Methods for prevention of tumor recurrence

Abstract

Provided herein are methods of cancer treatment using plant viral nanoparticles, such as Cowpea Mosaic Virus nanoparticles.

Description

METHODS FOR PREVENTION OF TUMOR RECURRENCECROSS REFERENCE TO RELATED PATENT APPLICATIONS

[0001] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application Nos. 63 / 521313, filed June 15, 2023, and 63 / 521615, filed June 16, 2023, the entire contents of which are incorporated herein by reference.STATEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with government support under Grant Nos. CA274640, CA224605, and CA253615 awarded by the National Institutes of Health (NIH). The government has certain rights in the invention.BACKGROUND

[0003] Throughout this disclosure reference is made to technical and patent literature to more fully describe the state of the art by an identifying citation or an Arabic numeral. The full bibliographic citation for the references identified by an Arabic numeral is provided immediately preceding the claims.

[0004] Metastasis is defined as the dissemination of cancer cells from the primary tumor followed by trafficking through the circulatory or lymphatic systems leading to invasion and tumor growth within distal organs[4]. In the clinic, this is denoted as stage IV cancer, and once metastasis has occurred, treatment options become limited and ineffective162 64L Metastatic cancers tend to be resistant to therapeutics, more aggressive, and demonstrate increased plasticity (e.g. by instigating angiogenesis in hypoxic conditions)11'31- the difficulty in treating metastatic cancer is evidenced by the fact that 66.7 - 90% of all cancer-related deaths are attributed to metastases14, 5]. Therefore, new interventions that either treat or protect against metastatic cancers are of dire need.

[0005] Prophylactic strategies that prevent onset and outgrowth of metastatic disease and those that prevent recurrence of disease after initial treatment (termed adjuvant therapy) are urgently needed16, 7]. Successful surgical removal of localized, primary tumors improves the possibility of a favorable outcome; however, in many cases the presence of cancer stem cellsor pre- disseminated cells may drive recurrence and metastasis. At times, even the surgery itself can lead to cancer metastasi s

[0065] . Therefore, adjuvant therapy is vital in the management of metastatic disease. Adjuvant therapy has already proven to be extremely successful in the prevention of breast and colon cancer metastases18, 9]; however, the current gold standard adjuvants are combinations of chemotherapeutics, which harbor harsh side effects and reduce quality of life

[0010] .

[0006] The chemotherapeutic regimens can also be taxing and require adjuvant therapy for up to months at a time at high dose density111]. Additionally, in patients that develop metastases regardless of adjuvant therapy, the cancer acquires adjuvant chemotherapy -induced resistance, and patient life expectancy drops compared to patients not provided any adjuvant therapy112, 13].

[0007] Immunotherapy has now been recognized as a pillar of cancer therapy and is a powerful approach. Immunotherapy can be specifically targeted to molecular markers, such as Herceptin used for treatment of human epidermal growth factor receptor 2 (HER2)-positive breast cancer

[0018] . More recent approaches focus on the checkpoint inhibitors (e.g. pembrolizumab and nivolumab

[0066] or small molecule immunostimulatory agents (e.g. TLR or STING agonists167, 68]). Nevertheless, these drugs are often effective only in a small subset of patients (e.g. only in patients with high programmed cell death protein-1 (PD-1) levels), can cause significant adverse effects, and require continuous or large numbers of doses169 71L Metastatic cancers, which are highly plastic, can also evade or decrease the effectiveness of these immunotherapeutics through the upregulation and recruitment of immunosuppressive cytokines and cells, respectively, as well as through downregulation of the targeted receptors

[0072]

[0008] Although moderately efficacious, current vaccines are therapeutic and designed to treat active disease. Provided herein are vaccines designed to be delivered during remission, when disease is clinically undetectable, an approach that termed “remission-stage vaccines” that can be modeled with prophylactic vaccines.SUMMARY OF THE DISCLOSURE

[0009] Herein, Applicant demonstrates that CPMV functions as an immunoprophylacticadjuvant therapy and treatment that rejects metastatic cancers. Longitudinal analyses demonstrate long-lasting adjuvanticity after a single administration of CPMV and development of systemic immune memory. Multiple metastatic models were used to demonstrate the wide scope and applicability of CPMV adjuvant therapy.

[0010] This disclosure demonstrates that a plant viral nanoparticle can be used as a therapy for the protection against metastatic cancers. In multiple models of metastatic cancer, administration of plant viral nanoparticle leads to potent upregulation of immunostimulatory cytokines leading to the accumulation and activation of innate immune cells. Innate immune cells are known in the art, and include macrophages, monocytes, neutrophils, and innate lymphoid cells.

[0074] This systemic immunostimulation leads to tumor cell death leading to the prevention of metastatic outgrowths within the intraperitoneal space and the lungs. Following the rejection of metastatic disease, systemic immune memory develops that is tumor-specific, but tissue-agnostic - re-challenge with the same cell line leads to rejection of the cancer in both the original and distant injection sites while re-challenge with another cell line lead to zero survivors

[0011] Thus, in one aspect, provided herein is a cancer vaccine, comprising a plant viral nanoparticle such as Cowpea Mosaic Virus (CPMV) (or CPMV particle as referred to herein), as well as related compositions and methods. In some embodiments, the disclosure relates to therapy and prophylaxis of cancer as well as methods to improve outcomes. In some embodiments, the cancer is a solid tumor, carcinoma, sarcoma, or a blood cancer. In one aspect the cancer is a metastatic cancer, e.g., intraperitoneal metastatic cancer.

[0012] In one aspect, provided is a method for inhibiting, delaying, slowing down, or preventing relapse of cancer in a subject in need thereof. The method comprises, consists essentially of, or consist of administering to the subject a plant viral nanoparticle such as cowpea mosaic virus (CPMV) particle, thereby inhibiting, delaying, slowing down, or preventing the relapse of the cancer in the subject. In some embodiments, the cancer is a solid tumor, carcinoma, sarcoma, or a blood cancer. In one aspect the cancer is a carcinoma. In some aspects, the compositions and methods extend the likelihood of progression free survival, overall survival, and reducing metastasis of a cancer patient after treatment and as compared to a subject not having received the composition or therapeutic method.

[0013] In another aspect, provided is a method for treating cancer in a subject in need thereof. The method comprises, consists essentially of, or consist of administering to the subject a plant viral nanoparticle such as a cowpea mosaic virus (CPMV) particle, thereby treating the cancer in the subject. In some embodiments, the cancer is a solid tumor, carcinoma, sarcoma, or a blood cancer. In one aspect the cancer is a carcinoma. In one aspect the cancer is a metastatic cancer, e.g., intraperitoneal metastatic cancer.

[0014] In yet another aspect, provided is a method for triggering or enhancing one or more of the following in a subject in need thereof: an immune response, immunostimulatory cytokines, increase in the ratio of Ml (antitumor) to M2 (protumor) macrophages, natural killer cells (NK cells), Toll Like Receptor (TLR)s 2, 4 and 7. The method comprises, consists essentially of, or consist of administering to the subject of a plant viral nanoparticle such as a cowpea mosaic virus (CPMV) particle cowpea mosaic virus (CPMV), thereby triggering or enhancing one or more of the following in the subject: an immune response, immunostimulatory cytokines, increase in the ratio of Ml (antitumor) to M2 (protumor) macrophages, natural killer cells (NK cells), TLRs 2, 4 and 7, chemokines, or myeloid- derived suppressor cells.

[0015] In one aspect, provided is a kit for use in a method as disclosed herein. The method comprises, consists essentially of, or consists of (a) an adjuvant which comprises, or consists essentially of, or consists of a plant viral nanoparticle, e.g., a cowpea mosaic virus (CPMV) particle; and an optional instruction for use. In one aspect, the cowpea mosaic virus (CPMV) particle is the only active agent in the adjuvant. In some embodiments, the kit further comprises (b) one or more antigen(s), or a cell comprising the one or more antigen(s). In some embodiments, the antigen(s) is a cancer antigen(s). In further embodiments, the cell comprising the one or more antigen(s) is a cancer cell. In some embodiments, the cancer is a solid tumor, carcinoma, sarcoma, or a blood cancer. In one aspect the cancer is a carcinoma. In one aspect the cancer is a metastatic cancer, e.g., intraperitoneal metastatic cancer.

[0016] This disclosure shows that plant viral nanoparticles such as CPMV are effective within the intraperitoneal space for up to 2 weeks off a single injection. Compared to small molecule therapeutics, which suffer from rapid clearance or drug washout effects, plant viral nanoparticles such as CPMV are able to withstand degradation for extended periods of timelowering the required number of doses. Indeed, using CPMV as an example, within the intraperitoneal space, CPMV was detectable for up to 2 weeks using RT-PCR. When compared to other adjuvants such as imiquimod and diABZI, CPMV can significantly reduce the number of tumor nodules in a metastatic lung cancer model. In vitro immunogenicity assays validated the increased immunogenicity of CPMV compared to imiquimod and diABZI.

[0017] Another advantage of plant viral nanoparticles such as CPMV, is the triple-pronged mechanism of CPMV. CPMV can activate TLRs 2, 4, and 7 meaning it has multimodal mechanisms for tumor clearance. While other adjuvants activate singular pathways, which are prone to become ineffective due to tumor downregulation of the pathways / resistance mechanisms, CPMV can bypass this issue due to the activation of multiple TLRs.

[0018] Once injected into the body, plant viral nanoparticles such as CPMV upregulates the expression of immunostimulatory cytokines and chemokines. Of note, the cytokines IFNy, IFNa, IL-12, and TNFa and the chemokines IP-10, MIP-la, MIP-ip, RANTES, MCP-1, and KC / GRO are upregulated. The chemokines especially lead to significant recruitment of immune cells such as dendritic cells, neutrophils, macrophages, and natural killer cells, and the cells that are recruited are polarized and activated into antitumor phenotypes. The ratio of Ml (antitumor) to M2 (protumor) macrophages is significantly increased following CPMV injection. This immunostimulatory state is active within the intraperitoneal space for up to 2 weeks leading to the rejection of tumor following intraperitoneal injection.

[0019] Following the rejection of the tumors, systemic immune memory is formed. The immune memory is dependent on the T cell response, as neutralization of both CD4+ and CD8+ T cells leads to a complete abrogation of immune memory. The memory is systemic, as re-challenge of mice in a distant area (e.g. subcutaneous re-challenge following initial intraperitoneal challenge) still leads to rejection of the tumors. ELISPOT analyses also demonstrate that the response is mostly Thl biased, as stimulation of splenocytes harvested from surviving mice leads to significant IFNy production. The memory is also tumor specific, as stimulation of the splenocytes with another cell line does not lead to production of IFNy orIL-4, and re-challenge of mice with a different tumor cell line lead to eventual death of all mice.BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figures la-lh: Characterization of CPMV nanoparticles. FIG. la) Structure of CPMV. CPMV consists of a bipartite genome of RNA-1 and RNA-2 encapsulated in identical, but separate capsids. The CPMV capsid (PDB ID: 1NY7) was reconstructed using UCSF Chimera software. FIG. lb) UV-Vis spectra of CPMV. The black inset of -1.73 indicates the A260 / A280 ratio of CPMV, which is within range of the ideal value of 1.8. FIG. 1c) SDS-PAGE of CPMV showing its small (S) and large (L) CP of 24 and 42 kDa, respectively, of which it has 60 copies each. FIG. Id) Agarose gel electrophoresis of CPMV. The co-migration of the RNA and the capsid shows that the CPMV nanoparticles are intact and stable. FIG. le) TEM of CPMV. The particles are icosahedral in shape and are - 30 nm. The scale bar represents 100 nm. FIG. If) DLS of CPMV. Size and poly dispersity index (PDI) are shown. FIG. 1g) FPLC of CPMV. The CPMV elutes at - 11 mL from the Superose 6 Increase column, and the A260 / A280 value at the peak is -1.7 mirroring the UV-Vis data from FIG. lb). FIG. Ih) RAW264.7-Luc NFKB reporter assay. CPMV demonstrates unique immunostimulatory properties, while control VNPs are inert. Both Q0 and PhMV are expressed as virus-like particles (VLPs). ** = p < 0.01, *** = p < 0.001. The data is displaying the average luminescence, and the error bars represent the standard deviation. The experiment was run in triplicate. The graph in FIG. Ih) was analyzed using one-way ANOVA. CCMV = cowpea chlorotic mottle virus; Q0 is a bacteriophage, here used as a VLP; TMV = tobacco mosaic virus, PhMV = physalis mottle virus, here used as a VLP.

[0021] Figures 2a-2j: CPMV prophylaxis protects against CT26-Luc metastasis. FIG. 2a) Injection schedule. BALB / C mice were given a single dose adjuvant therapy of CPMV (n = 35) followed by inoculation of 500,000 CT26-Luc cells i.p. Survivors were then rechallenged at 40 days, and some of the survivors were also injected every 3 days with a-CD4 and a-CD8 NAbs. FIG. 2b) Survival of mice following CT26-Luc challenge, n = 30 for CPMV and n = 5 for PBS. FIG. 2c) Average circumference fold change following CT26-Luc challenge (left) and individual circumference fold change of CPMV (middle) and PBS (right)mice, n = 30 for CPMV and n = 5 for PBS FIG. 2d) Average body weight fold change following CT26-Luc challenge (left) and individual body weight fold change curves of CPMV (middle) and PBS (right) mice, n = 30 for CPMV and n = 5 for PBS FIG. 2e) Average circumference fold change following re-challenge. Age-matched mice, which were not pre-exposed to either CPMV or CT26-Luc were used as the negative control. The *s represent the p-value comparisons between the survivors and the age-matched mice while the #s represent the p-value comparisons between the survivors and the a-CD4 / a-CD8 NAbs- injected mice (n = 5-7). FIG. 2f) Survival of mice following re-challenge. FIG. 2g) Individual circumference fold change graphs of the survivors (left), a-CD4 / a-CD8 NAb- injected mice (middle), and age-matched controls (right). FIGS. 2h-2i) ELISPOT data from splenocytes of survivors following initial challenge (n = 3). FIG. 2h) The splenocytes were stimulated with media, 4T1-Luc (negative control), CT26-Luc, and PMA / iono (positive control), and the number of spots were plotted. FIG. 2i) Qualitative analysis of individual wells from the ELISPOT. FIG. 2j) Survival curve of mice re-challenged with 4T1-Luc s.c. * / # = p < 0.05, ** = p < 0.01, ### = p < 0.001, **** = p < 0.0001, ns = not significant. The values displayed are all average values while the error bars represent the standard deviations. Graphs FIGS. 2c), 2d), and 2e) were analyzed using two-way ANOVA, FIGS. 2b) and 2f) were analyzed with a Log-rank (Mantel-Cox) test, and FIG. 2h) was analyzed with Student’s T-test. The schematic in FIG. 2a) was created on Biorender.com.

[0022] Figures 3a-3f: Longitudinal analysis of CPMV mediated CT26-Luc protection.FIG. 3a) Injection schedule. CPMV was injected i.p. in BALB / C mice followed by injection of CT26-Luc cells i.p. after 7, 14, 21, and 28 days. FIGS. 3b-e) Average circumference fold change of mice with FIG. 3b) 7, FIG. 3c) 14, FIG. 3d) 21, and FIG. 3e) 28 days between CPMV and CT26-Luc injection (left), and the individual circumference fold change curves for CPMV (middle) and PBS (right). FIG. 3f) Survival curves of mice with 7 (left), 14 (middle left), 21 (middle right), and 28 (right) days between the CPMV and CT26-Luc injection. Curves FIGS. 3b-3e) were analyzed by two-way ANOVA and are displaying the average circumference fold change with the error bars representing the standard deviation. Graph FIG. 3f) was analyzed using a log-rank (Mantel-Cox) test. ** = p < 0.01, **** = p < 0.0001, ns = not significant. All experiments were done with n = 5. The schematic in a) was created on Biorender.com.

[0023] Figures 4a-4e: Mechanism of action of CPMV prophylaxis. FIG. 4a) Flow cytometry data of i.p. fluid following PBS, CPMV i.v., CPMV s.c., and CPMV i.p. injection. The samples were read in triplicate from the combined i.p. fluid of n = 3 mice. FIG. 4b) Spider plot of the data in FIG. 4a. The groups were plotted as fold-changes against the group that had the highest level of that specific cell. FIG. 4c) Heat map showcasing the fold change in expression of the MSD data in FIG. 4d) and FIG. 4e) compared to PBS. * is comparing any of the i.p., i.p. + T, or i.v. data to PBS (p < 0.05) while the # is comparing the i.p. to i.v. (p < 0.05). FIG. 4d) MSD data of a select group of immunostimulatory cytokines (n = 4-5). FIG. 4e) MSD data of chemokines (n = 4-5). The data in FIGS. 4a), 4d), and 4e) represent the average values, and the errors bars are showing the standard deviation. The groups were compared using one way-ANOVA. The flow data was run in triplicate while the MSD was run at n = 4-5. * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001, ns = not significant. The MSD analysis of additionally investigated cytokines can be found in FIG.16

[0024] Figures 5a-5m: CPMV reduces lung B16F10 and 4T1-Luc tumor seeding prophylactically. FIG. 5a) Injection schedule for B16F10. CPMV is injected i.v. prophylactically in C57BL / 6J mice (n = 6-10) followed by injection of B16F10 cells i.v. after one week. The lungs are harvested 3 weeks PTI. FIG. 5b) Quantitative analysis of counted tumor nodules following lung harvesting. CPMV adjuvant therapy is administered s.c., i.v., or i.p. while PBS was injected i.v. (n = 6-10) FIG. 5c) Qualitative imaging of lungs from FIG. 5b) following lung harvesting. The lungs are fixed and then manually counted after harvesting. FIG. 5d) CPMV prophylaxis is compared to other adjuvants such as CCMV, diABZI, and imiquimod when injected i.v. CCMV is a similar plant virus to CPMV, diABZI is a STING agonist, and imiquimod is a TLR7 agonist, (n = 5-8) FIG. 5e) Qualitative imaging of lungs from FIG. 5d) following lung harvesting. FIG. 5f) Quantitative analysis of tumor nodules comparing CPMV to CCMV, diABZI, and imiquimod when injected i.p. (n = 6-8) FIG. 5g) Qualitative imaging of lungs from FIG. 5f) following lung harvesting. FIG. 5h) Injection schedule for 4T1-Luc. CPMV is injected in BALB / C mice (n = 9-10) followed by injection of 4T1-Luc i.v. after one week. The lungs are harvested 3 weeks after initial i.v. injection. FIG. 5i) Luminescent outgrowths of the 4T1-Luc within the lungs as measured by IVIS. The figure is showing average luminescence (left), individual curves for CPMV(middle), and PBS (right). FIG. 5j) Weight fold change in mice with respect to time. The figure is showing average weight fold change (left), individual curves for CPMV (middle), and individual curves for PBS (right). FIG. 5k) IVIS images of the mice. The colored areas represent areas of tumor development. FIG. 51) Tumor nodule counting. The tumor nodules were manually counted as with the Bl 6F 10 mice following Bouin staining. FIG. 5m) Qualitative images of the lungs counted in FIG. 51). The red arrows are pointing to the areas of tumor nodule development. The schematics in FIGS. 5a) and 5h) were created on Biorender.com. The lung tumor nodules for the B16F10 were analyzed using one-way ANOVA, and the 4T1-Luc were analyzed using Student’s T-Test. The luminescence and weight measurements were analyzed using two-way ANOVA. The data in FIGS. 5b), 5d), 5f), 5i), 5j), and 51) represent the average values, and the error bars are showing the standard deviations. * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001, and ns = not significant. The lungs in FIGS. 5c), 5e), 5g), and 5m) are representative images taken and are not the only lungs analyzed.

[0025] Figures 6a-6d: Mechanism of action of CPMV immunoprophylaxis. FIG. 6a) MSD data of immunostimulatory cytokines following i.v. injection of CPMV or PBS. FIG. 6b) MSD data of chemokines following i.v. injection of CPMV or PBS. FIG. 6c) Heat map showcasing the fold change of cytokines / chemokines in FIG. 6a) and FIG. 6b) comparing CPMV i.v. to PBS. MSD data of other cytokines can be found in FIG. 20. FIG. 6d) Flow cytometry data of the blood of mice following CPMV s.c., i.v., and i.p. injection. The MSD data was analyzed using Student’s T-test (n = 2-3), and the flow cytometry was analyzed using one-way ANOVA and run in triplicate from a combined pool of blood from n = 3 mice. The values represent the average values, and error bars are representing the standard deviation. * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001, ns = not significant.

[0026] Figures 7a-7d: 4T1-Luc primary surgery study combined with CPMV adjuvant therapy. FIG. 7a) Injection schedule schematic. Mice were injected with CPMV i.v. one day after surgical removal of a primary TNBC tumor (n = 10). FIG. 7b) Survival curve of mice given either CPMV or PBS adjuvant therapy. FIG. 7c) Luminescence measured by IVIS of mice. The figure is showing the average luminescence (left), individual luminescent curvesfor CPMV (middle), and individual luminescent curves for PBS (right). The values represent the average values, and the error bars represent the standard deviation. FIG. 7d) Representative IVIS images of the mice. The colored areas represent areas of tumor growth. The survival was analyzed using a log-rank (Mantel-Cox) test. * = p < 0.05. The graphic in FIG. 7a) was created on Biorender.com.

[0027] Figures 8a-8b: Weight of mice following re-challenge. FIG. 8a) Fold change in weight of mice compared to their weights at the time of re-challenge. The weight of the mice from the re-challenge experiment in FIGS 2e-2g were measured every two days. FIG. 8b) The individual weight fold changes from FIG. 8a. The data in FIG. 8a) is the average weight fold change, and the error bars represent the standard deviation (n = 5-7).

[0028] Figure 9: ELISPOT on surviving mice that did not survive re-challenge. Two mice that were re-challenged with CT26-Luc, but showed clear signs of tumor growth, were sacrificed before clinical endpoints and their splenocytes were used for ELISPOT analysis. While splenocytes from mice that survived re-challenge (FIGS. 2h, 2i) demonstrated potent IFNy production against CT26-Luc cells, these mice did not demonstrate significant IFNy production indicating loss of IFNy-producing splenocytes and thereby the importance of the memory T cell response in the clearance of re-challenged i.p. -disseminated tumors. The data is showing the average number of spots, and the error bars are showing the standard deviation (n = 2). The data was analyzed using Student’s T-test. ns = not significant.

[0029] Figures lOa-lOi: Formation of immune memory is tumor specific, but tissue agnostic. FIG. 10a) Injection schedule of CT26-Luc and 4T1-Luc re-challenge. BALB / C mice were injected with CPMV (n = 40) prophylactically followed by CT26-Luc injection i.p. Survivors were re-challenged with CT26-Luc i.p. or 4T1-Luc s.c. Survival curve FIG. 10b), circumference fold change FIG. 10c), and weight fold change FIG. lOd) following initial challenge after CPMV prophylaxis. The results closely mirror the data in FIGS. 2b-2d. FIG. lOe) Circumference fold change (left), weight fold change (middle), and survival of mice following re-challenge with CT26-Luc i.p. (n = 6). The results closely mirror the data from FIGS. 2e-2g. FIG. lOf) Tumor volume curve (left) and individual tumor growth curves (right) of mice (n = 5) following 4T1-Luc s.c. re-challenge. There were no long-term survivors, and all tumors grew unabated indicating that immune memory is tumor specific.Survival of the mice can be found on FIG. 2j. FIG. 10g) Injection schedule of CT26-Luc i.p. followed by CT26-Luc re-challenge s.c. to demonstrate that immune memory is tissue agonistic. FIG. lOh) Survival and FIG. lOi) tumor growth curves following CT26-Luc s.c. re-challenge. The initial circumference FIG. 10c) and weight fold changes FIG. lOd) as well as the tumor volume curves FIG. lOi) were analyzed with two-way ANOVA. The data is showing the average circumference and weight fold changes, and the tumor volume, and the error bars represent the standard deviation. The survival curves FIG. 10b) and FIG. lOh) were analyzed with a Log-rank (Mantel-Cox) test. ** = p < 0.01, **** = p < 0.0001. The schematics were created on Biorender.com.

[0030] Figures 1 la-1 Id: CPMV adjuvant therapy in pre-metastatic mice. FIG. Ila) Injection schedule. Female BALB / C mice are injected with 50,000 CT26-Luc cells i.p. prior to CPMV adjuvant treatment (n = 18) followed by injection of 500,000 CT26-Luc cells i.p. to stimulate patients with pre-disseminated disease. FIG. 11b) Survival curve of mice following CPMV adjuvant treatment. FIG. 11c) Circumference fold change of all mice (left), individual curves in the CPMV group (middle), and individual curves in the PBS group (right). FIG. lid) Weight fold changes of all mice (left), individual curves in the CPMV group (middle), and individual curves in the PBS group (right). The circumference and body weight graphs were analyzed using two-way ANOVA, and the survival curve was analyzed using a log-rank (Mantel-Cox) test. The data represents the average values, and the error bars represent the standard deviation. * = p < 0.05, ns = not significant.

[0031] Figures 12a-12e: Longitudinal analysis of body weight measurements in mice challenged with CT26-Luc after CPMV injection. FIG. 12a) Injection schedule. BALB / C mice (n = 5) were injected initially with CPMV followed by injection with 500,000 CT26- Luc cells i.p. after 7, 14, 21, and 28 days. FIG. 12b-e) Body weight fold change after injection of CPMV following CT26-Luc for FIG. 12b) 7 days, FIG. 12c) 14 days, FIG. 12d) 21 days, and FIG. 12e) 28 days. The body weight statistics were analyzed using two-way ANOVA. The curves represent the average body weigh fold change, and the error bars represent the standard deviation. **** = p < 0.0001, ns = not significant. The schematic in FIG. 12a) was created on Biorender.com.

[0032] Figure 13. Biodistribution and clearance of CPMV. CPMV biodistribution and clearance 1 and 2 weeks after i.p. injection were measured using qRT-PCR. CPMV biodistribution was investigated within the lungs, liver, kidney, spleen, and i.p. fluid, and normalized to mice injected with PBS (data not shown). CPMV is stable for extended periods of time within the i.p. space and was detectable up to 2 weeks after i.p. injection further demonstrating the long-lasting efficacy of CPMV after only a single injection. The data is showing the average ng of CPMV RNA, and the error bars represent the standard deviation (n = 3), ND = not detectable.

[0033] Figures 14a-14d: Route of administration of CPMV in CT26-Luc i.p. protection. FIG. 14a) Circumference fold change in BALB / C mice injected with PBS, CPMV s.c., CPMV i.v., and CPMV i.p. (n = 10). FIG. 14b) Weight fold change. FIG. 14c) Survival of mice. FIG. 14d) Individual circumference growth curves in mice injected with PBS (left), CPMV s.c. (left, middle), CPMV i.v. (right, middle), and CPMV i.p. (right). Only CPMV injected i.p. showed significant benefit in reducing tumor growth and extending survival. The circumference and weight fold change graphs were analyzed using two-way ANOVA. The values represent the average circumference and weight fold changes, and the error bars represent the standard deviation The survival curve was analyzed using a log-rank (Mantel- Cox) test. * = p < 0.05, *** = p < 0.001, **** = p < 0.0001, ns = not significant.

[0034] Figures 15a-15b: Flow cytometry gating strategy. FIG. 15a) Flow cytometry gating. All the flow cytometry was accomplished using a FACSCelesta, and analyzed using Flow Jo. For NK cells, either NKp46 or NK1.1 was used as the phenotypic marker depending on the mouse strain of BALB / C or C57BL / 6J, respectively. SSC = side scatter, FSC = forward scatter, FSC-H = forward scatter height, FSC-A = forward scatter area, CD = cluster of differentiation, PB = Pacific Blue, BV = Brilliant Violet, SB = SuperBright, NK = Natural Killer, APC = allophycocyanin, Cy7 = Cyanine7, MHC = Major Histocompatibility Complex, Cy5.5 = Cyanine 5.5, G-MDSC = granulocytic myeloid derived suppressor cell, M-MDSC = monocytic myeloid derived suppressor cell, PE = phycoerythrin, DC = dendritic cell. FIG. 15b) Table showcasing the phenotype used to analyze each cell.

[0035] Figures 16a-16b: MSD analysis of other cytokines not included in FIG. 4. FIG.16a) MSD analysis of immunosuppressive cytokines (n = 4-5). FIG. 16b) MSD analysis ofcytokines with both immunostimulatory and immunosuppressive functions or allergic reactions (n = 4-5). The groups were compared with one-way ANOVA. The data represents the average values, and the error bars represent the standard deviation. * = p < 0.05, *** = p < 0.001, **** = p < 0.0001, ns = not significant.

[0036] Figures 17a-17d: Tandem mass tag (TMT) analysis of i.p. fluid following CPMV injection. FIG. 17a) Volcano plot of the identified proteins within the i.p. fluid comparing injection of PBS vs. CPMV one week after injection. Proteins were considered significant above a significance value of 15 and a ratio < 0.5 or > 2. A ratio < 0.5 and significance > 15 indicated that the proteins were upregulated following CPMV injection while a ratio > 2 indicates downregulation. FIG. 17b) Proteins that were found within the PBS samples, but not the CPMV samples are shown. These samples could not be graphed in FIG. 17a) due to the inability to calculate a ratio. FIG. 17c) Heat map of the intensities from fibrinogen and hemoglobin-related proteins. The intensities were much greater than that of the other proteins and so were plotted on a separate heat map. FIG. 17d) Heat map of the intensities of all the other significant proteins found within the i.p. fluid.

[0037] Figures 18a-18f: IGURE 18. CPMV adjuvant therapy against an ID8-Defb29 / Vegf-A- c ovarian cancer model. FIG. 18a) Injection schedule. C57BL / 6J mice (n = 20) were injected i.p. with 200 pg of CPMV followed by injection of 2 x 106ID8- Defb29 Ve f-A-Luc cells i.p. after one week. FIG. 18b) Survival curve of mice. FIG. 18c) Circumference fold change of CPMV and PBS mice (left). Individual curves for CPMV (middle) and PBS (right) are also shown. FIG. 18d) Body weight fold change of CPMV and PBS mice (left). Individual curves for CPMV (middle) and PBS (right) are also shown. FIG. 18e) ELISPOT of the survivors in the CPMV group (n = 3). Splenocytes were harvested and stimulated with media only, B16F10 (negative control), VA -l)efb29 Ve j-A- wz, and PMA / iono (positive control). FIG. 18f) Qualitative images of the ELISPOT data in e). The red spots are indicative of IFNy production while the blue spots indicate IL-4 production corresponding to a Th 1 and Th2 response, respectively. The survival curve was analyzed using a log-rank (Mantel-Cox) test, the circumference and body weight were analyzed using two-way ANOVA, and the ELISPOT data was analyzed using a Student’s T-test. The data in FIGS. 18c-18e represent the average values, and the error bars represent the standarddeviation. * = p < 0.05, **** = p < 0.0001. The schematic in FIG. 18a) was created on Biorender.com.

[0038] Figure 19. RAW264.7-Luc NFKB signaling of CPMV and other adjuvants.100,000 RAW264.7-Luc cells were incubated with 10 x 106molecules of each reagent per cell and incubated for 24 h at 37°C. The luminescent output was measured using SteadyGlo (Promega) and read on a plate reader. The luminescence was analyzed using one-way ANOVA. The data represents the average luminescence, and the error bars represent the standard deviation. The readings were read in triplicate. *** = p < 0.001, **** = p < 0.0001. CCMV = cowpea chlorotic mottle virus.

[0039] Figures 20a-20b: MSD analysis of cytokines not shown in FIG. 6. FIG. 20a) MSD analysis of immunosuppressive cytokines (n = 2-3). FIG. 20b) MSD analysis of cytokines with both immunostimulatory and immunosuppressive functions or allergic reactions (n = 2- 3). The groups were compared with Student’s T-test. The values represent the average values, and the error bars represent the standard deviation. * = p < 0.05, ** = p < 0.01, ns = not significant.

[0040] Figures 21a-21b: Flow cytometry analysis of lungs and spleens following CPMV injection. FIG. 21a) Flow cytometry of the lungs. FIG. 21b) Flow cytometry of the spleen. Analyses in both the lungs and the spleen of the mice indicate minimal differences in the immune cell populations thereby demonstrating that clearance of the CTCs is most likely mediated by increased immune cell populations found within the blood of the mice as found in Fig. 6d. All samples in FIG. 21a) and FIG. 21b) were run in triplicate and were compared using one-way ANOVA. The values represent the average values, and the error bars represent the standard deviation. * = p < 0.05, ** = p < 0.01, *** = p < 0.001, ns = not significant. From left to right in every graph: PBS, s.c., i.v., and i.p..

[0041] Figures 22a-22c. :Transcriptomics profile of human PBMCs exposed to CPMV (20 ug / mL for 24 hours) using Chromium Next GEM Single Cell 5' Kit v2 (10X Genomics), Illumina NovaSeq X plus platform (Novogene), and lOx Genomics Cellranger v7.1.0 software. FIG. 22a) Representative cell-type annotation of PBMC of one donor. Nine individual cell populations were identified and highlighted in different colors, asshown to the right of the cell ID plot. Cell populations positive for the CPMV genome are highlighted in purple on the far-right plot. FIG. 22b) Volcano plots highlight changes in the expression of genes involved in interferon responses (left) and metabolic reprogramming (right). Triangles emphasize upregulated genes involved in the interferon and metabolic reprogramming pathways on the left and right plots, respectively. The list of the top eleven pathways is shown to the right. FIG. 22c) Representative violin plots highlight cell population-specific gene expression involved in epigenetic cell reprogramming. The list of the top sixteen long non-coding RNA genes is shown. Cell types from left to right in each graph in FIG. 22c: CD4+ T cell, CD8+ T cell, CXCR6+ / CD4+ / CD8- T cell, CXCR6+ / CD4- / CD8+ T cell, CXCR6+ / CD4- / CD8- T cell, B cell, NK cell, Monocyte, DC cell.DETAILED DESCRIPTIONDefinitions

[0042] As it would be understood, the section or subsection headings as used herein is for organizational purposes only and are not to be construed as limiting or separating or both limiting and separating the subject matter described.

[0043] Throughout this disclosure, various publications, patents and published patent specifications are referenced by an identifying citation. The disclosures of these publications, patents and published patent specifications are hereby incorporated by reference into the present disclosure in their entireties to more fully describe the state of the art to which this invention pertains.

[0044] The practice of the present technology will employ, unless otherwise indicated, conventional techniques of organic chemistry, pharmacology, immunology, molecular biology, microbiology, cell biology and recombinant DNA, which are within the skill of the art. See, e.g., Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual, 2ndedition (1989); Current Protocols In Molecular Biology (F. M. Ausubel, et al. eds., (1987)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (M.J. MacPherson, B.D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, a Laboratory Manual, and Animal Cell Culture (R.I. Freshney, ed. (1987)).

[0045] As used in the specification and claims, the singular form “a,” “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes a plurality of cells, including mixtures thereof.

[0046] As used herein, the term “comprising” is intended to mean that the compounds, compositions and methods include the recited elements, but not exclude others. “Consisting essentially of’ when used to define compounds, compositions and methods, shall mean excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants, e.g., from the isolation and purification method and pharmaceutically acceptable carriers, preservatives, and the like. “Consisting of’ shall mean excluding more than trace elements of other ingredients. Embodiments defined by each of these transition terms are within the scope of this technology.

[0047] “Optional” or “optionally” means that the subsequently described circumstance may or may not occur, so that the description includes instances where the circumstance occurs and instances where it does not.

[0048] As used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0049] All numerical designations, e.g., pH, temperature, time, concentration, and molecular weight, including ranges, are approximations which are varied (+) or (-) by increments of 1, 5, or 10%. It is to be understood, although not always explicitly stated that all numerical designations are preceded by the term “about.” It also is to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art.

[0050] As used herein, the term “about” is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value. The term “about” when used before a numerical designation, e.g., temperature, time, amount, and concentration, including range, indicates approximations which may vary by (+) or (-) 15%, 10%, 5%, 3%, 2%, or 1 %.

[0051] “Substantially” or “essentially” means nearly totally or completely, for instance, 95% or greater of some given quantity. In some embodiments, “substantially” or “essentially” means 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%.

[0052] As used herein, the term “animal” refers to living multi-cellular vertebrate organisms, a category that includes, for example, mammals and birds. The term “mammal” includes both human and non-human mammals.

[0053] The term “subject,” “host,” “individual,” and “patient” are as used interchangeably herein to refer to animals, typically mammalian animals. Any suitable mammal can be treated by a method described herein. Non-limiting examples of mammals include humans, non- human primates (e.g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, and the like), domestic animals (e.g., dogs and cats), farm animals (e.g., horses, cows, goats, sheep, pigs) and experimental animals (e.g., mouse, rat, rabbit, guinea pig). In some embodiments, a mammal is a human. A mammal can be any age or at any stage of development (e.g., an adult, teen, child, infant, or a mammal in utero). A mammal can be male or female. In some embodiments, a subject is a human.

[0054] A “composition” as used herein, refers to an active agent, such as a compound as disclosed herein and a carrier, inert or active. The carrier can be, without limitation, solid such as a bead or resin, or liquid, such as phosphate buffered saline.

[0055] Carriers also include pharmaceutical excipients and additives proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars, including monosaccharides, di-, tri, tetraoligosaccharides, and oligosaccharides; derivatized sugars such as alditols, aldonic acids, esterified sugars and the like; and polysaccharides or sugar polymers), which can be present singly or in combination, comprising alone or in combination 1-99.99% by weight or volume. Exemplary protein excipients include serum albumin such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and the like. Representative amino acid / antibody components, which can also function in a buffering capacity, include alanine, arginine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and the like. Carbohydrate excipients are also intended within the scope of this technology, examples of which includebut are not limited to monosaccharides such as fructose, maltose, galactose, glucose, D- mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol) and myoinositol.

[0056] A “pharmaceutical composition” is intended to include the combination of an active agent with a carrier, inert or active, making the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.

[0057] “Pharmaceutically acceptable carriers” refers to any diluents, excipients, or carriers that can be used in the compositions disclosed herein. Pharmaceutically acceptable carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances, such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, a standard reference text in this field. They may be selected with respect to the intended form of administration, that is, oral tablets, capsules, elixirs, syrups and the like, and consistent with conventional pharmaceutical practices.

[0058] The compositions used in accordance with the disclosure can be packaged in dosage unit form for ease of administration and uniformity of dosage. The term "unit dose" or "dosage" refers to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of the composition calculated to produce the desired responses in association with its administration, i.e., the appropriate route and regimen. The quantity to be administered, both according to number of treatments and unit dose, depends on the result and / or protection desired. Precise amounts of the composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dose include physicaland clinical state of the subject, route of administration, intended goal of treatment (alleviation of symptoms versus cure), and potency, stability, and toxicity of the particular composition. Upon formulation, solutions are administered in a manner compatible with the dosage formulation and in such amount as is therapeutically or prophylactically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described herein.

[0059] As used herein, the phrase “immune response” or its equivalent “immunological response” refers to the development of a cell-mediated response (e.g. mediated by antigenspecific T cells or their secretion products). A cellular immune response is elicited by the presentation of polypeptide epitopes in association with Class I or Class II MHC molecules, to treat or prevent a viral infection, expand antigen-specific B-reg cells, TCI, CD4+ T helper cells and / or CD8+ cytotoxic T cells and / or disease generated, autoregulatory T cell and B cell “memory” cells. The response may also involve activation of other components. In some aspect, the term “immune response” may be used to encompass the formation of a regulatory network of immune cells. Thus, the term “regulatory network formation” may refer to an immune response elicited such that an immune cell, preferably a T cell, more preferably a T regulatory cell, triggers further differentiation of other immune cells, such as but not limited to, B cells or antigen-presenting cells - non-limiting examples of which include dendritic cells, monocytes, and macrophages. In certain embodiments, regulatory network formation involves B cells being differentiated into regulatory B cells; in certain embodiments, regulatory network formation involves the formation of tolerogenic antigen-presenting cells.

[0060] The term “immune cells” includes, e.g., white blood cells (leukocytes) which are derived from hematopoietic stem cells (HSC) produced in the bone marrow, lymphocytes (T cells, B cells, natural killer (NK) cells) and myeloid-derived cells (neutrophil, eosinophil, basophil, monocyte, macrophage, dendritic cells). “T cell” includes all types of immune cells expressing CD3 including T-helper cells (CD4+ cells), cytotoxic T-cells (CD8+ cells), natural killer T-cells, T-regulatory cells (Treg) and gamma-delta T cells. A “cytotoxic cell” includes CD8+ T cells, natural-killer (NK) cells, and neutrophils, which cells are capable of mediating cytotoxicity responses. Cytokines are small secreted proteins released by immune cells that have a specific effect on the interactions and communications between the immunecells. Cytokines can be pro-inflammatory or anti-inflammatory. Non-limiting example of a cytokine is Granulocyte-macrophage colony-stimulating factor (GM-CSF), which stimulates stem cells to produce granulocytes (neutrophils, eosinophils, and basophils) and monocytes.

[0061] Administration or treatment in “combination” refers to administering two agents such that their pharmacological effects are manifest at the same time. Combination does not require administration at the same time or substantially the same time, although combination can include such administrations.

[0062] An “effective amount” is an amount sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications or dosages. Such delivery is dependent on a number of variables including the time period for which the individual dosage unit is to be used, the bioavailability of the therapeutic agent, the route of administration, etc. It is understood, however, that specific dose levels of the therapeutic agents disclosed herein for any particular subject depends upon a variety of factors including the activity of the specific compound employed, bioavailability of the compound, the route of administration, the age of the animal and its body weight, general health, sex, the diet of the animal, the time of administration, the rate of excretion, the drug combination, and the severity of the particular disorder being treated and form of administration. In general, one will desire to administer an amount of the compound that is effective to achieve a serum level commensurate with the concentrations found to be effective in vivo. These considerations, as well as effective formulations and administration procedures are well known in the art and are described in standard textbooks.

[0063] As used herein, “treating” or “treatment” of a disease in a subject refers to (1) preventing the symptoms or disease from occurring in a subject that is predisposed or does not yet display symptoms of the disease; (2) inhibiting the disease or arresting its development; or (3) ameliorating or causing regression of the disease or the symptoms of the disease. As understood in the art, “treatment” is an approach for obtaining beneficial or desired results, including clinical results. For the purposes of the present technology, beneficial or desired results can include one or more, but are not limited to, alleviation or amelioration of one or more symptoms, diminishment of extent of a condition (including a disease), stabilized (z.e., not worsening) state of a condition (including disease), delay orslowing of condition (including disease), progression, amelioration or palliation of the condition (including disease), states and remission (whether partial or total), whether detectable or undetectable. When the disease is cancer, the following clinical end points are non-limiting examples of treatment: reduction in tumor burden, slowing of tumor growth, longer overall survival, longer time to tumor progression, inhibition of metastasis or a reduction in metastasis of the tumor, or delay, slowing, or prevent of relapse. In one aspect, treatment excludes prophylaxis. In one aspect, treatment provides a longer progression free survival or a longer overall survival.

[0064] In one embodiment, the term “disease” or “disorder” as used herein refers to a cancer or a tumor (which are used interchangeably herein), a status of being diagnosed with such disease, a status of being suspect of having such disease, or a status of at high risk of having such disease. In one aspect, the cancer is ovarian cancer such as ovarian serous carcinoma.

[0065] " Cancer" or "malignancy" are used as synonymous terms and refer to any of a number of diseases that are characterized by uncontrolled, abnormal proliferation of cells, the ability of affected cells to spread locally or through the bloodstream and lymphatic system to other parts of the body (i.e., metastasize) as well as any of a number of characteristic structural and / or molecular features. In some embodiments, the term “cancer” is used interchangeably with the term “tumor”. In one aspect, the cancer is solid tumor or blood cancer. In one aspect, the solid tumor is a carcinoma or sarcoma. In one aspect, the cancer is a lung cancer, an intraperitoneal cancer, a colorectal cancer, a colon cancer, an ovarian cancer, a melanoma, and breast cancer.

[0066] As used herein, a “metastatic” cancer cell refers to a cancer cell that is translocated from a primary cancer site (i.e., a location where the cancer cell initially formed from a normal, hyperplastic or dysplastic cell) to a site other than the primary site, where the translocated cancer cell lodges and proliferates.

[0067] As used herein, the terms “reduce” and “inhibit” are used together because it is recognized that, in some cases, a decrease can be reduced below the level of detection of a particular assay, for example by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 85%, about90%, about 95%, about 99%, or about 100% of the reference level. As such, it may not always be clear whether the expression level or activity is “reduced” below a level of detection of an assay or is completely “inhibited.” Nevertheless, it will be clearly determinable, following a treatment according to the present methods.

[0068] The phrase “first-line” or “second-line” or “third-line” refers to the order of treatment received by a patient. First-line therapy regimens are treatments given first, whereas seconder third-line therapy are given after the first-line therapy or after the second-line therapy, respectively. The National Cancer Institute defines first-line therapy as “the first treatment for a disease or condition. In patients with cancer, primary treatment can be surgery, chemotherapy, radiation therapy, or a combination of these therapies. First-line therapy is also referred to those skilled in the art as “primary therapy and primary treatment”. Typically, a patient is given a subsequent chemotherapy regimen because the patient did not show a positive clinical or sub-clinical response to the first-line therapy or the first-line therapy has stopped.

[0069] A “tumor response” (TR) refers to a tumor’s response to therapy. A “complete response” (CR) to a therapy refers to the clinical status of a patient with evaluable but non- measurable disease, whose tumor and all evidence of disease have disappeared following administration of the therapy. In this context, a “partial response” (PR) refers to a response that is anything less than a complete response. “Stable disease” (SD) indicates that the patient is stable following the therapy. “Progressive disease” (PD) indicates that the tumor has grown (i.e. become larger) or spread (i.e. metastasized to another tissue or organ) or the overall cancer has gotten worse following the therapy. For example, tumor growth of more than 20 percent since the start of therapy typically indicates progressive disease. “Non-response” (NR) to a therapy refers to status of a patient whose tumor or evidence of disease has remained constant or has progressed.

[0070] “ Overall Survival” (OS) refers to the length of time of a cancer patient remaining alive following a cancer therapy.

[0071] “Progression free survival” (PFS) or “Time to Tumor Progression” (TTP) refers to the length of time following a therapy, during which the tumor in a cancer patient does not grow.Progression-free survival includes the amount of time a patient has experienced a complete response, partial response or stable disease.

[0072] “Disease free survival” refers to the length of time following a therapy, during which a cancer patient survives with no signs of the cancer or tumor.

[0073] “ Time to Tumor Recurrence (TTR)” refers to the length of time, following a cancer therapy such as surgical resection or chemotherapy, until the tumor has reappeared (come back). The tumor may come back to the same place as the original (primary) tumor or to another place in the body.

[0074] As used herein, an anticancer agent refers to any drug or compound used for anticancer treatment. These include any drug that renders or maintains a clinical symptom or diagnostic marker of tumors and cancer, alone or in combination with other compounds, that reduces or maintains a state of remission, or reduction or prevention or remission. In some embodiments, the agent is an RNA and / or a DNA. In some embodiments, the agent is a protein or a polypeptide. In some embodiments, the agent is a chemical compound. Examples of anticancer agents include angiogenesis inhibitors such as angiostatin Kl-3, DL- adifluoromethyl-omithine, endostatin, fumagillin, genistein, minocycline, staurosporine, and (+)-thalidomide; DNA intercalating or cross-linking agents such as bleomycin, carboplatin, carmustine, chlorambucil, cyclophosphamide, cisplatin, melphalan, mitoxantrone, and oxaliplatin; DNA synthesis inhibitors such as methotrexate, 3-Amino-l,2,4-benzotriazine 1,4- dioxide, aminopterin, cytosine b-D-arabinofuranoside, 5-Fluoro-5’ -deoxyuridine, 5- Fluorouracil, gaciclovir, hydroxyurea, and mitomycin C; DNA-RNA transcription regulators such as actinomycin D, daunorubicin, doxorubicin, homoharringtonine, and idarubicin; enzyme inhibitors such as S(+)-camptothecin, curcumin, (-)-deguelin, 5,6- dichlorobenzimidazole I -b-D-ribofuranoside, etoposine, formestane, fostriecin, hispidin, cyclocreatine, mevinolin, trichostatin A, tyrophostin AG 34, and tyrophostin AG 879, Gene Regulating agents such as 5-aza-2’-deoxycitidine, 5-azacytidine, cholecalciferol, 4- hydroxytamoxifen, melatonin, mifepristone, raloxifene, all trans-retinal, all trans retinoic acid, 9-cis-retinoic acid, retinol, tamoxifen, and troglitazone; Microtubule Inhibitors such as colchicine, dolostatin 15, nocodazole, paclitaxel, podophyllotoxin, rhizoxin, vinblastine, vincristine, vindesine, and vinorelbine; humanised or mouse / human chimeric monoclonalantibodies against defined cancer associated structures (such as Trastuzumab, Rituximab, Cetuximab, Bevacizumab, Alemtuzumab); and various other antitumor agents such as 17- (allylamino)-17- demethoxygeldanamycin, 4-Amino-l,8-naphthalimide, apigenin, brefeldin A, cimetidine, dichloromethylene-diphosphonic acid, leuprolide, luteinizing-hormone- releasing hormone, pifithrin-a, rapamycin, thapsigargin, and bikunin, and derivatives (as defined for imaging agents) thereof.

[0075] As used herein, an ablative therapy is a treatment destroying or ablating cancer tumors. In one embodiment, the ablative therapy does not require invasive surgery. In other embodiments, the ablative therapy refers to removal of a tumor via surgery. In some embodiments, the step ablating the cancer includes immunotherapy of the cancer. Cancer immunotherapy is based on therapeutic interventions that aim to utilize the immune system to combat malignant diseases. It can be divided into unspecific approaches and specific approaches. Unspecific cancer immunotherapy aims at activating parts of the immune system generally, such as treatment with specific cytokines known to be effective in cancer immunotherapy (e.g. IL-2, interferon's, cytokine inducers).

[0076] The terms “oligonucleotide” or “polynucleotide” or “portion,” or “segment” thereof refer to a stretch of polynucleotide residues which is long enough to use in PCR or various hybridization procedures to identify or amplify identical or related parts of mRNA or DNA molecules. The polynucleotide compositions of this invention include RNA, cDNA, genomic DNA, synthetic forms, and mixed polymers, both sense and antisense strands, and may be chemically or biochemically modified or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those skilled in the art. Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.), charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), pendent moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen, etc.), chelators, alkylators, and modified linkages (e.g., alpha anomeric nucleic acids, etc.). Also included are synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen bonding and other chemical interactions. Such molecules are known in the art and include, for example,those in which peptide linkages substitute for phosphate linkages in the backbone of the molecule.

[0077] The term “contacting” means direct or indirect binding or interaction between two or more. A particular example of direct interaction is binding. A particular example of an indirect interaction is where one entity acts upon an intermediary molecule, which in turn acts upon the second referenced entity. Contacting as used herein includes in solution, in solid phase, in vitro, ex vivo, in a cell and in vivo. Contacting in vivo can be referred to as administering, or administration.

[0078] As used herein, the term “plant virus” includes viruses that infect plants or plant systems, e.g., leaves, root and / or stems. Plant viruses can be stably stored (and are stable without cold chain requirements). Plant viruses do not infect or replicate in mammalian cells, thus adding another layer of safety compared to oncolytic viral therapies. Non-limiting examples include tobacco mosaic virus (TMV), tobacco mold green mottle virus (TMGMV), physalis mottle virus like particle (PhMV), cowpea chlorotic mottle virus (CCMV), and cowpea mosaic virus (CPMV). Method of replicating and producing virus for therapeutic application are known in the art and described in WO 2022 / 221692, published October 20, 2022, and incorporated herein by reference.

[0079] As used herein, the term “plant viral nanoparticle,” “plant virus nanoparticle,” or “VLP” refers to a non-replicating, viral shell, derived from one or more plant viruses (e.g., one or more plant viruses described herein). VLPs are generally composed of one or more viral proteins, such as, but not limited to, those proteins referred to as capsid, coat, shell, surface and / or envelope proteins, or particle-forming polypeptides derived from these proteins. VLPs can form spontaneously upon recombinant expression of the protein in an appropriate expression system. VLPs can also be engineered, e.g., comprising, or consisting essentially of, or yet further consisting of, one or more viral proteins that comprise, or consists essentially of, or yet further consists of, a modification. Methods for producing VLPs are known in the art. The presence of VLPs following recombinant expression of viral proteins can be detected using conventional techniques known in the art, such as by electron microscopy, biophysical characterization, and the like. Further, VLPs can be isolated by known techniques, e.g., density gradient centrifugation and identified by characteristicdensity banding. See, for example, Baker et al. (1991) Biophys. J. 60: 1445-1456; and Hagensee et al. (1994) J. Viral. 68:4503-4505; Vincente, J Invertebr Pathol., 2011; Schneider Ohrum and Ross, Curr. Top. Microbial. Immunol., 354: 53073, 2012). As used herein, VLP intends naturally occurring (wild-type or native) VLP and engineered VLP, unless explicitly stated otherwise.

[0080] Virus-like Particles (VLPs) are generally composed of one or more viral proteins, such as, but not limited to, those proteins referred to as capsid, coat, shell, surface and / or envelope proteins, or particle-forming polypeptides derived from these proteins. VLPs can form spontaneously upon recombinant expression of the protein in an appropriate expression system. VLPs can also be engineered, e.g., comprising, or consisting essentially of, or yet further consisting of, one or more viral proteins that comprise, or consists essentially of, or yet further consists of, a modification. Methods for producing VLPs are known in the art. The presence of VLPs following recombinant expression of viral proteins can be detected using conventional techniques known in the art, such as by electron microscopy, biophysical characterization, and the like. Further, VLPs can be isolated by known techniques, e.g., density gradient centrifugation and identified by characteristic density banding. See, for example, Baker et al. (1991) Biophys. J. 60: 1445-1456; and Hagensee et al. (1994) J. Viral. 68:4503-4505; Vincente, J Invertebr Pathol., 2011; Schneider Ohrum and Ross, Curr. Top. Microbial. Immunol., 354: 53073, 2012).

[0081] In some embodiments, the plant viral nanoparticle or VLP is derived from Cowpea mosaic virus (CPMV). CPMV is a non-enveloped plant virus that belongs to the Comovirus genus. CPMV strains include, but are not limited to, SB (Agrawal, H.O. (1964). Meded. Landb. Hoogesch. Wagen. 64: 1) and Vu (Agrawal, H.O. (1964). Meded. Landb. Hoogesch. Wagen. 64: 1). Cowpea mosaic virus (CPMV) is a VLP and a plant-infecting member of the order Picornavirales, with a relatively simple, non-enveloped capsid that has been extensively studied and a positive-sense, single-stranded RNA genome. For CPMV, the genome is bipartite, with RNA-1 (6 kb) and RNA-2 (3.5 kb) being separately encapsidated. CPMV has an icosahedral capsid structure, which is ~30 nm in diameter and is formed from 60 copies each of a Large (L) and Small (S) coat protein. These two coat proteins are processed from a single RNA-2-encoded precursor polyprotein (VP60) by the action of the24 K viral proteinase which is encoded by RNA-1. Thus capsid assembly, as well as viral infection, is dependent on the presence of both genomic segments in an infected plant cell. In some embodiments, the VLP particles have been treated, prepared and / or inactivated by methods known in the art. In some instances, CPMV produces a large capsid protein and a small capsid protein precursor (which generates a mature small capsid protein). In some cases, CPMV capsid is formed from a plurality of large capsid proteins and mature small capsid proteins. In some cases, the large capsid protein is a wild-type large capsid protein, optionally expressed by SB or Vu strain. In other instances, the large capsid protein is a modified large capsid protein, e.g., comprising, or consisting essentially of, or yet further consisting of, one or more substitutions, insertions, and / or deletions. In some cases, the large capsid protein comprises, or consists essentially of, or yet further consists of, the sequence as set forth in the UniProtKB ID P03599 (residues 460-833):MEQNLFALSLDDTSSVRGSLLDTKFAQTRVLLSKAMAGGDVLLDEYLYDVVNGQD FRAT VAFLRTHVITGKIKVTATTNI SDNS GCCLMLAINSGVRGKYSTDVYTICSQDSM TWNPGCKKNFSFTFNPNPCGDSWSAEMISRSRVRMTVICVSGWTLSPTTDVIAKLDW SIVNEKCEPTIYHLADCQNWLPLNRWMGKLTFPQGVTSEVRRMPLSIGGGAGATQA FLANMPNSWISMWRYFRGELHFEVTKMSSPYIKATVTFLIAFGNLSDAFGFYESFPHR IVQFAEVEEKCTLVFSQQEFVTAWSTQVNPRTTLEADGCPYLYAIIHDSTTGTISGDF NLGVKLVGIKDFCGIGSNPGIDGSRLLGAIAQ (SEQ ID NO: 1), or an equivalent thereof.

[0082] In some cases, the mature small capsid protein is a wild-type mature small capsid protein, optionally expressed by SB or Vu strain. In other instances, the mature small capsid protein is a modified mature small capsid protein, e.g., comprising, or consisting essentially of, or yet further consisting of, one or more substitutions, insertions, and / or deletions. In some cases, the mature small capsid protein comprises, or consists essentially of, or yet further consists of, the sequence as set forth in the UniProtKB ID P03599 (residues 834- 1022):GPVCAEASDVYSPCMIASTPPAPFSDVTAVTFDLINGKITPVGDDNWNTHIYNPPIMN VLRTAAWKSGTIHVQLNVRGAGVKRADWDGQVFVYLRQSMNPESYDARTFVISQP GSAMLNFSFDIIGPNSGFEFAESPWANQTTWYLECVATNPRQIQQFEVNMRFDPNFR VAGNILMPPFPLSTETPPL (SEQ ID NO: 2), or an equivalent thereof.

[0083] Cowpea mosaic virus (CPMV) is a plant-infecting member of the order Picornavirales, with a relatively simple, non-enveloped capsid that has been extensively studied and a positive-sense, single-stranded RNA genome. For CPMV, the genome is bipartite, with RNA-1 (6 kb) and RNA-2 (3.5 kb) being separately encapsidated. CPMV has an icosahedral capsid structure, which is ~30 nm in diameter and is formed from 60 copies each of a Large (L) and Small (S) coat protein. These two coat proteins are processed from a single RNA-2-encoded precursor polyprotein (VP60) by the action of the 24 K viral proteinase which is encoded by RNA-1. Thus capsid assembly, as well as viral infection, is dependent on the presence of both genomic segments in an infected plant cell.

[0084] The terms “CPMV” “CPMV virus” or “CPMV particles” are used interchangeably, referring to a CPMV comprising, or alternatively consisting essentially of, or yet consisting of a capsid and an RNA genome (which is also referred to herein as a viral genome) encapsidated in the capsid. In some embodiments, the CPMV particles have been treated, prepared and / or inactivated by a method as disclosed herein. In some embodiments, the CPMV particle further comprises a heterologous RNA, which is heterologous to (i.e., not naturally presented in) a native CPMV free of any human intervention.

[0085] In some embodiments, the CPMV can be substitute by another plant virus, for example another plant retrovirus. However, a bacteriophage or mammalian virus can be used in some embodiments of the invention. When a plant viral nanoparticle is used, in some embodiments the plant virus is a plant picomavirus. A plant picomavirus is a virus belonging to the family Secoaviridae, which together with mammalian picornaaviruses belong to the order of the Picornavirales. Plant picornaviruses are relatively small, nonenveloped, positive- stranded RNA viruses with an icosahedral capsid. Plant picornaaviruses have a number of additional properties that distinguish them from other picornaaviruses, and are categorized as the subfamily secoviridae. In some embodiments, the virus particles are selected from the Comovirinae virus subfamily. Examples of viruses from the Comovirinae subfamily include Cowpea mosaic virus, Broad bean wilt virus 1, and Tobacco ringspot virus. In a further embodiment, the virus particles are from the Genus comovirus. A preferred example of a comovirus is the cowpea mosaic virus particles. Other suitable plant virus includes, but is notlimited to bean pod mottle virus (BPMV), cowpea severe mosaic virus or rice tungro spherical virus.

[0086] In some embodiments, the plant viral nanoparticle or VLP is derived from Cowpea chlorotic mottle virus (CCMV). CCMV is a spherical plant virus that belongs to the Bromovirus genus. Several strains have been identified and include, but not limited to, Carl (Ali, et al., 2007. J. Virological Methods 141 :84-86), Car2 (Ali, et al., 2007. J. Virological Methods 141 :84-86, 2007), type T (Kuhn, 1964. Phytopathology 54:1441-1442), soybean (S) (Kuhn, 1968. Phytopathology 58: 1441-1442), mild (M) (Kuhn, 1979. Phytopathology 69:621-624), Arkansas (A) (Fulton, et al., 1975. Phytopathology 65: 741-742), bean yellow stipple (BYS) (Fulton, et al., 1975. Phytopathology 65: 741-742), R (Sinclair, ed. 1982. Compendium of Soybean Diseases. 2nded. The American Phytopathological Society, St. Paul. 104 pp.), and PSM (Paguio, et al., 1988. Plant Diseases 72(9): 768-770).

[0087] In some instances, the plant viral nanoparticle or VLP from CCMV comprise, or consists essentially of, or yet further consists of, a plurality of capsid proteins. In some instances, the capsid protein is a wild-type CCMV capsid, optionally expressed by Carl, Car2, type T, soybean (S), mild (M), Arkansas (A), bean yellow stipple (BYS), R, or PSM strain. In other instances, the capsid protein is a modified capsid protein, e.g., comprising, or consisting essentially of, or yet further consisting of, one or more substitutions, insertions, and / or deletions. In some cases, the CCMV capsid comprises, or consists essentially of, or yet further consists of, the sequence as set forth in the UniProtKB ID P03601 :MSTVGTGKLTRAQRRAAARKNKRNTRVVQPVIVEPIASGQGKAIKAWTGYSVSKW TASCAAAEAKVTSAITISLPNELSSERNKQLKVGRVLLWLGLLPSVSGTVKSCVTETQ TTAAASFQVALAVADNSKDVVAAMYPEAFKGITLEQLTADLTIYLYSSAALTEGDVI VHLEVEHVRPTFDDSFTPVY (SEQ ID NO: 3), or an equivalent thereof.

[0088] In some cases, the virus or VLP from CCMV is prepared by the method as described in Ali et al., “Rapid and efficient purification of Cowpea chlorotic mottle virus by sucrose cushion ultracentrifugation,” Journal of Virological Methods 141 : 84-86 (2007).

[0089] In some embodiments, the plant viral nanoparticle or VLP is derived from Physalis mottle virus (PhMV). PhMV is a single stranded RNA virus that belongs to the genus Tymovirus. In some instances, the plant viral nanoparticle or VLP from PhMV comprises, orconsists essentially of, or yet further consists of, a plurality of coat proteins. In some instances, the coat protein is a wild-type PhMV coat protein. In other instances, the coat protein is a modified coat protein, e.g., comprising, or consisting essentially of, or yet further consisting of, one or more substitutions, insertions, and / or deletions. In some cases, the PhMV coat comprise, or consists essentially of, or yet further consists of, the sequence as set forth in the UniProtKB ID P36351 :MDSSEVVKVKQASIPAPGSILSQPNTEQSPAIVLPFQFEATTFGTAETAAQVSLQTADP ITKLTAPYRHAQIVECKAILTPTDLAVSNPLTVYLAWVPANSPATPTQILRVYGGQSF VLGGAISAAKTIEVPLNLDSVNRMLKDSVTYTDTPKLLAYSRAPTNPSKIPTASIQISG RIRLSKPMLIAN (SEQ ID NO: 4), or an equivalent thereof.

[0090] In some embodiments, the plant viral nanoparticle or VLP is derived from Sesbania mosaic virus (SeMV). SeMV is a positive stranded RNA virus that belongs to the genus Sobemovirus. In some instances, the virus or VLP from SeMV comprise, or consists essentially of, or yet further consists of, a plurality of capsid proteins. In some instances, the capsid protein is a wild-type SeMV capsid protein. In other instances, the capsid protein is a modified capsid protein, e.g., comprising, or consisting essentially of, or yet further consisting of, one or more substitutions, insertions, and / or deletions. In some cases, the SeMV capsid comprises, or consists essentially of, or yet further consists of, the sequence as set forth in the UniProtKB ID Q9EB06:MAKRLSKQQLAKAIANTLETPPQPKAGRRRNRRRQRSAVQQLQPTQAGISMAPSAQ GAMVRIRNPAVSSSRGGITVLTHSELSAEIGVTDSIVVSSELVMPYTVGTWLRGVAA NWSKYSWLSVRYTYIPSCPSSTAGSIHMGFQYDMADTVPVSVNQLSNLRGYVSGQV WSGSAGLCFINGTRCSDTSTAISTTLDVSKLGKKWYPYKTSADYATAVGVDVNIATP LVPARLVIALLDGSSSTAVAAGRIYCTYTIQMIEPTASALNN (SEQ ID NO: 5), or an equivalent thereof.

[0091] The virus can be obtained according to various methods known to those skilled in the art. In embodiments where plant virus particles are used, the virus particles can be obtained from the extract of a plant infected by the plant virus or using the method disclosed herein. For example, cowpea mosaic virus can be grown in black eyed pea plants, which can be infected within 10 days of sowing seeds. Plants can be infected by, for example, coating theleaves with a liquid containing the virus, and then rubbing the leaves, preferably in the presence of an abrasive powder which wounds the leaf surface to allow penetration of the leaf and infection of the plant. Within a week or two after infection, leaves are harvested and plant viral nanoparticles are extracted. In the case of cowpea mosaic virus, 100 mg of virus can be obtained from as few as 50 plants. Procedures for obtaining plant picornavirus particles using extraction of an infected plant are known to those skilled in the art. See Wellink J., Meth Mol Biol, 8, 205- 209 (1998). Procedures are also available for obtaining virus-like particles. Saunders et al., Virology, 393(2):329-37 (2009). The disclosures of both of these references are incorporated herein by reference.

[0092] In some embodiments, the plant viral nanoparticle, e.g., CPMV or CMPV particle has a diameter of from about 15 nm to about 60 nm, or from about 15 nm to about 50 nm, or from about 10 nm to about 40 nm, or from about 20 or 25 nm to about 50 nm, or from about 20 or 25 nm to about 40 nm, or from about 15 nm to about 35 nm, or from about 15 nm to about 40 nm, or from about 15 nm to about 45 nm, or alternatively about 15 nm, or about 20 nm, or about 25 nm, or about 30 nm, or about 35 nm, or about 40 nm, or about 45nm, or about 50 nm, or about 55 nm, or about 60 nm.

[0093] An “effective amount” is an amount sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications or dosages. Such delivery is dependent on a number of variables including the time period for which the individual dosage unit is to be used, the bioavailability of the therapeutic agent, the route of administration, etc. It is understood, however, that specific dose levels of the therapeutic agents disclosed herein for any particular subject depends upon a variety of factors including the activity of the specific compound employed, bioavailability of the compound, the route of administration, the age of the animal and its body weight, general health, sex, the diet of the animal, the time of administration, the rate of excretion, the drug combination, and the severity of the particular disorder being treated and form of administration. In general, one will desire to administer an amount of the compound that is effective to achieve a serum level commensurate with the concentrations found to be effective in vivo. These considerations, as well as effective formulations and administration procedures are well known in the art and are described in standard textbooks.

[0094] “Therapeutically effective amount” of a drug or an agent refers to an amount of the drug or the agent that is an amount sufficient to obtain a pharmacological response such as passive immunity; or alternatively, is an amount of the drug or agent that, when administered to a patient with a specified disorder or disease, is sufficient to have the intended effect, e.g., treatment, alleviation, amelioration, palliation or elimination of one or more manifestations of the specified disorder or disease in the patient. A therapeutic effect does not necessarily occur by administration of one dose and may occur only after administration of a series of doses. Thus, a therapeutically effective amount may be administered in one or more administrations.

[0095] “Administration” can be effected in one dose, continuously or intermittently throughout the course of treatment. Methods of determining the most effective means and dosage of administration are known to those of skill in the art and will vary with the composition used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician. Suitable dosage formulations and methods of administering the agents are known in the art. Route of administration can also be determined and method of determining the most effective route of administration are known to those of skill in the art and will vary with the composition used for treatment, the purpose of the treatment, the health condition or disease stage of the subject being treated, and target cell or tissue. Non-limiting examples of route of administration include systemic or localized administration, e.g., oral administration, intratumorally, or localized at the site of the cancer or tumor, nasal administration, injection, and topical application. Additional non-limiting routes of administration include transdermal, intranasal, vaginal, rectal, subcutaneous intravenous, intravenous, intraarterial, intramuscular, intraosseous, intraperitoneal, intraocular, subconjunctival, sub-Tenon’s, intravitreal, retrobulbar, intracameral, intratumoral, epidural and intrathecal. In one aspect, administration is systemic or intraperitoneal.Modes for Carrying Out the DisclosureTherapeutic Methods and Kits

[0096] In one aspect, provided is a method for inhibiting, delaying, slowing down, orpreventing relapse of cancer in a subject in need thereof. The method comprises, consists essentially of, or consists of administering to the subject a plant viral nanoparticle such as cowpea mosaic virus (CPMV) particle, thereby inhibiting, delaying, slowing down, or preventing the relapse of the cancer in the subject. In some embodiments, the cancer is a solid tumor, carcinoma, sarcoma, or a blood cancer. In one aspect the cancer is a carcinoma. In some aspects, the compositions and methods extend the likelihood of progression free survival, overall survival, and reducing metastasis of a cancer patient after treatment and as compared to a subject not having received the composition or therapeutic method.

[0097] For example, FIG. 2 shows the extended survival and reduced tumor circumference in mice treated with CPMV as compared to a control. Further, FIG. 5 shows tumor nodule reducing potential of CPMV.

[0098] The method can further comprise, consist essentially of, or yet consist of tumor resection of the tumor or cancer prior to, or after administration of the plant viral nanoparticle or CPMV. Alternatively, a different cancer therapy can be administered before, after or at the same time as the disclosed nanoparticles.

[0099] In one aspect the plant viral nanoparticle is administered subsequent to primary treatment to prevent recurrence or relapse of cancer. In another aspect, the plant viral nanoparticle is administered by a method selected from intraperitoneal or intravenously subsequent to primary treatment to prevent recurrence or relapse of cancer.

[0100] In one aspect the plant viral nanoparticle is a CMPV nanoparticle that is administered subsequent to primary treatment to prevent recurrence or relapse of cancer. In another aspect, the plant viral nanoparticle is a CMPV nanoparticle that is administered by a method selected from intraperitoneal or intravenously subsequent to primary treatment to prevent recurrence or relapse of cancer.

[0101] In other aspects, the plant viral nanoparticle is selected from a plant Picornavirus nanoparticle, a Comovirus nanoparticle, a Cowpea mosaic viral nanoparticle, a Broad bean wilt virus 1 nanoparticle, a Tobacco ringspot viral nanoparticle, a cowpea severe mosaic viral nanoparticle, a bean pod mottle viral nanoparticle or a rice tungro spherical viral nanoparticle

[0102] In one aspect the plant viral nanoparticle is non-replicating and non-infectious in the subject.

[0103] In some embodiments, the plant viral nanoparticle or CPMV particle has a diameter of from about 15 nm to about 60 nm, or from about 15 nm to about 50 nm, or from about 10 nm to about 40 nm, or from about 20 or 25 nm to about 50 nm, or from about 20 or 25 nm to about 40 nm, or from about 15 nm to about 35 nm, or from about 15 nm to about 40 nm, or from about 15 nm to about 45 nm, or alternatively about 15 nm, or about 20 nm, or about 25 nm, or about 30 nm, or about 35 nm, or about 40 nm, or about 45nm, or about 50 nm, or about 55 nm, or about 60 nm.

[0104] In addition, a plurality of the viral plant nanoparticle can be administered to the subject, wherein the viral plant nanoparticles are the same or different from each other.

[0105] The subject can be an animal or mammal, e.g., a human patient. In some embodiments, the subject is in remission.

[0106] In another aspect, provided is a method for treating cancer in a subject in need thereof. The method comprises, consists essentially of, or consist of administering to the subject a plant viral nanoparticle such as a cowpea mosaic virus (CPMV) particle, thereby treating the cancer in the subject. In some embodiments, the cancer is a solid tumor, carcinoma, sarcoma, or a blood cancer. In one aspect the cancer is a carcinoma. In one aspect the cancer is a metastatic cancer, e.g., intraperitoneal metastatic cancer.

[0107] In one aspect, the plant viral nanoparticle or CPMV is administered intraperitoneally.

[0108] The method can further comprise, consist essentially of, or yet consist of tumor resection of the tumor or cancer prior to, or after administration of the plant viral nanoparticle or CPMV. Alternatively, a different cancer therapy can be administered before, after or at the same time as the disclosed nanoparticles.

[0109] In one aspect the plant viral nanoparticle is administered after primary treatment to prevent recurrence or relapse of cancer thereby treating the cancer. In another aspect, the plant viral nanoparticle is administered by a method selected from intraperitoneal orintravenously subsequent to primary treatment to prevent recurrence or relapse of cancer thereby treating the cancer.

[0110] In one aspect the plant viral nanoparticle is a CMPV nanoparticle that is administered subsequent to primary treatment to prevent recurrence or relapse of cancer. In some embodiments, the subject is in remission. In another aspect, the plant viral nanoparticle is a CMPV nanoparticle that is administered by a method selected from intraperitoneal or intravenously subsequent to primary treatment to prevent recurrence or relapse of cancer thereby treating the cancer. In some embodiments, the plant viral nanoparticle or CPMV particle has a diameter of from about 15 nm to about 60 nm, or from about 15 nm to about 50 nm, or from about 10 nm to about 40 nm, or from about 20 or 25 nm to about 50 nm, or from about 20 or 25 nm to about 40 nm, or from about 15 nm to about 35 nm, or from about 15 nm to about 40 nm, or from about 15 nm to about 45 nm, or alternatively about 15 nm, or about 20 nm, or about 25 nm, or about 30 nm, or about 35 nm, or about 40 nm, or about 45nm, or about 50 nm, or about 55 nm, or about 60 nm.[OHl] In addition, a plurality of the viral plant nanoparticle can be administered to the subject, wherein the viral plant nanoparticles are the same or different from each other.

[0112] In one aspect, the plant viral nanoparticle or CPMV is administered intraperitoneally or intravenously.

[0113] The subject can be an animal or mammal, e.g., a human patient.

[0114] In yet another aspect, provided is a method for triggering or enhancing one or more of the following in a subject in need thereof: an immune response, immunostimulatory cytokines, increase in the ratio of Ml (antitumor) to M2 (protumor) macrophages, natural killer cells (NK cells), Toll Like Receptor (TLR)s 2, 4 and 7. The method comprises, consists essentially of, or consist of administering to the subject of a plant viral nanoparticle such as a cowpea mosaic virus (CPMV) particle cowpea mosaic virus (CPMV), thereby triggering or enhancing one or more of the following in the subject: an immune response, immunostimulatory cytokines, increase in the ratio of Ml (antitumor) to M2 (protumor) macrophages, natural killer cells (NK cells), TLRs 2, 4 and 7, chemokines, or myeloid- derived suppressor cells.

[0115] In one aspect, the immunostimulatory cytokines are selected from at least one of IFNy, IFNa, TNF-a, IL-12p70, IL-12 / IL-23p40, IL-12 / IL-23p4, IL-15, GM-CSF, IL-2, and IL-17. In one aspect, the chemokines are selected from at least one of IP-10, MIP-la, MIP- 10, RANTES, MCP-1, and KC / GRO. FIGS. 4 and 6 shows immunostimulatory cytokines and chemokines enhanced with the application of CPMV.

[0116] In one aspect, the plant viral nanoparticle or CPMV is administered intraperitoneally or intravenously.

[0117] The method can further comprise, consist essentially of, or yet consist of tumor resection of the tumor or cancer prior to, or after administration of the plant viral nanoparticle or CPMV. Alternatively, a different cancer therapy can be administered before, after or at the same time as the disclosed nanoparticles. For example, FIG. 7 shows increased survival in mice injected with CPMV after removal of a primary tumor, as compared the control mice.

[0118] In some embodiments, the plant viral nanoparticle or CPMV particle has a diameter of from about 15 nm to about 60 nm, or from about 15 nm to about 50 nm, or from about 10 nm to about 40 nm, or from about 20 or 25 nm to about 50 nm, or from about 20 or 25 nm to about 40 nm, or from about 15 nm to about 35 nm, or from about 15 nm to about 40 nm, or from about 15 nm to about 45 nm, or alternatively about 15 nm, or about 20 nm, or about 25 nm, or about 30 nm, or about 35 nm, or about 40 nm, or about 45nm, or about 50 nm, or about 55 nm, or about 60 nm.

[0119] In addition, a plurality of the viral plant nanoparticle can be administered to the subject, wherein the viral plant nanoparticles are the same or different from each other.

[0120] In one aspect, the plant viral nanoparticle or CPMV is administered intraperitoneally or intravenously.

[0121] The subject can be an animal or mammal, e.g., a human patient. In some embodiments, the subject is in remission.

[0122] The methods as disclosed herein can further comprise, consist essentially of, or yet consist of tumor resection of the tumor or cancer prior to, or after administration of the plantviral nanoparticle or CPMV. Alternatively, a different cancer therapy can be administered before, after or at the same time as the disclosed nanoparticles.

[0123] As is apparent to the skilled artisan, in one aspect, an effective amount is administered which is determined by the treating physician or veterinarian.

[0124] When administered to an animal, the methods provide an animal model to test combination therapy or personalized therapies. For example, the animal can be inoculated with tumor cells from a human patient. Alternatively, when the subject is a non-human animal, the methods provide veterinarian therapies. When the subject is a human patient, the methods provide methods to achieve one or more of the disclosed therapeutic benefits.

[0125] Also provided are kits comprising the viral plant nanoparticle or CPMV alone or in combination with a carrier such as a pharmaceutically acceptable carrier and instructions for use in the disclosed methods.

[0126] In some embodiments relating to any method(s), any kit(s), any composition(s), any aspect(s), or any other embodiment s) as disclosed herein, the CPMV particle is nonreplicating and noninfectious in the subject.

[0127] In some embodiments, a method as disclosed herein further comprises isolating a cancer cell from the subject prior to the administration of the plant viral nanoparticle or CMPV or a plurality of same to determine the phenotype or genotype of the tumor or cancer

[0128] In some embodiments relating to any method(s), any kit(s), any composition(s), any aspect(s), or any other embodiment(s) as disclosed herein, the plant viral nanoparticles or CPMV as disclosed herein are administrated in one dose or in two separate doses.

[0129] In some embodiments relating to any method(s), any kit(s), any composition(s), any aspect(s), or any other embodiment(s) as disclosed herein, the subject is a cancer patient in a remission stage. Additionally or alternatively, the cancer in the subject is clinically undetectable, prior to or after primary, secondary, or other prior therapy.

[0130] In some embodiments relating to any method(s), any kit(s), any composition(s), any aspect(s), or any other embodiment s) as disclosed herein, the subject is a cancer patient whohas been treated by one or more of: an ablative therapy, a chemotherapy, a radiation therapy, an immune checkpoint blockade therapy, or another anti-cancer therapy.

[0131] In some embodiments, a method as disclosed herein further comprises treating the subject with one or more of: an ablative therapy, a chemotherapy, a radiation therapy, an immune checkpoint blockade therapy, or another anti-cancer therapy.

[0132] In some embodiments relating to any method(s), any kit(s), any composition(s), any aspect(s), or any other embodiment s) as disclosed herein, as disclosed herein are administered as a first line therapy, a second line therapy, a third line therapy or a fourth line therapy. In one aspect, the methods are only provided after the subject has undergone a primary therapy, e.g., as an adjuvant therapy.

[0133] In one aspect, provided is a kit for use in a method as disclosed herein. The method comprises, consists essentially of, or consists of (a) an adjuvant which comprises, or consists essentially of, or consists of a cowpea mosaic virus (CPMV) particle; and an optional instruction for use.Compositions

[0134] In some embodiments relating to any method(s), any kit(s), any composition(s), any aspect(s), or any other embodiment(s) as disclosed herein, the plant viral nanoparticle or CPMV particle is non-replicating and noninfectious in a subject.

[0135] In another aspect, provided is a vaccine composition comprising, consisting essentially of, or consisting of a plant viral nanoparticle such as a cowpea mosaic virus (CPMV) particle alone or in combination with a carrier, such as a pharmaceutically acceptable carrier and optionally, one or more of a stabilizer or preservative.

[0136] Compositions, including pharmaceutical compositions comprising, consisting essentially of, or consisting of a component or a combination as described herein, can be manufactured by means of conventional mixing, dissolving, granulating, dragee-making levigating, emulsifying, encapsulating, entrapping, or lyophilization processes. The component or combination can be formulated in conventional manner using one or morephysiologically acceptable carriers, diluents, excipients, or auxiliaries which facilitate processing of the component or combination provided herein into preparations which can be used pharmaceutically.

[0137] The component or combination of the present disclosure can be administered by parenteral (e.g., intramuscular, intraperitoneal, intravenous, ICV, intraci sternal injection or infusion, subcutaneous injection, or implant), oral, by inhalation spray nasal, vaginal, rectal, sublingual, urethral (e.g., urethral suppository) or topical routes of administration (e.g., gel, ointment, cream, aerosol, etc.) and can be formulated in suitable dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants, excipients, and vehicles appropriate for each route of administration.

[0138] In one embodiment, this technology relates to a composition comprising a component or a combination as described herein and a carrier.

[0139] In another embodiment, this technology relates to a pharmaceutical composition comprising a component or a combination as described herein and a pharmaceutically acceptable carrier.

[0140] In another embodiment, this technology relates to a pharmaceutical composition comprising a therapeutically effective amount of a component or a combination as described herein and a pharmaceutically acceptable carrier.

[0141] The pharmaceutical compositions for the administration of a component or a combination as disclosed herein can be conveniently presented in dosage unit form and can be prepared by any of the methods well known in the art of pharmacy. The pharmaceutical compositions can be, for example, prepared by uniformly and intimately bringing the compounds provided herein into association with a liquid carrier, a finely divided solid carrier or both, and then, if necessary, shaping the product into the desired formulation. In the pharmaceutical composition, each component provided herein is included in an amount sufficient to produce the desired effect. For example, pharmaceutical compositions of the present technology may take a form suitable for virtually any mode of administration, including, for example, topical, ocular, oral, buccal, systemic, nasal, injection, infusion,transdermal, rectal, and vaginal, or a form suitable for administration by inhalation or insufflation. In one aspect, administration is intraperitoneal.

[0142] For topical administration, the component or the combination can be formulated as solutions, gels, ointments, creams, suspensions, etc., as is well-known in the art.

[0143] Systemic formulations include those designed for administration by injection (e.g., subcutaneous, intravenous, infusion, intramuscular, intrathecal, or intraperitoneal injection) as well as those designed for transdermal, transmucosal, oral, or pulmonary administration.

[0144] Useful injectable preparations include sterile suspensions, solutions, or emulsions of the compounds provided herein in aqueous or oily vehicles. The compositions may also contain formulating agents, such as suspending, stabilizing, and / or dispersing agents. The formulations for injection can be presented in unit dosage form, e.g., in ampules or in multidose containers, and may contain added preservatives.

[0145] Alternatively, the injectable formulation can be provided in powder form for reconstitution with a suitable vehicle, including but not limited to sterile pyrogen free water, buffer, and dextrose solution, before use. To this end, the component or the combination provided herein can be dried by any art-known technique, such as lyophilization, and reconstituted prior to use.

[0146] For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are known in the art.

[0147] For oral administration, the pharmaceutical compositions may take the form of, for example, lozenges, tablets, or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose, or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc, or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate). The tablets can be coated by methods well known in the art with, for example, sugars, films, or enteric coatings.

[0148] Compositions intended for oral use can be prepared according to any method known to the art for the manufacture of pharmaceutical compositions, and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents, and preserving agents in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the combination of compounds provided herein in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients can be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents (e.g., com starch or alginic acid); binding agents (e.g. starch, gelatin, or acacia); and lubricating agents (e.g., magnesium stearate, stearic acid, or talc). The tablets can be left uncoated or they can be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate can be employed. They may also be coated by the techniques well known to the skilled artisan. The pharmaceutical compositions of the present technology may also be in the form of oil-in-water emulsions.

[0149] Liquid preparations for oral administration may take the form of, for example, elixirs, solutions, syrups, or suspensions, or they can be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations can be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats); emulsifying agents (e.g., lecithin, or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, cremophore™, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, preservatives, flavoring, coloring, and sweetening agents as appropriate.Dosages and Dosing Regimens

[0150] The appropriate amount and dosing regimen of the component or the combination, when present to be administered to the subject according to any of the methods disclosed herein, may be determined by one of ordinary skill in the art.

[0151] In some embodiments, the component or the combination as disclosed herein, may be administered to a subject in need thereof, either alone or as part of a pharmaceutically acceptable formulation, once a week, once a day, twice a day, three times a day, or four times a day, or even more frequently.

[0152] Administration of the component or the combination as disclosed herein may be effected by any method that enables delivery of the component or the combination to the site of action. These methods include oral routes, intraduodenal routes, parenteral injection (including intravenous, subcutaneous, intramuscular, intravascular or infusion), topical, and rectal administration. Bolus doses can be used, or infusions over a period of 1, 2, 3, 4, 5, 10, 15, 20, 30, 60, 90, 120 or more minutes, or any intermediate time period can also be used, as can infusions lasting 3, 4, 5, 6, 7, 8, 9, 10, 12, 14 16, 20, 24 or more hours or lasting for 1-7 days or more. Infusions can be administered by drip, continuous infusion, infusion pump, metering pump, depot formulation, or any other suitable means.

[0153] Dosage regimens may be adjusted to provide the optimum desired response. For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form, as used herein, refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the disclosure are dictated by and directly dependent on (a) the unique characteristics of the chemotherapeutic agent and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.

[0154] Thus, the skilled artisan would appreciate, based upon the disclosure provided herein, that the dose and dosing regimen is adjusted in accordance with methods well-known in the therapeutic arts. That is, the maximum tolerable dose can be readily established, and the effective amount providing a detectable therapeutic benefit to a patient may also bedetermined, as can the temporal requirements for administering each agent to provide a detectable therapeutic benefit to the patient. Accordingly, while certain dose and administration regimens are exemplified herein, these examples in no way limit the dose and administration regimen that may be provided to a patient in practicing the present disclosure.

[0155] It is to be noted that dosage values may vary with the type and severity of the condition to be alleviated, and may include single or multiple doses. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition. For example, doses may be adjusted based on pharmacokinetic or pharmacodynamic parameters, which may include clinical effects such as toxic effects and / or laboratory values. Thus, the present disclosure encompasses intra-patient dose-escalation as determined by the skilled artisan. Determining appropriate dosages and regimens for administration of the chemotherapeutic agent are well-known in the relevant art and would be understood to be encompassed by the skilled artisan once provided the teachings disclosed herein.Experimental MethodsExample 1

[0156] The key challenge in cancer treatment is prevention of metastatic disease which is therapeutically resistant and carries poor prognoses necessitating efficacious prophylactic approaches that prevent metastasis and recurrence. Applicant previously demonstrated that cowpea mosaic virus (CPMV) induces durable antitumor responses when used in situ, i.e. intratumoral injection. As a new direction, Applicant show that CPMV demonstrates widespread effectiveness as an immunoprophylactic agent - potent efficacy is demonstrated in four metastatic models of colon, ovarian, melanoma, and breast cancer. Systemic administration of CPMV stimulates the innate immune system, enabling attack of cancer cells; processing of the cancer cells and associated antigens leads to systemic, durable, andadaptive antitumor immunity. Overall, CPMV demonstrated broad efficacy as an immunoprophylactic agent in the rejection of metastatic cancer.

[0157] Materials and Methods

[0158] Materials and cells

[0159] Potassium phosphate monobasic, potassium phosphate dibasic anhydrous, GlutaMAX™, HEPES, sodium pyruvate, Tween-20, and 2-mercaptoethanol were purchased from Fisher Scientific. Urea, Tris buffer, tris(2-carboxyethyl)phosphine (TCEP), chloroacetamide, Lys C, trypsin, trifluoroacetic acid (TFA), acetonitrile (ACN), and formic acid were purchased from Thermo Fisher. PBS was purchased from G Biosciences. PMA was purchased from Millipore Sigma. lonomycin, gadolinium(iii) chloride hexahydrate, and EDTA were purchased from Sigma-Aldrich. Paraformaldehyde 32% (v / v) was purchased from Electron Microscopy Sciences. Fetal bovine serum (FBS) was purchased from R&D Systems. Ethanol (EtOH) was purchased from VWR. Penicillin / streptomycin (P / S) was purchased from Cytiva. RPMI-1640 and DMEM were purchased from Coming.

[0160] CT26-Luc and 4T1-Luc cells were grown in RPMI-1640 supplemented with 10% (v / v) FBS and 1% (v / v) P / S. B16F10 cells were grown in DMEM supplemented with 10% (v / v) FBS and 1% (v / v) P / S. D?>-Defb29 / Vegf-A cells were grown in RPMI supplemented with 10% (v / v) FBS, 1% (v / v) P / S, 2 mM L-glutamine, 1 mM sodium pyruvate, and 0.05 mM 2-mercaptoethanol. RAW264.7-Luc cells were thawed in DMEM supplemented with 10% (v / v) heat-inactivated FBS, 1% (v / v) GlutaMAX (Gibco), and 1% P / S (Thaw Medium). After the first passage, the cells were moved to media consisting of the Thaw Medium plus 700 pg mL'1of genecticin (Thermo Fisher) (Growth Medium 11 A). All cells were grown at 37°C and 5% CO2.

[0161] CPMV purification and characterization

[0162] UV-Vis Spectroscopy

[0163] CPMV was produced and purified in infected black eyed pea plants as done previously

[0023] . The isolated CPMV was stored in 0.1 M KP buffer and stored at 4°C until use.To quantify the concentration of CPMV in the solution, UV-Vis spectroscopy (Nanodrop) and Beer’s law was used to measure the concentration: A = s * 1 * c, where A = absorbance, 8 = the extinction coefficient of CPMV (8.1 mL mg'1cm'1), 1 = path length, and c = concentration. An A260 / A280 value of ~1.8 indicated pure particles.

[0164] SDS-PAGE

[0165] CPMV (10 pg) was diluted in lx lithium dodecyl sulfate sample buffer (Life Technologies) and 0.1 M KP, and heated for 5 min at 95°C. The CPMV was loaded on to a 12% NuPAGE Gel (Thermo Fisher Scientific) and ran under the following conditions: 200 V, 120 mA, and 25 W in lx MOPS buffer before imaging. The gel was run for 40 min and then imaged under white light with an Alphaimager (Protein Simple) following protein staining with GelCode Blue Stain reagent (Thermo Fisher Scientific).

[0166] Agarose gel electrophoresis

[0167] For native gel electrophoresis, 10 pg of CPMV was diluted in lx Gel Loading Purple Dye (Biolabs) and 0.1 M KP, and loaded into 0.8% (w / v) agarose gels with lx GelRed nucleic acid gel stain (Gold Biotechnology). The samples were run at 120 V and 400 mA for 30 min. RNA was visualized under UV light, and protein was visualized under white light following 0.25% (w / v) Coomassie Blue staining.

[0168] DLS

[0169] CPMV was diluted to 0.1 mg mL'1in DI water, and run at RT with a Zetasizer Nano ZSP / Zen5600 (Malvern Panalytical) using the Smolvchowski method.

[0170] SEC

[0171] SEC was carried out on a Superose 6 SEC column (column dimensions: 10 x 300 mm, exclusion limit 4 x 107Mr, Cytiva) connected to an Akta Explorer FPLC machine (Cytiva). The CPMV was diluted to 0.5 mg mL'1in 0.1 M KP buffer, and run using an isocratic profile at 0.5 mL min'1. Absorbance measurements were carried out at 260 and 280 nm, and particle integrity was measured by the A26o / A28o ratio at the elution peak.

[0172] TEM

[0173] Formvar coated carbon films (VWR International) were coated with 20 pL of 0.1 mg mL'1CPMV diluted in DI water for 2 min. The CPMV solution was then replaced with 2% uranyl acetate for 2 min and allowed to dry before imaging on a Joel 1400 TEM.

[0174] CPMV is uniquely immunogenic compared to other viruses

[0175] The immunogenicity of CPMV was compared to the immunogenicity of other viruses using a RAW264.7-Luc NFKB reporter line (RAW264.-7). The RAW264.7-Luc cells were plated at a concentration of 30,000 cells in 100 pL of Growth Media 11 A, and 10 pg of CPMV, CCMV, QP, TMV, PhMV, and media were incubated for 48 hours. Following incubation, the cells were allowed to cool to RT for 30 min before the addition of 100 pL of the SteadyGlo Reagent (Promega) for 15 min at RT according to the manufacturer’s instructions. The luminescent output was measured on a plate reader (Tecan). CCMV and TMV were produced in black eyed peas and Nicotiana benthamiana plants, as done previously I24’25]. Q0 and PhMV were expressed in E. coli as done previously I26’27].Luminescence was read using SteadyGlo Reagent (Promega) according to the manufacturer’s instructions. The luminescent output was measured on a plate reader (Tecan).

[0176] CPMV adjuvant therapy against metastatic CT26-Luc colon cancerAll mice experiments were carried out in accordance with the guidelines set out by the Institutional Animal Care and Use Committee at the University of California San Diego, according to protocol SI 8021. All animals were purchased from the Jackson Laboratory and stored at the Moores Cancer Center and provided with unlimited food and drink.

[0177] The prophylactic ability of CPMV to prevent the onset of i.p. metastatic CT26-Luc growth was assessed. 6- to 8-week-old female BALB / C mice were purchased, and 200 pg of CPMV or PBS were injected i.p. in 200 pL of PBS. One week later, 500,000 CT26-Luc cells in 200 pL of PBS was injected i.p., and tumor growth was measured every 2 days by measuring the circumference and body weight of the mice. On day 40, the surviving mice inthe CPMV groups (there were no survivors in the PBS group) were split into 2 groups and rechallenged i.p. with 500,000 CT26- Luc cells in 200 pL of PBS. Naive mice that were age- matched to be the same age as the mice at the time of re-challenge were also challenged with 500,000 CT26-Luc cells. Tumor growth was monitored every 2 days through body weight and circumference measurements - mice were euthanized with the circumference increased > 60% over the original circumference or when the body weight reached > 75% over the original body weight or 30 g.

[0178] A separate group (n = 5) of the surviving mice following initial CT26-Luc challenge were instead used for ELISPOT analysis. In brief, a 96-well ELISPOT plate (Cellular Technology LTD) was coated with a-mouse IFNy and IL-4 capture antibodies and stored overnight (ON) at 4°C. The next day, the spleens of the mice were collected and digested into single-cell suspensions using a spleen dissociation kit (Militenyi Biotec) according to the manufacturer’s instructions. The splenocytes were then diluted to 500,000 cells / 100 pL in the CTL-Test medium supplemented with 1% (v / v) GlutaMax. The coated ELISPOT plate was washed 3x with PBS, and 100 pL of the cells were added to each well. The cells were then stimulated with 100 pL of media only, 500,000 4T1-Luc cells, 50 ng mL’1PMA and 1 pg mL'1iono, and 500,000 CT26- Luc cells, and incubated at 37°C and 5% CO2 for 48 h. All samples were run in duplicate.

[0179] Following incubation, the cells were washed 3x with PBS + 0.05% (v / v) Tween- 20 (PBST) followed by incubating the cells in 50 mM of EDTA in PBS for 20 min at 37°C. After washing with PBST, 100 pL of 1 : 1000 FITC-labeled a-fFNy and 1 :666 biotin-labeled a-IL4 were incubated for 2 hrs at RT followed by the addition of 100 pL of 1 : 1000 anti-FITC HRP and streptavidin-alkaline phosphatase secondary antibodies for 1 hr at RT. Lastly, a blue developer solution was incubated for 20 min followed by incubation with a red developer solution for 15 min. The plate was washed with tap water and dried ON at RT. The plate was imaged and the spots were quantified using an immunospot S6 ENTRY analyzer.

[0180] Re-challenge following CPMV adjuvant therapy

[0181] The re-challenge experiment was repeated once more with the same conditions except this time, the surviving mice were split into two groups. One group was re-challenged with 500,000 CT26- Luc cells i.p. like before while the other group was re-challenged s.c. with 200,0004T1-Luc cells diluted 1 : 1 with Matrigel (Coming). The mice in the CT26-Luc group were measured like before while the tumors in the 4T1-Luc group were measured every 2 days using digital calipers. Mice were euthanized when tumors reached 1000 mm3. In the CT26-Luc re-challenged mice, some mice that demonstrated clear signs of tumor growth were euthanized before reaching clinical endpoints, and their spleens were collected for ELISPOT analysis like before.

[0182] In a separate study, the mice were provided CPMV adjuvant therapy and CT26-Luc challenge like before. The survivors were then re-challenged with 100,000 CT26-Luc s.c. in a 50:50 mixture with Matrigel in a total volume of 100 pL. The tumors were measured every 2 days using digital calipers, and mice were euthanized when tumor volumes reached clinical endpoint volumes of 1,000 mm3.

[0183] Adjuvant therapy in pre-metastatic mice

[0184] To simulate the presence of metastatic lesions already present within the i.p. space, BALB / C mice were first injected with 50,000 CT26-Luc cells i.p. 3 days later, 200 pg of CPMV or PBS were injected i.p. followed by injection of 500,000 CT26-Luc cells after 7 days. The circumference and weight of the mice were measured every 2 days.

[0185] Longitudinal analysis of CPMV prophylaxis i.p.

[0186] The longitudinal ability of CPMV to protect against CT26-Luc challenge was carried out by incrementally increasing the time until CT26-Luc i.p. tumor challenge. Mice were injected with 200 pg of CPMV i.p., and then injected with 500,000 CT26-Luc cells 1, 2, 3, and 4 weeks following CPMV injection. Tumor growth was monitored by measuring the body weight and circumference.

[0187] To analyze CPMV biodistribution longitudinally, 200 pg of CPMV was injected i.p., and the i.p. fluid as well as the spleens, liver, kidneys, and lungs were collected after 1 and 2 weeks. Total RNA was extracted and purified from the solid organs using RNAqueous™Total RNA Isolation Kit (Invitrogen). Briefly, harvested organs were pooled and lysed using lysis buffer on a gentleMACS™ Dissociator (Miltenyi Biotec) and centrifuged at max speed to isolate supernatant. The supernatant was mixed with 1 : 1 with water with 64% (v / v) EtOH before loading onto the filter column twice to bind RNA. After this, the RNA bound to the column was washed subsequently with Wash 1 and Wash 2 / 3 concentrate and eluted with elution buffer at 70°C.

[0188] Total RNA from i.p. fluid was extracted using QIAamp Viral RNA mini kit (Qiagen). Briefly, i.p. fluid was pooled and incubated with carrier RNA in Buffer AVL for 10 minutes at RT. This solution was mixed with 100% EtOH and then loaded onto the QIAamp Mini column twice. The RNA bound to the column was washed subsequently with Buffer AW1 and AW2 before elution with Buffer AVE.

[0189] 5 ng of total RNA extracted from organs and i.p. fluid was probed from CPMV RNA using TaqMan Fast Virus 1-Step Master Mix (Applied Biosystems). Serial dilutions (100 ng, 10 ng, 1 ng, 0.1 ng, 0.01 ng CPMV RNA) were used as templates to graph a standard curve. All experiments included a no template control (NTC). CPMV Primers were designed and purchased from Integrated DNA Technologies:

[0190] Probe 5’- / 56FAM / TCGGGTTGT / ZEN / TGTTTGATGTTGGCC / 3IABkFQ / -3’ (SEQ ID NO: 6)

[0191] Primer 1 (RV) 5 ’-CAT GGA GTC TTG AGA GCA GAT AG-3’ (SEQ IDNO: 7)

[0192] Primer 2 (FW) 5’-ACA GCT ACC ACC AAC ATT TCT-3’ (SEQ ID NO: 8)

[0193] This experiment was performed using a CFX96 touch real-time PCR detection system (BioRad).

[0194] Administration route analysis and mechanism of action of CPMV prophylaxis against CT26-Luc i.p.

[0195] To determine whether CPMV had to be injected locally i.p. to protect against CT26-Luc, the route of administration was altered prior to CT26-Luc challenge. Inseparate mice, 200 pg of CPMV was injected i.p. as before, in 50 pL i.v. retro-orbitally, and in 100 pL s.c. One week following CPMV administration, the mice were challenged with 500,000 CT26-Luc cells, and the tumor growth and survival was followed as had been done previously.

[0196] In a separate experiment, CPMV was injected i.p., i.v., and s.c. as before. One week following injection, the i.p. fluid was collected with i.p. gavage, and the collected cells were readied for flow cytometry analysis. In brief, the fluid was first centrifuged down to collect the cells at 300 x g for 5 min at 4°C, and the cells were strained over a 70 pm cell strainer. The cells were centrifuged down, and incubated with lx RBC lysis buffer (eBioscience) for 5 min. Following washing, the cells were diluted to 1 x 107cells ml1, and 100 pL was added to a 96-well V- bottom plate. Next, the cells were stained with a 1 : 1000 dilution of a LIVE / DEAD Aqua (Thermo Scientific) solution and incubated at RT for 20 min followed by washing with PBS. The cells were then fixed for 20 min with 2% (v / v) PF A. Following washing, the Fcregions of the cells were then blocked with an a-CD 16 / 32 blocking antibody (Biolegend) for 20 min at 4°C. The cells were stained with the following antibodies (all from Biolegend) at a 1 :200 dilution for 1 hr at 4°C: Pacific Blue - CD45; APC-Cy7 - Ly6G; PE-Cy7 - Ly6C; SuperBright 645 - CD1 lb; Brilliant Violet 605 - NKp46; PE - F4 / 80; PerCP-Cy5.5 - CD86; APC - MHCII; SuperBright 780 - CD11c. Following washing, the cells were stored at 4°C in FACS buffer (PBS+ 0.5 M EDTA + FBS + IM HEPES) until use. Flow cytometry was carried out on a FACSCelesta, and data analysis was implemented using FlowJo.

[0197] The i.p. fluid was also analyzed via MSD using a customizable U-PLEX analyzing the following cytokines / chemokines: GM-CSF, IFNy, IL-2, IL-4, IL-5, IL-10, IL-12p70, IL-13, KC / GRO, eotaxin, IFN-p, IL-9, IL-15, IP-10, MCP-1, MIP-la, MIP-lp, TNF-a, VEGF-A, IFN-a, IL-12 / IL-23p40, IL-17A, and RANTES. Before MSD analysis, the i.p. fluid was concentrated down to 150 pL using a 3 kDa MWCO spin filter and stored at -80°C. The day of, an assigned U- PLEX linker was bound to a biotinylated U-PLEX capture antibody and incubated for 30 min.

[0198] 200 pL of the stop solution was then added and incubated for 30 min. The antibodies were combined into one solution, and 50 pL of the solution was coated per well for 1 hr at RT. The coating solution was discarded and the plate was then washed 3 x with the wash buffer. 25 pL of Diluent 41 was added to the plate followed by the addition of 25 pL of the i.p. wash samples, which was incubated at RT for 2 hrs. During the incubation, the detection antibody solution was prepared by diluting the detection antibody to the working concentration with Diluent 45. After the 2 hrs, the samples were discarded and the plate was washed 3x with wash buffer. The detection antibody (50 pL) was added to the plate and incubated for 1 hr at RT followed by washing and addition of 150 pL of MSD GOLD Read buffer. The plate was read using a MESO QuickPlex SQ 120 instrument and analyzed using MSD Workbench 4.0 software.

[0199] Tandem Mass Tag (TMT)

[0200] TMT was carried out with the help of the Biomolecular and Proteomics Mass Spectrometry Facility at UC San Diego. Female BALB / C mice were injected i.p. with 200 pg of CPMV or 200pL of PBS, and the i.p. fluid was collected and stored at -80°C.The protein fractions following i.p. injection of CPMV were first digested through pelleting of the samples. The pellet was resuspended in 600 pl of 8 M urea made in 100 mM Tris pH 8.0 and by vortexing for 5-10 min. TCEP reducing agent was added to a final concentration of 10 mM. The samples were then frozen ON at -20°C. The next day, the solution was thawed and vortexed for another 5 min until the solution became clear. Chloro-acetamide solution was added to a final concentration of 40 mM and vortexed for 5 min. An equal volume of 50 mM Tris pH 8.0 was added to the sample to reduce the urea concentration to 4 M followed by the addition of Lys C at a 1 :500 ratio of LysC to protein content. The solution was incubated at 37°C in a rotating incubator for 4-6 h, and 50 mM Tris pH 8.0 was added to reduce the urea concentration to 2 M. Trypsin was added at a 1:50 ratio of trypsin to protein content and incubated ON. The next day, the solution was acidified using TFA to a final concentration of 0.5% (v / v) TFA and vortexed for 5 min. The sample was centrifuged at 15,700 x g for 5 min to obtain aqueous and organic phases. The lower aqueous phase was collected and desalted using 100 mg C18-StageTips as described by the manufacturer’s protocol. The peptideconcentration of the sample was measured using BCA after resuspension in iTRAQ dissolution buffer.

[0201] 4 tags of Thermo Scientific™ TMTpro™ 16plex (Catalog number: A52045) was used for the labeling of the samples. The labeling protocol was followed according to the manufacturer’s instructions. The CPMV i.p. sample was tagged with TMT16-133N, and the PBS i.p. sample was tagged with TMT16-134N. Pierce™ High pH Reversed-Phase Peptide Fractionation Kit (Catalog number: 84868) was used for the high pH fractionation according to the manufacturer’s instructions with the exception that 12 fractions were generated.

[0202] Each fraction was analyzed by ultra-high-pressure liquid chromatography (UPLC) coupled with tandem mass spectroscopy (LC-MS / MS) using nano-spray ionization. The nano-spray ionization experiments were performed using an Orbitrap fusion Lumos hybrid mass spectrometer (Thermo Fisher) interfaced with nano-scale reversed-phase UPLC (Thermo Dionex UltiMate™ 3000 RSLC nano System) using a 25 cm, 75 pm ID glass capillary packed with 1.7 pm C18 (130) BEH™ beads (Waters Corporation). Peptides were eluted from the Cl 8 column into the mass spectrometer using a linear gradient (5-80%) of ACN at a flow rate of 375 pl min'1for 120 min. The buffers used to create the ACN gradient were: Buffer A (98% H2O, 2% ACN, 0.1% formic acid) and Buffer B (100% ACN, 0.1% formic acid). Mass spectrometer parameters are as follows: an MSI survey scan using the orbitrap detector (mass range (m / z): 400-1500 using quadrupole isolation, 60000 resolution setting, spray voltage of 2200 V, ion transfer tube temperature of 275°C, AGC target of 400000, and maximum injection time of 50 ms) was followed by data dependent scans at which the top speed for the most intense ions with charge states set to only include +2-5 ions, 5 s exclusion time, and while selecting ions with minimal intensities of 50000 at in which the collision event was carried out in the high energy collision cell (HCD Collision Energy of 38%). The first quadrupole isolation window was set at 0.7 (m / z). The fragment masses were analyzed in the Orbi-trap mass analyzer with mass resolution setting of 15000 and the following parameters: ion trap scan rate of turbo, first mass m / z of 100, AGC Target 20000 and maximum injection time of 22 ms). Protein identification and quantification was carried out using Peaks Studio 8.5 (Bioinformatics Solutions Inc.).

[0203] Cell depletion of innate immune cells

[0204] The importance of each innate immune cell in the prophylactic response was investigated through the depletion of each immune cell using NAbs. All NAbs were purchased from BioXCell - anti-mouse CD122 (IL-2R0) (clone 5H4; Catalog #: BE0272), anti-mouse CD317 (BST2, PDCA-1) (clone 927; Catalog #: BE0311), anti-mouse F4 / 80 (clone CEA3-1, Catalog #: BE0206), and anti-mouse Ly6G (clone 1A8; Catalog #: BE0075- 1) were used to deplete NK cells, pDCs, macrophages / monocytes, and neutrophils, respectively. Due to the absence of a pan- DC neutralization marker, Applicant focused on the neutralization of DCs to pDCs, which are the main DCs that are activated following viral infection and release copious amounts of type I IFNs

[0073] .

[0205] Female BALB / C mice (n = 7) were initially injected with 200 pg of CPMV i.p. followed by the injection of 200 pg of the NAbs i.p. after 6 days. 500,000 CT26-Luc cells were injected on day 7 i.p., and tumor growth was monitored using circumference and body weight measurements. The NAbs were injected every 5 days until the termination of the study.

[0206] ID8-Defb29 / Vegf-A-Luc prophylaxis with CPMV

[0207] To demonstrate the broad applicability of CPMV prophylaxis in multiple tumor types, an \V) -l)efb29 Vegj-A- wc ovarian cancer murine model was also tested. 6-8 week old female C57BL / 6J mice were purchased and injected with 200 pg of CPMV i.p. followed by injection of 2 x 106^ -Dejh29 / Ve f-AAwx i.p. Mice were euthanized when the body weight reached > 75% of the original body weight or the body weight totaled over 30 g. Mice were also euthanized when the abdominal circumference reached > 60% of the original circumference or reached a circumference of 9 cm. The circumference and body weight are used as indicators of tumor growth following i.p. tumor dissemination and ascites development. At day 67, the spleens of the surviving mice were collected and analyzed by ELISPOT. The splenocytes were stimulated with either media only, 500,000 B16F10 cells, 500,000 D’&-Defb29 / Vegf-A-Vuc cells, or PMA / iono.

[0208] CPMV prophylaxis against B16F 10 melanoma and 4T1-Luc TNBC metastasis to the lungs

[0209] The ability of CPMV to protect against metastasis to the lungs was also investigated in a B16F10 murine i.v. model. Female 6-8 week old C57BL / 6J mice were immunized with 200 pg of CPMV s.c., i.v., and i.p followed by injection of 100,000 B16F10 cells i.v. After 3 weeks, the lungs were harvested from the mice and the tumor nodules were manually counted. In a comparative study, CPMV was investigated head-to-head against other potential adjuvants and immunomodulatory small molecules. Mice were injected i.p. or i.v. with either 200 pg of CPMV or CCMV, or 5 pg of diABZI or imiquimod. 100,000 B16F10 cells were injected i.v., and the lungs were harvested and tumor nodules were manually counted after 2 weeks.

[0210] The immunogenicity of the CPMV and other reagents was also compared using the RAW264.7-Luc reporter line as indicated above. 100,000 cells were seeded, and the reagents were standardized to 10 x 106molecules / cell and incubated for 24 h at 37°C. The SteadyGlo Reagent was incubated as before and luminescence was measured on a Tecan plate reader.

[0211] For the 4T1-Luc prophylaxis studies, 6-8 week old female BALB / C mice were purchased. They were injected i.p. with 200 pg of CPMV followed by the injection of 100,000 4T1-Luc cells i.v. The growth of the 4T1-Luc cells was monitored using an IVIS (Xenogen) every 2-4 days. To measure luminescence, 200 pL of a 15 mg mL'1solution of D- luciferin (Gold Biotechnologies) was injected i.p, and the luminescence was measured using ROI measurements on the Living Image 3.0 software. The weight of the mice and luminescence within the lungs were measured every 2 days. 3 weeks following tumor inoculation, the lungs were collected and stored in Bouin’s solution (Sigma Aldrich) ON followed by storage in 70% (v / v) EtOH. The tumor nodules were then manually counted.

[0212] Mechanism of action of CPMV protection against lung metastatic tumor nodules

[0213] Blood of C57BL / 6J mice following i.v. injection of 200 pg of CPMV or PBS was collected by retro-orbital bleeding. Sera was collected from the mice by spinning down theblood at 2,000 x g and collecting the supernatant. The supernatant was stored at -80°C until MSD analysis, which was carried out like before with the i.p. fluid.

[0214] The blood of C57BL / 6J mice was also collected for flow cytometry analysis of the blood. The blood was incubated with 10 mL of lx RBC lysis buffer for 5 min at RT followed by the deactivation of the lysis buffer with 20 mL of PBS. Cells were collected by centrifugation at 500 x g for 5 min, and the pellet was resuspended with 10 mL of lx RBC lysis buffer for an additional lysis step. Following lysis and centrifugation, the cells were subjected to the same protocol as with the i.p. fluid flow cytometry analysis except that instead of a Brilliant Violet 605 NKp46 antibody, a Brilliant Violet 605 NK1.1 antibody was used. To corroborate that the main cellular recruitment was happening within the blood, the spleens and lungs of C57BL / 6J mice were also analyzed by flow cytometry. The spleens and lungs were digested using a spleen and lung dissociation kit, respectively, and then strained through a 70 pm cell strainer. The single cell suspension was then incubated with 5 mL of a lx RBC lysis buffer solution one time and processed for flow cytometry identically as with the blood.

[0215] Surgical removal of 4T1-Luc followed by CPMV adjuvant therapy

[0216] To ascertain that CPMV is effective in a more clinically-relevant model, 100,000 4T1-Luc TNBC cells were injected s.c. in the left flank of female BALB / C mice and surgically removed after two weeks. The mice were given anesthetic lidocaine (Vet One) according to the manufacturer’s instructions. The following day, the mice were injected i.v. with 200 pg of CPMV or 200 pL of PBS. The metastasis and recurrence of the 4T1-Luc cells were monitored using an IVIS every 2-4 days. To measure luminescence, 200 L of a 15 mg mL-1 solution of D-luciferin (Gold Biotechnologies) was injected i.p, and the luminescence was measured using ROI measurements on the Living Image 3.0 software. The mice weight was also measured, and mice were sacrificed when their weight decreased more than 15% from their heaviest recorded weight. Weight loss is used as an indicator of lowered quality of life in the mice due to tumor burden with 15% being the clinical endpoint according to IACUC regulations.

[0217] Statistical analysis

[0218] All statistical analyses were carried out using GraphPad Prism. When comparing between multiple groups such as when comparing tumor nodule counts, one-way ANOVA was utilized. When only two groups were present, a Student’s T-test was employed. A comparative analysis with time utilized a two-way ANOVA, and survival curves were analyzed using a log-rank (Mantel Cox) test. All graphs are showing the mean ± SD, and the number of samples included in each statistical analysis is written out within the figure captions. A p-value of < 0.05 was deemed significant.

[0219] Results and Discussion

[0220] CPMV production and characterization

[0221] CPMV is a 30 nm, positive-sense RNA plant virus with a bipartite genome (FIG. la) with two distinct RNAs (RNA-1 and RNA-2)|2S|. UV-Vis of the CPMV shows A260 / A280 values near 1.8 indicating pure particles (FIG. lb). Analysis of the proteins by SDS-PAGE shows the small (S) and large (L) coat proteins (CPs) of - 24 and 42 kDa, respectively (FIG. 1c); 60 copies each form an intact CPMV particle. Native gel electrophoresis of the CPMV shows that the genomic RNA and capsid co-migrate indicating stable, unbroken particles (FIG. Id). TEM, DLS, and SEC highlight that monodisperse and pure CPMV preparations were obtained (FIGS, le-lg).

[0222] As part of the quality control and assurance, Applicant confirmed the immunostimulatory nature of CPMV and also compared it to other viral nanoparticles (VNPs) using a RAW264.7 macrophage NFKB luminescent reporter cell line (RAW264.7- Luc). While CPMV showed significant luminescent signal, indicating innate immune stimulation over the media-only control, the other VNPs did not elicit stimulation (FIG. Ih). This confirms the unique nature of CPMV in its immunostimulatory capabilities

[0029] .

[0223] CPMV adjuvant therapy against CT26-Luc colon cancer disseminated in the i.p. space

[0224] Metastatic cancers in the i.p. space have poor prognoses and when at advanced stages are one of the deadliest forms of cancer[30 32Therefore, as an initial study, Applicant investigated whether CPMV could prevent settling and dissemination of i.p. metastases from i.p. challenge using CT26-Luc colon cancer cells (FIG. 2). CPMV was administered into the i.p. space (200 pg), and mice were challenged after 1 week with CT26-Luc tumors (500,000 cells, i.p.) (FIG. 2a). Mice that received CPMV treatment demonstrated significantly extended survival compared to the PBS mice with 56% of mice surviving the initial tumor challenge while all control mice succumbed to the disease within 25 days (FIG. 2b). The growth of the CT26-Luc within the i.p. space can be followed by measuring the abdominal circumference and body weight of the mice, which increase following ascites development and enlargement of the mouse peritoneal cavity. By day 20 post tumor injection (PTI), the average circumference and body weight of the mice in the CPMV group increased by only 4.4 and 4.6%, respectively, while in the PBS group the circumference and body weight had increased to 54.2 and 27.4%, respectively (FIGS. 2c, 2d) corresponding to a comparative increase of 12.3 and 6-fold compared to treated mice.

[0225] At day 40, the surviving mice were re-challenged with CT26-Luc (500,000 cells, i.p.) (FIG. 2a) - 5 of the mice were not re-challenged and used for ELISPOT analysis (see below). T cell depletion studies were simultaneously carried out, and T cells were depleted using a-CD4 and a-CD8 neutralizing antibodies (NAbs) every 3 days to elucidate the importance of the T cell response following re-challenge. Age-matched mice with no prior CT26-Luc exposure served as a control; age-matching was utilized to ensure that age does not play a factor in the response against the re-challenge. Similar to the initial challenge, the circumference increased quickly in the control mice (FIGS. 2e, 2g); in comparison, the tumors in the survivors of the initial challenge (from here on out referred to as the “survivors”) showed markedly diminished tumor growth. The importance of the T cell response was also demonstrated, as the tumors in the NAb-administered mice grew at similar rates as with the age-matched controls. By day 20 following the re-challenge, the circumference for the survivors, NAb-injected mice, and age-matched mice were 8.9, 38.4, and 39.2% greater than before the re-challenge, respectively. Survival following re-challenge was also markedly improved in the survivor group compared to controls (FIG. 2f). A median survival in the survivor’s group could not be determined due to there being > 50% survivalwhile in the NAb-injected mice and the age-matched controls, the median survival was 22 and 20 days, respectively. The weight of the mice following re-challenge is found in FIG. 8. This data indicates that boosting the immune system with CPMV prevents onset of metastases in the i.p. space and primes long-lasting T cell-dependent antitumor immunity.

[0226] To corroborate the importance of the T cell response as well as demonstrate the formation of immune memory, splenocytes from 5 survivors were harvested and subjected to an ELISPOT assay (FIGS. 2h, 2i). The splenocytes were isolated and stimulated with media, 4T1-Luc triple negative breast cancer (TNBC) (negative control), CT26-Luc, and phorbol 12- myristate 13-acetate (PMA) / ionomycin (iono) (positive control). The number of red spots, which measures IFNy production, produced by the CT26-Luc-stimulated splenocytes was significantly higher than in the media or 4T1-Luc signifying a potent cytotoxic T lymphocyte (CTL) / Thl response which was tumor specific. The CT26-Luc stimulation produced 539.8 spots, while the media and 4T1-Luc produced 31.4 and 48.2 spots, respectively. IL-4 production, measured by blue spots and which signifies a Th2 response, was significantly greater in the CT26-Luc samples when compared to media. The specific response to CT26- Luc stimulation demonstrates that following initial tumor killing, immune memory against the initial tumor is established - stimulation with a control 4T1-Luc TNBC cell line induced no IFNY production. As shown with the neutralization of CD4 and CD8 (FIGS. 2e-2g), this T cell response seems to be vital. Indeed, when ELISPOT was ran on splenocytes of mice that survived an initial challenge, but not a re-challenge, T cell response against the CT26-Luc was nonexistent (FIG. 9).

[0227] To demonstrate that the adaptive immune response elicited was indeed tumorspecific, Applicant performed another re-challenge experiment comparing re-challenge with CT26-Luc (as described above) and 4T1-Luc. The initial tumor prevention data were in good agreement with the FIG. 2 demonstrating good reproducibility (FIG. lOa-lOd). The survivors were then split into 2 groups: one re-challenged with CT26-Luc i.p. and the other with 4T1-Luc s.c. Re-challenge with CT26-Luc again led to long-term survivors due to the formation of immune memory (FIG. lOe). However, in the mice injected with the 4T1-Luc, the tumors grew unabated, and there were no survivors (FIG. 2j and FIG. lOf). To demonstrate that the immune memory is systemic and tissue agnostic, the experiment wasrepeated and surviving mice were re-challenged with 100,000 CT26-Luc cells s.c. away from the original injection site which was i.p. (FIG. 10g). By day 50 PTI, 9 / 10 of the survivors remained alive with complete tumor rejection while 5 / 7 of the mice in the age-matched mice had succumbed to the CT26-Luc (FIG. lOh). At day 27 PTI, the average tumor volume was 0 mm3for the survivors and 639.4 mm3for the age-matched mice (FIG. lOi). Overall, the rechallenge experiments demonstrate that CPMV prophylaxis generates potent immune memory after initial challenge that is tumor-specific, but tissue-agnostic.

[0228] Lastly, patients oftentimes are diagnosed with i.p. metastases already present, as current imaging techniques are not well suited for early diagnosis and screening of i.p. tumor nodules

[0030] . Therefore, to better mirror a clinical situation, mice were challenged with a low number of CT26-Luc cells (50,000 cells, i.p.) prior to CPMV prophylactic treatment, which was then followed by CT26-Luc challenge as described above (FIG. Ila). In this scenario, CPMV adjuvant therapy remained effective even with metastatic cells already present within the i.p. space (FIGS, llb-lld). Compared to PBS, CPMV increased the median survival from 19 to 39 days (FIG. 11b), and decreased circumference by 3.7-fold 19 days PTI (FIG.11c)

[0229] Contemporary treatment strategies, such as systemically administered chemotherapeutics and even localized i.p. chemotherapeutics are largely ineffective[33 3S |. Furthermore, peritoneal metastases are difficult to diagnose with common imaging techniques due to their small size and oftentimes go unnoticed

[0030] . Therefore, a prophylactic approach such as the one proposed with CPMV that immediately prevents such outgrowths could be vital in the treatment of these cancers.

[0230] Longitudinal analysis of CPMV prophylaxis against CT26-Luc colon cancer

[0231] To test the duration of immune protection after CPMV prophylaxis, a longitudinal analysis of CT26-Luc was carried out with the tumors injected after 7, 14, 21, and 28 days following CPMV prophylactic treatment (FIGS. 3a and 12a). CPMV treatment protected from tumor challenge 7 days post treatment as demonstrated in FIG. 2 (FIGS. 3b and 12b). Impressively, after only a single injection, efficacy continued to remain steadfast after 14 days (FIGS. 3c and 12c). In the 14-day mice, compared to the PBS, CPMV reduced thecircumference and body weight by 96.5- and 10.4-fold, respectively, 12 days PTI (FIGS. 3c and 12c). Additionally, CPMV extended survival with 3 / 5 mice showcasing long-term survival up to 75 days PTI, which was when the experiment was concluded, while all PBS mice reached clinical endpoints by day 20 (FIG. 3f).

[0232] However, in mice challenged with tumors at 21- or 28-days post CPMV treatment, the efficacy of the CPMV prophylaxis was completely abolished (FIGS. 3d-3f and FIGS. 12d, 12e), and there were no significant differences in the circumference and body weight changes between groups. One mouse in the CPMV and 21 -day tumor challenge group did survive long-term, which was considered statistically insignificant (FIG. 3f).

[0233] Regardless, CPMV was able to significantly delay tumor growth and extend survival when tumor challenge was carried within a 14-day window, indicating durable effects of the CPMV prophylactic treatment. The persistence of CPMV may be attributed to its nanoparticle character providing advantages over small molecules therapeutics, which may suffer from rapid clearance

[0036] . Additionally, CPMV is rather stable, which may aid its long- lasting efficacy. CPMV has been shown to be stable in a wide range of pH values (mirroring the pH of the GI tract) and temperatures thus demonstrating efficacy after a single injection [37,38] Tebiodistribution of CPMV was investigated 7 and 14-days following CPMV i.p. injection, which demonstrated that indeed CPMV remained detectable within the i.p. space within the 14-day window (FIG. 13). Future studies could elucidate whether tumor protection could be extended further following multiple injections of CPMV.

[0234] Route of administration of CPMV affects efficacy against i.p. metastases

[0235] Applicant then further investigated whether CPMV could be administered through different routes and still provide broad protection against CT26-Luc i.p. challenge. CPMV was injected either s.c., i.v., or i.p., and mice were challenged with CT26-Luc cells i.p. as before. While i.p. injection of CPMV demonstrated significant protection, s.c. and i.v. injection of CPMV did not slow tumor growth or extend survival (FIG. 14). There were no significant differences between PBS and mice injected with CPMV i.v. or s.c., which demonstrated that for i.p. prophylaxis, the CPMV route of administration has to be localized to the area of tumor development.

[0236] To probe the mechanism of action and to gain understanding why i.p. but not i.v. or s.c. administration elicited protection against tumor challenge, flow cytometry and cytokine multiplexed analyses were carried out using i.p. fluid collected 1 week post CPMV injection. While i.p. administration of CPMV significantly improved the recruitment of antitumor immune cells in the i.p. space (FIGS. 4a, 4b), this was not observed when CPMV was administered i.v. or s.c. Compared to controls, i.p. administration of CPMV increased the population of CD45+ cells within the i.p. space, specifically dendritic cells (DCs), neutrophils, activated neutrophils, monocytes, and natural killer (NK) cells were increased. Surprisingly, the percentage of macrophages was significantly decreased following i.p. treatment with CPMV, but when dissecting the phenotypic makeup of these macrophages, the i.p. injection of CPMV led to the polarization / recruitment of Ml macrophages and reduction of M2 macrophages thus displaying a greater M1 / M2 ratio - this was in stark contrast to the mice receiving PBS or CPMV i.v. or s.c. Tumor-associated macrophages (TAMs) are one of the major causes of immunosuppression within the TME leading to T cell exhaustion, release of immunosuppressive cytokines, and increase in metastasis

[0039] - therefore, decreasing the overall macrophage population while polarizing the remaining macrophages to antitumor phenotypes may be a key factor toward the successful immunoprevention of tumor growth in mice receiving the i.p. CPMV treatment.

[0237] It must also be noted that Applicant did observe increases in both the monocytic and granulocytic myeloid-derived suppressor cell (M / G-MDSC) populations. This may be a byproduct of the CPMV immune cascade - previous studies have demonstrated that CPMV causes significant release of IL-6, which has both immunostimulatory and immunosuppressive effects[17,40]. While IL-6 can improve the T cell response, it also causes the recruitment of MDSC populations[40,41]. Regardless, the increase in the G / M-MDSC populations do not seem to affect the potency of CPMV prophylaxis, and MDSCs can also be polarized into antitumor phenotypes. In summary, the injections of CPMV i.v. and s.c. did not induce immune cell recruitment into the i.p. space compared to CPMV i.p. injection - this aligns with the efficacy data (FIG. 14) in that CPMV i.p. and not i.v. or s.c. offers protection against CT26-Luc challenge. The gating strategy can be found in FIG. 15.

[0238] To probe the cytokine / chemokine response, MSD was carried out on the i.p. fluid one week after CPMV i.p. and i.v. injection (FIGS. 4c-4e, FIG. 16). A second study was done where CPMV was administered i.p. followed by i.p. CT26-Luc tumor challenge 1 week post CPMV therapy; i.p. fluid was then collected 24 hours PTI (this sample is labeled i.p. + T). Similar to previous Luminex data

[0020] , CPMV was able to induce upregulation of immunostimulatory cytokines notably fFNy, IFNa, TNF-a, IL-12p70, and IL-12 / IL-23p4. While non-infectious toward mammals, CPMV is recognized by PRRs and stimulates an antiviral response inducing IFN expression

[0042] . Although not significant, Applicant did notice trends towards increased IL-12p70 and IL-12 / IL-23p4 when tumor challenge was performed. Additionally, chemokines were significantly upregulated following CPMV i.p. injection (FIG. 4e). Compared to PBS, CPMV increased the production of IP-10, MIP-la, MIP-ip, RANTES, MCP-1, and KC / GRO by 9.4-, 9-, 10.6-, 9.8-, 3-, and 3.8-fold. These chemokines act as chemoattractants for a wide range of both innate and adaptive immune cells, which mirrors the finding of increased immune cell infiltration in the i.p. space upon CPMV treatment (FIGS. 4a, 4b). For instance, MCP-1 has been demonstrated to induce monocyte and DC recruitment, and the flow data corroborates that only CPMV i.p. increases both the monocyte and DC populations. The i.v. CPMV dose does not induce expression of cytokines or chemokines (FIGS. 4c-4e), consistent with lack of immune cell recruitment (FIG. 4a) and lack of efficacy (FIG. 14).

[0239] Proteomic studies using TMT were also performed; significant changes were denoted as any protein with a significance value above 15 and an expression ratio either < 0.5 or > 2. 28 proteins were upregulated within the i.p. space following CPMV injection, and 5 were downregulated (FIG. 17). Of note, IgG2a and IgG2b were one of the most upregulated proteins after CPMV injection - both IgG subtypes are vital in viral clearance

[0043] . However, TMT does not provide any information on the antigen specificity of the IgG2 although it is understood that i.p. administration of CPMV leads to generation of a-CPMV antibodies

[0044] . Others have demonstrated that injection of tumor-independent IgG can induce tumor regression in both prophylactic and treatment settings I45-47]. Therefore, it may be that the increase in IgGs is contributing to tumor rejection although this has not been experimentally validated. Indeed, others show that supernatant from IgG-stimulated macrophages can induce considerable tumor cell death

[0048] . CPMV injection also led to increased production offibronectin and peroxiredoxin- 1, which have been shown to improve chemotaxis and leukocyte migration and induces IFN production / aids in the antiviral activity of CD8+ T cells [49-52] Lastly, proteins related to amino acid and lipid metabolism (e.g. betaine-homocysteine S-methyltransferase and fatty acid-binding protein) as well as ATP-generating pathways such as glycolysis (e.g. S-adenosylmethionine synthase isoform type-1 and fructose-biphosphate aldolase B) were also increased.

[0240] Overall, CPMV led to significant increases in cyto / chemokines and immune cells into the i.p. space. While this may not directly lead to tumor killing, without wishing to be bound by any particular theory, Applicant hypothesizes that the recruited immune cells can home at an improved rate into the tumor lesions found within the peritoneal cavity due to their now proximity to the metastatic tumors. Additionally, these cells are better geared for tumor killing due to the immunostimulatory nature of the i.p. microenvironment as seen by the increased levels of activated neutrophils and the M1 / M2 ratio (FIG. 4a). Lastly, the recruited immune cells release additional immunostimulatory cytokines such as IFNy and TNFa that can have direct and indirect antitumor effects.

[0241] CPMV prophylaxis is applicable across tumor types

[0242] To demonstrate broad applicability of the CPMV prophylactic treatment, Applicant also tested efficacy of i.p. administered CPMV against i.p. challenge with D -Defb29 / Vegf- A cells, a model of ovarian cancer (FIG. 18a). Again, mice receiving the CPMV adjuvant therapy demonstrated significantly improved survival rates with a median survival of 52 for the CPMV group vs. 45 days for the control group (FIG. 18b). There were also long-term survivors for the CPMV group, which were not seen in the PBS-injected mice. With CPMV, the circumference and weight of the mice 45 days PTI increased an average of 22 and 20.1%, respectively (FIGS. 18c, 18d). However, in the PBS mice, the circumference and weight increased 62.2 and 42.6%, a comparative 2.8- and 2.1-fold increase compared to CPMV, respectively. The survivors in the CPMV group were analyzed via ELISPOT on day 75 PTI (FIGS. 18e, 18f), which indicated that the splenocytes from the surviving mice were able to produce IFNY following ID8-Defb29 / Vegf-A stimulation indicating immune memory and a Th 1 -dependent T cell response against the original tumor. On the other hand, stimulationwith a control B16F10 cell line did not produce any IFNy spots. Representative images of the ELISPOT can be found in FIG. 18f.

[0243] B16F10 melanoma lung metastasis prophylaxis with CPMV

[0244] Another major hurdle in the treatment of cancer is the metastasis of cancer cells to the lungs

[0053] . In fact, lung metastases are extremely common, and can be found in a myriad of cancer types ranging from breast, colorectal, uterine and melanoma

[0054] . Prognosis is poor, and survival rates are low for lung metastatic tumors; systemically administered therapeutics are oftentimes ineffective due to dose-limiting toxicity and drug resistance155 57Therefore, Applicant evaluated efficacy of CPMV as a prophylactic measure against Bl 6F 10 melanoma lung metastases. CPMV (200 pg) was administered s.c., i.v., and i.p., and tumor challenge was performed one week post treatment using B16F10 melanoma cells (100,000 cells, injected i.v.) (FIG. 5a). The lungs were harvested 3 weeks PTI, and the tumor nodules were manually counted. Interestingly, unlike with the i.p. prophylaxis, the route of administration was insignificant, and all administration routes considerably depreciated the number of metastatic tumor nodules in the lung (FIGS. 5b, 5c). Compared to PBS, there was a noticeable 3.3-, 3.8-, and 4.8-fold decrease in the number of tumor nodules following s.c., i.p., and i.v. injection, respectively. Qualitatively, the difference is also appreciable as seen in FIG. 5c

[0245] CPMV is unique in its ability to instigate an immune response - compared to other plant viruses, bacteriophages, and some mammalian viruses, it performs significantly better as a cancer immunotherapy

[0029] . Therefore, the ability of CPMV was compared head-to-head against other adjuvants to see whether the prophylactic ability was unique to CPMV (FIGS. 5d-5g). diABZI is a stimulator of interferon genes (STING) agonist while imiquimod is a TLR7 agonist l58-59l. As a negative control, the cowpea chlorotic mottle virus (CCMV), which has the same size and shape, is negatively charged, and is grown in the same plants as CPMV but is non-immunomodulatory in the context of cancer immunotherapy

[0025] , was utilized. The reagents were compared in both an i.p. and i.v. setting, and with both injection routes, CPMV fared better in reducing tumor nodules compared to any other adjuvant tested. When injectedi.v., CPMV decreased tumor nodule counts compared to PBS, CCMV, diABZI, and imiquimod by 2.7-, 2.6-, 2.7-, and 2.2-fold, respectively (FIGS. 5d, 5e). When injected i.p., CPMV improved tumor nodule rejection with 3.4-, 2.7-, 2-, and 3.2-fold reductions compared to PBS, CCMV, diABZI, and imiquimod, respectively (FIGS. 5f, 5g). Unlike diABZI and imiquimod, which are small molecule agonists, CPMV may not suffer from immediate washout and rapid degradation thereby prolonging its immunostimulatory effects. Additionally, diABZI and imiquimod display significant immunotoxicity as systemically administered agents while CPMV has been injected at doses up to 100 mg kg'1with no observed toxi cities

[0060] . In the clinic, one might expect that longer therapeutic windows such as with CPMV may be preferred to prolong protection against metastatic outgrowths with the least number of doses.

[0246] The immunostimulatory nature of the reagents was also compared using the RAW264.7-Luc NFKB reporter line. Data are in agreement and support the in vivo study; CPMV significantly outperformed the other reagents in the stimulation of the RAW264.7 macrophages (FIG. 19). The luminescent output of CPMV was 566.7 compared to 338, 399.7, 432, and 394.7 of media, CCMV, diABZi, and imiquimod, respectively. Therefore, while the other reagents could induce NFKB signaling compared to PBS, it was significantly lower than that of CPMV. The triple-pronged TLR activation of CPMV along with its multivalent nature likely leads to avidity effects and enhanced presentation of T helper epitopes

[0061] ; the nanoparticle character may explain the unmatched efficacy of CPMV compared to other adjuvants.

[0247] CPMV prophylaxis efficacy in a 4T1-Luc TNBC lung metastatic tumor model

[0248] To verify that CPMV is efficacious in a different tumor model, a 4T1-Luc TNBC cell line was utilized in BALB / C mice. Following i.v. treatment using CPMV, mice were challenged with 4T1-Luc cells (100,000 cells, i.v.) Disease burden was followed by luminescent imaging every 2 days. After 3 weeks, the lungs were harvested and the tumor nodules were manually counted (FIG. 5h). In the CPMV mice, the lungs displayed little to no tumor development, while in the PBS mice, the luminescence increased rapidly (FIG. 5i) indicating tumor development, which led to weight loss (FIG. 5j). By day 20 PTI, the luminescent counts within the PBS mice had reached 468.3 while the CPMV mice were 18.2,a 25.7-fold decrease. Representative imaging of mice is found in FIG. 5k. Manual counting of the tumor nodules from the harvested lungs also demonstrated that CPMV adjuvant therapy decreased the average number of nodules from 104 to 24, a 4.3-fold decrease (FIGS. 51, 5m) Even qualitatively, the differences are pronounced as can be seen by the red arrows in the first two lungs, which are pointing to areas of tumor growth (FIG. 5m).

[0249] CPMV elicits significant systemic immunostimulation causing the reduction of tumor nodules

[0250] The mechanism of action of the CPMV-induced reduction of tumor nodules was further characterized by MSD analysis of blood samples collected 1-day after i.v. CPMV treatment (FIGS. 6a-6c, FIG. 20). CPMV administration led to significant increases in immunostimulatory cytokines and chemokines within the plasma of the mice. Notably, there were significant increases in IFNy, IFNa, and TNF-a with 48.1-, 1.8-, and 2.5-fold increases, respectively. With the chemokines, CPMV injection led to significant increases in all tested chemokines (FIG. 6b). A heat map showing the fold-changes of the cytokines / chemokines following CPMV i.v. injection compared to PBS is shown in FIG. 6c. Overall this data was comparable to the observations made after i.p. administration (see FIG. 4).

[0251] Next, changes in immune cell populations within the blood were assayed; here, Applicant considered multiple routes of administration: s.c., i.v., and i.p. (FIG. 6d). While only i.p. administration leads to an increase of immune cells within the i.p. space (FIG. 4), trends in immune profiles within the blood are distinct, but overall increased independent of route of administration. For instance, neutrophil populations were increased significantly following s.c. injection, but not i.p. or i.v. On the other hand, NK cells were only increased by i.p. and i.v. injection, but not s.c. Notably, the M1 / M2 ratio is increased independent of injection route. Overall, the panels indicate significant activation of immune cells in blood; in stark contrast, flow cytometry of the spleens or lungs of the mice only showcased minimal differences in immune cell populations (FIG. 21). This data suggests that CPMV may assert efficacy through the clearance and processing of circulating tumor cells (CTCs) within the blood. The data also mirrors the metastatic tumor prophylaxis data (FIGS. 5d-5g) in that CPMV is effective regardless of administration route. However, for different indications,different administration routes may be more effective, and future studies should probe how the differences that are seen in the recruited cell type could affect tumor clearance.

[0252] Surgical removal of primary tumors followed by CPMV adjuvant therapy

[0253] To test the efficacy of CPMV in a more clinically relevant model, a surgery study was performed. Female BALB / C mice were inoculated with 4T1-Luc (100,000 cells, s.c.) followed by surgical removal of the tumor after 2 weeks (FIG. 7a). The following day, the mice were injected i.v. with CPMV as adjuvant therapy, and mice were monitored by IVIS for signs of metastasis and recurrence. Compared to PBS mice, CPMV administration led to a significant improvement in survival with a median survival of 36 days for CPMV and 29 days for PBS (FIG. 7b). CPMV prophylaxis also improved the number of long-term survivors by 400%. The luminescence of the 4T1-Luc tumors was measured, which demonstrated that the adjuvant therapy decreased the growth of the tumors albeit insignificantly (FIG. 7c). The luminescent imaging of the mice can be found in FIG. 7d, which highlights that CPMV adjuvant therapy decreased rates of tumor growth both in terms of recurrence and metastasis.

[0254] Together, data indicate that the CPMV prophylactic adjuvant therapy has broad efficacy and is mouse strain independent (C57BL / 6J and BALB / C mice), applicable to various tumor types (colon, ovarian, melanoma, and TNBC), tumor site independent (i.p. metastases, lung metastases, and s.c. tumors), able to generate systemic immune memory (both i.p. and s.c. re-challenge and confirmed through ELISPOT) and clinically relevant (predisseminated i.p. disease, surgical removal of primary tumors).

[0255] Conclusion

[0256] Metastatic cancers contribute the highest percentage of cancer-related deaths, and current treatment options for metastatic cancers remain limited. Adjuvant therapy has greatly improved outcomes in breast and colon cancer, and immunotherapies may play a significant role in future adjuvant treatment. CPMV is an immunostimulatory adjuvant that when injected intratumorally leads to recruitment of immunostimulatory cells, release of antitumorcytokines, and potent tumor cell death. Applicant shows here that CPMV also demonstrates significant utility in the adjuvant treatment and rejection of 4 metastatic models of murine cancer. When injected i.p. or i.v., CPMV causes the recruitment of a wide range of immune cells into either the i.p. space or blood, respectively, including DCs, neutrophils, and NK cells, which go on to release antitumor cytokines such as IFNy, IFNa, TNF-a, and IL-12. This leads to the rejection of i.p. and i.v. metastases leading to long-term survivors and building immune memory to protect against re-challenge. The broad applicability and effectiveness of CPMV adjuvant therapy across a range of cancer types paired with the long- lasting efficacy off of a single injection confers CPMV with the potential to be used as an adjuvant treatment in metastatic cancers.

[0257] Example 2

[0258] Trained Immunity

[0259] CPMV primes the innate immune system and weeks after priming, innate immune cells reject metastatic cancer (innate memory from training), and the processing of the cancer cells and associated antigens leads to systemic, durable, and adaptive antitumor immunity (long-term adaptive memory).

[0260] As disclosed herein, Applicant discovered that systemic CPMV administration prior to tumor challenge protects mice from onset of tumor growth (FIG. 3F). Applicant performed longitudinal analysis using a metastatic mouse model of colon cancer with intraperitoneal (IP) dissemination of CT26 metastases in BALB / c mice. CPMV (0.2 mg, IP) was administered 7, 14, 21, and 28 days prior to tumor challenge: CPMV immunoprevention was durable and animals were protected from settling and dissemination of IP metastases from IP CT26 colon cancer challenge - the immunoprevention effect was maintained over a 14-day window; this was accompanied with significantly extended survival (FIG. 3F). Efficacy of the CPMV ‘immunoprevention strategy’ was also demonstrated in mouse models of IP disseminated ovarian cancer (ID8-Defb29 / Vegf-A cells, data not shown), lung metastasis from intravenous (IV) challenge with melanoma cells (B16F10, FIGS. 5B-5C) or breast cancer cells (4T1, not shown). CPMV immunoprevention against systemic B16F10 tumor challenge was potent and independent of the route of administration - IP, IV,subcutaneous administration of CPMV primed an immunoprevention effect and prevented lung metastasis to form (FIGS. 5B-5C). Applicant compared efficacy of the immunomodulatory agents CPMV head-to-head against other adjuvants and CPMV demonstrated superior protection against tumor challenge compared to diABZI (STING agonist), and imiquimod (TLR7 agonist), and CCMV (cowpea chlorotic mottle virus) (FIGS. 5D-5E)

[0261] Collectively the results presented for example in FIGS. 3 and 5 indicate that CPMV acts as a training agent and induces heterologous protection against tumor challenge. Heterologous protection (i.e. immunity) is the induction or modification of a subject’s immune response upon exposure to a pathogen.

[0082] , The innate immune priming is durable and lasts for weeks after CPMV exposure when innate cells would have returned to a homeostasis state; therefore, data are consistent with in-duction of trained immunity. As used herein, “trained immunity” refers to a phenomenon of ‘innate immune memory’ accompanied by epigenetic changes and metabolic rewiring of innate immune cells. Indeed, single cell sequencing analysis of human PBMCs after CPMV exposure indicates stimulation of interferon signaling pathways along with metabolic changes, and epigenetic rewiring (FIG. 22).

[0262] Hallmarks of trained immunity are: epigenetic changes in promotors of pro- inflammatory genes of the innate immune cells and metabolic rewiring of the innate immune cells. The metabolic rewiring of innate immune cells or their progenitors regulates their plasticity and epigenomic reprogramming; for example, aerobic glycolysis is a hallmark of P- glucan-trained monocytes mediated by the AKT-mTOR-HIF la pathway [75, 76], Main epigenetic events during induction of trained immunity are the chromosomal reorganization on the level of topologically associated domains (TADs), induction of long noncoding RNA (IncRNA) activity, histone modifications and chromatin accessibility, and DNA (de)methylation [77-79], Indeed, single cell sequencing analysis of human PBMCs after 24- hour CPMV exposure indicates stimulation of interferon signaling pathways, and importantly metabolic changes and epigenetic rewiring (consistent with the upregulation of several long non-coding RNAs (IncRNAs) (FIG. 22). Trained immunity involves myeloid cells, natural killer (NK) cells, innate lymphoid cells (ILCs) and germline encoded recognition and effectormolecules (e.g. pattern recognition receptors, cytokines) - and as such is distinct from adaptative immune memory. It is important to note the role of neutrophils and NK cells in innate immunity (amongst innate lymphoid cells and non-immune cells)

[0080] , and neutrophils and NK cells play a significant role in immunomodulation mediated by CPMV [14, 81],

[0263] Numbered EmbodimentsAdditional embodiments include the following numbered embodiments:1. A method for one or more of: inhibiting, delaying, slowing down, or preventing relapse of cancer in a subject in need thereof, comprising administering to the subject a plant viral nanoparticle, thereby delaying, slowing down, or preventing the relapse of the cancer in the subject, optionally wherein the plant viral nanoparticle is administered after the subject has received a primary therapy.2. A method for one or more of: extending progression free survival, increasing overall survival, or reducing cancer metastasis in a subject in need thereof comprising administering to the subject a plant viral nanoparticle, thereby extending progression free survival, increasing overall survival, or reducing cancer metastasis in the subject, optionally wherein the plant viral nanoparticle is administered after the subject has received a primary therapy.3. A method for treating cancer in a subject in need thereof, comprising administering to the subject a plant viral nanoparticle, thereby treating the cancer in the subject, optionally wherein the plant viral nanoparticle is administered after the subject has received a primary therapy.4. A method for one or more of: triggering or enhancing one or more of the following in a subject in need thereof: an immune response, immunostimulatory cytokines, increase in the ratio of Ml (antitumor) to M2 (protumor) macrophages, natural killer cells (NK cells), Toll Like Receptor (TLR)s 2, 4 and 7, chemokines, or suppressor cells, comprising administering to the subject a plant viral nanoparticle, thereby triggering or enhancing one or more of the following in the subject in need thereof: an immune response, immunostimulatory cytokines, increase in the ratio of Ml (antitumor) to M2 (protumor) macrophages, natural killer cells(NK cells), Toll Like Receptor (TLR)s 2, 4 and 7, chemokines, or myeloid-derived suppressor cells.5. The method of embodiment 4, wherein the immunostimulatory cytokines are selected from at least one of IFNy, IFNa, TNF-a, IL-12p70, IL-12 / IL-23p40, IL-12 / IL-23p4, IL-15, GM-CSF, IL-2, and IL-17.6. The method of embodiment 4, wherein the chemokines are selected from at least one of IP-10, MIP-la, MIP-lp, RANTES, MCP-1, and KC / GRO.7. The method of embodiment 4, wherein the myeloid-derived suppressor cells are selected from monocytic myeloid-derived suppressor cells and granulocytic myeloid-derived suppressor cells.8. A method of priming trained immunity in a subject, comprising administering the subject a plant viral nanoparticle, thereby providing heterologous protection from disease, optionally wherein the plant viral nanoparticle is a CPMV nanoparticle, optionally wherein the disease is a cancer.9. The method of any one of embodiments 1-8, wherein the plant viral nanoparticle is selected from a plant Picomavirus nanoparticle, a Comovirus nanoparticle, a Cowpea mosaic viral nanoparticle, a Broad bean wilt virus 1 nanoparticle, a Tobacco ringspot viral nanoparticle, a cowpea severe mosaic viral nanoparticle, a bean pod mottle viral nanoparticle or a rice tungro spherical viral nanoparticle.10. The method of any of embodiments 1-9, wherein the plant viral nanoparticle is a CPMV nanoparticle, and optionally wherein the CPMV nanoparticle is non-replicating and noninfectious in the subject.11. The method of any of embodiments 1-7, 9 or 10, wherein the cancer is selected from a solid tumor, a carcinoma, a sarcoma, or a blood cancer.12. The method of any of embodiments 1-10, wherein the cancer is a primary cancer or a metastatic cancer.13. The method of any of embodiments 1-7 or 9-12, wherein the cancer is selected from a blood cancer, a carcinoma, a sarcoma, a lung cancer, an intraperitoneal cancer, a colorectal cancer, a colon cancer, an ovarian cancer, a melanoma, or a breast cancer.14. The method of any one of embodiments 1-7 or 9-13, wherein the subject is in remission.15. The method of any one of embodiments 1-7 or 9-14, further comprising isolating a cancer cell from the subject prior to the administration.16. The method of any one of embodiments 1-7 or 9-14, wherein the administration is subsequent to tumor resection.17. The method of any one of embodiments 1-7 or 9-16, wherein the cancer in the subject is clinically undetectable.18. The method of any one of embodiments 1-7 or 9-17, wherein the subject is a cancer patient who has been treated by one or more of: an ablative therapy, a chemotherapy, a radiation therapy, an immune checkpoint blockade therapy, or another anti-cancer therapy.19. The method of any one of embodiments 1-7 or 9-18, further comprising treating the subject with one or more of: an ablative therapy, a chemotherapy, a radiation therapy, an immune checkpoint blockade therapy, or another anti-cancer therapy subsequent to administration of the plant viral nanoparticle.20. The method of any one of embodiments 1-19, wherein the plant viral nanoparticle is administered as a second line therapy, a third line therapy or a fourth line therapy.21. The method of any one of embodiments 1-20, wherein the plant viral nanoparticle is administered locally or systemically.22. The method of any one of embodiments 1-21, wherein the plant viral nanoparticle is administered to the patient by oral, intratumoral, nasal, topical, transdermal, intranasal, vaginal, rectal, subcutaneous intravenous, intravenous, intraarterial, intramuscular,intraosseous, intraperitoneal, intraocular, subconjunctival, sub-Tenon’s, intravitreal, retrobulbar, intracameral, epidural or intrathecal administration.23. A kit for use in a method of any one of embodiments 1-22, comprising a plant viral nanoparticle and an optional instruction for use.24. The method of any of embodiments 1-22, wherein the plant viral nanoparticle is administered in combination with a carrier.25. The method of embodiment 24, wherein the carrier is a pharmaceutically acceptable carrier.26. A method for treating or preventing cancer in a subject in need thereof, comprising administering to the subject a plant viral nanoparticle, wherein the plant viral nanoparticle is a CPMV nanoparticle.27. The method of embodiment 26, wherein the cancer is a lung cancer or a colon cancer.28. The method of embodiment 26, wherein the plant viral nanoparticle is administered intraperitoneally, intravenously, or subcutaneously.29. The method of embodiment 26, wherein the plant viral nanoparticle is administered intratum orally.Equivalents

[0264] 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 technology belongs.

[0265] The present technology illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising,” “including,” “containing,” etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shownand described or portions thereof, but it is recognized that various modifications are possible within the scope of the present technology claimed.

[0266] Thus, it should be understood that the materials, methods, and examples provided here are representative of preferred aspects, are exemplary, and are not intended as limitations on the scope of the present technology.

[0267] It should be understood that although the present invention has been specifically disclosed by certain aspects, embodiments, and optional features, modification, improvement and variation of such aspects, embodiments, and optional features can be resorted to by those skilled in the art, and that such modifications, improvements and variations are considered to be within the scope of this disclosure.

[0268] The present technology has been described broadly and generically herein. Each of the narrower species and sub-generic groupings falling within the generic disclosure also form part of the present technology. This includes the generic description of the present technology with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0269] In addition, where features or aspects of the present technology are described in terms of Markush groups, those skilled in the art will recognize that the present technology is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0270] All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entireties, to the same extent as if each were incorporated by reference individually. In case of conflict, the present specification, including definitions, will control.

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Claims

What is claimed is:

1. A method for one or more of: inhibiting, delaying, slowing down, or preventing relapse of cancer in a subject in need thereof, comprising administering to the subject a plant viral nanoparticle, thereby delaying, slowing down, or preventing the relapse of the cancer in the subject, optionally wherein the plant viral nanoparticle is administered after the subject has received a primary therapy.

2. A method for one or more of: extending progression free survival, increasing overall survival, or reducing cancer metastasis in a subject in need thereof comprising administering to the subject a plant viral nanoparticle, thereby extending progression free survival, increasing overall survival, or reducing cancer metastasis in the subject, optionally wherein the plant viral nanoparticle is administered after the subject has received a primary therapy.

3. A method for treating cancer in a subject in need thereof, comprising administering to the subject a plant viral nanoparticle, thereby treating the cancer in the subject, optionally wherein the plant viral nanoparticle is administered after the subject has received a primary therapy.

4. A method for one or more of: triggering or enhancing one or more of the following in a subject in need thereof: an immune response, immunostimulatory cytokines, increase in the ratio of Ml (antitumor) to M2 (protumor) macrophages, natural killer cells (NK cells), Toll Like Receptor (TLR)s 2, 4 and 7, chemokines, or suppressor cells, comprising administering to the subject a plant viral nanoparticle, thereby triggering or enhancing one or more of the following in the subject in need thereof: an immune response, immunostimulatory cytokines, increase in the ratio of Ml (antitumor) to M2 (protumor) macrophages, natural killer cells (NK cells), Toll Like Receptor (TLR)s 2, 4 and 7, chemokines, or myeloid-derived suppressor cells, optionally wherein the immunostimulatory cytokines are selected from at least one of IFNy, IFNa, TNF-a, IL-12 / p70, IL-12 / IL-23p40, IL-12 / IL-23p4, IL-15, GM- CSF, IL-2, and IL-17, further optionally wherein, the chemokines are selected from at least one of IP- 10, MIP-la, MIP-lp, RANTES, MCP-1, and KC / GRO, and further optionallywherein the myeloid-derived suppressor cells are selected from monocytic myeloid-derived suppressor cells and granulocytic myeloid-derived suppressor cells.

5. A method of priming trained immunity in a subject, comprising administering the subject a plant viral nanoparticle, thereby providing heterologous protection from disease, optionally wherein the plant viral nanoparticle is a CPMV nanoparticle, optionally wherein the disease is a cancer.

6. The method of any one of claims 1-5, wherein the plant viral nanoparticle is selected from a plant Picornavirus nanoparticle, a Comovirus nanoparticle, a Cowpea mosaic viral nanoparticle, a Broad bean wilt virus 1 nanoparticle, a Tobacco ringspot viral nanoparticle, a cowpea severe mosaic viral nanoparticle, a bean pod mottle viral nanoparticle or a rice tungro spherical viral nanoparticle.

7. The method of any of claims 1-5, wherein the plant viral nanoparticle is a CPMV nanoparticle, optionally wherein the CPMV nanoparticle is non-replicating and noninfectious in the subj ect.

8. The method of any of claims 1-4 or 6-7 wherein the cancer is a primary cancer or a metastatic cancer.

9. The method of any of claims 1-4 or 6-8 wherein the cancer is selected from a blood cancer, a carcinoma, a sarcoma, a lung cancer, an intraperitoneal cancer, a colorectal cancer, a colon cancer, an ovarian cancer, a melanoma, or a breast cancer.

10. The method of any one of claims 1-4 or 6-9, wherein the subject is in remission.

11. The method of any one of claims 1-4 or 6-10, further comprising isolating a cancer cell from the subject prior to the administration.

12. The method of any one of claims 1-4 or 6-11, wherein the administration is subsequent to tumor resection.

13. The method of any one of claims 1-4 or 6-12, wherein the cancer in the subject is clinically undetectable.

14. The method of any one of claims 1-4 or 6-13, wherein the subject is a cancer patient who has been treated by one or more of: an ablative therapy, a chemotherapy, a radiation therapy, an immune checkpoint blockade therapy, or another anti-cancer therapy.

15. The method of any one of claims 1-4 or 6-14, further comprising treating the subject with one or more of: an ablative therapy, a chemotherapy, a radiation therapy, an immune checkpoint blockade therapy, or another anti-cancer therapy subsequent to administration of the plant viral nanoparticle.

16. The method of any one of claims 1-15, wherein the plant viral nanoparticle is administered as a second line therapy, a third line therapy or a fourth line therapy.

17. The method of any one of claims 1-16, wherein the plant viral nanoparticle is administered locally or systemically.

18. The method of any one of claims 1-17, wherein the plant viral nanoparticle is administered to the patient by oral, intratumoral, nasal, topical, transdermal, intranasal, vaginal, rectal, subcutaneous intravenous, intravenous, intraarterial, intramuscular, intraosseous, intraperitoneal, intraocular, subconjunctival, sub-Tenon’s, intravitreal, retrobulbar, intracameral, epidural or intrathecal administration.

19. A kit for use in a method of any one of claims 1-18, comprising a plant viral nanoparticle and an optional instruction for use.

20. The method of any of claims 1-18, wherein the plant viral nanoparticle is administered in combination with a carrier, optionally wherein the carrier is a pharmaceutically acceptable carrier.

21. A method for treating or preventing cancer in a subject in need thereof, comprising administering to the subject a plant viral nanoparticle, wherein the plant viral nanoparticle is a CPMV nanoparticle, optionally wherein the cancer is a lung cancer or a colon cancer, optionally wherein the. the plant viral nanoparticle is administered intraperitoneally, intravenously, subcutaneously, or intratum orally.

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