Anti-il-15 antibodies and use thereof

Abstract

Provided are a new antibody and antibody fragment that specifically bind to IL-15, and a composition containing the antibody or antibody fragment. Furthermore, provided are a nucleic acid encoding the antibody or antibody fragment thereof, a host cell containing same, and the related use. In addition, provided are the therapeutic and diagnostic uses of the antibodies and antibody fragments.

Description

Anti-il-15 antibodies and uses thereof

[0001] Cross-reference to Related Applications

[0002] This application is based on and claims priority to Chinese Patent Application No. 202410444282.8, filed on April 12, 2024, the entire contents of which are incorporated herein by reference.

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

[0004] BACKGROUND

[0005] The cytokine interleukin 15 (IL-15) has multiple immune roles 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 can bind to a heterotrimeric receptor composed of a unique a subunit (IL-15Ra) that confers receptor specificity and the shared receptors IL2Rß (CD122) and common y (yc) chain (CD132) with IL2. 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 sustain the survival of NK, effector CD8(+) and memory phenotype CD8(+) T cells.

[0006] IL-15Ra contains three protein domains: (i) an extracellular domain of 175 amino acids, (ii) a single 23 amino acid transmembrane region, and (iii) a cytoplasmic domain of 39 amino acids and is widely expressed in humans and mice independently of IL2R / IL-15Rßy. IL-15Ra binds IL-15 with extremely high affinity (Kd<10 -11M) binds to IL-15, retaining it on the cell surface. In this way, IL-15Ra can present IL-15 to IL2R / IL-15Rbyc on neighboring NK and T cells through an immunological synapse. This synapse mechanism limits the exposure of circulating IL-15, reducing the risk of autoimmunity. IL-15 can also persist in a membrane-bound form for several days through recycling as endosomal vesicles complexed with IL-15Ra. IL-15 is listed as the first drug in the list of drugs with high potential for use as immunotherapeutic drugs by the NCI Immunotherapeutic Drug Workshop.

[0007] 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). However, these anti-IL-15 antibodies have drawbacks in terms of affinity, epitope, blocking IL-15 signaling function, etc., and there is room for improvement.

[0008] Therefore, there is still a need in the art to obtain IL-15 antibodies with better function in terms of affinity, epitope or blocking IL-15 signaling.

[0009] SUMMARY

[0010] The present application provides a new antibody that specifically binds to IL-15, which selectively blocks the signaling pathways involved in IL-15.

[0011] In some embodiments, the present application provides a new monoclonal antibody targeting IL-15, which has stronger affinity, targets a different epitope (on the interface of IL-15 and IL2Ry) than some of the prior art anti-IL-15 antibodies, and better IL-15 signaling blocking function. In particular, the different epitope of the IL-15 antibody described in the present application helps to stably block a variety of IL-15 signals, including signals induced by IL-15 monomers and IL-15 / IL-15Ra complexes. Therefore, the anti-IL-15 antibody described in the present application exhibits stronger IL-15 signal blocking function in in vitro and in vivo experiments.

[0012] Accordingly, the present application relates to an antibody or fragment, e.g., antigen binding fragment, that specifically binds IL-15, e.g., human or cynomolgus IL-15. In some embodiments, the antibody or antigen binding fragment thereof of the present application specifically binds to human IL-15, e.g., a wild type IL-15 monomer or a complex thereof with a receptor, e.g., an IL-15 / IL-15Rα complex. In some embodiments, the antibody or antigen binding fragment thereof of the present application 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., having an H105R mutation. In some embodiments, the antibody or antigen binding fragment thereof of the present application binds to an IL-15 monomer or a complex thereof with a receptor, e.g., an IL-15 / IL-15Rα complex, having a mutation at position 108 of IL-15, e.g., having a Q108A mutation.

[0013] In some embodiments, the affinity of the antigen antibody binding is detected by ForteBio.

[0014] In some embodiments, the antibody or antigen binding fragment thereof of the present application binds to human IL-15 monomer (wild type or a variant that is not mutated at position 105, e.g., Q108A of IL-15) with a Kd D less than about 4 nM, 3.5 nM, 3 nM, 2.5 nM, or 2 nM, or greater than about 1 or 1.5 nM, or between any of the numerical ranges. In some embodiments, the Kd D between about 1 nM and 3.5 nM.

[0015] In some embodiments, the antibody or antigen binding fragment thereof of the present application binds to cynomolgus IL-15 with a Kd D less than about 4 nM, 3.5 nM, 3 nM, 2.5 nM, or 2 nM, or greater than about 1 or 1.5 nM, or between any of the numerical ranges. In some embodiments, the Kd D between about 1 nM and 2.5 nM, or between about 1.5 nM and 2 nM.

[0016] In some embodiments, the antibody or antigen binding fragment thereof of the present application binds to a complex of IL-15 with its receptor, e.g., an IL-15 / IL-15Rα complex (wild type or a variant that is not mutated at position 105 of the IL-15, e.g., an IL-15 / IL-15Rα complex comprising Q108A) with a Kd 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 between any of the numerical ranges. In some embodiments, the Kd D between about 0.1 nM and 1.5 nM, or between about 0.3 nM and 1 nM.

[0017] The antibodies or antigen-binding fragments thereof of the present application are effective in blocking downstream signaling pathways stimulated by IL-15 monomer or its complex with its receptor, e.g., IL-15 / IL-15Ra complex, e.g., in IL-15 reporter cell lines, e.g., in human erythroid leukemia cell-IL-15 reporter cell lines. In some embodiments, the antibodies or antigen-binding fragments thereof of the present application are effective in blocking downstream signaling pathway activation and / or inhibiting immune cell proliferation mediated by IL-15 monomer or its complex with its receptor, e.g., IL-15 / IL-15Ra complex, e.g., in PBMC cells. In some embodiments, the antibodies or antigen-binding fragments thereof of the present application are effective in blocking downstream signaling pathway activation mediated by IL-15 monomer or its complex with its receptor, e.g., IL-15 / IL-15Ra complex, thereby inhibiting CD8 T cell activation and / or IFNy cytokine secretion mediated by IL-15 or its complex with receptor, e.g., IL-15 / IL-15Ra, e.g., in CD8 T cells. In some embodiments, the antibodies or antigen-binding fragments thereof of the present application are effective in blocking T cell proliferation, T cell infiltration, and / or T cell activation (e.g., decrease in NKG2D expression level or CD69 expression level) mediated by IL-15 monomer or its complex with its receptor, e.g., IL-15 / IL-15Ra. In some embodiments, the T cells are a T cell population, e.g., comprising a T cell and / or a T cell.

[0018] In some embodiments, the antibodies or antigen-binding fragments thereof of the present application have good pharmacokinetic profiles, e.g., have long half-lives, and / or low clearance rates. In some embodiments, the antibodies or antigen-binding fragments thereof of the present application, when administered (e.g., subcutaneously injected), 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, e.g., about 250-280 hours, or a half-life of greater than 400 hours, e.g., greater than about 405 hours or greater than about 410 hours, e.g., a half-life of about 412 hours. In some embodiments, the antibodies or antigen-binding fragments thereof of the present application, when administered (e.g., subcutaneously injected), have a clearance rate 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.

[0019] In some embodiments, the antibodies or antigen-binding fragments thereof of the present application have good physicochemical properties, e.g., have good solubility or stability, e.g., have good colloidal stability and / or good thermal stability.

[0020] In some embodiments, the antibody or antigen-binding fragment thereof of the application is effective in preventing or treating inflammation, e.g., skin inflammation or small intestine inflammation. In some embodiments, the antibody or antigen-binding fragment thereof of the application is effective in preventing or treating an autoimmune disease, e.g., graft versus host disease, e.g., graft versus host disease with enhanced IL-15 signaling.

[0021] Accordingly, the present application relates to the following specific aspects.

[0022] 1. An anti-IL-15 antibody or antigen-binding fragment thereof, comprising:

[0023] HCDR1, HCDR2, and HCDR3 as comprised by a VH as set forth in SEQ ID NO: 15, 16, or 17, and LCDR1, LCDR2, and LCDR3 as comprised by a VL as set forth in SEQ ID NO: 19, 20, or 21;

[0024] HCDR1, HCDR2, and HCDR3 as comprised by a VH as set forth in SEQ ID NO: 15 or 16, and LCDR1, LCDR2, and LCDR3 as comprised by a VL as set forth in SEQ ID NO: 19 or 20;

[0025] HCDR1, HCDR2, and HCDR3 as comprised by a VH as set forth in SEQ ID NO: 16 or 17, and LCDR1, LCDR2, and LCDR3 as comprised by a VL as set forth in SEQ ID NO: 20 or 21;

[0026] HCDR1, HCDR2, and HCDR3 as comprised by a VH as set forth in SEQ ID NO: 15, and LCDR1, LCDR2, and LCDR3 as comprised by a VL as set forth in SEQ ID NO: 19;

[0027] HCDR1, HCDR2, and HCDR3 as comprised by a VH as set forth in SEQ ID NO: 16, and LCDR1, LCDR2, and LCDR3 as comprised by a VL as set forth in SEQ ID NO: 20;

[0028] HCDR1, HCDR2, and HCDR3 as comprised by a VH as set forth in SEQ ID NO: 17, and LCDR1, LCDR2, and LCDR3 as comprised by a VL as set forth in SEQ ID NO: 21; or

[0029] HCDR1, HCDR2, and HCDR3 contained in a VH as set forth in SEQ ID NO: 18, and LCDR1, LCDR2, and LCDR3 contained in a VL as set forth in SEQ ID NO: 22.

[0030] 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

[0031] the HCDR1, HCDR2, HCDR3, and LCDR1, LCDR2, and LCDR3 comprise or consist of the amino acid sequences set forth 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, respectively; or

[0032] the HCDR1, HCDR2, HCDR3, and LCDR1, LCDR2, and LCDR3 comprise or consist of the amino acid sequences set forth 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, respectively; or

[0033] the HCDR1, HCDR2, HCDR3, and LCDR1, LCDR2, and LCDR3 comprise or consist of the amino acid sequences set forth 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, respectively; or

[0034] the HCDR1, HCDR2, HCDR3, and LCDR1, LCDR2, and LCDR3, respectively, comprise or consist of the amino acid sequences set forth 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.

[0035] 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.

[0036] 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 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: 19-22, or comprises or consists of an amino acid sequence selected from any one of SEQ ID NOs: 19-22.

[0037] 5. An anti-IL-15 antibody or antigen-binding fragment thereof, comprising

[0038] (i) a VH comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 15, 16, or 17, or an amino acid sequence that is 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: 19, 20, or 21, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto;

[0039] (ii) a VH comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 15 or 16 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and a VL comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 19 or 20 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto;

[0040] (iii) a VH comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 16 or 17 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and a VL comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 20 or 21 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto;

[0041] (iv) a VH comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 15 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and a VL comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 19 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto;

[0042] (v) a VH comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 16 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and a VL comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 20 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto;

[0043] (vi) a VH comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 17, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and a VL comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 21, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; or

[0044] (vii) a VH comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 18, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and a VL comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 22, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto

[0045] 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

[0046] a) the heavy chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 15, 16, or 17, and the light chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 19, 20, or 21;

[0047] b) the heavy chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 15 or 16, and the light chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 19 or 20;

[0048] c) the heavy chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 16 or 17, and the light chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 20 or 21;

[0049] d) the heavy chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 15, and the light chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 19;

[0050] e) the heavy chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 16 and the light chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 20;

[0051] f) the heavy chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 17 and the light chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21 ; or

[0052] g) the heavy chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 18 and the light chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22.

[0053] 7. The anti-IL-15 antibody or antigen-binding fragment thereof of any one of embodiments 1-6, which comprises a Fc region, e.g., the Fc region is from an IgGl, IgG2, IgG3, or IgG4 Fc region, e.g., a human IgGl, IgG2, IgG3, or IgG4 Fc region, e.g., the IgGl Fc region

[0054] (i) comprises or consists of the amino acid sequence of SEQ ID NO: 41 ; or

[0055] (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.

[0056] 8. The anti-IL-15 antibody or antigen-binding fragment thereof of any one of embodiments 1-7, which comprises a heavy chain constant region, e.g., the heavy chain constant region is from an IgGl, IgG2, IgG3, or IgG4 constant region, e.g., a human IgGl, IgG2, IgG3, or IgG4 constant region, e.g., the IgGl heavy chain constant region

[0057] (i) comprises or consists of the amino acid sequence of SEQ ID NO: 25; or

[0058] (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: 25.

[0059] 9. The anti-IL-15 antibody or antigen-binding fragment thereof of embodiments 7 or 8, wherein the heavy chain constant region or Fc region comprises a mutation that reduces binding to Fcy receptors, e.g., a L234A / L235A mutation, e.g., a L234A / L235A mutation, e.g., the heavy chain constant region

[0060] (i) comprises or consists of the amino acid sequence of SEQ ID NO: 26; or

[0061] (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: 26.

[0062] 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 increases binding to FcRn receptors, e.g., a YTE mutation (M252Y / S254T / T256E).

[0063] 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 a mutation that reduces binding to Fcy receptors and a mutation that increases binding to FcRn receptors, e.g., comprises a L234A / L235A mutation and a YTE mutation, e.g., the heavy chain constant region

[0064] the Fc region

[0065] (i) comprises or consists of the amino acid sequence of SEQ ID NO: 27; or

[0066] (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

[0067] the Fc region

[0068] (i) comprises or consists of the amino acid sequence of SEQ ID NO: 42; or

[0069] (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: 42.

[0070] 12. The anti-IL-15 antibody or antigen-binding fragment thereof of any one of embodiments 1-11, comprising a light chain constant region, which is a lambda or Kappa light chain constant region, e.g., a human lambda or Kappa light chain constant region, preferably, the light chain constant region

[0071] (i) comprises or consists 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

[0072] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 28.

[0073] 13. 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;

[0074] Optionally, the heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23 or an amino acid sequence that is 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 set forth in SEQ ID NO: 24 or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto;

[0075] Optionally, the heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23 and the light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 24.

[0076] 14. The anti-IL-15 antibody or antigen-binding fragment thereof of any one of embodiments 1-13, wherein the antibody is a monoclonal antibody.

[0077] 15. 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.

[0078] 16. 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, bibody, or linear antibody.

[0079] 17. 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:

[0080] a) binds to a human or cynomolgus IL-15 monomer or a complex thereof with its receptor, such as an IL-15 / IL-15Ra complex, with high affinity, wherein the amino acid at position 105 of IL-15 in the monomer or complex is H;

[0081] b) effectively blocks downstream signaling pathways stimulated by IL-15 monomer or a complex thereof with its receptor, such as an IL-15 / IL-15Ra complex;

[0082] c) inhibits IL-15 or a complex thereof with a receptor, such as IL-15 / IL-15Ra, mediated CD8 T cell activation and / or IFNg cytokine secretion, e.g., in CD8 T cells;

[0083] d) effectively blocks IL-15 monomer or a complex thereof with its receptor, such as an IL-15 / IL-15Ra complex, mediated T cell proliferation, T cell infiltration, and / or T cell activation (e.g., decreased NKG2D expression level or CD69 expression level);

[0084] e) has good physicochemical properties and / or pharmacokinetic characteristics, e.g., good stability (e.g., colloidal stability and / or thermal stability), good solubility, long half-life, and / or low clearance;

[0085] f) is effective in preventing or treating inflammation, e.g., skin inflammation or small intestine inflammation;

[0086] g) is capable of effectively preventing or treating an autoimmune disease, e.g., graft versus host disease, e.g., effectively blocking IL-15 signaling enhanced graft versus host disease.

[0087] 18. An isolated nucleic acid encoding the anti-IL-15 antibody or antigen-binding fragment thereof of any one of embodiments 1-17.

[0088] 19. A vector comprising the nucleic acid of embodiment 18, preferably the vector is an expression vector.

[0089] 20. 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 a yeast cell, a mammalian cell (e.g. a 293 cell or a CHO cell, e.g. a CHO-K cell or a HEK293 cell) or other cell suitable for the production of an antibody or antigen binding fragment thereof.

[0090] 21. A method of producing an anti-IL-15 antibody or antigen binding fragment thereof, the method comprising

[0091] a) culturing the host cell of embodiment 20 under conditions suitable for the expression of the nucleic acid encoding the anti-IL-15 antibody or antigen binding fragment thereof of any one of embodiments 1-17,

[0092] b) optionally isolating the antibody or antigen binding fragment thereof,

[0093] 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, e.g. by Protein A purification.

[0094] 22. An immunoconjugate comprising the anti-IL-15 antibody or antigen binding fragment thereof of any one of embodiments 1-17 and another agent, e.g. a toxin, a small molecule drug, a cytotoxic agent, an apoptotic agent, a chelating agent, an immunomodulatory agent, e.g. an anti-inflammatory agent or an immunosuppressive agent.

[0095] 23. A pharmaceutical composition 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 optionally a pharmaceutical excipient.

[0096] 24. A pharmaceutical combination product 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 agent is selected from a cytokine, another antibody, a small molecule drug or an immunomodulatory agent, e.g. an anti-inflammatory agent or an immunosuppressive agent.

[0097] 25. A method of preventing or treating an IL-15 related disease and / or disorder 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.

[0098] 26. The method of embodiment 25, wherein the subject has a disease or condition associated with abnormal expression of IL-15 or its receptor complex, e.g., IL-15 / IL-15Ra complex, or abnormal activation of the signaling pathway mediated thereby, as compared to a healthy individual.

[0099] 27. The method of embodiment 25 or 26, wherein the disease or condition is selected from a tumor, e.g., a cancer, or an inflammation or inflammatory condition, e.g., an autoinflammatory disease, e.g., skin inflammation or small intestine inflammation; or an autoimmune disease, e.g., graft versus host disease.

[0100] 28. The method of any one of embodiments 25-27, wherein the method further comprises administering one or more other therapies, e.g., a therapeutic modality and / or other therapeutic agent, e.g., the therapeutic agent is selected from a cytokine, another antibody, a small molecule drug, or an immunomodulatory agent, e.g., an anti-inflammatory agent or an immunosuppressive agent. BRIEF DESCRIPTION OF DRAWINGS

[0101] Figure 1 shows the inhibition of IL-15 reporter cell line signaling by anti-IL-15 chimeric antibodies and control antibodies.

[0102] Figure 2 shows the inhibition of IL-15 reporter cell line signaling by anti-IL-15 antibodies.

[0103] Figure 3 shows the inhibition of IL-15 reporter cell line signaling by anti-IL-15 antibodies.

[0104] Figure 4 shows the inhibition of IL-15-induced immune cell proliferation by anti-IL-15 antibodies.

[0105] Figure 5 shows the inhibition of IL-15-induced immune cell proliferation by anti-IL-15 antibodies.

[0106] Figure 6 shows the inhibition of IL-15-induced immune cell proliferation by anti-IL-15 antibodies.

[0107] Figure 7 shows the inhibition of IL-15-induced CD8 T cell activation by anti-IL-15 antibodies.

[0108] Figure 8 shows the inhibition of IL-15-induced CD8 T cell activation by anti-IL-15 antibodies.

[0109] Figure 9 shows the inhibition of IL-15-induced skin T cell proliferation by anti-IL-15 antibodies.

[0110] Figure 10 shows the inhibition of IL-15-induced skin T cell activation by anti-IL-15 antibodies.

[0111] Figure 11 shows the inhibition of IL-15-induced small intestine CD8 T cell activation by anti-IL-15 antibodies.

[0112] Figure 12 shows the inhibition of IL-15 induced splenocyte CD8 T cell activation by anti-IL-15 antibodies.

[0113] Figure 13 shows the inhibition of IL-15 enhanced GvHD by anti-IL-15 antibodies.

[0114] Figure 14 shows the inhibition of IL-15 enhanced GvHD by anti-IL-15 antibodies.

[0115] Figure 15 shows the pharmacokinetic (PK) studies of antibodies in BALB / c mice.

[0116] Figure 16 shows the inhibition of IL-15 reporter cell line signaling by anti-IL-15 antibodies.

[0117] Figure 17 shows the inhibition of IL-15 reporter cell line signaling by anti-IL-15 antibodies.

[0118] Figure 18 shows the inhibition of IL-15 induced immune cell proliferation by anti-IL-15 antibodies.

[0119] Figure 19 shows the inhibition of IL-15 induced immune cell proliferation by anti-IL-15 antibodies.

[0120] Figure 20 shows the pharmacokinetic (PK) studies of antibodies in BALB / c mice.

[0121] DETAILED DESCRIPTION

[0122] Before the present application is described in detail, it is to be understood that this application is not limited to the particular methodology, protocols and reagents described herein as these can vary.

[0123] I. DEFINITIONS

[0124] 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 since the scope of the present application will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0125] For the purposes of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will include the plural and vice versa. It is further to be understood that all terminology used herein is for the purpose of describing only the particular embodiments of the application and is not intended to limit the scope of the application.

[0126] The term "about," when used in connection with a numerical value, means a range of numerical values that includes a lower limit of 5% (e.g., 4%, 3%, 2%, or 1%) less than the specified numerical value and an upper limit of 5% (e.g., 4%, 3%, 2%, or 1%) greater than the specified numerical value.

[0127] As used herein, the term "and / or" means any one of the items, or any two or more of the items, or all of the items.

[0128] As used herein, the term "comprising" or "including," means including, but not limited to, the recited items, integers, or steps, but does not mean that any other non-recited items, integers or steps are excluded. When the term "comprising" or "including" is used in this document, unless otherwise indicated, it is also contemplated that the recited items, integers or steps can also consist of the recited items, integers or steps. For example, when referring to an antibody variable region "comprising" a particular sequence, it is also intended to encompass an antibody variable region consisting of the particular sequence.

[0129] An "isolated" antibody or molecule is one which has been separated from a component of its natural environment. In some embodiments, an antibody or molecule is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC).

[0130] As used herein, "IL-15" refers to any native IL-15 polypeptide (e.g., a human IL-15 polypeptide) or variant thereof. The term "IL-15" encompasses "full-length," unprocessed IL-15 polypeptides as well as any form of IL-15 polypeptide produced by processing within the 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 a human or from another source, e.g., cynomolgus monkey, or prepared by recombinant or synthetic methods. In one embodiment of the application, a human IL-15 protein comprises or consists of the amino acid sequence set forth in SEQ ID NO: 39. In one embodiment of the application, a cynomolgus monkey IL-15 protein comprises or consists of the amino acid sequence set forth in SEQ ID NO: 40.

[0131] The terms "whole antibody" or "full-length antibody" are used interchangeably herein to refer to an antibody molecule having a native immunoglobulin molecule structure. In the case of a conventional four-chain IgG antibody, the full-length antibody comprises two heavy chains (H) and two light chains (L) which are interconnected by disulfide bonds. In the case of a heavy chain antibody which has only heavy chains but lacks light chains, the full-length antibody comprises two heavy chains (H) which are interconnected by disulfide bonds. For a conventional four-chain IgG antibody, the full-length antibody heavy chain is typically 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 comprises one domain, CL. Each heavy chain variable region VH and each light chain variable region VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: 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.

[0132] The term "antigen-binding fragment" of an antibody is a molecule other than a full-length antibody that comprises a portion of a full-length antibody that is capable of binding to an antigen bound by the full-length antibody or competes with the full-length antibody (i.e., with the full-length antibody from which the antigen-binding fragment is derived) for binding to the antigen. Antigen-binding fragments can be produced by recombinant DNA techniques, 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, bibodies or fragments thereof, or camelid antibodies, diabodies, single-domain antibodies (sdAb), nanobodies. For example, Fab fragments can be obtained by papain digestion of a full-length antibody. In addition, pepsin digestion of a full-length antibody under denaturing conditions generates F(ab')2, which is a dimer of two Fab' fragments linked by disulfide bonds. F(ab')2 can be reduced under non-denaturing conditions to break disulfide linkages for conversion to Fab' monomers. Fab' monomers are essentially Fab fragments with the additional hinge region. Fv fragments are composed of the VL and VH domains of a single arm of an antibody. The two domains of the Fv fragment, VL and VH, can be coded for by separate genes, but can also be produced using recombinant methods using a synthetic linker to connect the two domains so that they are produced as a single protein chain, and in which the VL and VH regions pair to form a single chain Fv (scFv) in the single protein chain.

[0133] The term "single chain antibody (scAb)" is used herein in the broadest sense, and specifically covers antibodies with mono- or multi- specificity (e.g., bispecificity) that are produced initially as a single, continuous polypeptide chain. Such single chain antibodies include, but are not limited to, those having two linked VL and VH regions. In one embodiment, the single chain antibody is a scFv.

[0134] A "diabody" is a bivalent small antibody constructed by gene fusion, e.g., it is a dimer of two polypeptide chains. The VL and VH domains of each polypeptide chain of a diabody are joined by a linker, such that the VL and VH encoded in the same polypeptide chain form a dimer with different single chain variable fragments. Diabodies generally have two antigen binding sites.

[0135] A "complementarity determining region" or "CDR region" or "CDR" is a region in an antibody variable domain that is hypervariable in sequence and forms structurally defined loops ("hypervariable loops") and / or contains antigen contact residues ("antigen contacts"). The CDRs are primarily responsible for binding to an epitope of an antigen. The CDRs of the heavy and light chains are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially from the N-terminus. CDRs located within the variable domain of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3, while CDRs located within the variable domain of the light chain are referred to as LCDR1, LCDR2, and LCDR3. The precise amino acid sequence boundaries of each CDR in a given light chain variable region or heavy chain variable region amino acid sequence can be determined using any of a number of well-known antibody CDR assignment schemes, or combinations thereof, including, for example: Chothia based on the three-dimensional structure of the antibody 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 Ed. U.S. Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), the international ImMunoGene Tics database (IMGT) (on the World Wide Web at imgt.cines.fr / ), and the North CDR definition based on affinity propagation clustering with a large number of crystal structures.

[0136] The following are exemplary schemes for the range of regions of CDRs defined using the kabat, Chothia, Kabat & Chothia (combination Kabat and Chothia scheme determined) schemes.

[0137] Unless otherwise indicated, in the application, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the manners described above. 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 application.

[0138] Unless otherwise indicated, in the application, when referring to residue positions in an antibody variable region, including heavy chain variable region residues and light chain variable region residues, the numbering of the positions is 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)).

[0139] In some embodiments, CDRl of the heavy chain variable region of an antibody in the application is determined according to the Kabat & Chothia scheme, CDR2 and CDR3 are determined according to the Kabat scheme. In some embodiments, CDRs of the light chain variable region of an antibody in the application are determined according to the Kabat scheme. In some embodiments, CDRs of the heavy chain variable region of an antibody in the application are determined according to the Kabat scheme.

[0140] HCDRl is determined according to the Kabat & Chothia scheme, HCDR2 and HCDR3 are determined according to the Kabat scheme, respectively;

[0141] LCDRl, LCDR2 and LCDR3 are determined according to the Kabat scheme, respectively.

[0142] An antibody that "binds to the same or overlapping epitope" as a reference antibody refers to 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, and vice versa.

[0143] An antibody that "competes for binding to its antigen" with a reference antibody refers to 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. And vice versa, 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 radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay.

[0144] An antibody that inhibits (e.g., competitively inhibits) the binding of a reference antibody to its antigen refers to an antibody that inhibits 50%, 60%, 70%, 80%, 90%, or 95% or more of the binding of the reference antibody to its antigen. Conversely, a reference antibody inhibits 50%, 60%, 70%, 80%, 90%, or 95% or more of the binding of the antibody to its antigen. Binding of an antibody to its antigen can be measured in terms of affinity (e.g., equilibrium dissociation constant). Methods of determining affinity are known in the art.

[0145] An antibody that exhibits the same or similar binding affinity and / or specificity as a reference antibody refers to an antibody that can have at least 50%, 60%, 70%, 80%, 90%, or 95% or more 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.

[0146] The term "chimeric antibody" is an antibody molecule in which (a) the constant region or portions thereof is altered, replaced or exchanged to create a different or altered class, effector function and / or species of constant region, or to confer new properties (e.g., enzymatic, toxic, hormonal, growth factor, drug, etc.) to the chimeric antibody, or (b) the variable region or portions thereof are altered, replaced or exchanged to create a different or altered antigen specificity. For example, a mouse antibody can be modified by replacing its constant region with a constant region from a human immunoglobulin. The chimeric antibody can retain its specificity in recognizing the antigen, while having reduced immunogenicity in humans as compared to the original mouse antibody, due to the replacement with human constant regions.

[0147] 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, e.g., when administered to humans as a therapeutic. This can be achieved, for example, by retaining the non-human antigen binding site and replacing the remainder of the antibody with their human counterparts (i.e., replacing the constant regions as well as the portions of the variable regions not involved in binding to the corresponding portions of a human antibody).

[0148] The term "Fc domain" or "Fc region" is used herein to define a 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. Native immunoglobulin "Fc domains" comprise two or three constant domains, i.e., CH2 domains, CH3 domains, and optionally, CH4 domains. For example, in native antibodies, an immunoglobulin Fc domain comprises the second and third constant domains (CH2 and CH3 domains) of a heavy chain derived from an IgG, IgA, and IgD class antibody; or the second, third and fourth constant domains (CH2, CH3 and CH4 domains) of a heavy chain derived from an IgM and IgE class antibody. Unless otherwise specified herein, amino acid residue positions in an Fc region or heavy chain constant region are numbered according to the EU numbering system (also referred to as EU index) as set forth in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0149] "Conservative modifications" as described herein include substitutions, deletions or additions to a polypeptide sequence, but do not substantially alter the functional activity of the polypeptide sequence. In some embodiments, a conservative modification is a conservative substitution. Conservative substitutions refer to the substitution of one amino acid for another within the same class, e.g., one acidic amino acid for another acidic amino acid, one basic amino acid for another basic amino acid, or one neutral amino acid for another neutral amino acid. For example, conservative substitutions often result in the substitution of one amino acid for another that is chemically similar. Tables of conservative substitutions are well known in the art. The following eight groups contain amino acids that are conservative substitutions for one another: 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 modifications" when applied to amino acid sequences of antibody molecules is used to refer to amino acid modifications that do not significantly affect or alter the desired antigen binding characteristics of the antibody molecules of the application containing the amino acid sequence. For example, a conservatively modified variant retains at least 80%, 85%, 90%, 95%, 98%, 99% or more, e.g., 100-110% or more, of the binding affinity of the parent antibody to the antigen of interest.

[0150] 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 the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has 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."

[0151] An "immunoconjugate" is an antibody conjugated to one or more other substances, including but not limited to a label. In some embodiments, immunoconjugates encompass antibody-drug conjugates (ADCs).

[0152] The term "therapeutic agent" as described herein encompasses any agent effective in preventing or treating a disease associated with inappropriate activation of pathways mediated by IL-15 or IL-15 receptor complex, including cytokines, other antibodies, small molecule drugs, or immunomodulatory agents (e.g., anti-inflammatory agents or immunosuppressive agents).

[0153] The term "small molecule drug" refers to an organic compound of low molecular weight that is capable of modulating a biological process. A "small molecule" is defined as a molecule having a molecular weight of less than 10 kD, typically less than 2 kD, and preferably less than 1 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 mimetics. As therapeutic agents, small molecules can be more able to penetrate cells, less susceptible to degradation, and less likely to elicit an immune response than macromolecules.

[0154] The term "immunomodulatory agent" as used herein refers to a natural or synthetic active agent or drug that suppresses or modulates an immune response. The immune response can be a humoral response or a cellular response. Immunomodulatory agents include anti-inflammatory agents or immunosuppressive agents.

[0155] "Immunosuppressive agent," "immunosuppressive drug," or "immunosuppressive agent" as used herein is a therapeutic agent used in immunosuppressive therapy to suppress or prevent the activity of the immune system.

[0156] The term "effective amount" refers to the amount or dose of an antibody or fragment or conjugate or composition or combination of the application, which, upon single or multiple dose administration to a patient, exerts a desired effect in the patient being treated or prevented.

[0157] A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody fragment or conjugates or compositions or combinations thereof are outweighed by the therapeutically beneficial effects. A "therapeutically effective amount" preferably inhibits a measurable parameter (e.g., modulation of IL-15 signaling) by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 50%, 60%, or 70% relative to an untreated subject. The term "therapeutically effective amount" as used herein in some embodiments is intended to qualify the amount of treatment necessary to prevent a condition (e.g., inflammation) or to reduce or eliminate an immune response in a treatment regimen.

[0158] A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Generally, since a prophylactic dose is used in subjects prior to or at the earlier stages of disease, the prophylactically effective amount will be less than the therapeutically effective amount. In some embodiments, the term "prophylactically effective amount" as used herein is intended to qualify the amount of prophylaxis necessary to prevent the progression and symptoms of a condition (e.g., inflammation) in a treatment regimen,

[0159] The terms "host cell", "host cell line", and "host cell culture" are used interchangeably and refer to cells in which an exogenous nucleic acid has been introduced, including the progeny of the cell. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages. Progeny can not be completely identical to the parent cell both in nucleic acid content and in physical identity, but rather can contain mutations. Mutant progeny that have the same function or biological activity as screened or selected in the originally transformed cell are included herein.

[0160] The term "label" as used herein refers to a compound or composition that is conjugated or fused directly or indirectly to a reagent, such as a polynucleotide probe or antibody, and that facilitates 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, in the case of an enzymatic label, can catalyze chemical changes to a detectable substrate compound or composition. The term is intended to encompass direct labeling of a probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody and indirect labeling by conjugating the probe or antibody to a secondary reagent which itself is labeled with a detectable substance.

[0161] The terms "individual," "subject," or "subject" are used interchangeably herein and include mammals. Mammals include, but are not limited to, domesticated animals (e.g., cows, 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.

[0162] The term "subject / patient / individual sample" as used herein refers to a collection of cells or fluids obtained from a patient or subject. The source of the tissue or cell sample can be a solid tissue, such as from a fresh, frozen and / or preserved organ or tissue sample or biopsy sample or a puncture sample; blood or any blood component; a body fluid, such as cerebrospinal fluid, amniotic fluid (amniotic water), peritoneal fluid (ascites), or interstitial fluid; a cell from a subject at any time of gestation or development. The tissue sample can contain compounds not naturally admixed with the tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and the like.

[0163] "nucleic acid encoding an anti-IL-15 antibody or fragment thereof" means one or more nucleic acid molecules that encode an antibody heavy chain or light chain (or fragment thereof, e.g., a heavy chain variable region or a light chain variable region), including such nucleic acid molecule(s) in a single vector or separate vectors, and such nucleic acid molecule(s) present at one or more locations within a host cell.

[0164] "Percent (%) identity" of an amino acid sequence refers to the percentage of amino acid residues in a candidate sequence that are identical with a specific amino acid sequence as shown in the specification, after aligning the candidate sequence with the specific amino acid sequence and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative changes as part of the sequence identity. In some embodiments, the present application contemplates variants of the antibody molecules of the present application that have a substantial degree of identity, e.g., at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% or more, to the antibody molecules specifically disclosed herein and their sequences. The variants can include conservative changes.

[0165] The term "pharmaceutically acceptable excipient" refers to a diluent, adjuvant (e.g., Freund's adjuvant (complete and incomplete)), excipient, carrier or stabilizer, etc., that is administered with the active ingredient.

[0166] The term "pharmaceutical composition" refers to a composition that is in a form suitable for its administration to a subject or individual and that does not contain additional ingredients that are unacceptable with respect to toxicity to the subject or individual to which the composition is administered.

[0167] As used herein, the term "pharmaceutical combination or combination product" refers to non-fixed combinations or fixed combinations, including but not limited to a kit, a pharmaceutical composition. The term "non-fixed combination" means that the active ingredients (e.g., (i) an antibody of the present application, and (ii) other therapeutic agents) are presented in a separate entity but with instructions to use, simultaneously, sequentially or separately for use in the combination according to the invention. The term "fixed combination" means that the active ingredients (e.g., (i) an antibody of the present application, and (ii) other therapeutic agents) are presented together in a single entity. Preferably, the dosages of the two or more active agents and / or the time interval of administration are selected so that a synergistic effect is achieved in treating the disease or condition being treated. The individual components can each be present in a separate formulation, which formulations can be the same or different.

[0168] The term "combination therapy" refers to the administration of two or more therapeutic agents, or treatment modalities (e.g., radiotherapy or surgery), to treat a disease described herein. Such administration includes co-administration of these therapeutic agents in a substantially simultaneous manner, such as in a single capsule having a fixed ratio of active ingredients. Alternatively, such administration

[0169] As used herein, "treatment" refers to slowing, interrupting, arresting, remitting, stopping, reducing, or reversing the progression of, alleviating a symptom of, or preventing or inhibiting further development of a disease, condition, or disorder.

[0170] As used herein, "prevention" includes inhibition of the onset or progression of a disease or disorder, or a symptom of a particular disease or disorder.

[0171] II antibody

[0172] In some embodiments, the anti-IL-15 antibody or antigen-binding fragment thereof of the present application comprises 3 complementarity determining regions from a heavy chain variable region (HCDR), HCDR1, HCDR2, and HCDR3.

[0173] In some embodiments, the anti-IL-15 antibody or antigen-binding fragment thereof of the present application comprises 3 complementarity determining regions from a light chain variable region (LCDR), LCDR1, LCDR2, and LCDR3.

[0174] In some embodiments, the anti-IL-15 antibody or antigen-binding fragment thereof of the present application comprises 3 complementarity determining regions from a heavy chain variable region (HCDR) and 3 complementarity determining regions from a light chain variable region (LCDR).

[0175] In some aspects, the anti-IL-15 antibody or antigen-binding fragment thereof of the present application comprises a heavy chain variable region (VH). In some aspects, the anti-IL-15 antibody or antigen-binding fragment thereof of the present application comprises a light chain variable region (VH). In some aspects, the anti-IL-15 antibody or antigen-binding fragment thereof of the present application comprises a heavy chain variable region and a light chain variable region (VH). In some embodiments, the heavy chain variable region comprises 3 complementarity determining regions from a heavy chain variable region (CDR), HCDR1, HCDR2 and HCDR3. In some embodiments, the light chain variable region comprises 3 complementarity determining regions from a light chain variable region (CDR), LCDR1, LCDR2 and LCDR3.

[0176] In some embodiments, the anti-IL-15 antibody or antigen-binding fragment thereof of the present application further comprises an antibody heavy chain constant region. In some embodiments, the anti-IL-15 antibody or antigen-binding fragment thereof of the present application further comprises an antibody light chain constant region. In some embodiments, the anti-IL-15 antibody or antigen-binding fragment thereof of the present application further comprises a heavy chain constant region and a light chain constant region.

[0177] In some embodiments, the heavy chain variable region of the present application:

[0178] (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 the amino acid sequence set forth in any one of SEQ ID NOs: 15-18; or

[0179] (ii) comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOs: 15-18; or

[0180] (iii) comprises or consists of an amino acid sequence that has 1 or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid alterations (preferably amino acid substitutions, more preferably amino acid conservative substitutions) compared to the amino acid sequence set forth in any one of SEQ ID NOs: 15-18, preferably the amino acid alterations do not occur in the CDR regions.

[0181] In some embodiments, the light chain variable region of the present application:

[0182] (i) comprises or consists of an amino acid sequence which has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to an amino acid sequence selected from the group consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 19-22; or

[0183] (ii) comprises or consists of an amino acid sequence selected from the group consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 19-22; or

[0184] (iii) comprises or consists of an amino acid sequence which has 1 or more, preferably not more than 10, more preferably not more than 5, 4, 3, 2, 1, amino acid alterations, preferably amino acid substitutions, more preferably amino acid conservative substitutions, compared to an amino acid sequence selected from the group consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 19-22, preferably the amino acid alterations do not occur in the CDR regions.

[0185] In some embodiments, the 3 complementarity determining regions from the heavy chain variable region (HCDRs), HCDR1, HCDR2 and HCDR3, according to the present application are selected from the group consisting of

[0186] (i) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in a VH as set forth in any one of SEQ ID NOs: 15-18;

[0187] (ii) a sequence which comprises collectively at least one and not more than 5, 4, 3, 2, or 1 amino acid alterations, preferably amino acid substitutions, preferably conservative substitutions, in the three HCDR regions, relative to the sequence of any one of (i),

[0188] wherein the HCDRs can be determined according to any CDR-determining scheme, respectively, for example according to the kabat, AbM, Chothia, Contact or IMGT scheme, respectively, or a combination thereof;

[0189] For example, the HCDR1 is determined according to the Kabat & Chothia scheme, and the HCDR2 and HCDR3 are determined according to the Kabat scheme, respectively.

[0190] In some embodiments, the 3 complementarity determining regions from the light chain variable region (LCDRs), LCDR1, LCDR2 and LCDR3, according to the present application are selected from the group consisting of

[0191] (i) three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in a VL as set forth in any one of SEQ ID NOs: 19-22, or

[0192] (ii) a sequence comprising collectively at least one and not more than 5, 4, 3, 2, or 1 amino acid alteration(s) (preferably amino acid substitution(s), preferably conservative substitution(s)) relative to the sequence of any of (i) in the three LCDR regions,

[0193] wherein the HCDRs can be determined according to any of the schemes to determine CDRs, respectively, e.g., according to the kabat, AbM, Chothia, Contact, or IMGT scheme, respectively, or a combination thereof;

[0194] For example, the LCDR1, 2 and 3 are determined according to the Kabat scheme, respectively.

[0195] In some embodiments, the HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 1, 2 or 3, or an amino acid sequence having one, two or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 1, 2 or 3.

[0196] In some embodiments, the HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 4, 5 or 6, or an amino acid sequence having one, two or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 4, 5 or 6.

[0197] In some embodiments, the HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 7, 8 or 9, or an amino acid sequence having one, two or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 7, 8 or 9.

[0198] In some embodiments, the LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 10, 1 1 or 12, or an amino acid sequence having one, two or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 10, 1 1 or 12.

[0199] In some embodiments, the LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 13, or an amino acid sequence having one, two or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 13.

[0200] In some embodiments, the LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 14 or 43, or an amino acid sequence having one, two or three alterations, preferably amino acid substitutions, preferably conservative substitutions, compared to the amino acid sequence of SEQ ID NO: 14 or 43.

[0201] In some embodiments, the antibody heavy chain constant region according to the application is a heavy chain constant region from IgGl, IgG2, IgG3 or IgG4, e.g. a constant region from human IgGl, IgG2, IgG3 or IgG4, e.g. is a constant region of human IgGl, IgG2, IgG3 or IgG4. In some embodiments, the constant region has a modification in the Fc region thereof. In some embodiments, the Fc region is a Fc region from IgGl, IgG2, IgG3 or IgG4, e.g. a Fc region from human IgGl, IgG2, IgG3 or IgG4, e.g. is a Fc region of human IgGl, IgG2, IgG3 or IgG4.

[0202] In one embodiment, the Fc region is modified in a property of an effector function of the Fc region, e.g. a complement-activating function of the Fc region. In one embodiment, the effector function has been reduced or eliminated relative to a wild-type isotype Fc region. In one embodiment, the effector function is reduced or eliminated by a method selected from the group consisting of: use of an Fc isotype that naturally has reduced or eliminated effector function, and Fc region modification. The Fc region can also comprise a modification that alters binding affinity to one or more Fc receptors.

[0203] In one embodiment, the Fc receptor is an Fc gamma receptor, in particular a human Fc gamma receptor. In some embodiments, the Fc region comprises a mutation that reduces binding to an Fc gamma receptor. In some embodiments, the Fc region for use in the application has a L234A / L235A mutation that reduces binding to an Fc gamma receptor. In some embodiments, the heavy chain constant region of the application comprises a mutation that reduces binding to an Fc7 receptor, e.g. a L234A / L235A mutation.

[0204] In some embodiments, the Fc receptor is an FcRn, in particular a human FcRn receptor. In some embodiments, the Fc region has a mutation that increases binding to an FcRn receptor. In some embodiments, the Fc region for use in the application has a YTE mutation (M252Y / S254T / T256E) that increases binding to an FcRn receptor. In some embodiments, the heavy chain constant region of the application comprises a mutation that increases binding to an FcRn receptor, e.g. a YTE mutation (M252Y / S254T / T256E).

[0205] In some embodiments, the Fc region or heavy chain constant region of the application comprises both a mutation that reduces binding to Fcy receptors and a mutation that increases binding to FcRn receptors, for example comprising L234A / L235A mutations and YTE mutations.

[0206] In some embodiments, the heavy chain constant region

[0207] (i) comprises or consists of an amino acid sequence selected from SEQ ID NO: 25, 26 or 27;

[0208] (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: 25, 26 or 27;

[0209] (iii) 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: 26 and has L234A / L235A mutations; or

[0210] (iv) 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 and has L234A / L235A mutations and YTE mutations.

[0211] In some embodiments, the heavy chain constant region of the application lacks the terminal lysine.

[0212] In some embodiments, the Fc region

[0213] (i) comprises or consists of an amino acid sequence selected from SEQ ID NO: 41 or 42;

[0214] (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

[0215] (iii) 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 and has L234A / L235A mutations and YTE mutations.

[0216] In some embodiments, the antibody light chain constant region of the present application is a light chain constant region from a lambda or Kappa light chain constant region, preferably from a Kappa light chain constant region, e.g., 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

[0217] (i) comprises or consists 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

[0218] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 28.

[0219] In some specific embodiments, the anti-IL-15 antibody of the present application comprises

[0220] HCDR1, HCDR2, and HCDR3 as comprised by a VH as set forth in SEQ ID NO: 15, 16, or 17, and LCDR1, LCDR2, and LCDR3 as comprised by a VL as set forth in SEQ ID NO: 19, 20, or 21;

[0221] HCDR1, HCDR2, and HCDR3 as comprised by a VH as set forth in SEQ ID NO: 15 or 16, and LCDR1, LCDR2, and LCDR3 as comprised by a VL as set forth in SEQ ID NO: 19 or 20;

[0222] HCDR1, HCDR2, and HCDR3 as comprised by a VH as set forth in SEQ ID NO: 16 or 17, and LCDR1, LCDR2, and LCDR3 as comprised by a VL as set forth in SEQ ID NO: 20 or 21;

[0223] HCDR1, HCDR2, and HCDR3 as comprised by a VH as set forth in SEQ ID NO: 15, and LCDR1, LCDR2, and LCDR3 as comprised by a VL as set forth in SEQ ID NO: 19;

[0224] HCDR1, HCDR2, and HCDR3 as comprised by a VH as set forth in SEQ ID NO: 16, and LCDR1, LCDR2, and LCDR3 as comprised by a VL as set forth in SEQ ID NO: 20;

[0225] HCDR1, HCDR2, and HCDR3 contained in a VH as set forth in SEQ ID NO: 17, and LCDR1, LCDR2, and LCDR3 contained in a VL as set forth in SEQ ID NO: 21; or

[0226] HCDR1, HCDR2, and HCDR3 contained in a VH as set forth in SEQ ID NO: 18, and LCDR1, LCDR2, and LCDR3 contained in a VL as set forth in SEQ ID NO: 22; or

[0227] wherein the HCDRs can each be determined according to any defined CDR scheme, e.g., according to the kabat, AbM, Chothia, Contact, or IMGT scheme, respectively, or a combination thereof.

[0228] 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.

[0229] In some specific embodiments, the anti-IL-15 antibody of the present application 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

[0230] the HCDR1, HCDR2, HCDR3, and LCDR1, LCDR2, and LCDR3 comprise or consist of the amino acid sequences set forth 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, respectively; or

[0231] the HCDR1, HCDR2, HCDR3, and LCDR1, LCDR2, and LCDR3 comprise or consist of the amino acid sequences set forth 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, respectively; or

[0232] the HCDR1, HCDR2, HCDR3, and LCDR1, LCDR2, and LCDR3 comprise or consist of the amino acid sequences set forth 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, respectively; or

[0233] the HCDR1, HCDR2, HCDR3, and LCDR1, LCDR2, and LCDR3 comprise or consist of the amino acid sequences set forth 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, respectively.

[0234] In some embodiments, the VH of the present application comprises a HCDRl, HCDR2, HCDR3, and the VL comprises a LCDR1, LCDR2, and LCDR3, wherein

[0235] the HCDR1, HCDR2, HCDR3, and LCDR1, LCDR2, and LCDR3 comprise or consist of the amino acid sequences set forth 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, respectively; or

[0236] the HCDR1, HCDR2, HCDR3, and LCDR1, LCDR2, and LCDR3 comprise or consist of the amino acid sequences set forth 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, respectively; or

[0237] the HCDR1, HCDR2, HCDR3, and LCDR1, LCDR2, and LCDR3 comprise or consist of the amino acid sequences set forth 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, respectively; or

[0238] the HCDR1, HCDR2, HCDR3, and LCDR1, LCDR2, and LCDR3 comprise or consist of the amino acid sequences set forth 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, respectively.

[0239] In some embodiments, the antibody or antigen-binding fragment thereof of the present application comprises:

[0240] (i) a VH comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 15, 16, or 17, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and / or a VL comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 19, 20, or 21, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto;

[0241] (ii) a VH comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 15 or 16, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and / or a VL comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 19 or 20, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto;

[0242] (iii) a VH comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 16 or 17, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and / or a VL comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 20 or 21, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto;

[0243] (iv) a VH comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 15, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and / or a VL comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 19, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto;

[0244] (v) a VH comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 16, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and / or a VL comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 20, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto;

[0245] (vi) a VH comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 17, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and / or a VL comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 21, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; or

[0246] (vii) a VH comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 18, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and / or a VL comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 22, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto

[0247] In some embodiments, the antibody or antigen-binding fragment thereof of the present application comprises a heavy chain variable region and a light chain variable region, wherein

[0248] (i) the heavy chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 15, 16, or 17, and the light chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 19, 20, or 21;

[0249] (ii) the heavy chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 15 or 16, and the light chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 19 or 20;

[0250] (iii) the heavy chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 16 or 17, and the light chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 20 or 21 ;

[0251] (iv) the heavy chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 15, and the light chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 19;

[0252] (v) the heavy chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 16, and the light chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 20;

[0253] (vi) the heavy chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 17, and the light chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21 ; or

[0254] (vii) the heavy chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 18, and the light chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22.

[0255] In some embodiments, the anti-IL-15 antibody or antigen-binding fragment thereof of the present application further comprises an antibody heavy chain. In some embodiments, the anti-IL-15 antibody or antigen-binding fragment thereof of the present application further comprises an antibody light chain. In some embodiments, the anti-IL-15 antibody or antigen-binding fragment thereof of the present application further comprises both the heavy chain and the light chain. In some embodiments, the antibody heavy chain of the present application comprises or consists of the heavy chain variable region of the present application and the heavy chain constant region of the present application. In some embodiments, the antibody light chain of the present application comprises or consists of the light chain variable region of the present application and the light chain constant region of the present application. In some embodiments, the antibody of the present application comprises or consists of two of the heavy chain and two of the light chain.

[0256] In some embodiments, the heavy chain of an antibody of the application comprises, consists of or consists essentially of the amino acid sequence set forth in 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 application comprises, consists of or consists essentially of the amino acid sequence set forth in 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.

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

[0258] In some embodiments, an antibody or antigen-binding fragment thereof of the application comprises a heavy chain and a light chain, wherein the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 23 and the light chain consists of the amino acid sequence set forth in SEQ ID NO: 24.

[0259] In one embodiment of the application, the amino acid changes described herein include substitutions, insertions, or deletions of amino acids. Preferably, the amino acid changes described herein are substitutions, preferably conservative substitutions. In preferred embodiments, the amino acid changes described in the application occur in regions outside the CDRs (e.g. in the FRs). More preferably, the amino acid changes described in the application occur in regions outside the heavy chain variable region and / or outside the light chain variable region.

[0260] In certain embodiments, the antibodies provided herein are altered to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites of an antibody can be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites is created or removed. When the antibody comprises an Fc region, carbohydrate attached to the Fc region can be altered. In some applications, it can be useful to remove a unwanted glycosylation site, for example to remove a fucosyl motif to increase antibody dependent cellular cytotoxicity (ADCC) function. In other applications, a galactosylation modification can be made to modify complement dependent cytotoxicity (CDC). In certain embodiments, it can be desirable to create cysteine engineered antibodies, e.g., "thioMAbs," in which one or more residues of an antibody are substituted with cysteine residues. In certain embodiments, the antibodies provided herein can be further modified to contain other nonproteinaceous moieties known in the art and readily available. Suitable modifications include, but are not limited to, amputation of the antibody. Non-limiting examples of water-soluble polymers suitable for amputation of the antibody include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymer, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-l,3-dioxane, poly-l,3,6-trioxane, ethylene / maleic anhydride copolymer, polyamino acids (homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.

[0261] In some embodiments, the anti-IL-15 antibodies or antigen-binding fragments thereof of the application have one or more of the following properties:

[0262] (i) exhibit the same or similar binding affinity and / or specificity for IL-15 as an antibody of the application (e.g., ch25B10, ch56G9, hz56G9.25L, or ch60G8);

[0263] (ii) inhibit (e.g., competitively inhibit) binding of an antibody of the application (e.g., ch25B10, ch56G9, hz56G9.25L, or ch60G8) to IL-15;

[0264] (iii) bind to the same or overlapping epitope as an antibody of the application (e.g., ch25B10, ch56G9, hz56G9.25L, or ch60G8);

[0265] (iv) competes with an antibody of the application (e.g., ch25B10, ch56G9, hz56G9.25L, or ch60G8) for binding to IL-15;

[0266] (v) one or more biological properties of an antibody of the application (e.g., ch25B10, ch56G9, hz56G9.25L, or ch60G8).

[0267] In some embodiments, the anti-IL-15 antibody of the application is an antibody of the IgGl form or an antibody of the IgG2 form or an antibody of the IgG3 form or an antibody of the IgG4 form, preferably an antibody of the IgGl form.

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

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

[0270] In some embodiments, the anti-IL-15 antibody is a chimeric antibody.

[0271] In some embodiments, the anti-IL-15 antibody of the application is a full-length antibody.

[0272] In some embodiments, the anti-IL-15 antibody of the application also encompasses a multispecific antibody or a bispecific antibody that specifically binds IL-15.

[0273] In one embodiment, the anti-IL-15 antibody of the application also encompasses antibody fragments (e.g., antigen binding fragments) thereof, preferably an antibody fragment selected from the group consisting of a Fab, a Fab', a Fab'-SH, a Fv, a single chain antibody (e.g., scFv), a (Fab')2, a single domain antibody such as a VHH, a dAb (domain antibody), a diabody, a dibody, or a linear antibody.

[0274] III. Nucleic acids of the application and host cells comprising the same

[0275] In one aspect, the application provides a nucleic acid encoding any one chain or any monomer or domain of an antibody or antigen binding fragment thereof of the application. The polynucleotide sequences encoding each chain can be generated using methods well known in the art. For example, the polypeptides encoded by the nucleic acids are capable of exhibiting the binding ability of human IL-15 (e.g., an IL-15 monomer or a complex thereof with a receptor) when expressed from a suitable expression vector. For example, in some embodiments, the nucleic acids encoding the variable regions of the heavy and / or light chains are operably linked in-frame to nucleic acids encoding the constant regions of the heavy and / or light chains, such that the nucleic acids encoding the heavy and / or light chains of the antibody are produced when expressed from a suitable expression vector.

[0276] In one aspect, the present application provides a nucleic acid encoding any of the anti-IL-15 antibodies or fragments thereof described herein. The nucleic acid can comprise a nucleic acid encoding the amino acid sequence of the variable region of the light chain and / or the variable region of the heavy chain of the antibody, or a nucleic acid encoding the amino acid sequence of the light chain and / or the heavy chain of the antibody.

[0277] For example, the nucleic acid of the present application comprises a nucleic acid encoding an amino acid sequence selected from the amino acid sequence set forth in any one of SEQ ID NOs: 15-24, or a nucleic acid 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 the amino acid sequence set forth in any one of SEQ ID NOs: 15-24. As will be apparent to those skilled in the art, because of the degeneracy of the code, each antibody or polypeptide amino acid sequence can be encoded by multiple nucleic acid sequences. Nucleic acid sequences encoding molecules of the present application can be produced using methods well known in the art, for example, by de novo solid phase DNA synthesis, or by PCR amplification. To facilitate production and purification, a secretory signal peptide at the N-terminus of the heavy chain and / or light chain of the antibody, and / or a tag peptide facilitating purification can be fused

[0278] The present application also provides a vector comprising a nucleic acid of the present application. In one embodiment, the vector is an expression vector, for example, a eukaryotic expression vector. Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phage, or yeast artificial chromosomes (YACs). In a preferred embodiment, the expression vector of the present application is a pcDNA vector, for example, a pcDNA3.1 and / or pcDNA3.4 expression vector.

[0279] In one embodiment, a host cell comprising the vector is provided. The present application also provides a host cell comprising the nucleic acid or the vector. Host cells suitable for replication and support of expression of antibodies of the present application are well known in the art. Such cells can be transfected or transduced with a particular expression vector, and large quantities of vector-containing cells can be grown for seeding large-scale fermenters to obtain sufficient quantities of the antibody for clinical applications. Suitable host cells for cloning or expression of antibody-encoding vectors include prokaryotic or eukaryotic cells described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector function are not needed. After expression, the antibody can be isolated from the bacterial cell paste in a soluble fraction, and can be further purified.

[0280] In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from a yeast cell, a mammalian cell (e.g., a CHO cell (e.g., CHO-S or CHO-K) or a 293 cell (e.g., 293F or HEK293 cell)), or other cell suitable for making an antibody or fragment thereof. In one embodiment, the host cell is prokaryotic, e.g., is a bacterium, e.g., E. coli.

[0281] For example, eukaryotic microorganisms such as filamentous fungi or yeasts are suitable cloning or expression hosts for vectors encoding antibodies. For example, fungal and yeast strains in which the glycosylation pathway has been "humanized" result in the production of antibodies having a partial or complete human glycosylation pattern. Host cells suitable for expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Vertebrate cells can also be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension can be used. Other examples of useful mammalian host cell lines are monkey kidney CV1 line (COS-7); human embryonic kidney line (HEK 293, 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 production of antibodies are known in the art.

[0282] IV. Production and purification of antibody molecules of the application

[0283] In one embodiment, the application provides a method of making an antibody molecule or fragment (preferably an antigen binding fragment) of the application, wherein the method comprises culturing the host cell under conditions suitable for expression of a nucleic acid encoding an antibody molecule or fragment (preferably an antigen binding fragment) of the application, and optionally isolating the antibody or fragment (e.g. antigen binding fragment) thereof. In certain embodiments, the method further comprises recovering an antibody molecule or fragment (e.g. antigen binding fragment) of the application from the host cell.

[0284] Polynucleotides encoding the polypeptide chains of the antibodies of the application 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 host cells. Methods well known to those skilled in the art can be used to construct the expression vectors. Once expression vectors comprising one or more nucleic acid molecules of the application for expression have been prepared, the expression vectors can be transfected or introduced into appropriate host cells. A variety of techniques can be used for this purpose, such as, for example, protoplast fusion, calcium phosphate precipitation, electroporation, transduction with retroviruses, viral infection, biolistics, liposome-based transfection or other conventional techniques.

[0285] Antibody molecules prepared as described herein can be purified by known art 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 also depend on factors such as net charge, hydrophobicity, hydrophilicity, and the like, and these will be apparent to those skilled in the art. The purity of the antibody molecules of the application 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.

[0286] V. Assays

[0287] 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.

[0288] The present application also provides assays for identifying anti-IL-15 antibodies having biological activities. The biological activities can include, for example, binding to IL-15 (e.g., binding to human IL-15), inhibition of IL-15 / IL-15Ra complex-mediated signaling pathway, activation of T cells, prevention or treatment of inflammation, etc. Antibodies having such biological activities in vivo and / or in vitro are also provided.

[0289] For assays of the above biological activities, reference can be made to the exemplary assay methods given in the Examples.

[0290] It is understood that any of the above assays can be performed using the immunoconjugates of the present application in place of or in addition to the anti-IL-15 antibodies.

[0291] It is understood that any of the above assays can be performed using a combination of the anti-IL-15 antibodies and another therapeutic agent.

[0292] VI. Immunoconjugates

[0293] In some embodiments, the present application provides immunoconjugates comprising any of the anti-IL-15 antibodies or antigen-binding fragments thereof provided herein and other agents, e.g., any active agents or labels suitable for forming an immunoconjugate with the IL-15 antibodies or antigen-binding fragments thereof.

[0294] In some embodiments, the active agents suitable for forming an immunoconjugate with the IL-15 antibodies or antigen-binding fragments thereof can be, for example, chemotherapeutic agents, toxins, small molecule drugs, cytotoxic agents, apoptosis agents, chelating agents, immunomodulatory agents, e.g., anti-inflammatory agents or immunosuppressive agents.

[0295] In some embodiments, the immunoconjugate is an antibody drug conjugate, e.g., an ADC.

[0296] VII. Pharmaceutical compositions and pharmaceutical formulations

[0297] In some embodiments, the present application 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 the composition is 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 application in combination with one or more other therapeutic agents. In some embodiments, the composition is a pharmaceutical formulation.

[0298] The present application also includes compositions, including pharmaceutical compositions or pharmaceutical formulations, comprising an anti-IL-15 antibody or antigen-binding fragment thereof or immunoconjugate thereof, or compositions, including pharmaceutical compositions or pharmaceutical formulations, comprising a polynucleotide encoding an anti-IL-15 antibody. In certain embodiments, the composition comprises one or more antibodies or fragments thereof that bind IL-15, or one or more polynucleotides encoding one or more antibodies or fragments thereof that bind IL-15. These compositions can further comprise suitable pharmaceutical excipients, such as pharmaceutical carriers, pharmaceutical excipients, including buffers, as known in the art.

[0299] 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.

[0300] For the use of pharmaceutical excipients and their uses, see also "Handbook of Pharmaceutical Excipients", Eighth Edition, R.C. Rowe, P.J. Seskey and S.C. Owen, Pharmaceutical Press, London, Chicago.

[0301] The compositions of the present application can be in a variety of forms. These forms include, for example, liquid, semi-solid and solid dosage

[0302] Pharmaceutical formulations comprising the antibodies described herein can be prepared by mixing antibodies of the present application having the desired degree of purity with one or more optional pharmaceutical excipients, preferably in the form of a lyophilized formulation or aqueous solution.

[0303] The pharmaceutical compositions or formulations of the application can also comprise more than one active ingredient, which is desirable for the particular indication being treated, preferably those that complement the activity of the antibody of the application. For example, it can be desirable to further provide other therapeutic agents, such as cytokines, small molecule drugs, immunomodulatory agents (e.g., immunosuppressants or anti-inflammatory agents), or other antibodies, etc. The active ingredients are present in combination in amounts that are effective for the purpose intended.

[0304] Sustained-release formulations can be prepared. Suitable examples of sustained-release formulations include semi-permeable matrices of solid hydrophobic polymers containing the antibody in which the drug is released from the matrix by diffusion through the matrix and possibly by swelling and increasing the porosity of the matrix or by a combination of both mechanisms.

[0305] VIII. Pharmaceutical combinations and kits

[0306] In some embodiments, the application also provides a pharmaceutical combination or combination product comprising an anti-IL-15 antibody or fragment thereof, preferably an antigen binding fragment thereof, or an immunoconjugate thereof, of the application, or a pharmaceutical composition comprising the same, and one or more other therapeutic agents (e.g., cytokines, chemotherapeutic agents, small molecule drugs, immunomodulatory agents (e.g., immunostimulatory or immunosuppressive agents or anti-inflammatory agents), or other antibodies, etc.).

[0307] It is another object of the application to provide a kit-of-parts comprising a pharmaceutical combination of the application, preferably in the form of pharmaceutical dosage units. Thereby dosage units can be provided according to a dosing regimen or pharmaceutical administration interval.

[0308] In one embodiment, the kit-of-parts of the application comprises in the same package:

[0309] - a first container containing a pharmaceutical composition comprising an anti-IL-15 antibody or fragment thereof or an immunoconjugate thereof;

[0310] - a second container containing a pharmaceutical composition comprising the other therapeutic agent.

[0311] The choice of other therapeutic agents that can be combined with the molecules of the application depends on the use of the molecules of the application. For example, when the molecules of the application are used for the treatment of autoimmune or autoinflammatory diseases, the other therapeutic agents are any active agents suitable for the treatment of autoimmune or autoinflammatory diseases, including but not limited to cytokines, small molecule drugs, other antibodies or immunomodulatory agents (e.g., immunosuppressive or anti-inflammatory agents). For example, when the molecules of the application are used for the treatment of tumors, the other therapeutic agents are any active agents suitable for the treatment of tumors, such as anti-tumor or anti-cancer agents, including but not limited to cytokines, chemotherapeutic agents, small molecule drugs, other antibodies or immunomodulatory agents (e.g., immunostimulatory agents).

[0312] IX. Uses and methods

[0313] In some embodiments, the antibodies or fragments thereof or immunoconjugates thereof of the application are provided for use as a monotherapy or in combination therapy for preventing or treating a disease or disorder in an individual, comprising administering to the individual a molecule of the application (e.g., an anti-IL-15 antibody or antigen-binding fragment thereof or immunoconjugate thereof of the application), a pharmaceutical composition, a pharmaceutical combination, or a pharmaceutical kit.

[0314] In some embodiments, the application relates to a molecule of the application (e.g., an anti-IL-15 antibody or antigen-binding fragment thereof or immunoconjugate thereof of the application), a pharmaceutical composition, a pharmaceutical combination, or a pharmaceutical kit for use in therapy, e.g., for use in the treatment of a disease or disorder mentioned herein.

[0315] In some embodiments, the application relates to a method of treating a disease or disorder with a molecule of the application (e.g., an anti-IL-15 antibody or antigen-binding fragment thereof or immunoconjugate thereof of the application), a pharmaceutical composition, a pharmaceutical combination, or a pharmaceutical kit, or to the use for such a treatment, or to the use for the manufacture of a medicament for such a treatment.

[0316] In some embodiments, the disease or disorder is an IL-15 related disease and / or disorder. The term "IL-15 related disease and / or disorder" relates to an abnormal activation of the signaling pathway mediated by IL-15 or its receptor complex, e.g., the IL-15 / IL-15Ra complex, or to an overexpression of IL-15 and / or an increased level of IL-15 and / or an abnormal IL-15 expression by cells or organs or tissues and / or an abnormal expression of IL-15 variants by cells or organs. Such diseases or disorders include, for example, autoimmune diseases and / or inflammatory disorders, or tumors. The term "autoimmune diseases and / or inflammatory disorders" is generally defined herein as diseases or disorders caused by an abnormal immune response of the body of an individual to substances and tissues normally present in the body, and which can or can not involve an abnormal inflammation of the immune system. In some embodiments, the inflammatory disorder is an autoinflammatory disease.

[0317] In some embodiments, the disease or condition is associated with abnormal activation of a signaling pathway mediated by IL-15 or its complex with IL-15 receptor (e.g., IL-15 / IL-15Ra complex) in a patient. In some embodiments, the disease or condition is associated with abnormal expression (e.g., overexpression, or increased level of expression, or expression of abnormal variants) of IL-15 or its complex with IL-15 receptor (e.g., IL-15 / IL-15Ra complex) in a patient, e.g., compared to a healthy individual. In some embodiments, the disease or condition is associated with expression of IL-15, e.g., moderate or high expression of IL-15, in a cell or tissue or organ in a patient. In some embodiments, the patient has (e.g., elevated levels of, e.g., nucleic acid or protein levels or activity of) IL-15 (e.g., compared to a healthy individual). In some embodiments, the patient has (e.g., elevated levels of, e.g., nucleic acid or protein levels or activity of) IL-15 in a biological sample (e.g., a cell or tissue) (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 a proximal healthy tissue or cell in the patient).

[0318] In some embodiments, the antibodies of the application are capable of preventing and / or treating a disease or condition associated with IL-15 by blocking the activation of downstream signaling pathways by IL-15 or its complex with IL-15 receptor (e.g., IL-15 / IL-15Ra complex).

[0319] In some embodiments, the disease or condition is selected from an inflammation or an inflammatory condition (e.g., an autoinflammatory disease), e.g., skin inflammation or small intestine inflammation. In some embodiments, the disease or condition is an autoimmune disease, e.g., graft versus host disease.

[0320] In some embodiments, the disease or condition suitable for prevention and / or treatment by the application is a tumor, e.g., a 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 an early, intermediate, or advanced stage or be metastatic. In some embodiments, the tumor is tumor immune escape.

[0321] In some embodiments, the signaling pathway mediated by IL-15 or its receptor complex, e.g., IL-15 / IL-15Ra complex, is abnormally activated in the tumor or tumor cells, e.g., compared to a healthy individual or a proximal healthy cell of the tumor cell.

[0322] 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 an individual having a tumor cell expressing IL-15 among all tumor cancers. In some embodiments, the individual has tumor cells expressing IL-15, e.g., moderately or highly expressing IL-15. In some embodiments, the individual has IL-15 (e.g., elevated levels, e.g., nucleic acid or protein levels or activity) in the individual, e.g., compared to a healthy individual. In some embodiments, the individual has IL-15 (e.g., elevated levels, e.g., nucleic acid or protein levels or activity) in a biological sample (e.g., tumor cell or tumor tissue) of the individual, 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 a proximal healthy tissue or cell of the individual.

[0323] In some embodiments, the antibody or antibody fragment or immunoconjugate or composition or product of the application will delay the onset of a disorder and / or symptoms associated with a disorder.

[0324] In some embodiments, the present application provides use of an anti-IL-15 antibody or fragment thereof of the application or an immunoconjugate or composition comprising the same in the manufacture or preparation of a medicament for a use described herein, e.g., for preventing or treating a relevant disease or disorder mentioned herein.

[0325] In some embodiments, the prophylactic or therapeutic methods described herein further comprise administering to the subject or individual an antibody molecule or pharmaceutical composition or immunoconjugate disclosed herein in combination with one or more other therapies, e.g., 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, e.g., therapeutic modalities and / or other therapeutic agents, for a use described herein, e.g., for preventing and / or treating a relevant disease or disorder mentioned herein.

[0326] The choice of additional therapeutic agent(s) depends on the use of the molecule of the application. For example, when the molecule of the application is used to treat an autoimmune or autoinflammatory disease, the additional therapeutic agent(s) is any agent active in the treatment of autoimmune diseases or inflammatory conditions, such as autoinflammatory diseases, including but not limited to cytokines, small molecule drugs, other antibodies or immunomodulatory agents (e.g., immunosuppressive or anti-inflammatory agents). For example, when the molecule of the application is used to treat a tumor, the additional therapeutic agent(s) is any agent active in the treatment of tumors, such as anti-tumor or anti-cancer agents, including but not limited to cytokines, chemotherapeutic agents, small molecule drugs, other antibodies or immunomodulatory agents (e.g., immunostimulatory agents); the additional therapeutic modality is, for example, selected from surgery or radiotherapy.

[0327] In some embodiments, the antibodies described herein can be combined with other antibodies for separate administration, e.g., separate administration as separate antibodies, or administration linked (e.g., as a bispecific or multispecific antibody molecule).

[0328] Such combination therapy encompasses combined administration (e.g., two or more therapeutic agents are included in the same formulation or separate formulations), and separate administration, in which case, administration of the antibody of the application can occur prior to, simultaneously with, and / or following, administration of the other therapeutic agent and / or therapy.

[0329] The antibodies of the application or fragments thereof (as well as immunoconjugates, compositions, pharmaceutical compositions, formulations, combination products, etc. comprising the same) can be administered by any suitable method, including parenteral administration, and, if local treatment is desired, intralesional administration. Parenteral injections or infusions include intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous injections or infusions.

[0330] X. Methods and compositions for diagnosis and detection

[0331] In one aspect, the application also relates to methods for diagnosis and detection of the antibodies of the application or antigen-binding fragments thereof, and compositions for diagnosis and detection comprising the same.

[0332] 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 its complex with IL-15 receptor (e.g., IL-15 / IL-15Rα complex) in a biological sample.

[0333] The term "detecting" as used herein includes quantitative or qualitative detection. Exemplary detection methods can involve immunohistochemistry, immunocytochemistry, flow cytometry (e.g., FACS), magnetic beads complexed with antibody molecules, ELISA assays, PCR techniques (e.g., RT-PCR). In certain embodiments, the biological sample is blood, serum, or other liquid sample of biological origin. In certain embodiments, the biological sample comprises cells or tissue.

[0334] In one embodiment, an anti-IL-15 antibody or fragment thereof is provided for use in a diagnostic or detection method.

[0335] In another aspect, a method of detecting the presence of IL-15 or its complex with an IL-15 receptor complex (e.g., IL-15 / IL-15Ra complex) in a biological sample is provided. In certain embodiments, the method comprises detecting the presence of IL-15 protein or its complex with an IL-15 receptor complex (e.g., IL-15 / IL-15Ra 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 its complex with an IL-15 receptor complex (e.g., IL-15 / IL-15Ra complex), and detecting whether an antigen-antibody complex is formed between the anti-IL-15 antibody or fragment thereof and IL-15 or its complex with an IL-15 receptor complex (e.g., IL-15 / IL-15Ra complex). Formation of an antigen-antibody complex is indicative of the presence of IL-15. The method can be an in vitro or in vivo method. In one embodiment, the 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 its complex with an IL-15 receptor complex (e.g., IL-15 / IL-15Ra complex) is a biomarker used to select the subject.

[0336] In some embodiments, a labeled anti-IL-15 antibody or fragment thereof is provided. Labels include, but are not limited to, labels or moieties that are directly detected, such as fluorescent labels, chromophoric labels, electron-dense labels, chemiluminescent labels, and radioactive labels, as well as moieties that are indirectly detected, such as enzymes or ligands, e.g., by enzymatic reaction or molecular interaction.

[0337] 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.

[0338] In some embodiments, IL-15 is detected prior to treatment, e.g., prior to initiation of treatment or prior to a treatment at an interval after treatment.

[0339] In some embodiments, a method of treating a disease of the application is provided, the method comprising assaying a subject (e.g., a sample) (e.g., a subject sample) for the presence of IL-15 or a complex thereof with an IL-15 receptor (e.g., an IL-15 / IL-15Ra complex), thereby determining a value for IL-15 or a complex thereof with an IL-15 receptor (e.g., an IL-15 / IL-15Ra complex), comparing the value for IL-15 or a complex thereof with an IL-15 receptor (e.g., an IL-15 / IL-15Ra complex) to a control value, and 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 other therapies, if the value for IL-15 or a complex thereof with an IL-15 receptor (e.g., an IL-15 / IL-15Ra complex) is greater than the control value, thereby treating the disease.

[0340] These and other aspects and embodiments of the application are described in and illustrated by the accompanying drawings (a brief description of which follows) and the following detailed description of the application. Any or all of the features discussed above and throughout this application can be combined in various embodiments of the application. The following examples further illustrate the application, however, it is understood that the examples are described in illustrative, and not restrictive, manner, and that modifications can be made by those skilled in the art. Examples

[0341] Example 1. Preparation of Anti-IL-15 Antibodies by Hybridoma

[0342] The present study utilized a hybridoma technology platform for antibody discovery.

[0343] 1.1. Immunization

[0344] Human IL-15 / IL-15Ra protein (Novoprotein, Cat# C15Y) was mixed with an equal volume of adjuvant TiterMax (sigma, Cat# T2684) and used to immunize Balb / c mice (from Beijing Vital River) by subcutaneous injection (50 μg protein per mouse) every two weeks. Three immunizations were performed, and blood was collected one week after the third immunization to test serum titers.

[0345] 1.2. Cell fusion and high-throughput screening

[0346] When the serum titer meets the requirements, the mouse spleen 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 electrofusion is performed. The fused cells are transferred from the electrofusion tank into a 50 mL centrifuge tube, and the cells are diluted to 1-2 x 10 4

[0347] Hybridoma cells specifically expressing anti-IL-15 antibodies are screened by enzyme-linked immunosorbent assay (ELISA). Human IL-15 (Novoprotein, Cat#C016) is diluted with coating solution (Thermo, Ca#28382) to 0.5 μg / mL, 100 μL / well is added to the enzyme-labeled plate, 37°C incubation for 1 hour, PBST (1X Phosphate-Buffered Saline, 0.5% 20 Detergent) is washed three times, and the plate is spun to remove the liquid; 200 μL / well of 3% NON-Fat Powdered Milk (Shenguo, Cat#A600669-0250) is added to block the enzyme-labeled plate, 37°C incubation for 1 hour, PBST is washed three times, and the plate is spun to remove the liquid; 50 μL / well of fused hybridoma supernatant is added, 37°C incubation for 1 hour, PBST is washed three times, and the plate is spun to remove the liquid; 100 μL / well of goat anti-mouse HRP (BIOLEGEND, Ca#405306) labeled secondary antibody (1:5000 diluted in PBST) is added, 37°C incubation for 1 hour, PBST is washed three times, and the plate is spun to remove the liquid; 100 μL / well of color developing solution tetramethylbenzidine (TMB) (INVITROGEN, Cat#002023) is added, and color development is carried out in the dark for 3 minutes; 50 μL / well of ELISA stop solution (Solarbio, Cat#C1058) is added, and the luminosity value at OD450 is read by an enzyme-labeled instrument. Positive clones against human IL-15 antigen are obtained after screening, and their binding to cynomolgus IL-15 is detected by the same method as described above. 20 Detergent) is washed three times, and the plate is spun to remove the liquid; 200 μL / well of 3% NON-Fat Powdered Milk (Shenguo, Cat#A600669-0250) is added to block the enzyme-labeled plate, 37°C incubation for 1 hour, PBST is washed three times, and the plate is spun to remove the liquid; 50 μL / well of fused hybridoma supernatant is added, 37°C incubation for 1 hour, PBST is washed three times, and the plate is spun to remove the liquid; 100 μL / well of goat anti-mouse HRP (BIOLEGEND, Ca#405306) labeled secondary antibody (1:5000 diluted in PBST) is added, 37°C incubation for 1 hour, PBST is washed three times, and the plate is spun to remove the liquid; 100 μL / well of color developing solution tetramethylbenzidine (TMB) (INVITROGEN, Cat#002023) is added, and color development is carried out in the dark for 3 minutes; 50 μL / well of ELISA stop solution (Solarbio, Cat#C1058) is added, and the luminosity value at OD450 is read by an enzyme-labeled instrument. Positive clones against human IL-15 antigen are obtained after screening, and their binding to cynomolgus IL-15 is detected by the same method as described above.

[0348] 1.3. Subcloning of positive hybridoma cells

[0349] ​Limited dilution subcloning step: Prepare a 96-well plate, add 200 μL of medium to each well, which is the same as the screening medium, but replace HAT with HT (hypoxanthine and thymidine) (Gibco, Cat#11067-030). Prepare a cell suspension of the positive wells screened in the above fusion, and add 100 μL to each well in the first row, mix well, then take 100 μL of the cell suspension in the first row to the second row, mix well, and then take 100 μL to the next row; repeat the above steps, and let the 96-well plate stand for 30 minutes. Count under a microscope. Take 100 cells and add them to 20 mL of medium, mix well, and plate at 200 μL per well. After one week, observe under a microscope, and mark the single clone wells.

[0350] When the confluence of cells in each well reaches more than 50%, detect them by the screening method of ELISA as described above, pick out the target positive wells, expand the culture, and then freeze the cells (finally select 18 clones).

[0351] Example 2. Preparation of chimeric antibodies

[0352] The present application uses molecular biology techniques to obtain the antibody sequence in the anti-IL-15 positive hybridoma cells, and uses it to construct human-mouse chimeric antibodies. The antibody light and heavy chain gene sequences of the 18 hybridoma candidate clones obtained in Example 1 are retrieved, and human-mouse chimeric antibodies are constructed.

[0353] Take about 5 x 10 6 extract RNA (Macherey-Nagel, Ca#740984.250). Reverse transcribe the cDNA using PrimeScript II 1st Strand cDNA Synthesis Kit (Takara). Design the upstream primer based on the base sequence in the 5' end FRi region, and the downstream primer based on the base in the antibody constant region or FR4 region, to amplify the antibody light and heavy chain variable region gene fragments. Connect them to the T vector (Mighty TA-cloning Kit, Takara), pick single clones for sequencing, and analyze the sequencing results using MEGA7 software. After alignment, 3 pairs of candidate clone sequences are obtained according to sequence homology.

[0354] Respectively, the light and heavy chain variable region gene fragments of the 3 pairs of candidate clones are connected to the T vector (Mighty TA-cloning Kit, Takara), and single clones are picked for sequencing, and the sequencing results are analyzed using MEGA7 software. After alignment, 3 pairs of candidate clone sequences are obtained according to sequence homology. II, Cat# C112-01) into pcDNA3.1 vector (containing heavy chain constant region: SEQ ID NO: 26; light chain constant region: SEQ ID NO: 27, respectively) to obtain the expression plasmids of light chain and heavy chain antibodies. Then the light chain plasmid of the same antibody was mixed with the heavy chain plasmid at a molar ratio of 1:1, and 293F cells were transfected with polyethyleneimine (PEI) (Polysciences, Ca# 23966). After 5-7 days of culture, when the cell viability was less than 60%, the cell culture supernatant was collected and the monoclonal antibody was purified with a Protein A affinity column. The specific purification method is described below in Example 3.2.

[0355] Three chimeric antibodies were obtained, including ch25B10, ch56G9, and ch60G8.

[0356] The sequences of control antibodies Amg714 and Caly002 are shown in Table 1 and Table 2, which were prepared in a similar manner as described above.

[0357] Table 1. Protein sequence of control antibody Amg714

[0358] Table 2. Protein sequence of control antibody Caly002

[0359] Example 3. Expression and purification of antibody protein and IL-15 / IL-15Rα-Fc protein

[0360] Human IL-15 / IL-15Rα-Fc, human Q108A IL-15 / IL-15Rα-Fc, and human H105R IL-15 / IL-15Rα-Fc proteins were used for subsequent affinity analysis and in vivo and in vitro functional experiments, and the sequences are shown in Table 3, Table 4, and Table 5.

[0361] Table 3. Protein sequence of human IL-15 / IL-15Rα-Fc

[0362] Table 4. Protein sequence of human Q108A IL-15 / IL-15Rα-Fc

[0363] Table 5. Protein sequence of human H105R IL-15 / IL-15Rα-Fc

[0364] 3.1. Protein preparation

[0365] Expi293F cells (purchased from Gibco) were cultured with Expi293F medium (Gibco, REF#A14351-01), and the cell density was checked (viability should be greater than 95%) one day before transfection, and adjusted to 3x10 6 cells / mL with fresh Expi293F medium, and the cell density was adjusted to 3x10 6 cells / mL on the day of transfection.

[0366] Take 1 / 10 of the final transfection volume of Opti-MEM medium (Gibco, REF#31985-070) as transfection buffer, add the plasmid to be transfected (where the plasmid is a vector connected by nucleic acid encoding IL-15-Fc chain (or its variant IL-15-Fc chain) or IL-15Rα_sushi_Fc chain to pcDNA3.1) at a ratio of 1 mg / L, and mix well, add PEI Max (Polysciences Inc. Cat#24765-1) at a ratio of DNA:PEI 1:3, mix well, incubate at room temperature for 20 minutes, then gently pour the mixture into the Expi293F cell suspension, shake while pouring, and culture the cells on a shaker, with a culture condition of 8% CO2, 36.5°C, 120 rpm.

[0367] After 16-18 hours of culture, add 2% (volume ratio) of Feed (100 g / L Phytone Peptone + 100 g / L Difco Select Phytone) with a concentration of 200 g / L, a final concentration of 5 g / L glucose solution, and a final concentration of 2.2 mM Valproic acid sodium salt (Merk, Cat#P4543-100G) to the cell suspension, mix gently, continue to culture at 8% CO2, 36.5°C, 120 rpm for 7 days, and then collect the sample. Mix the cell slurry with diatomite (Sartorius, Cat 1000037025) (40 g diatomite for 1 L cell slurry), and filter with a 0.22 μM disposable vacuum filter device.

[0368] 3.2. Protein purification

[0369] The HiTrap MabSelect Prism A (GE Healthcare, Cat# 17549853) affinity chromatography column was selected for affinity capture. Before purification, 10-20 column volumes of 0.1 M NaOH were passed through the pipeline and the affinity chromatography column, then the pipeline and the column were washed with 10-20 column volumes of distilled water, and the column was equilibrated with 5 column volumes of 1x PBS (Gibco). The filtered cell slurry was passed through the column, and the column was washed with 10 column volumes of 1x PBS to remove non-specific binding proteins. The column was washed with 5 column volumes of elution buffer (100 mM sodium citrate, pH 3.5), and the eluate was collected. The pH was adjusted to 6.0 with 2 M Tris, and the sample was filtered to remove bacteria.

[0370] The purity of the sample was detected by size exclusion chromatography (SEC).

[0371] The protein sample with satisfactory purity was centrifuged at 4000 rpm for 10 minutes using a 15 mL ultrafiltration centrifuge tube. After diluting the protein with PBS, the centrifugation was continued at 4000 rpm for 10 minutes. This operation was repeated several times to replace the storage buffer of the protein. After the buffer replacement was completed, the sample was filtered to remove bacteria, the antibody concentration was detected, and the protein was delivered.

[0372] Example 4. Detection of biological activity of chimeric antibodies using IL-15 reporter cell lines

[0373] The IL-15 reporter cell line was used to detect the function of three candidate chimeric antibodies. Anti-IL-15 antibodies can directly target IL-15 cytokines, block IL-15 downstream signaling pathways, and thus inhibit cell proliferation and activation. In this study, the fluorescent reporter cell line provided by Shanghai Jiman Biotechnology Co., Ltd. was used: 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 determine the proliferation and differentiation of the cells, and thus the inhibitory effect of the antibodies on IL-15 signaling was detected.

[0374] Cell recovery: 1) Take out the cells from liquid nitrogen tank, put into 37°C water bath, shake gently until thawing, keep the seal ring and bottle cap above the water surface to prevent contamination; 2) After thawing, sterilize with 75% alcohol, then transfer to the clean bench, follow the subsequent aseptic operation; 3) Transfer the cell suspension in the cryopreservation tube to a preheated 15 mL centrifuge tube, mix gently, centrifuge at 1000 rpm for 5 minutes to precipitate the cells, discard the supernatant; 4) Prepare complete 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 with 1 mL of complete medium, take a portion to count the viable cells with trypan blue (Gibco, 15250-061); 6) Adjust the viable cell density to 3-5 x 10 5 Cells / mL, inoculate the cell suspension into a T25 cell culture flask; 7) Incubate in a 37°C, 5% CO2 incubator.

[0375] Cell culture: Maintain 0.25 ug / mL puromycin (Gibco, A11138-02) and 3 ug / mL Blastincidin (Gibco, A11139-03) selection pressure during subculture, subculture every 2-3 days, control the subculture density at 0.5 x 10 6 Cells / mL.

[0376] Activity detection: 1) Cell plating: count the cells, adjust the cell density to 2 x 10 62) Preparation of antibodies and cytokines: IL-15 / IL-15Rα-Fc stimulator was prepared using 1640 culture medium without GM-CSF cytokine, the concentration was 0.01 ug / mL, 25 uL per well was added to the cell culture plate. Antibodies (of which the isotype sequences are SEQ ID NO: 37 and SEQ ID NO: 38) were diluted using sterile 96-well V-bottom plates (Bi Yun Tian, FPT019), 300 nM, 4-fold dilution, a total of 12 gradients, 25 uL per well was added to the cell culture plate, 5% CO2, 37°C incubation for 16 h. 3) Fluorescence detection: The Bio-Lite Luciferase Assay system reagent (Vazyme, DD1201-02) and 96-well plates were taken out in advance, and equilibrated at room temperature for 30 minutes, an equal volume of Bio-Lite Luciferase Assay system 1X lysis solution was added to each well, shaken gently, and then incubated for 10 minutes, and then detected by instrument.

[0377] The experimental results are shown in Figure 1, and the chimeric antibodies ch25B10, ch56G9 and ch60G8 can effectively block the downstream signal stimulated by IL-15 / IL-15Rα-Fc, and the inhibitory effect is better than that of the control anti-IL-15 antibody.

[0378] Example 5. Analysis of physicochemical properties of chimeric antibodies

[0379] The physicochemical properties of chimeric antibodies ch25B10, ch56G9 and ch60G8 were detected.

[0380] 5.1. SMAC column detection of antibody colloidal stability

[0381] The colloidal stability of the antibody was detected by recording the retention time of the antibody in the Zenix-HPLC column.

[0382] 5.2. HIC column detection of antibody colloidal hydrophobicity

[0383] The hydrophobicity of the antibody was detected by recording the retention time of the antibody in the HIC-HPLC column.

[0384] 5.3. CIC detection of antibody non-specificity

[0385] IgG from human serum was coated on an NHS-activated chromatography column, and the retention time of the antibody on the column was investigated by HPLC, which was significantly negatively correlated with its solubility. Therefore, this method can be used to screen antibodies with better solubility.

[0386] 5.4. DLS to detect thermal stability of antibodies

[0387] Dynamic light scattering (DLS) utilizes laser irradiation of small particles in solution doing Brownian motion, and detects the change of scattered light intensity. DLS can be used to detect the particle size of proteins in solution, and the stability under different temperatures or different concentrations.

[0388] The experimental results are shown in Table 6, and the antibodies ch25B10 and ch56G9 showed relatively good physicochemical properties.

[0389] Table 6. Results of thermal stability detection of antibodies

[0390] Example 6. Bio-Layer Interferometry (ForteBio) to determine the binding kinetics of the antibodies of the application to antigens

[0391] Bio-Layer Interferometry (ForteBio) was used to determine the equilibrium dissociation constant (KD) of the antibodies of the application to bind to human and cynomolgus IL-15. The ForteBio affinity assay was performed according to the existing method (Estep, P et al. High throughput solution Based measurement of antibody antigen affinity and epitope binning. MAbs, 2013. 5(2): p. 2708).

[0392] Half an hour before the start of the experiment, the appropriate number of AHC (18-5060, Sartorius) sensors were immersed in SD buffer (PBS 1x, BSA 0.1%, Tween 20 0.05%) according to the number of samples.

[0393] Take 100 μL of SD buffer, antibody, antigen

including human IL-15 (IL5-H52H8, Acro biosystems), cynomolgus IL-15 (IL5-C52H4, Acro biosystems), human IL-15 / IL-15Rα-Fc, human Q108A IL-15 / IL-15Rα-Fc, human H105R IL-15 / IL-15Rα-Fc

[0394] Example 7. Humanization of antibodies

[0395] Based on Discovery Studio (Dassault Systèmes) and PyMOL software, the chimeric antibodies ch25B10 and ch56G9 obtained by hybridoma screening were designed for humanization by the following steps:

[0396] (1) Determine the CDR region of the chimeric antibody;

[0397] (2) Screen the closest homologous sequences to the V / J regions of the heavy and light chains of the chimeric antibody in the human germline sequence database, respectively;

[0398] (3) Construct the CDR region of the chimeric antibody to the human Germline skeleton;

[0399] (4) Based on sequence and structural characteristics, determine the amino acid positions in the skeleton region that maintain the function of CDR, and perform back mutation (return to the input amino acid type) at the determined important sequence positions;

[0400] (5) Synthesize the sequence, prepare the antibody, and then determine the affinity of the humanized molecule to human IL-15;

[0401] (6) Given that the sequences of ch25B10 and ch56G9 are very similar, and in order to avoid the risk of drugability caused by the sequence in the light chain of ch56G9, combine the heavy chain sequence of humanized ch56G9 and the light chain sequence of humanized ch25B10 to obtain the final humanized molecule, hz56G9.25L.

[0402] The equilibrium dissociation constant (KD) of the antibody binding to human IL-15 was determined using the ForteBio assay as described in Example 6.

[0403] Table 7. Equilibrium dissociation constants of antibodies

[0404] From the affinity data shown in Table 7, it can be concluded that the humanized antibody, hz56G9.25L, retains comparable affinity to the chimeric antibody. The CDR, VH, VL amino acid sequences of the chimeric antibody and the humanized antibody are shown in Table 8, Table 9, Table 10.

[0405] Table 8. Amino acid sequences of CDRs of exemplary antibodies of the present application

[0406] Table 9. Heavy chain variable region (VH) of exemplary antibodies of the present application

[0407] Table 10. Light chain variable region (VL) of exemplary antibodies of the present application

[0408] Example 8. ForteBio assay for determining the binding kinetics of hz56G9.25L and control antibodies to antigen

[0409] The equilibrium dissociation constant (KD) of the humanized antibodies of the present application binding to human IL-15, cynomolgus IL-15, human IL-15 / IL-15Rα-Fc, human Q108A IL-15 / IL-15Rα-Fc, human H105R IL-15 / IL-15Rα-Fc was determined using the ForteBio assay. The ForteBio affinity assay method is the same as in Example 6. The results are shown in Tables 11 to 15 below.

[0410] From the results in Tables 11, 12, it can be seen that hz56G9.25L has higher affinity compared to Amg714.

[0411] From the results in Table 13, Table 14, and Table 15, it can be seen that hz56G9.25L targets a different epitope on the IL-15 protein than 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 acid 105 of IL-15 and does not target amino acid 108 of IL-15. It is reported in the literature 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 IL-15 / IL2Rg interface. Prior art references indicate that Amg714 targets the IL-15 / IL2Rg interface and Caly002 targets the IL-15 / IL2Rb interface, which is consistent with the results in Table 13, Table 14, and Table 15. Overall, hz56G9.25L targets an epitope that is unique compared to Amg714 and Caly002.

[0412] Table 11. Affinity constants (M) of antigen-antibody binding detected by ForteBio

[0413] Table 12. Affinity constants (M) of antigen-antibody binding detected by ForteBio

[0414] Table 13. Affinity constants (M) of antigen-antibody binding detected by ForteBio

[0415] Table 14. Affinity constants (M) of antigen-antibody binding detected by ForteBio

[0416] Table 15. Affinity constants (M) of antigen-antibody binding detected by ForteBio

[0417] Example 9. Biological activity of antibodies detected using IL-15 reporter cell line

[0418] Anti-IL-15 antibodies can directly target IL-15 cytokines, block IL-15 downstream signaling pathways, and thus inhibit cell proliferation and activation. In this study, the fluorescence reporter cell line provided by Shanghai Jiman Biotechnology Co., Ltd.: IL-15 pathway activity reporter cell (human erythroid leukemia cell-IL-15 reporter cell line, GM-C25386) was used. According to the method provided in the instructions, the expression of the fluorescence reporter gene was detected to detect the proliferation and differentiation of the cells, so as to detect the inhibition of the antibody on the IL-15 signal.

[0419] Cell recovery: see Example 4.

[0420] Cell culture: see Example 4.

[0421] Activity detection: 1) cell plating: cell counting, adjusting the cell density to 2x10 6 Cell culture: see Example 4.

[0422] The experimental results are shown in Figure 2 (IL-15 stimulation) and Figure 3 (IL-15 / IL-15Rα-Fc stimulation). The antibody hz56G9.25L can effectively block the activation of the downstream signaling pathway mediated by IL-15, and the inhibition effect is better than that of the control anti-IL-15 antibody.

[0423] Example 10. Detection of the inhibition of antibody on IL-15 signal-induced immune cell proliferation

[0424] Cell recovery: 1) The peripheral blood mononuclear cell (PBMC) cells (Shanghai Miaoshun TPCS, PB050C-W) were taken out of the liquid nitrogen tank, placed in a 37°C water bath, and gently shaken to melt. The sealing ring and bottle cap were kept above the water surface to prevent contamination; 2) After the cells were melted, they should be immediately disinfected with 75% alcohol and then moved to a clean bench for subsequent strict aseptic operation; 3) Prepare 1640 complete 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 frozen tube to the preheated 1640 complete medium containing DNase1 (SIGMA, D5025-375KU), mix gently, centrifuge at 300g for 6 minutes to precipitate the cells, and discard the supernatant; 4) Resuspend the cell pellet with 15mL complete medium, and take a portion for counting the viable cells using trypan blue (Gibco, 15250-061).

[0425] Activity detection: 1) Cell plating: Adjust the viable cell density to 1x10 6 Cell / mL, inoculate the cell suspension into a TC-treated white-bottom transparent cover 96-well flat-bottom luminescence plate (Shanghai Wohong, WHB-96-03) at 100 microliters per well, with 100,000 cells. 2) Prepare antibodies and cytokines: Use 1640 medium to prepare IL-15 (Novoprotein, C016) and IL-15 / IL-15Rα-Fc stimulators at concentrations of 0.01 and 0.5 μg / ml (or 5ug / ml), respectively, and add 50uL per well to the cell culture plate; dilute the antibody (Biyin, 2K7D4D001) in a sterile 96-well V-bottom plate (Bi Yun Tian, FPT019). Under IL-15 stimulation, the antibody is diluted 2.5 times at 300nM, with a total of 12 gradients, and under IL-15 / IL-15Rα-Fc stimulation, the antibody is diluted 2 times at 300nM, with a total of 12 gradients, and added to the cell culture plate at 50uL per well, incubated at 5% CO2 37°C for 72 hours. 3) Fluorescence detection: Take out the Cell Counting-Lite2.0 reagent (Vazyme, DD1101-02) and 96-well plate in advance, equilibrate at room temperature for 20-30 minutes, add an equal volume of Cell Counting-Lite2.0 lysis solution to each well, shake gently, and then incubate for 15 minutes. Detect with the instrument.

[0426] The experimental results are shown in Figure 4 (IL-15 stimulation), Figure 5 (IL-15 / IL-15Rα-Fc stimulation, 0.5 ug / ml), and Figure 6 (IL-15 / IL-15Rα-Fc stimulation, 5 ug / ml). The antibody hz56G9.25L can effectively block the activation of the downstream signaling pathway mediated by IL-15, inhibit the proliferation of immune cells, and the inhibitory effect is better than that of the control anti-IL-15 antibody.

[0427] Example 11. Detection of the inhibitory effect of the antibody on IL-15 signal-induced CD8 T cell activation

[0428] Isolation of CD8 T cells: 1) Take out the PBMC cells from the liquid nitrogen tank (Shanghai Miaoshun TPCS, Catalog No. PB050C-W), and place them in a 37°C water bath to melt gently. Keep the sealing ring and bottle cap above the water surface to prevent contamination; 2) After the cells are melted, they should be immediately disinfected with 75% alcohol and then transferred to a clean bench for subsequent aseptic operation; 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); 4) Transfer the cell suspension in the frozen tube to the preheated 1640 complete culture medium containing DNase1 (SIGMA, D5025-375KU), mix gently, centrifuge at 300g for 6 minutes to precipitate the cells, and discard the supernatant; 4) Resuspend the cell pellet with 15mL complete culture medium, and take a portion for counting the viable cells using trypan blue (Gibco, 15250-061); 5) Use CD8 T cell isolation kit (stemcell, 19053) to isolate CD8 T cells from PBMC.

[0429] CD8 T cell activation experiment: 1) Coat a 6-well plate with 1 ug / mL anti-human CD3 (Acro, CDE-H5223) and incubate at 4°C overnight; 2) Take the isolated CD8 T cells and add them to the 6-well plate coated with CD3 antibody to activate the CD8 T cells. Add 1 ug / mL anti-CD28 (Invitrogen, 16-0289-85) to each well with a volume of 3mL, and incubate at 5% CO2 37°C for 72 hours; 3) Collect the activated CD8 T cells after 3 days, adjust the cell density to 1x10 6 / ml, using TC to process white bottom transparent cover 96 well flat bottom luminescence plate (Shanghai Wohong, WHB-96-03), middle 60 holes, 100 microliters per hole, 100,000 cells. 4) Preparation of IL-15 (Novoprotein, C016) and IL-15 / IL-15Rα-Fc stimulator, concentration 0.01 and 0.5ug / mL, 50uL per hole to cell culture plate; 5) using sterile 96 well V bottom plate (Biyun Tian, FPT019) to dilute the antibody. Under the stimulation of IL-15, the antibody is diluted by 2.5 times with 300nM, a total of 12 gradients, and under the stimulation of IL-15 / IL-15Rα-Fc, the antibody is diluted by 2 times with 300nM, a total of 12 gradients, 50uL per hole to cell culture plate, 5% CO2 37℃ incubation for 72 hours; 6) collect supernatant, ELISA (MabTech, 3420-1H-20) to detect the secretion of IFNg.

[0430] The above experimental results are shown in Figure 7 (IL-15 stimulation), Figure 8 (IL-15 / IL-15Rα-Fc stimulation), the antibody hz56G9.25L can effectively block the activation of IL-15 mediated downstream signal pathway, inhibit IL-15 mediated CD8 T cell activation and IFNγ cytokine secretion, and the inhibition effect is better than that of the control anti-IL-15 antibody.

[0431] Example 12. Analysis of the physicochemical properties of the antibody hz56G9.25L

[0432] The physicochemical properties of the antibody hz56G9.25L were detected.

[0433] 12.1. SMAC column detection of antibody colloidal stability

[0434] The colloidal stability of the antibody was detected by recording the retention time of the antibody in the Zenix-HPLC column.

[0435] 12.2. HIC column detection of antibody colloidal hydrophobicity

[0436] The hydrophobicity of the antibody was detected by recording the retention time of the antibody in the HIC-HPLC column.

[0437] 12.3. CIC detection of antibody non-specificity

[0438] IgG from human serum was coated on an NHS activated chromatography column, and the retention time of the antibody on the column was investigated by HPLC, which was significantly negatively correlated with its solubility. Therefore, this method can be used to screen antibodies with better solubility.

[0439] 12.4. DLS detection of antibody thermal stability

[0440] Dynamic light scattering (DLS) utilizes a laser to irradiate small particles in solution doing Brownian motion, and detects the change of scattered light intensity. DLS can be used to detect the particle size of proteins in solution, and the stability under different temperatures or different concentrations.

[0441] The experimental results are shown in Table 16, and the antibody hz56G9.25L shows the expected physicochemical properties.

[0442] Table 16. Physicochemical properties of antibodies

[0443] Example 13. Detection of the inhibitory effect of antibodies on IL-15 signal-induced inflammation of mouse skin

[0444] Female C57BL / 6N mice, 6-9 weeks old, 16-19 g, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., grade SPF, quality inspection unit Beijing Vital River Laboratory Animal Technology Co., Ltd., certificate number 110011231110488161.

[0445] After 1 week of adaptive feeding, they were randomly divided into four groups: unmodeled group (i.e. PBS+Isotype), modeled group (i.e. IL-15 / IL-15Rα-Fc+Isotype), Amg714 treatment group and hz56G9.25L treatment group.

[0446] The mice were shaved on the dorsal side, and two 1cm*1cm frames were drawn on the skin with a marker pen, labeled as site 1 and site 2. The latter three groups were intradermally injected with IL-15 / IL-15Rα-Fc, 2.5ug / 50uL per frame, i.e. 5ug / 100uL per mouse, in the two frames on days 1, 3, 5, 7, 9, and 11 to induce a mouse dermatitis model, while the unmodeled group was intradermally injected with an equal volume of PBS.

[0447] The four groups of mice were intraperitoneally injected with Isotype (100ug / mouse), Isotype (100ug / mouse), Amg714 (100ug / mouse), and hz56G9.25L (100ug / mouse) on days 0, 3, 6, and 10, respectively. The skin thickness of the mice was monitored on days 1, 3, 5, 7, 10, and 14, and the skin thickness of the mice was measured using a vernier caliper and the degree of skin thickening was calculated (Change% = Day X skin thickness / Day 0 skin thickness x 100%-100%).

[0448] At the end of the experiment on day 14, the mice were euthanized, and the site 1 mouse skin was collected, fixed with formalin, and subjected to IHC staining to detect T cell infiltration; the site 2 mouse skin was collected in PBS, and flow cytometry was used to detect T cell infiltration and activation (NKG2D expression level) (the NKG2D flow cytometry analysis antibody used was BD 562800).

[0449] The flow cytometry analysis results are shown in FIGS. 9 and 10, and the antibody hz56G9.25L can effectively block IL-15-mediated T cell proliferation and activation. As shown in the figures, compared with Amg714, the antibody hz56G9.25L can more effectively inhibit the expression level of NKG2D.

[0450] Example 14. Detection of the inhibitory effect of the antibody on IL-15 signal-induced small intestine inflammation

[0451] The female C57BL / 6N mice, 6-9 weeks old, 16-19 g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., and were of SPF level, with the quality inspection unit being Beijing Vital River Laboratory Animal Technology Co., Ltd., and the certificate number being 110011231107923856.

[0452] After one week of adaptive feeding, the mice were randomly divided into four groups: the non-model group (i.e., PBS+Isotype), the model group (i.e., IL-15 / IL-15Rα-Fc+Isotype), the Amg714 treatment group, and the hz56G9.25L treatment group.

[0453] The latter three groups were intraperitoneally injected with IL-15 / IL-15Rα-Fc at 15 ug / 200 uL per mouse at -1 h, to induce a mouse intestinal inflammation model, and the non-model group was intraperitoneally injected with an equal volume of PBS.

[0454] The four groups of mice were intraperitoneally injected with Isotype (200 ug / mouse), Isotype (200 ug / mouse), Amg714 (200 ug / mouse), and hz56G9.25L (200 ug / mouse) at 0 h, 24 h, and 48 h, respectively. At the end of the experiment on day 72, the mice were euthanized, and the mouse small intestine (10 cm below the stomach) and the mouse spleen were collected and weighed, and flow cytometry was used to detect the T cell activation (CD69 expression level) of the small intestine (FIG. 11) and the spleen (FIG. 12).

[0455] The results of the above flow analysis experiment are shown in FIG. 11 and FIG. 12. Antibody hz56G9.25L can effectively block IL-15-mediated T cell activation, and has a trend of showing an advantage compared with Amg714. In FIG. 11, two T cell groups that exist in large quantities in the small intestine are detected, including αβ T cells and γδ T cells. Antibody hz56G9.25L has an obvious inhibitory effect on the activation of both groups of T cells.

[0456] Example 15. Detection of the inhibitory effect of the antibody on IL-15 signal enhanced graft-versus-host disease (GvHD)

[0457] Female NOG mice, 6-9 weeks old, 16-19 g, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., grade SPF, quality inspection unit Beijing Vital River Laboratory Animal Technology Co., Ltd., license number 110011231109256251. After 1 week of adaptive feeding, they were randomly divided into four groups: GvHD group (i.e. PBS+Isotype), GvHD accelerated group (i.e. IL-15 / IL-15Rα-Fc+Isotype), Amg714 treatment group, and hz56G9.25L treatment group.

[0458] All mice were intravenously injected with PBMC (1*10^7 / each mouse, All cells, LP230809010, LP221115011) on day 1 to induce a mouse GvHD model. The latter 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 to accelerate GvHD, while the first group was intraperitoneally injected with the same volume of PBS.

[0459] The four groups of mice were intraperitoneally injected with Isotype (100ug / mouse), Isotype (100ug / mouse), Amg714 (100ug / mouse), and hz56G9.25L (100ug / mouse) on days 0, 3, 7, 10, and 14. The mice were monitored for body weight on days 0, 3, 7, 10, 12, 14, 16, and 18, and the body weight change was calculated (Change%=Day X body weight / Day 0 body weight x 100%-100%). The mice were monitored for death throughout the experiment, and those with a body weight decrease of more than 20% were euthanized.

[0460] The results of the above flow analysis experiment are shown in FIG. 13 (PBMC donor 1) and FIG. 14 (PBMC donor 2). Antibody hz56G9.25L can effectively block IL-15 signal enhanced graft-versus-host disease (GvHD).

[0461] As shown in the figure, compared with the PBS group, IL-15 / IL-15Rα significantly accelerated the progress of GvHD, causing the mice to lose weight and die. In the treatment group, compared with Amg714, the antibody hz56G9.25L can more effectively inhibit IL-15 / IL-15Rα, thereby prolonging the survival of mice and slowing down the trend of weight loss in mice.

[0462] Example 16. Antibody pharmacokinetics (PK) study in BALB / c mice

[0463] The PK behavior of hz56G9.25L in vivo was observed through mouse pharmacokinetics (PK) experiments.

[0464] BALB / c mice were subcutaneously injected with 10 mpk of hz56G9.25L, and mouse serum samples were collected 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.

[0465] The ELISA method was used for detection: the plate was coated with IL-15 antigen (coastal protein, GMP-C016), then the serum to be detected was added, then the anti-human Fc secondary antibody (BETHYL, A80-104P) was detected, and finally TMB was developed. The PK detection results are shown in Figure 15 and Table 15, and the PK profile shows an IgG-like PK profile, with a half-life of 258 hours and a clearance rate of 0.21 ml / kg / h.

[0466] Table 17. Antibody pharmacokinetics (PK) study in BALB / c mice

[0467] Example 17. Antibody Fc engineering

[0468] Through the circulation mediated by human neonatal Fc receptor (FcRn), the antibody can maintain a relatively long half-life in serum. The Fc of the antibody has a pH-dependent interaction with FcRn. The present application performs Fc engineering on the hz56G9.25L monoclonal antibody, and by changing the amino acid sequence, the goal is to enhance the in vivo half-life of the drug. Specifically, the present application performs site-directed mutagenesis on three sites of the Fc segment of hz56G9.25L (M252Y / S254T / T256E) to improve the binding ability to FcRn, so as 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.

[0469] Table 18. Amino acid sequence of CDR of hz56G9.25L-YTE antibody

[0470] Table 19. Heavy chain variable region (VH) of hz56G9.25L-YTE antibody

[0471] Table 20. Light chain variable region (VL) of hz56G9.25L-YTE antibody

[0472] Table 21. Heavy chain of hz56G9.25L-YTE antibody

[0473] Table 22. Light chain of hz56G9.25L-YTE antibody

[0474] Example 18. Biological activity of hz56G9.25L-YTE detected by IL-15 reporter cell line

[0475] Cell recovery, cell culture and activity detection method refer to Example 9.

[0476] The experimental results are shown in Figure 16 (IL-15), Figure 17 (IL-15 / IL-15Rα-Fc), hz56G9.25L-YTE and hz56G9.25L have similar functions, both can effectively block the activation of downstream signaling pathway mediated by IL-15, and the inhibition effect is better than that of the control anti-IL-15 antibody.

[0477] Example 19. Inhibition of IL-15 signal-induced immune cell proliferation by hz56G9.25L-YTE

[0478] Cell recovery: refer to Example 10.

[0479] Activity detection: refer to Example 10. The difference is that the IL-15 (Novoprotein, C016) and IL-15 / IL-15Rα-Fc stimulators have a concentration of 0.01 and 0.5 ug / mL.

[0480] The experimental results are shown in Figure 18 (IL-15), Figure 19 (IL-15 / IL-15Rα-Fc), hz56G9.25L-YTE and hz56G9.25L have similar functions, both can effectively block the activation of downstream signaling pathway mediated by IL-15, and the inhibition effect is better than that of the control anti-IL-15 antibody.

[0481] Example 20. In vivo pharmacokinetic (PK) study of antibody in hFcRn mice

[0482] The PK behavior of hz56G9.25L and hz56G9.25L-YTE in vivo was observed by mouse pharmacokinetic (PK) experiment.

[0483] The hFcRn mice were injected with 10 mpk of hz56G9.25L and hz56G9.25L-YTE via tail vein, and the mouse serum samples were collected at 5 min, 2 h, 6 h, 24 h, 48 h, 96 h, 168 h, 336 h, 504 h, 672 h and 1008 h for detection.

[0484] The detection was performed by ELISA: the plate was coated with IL-15 antigen (Rhinovirus protein, GMP-C016), then the serum to be detected was added, then the secondary antibody (BETHYL, A80-104P) against human Fc was used for detection, and finally TMB was used for color development. The PK detection results are shown in Figure 20 and Table 23. The PK profiles of hz56G9.25L and hz56G9.25L-YTE both showed IgG-like PK profile, with half-lives of 277 and 412 hours, and clearance rates of 0.27 and 0.17 ml / kg / h, respectively. It can be seen that the PK behavior of hz56G9.25L-YTE molecule is better than that of hz56G9.25L molecule.

[0485] Table 23. Antibody pharmacokinetic (PK) study in BALB / c mice

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 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; 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; 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 an amino acid sequence selected from any one of SEQ ID NOs: 19-22, or consists of said amino acid sequence, or comprises an amino acid sequence selected from any one of SEQ ID NOs: 19-22, or consists of said 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 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, (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 (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.

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 of SEQ ID NO: 16, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 20, or the heavy chain variable region consists of the amino acid sequence of SEQ ID NO: 16, and the light chain variable region consists of the amino acid sequence of 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, e.g., an Fc region from an IgG1, IgG2, IgG3, or IgG4, e.g., a human IgG1, IgG2, IgG3, or IgG4, e.g., an IgG1 Fc region comprising or consisting of the amino acid sequence of SEQ ID NO: 41, or 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, e.g., the heavy chain constant region is derived from an IgG1, 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 comprises or consists of the amino acid sequence of SEQ ID NO: 25; 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: 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 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: 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 d) 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, e) optionally isolating the antibody or antigen-binding fragment thereof, 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.

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 small bowel inflammation; or an autoimmune disease, 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).

Images