WO2013122545A1 - Synergistic combinations for use in treating cancer - Google Patents

Abstract

The present invention relates to treating lymphoma or leukemia in a patient (e.g. a human patient) by the co-administration of: (i) at least one of: a TLR1 agonist, a TLR2 agonist, a TLR4 agonist, and a TLR9 agonist; and (ii) at least one chemotherapeutic agent which is a DNA damaging anticancer agent.

Description

SYNERGISTIC COMBINATIONS FOR USE IN TREATING CANCER

FIELD OF THE INVENTION

[001] The present invention relates to the fields of medicine and biochemistry. In particular, the present invention refers the identification of pharmaceutical compositions comprising a TLR agonist and a chemotherapeutic agent that are useful in treating cancer.

BACKGROUND OF THE INVENTION

[002] Toll like receptors (TLRs) play an essential role in immediate immune defence through the recognition of conserved motifs in pathogen-associated molecular patterns (PAMPs) and endogenous danger associated molecular patterns (DAMPs). Until now, 10 different types of TLRs have been discovered in various types of human cells such as macrophages, dendritic cells, neutrophils, epithelial cells, endothelial cells, NK cells, B and T lymphocytes. Despite the similarity of the shared Toll/IL-1 receptor (TIR) domain in their cytoplasmic region, each TLR recognizes unique molecules leading to the activation of the innate immune system to trigger antigen specific adaptive immune response.

[003] Immunomodulatory effects of TLR activation in cancer have been intensively studied. Natural TLR ligands as well as synthetic TLR agonists have caught attention as anti-infective and anti-tumor agents. TLR9 and TLR7 ligands such as CpG DNA and R-848 are being developed as adjuvant in cancer vaccination and immunomodulator for non-small cell lung cancer, melanoma and B cell leukemia. TLR mediated NK cells and T cells immune activation have been observed in immunosurveillance and immunoregulation in tumor and pathogen clearance. Specifically, systemic injection of a TLR1/2 agonist bacterial lipoprotein induces leukemia remission by reciprocal down-regulation of regulatory T cell (Treg) and upregulation of CTL function. Although stimulation of immune cells has been thought as the mechanism of anti-cancer effect of TLR ligands, expressions of TLRs in leukemia cells themselves and their involvement in leukemia cell proliferation have been reported.

[004] Non-Hodgkin lymphomas (NHLS) compose pf_a_diyerse range, of hematological malignancies. B cell non-Hodgkin lymphomas include Burkitt lymphoma, chronic lymphocytic leukemia and precursor B -lymphoblastic lymphoma. Although chemotherapeutic drugs exert therapeutic effects, long term tumor-free survival rate is still low in B cell NHLS patients. This is due to increased drug resistance, which is correlated with multiple relapses.

[005] Current cancer therapies such as surgical therapy, radiotherapy, chemotherapy, and immunotherapy have either been of limited success or have been accompanied by serious side effects with high relapse rates. To prevent remission after the surgical removal of cancer and to increase the efficacy of chemo/radiotherapy, immunotherapy has been used to activate host immune system against cancer cells. However, the low immunogenicity of cancer cells has been an obstacle for development of efficacious anti-cancer immunotherapy.

[006] Many times after surgery and after some delay period, the original tumor is observed to have metastasized so that secondary sites of cancer invasion have spread throughout the body and the patient subsequently dies of the secondary cancer growth. Although chemotherapy is widely used in the treatment of cancer, it is a systemic treatment based usually on the prevention of cell proliferation. Accordingly, chemotherapy is a non-specific treatment modality affecting all proliferating cells, including normal cells, leading to undesirable and often serious side effects.

[007] Thus, a need exists for new compositions for treating cancer, particularly cancer with rapidly proliferating cells.

SUMMARY OF THE INVENTION

[008] In a first aspect, there is provided an immunogenic composition comprising at least one Toll-like receptor (TLR) agonist and a least one chemotherapeutic agent.

[009] In a second aspect, there is provided a method of treating leukemia and lymphoma, the method comprising administering in a patient in need thereof (i)at least one TLR agonist and (ii) at least one chemotherapeutic agent.

[0010] In a third aspect, there is provided the use of (i) at least one TLR agonist, selected from the group consisting of: a TLR1 agonist, a TLR2 agonist, a TLR4 agonist, and a TLR9 agonist and (ii) at least one chemotherapeutic agent which is a DNA damaging ^ anticancer agent _mjhe_mjuiufac^

use in treating leukemia and lymphoma. [0011] In a fourth aspect, there is provided a method for determining susceptibility of a patient suffering from leukemia and/or lymphoma to a treatment with a composition as defined herein, wherein the method comprises: (i) administering at least one chemotherapeutic agent to said patient, (ii) comparing a first level of TLR selected from the group consisting of TLR mRNA level in a tumor sample, TLR cDNA level made from mRNA from said tumor sample, and TLR protein level from said tumor sample with the level of TLR selected from the group consisting of TLR mRNA level from a non-tumor sample from said patient, TLR cDNA level made from mRNA from said non-tumor sample, and TLR protein level from said non-tumor sample; and wherein a patient characterized by an increased level of TLR in said tumor sample is susceptible to a treatment with the at least one TLR agonist in said pharmaceutical composition.

BRIEF DESCRIPTION OF DRAWINGS

[0012] Those of skill in the art will understand that the drawings, described below, are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way.

[0013] Figure 1 A is a series of histogram plots showing expression of ligands for activating immunoreceptors b flow cytometry at the surface of B Leukemia cell line (ΕμΜΙ) after exposure in vitro to a TLR agonist and a chemotherapeutic agent, either alone or in combination. A composition comprising

Pam3CSK4, a TLR1/2 agonist and Ara-C, an antimetabolite increases immune stimulatory molecule expressions in vitro, thus increase the susceptibility of these cells to immunotherapy. EuMl cells subjected to indicated treatments were assessed for surface expression levels of MHC Class I (H-2K^b), CD40, CD69, CD80, CD86 and ICAM-1 by flow cytometry (black line), and were compared to their respective isotype control stainings (filled histograms).

[0014] Figure 1 B is series of bar graph showing expression of cytokines by ELISA in leukemia cells after in vitro exposure to a TLR agonist and a chemotherapeutic agent. A composition comprising Pam3CSK4 and Ara-C increases expression of cytokines thus increases the vulnerability of these cells to NK cells and activates an immune response^ ΕμΜ 1 cejls_^we e freated^

Ara-C, 1 μ^πιΐ of Pam₃CSK₄ or 1 μg/ml Pam₃CSK4 followed by 1 μΜ Ara-C for another 16 h. Supernatants were collected and analyzed for amounts of IL-6, TNF-a, RANTES and ΜΙΡ-Ια by ELISA. The data represent means ± SE of three independent experiments.

[0015] Figure 1 C is a series of bar graph showing expression of cytokines mRNA involved in cellular immune and antibody responses by real-time PCR (q-PCR) in B Leukemia cell line (ΕμΜΙ) after exposure in vitro to a TLR agonist and a chemotherapeutic agent, either alone or in combination. ΕμΜΙ cells subjected to indicated treatments were assessed for expression levels of IL-Ιβ, ΓΕΝ-α, TGFP3 and IL-4 by analysis. Data represent means ± s.d., n = 3 independent experiments.

[0016] Figure 1 D is a pair of histogram plots showing titration of the amount of chemotherapeutic drug necessary to activate NK cells by flow cytometry after pretreatment of B Leukemia cell line (ΕμΜΙ) exposed in vitro to a TLR agonist. A composition comprising Pam3CSK4, and two different doses of Ara-C (ΙμΜ and 10μΜ), were simultaneously used to treat EuMl cells. ΕμΜΙ cells were treated with Pam3CSK3 followed by either 1 or 10 μΜ Ara-C for 16 hours. Cells were stained for Rae-1 expression by flow cytometry after the following treatment: DMSO (dotted line;— ); Ara-C (full line; ); Pam3CSK4 followed by Ara-C (plus line;++-H-i-).

[0017] Figure 1 E is a series of histogram plots showing expression of ligands for activating immunoreceptors by flow cytometry at the surface of B Leukemia cell line (ΕμΜΙ) after exposure in vitro to a TLR agonist and a chemotherapeutic agent, either alone or in combination. ΕμΜΙ cells subjected to indicated treatments were assessed for surface expression levels of TLR 1, TLR2, DNAM-1, NGK2D and LFA-1 by flow cytometry (black line), and were compared to their respective isotype control stainings (filled histograms).

[0018] Figure 2 A is a dot plot showing tumor regression effect in blood by flow cytometry into recipient C57BL/6 intravenously injected with EuMl cells previously exposed to various compositions. The tumor burden is the smallest in mice that were injected with ΕμΜΙ cells pre-treated with Pam3CSK4 (Pam3) and Ara-C. 5 x 10⁵ DMSO, Ara-C, Pam₃CSK- or Pam₃CSK_-4 + Ara-C-treated ΕμΜΙ cells were injected intravenously into recipient C57BL/6 mice and analyzed for tumor burden in the blood by flow cytometry on day 11 post injection. *** p < 0.001. Sjafetical c^

[0019] Figure 2 B is a bar graph showing tumor regression effect in the spleen by flow cytometry into recipient C57BL/6 intravenously injected with EuMl cells previously exposed to various compositions. The tumor burden is the smallest in mice that were injected with ΕμΜΙ cells pre-treated with Pam3CSK4 (Pam3) and Ara-C. 5 x 10⁵ DMSO, Ara-C, Pam₃CSK4 or Pam₃CSK₄ + Ara-C-treated ΕμΜΙ cells were injected intravenously into recipient C57BL/6 mice and analyzed for tumor burden in the spleen by flow cytometry on day 1 1 post injection. *** p < 0.001. Statistical comparisons were conducted using the Student's t-test.

[0020] Figure 2 C is a Kaplan-Meier survival curve of C57BL/6 mice injected with Pam₃CSK₄ and/or Ara-C-treated EuMl cells showing the anti-tumor effect of Ara-C is enhanced by Pam₃CSK₄. Statistical comparisons were conducted using the log-rank test, p < 0.05; Ara-C vs Ara-C + PanvjCS *.

[0021] Figure 2 D is a bar graph showing apoptosis analysis by flow cytometry in EuMl cells exposed to the compositions having the indicated compounds. The regression of the tumor burden observed in mice injected with Pam3CSK4 and Ara-C treated ΕμΜΙ cells is not attributable to an increase in apoptosis prior to injection. Apoptotic cell death of ΕμΜΙ cells treated with DMSO, Pam3CSK4 (Pam3), Ara-C or Pam3CSK4 combined Ara-C (Pam3+Ara-C) was measured by flow cytometry analysis of annexin V and PI stained cells. *: p<0.05.

[0022] Figure 3 A and B are a pair of bar graphs showing the tumor burden in the blood (A) and spleen (B) of mice intravenously injected Pam3CSK4 and Ara-C-treated Thyl.l⁺ EuMl cells one day after receiving control antibodies, depleting anti-NKl.l (PK136, 500 μg/mouse) and/or anti-CD4 (GK1.5, 500 μg/mouse) or CD8 (2.43, 250 μg/mo se) antibodies. The effect of NK cells depletion, either alone (A) or in combination with the depletion of either CD4+ cells or CD8+ cells on tumor burden was assessed by detecting Thy 1.1+ by flow cytometry. This result show that NK cells and CD4+ cells play a role in the suppression of tumorigenesis in B leukemia cells exposed to Pam3CSK4 and Ara-C. 11 days after the administration of tumor cells, the percentage of Thy 1.1 ⁺ cells in the blood and spleen was determined by flow cytometry. ** p < 0.01, *** p < 0.001..

[0023] Figure 3 C is a dot plot demonstrating the depletion of NK cells by flow cytometry in mice injected with a PK136 antibody in vivo. The efficiency of NK cells depletion in mice intravenously injected with a NK cells-

specific CD3 antibodies. [0024] Figure 4 A is a scatter graph that shows the effect of different treatments on common lymphoid progenitor (CLP) cell populations by flow cytometry. Since leukemia is characterized as a bone marrow disorder, the effect of different treatments on CLP in lymph node of ΕμΜΙ cell xenograft mouse model. 5 x 10⁵ DMSO, Ara-C, Pam₃CSK or Pam₃CSK₄ + Ara-C treated Thyl.l⁺ EuMl cells were intravenously injected into the recipient wild-type C57BL/6 mice. Eleven days after injection, mice were analysed for the number of CLPs (Lin^" IL-7Ra⁺Sca-l^loc- kit'°AA4⁺Flt3^hi) in the bone marrow. This result shows that the treatment with the Pam3 and Ara-C is less toxic to CLP than other treatments. The combined treatment ameliorated the reduction of CLP by more than 2 fold compared to the other single treatments **: pO.01.

[0025] Figure 4 B is a pair of bar graphs showing by flow cytometry the cell populations of cells which are crucial for anti-cancer immunity in the spleen of mice intravenously injected with ΕμΜΙ cells exposed to various compounds as indicated on the X-scale. Fig. 4 A shows that in the recipient wild-type C57BL/6 mice intravenously injected with Thyl.l⁺ EuMl cells treated with DMSO, Ara-C or Pam3CSK4 (Pam3), the percentage of the CD4+ and CD8+T cell population is depleted. Surprisingly when mice were injected with Thyl.l⁺ ΕμΜΙ cells treated with Pam3CSK4 and Ara-C, the decrease in the CD4+ and CD8+T cell population was not as pronounced. The level of K cells did not significantly vary among the mice treated with the various compositions. DMSO, Ara-C, Pam3CSK4 (Pam3) or a combination of Pam3CSK4 and Ara-C (Pam3+Ara-C) treated Thyl.l⁺ ΕμΜΙ cells injected mice were euthanized at 11 days post injection and then CD4+T cells, CD8+T cells and NK cell populations were compared with PBS injected mouse spleen cells. ** p<0.01.

[0026] Figure 4 C is a series of dot plot demonstrating the effect of different treatments on common lymphoid progenitor (CLP) cell populations by flow cytometry. Since leukemia is characterized as a bone marrow disorder, the effect of different treatments on CLP in lymph node of ΕμΜΙ cell xenograft mouse model. 5 x 10⁵ DMSO, Ara-C, Pam₃CSK4 or Pam₃CSK4 + Ara-C treated Thyl.l⁺ ΕμΜΙ cells were intravenously injected into the recipient wild-type C57BL/6 mice. Eleven days after injection, mice were analysed for the number of CLPs jLjn^" IL-7Ra⁺Sca-l'°c- kit^l0AA4⁺Flt3^hi) in the bone marrow. This result shows that the treatment with the Pam3 and Ara-C is less toxic to CLP than other treatments. The combined treatment ameliorated the reduction of CLP by more than 2-fold compared to the other single treatments **: p<0.01.

[0027] Figure 5 A, B and C are a series of immunoblot exposure and bar graph analysis of EuMl cells treated with combination of Pam₃CS and Ara-C or DMSO and probed with antibodies against proteins involved in the DNA damage response and against NF-κΒ markers (A). The levels were quantified and normalized to DMSO-treated cells and plotted on a bar graph. (B). One out of three representative experiments is shown. (C) Time-course immunoblot analysis of ΙκΒ-α level at indicated time points after treatment with combination of Pam₃CSK₄ and Ara-C or DMSO. These figures show that Pam₃CSK₄ modulates Ara-C induced p53 and NF-KB activation.

[0028] Figure 6 A and B are a series of histogram plots showing expression of ligands for activating immunoreceptors by flow cytometry at the surface of ΕμΜΙ cells transduced with ΙκΒα super repressor (ΙκΒα-SR) mutant (A) or p53- specific shRNA (B) (thick line) or control plasmids (thin line) and subsequently exposed to the indicated compounds. (A and B) ΕμΜΙ cells transduced with ΙκΒα super repressor (ΙκΒα-SR) mutant (A) or p53-specific shRNA (B) (thick line) or control plasmids (thin line), were either treated with vehicle (DMSO) or Ara-C for 16 h or Pam₃CSK4 for 24 h or were pre-treated with 1 μg/ml Pam₃CSK₄, for 24 h followed by 1 μΜ Ara-C for 16h. Cell surface expression levels of MHC Class I (H- 2 ^b), CD69, CD80, CD86 and ICAM-1 were assessed by flow cytometry. Filled histograms show isotype control staining of control vector-transduced cells. Dotted lines indicate isotype control staining of ΙκΒα-SR or p53-specific shRNA transduced cells. These figures demonstrate that Pan^CSICi effect on Ara-C-induced upregulation of immunomodulatory molecules depends on NF-κΒ, but not p53.

DETAILED DESCRIPTION OF THE INVENTION

[0029] Leukemias are cancers of the white blood cells (WBCs) involving bone marrow, circulating WBCs and organs such as the spleen and lymph nodes. Acute leukemias consist of predominantly immature, poorly differentiated cells usually in the "blast" form. Chronic leukemias have more mature cells. They are described as lymphocytic (CLL) or myelocytic (CML). [0030] Lymphomas are a heterogeneous group of neoplasms arising in the reticuloendothelial and lymphatic systems. The major types are Hodgkin lymphoma and non-Hodgkin lymphoma (NHL). Lymphomas were once thought to be absolutely distinct from leukemias. However, better understanding of cell markers now show that the differentiation between the two cancers is often vague. The notion that lymphoma is relatively restricted to the lymphatic system and leukemias to the bone marrow, at least in early stages, is not always true. Most NHLs arise from B cells with the remainder arising from T cells or natural killer cells. In all cases, either precursor or mature cells may be involved.

[0031] Cytotoxic drugs work by damaging DNA or microtubules and are believed to gain much or all of their specificity in the human body from their ability to kill rapidly proliferating cells. However, the ability of cytotoxic drugs to kill rapidly proliferating cells is also the cause of their serious side effects. Proliferating tissues, such as bone marrow, gut and hair follicles are also affected during treatment of cancer with rapidly proliferating cells. Hence, there is a need to find treatment that would exploit the ability of chemotherapeutic drugs to kill rapidly proliferating cancer cells without affecting the cells of non-cancerous proliferating tissues.

[0032] In particular, elucidation of the factors that facilitate the activation of the host immune system against cancer cells would allow to enhance a tumor-specific immune response and to increase the therapeutic effect of the chemotherapeutic agents.

[0033] Traditionally, leukemia and lymphoma treatments include chemotherapy, radiotherapy, stem cells or bone marrow transplantation to alleviate the side effects of chemotherapy and immunotherapy or biological therapy. As with other types of cancer therapy, side effects of immunotherapy vary from person to person and will depend on the type of treatment, how it is administered, and what other therapies are given at the same time. Thus, it would be very beneficial to elucidate which combination of chemotherapy and immunotherapy is best adapted to patients according to their response to given treatments.

[0034] Methods and therapies for the modulation of the innate immune system response through the selection of specific factors will be useful in the

[0035] The goal of treatment of leukemias is complete remission, including resolution of abnormal clinical features, restoration of normal blood counts and normal hematopoiesis with less than 5% of blast cells and elimination of the leukemic clone. The complex nature of a patient's clinical situation necessitates the development of specific treatment protocols and drug regimens. Prognostic factors help determine treatment protocol and intensity.

[0036] Chemotherapeutic drugs such as cytotoxic drugs are often used in the treatment of leukemias, however they have serious side effects and high relapse rates in leukemias and lymphomas.

[0037] To prevent remission after the surgical removal of cancer and to increase the efficacy of chemotherapy and/or radiotherapy, immunotherapy has been used to activate host immune system against cancer cells. Some types of immunotherapy used to treat leukemia and lymphoma may include but are not limited to, interferons and other cytokines, monoclonal Antibodies, cancer vaccines, donor lymphocyte infusion and reduced-intensity allogeneic stem cell transplant. However, the low immunogenicity of cancer cells has been an obstacle for development of efficacious anti-cancer immunotherapy.

[0038] The present invention relates to the discovery that chemotherapeutic agents can be used to identify upregulatipn of a wide range of . factors involved in innate immune system. Of particular interest is the finding that expression of ligands of Toll-like receptors (i.e. TLR agonists) is upregulated in cancer cells treated with cytotoxic drugs. The present disclosure also relates to the discovery that TLR agonists can be used to induce expression of molecules that sense DNA damage such as ATM and Chk2.

[0039] Advantageously, chemotherapeutic agents render cells more immunogenic by inducing the expression of ligands for activating immune receptors. For example, DNA damage induced NKG2D ligand expression sensitizes ovarian cancer cell to natural killer (NK) cells mediated tumor cell lysis. NK cells are essential granular lymphocytes of the innate immune system which play a pivotal role in antitumor activity through the recognition of ligands expressed on abnormal cells.

[0040] DNA damage induces cell cycle arrest and activation of the DNA repair system in normal cells, whereas in cancer ceils it induces genetic instability because of defects in the various cell-cycle checkpoint pathway mediators. Mediators such as ATM^ATR and_Chkl,_which are ^ r^quired_f r_ NKG2D. ligand_ expression, are absent or poorly expressed in normal cells but highly expressed in stressed, virus-infected and cancer cells. NK cell activation is regulated by a balance between activating receptors such as NKG2D, NKp46 and DNAM1 and inhibitory receptors KIR, NKR-Pl and CD94/NKG2 (A/B). The critical role of DNAM-1, NKG2D and natural cytotoxicity receptors (NCRs) in NK cell mediated control of tumor metastases has been shown in several human cancers.

[0041] The innate immune system utilizes a variety of transmembrane or secreted pattern-recognition receptors (PRRs), which are vital for activation of complement and coagulation cascades, opsonisation, phagocytosis, apoptosis and induction of proinflammatory mediators. Toll-like receptors (TLRs) are important members of PRRs. Toll-like receptors (TLRs) play an essential role in immediate immune defenses through the recognition of conserved motifs in pathogen-associated molecular patterns (PAMPs) and endogenous danger associated molecular patterns (DAMPs). The naturally powerful immunostimulatory property of TLR agonists can be exploited for active immunotherapy against cancer.

[0042] The subsequent identification of specific factors involved in innate immune system, combined with bioinformatic analysis, enables the creation of a detailed molecular signature of potential candidate to increase the therapeutic effect of chemotherapeutic agent and to decrease side effects of said agent by allowing the use of lower dosage. This signature can guide the development of combination therapies that are optimally effective for a specific patient suffering from cancer. Such a molecular map has certain advantages over the more common genetic signature approach because most anti-cancer agents are small molecules that target proteins and not genes, and many small molecules targeting specific molecular alterations are currently in pharmaceutical development.

[0043] , Accordingly there is provided an immunogenic composition comprising at least one TLR (Toll-like receptor) agonist and at least one chemotherapeutic agent.

[0044] The term "immunogenicity" (and grammatical variants thereof) as defined herein is any compound or substance that has the ability to provoke an immune response in the body of a human or an animal.

[0045] The disclosure provides at least one TLR agonist, which may include but is not limited to a TLR1 agonist, a TLR2 agonist, a TLR4 agonist, and a TLR9 agonist.

[0046] A "TLR agonist" as defined herein is any molecule, which activates a toll-like receptor ("TLR"). As such, a TLR agonist includes a reference to any compound, ligand or substance that promotes the activation of a TLR, e.g., to induce a signalling event mediated by a TLR signal transduction pathway. The at least one TLR agonist may include but is not limited a TLRl agonist, a TLR2 agonist and a TLR4 agonist. Alternatively, the at least one TLR agonist include but is not limited to a TLRl agonist and a TLR2 agonist.

[0047] The choice of a specific TLR agonist may be determined by assessing the expression level of the Toll-like receptors (TLR) in the blood or spleen of the patient. The level of expression may be determined by comparing the level of TLR which may include but is not limited to TLR mRNA level in a tumor sample, TLR cDNA level made from mRNA from said tumor sample, and TLR protein level from said tumor sample with the level of TLR selected from the group consisting of TLR mRNA level from a non-tumor sample from said patient, TLR cDNA level made from mRNA from said non-tumor sample, and TLR protein level from said non-tumor sample.

[0048] The term "treatment" (and grammatical variants thereof) as used herein is intended to be construed broadly and includes a reference to any and all uses which remedy a disease state or symptoms, prevent the establishment of disease, or otherwise prevent, hinder, retard, or reverse the progression of disease or other undesirable symptoms in any way whatsoever. Accordingly, the term "treatment" can include any of the following (i) the prevention or inhibition of the leukemia or lymphoma, or recurrence thereof, (ii) the reduction or elimination of symptoms or leukemic or lymphoma cells, (iii) the substantial or complete elimination of the leukemia or lymphoma, and (iv) the stabilization (i. e. not worsening) of the leukemia or lymphoma.

[0049] As used herein, the singular form "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "an agent" includes a plurality of agents, including mixtures thereof and reference to "the nucleic acid sequence" generally includes reference to one or more nucleic acid sequences and equivalents thereof known to those skilled in the art, and so forth.

[0050] As used herein, the term "comprising" means "including".

Thus, for example, a composition "comprising" X may consist exclusively of X or may include one or more additional components.

[0051] As used herein, the term "chemotherapy" (and grammatical variants thereof) is the treatment of diseases including cancer with chemicals. In particular, it can be the treatment of cancer with one or more cytotoxic antineoplastic drugs ("chemotherapeutic agents") as part of a standardized regimen. Chemotherapy may be given with a curative intent or it may aim to prolong life or to palliate symptoms.

[0052] The TLR may be from any species but in at least some examples (e.g. where the patient is a human patient), the TLR is of human origin and, as such, the TLR agonist is an agonist of a human TLR. TLR agonists may be naturally expressed ligands of TLR or synthetic TLR agonist. TLR agonists can include but are not limited to Pam(3)CysSK(4) ((S)-[2,3-5is(palmitoyloxy)-(2-RS)- propyl]-N-palmitoyl-(R)-Cys-(S)-Ser-(S)-Lys₄-OH or Pam₃-Cys-Ser-(Lys)₄), Pam3Cys (S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-N-palmitoyl-(R)-cysteine or tripalmitoyl-S-glyceryl cysteine), Cadi-05, ODN 1585, lipoteichoic acids, zymosan, synthetic triacylated and diacylated lipopeptides, MALP-2, tripalmitoylated lipopeptides, a compound having a 2-aminopyridine fused to a five membered nitrogen-containing heterocyclic ring, lipopolysaccharide (LPS), Polyriboinosinic- polyribocytidylic acid (poly:IC), a CpG oligodeoxynucleotides (ODNs), monophosphoryl lipid A ("MPL"), an imidazoquinoline compound (e.g. an amide substituted imidazoquinoline amine), a beiizimidazolei derivative, a C8-substituted guanine ribonucleotide, an N7, C8-substituted guanine ribonucleotide, bacteria heat shock protein-60 (Hsp60), peptidoglycans, fiagellins, mannuronic acid polymers, flavolipins, teichuronic acids, ssRNA. (single stranded RNA), dsRNA (double stranded RNA), or a combination of the foregoing. Derivatives or analogues of the aforementioned are also included within the scope of the invention as are TLR agonists attached directly or indirectly to a second compound e.g. a carrier, microparticle or nanoparticle etc. Derivatives of Pam(3)CysSK(4) include, for example, diastereomers of Pam(3)CS (4).

[0053] Where the tumor cells of patient express a specific TLR, the corresponding TLR agonist may be selected. For example, where the at least one TLR agonist comprises a TLR 1 agonist, the leukemia or lymphoma expresses TLR1. Additionally, where the at least one TLR agonist comprises a TLR2 agonist, the leukemia or lymphoma expresses TLR2. Also, where the at least one TLR agonist comprises a TLR1 agonist aj^ a TL^

moieties), the leukemia or lymphoma expresses TLR1 and TLR2. In some examples, where the at least one TLR agonist comprises a TLR4 agonist, the leukemia or lymphoma expresses TLR4 or where the at least one TLR agonist comprises a TLR9 agonist, the leukemia or lymphoma expresses TLR9.

[0054] Chemotherapeutic agents, such as cytotoxic agents can include drugs traditionally used in the treatment of lymphoma and/or leukemia. Accordingly, the chemotherapeutic agent can include but is not limited to arabinosyl cytosine (ara- C or cytarabine), alkylating agents such as nitrosoureas and mechlorethamine, antimetabolites such as purine antimetabolites, hydroxyurea, capecitabine and 5- fluorouracil (5-FU), 6-mercaptopurine, 6-thioguanine, plant alkaloids such as vinblastine, vincristine and paclitaxel, plant extracts, radioisotopes, steroid hormones, S-l (Tegafur, 5-chloro-2,4-dihydroxypyridine and oxonic acid), 5-ethynyluracil, 5- azacytidine (5-AC), 2',2'-difluoro-2'-deoxycytidine (dFdC), gemcitabine, bortezomib, pentostatin, allopurinol, 2-flupro-arabinosyl-adenine (2F-ara-A), sulfur mustard (bischloroetyhylsulfide), melphalan, melpharan, chlorambucil, cyclophosphamide, ifosfamide, thiotepa, AZQ, mitomycin C, dianhydrogalactitol, dibromdducitol, alkyl sulfonate (busulfan), procarbazine, decarbazine, rebeccamycin, anthracyclins, non- intercalating topoisom erase inhibitors such as irinotecan (CPT-11) and topotecan, compounds that form adducts with nucleic acid including platinum derivatives, camptothecin and SN-38. Derivatives or analogues of the aforementioned are also included within the scope of the invention (e.g. in the case of Ara-C, N4-behenoyl Ara-C and the purine analogues fludarabine and cladribine might be considered instead of the pyrimidine analogue Ara-C) as are DNA damaging agents attached directly or indirectly to a second compound e.g. a carrier, microparticle or nanoparticle etc. Histone deacetylase inhibitors may also be employed in combination with the at least one TLR agonist, as well other moieties which may activate the DNA damage response.

[0055] Chemotherapeutic drugs can be selected according to their ability when tested in vitro and/or in vivo to upregulate factors involved in the innate immune system. Advantageously, chemotherapeutics drugs that induce expression of TLR agonists can be selected. The specific compositions may be selected according to the expression patterns of innate immune system factors seen in the patients. For example, if a specific chemotherapeutic drug used in the drug regimen induces expression of a TLR1 agonist, then it would be advantage usjo usje_a_TJJll agoms in the composition. In some specific compositions, the chemotherapeutic drug is a DNA damaging agent anti-cancer agent. [0056] Compositions may include Ara-C and Pam3CSK4, Ara-C and

Pam3Cys, Ara-C and Cadi-05, Ara-C and ODN 1585, Ara-C and lipoteichoic acids, Ara-C and zymosan, Ara-C and LPS (TLR4 agonist), Ara-C and R848 (TLR7/8), Ara-C and CpG (TLR9). The pharmaceutical compositions described herein for use in therapy. For example, the pharmaceutical composition may be for use in cancer, particularly for use in treating leukemia or lymphoma in a patient in need thereof.

[0057] Surprisingly, the amount of chemotherapeutic drug in the present composition may be less than the amount of chemotherapeutic drug when used alone. The amount of chemotherapeutic agent in the composition can be from about 0.1 mg to about 500 mg of the composition. In some variations, the amount of chemotherapeutic agent can be from about 1 mg to about 300 mg of the composition. In some variations, the amount of chemotherapeutic agent can be from about 10 mg to about 200 mg of the composition. In some variations, the amount of chemotherapeutic agent can be about 25 mg of the composition. In other variations, the amount of chemotherapeutic agent can be about 75 mg of the composition. In still other variations, the amount of chemotherapeutic agent can be about 150 mg of the composition. In further variations, the amount of chemotherapeutic agent can be from about 0.1 mg to about 30 mg of the composition. In some variations, the amount of chemotherapeutic agent can be from about 0.5 mg to about 20 mg of the composition. In some variations, the amount of chemotherapeutic agent can be from about 1 mg to about 15 mg of the composition. In some variations, the amount of chemotherapeutic agent can be from about 1 mg to about 10 mg of the composition. In some variations, the amount of chemotherapeutic agent can be from about 1 mg to about 5 mg of the composition.

[0058] Surprisingly, the amount of TLR agonist in the present composition may be less than the amount of TLR agonist when used alone. The amount of TLR agonist can be from about 0.1 mg to about 500 mg of the composition. In some variations, the amount of TLR agonist can be from about 1 mg to about 300 mg of the composition. In some variations, the amount of TLR agonist can be from about 10 mg to about 200 mg of the composition. In some variations, the amount of TLR agonist can be about 25 mg of the composition. In other variations, the amount of

variations, the amount of TLR agonist can be about 150 mg of the composition. In further variations, the amount of TLR agonist can be from about 0.1 mg to about 30 mg of the composition. In some variations, the amount of TLR agonist can be from about 0.5 mg to about 20 mg of the composition. In some variations, the amount of TLR agonist can be from about 1 mg to about 15 mg of the composition. In some variations, the amount of TLR agonist can be from about 1 mg to about 10 mg of the composition. In some variations, the amount of TLR agonist can be from about 1 mg to about 5 mg of the composition. It was found that the dose of chemotherapeutic agent can be lowered by 10 fold in case it is used together with a TLR agonist as defined herein.

[0059] Components (i) and (ii) of the immunogenic composition may be present in a single pharmaceutical composition (i.e. as a mixture of the two components) or may be present as separate pharmaceutical compositions.

[0060] The pharmaceutical composition(s) may comprise in addition to the at least one TLR agonist and/or the at least one chemotherapeutic agent one or more pharmaceutical excipients and/or additives. Optionally, the combined pharmaceutical preparation can be a kit of parts comprising components (i), (ii) and instructions for use in a method of the present invention. Optionally, the combined pharmaceutical preparation may comprise one or more further substances such as one or more further active agents (e.g. one or more further anticancer agents).

[0061] The pharmaceutically effective amount of the at least one TLR agonist in the composition to be used for treatment of lymphoma and/or leukemia can be a daily dose is 0.01 - 25 mg of the at least one TLR agonist per kg of body weight. In some variations, the daily dose is 0.05 - 20 mg of the at least one TLR agonist per kg of body weight. In some variations, the daily dose is 0.1 - 10 mg of the at least one TLR agonist per kg of body weight, or 1 - 10 mg of the at least one TLR agonist per kg of body weight. In some variations, the daily dose is 0.1 - 5 mg of the at least one TLR agonist per kg of body weight. In some variations, the daily dose is 0.1 - 2.5 mg of the at least one TLR agonist per kg of body weight.

[0062] The pharmaceutically effective amount of the at least one chemotherapeutic agent in the composition to be used for treatment of lymphoma and/or leukemia can be a daily dose is 100 - 10000 mg of the at least one chemotherapeutic agent per m² of body surface area. In some variations, the daily dose is 200 - 7500 mg of the at _Jeast one_ch^m therapeutic_agent per_mLof body- surface area. In some variations, the daily dose is 400 - 5000 mg of the at least one chemotherapeutic agent per m² of body surface area, or 500 - 5000 mg of the at least 2

one chemotherapeutic agent per m of body surface area. In some variations, the daily dose is 750- 4000 mg of the at least one chemotherapeutic agent per m of body surface area. In some variations, the daily dose is 1000 - 3000 mg of the at least one chemotherapeutic agent per m² of body surface area.

[0063] Determination of the expression level of TLR agonist after treatment of the cells with a chemotherapeutic drug may help select the most appropriate TLR agonist. Hence, the disclosure also relates to the use of (i) at least one TLR agonist which can include but is not limited to a TLR1 agonist, a TLR2 agonist, a TLR4 agonist, and a TLR9 agonist; and (ii) at least one chemotherapeutic agent in the manufacture of a medicament for use in treating leukemia or lymphoma.

[0064] Accordingly a method of treating leukemia or lymphoma can be useful. The method may include administering to the patient at least one TLR agonist as described above and at least one chemotherapeutic agent.

[0065] Accordingly, the invention relates to the method and use as described above where different modes of administration of the components of the pharmaceutical composition described herein are envisaged. Surprisingly, it has been found that the the at least one TLR agonist as described herein for use in the treatment of leukemia or lymphoma in a patient, wherein the treatment can include administering the patient at least one chemotherapeutic agent with the at least one TLR agonist is advantageous. The disclosure also relates to the use of at least one TLR agonist as described herein in the manufacture of a medicament for use in treating leukemia or lymphoma in a patient, wherein the treating comprises administering the patient at least one chemotherapeutic agent with the at least one TLR agonist.

[0066] Also disclosed herein is a chemotherapeutic agent for use in treating leukemia or lymphoma in a patient, wherein the treating comprises coadministering the patient at least one TLR agonist with the at least one a chemotherapeutic agent. The disclosure also relates to the use of at least one chemotherapeutic agent in the manufacture of a medicament for use in treating leukemia or lymphoma in a patient, wherein the treating comprises co-administering the patient at least one TLR agonist as described herein with the at least one chemotherapeutic agent.

[0067] The two components of the invention (i.e. the at least one TLR agonist and the at least one chemotherapeutic agent) may be co-administered at the same time (e.g. simultaneously) or at different times (e.g. sequentially) and over different periods of time, which may be separate from one another or overlapping. Where the two components are co- administered simultaneously they may be provided in one or more pharmaceutical compositions. Where the two components are provided in the form of separate compositions the two components may be co-administered separately, sequentially or simultaneously. The two components may be administered in any order but in a preferred embodiment the at least one TLR agonist is administered prior to the at least one chemotherapeutic agent.

[0068] In the present disclosure, the two components may be administered by the same or different routes. For example, the composition is administered locally. The present disclosure also, envisages administering the compounds systemically. In some instances, the compounds may be administered orally, intraadiposally, intraarterially, intraarticularly, intracranially, intradermally, intralesionally, intramuscularly, intranasally, intraocularally, intrapericardially, intraperitoneally, intrapleurally, intraprostatically, intrarectally, intrathecally, intratracheally, intratumorally, intraumbilically, intravaginally, intravenously, intravesicularlly, intravitreally, liposomally, locally, mucosally, orally, parenterally, rectally, subconjunctivally, subcutaneously, sublingually, topically, transbuccally, transdermally, vaginally, in cremes, in lipid compositions, via a catheter, via a lavage, via continuous infusion, via infusion, via inhalation, via injection, via local delivery, via localized perfusion, bathing target cells directly, or any combination thereof. For example, in some variations, the compounds are administered intravenously, intraarterially or orally. For example, in some variations, the compounds are administered intravenously.

[0069] Where the two components are administered as separate compositions, they may be administered within 120, 96, 84, 72, 48, 36, 24, 12, 6, 3, 2, 1, 0.5, 0.25 or 0.125 hours of each other. For example, the at least one TLR agonist is administered within 84, 72, 48, 36, 24, 12, 6, 3, 2, 1, 0.5, 0.25 or 0.125 hours prior to administration of the at least one chemotherapeutic agent. It may be useful to administer the chemotherapeutic drug prior the TLR agonist to assess the effect of the chemotherapeutic drug on the expression of factors involved in innate immune response and thus adap^the jmount of ^drug n^ed J¾dJhe_TLR_agonistJto-be -used in- a given patient. [0070] The present disclosure also relates to treating lymphoma or leukemia in a patient (e.g. a human patient) by the administration of: (i) at least one of: a TLRl agonist, a TLR2 agonist, a TLR4 agonist, and a TLR9 agonist; and (ii) at least one chemotherapeutic agent. The at least one TLR agonist and the at least one chemotherapeutic agent may function synergistically together and it is envisaged that this may allow a lowering of the dosage of the chemotherapeutic agent.

[0071] The leukemia can include but is not limited to aggressive leukemia, indolent leukemia, non-acute leukemia, acute leukemia, chronic leukemia, myelogenous leukemia, lymphocytic leukemia, T cell leukemia, B cell leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, and acute myeloid leukemia.

[0072] The lymphoma can include but is not limited to Hodgkin's

Lymphoma or non-Hodgkin's lymphoma (NHL) (e.g. B cell NHL), Burkitt lymphoma, chronic lymphocytic leukemia, precursor B lymphoblastic lymphoma, natural killer (NK) lymphoma, T cell lymphoma or B cell lymphoma.

[0073] A method of treating B cell leukemia in a human is disclosed which may include administering to the patient simultaneously, sequentially or separately: (i) Pam(3)CysSK(4); and (ii) Ara-C, and wherein the Pam(3)CysSK(4) is administered to the patient within 72, 48, 24, 12, 6, 3, 2 or 1 hours of the Ara-C.

[0074] It has been shown herein that chemotherapeutic agents render cells more immunogenic by inducing the expression of ligands for activating immune receptors. It is envisaged in the present disclosure that co-treatment of TLR2, TLR4 or TLR9 agonists with the chemotherapeutic agent Ara-C induce the expression of ligands for activating immune receptors, cell adhesion molecules, cytokines and chemokines in leukemia cells. Hence, co-treatement with the pharmaceutical compositions described herein renders tumor cells more immunogenic.

EXAMPLES

[0075] Example 1:

[0076] To demonstrate the efficiency and advantages of compositions comprising a TLR agonist and a chemotherapeutic drug for use in the treatment of leukemia or lympjiornajrome specific cimbinations_are_disclosed-below.-

[0077] To investigate direct effect of Pam3CSK4 pre-treatment on B leukemia cells in vivo, B leukemia cell line ΕμΜΙ which was generated from lymph node cell of Εμ-myc mice, a mouse model for Burkitt-like B cell lymphoma. Treatment of ΕμΜΙ cells with the TLR agonist Pam3CSK4 and to a lesser degree other TLR agonists significantly increased Ara-C induced expression of the CD40, CD69, CD80, CD86, ICAM-1 and H-2K^b (Fig. 1A). Treatment of ΕμΜΙ cells with Pam3CSK4 and Ara-C enhanced the secretion of IL-6, TNFa, RANTES and MlPla (Fig. IB). In addition, mRNA expression levels of cytokines involved in the innate and adaptive immune response were also increased (Fig. 1C, IL-Ιβ, IFN-a, TGFP3 and IL-4). No secretion of IFNp, IFNy, IL-12p70, and GM-CSF was detected.

[0078] Pre-treatment of ΕμΜΙ cells with Pam3CSK4 followed by either ΙμΜ or 10 μΜ of Ara-C for 16 hours indicated that when used simultaneously, the dose of chemotherapeutic agent can be lowered by 10 fold to obtain the same level of NK cells activation (Fig I D).

[0079] Expression of ligands for activating immunoreceptors by flow cytometry at the surface of B Leukemia cell line (ΕμΜΙ) after exposure in vitro to a TLR agonist and a chemotherapeutic agent, either alone or in combination was assessed. ΕμΜΙ cells subjected to the indicated treatments were assessed for surface expression levels of TLR1, TLR2, DNAM-1 , NGK2D and LFA-1 by flow cytometry, and were compared to their respective isotype control stainings.

[0080] These results suggest that Pam3CSK4 pretreatment sensitizes B leukemia cell to Ara-C by increasing the expression of co-stimulatory molecules, which may make the cells vulnerable to NK cells and activate an immune response.

[0081] Example 2:

[0082] Based on in vitro study data, the effect of Pam3CSK4 combined with Ara-C on tumor burden using a leukemia xenograft mouse model.

[0083] Thy 1.1+ expressing LN cells were treated with DMSO, Ara-C,

Pam3CSK4 and Pam3CSK4 followed by Ara-C. Transfer of DMSO, Ara-C, Pam3CSK4 or Pam3CSK4 followed by Ara-C-treated ΕμΜΙ cells expressing the genetic marker Thy 1.1+ resulted in tumor formation in all mice (Fig. 2). However, Pam3CSK4 and Ara-C treatment of ΕμΜΙ cells resulted in lower tumor burden in the blood and spleen of the injected mice when compared to Ara-C or Pam3CSK4-treated cells injected mice (Fig. 2A and B). Consistent with this finding, mice that received Pam3 CSK4_and Ara₌C-treated. cells_also_sur^ived_longer-SUggesting mat the combined- treatment interferes with tumorigenesis in vivo (Fig. 2C). [0084] , To analyze if Pam3CSK4 enhances Ara-C induced apoptosis of Thy 1.1+ ΕμΜΙ cells, we determined the rate of apoptosis before injections. Pam3CSK4 did not induce significant changes in the rate of apoptosis or proliferation of Ara-C-treated ΕμΜΙ cells as indicated by similar percentage of Annexin V+/PI+ cells (Fig. 2D) and cell numbers. This result suggests that the lower tumor burden observed in mice injected with Pam3CS 4 and Ara-C treated ΕμΜΙ cells is not attributed to changes in apoptosis or proliferation prior to injection.

[0085] Example 3:

[0086] Immunosurveillance in Εμ-myc mice depends on NK and T cells. To investigate if the Pam3CSK4 effect on Ara-C treated cells depends on immune cell subsets, NK cells were depleted prior to the transfer of treated ΕμΜΙ cells (Fig. 3C). Depletion of NK cells resulted in a modest increase in tumor burden in blood and spleen when compared to control antibody-injected mice. These results imply that NK cells play a role in the suppression of tumorigenesis in Pam3CSK4 combined with Ara-C treated B leukemia cells.

[0087] To address if immune cell subsets mediate the decrease of tumor cells in the blood of Εμ-myc mice, NK and/or T cells were depleted by administration of corresponding antibody one day before administration of treated ΕμΜΙ cells. Depletion of NK1.1⁺, CD4⁺ or CD 8⁺ cells had no significant impact on the tumor load in blood and spleen 11 days post injection (Figure 3 A and B). Simultaneous depletion of CD4⁺ and CD8⁺ increased the tumor levels although tumor burden was still lower than in mice injected with control-treated ΕμΜΙ cells possibly due to cell intrinsic effects of Ara-C. Depletion of NK cells and T cell subset resulted in a lower tumor load when compared to T cell depleted mice (Fig. 3B). Hence T cells and to a lesser degree NK cells contribute to rejection of ΕμΜΙ cells.

[0088] Example 4:

[0089] Since leukemia is characterized as a bone marrow disorder, we investigated whether the different treatments of leukemia cells affect the percentage of hematopoietic precursor cells in the bone marrow (41). A 99% reduction of Lin-IL- 7Ra+sca-lloc-kitloAA4+Flt3hi common lymphocyte progenitor cells (CLPs), but not other progenitors, was observed at 1 1 days after injection of ΕμΜΙ cells (Fig. 4 A and C). Pam3CSK4 or Ara-C treated _ Εμ_Μ1_ cejls induce^d similar

4A). In contrast, a higher number of CLPs was present in the bone marrow of mice that received Pam3CSK4 and Ara-C-treated ΕμΜΙ cells (Fig. 4A). [0090] CPLs give rise to NK and T cells among other immune cells.

Consistent with the loss of CLPs, the percentage of CD4+ and CD8+ T cells decreased in the blood of mice that received ΕμΜΙ cells (Fig. 4B). Similar to the effect on CLPs, the decrease of the percentage of T cells in the blood was less severe in mice that received of Pam3CSK4 and Ara-C-treated ΕμΜΙ cells when compared to single treated ΕμΜΙ cells..

[0091] Example 5:

[0092] To understand how PamsCSIQ renders Ara-C treated cells less tumorigenic, we analyzed the expression of the DNA damage marker p-H2AX, p53 and NF-κΒ. p53 but not H2AX phosphorylation, was modestly enhanced by PanvjCSlLi treatment prior to. Ara-C stimulation (Fig. 5 A). Pretreatment of ΕμΜΙ with Pam3CSK4 enhanced and prolonged the phosphorylation of p65 and ΙΚΚα/β (Fig. 5B).

[0093] To test if the enhanced activation of the two transcription factors p53 and NF-κΒ are important for the upregulation of costimulatory factors and cytokines, we impaired the expression by overexpressing an ΙκΒ-α super repressor or a p53-specific small hairpin R A (shRNA) (Fig. 6). Inhibition of NF-κΒ abrogated the effects of Pam₃CSK₄ on Ara-C-treated cells (Fig. 6A). In contrast, decreased p53 expression had no effect on the expression of the immunomodulatory molecules (Fig. 6B). In summary, our results suggest that Pam3CSK-4 enhances the anti-cancer effects of Ara-C by enhancing and prolonging the NF-i B-mediated proinflammatory signals.

[0094] The use of immune cells has been an important strategy to fight against cancer. In this study, we demonstrate that activation of the TLR1/2 receptor on B leukemia cells primes the cells and leads to an increase in expression of immune stimulating factors, which are further induced by a DNA damage inducing chemotherapeutic agent. Expression of TLR 1 and 2 in LN cells and their upregulation by Ara-C and Pam3CSK4 imply that TLR pathways are activated in B leukemia cells upon DNA damage as well as by their classical ligand.

[0095] p53 mediated induction of most TLR genes occurs in human T lymphocytes by DNA metabolic stressors such as ionizing radiation, 5-FU and doxorubicin. However, the same treatments do not induce TLRs in mouse

humans and mice. This finding suggests that the induction of TLR 1 and 2 in lymph node cells by Ara-C might not be mediated by p53. Although whether the inductions of TLR 1 and 2 by both Ara-C and Pam3CSK4 in lymph node cells share a common mechanism or not remains to be elucidated, the differential expression of co- stimulatory molecules depending on the type of treatment suggest that Pam3CSK4 and Ara-C stimulate the cells by different mechanisms and their combination results in an surprising increase in immunogenicity.

[0096] Among the surface molecules specifically increased in lymph nodes cells by Pam3CSK4 combined with Ara-C treatment are CD80 and IL-6. The critical role of CD80 expression in anti-leukemia immune responses mediated by NK and T cells has been well established. A murine T cell lymphoma cell line manipulated to express high level of IL-6 does not proliferate well in vivo compared to cells expressing IL-6 at low level, despite a similar proliferation rate in vitro. When acute myeloid leukaemia cells are transduced with lentivirus to express CD 80 and IL- 2 concurrently, there was an increase in NK and T cell cytotoxicity. Therefore, it is highly likely that CD80 and IL-6, which are expressed exclusively by Pam3CSK4 combined with Ara-C treatment, are the key molecules, which led to enhanced antitumor immune responses. TLR agonists upregulate a number of molecules that are associated with the response to damaged DNA in response to chemotherapy. Hence, it appears that TLR agonists render tumor cells more sensitive to chemotherapy drugs.

[0097] Blood analysis showed that the numbers of T and NK cells as well as the expression levels of DNAM-1 and NKG2D were significantly decreased in the LN cell xenograft. However, treatment of the cells with Pam3CSK4 combined Ara-C alleviated the decrease. NK cell activating receptor, DNAM-1 suppresses tumorigenesis through perforin mediated cytotoxicity. Soluble DNAM-1 ligand and NKG2D ligand inhibits immune function of NK cell receptor by promoting immune evasion of tumor cells. Although CD8+ CTL is critical for cancer immunotherapy, an important role of CD4+T cells in cancer elimination and various virus infections have been discovered. The killing activity of CD4+ CTL is mediated in a perforin dependent manner that triggers both necrosis and apoptosis in cancer cells. By this mechanism, TLR endogenous ligands released from dying tumor cells can be recognized by immune cells resulting in the modulation of host immunity.

[0098] Activation of T cells occurs through the engagement of the T

_cell receptor (TCR) and CD28. TCRs^_CD4+T cdlsjiaye^ Class_ II, which is expressed on activated APC like DCs, macrophage and B cells. Of note, MHC Class II was constitutively expressed on lymph node cells and its expression level was not altered by any treatments in our study. However, double depletion of NK and CD4+T cells further increased tumor burden in the mice injected with Pam3CSK4 combined Ara-C treated LN cell. It is possible that increased expression of co- stimulatory molecules along with cytokine secretion in lymph node cells in response to Pam3CSK4 combined with Ara-C recruit effector cells, mainly NK and CD4+T cells, to induce tumor cell death.

[0099] TLR tolerance studies to enhance efficacy of chemotherapy agents in cancer have been emerging, demonstrating TLR agonist pre-treatment increases the susceptibility of tumor cells to effector cell cytotoxicity. Our data presented here suggests that increased immunogenicity of B leukemia cells by the combination of a TLR agonist with a chemotherapeutic agent in vitro functionally corresponds to inhibition of tumorigenesis in vivo. A complex mechanism mediated by many factors is presumed to be activated by the combination of TLR1/2 agonist and DNA damage inducing agent.

[00100] Mice and cells. C57BL/6 mice were purchased from the Centre for Animal Resources at the National University of Singapore. Εμ-myc hemizygous mice on a C57BL/6 background were obtained from Jackson Laboratory, USA. Secondary B -lymphoma cells (LN) were prepared from lymph node of 108 days old Εμ-myc transgenic C57BL/6 mouse. LN cells were maintained in RPMI1640 medium containing 20 mM Hepes buffer, 10% heat-inactivated fetal bovine serum (FBS; Gibco/Invitrogen), 50 μΜ 2-mercaptoethanol, 200 μΜ asparagine, and 100 U/mL penicillin-streptomycin. All animal experiments were performed with the approval of the Institutional Animal Care and Use Committee of the National University of Singapore.

[00101] In vitro cell stimulation and ELISA. LN cells (1.6xl0⁵/ ml) were treated with 0.1% DMSO or 1 μg/ml Ara-C (Sigma) for 16-17 h. For the combination treatment, the cells were treated with 1 μg/ml Pam3CSK4 (Invivogen) for 24 h followed by 1 μg/ml Ara-C for 16 h. Cell free supernatant was harvested, frozen in liquid nitrogen and stored at -80°C until measurement. The quantification of Ml la (CCL3) and IL-6 in culture supernatants was determined using ELISA kits (R&D system, USA, eBioscience, USA) according to manufacturer's instructions.

leukemia cells were stained with anti-CD112 (6A606, Santa Cruz Biotechnology, USA), anti-CD 155 (Hycult biotechnology, USA), anti-TLRl (CD281, eBioscience, USA), TLR2 (CD282, from eBioscience, USA), anti-CD40 (eBioscience, USA), Anti-Rae-Ιβδ (R&D, USA), and APC donkey anti-rat IgG (eBioscience, USA). Blood, spleen and bone marrow cells were harvested from leukemia bearing C57BL/6 mice and red blood cells were removed using lysis buffer (0.15 M NH₄C1, 10 mM KHCO3, 0.1 mM Na₂EDTA). The cells were incubated with Fc receptor blocking antibody (eBioscience, U.S. A) and directly labeled with monoclonal antibodies APC anti-CD86 (B7-2), PE anti-CD80, FITC anti-MHC class I or II, Pacific blue anti- ICAM-1, PE-cy7 anti-NKG2D, PE anti-DNAM-1 which were purchased from eBioscience (USA). The percentage of effector cells (CD4+ and CD8+T cells, NK cells) in the total lymphocyte population was analyzed using Perepcy5.5-anti-CD3, APC-anti-NKl.l, Pacific blue-anti-CD8, PE-anti-CD4 purchased from eBioscience (U.S.A). The tumor burden was determined by staining cells with FITC anti-Thyl .l, Percpcy5.5 anti-B220, APC anti-IgM (eBioscence, U.S.A.). For the analysis of CLP (LinTL-7Ra⁺sca-l^,0ckit^l0AA4⁺Flt3^hi) population, bone marrow cells were isolated and stained with Lin antibody, Percpcy5.5 anti-sca-1, APCcy7 anti-c-kit, PE anti- AA4,FITC anti-IL-7Ra⁺, Pacific blue anti-streptoavidin, APC anti-Fit3 (eBioscience, USA). Analysis of stained cells was conducted using a FACSCalibur system (BD Biosciences, USA) and FlowJo 8.8.7 (Treestar, USA) programme.

[00103] Leukemia xenograft mice models and effector cell depletion. Thyl.l⁺ LN cells were cultured and divided into four groups. The cells were treated with combination of Pam3CSK4 with Ara-C, DMSO, Ara-C or Pam3CSK4 as described in the mice and cells section. Live cells were collected by Ficoll gradient centrifugation (1700 rpm, 20 min, 18°C). After washing three times with RPMI, (5x10⁵) LN cells suspended in 100 μΐ of PBS were intravenously injected into mice. On day 11 post injection, bone marrow cells were isolated for common progenitor cell staining and blood, spleen were used for surface molecules staining. On day -1, 4 and 9 of LN cell injection NK1.1 depleting antibody (PK136, 250 μg/mouse), CD8+T cell depleting antibody (TIB210, 250μ^ mouse) or CD4+T cell depleting antibody (GK1.5, 500 μg/mouse) were intraperitoneally injected. On day 11 or 12, blood and spleen cells were collected to determine leukemia tumor burden. For the survival curve of xenograft mice, the mice were euthanized at the terminal stage of disease when weight loss was jtnpre __than_ 15% _or_ .when . the_mice -became- unconscious after exhibiting clinical signs such as ruffled hair coat, reduced locomotor activity and hunched back. PBS was injected as a control. [00104] Annexin V and PI Assay. Apoptosis of DMSO, Ara-C, Pam3CSK4, and AraC and Pam3CSK4 combined treated cells were determined by Annexin V/Propidium Iodide (PI) staining using the Annexin V-APC apoptosis detection kit (eBioscience, USA) according to manufacturer's instructions. Briefly, LN cells were treated with 0.1% DMSO, 1 μΜ Ara-C, 1 μ^ιηΐ Pam3CSK4 or combination of Pam3CSK4 with Ara-C. Cells were stained subsequently stained for Annexin V and PL The blood cells were analyzed on Beckman Coulter's CyAn™ Flow Cytometer. All flow cytometry results were analyzed on Flowjo 8.8.6 (Treestar, USA).

[00105] Statistics. Statistical analyses were performed using the two- tailed Student's rtest with the exception of survival time, which was instead analyzed by the logrank (Cox-Mantel) test.

Claims

CLAIMS:

1. An immunogenic composition comprising:

i. at least one TLR (Toll like receptor) agonist; and

ii. at least one chemotherapeutic agent.

2. The composition according to claim 1, wherein the at least one TLR agonist is selected from the group consisting of: a TLRl agonist, a TLR2 agonist, a TLR4 agonist, and a TLR9 agonist.

3. The composition according to claim 1, wherein the at least one chemotherapeutic agent is selected from the group consisting of: a cytotoxic agent, a hormonal therapeutic agent and a monoclonal antibody.

4. The composition according to any one of claims 1 to 3, wherein the at least one chemotherapeutic agent is a DNA damaging agent.

5. The composition according to claim 1, wherein

i. the at least one TLR agonist is selected from the group consisting of: a TLRl agonist, a TLR2 agonist, a TLR4 agonist, and a TLR9 agonist; and

ii. the at least one chemotherapeutic agent is a DNA damaging agent.

6. The composition according to any one of claim 1 to 5, wherein the amount of the at least one TLR agonist is about 0.1 mg to about 500 mg.

7. The composition according to any one of claim 1 to 5, wherein the amount of the at least one chemotherapeutic agent is about 0.1 mg to about 500 mg.

8. The composition according to any one of claim 1 to 7 for use in therapy.

9. The composition according to any one of claim 1 to 8 for use in treating leukemia or lymphoma in a patient in need thereof.

10. A method of treating leukemia or lymphoma, the method comprising administering to a patient in need thereof a pharmaceutically effective amount of:

i. at least one TLR agonist; and

ii. at least one chemotherapeutic agent.

11. The method according to claim 10, wherein the at least one TLR agonist is selected from the group consisting of: a TLRl agonist, a TLR2 agonist, a TLR4 agonist, and a TLR9 agonist.

12. The method according to claim 10, wherein the at least one TLR agonist is selected from the group consisting of: a TLRl agonist, a TLR2 agonist, and a TLR4 agonist.

13. The method according to claim 10, wherein the at least one TLR agonist is selected from the group consisting of: a TLRl agonist and a TLR2 agonist.

14. The method according to claim 13, wherein the TLR agonist is an agonist of a human TLR.

15. The method according to any one of claims 10 to 14, wherein the TLR agonist is selected from the group consisting of: Pam(3)CysSK(4) ((S)-[2,3- 5w(palmitoyloxy)-(2-RS)-propyl]-N-palmitoyl-(R)-Cys-(S)-Ser-(S)-Lys₄-OH or Pam₃-Cys-Ser-(Lys) ), Pam3Cys (S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-N- palmitoyl-(R)-cysteine or tripalmitoyl-S-glyceryl cysteine), Cadi-05, ODN 1585, lipoteichoic acids, zymosan, synthetic triacylated and diacylated lipopeptides, MALP- 2, tripalmitoylated lipopeptides, a compound having a 2-aminopyridine fused to a five membered nitrogen-containing heterocyclic ring, lipopolysaccharide (LPS), Polyriboinosinic-polyribocytidylic acid (poly:IC), a CpG oligodeoxynucleotides (ODNs), monophosphoryl lipid A ("MPL"), an imidazoquinoline compound (e.g. an amide substituted imidazoquinoline amine), a benzimidazole derivative, a C8- substituted guanine ribonucleotide, an N7. C8-substituted guanine ribonucleotide, bacteria heat shock protein-60 (Hsp60), peptidoglycans, flagellins, mannuronic acid polymers, flavolipins, teichuronic acids, ssRNA (single stranded RNA), dsR A (double stranded RNA), or a combination thereof.

16. The method according to claim 15, wherein the group further comprises a derivative of the at least one TLR agonist.

17. The method according to claim 16, wherein the derivative is a diastereoisomer ofPam(3)CysSK(4).

18. The method according to claim 10, wherein the at least one chemotherapeutic agent is a compound selected from the group consisting of: : arabinosyl cytosine (ara- C or cytarabine), alkylating agents such as nitrosoureas and mechlorethamine, antimetabolites such as purine antimetabolites, hydroxyurea, capecitabine and 5- fluorouracil (5-FU), 6-mercaptopurine, 6-thioguanine, plant alkaloids such as vinblastine, vincristine and paclitaxel, plant extracts, radioisotopes, steroid hormones, S-l (Tegafur, 5-chloro-2,4-dihydroxypyridine and oxonic acid), 5-ethynyluracil, 5- azacytidine (5-AC), 2',2'-difluoro-2'-deoxycytidine (dFdC), gemcitabine, bortezomib, pentostatin, allopurinol, 2-fluoro-arabinosylⁱadenine (2F-ara-A), sulfur mustard (bischloroetyhylsulfide), melphalan, melpharan, chlorambucil, cyclophosphamide, ifosfamide, thiotepa, AZQ, mitomycin C, dianhydrogalactitol, dibromoducitol, alkyl sulfonate (busulfan), procarbazine, decarbazine, rebeccamycin, anthracyclins, non- intercalating topoisomerase inhibitors such as irinotecan (CPT-11) and topotecan, compounds that form adducts with nucleic acid including platinum derivatives, camptothecin and SN-38.

19. The method according to claim 18, wherein the group further comprises a derivative and/or an analogue of the at least one chemotherapeutic agent.

20. The method according to claim 19, wherein the analogue comprises a purine analogue of Ara-C selected from the group consisting of fludarabine and cladribine.

The_metho.d_ according_to_claim_l 9,_ wherein_the_derivative_is_N4-behenoyl-

22. The method according to claim 10, wherein the pharmaceutically effective amount of the at least one TLR agonist is a daily dose of about 0.01- 25 mg of TLR agonist per kg of body weight.

23. The method according to any one of claims 10 to 22, wherein the leukemia is selected from the group consisting of aggressive leukemia, indolent leukemia, non- acute leukemia, acute leukemia, chronic leukemia, myelogenous leukemia, lymphocytic leukemia, T cell leukemia, B cell leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, and acute myeloid leukemia.

24. The method according to any one of claims 10 to 22, wherein the lymphoma is selected from the group consisting of Hodgkin's Lymphoma or non-Hodgkin's lymphoma (NHL) (e.g. B cell NHL), Burkitt lymphoma, chronic lymphocytic leukemia, precursor B lymphoblastic lymphoma, natural killer (NK) lymphoma, T cell lymphoma or B cell lymphoma.

25. Use of

i. at least one TLR agonist, selected from the group consisting of: a TLR1 agonist, a TLR2 agonist, a TLR4 agonist, and a TLR9 agonist; and

ii. at least one chemotherapeutic agent

in the manufacture of a composition for the treatment of leukemia and/or lymphoma in a patient in need thereof.

26. A method for determining susceptibility of a patient suffering from leukemia and/or lymphoma to a treatment with a composition as defined in any one of claims 1 to 9, wherein the method comprises:

i. administering at least one chemotherapeutic agent to said patient ii. comparing a first level of TLR selected from the group consisting of

TLR mRNA level in a tumor sample, TLR cDNA level made from jmRNA Jrom_ s_aid^mor_s_ample,_ and__TLR .protein _le _el from. said_ tumor sample with the level of TLR selected from the group consisting of TLR mRNA level from a non-tumor sample from said patient, TLR cDNA level made from mRNA from said non-tumor sample, and TLR protein level from said non-tumor sample; and

wherein a patient characterized by an increased level of TLR in said tumor sample is susceptible to a treatment with the at least one TLR agonist in said pharmaceutical composition