Isolated antibody and use thereof

Abstract

The present invention relates to the field of biomedicine, and in particular to an antibody specifically binding to IL35 and IL27, and a use of the antibody. The antibody can inhibit binding of IL35 to a receptor thereof, and can also inhibit binding of IL27 to a receptor thereof. Moreover, the present invention further relates to a nucleic acid sequence encoding the antibody, a vector and cell comprising the nucleic acid sequence, a pharmaceutical composition comprising the antibody, and a use of the antibody or the composition comprising same for preparing a drug for treating an immune-related disease and a cancer.

Description

A separated antibody and its application

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to PCT application No. PCT / CN2023 / 114598, application date 2023.08.24, and invention name “A separated antibody and its use”, and the entire contents of that application are incorporated herein by reference.

[0003] References to electronic sequence listings

[0004] The contents of the electronic sequence listing (text name: IL35-27.xml, recording date: 2023.07.24, size: 58 KB) are incorporated herein by reference in their entirety. Technical Field

[0005] The present invention relates to the field of medicine, and in particular to a separated antibody or antigen-binding fragment, and a preparation method and use thereof. Background Art

[0006] Interleukin-35 (IL35) is a recently discovered heterodimeric cytokine in the IL12 family. It is composed of IL12 p35 (p35) and Epstein-Barr virus-induced gene 3 (EBI3) (Collison, Lauren W et al. Nature vol. 450, 7169 (2007): 566-9.; Niedbala, Wanda et al. European journal of immunology vol. 37, 11 (2007): 3021-9.). Unlike other IL12 family members, it signals through four unconventional receptors: IL12Rβ2 / WSX-1 (also known as IL27Rα), IL12Rβ2 / IL12Rβ2, IL12Rβ2 / GP130, and GP130 / GP130. The composition of the IL35 receptor varies depending on the cell type. In T cells, IL35 binds to gp130 and IL12Rβ2, and transmits signals through IL12Rβ2 / GP130 heterodimers or homodimers of each subunit (Collison, Lauren W, and Dario AA Vignali. Immunological reviews vol. 226 (2008): 248-62.; Collison, Lauren W et al. Nature immunology vol. 13, 3 290-9.5 Feb. 20). In B cells, IL35 transmits signals through IL12Rβ2 and WSX-1 (Shen, Ping et al. Nature vol. 507, 7492 (2014): 366-370.). Different receptor chains involved in IL35 trigger different STAT activation. For example, IL35 binds to IL12Rβ2 / WSX-1, leading to phosphorylation of STAT1 and STAT3 (Shen, Ping et al. Nature vol. 507, 7492 (2014): 366-370.), and binds to IL12Rβ2 / gp130, inducing the formation of STAT1-STAT4 heterodimers. IL35 signals through IL12Rβ2 homodimers or gp130 homodimers, leading to phosphorylation of STAT4 or STAT1, respectively (Collison, Lauren W et al. Nature immunology vol. 13, 3 290-9.5 Feb. 20).

[0007] IL35 exerts an immunosuppressive function by inhibiting the differentiation of helper T cells 17 and promoting the proliferation of Treg cells. Interleukin-35 plays a key role in a variety of immune-related diseases (such as autoimmune diseases, viral and bacterial infections) and tumors (Zhang, Junfeng et al. Immunology vol. 157, 1 (2019): 13-20.). It has been confirmed that IL35 is highly expressed in patients with chronic hepatitis, and its expression level is positively correlated with the severity of infection (Dong, Yuejiao et al. Life sciences vol. 252 (2020): 117663.). In addition, IL35 can also inhibit liver regeneration and the proliferation of hepatocytes in vivo and in vitro (Yang, Lanlan et al. Digestive diseases and sciences vol. 65, 12 (2020): 3614-3623.). IL35 is highly expressed in tumor tissues of human nasopharyngeal carcinoma, melanoma, and malignant B-cell lymphoma. These tumors produce IL35 that activates CD11b in the tumor microenvironment. + GR1 + Bone marrow-derived immunosuppressive cells help tumor cells escape immune evasion (Wang, Zhihui et al. Journal of immunology (Baltimore, Md.: 1950) vol. 190, 5 (2013): 2415-23.). Turnis et al. believe that IL35 present in the tumor microenvironment leads to reduced lymphocyte infiltration, reduced effector cell proliferation, increased tumor load, and reduced survival rate of immune-competent hosts. In addition, Treg-derived IL35 promotes the expression of multiple inhibitory receptors (e.g., programmed death receptor 1 (PD-1), lymphocyte activation gene 3 (LAG-3), T cell immunoglobulin and mucin domain-3 (TIM-3)), thereby promoting T cell exhaustion in tumors (Turnis, Meghan E et al. Immunity vol. 44, 2 (2016): 316-29.). Therefore, neutralizing IL35 with monoclonal antibodies may be an attractive immunotherapy approach.

[0008] IL27 is also a heterodimeric cytokine in the IL12 family, composed of IL27p28 and EBI3 subunits (Pflanz, Stefan et al. Immunity vol. 16, 6 (2002): 779-90). IL27 signals through the cell surface heterodimeric receptor WSX-1 / gp130 (Pflanz, Stefan et al. Journal of Immunology (Baltimore, Md.: 1950) vol. 172, 4 (2004): 2225-31). When IL27 binds to its receptor, STAT1 and STAT3 become phosphorylated. Cell type and activation state may affect IL27 signaling, thereby changing the corresponding STAT activation levels: mast cells (STAT3) (Pflanz, Stefan et al. Journal of immunology (Baltimore, Md.: 1950) vol. 172, 4 (2004): 2225-31.), monocytes (STAT1, STAT3 and NF-κB) (Guzzo, Christina et al. The Journal of biological chemistry vol. 285, 32 (2010): 24404-11.), macrophages (STAT1 and STAT3) (Greenwell-Wild, Teresa et al. Blood vol. 114, 9 (2009): 1864-74.), naive CD4+ T cells (TYK2, JAK1, KAK2, STAT1, STAT2, STAT3, STAT4) (Kamiya, Sadahiro et al. Journal of Immunology (Baltimore, Md.: 1950) vol. 173, 6 (2004): 3871-7.), and B cells (STAT1 and STAT3) (Larousserie, Frédérique et al. Journal of Immunology (Baltimore, Md.: 1950) vol. 176, 10 (2006): 5890-7.). IL27 was originally considered an immunostimulatory cytokine because of its ability to induce Th1 differentiation.However, in recent years, many studies have also discovered various immunosuppressive effects of IL27, including inhibiting Th17 cell differentiation and inducing the expression of co-inhibitory receptors on T cells (for example, PD-L1, LAG-3, TIM-3, cytotoxic T lymphocyte-associated protein 4 (CTLA-4) and Ig and ITIM domain protein T cell immunoreceptors (Hirahara, Kiyoshi et al. Immunity vol. 36, 6 (2012): 1017-30.; Do, JS et al. Mucosal immunology vol. 9, 1 (2016): 137-45.; Chihara, Norio et al. Nature vol. 558, 7710 (2018): 454-459.).

[0009] Studies have shown that in a mouse sepsis model induced by subcutaneous injection of Escherichia coli, IL27 can improve the survival rate of mice after being blocked (Seman, Brittany G et al. Infection and immunity vol.88, 3e00828-19.20Feb.2020). Similarly, in an acute lung injury model caused by cecal ligation and puncture (CLP), treatment with IL27 neutralizing antibodies can reduce lung inflammation and improve the survival rate of mice (Xu, Fang et al. Journal of clinical immunology vol.33, 7 (2013): 1257-68.). In a mouse model of secondary Staphylococcus aureus pneumonia after influenza infection, inhibiting IL27 can also accelerate bacterial clearance (Robinson, Keven M et al. "The role of IL27 in susceptibility to post-influenza Staphylococcus aureus pneumonia." Respiratory research vol.16, 1 10.5Feb.2015). In addition, studies have found that IL27 is associated with promoting cancer progression because it can promote the proliferation of human leukemia cell lines (Jia, Haiyan et al. Journal of interferon & cytokine research: the official journal of the International Society for Interferon and Cytokine Research vol. 36, 5 (2016): 302-16.), and tumor cells can secrete IL27, and high levels of IL27 are associated with advanced cancer (Larousserie, F et al. The Journal of pathology vol. 209, 3 (2006): 360-8.; Diakowska, Dorota et al. Advances in clinical and experimental medicine: official organ Wroclaw Medical University vol. 22, 5 (2013): 683-91.; Gonin, Julie et al. PloS one vol. 8, 10e75694. 10Oct. 2013). Therefore, IL27 may serve as a potential target for the treatment of immune-related diseases and tumors.

[0010] EBI3 is a subunit shared by IL27 and IL35 cytokines. It is a transcript induced by Epstein-Barr virus infection of B cells, hence the name EBI3 (Devergne, O et al. Journal of virology vol. 70, 2 (1996): 1143-53). EBI3 can also be expressed and secreted in other immune cells, including activated dendritic cells, regulatory T cells, and regulatory B cells. Studies have shown that in MCMV-infected EBI3-deficient mice, NK cell production of IL10 is reduced, dendritic cell maturation and CD8 + Increased T cell activation and significantly reduced viral loads in salivary gland and oral lavage fluids suggest that EBI3 plays an important role in the establishment of MCMV latency (Jensen, Helle et al. Proceedings of the National Academy of Sciences of the United States of America vol. 114, 7 (2017): 1625-1630). Furthermore, studies have shown that EBI3 is highly expressed in cancer tissues and correlates with poor prognosis and tumor progression in breast cancer (Jiang, Jinyan, and Xiangyong Liu. Oncology research and treatment vol. 41, 3 (2018): 111-115). Therefore, EBI3 also plays an important role in immune-related diseases and tumors.

[0011] Currently, there are reports of antibodies targeting IL27. For example, patent application CN115087671A discloses anti-IL27 antibodies (the anti-IL27 Ab1 in the patent application serves as the control antibody in the Examples section of this application) and their applications. However, no reports of antibodies targeting IL35 have been found. IL35 and IL27 share the same EBI3 subunit, and both can act as immunosuppressive molecules in immune-related diseases and tumors. Therefore, there is a need to develop antibody molecules that bind to IL35, IL27, or EBI3 to effectively treat related diseases.

[0012] The disclosures of all publications, patents, patent applications, and published patent applications mentioned herein are incorporated by reference in their entirety.

[0013] Application Overview

[0014] In some embodiments, an isolated antibody is provided comprising a heavy chain variable domain (V H ), the V Hcomprising: a heavy chain complementarity determining region (HC-CDR) 1 comprising SDYAWN (SEQ ID NO: 1); HC-CDR2 comprising SISYSGSTDYNPSLKS (SEQ ID NO: 4); and HC-CDR3 comprising SPIYYDYEEGDY (SEQ ID NO: 8); and a light chain variable domain (V L ), the V L It comprises: a light chain complementary determining region (LC-CDR) 1 comprising KASQDIX1X2YLS (SEQ ID NO:25), wherein X1 is A, N, Q or S, and X2 is A or S; a LC-CDR2 comprising RANRLVX1 (SEQ ID NO:26), wherein X1 is D, E or S; and a LC-CDR3 comprising LQYDEFPLT (SEQ ID NO:22).

[0015] In some embodiments, an isolated antibody is provided, comprising: (i) V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 4, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 8; and V L , the V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 18, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 22; (ii) V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 4, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 8; and V L , the V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 22; (iii) V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 4, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 8; and V L, the V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 20, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 22; (iv) V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 4, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 8; and V L , the V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 13, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 20, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 22; (v) V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 4, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 8; and V L , the V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 14, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 18, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 22; (vi) V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 4, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 8, or the V H A variant comprising up to about 5 amino acid substitutions in its HC-CDRs; and V L , the V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 14, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 22; (vii) V H , the V Hcomprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 4, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 8; and V L , the V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 14, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 20, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 22; (viii) V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 4, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 8; and V L , the V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 15, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 18, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 22; (ix) V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 4, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 8; and V L , the V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 16, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 20, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 22; (x) V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 4, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 8; and V L , the V L comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 16, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 18, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 22; or (xi) V H , the V Hcomprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 4, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 8; and V L , the V L It comprises: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 13, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 18, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 22.

[0016] In some embodiments, an isolated antibody is provided comprising: (i) V H , comprising the amino acid sequence of SEQ ID NO: 28 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 28; and V L , comprising the amino acid sequence of SEQ ID NO: 44 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 44; (ii) V H , comprising the amino acid sequence of SEQ ID NO: 29 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 29; and V L , comprising the amino acid sequence of SEQ ID NO: 45 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 45; (iii) V H , comprising the amino acid sequence of SEQ ID NO: 28 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 28; and V L , comprising the amino acid sequence of SEQ ID NO: 46 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 46; (iv) V H , comprising the amino acid sequence of SEQ ID NO: 29 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 29; and V L , comprising the amino acid sequence of SEQ ID NO: 47 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 47; (v) V H , comprising the amino acid sequence of SEQ ID NO: 28 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 28; and V L, comprising the amino acid sequence of SEQ ID NO: 48 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 48; (vi) V H , comprising the amino acid sequence of SEQ ID NO: 29 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 29; and V L , comprising the amino acid sequence of SEQ ID NO: 49 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 49; (vii) V H , comprising the amino acid sequence of SEQ ID NO: 28 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 28; and V L , comprising the amino acid sequence of SEQ ID NO: 50 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 50; or (viii) V H , comprising the amino acid sequence of SEQ ID NO: 29 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 29; and V L , comprising the amino acid sequence of SEQ ID NO: 51 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 51.

[0017] In some embodiments, the antibodies of the present application can bind to IL35, IL27, or EBI3.

[0018] In some embodiments, the isolated antibody binds to the target (eg, IL35, IL27, or EBI3) with a Kd value of 1 pM to 10 nM.

[0019] In some embodiments, a separated antibody is provided that competes with any of the above separated antibodies for binding to a target (e.g., IL35, IL27, or EBI3). In some embodiments, a separated antibody is provided that binds to the same epitope as any of the above separated antibodies.

[0020] In certain embodiments, any of the separated antibodies described above comprises an Fc fragment. In certain embodiments, the separated antibody is a full-length IgG antibody. In certain embodiments, the separated antibody is a full-length IgG1, IgG2, IgG3 or IgG4 antibody. In certain embodiments, the separated antibody is a chimeric, fully human or humanized antibody. In certain embodiments, the separated antibody is an antigen-binding fragment selected from Fab, Fab', F(ab)'2, Fab'-SH, single-chain Fv (scFv), Fv fragment, dAb, Fd, nanobody, diabody and linear antibody.

[0021] In some embodiments, an isolated nucleic acid molecule is provided, encoding any of the isolated antibodies described above. In some embodiments, a vector is provided, comprising any of the nucleic acid molecules described above. In some embodiments, a host cell is provided, comprising any of the isolated antibodies described above, any of the nucleic acid molecules described above, or any of the vectors described above. In some embodiments, a method for preparing an antibody is provided, comprising: a) culturing any of the host cells described above under conditions effective for expressing the antibody; and b) obtaining the expressed antibody from the host cell.

[0022] In some embodiments, a method for treating a desired individual disease or condition is provided, comprising administering to the individual an effective amount of any of the isolated antibodies described above. In some embodiments, the use of any of the isolated antibodies described above in the preparation of a pharmaceutical composition for treating a desired individual disease or condition is provided. In some embodiments, the use of any of the isolated antibodies described above or a pharmaceutical composition comprising the antibody in the preparation of a medicament for treating a disease or condition is provided. In some embodiments, the disease or condition is related to the IL35 and / or IL27 signaling pathway, including immune-related diseases, tumors, and the like. In some embodiments, the disease or condition is selected from, for example, viral infection, bacterial infection, and tumor.

[0023] Also provided are pharmaceutical compositions, kits, and manufactured articles comprising any of the antibodies described above. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 shows the binding curves of exemplary antibodies hum-m58-1, hum-m58-2, hum-m58-D56E-1, hum-m58-N30QD56S-2, and hum-m58-N30AD56S-2 to EBI3.

[0025] FIG2 shows the plasma concentration curves of exemplary antibodies hum-m58-D56E-1, hum-m58-N30QD56S-2, or hum-m58-N30AD56S-2.

[0026] FIG3 shows the results showing that the exemplary antibody hum-m58-N30AD56S-2 can effectively inhibit IL27-induced STAT1 phosphorylation in monkeys.

[0027] Detailed description of this application

[0028] In one aspect, the present application provides isolated antibody molecules. Through screening of a constructed scFv phage library and a combination of appropriately designed biochemical and biological experiments, antibody molecules capable of binding to human IL35, IL27 or EBI3 and inhibiting the interaction of human IL35 or IL27 with their receptors have been identified.

[0029] The antibodies provided herein include, for example, full-length antibodies, single-chain antibodies (scFvs), Fc fusion proteins, multispecific (eg, bispecific) antibodies, immunoconjugates, and the like.

[0030] In another aspect, the present application provides an isolated antibody comprising: a heavy chain variable domain (V H ), the V H comprising: HC-CDR1 comprising SDYAWN (SEQ ID NO: 1); HC-CDR2 comprising SISYSGSTDYNPSLKS (SEQ ID NO: 4); and HC-CDR3 comprising SPIYYDYEEGDY (SEQ ID NO: 8); and a light chain variable domain (V L ), the V L comprising: an LC-CDR1 comprising KASQDIX1X2YLS (SEQ ID NO: 25), wherein X1 is A, N, Q or S, and X2 is A or S; an LC-CDR2 comprising RANRLVX1 (SEQ ID NO: 26), wherein X1 is D, E or S; and an LC-CDR3 comprising LQYDEFPLT (SEQ ID NO: 22).

[0031] In another aspect, the present application provides an isolated antibody comprising: a heavy chain variable domain (V H ), the V Hcomprising: HC-CDR1 comprising NSGMS (SEQ ID NO: 2); HC-CDR2 comprising TINSNGGNTYYPDSVKG (SEQ ID NO: 5); and HC-CDR3 comprising DNGELYYDNWYFDV (SEQ ID NO: 9); and a light chain variable domain (V L ), the V L It comprises: an LC-CDR1 comprising RASESVDSYGNSFMH (SEQ ID NO: 17); an LC-CDR2 comprising RASNLES (SEQ ID NO: 21); and an LC-CDR3 comprising QQSNDDPPT (SEQ ID NO: 23).

[0032] In another aspect, the present application provides an isolated antibody comprising: a heavy chain variable domain (V H ), the V H comprising: HC-CDR1 comprising SDYAWN (SEQ ID NO: 1); HC-CDR2 comprising SIDYSGDSNYNPSLKS (SEQ ID NO: 6); and HC-CDR3 comprising SPIYYDYDEGDY (SEQ ID NO: 10); and a light chain variable domain (V L ), the V L comprising: an LC-CDR1 comprising KASQDINSYLS (SEQ ID NO: 12); an LC-CDR2 comprising RANRLVD (SEQ ID NO: 18); and an LC-CDR3 comprising LQYDEFPLT (SEQ ID NO: 22).

[0033] In another aspect, the present application provides an isolated antibody comprising: a heavy chain variable domain (V H ), the V H comprising: HC-CDR1 comprising SNGMS (SEQ ID NO: 3); HC-CDR2 comprising TINSNGGSTYYPDSVKG (SEQ ID NO: 7); and HC-CDR3 comprising DNGELYYANWYFDV (SEQ ID NO: 11); and a light chain variable domain (V L ), the V L It comprises: an LC-CDR1 comprising RASESVDSYGNSFMH (SEQ ID NO: 17); an LC-CDR2 comprising RASNLES (SEQ ID NO: 21); and an LC-CDR3 comprising QQSIEDPPT (SEQ ID NO: 24).

[0034] In some embodiments, the antibodies of the present application can bind to IL35, IL27, or EBI3.

[0035] Also provided are nucleic acids encoding the above antibodies, compositions comprising the above antibodies, and methods for preparing and using the above antibodies.

[0036] definition

[0037] As described herein, "treatment" or "treating" is a method for obtaining beneficial or desired results, including clinical results. In view of the purposes of this application, the beneficial or desired clinical results, include but are not limited to one or more of the following: alleviating one or more symptoms caused by the disease, alleviating the degree of the disease, stabilizing the disease (e.g., preventing or delaying disease worsening), preventing or delaying the spread of the disease (e.g., metastasis, spread of bacteria / viruses), preventing or delaying disease recurrence, delaying or slowing disease progression, improving the disease state, alleviating the disease (partial or complete), reducing the dosage of one or more other drugs required for treating the disease, delaying disease progression, improving or improving quality of life, increasing weight, and / or prolonging survival. At the same time, "treatment" also includes the reduction of disease pathology results (e.g., for cancer, tumor volume; for infectious diseases, pathogen load). The method of the present application takes into account any one or more aspects of these treatments.

[0038] The term "antibody" includes full-length antibodies and antigen-binding fragments thereof. Full-length antibodies include two heavy chains and two light chains. The variable regions of the light and heavy chains are responsible for antigen binding. The variable regions in both chains typically include three hypervariable loops, known as complementarity determining regions (CDRs) (light chain (LC) CDRs include LC-CDR1, LC-CDR2, and LC-CDR3, and heavy chain (HC) CDRs include HC-CDR1, HC-CDR2, and HC-CDR3). The CDR boundaries of the antibodies or antigen-binding fragments disclosed herein can be defined or identified by the Kabat, Chothia, or Al-Lazikani conventions (Al-Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Kabat 1987; Kabat 1991). The three CDR regions of the heavy or light chain are inserted between flanking segments called framework regions (FRs), which are more highly conserved than the CDR regions and form a scaffold that supports the hypervariable loops. The constant regions of the heavy and light chains are not involved in antigen binding, but exhibit various effector functions. Antibodies are classified based on the amino acid sequence of their heavy chain constant regions. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by having α, δ, ε, γ, and μ heavy chains, respectively. Several major antibody classes are divided into subclasses, such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain).

[0039] As used herein, the term "antigen-binding fragment" includes antibody fragments, such as diabodies, Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabodies (dsdiabodies), single-chain Fv (scFv), scFv dimers (divalent diabodies), multispecific antibodies composed of antibody fragments comprising one or more CDRs, single-domain antibodies, nanobodies, domain antibodies, bivalent domain antibodies, or any other antibody fragment that can bind to an antigen but does not contain a complete antibody structure. Antigen-binding fragments can bind to the same antigen as a parent antibody or parent antibody fragment (such as a parent scFv). Antigen-binding fragments also include fusion proteins comprising the above-mentioned antibody fragments. In some embodiments, an antigen-binding fragment may include one or more CDRs from a specific human antibody that are transplanted to a framework region from one or more different human antibodies.

[0040] As used herein, the term "epitope" refers to a specific group of atoms or amino acids on an antigen to which an antibody or antibody portion binds. If two antibodies or antibody portions exhibit competitive binding to an antigen, they likely bind to the same epitope on the antigen.

[0041] As used herein, a first antibody "competes" for binding to a target (e.g., IL35, IL27, or EBI3) with a second antibody when the first antibody inhibits binding of the second antibody to the target (e.g., IL35, IL27, or EBI3) by at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) at equimolar concentrations, and vice versa. PCT Publication WO 03 / 48731 describes a high-throughput antibody "epitope binning" method based on cross-competition.

[0042] As used herein, the terms "specifically bind," "specifically recognize," or "specific for" refer to a measurable and reproducible interaction, e.g., binding of an antibody to a target that can confirm the presence of the target in a heterogeneous population of molecules, including biomolecules. For example, an antibody that specifically recognizes a target (which may be an epitope) means that the antibody binds to the target with greater affinity, avidity, more readily, and / or more persistently than it binds to other targets. In some embodiments, an antibody that specifically recognizes an antigen reacts with one or more antigenic determinants of the antigen with an affinity that is at least 10 times greater than its binding affinity to other targets.

[0043] As used herein, an "isolated" antibody is one that (1) is not related to naturally occurring proteins, (2) does not contain other proteins from the same source, (3) is expressed by cells of a different species, or (4) does not occur in nature.

[0044] As used herein, the term "isolated nucleic acid" refers to a nucleic acid of genomic, cDNA, or synthetic origin, or a combination thereof. Depending on its origin, the "isolated nucleic acid" is (1) unrelated to all or part of the polynucleotide sequence found in nature, (2) operably linked to a polynucleotide sequence with which it is not naturally associated, or (3) not found in nature as part of a longer sequence.

[0045] As used herein, the term "CDR" or "complementarity determining region" refers to the non-contiguous antigen binding sites found within the variable domains of heavy and light chain polypeptides. In the literature Kabat et al., J. Biol. Chem. 252: 6609-6616 (1977); Kabat et al., USDept. of Health and Human Services, "Sequences of proteins of immunological interest" (1991); Chothia et al., J. Mol. Biol. 196: 901-917 (1987); Al-Lazikani B. et al. al., J. Mol. Biol., 273: 927-948 (1997); MacCallum et al., J. Mol. Biol. 262: 732-745 (1996); Abhinandan and Martin, Mol. Immunol., 45: 3832-3839 (2008); Lefranc MPet These specific regions are described in, for example, Desmond et al., Dev. Comp. Immunol., 27:55-77 (2003); and Honegger and Plückthun, J. Mol. Biol., 309:657-670 (2001), where these definitions include overlap or subsets of amino acid residues when compared to one another. However, any definition used to designate a CDR of an antibody, transplanted antibody, or variant thereof is included within the scope of the term as defined and used herein. Table 1 lists the positions of the amino acid residues included in the CDRs defined by each of the above-cited references for comparison. Algorithms and binding interfaces for CDR prediction are known in the art, including, for example, Abhinandan and Martin, Mol. Immunol., 45: 3832-3839 (2008); Ehrenmann F. et al., Nucleic Acids Res., 38: D301-D307 (2010); and Adolf-Bryfogle J. et al., Nucleic Acids Res., 43: D432-D438 (2015). The contents of the references cited in this paragraph are incorporated herein by reference in their entirety for use in this application and in one or more claims that may be included in this article.

[0046] Table 1: CDR definitions

[0047] 1The amino acid residue numbers refer to the nomenclature of Kabat et al.

[0048] 2 The amino acid residue numbers refer to the nomenclature of Chothia et al.

[0049] 3 The amino acid residue numbers refer to the nomenclature of MacCallum et al.

[0050] 4 The amino acid residue numbers refer to the nomenclature of Lefranc et al.

[0051] 5 The amino acid residue numbers refer to the nomenclature of Honegger and Plückthun.

[0052] The term "chimeric antibody" refers to an antibody in which a portion of the heavy chain and / or light chain is identical or homologous to the corresponding sequence in an antibody from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical or homologous to the corresponding sequence in an antibody from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they have the biological activity described in the present application (see US Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984)).

[0053] "Fv" is the smallest antibody fragment that contains a complete antigen recognition and binding site. This fragment is a dimer formed by a heavy chain variable domain and a light chain variable domain tightly non-covalently linked. Six hypervariable loops (3 loops each in the light chain and heavy chain) are derived from the folding of these two domains. The hypervariable loops provide the antibody with amino acid residues for binding to the antigen and confer specificity for binding to the antibody. However, even a single variable domain (or half of an Fv fragment, which only contains 3 CDRs specific for the antigen) has the ability to recognize and bind to the antigen, although its affinity is lower than that of the complete binding site.

[0054] "Single-chain Fv", also abbreviated as "sFv" or "scFv", is a Fv that contains V molecules linked into a single polypeptide chain. H and V L In some embodiments, the scFv polypeptide further comprises a V H and V LThe linker polypeptide between the domains allows the scFv to form an ideal structure for antigen binding. For an overview of scFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0055] The term "diabody" is used in V H and V L A small antibody fragment is prepared by constructing scFv fragments (see above) using short linkers (e.g., 5-10 residues) between the domains. This allows the variable domains to pair between chains rather than within the chain, resulting in a bivalent fragment, that is, a fragment with two antigen-binding sites. Bispecific diabodies are heterodimers of two "crossover" scFv fragments, in which the V domains of the two antibodies are H and V L The domains are located on different polypeptide chains. Diabodies are fully described in EP 404,097; WO 93 / 11161; Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

[0056] The "humanized" form of a non-human (such as a rodent) antibody is a chimeric antibody that includes minimal sequences from a non-human antibody. In most cases, a humanized antibody is a human immunoglobulin (recipient antibody) in which the hypervariable region (HVR) residues of the recipient antibody are replaced by hypervariable region residues from a non-human species such as a mouse, rat, rabbit or non-human mammal with ideal antibody specificity, affinity and performance (donor antibody). In some cases, residues in the human immunoglobulin framework region are replaced by corresponding non-human residues. In addition, a humanized antibody may include residues that are not present in either the recipient antibody or the donor antibody. These modifications can further improve the performance of the antibody. Typically, a humanized antibody will comprise substantially all, at least one, and usually two variable domains, wherein all or substantially all of the hypervariable loops correspond to the hypervariable loops of a non-human immunoglobulin, and all or substantially all of the framework regions are human immunoglobulin sequences. The human antibody optionally also comprises at least a portion of an immunoglobulin constant region (Fc), typically a constant region of a human immunoglobulin. For details, see Jones et al., Nature 321: 522-525 (1986); Riechmann et al., Nature 332: 323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2: 593-596 (1992).

[0057] The "percent (%) amino acid sequence identity" or "homology" of the polypeptide and antibody sequences identified herein is defined as the percentage of identical amino acid residues in a candidate sequence and the polypeptide sequence being compared, when the sequences are aligned, considering conservative substitutions as part of the sequence identity. Percent amino acid sequence identity can be determined by various alignment methods within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), or MUSCLE software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm needed to maximize alignment over the full length of the sequences being compared. However, for the purposes of this article, percent amino acid sequence identity values ​​are generated using the sequence alignment computer program MUSCLE (Edgar, RC, Nucleic Acids Research 32(5):1792-1797, 2004; Edgar, RC, BMC Bioinformatics 5(1):113, 2004).

[0058] The term "Fc receptor" or "FcR" is used to describe a receptor that binds to the Fc region of an antibody. In some embodiments, the FcR described herein is an FcR that binds to an IgG antibody (a gamma receptor), including receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), which have similar amino acid sequences and differ primarily in the cytoplasmic domain. The cytoplasmic domain of the activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM). The cytoplasmic domain of the inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) (see M.in Annu. Rev. Immunol. 15:203-234 (1997). The term also includes allotypes, such as the FcγRIIIA allotypes: FcγRIIIA-Phe158, FcγRIIIA-Val158, FcγRIIA-R131, and / or FcγRIIA-H131. FcRs are described in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991) and Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). The term FcR in this application encompasses other types of FcRs, including those identified in the future. The term FcR also includes the neonatal receptor FcRn, which is responsible for the transfer of maternal IgGs to the newborn (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)).

[0059] The term "FcRn" refers to the neonatal Fc receptor (FcRn). FcRn is structurally similar to the major histocompatibility complex (MHC) and consists of an α chain non-covalently bound to β2 microglobulin. The various functions of the neonatal Fc receptor FcRn are reviewed in Ghetie and Ward (2000) Annu. Rev. Immunol. 18, 739-766. FcRn plays an important role in the passive transport of immunoglobulins (IgGs) from mother to newborn and in regulating serum IgG levels. As a salvage receptor, FcRn can bind and transport endocytosed IgGs in their intact form within and between cells, protecting them from the default degradation pathway.

[0060] The "CH1 domain" of a human IgG heavy chain constant region typically stretches from amino acid 118 to amino acid 215 (EU numbering system).

[0061] The "hinge region" is generally defined as stretching from Glu 216 to Pro 230 of human IgG1 (Burton, Molec. Immunol. 22: 161-206 (1985)). By placing the first and last cysteine ​​residues that form inter-heavy chain disulfide bonds in the same positions as in IgG1, the hinge regions of other IgG isotypes can be aligned with the IgG1 sequence.

[0062] The "CH2 domain" of the human IgG Fc region typically extends from amino acids 231 to 340. The CH2 domain is unique in that it does not closely pair with another domain. Instead, two N-terminally linked branched sugar chains are inserted between the two CH2 domains of the intact native IgG molecule. It is hypothesized that carbohydrates may serve as a substitute for domain-to-domain pairing, helping to maintain CH2 domain stability. Burton, Molec. Immunol. 22:161-206 (1985).

[0063] The "CH3" domain includes the CH2 domain extending from the C-terminal residue within the Fc region (from amino acid 341 to the C-terminus of the antibody sequence, usually amino acid residue 446 or 447 in IgG).

[0064] A "functional Fc fragment" possesses the "effector functions" of a native Fc region sequence. Exemplary "effector functions" include C1q binding; complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; and downregulation of cell surface receptors (e.g., B cell receptor; BCR). Such effector functions generally require the Fc region to be bound to a binding domain (e.g., an antibody variable region) and can be assessed using a variety of experimental methods known in the art.

[0065] IgG Fc variant antibodies with "altered" FcR binding affinity or ADCC activity have increased or decreased FcR binding activity and / or ADCC activity compared to the parent polypeptide or a polypeptide comprising a native Fc sequence. Fc variants that exhibit "enhanced binding" to FcRs have a higher binding affinity (e.g., lower apparent Kd or IC) to at least one FcR compared to the parent polypeptide or a polypeptide comprising a native IgG Fc sequence. 50In some embodiments, the binding capacity is enhanced by 3-fold, e.g., 5-, 10-, 25-, 50-, 60-, 100-, 150-, 200-, or even up to 500-fold, or the binding capacity is increased by 25% to 1000%, compared to the parent polypeptide. An Fc variant that exhibits "reduced binding" to an FcR has a lower affinity for at least one FcR (e.g., a higher apparent Kd or IC) than the parent polypeptide. 50 Compared with the parent polypeptide, its binding capacity is reduced by 40% or more.

[0066] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" is a form of cytotoxicity that refers to the binding of secreted Ig to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages), enabling these cytotoxic effector cells to specifically bind to target cells bearing antigen and subsequently kill the target cells using cytotoxins. Antibodies "arm" the cytotoxic cells and are required for this killing. Of the major cell types that mediate ADCC, NK cells express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. The expression of FcRs on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess the ADCC activity of a target molecule, an in vitro ADCC assay can be performed, as described in U.S. Patent Nos. 5,500,362 or 5,821,337. Effector cells suitable for such experiments include peripheral blood mononuclear cells (PBMC) and natural killer cells (NK). Alternatively, or in addition, the ADCC activity of the target molecule can also be assessed in vivo, for example, as described in the animal model disclosed in Clynes et al. PNAS (USA) 95: 652-656 (1998).

[0067] Polypeptides comprising Fc region variants exhibit "enhanced ADCC activity" or are capable of more effectively mediating ADCC in the presence of human effector cells, compared to polypeptides comprising wild-type IgG Fc polypeptides or parent polypeptides. When tested in substantially the same quantity as polypeptides comprising wild-type IgG Fc polypeptides (or parent polypeptides), such polypeptides comprising Fc region variants are capable of more effectively mediating ADCC, both in vitro and in vivo. Such variants are generally identified using any in vitro ADCC assay known in the art, such as assays or methods for identifying ADCC activity, such as in animal models. In some embodiments, such variants mediate ADCC 5- to 100-fold more efficiently, such as 25- to 50-fold more efficiently, than wild-type Fc (or parent polypeptide).

[0068] "Complement-dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (Clq) to an antibody (of the appropriate structural subclass) that binds to the cognate antigen. To assess complement activation, a CDC assay, such as that described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), can be performed. Polypeptide variants with altered Fc region amino acid sequences and increased or decreased Clq binding capacity are described in U.S. Patent No. 6,194,551 Bl and WO99 / 51642. The contents of these patent publications are expressly incorporated herein by reference. See also Idusogie et al. J. Immunol. 164:4178-4184 (2000).

[0069] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate forms of each other and encode the same amino acid sequence. A nucleotide sequence encoding a protein or RNA may also include introns, for example, a nucleotide sequence encoding a protein may contain introns in some forms.

[0070] The term "operably linked" refers to a functional connection between a regulatory sequence and a heterologous nucleotide sequence, thereby enabling expression of the latter. For example, a first nucleotide sequence is operably linked to a second nucleotide sequence when the first nucleotide sequence is in a functional relationship with the second nucleotide sequence. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, if necessary, can link two protein coding regions in the same reading frame.

[0071] "Homologous" refers to the sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. If the same base or amino acid monomer subunit is present at the same position in two compared sequences, for example, adenine is present at the same position in two DNA molecules, then the two DNA molecules are homologous at that position. The percentage homology between two sequences refers to the ratio of the number of matching or homologous positions shared by the two sequences to the total number of positions multiplied by 100. For example, if 6 out of 10 positions in two sequences are matched or homologous, then the homology between the two sequences is 60%. For example, the DNA sequences ATTGCC and TATGGC have 50% homology. Generally speaking, when aligning two sequences, the comparison is performed with the goal of obtaining maximum homology.

[0072] An "effective amount" of an antibody or composition disclosed herein is an amount sufficient to achieve a specific purpose. An "effective amount" can be determined empirically and by known methods related to the purpose.

[0073] The term "therapeutically effective amount" refers to an amount of an antibody or composition thereof disclosed herein that is effective in treating an individual's disease or symptom. For example, in the case of cancer, a therapeutically effective amount of an antibody or composition thereof refers to an amount that can reduce the number of cancer cells; reduce the size or weight of the tumor; inhibit (i.e., slow down or preferably stop to some extent) the infiltration of tumor cells into peripheral organs; inhibit (i.e., slow down or preferably stop to some extent) tumor metastasis; inhibit the growth of the tumor to some extent, and / or alleviate one or more symptoms associated with cancer to some extent. The antibodies or compositions thereof disclosed herein are capable of preventing and / or killing existing tumor cells to some extent, and it can be cytostatic or cytotoxic. In some embodiments, a therapeutically effective amount refers to an amount that can prolong the patient's survival. In some embodiments, a therapeutically effective amount refers to an amount that can improve the patient's progression-free survival.

[0074] As used herein, "pharmaceutically acceptable" or "pharmacologically compatible" refers to a material that is free of biological activity or other undesirable properties, e.g., a material that can be added to a pharmaceutical composition administered to a patient without causing a significant adverse biological reaction, or that does not interact in a deleterious manner with any other component contained in the composition. Pharmaceutically acceptable carriers or excipients preferably meet the required standards for toxicology or manufacturing testing and / or are included in the inactive ingredient guide compiled by the U.S. Food and Drug Administration.

[0075]

[0066] The embodiments of the present application described herein should be understood to include "consisting of" and / or "consisting essentially of" embodiments.

[0076] Reference herein to "about" for a value or parameter includes (and describes) variations of the value or parameter itself. For example, a description involving "about X" includes a description of "X".

[0077] As used herein, reference to "not" a value or parameter generally indicates and describes "other than" a value or parameter. For example, "the method cannot be used to treat type X cancer" means that the method is generally used to treat other types of cancer besides type X cancer.

[0078] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0079] Antibody

[0080] On the one hand, the application provides separated antibodies, including, but not limited to, humanized antibodies, chimeric antibodies, mouse antibodies, human antibodies, and antibody molecules comprising heavy and / or light chain CDRs as described herein. In certain embodiments, the application provides separated antibodies that bind to IL35, IL27 or EBI3. Expected antibodies include, for example, full-length antibodies (such as full-length IgG1 or IgG4), single-chain antibodies, Fc fusion proteins, multispecific (such as bispecific) antibodies, immunoconjugates, and the like. In certain embodiments, the separated antibody is a full-length antibody (such as full-length IgG1 or IgG4) or an antigen-binding fragment thereof that binds to IL35, IL27 or EBI3. In certain embodiments, the separated antibody is Fab, Fab', F(ab)'2, Fab'-SH, single-chain Fv (scFv), Fv fragment, dAb, Fd, nanobody, double-chain antibody (diabody) or linear antibody. In some embodiments, an isolated antibody is one that binds to a target (e.g., IL35, IL27, or EBI3) with an affinity that is at least 10 times greater (including, for example, 10 ... 2 , 10 3 , 10 4 , 10 5 , 10 6 , or 10 7 times). In some embodiments, a non-target refers to an antigen that is not IL35, IL27, EBI3, or does not contain an EBI3 subunit. Binding affinity can be determined by methods known in the art, such as ELISA, fluorescence-activated cell sorting (FACS) analysis, or radioimmunoprecipitation assay (RIA). Kd values ​​can be determined by methods known in the art, such as surface plasmon resonance (SPR) or biolayer interferometry (BLI).

[0081] Although antibodies comprising human sequences (e.g., human heavy and light chain variable domains comprising human CDR sequences) are discussed extensively herein, non-human antibodies are also contemplated. In some embodiments, non-human antibodies include human CDR sequences and non-human framework sequences of the antibodies described herein. In some embodiments, non-human framework sequences include any sequence for generating heavy and / or light chain variable domains using one or more human CDR sequences as described herein, including, for example, mammals such as mice, rats, rabbits, pigs, cattle (e.g., cattle, bulls, buffaloes), deer, sheep, goats, chickens, cats, dogs, ferrets, primates (e.g., small apes, macaques), etc. In some embodiments, non-human antibodies include antibodies produced by transplanting one or more human CDR sequences described herein into non-human framework sequences (e.g., mouse or chicken framework sequences). In some embodiments, the above antibodies bind to IL35, IL27, or EBI3.

[0082] The amino acid sequence of an exemplary human IL35 p35 subunit comprises or consists of the amino acid sequence set forth in SEQ ID NO: 59. The amino acid sequence of an exemplary human EBI3 subunit comprises or consists of the amino acid sequence set forth in SEQ ID NO: 60. The amino acid sequence of an exemplary human IL27 p28 subunit comprises or consists of the amino acid sequence set forth in SEQ ID NO: 61.

[0083] In some embodiments, the isolated antibodies described herein recognize an epitope in a target (e.g., IL35, IL27, or EBI3). In some embodiments, the antibodies described herein recognize an epitope in EBI3. In some embodiments, the antibodies cross-react with IL35, IL27, or EBI3 of species other than human. In some embodiments, the antibodies are completely specific for human IL35, IL27, or EBI3 and do not cross-react with other non-human species.

[0084] In some embodiments, the antibody cross-reacts with at least one allelic variant of its target (e.g., IL35, IL27, or EBI3) protein (or fragment thereof). In some embodiments, the allelic variant has up to 30 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30) amino acid substitutions (e.g., conservative substitutions) compared to a naturally occurring target (e.g., IL35, IL27, or EBI3) protein (or fragment thereof). In some embodiments, the antibody does not cross-react with any allelic variant of its target (e.g., IL35, IL27, or EBI3) protein (or fragment thereof).

[0085] In some embodiments, the antibody cross-reacts with at least one interspecies variant of its target (e.g., IL35, IL27, or EBI3) protein. In some embodiments, for example, the IL35 protein (or its fragment) is human IL35, the IL27 protein (or its fragment) is human IL27, the EBI3 subunit (or its fragment) is human EBI3, and the interspecies variant of the IL35, IL27, or EBI3 protein (or its fragment) is a variant in cynomolgus monkeys. In some embodiments, the antibody does not cross-react with any interspecies variant of its target (e.g., IL35, IL27, or EBI3) protein.

[0086] In some embodiments, any antibody as described herein comprises an antibody heavy chain constant region and an antibody light chain constant region. In some embodiments, the antibody comprises an IgG1 type heavy chain constant region. In some embodiments, the antibody comprises an IgG2 type heavy chain constant region. In some embodiments, the antibody comprises an IgG3 type heavy chain constant region. In some embodiments, the antibody comprises an IgG4 type heavy chain constant region. In some embodiments, the heavy chain constant region comprises (includes, consists of, or consists essentially of) the amino acid sequence SEQ ID NO: 55. In some embodiments, the heavy chain constant region comprises (includes, consists of, or consists essentially of) the amino acid sequence SEQ ID NO: 56. In some embodiments, the antibody comprises a kappa light chain constant region. In some embodiments, the light chain constant region comprises (includes, consists of, or consists essentially of) the amino acid sequence SEQ ID NO: 57. In some embodiments, the antibody comprises a lambda light chain constant region. In some embodiments, the light chain constant region comprises (includes, consists of, or consists essentially of) the amino acid sequence SEQ ID NO: 58. In some embodiments, the antibody comprises an antibody heavy chain variable domain and an antibody light chain variable domain. In some embodiments, any antibody as described herein binds to IL35, IL27, or EBI3.

[0087] In some embodiments, the antibody comprises a heavy chain variable domain (V H ), the V H comprising: HC-CDR1 comprising SDYAWN (SEQ ID NO: 1); HC-CDR2 comprising SISYSGSTDYNPSLKS (SEQ ID NO: 4); and HC-CDR3 comprising SPIYYDYEEGDY (SEQ ID NO: 8); and a light chain variable domain (V L ), the V L comprising: an LC-CDR1 comprising KASQDIX1X2YLS (SEQ ID NO: 25), wherein X1 is A, N, Q or S, and X2 is A or S; an LC-CDR2 comprising RANRLVX1 (SEQ ID NO: 26), wherein X1 is D, E or S; and an LC-CDR3 comprising LQYDEFPLT (SEQ ID NO: 22).

[0088] In some embodiments, the antibody comprises a V H , the V Hcomprising: HC-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 4, and HC-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 8, or the V H A variant comprising up to about 5 amino acid substitutions in its HC-CDRs; and V L , the V L comprising: LC-CDR1 comprising the amino acid sequence shown in any one of SEQ ID NOs: 12-16, LC-CDR2 comprising the amino acid sequence shown in any one of SEQ ID NOs: 18-20, and LC-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 22, or the V L A variant comprising up to about 5 amino acid substitutions in its LC-CDRs.

[0089] In some embodiments, the antibody comprises a V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 4, HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 8, or the V H A variant comprising up to about 5 amino acid substitutions in its HC-CDRs; and V L , the V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 18, LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 22, or the V L A variant comprising up to about 5 amino acid substitutions in its LC-CDRs.

[0090] In some embodiments, the antibody comprises a V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 4, HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 8, or the V H A variant comprising up to about 5 amino acid substitutions in its HC-CDRs; and V L , the V Lcomprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 19, LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 22, or the V L A variant comprising up to about 5 amino acid substitutions in its LC-CDRs.

[0091] In some embodiments, the antibody comprises a V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 4, HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 8, or the V H A variant comprising up to about 5 amino acid substitutions in its HC-CDRs; and V L , the V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 20, LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 22, or the V L A variant comprising up to about 5 amino acid substitutions in its LC-CDRs.

[0092] In some embodiments, the antibody comprises a V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 4, HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 8, or the V H A variant comprising up to about 5 amino acid substitutions in its HC-CDRs; and V L , the V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 13, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 20, LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 22, or the V L A variant comprising up to about 5 amino acid substitutions in its LC-CDRs.

[0093] In some embodiments, the antibody comprises a V H , the V Hcomprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 4, HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 8, or the V H A variant comprising up to about 5 amino acid substitutions in its HC-CDRs; and V L , the V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 14, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 18, LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 22, or the V L A variant comprising up to about 5 amino acid substitutions in its LC-CDRs.

[0094] In some embodiments, the antibody comprises a V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 4, HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 8, or the V H A variant comprising up to about 5 amino acid substitutions in its HC-CDRs; and V L , the V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 14, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 19, LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 22, or the V L A variant comprising up to about 5 amino acid substitutions in its LC-CDRs.

[0095] In some embodiments, the antibody comprises a V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 4, HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 8, or the V H A variant comprising up to about 5 amino acid substitutions in its HC-CDRs; and V L , the V Lcomprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 14, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 20, LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 22, or the V L A variant comprising up to about 5 amino acid substitutions in its LC-CDRs.

[0096] In some embodiments, the antibody comprises a V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 4, HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 8, or the V H A variant comprising up to about 5 amino acid substitutions in its HC-CDRs; and V L , the V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 15, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 18, LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 22, or the V L A variant comprising up to about 5 amino acid substitutions in its LC-CDRs.

[0097] In some embodiments, the antibody comprises a V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 4, HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 8, or the V H A variant comprising up to about 5 amino acid substitutions in its HC-CDRs; and V L , the V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 16, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 20, LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 22, or the V L A variant comprising up to about 5 amino acid substitutions in its LC-CDRs.

[0098] In some embodiments, the antibody comprises a V H , the V Hcomprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 4, HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 8, or the V H A variant comprising up to about 5 amino acid substitutions in its HC-CDRs; and V L , the V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 16, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 18, LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 22, or the V L A variant comprising up to about 5 amino acid substitutions in its LC-CDRs.

[0099] In some embodiments, the antibody comprises a V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 4, HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 8, or the V H A variant comprising up to about 5 amino acid substitutions in its HC-CDRs; and V L , the V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 13, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 18, LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 22, or the V L A variant comprising up to about 5 amino acid substitutions in its LC-CDRs.

[0100] In some embodiments, the antibody comprises: V H , the V H comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 27-29 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence as set forth in any one of SEQ ID NOs: 27-29; and V L , the V LComprising the amino acid sequence shown in any one of SEQ ID NOs:33-51 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence shown in any one of SEQ ID NOs:33-51.

[0101] In some embodiments, the antibody comprises: V H , the V H comprising the amino acid sequence of SEQ ID NO: 27 or a variant thereof having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 27; and V L , the V L comprising the amino acid sequence of SEQ ID NO: 33 or a variant thereof having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 33.

[0102] In some embodiments, the antibody comprises: V H , the V H comprising the amino acid sequence of SEQ ID NO: 27 or a variant thereof having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 27; and V L , the V L comprising the amino acid sequence of SEQ ID NO: 34 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 34.

[0103] In some embodiments, the antibody comprises: V H , the V H comprising the amino acid sequence of SEQ ID NO: 27 or a variant thereof having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 27; and V L , the V Lcomprising the amino acid sequence of SEQ ID NO: 35 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 35.

[0104] In some embodiments, the antibody comprises: V H , the V H comprising the amino acid sequence of SEQ ID NO: 27 or a variant thereof having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 27; and V L , the V L comprising the amino acid sequence of SEQ ID NO: 36 or a variant thereof having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 36.

[0105] In some embodiments, the antibody comprises: V H , the V H comprising the amino acid sequence of SEQ ID NO: 27 or a variant thereof having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 27; and V L , the V L comprising the amino acid sequence of SEQ ID NO: 37 or a variant thereof having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 37.

[0106] In some embodiments, the antibody comprises: V H , the V H comprising the amino acid sequence of SEQ ID NO: 27 or a variant thereof having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 27; and V L , the V L comprising the amino acid sequence of SEQ ID NO: 38 or a variant thereof having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 38.

[0107] In some embodiments, the antibody comprises: V H , the V H comprising the amino acid sequence of SEQ ID NO: 27 or a variant thereof having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 27; and V L , the V L comprising the amino acid sequence of SEQ ID NO: 39 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 39.

[0108] In some embodiments, the antibody comprises: V H , the V H comprising the amino acid sequence of SEQ ID NO: 27 or a variant thereof having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 27; and V L , the V L comprising the amino acid sequence of SEQ ID NO:40 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO:40.

[0109] In some embodiments, the antibody comprises: V H , the V H comprising the amino acid sequence of SEQ ID NO: 27 or a variant thereof having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 27; and V L , the V L comprising the amino acid sequence of SEQ ID NO:41 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO:41.

[0110] In some embodiments, the antibody comprises: V H , the V Hcomprising the amino acid sequence of SEQ ID NO: 27 or a variant thereof having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 27; and V L , the V L comprising the amino acid sequence of SEQ ID NO:42 or a variant thereof having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO:42.

[0111] In some embodiments, the antibody comprises: V H , the V H comprising the amino acid sequence of SEQ ID NO: 27 or a variant thereof having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 27; and V L , the V L comprising the amino acid sequence of SEQ ID NO: 43 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 43.

[0112] In some embodiments, the antibody comprises: V H , the V H comprising the amino acid sequence of SEQ ID NO: 28 or a variant thereof having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 28; and V L , the V L comprising the amino acid sequence of SEQ ID NO: 44 or a variant thereof having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 44.

[0113] In some embodiments, the antibody comprises: V H , the V H comprising the amino acid sequence of SEQ ID NO: 29 or a variant thereof having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 29; and VL , the V L comprising the amino acid sequence of SEQ ID NO: 45 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 45.

[0114] In some embodiments, the antibody comprises: V H , the V H comprising the amino acid sequence of SEQ ID NO: 28 or a variant thereof having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 28; and V L , the V L comprising the amino acid sequence of SEQ ID NO: 46 or a variant thereof having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 46.

[0115] In some embodiments, the antibody comprises: V H , the V H comprising the amino acid sequence of SEQ ID NO: 29 or a variant thereof having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 29; and V L , the V L comprising the amino acid sequence of SEQ ID NO: 47 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 47.

[0116] In some embodiments, the antibody comprises: V H , the V H comprising the amino acid sequence of SEQ ID NO: 28 or a variant thereof having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 28; and V L , the V Lcomprising the amino acid sequence of SEQ ID NO: 48 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 48.

[0117] In some embodiments, the antibody comprises: V H , the V H comprising the amino acid sequence of SEQ ID NO: 29 or a variant thereof having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 29; and V L , the V L comprising the amino acid sequence of SEQ ID NO: 49 or a variant thereof having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 49.

[0118] In some embodiments, the antibody comprises: V H , the V H comprising the amino acid sequence of SEQ ID NO: 28 or a variant thereof having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 28; and V L , the V L comprising the amino acid sequence of SEQ ID NO: 50 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 50.

[0119] In some embodiments, the antibody comprises: V H , the V H comprising the amino acid sequence of SEQ ID NO: 29 or a variant thereof having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 29; and V L , the V L comprising the amino acid sequence of SEQ ID NO: 51 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 51.

[0120] In some embodiments, the antibody comprises a V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 2, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 9, or the V H A variant comprising up to about 5 amino acid substitutions in its HC-CDRs; and V L , the V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 17, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 21, LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 23, or the V L A variant comprising up to about 5 amino acid substitutions in its LC-CDRs.

[0121] In some embodiments, the antibody comprises: V H , the V H comprising the amino acid sequence of SEQ ID NO: 30 or a variant thereof having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 30; and V L , the V L comprising the amino acid sequence of SEQ ID NO:52 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO:52.

[0122] In some embodiments, the antibody comprises a V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 6, HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 10, or the V H A variant comprising up to about 5 amino acid substitutions in its HC-CDRs; and V L , the V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 18, LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 22, or the VL A variant comprising up to about 5 amino acid substitutions in its LC-CDRs.

[0123] In some embodiments, the antibody comprises: V H , the V H comprising the amino acid sequence of SEQ ID NO: 31 or a variant thereof having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 31; and V L , the V L comprising the amino acid sequence of SEQ ID NO: 53 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 53.

[0124] In some embodiments, the antibody comprises a V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 3, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 7, HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 11, or the V H A variant comprising up to about 5 amino acid substitutions in its HC-CDRs; and V L , the V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 17, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 21, LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 24, or the V L A variant comprising up to about 5 amino acid substitutions in its LC-CDRs.

[0125] In some embodiments, the antibody comprises: V H , the V H comprising the amino acid sequence of SEQ ID NO: 32 or a variant thereof having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 32; and V L , the V Lcomprising the amino acid sequence of SEQ ID NO: 54 or a variant thereof having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 54.

[0126] In some embodiments, the above-mentioned amino acid substitutions are limited to the "exemplary substitutions" shown herein in Table 4. In some embodiments, the amino acid substitutions are limited to the "preferred substitutions" shown herein in Table 4.

[0127] In some embodiments, functional epitopes can be resolved by combinatorial alanine scanning. In this process, combinatorial alanine scanning technology can be used to identify amino acids in the target (e.g., IL35, IL27, or EBI3) protein that are necessary for interaction with the antibody. In some embodiments, the epitope is conformational and can be identified using a crystal structure of an antibody bound to the target (e.g., IL35, IL27, or EBI3) protein.

[0128] In some embodiments, the present application provides antibodies that compete with any of the antibodies described herein for binding to a target (e.g., IL35, IL27, or EBI3). In some embodiments, antibodies are provided that can competitively bind to an epitope on a target (e.g., IL35, IL27, or EBI3) with any of the antibodies described herein. In some embodiments, a separated antibody is provided that is combined with a V H and V L The antibody molecules bind to the same epitope, wherein the V H comprising the amino acid sequence shown in any one of SEQ ID NOs: 27-32, and said V L In some embodiments, a separate antibody is provided, which is amplified by a method comprising: H and V L The antibody competitively binds to the target (e.g., IL35, IL27 or EBI3), wherein the V H comprising the amino acid sequence shown in any one of SEQ ID NOs: 27-32, and said V L Comprising the amino acid sequence shown in any one of SEQ ID NOs: 33-54.

[0129] In some embodiments, competition experiments can be used to identify monoclonal antibodies that compete with the antibodies described herein for binding to a target (e.g., IL35, IL27, or EBI3). Competition experiments can determine whether two antibodies bind to the same epitope by recognizing identical or spatially overlapping epitopes or by competitively inhibiting the binding of one antibody to the antigen by another. In certain embodiments, such competing antibodies bind to the same epitope as the antibodies described herein. Some exemplary competition experiments include, but are not limited to, conventional experiments as described in Harlow and Lane (1988) Antibodies: A Laboratory Manual ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). Detailed exemplary methods for parsing epitopes bound by antibodies are described in Morris (1996) "Epitope Mapping Protocols," in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ). In some embodiments, if each antibody blocks 50% or more of the binding of another antibody, it is said to bind to the same epitope. In some embodiments, the antibody that competes with the antibodies described herein is a chimeric antibody, a humanized antibody, or a fully human antibody.

[0130] Exemplary antibody sequences are shown in Tables 2 and 3, where CDR numbering is performed according to the Kabat definition. Those skilled in the art will recognize that there are a variety of known algorithms for predicting the positions of CDRs and defining antibody light and heavy chain variable regions. H and / or V L Sequences, but based on prediction algorithms other than those exemplified in the table below, are also within the scope of this application.

[0131] Table 2 Exemplary Antibody CDR Sequences

[0132] Table 3 Exemplary sequences

[0133] IL35 and IL27

[0134] The IL12 (IL-12) cytokine family belongs to the IL6 (IL-6) superfamily of type I cytokines. Unlike IL6 family members, which are secreted as single-subunit monomers, IL12 family members are secreted as heterodimers. The IL12 cytokine family includes IL12, IL23 (IL-23), IL27 (IL-27), and IL35 (IL-35), which share structural similarities but mediate distinct immune functions. Each member is composed of an α chain (p19, p28, or p35) and a β chain (p40 or EBI3). IL12 (p35 / p40) and IL23 (p19 / p40) are mainly proinflammatory factors, while IL27 (p28 / EBI3) and IL35 (p35 / EBI3) are mainly inhibitory cytokines (Vignali, Dario AA, and Vijay K Kuchroo. Nature immunology vol.13, 8 722-8.19 Jul.2012, doi:10.1038 / ni.2366). In addition, there are reports on monomers and homodimers that they act as natural antagonists of cytokine signaling (Jones, Lindsay L, and Dario AA Vignali. Immunologic research vol.51, 1 (2011): 5-14.).

[0135] These cytokines signal through unique pairings of five receptor chains: IL12Rβ1, IL12Rβ2, IL23R, WSX-1, and gp130. IL12 signals through IL12Rβ1 and IL12Rβ2, IL23 signals through IL-23R and IL12Rβ1, and IL27 utilizes gp130 and WSX-1. In T cells, IL-35 signals through IL12Rβ2 and gp130, although it can also signal through IL12Rβ2 / IL12Rβ2 and gp130 / gp130 homodimers. These receptor chains mediate signaling through the JAK-STAT pathway. The key Janus kinases mediating STAT phosphorylation by IL-12 family members are JAK2 and JAK1 / TYK2. Different receptor chains utilize different STATs, such as IL-12 signal transduction, which is mainly through pSTAT4, IL-23 through pSTAT3 and pSTAT4, and IL-27 through pSTAT1 and pSTAT3. IL-35 carries out signal transduction through gp130 and IL12Rβ2 homodimers and is mediated by pSTAT1 and pSTAT4 respectively. These homodimer receptors can inhibit T cell proliferation, but cannot induce iTr35 conversion. In contrast, T cell suppression and iTr35 induction can be mediated by IL12Rβ2 / gp130 heterodimer receptors, which drive the formation of pSTAT1:pSTAT4 heterodimers, which are sequentially bound to unique sites of EbI3 and IL12a promoters (Collison, Lauren W et al. Nature immunology vol. 13, 3 290-9.5 Feb. 2012). Recent studies have also shown that in B cells, IL-35 mediates signaling through the IL12Rβ2 / WSX-1 heterodimer and induces STAT1 and STAT3 phosphorylation (Shen, Ping et al. Nature vol. 507, 7492 (2014): 366-370).

[0136] In general, IL-35 inhibits the proliferation of conventional T cells and converts conventional T cells into regulatory T cell groups known as iTr35. It also suppresses immune response by strengthening Treg development and inhibiting Th17 cell growth (Xue, Wenhua et al. Journal of Cancer vol.10,9 2074-2082.12May.2019). There are multiple pieces of evidence showing that IL-35 plays a vital role in the pathogenesis of many diseases, including autoimmune diseases, bacterial or viral infections, inflammatory diseases and tumors. Unlike other IL-12 family members, they are mainly secreted by activated antigen-presenting cells, while IL-35 is mainly expressed in regulatory T cells and acts as an immunosuppressive factor. In recent years, a large number of studies have shown that IL-35 is secreted in a wide range of tissues and different cell types, including regulatory B cells, dendritic cells (DCs), endothelial cells, smooth cells, muscle cells, and monocytes (Zhao, Na et al. Clinics and research in hepatology and gastroenterology vol. 45, 1 (2021): 101518). Recently, it has been reported that tumor cells can secrete IL-35, which may play a key role in tumor progression by recruiting and reshaping other immune cells in the microenvironment (Huang, Chongbiao et al. Gastroenterology vol. 154, 3 (2018): 675-688.).

[0137] IL-27 has multiple dual functions, including both pro-inflammatory and anti-inflammatory effects. + T cell proliferation, Th1 cell polarization, cytotoxic T cell activation, production of natural killer cells, activation of macrophages and dendritic cells to promote inflammation. IL-27 can also inhibit inflammation by inducing programmed cell death and inhibiting effector cells (Beizavi, Zahra et al. International reviews of immunology vol. 40, 5 (2021): 319-329).

[0138] Full-length antibody

[0139] In some embodiments, the antibody is a full-length antibody. In some embodiments, the full-length antibody is IgA, IgD, IgE, IgG or IgM. In some embodiments, the full-length antibody comprises an IgG constant region, such as a constant region of IgG1, IgG2, IgG3, IgG4 or a variant thereof. In some embodiments, the full-length antibody comprises a lambda light chain constant region. In some embodiments, the long antibody comprises a kappa light chain constant region. In some embodiments, the full-length antibody is a human full-length antibody. In some embodiments, the full-length antibody comprises a mouse immunoglobulin Fc sequence. In some embodiments, the full-length antibody comprises an altered or otherwise modified Fc sequence such that it has enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) effector functions.

[0140] Thus, for example, in some embodiments, a full-length antibody comprising an IgG1 constant region is provided. In some embodiments, the IgG1 is a human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 55. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 57. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 58. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 55 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 57. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 55 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 58.

[0141] In some embodiments, a full-length antibody comprising an IgG2 constant region is provided. In some embodiments, the IgG2 is human IgG2. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 57. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 58.

[0142] In some embodiments, a full-length antibody comprising an IgG3 constant region is provided. In some embodiments, the IgG3 is a human IgG3. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 57. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 58.

[0143] In some embodiments, a full-length antibody comprising an IgG4 constant region is provided. In some embodiments, the IgG4 is a human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 56. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 57. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 58. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 56 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 57. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 56 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 58.

[0144] In some embodiments, any of the antibodies described above binds to IL35, IL27, or EBI3.

[0145] Binding affinity

[0146] Binding affinity is expressed as Kd, Koff, Kon or Ka. As used herein, the term Koff refers to the rate constant for the dissociation of an antibody from an antigen / antibody complex, as measured by a kinetic selection apparatus. The term Kon refers to the association rate constant for the binding of an antibody to an antigen to form an antigen / antibody complex. The equilibrium dissociation constant, Kd, ​​used herein refers to the dissociation constant for a specific antibody-antigen interaction, and is the concentration of antigen required for the antigen to occupy half of all antibody binding sites in a solution of antibody molecules and to reach equilibrium, which is equal to Koff / Kon. The determination of Kd assumes that all binding molecules are in solution. In cases where the antibody is attached to the cell wall, such as in a yeast expression system, the corresponding equilibrium dissociation rate constant is expressed as EC 50 It is expressed as , which is a good approximation of Kd. The affinity binding constant Ka is the reciprocal of the dissociation constant Kd.

[0147] The dissociation constant (Kd) can be used as an indicator of the affinity of the reactive antibody portion for the antigen. For example, a simple analysis can be performed using antibodies labeled with various markers using the Scatchard method and a Biacore instrument (manufactured by Amersham Biosciences) to analyze the interaction between biomolecules by surface plasmon resonance according to the user manual or the accompanying kit. The Kd values ​​obtained using these methods are expressed in units of M. An antibody that specifically binds to a target may have, for example, a Kd of ≤10 -7 M, ≤10 -8 M, ≤10 -9 M, ≤10 -10 M, ≤10 -11M, ≤10 -12 M or ≤10 -13 Kd value of M.

[0148] The binding specificity of an antibody can be determined experimentally by methods known in the art, including, but not limited to, Western blots, ELISA, RIA, ECL, IRMA, EIA, BIAcore assays, and peptide scanning.

[0149] In some embodiments, the antibody binds to the target (e.g., IL35, IL27, or EBI3) with a Kd value of 10 -7 M to 10 - 13 M (e.g. 10 -7 M to 10 -13 M, 10 -8 M to 10 -13 M, 10 -9 M to 10 -13 M or 10 -10 M to 10 -12 M). Thus, in some embodiments, the Kd value of the binding between the antibody and the target (e.g., IL35, IL27 or EBI3) is 10 -7 M to 10 - 13 M, 1×10 -7 M to 5×10 -13 M, 10 -7 M to 10 -12 M, 10 -7 M to 10 -11 M, 10 -7 M to 10 -10 M, 10 -7 M to 10 -9 M, 10 -8 M to 10 -13 M, 1×10 -8 M to 5×10 -13 M, 10 -8 M to 10 -12 M, 10 -8 M to 10 -11 M, 10 -8 M to 10 -10 M, 10 -8 M to 10 -9 M, 5×10 -9 M to 1×10 -13 M, 5×10 -9 M to 1×10 -12 M, 5×10 -9 M to 1×10 -11M, 5×10 -9 M to 1×10 -10 M, 10 -9 M to 10 -13 M, 10 -9 M to 10 -12 M, 10 -9 M to 10 -11 M, 10 -9 M to 10 -10 M, 5×10 -10 M to 1×10 -13 M, 5×10 -10 M to 1×10 -12 M, 5×10 -10 M to 1×10 -11 M, 10 -10 M to 10 -13 M, 1×10 - 10 M to 5×10 -13 M, 1×10 -10 M to 1×10 -12 M, 1×10 -10 M to 5×10 -12 M, 1×10 -10 M to 1×10 -11 M, 10 - 11 M to 10 -13 M, 1×10 -11 M to 5×10 -13 M, 10 -11 M to 10 -12 M, 10 -12 M to 10 -13 In some embodiments, the Kd value of the binding between the antibody and the target (eg, IL35, IL27 or EBI3) is 10 -7 M to 10 -13 M.

[0150] In some embodiments, the Kd value of the antibody binding to the non-target is higher than the Kd value of the antibody binding to the target, and in some embodiments cited herein, the binding affinity of the antibody to the target (e.g., IL35, IL27, or EBI3) is higher than the binding affinity of the antibody to the non-target. In some embodiments, the non-target refers to an antigen that is not IL35, not IL27, not EBI3, or does not contain an EBI3 subunit. In some embodiments, the Kd value of the antibody binding to the non-target differs by at least 10 times, for example, 10-100 times, 100-1000 times, 10 3 -10 4 times, 10 4 -10 5times, 10 5 -10 6 times, 10 6 -10 7 times, 10 7 -10 8 times, 10 8 -10 9 times, 10 9 -10 10 times, 10 10 -10 11 times, 10 11 -10 12 times.

[0151] In some embodiments, the antibody has a Kd value of 10 for binding to a non-target -1 M to 10 -6 M (e.g. 10 -1 M to 10 -6 M, 10 -1 M to 10 -5 M, 10 -2 M to 10 -4 M). In some embodiments, the non-target is an antigen that is not IL35, IL27, or EBI3, or does not contain an EBI3 subunit. Therefore, in some embodiments, the Kd value of the binding between the antibody and the non-target is 10 -1 M to 10 -6 M, 1×10 -1 M to 5×10 -6 M, 10 -1 M to 10 -5 M, 1×10 -1 M to 5×10 -5 M, 10 -1 M to 10 -4 M, 1×10 -1 M to 5×10 -4 M, 10 -1 M to 10 -3 M, 1×10 -1 M to 5×10 -3 M, 10 -1 M to 10 -2 M, 10 -2 M to 10 -6 M, 1×10 -2 M to 5×10 -6 M, 10 -2 M to 10 -5 M, 1×10 -2 M to 5×10 -5 M, 10 -2 M to 10 -4 M, 1×10-2 M to 5×10 -4 M, 10 -2 M to 10 -3 M, 10 -3 M to 10 -6 M, 1×10 -3 M to 5×10 -6 M, 10 -3 M to 10 -5 M, 1×10 -3 M to 5×10 -5 M, 10 -3 M to 10 -4 M, 10 -4 M to 10 -6 M, 1×10 -4 M to 5×10 -6 M, 10 -4 M to 10 -5 M, 10 -5 M to 10 -6 M.

[0152] In some embodiments, when an antibody as described herein recognizes a target (e.g., IL35, IL27, or EBI3) with high binding affinity and binds to a non-target with low binding affinity, the antibody binds to the target with a Kd value of 10 -7 M to 10 -13 M (e.g. 10 -7 M to 10 -13 M, 10 -8 M to 10 -13 M, 10 -9 M to 10 -13 M, 10 -10 M to 10 -12 M), and the Kd value for binding to non-target is 10 -1 M to 10 -6 M (e.g. 10 -1 M to 10 -6 M, 10 -1 M to 10 -5 M, 10 -2 M to 10 -4 M).

[0153] In some embodiments, when the antibodies mentioned herein recognize a target (e.g., IL35, IL27, or EBI3), the binding affinity of the antibody is compared to the binding affinity of a control antibody that specifically binds to IL35 and / or IL27. In some embodiments, the Kd value of the binding between the control antibody that specifically binds to IL35 and / or IL27 and IL35, IL27, or EBI3 can be at least 2 times, for example, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 10-100 times, 100-1000 times, 1 ... 3 -10 4 times.

[0154] Nucleic Acids

[0155] Nucleic acid molecules encoding the above-mentioned antibodies are also contemplated. In some embodiments, a nucleic acid (or a group of nucleic acids) encoding the above-mentioned full-length antibodies is provided, including any of the full-length antibodies described herein. In some embodiments, any of the antibodies described herein binds to IL35, IL27, or EBI3. In some embodiments, the nucleic acid (or a group of nucleic acids) of the antibodies described herein may further include a nucleic acid sequence encoding a polypeptide tag (e.g., a protein purification tag, a His tag, an HA tag).

[0156] Also contemplated herein are isolated host cells comprising the above-mentioned antibodies, isolated nucleic acids encoding the polypeptide components of the antibodies, or vectors comprising nucleic acids encoding the polypeptide components of the antibodies described herein.

[0157] The present application also includes variants of these nucleic acid sequences. For example, variants include nucleotide sequences that hybridize to the nucleic acid sequences encoding the antibodies of the present application under at least moderately stringent hybridization conditions.

[0158] The present application also provides a vector into which the nucleic acid sequence of the present application can be inserted.

[0159] In brief, a natural or synthetic nucleic acid encoding an antibody described herein is inserted into a suitable expression vector such that the nucleic acid is operably linked to 5' and 3' regulatory elements, for example, a promoter (e.g., a lymphocyte-specific promoter) and a 3' untranslated region (UTR), and the antibody described herein (e.g., a full-length antibody) can be expressed. The vector can be suitable for replication and integration in eukaryotic host cells. Typical cloning and expression vectors contain transcriptional and translational terminators, initiation sequences, and promoters that regulate the expression of the target nucleic acid sequence.

[0160] The nucleic acids described herein can also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Nucleic acid delivery methods are known in the art. For example, see US Pat. Nos. 5,399,346, 5,580,859, and 5,589,466, which are incorporated herein by reference in their entirety. In some embodiments, the present application also provides gene therapy vectors.

[0161] Nucleic acids can be cloned into many types of vectors. For example, nucleic acids can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0162] In addition, the expression vector can be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art and is described in, for example, Green and Sambrook (2013, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and other virology or molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and slow viruses. Typically, suitable vectors include a replication origin that works in at least one organism, a promoter sequence, a convenient restriction endonuclease site, and one or more selection markers (see, for example, WO 01 / 96584; WO 01 / 29058; and US Pat. No. 6,326,193).

[0163] Many virus-based systems have been developed for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. Techniques known in the art can be applied to insert the selected gene into a vector and package it in retroviral particles. The recombinant virus is then isolated and delivered to the cells of the subject in vivo or in vitro. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In some embodiments, lentiviral vectors are used. Vectors derived from retroviruses, such as lentiviruses, are suitable tools for achieving long-term gene transfer because they allow for long-term stable integration of transgenes and their propagation in daughter cells. Lentiviral vectors have additional advantages over tumor-derived retroviruses, such as mouse leukemia viruses, because they can transduce non-dividing cells, such as hepatocytes. At the same time, they also have the additional advantage of low immunogenicity.

[0164] Other promoter elements, such as enhancers, regulate the frequency of transcription initiation. These are typically located 30-110 bp upstream of the start site, although many promoters have recently been found to contain functional elements downstream of the start site. The spacing between promoter elements is often flexible, so that promoter function is maintained even when elements are interchanged or moved. In the thymidine kinase (Tk) promoter, activity only begins to decline when the spacing between promoter elements increases to 50 bp.

[0165] An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a very strong constitutive promoter sequence that can drive high-level expression of any polynucleotide sequence operably linked thereto. Another example of a suitable promoter is the elongation factor 1α (EF-1α) promoter. However, other constitutive promoters may also be used, including but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus long terminal repeat (HIV-LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters, including but not limited to the actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. In addition, this application should not be limited to the use of only constitutive promoters, as inducible promoters are also considered in this application. The use of an inducible promoter provides a molecular switch that can turn on expression of an operably linked polynucleotide sequence when such expression is desired and turn off expression when it is not desired. Inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0166] In some embodiments, expression of the antibodies described herein is inducible. In some embodiments, the nucleic acid sequence encoding the antibodies described herein is operably linked to an inducible promoter, including any of the inducible promoters described herein.

[0167] Inducible promoter

[0168] The use of an inducible promoter provides a molecular switch that turns on the expression of an operably linked polynucleotide sequence when expression is desired, and turns off expression when expression is not desired. Exemplary inducible promoters suitable for use in eukaryotic cells include, but are not limited to, hormone-regulated elements (e.g., see Mader, S. and White, JH (1993) Proc. Natl. Acad. Sci. USA 90:5603-5607), synthetic ligand-regulated elements (see Spencer, DM et al (1993) Science 262:1019-1024), and ionizing radiation-regulated elements (see Manome, Y. et al (1993) Biochemistry 32:10607-10613; Datta, R. et al (1992) Proc. Natl. Acad. Sci. USA 89:1014-10153). Other exemplary inducible promoters suitable for use in in vivo or in vitro mammalian systems are described in Gingrich et al. (1998) Annual Rev. Neurosci 21: 377-405. In some embodiments, the inducible promoter system used to express the antibodies described herein is the Tet system. In some embodiments, the inducible promoter system used to express the antibodies described herein is the E. coli lac repression system.

[0169] An exemplary inducible promoter system used in this application is the Tet system. This system is based on the Tet system described by Gossen et al. (1993). In an exemplary embodiment, the target polynucleotide is controlled by a promoter comprising one or more Tet operator (TetO) sites. In the inactive state, the Tet repressor (TetR) binds to the TetO site and inhibits transcription of the promoter. In the active state, for example, in the presence of an inducer such as tetracycline (Tc), anhydrotetracycline, doxycycline (Dox) or its active analogs, the inducer releases TetR from TetO, thereby causing transcription to occur. Doxycycline is a member of the tetracycline antibiotic family, and its chemical name is 1-dimethylamino-2,4a,5,7-pentahydroxy-11-methyl-4,6-dioxy-1,4a,11,11a,12,12a-hexahydrotetraene-3-carboxamide.

[0170] In one embodiment, TetR is codon-optimized for expression in mammalian cells, such as mouse or human cells. Due to the degeneracy of the genetic code, most amino acids are encoded by more than one codon, so that the sequence of a given nucleic acid has a large number of variants without any change in the amino acid sequence it encodes. However, many organisms differ in codon usage, also known as "codon preference" (i.e., the preference for using a specific codon for a given amino acid). Codon preference is generally associated with the presence of a dominant tRNA species for a particular codon, which in turn improves the efficiency of mRNA translation. Therefore, coding sequences derived from specific species (e.g., prokaryotes) can be customized by codon optimization to improve their expression in different species (e.g., eukaryotes).

[0171] Other specific variants of the Tet system include the following "Tet-Off" and "Tet-On" systems. In the Tet-off system, transcription is inactivated in the presence of Tc or Dox. In this system, a tetracycline-regulated transcription activator protein (tTA), composed of a fusion of TetR and the strong transcriptional activation domain of herpes simplex virus VP16, regulates the expression of the target nucleic acid under the transcriptional control of a tetracycline-responsive promoter element (TRE). The TRE element consists of a TetO sequence fused in series with a promoter (usually a minimal promoter sequence derived from the immediate early promoter of human cytomegalovirus). In the absence of Tc or Dox, tTA binds to TRE and activates transcription of the target gene. In the presence of Tc or Dox, tTA cannot bind to TRE and the target gene cannot be expressed.

[0172] In contrast, in the Tet-On system, transcription is activated in the presence of either Tc or Dox. The Tet-On system is based on the reverse tetracycline-regulated transcriptional activator rtTA. Like tTA, rtTA is a fusion protein consisting of the TetR repressor and the VP16 transcriptional activation domain. However, a four-amino acid change in the DNA-binding region of TetR alters rtTA's binding properties, allowing it to only recognize the tetO sequence within the target transgene's TRE in the presence of Dox. Therefore, in the Tet-On system, rtTA can activate transcription of its TRE-regulated target gene only in the presence of Dox.

[0173] Another inducible promoter system is the lac repressor system of Escherichia coli (see Brown et al., Cell 49: 603-612 (1987)). The lac repressor system functions by regulating the transcription of a target polynucleotide operably linked to a promoter comprising the lac operator (lacO). The lac repressor (lacR) binds to LacO, thereby preventing transcription of the target polynucleotide. Expression of the target polynucleotide is induced by a suitable inducing agent, for example, isopropyl-β-D-thiogalactopyranoside (IPTG).

[0174] In order to evaluate the expression of a polypeptide or portion thereof, the expression vector to be introduced into the cell may also contain a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from a cell population transfected or infected by a viral vector. In other aspects, the selectable marker can be carried on a separate DNA fragment and used in co-transfection experiments. Either the selectable marker gene or the reporter gene can be flanked by suitable regulatory sequences to enable expression in the host cell. Useful selectable markers include, for example, antibiotic resistance genes such as neo and similar genes.

[0175] Reporter genes can be used to identify potential transfected cells and evaluate the function of regulatory sequences. Generally, a reporter gene is a gene that is not present in or expressed by a recipient organism or tissue, and that encodes a polypeptide whose expression is expressed as some easily detectable properties, such as enzymatic activity. After the DNA is introduced into the recipient cell, the expression of the reporter gene is detected at an appropriate time. Suitable reporter genes can include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein (see, Ui-Tel et al., 2000 FEBS Letters 479: 79-82). Suitable expression systems are well known and can be prepared by known techniques or obtained commercially. Generally, the construct of the minimum 5' flanking region that can show the highest expression level of the reporter gene is identified as a promoter. Such a promoter region can be connected to a reporter gene and used to assess the ability of certain substances to regulate promoter-driven transcription.

[0176] In some embodiments, nucleic acid encoding any one of the full-length antibodies described herein is provided. In some embodiments, the nucleic acid includes one or more nucleic acid sequences encoding the full-length antibody heavy and light chains described herein. In some embodiments, each of the one or more nucleic acid sequences is contained in a separate vector. In some embodiments, at least some nucleic acid sequences are contained in the same vector. In some embodiments, all nucleic acid sequences are contained in the same vector. The vector can be selected from, for example, mammalian expression vectors and viral vectors (such as vectors derived from retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses).

[0177] Methods for introducing genes into cells and expressing them are known in the art. In the context of expression vectors, the vectors can be readily introduced into host cells, such as mammalian cells, bacteria, yeast, or insect cells, by any method known in the art. For example, expression vectors can be introduced into host cells by physical, chemical, or biological methods.

[0178] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, biolistic methods, microinjection, electroporation, and the like. Methods for preparing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Green and Sambrook (2013, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). In some embodiments, polynucleotides are introduced into host cells by calcium phosphate transfection.

[0179] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian cells, such as human cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus type 1, adenoviruses, and adeno-associated viruses, among others. See, for example, US Pat. Nos. 5,350,674 and 5,585,362.

[0180] Chemical methods for introducing polynucleotides into host cells include colloidal dispersion systems, such as polymer complexes, nanocapsules, microspheres, magnetic beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system used as a delivery vehicle in vivo and in vitro is a liposome (e.g., an artificial membrane vesicle).

[0181] In the case of using a non-viral delivery system, an exemplary delivery vehicle is a liposome. It is contemplated that a lipid formulation is used to import nucleic acid into a host cell (in vitro, in vitro or in vivo). On the other hand, the nucleic acid can be combined with lipids. The nucleic acid combined with lipids can be wrapped into the aqueous interior of the liposome, dispersed in the lipid bilayer of the liposome, connected to the liposome by a linker molecule combined with the liposome and the oligonucleotide, embedded in the liposome, form a complex with the liposome, be dispersed in a solution containing lipids, mix with lipids, combine with lipids, be suspended in lipids, be contained in micelles or mix with micelles, or otherwise combine with lipids. The compositions related to lipids, lipid / DNA or lipid / expression vectors are not limited to any particular structure in solution. For example, they may exist with a bilayer structure, with micelles or with a "collapsed" structure. They can also simply be dispersed in solution and may form aggregates of uneven size or shape. Lipids are fatty substances and can be naturally occurring or synthetic lipids. For example, lipids include fat droplets naturally present in the cytoplasm, as well as a class of compounds containing long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0182] Regardless of the method used to introduce the exogenous nucleic acid into the host cell or otherwise expose the cell to the inhibitor of the present application, a variety of experiments can be performed to confirm the presence of the recombinant DNA sequence in the host cell. Such experiments include, for example, "molecular biology" experiments well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR, and PCR; "biochemical" experiments, such as detecting the presence or absence of a particular polypeptide, such as by immunological methods (ELISAs and Western blots) or identification by the assays described herein, all fall within the scope of the present application.

[0183] Antibody preparation

[0184] In some embodiments, the antibodies described herein are monoclonal antibodies or are derived from monoclonal antibodies. In some embodiments, the antibodies comprise V from a monoclonal antibody. H and V L , or variants thereof. In some embodiments, the antibody further comprises a C from a monoclonal antibody H 1 and C L Region, or variants thereof. Monoclonal antibodies can be prepared using methods known in the art, such as hybridoma cell methods, phage display methods, or recombinant DNA methods. In addition, exemplary phage display methods are described herein and in the following examples.

[0185] In the hybridoma method, an immunizing agent is usually used to immunize hamsters, mice or other suitable host animals to induce lymphocytes that produce or are capable of producing antibodies that specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro. The immunizing agent may include a polypeptide or fusion protein of the target protein. Typically, if human cells are needed, peripheral blood lymphocytes (PBLs) are used, and if non-human mammalian derived cells are needed, spleen cells or lymph node cells are used. Lymphocytes are fused with immortalized cell lines using an appropriate fusion agent, such as polyethylene glycol, to form hybridoma cells. Immortalized cell lines are typically transformed mammalian cells, especially myeloma cells of rodent, bovine and human origin. Rat or mouse myeloma cell lines are typically used. Hybridoma cells can be cultured in a suitable culture medium, which preferably contains one or more substances that inhibit the growth or survival of unfused immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT or HPRT), hybridoma cell culture medium typically includes hypoxanthine, aminopterin, and thymidine (HAT medium), which prevents the growth of HGPRT-deficient cells.

[0186] In some embodiments, the immortalized cell line is effectively fused, ensures high-level stable expression of the antibody by the selected antibody-producing cells, and is sensitive to certain culture media, such as HAT medium. In some embodiments, the immortalized cell line is a mouse myeloma cell line, which can be obtained from, for example, the Salk Cell Collection in San Diego, California and the American Type Culture Collection in Manassas, Virginia. Human myeloma and mouse-human hybrid myeloma cell lines are also described for use in preparing human monoclonal antibodies.

[0187] The presence of monoclonal antibodies against the polypeptide in the culture medium of the hybridoma cells can then be determined. The binding specificity of the monoclonal antibodies produced by the hybridoma cells can be determined by immunoprecipitation or in vitro binding experiments, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such techniques or analytical methods are known in the art. The binding affinity of the monoclonal antibodies can be determined by Scatchard analysis as described in, for example, Munson and Pollard, Anal. Biochem., 107:220 (1980).

[0188] After identifying the desired hybridoma cells, the target clones can be subcloned by limiting dilution and cultured by standard methods. Suitable culture media for this purpose include, for example, modified Eagle medium (DMEM) and RPMI-1640 culture medium. Alternatively, hybridoma cells can be grown in mammalian ascites.

[0189] The monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures, such as protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0190] In some embodiments, according to any of the antibodies described herein, the antibody comprises a sequence selected from a clone of an antibody library (e.g., a phage library displaying scFv or Fab fragments). The clone can be identified by screening combinatorial libraries of antibody fragments having the desired activity. For example, various methods are known in the art for generating phage display libraries and screening these libraries to obtain antibodies with the desired binding properties. These methods are reviewed, for example, in Hoogenboom et al., Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001), and in, for example, McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352:624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1992); Marks and Bradbury, Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al. It is further described in Fellouse, Proc. Natl. Acad. Sci. USA 101(34):12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2):119-132 (2004).

[0191] In some phage display methods, V H and V LAll components of the gene are randomly recombined in a phage library, and then phage that can bind to the antigen is screened, as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). Phage usually display antibody fragments in the form of scFv fragments or Fab fragments. Library phage of immune origin provides high affinity antibodies to immunogens without the need to construct hybridoma cells. Alternatively, natural libraries (e.g., from humans) can be cloned to provide a single source of antibodies to a variety of non-self antigens and self antigens without any immunization, as described in Griffiths et al., EMBO J, 12: 725-734 (1993). Finally, natural libraries can also be prepared by cloning non-rearranged V-gene fragments from stem cells and using PCR primers containing random sequences to encode CDR3 hypervariable regions and complete rearrangement in vitro, as described in Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example, US Pat. No. 5,750,373 and US Patent Publication Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936 and 2009 / 0002360.

[0192] The antibody is prepared by screening the antibody portion of the library for binding to the target (e.g., IL35, IL27, or EBI3) through phage display. The library can be a human scFv phage display library with at least 1×10 9 (For example, at least 1×10 9 , 2.5×10 9 , 5×10 9 , 7.5×10 9 , 1×10 10 , 2.5×10 10 , 5×10 10 , 7.5×10 10 or 1×10 11) kinds of unique human antibody fragments with different diversity. In some embodiments, the library is a human natural library constructed by DNA extracted from PMBCs and spleens of healthy subjects, comprising all heavy and light chain subfamilies. In some embodiments, the library is a human natural library constructed by DNA extracted from PMBCs isolated from patients with various diseases, such as patients with autoimmune diseases, cancer patients, and patients with infectious diseases. In some embodiments, the library is a semisynthetic human library, wherein the heavy chain CDR3 is completely random, and all amino acids (except cysteine) are present at any given position with the same probability. (See, for example, Hoet, RM et al., Nat. Biotechnol. 23 (3): 344-348, 2005). In some embodiments, the heavy chain CDR3 length of the semisynthetic human library is between 5 and 24 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24) amino acids. In some embodiments, the library is a fully synthetic phage display library. In some embodiments, the library is a non-human phage display library.

[0193] With target (for example, IL35, IL27 or EBI3) there is a phage clone with high affinity can be screened by iterative combination of phage and target (for example, IL35, IL27 or EBI3), described target (for example, IL35, IL27 or EBI3) is combined with solid support (for example, the bead for solution panning or the mammalian cell for cell panning), next remove unbound phage, and elute specific binding phage. Subsequently, the phage clone of elution binding is also used to infect suitable host cells, such as E.coli XL1-Blue, express and purify. Can be by multiple rounds of panning (for example, 2,3,4,5,6 or more rounds), such as solution panning, cell panning or both are combined to enrich for the phage clone in conjunction with IL35, IL27 and / or EBI3. Binding of the enriched phage clones to the target (eg, IL35, IL27, or EBI3) can be detected by any method known in the art, including, for example, ELISA and FACS.

[0194] Another method for screening antibody libraries is to display proteins on the surface of yeast cells. Wittrup et al. (U.S. Patents 6,699,658 and 6,696,251) developed a method for displaying libraries on yeast cells. In this yeast display system, one component includes a yeast lectin protein (Aga1) anchored to the yeast cell wall, and the other component includes the second subunit of the lectin protein Aga2, which can bind to the Aga1 protein through a disulfide bond and then be displayed on the yeast cell surface. The Aga1 protein is expressed by integrating the Aga1 gene into the yeast chromosome. A single-chain variable fragment (scFv) library is fused to the Aga2 gene in a yeast display plasmid, and after transformation, the library can be retained in yeast due to the presence of an additional nutritional marker. Both Aga1 and Aga2 proteins are expressed under the control of a galactose-inducible promoter.

[0195] Human antibody V gene library (V H and V K The fragments were obtained by PCR using a set of degenerate primers (Sblattero, D. and Bradbury, A. Immunotechnology 3, 271-278 1998). The PCR templates were obtained from commercially available RNA or cDNA, including PBMC, spleen, lymph node, bone marrow and tonsil. H and V K After the PCR libraries were combined, they were assembled into scFv formats by overlap extension PCR (Sheets, MD et al, Proc. Natl. Acad. Sci. USA 95, 6157–6162 1998). To construct a yeast scFv display library, the resulting scFv PCR products were cloned into a yeast display plasmid in yeast by homologous recombination. (Chao, G, et al, Nat Protoc. 2006; 1(2): 755-68. Miller KD, et al. Current Protocols in Cytometry 4.7.1-4.7.30, 2008).

[0196] Mammalian cell display systems can be used to screen the antibodies described herein, wherein the antibody portion is displayed on the cell surface and antibodies targeting IL35, IL27 and / or EBI3 are isolated by antigen-directed screening methods (as described in US patent No. 7,732,195B2). A Chinese hamster ovary (CHO) cell library displaying a large number of human IgG antibody genes can be established and used to find clones expressing high-affinity antibody genes. Another display system has been developed that allows the same protein to be displayed and secreted simultaneously on the cell surface through alternative splicing, wherein the displayed protein phenotype remains associated with the genotype, making it possible to characterize the secreted soluble antibody in both biophysical and cell function-based assays. This method overcomes many limitations of previous mammalian cell display and enables direct screening and maturation of full-length, glycosylated IgGs (Peter M. Bowers, et al, Methods 2014, 65: 44-56). Transient expression systems are suitable for a single round of antigen selection before antibody gene recovery and are therefore most useful for selecting antibodies from smaller libraries. Stable exosome vectors offer an attractive alternative. Exosome vectors can be efficiently transfected and stably maintained at low copy numbers, allowing for multiple rounds of panning and the resolution of more complex antibody repertoires.

[0197] The IgG library was constructed based on the ligation of germline sequence V gene segments isolated from a group of human donors and rearranged (D)J regions. RNA collected from 2000 human blood samples was reverse transcribed into cDNA. H and V K Specific primers amplify V H and V K The fragment was purified by gel extraction. H and V K The fragments were subcloned into display vectors containing IgG1 or K constant regions, and then electroporated or transduced into 293T cells to prepare IgG libraries. H and V K To generate scFv, the fragments are then subcloned into a display vector and electroporated or transduced into 293T cells. As is well known, IgG libraries are constructed based on germline sequence V gene segments and rearranged (D)J regions isolated from a group of donors, which can be mice, rats, rabbits or monkeys.

[0198] Monoclonal antibodies can also be prepared by recombinant DNA methods, such as described in US Patent No. 4,816,567. The DNA encoding the monoclonal antibodies described in this application can be easily separated and sequenced by conventional methods (e.g., by oligonucleotide probes that can specifically bind to the light and heavy chain genes encoding mouse antibodies). Hybridoma cells as described above or phage clones of the binding targets of this application (e.g., IL35, IL27, or EBI3) can be used as the source of this DNA. After separation, the DNA can be placed in an expression vector, which is then transfected into a host cell, such as a monkey COS cell, a Chinese hamster ovary carcinoma (CHO) cell, or a myeloma cell that does not produce immunoglobulins, to obtain the monoclonal antibodies synthesized in the recombinant host cell. The DNA may also be modified, for example, by replacing the coding sequence for human heavy and light chain constant regions and / or replacing homologous non-human sequences with framework regions (US Patent No. 4,816,567; Morrison et al., supra), or by covalently linking the coding sequence for all or part of a non-immunoglobulin polypeptide to the immunoglobulin coding sequence. Such a non-immunoglobulin polypeptide may replace the constant region of the antibody of the present application, or may replace one of the antigen binding sites in the variable domain of the antibody of the present application to form a chimeric bivalent antibody.

[0199] The antibody can be a monovalent antibody. Methods for preparing monovalent antibodies are known in the art. For example, a method involves recombinant expression of immunoglobulin light chains and modified heavy chains. The heavy chain is typically truncated at any position in the Fc region to prevent cross-linking of the heavy chains. Alternatively, the relevant cysteine ​​residues are replaced with other amino acid residues or deleted to prevent cross-linking.

[0200] In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce antibody fragments, particularly Fab fragments, can be accomplished using any method known in the art.

[0201] The antibody variable domains with the desired binding specificity (antibody-antigen binding site) can be fused to an immunoglobulin constant region. Preferably, the fusion is performed with an immunoglobulin heavy chain constant region, which includes at least a portion of the hinge, CH2, and CH3 regions. In some embodiments, the first heavy chain constant region (CH1) containing the necessary sites for light chain binding is present in at least one fusion. The DNA encoding the immunoglobulin heavy chain fusion, and if desired, the DNA encoding the immunoglobulin light chain, is inserted into a separate expression vector and co-transfected into a suitable host organism.

[0202] Fully human and humanized antibodies

[0203] Antibodies as described herein (such as full-length antibodies) can be fully human antibodies or humanized antibodies. The humanized form of non-human (such as mouse) antibody part is a chimeric immunoglobulin, immunoglobulin chain or its fragment (such as Fv, Fab, Fab', F(ab')2, scFv or other antigen-binding subsequences of antibody), which generally includes a minimum sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins, immunoglobulin chains or its fragment (receptor antibody), wherein the residues of the receptor CDR are replaced by non-human (donor antibody) CDR residues with desired specificity, affinity and performance, such as mouse, rat or rabbit CDR. In certain embodiments, human immunoglobulin Fv framework region residues are replaced by corresponding non-human residues. Humanized antibodies can also include amino acid residues that neither belong to the receptor antibody nor in the CDR or framework region sequences introduced. Generally, humanized antibodies include at least one, usually two variable domains, wherein all or substantially all CDR regions correspond to the CDR regions of non-human immunoglobulins, and all or substantially all framework regions are human immunoglobulin consensus sequences.

[0204] Typically, a humanized antibody contains one or more amino acid residues introduced from a non-human source. Those non-human amino acid residues are generally referred to as "imported" residues, typically from the "imported" variable domain. According to some embodiments, humanization can be performed essentially according to the following method of Winter and colleagues (Jones et al., Nature, 321: 522-525 (1986); Riechmann et al., Nature, 332: 323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)), by replacing the corresponding sequence of a human antibody with rodent CDRs or CDR sequences. Therefore, this "humanized" antibody portion (US Patent No. 4,816,567) is substantially less than a complete human antibody, and its variable domains have been replaced by corresponding sequences from a non-human source. In practice, the humanized antibody portion is a typical human antibody portion in which some CDR residues and possibly some framework region residues are substituted by residues from analogous sites in rodent antibodies.

[0205] Fully human antibodies are an alternative to humanization. For example, it is now possible to prepare transgenic animals (e.g., mice) that can produce a complete library of fully human antibodies after immunization without producing endogenous immunoglobulins. For example, it has been reported that homozygous deletion of the antibody heavy chain joining region (JH) gene in chimeric and germline mutant mice completely inhibits endogenous antibody production. Transferring the human germline immunoglobulin gene array into such germline mutant mice can produce human antibodies upon antigen stimulation, see, for example, akobovits et al., PNAS USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggemann et al., Year in Immunol., 7:33 (1993); US Patent Nos. 5,545,806, 5,569,825, 5,591,669, 5,545,807; and WO 97 / 17852. Alternatively, fully human antibodies can be prepared by introducing human immunoglobulin loci into transgenic animals (e.g., mice in which endogenous immunoglobulin genes have been partially or completely silenced). Upon antigen stimulation, the production of fully human antibodies is found to be very similar to that produced in humans in all aspects, including gene rearrangement, assembly, and antibody libraries. This method is described in, for example, US Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016, and Marks et al., Bio / Technology, 10:779-783 (1992); Lonberg et al., Nature, 368:856-859 (1994); Morrison, Nature, 368:812-813 (1994); Fishwild et al., Nature Biotechnology, 14:845-851 (1996); Neuberger, Nature Biotechnology, 14:826 (1996); Lonberg and Described in Huszar, Intern. Rev. Immunol., 13:65-93 (1995).

[0206] Fully human antibodies can also be produced by in vitro activated B cells (see US Patents 5,567,610 and 5,229,275) or by using various techniques known in the art, including phage display libraries. Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). The techniques of Cole et al. and Boerner et al. can also be used to prepare fully human monoclonal antibodies. See Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p.77 (1985) and Boerner et al., J. Immunol., 147 (1):86-95 (1991).

[0207] Antibody variants

[0208] In some embodiments, the amino acid sequence of the antibody variants (e.g., full-length antibodies) provided herein is also under consideration. In some embodiments, the antibodies or variants thereof provided herein are combined with IL35, IL27 or EBI3. For example, it may be necessary to improve the binding affinity and / or other biological activities of the antibody. The amino acid sequence of the antibody variant can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions and / or insertions and / or substitutions of residues in the antibody amino acid sequence. The final construction can be completed by any combination of amino acid residue deletions, insertions and substitutions to give the desired characteristics. For example, antigen binding.

[0209] In some embodiments, antibody variants having one or more amino acid substitutions are provided. The target sites of the substitution mutations include hypervariable regions (HVRs) and framework regions (FRs). Amino acid substitutions can be introduced into the target antibody and the product screened for desired activity, for example, improved biological activity, maintained / improved antigen binding ability, reduced immunogenicity, or improved ADCC or CDC.

[0210] Conservative substitutions are shown in Table 4 below.

[0211] Table 4 Conservative substitutions

[0212] Amino acids are divided into different categories based on the properties of their side chains:

[0213] a. Hydrophobic amino acids: Norleucine, Met, Ala, Val, Leu, Isoleucine;

[0214] b. Neutral hydrophilic amino acids: cysteine ​​Cys, serine Ser, threonine Thr, asparagine Asn, glutamine Gln;

[0215] c. Acidic amino acids: Aspartic acid Asp, glutamic acid Glu;

[0216] d. Basic amino acids: histidine His, lysine Lys, arginine Arg;

[0217] e. Contains amino acids that affect chain direction: glycine Gly, proline Pro;

[0218] f. Aromatic amino acids: tryptophan Trp, tyrosine Tyr, phenylalanine Phe.

[0219] Non-conservative amino acid substitutions include substituting one class for another.

[0220] An exemplary substitution variant is an affinity-matured antibody, which can be conveniently produced using, for example, affinity maturation techniques based on phage display. In short, one or more CDR residues are mutated, the variant antibody portion is displayed on phage, and variants with specific biological activity (e.g., improved affinity with the antigen) are screened. Changes (e.g., substitutions) can be made in the HVRs region to obtain improved affinity. Changes can be made in the "hotspots" of the HVR, i.e., residues encoded by codons that undergo high-frequency mutations during somatic maturation (see, e.g., Chowdhury, Methods Mol. Biol. 207: 179-196 (2008)), and / or at specific deterministic residues (SDRs), and the resulting variant V can be detected. H and V L Methods for constructing and reselecting affinity maturation from secondary libraries have been described in some literature, for example, Hoogenboom et al. in Methods in Molecular Biology 178: 1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)).

[0221] In some affinity maturation embodiments, diversity is introduced into the variable genes selected for affinity maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide directed mutagenesis). A secondary library is then created. The library is screened to identify antibody variants with the desired affinity. Another method for introducing diversity includes HVR-mediated methods, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding are specifically identified, for example, using alanine scanning mutagenesis or modeling. Typically, CDR-H3 and CDR-L3 regions are particularly key targets.

[0222] In some embodiments, substitutions, insertions or deletions may occur within one or more HVRs, as long as such changes do not substantially reduce the ability of the antibody to bind to the antigen. For example, conservative changes that do not substantially reduce binding affinity (e.g., conservative substitutions provided herein) may be produced in HVRs. These changes may occur outside of HVR "hot spots" or SDRs regions. In some embodiments, the variant V provided above H and V L Sequences, each HVR is either unchanged or contains no more than 1, 2 or 3 amino acid substitutions.

[0223] A useful method for identifying amino acid residues or regions in antibodies that can be targeted for mutation is called "alanine scanning mutagenesis," as described in Cunningham and Wells (1989) Science, 244: 1081-1085. In this method, one or a group of target residues (e.g., charged residues such as arginine, aspartic acid, histidine, lysine, and glutamic acid) are replaced with neutral or negatively charged amino acids (e.g., alanine or glutamic acid) to determine whether the antibody-antigen interaction is affected. Substitutions can be further introduced at the amino acid position to demonstrate that the position has functional sensitivity to the initial substitution. Alternatively or additionally, the contact sites between the antibody and the antigen are identified by the crystal structure of the antigen-antibody complex. These contact site residues and adjacent residues can be targeted or eliminated as substitution candidates. Variants are screened to determine whether they have the desired properties.

[0224] Insertions of amino acid sequences include fusions at the amino and / or carboxyl termini ranging in length from one residue to polypeptides comprising 100 or more residues, and also include insertions of one or more amino acid residues within a sequence. Examples of terminal insertions include antibodies having a methionyl residue at the N-terminus. Other insertion variants of antibody molecules include fusions of an enzyme (e.g., ADEPT) or a polypeptide that increases the serum half-life of the antibody molecule to the N- or C-terminus of the antibody molecule.

[0225] Fc region variants

[0226] In some embodiments, one or more amino acid modifications are introduced into the Fc region of an antibody described herein (e.g., a full-length antibody or antibody fusion protein), thereby generating an Fc region variant. In some embodiments, the Fc region variant has enhanced ADCC potency, typically associated with receptors that bind to Fc (FcRs). In some embodiments, the Fc region variant has reduced ADCC potency. There are many examples of how changes or mutations in the Fc sequence affect its potency, for example, WO 00 / 42072 and Shields et al. J Biol. Chem. 9(2): 6591-6604 (2001) describe antibody variants with enhanced or reduced binding to FcRs. The contents of these publications are incorporated herein by reference.

[0227] Antibody-dependent cell-mediated cytotoxicity (ADCC) is the mechanism of action of therapeutic antibodies against tumor cells. ADCC is a cell-mediated immune defense in which effector cells of the immune system actively lyse target cells (e.g., cancer cells) when antigens on the target cell membrane are bound by specific antibodies. The ADCC effect typically involves NK cells activated by antibodies. NK cells express the Fc receptor CD16. This receptor recognizes and binds to the Fc portion of antibody molecules bound to the surface of target cells. The most common Fc receptors on the surface of NK cells are CD16 or FcγRIII. Binding of Fc receptors to the Fc region of antibodies leads to activation of NK cells, release of cytolytic granules, and subsequent apoptosis of the target cells. The specificity of ADCC against tumor cells can be determined using NK-92 cells transfected with high-affinity FcRs. The results are compared with wild-type NK-92 cells that do not express FcRs.

[0228] In some embodiments, the present application also provides a variant of an antibody described herein (e.g., a full-length antibody variant) comprising an Fc region with some but not all effector functions, such that it has an extended half-life in vivo, but a specific effector function (e.g., CDC or ADCC) is unnecessary or harmful, making such an antibody an ideal candidate for the present application. The reduction / elimination of CDC and / or ADCC activity is confirmed by performing cytotoxicity assays in vitro and / or in vivo. For example, an Fc receptor (FcR) binding assay is used to confirm that the antibody lacks FcγR binding ability (and therefore may lack ADCC activity) but still retains FcRn binding ability. Among the main cells mediating ADCC, NK cells express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. The expression of FcR on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet Annu. Rev. Immunol. 9: 457-492 (1991). Non-limiting examples of in vitro assessment of ADCC activity of a molecule of interest are described in US Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); US Pat. No. 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive detection methods can be used (see, e.g., ACTI TM Flow cytometry nonradioactive cytotoxicity assay (Cell Technology, Inc. Mountain View, Calif.) and CYTOTOX 96 TMNon-radioactive cytotoxicity assays (Promega, Madison, Wis.) can be used. Effector cells used in such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, ADCC activity of the target molecule can be tested in vivo, for example, in animal models as described in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). A C1q binding assay can also be performed to confirm that the antibody does not bind to C1q and thus lacks CDC activity. See, for example, C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay can be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life can be determined using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0229] Antibodies with reduced effector function comprising one or more substitutions at residues 238, 265, 269, 270, 297, 327, and 329 in the Fc region ( US Pat. No. 6,737,056 ). These Fc variants include Fc variants with substitutions at two or more residues at positions 265, 269, 270, 297, and 327, including an Fc variant known as "DANA" in which residues 265 and 297 are substituted with alanine ( US Pat. No. 7,332,581 ).

[0230] Such antibody variants with increased or decreased binding to FcRs have been described (see, eg, US Pat. No. 6,737,056; WO 2004 / 056312, and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001)).

[0231] In some embodiments, a variant of an antibody (e.g., a full-length antibody) described herein is provided, comprising an Fc region variant having one or more amino acid substitutions that enhance ADCC effects. In some embodiments, the Fc region variant comprises one or more amino substitutions that enhance ADCC effects, the positions of these substitutions being at positions 298, 333, and / or 334 (EU residue numbering) in the Fc region. In some embodiments, the antibody (e.g., full-length antibody) variant comprises amino acid substitutions at positions S298A, E333A, and K334A in the Fc region.

[0232] In some embodiments, alterations in the Fc region result in altered (ie, enhanced or reduced) C1q binding and / or complement-dependent cytotoxicity (CDC), as described in US Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al., J. Immunol. 164:4178-4184 (2000).

[0233] In some embodiments, a variant of an antibody described herein (e.g., a full-length antibody) is provided, comprising an Fc region variant with one or more amino acid substitutions that can extend half-life or enhance binding to an Fc receptor (FcRn). Antibodies with extended half-life and improved FcRn binding are described in US 2005 / 0014934A1 (Hinton et al.). These antibody Fc regions comprise one or more amino acid substitutions that enhance binding of the Fc region to FcRn. These Fc variants comprise one or more substitutions in the Fc region at residues 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434, such as substitutions at residue 434 in the Fc region (US Pat. No. 7,371,826).

[0234] See also Duncan & Winter, Nature 322:738-40 (1988); US Pat. No. 5,648,260; US Pat. No. 5,624,821 and WO 94 / 29351 for additional examples of Fc region variants.

[0235] The present application contemplates antibodies (eg, full-length antibodies that bind to IL35, IL27, or EBI3) comprising any one of the Fc variants described herein, or a combination thereof.

[0236] Glycosylation variants

[0237] In some embodiments, the antibodies provided herein (e.g., full-length antibodies that bind to IL35, IL27, or EBI3) are altered to increase or decrease the degree of glycosylation of the anti-NGF antibody. Adding or deleting glycosylation sites on an antibody can be conveniently achieved by altering the amino acid sequence of the antibody or its polypeptide portion to add or remove one or more glycosylation sites.

[0238] Wherein the antibody described herein comprises Fc district, can change the sugar connected thereto.Natural antibodies produced by mammalian cells generally comprise branched biantennary oligosaccharides, and this oligosaccharide is generally connected to Fc district CH2 domain Asn297 by N-connection, see for example Wright et al., TIBTECH 15:26-32 (1997).Described oligosaccharide can comprise a variety of carbohydrates, for example mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid, and trehalose connected to the GlcNAc of biantennary oligosaccharide structure " stem " portion.In certain embodiments, can carry out oligosaccharide modification to the antibody of the present application, thereby produce antibody variant with certain improved characteristics.

[0239] The N-glycans attached to the CH2 domain of the Fc region are heterogeneous. Antibodies or Fc fusion proteins produced in CHO cells are fucosylated by fucosyltransferase activity (see Shoji-Hosaka et al., J. Biochem. 2006, 140:777-83). Typically, a small fraction of naturally occurring non-fucosylated IgGs can be detected in human serum. N-glycosylation of the Fc region is important for binding to FcγRs; however, non-fucosylated N-glycans enhance the binding of Fc to FcγRIIIa. Enhanced binding to FcRIIIa results in enhanced ADCC, which is advantageous in certain antibody therapeutic applications requiring cytotoxicity.

[0240] In some embodiments, when Fc-mediated cytotoxicity is not desired, enhanced effector function may be detrimental. In some embodiments, the Fc fragment or CH2 domain is non-glycosylated. In some embodiments, glycosylation is prevented by mutating the N-glycosylation site in the CH2 domain.

[0241] In some embodiments, variants of antibodies described herein (e.g., full-length antibodies) are provided, which comprise an Fc region, wherein the carbohydrate structure attached to the Fc region has reduced fucose or lacks fucose, which may enhance ADCC function. Specifically, the antibodies provided herein have reduced fucose relative to the same antibodies produced by wild-type CHO cells. That is, they are characterized in that they have less fucose than antibodies produced by natural CHO cells (e.g., CHO cells that produce natural glycosylated forms, CHO cells containing natural FUT8 genes). In some embodiments, the N-linked glycans of the antibody have less than 50%, 40%, 30%, 20%, 10% or 5% fucose. For example, the fucose content of the antibody may be 1%-80%, 1%-65%, 5%-65% or 20%-40%. In some embodiments, the N-linked glycans of the antibody do not contain fucose, that is, the antibody is completely free of fucose, or has no fucose or is defucosylated. The fucose content is determined by calculating the average fucose content within the sugar chains attached to Asn297 relative to the total amount of all sugar structures (such as complex, hybrid, or mannose structures) attached to Asn297 as measured by MALDI-TOF mass spectrometry, as described in WO 2008 / 077546. Asn297 refers to the asparagine residue at position 297 in the Fc region (EU Fc region residue numbering system). However, due to minor sequence variations in antibodies, Asn297 may also be located ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. These fucosylation variants may have enhanced ADCC function. See, for example, US Patent Publication Nos. US2003 / 0157108 (Presta, L.), US2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.).Examples of publications related to "defucosylated" or "fucose-deficient" antibody variants include US 2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; US 2003 / 0115614; US 2002 / 0164328; US 2004 / 0093621; US ​​2004 / 0132140; US 2004 / 0110704; US 2004 / 0110282; US 2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO 2005 / 035778; WO 2005 / 053742; WO 2002 / 031140; Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004). Cell lines capable of producing defucosylated antibodies include Lec13 CHO cells lacking protein fucosylation function (Ripka et al. Arch. Biochem. Biophys. 249: 533-545 (1986); US Pat Appl No US2003 / 0157108 A1, Presta, L; and WO 2004 / 056312 A1, Adams et al., especially Example 11), and gene knockout cell lines, such as CHO cells in which the α-1,6-fucosyltransferase gene, FUT8, is knocked out (see Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4): 680-688 (2006); and WO2003 / 085107).

[0242] Variants of the antibodies described herein (e.g., full-length antibodies) further provide bisected oligosaccharides, for example, wherein the biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibody (e.g., full-length antibody) variants may have reduced fucosylation and / or enhanced ADCC function. Examples of such antibody variants are described in WO 2003 / 011878 (Jean-Mairet et al.); US Pat. No. 6,602,684 (Umana et al.); US 2005 / 0123546 (Umana et al.), and Ferrara et al., Biotechnology and Bioengineering, 93(5):851-861 (2006). Variants of the antibodies described herein (e.g., full-length antibodies) are also provided, which have at least one galactose residue in the oligosaccharide attached to the Fc region. Such antibody variants may have enhanced CDC function. Such variants are described, for example, in WO 1997 / 30087 (Patel et al.); WO 1998 / 58964 (Raju, S.); and WO 1999 / 22764 (Raju, S.).

[0243] In some embodiments, the variants of the antibodies (e.g., full-length antibodies) comprise an Fc region that binds to FcγRIII. In some embodiments, the variants of the antibodies (e.g., full-length antibodies) comprising the Fc region have ADCC activity in the presence of human effector cells (e.g., T cells), or have enhanced ADCC activity in the presence of human effector cells compared to an otherwise identical antibody (e.g., full-length antibody) having a human wild-type IgG1 Fc region.

[0244] Cysteine ​​engineered variants

[0245] In some embodiments, it is desirable to prepare cysteine ​​engineered antibodies (e.g., full-length antibodies) in which one or more amino acid residues are substituted with cysteine ​​residues. In some embodiments, the substituted residues occur at accessible sites of the antibody. By replacing those residues with cysteine, reactive sulfhydryl groups are located at accessible sites of the antibody, which can be used to couple the antibody to other moieties, such as drug moieties or linker-drug moieties, to prepare immunoconjugates as further described herein. Cysteine ​​engineered antibodies (e.g., full-length antibodies) can be prepared as described, for example, in U.S. Pat. No. 7,521,541.

[0246] derivative

[0247] In some embodiments, the antibodies provided herein (e.g., full-length antibodies) can be further modified to include other non-protein moieties known in the art and readily available. Suitable portions for derivatized antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), dextran or poly-(n-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, propylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol and mixtures thereof. Polyethylene glycol propionaldehyde has advantages in manufacturing due to its stability in water. Polymers can have any molecular weight and can be branched or unbranched. The number of polymers attached to the antibody can vary, and if more than one polymer is attached, they can be the same or different molecules. Generally, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the desired properties or function of the antibody, whether the antibody derivative is intended for treatment of a particular condition, etc.

[0248] Pharmaceutical composition

[0249] Also provided herein are compositions (e.g., pharmaceutical compositions, also referred to herein as formulations) comprising any of the antibodies (e.g., full-length antibodies), nucleic acids encoding the antibodies, vectors comprising nucleic acids encoding the antibodies, or host cells comprising nucleic acids or vectors described herein. In some embodiments, a pharmaceutical composition is provided comprising any of the antibodies described herein and a pharmaceutically acceptable carrier.

[0250] Suitable antibody formulations can be obtained by mixing an antibody having the desired purity with an optional pharmaceutically acceptable carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)) and prepared in the form of a lyophilized formulation or a liquid formulation. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations used, and include buffers such as phosphates, citric acid, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethylammonium chloride; benzalkonium chloride; benzethonium chloride; phenol; butyl alcohol or benzyl alcohol; alkyl parabens such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol); low molecular weight (less than 1 0 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (such as zinc-protein complexes); and / or nonionic surfactants such as TWEEN TM , PLURONICS TM Or polyethylene glycol (PEG); Exemplary formulations are described in WO98 / 56418 and expressly incorporated herein by reference. Lyophilized formulations suitable for subcutaneous administration are described in WO97 / 04801. Such lyophilized formulations can be reconstituted with a suitable diluent to a high protein concentration formulation, and the reconstituted formulation can be administered subcutaneously to the subject to be treated herein. Cationic liposomes or liposomes can be used to deliver the antibodies in this application to cells.

[0251] In addition to the antibodies described herein (full-length antibodies), the formulations described herein may also contain one or more other active substances necessary for the treatment of a specific condition, preferably substances with complementary activities and no adverse reactions to each other. For example, in addition to the antibodies, it may be necessary to further include an anti-tumor agent, a growth inhibitory agent, a cytotoxic agent, a chemotherapeutic agent, an immunomodulatory agent, or an anti-infective agent. These molecules are present in combination in an amount effective for the intended purpose. The effective amount of the other substances depends on the amount of the antibody in the formulation, the type of disease or condition or treatment, and other factors as described above. These drugs are generally used in the same dosage and route of administration as described herein, or at 1% to 99% of the currently used dosage.

[0252] Antibodies described herein (e.g., full-length antibodies) can also be entrapped in microcapsules prepared, for example, by coacervation techniques and interfacial polymerization, such as hydroxymethylcellulose or gelatin-microcapsules and poly(methyl methacrylate) microcapsules in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or in macroemulsions, respectively. Sustained-release formulations can be prepared.

[0253] Sustained-release formulations with antibodies described herein (e.g., full-length antibodies) can be prepared. Suitable examples of sustained-release formulations include solid hydrophobic polymer semipermeable matrices containing antibodies (or fragments thereof), which are in the form of molded articles, such as films or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate) or poly(vinyl alcohol)), polylactic acid (US Pat. No. 3,773,919), L-glutamic acid and L-glutamic acid ethyl ester copolymers, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprorelin acetate) and poly-D (-) -3-hydroxybutyric acid. Although polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid can release molecules for more than 100 days, certain hydrogels can release proteins in a shorter period of time. When encapsulated antibodies remain in the body for a long time, they can denature or aggregate due to exposure to a humid environment at 37°C, potentially leading to loss of biological activity or altered immunogenicity. Rational strategies can be designed to stabilize antibodies based on the corresponding mechanisms. For example, if the aggregation mechanism is found to be through the formation of intermolecular SS bonds via thiodisulfide exchange, stabilization can be achieved by modifying sulfhydryl residues, lyophilizing in acidic solutions, controlling water content, using appropriate additives, and developing specific polymer matrix compositions.

[0254] In some embodiments, the antibody (eg, full-length antibody) is formulated in a buffer containing citrate, sodium chloride, acetate, succinate, glycine, polysorbate 80 (Tween 80), or any combination thereof.

[0255] Preparations for in vivo administration must be sterile. This can be readily achieved, for example, by filtration through sterile filtration membranes.

[0256] Treatments using antibodies

[0257] The antibodies described herein (e.g., full-length antibodies) and / or compositions described herein can be administered to individuals (e.g., mammals, such as humans) to treat diseases and / or conditions (e.g., immune-related diseases, tumors) associated with the IL35 and / or IL27 signaling pathways, including but not limited to viral infections, bacterial infections, and tumors. Therefore, the present application, in some embodiments, provides a method for treating diseases and / or conditions (e.g., immune-related diseases, tumors) associated with the IL35 and / or IL27 signaling pathways, comprising administering to the individual an effective amount of a composition (e.g., a pharmaceutical composition) comprising an antibody described herein (e.g., a full-length antibody), and / or any of the antibodies described herein (e.g., full-length antibodies). In some embodiments, the individual is human.

[0258] For example, in some embodiments, a method for treating an individual with a disease associated with the IL35 and / or IL27 signaling pathway (e.g., an immune-related disease, a tumor) is provided, comprising administering to the individual an effective amount of a pharmaceutical composition comprising an antibody (e.g., a full-length antibody) that binds to an epitope on a target (e.g., IL35, IL27, or EBI3). In some embodiments, the antibody is a full-length antibody. In some embodiments, the full-length antibody is an IgG1 or IgG4 antibody. In some embodiments, the disease or condition is selected from, for example, a viral infection, a bacterial infection, or a tumor. In some embodiments, the individual is human.

[0259] In some embodiments, the antibody binds to IL35, IL27, or EBI3.

[0260] In certain embodiments, the individual is a mammal (e.g., a human, non-human primate, rat, mouse, cattle, horse, pig, sheep, goat, dog, cat, etc.). In certain embodiments, the individual is a human. In certain embodiments, the individual is a clinical patient, a clinical trial volunteer, an experimental animal, etc. In certain embodiments, the individual is less than 60 years old (including, for example, less than 50, 40, 30, 25, 20, 15, or 10 years old). In certain embodiments, the individual is greater than 60 years old (including, for example, greater than 70, 80, 90, or 100 years old). In certain embodiments, the individual is diagnosed as or is susceptible to one or more diseases or conditions described herein (e.g., immune-related diseases, tumors) from a genetic perspective. In certain embodiments, the individual has one or more risk factors associated with one or more diseases or conditions described herein.

[0261] In some embodiments, the present application provides a method of delivering an antibody (eg, any of the antibodies described herein) to cells expressing IL35 and / or IL27 in an individual, the method comprising administering to the individual a composition comprising the antibody.

[0262] Many diagnostic methods and clinical descriptions of immune-related diseases, tumors, or any other diseases that exhibit abnormalities in the IL35 and / or IL27 signaling pathways are known in the art. Such methods include, but are not limited to, immunohistochemistry, PCR, and fluorescence in situ hybridization (FISH).

[0263] In some embodiments, the antibodies (e.g., full-length antibodies) and / or compositions described herein are used in combination with a second, third, or fourth agent (including, for example, an anti-tumor agent, a growth inhibitory agent, a cytotoxic agent, a chemotherapeutic agent, an immunomodulatory agent, or an anti-infective agent) to treat diseases associated with the IL35 and / or IL27 signaling pathway.

[0264] Dosage and method of administration of antibodies

[0265] The dosage of an antibody (e.g., an antibody as described herein) composition administered to an individual (e.g., a person) may vary depending on the specific composition, mode of administration, and type of disease being treated. In some embodiments, the amount of a composition (e.g., a composition comprising an antibody as described herein) can effectively produce an objective response (e.g., a partial response or a complete response) in immune-related diseases or tumor treatment. In some embodiments, the amount of the antibody composition is sufficient to produce a complete response in an individual. In some embodiments, the amount of the antibody composition is sufficient to produce a partial response in an individual. In some embodiments, the dosage of the antibody composition (e.g., when administered alone) is sufficient to produce an overall response rate higher than 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 64%, 65%, 70%, 75%, 80%, 85% or 90% in an individual population treated with the antibody composition. An individual's response to the methods described herein can be determined, for example, by pathogen load.

[0266] In some embodiments, the amount of the composition (e.g., a composition comprising an antibody described herein) is sufficient to extend the progression-free survival of an individual. In some embodiments, the amount of the composition is sufficient to extend the overall survival of an individual. In some embodiments, the amount of the composition (e.g., when administered alone) is sufficient to produce a clinical benefit greater than 50%, 60%, 70%, or 77% in a population of individuals treated with the antibody composition.

[0267] In some embodiments, the amount of a composition (e.g., a composition comprising an antibody described herein), when used alone or in combination with a second, third, and / or fourth agent, is an amount sufficient to control symptoms and reduce the risk of exacerbations compared to the corresponding activity in other subjects not receiving treatment before treatment. The magnitude of the therapeutic effect can be measured using standard methods, such as in vitro assays using purified enzymes, cell-based assays, animal models, or human trials.

[0268] In some embodiments, the amount of an antibody described herein (e.g., a full-length antibody) in the composition is below a level that causes a toxic effect (i.e., an effect above a clinically acceptable toxicity level) when the composition is administered to a subject, or is at a level at which potential side effects are manageable or tolerable.

[0269] In some embodiments, following the same dosing regimen, the amount of the composition is close to the maximum tolerated dose (MTD) of the composition. In some embodiments, the amount of the composition is greater than 80%, 90%, 95% or 98% of the MTD.

[0270] In some embodiments, the amount of an antibody described herein (eg, a full-length antibody) in the composition is in the range of 0.001 μg to 1000 μg.

[0271] In any of the above embodiments, the effective amount of the antibody described herein (eg, full-length antibody) in the composition is in the range of 0.1 μg / kg to 100 mg / kg, calculated based on body weight.

[0272] Compositions containing antibodies described herein can be administered to individuals (e.g., humans) by a variety of routes, including, for example, intravenous injection, intraarterial administration, intraperitoneal injection, intrapulmonary administration, oral administration, inhalation administration, intravascular administration, intramuscular injection, intratracheal administration, subcutaneous injection, intraocular administration, intrathecal administration, mucosal administration, or transdermal administration. In some embodiments, a sustained-release formulation of the composition is used. In some embodiments, the composition is administered intravenously. In some embodiments, the composition is administered by arterial administration. In some embodiments, the composition is administered intraperitoneally. In some embodiments, the composition is administered intrahepatically. In some embodiments, the composition is administered by hepatic artery infusion. In some embodiments, the composition is administered at a location away from the first lesion.

[0273] In some embodiments, the antibody binds to IL35, IL27, or EBI3.

[0274] Products and kits

[0275] In some embodiments of the present application, a product is provided, comprising a substance that can be used to treat diseases associated with the IL35 and / or IL27 signaling pathway (e.g., immune-related diseases, tumors), or for delivering antibodies (e.g., full-length antibodies) to cells expressing IL35 and IL27. The product may include a container and a label or package insert on or accompanying the container. Suitable containers include, for example, bottles, vials, syringes, etc. The container can be made of a variety of materials, such as glass or plastic. Typically, the container contains a composition that can effectively treat the disease or condition described herein and has a sterile port (e.g., the container can be an intravenous infusion bag or a vial with a lid that can be pierced by a hypodermic injection needle). At least one active substance in the composition is the antibody described herein. The label or package insert indicates the specific condition that the composition can be used to treat. The label or package insert further includes instructions for administering the antibody composition to a patient. Products and kits including combination therapies are within the scope of this invention.

[0276] Package inserts are instructions typically included in the commercial packaging of therapeutic products that contain information regarding the indications, usage, dosage, administration, contraindications, and / or warnings related to the use of these therapeutic products. In some embodiments, the package insert indicates that the composition can be used to treat diseases related to the IL35 and / or IL27 signaling pathways (e.g., immune-related diseases, tumors). In some embodiments, the package insert indicates that the composition can be used to treat the following diseases, including viral infections, bacterial infections, and tumors.

[0277] In addition, the article of manufacture may further comprise a second container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, or dextrose solution. Other materials desirable from a commercial and user perspective may also be included, including other buffers, diluents, filters, needles, and syringes.

[0278] It also relates to kits that can be used for various purposes, such as for treating diseases associated with IL35 and / or IL27 signaling pathways (e.g., immune-related diseases, tumors), or for delivering antibodies (e.g., full-length antibodies) to cells expressing IL35 and IL27, optionally in combination with products. The kit of the present application includes one or more containers comprising an antibody composition (or single-dose form and / or product), and in some embodiments, further comprising another agent (e.g., an agent described herein) and / or instructions for use consistent with any of the methods described herein. The kit may further include a description of selecting an individual suitable for treatment. The instructions for use included in the kit in the present application are typically written instructions on a label or package insert (e.g., a paper sheet contained in the kit), and machine-readable instructions (e.g., instructions on a magnetic or optical storage disc) are also acceptable.

[0279] For example, in some embodiments, the kit includes a composition comprising an antibody described herein (e.g., a full-length antibody). In some embodiments, the kit includes: a) a composition comprising any of the antibodies described herein, and b) at least one effective amount of other agent that can enhance the effect of the antibody (e.g., therapeutic effect, detection effect). In some embodiments, the kit includes: a) a composition comprising any of the antibodies described herein, and b) instructions for administering the antibody composition to an individual for treating a disease associated with the IL35 and / or IL27 signaling pathway (e.g., immune-related disease, tumor). In some embodiments, the kit includes: a) a composition comprising any of the antibodies described herein, and b) at least one effective amount of other agent that can enhance the effect of the antibody (e.g., therapeutic effect, detection effect), and c) instructions for administering the antibody composition and other substances to an individual for treating a disease associated with the IL35 and / or IL27 signaling pathway (e.g., immune-related disease, tumor). The antibodies and other substances may be present in separate containers or in the same container. For example, the kit may include one specific composition or two or more compositions, wherein one composition includes an antibody and another composition includes another agent.

[0280] In some embodiments, the kit comprises one (or a set of) nucleic acids encoding an antibody described herein (e.g., a full-length antibody). In some embodiments, the kit comprises: a) one (or a set of) nucleic acids encoding an antibody described herein (e.g., a full-length antibody), and b) a host cell expressing the nucleic acid (or a set of nucleic acids). In some embodiments, the kit comprises: a) one (or a set of) nucleic acids encoding an antibody described herein (e.g., a full-length antibody), and b) instructions for use, suitable for: i) expressing the antibody in a host cell, ii) preparing a composition comprising the antibody, and iii) administering a composition comprising the antibody to an individual to treat a disease associated with the IL35 and / or IL27 signaling pathway (e.g., an immune-related disease, a tumor). In some embodiments, the kit comprises: a) a nucleic acid (or a set of nucleic acids) encoding an antibody described herein (e.g., a full-length antibody), b) a host cell that expresses the nucleic acid (or a set of nucleic acids), and c) instructions for use, suitable for: i) expressing the antibody in the host cell, ii) preparing a composition comprising the antibody, and iii) administering the composition comprising the antibody to an individual to treat a disease associated with the IL35 and / or IL27 signaling pathway (e.g., an immune-related disease, a tumor).

[0281] The test kits described herein are packaged in a suitable form. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed polyester film or plastic bags), etc. The test kits may optionally provide other components, such as buffers and instructional information. Therefore, the application also provides articles, which include vials, bottles, jars, flexible packaging (e.g., sealed polyester film or plastic bags), etc.

[0282] The instructions for use of the antibody composition generally include information such as dosage, administration cycle and route of administration. The container can be a unit dose, a bulk package (e.g., a multi-dose package) or a subunit dose. For example, a test kit comprising an antibody as described herein (e.g., a full-length antibody) in sufficient dosage is provided to treat an individual for a long period of time, e.g., one week, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 3 months, 4 months, 5 months, 7 months, 8 months, 9 months or longer. The test kit may also include multiple unit doses of the antibody, pharmaceutical composition and instructions for use, and packaged in an amount sufficient for storage and use in a pharmacy, e.g., a hospital pharmacy or compounding pharmacy.

[0283] Those skilled in the art will recognize that several embodiments are possible within the scope and purpose of this application. The application will now be described in more detail with reference to the following non-limiting examples. The following examples further illustrate the application, but should not be construed as limiting its scope in any way. DETAILED DESCRIPTION

[0284] Example 1: Preparation of IL27 and IL35 recombinant proteins

[0285] The cDNA fragments of the human EBI3 subunit (GenBank ID: 10148) and the p28 subunit (GenBank ID: 246778) with the N-terminal signal peptide removed were amplified by PCR. A His tag was added to the C-terminus of the p28 subunit. The EBI3 and p28 cDNAs were seamlessly cloning and connected using a linker. The resulting plasmid was constructed into a eukaryotic expression vector to generate a human IL27 expression plasmid. The recombinant protein obtained using this plasmid was named hIL27-His, where "His" represents the His tag.

[0286] The cDNA fragments of the human EBI3 subunit (GenBank ID: 10148) and the human p35 subunit (GenBank ID: 3592) with the N-terminal signal peptide removed were amplified by PCR, and either an hFc or mFc tag was fused to the C-terminus of the p35 subunit. The EBI3 and p35 cDNAs were linked using a linker via seamless cloning and constructed into eukaryotic expression vectors to generate human IL35 expression plasmids. The recombinant proteins obtained using these plasmids were designated hIL35-hFc or hIL35-mFc, respectively, where "hFc" represents the human Fc fragment and "mFc" represents the mouse Fc fragment. A monkey IL35 expression plasmid was constructed using the same method, and the recombinant protein obtained using this plasmid was designated cynoIL35-hFc, where "hFc" represents the human Fc fragment.

[0287] cDNA fragments of the human EBI3 subunit (GenBank ID: 10148), the human p35 subunit (GenBank ID: 3592) without the N-terminal signal peptide, and the internal ribosome entry site (IRES) sequence or 2A peptide sequence were amplified by PCR. A His tag was added to the C-terminus of EBI3, and a Flag tag was added to the C-terminus of the p35 subunit. The EBI3 and p35 cDNAs were linked by seamless cloning using the IRES sequence or 2A peptide cDNA and cloned into a eukaryotic expression vector to generate a human IL35 expression plasmid. This plasmid can simultaneously express both IL35 subunits, p35 and EBI3, and assemble them into a complete IL35. The expression product of the plasmid was purified using a nickel column and validated by ELISA using anti-Flag-HRP and anti-His-HRP. The results were all positive, indicating that the p35 and EBI3 subunits can assemble into a complete IL35. The recombinant protein obtained using the above plasmid was named hIL35-His, where "His" represents a His tag.

[0288] A cDNA fragment of the human IL12Rβ2 extracellular domain (GenBank ID: 3595) was amplified by PCR, and an hFc tag was fused to its C-terminus. The IL12Rβ2 extracellular domain was constructed into a eukaryotic expression vector by seamless cloning to generate a human IL12Rβ2 extracellular domain expression plasmid. The recombinant protein obtained using this plasmid was named hIL12Rβ2-hFc, where "hFc" stands for human Fc fragment.

[0289] A cDNA fragment of the human gp130 subunit (GenBank ID: 3572) was amplified by PCR and fused to the C-terminus with an hFc tag. The gp130 subunit was seamlessly cloned into a eukaryotic expression vector to generate a human gp130 subunit expression plasmid. The recombinant protein obtained using this plasmid was named gp130-hFc, where "hFc" stands for human Fc fragment.

[0290] The above expression vectors were transfected into 293F cells and cultured at 37°C, 5% CO2, and 120 rpm for 5 days. The cell culture medium was collected. The His-tagged recombinant protein was purified using a nickel column (Ni) according to the manufacturer's instructions, and the Fc-tagged recombinant protein was purified using a Protein A affinity column.

[0291] Example 2: Screening for single-chain antibodies (scFv) binding to IL35 / IL27

[0292] 2.1 Construction of scFv antibody yeast display library:

[0293] The hIL35-mFc prepared in Example 1 was used as an antigen and mixed with an equal volume (v / v) of adjuvant to immunize 6-8 week old BALB / c mice by subcutaneous multi-point injection. After several rounds of immunization, the total IgG titer in the serum of the immunized mice was detected by ELISA. Mice with high IgG titers were selected and intraperitoneally immunized with equal amounts of hIL35-mFc and cynoIL35-hFc. After 3 days, a phage display library was established using the spleen of the mouse: Briefly, the spleen of the immunized mouse was taken, RNA was extracted, and cDNA was obtained by reverse transcription. V H and V K Specific primers amplify V H and V K The fragment was purified by gel recovery and ligated to V H and V K , scFv was constructed and cloned into the phage display plasmid pDAN3X. Subsequently, the plasmid was electroporated into Escherichia coli TG1, and the phage was used to infect Escherichia coli TG1 to obtain the scFv antibody phage display library.

[0294] 2.2 Screening of single-chain antibodies (scFv) binding to IL35 / IL27:

[0295] In multiple rounds of panning, a solid phase screening strategy was used to isolate phage-displayed antibodies that bind to human IL35 from the phage display library. Briefly, hIL35-His antigen was coated on an ELISA plate, and the constructed phage display library was added to the plate at an input of 4 × 10 11 pfu / well and incubate at 37°C for 2 hours. Wash the plate to remove nonspecific and weak-affinity phages, retain the positive phages, and then elute the positive phages with an acidic elution solution. Repeat this process for three rounds of screening to enrich the positive phages that bind to hIL35-His. After infecting Escherichia coli with the positive phages, spread them onto culture dishes, pick single clones and culture them, and then further test the binding activity of the individual positive clones with the hIL35-His antigen, cynoIL35-hFc antigen, or hIL27-His antigen prepared in Example 1 by ELISA. Positive clones that can bind to human IL35, cynomolgus monkey IL35, and human IL27 are selected for subsequent experiments.

[0296] Example 3: Preparation and characterization of chimeric antibodies

[0297] 3.1 Preparation of chimeric antibodies

[0298] The positive clones screened initially were sequenced and V was amplified from the phage display vector. L and V H , respectively, into the eukaryotic expression vectors pTTa1-L (containing the human kappa constant region) and pTTa1-H1 (containing the human IgG1 heavy chain constant region), or pTTa1-H4 (containing the human IgG4 heavy chain constant region). The light chain-expressing plasmids and heavy chain-expressing plasmids were co-transfected into 293F cells and incubated at 37°C, 5% CO2, and 120 rpm for 6 days. Antibodies were purified using a Protein A affinity chromatography column according to the manufacturer's instructions, and the antibody concentration was determined.

[0299] 3.2 ELISA binding assay

[0300] The purified chimeric antibody (reconstructed into adult IgG1 form) was subjected to binding experiments with human IL35 and IL27, respectively, to identify the binding activity of the antibody. Briefly, the hIL35-hFc antigen or hIL27-His antigen prepared in Example 1 was diluted to 2 μg / ml, and 100 μl of antigen was added to each well of a 96-well plate and incubated at 4°C overnight. Wash with PBST solution 6 times and add 50 μL of 1% BSA. The antibody sample to be tested was diluted to 50 μg / ml, followed by 5-fold gradient dilution for a total of 8 concentrations. The gradient diluted samples were added to 96-well plates, 50 μl per well, and incubated at 37°C for 1 hour. Subsequently, it was washed 6 times with PBST solution. 100 μl of 4000-fold diluted goat anti-human Kappa-AP (Sothernbiotech, Cat#2060-04) secondary antibody was added to each well and incubated at 37°C for 1 hour. Wash with PBST solution 6 times. 100 μl of pNpp was added to each well for color development and incubated at 37°C for 5-10 minutes. OD405nm was measured using a microplate reader. Binding curves were generated using PRISM software to calculate the EC values ​​for each antibody molecule binding to human IL35 and IL27. 50 value.

[0301] The results are shown in Table 5-1. The obtained chimeric antibodies m24, m57, m58, and m62 can bind to both IL35 and IL27, while the control antibody anti-IL27 Ab1 only binds to IL27 and not to IL35.

[0302] Table 5-1: EC values ​​of chimeric antibodies binding to human IL35 and IL27 50 value

[0303] Note: “ / ” indicates no binding activity

[0304] IL35 and IL27 share a common subunit, EBI3. ELISA was performed to verify binding of the above antibodies to the EBI3 subunit. Briefly, EBI3 antigen (R&D, Catalog #: 11084-EI) was diluted to 2 μg / ml, and 100 μl of antigen was added to each well of a 96-well plate and incubated overnight at 4°C. The plate was washed six times with PBST, and 50 μl of 1% BSA was added. The antibody sample to be tested was diluted to 200 μg / ml and then serially diluted 5-fold to a total of eight concentrations. 50 μl of the serially diluted sample was added to each well of a 96-well plate and incubated at 37°C for 1 hour. The plate was then washed six times with PBST. 100 μl of a 4000-fold diluted goat anti-human Kappa-AP (Sothernbiotech, Cat #2060-04) secondary antibody was added to each well and incubated at 37°C for 1 hour. The plate was washed six times with PBST. 100 μl of pNpp was added to each well for color development and incubated at 37°C for 5-10 minutes. OD405nm was measured using a microplate reader. Binding curves were generated using PRISM software to calculate the EC values ​​for each antibody molecule binding to human IL35 and IL27. 50 value.

[0305] The results are shown in Table 5-2. The obtained chimeric antibodies m24, m57, m58, and m62 were all able to bind to EBI3, while the control antibody anti-IL27 Ab1 did not bind to EBI3.

[0306] Table 5-2: EC values ​​of chimeric antibodies binding to human IL35 and IL27 50 value

[0307] 3.3 Determination of chimeric antibody activity using the IL27-STAT3 reporter gene assay:

[0308] After IL27 binds to its receptor, it activates the transcription factor STAT3. STAT3 forms a dimer and enters the cell nucleus, recognizes and binds to response elements (such as SIE), and induces the expression of downstream reporter genes (such as luciferase). After the antibody binds to IL27, it inhibits the binding of IL27 to its receptor, thereby inhibiting the expression of the reporter gene. Based on the above principle, the plasmid pGL4.47 [luc2P / SIE / puro] containing the STAT3 response element SIE, the luciferase gene, and the puromycin selection marker gene was transfected into HekblueIL6 (In vivogene, Cat#hkb-hil6) cells, and puromycin was added for resistance screening to obtain a stable monoclonal cell line. Finally, the clone HekblueIL6-3C with high reporter gene activity was selected to detect the reporter gene expression activity, thereby characterizing the inhibitory activity of the antibody on the IL27 signaling pathway.

[0309] Briefly, the stable cell line HekblueIL6-3C was seeded into 96-well culture plates at a rate of 4 × 10 cells per well. 4 cells. Dilute the hIL27-His protein prepared in Example 1 to 30 ng / ml with complete culture medium. Dilute the test antibody concentration to 9 μg / ml with PBS (dilute to 300 μg / ml for single-point initial screening, no need for gradient dilution), followed by 3-fold gradient dilution, for a total of 10 concentrations. Add 50 μl of diluted test antibody and 50 μl of hIL27-His protein to a 96-well plate, shake gently to mix, and incubate at 37°C, 5% CO2 for 24 hours. Take out the 96-well plate, equilibrate to room temperature, and add 50 μl of ONE-Glo to each well. TM (Promega, Cat#E6120). After shaking in the dark for 10 minutes, the fluorescence intensity of each well was measured using a microplate reader. The fluorescence intensity-antibody concentration logarithmic curve was generated using PRISM software to calculate the IC of each antibody to inhibit IL27 activation of STAT3 signaling. 50 .

[0310] The results in Table 6 show that m24, m58, and m62 can effectively block the IL27 signaling pathway. m57 was only screened at a single point, and its IC 50 The results showed that it could also effectively block the IL27 signaling pathway (data not shown).

[0311] Table 6: Inhibitory activity of chimeric antibodies

[0312] 3.4 Chimeric antibodies inhibit the binding of human IL35 to HekblueIL6 cells:

[0313] HekbuleIL6 cells express IL35 receptors, so IL35 can bind to these cells. The activity of the test antibody in inhibiting the binding of IL35 to its receptor can be evaluated by detecting the binding of IL35 to cells. Briefly, HekblueIL6 cells were seeded into 96-well culture plates, and 50 μL (4x10 6cells / mL). Dilute the antibody to be tested to 800 μg / ml (no gradient dilution is required for single-point initial screening), followed by 3-fold gradient dilution for a total of 8 concentrations. Dilute the hIL35-hFc protein prepared in Example 1 to 160 μg / ml. Add 25 μl of the diluted antibody to be tested and 25 μL of hIL35-hFc protein to a 96-well plate, mix well, and incubate at 37°C, 5% CO2 for 30 minutes. After incubation, add 100 μL of PBS (containing 1% BSA) to each well, centrifuge at 600g for 5 minutes, and discard the supernatant. Add 100 μL of PBS (containing 1% BSA) to each well to resuspend the cells, centrifuge and remove the supernatant. Add 100 μL of mouse anti-human IgG Fc-FITC (1:200 dilution) to each well, mix well, and incubate at 4°C for 30 minutes. After incubation, 100 μL of PBS (containing 1% BSA) was added to each well, and the cells were centrifuged at 600 g for 5 minutes. The supernatant was discarded. The cells were resuspended in 100 μL of PBS (containing 1% BSA) per well, and FITC fluorescence was measured by flow cytometry. The proportion of IL35-positive cells in the main cell population was analyzed, and the inhibitory activity of the antibody against IL35 binding to HekBlueIL6 cells was analyzed by plotting the positive cell proportion against the logarithm of the antibody concentration.

[0314] The results in Table 7 show that m57, m58, and m62 can effectively inhibit the binding of IL35 to HekblueIL6 cells. m24 was only screened at a single point, and its IC 50 The results showed that it could also inhibit the binding of IL35 to HekblueIL6 cells (data not shown).

[0315] Table 7: Activity of chimeric antibodies in inhibiting the binding of IL35 to HekblueIL6 cells

[0316] 3.5 Chimeric Antibodies Inhibit Binding of Human IL35 to PBMCs:

[0317] Activated PBMC cells also express IL35 receptors. The activity of exemplary antibodies m57 and m58 in primary cells can be further evaluated by detecting the binding of IL35 and activated PBMC. Briefly, PBMC cells were cultured, and IL2 and CD3 / CD28 magnetic beads (Gibco, Cat#11131D) were added to the culture medium to induce PBMC cell activation. Thereafter, the activated PBMC cells were seeded into 96-well culture plates, with 50 μL (4x10 6cells / mL). Dilute the test antibody to 800 μg / ml, then serially dilute 3-fold for a total of 8 concentrations. Dilute the hIL35-hFc protein prepared in Example 1 to 160 μg / ml. Add 25 μl of the diluted test antibody and 25 μl of hIL35-hFc protein to a 96-well plate, mix well, and incubate at 37°C, 5% CO2 for 30 minutes. After incubation, add 100 μl of PBS (containing 1% BSA) to each well, centrifuge at 600g for 5 minutes, and discard the supernatant. Add 100 μl of PBS (containing 1% BSA) to each well to resuspend the cells, centrifuge, and remove the supernatant. Add 100 μl of mouse anti-human IgG Fc-FITC (1:200 dilution) to each well, mix well, and incubate at 4°C for 30 minutes. After incubation, add 100 μl of PBS (containing 1% BSA) to each well, centrifuge at 600g for 5 minutes, and discard the supernatant. Resuspend the cells in 100 μL of PBS (containing 1% BSA) per well and measure FITC fluorescence by flow cytometry. Analyze the proportion of IL35-positive cells in the main cell population and plot the positive cell proportion against the logarithm of the antibody concentration to analyze the inhibitory activity of the antibody against IL35 binding to PBMCs.

[0318] The results are shown in Table 8. In activated primary cells PBMC, the exemplary antibodies m57 and m58 can still effectively inhibit the binding of IL35 to its receptor.

[0319] Table 8: Activity of chimeric antibodies in inhibiting the binding of IL35 to activated PBMCs

[0320] 3.6 Affinity of chimeric antibodies:

[0321] The binding affinity of m57 and m58 antibodies to human IL-27 or human IL-35 was characterized using Biacore 8K (GE). Briefly, m57 or m58 antibodies were serially diluted and immobilized on a CM5 sensor chip. The affinity of the antibodies to human IL-27 or human IL-35 antigens was measured at different concentrations. The association and dissociation rates of the antibodies were measured using SPR technology to determine the binding affinity.

[0322] The results are shown in Table 9. Both m57 and m58 can bind to human IL27 and human IL35, and their binding affinity is on the order of 10 -10 M~10 -11 M.

[0323] Table 9: Affinity of chimeric antibodies

[0324] 3.7 Chimeric antibodies inhibit PDL1 expression in PBMC cells induced by human IL27 or IL35:

[0325] Preliminary experimental results showed that both IL27 and IL35 can induce T cells to express PD-L1, so the activity of the test antibody can be analyzed by detecting the expression level of PD-L1.

[0326] IL35-induced PD-L1 expression: PBMC cells were seeded into 96-well culture plates, with 100 μL (4×10 6 cells / mL). The test antibody was diluted to 1600 μg / ml, followed by 3-fold serial dilution for a total of 10 concentrations. The hIL35-hFc protein prepared in Example 1 was diluted to 80 μg / ml. 50 μL of the diluted test antibody and 50 μL of hIL35-hFc were added to a 96-well plate, mixed, and incubated at 37°C and 5% CO2 for 72 h. After gentle shaking at 600 rpm for 30 s, the cells were centrifuged at 600 g for 5 min, the supernatant was discarded, and 150 μL of PBS (containing 2% BSA) was added to each well to resuspend the cells, centrifuge, and discard the supernatant. 100 μL of APC anti-human PD-L1 antibody (1:100 dilution) was added to each well, mixed, incubated in the dark at 4°C for 30 minutes, centrifuged at 600 g for 5 min, and the supernatant was discarded. 150 μL of PBS (containing 2% BSA) was added to each well to resuspend and wash the cells. Add 50 μL of fixative solution to each well, mix thoroughly, and incubate at room temperature for 10 minutes. Add 100 μL of PBS (containing 2% BSA) to each well, centrifuge, and discard the supernatant. Resuspend the cells in 100 μL of PBS (containing 1% BSA) per well, and measure APC fluorescence by flow cytometry. The main cell population was selected to analyze the proportion of PD-L1-positive cells. The positive cell proportion was plotted against the logarithm of the antibody concentration to analyze the inhibitory activity of the antibody against IL35-induced PD-L1 expression in PBMCs.

[0327] IL27-induced PD-L1 expression: PBMC cells were seeded into 96-well culture plates, with 100 μL (4×10 6cells / mL). The test antibody was diluted to 30 μg / ml, followed by 3-fold serial dilution for a total of 9 concentrations. The hIL27-His protein prepared in Example 1 was diluted to 200 ng / ml. 50 μL of the diluted test antibody and 50 μL of hIL27-His were added to a 96-well plate, mixed, and incubated at 37°C and 5% CO2 for 72 h. After gentle shaking at 600 rpm for 30 s, the plate was centrifuged at 600 g for 5 min, the supernatant was discarded, and 150 μL of PBS (containing 2% BSA) was added to each well to resuspend the cells, centrifuge, and discard the supernatant. 100 μL of APC anti-human PD-L1 antibody (1:100 dilution) was added to each well, mixed, incubated at 4°C in the dark for 30 min, centrifuged at 600 g for 5 min, and the supernatant was discarded. 150 μL of PBS (containing 2% BSA) was added to each well to resuspend and wash the cells. 50 μL of fixative was added to each well, mixed, and allowed to stand at room temperature for 10 min. Add 100 μL of PBS (containing 2% BSA) to each well, centrifuge, and discard the supernatant. Resuspend the cells in 100 μL of PBS (containing 1% BSA) per well, and measure APC fluorescence by flow cytometry. The main cell population was selected to analyze the proportion of PD-L1-positive cells. The inhibitory activity of the antibody against IL27-induced PD-L1 expression in PBMCs was analyzed by plotting the positive cell proportion against the logarithm of the antibody concentration.

[0328] The results are shown in Table 10. In PBMC cells, the exemplary antibody m58 can effectively inhibit PD-L1 expression induced by IL27 or IL35.

[0329] Table 10: Chimeric antibodies inhibit IL35 / IL27-induced PD-L1 expression in PBMC cells

[0330] Example 4: Design, preparation and characterization of m58 variants

[0331] Design and preparation of 4.1m58 variants:

[0332] After analyzing the screened antibody m58, we found that there are potential NS deamidation sites and aspartic acid isomerization sites in its light chain variable domain, which may affect the stability, structure and function of the antibody due to deamidation and / or isomerization. L Site-directed mutagenesis was performed in the CDR region to eliminate potential deamidation risk points and / or aspartic acid isomerization risk points. In this example, a total of 10 mutation schemes were designed, namely: N30A, N30S, N30Q, S31A, D56E, D56S, N30AD56S, N30QD56E, N30QD56S, S31AD56S. The mutated antibodies were expressed and purified, and their activity was further tested. The amino acid sequences of the above-mentioned mutated antibodies are shown in Tables 2 and 3.

[0333] 4.2 IL27-STAT3 reporter gene assay for the activity of the m58 variant antibody:

[0334] The method in Example 3.3 was used to detect the activity of various m58 variants in blocking the IL27 signaling pathway.

[0335] The results are shown in Table 11. The m58 variants N30A, N30S, N30Q, S31A, D56E, D56S, N30AD56S, N30QD56E, N30QD56S, and S31AD56S can all block the IL27 signaling pathway, and their blocking activity is basically equivalent to that of the parent antibody m58.

[0336] Table 11: Inhibitory activity of m58 variants

[0337] The 4.3m58 variant inhibits the binding of human IL35 to HekblueIL6 cells:

[0338] The method in Example 3.4 was used to detect the inhibitory activity of each m58 variant on the binding of IL35 to HekblueIL6 cells.

[0339] The results are shown in Table 12. The m58 variants N30A, N30S, N30Q, S31A, D56E, D56S, N30AD56S, N30QD56E, N30QD56S, and S31AD56S can all inhibit the binding of human IL35 to HekblueIL6 cells, and their inhibitory activity is significantly better than or equivalent to that of the parent antibody m58.

[0340] Table 12: Activity of m58 variants in inhibiting IL35 binding to HekblueIL6 cells

[0341] In addition, capillary isoelectric focusing and size exclusion chromatography were used to assess changes in charge heterogeneity and aggregation after pressurization. The results showed that the acidic peaks, basic peaks, and aggregation of the above-mentioned variants were improved to varying degrees (results not shown). This indicates that the above-mentioned mutations reduce the risk of deamidation and / or isomerization without affecting activity and binding, which is beneficial for the subsequent development of antibodies.

[0342] Example 5: Humanization of antibodies and characterization of humanized antibodies

[0343] Based on the typical structures of the light chain and heavy chain variable region CDRs of the chimeric antibody m58 or its variants m58-D56E, m58-N30QD56S, and m58-N30AD56S, the light chain and heavy chain variable region sequences were compared with the sequences in the antibody germline database to obtain human germline templates with high homology. The CDR regions of the mouse antibody were transplanted onto the selected human germline templates to produce humanized variable regions, which were then recombined with the corresponding human IgG constant regions (preferably IgG1 heavy chain and κ light chain). Based on the three-dimensional structure of the mouse antibody, the buried residues, residues that directly interact with the CDR region, and residues for V L and V H By backmutating residues that significantly impact the conformation of the m58, different light and heavy chains were designed and combined to generate a series of humanized molecules. Ultimately, two humanized molecules, m58 and its variants m58-D56E, m58-N30QD56S, and m58-N30AD56S, were obtained, designated hum-m58-1, hum-m58-2, hum-m58-D56E-1, hum-m58-D56E-2, hum-m58-N30QD56S-1, hum-m58-N30QD56S-2, hum-m58-N30AD56S-1, and hum-m58-N30AD56S-2, respectively. The sequences of these humanized antibodies are shown in Tables 2 and 3.

[0344] 5.1 Determination of humanized antibody activity using the IL27-STAT3 reporter gene assay:

[0345] The method in Example 3.3 was used to test the inhibitory activity of humanized antibodies hum-m58-1, hum-m58-2, hum-m58-D56E-1, hum-m58-D56E-2, hum-m58-N30QD56S-1, hum-m58-N30QD56S-2, hum-m58-N30AD56S-1, and hum-m58-N30AD56S-2 against the IL27 signaling pathway.

[0346] As shown in Table 13, hum-m58-1, hum-m58-2, hum-m58-D56E-1, hum-m58-D56E-2, hum-m58-N30QD56S-1, hum-m58-N30QD56S-2, hum-m58-N30AD56S-1, and hum-m58-N30AD56S-2 were all able to block the IL27 signaling pathway, and the inhibitory activity of the mutant humanized molecules was significantly superior to that of the parent humanized molecules.

[0347] Table 13: Inhibitory activity of humanized antibodies

[0348] 5.2 Humanized antibodies inhibit the binding of human IL35 to HekblueIL6 cells:

[0349] The method in Example 3.4 was used to test the inhibitory activity of humanized antibodies hum-m58-1, hum-m58-2, hum-m58-D56E-1, hum-m58-D56E-2, hum-m58-N30QD56S-1, hum-m58-N30QD56S-2, hum-m58-N30AD56S-1, and hum-m58-N30AD56S-2 against the binding of IL35 to HekblueIL6 cells.

[0350] As shown in Table 14, hum-m58-1, hum-m58-2, hum-m58-D56E-1, hum-m58-D56E-2, hum-m58-N30QD56S-1, hum-m58-N30QD56S-2, hum-m58-N30AD56S-1, and hum-m58-N30AD56S-2 were able to inhibit the binding of IL35 to HekBlueIL6 cells.

[0351] Table 14: Activity of humanized antibodies in inhibiting the binding of IL35 to HekblueIL6 cells

[0352] 5.3 Humanized antibodies inhibit the binding of human IL35 to PBMC:

[0353] The method in Example 3.5 was used to further test the inhibitory activity of exemplary humanized antibodies hum-m58-D56E-1, hum-m58-D56E-2, hum-m58-N30QD56S-1, hum-m58-N30QD56S-2, hum-m58-N30AD56S-1, and hum-m58-N30AD56S-2 against the binding of IL35 to activated PBMCs.

[0354] As shown in Table 15, the exemplary humanized antibodies hum-m58-D56E-1, hum-m58-D56E-2, hum-m58-N30QD56S-1, hum-m58-N30QD56S-2, hum-m58-N30AD56S-1, and hum-m58-N30AD56S-2 were all able to inhibit the binding of human IL35 to activated PBMCs.

[0355] Table 15: Activity of humanized antibodies in inhibiting the binding of IL35 to activated PBMC

[0356] 5.4 Humanized antibodies inhibit the binding of IL27 or IL35 to their receptors:

[0357] IL35 signals through four unconventional receptors: IL12Rβ2 / WSX-1, IL12Rβ2 / IL12Rβ2, IL12Rβ2 / GP130, and GP130 / GP130. In contrast, the IL27 receptor has only one form: WSX-1 / gp130. The exemplary humanized antibodies hum-m58-D56E-1, hum-m58-D56E-2, hum-m58-N30QD56S-1, hum-m58-N30QD56S-2, hum-m58-N30AD56S-1, and hum-m58-N30AD56S-2 were tested for their inhibitory activity against the binding of IL27 or IL35 to their receptor subunits using ELISA.

[0358] Determination of the activity of humanized antibodies in inhibiting the binding of IL35 to gp130, IL12Rb2, and WSX-1 receptor subunits: The gp130-hFc, hIL12Rβ2-hFc, or WSX-1 antigen prepared in Example 1 was dissolved in PBS solution and coated on a 96-well plate at 0.5 μg / well at 4°C overnight. Wash with PBST solution six times, add 50 μl of 2% BSA, block at 37°C for 1 hour, and wash six times with PBST solution. The antibody sample to be tested was diluted to 100 μg / ml and then serially diluted 5-fold for a total of 8 concentrations. The serially diluted samples were added to each well of a 96-well plate, 50 μl per well, and 50 μl of 20 μg / ml hIL35-hFc was added at the same time. The plates were incubated at 37°C for 2 hours. The plates were then washed six times with PBST solution. Add 100 μl of 1:2000 diluted biotin-labeled anti-IL12 antibody (R&D, Cat#BAF219) to each well and incubate at 37°C for 2 h. Wash 6 times with PBST solution. Add 100 μl SA-HRP (1:5000 diluted in PBS) to each well and incubate at 37°C for 1 h. Wash 6 times with PBST solution. Add 100 μl TMB to each well for color development and incubate at 37°C for 5-10 min. Terminate the reaction with 2M H2SO4. Read the absorbance at 450 nm using a microplate reader. Generate inhibition curves using PRISM and calculate IC 50 value.

[0359] To determine the inhibitory activity of humanized antibodies against IL27 binding to the WSX-1 receptor subunit, WSX-1 antigen (purchased from Sino Biological, Cat. No. 10489-H02H) was dissolved in PBS and coated onto 96-well plates at 0.2 μg / well at 4°C overnight. The plates were washed six times with PBST, blocked with 50 μl of 2% BSA at 37°C for 1 hour, and washed six times with PBST. The antibody sample to be tested was diluted to 100 μg / ml and then serially diluted five-fold to a total of eight concentrations. An equal volume of 0.32 μg / ml hIL27-His was added to each diluted antibody sample and allowed to stand at room temperature for 30 minutes. The resulting mixture was added to a 96-well plate and incubated at 37°C for 1 hour. The plates were then washed six times with PBST. 100 μl of anti-His-HRP antibody (1:1000 dilution) was added to each well and incubated at 37°C for 1 hour. The plates were then washed six times with PBST. Add 100 μl of TMB to each well for color development and incubate at 37°C for 5-10 min. Terminate the reaction with 2 M H₂SO₄. Read the absorbance at 450 nm using a microplate reader. Generate inhibition curves using PRISM and calculate the IC₂. 50 value.

[0360] As shown in Table 16, the exemplary humanized antibodies hum-m58-D56E-1, hum-m58-D56E-2, hum-m58-N30QD56S-1, hum-m58-N30QD56S-2, hum-m58-N30AD56S-1, and hum-m58-N30AD56S-2 were able to inhibit the binding of IL27 or IL35 to their receptor subunits.

[0361] Table 16: Activity of humanized antibodies in inhibiting the binding of IL27 or IL35 to their receptor subunits

[0362] Note: “ / ” means not detected

[0363] 5.5 Humanized antibodies inhibit PDL1 expression in PBMC cells induced by human IL27 or IL35:

[0364] The method in Example 3.7 was used to test the inhibitory activity of exemplary humanized antibodies hum-m58-D56E-1, hum-m58-D56E-2, hum-m58-N30QD56S-1, hum-m58-N30QD56S-2, hum-m58-N30AD56S-1, and hum-m58-N30AD56S-2 against IL27-induced PDL1 expression in PBMC cells.

[0365] As shown in Table 17-1, the exemplary humanized antibodies hum-m58-D56E-1, hum-m58-D56E-2, hum-m58-N30QD56S-1, hum-m58-N30QD56S-2, hum-m58-N30AD56S-1, and hum-m58-N30AD56S-2 were able to inhibit human IL27-induced PDL1 expression in PBMC cells.

[0366] Table 17-1: Humanized antibodies inhibit IL27-induced PD-L1 expression in PBMC cells

[0367] The method described in Example 3.7 was used to test the inhibitory activity of exemplary humanized antibodies hum-m58-D56E-1 and hum-m58-N30QD56S-2 on IL35-induced PDL1 expression in PBMC cells.

[0368] The results are shown in Table 17-2. The exemplary humanized antibodies hum-m58-D56E-1 and hum-m58-N30QD56S-2 were both able to inhibit the expression of PDL1 in PBMC cells induced by human IL35.

[0369] Table 17-2: Humanized antibodies inhibit IL35-induced PD-L1 expression in PBMC cells

[0370] 5.6 Affinity of humanized antibodies:

[0371] The method described in Example 3.6 was used to test the affinity of exemplary humanized antibodies hum-m58-N30QD56S-2 and hum-m58-N30AD56S-2 for human IL27 or human IL35 antigens.

[0372] The results are shown in Table 18. The exemplary humanized antibodies hum-m58-N30QD56S-2 and hum-m58-N30AD56S-2 can bind to human IL27 and human IL35 with a binding affinity of the order of 10 -10 M~10 -12 M.

[0373] Table 18: Affinity of humanized antibodies

[0374] 5.7 Binding of Humanized Antibodies to EBI3:

[0375] The affinity of exemplary humanized antibodies hum-m58-1, hum-m58-2, hum-m58-D56E-1, hum-m58-N30QD56S-2, and hum-m58-N30AD56S-2 to human EBI3 antigen was tested using the method in Example 3.2.

[0376] As shown in FIG1 , the exemplary humanized antibodies hum-m58-1, hum-m58-2, hum-m58-D56E-1, hum-m58-N30QD56S-2, and hum-m58-N30AD56S-2 were all able to bind to EBI3.

[0377] Example 6: Pharmacokinetics of Antibodies in Rats

[0378] Twelve SD rats were randomly divided into three groups, with 4 rats in each group, half male and half female. Each group of rats was intravenously injected with 10 mpk of the exemplary antibody hum-m58-D56E-1, hum-m58-N30QD56S-2, or hum-m58-N30AD56S-2. Blood was collected before administration, and 5 minutes, 1 hour, 5 hours, 1 day, 3 days, 6 days, 10 days, and 14 days after administration. After blood centrifugation, plasma was collected and the blood drug concentration in each sample was measured by ELISA. Pharmacokinetic parameters were determined using the non-compartmental model (NCA) using the software version 6.0. The PK parameters, including maximum serum concentration (Cmax), half-life (T), and serum saturation (S) were determined using the linear trapezoidal rule, linear interpolation, and uniform weighting using the mean concentration values ​​of each antibody. 1 / 2 ), apparent volume of distribution (Vz), clearance (Cl), and the area under the curve from the time of administration to the last measurable concentration (AUClast).

[0379] The results showed that no drug-related abnormal clinical manifestations were observed during the experimental period. The drug serum concentration curve is shown in Figure 2.

[0380] Example 7: Pharmacodynamics of Antibodies in Monkeys

[0381] IL27 binds to its receptor (WSX-1 / gp130) and induces STAT1 phosphorylation in T cells. Therefore, the in vivo pharmacological properties of antibodies can be studied by analyzing changes in IL27-induced STAT1 phosphorylation levels after intravenous administration of antibodies.

[0382] Two monkeys were injected intravenously with the exemplary antibody hum-m58-N30AD56S-2 (10 mpk). Whole blood was collected before dosing (day 0) and on days 3, 7, 10, 14, and 21 after dosing, and heparin was added for anticoagulation. The collected whole blood was added to a 96-well plate at 50 μl / well and incubated at 37°C, 5% CO2 in a humidified incubator for 30 minutes. 50 μl of IL-27 was added to each well and incubated at 37°C, 5% CO2 in a humidified incubator for 30 minutes. 100 μl of fixative was added to each well and incubated at 37°C in the dark for 12 minutes. The supernatant was discarded after centrifugation. 200 μl of 1% BSA was added to each well to resuspend the cells and the supernatant was discarded after centrifugation. 200 μl of red blood cell lysis buffer (Tianjin Haoyang Biological, Cat#: NH4CL2009) was added and the cells were lysed at 4°C for 5 minutes. The cells were centrifuged at 600 g for 5 minutes and the supernatant discarded. Add 10 μl PBS (without serum) to each well to resuspend the cells to prevent the cells from clumping after adding the membrane permeabilization agent. Add 150 μl / well pre-cooled Phosflow TM Permeabilize the membrane with Perm Buffer III, pipette several times, incubate on ice for 30 minutes, and centrifuge to discard the supernatant. Add 200 μL of 1% BSA to each well to resuspend the cells, centrifuge, and discard the supernatant. Add Alexa Fluor 647-labeled phospho-STAT1 (Ser727) antibody, mix thoroughly, and incubate at 4°C in the dark for 30 minutes. After incubation, centrifuge at 600 g for 5 minutes and discard the supernatant. Wash the cells with 1% BSA and resuspend them in 120 μL of 1% BSA to each well. Fluorescence intensity of blood samples at different time points was measured by flow cytometry.

[0383] The results are shown in FIG3 . On day 3 after administration of the exemplary antibody hum-m58-N30AD56S-2, IL27-induced T cell STAT1 phosphorylation was completely inhibited, and the inhibitory effect of the antibody was still observed until day 21.

Claims

1. An isolated antibody comprising: Heavy chain variable domain (V H ), the V H Include: HC-CDR1 comprising SDYAWN (SEQ ID NO: 1); HC-CDR2 comprising SISYSGSTDYNPSLKS (SEQ ID NO:4); and HC-CDR3 comprising SPIYYDYEEGDY (SEQ ID NO: 8); and the light chain variable domain (V L ), the V L Include: LC-CDR1 comprising KASQDIX1X2YLS (SEQ ID NO: 25), wherein X1 is A, N, Q or S, and X2 is A or S; LC-CDR2 comprising RANRLVX1 (SEQ ID NO: 26), wherein X1 is D, E or S; and LC-CDR3 comprising LQYDEFPLT (SEQ ID NO: 22).

2. The isolated antibody according to claim 1, comprising: (i)V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 4, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 8; and V L , the V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 18, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 22; (ii) V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 4, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 8; and V L , the V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 22; (iii)V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 4, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 8; and V L , the V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 20, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 22; (iv)V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 4, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 8; and V L , the V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 13, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 20, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 22; (v)V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 4, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 8; and V L , the V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 14, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 18, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 22; (vi)V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 4, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 8; and V L , the V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 14, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 22; (vii) V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 4, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 8; and V L , the V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 14, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 20, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 22; (viii)V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 4, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 8; and V L , the V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 15, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 18, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 22; (ix)V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 4, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 8; and V L , the V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 16, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 20, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 22; (x)V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 4, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 8; and V L , the V L comprising: a LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 16, a LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 18, and a LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 22; or (xi)V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 4, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 8; and V L , the V L It comprises: LC-CDR1, which comprises the amino acid sequence of SEQ ID NO:13, LC-CDR2, which comprises the amino acid sequence of SEQ ID NO:18, and LC-CDR3, which comprises the amino acid sequence of SEQ ID NO:

22.

3. The isolated antibody according to claim 1 or 2, comprising: (i)V H , the V H comprising the amino acid sequence SEQ ID NO: 28 or a variant thereof, wherein the variant has the same amino acid sequence as SEQ ID NO:28 has at least about 80% sequence identity; and V L , the V L comprising the amino acid sequence of SEQ ID NO:44 or a variant thereof, wherein the variant has at least about 80% sequence identity to the amino acid sequence of SEQ ID NO:44; (ii) V H , the V H comprising the amino acid sequence of SEQ ID NO: 29 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 29; and V L , the V L comprising the amino acid sequence of SEQ ID NO:45 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO:45; (iii)V H , the V H comprising the amino acid sequence of SEQ ID NO: 28 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 28; and V L , the V L comprising the amino acid sequence of SEQ ID NO:46 or a variant thereof, wherein the variant has at least about 80% sequence identity to the amino acid sequence of SEQ ID NO:46; (iv)V H , the V H comprising the amino acid sequence of SEQ ID NO: 29 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 29; and V L , the V L comprising the amino acid sequence of SEQ ID NO:47 or a variant thereof, wherein the variant has at least about 80% sequence identity to the amino acid sequence of SEQ ID NO:47; (v)V H , the V H comprising the amino acid sequence of SEQ ID NO: 28 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 28; and V L , the V L comprising the amino acid sequence of SEQ ID NO:48 or a variant thereof, wherein the variant has at least about 80% sequence identity to the amino acid sequence of SEQ ID NO:48; (vi)V H , the V H comprising the amino acid sequence of SEQ ID NO: 29 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 29; and V L , the V L comprising the amino acid sequence of SEQ ID NO:49 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO:49; (vii) V H , the V H comprising the amino acid sequence of SEQ ID NO: 28 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 28; and V L , the V L comprising the amino acid sequence of SEQ ID NO:50 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO:50; or (viii)V H , the V H comprising the amino acid sequence of SEQ ID NO: 29 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 29; and V L , the V L Contains amino acid sequence SEQ ID NO:51 or a variant thereof, wherein the variant has at least about 80% sequence identity to the amino acid sequence of SEQ ID NO:

51.

4. The isolated antibody of any one of claims 1-3, wherein the antibody binds to IL35, IL27 or EBI3.

5. The isolated antibody of any one of claims 1-4, wherein the antibody comprises an Fc fragment.

6. The isolated antibody of claim 5, wherein the antibody is a full-length IgG antibody.

7. The isolated antibody of claim 6, wherein the antibody is a full-length IgG1, IgG2, IgG3 or IgG4 antibody.

8. The isolated antibody of any one of claims 1-7, wherein the antibody is a chimeric, humanized, or fully human antibody.

9. The isolated antibody according to any one of claims 1 to 4 is an antigen binding fragment, wherein the antigen binding fragment is selected from Fab, Fab', F(ab)'2, Fab'-SH, single-chain antibody (scFv), Fv fragment, dAb, Fd, nanobody, double-chain antibody (diabody) and linear antibody.

10. An isolated nucleic acid molecule encoding the isolated antibody of any one of claims 1-9.

11. A vector comprising the isolated nucleic acid molecule of claim 10.

12. An isolated host cell comprising the isolated antibody of any one of claims 1-9, the isolated nucleic acid molecule of claim 10, or the vector of claim 11.

13. A method for preparing an antibody, comprising: a) culturing the host cell of claim 12 under conditions effective to express the antibody; and b) obtaining the expressed antibody from the host cell.

14. A pharmaceutical composition comprising the isolated antibody of any one of claims 1 to 9, the isolated nucleic acid molecule of claim 10, the vector of claim 11, the isolated host cell of claim 12, or the antibody prepared by the method of claim 13, and a pharmaceutically acceptable carrier.

15. Use of the isolated antibody of any one of claims 1-9, the isolated nucleic acid molecule of claim 10, the vector of claim 11, the isolated host cell of claim 12, the antibody prepared by the method of claim 13, or the pharmaceutical composition of claim 14 in the preparation of a medicament for treating a disease or condition in an individual in need thereof.

16. A method for treating a disease or condition, the method comprising administering to a subject in need thereof an effective amount of the isolated antibody of any one of claims 1-9, the isolated nucleic acid molecule of claim 10, the vector of claim 11, the isolated host cell of claim 12, the antibody prepared by the method of claim 13, or the pharmaceutical composition of claim 14.

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