGATTCCAAGGACTCGACCTACAGTCTAAGCTCCACCCTCACACTGTCGAAGGCGGACTACGAGAAGCACAAGGTGTACGCCTGCGAGGTCACCCACCAGGGCCTGAGCAGCCCGGTCACCAAGTCCTTCAACCGGGGCGAGTGCTGA(SEQ ID NO:42)。

[0593] Light chain Ab7-V L1 -κ has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR 3-FR4-constant region):

[0594] DIQMTQSPSSSLSASVGDRVTITCRASENIYSYLAWYQQKQGKAPKLLVYNAETLTEGVPSRFSGSGSGTDFTLTISSLQPEDFANYYCQHHYGTPLTFGQGTKLDIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:43).

[0595] Encoding light chain Ab7-V L1 -The nucleic acid sequence of κ has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-constant region):

[0596] GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTGAGAACATTTACAGCTATTTAGCATGGTATCAGCAGAAACAGGGGAAAGCCCCTAAGCTCCTGGTCTATAATGCAGAAACCTTGACAGAAGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAAATTACTACTGTCAACATCATTACGGTACCCCGCTCACATTCGGCCAAGGGACCAAGCTGGATATCAAACGAACGGTGGCCGCGCCGAGCGTCTTCATCTTCCCGCCTTCCGACGAGCAGCTCAAGTCCGGGACCGCCTCCGTAGTATGCCTCCTCAATAACTTCTACCCCCGGGAGGCGAAGGTCCAGTGGAAGGTCGACAACGCCCTCCAATCGGGCAACTCCCAGGAGTCGGTGACCGAGCAGGATTCCAAGGACTCGACCTACAGTCTAAGCTCCACCCTCACACTGTCGAAGGCGGACTACGAGAAGCACAAGGTGTACGCCTGCGAGGTCACCCACCAGGGCCTGAGCAGCCCGGTCACCAAGTCCTTCAACCGGGGCGAGTGCTGA(SEQ ID NO:44).

[0597] Light chain Ab7-V L2 -κ has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR 3-FR4-constant region):

[0598] DIQMTQSPSSSLSASVGDRVTITCRASENIYSYLAWYQQKPGKAPKLLVYNAETLTEGVPSRFSGSGSGTDFTLTISSLQPEDFANYYCQHHYGTPLTFGQGTKLEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:45).

[0599] Encoding light chain Ab7-V L2 -The nucleic acid sequence of κ has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-constant region):

[0600] GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTGAGAACATTTACAGCTATTTAGCATGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGGTCTATAATGCAGAAACCTTGACAGAAGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAAATTACTACTGTCAACATCATTACGGTACCCCGCTCACATTCGGCCAAGGGACCAAGCTGGAAATCAAACGAACGGTGGCCGCGCCGAGCGTCTTCATCTTCCCGCCTTCCGACGAGCAGCTCAAGTCCGGGACCGCCTCCGTAGTATGCCTCCTCAATAACTTCTACCCCCGGGAGGCGAAGGTCCAGTGGAAGGTCGACAACGCCCTCCAATCGGGCAACTCCCAGGAGTCGGTGACCGAGCAGGATTCCAAGGACTCGACCTACAGTCTAAGCTCCACCCTCACACTGTCGAAGGCGGACTACGAGAAGCACAAGGTGTACGCCTGCGAGGTCACCCACCAGGGCCTGAGCAGCCCGGTCACCAAGTCCTTCAACCGGGGCGAGTGCTGA(SEQ ID NO:46).

[0601] Light chain Ab7-V L3 -κ has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR 3-FR4-constant region):

[0602] DIQMTQSPSSSLSASVGDRVTITCRASENIYSYLAWYQQKPGKAPKLLVYNAETLTEGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYGTPLTFGQGTKLEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:47).

[0603] Encoding light chain Ab7-V L3 -The nucleic acid sequence of κ has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-constant region):

[0604] GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTGAGAACATTTACAGCTATTTAGCATGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGGTCTATAATGCAGAAACCTTGACAGAAGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACATCATTACGGTACCCCGCTCACATTCGGCCAAGGGACCAAGCTGGAAATCAAACGAACGGTGGCCGCGCCGAGCGTCTTCATCTTCCCGCCTTCCGACGAGCAGCTCAAGTCCGGGACCGCCTCCGTAGTATGCCTCCTCAATAACTTCTACCCCCGGGAGGCGAAGGTCCAGTGGAAGGTCGACAACGCCCTCCAATCGGGCAACTCCCAGGAGTCGGTGACCGAGCAGGATTCCAAGGACTCGACCTACAGTCTAAGCTCCACCCTCACACTGTCGAAGGCGGACTACGAGAAGCACAAGGTGTACGCCTGCGAGGTCACCCACCAGGGCCTGAGCAGCCCGGTCACCAAGTCCTTCAACCGGGGCGAGTGCTGA(SEQ ID NO:48).

[0605] Light chain Ab7-V L4 -κ has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR 3-FR4-constant region):

[0606] DIQMTQSPSSSLSASVGDRVTITCRASENIYSYLNWYQQKPGKAPKLLVYNAETLTEGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYGTPLTFGQGTKLEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:49).

[0607] Encoding light chain Ab7-V L4 -The nucleic acid sequence of κ has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-constant region):

[0608] (SEQ ID NO:50).

[0609] It is also contemplated that the variable region sequences may be fused to other antibody constant region sequences to produce full-length immunoglobulin heavy and light chains. H0 、Ab7-V H1 、Ab7-V H 2. Ab7-V H 3. Ab7-V H 4 or Ab7-V H The heavy chain variable region of Ab7-V can be combined with human IgG2, IgG3, IgG4, or IgG4 containing one or more amino acid substitutions in the constant region (e.g., IgG4mt or IgG4mt2). L 0. Ab7-V L 1. Ab7-V L 2. Ab7-V L3 or Ab7-V L 4 light chain variable regions combined with human lambda constant regions.

[0610] The DNA fragments encoding the heavy and light chain variable regions of the above-mentioned Ab7 and humanized Ab7 variants were synthesized using flanking restriction enzyme sites to be cloned into the pANT expression vector (Antitope) system for IgG1 heavy chain and κ light chain. All constructs were confirmed 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.

[0611] Table 2

[0612]

[0613]

[0614] Antibodies were purified from cell culture supernatants on protein A-conjugated sepharose columns (GE Healthcare, Little Chalfont, UK), buffer exchanged into 1× DPBS pH 7.2, and quantified using the extinction coefficient (Ec (0.1％) )Through OD 280nm Quantification was performed. 1 μg of each antibody was analyzed by SDS-PAGE, and bands corresponding to the typical antibody profile were observed (data not shown).

[0615] In vitro characterization of anti-EBI3 antibodies

[0616] The Ab7 antibody and Ab7 antibody variants generated as described in Table 2 were tested in a series of in vitro assays to determine their biological properties and activities.

[0617] To assess the binding of humanized Ab7 antibody variants relative to chimeric Ab7.1 antibodies, a binding competition ELISA was established. The assay plate was coated with 1 μg / mL of 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 on the plate to generate an eight-point binding curve. The antibody diluent was premixed with biotinylated Ab7.1 antibody to a final constant concentration of 0.08 μg / mL. The antibody mixture was then transferred to the coated assay plate and incubated at room temperature for 1 hour. The binding of the 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.

[0618] Determine the absolute IC of the tested antibodies using a four-parameter logistic curve 50 Values ​​relative to the IC of the chimeric antibody on each plate 50 Normalized IC 50 The results are summarized in Table 3. L4 With the exception of variants of the light chain, all generated humanized Ab7 variants had similar binding profiles to the chimeric Ab7.1 antibody, with most variants competing within two-fold of the chimeric Ab7.1 antibody.

[0619] Table 3

[0620]

[0621]

[0622] The antibodies were further characterized by surface plasmon resonance (SPR). Kinetic experiments were performed on a Biacore T200 (GE Healthcare, Uppsala, Sweden). All experiments were performed using hIL-27 (R&D Systems, Abingdon, UK) in HBS-P+ running buffer (pH 7.4) (GE Healthcare, Little Chalfont, UK) at 25°C. Human IL-27 (hIL-27) antigen was captured to approximately 16RU on a CM5 chip. Immobilization was performed at a protein concentration of 1 μg / mL in 10 mM acetate buffer (pH 5.0). A three-point three-fold dilution range of 3.3 nM to 30 nM of antibody diluted in HBS-P+ buffer was used, with no regeneration between each concentration. The association phase of three injections of increasing concentrations of antibody was monitored for 75 seconds each time, and a single dissociation phase of 250 seconds was measured after the last antibody injection. A single injection of 2 M MgCl2 was used for 120 seconds to regenerate the hIL-27 surface. Multiple replicates (n=4) of the chimeric antibody were performed throughout the assay to examine the stability of the surface and analyte during the kinetic cycles.

[0623] 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 from the 1:1 binding model analysis are summarized in Table 4, and the results from the bivalent analyte model are summarized in Table 5.

[0624] Table 4

[0625]

[0626] Table 5

[0627]

[0628]

[0629] Single cycle kinetics using a 1:1 model (Table 4) showed that Ab7-V L4 The humanized variants did not bind to hIL-27, and the remaining variants bound within two-fold of the chimeric antibody, consistent with the results from the bivalent analyte model (Table 5) and the competition ELISA.

[0630] In summary, the binding affinity of the Ab7.1 chimeric antibody was 1 nM as determined by single cycle kinetics and using a 1:1 binding model. All humanized variants had a KD of 1.5 nM or lower, but the Ab7-V L4 These results demonstrate that Ab7 antibody and Ab7 antibody variants bind to the EBI3 subunit of IL-27 with high affinity.

[0631] Example 2: Production of anti-IL-27 antibodies that specifically bind to the EBI3 and / or P28 subunits of human IL-27 in yeast

[0632] Using the methods described below, additional anti-IL-27 monoclonal antibodies representing multiple epitope bins were selected from eight original human synthetic yeast libraries.

[0633] Materials and methods

[0634] Produce approximately 10 of each as described previously 9 Eight initial human synthetic yeast libraries of diverse diversity (see, e.g., Xu et al., (2013) Protein Eng Des Sel 26(10):663-670; WO2009036379; WO2010105256; and WO2012009568, all of which are incorporated herein by reference in their entirety). For the first two rounds of selection, magnetic bead sorting using the Miltenyi MACS system was performed as described previously (see, e.g., Siegel et al. (2004) J Immunol Methods 286(1-2):141-153, which are incorporated herein by reference in their entirety).

[0635] Briefly, yeast cells (approximately 10 10 cells / library) were incubated with 3 ml of 100 nM biotinylated antigen (recombinant human IL-27; R&D Systems) in wash buffer (phosphate buffered saline (PBS) / 0.1% bovine serum albumin (BSA)) for 30 minutes. After washing once with 40 ml of ice-cold wash buffer, the cell pellet was resuspended in 20 mL of wash buffer, and streptavidin microbeads (500 μl) were added to the yeast and incubated at 4°C for 15 minutes. Next, the yeast cells were pelleted, resuspended in 20 mL of wash buffer, and loaded onto a Miltenyi LS column. After loading 20 mL, the column was washed 3 times with 3 ml of wash buffer. The column was then removed from the magnetic field, and the yeast cells were eluted with 5 mL of growth medium and then grown overnight. The following rounds of selection were performed using flow cytometry. Approximately 2×10 7 Yeast cells were pelleted, washed three times with wash buffer, and incubated at 30°C under equilibrium conditions with decreasing concentrations of biotinylated antigen (100 to 1 nM), 30 nM biotinylated antigen from different species to obtain species cross-reactivity, or with polyspecific depletion reagent (PSR) to remove nonspecific antibodies from the selection. For PSR depletion, the library was incubated with a 1:10 dilution of the biotinylated PSR reagent.

[0636] The yeast cells were then washed twice with wash buffer and stained with LC-FITC (1:100 dilution) and SA-633 (1:500 dilution) or EAPE (1:50 dilution) secondary reagents at 4°C for 15 minutes. After washing twice with wash buffer, the cell pellet was resuspended in 0.3 mL of wash buffer and transferred to a sorting tube with a filter. Sorting was performed using a FACS ARIA sorter (BD Biosciences), and the sorting gate was determined to select antibodies with the desired characteristics. The selection rounds were repeated until a population with all the desired characteristics was obtained. After the final round of sorting, the yeast cells were plated and single colonies were picked for ...

Claims

1. Use of a detection antibody in the preparation of a kit for use in a method for detecting p28 in a sample from a subject, wherein the method comprises the following steps: (a) contacting a sample from the subject with a detection antibody under conditions that allow the detection antibody to form a detection antibody-p28 complex in the presence of IL-27 or p28 in the sample; and (b) detecting the presence of the complex produced in step (a); wherein the detection antibody comprises a monoclonal antibody or an antigen-binding portion thereof that specifically binds to human IL-27, wherein the antibody or antigen-binding portion thereof comprises a heavy chain CDR and a light chain CDR selected from the group consisting of: (i) the heavy chain CDR1, CDR2 and CDR3 sequences as shown in SEQ ID NOs: 706, 707 and 708, respectively, and the light chain CDR1, CDR2 and CDR3 sequences as shown in SEQ ID NOs: 714, 715 and 716, respectively, which are numbered according to IMGT.

2. Use of a detection antibody in the preparation of a kit for use in a method for detecting an IL-27-related cancer in a subject, wherein the method comprises the following steps: (a) contacting a sample from the subject with a detection antibody under conditions that allow the detection antibody to form a detection antibody-p28 complex in the presence of IL-27 or p28 in the sample; and (b) detecting the presence of the complex produced in step (a); wherein the detection antibody comprises a monoclonal antibody or an antigen-binding portion thereof that specifically binds to human IL-27, wherein the antibody or antigen-binding portion thereof comprises a heavy chain CDR and a light chain CDR selected from the group consisting of: (i) the heavy chain CDR1, CDR2 and CDR3 sequences as shown in SEQ ID NOs: 706, 707 and 708, respectively, and the light chain CDR1, CDR2 and CDR3 sequences as shown in SEQ ID NOs: 714, 715 and 716, respectively, which are numbered according to IMGT.

3. The use according to claim 1 or 2, wherein the method further comprises contacting the sample with a capture antibody to produce a complex comprising IL-27 or EBI3 and the capture antibody in the presence of IL-27 or EBI3 in the sample.

4. The use according to claim 3, wherein the capture antibody comprises: (i) the heavy chain CDR1 sequence as shown in SEQ ID NO: 23; (ii) the heavy chain CDR2 sequence as shown in SEQ ID NO: 24; (iii) the heavy chain CDR3 sequence as shown in SEQ ID NO: 25; (iv) the light chain CDR1 sequence as shown in SEQ ID NO: 26; (v) the light chain CDR2 sequence as shown in SEQ ID NO: 27; and (vi) the light chain CDR3 sequence as shown in SEQ ID NO:

28.

5. The use according to claim 4, wherein (a) the capture antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 5, 7, 9, 11 and 13, and the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 17, 19 and 21; (b) the detection antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises the amino acid sequence as shown in SEQ ID NO: 712, and the light chain variable region comprises the amino acid sequence as shown in SEQ ID NO: 720; or (c) Both (a) and (b).

6. The use according to claim 2, wherein the IL-27-related cancer is selected from renal cell carcinoma (RCC), hepatocellular carcinoma (HCC), lung cancer, gastroesophageal cancer, ovarian cancer, endometrial cancer, melanoma, leukemia and lymphoma.

7. A monoclonal antibody or antigen binding portion thereof that specifically binds to human IL-27, wherein the antibody or antigen binding portion thereof comprises a heavy chain CDR and a light chain CDR selected from the group consisting of: (i) the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 706, 707 and 708, respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 714, 715 and 716, respectively, which are numbered according to IMGT.

8. The use according to any one of claims 1, 2, 4 or 5 or the monoclonal antibody or antigen binding portion thereof according to claim 7, wherein the antibody or antigen binding portion thereof that specifically binds to human IL-27 comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region and the light chain variable region have the amino acid sequences shown in SEQ ID NO: 712 and 720, respectively.

9. The use according to any one of claims 1, 2, 4 or 5 or the monoclonal antibody or antigen binding portion thereof according to claim 7, wherein the antibody or antigen binding portion thereof that specifically binds to human IL-27 comprises a heavy chain and a light chain, and the heavy chain and the light chain have the amino acid sequences shown in SEQ ID NO: 722 and 724, respectively.

10. The use according to any one of claims 1, 2, 4 or 5 or the monoclonal antibody or antigen binding portion thereof according to claim 7, wherein the antibody or antigen binding portion thereof that specifically binds to human IL-27 comprises a heavy chain and a light chain, and the heavy chain and the light chain have the amino acid sequences shown in SEQ ID NO: 726 and 724, respectively.

11. The use according to any one of claims 1, 2, 4 or 5 or the monoclonal antibody or antigen binding portion thereof according to claim 7, wherein the antibody or antigen binding portion thereof that specifically binds to human IL-27 antagonizes IL-27.

12. The use according to any one of claims 1 or 2 or the monoclonal antibody or antigen binding portion thereof according to claim 7, wherein the antibody that specifically binds to human IL-27 is selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD and IgE antibodies.

13. The use according to any one of claims 1 or 2 or the monoclonal antibody or antigen binding portion thereof according to claim 7, wherein the antibody that specifically binds to human IL-27 is an IgG1 antibody or an IgG4 antibody.

14. The use according to any one of claims 1 or 2 or the monoclonal antibody or antigen binding portion thereof according to claim 7, wherein the antibody that specifically binds to human IL-27 comprises a wild-type IgG1 heavy chain constant region or a wild-type IgG4 heavy chain constant region.

15. The use according to any one of claims 1 or 2 or the monoclonal antibody or antigen binding portion thereof according to claim 7, wherein the antibody that specifically binds to human IL-27 comprises an Fc domain comprising at least one mutation.

16. The use or monoclonal antibody or antigen binding portion thereof according to claim 15, comprising a mutant IgG1 heavy chain constant region or a mutant IgG4 heavy chain constant region.

17. The use or monoclonal antibody or antigen binding portion thereof according to claim 16, wherein the mutant IgG4 heavy chain constant region comprises a substitution selected from S228P, L235E, L235A and a combination thereof, according to EU numbering.

18. A kit comprising the monoclonal antibody or antigen-binding portion thereof according to any one of claims 7 to 17 and instructions for use for detecting IL-27 in a sample from a subject.

19. A nucleic acid comprising a nucleotide sequence encoding both the light chain and the heavy chain of the monoclonal antibody or antigen binding portion thereof of any one of claims 7-17.

20. An expression vector comprising the nucleic acid of claim 19.

21. A host cell transformed with the expression vector according to claim 20.

22. A method for producing a monoclonal antibody or an antigen binding portion thereof that specifically binds to human IL-27, the method comprising maintaining the cell according to claim 21 under conditions that allow expression of the monoclonal antibody or the antigen binding portion thereof.

23. The method of claim 22, further comprising obtaining the monoclonal antibody or antigen-binding portion thereof.

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