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 ameliorating one or more symptoms of a disease such as cancer by administering an antibody or an antigen-binding portion 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 / 646,496, filed March 22, 2018, the entire contents of which are incorporated herein by reference. Invention Field

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

[0005] In recent years, increasing evidence has shown that the immune system is a crucial barrier to tumor formation and development. The existence of naturally occurring T cells with anti-tumor potential or activity in cancer patients rationalizes the development of immunotherapies in oncology. Immune cells, such as T cells, macrophages, and natural killer cells, can exhibit anti-tumor activity and effectively control the occurrence and growth of malignant tumors. Tumor-specific or related antigens can induce immune cells to recognize and eliminate malignant tumors (Chen & Mellman, (2013) Immunity 39(1): 1-10). Despite the presence of tumor-specific immune responses, malignant tumors often evade or avoid immune attacks through various immunomodulatory mechanisms, leading to uncontrolled tumor occurrence and progression (Motz & Coukos, (2013) Immunity 39(1): 61-730). In fact, a hallmark of emerging cancers is the exploitation of these immunomodulatory mechanisms and the failure of anti-tumor immune responses, leading to tumor evasion and escape from immune killing (Hanahan and Weinberg (2011) Cell 144(5): 646-674).

[0006] IL-27 is a heterodimeric cytokine composed of two subunits (EBI3 and IL-27p28). Structurally, IL-27 is associated with the IL-12 and IL-6 cytokine families. IL-27 binds to a heterodimeric receptor composed of the IL-27Rα (WSX1) and gp130 chains and mediates signal transduction via this receptor, which primarily mediates signal transduction through STAT1 and STAT3. Initial reports described IL-27 as an immune-enhancing cytokine that supports CD4+ T cell proliferation, helper T(Th)1 cell differentiation, and IFN-γ production, typically acting synergistically with IL-12. Subsequent studies have shown that IL-27 exhibits complex immunomodulatory functions, producing pro-inflammatory or anti-inflammatory effects depending on the biological environment and the experimental models used. IL-27 can drive the expression of various immunomodulatory molecules in human cancer cells, which can support local dysregulation of the immune response in vivo (Fabbi et al., (2017) Mediators Inflamm 3958069. Published online on February 1, 2017 doi: 10.1155 / 2017 / 3958069, and the references contained therein).

[0007] Despite significant advances in cancer treatment and management, there remains a persistent need for new and effective therapies for the treatment and management of cancer. Summary of the Invention

[0008] This document discloses antibodies or their antigen-binding portions that specifically bind to and antagonize human IL-27 (interleukin 27) with high affinity and specificity. Nucleic acid molecules encoding antibody molecules, expression vectors, host cells, and methods for preparing antibody molecules are also provided. Pharmaceutical compositions comprising antibody molecules are also provided. The anti-IL-27 antibodies or their antigen-binding portions disclosed herein can be used (alone or in combination with other therapeutic agents or procedures) to treat, prevent, and / or diagnose conditions, including immune disorders and cancer. Therefore, this document discloses compositions and methods for treating and / or diagnosing various diseases, including cancer and immune disorders, using anti-IL-27 antibody molecules.

[0009] In some embodiments, this disclosure provides isolated monoclonal antibodies or antigen-binding moieties thereof that specifically bind to and antagonize human IL-27, wherein said antibody or antigen-binding moieties exhibit at least one or more of the following properties: (i) having an equilibrium dissociation constant (K0) of 15 nM or less. D(i) binding to human IL-27; (ii) blocking the binding of IL-27 to the IL-27 receptor; (iii) inhibiting or reducing STAT1 and / or STAT3 phosphorylation in cells; (iv) inhibiting or reducing the inhibition of CD161 expression in cells; (v) inhibiting or reducing PD-L1 and / or TIM-3 expression in cells; (vi) inducing or increasing the secretion of one or more PD-1-mediated cytokines from cells; and (vii) combinations of (i) to (vi).

[0010] In some implementations, the isolated monoclonal antibody or its antigen-binding moiety has an equilibrium dissociation constant (K0) of 15 nM or less. D It binds to human IL-27.

[0011] In some implementations, the isolated monoclonal antibody or its antigen-binding moiety binds to recombinant human IL-27 or mouse IL-27.

[0012] On the one hand, this disclosure provides isolated monoclonal antibodies or antigen-binding moieties thereof that specifically bind to and antagonize human IL-27, wherein the antibody or antigen-binding moieties thereof have heavy chain and light chain CDRs, wherein the heavy chain and light chain CDRs are: (i) heavy chain CDR1 is N-GFTFXXXX-C (SEQ ID NO: 408), heavy chain CDR2 is N-ISSSXXYI-C (SEQ ID NO: 409), and heavy chain CDR3 is SEQ ID NO: 163; the light chain CDR1, CDR2, and CDR3 sequences are SEQ ID NO: 169, 170, and 171, respectively; or (ii) heavy chain CDR1 is N-FTFXXXXMN-C (SEQ ID NO: 410), heavy chain CDR2 is N-XISSSXXYIXYADSVKG-C (SEQ ID NO: 411), and heavy chain CDR3 is SEQ ID NO: 166; the light chain CDR1, CDR2, and CDR3 sequences are SEQ ID NO: 169, 170, and 171, respectively. NO: 172, 173 and 174.

[0013] In one implementation, heavy chain CDR1 is N-GFTF[S / A / R][S / R][T / Y][G / S]-C (SEQ ID NO: 412) and heavy chain CDR2 is N-ISSS[S / G][S / A]YI-C (SEQ ID NO: 413); or heavy chain CDR1 is N-FTF[S / A / R][S / R][T / Y][G / S]MN-C (SEQ ID NO: 414) and heavy chain CDR2 is N-[G / S]ISSS[S / G][S / A]YI[L / Y]YADSVKG-C (SEQ ID NO: 415).

[0014] In another embodiment, the corresponding heavy chain and light chain CDRs are: (i) the heavy chain CDR1, CDR2, and CDR3 sequences are SEQ ID NO: 161, 162, and 163, respectively, and the light chain CDR1, CDR2, and CDR3 sequences are SEQ ID NO: 169, 170, and 171, respectively; (ii) the heavy chain CDR1, CDR2, and CDR3 sequences are SEQ ID NO: 164, 165, and 166, respectively, and the light chain CDR1, CDR2, and CDR3 sequences are SEQ ID NO: 172, 173, and 174, respectively; (iii) the heavy chain CDR1, CDR2, and CDR3 sequences are SEQ ID NO: 73, 74, and 75, respectively, and the light chain CDR1, CDR2, and CDR3 sequences are SEQ ID NO: 81, 82, and 83, respectively; (iv) the heavy chain CDR1, CDR2, and CDR3 sequences are SEQ ID NO: 169, 170, and 171, respectively. NO: 76, 77, and 78, and the light chain CDR1, CDR2, and CDR3 sequences are SEQ ID NO: 84, 85, and 86, respectively; (v) the heavy chain CDR1, CDR2, and CDR3 sequences are SEQ ID NO: 95, 96, and 97, and the light chain CDR1, CDR2, and CDR3 sequences are SEQ ID NO: 103, 104, and 105, respectively; (vi) the heavy chain CDR1, CDR2, and CDR3 sequences are SEQ ID NO: 98, 99, and 100, and the light chain CDR1, CDR2, and CDR3 sequences are SEQ ID NO: 106, 107, and 108, respectively; (vii) the heavy chain CDR1, CDR2, and CDR3 sequences are SEQ ID NO: 117, 118, and 119, and the light chain CDR1, CDR2, and CDR3 sequences are SEQ ID NO: 84, 85, and 86, respectively. NO: 125, 126 and 127; (viii) the heavy chain CDR1, CDR2 and CDR3 sequences are SEQ ID NO: 120, 121 and 122 respectively, and the light chain CDR1, CDR2 and CDR3 sequences are SEQ ID NO: 128, 129 and 130 respectively; (ix) the heavy chain CDR1, CDR2 and CDR3 sequences are SEQ ID NO: 139, 140 and 141 respectively, and the light chain CDR1, CDR2 and CDR3 sequences are SEQ ID NO: 147, 148 and 149 respectively; (x) the heavy chain CDR1, CDR2 and CDR3 sequences are SEQ ID NO: 142, 143 and 144 respectively, and the light chain CDR1, CDR2 and CDR3 sequences are SEQ ID NO: 150, 151 and 152 respectively;(xi) The heavy chain CDR1, CDR2, and CDR3 sequences are SEQ ID NO: 51, 52, and 53, respectively, and the light chain CDR1, CDR2, and CDR3 sequences are SEQ ID NO: 59, 60, and 61, respectively; or (xii) The heavy chain CDR1, CDR2, and CDR3 sequences are SEQ ID NO: 54, 55, and 56, respectively, and the light chain CDR1, CDR2, and CDR3 sequences are SEQ ID NO: 62, 63, and 64, respectively.

[0015] Another aspect of this disclosure provides isolated monoclonal antibodies or antigen-binding moieties thereof that specifically bind to and antagonize human IL-27, said antibody or antigen-binding moieties comprising heavy chain and light chain CDRs, said heavy chain and light chain CDRs being: (i) heavy chain CDR1 being N-GGSFSXYX-C (SEQ ID NO: 416), heavy chain CDR2 being N-IDXSGXT-C (SEQ ID NO: 417), and heavy chain CDR3 being N-ARXXXYY[X]. n＝0-1 DSS[X] n＝4-6 DX-C (SEQ ID NO: 418); and light chain CDR1 is N-QXXSXY-C (SEQ ID NO: 419), light chain CDR2 is N-DXS-C (SEQ ID NO: 420) and light chain CDR3 is N-QQXXDXPIT-C (SEQ ID NO: 421); or (ii) heavy chain CDR1 is N-GSFSXYXWS-C (SEQ ID NO: 422), heavy chain CDR2 is N-SIDXSGXTXYNPSLKS-C (SEQ ID NO: 423) and heavy chain CDR3 is N-ARXXXYY[X] n＝0-1 DSS[X] n＝4-6 DX-C (SEQ ID NO: 418); and light chain CDR1 is N-XASQXXSXYLX-C (SEQ ID NO: 424), light chain CDR2 is N-DXSNXXT-C (SEQ ID NO: 425) and light chain CDR3 is N-QQXXDXPIT-C (SEQ ID NO: 421).

[0016] In some embodiments, (i) respectively, heavy chain CDR1 is N-GGSFS[R / D]Y[E / Y]-C (SEQ ID NO: 426), heavy chain CDR2 is N-ID[W / Y]SG[I / S]TC (SEQ ID NO: 427), heavy chain CDR3 is N-AR[D / L][P / G][M / V]YY[- / Y]DSS[VSTGSV / DLGF]D[V / I]-C (SEQ ID NO: 428); and light chain CDR1 is NQ[S / D][V / I]S[S / N]YC (SEQ ID NO: 429), light chain CDR2 is ND[S / A]SC (SEQ ID NO: 430) and light chain CDR3 is N-QQ[D / Y][S / D]D[H / L]PIT-C (SEQ ID NO: 428). NO: 431); or (ii) respectively, heavy chain CDR1 is N-GSFS[R / D]Y[E / Y]WS-C (SEQ ID NO: 432), heavy chain CDR2 is N-SID[W / Y]SG[I / S]T[N / E]YNPSLKS-C (SEQ ID NO: 433), heavy chain CDR3 is N-AR[D / L][P / G][M / V]YY[- / Y]DSS[VSTGSV / DLGF]D[V / I]-C (SEQ ID NO: 428); and light chain CDR1 is N-[Q / R]ASQ[S / D][V / I]S[S / N]YL[N / A]-C (SEQ ID NO: 434), light chain CDR2 is ND[S / A]SN[R / L][A / E]TC (SEQ ID NO: 432). NO: 435) and the light chain CDR3 is N-QQ[D / Y][S / D]D[H / L]PIT-C (SEQ ID NO: 431).

[0017] In some implementations, (i) the heavy chain CDR1, CDR2, and CDR3 sequences are SEQ ID NO: 229, 230, and 231, respectively, and the light chain CDR1, CDR2, and CDR3 sequences are SEQ ID NO: 237, 238, and 239, respectively; or (ii) the heavy chain CDR1, CDR2, and CDR3 sequences are SEQ ID NO: 232, 233, and 234, respectively, and the light chain CDR1, CDR2, and CDR3 sequences are SEQ ID NO: 240, 241, and 242, respectively.

[0018] In some embodiments, (i) the heavy chain CDR1, CDR2 and CDR3 sequences are SEQ ID NO: 251, 252 and 253, respectively, and the light chain CDR1, CDR2 and CDR3 sequences are SEQ ID NO: 259, 260 and 261, respectively; or (ii) the heavy chain CDR1, CDR2 and CDR3 sequences are SEQ ID NO: 254, 255 and 256, respectively, and the light chain CDR1, CDR2 and CDR3 sequences are SEQ ID NO: 262, 263 and 264, respectively.

[0019] Another aspect of this disclosure provides isolated monoclonal antibodies or antigen-binding moieties thereof that specifically bind to and antagonize human IL-27, wherein the antibody or antigen-binding moieties thereof comprise heavy chain and light chain CDRs, the heavy chain and light chain CDRs being heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 23, 24 and 25, respectively, and CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 26, 27 and 28, respectively.

[0020] Another aspect of this disclosure provides isolated monoclonal antibodies or antigen-binding moieties thereof that specifically bind to and antagonize human IL-27, wherein the antibody or antigen-binding moieties comprise heavy and light chain CDRs, the heavy and light chain CDRs being: (i) heavy chain CDR1, CDR2, and CDR3 sequences SEQ ID NO: 339, 340, and 341, respectively, and light chain CDR1, CDR2, and CDR3 sequences SEQ ID NO: 347, 348, and 349, respectively; or (ii) heavy chain CDR1, CDR2, and CDR3 sequences SEQ ID NO: 342, 343, and 344, respectively, and light chain CDR1, CDR2, and CDR3 sequences SEQ ID NO: 350, 351, and 352, respectively.

[0021] Another aspect of this disclosure provides isolated monoclonal antibodies or antigen-binding moieties thereof that specifically bind to and antagonize human IL-27, wherein the antibody or antigen-binding moieties comprise heavy and light chain CDRs, the heavy and light chain CDRs being: (i) heavy chain CDR1, CDR2, and CDR3 sequences SEQ ID NO: 185, 186, and 187, respectively, and light chain CDR1, CDR2, and CDR3 sequences SEQ ID NO: 193, 194, and 195, respectively; or (ii) heavy chain CDR1, CDR2, and CDR3 sequences SEQ ID NO: 188, 189, and 190, respectively, and light chain CDR1, CDR2, and CDR3 sequences SEQ ID NO: 196, 197, and 198, respectively.

[0022] One aspect of this disclosure provides an isolated monoclonal antibody or antigen-binding moiety thereof that specifically binds to and antagonizes human IL-27, wherein the antibody or antigen-binding moiety comprises a heavy chain and a light chain CDR, the heavy chain and the light chain CDR being: (i) heavy chain CDR1, CDR2 and CDR3 sequences SEQ ID NO: 207, 208 and 209 respectively, and light chain CDR1, CDR2 and CDR3 sequences SEQ ID NO: 215, 216 and 217 respectively; or (ii) heavy chain CDR1, CDR2 and CDR3 sequences SEQ ID NO: 210, 211 and 212 respectively, and light chain CDR1, CDR2 and CDR3 sequences SEQ ID NO: 218, 219 and 220 respectively.

[0023] Another aspect of this disclosure provides isolated monoclonal antibodies or antigen-binding moieties thereof that specifically bind to and antagonize human IL-27, wherein the antibody or antigen-binding moieties comprise heavy and light chain CDRs, the heavy and light chain CDRs being: (i) heavy chain CDR1, CDR2, and CDR3 sequences SEQ ID NO: 273, 274, and 275, respectively, and light chain CDR1, CDR2, and CDR3 sequences SEQ ID NO: 281, 282, and 283, respectively; or (ii) heavy chain CDR1, CDR2, and CDR3 sequences SEQ ID NO: 276, 277, and 278, respectively, and light chain CDR1, CDR2, and CDR3 sequences SEQ ID NO: 284, 285, and 286, respectively.

[0024] One aspect of this disclosure provides an isolated monoclonal antibody or antigen-binding moiety thereof that specifically binds to and antagonizes human IL-27, wherein said antibody or antigen-binding moiety comprises heavy chain and light chain CDRs, said heavy chain and light chain CDRs being: (i) respectively, heavy chain CDR1 being N-GFTFSSYG-C (SEQ ID NO: 361), heavy chain CDR2 being N-IXXDGSXK-C (SEQ ID NO: 436) and heavy chain CDR3 being N-ARXAP[X]. n＝3-8 DV-C (SEQ ID NO: 437); and light chain CDR1 is N-QSXSSY-C (SEQ ID NO: 438), light chain CDR2 is N-XXS-C (SEQ ID NO: 439) and light chain CDR3 is N-QQXXXXP[X] n＝0-1TC (SEQ ID NO: 440); or (ii) respectively, heavy chain CDR1 is N-FTFSSYGMX-C (SEQ ID NO: 441), heavy chain CDR2 is N-XIXXDGSXKYYXDSVKG-C (SEQ ID NO: 442) and heavy chain CDR3 is N-ARXAP[X] n＝3-8 DV-C (SEQ ID NO: 437); and light chain CDR1 is N-RASQSXSSYLX-C (SEQ ID NO: 443), and light chain CDR2 is N-[X]. n＝1-2 SS[X] n＝3-4 -C (SEQ ID NO: 444) and the light chain CDR3 are N-QQXXXXP[X] n＝0-1 TC (SEQ ID NO: 440).

[0025] In some embodiments, (i) respectively, heavy chain CDR1 is N-GFTFSSYG-C (SEQ ID NO: 361), heavy chain CDR2 is NI[K / W][Q / Y]DGS[E / N]KC (SEQ ID NO: 445) and heavy chain CDR3 is N-AR[D / G]AP[WDIYDYYM / EYV]DV-C (SEQ ID NO: 446); and light chain CDR1 is N-QS[I / V]SSY-C (SEQ ID NO: 447), light chain CDR2 is N-[A / D][A / S]SC (SEQ ID NO: 448) and light chain CDR3 is N-QQ[S / Y][Y / S][V / L][P / Y]P[W / -]TC (SEQ ID NO: 449); or (ii) heavy chain CDR1 is N-FTFSSYGM[S / H]-C (SEQ ID NO: 361). SEQ ID NO: 450); heavy chain CDR2 is N-[N / V]I[K / W][Q / Y]DGS[E / N]KYY[V / A]DSVKG-C (SEQ ID NO: 451) and heavy chain CDR3 is N-AR[D / G]AP[WDIYDYYM / EYV]DV-C (SEQ ID NO: 446); and light chain CDR1 is N-RASQS[I / V]SSYL[N / A]-C (SEQ ID NO: 452), light chain CDR2 is N-[AA / D]SS[LQS / NRAT]-C (SEQ ID NO: 453) and light chain CDR3 is N-QQ[S / Y][Y / S][V / L][P / Y]P[W / -]TC (SEQ ID NO: 449).

[0026] In some implementations, (i) the heavy chain CDR1, CDR2 and CDR3 sequences are SEQ ID NO: 361, 362 and 363, respectively, and the light chain CDR1, CDR2 and CDR3 sequences are SEQ ID NO: 369, 370 and 371, respectively; or (ii) the heavy chain CDR1, CDR2 and CDR3 sequences are SEQ ID NO: 364, 365 and 366, respectively, and the light chain CDR1, CDR2 and CDR3 sequences are SEQ ID NO: 372, 373 and 374, respectively.

[0027] In one embodiment, (i) the heavy chain CDR1, CDR2 and CDR3 sequences are SEQ ID NO: 383, 384 and 385, respectively, and the light chain CDR1, CDR2 and CDR3 sequences are SEQ ID NO: 391, 392 and 393, respectively; or (ii) the heavy chain CDR1, CDR2 and CDR3 sequences are SEQ ID NO: 386, 387 and 388, respectively, and the light chain CDR1, CDR2 and CDR3 sequences are SEQ ID NO: 394, 395 and 396, respectively.

[0028] Another aspect of this disclosure provides isolated monoclonal antibodies or antigen-binding moieties thereof that specifically bind to and antagonize human IL-27, said antibody or antigen-binding moieties comprising heavy chain and light chain CDRs, said heavy chain and light chain CDRs being: (i) respectively, heavy chain CDR1 being N-GFTFXXXX-C (SEQ ID NO: 408), heavy chain CDR2 being N-IXXXXXXX-C (SEQ ID NO: 456) and heavy chain CDR3 being N-AR[X]. n＝6-15 DX-C (SEQ ID NO: 458); and the light chain CDR1 is N-QS[X]. n＝1-3 SS[X] n＝0-4 YC (SEQ ID NO: 460), light chain CDR2 is N-XXS-C (SEQ ID NO: 462) and light chain CDR3 is N-QQXXXXP[X] n＝0-1 TC (SEQ ID NO: 464); or (ii) respectively, heavy chain CDR1 is N-FTFXXXXMX-C (SEQ ID NO: 466), heavy chain CDR2 is N-XIXXXXXXXXYXDSVKG-C (SEQ ID NO: 468) and heavy chain CDR3 is N-AR[X] n＝6-15 DX-C (SEQ ID NO: 470); and light chain CDR1 is N-RASQSXSSYLX-C (SEQ ID NO: 472), and light chain CDR2 is N-[X]. n＝1-2 S[X]n＝4-5 -C (SEQ ID NO: 474) and the light chain CDR3 are N-QQXXXXP[X] n＝0-1 TC (SEQ ID NO: 476).

[0029] In some embodiments, (i) respectively, heavy chain CDR1 is N-GFTF[S / A / R][S / R][T / Y][G / S]-C (SEQ ID NO: 454), heavy chain CDR2 is NI[S / K / W][S / Q / Y][S / D][S / G][S / A][Y / E / N][I / K]-C (SEQ ID NO: 455) and heavy chain CDR3 is N-AR[DGGRTSYTATAHNWF / DAPWDIYDYYM / GAPEYV]D[P / V]-C (SEQ ID NO: 457); and light chain CDR1 is N-QS[VLF / I / V]SS[NNKN / -]YC (SEQ ID NO: 459), and light chain CDR2 is N-[W / A / D][A / S]SC (SEQ ID NO: 459). NO: 461) and light chain CDR3 is N-QQ[H / S / Y][A / Y / S][S / V / L][A / P / Y]P[P / W / -]TC (SEQ ID NO: 463); or (ii) respectively, heavy chain CDR1 is N-FTF[S / A / R][S / R][T / Y][G / S]M[N / S / H]-C (SEQ ID NO: 465), heavy chain CDR2 is N-[G / S / N / V]I[S / K / W][S / Q / Y][S / D][S / G][S / A][Y / E / N][I / K][L / Y]Y[V / A]DSVKG-C (SEQ ID NO: 467) and heavy chain CDR3 is N-AR[D / G][GGRTSYTATAHNWF / APWDIYDYYM / APEYV]D[P / V]-C (SEQ ID NO: 463). NO: 469); and light chain CDR1 is N-RASQS[I / V]SSYL[N / A]-C (SEQ ID NO: 471), light chain CDR2 is N-[WA / AA / D]S[TRES / SLQS / SNRAT]-C (SEQ ID NO: 473) and light chain CDR3 is N-QQ[H / S / Y][A / Y / S][S / V / L][A / P / Y]P[P / W / -]TC (SEQ ID NO: 475).

[0030] In some embodiments, the isolated monoclonal antibody or its antigen-binding portion inhibits or reduces STAT1 and / or STAT3 phosphorylation in cells. In some embodiments, the cells are immune cells. In some embodiments, the cells are cancer cells.

[0031] In some embodiments, the isolated monoclonal antibody or its antigen-binding portion inhibits or reduces the inhibition of CD161 expression in cells (e.g., improves or alleviates the inhibition of CD161 expression in cells). In some embodiments, the cells are immune cells.

[0032] In some embodiments, the isolated monoclonal antibody or its antigen-binding portion inhibits or reduces IL-27-mediated PD-L1 and / or TIM-3 expression in cells. 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 and TIM-3 expression are reduced. In some embodiments, the cells are immune cells.

[0033] In some embodiments, the isolated monoclonal antibody or its antigen-binding portion induces or enhances the secretion of one or more PD-1-mediated cytokines from cells. 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 cells are immune cells.

[0034] In some embodiments, the isolated monoclonal antibody or its antigen-binding moiety 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, the mutant IgG4 heavy chain constant region comprises any one or a combination of the substitutions S228P, L235E, and L235A according to EU designations.

[0035] In some embodiments, this disclosure provides isolated monoclonal antibodies or antigen-binding portions thereof that bind substantially the same epitopes on IL-27 as the antibody or antigen-binding portion thereof according to any of the foregoing embodiments.

[0036] In some embodiments, this disclosure provides an isolated monoclonal antibody or its antigen-binding portion thereof, said isolated monoclonal antibody or its antigen-binding portion thereof being bound to at least one of the amino acid residues containing IL-27 by an antibody or its antigen-binding portion according to any of the foregoing embodiments.

[0037] In some embodiments, this disclosure provides isolated monoclonal antibodies or antigen-binding portions thereof, wherein mutations in the epitopes on IL-27 bound by the antibody or antigen-binding portion thereof inhibit, reduce, or block binding to the antibody or antigen-binding portion thereof and to the antibody or antigen-binding portion thereof according to any of the foregoing embodiments.

[0038] In some embodiments, this disclosure provides isolated monoclonal antibodies or antigen-binding portions thereof that bind to epitopes on IL-27, wherein the epitopes are the same as or similar to the epitopes bound to the antibody molecules described in Table 12.

[0039] In some embodiments, this disclosure provides isolated monoclonal antibodies or antigen-binding moieties thereof that specifically bind to and antagonize human IL-27, wherein said antibody or antigen-binding moieties comprise heavy and light chain CDRs selected from the group consisting of:

[0040] (i) The heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 51, 52 and 53 respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 59, 60 and 61 respectively;

[0041] (ii) The heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 73, 74 and 75 respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 81, 82 and 83 respectively;

[0042] (iii) The heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 95, 96 and 97 respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 103, 104 and 105 respectively;

[0043] (iv) The heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 117, 118 and 119 respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 125, 126 and 127 respectively;

[0044] (v) The heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 139, 140 and 141, respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 147, 148 and 149, respectively;

[0045] (vi) The heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 161, 162 and 163 respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 169, 170 and 171 respectively;

[0046] (vii) The heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 185, 186 and 187 respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 193, 194 and 195 respectively;

[0047] (viii) The heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 207, 208 and 209 respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 215, 216 and 217 respectively;

[0048] (ix) The heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 229, 230 and 231 respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 237, 238 and 239 respectively;

[0049] (x) The heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 251, 252 and 253 respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 259, 260 and 261 respectively;

[0050] (xi) are the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 273, 274 and 275, respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 281, 282 and 283, respectively;

[0051] (xii) The heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 295, 296 and 297 respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 303, 304 and 305 respectively;

[0052] (xiii) The heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 317, 318 and 319 respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 325, 326 and 327 respectively;

[0053] (xiv) shows the heavy chain CDR1, CDR2 and CDR3 sequences in SEQ ID NO: 339, 340 and 341, respectively, and the light chain CDR1, CDR2 and CDR3 sequences in SEQ ID NO: 347, 348 and 349, respectively;

[0054] (xv) The heavy chain CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 361, 362, and 363, respectively, and the light chain CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 369, 370, and 371, respectively; and

[0055] (xvi) are the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 383, 384 and 385, respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 391, 392 and 393, respectively.

[0056] In some embodiments, this disclosure provides isolated monoclonal antibodies or antigen-binding moieties thereof that specifically bind to and antagonize human IL-27, wherein the antibody or antigen-binding moieties comprise heavy chain and light chain CDRs, wherein the heavy chain CDR1, CDR2 and CDR3 sequences are shown in SEQ ID NO: 161, 162 and 163, respectively, and the light chain CDR1, CDR2 and CDR3 sequences are shown in SEQ ID NO: 169, 170 and 171, respectively.

[0057] In some embodiments, this disclosure provides isolated monoclonal antibodies or antigen-binding moieties thereof that specifically bind to and antagonize human IL-27, wherein said antibody or antigen-binding moieties comprise heavy and light chain CDRs selected from the group consisting of:

[0058] (i) the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 54, 55 and 56 respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 62, 63 and 64 respectively;

[0059] (ii) The heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 76, 77 and 78 respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 84, 85 and 86 respectively;

[0060] (iii) The heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 98, 99 and 100 respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 106, 107 and 108 respectively;

[0061] (iv) The heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 120, 121 and 122 respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 128, 129 and 130 respectively;

[0062] (v) The heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 142, 143 and 144 respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 150, 151 and 152 respectively;

[0063] (vi) The heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 164, 165 and 166 respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 172, 173 and 174 respectively;

[0064] (vii) The heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 188, 189 and 190 respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 196, 197 and 198 respectively;

[0065] (viii) The heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 210, 211 and 212 respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 218, 219 and 220 respectively;

[0066] (ix) The heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 232, 233 and 234 respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 240, 241 and 242 respectively;

[0067] (x) The heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 254, 255 and 256 respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 262, 263 and 264 respectively;

[0068] (xi) are the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 276, 277 and 278, respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 284, 285 and 286, respectively;

[0069] (xii) The heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 298, 299 and 300 respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 306, 307 and 308 respectively;

[0070] (xiii) The heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 320, 321 and 322 respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 328, 329 and 330 respectively;

[0071] (xiv) shows the heavy chain CDR1, CDR2 and CDR3 sequences in SEQ ID NO: 342, 343 and 344, respectively, and the light chain CDR1, CDR2 and CDR3 sequences in SEQ ID NO: 350, 351 and 352, respectively;

[0072] (xv) The heavy chain CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 364, 365, and 366, respectively, and the light chain CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 372, 373, and 374, respectively; and

[0073] (xvi) are the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 386, 387 and 388, respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 394, 395 and 396, respectively.

[0074] In some embodiments, this disclosure provides a species-specific monoclonal antibody or antigen-binding moiety thereof that specifically binds to and antagonizes human IL-27, wherein the antibody or antigen-binding moiety comprises heavy chain and light chain CDRs, wherein the heavy chain CDR1, CDR2 and CDR3 sequences are shown in SEQ ID NO: 164, 165 and 166, respectively, and the light chain CDR1, CDR2 and CDR3 sequences are shown in SEQ ID NO: 172, 173 and 174, respectively.

[0075] In some embodiments, this disclosure provides isolated monoclonal antibodies or antigen-binding moieties thereof that specifically bind to and antagonize human IL-27, wherein the antibody or antigen-binding moieties comprise heavy and light chain CDRs, wherein the amino acid sequences of the heavy chains 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 chains CDR1, CDR2, and CDR3 are RASENIYSYLA (SEQ ID NO: 26), NAETLTE (SEQ ID NO: 27), and QHHYGTPLT (SEQ ID NO: 28), respectively.

[0076] In some embodiments, this disclosure provides isolated monoclonal antibodies or antigen-binding moieties thereof that specifically bind to and antagonize human IL-27, wherein the antibody or antigen-binding moieties comprise heavy chain and light chain variable regions, wherein the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 57, 79, 101, 123, 145, 167, 191, 213, 235, 257, 279, 301, 323, 345, 367 and 389; and wherein the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 65, 87, 109, 131, 153, 175, 199, 221, 243, 265, 287, 309, 331, 353, 375 and 397.

[0077] In some embodiments, this disclosure provides isolated monoclonal antibodies or antigen-binding moieties thereof that specifically bind to and antagonize human IL-27, wherein said antibody or antigen-binding moieties comprise heavy chain and light chain variable regions, said heavy chain and light chain variable regions each comprising an amino acid sequence selected from the group consisting of:

[0078] (i)SEQ ID NO: 57 and 65;

[0079] (ii) SEQ ID NO: 79 and 87;

[0080] (iii) SEQ ID NO: 101 and 109;

[0081] (iv)SEQ ID NO: 123 and 131;

[0082] (v)SEQ ID NO: 145 and 153;

[0083] (vi)SEQ ID NO: 167 and 175;

[0084] (vii)SEQ ID NO: 191 and 199;

[0085] (viii) SEQ ID NO: 213 and 221;

[0086] (ix)SEQ ID NO: 235 and 243;

[0087] (x)SEQ ID NO: 257 and 265;

[0088] (xi)SEQ ID NO: 279 and 287;

[0089] (xii)SEQ ID NO: 301 and 309;

[0090] (xiii) SEQ ID NO: 323 and 331;

[0091] (xiv)SEQ ID NO: 345 and 353;

[0092] (xv)SEQ ID NO: 367 and 375; and

[0093] (xvi)SEQ ID NO: 389 and 397.

[0094] In some embodiments, this disclosure provides isolated monoclonal antibodies or antigen-binding moieties thereof that specifically bind to and antagonize human IL-27, wherein the antibody or antigen-binding moieties comprise heavy and light chain variable regions, wherein the heavy chain variable region comprises an amino acid sequence having at least 90% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 57, 79, 101, 123, 145, 167, 191, 213, 235, 257, 279, 301, 323, 345, 367, and 389; and wherein the light chain variable region comprises an amino acid sequence having at least 90% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 65, 87, 109, 131, 153, 175, 199, 221, 243, 265, 287, 309, 331, 353, 375, and 397.

[0095] In some embodiments, this disclosure provides isolated monoclonal antibodies or antigen-binding moieties thereof that specifically bind to and antagonize human IL-27, wherein said antibody or antigen-binding moieties comprise heavy and light chain variable regions, said heavy and light chain variable regions each comprising an amino acid sequence having at least 90% identity with an amino acid sequence selected from the group consisting of:

[0096] (i)SEQ ID NO: 57 and 65;

[0097] (ii) SEQ ID NO: 79 and 87;

[0098] (iii) SEQ ID NO: 101 and 109;

[0099] (iv)SEQ ID NO: 123 and 131;

[0100] (v)SEQ ID NO: 145 and 153;

[0101] (vi)SEQ ID NO: 167 and 175;

[0102] (vii)SEQ ID NO: 191 and 199;

[0103] (viii) SEQ ID NO: 213 and 221;

[0104] (ix)SEQ ID NO: 235 and 243;

[0105] (x)SEQ ID NO: 257 and 265;

[0106] (xi)SEQ ID NO: 279 and 287;

[0107] (xii)SEQ ID NO: 301 and 309;

[0108] (xiii) SEQ ID NO: 323 and 331;

[0109] (xiv)SEQ ID NO: 345 and 353;

[0110] (xv)SEQ ID NO: 367 and 375; and

[0111] (xvi)SEQ ID NO: 389 and 397.

[0112] In some embodiments, this disclosure provides isolated monoclonal antibodies or antigen-binding moieties thereof that specifically bind to and antagonize human IL-27, wherein the antibody or antigen-binding moieties thereof comprise heavy chain and light chain variable regions, the heavy chain and light chain variable regions comprising the amino acid sequences shown in SEQ ID NO: 167 and 175, respectively.

[0113] In some embodiments, this disclosure provides isolated monoclonal antibodies or antigen-binding moieties thereof that specifically bind to and antagonize human IL-27, wherein the antibody or antigen-binding moieties thereof comprise heavy chain and light chain variable regions, the heavy chain and light chain variable regions comprising amino acid sequences having at least 90% identity with the amino acid sequences shown in SEQ ID NO: 167 and 175, respectively.

[0114] In some embodiments, this disclosure provides isolated monoclonal antibodies or antigen-binding moieties thereof that specifically bind to and antagonize human IL-27, wherein the antibody or antigen-binding moieties comprise a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 67, 89, 111, 133, 155, 177, 201, 223, 245, 267, 289, 311, 333, 355, 377, and 399; and wherein the light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 69, 91, 113, 135, 157, 179, 203, 225, 247, 269, 291, 313, 335, 357, 379, and 401.

[0115] In some embodiments, this disclosure provides isolated monoclonal antibodies or antigen-binding moieties thereof that specifically bind to and antagonize human IL-27, wherein said antibody or antigen-binding moieties comprise a heavy chain and a light chain, wherein said heavy chain comprises an amino acid sequence having at least 90% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 67, 89, 111, 133, 155, 177, 201, 223, 245, 267, 289, 311, 333, 355, 377, and 399; and wherein said light chain comprises an amino acid sequence having at least 90% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 69, 91, 113, 135, 157, 179, 203, 225, 247, 269, 291, 313, 335, 357, 379, and 401.

[0116] In some embodiments, this disclosure provides isolated monoclonal antibodies or antigen-binding moieties thereof that specifically bind to and antagonize human IL-27, wherein the antibody or antigen-binding moieties comprise a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 71, 93, 115, 137, 159, 181, 205, 227, 249, 271, 293, 315, 337, 359, 381, and 403; and wherein the light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 69, 91, 113, 135, 157, 179, 203, 225, 247, 269, 291, 313, 335, 357, 379, and 401.

[0117] In some embodiments, this disclosure provides isolated monoclonal antibodies or antigen-binding moieties thereof that specifically bind to and antagonize human IL-27, wherein the antibody or antigen-binding moieties comprise a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence having at least 90% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 71, 93, 115, 137, 159, 181, 205, 227, 249, 271, 293, 315, 337, 359, 381, and 403; and wherein the light chain comprises an amino acid sequence having at least 90% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 69, 91, 113, 135, 157, 179, 203, 225, 247, 269, 291, 313, 335, 357, 379, and 401.

[0118] In some embodiments, this disclosure provides isolated monoclonal antibodies or antigen-binding moieties thereof that specifically bind to and antagonize human IL-27, wherein said antibody or antigen-binding moieties comprise heavy and light chains, said heavy and light chains each comprising an amino acid sequence selected from the group consisting of:

[0119] (i)SEQ ID NO: 67 and 69;

[0120] (ii) SEQ ID NO: 89 and 91;

[0121] (iii) SEQ ID NO: 111 and 113;

[0122] (iv)SEQ ID NO: 133 and 135;

[0123] (v)SEQ ID NO: 155 and 157;

[0124] (vi)SEQ ID NO: 177 and 179;

[0125] (vii)SEQ ID NO: 201 and 203;

[0126] (viii) SEQ ID NO: 223 and 225;

[0127] (ix)SEQ ID NO: 245 and 247;

[0128] (x)SEQ ID NO: 267 and 269;

[0129] (xi)SEQ ID NO: 289 and 291;

[0130] (xii)SEQ ID NO: 311 and 313;

[0131] (xiii) SEQ ID NO: 333 and 335;

[0132] (xiv)SEQ ID NO: 355 and 357;

[0133] (xv)SEQ ID NO: 377 and 379; and

[0134] (xvi)SEQ ID NO: 399 and 401.

[0135] In some embodiments, this disclosure provides isolated monoclonal antibodies or antigen-binding moieties thereof that specifically bind to and antagonize human IL-27, wherein said antibody or antigen-binding moieties comprise a heavy chain and a light chain, said heavy chain and light chain each comprising an amino acid sequence having at least 90% identity with an amino acid sequence selected from the group consisting of:

[0136] (i)SEQ ID NO: 67 and 69;

[0137] (ii) SEQ ID NO: 89 and 91;

[0138] (iii) SEQ ID NO: 111 and 113;

[0139] (iv)SEQ ID NO: 133 and 135;

[0140] (v)SEQ ID NO: 155 and 157;

[0141] (vi)SEQ ID NO: 177 and 179;

[0142] (vii)SEQ ID NO: 201 and 203;

[0143] (viii) SEQ ID NO: 223 and 225;

[0144] (ix)SEQ ID NO: 245 and 247;

[0145] (x)SEQ ID NO: 267 and 269;

[0146] (xi)SEQ ID NO: 289 and 291;

[0147] (xii)SEQ ID NO: 311 and 313;

[0148] (xiii) SEQ ID NO: 333 and 335;

[0149] (xiv)SEQ ID NO: 355 and 357;

[0150] (xv)SEQ ID NO: 377 and 379; and

[0151] (xvi)SEQ ID NO: 399 and 401.

[0152] In some embodiments, this disclosure provides isolated monoclonal antibodies or antigen-binding moieties thereof that specifically bind to and antagonize human IL-27, wherein said antibody or antigen-binding moieties comprise heavy and light chains, said heavy and light chains each comprising an amino acid sequence selected from the group consisting of:

[0153] (i)SEQ ID NO: 71 and 69;

[0154] (ii) SEQ ID NO: 93 and 91;

[0155] (iii) SEQ ID NO: 115 and 113;

[0156] (iv)SEQ ID NO: 137 and 135;

[0157] (v)SEQ ID NO: 159 and 157;

[0158] (vi)SEQ ID NO: 181 and 179;

[0159] (vii)SEQ ID NO: 205 and 203;

[0160] (viii) SEQ ID NO: 227 and 225;

[0161] (ix)SEQ ID NO: 249 and 247;

[0162] (x)SEQ ID NO: 271 and 269;

[0163] (xi)SEQ ID NO: 293 and 291;

[0164] (xii)SEQ ID NO: 315 and 313;

[0165] (xiii) SEQ ID NO: 337 and 335;

[0166] (xiv)SEQ ID NO: 359 and 357;

[0167] (xv)SEQ ID NO: 381 and 379; and

[0168] (xvi)SEQ ID NO: 403 and 401.

[0169] In some embodiments, this disclosure provides isolated monoclonal antibodies or antigen-binding moieties thereof that specifically bind to and antagonize human IL-27, wherein said antibody or antigen-binding moieties comprise a heavy chain and a light chain, said heavy chain and light chain each comprising an amino acid sequence having at least 90% identity with an amino acid sequence selected from the group consisting of:

[0170] (i)SEQ ID NO: 71 and 69;

[0171] (ii) SEQ ID NO: 93 and 91;

[0172] (iii) SEQ ID NO: 115 and 113;

[0173] (iv)SEQ ID NO: 137 and 135;

[0174] (v)SEQ ID NO: 159 and 157;

[0175] (vi)SEQ ID NO: 181 and 179;

[0176] (vii)SEQ ID NO: 205 and 203;

[0177] (viii) SEQ ID NO: 227 and 225;

[0178] (ix)SEQ ID NO: 249 and 247;

[0179] (x)SEQ ID NO: 271 and 269;

[0180] (xi)SEQ ID NO: 293 and 291;

[0181] (xii)SEQ ID NO: 315 and 313;

[0182] (xiii) SEQ ID NO: 337 and 335;

[0183] (xiv)SEQ ID NO: 359 and 357;

[0184] (xv)SEQ ID NO: 381 and 379; and

[0185] (xvi)SEQ ID NO: 403 and 401.

[0186] In some embodiments, this disclosure provides isolated monoclonal antibodies or antigen-binding moieties thereof that specifically bind to and antagonize human IL-27, wherein the antibody or antigen-binding moieties comprise heavy and light chains comprising the amino acid sequences shown in SEQ ID NO: 177 and 179, respectively.

[0187] In some embodiments, this disclosure provides isolated monoclonal antibodies or antigen-binding moieties thereof that specifically bind to and antagonize human IL-27, wherein the antibody or antigen-binding moieties comprise heavy and light chains, the heavy and light chains comprising amino acid sequences having at least 90% identity with the amino acid sequences shown in SEQ ID NO: 177 and 179, respectively.

[0188] In some embodiments, this disclosure provides isolated monoclonal antibodies or antigen-binding moieties thereof that specifically bind to and antagonize human IL-27, wherein the antibody or antigen-binding moieties comprise heavy and light chains comprising the amino acid sequences shown in SEQ ID NO: 181 and 179, respectively.

[0189] In some embodiments, this disclosure provides isolated monoclonal antibodies or antigen-binding moieties thereof that specifically bind to and antagonize human IL-27, wherein the antibody or antigen-binding moieties comprise heavy and light chains, the heavy and light chains comprising amino acid sequences having at least 90% identity with the amino acid sequences shown in SEQ ID NO: 181 and 179, respectively.

[0190] In some embodiments, this disclosure provides a method for inhibiting or reducing STAT1 and / or STAT3 phosphorylation in cells, the method comprising contacting the cells with an isolated monoclonal antibody or antigen-binding fragment provided by this disclosure, wherein the antibody or its antigen-binding portion inhibits or reduces STAT1 and / or STAT3 phosphorylation in the cells.

[0191] In some embodiments, this disclosure provides a method for inhibiting or reducing IL-27-mediated inhibition of CD161 expression in cells, the method comprising contacting the cells with an isolated monoclonal antibody or antigen-binding fragment provided herein, wherein the antibody or its antigen-binding portion inhibits or reduces the inhibition of CD161 expression in cells.

[0192] In some embodiments, this disclosure provides a method for inhibiting or reducing IL-27-mediated PD-L1 and / or TIM-3 expression in cells, the method comprising contacting the cells with an isolated monoclonal antibody or antigen-binding fragment provided by this disclosure, wherein the antibody or its antigen-binding portion inhibits or reduces the expression of PD-L1 and / or TIM-3 in the cells.

[0193] In some embodiments, this disclosure provides a method for inducing or enhancing the secretion of one or more cytokines from a cell, the method comprising contacting the cell with an isolated monoclonal antibody or antigen-binding fragment provided by this disclosure, wherein the antibody or antigen-binding portion thereof induces or enhances the secretion of one or more PD-1-mediated cytokines from the cell.

[0194] In some embodiments, this disclosure provides a method for stimulating an immune response in a subject, the method comprising administering to the subject an effective amount of an isolated monoclonal antibody or antigen-binding fragment that specifically binds to and antagonizes human IL-27, provided in this disclosure, or a pharmaceutical composition comprising said antibody or its antigen-binding portion and a pharmaceutically acceptable carrier.

[0195] In some embodiments, this disclosure provides a method of treating a subject with cancer, the method comprising administering to the subject an effective amount of an isolated monoclonal antibody or antigen-binding portion thereof that specifically binds to and antagonizes IL-27, provided in this disclosure, or a pharmaceutical composition comprising said antibody or antigen-binding portion thereof and a pharmaceutically acceptable carrier.

[0196] In some embodiments, this disclosure provides a method for stimulating an immune response in a subject or treating a subject’s cancer, the method comprising administering to the subject an effective amount of an isolated monoclonal antibody or antigen-binding fragment provided in this disclosure, or a pharmaceutical composition comprising the antibody or its antigen-binding portion and a pharmaceutically acceptable carrier, wherein the antibody or its antigen-binding portion or the pharmaceutical composition inhibits or reduces STAT1 and / or STAT3 phosphorylation in cells, thereby stimulating the immune response or treating the cancer.

[0197] In some embodiments, this 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 in this disclosure, or a pharmaceutical composition comprising said antibody or its antigen-binding portion and a pharmaceutically acceptable carrier, wherein said antibody or its antigen-binding portion or the pharmaceutical composition inhibits or reduces IL-27-mediated inhibition of CD161 expression in cells, thereby stimulating the immune response or treating the cancer.

[0198] In some embodiments, this 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 in this disclosure, or a pharmaceutical composition comprising said antibody or its antigen-binding portion and a pharmaceutically acceptable carrier, wherein said antibody or its antigen-binding portion or the pharmaceutical composition inhibits or reduces IL-27-mediated PD-L1 expression and / or TIM-3 expression in cells, thereby stimulating the immune response or treating the cancer.

[0199] In some embodiments, this 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 in this disclosure, or a pharmaceutical composition comprising said antibody or its antigen-binding portion and a pharmaceutically acceptable carrier, wherein said antibody or its antigen-binding portion or said pharmaceutical composition induces or enhances the secretion of one or more PD-1-mediated cytokines from cells, thereby stimulating said immune response or treating said cancer.

[0200] In some implementations, the cancer is selected from the group consisting of: 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 (including, for example, uveal 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, such as clear cell renal cell carcinoma).

[0201] In some embodiments, this disclosure provides a method of treating a subject with cancer, the method comprising administering to the subject an effective amount of a separate monoclonal antibody or antigen-binding portion thereof that specifically binds to and antagonizes IL-27, provided by this 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 anticancer therapies (including, for example, tyrosine kinase inhibitors (TKIs)), oncolytic agents, cytotoxic agents, immunotherapy-based therapies, cytokines, surgical procedures, radiation procedures, activators of co-stimulatory molecules, inhibitors of inhibitory molecules, vaccines, or cell immunotherapy, or combinations thereof.

[0202] In some implementations, one or more additional therapeutic agents are PD-1 antagonists, PD-L1 inhibitors, TIM-3 inhibitors, LAG-3 inhibitors, TIGIT inhibitors, CD112R inhibitors, TAM inhibitors, STING agonists, 4-1BB agonists, or combinations thereof.

[0203] In some implementations, one or more additional therapeutic agents are PD-1 antagonists. In some implementations, the PD-1 antagonist is selected from the group consisting of: PDR001, nivolumab, pebuprofen, pildizumab, MEDI0680, REGN2810, TSR-042, PF-06801591, and AMP-224.

[0204] In some embodiments, 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, avermab, durvalumab, and BMS-936559. In some embodiments, this disclosure provides a method for enhancing one or more activities of an anti-PD-1 antibody (e.g., enhancing PD-1-mediated cytokine secretion; enhancing anti-PD-1-mediated TNF secretion; enhancing anti-PD-1-mediated IL-6 secretion from cells exposed to an anti-PD-1 antibody), said method comprising exposing cells to an antibody or its antigen-binding portion provided by this disclosure, simultaneously or sequentially exposed to an anti-PD-1 antibody, thereby enhancing one or more activities of the anti-PD-1 antibody.

[0205] In some embodiments, this disclosure provides pharmaceutical compositions comprising an anti-PD-1 antibody and / or an anti-PD-L1 antibody, as well as an antibody or its antigen-binding moiety disclosed herein (e.g., an anti-IL-27 antibody) and a pharmaceutically acceptable carrier.

[0206] In relevant embodiments, this disclosure provides a kit containing an anti-PD-1 antibody and / or an anti-PD-L1 antibody for simultaneous or sequential administration, as well as the antibody disclosed herein or its antigen-binding portion (e.g., anti-IL-27 antibody), and instructions for use thereof.

[0207] In some implementations, one or more additional therapeutic agents are TIM-3 inhibitors, optionally wherein the TIM-3 inhibitor is MGB453 or TSR-022.

[0208] In some embodiments, one or more additional therapeutic agents are LAG-3 inhibitors, optionally wherein the LAG-3 inhibitor is selected from the group consisting of LAG525, BMS-986016 and TSR-033.

[0209] 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.

[0210] In some embodiments, this disclosure provides a method for detecting IL-27 in a sample from a subject, the method comprising (a) if IL-27 is present in the sample from the subject, contacting the sample with a detection antibody, wherein the detection antibody is an antibody or an antigen-binding fragment thereof provided in this disclosure, under conditions allowing the formation of a detection antibody-IL-27 complex; and (b) detecting the presence of the complex generated in step (a), if any.

[0211] In some embodiments, this disclosure provides a method for detecting IL-27-related cancer in a subject, the method comprising the steps of: (a) if IL-27 is present in a sample from a subject suspected of having IL-27-related cancer, contacting the sample with a detection antibody, wherein the detection antibody is an antibody provided by this disclosure or its antigen-binding portion thereof, under conditions allowing the formation of a detection antibody-IL-27 complex; and (b) detecting the presence of the complex generated in step (a), if any. In some embodiments, the detection antibody is conjugated to a detectable marker. In some embodiments, the method further includes, if IL-27 is present in the sample, contacting the sample with a capture antibody to generate a complex comprising IL-27 and the capture antibody, wherein the capture antibody is an antibody provided by this disclosure or its antigen-binding portion thereof.

[0212] In some embodiments, the detection antibody or capture antibody comprises heavy chain and light chain CDRs, wherein the amino acid sequences of the heavy chains 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 chains CDR1, CDR2 and CDR3 are RASENIYSYLA (SEQ ID NO: 26), NAETLTE (SEQ ID NO: 27) and QHHYGTPLT (SEQ ID NO: 28), respectively.

[0213] In some embodiments, the detection antibody or capture antibody comprises heavy chain and light chain variable regions, the heavy chain and light chain variable regions comprising the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 3, respectively.

[0214] In some embodiments, the capture antibody is immobilized on a solid support. In some embodiments, the sample is contacted with the capture antibody and then with a detection antibody. In some embodiments, the sample is a body fluid sample. In some embodiments, the fluid sample is blood, serum, plasma, cell lysate, or tissue lysate.

[0215] 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. 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).

[0216] definition

[0217] Unless otherwise stated, the terms used in the claims and description are defined as follows.

[0218] It must be noted that, unless the context clearly specifies otherwise, the singular forms “a,” “an,” and “the” as used in the specification and appended claims include plural references.

[0219] As used herein, “about” will be understood by those skilled in the art and will vary to some extent depending on the context in which it is used. If the use of the term is unclear to those skilled in the art, given the context in which it is used, “about” will mean plus or minus 10% of a specific value.

[0220] As used herein, the term "agonist" means any molecule that partially or completely promotes, induces, increases, and / or activates the biological activity of the natural polypeptides disclosed herein. Suitable agonist molecules particularly include agonist antibodies or antibody fragments, fragments or amino acid sequence variants of natural polypeptides, peptides, or proteins. In some embodiments, activation in the presence of an agonist is observed in a dose-dependent manner. In some embodiments, the measured signal (e.g., biological activity) is at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% higher than the signal measured using a negative control under comparable conditions. Methods for identifying agonists suitable for the methods of this disclosure are also disclosed herein. For example, these methods include, but are not limited to, combination assays such as enzyme-linked immunosorbent assay (ELISA) and Forte. Systemic and radioimmunoassays (RIA) are used. These assays determine the ability of an agonist to bind to a target peptide (e.g., a receptor or ligand) and thus indicate the agonist's ability to promote, increase, or activate peptide activity. Functional assays can also be used to determine the potency of an agonist, such as its ability to activate or promote peptide function. For example, a functional assay may include contacting the peptide with a candidate agonist molecule and measuring detectable changes in one or more biological activities typically associated with the peptide. The potency of an agonist is typically determined by its EC... 50 Defined by the value (the concentration required to activate a 50% agonist response). EC 50 The lower the value, the greater the efficacy of the agonist, and the lower the concentration required to activate the maximum biological response.

[0221] As used herein, the term "alanine scan" refers to a technique used to determine 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 peptide. This technique involves replacing a wild-type residue in a peptide with an alanine residue, then evaluating the stability or one or more functions (e.g., binding affinity) of the alanine-substituted derivative or mutant peptide, and comparing it to the wild-type peptide. Techniques for replacing wild-type residues in peptides with alanine are known in the art.

[0222] The term “improvement” refers to any therapeutically beneficial outcome in the treatment of a disease state (e.g., cancer), including its prevention, reduction, relief, or cure.

[0223] As used herein, the term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that have been modified, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs are compounds that have the same basic chemical structure as naturally occurring amino acids (i.e., carbon atoms bound to hydrogen, carboxyl, amino, and R groups), such as homoserine, ortholeucine, methionine sulfoxide, and methionine methylsulfonium. These analogs have modified R groups (e.g., ortholeucine) or modified peptide backbones but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimics are compounds whose structure differs from the general chemical structure of amino acids but whose function is similar to that of naturally occurring amino acids.

[0224] Amino acids in this article can be referred to by their commonly known three-letter symbols or by a one-letter symbol recommended by the IUPAC-IUB Biochemical Nomenclature Committee. Similarly, nucleotides can be referred to by their generally accepted single-letter codes.

[0225] As used herein, “amino acid substitution” refers to replacing 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 incorporating at least one additional amino acid into a predetermined amino acid sequence. While insertions typically consist of the insertion of one or two amino acid residues, larger “peptide insertions” can also be made, such as inserting about 3 to about 5 amino acid residues or even up to about 10, 15, or 20 amino acid residues. As disclosed above, one or more inserted residues can be naturally occurring or non-naturally occurring. “Amino acid deletion” refers to removing at least one amino acid residue from a predetermined amino acid sequence.

[0226] As used herein, the terms “amount” or “level” are used in the broadest sense and refer to the quantity, concentration, or abundance of a substance (e.g., metabolites, small molecules, proteins, mRNA, biomarkers). When referring to metabolites or small molecules (e.g., drugs), the terms “amount,” “level,” and “concentration” are often used interchangeably and generally refer to a detectable amount in a biological sample. An “elevated level” or “increased level” refers to an increase in the quantity, concentration, or abundance of a substance in a sample relative to a control sample (such as one or more individuals without a disease or condition (e.g., cancer)) or an internal control. In some embodiments, the level of elevation of a substance (e.g., a drug) in a sample refers to an increase in the amount of the substance relative to the amount in a control sample of about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%, as determined by techniques known in the art (e.g., HPLC). A “reduced level” refers to a reduction 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 without a disease or condition (e.g., cancer)) or an internal control. In some embodiments, the reduced level is little or an undetectable amount, concentration, or abundance. In some embodiments, the level of reduction of a substance (e.g., a drug) in a sample refers to a reduction 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).

[0227] When referring to proteins, mRNAs, or biomarkers (such as those described herein), the terms “level of expression” or “expression level” are generally used interchangeably and typically refer to the detectable amount of a protein, mRNA, or biomarker in a biological sample. In some respects, the detectable amount or level of a protein, mRNA, or biomarker is related to the likelihood of a response to an agent (such as those described herein). “Expression” generally refers to the process by which information contained in a gene is translated into a structure present and functioning in the cell (e.g., a protein biomarker, such as PD-L1). Thus, as used herein, “expression” can 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 modifications of a polypeptide) should also be considered expressed, whether they originate from transcripts produced by alternative splicing or degradation of transcripts or from post-translational processing of polypeptides (e.g., via proteolysis). "Expressed genes" include those genes that are transcribed into polynucleotides (as mRNA) and then translated into polypeptides, as well as those genes that are transcribed into ribonucleic acid but not translated into polypeptides (e.g., transfer RNA and ribosomal RNA). "Elevated expression," "elevated expression level," or "elevated level" refers to an increase or higher level of expression of a substance in a sample relative to a control sample (such as one or more individuals without disease or condition (e.g., cancer)) or an internal control. In some embodiments, elevated expression of a substance (e.g., a protein biomarker, such as PD-L1) in a sample refers to an increase in the amount of the substance by approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% relative to the amount of the substance in a control sample, as determined by techniques known in the art (e.g., FACS). "Decreased expression," "decreased expression level," or "decreased level" refers to a reduced or decreased level of expression of a substance (e.g., a protein biomarker) in an individual relative to a control (such as one or more individuals without a disease or condition (e.g., cancer)) or an internal control. In some embodiments, reduced expression is little or no expression. In some embodiments, reduced expression of a substance (e.g., a protein biomarker) in a sample refers to a reduction in the amount of the substance relative to the amount in a control sample of approximately 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).

[0228] As used in this article, the term “angiogenesis” or “neovascularization” refers to the process by which new blood vessels develop from previously 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 vascular or circulating endothelial stem cells (Takahashi et al., (1995) Nat. Med. 5: 434-438; Isner et al., (1999) J. Clin. Invest. 103: 1231-1236) are activated, migrate, proliferate, and differentiate into luminal structures in response to growth factor or hormone signals, or to hypoxia or local ischemia, forming new blood vessels. During local ischemia (as occurs in cancer), the increased need for oxygenation and nutrient delivery apparently induces the affected tissue to secrete angiogenic factors; these factors stimulate the formation of new blood vessels. Several other terms are associated with angiogenesis.

[0229] As used herein, the term "antagonist" refers to any molecule that partially or completely blocks, inhibits, or neutralizes the biological activity of the natural polypeptides disclosed herein. Suitable antagonistic molecules particularly include antagonistic antibodies or antibody fragments, natural polypeptides, peptides, or fragments or amino acid sequence variants of proteins. In some embodiments, inhibition in the presence of the 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. Methods for identifying antagonists suitable for the methods of this disclosure are also disclosed herein. For example, these methods include, but are not limited to, combination assays such as enzyme-linked immunosorbent assay (ELISA), Fore... Systems, radioimmunoassay (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 peptide (e.g., a receptor or ligand) and thus indicate the antagonist's ability to inhibit, neutralize, or block peptide activity. Functional assays can also be used to determine the potency of an antagonist, such as its ability to inhibit peptide or agonist function. For example, a functional assay may include contacting the peptide with a candidate agonist molecule and measuring a detectable change in one or more biological activities typically associated with the peptide. The potency of an antagonist is typically determined by its EC50. 50 The IC (Induced Concentration) is defined as the concentration required to suppress a 50% agonist response. 50 The lower the value, the greater the efficacy of the antagonist, and the lower the concentration required to inhibit the maximum biological response.

[0230] As used herein, the phrase “antibody or antigen-binding moiety of human IL-27” refers to an antibody that antagonizes the activity of at least one known 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, an antibody associated with a reduction (or decrease) of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of human IL-27 activity. Further 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.

[0231] As used herein, the term "anti-IL-27 antagonist antibody" (which may be interchangeably referred to as "anti-IL-27 antibody") means an antibody that specifically binds to and antagonizes the biological activity of IL-27 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, inhibit, suppress, or reduce the biological activity of IL-27 (e.g., ligand binding, enzyme activity) (including downstream pathways mediated by IL-27 signaling or function, such as receptor binding and / or eliciting cellular responses to IL-27 or its metabolites). In some embodiments, the anti-IL-27 antagonist antibodies provided in this disclosure bind to human IL-27 and prevent, block, or inhibit the binding of human IL-27 to its homologous 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 antagonist antibody specifically binds to EBI3 monomers. In some embodiments, the anti-IL-27 antagonist antibody specifically binds to IL-27p28 monomers. In some embodiments, the anti-IL-27 antagonist antibody specifically binds to both IL-27 monomers. In some embodiments, the anti-IL-27 antagonist antibody specifically binds to discontinuous epitopes containing both EBI3 and p28. In some embodiments, the anti-IL-27 antagonist antibody inhibits or reduces STAT1 and / or STAT3 phosphorylation in cells. In some embodiments, the anti-IL-27 antagonist antibody inhibits or reduces the inhibition of CD161 expression in cells (e.g., improves or alleviates IL-27-mediated inhibition of CD161 expression in cells). In some embodiments, the anti-IL-27 antagonist antibody inhibits or reduces the expression of PD-L1 and / or TIM-3 in cells. In some embodiments, the anti-IL-27 antagonist antibody induces or enhances the secretion of one or more PD-1-mediated cytokines from cells. In some embodiments, the anti-IL-27 antagonist antibody binds to human IL-27 and stimulates or enhances an antitumor response. In some embodiments, the anti-IL-27 antagonist antibody binds to human IL-27 with an affinity of 15 nm or less. In some embodiments, the anti-IL-27 antagonist antibody binds to human IL-27 and contains 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.

[0232] As used herein, the term "antibody" refers to a complete antibody comprising two light-chain polypeptides and two heavy-chain polypeptides. Complete antibodies include various antibody isotypes, including IgM, IgG, IgA, IgD, and IgE antibodies. The term "antibody" includes polyclonal antibodies, monoclonal antibodies, chimerized or chimeric antibodies, humanized antibodies, primatized antibodies, deimmunized antibodies, and fully human antibodies. Antibodies can be prepared in or derived from any of a variety of species, such as mammals like 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. As used herein, the terms "antibody fragment," "antigen-binding fragment," or similar terms refer to the ability to retain binding to a target antigen (e.g., IL-27) and inhibit the activity of said target antigen. Such fragments include, for example, single-chain antibodies, single-chain Fv fragments (scFv), Fd fragments, Fab fragments, Fab' fragments, or F(ab')2 fragments. An scFv fragment is a single polypeptide chain that includes both the heavy and light chain variable regions of the antibody from which the scFv is derived. Additionally, the definition of an antibody also includes intracellular antibodies, microantibodies, triabodies, and diabodies, which are applicable to the methods described herein. See, for example, Todorovska et al., (2001) J. Immunol. Methods 248(1): 47-66; Hudson and Kortt, (1999) J. Immunol. Methods 231(1): 177-189; Poljak, (1994) Structure 2(12): 1121-1123; Rondon and Marasco, (1997) Annu. Rev. Microbiol. 51: 257-283, the contents of each of these documents are incorporated herein by reference in their entirety.

[0233] As used herein, the term "antibody fragment" also includes, for example, single-domain antibodies, such as camelified 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 applications 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, this disclosure provides single-domain antibodies comprising two modified VH domains (enabling the formation of single-domain antibodies).

[0234] In some embodiments, the antigen-binding fragment includes a variable region of a heavy chain polypeptide and a variable region of a light chain polypeptide. In some embodiments, the antigen-binding fragment described herein comprises the CDRs of the light chain of the antibody and the heavy chain polypeptide.

[0235] The term "antigen-presenting cell" or "APC" refers to cells that display foreign antigens complexed with MHC on their surface. T cells recognize this complex via T cell receptors (TCRs). Examples of APC cells include, but are not limited to: B cells, dendritic cells (DCs), peripheral blood monocytes (PBMCs), monocytes (such as THP-1), B lymphoblast-like cells (such as C1R.A2, 1518B-LCL), and monocyte-derived dendritic cells (DCs). Some APCs internalize antigens through phagocytosis or receptor-mediated endocytosis.

[0236] The term “antigen presentation” refers to the process by which APCs capture antigens and make them recognizable by T cells (e.g., as a component of MHC-I and / or MHC-II conjugates).

[0237] As used herein, the term "apoptosis" refers to the programmed cell death process that occurs in multicellular organisms, such as humans. Highly regulated biochemical and molecular events leading to apoptosis result in observable and cell-specific morphological changes, including vesicle formation, cell shrinkage, chromosomal DNA condensation and breakage, and mRNA decay. A common method for identifying cells undergoing apoptosis (including T cells) is to expose them to a fluorophore-conjugated protein (annexin V). Annexin V is frequently used to detect apoptotic cells by its ability to bind to phosphatidylserine residues on the extracellular lobules of the plasma membrane, an early indicator that the cell is undergoing apoptosis.

[0238] As used herein, the term "B cell" (or "B lymphocyte") refers to a type of white blood cell subtype of lymphocytes. B cells play a role in the humoral immune component of the adaptive immune system by secreting antibodies. B cells also present antigens and secrete cytokines. Unlike the other two types of lymphocytes (T cells and natural killer cells), B cells express B cell receptors (BCRs) on their cell membranes. BCRs allow B cells to bind to specific antigens, which will initiate an antibody response against that antigen.

[0239] As used herein, the term “binding to immobilized IL-27” refers to the ability of the antibody of this disclosure to bind to IL-27 (e.g., IL-27 expressed on the cell surface or attached to a solid support).

[0240] As used herein, the term "bispecific" or "bifunctional antibody" refers to an artificial hybrid antibody having two distinct heavy / light chain pairs and two distinct binding sites. Bispecific antibodies can be produced by a variety of methods, including hybridoma fusion or Fab' fragment linkage. See, for example, Songsivilai & Lachmann, (1990) Clin. Exp. Immunol. 79: 315-321; Kostelny et al., (1992) J. Immunol. 148: 1547-1553.

[0241] Conventionally, the recombinant generation of bispecific antibodies is based on the co-expression of two immunoglobulin heavy / light chain pairs, where the two heavy / light chain pairs have different specificities (Milstein and Cuello, (1983) Nature 305: 537-539). An antibody variable domain with the desired binding specificity (antibody-antigen binding site) can be fused to an immunoglobulin constant domain sequence. Preferably, the heavy chain variable region is fused to an immunoglobulin heavy chain constant region (including at least a portion of the hinge region, CH2 region, and CH3 region). For further details on currently known illustrative methods for generating bispecific antibodies, see, for example, 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, along with many cross-linking techniques, in U.S. Patent No. 4,676,980.

[0242] Various techniques for preparing and isolating bispecific antibody fragments directly from recombinant cell cultures are also 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 linked to the Fab' portion of two different antibodies via gene fusion. Antibody homodimers can be reduced to monomers in the hinge region and then oxidized to form antibody heterodimers. This method can also be used to produce antibody homodimers. The “biantibody” technique described by Hollinger et al. (1993) Proc Natl Acad Sci USA 90: 6444-6448 provides an alternative mechanism for preparing bispecific antibody fragments. The fragments contain heavy chain variable domains (VH) linked to light chain variable domains (VL) via linkers that are too short to allow pairing between the two domains on the same chain. Therefore, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thus forming two antigen-binding sites. Another strategy for preparing bispecific antibody fragments using single-chain Fv (scFv) dimers has also been reported. See, for example, Gmber 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 short, these antibodies contain 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.

[0243] Antibodies with more than two titers (e.g., trispecific antibodies) are envisioned and described, for example, in Tutt et al. (1991) J Immunol 147:60.

[0244] This disclosure also includes variant forms of multispecific antibodies, such as the dual variable domain immunoglobulin (DVD-Ig) molecule described in Wu et al. (2007) Nat Biotechnol 25(11):1290-1297. The DVD-Ig molecule is designed such that two distinct light chain variable domains (VL) from two different parental antibodies are directly tandemly linked or linked via short adapters using recombinant DNA technology, followed by a light chain constant domain. Similarly, the heavy chain comprises two distinct heavy chain variable domains (VH) tandemly linked, followed by constant regions CH1 and Fc. Methods for preparing DVD-Ig molecules from two parental antibodies are further described, for example, in PCT publications WO 08 / 024188 and WO 07 / 024715. In some embodiments, the bispecific antibody is a Fab tandem immunoglobulin in which a light chain variable region having a second specificity is fused to the heavy chain variable region of the intact antibody. Such antibodies are described, for example, in International Patent Application Publication No. WO 2015 / 103072.

[0245] As used herein, “cancer antigen” or “tumor antigen” means (i) tumor-specific antigen, (ii) 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.

[0246] As used herein, the term "cancer-specific immune response" refers to an immune response induced by the presence of a tumor, cancer cells, or cancer antigen. In some embodiments, the response includes the proliferation of cancer antigen-specific lymphocytes. In some embodiments, the response includes the expression and upregulation of antibodies and T-cell receptors, as well as the formation and release of lymphokines, chemokines, and cytokines. The innate and adaptive immune systems interact to initiate an antigenic response against a tumor, cancer cells, or cancer antigen. In some embodiments, the cancer-specific immune response is a T-cell response.

[0247] The term "cancer" is recognized in this art as referring to a malignant tumor of epithelial or endocrine tissue, including respiratory cancers, gastrointestinal cancers, genitourinary cancers, testicular cancers, breast cancers, prostate cancers, endocrine cancers, and melanomas. The anti-IL-27 antibody described herein may be used to treat patients who have, are suspected of having, any type of cancer (including kidney cancer or melanoma), or any viral disease, or who may be at high risk of developing said cancer or viral disease. Exemplary cancers include those arising from tissues of the cervix, lungs, prostate, breast, head and neck, colon, and ovaries. The term also includes carcinosarcomas, which include malignant tumors composed of cancerous and sarcomatous tissues. "Adenocarcinoma" refers to cancer originating from glandular tissue or in which tumor cells form identifiable glandular structures.

[0248] As used herein, the term "CD112R" refers to a member of the poliovirus receptor-like protein family and a co-inhibitory receptor for human T cells. CD112R is preferentially expressed on T cells and inhibits T cell receptor-mediated signaling. CD112 is widely expressed on antigen-presenting cells and tumor cells and is a ligand for CD112R. CD112R competitively binds to CD112 with CD226. Disruption of the CD112R-CD112 interaction enhances the response of human T cells. CD112R serves as a novel checkpoint for human T cells through its interaction with CD112. As used herein, the term "CD112R inhibitor" refers to an agent that disrupts, blocks, or inhibits the biological function or activity of CD112R.

[0249] As used herein, the term "CD137" (or "4-1BB") refers to a member of the tumor necrosis factor (TNF) receptor superfamily. 4-1BB is a costimulatory immune checkpoint molecule that primarily targets activated T cells. Crosslinking of CD137 enhances T cell proliferation, IL-2 secretion, survival, and cytolytic activity. As used herein, the term "4-1BB agonist" refers to an agent that stimulates, induces, or enhances one or more functions of 4-1BB. An exemplary 4-1BB agonist is utomilumab (PF-05082566), a fully human IgG2 monoclonal antibody that targets this 4-1BB to stimulate T cells.

[0250] As used herein, the term “CD161” (or cytotoxic lectin-like receptor subfamily B, member 1 (KLRB1); NK1.1, or NKR-P1A) refers to a member of the C-type lectin superfamily. CD161 is a marker of T cells, and its expression is associated with T cell infiltration into the tumor microenvironment of many different cancer types. CD161 is further described in Fergusson et al., ((2014) Cell Reports 9(3): 1075-1088, which is incorporated herein by reference in its entirety).

[0251] As used herein, the term “IL-27” or “interleukin 27” refers to the IL-27 cytokine. IL-27 is associated with the IL-6 / IL-12 cytokine family and is a heterodimeric cytokine containing a first subunit called Epstein-Barr virus-induced gene 3 (EBI3; also known as IL-27 subunits β and IL-27B) and a second subunit called IL-27p28 (also known as IL30, IL-27 subunits 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 its 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) ibid.). While initially described as a factor promoting the initiation of the Th1 response, it has been found that IL-27 exerts its primary T-cell suppressive function by limiting Th1 responses, inhibiting Th2 and Th17 cell differentiation, and regulating the development of Tr1 and other regulatory T cell populations (Dietrich et al. (2014) J. Immunol. 192: 5382-5389). In addition to its role as an immunomodulator, IL-27 also regulates angiogenesis, hematopoiesis, and osteoclastogenesis (ibid.).

[0252] IL-27 signals via the heterodimer type I cytokine receptor (IL-27 receptor or IL-27R), which contains 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 oncosin M receptor). p130 is also a receptor subunit for IL-6 family cytokines (Liu et al. (2008) Scan. J. Immunol. 68: 22-299, Diakowski (2013) ibid.). IL-27 signals via IL-27R to activate multiple signaling cascades, including the JAK-STAT and p38 MAPK pathways.

[0253] EBI3 is also believed to have biological functions independent of p28 or IL-27 heterodimers. For example, EBI3 also interacts with p35 to form the heterodimer cytokine IL-35 (Yoshida et al. (2015) Annu. Rev Immunol. 33: 417-43) and has been shown to selectively overexpress in certain cell types without a corresponding increase in p28 or IL-27 (Larousserie et al. (2005) Am. J. Pathol. 166(4): 1217-28).

[0254] The amino acid sequence of an exemplary human EBI3 protein is provided in SEQ ID NO: 698 (NCBI reference sequence: NP_005746.2; N-MTPQLLLALVLWASCPPCSGRKGPPAALTLPRVQCRASRYPIAVDCSWTLPPAPNSTSPVSFIATYRLGMAARGHSWPCLQQTPTSTSCTITDVQLFSMAPYVLNVTAVHPWGSSSSFVPFITEHIIKPDPPEGVRLSPLAERQLQVQWEPPGSWPFPEIFSLKYWIRYKRQGAARFHRVGPIEATSFILRAVRPRARYYVQVAAQDLTDYGELSDWSLPATATMSLGK-C). An exemplary amino acid sequence of 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-MRGGRGAPFWLWPLPKLALLPLLWVLFQRTRPQGSAGPLQCYGVGPLGDLNCSWEPLGDLGAPSELHLQSQKYRSNKTQTVAVAAGRSWVAIPREQLTMSDKLLVWGTKAGQPLWPPVFVNLETQMKPNAPRLGPDVDFSEDDPLEATVHWAPPTWPSHKVLICQFHYRRCQEAAWTLLEPELKTIPLTPVEIQDLELATGYKVYGRCRMEKEEDLWGEWSPILSFQTPPSAPKDVWVSGNLCGTPGGEEPLLLWKAPGPCVQVSYKVWFWVGGRELSPEGITCCCSLIPSGAEWARVSAVNATSWEPLTNLSLVCLDSASAPRSVAVSSIAGSTELLVTWQPGPGEPLEHVVDWARDGDPLEKLNWVRLPPGNLSALLPGNFTVGVPYRITVTAVSASGLASASSVWGFREELAPLVGPTLWRLQDAPPGTPAIAWGEVPRHQLRGHLTHYTLCAQSGTSPSVCM[NVSGNTQSVTLPDLPWGPCELWVTASTIAGQGPPGPILRLHLPDNTLRWKVLPGILFLWGLFLLGCGLSLATSGRCYHLRHKVLPRWVWEKVPDPANSSSGQPHMEQVPEAQPLGDLPILEVEEMEPPPVMESSQPAQATAPLDSGYEKHFLPTPEELGLLGPPRPQVLA-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).

[0255] As used herein, the term “competition”, when used in the context of antigen-binding proteins (e.g., immunoglobulins, antibodies, or antigen-binding fragments thereof) competing for binding to the same epitope, refers to the interaction between antigen-binding proteins as determined by assays (e.g., competitive binding assays; cross-blocking assays) in which the test antigen-binding protein (e.g., test antibody) inhibits (e.g., reduces or blocks) the specific binding of a reference antigen-binding protein (e.g., reference antibody) to a common antigen (e.g., IL-27 or a fragment thereof).

[0256] The term "derived from" a polypeptide or amino acid sequence refers to the source of the polypeptide. Preferably, the polypeptide or amino acid sequence derived from a specific sequence 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 those skilled 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, for example, it may have been substituted with another amino acid residue or may have one or more amino acid residues inserted or deleted.

[0257] The polypeptide may contain amino acid sequences that are not naturally occurring. Such variants must have less than 100% sequence identity or similarity to the starting molecule. In some embodiments, the variant will have, for example, an amino acid sequence that has 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% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%), and most preferably about 95% to less than 100% amino acid sequence identity or similarity to the starting polypeptide in length.

[0258] In some embodiments, the starting polypeptide sequence differs from the sequence derived therefrom by one amino acid. Identity or similarity relative to this sequence is defined herein as the percentage of amino acid residues in the candidate sequence that are identical (i.e., identical residues) to the initial sequence after sequence alignment and the introduction of vacancies (if desired) to achieve the maximum percentage of sequence identity. In some embodiments, the polypeptide consists of, is substantially composed of, or contains an amino acid sequence selected from the sequences shown in Table 12. In some embodiments, the polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from the sequences shown in Table 12. In some embodiments, the polypeptide comprises a continuous amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with a continuous amino acid sequence selected from the sequences shown in Table 12. In some 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 of these numbers) continuous amino acids selected from the sequences shown in Table 12.

[0259] In some embodiments, the antibodies of this disclosure are encoded by nucleotide sequences. The nucleotide sequences of this invention can be used in a variety of applications, including cloning, gene therapy, protein expression and purification, mutation introduction, DNA vaccination of hosts in need, antibody generation (for example, passive immunization), PCR, primer and probe generation, etc. In some embodiments, the nucleotide sequences of this invention comprise, or are substantially comprised of, the nucleotide sequences selected from those shown in Table 12. In some embodiments, the nucleotide sequences comprise nucleotide sequences having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with nucleotide sequences selected from those shown in Table 12. In some embodiments, the nucleotide sequence comprises a continuous nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with a continuous nucleotide sequence selected from the sequences shown in Table 12. In some 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 of these numbers) continuous nucleotides from a nucleotide sequence selected from the sequences shown in Table 12.

[0260] Those skilled in the art will also understand that antibodies suitable for the methods disclosed herein can be modified to differ in sequence from their naturally occurring or native origin, while retaining the desired activity of the native sequence. For example, nucleotide or amino acid substitutions resulting in conserved substitutions or alterations at “non-essential” amino acid residues can be performed. Mutations can be introduced using standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis.

[0261] Antibodies suitable for the methods disclosed herein may contain conserved amino acid substitutions at one or more amino acid residues, such as essential or non-essential amino acid residues. A “conserved amino acid substitution” is an amino acid substitution in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, non-essential amino acid residues in the binding polypeptide are preferably replaced by another amino acid residue from the same side chain family. In some embodiments, an amino acid segment may be replaced by a structurally similar segment that differs in sequence and / or the composition of side chain family members. Alternatively, in some embodiments, mutations may be randomly introduced along all or part of the coding sequence, for example by saturation mutagenesis, and the resulting mutants may be incorporated into the binding peptides of the present invention, and their ability to bind to desired targets may be screened.

[0262] As used in this article, the term antigen “cross-presentation” refers to the presentation of exogenous protein antigens to T cells via MHC class I and II molecules on APCs.

[0263] As used herein, the term "cross-reactivity" refers to the ability of an antibody of this disclosure to bind to IL-27 from a different species. For example, an antibody of this 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 antigens in a binding assay (e.g., SPR, ELISA), or by detecting binding to cells physiologically expressing IL-27 or otherwise functional interactions with said cells. Methods for determining cross-reactivity include the 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.

[0264] As used in this article, the term "cytotoxic T lymphocyte (CTL) response" refers to an immune response induced by cytotoxic T cells. The CTL response is primarily driven by CD8+ cells. + T cell mediated.

[0265] 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 the most potent antigen-presenting cell type. DCs capture and process antigens, converting proteins into peptides that are presented on the major histocompatibility complex (MHC) molecules recognized by T cells. DCs are heterogeneous, including myeloid and plasmacytoid DCs; although all DCs are capable of taking up, processing, and presenting antigens to naïve T cells, DC subtypes differ in their markers and vary in localization, migration pathways, detailed immune functions, and dependence on infectious or inflammatory stimuli (for which they are generated). In the development of adaptive immune responses, the phenotype and function of DCs play a role in initiating tolerance, memory, and polarization differentiation of T-helper cells 1 (Th1), Th2, and Th17.

[0266] As used herein, the term "dendritic cell activation" refers to the transformation from immature dendritic cells to mature dendritic cells; and activated dendritic cells include both mature dendritic cells and those undergoing transformation, wherein the expression of CD80 and CD86, which induce co-stimulatory signals, is increased by activation stimulation. Mature human dendritic cells are cells that are positive for the expression of CD40, CD80, CD86, and HLA class II (e.g., HLA-DR). For example, immature dendritic cells can be distinguished from mature dendritic cells based on markers selected from the group consisting of CD80 and CD86. Immature dendritic cells are weakly positive for these markers, preferably negative, while mature dendritic cells are positive. The differentiation of mature dendritic cells is routinely performed by those skilled in the art, and the respective markers and methods for measuring their expression are well known to those skilled in the art.

[0267] As used in this article, the term "EC" 50 "" refers to the concentration of an antibody or its antigen-binding moiety that induces a response of 50% of the maximum response (i.e., halfway between the maximum response and the baseline) in an in vitro or in vivo assay.

[0268] 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 "therapeutic effective dose" is defined as an amount sufficient to cure or at least partially stop the disease and its complications in a patient who already has the disease. The effective amount for this purpose depends on the severity of the condition being treated and the general state of the patient's own immune system.

[0269] As used herein, the term "epitope" or "antigenic determinant" refers to a site on an antigen where an immunoglobulin or antibody specifically binds. The term "epitope mapping" refers to a process or method for identifying binding sites or epitopes of an antibody or its antigen-binding fragment on its target protein antigen. This document provides methods and techniques for epitope mapping. Epitopes can be formed from consecutive amino acids or from non-consecutive amino acids arranged closely together by the ternary folding of a protein. Epitopes formed from consecutive amino acids are generally preserved upon exposure to denaturing solvents, while epitopes formed from ternary folds are generally lost upon treatment with denaturing solvents. Epitopes typically comprise at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in a distinctive spatial conformation. Methods for determining which epitopes a given antibody binds (i.e., epitope mapping) are well known in the art, including, for example, immunoblotting and immunoprecipitation assays, in which the reactivity of overlapping or consecutive peptides of IL-27 with a given anti-IL-27 antibody is tested. Methods for determining epitope spatial conformations include those in the art and those described herein, such as X-ray crystallography and two-dimensional nuclear magnetic resonance (see, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GEMorris, ed. (1996)).

[0270] This disclosure also includes antibodies that bind to epitopes on IL-27, said epitopes comprising all or part of the epitopes recognized by the specific antibodies described herein (e.g., the same or overlapping regions or regions between or across said regions).

[0271] This 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 demonstrate 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 an immunoglobulin-inhibiting reference antibody being tested specifically binds to a common antigen, such as IL-27. Many types of competitive binding assays are known, such as: solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), sandwich competitive assay (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 labeling assay, solid-phase direct labeling sandwich assay (see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988)); solid-phase direct labeling RIA using I-125 label (see Morel et al., Mol. Immunol. 25(1):7 (1988)); solid-phase direct biotin-avidin EIA (Cheung et al., Virology 176:546 (1990)); and directly labeled RIA. (Moldenhauer et al., Scand. J. Immunol. 32: 77 (1990)). Typically, this assay involves using a purified antigen or cells with one of these bound to a solid surface, an unlabeled test immunoglobulin, and a labeled reference immunoglobulin. Competitive inhibition is measured by determining the amount of the label bound to the solid surface or cells in the presence of the test immunoglobulin. An excess of the test immunoglobulin is usually present. Typically, when an excess of the competing antibody is present, it will inhibit the 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.

[0272] Other techniques include, for example, epitope mapping methods, such as X-ray analysis of antigen:antibody complex crystals, which provides atomic resolution of the epitopes, and mass spectrometry analysis with hydrogen / deuterium (H / D) exchange combinations, which studies the conformation and kinetics of antigen:antibody interactions. Other methods monitor the binding of antibodies to antigen fragments or mutated forms of antigens, where loss of binding due to modifications of amino acid residues in the antigen sequence is often considered an indicator of epitope components. Additionally, computational combinatorial methods for epitope mapping are also available. These methods rely on the ability of a target antibody to affinity-isolate specific short peptides from a combinatorial phage-displayed peptide library. The peptide is then considered as a precursor corresponding to an epitope definition used to screen the peptide library. Computational algorithms have also been developed for epitope mapping, which have been shown to map conformationally discontinuous epitopes.

[0273] As used herein, the term "Fc-mediated effector function" or "Fc effector function" refers to the biological activity of an antibody other than its primary function and purpose. 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 the Fcγ receptor. In some embodiments, tumor antigen-targeting antibodies have effector functions, such as ADCC activity. In some embodiments, the tumor antigen-targeting 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.

[0274] As used herein, the term "Fc receptor" refers to a polypeptide found on the surface of immune effector cells that is bound to the Fc region of an antibody. In some embodiments, the Fc receptor is the Fcγ receptor. There are three subclasses of Fcγ receptors: FcγRI (CD64), FcγRII (CD32), and FγcRIII (CD16). All four IgG isotypes (IgG1, IgG2, IgG3, and IgG4) bind to and activate Fc receptors FcγRI, FcγRII, and FcγRIIIA. FcγRII is an inhibitory receptor, therefore antibodies bound to this receptor do not activate complement and cellular responses. FcγRI is a high-affinity receptor that binds to IgG in monomeric form, while FcγRII and FcγRII are low-affinity receptors that bind to IgG only in polymeric form with slightly lower affinity. The binding of an antibody to the Fc receptor and / or C1q is controlled by specific residues or domains within the Fc region. Binding also depends on residues located within the hinge region and the CH2 portion of the antibody. In some embodiments, the agonistic and / or therapeutic activity of the antibodies described herein depends on the binding of the Fc region to an Fc receptor (e.g., FcγR). In some embodiments, the agonistic and / or therapeutic activity of the antibodies described herein is enhanced by the binding of the Fc region to an Fc receptor (e.g., FcγR).

[0275] A list of some Fc receptor sequences used in this disclosure is shown in Table 13 below.

[0276] As used herein, the term "glycosylation pattern" is defined as a pattern covalently linked to carbohydrate units of a protein, and more specifically, to an immunoglobulin. The glycosylation pattern of a heterologous antibody can be characterized as substantially similar to the glycosylation pattern naturally present on antibodies produced from non-human transgenic animal species, whereby those skilled in the art will recognize that the similarity of the heterologous antibody's glycosylation pattern to the glycosylation pattern in non-human transgenic animal species is greater than its similarity to the species from which the transgenic CH gene originates.

[0277] As used herein, the term “human antibody” includes antibodies having variable and constant regions (if any) of a human immunoglobulin sequence. Human antibodies of this disclosure may include amino acid residues not encoded by a human immunoglobulin sequence (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo) (see, for example, 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 the CDR sequence of a lineage derived from another mammal species (such as a mouse) has been transplanted onto a human frame sequence (i.e., humanized antibodies).

[0278] As used herein, the term "heterologous antibody" is defined relative to the transgenic nonhuman organism from which such antibody is produced. The term refers to an antibody having an amino acid sequence or a nucleic acid-coding sequence that corresponds to an amino acid sequence or nucleic acid-coding sequence found in an organism not composed of transgenic nonhuman animals, and typically originates from a species other than transgenic nonhuman animals.

[0279] The terms “inducing an immune response” and “enhancing an immune response” are used interchangeably and refer to stimulation (i.e., passive or adaptive) of an immune response to a specific antigen. The term “induction” used in relation to inducing CDC or ADCC refers to stimulation of a specific direct cell-killing mechanism.

[0280] As used herein, the term "immunogenic cell death" (or "immunogenic apoptosis") refers to a mode of cell death associated with the activation of one or more signaling pathways that induce the pre-death expression and release of damage-associated molecular pattern (DAMP) molecules (e.g., adenosine triphosphate, ATP) from tumor cells, leading to enhanced tumor cell immunogenicity and tumor cell death in an immunogenic manner (e.g., via phagocytosis). As used herein, the term "immunogenic cell death inducer" refers to a chemical, biological, or pharmaceutical agent that induces a process, pathway, or mode of immunogenic cell death.

[0281] As used herein, the terms “inhibition,” “reduction,” or “blocking” (e.g., referring to the inhibition or reduction of human IL-27-mediated STAT1 and / or STAT3 phosphorylation in cells) are used interchangeably and include partial and complete inhibition / blocking. Inhibition / blocking of IL-27 reduces or alters the normal level or type of activity that occurs in the absence of inhibition or blocking. Inhibition and blocking are also intended to include any measurable reduction in the binding affinity of IL-27 when contacted with an anti-IL-27 antibody compared to IL-27 not contacted with an anti-IL-27 antibody, for example, inhibition of IL-27 binding by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0282] As used herein, the terms “inhibit angiogenesis,” “reduce angiogenesis,” and “reduce angiogenesis” refer to reducing the level of angiogenesis in a tissue to an amount that is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or lower than that in a corresponding control tissue, and most preferably the same as the level observed in a control tissue.

[0283] As used herein, the term “growth inhibition” (e.g., involving cells) is intended to include any measurable reduction 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%.

[0284] As used herein, “preventable,” “treatment-required,” or “required” subjects are those who, according to the judgment of an appropriate practicing physician (e.g., a doctor, nurse, or caregiver in the case of a human; a veterinarian in the case of a nonhuman mammal), would reasonably benefit from a given treatment (such as treatment with a composition containing an anti-IL-27 antibody).

[0285] The term "in vivo" refers to processes that occur within a living organism.

[0286] As used herein, the term "isolated antibody" refers to an antibody that is substantially free of other antibodies with different antigen specificities (e.g., an isolated antibody that specifically binds to and antagonizes human IL-27 is substantially free of antibodies that specifically bind to antigens other than IL-27). However, isolated antibodies that specifically bind to epitopes may be cross-reactive with other IL-27 proteins from different species. However, in the specific binding assays described herein, the antibody continues to show specific binding to human IL-27. Additionally, isolated antibodies are generally substantially free of other cellular material and / or chemicals. In some embodiments, combinations of "isolated" antibodies with different IL-27 specificities are combined in a well-defined composition.

[0287] As used herein, the term "isolated nucleic acid molecule" refers to an antibody or antibody motif that encodes an antibody or antibody that binds to IL-27 (e.g., V). H V L Nucleic acids (CDR3) are defined as those in which the nucleotide sequence encoding an antibody or antibody moiety does not contain nucleotide sequences encoding an antibody or antibody moiety that binds to an antigen other than IL-27, said other sequences being naturally located flanking the nucleic acid in human genomic DNA. For example, sequences selected from those shown in Table 12 correspond to the heavy chain (V) of a monoclonal antibody containing the anti-IL-27 antibody described herein. H ) and light chains (V L ) The nucleotide sequence of the variable region.

[0288] As used herein, “isotype” refers to an antibody class (such as IgM or IgG1) encoded by a heavy chain constant region gene. In some embodiments, the human monoclonal antibody of this disclosure is an IgG1 isotype. In some embodiments, the human monoclonal antibody of this disclosure is an IgG2 isotype. In some embodiments, the human monoclonal antibody of this disclosure is an IgG3 isotype. In some embodiments, the human monoclonal antibody of this disclosure is an IgG4 isotype. As will be apparent to those skilled in the art, the identification of antibody isotypes (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE) is routine in the art and typically involves a combination of sequence alignments with known antibodies, disclosed Fc variant sequences, and conserved sequences.

[0289] As used in this article, the term "isotype switching" refers to the phenomenon where the type or isotype of an antibody changes from one type of Ig to another.

[0290] As used in this article, the term "KD" or "K" refers to... D "K" refers to the equilibrium dissociation constant of the binding reaction between antibody and antigen. DThe value of K is a numerical representation of the ratio of the antibody dissociation rate constant (kd) to the antibody binding rate constant (ka). D The value of K is inversely proportional to the antibody's affinity for the antigen. D The smaller the value, the greater the affinity of the antibody for its antigen. Affinity is the strength of the binding between a single molecule and its ligand, usually determined by the equilibrium dissociation constant (K0). D This constant is used to measure and report the ordering strength of bimolecular interactions.

[0291] As used in this article, the term "kd" or "k" d (or "koff" or "k") off The value of kd is the dissociation rate constant of an antibody from an antibody / antigen complex. kd is expressed as a numerical value representing the fraction of the complex that decays or dissociates per second, in seconds. -1 Expressed in units.

[0292] As used in this article, the term "ka" or "k" a (or “kon” or “k”) on The term "ka" refers to the binding rate constant of antibody-antigen association. The value of ka is a numerical representation of the number of antibody / antigen complexes formed per second in 1 mole (1M) of antibody and antigen solution, expressed in M. -1 Second -1 Expressed in units.

[0293] As used in this article, the term "leukocyte" refers to a type of white blood cell that participates in protecting the body from infectious organisms and foreign substances. Leukocytes are produced in the bone marrow. There are five main types of leukocytes, divided into two main categories: polymorphonuclear leukocytes (neutrophils, eosinophils, and basophils) and monocytes (monocytes and lymphocytes).

[0294] As used in this article, the term "lymphocyte" refers to a type of white blood cell or leukocyte that participates in the body's immune defense. There are two main types of lymphocytes: B cells and T cells.

[0295] As used herein, the terms “connected,” “fused,” or “fusion” are used interchangeably. These terms refer to the linking of two or more elements, components, or domains together by any means, including chemical conjugation or recombination. Methods of chemical conjugation (e.g., using heterobifunctional crosslinking agents) are known in the art.

[0296] As used herein, “topical application” or “topical delivery” refers to the delivery of a drug that does not rely on the transport of the composition or agent through the vascular system to its intended target tissue or site. For example, the composition or agent may be delivered by injection or implantation, or by injection or implantation of a device containing the composition or agent. After topical application near the target tissue or site, the composition or agent, or one or more components thereof, may diffuse to the intended target tissue or site.

[0297] As used in this article, "MHC molecules" refers to two types of molecules: MHC class I and MHC class II. MHC class I molecules present antigens to specific CD8+ T cells, while MHC class II molecules present antigens to specific CD4+ T cells. Exogenously delivered antigens to APCs are primarily processed for association with APC class II. Conversely, endogenously delivered antigens to APCs are primarily processed for association with MHC class I.

[0298] As used herein, the term "monoclonal antibody" refers to an antibody that exhibits a single binding specificity and affinity for a specific epitope. Therefore, the term "human monoclonal antibody" refers to an antibody that exhibits a single binding specificity and has variable regions and optional constant regions derived from human germline immunoglobulin sequences. In some embodiments, the human monoclonal antibody is produced by a hybridoma comprising B cells derived from a transgenic nonhuman animal (e.g., a transgenic mouse) fused with immortalized cells, the animal having a genome containing human heavy chain transgenes and light chain transgenes.

[0299] As used in this article, the term "monocyte" refers to a type of white blood cell that can differentiate into macrophages and dendritic cells to achieve an immune response.

[0300] As used in this article, the term "natural killer (NK) cells" refers to a type of cytotoxic lymphocyte. These are large, typically granular, non-T, non-B lymphocytes that kill certain tumor cells and play an important role in innate immunity against viruses and other intracellular pathogens, as well as in antibody-dependent cell-mediated cytotoxicity (ADCC).

[0301] As used herein, the term "naturally occurring" applied to objects refers to the fact that an object can exist in nature. For example, polypeptide or polynucleotide sequences present in organisms (including viruses) are naturally occurring, meaning they can be isolated from natural sources and have not been intentionally modified in a laboratory setting.

[0302] As used herein, the term "non-converting isotype" refers to the isotype class of the heavy chain produced when allotype conversion has not occurred; the CH gene encoding the non-converting isotype is typically the first CH gene immediately downstream of the functionally rearranged VDJ gene. Allotype conversion is classified into classical and non-classical allotype conversion. Classical allotype conversion occurs through recombination events involving at least one conversion sequence region in the transgene. Non-classical allotype conversion can occur, for example, through human σ... μ With people ∑ μ Homologous recombination (δ-related deletion) occurs between genes. Alternative non-classical conversion mechanisms, such as intergenetic and / or interchromosomal recombination, can occur and achieve isotype conversion.

[0303] As used herein, the term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides and their polymers existing in single-stranded or double-stranded form. Unless specifically defined, the term includes nucleic acids containing known natural nucleotide analogs, having similar binding properties to a reference nucleic acid, and being metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly includes variants of its conserved modifications (e.g., degenerate codon substitutions) and complementary sequences, as well as explicitly stated sequences. 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 a mixture of 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 can also be conserved. The term nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene.

[0304] The polynucleotides used herein can consist of any polynucleotide or polydeoxynucleotide, which can be unmodified RNA or DNA or modified RNA or DNA. For example, polynucleotides can consist of single-stranded and double-stranded DNA, DNA consisting of a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, and RNA consisting of a mixture of single-stranded and double-stranded regions, or hybrid molecules containing DNA and RNA that may be single-stranded regions or, more commonly, double-stranded regions or a mixture of single-stranded and double-stranded regions. Additionally, polynucleotides can consist of triple-stranded regions containing RNA or DNA or RNA and DNA. Polynucleotides may also contain one or more modified bases or a DNA or RNA backbone modified for stability or other reasons. "Modified" bases include, for example, triphenylmethylated bases and rare bases such as inosine. DNA and RNA can be modified in various ways; therefore, "polynucleotide" includes forms with chemical, enzymatic, or metabolic modifications.

[0305] When a nucleic acid is in a functional relationship with another nucleic acid sequence, it is "operably linked." For example, if a promoter or enhancer affects the transcription of a sequence, it is operably linked to a coding sequence. In the case of transcriptional regulatory sequences, operably linked means that the linked DNA sequences are contiguous and, where necessary, two protein-coding regions are linked (the two protein-coding regions are contiguous and within the reading frame). For conversion sequences, operably linked means that the sequence is capable of conversion and recombination.

[0306] As used in this article, “parenteral application”, “for parenteral”, and other grammatically equivalent phrases refer to administration methods other than enteral and local application, usually by injection, including but not limited to intravenous, intranasal, intraocular, intramuscular, intraarterial, intrasheath, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, spinal, epidural, intracerebral, intracranial, intracarotid artery, and intrasternal injection and infusion.

[0307] As used in this article, the term "patient" includes people receiving preventative or therapeutic treatment and other mammalian subjects.

[0308] As used herein, the term "PD-1 antagonist" refers to any compound or biomolecule that inhibits the PD-1 signaling pathway or otherwise suppresses PD-1 function in cells (e.g., immune cells). In some embodiments, PD-1 antagonists block the binding of PD-L1 to PD-1 and / or PD-L2 to PD-1. In some embodiments, PD-1 antagonists specifically bind to PD-1. In some embodiments, PD-1 antagonists specifically bind to PD-L1.

[0309] In the context of two or more nucleic acid or polypeptide sequences, the term "percentage of identity" refers to two or more sequences or subsequences that, when compared and aligned for maximum similarity, have a specific percentage of identical nucleotides or amino acids, as measured visually using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to those skilled in the art). Depending on the application, the "percentage of identity" may exist over a region of the sequences being compared, such as a functional domain, or over the full length of the two sequences to be compared. For sequence comparison, typically, one sequence serves as a reference sequence to be compared with the test sequence. When using a sequence comparison algorithm, the test and reference sequences are input into a computer, and if necessary, subsequence coordinates are specified, along with the sequence algorithm program parameters. The sequence comparison algorithm then calculates the percentage of sequence identity of one or more test sequences relative to the reference sequence based on the specified program parameters.

[0310] The best 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 similarity search method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85: 2444 (1988), by the computerized execution 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., hereinafter).

[0311] One example of an algorithm suitable for determining sequence identity percentage and sequence similarity is the BLAST algorithm, described in Altschul et al., J.Mol.Biol.215:403-410 (1990). Software for performing BLAST analysis is publicly available from the website of the National Center for Biotechnology Information.

[0312] As is commonly used in this article, “pharmaceutical acceptable” means, to the extent reasonably medically permissible, compounds, materials, compositions and / or dosage forms that are suitable for use in contact with the tissues, organs and / or body fluids of humans and animals without excessive toxicity, irritation, allergic reactions or other problems or complications commensurate with a reasonable benefit / risk ratio.

[0313] As used herein, “pharmaceutically acceptable carrier” means and includes any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delay agents. The composition may include pharmaceutically acceptable salts, such as acid addition salts or base addition salts (see, for example, Berge et al. (1977) J Pharma Sci.). 66 (1-19)

[0314] As used herein, the terms “polypeptide,” “peptide,” and “protein” are used interchangeably to refer to polymers of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acids, as well as to both naturally occurring and non-naturally occurring amino acid polymers.

[0315] As used herein, the term “prevention”, when used in conjunction with disease, refers to the administration of a composition that reduces the frequency of symptoms of a subject’s disease or delays the onset of such symptoms relative to a subject who has not received the composition.

[0316] As used herein, the terms “purified” or “isolated” for any protein (antibody or fragment) described herein refer to a polypeptide isolated or purified from its naturally occurring accompanying components (e.g., proteins or other naturally occurring biological or organic molecules) (e.g., other proteins, lipids, and nucleic acids in prokaryotes expressing said protein). Typically, a polypeptide is purified when it constitutes at least 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, or 99% by weight) of the total protein in a sample.

[0317] As used herein, the term “programmed cell death protein 1” or “PD-1” refers to the programmed cell death protein 1 polypeptide (an immunosuppressive receptor belonging to the CD28 family) 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, isotypes, and species homologs of hPD-1, as well as analogs that share at least one common epitope with hPD-1. The complete hPD-1 sequence is available in GenBank accession number AAC51773.

[0318] As used herein, the term “programmed death-ligand-1” or “PD-L1” refers to one of the two cell surface glycoprotein ligands of PD-1 (the other being PD-L2), which downregulates T cell activation and cytokine secretion upon binding to PD-1. Alternative names and synonyms for PD-L1 include: PDCD1L1, PDL1, B7H1, B7-4, CD274, and B7-H. The term “PD-L1” as used herein includes human PD-L1 (hPD-L1), variants, isotypes, and species homologs of hPD-L1, as well as analogs sharing at least one common epitope with hPD-L1. The complete hPD-L1 sequence is available in GenBank accession number Q9NZQ7.

[0319] 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 29 December 2006) 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 between PD-1 and PD-L1 or PD-L2; when the interaction between PD-1 and PD-L2 is also blocked, the effect is additive (Iwai et al. (2002) Proc. Nat'l. Acad. Sci. USA 99: 12293-7; Brown et al. (2003) J. Immunol. 170: 1257-66).

[0320] For several cancers, tumor survival and proliferation are maintained by tumor-mediated immune checkpoint regulation. This regulation can lead to the disruption of the anti-cancer immune system. For example, recent studies have shown that tumor cells expressing immune checkpoint receptor ligands, such as PD-L1 or PD-L2, can downregulate immune system activity in the tumor microenvironment and promote cancer immune evasion, specifically by suppressing T cells. PD-L1 is highly expressed in a variety of human cancers (Dong et al., (2002) Nat Med 8: 787-789). The PD-L1 receptor PD-1 is expressed on lymphocytes (e.g., activated T cells) and is generally involved in downregulating the immune system (specifically by suppressing T cells) and promoting self-tolerance. However, when the PD-1 receptor expressed on T cells binds to its homologous PD-L1 ligand on tumor cells, the resulting T cell suppression leads to impaired immune responses against the tumor (e.g., reduced tumor-infiltrating lymphocytes or cancer cells establishing immune evasion).

[0321] In large-scale studies of cancers such as ovarian cancer, renal cancer, colorectal cancer, pancreatic cancer, liver cancer, and melanoma, PD-L1 expression has been shown to be associated with poor prognosis and reduced overall survival, regardless of subsequent treatment (see, for example, 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, the discovery of PD-1 expression on tumor lymphocytes has been shown to be a marker of 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 new class of antitumor inhibitors that act by modulating immune cell-tumor cell interactions.

[0322] As used herein, the term "rearranged" refers to the configuration of a heavy or light chain immunoglobulin locus in which the V segment encodes substantially complete V. H or V L The conformation of the domain is adjacent to the DJ or J segment. Rearranged immunoglobulin loci can be identified by comparison with germline DNA; the rearranged loci will have at least one recombinant heptamer / nonamer homologous element.

[0323] 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 this term refers not only to the specific test cell but also to the progeny of such cells. Because certain modifications can 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.

[0324] 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, which are transgenic or transchromosomal with respect to human immunoglobulin genes, (b) antibodies isolated from host cells transformed to express the antibody, such as from transfected tumors, (c) antibodies isolated from recombinant human antibody libraries, and (d) antibodies prepared, expressed, generated, or isolated by any other means involving splicing human immunoglobulin gene sequences into other DNA sequences. Such recombinant human antibodies contain variable and constant regions utilizing specific human germline immunoglobulin sequences encoded by germline genes, but including rearrangements and mutations that subsequently occur, for example, during antibody maturation. As is known in the art (see, for example, Lonberg (2005) Nature Biotech. 23(9): 1117-1125), the variable region contains an antigen-binding domain encoded by various genes that rearrange to form antibodies specific to foreign antigens. In addition to rearrangements, the variable region can be further modified by multiple single amino acid changes (called somatic mutations or hypermutations) to increase the antibody's affinity for foreign antigens. 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 respond to antigen rearrangements and somatic mutations may not have exactly the same sequence as the original nucleic acid molecule, but rather have substantially the same or similar sequence (i.e., at least 80% identity).

[0325] As used herein, the term "reference antibody" (which may be used interchangeably 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 serves to establish a relationship between itself and one or more different antibodies, wherein said relationship is the binding of the reference antibody and one or more different antibodies to the same epitope on IL-27. As used herein, the term refers to an anti-IL-27 antibody that can be used to test or assay, such as those described herein (e.g., competitive binding assays), as a competitor, wherein said assays can be used to discover, identify, or develop one or more different antibodies that bind to the same epitope.

[0326] 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. Generally, when recombinant human IL-27 is used as the analyte and the antibody as the ligand in a BIACORE 2000 instrument via surface plasmon resonance (SPR) technology, the antibody binds at approximately 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 The antibody binds to a predetermined antigen with an affinity at least twice that of a nonspecific antigen (e.g., BSA, casein) other than the predetermined antigen or closely related antigens. In some embodiments, when using recombinant human IL-27 as an analyte and the antibody as a ligand to be measured in a BIACORE 2000 instrument by surface plasmon resonance (SPR) technology, the antibody specifically bound to IL-27 at a concentration of less than about 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 10⁻⁶ m). -8 M), optionally less than about 10 nM (10 -8 M), optionally less than about 5nM (5x10 -9 M), optionally less than about 1 nM (10 -9 M), optionally less than about 0.1nM(10 -10 M), optionally less than about 0.01nM (10 -11 M) or even smaller equilibrium dissociation constant (K) D The binding of an antibody to a predetermined antigen occurs at least twice 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 “antibody that recognizes an antigen” and “antibody specific to an antigen” are used interchangeably herein with the term “antibody that specifically binds to an antigen.”

[0327] As used herein, the term “STAT1 phosphorylation” refers to the phosphorylation of the signal transducer and transcription activator 1 (STAT1) polypeptide (a transcription factor encoded by the human STAT1 gene). STAT molecules are phosphorylated by receptor-associated kinases, leading to activation and dimerization by forming homodimers or heterodimers, which translocate to the cell nucleus to function as transcription factors. STAT1 can be activated (i.e., phosphorylated) in response to signal transduction by several ligands, including IL-27. IL-27 signals via IL-27R, resulting in STAT1 phosphorylation (pSTAT1). STAT1 plays a crucial role in gene expression involved in cell survival, viability, or pathogen response. Methods for determining STAT1 phosphorylation as a result of IL-27 signaling include, but are not limited to, flow cytometry analysis of cells labeled with antibodies that specifically recognize phosphorylated STAT1 (see, for example, Tochizawa et al., (2006) J Immunol Methods 313(1-2): 29-37).

[0328] As used herein, the term “STAT3 phosphorylation” refers to the phosphorylation of the signal transducer and activator of transcription 3 (STAT3) polypeptide (a transcription factor encoded by the human STAT3 gene). STAT3 mediates the expression of a variety of genes in response to cellular stimuli and thus plays a key role in many cellular processes such as cell growth and apoptosis. Methods for determining STAT3 phosphorylation as a result of IL-27 signaling include, but are not limited to, analysis of cells or cell extracts labeled with antibodies that specifically recognize phosphorylated STAT3 (see, for example, Fursov et al., (2011) Assay Drug Dev Technol 9(4): 420-429).

[0329] As used herein, the term "transformation sequence" refers to those DNA sequences responsible for the transformation recombination. The "transformation donor" sequence, typically the μ-transformation region, will be the 5' (i.e., upstream) of the construct region that is deleted during the transformation recombination process. The "transformation acceptor" region will be located between the construct region to be deleted and the replacement constant region (e.g., γ, ε, etc.). Because there is no specific site where recombination always occurs, the final gene sequence is generally unpredictable from the construct.

[0330] As used herein, the term "subject" includes any human or non-human animal. For example, the methods and compositions of the present invention can be used to treat subjects suffering from immune disorders. The term "non-human animal" includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cattle, chickens, amphibians, reptiles, etc.

[0331] For nucleic acids, the term "substantial homology" means that two nucleic acids or their designated sequences are identical in at least about 80% of their nucleotides, typically at least about 90% to 95%, more preferably at least about 98% to 99.5%, when optimally aligned and compared, with appropriate nucleotide insertions or deletions. Alternatively, substantial homology exists when a segment hybridizes with a complementary sequence of the strand under selective hybridization conditions.

[0332] The percentage of identity between 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. The comparison of sequences and the determination of the percentage of identity between two sequences can be accomplished using mathematical algorithms as described in the following non-limiting examples.

[0333] The percentage of identity between two nucleotide sequences can be determined using the GAP procedure in the GCG software package (available at http: / / www.gcg.com), using the NWSgapdna.CMP matrix and gap weights of 40, 50, 60, 70, or 80 and length weights of 1, 2, 3, 4, 5, or 6. The percentage of 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 weighted residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the percentage of identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. (48): 444-453 (1970)) algorithm in the GAP program incorporated into the GCG software package (available at http: / / www.gcg.com), using a Blossum62 matrix or a PAM250 matrix, gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0334] 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 relevant sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al. (1990) J.Mol.Biol.215:403-10. BLAST nucleotide searches can be performed using the NBLAST program with a score of 100 and a word length of 12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the present invention. BLAST protein searches can be performed using the XBLAST program with a score of 50 and a word length of 3 to obtain amino acid sequences homologous to the protein molecules of the present invention. To obtain nicked alignments for comparative purposes, nicked BLAST can be used as described in Altschul et al., (1997) Nucleic Acids Res.25(17):3389-3402. When using BLAST and nicked BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http: / / www.ncbi.nlm.nih.gov.

[0335] Nucleic acids can exist in whole cells, cell lysates, or in partially purified or substantially pure forms. Nucleic acids are “isolated” or “substantially purified” when purified from other cellular components or other contaminants (e.g., other cellular nucleic acids or proteins) using 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).

[0336] The nucleic acid compositions of this disclosure (although typically natural sequences, except for modified restriction sites, etc.) derived from cDNA, genome, or mixtures thereof can be mutated using standard techniques that provide the gene sequence. For coding sequences, these mutations may affect the amino acid sequence as needed. In particular, DNA sequences substantially homologous to or derived from natural V, D, J, constant, transformed sequences, and other such sequences described herein are contemplated (where “derived from” means the sequence is identical to or modified from another sequence).

[0337] As used herein, the term “STING” (or TMEM173) refers to the stimulator of the interferon gene, a protein that functions as both a direct cytosol DNA sensor and an adaptor protein. In humans, STING is encoded by the TMEM173 gene. STING plays an important role in innate immunity. When cells are infected by 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 and nearby cells from local infection by binding to the same cell that secretes it and nearby cells. An exemplary amino acid sequence of STING is available in the NCBI Genbank database with accession number NP_001288667.

[0338] The term "T cell" refers to a type of white blood cell that is distinguished from other white blood cells by the presence of T cell receptors on its cell surface. There are several subgroups 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 cells 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 cells) C Cells, CD8 + T cells, cytotoxic T lymphocytes, T-killers, cytotoxic T cells, memory T cells, and subtypes, including central memory T cells (T cells). CM T cells), effector memory T cells (T cells) EM and T ERMA (cells) and resident memory T cells (T cells) RM T cells), regulatory T cells (also known as T cells) reg Cells (or suppressor T cells) and their subtypes, including CD4 + FOXP3 + T reg Cells, CD4 + FOXP3 - T reg Cells, Tr1 cells, Th3 cells and T cells 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 / Vδ2 T cells. Any one or more of the foregoing or not mentioned T cells may be the target cell type for use in the methods of use of this invention.

[0339] 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+ cells) and their components. + Any response mediated by a T cell. T cell-mediated responses include, for example, T cell cytotoxicity and proliferation.

[0340] As used herein, the terms “therapeutic effective amount” or “therapeutic effective dose” or similar terms used herein are intended to indicate the amount of an agent (e.g., an anti-IL-27 antibody or its antigen-binding fragment) that will elicit a desired biological or medical response (e.g., improvement of one or more symptoms of cancer).

[0341] 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 role, controlling the initiation and progression of tumor development while simultaneously controlling the relevant antitumor responses of different immune cells. Further description of TAM receptors can be 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.

[0342] As used herein, unless otherwise stated, the term "TIGIT" or "T-cell immune receptor with Ig and ITIM domains" refers to any naturally occurring TIGIT from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). TIGIT is also known in the art as DKFZp667A205, FLJ39873, protein 9 containing V-set and immunoglobulin domains, protein 3 containing V-set and transmembrane domains, VSIG9, VSTM3, and WUCAM. The term also includes naturally occurring variants of TIGIT, such as splice variants or allelic variants. An exemplary amino acid sequence of human TIGIT can be found under UniProt accession number Q495A1.

[0343] As used herein, the terms “treat,” “treating,” and “treatment” refer to treatment or preventative measures. A method of “treatment” involves administering the human antibody of this disclosure to a subject who requires such treatment (e.g., a subject requiring an enhanced immune response against a specific antigen or a subject who may eventually acquire the condition) to prevent, cure, delay, reduce the severity of said condition or recurrent condition, or improve one or more of its symptoms, or prolong the subject’s survival beyond what would have been expected without such treatment.

[0344] 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 growth 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 the extracellular matrix also constitute the TME. Tumors and their surrounding microenvironment are closely related and constantly interact. Tumors can influence the microenvironment by releasing extracellular signals, promoting tumor angiogenesis, and inducing peripheral immune tolerance, while immune cells within the microenvironment can influence the growth and evolution of tumor cells.

[0345] As used herein, the term “unrearranged” or “phylogenetic configuration” refers to the configuration of the V segment in which the V segment does not recombine and is thus closely adjacent to the D or J segment.

[0346] As used herein, the term "vector" is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a "plasmid," which is a circular double-stranded DNA loop into which an additional DNA segment can be linked. Another type of vector is a viral vector, in which an additional DNA segment can be linked to a viral genome. Some vectors are capable of autonomous replication in host cells to which they are introduced (e.g., bacterial vectors with bacterial replication origin sites and free mammalian vectors). Other vectors (e.g., non-free mammalian vectors) can be integrated into the host cell's genome upon introduction into the host cell, thereby replicating along with the host genome. Furthermore, some vectors are capable of directing the expression of genes operatively linked to them. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). Typically, expression vectors useful in recombinant DNA technology are in the form of plasmids. In this specification, "plasmid" and "vector" are used interchangeably because plasmids are the most commonly used form of vector. However, the present invention is intended to include other forms of expression vectors, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), which have equivalent functions.

[0347] 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 pertains. Preferred methods and materials are described below, although similar or equivalent methods and materials may also be used in the practice or testing of the methods and compositions currently disclosed. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. Attached Figure Description

[0348] Figure 1 This is a table providing affinity data for the anti-IL-27 antibody, as indicated. Affinity measurements were performed using the ForteBio and MesoScale Discovery methods.

[0349] Figure 2 It is a graph depicting the binding of anti-IL-27 antibody to recombinant IL-27, as indicated and as measured by ELISA.

[0350] Figures 3A-3E These are bar charts, two-graphs, bar charts, and line charts. Figure 3A This is a graph depicting the inhibition of IL-27-mediated STAT1 phosphorylation in human whole blood by anti-IL-27 antibodies, as indicated and measured by flow cytometry. Figure 3B This is a graph depicting the inhibition of IL-27-mediated STAT1 phosphorylation in human PBMCs by anti-IL-27 antibodies, as indicated and measured by flow cytometry. Figure 3C This is a graph depicting the inhibition of IL-27-mediated STAT1 phosphorylation in U937 cells by anti-IL-27 antibody, as indicated and measured by flow cytometry. Figure 3D This is a graph depicting the inhibition of IL-27-mediated STAT1 phosphorylation in HUT-78 cells by anti-IL-27 antibody, as indicated and measured by flow cytometry. Figure 3E This is a diagram showing SRF388 inhibiting IL-27-mediated pSTAT1 in human whole blood T cells.

[0351] Figure 4 This is a graph depicting the reversal of IL-27-mediated inhibition of CD161 expression in T cells by a series of anti-IL-27 antibody concentrations as indicated. CD161 expression was measured by flow cytometry.

[0352] Figure 5A This 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 5BThis is a graph depicting the extent to which anti-IL-27 antibodies, as measured by ELISA, increase PD-1-mediated IL-6 secretion in human PBMCs. Figure 5C This is a dot plot showing that SRF388 combined with PD-1 blockade leads to increased cytokine production in PBMCs of healthy donors and RCC patients (abbreviations: CBA = flow cytometry bead array, IFNγ = interferon-γ, MSD = Mesoscale Discovery, PBMC = peripheral blood mononuclear cells, PD-1 = programmed death receptor-1, RCC = renal cell carcinoma, TNFα = tumor necrosis factor-α). Figure 5D The results showed that IL-27 inhibited cytokine production after PD-1 blockade, and this inhibition was restored when combined with SRF388 (abbreviations: Ctrl = control, ns = not significant, PBMC = peripheral blood mononuclear cells, rhIL-27 = recombinant human IL-27). Figure 5E This paper summarizes the observed cytokine induction (specifically, TNFα, IFNγ, IL-6, and IL-17A) in PBMC cultures of various specified cell types when exposed to SRF388 antibody, αPD-1 antibody, or a combination of SRF388 and αPD-1 antibody. Figure 5F The effects of different concentrations of designated single antibodies (SRF405, SRF410, SRF411, SRF414, SRF416, SRF536, SRF543, SRF529, SRF381, SRF388 and Ab7) on pSTAT1 signaling in U937 (lymphoma) cells were shown. Figure 5G The effects of different concentrations of these individual antibodies on pSTAT1 signaling in PBMCs (peripheral blood mononuclear cells) were shown. Figure 5H The effects of different concentrations of these individual antibodies on CD161 signaling in PBMCs (peripheral blood mononuclear cells) were shown. Figure 5I The effects of different concentrations of these individual antibodies (excluding SRF414) on PD-L1 signaling in CD4 T lymphocytes (CD4 cells) were shown. Figure 5J The effects of different concentrations of these individual antibodies (excluding SRF529) on PD-L1 signaling in monocytes were shown. Figure 5K The effects of different concentrations of these individual antibodies (excluding SRF529) on TIM-3 signaling in monocytes were shown.

[0353] Figure 6A The figure depicts the inhibition of IL-27-mediated PD-L1 expression produced by treating human monocytes with anti-IL-27 antibodies, as measured by flow cytometry. Figure 6BThis is a graph depicting the dose-dependent inhibition of IL-27-mediated PD-L1 expression produced by treating human monocytes with a series of concentrations of anti-IL-27 antibodies that specifically bind to EBI3 monomers, as determined by flow cytometry. Figure 6C It is a graph depicting the inhibition of IL-27-mediated TIM3 expression produced by treating human monocytes with anti-IL-27 antibodies, as measured by flow cytometry. Figure 6D The figure depicts the inhibition of IL-27-mediated PD-L1 expression produced by treating resting human T cells with an anti-IL-27 antibody, as measured by flow cytometry.

[0354] Figure 7A This is a dot plot that depicts, as indicated, 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 a combination of αPD-1 and αCTLA-4 antibodies, as determined by visual counting of nodules from the lungs isolated from mice. Figure 7B A graph is provided depicting the growth kinetics of bioluminescent B16-Luc tumors in mice treated with anti-IL-27 antibody (SRF388) or an isotype control antibody, as determined by bioluminescence imaging analysis. Figure 7C Images of a series of fixed, sectioned lung tissues stained with hematoxylin and eosin, as indicated, are shown, isolated from B16F10 tumor-bearing mice treated with anti-IL27 antibody (SRF388), isotype control antibody, αWSX-1 antibody, or a combination of αPD-1 and αCTLA-4 antibodies. Figure 7D This is a dot plot depicting, as indicated, the total tumor area, as determined by image analysis software, expressed as a percentage of the total tissue area of ​​fixed, eosin-stained, B16F10 lung tissue sections containing B16F10 tumor tissue isolated from B16F10 tumor-bearing mice treated with anti-IL27 antibody (SRF388), an allotype control antibody, αWSX-1 antibody, or a combination of αPD-1 and αCTLA-4 antibodies. Similar reductions in the number of surface lung metastases and total tumor area were observed for IL-27RA (WSX-1)-mediated antibody blockade and for the combination therapy of anti-PD-1 and anti-CTLA-4.

[0355] Figure 8A A scatter plot is provided, depicting microarray data of genes whose expression changes were greater than >1.0 log2 fold (black dots) in spleen cells isolated from IL-27-overexpressing mice after treatment with IL-27 microloops. Figure 8B Provided a description as indicated, such as Figure 8AA graph showing the expression levels of selected immune regulatory genes in spleen cells. Figure 8C Ectopic expression of human IL-27 induced the expression of inhibitory receptors on mouse T cells in vivo, and SRF388 reduced inhibitory receptor expression on T cells in vivo after treatment with IL-27 microcircuits. Six-week-old female Balb / c mice were injected with either an empty vector (control) or hIL-27 microcircuits. Five days post-transfection, PBMCs (top left and top right images) and total spleen cells (bottom left and bottom right images) were collected, stained, and analyzed by flow cytometry. The expression of indicated markers was analyzed on CD4+ T cells (top left and bottom left images) and CD8+ T cells (top right and bottom right images). Analysis was performed using FlowJo software. Figure 8D The results showed that SRF388 inhibited the detection of microcyclic human IL-27 in mouse plasma.

[0356] Figure 9 An overview of the selected monoclonal antibody properties is presented.

[0357] Figure 10A A graph of the antibody sequence is presented, where the sequence partitions reflect the NT number. Figure 10B A graph showing the antibody sequence (corresponding to) Figure 10A (Sequence chart), where sequence partitions reflect ImMunoGeneTics (IMGT) numbers. Figure 10A and Figure 10B In both cases, the amino acids highlighted in the CDR sequence indicate mutations from germline-coded sequences. As will be apparent to those skilled in the art, antibody numbering, including the determination of the CDR sequence, framework sequence, etc., can be performed in a variety of art-recognized methods, including through... Figure 10A and Figure 10B The NT and IMGT numbering systems presented here and used elsewhere in this document. Detailed Implementation

[0358] This disclosure provides, at least in part, antibody molecules that bind to human IL-27 with high affinity and specificity. In one embodiment, a human antibody binding to IL-27 is disclosed herein. As used herein, the terms "IL-27" and "IL27" interchangeably refer to the heterodimeric cytokine IL-27, which consists of two distinct subunits encoded by two distinct genes: Epstein-Barr virus-inducible gene 3 (EBI3) and IL-27p28. IL-27 possesses both pro-inflammatory and anti-inflammatory properties and has different effects on hematopoietic and non-hematopoietic cells.

[0359] Therefore, in one aspect, this disclosure provides a monoclonal antibody or antigen-binding moiety thereof that specifically binds to and antagonizes human IL-27, wherein said antibody or antigen-binding moiety thereof exhibits at least one or more of the following properties:

[0360] (i) with an equilibrium dissociation constant of 15 nM or less (K D It binds to human IL-27;

[0361] (ii) Block the binding of IL-27 to its receptor;

[0362] (iii) Inhibit or reduce STAT1 and / or STAT3 phosphorylation in cells;

[0363] (iv) Inhibit or reduce IL-27-mediated inhibition of CD161 expression in cells;

[0364] (v) Inhibit or reduce IL-27-mediated PD-L1 and / or TIM-3 expression in cells;

[0365] (vi) Inducing or enhancing the secretion of one or more PD-1-mediated cytokines from cells; and

[0366] Combinations of (vii)(i) to (vi).

[0367] In other respects, this 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.

[0368] Other aspects of the invention include nucleic acid molecules encoding antibody molecules, expression vectors, host cells, and methods for preparing antibody molecules. Immunoconjugates, multispecific 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., leukemia, e.g., lymphoma, e.g., AML), lung cancer (e.g., non-small cell lung cancer), pancreatic cancer, breast cancer (e.g., triple-negative breast cancer), melanoma, testicular cancer, sarcoma, head and neck cancer (e.g., squamous cell 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, e.g., clear cell renal carcinoma).

[0369] Anti-IL-27 antibody and its antigen-binding fragment

[0370] This disclosure provides antibodies that specifically bind to and antagonize IL-27, particularly human IL-27, and their antigen-binding moieties. This document provides isolated monoclonal antibodies that specifically bind to human IL-27, or their antigen-binding moieties, comprising heavy and light chain CDRs as shown in Table 12, and variable sequences.

[0371] In some embodiments, this disclosure provides isolated monoclonal antibodies or antigen-binding moieties thereof that specifically bind to and antagonize human IL-27, wherein said antibody or antigen-binding moieties exhibit at least one or more of the following properties: (i) having an equilibrium dissociation constant (K0) of 15 nM or less. D (i) binding to human IL-27; (ii) blocking the binding of IL-27 to the IL-27 receptor; (iii) inhibiting or reducing STAT1 and / or STAT3 phosphorylation in cells; (iv) inhibiting or reducing CD161 expression in cells; (v) inhibiting or reducing PD-L1 and / or TIM-3 expression in cells; (vi) inducing or increasing the secretion of one or more PD-1-mediated cytokines from cells; and (vii) combinations of (i) to (vi).

[0372] In some implementations, the isolated monoclonal antibody or its antigen-binding moiety has an equilibrium dissociation constant (K0) of 15 nM or less. D It binds to human IL-27.

[0373] In some implementations, the isolated monoclonal antibody or its antigen-binding moiety binds to recombinant human IL-27 or mouse IL-27.

[0374] In some embodiments, the isolated monoclonal antibody or its antigen-binding portion inhibits or reduces STAT1 and / or STAT3 phosphorylation in cells. In some embodiments, the cells are immune cells. In some embodiments, the cells are cancer cells.

[0375] In some embodiments, the isolated monoclonal antibody or its antigen-binding portion inhibits or reduces the inhibition of CD161 expression in cells (e.g., improves or alleviates the inhibition of CD161 expression in cells). In some embodiments, the cells are immune cells.

[0376] In some embodiments, the isolated monoclonal antibody or its antigen-binding portion inhibits or reduces the expression of PD-L1 and / or TIM-3 in cells. 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 and TIM-3 expression are reduced. In some embodiments, the cells are immune cells.

[0377] In some embodiments, the isolated monoclonal antibody or its antigen-binding portion induces or enhances the secretion of one or more PD-1-mediated cytokines from cells. 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 cells are immune cells.

[0378] In some embodiments, the isolated monoclonal antibody or its antigen-binding moiety 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, the mutant IgG4 heavy chain constant region comprises any one or a combination of the substitutions S228P, L235E, and L235A according to EU designations.

[0379] In some embodiments, this disclosure provides isolated monoclonal antibodies or antigen-binding portions thereof that bind substantially the same epitopes on IL-27 as the antibodies or antigen-binding portions thereof according to any of the foregoing embodiments.

[0380] In some embodiments, this disclosure provides isolated monoclonal antibodies or antigen-binding portions thereof, said monoclonal antibodies or antigen-binding portions thereof being bound to at least one of the amino acid residues containing IL-27 by an antibody or antigen-binding portion thereof according to any of the foregoing embodiments.

[0381] In some embodiments, this disclosure provides isolated monoclonal antibodies or antigen-binding portions thereof, wherein mutations in the epitopes on IL-27 bound by the antibody or antigen-binding portion thereof inhibit, reduce, or block binding to the antibody or antigen-binding portion thereof and to the antibody or antigen-binding portion thereof according to any of the foregoing embodiments.

[0382] In some embodiments, this disclosure provides isolated monoclonal antibodies or antigen-binding portions thereof that bind to epitopes on IL-27, wherein the epitopes are the same as or similar to the epitopes bound to the antibody molecules described in Table 12.

[0383] In some embodiments, this disclosure provides isolated monoclonal antibodies or antigen-binding moieties thereof that specifically bind to and antagonize human IL-27, wherein said antibody or antigen-binding moieties comprise heavy and light chain CDRs selected from the group consisting of:

[0384] (i) The heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 51, 52 and 53 respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 59, 60 and 61 respectively;

[0385] (ii) The heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 73, 74 and 75 respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 81, 82 and 83 respectively;

[0386] (iii) The heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 95, 96 and 97 respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 103, 104 and 105 respectively;

[0387] (iv) The heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 117, 118 and 119 respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 125, 126 and 127 respectively;

[0388] (v) The heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 139, 140 and 141, respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 147, 148 and 149, respectively;

[0389] (vi) The heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 161, 162 and 163 respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 169, 170 and 171 respectively;

[0390] (vii) The heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 185, 186 and 187 respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 193, 194 and 195 respectively;

[0391] (viii) The heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 207, 208 and 209 respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 215, 216 and 217 respectively;

[0392] (ix) The heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 229, 230 and 231 respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 237, 238 and 239 respectively;

[0393] (x) The heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 251, 252 and 253 respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 259, 260 and 261 respectively;

[0394] (xi) are the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 273, 274 and 275, respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 281, 282 and 283, respectively;

[0395] (xii) The heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 295, 296 and 297 respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 303, 304 and 305 respectively;

[0396] (xiii) The heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 317, 318 and 319 respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 325, 326 and 327 respectively;

[0397] (xiv) shows the heavy chain CDR1, CDR2 and CDR3 sequences in SEQ ID NO: 339, 340 and 341, respectively, and the light chain CDR1, CDR2 and CDR3 sequences in SEQ ID NO: 347, 348 and 349, respectively;

[0398] (xv) The heavy chain CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 361, 362, and 363, respectively, and the light chain CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 369, 370, and 371, respectively; and

[0399] (xvi) are the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 383, 384 and 385, respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 391, 392 and 393, respectively.

[0400] In some embodiments, this disclosure provides a species-specific monoclonal antibody or antigen-binding moiety thereof that specifically binds to and antagonizes human IL-27, wherein the antibody or antigen-binding moiety comprises heavy chain and light chain CDRs, wherein the heavy chain CDR1, CDR2 and CDR3 sequences are shown in SEQ ID NO: 161, 162 and 163, respectively, and the light chain CDR1, CDR2 and CDR3 sequences are shown in SEQ ID NO: 169, 170 and 171, respectively.

[0401] In some embodiments, this disclosure provides isolated monoclonal antibodies or antigen-binding moieties thereof that specifically bind to and antagonize human IL-27, wherein said antibody or antigen-binding moieties comprise heavy and light chain CDRs selected from the group consisting of:

[0402] (i) the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 54, 55 and 56 respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 62, 63 and 64 respectively;

[0403] (ii) The heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 76, 77 and 78 respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 84, 85 and 86 respectively;

[0404] (iii) The heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 98, 99 and 100 respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 106, 107 and 108 respectively;

[0405] (iv) The heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 120, 121 and 122 respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 128, 129 and 130 respectively;

[0406] (v) The heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 142, 143 and 144 respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 150, 151 and 152 respectively;

[0407] (vi) The heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 164, 165 and 166 respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 172, 173 and 174 respectively;

[0408] (vii) The heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 188, 189 and 190 respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 196, 197 and 198 respectively;

[0409] (viii) The heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 210, 211 and 212 respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 218, 219 and 220 respectively;

[0410] (ix) The heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 232, 233 and 234 respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 240, 241 and 242 respectively;

[0411] (x) The heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 254, 255 and 256 respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 262, 263 and 264 respectively;

[0412] (xi) are the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 276, 277 and 278, respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 284, 285 and 286, respectively;

[0413] (xii) The heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 298, 299 and 300 respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 306, 307 and 308 respectively;

[0414] (xiii) The heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 320, 321 and 322 respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 328, 329 and 330 respectively;

[0415] (xiv) shows the heavy chain CDR1, CDR2 and CDR3 sequences in SEQ ID NO: 342, 343 and 344, respectively, and the light chain CDR1, CDR2 and CDR3 sequences in SEQ ID NO: 350, 351 and 352, respectively;

[0416] (xv) The heavy chain CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 364, 365, and 366, respectively, and the light chain CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 372, 373, and 374, respectively; and

[0417] (xvi) are the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 386, 387 and 388, respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 394, 395 and 396, respectively.

[0418] In some embodiments, this disclosure provides a species-specific monoclonal antibody or antigen-binding moiety thereof that specifically binds to and antagonizes human IL-27, wherein the antibody or antigen-binding moiety comprises heavy chain and light chain CDRs, wherein the heavy chain CDR1, CDR2 and CDR3 sequences are shown in SEQ ID NO: 164, 165 and 166, respectively, and the light chain CDR1, CDR2 and CDR3 sequences are shown in SEQ ID NO: 172, 173 and 174, respectively.

[0419] In some embodiments, this disclosure provides isolated monoclonal antibodies or antigen-binding moieties thereof that specifically bind to and antagonize human IL-27, wherein the antibody or antigen-binding moieties comprise heavy and light chain CDRs, wherein the amino acid sequences of the heavy chains 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 chains CDR1, CDR2, and CDR3 are RASENIYSYLA (SEQ ID NO: 26), NAETLTE (SEQ ID NO: 27), and QHHYGTPLT (SEQ ID NO: 28), respectively.

[0420] In some embodiments, this disclosure provides isolated monoclonal antibodies or antigen-binding moieties thereof that specifically bind to and antagonize human IL-27, wherein the antibody or antigen-binding moieties comprise heavy chain and light chain variable regions, wherein the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 57, 79, 101, 123, 145, 167, 191, 213, 235, 257, 279, 301, 323, 345, 367 and 389; and wherein the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 65, 87, 109, 131, 153, 175, 199, 221, 243, 265, 287, 309, 331, 353, 375 and 397.

[0421] In some embodiments, this disclosure provides isolated monoclonal antibodies or antigen-binding moieties thereof that specifically bind to and antagonize human IL-27, wherein said antibody or antigen-binding moieties comprise heavy chain and light chain variable regions, said heavy chain and light chain variable regions each comprising an amino acid sequence selected from the group consisting of:

[0422] (i)SEQ ID NO: 57 and 65;

[0423] (ii) SEQ ID NO: 79 and 87;

[0424] (iii) SEQ ID NO: 101 and 109;

[0425] (iv)SEQ ID NO: 123 and 131;

[0426] (v)SEQ ID NO: 145 and 153;

[0427] (vi)SEQ ID NO: 167 and 175;

[0428] (vii)SEQ ID NO: 191 and 199;

[0429] (viii) SEQ ID NO: 213 and 221;

[0430] (ix)SEQ ID NO: 235 and 243;

[0431] (x)SEQ ID NO: 257 and 265;

[0432] (xi)SEQ ID NO: 279 and 287;

[0433] (xii)SEQ ID NO: 301 and 309;

[0434] (xiii) SEQ ID NO: 323 and 331;

[0435] (xiv)SEQ ID NO: 345 and 353;

[0436] (xv)SEQ ID NO: 367 and 375; and

[0437] (xvi)SEQ ID NO: 389 and 397.

[0438] In some embodiments, this disclosure provides isolated monoclonal antibodies or antigen-binding moieties thereof that specifically bind to and antagonize human IL-27, wherein the antibody or antigen-binding moieties comprise heavy and light chain variable regions, wherein the heavy chain variable region comprises an amino acid sequence having at least 90% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 57, 79, 101, 123, 145, 167, 191, 213, 235, 257, 279, 301, 323, 345, 367, and 389; and wherein the light chain variable region comprises an amino acid sequence having at least 90% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 65, 87, 109, 131, 153, 175, 199, 221, 243, 265, 287, 309, 331, 353, 375, and 397.

[0439] In some embodiments, this disclosure provides isolated monoclonal antibodies or antigen-binding moieties thereof that specifically bind to and antagonize human IL-27, wherein said antibody or antigen-binding moieties comprise heavy chain and light chain variable regions, said heavy chain and light chain variable regions each comprising an amino acid sequence having at least 90% identity with an amino acid sequence selected from the group consisting of:

[0440] (i)SEQ ID NO: 57 and 65;

[0441] (ii) SEQ ID NO: 79 and 87;

[0442] (iii) SEQ ID NO: 101 and 109;

[0443] (iv)SEQ ID NO: 123 and 131;

[0444] (v)SEQ ID NO: 145 and 153;

[0445] (vi)SEQ ID NO: 167 and 175;

[0446] (vii)SEQ ID NO: 191 and 199;

[0447] (viii) SEQ ID NO: 213 and 221;

[0448] (ix)SEQ ID NO: 235 and 243;

[0449] (x)SEQ ID NO: 257 and 265;

[0450] (xi)SEQ ID NO: 279 and 287;

[0451] (xii)SEQ ID NO: 301 and 309;

[0452] (xiii) SEQ ID NO: 323 and 331;

[0453] (xiv)SEQ ID NO: 345 and 353;

[0454] (xv)SEQ ID NO: 367 and 375; and

[0455] (xvi)SEQ ID NO: 389 and 397.

[0456] In some embodiments, this disclosure provides isolated monoclonal antibodies or antigen-binding moieties thereof that specifically bind to and antagonize human IL-27, wherein the antibody or antigen-binding moieties thereof comprise heavy chain and light chain variable regions, the heavy chain and light chain variable regions comprising the amino acid sequences shown in SEQ ID NO: 167 and 175, respectively.

[0457] In some embodiments, this disclosure provides isolated monoclonal antibodies or antigen-binding moieties thereof that specifically bind to and antagonize human IL-27, wherein the antibody or antigen-binding moieties thereof comprise heavy chain and light chain variable regions, the heavy chain and light chain variable regions comprising amino acid sequences having at least 90% identity with the amino acid sequences shown in SEQ ID NO: 167 and 175, respectively.

[0458] In some embodiments, this disclosure provides isolated monoclonal antibodies or antigen-binding moieties thereof that specifically bind to and antagonize human IL-27, wherein the antibody or antigen-binding moieties comprise a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 67, 89, 111, 133, 155, 177, 201, 223, 245, 267, 289, 311, 333, 355, 377, and 399; and wherein the light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 69, 91, 113, 135, 157, 179, 203, 225, 247, 269, 291, 313, 335, 357, 379, and 401.

[0459] In some embodiments, this disclosure provides isolated monoclonal antibodies or antigen-binding moieties thereof that specifically bind to and antagonize human IL-27, wherein said antibody or antigen-binding moieties comprise a heavy chain and a light chain, wherein said heavy chain comprises an amino acid sequence having at least 90% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 67, 89, 111, 133, 155, 177, 201, 223, 245, 267, 289, 311, 333, 355, 377, and 399; and wherein said light chain comprises an amino acid sequence having at least 90% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 69, 91, 113, 135, 157, 179, 203, 225, 247, 269, 291, 313, 335, 357, 379, and 401.

[0460] In some embodiments, this disclosure provides isolated monoclonal antibodies or antigen-binding moieties thereof that specifically bind to and antagonize human IL-27, wherein the antibody or antigen-binding moieties comprise a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 71, 93, 115, 137, 159, 181, 205, 227, 249, 271, 293, 315, 337, 359, 381, and 403; and wherein the light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 69, 91, 113, 135, 157, 179, 203, 225, 247, 269, 291, 313, 335, 357, 379, and 401.

[0461] In some embodiments, this disclosure provides isolated monoclonal antibodies or antigen-binding moieties thereof that specifically bind to and antagonize human IL-27, wherein the antibody or antigen-binding moieties comprise a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence having at least 90% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 71, 93, 115, 137, 159, 181, 205, 227, 249, 271, 293, 315, 337, 359, 381, and 403; and wherein the light chain comprises an amino acid sequence having at least 90% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 69, 91, 113, 135, 157, 179, 203, 225, 247, 269, 291, 313, 335, 357, 379, and 401.

[0462] In some embodiments, this disclosure provides isolated monoclonal antibodies or antigen-binding moieties thereof that specifically bind to and antagonize human IL-27, wherein said antibody or antigen-binding moieties comprise a heavy chain and a light chain, said heavy chain and light chain each comprising an amino acid sequence selected from the group consisting of:

[0463] (i)SEQ ID NO: 67 and 69;

[0464] (ii) SEQ ID NO: 89 and 91;

[0465] (iii) SEQ ID NO: 111 and 113;

[0466] (iv)SEQ ID NO: 133 and 135;

[0467] (v)SEQ ID NO: 155 and 157;

[0468] (vi)SEQ ID NO: 177 and 179;

[0469] (vii)SEQ ID NO: 201 and 203;

[0470] (viii) SEQ ID NO: 223 and 225;

[0471] (ix)SEQ ID NO: 245 and 247;

[0472] (x)SEQ ID NO: 267 and 269;

[0473] (xi)SEQ ID NO: 289 and 291;

[0474] (xii)SEQ ID NO: 311 and 313;

[0475] (xiii) SEQ ID NO: 333 and 335;

[0476] (xiv)SEQ ID NO: 355 and 357;

[0477] (xv)SEQ ID NO: 377 and 379; and

[0478] (xvi)SEQ ID NO: 399 and 401.

[0479] In some embodiments, this disclosure provides isolated monoclonal antibodies or antigen-binding moieties thereof that specifically bind to and antagonize human IL-27, wherein said antibody or antigen-binding moieties comprise a heavy chain and a light chain, said heavy chain and light chain each comprising an amino acid sequence having at least 90% identity with an amino acid sequence selected from the group consisting of:

[0480] (i)SEQ ID NO: 67 and 69;

[0481] (ii) SEQ ID NO: 89 and 91;

[0482] (iii) SEQ ID NO: 111 and 113;

[0483] (iv)SEQ ID NO: 133 and 135;

[0484] (v)SEQ ID NO: 155 and 157;

[0485] (vi)SEQ ID NO: 177 and 179;

[0486] (vii)SEQ ID NO: 201 and 203;

[0487] (viii) SEQ ID NO: 223 and 225;

[0488] (ix)SEQ ID NO: 245 and 247;

[0489] (x)SEQ ID NO: 267 and 269;

[0490] (xi)SEQ ID NO: 289 and 291;

[0491] (xii)SEQ ID NO: 311 and 313;

[0492] (xiii) SEQ ID NO: 333 and 335;

[0493] (xiv)SEQ ID NO: 355 and 357;

[0494] (xv)SEQ ID NO: 377 and 379; and

[0495] (xvi)SEQ ID NO: 399 and 401.

[0496] In some embodiments, this disclosure provides isolated monoclonal antibodies or antigen-binding moieties thereof that specifically bind to and antagonize human IL-27, wherein said antibody or antigen-binding moieties comprise a heavy chain and a light chain, said heavy chain and light chain each comprising an amino acid sequence selected from the group consisting of:

[0497] (i)SEQ ID NO: 71 and 69;

[0498] (ii) SEQ ID NO: 93 and 91;

[0499] (iii) SEQ ID NO: 115 and 113;

[0500] (iv)SEQ ID NO: 137 and 135;

[0501] (v)SEQ ID NO: 159 and 157;

[0502] (vi)SEQ ID NO: 181 and 179;

[0503] (vii)SEQ ID NO: 205 and 203;

[0504] (viii) SEQ ID NO: 227 and 225;

[0505] (ix)SEQ ID NO: 249 and 247;

[0506] (x)SEQ ID NO: 271 and 269;

[0507] (xi)SEQ ID NO: 293 and 291;

[0508] (xii)SEQ ID NO: 315 and 313;

[0509] (xiii) SEQ ID NO: 337 and 335;

[0510] (xiv)SEQ ID NO: 359 and 357;

[0511] (xv)SEQ ID NO: 381 and 379; and

[0512] (xvi)SEQ ID NO: 403 and 401.

[0513] In some embodiments, this disclosure provides isolated monoclonal antibodies or antigen-binding moieties thereof that specifically bind to and antagonize human IL-27, wherein said antibody or antigen-binding moieties comprise a heavy chain and a light chain, said heavy chain and light chain each comprising an amino acid sequence having at least 90% identity with an amino acid sequence selected from the group consisting of:

[0514] (i)SEQ ID NO: 71 and 69;

[0515] (ii) SEQ ID NO: 93 and 91;

[0516] (iii) SEQ ID NO: 115 and 113;

[0517] (iv)SEQ ID NO: 137 and 135;

[0518] (v)SEQ ID NO: 159 and 157;

[0519] (vi)SEQ ID NO: 181 and 179;

[0520] (vii)SEQ ID NO: 205 and 203;

[0521] (viii) SEQ ID NO: 227 and 225;

[0522] (ix)SEQ ID NO: 249 and 247;

[0523] (x)SEQ ID NO: 271 and 269;

[0524] (xi)SEQ ID NO: 293 and 291;

[0525] (xii)SEQ ID NO: 315 and 313;

[0526] (xiii) SEQ ID NO: 337 and 335;

[0527] (xiv)SEQ ID NO: 359 and 357;

[0528] (xv)SEQ ID NO: 381 and 379; and

[0529] (xvi)SEQ ID NO: 403 and 401.

[0530] In some embodiments, this disclosure provides isolated monoclonal antibodies or antigen-binding moieties thereof that specifically bind to and antagonize human IL-27, wherein the antibody or antigen-binding moieties comprise heavy and light chains comprising the amino acid sequences shown in SEQ ID NO: 177 and 179, respectively.

[0531] In some embodiments, this disclosure provides isolated monoclonal antibodies or antigen-binding moieties thereof that specifically bind to and antagonize human IL-27, wherein the antibody or antigen-binding moieties comprise heavy and light chains, the heavy and light chains comprising amino acid sequences having at least 90% identity with the amino acid sequences shown in SEQ ID NO: 177 and 179, respectively.

[0532] In some embodiments, this disclosure provides isolated monoclonal antibodies or antigen-binding moieties thereof that specifically bind to and antagonize human IL-27, wherein the antibody or antigen-binding moieties comprise heavy and light chains comprising the amino acid sequences shown in SEQ ID NO: 181 and 179, respectively.

[0533] In some embodiments, this disclosure provides isolated monoclonal antibodies or antigen-binding moieties thereof that specifically bind to and antagonize human IL-27, wherein the antibody or antigen-binding moieties comprise heavy and light chains, the heavy and light chains comprising amino acid sequences having at least 90% identity with the amino acid sequences shown in SEQ ID NO: 181 and 179, respectively.

[0534] Methods for generating anti-IL-27 antibodies and their antigen-binding fragments

[0535] This disclosure also describes methods for generating any of the anti-IL-27 antibodies or antigen-binding fragments thereof described herein. In some embodiments, the method for preparing the antibodies described herein may include immunizing a subject (e.g., a non-human mammal) with a suitable immunogen. Suitable immunogens for generating any of the antibodies described herein are described herein. For example, to generate an antibody that binds to IL-27, a person skilled in the art may immunize a suitable subject (e.g., a non-human mammal such as a rat, mouse, gerbil, hamster, dog, cat, pig, goat, horse, or non-human primate) with IL-27. In some embodiments, a 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, a full-length human IL-27p28 monomeric polypeptide comprising the amino acid sequence shown in SEQ ID NO: 98 is used as an immunogen.

[0536] A suitable antigen can be used to immunize a suitable subject (e.g., a non-human mammal), followed by multiple booster immunizations sufficient to induce antibody production in the mammal. The immunogen can be administered to the subject (e.g., a non-human mammal) together with an adjuvant. Adjuvants used to induce antibody production in a subject include, but are not limited to, protein adjuvants; bacterial adjuvants, such as intact bacteria (BCG, Corynebacterium parvum, or Salmonella minnesota) and bacterial components, including cell wall skeletons, trehalose dimethicone, monophosphoryl lipid A, methanol extractable residue (MER) of Mycobacterium tuberculosis, complete or incomplete Freund's adjuvants; viral adjuvants; chemical adjuvants, such as aluminum hydroxide, and iodoacetate and cholesterol hemisuccinate. Other adjuvants that can be used in methods for inducing an immune response include, for example, cholera toxin and parapoxovirus proteins. See also Bieg et al. (1999) Autoimmunity 31(1) : 15-24. See also, for example, Lodmell et al. (2000) Vaccine 18 : 1059-1066; Johnson et al. (1999) J Med Chem 42 : 4640-4649; Baldridge et al. (1999) Methods 19 : 103-107 and Gupta et al. (1995) Vaccine 13(14) :1263-1276.

[0537] In some embodiments, the method includes 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 the aforementioned IL-27 peptide. Antibody-producing cells (e.g., B cells from the spleen) of 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 from 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 / O-Ag 14. After fusion, the cells are expanded in a suitable culture medium, with selective medium, such as HAT medium, replenished at regular intervals to prevent normal myeloma cells from growing beyond the desired number of hybridoma cells. The desired antibody (e.g., an antibody that binds to human IL-27) is then screened for secretion in the obtained hybrid cells, and in some embodiments, the person skilled in the art can obtain it from U.S. Patent No. 6,300,064 (belonging to Knappik et al.; Morphosys AG) and Schoonbroodt et al. (2005) Nucleic Acids Res 33(9) Anti-IL-27 antibodies were identified in non-immune-biased libraries described in e81.

[0538] In some implementations, the methods described herein may involve, for example, phage display, bacterial display, yeast surface display, eukaryotic virus display, mammalian cell display, and cell-free (e.g., ribosome display) antibody screening techniques, or a combination thereof (see, for example, Etz et al. (2001) J Bacteriol). 183 :6924-6935;Cornelis(2000)Curr Opin Biotechnol 11 : 450-454; Klemm et al. (2000) Microbiology 146 :3025-3032; Kieke et al. (1997) Protein Eng 10 : 1303-1310; Yeung et al. (2002) Biotechnol Prog 18 :212-220; Boder et al. (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).

[0539] 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 a phage particle carrying a polynucleotide sequence encoding them. Such phages can be used to display antigen-binding domains of antibodies, such as Fab, Fv, or disulfide-bonded Fv antibody fragments, expressed from a library or a combined antibody library (e.g., human or mouse). The phages used in these methods are typically filamentous phages, such as fd and M13. The antigen-binding domain is expressed as a protein recombinantly fused to any 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 described herein for the preparation of immunoglobulins or fragments thereof include those disclosed in the following literature: 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, for example, in U.S. Patent Nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,743, and 5,969,108.

[0540] In some implementations, the phage display antibody library can be generated using mRNA collected from B cells of immunized mammals. For example, a spleen cell sample containing B cells can be isolated from mice immunized with the aforementioned IL-27 peptide. The 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), ibid.; Benny KCLo (2004), ibid.; and Borrebaek (1995), ibid. The cDNA encoding the variable regions of immunoglobulin heavy and light chain peptides is used to construct the phage display library. Methods for generating such libraries are described, for example, Merz et al. (1995), J Neurosci Methods. 62(1-2) : 213-9; Di Niro et al. (2005) Biochem J 388(Pt 3) : 889-894 and Engberg et al. (1995) Methods Mol Biol 51 :355-376.

[0541] In some implementations, a combination of selection and screening can be used to identify target antibodies from, for example, antibody populations generated by hybridomas or phage-displayed antibody libraries. Suitable methods are known in the art and described, for example, in Hoogenboom (1997) Trends in Biotechnology. 15:62-70; Brinkman et al. (1995), ibid.; Ames et al. (1995), ibid.; Kettleborough et al. (1994), ibid.; Persic et al. (1997), ibid. and Burton et al. (1994), ibid. For example, multiple phage vectors (each encoding a fusion protein of a phage coat protein (e.g., pIII, pVIII, or pIX of M13 phage) with a different antigen-binding region) are generated using standard molecular biology techniques and then introduced into a bacterial population (e.g., *E. coli*). In some embodiments, phage expression in bacteria may require the use of helper phages. In some embodiments, helper phages are not required (see, for example, Chasteen et al., (2006) Nucleic Acids Res 34(21):e145). Phages generated from bacteria are recovered and then contacted with (immobilized) target antigens, for example, bound to a solid support. It can also bring phages into contact with antigens in solution, and then allow the complex to bind to a solid support.

[0542] The specificity and binding affinity of antibody subsets screened using the methods described above for a specific antigen (e.g., human IL-27) can be characterized using any immunological or biochemical method known in the art. For example, the specific binding of an antibody to IL-27 can be determined, for instance, using immunological or biochemical methods such as, but not limited to, ELISA assays, SPR assays, immunoprecipitation assays, affinity chromatography, and equilibrium dialysis as described above. Immunoassays that can be used to analyze the immunospecific binding and cross-reactivity of antibodies include, but are not limited to, competitive and non-competitive assay systems that utilize techniques such as Western blotting, RIA, ELISA (enzyme-linked immunosorbent assay), sandwich immunoassays, immunoprecipitation assays, immunodiffusion assays, agglutination assays, complement fixation assays, immunoradioassays, fluorescence immunoassays, and protein A immunoassays. Such assays are routine and well-known in the art.

[0543] It should be understood that the above methods can also be used to determine, for example, whether anti-IL-27 antibodies do not bind to full-length human IL-27 and / or IL-27 protein.

[0544] In embodiments where the selected CDR amino acid sequence is a short sequence (e.g., less than 10-15 amino acids in length), it can be, for example, as in the Nucleic Acids Symposium Series by Shiraishi et al. (2007). 51(1)As described in U.S. Patent Nos. 129-130 and 6,995,259, nucleic acids encoding CDRs are chemically synthesized. For a given nucleic acid sequence encoding a recipient antibody, the region of the nucleic acid sequence encoding the CDR can be replaced using standard molecular biology techniques with chemically synthesized nucleic acids. 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.

[0545] In some embodiments, the anti-IL-27 antibody described herein comprises an altered heavy chain constant region having reduced (or no) effector function relative to its corresponding unaltered constant region. The effector function of the constant region involving the anti-IL-27 antibody can be modulated by altering the properties of the constant region or Fc region. Altered effector functions include, for example, regulation of one or more of the following activities: antibody-dependent cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), apoptosis, binding to one or more Fc receptors, and pro-inflammatory responses. Regulation refers to an enhancement, reduction, or elimination of the effector functional activity exhibited by the test antibody comprising the altered constant region compared to the activity of the unaltered form of the constant region. In certain embodiments, regulation includes cases where the activity is eliminated or completely absent.

[0546] In one embodiment, the anti-IL-27 antibody described herein comprises an IgG4 heavy chain constant region. In one embodiment, the IgG4 heavy chain constant region is a wild-type IgG4 heavy chain constant region. In another embodiment, for example, according to EU designations (Kabat, EA, et al., ibid.), 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 the antibodies used in this disclosure are shown in Table 12. In one embodiment, the anti-IL-27 antibody described herein comprises an IgG1 constant region. In one embodiment, the IgG1 heavy chain constant region is a wild-type IgG1 heavy chain constant region. In another embodiment, the IgG1 heavy chain constant region comprises a mutation. Representative sequences of wild-type and mutant IgG4 constant regions for the antibodies used in this disclosure are shown in Table 12.

[0547] A modified constant region exhibiting 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 its unaltered form. Modified constant regions exhibiting enhanced FcR binding bind at least one FcR with a greater affinity than the unaltered polypeptide. Modified constant regions exhibiting decreased FcR binding bind at least one FcR with a lower affinity than the unaltered form. Such variants exhibiting decreased FcR binding may have little or no binding to FcR, for example, 0 to 50% (e.g., less than 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 3) compared to the binding level of the native sequence immunoglobulin constant region or Fc region with FcR. Binding to FcR at concentrations of 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%. Similarly, constant regions showing altered ADCC and / or CDC activity compared to unchanged constant regions may exhibit enhanced or decreased ADCC and / or CDC activity. For example, in some embodiments, anti-IL-27 antibodies containing altered constant regions may exhibit approximately 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 unaltered form of the constant region for ADCC and / or CDC activity. Anti-IL-27 antibodies described herein that contain altered constant regions exhibit reduced ADCC and / or CDC activity may exhibit reduced ADCC and / or CDC activity or no ADCC and / or CDC activity.

[0548] In some embodiments, the anti-IL-27 antibodies described herein exhibit reduced effector function or no effector function. In some embodiments, the anti-IL-27 antibody comprises a hybridization constant region or a portion thereof, such as a G2 / G4 hybridization constant region (see, for example, Burton et al. (1992) Adv Immun).51 : 1-18; Canfield et al. (1991) J Exp Med 173 :1483-1491 and Mueller et al. (1997) Mol Immunol 34(6) (441-452). See above.

[0549] In some embodiments, the anti-IL-27 antibody may include a constant region exhibiting altered complement-dependent cytotoxicity (CDC) that is enhanced or reduced. CDC activity can be modulated 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. Optionally or additionally, one or more cysteine ​​residues may be introduced into the Fc region, thereby allowing interchain disulfide bonds to form in that region. The resulting homodimeric antibody may have enhanced or reduced internalization capacity and / or enhanced or reduced complement-mediated cell killing. See, for example, 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 U.S. Patent Nos. 738-40; and U.S. Patent Nos. 5,648,260 and 5,624,821.

[0550] Expression and purification of recombinant antibodies

[0551] The antibodies or antigen-binding fragments described herein can be produced using a variety of techniques known in the fields of molecular biology and protein chemistry. For example, one or both of the nucleic acids encoding the heavy and light chain polypeptides of the antibody can be inserted into an expression vector containing transcriptional and translational regulatory sequences, such as promoter sequences, ribosome binding sites, transcription initiation and termination sequences, translation initiation and termination sequences, transcription terminator signals, polyadenylation signals, and enhancer or activator sequences. The regulatory sequences include promoters and transcription initiation and termination sequences. Furthermore, the expression vector may include more than one replication system, allowing it to be maintained in two different organisms, such as for expression in mammalian or insect cells, and for cloning and amplification in a prokaryotic host.

[0552] Several possible vector systems can be used to express heavy and light chain polypeptides from nucleic acid clones in mammalian cells. One class of vectors relies on integrating the desired gene sequence into the host cell genome. Cells with stably integrated DNA can be used to simultaneously introduce drug resistance genes such as *E. coli* gpt (Mulligan and Berg (1981) Proc Natl Acad Sci USA). 78 :2072) or Tn5 neo (Southern and Berg (1982) Mol Appl Genet 1 The selectable marker gene can be selected by linking it to the DNA sequence to be expressed, or by co-transfection to introduce the selectable marker gene into the same cell (Wigler et al. (1979) Cell). 16 :77). The second type of vector utilizes DNA elements that confer autonomous replication ability 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 SciUSA) 81 :1292) or SV40 virus (Lusky and Botchan (1981) Nature) 293 :79).

[0553] Expression vectors can be introduced into cells in a manner suitable for subsequent nucleic acid expression. The introduction methods discussed below are primarily determined by the target cell type. Exemplary methods include CaPO4 precipitation, liposome fusion, cationic liposomes, electroporation, viral infection, dextran-mediated transfection, amine-mediated transfection, protoplast fusion, and direct microinjection.

[0554] Suitable host cells for expressing antibodies or their antigen-binding fragments 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.

[0555] In some implementations, the antibody or a fragment thereof may be expressed in and purified from a transgenic animal (e.g., a transgenic mammal). For example, as in Houdebine (2002) Curr Opin Biotechnol 13(6)Antibodies can be generated in transgenic nonhuman mammals (e.g., rodents) and isolated from milk, as described in 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.

[0556] Antibodies and their fragments can be produced from cells by culturing host cells transformed with an expression vector containing nucleic acid encoding the antibody or fragment for a certain period of time under conditions sufficient to allow protein expression. Such conditions for protein expression will vary depending on the choice of expression vector and host cell, and will be readily determined by those skilled in the art through routine experiments. For example, antibodies expressed in *E. coli* can be refolded from inclusion bodies (e.g., see Hou et al. (1998) Cytokine). 10 : 319-30). Bacterial expression systems and their methods of use 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 depending on many factors and can be easily optimized as needed. The antibodies (or fragments thereof) described herein can be expressed in mammalian cells or other expression systems (including but not limited to yeast, baculoviruses) and in vitro expression systems (see, for example, Kaszubska et al. (2000) Protein Expression and Purification). 18 It is expressed in : 213-220).

[0557] After expression, antibodies and their fragments can be isolated. Antibodies or their fragments can be isolated 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 electrophoresis, molecular techniques, immunological techniques, and chromatographic techniques, including ion-exchange chromatography, hydrophobic chromatography, affinity chromatography, and reversed-phase HPLC. For example, antibodies can be purified using standard anti-antibody columns (e.g., protein-A or protein-G columns). Ultrafiltration and percolation 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 level of purification will vary depending on the intended use. In some cases, purification of the expressed antibody or its fragments is not necessary.

[0558] Methods for determining the yield or purity of purified antibodies or fragments thereof are known in the art, including, for example, the Bradford assay, ultraviolet spectroscopy, biuret protein assay, Lowry protein assay, aminomelin assay, high-performance liquid chromatography (HPLC), mass spectrometry (MS), and gel electrophoresis methods (e.g., staining with protein staining agents such as Coomassie blue or colloidal silver).

[0559] Modification of antibodies or their antigen-binding fragments

[0560] Antibodies or their antigen-binding fragments can be modified after expression and purification. Modifications can be covalent or non-covalent. Such modifications can be introduced into the antibody or fragment by reacting, for example, the target amino acid residues of the peptide with an organic derivatizing agent capable of reacting with selected side-chain or terminal residues. A suitable modification site can be selected using any of several criteria, including, for example, structural analysis or amino acid sequence analysis of the antibody or fragment.

[0561] In some embodiments, an antibody or its antigen-binding fragment may be conjugated to a heterologous moiety. The heterologous moiety may be, for example, a heterologous peptide, a therapeutic agent (e.g., a toxin or drug), or a detectable marker, such as, but not limited to, a radiolabel, an enzyme-catalyzed marker, a fluorescent marker, a heavy metal marker, a luminescent marker, or an affinity tag such as biotin or streptavidin. Suitable heterologous peptides 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 purifying the antibody or fragment. Heterologous peptides also include peptides (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 markers include, but are not limited to, fluorescein, FITC, green fluorescent protein (GFP), and DyLight. TM 488, Phycoerythrin (PE), Propidium Iodide (PI), PerCP, PE-Alexa 700, Cy5, allophycocyanin, and Cy7. Luminescent markers include, for example, any of a variety of luminescent lanthanide chelates (e.g., europium or terbium). Suitable europium chelates, for example, include europium chelates of diethylenetriaminepentaacetic acid (DTPA) or tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). Enzymatic markers include, for example, alkaline phosphatase, CAT, luciferase, and horseradish peroxidase.

[0562] 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 through a "hindered" disulfide bond. In these linkages, the disulfide bond within the cross-linking unit is protected (by hindering the groups on either side of the disulfide bond) from reduction by, for example, the action of reduced glutathione or disulfide reductases. A suitable reagent, 4-succinimideoxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene (SMPT), utilizes a terminal lysine residue on one protein and a terminal cysteine ​​residue on another protein to form such a linkage between two proteins. Heterobifunctional reagents that are cross-linked through different coupling moieties on each protein can also be used. Other useful crosslinking agents include, but are not limited to, agents that link two amino groups (e.g., N-5-azido-2-nitrobenzoyloxysuccinimide), two thiol groups (e.g., 1,4-bis-maleimide butane), an amino group and a thiol group (e.g., m-maleimide benzoyl-N-hydroxysuccinimide ester), an amino group and a carboxyl group (e.g., 4-[p-azidosalicylic acid]butane), and an amino group and a guanidine group present in the side chain of arginine (e.g., p-azidophenylglyoxal monohydrate).

[0563] In some implementations, the radiolabel can be directly conjugated to the amino acid backbone of the antibody. Alternatively, the radiolabel can be part of a larger molecule (e.g., meta-[ 125 I] Iodophenyl-N-hydroxysuccinimide ([ 125 In I]mIPNHS) 125 I) contains m-iodophenyl (mIP) derivatives that bind to a free amino group to form the associated protein (see, for example, Rogers et al. (1997) J Nucl Med 38 The radiolabel (e.g., chelates with DOTA or DTPA) is then bound to the protein backbone. Methods for conjugating radiolabels or larger molecules / chelates containing them to antibody or antigen-binding fragments described herein are known in the art. Such methods involve incubating the protein with the radiolabel under conditions favorable to the binding of the radiolabel or chelating agent to the protein (e.g., pH, salt concentration, and / or temperature) (see, for example, U.S. Patent No. 6,001,329).

[0564] Methods for conjugating fluorescent labels (sometimes referred to as “fluorophores”) to proteins (e.g., antibodies) are known in the field of protein chemistry. For example, a fluorophore can be conjugated to a free amino group (e.g., lysine) or a thiol group (e.g., cysteine) of a protein using a succinimide (NHS) ester or tetrafluorophenyl (TFP) ester moiety attached to the fluorophore. In some embodiments, the fluorophore can be conjugated to a heterobifunctional crosslinking agent moiety (such as sulfonyl-SMCC). Suitable conjugation methods involve incubating an antibody protein or a fragment thereof with the fluorophore under conditions that promote binding between the fluorophore and the protein. See, for example, Welch and Redvanly (2003), “Handbook of Radiopharmaceuticals: Radiochemistry and Applications,” John Wiley and Sons (ISBN 0471495603).

[0565] In some implementations, the antibody or fragment may be modified, for example, with portions that improve the stability and / or retention of the antibody in circulation (e.g., in blood, serum, or other tissues). For example, this can be achieved as described by Lee et al. (1999) Bioconjug Chem. 10(6) :973-8; Kinstler et al. (2002) Advanced Drug Deliveries Reviews 54 : 477-485; and Roberts et al. (2002) Advanced Drug Delivery Reviews 5 4:459-476 describes the PEGylation of the antibody or fragment, or the hydroxyethylation (HESylated) of the antibody or fragment (Fresenius Kabi, Germany; see, for example, et al. (2010) Int J Pharm 387(1-2) (110-119). The stabilization fraction can increase the stability or retention of the antibody (or fragment) by at least 1.5-fold (e.g., at least 2, 5, 10, 15, 20, 25, 30, 40, or 50 times or more).

[0566] In some embodiments, the antibodies or antigen-binding fragments described herein may be glycosylated. In some embodiments, the antibodies or antigen-binding fragments described herein may be enzymatically or chemically treated, or the antibodies or antigen-binding fragments may be generated from cells, such that the antibodies or fragments have reduced glycosylation or no glycosylation. Methods for generating 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.

[0567] Pharmaceutical compositions and formulations

[0568] In some 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.

[0569] In some embodiments, the acceptable formulation material is preferably non-toxic to the recipient at the dose and concentration used. In some embodiments, one or more formulation materials are for subcutaneous and / or intravenous administration. In some embodiments, the pharmaceutical composition may contain formulation materials for altering, maintaining, or preserving, for example, the composition's pH, molality, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption, or permeation. In some embodiments, suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine, or lysine); antibacterial agents; antioxidants (such as ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (such as borates, bicarbonates, Tris-HCl, citrates, phosphates, or other organic acids); fillers (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, flavoring agents, and diluents; emulsifiers; and hydrophilic polymers (such as poly… Vinylpyrrolidone; low molecular weight peptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide); solvents (such as glycerol, propylene glycol, or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as prolind, PEG, dehydrated sorbitol esters, polysorbates such as polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol, terpineol); stability enhancers (such as sucrose or sorbitol); tension enhancers (such as alkali metal halides, preferably sodium chloride or potassium chloride, mannitol or sorbitol); delivery media; diluents; excipients and / or pharmaceutical adjuvants. (Remington's Pharmaceutical Sciences, 18th edition, ARGennaro, editor, Mack Publishing Company (1995). In some 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 some embodiments, the optimal pharmaceutical composition will be determined by those skilled in the art based on, for example, the intended route of administration, delivery method, and required dosage. See, for example, Remington's Pharmaceutical Sciences, ibid.)In some embodiments, such compositions can affect the physical state, stability, in vivo release rate, and / or in vivo clearance rate of anti-IL-27 antibodies.

[0570] In some embodiments, the primary medium or carrier in the pharmaceutical composition may be aqueous or non-aqueous in nature. For example, in some embodiments, a suitable medium or carrier may be water for injection, physiological saline, or artificial cerebrospinal fluid, which may be supplemented with other materials commonly used in compositions for parenteral administration. In some embodiments, the saline comprises isotonic phosphate-buffered saline. In some embodiments, neutral buffered saline or saline mixed with serum albumin are further exemplary mediums. In some embodiments, the pharmaceutical composition comprises Tris buffer with a pH of about 7.0-8.5, or acetate buffer with a pH of about 4.0-5.5, which may also contain sorbitol or a suitable alternative thereof. In some embodiments, a composition containing anti-IL-27 antibodies may be prepared for storage by mixing a selected composition having the desired purity with an optional formulation (Remington's Pharmaceutical Sciences, ibid.) in the form of a lyophilized cake or an aqueous solution. Additionally, in some embodiments, a suitable excipient such as sucrose may be used to formulate the composition containing anti-IL-27 antibodies into a lyophilized product.

[0571] In some embodiments, the pharmaceutical composition may be selected for parenteral delivery. In some embodiments, the composition may be selected for inhalation or delivery via the digestive tract (such as oral administration). The preparation of such pharmaceutically acceptable compositions is within the capabilities of those skilled in the art.

[0572] In some embodiments, the formulation component is present at a concentration acceptable at the application site. In some embodiments, a buffer solution is used to maintain the composition at a physiological pH or slightly lower, typically in the pH range of about 5 to about 8.

[0573] In some embodiments, when parenteral administration is envisioned, the therapeutic composition may be present in a pharmaceutically acceptable medium in the form of a pyrogen-free, parenteral-acceptable aqueous solution containing an anti-IL-27 antibody. In some embodiments, the medium for parenteral injection is sterile distilled water, wherein the anti-IL-27 antibody is formulated as a sterile isotonic solution and appropriately stored. In some embodiments, the preparation may involve formulating the desired molecule with an agent (such as injectable microspheres, bio-erosive particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads, or liposomes) that provides controlled or sustained release of the product, which can then be delivered via reservoir injection. In some embodiments, hyaluronic acid may also be used, which may have the effect of promoting duration of circulation. In some embodiments, an implantable drug delivery device may be used to introduce the desired molecule.

[0574] In some embodiments, the pharmaceutical composition may be formulated for inhalation. In some embodiments, the anti-IL-27 antibody may be formulated as a dry powder for inhalation. In some embodiments, an inhalation solution containing the anti-IL-27 antibody may be formulated using a propellant for aerosol delivery. In some embodiments, the solution may be nebulized. Lung administration is further described in PCT application number PCT / US94 / 001875 (which describes pulmonary delivery of chemically modified proteins).

[0575] In some embodiments, oral administration of the formulation is envisioned. In some embodiments, the anti-IL-27 antibody administered in this manner may or may not be formulated with those carriers typically used to formulate solid dosage forms such as tablets and capsules. In some embodiments, the capsule may be designed to release the active portion of the formulation at a point in the gastrointestinal tract when bioavailability is maximized and pre-degradation is minimized. In some embodiments, at least one additional agent may be included to promote the absorption of the anti-IL-27 antibody. In some embodiments, diluents, flavoring agents, low-melting-point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrants, and binders may also be used.

[0576] In some embodiments, the pharmaceutical composition may comprise an effective amount of anti-IL-27 antibody mixed with a non-toxic excipient suitable for tablet production. In some embodiments, a solution may be prepared in unit dose form by dissolving the tablet in sterile water or another suitable medium. In some 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 gum arabic; or lubricants such as magnesium stearate, stearic acid, or talc.

[0577] Other pharmaceutical compositions will be apparent to those skilled in the art, including formulations containing an anti-IL-27 antibody in sustained- or controlled-release formulations. In some embodiments, techniques for formulating a variety of other sustained- or controlled-release components, such as liposome carriers, biodegradable microparticles or porous beads, and reservoir injections, are also known to those skilled in the art. See, for example, PCT application number PCT / US93 / 00829, which describes the controlled release of porous polymeric microparticles for delivering pharmaceutical compositions. In some embodiments, sustained-release formulations may comprise a semi-permeable polymer matrix in the form of a molded article (e.g., a film or microcapsule). The sustained-release matrix may include polyesters, hydrogels, polylactide (US Patent Nos. 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., ibid.), or poly-D(-)-3-hydroxybutyric acid (EP 133,988). In some embodiments, the sustained-release composition may also comprise liposomes, which may 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; EP088,046 and EP 143,949.

[0578] Pharmaceutical compositions intended for internal administration are typically sterile. In some embodiments, this can be achieved through filtration using a sterile filter membrane. In some embodiments, when lyophilizing the composition, sterilization using this method can be performed before or after lyophilization and reconstitution. In some embodiments, compositions intended for parenteral administration can be stored in lyophilized or solution form. In some embodiments, parenteral compositions are typically placed in a container with a sterile inlet, such as an intravenous solution bag or vial with a stopper that can be punctured by a hypodermic needle.

[0579] In some embodiments, once a pharmaceutical composition is formulated, it can be stored in sterile vials in the form of a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder. In some embodiments, such formulations can be stored in ready-to-use or administration-reconstituted forms (e.g., lyophilized).

[0580] In some embodiments, a cartridge for producing a single-dose administration unit is provided. In some embodiments, the cartridge may include a first container containing dried protein and a second container containing an aqueous formulation. In some embodiments, a cartridge is included that contains single-chamber and multi-chamber pre-filled syringes (e.g., liquid syringes and lyophilized syringes).

[0581] In some embodiments, the effective amount of the pharmaceutical composition containing an anti-IL-27 antibody for therapeutic use will depend, for example, on the treatment context and objectives. Those skilled in the art will understand that, according to some embodiments, the appropriate dose level for treatment will therefore vary in part depending on the molecule delivered, the indication for the use of the anti-IL-27 antibody, the route of administration, and the patient's size (weight, body surface area, or organ size) and / or condition (age and general health status). In some embodiments, clinicians may titrate the dose and modify the route of administration to obtain optimal therapeutic effect.

[0582] In some embodiments, the dosing frequency will take into account the pharmacokinetic parameters of the anti-IL-27 antibody in the formulation used. In some embodiments, the clinician will administer the composition until a dose is reached to achieve the desired effect. In some embodiments, the composition may therefore be administered over time as 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 implanted device or catheter. Further refinement of the appropriate dose is routinely performed by those skilled in the art and within the scope of their routine duties. In some embodiments, the appropriate dose can be determined by using appropriate dose-response data.

[0583] In some embodiments, the pharmaceutical composition is administered via known methods, such as oral administration, injection via intravenous, intraperitoneal, intracerebral (parenchymal), intraventricular, intramuscular, subcutaneous, intraocular, intraarterial, portal vein, or intralesional routes; via a continuous-release system or implantable device. In some embodiments, the composition may be administered by bolus injection or continuous infusion, or via an implantable device. In some embodiments, individual components of the combination therapy may be administered via different routes.

[0584] In some embodiments, the composition can be applied topically by implanting a membrane, sponge, or other suitable material on which the desired molecule has been adsorbed or encapsulated. In some embodiments, when using an implantable device, the device can be implanted into any suitable tissue or organ, and delivery of the desired molecule can be achieved by diffusion, timed release bolus, or continuous administration. In some embodiments, it may be desirable to use the pharmaceutical composition containing anti-IL-27 antibodies in an ex vivo manner. In such cases, cells, tissues, and / or organs removed from a patient are exposed to the pharmaceutical composition containing anti-IL-27 antibodies, after which the cells, tissues, and / or organs are implanted back into the patient.

[0585] In some embodiments, anti-IL-27 antibodies can be delivered by implanting genetically engineered cells to express and secrete peptides using methods such as those described herein. In some embodiments, such cells can be animal or human cells and can be autologous, allogeneic, or xenogeneic. In some embodiments, the cells can be immortalized. In some embodiments, to reduce the chance of an immune response, the cells can be encapsulated to prevent infiltration into surrounding tissues. In some embodiments, the encapsulating material is typically a biocompatible, semi-permeable 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.

[0586] application

[0587] The compositions described herein can be used in a variety of diagnostic and therapeutic applications. For example, detectable labeled antigen-binding molecules can be used to determine the presence or amount of a target antigen in a sample (e.g., a biological sample). The compositions can be used in in vitro assays to study inhibition of target antigen function. In some embodiments, such as those where the compositions bind to and inhibit complement proteins, the compositions can be used as a positive control in an assay designed to identify other novel compounds that inhibit complement activity or are otherwise used to treat complement-related conditions. For example, IL-27 inhibitory compositions can be used as a positive control in an assay to identify other compounds (e.g., small molecules, aptamers, or antibodies) that reduce or eliminate IL-27 production. The compositions can also be used in the therapeutic methods detailed below.

[0588] In some embodiments, this disclosure provides a method for detecting IL-27 in a biological sample or subject, comprising (i) contacting the sample or subject (and optionally a reference sample or subject) with any antibody described herein under conditions that allow the antibody molecule and IL-27 to interact, and (ii) detecting the formation of a complex between the antibody molecule and the sample or subject (and optionally a reference sample or subject).

[0589] medicine box

[0590] The kit may include the anti-IL-27 antibody disclosed herein and instructions for use. The kit may contain the anti-IL-27 antibody, one or more controls, and various buffers, reagents, enzymes, and other standard components known in the art in a suitable container. In some aspects, this disclosure provides a kit containing the anti-IL-27 antibody or antigen-binding portion disclosed herein, and instructions for use to stimulate an immune response in a subject or to treat a cancer in a subject, optionally in combination with one or more additional therapeutic agents or procedures described herein.

[0591] 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 is placed and, in some cases, appropriately aliquoted. If additional components are provided, the kit may include another container into which the component 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 that hold the desired vials within them. The container and / or kit may include labels with instructions for use and / or warnings.

[0592] How to use

[0593] The compositions of the present invention have numerous in vitro and in vivo uses, including the detection and / or quantification of IL-27 and / or antagonism of IL-27 function.

[0594] In some embodiments, this disclosure provides a method for inhibiting or reducing STAT1 and / or STAT3 phosphorylation in cells, the method comprising contacting the cells with an isolated monoclonal antibody or antigen-binding fragment provided by this disclosure, wherein the antibody or its antigen-binding portion inhibits or reduces STAT1 and / or STAT3 phosphorylation in the cells.

[0595] In some embodiments, this disclosure provides a method for inhibiting or reducing the inhibition of CD161 expression in cells, the method comprising contacting cells with an isolated monoclonal antibody or antigen-binding fragment provided by this disclosure, wherein the antibody or its antigen-binding portion inhibits or reduces the inhibition of CD161 expression in cells.

[0596] In some embodiments, this disclosure provides a method for inhibiting or reducing the expression of PD-L1 and / or TIM-3 in cells, the method comprising contacting cells with an isolated monoclonal antibody or antigen-binding fragment provided by this disclosure, wherein the antibody or its antigen-binding portion inhibits the expression of PD-L1 and / or TIM-3 in the cells.

[0597] In some embodiments, this disclosure provides a method for inducing or enhancing the secretion of one or more cytokines from a cell, the method comprising contacting the cell with an isolated monoclonal antibody or antigen-binding fragment provided by this disclosure, wherein the antibody or antigen-binding portion thereof induces or enhances the secretion of one or more PD-1-mediated cytokines from the cell.

[0598] In some embodiments, this disclosure provides a method for stimulating an immune response in a subject, the method comprising administering to the subject an effective amount of an isolated monoclonal antibody or antigen-binding fragment that specifically binds to and antagonizes human IL-27, provided in this disclosure, or a pharmaceutical composition comprising said antibody or its antigen-binding portion and a pharmaceutically acceptable carrier.

[0599] In some embodiments, this disclosure provides a method of treating a subject with cancer, the method comprising administering to the subject an effective amount of an isolated monoclonal antibody or antigen-binding portion thereof that specifically binds to and antagonizes IL-27, provided in this disclosure, or a pharmaceutical composition comprising said antibody or antigen-binding portion thereof and a pharmaceutically acceptable carrier.

[0600] In some embodiments, this 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 in this disclosure, or a pharmaceutical composition comprising said antibody or its antigen-binding portion and a pharmaceutically acceptable carrier, wherein said antibody or its antigen-binding portion or the pharmaceutical composition inhibits or reduces STAT1 and / or STAT3 phosphorylation in cells, thereby stimulating the immune response or treating the cancer.

[0601] In some embodiments, this 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 in this disclosure, or a pharmaceutical composition comprising said antibody or its antigen-binding portion and a pharmaceutically acceptable carrier, wherein said antibody or its antigen-binding portion or said pharmaceutical composition inhibits or reduces the inhibition of CD161 expression in cells, thereby stimulating the immune response or treating said cancer.

[0602] In some embodiments, this 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 in this disclosure, or a pharmaceutical composition comprising said antibody or its antigen-binding portion and a pharmaceutically acceptable carrier, wherein said antibody or its antigen-binding portion or the pharmaceutical composition inhibits or reduces the expression of PD-L1 and / or TIM-3 in cells, thereby stimulating the immune response or treating the cancer.

[0603] In some embodiments, this 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 in this disclosure, or a pharmaceutical composition comprising said antibody or its antigen-binding portion and a pharmaceutically acceptable carrier, wherein said antibody or its antigen-binding portion or said pharmaceutical composition induces or enhances the secretion of one or more PD-1-mediated cytokines from cells, thereby stimulating said immune response or treating said cancer.

[0604] In some implementations, 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, such as clear cell renal cell carcinoma).

[0605] The above-described compositions are particularly useful for methods of treating or preventing various cancers in subjects. The compositions can be administered to subjects (e.g., human subjects) using various methods, depending on the route of administration. These routes can be, for example, intravenous injection or infusion (IV), subcutaneous injection (SC), intraperitoneal injection (IP), intramuscular injection (IM), or intrathecal injection (IT). Injection can be a bolus or a continuous infusion.

[0606] Administration can be achieved, for example, by local infusion, injection, or implantation. The implant can be a porous, non-porous, or gel-like material, including membranes such as salivary elastic membranes or fibers. The implant can be configured to continuously or periodically release the composition to a subject. See, for example, U.S. Patent Application Publication No. 20080241223; U.S. Patents 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. The composition can be delivered to a subject via an implantable device based on, for example, a diffusion system, an erosive system, or a convection system (e.g., an osmotic pump, a biodegradable implant, an electrodiffusion system, an electroosmotic system, a vapor pressure pump, an electrolytic pump, a foaming pump, a piezoelectric pump, an erosion-based system, or an electromechanical system).

[0607] In some implementations, anti-IL-27 antibodies or their antigen-binding fragments are therapeutically delivered to the subject via topical application.

[0608] The appropriate dose of the antibody or its fragment described herein (a dose sufficient to treat or prevent 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 inhibitory compound used. For example, treating a subject with cancer may require a different dose of the complete anti-IL-27 antibody compared to the dose of the IL-27-binding Fab' antibody fragment required to treat the same subject. Other factors influencing the dose administered to a subject include, for example, the type or severity of the cancer. For example, a subject with metastatic melanoma may require a different dose of anti-IL-27 antibody compared to a subject with glioblastoma. Other factors may include, for example, other medical conditions affecting the subject concurrently or previously, the subject's general health condition, the subject's genetic predisposition, diet, timing of administration, excretion rate, drug combination, and any other additional therapeutic agents administered to the subject. It should also be understood that the specific dose and treatment regimen for any particular subject will also depend on the judgment of the practicing physician (e.g., a doctor or nurse). Appropriate doses are described herein.

[0609] Pharmaceutical compositions may comprise a therapeutically effective amount of the anti-IL-27 antibody or its antigen-binding fragment described herein. If more than one agent is used, such an effective amount can be readily determined by those skilled in the art, in part based on the effect of the administered antibody or the combined effect of the antibody with one or more other active agents. The therapeutically effective amount of the antibody or its fragment described herein may also vary depending on factors such as an individual's disease state, age, sex, and weight, and the ability of the antibody (and one or more other active agents) to elicit a desired response (e.g., reduced tumor growth) in the individual. For example, a therapeutically effective amount of the anti-IL-27 antibody may inhibit (reduce its severity or eliminate its occurrence) 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 the amount by which any toxic or adverse effects of the composition are outweighed by the beneficial therapeutic effect.

[0610] Appropriate human doses of any of the antibodies or fragments described herein may be further evaluated, for example, in 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.

[0611] In some embodiments, the composition comprises any antibody or antigen-binding fragment thereof described herein, and one or more (e.g., two, three, four, five, six, seven, eight, nine, ten, or eleven 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 respective concentrations of the antibody and the agent, when combined, are therapeutically effective for treating or preventing cancer (e.g., melanoma) in the subject.

[0612] The toxicity and therapeutic effects of such compositions can be determined by known pharmaceutical procedures in cell cultures or laboratory animals (e.g., animal models of any of the cancers described herein). These procedures can be used, for example, to determine the LD50. 50 (Lethal dose for 50% of the population) and ED 50 (Therapeutic effective dose for 50% of the population). The dose-to-toxicity ratio is the therapeutic index, which can be expressed as LD50. 50 / ED 50 The ratio. Antibodies or their antigen-binding fragments exhibiting a high therapeutic index are preferred. Although compositions showing toxic side effects can be used, care should be taken to design delivery systems that target such compounds to the affected tissue sites and minimize potential damage to normal cells, thereby reducing side effects.

[0613] Data obtained from cell culture assays and animal studies can be used to formulate a range of doses for human use. The dosage of such antibodies or their antigen-binding fragments is typically within the range of ED. 50 The antibody or fragment is within the circulating concentration range and has little or no toxicity. The dosage can vary within this range, depending on the dosage form and route of administration. For the anti-IL-27 antibody described herein, the therapeutically effective dose can be initially estimated from cell culture assays. Doses can be formulated in animal models to achieve IC50 values ​​as determined in cell cultures. 50 The range of circulating plasma concentrations (i.e., the antibody concentration at which half-maximal inhibition of symptoms is achieved). This information can be used to more accurately determine the effective dose in the human body. Plasma levels can be measured, for example, by high-performance liquid chromatography. In some embodiments, such as where local application is required (e.g., to the eye or joint), cell cultures or animal modeling can be used to determine the dose required to achieve a therapeutically effective concentration at the local site.

[0614] In some implementations, the method may be combined with other cancer treatments. For example, the composition may be administered to a subject concurrently 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 therapies.

[0615] As described above, the compositions described herein (e.g., anti-IL-27 compositions) can be used to treat a variety of cancers, such as, but not limited to: Kaposi's sarcoma, leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, myeloblasts promyelocyte myelomonocytic monocytic erythroleukemia, 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, Waldenström macroglobulinemia, heavy chain disease, solid tumors, sarcomas and carcinomas, fibrosarcoma, myxosarcoma, liposarcoma, etc. Chondrosarcoma, Osteosarcoma, Osteosarcoma, Chordoma, Angiosarcoma, Endothelial Sarcoma, Lymphangiosarcoma, Lymphangioendothelial Sarcoma, Synovial Tumor, Mesothelioma, Ewing's Tumor, Leiomyosarcoma, Rhabdomyosarcoma, Colon Sarcoma, Colorectal Cancer, Pancreatic Cancer, Breast Cancer, Ovarian Cancer, Prostate Cancer, Squamous Cell Carcinoma, Basal Cell Carcinoma, Adenocarcinoma, Hidradenoma, Sebaceous Gland Carcinoma, Papillary Carcinoma, Papillary Adenoma, Cystic Adenoma, Medullary Carcinoma, Bronchial Carcinoma, Renal Cell Carcinoma, Hepatocellular Carcinoma (HCC), Hepatocellular Tumor, Bile Duct Carcinoma, Choriocarcinoma Pilocarcinoma, seminoma, embryonal carcinoma, nephroblastoma, cervical cancer, uterine cancer, testicular tumor, lung cancer, small cell lung cancer, non-small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, nasopharyngeal carcinoma, esophageal cancer, basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and central nervous system cancer. (CNS) cancers, including cervical cancer, choriocarcinoma, colorectal cancer, connective tissue cancer, digestive system cancers, 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 cancers (e.g., lip, tongue, mouth and pharynx), ovarian cancer, pancreatic cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer; respiratory system cancers, sarcomas, skin cancer, stomach cancer, testicular cancer, thyroid cancer, uterine cancer, and urinary system cancers.

[0616] Combination therapy

[0617] In some embodiments, the anti-IL-27 antibody or its antigen-binding portion provided by this disclosure may be combined with one or more other therapeutic agents or treatments (e.g., another therapeutic agent or treatment for cancer). For example, the anti-IL-27 antibody or its antigen-binding portion may be administered to a subject (e.g., a human patient) in combination with one or more other therapeutic agents, wherein said combination provides therapeutic benefit to a subject who has cancer or is at risk of developing cancer.

[0618] In some embodiments, an anti-IL-27 antibody or its antigen-binding portion and one or more additional therapeutic agents are administered at the same time (e.g., simultaneously). In other embodiments, an anti-IL-27 antibody or its antigen-binding portion is administered at a first time, followed by one or more additional therapeutic agents at a second time (e.g., sequentially). In some embodiments, one or more additional therapeutic agents are administered at a first time, followed by an anti-IL-27 antibody at a second time.

[0619] The anti-IL-27 antibody or its antigen-binding fragment described herein may replace or enhance previously or currently administered therapy. For example, after treatment with an anti-IL-27 antibody or its antigen-binding fragment, the administration of one or more additional therapeutic agents may be discontinued or reduced, for example, by administering one or more additional therapeutic agents at a lower level. In some embodiments, the administration of previous therapy may be maintained. In some embodiments, previous therapy will be maintained until the level of the anti-IL-27 antibody reaches a level sufficient to provide a therapeutic effect.

[0620] In some embodiments, this disclosure provides a method of treating a subject with cancer, the method comprising administering to the subject an effective amount of a monoclonal antibody or antigen-binding portion thereof that specifically binds to and antagonizes IL-27 isolate, provided by this 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 anticancer therapy, oncolytic drugs, cytotoxic agents, immunotherapy, cytokines, surgical procedures, radiation procedures, activators of co-stimulatory molecules, inhibitors of inhibitory molecules, vaccines, or cell immunotherapy, or combinations thereof.

[0621] In some implementations, one or more additional therapeutic agents are PD-1 antagonists, TIM-3 inhibitors, LAG-3 inhibitors, TIGIT inhibitors, CD112R inhibitors, TAM inhibitors, STING agonists, 4-1BB agonists, or combinations thereof.

[0622] In some embodiments, 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, pebuprofen, pildizumab, MEDI0680, REGN2810, TSR-042, PF-06801591, and AMP-224. In some embodiments, 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, acitumab, durvalumab, and BMS-936559. In some embodiments, this disclosure provides a method for enhancing one or more activities of anti-PD-1 antibodies (e.g., enhancing PD-1-mediated cytokine secretion; enhancing anti-PD-1-mediated TNFα secretion; enhancing anti-PD-1-mediated IL-6 secretion from cells exposed to anti-PD-1 antibodies), said method comprising exposing cells to an antibody or its antigen-binding portion provided by this disclosure, simultaneously or sequentially exposed to an anti-PD-1 antibody, thereby enhancing one or more activities of the anti-PD-1 antibody.

[0623] In some implementations, one or more additional therapeutic agents are sunitinib. Carbotinib Axitinib Lenvatinib Everolimus bevacizumab Iparstat, NKTR-214 (CD-122-biased agonist), Tevozani abexinosta, ipilimumab Trimelimumab, Pazopanib Sorafenib Tissirolimus Ramucirumab Niraparib, Savolitinib, Vorolanib (X-82), Regorafenib Donafenib (multi-kinase inhibitor), Camrelizumab (SHR-1210), Pexastimogened Evacirepvec (JX-594), Ramucirumab Apatinib (YN968D1), Doxorubicin Capsules Tevantinib (ARQ197), ADI-PEG 20, Binitinib, Apatinib Mesylate, Nintedanib, Rilurumab, Nivolumab Pebuprocilbazine Pidizhumab Acimenone Dvalumab cemiplimab-rwlc Tislelizumab and Spartazumab.

[0624] In some implementations, one or more additional therapeutic agents are TIM-3 inhibitors, optionally wherein the TIM-3 inhibitor is MGB453 or TSR-022.

[0625] In some embodiments, one or more additional therapeutic agents are LAG-3 inhibitors, optionally wherein the LAG-3 inhibitor is selected from the group consisting of LAG525, BMS-986016 and TSR-033.

[0626] 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.

[0627] Combination with chemotherapy agents

[0628] Chemotherapy agents suitable for use in combination with and / or co-administered with the compositions of the present invention include, for example: paclitaxel, cytochalasin B, bacitracin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthrancindione, mitoxantrone, photomycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and their analogues or homologues. Other agents include, for example, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil aminoimide), alkylating agents (e.g., nitrogen mustard, thiotepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, cis-dichlorodiamineplatin(II) (DDP), procarbazine, hexamethylmelamine, cisplatin, carboplatin, oxaliplatin, nedaplatin, ceterplatin, or triplatin tetranitrate), anthracyclines (e.g., daunorubicin (formerly known as doxorubicin) and doxorubicin), antibiotics (e.g., dermatomycin (formerly known as actinomycin), bleomycin, styracin, and amiodarone (AMC)), and antimitotic agents (e.g., vincristine and vinblastine), and temozolomide.

[0629] Combination with PD-1 / PD-L1 antagonists

[0630] In some embodiments, the anti-IL-27 antibody or its antigen-binding portion provided by this disclosure is combined with one or more PD-1 antagonists (e.g., combined administration), said PD-1 antagonists specifically bind to human PD-1 or PD-L1 and inhibit PD-1 / PD-L1 bioactivity and / or one or more downstream pathways and / or cellular processing or other human PD-1 / PD-L1-mediated functions mediated by human PD-1 / PD-L1 signaling.

[0631] Therefore, this article provides PD-1 antagonists that can directly or allosterically block, antagonize, inhibit, suppress, or reduce the biological activity of PD-1 / PD-L1, including downstream pathways and / or cellular processes mediated by PD-1 / PD-L1 signaling, such as receptor binding and / or initiation of cellular responses to PD-1 / PD-L1. This article also provides PD-1 antagonists that reduce the number or amount of human PD-1 or PD-L1 produced by cells or subjects.

[0632] In some embodiments, this 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 antagonist binds to mRNA encoding PD-1 or PD-L1 and blocks translation. In some embodiments, the PD-1 antagonist binds to mRNA encoding PD-1 or PD-L1 and causes degradation and / or turnover.

[0633] In some embodiments, PD-1 antagonists inhibit PD-1 signaling or function. In some embodiments, PD-1 antagonists block the binding of PD-1 to PD-L1, PD-L2, or both PD-L1 and PD-L2. In some embodiments, PD-1 antagonists block the binding of PD-1 to PD-L1. In some embodiments, PD-1 antagonists block the binding of PD-1 to PD-L2. In some embodiments, PD-1 antagonists block the binding of PD-1 to both PD-L1 and PD-L2. In some embodiments, PD-1 antagonists specifically bind to PD-1. In some embodiments, PD-1 antagonists specifically bind to PD-L1. In some embodiments, PD-1 antagonists specifically bind to PD-L2.

[0634] In some embodiments, PD-1 antagonists inhibit the binding of PD-1 to its homologous ligands. In some embodiments, PD-1 antagonists inhibit the binding of PD-1 to PD-L1, PD-1 to PD-L2, or PD-1 to both PD-L1 and PD-L2. In some embodiments, PD-1 antagonists do not inhibit the binding of PD-1 to its homologous ligands.

[0635] In some embodiments, the PD-1 antagonist is a separated monoclonal antibody (mAb) or its antigen-binding fragment that specifically binds to PD-1 or PD-L1. In some embodiments, the PD-1 antagonist is an antibody or its antigen-binding fragment that specifically binds to human PD-1. In some embodiments, the PD-1 antagonist is an antibody or its antigen-binding fragment that specifically binds to human PD-L1. In some embodiments, the PD-1 antagonist is an antibody or its antigen-binding fragment that binds to human PD-L1 and inhibits the binding of PD-1 to PD-1. In some embodiments, the PD-1 antagonist is an antibody or its antigen-binding fragment that binds to human PD-1 and inhibits the binding of PD-L1 to PD-1.

[0636] 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 for use by clinicians to treat cancer.

[0637] Examples of anti-human PD-1 monoclonal antibodies or antigen-binding fragments thereof that may contain a PD-1 antagonist in any of the compositions, methods and uses provided by this disclosure include, but are not limited to: (Pebutropuzumab, 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 incorporated herein by reference in their entirety; Bristol Myers Squibb), MEDI0680 (AMP-514), BGB-A317 and BGB-108 (BeiGene), 244C8 and 388D4 (see WO2016106159, which is incorporated herein by reference in its entirety; Enumeral Biomedical), PDR001 (Novartis) and REGN2810 (Regeneron). Therefore, in some embodiments, the PD-1 antagonist is peburolizumab. In some implementations, the PD-1 antagonist is nivolumab.

[0638] Examples of anti-human PD-L1 monoclonal antibodies or antigen-binding fragments thereof that may contain a PD-1 antagonist in any of the compositions, methods and uses provided by this disclosure include, but are not limited to: (Acimetidine, MSB0010718C, see WO2013 / 79174, which is incorporated herein by reference in its entirety; Merck / Pfizer) (Dvalumab, 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). Therefore, in some embodiments, the PD-1 antagonist is avermab. In some embodiments, the PD-1 antagonist is devalumab. In some embodiments, the PD-1 antagonist is atezolizumab.

[0639] 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 fused to a constant region (such as the Fc region) of an immunoglobulin molecule, or a PD-1 binding portion. Examples of PD-1-specifically binding immunoadhesins 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.

[0640] Those skilled 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.

[0641] In some implementations, the PD-1 / PD-L1 antagonist is a small molecule, nucleic acid, peptide, peptide mimic, protein, carbohydrate, carbohydrate derivative, or sugar polymer. Exemplary small molecule PD-1 inhibitors are described in Zhan et al., (2016) Drug Discov Today 21(6): 1027-1036.

[0642] Combination with TIM-3 inhibitors

[0643] In some embodiments, the anti-IL-27 antibody or its antigen-binding portion provided in this disclosure is combined with a TIM-3 inhibitor (e.g., administered in combination). The TIM-3 inhibitor may be an antibody, its antigen-binding fragment, an immunoadhesive, 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, the anti-IL-27 antibody or its antigen-binding portion is administered in combination with MGB453. In some embodiments, the anti-IL-27 antibody or its antigen-binding portion is administered in combination with TSR-022.

[0644] Combination with LAG-3 inhibitors

[0645] In some embodiments, the anti-IL-27 antibody or its antigen-binding portion provided in this disclosure is combined with a LAG-3 inhibitor (e.g., combined administration). The LAG-3 inhibitor may be an antibody, its antigen-binding fragment, 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).

[0646] Other combinations

[0647] In some embodiments, the anti-IL-27 antibody or its antigen-binding portion provided by this disclosure is combined with a TIGIT inhibitor, a kinase inhibitor (e.g., a tyrosine kinase inhibitor (TKI)), a CD112R inhibitor, a TAM receptor inhibitor, a STING agonist and / or a 4-1BB agonist or a combination thereof (e.g., combined administration).

[0648] Detection methods

[0649] In some embodiments, the anti-IL-27 antibody or its antigen-binding fragment described herein can be used in methods for detecting and / or quantifying human IL-27 in biological samples. Therefore, the anti-IL-27 antibody or its antigen-binding fragment described herein can be used to diagnose, predict, and / or determine the progression of a patient's disease (e.g., cancer).

[0650] As defined herein, monitoring cancer improvement in a subject (e.g., a human patient) means assessing changes in the subject's disease parameters, such as a reduction in tumor growth. In some embodiments, the assessment is performed at least one (1) hour after administration, such as at least 2, 4, 6, 8, 12, 24, or 48 hours, or at least 1 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. Subjects may be assessed at one or more of the following times: before treatment begins; during treatment; or after one or more of the criteria for treatment have been administered. The assessment may include evaluating the need for further treatment, such as whether the dose, frequency of administration, or duration of treatment should be changed. It may also include evaluating the need to add or withdraw selected modalities of treatment, such as adding or withdrawing any cancer treatment described herein.

[0651] In some embodiments, this disclosure provides a method for detecting IL-27 in a sample from a subject, the method comprising (a) if IL-27 is present in the sample from the subject, contacting the sample with a detection antibody, wherein the detection antibody is an antibody or an antigen-binding fragment thereof provided in this disclosure, under conditions allowing the formation of a detection antibody-IL-27 complex; and (b) detecting the presence of the complex generated in step (a), if any.

[0652] In some embodiments, this disclosure provides a method for detecting IL-27-related cancer in a subject, the method comprising the steps of: (a) if IL-27 is present in a sample from a subject suspected of having IL-27-related cancer, contacting the sample with a detection antibody, wherein the detection antibody is an antibody provided by this disclosure or its antigen-binding portion thereof, under conditions allowing the formation of a detection antibody-IL-27 complex; and (b) detecting the presence of the complex generated in step (a), if any. In some embodiments, the detection antibody is conjugated to a detectable marker. In some embodiments, the method further includes, if IL-27 is present in the sample, contacting the sample with a capture antibody to generate a complex comprising IL-27 and the capture antibody, wherein the capture antibody is an antibody provided by this disclosure or its antigen-binding portion thereof.

[0653] In some embodiments, the capture antibody is immobilized on a solid support. In some embodiments, the sample is contacted with the capture antibody before detection. In some embodiments, the sample is a body fluid sample. In some embodiments, the fluid sample is blood, serum, plasma, cell lysate, or tissue lysate.

[0654] 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).

[0655] Example

[0656] Although this disclosure has been described with reference to its specific embodiments, those skilled in the art will understand that various changes and equivalent substitutions can be made without departing from the true spirit and scope of this disclosure. Furthermore, many modifications can be made to adapt specific circumstances, materials, material composition, processes, and process steps to the objectives, spirit, and scope of this disclosure. All such modifications are intended to be within the scope of this disclosure.

[0657] Example 1: Generation and characterization of anti-IL-27 antibodies that specifically bind to IL-27 EBI3 monomers

[0658] This embodiment describes the generation of anti-IL-27 antibodies that specifically bind to the EBI3 subunit of human IL-27. Briefly, BALB / c mice were immunized with a human EBI3 immunovector (Aldevron) to generate and isolate hybridomas expressing anti-EBI3 monoclonal antibodies. The isolated hybridomas included those expressing anti-IL-27 antibody molecules (referred to herein as Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, and Ab8). Hybridoma supernatants were analyzed by flow cytometry on mammalian cells expressing the surface-targeting human EBI3. Supernatants from all tested hybridoma clones were bound to cells expressing EBI3 (data not shown).

[0659] The exemplary isolated anti-EBI3 antibody Ab7 (hereinafter referred to as "Ab7") contains the immunoglobulin heavy chain variable region (hereinafter referred to as "Ab7-V"). H0 ") and the variable region of the immunoglobulin light chain (hereinafter referred to as "Ab7-V") L0 Sequencing was performed and further characterized below (the N-terminal signal peptide sequence is not shown).

[0660] The heavy chain variable region (Ab7-V) of isolated Ab7 antibody H0 It has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-constant region):

[0661]

[0662] Encoding heavy chain variable region Ab7-V H0 The nucleic acid sequence has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4):

[0663]

[0664] The light chain variable region (Ab7-V) of the isolated Ab7 antibody L0 It has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-constant region):

[0665]

[0666] The heavy chain of isolated Ab7 antibody (Ab7-V) H0 -mIgG2a) has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-constant region):

[0667]

[0668] Encoding the light chain variable region Ab7-V L0 The nucleic acid sequence has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4):

[0669]

[0670] The light chain of isolated Ab7 antibody (Ab7-V) L0 -mκ) has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-constant region):

[0671]

[0672] Humanized Ab7 antibodies were designed using methods known in the art. In short, the V region gene sequence encoding a mouse monoclonal Ab7 antibody was used to construct a series of fully humanized antibodies. The variable region gene was cloned into a vector encoding the human IgG1 heavy chain constant domain and the human κ light chain constant domain. The chimeric and humanized antibodies were transiently expressed in mammalian cells. Humanization of the isolated Ab7 heavy chain variable region resulted in five humanized heavy chain variable region variants (hereinafter referred to as "Ab7-V"). H1 “Ab7-V” H2 “Ab7-V” H3 “Ab7-V” H4 "and Ab7-V"H5 The humanization of the separated Ab7 light chain variable region resulted in four humanized light chain variable region variants (hereinafter referred to as "Ab7-V"). L1 “Ab7-V” L2 “Ab7-V” L3 "and Ab7-V" L4 ”).

[0673] The protein sequence defining the variable region of the humanized Ab7 variant and the nucleotide sequence encoding the variable region of the humanized Ab7 variant are summarized below (the N-terminal signal peptide sequence is not shown).

[0674] Heavy chain variable region Ab7-V H1 It has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4):

[0675]

[0676] Encoding heavy chain variable region Ab7-V H1 The nucleic acid sequence has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4):

[0677]

[0678] Heavy chain variable region Ab7-V H2 It has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4):

[0679]

[0680] Encoding heavy chain variable region Ab7-V H2 The nucleic acid sequence has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4):

[0681]

[0682] Heavy chain variable region Ab7-V H3 It has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4):

[0683]

[0684] Encoding heavy chain variable region Ab7-V H3 The nucleic acid sequence has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4):

[0685]

[0686]

[0687] Heavy chain variable region Ab7-V H4 It has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4):

[0688]

[0689] Encoding heavy chain variable region Ab7-V H4 The nucleic acid sequence has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4):

[0690]

[0691] Heavy chain variable region Ab7-V H5 It has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4):

[0692]

[0693] Encoding heavy chain variable region Ab7-V H5 The nucleic acid sequence has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4):

[0694]

[0695] Light chain variable region Ab7-V L1 It has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4):

[0696]

[0697] Encoding the light chain variable region Ab7-V L1 The nucleic acid sequence has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4):

[0698]

[0699] Light chain variable region Ab7-V L2 It has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4):

[0700]

[0701] Encoding the light chain variable region Ab7-V L2 The nucleic acid sequence has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4):

[0702]

[0703] Light chain variable region Ab7-V L3 It has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4):

[0704]

[0705] Encoding the light chain variable region Ab7-V L3 The nucleic acid sequence has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4):

[0706]

[0707] Light chain variable region Ab7-V L4 It has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4):

[0708]

[0709] Encoding the light chain variable region Ab7-V L4 The nucleic acid sequence has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4):

[0710]

[0711] The heavy and light chain CDR amino acid sequences (Kabat definition) of the isolated parental Ab7 antibody are shown in Table 1.

[0712] Table 1

[0713]

[0714] To generate complete chimeric and humanized heavy or light chain antibody sequences, each of the above-mentioned heavy chain variable regions is combined with a human IgG1 constant region, and each of the above-mentioned light chain variable regions is combined with a human κ constant region.

[0715] The complete heavy and light chain protein sequences defining the chimeric and humanized Ab7 variants are summarized below (the N-terminal signal peptide sequence is not shown).

[0716] Heavy chain Ab7-V H0 -IgG1 has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-constant region):

[0717]

[0718]

[0719] Encoding heavy chain Ab7-V H0 -IgG1 nucleic acid sequence has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-constant region):

[0720]

[0721]

[0722] Heavy chain Ab7-V H1 -IgG1 has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-constant region):

[0723]

[0724] Encoding heavy chain Ab7-V H1 -IgG1 nucleic acid sequence has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-constant region):

[0725]

[0726]

[0727] Heavy chain Ab7-V H2 -IgG1 has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-constant region):

[0728]

[0729] Encoding heavy chain Ab7-V H2 -IgG1 nucleic acid sequence has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-constant region):

[0730]

[0731]

[0732] Heavy chain Ab7-VH3 -IgG1 has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-constant region):

[0733]

[0734]

[0735] Encoding heavy chain Ab7-V H3 -IgG1's nucleic acid sequence has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-constant region):

[0736]

[0737]

[0738] Heavy chain Ab7-V H4 -IgG1 has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-constant region):

[0739]

[0740] Encoding heavy chain Ab7-V H4 -IgG1's nucleic acid sequence has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-constant region):

[0741]

[0742]

[0743] Heavy chain Ab7-V H5 -IgG1 has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-constant region):

[0744]

[0745] Encoding heavy chain Ab7-V H5 -IgG1's nucleic acid sequence has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-constant region):

[0746]

[0747]

[0748] Light chain Ab7-V L0-κ has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-constant region):

[0749]

[0750] Encoding light chain Ab7-V L0 The -κ nucleic acid sequence has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-constant region):

[0751]

[0752] Light chain Ab7-V L1 -κ has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-constant region):

[0753]

[0754] Encoding light chain Ab7-V L1 The -κ nucleic acid sequence has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-constant region):

[0755]

[0756] Light chain Ab7-V L2 -κ has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-constant region):

[0757]

[0758] Encoding light chain Ab7-V L2 The -κ nucleic acid sequence has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-constant region):

[0759]

[0760]

[0761] Light chain Ab7-V L3 -κ has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-constant region):

[0762]

[0763] Encoding light chain Ab7-V L3The -κ nucleic acid sequence has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-constant region):

[0764]

[0765]

[0766] Light chain Ab7-V L4 -κ has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-constant region):

[0767]

[0768] Encoding light chain Ab7-V L4 The -κ nucleic acid sequence has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-constant region):

[0769]

[0770]

[0771] It is also anticipated that variable region sequences can be fused with other antibody constant region sequences to generate full-length immunoglobulin heavy and light chains. For example, Ab7-V... H0 Ab7-V H1 Ab7-V H 2. Ab7-V H 3. Ab7-V H 4 or Ab7-V H The 5-heavy chain variable region 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). Similarly, Ab7-V can be combined. L 0, Ab7-V L 1. Ab7-V L 2. Ab7-V L 3 or Ab7-V L 4. Combination of the variable region of the light chain with the constant region of human λ.

[0772] DNA fragments encoding the heavy and light chain variable regions of the Ab7 and humanized Ab7 variants described above were synthesized using flanking restriction enzyme sites and cloned into the pANT expression vector (Antitope) system for the IgG1 heavy and κ light chains. All constructs were confirmed by sequencing. The heavy and light chain combinations shown in Table 2 were transiently transfected into HEK293EBNA adherent cells (LGC Standards, Teddington, UK) using the PEI transfection method and incubated for 7 days post-transfection.

[0773] Table 2

[0774]

[0775]

[0776] Antibodies were purified from cell culture supernatant on a protein A-conjugated agarose column (GE Healthcare, Little Chalfont, UK). The buffer was exchanged into 1x DPBS (pH 7.2), and the extinction coefficient (Ec) was used based on the predicted amino acid sequence. (0.1％) ) through OD 280nm Quantification was performed. SDS-PAGE analysis of 1 μg of each antibody revealed bands corresponding to typical antibody patterns (data not shown).

[0777] In vitro characterization of anti-EBI3 antibody

[0778] The generated Ab7 antibodies and Ab7 antibody variants, as described in Table 2, were tested in a series of in vitro assays to determine their biological characteristics and activities.

[0779] To evaluate the binding of the humanized Ab7 antibody variant to the chimeric Ab7.1 antibody, a binding competition ELISA was established. The assay plate was coated with 1 μg / mL of human IL-27 (hIL-27) diluted in 1x DPBS (pH 7.2) (R&D Systems, Abingdon, UK) and incubated overnight at 4°C. All antibodies were diluted to 25 μg / mL in 2% BSA / DPBS and serially diluted 3-fold along the plate to generate an eight-point binding curve. The antibody dilution was premixed with the biotinylated Ab7.1 antibody at a constant final 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. 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 finally stopped with 1M HCl, and the absorbance at 450 nm was read on a Dynex Technologies MRX TC II plate reader.

[0780] The absolute IC50 of the test antibody was determined using a four-parameter logic curve. 50 Values. Table 3 summarizes the IC50 values ​​relative to the chimeric antibody on each plate. 50 Standardized IC 50 The results showed that, in addition to containing Ab7-V L4 Apart from the light chain variants, all the resulting humanized Ab7 variants have similar binding profiles to the chimeric Ab7.1 antibody, with most variants competing within twice the range of the chimeric Ab7.1 antibody.

[0781] Table 3

[0782]

[0783]

[0784] The antibody was further characterized by surface plasmon resonance (SPR). Kinetic experiments were performed on a Biacore T200 (GE Healthcare, Uppsala, Sweden). All experiments were run at 25°C using hIL-27 (R&D Systems, Abingdon, UK) and HBS-P+ run buffer (pH 7.4) (GE Healthcare, Little Chalfont, UK). Human IL-27 (hIL-27) antigen was captured on a CM5 chip up to approximately 16 RU. Immobilization was performed at a protein concentration of 1 μg / mL in 10 mM acetate buffer (pH 5.0). Antibody was diluted three times in HBS-P+ buffer at three-point 3-fold dilutions ranging from 3.3 nM to 30 nM, with no regeneration between each concentration. The association phase, lasting 75 seconds, was monitored with each injection of antibody at three increasing concentrations, and a single dissociation phase, lasting 250 seconds, was measured after the last antibody injection. The hIL-27 surface was regenerated using a single injection of 2M MgCl2 lasting 120 s. Multiple replicates of the chimeric antibody (n=4) were performed throughout the assay to examine the stability of the surface and analyte during kinetic cycling.

[0785] Due to the bivalent nature of antibodies, both the 1:1 binding model and the bivalent analyte model were used to analyze the data. Table 4 summarizes the results of the 1:1 binding model analysis, and Table 5 summarizes the results of the bivalent analyte model analysis.

[0786] Table 4

[0787]

[0788] Table 5

[0789]

[0790]

[0791] The single-cycle dynamics of Ab7-V are demonstrated using a 1:1 model (Table 4). L4 Humanized variants did not bind to hIL-27, and the remaining variants bound within twice the amount of the chimeric antibody, consistent with the results of the bivalent analyte model (Table 5) and competitive ELISA.

[0792] In summary, the binding affinity of the Ab7.1 chimeric antibody, as determined by single-cycle kinetics and using a 1:1 binding model, is 1 nM. This excludes the Ab7-V-containing antibodies whose binding is eliminated. L4Apart from the variants, all humanized variants had a KD of 1.5 nM or lower. These results indicate that the Ab7 antibody and Ab7 antibody variants bind to the EBI3 subunit of IL-27 with high affinity.

[0793] Example 2: Production of anti-IL-27 antibodies in yeast that specifically bind to the EBI3 and / or P28 subunits of human IL-27.

[0794] Using the method described below, additional anti-IL-27 monoclonal antibodies representing multiple epitope bins were selected from eight initial human synthetic yeast libraries.

[0795] Materials and methods

[0796] As mentioned earlier, eight offspring, each with approximately 10 [units of something], were bred. 9 A diverse initial human synthetic yeast library (see, e.g., see 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, as previously described, magnetic bead sorting was performed using the Miltenyi MACS system (see, e.g., Siegel et al., (2004) J Immunol Methods 286(1-2): 141-153, which is incorporated herein by reference in its entirety).

[0797] In short, yeast cells (approximately 10) are incubated at 30°C. 10 Yeast cells (100 cells / library) were incubated with 3 ml of 100 nM biotinylated antigen (recombinant human IL-27; R&D Systems) in wash buffer (phosphate-buffered saline (PBS) / 0.1% bovine serum albumin (BSA)) for 30 min. After washing once with 40 ml of ice-cold wash buffer, the cell pellet was resuspended in 20 ml of wash buffer, and streptoacidin microbeads (500 μl) were added to the yeast pellet, and incubated at 4 °C for 15 min. Next, the yeast cell pellet was resuspended in 20 ml of wash buffer and loaded onto a Miltenyi LS column. After loading 20 mL, the column was washed three times with 3 ml of wash buffer. The column was then removed from the magnetic field, 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 × 10⁶ cells / library were selected. 7Yeast cell pellets were washed three times with washing buffer and incubated at 30°C under equilibration conditions with decreasing concentrations of biotinylated antigen (100 to 1 nM) and 30 nM of biotinylated antigen from different species to obtain species cross-reactivity, or with a multispecific depletion reagent (PSR) to remove nonspecific antibodies from the selection. For PSR depletion, the library was incubated with a 1:10 dilution of biotinylated PSR reagent.

[0798] Yeast cells were then washed twice with wash buffer and stained at 4°C for 15 minutes with LC-FITC (1:100 dilution) and SA-633 (1:500 dilution) or EAPE (1:50 dilution) secondary reagents. After washing twice with wash buffer, the cell pellet was resuspended in 0.3 mL of wash buffer and transferred to a filter-lined sorting tube. Sorting was performed using a FACS ARIA sorter (BD Biosciences), and sorting gates were determined to select antibodies with the desired characteristics. The selection rounds were repeated until a population with all desired characteristics was obtained. After the final round of sorting, yeast cells were plated, and individual colonies were selected for characterization.

[0799] Light chain diversification

[0800] A light chain diversification strategy was used in the initial discovery phase to further discover and improve antibodies.

[0801] Light chain batch diversification solution: Extracting yeast from initial selection output through crushing and grabbing ( The heavy chain plasmid (selection output) was propagated in E. coli and subsequently purified from E. coli, and then transformed into a plasmid with a 5x10⁻¹⁰ molecule. 6 The selection was conducted using the same conditions as the initial findings, employing one round of MACS and four rounds of FACS.

[0802] Antibody optimization

[0803] As described below, antibody optimization is achieved by introducing diversity into the variable regions of the heavy and light chains.

[0804] CDRH1 and CDRH2 selection: Recombining single antibody CDRH3 to a diversity of 1x10 8 In pre-constituted libraries containing CDRH1 and CDRH2 variants, and as described in the initial findings, selection was performed using one round of MACS and four rounds of FACS. In different rounds of FACS, PSR binding, species cross-reactivity, and affinity pressure of the libraries were observed by titration or parental Fab pre-complexation, and sorting was performed to obtain populations with the desired characteristics.

[0805] Antibody production and purification

[0806] Yeast clones were grown to saturation and then induced with shaking at 30°C for 48 hours. After induction, the yeast cells were precipitated, and the supernatant was harvested for purification. IgG was purified using a Protein A column and eluted with acetic acid (pH 2.0). Fab fragments were generated by papain digestion and then purified using KappaSelect (GE Healthcare LifeSciences).

[0807] ForteBio K D Measurement

[0808] As previously mentioned, ForteBio affinity measurements are typically performed on Octet RED384 (see, e.g., Estep et al., High throughput solution-based measurement of antibody-antigen affinity and epitope binning. Mabs 5(2), 270-278 (2013), incorporated herein by reference in its entirety). In short, ForteBio affinity measurements were performed by loading IgG online onto the AHQ sensor. The sensor was equilibrated offline in assay buffer for 30 min, then monitored online for 60 s to establish a baseline. The IgG-loaded sensor was exposed to 100 nM antigen for 3 min, then transferred to assay buffer for 3 min to measure the dissociation rate. All kinetics were analyzed using a 1:1 binding model. Recombinant human IL-27 protein (R&D Systems Cat: 2526-IL) was used as the antigen. Affinity measurements of the anti-IL-27 antibody were performed as follows: Figure 1 As shown.

[0809] ForteBio Epitope Grouping / Ligand Blockade

[0810] Epitope grouping / ligand blocking was performed using a standard sandwich cross-blocking assay. A control anti-target IgG was loaded onto the AHQ sensor, and unoccupied Fc-binding sites on the sensor were blocked with an unrelated human IgG1 antibody. The sensor was then exposed to 100 nM of the target antigen, followed by exposure to a secondary anti-target antibody or ligand. Additional binding of the secondary antibody or ligand after antige...

Claims

1. An isolated 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, wherein: The heavy chain CDR1, CDR2 and CDR3 sequences are shown in SEQ ID NOs: 161, 162 and 163, respectively, and the light chain CDR1, CDR2 and CDR3 sequences are shown in SEQ ID NOs: 169, 170 and 171, respectively, according to IMGT nomenclature.

2. An isolated 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, wherein The heavy chain CDR1, CDR2 and CDR3 sequences are shown in SEQ ID NOs: 164, 165 and 166, respectively, and the light chain CDR1, CDR2 and CDR3 sequences are shown in SEQ ID NOs: 172, 173 and 174, respectively, according to NT nomenclature.

3. The isolated monoclonal antibody or antigen binding portion thereof of claim 1 or 2, wherein the antibody or antigen binding portion thereof antagonizes IL-27.

4. The isolated monoclonal antibody or antigen binding portion thereof of claim 1 or 2, wherein the antibody or antigen binding portion thereof inhibits or reduces STAT1 phosphorylation in a cell.

5. The isolated monoclonal antibody or antigen binding portion thereof of claim 4, wherein the cell is an immune cell.

6. The isolated monoclonal antibody or antigen binding portion thereof of claim 4, wherein the cell is a cancer cell.

7. The isolated monoclonal antibody or antigen binding portion thereof of claim 1 or 2, wherein the antibody or antigen binding portion thereof inhibits or reduces the inhibition of CD161 expression in a cell.

8. The isolated monoclonal antibody or antigen binding portion thereof of claim 7, wherein the cell is an immune cell.

9. The isolated monoclonal antibody or antigen-binding portion thereof of claim 1 or 2, wherein the antibody or antigen-binding portion thereof inhibits or reduces PD-L1 and / or TIM-3 expression in a cell.

10. The isolated monoclonal antibody or antigen binding portion thereof of claim 9, wherein the cell is an immune cell.

11. The isolated monoclonal antibody or antigen binding portion thereof of claim 9, wherein the cell is a cancer cell.

12. The isolated monoclonal antibody or antigen binding portion thereof of claim 9, wherein the cell is a cancer cell, wherein the antibody or antigen binding portion thereof inhibits or reduces PD-L1 expression in the cancer cell.

13. The isolated monoclonal antibody or antigen binding portion thereof of claim 1 or 2, wherein the antibody or antigen binding portion thereof induces or enhances PD-1-mediated secretion of one or more cytokines from cells.

14. The isolated monoclonal antibody or antigen binding portion thereof of claim 13, wherein the one or more cytokines are IFNg, IL-17, TNFα or IL-6.

15. The isolated monoclonal antibody or antigen binding portion thereof of claim 13, wherein the one or more cytokines is TNFα.

16. The isolated monoclonal antibody, or antigen binding portion thereof, of claim 1 or 2, wherein the antibody is selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD and IgE antibodies.

17. The isolated monoclonal antibody or antigen binding portion thereof according to claim 16, wherein the antibody is an IgG1 antibody or an IgG4 antibody.

18. The isolated monoclonal antibody, or antigen binding portion thereof, of claim 17, wherein the antibody comprises a wild-type IgG1 heavy chain constant region.

19. The isolated monoclonal antibody, or antigen binding portion thereof, of claim 17, wherein the antibody comprises a wild-type IgG4 heavy chain constant region.

20. The isolated monoclonal antibody, or antigen binding portion thereof, of claim 1 or 2, wherein the antibody comprises an Fc domain comprising at least one mutation.

21. The isolated monoclonal antibody, or antigen binding portion thereof, of claim 20, wherein the antibody comprises a mutant IgG1 heavy chain constant region.

22. The isolated monoclonal antibody, or antigen binding portion thereof, of claim 20, wherein the antibody comprises a mutant IgG4 heavy chain constant region.

23. The isolated monoclonal antibody, or antigen binding portion thereof, of claim 22, wherein the mutant IgG4 heavy chain constant region comprises any one of the substitutions S228P, L235E, L235A, or a combination thereof according to EU numbering.

24. An isolated monoclonal antibody or antigen binding portion thereof that specifically binds to and antagonizes human IL-27, wherein the antibody or antigen binding portion thereof comprises heavy and light chain variable regions having the amino acid sequences shown in SEQ ID NOs: 167 and 175, respectively.

25. 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 having the amino acid sequences shown in SEQ ID NOs: 177 and 179, respectively.

26. 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 having the amino acid sequences shown in SEQ ID NOs: 181 and 179, respectively.

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

28. A nucleic acid comprising a nucleotide sequence encoding the light chain, the heavy chain, or both the light chain and the heavy chain of the isolated monoclonal antibody or antigen binding portion thereof of any one of claims 1 to 26. An expression vector comprising the nucleic acid of claim 28 .

30. A cell transformed with the expression vector of claim 29.

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

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

33. Use of the isolated monoclonal antibody or antigen-binding fragment of any one of claims 1 to 26 or the pharmaceutical composition of claim 27 in the preparation of a medicament for treating melanoma in a subject.

34. A kit comprising an anti-PD-1 antibody and the antibody or antigen-binding portion thereof of any one of claims 1 to 26 for simultaneous or sequential administration, and instructions for use thereof.

35. The use of claim 33, wherein the isolated monoclonal antibody, or antigen binding portion thereof, is administered in combination with one or more additional therapeutic agents or procedures, wherein the one or more additional therapeutic agents or procedures are selected from the group consisting of chemotherapy, immune-based therapy, surgical procedures, radiation procedures, or a combination thereof.

36. The use of claim 35, wherein the one or more additional therapeutic agents or procedures is a chemotherapy selected from the group consisting of targeted anti-cancer therapy, oncolytic drugs, cytotoxic agents, or a combination thereof.

37. The use of claim 35, wherein the one or more additional therapeutic agents or procedures are immune-based therapies selected from the group consisting of cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules, vaccines, cellular immunotherapy, or a combination thereof.

38. The use of claim 35, wherein the one or more additional therapeutic agents or procedures is a surgical procedure.

39. The use of claim 35, wherein the one or more additional therapeutic agents or procedures are radiation procedures.

40. The use of claim 35, wherein the one or more additional therapeutic agents are a PD-1 antagonist, a PD-L1 inhibitor, a TIM-3 inhibitor, a LAG-3 inhibitor, a TIGIT inhibitor, a CD112R inhibitor, a TAM inhibitor, a STING agonist, a 4-1BB agonist, or a combination thereof.

41. The use of claim 40, wherein the one or more additional therapeutic agents is a PD-1 antagonist.

42. The use according to claim 41, wherein the PD-1 antagonist is selected from the group consisting of: PDR001, nivolumab, pembrolizumab, pidilizumab, MEDI0680, REGN2810, TSR-042, PF-06801591 and AMP-224.

43. The use of claim 40, wherein the PD-L1 inhibitor is selected from the group consisting of: FAZ053, atezolizumab, avelumab, durvalumab, and BMS-936559.

44. The use of claim 40, wherein the one or more additional therapeutic agents are selected from the group consisting of sunitinib, cabozantinib, axitinib, lenvatinib, everolimus, bevacizumab, epalrestat, NKTR-214, tivozanib, abenotat, ipilimumab, tremelimumab, pazopanib, sorafenib, temsirolimus, ramucirumab, niraparib, sovolitinib, vorolanib, regorafenib, donafenib, carrelizumab, pemudevi, ramucirumab, apatinib, doxorubicin capsules, tivantinib, ADI-PEG 20. Binitinib, apatinib mesylate, nintedanib, rilutumab, nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab, cemiplimab-rwlc, tislelizumab, and spartalizumab.

45. The use of claim 40, wherein the one or more additional therapeutic agents is a TIM-3 inhibitor.

46. ​​The use of claim 45, wherein the TIM-3 inhibitor is MGB453 or TSR-022.

47. The use of claim 40, wherein the one or more additional therapeutic agents is a LAG-3 inhibitor.

48. The use of claim 47, wherein the LAG-3 inhibitor is selected from the group consisting of LAG525, BMS-986016 and TSR-033.

49. The use of claim 40, wherein the one or more additional therapeutic agents is a TIGIT inhibitor.

50. The use of claim 40, wherein the one or more additional therapeutic agents is a CD112R inhibitor.

51. The use of claim 40, wherein the one or more additional therapeutic agents is a TAM inhibitor.

52. The use of claim 40, wherein the one or more additional therapeutic agents is a 4-1BB agonist.

53. The use of claim 35, wherein the one or more additional therapeutic agents is a tyrosine kinase inhibitor (TKI).

54. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 26 or a pharmaceutical composition according to claim 27 in the preparation of a kit for use in a method for detecting IL-27 or EBI3 in a sample from a subject, wherein the method comprises (a) if IL-27 or EBI3 is present in the sample from the subject, contacting the sample with a detection antibody under conditions that allow the detection antibody to form a detection antibody-EBI3 complex; and (b) detecting the presence of the complex produced in step (a), if any, and the method further comprises contacting the sample with a capture antibody to produce a complex comprising IL-27 and the capture antibody if IL-27 is present in the sample, wherein the capture antibody is an antibody or antigen-binding portion thereof according to any one of claims 1 to 26, and contacting the sample with the capture antibody and then with the detection antibody.

55. The use of claim 54, wherein the detection antibody is coupled to a detectable label.

56. The use of claim 54, wherein the capture antibody is immobilized on a solid support.

57. The use of claim 54, wherein the sample is a body fluid sample.

58. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 26 or the pharmaceutical composition according to claim 27 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) if IL-27 or EBI3 is present in the sample from the subject suspected of having an IL-27-associated cancer, contacting the sample with a detection antibody under conditions that allow the detection antibody to form a detection antibody-EBI3 complex; and (b) detecting the presence of the complex produced in step (a), if any, and the method further comprises contacting the sample with a capture antibody if IL-27 is present in the sample to produce a complex comprising IL-27 and the capture antibody, wherein the capture antibody is an antibody or antigen-binding portion thereof according to any one of claims 1 to 26, and contacting the sample with the capture antibody and then with the detection antibody.

59. The use of claim 58, wherein the detection antibody is coupled to a detectable label.

60. The use of claim 58, wherein the capture antibody is immobilized on a solid support.

61. The use of claim 58, wherein the sample is a body fluid sample.

62. The use of claim 61, wherein the body fluid sample is blood, serum, plasma, cell lysate or tissue lysate.

63. The use of claim 58, 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.

64. The use of claim 63, wherein the cancer is renal cell carcinoma (RCC).

65. The use of claim 63, wherein the cancer is hepatocellular carcinoma (HCC).

66. The use of claim 63, wherein the cancer is selected from leukemia and lymphoma.

67. The use of claim 63, wherein the cancer is ovarian cancer.

68. A kit comprising the isolated monoclonal antibody or antigen binding portion thereof of any one of claims 1 to 26 or the pharmaceutical composition of claim 27 and instructions for use in treating an IL-27-related cancer in a subject.

69. The kit of claim 68, further comprising instructions for use in combination with one or more additional therapeutic agents or procedures.

70. A kit comprising the isolated monoclonal antibody or antigen binding portion thereof of any one of claims 1 to 26 or the pharmaceutical composition of claim 27, and instructions for use in detecting IL-27 in a sample from a subject.

71. The kit of claim 70, further comprising instructions for detecting an IL-27-associated cancer in a subject.

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