WO2019213686A1

WO2019213686A1 - Therapeutic compositions and uses therefor

Info

Publication number: WO2019213686A1
Application number: PCT/AU2018/050437
Authority: WO
Inventors: Mark Smyth; Kyohei Nakamura
Original assignee: The Council Of The Queensland Institute Of Medical Research
Priority date: 2018-05-10
Filing date: 2018-05-10
Publication date: 2019-11-14

Abstract

Disclosed are compositions and methods for treating cancer in a subject. More particularly, the invention relates to compositions comprising an inhibitor of IL-18 function and their use in therapy. In another aspect, the present invention provides methods of treating multiple myeloma in a subject.

Description

TITLE OF THE INVENTION

“THERAPEUTIC COMPOSITIONS AND USES THEREFOR”

FIELD OF THE INVENTION

[0001] This invention relates generally to compositions and methods for treating cancer . More particularly, the present invention relates to compositions comprising an inhibitor of IL-18 function, and methods of treating multiple myleoma.

BACKGROUND OF THE INVENTION

[0002] Multiple myeloma is characterized by clonal expansion of malignant plasma cells (PCs) in the bone marrow (BM), paraproteinemia, and clinical symptoms including anemia, bone destruction, and kidney injury (Palumbo, 2011). Although overall survival has been considerably improved by therapeutic agents such as proteasome inhibitors, immunomodulatory drugs, and monoclonal antibodies (mAbs) against myeloma surface proteins (e.g., elotuzumab and daratumumab) (Rajkumar, 2016), primary and acquired resistance against these anti-myeloma therapies remains a major barrier to cure. Due to intraclonal heterogeneity of malignant PCs alone might not be sufficient to achieve profound and durable clinical responses (Robiou du Pont et al., 2017). Thus, in-depth understanding of the multiple myeloma microenvironment is warranted to further improve patient outcomes.

[0003] It is recognized that tumour-promoting inflammation is a hallmark of cancer (Hanahan, 2011). Dysregulated inflammation in the tumour microenvironment promotes tumour growth, directly through cytokine-induced stimulation of malignant cells and/or indirectly by inducing growth factors, angiogenesis, and tissue

remodeling. More importantly, tumour-promoting inflammation is tightly associated with another hallmark of cancer, avoiding immune destruction, by mobilization of myeloid-derived suppressor cells (MDSCs) and tumour-associated macrophages (see, Ugel et al., 2015). Although tumour-promoting inflammation is orchestrated by the complex crosstalk between intrinsic and extrinsic pathways, pattern recognition receptors (PRRs), such as Toll-like receptors and nucleotide oligomerization domain- like receptors (NOD-like receptors; NLRs), play pivotal roles in inflammatory responses through recognition of various damage-associated molecular patterns (DAMPs) (Karki et al., 2017; Nakamura, 2017). In this context, interleukin (IL)-1 b plays pleiotropic roles in cancer progression by its potent pro-inflammatory property, whereas IL-18, another member of the IL-1 cytokine family, is characterized by its ability to induce IFN-g (IFN-g) from natural killer (NK) and T helper 1 (Th1 ) cells in synergy with IL-12 (see, Garlanda et al., 2013; Nakanishi et al., 2001).

[0004] Notably, among various types of malignancies, multiple myeloma is particularly characterized by the presence of an inflammatory network in its microenvironment. Yet, it remains to be determined what kinds of PRRs and inflammatory mediators play a dominant role in multiple myeloma-associated inflammation contributes to immunosuppression. Historically, IL-6 has long been recognized as a central cytokine for myeloma survival, proliferation, and

chemotherapy resistance (Guillerey et al., 2016). Nevertheless, recent clinical trials have shown the limited clinical efficacy of the neutralizing anti-IL-6 mAb situximab against myeloma (see, San-Miguel et al., 2014, Voorhees et al., 2013), highlighting the need for a better understanding of the multiple myeloma inflammatory

environment. In particular, the inflammasome, a key regulator of IL-1 family cytokines, might critically drive multiple myeloma-associated inflammation, as several lines of evidence suggest the involvement of IL-1 family cytokines in the multiple myeloma pathology (see, Alexandrakis et al., 2004; Calcinotto et al., 2015; and Lust et al., 2009). In this study, we aim to determine the role of the

inflammasome and IL-1 family cytokines in the multiple myeloma immune

microenvironment.

SUMMARY OF THE INVENTION

[0005] The present invention was predicated, at least in part, on the surprising realization by the present inventors that IL-18 antagonists can be used to treat subjects with multiple myeloma.

[0006] Thus, in one aspect, the present invention provides method of treating multiple myeloma in a subject, the method comprising administering to the subject a composition that comprises an inhibitor of IL-18 function, to thereby treat the multiple myeloma in the subject. Typically, the inhibitor of IL-18 function comprises a peptide, nucleic acid, antigen-binding molecule, or small molecule inhibitor.

[0007] In preferred embodiments, the inhibitor of IL-18 function binds specifically to a polypeptide selected from the group comprising: an IL-18

polypeptide, an IL18R polypeptide (i.e., an IL18R1 polypeptide and/or an IL18RAP polypeptide), or an IL18BP polypeptide.

[0008] In some embodiments, the multiple myeloma is resistant or not responsive to chemotherapy treatment and/or radiotherapy treatment. Typically, the subject has previously been exposed to chemotherapy and/or radiotherapy treatment regimens, which have not succeeded in treating the multiple myeloma.

[0009] In some embodiments, the method further comprises concurrently administering a chemotherapeutic agent to the subject. Suitable chemotherapeutic agents may be selected from any chemotherapeutic agent known to be at least partially effective for treating multiple myeloma. In some embodiments the

compositions of the invention are co-administered with a chemotherapeutic agent selected from the group comprising: melphalan, prednisone, vincristine, doxorubicin, decadron, BCNU, cyclophosphamide, adriamycin, dexamethasone, thalidomide, bortezomib, pamidronate, and zoledronic acid.

[0010] In some embodiments of this type, the inhibitor of IL-18 function and the chemotherapeutic agent are administered to the subject simultaneously, sequentially, or separately.

[0011] In some embodiments, the inhibitor of IL-18 function is an antigen- binding molecule. In some embodiments of this type, the antigen-binding molecule specifically binds to at least a portion of the IL-18 polypeptide set forth in SEQ ID NO: 1. In some alternative embodiments, the antigen binding molecule specifically binds to at least a portion of an IL-18 Receptor 1 (IL18R1 ) polypeptide, such as the IL18R1 amino acid sequence set forth in SEQ ID NO: 3. In some other

embodiments, the antigen-binding molecule specifically binds to at least a portion of an IL-18 Receptor Accessory Protein (IL18RAP) polypeptide, such as the IL18RAP amino acid sequence set forth in SEQ ID NO: 5. In some other embodiments, the antigen-binding molecule specifically binds to at least a portion of an IL18BP polypeptide, such as the IL18BP amino acid sequence set forth in SEQ ID NO: 7. [0012] In some embodiments, the inhibitor of IL-18 function may comprise a peptide. Suitably, the peptide may comprise, consist, or consist essentially of between around 5 and 30 amino acid residues.

[0013] In some of the same embodiments and other embodiments, the compositions of the invention further comprise a targeting agent. The targeting agent is useful for delivering the IL-18 inhibitor to the memory T cells located in the bone marrow. Accordingly, in some preferred embodiments, the targeting agent binds to a cell-surface receptor on the memory T cell (e.g., a CD8⁺ T cell).

[0014] In some embodiments, the targeting agent specifically binds to a polypeptide selected from: CD38 polypeptide, CAR-MIL polypeptide, CAR-T polypeptide, BCMA polypeptide, CD137 polypeptide, CD319, polypeptide CD46 polypeptide, CD47 polypeptide, MCL-1 polypeptide, CD229 polypeptide, CD54 polypeptide, CD56 polypeptide, CD3 polypeptide, CD200 polypeptide, CD16A polypeptide, CGEN-928 polypeptide, CD48 polypeptide, CD319 polypeptide, LMA polypeptide, KMA polypeptide, FcRH5 polypeptide, hTfR IgA polypeptide, Dkk1 polypeptide, APRIL polypeptide, CD95 polypeptide, KIR polypeptide, CS1

polypeptide, CD19 polypeptide, CD20 polypeptide, CD74 polypeptide, SDF-1 polypeptide, IL-2 polypeptide, IL-6 polypeptide, PSGL-1 polypeptide, and CD40 polypeptide.

[0015] In some preferred embodiments of this type, the targeting agent comprises an antigen-binding molecule that specifically binds to a cell surface receptor on a memory T cell. By way of an illustrative example, the cell surface receptor may be a CD38 polypeptide.

[0016] In some alternative embodiments, the inhibitor of IL-18 function is an siRNA molecule.

[0017] In another aspect, the present invention provides the use of an inhibitor of IL-18 function in the treatment of multiple myeloma.

[0018] In another aspect, the present invention provides the use of an inhibitor of IL-18 function in the manufacture of a medicament for the treatment of multiple myeloma. [0019] In yet another aspect, the present invention provides compositions for treating multiple myeloma, the compositions comprising an inhibitor of IL-18 function, and a chemotherapeutic agent.

[0020] Preferably, the chemotherapeutic agent is selected from any agent known to be effective for at least partially treating multiple myeloma. Suitable chemotherapeutic agents include at least one of melphalan, prednisone, vincristine, doxorubicin, decadron, BCNU, cyclophosphamide, adriamycin, dexamethasone, thalidomide, bortezomib, pamidronate, and zoledronic acid.

[0021] In some embodiments, the inhibitor of IL-18 function binds specifically to a polypeptide selected from an IL-18 polypeptide, IL18R polypeptide, or IL18BP polypeptide.

[0022] Typically, the inhibitor of IL-18 function is selected from the group comprising: a peptide, nucleic acid, antigen-binding molecule, and small molecule inhibitor.

[0023] The inhibitor of IL-18 function and the chemotherapeutic agent are delivered to the subject simultaneously, sequentially, or separately.

[0024] In some embodiments, the inhibitor of IL-18 function is an antigen- binding molecule. In some embodiments of this type, the antigen-binding molecule specifically binds to at least a portion of the IL-18 polypeptide set forth in SEQ ID NO: 1. In some alternative embodiments, the antigen binding molecule specifically binds to at least a portion of an IL-18 Receptor 1 (IL18R1 ) polypeptide, such as the IL18R1 amino acid sequence set forth in SEQ ID NO: 3. In some other

embodiments, the antigen-binding molecule specifically binds to at least a portion of an IL-18 Receptor Accessory Protein (IL18RAP) polypeptide, such as the IL18RAP amino acid sequence set forth in SEQ ID NO: 5. In some other embodiments, the antigen-binding molecule specifically binds to at least a portion of an IL18BP polypeptide, such as the IL18BP amino acid sequence set forth in SEQ ID NO: 7.

[0025] In some other embodiments, the inhibitor of IL-18 function is a peptide.

[0026] In some alternative embodiments, the inhibitor of IL-18 function comprises, consists, or consists essentially of, a soluble IL18R1 polypeptide (e.g., such as the amino acid sequence set forth in SEQ ID NO: 3), or a soluble IL18RAP polypeptide (such as the amino acid sequence set forth in SEQ ID NO: 5). In some other embodiments, the inhibitor of IL-18 function comprises, consists, or consists essentially of a soluble IL18BP (such as the amino acid sequence set forth in SEQ ID NO: 7).

[0027] In some of the same embodiments and other embodiments, the composition further comprises a targeting agent. Preferably, the targeting agent binds to a cell surface receptor present on a memory T cell.

[0028] By way of an illustration, the targeting agent specifically binds to a polypeptide selected from: CD38 polypeptide, CAR-MIL polypeptide, CAR-T polypeptide, BCMA polypeptide, CD137 polypeptide, CD319, polypeptide CD46 polypeptide, CD47 polypeptide, MCL-1 polypeptide, CD229 polypeptide, CD54 polypeptide, CD56 polypeptide, CD3 polypeptide, CD200 polypeptide, CD16A polypeptide, CGEN-928 polypeptide, CD48 polypeptide, CD319 polypeptide, LMA polypeptide, KMA polypeptide, FcRH5 polypeptide, hTfR IgA polypeptide, Dkk1 polypeptide, APRIL polypeptide, CD95 polypeptide, KIR polypeptide, CS1

[0029] In some preferred embodiments of this type, the targeting agent comprises an antigen-binding molecule that specifically binds to a cell surface receptor on a memory T cell. By way of an illustrative example, the cell surface receptor may be a CD38 polypeptide.

[0030] In some alternative embodiments, the inhibitor of IL-18 reduces or prevents the production of IL-18 polypeptide. For example, the inhibitor of IL-18 function may be an siRNA molecule (e.g., an antisense RNA molecule).

[0031] In still another aspect, the present invention provides a composition for treating multiple myeloma in a subject, the composition comprising an inhibitor of IL-18 function and a memory T cell targeting agent.

[0032] Preferably, the inhibitor of IL-18 function binds specifically to a polypeptide selected from an IL-18 polypeptide, IL18R polypeptide (i.e., IL18R1 polypeptide or IL18RAP polypeptide), and IL18BP polypeptide.

[0033] Typically, the inhibitor of IL-18 function is selected from a peptide, nucleic acid molecule, antigen-binding molecule, or small molecule inhibitor. [0034] In some preferred embodiments, the inhibitor of IL-18 is an antigen- binding molecule. In some embodiments, the antigen-binding molecule specifically binds to at least a portion of an IL-18 polypeptide (as set forth in SEQ ID NO: 1 ). In other embodiments, the antigen binding molecule specifically binds to at least a portion of an IL18R1 polypeptide (as set forth in SEQ ID NO: 3). In other

embodiments, the antigen-binding molecule specifically binds to at least a portion of an IL18RAP polypeptide (as set forth in SEQ ID NO: 5). In other embodiments, the antigen-binding molecule specifically binds to at least a portion of an IL18BP polypeptide (as set forth in SEQ ID NO: 7).

[0035] Typically, the targeting agent binds to a cell surface receptor present on a memory T cell.

[0036] The composition of any one of claim 50, wherein the targeting agent specifically binds to a polypeptide selected from: CD38 polypeptide, CAR-MIL polypeptide, CAR-T polypeptide, BCMA polypeptide, CD137 polypeptide, CD319, polypeptide CD46 polypeptide, CD47 polypeptide, MCL-1 polypeptide, CD229 polypeptide, CD54 polypeptide, CD56 polypeptide, CD3 polypeptide, CD200 polypeptide, CD16A polypeptide, CGEN-928 polypeptide, CD48 polypeptide, CD319 polypeptide, LMA polypeptide, KMA polypeptide, FcRH5 polypeptide, hTfR IgA polypeptide, Dkk1 polypeptide, APRIL polypeptide, CD95 polypeptide, KIR polypeptide, CS1 polypeptide, CD19 polypeptide, CD20 polypeptide, CD74 polypeptide, SDF-1 polypeptide, IL-2 polypeptide, IL-6 polypeptide, PSGL-1 polypeptide, and CD40 polypeptide.

[0037] The method of any one of claims 41 to 51 , wherein the targeting agent comprises an antigen-binding molecule that specifically binds to a receptor presented on the surface of a memory T cell.

[0038] The composition of any one of claims 41 to 52, wherein the targeting agent is an antigen-binding molecule that specifically binds to a CD38 polypeptide.

[0039] The composition of any one of claims 41 to 52, wherein the

composition is a bispecific antibody.

[0040] A method of treating multiple myeloma in a subject, the method comprising administering a composition that comprises an IL-18 antagonist and a targeting agent. [0041] The present invention also provide particles, nanoparticles and/or polymeric nanoparticles that can encapsulate one or more composition of the present invention. The nanodelivery system of the present invention improves pharmacokinetics, targeting of tissues and cells to enhance efficacy, specificity and lower toxicity. The present conjugates designed for increasing immune response, and particles comprising such compositions provide more specific compositions and methods to treat multiple myeloma. The active agents of the conjugates in the nanoparticle are then released inside the APCs. In some embodiments, the active agents are only released within certain environments. In some embodiments, particles, nanoparticles and/or polymeric nanoparticles target bone marrow and delivers the compositions of the invention to the bone marrow. Such solid

nanoparticles and their preparation are taught in, for example, International Patent Publication No. WO2014/106208, the contents of which are incorporated herein in their entirety.

BRIEF DESCRIPTION OF THE FIGURES

[0042] An example of the present invention will now be described with reference to the accompanying drawings, in which:

[0043] Figure 1 shows that IL-18 is critically required for multiple myeloma progression. (A-F) C57BL/6 WT, 111 r , and 1118^~ mice were injected intravenously with 2 x 10⁶ Vk12653 multiple myeloma cells. Graphs showing the serum y-globulin levels on day 21 (A) and day 35 (B) post-multiple myeloma challenge. Serum protein electrophoresis results showing paraproteinemia on day 35 post-multiple myeloma challenge (C). Representative flow cytometry plots showing the frequency of CD138⁺B220^_ plasma cells (PCs) (D), and graphs showing the percentages (E) and the numbers (F) of PCs in the BM on day 35 post-multiple myeloma challenge. Data are shown as means ± SEM of 10 individual mice pooled from two experiments. (G and H) Kaplan-Meier survival curves of mice injected with 2 x 10⁶ Vk12653 multiple myeloma cells; wherein filled circles are WT; empty circles are //7r^A; and triangles are 1118^A; (G) or 5 x 10⁵ Vk12598 MM cells, wherein WT (n = 25); //7r^A (n = 19); and 1118^~ (n = 25) (H). Results are pooled from three (G) or two (H) experiments and 17- 25 mice per group are shown. Differences were tested for statistical significance using a Kruskal-Wallis test with post hoc Dunn’s test (A, B, E, and F) or a Mantel- Cox test (G and H), *p < 0.05, ^**p < 0.01 , ^***p < 0.001 , ^****p < 0.0001.

[0044] Figure 2 shows that H18^~/~ mice are protected from multi progression.

(A, B) Representative flow plots (A) and graphs (B) showing the percentages of plasma cells (PCs) in the naive multiple myeloma-free BM in WT, IHr^, and IH8^~/~ mice. Data are shown as mean ± SEM of four individual mice. (C, D) Graphs showing the percentages (C) and the numbers (D) of spleen PCs in indicated mice on day 35 post-multiple myeloma challenge. Data are shown as mean ± SEM of 10 individual mice pooled from two experiments. Differences were tested for statistical significance using a Kruskal-Wallis test with post-hoc Dunn’s test. ^*p < 0.05, ^****, p < 0.0001. (E-H) WT mice were co-housed with 1118^ mice for two weeks prior to the challenge with Vk12653 multiple myeloma. Graphs showing the mean y-globulin levels in mice on day 20 (E) and on day 30 (F) post-multiple myeloma challenge, and graphs showing the percentages (G) and the numbers (FI) of PCs on day 30 post- multiple myeloma challenge. Data are shown as mean ± SEM of 8-10 mice per group from one experiment. Differences between co-housed mice and non-co- housed counterparts were tested for statistical significance using a Mann-Whitney U test. NS: not significant https://mk.com.au/our-people/mark-metzeling/

[0045] Figure 3 shows that NLRP3 and ASC are partially involved in multiple myeloma progression. (A-D) Graphs showing the mean g-globulin levels ± SEM in WT, Nlrp3^~/~, and ASC mice on day 21 (A) and day 35 (B) after Vk12653 multiple myeloma challenge, and graphs showing the percentages (C) and the numbers (D) of PCs on day 35 post-multiple myeloma challenge. Data are shown as mean ± SEM of 9-10 mice per group pooled from two experiments. (E) Kaplan-Meier survival curves of indicated mice injected with 2 x 10⁶ Vk12653 multiple myeloma cells.

Results are pooled from two experiments and 19-20 mice per group are shown. Differences were tested for statistical significance using a Kruskal- Wallis test with post-hoc Dunn’s test (A-D) and a Mantel-Cox test (E). ^*p < 0.05, ^***p < 0.001.

[0046] Figure 4 shows that NLRP1 is critically required for multiple myeloma progression. (A-D) Graphs showing the serum g-globulin levels in WT and Nlrpl^ mice on day 21 (A) and day 35 (B) after Vk12653 multiple myeloma challenge and the percentages (C) and the numbers (D) of PCs in the BM on day 35 post-multiple myeloma challenge. Data are shown as means ± SEM of 9-10 individual mice pooled from two experiments. (E and F) Kaplan-Meier survival curves of mice injected with Vk12653 multiple myeloma cells; black circles represent WT; empty circles represent /V/rp7 ^A (E) or Vk12598 multiple myeloma cells (F). Results are pooled from three (E) or two (F) experiments and 15-20 mice per group are shown. (G-J) Graphs showing the percentages (G and I) and the numbers (H and J) of BM PCs in the indicated BM chimeric mice on day 30 after Vk12653 multiple myeloma challenge. Data are shown as means ± SEM of 9-10 individual mice pooled from two experiments. Differences were tested for statistical significance using a Mann- Whitney U test (A-D), a Mantel-Cox test (E and F), and a Kruskal-Wallis test with post hoc Dunn’s test (G-J), ^*p < 0.05, ^**p < 0.01 , ^***p < 0.001 , ^****p < 0.0001 .

[0047] Figure 5 shows that CD8⁺ T cells are essentially required for the control of myeloma in Nlrp1^~/~ and 1118^~' ^~ mice. (A) Schematic illustrating the experimental design of antibody treatment (clg: control Ig; i.p.: intraperitoneal; i.v.: intravenous). (B-C) Representative flow plots showing the percentages of CD8⁺ T cells (B) and NK cells (C) in the peripheral blood (PB) and BM on day 5 after treatment of indicated antibodies in naive WT, /V/rpT⁷ , and // S⁷ mice. (D-F) Graphs showing the mean g- globulin levels ± SEM (left) and Kaplan-Meier survival curves (right) in WT (D), Nlrp1^~l^~ (E), and II18^~I^~ (F) mice with Vk12653 multiple myeloma treated with indicated antibodies. Data are shown as means ± SEM of 8-1 1 individual mice pooled from two experiments. Differences were tested for statistical significance using a Kruskal-Wallis test with post hoc Dunn’s test (bar graphs) and Mantel-Cox test (survival curves). ^*p < 0.05, ^**p < 0.01 , ^***p < 0.001 , ^****p < 0.0001 .

[0048] Figure 6 shows that IL-18 acts as an immunosuppressive switch that drives multiple myeloma progression. (A-C) wild-type (WT) BM cells (10⁶) were cultured in the presence or absence of indicated cytokines for four days. Graphs showing the mean number of CD11 b⁺Gr-1 ⁺ cells (A), PMN subset (CD1 1 b⁺Ly6G⁺) (B), and MO subset (CD1 1 b⁺Ly6C^high) (C) after four days of culture. Data are shown as mean ± SD of three experiments. (D and E) Representative histograms and graphs showing the mean percentage of proliferating CD8⁺ T cells co-cultured with indicated cytokine-stimulated BM CD11 b⁺Gr-1⁺ cells in a 0.25:1 ratio (CD1 1 b⁺Gr-1 ⁺ cells:CD8⁺ T cells) (D) and the mean levels of IFN-g in culture supernatants (E). (F) Graphs showing the T cell proliferation-suppressing activity of PMN-MDSCs (left) and MO-MDSCs (right) induced by GM-CSF with or without rlL-18. (G)

Representative immunoblots for C/EBRb, NOS2, arginase I, and b-actin in

differentially induced MDSCs and freshly isolated control BM CD1 1 b⁺ cells. (FI)

Graph showing the mean nitrite levels in culture supernatants from differentially induced MDSCs after four days of culture with indicated cytokines. Data are shown as mean ± SD of two experiments. (I) Graph showing the mean arginase activity levels in cell lysates from differentially induced MDSCs after four days of culture with indicated cytokines. Data are shown as mean ± SD of two experiments. (J) Graph showing the mean reactive oxygen species (ROS) levels in differentially induced MDSCs after four days of culture with indicated cytokines. Data are shown as means ± SD of two experiments. (K) Graph showing the effect of following inhibitors on T cell-suppressing activity of IL-18-induced MDSCs: catalase (ROS inhibitor), L-NMMA (inducible nitric oxide synthase inhibitor), and nor-NOFIA (arginase inhibitor). (L and M) Graphs showing the levels of IL-18 in blood serum (L) and BM fluids (M) in naive WT mice and WT mice challenged with Vk12653 multiple myeloma at indicated days after injection (n = 9 mice per group). (N) Graph showing the levels of IL-18 in the cell lysate derived from MACS-isolated CD138⁺ PCs from spleens containing

Vk12653 multiple myeloma or Vk12598 multiple myeloma. Error bars represent mean ± SEM of triplicate. (O-Q) Graphs showing the percentage of indicated cells among total CD45⁺ leukocytes in BM (O) and peripheral blood (PB) (P and Q) (n = 7 - 9 mice per group). (S) Graph showing T cell-suppressing activity of BM CD1 1 b⁺Gr- 1 ⁺ cells isolated from

mice on days 35-40 after Vk12653 multiple myeloma injection (n = 6 mice per group). (R) Graphs showing the frequency of spleen CD1 1 b⁺Gr-1 ⁺ cells and their subset in WT, /V/rpT⁷-, and //78^A mice challenged with Vk12653 multiple myeloma from the same cohort of mice described in Figure 1A-E and Figure 3A-D. Data are shown as means ± SEM. (S) Graph showing T cell- suppressing activity of BM CD11 b⁺Gr-1 ⁺ cells isolated from WT and //7S^_/ mice on days 35-40 after Vk12653 multiple myeloma injection (n = 6 mice per group). (T) Schematic illustrating the experimental design of anti-Ly6G treatment and Kaplan- Meier survival curves of Vk12653 multiple myeloma-bearing WT mice treated with indicated mAb. Pooled data from two independent experiments are shown. (6U) Schematic illustrating the experimental design of rlL-18 treatment in WT and gene- targeted mice with Vk12653 multiple myeloma (U). i.p., intraperitoneal; i.v., intravenous. Graph showing the numbers of total CD11 b⁺Gr-1⁺ cells, PMN subset, and MO subset in BM and spleen after indicated treatment (V). Data are shown as mean ± SEM of 6-7 individual mice pooled from two experiments. Graph showing T cell suppressing activity of isolated BM CD11b⁺Gr-1⁺ cells from rlL-18- or PBS- treated mice (W). Data are shown as means ± SEM. Pooled data from two independent experiments are shown. (X) Immunoblots showing C/EBRb, NOS2, arginase I, and b-actin expression in isolated BM CD1 ^⁺Gr-1⁺ cells from rlL-18- or PBS-treated mice. (Y) Schematic illustrating the experimental design of rlL-18 treatment in WT and gene-targeted mice with Vk12653 multiple myeloma.

[0049] Figure 7 (A-C) Schematic illustrating the experimental design of rlL-18 treatment in WT and gene-targeted mice with Vk12653 MM (L). (D-H) Graphs showing the frequency of indicated immune cells in the CD45-gated population from naive WT, Nlrp1^~/~ and II18 mice. Data are shown as mean ± SEM of 6 individual mice pooled from two experiments. (I) Graph showing CD8⁺ T cell suppressing activity of BM CD11 bC3G-1 ⁺ cells isolated from WT, Nlrp ^A, and 1118^~A mice treated with rlL-18 for 4 consecutive days. Data are shown as means ± SEM Data are shown as means ± SEM, pooled from two experiments. Differences were tested for statistical significance using a one-way ANOVA with post hoc Tukey’s test (6D, E, and 6K-6Q), an unpaired two-tailed Student’s t test (6F and 6SK), a Mann-Whitney U test for g-globulin levels (7F-7Q), and a Mantel-Cox test for survival curves (7A- 7C, and 7J). *p < 0.05, *^*p < 0.01 , ***p < 0.001 , ^****p < 0.0001. NS, not significant.

[0050] Figure 8 shows that MDSCs limit T cell responses in multiple myeloma patients. (A and B) A global transcriptomic analysis of CD138 BM aspirates from 73 multiple myeloma patients at diagnosis was performed by RNA sequencing.

Fleatmap showing the inverse correlation between MDSC and cytotoxic lymphocyte signature genes within BM aspirates from multiple myeloma patients (A). Patient’s log-normalized gene expression heatmap showing the hierarchical clustering of multiple myeloma patients according to MDSC and cytotoxic lymphocyte signature genes (B). (E-J) BM CD33⁺CD11 b⁺HLADR CD15⁺ PMN-MDSCs and CD33⁺CD11 b⁺CD14⁺HLA-DR⁺ monocytes were isolated from multiple myeloma patients and co-cultured with healthy donor (HD) (E-H) or autologous multiple myeloma patient (I and J) CD3⁺ T cells stimulated by anti-CD3/CD28 microbeads. Representative histograms showing the proliferation of CD4⁺ T cells after 5 days (E and I) and graphs recapitulating the percentage of inhibition from the same culture (F and J). Graphs showing cytokine levels in the supernatant from HD CD3⁺ T cell and PMN-MDSC or monocyte co-cultures (E). Sup, suppressor cells. (G) BM

CD33⁺CD11 b⁺HLA-DR CD15⁺ PMN-MDSCs and CD33⁺CD11 b⁺CD14⁺ HLA-DR⁺ monocytes were isolated from multiple myeloma patients, and co-cultured with healthy donor (HD) CD3⁺ T cells stimulated by anti-CD3/CD28 microbeads.

Representative histograms showing the proliferation of CD8⁺ T cells after 5 days and graphs recapitulating the percentage of inhibition from the same culture. Data are shown as means ± SEM from n = 15. (K) BM CD33⁺CD11 b⁺HLA-DR CD15⁺ PMN- MDSCs and CD33⁺CD11 b⁺CD14⁺ HLA-DR⁺ monocytes were isolated from multiple myeloma patients, and co-cultured with autologous multiple myeloma patient CD3⁺ T cells stimulated by anti-CD3/CD28 microbeads. Representative histograms showing the proliferation of CD8⁺ T cells after 5 days and graphs recapitulating the

percentage of inhibition from the same culture. Data are shown as means ± SEM from n = 9 multiple myeloma patients (D). Data are shown as means ± SEM from n = 15 (C and D), n = 5 (E), and n = 9 (F and G). Unpaired two-tailed Student’s t test with Holm-Sidak multiple test correction, ^*p < 0.05, ^**p < 0.01 , ^***p < 0.001.

[0051] Figure 9 shows that IL-18 augments the immunosuppressive activity of multiple myeloma MDSCs. (A) Heatmap showing the hierarchical clustering of multiple myeloma patients according to genes highly correlated with IL18 (correlation > 0.7). (B) Heatmap showing the correlation between IL18 and classical PMN-MDSC signature genes within multiple myeloma patient BM aspirates. (C) Heatmap showing the correlation between MDSC signature genes and genes encoding indicated cytokines within multiple myeloma patient BM aspirates. (D) BM

CD33⁺CD11 b HLADR CD14 CD15 myeloid precursor cells were isolated from multiple myeloma patients and cultured in the presence or absence of rlL-18 (50 ng/mL) for 6 days. Representative flow cytometry plots showing the percentage of viable CD33⁺CD11 b⁺HLA-DR MDSCs after 6 days of culture with or without rlL-18. (E and F) Freshly isolated CD33⁺CD11 b HLADR myeloid precursor cells or IL-18 induced CD33⁺CD11 b HLA-DR MDSCs were co-cultured with healthy donor CD3⁺ T cells stimulated by anti-CD3/CD28 microbeads at the indicated ratios.

Representative histograms from 13 multiple myeloma patients showing the proliferation of CD4⁺ T cells in the presence of the indicated cells (E) and graph recapitulating the percentage of inhibition of CD4⁺ T cell proliferation (F). Sup, supernatant. Data are shown as means ± SEM from n = 13 multiple myeloma patients. Unpaired two-tailed Student’s t test with Holm-Sidak multiple test correction, ***p < 0.001. (G, H) Freshly isolated CD33⁺CD11 b HLA-DR myeloid precursor cells or IL-18-induced CD33⁺CD11 b HLA-DR MDSCs were co-cultured with HD CD3⁺ T cells stimulated by anti-CD3/CD28 microbeads at the indicated ratios. Representative histograms showing the proliferation of CD8⁺ T cells in the presence of the indicated cells (G) and graph recapitulating the percentage of inhibition of CD8⁺ T cell proliferation (H). Data are shown as means ± SEM from n = 13 multiple myeloma patients.

[0052] Figure 10 shows that a high level of BM IL-18 is an independent determinant of poor prognosis in multiple myeloma patients. (A and B) Graph showing BM plasma IL-18 levels in a retrospective cohort of 152 multiple myeloma patients at diagnosis (A) and Kaplan-Meier survival estimates over more than 80 months of follow-up for I L-18^high (> median value) and I L-18^|0W (% median value) multiple myeloma patients (B). (C) Kaplan-Meier survival curves over more than 80 months of follow-up for 152 multiple myeloma Graph showing the relationship between BM IL-18 levels and serum b2 microglobulin (P2MG) levels in 144 multiple myeloma patients with a high (> median value) or a low level (< median value) of the indicated cytokines. Differences were tested for statistical significance using a Mantel-Cox test. (D) Multivariate analysis showing hazard ratio and associated p value of the indicated variables for 145 multiple myeloma patients’ overall survival. (E) Graph showing BM plasma IL-18 levels in multiple myeloma patients separated according clinical parameters including International Staging System (ISS), high-risk cytogenetics, gender, and age. Data are shown as mean ± SD. (F) Graph showing the relationship between BM IL-18 levels and serum b2 microglobulin (b2Mΰ) levels in 144 multiple myeloma patients. (G) Kaplan-Meier survival curves in I L-18^h'^9h (> 245 pg/ml; n = 41 ) and IL-18^|0W patients (n = 52) who subsequently received bortezomib (velcade), dexamethasone and melphalan (VD-MEL200) in IFM2005-01 and IFM2007-01 clinical trials. Differences were tested for statistical significance using a Mantel-Cox test.

[0053] Figure 11 shows that I L-18 is a potential therapeutic target in the multiple myeloma BM microenvironment. (A and B) Schematics illustrating the experimental design of anti-l L-18 mAb treatment in WT mice with Vk12653 multiple myeloma (A). Graph showing the serum g-globulin levels on day 21 after Vk12653 multiple myeloma challenge and Kaplan-Meier survival curves of WT mice treated with anti-IL-18 or control Ig (clg) (n = 10 per group) (B). i.v., intravenous. (C-F) Schematics illustrating the experimental design and Kaplan-Meier survival curves of WT mice treated with indicated therapy after injection with Vk12653 (C) or Vk12598 multiple myeloma (D). Results are pooled from two (C) or three (D) experiments and 14-24 mice per group are shown. Graphs showing the ratio of CD8⁺⁺T cells to MDSCs in peripheral blood (PB) in mice with Vk12653 (E) or Vk12598 multiple myeloma (F). Results are pooled from two experiments and 14-16 mice per group are shown. (G) Schematics illustrating the experimental design and Kaplan-Meier survival curves of WT mice with Vk12653 multiple myeloma treated with indicated therapy with or without anti-CD8 (n = 6-8 per group). Data are shown as means ± SEM. Differences were tested for statistical significance using a Mann-Whitney U test (B), a Mantel-Cox test (B, C, D, and G), a one- way ANOVA with post hoc Tu key’s test (E and F). ^*p < 0.05, ^**p < 0.01 , ^***p < 0.001 , ^****p < 0.0001.

DETAILED DESCRIPTION OF THE INVENTION

[0054] The following description of the embodiments of the invention is not intended to limit the invention to these embodiments, but rather to enable any person skilled in the art to make and use this invention.

1. Definitions

[0055] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described. For the purposes of the present invention, the following terms are defined below.

[0056] The articles“a” and“an” are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element. Thus, for example, the term“cis- acting sequence” also includes a plurality of c/s-acting sequences.

[0057] The terms“administration concurrently” or“administering concurrently” or“co-administering” and the like refer to the administration of a single composition contains two or more actives, or the administration of each active as separate compositions and/or delivered by separate routes either contemporaneously or simultaneously or sequentially within a short enough period of time that the effective result is equivalent to that obtained when all such actives are administered as a single composition. By“simultaneously” is meant that the active agents are administered at substantially the same time, and desirably together in the same formulation. By“contemporaneously” it is meant that the active agents are

administered closely in time (e.g., one agent is administered within from about 1 min to within about 1 day before or after the other). Any contemporaneous time is useful. However, it will often be the case that when not administered simultaneously, the agents will be administered within about 1 min to within about 8 hrs and preferably within less than about 1 hr to about 4 hrs. When administered contemporaneously, the agents are suitably administered at the same site on the subject. The term“same site” includes the exact location, but can be within about 0.5 cm to about 15 cm, preferably from within about 0.5 cm to about 5 cm. The term“separately” as used herein means that the agents are administered at an interval, for example at an interval of about a day to several weeks or months. The active agents may be administered in either order. The term“sequentially” as used herein means that the agents are administered in sequence, for example at an interval or intervals of minutes, hours, days or weeks. If appropriate, the active agents may be

administered in a regular repeating cycle. [0058] As used herein,“and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (or).

[0059] Further, the term“about”, as used herein when referring to a

measurable value such as an amount, dose, time, temperature, activity, level, number, frequency, percentage, dimension, size, amount, weight, position, length and the like, is meant to encompass variations of ± 20%, ± 10%, ± 5%, ± 1%, ±

0.5%, or even ± 0.1 % of the specified amount, dose, time, temperature, activity, level, number, frequency, percentage, dimension, size, amount, weight, position, length and the like.

[0060] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either both of those included limits are also included in the invention.

[0061] The terms“antagonist” and“inhibitor” are used interchangeably herein to refer to any molecule that partially or fully blocks, inhibits, stops, diminishes, reduced, impedes, impairs or neutralizes one or more biological activities or functions of IL-8 or a receptor to which it binds (e.g., IL-8R) in any setting including in vitro, in situ, or in vivo. Likewise, the terms“antagonize”,“antagonizing”, inhibit”, “inhibiting” and the like are used interchangeably herein to refer to blocking, inhibiting, stopping, diminishing, reducing, impeding, impairing, or neutralizing an activity or function as described for example above or elsewhere herein. By way of an example, the terms“antagonize” and“inhibit” can refer to a decrease of about 10%, 20%, 30%, 40%, 50%, 60%, 60%, 70%, 80%, 90%, or 100% in an activity or function.

[0062] By“antigen-binding molecule” is meant a molecule that has binding affinity for a target antigen. It will be understood that this term extends to immunoglobulins, immunoglobulin fragments, and non-immunoglobulin derived protein frameworks that exhibit antigen-binding activity. Representative antigen- binding molecules that are useful in the practice of the present invention include polyclonal and monoclonal antibodies, as well as their fragments (such as Fab, Fab’, F(ab’)2, Fv), single chain (scFv) and domain antibodies (including, for example, shark and camelid antibodies), and fusion proteins comprising an antibody, and any other modified configuration of the immunoglobulin molecule that comprises an antigen- binding/recognition site. An antibody includes an antibody of any class, such as IgG, IgA, or IgM (or sub-class thereof), and the antibody need not be of any particular class. Depending on the antibody amino acid sequence of the constant region of its heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes) (e.g., lgG1 , lgG2, lgG3, lgG4, lgA1 , lgA2). The heavy-chain constant regions that correspond to the different classes of immunoglobulins are called a, d, e, y, and m, respectively. The subunit structures and three-dimensional configurations of different classes of

immunoglobulins and well known. Antigen-binding molecules also encompass dimeric antibodies, as well as multivalent forms of antibodies. In some embodiments, the antigen-binding molecules are chimeric antibodies, in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies so long as they exhibit the desired biological activity (see, for example, U.S. Patent No. 4,816,567; and Morrison et al., 1984, Proc. Natl. Acad. Sci. USA 81 : 6851-6855). Flumanised antibodies are also contemplated, which are generally produced by transferring complementarity determining regions (CDRs) from heavy and light variable chains of a non-human (e.g., rodent, preferably mouse) immunoglobulin into a human variable domain. Typical residues of human antibodies are then substituted in the framework regions of the non-human counterparts. The use of antibody components derived from humanized antibodies obviates potential problems associated with the immunogenicity of non-human constant regions. General techniques for cloning non-human, particularly murine, immunoglobulin variable domains are described, for example, by Orlandi et al., (1989, Proc. Natl. Acad. Sci. USA 86:3833). Techniques for producing humanized monoclonal antibodies are described, for example, by Jones et al., (1986, Nature 321 :522), Carter et al., (1992, Proc. Natl. Acad. Sci. USA 89 4285), Sandhu (1992, Crit. Rev. Biotech. 12 : 437), Singer et al., (1993, J. Immunol. 150: 2844), Sudhir (ed. Antibody Engineering Protocols, Humana Press, Inc., 1995), Kelley (“Engineering Therapeutic Antibodies” in Protein Engineering: Principles and Practice, pages 399-434, 1996), and Queen et al., U.S. Patent No. 5,693,762 (1997). Humanised antibodies include“primatised” antibodies in which the antigen-binding region of the antibody is derived from an antibody produced by immunizing macaque monkeys with the antigen of interest.

[0063] By“coding sequence” is meant any nucleic acid sequence that contributes to the code for the polypeptide product of a gene or for the final mRNA product of a gene (e.g., the mRNA product of a gene following splicing). By contrast, the term“non-coding sequence” refers to any nucleic acid sequence that does not contribute to the code for the polypeptide product of a gene or for the final mRNA product of a gene.

[0064] By "corresponds to" or "corresponding to" is meant a nucleic acid or amino acid sequence w that displays substantial sequence identity or similarity to an nucleic acid or amino acid sequence in a reference sequence. In general, the polynucleotide or polypeptide will display at least about 30, 40, 50, 55, 60, 65, 70,

75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 % identity or similarity to at least a portion of the reference sequence.

[0065] Throughout this specification, unless the context requires otherwise, the words“comprise”,“comprises” and“comprising” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. Thus, use of the term“comprising” and the like indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present. By “consisting of is meant including, and limited to, whatever follows the phrase “consisting of. Thus, the phrase“consisting of indicates that the listed elements are required or mandatory, and that no other elements may be present. By“consisting essentially of is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase“consisting essentially of indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements.

[0066] By“corresponds to” or“corresponding to” is meant an amino acid sequence that displays substantial sequence identity or similarity to an amino acid sequence in a target antigen. In general, the antigen will display at least about 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or similarity to at least a portion of the target antigen.

[0067] An“effective amount”, in the context of preventing or treating multiple myeloma, is meant the administration of an amount of composition to an individual in need thereof, either in a single dose or as part of a series, that is effective for that prevention or treatment. The effective amount will vary depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated, the formulation of the composition, the assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determine through routine trials.

[0068] By "expression vector" is meant any autonomous genetic element capable of directing the synthesis of a protein encoded by the vector. Such expression vectors are known by practitioners in the art.

[0069] The term“gene” is used in its broadest context to include both a genomic DNA region corresponding to the gene as well as a cDNA sequence corresponding to exons or a recombinant molecule engineered to encode a functional form of a product.

[0070] The terms“IL-18” and“IL-18 polypeptide” (also known as Iboctadekin, interferon-y-inducing factor (IFN-y-inducing factor), and I L-1 y), as used herein means “interleukin-18,” a polypeptide having a sequence according to UniProtKB Accession No. Q141 16, as set forth in SEQ ID NO: 1 , the product of an IL18 gene (e.g., the human IL18 gene (identified by GenBank Accession No. U90434)), and includes all of the variants, isoforms and species homologues of IL-18.

[0071] The term“inhibitor of IL-18” within the context of this invention refers to any molecule modulating IL-18 production and/or action in such a way that IL-18 production and/or action is attenuated, reduced, or partially, substantially or completely prevented or blocked.

[0072] The term“IL-18 receptor” as used herein means a receptor or a receptor complex mediating IL-18 signalling. IL-18 signalling requires two receptors, IL-18 receptor 1 (IL18R1) and IL-18 receptor accessory protein (IL18RAP). Thus, “IL-18 receptor” contemplates both IL18R1 and IL18RAP. The term“IL18R1” (also known as CD218a, CDw218a, I L1 Rrp, and IL18RA) as used herein means “interleukin 18 receptor 1 ,” a polypeptide having an amino acid sequence according to UniProtKB Accession No. Q13478, the product of an IL18R1 gene (e.g., a human IL18R1 gene (identified by GenBank Accession No. U43672)), and includes all of the variants, isoforms and species homologues of IL18R1. The term“IL18RAP” (also known as Accessory protein-like (AcPL), CD218b, CDw218b, IL-1 receptor 7

(IL1 R7), IL18Rp, and IL18Rb) as used herein means“interleukin 18 receptor accessory protein,” a polypeptide having an amino acid sequence according to UniProtKB Accession No. 095256, the product of an IL18RAP gene (e.g., a human IL18RAP gene (identified by GenBank Accession No. AF077346)), and includes all of the variants, isoforms, and species homologues of IL18RAP. Variants of IL18R1 and IL18RAP also include soluble mature receptors.

[0073] “IL-18 signalling” as used herein means the processes initiated by IL- 18 or another IL-18 receptor on the cell surface, resulting in measurable changes in cell function. The IL-18 receptor complex includes IL18R1 and IL18RAP, and ligand binding activates downstream signal transduction pathways for example, NFKB, leading to the production of cytokines and chemokines. IL-18 signalling can be measured, for example, by assessing the amount of cytokines and chemokines produced upon induction with an IL-18 receptor ligand, for example, measuring production of CXCL-8, IL-6, G-CSF, MCP-1 , MIP-1a, RANTES, or CCL2 (as described in Cai et al., Cytokine, 2001 , 167: 6559-67; Wong et al., Am. J. Respir. Cell Mol. Biol., 2005, 33: 186-194). The methods and suitable readout systems are well known in the art and are commercially available.

[0074] The terms“patient”,“subject” and“individual” are used interchangeably herein, and refer to an animal, particularly a vertebrate. This includes human and non-human animals. The term“non-human animals” and“non-human mammals” are used interchangeably herein includes all vertebrates, e.g., mammals, such as non- human primates, (particularly higher primates), sheep, dog, rodent (e.g. mouse or rat), guinea pig, goat, pig, cat, rabbits, cows. In some embodiments, the subject is human. In another embodiment, the subject is an experimental animal or animal substitute as a disease model. Preferably, the subject is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but are not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of disorders associated with unwanted neuronal activity. In addition, the methods and compositions described herein can be used to treat domesticated animals and/or pets. A subject can be male or female. A subject can be a fully developed subject (e.g., an adult) or a subject undergoing the developmental process (e.g., a child, infant or fetus).

[0075] By“pharmaceutically-acceptable carrier” is meant a solid or liquid filler, diluent, or encapsulating substance that may be safely used in topical or systemic administration.

[0076] The term“pharmaceutically compatible salt” as used herein refers to a salt which is toxicologically safe for human and animal administration. This salt may be selected from a group including hydrochlorides, hydrobromides, hydroiodides, sulfates, bisulfates, nitrates, citrates, tartrates, bitartrates, phosphates, malates, maleates, napsylates, fumarates, succinates, acetates, terephthalates, pamoates, and pectinates.

[0077] The term“polynucleotide” or“nucleic acid” as used herein designates mRNA, RNA, cRNA, c DNA, or DNA. The term typically refers to oligonucleotides greater than 30 nucleotides in length.

[0078] “Polypeptide,”“peptide,” and“protein” are used interchangeably herein to refer to a polymer of amino acid residues and to variants and synthetic analogues of the same. Thus, these terms apply to amino acid polymers in which one or more amino acid residues is a synthetic non-naturally occurring amino acid, such as a chemical analogue or a corresponding naturally occurring amino acid, as well as to naturally-occurring amino acid polymers.

[0079] The term“peptide variant,”“polypeptide variant,” and“variant” refer to polypeptides which vary from a reference polypeptide by the addition, deletion, or substitution of at least one amino acid. It is well understood in the art that some amino acids may be changed to others with broadly similar properties without changing the nature of the activity of the polypeptide. Preferred variant polypeptides comprise conservative amino acid substitutions. Exemplary conservative

substitutions in a polypeptide may be made according to Table 1 .

TABLE 1

Phe Met Leu T r

[0080] The term“inactive variant” refers to a polypeptide which varies from a referenced polypeptide by the addition, deletion, or substitution of at least one amino acid that is important in conferring activity to the polypeptide. It is well understood in the art that some amino acids may be charged to disrupt critical interactions and therefore change the nature of the activity of the polypeptide. Suitably, an inactive variant polypeptide will have only around 50%, 45%, 40%, 35%, 30%, 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11 %, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1 %, less than 1 %, or 0% activity of the corresponding wild-type polypeptide.

[0081] Substantial changes in function are made by selecting substitutions that are less conservative than those shown in Table 1. Other replacements would be non-conservative substitutions and relatively fewer of these may be tolerated. Generally, the substitutions which are likely to produce the greatest changes in a polypeptide’s properties are those in which (a) a hydrophilic residue (e.g., Ser or Asn) is substituted for, or by, a hydrophobic residue (e.g., Ala, Leu, lie, Phe, or Val); (b) a cysteine or proline is substituted for, or by, any other residue; (c) a residue having an electropositive side chain (e.g., Arg, His, or Lys) is substituted for, or by, an electronegative residue (e.g., Glu or Asp); or (d) a residue having a smaller side chain (e.g., Ala, Ser) or no side chain (e.g., Gly) is substituted for, or by, one having a bulky side chain (e.g., Phe or Trp).

[0082] By“treatment,”“treat,”“treated” and the like is meant to include both prophylactic and therapeutic treatment, including, but not limited to preventing, relieving, altering, reversing, affecting, inhibiting, the development or progression of, ameliorating, or curing (1 ) multiple myeloma; or (2) a symptom of multiple myeloma; or (3) a predisposition toward multiple myeloma.

[0083] Persons skilled in the art will appreciate that numerous variations and modifications will become apparent. All such variations and modifications which become apparent to persons skilled in the art, should be considered to fall within the spirit and scope of the invention broadly before described.

[0084] Thus, for example, it will be appreciated that features from different examples above and elsewhere herein may be used interchangeably where appropriate.

2. Compositions

[0085] The present invention is predicated at least in part on the determination that IL-18 plays an important role as an immunosuppressive switch that drives multiple myeloma progression, and that progression of multiple myeloma can be reduced or reversed through inhibition of IL-18 function. These determinations lead the inventors to discover that multiple myeloma may be treated by removal, inhibition or neutralization of IL-18 production and/or function.

[0086] Based on these observations, the present inventors proposed that more efficacious prophylactic or therapeutic treatment regiments can be achieved using compositions that comprise an inhibitor of IL-18 function.

2.1 Inhibitors of IL-18 function

[0087] The term“inhibitor of IL-18 function” encompasses any molecule modulating the production or an activity of an IL-18 polypeptide in such a way that IL-18 polypeptide production and/or activity (or signaling through the IL-18 receptor (IL18R)) is attenuated, reduced, or partially, substantially or completely prevented or blocked.

[0088] The inhibitors of IL-18 function can either bind to or sequester the IL-18 polypeptide directly with sufficient affinity and specificity to partially or substantially neutralize the IL-18 or IL-18 binding site(s) responsible for IL-18 polypeptide binding to the IL18R polypeptide or IL18BP polypeptide. The inhibitor of IL-18 function may also inhibit the IL-18 signaling pathway, which is activated within the cells upon an IL-18 polypeptide binding to an IL-18R polypeptide. In some embodiments, the inhibitor of IL-18 function is selected from a neutralizing antibody directed against IL-18, a neutralizing antibody directed against any of the IL-18 receptor subunits, a neutralizing antibody directed against the IL-18 binding protein, an inhibitor of the IL- 18 signaling pathway, an antagonist of IL-18 which competes with IL-18 and blocks the IL-18 receptor, an inhibitor of caspase-1 (ICE), and/or an IL18R or IL18BP polypeptide, isoform, functional derivative, active fraction or circularly permutated derivatives thereof inhibiting the biological activity of IL-18.

[0089] An inhibitor of IL-18 production can be any molecule negatively affecting the synthesis, processing or maturation of IL-18. In some embodiments, suppressors of gene expression of IL-18, antisense mRNAs reducing or preventing the transcription of the IL-18 mRNA or leading to degradation of the mRNA, proteins impairing correct folding, or partially or substantially preventing secretion of IL-18, proteases degrading IL-18, once it has been synthesised, inhibitors of proteases cleaving pro-IL-18 in order to generate mature IL-18, such as inhibitors of caspase-1 and the like. In some embodiments of the invention, an antisense nucleic acid sequence which is expressed by a plasmid vector is used to inhibit IL-18, IL-18R1 , or IL18BP expression. The antisense expression vector is used to transfect a mammalian cell or the mammal itself, thereby causing reduced endogenous expression of IL-18, IL18R1 , or IL18BP.

[0090] Antisense molecules and their use for inhibiting gene expression are well known in the art (see, for example, Cohen, 1989, In: Oligodeoxyribonucleotides, Antisense Inhibitors of Gene Expression, CRC Press). Antisense nucleic acids are DNA or RNA molecules that are complementary, as that term is defined elsewhere herein, to at least a portion of a specific mRNA molecule (Weintraub, 1990, Scientific American 262:40). In the cell, antisense nucleic acids hybridize to the corresponding mRNA, forming a double-stranded molecule thereby inhibiting the translation of genes.

[0091] The use of antisense methods to inhibit the translation of genes is known in the art, and is described, for example, in Marcus-Sakura (1988, Anal.

Biochem. 172:289). Such antisense molecules may be provided to the cell via genetic expression using DNA encoding the antisense molecule as taught by Inoue, 1993, U.S. Patent No. 5,190,931.

[0092] Alternatively, antisense molecules of the invention may be made synthetically and then provided to the cell. Antisense oligomers of between about 10 nucleotides to about 30 nucleotides, and more preferably about 15 nucleotides, are preferred, since they are easily synthesized and introduced into a target cell.

Synthetic antisense molecules contemplated by the invention include oligonucleotide derivatives known in the art which have improved biological activity compared to unmodified oligonucleotides (see, for example, U.S. Patent No. 5,023,243).

[0093] An inhibitor of IL-18 action can be an IL-18 antagonist, for example. Antagonists can either bind to or sequester the IL-18 molecule itself with sufficient affinity and specificity to partially or substantially neutralize the IL-18 or IL-18 binding site(s) responsible for IL-18 binding to its ligands (e.g., the IL-18 receptors). The inhibitor of IL-18 function includes any molecule or compound that directly or indirectly binds or physically associates with IL-18 or its receptor(s) (e.g., IL18R1) and that suitably blocks, inhibits, or otherwise antagonizes at least one of its functions or activities (e.g., binding to or interacting with one or more surface molecules (e.g., receptors) present on white blood cells, especially lymphocytes, and more especially NK cells or T cells. The binding or association may involve the formation of an induced magnetic field or paramagnetic field, covalent bond formation, an ionic interaction such as, for example, an ionic lattice, a hydrogen bond, or alternatively, a van de Waals interaction such as, for example, a dipole- dipole interaction, dipole-induced-dipole interaction, induced-dipole-induced-dipole interaction or a repulsive interaction or any combination of the above forces of attraction.

[0094] In certain embodiments, the inhibitor of IL-18 function is an inactive variant form of IL-18. For example, an inactive variant IL-18 polypeptides may be distinguished from a wild-type IL-18 polypeptide by one or more amino acids. By way of an illustrative example, a suitable wild-type polypeptide is the human IL-18 polypeptide, with an amino acid sequence set forth in SEQ ID NO: 1.

[0095] In some embodiments, the recombinant soluble IL-18 polypeptide is encoded by the nucleic acid sequence corresponding to the sequence set forth in SEQ ID NO: 2. Suitably, the inhibitor of IL-18 function is encoded in an expression vector that comprises a IL-18 coding sequence comprising a polynucleotide sequence corresponding to the sequence set forth in SEQ ID NO:2.

[0096] Inhibitors of IL-18 function may be soluble IL-18 receptors or molecules mimicking the receptors, or agent blocking the IL-18 receptors, or IL-18 antigen- binding molecules, such as polyclonal or monoclonal antibodies, or any other agent or molecule preventing the binding of IL-18 to its targets, thus diminishing or preventing triggering of the intra- or extracellular reactions mediated by 11-18. In some embodiments, the inhibitor of IL-18 function is any molecule capable of specifically preventing activation of cellular receptors for IL-18. For example, inhibitors of this type can be selected from soluble, membrane-bound or defective IL-18 receptors or soluble IL-18 receptor subunits, including but not limited to IL-18 receptor 1 (IL18R1) and IL-18 receptor accessory protein (IL18RAP) polypeptides. [0097] In some embodiments, the inhibitor of IL-18 function comprises an amino acid sequence corresponding to the IL18R1 polypeptide sequence set forth below, and deposited as UniProtKB Accession No. Q13478:

MNCRELPLTLWVLISVSTAESCTSRPHITWEGEPFYLKHCSCSLAHEIETTTK

SWYKSSGSQEHVELNPRSSSRIALHDCVLEFWPVELNDTGSYFFQMKNYTQ

KWKLNVIRRNKHSCFTERQVTSKIVEVKKFFQITCENSYYQTLVNSTSLYKNC

KKLLLENNKNPTIKKNAEFEDQGYYSCVHFLHHNGKLFNITKTFNITIVEDRSNI

VPVLLGPKLNHVAVELGKNVRLNCSALLNEEDVIYWMFGEENGSDPNIHEEK

EMRIMTPEGKWHASKVLRIENIGESNLNVLYNCTVASTGGTDTKSFILVRKAD

MADIPGHVFTRGMIIAVLILVAWCLVTVCVIYRVDLVLFYRHLTRRDETLTDGK

TYDAFVSYLKECRPENGEEHTFAVEILPRVLEKHFGYKLCIFERDWPGGAVV

DEIHSLIEKSRRLIIVLSKSYMSNEVRYELESGLHEALVERKIKIILIEFTPVTDFT

FLPQSLKLLKSHRVLKWKADKSLSYNSRFWKNLLYLMPAKTVKPGRDEPEVL

PVLSES [SEQ ID NO: 3]

[0098] In some embodiments, a suitable soluble IL-18R polypeptide may lack the native signal sequence (i.e., residues 1-18 of the amino acid sequence set forth in SEQ ID NO: 3).

[0099] In some embodiments, the IL18R1 polypeptide is encoded by the nucleic acid sequence set forth in SEQ ID NO: 4. Suitably, the inhibitor of IL-18 function is encoded in an expression vector that comprises a IL18R1 coding sequence comprising a polynucleotide sequence corresponding to the sequence set forth in SEQ ID NO:4.

[0100] IL18RAP and its isoforms are known in the art, such as that disclosed and described in Born et al., J. Biol. Chem., 46: 6, 29445-29450 (1998). In some embodiments, the inhibitor of IL-18 function comprises an amino acid sequence corresponding to the IL17RAP polypeptide deposited as UniProtKB Accession No. 095256, and set forth below:

MLCLGWIFLWLVAGERIKGFNISGCSTKKLLWTYSTRSEEEFVLFCDLPEPQK

SHFCHRNRLSPKQVPEHLPFMGSNDLSDVQWYQQPSNGDPLEDIRKSYPHII

QDKCTLHFLTPGVNNSGSYICRPKMIKSPYDVACCVKMILEVKPQTNASCEYS

ASHKQDLLLGSTGSISCPSLSCQSDAQSPAVTWYKNGKLLSVERSNRIWDE

VYDYHQGTYVCDYTQSDTVSSWTVRAWQVRTIVGDTKLKPDILDPVEDTLE VELGKPLTISCKARFGFERVFNPVIKWYIKDSDLEWEVSVPEAKSIKSTLKDEII ERNIILEKVTQRDLRRKFVCFVQNSIGNTTQSVQLKEKRGWLLYILLGTIGTLV AVLAASALLYRHWIEIVLLYRTYQSKDQTLGDKKDFDAFVSYAKWSSFPSEAT SSLSEEHLALSLFPDVLENKYGYSLCLLERDVAPGGVYAEDIVSIIKRSRRGIFI LSPNYVNGPSIFELQAAVNLALDDQTLKLILIKFCYFQEPESLPHLVKKALRVLP TVTWRGLKSVPPNSRFWAKMRYHMPVKNSQGFTWNQLRITSRIFQWKGLSR TETTGRSSQPKEW [SEQ ID NO: 5]

[0101] More specifically, a particularly suitable soluble IL-18R polypeptide may lack the native transmembrane domain (i.e., lacking amino acids 357-377 of the wild-type human IL18RAP polypeptide sequence set forth in SEQ ID NO: 5).

Accordingly, in some embodiments of this type, the inhibitor of IL-12 function is an IL18RAP polypeptide that comprises the extracellular domain (corresponding to amino acid residues 20-356 of the IL18RAP sequence set forth in SEQ ID NO: 5) or a fragment thereof. Optionally, the IL18RAP polypeptide may also comprise the native signal sequence (i.e., residues 1-20 of the amino acid sequence set forth in SEQ ID NO: 5).

[0102] In some embodiments, the IL18RAP polypeptide is encoded by the nucleic acid sequence set forth in SEQ ID NO: 6. Suitably, the inhibitor of IL-18 function is encoded in an expression vector that comprises a IL18RAP coding sequence comprising a polynucleotide sequence corresponding to the sequence set forth in SEQ ID NO:6.

2.2 IL-18 binding protein

[0103] The term“IL-18 binding proteins” is used herein synonymously with “IL-18 binding protein” or“IL18BP”. It comprises IL-18 binding proteins as defined in WO 99/09063 and/or in Novick et al., 1999, including splice variants and/or isoforms of IL-18 binding proteins, as defined in Kim et al., 2000, which bind to IL-18. In some embodiments, the human IL18BP isoforms a and c of are particularly useful. The proteins of these embodiments may be glycosylated or non-glycosylated.

Furthermore, they may be derived from natural sources, or they may preferably be produced recombinantly. Recombinant expression may be carried out in prokaryotic expression systems such as E. coli, or in eukaryotic expression systems

(preferably, mammalian expression systems).

[0104] In some embodiments, the inhibitor of IL-18 function is any molecule capable of specifically preventing the binding of an IL-18 polypeptide to an IL-18BP polypeptide. For example, inhibitors of this type can be selected from soluble, membrane-bound or inactive IL18BP polypeptides. In some embodiments, the inhibitor of IL-18 function comprises an amino acid sequence corresponding to the IL18BP polypeptide sequence below, and deposited as UniProtKB Accession No. 095998:

MTMRHNWTPDLSPLWVLLLCAHWTLLVRATPVSQTTTAATASVRSTKDPCP

SQPPVFPAAKQCPALEVTWPEVEVPLNGTLSLSCVACSRFPNFSILYWLGNG

SFIEHLPGRLWEGSTSRERGSTGTQLCKALVLEQLTPALHSTNFSCVLVDPE

QWQRHWLAQLWAGLRATLPPTQEALPSSHSSPQQQG [SEQ ID NO: 7]

[0105] Optionally, the IL18BP polypeptide may comprise the native signal sequence (i.e., residues 1-30 of the amino acid sequence set forth in SEQ ID NO: 7).

[0106] Polypeptide variants may be prepared by known synthesis and/or site- directed mutagenesis techniques, or any other technique known in the art for changing amino acid sequences.

[0107] In some embodiments, the IL18BP polypeptide is encoded by the nucleic acid sequence set forth in SEQ ID NO: 8. Suitably, the inhibitor of IL-18 function is encoded in an expression vector that comprises a IL18BP coding sequence comprising a polynucleotide sequence corresponding to the sequence set forth in SEQ ID NO:8.

[0108] In some embodiments, the inhibitor of IL-18 function comprises an IL18BP polypeptide variant. Polypeptide variants in accordance with the present invention include proteins encoded by a nucleic acid, such as DNA or RNA, which hybridizes to DNA or RNA that encodes an IL18BP, at least under low stringency conditions.

[0109] Preferably, IL18BP polypeptide variants comprise an amino acid sequence with high similarity to a wild-type IL18BP polypeptide sequence, such as to retain an activity comparable to the wild-type IL18BP. For example, one activity of a wild-type IL8BP is its ability to bind to an IL-18 polypeptide. As long as the polypeptide variant has substantial binding activity to IL-18, it can be used in the purification of IL-18, such as by means of affinity chromatography, and thus can be considered to have substantially similar activity to IL-18BP. It can be determined whether any given IL18BP polypeptide variant has substantially the same activity as IL18BP by any means of routine experimentation (e.g., competition assay to determine binding to an appropriately labelled IL-18, such as radioimmunoassay or ELISA assay).

[0110] In some embodiments, the IL18BP polypeptide variant has at least 40% identity or homology with the sequence a wild-type IL18BP, as defined in WO 99/09063. More preferably, it has at least 50%, at least 60%, at least 70%, at least 80% or, most preferably, at least 90% identity or homology thereto.

[0111] Examples of the methodologies for introducing amino acid substitutions into polypeptides include the method steps such as presented in U.S. Patent Nos. 4,959,314, 4,588,585, 4,737,462, 5,116,943, 4,965,195, 4,879,111 , and 5,017,691 ; with lysine substituted proteins presented in U.S. Patent No. 4,904,584.

2.3 Fusion Proteins

[0112] In some embodiments, the antagonist of IL-18 function is a fusion protein, the fusion protein comprising, consisting, or consisting essentially of an IL18BP polypeptide conjugated or linked to another polypeptide sequence that, for example, has an enhanced bioavailability upon administration to the subject. An IL18BP may therefore be conjugated to another polypeptide (for example, an immunoglobulin or a fragment thereof).

2.4 Functional derivatives

[0113] In some embodiments, the inhibitor of IL-18 function comprises a functional derivative of a IL18BP polypeptide, or a variant or fusion thereof. Suitable functional derivatives thereof may be prepared from the functional groups that occur as side chains on the residues, or the N- or C-terminal groups, by any means known in the art. By way of an illustrative example, functional derivatives may include polyethylene glycol side-chains, which may mask antigenic sites and extend the residence of an IL18BP after administration. Other derivatives include aliphatic esters of the carboxyl groups, amides of the carboxyl groups by reaction with ammonia or with primary or secondary amines, N-acyl derivatives of free amino groups of the amino acid residues formed with acyl moieties (e.g., alkanoyl or carbocydic aroyl groups) or O-acyl derivatives of free hydroxyl groups (for example that of seryl or threonyl residues) formed with acyl moieties.

[0114] In some of the same embodiments and other embodiments, the inhibitor of IL-18 function may include an biologically active fragment of a wild-type IL18BP polypeptide. In embodiments of this type, the present invention covers any fragment or precursor of the polypeptide chain of the protein molecule alone or together with associated molecules or residues linked thereto (e.g., sugar or phosphate residues, or aggregates of the protein molecule or the sugar residues by themselves) provided said fraction has substantially similar activity to IL-18BP.

[0115] In some embodiments, the present invention is drawn to agents (e.g., antigen-binding molecules) that block the association of IL18R1 and IL18RAP

(and/or with additional receptor subunits) and thereby prevent formation of a functional receptor complex (i.e., a receptor complex that is capable of being activated).

2.5 Antigen-binding molecules

[0116] In a further preferred embodiment of the invention, the inhibitor of IL-18 function is an antigen-binding molecule directed against IL-18 or its receptors, IL18R. Antigen-binding molecule directed to any of the IL-18R subunits, including IL18R1 and IL18RAP, may be used in accordance with these embodiments.

[0117] In embodiments of this type, the antigen-binding molecules may be polyclonal antibodies, monoclonal antibodies, single chain antibodies (Fc antibodies) chimeric antibodies, humanized antibodies, or fully human antibodies Recombinant antibodies and fragments thereof are characterized by high affinity binding to IL-18 or IL-18R in vivo and low toxicity. The antibodies which can be used in the invention are characterized by their ability to treat patients for a period sufficient to have good to excellent regression or alleviation of the multiple myeloma or any symptom or group of symptoms related to the multiple myeloma, together with a low toxicity. [0118] Neutralizing antibodies are readily raised in animals such as rabbits, goat or mice by immunization with a polypeptide selected from an IL-18

polypeptide, IL18R1 polypeptide, and an IL18RAP polypeptide. Immunized mice are particularly useful for providing sources of B cells for the manufacture of hybridomas, which in turn may be cultured to produce large quantities of anti-IL-18 monoclonal antibodies.

IL-18 Antigen-Binding Molecules

[0119] In some embodiments, aspects of the invention provide an antigen- binding molecule specific for IL-18 which inhibits the binding of IL-18 to one or both of IL-18R and IL-18BP and thereby reduces IL-18 activity.

[0120] In some embodiments, the antigen-binding molecule is a single chain antibody. Techniques described for the production of single chain antibodies (see, U.S. Patent No. 4,946,778; Bird, 1988, Science 242:423-426; Huston et al., 1988, Proc. Natl. Acad, Sci. USA 85:5879-5883; and Ward et al., 1989, Nature 334:544- 546) can also be adapted to produce single chain antibodies against IL-18 gene products. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide.

[0121] An isolated antibody may bind to an epitope on an IL-18 polypeptide which wholly or partially overlaps the IL18R1 polypeptide binding site and/or the IL18BP polypeptide binding site. For example, an antibody specific for IL-18 may bind to an epitope of IL-18 which comprises one or more of amino acid residues Tyr1 , Gly3, Leu5, Glu6, Lys8, Met51 , Lys53, Asp54, Ser55, Gln56, Pro57, Arg58, Gly59, Met60, Arg104, Ser105 and Pro107 of the human wild-type IL-18 polypeptide sequence, or the corresponding residues from IL-18 of other species (for example a primate such as Rhesus macaque).

[0122] An antibody for IL-18 may bind to an IL-18 epitope which comprises 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, or all 17 residues selected from the group consisting of Tyr1 , Gly3, Leu5, Glu6, Lys8, Met51 , Lys53, Asp54, Ser55, Gln56, Pro57, Arg58, Gly59, Met60, Arg104, Ser105, and Pro107 of a human wild- type IL-18 polypeptide sequence. For example, the IL-18 epitope may comprise, consist, or consist essentially of, residues Tyr1 , Gly3, Leu5, Met51 , Lys53, Asp54, Ser55, Gln56, Pro57, Arg58, Gly59, Met60, Arg104, and Ser105. Optionally, the epitope may additionally comprise residues Glu6, Lys8 and Pro107.

[0123] In some preferred embodiments, an antibody for IL-18 may bind to an IL-18 epitope which consists of Tyr1 , Gly3, Leu5, Met51 , Lys53, Asp54, Ser55,

Gln56, Pro57, Arg58, Gly59, Met60, Arg104 and Ser105.

[0124] Alternatively, suitable IL-18 antibodies are described in detail in

European Patent No. 1163271 , the entire contents of which is incorporated herein by reference. Specifically, antigen-binding molecules considered to be suitable for use in the present invention include those comprising a light chain variable region having an amino acid sequence as set forth in SEQ ID NO. 39. In some of the same embodiments and other embodiments, the IL-18 antigen-binding molecules comprise a heavy chain variable region having an amino acid sequence of SEQ ID NO. 40.

[0125] Suitably, the antigen-binding molecules specific for IL-18 have CDR sequences comprising, consisting, or consisting essentially of the following amino acid sequences:

TABLE 2

[0126] Alternatively, some IL-18 antibodies are described in detail in U.S. Patent Publication No. 9,255,144. The antibodies identified in that patent publication are therefore particularly suitable for use with the present invention and are incorporated herein.

[0127] In some alternative embodiments, antibodies which specifically bind to IL-18 as described herein may comprise: (a) a V_HCDRI having an amino acid sequence identical to or comprising 1 , 2, 3 or 4 amino acid residue substitutions relative to SEQ ID NO: 9; (b) a V_HCDR2 having an amino acid sequence identical to or comprising 1 , 2, 3 or 4 amino acid residue substitutions relative to SEQ ID NO: 10; (c) a V_HCDR3 having an amino acid sequence identical to or comprising 1 , 2, 3, 4, or 5 amino acid residue substitutions relative to SEQ ID NO: 11 ; (d) a VLCDRI having an amino acid sequence identical to or comprising 1 , 2, 3 or 4 amino acid residue substitutions relative to SEQ ID NO: 12; (e) a Vi_CDR2 having an amino acid sequence identical to or comprising 1 , 2, 3 or 4 amino acid residue substitutions relative to SEQ ID NO: 13; and (f) a Vi_CDR3 having an amino acid sequence identical to or comprising 1 , 2, 3, 4, 5, 6, 7, 8, or 9 amino acid residue substitutions relative to SEQ ID NO: 14.

[0128] Alternatively, the V_HCDR and V_LCDR sequences can be selected from Table 3.

TABLE 3

2 SGGYY SIYYSGSTYYNPS TPAYDARADFFD RASQGISSW KASTL QDISFPPW WS LKS V LA ES T

3 S

4 SGGYY SIYYSGSTYYNPS TPAYDGDARADF RASQGISSW KASTL QQSHHPN WS LKS FDV LA ES WD

5 QSLIPQW

6 SGGYY SIYYSGSTYYDPL TPAYFGQDTDFF RASQQGISS KASTL ANIAFPPW WS KS AV WLA ES T

7 NIAFPPW

8 ADGYY SLYYSGSTYYNPS TPAYFGQDRTDF RASQGISSW KASTL QQSHHPP WS LRG FDV LA ES WT

9 SHHPP

2.6 IL18R Antigen-binding Molecules

[0129] By way of an illustrative example of this type, suitable IL18R antigen- binding molecules include those described in International PCT Patent Publication No. W02006/009114, which is incorporated herein by reference in its entirety. In some embodiments, the antigen-binding molecule binds specifically to the IL18R1 subunit, for example, to a polypeptide comprising, consisting, or consisting essentially of amino acids 120-140 of the IL18R1 polypeptide sequence set forth in SEQ ID NO: 3. In some alternative embodiments, the IL18R antigen-binding molecule binds to a polypeptide comprising, consisting, or consisting essentially of amino acids 142-162 of the IL18R1 polypeptide sequence set forth in SEQ ID NO: 3.

[0130] Antibodies may be prepared by methods widely known in the relevant field (see, for example, Chow, M. et al., Proc. Natl. Acad. Sci. USA 82: 910-914; and Bittle, F.J. et al., J. Gen. Virol. 66: 2347-2354 (1985)). Generally, an animal can be immunized with a free peptide; however, an anti-peptide antibody titre can be boosted by coupling the peptide to a polymeric carrier (for example, keyhole limpet hemocyanin (KLH) or tetanus toxoid). For example, a cysteine containing peptide can be coupled to a carrier using a linker like m-maleimidobenzoyl-N- hydroxysuccinimide ester (MBS), whereas other peptides can be coupled to a carrier using a more common linking agent like glutaraldehyde. Animals like rabbits, rats, and mice can be immunized with any of a free or carrier-coupled peptide by, for example, intraperitoneal and/or intradermal injection of an emulsion comprising about 100 pg of peptide or carrier protein and Freund's adjuvant. Some booster injections can be required at intervals of, for example, about two weeks, in order to provide, for example, an anti-peptide antibody of useful titre that can be detected by ELISA assay using a free peptide adsorbed to a solid surface. The titre of the anti- peptide antibody in serum from an immunized animal can be increased by choosing an anti-peptide antibody, for example, using adsorption to the peptide on a solid support and elution of the antibody chosen by a method widely known in the relevant field.

[0131] It is evident that Fab and F(ab')2 and other fragments of an antibody according to the present invention can be used according to a method disclosed herein. Such a fragment is produced representatively by cleavage due to proteolysis using an enzyme like papain (resulting in an Fab fragment) or pepsin (resulting in an F(ab')2 fragment). Alternatively, an IL-18 receptor binding fragment can be produced by applying recombinant DNA technology or by synthetic chemistry. Hence, it can be said that an antibody according to the present invention only need to have at least an antibody fragment that recognizes an IL-18 receptor peptide antigen according to the present invention (for example, Fab and F(ab')2 fragments).

Therefore, it should be noted that an immunoglobulin consisting of an antibody fragment that recognizes an IL-18 receptor peptide antigen according to the present invention and an Fc fragment of a different antibody molecule is also included in the present invention.

[0132] As used herein, the term“antibody” encompasses both a complete antibody molecule and antibody fragment (for example, Fab and F(ab')2 fragments) capable of binding specifically to its polypeptide target. The Fab and F(ab')2 fragments lack the Fc portion of the complete antibody, is more quickly eliminated by circulation, and can hardly have the nonspecific tissue binding of the complete antibody (Wahl et al., J. Nucl. Med. 24: 316-325 (1983)). Therefore, in some embodiments these fragments are preferable.

[0133] Furthermore, an additional antibody capable of binding to an IL18R antigen peptide antigen can be produced by two-step procedures through the use of an anti-ideotype antibody. Such a method utilizes the fact that an antibody per se is an antigen, and therefore enables to obtain an antibody that binds to a secondary antibody. According to this method, an antibody that binds specifically to an IL-18 receptor is used to immunize an animal (for example, a mouse). Next, splenocytes of such an animal are used to produce hybridoma cells, and the hybridoma cells are screened to identify a clone that produces an antibody whose capability of binding to an antibody that binds specifically to an IL-18 receptor can be blocked by an IL-18 receptor peptide antigen. Such antibodies include anti-ideotype antibodies against an antibody that binds specifically to an IL-18 receptor, and can be used to immunize an animal for inducing the formation of an antibody that binds specifically to an additional IL-18 receptor.

[0134] It is evident that Fab and F(ab')2 and other fragments of an antibody according to the present invention can be used according to the methods disclosed herein. Such a fragment is produced representatively by cleavage due to proteolysis using an enzyme like papain (resulting in an Fab fragment) or pepsin (resulting in an F(ab')2 fragment). Alternatively, an IL-18 receptor binding fragment can be produced by applying recombinant DNA technology or by synthetic chemistry. Hence, it can be said that an antibody according to the present invention only need to have at least an antibody fragment that recognizes an IL-18 receptor peptide antigen according to the present invention (for example, Fab and F(ab')2 fragments). Therefore, it should be noted that an immunoglobulin consisting of an antibody fragment that recognizes an IL-18 receptor peptide antigen according to the present invention and an Fc fragment of a different antibody molecule is also included in the present invention.

[0135] Chimeric antibodies are immunoglobulin molecules characterized by two or more segments or portions derived from different animal species. Generally, the variable region of the chimeric antibody is derived from a non-human mammalian antibody, such as murine monoclonal antibody, and the immunoglobulin constant region is derived from a human immunoglobulin molecule. Preferably, both regions and the combinat m-maleimidobenzoyl ion have low immunogenicity as routinely determined (Elliott et al., 1994).

[0136] Flumanized antibodies are immunoglobulin molecules created by genetic engineering techniques in which the murine constant regions are replaced with human counterparts while retaining the murine antigen binding regions. The resulting mouse-human chimeric antibodies preferably have a reduced

immunogenicity and improved pharmacokinetics in humans as compared to murine antibodies (Knight et al., 1993).

[0137] In some of the same embodiments and some other embodiments, the IL-18 or IL-18R antibodies of the invention are humanized antibodies. Preferred examples of humanized anti-IL-18 antibodies are described in, for example

European Patent Application No. EP 0974600, which is herby incorporated by reference in its entirety.

[0138] In yet a further preferred embodiment, the antibody is fully human. The technology for producing human antibodies is described in detail e.g., in International PCT Publication Nos. WO 00/76310 and WO 99/153049, U.S. Patent No. 6,162,963 or Australian Patent No. 5,336,100.

[0139] One method for the preparation of fully human antibodies consist of “humanization” of the mouse humoral immune system, i.e., production of mouse strains able to produce human Ig (Xenomice), by the introduction of human immunoglobulin (Ig) loci into mice in which the endogenous Ig genes have been inactivated. The Ig loci are complex in terms of both their physical structure and the gene rearrangement and expression processes required to ultimately produce a broad immune response. Antibody diversity is primarily generated by combinatorial rearrangement between different V, D, and J genes present in the Ig loci. These loci also contain the interspersed regulatory elements, which control antibody

expression, allelic exclusion, class switching and affinity maturation. Introduction of un-rearranged human Ig transgenes into mice has demonstrated that the mouse recombination machinery is compatible with human genes. Furthermore, hybridomas secreting antigen specific humanized mAbs of various isotypes can be obtained by Xenomice immunisation with antigen.

[0140] Fully human antibodies and methods for their production are known in the art (Mendez et al., (1997); Buggemann et al., (1991 ); Tomizuka et al., (2000), and International PCT Patent Publication No. W098/124893).

3. Targeting the inhibitor o†IL-18 function

[0141] In one aspect, the compositions of the present invention include a targeting agent, to deliver the inhibitor of IL-18 function to a target cell (e.g., a multiple myeloma cell). The term“target cell” as used herein means any undesirable cell in a subject that can be targeted by a composition of the present invention.

[0142] In some embodiments, the targeting agent binds to a multiple myeloma polypeptide cell surface receptor polypeptide selected from the group comprising: CD38, CAR-MIL, CAR-T, BCMA, CD137, CD319, CD46, CD47, MCL-1 , CD229, CD54, CD56, CD3, CD200, CD16A, CGEN-928, CD48, CD319, LMA, KMA, FcRH5, hTfR IgA, Dkk1 , APRIL, CD95, KIR, CS1 , CD19, CD20, CD74, SDF-1 , IL-2, IL-6, PSGL-1 , and CD40.

[0143] In some preferred embodiments, the targeting agent is conjugated, fused or otherwise linked to the inhibitor of IL-18 function.

[0144] In some of the same embodiments and some other embodiments, the targeting agent is an antigen-binding molecule. Exemplary embodiments of this type include antigen-binding molecules that specifically bind a cell surface receptor polypeptide selected from the group comprising: CD38 polypeptide, CAR-MIL polypeptide, CAR-T polypeptide, BCMA polypeptide, CD137 polypeptide, CD319, polypeptide CD46 polypeptide, CD47 polypeptide, MCL-1 polypeptide, CD229 polypeptide, CD54 polypeptide, CD56 polypeptide, CD3 polypeptide, CD200 polypeptide, CD16A polypeptide, CGEN-928 polypeptide, CD48 polypeptide, CD319 polypeptide, LMA polypeptide, KMA polypeptide, FcRH5 polypeptide, hTfR IgA polypeptide, Dkk1 polypeptide, APRIL polypeptide, CD95 polypeptide, KIR polypeptide, CS1 polypeptide, CD19 polypeptide, CD20 polypeptide, CD74 polypeptide, SDF-1 polypeptide, IL-2 polypeptide, IL-6 polypeptide, PSGL-1 polypeptide, and CD40 polypeptide.

[0145] In some embodiments, the target cell is a cell expressing or

overexpressing a multiple myeloma cell marker (e.g., CD38).

[0146] In some preferred embodiments, the targeting agent comprises an antigen-binding molecules that specifically binds to a CD38 polypeptide. Suitable CD38 antigen-binding molecules include those disclosed in U.S Patent No.

7,829,673, the entire contents of which is incorporated herein by reference.

[0147] In some exemplary embodiments, the targeting agent comprises a CD38 antigen-binding fragment comprising a V_L region consisting essentially of the sequence set forth in SEQ ID NO: 15.

[0148] In some embodiments, the targeting agent comprises a CD38 antigen- binding fragment comprising a V_H region consisting essentially of the sequence set forth in SEQ ID NO: 19.

[0149] In some embodiments, the targeting agent comprises a CD38 antigen- binding fragment comprising a V_L region consisting essentially of the sequence set forth in SEQ ID NO: 15 and a V_H region consisting essentially of the sequence set forth in SEQ ID NO: 19.

[0150] In some embodiments, the targeting agent comprises ides a CD38 antigen-binding fragment comprising a V_LCDR1 consisting essentially of the sequence set forth in SEQ ID NO: 16.

[0151] In some embodiments, the targeting agent comprises a CD38 antigen- binding fragment comprising a V_LCDR2 consisting essentially of the sequence set forth in SEQ ID NO: 17 [0152] In some embodiments, the targeting agent comprises a CD38 antigen- binding fragment comprising a V_LCDR3 consisting essentially of the sequence set forth in SEQ ID NO: 18.

[0153] In some embodiments, the targeting agent comprises a CD38 antigen- binding fragment comprising a V_HCDR1 consisting essentially of the sequence set forth in SEQ ID NO: 20.

[0154] In some embodiments, the targeting agent comprises a CD38 antigen- binding fragment comprising a V_HCDR2 consisting essentially of the sequence set forth in SEQ ID NO: 21.

[0155] In some embodiments, the targeting agent comprises a CD38 antigen- binding fragment comprising a V_HCDR3 consisting essentially of the sequence set forth in SEQ ID NO: 22.

[0156] In some embodiments, the targeting agent comprises a CD38 antigen- binding fragment comprising V_LCDRs (V_LCDR1 , CDR2, and CDR3) consisting essentially of the sequences set forth in SEQ ID NO: 16 SEQ ID NO: 17 and SEQ ID NO: 18, respectively.

[0157] In some embodiments, the targeting agent comprises a CD38 antigen- binding fragment that comprises V_HCDRs (V_HCDR1 , CDR2, and CDR3) consisting essentially of the sequences set forth in SEQ ID NO: 20, SEQ ID NO: 21 , and SEQ ID NO: 22, respectively.

[0158] In some embodiments, the targeting agent comprises a CD38 antigen- binding fragment comprising a V_L region consisting essentially of the sequence set forth in SEQ ID NO: 23.

[0159] In some embodiments, the targeting agent comprises a CD38 antigen- binding fragment comprising a V_H region consisting essentially of the sequence set forth in SEQ ID NO: 27

[0160] In some embodiments, the targeting agent comprises a CD38 antigen- binding fragment comprising a V_L region consisting essentially of the sequence set forth in SEQ ID NO: 23, and a V_H region consisting essentially of the sequence set forth in SEQ ID NO: 27. [0161] In some embodiments, the targeting agent comprises a CD38 antigen- binding fragment comprising a V_LCDR1 consisting essentially of the sequence set forth in SEQ ID NO: 24.

[0162] In some embodiments, the targeting agent comprises a CD38 antigen- binding fragment comprising a V_LCDR2 consisting essentially of the sequence set forth in SEQ ID NO: 25.

[0163] In some embodiments, the targeting agent comprises a CD38 antigen- binding fragment comprising a V_LCDR3 consisting essentially of the sequence set forth in SEQ ID NO: 26.

[0164] In some embodiments, the targeting agent comprises a CD38 antigen- binding fragment comprising a V_HCDR1 consisting essentially of the sequence set forth in SEQ ID NO: 28.

[0165] In some embodiments, the targeting agent comprises a CD38 antigen- binding fragment comprising a V_HCDR2 consisting essentially of the sequence set forth in SEQ ID NO: 29.

[0166] In some embodiments, the targeting agent comprises a CD38 antigen- binding fragment comprising a V_HCDR3 consisting essentially of the sequence set forth in SEQ ID NO: 30.

[0167] In some embodiments, the targeting agent comprises a CD38 antigen- binding fragment comprising V_LCDRs (V_LCDR1 , CDR2, and CDR3) consisting essentially of SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26, respectively

[0168] In some embodiments, the targeting agent comprises a CD38 antigen- binding fragment that comprises V_HCDRs (V_HCDR1 , CDR2, and CDR3) consisting essentially of SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30, respectively.

[0169] In some embodiments, the targeting agent comprises a CD38 antigen- binding fragment comprising a V_L region consisting essentially of the sequence set forth in SEQ ID NO: 31.

[0170] In some embodiments, the targeting agent comprises a CD38 antigen- binding fragment comprising a V_H region consisting essentially of the sequence set forth in SEQ ID NO: 35.

[0171] In some embodiments, the targeting agent comprises a CD38 antigen- binding fragment comprising a V_L region consisting essentially of the sequence set forth in SEQ ID NO: 31 and a V_H region consisting essentially of the sequence set forth in SEQ ID NO: 35.

[0172] In some embodiments, the targeting agent comprises a CD38 antigen- binding fragment comprising a V_LCDR1 consisting essentially of the sequence set forth in SEQ ID NO: 32.

[0173] In some embodiments, the targeting agent comprises a CD38 antigen- binding fragment comprising a V_LCDR2 consisting essentially of the sequence set forth in SEQ ID NO: 33.

[0174] In some embodiments, the targeting agent comprises a CD38 antigen- binding fragment comprising a V_LCDR3 consisting essentially of the sequence set forth in SEQ ID NO: 34.

[0175] In some embodiments, the targeting agent comprises a CD38 antigen- binding fragment comprising a V_HCDR1 consisting essentially of the sequence set forth in SEQ ID NO: 36.

[0176] In some embodiments, the targeting agent comprises a CD38 antigen- binding fragment comprising a V_HCDR2 consisting essentially of the sequence set forth in SEQ ID NO: 37.

[0177] In some embodiments, the targeting agent comprises a CD38 antigen- binding fragment comprising a V_HCDR3 consisting essentially of the sequence set forth in SEQ ID NO: 38.

[0178] In some embodiments, the targeting agent comprises a CD38 antigen- binding fragment comprising V_LCDRs (V_LCDR1 , CDR2, and CDR3) consisting essentially of the sequences set forth in SEQ ID NO: 32, SEQ ID NO: 33, and SEQ ID NO: 34, respectively.

[0179] In some embodiments, the present invention provides a CD38 antigen- binding fragment that comprises V_HCDRs (V_HCDR1 , CDR2, and CDR3) consisting essentially of the sequences set forth in SEQ ID NO: 36, SEQ ID NO: 37 and SEQ ID NO: 38, respectively.

[0180] In some of the same embodiments and other embodiments, the targeting agent comprises a CD38 antigen-binding fragment that comprises a flexible linker positioned between the V_L region and V_H region of the CD38 antigen-binding fragment.ln some of the same embodiments and other embodiments, the targeting agent comprises a CD38 antigen-binding fragment wherein the VL and VH regions are presented on separate chains in the context of an immunoglobulin fold protein and oriented such that the VLCDRI , CDR2, CDR3 and V_HCDRI , CDR2, and CDR3 cooperatively associate to contribute in selectively and/or specifically bind an antigenic determinant on CD38. In some embodiments, the targeting agent comprises a CD38 antigen-binding fragment comprising two sets of variable domains (sets of associated VL and VH domains on associated separate chains), such that the CD38 antigen-binding fragment comprises two identical antigenic determinant binding sites. In some embodiments of this type, the composition comprises an IL-18-CD38 bispecific antibody.

[0183] Alternatively, in some embodiments the targeting agent specifically binds to a BCMA polypeptide. Preferably, the targeting agent is an antigen-binding molecule that specifically binds to BCMA. U.S. Patent Application Publication No. 2017/0051068, the entire contents of which is incorporated herein by reference, describes a number of suitable antigen-binding molecules that are suitable for use with the present invention.

[0184] By way of an illustrative example, Table 4 (below) provides a summary of examples of some BCMA polypeptide-specific antigen binding molecules.

TABLE 4

[0185] In some embodiments of this type, the composition comprises an IL-18-BCMA bispecific antibody.

[0186] In some embodiments, the targeting agent is a peptide that binds to a surface polypeptide present on a multiple myeloma cell, and is conjugated or otherwise linked to the inhibitor of IL-18 function.

4. Pharmaceutical compositions

[0187] In accordance with the present invention, the compositions are useful in compositions and methods for the treatment or prevention of a condition involving IL-18 function, such as a multiple myeloma.

[0188] Thus, in some embodiments, the compositions of the present invention may be in the form of a pharmaceutical composition, wherein the pharmaceutical composition comprises a composition of the invention and a pharmaceutically acceptable carrier or diluent.

[0189] The proteinaceous molecules of the invention may be formulated into the pharmaceutical compositions as neutral or salt forms.

[0190] As will be appreciated by those skilled in the art, the choice of pharmaceutically acceptable carrier or diluent will be dependent on the route of administration and on the nature of the condition and the subject to be treated. The particular carrier or delivery system and route of administration may be readily determined by a person skilled in the art. The carrier or delivery system and route of administration should be carefully selected to ensure that the activity of the composition is not depleted during preparation of the formulation and the

composition is able to reach the site of action intact. The pharmaceutical

compositions of the invention may be administered through a variety of routes including, but not limited to, oral, rectal, topical, intranasal, intraocular, transmucosal, intestinal, enteral, intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intracerebral, intravaginal, intravesical, intravenous or intraperitoneal administration.

[0191] The pharmaceutical forms suitable for injectable use include sterile injectable solutions or dispersions and sterile powders for the preparation of sterile injectable solutions. Such forms should be stable under the conditions of

manufacture and storage and may be preserved against reduction, oxidation and microbial contamination.

[0192] A person skilled in the art will readily be able to determine appropriate formulations for the compositions of the invention using conventional approaches. Techniques for formulation and administration may be found in, for example, Remington (1980) Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., latest edition.

[0193] Identification of preferred pH ranges and suitable excipients, such as antioxidants, is routine in the art, for example, as described in Katdare and Chaubel (2006) Excipient Development for Pharmaceutical, Biotechnology and Drug Delivery Systems (CRC Press). Buffer systems are routinely used to provide pH values of a desired range and may include, but are not limited to, carboxylic acid buffers, such as acetate, citrate, lactate, tartrate and succinate; glycine; histidine; phosphate; tris(hydroxymethyl)aminomethane (Tris); arginine; sodium hydroxide; glutamate; and carbonate buffers. Suitable antioxidants may include, but are not limited to, phenolic compounds such as butylated hydroxytoluene (BHT) and butylated hydroxyanisole; vitamin E; ascorbic acid; reducing agents such as methionine or sulphite; metal chelators such as ethylene diamine tetraacetic acid (EDTA); cysteine hydrochloride; sodium bisulfite; sodium metabisulfite; sodium sulphite; ascorbyl palmitate; lecithin; propyl gallate; and alpha-tocopherol.

[0194] For injection, the compositions of the invention may be formulated in aqueous solutions, suitably in physiologically compatible buffers such as Hanks' solution, Ringer's solution or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

[0195] The compositions of the present invention may be formulated for administration in the form of liquids, containing acceptable diluents (such as saline and sterile water), or may be in the form of lotions, creams or gels containing acceptable diluents or carriers to impart the desired texture, consistency, viscosity and appearance. Acceptable diluents and carriers are familiar to those skilled in the art and include, but are not restricted to, ethoxylated and non-ethoxylated

surfactants, fatty alcohols, fatty acids, hydrocarbon oils (such as palm oil, coconut oil, and mineral oil), cocoa butter waxes, silicon oils, pH balancers, cellulose derivatives, emulsifying agents such as non-ionic organic and inorganic bases, preserving agents, wax esters, steroid alcohols, triglyceride esters, phospholipids such as lecithin and cephalin, polyhydric alcohol esters, fatty alcohol esters, hydrophilic lanolin derivatives and hydrophilic beeswax derivatives.

[0196] Alternatively, the compositions of the present invention can be formulated readily using pharmaceutically acceptable carriers well known in the art into dosages suitable for oral administration, which is also contemplated for the practice of the present invention. Such carriers enable the bioactive agents of the invention to be formulated in dosage forms such as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. These carriers may be selected from sugars, starches, cellulose and its derivatives, malt, gelatin, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffered solutions, emulsifiers, isotonic saline and pyrogen- free water.

[0197] Pharmaceutical formulations for parenteral administration include aqueous solutions of the proteinaceous molecules of the invention in water-soluble form. Additionally, suspensions of the compositions of the invention may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

[0198] Sterile solutions may be prepared by combining the active compounds in the required amount in the appropriate solvent with other excipients as described above as required, followed by sterilization, such as filtration. Generally, dispersions are prepared by incorporating the various sterilized active compounds into a sterile vehicle which contains the basic dispersion medium and the required excipients as described above. Sterile dry powders may be prepared by vacuum- or freeze-drying a sterile solution comprising the active compounds and other required excipients as described above.

[0199] Pharmaceutical preparations for oral use can be obtained by combining the compositions of the invention with solid excipients and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatine, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Such compositions may be prepared by any of the methods of pharmacy but all methods include the step of bringing into association one or more therapeutic agents as described above with the carrier which constitutes one or more necessary ingredients. In general, the pharmaceutical compositions of the present invention may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

[0200] Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of particle doses.

[0201] Pharmaceuticals which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added.

[0202] The compositions of the invention may be incorporated into modified- release preparations and formulations, for example, polymeric microsphere formulations, and oil- or gel-based formulations.

[0203] In particular embodiments, the compositions of the invention may be administered in a local rather than systemic manner, such as by injection of the proteinaceous molecule directly into a tissue, which is preferably subcutaneous or omental tissue, often in a depot or sustained release formulation.

[0204] Furthermore, the compositions of the invention may be administered in a targeted drug delivery system, such as in a particle which is suitable targeted to and taken up selectively by a cell or tissue. In some embodiments, the compositions of the invention are contained or otherwise associated with a vehicle selected from liposomes, micelles, dendrimers, biodegradable particles, artificial DNA

nanostructure, lipid-based nanoparticles and carbon or old nanoparticles. In illustrative examples of this type, the vehicle is selected from poly(lactic acid) (PLA), poly(glycolic acid) (PGA), poly(lactic-co-glycolic acid) (PLGA), poly(ethylene glycol) (PEG), PLA-PEG copolymers and combinations thereof.

[0205] In cases of local administration or selective uptake, the effective local concentration of the agent may not be related to plasma concentration.

[0206] It is advantageous to formulate the compositions in dosage unit form for ease of administration and uniformity of dosage. The determination of the novel dosage unit forms of the present invention is dictated by and directly dependent on the unique characteristics of the active material, the particular therapeutic effect to be achieved and the limitations inherent in the art of compounding active materials for the treatment of disease in living subjects having a diseased condition in which bodily health is impaired as herein disclosed in detail.

[0207] While the compositions of the invention may be the sole active ingredient administered to the subject, the administration of other cancer therapies concurrently with said compositions is within the scope of the invention. For example, the compositions or variants described herein may be administered concurrently with one or more cancer therapies, non-limiting examples of which include radiotherapy, surgery, chemotherapy, hormone ablation therapy, pro- apoptosis therapy and immunotherapy; particularly chemotherapy. The compositions of the invention may be therapeutically used prior to treatment with the cancer therapy, may be therapeutically used after the cancer therapy, or may be

therapeutically used simultaneously with the cancer therapy.

[0208] Suitable radiotherapies include radiation and waves that induce DNA damage, for example, g-irradiation, X-rays, UV irradiation, microwaves, electronic emissions and radioisotopes. Typically, therapy may be achieved by irradiating the localized tumor site with the above described forms of radiations. It is most likely that all of these factors cause a broad range of damage to DNA, on the precursors of DNA, on the replication and repair of DNA and on the assembly and maintenance of chromosomes.

[0209] The dosage range for X-rays ranges from daily doses of 50-200 roentgens for prolonged periods of time such as 3-4 weeks, to single doses of 2000- 6000 roentgens. Dosage ranges for radioisotopes vary widely and depend on the half-life of the isotope, the strength and type of radiation emitted and the uptake by the neoplastic cells. Suitable radiotherapies may include, but are not limited to, conformal external beam radiotherapy (50-100 Gray given as fractions over 4-8 weeks), either single shot or fractionated high dose brachytherapy, permanent interstitial brachytherapy and systemic radioisotopes such as Strontium 89. In some embodiments, the radiotherapy may be administered with a radiosensitizing agent. Suitable radiosensitizing agents may include, but are not limited to, efaproxiral, etanidazole, fluosol, misonidazole, nimorazole, temoporfin and tirapazamine.

[0210] Suitable chemotherapeutic agents may include, but are not limited to, antiproliferative/antineoplastic drugs and combinations thereof including alkylating agents (for example cisplatin, carboplatin, cyclophosphamide, nitrogen mustard, melphalan, chlorambucil, busulphan and nitrosoureas), antimetabolites (for example antifolates such as fluoropyri dines like 5-fluorouracil and tegafur, raltitrexed, methotrexate, cytosine arabinoside and hydroxyurea), anti -tumor antibiotics (for example anthracyclines like adriamycin, bleomycin, doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C, dactinomycin and mithramycin), antimitotic agents (for example Vinca alkaloids like vincristine, vinblastine, vindesine and vinorelbine and taxoids like paclitaxel and docetaxel), and topoisomerase inhibitors (for example epipodophyllotoxins like etoposide and teniposide, amsacrine, topotecan and camptothecin); cytostatic agents such as antiestrogens (for example tamoxifen, toremifene, raloxifene, droloxifene and idoxifene), estrogen receptor down regulators (for example fulvestrant), antiandrogens (for example bicalutamide, flutamide, nilutamide and cyproterone acetate), UH antagonists or LHRH agonists (for example goserelin, leuprorelin and buserelin), progestogens (for example megestrol acetate), aromatase inhibitors (for example as anastrozole, letrozole, vorozole and exemestane) and inhibitors of 5-reductase such as finasteride; agents which inhibit cancer cell invasion (for example metalloproteinase inhibitors like marimastat and inhibitors of urokinase plasminogen activator receptor function); inhibitors of growth factor function, for example such inhibitors include growth factor antibodies, growth factor receptor antibodies (for example the anti-erbb2 antibody trastuzumab [HERCEPTIN™] and the anti-ErbB1 antibody Cetuximab [C225]), farnesyl transferase inhibitors, MEK inhibitors, tyrosine kinase inhibitors and serine/threonine kinase inhibitors, for example other inhibitors of the epidermal growth factor family (for example other EGFR family tyrosine kinase inhibitors such as N-(3-chloro-4-fluorophenyl)-7-methoxy-6-(3-morpholinopropoxy)quinazolin-4- amine (Gefitinib, AZD1839), N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy) quinazolin-4-amine (Erlotinib, OSI-774) and 6-acrylamido-N-(3-chloro-4- fluorophenyl)-7-(3-mo holinopropoxy)quinazoli-n-4-amine (Cl 1033)), for example inhibitors of the platelet-derived growth factor family and for example inhibitors of the hepatocyte growth factor family; anti -angiogenic agents such as those which inhibit the effects of vascular endothelial growth factor, (for example the anti-vascular endothelial cell growth factor antibody bevacizumab [Avastin™], compounds such as those disclosed in International Patent Applications WO 97/22596, WO 97/30035, WO 97/32856 and WO 98/13354) and compounds that work by other mechanisms (for example linomide, inhibitors of integrin anb3 function and angiostatin); vascular damaging agents such as Combretastatin A4 and compounds disclosed in

International Patent Applications WO 99/02166, WO00/40529, WO 00/41669, WOO 1/92224, W002/04434 and W002/08213; antisense therapies, for example those which are directed to the targets listed above, such as ISIS 2503, an anti-ras antisense; and gene therapy approaches, including for example approaches to replace aberrant genes such as aberrant p53 or aberrant GDEPT (gene-directed enzyme pro-drug therapy) approaches such as those using cytosine deaminase, thymidine kinase or a bacterial nitroreductase enzyme and approaches to increase patient tolerance to chemotherapy or radiotherapy such as multi-drug resistance gene therapy.

[0211] Suitable immunotherapy approaches may include, but are not limited to ex wVo and in vivo approaches to increase the immunogenicity of patient tumor cells such as transfection with cytokines including IL-2, IL-4 or granulocyte-macrophage colony stimulating factor; approaches to decrease T-cell anergy; approaches using transfected immune cells such as cytokine-transfected dendritic cells; approaches using cytokine-transfected tumor cell lines; and approaches using anti -idiotypic antibodies. These approaches generally rely on the use of immune effector cells and molecules to target and destroy cancer cells. The immune effector may be, for example, an antibody specific for some marker on the surface of a malignant cell.

The antibody alone may serve as an effector of therapy or it may recruit other cells to actually facilitate cell killing. The antibody also may be conjugated to a drug or toxin (chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve merely as a targeting agent. Alternatively, the effector may be a

lymphocyte carrying a surface molecule that interacts, either directly or indirectly, with a malignant cell target. Various effector cells include cytotoxic T cells and NK cells.

[0212] Examples of other cancer therapies include phytotherapy, cryotherapy, toxin therapy or pro-apoptosis therapy. A person skilled in the art would appreciate that this list is not exhaustive of the types of treatment modalities available for cancer and other hyperplastic lesions.

[0213] It is well known that chemotherapy and radiation therapy target rapidly dividing cells and/or disrupt the cell cycle or cell division. These treatments are offered as part of the treating several forms of cancer, aiming either at slowing their progression or reversing the symptoms of disease by means of a curative treatment. However, these cancer treatments may lead to an immunocompromised state and ensuing pathogenic infections and, thus, the present invention also extends to combination therapies, which employ a composition as described herein, a cancer therapy and an anti-infective agent that is effective against an infection that develops or that has an increased risk of developing from an immunocompromised condition resulting from the cancer therapy. The anti-infective drug is suitably selected from antimicrobials, which may include, but are not limited to, compounds that kill or inhibit the growth of microorganisms such as viruses, bacteria, yeast, fungi, protozoa, etc. and, thus, include antibiotics, amebicides, antifungals, antiprotozoals, antimalarials, antituberculotics and antivirals. Anti-infective drugs also include within their scope anthelmintics and nematocides. Illustrative antibiotics include quinolones (e.g., amifloxacin, cinoxacin, ciprofloxacin, enoxacin, fleroxacin, flumequine, lomefloxacin, nalidixic acid, norfloxacin, ofloxacin, levofloxacin, lomefloxacin, oxolinic acid, pefloxacin, rosoxacin, temafloxacin, tosufloxacin, sparfloxacin, clinafloxacin, gatifloxacin, moxifloxacin; gemifloxacin; and garenoxacin), tetracyclines,

glycylcyclines and oxazolidinones (e.g., chlortetracycline, demeclocycline, doxycycline, lymecycline, methacycline, minocycline, oxytetracycline, tetracycline, tigecycline; linezolide, eperezolid), glycopeptides, aminoglycosides (e.g., amikacin, arbekacin, butirosin, dibekacin, fortimicins, gentamicin, kanamycin, menomycin, netilmicin, ribostamycin, sisomicin, spectinomycin, streptomycin, tobramycin), b- lactams (e.g., imipenem, meropenem, biapenem, cefaclor, cefadroxil, cefamandole, cefatrizine, cefazedone, cefazolin, cefixime, cefmenoxime, cefodizime, cefonicid, cefoperazone, ceforanide, cefotaxime, cefotiam, cefpimizole, cefpiramide, cefpodoxime, cefsulodin, ceftazidime, cefteram, ceftezole, ceftibuten, ceftizoxime, ceftriaxone, cefuroxime, cefuzonam, cephacetrile, cephalexin, cephaloglycin, cephaloridine, cephalothin, cephapirin, cephradine, cefinetazole, cefoxitin, cefotetan, azthreonam, carumonam, flomoxef, moxalactam, amdinocillin, amoxicillin, ampicillin, azlocillin, carbenicillin, benzylpenicillin, carfecillin, cloxacillin, dicloxacillin, methicillin, mezlocillin, nafcillin, oxacillin, penicillin G, piperacillin, sulbenicillin, temocillin, ticarcillin, cefditoren, SC004, KY-020, cefdinir, ceftibuten, FK-312, S-1090, CP-0467, BK-218, FK-037, DQ-2556, FK-518, cefozopran, ME1228, KP-736, CP-6232, Ro 09- 1227, OPC-20000, LY206763), rifamycins, macrolides (e.g., azithromycin, clarithromycin, erythromycin, oleandomycin, rokitamycin, rosaramicin, roxithromycin, troleandomycin), ketolides (e.g., telithromycin, cethromycin), coumermycins, lincosamides (e.g., clindamycin, lincomycin) and chloramphenicol.

[0214] Illustrative antivirals include abacavir sulfate, acyclovir sodium, amantadine hydrochloride, amprenavir, cidofovir, delavirdine mesylate, didanosine, efavirenz, famciclovir, fomivirsen sodium, foscarnet sodium, ganciclovir, indinavir sulfate, lamivudine, lamivudine/zidovudine, nelfinavir mesylate, nevirapine, oseltamivir phosphate, ribavirin, rimantadine hydrochloride, ritonavir, saquinavir, saquinavir mesylate, stavudine, valacyclovir hydrochloride, zalcitabine, zanamivir and zidovudine.

[0215] Suitable amebicides or antiprotozoals include, but are not limited to, atovaquone, chloroquine hydrochloride, chloroquine phosphate, metronidazole, metronidazole hydrochloride and pentamidine isethionate. Anthelmintics can be at least one selected from mebendazole, pyrantel pamoate, albendazole, ivermectin and thiabendazole. Illustrative antifungals can be selected from amphotericin B, amphotericin B cholesteryl sulfate complex, amphotericin B lipid complex, amphotericin B liposomal, fluconazole, flucytosine, griseofulvin microsize, griseofulvin ultramicrosize, itraconazole, ketoconazole, nystatin and terbinafine hydrochloride. Suitable antimalarials include, but are not limited to, chloroquine hydrochloride, chloroquine phosphate, doxycycline, hydroxychloroquine sulfate, mefloquine hydrochloride, primaquine phosphate, pyrimethamine and pyrimethamine with sulfadoxine. Antituberculotics include but are not restricted to clofazimine, cycloserine, dapsone, ethambutol hydrochloride, isoniazid, pyrazinamide, rifabutin, rifampin, rifapentine and streptomycin sulfate.

[0216] As previously described, the compositions may be compounded for convenient and effective administration in effective amounts with a suitable pharmaceutically acceptable carrier in dosage unit form. In some embodiments, a unit dosage form may comprise the active component of the invention in amount in the range of from about 2 mg to about 2000 mg. The active component of the invention may be present in an amount of from about 1 mg to about 2000 mg/mL of carrier. In embodiments where the pharmaceutical composition comprises one or more additional active ingredients, the dosages are determined by reference to the usual dose and manner of administration of the said ingredients.

5. Methods.

[0217] In accordance with the present invention, the compositions of the invention are useful in methods for the treatment or prevention of conditions associated with IL-18 activity, particularly conditions in respect of which inhibitor of IL-18 activity is associated with effective prevention or treatment. Thus, the

compositions of the invention are useful for the treatment and/or prevention of conditions such as multiple myeloma.

[0218] Thus, in one aspect of the present invention, there is provided a method of treating or preventing multiple myeloma in a subject, the method comprising administration of a composition of the invention to the subject.

[0219] While the compositions of the invention may be the sole active agent administered to the subject, the administration of other active agents is within the scope of the invention. For example, the compositions of the invention may be administered with one or more therapeutic agents, such as a chemotherapeutic agent. The compositions of the invention, and the one or more therapeutic agents may be administered separately, simultaneously, or sequentially.

[0220] Accordingly, in another aspect of the present invention, there is provided the use of the compositions of the invention, for therapy.

[0221] In yet another aspect of the present invention, there is provided the use of the compositions of the invention, in the manufacture of a medicament for therapy.

[0222] In yet another aspect of the invention, there is provided a method of inhibiting or reducing the IL-18 function in a lymphocyte (e.g., CD8⁺ T cell), comprising contacting the lymphocyte with a composition of the invention. In preferred embodiments, the lymphocyte is a memory T cell. In some embodiments, the memory T cell is a CD8⁺ T cell. Furthermore, in some preferred embodiments, the memory T cell is localized to the bone marrow.

[0223] In still another aspect of the invention, there is provided the use of an composition of the invention, in the manufacture of a medicament for the treatment of multiple myeloma. [0224] In some embodiments, the proteinaceous molecules of the invention result in a reduction, impairment, abrogation of IL-18 function in bone marrow derived lymphocytes. Suitable bone marrow-derived lymphocytes may include, but are not limited to, T cells (e.g., CD8⁺ T cells) and natural killer (NK) cells.

[0225] In some embodiments, the compositions of the invention are used for treating, preventing and/or relieving the symptoms of a multiple myeloma.

[0226] The compositions of the invention are suitable for treating an individual who has been diagnosed with multiple myeloma, who is suspected of having a multiple myeloma, who is known to be susceptible and who is considered likely to develop multiple myeloma, or who is considered likely to develop a recurrence of previously treated multiple myeloma.

[0227] In another aspect, the compositions of the invention are also suitable for treating an individual who has been diagnosed with multiple myeloma that is resistant to chemotherapy and/or radiotherapy.

[0228] In particular embodiments, the methods and uses involve the

administration of one or more further active agents as described in Section 3 supra, such as an additional cancer therapy and/or anti-infective agent; particularly a cancer therapy; especially a chemotherapeutic. The one or more further active agents and compositions of the invention may be administered separately, simultaneously or sequentially.

6. Particle embodiments

[0229] In some embodiments, an inhibitor of IL-18 function, and optionally a targeting agent, is provided in particulate form. In embodiments in which an inhibitor or IL-18 function and a targeting agent are employed, they may be contained in or otherwise associated with the same particle or with different particles. A variety of particles may be used in the invention, including but not limited to, liposomes, micelles, lipidic particles, ceramic/inorganic particles and polymeric particles, and are typically selected from nanoparticles and microparticles.

[0230] In illustrative examples, the particles have a dimension of less than about 100 pm, more suitably in the range of less than or equal to about 500 nm, although the particles may be as large as about 10 pm, and as small as a few nm. Liposomes consist basically of a phospholipid bilayer forming a shell around an aqueous core. Advantages include the lipophilicity of the outer layers which“mimic” the outer membrane layers of cells and that they are taken up relatively easily by a variety of cells. Polymeric vehicles typically consist of micro/nanospheres and micro/nanocapsules formed of biocompatible polymers, which are either

biodegradable (for example, polylactic acid) or non-biodegradable (for example, ethylenevinyl acetate). Some of the advantages of the polymeric devices are ease of manufacture and high loading capacity, range of size from nanometer to micron diameter, as well as controlled release and degradation profile.

[0231] In some embodiments, the particles comprise an antigen-binding molecule on their surface, which is immuno-interactive with a marker that is expressed at higher levels on memory T cells than on cells that are not memory T cells. Illustrative markers of this type include but are not limited to the group comprising: a CD38 polypeptide, CAR-MIL polypeptide, CAR-T polypeptide, BCMA polypeptide, CD137 polypeptide, CD319, polypeptide CD46 polypeptide, CD47 polypeptide, MCL-1 polypeptide, CD229 polypeptide, CD54 polypeptide, CD56 polypeptide, CD3 polypeptide, CD200 polypeptide, CD16A polypeptide, CGEN-928 polypeptide, CD48 polypeptide, CD319 polypeptide, LMA polypeptide, KMA polypeptide, FcRH5 polypeptide, hTfR IgA polypeptide, Dkk1 polypeptide, APRIL polypeptide, CD95 polypeptide, KIR polypeptide, CS1 polypeptide, CD19

polypeptide, CD20 polypeptide, CD74 polypeptide, SDF-1 polypeptide, IL2 polypeptide, IL6 polypeptide, PSGL-1 , polypeptide and CD40 polypeptide..

[0232] The particles can be prepared from a combination of the inhibitors of IL-18 function and optionally a targeting agent, and a surfactant, excipient or polymeric material. In some embodiments, the particles are biodegradable and biocompatible, and optionally are capable of biodegrading at a controlled rate for delivery of a the therapeutic agents. The particles can be made of a variety of materials. Both inorganic and organic materials can be used. Polymeric and non- polymeric materials, such as fatty acids, may be used. Other suitable materials include, but are not limited to, gelatin, polyethylene glycol, trehalose, dextran, and chitosan. Particles with degradation and release times ranging from seconds to months can be designed and fabricated, based on factors such as the particle material. 6.1 Polymeric Particles

[0233] Polymeric particles may be formed from any biocompatible and desirably biodegradable polymer, copolymer, or blend. The polymers may be tailored to optimize different characteristics of the particle including: i) interactions between the bioactive agents to be delivered and the polymer to provide stabilization of the bioactive agents and retention of activity upon delivery; ii) rate of polymer degradation and, thereby, rate of agent release profiles; iii) surface characteristics and targeting capabilities via chemical modification; and iv) particle porosity.

[0234] Surface eroding polymers such as poly anhydrides may be used to form the particles. For example, polyanhydrides such as poly[(p-carboxyphenoxy)- hexane anhydride] (PCPH) may be used. Biodegradable polyanhydrides are described in, for example, U.S. Patent No. 4,857,311.

[0235] In other embodiments, bulk eroding polymers such as those based on polyesters including poly(hydroxy acids) or poly(esters) can be used. For example, polyglycolic acid (PGA), polylactic acid (PLA), or copolymers thereof may be used to form the particles. The polyester may also have a charged or functionalizable group, such as an amino acid. In illustrative examples, particles with controlled release properties can be formed of poly(D,L-lactic acid) and/or poly(D,L-lactic-co-glycolic acid) ("PLGA") which incorporate a surfactant such as DPPC.

[0236] Other polymers include poly(alkylcyanoacrylates), polyamides, polycarbonates, polyalkylenes such as polyethylene, polypropylene, poly(ethylene glycol), polyethylene oxide), polyethylene terephthalate), poly vinyl compounds such as polyvinyl alcohols, polyvinyl ethers, and polyvinyl esters, polymers of acrylic and methacrylic acids, celluloses and other polysaccharides, and peptides or proteins, or copolymers or blends thereof. Polymers may be selected with or modified to have the appropriate stability and degradation rates in vivo for different controlled drug delivery applications.

[0237] In some embodiments, particles are formed from functionalized polyester-graft copolymers, as described in Hrkach et al., ( 1995, Macromolecules 28:4736-4739; and "Poly(L-Lactic acid-co-amino acid) Graft Copolymers: A Class of Functional Biodegradable Biomaterials" in Hydrogels and Biodegradable Polymers for Bioapplications, ACS Symposium Series No. 627, Raphael M. Ottenbrite et al., Eds., American Chemical Society, Chapter 8, pp. 93-101 , 1996.)

[0238] Materials other than biodegradable polymers may be used to form the particles. Suitable materials include various non-biodegradable polymers and various excipients. The particles also may be formed of the bioactive agent(s) and surfactant alone.

[0239] Polymeric particles may be prepared using single and double emulsion solvent evaporation, spray drying, solvent extraction, solvent evaporation, phase separation, simple and complex coacervation, interfacial polymerization, and other methods well known to those of ordinary skill in the art. Particles may be made using methods for making microspheres or microcapsules known in the art, provided that the conditions are optimized for forming particles with the desired diameter.

[0240] Methods developed for making microspheres for delivery of

encapsulated agents are described in the literature, for example, as described in Doubrow, M., Ed.,“Microcapsules and Nanoparticles in Medicine and Pharmacy,” CRC Press, Boca Raton, 1992. Methods also are described in Mathiowitz and Langer (1987, J. Controlled Release 5, 13-22); Mathiowitz et al., (1987, Reactive Polymers 6, 275-283); and Mathiowitz et al., (1988, J. Appl. Polymer Sci. 35, 755- 774) as well as in U.S. Patent Nos. 5,213,812, 5,417,986, 5,360,610, and 5,384,133. The selection of the method depends on the polymer selection, the size, external morphology, and crystallinity that is desired, as described, for example, by

Mathiowitz et al., (1990, Scanning Microscopy 4: 329- 340; 1992, J. Appl. Polymer Sci. 45, 125-134); and Benita et al., (1984, J. Pharm. Sci. 73, 1721-1724). [0204] In solvent evaporation, described for example, in Mathiowitz et al., (1990), Benita; and U.S. Patent No. 4,272,398 to Jaffe, the polymer is dissolved in a volatile organic solvent, such as methylene chloride. Several different polymer concentrations can be used, for example, between 0.05 and 2.0 g/mL. The bioactive agent(s), either in soluble form or dispersed as fine particles, is (are) added to the polymer solution, and the mixture is suspended in an aqueous phase that contains a surface-active agent such as poly (vinyl alcohol). The aqueous phase may be, for example, a concentration of 1 % poly(vinyl alcohol) w/v in distilled water. The resulting emulsion is stirred until most of the organic solvent evaporates, leaving solid microspheres, which may be washed with water and dried overnight in a lyophilizer. Microspheres with different sizes (between 1 and 1000 pm) and morphologies can be obtained by this method. Solvent removal was primarily designed for use with less stable polymers, such as the polyanhydrides. In this method, the agent is dispersed or dissolved in a solution of a selected polymer in a volatile organic solvent like methylene chloride. The mixture is then suspended in oil, such as silicon oil, by stirring, to form an emulsion. Within 24 hours, the solvent diffuses into the oil phase and the emulsion droplets harden into solid polymer microspheres. Unlike the hot- melt microencapsulation method described for example in Mathiowitz et al., (1987, Reactive Polymers 6:275), this method can be used to make microspheres from polymers with high melting points and a wide range of molecular weights.

Microspheres having a diameter for example between one and 300 microns can be obtained with this procedure.

[0241] With some polymeric systems, polymeric particles prepared using a single or double emulsion technique, vary in size depending on the size of the droplets. If droplets in water-in-oil emulsions are not of a suitably small size to form particles with the desired size range, smaller droplets can be prepared, for example, by sonication or homogenation of the emulsion, or by the addition of surfactants.

[0242] If the particles prepared by any of the above methods have a size range outside of the desired range, particles can be sized, for example, using a sieve, and further separated according to density using techniques known to those of skill in the art.

[0243] The polymeric particles can be prepared by spray drying. Methods of spray drying, such as that disclosed in International PCT Patent Publication No.

WO 96/09814 by Sutton and Johnson, disclose the preparation of smooth, spherical microparticles of a water-soluble material with at least 90% of the particles

possessing a mean size between 1 and 10 pm.

6.2 Ceramic Particles

[0244] Ceramic particles may also be used to deliver the bioactive agents of the invention. These particles are typically prepared using processes similar to the well-known sol-gel process and usually require simple and room temperature conditions as described for example in Brinker et al., ("Sol-Gel Science: The Physics and Chemistry of Sol-Gel Processing;" Academic Press: San Diego, 1990, p-60), and Avnir et al., (1994, Chem. Mater. 6, 1605). Ceramic particles can be prepared with desired size, shape and porosity, and are extremely stable. These particles also effectively protect doped molecules (polypeptides, drugs etc.) against denaturation induced by extreme pH and temperature (Jain et al., 1998, J. Am. Chem. Soc. 120, 1 1092-11095). In addition, their surfaces can be easily

functionalized with different groups (Lai et al., 2000, Chem. Mater. 12, 2632-2639; Badley et al., 1990, Langmuir 6, 792-801 ), and therefore they can be attached to a variety of monoclonal antibodies and other ligands in order to target them to desired sites in vivo.

[0245] Various ceramic particles have been described for delivery in vivo of active agents. For example, British Patent No. 1 ,590,574 discloses incorporation of biologically active components in a sol-gel matrix. International Patent Publication No. WO 97/45367 discloses controllably dissolvable silica xerogels prepared via a sol-gel process, into which a biologically active agent is incorporated by

impregnation into pre-sintered particles (1 to 500 pm) or disks. International

Publication No. WO 00/50349 discloses controllably biodegradable silica fibres prepared via a sol-gel process, into which a biologically active agent is incorporated during synthesis of the fibre. U.S. Patent Application Publication No. 2004/0180096 describes ceramic nanoparticles in which a bioactive substance is entrapped. The ceramic nanoparticles are made by formation of a micellar composition of the dye. The ceramic material is added to the micellar composition and the ceramic nanoparticles are precipitated by alkaline ' hydrolysis. U.S. Patent Application Publication No. 2005/0123611 discloses controlled release ceramic particles comprising an active material substantially homogeneously dispersed throughout the particles. These particles are prepared by mixing a surfactant with an apolar solvent to prepare a reverse micelle solution; (b) dissolving a gel precursor, a catalyst, a condensing agent and a soluble active material in a polar solvent to prepare a precursor solution; (c) combining the reverse micelle solution and the precursor solution to provide an emulsion and (d) condensing the precursor in the emulsion. U.S. Patent Application Publication No. 2006/0210634 discloses adsorbing bioactive substances onto ceramic particles comprising a metal oxide (e.g., titanium oxide, zirconium oxide, scandium oxide, cerium oxide and yttrium oxide) by evaporation. Kortesuo et al., (2000, Int J Pharm. 200(2):223-229) disclose a spray drying method to produce spherical silica gel particles with a narrow particle size range for controlled delivery of drugs such as toremifene citrate and dexmedetomidine HC1. Wang et al., (2006, Int J Pharm. 308(1-2): 160- 167) describe the combination of adsorption by porous CaC03 microparticles and encapsulation by polyelectrolyte multilayer films for delivery of bioactive substances.

6.3 Liposomes

[0246] Liposomes can be produced by standard methods such as those reported by Kim et al., (1983, Biochim. Biophys. Acta 728, 339-348); Liu et al.,

(1992, Biochim: Biophys. Acta 1104, 95- 101 ); Lee et al., ( 1992, Biochim. Biophys. Acta. 1103, 185- 197), Brey et al., (U.S. Patent Application Publication No.

20020041861), Hass et al., (U.S. Patent Application Publication No. 2005/0232984), Kisak et al., (U.S. Patent Application Publication No. 2005/0260260) and Smyth- Templeton et al., (U.S. Patent Application Publication No. 2006/0204566).

Additionally, reference may be made to Copeland et al., (2005, Immunol. Cell Biol. 83: 95-105) who review lipid based particulate formulations for the delivery of antigen, and to Bramwell et al., (2005, Crit. Rev. Ther. Drug Carrier Syst. 22(2): 151- 214; 2006, J Pharm Pharmacol. 58(6):717-728) who review particulate delivery systems for therapeutic agents, including methods for the preparation of protein- loaded liposomes. Many liposome formulations using a variety of different lipid components have been used in various in vitro cell culture and animal experiments. Parameters have been identified that determine liposomal properties and are reported in the literature, for example, by Lee et al., (1992, Biochim. Biophys. Acta. 1103, 185-197); Liu et al., (1992, Biochim. Biophys. Acta. 1104, 95-101); and Wang et al., (1989, Biochem. 28, 9508-951).

[0247] Briefly, the lipids of choice (and any organic-soluble bioactive), dissolved in an organic solvent, are mixed and dried onto the bottom of a glass tube under vacuum. The lipid film is rehydrated using an aqueous buffered solution containing any water-soluble bioactives to be encapsulated by gentle swirling. The hydrated lipid vesicles can then be further processed by extrusion, submitted to a series of freeze-thawing cycles or dehydrated and then rehydrated to promote encapsulation of bioactives. Liposomes can then be washed by centrifugation or loaded onto a size-exclusion column to remove unentrapped bioactive from the liposome formulation and stored at 4 °C. The basic method for liposome preparation is described in more detail in Thierry et al., (1992, Nuc. Acids Res. 20:5691-5698).

[0248] A particle carrying a payload of bioactive agent(s) can be made using the procedure as described in: Pautot et al., (2003, Proc. Natl. Acad. Sci. USA 100(19): 10718-21 ). Using the Pautot et al. technique, streptavidin-coated lipids (DPPC, DSPC, and similar lipids) can be used to manufacture liposomes. The drug encapsulation technique described by Needham et al., (2001 , Advanced Drug Delivery Reviews 53(3): 285-305) can be used to load these vesicles with one or more active agents.

[0249] The liposomes can be prepared by exposing chloroformic solution of various lipid mixtures to high vacuum and subsequently hydrating the resulting lipid films (DSPC/CHOL) with pH 4 buffers, and extruding them through polycarbonated filters, after a freezing and thawing procedure. It is possible to use DPPC

supplemented with DSPC or cholesterol to increase encapsulation efficiency or increase stability, etc. A transmembrane pH gradient is created by adjusting the pH of the extravesicular medium to 7.5 by addition of an alkalinization agent. A bioactive agent (e.g., an inhibitor of IL-18 function and optionally targeting that delivers the inhibitor to memory T cells in the bone marrow) can be subsequently entrapped by addition of a solution of the bioactive agent in small aliquots to the vesicle solution, at an elevated temperature, to allow accumulation of the bioactive agent inside the liposomes.

[0250] Other lipid-based particles suitable for the delivery of the bioactive agents of the present invention such as niosomes are described by Copeland et al., (2005, Immunol. Cell Biol. 83: 95-105).

6.4 Ballistic particles

[0251] The bioactive agents of the present invention (e.g. , an inhibitor of IL-18 function and optionally targeting agent that delivers the inhibitor to memory T cells in the bone marrow) may be attached to (e.g., by coating or conjugation) or otherwise associated with particles suitable for use in needleless or "ballistic" (biolistic) delivery. Illustrative particles for ballistic delivery are described, for example, in: International Patent Publication Nos. WO 02/101412; WO 02/100380; WO 02/43774; WO 02/19989; WO 01/93829; WO 01/83528; WO 00/63385; WO 00/26385;

WO 00/19982; WO99/01 168; WO 98/10750; and WO 97/48485. It shall be

understood, however, that such particles are not limited to their use with a ballistic delivery device and can otherwise be administered by any alternative technique (e.g., injection or microneedle delivery) through which particles are deliverable to memory T cells.

[0252] The compositions of the invention can be coated or chemically coupled to carrier particles (e.g., core carriers) using a variety of techniques known in the art. Carrier particles are selected from materials which have a suitable density in the range of particle sizes typically used for intracellular delivery. The optimum carrier particle size will, of course, depend on the diameter of the target cells. Illustrative particles have a size ranging from about 0.01 to about 250 pm, from about 10 to about 150 pm, and from about 20 to about 60 pm; and a particle density ranging from about 0.1 to about 25 g/cm³, and a bulk density of about 0.5 to about 3.0 g/cm³, or greater. Non-limiting particles of this type include metal particles such as, tungsten, gold, platinum and iridium carrier particles. Tungsten particles are readily available in average sizes of 0.5 to 2.0 pm in diameter. Gold particles or

microcrystalline gold (e.g., gold powder A1570, available from Engelhard Corp., East Newark, N.J.) may also be used. Gold particles provide uniformity in size (available from Alpha Chemicals in particle sizes of 1 -3 pm, or available from Degussa, South Plainfield, N.J. in a range of particle sizes including 0.95 pm) and low toxicity.

Microcrystalline gold provides a diverse particle size distribution, typically in the range of 0.1-5 pm. The irregular surface area of microcrystalline gold provides for highly efficient coating with the active agents of the present invention.

[0253] Many methods are known and have been described for adsorbing, coupling or otherwise attaching bioactive molecules (e.g., hydrophilic molecules such as proteins and nucleic acids) onto particles such as gold or tungsten particles. In illustrative examples, such methods combine a predetermined amount of gold or tungsten with the bioactive molecules, CaCI₂ and spermidine. In other examples, ethanol is used to precipitate the bioactive molecules onto gold or tungsten particles (see, for example, Jumar et al., 2004, Phys Med. Biol. 49: 3603-3612). The resulting solution is suitably vortexed continually during the coating procedure to ensure uniformity of the reaction mixture. After attachment of the bioactive

molecules, the particles can be transferred for example to suitable membranes arid allowed to dry prior to use, coated onto surfaces of a sample module or cassette, or loaded into a delivery cassette for use in particular particle-mediated delivery instruments.

[0254] The formulated compositions may suitably be prepared as particles using standard techniques, such as by simple evaporation (air drying), vacuum drying, spray drying, freeze drying (lyophilization), spray-freeze drying, spray coating, precipitation, supercritical fluid particle formation, and the like. If desired, the resultant particles can be dandified using the techniques described in International Patent Publication No. WO 97/48485.

6.5 Surfactants

[0255] Surfactants which can be incorporated into particles include

phosphoglycerides. Exemplary phosphoglycerides include phosphatidylcholines, such as the naturally occurring surfactant, L-a-phosphatidylcholine dipalmitoyl ("DPPC"). The surfactants advantageously improve surface properties by, for example, reducing particle- particle interactions, and can render the surface of the particles less adhesive. The use of surfactants endogenous to the lung may avoid the need for the use of non-physiologic surfactants.

[0256] Providing a surfactant on the surfaces of the particles can reduce the tendency of the particles to agglomerate due to interactions such as electrostatic interactions, Van der Waals forces, and capillary action. The presence of the surfactant on the particle surface can provide increased surface rugosity

(roughness), thereby improving aerosolization by reducing the surface area available for intimate particle-particle interaction.

[0257] Surfactants known in the art can be used including any naturally occurring surfactant. Other exemplary surfactants include diphosphatidyl glycerol (DPPG); hexadecanol; fatty alcohols such as polyethylene glycol (PEG);

polyoxyethylene-9-lauryl ether; a surface active fatty acid, such as palmitic acid or oleic acid; sorbitan trioleate (Span 85); glycocholate; surfactin; a poloxamer; a sorbitan fatty acid ester such as sorbitan trioleate; tyloxapol and a phospholipid.

7. Kits

[0258] The present invention also provides kits comprising an inhibitor of IL-18 function as broadly described above and elsewhere herein. Such kits may comprise additionally alternate agents for concurrent use with the compositions of the invention.

[0259] In some embodiments, in addition to the compositions of the invention the kit may include suitable components to assist in performing the methods of the invention, such as, for example, administration device(s), buffer(s), and/or diluent(s). The kits may include containers for housing the various components, and

instructions using the kit components in the methods of the present invention.

[0260] In order that the invention may be readily understood and put into practical effect, particular preferred embodiments will now be described by way of the following non-limiting examples.

EXAMPLES

EXAMPLE 1

IL-18 Is CRITICALLY REQUIRED FOR MULTIPLE MYELOMA PROGRESSION

[0261] IL-1 family cytokines are known to function as crucial mediators in sterile inflammation. The present inventors aimed, therefore, to understand the role of IL-1 family cytokines, particularly I L-1 b, in the multiple myeloma inflammatory microenvironment. To this end, transplantable myeloma cell lines derived from Vk*MYC transgenic mice, which have been established as reliable preclinical models to test the efficacy of anti-myeloma agents. The Vk^*MYC-derived multiple myeloma cell line, Vk12653 multiple myeloma, into wild-type (WT), 111 r^A, and 1118^~A mice. As previously reported, WT mice challenged with Vk^*MYC-derived multiple myeloma cells showed progressive paraproteinemia (see, Figures 1A-1 B) and expansion of malignant B220 CD138⁺ PCs in the BM on day 35 after injections (Figures 1 D-1 F), suggesting that host IL-1 signalling has a limited role in multiple myeloma.

Surprisingly, however, 1118^~A mice did not show obvious paraproteinemia (see,

Figures 1A-1C) and expansion of PCs in the BM by day 35 post-myeloma challenge (see, Figure 1 D-1 F, 2A, and 2B). Unexpectedly, compared with WT mice, 111 r mice were completely protected from extramedullary dissemination (Figures 2C-2D) that is seen in mice with Vk12653 multiple myeloma at advanced stages. In line with these results, H18^~/~ mice showed delayed multiple myeloma progression and prolonged survival time was 48 days in WT and 111 r^A mice (Figure 1 G). Notably, approximately 50% of the 1118^~ mice were protected from the lethal multiple myeloma progression (Figure 1 G).

[0262] An independent multiple myeloma cell line derived from Vk^*MYC transgenic mice (Vk12598 multiple myeloma) was then tested. The majority of WT and 111 r mice succumbed to paralysis or other multiple myeloma lethal symptoms within two months after injection of Vk12598 multiple myeloma cells (Figure 1 H). Surprisingly, II18 mice were completely protected during a four month observation period (Figure 1 H), further supporting that IL-18 is crucial for multiple myeloma progression. [0263] An aberrant gut microbiota in 1118^~ mice is responsible for susceptibility to inflammation-induced colorectal carcinogenesis, which is transferable to WT mice by co-housing (see, Hu et al., 2013). However, co-housing of WT and IL18^~ mice did not alter levels of paraproteinemia or BM myeloma burden compared with non- co-housed counterparts, indicating that susceptibility and/or resistance to myeloma is not transferable (Figures 2E-2H). Overall, our results revealed IL-18 as an

indispensable factor that fosters multiple myeloma progression.

Materials & Methods

Mice

[0264] C57BL/6 wild type (WT), Rag2^~ H2rg^~ , and Ptprc^a mice were

purchased from Walter and Eliza Hall Institute for Medical Research or bred in-house at the QIMR Berghofer Medical Research Institute. C57BL/6 111 r (Thomas et al., 2004), 1118^~A (Takeda et al., 1998), Nlrp3^~A (Martinon et al., 2006), Asc^~A (Mariathasan et al., 2004), and Nlrp1^~ (Masters et al., 2012) mice were described before. All experiments were approved by the QIMR Berghofer Medical Research Institute Animal Ethics Committee.

Vk*MYC Transplantable Models

[0265] Transplantable Vk*MYC multiple myeloma cell lines (Vk12653 and Vk12598) were generated and expanded as previously described (Chesi et al., 2012; Guillerey et al., 2015). Briefly, these cell lines were maintained in Rag2^~ H2rg^~ mice to avoid contamination with host-derived lymphocytes. Rag2^~ H2rg^~ mice usually develop massive splenic infiltration of malignant plasma cells within 4-5 weeks after injection of Vk*MYC multiple myeloma cells. Splenocytes containing > 50% of malignant PCs were frozen and used for experiments. Vk12653 multiple myeloma cells (2 x 10⁶) or Vk12598 multiple myeloma cells (5 x 10⁵) were injected i.v. into tail vein of indicated strains of mice. The percentage of myeloma monoclonal Ig in the serum was quantified by serum protein electrophoresis (Sebia Hydrasys system). To evaluate multiple myeloma burden in the BM, left femurs were flashed with PBS, using a 10 ml syringe with a 23G needle. The percentage and number of

B220 CD138⁺ PCs in BM were analysed by flow cytometry at indicated time points. For survival analysis, mice were monitored daily according to institutional ethic guidelines, and were euthanized when mice developed signs of reduced mobility including paralysis, hunched posture, or respiratory distress.

Flow Cytometry

[0266] Immunostaining of single cell suspensions was performed according to standard protocols. Cells were stained with fluorochrome-conjugated mAbs for 30 min on ice in the presence of anti-CD 16/32 mAb (2.4G2). Cell number was calculated by using BD Liquid Counting Beads (BD Biosciences). Data acquisition was performed using BD FACSCANTO II or LSRFortessa Flow Cytometer (BD Biosciences). Flow cytometric analysis was performed using Flowjo software

(Treestar).

Statistics

[0267] Statistical analyses were performed using GraphPad Prism 7 Software. Mann-Whitney U test or unpaired student t test was used for single comparisons between two groups. For comparison of three or more groups, one-way ANOVA with Tukey’s multiple comparison test, Holm-Sidak multiple test correction, or non- parametric Kruskal-Wallis test with Dunn’s multiple comparison post-test were used. Differences in survival were evaluated with the Mantel-Cox test p < 0.05 was considered statistically significant (p < 0.05 = ^*; p < 0.01 = ^**; p < 0.001 = ^***; p < 0.0001 = ^****).

EXAMPLE 2

NLRP1 Is CRITICALLY REQUIRED FOR MULTIPLE MYELOMA PROGRESSION

[0268] The secretion of I L-1 b and IL-18 is tightly regulated in a post- transcriptional manner, mainly by inflammasomes. In particular, the NLRP3 inflammasome has been implicated in a wide variety of inflammatory diseases, including cancer (see, Karki et al, 2017). To assess the involvement of NLRP3 and the adaptor protein ASC, WT, Nlrp3^/, and Asc^~/~ mice were challenged with Vk12653 multiple myeloma cells. Both Nlrp3^~ , and Asc^~ mice showed reduced levels of paraproteinemia (Figures 3A and 3B) and BM myeloma burden (Figures 3C and 3D), compared with WT mice. However, neither A//rp3^_A nor Asc^A mice represented the strong multiple myeloma-resistant phenotype seen in IH8^~/~ mice, as only a modest survival benefit was observed in Asc⁷ mice (Figure 3E). These data suggest that NLRP3- and ASC-independent pathways might be responsible for IL18-driven multiple myeloma progression.

[0269] ASC is known to be dispensable for the NLRP1 inflammasome activation (see, Masters et al., 2012). More recently, it has been demonstrated that the NLRP2 inflammasome is predominantly responsible for IL-18 production in the context of obesity and metabolic syndrome (see, Murphy et al., 2016). Based on the fact that mice lacking the NLRP1 inflammasome phenocopy those lacking IL-18 (see, Murphy et al, 2016), it was investigated whether mice lacking NLRP1 could be protected from multiple myeloma progression.

[0270] To this end, Vk12653 multiple myeloma cells were injected into mice lacking all three isoforms of NLRP1 (Nlrp1a^~ Nlrp1b^/ Nlrplc^, collectively referred to as /V/rpT^7·). Strikingly, it was observed that Nlrp1^~/~ mice were almost completely protected from paraproteinemia (Figures 4A and 4B) and expansion of PCs in the BM (Figures 4C and 4D) by day 35 post-multiple myeloma challenge. Furthermore, similarly to 1118^ mice, Nlrp ^ mice showed remarkably prolonged survival after the challenge with Vk12653 (Figure 4E). and Vk12598 (Figure 4F) multiple myeloma cells, providing further evidence of the link between NLRP1 and IL-18.

[0271] To gain insight into the cell type that is responsible for IL-18 production, BM chimeras were generated between WT and Nlrp1^~/~ mice (Figures 2G and 2H), and WT and 1118^~ mice (Figures 4I and 4J), followed by Vk12653 multiple myeloma challenge. All mice were reconstituted effectively with more than 90% of donor- derived hematopoietic cells (data not shown).

[0272] In either chimera, the contribution of radio-resistant cells tended to be greater, although lack of NLRP1 or IL-18 in both radio-sensitive and radio-resistant cellular compartments was required for the multiple myeloma-resistant phenotype (Figures 4G-4J). Thus, these data conclude that IL-18, possibly driven by NLRP1 , plays a crucial role for the multiple myeloma progression.

Materials and Methods

BM Chimeras

[0273] WT, Nlrp1 , or 1118^~ mice received two doses of 5.5 Gy, 3 hr apart, and were immediately injected with 5 x 10⁶ BM cells from indicated donor mice. Neomycin sulfate (Sigma) were added to drinking water (1 g/L) for two weeks and used for experiments eight weeks after transplantation.

EXAMPLE 3 CD8⁺ T CELL-DEPENDENT CONTROL OF MULTIPLE MYELOMA IN NLRP1 AND IL18 MICE

[0274] To understand how NLRP1 and IL-18 contribute to multiple myeloma progression, the role of effector lymphocytes in the control of Vk12653 multiple myeloma was investigated. WT, Nlrp1 , and II18 mice were treated with anti- asialoGM-1 or anti-CD8P to deplete NK and CD8⁺ T cells, respectively (Figures 5A- 5C). Depletion of either NK cells or CD8⁺ T cells exacerbated paraproteinemia and shortened survival in WT mice (Figure 5D), supporting that both NK cells and CD8⁺ T cells are implicated in immune-mediated multiple myeloma control. Similarly, depletion of either NK cells or CD8⁺ T cells increased levels of paraproteinemia in Nlrp1^~ (Figure 5E) and 1118^ mice (Figure 5F) at day 21 post-multiple myeloma challenge. Intriguingly, only depletion of CD8⁺ T cells abrogated the resistance against multiple myeloma progression in Nlrp1 (Figure 5E) or 1118^~A mice (Figure 5F). These data indicate that CD8⁺ T cells are required for the multiple myeloma- resistant phenotype in Nlrp and II18^~A mice.

Materials & Methods

In Vivo Treatment

[0275] Depleting anti-CD8p mAb (53.5.8, Bio X Cell), anti-asialoGM-1 polyclonal Ab (Wako), or control Ig (1 -1 , Bio X Cell) were injected i.p. (100 mg) on day -1 and 0, and then weekly for four weeks. Depleting anti-Ly6G mAb (1 A8, Bio X Cell) was injected i.p. (10 mg/kg) every other day. The anti-IL-18 neutralizing mAb (SK113AE4, kindly provided by Irmgard Forster, University of Bonn) (Lochner et al., 2002) was administered at 10 mg/kg with or without bortezomib (0.5 mg/kg,

Calbiochem). EXAMPLE 4

IL-18 ACTS AS AN IMMUNOSUPPRESSIVE SWITCH THAT DRIVES MULTIPLE MYELOMA

PROGRESSION

[0276] The critical requirement of CD8⁺ T cells raised a possibility that host- derived IL-18 might limit immune-mediated multiple myeloma control, rather than act as an essential pro-survival factor for multiple myeloma cells per se. Given that various pro-inflammatory cytokines can induce MDSCs in vitro, we postulated that the myeloma-niche-derived IL-18 gave rise to MDSCs in the BM microenvironment. To address this possibility, we first tested the effect of IL-18 on cytokine-induced generation of MDSCs in vitro. Consistent with previous reports (see, Marigo et al., 2010; Ugel et al., 2015), granulocyte-macrophage colony stimulating factor

(GM-CSF) sufficiently expanded CD1 1 b⁺Gr-1 ⁺ cells during four days of culture of BM cells (Figure S4A). In contrast, recombinant IL-18 (rlL-18) alone had a negligible impact on the total number of CD1 1 b⁺Gr-1 ⁺ cells (Figure 6A) or their subsets, namely Ly6G⁺ polymorphonuclear (PMN) subset (Figure 6B) and Ly6G Ly6Chigh monocyte (MO) subset (Figure 6C). Furthermore, rlL-18 did not show additional effects on the number of CD1 1 b⁺Gr-1 ⁺ cells even in combination with GM-CSF (Figures 6A-6C). Thus, IL-18 had a limited role in the expansion of MDSC-like cells.

[0277] The functional impact of IL-18 on BM myeloid cells was then examined, by isolating CD1 1 b⁺Gr-1 ⁺ cells after four days culture of cytokine-stimulated BM cells, followed by co-culture with CD8⁺ T cells on anti-CD3-coated plates. Strikingly, CD1 1 b⁺Gr-1 ⁺ cells derived from rlL-18-stimulated BM cells potently suppressed CD8⁺ T cell proliferation (Figure 6D) and IFN-g production (Figure 6E), compared with freshly isolated naive BM CD1 1 b⁺Gr-1 ⁺ cells or GM-CSF-expanded

CD1 1 b⁺Gr-1 ⁺ cells, indicating that rlL-18 induced MDSCs. The IL-18-driven immunosuppressive activity was observed in both PMN-MDSCs and MO-MDSCs (Figure 4G). Next, how IL-18 conferred its immunosuppressive activities was determined. Recently, the C/EBRb transcription factor has been identified as a critical factor for functional maturation of MDSCs (Marigo et al., 2010). Indeed, the expression of C/EBRb was induced in the MDSCs derived from rlL-18-stimulated BM cells (Figure 4G). Furthermore, the IL-18-induced MDSCs expressed nitric oxide synthase 2 (NOS2) and arginase 1 (ARG1 ) (Figure 4G), both of which are key immunosuppressive mediators regulated by C/EBRb. Consistent with the

upregulation of these proteins, enzymatic activity of NOS2 and ARG1 was augmented in the IL-18- induced MDSCs (Figures 6H and 6I). Although rlL-18 treatment also increased the levels of reactive oxygen species (ROS) (Figure 6J), inhibitors for NOS2 (L-NMMA) and ARG1 (Nor-NOFIA), but not for ROS (catalase), attenuated their suppressive activity (Figure 4K), indicating that NOS2 and ARG1 are mainly implicated in their immunosuppression. Overall, these results indicate that IL-18 alone is sufficient to induce MDSCs.

[0278] The levels of IL-18 in multiple myeloma-bearing mice were then examined, and it was found that IL-18 levels were increased in the BM, but not in the blood serum, at late stages of Vk12653 multiple myeloma (Figures 6L and 6M). These data suggest that multiple myeloma progression triggers IL-18 release locally in the BM niche. By contrast, isolated malignant PCs contained negligible levels of IL-18, excluding the possibility that malignant PCs can be a source of IL-18 (Figure 6N). The expansion of CD11b⁺Gr-1⁺ cells was hardly detected in the BM, as the naive BM harbours a significant proportion of normal myeloid cells, which were phenotypically indistinguishable from MDSCs (Figure 60). However, an increase of CD11 b⁺Gr-1⁺ cells, particularly the PMN subset, in peripheral blood (Figures 6P and 6Q) and spleen (Figure 6R) was observed in the late stages of multiple myeloma development, suggesting that MDSCs were propagated during the course of multiple myeloma progression. Indeed, BM CD11 b⁺Gr-1⁺ cells isolated from WT mice with Vk12653 multiple myeloma at late stages showed T cell suppressive activity, compared with those generated in 1118^~A mice, indicating that the multiple myeloma progression led to generation of functional MDSCs (Figure 6S). Furthermore, anti- Ly6G mAb treatment prolonged survival in mice with Vk12653 multiple myeloma (Figure 6T), supporting that MDSCs promote multiple myeloma progression.

[0279] To investigate the impact of IL-18 on the BM myeloid cells in vivo, naive WT mice were treated with rlL-18 for four consecutive days (Figure 6U). In concert with in vitro findings (Figures 6A-6C), the systemic administration of rlL-18 had negligible effects on the number of total CD11 b⁺Gr-1⁺ cells or their subsets (Figure 6V). Notably, the ex-vivo isolated BM CD11 b⁺Gr-1⁺ cells derived from rlL-18- treated mice possessed suppressive activity against T cell proliferation (Figure 6W), which was associated with upregulation of C/EBRb, NOS2, and ARG1 (Figure 6X). Again, these results support the hypothesis that IL-18 gave rise to functionally competent MDSCs in the BM. To investigate the effect of IL-18 on multiple myeloma progression, mice were pre-treated with rlL-18 for four consecutive days before Vk12653 multiple myeloma challenge, followed by maintenance treatment (twice per week for four weeks) (Figure 6Y). It was observed that the rlL-18 treatment exacerbated paraproteinemia and slightly shortened survival in WT mice with

Vk12653 multiple myeloma (Figure 7 A). More strikingly, the detrimental effect of IL- 18 was exaggerated in Nlrp1 and 1118^~A mice, as rlL-18 treatment abrogated the multiple myeloma resistance in these mice (Figures 7B and 7C). Of note, no significant differences were seen in the frequency of BM myeloid and lymphocyte subsets among naive WT, Nlrp1^~/~, and IH8^~/~ mice (Figure 7D-7H). In addition, we observed similar levels of suppressive activity in the IL-18-induced MDSCs derived from WT, Nlrp1^~/~, and 1118^~ mice (Figure 7I), excluding the possibility of intrinsic functional defects in Nlrp1^~/~ or H18^~/~ MDSCs.

[0280] Finally, it was investigated whether systemic administration of IL-18 could directly fuel malignant PC growth. To assess this, we tested the similar rlL-18 treatment in Rag2-^/~ll2rg^~/~ mice with Vk12653 multiple myeloma. These results show that rlL-18 treatment did not affect levels of paraproteinemia or survival in Rag2^~/~ Ifcrg^ mice (Figure 7J) strongly suggesting that IL-18 accelerates multiple myeloma progression only in the presence of effector lymphocytes. Taken together, these results demonstrate that IL-18 turns on an immunosuppressive switch in the BM niche through the generation of functional MDSCs, which allows multiple myeloma cells to evade immune-mediated control.

Materials & Methods

In Vitro MDSC Generation

[0281] MDSCs were generated as described with minor modifications (Marigo et al., 2010; Youn et al., 2008). Briefly, WT BM cells were obtained from femur and tibia, and cultured in Dulbecco modified Eagle medium (Gibco) containing 10% fetal calf serum, 2 mM glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin, and 50 mM 2-mercaptoethanol. GM-CSF (10 ng/ml, Biolegend) and/or recombinant IL-18 (50 ng/ml, provided by Glaxo Smith Kline) were added to induce MDSCs. After four days, non-adherent cells were collected, and CD1 1 b⁺ cells were positively selected by magnetic-activated cell sorting (MACS), using PE-conjugated anti-CD11 b mAb and anti-PE microbeads (Miltenyi Biotec). The percentage of CD11 b⁺Gr-1 ⁺ cells was usually > 94% after positive selection. In some experiments, CD1 1 b⁺Ly6G⁺ PMN- MDSCs and CD1 1 b⁺Ly6C^hi9h MO-MDSCs were sorted by BD FACSAria II cell sorter (BD Biosciences).

T Cell Proliferation Assay

[0282] CD8⁺ T cells were isolated from spleen by MACS using anti-CD8 microbeads, and stained with Cell Trace Violet (CTV, Thermo Fisher Scientific) according to manufacturer’s instruction. Isolated CD8⁺ T cells were co-cultured with MDSCs at indicated ratios on 96-well plates coated with anti-CD3 (2.5 mg/ml) for 72 hr. The percentages of proliferating CD8⁺ T cells were determined by

CTV-dilution. IFN-g levels in culture supernatant were measured by cytometric bead arrays (CBA, BD Biosciences). In some experiments, catalase (1000 U/mL; Sigma), L-NMMA (NG-Monomethyl-L-arginine, 0.5 mM; Sigma), or nor-NOFIA (Nu-hydroxy- nor-Arginine, 0.5 mM; Calbiochem) were added before co-culture to inhibit ROS, arginase, or NOS respectively (Youn et al., 2008).

Immunoblot

[0283] MACS-isolated CD1 1 b⁺ cells were homogenized in RIPA lysis buffer (Sigma-Aldrich) supplemented with complete protease inhibitor tablet (Roche). Cell extracts were separated by SDS-PAGE and transferred onto PVDF membranes (Bio-Rad). For blotting, antibodies against C/EBRb (C-19, Santa Cruz), arginase 1 (FI-52, Santa Cruz), NOS (C-1 1 , Santa Cruz), and b-actin (13E5, Cell Signaling Technology) were used followed by appropriate HRP-conjugated secondary antibodies (Cell Signalling technology). The proteins were visualized by ECL-Plus (GE Flealthcare) using the Syngene G Box system (Syngene) or ImageQuant LAS 500 (GE Flealthcare Life Sciences).

Arginase Assay

[0284] Arginase activity of MDSCs was determined by arginase activity assay kit (Sigma) according to manufacturer’s instruction. Briefly, cells lysates were incubated with substrate buffer containing manganese for 2 hr at 37 °C. Urea production was measured by colorimetry to determine arginase activity.

Nitrite Production Assay

[0285] Nitrite levels in culture supernatants were measured by Griess reagent system (Promega). Briefly, MACS-isolated MDSCs were cultured with LPS

(Lipopolysaccharides from Escherichia coli 0127:B8, Sigma-Aldrich, 100 ng/ml) for 24 hr, and equal volumes of supernatants were mixed with Greiss Reagent (1 % sulfanilamide in 5% phosphoric acid and 0.1 % N-1 -naphthyl-ethylenediamine dihydrochloride) for 20 min. The absorbance at 540 nm was measured to determine nitrite levels.

ROS Measurement

[0286] Differentially induced MDSCs were incubated with phorbol 12-myristate 13-acetate (PMA) (50 ng/ml, Sigma) for 20 min at 37 °C in the presence of CellROX green reagent (Thermo Fisher Scientific). ROS levels in CD1 1 b⁺Ly6G⁺ cells were calculated by subtracting the MFI of an unstained sample from the MFI of a stained sample.

EXAMPLE 5

MDSCs LIMIT T CELL RESPONSES IN MULTIPLE MYELOMA PATIENTS

[0287] To obtain a comprehensive view of the immune microenvironment in multiple myeloma patients, we performed a global transcriptomic analysis of CD138 BM aspirates from 73 newly diagnosed multiple myeloma patients through next- generation RNA sequencing. Intriguingly, we found an inverse correlation between PMN-MDSC signature genes ( ITGAM , ARG1, CYBB, OLR1, FUT4, CEACAM8, S100A8, and S100A9) and cytotoxic lymphocyte signature genes (CD2, CD3E, CD3D, TBX21, CD8B, PRF1, GZMA, and GZMB) (Figure 8A). In addition, unsupervised hierarchical clustering revealed that MDSC gene signature and cytotoxic gene signature segregate multiple myeloma patients into two groups:

MDSC^hi9hcytotoxic^low group and MDSC^lowcytotoxic^hi9h group (Figure 8B). These data suggested that PMN-MDSCs might contribute to limiting cytotoxic T cell functions in the multiple myeloma microenvironment. In concert with this hypothesis, a considerable fraction of cells had a phenotype consistent with PMN-MDSCs (CD33⁺CD11 b⁺ HLA-DR CD15⁺) within the BM of multiple myeloma patients (Figures 8C and 8D). Of note, only a minor fraction of BM cells had an MO-MDSC phenotype (CD33⁺CD11 b⁺HLA-DR CD14⁺) (Figures 8C and 8D). To verify whether multiple myeloma PMN-MDSCs possess suppressive activity, we isolated BM PMN- MDSCs (CD11b⁺CD15⁺HLA-DR- cells) and control BM MOs (CD11b⁺CD14⁺HLA- DR⁺ cells) from newly diagnosed multiple myeloma patients, followed by co-culture with healthy donor-derived CD3⁺ T cells. We observed that PMN-MDSCs, but not control MOs, potently suppressed T cell receptor (TCR)-induced CD4⁺ T cell and CD8⁺ T cell proliferation in a ratio-dependent manner (Figures 8E-8G). In line with these results, PMN-MDSCs also inhibited IL-2 and IFN-g secretion by CD3⁺ T cells (Figure 8H). Finally, we confirmed that PMN-MDSCs exhibited suppressive activity against autologous multiple myeloma patient-derived CD4⁺ and CD8⁺ T cells (Figures 8I-8K). Collectively, these results highlight prominent immunosuppressive properties of PMN-MDSCs that limit T cell responses in multiple myeloma patients.

Materials & Methods

Multiple Myeloma Patients

[0288] All patients gave written informed consent before screening and being enrolled in the study and collection was approved by lUCT-Oncopole and the CFIU- Toulouse review boards. Fresh BM aspirates from multiple myeloma patients routinely collected in the lUC-Toulouse between 2013 and 2017 were depleted from malignant plasma cells using anti-CD 138-coated magnetic beads (Miltenyi Biotec, Paris, France) and used for RNA-sequencing and MDSC functional assays. BM sera from 152 multiple myeloma patients with measurable disease were collected at diagnosis between October 2007 and October 2009 in different French hematology units. All patients except three were enrolled into IFM (Intergroupe Francophone du Myelome) protocols including IFM 99-06, IFM 2005-01 , IFM 2007-02 and IFM 2008 (Facon et al., 2007; Flarousseau et al., 2010; Moreau et al., 2011). Among the three patients not included in one of these protocols, two received VTD Velcade

Thalidomide Dexamethasone) and one VMP (velcade melphalan prednisone) without autologous stem-cell transplantation (ASCT). multiple myeloma patients’ risk factors including age, ISS score, and cytogenetic abnormalities such as t (4; 14) and del(17p) were investigated.

Cytokine Analysis

[0289] Mouse blood serum and BM IL-18 levels were determined by mouse IL-18 ELISA Kit (MBL International) according to manufacturer’s instruction. IL-18, M-CSF, GM-CSF, IL-6 and I L-1 b levels were evaluated in the BM plasma of multiple myeloma patients by Luminex assay technology (Hu immune monitoring 65 Plex, Thermo Fisher Scientific) according to manufacturer’s instruction. We discriminated patients with a high (I L-18^high) or a low (IL-18^|0W) level of IL-18 using the median value (245.59 pg/ml) as cut-off.

Multiple Myeloma Patient MDSC Analysis

[0290] CD3⁺ T cells were isolated from healthy donor PBMCs by negative selection using Pan T cell Isolation Kit (Miltenyi Biotec), according to manufacturer’s instructions.

[0291] CD33⁺CD1 1 b⁺HLA-DR CD15⁺ PMN-MDSCs, CD33⁺CD1 1 b⁺HLA- DR⁺CD14⁺ monocytes, CD33⁺CD1 1 b HLA-DR BM precursor cells and autologous BM CD3⁺ T cells were sorted from CD138 multiple myeloma patient BM samples using MoFLo Astrios cell sorter (Beckman Coulter). BM precursor cells (1 x 10⁵) were cultured in the presence or absence of 50 ng/ml of rlL-18 (MBL international) for 6 days, analysed for the presence of MDSCs by flow cytometry and used in suppression assays. Suppression assays were performed using healthy donor or multiple myeloma patient’s autologous CD3⁺ T cells stained with Cell Trace Violet (CTV, Thermo Fisher Scientific), stimulated with aCD3/aCD28/ctCD2 microbeads (T cell activation/expansion kit, Miltenyi Biotec), in the presence of multiple myeloma patient’s PMN-MDSCs, monocytes, freshly isolated or rlL-18 treated BM precursor cells at the indicated ratio. CTV dilution of CD4⁺ and CD8⁺ T cells was analysed after five days by flow cytometry. IFN-g and IL-2 levels were measured in the

corresponding cell culture supernatants by CBA (BD Biosciences).

Library Construction for RNA Sequencing

[0292] Total RNA was isolated from the CD138 fractions of multiple myeloma patient BM aspirates using RNeasy mini kit (Qiagen) according to the manufacturer’s instructions. The quality of the isolated total RNA from each sample was checked on 2200 Tapestation System (Agilent, Santa Clara, Calif) using the RNA ScreenTape assay and the quantitation was performed using the NanoVue Plus

Spectrophotometer (Biochrom Ltd, Cambridge, UK). 400 ng of total RNA per sample with a RNA integrity number > 8 were used for library preparation. The RNA- Seq library preparation was performed using TruSeq Stranded mRNA Sample Preparation kit (lllumina, San Diego, Calif) according to the manufacturer’s instructions (High Sample Protocol). The RNA-Seq libraries were checked on the 2200 Tapestation System (Agilent) using the High-Sensitivity DNA 1000 ScreenTape assay. The average fragment size of the final libraries was found to be 230 to 327 bps. Libraries were quantified by qPCR on a LightCycler 480 System (Roche

Molecular Diagnostics, Pleasanton, Calif) using KAPA Library Quantification Kit for lllumina platforms (Kapa Biosystems, Boston, Massachusetts). Libraries were then pooled in equal quantities, checked and quantified using same techniques (2200 Tapestation and LightCycler 480). Paired-end sequencing (2 x 75 bps) of these libraries was performed on a NextSeq 500 lllumina sequencing platform (lllumina) by five successive runs using NextSeq 500 High Output kit v2 (lllumina) generating in average 20 million pairs of reads per sample.

RNA-Seq Bioinformatic Analysis

[0293] Fastq files were aligned to the Ensembl GRCh38 human reference genome using STAR (version 2.3.0, (Dobin et al., 2013)) and gene expression summary was obtained using the Subread feature count algorithm (version 1.4.6, (Liao et al., 2014)). Patient gene expression was then normalized using standard procedure from the limma R package (Ritchie et al., 2015), retaining only genes expressed (more than 15 aligned reads) in at least 50% of patients. Following normalization, correlation between individual genes was computed using Pearson’s correlation. Following normalization, the correlation of each individual gene to IL-18 was computed using Pearson’s correlation. Genes with a correlation higher than 0.7 were retained to cluster patients. Gene set enrichment analysis was performed using the Reactome Knowledgebase (www.reactome.org) (Fabregat et al., 2016). RNA seq data can be found at GSE104171 using the token (cfihcuoshxirzmr). EXAMPLE 6

IL-18 AUGMENTS IMMUNOSUPPRESSIVE ACTIVITY OF MULTIPLE MYELOMA MDSCS

[0294] We next addressed whether IL-18 was implicated in the functional maturation of multiple myeloma MDSCs as we observed in mouse BM MDSCs. To this end, we took advantage of our global transcriptomic analysis of the multiple myeloma microenvironment and identified a list of 308 genes strongly associated with IL18 gene expression (correlation > 0.70, p < 10 ⁸) within multiple myeloma patient BM (Figure 9A). Intriguingly, the gene set enrichment analysis of Table 5 revealed a significant enrichment in gene network related to neutrophil/PMN-MDSC.

TABLE 5

[0295] Indeed, within multiple myeloma patient BM samples, IL-18 mRNA levels strongly correlated with expression of genes related to classical PMN-MDSCs (ITGAM, ARG1, S100A9, CEACAM8, and MMP9 ), including recently identified human cancer PMN-MDSC-specific genes ( OLR1 , RETN, LCN2, CD24, MMP8, and COL17A1) (Condamine et al., 2016), confirming the link between IL-18 and PMN- MDSCs in the BM niche of multiple myeloma patients (Figures 9B and 9C, and Table 4). Of note, IL1B, VEGFA, and CSF1 that were previously associated with MDSC accumulation or functions in cancer patients did not correlate with MDSC signature genes within multiple myeloma patients’ BM (Table 6).

TABLE 6

[0296] To further understand the functional role of IL-18, we isolated

CD33⁺CD11 b HLADR CD14 CD15 BM myeloid precursor cells from multiple myeloma patients, followed by six days of culture in the presence or absence of rlL-18. We observed that rlL-18 modestly increased the total viable

CD33⁺CD11 b⁺CD15+HLA-DR- cells (Figure 9D). Notably, rlL-18-treated HLA-DR- CD11 b CD33⁺ cells, but not untreated counterparts, potently suppressed CD4⁺ and CD8+ T cell proliferation (Figures 9E-9FI). Together, these data showed that IL-18 contributes to the generation of functionally competent MDSCs in the multiple myeloma microenvironment.

EXAMPLE 7

HIGH LEVELS OF BM PLASMA IL-18 PREDICT POOR PROGNOSIS IN MULTIPLE MYELOMA

PATIENTS

[0297] To further gain insight into the clinical importance of our findings, we next analysed IL-18 levels within the BM plasma of a retrospective cohort of 152 multiple myeloma patients at diagnosis. We observed a wide range of BM IL-18 levels among multiple myeloma patients (median value 245.6 pg/mL, range 52.2 to 1829 pg/mL) (Figure 10A). Interestingly, we found that multiple myeloma patients with low IL-18 levels (IL-18^|0W < median) had a significantly (p = 0.0026) longer overall survival than patients with high IL-18 levels (IL-18^high > median; Figure 10B). By contrast, BM levels of other cytokines such as IL-1 b, IL-6, M-CSF, GM-CSF, and VEGF-A did not have any significant impact on multiple myeloma patient overall survival (Figure 10C). Notably, BM IL-18 levels also showed a significant correlation with overall survival rates in multivariate analysis after adjustments using the most relevant variables, including age, gender, and the presence of cytogenetic abnormalities such as t(4;14) and del(17p) (Figure 10D). The adjusted hazard ratio of overall survival rates for patients with IL-18^high levels versus IL-18^|0W levels was 1.84 (95% Cl 1.15-2.94; p = 0.010). Indeed, we did not find an association between BM IL-18 levels and classical myeloma risk factors or serum b2-ΐΎΐΐa^K^uIΐ^h levels (Figures 10E and 10F). To exclude the possibility that different treatment regimens affected the prognostic impact of IL-18 that we observed, we performed a univariate analysis of 93 multiple myeloma patients who subsequently received bortezomib (VELCADE™), dexamethasone, and high-dose melphalan (namely, the VD-MEL200 regimen). Using the same IL-18 cut-off level of 245.6 pg/mL (Figure 10A), we found that patients with IL-18^high had a significantly lower survival than IL-18^|0W patients (p = 0.009) (Figure 10G). Altogether our results suggest that a high BM IL-18 level is an independent determinant of poor overall survival in multiple myeloma patients, further supporting the hypothesis that IL-18 contributes to multiple myeloma progression.

Survival Analysis

[0298] Overall survival was calculated from multiple myeloma diagnosis to death from any cause.

[0299] Kaplan-Meier curves and p-values from log-rank tests were computed using the survival package in R (version 2.38). Multivariate analysis was adjusted using three of the most relevant high-risk prognosis variables: age, presence of del(17p), and presence of t(4; 14) translocation evaluated by fluorescence in situ hybridization (FISH), to which we added the gender covariate. Seven patients with missing information for at least one of these variables were excluded from the analysis. Cox proportional hazard models were adjusted using the survival package in R (Version 2.41-3; https://github.com/therneau/survival) and effects tested using ANOVA from the limma package. EXAMPLE 8

DYSREGULATED IL-18 IS A POTENTIAL THERAPEUTIC TARGET IN THE MULTIPLE MYELOMA

MICROENVIRONMENT

[0300] To understand whether dysregulated IL-18 could be a therapeutic target, we performed pre-treatment with IL-18 neutralizing mAb followed by five weeks of maintenance treatment in WT mice with Vk12653 multiple myeloma (Figure 11A). Indeed, we observed that the long-term blockade of IL-18 delayed multiple myeloma progression (Figure 11 B), supporting that IL-18 is a potential therapeutic target. We were intrigued by our finding that BM IL-18 levels separated prognostic subgroups among multiple myeloma patients who subsequently received the same VD-MEL200 regimen (Figure 10G). The proteasome inhibitor bortezomib (Btz) is known to elicit immunogenic cell death in multiple myeloma cells, leading to enhanced anti-myeloma immune responses (Spisek et al., 2007). Indeed, we recently demonstrated that CD226, an adhesion molecule involved in NK cell- and T cell-mediated cytotoxicity, is indispensable for the optimal Btz-mediated anti- myeloma efficacy (Guillerey et al., 2015), thus supporting the importance of immune- mediated anti-myeloma responses by Btz. These new data described above led us to hypothesize that therapeutic blockade of immunosuppressive IL-18 in combination with Btz might be a rational treatment strategy. The Vk12598 and Vk12653 multiple myeloma cell lines were originally established as Btz resistant (Chesi et al., 2012). In concert, two cycles of Btz monotherapy failed to improve survival in mice with Vk12653 (Figure 11C) or Vk12598 multiple myeloma (Figure 8D). In addition, short- term IL-18 mAb was insufficient to improve survival against established multiple myeloma (Figures 11C and 1 D). However, strikingly, when combined with IL-18 mAb, we observed that Btz significantly prolonged survival in both models (Figures 11C and 11 D). The therapeutic efficacy was associated with an increased CD8⁺ T cell/MDSC ratio in peripheral blood (Figures 11 E and 11 F) and dependent on CD8⁺

T cells (Figure 11 G). Taken together, these data suggest that dysregulated IL-18 is a potential target in the multiple myeloma niche to overcome immunosuppression. DISCUSSION

[0301] We demonstrated that the pro-inflammatory cytokine IL-18 was critically involved in multiple myeloma-associated inflammation and

immunosuppression, and provided evidence that dysregulated IL-18 was a potential therapeutic target in the multiple myeloma niche. Using Vk^*MYC preclinical multiple myeloma models, we showed that 1118-/- mice were protected from multiple myeloma progression in a CD8+ T cell-dependent manner. Our data suggest that IL-18 acts as a key driver for multiple myeloma-associated immunosuppression through

generation of functional MDSCs. This hypothesis was supported by the

comprehensive analysis of the transcriptional landscape of the immune

microenvironment in multiple myeloma patients, which revealed the inverse correlation between PMN-MDSC signature and cytotoxic signature, and the positive correlation between 1118 and PMN-MDSC signature genes. Moreover, we found that a high level of BM IL-18 was an independent determinant of poor prognosis in multiple myeloma patients.

[0302] IL-18 was originally identified as an IFN-y-inducing factor in liver extracts of mice treated with Propionibacterium acnes and lipopolysaccharide

(Nakanishi et al., 2001 ), and thereby has been recognized as an immunostimulatory cytokine. In fact, 1118-/- mice have impaired NK cell activity (see, Takeda et al., 1998) and are susceptible to experimental metastasis (see, Dupaul- Chicoine et al., 2015). It is noteworthy that the IL-18 single-cytokine-deficient mice were remarkably protected from multiple myeloma, an exquisitely inflammatory niche-dependent cancer, although growing evidence suggests that IL-18 also possesses pro-tumour effects such as angiogenesis (see, Fabbi et al., 2015). Our data, showing the critical requirement of CD8+ T cells for multiple myeloma resistance and the pro-myeloma properties of rlL-18 in immunocompetent, but not in lymphocyte-deficient mice, suggested that IL-18 hampers immune-mediated control of multiple myeloma, rather than acting as a growth factor for malignant PCs. Indeed, IL-18 conferred potent immunosuppressive activity toward BM myeloid cells in mice and multiple myeloma patients, highlighting the negative impact of IL-18 on anti-tumour immune responses.

[0303] Due to its ability to stimulate IL-6 production, IL-1 b has been well studied in the context of multiple myeloma (see, Lust et al., 2009), while the role of IL-18 in multiple myeloma immunopathology has been poorly studied. Notably, in a phase II clinical trial, IL-1 receptor antagonist in combination with low-dose dexamethasone delayed progression from smoldering or indolent multiple myeloma to active multiple myeloma, indicating that IL-1 signalling does contribute to multiple myeloma progression at least in the asymptomatic early stage (Lust et al., 2009). Surprisingly, we observed that 111 r -/- mice showed a negligible survival benefit in the aggressive Vk^*MYC transplantable multiple myeloma models. The phenotypic difference between 111 r -/- and 1118-/- mice might be explained, at least in part, by their differential regulation at the transcriptional level and by distinct cellular distribution of I L-1 b and IL-18. In the multiple myeloma microenvironment, macrophages are a dominant source of I L-1 b (see, Hope et al., 2014). Whereas a DAMP-mediated priming signal is required to induce pro-IL-1 b prior to proteolytic processing, pro-IL- 18 is constitutively expressed in various cell types, including macrophages, osteoblasts, and mesenchymal stem cells (see, Arend et al., 2008). In fact, our BM chimera experiments showed that lack of IL-18 in both radio-sensitive and radio- resistant cells was necessary to protect mice from multiple myeloma progression, suggesting that a range of cells were responsible for IL-18 production.

[0304] Thus, we postulated that the devastating progression of multiple myeloma triggers an abundant IL-18 release in the BM microenvironment. Indeed, we observed considerable levels of IL-18 in the BM of multiple myeloma patients, given that reported circulating serum IL-18 levels in healthy subjects are 80 - 120 pg/mL (see, Dinarello et al., 2013). Notably, it was previously shown that blood circulating IL-18 levels were elevated in multiple myeloma patients with advanced- stage disease; however, there was no significant difference in overall survival between blood I L-18high and IL-18low multiple myeloma patients (Alexandrakis et al., 2004). By contrast, we found that an increased level of BM IL-18 is a powerful indicator of poor prognosis, independent of age, high-risk cytogenetics, or treatment. As many pro-inflammatory cytokines are produced and consumed at the site of inflammation, it is likely that the BM IL-18 levels, rather than circulating IL-18 levels, are highly relevant to the multiple myeloma disease pathology.

[0305] At the level of post-transcriptional processing, our data suggested that NLRP1 , rather than NLRP3, contributes to multiple myeloma progression. In accordance with a previous study that revealed the phenotypic similarity between /V/rp7 ^A and 1118^A mice (Murphy et al., 2016), Nlrpl^ mice displayed an multiple myeloma-resistant phenotype similar to that seen in 1118^~ mice. Although the NLRP1 inflammasome is activated in response to anthrax lethal toxin, Toxoplasma infection, and hematopoietic stress (Chavarria-Smith and Vance, 2015), it remains largely unknown whether NLRP1 directly recognizes certain DAMPs or the

disturbance of intracellular homeostasis, recently termed homeostasis-altering molecular processes (see, Liston and Masters, 2017). Despite the lack of

understanding of its activation mechanism(s), our results demonstrate that the specific inhibition of NLRP1 might be a potential approach for anti-myeloma therapy. However, the possibility remains that functional redundancy might exist among the inflammasome members in the niche of multiple myeloma patients, which could limit the therapeutic efficacy by single blockade of NLRP1. Thus, we tested a therapeutic approach to target IL-18 in this study, and showed that anti-IL-18 neutralizing mAb in combination with Btz improved survival in the Vk^*MYC multiple myeloma models. This result has clinical implications, as IL-18 neutralization mAb showed a good safety profile in type 2 diabetes mellitus patients (see, McKie et al., 2016).

[0306] Multiple myeloma-associated immunosuppression has gained prominence in the era of novel anti-myeloma agents and immunotherapy. The presence of functional MDSCs in multiple myeloma has been well documented (see, Gorgun et al., 2013; and Ramachandran et al., 2013); however, the exact impact of MDSCs in the multiple myeloma immune niche has not been defined. We provided robust evidence that MDSCs negatively regulate T cell responses in multiple myeloma by our comprehensive transcriptional analysis. Generally, in solid malignancies, MDSCs are generated in the BM in response to proinflammatory cytokines and growth factors, followed by recruitment to tumour sites and peripheral lymphoid organs, where MDSCs exert potent immunosuppressive activity (see, Ugel et al., 2015). By contrast, in marrow-devastating multiple myeloma, it is plausible that multiple myeloma- associated inflammation is tightly associated with the conversion of neighbouring normal BM myeloid cells into MDSCs, leading to progressive generation of an immunosuppressive niche. Our results strongly suggest that IL-18 critically contributes to this process. Although a wide variety of pro-inflammatory cytokines, including IL-1 family cytokines, can induce MDSCs in vitro (see, Lechner et al., 2010; and Lim et al., 2014), it had remained largely unknown what types of cytokines played a pivotal role in the suppressive activity of multiple myeloma MDSCs and disease progression. We now propose that IL-18 is a key driver for MDSC function in the multiple myeloma BM niche.

[0307] It is now appreciated that a wide variety of anti-cancer drugs elicit immunogenic cell death in cancer cells, which augments anti-tumour immune responses (see, Galluzzi et al., 2017). Indeed, we have previously demonstrated that effector lymphocytes are critically required for the optimal anti-tumour efficacy of the histone deacetylase inhibitor vorinostat or Btz (see, Christiansen et al., 2011 ;

Guillerey et al., 2015). Therefore, targeting multiple myeloma-associated

immunosuppression in combination with anti-myeloma agents could be a rational approach. Overall, our results revealed IL-18 as a potential therapeutic target to overcome immunosuppression, which will provide important insights into the therapeutic strategies in multiple myeloma.

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Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. A method of treating multiple myeloma in a subject, the method comprising administering to the subject a composition that comprises an inhibitor of IL-18 function to the subject, to thereby treat the multiple myeloma in the subject.

2. The method of claim 1 , wherein the inhibitor of IL-18 function binds

specifically to an IL-18 polypeptide, an IL18R polypeptide, or an IL18BP polypeptide.

3. The method of claim 1 or claim 2, wherein the inhibitor of IL-18 function is a peptide, nucleic acid, antigen-binding molecule, or small molecule inhibitor.

4. The method of any one of claims 1 to 3, wherein the multiple myeloma is resistant to chemotherapy treatment and/or radiotherapy treatment.

5. The method of any one of claim 1 to 4, wherein the method further comprises administering a chemotherapeutic agent to the subject.

6. The method of claim 5, wherein the chemotherapeutic agent is selected from at least one of melphalan, prednisone, vincristine, doxorubicin, decadron, BCNU, cyclophosphamide, adriamycin, dexamethasone, thalidomide, bortezomib, pamidronate, and zoledronic acid.

7. the method of claim 5 or claim 6, wherein the inhibitor of IL-18 function and the chemotherapeutic agent are delivered to the subject simultaneously, sequentially, or separately.

8. The method of any one of claims 1 or claim 7, wherein the inhibitor of IL-18 is an antigen-binding molecule.

9. The method of claim 8, wherein the antigen-binding molecule specifically binds to at least a portion of an IL-18 polypeptide (as set forth in SEQ ID NO: 1 ).

10. The method of claim 8, wherein the antigen binding molecule specifically binds to at least a portion of an IL18R1 polypeptide (as set forth in SEQ ID NO: 3).

11. The method of claim 8, wherein the antigen-binding molecule specifically binds to at least a portion of an IL18RAP polypeptide (as set forth in SEQ ID NO: 5).

12. The method of claim 8, wherein the antigen-binding molecule specifically binds to at least a portion of an IL18BP polypeptide (as set forth in SEQ ID NO: 7).

13. The method of any one of claims 1 to 7, wherein the inhibitor of IL-18 function is a peptide.

14. The method of any one of claims 1 to 14, wherein the composition further comprises a targeting agent.

15. The method of claim 14, wherein the targeting agent binds to a cell surface receptor polypeptide present on a memory T cell.

16. The method of claim 15, wherein the cell surface receptor polypeptide is

selected from the group comprising: a CD38 polypeptide, CAR-MIL

polypeptide, CAR-T polypeptide, BCMA polypeptide, CD137 polypeptide, CD319, polypeptide CD46 polypeptide, CD47 polypeptide, MCL-1

polypeptide, CD229 polypeptide, CD54 polypeptide, CD56 polypeptide, CD3 polypeptide, CD200 polypeptide, CD16A polypeptide, CGEN-928 polypeptide, CD48 polypeptide, CD319 polypeptide, LMA polypeptide, KMA polypeptide, FcRH5 polypeptide, hTfR IgA polypeptide, Dkk1 polypeptide, APRIL polypeptide, CD95 polypeptide, KIR polypeptide, CS1 polypeptide, CD19 polypeptide, CD20 polypeptide, CD74 polypeptide, SDF-1 polypeptide, IL-2 polypeptide, IL-6 polypeptide, PSGL-1 polypeptide, and a CD40 polypeptide.

17. The method of any one of claims 14 to 16, wherein the targeting agent

comprises an antigen-binding molecule that specifically binds to a receptor presented on the surface of a memory T cell.

18. The method of any one of claims 14 to 17, wherein the targeting agent is an antigen-binding molecule that specifically binds to a CD38 polypeptide.

19. The method of any one of claims 1 to 7, wherein the inhibitor of IL-18 function is an siRNA molecule.

20. Use of an inhibitor of IL-18 function in the treatment of multiple myeloma.

21. Use of an inhibition or IL-18 function in the manufacture of a medicament for the treatment of multiple myeloma.

22. A composition for treating multiple myeloma, the composition comprising an IL-18 antagonist and a chemotherapeutic agent.

23. The composition of claim 22, wherein the chemotherapeutic agent is selected from at least one of melphalan, prednisone, vincristine, doxorubicin, decadron, BCNU, cyclophosphamide, adriamycin, dexamethasone, thalidomide, bortezomib, pamidronate, and zoledronic acid.

24. The composition of claim 22 or claim 23, wherein the inhibitor of IL-18 function binds specifically to an IL-18 polypeptide, an IL18R polypeptide, or an IL18BP polypeptide.

25. The composition of any one of claims 22 to 24, wherein the inhibitor of IL-18 function is a peptide, nucleic acid, antigen-binding molecule, or small molecule inhibitor.

26. The composition of any one of claims 22 to 25, wherein the inhibitor of IL-18 function and the chemotherapeutic agent are delivered to the subject simultaneously, sequentially, or separately.

27. The composition of any one of claims 22 or claim 26, wherein the inhibitor of IL-18 is an antigen-binding molecule.

28. The composition of claim 27, wherein the antigen-binding molecule

specifically binds to at least a portion of an IL-18 polypeptide (as set forth in SEQ ID NO: 1 ).

29. The composition of claim 27, wherein the antigen binding molecule

specifically binds to at least a portion of an IL18R1 polypeptide (as set forth in SEQ ID NO: 3).

30. The composition of claim 27, wherein the antigen-binding molecule

specifically binds to at least a portion of an IL18RAP polypeptide (as set forth in SEQ ID NO: 5).

31. The composition of claim 27, wherein the antigen-binding molecule

specifically binds to at least a portion of an IL18BP polypeptide (as set forth in SEQ ID NO: 7).

32. The composition of any one of claims 22 to 26, wherein the inhibitor of IL-18 function is a peptide.

33. The composition of any one of claims 22 to 32, wherein the composition

further comprises a targeting agent.

34. The composition of claim 33, wherein the targeting agent binds to a cell surface receptor polypeptide present on a memory T cell.

35. The composition of claim 34, wherein the cell surface receptor polypeptide is selected from the group comprising: a CD38 polypeptide, CAR-MIL

polypeptide, CD229 polypeptide, CD54 polypeptide, CD56 polypeptide, CD3 polypeptide, CD200 polypeptide, CD16A polypeptide, CGEN-928 polypeptide, CD48 polypeptide, CD319 polypeptide, LMA polypeptide, KMA polypeptide, FcRH5 polypeptide, hTfR IgA polypeptide, Dkk1 polypeptide, APRIL polypeptide, CD95 polypeptide, KIR polypeptide, CS1 polypeptide, CD19 polypeptide, CD20 polypeptide, CD74 polypeptide, SDF-1 polypeptide, IL-2 polypeptide, IL-6 polypeptide, PSGL-1 polypeptide, and CD40

polypeptide.The method of any one of claims 33 to 35, wherein the targeting agent comprises an antigen-binding molecule that specifically binds to a receptor presented on the surface of a T cell (e.g., a memory T cell).

36. The composition of any one of claims 33 to 36, wherein the targeting agent is an antigen-binding molecule that specifically binds to a CD38 polypeptide.

37. The composition of any one of claims 22 to 37, wherein the inhibitor of IL-18 reduces or prevents the production of IL-18 polypeptide.

38. The composition of claim 38, wherein the inhibitor of IL-18 function is an

siRNA molecule.

39. Use of the composition as defined in any one of claims 22 to 39, in the

manufacture of a medicament for treating multiple myeloma in a subject.

40. A composition for treating multiple myeloma, the composition comprising an inhibitor of IL-18 function and a memory T cell targeting agent.

41.The composition of claim 41 , wherein the inhibitor of IL-18 function binds

42. The composition of claim 41 or claim 42, wherein the inhibitor of IL-18 function is a peptide, nucleic acid, antigen-binding molecule, or small molecule inhibitor.

43. The composition of any one of claims 41 to claim 43, wherein the inhibitor of IL-18 is an antigen-binding molecule.

44. The composition of claim 44, wherein the antigen-binding molecule

45. The composition of claim 44, wherein the antigen binding molecule

46. The composition of claim 44, wherein the antigen-binding molecule

47. The composition of claim 44, wherein the antigen-binding molecule

48. The composition of any one of claims 41 to 43, wherein the inhibitor of IL-18 function is a peptide.

49. The composition of any one of claims 41 to 49, wherein the targeting agent binds to a cell surface receptor polypeptide present on a memory T cell.

50. The composition of claim 50, wherein the cell surface receptor polypeptide is selected from the group comprising: a CD38 polypeptide, CAR-MIL

polypeptide, CD229 polypeptide, CD54 polypeptide, CD56 polypeptide, CD3 polypeptide, CD200 polypeptide, CD16A polypeptide, CGEN-928 polypeptide, CD48 polypeptide, CD319 polypeptide, LMA polypeptide, KMA polypeptide, FcRH5 polypeptide, hTfR IgA polypeptide, Dkk1 polypeptide, APRIL polypeptide, CD95 polypeptide, KIR polypeptide, CS1 polypeptide, CD19 polypeptide, CD20 polypeptide, CD74 polypeptide, SDF-1 polypeptide, IL-2 polypeptide, IL-6 polypeptide, PSGL-1 polypeptide and CD40 polypeptide.

51.The method of any one of claims 41 to 51 , wherein the targeting agent

52. The composition of any one of claims 41 to 52, wherein the targeting agent is an antigen-binding molecule that specifically binds to a CD38 polypeptide.

53. The composition of any one of claims 41 to 52, wherein the composition is a bispecific antibody.

54. A method of treating multiple myeloma in a subject, the method comprising administering a composition that comprises an IL-18 antagonist and a targeting agent, to thereby treat the multiple myeloma in the subject.

55. The method of claim 53, further comprising administering to the subject at least one dose of a chemotherapeutic agent.

56. A bispecific antigen-binding molecule that specifically binds to an IL-18

polypeptide and a T cell surface receptor polypeptide.

57. A bispecific antigen-binding molecule that specifically binds to an IL18R

polypeptide and a T cell surface receptor polypeptide.

58. The antigen-binding molecule of claim 56, wherein the IL18R polypeptide is selected from an IL18R1 polypeptide or a an IL18RAP polypeptide.

59. A bispecific antigen-binding molecule that specifically binds to an IL18BP

polypeptide and a T cell surface receptor polypeptide.

60. The antigen-binding molecule of any one of claims 55 to 58, wherein the T cell surface receptor polypeptide is selected from the group comprising: a CD38 polypeptide, CAR-MIL polypeptide, CAR-T polypeptide, BCMA polypeptide, CD137 polypeptide, CD319, polypeptide CD46 polypeptide, CD47

polypeptide, MCL-1 polypeptide, CD229 polypeptide, CD54 polypeptide,

CD56 polypeptide, CD3 polypeptide, CD200 polypeptide, CD16A polypeptide, CGEN-928 polypeptide, CD48 polypeptide, CD319 polypeptide, LMA polypeptide, KMA polypeptide, FcRH5 polypeptide, hTfR IgA polypeptide,

Dkk1 polypeptide, APRIL polypeptide, CD95 polypeptide, KIR polypeptide, CS1 polypeptide, CD19 polypeptide, CD20 polypeptide, CD74 polypeptide, SDF-1 polypeptide, IL-2 polypeptide, IL-6 polypeptide, PSGL-1 polypeptide, and CD40 polypeptide.

61. A pharmaceutical composition comprising the bispecific antigen-binding

molecule of any one of claims 55 to 59, and a pharmaceutically acceptable carrier, excipient or diluent.