Cascade amplification of therapeutic payloads for cancer immunotherapy

Abstract

Achieving sufficient therapeutic payload delivery remains a significant challenge in gene therapy, particularly for cancer immunotherapy, where payload thresholds are critical for efficacy. To address this, we developed a Cascade Amplification of Therapeutic Payloads (CATP) system, leveraging lipid nanoparticles (LNPs) to co-deliver self-amplifying mRNA (SamRNA) and modified mRNA encoding alphavirus capsids and envelopes. The CATP system initiates a dual-amplification process: SamRNA amplifies therapeutic payloads within transfected cells, while capsid and envelope proteins package SamRNA into defective viral particles to infect neighboring cells, enabling secondary payload amplification. This single-cycle infection ensures enhanced efficacy while maintaining safety. In vitro and in vivo studies demonstrated the CATP system's superiority over conventional SamRNA delivery. In a B16F10 melanoma model, CATP achieved a 525-fold increase in intratumoral IL-12 levels, resulting in tumor regression and long-term immune memory. The platform also showed broad applicability, effectively treating MC38 colorectal cancer, CT26 colon cancer, and P53^null Kras^G12D pancreatic ductal adenocarcinoma. Additionally, optimization of therapeutic payloads with mutant IL-18 further enhanced anti-tumor efficacy. The CATP system represents a transformative approach to gene therapy, providing a scalable, safe, and potent platform for cancer immunotherapy. Its dual-amplification strategy offers new opportunities for overcoming payload limitations across diverse malignancies.

Fig. 1. Cascade amplifications of therapeutic payloads encoding mouse IL-12 on B16F10 melanoma therapy.

a Illustration of the cascade amplification of therapeutic payloads (CATP) was created in BioRender. Li, Y. (2025) https://BioRender.com/g50p020. b Illustrations of constructs for CATP. c Effects of CATP in HEK293 cells. HEK293 cells were treated by LNP encapsulated SamRNA mRNA encoding with eGFP and modified mRNA encoding with envelop and capsids from VEE or LNP encapsulated SamRNA mRNA encoding with eGFP and modified mRNA encoding firefly luciferase. Then the GFP percentages were determined by flow cytometer at days 1, 2, 3, and 4 post transfection. The shown are the changes of GFP versus days post transfection. d Scheme of tumor inoculation and treatment: Six- to eight-week-old C57BL/6 mice (n = 5 per group, a cage of animal) were subcutaneously inoculated with 1 million B16F10 melanoma cells. Seven days post-inoculation, mice were intratumorally treated with PBS (Control group of basal line), or LNP encapsulating 10 µg SamRNA encoding with mouse IL-12 (mIL-12) and 1 µg modified mRNA encoding with firefly luciferase (Control group of treated group) or LNP encapsulated 10 µg of SamRNA encoding mIL-12 and 1 µg modified mRNA encoding capsids/envelops from VEE, following the principle that molar ratio of modified mRNA (1 µg) is smaller than it of samRNA (10 µg). e mIL-12 expression in sera and tumors. The P-Values labeled in e were determined by Tukey’s multiple comparisons test.

Fig. 2. In vivo synthesis of defective viruses significantly enhances therapeutic efficacy against B16F10 melanoma.

Six- to eight-week-old C57BL/6 mice (n = 5 per group, a cage of animal) were subcutaneously inoculated with 1 million B16F10 melanoma cells. Seven days post-inoculation, mice were intratumorally treated with PBS (Control group of basal line), or LNP encapsulating 10 µg SamRNA encoding with mouse IL-12 (IL-12) and 1 µg modified mRNA encoding with firefly luciferase (Control group of treated group) or LNP encapsulated 10 µg of SamRNA encoding IL-12 and 1 µg modified mRNA encoding capsids/envelops from VEE. Results are shown as: Tumor areas (Y-axis), Survival rates (Y-axis), and Body weight changes (Y-axis) versus days post-B16F10 melanoma cell inoculation (X-axis) a, b, and c, respectively. The P-Values label in b were determined by a Comparison of Survival Curves (Kaper-myer) test.

Fig. 3. Comparison of therapeutic efficacies by CATP with VEE, SIN, and SFV4 capsids/envelop.

Six- to eight-week-old C57BL/6 mice (n = 5 per group, a cage of animal) were subcutaneously inoculated with 1 million B16F10 melanoma cells. Seven days post-inoculation, mice were intratumorally treated with PBS (Control group of basal line), or LNP encapsulated 10 µg of SamRNA encoding mIL-12 and 1 µg modified mRNA encoding capsids/envelops from VEE (Control group of treated group), SIN, and SFV4 as indicated. Results are shown as: Tumor areas (Y-axis), Survival rates (Y-axis), and Body weight changes (Y-axis) versus days post-B16F10 cell inoculation (X-axis) (a, b, c), respectively. d Illustrations of CATP with oncolytic effects by the capsids/envelop such as SFV4 was created in BioRender. Li, Y. (2025) https://BioRender.com/bguaszo. The SamRNA, regular mRNA, therapeutic payloads, capsids/envelop, defective viruses, tumor associated antigens (TAA), and inflammasomes triggered by defective viruses and LNP-mRNA are indicated. The P-Values labeled in a and c were determined by a two-way ANOVA test and Comparison of Survival Curves (Kaper-myer) test.

Fig. 4. Optimization of therapeutic payloads of CATP in B16F10 melanoma model.

Six- to eight-week-old C57BL/6 mice (n = 5 per group, a cage of animal) were subcutaneously inoculated with 1 million B16F10 melanoma cells. Seven days post-inoculation, mice were intratumorally treated with PBS (Control group of basal line), or LNP encapsulating 5 µg SamRNA encoding with mouse IL-12 (IL-12), 5 µg SamRNA encoding with firefly Luciferase, and 1 µg modified mRNA encoding capsids/envelops from SFV4 (Control group of treated group); or LNP encapsulating 5 µg SamRNA encoding with mouse wild type IL-18 (wtIL-18) or mutant IL-18 (mtIL-18), 5 µg SamRNA encoding with firefly Luciferase, and 1 µg modified mRNA encoding capsids/envelops from SFV4; or LNP encapsulating 5 µg SamRNA encoding with mouse IL-12 (IL-12), 5 µg SamRNA encoding with wild type IL-18 (wtIL-18) or mutant IL-18 (mtIL-18), and 1 µg modified mRNA encoding capsids/envelops from SFV4 as indicated. Results are shown as: Tumor areas (Y-axis), Survival rates (Y-axis), and Body weight changes (Y-axis) versus days post B16F10 melanoma cell inoculation (X-axis) (a–c), respectively. The P-Values labeled was determined by a two-way ANOVA test or Comparison of Survival Curves (Kaper-myer) test.

Fig. 5. Therapeutic efficacy of CATP with optimized therapeutic payloads and SFV4 capsids in KPC (P53^nullKRas^G12D) pancreatic duct cancer model.

Six- to eight-week-old C57BL/6 mice (n = 5 per group, a cage of animal) were subcutaneously inoculated with cancer cells as indicated. Seven days post inoculation, the tumor was intratumorally treated with the LNP encapsulated with mRNA as indicated, with PBS (Control group of basal line), or LNP encapsulating 5 µg SamRNA encoding with mouse IL-12 (IL-12), 5 µg SamRNA encoding with firefly Luciferase, and 1 µg modified mRNA encoding capsids/envelops from SFV4 (Control group of treated group); or LNP encapsulating 5 µg SamRNA encoding with mouse IL-12 (IL-12), 5 µg SamRNA encoding with mutant IL-18 (mtIL-18), and 1 µg modified mRNA encoding capsids/envelops from SFV4. Results are shown as: Tumor areas (Y-axis), Survival rates (Y-axis), and Body weight changes (Y-axis) versus days post KPC (P53^null KRas^G12D) pancreatic duct cancer cell inoculation (X-axis) (a–c), respectively. The P-Values labeled in a and b were determined by a two-way ANOVA test or Comparison of Survival Curves (Kaper-myer) test.

Fig. 6. CATP with mouse IL-12 and mutant IL-18 induces long-term immune memory against tumor recurrences.

a, b Re-challenges of fully tumor regressed mice in the group LNP encapsulated 5 µg of SamRNA encoding with mouse mutant IL-18 plus 5 µg of SamRNA encoding mouse IL-12 and 1 µg of modified mRNA encoding with SFV4 capsids/envelop proteins. The treated mice (n = 10) and naïve mice (n = 7, Control group) matched with age and sex and were challenged with 0.1 million B16F10 cells. Results are shown as: Tumor areas (Y-axis) and Survival rates (Y-axis) versus days post B16F10 melanoma cancer cell inoculation (X-axis). c Scheme of tumor inoculation and treatment. d, e Phenotyping of memory precursor of CD8 T cells and cytotoxic CD8 T cells in spleen. Six- to eight-week-old C57BL/6 mice (n = 5 per group, a cage of animal) were subcutaneously inoculated with 1 million B16F10 cells. Seven days post-inoculation, mice received a single intratumorally injection of PBS (Control group of basal line), LNP encapsulated 10 µg of SamRNA encoding with mIL-12 and 1 µg of modified mRNA encoding with luciferase (Control group of treated group) or LNP encapsulated 10 µg SamRNA encoding with mIL-12 and 1 µg of modified mRNA encoding VEE capsids and envelop proteins. On day 7 injections of LNP-mRNA, the mice were sacrificed. Shown are the numerates of the CD8⁺ CD62L⁺ CD122⁺ and CD8⁺ CD62L^- KLRG1⁺ in draining lymph nodes (DLN) (d) and spleens (e), respectively. The P-Values labeled in a, b, d were determined by a two-way ANOVA test for a Comparison of Survival Curves (Kaper-myer) test for b or a two-way ANOVA test for d.