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CN120818052A - Anti-IL-15 antibodies and uses thereof - Google Patents

Anti-IL-15 antibodies and uses thereof

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Publication number
CN120818052A
CN120818052A CN202510453244.3A CN202510453244A CN120818052A CN 120818052 A CN120818052 A CN 120818052A CN 202510453244 A CN202510453244 A CN 202510453244A CN 120818052 A CN120818052 A CN 120818052A
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China
Prior art keywords
seq
amino acid
acid sequence
antibody
heavy chain
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Pending
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CN202510453244.3A
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Inventor
熊慧中
李津旸
李莉
倪萌
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Innovent Biologics Suzhou Co Ltd
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Innovent Biologics Suzhou Co Ltd
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Publication of CN120818052A publication Critical patent/CN120818052A/en
Pending legal-status Critical Current

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Abstract

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

Description

Anti-IL-15 antibodies and uses thereof
Cross Reference to Related Applications
The present application is based on and claims priority from chinese patent application number 202410444282.8, day 2024, month 4, and day 12, which is incorporated herein by reference in its entirety.
The present invention relates to novel antibodies and antibody fragments that specifically bind to IL-15 and compositions containing the antibodies or antibody fragments. Furthermore, the invention relates to nucleic acids encoding said antibodies or antibody fragments thereof and host cells comprising the same, as well as related uses. Furthermore, the invention relates to therapeutic and diagnostic uses of these antibodies and antibody fragments.
Background
The cytokine interleukin 15 (IL-15) has a variety of immune roles, playing an important role in the development, homeostasis and function of memory cd8+ T cells, NK cells and other immune cells. IL-15 is a 14-15kDa glycoprotein that binds to a heterotrimeric receptor consisting of a unique alpha subunit (IL-15 Rα) that confers receptor specificity and the receptor IL2Rβ (CD 122) and a common gamma (yc) chain (CD 132) that is shared with IL 2. However, unlike IL2, IL-15 does not (i) activate T regulatory cells (tregs) (ii) induce activation-induced cell death (AICD) in CD8 (+) T cells (iii) cause vascular capillary leakage and (iv) IL-15 can maintain survival of NK, effector CD8 (+) and memory phenotype CD8 (+) T cells.
IL-15Rα comprises three protein domains, (i) 175 amino acid extracellular domain, (ii) a single 23 amino acid transmembrane region, and (iii) 39 amino acid cytoplasmic domain, and is widely expressed in humans and mice independently of IL2R/IL-15Rβγc. IL-15Rα binds IL-15 with extremely high affinity (Kd <10 -11 M) such that it retains IL-15 on the cell surface. In this way, IL-15Rα can be through the immune synapse IL-15 into adjacent NK and T cells on IL2R/IL-15Rβγc. This synaptic mechanism limits exposure of circulating IL-15, thereby reducing the risk of autoimmunity. IL-15 can also be in membrane-bound form for several days by recovery as an endosomal vesicle forming a complex with IL-15Rα. IL-15 was listed by the NCI immunotherapeutic Congress as the first in the list of drugs with high potential for use as immunotherapeutic drugs.
Although several IL-15 antibodies have been developed in the prior art (US 7329405B2, US11130806B2 or Richmond JM et a1.,Antibody blockade ofIL-15signaling hasthe potential to durably reverse vitiligo.Sci TranslMed.2018Jul 18;10(450)：eaam7710.doi：10.1126/scitranslmed.aam7710.PMID：30021889;PMCID：PMC6495055).), these anti-IL-15 antibodies have drawbacks in terms of affinity, epitope, blocking IL-15 signaling functions, etc., and there is room for improvement.
Thus, there remains a need in the art to obtain IL-15 antibodies that function better in terms of affinity, epitope, or block IL-15 signaling.
Summary of The Invention
The present invention provides a novel antibody that specifically binds to IL-15, which selectively blocks a signaling pathway to which IL-15 is directed.
In some embodiments, the invention provides a novel IL-15 targeting monoclonal antibody with greater affinity, targeting a differentiated epitope (on the interface of IL-15 and IL2 Rgamma interactions) with a portion of the prior art anti-IL-15 antibodies, and better IL-15 signaling blocking function. In particular, the differential epitopes of the IL-15 antibodies described herein contribute to a stable blocking function against a variety of IL-15 signals, including those induced by IL-15 monomers and IL-15/IL-15Rα complexes. Thus, the anti-IL-15 antibodies described herein exhibit greater IL-15 signaling blocking function in vitro and in vivo experiments.
Thus, the present invention relates to an antibody or fragment, e.g. an antigen binding fragment, that specifically binds IL-15, e.g. human or cynomolgus monkey IL-15. In some embodiments, the antibodies or antigen binding fragments thereof of the invention specifically bind to human IL-15 (e.g., wild-type IL-15 monomer or its complex with a receptor (e.g., IL-15/IL-15Rα complex), in some embodiments, the antibodies or antigen binding fragments thereof of the invention bind to an IL-15 monomer or complex thereof with a mutation at position 108 of IL-15 (e.g., with a Q108A mutation) (e.g., an IL-15/IL-15Rα complex) with a receptor.
In some embodiments, the affinity of antigen-antibody binding is detected by ForteBio.
In some embodiments, the antibodies or antigen binding fragments thereof of the invention have a K D of less than about 4nM, 3.5nM, 3nM, 2.5nM, or 2nM, or greater than about 1 or 1.5nM, or between any range of values, with human IL-15 monomer (wild-type or variant that is not mutated at position 105, e.g., IL-15 of Q108A). In some embodiments, the K D is between about 1nM and 3.5 nM.
In some embodiments, an antibody or antigen binding fragment thereof of the invention has a K D to cynomolgus monkey IL-15 of less than about 4nM, 3.5nM, 3nM, 2.5nM, or 2nM, or greater than about 1 or 1.5nM, or between any range of values. In some embodiments, the K D is between about 1nM and 2.5nM, or between about 1.5nM and 2 nM.
In some embodiments, an antibody or antigen binding fragment thereof of the invention binds to a complex of IL-15 and its receptor (e.g., an IL-15/IL-15 ra complex) (wild-type or a variant that is H at position 105 of the IL-15, i.e., a variant that is not mutated, e.g., an IL-15/IL-15 ra complex comprising Q108A) with K D of less than about 2nM, 1.5nM, 1nM, or 0.5nM, or greater than about 0.1, 0.2, or 0.3nM, or between any range of values. In some embodiments, the K D is between about 0.1nM and 1.5nM, or between about 0.3nM and 1 nM.
The antibodies or antigen binding fragments thereof of the invention are effective in blocking downstream signaling pathways stimulated by IL-15 monomer or its complex with its receptor, e.g., IL-15/IL-15Rα complex, e.g., in IL-15reporter 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 invention are effective to block activation of downstream signaling pathways mediated by an IL-15 monomer or a complex thereof with its receptor, e.g., an IL-15/IL-15Rα complex, and/or to inhibit immune cell proliferation, e.g., in PBMC cells. In some embodiments, the antibodies or antigen binding fragments thereof of the invention are effective to block downstream signaling pathway activation mediated by IL-15 monomer or its complex with its receptor, e.g., IL-15/IL-15Rα complex, thereby inhibiting CD8T cell activation and/or IFN gamma cytokine secretion mediated by IL-15 or its complex with its receptor, e.g., IL-15/IL-15Rα, e.g., in CD8T cells. In some embodiments, the antibodies of the invention, or antigen binding fragments thereof, are capable of effectively blocking T cell proliferation, T cell infiltration, and/or T cell activation (e.g., reduced NKG2D expression levels or CD69 expression levels) mediated by IL-15 monomers or complexes thereof with their receptors, e.g., IL-15/IL-15 ra complexes. In some embodiments, the T cell is a population of T cells, e.g., comprising αβ T cells and/or γδ T cells.
In some embodiments, the antibodies or antigen binding fragments thereof of the invention have good pharmacokinetic characteristics in vivo, e.g., have a long half-life, and/or lower clearance. In some embodiments, the antibodies of the invention, or antigen binding fragments thereof, 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, such as about 250-280 hours, or a half-life of greater than 400 hours, such as greater than about 405 hours or greater than about 410 hours, such as up to about 412 hours, when administered (e.g., subcutaneously). In some embodiments, the antibodies or antigen-binding fragments thereof of the invention have a clearance of less than about 0.3mg/kg/h, such as less than about 0.27 or less than about 0.23, 0.22, 0.21, 0.2, 0.19, or 0.18, when administered (e.g., subcutaneously).
In some embodiments, the antibodies or antigen binding fragments thereof of the invention have good physicochemical properties, e.g., have good solubility or stability, e.g., have good colloidal stability and/or good thermal stability.
In some embodiments, the antibodies or antigen binding fragments thereof of the invention are effective in preventing or treating inflammation, such as skin inflammation or small intestine inflammation. In some embodiments, the antibodies of the invention, or antigen-binding fragments thereof, are effective in preventing or treating an autoimmune disease, such as graft versus host disease, e.g., graft versus host disease that blocks IL-15 signaling enhancement.
Accordingly, the present invention relates to the following specific aspects.
1. An anti-IL-15 antibody or antigen-binding fragment thereof, comprising:
HCDR1, HCDR2 and HCDR3 comprised by VH as shown in SEQ ID No. 15, 16 or 17, LCDR1, LCDR2 and LCDR3 comprised by VL as shown in SEQ ID No. 19, 20 or 21;
HCDR1, HCDR2 and HCDR3 comprised by VH as shown in SEQ ID No. 15 or 16, LCDR1, LCDR2 and LCDR3 comprised by VL as shown in SEQ ID No. 19 or 20;
HCDR1, HCDR2 and HCDR3 comprised by VH as shown in SEQ ID No. 16 or 17, LCDR1, LCDR2 and LCDR3 comprised by VL as shown in SEQ ID No. 20 or 21;
HCDR1, HCDR2 and HCDR3 comprised by VH as shown in SEQ ID No. 15, LCDR1, LCDR2 and LCDR3 comprised by VL as shown in SEQ ID No. 19;
HCDR1, HCDR2 and HCDR3 comprised by VH as shown in SEQ ID No. 16, LCDR1, LCDR2 and LCDR3 comprised by VL as shown in SEQ ID No. 20;
HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:17, LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:21, or
HCDR1, HCDR2 and HCDR3 contained in VH shown in SEQ ID NO. 18 and LCDR1, LCDR2 and LCDR3 contained in VL 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 (HCDR 1), a second heavy chain complementarity determining region (HCDR 2), a third heavy chain complementarity determining region (HCDR 3), and a first light chain complementarity determining region (LCDR 1), a second light chain complementarity determining region (LCDR 2), and a third light chain complementarity determining region (LCDR 3), wherein the antibody or antigen-binding fragment thereof
The HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 respectively comprise or consist of the amino acid sequences shown as 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
The HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 respectively comprise or consist of the amino acid sequences shown as 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
The HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 respectively comprise or consist of the amino acid sequences shown as 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
The HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 respectively comprise or 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, respectively, 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 embodiment 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 having at least 90% identity to an amino acid sequence selected from any one of SEQ ID NOs 15-18.
4. The anti-IL-15 antibody or antigen-binding fragment thereof of any one of embodiments 1-3, comprising a light chain variable region (VL), wherein the light chain variable region comprises, consists of, or consists of an amino acid sequence having at least 90% identity to an amino acid sequence set forth in any one of SEQ ID NOs 19-22.
5. An anti-IL-15 antibody or antigen-binding fragment thereof comprising
(I) An amino acid sequence comprising or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence shown in SEQ ID No. 15, 16 or 17, or a VH consisting of said amino acid sequence, and an amino acid sequence comprising or consisting of an amino acid sequence shown in SEQ ID No. 19, 20 or 21 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence thereof;
(ii) An amino acid sequence comprising or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID No. 15 or 16, or a VH consisting of said amino acid sequence, and an amino acid sequence comprising or consisting of an amino acid sequence shown in SEQ ID No. 19 or 20 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto;
(iii) An amino acid sequence comprising or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID No. 16 or 17 or a VH consisting of said amino acid sequence, and an amino acid sequence comprising or consisting of an amino acid sequence shown in SEQ ID No. 20 or 21 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto;
(iv) An amino acid sequence comprising or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID No. 15 or a VH consisting of said amino acid sequence, and an amino acid sequence comprising or consisting of an amino acid sequence shown in SEQ ID No. 19 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto;
(v) An amino acid sequence comprising or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID No. 16 or a VH consisting of said amino acid sequence, and an amino acid sequence comprising or consisting of an amino acid sequence shown in SEQ ID No. 20 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence;
(vi) Comprising or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO. 17, or a VH consisting of said amino acid sequence, and comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO. 21, or a VL consisting of said amino acid sequence
(Vii) Comprising or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO. 18, or a VH consisting of said amino acid sequence, and comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO. 22.
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
A) The heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO. 15, 16 or 17, and the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO. 19, 20 or 21;
b) The heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO. 15 or 16, and the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO. 19 or 20;
c) The heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO. 16 or 17, and the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO. 20 or 21;
d) The heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO. 15, and the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO. 19;
e) The heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO. 16, and the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO. 20;
f) The heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO. 17, and the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO. 21, or
G) The heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO. 18, and the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO. 22.
7. The anti-IL-15 antibody or antigen-binding fragment thereof of any one of embodiments 1-6 comprising an Fc region, e.g., the Fc region is from an Fc region of IgG1, igG2, igG3, or IgG4, e.g., a Fc region of human IgG1, igG2, igG3, or IgG4, e.g., the IgG1 Fc region
(I) Comprising or consisting of the amino acid sequence of SEQ ID NO. 41, or
(Ii) Comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO. 41.
8. The anti-IL-15 antibody or antigen-binding fragment thereof of any one of embodiments 1-7 comprising a heavy chain constant region, e.g., a constant region from IgG1, igG2, igG3, or IgG4, e.g., a human IgG1, igG2, igG3, or IgG4, e.g., the IgG1 heavy chain constant region
(I) An amino acid sequence comprising or consisting of SEQ ID NO 25, or
(Ii) Comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO. 25.
9. The anti-IL-15 antibody or antigen-binding fragment thereof of embodiment 7 or 8, wherein the heavy chain constant region or Fc region comprises a mutation, e.g., an L234A/L235A mutation, e.g., the heavy chain constant region, having reduced binding to an fcγ receptor
(I) An amino acid sequence comprising or consisting of SEQ ID NO. 26, or
(Ii) Comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO. 26.
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 receptor, such as a YTE mutation (M252Y/S254T/T256E).
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 comprises both a mutation that reduces binding to an fcγ receptor and a mutation that increases binding to an FcRn receptor, e.g., comprises an L234A/L235A mutation and a YTE mutation, e.g.
The heavy chain constant region
(I) An amino acid sequence comprising or consisting of SEQ ID NO 27, or
(Ii) Comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO 27, or
The Fc region
(I) Comprising or consisting of the amino acid sequence of SEQ ID NO. 42, or
(Ii) Comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO. 42.
12. The anti-IL-15 antibody or antigen-binding fragment thereof of any one of embodiments 1-11, comprising a light chain constant region that 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
(I) Comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity 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.
20. The anti-IL-15 antibody or antigen-binding fragment thereof of any one of embodiments 1-12 comprising or consisting of a heavy chain and/or a light chain, wherein the heavy chain comprises or consists of the VH and heavy chain constant regions of any one of embodiments 1-12 and/or the light chain comprises or consists of the VL and light chain constant regions of any one of embodiments 1-12;
optionally, the heavy chain comprises or consists of the amino acid sequence shown 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, and/or the light chain comprises or consists of the amino acid sequence shown 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;
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.
13. The anti-IL-15 antibody or antigen-binding fragment thereof of any one of embodiments 1-13, wherein the antibody is a monoclonal antibody.
14. The anti-IL-15 antibody or antigen-binding fragment thereof of any one of embodiments 1-14, wherein the antibody is a humanized antibody or chimeric antibody.
15. The anti-IL-15 antibody or antigen-binding fragment thereof of any one of embodiments 1-15, wherein the antigen-binding fragment is an antibody fragment selected from the group consisting of Fab, fab '-SH, fv, single chain antibody (e.g., scFv), (Fab') 2, dAb (domainantibody), diabody, or linear antibody.
16. The anti-IL-15 antibody or antigen-binding fragment thereof of any one of embodiments 1-16, wherein the antibody or antigen-binding fragment thereof has one or more of the following properties:
a) An IL-15 monomer that binds with high affinity to a human or cynomolgus monkey or a complex thereof with its receptor, e.g., an IL-15/IL-15Rα complex, wherein in the monomer or complex, the amino acid at position 105 of IL-15 is H;
b) Effectively block downstream signaling pathways stimulated by IL-15 monomer or its complex with its receptor, e.g., IL-15/IL-15Rα complex;
c) Inhibiting IL-15 or its complex with a receptor, such as IL-15/IL-15 ra mediated activation of CD8T cells and/or IFNg cytokine secretion, e.g. in CD8T cells;
d) Effectively blocking T cell proliferation, T cell infiltration and/or T cell activation (e.g., decreased NKG2D expression levels or CD69 expression levels) mediated by IL-15 monomer or its complex with its receptor, e.g., IL-15/IL-15 ra complex;
e) Has good physicochemical properties and/or pharmacokinetic characteristics, for example, has good stability (e.g., colloidal stability and/or thermal stability), good solubility, long half-life, and/or low clearance;
f) Effective in preventing or treating inflammation, such as skin inflammation or small intestine inflammation;
g) Can effectively prevent or treat autoimmune diseases such as graft versus host diseases, such as graft versus host diseases which effectively block IL-15 signal enhancement.
17. An isolated nucleic acid encoding the anti-IL-15 antibody or antigen-binding fragment thereof of any one of embodiments 1-17.
18. A vector comprising the nucleic acid of embodiment 18, preferably the vector is an expression vector.
19. A host cell comprising the nucleic acid of embodiment 18 or the vector of embodiment 19, preferably the host cell is prokaryotic or eukaryotic, more preferably selected from a yeast cell, a mammalian cell (e.g., 293 cell or CHO cell, e.g., CHO-K cell or HEK293 cell) or other cell suitable for the production of antibodies or antigen-binding fragments thereof.
20. A method of preparing an anti-IL-15 antibody or antigen-binding fragment thereof, comprising
D) Culturing the host cell of embodiment 20 under conditions suitable for expression of a nucleic acid encoding the anti-IL-15 antibody or antigen-binding fragment thereof of any one of embodiments 1-17,
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, e.g. by protein a.
21. An immunoconjugate comprising the anti-IL-15 antibody or antigen-binding fragment thereof of any one of embodiments 1-17 and other substances, e.g., toxins, small molecule drugs, cytotoxic agents, apoptotic agents, chelators, immunomodulators, e.g., anti-inflammatory agents or immunosuppressants.
22. 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.
23. A pharmaceutical combination comprising an 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., selected from cytokines, other antibodies, small molecule drugs, or immunomodulators (e.g., anti-inflammatory agents or immunosuppressants).
24. A method of preventing or treating an IL-15-associated disease and/or disorder in a subject, 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 of embodiment 24.
25. The method of embodiment 25, wherein the subject has abnormal expression of IL-15 or a receptor complex thereof, e.g., IL-15/IL-15 ra complex, or a disease or disorder associated with abnormal activation of a mediated signaling pathway, in comparison to a healthy individual.
26. The method of embodiment 25 or 26, wherein the disease or disorder is selected from a tumor, such as cancer, or an inflammatory or inflammatory disorder, such as an auto-inflammatory disease, e.g., skin inflammation or small intestine inflammation, or an autoimmune disease, e.g., graft versus host disease.
27. The method of any one of embodiments 25-27, wherein the method further comprises administering one or more other therapies, such as therapeutic modalities, and/or other therapeutic agents, such as selected from cytokines, other antibodies, small molecule drugs, or immunomodulators (e.g., anti-inflammatory agents or immunosuppressants).
Drawings
FIG. 1 shows the inhibition of IL-15 reporter cell line signaling by anti-IL-15 chimeric antibodies and control antibodies.
FIG. 2 shows the inhibition of IL-15 reporter cell line signaling by anti-IL-15 antibodies.
FIG. 3 shows the inhibition of IL-15 reporter cell line signaling by anti-IL-15 antibodies.
FIG. 4 shows inhibition of IL-15 induced immune cell proliferation by anti-IL-15 antibodies.
FIG. 5 shows inhibition of IL-15 induced immune cell proliferation by anti-IL-15 antibodies.
FIG. 6 shows inhibition of IL-15 induced immune cell proliferation by anti-IL-15 antibodies.
FIG. 7 shows inhibition of IL-15 induced activation of CD8T cells by anti-IL-15 antibodies.
FIG. 8 shows inhibition of IL-15-induced activation of CD8T cells by anti-IL-15 antibodies.
FIG. 9 shows inhibition of IL-15 induced skin T cell proliferation by anti-IL-15 antibodies.
FIG. 10 shows inhibition of IL-15-induced skin T cell activation by anti-IL-15 antibodies.
FIG. 11 shows inhibition of IL-15-induced activation of small intestine CD8T cells by anti-IL-15 antibodies.
FIG. 12 shows inhibition of IL-15-induced activation of spleen CD8T cells by anti-IL-15 antibodies.
FIG. 13 shows the IL-15 enhanced GvHD inhibition by anti-IL-15 antibodies.
FIG. 14 shows the IL-15 enhanced GvHD inhibition by anti-IL-15 antibodies.
FIG. 15 shows in vivo Pharmacokinetic (PK) studies of antibodies in BALB/c mice.
FIG. 16 shows inhibition of IL-15 reporter cell line signaling by anti-IL-15 antibodies.
FIG. 17 shows IL-15 reporter cell line signal inhibition by anti-IL-15 antibodies.
FIG. 18 shows inhibition of IL-15 induced immune cell proliferation by anti-IL-15 antibodies.
FIG. 19 shows inhibition of IL-15 induced immune cell proliferation by anti-IL-15 antibodies.
FIG. 20 shows in vivo Pharmacokinetic (PK) studies of antibodies in BALB/c mice.
Detailed Description
Before the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein as these may vary.
I. Definition of the definition
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which 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 invention belongs.
For purposes of explaining the present specification, the following definitions will be used, and terms used in the singular form may also include the plural, and vice versa, as appropriate. 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.
The term "about" when used in conjunction with a numerical value is intended to encompass numerical values within a range having 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.
As used herein, the term "and/or" means any one of the selectable items or two or more or all of the selectable items.
As used herein, the terms "comprises" or "comprising" are intended to include the stated elements, integers or steps but do not exclude any other elements, integers or steps. In this document, the terms "comprises" or "comprising" when used herein, unless otherwise indicated, also encompass the instances of the recited elements, integers, or steps in combination. For example, when referring to an antibody variable region "comprising" a particular sequence, it is also intended to encompass antibody variable regions consisting of that particular sequence.
An "isolated" antibody or molecule is an antibody or molecule that has been separated from components of its natural environment. In some embodiments, the antibody or molecule is purified to greater than 95% or 99% purity, as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC).
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" untreated IL-15 polypeptides and any form of IL-15 polypeptide resulting from intracellular processing. 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 invention, the human IL-15 protein comprises or consists of the amino acid sequence shown in SEQ ID NO. 39. In one embodiment of the invention, the cynomolgus monkey IL-15 protein comprises or consists of the amino acid sequence shown in SEQ ID NO. 40.
The terms "whole antibody" or "full length antibody" are used interchangeably herein to refer to an antibody molecule having the molecular structure of a native immunoglobulin. In the case of conventional four-chain IgG antibodies, the full-length antibody comprises two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. In the case of heavy chain antibodies having only heavy chains but lacking light chains, the full length antibody comprises two heavy chains (H) interconnected by disulfide bonds. For conventional four-chain IgG antibodies, the full-length antibody heavy chain typically consists 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 3 domains CH1, CH2 and CH3. Full length antibody light chains consist of a light chain variable region (abbreviated herein as VL) and a light chain constant region, wherein the light chain constant region consists of one domain CL. Each heavy chain variable region VH and each light chain variable region consists of three CDRs and 4 FRs, arranged from amino-terminus to carboxy-terminus in the 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.
The term "antigen-binding fragment" of an antibody is a different molecule than a full-length antibody, which comprises a portion of a full-length antibody, but which is capable of binding to the antigen of the full-length antibody or competing with the full-length antibody (i.e., with the full-length antibody from which the antigen-binding fragment was derived) for binding to the antigen. Antigen binding fragments may be prepared 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 or fragments thereof, or camelid antibodies, diabodies, single domain antibodies (sdabs), nanobodies. For example, fab fragments can be obtained by papain digestion of full length antibodies. In addition, complete antibody production F (ab ') 2, which is a dimer of Fab', is a bivalent antibody fragment by pepsin digestion under the disulfide bonds of the hinge region. F (ab ') 2 can be reduced under neutral conditions by breaking disulfide bonds in the hinge region, thereby converting F (ab ') 2 dimers to Fab ' monomers. The Fab' monomer is essentially a Fab fragment with a hinge region. Fv fragments consist 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 encoded by separate genes, but recombinant methods can also be used, using a synthetic linker peptide to join the two domains so that they are produced as a single protein chain in which the VL and VH regions pair to form a single chain Fv (scFv).
The term "single chain antibody (scAb)" is used herein in its broadest sense and specifically covers antibodies with mono-or multi-specificity (e.g., bispecific) that are initially produced as a single continuous polypeptide chain. Such single chain antibodies include, but are not limited to, having two linked VL and VH regions. In one embodiment, the single chain antibody is an scFv.
A "diabody" is a bivalent minibody constructed by gene fusion, for example, which is a dimer composed of two polypeptide chains. The VL and VH domains of each polypeptide chain of a diabody are joined by a linker, whereby the VL and VH encoded in the same polypeptide chain form dimers having different single chain variable region fragments. Diabodies generally have two antigen binding sites.
"Complementarity determining regions" or "CDR regions" or "CDRs" are regions of an antibody variable domain that are hypervariable in sequence and form structurally defined loops ("hypervariable loops") and/or contain antigen-contacting residues ("antigen-contacting points"). CDRs are mainly responsible for binding to the epitope. CDRs of the heavy and light chains are commonly referred to as CDR1, CDR2, and CDR3, numbered sequentially from the N-terminus. CDRs located within the antibody heavy chain variable domain are referred to as HCDR1, HCDR2 and HCDR3, while CDRs located within the antibody light chain variable domain are referred to as LCDR1, LCDR2 and LCDR3. In a given light chain variable region or heavy chain variable region amino acid sequence, the exact amino acid sequence boundaries of each CDR can be determined using any one or a combination of a number of well-known antibody CDR assignment schemes including, for example, chothia (Chothia et al (1989) Nature 342:877-883, al-Lazikani et al ,"Standard conformationsfor the canonical structures of immunoglobulins",Journal of Molecular Biology,273,927-948(1997)), based on Kabat (Kabat et al Sequences of Proteins of Immunological Interest, 4th edition ,U.S.Department of Health and Human Services,National Institutes of Health(1987)),AbM(University of Bath),Contact(University College London), International ImMunoGene Tics database (IMGT) (on the world Wide Web. Impulse. CInes. Fr/on) of antibody three-dimensional structure and CDR loop topology, and North definition based on neighbor-propagating clustering (affinity propagation clustering) using a large number of crystal structures.
The following is an exemplary scheme for the range of regions of CDRs defined using the Kabat, chothia, kabat & Chothia (determined by combining Kabat and Chothia schemes) scheme.
CDR Kabat protocol Chothia protocol Kabat&Chothia
LCDR1 L24-L34 L26-L32
LCDR2 L50-L56 L50-L52
LCDR3 L89-L97 L91-L96
HCDR1 H31-H35 H26-H32 H26-H35
HCDR2 H50-H65 H53-H55
HCDR3 H95-H102 H96-H101
In the present invention, unless otherwise indicated, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the above-described ways. CDRs may also be determined based on having the same Kabat numbering positions as the reference CDR sequences (e.g., any of the exemplary CDRs of the invention).
In the present invention, unless otherwise indicated, when referring to residue positions in the antibody variable region (including heavy chain variable region residues and light chain variable region residues) it is meant a numbering position according to the Kabat numbering system (Kabat et al ,Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md.(1991)).
In some embodiments, the CDR1 of the heavy chain variable region of an antibody of the invention is determined according to the Kabat & Chothia protocol, and CDR2 and CDR3 are determined according to the Kabat protocol. In some embodiments, the CDRs of the light chain variable region of an antibody of the invention are determined according to the Kabat protocol. In some embodiments, antibodies of the invention
HCDR1 was determined according to the Kabat & Chothia protocol and HCDR2 and HCDR3 were each determined according to the Kabat protocol;
LCDR1, LCDR2 and LCDR3 were each determined according to the Kabat protocol.
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 conversely, a reference antibody blocks 50%, 60%, 70%, 80%, 90% or 95% or more of the binding of the antibody to its antigen in a competition assay.
An antibody that competes with a reference antibody for binding to its antigen refers to an antibody that blocks 50%, 60%, 70%, 80%, 90% or more than 95% of the binding of the reference antibody to its antigen in a competition assay. In contrast, the reference antibody blocks 50%, 60%, 70%, 80%, 90% or 95% or more of the binding of the antibody to its antigen in a competition assay. Numerous types of competitive binding assays can be used to determine whether an antibody competes with another, e.g., solid phase direct or indirect Radioimmunoassay (RIA), solid phase direct or indirect Enzyme Immunoassay (EIA), sandwich competition assays.
An antibody that inhibits (e.g., competitively inhibits) binding of a reference antibody to its antigen refers to an antibody that inhibits 50%, 60%, 70%, 80%, 90% or more than 95% of binding of the reference antibody to its antigen. Conversely, a reference antibody inhibits the binding of 50%, 60%, 70%, 80%, 90% or 95% or more of the antibody to its antigen. The binding of an antibody to its antigen can be measured by affinity (e.g., equilibrium dissociation constant). Methods for determining affinity are known in the art.
An antibody that exhibits the same or similar binding affinity and/or specificity as a reference antibody refers to an antibody that is capable of having at least 50%, 60%, 70%, 80%, 90% or more than 95% of the binding affinity and/or specificity of the reference antibody. This can be determined by any method known in the art for determining binding affinity and/or specificity.
The term "chimeric antibody" is an antibody molecule in which (a) a constant region or portion thereof is altered, substituted, or exchanged such that the antigen binding site is linked to a constant region of a different or altered class, effector function, and/or species or a completely different molecule (e.g., enzyme, toxin, hormone, growth factor, drug) or the like that confers novel properties to the chimeric antibody, or (b) a variable region or portion thereof is altered, substituted, or exchanged with a variable region having different or altered antigen specificity. For example, a mouse antibody may be modified by replacing its constant region with a constant region derived from a human immunoglobulin. Due to the replacement with human constant regions, the chimeric antibody can retain its specificity in recognizing antigen while having reduced immunogenicity in humans as compared to the original mouse antibody.
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, for example, when administered to a human as a therapeutic. This can be accomplished, for example, by retaining non-human antigen binding sites and replacing the remainder of the antibody with their human counterpart (i.e., replacing the constant and non-binding portions of the variable regions with the counterpart of the human antibody).
The term "Fc domain" or "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. The native immunoglobulin "Fc domain" comprises two or three constant domains, namely a CH2 domain, a CH3 domain, and optionally a CH4 domain. For example, in natural antibodies, the immunoglobulin Fc domain comprises the second and third constant domains (CH 2 domain and CH3 domain) derived from the two heavy chains of antibodies of the IgG, igA and IgD classes, or comprises the second, third and fourth constant domains (CH 2 domain, CH3 domain and CH4 domain) derived from the two heavy chains of antibodies of the IgM and IgE classes. Unless otherwise indicated herein, amino acid residue numbering in the Fc region or heavy chain constant region is according to the EU numbering system (also referred to as the EU index) as described in Kabat et al Sequences of Proteins of Immunological Interes, 5 th edition, public HEALTH SERVICE, national Institutes of Health, bethesda, MD, 1991.
"Conservative changes" as described herein include substitutions, deletions or additions to the polypeptide sequence, but do not substantially alter the desired functional activity of the polypeptide sequence. In some embodiments, the conservative change is a conservative substitution. Conservative substitutions refer to the substitution of one amino acid with another within the same class, e.g., the substitution of one acidic amino acid with another acidic amino acid, the substitution of one basic amino acid with another basic amino acid, or the substitution of one neutral amino acid with another neutral amino acid. For example, conservative substitutions often result in the substitution of an amino acid for a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. The following list 8 groups contain amino acids with conservative substitutions for each other, 1) alanine (A), glycine (G), 2) aspartic acid (D), glutamic acid (E), 3) asparagine (N), glutamine (Q), 4) arginine (R), lysine (K), 5) isoleucine (I), leucine (L), methionine (M), valine (V), 6) phenylalanine (F), tyrosine (Y), tryptophan (W), 7) serine (S), threonine (T), and 8) cysteine (C), methionine (M). In some embodiments, the term "conservative change" when applied to an antibody molecule amino acid sequence is used to refer to an amino acid modification that does not significantly affect or alter the antigen binding characteristics of interest of the antibody molecule of the invention that contains the amino acid sequence. For example, conservatively altered variants retain at least 80%,85%,90%,95%,98%,99% or more, e.g., 100 to 110% or more, of the binding affinity for the antigen of interest relative to the parent antibody.
The term "vector" as used herein refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures and that bind to the genome of a host cell into which they have been introduced. Some vectors are capable of directing the expression of a nucleic acid to which they are operably linked. Such vectors are referred to herein as "expression vectors".
An "immunoconjugate" is an antibody conjugated to one or more other substances, including but not limited to a label. In some embodiments, the immunoconjugate encompasses an antibody-drug conjugate (ADC).
The term "therapeutic agent" as described herein encompasses any substance that is effective in preventing or treating a disease associated with inappropriate activation of an IL-15 or IL-15 receptor complex mediated pathway, including cytokines, other antibodies, small molecule drugs, or immunomodulators (e.g., anti-inflammatory agents or immunosuppressants).
The term "small molecule drug" refers to a low molecular weight organic compound capable of modulating biological processes. A "small molecule" is defined as a molecule having a molecular weight of less than 10kD, typically less than 2kD and preferably less than 1 kD. Small molecules include, but are not limited to, inorganic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptidomimetics, and antibody mimetics. As a therapeutic agent, small molecules may be more cell permeable, less susceptible to degradation, and less prone to eliciting an immune response than large molecules.
The term "immunomodulator" as used herein refers to a natural or synthetic active agent or drug that inhibits or modulates an immune response. The immune response may be a humoral response or a cellular response. Immunomodulators include anti-inflammatory agents or immunosuppressants.
As used herein, an "immunosuppressant," "immunosuppressant drug," or "immunosuppressant" is a therapeutic agent used in immunosuppressive therapy to inhibit or prevent the activity of the immune system.
The term "effective amount" refers to an amount or dose of an antibody or fragment or conjugate or composition or combination of the invention that, upon administration to a patient in single or multiple doses, produces a desired effect in a patient in need of treatment or prevention.
"Therapeutically effective amount" means an amount effective to achieve the desired therapeutic result at the desired dosage and for the desired period of time. A therapeutically effective amount is also an amount in which any toxic or detrimental effect of the antibody or antibody fragment or conjugate or composition or combination thereof is less than a therapeutically beneficial effect. A "therapeutically effective amount" preferably inhibits a measurable parameter (e.g., for IL-15 signaling pathway) 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 define the amount of treatment necessary to treat a condition (e.g., inflammation) or reduce or eliminate an immune response in a treatment regimen.
"Prophylactically effective amount" means an amount effective to achieve the desired prophylactic result at the desired dosage and for the desired period of time. Typically, since the prophylactic dose is administered in the subject prior to or at an earlier stage of the 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 define the amount of prophylaxis necessary to prevent the progression of a condition or disease and symptoms (e.g., inflammation) in a therapeutic regimen,
The terms "host cell", "host cell line" and "host cell culture" are used interchangeably and refer to a cell into which exogenous nucleic acid is introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells" which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages. The progeny may not be exactly identical in nucleic acid content to the parent cell, but may comprise the mutation. Included herein are mutant progeny selected or selected for the same function or biological activity in the initially transformed cells.
The term "label" as used herein refers to a compound or composition that is directly or indirectly conjugated or fused to and facilitates detection of an agent (such as a polynucleotide probe or antibody) to which it is conjugated or fused. The label itself may be detectable (e.g., radioisotope labels or fluorescent labels) or in the case of enzymatic labels may catalyze chemical alteration of a substrate compound or composition which is detectable. 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 as well as indirect labeling of the probe or antibody by reaction with another reagent that is directly labeled.
The terms "individual," "subject," or "subject" as used herein are used interchangeably and include mammals. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is a human.
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 may be solid tissue, like a tissue sample or biopsy or puncture from a fresh, frozen and/or preserved organ or tissue, blood or any blood component, body fluids such as cerebrospinal fluid, amniotic fluid (amniotic fluid), peritoneal fluid (ascites), or interstitial fluid, cells from a subject at any time during pregnancy or development. Tissue samples may contain compounds that are not naturally intermixed with the tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and the like.
"Nucleic acid encoding an anti-IL-15 antibody or fragment thereof" refers to one or more nucleic acid molecules encoding an antibody heavy or light chain (or fragment thereof, e.g., a heavy chain variable region or a light chain variable region), including such nucleic acid molecules in a single vector or in separate vectors, as well as such nucleic acid molecules present at one or more positions in a host cell.
"Percent (%) identity" of an amino acid sequence refers to the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues of a particular amino acid sequence shown in the present specification, after aligning the candidate sequence to the particular amino acid sequence shown in the present specification and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and without regard to any conservative substitutions as part of the sequence identity. In some embodiments, the invention contemplates variants of the antibody molecules of the invention that have substantial identity, e.g., at least 80%,85%,90%,95%,97%,98% or 99% or more identity, to the antibody molecules specifically disclosed herein and sequences thereof. The variant may comprise a conservative change.
The term "pharmaceutical adjuvant" refers to diluents, adjuvants (e.g., freund's adjuvant (complete and incomplete)), excipients, carriers or stabilizers, etc. for administration with the active substance.
The term "pharmaceutical composition" refers to a composition that exists in a form that is effective to allow the biological activity of the active ingredient contained therein, and that does not contain additional ingredients that have unacceptable toxicity to the subject or individual to whom the composition is administered.
As used herein, the term "pharmaceutical combination or combination product" refers to a non-fixed combination product or fixed combination product, including but not limited to a kit/kit, pharmaceutical composition. The term "non-fixed combination" means that the active ingredients (e.g., (i) an antibody of the invention, and (ii) other therapeutic agent) are administered to a patient simultaneously, without specific time constraints, or sequentially at the same or different time intervals, in separate entities, wherein such administration provides prophylactically or therapeutically effective levels of two or more active agents in the patient. The term "fixed combination" means that two or more active agents are administered to a patient simultaneously in the form of a single entity. The dosages and/or time intervals of the two or more active agents are preferably selected so that the combined use of the parts will produce an effect in the treatment of a disease or condition that is greater than that achieved by either component alone. The components can be in the form of separate preparations, and the preparations can be the same or different.
The term "combination therapy" refers to the administration of two or more therapeutic agents or modes of treatment (e.g., radiation therapy or surgery) to treat the diseases described herein. Such administration includes co-administration of the therapeutic agents in a substantially simultaneous manner, e.g., in a single capsule with a fixed ratio of active ingredients. Or such administration includes co-administration of the individual active ingredients in multiple or separate containers (e.g., tablets, capsules, powders, and liquids). The powder and/or liquid may be reconstituted or diluted to the desired dosage prior to administration. In addition, such administration also includes the use of each type of therapeutic agent in a sequential manner at about the same time or at different times. In either case, the treatment regimen will provide a beneficial effect of the pharmaceutical combination in treating the disorders or conditions described herein.
As used herein, "treating" refers to slowing, interrupting, blocking, alleviating, stopping, reducing, or reversing the onset of symptoms, complications, or biochemical indicators of a disease, alleviating symptoms, or preventing or inhibiting the further development of a disease, condition, or disorder.
As used herein, "preventing" includes inhibition of the occurrence or progression of a disease or disorder or a symptom of a particular disease or disorder.
II antibodies
In some embodiments, the anti-IL-15 antibodies or antigen-binding fragments thereof of the invention comprise 3 complementarity determining regions (HCDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3.
In some embodiments, the anti-IL-15 antibodies or antigen-binding fragments thereof of the invention comprise 3 complementarity determining regions (LCDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3.
In some embodiments, the anti-IL-15 antibodies or antigen-binding fragments thereof of the invention comprise 3 complementarity determining regions (HCDRs) from the heavy chain variable region and 3 complementarity determining regions (LCDRs) from the light chain variable region.
In some aspects, the anti-IL-15 antibodies or antigen-binding fragments thereof of the invention comprise a heavy chain variable region (VH). In some aspects, the anti-IL-15 antibodies or antigen-binding fragments thereof of the invention comprise a light chain variable region (VH). In some aspects, the anti-IL-15 antibodies or antigen-binding fragments thereof of the invention comprise 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 (CDRs) from the heavy chain variable region, HCDR1, HCDR2 and HCDR3. In some embodiments, the light chain variable region comprises 3 Complementarity Determining Regions (CDRs) from the light chain variable region, LCDR1, LCDR2 and LCDR3.
In some embodiments, the anti-IL-15 antibodies or antigen-binding fragments thereof of the invention further comprise an antibody heavy chain constant region. In some embodiments, the anti-IL-15 antibodies or antigen-binding fragments thereof of the invention further comprise an antibody light chain constant region. In some embodiments, the invention of the anti-IL-15 antibody or antigen binding fragment thereof also contains heavy chain constant region and light chain constant region.
In some embodiments, the heavy chain variable regions described herein:
(i) Comprising or consisting of an amino acid sequence having 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 any of SEQ ID NOs 15 to 18, or
(Ii) Comprising or consisting of an amino acid sequence selected from any one of SEQ ID NOs 15 to 18, or
(Iii) An amino acid sequence comprising 1 or more (preferably NO more than 10, more preferably NO more than 5, 4, 3, 2, 1) amino acid changes (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 to 18 consists of said amino acid sequence, preferably said amino acid changes do not occur in the CDR regions.
In some embodiments, the light chain variable regions of the invention
(I) Comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from any of SEQ ID NOs 19 to 22, or
(Ii) Comprising or consisting of an amino acid sequence selected from any one of SEQ ID NOS.19-22, or
(Iii) An amino acid sequence comprising 1 or more (preferably NO more than 10, more preferably NO more than 5, 4, 3, 2, 1) amino acid changes (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.19-22, preferably,
The amino acid changes do not occur in the CDR regions.
In some embodiments, the 3 complementarity determining regions (HCDRs) from the heavy chain variable region described herein, HCDR1, HCDR2 and HCDR3 are selected from
(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;
(ii) A sequence comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) in total on the three HCDR regions relative to the sequence of any of (i),
Wherein the HCDR may be determined according to any CDR-determining scheme, e.g. kabat, abM, chothia, contact or IMGT schemes, respectively, or a combination thereof;
for example, the HCDR1 is determined according to the Kabat & Chothia protocol, and the HCDR2 and HCDR3 are each determined according to the Kabat protocol.
In some embodiments, the 3 complementarity determining regions (LCDRs) from the light chain variable region described herein, LCDR1, LCDR2 and LCDR3 are selected from
(I) Three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as set forth in any one of SEQ ID NOS.19-22, or
(Ii) A sequence comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) in total on the three LCDR regions relative to the sequence of any one of (i),
Wherein the HCDR may be determined according to any CDR-determining scheme, e.g. kabat, abM, chothia, contact or IMGT schemes, respectively, or a combination thereof;
for example, the LCDR1, 2 and 3 are each determined according to the Kabat protocol.
In some embodiments, the HCDR1 comprises or consists of the amino acid sequence of SEQ ID No. 1,2 or 3, or the HCDR1 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID No. 1,2 or 3.
In some embodiments, the HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO. 4, 5 or 6, or the HCDR2 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) as compared to the amino acid sequence of SEQ ID NO. 4, 5 or 6.
In some embodiments, the HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO. 7, 8 or 9, or the HCDR3 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) as compared to the amino acid sequence of SEQ ID NO. 7, 8 or 9.
In some embodiments, the LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO. 10, 11 or 12, or LCDR1 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) as compared to the amino acid sequence of SEQ ID NO. 10, 11 or 12.
In some embodiments, the LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO. 13, or LCDR2 comprises an amino acid sequence having one, two, or three changes (preferably amino acid substitutions, preferably conservative substitutions) as compared to the amino acid sequence of SEQ ID NO. 13.
In some embodiments, the LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO. 14 or 43, or LCDR3 comprises an amino acid sequence having one, two, or three changes (preferably amino acid substitutions, preferably conservative substitutions) as compared to the amino acid sequence of SEQ ID NO. 14 or 43.
In some embodiments, the antibody heavy chain constant region of the invention is a heavy chain constant region from IgG1, igG2, igG3, or IgG4, e.g., a constant region from human IgG1, igG2, igG3, or IgG 4. In some embodiments, the constant region has a modification in the Fc region thereof. In some embodiments, the Fc region is an Fc region from IgG1, igG2, igG3, or IgG4, e.g., an Fc region from human IgG1, igG2, igG3, or IgG 4.
In one embodiment, the Fc region is modified in the nature of its effector function (e.g., complement activation 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, effector function is reduced or eliminated by a method selected from the group consisting of using an Fc isoform naturally having reduced or eliminated effector function, and Fc region modification. The Fc region may also comprise modifications that alter the binding affinity for one or more Fc receptors.
In one embodiment, the Fc receptor is an fcγ receptor, in particular a human fcγ receptor. In some embodiments, the Fc region comprises a mutation that reduces binding to an fcγ receptor. In some embodiments, the Fc region for use in the present invention has an L234A/L235A mutation that reduces binding to fcγ receptors. In some embodiments, the heavy chain constant regions of the invention comprise mutations that have reduced binding to fcγ receptors, such as the L234A/L235A mutation.
In some embodiments, the Fc receptor is FcRn, particularly the human FcRn receptor. In some embodiments, the Fc region has a mutation that increases binding to FcRn receptor. In some embodiments, the Fc region used in the present invention has YTE mutations (M252Y/S254T/T256E) that bind to the FcRn receptor. In some embodiments, the heavy chain constant regions of the invention comprise mutations with increased binding to FcRn receptor, such as YTE mutations (M252Y/S254T/T256E).
In some embodiments, the Fc region or heavy chain constant region of the invention comprises both a mutation that reduces binding to fcγ receptor and a mutation that increases binding to FcRn receptor, e.g., comprising an L234A/L235A mutation and a YTE mutation.
In some embodiments, the heavy chain constant region
(I) Comprising or consisting of an amino acid sequence selected from SEQ ID NO. 25, 26 or 27;
(ii) Comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID No. 25, 26 or 27;
(iii) Comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO. 26 and having an L234A/L235A mutation, or
(Iv) Comprising an amino acid sequence having at least 85%,90%, 91%, 92%, 93%, 94%, 95%,96%,97%,98% or 99% identity to the amino acid sequence of SEQ ID NO. 27, and having an L234A/L235A mutation and a YTE mutation.
In some embodiments, the heavy chain constant regions of the invention lack a terminal lysine.
In some embodiments, the Fc region
(I) Comprising or consisting of an amino acid sequence selected from SEQ ID NO. 41 or 42;
(ii) Comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO. 41 or 42, or
(Iii) Comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO. 42, and having an L234A/L235A mutation and a YTE mutation.
In some embodiments, the antibody light chain constant regions of the invention are light chain constant regions from lambda or Kappa light chain constant regions, preferably from Kappa light chain constant regions, e.g., lambda or Kappa light chain constant regions, such as human lambda or Kappa light chain constant regions. In some embodiments, the light chain constant region
(I) Comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity 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.
In some specific embodiments, the invention of the anti IL-15 antibody contains
HCDR1, HCDR2 and HCDR3 comprised by VH as shown in SEQ ID No. 15, 16 or 17, LCDR1, LCDR2 and LCDR3 comprised by VL as shown in SEQ ID No. 19, 20 or 21;
HCDR1, HCDR2 and HCDR3 comprised by VH as shown in SEQ ID No. 15 or 16, LCDR1, LCDR2 and LCDR3 comprised by VL as shown in SEQ ID No. 19 or 20;
HCDR1, HCDR2 and HCDR3 comprised by VH as shown in SEQ ID No. 16 or 17, LCDR1, LCDR2 and LCDR3 comprised by VL as shown in SEQ ID No. 20 or 21;
HCDR1, HCDR2 and HCDR3 comprised by VH as shown in SEQ ID No. 15, LCDR1, LCDR2 and LCDR3 comprised by VL as shown in SEQ ID No. 19;
HCDR1, HCDR2 and HCDR3 comprised by VH as shown in SEQ ID No. 16, LCDR1, LCDR2 and LCDR3 comprised by VL as shown in SEQ ID No. 20;
HCDR1, HCDR2 and HCDR3 comprised by VH as shown in SEQ ID NO:17, LCDR1, LCDR2 and LCDR3 comprised by VL as shown in SEQ ID NO:21, or
HCDR1, HCDR2 and HCDR3 comprised by VH as shown in SEQ ID No. 18, LCDR1, LCDR2 and LCDR3 comprised by VL as shown in SEQ ID No. 22;
wherein the HCDR may be determined according to any CDR-determining scheme, e.g. kabat, abM, chothia, contact or IMGT schemes, respectively, or a combination thereof;
For example, the HCDR1 is determined according to the Kabat & Chothia protocol, and the HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are each determined according to the Kabat protocol.
In some embodiments, the anti-IL-15 antibodies of the invention comprise a first heavy chain complementarity determining region (HCDR 1), a second heavy chain complementarity determining region (HCDR 2), a third heavy chain complementarity determining region (HCDR 3), and a first light chain complementarity determining region (LCDR 1), a second light chain complementarity determining region (LCDR 2), and a third light chain complementarity determining region (LCDR 3), wherein
The HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 respectively comprise or consist of the amino acid sequences shown as 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
The HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 respectively comprise or consist of the amino acid sequences shown as 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
The HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 respectively comprise or consist of the amino acid sequences shown as 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
The HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 respectively comprise or 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, respectively, SEQ ID NO 3, SEQ ID NO 6, SEQ ID NO 9, SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 43.
In some embodiments, a VH of the invention comprises HCDR1, HCDR2, HCDR3, and the VL comprises LCDR1, LCDR2, and LCDR3, wherein
The HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 respectively comprise or consist of the amino acid sequences shown as 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
The HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 respectively comprise or consist of the amino acid sequences shown as 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
The HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 respectively comprise or consist of the amino acid sequences shown as 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
The HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 respectively comprise or 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, respectively, SEQ ID NO 3, SEQ ID NO 6, SEQ ID NO 9, SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 43.
In some embodiments, an antibody or antigen binding fragment thereof of the invention comprises:
(i) Comprising or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO. 15, 16 or 17, and/or comprising or consisting of an amino acid sequence shown in SEQ ID NO. 19, 20 or 21 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO. 19, 20 or 21;
(ii) Comprising or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO. 15 or 16, and/or comprising or consisting of an amino acid sequence shown in SEQ ID NO. 19 or 20 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence;
(iii) Comprising or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO. 16 or 17, and/or comprising or consisting of an amino acid sequence shown in SEQ ID NO. 20 or 21 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence;
(iv) Comprising or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO. 15, or a VH consisting of said amino acid sequence, and/or comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO. 19;
(v) Comprising or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO. 16, or a VH consisting of said amino acid sequence, and/or comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO. 20;
(vi) Comprising or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO. 17 and/or comprising or consisting of an amino acid sequence shown in SEQ ID NO. 21 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence thereof or consisting of a VL of said amino acid sequence
(Vii) Comprising or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO. 18, or a VH consisting of said amino acid sequence, and/or comprising or consisting of the amino acid sequence shown in SEQ ID NO. 22, having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.
In some embodiments, an antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region and a light chain variable region, wherein
(I) The heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO. 15, 16 or 17, and the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO. 19, 20 or 21;
(ii) The heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO. 15 or 16, and the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO. 19 or 20;
(iii) The heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO. 16 or 17, and the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO. 20 or 21;
(iv) The heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO. 15, and the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO. 19;
(v) The heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO. 16, and the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO. 20;
(vi) The heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO. 17, and the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO. 21, or
(Vii) The heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO. 18, and the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO. 22.
In some embodiments, the anti-IL-15 antibodies or antigen-binding fragments thereof of the invention further comprise an antibody heavy chain. In some embodiments, the anti-IL-15 antibodies or antigen-binding fragments thereof of the invention further comprise an antibody light chain. In some embodiments, the invention of the anti IL-15 antibody or antigen binding fragments thereof also contains the heavy chain and light chain. In some embodiments, an antibody heavy chain of the invention comprises or consists of a heavy chain variable region of the invention and a heavy chain constant region of the invention. In some embodiments, an antibody light chain of the invention comprises or consists of a light chain variable region of the invention and a light chain constant region of the invention. In some embodiments, an antibody of the invention comprises or consists of two said heavy chains and two said light chains.
In some embodiments, the heavy chain of an antibody of the invention comprises or consists 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 invention comprises or consists 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.
In some embodiments, an antibody or antigen-binding fragment thereof of the invention 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.
In some embodiments, an antibody or antigen-binding fragment thereof of the invention 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.
In one embodiment of the invention, the amino acid changes described herein include substitutions, insertions or deletions of amino acids. Preferably, the amino acids described herein are changed to amino acid substitutions, preferably conservative substitutions. In a preferred embodiment, the amino acid changes described in the present invention occur in regions outside the CDRs (e.g., in the FR). More preferably, the amino acid changes described herein occur in regions outside the heavy chain variable region and/or outside the light chain variable region.
In certain embodiments, the antibodies provided herein are altered to increase or decrease the degree to which the antibodies are glycosylated. The addition or deletion of glycosylation sites to an antibody can be conveniently accomplished by altering the amino acid sequence so as to create or remove one or more glycosylation sites. When an antibody contains an Fc region, the saccharide attached thereto may be changed. In some applications, modifications that remove unwanted glycosylation sites may be useful, for example, removal of fucose motifs to enhance Antibody Dependent Cellular Cytotoxicity (ADCC) function. In other applications, galactosylation modifications may be performed to modify Complement Dependent Cytotoxicity (CDC). In certain embodiments, it may be desirable to produce cysteine engineered antibodies, such as "thioMAbs," in which one or more residues of the antibody are replaced with cysteine residues. In certain embodiments, the antibodies provided herein can be further modified to contain other non-protein moieties known and readily available in the art. Moieties suitable for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1, 3-dioxane, poly-1, 3, 6-trioxane, ethylene/maleic anhydride copolymers, polyaminoacids (homo-or random copolymers), and dextran or poly (n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.
In some embodiments, the anti-IL-15 antibodies or antigen binding fragments thereof of the invention have one or more of the following properties:
(i) Shows the same or similar binding affinity and/or specificity as the antibodies of the invention for IL-15 (e.g. ch25B10, ch56G9, hz56G9.25l or ch60G 8);
(ii) Inhibit (e.g., competitively inhibit) binding of an antibody of the invention (e.g., ch25B10, ch56G9, hz56G9.25l, or ch60G 8) to IL-15;
(iii) Binding to the same or overlapping epitope as the antibodies of the invention (e.g. ch25B10, ch56G9, hz56G9.25l or ch60G 8):
(iv) Competing with an antibody of the invention (e.g., ch25B10, ch56G9, hz56G9.25l, or ch60G 8) for binding to IL-15;
(v) Having one or more of the biological properties of the antibodies of the invention (e.g., ch25B10, ch56G9, hz56G9.25l, or ch60G 8).
In some embodiments, the anti-IL-15 antibodies of the invention are antibodies in the IgG1 format or in the IgG2 format or in the IgG3 format or in the IgG4 format, preferably in the IgG1 format.
In some embodiments, the anti-IL-15 antibody is a monoclonal antibody.
In some embodiments, the anti-IL-15 antibody is humanized.
In some embodiments, the anti-IL-15 antibody is a chimeric antibody.
In some embodiments, the anti-IL-15 antibodies of the invention are full length antibodies.
In some embodiments, the invention of the anti IL-15 antibodies also cover specific binding IL-15 multi-specific antibodies or bispecific antibodies.
In one embodiment, the anti-IL-15 antibodies of the invention also encompass antibody fragments thereof (e.g., antigen binding fragments), preferably antibody fragments selected from the group consisting of Fab, fab '-SH, fv, single chain antibodies (e.g., scFv), (Fab') 2, single domain antibodies such as VHH, dAb (domain antibody), diabody, or linear antibodies.
Nucleic acids of the invention and host cells comprising the same
In one aspect, the invention provides nucleic acids encoding either chain or any monomer or domain of an antibody or antigen binding fragment thereof of the invention. The polynucleotide sequences encoding each strand may be generated using methods well known in the art. For example, the polypeptide encoded by the nucleic acid is capable of exhibiting the binding capacity of human IL-15 (e.g., IL-15 monomer or its complex with a receptor) when expressed from a suitable expression vector. For example, in some embodiments, the nucleic acid encoding the variable region of the heavy and/or light chain is operably linked in frame to a nucleic acid encoding the constant region of the heavy and/or light chain, such that when expressed from a suitable expression vector, a nucleic acid encoding the heavy and/or light chain of the antibody is produced.
In one aspect, the invention provides a coding any of the anti-IL-15 antibodies or fragments thereof described herein. The nucleic acid may comprise a nucleic acid encoding an amino acid sequence of a light chain variable region and/or a heavy chain variable region of an antibody, or a nucleic acid encoding an amino acid sequence of a light chain and/or a heavy chain of an antibody.
For example, a nucleic acid of the invention comprises a nucleic acid encoding an amino acid sequence selected from 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 any one of SEQ ID NOS.15-24. As will be apparent to those of skill in the art, because of the degeneracy of the codons, each antibody or polypeptide amino acid sequence may be encoded by a variety of nucleic acid sequences. Nucleic acid sequences encoding the molecules of the invention may be produced by methods well known in the art, for example by de novo solid phase DNA synthesis, or by PCR amplification, to facilitate production and purification, secretion signal peptides may be fused to the N-terminus of the heavy and/or light chain of an antibody, and/or tag peptides facilitating purification
The invention also provides vectors comprising the nucleic acids of the invention. In one embodiment, the vector is an expression vector, such as a eukaryotic expression vector. Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phage, or Yeast Artificial Chromosomes (YACs). In a preferred embodiment, the expression vector of the invention is a pcDNA vector, such as a pcDNA3.1 and/or pcDNA3.4 expression vector.
In one embodiment, a host cell comprising the vector is provided. The invention also provides a host cell comprising said nucleic acid or said vector. Host cells suitable for replication and supporting expression of the antibodies of the invention are well known in the art. Such cells can be transfected or transduced with specific expression vectors and large numbers of vector-containing cells can be grown for inoculation of large-scale fermenters to obtain sufficient amounts of antibodies for clinical use. Suitable host cells for cloning or expressing the antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector function are not required. After expression, the antibodies may be isolated from the bacterial cell paste in the soluble fraction and may be further purified.
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 the production of antibodies or fragments thereof. In one embodiment, the host cell is prokaryotic, e.g., is a bacterium, e.g., E.coli.
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 whose glycosylation pathways have been "humanized" result in the production of antibodies with a partially or fully human glycosylation pattern. Host cells suitable for expressing glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Vertebrate cells can also be used as hosts. For example, mammalian cell lines engineered to be suitable for suspension growth may be used. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed with SV40 (COS-7), human embryonic kidney line (HEK 293, 293F or 293T cells), and the like. Other useful mammalian host cell lines include Chinese Hamster Ovary (CHO) cells, including DHFR-CHO cells, CHO-S cells, expiCHO, and the like, and myeloma cell lines such as Y0, NS0 and Sp2/0. Mammalian host cell lines suitable for antibody production are known in the art.
Production and purification of antibody molecules of the invention
In one embodiment, the invention provides a method of preparing an antibody molecule of the invention or a fragment thereof (preferably an antigen binding fragment) wherein the method comprises culturing the host cell under conditions suitable for expression of a nucleic acid encoding an antibody molecule of the invention or a fragment thereof (preferably an antigen binding fragment) and optionally isolating the antibody or fragment thereof (e.g., an antigen binding fragment). In a certain embodiment, the method further comprises recovering the antibody molecule of the invention or fragment thereof (e.g., antigen binding fragment) from the host cell.
Polynucleotides encoding the polypeptide chains of the antibodies of the invention may be inserted into one or more vectors (e.g., pcdna3.1 and/or pcdna3.4 expression vectors) for further cloning and/or expression in a host cell. Methods well known to those skilled in the art can be used to construct expression vectors. Once an expression vector comprising one or more nucleic acid molecules of the invention has been prepared for expression, the expression vector can be transfected or introduced into a suitable host cell. Various techniques may be used to achieve this, such as protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, liposome-based transfection, or other conventional techniques.
Antibody molecules prepared as described herein may be purified by known prior art techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like. The actual conditions used to purify a particular protein 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 invention may 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.
V. assay
The anti-IL-15 antibodies provided herein can be identified, screened, or characterized for physical/chemical properties and/or biological activity by a variety of assays known in the art.
The invention also provides assays for identifying anti-IL-15 antibodies that are biologically active. Biological activity may include, for example, binding to IL-15 (e.g., binding to human IL-15), inhibition of IL-15/IL-15 ra complex-mediated signaling pathways, activation of T cells, prevention or treatment of inflammation, and the like. Antibodies having such biological activity in vivo and/or in vitro are also provided.
For the determination of the above biological activity, reference may be made in particular to the exemplary determination methods given in the examples.
It will be appreciated that any of the above assays can be performed using the immunoconjugates of the invention in place of or in addition to anti-IL-15 antibodies.
It will be appreciated that any of the above assays can be performed using a combination of an anti-IL-15 antibody and another therapeutic agent.
VI immunoconjugates
In some embodiments, the invention provides immunoconjugates comprising any of the anti-IL-15 antibodies or antigen-binding fragments thereof provided herein and other substances, e.g., any active agent or label suitable for forming an immunoconjugate with the IL-15 antibody or antigen-binding fragment thereof.
In some embodiments, the active agent suitable for forming an immunoconjugate with an IL-15 antibody or antigen-binding fragment thereof may be, for example, a chemotherapeutic agent, a toxin, a small molecule drug, a cytotoxic agent, an apoptotic agent, a chelator, an immunomodulatory agent, for example, an anti-inflammatory agent or an immunosuppressant.
In some embodiments, the immunoconjugate is an antibody drug conjugate, e.g., an ADC.
VII pharmaceutical composition and pharmaceutical preparation
In some embodiments, the invention provides a composition comprising any of the anti-IL-15 antibodies or fragments thereof (preferably antigen-binding fragments thereof) or immunoconjugates thereof described herein, preferably the composition is a pharmaceutical composition. In one embodiment, the composition further comprises a pharmaceutical excipient. In one embodiment, a composition, e.g., a pharmaceutical composition, comprises a combination of an anti-IL-15 antibody or fragment thereof of the invention or an immunoconjugate thereof, and one or more other therapeutic agents. In some embodiments, the composition is a pharmaceutical formulation.
The invention also includes compositions (including pharmaceutical compositions or pharmaceutical formulations) comprising an anti-IL-15 antibody or antigen-binding fragment thereof or an immunoconjugate thereof, or compositions (including pharmaceutical compositions or pharmaceutical formulations) comprising a polynucleotide encoding an anti-IL-15 antibody. In certain embodiments, the compositions comprise one or more antibodies or fragments thereof that bind to IL-15, or one or more polynucleotides encoding one or more antibodies or fragments thereof that are anti-IL-15. These compositions may also contain suitable pharmaceutical excipients, such as pharmaceutically acceptable carriers, pharmaceutically acceptable excipients as known in the art, including buffers.
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.
For the use of pharmaceutical excipients and their use, see also "Handbook of Pharmaceutical Excipients", eighth edition, R.C.Rowe, P.J.Seskey and s.c. owen, pharmaceutical Press, london, chicago.
The compositions of the present invention may be in a variety of forms. These include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injectable solutions and infusible solutions), powders or suspensions, liposomes, and suppositories. The preferred form depends on the intended mode of administration and the therapeutic use.
Pharmaceutical formulations comprising the antibodies of the invention, preferably in the form of lyophilized formulations or aqueous solutions, may be prepared by mixing an antibody of the invention or fragment thereof of the desired purity with one or more optional pharmaceutical excipients.
The pharmaceutical compositions or formulations of the present invention may also contain more than one active ingredient which is required for the particular indication being treated, preferably those active ingredients having complementary activities which do not adversely affect each other. For example, it may be desirable to also provide other therapeutic agents, such as cytokines, small molecule drugs, immunomodulators (e.g., immunosuppressants or anti-inflammatory agents), or other antibodies, and the like. The active ingredients are suitably present in combination in an amount effective for the intended use.
Sustained release formulations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules.
VIII pharmaceutical composition and kit
In some embodiments, the invention also provides a pharmaceutical combination or pharmaceutical combination product comprising an anti-IL-15 antibody of the invention or fragment thereof (preferably an antigen-binding fragment), or an immunoconjugate thereof, or a pharmaceutical composition comprising the same, and one or more other therapeutic agents (e.g., cytokines, chemotherapeutic agents, small molecule drugs, immunomodulators (e.g., immune activators or immunosuppressants or anti-inflammatory agents), or other antibodies, etc.).
It is a further object of the present invention to provide a kit comprising the pharmaceutical combination of the present invention, preferably in the form of a pharmaceutical dosage unit. The dosage units may thus be provided according to the dosing regimen or the interval between drug administrations.
In one embodiment, the kit of parts of the invention comprises, in the same package:
-a first container containing a pharmaceutical composition comprising an anti-IL-15 antibody or fragment thereof or an immunoconjugate thereof;
-a second container containing a pharmaceutical composition comprising the other therapeutic agent.
The choice of other therapeutic agents that can be combined with the molecules of the invention depends on the use of the molecules of the invention. For example, when the molecules of the invention are used to treat autoimmune or autoimmune diseases, the additional therapeutic agent is any active agent suitable for treating autoimmune or autoimmune diseases, including but not limited to cytokines, small molecule drugs, other antibodies or immunomodulators (e.g., immunosuppressants or anti-inflammatory agents). For example, when the molecules of the invention are used to treat tumors, the additional therapeutic agent is any active agent suitable for treating tumors, such as an anti-tumor or anti-cancer agent, including but not limited to cytokines, chemotherapeutic agents, small molecule drugs, other antibodies or immunomodulatory agents (e.g., immune activators).
IX. uses and methods
In some embodiments, there is provided a method of preventing or treating a disease or disorder in an individual as a single agent or in combination with an antibody or fragment thereof of the invention or an immunoconjugate thereof, comprising administering to the individual a molecule of the invention (e.g., an anti-IL-15 antibody or antigen-binding fragment thereof or immunoconjugate thereof), a pharmaceutical composition, a pharmaceutical combination, or a kit of parts of the invention.
In some embodiments, the invention relates to a molecule of the invention (e.g., an anti-IL-15 antibody of the invention or an antigen-binding fragment thereof or an immunoconjugate thereof), a pharmaceutical composition, a pharmaceutical combination, or a kit for use in therapy, e.g., for treating a disease or disorder mentioned herein.
In some embodiments, the invention relates to a method of treating a disease or disorder with a molecule of the invention (e.g., an anti-IL-15 antibody of the invention or an antigen-binding fragment thereof or an immunoconjugate thereof), a pharmaceutical composition, a pharmaceutical combination, or a kit, or for use in such treatment, or for use in the manufacture of a medicament for use in such treatment.
In some embodiments, the disease or disorder is an IL-15 related disease and/or disorder. The term "IL-15 related diseases and/or disorders" relates to abnormal activation of IL-15 or its receptor complex, e.g. IL-15/IL-15Rα complex mediated signaling pathways, or to overexpression of IL-15 and/or increased levels of IL-15 and/or abnormal IL-15 expression and/or abnormal expression of IL-15 variants of a cell or organ or tissue. Such diseases or disorders include, for example, autoimmune diseases and/or inflammatory disorders, or tumors. The term "autoimmune disease and/or inflammatory disorder" is defined herein generally as a disease or disorder caused by an abnormal immune response of an individual's body to substances and tissues normally present in the body, and may or may not involve an inflammatory abnormality of the immune system. In some embodiments, the inflammatory disorder is an auto-inflammatory disease.
In some embodiments, the disorder or condition is a disease or disorder in which IL-15 or its aberrant activation of a signaling pathway mediated by an IL-15 receptor complex (e.g., IL-15/IL-15Rα complex) is compared to a healthy individual. In some embodiments, the disease or disorder is in a patient with, or has an increase in the level of nucleic acid in an IL-15 or its complex with an IL-15 receptor (e.g., IL-15/IL-15 ra complex) that is aberrantly expressed (e.g., overexpressed, or expressed at increased levels, or aberrantly variant expressed) as compared to a healthy individual. In some embodiments, in a patient with the disease or disorder, the cell or tissue or organ expresses IL-15, e.g., moderately or higher expresses IL-15. In some embodiments, the patient has (e.g., an elevated level, e.g., nucleic acid or protein level or activity) IL-15 (e.g., as compared to a healthy individual). In some embodiments, the patient's biological sample (e.g., cells or tissues) has (e.g., elevated levels of, e.g., nucleic acid or protein levels or activity) IL-15 therein (e.g., as compared to a biological sample of a healthy individual (e.g., corresponding tissue or cells in a healthy individual), or as compared to IL-15 in adjacent healthy tissue or cells of the patient).
In some embodiments, the antibodies of the invention are capable of preventing and/or treating diseases or disorders associated with IL-15 by blocking activation of downstream signaling pathways by IL-15 or its receptor complex with IL-15 (e.g., IL-15/IL-15Rα complex).
In some embodiments, the disease or disorder is selected from inflammation or an inflammatory disorder (e.g., an autoinflammatory disease), such as skin inflammation or small intestine inflammation. In some embodiments, the disease or disorder is an autoimmune disease such as graft versus host disease.
In some embodiments, the disease or disorder suitable for prophylaxis and/or treatment of the present invention is a tumor, such as a cancer. In some embodiments, the tumor is a solid tumor or a hematological tumor, and the metastatic lesion. In one embodiment, examples of solid tumors include malignant tumors. The cancer may be in early, intermediate or late stages or metastatic cancer. In some embodiments, the tumor is tumor immune escape.
In some embodiments, the IL-15 or receptor complex thereof, e.g., IL-15/IL-15Rα complex, in the tumor or tumor cell is abnormally activated, e.g., by healthy cells that are in close proximity to healthy individuals or tumor cells.
In some embodiments, the tumor is an IL-15 positive tumor or cancer. In some embodiments, the tumor is associated with aberrant expression or aberrant activity of IL-15. In some embodiments, an IL-15 positive tumor or cancer refers to having tumor cells that express IL-15 in an individual with all tumor cancers. In some embodiments, the tumor cells of the individual express IL-15, e.g., moderately or higher expressing IL-15. In some embodiments, the individual has (e.g., an elevated level, e.g., nucleic acid or protein level or activity) IL-15 (e.g., as compared to a healthy individual). In some embodiments, the individual has (e.g., elevated levels of, e.g., nucleic acid or protein levels or activity) IL-15 in a biological sample (e.g., tumor cells or tumor tissue) of the individual (e.g., as compared to a biological sample of a healthy individual (e.g., corresponding tissue or cells in a healthy individual), or as compared to IL-15 in adjacent healthy tissue or cells of the individual).
In some embodiments, an antibody or antibody fragment or immunoconjugate or composition or product of the invention delays the onset of the disorder and/or symptoms associated with the disorder.
In some embodiments, the invention provides the use of an anti-IL-15 antibody or fragment thereof of the invention, or an immunoconjugate or composition comprising the same, in the manufacture or manufacture of a medicament for use as described herein, e.g., for the prevention or treatment of a related disease or disorder as referred to herein.
In some embodiments, the methods of prevention or treatment described herein further comprise administering to the subject or individual a combination of an antibody molecule or pharmaceutical composition or immunoconjugate disclosed herein, and one or more other therapies, such as a therapeutic modality and/or other therapeutic agent. In some embodiments, anti-IL-15 antibodies or fragments 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 the uses described herein, e.g., for the prevention and/or treatment of the related diseases or disorders mentioned herein.
Therapeutic modes the choice of other therapeutic agents depends on the use of the molecules of the invention. For example, when the molecules of the invention are used to treat autoimmune or autoimmune diseases, the other therapeutic agent is any active agent suitable for treating autoimmune or inflammatory disorders such as autoinflammatory diseases, including but not limited to cytokines, small molecule drugs, other antibodies or immunomodulators (e.g., immunosuppressants or anti-inflammatory agents). For example, when the molecules of the invention are used to treat tumors, the additional therapeutic agent is any agent suitable for treating tumors, such as an anti-tumor or anti-cancer agent, including but not limited to cytokines, chemotherapeutic agents, small molecule drugs, other antibodies or immunomodulators (e.g., immune activators), the additional therapeutic modality being selected, for example, from surgery or radiation therapy.
In some embodiments, the antibodies described herein can be combined with other antibodies for separate administration, e.g., as separate antibodies, or administered when linked (e.g., as a bispecific or multispecific antibody molecule).
Such combination therapies encompass the administration of a combination (e.g., two or more therapeutic agents contained in the same formulation or separate formulations), and the administration of a separate, in which case the administration of an antibody of the invention may occur prior to, concurrent with, and/or subsequent to the administration of the other therapeutic agent and/or therapy.
The antibodies or fragments thereof (as well as immunoconjugates, compositions, pharmaceutical compositions, formulations, combinations, etc. comprising the same) of the invention may be administered by any suitable method, including parenteral administration, and, if desired for topical treatment, intralesional administration. Parenteral injection or infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous injection or infusion.
Methods and compositions for diagnosis and detection
In one aspect, the invention also relates to methods for diagnosis and detection of the antibodies of the invention or antigen binding fragments thereof and compositions for diagnosis and detection comprising the same.
In certain embodiments, any of the anti-IL-15 antibodies or fragments thereof provided herein (preferably antigen binding fragments) can be used to detect the presence of IL-15 or its complex with an IL-15 receptor (e.g., IL-15/IL-15Rα complex) in a biological sample.
The term "detection" as used herein, including quantitative or qualitative detection, exemplary detection methods may 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 a cell or tissue.
In one embodiment, anti-IL-15 antibodies or fragments thereof are provided for use in diagnostic or detection methods.
In another aspect, methods for detecting the presence of IL-15 or its complex with an IL-15 receptor (e.g., IL-15/IL-15Rα complex) in a biological sample are provided. In certain embodiments, the methods comprise detecting the presence of an IL-15 protein or its complex with an IL-15 receptor (e.g., IL-15/IL-15Rα complex) in a biological sample. In certain embodiments, IL-15 is human IL-15. In certain embodiments, the methods comprise 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 to an IL-15 receptor complex (e.g., IL-15/IL-15 ra complex), and detecting whether an antigen-antibody complex is formed between the anti-IL-15 antibody or fragment thereof and IL-15 or to an IL-15 receptor complex (e.g., IL-15/IL-15 ra complex). The formation of an antigen-antibody complex indicates the presence of IL-15. The method may be an in vitro or an in vivo method. In one embodiment, an anti-IL-15 antibody or fragment thereof is used to select a subject suitable for treatment with an anti-IL-15 antibody or fragment thereof, e.g., wherein IL-15 or its complex with an IL-15 receptor (e.g., IL-15/IL-15Rα complex) is a biomarker for selecting the subject.
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 detected directly (e.g., fluorescent labels, chromophore labels, electron dense labels, chemiluminescent labels, and radiolabels), as well as moieties that are detected indirectly, such as enzymes or ligands, e.g., by enzymatic reactions or molecular interactions.
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 use with other therapies. In some embodiments, the sample is obtained during or after treatment with other therapies.
In some embodiments, IL-15 is detected prior to treatment, e.g., prior to initiation of treatment or prior to a treatment after a treatment interval.
In some embodiments, a method of treating a disease of the invention is provided, the method comprising assaying a subject (e.g., a sample) (e.g., a subject sample) for the presence of IL-15 or its complex with an IL-15 receptor (e.g., IL-15/IL-15Rα complex), thereby determining the IL-15 or its complex with an IL-15 receptor (e.g., IL-15/IL-15Rα complex) value, comparing the IL-15 or its complex with an IL-15 receptor (e.g., IL-15/IL-15Rα complex) value with a control value, and if the IL-15 or its complex with an IL-15 receptor (e.g., IL-15/IL-15Rα complex) value is greater than the control value, administering to the subject a therapeutically effective amount of an anti-IL-15 antibody or fragment thereof (e.g., an anti-IL-15 antibody or fragment thereof described herein), optionally in combination with one or more other therapies, thereby treating the disease.
These and other aspects and embodiments of the application are described in and exemplified by the following examples in the figures (which are briefly described to follow) and in the following detailed description. Any or all of the features discussed above and throughout the present application may be combined in various embodiments of the application. The following examples further illustrate the application, however, it is to be understood that the examples are presented by way of illustration and not limitation, and that various modifications may be made by those skilled in the art.
Examples
EXAMPLE 1 preparation of anti-IL-15 antibodies by hybridomas
The present study utilized the hybridoma technology platform for antibody discovery.
1.1. Immunization
Human IL-15/IL-15Rα protein (Novoprotein, cat#C15Y) was mixed with the adjuvant TiterMax (sigma, cat#T2684) in equal volumes and immunized into Balb/C mice (from Beijing Vetong rituximab) once every two weeks (50 μg protein per mouse) and serum titers were detected by blood sampling every 3 weeks.
1.2. Cell fusion and high throughput screening
When the serum titer meets the requirement, the spleen of the mouse is picked to prepare B lymphocyte suspension, and the B lymphocyte suspension is mixed with SP2/0 myeloma cells (ATCC, CRL-1581) in a ratio of 1:2-1:1 for electrofusion. Transferring the fused cells from the electrofusion tank into a 50mL centrifuge tube, diluting the cells to 1-2X 10 4 cells/mL by using a culture medium containing HAT (hypoxanthine, aminopterin and thymidine), and adding 100 mu l of cell suspension into each well of a 96-well plate. Screening media was changed at 7 th day after fusion, and enzyme-linked immunosorbent assay (ELISA) was performed after 10 th day (or longer depending on the cell growth state) of culture, and positive clones were screened.
Hybridoma cells specifically expressing anti-IL-15 antibodies were screened by enzyme-linked immunosorbent assay (ELISA). Human IL-15 (Novoprotein, cat#C016) was diluted to 0.5. Mu.g/mL with coating solution (Thermo, ca#28382), 100. Mu.L/well was added to the ELISA plate, respectively, and incubated at 37℃for 1 hour, PBST (1X Phosphate-Buffered Saline, 0.5%)Detergent) three washes, with plate liquid removed, 200. Mu.L/well of 3% NON-Fat Powdered Milk (In, cat#A 600669-0250) blocking the ELISA plate, 37℃incubation for 1 hour, PBST three washes, plate liquid removed, 50. Mu.L/well of fused hybridoma supernatant, 37℃incubation for 1 hour, PBST three washes, plate liquid removed, 100. Mu.L/well of goat anti-mouse HRP (BIOLEGEND, ca# 405306) labeled secondary antibody (1:5000 diluted in PBST), 37℃incubation for 1 hour, PBST three washes, plate liquid removed, 100. Mu.L/well of chromogenic solution tetramethyl benzidine (TMB) (INVITROGEN, cat# 002023), light development for 3 minutes, 50. Mu.L/well of stop ELISA stop solution (Solarbio, cat#C 8), and ELISA plate reading of the value at 105450. Screening to obtain clone positive to human IL-15 antigen, and detecting its binding with cynomolgus monkey IL-15.
1.3. Positive hybridoma subcloning
Limiting dilution subcloning step A96-well plate was prepared, 200. Mu.L of medium was added to each well, and HAT was replaced with HT (hypoxanthine and thymidine) based on the selection medium (Gibco, cat # 11067-030), and the remaining formulations were identical. Preparing cell suspension from the cells in the positive holes selected by the fusion sieve, adding 100 mu L of the cell suspension into each hole in the first row, uniformly mixing, adding 100 mu L of the cell suspension in the first row into the second row, fully mixing, adding 100 mu L of the cell suspension into the next row, repeating the steps, standing for 96 hole plates for 30 minutes, and observing and counting under a microscope. A volume corresponding to 100 cells was added to 20mL of medium and mixed well to plate 200. Mu.L per well. After one week, the monoclonal wells were judged and labeled by microscopic observation.
When the confluence of cells in each hole reaches more than 50%, the screening method is used for detecting the cells by the ELISA, the target positive holes are selected, and the cells are frozen after the expanded culture (18 clones are finally selected for strain fixation).
EXAMPLE 2 preparation of chimeric antibodies
The invention utilizes molecular biology technology to obtain antibody sequence in anti-IL-15 positive hybridoma cell, and utilizes it to construct human-mouse chimeric antibody. The 18 hybridoma candidate clones obtained in example 1 were subjected to the modulation of antibody light and heavy chain gene sequences, and were constructed into human mouse chimeric antibodies.
About 5X 10 6 of each hybridoma cell line freshly cultured was taken and RNA was extracted (Macherey-Nagel, ca# 740984.250). cDNA was obtained by reverse transcription using PRIMESCRIPT II.sup.1 st Strand cDNA Synthesis Kit (Takara). The upstream primer is designed by the base sequence of FR1 region at the 5' end, and the downstream primer is designed by the base of the constant region or FR4 region of the antibody, so that the light chain and heavy chain variable region gene fragments of the antibody are amplified. The obtained product was ligated to a T vector (Mighty TA-cloning Kit, takara), and the obtained product was sequenced by picking up a monoclonal antibody, and the sequencing result was analyzed and aligned by MEGA7 software. And obtaining 3 pairs of candidate clone sequences according to sequence homology through comparison.
Respectively passing 3 pairs of candidate cloned light and heavy chain variable region gene fragments through homologous recombination enzymes of Nanjinofuzan companyCatalog number C112-01) was ligated into pcDNA3.1 vector (comprising heavy chain constant region: SEQ ID NO:26; light chain constant region: SEQ ID NO:27, respectively) to obtain expression plasmids for light and heavy chain antibodies. The light chain plasmid and the heavy chain plasmid of the same antibody were then mixed in a molar ratio of 1:1, 293F cells were transfected with Polyethylenimine (PEI) (Polysciences, ca# 23966), and after 5-7 days of culture, when the cell viability was below 60%, the cell culture supernatants were collected and the monoclonal antibodies were purified using a ProteinA affinity column. Specific purification methods are shown in example 3.2 below.
A total of 3 chimeric antibodies were obtained, including ch25B10, ch56G9, ch60G8.
The sequences of control antibodies Amg, 714 and Caly002 are shown in tables 1 and 2, prepared similarly to that described above.
TABLE 1 control antibody Amg protein sequences 714
Name of the name Amino acid sequence
Amg714VH SEQ ID NO:33
Amg714VL SEQ ID NO:34
TABLE2 control antibody Caly protein sequences
Name of the name Amino acid sequence
Caly002VH SEQ ID NO:35
Caly002VL SEQ ID NO:36
EXAMPLE 3 expression and purification of antibody proteins and IL-15/IL-15Rα -Fc proteins
Human IL-15/IL-15Rα -Fc, human Q108AIL-15/IL-15Rα -Fc, human H105RIL-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 tables 3,4, and 5.
TABLE 3 human IL-15/IL-15Rα -Fc protein sequences
Name of the name Amino acid sequence
IL-15(WT)-Fc SEQ ID NO:29
IL-15Rα_sushi-Fc SEQ ID NO:30
TABLE 4 human Q108A IL-15/IL-15Rα -Fc protein sequences
TABLE 5 human H105R IL-15/IL-15R alpha-Fc protein sequences
Name of the name Amino acid sequence
IL-15(H105R)-Fc SEQ ID NO:32
IL-15Rα_suShi-Fc SEQ ID NO:30
3.1. Protein preparation
The extrap 293F cells (purchased from Gibco) were cultured with extrap 293F medium (Gibco, REF#A14351-01), the cell density was measured one day before transfection (viability required to be greater than 95%) and continued with fresh extrap 293F medium adjusted to 3X 10 6 cells/mL and the cell density was adjusted to 3X 10 6 cells/mL on the day of transfection.
1/10 Of the final transfection volume of Opti-MEM medium (Gibco, REF#31985-070) was used as transfection buffer, the plasmid to be transfected (in which the plasmid was linked to the vector of pcDNA3.1 by the nucleic acid encoding the IL-15-Fc chain (or variant IL-15-Fc chain thereof) or IL-15Rα_sushi_Fc chain, respectively) was added in a ratio of 1mg/L, wherein the two chain plasmids were 1:1 in ratio, mixed, added PEIMax (Polysciences Inc. Cat# 24765-1) in a mass ratio of DNA: PEI 1:3, mixed, incubated at room temperature for 20 minutes, and the mixture was gently poured into an suspension of Expi293F cells, shaking while shaking, and the cells were placed in shaking culture under conditions of 8% CO2, 36.5℃and 120rpm.
After 16 to 18 hours of culture, the cell suspension was supplemented with 2% (volume ratio) of Feed (100G/LPhytone Peptone +100G/L Difco Select Phytone) at a concentration of 200G/L, a glucose solution at a final concentration of 5G/L and Valproic acid sodium salt (Merk, cat#P4543-100G) at a final concentration of 2.2mM, gently mixed, and further cultured at 36.5℃at 120rpm for 7 days, followed by sample collection. The cell feed was mixed with celite (Sartorius, cat 1000037025) (1L cell feed plus 40g celite) and filtered using a 0.22 μm disposable vacuum filter.
3.2. Protein purification
Performing affinity capture by using HiTrap MabSelect PrismA (GE HEALTHCARE, cat # 17549853) affinity chromatography column, before purification, flowing 0.1MNaOH (10-20 times of column volume) through the pipeline and the affinity chromatography column, washing the pipeline and the column with distilled water (10-20 times of column volume), balancing the packed column with 1 XPBS (Gibco) (5 times of column volume), passing the filtered cell feed liquid through the column, washing the packed column with 1 XPBS (10 times of column volume) to remove non-specific binding proteins, washing the packing with eluting buffer (100mM sodium citrate,pH 3.5) (5 times of column volume), collecting the eluent, adjusting pH to 6.0 with 2M Tris, filtering and sterilizing.
The purity of the samples was checked by size exclusion chromatography (size exclusion chromatography; SEC).
Protein samples meeting the purity standards were centrifuged for 10 minutes at 4000 rpm using a 15mL ultrafiltration centrifuge tube, the protein was diluted with PBS and centrifuged again, and the procedure was repeated several times at 4000 rpm for 10 minutes to replace the protein storage buffer. After the liquid exchange is completed, filtering and sterilizing are carried out, the concentration of the antibody is detected, and the protein is delivered.
EXAMPLE 4 detection of biological Activity of chimeric antibodies Using IL-15 reporter cell lines
The IL-15 reporter cell line was used to perform a functional assay on 3 candidate chimeric antibodies. anti-IL-15 antibodies can directly target IL-15 cytokines, block IL-15 downstream signaling pathways, and thereby inhibit proliferation and activation of cells. The inhibition of IL-15 signal by antibodies was detected by detecting the expression response cell proliferation and differentiation of the fluorescent reporter gene according to the methods provided in the specification using the fluorescent reporter cell line IL-15 pathway activity reporter cell (human erythroid leukemia cell-IL-15 reporter cell line, GM-C25386) provided by Shanghai Jiman biosystems.
Cell recovery, namely 1) taking out cells from a liquid nitrogen tank, putting the cells into a 37 ℃ water bath kettle, slightly shaking the liquid nitrogen tank until the cells melt, keeping a sealing ring and a bottle cap above the water surface to prevent bacterial contamination, 2) immediately sterilizing the cells by using 75% alcohol, transferring the cells to an ultra clean bench, strictly performing aseptic operation afterwards, 3) transferring the cell suspension of a cryopreservation tube into a preheated 15mL centrifuge tube, slightly mixing the cells at 1000rpm, centrifuging the tubes for 5 minutes to precipitate the cells, discarding the supernatant, 4) preparing a complete culture medium ：1640(Gibco,22400-071)、10％FBS(Gibco,10099-141C)、1％pen/strep(Hyclone,SV30010)、2ng/mL GM-CSF(R&D,215-GM-010);5) in advance, re-suspending the cell precipitation by using 1mL of complete culture medium, taking out the part, counting living cells by using trypan blue (Gibco, 15250-061), 6) adjusting the density of the living cells to 3-5x10 5 cells/mL, inoculating the cell suspension into a T25 cell culture bottle, and 7) standing and culturing the cells in a constant temperature incubator at 37 ℃ and 5% CO 2.
Cell culture, in which the screening pressures of 0.25ug/mLpuromycin (Gibco, A11138-02) and 3ug/mLBlastincidin (Gibco, A11139-03) were maintained at the time of passage, and the passage density was controlled at 0.5X10 6 cells/mL at 2-3 days.
Activity detection 1) cell plating, cell counting, cell density adjustment to 2X10 6 cells/mL, removal of 50uL cells, addition to TC treated white bottom clear cap 96 well flat bottom light emitting plate (Shanghai macromacro, WHB-96-03), 5% CO 2, 37℃starvation treatment for 4h. 2) Antibodies and cytokines were formulated using 1640 medium without GM-CSF cytokine to formulate IL-15/IL-15Rα -Fc stimulator at a concentration of 0.01 ug/mL, 25uL per well was added to the cell culture plate. Antibodies (of which Isotype sequences were SEQ ID NO:37 and SEQ ID NO: 38) were diluted using a sterile 96-well V-bottom plate (Biyun, FPT 019), 300nM, 4-fold dilution, total of 12 gradients, 25uL per well added to the cell culture plate, 5% CO 2, and incubated at 37℃for 16h. 3) Fluorescence detection, taking out the Bio-Lite Luciferase ASSAY SYSTEM reagent (Vazyme, DD 1201-02) and a 96-well plate in advance, balancing for 30 minutes at room temperature, adding an equal volume of Bio-Lite Luciferase ASSAY SYSTEM X lysate into each well, shaking gently, standing for cracking for 10 minutes, and detecting by an instrument.
As shown in FIG. 1, the chimeric antibodies ch25B10, ch56G9 and ch60G8 can effectively block the downstream signal of IL-15/IL-15Rα -Fc stimulation, and the inhibition effect is superior to that of the control anti-IL-15 antibody.
EXAMPLE 5 physicochemical Property analysis of chimeric antibodies
The physicochemical properties of the chimeric antibody ch25B10, ch56G9, ch60G8 were examined.
SMAC column detection of antibody colloidal stability
Antibody colloidal stability was detected by recording the retention time of the antibody in Zenix-HPLC column.
HIC column detection of antibody colloidal hydrophobicity
The hydrophobicity of the antibodies was detected by recording the retention time of the antibodies in the HIC-HPLC column.
CIC detection antibody nonspecific
IgG from human serum was coated onto NHS activated chromatography column and the retention time of the antibody on this column was examined by HPLC and showed a significant negative correlation with its solubility. Therefore, the method can be used for screening out the antibodies with better solubility.
Thermal stability of DLS detection antibodies
Dynamic Light Scattering (DLS) irradiates small particles making brownian motion in a solution with laser light, and detects a change in scattered light intensity. DLS can be used to detect protein particle size in solution, as well as stability at different temperatures or different concentrations.
The experimental results are shown in Table 6, and the antibody ch25B10, ch56G9 showed relatively excellent physicochemical properties.
TABLE 6 thermal stability test results of antibodies
Example 6 biological optical interferometry (ForteBio) determination of binding kinetics of antibodies of the invention to antigen
The equilibrium dissociation constant (KD) of the antibodies of the invention for binding human, cynomolgus monkey IL-15 was determined using a biological optical interferometry (ForteBio). ForteBio affinity assays were performed according to the existing methods (Estep, P et al ,Highthroughput solutionBasedmeasurement of antibody antigen affinity and epitope binning.MAbs,2013.5(2)：, page 2708).
Half an hour before the start of the experiment, an appropriate number of AHC (18-5060, sartorius) sensors were immersed in SD buffer (PBS 1×, BSA0.1%, tween 200.05%) based on the number of samples.
100. Mu.L of SD buffer, antibody, antigen [ including human IL-15 (IL 5-H52H8, acro biosystems), cynomolgus monkey IL-15 (IL 5-C52H4, acro biosystems), human IL-15/IL-15Rα -Fc, human Q108A IL-15/IL-15Rα -Fc, human H105RIL-15/IL-15Rα -Fc ] were added to 96-well black polystyrene half-microplates (Greiner, 675076), respectively. The sensor positions are selected according to the sample position layout. The instrument setting parameters are as follows, the operation steps are Baseline, loading-1 nm, baseline, association and Dissociation, the operation time of each step depends on the combination and dissociation speed of the sample, the rotating speed is 1000rpm, and the temperature is 30 ℃. KD values were analyzed using ForteBio analysis software. The results of the experiments are shown in Table 7 below and tables 11-15.
Example 7 antibody humanization
Chimeric antibodies ch25B10 and ch56G9 obtained by hybridoma selection were humanized based on Discovery Studio (Dassault Syst. Mu. Mes) and PyMOL software, respectively, by the following steps:
(1) Determining CDR regions of the chimeric antibody;
(2) Screening human germline sequence databases for homologous sequences closest to the V/J regions of the heavy and light chains of the chimeric antibodies, respectively;
(3) Constructing the CDR regions of the chimeric antibody onto the human Germline framework;
(4) Determining amino acid positions in the framework region that function to maintain the CDR based on sequence and structural features, and performing back mutation (return to the input amino acid type) at the sequence positions determined to be important;
(5) Synthesizing a sequence, and determining the affinity of the humanized molecule and human IL-15 after preparing the antibody;
(6) In view of the close similarity of the ch25B10 and ch56G9 sequences, and in order to avoid the risk of patency associated with the sequences in the ch56G9 light chain, the humanized heavy chain sequence of ch56G9 and the humanized light chain sequence of ch25B10 were combined to give the final humanized molecule, hz56G9.25l.
The equilibrium dissociation constant (KD) of antibodies binding human IL-15 was determined using the ForteBio assay as described in example 6.
TABLE 7 equilibrium dissociation constant of antibodies
From the affinity data presented in table 7, it can be seen that humanized antibody, hz56g9.25l, maintained affinity comparable to chimeric antibody. The CDR, VH, VL amino acid sequences of the chimeric antibodies and the humanized antibodies are shown in tables 8, 9 and 10.
TABLE 8 amino acid sequences of CDRs of exemplary antibodies of the invention
TABLE 9 heavy chain variable regions (VH) of exemplary antibodies of the invention
TABLE 10 light chain variable region (VL) of exemplary antibodies of the invention
EXAMPLE 8 ForteBio assay of binding kinetics of hz56G9.25L and control antibodies to antigen
The equilibrium dissociation constants (KD) of the humanized antibodies of the invention for binding to human IL-15, cynomolgus monkey IL-15, human IL-15/IL-15Rα -Fc, human Q108A IL-15/IL-15Rα -Fc, human H105R IL-15/IL-15Rα -Fc were determined using the ForteBio assay. The ForteBio affinity assay was the same as in example 6. The experimental results are shown in tables 11 to 15 below.
From the results in tables 11 and 12, it can be seen that hz56G9.25L has a higher affinity than Amg 714.
From the results in Table 13, table 14, table 15, it can be seen that hz56G9.25L targets a different epitope on the IL-15 protein than Amg714 and Caly 002. Specifically, amg714,714 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 and does not target amino acid 108 of IL-15. The literature reports that IL-15 or IL-15/IL-15Rα complexes activate IL-15 signaling pathways by binding to the IL2Rb/IL2Rg complex. The amino acid at position 105 and 108 of IL-15 are both located at the interface between IL-15 and IL2 Rg. The prior art references indicate that Amg714,714 targets the IL-15 to IL2Rg interface and Caly002 targets the IL-15 to IL2Rb interface, consistent with the results in tables 13, 14, 15. Overall, hz56g9.25l targets an epitope that is unique compared to Amg714 and Caly 002.
Table 11 affinity constants for fortebio detection of antigen-antibody binding (M)
Table 12 affinity constants for fortebio detection of antigen-antibody binding (M)
TABLE 13 affinity constant for ForteBio detection of antigen-antibody binding (M)
Table 14 affinity constants for fortebio detection of antigen-antibody binding (M)
Table 15 affinity constants for fortebio detection of antigen-antibody binding (M)
Example 9 detection of biological Activity of antibodies Using IL-15 reporter cell lines
Anti-IL-15 antibodies can directly target IL-15 cytokines, block IL-15 downstream signaling pathways, and thereby inhibit proliferation and activation of cells. The inhibition of IL-15 signal by antibodies was detected by detecting the expression response cell proliferation and differentiation of the fluorescent reporter gene according to the methods provided in the specification using the fluorescent reporter cell line IL-15 pathway activity reporter cell (human erythroid leukemia cell-IL-15 reporter cell line, GM-C25386) provided by Shanghai Jiman biosystems.
Cell resuscitation see example 4.
Cell culture see example 4.
Activity detection 1) cell plating, cell counting, cell density adjustment to 2X10 6 cells/mL, removal of 50uL of cells and addition to TC treated white bottom clear cap 96 well flat bottom light emitting plate (Shanghai macromacro, WHB-96-03), starvation treatment at 5% CO 2, 37℃for 4 hours. 2) Antibodies and cytokines were formulated using 1640 medium without GM-CSF cytokine to formulate IL-15 (Novoprotein, C016) and IL-15/IL-15Rα -Fc stimulators at concentrations of 0.001 and 0.01 ug/mL, respectively, and 25uL per well was added to the cell culture plate. Antibodies were diluted using a sterile 96-well V-bottom plate (bi-cloud, FPT 019), 300nm, 4-fold dilution, total of 12 gradients, 25uL per well added to cell culture plates, 5% co 2, and incubated at 37 ℃ for 16h. 3) Fluorescence detection, namely taking out the Bio-Lite LuciferaseAssay system reagent (Vazyme, DD 1201-02) and a 96-well plate in advance, balancing for 30 minutes at room temperature, adding an equal volume of Bio-Lite LuciferaseAssay system X lysate into each well, shaking gently, standing for cracking for 10 minutes, and detecting by an instrument
The results of the experiments are shown in FIG. 2 (IL-15 stimulation) and FIG. 3 (IL-15/IL-15 Rα -Fc stimulation), and antibody hz56G9.25L can effectively block IL-15 mediated downstream signaling pathway activation, and has better inhibition effect than control anti-IL-15 antibody.
Example 10 detection of the inhibitory Effect of antibodies on IL-15 Signal induced proliferation of immune cells
Cell recovery 1) Peripheral Blood Mononuclear Cells (PBMC) are taken out from a liquid nitrogen tank (Shanghai Miao along TPCS, product number PB 050C-W), put into a 37 ℃ water bath kettle to gently shake until melting, keep a sealing ring and a bottle cap above the water surface to prevent bacterial contamination, 2) after cell melting, 75% alcohol is immediately used for sterilization, the cells are moved to an ultra clean bench and then strictly sterilized, 3) 1640 complete medium ：1640(Gibco,22400-071)、10％FBS(Gibco,10099-141C)、1％pen/strep(Hyclone,SV30010)、1％Sodium Pyruvate(Gibco,1136-070) and 0.1% beta-ME (Gibco, 21985-023) are prepared in advance, 3) cell suspension of a cryopreservation tube is transferred into the pre-preheated 1640 complete medium containing DNase1 (SIGMA, D5025-375 KU), gently mixed, 300g is centrifuged for 6 minutes to precipitate cells, supernatant is discarded, 4) 15mL complete medium is used for cell precipitation, and trypan blue (Gibc0, 50-061) is used for counting living cells.
Activity assay 1) cell plating by adjusting the viable cell density to 1X10 6 cells/mL, inoculating the cell suspension into a TC-treated white bottom clear cover 96-well flat bottom light emitting plate (Shanghai horizontal macro, WHB-96-03), 100 microliters per well, 100,000 cells. 2) The antibodies and cytokines were formulated using 1640 medium to formulate IL-15 (Novoprotein, C016) and IL-15/IL-15Rα -Fc stimulators at concentrations of 0.01 and 0.5 μg/ml (or 5 ug/ml), respectively, and 50 μl per well was added to the cell culture plate, and the antibodies (Baiying organism, 2K7D4D 001) were diluted with sterile 96-well V-bottom plate (Biyun, FPT 019). The antibody was diluted 300nM, 2.5-fold with 12 gradients and the antibody was diluted 300nM, 2-fold with IL-15/IL-15Rα -Fc, and 50uL per well was added to the cell culture plate and incubated at 5% CO 2 37 ℃for 72 hours. 3) And (3) fluorescence detection, namely taking out the Cell Counting-Lite2.0 reagent (Vazyme, DD 1101-02) and a 96-well plate in advance, balancing for 20-30 minutes at room temperature, adding an equal volume of Cell Counting-Lite2.0 lysate into each well, shaking gently, standing for cracking for 15 minutes, and detecting by an instrument.
The results of the above experiments are shown in FIG. 4 (IL-15 stimulation), FIG. 5 (IL-15/IL-15 Rα -Fc stimulation, 0.5 ug/ml), and FIG. 6 (IL-15/IL-15 Rα -Fc stimulation, 5 ug/ml), and antibody hz56G9.25L can effectively block IL-15-mediated downstream signaling pathway activation, inhibit immune cell proliferation, and have an inhibitory effect superior to that of the control anti-IL-15 antibody.
Example 11 detection of the inhibitory Effect of antibodies on IL-15 Signal induced activation of CD8T cells
Separating CD8T cells, 1) taking out PBMC cells (Shanghai Miao along TPCS, product number PB 050C-W) in a liquid nitrogen tank, putting into a 37 ℃ water bath kettle, gently shaking until melting, keeping a sealing ring and a bottle cap above the water surface to prevent bacterial contamination, 2) immediately sterilizing the cells by 75% alcohol, transferring to an ultra clean bench, and subsequently strictly performing aseptic operation, 3) preparing 1640 complete medium ：1640(Gibco,22400-071)、10％FBS(Gibco,10099-141C)、1％pen/strep(Hyclone,SV30010)、1％SodiumPyruvate(Gibco,1136-070) and 0.1% beta-ME (Gibco, 21985-023) in advance, 4) transferring the cell suspension of a cryopreservation tube into the previously preheated 1640 complete medium containing DNase1 (SIBCO, D5025-375 KU), gently mixing, centrifuging for 6 minutes to precipitate the cells, discarding the supernatant, 4) re-suspending the cell pellet by using 15mL complete medium, taking out part and counting the viable cells by using trypan blue (Gibco, 15250-061), and 5) separating CD8T cells from the PBMC by using a CD8T cell separation kit (stemcell, 19053).
CD8T cell activation experiments 1) coating 6 well plates with 1 ug/mL anti-human CD3 (Acro, CDE-H5223) overnight at 4 degrees, 2) adding isolated CD8T cells to CD3 antibody coated 6 well plates, activating CD8T cells, adding 1 ug/mL anti-CD28 (Invitrogen, 16-0289-85), incubating for 72 hours at 5% CO 2 ℃ per well volume 3mL, 3) collecting activated CD8T cells after 3 days, adjusting cell density to 1X10 6/mL, using TC to treat white bottom clear cover 96 well flat bottom light emitting plates (Shanghai macro, WHB-96-03), plating middle 60 wells, 100 microliters per well, 100,000 cells. 4) IL-15 (Novoprotein, C016) and IL-15/IL-15Rα -Fc stimulators were formulated at concentrations of 0.01 and 0.5ug/mL, 50uL per well was added to the cell culture plate, and 5) the antibodies were diluted using a sterile 96-well V-plate (Biyun, FPT 019). Antibody was diluted 300nM, 2.5-fold with 12 gradients, IL-15/IL-15Rα -Fc-stimulated, antibody was diluted 300nM, 2-fold with 12 gradients, 50uL per well was added to cell culture plates, incubated at 5% CO237℃for 72 hours, 6) supernatant was collected and assayed for secretion by ELISA (MabTech, 3420-1H-20).
The results of the above experiments are shown in FIG. 7 (IL-15 stimulation) and FIG. 8 (IL-15/IL-15 Rα -Fc stimulation), and the antibody hz56G9.25L can effectively block the activation of IL-15 mediated downstream signaling pathway, inhibit the activation of IL-15 mediated CD8T cells and secretion of IFNγ cytokines, and has better inhibition effect than the control anti-IL-15 antibody.
Example 12 physicochemical Property analysis of antibody hz56G9.25L
Physicochemical properties of antibody hz56G9.25L were examined.
SMAC column detection of antibody colloidal stability
Antibody colloidal stability was detected by recording the retention time of the antibody in Zenix-HPLC column.
HIC column detection of antibody colloidal hydrophobicity
The hydrophobicity of the antibodies was detected by recording the retention time of the antibodies in the HIC-HPLC column.
CIC detection antibody nonspecific
IgG from human serum was coated onto NHS activated chromatography column and the retention time of the antibody on this column was examined by HPLC and showed a significant negative correlation with its solubility. Therefore, the method can be used for screening out the antibodies with better solubility.
Thermal stability of DLS detection antibodies
Dynamic Light Scattering (DLS) irradiates small particles making brownian motion in a solution with laser light, and detects a change in scattered light intensity. DLS can be used to detect protein particle size in solution, as well as stability at different temperatures or different concentrations.
The experimental results are shown in Table 16, and antibody hz56G9.25L shows physicochemical properties in accordance with expectations.
TABLE 16 physicochemical Properties of antibodies
Example 13 detection of the inhibitory Effect of antibodies on IL-15 Signal-induced murine skin inflammation
Female C57BL/6N mice, 6-9 weeks old, 16-19g, purchased from Beijing vitamin Toril laboratory animal technology Co., ltd., grade SPF, quality control unit of Beijing vitamin Toril laboratory animal technology Co., ltd., qualification number 110011231110488161.
After 1 week of adaptive feeding, the animals were randomly divided into four groups, i.e., non-modular (i.e., PBS+ Isotype), modular (i.e., IL-15/IL-15Rα -Fc+ Isotype), amg treatment group and hz56G9.25L treatment group.
Mice were shaved on the dorsal-ventral side, two 1em frames were drawn on the skin with a marker, recorded as site1 and site2, and the latter three groups were injected intradermally with IL-15/IL-15Rα -Fc,2.5ug/50uL each frame, i.e., 5ug/100uL each mouse, on days 1,3,5,7,9,11, respectively, to induce a murine dermatitis model without molding an intradermal injection of equal volumes of PBS.
Four groups of mice were injected intraperitoneally Isotype (100 ug/mouse), isotype (100 ug/mouse), amg (100 ug/mouse), hz56G9.25L (100 ug/mouse), on day 0,3,6,10, respectively. The skin thickness of the mice was monitored on Day 1,3,5,7,10,14, measured with a vernier caliper and the skin thickening was calculated (Change% = Day X skin thickness/Day 0 skin thickness X100% -100%).
After the experiment is finished on day 14, mice are euthanized, local skin of mice site1 modeling is collected, IHC staining is carried out after formalin fixation to detect the T cell infiltration condition, local skin of mice site2 modeling is collected in PBS, and the T cell infiltration and activation (NKG 2D expression level) condition is detected in a flow manner (the NKG2D flow analysis antibody used is BD 562800).
The results of the above flow assay are shown in FIG. 9 and FIG. 10, and antibody hz56G9.25L can effectively block IL-15 mediated T cell proliferation and activation. As shown, antibody hz56g9.25l can more effectively inhibit the expression level of NKG2D than Amg D714.
Example 14 detection of the inhibitory Effect of antibodies on IL-15 Signal induced inflammation of the small intestine
Female C57BL/6N mice, 6-9 weeks old, 16-19g, purchased from Beijing vitamin Toril laboratory animal technology Co., ltd., grade SPF, quality control unit of Beijing vitamin Toril laboratory animal technology Co., ltd., qualification number 110011231107923856.
After 1 week of adaptive feeding, the animals were randomly divided into four groups, i.e., non-modular (i.e., PBS+ Isotype), modular (i.e., IL-15/IL-15Rα -Fc+ Isotype), amg treatment group and hz56G9.25L treatment group.
The latter three groups were intraperitoneally injected with IL-15/IL-15Rα -Fc, i.e., 15ug/200uL per mouse, at the first 1 hour to induce a model of enteritis in the mice, while the non-modular group was intraperitoneally injected with an equal volume of PBS.
Four groups of mice were injected Isotype (200 ug/mouse), isotype (200 ug/mouse), amg714 (200 ug/mouse), hz56G9.25L (200 ug/mouse) intraperitoneally at 0h,24h,48h, respectively. At the end of the experiment at 72 hours, mice were euthanized, mice were collected from their small intestine (10 cm section of intestine below the stomach), mice were collected from their spleen and weighed, and the conditions of T cell activation (CD 69 expression level) of the small intestine (fig. 11) and spleen (fig. 12) were flow tested (the CD69 flow assay antibody used was Biolegend 104508).
The results of the above flow assay are shown in FIGS. 11 and 12, and antibody hz56G9.25L is effective in blocking IL-15 mediated T cell activation and has a tendency to be advantageous over Amg714,714. Among them, two T cell populations present in large numbers in the small intestine, including αβ T cells and γδ T cells, were detected in fig. 11, and antibody hz56g9.25l had a remarkable inhibitory effect on activation of T cells of both populations.
Example 15 detection of the inhibitory Effect of antibodies on IL-15 Signal-enhanced graft versus host disease (GvHD)
Female NOG mice, 6-9 weeks old, 16-19g, were purchased from Peking Violet laboratory animal technologies Co., ltd., grade SPF, quality control unit was Peking Violet laboratory animal technologies Co., ltd., and the eligibility number was 110011231109256251. After 1 week of adaptive feeding, the animals were randomized into four groups, gvHD group (i.e., pbs+ Isotype), gvHD accelerated group (i.e., IL-15/IL-15 ra-fc+ Isotype), amg714 treatment group, and hz56g9.25l treatment group.
All mice were injected intravenously with PBMC (1X 10≡7/mouse, all cells, LP230809010, LP 221115011) on day 1 to induce the mouse GvHD model, and the latter three groups were injected intraperitoneally with IL-15/IL-15Rα -Fc,5ug/200uL each on day 1,3,5,7,10,12,14 to accelerate GvHD, while the first group was intraperitoneally injected with an equal volume of PBS.
Four groups of mice were injected intraperitoneally Isotype (100 ug/mouse), isotype (100 ug/mouse), amg (100 ug/mouse), and hz56G9.25L (100 ug/mouse), respectively, on day 0,3,7,10,14. Mice were monitored for weight on Day 0,3,7,10,12,14,16,18, measured using an electronic balance and weight changes were calculated (Change% = Day X body weight/Day 0 body weight X100% -100%), mice were monitored for death throughout the experiment, and mice with weight loss exceeding 20% were euthanized.
As shown in fig. 13 (PBMC donor 1) and fig. 14 (PBMC donor 2), the antibody hz56g9.25l can effectively block graft versus host disease (GvHD) with IL-15 signaling enhancement.
As shown, IL-15/IL-15 ra significantly accelerated the progression of GvHD compared to PBS group, resulting in weight loss and death of mice. In the treated group, antibody hz56G9.25L was more effective in inhibiting IL-15/IL-15Rα than Amg714,714, thereby prolonging survival and slowing down the weight loss in mice.
EXAMPLE 16 in vivo Pharmacokinetic (PK) study of antibodies in BALB/c mice
The in vivo PK behaviour of hz56g9.25l was observed by mouse Pharmacokinetic (PK) experiments.
BALB/c mice were subcutaneously injected with 10mpk of hz56G9.25L, each at 0.5 hours, 2 hours, 6 hours, 24 hours, 48 hours, 96 hours, 168 hours, 336 hours, 504 hours and 840 hours, and mice serum samples were taken for testing.
ELISA was used to test the plates coated with IL-15 antigen (off-shore protein, GMP-C016), followed by the addition of serum to be tested, followed by detection with anti-human Fc secondary antibody (BETHYL, A80-104P) and finally TMB development. The PK detection results are shown in FIG. 15 and Table 15, the PK curve shows PKprofile of IgG sample, the half-life reaches 258 hours, and the clearance rate is 0.21ml/kg/h.
TABLE 17 in vivo Pharmacokinetic (PK) studies of antibodies in BALB/c mice
Example 17 antibody Fc engineering
Antibodies can maintain a relatively long half-life in serum through human neonatal Fc receptor (FcRn) mediated circulation. Antibody Fc has a pH-dependent interaction with FcRn. The invention aims at the hz56G9.25L monoclonal antibody to carry out Fc reconstruction, and the in vivo half life of the drug is enhanced by changing the amino acid sequence. Specifically, the invention performs site-directed mutagenesis (M252Y/S254T/T256E) on 3 sites of the Fc segment of hz56G9.25L to improve the binding capacity to FcRn in order to prolong the half-life of the FcRn in vivo. The obtained antibody was named hz56G9.25L-YTE, and the CDR, VH, VL, heavy chain and light chain amino acid sequences of the antibody are shown in Table 18, table 19, table 20, table 21 and Table 22.
TABLE 18 amino acid sequences of CDRs of hz56G9.25L-YTE antibody
Table 19. Heavy chain variable region (VH) of hz56G9.25L-YTE antibody
Antibody name VH proteins
hz56G9.25L-YTE SEQ ID NO:17
Table 20.Hz56G9.25L-YTE antibody light chain variable region (VL)
Antibody name VL proteins
hz56G9.25L-YTE SEQ ID NO:21
TABLE 21 heavy chain of hz56G9.25L-YTE antibody
TABLE 22 light chain of hz56G9.25L-YTE antibody
Antibody name Light chain proteins
hz56G9.25L-YTE SEQ ID NO:24
EXAMPLE 18 detection of biological Activity of hz56G9.25L-YTE Using IL-15 reporter cell lines
Cell resuscitation, cell culture and activity detection methods are described in example 9.
The experimental results are shown in FIG. 16 (IL-15) and FIG. 17 (IL-15/IL-15 Rα -Fc), and hz56G9.25L-YTE and hz56G9.25L have similar functions, can effectively block the activation of the IL-15 mediated downstream signal pathway, and have better inhibition effect than the control anti-IL-15 antibody.
Example 19 detection of the inhibitory Effect of hz56G9.25L-YTE on IL-15 Signal induced proliferation of immune cells
Cell resuscitation see example 10.
Activity assays, see example 10. The concentrations were 0.01 and 0.5ug/mL for IL-15 (Novoprotein, C016) and IL-15/IL-15Rα -Fc stimulators.
The experimental results are shown in FIG. 18 (IL-15), FIG. 19 (IL-15/IL-15 Rα -Fc), and hz56G9.25L-YTE and hz56G9.25L have similar functions, can effectively block the activation of the IL-15 mediated downstream signal pathway, and have better inhibition effect than the control anti-IL-15 antibody.
EXAMPLE 20 in vivo Pharmacokinetic (PK) study of antibodies in hFcRn mice
PK behaviour of hz56g9.25l and hz56g9.25l-YTE in vivo was observed by mouse Pharmacokinetic (PK) experiments.
Mice serum samples were taken at 5 minutes, 2 hours, 6 hours, 24 hours, 48 hours, 96 hours, 168 hours, 336 hours, 504 hours, 672 hours and 1008 hours, respectively, for 10mpk of hz56G9.25L and hz56G9.25L-YTE intravenously from the tail of the hFcRn mice.
ELISA was used to test the plates coated with IL-15 antigen (off-shore protein, GMP-C016), followed by the addition of serum to be tested, followed by detection with anti-human Fc secondary antibody (BETHYL, A80-104P) and finally TMB development. The PK detection results are shown in FIG. 20 and Table 23, wherein PK curves of hz56G9.25L and hz56G9.25L-YTE show PKprofile of IgG sample, half-lives are 277 and 412 hours respectively, and clearance rates are 0.27 and 0.17ml/kg/h respectively. It can be seen that the PK behavior of the hz56G9.25L-YTE molecule is better than that of the hz56G9.25L molecule.
TABLE 23 in vivo Pharmacokinetic (PK) studies of antibodies in BALB/c mice

Claims (19)

1. An anti-IL-15 antibody or antigen-binding fragment thereof, comprising:
HCDR1, HCDR2 and HCDR3 comprised by VH as shown in SEQ ID No. 17, LCDR1, LCDR2 and LCDR3 comprised by VL as shown in SEQ ID No. 21;
HCDR1, HCDR2 and HCDR3 comprised by VH as shown in SEQ ID No. 15, 16 or 17, LCDR1, LCDR2 and LCDR3 comprised by VL as shown in SEQ ID No. 19, 20 or 21;
HCDR1, HCDR2 and HCDR3 comprised by VH as shown in SEQ ID No. 15 or 16, LCDR1, LCDR2 and LCDR3 comprised by VL as shown in SEQ ID No. 19 or 20;
HCDR1, HCDR2 and HCDR3 comprised by VH as shown in SEQ ID No. 16 or 17, LCDR1, LCDR2 and LCDR3 comprised by VL as shown in SEQ ID No. 20 or 21;
HCDR1, HCDR2 and HCDR3 comprised by VH as shown in SEQ ID No. 15, LCDR1, LCDR2 and LCDR3 comprised by VL as shown in SEQ ID No. 19;
HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO. 16, LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO. 20, or
HCDR1, HCDR2 and HCDR3 contained in VH shown in SEQ ID NO. 18 and LCDR1, LCDR2 and LCDR3 contained in VL 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 (HCDR 1), a second heavy chain complementarity determining region (HCDR 2), a third heavy chain complementarity determining region (HCDR 3), and a first light chain complementarity determining region (LCDR 1), a second light chain complementarity determining region (LCDR 2), and a third light chain complementarity determining region (LCDR 3), wherein the antibody or antigen-binding fragment thereof
The HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 respectively comprise or consist of the amino acid sequences shown as 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 amino acid sequences shown as 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
The HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 respectively comprise or consist of the amino acid sequences shown as 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
The HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 respectively comprise or consist of the amino acid sequences shown as 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
The HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 respectively comprise or 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, respectively, 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 according to claim 1 or 2, comprising a heavy chain variable region (VH), wherein the heavy chain variable region comprises or consists of an amino acid sequence having at least 90% identity to an amino acid sequence selected from any one of SEQ ID NOs 15 to 18 or comprises or consists of an amino acid sequence selected from any one of SEQ ID NOs 15 to 18, and/or
Comprising a light chain variable region (VL), wherein said light chain variable region comprises or consists of an amino acid sequence having at least 90% identity to an amino acid sequence set forth in any one of SEQ ID NOs 19 to 22 or comprises or consists of an amino acid sequence set forth in any one of SEQ ID NOs 19 to 22.
4. An anti-IL-15 antibody or antigen-binding fragment thereof
(I) An amino acid sequence comprising or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence shown in SEQ ID No. 15, 16 or 17, or a VH consisting of said amino acid sequence, and an amino acid sequence comprising or consisting of an amino acid sequence shown in SEQ ID No. 19, 20 or 21 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence thereof;
(ii) An amino acid sequence comprising or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID No. 15 or 16, or a VH consisting of said amino acid sequence, and an amino acid sequence comprising or consisting of an amino acid sequence shown in SEQ ID No. 19 or 20 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto;
(iii) An amino acid sequence comprising or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID No. 16 or 17 or a VH consisting of said amino acid sequence, and an amino acid sequence comprising or consisting of an amino acid sequence shown in SEQ ID No. 20 or 21 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto;
(iv) An amino acid sequence comprising or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID No. 15 or a VH consisting of said amino acid sequence, and an amino acid sequence comprising or consisting of an amino acid sequence shown in SEQ ID No. 19 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto;
(v) An amino acid sequence comprising or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID No. 16 or a VH consisting of said amino acid sequence, and an amino acid sequence comprising or consisting of an amino acid sequence shown in SEQ ID No. 20 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence;
(vi) Comprising or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO. 17, or a VH consisting of said amino acid sequence, and comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO. 21, or a VL consisting of said amino acid sequence
(Vii) Comprising or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO. 18, or a VH consisting of said amino acid sequence, and comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO. 22.
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 shown in SEQ ID NO. 17 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO. 21, or the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO. 17 and the light chain variable region consists of the amino acid sequence shown in SEQ ID NO. 21;
b) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO. 15, 16 or 17 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO. 19, 20 or 21, or the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO. 15, 16 or 17 and the light chain variable region consists of the amino acid sequence shown in SEQ ID NO. 19, 20 or 21;
c) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO. 15 or 16 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO. 19 or 20, or the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO. 15 or 16 and the light chain variable region consists of the amino acid sequence shown in SEQ ID NO. 19 or 20;
d) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO. 16 or 17 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO. 20 or 21, or the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO. 16 or 17 and the light chain variable region consists of the amino acid sequence shown in SEQ ID NO. 20 or 21;
e) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO. 15 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO. 19, or the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO. 15 and the light chain variable region consists of the amino acid sequence shown in SEQ ID NO. 19;
f) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO. 16 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO. 20 or the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO. 16 and the light chain variable region consists of the amino acid sequence shown in SEQ ID NO. 20, or
G) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO. 18 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO. 22 or the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO. 18 and the light chain variable region consists of the amino acid sequence shown in SEQ ID NO. 22.
6. The anti-IL-15 antibody or antigen-binding fragment thereof of any one of claims 1-5, wherein
The anti-IL-15 antibody or antigen binding fragment thereof comprises or consists of an Fc region, e.g., the Fc region is from an Fc region of IgG1, igG2, igG3, or IgG4, e.g., an Fc region of human IgG1, igG2, igG3, or IgG4, e.g., the IgG1 Fc region comprises or consists of the amino acid sequence of SEQ ID No. 41, or comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID No. 41;
Or alternatively
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 IgG1,
A constant region of IgG2, igG3, or IgG4, e.g., a human IgG1, igG2, igG3, or IgG4, 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 having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO. 25;
optionally, the heavy chain constant region or Fc region comprises or consists of an amino acid sequence comprising SEQ ID No. 26, e.g., a mutation, e.g., an L234A/L235A mutation, having reduced binding to an fcγ receptor;
or an amino acid sequence comprising at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO. 26;
Optionally, the heavy chain constant region or Fc region comprises a mutation that increases binding to FcRn receptor, such as a YTE mutation (M252Y/S254T/T256E);
Optionally, the heavy chain constant region or Fc region comprises a mutation that simultaneously comprises a mutation that reduces binding to an fcγ receptor and a mutation that enhances binding to an FcRn receptor, e.g., comprises an L234A/L235A mutation and a YTE mutation, e.g.
The heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO. 27, or comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity 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 having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO. 42.
7. The anti-IL-15 antibody or antigen-binding fragment thereof of any one of claims 1-6, comprising a light chain constant region that 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 having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity 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 or consisting of a heavy chain and/or a light chain, wherein the heavy chain comprises or consists of the VH and heavy chain constant regions of any one of claims 1-7, and/or the light chain comprises or consists of the VL and light chain constant regions of any one of claims 1-7;
optionally, the heavy chain comprises or consists of the amino acid sequence shown 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, and/or the light chain comprises or consists of the amino acid sequence shown 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;
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 of any one of claims 1-8, wherein the antibody is a monoclonal antibody, or the antibody is a humanized antibody or chimeric antibody.
10. The anti-IL-15 antibody or antigen-binding fragment thereof of any one of claims 1-9, wherein the antigen-binding fragment is an antibody fragment selected from the group consisting of Fab, fab '-SH, fv, single chain antibody (e.g., scFv), (Fab') 2, dAb (domainantibody), diabody, or linear antibody.
11. The anti-IL-15 antibody or antigen-binding fragment thereof of any one of claims 1-10, wherein the antibody or antigen-binding fragment thereof has one or more of the following properties:
a) An IL-15 monomer that binds with high affinity to a human or cynomolgus monkey or a complex thereof with its receptor, e.g., an IL-15/IL-15Rα complex, wherein in the monomer or complex, the amino acid at position 105 of IL-15 is H;
b) Effectively block downstream signaling pathways stimulated by IL-15 monomer or its complex with its receptor, e.g., IL-15/IL-15Rα complex;
c) Inhibiting IL-15 or its complex with a receptor, such as IL-15/IL-15 ra mediated activation of CD8T cells and/or IFNg cytokine secretion, e.g. in CD8T cells;
d) Effectively blocking T cell proliferation, T cell infiltration and/or T cell activation (e.g., decreased NKG2D expression levels or CD69 expression levels) mediated by IL-15 monomer or its complex with its receptor, e.g., IL-15/IL-15 ra complex;
e) Has good physicochemical properties and/or pharmacokinetic characteristics, for example, has good stability (e.g., colloidal stability and/or thermal stability), good solubility, long half-life, and/or low clearance;
f) Effective in preventing or treating inflammation, such as skin inflammation or small intestine inflammation;
g) Can effectively prevent or treat autoimmune diseases such as graft versus host diseases, such as graft versus host diseases which effectively block IL-15 signal enhancement.
12. An isolated nucleic acid encoding the anti-IL-15 antibody or antigen-binding fragment thereof of any one of claims 1-11.
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 a yeast cell, a mammalian cell (e.g. 293 cell or CHO cell, such as CHO-K cell or HEK293 cell) or other cell suitable for the preparation of antibodies or antigen binding fragments thereof.
15. A method of preparing an anti-IL-15 antibody or antigen-binding fragment thereof, comprising
A) Culturing the host cell of claim 14 under conditions suitable for expression of a nucleic acid encoding the anti-IL-15 antibody or antigen-binding fragment thereof of any one of claims 1-11,
B) Optionally isolating the antibody or antigen binding fragment thereof,
C) Optionally the method further comprises recovering the anti-IL-15 antibody or antigen-binding fragment thereof from the host cell, optionally the antibody is purified, e.g. by protein a.
16. An immunoconjugate comprising the anti-IL-15 antibody or antigen-binding fragment thereof of any one of claims 1-11 and other substances, such as toxins, small molecule drugs, cytotoxic agents, apoptotic agents, chelators, immunomodulators, such as anti-inflammatory agents or immunosuppressants.
17. A pharmaceutical composition comprising the anti-IL-15 antibody or antigen-binding fragment thereof of any one of claims 1-11 or the immunoconjugate of claim 16, and optionally a pharmaceutical excipient.
18. A pharmaceutical combination comprising an anti-IL-15 antibody or antigen-binding fragment thereof according to any one of claims 1-11 or an immunoconjugate according to claim 16, and one or more other therapeutic agents, e.g. selected from cytokines, other antibodies, small molecule drugs or immunomodulators (e.g. anti-inflammatory or immunosuppressant).
19. A method of preventing or treating an IL-15-associated disease and/or disorder in a subject, 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 claims 1-11, or the immunoconjugate of claim 16, or the pharmaceutical composition of claim 17, or the pharmaceutical combination of claim 18;
Optionally, in the subject, a disease or disorder associated with aberrant expression of IL-15 or a receptor complex thereof, such as an IL-15/IL-15 ra complex, or aberrant activation of a mediated signaling pathway, as compared to a healthy individual;
optionally, the disease or disorder is selected from a tumor, such as a cancer, or an inflammation or inflammatory disorder, such as an autoinflammatory disease,
Such as skin inflammation or small intestine inflammation, or autoimmune diseases such as graft versus host disease, or
Optionally, the method further comprises administering one or more other therapies, such as therapeutic modalities, and/or other therapeutic agents, such as selected from cytokines, other antibodies, small molecule drugs, or immunomodulators (e.g., anti-inflammatory agents or immunosuppressants).
CN202510453244.3A 2024-04-12 2025-04-11 Anti-IL-15 antibodies and uses thereof Pending CN120818052A (en)

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