Compositions of virus-like particles and microcrystalline tyrosine

Abstract

The present invention relates to compositions comprising virus-like particles and microcrystalline tyrosine, and their use for treating cancer.

Description

[Technical field]

[0001] The present invention relates to compositions comprising virus-like particles and microcrystalline tyrosine, and their use for treating cancer. [Background technology]

[0002] Cancer immunotherapy has brought significant improvements to patients in terms of survival and quality of life in recent decades and has established itself as a mainstay of cancer care (Esfahani K et al. Curr Oncol. 2020 April:27(S2)87-97).

[0003] Among them, cancer vaccines have been developed herewith, aiming to avoid oncogenic infection or destroy cancer cells, as well as classical vaccination to train the immune system to fight off pathogenic pathogens.Great success has been achieved through preventive cancer vaccines against human papillomavirus (HPV), which causes cervical cancer, and hepatitis B virus (HBV), which causes liver cancer (Stanley M., Philos Trans R Soc Lond B Biol Sci.2017;372(1732); Liu MA., Philos Trans R Soc Lond B Biol Sci.2011;366(1579):2823-6).

[0004] In contrast to prevention, the development of vaccines against established cancers is more challenging because tumors are often immunosuppressive and suitable targets are difficult to identify. Moreover, vaccines used so far have rather low immunogenicity (Hollingsworth RE, Jansen K., NPJ Vaccines. 2019; 4: 7). Previous attempts to develop therapeutic cancer vaccines have been based mainly on targeting tumor-associated antigens (TAA) and more recently tumor-specific antigens (TSA). Targeting of TAA is likely associated with immune tolerance to self-antigens, but has only shown moderate efficacy. High-affinity T cells against self-antigens are eliminated during T cell development, and further peripheral tolerance mechanisms are responsible for keeping the immune response at a low level. In contrast, TSAs are either virus-derived or mutated self-peptides, for which T cell tolerance is not induced or is not very expected. Nevertheless, the immune system may not respond adequately to these tumor antigens, and the immune system may be weakened due to side effects of cancer treatment, patient age and T cell exhaustion (Hollingsworth RE, Jansen K., NPJ Vaccines. 2019;4:7).

[0005] The use of virus-like particles (VLPs) for vaccination against cancer has recently attracted increasing interest due to their ability to induce combined effective cellular and humoral immune responses (Mohsen MO et al., WIREs Nanomed Nanobiotechnol. 2019; e1579). To further increase CTL responses upon vaccination with VLPs, physiological properties of the lymphatic system were exploited. When Cucumber Mosaic Virus-Like Particles presenting LCMV-gp33 peptides were formulated with micron-sized microcrystalline adjuvant (MCT), it was shown that not only did CuMVTT-p33 nanoparticles decorate the surface of the micron-sized MCT adjuvant and form local depots, but vaccination with this combination enhanced specific CTL responses and mediated strong antitumor effects in a stringent B16F10p33 mouse tumor model (Mohsen, Heath et al., 2019; Heath MH et al., 2020, Frontiers in Immunology 11:594911).

[0006] In situ (intratumoral) immunotherapy has also attracted interest in recent years in the treatment of solid tumors, aiming to produce local and systemic antitumor effects (Marabelle A et al., Ann Oncol. 2017;28(suppl_12):xii33-xii43; Nobuoka D et al., Hum Vaccin Immunother. 2013;9(6):1234-6). In particular, the therapeutic accessibility of malignant melanoma lesions has increased interest towards the development of in situ and localized therapies (Middleton MR et al., Br J Cancer. 2020;123(6):885-97). Some recent examples of intratumoral treatments in melanoma include CMP-001, a virus-like particle (VLP) loaded with A-type CpG in combination with systemic anti-PD-1 (Ribas A et al., Cancer Discov. 2021, 2998). Furthermore, Lizotte et al. showed that intratumoral administration of eCPMV was effective in treating cutaneous B16F10 melanoma, forming central tumor necrosis (Lizotte PH et al., Nat Nanotechnol. 2016;11(3):295-303). Another study combined flexible plant VLPs (PVX) with the chemotherapy drug doxorubicin (DOX), either packaged in the VLPs or mixed together, to treat B16F10 melanoma, showing that intratumoral injection of PVX-VLPs could delay tumor progression, and co-administration of VLPs with DOX enhanced the induced antitumor response and increased survival (Lee KL et al., Nano Lett. 2017;17(7):4019-28).

[0007] In light of the still substantial challenges to improving immunotherapy for cancer, there is a need for effective, affordable, rapid and safe immunotherapies that can be broadly used for various types of tumors, especially solid tumors. Summary of the Invention

[0008] The present inventors have surprisingly found that microcrystalline tyrosine (MCT) formulated and decorated with virus-like particles (VLPs) results in a robust and efficient antitumor response and inhibits local and distant tumor growth. Importantly, the immunotherapy of the present invention does not require identification of the relevant antigens of the individual patient, and furthermore, the compositions of the present invention do not contain tumor-associated or tumor-specific antigens associated or covalently bound to the VLPs.

[0009] In particular, a preferred composition of the invention comprising microcrystalline tyrosine (MCT) formulated and decorated with a virus-like particle (VLP) has been found to protect against murine melanoma and inhibit B16F10 tumor growth locally and systemically, as shown in a B16F10 xenograft melanoma mouse model. Furthermore, intratumoral injection of the composition of the invention results in the formation of an immunogenic depot in the injected tumor, and induces the uptake of polyfunctional CD8 + and CD4 + T cells were enhanced, supporting the induction of systemic protection (from uninjected distant tumors) via recirculating T cells. In addition, local inflammatory and immune responses were associated with upregulation of genes involved in complement activation and collagen formation.

[0010] Thus, in a first aspect, the present invention provides a composition comprising particles, each particle comprising: (a) at least one virus-like particle (VLP), preferably a recombinant VLP; (b) at least one microcrystalline tyrosine, preferably microcrystalline L-tyrosine, having a median particle size of 0.1 μm to 50 μm, preferably 1 μm to 10 μm, preferably as determined by Flow Particle Image Analyser (FPIA), and more preferably as described in Example 2; and preferably consisting of the VLP does not comprise a tumor-associated or tumor-specific antigen covalently bound to the VLP, and more preferably the VLP does not comprise an antigen (such as an allergen) covalently bound to the VLP; A composition is provided.

[0011] Preferably, the composition is capable of treating a tumor, preferably a malignant tumor, more preferably, a solid tumor, preferably a malignant solid tumor, and even more preferably, the composition is capable of treating a malignant solid tumor intratumorally.

[0012] In a further aspect, the present invention provides a method for producing a composition comprising the steps of: (a) at least one virus-like particle (VLP), preferably a recombinant VLP; (b) at least one microcrystalline tyrosine, preferably microcrystalline L-tyrosine, having a median particle size of 0.1 μm to 50 μm, preferably 1 μm to 10 μm, preferably as determined by Flow Particle Image Analyser (FPIA), and more preferably as described in Example 2; and preferably consisting of the VLP does not comprise a tumor-associated or tumor-specific antigen covalently bound to the VLP, and more preferably the VLP does not comprise an antigen (such as an allergen) covalently bound to the VLP; A composition is provided.

[0013] Preferably, the composition is capable of treating a tumor, preferably a malignant tumor, more preferably, a solid tumor, preferably a malignant solid tumor, and even more preferably, the composition is capable of treating a malignant solid tumor intratumorally.

[0014] In a still further aspect, the invention provides a composition comprising particles, each particle comprising: (a) at least one virus-like particle (VLP), preferably a recombinant VLP; (b) at least one microcrystalline tyrosine, preferably microcrystalline L-tyrosine, having a median particle size of 0.1 μm to 50 μm, preferably 1 μm to 10 μm, preferably as determined by Flow Particle Image Analyser (FPIA), and more preferably as described in Example 2; and preferably consisting of the VLP does not comprise a tumor associated antigen covalently bound to the VLP, and more preferably the VLP does not comprise an antigen (such as an allergen) covalently bound to the VLP; A composition is provided.

[0015] Preferably, the composition is capable of treating a tumor, preferably a malignant tumor, more preferably, a solid tumor, preferably a malignant solid tumor, and even more preferably, the composition is capable of treating a malignant solid tumor intratumorally.

[0016] In a still further aspect, the present invention provides a method for producing a composition comprising the steps of: (a) at least one virus-like particle (VLP), preferably a recombinant VLP; (b) at least one microcrystalline tyrosine, preferably microcrystalline L-tyrosine, having a median particle size of 0.1 μm to 50 μm, preferably 1 μm to 10 μm, preferably as determined by Flow Particle Image Analyser (FPIA), and more preferably as described in Example 2; and preferably consisting of the VLP does not comprise a tumor associated antigen covalently bound to the VLP, and more preferably the VLP does not comprise an antigen (such as an allergen) covalently bound to the VLP; A composition is provided.

[0017] Preferably, the composition is capable of treating a tumor, preferably a malignant tumor, more preferably, a solid tumor, preferably a malignant solid tumor, and even more preferably, the composition is capable of treating a malignant solid tumor intratumorally.

[0018] Further aspects and embodiments of the invention will become apparent as this description continues. [Brief description of the drawings]

[0019] [Figure 1A] A sketch of a difficult-to-treat mouse melanoma model. [Figure 1B] FIG. 1 shows vaccination regimens and groups using intratumoral injection. [Figure 1C] FIG. 1 shows the percentage of CD11bHi Ly6CHi (mean±SEM) in the blood of each group of mice implanted with B16F10 melanoma tumors on day 12. [Figure 1D] Tumor volume in mm3 (mean ± SEM) measured 14 days after tumor implantation for groups treated with PBS, CuMVtt-VLP, or CuMVtt-VLP formulated with MCT, with each dot representing a tumor. [Figure 1E] Figure 1 shows the density of CD8+ T cells (mean ± SEM) in tumors pre-gated on TILs (tumor infiltrating lymphocytes). Density was measured by dividing the total number of CD8+ T cells in each tumor by its volume. [Figure 1F] FIG. 1 shows the correlation between density of CD8+ T cells and tumor volume in mm3. [Figure 1G] Representative FACS plots showing total numbers of CD8+ T cells in tumors pre-gated on TILs. Statistical analysis by Student's t-test (n=11 in control group, n=10 in other groups). [Figure 1H]Figure 1 shows the density of CD8+IFN-γ+ T cells (mean ± SEM) in tumors pre-gated for TILs. Density was measured by dividing the total number of CD8+IFN-γ+ T cells in each tumor by its volume. For (n=5). One representative of two experiments is shown. *P<0.05: **P<0.01: ***P<0.0001 [Figure 1I] Representative FACS plot showing total numbers of CD8+IFN-γ+ producing cells pre-gated on TILs. [Figure 2A] FIG. 1 shows total IgG antibodies against CuMVtt-VLP in the serum of mice treated intratumorally with PBS, CuMVtt-VLP, or CuMVtt-VLP formulated with MCT, as measured by OD450. [Figure 2B] Figure 1 shows a 1:20 dilution of total IgG antibodies against CuMVtt-VLP in serum from mice treated intratumorally with PBS, CuMVtt-VLP, or CuMVtt-VLP formulated with MCT, as measured by OD450. Statistical analysis by Student's t-test (n=5). One representative of two experiments is shown. *P<0.05: **P<0.01: ***P<0.0001. [Figure 3A] FIG. 13 shows the PBS-treated control group, where necrosis constituted 30% of the tumor section. Necrosis is shown as grey areas with a white lining (*). [Figure 3B] Figure 1 shows the group treated with CuMVtt-VLP+MCT, where necrosis constituted 2% of the tumor section, shown as grey areas with a white lining (*). [Figure 3C] FIG. 1 shows the PBS-treated control group, which features the highest tumor resistance (TMR). Each white dot corresponds to a mitotic cell figure. Note that only the left side of the tumor (approximately 60%) was evaluated for TMR. [Figure 3D] Figure 1 shows the group treated with CuMVtt-VLP+MCT. The tumor area was 17 mm2, and the dividing cell ratio (TMR) was 47 / mm2. Each white dot corresponds to a dividing cell image. The TMR of the entire tumor cross section was evaluated. H&E staining. [Figure 4A] FIG. 1 shows the experimental design. [Figure 4B] FIG. 1 shows the % of CD8+ T cells in the blood of naive C57 / BL / 6 mice before and after CD8+ T cell depletion using anti-CD8a mAb, with depletion efficacy of approximately 95%. [Figure 4C] Representative FACS plots showing CD8+ T cell populations before and after depletion. [Figure 4D] FIG. 13 shows the % of CD8+ T cells in the blood (after CD8+ T cell depletion) of C57 / BL / 6 mice bearing B16F10 tumors and treated with PBS, CuMVtt-VLP+MCT or intratumoral injection of CuMVtt-VLP+MCT. [Figure 4E] Tumor volume mm3 (mean + / - SEM) measured 14 days after tumor implantation for groups treated with PBS, CuMVtt-VLP+MCT or CuMVtt-VLP+MCT (after CD8+ T cell depletion). Each dot represents a tumor, statistical analysis by Student's t-test (n=5). One representative of two experiments is shown. *P<0.05: **P<0.01: ***P<0.0001. [Figure 5A] 1 is a sketch of a difficult-to-treat mouse melanoma model for abscopal effect experiments, including groups using vaccination regimens and intratumoral injections. [Figure 5B] FIG. 1 shows the tumor volume in mm3 (mean ± SEM) of left and right tumors measured 14 days after tumor implantation for groups treated with CuMVtt-VLP formulated with PBS or MCT, with each dot representing a tumor. [Figure 5C] Representative right (treated) and left (untreated) mice bearing B16F10 melanoma tumors on day 14. [Figure 5D] Figure 1 shows the density of CD8+ T cells (mean ± SEM) in left and right tumors pre-gated on TILs (tumor infiltrating lymphocytes). Density was measured by dividing the total number of CD8+ T cells in each tumor by its volume. [Figure 5E]Representative FACS plots showing total numbers of CD8+ T cells in left and right tumors pre-gated for TILs. Statistical analysis by Student's t-test (n=5). One representative of two experiments is shown. *P<0.05: **P<0.01: ***P<0.0001. [Figure 6] FIG. 1 shows tumor volume mm3 (mean±SEM) measured 14 days after tumor implantation for groups treated with PBS, Qb-VLP formulated with MCT, CuMVtt-VLP formulated with MCT, or CCMVtt-VLP formulated with MCT, where each dot represents a tumor. Due to internal variation with each group and small sample numbers, we combined different VLPs+MCT and compared it with the PBS group. Statistical analysis by Student's t-test (n=5). One representative experiment is shown. *P<0.05: **P<0.01: ***P<0.0001. [Figure 7A] FIG. 1 shows the surgical dissection of a preputial gland melanoma mass in a gray stallion, including amputation of the male genitalia. [Figure 7B] The outer solid melanoma lesion on the lower part of the tail is circled. [Figure 7C] FIG. 10: 8 days after intratumoral injection of the novel immune enhancer. Note the softening of the lesion and the secretion of the black melanoma. [Figure 7D] FIG. 1 shows a melanoma lesion two days after the third intratumoral injection; note the sloughing off of the solid tumor. [Figure 7E] FIG. 1 shows a melanoma lesion two days after the third intratumoral injection; note the sloughing off of the solid tumor. [Figure 7F] The same lesion is shown one year after treatment, with no melanoma progression noted. [Figure 7G] FIG. 1 shows an untreated melanoma nodule in the upper part of the tail, indicated by an arrow. [Figure 7H] The untreated nodules disappeared after 1 year. [Figure 7I] FIG. 1 shows an ultrasound image of a grey mare with an untreated mammary mass measuring 4.61 cm by 6.12 cm. [Figure 7J] FIG. 1 shows an ultrasound image of a mammary mass after a single dose of intratumoral guided injection of a novel immune enhancer, measuring 3.02 cm×5.07 cm. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0021] As used herein, the term "about" shall mean + / - 10%. For example, about 50% shall mean 45% to 55%. Preferably, as used herein, the term "about" shall mean + / - 5%. For example, about 50% shall mean 47.5% to 52.5%.

[0022] As used herein, the phrase "number X to number Y" includes number X and number Y. For example, the phrase "0.1 μm to 50 μm" refers to 0.1 μm and 50 μm as well as values ​​therebetween. The same applies to the phrase "approximate number X to approximately number Y."

[0023] As used herein, the term "% (w / v)" refers to (mass of component / total volume of composition) x 100. As an example, 4% by weight L-tyrosine refers to 4 g of L-tyrosine per 100 ml of composition.

[0024] The terms "a" or "an" as used herein mean "at least one," unless otherwise specified.

[0025] Virus-like particle (VLP): As used herein, the term "virus-like particle (VLP)" refers to a non-replicative or non-infectious, preferably non-replicative and non-infectious, virus particle, or a non-replicative or non-infectious, preferably non-replicative and non-infectious structure resembling a virus particle, preferably a viral capsid. As used herein, the term "non-replicative" refers to the inability to replicate the genome contained in the VLP. As used herein, the term "non-infectious" refers to the inability to enter a host cell. Virus-like particles according to the invention are non-replicative and non-infectious because they lack all or part of the viral genome or genome function. Virus-like particles according to the invention may contain nucleic acids different from their genome. Recombinantly produced virus-like particles typically contain host cell-derived RNA. An exemplary and preferred embodiment of a virus-like particle according to the invention is a viral capsid composed of a polypeptide of the invention. Virus-like particles are typically macromolecular assemblies composed of viral coat proteins, typically containing 60, 120, 180, 240, 300, 360 or more than 360 protein subunits per virus-like particle. Typically and preferably, the interaction of these subunits results in the formation of a viral capsid or viral capsid-like structure with a unique repetitive organization. One characteristic of virus-like particles is the highly ordered repetitive arrangement of their subunits.

[0026] Virus-like particle of RNA bacteriophage: As used herein, the term "virus-like particle of RNA bacteriophage" refers to a virus-like particle that comprises, or preferably consists essentially of, coat proteins, variants or fragments thereof of RNA bacteriophage. In addition, the virus-like particle of RNA bacteriophage is similar in structure to RNA bacteriophage, is non-replicative and / or non-infectious, lacks at least one gene or genes encoding the replication mechanism of RNA bacteriophage, and typically also lacks one or more genes encoding one or more proteins involved in viral attachment to or invasion of the host. Also included are virus-like particles of RNA bacteriophage, where one or more genes are still present but inactive, thus resulting in non-replicative and / or non-infectious virus-like particles of RNA bacteriophage. Preferred VLPs derived from RNA bacteriophage exhibit icosahedral symmetry and consist of 180 subunits (monomers). Preferred methods for rendering RNA bacteriophage virus-like particles non-replicative and / or non-infectious are by physical or chemical inactivation, e.g., UV irradiation, formaldehyde treatment, or, typically and preferably, by genetic manipulation.

[0027] CuMV virus-like particle: The term "CuMV virus-like particle" or CuMV VLP refers to a virus-like particle that comprises, or preferably consists essentially of, or preferably consists of at least one CuMV polypeptide. Preferably, the CuMV virus-like particle comprises said CuMV polypeptide as the major, and even more preferably, the only protein component of the capsid structure. Typically and preferably, the CuMV virus-like particle resembles the structure of the CuMV capsid. The CuMV virus-like particle is non-replicative and / or non-infectious, lacking at least one gene or genes encoding at least the replication machinery of CuMV, and typically also lacking one or more genes encoding one or more proteins involved in viral attachment to or entry into the host. This definition also includes virus-like particles in which the above-mentioned one or more genes are still present but inactive. A preferred method of rendering CuMV virus-like particles non-replicative and / or non-infectious is by physical or chemical inactivation, such as UV irradiation, formaldehyde treatment. Preferably, the CuMV VLP lacks one or more genes encoding the CuMV replication machinery and also lacks one or more genes encoding one or more proteins involved in viral attachment to or entry into the host. Even more preferably, the non-replicative and / or non-infectious virus-like particles are obtained by recombinant gene technology. The recombinantly produced CuMV virus-like particles according to the invention are typically and preferably free of viral genomes. The present invention also encompasses virus-like particles comprising two or more polypeptides, often referred to as mosaic VLPs. Thus, in one embodiment, the virus-like particles according to the present invention comprise at least two different species of polypeptides, at least one of said species of polypeptides being a CuMV polypeptide. Preferably, the CuMV VLP is a macromolecular assembly composed of CuMV coat protein, typically comprising 180 coat protein subunits per VLP.Typically and preferably, a CuMV VLP as used herein comprises, consists essentially of, or alternatively consists of at least one CuMV polypeptide comprising, or preferably consisting of, (i) an amino acid sequence of a CuMV coat protein, or (ii) a mutated amino acid sequence, wherein the mutated amino acid sequence is an amino acid sequence of a CuMV coat protein, and wherein the mutated amino acid sequence and the mutated amino acid sequence exhibit at least 90%, preferably at least 95%, more preferably at least 98%, and even more preferably at least 99% sequence identity.

[0028] Tumor-associated antigen (TAA) / tumor-specific antigen (TSA): The terms "tumor-associated antigen (TAA)" and "tumor-specific antigen (TSA)" are known to those skilled in the art. They are used herein as defined and further described in Hollingsworth RE, Jansen K., NPJ Vaccines. 2019;4:7. (See especially FIG. 1).

[0029] Solid tumor: A solid tumor can be defined as an abnormal mass of solid tissue. Solid tumors can be benign or malignant and are named according to the cell type of origin. Examples of malignant tumors are carcinoma (derived from epithelial cells), sarcoma (derived from mesenchymal cells) or lymphoma (derived from lymphocytes). In contrast, so-called "liquid tumors" (e.g., leukemia) do not form solid tumors. Thus, solid malignant tumors and cancers are defined as abnormal cell proliferation in "parenchymal" organs such as the breast or prostate, respectively, in contrast to leukemia, which is a cancer that affects the blood, which is liquid. The terms "solid tumor" and "solid malignant tumor" are used interchangeably herein.

[0030] Antigen: As used herein, the term "antigen" refers to a molecule that, when presented by an MHC molecule, can be bound by an antibody or a T cell receptor (TCR). An antigen can further be recognized by the immune system and / or can induce a humoral and / or cellular immune response that results in the activation of B and / or T lymphocytes. However, the term "antigen" as used herein does not refer to the virus-like particles contained in the compositions and / or pharmaceutical compositions of the present invention, nor to the Th cell epitopes contained in the modified polypeptides that can assemble to form said VLPs. Moreover, the term "antigen" also does not refer to any component that forms the particles or compositions of the present invention, such as the coat protein or parts thereof.

[0031] Coat protein: The term "coat protein" refers to a viral protein, preferably a subunit of the native capsid of a virus, preferably a subunit of an RNA bacteriophage or plant virus, that can be incorporated into a viral capsid or VLP. The term coat protein encompasses naturally occurring coat proteins as well as recombinantly expressed coat proteins. Further encompassed are mutants and fragments of coat proteins, which mutants and fragments retain the ability to form VLPs.

[0032] Polypeptide: As used herein, the term "polypeptide" refers to a polymer composed of amino acid monomers linearly linked by peptide bonds (also known as amide bonds). The term polypeptide refers to a continuous chain of amino acids and does not refer to a specific length of the product. Thus, peptides and proteins are included within the definition of a polypeptide.

[0033] Cucumber Mosaic Virus (CuMV) Polypeptide: As used herein, the term "CuMV polypeptide" refers to a polypeptide comprising, or preferably consisting of, (i) an amino acid sequence of a coat protein of CuMV, or (ii) a mutated amino acid sequence, wherein the mutated amino acid sequence is an amino acid sequence of a coat protein of CuMV, and wherein the mutated amino acid sequence and the mutated amino acid sequence, i.e., the coat protein of CuMV, exhibit at least 90%, preferably at least 95%, more preferably at least 98%, and even more preferably at least 99% sequence identity. Typically and preferably, the CuMV polypeptide is capable of forming virus-like particles of CuMV upon expression by self-assembly.

[0034] Cucumber Mosaic Virus (CuMV) Coat Protein (CP): As used herein, the term "CuMV Coat Protein (CP)" refers to the naturally occurring Cucumber Mosaic Virus coat protein. Due to the extremely broad host range of CuMV, many different strains and isolates of CuMV are known, and the coat proteins of said strains and isolates have been sequenced and are therefore known to those skilled in the art. The sequences of said CuMV coat proteins (CP) are listed and retrievable in known databases such as Genbank, www.dpvweb.net or www.ncbi.nlm.nih.gov / protein / . As an example, they are listed in European Patent Application No. 14189897.3. Further examples of CuMV coat proteins are provided in SEQ ID NOs: 1, 10 and 13. It is noteworthy that these strains and isolates have very similar coat protein sequences in different protein domains, including the N-terminus of the coat protein. In particular, 98.1% of all fully sequenced CuMV isolates share greater than 85% sequence identity within the first 28 amino acids of their coat protein sequence, and still 79.5% of all fully sequenced CuMV isolates share greater than 90% sequence identity within the first 28 amino acids of their coat protein sequence. Typically and preferably, the coat protein of CuMV used in the present invention is capable of forming CuMV virus-like particles upon expression by self-assembly. Preferably, the coat protein of CuMV used in the present invention is capable of forming CuMV virus-like particles upon expression by self-assembly in E. coli.

[0035] Modified virus-like particle (VLP) of Cucumber Mosaic Virus (CuMV): As used herein, the term "modified virus-like particle (VLP) of CuMV" refers to a VLP of CuMV that is modified, i.e. comprises, or preferably consists essentially of, or preferably consists of at least one modified CuMV polypeptide, which comprises, or preferably consists of, a CuMV polypeptide and a helper T cell epitope. Typically and preferably, the helper T cell epitope (i) is fused to the N-terminus of the CuMV polypeptide, (ii) is fused to the C-terminus of the CuMV polypeptide, (iii) replaces a stretch of consecutive amino acids of the CuMV polypeptide, wherein the sequence identity between the substituted stretch of consecutive amino acids of the CuMV polypeptide and the helper T cell epitope is at least 15%, preferably at least 20%, or (iv) replaces an N-terminal region of the CuMV polypeptide, wherein the substituted N-terminal region of the CuMV polypeptide consists of 5 to 15 consecutive amino acids. Preferably, the helper T cell epitope replaces an N-terminal region of the CuMV polypeptide, wherein the substituted N-terminal region of the CuMV polypeptide consists of 5 to 15 consecutive amino acids, preferably 9 to 14 consecutive amino acids, more preferably 11 to 13 consecutive amino acids, and most preferably 11, 12 or 13 consecutive amino acids. Preferably, said modified VLP of CuMV of the present invention is a recombinant modified VLP of CuMV.

[0036] Modified CuMV polypeptide: As used herein, the term "modified CuMV polypeptide" refers to a CuMV polypeptide that has been modified as defined herein, the modified CuMV polypeptide comprising, or preferably consisting of, a CuMV polypeptide and a helper T cell epitope. Typically, the modified CuMV polypeptide is capable of forming a CuMV virus-like particle upon expression by self-assembly. Preferably, the modified CuMV polypeptide is a recombinant modified CuMV polypeptide that is capable of forming a CuMV virus-like particle upon expression by self-assembly in E. coli.

[0037] N-terminal region of a CuMV polypeptide: As used herein, the term "N-terminal region of a CuMV polypeptide" refers either to the N-terminus of the CuMV polypeptide, in particular to the N-terminus of a coat protein of CuMV, or to a region at the N-terminus of the CuMV polypeptide or coat protein of CuMV, but starting from the second amino acid at the N-terminus of the CuMV polypeptide or coat protein of CuMV, if the CuMV polypeptide or coat protein comprises an N-terminal methionine residue. Preferably, if the CuMV polypeptide or coat protein comprises an N-terminal methionine residue, from a practical point of view, the start codon encoding the methionine is usually deleted and added to the N-terminus of a Th cell epitope. More preferably, one, two or three further amino acids, preferably one amino acid, may optionally be inserted between the start methionine and the Th cell epitope for cloning purposes. The term "N-terminal region of a mutant amino acid sequence of a CuMV polypeptide or a CuMV coat protein" as used herein refers either to the N-terminus of the mutant amino acid sequence of the CuMV polypeptide or the coat protein of CuMV, or to the region of the N-terminus of the mutant amino acid sequence of the CuMV polypeptide or the coat protein of CuMV, but starting from the second amino acid at the N-terminus of the mutant amino acid sequence of the CuMV polypeptide or the coat protein of CuMV, if the mutant amino acid sequence contains an N-terminal methionine residue. Preferably, if the CuMV polypeptide or the coat protein contains an N-terminal methionine residue, from a practical point of view, the start codon encoding the methionine is usually deleted and added to the N-terminus of the Th cell epitope. More preferably, one, two or three further amino acids, preferably one amino acid, may optionally be inserted between the start methionine and the Th cell epitope for cloning purposes.

[0038] Recombinant Polypeptide: In the context of the present invention, the term "recombinant polypeptide" refers to a polypeptide obtained by a process that includes at least one step of recombinant DNA technology. Typically and preferably, recombinant polypeptides are produced in prokaryotic expression systems. It is clear to the skilled artisan that recombinantly produced polypeptides expressed in prokaryotic expression systems, such as E. coli, may contain an N-terminal methionine residue. The N-terminal methionine residue is typically cleaved from the recombinant polypeptide in the expression host during maturation of the recombinant polypeptide. However, cleavage of the N-terminal methionine may be incomplete. Thus, a preparation of recombinant polypeptides may contain a mixture of otherwise identical polypeptides with and without an N-terminal methionine residue. Typically and preferably, a preparation of recombinant polypeptides contains less than 10%, more preferably less than 5%, and even more preferably less than 1% of recombinant polypeptides with an N-terminal methionine residue.

[0039] Recombinant CuMV polypeptide: The term "recombinant CuMV polypeptide" refers to a CuMV polypeptide as defined above obtained by a process comprising at least one step of recombinant DNA technology. Typically and preferably, a preparation of recombinant CuMV polypeptide contains less than 10%, more preferably less than 5%, and even more preferably less than 1% of recombinant CuMV polypeptides having an N-terminal methionine residue. As a result, a recombinant virus-like particle of the invention may contain recombinant polypeptides that are otherwise identical, both with and without an N-terminal methionine residue.

[0040] Recombinant modified CuMV polypeptide: The term "recombinant modified CuMV polypeptide" refers to a modified CuMV polypeptide as defined above obtained by a process comprising at least one step of recombinant DNA technology. Typically and preferably, a preparation of recombinant modified CuMV polypeptide contains less than 10%, more preferably less than 5%, and even more preferably less than 1% of recombinant modified CuMV polypeptides having an N-terminal methionine residue. As a result, a recombinant virus-like particle of the invention may contain recombinant polypeptides that are otherwise identical, both with and without an N-terminal methionine residue.

[0041] Recombinant virus-like particle: In the context of the present invention, the term "recombinant virus-like particle" refers to a virus-like particle (VLP) obtained by a process that includes at least one step of recombinant DNA technology. Typically and preferably, the recombinant VLP is obtained by expression of a recombinant viral coat protein in a host, preferably a bacterial cell. Typically and preferably, the recombinant virus-like particle comprises at least one recombinant polypeptide, preferably a recombinant CuMV polypeptide or a recombinant modified CuMV polypeptide. Most preferably, the recombinant virus-like particle is composed of or consists of a recombinant CuMV polypeptide or a recombinant modified CuMV polypeptide. As a result, in the context of the present invention, when the definition of the recombinant VLPs of the present invention is achieved with reference to a specific amino acid sequence that includes an N-terminal methionine residue, the scope of these recombinant VLPs of the present invention includes VLPs formed by said specific amino acid sequence without said N-terminal methionine residue, but also includes VLPs formed by said specific amino acid sequence with said N-terminal methionine, even if typically in a small amount as shown herein. Furthermore, where the definition of a recombinant VLP of the present invention is achieved with reference to a particular amino acid sequence that includes an N-terminal methionine residue, it is still within the scope of the present invention to include VLPs that include both that amino acid sequence and an amino acid sequence that lacks an N-terminal methionine residue.

[0042] Mutant amino acid sequence: the term "mutated amino acid sequence" refers to an amino acid sequence obtained by introducing a defined set of mutations into a mutated amino acid sequence. In the context of the present invention, the mutated amino acid sequence is typically and preferably the amino acid sequence of the coat protein of CuMV. Thus, the mutated amino acid sequence differs from the amino acid sequence of the coat protein of CuMV by at least one amino acid residue, and the mutated amino acid sequence and the mutated amino acid sequence show at least 90% sequence identity. Typically and preferably, the mutated amino acid sequence and the mutated amino acid sequence show at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity. Preferably, the mutated amino acid sequence and the mutated sequence differ by at most 11, 10, 9, 8, 7, 6, 4, 3, 2 or 1 amino acid residues, more preferably the differences are selected from insertions, deletions and amino acid exchanges. Preferably, the variant amino acid sequence differs from the amino acid sequence of the coat protein of CuMV by at least one amino acid, preferably the difference is an amino acid exchange.

[0043] The position on an amino acid sequence corresponding to a given residue of another amino acid sequence can be identified by sequence alignment, typically and preferably using the BLASTP algorithm, most preferably using standard settings. Typical and preferred standard settings are: expectation threshold: 10; word size: 3; max match within query: 0; matrix: BLOSUM62; gap cost: presence 11, extension 1; composition adjustment: conditional composition score matrix adjustment.

[0044] Sequence identity: The sequence identity of two given amino acid sequences is determined based on the alignment of both sequences. Algorithms for determining sequence identity are available to those skilled in the art. Preferably, the sequence identity of two amino acid sequences is determined using publicly available computer homology programs such as the "BLAST" program (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi) or "CLUSTALW" (http: / / www.genome.jp / tools / clustalw / ), preferably by the "BLAST" program provided at the NCBI homepage at http: / / blast.ncbi.nlm.nih.gov / Blast.cgi, using the default settings provided therein. Typical and preferred standard settings are: expectation threshold: 10; word size: 3; maximum match within query range: 0; matrix: BLOSUM62; gap cost: presence 11, extension 1; composition adjustment: conditional composition score matrix adjustment.

[0045] Amino acid exchange: The term amino acid exchange refers to the replacement of a given amino acid residue in an amino acid sequence by any other amino acid residue with a different chemical structure, preferably by another proteinogenic amino acid residue. Thus, in contrast to the insertion or deletion of an amino acid, an amino acid exchange does not change the total number of amino acids of the amino acid sequence. Highly preferred in the context of the present invention is the replacement of an amino acid residue of the amino acid sequence to be mutated by a lysine or cysteine ​​residue.

[0046] Epitope: The term epitope refers to a continuous or discontinuous portion of an antigen, preferably a polypeptide, which can be specifically bound by an antibody or a T-cell receptor in the context of an MHC molecule. With respect to antibodies, specific binding excludes non-specific binding, but does not necessarily exclude cross-reactivity. An epitope typically comprises 5-20 amino acids in a spatial conformation unique to the antigenic site.

[0047] Helper T (Th) cell epitope: As used herein, the term "helper T (Th) cell epitope" refers to an epitope capable of recognition by helper Th cells. In another preferred embodiment, the helper T cell epitope is a universal helper T cell epitope. Universal Th cell epitope: As used herein, the term "universal Th cell epitope" refers to a Th cell epitope capable of binding to at least one, preferably two or more MHC class II molecules. The simplest way to determine whether a peptide sequence is a universal Th cell epitope is to measure the ability of the peptide to bind to individual MHC class II molecules. This can be measured by the ability of the peptide to compete with the binding of a known Th cell epitope peptide to an MHC class II molecule. A representative selection of HLA-DR molecules is described, for example, in Alexander J et al., Immunity (1994) 1:751-761. The affinity of a Th cell epitope for an MHC class II molecule is at least 10 -5M. An alternative, more laborious but more relevant method for determining the "universality" of a Th cell epitope is the demonstration that a larger proportion of people (more than 30%) generate a measurable T cell response upon immunization and boosting one month later with a protein containing the Th cell epitope formulated in IFA. A representative collection of MHC class II molecules present in different individuals is shown in Panina-Bordignon P et al., Eur J Immunol (1989) 19:2237-2242. As a result, the term "universal Th cell epitope" as used herein preferably refers to a Th cell epitope that generates a measurable T cell response upon immunization and boosting (one month later with a protein containing the Th cell epitope formulated in IFA) in more than 30% of a selected population, as described in Panina-Bordignon P et al., Eur J Immunol (1989) 19:2237-2242. Furthermore, and even more preferably, the term "universal Th cell epitope" as used herein refers to a Th cell epitope capable of binding to at least one, preferably at least two, even more preferably at least three DR alleles selected from DR1, DR2w2b, DR3, DR4w4, DR4w14, DR5, DR7, DR52a, DRw53, DR2w2a, preferably selected from DR1, DR2w2b, DR4w4, DR4w14, DR5, DR7, DRw53, DR2w2a with an affinity of at least 500 nM (as described in Alexander J et al., Immunity (1994) 1:751-761 and references cited therein), and a preferred binding assay for assessing said affinity is that described in Sette A et al., J Immunol (1989) 142:35-40.In an even more preferred manner, the term "universal Th cell epitope" as used herein refers to a Th cell epitope capable of binding to at least one, preferably at least two, even more preferably at least three DR alleles selected from DR1, DR2w2b, DR4w4, DR4w14, DR5, DR7, DRw53, DR2w2a, preferably with an affinity of at least 500 nM (as described in Alexander J et al., Immunity (1994) 1:751-761 and references cited therein), and a preferred binding assay for assessing said affinity is that described in Sette A et al., J Immunol (1989) 142:35-40.

[0048] Universal Th cell epitopes have been described by Alexander J et al., Immunity (1994) 1:751-761, Panina-Bordignon P et al., Eur J Immunol (1989) 19:2237-2242, Calvo-Calle JM et al., J Immunol (1997) 159:1362-1373, and Valmori D et al., J Immunol (1992) 149:717-721, and are known to those skilled in the art.

[0049] Effective amount: As used herein, the term "effective amount" refers to an amount necessary or sufficient to realize a desired biological effect. An effective amount of a composition or alternatively a pharmaceutical composition is an amount that achieves this selected result, and such an amount can be routinely determined by one of ordinary skill in the art. The effective amount can vary depending on the particular composition administered and the size of the subject. One of ordinary skill in the art can empirically determine the effective amount of a particular composition of the present invention without necessitating undue experimentation.

[0050] Treatment: As used herein, the terms "treatment", "treat", "treated" or "treating" refer to prophylaxis and / or therapy. In one embodiment, the terms "treatment", "treat", "treated" or "treating" refer to therapeutic treatment. In another embodiment, the terms "treatment", "treat", "treated" or "treating" refer to prophylactic treatment.

[0051] Immunostimulatory agent: As used herein, the term "immunostimulatory agent" refers to an agent capable of inducing and / or enhancing an immune response. As used herein, immunostimulatory agents include, but are not limited to, Toll-like receptor activators and agents that induce cytokine secretion. Toll-like receptor activators include, but are not limited to, immunostimulatory nucleic acids.

[0052] Immunostimulatory Nucleic Acid: As used herein, the term immunostimulatory nucleic acid refers to a nucleic acid capable of inducing and / or enhancing an immune response. Immunostimulatory nucleic acids include ribonucleic acid and desoxyribonucleic acid, both of which may be either double-stranded or single-stranded. A preferred ISS-NA is a ribonucleic acid, and more preferably the ribonucleic acid is single-stranded.

[0053] Packaging: The term "packaged" as used herein refers to the state of an immunostimulatory agent relative to a VLP. The term "packaged" as used herein includes binding, which may be covalent, for example by chemical coupling, or non-covalent, for example ionic interactions, hydrophobic interactions, hydrogen bonds, etc. The term also includes encapsulation or partial encapsulation of an immunostimulatory agent. Thus, an immunostimulatory agent may be encapsulated by a VLP without the presence of an actual bond, in particular a covalent bond. In a preferred embodiment, at least one immunostimulatory agent is packaged inside a VLP, most preferably in a non-covalent manner. When the immunostimulatory agent is a nucleic acid, preferably an RNA, the term packaged means that the nucleic acid is not accessible to nuclease hydrolysis, preferably to RNAse hydrolysis.

[0054] Several aspects of the invention are disclosed herein, and each of the embodiments and preferred embodiments further mentioned herein is applicable to each and every aspect of the invention disclosed herein, even if not explicitly mentioned.

[0055] In a first aspect, the present invention provides a composition comprising particles, each particle comprising: (a) at least one virus-like particle (VLP), preferably a recombinant VLP; (b) at least one microcrystalline tyrosine, preferably microcrystalline L-tyrosine, having a median particle size of 0.1 μm to 50 μm, preferably 1 μm to 10 μm, preferably as determined by Flow Particle Image Analyser (FPIA), and more preferably as described in Example 2; and preferably consisting of the VLP does not contain a tumor-associated or tumor-specific antigen associated or covalently bound to the VLP, and more preferably the VLP does not contain an antigen (such as an allergen) associated or covalently bound to the VLP; A composition is provided.

[0056] Preferably, the composition is capable of treating a tumor, preferably a malignant tumor, more preferably, a solid tumor, preferably a malignant solid tumor, and even more preferably, the composition is capable of treating a malignant solid tumor intratumorally.

[0057] In a further aspect, the present invention provides a method for producing a composition comprising the steps of: (a) at least one virus-like particle (VLP), preferably a recombinant VLP; (b) at least one microcrystalline tyrosine, preferably microcrystalline L-tyrosine, having a median particle size of 0.1 μm to 50 μm, preferably 1 μm to 10 μm, preferably as determined by Flow Particle Image Analyser (FPIA), and more preferably as described in Example 2; and preferably consisting of the VLP does not contain a tumor-associated or tumor-specific antigen associated or covalently bound to the VLP, and more preferably the VLP does not contain an antigen (such as an allergen) associated or covalently bound to the VLP; A composition is provided.

[0058] Preferably, the composition is capable of treating a tumor, preferably a malignant tumor, more preferably, a solid tumor, preferably a malignant solid tumor, and even more preferably, the composition is capable of treating a malignant solid tumor intratumorally.

[0059] In a still further aspect, the invention provides a composition comprising particles, each particle comprising: (a) at least one virus-like particle (VLP), preferably a recombinant VLP; (b) at least one microcrystalline tyrosine, preferably microcrystalline L-tyrosine, having a median particle size of 0.1 μm to 50 μm, preferably 1 μm to 10 μm, preferably as determined by Flow Particle Image Analyser (FPIA), and more preferably as described in Example 2; and preferably consisting of the VLP does not contain a tumor-associated antigen associated or covalently bound to the VLP, and more preferably the VLP does not contain an antigen (such as an allergen) associated or covalently bound to the VLP; A composition is provided.

[0060] Preferably, the composition is capable of treating a tumor, preferably a malignant tumor, more preferably, a solid tumor, preferably a malignant solid tumor, and even more preferably, the composition is capable of treating a malignant solid tumor intratumorally.

[0061] In a still further aspect, the present invention provides a method for producing a composition comprising the steps of: (a) at least one virus-like particle (VLP), preferably a recombinant VLP; (b) at least one microcrystalline tyrosine, preferably microcrystalline L-tyrosine, having a median particle size of 0.1 μm to 50 μm, preferably 1 μm to 10 μm, preferably as determined by Flow Particle Image Analyser (FPIA), and more preferably as described in Example 2; and preferably consisting of the VLP does not contain a tumor-associated antigen associated or covalently bound to the VLP, and more preferably the VLP does not contain an antigen (such as an allergen) associated or covalently bound to the VLP; A composition is provided.

[0062] Preferably, the composition is capable of treating a tumor, preferably a malignant tumor, more preferably, a solid tumor, preferably a malignant solid tumor, and even more preferably, the composition is capable of treating a malignant solid tumor intratumorally.

[0063] In a preferred embodiment, the microcrystalline tyrosine has a median particle size of 0.2 μm to 30 μm, more preferably 0.5 μm to 25 μm, as determined by Flow Particle Image Analyser (FPIA) and more preferably as described in Example 2. In a more preferred embodiment, the microcrystalline tyrosine has a median particle size of 0.75 μm to 20 μm, more preferably 1 μm to 10 μm, as determined by Flow Particle Image Analyser (FPIA) and more preferably as described in Example 2. In a preferred embodiment, the microcrystalline tyrosine has a median particle size of 2 μm to 8 μm, more preferably 3 μm to 7 μm, as determined by Flow Particle Image Analyser (FPIA) and more preferably as described in Example 2. In a preferred embodiment, the microcrystalline tyrosine has a median particle size of 3.5 μm to 6 μm, more preferably 3.5 μm to 5.5 μm, preferably as determined by Flow Particle Image Analyser (FPIA), and more preferably as described in Example 2.

[0064] In a preferred embodiment, the microcrystalline tyrosine has a 10th percentile particle size of 0.01 μm to 5 μm, more preferably 0.02 μm to 3 μm, and even more preferably 0.05 μm to 2.5 μm, as determined by a Flow Particle Image Analyser (FPIA) and more preferably as described in Example 2. In a more preferred embodiment, the microcrystalline tyrosine has a 10th percentile particle size of 0.075 μm to 2 μm, and even more preferably 0.1 μm to 1 μm, as determined by a Flow Particle Image Analyser (FPIA) and more preferably as described in Example 2. In a preferred embodiment, the microcrystalline tyrosine has a 10th percentile particle size of 0.2 μm to 0.9 μm, and even more preferably 0.3 μm to 0.8 μm, as determined by a Flow Particle Image Analyser (FPIA) and more preferably as described in Example 2. In a preferred embodiment, the microcrystalline tyrosine has a 10th percentile particle size of 0.4 μm to 0.8 μm, more preferably 0.5 μm to 0.75 μm, preferably as determined by a Flow Particle Image Analyser (FPIA), and more preferably as described in Example 2.

[0065] In a preferred embodiment, the microcrystalline tyrosine has a 90th percentile particle size of 0.5 μm to 250 μm, more preferably 1 μm to 150 μm, and even more preferably 2.5 μm to 125 μm, as determined by a Flow Particle Image Analyser (FPIA) and more preferably as described in Example 2. In a more preferred embodiment, the microcrystalline tyrosine has a 90th percentile particle size of 3.75 μm to 100 μm, and even more preferably 5 μm to 50 μm, as determined by a Flow Particle Image Analyser (FPIA) and more preferably as described in Example 2. In a preferred embodiment, the microcrystalline tyrosine has a 90th percentile particle size of 10 μm to 40 μm, more preferably 10 μm to 35 μm, and even more preferably 10 μm to 30 μm, as determined by a Flow Particle Image Analyser (FPIA) and more preferably as described in Example 2. In a more preferred embodiment, the microcrystalline tyrosine has a 90th percentile particle size of between 15 μm and 30 μm, more preferably between 17.5 μm and 27.5 μm, preferably as determined by Flow Particle Image Analyser (FPIA) and more preferably as described in Example 2.

[0066] In a preferred embodiment, the microcrystalline tyrosine has a maximum particle size of 750 μm, more preferably 500 μm, even more preferably 400 μm, and even more preferably 300 μm, preferably as determined by a Flow Particle Image Analyser (FPIA) and more preferably as described in Example 2. In a further preferred embodiment, the microcrystalline tyrosine has a maximum particle size of 250 μm, and even more preferably 225 μm, preferably as determined by a Flow Particle Image Analyser (FPIA) and more preferably as described in Example 2. In a preferred embodiment, the microcrystalline tyrosine has a maximum particle size of 200 μm, more preferably 175 μm, and even more preferably 150 μm, preferably as determined by a Flow Particle Image Analyser (FPIA) and more preferably as described in Example 2. In a preferred embodiment, the microcrystalline tyrosine has a maximum particle size of 125 μm, more preferably 100 μm, preferably as determined by Flow Particle Image Analyser (FPIA), and more preferably as described in Example 2.

[0067] In a further preferred embodiment, the microcrystalline tyrosine has a maximum particle size of 750 μm, preferably the microcrystalline tyrosine has a maximum particle size of 150 μm.

[0068] In a further preferred embodiment, the VLPs are adsorbed to the microcrystalline tyrosine.

[0069] In a preferred embodiment, the composition is an aqueous composition, preferably the composition comprises a buffer, more preferably phosphate buffered saline. In a preferred embodiment, the composition is an aqueous composition, preferably the composition comprises phosphate buffered saline. In a preferred embodiment, the composition is an aqueous composition comprising phosphate buffered saline.

[0070] In a further preferred embodiment, the composition is an aqueous composition, preferably the concentration of the microcrystalline tyrosine, preferably the microcrystalline L-tyrosine, is from 0.5% to 10% (weight tyrosine / volume of solution), preferably the concentration of the microcrystalline tyrosine, preferably the microcrystalline L-tyrosine, is from 1% to 7.5% (weight tyrosine / volume of solution).

[0071] In a preferred embodiment, the composition is an aqueous composition, and the concentration of the microcrystalline tyrosine, preferably the microcrystalline L-tyrosine, is 0.5% to 10% (weight of tyrosine / volume of solution), preferably 1% to 7.5%, more preferably 2% to 7%. In another preferred embodiment, the composition is an aqueous composition, and the concentration of the microcrystalline tyrosine is 2.5% to 6% (weight of tyrosine / volume of solution), preferably 3% to 5%. In another preferred embodiment, the concentration of the microcrystalline tyrosine is 3.5% to 4.5% (weight of tyrosine / volume of solution), even about 4% (weight of tyrosine / volume of solution). In another preferred embodiment, the concentration of the microcrystalline tyrosine is about 1% (weight of tyrosine / volume of solution). In another preferred embodiment, the concentration of the microcrystalline tyrosine is about 1.5% (weight of tyrosine / volume of solution). In another preferred embodiment, the concentration of the microcrystalline tyrosine is about 2% (weight of tyrosine / volume of solution). In another preferred embodiment, the concentration of the microcrystalline tyrosine is about 2.5% (weight tyrosine / volume of solution). In another preferred embodiment, the concentration of the microcrystalline tyrosine is about 3% (weight tyrosine / volume of solution). In another preferred embodiment, the concentration of the microcrystalline tyrosine is about 3.5% (weight tyrosine / volume of solution). In another preferred embodiment, the concentration of the microcrystalline tyrosine is about 4% (weight tyrosine / volume of solution). In another preferred embodiment, the concentration of the microcrystalline tyrosine is about 4.5% (weight tyrosine / volume of solution). In another preferred embodiment, the concentration of the microcrystalline tyrosine is about 5% (weight tyrosine / volume of solution). In another preferred embodiment, the concentration of the microcrystalline tyrosine is about 5.5% (weight tyrosine / volume of solution). In another preferred embodiment, the concentration of the microcrystalline tyrosine is about 6% (weight tyrosine / volume of solution).

[0072] In a further preferred embodiment, the microcrystalline tyrosine is (i) providing an aqueous NaOH solution, the aqueous NaOH solution having a molar concentration of 1 M to 5 M, preferably 3 M to 4 M, more preferably 3.2 M to 3.8 M, and even more preferably 3.2 M or 3.8 M; (ii) providing an aqueous HCl solution, the aqueous HCl solution having a molar concentration of 1M to 5M, preferably 3M to 4M, more preferably 3.8M; (iii) dissolving tyrosine, preferably L-tyrosine, in the aqueous HCl solution to obtain an HCl solution containing tyrosine, the concentration of the tyrosine in the HCl solution being 5-40% (weight of tyrosine / volume of HCl solution), preferably 10-30% (weight of tyrosine / volume of HCl solution), more preferably 24% (weight of tyrosine / volume of HCl solution); (iv) co-precipitating the microcrystalline tyrosine by combining and homogenizing the aqueous NaOH solution and the HCl solution containing tyrosine, preferably in equal amounts, preferably the co-precipitating step further comprises adding Evans' solution in the presence of a phosphate buffer; (v) isolating the microcrystalline tyrosine and optionally purifying the microcrystalline tyrosine; (vi) optionally suspending the microcrystalline tyrosine in a buffer solution, preferably phosphate buffered saline, to a concentration of 2% to 5% (weight of tyrosine / volume of solution), preferably to a concentration of 3.5% to 4.5% (weight of tyrosine / volume of solution), and more preferably to a concentration of 4% (weight of tyrosine / volume of solution); The method is obtained by a method comprising the steps of:

[0073] In a preferred embodiment, the aqueous HCl and NaOH solutions containing tyrosine are sterile filtered prior to the co-precipitation. In a preferred embodiment, the molarity of the aqueous HCl and NaOH solutions and the volumes used for co-precipitation are selected such that the final pH of the microcrystalline tyrosine suspension is between 5.0 and 7.0.

[0074] In a preferred embodiment, the VLP is derived from a plant virus or is a VLP of an RNA bacteriophage.

[0075] In a preferred embodiment, the VLP is derived from a plant virus. In a preferred embodiment, the VLP is a VLP of an RNA bacteriophage. In a preferred embodiment, the VLP is derived from a plant virus selected from the group consisting of Cucumber Mosaic Virus (CuMV), Cowpea Chlorotic Mottle Virus (CCMV) and Tobacco Mosaic Virus (TMV). In a preferred embodiment, the VLP is derived from Cucumber Mosaic Virus (CuMV). In a preferred embodiment, the VLP is derived from Cowpea Chlorotic Mottle Virus (CCMV). In a preferred embodiment, the VLP is a VLP of an RNA bacteriophage. In a preferred embodiment, the VLP is a VLP of an RNA bacteriophage, the RNA bacteriophage being selected from the group consisting of Qβ and AP205. In a preferred embodiment, the VLP is a VLP of RNA bacteriophage Qβ. In a preferred embodiment, the VLP is a VLP of RNA bacteriophage AP205.

[0076] In a preferred embodiment, the VLP is derived from a plant virus or is a VLP of an RNA bacteriophage, preferably the plant virus is selected from the group consisting of Cucumber Mosaic Virus (CuMV), Cowpea Chlorotic Mottle Virus (CCMV) and Tobacco Mosaic Virus (TMV), and preferably the VLP of an RNA bacteriophage is a VLP of RNA bacteriophage Qβ or a VLP of RNA bacteriophage AP205.

[0077] In a further preferred embodiment, the VLP comprises, consists essentially of, or alternatively consists of recombinant coat proteins of an RNA bacteriophage, preferably the VLP comprises, preferably consists of recombinant coat proteins of RNA bacteriophage Qβ or RNA bacteriophage AP205, more preferably the VLP comprises, preferably consists of recombinant coat proteins of RNA bacteriophage Qβ. In a further preferred embodiment, the VLP comprises, preferably consists of recombinant coat proteins comprising, or preferably consisting of, an amino acid sequence selected from (a) SEQ ID NO: 14, (b) a mixture of SEQ ID NO: 14 and SEQ ID NO: 15, or (c) SEQ ID NO: 16. In a further preferred embodiment, the VLP is a VLP of RNA bacteriophage Qβ. In a further preferred embodiment, the VLP comprises, consists essentially of, or alternatively consists of recombinant coat proteins of RNA bacteriophage Qβ. In a further preferred embodiment, the VLP comprises, consists essentially of, or alternatively consists of a recombinant coat protein comprising, or preferably consisting of, SEQ ID NO:14.

[0078] In another preferred embodiment, the core particle is a virus-like particle (VLP), the VLP being a VLP of RNA bacteriophage Qβ, the VLP comprising, consisting essentially of, or alternatively consisting of, a recombinant coat protein of RNA bacteriophage Qβ, the recombinant coat protein comprising, or preferably consisting of, SEQ ID NO:14.

[0079] In a preferred embodiment, the VLP is a Cucumber Mosaic Virus (CuMV) VLP, preferably the Cucumber Mosaic Virus (CuMV) VLP comprises, preferably consists of, at least one recombinant coat protein or mutant amino acid sequence, wherein the mutant amino acid sequence and the coat protein of CuMV exhibit at least 90%, preferably at least 95%, even more preferably at least 98%, and more preferably at least 99% sequence identity.

[0080] In a further preferred embodiment, the VLP is a Cucumber Mosaic Virus (CuMV) VLP, preferably said Cucumber Mosaic Virus (CuMV) VLP comprises, preferably consists of, at least one recombinant coat protein or mutant amino acid sequence of SEQ ID NO:1, wherein said mutant amino acid sequence and said coat protein of CuMV of SEQ ID NO:1 exhibit at least 90%, preferably at least 95%, even more preferably at least 98% and more preferably at least 99% sequence identity.

[0081] In a further preferred embodiment, the VLP is a modified VLP, the modified VLP comprising, and preferably consisting of, at least one modified VLP polypeptide, the modified VLP polypeptide comprising: (a) a VLP polypeptide; (b) helper T cell epitopes and Including, The VLP polypeptide comprises: (i) the amino acid sequence of a viral coat protein, preferably the amino acid sequence of a plant virus coat protein, or (ii) a mutated amino acid sequence, wherein the mutated amino acid sequence and the coat protein of the virus exhibit a sequence identity of at least 90%, preferably at least 95%, more preferably at least 98% and even more preferably at least 99%. It comprises or preferably consists of:

[0082] In a further preferred embodiment, the VLP is a modified VLP of Cucumber Mosaic Virus (CuMV), the modified VLP of CuMV comprising, and preferably consisting of, at least one modified CuMV polypeptide, the modified CuMV polypeptide being (a) a CuMV polypeptide; (b) helper T cell epitopes and comprising, or preferably consisting of, The CuMV polypeptide is (i) the amino acid sequence of the coat protein of CuMV, or (ii) a mutant amino acid sequence, wherein said mutant amino acid sequence and said coat protein of CuMV show a sequence identity of at least 90%, preferably at least 95%, more preferably at least 98% and even more preferably at least 99%. It comprises or preferably consists of:

[0083] In a preferred embodiment, the CuMV polypeptide is a coat protein of CuMV or an amino acid sequence having at least 75%, preferably 85%, sequence identity to SEQ ID NO: 1. In a preferred embodiment, the CuMV polypeptide is a coat protein of CuMV or an amino acid sequence having at least 90%, preferably 95%, sequence identity to SEQ ID NO: 1. In a preferred embodiment, the CuMV polypeptide is a coat protein of CuMV having SEQ ID NO: 1. In a preferred embodiment, the coat protein of CuMV comprises SEQ ID NO: 1. In a preferred embodiment, the coat protein of CuMV consists of SEQ ID NO: 1. In a preferred embodiment, the CuMV polypeptide comprises a coat protein of CuMV. In a preferred embodiment, the CuMV polypeptide consists of a coat protein of CuMV. In a preferred embodiment, the CuMV polypeptide comprises a coat protein of CuMV, and the coat protein of CuMV comprises SEQ ID NO: 1. In a preferred embodiment, the CuMV polypeptide comprises a coat protein of CuMV, and the coat protein of CuMV consists of SEQ ID NO: 1. In a preferred embodiment, said CuMV polypeptide consists of a coat protein of CuMV, said coat protein of CuMV consisting of SEQ ID NO:1.

[0084] In a preferred embodiment, the CuMV polypeptide comprises SEQ ID NO:2 or an amino acid sequence region, which has at least 75% sequence identity with SEQ ID NO:2. In a preferred embodiment, the CuMV polypeptide comprises SEQ ID NO:2 or an amino acid sequence region, which has at least 80% sequence identity with SEQ ID NO:2. In a preferred embodiment, the CuMV polypeptide comprises SEQ ID NO:2 or an amino acid sequence region, which has at least 85% sequence identity with SEQ ID NO:2. In a preferred embodiment, the CuMV polypeptide comprises SEQ ID NO:2 or an amino acid sequence region, which has at least 90% sequence identity with SEQ ID NO:2. In a preferred embodiment, the CuMV polypeptide comprises SEQ ID NO:2 or an amino acid sequence region, which has at least 95% sequence identity with SEQ ID NO:2. In a preferred embodiment, the CuMV polypeptide comprises SEQ ID NO:2 or an amino acid sequence region, which has at least 98% sequence identity with SEQ ID NO:2. In a preferred embodiment, the CuMV polypeptide comprises SEQ ID NO:2, or an amino acid sequence region, which has at least 99% sequence identity to SEQ ID NO:2.

[0085] In a preferred embodiment, the CuMV polypeptide comprises or preferably consists of (i) an amino acid sequence of a coat protein of CuMV, which amino acid sequence comprises, or preferably consists of, SEQ ID NO:1, or (ii) an amino acid sequence having at least 90% sequence identity to SEQ ID NO:1, and the amino acid sequence defined in (i) or (ii) comprises SEQ ID NO:2 or an amino acid sequence region having at least 90% sequence identity to SEQ ID NO:2. In a preferred embodiment, the CuMV polypeptide comprises or preferably consists of (i) an amino acid sequence of a coat protein of CuMV, which amino acid sequence comprises, or preferably consists of, SEQ ID NO:1, or (ii) an amino acid sequence having at least 95% sequence identity to SEQ ID NO:1, and the amino acid sequence defined in (i) or (ii) comprises SEQ ID NO:2 or an amino acid sequence region having at least 95% sequence identity to SEQ ID NO:2. In a preferred embodiment, the CuMV polypeptide comprises (i) an amino acid sequence of a coat protein of CuMV, which comprises, or preferably consists of, an amino acid sequence of SEQ ID NO:1, or (ii) an amino acid sequence having at least 90% sequence identity to SEQ ID NO:1, wherein the amino acid sequence defined in (i) or (ii) comprises, or preferably consists of, SEQ ID NO:2.

[0086] In a preferred embodiment, the modified CuMV polypeptide further comprises a helper T cell epitope, which replaces the N-terminal region of the CuMV polypeptide.

[0087] In a preferred embodiment, the number of amino acids substituted in the N-terminal region is equal to or less than the number of amino acids constituting the helper T cell epitope. In a preferred embodiment, the substituted N-terminal region of the CuMV polypeptide consists of 5 to 15 consecutive amino acids. In a preferred embodiment, the substituted N-terminal region of the CuMV polypeptide consists of 9 to 14 consecutive amino acids. In a preferred embodiment, the substituted N-terminal region of the CuMV polypeptide consists of 11 to 13 consecutive amino acids. In a preferred embodiment, the N-terminal region of the CuMV polypeptide corresponds to amino acids 2 to 12 of SEQ ID NO: 1. In a preferred embodiment, the modified CuMV polypeptide further comprises a helper T cell epitope, which replaces the N-terminal region of the CuMV polypeptide, and which corresponds to amino acids 2 to 12 of SEQ ID NO: 1. In a preferred embodiment, the helper T cell epitope is a universal helper T cell epitope. In a preferred embodiment, the helper T cell epitope consists of a maximum of 20 amino acids.

[0088] In a preferred embodiment, the Th cell epitope is selected from TT830-843 (SEQ ID NO: 3), PADRE (SEQ ID NO: 4), TT947-967 (SEQ ID NO: 5), HA307-319 (SEQ ID NO: 6), HBVnc50-69 (SEQ ID NO: 7), CS378-398 (SEQ ID NO: 8) and MT17-31 (SEQ ID NO: 9). In a highly preferred embodiment, the Th cell epitope is a Th cell epitope derived from tetanus toxin or is the PADRE sequence. In a preferred embodiment, the helper T cell epitope is derived from a human vaccine. In a highly preferred embodiment, the Th cell epitope is a Th cell epitope derived from tetanus toxin. In a preferred embodiment, the Th cell epitope is the PADRE sequence. In a highly preferred embodiment, the Th cell epitope comprises the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4. In a highly preferred embodiment, the Th cell epitope consists of the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4. In a highly preferred embodiment, the Th cell epitope comprises the amino acid sequence of SEQ ID NO: 3. In a preferred embodiment, the Th cell epitope consists of the amino acid sequence of SEQ ID NO: 3. In a highly preferred embodiment, the Th cell epitope comprises the amino acid sequence of SEQ ID NO: 4. In a highly preferred embodiment, the Th cell epitope consists of the amino acid sequence of SEQ ID NO: 4.

[0089] In a preferred embodiment, the CuMV polypeptide comprises, or preferably consists of, the amino acid sequence of a coat protein of CuMV, the amino acid sequence comprising, or preferably consists of, SEQ ID NO:1 or an amino acid sequence having at least 95% sequence identity to SEQ ID NO:1, the amino acid sequence comprising SEQ ID NO:2, the helper T cell epitope replaces the N-terminal region of the CuMV polypeptide, the substituted N-terminal region of the CuMV polypeptide consists of 11 to 13 consecutive amino acids, preferably 11 consecutive amino acids, and more preferably the N-terminal region of the CuMV polypeptide corresponds to amino acids 2 to 12 of SEQ ID NO:1.

[0090] In another highly preferred embodiment, the modified CMV polypeptide comprises the amino acid sequence of SEQ ID NO: 11. In another highly preferred embodiment, the modified CMV polypeptide consists of the amino acid sequence of SEQ ID NO: 11. In another highly preferred embodiment, the modified CMV polypeptide comprises the amino acid sequence of SEQ ID NO: 12. In another highly preferred embodiment, the modified CMV polypeptide consists of the amino acid sequence of SEQ ID NO: 12.

[0091] In a further highly preferred embodiment, the present invention relates to a composition comprising particles, each particle comprising: (a) at least one virus-like particle (VLP), preferably a recombinant VLP, said VLP being a modified VLP of Cucumber Mosaic Virus (CuMV), said modified VLP of CuMV comprising, preferably consisting of, at least one modified CuMV polypeptide, said modified CuMV polypeptide comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 11; (b) at least one microcrystalline tyrosine, preferably microcrystalline L-tyrosine, having a median particle size of 0.1 μm to 50 μm, preferably 1 μm to 10 μm, preferably as determined by Flow Particle Image Analyser (FPIA), and more preferably as described in Example 2. and preferably consisting of the VLP does not comprise a tumor-associated or tumor-specific antigen covalently bound to the VLP, and more preferably the VLP does not comprise an antigen covalently bound to the VLP; A composition is provided.

[0092] In a further highly preferred embodiment, the present invention comprises: (a) at least one virus-like particle (VLP), preferably a recombinant VLP, said VLP being a modified VLP of Cucumber Mosaic Virus (CuMV), said modified VLP of CuMV comprising, preferably consisting of, at least one modified CuMV polypeptide, said modified CuMV polypeptide comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 11; (b) at least one microcrystalline tyrosine, preferably microcrystalline L-tyrosine, having a median particle size of 0.1 μm to 50 μm, preferably 1 μm to 10 μm, preferably as determined by Flow Particle Image Analyser (FPIA), and more preferably as described in Example 2. and preferably consisting of the VLP does not comprise a tumor-associated or tumor-specific antigen covalently bound to the VLP, and more preferably the VLP does not comprise an antigen covalently bound to the VLP; A composition is provided.

[0093] In a further highly preferred embodiment, the present invention relates to a composition comprising particles, each particle comprising: (a) at least one virus-like particle (VLP), preferably a recombinant VLP, said VLP being a modified VLP of Cucumber Mosaic Virus (CuMV), said modified VLP of CuMV comprising, preferably consisting of, at least one modified CuMV polypeptide, said modified CuMV polypeptide comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 11; (b) at least one microcrystalline tyrosine, preferably microcrystalline L-tyrosine, having a median particle size of 0.1 μm to 50 μm, preferably 1 μm to 10 μm, preferably as determined by Flow Particle Image Analyser (FPIA), and more preferably as described in Example 2. and preferably consisting of the VLP does not comprise a tumor associated antigen covalently bound to the VLP, more preferably the VLP does not comprise an antigen covalently bound to the VLP; A composition is provided.

[0094] In a further highly preferred embodiment, the present invention comprises: (a) at least one virus-like particle (VLP), preferably a recombinant VLP, said VLP being a modified VLP of Cucumber Mosaic Virus (CuMV), said modified VLP of CuMV comprising, preferably consisting of, at least one modified CuMV polypeptide, said modified CuMV polypeptide comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 11; (b) at least one microcrystalline tyrosine, preferably microcrystalline L-tyrosine, having a median particle size of 0.1 μm to 50 μm, preferably 1 μm to 10 μm, preferably as determined by Flow Particle Image Analyser (FPIA), and more preferably as described in Example 2. and preferably consisting of the VLP does not comprise a tumor associated antigen covalently bound to the VLP, more preferably the VLP does not comprise an antigen covalently bound to the VLP; A composition is provided.

[0095] In a further preferred embodiment, the composition does not comprise a specific cancer antigen, in a further preferred embodiment, the composition does not comprise a tumor-associated antigen, in a further preferred embodiment, the composition does not comprise a tumor-specific antigen.

[0096] In a further highly preferred embodiment, the VLP does not comprise a specific cancer antigen covalently bound to the VLP, more preferably the VLP does not comprise an antigen covalently bound to the VLP. In a further highly preferred embodiment, the VLP does not comprise a specific tumor associated antigen covalently bound to the VLP, more preferably the VLP does not comprise an antigen covalently bound to the VLP. In a further highly preferred embodiment, the VLP does not comprise a specific tumor specific antigen covalently bound to the VLP, more preferably the VLP does not comprise an antigen covalently bound to the VLP. In a further highly preferred embodiment, the VLP does not comprise a CTL epitope p33 derived from lymphocytic choriomeningitis virus glycoprotein covalently bound to the VLP, more preferably the VLP does not comprise an antigen covalently bound to the VLP. In a further highly preferred embodiment, the VLP, preferably the recombinant VLP, comprises an immunostimulatory agent packaged within the VLP, preferably the immunostimulatory agent is a Toll-like receptor activator. In a further highly preferred embodiment, the Toll-like receptor activator is a Toll-like receptor 7 and / or 8 activator, preferably the Toll-like receptor 7 and / or 8 activator is ssRNA. In a further highly preferred embodiment, the Toll-like receptor activator is an immunostimulatory nucleic acid, preferably the immunostimulatory nucleic acid is ssRNA. In a further highly preferred embodiment, the composition is capable of treating a tumor, preferably a malignant tumor, more preferably the composition is capable of treating a solid tumor, preferably a malignant solid tumor, and even more preferably the composition is capable of treating a malignant solid tumor intratumorally. In a further highly preferred embodiment, the malignant solid tumor is selected from breast, prostate, colorectal, lung and melanoma. In a further highly preferred embodiment, the malignant solid tumor is a breast malignant tumor. In a further highly preferred embodiment, the malignant solid tumor is a prostate malignant tumor. In a further highly preferred embodiment, the malignant solid tumor is a colorectal malignant tumor. In a further highly preferred embodiment, the malignant solid tumor is a lung malignant tumor. In a further highly preferred embodiment, the malignant solid tumor is a melanoma malignancy.

[0097] In a further aspect, the present invention provides a pharmaceutical composition comprising a composition of the present invention and a pharma- ceutically acceptable carrier.

[0098] In a further aspect, the present invention provides a composition as described and claimed herein for use in a method of treating a solid tumor in a patient, wherein the patient is a human patient or an animal patient, preferably the animal patient is an equine animal, preferably selected from a horse, a dog, or a cat, more preferably the animal patient is a horse. In a highly preferred embodiment, the method of treating a solid tumor in a patient is a method of treating the solid tumor in a patient intratumorally. In a further highly preferred embodiment, the solid tumor is selected from breast cancer, prostate cancer, colorectal cancer, lung cancer, and melanoma cancer. In a further highly preferred embodiment, the solid tumor is breast cancer. In a further highly preferred embodiment, the solid tumor is prostate cancer. In a further highly preferred embodiment, the solid tumor is colorectal cancer. In a further highly preferred embodiment, the solid tumor is lung cancer. In a further highly preferred embodiment, the solid tumor is melanoma cancer. In a further highly preferred embodiment, the method comprises administration of the composition of the present invention to the patient, wherein the administration is intratumor administration of the composition to the patient. In a further highly preferred embodiment, the method comprises administering to the patient a composition of the invention, wherein said administration of said composition occurs within a tumor of the patient or substantially adjacent to a tumor of the patient.

[0099] In a further aspect, the invention provides the use of a composition as described and claimed herein for the manufacture of a medicament for treating a solid tumor in a patient, said patient being a human or animal patient, preferably said animal patient being an equine animal, preferably selected from horses, dogs or cats, more preferably said animal patient being a horse. In a highly preferred embodiment, said treating a solid tumor in a patient is treating said solid tumor in a patient intratumorally. In a further highly preferred embodiment, said solid tumor is selected from breast cancer, prostate cancer, colorectal cancer, lung cancer and melanoma cancer. In a further highly preferred embodiment, said solid tumor is breast cancer. In a further highly preferred embodiment, said solid tumor is prostate cancer. In a further highly preferred embodiment, said solid tumor is colorectal cancer. In a further highly preferred embodiment, said solid tumor is lung cancer. In a further highly preferred embodiment, said solid tumor is melanoma cancer. In a further highly preferred embodiment, said treating a solid tumor in a patient is intratumorally administering said composition to said patient. In a further highly preferred embodiment, said treating a solid tumor in a patient comprises administering said composition intratumorally or substantially adjacent to a tumor in said patient.

[0100] In yet a further aspect, the present invention provides a method of treating a solid tumor in a patient, the patient being a human or animal patient, preferably the animal patient is an equine animal, preferably selected from horses, dogs or cats, more preferably the animal patient is a horse, comprising administering to the human or animal patient an effective amount of a composition or pharmaceutical composition as described and claimed herein. In a highly preferred embodiment, the method of treating a solid tumor in a patient is a method of treating the solid tumor in a patient intratumorally. In a further highly preferred embodiment, the solid tumor is selected from breast cancer, prostate cancer, colorectal cancer, lung cancer and melanoma cancer. In a further highly preferred embodiment, the solid tumor is breast cancer. In a further highly preferred embodiment, the solid tumor is prostate cancer. In a further highly preferred embodiment, the solid tumor is colorectal cancer. In a further highly preferred embodiment, the solid tumor is lung cancer. In a further highly preferred embodiment, the solid tumor is melanoma cancer. In a further highly preferred embodiment, the method comprises administering to the patient a composition of the present invention, the administering being an intratumor administration of the composition to the patient. In a further highly preferred embodiment, the method comprises administering to the patient a composition of the invention, wherein said administration of said composition occurs within a tumor of the patient or substantially adjacent to a tumor of the patient.

[0101] example A preferred virus-like particle (VLP), particularly a recombinant VLP, used herein is prepared as described. Thus, in one embodiment, the VLP is a VLP of RNA bacteriophage Qβ, comprising, preferably consisting of, the recombinant coat protein of RNA bacteriophage Qβ of SEQ ID NO: 14. Such virus-like particles of RNA bacteriophage are disclosed in WO 02 / 056905, the disclosure of which is incorporated herein by reference in its entirety. In particular, Example 18 of WO 02 / 056905 contains a detailed description of the preparation of VLP particles of RNA bacteriophage Qβ. In a further embodiment, the VLP is a VLP of cowpea chlorotic mottle virus (CCMV), particularly a modified VLP of CCMV, in which a helper T cell epitope is incorporated at the N-terminus or C-terminus. In a further embodiment, the VLP is CCMVtt830 as described in Zinkhan S et al., Journal of Controlled Release (2021) 331:296-308, the disclosure of which is incorporated herein by reference in its entirety. In a highly preferred embodiment, the VLP is a Cucumber Mosaic Virus (CuMV) VLP, in particular a modified CuMV VLP, in which a helper T cell epitope replaces the N-terminal region of the CuMV polypeptide. In a highly preferred embodiment, the VLP is CuMVtt830 comprising a modified CuMV polypeptide of SEQ ID NO: 11, as described herein and disclosed in WO 2016 / 062720, or CuMV-Npadr comprising a modified CuMV polypeptide of SEQ ID NO: 12, preferably CuMVtt830 comprising a modified CuMV polypeptide of SEQ ID NO: 11. It is noted that in WO 2016 / 062720, CuMV mosaic virus is abbreviated as CMV. In particular, Examples 1-6 of WO 2016 / 062720 contain detailed descriptions of the preparation of VLP particles of modified CuMV polypeptides of SEQ ID NO:11 and SEQ ID NO:12.

[0102] Example 1 Preparation of microcrystalline tyrosine (MCT) Microcrystalline tyrosine (MCT) was prepared similarly as described by Bell AJ et al., 2015, Journal of Inorganic Biochemistry 152:147-153). Briefly, microcrystalline tyrosine (MCT) is produced by co-precipitation of a 24% w / v solution of L-tyrosine in 3.8 M hydrochloric acid with 3.2 M sodium hydroxide. The co-precipitation is completed in Evans solution in the presence of phosphate buffer. The product is washed with phosphate buffered saline through a stainless steel sintered filter to reduce excess chloride content. The product is recovered as a microcrystalline suspension in phosphate buffered saline at a concentration of 4% w / v L-tyrosine.

[0103] The following two formulations were prepared, as described in Example 2, with the following particle sizes as measured by FPIA: [Table 1]

[0104] Example 2 Particle size analysis using the Malvern Flow Particle Image Analyser 3000 A standard and preferred analytical method for determining and analyzing the particle size and particle distribution of microcrystalline tyrosine, preferably microcrystalline L-tyrosine, according to the present invention is performed by using a Malvern Instruments FPIA 3000, where FPIA stands for Flow Particle Image Analyser (FPIA).

[0105] Traditional particle size methods such as laser diffraction can view monodisperse samples as polydisperse due to random particle orientation. FPIA instruments are image analysis devices that utilize the "sheath flow" principle to orient and align particles so that more uniform and accurate results can be found for particle shape and size. This is accomplished by ensuring that the largest area of ​​the particle faces the image detector. FPIA utilizes a charge-coupled device (CCD) camera to take pictures of the particles as they pass through the flow cell in this controlled manner and analyze them in "real time."

[0106] The FPIA is capable of detecting a range of 1.5μm-160μm by using two different lenses, one that shows a quoted detection range of 1.5μm-40μm using a high power field (HPF) and the other that shows a quoted detection range of 8μm-160μm using a low power field (LPF). Up to 36,000 particles can be imaged and measured in a single run with a total analysis time of 2 minutes 30 seconds.

[0107] The particle size of the microcrystalline tyrosine, preferably microcrystalline L-tyrosine, as used and described herein is determined by the equivalent circumferential diameter D as described and defined by Li et al. (Li et al., Particulate Science and Technology, 23:265-284, 2005; see in particular Figure 2(b) and formula (2) on page 271). p As used herein, median particle size refers to the particle size at which 50% of all particle sizes measured in a distribution and composition are typically and preferably measured by FPIA as described in the present examples. p Further values ​​measured by FPIA for microcrystalline tyrosine, preferably microcrystalline L-tyrosine, are typically and preferably the 10th percentile, the 90th percentile and the maximum particle size. The 10th percentile typically and preferably refers to the particle size determined by FPIA for which 10% of the total particle sizes measured, respectively, in the distribution and composition are D by FPIA, as described in the present example. pThe 90th percentile refers to a size equal to or less than the size determined as D by FPIA, typically and preferably equal to 90% of all particle sizes measured in the distribution and composition, respectively, as described in this example. p and maximum particle size refers to the largest particle size value measured in the distribution and composition, respectively.

[0108] Example 3 Preparation of a composition containing CuMVtt830-VLPs and MCTs A composition of CuMVtt830-VLP with 4% w / v MCT was prepared by mixing both components in a shaker at room temperature for 1 h and allowing the nano-sized VLPs to decorate the micron-sized MCT crystals, similar to that described in Mohsen MO et al. (Journal for ImmunoTherapy of Cancer (2020) 7(1):114). Accordingly, Qβ-VLP formulated with 4% w / v MCT or CuMV formulated with 4% w / v MCT were TT -VLPs were prepared.

[0109] Example 4 Antitumor effect of a composition containing CuMVtt830-VLP and MCT A composition comprising CuMVtt830-VLPs and MCT was incubated with 1×10 cells of the B16F10 melanoma cell line as shown in FIG. 1A and described as follows: 6 cells, RAG2 - / - The tumors were tested in an invasive transplantation mouse melanoma model by injection into the flank of C57 / BL / 6 mice. After 12 days, growing tumors were harvested and cut to approximately 2 mm diameter for implantation into the flank of WT C57 / BL / 6J mice (8–12 weeks old; Harlan) under full anesthesia. 2 was processed into fragments.

[0110] Implanted WT C57 / BL / 6 mice received three intratumoral injections over a 14-day period on the schedule shown in Figure 1B. Thus, on days 2, 7, and 11 after tumor implantation, mice received intratumoral injections with 100 μl phosphate-buffered saline (PBS), 50 μg CuMVtt830-VLPs alone diluted in PBS in a final volume of 100 μl, or 50 μg CuMVtt830-VLPs formulated with 4% w / v MCT (40 mg / ml) in a final volume of 100 μl / dose (CuMVtt830-VLPs are abbreviated as CuMVtt-VLPs in Figure 1B).

[0111] Tumor growth was followed every 2 days and calculated using the formula V = (WxWxL) / 2, where V = mm 3The final tumor volume at 10 min was measured using calipers according to L=tumor length and W=tumor width. Tumors were harvested and measured on day 14. Tumors were harvested in DMEM medium containing 10% FBS and 1% penicillin / streptomycin on ice. Tumor infiltrating lymphocytes (TILs) were harvested by dissecting the tumor into small pieces, crushing using a 70 μM cell strainer, and washing the cells in the process using DMEM medium containing 10% FBS and 1% penicillin / streptomycin in a 50 ml Falcon tube as follows: The harvested cells were slowly added to a 15 ml tube containing 2 ml of 35% Percoll. The tube was centrifuged at 1800 rpm for 25 min at room temperature to isolate the TILs. The TILs were then resuspended in 200 μl of PBS, 0.1% BSA, and 100 μl was transferred to a 96-well plate v-bottom and centrifuged at 1200 rpm for 5 min. The supernatant was discarded and the RBCs were lysed using 500 μl of ACK buffer (Sigma-Aldrich) for 2–3 min on ice. TILs were stained with anti-mouse CD16 / CD32 (mouse BD Fc block) mAb clone 2.4G2 (BD Bioscience) for 10 min in the dark, centrifuged as above, and stained with PE anti-mouse CD8α mAb clone 53-6.7 (BD Bioscience). The plates were centrifuged at 1200 rpm for 5 min, the supernatant was discarded, and the TILs were resuspended in PBS, 0.1% BSA and added to a 5 ml round-bottom tube with a cell strainer to remove excess tumor debris. Samples were read by FACSCaliber and analysis was performed using GraphPad Prism version 8.4.2 (464). Furthermore, the CD8 + CD8 is determined by dividing the total number of T cells by their volume. + T cell density was measured.

[0112] On day 12, whole blood was collected in heparin to identify myeloid-derived suppressor cells (MDSCs), particularly CD11b, in the blood of treated mice. Hi Ly6C HiThe percentage of RBCs was evaluated. In detail, blood was collected from the control and treatment groups on day 12 after tumor implantation. An incision was made in the tail vein of the mice to collect blood. 150 μl of blood was collected in 500 μl of 1×PBS containing heparin and kept on ice. Cells were centrifuged at 1200 rpm for 5 min and the supernatant was aspirated. RBCs were lysed using 500 μl of ACK buffer (Sigma-Aldrich) for 2-3 min on ice. Cells were collected by centrifugation at 1200 rpm for 5 min. The supernatant was aspirated and cells were resuspended in 1×PBS containing 0.1% BSA and centrifuged again. Pelleted cells were stained with anti-mouse CD16 / CD32 (mouse BD Fc block) mAb clone 2.4G2 (BD Bioscience) for 10 min in the dark, centrifuged as above, and stained with PE anti-mouse CD8α mAb clone 53-6.7 (BD Bioscience) or FITC anti-mouse Ly6C clone HK1.4 (BioLegend) and APC / Cyanine7 anti-mouse CD11b clone M / 170 (BioLegend) according to the experiment. Samples were read by FACSCaliber and analysis was performed using GraphPad Prism version 8.4.2 (464).

[0113] result A significant decrease in the % of CD11bHi Ly6CHi population in the group treated with CuMVtt830-VLP+MCT was observed (Figure 1C). On day 14, mice were sacrificed and tumor analysis was performed. Tumor volume mm 3was measured for all mice (Fig. 1D). CuMVtt830-VLP alone showed a significant reduction in tumor size when compared to the group treated with PBS. However, the antitumor effect was much more pronounced in the group treated with CuMVtt830-VLP formulated with MCT, as described above, when compared to all other groups. The effect of intratumoral treatment on tumor lymphocytic composition, especially CD8+ T cells, was further examined. It is readily recognized that CD8+ T cell infiltration into tumors correlates with better prognosis and is essential evidence for an effective immune response (Haanen JB et al., Melanoma-specific tumor-infiltrating lymphocytes but not circulating melanoma-specific T cells may predict survival in resected advanced-stage melanoma patients. Cancer Immunol Immunother. 2006;55(4):451-8.4). The composition containing CuMVtt830-VLP and MCT particles significantly increased the density of CD8+ T cell infiltration into tumors when compared to other groups (Figure 1E). The measured density of CD8+ T cells was significantly higher than that of tumor volume mm 3 The results were significantly correlated with the CD8+IFN-γ+ T cell density in the tumors (Fig. 1F). The inventors also evaluated the density of CD8+IFN-γ+ T cells in the tumors, and the data showed that the group treated with the composition containing CuMVtt-VLP and MCT showed the strongest cytokine production (Fig. 1G and H).

[0114] In addition, an intracellular cytokine (ICS) staining protocol was performed using specifically anti-CD8α mAb as a surface marker and IFN-γ mAb as an intracellular cytokine marker. In detail, 100 μl of TILs as above were transferred to sterile 96-well flat-bottom plates for ICS experiments. TILs were incubated with 100 U / ml mouse IL-2 (mIL2-Ref:11271164001-MERCK) in DMEM medium containing 10% FBS and 1% penicillin / streptomycin for 2 days at 37°C. TILs were washed three times with DMEM medium containing 10% FBS and 1% penicillin / streptomycin, and a stimulation cocktail was added (2 μg / ml ionomycin, 2 μg / ml PMA, and brefeldin and monensin (1:1000)) for 6 hours at 37°C. TILs were washed three times with DMEM medium to remove the stimulation cocktail, and then transferred to 96-well v-bottom plates for staining. TILs were stained with anti-mouse CD16 / CD32 (mouse BD Fc block) mAb clone 2.4G2 (BD Bioscience) for 10 min in the dark, centrifuged as above, and stained with PE anti-mouse CD8α mAb clone 53-6.7 (BD Bioscience). Plates were centrifuged at 1200 rpm for 5 min, the supernatant was discarded, and TILs were fixed for 15 min at 4°C using 100 μl of fixation buffer (BD Cytofix). Plates were centrifuged at 1200 rpm for 5 min, the supernatant was discarded, and TILs were washed with 100 μl of 1x permeabilization wash buffer (BioLegend), immediately centrifuged at 1200 rpm for 5 min, and the supernatant was discarded. TILs were then stained with APC anti-mouse IFN-γ mAb clone XMG1.2 (MERCK) and PerCP-Cyanine5.5 anti-mouse TNF-α mAb clone MP6-XT22 (BioLegend). Plates were centrifuged at 1200 rpm for 5 min, the supernatant was discarded, and TILs were resuspended in PBS, 0.1% BSA and added to a 5 ml round-bottom tube with a cell strainer to remove excess tumor debris. Samples were read by FACSCaliber and analysis was performed using GraphPad Prism version 8.4.2 (464).

[0115] A representative FACS plot showing the total number of CD8+IFN-γ+ producing cells pre-gated on TILs is shown in FIG. 1I.

[0116] Example 5 Systemic effects induced upon intratumoral injection of a composition containing CuMVtt830-VLP and MCT To study the systemic response of the composition of CuMVtt830-VLPs and MCT, the antibody titers induced against CuMVtt830-VLPs in the serum of treated mice were measured as a biomarker.

[0117] Here, serum was collected on day 12 by warming the mice for 15 min and collecting 100 μl of whole blood from the mouse tail in a BD Microtainer tube as follows: Serum was separated by centrifugation at 8000 rpm for 1 min and stored at -20°C. To determine total IgG antibody titers against CuMVtt830-VLP, ELISA plates were coated overnight with CuMVtt830-VLP at a concentration of 2 μg / ml. Plates were washed with PBS-0.01% Tween and blocked for 2 h using 100 μl of PBS-casein 0.15%. Serum from treated mice was first diluted 1 / 20 and serially diluted 1 / 3. Plates were incubated for 1 h at room temperature. After washing with PBS-0.01% Tween, goat anti-mouse IgG conjugated to horseradish peroxidase (HRP) was added 1 / 1000 and incubated for 1 h at room temperature. Plates were developed and OD450 readings were taken.

[0118] As described above, the groups treated with CuMVtt830-VLP or cCuMVtt830-VLP formulated with MCT had anti-VLP antibodies as expected, whereas no antibodies were detected in the control group (Figures 2A and 2B). The results obtained indicate that intratumoral injection of our novel treatment can induce a systemic effect.

[0119] Example 6 A composition comprising CuMVtt830-VLPs and MCT reduces tumor dividing ratio (TMR) and necrosis To further investigate the effect of intratumoral injection, experiments similar to those described in Example 4 were performed.

[0120] On day 14, tumors were harvested from each group, i.e., compositions of CuMVtt830-VLP and MCT, and PBS as a control, and the median size tumor from each group was stored in 4% paraformaldehyde for 1 day before histological analysis. One complete section of each harvested tumor was examined after melanin bleaching and subsequent H&E staining. Table 1 shows the general characteristics of the harvested tumors, specifically, tumor diameter and depth (mm). The composition containing CuMVtt830-VLP and MCT showed the smallest diameter and depth. [Table 2]

[0121] Various studies have previously shown that the impact of tumor necrosis on prognosis may represent a paradoxical relationship in which increased tumor cell death indicates a more aggressive cancer. However, this relationship can be explained by rapid tumor growth, where the tumor outgrows its own blood supply, resulting in the formation of a hypoxic microenvironment, which subsequently causes tumor cell death. The presence of necrosis was significantly associated with advanced stage, poor differentiation, venous invasion, and large tumor size (Swinson DEB et al., Lung Cancer. 2002, 37(3):235-40; Pollheimer MJ et al., Hum Pathol. 2010, 41(12):1749-57). The tumor mitotic ratio (TMR) is considered an important and independent predictor of survival in melanoma patients. Studies from different centers have shown that TMR may improve the accuracy of melanoma staging (Azzola MF et al., Cancer. 2003, 97(6):1488-98; Thompson JF et al., J Clin Oncol. 2011, 29(16):2199-205). It has also been shown that TMR possibly indicates tumor depth (Basir HRG et al., Clin Cosmet Inv Derm. 2018, 11:125-30). Therefore, digital histological evaluation was performed and the results show a significant reduction in the percentage of necrosis in the group treated with a composition containing CuMVtt830-VLP and MCT (Figure 3A, Figure 3B). This is consistent with the 1mm 3 This is also true when comparing the number of mitoses per 100 cells (Figures 3C and 3D). These findings are shown in Table 2. [Table 3]

[0122] Example 7 The composition comprising CuMVtt830-VLP and MCT is CD8 + Increase T cells Mechanism of action of intratumoral injection of a composition containing CuMVtt830-VLP and MCT on CD8 + To test the role of T cells, we performed experiments as summarized in FIG. 4A using three groups of mice: [Table 4]

[0123] In group 3, CD8 mAb was isolated from C57BL / 6 blood by using anti-CD8a mAb (clone 53-6.7). + T cells were depleted. Briefly, 10ug / dose of anti-CD8a mAb was administered intravenously 2 days before tumor implantation and every 2 days thereafter. + T cells were identified before tumor implantation and on day 12 after tumor implantation. Tumor-bearing mice were treated with PBS or a composition containing CuMVtt830-VLP and MCT on days 2, 7, and 11 (CD8 + Mice were treated three times over a 14-day period. + T cell depletion was approximately 95% efficient (Figures 4B and 4C). + The frequency of T cells was checked again, and the results showed that intratumoral injection of CuMVtt830-VLP+MCT significantly increased the CD8 T cell count in the blood of group 2 compared to the control group or group 3. + The results show that the IL-16 expression of CD8 T cells in the IL-16 mice increased the % of CD8 T cells (Figure 4D), which can be used as a prognostic marker. On day 14, tumors were harvested and tumor volumes were measured. The results showed that the IL-16 expression of CD8 T cells in the IL-16 mice increased the % of CD8 T cells in the IL-16 mice (Figure 4D). This can be used as a prognostic marker. + showed that depletion of T cells prevented the antitumor effect of the treatment, and CD8 + indicating an essential role for T cells ( Fig. 4E ).

[0124] Example 8 A composition containing CuMVtt830-VLPs and MCT induces the abscopal effect upon intratumoral injection To study the abscopal effect of intratumoral treatment, we established an experiment as shown in Figure 5A, in which control PBS and CuMV formulated with MCT were administered intratumorally. TT -VLP(CuMV TTTwo treatment groups were used: one with CuMV-VLP 50ug / dose and the other with 4% MCT 100ul / dose. The right implanted tumor was treated by intratumoral injection, while the left tumor was left untreated. Due to the high tumor burden in this experiment, we decided to increase the intratumoral injection to twice a week. We also used CuMV formulated with MCT. TT The effect of anti-PD1 (intravenous injection) in combination with CuMV-VLPs was tested. However, the results did not show any additional antitumor effect, which could be explained by the fact that B16F10 melanoma tumors are unresponsive to anti-PD1 treatment (data not shown) (3). The results obtained were consistent with the results shown in Table 1. TT We demonstrated that intratumoral injection of the combination of -VLP+MCT could result in an antitumor response in the injected tumor as well as an abscopal effect (Figure 5B and Figure 5C). These findings were supported by the CD8 + This is also confirmed when measuring the density of T cells. These findings correlate with the results obtained in Example 2 and show that a systemic effect can be induced by intratumoral injection.

[0125] Example 9 Antitumor effects of various VLPs formulated with MCT upon intratumoral injection Various compositions of the invention containing various VLPs were prepared and tested for their antitumor effects. Thus, experiments similar to those described in Example 4 were performed using the following groups: PBS (control), CuMV-VLP formulated with MCT, Qβ-VLP formulated with MCT, or CCMV-VLP formulated with MCT. Tumors were treated intratumorally three times over a period of 14 days. Tumors were harvested on day 14 and tumor volumes were measured as described above. The results show that formulating various VLPs with MCT can induce antitumor effects in the B16F10 xenograft melanoma model (Figure 6).

[0126] Example 10 Potent antitumor responses and long-term protection of CuMVtt-VLPs formulated with MCT adjuvant in an equine melanoma model The efficacy of the novel treatment with a composition containing particles of CuMVtt830-VLPs and MCT was tested in an equine melanoma model. Two privately owned Arabian grey / white horses with severe melanoma metastases, already scheduled for euthanasia, were treated with a composition containing CuMVtt830-VLPs and MCT formulations. The first horse was a FEI Endurance grey stallion with an infiltrating melanoma in the area surrounding the uropygial gland and multiple melanoma external lesions in the tail. The internal melanoma mass was surgically removed and the penis had to be amputated due to a large preputial mass (Figure 7A). After one week, intratumoral treatment of the external melanoma lesions was started, with a total of four injections (10 days apart) using a pressure syringe. The induced antitumor response was followed weekly, in addition to temperature measurements, complete blood counts (CBC) and general health assessment. The consistency of the firm outer melanoma lesions changed over the treatment period, becoming softer and leaking a black secretion. Adverse effects recorded included mild itching, but no fever, and a normal CBC. Thus, the horse responded well to treatment, with a reduction in tumor size, indicating the efficacy of the novel treatment in eliminating large melanoma lesions in gray horses (Figures 7B-7F). The horse was followed for one year with no signs of recurrence. Furthermore, a distant ("abscopal") effect was observed in an untreated melanoma nodule on the top of the tail (Figures 7G and H). The second equine case is a pregnant gray Arabian mare diagnosed with a mammary mass. Ultrasound-guided intratumoral injections (two injections in total so far) of the novel immune enhancer were administered. No side effects were reported, and there was a notable reduction in the internal mass, as shown in Figures 7I and J. Treatment could not be continued as the horse was scheduled for euthanasia.

Claims

1. A composition comprising particles, each particle comprising: (a) at least one virus-like particle (VLP), preferably a recombinant VLP; (b) at least one microcrystalline tyrosine, preferably microcrystalline L-tyrosine, said microcrystalline tyrosine having a median particle size of 0.1 μm to 50 μm, preferably as determined by Flow Particle Image Analyzer (FPIA), and more preferably as described in Example 2; and preferably consisting of the VLP does not comprise a tumor-associated or tumor-specific antigen covalently bound to the VLP, and more preferably the VLP does not comprise an antigen covalently bound to the VLP; composition.

2. 2. The composition of claim 1, wherein the microcrystalline tyrosine has a maximum particle size of 750 μm, preferably the microcrystalline tyrosine has a maximum particle size of 150 μm.

3. The composition of claim 1 or 2, wherein the VLPs are adsorbed to the microcrystalline tyrosine.

4. 4. The composition according to any one of claims 1 to 3, wherein the composition is an aqueous composition, preferably the concentration of said microcrystalline tyrosine, preferably said microcrystalline L-tyrosine, is from 0.5% to 10% (weight tyrosine / volume of solution), preferably the concentration of said microcrystalline tyrosine, preferably said microcrystalline L-tyrosine, is from 1% to 7.5% (weight tyrosine / volume of solution).

5. wherein said VLP is a modified VLP, said modified VLP comprising, and preferably consisting of, at least one modified VLP polypeptide, said modified VLP polypeptide comprising: (a) a VLP polypeptide; (b) a helper T cell epitope; Including, The VLP polypeptide comprises: (i) the amino acid sequence of a viral coat protein, preferably the amino acid sequence of a plant virus coat protein, or (ii) a mutated amino acid sequence, wherein said mutated amino acid sequence and said coat protein of a virus exhibit a sequence identity of at least 90%, preferably at least 95%, more preferably at least 98% and also more preferably at least 99%. comprising, or preferably consisting of, A composition according to any one of claims 1 to 4.

6. 6. The composition according to any one of claims 1 to 5, wherein the VLP is derived from a plant virus or is a VLP of an RNA bacteriophage, preferably the plant virus is selected from the group consisting of Cucumber Mosaic Virus (CuMV), Cowpea Chlorotic Mottle Virus (CCMV) and Tobacco Mosaic Virus (TMV), and preferably the VLP of an RNA bacteriophage is a VLP of RNA bacteriophage Qβ or a VLP of RNA bacteriophage AP205.

7. The VLP is a modified VLP of Cucumber Mosaic Virus (CuMV), the modified VLP of CuMV comprising, and preferably consisting of, at least one modified CuMV polypeptide, the modified CuMV polypeptide comprising: (c) a CuMV polypeptide; and (d) a helper T cell epitope; comprising, or preferably consisting of, The CuMV polypeptide is (i) the amino acid sequence of the coat protein of CuMV, or (ii) a variant amino acid sequence, wherein said variant amino acid sequence and said coat protein of CuMV exhibit a sequence identity of at least 90%, preferably at least 95%, more preferably at least 98% and even more preferably at least 99%. comprising, or preferably consisting of, A composition according to any one of claims 1 to 6.

8. The composition of claim 7, wherein the CuMV polypeptide is a coat protein of CuMV or an amino acid sequence having at least 90%, preferably 95%, sequence identity to SEQ ID NO:

1.

9. 9. The composition of claim 7 or claim 8, wherein the helper T cell epitope replaces the N-terminal region of the CuMV polypeptide, the N-terminal region of the CuMV polypeptide corresponding to amino acids 2 to 12 of SEQ ID NO: 1, preferably the Th cell epitope is derived from tetanus toxin or is the PADRE sequence.

10. 10. The composition of any one of claims 5 to 9, wherein the Th cell epitope comprises the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:

4.

11. A composition for use according to any one of claims 7 to 10, wherein the modified CMV polypeptide comprises, preferably consists of, the amino acid sequence of SEQ ID NO:11 or SEQ ID NO:

12.

12. The composition of any one of claims 1 to 11, wherein the VLP, preferably the recombinant VLP, comprises an immunostimulatory substance packaged within the VLP, preferably the immunostimulatory substance is a Toll-like receptor activator, more preferably the Toll-like receptor activator is an immunostimulatory nucleic acid, and even more preferably the immunostimulatory nucleic acid is ssRNA.

13. 13. The composition of any one of claims 1 to 12 for use in a method of treating a solid tumour in a patient, wherein the patient is a human patient or an animal patient, preferably wherein the animal patient is an equine animal, preferably selected from a horse, a dog or a cat, more preferably wherein the animal patient is a horse.

14. 14. The composition for use according to claim 13, wherein said method of treating a solid tumor in a patient is a method of treating said solid tumor in a patient intratumorally.

15. The composition for use according to claim 13 or claim 14, wherein the solid cancer is selected from breast cancer, prostate cancer, colorectal cancer, lung cancer and melanoma cancer, preferably melanoma cancer.

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