A combination for cancer therapy.

Field of the invention

The present invention relates to the fields of medicine and immunology. In particular, it relates to a novel combination for use in the treatment, prevention and/or delay of cancer.

Background of the invention

Immunotherapy for cancer is a promising approach to employ the body's own immune system to recognize and eradicate cancer cells. Many immune adjuvants/cancer vaccines, for instance nucleic acid based adjuvants such as various CpG, have been developed that aim at the induction of a potent anti-tumor immune response. Using this approach, immune cells that specifically can kill tumor cells can be induced and activated. However, tumor cells feature several mechanisms to suppress the function of immune cells and therefore largely hamper the efficacy of cancer immunotherapy. Cancer immunotherapy is a promising new treatment modality that enters the clinic right now. The last couple of years, several immunotherapy based treatments have been approved by the FDA such as sipuleucel-T, a dendritic cell (DC) based cancer vaccine, as well as ipilimumab, a monoclonal antibody against the cytotoxic T-lymphocyte antigen-4 (CTLA-4) immune checkpoint. In addition, other immune-based therapies, including blockade of programmed death 1 (PD-1) or its ligand PD-L1 as well as chimeric antigen- receptor carrying T cells, have achieved promising antitumor effects.

Therapeutic success of cancer immunotherapy however, is largely limited by said ability of tumor cells to suppress anti-tumor immune responses and to escape from the immune system at multiple levels. New compounds able to limit tumor immune suppression and boost immune responses are key for cancer immunotherapy efficacy.

A strategy for cancer immunotherapy efficacy involves inhibition of STAT3, a promoter of oncogenesis, in combination with CpG. STAT3 is a transcription factor that regulates gene expression, particularly expression of immunosuppressive genes. Hence STAT3 inhibition changes predominantly gene expression of target cells like immune cells and cancer cells. Therefore STAT3 inhibition reprograms immune cells directly and makes them more prone to activation by CpG (Molavi et al, 2008). However, there is evidence showing that STAT3 suppresses tumor growth (de la Iglesia et al, 2008; Lee et al., 2012; Musteanu et al., 2010). Therefore, for several tumor types, STAT3 inhibition may not be an option.

One strategy of tumor cells to evade the immune system involves the aberrantly high expression sialic acid sugars on tumor cell surface. First, sialic acids have a highly negative charge. Therefore, accumulation of sialic acids creates a net negative charge on the tumor cell surface that hinders interactions with immune cells. More specifically, sialic acids hinder the accessibility and recognition of structures on tumor cells such as antigens. Thereby, immune cells are physically hindered to screen the tumor cell surface with their recognition receptors and to recognize them as tumor cell.

Second, sialic acids have an immune suppressive activity and can dampen activation of the immune system. Sialic acids are expressed by virtually every cell. Eventually, sialic acids mark host cells as self in order to discriminate them from bacteria that do not express sialic acids. Therefore, cells of the (mammalian) immune system express specific sialic acid-recognizing receptors (Siglecs) to recognize host cells via their sialic acid sugars. Siglecs can inhibit activation of immune cells and even induce apoptosis among recognition of sialic acid ligands. It was hypothesized that tumor cells inhibit immune cell function by covering their membrane with sialic acid molecules via the interaction with immune suppressive receptors such as siglecs. As a consequence, tumor cells eventually escape from natural recognition and eradication by the immune system. Accordingly, there is a large demand for an improved immunotherapy for cancer, preferably involving agents that limit tumor immune suppression.

Description of the invention

One highly potent immune suppression strategy of tumor cells involves said aberrantly high expression sialic acid sugars. Sialic acids are located on proteins and lipids on the cell membrane and are commonly overexpressed on the membrane of cancer cells. Furthermore, sialic acids and sialic acid-carrying molecules can be secreted by tumor cells, thereby having a systemic immune suppressive effect. Immune cells express immunosuppressive receptors called Siglecs that specifically recognize these sialic acids and inhibit immune cell activation and function. Interfering with these immunosuppressive sialic acids might allow to boost cancer immunotherapy.

The inventors have recently found for the first time that a novel glycomimetic potently blocks sialic acid synthesis and expression in cancer cells (Biill et al, 2013). Therapeutic administration of this glycomimetic alone in a preclinical mouse melanoma model delayed tumor growth modestly. While CpG treatment alone had almost no effect on tumor growth, it has now surprisingly been demonstrated that combining CpG with the glycomimetic treatment provides a synergistic effect; tumor growth is strongly prevented.

Accordingly, in a first aspect, the present invention provides for a combination of a source of a sialic acid blocker and a source of an immune adjuvant. Such combination, sialic acid blocker and immune adjuvant are herein referred to as a combination, a sialic acid blocker and immune adjuvant according to the present invention, respectively. Within the context of the present invention 'a combination' means that a source of a sialic acid blocker and a source of an immune adjuvant are contemplated and encompassed. Each source may be together or present together or combined together or physically in contact with the other source forming one single composition. Each source may alternatively be comprised within a distinct composition. However the invention provides the insight that both sources of a sialic acid blocker and immune adjuvant are needed or are used in order to get an effect according to the present invention as defined herein. If each source is not present in a same single composition, each source and/or each distinct composition comprising a source of a combination according to the present invention may be used sequentially or simultaneously.

Within the context of the present invention, a source may be any source available to a person skilled in the art; the sialic acid blocker may be present as such, but it may also be present as a precursor or variant of a sialic acid blocker. Likewise, the source of the immune adjuvant may be any source available to the person skilled in the art; the immune adjuvant may be available as such, but may also be present as a precursor or variant of an immune adjuvant.

Within the context of the present invention, a sialic acid blocker is a compound that is capable of interfering with the sialic acid expression in and on tumor cells. Such sialic acid blocker may be any compound known to the person skilled in the art having the capability of interfering with the sialic acid expression in and on tumor cells. Preferably, a sialic acid blocker according to the present invention is a compound that prevents incorporation of endogenous sialic acids into cell surface glycoproteins and glycolipids. Preferably, a sialic acid blocker according to the present invention is a glycomimetic, preferably a carbocyclic mimic of sialic acid. Preferably, a sialic acid blocker according to the present invention is a compound that inhibits the sialoglycoprotein and/or sialoglycolipid synthesis pathway, preferably by inhibiting the function of a sialyltransferase, preferably by inhibiting Golgi-resident sialyltransferases. A preferred sialic acid blocker is P-3Fax-Neu5Ac (P-3Fax-Neu5Ac is a peracetylated analogue of natural occurring Neu5Ac that due to attachment of a fluorine atom to the sialic acid backbone inhibits sialyltransferase function; Figure 1) or a variant thereof that is active as a sialyltransferase inhibitor. As a result of the inhibition of the sialyltransferase, incorporation of endogenous sialic acid into cell surface glycoproteins and glycolipids is prevented and accumulating endogenous sialic acids in the cell trigger a negative feedback mechanism that reduces synthesis of endogenous sialic acids.

Within the context of the present invention, an immune adjuvant may be any relevant compound that is able to initiate or enhance an immune response. Particularly preferred adjuvants are adjuvants that trigger Pattern Recognition Receptors such as Toll-Like Receptors TLR3, 7, 8 and/or TLR9, preferably TLR9, such as, but not limited to nucleic acid based immune adjuvants such as dsRNA, poly(LC), Resiquimod (R-848), dendritic cell vaccines, ipilimumab (a monoclonal antibody against the cytotoxic T-lymphocyte antigen-4 (CTLA-4) immune checkpoint), blockade agents of programmed death 1 (PD- 1) or its ligand PD-L1, chimeric antigen-receptor carrying T cells and methylated or unmethylated CpG. Such adjuvants may be produced synthetically or may be produced from naturally occurring nucleic acids. Nucleic acid based immune adjuvants may comprise nucleic acid base analogues. A CpG in the context of the present invention is an oligonucleotide that comprises at least one CpG immunostimulatory motif, including pharmaceutically acceptable salts thereof; the person skilled knows relevant CpG's, e.g. from Bode et al, 2011, which is herein incorporated by reference. A CpG according to the present invention may comprise at least one internucleotide linkage that has a phosphate backbone modification such as a phosphorothioate or a phosphorodithioate modification and/or at least one stabilized internucleotide linkage. The use of phosphorothioate nucleotides enhances resistance to nuclease digestion when compared with native phosphodiester nucleotides, resulting in a substantially longer in vivo half life (30-60 min compared with 5-10 min for phosphodiester). In some embodiments, all internucleotide linkages have phosphate backbone modifications such as phosphorothioate or phosphorodithioate modifications. A CpG according to the present invention may comprise at least one nucleotide analogue providing enhanced immunostimulatory activity such as described in WO2008/068638 which is herein incorporated by reference. A preferred CpG according to the present invention is one selected from the ones described in Bode et al, 2011 and in EP2591787A1 which are herein incorporated by reference, such as but not limited to, class A CpG (also known as D-type CpG), class B CpG (also known as K-type CpG), class P CpG and class C CpG. A-class CpG ohgodeoxyribonucleotides (ODN) typically include nuclease-resistant (stabilized) base sequences comprised of three or more consecutive guanines (poly-G motifs) at one or both ends, and a central region comprised of one or more CpG dinucleotides contained in a self-complementary palindrome. Members of A-class CpG ODN activate natural killer (NK) cells and induce interferon-alpha (INF-[alpha]) secretion from plasmacytoid dendritic cells (pDC). B-class CpG oligodeoxyribonucleotides typically include a stabilized non-palindromic nucleotide sequence, which comprises one or more CpG dinucleotides. In contrast to A-class ODN, B-class CpG oligodeoxyribonucleotides strongly activate B cells, but induce comparatively weaker INF-[alpha] secretion. C-class CpG oligodeoxyribonucleotides typically include one or more CpG motifs, which are located within the 5'-region, and a palindromic sequence, which is located at or near the 3 '-end. They exhibit immunostimulatory activity that is characteristic of both A-class and B-class CpG ODN, including induction of INF-[alpha] secretion and activation of NK cells. At similar concentrations, C-class oligodeoxyribonucleotides generally exhibit B cell activation that is greater than what is observed with A-class CpG ODN, but is less than what is typically seen with B-class CpG ODN. Class P CpG contain two palindromic sequences, enabling them to form higher ordered structures. Class P CpG activate B cells and pDCs, and induce substantially greater IFN-a production when compared with class C CpG. Recent studies have shown that CpG oligodeoxyribonucleotides induce immunostimulatory activity through interaction with Toll-like receptor 9 (TLR9). See e.g. Krieg 2006; Jurk, et al, 2004; Vollmer, et al, 2004; Krieg, 2001; Rutz et al, 2004; Bauer et al., 2001; Latz et al., 2004 and WO 2003/015711. A more preferred CpG according to the present invention is an oligodeoxynucleotide with phosphodithioate backbone and sequence 5'- TCCATGACGTTCCTGATGCT-3 ' . A CpG according to the present invention may be optimized for a specific species such as mouse or human being; the person skilled in the art knows how to optimize a CpG for a specific species (see e.g. Bode et al, 2011). In a second aspect, the present invention provides for a single composition comprising a combination according to the first aspect of the present invention. In addition, the present invention provides for a first composition comprising a source of a sialic acid blocker according to the present invention and an a second composition comprising a source of an immune adjuvant according to the present invention. A composition according to the present invention may be in the liquid, solid or semi-liquid or semi-solid form. If each source is not present in a same single composition, each source and/or each distinct composition comprising a source of a combination according to the present invention may be used sequentially or simultaneously. Preferably, a composition according to the present invention comprises

a pharmaceutical acceptable excipient. Such composition is herein also referred to as a pharmaceutical composition according to the present invention. Pharmaceutically acceptable excipients are known and customary to the person skilled in the art and for instance described in Remington; The Science and Practice of Pharmacy, 21st Edition 2005, University of Sciences in Philadelphia.

Preferably, a composition according to the present invention further comprises a delivery vehicle. In said delivery vehicle, at least one of the source of the sialic acid blocker according to the present invention and the source of the immune adjuvant is contained in the delivery vehicle or is attached to the delivery vehicle. Accordingly, a source of the sialic acid blocker according to the present invention may be present in or attached to the delivery vehicle, a source of the immune adjuvant according to the present invention may be present in or attached to the delivery vehicle and the source of the sialic acid blocker according to the present invention and the source of the immune adjuvant may be present in or attached to the delivery vehicle. When both the source of the sialic acid blocker according to the present invention and the source of the immune adjuvant according to the present invention are present in or attached to a delivery vehicle, they may be present in or attached to separate delivery vehicles. A preferred delivery vehicle in a composition according to the present invention is a nanoparticle or an antibody or an antibody conjugate. In case the delivery vehicle is an antibody or an antibody conjugate, the sialic acid blocker or the immune adjuvant according to the present invention is attached to the delivery vehicle; the antibody is preferably an anti-tyrosinase related protein- 1 antibody. A nanoparticle according to the present invention is preferably a poly(lactic-co-glycolic acid) (PLGA) based nanoparticle. Preferably , a nanoparticle according to the present invention comprises a targeting device. Such targeting device may be any compound that is capable to target the delivery vehicle, in vitro, ex vivo or in vivo, to a predetermined target. The predetermined target may be a microbiological cell, preferably a tumor cell, more preferably a melanoma cell. A preferred targeting device according to the present invention is an antibody which may be polyclonal but preferably is monoclonal. A preferred antibody is an anti-tyro sinase related protein- 1 antibody to target the nanoparticle according to the invention to a melanoma cell.

In a third aspect, the present invention provides for the medical use of a combination according to the first aspect of the present invention or of a composition according to the second aspect according to the present invention.

Accordingly, there is provided for a combination according to the first aspect of the present invention or a composition according to second aspect of the present invention for use as a medicament, preferably for the use in treatment, prevention or delay of cancer. A combination according to the first aspect of the present invention or a composition according to the second aspect of the present invention when used as a medicament preferably in the treatment, prevention or delay of cancer can conveniently be combined with state of the art cancer therapies such as, but not limited to cancer medicaments, radiation, surgical procedures, chemotherapy, targeted therapies or a combination thereof.

There is also provided for the use of a combination according to the first aspect of the present invention or a composition according to second aspect of the present invention for the manufacture of a medicament, preferably a medicament for the treatment, prevention or delay of cancer.

There is also provided a method of treatment, prevention or delay of cancer, comprising administration of a combination according to the first aspect of the present invention or a composition according to second aspect of the present invention, to a subject in need thereof.

Formulation of medicaments, ways of administration and the use of pharmaceutically acceptable excipients are known and customary in the art and for instance described in Remington; The Science and Practice of Pharmacy, 21st Edition 2005, University of Sciences in Philadelphia.

The medical use and methods according to this aspect of the present invention can be used to treat human and animal subjects. The cancer is preferably neuroblastoma, glioma, leukaemia, lung cancer, bladder cancer, renal cancer, pancreatic cancer or epithelial cancer and more preferably melanoma. The treatment, prevention or delay of cancer is preferably the treatment, prevention or delay of cancer metastasis.

In the medical use and methods according to this aspect of the invention, administration may be performed through any suitable route including but not limited to: oral, aerosol or other device for delivery to the lungs, nasal spray, intravenous, intramuscular, subcutaneous, intradermal, intraperitoneal, intrathecal, vaginal, rectal, topical, lumbar puncture, intrathecal, intratumoral, peritumoral. A combination, a composition, compositions or a single composition according to the present invention may be administered to a subject or to a cell, tissue, tumor or organ of said subject for at least one week, one month, six month, one year or more. The frequency of administration of combination, a composition, compositions or a single composition according to the present invention may depend on several parameters such as the medical condition of the patient. The frequency may be ranged between at least once, two, three, four times a day, a week, or two weeks, or three weeks or four weeks or five weeks or a longer time period. The dosage of the immune adjuvant is preferably ranged from 0.1 and 30 mg/kg body weight, preferably from 0.5 and 20 mg/kg, more preferably from 1 and 10 mg/kg, more preferably from 2 and 5 mg/kg of the immune adjuvant, more preferably 3 mg/kg. The dosage of the sialic acid blocker is preferably ranged from 1 and 50 mg/kg body weight, preferably from 5 and 20 mg/kg, more preferably from 5 and 15 mg/kg, more preferably from 7 and 12mg/kg of the immune adjuvant, more preferably 10 mg/kg. In case the immune adjuvant and/or the sialic acid blocker are attached to or present in a delivery vehicle according to the present invention for targeting to the cells of interest, the dosage of the immune adjuvant and/or the sialic acid blocker can be significantly lower to achieve the same or even a better effect due to the specific targeting of the compounds.

In this document and in its claims, the verb "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".

The word "about" or "approximately" when used in association with a numerical value (e.g. about 10) preferably means that the value may be the given value (of 10) more or less 1% of the value.

The sequence information as provided herein should not be so narrowly construed as to require inclusion of erroneously identified bases. The skilled person is capable of identifying such erroneously identified bases and knows how to correct for such errors.

All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.

Figure legends Figure 1.

Structural representation of P-3Fax-Neu5Ac and mode of action.

A) Structural representation of P-3Fax-Neu5Ac.

B) P-3Fax-Neu5Ac can enter the sialoglycoprotein (SGP) or sialoglycolipid (SGL) synthesis pathway similar to endogenous sialic acids. Instead of being incorporated into sialoglycoproteins or sialoglycolipids, P-3Fax-Neu5Ac blocks the function of Golgi- resident sialyltransferases (STs). This blockade prevents incorporation of endogenous sialic acids into cell surface glycoproteins (GP) and glycolipids (GL). Furthermore, accumulating endogenous sialic acids in the cell trigger a negative feedback mechanism that deactivates de novo synthesis of endogenous sialic acids.

Figure 2.

Intratumoral inj ections of P-3Fax-Neu5 Ac hamper B 16-F 10 melanoma growth.

A) Timeline showing tumor growth experiment and treatment schedule. On day 0, 50.000 B16-F10 cells were inoculated subcutaneously into C57BL/6 mice. From day 10 onwards PBS, 0.3mg or lmg control P-Neu5Ac or 0.3mg or lmg P-3Fax-Neu5Ac were injected intratumoral three times per week until day 40 (black arrow heads). Tumor growth and survival times in the different treatment groups (n=9-12) was monitored until day 70.

B) Tumor growth in the different treatment groups is shown in time as mean tumor volume (mm3) ± SEM. Statistical significance was determined by ANOVA followed by Bonferroni's correction using Prism 5.03 (GraphPad Software, Inc., La Jolla, CA), p- values <0.05 were considered significant (p<0.05 *, p<0.01 **, p<0.001 ***).

C) Kaplan-Meyer curves represent percentage survival of the different treatment groups. Log-rank test was used to determine statistically significant differences between the different treatment groups compared to the PBS control group.

Figure 3.

Intratumoral injections with P-3Fax-Neu5Ac block sialic acid expression in the tumor mass.

A) Representative histograms showing sialic acid expression on single cells prepared from subcutaneous B16-F10 tumors following 3 intratumoral injections with 0.3mg or lmg P-3Fax-Neu5Ac, measured by flow cytometry using fluorescent MALII, SNA-I or PNA lectin.

B-D) Quantification of sialic acid expression on single cells prepared from subcutaneous B16-F10 tumors following 3 intratumoral injections with 0.3mg or lmg P-3Fax- Neu5 Ac, measured by flow cytometry using fluorescent MALII, SNA-I or PNA lectin. (n=6). Statistical significance was determined by ANOVA followed by Bonferroni's correction using Prism 5.03 (GraphPad Software, Inc., La Jolla, CA), p-values <0.05 were considered significant (p<0.05 *, p<0.01 **, p<0.001 ***). Figure 4.

Effect of P-3Fax-Neu5Ac on tumor growth is mediated by effector CD8+ immune cells.

A) Tumor infiltration of CD8+ T cells following 3 intratumoral injections with 0.3mg P- 3Fax-Neu5Ac or control P-Neu5Ac as determined by flow cytometry (n=3). Statistical significance was determined by ANOVA followed by Bonferroni's correction using Prism 5.03 (GraphPad Software, Inc., La Jolla, CA), p-values <0.05 were considered significant (p<0.05 *, p<0.01 **, p<0.001 ***).

B) On day 0, 50.000 B16-F10 cells were inoculated subcutaneously into C57BL/6 mice. On day 8 and 20 post inoculation, 330μg of isotype monoclonal antibody or depleting monoclonal antibody directed against CD4, CD8 or NKl . l was injected intraperitoneal. From day 10 onwards PBS or 0.3mg P-3Fax-Neu5Ac were injected intratumoral three times per week. Tumor growth in the different treatment groups is shown in time as mean tumor volume (mm3) ± SEM (n=6).

Figure 5.

Sialic acid blockade with P-3Fax-Neu5Ac synergizes with CpG immune adjuvant.

A) Timeline showing tumor growth experiment and treatment schedule. On day 0, 50.000 B16-F10 cells were inoculated subcutaneously into C57BL/6 mice and from day 10 onwards 3 intratumoral P-3Fax-Neu5Ac injection were administered per week. On day 14 and day 21 post inoculation, 100μg CpG was injected peritumoral.

B) Tumor growth in the different treatment groups is shown in time as mean tumor volume (mm3) ± SEM (n=l l-12). Statistical significance was determined by ANOVA followed by Bonferroni's correction using Prism 5.03 (GraphPad Software, Inc., La Jolla, CA), p-values <0.05 were considered significant (p<0.05 *, p<0.01 **, p<0.001 ***). C) Kaplan-Meyer curves represent percentage survival of the different treatment groups. Log-rank test was used to determine statistically significant differences between the P- 3Fax-Neu5Ac and the P-3Fax-Neu5Ac + CpG treatment groups. Figure 6.

Incorporation of P-3Fax-Neu5Ac into melanoma antigen TRP-1 -targeting nanoparticles.

A) Expression of the melanoma-specific antigen TRP-1 (tyrosinase-related protein- 1) on the surface of B16-F10 cells. Cells were stained with isotype control Phycoerythrin- conjugated antibodies (upper histogram) or Phycoerythrin-conjugated anti-TRP-1 monoclonal antibodies (lower histogram).

B) Schematic representation of TRP-1 -targeting nanoparticles containing P-3Fax- Neu5Ac. P-3Fax-Neu5Ac is incorporated into PLGA (poly(lactic-co-glycolic acid))- based nanoparticles with a diameter of 200nm. Nanoparticles are coated with a PEG (polyethylene glycol) lipid layer that allow conjugation with monoclonal anti-TRP-1 antibodies.

C) Confocal images showing uptake of isotype antibody (left) or anti-TRP-1 -targeting (right) P-3Fax-Neu5Ac nanoparticles by B16-F10 cells. Cells were incubated for 16 hours with lmg/ml nanoparticles containing FSA-P1 and fluorescent ATTO 647 Dye (green), fixed and nuclei were stained with DAPI (blue).

D) Sialic acid blockade in B16-F10 cells using TRP-1 -targeting P-3Fax-Neu5Ac nanoparticles. Cells were incubated for 3 days with lmg/ml isotype control or anti-TRP- 1 -targeting P-3Fax-Neu5Ac nanoparticles or an equal amount of soluble P-3Fax-Neu5Ac (FSA-P1) (23. ^g/ml). Sialic acid expression was quantified by flow cytometry using fluorescent MALII lectin (n=3). Statistical significance was determined by ANOVA followed by Bonferroni's correction using Prism 5.03 (GraphPad Software, Inc., La Jolla, CA), p-values <0.05 were considered significant (p<0.05 *, p<0.01 **, p<0.001 ***).

Figure 7.

Targeted delivery of P-3Fax-Neu5Ac precludes metastatic spread.

A) Treatment schedule. 20mg/kg isotype control antibody- or anti-TRP-1 antibody- coated P-3Fax-Neu5Ac NPs or empty anti-TRP-1 antibody-coated NPs were injected intravenously into the tail vein of mice. 1 hour later, 0.5x106 B16-F 10 cells were injected into the tail vein of mice (n=6). 16 hours following injection with B16-F10 cells, another dose of 20mg/kg NPs was administered intravenously and 14 days later, mice were sacrificed and lungs were collected and fixed overnight in Fekete's solution. Representative images show formation of B16-F10 nodules in the lung

(B). All nodules were enumerated and are presented in a scatter dot plot (C).

Figure 8

Intratumoral injections of P-3F _ax -Neu5Ac hamper 9464D neuroblastoma growth.

A) Tumor growth in volume v/s time

B) Kaplan-Meyer survival curve

Figure 9

Effect of CD8 ⁺ cell depletion on tumor growth suppression by P-3F _ax -Neu5 Ac.

A) Tumor growth in volume v/s time

B) Kaplan-Meyer survival curve

Figure 10

Sialic acid blockade using P-3F _ax -Neu5 Ac enhances APC maturation with TLR ligands. A) CD80 expression on P-3F _ax -Neu5Ac treated mouse dendritic cells, fold change in view of control

B) CD80 expression on P-3F _ax -Neu5Ac-treated dendritic cells of six human donors normalized to control

The present invention is further described by the following examples which should not be construed as limiting the scope of the invention.

Unless stated otherwise, the practice of the invention will employ standard

conventional methods of molecular biology, virology, microbiology or biochemistry. Such techniques are described in Sambrook et al. (1989) Molecular Cloning, A

Laboratory Manual (2 ^nd edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press; in Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, NY; in Volumes 1 and 2 of Ausubel et al. (1994) Current Protocols in Molecular Biology, Current Protocols, USA; and in Volumes I and II of Brown (1998) Molecular Biology LabFax, Second Edition, Academic Press (UK); Oligonucleotide Synthesis (N. Gait editor); Nucleic Acid Hybridization (Hames and Higgins, eds.).

Examples

Example 1. Synergy of a sialic acid blocker and immune adjuvant CpG in the treatment of cancer.

Introduction

Tumor cells of various origin have been described to cover their cell surface with aberrant levels of sialic acid sugars. Sialic acids are attached to cell surface proteins and lipids (glycoproteins and glycolipids). This process is carried out in the Golgi system by a specific enzyme family termed sialyltransferases. So far, more than 20 of these sialyltransferases have been discovered in mice and men. Each of them attaches sialic acid sugars to glycoproteins and glycolipids via a specific bond (glycosidic bond) to specific proteins and lipids. Tumor cells overexpress sialyltransferases and subsequently produce abnormally high amounts of sialic acid-carrying glycoproteins and lipids. High expression of sialyltransferases and sialic acids contributes to tumor growth and metastasis formation and correlates with a poor prognosis for patients. Therefore, specific strategies to block aberrant sialic acid production in tumors could be of high therapeutic value.

Whereas the role of sialic acids in tumor migration and metastasis formation is under extensive investigation, we started two years ago to study the immunomodulatory role of tumor sialic acids and defined them as targets to improve tumor immunotherapy. The rationale for this project bases on the biophysical properties of sialic acids and their potency not only to effect tumor cell/immune cell interactions but also to affect immune cells directly. First, sialic acids have a highly negative charge. Therefore, accumulation of sialic acids creates a net negative charge on the tumor cell surface that hinders interactions with immune cells. More specifically, sialic acids hinder the accessibility and recognition of structures on tumor cells such as antigens. Thereby, immune cells are physically hindered to screen the tumor cell surface with their recognition receptors and to recognize them as tumor cell.

Second, sialic acids have an immune suppressive activity and can dampen activation of the immune system. Sialic acids are expressed by virtually every cell. Eventually, sialic acids mark host cells as self in order to discriminate them from bacteria that do not express sialic acids. Therefore, all cells of the (mammalian) immune system express specific sialic acid-recognizing receptors (siglecs) to recognize host cells via their sialic acid sugars. Siglecs can inhibit activation of immune cells and even induce apoptosis among recognition of sialic acid ligands. We hypothesize that tumor cells inhibit immune cell function by covering their membrane with sialic acid molecules via the interaction with immune suppressive receptors such as siglecs. As a consequence, tumor cells eventually escape from natural recognition and eradication by the immune system. Furthermore, cross-talk between immune cell functions itself is regulated by sialic acid molecules and can be employed to boost immunity. Therefore, compounds that block sialylation via interference with sialyltransferase expression or function will be vaccine adjuvants alone or when combined with any type of immunotherapy aiming to activate the immune system to elicit an anti-tumor immune response. Objective

A solution to the aforementioned problem was hypothesized to be interference with sialic acid expression in tumor cells. Main targets are the sialyltransferases. Blocking the function of this enzyme family or hindering their expression would disable the production of sialic acids in tumor cells. Subsequently, the immune system could recognize tumor cells by screening their surface for antigens. Moreover the immune suppressive signals that sialic acids derived from tumor cells imposes on immune cells would be omitted. Combining this strategy with specific or unspecific activation of the immune system (e.g. via immune adjuvants, antibodies, cell therapy, tumor vaccines) would create a novel vaccine type that will pave the way for successful immunotherapy against cancer.

Results

Last year, Rillahan et al published the structure and synthesis of a specific inhibitor of sialyltransferases (P-3F _ax -Neu5Ac) and could show its ability to deplete sialic acids from mammalian cells without causing cellular cytotoxicity (Rillahan et al, 2012, Nat Chem Biol.) (Figure 1A-C). We have shown for the first time that this compound, P-3F _ax - Neu5Ac blocks the expression of sialic acids in mouse melanoma cells with high efficiency. Furthermore, we have described the kinetics of this sialyltransferase inhibitor and could show that the blocking effect lasts for almost four days. In accordance to literature, we observed that this inhibitor can impair adhesion and migration of tumor cells. Finally, melanoma cells that were treated with the inhibitor before injection into mice grow significantly slower (Biill et al, 2013).

We prompted to investigate the therapeutic potential of P-3F _ax -Neu5Ac in a mouse B 16- F10 melanoma model. Preliminary data clearly showed that injections of this inhibitor as a stand-alone treatment antagonizes tumor growth in a dose-dependent manner (Figure 2A-C). In this study we injected PBS, 0.3mg (lOmg/kg) or lmg (30mg/kg) non-blocking P-Neu5Ac control or 0.3mg (lOmg/kg) or lmg (30mg/kg) P-3F _ax -Neu5Ac directly into the tumor three times per week for four weeks. During this treatment period, the melanoma grew slower in the P-3F _ax -Neu5 Ac group in a dose-dependent manner.

In order to identify the mechanisms underlying the observed tumor remission, we have looked broadly at effects that occur after injections of P-3F _ax -Neu5 Ac into the tumor mass. After three injections with P-3F _ax -Neu5 Ac, mice were sacrificed and the sialic acid expression on the tumor tissue was assessed using fluorescent sialic acid binding lectins. We could show reduction of sialic acid expression in the isolated tumor tissue, but not other organs such as the spleen (Figure 3 A-D). Finally, we observed enhanced infiltration of immune cells, especially CD8 ⁺ cells in the tumor following treatment (Figure 4 A)

In order to confirm the involvement of the immune system in the effect of sialic acid blockade with P-3F _ax -Neu5Ac on tumor growth, we depleted effector immune cell populations (CD8 ⁺ , CD4 ⁺ , K1.1 ⁺ cells) capable of killing tumor cells in mice and tested if P-3F _ax -Neu5Ac can still effect tumor growth. Strikingly, while the effect of sialic acid blockade on tumor growth upon CD4 ⁺ cell and K1.1 ⁺ cell depletion was unaffected, CD8 ⁺ cell depletion abrogated the effect of sialic acid blockade on tumor growth (Figure 4B). These data demonstrate the involvement of the immune system and in particular CD8 ⁺ cells to mediate the effect of sialic acid blockade.

CD8 ⁺ cells mainly comprise effector T cells that can specifically activated by immunotherapy to recognize and eradicate tumor cells. Therefore, we assessed if sialic acid blockade can help to boost cancer immunotherapy. In addition to the intratumoral P-3F _ax -Neu5Ac injections (0.3mg) three times per week as described above, the immune adjuvant CpG (100μg) was injected peritumoral on day 14 and 21 post inoculation. While injections with CpG alone had no effect on tumor growth and median survival time, and P-3F _ax -Neu5Ac injections alone slightly prolonged the survival time in this stringent tumor model, co administration resulted in a strong synergistic effect leading to dramatic reduction in tumor growth and doubling of the median survival time (Figure 5A, B). For targeted delivery of P-3F _ax -Neu5Ac to tumor cells in vivo we have developed tumor- targeting nanoparticles made from FDA-approved poly(lactic-co-glycolic acid) that contain P-3F _ax -Neu5Ac and effectively and specifically block sialic acid expression in tumor cells (Figure 6A-D). These tumor-targeting nanoparticles allowed delivery of P- 3F _ax -Neu5 Ac to tumor cells in vivo and precluded metastasis formation in a mouse cancer metastasis model (Figure 7 A-C).

Next, we prompted to investigate whether sialic acid blockade can be applied to different tumor types. We used the autologous 9464D neuroblastoma model in C57B1/6 mice. On day 0, 10xl0 ⁶ 9464D cells were inoculated subcutaneously into C57BL/6 mice. From day 10 onwards, PBS, lOmg/kg or 20m/kg P-3F _ax -Neu5Ac were injected intratumoral three times per week until day 50. Tumor growth and survival times in the different treatment groups (n=12) was monitored until day 62. Tumor growth in the different treatment groups is shown in time as mean tumor volume (mm ³ ) ± SEM (Figure 8A). Statistical significance was determined by ANOVA followed by Bonferroni's correction using Prism 5.03 (GraphPad Software, Inc., La Jolla, CA), p-values <0.05 were considered significant (p<0.05 *, p<0.01 **, p<0.001 ***). Kaplan-Meyer curves represent percentage survival of the different treatment groups (Figure 8B). Log-rank test was used to determine statistically significant differences between the different treatment groups compared to the PBS control group.

The results demonstrate that sialic acid blockade alone has a suppressive effect on tumor growth in the 9464D neuroblastoma model. These data substantiate that sialic acid blockade can be applied to different tumor types.

Next, we set out to determine whether sialic acid blockade has an effect on CD8 ⁺ T cell mediated tumor growth suppression. We used the B16-F10 C57B1/6 mouse melanoma model and compared treatment with P-3F _ax -Neu5 Ac in CD8 ⁺ depleted- and non-depleted C56B1/6 mice. On day 0, 50 x 10 ³ B16-F10 cells were inoculated subcutaneously into C57BL/6 mice. On day 8, 300μ§ isotype control antibodies or CD8 ⁺ cell-depleting antibodies were injected intraperitoneal and from day 10 onwards, PBS or 0.3mg P-3F _ax -Neu5Ac were injected intratumoral three times per week until day 40. Tumor growth and survival times in the different treatment groups (n=6) was monitored until day 50. Tumor growth in the different treatment groups is shown in time as mean tumor volume (mm ³ ) ± SEM (Figure 9A). Statistical significance was determined by ANOVA followed by Bonferroni's correction using Prism 5.03 (GraphPad Software, Inc., La Jolla, CA), p-values <0.05 were considered significant (p<0.05 *, p<0.01 **, p<0.001 ***). Kaplan-Meyer curves represent percentage survival of the different treatment groups (Figure 9B). Log-rank test was used to determine statistically significant differences between the different treatment groups compared to the isotype + PBS control group.

The results demonstrate that CD8 ⁺ T cells mediate tumor growth suppression upon sialic acid blockade in the B16-F10 melanoma model. These data substantiate that sialic acid blockade has a positive effect on immunotherapy that induces a CD8 ⁺ T cell response.

Next, we set out to determine whether sialic acid blockade can enhance Toll-like receptor mediated maturation of professional antigen presenting cells.

Mouse bone marrow-derived dendritic cells were cultured for three days in the presence or absence of P-3F _ax -Neu5Ac and stimulated for 24 hours with toll-like receptor agonists LPS, CpG or polyl/C. Maturation of the dendritic cells was assessed by quantifying surface expression of the maturation marker CD80 using flow cytometry. The histogram (Figure 10A) shows CD80 expression on P-3Fax-Neu5Ac-treated dendritic cells normalized to control (n=6). Human monocyte-derived dendritic cells were incubated for four days with or without P-3F _ax -Neu5Ac and stimulated for 24 hours with the TLR agonists polyl/C, R848 and LPS. The histogram (Figure 10B) shows CD80 expression on P-3F _ax -Neu5 Ac-treated dendritic cells of six human donors normalized to control. The results demonstrate that sialic acid blockade strongly enhances activation of dendritic cells of human and mouse origin with different types of immune adjuvants. These data substantiate that next to CpG other TLR agonists and immune adjuvants can synergize with sialic acid blockade. Collectively, these data provide evidence that depletion of sialic acids from tumors and immune cells in vivo leads to activation of the immune system and potentially elucidation of a strong anti-tumor immune response. Strikingly, further boosting of anti-tumor immunity using the immune adjuvant CpG synergized the effect of sialic acid blockade. Therefore we suggest that strategies to deplete aberrant sialic acid expression in tumor cells and immune cells (e.g. with P-3Fax-Neu5Ac) can work synergistically and could in combination with immune activating approaches (e.g. CpG) lead to the development of strong tumor immune therapies. Here, the tumor-targeting nanoparticles could serve as co-delivery strategy for the save use in vivo.

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