Anti-IL-15 antibodies and uses thereof

Abstract

The present invention relates to novel antibodies and antibody fragments that specifically bind to IL-15 and to compositions containing said antibodies or antibody fragments. Furthermore, the present invention relates to nucleic acids encoding said antibodies or antibody fragments thereof and host cells comprising the same, as well as related uses. In addition, the invention relates to therapeutic and diagnostic uses of these antibodies and antibody fragments.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application is based on the Chinese patent application with application number 202410444282.8 and application date April 12, 2024, and claims the priority of the Chinese patent application. The entire content of the Chinese patent application is hereby introduced into this application as a reference.

[0003] The present invention relates to novel antibodies and antibody fragments that specifically bind to IL-15, as well as compositions containing such antibodies or antibody fragments. Furthermore, the present invention relates to nucleic acids encoding such antibodies or antibody fragments, host cells containing such antibodies, and related uses. Furthermore, the present invention relates to therapeutic and diagnostic uses of these antibodies and antibody fragments. Background of the Invention

[0004] The cytokine interleukin 15 (IL-15) has multiple immune effects and plays an important role in the development, homeostasis, and function of memory CD8+ T cells, NK cells, and other immune cells. IL-15 is a 14-15 kDa glycoprotein that binds to a heterotrimeric receptor composed of a unique α subunit (IL-15Rα) that confers receptor specificity and a shared receptor with IL2, IL2Rβ (CD122), and a common γ (γc) chain (CD132). However, unlike IL2, IL-15 does not (i) activate T regulatory cells (Tregs) (ii) induce activation-induced cell death (AICD) in CD8(+) T cells (iii) cause vascular capillary leakage and (iv) IL-15 can maintain the survival of NK, effector CD8(+), and memory phenotype CD8(+) T cells.

[0005] IL-15Rα consists of three protein domains: (i) a 175 amino acid extracellular domain, (ii) a single 23 amino acid transmembrane region, and (iii) a 39 amino acid cytoplasmic domain, and is widely expressed in humans and mice independently of IL2R / IL-15Rβγc. IL-15Rα binds to IL-15Rα with extremely high affinity (Kd < 10 -11 M) binds to IL-15, causing it to retain IL-15 on the cell surface. In this way, IL-15Rα can present IL-15 to IL2R / IL-15Rβγc on neighboring NK and T cells through the immune synapse. This synaptic mechanism limits the exposure of circulating IL-15, thereby reducing the risk of autoimmunity. IL-15 can also be recycled as a complex with IL-15Rα in endosomal vesicles, persisting in a membrane-bound form for several days. IL-15 was listed as the first on the list of drugs with high potential for use as immunotherapy drugs by the NCI Immunotherapeutic Drug Workshop.

[0006] Although several IL-15 antibodies have been developed in the prior art (US7329405B2, US11130806B2 or Richmond JM et al., Antibody blockade of IL-15 signaling has the potential to durably reverse vitiligo. Sci Transl Med. 2018 Jul 18; 10(450):eaam7710.doi: 10.1126 / scitranslmed.aam7710. PMID: 30021889; PMCID: PMC6495055), these anti-IL-15 antibodies have disadvantages in terms of affinity, epitope, and blocking IL-15 signaling function, leaving room for improvement.

[0007] Therefore, there is still a need in the art to obtain IL-15 antibodies with better functions in terms of affinity, epitope or blocking IL-15 signal transduction. SUMMARY OF THE INVENTION

[0008] The present invention provides a novel antibody that specifically binds to IL-15 and selectively blocks the signal pathway involved in IL-15.

[0009] In some embodiments, the present invention provides a novel monoclonal antibody targeting IL-15, which has stronger affinity, targets a differentiated epitope (at the interface between IL-15 and IL2Rγ) compared to some prior art anti-IL-15 antibodies, and has better IL-15 signal blocking function. Specifically, due to the differentiated epitope of the IL-15 antibody described in the present invention, it helps to have a stable blocking function for multiple IL-15 signals (including signals induced by IL-15 monomers and IL-15 / IL-15Rα complexes). Therefore, the anti-IL-15 antibody described in the present invention exhibits stronger IL-15 signal blocking function in both in vitro and in vivo experiments.

[0010] Therefore, the present invention relates to an antibody or fragment, such as an antigen-binding fragment, that specifically binds to IL-15, such as human or cynomolgus monkey IL-15. In some embodiments, the antibody or antigen-binding fragment thereof of the present invention specifically binds to human IL-15 (e.g., wild-type IL-15 monomer or its complex with a receptor (e.g., IL-15 / IL-15Rα complex). In some embodiments, the antibody or antigen-binding fragment thereof of the present invention does not bind to an IL-15 monomer or IL-15 / IL-15Rα complex having a mutation at position 105 of IL-15 (e.g., an H105R mutation). In some embodiments, the antibody or antigen-binding fragment thereof of the present invention binds to an IL-15 monomer or its complex with a receptor (e.g., an IL-15 / IL-15Rα complex) having a mutation at position 108 of IL-15 (e.g., a Q108A mutation).

[0011] In some embodiments, the affinity of antigen-antibody binding is detected by ForteBio.

[0012] In some embodiments, the K of an antibody or antigen-binding fragment thereof of the invention is greater than that of a human IL-15 monomer (wild type or a variant without a mutation at position 105, such as IL-15 with Q108A). D In some embodiments, the K D Between approximately 1 nM and 3.5 nM.

[0013] In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention are K-binding domains of cynomolgus monkey IL-15. D In some embodiments, the K D Between about 1 nM and 2.5 nM, or between about 1.5 nM and 2 nM.

[0014] In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention bind to the K of a complex of IL-15 and its receptor (e.g., an IL-15 / IL-15Rα complex) (wild type or a variant in which position 105 of IL-15 is H, i.e., not mutated, such as an IL-15 / IL-15Rα complex comprising Q108A). D Less than about 2 nM, 1.5 nM, 1 nM, or 0.5 nM, or greater than about 0.1, 0.2, or 0.3 nM, or any range therebetween. In some embodiments, the K D Between about 0.1 nM and 1.5 nM, or between about 0.3 nM and 1 nM.

[0015] The antibodies or antigen-binding fragments thereof of the present invention can effectively block downstream signaling pathways stimulated by IL-15 monomers or complexes thereof with their receptors, such as IL-15 / IL-15Rα complexes, for example, in IL-15 reporter cell lines, such as human erythroleukemia cell-IL-15reporter cell lines. In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention effectively block downstream signaling pathway activation mediated by IL-15 monomers or complexes thereof with their receptors, such as IL-15 / IL-15Rα complexes, and / or inhibit immune cell proliferation, such as in PBMC cells. In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention can effectively block downstream signaling pathway activation mediated by IL-15 monomers or complexes thereof with their receptors, such as IL-15 / IL-15Rα complexes, thereby inhibiting CD8 T cell activation and / or IFNγ cytokine secretion mediated by IL-15 or complexes thereof with their receptors, such as IL-15 / IL-15Rα, for example, in CD8 T cells. In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention can effectively block T cell proliferation, T cell infiltration and / or T cell activation (e.g., decreased NKG2D expression levels or CD69 expression levels) mediated by IL-15 monomers or complexes thereof with their receptors, such as IL-15 / IL-15Rα complexes. In some embodiments, the T cells are T cell populations, for example, including αβ T cells and / or γδ T cells.

[0016] In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention have good pharmacokinetic characteristics in vivo, for example, long half-life, and / or low clearance rate. In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention, when administered (e.g., subcutaneously), have a half-life of greater than about 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, or 410 hours, for example, a half-life of about 250-280 hours, or a half-life of greater than 400 hours, for example, greater than about 405 hours or greater than about 410 hours, for example, a half-life of about 412 hours. In some embodiments, the antibodies or antigen-binding fragments thereof of the invention, when administered (e.g., by subcutaneous injection), have a clearance of less than about 0.3 mg / kg / h, e.g., less than about 0.27, or less than about 0.23, 0.22, 0.21, 0.2, 0.19, or 0.18.

[0017] In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention have good physicochemical properties, such as good solubility or stability, such as good colloidal stability and / or good thermal stability.

[0018] In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention can effectively prevent or treat inflammation, such as skin inflammation or small intestinal inflammation. In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention can effectively prevent or treat autoimmune diseases such as graft-versus-host disease, such as graft-versus-host disease that effectively blocks IL-15 signaling enhancement.

[0019] Therefore, the present invention relates to the following specific aspects.

[0020] 1. An anti-IL-15 antibody or an antigen-binding fragment thereof, comprising:

[0021] HCDR1, HCDR2 and HCDR3 comprised by the VH as shown in SEQ ID NO: 15, 16 or 17, and LCDR1, LCDR2 and LCDR3 comprised by the VL as shown in SEQ ID NO: 19, 20 or 21;

[0022] HCDR1, HCDR2, and HCDR3 comprised by the VH as set forth in SEQ ID NO: 15 or 16, and LCDR1, LCDR2, and LCDR3 comprised by the VL as set forth in SEQ ID NO: 19 or 20;

[0023] HCDR1, HCDR2, and HCDR3 comprised by the VH as shown in SEQ ID NO: 16 or 17, and LCDR1, LCDR2, and LCDR3 comprised by the VL as shown in SEQ ID NO: 20 or 21;

[0024] HCDR1, HCDR2, and HCDR3 comprised by the VH as shown in SEQ ID NO: 15, and LCDR1, LCDR2, and LCDR3 comprised by the VL as shown in SEQ ID NO: 19;

[0025] HCDR1, HCDR2, and HCDR3 comprised by the VH as shown in SEQ ID NO: 16, and LCDR1, LCDR2, and LCDR3 comprised by the VL as shown in SEQ ID NO: 20;

[0026] HCDR1, HCDR2 and HCDR3 comprised by VH as shown in SEQ ID NO: 17, and LCDR1, LCDR2 and LCDR3 comprised by VL as shown in SEQ ID NO: 21; or

[0027] The HCDR1, HCDR2 and HCDR3 comprised by VH as shown in SEQ ID NO: 18, and the LCDR1, LCDR2 and LCDR3 comprised by VL as shown in SEQ ID NO: 22.

[0028] 2. An anti-IL-15 antibody or antigen-binding fragment thereof, comprising a first heavy chain complementarity determining region (HCDR1), a second heavy chain complementarity determining region (HCDR2), a third heavy chain complementarity determining region (HCDR3), and a first light chain complementarity determining region (LCDR1), a second light chain complementarity determining region (LCDR2), and a third light chain complementarity determining region (LCDR3), wherein the

[0029] The HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 respectively comprise or consist of the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 14; or

[0030] The HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 respectively comprise or consist of the amino acid sequences shown in SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO. 8, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 43; or

[0031] The HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 respectively comprise or consist of the amino acid sequences shown in SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO. 8, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 14; or

[0032] The HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 respectively contain the amino acid sequences shown in SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 43, or respectively consist of the amino acid sequences shown in SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 43.

[0033] 3. The anti-IL-15 antibody or antigen-binding fragment thereof of embodiment 1 or 2, comprising a heavy chain variable region (VH), wherein the heavy chain variable region comprises, or consists of, an amino acid sequence that is at least 90% identical to an amino acid sequence selected from any one of SEQ ID NOs: 15-18, or comprises, or consists of, an amino acid sequence selected from any one of SEQ ID NOs: 15-18.

[0034] 4. The anti-IL-15 antibody or antigen-binding fragment thereof of any one of embodiments 1-3, comprising a light chain variable region (VL), wherein the light chain variable region comprises, consists of, or comprises an amino acid sequence that is at least 90% identical to an amino acid sequence selected from any one of SEQ ID NOs: 19-22.

[0035] 5. An anti-IL-15 antibody or an antigen-binding fragment thereof, comprising

[0036] (i) a VH comprising, or consisting of, an amino acid sequence as set forth in SEQ ID NO: 15, 16 or 17, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and a VL comprising, or consisting of, an amino acid sequence as set forth in SEQ ID NO: 19, 20 or 21, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0037] (ii) a VH comprising, or consisting of, an amino acid sequence as set forth in SEQ ID NO: 15 or 16, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and a VL comprising, or consisting of, an amino acid sequence as set forth in SEQ ID NO: 19 or 20, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0038] (iii) a VH comprising, or consisting of, an amino acid sequence as set forth in SEQ ID NO: 16 or 17, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and a VL comprising, or consisting of, an amino acid sequence as set forth in SEQ ID NO: 20 or 21, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0039] (iv) a VH comprising, or consisting of, an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and a VL comprising, or consisting of, an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto,

[0040] (v) a VH comprising, or consisting of, an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and a VL comprising, or consisting of, an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto,

[0041] (vi) a VH comprising, or consisting of, an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and a VL comprising, or consisting of, an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, as set forth in SEQ ID NO: 21; or

[0042] (vii) a VH comprising, or consisting of, an amino acid sequence set forth in SEQ ID NO: 18, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and a VL comprising, or consisting of, an amino acid sequence set forth in SEQ ID NO: 22, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0043] 6. The anti-IL-15 antibody or antigen-binding fragment thereof of any one of embodiments 1-5, comprising a heavy chain variable region and a light chain variable region, wherein

[0044] a) the heavy chain variable region comprises or consists of the amino acid sequence of SEQ ID NO: 15, 16 or 17, and the light chain variable region comprises or consists of the amino acid sequence of SEQ ID NO: 19, 20 or 21;

[0045] b) the heavy chain variable region comprises or consists of the amino acid sequence of SEQ ID NO: 15 or 16, and the light chain variable region comprises or consists of the amino acid sequence of SEQ ID NO: 19 or 20;

[0046] c) the heavy chain variable region comprises or consists of the amino acid sequence of SEQ ID NO: 16 or 17, and the light chain variable region comprises or consists of the amino acid sequence of SEQ ID NO: 20 or 21;

[0047] d) the heavy chain variable region comprises or consists of the amino acid sequence of SEQ ID NO: 15, and the light chain variable region comprises or consists of the amino acid sequence of SEQ ID NO: 19;

[0048] e) the heavy chain variable region comprises or consists of the amino acid sequence of SEQ ID NO: 16, and the light chain variable region comprises or consists of the amino acid sequence of SEQ ID NO: 20;

[0049] f) the heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 17, and the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 21; or

[0050] g) the heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 18, and the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 22.

[0051] 7. The anti-IL-15 antibody or antigen-binding fragment thereof of any one of embodiments 1-6, comprising an Fc region, e.g., the Fc region is from an IgG1, IgG2, IgG3, or IgG4 Fc region, e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region, e.g., the IgG1 Fc region

[0052] (i) comprising or consisting of the amino acid sequence of SEQ ID NO: 41; or

[0053] (ii) comprising an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:41.

[0054] 8. The anti-IL-15 antibody or antigen-binding fragment thereof of any one of embodiments 1-7, comprising a heavy chain constant region, for example, the heavy chain constant region is derived from the constant region of IgG1, IgG2, IgG3, or IgG4, for example, the constant region of human IgG1, IgG2, IgG3, or IgG4, for example, the IgG1 heavy chain constant region

[0055] (i) comprising or consisting of the amino acid sequence of SEQ ID NO: 25; or

[0056] (ii) comprising an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 25.

[0057] 9. The anti-IL-15 antibody or antigen-binding fragment thereof of embodiment 7 or 8, wherein the heavy chain constant region or Fc region comprises a mutation that reduces binding to Fcγ receptors, such as L234A / L235A mutation, such as L234A / L235A mutation, such as the heavy chain constant region

[0058] (i) comprising or consisting of the amino acid sequence of SEQ ID NO: 26; or

[0059] (ii) comprising an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 26.

[0060] 10. The anti-IL-15 antibody or antigen-binding fragment thereof of any one of embodiments 7-9, wherein the heavy chain constant region or Fc region comprises a mutation that improves binding to the FcRn receptor, such as a YTE mutation (M252Y / S254T / T256E).

[0061] 11. The anti-IL-15 antibody or antigen-binding fragment thereof of any one of embodiments 7-10, wherein the heavy chain constant region or Fc region comprises both a mutation that reduces binding to Fcγ receptors and a mutation that increases binding to FcRn receptors, for example, a L234A / L235A mutation and a YTE mutation, for example

[0062] The heavy chain constant region

[0063] (i) comprising or consisting of the amino acid sequence of SEQ ID NO: 27; or

[0064] (ii) comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 27; or

[0065] The Fc region

[0066] (i) comprising or consisting of the amino acid sequence of SEQ ID NO: 42; or

[0067] (ii) comprising an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:42.

[0068] 12. The anti-IL-15 antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 11, comprising a light chain constant region, wherein the light chain constant region is a lambda or kappa light chain constant region, such as a human lambda or kappa light chain constant region, preferably, the light chain constant region

[0069] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 28; or

[0070] (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 28.

[0071] 20. The anti-IL-15 antibody or antigen-binding fragment thereof of any one of embodiments 1-12, comprising a heavy chain and / or a light chain, wherein the heavy chain comprises or consists of the VH of any one of embodiments 1-12 and a heavy chain constant region; and / or the light chain comprises or consists of the VL of any one of embodiments 1-12 and a light chain constant region;

[0072] Optionally, the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO:23, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; and / or the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO:24, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0073] Optionally, the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 23 and the light chain comprises the amino acid sequence shown in SEQ ID NO: 24; or the heavy chain consists of the amino acid sequence shown in SEQ ID NO: 23 and the light chain consists of the amino acid sequence shown in SEQ ID NO: 24.

[0074] 13. The anti-IL-15 antibody or antigen-binding fragment thereof of any one of embodiments 1-13, wherein the antibody is a monoclonal antibody.

[0075] 14. The anti-IL-15 antibody or antigen-binding fragment thereof of any one of embodiments 1-14, wherein the antibody is a humanized antibody or a chimeric antibody.

[0076] 15. The anti-IL-15 antibody or antigen-binding fragment thereof of any one of embodiments 1-15, wherein the antigen-binding fragment is an antibody fragment selected from the group consisting of: Fab, Fab', Fab'-SH, Fv, single-chain antibody (e.g., scFv), (Fab')2, dAb (domain antibody), diabody, diabody, or linear antibody.

[0077] 16. The anti-IL-15 antibody or antigen-binding fragment thereof of any one of embodiments 1-16, wherein the antibody or antigen-binding fragment thereof has one or more of the following properties:

[0078] a) binds with high affinity to a human or cynomolgus monkey IL-15 monomer or a complex with its receptor, such as an IL-15 / IL-15Rα complex, wherein amino acid 105 of IL-15 is H in the monomer or complex;

[0079] b) effectively blocking the downstream signaling pathways stimulated by IL-15 monomers or their complexes with their receptors, such as the IL-15 / IL-15Rα complex;

[0080] c) inhibiting IL-15 or its complex with a receptor, such as IL-15 / IL-15Rα, mediated CD8 T cell activation and / or IFNg cytokine secretion, for example in CD8 T cells;

[0081] d) effectively blocking T cell proliferation, T cell infiltration and / or T cell activation mediated by IL-15 monomer or its complex with its receptor, such as IL-15 / IL-15Rα complex (e.g., decreasing NKG2D expression level or CD69 expression level);

[0082] e) having good physicochemical properties and / or pharmacokinetic characteristics, such as good stability (e.g., colloidal stability and / or thermal stability), good solubility, long half-life and / or low clearance rate;

[0083] f) effectively preventing or treating inflammation, such as skin inflammation or small intestinal inflammation;

[0084] g) It can effectively prevent or treat autoimmune diseases such as graft-versus-host disease, for example, effectively block graft-versus-host disease enhanced by IL-15 signaling.

[0085] 17. An isolated nucleic acid encoding the anti-IL-15 antibody or antigen-binding fragment thereof of any one of embodiments 1-17.

[0086] 18. A vector comprising the nucleic acid of embodiment 18, preferably the vector is an expression vector.

[0087] 19. A host cell comprising the nucleic acid of embodiment 18 or the vector of embodiment 19, preferably, the host cell is prokaryotic or eukaryotic, more preferably selected from yeast cells, mammalian cells (e.g., 293 cells or CHO cells, such as CHO-K cells or HEK293 cells) or other cells suitable for preparing antibodies or antigen-binding fragments thereof.

[0088] 20. A method for preparing an anti-IL-15 antibody or an antigen-binding fragment thereof, the method comprising

[0089] d) culturing the host cell of embodiment 20 under conditions suitable for expression of a nucleic acid encoding the anti-IL-15 antibody or antigen-binding fragment thereof of any one of embodiments 1-17,

[0090] e) optionally isolating the antibody or antigen-binding fragment thereof,

[0091] f) Optionally, the method further comprises recovering the anti-IL-15 antibody or antigen-binding fragment thereof from the host cell. Optionally, the antibody is purified, for example, by Protein A purification.

[0092] 21. An immunoconjugate comprising the anti-IL-15 antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 17 and other substances, such as toxins, small molecule drugs, cytotoxic agents, apoptotic agents, chelating agents, immunomodulators, such as anti-inflammatory agents or immunosuppressants.

[0093] 22. A pharmaceutical composition comprising the anti-IL-15 antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 17 or the immunoconjugate according to embodiment 22, and optionally a pharmaceutically acceptable excipient.

[0094] 23. A pharmaceutical combination comprising the anti-IL-15 antibody or antigen-binding fragment thereof of any one of embodiments 1-17 or the immunoconjugate of embodiment 22, and one or more other therapeutic agents, e.g., the therapeutic agents are selected from cytokines, other antibodies, small molecule drugs, or immunomodulators (e.g., anti-inflammatory agents or immunosuppressants).

[0095] 24. A method for preventing or treating an IL-15-related disease and / or condition in an individual, the method comprising administering to the subject an effective amount of the anti-IL-15 antibody or antigen-binding fragment thereof of any one of embodiments 1-17, or the immunoconjugate of embodiment 22, or the pharmaceutical composition of embodiment 23, or the pharmaceutical combination product of embodiment 24.

[0096] 25. The method of embodiment 25, wherein the subject has a disease or condition associated with abnormal expression of IL-15 or its receptor complex, such as the IL-15 / IL-15Rα complex, or abnormal activation of a mediated signaling pathway compared to a healthy individual.

[0097] 26. The method of embodiment 25 or 26, wherein the disease or disorder is selected from a tumor, such as cancer, or inflammation or an inflammatory disorder, such as an autoinflammatory disease, such as skin inflammation or small bowel inflammation; or an autoimmune disease, such as graft-versus-host disease.

[0098] 27. The method of any one of embodiments 25-27, wherein the method further comprises administering one or more other therapies, such as treatment modalities and / or other therapeutic agents, for example, the therapeutic agents are selected from cytokines, other antibodies, small molecule drugs, or immunomodulators (e.g., anti-inflammatory agents or immunosuppressants). BRIEF DESCRIPTION OF THE DRAWINGS

[0099] Figure 1 The inhibition of IL-15 reporter cell line signaling by anti-IL-15 chimeric antibodies and control antibodies is shown.

[0100] Figure 2 Shown is the inhibition of signaling in IL-15 reporter cell lines by anti-IL-15 antibodies.

[0101] Figure 3 Shown is the inhibition of signaling in IL-15 reporter cell lines by anti-IL-15 antibodies.

[0102] Figure 4 The results show that anti-IL-15 antibodies inhibit IL-15-induced immune cell proliferation.

[0103] Figure 5 The results show that anti-IL-15 antibodies inhibit IL-15-induced immune cell proliferation.

[0104] Figure 6 The results show that anti-IL-15 antibodies inhibit IL-15-induced immune cell proliferation.

[0105] Figure 7 The results show that anti-IL-15 antibodies inhibit IL-15-induced CD8 T cell activation.

[0106] Figure 8 The results show that anti-IL-15 antibodies inhibit IL-15-induced CD8 T cell activation.

[0107] Figure 9 The results show that anti-IL-15 antibodies inhibit IL-15-induced skin T cell proliferation.

[0108] Figure 10 The results show that anti-IL-15 antibodies inhibit IL-15-induced skin T cell activation.

[0109] Figure 11 The results show that anti-IL-15 antibodies inhibit IL-15-induced small intestinal CD8 T cell activation.

[0110] Figure 12 The results show that anti-IL-15 antibodies inhibit IL-15-induced splenic CD8 T cell activation.

[0111] Figure 13 The results show that anti-IL-15 antibodies can inhibit IL-15-enhanced GvHD.

[0112] Figure 14 The results show that anti-IL-15 antibodies can inhibit IL-15-enhanced GvHD.

[0113] Figure 15 Shown is the pharmacokinetic (PK) study of the antibody in BALB / c mice.

[0114] Figure 16 Shown is the inhibition of signaling in IL-15 reporter cell lines by anti-IL-15 antibodies.

[0115] Figure 17 Shown is the inhibition of signaling in IL-15 reporter cell lines by anti-IL-15 antibodies.

[0116] Figure 18 The results show that anti-IL-15 antibodies inhibit IL-15-induced immune cell proliferation.

[0117] Figure 19 The results show that anti-IL-15 antibodies inhibit IL-15-induced immune cell proliferation.

[0118] Figure 20 Shown is the pharmacokinetic (PK) study of the antibody in BALB / c mice. Detailed Description of the Invention

[0119] Before the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described herein as these may vary.

[0120] I. Definition

[0121] It should be understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to limit the scope of the present invention, which will be limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs.

[0122] To interpret this specification, the following definitions will apply, and wherever appropriate, terms used in the singular may also include the plural, and vice versa. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0123] The term "about" when used in conjunction with a numerical value is meant to encompass a range of numerical values ​​having a lower limit that is 5% (e.g., 4%, 3%, 2% or 1%) less than the specified numerical value and an upper limit that is 5% (e.g., 4%, 3%, 2% or 1%) greater than the specified numerical value.

[0124] As used herein, the term "and / or" means any one of the alternatives or two or more or all of the alternatives.

[0125] As used herein, the terms "comprising" or "including" are intended to include the recited elements, integers, or steps, but do not exclude any other elements, integers, or steps. In this document, when the terms "comprising" or "including" are used, unless otherwise indicated, combinations of the recited elements, integers, or steps are also encompassed. For example, when reference is made to an antibody variable region "comprising" a specific sequence, it is intended to encompass an antibody variable region consisting of that specific sequence.

[0126] An "isolated" antibody or molecule is one that has been separated from a component of its natural environment. In some embodiments, the antibody or molecule is purified to greater than 95% or 99% purity, as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC).

[0127] As used herein, "IL-15" refers to any naturally occurring IL-15 polypeptide (e.g., a human IL-15 polypeptide) or variants thereof. The term "IL-15" encompasses "full-length" unprocessed IL-15 polypeptide as well as any form of the IL-15 polypeptide produced by processing within a cell. The term also encompasses naturally occurring variants of IL-15, such as those encoded by splice variants and allelic variants. The IL-15 polypeptides described herein can be isolated from a variety of sources, such as from humans or from another source, such as cynomolgus monkeys, or prepared by recombinant or synthetic methods. In one embodiment of the invention, the human IL-15 protein comprises or consists of the amino acid sequence set forth in SEQ ID NO: 39. In one embodiment of the invention, the cynomolgus monkey IL-15 protein comprises or consists of the amino acid sequence set forth in SEQ ID NO: 40.

[0128] The terms "whole antibody" or "full-length antibody" are used interchangeably herein and refer to antibody molecules with the structure of natural immunoglobulin molecules. In the case of conventional four-chain IgG antibodies, the full-length antibody comprises two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. In the case of heavy chain antibodies having only heavy chains and lacking light chains, the full-length antibody comprises two heavy chains (H) interconnected by disulfide bonds. For conventional four-chain IgG antibodies, the full-length antibody heavy chain is generally composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region, wherein the heavy chain constant region comprises at least three domains CH1, CH2 and CH3. The full-length antibody light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region, wherein the light chain constant region consists of one domain CL. Each heavy chain variable region VH and each light chain variable region are composed of three CDRs and four FRs, arranged in the following order from amino terminus to carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The term "antibody fragment" includes a portion of an intact antibody. In a preferred embodiment, the antibody fragment is an antigen-binding fragment.

[0129] The term "antigen-binding fragment" of an antibody is a molecule that is different from a full-length antibody and that contains a portion of a full-length antibody, but that can bind to the antigen of the full-length antibody or compete with the full-length antibody (i.e., the full-length antibody from which the antigen-binding fragment is derived) for antigen binding. Antigen-binding fragments can be prepared by recombinant DNA technology, or by enzymatic or chemical cleavage of intact antibodies. Antigen-binding fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, dAb (domain antibody), linear antibodies, single-chain antibodies (e.g., scFv); single-domain antibodies such as VHH, diabodies, diabodies or fragments thereof, or camelid antibodies, diabodies, single-domain antibodies (sdAb), and nanobodies. For example, Fab fragments can be obtained by digesting a full-length antibody with papain. In addition, digesting a full antibody below the disulfide bonds in the hinge region with pepsin produces F(ab')2, which is a dimer of Fab' and a divalent antibody fragment. F(ab')2 can be reduced under neutral conditions by destroying the disulfide bond in the hinge region, thereby converting the F(ab')2 dimer into a Fab' monomer. The Fab' monomer is basically a Fab fragment with a hinge region. The Fv fragment consists of the VL and VH domains of a single antibody arm. The two domains of the Fv fragment, VL and VH, can be encoded by independent genes, but recombinant methods can also be used to connect the two domains using a synthetic linker peptide to produce them as a single protein chain, and in the single protein chain, the VL region and the VH region are paired to form a single-chain Fv (scFv).

[0130] The term "single-chain antibody (scAb)" is used herein in the broadest sense and specifically covers antibodies with monospecificity or multispecificity (e.g., bispecificity) that are initially produced as a single continuous polypeptide chain. Such single-chain antibodies include, but are not limited to, antibodies having two linked VL and VH regions. In one embodiment, the single-chain antibody is an scFv.

[0131] "Diabodies" are small, bivalent antibodies constructed through gene fusion, for example, dimers composed of two polypeptide chains. The VL and VH domains of each polypeptide chain of a diabody are linked by a linker, so that the VL and VH encoded in the same polypeptide chain form a dimer with different single-chain variable region segments. Diabodies generally have two antigen-binding sites.

[0132] "Complementarity determining region" or "CDR region" or "CDR" is a region in an antibody variable domain that is highly variable in sequence and forms structurally determined loops ("hypervariable loops") and / or contains antigen contact residues ("antigen contact points"). CDRs are primarily responsible for binding to antigenic epitopes. The CDRs of the heavy and light chains are typically referred to as CDR1, CDR2, and CDR3, and are numbered sequentially starting from the N-terminus. The CDRs located within the antibody heavy chain variable domain are referred to as HCDR1, HCDR2, and HCDR3, while the CDRs located within the antibody light chain variable domain are referred to as LCDR1, LCDR2, and LCDR3. In a given light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any one or a combination of a number of well-known antibody CDR assignment schemes, including, for example, Chothia based on the three-dimensional structure of antibodies and the topology of the CDR loops (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, US Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), International ImmunoGene Tics database (IMGT) (on the World Wide Web at imgt.cines.fr / ), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures.

[0133] The following are exemplary schemes for defining the region extent of CDRs using the Kabat, Chothia, and Kabat & Chothia (defined by combining the Kabat and Chothia schemes) schemes.

[0134] CDR Kabat regimen Chothia Solution Kabat & Chothia LCDR1 L24-L34 L26-L32 LCDR2 L50-L56 L50-L52 LCDR3 L89-L97 L91-L96 HCDR1 H31-H35 H26-H32 H26-H35 HCDR2 H50-H65 H53-H55 HCDR3 H95-H102 H96-H101

[0135] Unless otherwise indicated, in the present invention, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the above ways. CDRs can also be determined based on having the same Kabat numbering position as a reference CDR sequence (e.g., any of the exemplary CDRs of the present invention).

[0136] Unless otherwise indicated, in the present invention, when referring to residue positions in the variable region of an antibody (including heavy chain variable region residues and light chain variable region residues), the numbering refers to the position according to the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0137] In some embodiments, the CDR1 of the heavy chain variable region of the antibody of the present invention is determined according to the Kabat & Chothia scheme, and the CDR2 and CDR3 are determined according to the Kabat scheme. In some embodiments, the CDR of the light chain variable region of the antibody of the present invention is determined according to the Kabat scheme. In some embodiments, the CDR of the light chain variable region of the antibody of the present invention is determined according to the Kabat scheme.

[0138] HCDR1 was determined according to the Kabat & Chothia scheme, and HCDR2 and HCDR3 were determined according to the Kabat scheme;

[0139] LCDR1, LCDR2 and LCDR3 were determined according to the Kabat protocol.

[0140] An "antibody that binds to the same or overlapping epitope as a reference antibody" is an antibody that blocks 50%, 60%, 70%, 80%, 90% or 95% or more of the binding of the reference antibody to its antigen in a competition assay, whereas conversely, the reference antibody blocks 50%, 60%, 70%, 80%, 90% or 95% or more of the binding of the antibody to its antigen in a competition assay.

[0141] An antibody that competes with a reference antibody for binding to its antigen is an antibody that blocks 50%, 60%, 70%, 80%, 90% or 95% or more of the binding of the reference antibody to its antigen in a competition assay. Conversely, a reference antibody blocks 50%, 60%, 70%, 80%, 90% or 95% or more of the binding of the antibody to its antigen in a competition assay. Numerous types of competitive binding assays can be used to determine whether one antibody competes with another, such as solid phase direct or indirect radioimmunoassays (RIAs), solid phase direct or indirect enzyme immunoassays (EIAs), and sandwich competition assays.

[0142] An antibody that inhibits (e.g., competitively inhibits) the binding of a reference antibody to its antigen is an antibody that inhibits the binding of the reference antibody to its antigen by 50%, 60%, 70%, 80%, 90%, or 95% or more. Conversely, the reference antibody inhibits the binding of the antibody to its antigen by 50%, 60%, 70%, 80%, 90%, or 95% or more. The binding of an antibody to its antigen can be measured by affinity (e.g., equilibrium dissociation constant). Methods for determining affinity are known in the art.

[0143] An antibody that exhibits the same or similar binding affinity and / or specificity as a reference antibody is an antibody that has at least 50%, 60%, 70%, 80%, 90% or more than 95% of the binding affinity and / or specificity of the reference antibody. This can be determined by any method known in the art for determining binding affinity and / or specificity.

[0144] The term "chimeric antibody" is an antibody molecule in which (a) the constant region or a portion thereof is changed, replaced or exchanged so that the antigen binding site is connected to a constant region of a different or altered class, effector function and / or species or a completely different molecule (e.g., enzyme, toxin, hormone, growth factor, drug) that imparts new properties to the chimeric antibody; or (b) the variable region or a portion thereof is changed, replaced or exchanged with a variable region having a different or altered antigenic specificity. For example, a mouse antibody can be modified by replacing its constant region with a constant region from a human immunoglobulin. Due to the replacement with a human constant region, the chimeric antibody can retain its specificity in recognizing the antigen while having reduced immunogenicity in humans as compared to the original mouse antibody.

[0145] A "humanized antibody" is an antibody that retains the antigen-specific reactivity of a non-human antibody (e.g., a mouse monoclonal antibody) while being less immunogenic when administered to humans, for example, as a therapeutic. This can be achieved, for example, by retaining the non-human antigen-binding site and replacing the remaining portions of the antibody with their human counterparts (i.e., replacing the constant region and portions of the variable region not involved in binding with the corresponding portions of a human antibody).

[0146] The term "Fc domain" or "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. A native immunoglobulin "Fc domain" comprises two or three constant domains, namely a CH2 domain, a CH3 domain, and an optional CH4 domain. For example, in a native antibody, an immunoglobulin Fc domain comprises the second and third constant domains (CH2 domain and CH3 domain) of two heavy chains derived from IgG, IgA, and IgD class antibodies; or the second, third, and fourth constant domains (CH2 domain, CH3 domain, and CH4 domain) of two heavy chains derived from IgM and IgE class antibodies. Unless otherwise indicated herein, amino acid residue numbering in the Fc region or heavy chain constant region is according to the EU numbering system as described in Kabat et al., Sequences of Proteins of Immunological Interes, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991 (also called the EU index).

[0147] As used herein, "conservative changes" include substitutions, deletions, or additions to a polypeptide sequence that do not substantially alter the desired functional activity of the polypeptide sequence. In some embodiments, conservative changes are conservative substitutions. A conservative substitution refers to the replacement of one amino acid with another within the same class, such as the replacement of an acidic amino acid with another acidic amino acid, the replacement of a basic amino acid with another basic amino acid, or the replacement of a neutral amino acid with another neutral amino acid. For example, conservative substitutions often result in the replacement of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. The following lists 8 groups of amino acids that are conservative substitutions for each other: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine ​​(C), Methionine (M). In some embodiments, the term "conservative change" is used to refer to amino acid modifications that do not significantly affect or change the target antigen binding characteristics of the antibody molecule of the present invention containing the amino acid sequence when applied to the antibody molecule amino acid sequence. For example, a conservatively changed variant maintains at least 80%, 85%, 90%, 95%, 98%, 99% or higher, such as 100-110% or higher binding affinity for the target antigen relative to the parent antibody.

[0148] The term "vector," as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which they have been introduced. Some vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0149] An "immunoconjugate" is an antibody conjugated to one or more other substances, including but not limited to a label. In some embodiments, the immunoconjugate encompasses an antibody-drug conjugate (ADC).

[0150] As used herein, the term "therapeutic agent" encompasses any substance effective in preventing or treating diseases associated with inappropriate activation of pathways mediated by IL-15 or the IL-15 receptor complex, including cytokines, other antibodies, small molecule drugs, or immunomodulators (e.g., anti-inflammatory agents or immunosuppressants).

[0151] The term "small molecule drug" refers to low molecular weight organic compounds that are capable of regulating biological processes. "Small molecules" are defined as molecules with a molecular weight of less than 10 kD, typically less than 2 kD, and preferably less than 10 kD. Small molecules include, but are not limited to, inorganic molecules, organic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptide mimetics, and antibody mimics. As therapeutic agents, small molecules can be more cell-permeable, less susceptible to degradation, and less prone to eliciting an immune response than macromolecules.

[0152] As used herein, the term "immunomodulator" refers to a natural or synthetic agent or drug that inhibits or modulates an immune response. The immune response can be a humoral response or a cellular response. Immunomodulators include anti-inflammatory agents or immunosuppressants.

[0153] As used herein, an "immunosuppressant," "immunosuppressive drug," or "immunosuppressant" is a therapeutic agent used in immunosuppressive therapy to suppress or prevent the activity of the immune system.

[0154] The term "effective amount" refers to an amount or dosage of an antibody or fragment or conjugate or composition or combination of the present invention that produces the desired effect in a patient in need of treatment or prevention after administration in single or multiple doses to the patient.

[0155] A "therapeutically effective amount" refers to an amount effective to achieve the desired therapeutic outcome at the required dosage and for the required period of time. A therapeutically effective amount is also an amount in which any toxic or deleterious effects of the antibody or antibody fragment or its conjugate or composition or combination are outweighed by the therapeutically beneficial effects. A "therapeutically effective amount" preferably inhibits a measurable parameter (e.g., inhibition of the IL-15 signaling pathway) by at least about 20%, more preferably at least about 40%, even more preferably at least about 50%, 60%, or 70% relative to an untreated subject. In some embodiments, the term "therapeutically effective amount" as used herein is intended to define the amount of treatment necessary to treat a condition (e.g., inflammation) or to reduce or eliminate an immune response in a treatment regimen.

[0156] A "prophylactically effective amount" refers to an amount effective to achieve the desired preventive result at the required dosage and for the required period of time. Typically, a prophylactic amount will be less than a therapeutically effective amount because a prophylactic dose is used in a subject prior to or at an earlier stage of disease. In some embodiments, the term "prophylactically effective amount" as used herein is intended to qualify as the amount necessary to prevent the progression and symptoms (e.g., inflammation) of a condition or disease in a treatment regimen.

[0157] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom, without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to the parent cell, but may contain mutations. Mutant progeny screened or selected for the same function or biological activity as the initially transformed cell are included herein.

[0158] The term "label" as used herein refers to a compound or composition that is directly or indirectly conjugated or fused to a reagent (such as a polynucleotide probe or antibody) and promotes the detection of the reagent to which it is conjugated or fused. The label itself can be detectable (e.g., a radioisotope label or a fluorescent label) or can catalyze a chemical change in a detectable substrate compound or composition in the case of an enzymatic label. The term is intended to encompass direct labeling of a probe or antibody by coupling (i.e., physically connecting) a detectable substance to the probe or antibody and indirect labeling of the probe or antibody by reacting with another reagent of the direct label.

[0159] As used herein, the terms "individual," "subject," or "subject" are used interchangeably and include mammals. Mammals include, but are not limited to, domestic animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is a human.

[0160] As used herein, the term "subject / patient / individual sample" refers to a collection of cells or fluids obtained from a patient or subject. The source of a tissue or cell sample can be solid tissue, such as an organ or tissue sample or a biopsy sample or a puncture sample from fresh, frozen and / or preserved; blood or any blood component; body fluids, such as cerebrospinal fluid, amniotic fluid (amniotic fluid), peritoneal fluid (ascites), or interstitial fluid; cells from any time during the subject's pregnancy or development. Tissue samples may contain compounds that are not naturally contaminated with tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and the like.

[0161] "Nucleic acid encoding an anti-IL-15 antibody or fragment thereof" refers to one or more nucleic acid molecules encoding an antibody heavy chain or light chain (or fragment thereof, such as a heavy chain variable region or a light chain variable region), including such nucleic acid molecules in a single vector or separate vectors, and such nucleic acid molecules present at one or more locations in a host cell.

[0162] "Percent identity (%)" of an amino acid sequence refers to the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the specific amino acid sequence set forth in this specification, after aligning the candidate sequence with the specific amino acid sequence set forth in this specification and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. In some embodiments, the present invention contemplates variants of the antibody molecules of the present invention that have a substantial degree of identity, e.g., at least 80%, 85%, 90%, 95%, 97%, 98% or 99% or more, relative to the antibody molecules and sequences thereof specifically disclosed herein. Such variants may comprise conservative changes.

[0163] The term "pharmaceutical excipient" refers to a diluent, adjuvant (eg, Freund's adjuvant (complete and incomplete)), excipient, carrier, stabilizer, or the like, which is administered together with the active substance.

[0164] The term "pharmaceutical composition" refers to a composition that is in a form that permits the biological activity of the active ingredient contained therein to be effective, and that contains no additional ingredients that are unacceptably toxic to the subject or individual to which the composition is administered.

[0165] As used herein, the term "pharmaceutical combination or combination product" refers to a non-fixed combination product or a fixed combination product, including but not limited to a kit / test kit, a pharmaceutical composition. The term "non-fixed combination" means that the active ingredients (e.g., (i) an antibody of the present invention, and (ii) other therapeutic agent) are administered to a patient simultaneously, without specific time restrictions, or at the same or different time intervals, in separate entities, wherein such administration provides two or more active agents with prophylactic or therapeutically effective levels in the patient's body. The term "fixed combination" means that two or more active agents are administered to a patient simultaneously in the form of a single entity. The dosage and / or time interval of the two or more active agents are preferably selected so that the combined use of the parts can produce an effect greater than that achieved by using any one component alone when treating a disease or condition. Each component can be in the form of a separate formulation, which can be the same or different.

[0166] The term "combination therapy" refers to the administration of two or more therapeutic agents or treatment modalities (e.g., radiotherapy or surgery) to treat diseases described herein. This administration includes co-administering these therapeutic agents in a substantially simultaneous manner, such as in a single capsule with a fixed ratio of active ingredients. Alternatively, this administration includes co-administration of each active ingredient in a variety of or separate containers (e.g., tablets, capsules, powders, and liquids). Powders and / or liquids can be reconstituted or diluted to the desired dose before administration. In addition, this administration also includes using each type of therapeutic agent in a sequential manner at approximately the same time or at different times. In either case, the therapeutic regimen will provide the beneficial effects of the drug combination in treating disorders or conditions described herein.

[0167] As used herein, "treat," ..."

[0168] As used herein, "prevention" includes the inhibition of the onset or development of a disease or condition or symptoms of a particular disease or condition.

[0169] II Antibodies

[0170] In some embodiments, the anti-IL-15 antibody or antigen-binding fragment thereof of the present invention comprises three complementarity determining regions (HCDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3.

[0171] In some embodiments, the anti-IL-15 antibody or antigen-binding fragment thereof of the present invention comprises three complementarity determining regions (LCDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3.

[0172] In some embodiments, the anti-IL-15 antibody or antigen-binding fragment thereof of the invention comprises three complementarity determining regions (HCDRs) from a heavy chain variable region and three complementarity determining regions (LCDRs) from a light chain variable region.

[0173] In some aspects, the anti-IL-15 antibodies or antigen-binding fragments thereof of the present invention comprise a heavy chain variable region (VH). In some aspects, the anti-IL-15 antibodies or antigen-binding fragments thereof of the present invention comprise a light chain variable region (VH). In some aspects, the anti-IL-15 antibodies or antigen-binding fragments thereof of the present invention comprise a heavy chain variable region and a light chain variable region (VH). In some embodiments, the heavy chain variable region comprises three complementary determining regions (CDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3. In some embodiments, the light chain variable region comprises three complementary determining regions (CDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3.

[0174] In some embodiments, the anti-IL-15 antibodies or antigen-binding fragments thereof of the present invention further comprise an antibody heavy chain constant region. In some embodiments, the anti-IL-15 antibodies or antigen-binding fragments thereof of the present invention further comprise an antibody light chain constant region. In some embodiments, the anti-IL-15 antibodies or antigen-binding fragments thereof of the present invention further comprise a heavy chain constant region and a light chain constant region.

[0175] In some embodiments, the heavy chain variable region of the present invention:

[0176] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from any one of SEQ ID NOs: 15-18; or

[0177] (ii) comprises or consists of an amino acid sequence selected from any one of SEQ ID NOs: 15-18; or

[0178] (iii) an amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence shown in any one of SEQ ID NOs: 15 to 18, consisting of said amino acid sequence. Preferably, said amino acid changes do not occur in the CDR regions.

[0179] In some embodiments, the light chain variable region of the present invention

[0180] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from any one of SEQ ID NOs: 19-22; or

[0181] (ii) comprises or consists of an amino acid sequence selected from any one of SEQ ID NOs: 19-22; or

[0182] (iii) an amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence shown in any one of SEQ ID NOs: 19-22, consisting of said amino acid sequence, preferably,

[0183] The amino acid changes did not occur in the CDR regions.

[0184] In some embodiments, the three complementarity determining regions (HCDRs) from the heavy chain variable region of the present invention, HCDR1, HCDR2 and HCDR3 are selected from

[0185] (i) three complementarity determining regions HCDR1, HCDR2, and HCDR3 contained in the VH of any one of SEQ ID NOs: 15-18;

[0186] (ii) a sequence comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid change (preferably an amino acid substitution, preferably a conservative substitution) in the three HCDR regions relative to the sequence of any one of (i),

[0187] wherein the HCDRs can be determined according to any scheme for determining CDRs, for example, according to the Kabat, AbM, Chothia, Contact or IMGT schemes or a combination thereof;

[0188] For example, the HCDR1 is determined according to the Kabat & Chothia scheme, and the HCDR2 and HCDR3 are each determined according to the Kabat scheme.

[0189] In some embodiments, the three complementarity determining regions (LCDRs) from the light chain variable region of the present invention, LCDR1, LCDR2 and LCDR3 are selected from

[0190] (i) three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in the VL as shown in any one of SEQ ID NOs: 19-22, or

[0191] (ii) a sequence comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid change (preferably an amino acid substitution, preferably a conservative substitution) in the three LCDR regions relative to the sequence of any one of (i),

[0192] wherein the HCDRs can be determined according to any scheme for determining CDRs, for example, according to the Kabat, AbM, Chothia, Contact or IMGT schemes or a combination thereof;

[0193] For example, the LCDRs 1, 2 and 3 are each determined according to the Kabat protocol.

[0194] In some embodiments, the HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 1, 2 or 3, or the HCDR1 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 1, 2 or 3.

[0195] In some embodiments, the HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 4, 5 or 6, or the HCDR2 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 4, 5 or 6.

[0196] In some embodiments, the HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 7, 8 or 9, or the HCDR3 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 7, 8 or 9.

[0197] In some embodiments, the LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 10, 11 or 12, or LCDR1 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 10, 11 or 12.

[0198] In some embodiments, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 13, or consists of the amino acid sequence, or LCDR2 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 13.

[0199] In some embodiments, the LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 14 or 43, or the LCDR3 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 14 or 43.

[0200] In some embodiments, the antibody heavy chain constant region of the present invention is a heavy chain constant region from IgG1, IgG2, IgG3 or IgG4, for example, a constant region from human IgG1, IgG2, IgG3 or IgG4, for example, a constant region from human IgG1, IgG2, IgG3 or IgG4. In some embodiments, the constant region has a modification in the Fc region therein. In some embodiments, the Fc region is an Fc region from IgG1, IgG2, IgG3 or IgG4, for example, an Fc region from human IgG1, IgG2, IgG3 or IgG4, for example, a Fc region from human IgG1, IgG2, IgG3 or IgG4.

[0201] In one embodiment, the Fc region is modified with respect to the properties of the effector functions of the Fc region (e.g., the complement activation function of the Fc region). In one embodiment, the effector functions have been reduced or eliminated relative to wild-type Fc regions. In one embodiment, the effector functions are reduced or eliminated by a method selected from the group consisting of: using an Fc isotype that naturally has reduced or eliminated effector functions, and Fc region modifications. The Fc region may also comprise modifications that alter binding affinity for one or more Fc receptors.

[0202] In one embodiment, the Fc receptor is an Fcγ receptor, particularly a human Fcγ receptor. In some embodiments, the Fc region comprises a mutation that reduces binding to the Fcγ receptor. In some embodiments, the Fc region used in the present invention comprises an L234A / L235A mutation that reduces binding to the Fcγ receptor. In some embodiments, the heavy chain constant region of the present invention comprises a mutation that reduces binding to the Fcγ receptor, such as the L234A / L235A mutation.

[0203] In some embodiments, the Fc receptor is FcRn, particularly a human FcRn receptor. In some embodiments, the Fc region has a mutation that improves binding to the FcRn receptor. In some embodiments, the Fc region used in the present invention has a YTE mutation (M252Y / S254T / T256E) that improves binding to the FcRn receptor. In some embodiments, the heavy chain constant region of the present invention comprises a mutation that improves binding to the FcRn receptor, such as a YTE mutation (M252Y / S254T / T256E).

[0204] In some embodiments, the Fc region or heavy chain constant region of the present invention comprises both a mutation that reduces binding to an Fcγ receptor and a mutation that increases binding to an FcRn receptor, for example, a L234A / L235A mutation and a YTE mutation.

[0205] In some embodiments, the heavy chain constant region

[0206] (i) comprising or consisting of an amino acid sequence selected from SEQ ID NO: 25, 26 or 27;

[0207] (ii) comprising an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 25, 26 or 27;

[0208] (iii) comprising an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 26 and has an L234A / L235A mutation; or

[0209] (iv) comprising an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 27, and having an L234A / L235A mutation and a YTE mutation.

[0210] In some embodiments, the heavy chain constant regions of the invention lack a terminal lysine.

[0211] In some embodiments, the Fc region

[0212] (i) comprising or consisting of an amino acid sequence selected from SEQ ID NO: 41 or 42;

[0213] (ii) comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 41 or 42; or

[0214] (iii) comprising an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 42, and having an L234A / L235A mutation and a YTE mutation.

[0215] In some embodiments, the antibody light chain constant region of the present invention is a light chain constant region from a lambda or kappa light chain constant region, preferably from a kappa light chain constant region, for example, a lambda or kappa light chain constant region, such as a human lambda or kappa light chain constant region. In some embodiments, the light chain constant region

[0216] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 28; or

[0217] (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 28.

[0218] In some specific embodiments, the anti-IL-15 antibody of the present invention comprises

[0219] HCDR1, HCDR2, and HCDR3 comprised by the VH as set forth in SEQ ID NO: 15, 16, or 17, and LCDR1, LCDR2, and LCDR3 comprised by the VL as set forth in SEQ ID NO: 19, 20, or 21;

[0220] HCDR1, HCDR2, and HCDR3 comprised by the VH as set forth in SEQ ID NO: 15 or 16, and LCDR1, LCDR2, and LCDR3 comprised by the VL as set forth in SEQ ID NO: 19 or 20;

[0221] HCDR1, HCDR2, and HCDR3 comprised by the VH as shown in SEQ ID NO: 16 or 17, and LCDR1, LCDR2, and LCDR3 comprised by the VL as shown in SEQ ID NO: 20 or 21;

[0222] HCDR1, HCDR2, and HCDR3 comprised by the VH as shown in SEQ ID NO: 15, and LCDR1, LCDR2, and LCDR3 comprised by the VL as shown in SEQ ID NO: 19;

[0223] HCDR1, HCDR2, and HCDR3 comprised by the VH as set forth in SEQ ID NO: 16, and LCDR1, LCDR2, and LCDR3 comprised by the VL as set forth in SEQ ID NO: 20;

[0224] HCDR1, HCDR2 and HCDR3 comprised by VH as shown in SEQ ID NO: 17, and LCDR1, LCDR2 and LCDR3 comprised by VL as shown in SEQ ID NO: 21; or

[0225] HCDR1, HCDR2, and HCDR3 comprised by the VH as set forth in SEQ ID NO: 18, and LCDR1, LCDR2, and LCDR3 comprised by the VL as set forth in SEQ ID NO: 22;

[0226] wherein the HCDRs can be determined according to any scheme for determining CDRs, for example, according to the Kabat, AbM, Chothia, Contact or IMGT schemes or a combination thereof;

[0227] For example, the HCDR1 is determined according to the Kabat & Chothia scheme, and the HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are each determined according to the Kabat scheme.

[0228] In some specific embodiments, the anti-IL-15 antibody of the invention comprises a first heavy chain complementarity determining region (HCDR1), a second heavy chain complementarity determining region (HCDR2), a third heavy chain complementarity determining region (HCDR3), and a first light chain complementarity determining region (LCDR1), a second light chain complementarity determining region (LCDR2), and a third light chain complementarity determining region (LCDR3), wherein

[0229] The HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 respectively comprise or consist of the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 14; or

[0230] The HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 respectively comprise or consist of the amino acid sequences shown in SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO. 8, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 43; or

[0231] The HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 respectively comprise or consist of the amino acid sequences shown in SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO. 8, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 14; or

[0232] The HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 respectively contain the amino acid sequences shown in SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 43, or respectively consist of the amino acid sequences shown in SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 43.

[0233] In some embodiments, the VH of the invention comprises HCDR1, HCDR2, HCDR3, and the VL comprises LCDR1, LCDR2, and LCDR3, wherein

[0234] The HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 respectively comprise or consist of the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 14; or

[0235] The HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 respectively comprise or consist of the amino acid sequences shown in SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO. 8, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 43; or

[0236] The HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 respectively comprise or consist of the amino acid sequences shown in SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO. 8, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 14; or

[0237] The HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 respectively contain the amino acid sequences shown in SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 43, or respectively consist of the amino acid sequences shown in SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 43.

[0238] In some embodiments, the antibody or antigen-binding fragment thereof of the present invention comprises:

[0239] (i) a VH comprising, or consisting of, an amino acid sequence as set forth in SEQ ID NO: 15, 16 or 17, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and / or a VL comprising, or consisting of, an amino acid sequence as set forth in SEQ ID NO: 19, 20 or 21, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0240] (ii) a VH comprising, or consisting of, an amino acid sequence as set forth in SEQ ID NO: 15 or 16, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and / or a VL comprising, or consisting of, an amino acid sequence as set forth in SEQ ID NO: 19 or 20, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0241] (iii) a VH comprising, or consisting of, an amino acid sequence as set forth in SEQ ID NO: 16 or 17, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and / or a VL comprising, or consisting of, an amino acid sequence as set forth in SEQ ID NO: 20 or 21, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0242] (iv) a VH comprising, or consisting of, an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and / or a VL comprising, or consisting of, an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto;

[0243] (v) a VH comprising, or consisting of, an amino acid sequence as set forth in SEQ ID NO:16, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and / or a VL comprising, or consisting of, an amino acid sequence as set forth in SEQ ID NO:20, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0244] (vi) a VH comprising, or consisting of, an amino acid sequence as set forth in SEQ ID NO:17, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and / or a VL comprising, or consisting of, an amino acid sequence as set forth in SEQ ID NO:21, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; or

[0245] (vii) a VH comprising, or consisting of, an amino acid sequence as set forth in SEQ ID NO: 18, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and / or a VL comprising, or consisting of, an amino acid sequence as set forth in SEQ ID NO: 22, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0246] In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention comprise a heavy chain variable region and a light chain variable region, wherein

[0247] (i) the heavy chain variable region comprises or consists of the amino acid sequence of SEQ ID NO: 15, 16 or 17, and the light chain variable region comprises or consists of the amino acid sequence of SEQ ID NO: 19, 20 or 21;

[0248] (ii) the heavy chain variable region comprises or consists of the amino acid sequence of SEQ ID NO: 15 or 16, and the light chain variable region comprises or consists of the amino acid sequence of SEQ ID NO: 19 or 20;

[0249] (iii) the heavy chain variable region comprises or consists of the amino acid sequence of SEQ ID NO: 16 or 17, and the light chain variable region comprises or consists of the amino acid sequence of SEQ ID NO: 20 or 21;

[0250] (iv) the heavy chain variable region comprises or consists of the amino acid sequence of SEQ ID NO: 15, and the light chain variable region comprises or consists of the amino acid sequence of SEQ ID NO: 19;

[0251] (v) the heavy chain variable region comprises or consists of the amino acid sequence of SEQ ID NO: 16, and the light chain variable region comprises or consists of the amino acid sequence of SEQ ID NO: 20;

[0252] (vi) the heavy chain variable region comprises or consists of the amino acid sequence of SEQ ID NO: 17, and the light chain variable region comprises or consists of the amino acid sequence of SEQ ID NO: 21; or

[0253] (vii) the heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 18, and the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 22.

[0254] In some embodiments, the anti-IL-15 antibodies or antigen-binding fragments thereof of the present invention further comprise an antibody heavy chain. In some embodiments, the anti-IL-15 antibodies or antigen-binding fragments thereof of the present invention further comprise an antibody light chain. In some embodiments, the anti-IL-15 antibodies or antigen-binding fragments thereof of the present invention further comprise the heavy and light chains described herein. In some embodiments, the heavy chains of the antibodies of the present invention comprise the heavy chain variable region described herein and the heavy chain constant region described herein, or consist of the heavy chain variable region described herein and the heavy chain constant region described herein. In some embodiments, the light chains of the antibodies of the present invention comprise the light chain variable region described herein and the light chain constant region described herein, or consist of the light chain variable region described herein and the light chain constant region described herein. In some embodiments, the antibodies of the present invention comprise two heavy chains and two light chains described herein, or consist of two heavy chains and two light chains described herein.

[0255] In some embodiments, the heavy chain of an antibody of the invention comprises or consists of the amino acid sequence of SEQ ID NO: 23, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto. In some embodiments, the light chain of an antibody of the invention comprises or consists of the amino acid sequence of SEQ ID NO: 24, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0256] In some embodiments, the antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 23 and the light chain comprises the amino acid sequence shown in SEQ ID NO: 24.

[0257] In some embodiments, the antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain and a light chain, wherein the heavy chain consists of the amino acid sequence shown in SEQ ID NO: 23 and the light chain consists of the amino acid sequence shown in SEQ ID NO: 24.

[0258] In one embodiment of the present invention, the amino acid changes described herein include amino acid substitutions, insertions, or deletions. Preferably, the amino acid changes described herein are amino acid substitutions, preferably conservative substitutions. In a preferred embodiment, the amino acid changes described herein occur in regions outside the CDRs (e.g., in the FRs). More preferably, the amino acid changes described herein occur in regions outside the heavy chain variable region and / or outside the light chain variable region.

[0259] In certain embodiments, the antibodies provided herein are modified to increase or decrease the degree of glycosylation of the antibody. The addition or deletion of glycosylation sites of the antibody can be conveniently achieved by changing the amino acid sequence to produce or remove one or more glycosylation sites. When the antibody comprises an Fc region, the carbohydrate attached thereto can be changed. In some applications, the modification of unwanted glycosylation sites can be useful, such as removing the fucose motif to improve antibody-dependent cellular cytotoxicity (ADCC) function. In other applications, galactosylation can be performed to modify complement-dependent cytotoxicity (CDC). In certain embodiments, it may be necessary to produce antibodies engineered with cysteine, such as "thio MAbs," in which one or more residues of the antibody are replaced with cysteine ​​residues. In certain embodiments, the antibodies provided herein can be further modified to contain other non-proteinaceous moieties known in the art and readily available. Suitable portions for antibody derivatization 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-dioxane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.

[0260] In some embodiments, the anti-IL-15 antibodies or antigen-binding fragments thereof of the present invention have one or more of the following properties:

[0261] (i) exhibiting the same or similar binding affinity and / or specificity as an antibody of the invention for IL-15 (e.g., ch25B10, ch56G9, hz56G9.25L, or ch60G8);

[0262] (ii) inhibiting (e.g., competitively inhibiting) the binding of an antibody of the invention (e.g., ch25B10, ch56G9, hz56G9.25L, or ch60G8) to IL-15;

[0263] (iii) binds to the same or overlapping epitope as an antibody of the invention (e.g., ch25B10, ch56G9, hz56G9.25L, or ch60G8):

[0264] (iv) competing with an antibody of the invention (e.g., ch25B10, ch56G9, hz56G9.25L, or ch60G8) for binding to IL-15;

[0265] (v) possessing one or more biological properties of an antibody of the invention (eg, ch25B10, ch56G9, hz56G9.25L, or ch60G8).

[0266] In some embodiments, the anti-IL-15 antibody of the present invention is an antibody in the form of IgG1, an antibody in the form of IgG2, an antibody in the form of IgG3, or an antibody in the form of IgG4, preferably, an antibody in the form of IgG1.

[0267] In some embodiments, the anti-IL-15 antibody is a monoclonal antibody.

[0268] In some embodiments, the anti-IL-15 antibody is humanized.

[0269] In some embodiments, the anti-IL-15 antibody is a chimeric antibody.

[0270] In some embodiments, the anti-IL-15 antibodies of the invention are full-length antibodies.

[0271] In some embodiments, the anti-IL-15 antibodies of the present invention also encompass multispecific antibodies or bispecific antibodies that specifically bind to IL-15.

[0272] In one embodiment, the anti-IL-15 antibodies of the present invention also encompass antibody fragments thereof (e.g., antigen-binding fragments), preferably antibody fragments selected from the group consisting of: Fab, Fab', Fab'-SH, Fv, single-chain antibodies (e.g., scFv), (Fab')2, single-domain antibodies such as VHH, dAb (domain antibody), diabodies, diabodies, or linear antibodies.

[0273] III. Nucleic acids of the present invention and host cells containing the same

[0274] In one aspect, the present invention provides nucleic acids encoding any chain or any monomer or domain of an antibody or antigen-binding fragment thereof of the present invention. Polynucleotide sequences encoding each chain can be generated using methods well known in the art. For example, when expressed from a suitable expression vector, the polypeptide encoded by the nucleic acid can exhibit binding ability to human IL-15 (e.g., an IL-15 monomer or a complex thereof with a receptor). For example, in some embodiments, the nucleic acid encoding the variable region of the heavy chain and / or light chain is operably linked in frame with the nucleic acid encoding the constant region of the heavy chain and / or light chain, thereby generating nucleic acids encoding the heavy and / or light chains of the antibody when expressed from a suitable expression vector.

[0275] In one aspect, the present invention provides nucleic acids encoding any of the anti-IL-15 antibodies or fragments thereof described herein. The nucleic acids may comprise nucleic acids encoding the amino acid sequences of the light chain variable region and / or heavy chain variable region of the antibody, or nucleic acids encoding the amino acid sequences of the light chain and / or heavy chain of the antibody.

[0276] For example, the nucleic acids of the present invention include nucleic acids encoding an amino acid sequence selected from any one of SEQ ID NOs: 15-24, or nucleic acids encoding an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from any one of SEQ ID NOs: 15-24. As will be appreciated by those skilled in the art, due to codon degeneracy, each antibody or polypeptide amino acid sequence can be encoded by multiple nucleic acid sequences. Nucleic acid sequences encoding the molecules of the present invention can be generated using methods well known in the art, such as de novo solid-phase DNA synthesis or PCR amplification. To facilitate production and purification, a secretory signal peptide and / or a tag peptide that facilitates purification can be fused to the N-terminus of the heavy and / or light chains of the antibody.

[0277] The present invention also provides vectors comprising the nucleic acid of the present invention. In one embodiment, the vector is an expression vector, such as a eukaryotic expression vector. Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phages, or yeast artificial chromosomes (YACs). In preferred embodiments, the expression vector of the present invention is a pcDNA vector, such as pcDNA3.1 and / or pcDNA3.4 expression vectors.

[0278] In one embodiment, a host cell comprising the vector is provided. The present invention also provides a host cell comprising the nucleic acid or the vector. Host cells suitable for replication and support expression of the antibodies of the present invention are well known in the art. Such cells can be transfected or transduced with specific expression vectors, and large quantities of vector-containing cells can be grown for inoculating large-scale fermenters, thereby obtaining sufficient amounts of antibodies for clinical applications. Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector functions are not required. After expression, the antibodies can be separated from the bacterial cell paste in the soluble fraction and can be further purified.

[0279] In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells (e.g., CHO cells (e.g., CHO-S or CHO-K) or 293 cells (e.g., 293F or HEK293 cells)) or other cells suitable for preparing antibodies or fragments thereof. In one embodiment, the host cell is prokaryotic, for example, a bacterium, such as Escherichia coli.

[0280] For example, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors. For example, fungal and yeast strains whose glycosylation pathways have been "humanized" result in the production of antibodies with partially or fully human glycosylation patterns. Host cells suitable for expressing glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Vertebrate cells can also be used as hosts. For example, mammalian cell lines modified to be suitable for suspension growth can be used. Other examples of useful mammalian host cell lines are monkey kidney CV1 lines (COS-7) transformed with SV40; human embryonic kidney lines (HEK293, 293F or 293T cells), etc. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells, CHO-S cells, ExpiCHO, etc.; and myeloma cell lines such as Y0, NS0 and Sp2 / 0. Mammalian host cell lines suitable for producing antibodies are known in the art.

[0281] IV. Production and Purification of Antibody Molecules of the Invention

[0282] In one embodiment, the present invention provides a method for preparing an antibody molecule or fragment thereof (preferably an antigen-binding fragment) of the present invention, wherein the method comprises culturing the host cell under conditions suitable for expressing a nucleic acid encoding an antibody molecule or fragment thereof (preferably an antigen-binding fragment) of the present invention, and optionally isolating the antibody or fragment thereof (e.g., an antigen-binding fragment). In a certain embodiment, the method further comprises recovering the antibody molecule or fragment thereof (e.g., an antigen-binding fragment) of the present invention from the host cell.

[0283] The polynucleotide encoding the polypeptide chain of the antibody of the present invention can be inserted into one or more vectors (e.g., pcDNA3.1 and / or pcDNA3.4 expression vectors) for further cloning and / or expression in a host cell. Methods well known to those skilled in the art can be used to construct expression vectors. Once an expression vector comprising one or more nucleic acid molecules of the present invention has been prepared for expression, the expression vector can be transfected or introduced into a suitable host cell. Various techniques can be used to achieve this purpose, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, liposome-based transfection or other conventional techniques.

[0284] Antibody molecules prepared as described herein can be purified by known techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like. The actual conditions used to purify a particular protein will also depend on factors such as net charge, hydrophobicity, hydrophilicity, and such factors, and these will be apparent to those skilled in the art. The purity of the antibody molecules of the invention can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, and the like.

[0285] V. Assay

[0286] The anti-IL-15 antibodies provided herein can be identified, screened, or characterized for their physical / chemical properties and / or biological activities by a variety of assays known in the art.

[0287] The present invention also provides assays for identifying anti-IL-15 antibodies with biological activity. Biological activity can include, for example, binding to IL-15 (e.g., binding to human IL-15), inhibition of signaling pathways mediated by the IL-15 / IL-15Rα complex, activation of T cells, prevention or treatment of inflammation, etc. Antibodies having such biological activity in vivo and / or in vitro are also provided.

[0288] For the determination of the above biological activity, please refer to the exemplary determination methods given in the Examples.

[0289] It will be appreciated that any of the above assays can be performed using the immunoconjugates of the invention in place of or in addition to the anti-IL-15 antibody.

[0290] It will be appreciated that any of the above assays can be performed using a combination of an anti-IL-15 antibody and an additional therapeutic agent.

[0291] VI. Immunoconjugates

[0292] In some embodiments, the present invention provides immunoconjugates comprising any of the anti-IL-15 antibodies or antigen-binding fragments thereof provided herein and other substances, such as any active agent or label suitable for forming an immunoconjugate with the IL-15 antibody or antigen-binding fragment thereof.

[0293] In some embodiments, the active agent suitable for forming an immunoconjugate with an IL-15 antibody or antigen-binding fragment thereof can be, for example, a chemotherapeutic agent, a toxin, a small molecule drug, a cytotoxic agent, an apoptotic agent, a chelating agent, an immunomodulatory agent, such as an anti-inflammatory agent or an immunosuppressant.

[0294] In some embodiments, the immunoconjugate is an antibody drug conjugate, such as an ADC.

[0295] VII. Pharmaceutical Compositions and Pharmaceutical Formulations

[0296] In some embodiments, the present invention provides a composition comprising any of the anti-IL-15 antibodies or fragments thereof (preferably antigen-binding fragments thereof) or immunoconjugates thereof described herein, preferably a pharmaceutical composition. In one embodiment, the composition further comprises a pharmaceutical excipient. In one embodiment, the composition, e.g., a pharmaceutical composition, comprises an anti-IL-15 antibody or fragment thereof or immunoconjugate thereof of the present invention, and a combination of one or more other therapeutic agents. In some embodiments, the composition is a pharmaceutical formulation.

[0297] The present invention also includes compositions (including pharmaceutical compositions or pharmaceutical preparations) comprising anti-IL-15 antibodies or antigen-binding fragments thereof or immunoconjugates thereof, or compositions (including pharmaceutical compositions or pharmaceutical preparations) comprising polynucleotides encoding anti-IL-15 antibodies. In certain embodiments, the compositions comprise one or more antibodies or fragments thereof that bind to IL-15, or one or more polynucleotides encoding one or more anti-IL-15 antibodies or fragments thereof. These compositions may also contain suitable pharmaceutical excipients, such as pharmaceutical carriers and pharmaceutical excipients known in the art, including buffers.

[0298] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like that are physiologically compatible.

[0299] For the use of pharmaceutical excipients and their uses, see also "Handbook of Pharmaceutical Excipients", 8th edition, RC Rowe, PJ Eskey and S C Owen, Pharmaceutical Press, London, Chicago.

[0300] The compositions of the present invention can be in a variety of forms. These forms include, for example, liquid, semisolid and solid dosage forms, such as liquid solutions (e.g., injectable solutions and infusible solutions), powders or suspensions, liposomes and suppositories. The preferred form depends on the intended mode of administration and therapeutic use.

[0301] Pharmaceutical formulations comprising the antibodies described herein can be prepared by mixing the antibodies of the invention or fragments thereof having the desired degree of purity with one or more optional pharmaceutical excipients, preferably in the form of a lyophilized formulation or an aqueous solution.

[0302] The pharmaceutical composition or preparation of the present invention can also include more than one active ingredient, the active ingredient being required for the specific indication being treated, preferably having those active ingredients of complementary activities that do not adversely affect each other. For example, it is desirable to also provide other therapeutic agents, such as cytokines, small molecule drugs, immunomodulators (such as immunosuppressants or anti-inflammatory agents) or other antibodies, etc. The active ingredients are suitably combined in an amount effective for the intended use.

[0303] Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, eg, films, or microcapsules.

[0304] VIII. Pharmaceutical Combinations and Kits

[0305] In some embodiments, the present invention also provides a pharmaceutical combination or pharmaceutical combination product comprising an anti-IL-15 antibody or fragment thereof (preferably an antigen-binding fragment) of the present invention, or an immunoconjugate thereof, or a pharmaceutical composition comprising the same, and one or more other therapeutic agents (e.g., cytokines, chemotherapeutic agents, small molecule drugs, immunomodulators (e.g., immune activators, immunosuppressants, or anti-inflammatory agents), or other antibodies, etc.).

[0306] Another object of the present invention is to provide a kit comprising the pharmaceutical combination of the present invention, preferably in the form of a pharmaceutical dosage unit, whereby dosage units can be provided according to a dosing regimen or a drug administration interval.

[0307] In one embodiment, the kit of parts of the present invention comprises in the same package:

[0308] - a first container containing a pharmaceutical composition comprising an anti-IL-15 antibody or fragment thereof or an immunoconjugate thereof;

[0309] - A second container containing a pharmaceutical composition comprising an additional therapeutic agent.

[0310] The selection of other therapeutic agents that can be combined with the molecules of the present invention depends on the use of the molecules of the present invention. For example, when the molecules of the present invention are used to treat autoimmune diseases or autoinflammatory diseases, the other therapeutic agents are any active agents suitable for treating autoimmune diseases or autoinflammatory diseases, including but not limited to cytokines, small molecule drugs, other antibodies or immunomodulators (such as immunosuppressants or anti-inflammatory agents). For example, when the molecules of the present invention are used to treat tumors, the other therapeutic agents are any active agents suitable for treating tumors, such as antitumor agents or anticancer agents, including but not limited to cytokines, chemotherapeutic agents, small molecule drugs, other antibodies or immunomodulators (such as immune activators).

[0311] IX. Use and Method

[0312] In some embodiments, provided are methods of preventing or treating a disease or condition in an individual using an antibody or fragment thereof or immunoconjugate thereof of the present invention as a single agent or in combination, comprising administering to the individual a molecule of the present invention (e.g., an anti-IL-15 antibody or antigen-binding fragment thereof or immunoconjugate thereof of the present invention), a pharmaceutical composition, a pharmaceutical combination, or a pharmaceutical kit.

[0313] In some embodiments, the invention relates to a molecule of the invention (e.g., an anti-IL-15 antibody or antigen-binding fragment thereof or immunoconjugate thereof), pharmaceutical composition, pharmaceutical combination or kit of the invention for use in therapy, e.g., for treating a disease or condition mentioned herein.

[0314] In some embodiments, the invention relates to methods of treating a disease or condition using a molecule of the invention (e.g., an anti-IL-15 antibody or antigen-binding fragment thereof, or immunoconjugate thereof), pharmaceutical composition, pharmaceutical combination, or kit of the invention, or use for such treatment, or use for the preparation of a medicament for such treatment.

[0315] In some embodiments, the disease or condition is an IL-15-related disease and / or condition. The term "IL-15-related disease and / or condition" relates to abnormal activation of a signaling pathway mediated by IL-15 or its receptor complex, such as an IL-15 / IL-15Rα complex, or to overexpression of IL-15 and / or increased levels of IL-15 in cells or organs or tissues and / or abnormal IL-15 expression and / or abnormal expression of IL-15 variants in cells or organs. Such diseases or conditions include, for example, autoimmune diseases and / or inflammatory conditions, or tumors. The term "autoimmune disease and / or inflammatory condition" is generally defined herein as a disease or condition caused by an abnormal immune response of an individual's body to substances and tissues normally present in the body, as well as inflammatory abnormalities that may or may not involve the immune system. In some embodiments, the inflammatory condition is an autoinflammatory disease.

[0316] In some embodiments, in patients with the disease or condition, compared to healthy individuals, IL-15 or its and IL-15 receptor complex (e.g., IL-15 / IL-15Rα complex) mediated abnormal activation of the signaling pathway. In some embodiments, in patients with the disease or condition, compared to healthy individuals, IL-15 or its and IL-15 receptor complex (e.g., IL-15 / IL-15Rα complex) is abnormally expressed (e.g., overexpressed, or increased level of expression, or abnormal variant expression) or has an increase in nucleic acid levels. In some embodiments, in patients with the disease or condition, cells or tissues or organs express IL-15, e.g., moderate or high expression of IL-15. In some embodiments, the patient has (e.g., elevated levels, e.g., nucleic acid or protein levels or activity) IL-15 (e.g., compared to healthy individuals). In some embodiments, the patient has (e.g., elevated levels, such as nucleic acid or protein levels or activity, of) IL-15 in a biological sample (e.g., cells or tissue) of the patient (e.g., compared to IL-15 in a biological sample of a healthy individual (e.g., a corresponding tissue or cell in a healthy individual), or compared to adjacent healthy tissue or cells of the patient).

[0317] In some embodiments, the antibodies of the present invention can prevent and / or treat diseases or conditions related to IL-15 by blocking the activation of downstream signaling pathways by IL-15 or its complex with the IL-15 receptor (e.g., IL-15 / IL-15Rα complex).

[0318] In some embodiments, the disease or disorder is selected from inflammation or an inflammatory disorder (eg, an autoinflammatory disease), such as skin inflammation or small intestinal inflammation. In some embodiments, the disease or disorder is an autoimmune disease such as graft-versus-host disease.

[0319] In some embodiments, the disease or condition suitable for prevention and / or treatment of the present invention is a tumor, such as cancer. In some embodiments, the tumor is a solid tumor or a hematological tumor and a metastatic lesion. In one embodiment, examples of solid tumors include malignant tumors. The cancer can be in the early, middle or late stages or be a metastatic cancer. In some embodiments, the tumor is a tumor that has escaped immune toxicity.

[0320] In some embodiments, the signaling pathway mediated by IL-15 or its receptor complex, such as the IL-15 / IL-15Rα complex, is abnormally activated in the tumor or tumor cell, for example, compared to healthy cells in a healthy individual or adjacent to the tumor cell.

[0321] In some embodiments, the tumor is an IL-15 positive tumor or cancer. In some embodiments, the tumor is associated with abnormal expression or abnormal activity of IL-15. In some embodiments, an IL-15 positive tumor or cancer refers to a tumor cell expressing IL-15 in an individual suffering from all tumor cancers. In some embodiments, the tumor cells of the individual express IL-15, such as moderate or high expression of IL-15. In some embodiments, the individual has (e.g., elevated levels of, such as nucleic acid or protein levels or activity) IL-15 (e.g., compared to healthy individuals). In some embodiments, the biological sample (e.g., tumor cell or tumor tissue) of the individual has (e.g., elevated levels of, such as nucleic acid or protein levels or activity) IL-15 (e.g., compared to a biological sample of a healthy individual (e.g., a corresponding tissue or cell in a healthy individual), or compared to IL-15 in adjacent healthy tissues or cells of the individual).

[0322] In some embodiments, the antibodies or antibody fragments or immunoconjugates or compositions or products of the invention delay the onset of a disorder and / or symptoms associated with the disorder.

[0323] In some embodiments, the present invention provides use of an anti-IL-15 antibody or fragment thereof, or an immunoconjugate or composition comprising the same, of the present invention in the production or preparation of a medicament for use as described herein, e.g., for preventing or treating a disease or condition as described herein.

[0324] In some embodiments, the methods of prevention or treatment described herein further comprise administering to the subject or individual an antibody molecule, pharmaceutical composition, or immunoconjugate disclosed herein in combination with one or more other therapies, such as therapeutic modalities and / or other therapeutic agents. In some embodiments, the anti-IL-15 antibody or fragment thereof (as well as immunoconjugates, compositions, pharmaceutical compositions, formulations, etc. comprising the same) can also be administered in combination with one or more other therapies, such as therapeutic modalities and / or other therapeutic agents, for the purposes described herein, for example, for preventing and / or treating the relevant diseases or conditions mentioned herein.

[0325] The choice of other therapeutic agents for treatment depends on the use of the molecules of the present invention. For example, when the molecules of the present invention are used to treat autoimmune diseases or autoinflammatory diseases, the other therapeutic agents are any active agents suitable for treating autoimmune diseases or inflammatory conditions such as autoinflammatory diseases, including but not limited to cytokines, small molecule drugs, other antibodies or immunomodulators (such as immunosuppressants or anti-inflammatory agents). For example, when the molecules of the present invention are used to treat tumors, the other therapeutic agents are any active agents suitable for treating tumors, such as anti-tumor agents or anti-cancer agents, including but not limited to cytokines, chemotherapeutic agents, small molecule drugs, other antibodies or immunomodulators (such as immune activators); the other treatment modalities are, for example, selected from surgery or radiotherapy.

[0326] In some embodiments, the antibodies described herein can be combined with other antibodies for separate administration, e.g., each as a separate antibody, or when linked (e.g., as a bispecific or multispecific antibody molecule).

[0327] Such combination therapies encompass combined administration (e.g., two or more therapeutic agents contained in the same formulation or separate formulations), and separate administration, in which case administration of the antibodies of the invention can occur prior to, concurrently with, and / or after administration of the other therapeutic agents and / or therapies.

[0328] The antibodies or fragments thereof of the present invention (as well as immunoconjugates, compositions, pharmaceutical compositions, preparations, combination products, etc. comprising the same) can be administered by any suitable method, including parenteral administration and, if desired for local treatment, intralesional administration. Parenteral injection or infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous injection or infusion.

[0329] X. Methods and Compositions for Diagnosis and Detection

[0330] In one aspect, the present invention also relates to methods for diagnosing and detecting the antibodies or antigen-binding fragments thereof of the present invention and compositions for diagnosing and detecting comprising the same.

[0331] In certain embodiments, any of the anti-IL-15 antibodies or fragments thereof (preferably antigen-binding fragments) provided herein can be used to detect the presence of IL-15 or a complex thereof with an IL-15 receptor (e.g., an IL-15 / IL-15Rα complex) in a biological sample.

[0332] The term "detection" as used herein includes quantitative or qualitative detection, and exemplary detection methods can involve immunohistochemistry, immunocytochemistry, flow cytometry (e.g., FACS), magnetic beads of antibody molecule complexes, ELISA assays, PCR-techniques (e.g., RT-PCR). In certain embodiments, the biological sample is blood, serum, or other liquid samples of biological origin. In certain embodiments, the biological sample comprises cells or tissues.

[0333] In one embodiment, an anti-IL-15 antibody or fragment thereof is provided for use in a diagnostic or detection method.

[0334] In another aspect, a method for detecting the presence of IL-15 or a complex thereof with an IL-15 receptor (e.g., an IL-15 / IL-15Rα complex) in a biological sample is provided. In certain embodiments, the method comprises detecting the presence of an IL-15 protein or a complex thereof with an IL-15 receptor (e.g., an IL-15 / IL-15Rα complex) in a biological sample. In certain embodiments, the IL-15 is human IL-15. In certain embodiments, the method comprises contacting a biological sample with an anti-IL-15 antibody or fragment thereof as described herein under conditions that allow the anti-IL-15 antibody or fragment thereof to bind to IL-15 or a complex thereof with an IL-15 receptor (e.g., an IL-15 / IL-15Rα complex), and detecting whether an antigen-antibody complex is formed between the anti-IL-15 antibody or fragment thereof and IL-15 or a complex thereof with an IL-15 receptor (e.g., an IL-15 / IL-15Rα complex). The formation of an antigen-antibody complex indicates the presence of IL-15. The method can be an in vitro or in vivo method. In one embodiment, an anti-IL-15 antibody or fragment thereof is used to select a subject suitable for treatment with the anti-IL-15 antibody or fragment thereof, e.g., wherein IL-15 or a complex thereof with an IL-15 receptor (e.g., an IL-15 / IL-15Rα complex) is a biomarker used to select the subject.

[0335] In some embodiments, a labeled anti-IL-15 antibody or fragment thereof is provided. Labels include, but are not limited to, directly detectable labels or moieties (e.g., fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, and radioactive labels), as well as moieties that are indirectly detected, such as enzymes or ligands, e.g., via an enzymatic reaction or molecular interaction.

[0336] In some embodiments provided herein, the sample is obtained prior to treatment with an anti-IL-15 antibody or fragment thereof. In some embodiments, the sample is obtained prior to treatment with another therapy. In some embodiments, the sample is obtained during treatment with another therapy, or after treatment with another therapy.

[0337] In some embodiments, IL-15 is detected prior to treatment, eg, prior to initiation of treatment or prior to a treatment after a treatment interval.

[0338] In some embodiments, a method of treating a disease of the present invention is provided, the method comprising: testing a subject (e.g., a sample) (e.g., a sample from a subject) for the presence of IL-15 or a complex thereof with an IL-15 receptor (e.g., an IL-15 / IL-15Rα complex), thereby determining a value for IL-15 or a complex thereof with an IL-15 receptor (e.g., an IL-15 / IL-15Rα complex), comparing the value for IL-15 or a complex thereof with an IL-15 receptor (e.g., an IL-15 / IL-15Rα complex) to a control value, and if the value for IL-15 or a complex thereof with an IL-15 receptor (e.g., an IL-15 / IL-15Rα complex) is greater than the control value, administering to the subject a therapeutically effective amount of an anti-IL-15 antibody or fragment thereof (e.g., an anti-IL-15 antibody or fragment thereof described herein), optionally in combination with one or more additional therapies, thereby treating the disease.

[0339] These and other aspects and embodiments of the present invention are described in the accompanying drawings (a brief description of the drawings follows) and the following detailed description of the invention and are exemplified in the following examples. Any or all of the features discussed above and throughout this application may be combined in various embodiments of the present invention. The following examples further illustrate the present invention, however, it should be understood that the examples are described in an illustrative and non-limiting manner, and that various modifications may be made by those skilled in the art. Example

[0340] Example 1. Production of anti-IL-15 antibodies by hybridomas

[0341] This study utilized a hybridoma technology platform for antibody discovery.

[0342] 1.1. Immunity

[0343] Human IL-15 / IL-15Rα protein (Novoprotein, Cat#C15Y) and adjuvant TiterMax (Sigma, Cat#T2684) were mixed in equal volumes and then immunized into Balb / c mice (from Beijing Weitonglihua) with a subcutaneous injection every two weeks (50 μg protein per mouse). Blood was collected one week after three immunizations to detect serum titers.

[0344] 1.2. Cell fusion and high-throughput screening

[0345] When the serum titer meets the requirements, the spleen of the mouse is removed to prepare a B lymphocyte suspension, which is mixed with SP2 / 0 myeloma cells (ATCC, CRL-1581) at a ratio of 1:2 to 1:1 and then electrofused. The fused cells are transferred from the electrofusion tank into a 50 mL centrifuge tube and diluted to 1 to 2 × 10 cells using a medium containing HAT (hypoxanthine, aminopterin, and thymidine). 4 Cells were cultured at a concentration of 100 μl cells / mL and 100 μl of cell suspension was added to each well of a 96-well plate. The screening medium was replaced on the 7th day after fusion. After the 10th day of culture (or longer, depending on the cell growth status), an enzyme-linked immunosorbent assay (ELISA) was performed to screen for positive clones.

[0346] Hybridoma cells that specifically express anti-IL-15 antibodies were screened by enzyme-linked immunosorbent assay (ELISA). Human IL-15 (Novoprotein, Cat#C016) was diluted to 0.5 μg / mL with coating solution (Thermo, Ca#28382), and 100 μL / well was added to the ELISA plate. The plate was incubated at 37°C for 1 hour and then diluted with PBST (1X Phosphate-Buffered Saline, 0.5% Detergent) for three times, and shake off the liquid in the plate; 200 μL / well 3% NON-Fat Powdered The ELISA plate was blocked with milk (Sanggong, Cat#A600669-0250), incubated at 37°C for 1 hour, washed three times with PBST, and the liquid in the plate was shaken off; 50 μL / well of the fused hybridoma supernatant was added, incubated at 37°C for 1 hour, washed three times with PBST, and the liquid in the plate was shaken off; 100 μL / well of the secondary antibody labeled with goat anti-mouse HRP (BIOLEGEND, Ca#405306) (diluted 1:5000 in PBST) was added, incubated at 37°C for 1 hour, washed three times with PBST, and the liquid in the plate was shaken off; 100 μL / well of the color developing solution tetramethylbenzidine (TMB) (INVITROGEN, Cat#002023) was added, and the color was developed in the dark for 3 minutes; 50 μL / well of the ELISA stop solution (Solarbio, Cat#C1058) was added, and the photometric value at OD450 was read with a microplate reader. After screening, clones positive for human IL-15 antigen were obtained and their binding to cynomolgus monkey IL-15 was tested using the same method and steps as described above.

[0347] 1.3. Subcloning of positive hybridoma cells

[0348] Limiting dilution subcloning steps: prepare a 96-well plate and add 200 μL of culture medium to each well. This culture medium is based on the screening culture medium, but HAT is replaced with HT (hypoxanthine and thymidine nucleoside) (Gibco, Cat#11067-030). The rest of the formula is the same. Prepare a cell suspension from the cells in the positive wells screened by the above fusion, take 100 μL and add it to each well in the first row and mix well. Then take 100 μL of the cell suspension in the first row and add it to the second row. After thorough mixing, take 100 μL and add it to the next row. Repeat the above steps, let the 96-well plate stand for 30 minutes, and observe and count under a microscope. Take the volume corresponding to 100 cells and add 20 mL of culture medium, mix well and plate, 200 μL per well. Observe under a microscope one week later to determine and mark the monoclonal wells.

[0349] When the confluence of cells in each well reached more than 50%, the cells were detected by the same ELISA screening method as above, and the target positive wells were picked out. After expansion culture, the cells were frozen (finally 18 clones were selected for final determination).

[0350] Example 2. Preparation of chimeric antibodies

[0351] The present invention utilizes molecular biological techniques to obtain antibody sequences from anti-IL-15 positive hybridoma cells and utilizes these sequences to construct human-mouse chimeric antibodies. The antibody light and heavy chain gene sequences of the 18 hybridoma candidate clones obtained in Example 1 were retrieved and human-mouse chimeric antibodies were constructed from these sequences.

[0352] Take about 5×10 freshly cultured hybridoma cells of each strain 6 RNA was extracted (Macherey-Nagel, Ca#740984.250). cDNA was obtained by reverse transcription using the PrimeScript II 1st Strand cDNA Synthesis Kit (Takara). Upstream primers were designed based on the base sequence located in the 5' FR1 region, and downstream primers were designed based on the bases located in the antibody constant region or FR4 region to amplify the antibody light chain and heavy chain variable region gene fragments. They were ligated into a T vector (Mighty TA-cloning Kit, Takara), single clones were picked for sequencing, and the sequencing results were analyzed and compared using MEGA7 software. After comparison, three pairs of candidate clone sequences were obtained based on sequence homology.

[0353] The light and heavy chain variable region gene fragments of three pairs of candidate clones were respectively transformed into the homologous recombinase ( The light and heavy chain antibody expression plasmids were obtained by ligating the heavy chain constant region (Catalog No. C112-01) into a pcDNA3.1 vector (containing the heavy chain constant region: SEQ ID NO: 26; the light chain constant region: SEQ ID NO: 27, respectively). The light chain and heavy chain plasmids of the same antibody were then mixed at a 1:1 molar ratio and transfected into 293F cells using polyethyleneimine (PEI) (Polysciences, Ca# 23966). After 5-7 days of culture, when cell viability dropped below 60%, the cell culture supernatant was collected and the monoclonal antibody was purified using a Protein A affinity column. The specific purification method is described in Example 3.2 below.

[0354] A total of three chimeric antibodies were obtained, including ch25B10, ch56G9, and ch60G8.

[0355] The sequences of control antibodies Amg714 and Caly002 are shown in Tables 1 and 2 and were prepared similarly to those described above.

[0356] Table 1. Protein sequence of control antibody Amg714

[0357] name Amino acid sequence Amg714VH SEQ ID NO: 33 Amg714VL SEQ ID NO: 34

[0358] Table 2. Control antibody Caly002 protein sequence

[0359] name Amino acid sequence Caly002VH SEQ ID NO: 35 Caly002VL SEQ ID NO: 36

[0360] Example 3. Expression and purification of antibody protein and IL-15 / IL-15Rα-Fc protein

[0361] Human IL-15 / IL-15Rα-Fc, human Q108AIL-15 / IL-15Rα-Fc, and human H105RIL-15 / IL-15Rα-Fc proteins were used for subsequent affinity analysis and in vivo and in vitro functional experiments. The sequences are shown in Tables 3, 4, and 5.

[0362] Table 3. Human IL-15 / IL-15Rα-Fc protein sequence

[0363] name Amino acid sequence IL-15(WT)-Fc SEQ ID NO: 29 IL-15Rα_sushi-Fc SEQ ID NO: 30

[0364] Table 4. Human Q108A IL-15 / IL-15Rα-Fc protein sequence

[0365]

[0366] Table 5. Human H105R IL-15 / IL-15Rα-Fc protein sequence

[0367] name Amino acid sequence IL-15(H105R)-Fc SEQ ID NO: 32 IL-15Rα_suShi-Fc SEQ ID NO: 30

[0368] Protein preparation

[0369] Expi293F cells (purchased from Gibco) were cultured with Expi293F medium (Gibco, REF#A14351-01). The cell density was checked one day before transfection (viability should be greater than 95%) and adjusted to 3 × 10 6 The cell density was adjusted to 3×10 cells / mL on the day of transfection. 6 cells / mL.

[0370] Opti-MEM medium (Gibco, REF#31985-070) was used as the transfection buffer at 1 / 10 the final transfection volume. The plasmid to be transfected (wherein the plasmid is composed of nucleic acids encoding the IL-15-Fc chain (or its variant IL-15-Fc chain) or the IL-15Rα_sushi_Fc chain ligated to the pcDNA3.1 vector) was added at a ratio of 1 mg / L. The two chain plasmids were mixed at a ratio of 1:1. PEIMax (Polysciences Inc. Cat#24765-1) was added at a DNA:PEI mass ratio of 1:3. The mixture was mixed and incubated at room temperature for 20 minutes. The mixture was then gently poured into the Expi293F cell suspension with shaking. The cells were cultured in a shaker at 8% CO2, 36.5°C, and 120 rpm.

[0371] After 16-18 hours of culture, the cell suspension was supplemented with 2% (v / v) of 200 g / L feed (100 g / L Phytone Peptone + 100 g / L Difco Select Phytone), a glucose solution with a final concentration of 5 g / L, and valproic acid sodium salt (Merk, Cat# P4543-100G) with a final concentration of 2.2 mM. The suspension was gently mixed and cultured for 7 days at 8% CO₂, 36.5°C, and 120 rpm before sampling. The cell suspension was then mixed with diatomaceous earth (Sartorius, Cat# 1000037025) (40 g of diatomaceous earth per 1 L of cell suspension) and filtered using a 0.22 μM disposable vacuum filter.

[0372] Protein purification

[0373] A HiTrap MabSelect PrismA (GE Healthcare, Cat#17549853) affinity chromatography column was used for affinity capture. Before purification, 10-20 column volumes of 0.1 M NaOH were passed through the tubing and affinity chromatography column, and then the tubing and column were washed with 10-20 column volumes of distilled water. The packed column was equilibrated with 5 column volumes of 1× PBS (Gibco). The filtered cell feed was passed through the column, and the packed column was washed with 10 column volumes of 1× PBS to remove nonspecific binding proteins. The packed column was rinsed with 5 column volumes of elution buffer (100 mM sodium citrate, pH 3.5), the eluate was collected, the pH was adjusted to 6.0 with 2 M Tris, and the column was sterilized by filtration.

[0374] The purity of the samples was checked by size exclusion chromatography (SEC).

[0375] Protein samples that meet the purity standards are centrifuged in 15 mL ultrafiltration centrifuge tubes at 4000 rpm for 10 minutes. The protein is diluted with PBS and centrifuged again at 4000 rpm for 10 minutes. Repeat this process several times to replace the protein storage buffer. After the buffer exchange, filter sterilize, test the antibody concentration, and deliver the protein.

[0376] Example 4. Detection of the biological activity of chimeric antibodies using IL-15 reporter cell lines

[0377] The functional testing of three candidate chimeric antibodies was performed using an IL-15 reporter cell line. Anti-IL-15 antibodies can directly target the IL-15 cytokine, blocking the IL-15 downstream signaling pathway, thereby inhibiting cell proliferation and activation. This study used a fluorescent reporter cell line provided by Shanghai Jiman Biotechnology Co., Ltd.: IL-15 pathway activity reporter cells (human erythroid leukemia cells-IL-15 reporter cell line, GM-C25386). According to the method provided in the instructions, the expression of the fluorescent reporter gene was detected to reflect cell proliferation and differentiation, thereby detecting the inhibitory effect of the antibody on IL-15 signaling.

[0378] Cell recovery: 1) Remove cells from the liquid nitrogen tank and gently shake them in a 37°C water bath until they melt. Keep the sealing ring and bottle cap above the water surface to prevent contamination. 2) After the cells melt, they should be immediately disinfected with 75% alcohol and moved to a clean bench. Subsequent sterile procedures should be strictly followed. 3) Transfer the cell suspension in the cryopreserved tube to a preheated 15 mL centrifuge tube, mix gently, and centrifuge at 1000 rpm for 5 minutes to pellet the cells. Discard the supernatant. 4) Prepare complete culture medium in advance: 1640 (Gibco, 22400-071), 10% FBS (Gibco, 10099-141C), 1% pen / strep (Hyclone, SV30010), 2 ng / mL GM-CSF (R&D, 215-GM-010); 5) Resuspend the cell pellet in 1 mL of complete culture medium, remove a portion and count the viable cells using trypan blue (Gibco, 15250-061); 6) Adjust the viable cell density to 3-5x10 5 cells / mL, and inoculate the cell suspension into a T25 cell culture flask; 7) culture in a 37°C, 5% CO2 constant temperature incubator.

[0379] Cell culture: Maintain 0.25ug / mL puromycin (Gibco, A11138-02) and 3ug / mL blastincidin (Gibco, A11139-03) screening pressure during subculturing, subculture every 2-3 days, and control the subculture density at 0.5x10 6 cells / mL.

[0380] Activity assay: 1) Cell plating: Count the cells and adjust the cell density to 2x10 6Cells were plated at 100 μg / mL. 50 μL of cells were removed and added to a TC-treated 96-well flat-bottom luminescent plate with a white bottom and transparent cover (Shanghai Wohong, WHB-96-03). The cells were starved at 37°C with 5% CO2 for 4 h. 2) Preparation of Antibodies and Cytokines: IL-15 / IL-15Rα-Fc stimulator was prepared in 1640 medium without GM-CSF at a concentration of 0.01 μg / mL. 25 μL was added to each well of the cell culture plate. Antibodies (with isotype sequences of SEQ ID NO: 37 and SEQ ID NO: 38) were diluted in a sterile 96-well V-bottom plate (Biyuntian, FPT019) at 300 nM in a 4-fold dilution series, with a total of 12 dilutions. 25 μL was added to each well of the cell culture plate. The cells were incubated at 37°C with 5% CO2 for 16 h. 3) Fluorescence detection: Remove the Bio-Lite Luciferase Assay system reagent (Vazyme, DD1201-02) and 96-well plate in advance and equilibrate at room temperature for 30 minutes. Add an equal volume of Bio-Lite Luciferase Assay system 1X lysis buffer to each well, shake gently, and let it stand for 10 minutes before detection.

[0381] The experimental results are as follows Figure 1 As shown, chimeric antibodies ch25B10, ch56G9, and ch60G8 can effectively block downstream signals stimulated by IL-15 / IL-15Rα-Fc, and the inhibitory effect is better than that of the control anti-IL-15 antibody.

[0382] Example 5. Analysis of physicochemical properties of chimeric antibodies

[0383] The physicochemical properties of chimeric antibodies ch25B10, ch56G9, and ch60G8 were tested.

[0384] 5.1. SMAC Column Detection of Antibody Colloidal Stability

[0385] The antibody colloidal stability was tested by recording the retention time of the antibody in the Zenix-HPLC column.

[0386] 5.2. Detection of Antibody Colloid Hydrophobicity Using HIC Column

[0387] The hydrophobicity of the antibody was detected by recording the retention time of the antibody in the HIC-HPLC column.

[0388] 5.3. Nonspecificity of CIC Detection Antibodies

[0389] IgG from human serum was coated onto an NHS-activated column, and the retention time of the antibody on the column was examined by HPLC. Retention time showed a significant negative correlation with solubility, thus enabling the screening of antibodies with better solubility.

[0390] 5.4. DLS Detection of Antibody Thermal Stability

[0391] Dynamic light scattering (DLS) uses laser light to illuminate small particles undergoing Brownian motion in a solution and detects changes in the intensity of the scattered light. DLS can be used to determine the particle size of proteins in solution and their stability under varying temperatures or concentrations.

[0392] The experimental results are shown in Table 6. Antibodies ch25B10 and ch56G9 exhibited relatively good physicochemical properties.

[0393] Table 6. Antibody thermal stability test results

[0394]

[0395]

[0396] Example 6. Determination of the binding kinetics between the antibodies of the present invention and antigens using biointerferometry (ForteBio)

[0397] The equilibrium dissociation constant (KD) of the antibodies of the present invention binding to human and cynomolgus monkey IL-15 was determined using biointerferometry (ForteBio). ForteBio affinity determination was performed according to existing methods (Estep, P et al., High throughput solution-based measurement of antibody antigen affinity and epitope binding. MAbs, 2013. 5(2): p. 2708).

[0398] Half an hour before the start of the experiment, according to the number of samples, an appropriate number of AHC (18-5060, Sartorius) sensors were taken and immersed in SD buffer (PBS 1×, BSA 0.1%, Tween 20 0.05%).

[0399] 100 μL of SD buffer, antibody, and antigen (including human IL-15 (IL5-H52H8, Acro biosystems), cynomolgus macaque IL-15 (IL5-C52H4, Acro biosystems), human IL-15 / IL-15Rα-Fc, human Q108A IL-15 / IL-15Rα-Fc, and human H105R IL-15 / IL-15Rα-Fc) were added to a 96-well black polystyrene half-well microplate (Greiner, 675076). The plate was arranged according to the sample position and the sensor position was selected. The instrument parameters were set as follows: Run steps: Baseline, Loading ~1 nm, Baseline, Association, and Dissociation; the run time for each step depends on the sample association and dissociation rates. The rotation speed was 1000 rpm and the temperature was 30°C. KD values ​​were analyzed using ForteBio analysis software. The experimental results are shown in Tables 7 and 11-15 below.

[0400] Example 7. Antibody humanization

[0401] Based on Discovery Studio (Dassault Systèmes) and PyMOL software, chimeric antibodies ch25B10 and ch56G9 obtained from hybridoma screening were humanized through the following steps:

[0402] (1) Determine the CDR region of the chimeric antibody;

[0403] (2) screening the human germline sequence database for the closest homologous sequences to the V / J regions of the chimeric antibody heavy and light chains, respectively;

[0404] (3) constructing the CDR region of the chimeric antibody onto the human Germline backbone;

[0405] (4) Based on the sequence and structural characteristics, determine the amino acid positions in the framework region that play a role in maintaining the CDR function, and perform back mutations (returning to the input amino acid type) at the sequence positions determined to be important;

[0406] (5) synthesizing the sequence, preparing the antibody, and then determining the affinity of the humanized molecule for human IL-15;

[0407] (6) Given that the sequences of ch25B10 and ch56G9 are very similar, and in order to avoid the drugability risks brought by the sequence in the ch56G9 light chain, the humanized heavy chain sequence of ch56G9 and the humanized light chain sequence of ch25B10 were combined to form the final humanized molecule, hz56G9.25L.

[0408] The equilibrium dissociation constant (KD) for antibody binding to human IL-15 was determined using the ForteBio assay as described in Example 6.

[0409] Table 7. Equilibrium dissociation constants of antibodies

[0410]

[0411] Based on the affinity data presented in Table 7, it can be concluded that the humanized antibody, hz56G9.25L, maintains comparable affinity to the chimeric antibody. The CDR, VH, and VL amino acid sequences of the chimeric and humanized antibodies are shown in Tables 8, 9, and 10.

[0412] Table 8. Amino acid sequences of CDRs of exemplary antibodies of the present invention

[0413]

[0414] Table 9. Heavy chain variable regions (VH) of exemplary antibodies of the present invention

[0415]

[0416] Table 10. Light chain variable regions (VL) of exemplary antibodies of the present invention

[0417]

[0418] Example 8. ForteBio determination of the binding kinetics of hz56G9.25L and control antibodies to antigens

[0419] The equilibrium dissociation constants (KD) of the humanized antibodies of the present invention for binding to human IL-15, cynomolgus monkey IL-15, human IL-15 / IL-15Rα-Fc, human Q108A IL-15 / IL-15Rα-Fc, and human H105R IL-15 / IL-15Rα-Fc were determined using the ForteBio assay. The ForteBio affinity assay method was the same as in Example 6. The experimental results are shown in Tables 11 to 15 below.

[0420] According to the results in Tables 11 and 12, it can be seen that hz56G9.25L has a higher affinity than Amg714.

[0421] Based on the results in Tables 13, 14, and 15, it can be seen that hz56G9.25L targets different epitopes on the IL-15 protein compared to Amg714 and Caly002. Specifically, Amg714 targets amino acids 108 and 105 of IL-15, Caly002 does not target amino acids 105 or 108 of IL-15, and hz56G9.25L targets amino acids 105 of IL-15 and does not target amino acids 108 of IL-15. Literature reports that IL-15 or the IL-15 / IL-15Rα complex activates the IL-15 signaling pathway by binding to the IL2Rb / IL2Rg complex. Amino acids 105 and 108 of IL-15 are both located at the interface between IL-15 and IL2Rg. Prior art references indicate that Amg714 targets the contact interface between IL-15 and IL2Rg, while Caly002 targets the contact interface between IL-15 and IL2Rb, which is consistent with the results in Tables 13, 14, and 15. Overall, hz56G9.25L targets a unique epitope compared to Amg714 and Caly002.

[0422] Table 11. Affinity constants (M) for antigen-antibody binding assays performed by ForteBio

[0423]

[0424] Table 12. Affinity constants (M) for antigen-antibody binding assays performed by ForteBio

[0425]

[0426] Table 13. Affinity constants (M) for antigen-antibody binding assays performed by ForteBio.

[0427]

[0428] Table 14. Affinity constants (M) for antigen-antibody binding assays by ForteBio

[0429]

[0430]

[0431] Table 15. Affinity constants (M) for antigen-antibody binding assays performed by ForteBio.

[0432]

[0433] Example 9. Detection of Antibody Biological Activity Using IL-15 Reporter Cell Line

[0434] Anti-IL-15 antibodies can directly target the IL-15 cytokine, blocking the IL-15 downstream signaling pathway, thereby inhibiting cell proliferation and activation. This study used a fluorescent reporter cell line provided by Shanghai Jiman Biotechnology Co., Ltd.: IL-15 pathway activity reporter cells (human erythroid leukemia cells - IL-15 reporter cell line, GM-C25386). According to the method provided in the instructions, the expression of the fluorescent reporter gene was detected to reflect cell proliferation and differentiation, thereby detecting the inhibitory effect of the antibody on IL-15 signaling.

[0435] Cell recovery: See Example 4.

[0436] Cell culture: See Example 4.

[0437] Activity assay: 1) Cell plating: Count the cells and adjust the cell density to 2x10 6 Cells were diluted to 0.001 μg / mL for 10 minutes. 50 μL of cells were removed and added to a TC-treated 96-well flat-bottom luminescent plate with a white bottom and transparent cover (Shanghai Wohong, WHB-96-03). The cells were starved at 37°C with 5% CO2 for 4 hours. 2) Preparation of antibodies and cytokines: IL-15 (Novoprotein, C016) and IL-15 / IL-15Rα-Fc stimulators were prepared in 1640 medium without GM-CSF cytokines at concentrations of 0.001 μg / mL and 0.01 μg / mL, respectively. 25 μL was added to each well of the cell culture plate. Antibodies were diluted in a sterile 96-well V-bottom plate (Biyuntian, FPT019) to a 300 nM concentration in 12 4-fold dilutions. 25 μL was added to each well of the cell culture plate. The cells were incubated at 37°C with 5% CO2 for 16 hours. 3) Fluorescence detection: Take out the Bio-Lite Luciferase Assay system reagent (Vazyme, DD1201-02) and 96-well plate in advance, equilibrate at room temperature for 30 minutes, add an equal volume of Bio-Lite Luciferase Assay system 1X lysis buffer to each well, shake gently, let it stand for 10 minutes, and then detect with the instrument.

[0438] The experimental results are as follows Figure 2 (IL-15 stimulation), Figure 3 (IL-15 / IL-15Rα-Fc stimulation) shows that the antibody hz56G9.25L can effectively block the activation of IL-15-mediated downstream signaling pathways, and the inhibitory effect is better than that of the control anti-IL-15 antibody.

[0439] Example 10. Detection of the inhibitory effect of antibodies on IL-15 signal-induced immune cell proliferation

[0440] Cell recovery: 1) Remove peripheral blood mononuclear cells (PBMC) (Shanghai Miaoshun TPCS, catalog number PB050C-W) from the liquid nitrogen tank and gently shake them in a 37°C water bath until they melt. Keep the sealing ring and bottle cap above the water surface to prevent contamination. 2) After the cells melt, they should be immediately disinfected with 75% alcohol and moved to a clean bench. Subsequent sterile procedures should be strictly followed. 3) Prepare 1640 complete culture medium in advance: 1640 (Gibco, 22400-071), 10% FBS (Gibco, 10099-141C), 1% pen / strep (Hyclone, SV30010), 1% Sodium Pyruvate (Gibco, 1136-070) and 0.1% β-ME (Gibco, 21985-023); 3) Transfer the cell suspension in the cryovial to pre-warmed 1640 complete medium containing DNase1 (SIGMA, D5025-375KU), mix gently, centrifuge at 300g for 6 minutes to pellet the cells, and discard the supernatant; 4) Resuspend the cell pellet in 15 mL of complete medium, remove a portion and count the viable cells using trypan blue (Gibco, 15250-061).

[0441] Activity assay: 1) Cell plating: Adjust the density of viable cells to 1x10 6 Cells / mL were plated in a TC-treated, white-bottomed, transparent-lid, 96-well flat-bottom luminescent plate (Shanghai Wohong, WHB-96-03) at a volume of 100 μL per well, containing 100,000 cells. 2) Preparation of antibodies and cytokines: IL-15 (Novoprotein, C016) and IL-15 / IL-15Rα-Fc stimulators were prepared in 1640 medium at concentrations of 0.01 and 0.5 μg / mL (or 5 μg / mL), respectively, and 50 μL was added to each well of the cell culture plate. Antibodies (Bio-Ying Bio, 2K7D4D001) were diluted in a sterile 96-well V-bottom plate (Beyotime, FPT019). Under IL-15 stimulation, the antibody was diluted 2.5-fold at 300 nM for a total of 12 steps. Under IL-15 / IL-15Rα-Fc stimulation, the antibody was diluted 2-fold at 300 nM for a total of 12 steps. 50 μL was added to each well of the cell culture plate and incubated at 37°C in 5% CO2 for 72 hours. 3) Fluorescence Detection: Remove the Cell Counting-Lite 2.0 reagent (Vazyme, DD1101-02) and 96-well plate in advance and equilibrate at room temperature for 20-30 minutes. Add an equal volume of Cell Counting-Lite 2.0 lysis buffer to each well, gently shake, and incubate for 15 minutes before detection.

[0442] The above experimental results are as follows Figure 4 (IL-15 stimulation), Figure 5(IL-15 / IL-15Rα-Fc stimulation, 0.5ug / ml), Figure 6 (IL-15 / IL-15Rα-Fc stimulation, 5ug / ml) shows that the antibody hz56G9.25L can effectively block the activation of IL-15-mediated downstream signaling pathways and inhibit the proliferation of immune cells, and the inhibitory effect is better than that of the control anti-IL-15 antibody.

[0443] Example 11. Detection of the inhibitory effect of antibodies on IL-15 signal-induced CD8 T cell activation

[0444] Isolation of CD8 T cells: 1) Remove PBMC cells (Shanghai Miaoshun TPCS, catalog number PB050C-W) from a liquid nitrogen tank and gently shake them in a 37°C water bath until they melt. Keep the sealing ring and bottle cap above the water surface to prevent contamination. 2) After the cells melt, they should be immediately disinfected with 75% alcohol and moved to a clean bench. Subsequent sterile procedures must be strictly followed. 3) Prepare 1640 complete culture medium in advance: 1640 (Gibco, 22400-071), 10% FBS (Gibco, 10099-141C), 1% pen / strep (Hyclone, SV30010), and 1% Sodium Pyruvate (Gibco, 1136-070) and 0.1% β-ME (Gibco, 21985-023); 4) Transfer the cell suspension in the cryovial to pre-warmed 1640 complete medium containing DNase1 (SIGMA, D5025-375KU), mix gently, centrifuge at 300g for 6 minutes to pellet the cells, and discard the supernatant; 4) Resuspend the cell pellet in 15 mL of complete medium, remove a portion and count the live cells using trypan blue (Gibco, 15250-061); 5) Use a CD8 T cell isolation kit (stemcell, 19053) to isolate CD8 T cells from PBMCs.

[0445] CD8 T cell activation assay: 1) Coat a 6-well plate with 1 μg / mL anti-human CD3 (Acro, CDE-H5223) at 4°C overnight; 2) Remove isolated CD8 T cells and add them to a 6-well plate coated with CD3 antibody to activate CD8 T cells. Add 1 μg / mL anti-CD28 (Invitrogen, 16-0289-85) to each well to a volume of 3 mL and incubate at 37°C in 5% CO2 for 72 hours; 3) Collect the activated CD8 T cells 3 days later and adjust the cell density to 1x10 6100,000 cells were plated in the middle 60 wells of a TC-treated, white-bottom, transparent-cover 96-well flat-bottom luminescent plate (Shanghai Wohong, WHB-96-03). 100 μL of IL-15 (Novoprotein, C016) and IL-15 / IL-15Rα-Fc stimulators were prepared at concentrations of 0.01 and 0.5 μg / mL, respectively, and 50 μL was added to each well of the cell culture plate. 5) Antibodies were diluted in a sterile 96-well V-bottom plate (Beyotime, FPT019). Under IL-15 stimulation, the antibody was diluted 2.5-fold at 300 nM for a total of 12 steps. Under IL-15 / IL-15Rα-Fc stimulation, the antibody was diluted 2-fold at 300 nM for a total of 12 steps. 50 uL was added to each well of the cell culture plate and incubated at 37°C with 5% CO2 for 72 hours. 6) The supernatant was collected and IFNg secretion was detected by ELISA (MabTech, 3420-1H-20).

[0446] The above experimental results are as follows Figure 7 (IL-15 stimulation), Figure 8 (IL-15 / IL-15Rα-Fc stimulation) shows that the antibody hz56G9.25L can effectively block IL-15-mediated downstream signaling pathway activation, inhibit IL-15-mediated CD8 T cell activation and IFNγ cytokine secretion, and the inhibitory effect is better than that of the control anti-IL-15 antibody.

[0447] Example 12. Analysis of the physicochemical properties of antibody hz56G9.25L

[0448] Detect the physicochemical properties of antibody hz56G9.25L.

[0449] 12.1. SMAC Column Testing of Antibody Colloidal Stability

[0450] The antibody colloidal stability was tested by recording the retention time of the antibody in the Zenix-HPLC column.

[0451] 12.2. Detection of Antibody Colloid Hydrophobicity Using HIC Column

[0452] The hydrophobicity of the antibody was detected by recording the retention time of the antibody in the HIC-HPLC column.

[0453] 12.3. Nonspecificity of CIC Detection Antibodies

[0454] IgG from human serum was coated onto an NHS-activated column, and the retention time of the antibody on the column was examined by HPLC. Retention time showed a significant negative correlation with solubility, thus enabling the screening of antibodies with better solubility.

[0455] 12.4. DLS Detection of Antibody Thermal Stability

[0456] Dynamic light scattering (DLS) uses laser light to illuminate small particles undergoing Brownian motion in a solution and detects changes in the intensity of the scattered light. DLS can be used to determine the particle size of proteins in solution and their stability under varying temperatures or concentrations.

[0457] The experimental results are shown in Table 16, and the antibody hz56G9.25L exhibited expected physicochemical properties.

[0458] Table 16. Physicochemical properties of antibodies

[0459]

[0460] Example 13. Detection of the inhibitory effect of antibodies on IL-15 signaling-induced mouse skin inflammation

[0461] Female C57BL / 6N mice, 6-9 weeks old, weighing 16-19 g, were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd., of SPF grade, and the quality inspection unit was Beijing Weitonglihua Experimental Animal Technology Co., Ltd., with the certificate number 110011231110488161.

[0462] After one week of adaptive feeding, the mice were randomly divided into four groups: non-modeling group (i.e., PBS+Isotype), modeling group (i.e., IL-15 / IL-15Rα-Fc+Isotype), Amg714 treatment group, and hz56G9.25L treatment group.

[0463] The dorsal and ventral sides of the mice were shaved, and two 1cm*1cm boxes were drawn on the skin with a marker and recorded as site1 and site2. The three groups were then intradermally injected with IL-15 / IL-15Rα-Fc in the two boxes on days 1, 3, 5, 7, 9, and 11, respectively, at 2.5ug / 50uL in each box, i.e., 5ug / 100uL in each mouse, to induce a mouse dermatitis model. At the same time, the non-modeling group was intradermally injected with an equal volume of PBS.

[0464] Four groups of mice were intraperitoneally injected with Isotype (100 μg / mouse), Isotype (100 μg / mouse), Amg714 (100 μg / mouse), and hz56G9.25L (100 μg / mouse) on days 0, 3, 6, and 10, respectively. Skin thickness was monitored on days 1, 3, 5, 7, 10, and 14 using a vernier caliper, and the degree of skin thickening was calculated (Change % = Day x Skin Thickness / Day 0 Skin Thickness x 100% - 100%).

[0465] The experiment was terminated on the 14th day, and the mice were euthanized. The local skin of the mouse site 1 model was collected and fixed with formalin, and T cell infiltration was detected by IHC staining. The local skin of the mouse site 2 model was collected and placed in PBS, and T cell infiltration and activation (NKG2D expression level) were detected by flow cytometry (the NKG2D flow cytometry antibody used was BD 562800).

[0466] The above flow cytometry results are as follows Figure 9 、 Figure 10 As shown in the figure, the antibody hz56G9.25L can effectively block IL-15-mediated T cell proliferation and activation. As shown in the figure, compared with Amg714, the antibody hz56G9.25L can more effectively inhibit the expression level of NKG2D.

[0467] Example 14. Detection of the inhibitory effect of antibodies on IL-15 signaling-induced small intestinal inflammation

[0468] Female C57BL / 6N mice, 6-9 weeks old, weighing 16-19 g, were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd., of SPF grade. The quality inspection unit was Beijing Weitonglihua Experimental Animal Technology Co., Ltd., and the certificate number was 110011231107923856.

[0469] After one week of adaptive feeding, the mice were randomly divided into four groups: non-modeling group (i.e., PBS+Isotype), modeling group (i.e., IL-15 / IL-15Rα-Fc+Isotype), Amg714 treatment group, and hz56G9.25L treatment group.

[0470] The last three groups were intraperitoneally injected with IL-15 / IL-15Rα-Fc (15ug / 200uL per mouse) within the first hour to induce a mouse enteritis model. At the same time, the non-modeling group was intraperitoneally injected with an equal volume of PBS.

[0471] The mice in the four groups were injected intraperitoneally with Isotype (200ug / mouse), Isotype (200ug / mouse), Amg714 (200ug / mouse), and hz56G9.25L (200ug / mouse) at 0h, 24h, and 48h, respectively. The experiment was terminated at 72h, and the mice were euthanized. The small intestine (10cm below the stomach) of the mice was collected, and the spleen of the mice was collected and weighed. The small intestine ( Figure 11 ) and spleen ( Figure 12 ) T cell activation (CD69 expression level) (the CD69 flow cytometry antibody used was Biolegend 104508).

[0472] The above flow cytometry results are as follows Figure 11 、 Figure 12As shown, antibody hz56G9.25L can effectively block IL-15-mediated T cell activation and has a trend of showing advantages compared with Amg714. Figure 11 Two types of T cells that exist in large numbers in the small intestine were detected, including αβT cells and γδT cells. The antibody hz56G9.25L had a significant inhibitory activation effect on both types of T cells.

[0473] Example 15. Detection of the inhibitory effect of antibodies on IL-15 signal-enhanced graft-versus-host disease (GvHD)

[0474] Female NOG mice, 6-9 weeks old and weighing 16-19 g, were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. They were SPF-grade and quality-inspected by Beijing Weitonglihua Laboratory Animal Technology Co., Ltd., with a certificate number of 110011231109256251. After one week of adaptive feeding, they were randomly divided into four groups: a GvHD group (PBS + isotype), a GvHD-accelerated group (IL-15 / IL-15Rα-Fc + isotype), an Amg714-treated group, and an hz56G9.25L-treated group.

[0475] All mice were intravenously injected with PBMC (1*10^7 / mouse, All cells, LP230809010, LP221115011) on day 1 to induce a mouse GvHD model; the last three groups were intraperitoneally injected with IL-15 / IL-15Rα-Fc, 5ug / 200uL per mouse on days 1, 3, 5, 7, 10, 12, and 14, respectively, to accelerate GvHD. At the same time, the first group was intraperitoneally injected with an equal volume of PBS.

[0476] Four groups of mice were intraperitoneally injected with Isotype (100 μg / mouse), Isotype (100 μg / mouse), Amg714 (100 μg / mouse), and hz56G9.25L (100 μg / mouse) on days 0, 3, 7, 10, 12, 14, 16, and 18, respectively. Mouse body weight was measured using an electronic balance, and weight change was calculated (% Change = Day × Weight / Day 0 Weight × 100% - 100%). Mice were monitored for mortality throughout the experiment, and mice with a weight loss exceeding 20% ​​were euthanized.

[0477] The above flow cytometry results are as follows Figure 13 (PBMC donor 1), Figure 14 (PBMC donor 2) As shown, the antibody hz56G9.25L can effectively block IL-15 signaling-enhanced graft-versus-host disease (GvHD).

[0478] As shown in the figure, compared to the PBS group, IL-15 / IL-15Rα significantly accelerated the progression of GvHD, causing weight loss and death in mice. In the treatment group, the antibody hz56G9.25L was more effective than Amg714 in inhibiting IL-15 / IL-15Rα, thereby prolonging the survival of mice and slowing their weight loss.

[0479] Example 16. Pharmacokinetic (PK) study of antibodies in BALB / c mice

[0480] The pharmacokinetic (PK) behavior of hz56G9.25L in vivo was observed through mouse pharmacokinetic (PK) experiments.

[0481] BALB / c mice were subcutaneously injected with 10 mpk of hz56G9.25L, and serum samples were collected from the mice at 0.5 hours, 2 hours, 6 hours, 24 hours, 48 ​​hours, 96 hours, 168 hours, 336 hours, 504 hours, and 840 hours for detection.

[0482] ELISA was used to detect the IL-15 antigen (nearshore protein, GMP-C016) on the plate, followed by the addition of the serum to be tested, followed by detection with an anti-human Fc secondary antibody (BETHYL, A80-104P), and finally TMB color development. Figure 15 As shown in Table 15, the PK curve showed an IgG-like PK profile, with a half-life of 258 hours and a clearance rate of 0.21 ml / kg / h.

[0483] Table 17. Pharmacokinetic (PK) study of antibodies in BALB / c mice

[0484]

[0485] Example 17. Antibody Fc modification

[0486] Through the circulation effect mediated by the human neonatal Fc receptor (FcRn), the antibody can maintain a relatively long half-life in the serum. The antibody Fc has a pH-dependent interaction with FcRn. The present invention performs Fc modification on the hz56G9.25L monoclonal antibody, and by changing the amino acid sequence, aims to enhance the half-life of the drug in vivo. Specifically, the present invention performs site-directed mutation (M252Y / S254T / T256E) on three sites of the Fc segment of hz56G9.25L to improve the binding ability to FcRn, in order to prolong its half-life in vivo. The obtained antibody is named hz56G9.25L-YTE, and the CDR, VH, VL, heavy chain, and light chain amino acid sequences of the antibody are shown in Tables 18, 19, 20, 21, and 22.

[0487] Table 18. Amino acid sequences of CDRs of hz56G9.25L-YTE antibody

[0488]

[0489]

[0490] Table 19. Heavy chain variable region (VH) of hz56G9.25L-YTE antibody

[0491] Antibody name VH proteins hz56G9.25L-YTE SEQ ID NO: 17

[0492] Table 20. Light chain variable region (VL) of hz56G9.25L-YTE antibody

[0493] Antibody name VL protein hz56G9.25L-YTE SEQ ID NO: 21

[0494] Table 21. Heavy chain of hz56G9.25L-YTE antibody

[0495]

[0496] Table 22. Light chain of hz56G9.25L-YTE antibody

[0497] Antibody name Light chain protein hz56G9.25L-YTE SEQ ID NO: 24

[0498] Example 18. Detection of the biological activity of hz56G9.25L-YTE using IL-15 reporter cell line

[0499] For cell recovery, cell culture and activity detection methods, see Example 9.

[0500] The experimental results are as follows Figure 16 (IL-15), Figure 17 As shown in (IL-15 / IL-15Rα-Fc), hz56G9.25L-YTE and hz56G9.25L have similar functions and can effectively block the activation of IL-15-mediated downstream signaling pathways, and the inhibitory effect is better than that of the control anti-IL-15 antibody.

[0501] Example 19. Detection of the inhibitory effect of hz56G9.25L-YTE on IL-15 signal-induced immune cell proliferation

[0502] Cell recovery: See Example 10.

[0503] Activity assay: see Example 10. The difference was IL-15 (Novoprotein, C016) and IL-15 / IL-15Rα-Fc stimulators at concentrations of 0.01 and 0.5 ug / mL.

[0504] The experimental results are as follows Figure 18 (IL-15), Figure 19 As shown in (IL-15 / IL-15Rα-Fc), hz56G9.25L-YTE and hz56G9.25L have similar functions and can effectively block the activation of IL-15-mediated downstream signaling pathways, and the inhibitory effect is better than that of the control anti-IL-15 antibody.

[0505] Example 20 Pharmacokinetic (PK) Study of Antibodies in hFcRn Mice

[0506] The pharmacokinetic (PK) behavior of hz56G9.25L and hz56G9.25L-YTE in vivo was observed through mouse pharmacokinetic (PK) experiments.

[0507] hFcRn mice were injected with 10 mpk of hz56G9.25L and hz56G9.25L-YTE through the tail vein, and serum samples were collected for detection at 5 minutes, 2 hours, 6 hours, 24 hours, 48 ​​hours, 96 hours, 168 hours, 336 hours, 504 hours, 672 hours and 1008 hours.

[0508] ELISA was used to detect the IL-15 antigen (nearshore protein, GMP-C016) on the plate, followed by the addition of the serum to be tested, followed by detection with an anti-human Fc secondary antibody (BETHYL, A80-104P), and finally TMB color development. Figure 20 As shown in Table 23, the PK profiles of hz56G9.25L and hz56G9.25L-YTE both exhibited IgG-like PK profiles, with half-lives of 277 and 412 hours, respectively, and clearances of 0.27 and 0.17 ml / kg / h, respectively. This suggests that the PK behavior of hz56G9.25L-YTE is superior to that of hz56G9.25L.

[0509] Table 23. Pharmacokinetic (PK) study of antibodies in BALB / c mice

[0510]

[0511]

[0512]

[0513]

[0514]

Claims

1. An anti-IL-15 antibody or an antigen-binding fragment thereof, comprising: HCDR1, HCDR2, and HCDR3 comprised by the VH as shown in SEQ ID NO: 17, and LCDR1, LCDR2, and LCDR3 comprised by the VL as shown in SEQ ID NO: 21; HCDR1, HCDR2 and HCDR3 comprised by the VH as shown in SEQ ID NO: 15, 16 or 17, and LCDR1, LCDR2 and LCDR3 comprised by the VL as shown in SEQ ID NO: 19, 20 or 21; HCDR1, HCDR2 and HCDR3 comprised by the VH as shown in SEQ ID NO: 15 or 16, and LCDR1, LCDR2 and LCDR3 comprised by the VL as shown in SEQ ID NO: 19 or 20; HCDR1, HCDR2 and HCDR3 comprised by the VH as shown in SEQ ID NO: 16 or 17, and LCDR1, LCDR2 and LCDR3 comprised by the VL as shown in SEQ ID NO: 20 or 21; HCDR1, HCDR2, and HCDR3 comprised by the VH as shown in SEQ ID NO: 15, and LCDR1, LCDR2, and LCDR3 comprised by the VL as shown in SEQ ID NO: 19; HCDR1, HCDR2 and HCDR3 comprised by VH as shown in SEQ ID NO: 16, and LCDR1, LCDR2 and LCDR3 comprised by VL as shown in SEQ ID NO: 20; or The HCDR1, HCDR2 and HCDR3 comprised by VH as shown in SEQ ID NO: 18, and the LCDR1, LCDR2 and LCDR3 comprised by VL as shown in SEQ ID NO:

22.

2. An anti-IL-15 antibody or antigen-binding fragment thereof, comprising a first heavy chain complementarity determining region (HCDR1), a second heavy chain complementarity determining region (HCDR2), a third heavy chain complementarity determining region (HCDR3), and a first light chain complementarity determining region (LCDR1), a second light chain complementarity determining region (LCDR2), and a third light chain complementarity determining region (LCDR3), wherein the The HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 respectively comprise or consist of the amino acid sequences shown in SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 14; or The HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 respectively comprise or consist of the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 14; or The HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 respectively comprise or consist of the amino acid sequences shown in SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 43; or The HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 respectively contain the amino acid sequences shown in SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 43, or respectively consist of the amino acid sequences shown in SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO:

43.

3. The anti-IL-15 antibody or antigen-binding fragment thereof of claim 1 or 2, comprising a heavy chain variable region (VH), wherein the heavy chain variable region comprises, consists of, or comprises an amino acid sequence that is at least 90% identical to an amino acid sequence selected from any one of SEQ ID NOs: 15-18; and / or It comprises a light chain variable region (VL), wherein the light chain variable region comprises an amino acid sequence that is at least 90% identical to the amino acid sequence shown in any one of SEQ ID NOs: 19-22, or consists of the amino acid sequence, or comprises an amino acid sequence shown in any one of SEQ ID NOs: 19-22, or consists of the amino acid sequence.

4. An anti-IL-15 antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof (i) a VH comprising, or consisting of, an amino acid sequence as set forth in SEQ ID NO: 15, 16 or 17, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and a VL comprising, or consisting of, an amino acid sequence as set forth in SEQ ID NO: 19, 20 or 21, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; (ii) a VH comprising, or consisting of, an amino acid sequence as set forth in SEQ ID NO: 15 or 16, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and a VL comprising, or consisting of, an amino acid sequence as set forth in SEQ ID NO: 19 or 20, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; (iii) a VH comprising, or consisting of, an amino acid sequence as set forth in SEQ ID NO: 16 or 17, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and a VL comprising, or consisting of, an amino acid sequence as set forth in SEQ ID NO: 20 or 21, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; (iv) a VH comprising, or consisting of, an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and a VL comprising, or consisting of, an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, (v) a VH comprising, or consisting of, an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to, or consisting of, the amino acid sequence of SEQ ID NO: 16, and a VL comprising, or consisting of, an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to, or consisting of, the amino acid sequence of SEQ ID NO: 20; (vi) a VH comprising, or consisting of, an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and a VL comprising, or consisting of, an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, as shown in SEQ ID NO: 21; or (vii) a VH comprising, or consisting of, an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and a VL comprising, or consisting of, an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

5. An anti-IL-15 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein a) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 17, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 21; or the heavy chain variable region consists of the amino acid sequence of SEQ ID NO: 17, and the light chain variable region consists of the amino acid sequence of SEQ ID NO: 21; b) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 15, 16 or 17, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 19, 20 or 21; or the heavy chain variable region consists of the amino acid sequence of SEQ ID NO: 15, 16 or 17, and the light chain variable region consists of the amino acid sequence of SEQ ID NO: 19, 20 or 21; c) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 15 or 16, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 19 or 20; or the heavy chain variable region consists of the amino acid sequence of SEQ ID NO: 15 or 16, and the light chain variable region consists of the amino acid sequence of SEQ ID NO: 19 or 20; d) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 16 or 17, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 20 or 21; or the heavy chain variable region consists of the amino acid sequence of SEQ ID NO: 16 or 17, and the light chain variable region consists of the amino acid sequence of SEQ ID NO: 20 or 21; e) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 15, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 19; or the heavy chain variable region consists of the amino acid sequence of SEQ ID NO: 15, and the light chain variable region consists of the amino acid sequence of SEQ ID NO: 19; f) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 16, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 20, or the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO: 16, and the light chain variable region consists of the amino acid sequence shown in SEQ ID NO: 20; or g) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 18, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 22, or the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO: 18, and the light chain variable region consists of the amino acid sequence shown in SEQ ID NO:

22.

6. The anti-IL-15 antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein The anti-IL-15 antibody or antigen-binding fragment thereof comprises an Fc region, for example, the Fc region is derived from an IgG1, IgG2, IgG3, or IgG4 Fc region, for example, a human IgG1, IgG2, IgG3, or IgG4 Fc region, for example, the IgG1 Fc region comprises or consists of the amino acid sequence of SEQ ID NO: 41; or comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 41; or The anti-IL-15 antibody or antigen-binding fragment thereof comprises a heavy chain constant region, for example, the heavy chain constant region is derived from IgG1, an IgG2, IgG3, or IgG4 constant region, e.g., a human IgG1, IgG2, IgG3, or IgG4 constant region, e.g., the IgG1 heavy chain constant region comprising or consisting of the amino acid sequence of SEQ ID NO: 25, or comprising an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 25; Optionally, the heavy chain constant region or Fc region comprises a mutation that reduces binding to Fcγ receptors, such as L234A / L235A mutations, for example, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 26; or comprising an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 26; Optionally, the heavy chain constant region or Fc region comprises a mutation that improves binding to the FcRn receptor, such as a YTE mutation (M252Y / S254T / T256E); Optionally, the heavy chain constant region or Fc region comprises both a mutation that reduces binding to Fcγ receptors and a mutation that increases binding to FcRn receptors, for example, a L234A / L235A mutation and a YTE mutation, for example The heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 27; or comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 27; or The Fc region comprises or consists of the amino acid sequence of SEQ ID NO: 42, or comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:

42.

7. The anti-IL-15 antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, comprising a light chain constant region, wherein the light chain constant region is a lambda or kappa light chain constant region, such as a human lambda or kappa light chain constant region, preferably, the light chain constant region (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 28; or (ii) comprising or consisting of the amino acid sequence of SEQ ID NO:

28.

8. The anti-IL-15 antibody or antigen-binding fragment thereof of any one of claims 1-7, comprising a heavy chain and / or a light chain, wherein the heavy chain comprises, or consists of, the VH of any one of claims 1-7 and a heavy chain constant region; and / or the light chain comprises, or consists of, the VL of any one of claims 1-7 and a light chain constant region; Optionally, the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 23, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; and / or the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 24, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; Optionally, the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 23 and the light chain comprises the amino acid sequence shown in SEQ ID NO: 24; or the heavy chain consists of the amino acid sequence shown in SEQ ID NO: 23 and the light chain consists of the amino acid sequence shown in SEQ ID NO:

24.

9. The anti-IL-15 antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, wherein the antibody is a monoclonal antibody; or the antibody is a humanized antibody or a chimeric antibody.

10. The anti-IL-15 antibody or antigen-binding fragment thereof of any one of claims 1 to 9, wherein the antigen-binding fragment is an antibody fragment selected from the group consisting of: Fab, Fab', Fab'-SH, Fv, single-chain antibody (e.g., scFv), (Fab')2, dAb (domain antibody), diabody, diabody, or linear antibody.

11. The anti-IL-15 antibody or antigen-binding fragment thereof of any one of claims 1 to 10, wherein the antibody or antigen-binding fragment thereof has one or more of the following properties: a) binds with high affinity to a human or cynomolgus monkey IL-15 monomer or a complex with its receptor, such as an IL-15 / IL-15Rα complex, wherein amino acid 105 of IL-15 is H in the monomer or complex; b) effectively blocking the downstream signaling pathways stimulated by IL-15 monomers or their complexes with their receptors, such as the IL-15 / IL-15Rα complex; c) inhibiting IL-15 or its complex with a receptor, such as IL-15 / IL-15Rα, mediated CD8 T cell activation and / or IFNg cytokine secretion, for example in CD8 T cells; d) effectively blocking T cell proliferation, T cell infiltration and / or T cell activation mediated by IL-15 monomer or its complex with its receptor, such as IL-15 / IL-15Rα complex (e.g., decreasing NKG2D expression level or CD69 expression level); e) having good physicochemical properties and / or pharmacokinetic characteristics, such as good stability (e.g., colloidal stability and / or thermal stability), good solubility, long half-life and / or low clearance rate; f) effectively preventing or treating inflammation, such as skin inflammation or small intestinal inflammation; g) It can effectively prevent or treat autoimmune diseases such as graft-versus-host disease, for example, effectively block graft-versus-host disease enhanced by IL-15 signaling.

12. An isolated nucleic acid encoding the anti-IL-15 antibody or antigen-binding fragment thereof of any one of claims 1 to 11.

13. A vector comprising the nucleic acid of claim 12, preferably said vector is an expression vector.

14. A host cell comprising the nucleic acid of claim 12 or the vector of claim 13, preferably, the host cell is prokaryotic or eukaryotic, more preferably selected from yeast cells, mammalian cells (e.g., 293 cells or CHO cells, e.g., CHO-K cells or HEK293 cells) or other cells suitable for preparing antibodies or antigen-binding fragments thereof.

15. A method for preparing an anti-IL-15 antibody or an antigen-binding fragment thereof, the method comprising a) culturing the host cell of claim 14 under conditions suitable for expressing a nucleic acid encoding the anti-IL-15 antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, b) optionally isolating said antibody or antigen-binding fragment thereof, c) Optionally, the method further comprises recovering the anti-IL-15 antibody or antigen-binding fragment thereof from the host cell. Optionally, the antibody is purified, for example, by Protein A purification.

16. An immunoconjugate comprising the anti-IL-15 antibody or antigen-binding fragment thereof according to any one of claims 1 to 11 and other substances, such as toxins, small molecule drugs, cytotoxic agents, apoptotic agents, chelating agents, immunomodulators, such as anti-inflammatory agents or immunosuppressants.

17. A pharmaceutical composition comprising the anti-IL-15 antibody or antigen-binding fragment thereof according to any one of claims 1 to 11 or the immunoconjugate according to claim 16, and optionally a pharmaceutically acceptable excipient.

18. A pharmaceutical combination comprising the anti-IL-15 antibody or antigen-binding fragment thereof of any one of claims 1 to 11 or the immunoconjugate of claim 16, and one or more other therapeutic agents, e.g., the therapeutic agent is selected from a cytokine, other antibody, small molecule drug, or immunomodulator (e.g., an anti-inflammatory agent or an immunosuppressant).

19. A method for preventing or treating an IL-15-related disease and / or condition in an individual, the method comprising administering to the subject an effective amount of the anti-IL-15 antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, or the immunoconjugate according to claim 16, or the pharmaceutical composition according to claim 17, or the pharmaceutical combination product according to claim 18; Optionally, the subject has a disease or condition associated with abnormal expression of IL-15 or its receptor complex, such as IL-15 / IL-15Rα complex, or abnormal activation of a mediated signaling pathway compared to a healthy individual; Optionally, the disease or disorder is selected from a tumor, such as cancer, or inflammation or an inflammatory disorder, such as an autoinflammatory disease, such as skin inflammation or intestinal inflammation; or autoimmune diseases such as graft-versus-host disease; or Optionally, the method further comprises administering one or more other therapies, such as treatment modalities and / or other therapeutic agents, for example, selected from cytokines, other antibodies, small molecule drugs, or immunomodulators (eg, anti-inflammatory agents or immunosuppressants).

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