Intratumoral immunotherapy with il-12 combined with CCL3

Abstract

The invention relates to methods of treating a tumor in a subject by administering to the subject the following active agents: (a) interleukin- 12 (IL-12), a nucleotide expressing IL-12, or combinations thereof; and (b) c-c motif chemokine ligand 3 (CCL3), a nucleotide expressing CCL3, or combinations thereof. The invention also relates to therapeutic compositions that include the following active agents; (a) IL-12, a nucleotide expressing IL-12, or combinations thereof; and (b) CCL3, a nucleotide expressing CCL3, or combinations thereof.

Description

PCT Application Attorney Docket No. AF60544.P059WODartmouth Ref. No. 2025-009TITLEINTRA TUMORAL IMMUNOTHERAPY WITH IL- 12 COMBINED WITH CCL3SEQUENCE DISCLOSURE STATEMENT

[0001] Pursuant to 37 C.F.R. § 1.834, Applicant has submitted a sequence listing in XML format (“Sequence Listing”). The name of the file containing the Sequence Listing is “AF60544.P059WO.xml”. The date of the creation of the Sequence Listing is October 13. 2025. The size of the Sequence Listing is 12,000 bytes. Applicant hereby incorporates by reference the material in the Sequence Listing.CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 706,531, filed on October 11, 2024. The entirety of the aforementioned application is incorporated herein by reference.BACKGROUND

[0003] A need exists for more effective therapeutic methods and compositions for eradicating tumors. Numerous embodiments of the present disclosure aim to address the aforementioned need.SUMMARY

[0004] In some embodiments, the present disclosure pertains to methods of treating a tumor in a subject. In some embodiments, such methods include administering to the subject the following active agents: (a) interleukin- 12 (IL-12), a nucleotide expressing IL-12, or combinations thereof; and (b) c-c motif chemokine ligand 3 (CCL3), a nucleotide expressing CCL3, or combinations thereof. Additional embodiments of the present disclosure pertain to therapeutic compositions that include the following active agents: (a) IL- 12, a nucleotide expressing IL- 12, or combinations thereof; and (b) CCL3, a nucleotide expressing CCL3, or combinations thereof.PCT Application Attorney Docket No. AF60544.P059WODartmouth Ref. No. 2025-009

[0005] The methods and therapeutic compositions of the present disclosure may be utilized to treat various tumors. For instance, in some embodiments, the tumor is a solid tumor. In some embodiments, the methods and therapeutic compositions of the present disclosure may be used to treat or prevent a cancer in a subject. In some embodiments, the cancer includes, without limitation, melanoma, breast cancer, lung cancer, pancreatic cancer, colon cancer, metastatic cancer, non-metastatic cancer, or combinations thereof.

[0006] In some embodiments, the methods and therapeutic compositions of the present disclosure may be utilized with additional treatments. For instance, in some embodiments, the methods and therapeutic compositions of the present disclosure may be used as a component of an intratumoral immunotherapy.BRIEF DESCRIPTION OF THE DRAWINGS

[0007] FIGS. 1A-1C illustrate that IL- 12 and CCL3 therapy eradicates treated tumors and elicits systemic responses in Bl 6F 10 treated mice.

[0008] FIGS. 2A-2C provide additional data illustrating that IL- 12 and CCL3 therapy eradicates treated tumors and elicits systemic responses in Bl 6F 10 treated mice.

[0009] FIGS. 3A-3C illustrate that IL- 12 and CCL3 therapy eradicate treated tumors and elicits systemic responses in MC38 treated mice.

[0010] FIGS 4A-4C illustrate that IL- 12 and CCL3 therapy is not totally dependent on crosspresentation by CD8 T cells.

[0011] FIGS. 5A-5B illustrate that IL- 12 and CCL3 create a local microenvironment that promotes IFN-driven activation of cytotoxic effectors cells, thereby shifting the tumor microenvironment (TME) away from neutrophil and myeloid- derived suppressor cells (MDSC)-induced angiogenesis.PCT Application Attorney Docket No. AF60544.P059WODartmouth Ref. No. 2025-009DETAILED DESCRIPTION

[0012] It is to be understood that both the foregoing general description and the following detailed description are illustrative and explanatory, and are not restrictive of the subject matter, as claimed. In this application, the use of the singular includes the plural, the word “a” or “an” means “at least one”, and the use of “or” means “and / or”, unless specifically stated otherwise. Furthermore, the use of the term “including”, as well as other forms, such as “includes” and “included”, is not limiting. Also, terms such as “element” or “component” encompass both elements or components comprising one unit and elements or components that include more than one unit unless specifically stated otherwise.

[0013] The section headings used herein are for organizational purposes and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including, but not limited to. patents, patent applications, articles, books, and treatises, are hereby expressly incorporated herein by reference in their entirety for any purpose. In the event that one or more of the incorporated literature and similar materials defines a term in a manner that contradicts the definition of that term in this application, this application controls.

[0014] Current therapeutic methods and compositions for eradicating tumors have numerous limitations. For instance, intratumoral immunotherapy is a therapeutic approach which strives to treat cancer with immuno-stimulatory agents injected locally. Intratumoral immunotherapy has the ability to not only stimulate local anti-tumor immunity to shrink the primary tumor but can also stimulate systemic anti-tumor immunity that can impact tumors at distant sites (abscopal effect).

[0015] Clinical studies have shown the success of intratumoral immunotherapy with many immune stimulatory compounds, including talimogene laherparepvec (T-VEC) and interleukin- 12 (IL-12). IL- 12 is a pro-inflammatory cytokine involved in the activation of T-cells and natural killer (NK) cells. Such activation leads to the increased production of interferon gamma (IFNy).

[0016] Previous data has shown an improved efficacy of IL- 12 anti-tumor effect in different solid tumors when combined with CD 154 (CD40L). However, a need exists for therapeutics with more enhanced antitumor effects.

[0017] In sum, a need exists for more effective therapeutic methods and compositions for eradicating tumors. Numerous embodiments of the present disclosure aim to address the aforementioned need.PCT Application Attorney Docket No. AF60544.P059WODartmouth Ref. No. 2025-009

[0018] In some embodiments, the present disclosure pertains to methods of treating a tumor in a subject. In some embodiments, such methods include administering to the subject the following active agents: (a) interleukin- 12 (IL-12), a nucleotide expressing IL-12, or combinations thereof; and (b) c-c motif chemokine ligand 3 (CCL3), a nucleotide expressing CCL3. or combinations thereof. Additional embodiments of the present disclosure pertain to therapeutic compositions that include the following active agents: (a) IL-12, a nucleotide expressing IL-12, or combinations thereof; and (b) CCL3, a nucleotide expressing CCL3, or combinations thereof. As set forth in more detail herein, the methods and therapeutic compositions of the present disclosure can include numerous embodiments.

[0019] Modes of administration

[0020] The methods of the present disclosure may be utilized to administer active agents in various manners. For instance, in some embodiments, the administration occurs by a method that includes, without limitation, intratumoral administration, oral administration, inhalation, subcutaneous administration, intravenous administration, intraperitoneal administration, intramuscular administration, direct injection into the tumor, intrathecal injection, intra-articular administration, topical administration, central administration, peripheral administration, aerosol-based administration, nasal administration, transmucosal administration, transdermal administration, parenteral administration, or combinations thereof.

[0021] In some embodiments, the administration includes intratumoral administration. In some embodiments, the intratumoral administration includes intratumoral electroporation.

[0022] Active agents

[0023] The methods of the present disclosure may be utilized to administer various active agents to subjects. For instance, in some embodiments, the active agents include (a) IL-12; and (b) CCL3. In some embodiments, the active agents include (a) a nucleotide expressing IL-12; and (b) a nucleotide expressing CCL3. In some embodiments, the active agents include (a) a nucleotide expressing IL-12; and (b) CCL3. In some embodiments, the active agents include (a) IL- 12: and (b) a nucleotide expressing CCL3.

[0024] In some embodiments, active agents may include a nucleotide expressing IL- 12, a nucleotide expressing CCL3, or combinations thereof. In some of such embodiments, the nucleotides may include, without limitation, DNA, RNA, mRNA, or combinations thereof. In some embodiments, the nucleotides may include DNA. In some embodiments, the DNA may be part of one or more plasmids.PCT Application Attorney Docket No. AF60544.P059WODartmouth Ref. No. 2025-009

[0025] In some embodiments, the active agents include IL- 12. In some embodiments, the active agents include IL-12A. In some embodiments, IL- 12 includes SEQ ID NO: 1. In some embodiments, IL- 12 includes a sequence with at least 65% sequence identity to SEQ ID NO: 1. In some embodiments, IL-12 includes a sequence with at least 70% sequence identity to SEQ ID NO: 1. In some embodiments, IL- 12 includes a sequence with at least 75% sequence identity to SEQ ID NO: 1. In some embodiments. IL- 12 includes a sequence with at least 80% sequence identity to SEQ ID NO: 1. In some embodiments, IL- 12 includes a sequence with at least 85% sequence identity to SEQ ID NO: 1. In some embodiments, IL- 12 includes a sequence with at least 90% sequence identity to SEQ ID NO: 1. In some embodiments, IL-12 includes a sequence with at least 95% sequence identity to SEQ ID NO: 1. In some embodiments, IL- 12 includes a sequence with at least 99% sequence identity to SEQ ID NO: 1.

[0026] In some embodiments, the active agents include a nucleotide sequence expressing IL- 12. In some embodiments, the active agents include a nucleotide sequence expressing IL- 12 A. In some embodiments, the nucleotide sequence expressing IL-12 includes SEQ ID NO: 2. In some embodiments, the nucleotide sequence expressing IL- 12 includes a sequence with at least 65% sequence identity to SEQ ID NO: 2. In some embodiments, the nucleotide sequence expressing IL- 12 includes a sequence with at least 70% sequence identity to SEQ ID NO: 2. In some embodiments, the nucleotide sequence expressing IL- 12 includes a sequence with at least 75% sequence identity to SEQ ID NO: 2. In some embodiments, the nucleotide sequence expressing IL- 12 includes a sequence with at least 80% sequence identity to SEQ ID NO: 2. In some embodiments, the nucleotide sequence expressing IL-12 includes a sequence with at least 85% sequence identity to SEQ ID NO: 2. In some embodiments, the nucleotide sequence expressing IL- 12 includes a sequence with at least 90% sequence identity to SEQ ID NO: 2. In some embodiments, the nucleotide sequence expressing IL- 12 includes a sequence with at least 95% sequence identity to SEQ ID NO: 2. In some embodiments, the nucleotide sequence expressing IL- 12 includes a sequence with at least 99% sequence identity to SEQ ID NO: 2.PCT Application Attorney Docket No. AF60544.P059WODartmouth Ref. No. 2025-009

[0027] In some embodiments, the active agents include IL-12B. In some embodiments, IL- 12 includes SEQ ID NO: 3. In some embodiments, IL-12 includes a sequence with at least 65% sequence identity to SEQ ID NO: 3. In some embodiments, IL-12 includes a sequence with at least 70% sequence identity to SEQ ID NO: 3. In some embodiments, IL- 12 includes a sequence with at least 75% sequence identity to SEQ ID NO: 3. In some embodiments, IL- 12 includes a sequence with at least 80% sequence identity to SEQ ID NO: 3. In some embodiments, IL-12 includes a sequence with at least 85% sequence identity to SEQ ID NO: 3. In some embodiments, IL-12 includes a sequence with at least 90% sequence identity to SEQ ID NO: 3. In some embodiments, IL- 12 includes a sequence with at least 95% sequence identity to SEQ ID NO: 3. In some embodiments. IL-12 includes a sequence with at least 99% sequence identity to SEQ ID NO: 3.

[0028] In some embodiments, the active agents include a nucleotide sequence expressing IL-12B. In some embodiments, the nucleotide sequence expressing IL- 12 includes SEQ ID NO: 4. In some embodiments, the nucleotide sequence expressing IL-12 includes a sequence with at least 65% sequence identity to SEQ ID NO: 4. In some embodiments, the nucleotide sequence expressing IL- 12 includes a sequence with at least 70% sequence identity to SEQ ID NO: 4. In some embodiments, the nucleotide sequence expressing IL- 12 includes a sequence with at least 75% sequence identity to SEQ ID NO: 4. In some embodiments, the nucleotide sequence expressing IL- 12 includes a sequence with at least 80% sequence identity to SEQ ID NO: 4. In some embodiments, the nucleotide sequence expressing IL-12 includes a sequence with at least 85% sequence identity to SEQ ID NO: 4. In some embodiments, the nucleotide sequence expressing IL- 12 includes a sequence with at least 90% sequence identity to SEQ ID NO: 4. In some embodiments, the nucleotide sequence expressing IL- 12 includes a sequence with at least 95% sequence identity to SEQ ID NO: 4. In some embodiments, the nucleotide sequence expressing IL- 12 includes a sequence with at least 99% sequence identity to SEQ ID NO: 4.PCT Application Attorney Docket No. AF60544.P059WODartmouth Ref. No. 2025-009

[0029] In some embodiments, the active agents include CCL3. In some embodiments, CCL3 includes SEQ ID NO: 5. In some embodiments, CCL3 includes a sequence with at least 65% sequence identity to SEQ ID NO: 5. In some embodiments, CCL3 includes a sequence with at least 70% sequence identity to SEQ ID NO: 5. In some embodiments. CCL3 includes a sequence with at least 75% sequence identity to SEQ ID NO: 5. In some embodiments, CCL3 includes a sequence with at least 80% sequence identity to SEQ ID NO: 5. In some embodiments, CCL3 includes a sequence with at least 85% sequence identity to SEQ ID NO: 5. In some embodiments, CCL3 includes a sequence with at least 90% sequence identity to SEQ ID NO: 5. In some embodiments, CCL3 includes a sequence with at least 95% sequence identity to SEQ ID NO: 5. In some embodiments, CCL3 includes a sequence with at least 99% sequence identity to SEQ ID NO: 5.

[0030] In some embodiments, the active agents include a nucleotide sequence expressing CCL3. In some embodiments, the nucleotide sequence expressing CCL3 includes SEQ ID NO: 6. In some embodiments, the nucleotide sequence expressing CCL3 includes a sequence with at least 65% sequence identity to SEQ ID NO: 6. In some embodiments, the nucleotide sequence expressing CCL3 includes a sequence with at least 70% sequence identity to SEQ ID NO: 6. In some embodiments, the nucleotide sequence expressing CCL3 includes a sequence with at least 75% sequence identity to SEQ ID NO: 6. In some embodiments, the nucleotide sequence expressing CCL3 includes a sequence with at least 80% sequence identity to SEQ ID NO: 6. In some embodiments, the nucleotide sequence expressing CCL3 includes a sequence with at least 85% sequence identity to SEQ ID NO: 6. In some embodiments, the nucleotide sequence expressing CCL3 includes a sequence with at least 90% sequence identity to SEQ ID NO: 6. In some embodiments, the nucleotide sequence expressing CCL3 includes a sequence with at least 95% sequence identity to SEQ ID NO: 6. In some embodiments, the nucleotide sequence expressing CCL3 includes a sequence with at least 99% sequence identity to SEQ ID NO: 6.

[0031] The active agents of the present disclosure may be administered to subjects at various concentrations. Moreover, the therapeutic compositions of the present disclosure may include various concentrations of active agents.PCT Application Attorney Docket No. AF60544.P059WODartmouth Ref. No. 2025-009

[0032] For instance, in some embodiments, each of the active agent concentrations may range from about 1 pg per dose to about 1 g per dose. In some embodiments, each of the active agent concentrations may range from about 10 pg per dose to about 1 g per dose. In some embodiments, each of the active agent concentrations may range from about 100 pg per dose to about 500 mg per dose. In some embodiments, each of the active agent concentrations may range from about 500 pg per dose to about 100 mg per dose. In some embodiments, each of the active agent concentrations may range from about 1 pg per dose to about 1 mg per dose. In some embodiments, each of the active agent concentrations may range from about 1 mg per dose to about 100 mg per dose.

[0033] In some embodiments where the active agents may be nucleic acids, each of the active agent concentrations may range from about 1 pg per dose to about 100 mg per dose. In some of such embodiments, each of the active agent concentrations may range from about 1 pg per dose to about 1 mg per dose.

[0034] Subjects

[0035] The active agents and therapeutic compositions of the present disclosure may be administered to various subjects. For instance, in some embodiments, the subject is a human being. In some embodiments, the subject is suffering from a cancer. In some embodiments, the subject is vulnerable to a cancer. In some embodiments, the subject is a non-human mammal, such as a rat, mouse, horse, cow, dog, cat, or cattle.

[0036] Applications

[0037] The methods and therapeutic compositions of the present disclosure may be utilized to treat various tumors. For instance, in some embodiments, the tumor is a solid tumor. In some embodiments, the methods and therapeutic compositions of the present disclosure may be used to treat or prevent tumor progression in a subject.

[0038] In some embodiments, the methods and therapeutic compositions of the present disclosure may be used to treat or prevent a cancer in a subject. In some embodiments, the cancer includes, without limitation, melanoma, breast cancer, lung cancer, pancreatic cancer, colon cancer, metastatic cancer, non-metastatic cancer, or combinations thereof.PCT Application Attorney Docket No. AF60544.P059WODartmouth Ref. No. 2025-009

[0039] In some embodiments, the methods and therapeutic compositions of the present disclosure may be utilized with additional treatments. For instance, in some embodiments, the methods and therapeutic compositions of the present disclosure may be used as a component of an intratumoral immunotherapy.

[0040] Therapeutic compositions

[0041] In addition to active agents, the therapeutic compositions of the present disclosure can include various additional components. For instance, in some embodiments, the additional components may include pharmaceutically acceptable compositions. In some embodiments, the pharmaceutically acceptable compositions may include one or more physiologically acceptable carriers or excipients. In some embodiments, the pharmaceutically acceptable compositions can also include formulation materials for modifying, maintaining, or preserving various conditions, including pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, and / or adsorption or penetration of the compounds. Suitable formulation materials include, without limitation, amino acids (e.g., glycine), antimicrobials, antioxidants (e.g., ascorbic acid), buffers (e.g., Tris-HCl), bulking agents (e.g., mannitol and glycine), chelating agents (e.g., EDTA), complexing agents (e.g., hydroxypropyl-beta-cyclodextrin), or combinations thereof.

[0042] The therapeutic compositions of the present disclosure may be suitable for various uses. For instance, in some embodiments, the therapeutic compositions of the present disclosure are suitable for use in treating or preventing tumor progression in a subject. In some embodiments, the therapeutic compositions of the present disclosure are suitable for use in treating or preventing a cancer in a subject. In some embodiments, the cancer includes, without limitation, melanoma, breast cancer, lung cancer, pancreatic cancer, colon cancer, metastatic cancer, non-metastatic cancer, or combinations thereof.

[0043] Additional embodiments

[0044] Reference will now be made to more specific embodiments of the present disclosure and experimental results that provide support for such embodiments. However, Applicant notes that the disclosure below is for illustrative purposes only and is not intended to limit the scope of the claimed subject matter in any way.

[0045] Example 1. Intratumoral introduction of IL-12 and CCL3 induces abscopal effects in tumorsPCT Application Attorney Docket No. AF60544.P059WODartmouth Ref. No. 2025-009

[0046] This Example aims to evaluate the combined anti-tumor effects of IL- 12 and CCL3 on mouse tumor models. The results establish that the combinations of IL- 12 and CCL3 can be expressed in or injected in tumors to stimulate antitumor immune responses against the treated tumor and stimulate systemic antitumor immunity that can eliminate or suppress metastatic disease.

[0047] Applicant electroporated an IL- 12 expressing plasmid in combination with CCL3 into mouse tumor models. The combined administration improved effector function of T cells, NK cells and macrophages in the tumor models.

[0048] Example 1.1. IL- 12 and CCL3 electroporation therapy improves abscopal effect in B16F10 models

[0049] As illustrated in FIGS. 1A-1C and FIGS. 2A-2C, Applicant demonstrated that IL-12 and CCL3 therapy eradicates treated tumors and elicits systemic responses in mice. C57BL / 6 mice bearing B16F10 tumors on both flanks were intratumorally electroporated at the right flank only with 30 ul of PBS, 50 ug of CCL3, 50 ug of IL-12, and 50 ug of IL-12+ 50 ug of CCL3 at days 0 and 7 (n=3). FIGS. 1A-1B show the growth of primary electroporated tumors (FIG. 1A) and untreated contralateral tumors (FIG. IB). FIG. 1C is a Kaplan-Meir curve showing survival. 1 out of 3 mice completely eradicated untreated tumors. (* P < 0.05; ** P <0.01; ***P < 0.001 in comparison to BS).

[0050] In another experiment, C57BL / 6 mice bearing B16F10 tumors on both flanks were intratumorally electroporated at the right flank only with 100 ug of BS, 50 ug of IL- 12 + 50 ug of BS and 50 ug of IL- 12 + 50 ug of CCL3 at days 0 and 7 (n=4). FIGS. 2A-2B show the growth of primary electroporated tumors (FIG. 2A) and untreated contralateral tumors (FIG. 2B). FIG. 2C is a Kaplan- Meir curve showing survival. 1 out of 4 completely eradicated the untreated tumor. (* P < 0.05; ** P <0.01; ***P < 0.001 in comparison to BS)

[0051] Example 1.2, IL-12 and CCL3 electroporation therapy improves abscopal effect in MC38 modelPCT Application Attorney Docket No. AF60544.P059WODartmouth Ref. No. 2025-009

[0052] As illustrated in FIGS. 3A-3C, IL- 12 and CCL3 therapy eradicates treated tumors and elicits systemic responses. C57BL / 6 mice bearing MC38 tumors on both flanks were intratumorally electroporated at the right flank only with 100 ug of BS, 50 ug of CCL3 + 50 ug of BS, 50 ug of IL- 12 + 50 ug of BS and 50 ug of IL- 12 + 50 ug of CCL3 at days 0 and 7 (n=4). FIGS. 3A-3B show growth of primary electroporated tumors (FIG. 3A) and untreated contralateral tumors (FIG. 3B). FIG. 3C shows a Kaplan-Meir curve showing survival. 3 out of 4 mice completely eradicated the untreated tumor. (* P < 0.05; ****P < 0.0001 in comparison to BS).

[0053] Example 1.3. IL-12 and CCL3 electroporation therapy is not totally dependent on CD8 T cells cross-presentation

[0054] As illustrated in FIGS. 4A-4C, IL-12 + CCL3 therapy is not totally dependent on CD8 T cells cross-presentation. C57BL / 6 mice bearing B16F10 tumors on both flanks were intratumorally electroporated at the right flank only with 100 ug of BS, 50 ug of CCL3 + 50 ug of BS, 50 ug of IL- 12 + 50 ug of BS and 50 ug of IL-12 + 50 ug of CCL3 at days 0 and 7 (n=5). FIGS. 4A-4B show the growth of primary electroporated tumors (FIG. 4A) and untreated contralateral tumors (FIG. 4B). FIG. 4C shows a Kaplan-Meir curve showing survival. 2 out of 5 mice completely eradicated the treated tumors.

[0055] Example 1.4, IL- 12 + CCL3 creates a local microenvironment that promotes IFN-driven activation of cytotoxic effectors cells

[0056] As illustrated in FIGS. 5A-5B, IL-12 + CCL3 creates a local microenvironment that promotes IFN-driven activation of cytotoxic effectors cells, which shifts the TME away from neutrophil / MDSC- supporting and angiogenesis. C57BL / 6 mice bearing B 16F10 tumors on both flanks were intratumorally electroporated at the right flank only with 100 ug of BS, 50 ug of IL- 12 + 50 ug of BS, and 50 ug of IL- 12 + 50 ug of CCL3 at days 0 and 7 (n=3). Tumors and tumor draining lymph node (TDLN) were harvested at day 4 and processed for 32-mouse Luminex. The data represent the pooled results from three samples in each group.

[0057] Example 1.5. Discussion

[0058] In sum, this Example demonstrated that IL- 12 and CCL3 can be expressed or injected in tumors to stimulate antitumor immune responses against the treated tumor, such as through stimulation of systemic antitumor immunity that can eliminate or suppress metastatic disease.PCT Application Attorney Docket No. AF60544.P059WODartmouth Ref. No. 2025-009

[0059] Without being bound by theory, the antitumor effect observed could be due to the ability of the combination of IL- 12 and CCL3 to massively recruit NK cells via IFNy and modulate T reg infiltration, but also due to the activation of eDC that are involved in antigen presentation, the increase in the effector function of CD8 T cells as shown with CCL3, or the modulation of the tumor microenvironment. The abscopal effect observed with IL- 12 and CCL3 reveals the systemic effect of immune cell activation and represents a therapy against metastasis, which is responsible for the spread and death of cancer patients.

[0060] Without further elaboration, it is believed that one skilled in the art can, using the description herein, utilize the present disclosure to its fullest extent. The embodiments described herein are to be construed as illustrative and not as constraining the remainder of the disclosure in any way whatsoever. While the embodiments have been shown and described, many variations and modifications thereof can be made by one skilled in the art without departing from the spirit and teachings of the invention. Accordingly, the scope of protection is not limited by the description set out above, but is only limited by the claims, including all equivalents of the subject matter of the claims. The disclosures of all patents, patent applications and publications cited herein are hereby incorporated herein by reference, to the extent that they provide procedural or other details consistent with and supplementary to those set forth herein.

Claims

PCT Application Attorney Docket No. AF60544.P059WODartmouth Ref. No. 2025-009CLAIMS1. A method of treating a tumor in a subject, said method comprising: administering to the subject the following active agents:(a) interleukin- 12 (IL-12), a nucleotide expressing IL-12, or combinations thereof; and(b) c-c motif chemokine ligand 3 (CCL3), a nucleotide expressing CCL3, or combinations thereof.

2. The method of claim 1, wherein the method comprises administering to the subject the following active agents:(a) IL- 12; and(b) CCL3.

3. The method of claim 1, wherein the method comprises administering to the subject the following active agents:(a) a nucleotide expressing IL- 12; and(b) a nucleotide expressing CCL3.

4. The method of claim 3, wherein the nucleotide is selected from the group consisting of DNA, RNA, mRNA, or combinations thereof.

5. The method of claim 3, wherein the nucleotide comprises DNA, and wherein the DNA is part of one or more plasmids.

6. The method of claim 1, wherein the administering comprises intratumoral administration.

7. The method of claim 1, wherein the method is used to treat or prevent tumor progression in the subject.

8. The method of claim 1, wherein the method is used to treat or prevent a cancer in a subject.PCT Application Attorney Docket No. AF60544.P059WODartmouth Ref. No. 2025-0099. The method of claim 8, wherein the cancer is selected from the group consisting of melanoma, breast cancer, lung cancer, pancreatic cancer, colon cancer, metastatic cancer, non-metastatic cancer, or combinations thereof.

10. The method of claim 9, wherein the method is used as a component of an intratumoral immunotherapy.

11. The method of claim 1, wherein the subject is a human being.

12. A therapeutic composition comprising the following active agents:(a) interleukin- 12 (IL-12), a nucleotide expressing IL-12, or combinations thereof: and(b) c-c motif chemokine ligand 3 (CCL3), a nucleotide expressing CCL3, or combinations thereof.

13. The therapeutic composition of claim 12, wherein the therapeutic composition is suitable for use in treating or preventing tumor progression in a subject.

14. The therapeutic composition of claim 12, wherein the therapeutic composition is suitable for use in treating or preventing a cancer in a subject, wherein the cancer is selected from the group consisting of melanoma, breast cancer, lung cancer, pancreatic cancer, colon cancer, metastatic cancer, non-metastatic cancer, or combinations thereof.

15. The therapeutic composition of claim 12, wherein the active agents comprise:(a) IL- 12; and(b) CCL3.

16. The therapeutic composition of claim 12, wherein the active agents comprise:(a) a nucleotide expressing IL- 12; and(b) a nucleotide expressing CCL3.PCT Application Attorney Docket No. AF60544.P059WODartmouth Ref. No. 2025-00917. The therapeutic composition of claim 16, wherein the nucleotide is selected from the group consisting of DNA, RNA, mRNA, or combinations thereof.

18. The therapeutic composition of claim 16, wherein the nucleotide comprises DNA, and wherein the DNA is part of one or more plasmids.

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