WO2019010566A1

WO2019010566A1 - Anti-s100a8/a9 for inhibiting immunosuppressive activity of mdscs cells

Info

Publication number: WO2019010566A1
Application number: PCT/CA2018/050747
Authority: WO
Inventors: Philippe Tessier; Joan DEFRENE; Malika LAOUEDJ; Frédéric BARABE
Original assignee: UNIVERSITé LAVAL
Priority date: 2017-07-10
Filing date: 2018-06-20
Publication date: 2019-01-17

Abstract

A solid tumor in a subject is treated with a composition comprising a carrier and one of an anti-S100A8, an anti-S100A9, or a combination thereof. The composition diminishes the numbers of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs), reduces the activity of MDSCs, and increases the numbers of CD8 cells. The composition furthermore increases the differentiation of MDSCs into mature myeloid cells in the subject.

Description

ANTI-S100A8/A9 FOR INHIBITING IMMUNOSUPPRESSIVE

ACTIVITY OF MDSCS CELLS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims benefit of U.S. Provisional Application No. 62/530,433 filed July 10, 2017, the content of which is hereby incorporated by reference in their entirety.

TECHNICAL FIELD

[0002] It is provided a method of treating a subject with a solid tumor comprising the use of an anti-S 100A8 and/or anti-S 1009 molecule.

BACKGROUND

[0003] Immunosuppressive cells, mainly T regulatory cells (Tregs) and myeloid- derived suppressor cells (MDSCs), negatively regulate immune responses in cancer. MDSCs are a major cell type utilized by tumors to escape immune surveillance. Increased numbers of MDSCs have been found in patients with solid tumors. MDSCs are a heterogeneous group of cells consisting of early myeloid progenitors/precursors of granulocytes, monocytes, and dendritic cells defined by their immunosuppressive functions. Two major populations of MDSCs have been described in the tumor environment: polymorphonuclear MDSCs (PMN-MDSCs) and monocytic MDSCs (Mo- MDSCs). In mice, PMN-MDSCs are CD1 1 b⁺Ly6C^l0WLy6G⁺ while Mo-MDSCs are CD1 1 b⁺Ly6C^highLy6G^". In humans, PMN-MDSCs are defined as CD33⁺CD15⁺CD14^" CD1 1 b⁺HLA-DR-"^0W while Mo-MDSCs are CD33⁺CD15 D14⁺CD1 1 b⁺HLA-DR^{" l0W}. They are known to accumulate in blood, spleen, lymph nodes, and tumors and are major regulators of immune responses in cancer by helping tumor cells to evade cytotoxic T cells by suppressing their activity. These cells account for approximately 20- 30% of bone marrow cells and 1 -5% of splenocytes in healthy humans and animals, but increase to 60-70% and 10-20%, respectively, in cancers.

[0004] There are differences in suppressive capacities and functional mechanisms between the 2 MDSC subsets, but the major roles of MDSCs is immune suppression by inducing regulatory T cells and inhibition of activation of CD4 and CD8 T cells by deprivation of amino acids (arginine) and the release of oxidizing molecules like NO. Differentiation of MDSCs into mature cells abolishes their immune-suppressive function.

[0005] MDSCs are produced in the bone marrow, and in healthy individuals, they differentiate in granulocytes, macrophages, and dendritic cells. Consequently, MDSCs are mildly immunosuppressive in healthy individuals. However, MDSCs fail to differentiate in cancer, thereby proliferating and mobilizing from bone marrow to peripheral organs and tumors where they suppress T cells.

[0006] Accordingly, MDSCs in solid tumors induce tumor development and growth by providing a tumor-supportive and immune-suppressive microenvironment.

[0007] Patients with metastatic, refractory or solid tumors have very poor prognosis. Accordingly, it is thus highly desired to be provided with improved methods for treating subjects with solid tumors.

SUMMARY

[0008] One aim of the present disclosure is to provide a composition comprising an anti-S100A8, an anti-S100A9, or a combination thereof, and a carrier for treating a subject with a solid tumor.

[0009] In an embodiment, the anti-S100A8 or anti-S100A9 is an antibody.

[0010] In another embodiment, the anti-S100A8 specifically binds to a portion of a S100A8 protein.

[0011] In a further embodiment, the anti-S100A9 specifically binds to a portion of a S100A9 protein.

[0012] In an embodiment, the anti-S100A8 specifically binds to a S100A8/S100A8 homodimer or a S100A8/S100A9 heterodimer.

[0013] In a further embodiment, the anti-S100A9 specifically binds to a S100A9/S100A9 homodimer or a S100A8/S100A9 heterodimer.

[0014] In another embodiment, the S100A8 protein is a human S100A8.

[0015] In a supplemental embodiment, the S100A9 protein is a human S100A9. [0016] In an alternate embodiment, the anti-S100A8 or anti-S100A9 is a monoclonal or a polyclonal antibody.

[0017] In another embodiment, the anti-S100A8 or anti-S100A9 is a mouse antibody, a goat antibody, a human antibody or a rabbit antibody.

[0018] In another embodiment, the anti-S100A8 or anti-S100A9 is a humanized antibody.

[0019] In a further embodiment, the anti-S100A8 or anti-S100A9 antibody comprises an epitope binding fragment selected from the group consisting of: Fv, F(ab'), or F(ab')2.

[0020] In another embodiment, the anti-S100A8 antibody comprises a heavy chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 1.

[0021] In another embodiment, the anti-S100A8 antibody comprises a heavy chain variable region consisting of SEQ ID NO: 2.

[0022] In a further embodiment, the anti-S100A8 antibody comprises a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 3.

[0023] In a further embodiment, the anti-S100A8 antibody comprises a light chain variable region consisting of SEQ ID NO: 4.

[0024] In another embodiment, the anti-S100A9 antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1 1 , 12, 13, 14, 15, 16 and 17.

[0025] In another embodiment, the anti-S100A9 antibody comprises a chain selected from: SEQ ID NO: 5, 6, 7, 8, 9 or 10.

[0026] In a further embodiment, the anti-S100A8 or anti-S100A9 is formulated for an injection.

[0027] In another embodiment, the composition described herein is formulated for an administration with a chemotherapeutic agent.

[0028] In an embodiment, the composition described herein is for an administration with an anti-PD1 , an anti-PDL1 , or a combination thereof. [0029] In another embodiment, the composition diminishes the numbers of PMN- MDSCs in the subject.

[0030] In an embodiment, the composition increases CD8 cells and reduces the activity of MDSCs in said subject.

[0031] In a further embodiment, the composition decreases the differentiation of MDSCs into mature myeloid cells in said subject.

[0032] In a particular embodiment, the solid tumor is a sarcoma, a carcinoma, or a lymphoma.

[0033] In addition, encompassed herein a solid tumor which is a fibrosarcoma, a myxosarcoma, a liposarcoma, a chondrosarcoma, an osteogenic sarcoma, a chordoma, an angiosarcoma, an endotheliosarcoma, a lymphangiosarcoma, a lymphangioendotheliosarcoma, a synovioma, a mesothelioma, an Ewing's tumor, a leiomyosarcoma, a rhabdomyosarcoma, a colon carcinoma, a pancreatic cancer, a breast cancer, an ovarian cancer, a prostate cancer, a squamous cell carcinoma, a basal cell carcinoma, an adenocarcinoma, a sweat gland carcinoma, a sebaceous gland carcinoma, a papillary carcinoma, a papillary adenocarcinomas, a cystadenocarcinoma, a medullary carcinoma, a bronchogenic carcinoma, a renal cell carcinoma, an hepatoma, a bile duct carcinoma, a choriocarcinoma, a seminoma, an embryonal carcinoma, a Wilms' tumor, a cervical cancer, a testicular tumor, a lung carcinoma, a small cell lung carcinoma, a bladder carcinoma, an epithelial carcinoma, a glioma, an astrocytoma, a medulloblastoma, a craniopharyngioma, an ependymoma, a pinealoma, an hemangioblastoma, an acoustic neuroma, an oligodendroglioma, a meningioma, a melanoma, a neuroblastoma, or a retinoblastoma.

[0034] In an embodiment, the subject is an animal or a human. Particularly, the animal is a mouse.

[0035] It is also provided a method of treating a subject with a solid tumor comprising the step of administering the composition described herein.

[0036] It is additionally provided the use of the composition described herein for treating a subject with a solid tumor.

[0037] It is further provided the use of the composition described herein in the manufacture of a medicament for treating a subject with a solid tumor. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Reference will now be made to the accompanying drawings.

[0039] Fig. 1 illustrates in (A) flow cytometry analyses of AML cells from representative mice treated with anti-S100A8. Contour plots are gated on EGFP+ cells; in (B) representative May-Grunwald-Giemsa stained cytospins of AML cells from anti- Si 00 A8-treated mice showing signs of granulocytic differentiation, scale bars = 20μηι; in (C) representative cytospins of AML cells from S100A9 protein-treated mice showing signs of granulocytic differentiation, scale bars, 20μηι; and in (D) cell-cycle profiles of bone marrow cells from control, anti-S100A8 recipients. Results are represented as the mean ± SEM, P-value was determined by student t-test.

[0040] Fig. 2 illustrates S100A9, but not S100A8 protein, promoting the differentiation of myeloid precursors/progenitors cells. Murine bone marrow cells from C57BL/6 mice were cultured with increasing concentrations (Mg/ml) of S100A8 and S100A9 proteins for 48h. The presence of neutrophils, monocytes and macrophages was analyzed by flow cytometry.

[0041] Fig. 3 illustrates the expression of S100A8 and S100A9 by murine B16-F10 melanoma cells. Expression of S100A8 and S100A9 was analyzed by flow cytometry in B16-F10 cells grown in vitro.

[0042] Fig. 4 illustrates tumor growth of B16-F10 cells injected subcutaneously in C57BL/6 mice. Data are the mean tumor volume +/- SEM of 9 mice.

[0043] Fig. 5 illustrates increased presence of MDSCs in the spleen and blood of mice with B16-F10 subcutaneous tumors. B16-F10 cells were injected s.c. in mice to induce tumors. Peripheral blood, tumors, and spleen were harvested 2 weeks later. Single cell suspensions were analyzed by flow cytometry.

[0044] Fig. 6 illustrates expression of S100A8/A9 in MDSCs of tumor-bearing mice. B16-F10 cells were injected s.c. in mice to induce tumors. Peripheral blood, tumors, and spleen were harvested 2 weeks later. Single cell suspensions were analyzed by flow cytometry.

[0045] Fig. 7 illustrates that antibodies against S100A8 or S100A9 inhibit B16-F10 tumor growth. B16-F10 melanoma cells were injected s.c. in the flanks of mice. Purified rabbit anti-S100A8 IgGs or purified normal rabbit IgGs (100 μg/mouse) were injected three times a week starting on day 2 post-injection of tumor cells. Tumor growth was monitored daily using a caliper. Data are the mean of 8 mice per groups.

[0046] Fig. 8 illustrates the presence of MDSCS in the blood of tumor-bearing mice treated with anti-S100A8 or anti-S100A9. B16-F10 cells were injected s.c. in mice to induce tumors. Spleens were harvested 2 weeks later. Single cell suspensions were analyzed by flow cytometry. Data are the mean +/- SEM of 5 mice. *, p<0,05.

[0047] Fig. 9 illustrates the presence of MDSCS in the spleen of tumor-bearing mice treated with anti-S100A8. B16-F10 cells were injected s.c. in mice to induce tumors. Spleens were harvested 2 weeks later. Single cell suspensions were analyzed by flow cytometry. Particularly, in (A) it is shown the presence of PMN-MDSCs in the spleen and differentiation of MDSCs in mature cells measured by expression of CD1 1 b; and in (B) the presence of Mo-MDSCs in the spleen and differentiation of MDSCs in mature cells measured by expression of CD1 1 b. Data are the mean +/- SEM of 5 mice.

[0048] Fig. 10 illustrates upregulation of immune responses in tumor-bearing mice treated with anti-S100A8 antibodies showing in (A) CD8 positive T cells in spleen of mice injected with B16-F10 cells; and in (B) proliferation of CD4+ T cells in co-culture with PMN-MDSCs isolated from spleens of tumor-bearing mice injected with anti- S100A8. Data are the mean +/- SEM of 5 mice.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0049] In accordance with the present invention, there is provided a method of treating solid tumor by administering anti-S100A8 and/or anti-S100A9 antibodies.

[0050] Because of their myeloid origin, MDSCs express the myeloid-derived proteins S100A8 and S100A9, and S100A8 and S100A9 are detected on their surface, indicating that they secrete S100A8 and S100A9.

[0051] S100A8 and S100A9 are damage-associated molecular patterns which form a subset of the S100 family of calcium-binding proteins. These intracellular proteins control protein phosphorylation, enzymatic activities, Ca²⁺ homeostasis, and intermediate filaments polymerisation. S100A8 and S100A9 are expressed abundantly in neutrophils (30% of cytosolic proteins) and monocytes, but are generally not expressed in tissue macrophages. Platelets epithelial, mesenchymal and endothelial cells also express and secrete these proteins under infectious or inflammatory conditions. S100A8 and S100A9 exist as non-covalently bonded homodimers. In addition, in the presence of calcium, S100A8 and S100A9 form a noncovalent heterodimer called S100A8/A9 or calprotectin.

[0052] S100A8 and S100A9 show opposite activities. Glucocorticoids and IL-10 induce S100A8 expression, supporting an anti-inflammatory function for S100A8. In addition, S100A8 is oxidized and nitrosylated on its cysteine residue, leading to the formation of covalent bounds between the monomers. Oxidized S100A8 is antiinflammatory, inhibiting mast cell degranulation and cytokine secretion induced by FcsR crosslinking. In addition, inhibition of S100A8 aggravates chronic inflammation, indicating that S100A8 dampens the immune response in chronic inflammation. In contrast, S100A9 is a potent inducer of phagocytosis and degranulation and a mild inducer of ROS production by neutrophils and monocytes. S100A9 also induces cytokine secretion by monocytes, by activating NF-κΒ and the inflammasome, and blocking S100A9 inhibits chronic inflammation. Thus S100A9 promotes inflammation by enhancing phagocyte migration and inducing the secretion of pro-inflammatory cytokines, as well as the release of tissue-degrading enzymes and ROS. Oxidized S100A8 presumably counterbalance these functions by its anti-inflammatory activities. S100A8 and S100A9 bind to RAGE, the scavenger receptor (CD36), CD33, EM MPRIN, neuroplastin-β, or the Toll-like receptor 4 (TLR4). In addition, antibodies against TLR2 inhibit the activation of NF-κΒ by S100A8 and S100A9 in THP-1 cells, indicating that TLR2 is also a receptor for both proteins.

[0053] Expression of S100A8/A9 either by tumor cells or tumor infiltrating phagocytes has been extensively reported. S100A8/A9 was reported to induce apoptosis in various tumor cell lines. S100A8/A9 has also been involved in the establishment of a favorable environment for metastases. Plasma S100A8/A9 correlates with cancer stages and the presence of MDSCs in gastric cancers. This heterodimer also binds to and activates NF-κΒ, and induces proliferation of colonic and prostate tumor cell lines. Pancreatic ductal adenocarcinoma cells express high levels of the S100A8/A9 complex which causes an expansion of Mo-MDSCs, suggesting that S100A8/A9 constitutes one of the immunosuppressive soluble mediators released by these cells. In addition, IRF7 regulates the development of granulocytic myeloid- derived suppressor cells by repressing expression of S100A9 in cancer. S100A8/A9 expression by lung phagocytes has also been linked to metastasis and migration to this organ, and expression of S100A8 and S100A9 regulates proliferation and differentiation of breast cancer cells and acute myeloid leukemia. [0054] Acute myeloid leukemia was used as a model to decipher the effects of S100A8 and S100A9 on the differentiation of myeloid cell precursors and progenitors, as precursors and progenitors are enriched in this disease. These studies demonstrated that S100A8 and S100A9, both produced by AML cells, regulate the differentiation and proliferation of AML cells. Blocking of S100A8 or addition of S100A9 induced the differentiation of AML cells and their growth arrest in mouse and human models of AML in primary cells from AML patients (see Fig. 1 ). S100A9 activates TLR4, leading to the phosphorylation of p38 MAPK, ERK1/2, and JNK, which in turn activate CREB, c-JUN, and NF-κΒ. These results highlight the opposite effects of S100A8 and S100A9: S100A8 favoring proliferation over differentiation, and S100A9 promoting the differentiation of myeloid cells.

[0055] The effects of S100A8 and S100A9 on the differentiation of myeloid precursors/progenitors was next confirmed using normal bone marrow. S100A9, but not S100A8 protein increases the production of neutrophils, monocytes, and macrophages from bone marrow cell cultures (see Fig. 2). These results suggest that, as in acute myeloid leukemia, S100A8 acts as a repressor of myeloid cell differentiation, and that S100A9 promotes the differentiation of myeloid cells. Therefore, modulating the S100A8:S100A9 ratios could influence the generation of MDSCs

[0056] The murine melanoma cell line B16-F10 was used as a tumor model in mice as they are routinely used to study MDSCs. B16-F10 cells expressed S100A8 and S100A9 when cultured in vitro (see Fig. 3). Subcutaneous injection of B16-F10 cells (250,000 cells per mouse) in the flank of mice caused tumors (Fig. 4). Two weeks later, the mice were sacrificed and the tumor, peripheral blood, bone marrow, and spleen, were harvested. The tumors were digested using collagenase, then single cell preparations of spleen, tumor, blood, and bone marrow were analyzed by flow cytometry using markers of myeloid cells and MDSCs. There was an increase of PMN- MDSCs and Mo-MDSCs-like cells in the bone marrow, blood, and spleen of tumor- bearing mice compared to naive mice (Fig. 5). Expression of S100A8, but not S100A9, was increased in PMN-MDSCs and Mo-MDSCs from the bone marrow in tumor- bearing mice (Fig. 6). However, expression of S100A8 was reduced in PMN-MDSCs and Mo-MDSCs in the blood of tumor-bearing mice. Interestingly, Mo-MDSCs did not express S100A9 in the blood or spleen of either naive or tumor-bearing mice. Thus, tumor cells induce the generation of MDSCs that show a reduced expression of S100A8 and S100A9. [0057] To decipher the role of S100A8 in tumor immunosuppression, tumor-bearing mice were injected with anti-S100A8 antibodies. Injections of anti-S100A8 or anti- Si 00A9 slowed down tumor growth (Fig. 7). Anti-S100A8 and anti-S100A9 diminished the numbers of PMN-MDSCs, but not Mo-MDSCs, and both antibodies promoted their differentiation in the blood of tumor-bearing mice (Fig. 8). Anti-S100A8 also decreased the numbers of PMN-MDSCs and Mo-MDSCs in the spleen compared to untreated tumor-bearing mice (Fig. 9). In addition, there was an increase of CD8 cells in the spleen and the activity of MDSCs was reduced in anti-S100A8- and anti-S100A9- injected mice (Fig. 10). These results indicate that S100A8 and S100A9 repress the differentiation of MDSCs into mature myeloid cells.

[0058] Accordingly, it is provided that cancer cells modify the ratio of S100A8 to S100A9 secreted, favoring the expression of the anti-inflammatory DAMP S100A8 over the expression of the pro-inflammatory S100A9. As S100A8 inhibits the differentiation of myeloid cell progenitors and precursors in mature myeloid cells like neutrophils, monocytes and dendritic cells, this leads to the accumulation of immature myeloid cells. These cells act as MDSCs to suppress the immune response toward the cancer cells. Injection of anti-S100A8 restores normal maturation of myeloid cells, thereby inhibiting the immunosuppressive activity of MDSCs.

[0059] Accordingly it is disclosed humanized antibodies and antibodies from non- human species against S100A8 and/or S100A9, and particularly human S100A8 and/or S100A9, whose protein sequences can be modified to increase their similarity to antibody variants produced naturally in humans. Humanization can be necessary when the process of developing a specific antibody involves generation in a non-human immune system (such as that in mice). Antibody humanization methods are designed to produce a molecule with minimal immunogenicity when applied to humans, while retaining the specificity and affinity of the parental non-human antibody. The protein sequences of antibodies produced in this way are partially distinct from homologous antibodies occurring naturally in humans, and are therefore potentially immunogenic when administered to human patients.

[0060] Humanized antibodies encompassed herein can be produced via enrichment technologies such as phage display or immunization of transgenic mice bearing the antibody human gene repertoire have provided powerful means to generate human antibodies. [0061] Humanized antibodies and antibodies from non-human species whose protein sequences have been modified to increase their similarity to antibody variants produced naturally in humans. Humanization can be necessary when the process of developing a specific antibody involves generation in a non-human immune system (such as that in mice). Antibody humanization methods are designed to produce a molecule with minimal immunogenicity when applied to humans, while retaining the specificity and affinity of the parental non-human antibody. The protein sequences of antibodies produced in this way are partially distinct from homologous antibodies occurring naturally in humans, and are therefore potentially immunogenic when administered to human patients.

[0062] More particularly, the antibody described herein specifically binds to an epitope on S100A8 and/or S100A9.

[0063] In a particular embodiment, the antibody comprises an epitope binding fragment that is selected from: Fv and/or F(ab') and/or F(ab')2. In particular, the antibody comprises an epitope-binding single chain antibody.

[0064] Particularly, the antibody encompassed herein comprises a heavy chain variable region encoded by nucleotide sequence:

GGATCCCAGGTTCAGCTGCAGCAGTCAGGGGCAGAGCTTGTGAAGCCAGGGGCCTCAGT CAAGTTGTCCTGCACAGCTTCTGGCTTCAACATTAAAGACACCTATTTGCACTGGGTGA AGCAGAGGCCTGAGCAGGGCCTGGAGTGGGTTGGAAGGATTGATCCTGCGAATGGTGAT ACTAAATATGACCCGAAGTTCCAGGCCAAGGCCACTATAACAGCTGACACAACCTCCAA CACAGCCTACGTGCACCTCAACAGCCTGACATCTGAGGACACTGCCGTCTATTTCTGTA CTGGGGGATGGCAGATGGGGGGCCGGTACTTCGATGTCTGGGGCGCAGGGACAACGGTC ACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATGGTGGCGGTGGTTCT (SEQ ID NO: 1 ).

[0065] In a particular embodiment, the antibody comprises a heavy chain variable region consisting of:

GSQVQLQQSGAELVKPGASVKLSCTASGFNIKDTYLHWVKQRPEQGLEWVGRIDPANGD TKYDPKFQAKATITADTTSNTAYVHLNSLTSEDTAVYFCTGGWQMGGRYFDVWGAGTTV TVSSAKTTPPSVYGGGGS (SEQ ID NO: 2). [0066] Particularly, the antibody encompassed herein comprises a light chain variable region encoded by nucleotide sequence:

GATGTTGTGATGACCCAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCAC CATCTCATACAGGGCCAGCAAAAGTGTCAGTACATCTGGCTATAGTTATATGCACTGGA ACCAACAGAAACCAGGACAGCCACCCAGACTCCTCATCTATCTTGTATCCAACCTAGAA TCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACAT CCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCACATTAGGGAGCTTA CACGTTCGGAGGGGGGACCAAGCTGGAAATAA (SEQ ID NO: 3).

[0067] In a particular embodiment, the antibody comprises a light chain variable region consisting of:

DWMTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLE SGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELTRSEGGPSWK (SEQ ID NO: 4).

[0068] In a particular embodiment, the antibody comprises a chain selected from: SEQ ID NO: 5, 6, 7, 8, 9 or 10.

[0069] In another embodiment, the antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1 1 , 12, 13, 14, 15, 16 and 17.

[0070] The anti-S100 Ab described herein may be employed in admixture with a suitable physiological or pharmaceutical carrier. Such compositions comprise a therapeutically effective amount of the antibody, and a physiologically or a pharmaceutically acceptable carrier or excipient. Such a carrier includes but is not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The formulation should suit the mode of administration.

[0071] An antibody as defined herein, acting as inhibitor or antagonist of S100A8 and/or S100A9 protein, can be administered alone or in combination with other antibodies directed toward other complementary targets, including but not limited to, other S100 polynucleotides or polypeptides.

[0072] The antibodies encompassed herein may be advantageously utilized in combination with other monoclonal or chimeric antibodies, with cytokines, or with S100 proteins. The antibodies may be administered alone or in combination with other types of treatments. For example, the antibody described herein can be administered in combination, simultaneously or separately, with for example a chemotherapeutic agent, such as daunorubicin (Cerubidine), doxorubicin (Adriamycin), and cytarabine. Generally, administration of products of a species origin or species reactivity (in the case of antibodies) that is the same species as that of the patient is preferred. Thus, in a preferred embodiment, human antibodies, fragments, derivatives, or analogs are administered to a human or animal patient for therapy or prophylaxis.

[0073] Alternatively, antibodies encompassed herein may be advantageously utilized in combination with anti-PD1 and/or anti-PDL1.

[0074] Programmed Cell Death 1 (PD-1 ) is a cell surface signaling receptor that plays a critical role in the regulation of T cell activation and tolerance. Programmed Death Ligand-1 (PD-L1 ) is expressed on antigen-presenting cells as well as many human cancers and has been shown to downregulate T cell activation and cytokine secretion upon binding to PD-1.

[0075] It is preferred to use high affinity and/or potent in vivo inhibiting and/or neutralizing antibodies against S100 polypeptides or polynucleotides encompassed herein, fragments or regions thereof, for therapy of disorders related to S100 polynucleotides or polypeptides, including fragments thereof. Such antibodies, fragments, or regions, will preferably have an affinity for S100 polypeptide encompassed herein.

[0076] As provided herein, anti-S100A8 and/or anti-S100A9 are used for treating solid tumors in a subject, such as an animal or a human.

[0077] A solid tumor is essentially considered to be an abnormal mass of tissue that usually does not contain cysts or liquid areas. Solid tumors may be benign (not cancer), or malignant (cancer). Different types of solid tumors are named for the type of cells that form them. Examples of solid tumors are sarcomas, carcinomas, and lymphomas. Leukemias (cancers of the blood) generally do not form solid tumors.

[0078] Encompassed herein, the antibodies described herein are used to treat solid tumors which include sarcomas and carcinomas such as, but not limited to, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

[0079] In another embodiment, the present disclosure provides a combination comprising a therapeutically effective amount of an anti-S100A8 and/or anti-S100A9, as defined herein, and a therapeutically effective amount of at least one or more therapeutic agents useful in the method of the present disclosure.

[0080] It will be clear to a person of ordinary skill that the amounts and/or ratios of therapeutic agents will be readily adjusted. It will be understood that the scope of combinations described herein is not particularly limited, but includes in principle any therapeutic agent useful for preventing or treating the diseases described herein.

[0081] It will also be appreciated that the amounts and/or ratios of therapeutic agents for use in treatment will vary not only with the particular agent selected but also with the route of administration, the nature of the condition for which treatment is required and the age and condition of the patient and will be ultimately at the discretion of the attendant physician.

[0082] The compounds defined herein can be administered concurrently to the one or more agents used herein in the methods and combinations. The desired doses may conveniently be presented in a single dose or as divided dose administered at appropriate intervals, for example as two, three, four or more doses per day or continuously such as in a perfusion. The compound can be administered on a dosage regimen distinct to the one or more agents used herein in the methods and combinations. Alternatively, the compound can be administered sequentially or concurrently in distinct formulations or in a common formulation.

[0083] Pharmaceutical compositions may comprise pharmaceutically acceptable carrier(s) and/or excipient(s). Many pharmaceutically acceptable carrier(s) and/or excipient(s) are known in the art. It will be understood by those in the art that a pharmaceutically acceptable carrier must be compatible with the other ingredients of the formulation and tolerated by a subject in need thereof. The proportion of each carrier is determined by the solubility and chemical nature of the agent(s), the route of administration, and standard pharmaceutical practice.

[0084] In order to ensure consistency of administration, in an embodiment of the present disclosure, the pharmaceutical composition is in the form of a discrete dosage unit and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing into association the active compound with a liquid carrier or solid carrier or both and then, if necessary, shaping the product into the desired formulation.

[0085] The compounds and combinations according to the disclosure may be formulated for parenteral administration (e.g. by injection, for example bolus injection or continuous infusion) and may be presented in unit dose form in ampoules, pre-f illed syringes, small volume infusion or in multi-dose containers with an added preservative. The compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilisation from solution, for constitution with a suitable vehicle, e.g. sterile, pyrogen-free water, before use.

EXAMPLE I

[0086] All experiments were carried out in accordance with the Universite Laval Animal Protection Committee (Quebec City, Quebec, Canada). Female C57BL/6 mice were obtained from Jackson Laboratories and used at 6-12 weeks old.

[0087] Bone marrow (BM) cells were harvested from 5-fluorouracil-treated (150 mg/kg) C57BL/6 wild-type (VVT) and then stimulated as previously described (Kroon et al. , 1998, The EMBO Journal, 17: 3714-3725). Cells were co-cultured for 2 days with polybrene (6 g/ml, Sigma-Aldrich) on irradiated (150 Gy) virus producer GP+E86 cells expressing MSC\Z-hoxa9-IRES-meis1a-hpgk-EGFP (mpgk-Neo cassette from the vector, kindly provided by Dr. G Sauvageau, Montreal, was replaced with hpgk-EGFP) as previously described (Barabe et al. , 2007, Science, 316: 600-604) or S100A8KO recipient mice were intravenously injected with 4 x 10⁵ infected murine BM cells. For secondary transplantation, 2 x 10⁵ BM cells from primary leukemia were injected into sub-lethally irradiated (4.5 Gy) recipients via the tail vein. For antibody treatment, mice received 10 mg/kg of body weight pAb anti-S100A8 or control immunoglobulin G (IgG) intraperitoneally three times per week starting on day 4 post-transplantation until mice were moribund. For protein treatment, recipient mice were injected intraperitoneally three times per week with 20 μg/mouse recombinant murine S100A9 (rmS100A9), or phosphate buffer saline (PBS) starting on day 4 post-transplantation. Mice received rmS100A9 (20 μg/mouse) or pAb anti-S100A8 (10 mg/kg) intraperitoneally three times per week until mice were moribund.

[0088] Single cell suspensions of bone marrow, spleen, and blood cells were stained for 30 minutes on ice with various antibodies (all antibodies were obtained from BD Bioscience): CD1 1 b-PerCPCy5.5 (clone M 1/70), G -APC (RB6 8C5), and Ly6G- APC (1 A8) Intracellular staining of S100A8 and S100A9 on human or murine AML cells was performed with polyclonal rabbit anti-S100A8 or anti-S100A9 IgG for 30 minutes. Cells were then incubated with Alexa647 goat anti-lgG rabbit (Life Technologies). Acquisition was performed on FACSCanto (BD Bioscience) and results were analyzed using FlowJo software (TreeStar Inc, Ashland, OR).

[0089] Male and female C57BL/6 mice (10-12 weeks old) were euthanized by dislocation. Bone marrow cells were obtained from femurs and tibias and were cultured for 2 days. Cells were plated on 6-well plates containing RPM I 1640 medium, supplemented with 10% heat-inactivated FBS, 1X Non-Essential Amino Acid, 50μ Μ β- mercaptoethanol, 0.2% Primocin, and different concentrations of S100A8 and S100A9 and incubated for 48h at 37^°C. Cells were then harvested, stained and analysed by flow cytometry.

[0090] To establish subcutaneous tumors, 5 ^χ 10⁵ B16-F10 cells in 100 μΙ_ of PBS were injected in the right flank of mice. Tumors were monitored daily. Tumor size was estimated using the following formula: Tumor volume =4/3π X (Length/2) X (Width/2)². Mice received 10 mg/kg of body weight pAb anti-S100A8, mAb anti-S100A9 or control immunoglobulin G (IgG) intraperitoneally three times per week starting on day 24 post- injection of tumor cells.

[0091] Cells were stained with Live/Dead Fixable Blue Dead Cell Stain (Invitrogen, Paisley, UK). Fc receptors on cells were blocked by incubation with Fc block (1 g per 10^s cells, eBioscience, San Diego, CA, USA) for 15 min on ice followed by incubation with a combination of anti-CD103-eFluor450, anti-CD1 15-PE-eFluor610, anti-CD1 1 b- eVolve605, anti-CD1 1 c-PE-Cy7, anti-CD86-PE-Cy5, anti-F4/80-eFluor660, anti-Ly-6C- Alexa488, anti-MHCI I-Alexa700, anti-Ly-6G-PerCP-eFluor710, anti-CD45RB- eVolve605 (eBioscience, San Diego, CA), anti-CD45-BUV661 (BD biosciences, Mississauga, ON, CA) for 45 min on ice and then washed two times with PBS1x + 2% FBS + 1 mM EDTA. Cell fluorescence was then analysed on a BD LSR/LSRI I (BD Biosciences, Mississauga, ON, CA) and data was analysed using FlowJo 10 software (Tree Star Inc. ).

[0092] While the present disclosure has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations, and including such departures from the present disclosure as come within known or customary practice within the art and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A composition comprising an anti-S100A8, an anti-S100A9, or a combination thereof, and a carrier for treating a subject with a solid tumor.

2. The composition of claim 1 , wherein said anti-S100A8 or anti-S100A9 is an antibody.

3. The composition of claim 1 or 2, wherein said anti-S100A8 specifically binds to a portion of a S100A8 protein.

4. The composition of claim 1 or 2, wherein said anti-S100A9 specifically binds to a portion of a S100A9 protein.

5. The composition of any one of claims 1 -3, wherein said anti-S100A8 specifically binds to a S100A8/S100A8 homodimer or a S100A8/S100A9 heterodimer.

6. The composition of any one of claims 1 , 2 and 4, wherein said anti-S100A9 specifically binds to a S100A9/S100A9 homodimer or a S100A8/S100A9 heterodimer.

7. The composition of claim 3 or 5, wherein the S 00A8 protein is a human S100A8.

8. The composition of claim 4 or 6, wherein the S100A9 protein is a human S100A9.

9. The composition of any one of claims 1 -8, wherein said anti-S100A8 or anti-S100A9 is a monoclonal or a polyclonal antibody.

10. The composition of any one of claims 1 -9, wherein said anti-S100A8 or anti-S100A9 is a mouse antibody, a goat antibody, a human antibody or a rabbit antibody.

1 1. The composition of any one of claims 1 -10, wherein said anti-S100A8 or anti-S100A9 is a humanized antibody.

12. The composition of claim 2, wherein said anti-S100A8 or anti-S100A9 antibody comprises an epitope binding fragment selected from the group consisting of: Fv, F(ab'), or F(ab')2.

13. The composition of claim 2 or 12, wherein said anti-S100A8 antibody comprises a heavy chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 1.

RECTIFIED SHEET (RULE 91.1)

14. The composition of claim 2 or 12, wherein said anti-S100A8 antibody comprises a heavy chain variable region consisting of SEQ ID NO: 2.

15. The composition of any one of claims 2 and 12-14, wherein said anti-S100A8 antibody comprises a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 3.

16. The composition of any one of claims 2 and 12-14, wherein said anti-S100A8 antibody comprises a light chain variable region consisting of SEQ ID NO: 4.

17. The composition of claim 2, wherein said anti-S100A9 antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 1 , 12, 13, 14, 15, 16 and 17.

18. The composition of claim 2, wherein said anti-S100A9 antibody comprises a chain selected from: SEQ ID NO: 5, 6, 7, 8, 9 or 10.

19. The composition of any one of claims 1-18, wherein said anti-S100A8 or anti-S100A9 is formulated for an injection.

20. The composition of any one of claims 1-19, formulated for an administration with a chemotherapeutic agent.

21 . The composition of any one of claims 1 -20, formulated for an administration with an anti- PD1 , an anti-PDL1 , or a combination thereof.

22. The composition of any one of claims 1 -21 , wherein said composition diminishes the numbers of PMN-MDSCs in the subject.

23. The composition of any one of claims 1 -22, wherein said composition increases CD8 cells and reduces the activity of MDSCs in said subject.

24. The composition of any one of claims 1 -23, wherein said composition decreases the differentiation of MDSCs into mature myeloid cells in said subject.

25. The composition of any one of claims 1-24, wherein said solid tumor is a sarcoma, a carcinoma, or a lymphoma.

RECTIFIED SHEET (RULE 91.1)

26. The composition of any one of claims 1-24, wherein said solid tumor is a fibrosarcoma, a myxosarcoma, a liposarcoma, a chondrosarcoma, an osteogenic sarcoma, a chordoma, an angiosarcoma, an endotheliosarcoma, a lymphangiosarcoma, a lymphangioendotheliosarcoma, a synovioma, a mesothelioma, an Ewing's tumor, a leiomyosarcoma, a rhabdomyosarcoma, a colon carcinoma, a pancreatic cancer, a breast cancer, an ovarian cancer, a prostate cancer, a squamous cell carcinoma, a basal cell carcinoma, an adenocarcinoma, a sweat gland carcinoma, a sebaceous gland carcinoma, a papillary carcinoma, a papillary adenocarcinomas, a cystadenocarcinoma, a medullary carcinoma, a bronchogenic carcinoma, a renal cell carcinoma, an hepatoma, a bile duct carcinoma, a choriocarcinoma, a seminoma, an embryonal carcinoma, a Wilms' tumor, a cervical cancer, a testicular tumor, a lung carcinoma, a small cell lung carcinoma, a bladder carcinoma, an epithelial carcinoma, a glioma, an astrocytoma, a medulloblastoma, a craniopharyngioma, an ependymoma, a pinealoma, an hemangioblastoma, an acoustic neuroma, an oligodendroglioma, a meningioma, a melanoma, a neuroblastoma, or a retinoblastoma.

27. The composition of any one of claims 1 -26, wherein said subject is an animal or a human.

28. The composition of claim 27, wherein said animal is a mouse.

29. A method of treating a subject with a solid tumor comprising the step of administering the composition of any one of claims 1 -28.

30. Use of the composition of any one of claims 1-28 for treating a subject with a solid tumor.

31 . Use of the composition of any one of claims 1 -28 in the manufacture of a medicament for treating a subject with a solid tumor.

RECTIFIED SHEET (RULE 91.1)