WO2020154786A1

WO2020154786A1 - Immunomodulatory virus-like particles, compositions and therapeutic use thereof

Info

Publication number: WO2020154786A1
Application number: PCT/BR2020/050016
Authority: WO
Inventors: Marcio Chaim BAJGELMAN; Soledad PALAMETA; Andrea Manrique RINCÓN
Original assignee: Centro Nacional De Pesquisa Em Energia E Materiais – Cnpem
Priority date: 2019-01-28
Filing date: 2020-01-27
Publication date: 2020-08-06
Also published as: BR112021014790A2

Abstract

This present application refers to novel virus-like particles (VLPs), which are useful as immunomodulatory agents, compositions and uses thereof, as well as methods of treatment. The VLPs disclosed herein are target-driven and have immunomodulatory properties provided by heterologous molecules attached, or anchored, thereto.

Description

IMMUNOMODULATORY VIRUS-LIKE PARTICLES, COMPOSITIONS AND

THERAPEUTIC USE THEREOF

TECHNICAL FIELD

[0001] The present disclosure is generally related to targeted virus-like particles having immunomodulatory properties, pharmaceutical compositions, therapeutic methods and uses thereof.

BACKGROUND

Virus-like particles (VLPs) are particles formed by structural viral proteins that have inherent property for self-assembling and mimicking the morphology of a native virus exhibiting a repetitive array of antigens. In contrast to viruses, VLPs are non-infectious and non-replicating because they lack the genetic material therefor. VLPs have a deriving virus-particle-size range between 22 nm and 150 nm, varying according to the incorporated viral proteins (Chroboczek, Szurgot and Szolajska (2014), Acta Biochimica Polonica, v. 61, n. 3; Grgacic and Anderson (2006), Methods, v. 40, n. 1, p. 60-65) .

[0002] VLPs may be divided into two categories, depending on the structure of their parental viruses: non-enveloped VLPs and enveloped VLPs. Non-enveloped VLPs are categorized as single or multiple capsid protein VLPs. These VLPs, composed of a single capsid protein, can be produced in prokaryotic and eukaryotic expression systems (Chen et al . (2011) PLoS One 6(9): e24671; Bundy and Swartz (2011), Journal of biotechnology, v. 154, n. 4, p. 230-239) . On the other hand, the VLPs of proteins of multiple non-enveloped capsids are more complex and difficult to produce. These complex VLPs are generally produced in eukaryotic hosts such as yeast (Rodriguez-Limas et al., (2011), Microbial cell factories, v. 10, n. 1, p. 33), insect cells (Fernandes et al . (2013), Expert review of vaccines 12, N. 2, p. 225-236) and plants (Scotti and Rybicki (2013), Expert review of vaccines, v. 12, n. 2, p. 211-224), which allow the coexpression of different capsid and complex assembly of the VLPs within a cell .

[0003] VLPs are useful as platforms for the development of immunomodulators , as they can display antigens, target- specific molecules with high specificity for the treatment of several diseases. Other molecules, such as immunomodulators, may be added to the VLP platform. Generally, in order to display such molecules on its surface, the gene sequences encoding the VLPs are modified by means of molecular biology.

[0004] VLPs have been shown to induce potent humoral and cellular responses, since they are generally more immunogenic than the subunits or immunogens of recombinant proteins. In particular, VLPs exhibit conformational epitopes and can activate B cell receptors and T cell- independent IgM responses (Zhang et al . (2009), Journal of immunotherapy (Hagerstown, Md. : 1997) 32, Nr. 2: 118) ; Zabel et al . (2004), The Journal of Immunology, V12, p.5499-5508; Ramani et al . (2017), Clinical and Vaccine Immunology, v.24, N. 5, p e00571-16) .

[0005] Therapeutic VLPs can be targeted by the presentation of target cell-specific tropism ligands. Thus, cancer cells, often over-express receptors that help promote their growth such as folate, epidermal growth factor, and transferrin receptors ( Toporkiewicz et al. (2015), International journal of nanomedicine 10: 1399). Therefore, VLPs exhibiting their respective ligands have been widely used for targeted delivery and uptake by various cancer cells (Galaway and Stockley (2012), Molecular pharmaceutics, v. 10, n. 1, p. 59-68) . However, it should be noted that these receptors are also expressed to a lesser extent in healthy cells, resulting in associated cytotoxicity and reduced efficiency due to competition with natural ligands found in the bloodstream (Allen (2002), Nature Reviews Cancer 2, Nr. 10: 750.

[0006] In order to improve target specificity and delivery efficiency, other targeting ligands have been tested. In a first instance, the use of tumor-specific antibodies fused with VLPs for producing target-directed VLPs has been addressed, because of their excellent binding and target specificity. Nonetheless, such system has several limitations and disadvantages, such as its large size, which can limit tissue penetration and increase production cost. Because of such limitations, VLPs were conjugated with other smaller and less expensive targeting ligands in the form of DNA aptamers (Cohen and Bergkvist 2013; Tong et al . , 2009) and peptides (Shishido et al . , 2010), which can achieve a binding specificity and affinity similar to the antibodies.

[0007] Although DNA aptamers still require conjugation to the VLP surface, targeting peptides have the option of being genetically fused to viral capsid proteins (Munch et al. (2013), Molecular therapy 21, Nr. 1: 109-118), greatly simplifying the production of VLPs. More importantly, large combinatorial libraries of aptamers and DNA peptides are easily created and tested for desired binding affinity and targeting specificity, even when the cellular receptor is unknown (Koivunen et al . (1999), Journal of Nuclear Medicine 40, Nr. 5: 883-888) . This highly productive engineering approach not only provides flexibility in the choice of targeted ligands, but it also allows for the adjustment of its binding affinity to cell surface receptors, which would provide a greater tissue penetration of VLPs .

[0008] Iimmunomodulation-based therapeutic strategies have revolutionized cancer treatment. Such strategies are usually designed for inhibiting mechanisms associated with immunological tolerance of tumor cells. Immunomodulation strategies include, for example, checkpoint inhibitory antibodies which block the cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and Programmed cell death protein 1 (PD1) receptors related to the maintenance of immunosuppression. Another immunomodulation strategy, in addition to blocking T-cell immunosuppressive receptors, consists of stimulating agonistic receptors, such as CD134 cell surface receptor (0X40) and CD137 cell surface receptor (4-1BB), potentiating lymphocyte activity and enhancing antitumor immune surveillance .

[0009] For example, receptors CD137 (also known as TNFRSF9 and 4-1BB) and CD134 (also known as TNFRSF4 and or 0X40) are present on the surface of activated T cells and act on the transduction of costimulatory signals associated with cell activation, survival and proliferation (Croft (2003) Cytokine Growth Factor Rev 14(3-4) : 265-273.; So et al .

( 2008 ) Cytokine Growth Factor Rev 19(3-4) : 253-262), co stimulation mediated by CD137 in CD8 cells leads to increased IL2 production and increased cell proliferation.

[0010] Literature also suggests that the binding of agonist antibody to CD134 receptor (0X40) induces inhibition of the Forkhead Box P3 (FoxP3) transcription factor, associated with the regulatory T cell immunosuppressive phenotype (So and Croft (2007) J Immunol 179(3) : 1427-1430) . Data from the literature also demonstrates that antitumor vaccines encoding TNFSF ligands 41BBL and OX40L induce T cell co- stimulation, regulatory T cell inhibition and potentiation of antitumor immune response (Manrique-Rincon et al . (2017) Front Immunol 8: 1150; Manrique-Rincon et al. (2018) J Biotechnol 284: 11-16) . Moreover, the use of a soluble 4-1BB ligand, agonist antibodies and multivalent aptamers directed to the CD137 receptor has led to the elimination of tumors in animal models (Melero et al. (1997) Nat Med 3(6) : 682- 685.; McNamara et al. (2008) J Clin Invest 118(1) : 376-386) . The CD134 receptor is associated with phenotypic changes in the activated CD4 cell. The binding of agonist antibodies results in increased cytokine production and maintenance of lymphocyte survival (Taraban et al. (2002) Eur J Immunol 32(12) : 3617-3627) .

[0011] Alternatives for the development of novel immunomodulatory drugs are disclosed herein. The advantages of the embodiments disclosed herein, which will be better explained below, include high target specificity of the disclosed VLPs, as well as a synergistic effect between protein ligands and immunomodulators attached thereto.

[0012] Target-specific peptides for use in therapy are described in the prior art. For example, WO2016164305 refers to the target-specific polypeptides which can be used as protein therapeutics to bind cells or soluble factors involved in diseases, such as cancer. However, the technology described in such document is based on the development of polypeptide sequences which target tumor antigens, or immunomodulation targets, aimed at the development of chimeric antigen receptor (CAR) , and vaccines used in VLPs, encoding polypeptide sequences and inducing the immune response against that sequence. In contrast, the presently disclosed technology does not involve developing CARs, nor cell lines included in CAR, nor developing vaccines used in VLPs. On the other hand, the embodiments disclosed herein refer to VLPs carrying immunomodulatory molecules target- driven to a tumor site. For the purposes of the embodiments disclosed herein, it is not necessary generating a CAR, or any immunomodulatory cell line, nor inducing the production of anti-tumor antibodies with VLPs. The present disclosure refers to VLPs which have such elements of immunomodulation and targeting on their surface. These VLPs lack any genome and don't harbor expression plasmids.

[0013] Another prior art document, namely W02016/ 127015 describes the generation of a vector that enables the production of co-stimulatory proteins simultaneously with the production of vaccine proteins. Such vector can be carried by a virus or VLP, so that expression occurs in cells. These co-stimulatory and vaccine proteins are secreted, rather than targeted, so they can have a systemic effect on the body, exacerbating the immune response and can trigger autoimmune responses. Nucleic acid vectors by itself also may represent a biosafety concern, since these vectors could induce heterologous gene expression or even causee a random integration in the host cell genome, activating or interfering undesirable gene expression. On the other hand, the target-directed particles disclosed herein enable delivering of immunomodulatory proteins directly to the tumor site. As previously mentioned, these particles lack genome. Such immunomodulators may stimulate cells of the immune system to eliminate the tumor. Therefore, the effect of immunomodulators is driven to the tumor site. Moreover, as explained below, the VLPs disclosed herein enable vehiculating soluble molecules, such as GM-CSF, anchored to their surface in order to deliver such molecules to the tumor site, which enhances immunomodulatory effects in addition to reducing undesired systemic effects. [0014] Chimeric proteins derived from IgA and IgM immunoglobulin chains that have T cell 0X40 co -stimulatory elements fused to tumor antigen binding proteins are also described in the prior art, for example in WO2018/017888. In contrast therewith, the embodiments disclosed herein do not require synthesizing chimeric proteins derived from immunoglobulins. Only particles derived from retroviral envelope (VLP) are decorated with immunomodulators , such as 0X40 ligand, 4-1BB ligand or soluble proteins such as GM-CSF that are anchored to the VLP surface. Furthermore, complicated processes for producing multivalent immunoglobulins are not required either. We use VLPs as a vector allows the delivery of simpler molecules, such as a peptide which recognize, for example, tumor cells. Instead of using agonist antibodies for co-stimulating 0X40 in T cells, an 0X40 ligand is attached to the surface of the VLP. Soluble immunomodulators may also be anchored to the surface of the VLPs disclosed herein.

BRIEF DISCLOSURE OF THE EMBODIMENTS

[0015] Novel virus-like particles (VLPs) useful as immunomodulatory agents are disclosed herein, which particularly enhance the immune response against tumors.

[0016] According to preferred embodiments, the capsids of the VLPs disclosed herein have one or more heterologous molecules attached thereto. According to such embodiments, the heterologous molecules may be one or more of antigens, peptide ligands which drive tropism to a specific target, immunomodulators, cell surface receptors, as well as combinations thereof. Such molecules may be herein described as being attached to the VLPs' surface, anchored, or surface- anchored, thereto. Alternatively, the VLPs may be described as being decorated with heterologous particles, or as harboring the same. [0017] Examples of peptide ligands, immunomodulators , cell surface receptors useful according to the present disclosure may be, for example, a synthetic ligand for the prostate- specific membrane antigen (PSMA) , which is expressed in positive tumor cells, and surface-anchored immunomodulators as granulocyte-macrophage colony stimulating factor (GM- CSF) , CD137 cell surface receptor ligand (4-1BB), CD134 cell surface receptor ligand (OX40L) and combinations thereof.

[0018] Other embodiments disclosed herein refer to medical uses, methods of treatment and pharmaceutical compositions related to or comprising the VLPs disclosed herein .

DESCRIPTION OF THE DRAWINGS

[0019] Some aspects of the disclosure can be better understood with reference to the following drawings. The components in the drawings are not necessarily to Scale, emphasis is being placed upon illustrating the principles of the present disclosure.

[0020] FIG. 1 depicts Polystyrene beads that were loaded with the VLP-Hygro or with VLP-Ox40L / 4-1BBL. The beads were then stained with antiOx40L-PE or anti4-lBBL-PE antibodies, following flow cytometry. Green curve represents beads stained with antibodies without the addition of VLPs. Red Curve represents beads incubated with VLPs and stained with antibodies.

[0021] FIG. 2 illustrates the effects of immunostimulatory VLPs on CD4 + T cells. The bar graph shows the mean increase in Interferon gamma (IFN-y) production and secretion in cells that were incubated with the 4-1BBL and OX40L ligands. The same cells were subjected to flow cytometry to evaluate proliferation by labeling with CFSE . Data was analyzed using ANOVA followed by the Turkey's multiple comparison test considering significant values *** p <0.05, mean + SEM.

[0022] FIG . 3 shows that immunomodulatory VLPs were able of inhibiting FoxP3 transcription factor in iTR. The bar graph represents the mean expression of Foxp3 in the iTR cells. Negative: non-treated iTR, Hygro: iTR treated with control VLPs. Other groups were treated with the indicated costimulatory VLP-OX40L or VLP 41BBL/OX40L. Data was analyzed using ANOVA followed by the Turkey's multiple comparison test considering significant values *** p <0.05 against the negative control, mean + SEM.

[0023] FIG . 4 illustrates that Peptide-decorated VLPs present binding-selectivity for PSMA positive cells. VLPs decorated with PSMA targeting peptide ligand (LM or LD peptides), and 4-1BBL, were incubated with 3T3 (green curve) and 3T3-PSMA (red curve) cells following antibody staining with the anti-4-lBBL-PE antibody. Binding of VLP by LM / LD peptides was detected by flow cytometry.

[0024] FIG . 5 illustrates that VLPs LD-GM-41BBL (LDGM41L) and the VLP LD-GM-OX40L (LDGMOxL) harbors association of immunomodulators . It is shown flow cytometry with indicated samples stained with anti-41bbL, anti-GM-CSF and anti-OX40L antibodies conjugated to the phycoerythrin (PE) fluorophore.

[0025] FIG . 6 illustrates the effects of inhibition assay of FoxP3 in iTRs cells. The bars represent the mean expression of Foxp3 in iTR cells. The iTR cells were treated with control VLPs (Hygro) and immuno-targetted VLPs. The data was analyzed using ANOVA, followed by the Turkey's multiple comparison test, considering significant values ** p <0.05 in relation to negative control, mean + SEM. [0026] FIG . 7 shows in vivo experiments made to investigate the immunomodulatory potential of immune-targeted VLPs harboring the LD tumor-specific ligand. Graphs show two independent in vivo experiments; tumors were injected on day 1, followed by administration of therapeutic VLPs on days 2, 5 and 8. After 25 days the animals were sacrificed, and the tumors analyzed. (A) animals were given LD-VLPs harboring just one immunomodulator . (B) animals were given LD-VLPs harboring two immunomodulators , in comparison with LD-41BB, harboring just a single immunomodulator. The Mann-Whitney test was used for statistical analysis considering significant values of * p <0.05 compared to the VLP LD-Hygro control .

[0027] FIG . 8 illustrates in vivo experiment made to evaluate target-driven elimination of B16-PSMA positive cells. Experimental groups: PBS (negative control without VLP) , LD-Hygro (group treated with irrelevant VLP without therapeutic function) and LD + GM + OX40L (group treated with therapeutic immune-targeted VLP) . Tumors were injected on day 1, followed by therapeutic VLPs on days 2, 5 and 8. After 20 or 28 days respectively, the animals were sacrificed, and the tumors were analyzed. Each dot represents an animal and the tumor volume reached at the end of the experiment, as well as the mean and SEM for each experimental group .

DE TAILED DE SCRIPTION OF THE EMBODIMENTS

[0028] A first embodiment disclosed herein refers to an immunomodulatory target-driven virus-like particle (VLP) comprising one or more heterologous molecules attached and/or anchored to its surface.

[0029] The heterologous molecule attached, or anchored, to the VLDs disclosed herein may be selected from an antigen, a target-specific molecule, e . g . a peptide, and an immunomodulator . Alternatively, The VLPs disclosed herein may comprise a combination of heterologous molecules, e.g. at least a target-specific peptide and at least an immunomodulator ligand attached and/or anchored thereto.

[0030] Such heterologous molecules may provide, for example, specific targeting properties to the VLP . Such specific targeting properties render several advantages to the VLP, such as avoiding systemic side-effects. Moreover, the more target-specific is the VLP, the lower amount thereof is required for reaching a desired effect, particularly a therapeutic effect in a subject.

[0031] According to the above embodiments, specific targeting of the VLPs disclosed herein may be mediated, for example, by one or more surface-anchored peptide sequences. Such sequences bind to targets, such as tumor cell membrane receptors, proteins, antigens and the like.

[0032] In a specific embodiment, a surface-anchored synthetic peptide sequence targets prostate-specific membrane antigen (PSMA) . PSMA is a known marker of prostate cancer (Lee et al. (2002) Mol Ther 6(3) : 415-421; Gosh and Heston (2004) J Cell Biochem 91(3) : 528-539) . The PSMA targeting peptide ligand (LD) is described the literature (Shen et al . (2013) PLoS One 8(7) : e68339) and comprises the amino acid sequence SHSFSVGSGDHSPFT . According to such embodiment, which may also be combined with any of the above- described ones, the sequence of the target-specific peptide comprised in the VLPs disclosed herein may be SHSFSVGSGDHSPFT, or a functionally equivalent variant thereof. According to such specific embodiment, target- specific peptide, also referred to as LD, is used for the targeting of VLP to PSMA positive tumor cells. [0033] The amino acid sequence SHSFSVGSGDHSPFT may be assembled, for example, into a cassette composed of the following elements: ATG-IgK-LD-PDGFR-TGA, where ATG: start codon, IG-K leader sequence to target protein to VLP surface, LD : targeting sequence to tumor cells, PDGFR: transmembrane domain of platelet-derived growth factor, which anchors the fusion protein to the surface of the VLP. All such elements are cloned into the same reading frame in order to obtain a cassette encoding a fusion protein. Such cassette is inserted into a plasmid between a CMV enhancer promoter at the 5' end and a polyadenylation signal at the 3' end.

[0034] Therefore, according to some of the preferred embodiments, the VLPs disclosed herein are driven to the tumor site due to an interaction of the ligand peptide LD, attached to its surface, with the PSMA surface protein of the tumor cell. Accordingly, in a preferred embodiment, the VLPs disclosed herein delivery GM-CSF to the tumor site locally, as it is target-specific, also stimulating the action of APC cells and potentiating the antitumor immune response .

[0035] In other embodiments, which may also be combined with any of the above-described ones, the VLPs comprise one or more immunomodulators attached, or anchored, to its surface. According to such embodiments, an immunomodulator ligand may be, for example, GM-CSF, 4-1BB ligand, 0X40 ligand and/or combinations thereof. According to such embodiments, immunomodulatory properties of the VLPs disclosed herein are provided by proteins attached, or anchored, to the VLP surface. Such proteins may be, for example, ligand proteins, cytokines and the like. For example, chimeric granulocyte- macrophage colony stimulating factor (GM-CSF) , 4-1BBL (also known as CD137 ligand), OX40L (also known as CD252), or combinations thereof. [0036] According to a specific embodiment, the chimeric GM- CSF protein is a preferred surface-anchored protein because it acts on the activation of antigen presenting cells (APC) whilst T-cell costimulatory ligand as OX40L or 4-1BBL, stimulates antitumor lymphocyte action and may also contribute to inhibiting regulatory T cells. The GM-CSF cytokine is widely described in the literature as an immunomodulator that stimulates and activates Antigen presenting cells (APCs), increasing the antitumor response (Dranoff et al. (1993), Proc Natl Acad Sci U S A 90(8) : 3539 - 3543; Eager and Nemunaitis (2005), Mol Ther 12(1) : 18-27; Lipson et al . (2015), J Transl Med 13: 214; Manrique-Rincon et al. (2017), Front Immunol 8: 1150) . Data from the literature demonstrate that antitumor vaccines harboring a membrane-bound GM-CSF induce comparable anti-tumor effects to soluble GM-CSF (Yei et al . (2002), Gene Ther 9(19) : 1302- 1311) . Therefore, the hypothesis of a synergistic effect resulting from the association of a GM-CSF anchored to a VLP as disclosed herein was tested.

[0037] The above-described preferred embodiments may be carried out, for example, by amplifying the GM-CSF sequence from C57 mouse cDNA. In the present case, the sequence was cloned without the start codon, into a cassette composed of the following elements: ATG-IgK-GMCSF-PDGFR-TGA, where ATG: start codon, IG-K leader sequence to target protein to VLP surface, GM-CSF: granulocyte-monocyte colony stimulating factor, PDGFR : transmembrane domain of platelet -derived growth factor, which anchors the fusion protein to the surface of the VLP. All these elements were cloned into the same reading frame in order to obtain a cassette encoding a fusion protein. This cassette is inserted into a plasmid between a CMV enhancer promoter at the 5 ' end and a polyadenylation signal at the 3' end. [0038] According to other embodiments, which may be combined with the ones disclosed above, VLPs comprising 0X40 ligand attached or anchored to its surface are also disclosed. The OX40L ligand, also known as CD252, is expressed on APC cells and can interact with the 0X40 receptor expressed on activated T cells. The 0X40 receptor mediates the transduction of costimulatory signals (So et al . (2008), Cytokine Growth Factor Rev 19(3-4) : 253-262) . The importance of 0X40 signaling in regulating the accumulation of CD8 T cells during the immune response was demonstrated (Bansal-Pakala et al . (2004), J Immunol 172(8) : 4821-4825) , as well as the generation and maintenance of memory cells (Croft et al . (2009), Annual review of immunology, v.28, p. 57-78) . In the case of CD4 (+) T cells, costimulation mediated by the binding of an agonist antibody (0X86) to the 0X40 receptor may induce an increase in the survival of activated CD4 T lymphocyte. Furthermore, literature data also demonstrate that binding of the 0X86 agonist antibody to the regulatory T cell receptor 0X40, can induce inhibition of the expression of the FoxP3 transcription factor, which is associated with maintenance of the Treg immunosuppressive phenotype (Fontenot et al . (2003), Nat Immunol 4(4): 330-336; Sakaguchi (2004), Annu Rev Immunol 22: 531-562; So and Croft (2007), J Immunol 179(3) : 1427-1430; Piconese et al. (2008), J Exp Med 205(4) : 825-839) .

[0039] Regulatory T cells have the unique property of inhibiting proliferation of effector T cells, which is essential to block the activity of autoreactive lymphocytes, maintaining a balance between immunotolerance and immunosurveillance (Sakaguchi et al. (1995), J Immunol 155(3) : 1151-1164; Shimizu et al. (1999), J Immunol 163 ( 10 ) : 5211-5218; Shimizu et al . (2002), Nat Immunol 3(2) : 135-142; Somasundaram et al . (2002) , Cancer Res 62 (18) : 5267-5272) . Data from the literature shows an increase in regulatory T cells infiltration into tumor sites (Betts et al . (2011), Gut; Chen et al . (2011), PLoS One 6(9) : e24671; D'Arena et al . (2011), Leuk Res 35(3) : 363-368) . These regulatory T cells can inhibit the proliferation of effector T cells in the tumor site, antagonizing the antitumor response. Because of this, the inhibition of Treg is been tracked as a strategy to increase antitumor response. Inactivation of regulatory T cells can be performed using the drug ONTAK ®, which is a chimera composed of a portion of antibody that recognizes the CD25 receptor, and the DTA subunit of diphtheria toxin. Ontak® binds to CD25 mediating the internalization of DTA, which exerts a toxic effect by eliminating the target cell (Olsen et al . (2001), J Clin Oncol 19(2) : 376-388; Dannull et al . (2005), J Clin Invest 115(12) : 3623-3633) . Despite eliminating regulatory T cells, Ontak® also eliminates activated CD4 (+) T cells, which also have the CD25 marker constitutively expressed on the cell surface. These activated CD4 T cells could be important in the fight against tumor cells. Another strategy described in the literature is the use of 0X86 agonist antibody driven to the 0X40 receptor. This strategy allowed the inhibition of regulatory T cells promoting potentiation of antitumor immunity (So and Croft (2007), J Immunol 179(3) : 1427-1430; Piconese et al. (2008), J Exp Med 205(4) : 825-839) .

[0040] In view of the non-specificity of Treg inhibitory strategies targeting CD25 receptor and consequent antagonism of the antitumor response, a novel proposal for inhibition of regulatory T cells through the use of VLPs decorated with 0X40 ligand is currently disclosed. Analogous to the 0X86 antibody, it was found that OX40L-coated VLP attenuates expression of the FoxP3 transcription factor in regulatory T cells. According to literature data, the inhibition of FoxP3 in regulatory T cells, mediated by 0X40 co-stimulation, is associated to enhancing antitumor immune response (So and Croft (2007), J Immunol 179(3) : 1427-1430; Piconese et al . (2008), J Exp Med 205 (4) : 825-839) .

[0041] Therefore, OX40L and/or 4-1BBL are herein shown to enhance T cell activation and contributed to inhibiting Treg immunosuppressive phenotype. Moreover, the association of such ligands with GM-CSF is also shown herein to boost antitumor effect of the disclosed VLPs . Therefore, specific embodiments disclosed herein refer to VLPs comprising several combinations of a targeted peptide ligand and immunomodulators attached to its surface. For example, two or more immunomodulators may be attached to a VLP, or a targeted ligand protein may be combined with one or more immunomodulators. In preferred embodiments, trivalent VLPs (i.e. VLPs comprising three heterologous molecules attached thereto are disclosed) .

[0042] An additional embodiment disclosed herein refers to therapeutic compositions comprising an effective amount of a VLP as disclosed herein and a pharmaceutically- acceptable carrier.

[0043] According to a specific embodiment, the VLPs disclosed herein have antitumor and immunomodulatory properties. Therefore, an additional embodiment disclosed herein is a method of treating an immune disease or condition, such as cancer, comprising administering an effective amount of the VLP as disclosed herein to a subject in need thereof. A further embodiment disclosed herein is the use of an effective amount of the VLP as disclosed herein for treating an immune disease or condition, such as cancer. An alternative embodiment is the use of an effective amount of the VLP as disclosed herein for preparing a pharmaceutical composition for treating an immune disease or condition, such as cancer. According to such embodiments, an immune disease or condition may be, for example cancer or even infectious diseases. According to such embodiments, cancer may be, for example, prostate cancer.

[0044] As previously mentioned, the VLPs disclosed herein may be derived from HIV-1 lentivirus, Moloney Murine Leukemia Virus, or a functionally equivalent variant thereof. According to an alternative specific embodiment, the VLP disclosed herein may be assembled by cloning of cassetes, for example, the following two ones, or a functionally equivalent variant thereof :

ATG-IgK-LD-PDGFR-TGA, where ATG: start codon, IG-K leader sequence to target protein to VLP surface, LD: targeting sequence to tumor cells, PDGFR: transmembrane domain of platelet-derived growth factor, which anchors the fusion protein to the surface of the VLP.

ATG-IgK-GMCSF-PDGFR-TGA, where ATG: start codon, IG-K leader sequence to target protein to VLP surface, GM- CSF: granulocyte-monocyte colony stimulating factor, PDGFR: transmembrane domain of platelet-derived growth factor, which anchors the fusion protein to the surface of the VLP.

[0045] The VLPs disclosed herein may be produced in cell culture by transfecting DNA vectors that separately encode the viral capsid, and combinations of LD peptide and immunomodulators as GM-CSF, OX40L ligand, 4-1BBL ligand. All DNA vectors lack packaging signaling, and in this way, VLPs lack genome. The VLPs are produced and released by cells directly into the culture medium, which can be harvested, filtered to remove cellular debris and frozen. This frozen culture medium contains the VLPs that can be used in later applications. It is possible to improve purification steps using Amicon columns (Millipore®) or dialysis membranes to exchange the culture medium for PBS or another vehicle of interest .

[0046] The examples below illustrate alternatives for the reproduction of the embodiments disclosed herein, as well as show the effectiveness thereof.

EXAMPLES

Example 1: VLPs can be decorated with immunomodulatory ligands on their surface

[0047] In order to verify if VLPs can be generated harboring immunomodulatory ligands on their surface, VLPs decorated with OX40L and 41BBL were generated. These VLPs were incubated with polystyrene beads and then labeled with secondary antibodies for flow cytometry. As seen in FIG. 1, the dual functionalized VLP labels for both OX40L and 4- 1BBL. The VLP hygro is a control-VLP that has no surface binders and has no labeling.

[0048] FIG. 1 depicts Polystyrene beads that were loaded with the VLP-Hygro or with VLP-Ox40L / 4-1BBL. The beads were then stained with antiOx40L-PE or anti4-lBBL-PE antibodies, following flow cytometry.

Example 2: Combination of immunomodulators enhance T cell activity and proliferation

[0049] In order to verify the immunomodulatory activity of VLPs, a control VLP (Hygro) was produced, a monovalent VLP decorated with the 4-1BB ligand (41bbL), a monovalent VLP decorated with the 0X40 ligand (Ox40L) and a divalent VLP 4- 1BBL + Ox40L. Such VLPs were incubated with CFSE-labeled CD4 positive T cells to measure the proliferation and secretion of the cytokine IFN-gamma.

[0050] As shown in FIG. 2, immunomodulatory VLPs induced increased cell proliferation and increased IFN-gamma production. Moreover, a synergistic effect was found in the combination of OX40L and 4-1BBL. In comparison with the antitumor vaccines that require using genetically modified autologous cells, the VLPs have the simplicity of not requiring cells, simply administering the particle to induce the biological effect.

Example 3: Immunomodulatory VLPs inhibit FoxP3 transcription factor of regulatory T cells

[0051] As mentioned above, regulatory T cells may antagonize the antitumor immune response (Sakaguchi et al . (1995), J Immunol 155(3) : 1151-1164; Shimizu et al. (1999), J Immunol 163(10): 5211-5218; Shimizu et al . (2002), Nat Immunol 3(2) : 135-142; Somasundaram et al . (2002), Cancer Res 62(18) : 5267-5272). The FoxP3 transcription factor is considered as a master key in the regulation of Treg immunosuppressive activity and its inactivation can inhibit the immunosuppressive phenotype and potentiate the antitumor effect (Fontenot et al . (2003), Nat Immunol 4(4) : 330-336; Sakaguchi (2004), Annu Rev Immunol 22: 531-562; So and Croft (2007), J Immunol 179(3) : 1427-1430; Piconese et al. (2008), J Exp Med 205(4) : 825-839) .

[0052] As seen in FIG. 3, immunomodulatory VLPs induced inhibition of FoxP3 expression in regulatory T cells.

Example 4 : VLPs can be decorated with peptides driving tropism to PSMA positive tumor cells [0053] Previous studies attempted to find strategies for systemic delivery of TNFSF receptor agonists, such as 4-1BB agonists, in order to reduce toxicity and adverse effects (Schrand et al. (2015), Oncoimmunology 4(3): e970918) . In a similar manner, it was hypothesized that the introduction of a ligand for targeting immunomodulatory VLPs could bring the added benefit of reducing cytotoxicity or adverse effects due systemic administration.

[0054] To this end, VLPs decorated with two different PSMA- targeting peptide ligands were generated, which are herein referred to as "LD" and "LM", in addition to an immunomodulatory 4-1BBL ligand. These LD and LM peptides have been previously described in the literature as showing affinity and selectivity for binding to PSMA (Shen et al . (2013) , PLoS One 8 (7) : e68339) . Therefore, idea was to generate bivalent VLPs containing one of these ligands in order to verify the possibility of driving VLP tropism to PSMA positive cells, in addition to the 4-1BBL ligand, which could be used to label the particle. Therefore, the VLP decorated with LM / LD ligands and the immunomodulator 4- 1BBL could bind to a PSMA positive cells, and since the VLP also has the 4-1BBL, it could be labelled with a flow cytometry antibody.

[0055] As shown in Fig. 4, the double-functionalized VLP was incubated with NIH-3T3 cells, or with NIH-3T3-PSMA cells, verifying the selectivity of LD / LM-coated VLPs targeting PSMA positive cells . The best result was observed for the LD ligand (left graph) .

Example 5: Generation and characterization of trivalent target-driven VLPs [0056] As shown in FIG. 2, the combination of immunomodulators may act in synergy, enhancing T cell stimulation. Thus, trivalent VLPs were generated, decorated with the PSMA ligand and immunomodulators GM-CSF, OX40L and 41BBL. As a proof of concept, two types of VLP were initially generated, which, in addition to the LD ligand, also had the immunomodulator GM-CSF or 4-1BBL. As shown in FIG. 5, a flow cytometry assay was performed, in which polystyrene beads were incubated with the indicated VLPs: LDGM41L (LD + GMCSF + 41BBL) or LDGMOxL (LD + GMCSF + OX40L) and stained with flow cytometry antibodies 41BBL-PE, anti OX40L-PE and anti GM-CSF-PE. The labelling confirms presence of immunomodulatory ligands on surface of immuno-targeted VLPs.

Example 6 : Trivalent target-driven VLPs show immunomodulatory activity

[0057] After verifying the possibility of generating trivalent VLPs, an assay to inhibit regulatory T cells was performed. Some additional

[0058] conditions were also tested, in which the generated VLP had only the PSMA ligand and a single immunomodulator for comparison with previously tested monovalent VLPs (FIG. 6) . As shown in FIG. 6, trivalent VLP induces FoxP3 inhibition comparable to the inhibition observed with a single immunomodulator.

Example 7 : Immunomodulatory VLPs potentiate tumor inhibition in immunocompetent mice.

[0059] An in vivo assay was performed to check antitumor activity of immunomodulatory VLPs. An immunocompetent model was used, in which C57 black mice were challenged with B16- PSMA tumor cells. These B16-PSMA tumor cells derived from the parental strain B16-F10 (ATCC CRL6475) were genetically modified to express the PSMA.

[0060] Animals were given tumors on day 1, following the treatment with three doses of immune-targeted VLPs administered on days 2, 5 and 8. The animals were sacrificed on day 25th after tumor inoculation. As shown in FIG. 7A, Animals were treated with LD-VLPs decorated with a single immunomodulator, observing the best biological effect in this group for animals treated with VLP-LD-41BBL . In FIG. 7B, the same assay was performed but, this time using the trivalent VLPs, harboring the LD ligand in association of two immunomodulators . In this experimental group, it was observed the best biological effect for animals treated with the VLP-LD-GM-OX40L.

[0061] In addition to the elimination of tumor cells, the vaccine combination was also found to induce protective immunity in re-challenged animals. Accordingly, VLPs harboring the same combination of immunomodulators may induce a similar antitumor protection, without employing genetically modified autologous cells and their readministration, reducing the complexity of the therapeutic proposal .

Example 8: Immunomodulatory VLPs decorated with LD ligand selectively potentiate elimination of PSMA positive cells

[0062] The aim of decorating VLPs with a PSMA ligand was to enable a targeting of the immunomodulatory VLP to the tumor site, concentrating the action of these immunomodulators in a more localized manner, also expecting to reduce the toxicity or inherent adverse effects of the systemic administration. To that end, a new in vivo assay was performed using the same model of C57 animals challenged with syngeneic cells. However, this time one group with B16 parental cells and a second group with B16-PSMA cells were challenged. As shown in FIG. 8, it was observed a more pronounced antitumor effect on B16-PSMA cells, suggesting a selectivity for elimination of PSMA positive cells.

Claims

1. A virus-like particle (VLP) comprising heterologous molecules attached thereto, wherein the heterologous molecules are selected from a target-specific ligand peptide, an immunomodulator and combinations thereof.

2. The VLP of claim 1, wherein the target-specific ligand peptide is a PSMA-targeting peptide ligand.

3. The VLP of claim 1, wherein the target-specific ligand peptide selectively potentiate elimination of PSMA positive cells .

4. The VLP of claim 1, wherein the target-specific ligand peptide comprises the amino acid sequence SHSFSVGSGDHSPFT .

5. The VLP of claim 1, wherein the immunomodulator is a cytokine .

6. The VLP of claim 1, wherein the immunomodulator is selected from GMCSF, 41BBL, Ox40L and combinations thereof.

7. The VLP of claim 1 comprising a target-specific ligand peptide, GMCSF and 41BBL attached to its surface.

8. The VLP of claim 1 wherein the heterologous molecules are a target-specific ligand peptide comprising the amino acid sequence SHSFSVGSGDHSPFT, GMCSF and OX40L.

9. A pharmaceutical composition comprising a VLP as defined in claim 1 and a pharmaceutically acceptable carrier.

10. Use of a VLP of claim 1 for preparing a composition as described in claim 9 for treating an immune disease.

11. The use of claim 10, wherein the immune disease is cancer .

12. The VLP described in claims 1 to 8 for use in a method for treating an immune disease.

13. The VLP of claim 12, wherein the immune disease is cancer .

14. A method of treating an immune disease comprising administering an effective amount of the VLP of claim 1 to a subject in need thereof.

15. The method of claim 14, wherein the immune disease is cancer .