Modular mRNA cocktail enables synergistic activation of antigen-specific T cells for cancer immunotherapy

Toan Van Le; Shota Imai; Iriya Fujitsuka; Makie Ueda; Sayuri Nakamae; Shusaku Mizukami; Rikinari Hanayama; Tomoyoshi Yamano

doi:10.1080/2162402X.2026.2736339

. 2026 Sep 22;15(1):2736339. doi: 10.1080/2162402X.2026.2736339

Modular mRNA cocktail enables synergistic activation of antigen-specific T cells for cancer immunotherapy

Toan Van Le ^a, Shota Imai ^a, Iriya Fujitsuka ^a, Makie Ueda ^a, Sayuri Nakamae ^b, Shusaku Mizukami ^b, Rikinari Hanayama ^a,^c,^*, Tomoyoshi Yamano ^a,^c,^*

PMCID: PMC13613922 PMID: 42773579

Abstract

mRNA-based therapeutics have demonstrated notable success in SARS-CoV-2 vaccines and are emerging in cancer immunotherapy. However, conventional mRNA cancer vaccines are limited in part by the low immunogenicity of tumor-associated and neoantigens. We addressed this limitation by formulating a modular, liposome-based mRNA cocktail comprising three distinct mRNAs encoding tumor antigens, the co-stimulatory molecule CD80, and membrane-tethered IL-2. Administration of this mRNA cocktail resulted in synergistic activation of tumor antigen-specific CD8⁺ T cells and robust anti-tumor immune responses. In addition, substituting IL-2 in the mRNA cocktail with membrane-tethered IL-12 led to the expansion and differentiation of endogenous antigen-specific Th1 helper T cells in vivo. Importantly, this platform activated NY-ESO-1-specific CD8⁺ T cells in HLA-A*02:01-transgenic mice, highlighting its translational potential. This modular mRNA cocktail provides a flexible and translatable platform for precision cancer immunotherapy by enabling coordinated activation of both CD8⁺ and CD4⁺ T cell responses.

Keywords: mRNA cancer vaccine, tumor antigen–specific T cells, CD8⁺ T cell activation, Th1 differentiation, NY-ESO-1

Introduction

mRNA-based therapeutics have dramatically accelerated the development of vaccines for infectious disease. This accelerated timeline is evident in the significant progress achieved in less than a year for SARS-CoV-2, a process that typically takes several years. ¹ ^, ² This advancement, highlighted by the practicality and safety of mRNA vaccines, has increased interest in mRNA therapeutics globally. As a result, the development of vaccines for various infectious diseases and cancers is being pursued. ³ ^, ⁴

In cancer immunotherapy, mRNA vaccines encoding cancer neoantigens are currently being evaluated in clinical trials. ⁴ ^, ⁵ Recent efforts have focused on vaccines that encode multiple neoantigen sequences found in cancer cells. ⁶ ^, ⁷ However, in the context of cancer vaccines, the effectiveness of mRNA alone remains limited, often necessitating the concurrent use of immune checkpoint inhibitors and other anti-cancer treatments. ⁸ This highlights the need for more efficient methods to activate CD8⁺ T cells to enhance antitumor responses.

The delivery of mRNA into cells triggers a cascade of events, starting with its translation into proteins, which are subsequently degraded by proteasomes. The resulting peptides are then complexed with major histocompatibility complex class I (MHC I) molecules and displayed on the cell surface for recognition by CD8⁺ T cells. ⁹ However, T cell activation is not solely dependent on T cell receptor (TCR) engagement but also requires co-stimulatory signals. ¹⁰ While professional antigen-presenting cells (APCs) express these co-stimulatory molecules, non-APCs lack them and are unable to directly activate naïve CD8⁺ T cells upon mRNA uptake. In such cases, cross-presentation by professional APCs is essential. ¹¹ Non-APCs that have taken up mRNA-encoded antigens rely on antigen transfer to dendritic cells, particularly XCR1⁺ conventional type 1 DCs (cDC1s), which are specialized for cross-presentation and subsequent activation of CD8⁺ T cells. ¹² This dependency implies that the therapeutic potential of mRNA vaccines may be constrained by the efficiency of antigen handover and functionality of cross-presenting DC subsets. To address this limitation, recent efforts have focused on the development of lipid nanoparticles (LNPs) that preferentially target dendritic cells, including both cDC1s and migratory cDC2s. By engineering ionizable lipids or formulating mRNA with DC-targeting delivery platforms, such as lipid nanoparticles (LNPs) or lipoplexes (LPX), it is now possible to selectively deliver mRNA into DCs. This targeted approach bypasses the need for antigen transfer from non-APCs and enables direct expression of antigens and co-stimulatory molecules within dendritic cells. ¹³ ^, ¹⁴ These delivery systems have demonstrated enhanced CD8⁺ T cell priming and improved therapeutic efficacy in preclinical cancer models, reinforcing the rationale for APC-directed mRNA delivery as a key component of next-generation cancer vaccines.

In this study, we propose a novel approach to enhance the efficacy of mRNA vaccines by introducing a modular mRNA cocktail that encodes not only tumor antigens but also co-stimulatory molecules and cytokines. This approach provides a means to amplify antigen-specific T cell responses by supplementing antigen delivery with co-stimulatory and cytokine signals, thereby overcoming the limited immunogenicity of conventional mRNA vaccines. The mRNA cocktail effectively induces robust activation of antigen-specific CD8⁺ T cells and orchestrates potent cytotoxic immune responses. Moreover, substituting IL-2 with cytokines such as IL-12 promotes in vivo differentiation of antigen-specific Th1 helper T cells, highlighting the potential of this platform to precisely modulate T cell differentiation in a context-dependent manner.

Materials and methods

Cell lines

The OVA-expressing murine lymphoma cell line, a derivative of EL4 (E.G7; ATCC, CRL-2113) was cultured in RPMI 1640 (Nacalai Tesque) supplemented with 10% fetal calf serum (FCS; Thermo Fisher Scientific), 1 × non-essential amino acids (Nacalai Tesque), 1 mM sodium pyruvate (Nacalai Tesque), 100 U/mL penicillin, 100 U/mL streptomycin (FUJIFILM Wako), and 0.05 μM 2-mercaptoethanol (Thermo Fisher Scientific). To generate a stable MC38 cell line expressing mutant Rpl18, we used a codon-optimized mouse Rpl18 gene containing the Q125R mutation, which was previously reported in MC38 cells, ¹⁵ but was absent in MC38 cells obtained from Kerafast. The mutated gene was fused to the transmembrane domain of mouse CD8 via a 2 A peptide and cloned into a third-generation lentiviral vector (pLJM1, Addgene #91980) under the CMV promoter. MC38 cells were transduced with lentiviral particles to generate cells stably expressing mutant Rpl18. Rpl18-MC38 cells were cultured in DMEM (Nacalai Tesque) supplemented with 10% FCS, 2 mM L-glutamine (Nacalai Tesque), 1 × non-essential amino acids, 1 mM sodium pyruvate, 10 mM HEPES (Nacalai Tesque), 50 μg/mL gentamicin sulfate (Nacalai Tesque), and 100 U/mL penicillin-streptomycin. Human embryonic kidney (HEK)-293T cells (ATCC, Cat# CRL-3216) were cultured in DMEM supplemented with 10% FCS, 100 U/mL penicillin, and 100 U/mL streptomycin. All the cells were maintained at 37 °C in a humidified atmosphere containing 5% CO₂.

Mice

C57BL/6 mice were purchased from Japan SLC. OT-I TCR transgenic mice ¹⁶ and HLA-A*02:01 transgenic mice ¹⁷ were housed in a specific pathogen-free facility. All animal experiments were performed following a protocol approved by Kanazawa University.

mRNA construct and in vitro transcription

Plasmids for mRNA preparation were codon-optimized for murine expression using VectorBuilder (VectorBuilder Ltd.). Each construct contained a consensus Kozak sequence upstream of the start codon and three stop codons downstream of the gene of interest. DNA fragments were synthesized by Eurofins Genomics and cloned into the vector, which contained a T7 RNA polymerase promoter, 5′UTR, coding region, 3′UTR, and 128-base poly(A) tail. The 5′UTR, coding region, and 3′UTR sequences were based on those described in a previous study. ¹⁸

mRNA was synthesized by in vitro transcription using the HiScribe T7 mRNA Kit with CleanCap Reagent AG (New England Biolabs), following the manufacturer’s protocol. Co-transcriptional incorporation of CleanCap Reagent AG was used to generate Cap1-capped transcripts, while uridine triphosphate was substituted with N1-methylpseudouridine-5′-triphosphate. The synthesized mRNA was subsequently purified with the Monarch RNA Cleanup Kit (New England Biolabs), and its integrity was assessed using an RNA ScreenTape assay (Agilent Technologies).

mRNA transfection in vitro

mRNA transfection was performed using the TransIT®-mRNA Transfection Kit (Mirus Bio), a cationic lipid-based reagent. Briefly, mRNA was diluted in Opti-MEM (Thermo Fisher Scientific) and mixed with TransIT-mRNA reagent according to the manufacturer's protocol. The resulting RNA-lipid complexes were then added to the cells. Target protein expression was evaluated using flow cytometry 18 hours after transfection.

In vitro T-cell proliferation assay

Lymph node T cells were isolated from OT-I transgenic mice and labeled with 1 μM CellTrace™ Violet (CTV; Thermo Fisher Scientific) at 37 °C for 3 minutes. A total of 2 × 10⁵ CTV-labeled OT-I T cells were co-cultured with MC38 cells transfected with mRNA encoding OVA-MITD, OVA-MITD + CD80, OVA-MITD + membrane-anchored IL-2, or OVA-MITD + CD80 + membrane-anchored IL-2 at a 4:1 T cell to MC38 cell ratio. The transfected MC38 cells were prepared according to the mRNA transfection method described above. After three days of culture, T cell proliferation was analyzed by flow cytometry.

mRNA formulation and intravenous administration

mRNA was formulated using in vivo-jetRNA+ (Polyplus) according to the manufacturer’s instructions. Briefly, mRNA was diluted in a suitable buffer and mixed with in vivo-jetRNA+ reagent. A total of 200 μL of the resulting nanoparticle suspension was intravenously injected into each mouse.

Tetramer staining

To evaluate antigen-specific CD8⁺ T cell responses, splenocytes were isolated from immunized mice and seeded into 96-well plates at a density of 3 × 10⁶ cells per well. Cells were incubated with H-2K^b/OVA (257-264), H-2K^b/Rpl18 (KILTFDRL), or HLA-A*02:01-H-2K^b/NY-ESO-1 (SLLMWITQC) tetramers at 37 °C for 30 min, and with I-A^b/OVA (323-339) tetramers at 37 °C for 1 h. Following incubation, cells were subjected to surface staining according to standard protocols. After washing with PBS containing 2% FCS, samples were acquired on a BD flow cytometer using FACSDiva software (BD Biosciences). Data analysis was performed using FlowJo software (v10.4.1; BD Biosciences).

In vivo tumor models and mRNA treatment regimen

A total of 1 × 10⁵ E.G7 tumor cells suspended in 200 μL of PBS (FUJIFILM Wako) were subcutaneously injected into the right flank of C57BL/6 mice. For the Rpl18-MC38 tumor model, 5 × 10⁵ Rpl18-MC38 cells suspended in a 1:1 mixture of 100 μL sterile PBS and 100 μL Matrigel (Corning) were injected into the same site. For both tumor models, when tumors reached approximately 100 mm³ in volume, mice received weekly intravenous injections of mRNA for three consecutive weeks. Treatment consisted of either OVA-MITD (5 μg) or Rpl18-MITD (5 μg) combined with control mRNA (10 μg), or a cocktail of OVA-MITD (5 μg) or Rpl18-MITD (5 μg), CD80 mRNA (5 μg), and membrane-anchored IL-2 mRNA (5 μg). As a negative control, control mRNA (15 μg) alone was administered. Akaluc-Venus mRNA was used as the control mRNA in all the treatment groups. ¹⁹ Tumor volumes were measured two to three times per week using digital calipers and calculated using the formula (length × width²)/2, where the length and width represent the longest and shortest tumor diameters, respectively. Mice were euthanized when tumors reached ≥2000 mm³, exhibited ulceration, or showed signs of severe distress or moribundity in accordance with predefined humane endpoint criteria.

Early initiation of mRNA treatment

To evaluate the effect of earlier treatment initiation on therapeutic efficacy, Rpl18-MC38 cells were implanted subcutaneously as described above. Mice were left untreated or received intravenous injections of control mRNA (15 μg), Rpl18-MITD mRNA (5 μg) supplemented with control mRNA (10 μg), or the Rpl18-MITD, CD80, and membrane-anchored IL-2 mRNA cocktail (5 μg each; total 15 μg) on days 3, 10, and 17 after tumor implantation. Treatment was therefore initiated before tumors reached the approximately 100 mm³ threshold used for treatment initiation in the standard therapeutic model. All mRNAs were formulated with in vivo-jetRNA⁺ as described above. Tumor growth and survival were monitored according to the procedures described above.

Combined intravenous and intratumoral administration

To evaluate the effect of combined systemic and local mRNA delivery on therapeutic efficacy, Rpl18-MC38 tumor-bearing mice received control mRNA (15 μg), Rpl18-MITD mRNA (5 μg) supplemented with control mRNA (10 μg), or a cocktail of Rpl18-MITD, CD80, and membrane-anchored IL-2 mRNAs (5 μg each; total 15 μg) on days 8, 15, and 22 after tumor implantation. All mRNAs were formulated with in vivo-jetRNA⁺ as described above. For each treatment, half of the total formulated mRNA dose was administered intravenously and the remaining half was administered intratumorally. Thus, the total mRNA dose per mouse remained 15 μg per treatment, identical to that used in the intravenous-only therapeutic regimen. Tumor growth and survival were monitored as described above.

Prophylactic vaccination and tumor rechallenge

To evaluate whether prophylactic vaccination could induce protective antitumor immunity, naïve C57BL/6 mice were left untreated or received three weekly intravenous injections of control mRNA (15 μg), Rpl18-MITD mRNA (5 μg) supplemented with control mRNA (10 μg), or a cocktail of Rpl18-MITD, CD80, and membrane-anchored IL-2 mRNAs (5 μg each; total 15 μg) on days −21, −14, and −7. On day 0, mice were subcutaneously challenged with 5 × 10⁵ Rpl18-MC38 cells. Tumor growth and survival were monitored as described above.

To assess durable antitumor immune memory, mice that remained tumor-free following either prophylactic vaccination or the combined intravenous and intratumoral therapeutic regimen described above were rechallenged with 5 × 10⁵ Rpl18-MC38 cells by subcutaneous injection into the contralateral flank without additional mRNA administration. Age-matched naïve mice challenged with the same number of Rpl18-MC38 cells served as controls. Tumor growth was monitored as described above.

Combined MHC-I- and MHC-II-targeted mRNA cocktail treatment

E.G7 tumor cells were implanted subcutaneously as described above. When tumors reached approximately 100 mm³, mice were assigned to one of four treatment groups. Mice received the MHC-I-targeted cocktail, comprising OVA-MITD, CD80, and membrane-anchored IL-2 mRNAs (5 μg each; total 15 μg), on days 8 and 15; the MHC-II-targeted cocktail, comprising OVA-MHC-II, CD80, and membrane-anchored IL-12 mRNAs (5 μg each; total 15 μg), on days 8 and 15; or both cocktails according to an interdigitated schedule, with the MHC-I-targeted cocktail administered on days 8 and 15 and the MHC-II-targeted cocktail administered on days 11 and 18. Untreated mice served as controls. Tumor growth and survival were monitored as described above.

To evaluate durable protective antitumor immunity, tumor-free mice from the combined-treatment group were rechallenged with 1 × 10⁵ E.G7 cells by subcutaneous injection without additional mRNA administration. Age-matched naïve mice challenged with the same number of E.G7 cells served as controls. Tumor growth was monitored as described above.

Isolation and preparation of tumor-infiltrating lymphocytes (TILs)

Tumors were harvested and minced into ~2–3 mm fragments. Tissue dissociation was performed using the Tumor Dissociation Kit, mouse (Miltenyi Biotec) following the manufacturer’s protocol. Briefly, tumor fragments were incubated with enzyme mix in RPMI 1640 medium and processed on the GentleMACS™ Octo Dissociator (Miltenyi Biotec). The resulting single-cell suspension was filtered through a 70 μm strainer to remove debris. Cells were washed twice with PBS containing 2% FCS and subsequently counted. Purified TILs were resuspended in complete RPMI 1640 medium containing 10% FCS for subsequent flow cytometric analysis.

In vivo killing assay

HLA-A*02:01 transgenic mice were intravenously administered in vivo-jetRNA⁺ complexes containing the NY-ESO-1-MITD, CD80, and membrane-anchored IL-2 mRNA cocktail, NY-ESO-1-MITD mRNA alone, or control mRNA. Seven days after mRNA administration, target splenocytes were prepared from syngeneic HLA-A*02:01 transgenic mice. Splenocytes were divided into two populations and pulsed with either the NY-ESO-1 peptide or an irrelevant Wilms tumor 1 (WT1) peptide at a concentration of 10 μM for 2 h at 37 °C. After two washes to remove unbound peptides, the NY-ESO-1 peptide-pulsed cells were labeled with CellTrace Violet (CTV), whereas the WT1 peptide-pulsed control cells were labeled with CFSE. Equal numbers of the two populations were mixed, and a total of 2 × 10⁶ target cells, comprising 1 × 10⁶ CTV-labeled NY-ESO-1 peptide-pulsed cells and 1 × 10⁶ CFSE-labeled WT1 peptide-pulsed cells, were transferred intravenously into each recipient mouse. Twenty-four hours after target-cell transfer, spleens were harvested and analyzed by flow cytometry. NY-ESO-1-specific cytotoxicity was quantified based on the preferential loss of CTV-labeled NY-ESO-1 peptide-pulsed cells relative to CFSE-labeled WT1 peptide-pulsed control cells.

Antibodies and flow cytometry

Antibody staining was performed according to standard procedures. Monoclonal antibodies for surface staining, including antibodies against mCD3 (clone: 17A2), mCD4 (clone: GK1.5), mCD8α (clone: 53-6.7), mCD44 (clone: IM7), mCD45 (clone: 30-F11), mCD62L (clone: MEL-14), mCD80 (clone: 16-10A1), mTCR-Vα2 (clone: B20.1), mH-2K^b bound to SIINFEKL-PE (clone: 25-D1.16), mIL-2 (clone: JES6-5H4), were purchased from BioLegend. Intracellular staining was performed using antibodies against mIFN-γ (clone: XMG1.2), mT-bet (clone: 4B.10) in combination with the True-Nuclear Transcription Factor Buffer Set (BioLegend). OVA-specific CD8⁺ T cells, Rpl18-specific CD8⁺ T cells, NY-ESO-1-specific CD8⁺ T cells, and OVA-specific CD4⁺ T cells were quantified using the following tetramers obtained from the NIH Tetramer Core Facility: H-2K^b/OVA (257-264), H-2K^b/Rpl18 (KILTFDRL), HLA-A*02:01-H2K^b/NY-ESO-1 (SLLMWITQC), and I-A^b/OVA (323-339), respectively. Staining was performed according to the manufacturer's instructions. For intracellular staining, cells were fixed and permeabilized using the True-Nuclear Transcription Factor Buffer Set (BioLegend) according to the manufacturer’s instructions. Briefly, cells were incubated with fixation buffer for 60 minutes at room temperature in the dark, followed by two washes with permeabilization buffer. Cells were then stained with antibodies diluted 1:50 in permeabilization buffer for 45 minutes at room temperature in the dark. After washing, cells were resuspended in FACS buffer and subjected to flow cytometry analysis. Data were acquired using the FACSDiva software (BD Biosciences). A FACSMelody (BD Biosciences) was used for cell sorting. Data analysis was performed using FlowJo v.10.4.1 software.

Statistical analysis

Statistical analyses were performed using GraphPad Prism 8.4.2 software (San Diego, CA, USA). Data are presented as mean ± SEM. Data normality was assessed using the Shapiro-Wilk test. For normally distributed data, comparisons between two groups were conducted using an unpaired two-tailed Student’s t-test, whereas non-normally distributed data were analyzed using the Mann-Whitney U test. For experiments with n = 3, statistical significance was evaluated using the Welch's t-test. A P value < 0.05 was considered statistically significant.

Results

Generation of mRNA cocktail-transfected cells for activation of antigen-specific CD8⁺ T cells

To evaluate the ability of an mRNA cocktail to activate antigen-specific CD8⁺ T cells, we synthesized a set of mRNAs encoding OVA-MITD (a model antigen containing OT-I and OT-II epitopes fused to a minimal MHC class I trafficking domain (MITD) to enhance antigen presentation), ²⁰ the costimulatory molecule CD80, and membrane-anchored IL-2 (generated by fusing IL-2 to the extracellular domain of CD8α). Plasmids used for in vitro transcription were designed to include a 128-nucleotide poly(A) tail for enhanced stability and incorporated N1-methylpseudouridine triphosphate (m¹ΨTP) instead of UTP (Figure 1a, Supplementary Figure 1a–c). ²¹ ^, ²² These mRNAs were transfected into MC38 cells, resulting in robust surface expression of MHC I, CD80, and IL-2, as confirmed by flow cytometry (Figure 1b, c). When co-cultured with OVA-specific CD8⁺ T cells (OT-I), MC38 cells expressing all three components (OVA-MITD, CD80, and IL-2) induced the most robust OT-I proliferation compared to cells expressing OVA-MITD alone or in combination with either CD80 or IL-2 (Figure 1d, e, Supplementary Figure 1d, e).

Five panels showing mRNA constructs, surface protein expression, and antigen-specific CD8+ T-cell proliferation. A five-panel figure demonstrates antigen-specific CD8 plus T cell activation by mRNA-transfected cells. Panel A shows three mRNA constructs: OVA-MITD, mouse CD80, and membrane-anchored mouse IL-2, each with CAP1 cap, open reading frame, and poly A tail. Panel B illustrates the experimental scheme where MC38 cells are transfected with mRNA at hour zero, cultured for 18 hours, treated with mitomycin C, then co-cultured with CellTrace Violet-labeled OT-I CD8 + T cells with analysis at 72 hours. Panel C Representative flow cytometry histograms showing expression of OVA-H-2Kᵇ, CD80, and membrane-anchored IL-2 in MC38 cells after transfection with different combinations of mRNAs: OVA-H2-Kb alone, OVA-H2-Kb plus CD80, OVA-H2-Kb plus IL2, and OVA-H2-Kb plus CD80 plus IL2. Each row shows three histograms measuring OVA-H2-Kb-PE, mCD80-FITC, and mIL2-APC, with filled gray histograms representing untransfected MC38 cells and open black histograms representing mRNA-transfected cells. Panel D presents a bar graph showing OT-I cell frequencies across six groups rising from approximately 15 percent in control mRNA to approximately 90 percent in combined OVA-MITD plus CD80 plus IL2 treatment, with p values less than 0.0001 marked between groups. Panel E shows CTV low OT-I cell frequencies across the same six groups, displaying similar trends increasing from approximately 1 percent in control to approximately 90 percent in combined treatment conditions, with p values ranging from 0. — Activation of antigen-specific CD8⁺ T cells by mRNA cocktail-transfected cells. (a) Schematics of the individual mRNA constructs encoding OVA-MITD, mouse CD80 or membrane-anchored mouse IL-2. 5′ cap, 5′ UTR, open reading frame, 3′ UTR and 128-nt poly(A) tail are indicated. (b) Co-culture scheme. MC38 cells were harvested 18 h after mRNA transfection, mitomycin C (MMC)-treated, and co-cultured with CellTrace Violet-labeled OT-I CD8⁺ T cells. (c) Flow-cytometric analysis of surface OVA-K^b complexes, CD80, and membrane-anchored IL-2 on MC38 cells 18 h after transfection. Filled gray histograms: untransfected MC38 cells; open black histograms: mRNA-transfected cells. Proliferation of OT-I cells was analyzed 72 h after co-culture. (d) Frequencies of OT-I cells. (e) Frequencies of CTV^low OT-I cells. Each dot represents one biological replicate. Data are shown as mean ± SEM (n = 6). The Shapiro-Wilk test was used to assess normality. Normally distributed data were analyzed using an unpaired two-tailed Student’s t-test, whereas non-normally distributed data were analyzed using the Mann-Whitney U test.

In vivo administration of an mRNA cocktail stimulates antigen-specific CD8⁺ T cells

We examined whether in vivo administration of an mRNA cocktail could promote clonal expansion of endogenous antigen-specific CD8⁺ T cells. Mice were administered control mRNA, OVA-MITD mRNA alone, or a cocktail of OVA-MITD, CD80, and IL-2 mRNAs once weekly for three consecutive weeks. All mRNAs were formulated using the in vivo-jetRNA⁺ liposomal delivery reagent for efficient systemic delivery. One week after the final injection, spleens were harvested and analyzed for OVA-specific CD8⁺ T cells using MHC class I tetramers (Figure 2a). Consistent with the in vitro results, OVA-tetramer⁺ CD8⁺ T cells expanded more robustly in the cocktail group (56%) than in the OVA-MITD group (24.3%) (Figure 2b, c, Supplementary Figure 2a). The expanded CD8⁺ T cells exhibited a CD44^hiCD62L^low phenotype, characteristic of effector or effector memory T cells, and expressed IFN-γ (Figure 2d, Supplementary Figure 2b, c). Total splenocyte numbers were comparable across all groups (Supplementary Figure 2d), suggesting that the observed expansion of OVA-specific CD8⁺ T cells was not due to nonspecific activation, but rather reflected a targeted immune response.

Ten panels showing mRNA vaccination schedules, OVA-specific CD8+ T-cell responses, tumor growth, and survival in mice. A ten-panel figure demonstrates mRNA immunotherapy effects in mice. Panel A shows a timeline of three weekly intravenous mRNA injections on days 0, 7, and 14 in C57BL/6 mice with analysis on day 21. Panel B representative flow cytometry dot plots showing increasing frequencies of OVA-specific CD8⁺ T cells across the treatment groups. . Panel C quantification of tetramer⁺ CD8⁺ T cells in the spleen, shown as cell numbers. Panel D quantification of IFN-γ⁺ CD8⁺ T cells, shown as cell numbers. Panel E shows a tumor model timeline with EG7 cell transplantation on day 0 and mRNA injections on days 7, 14, and 21. Panel F displays four tumor growth curves over 50 days: untreated mice show rapid growth, control mRNA shows moderate growth, OVA-MITD shows slower growth, and cocktail of OVA-MITD ,CD80, IL2 shows slowest growth. Panel G is a Kaplan-Meier survival curve with untreated and control mRNA showing early mortality around day 20, OVA-MITD showing intermediate survival, and cocktail of OVA-MITD, CD80,IL2 showing best survival beyond day 40, p less than 0.0001. Panel H illustrates a second tumor model timeline with EG7 transplantation, mRNA injection when tumors reach approximately 100 cubic millimeters, and tumor-infiltrating lymphocyte analysis one week later. Panel i representative flow cytometry plots of CD4⁺ and CD8⁺ tumor-infiltrating T cells. Panel j is quantification of tetramer⁺ CD8⁺ T cells among tumor-infiltrating lymphocytes. — In vivo administration of an mRNA cocktail stimulates antigen-specific CD8⁺ T cells. (a) C57BL/6 mice received three weekly intravenous injections on days 0, 7, and 14 of control mRNA, OVA-MITD mRNA (5 µg) supplemented with control mRNA (10 µg), or a cocktail of OVA-MITD, CD80, and membrane-anchored IL-2 mRNAs (5 µg each; total 15 µg). Spleens were collected 7 d after the final dose. (b) Representative dot plots of OVA-specific CD8⁺ T cells. (c) Number of tetramer⁺ CD8⁺ T cells in the spleens. (d) Number of IFN-γ⁺ CD8⁺ T cells. (e) Mice were subcutaneously inoculated with OVA-expressing EL4 cells (E.G7), followed by intravenous administration of mRNAs on days 7, 14, and 21. (f) Tumor growth curves. (g) Kaplan–Meier survival curve. (h) E.G7 cells were subcutaneously implanted into C57BL/6 mice. When tumors reached approximately 100 mm³, mice received intravenous injections of control mRNA, OVA-MITD mRNA supplemented with control mRNA, or a cocktail of OVA-MITD, CD80, and membrane-anchored IL-2 mRNAs. Tumor-infiltrating lymphocytes were analyzed one week after mRNA administration. (i) Representative dot plots of the TILs. (j) Number of tetramer⁺ CD8⁺ T cells. Unless otherwise indicated, n = 5 mice per group. Data in (c, d, and j) are presented as mean ± SEM. Data normality was assessed using the Shapiro–Wilk test. Normally distributed data were analyzed using an unpaired two-tailed Student’s t-test, whereas non-normally distributed data were analyzed using the Mann–Whitney U test. Statistical significance in (j) was evaluated using Welch’s t-test (n = 3 mice per group). Survival differences in (g) were analyzed using the log-rank (Mantel–Cox) test.

To assess therapeutic efficacy, we used a tumor model in which E.G7 cells (OVA-expressing EL-4 derivatives) were subcutaneously implanted into C57BL/6 mice. When tumors reached 100 mm³, mice received weekly intravenous injections of control mRNA, OVA-MITD mRNA alone, or the mRNA cocktail for three weeks (Figure 2e). While OVA-MITD mRNA monotherapy delayed tumor growth and extended survival relative to the control, the mRNA cocktail led to greater tumor suppression and prolonged survival (Figure 2f, g).

To elucidate the underlying mechanisms, we analyzed tumor-infiltrating lymphocytes (TILs) one week after mRNA administration (Figure 2h). CD8⁺ T cells constituted 4.6% of TILs in the OVA-MITD group but increased to 13.5% in the cocktail group (Figure 2i, j, Supplementary Figure 2e–g). Furthermore, a higher proportion of CD8⁺ T cells expressed IFN-γ in the cocktail group (Supplementary Figure 2h), indicating enhanced infiltration and activation of CD8⁺ T cells in the tumor microenvironment. Although the relative proportion of CD4⁺ TILs appeared lower in the vaccinated groups (Figure 2i), this likely reflected the marked increase in CD8⁺ TILs rather than an absolute reduction in CD4⁺ T-cell infiltration (Supplementary Figure 2i). In addition, among the groups analyzed for TILs, the cocktail group consistently exhibited the smallest tumor size, in agreement with the tumor growth data presented above (Figure 2f, Supplementary Figure 2j). These results demonstrate that in vivo administration of the mRNA cocktail expands antigen-specific CD8⁺ T cells and elicits potent antitumor immunity in the OVA antigen model.

In vivo administration of an mRNA cocktail expands neoantigen-specific CD8⁺ T cells

Using Rpl18 as a model neoantigen in the MC38 tumor system, we investigated whether in vivo administration of an mRNA cocktail could expand neoantigen-specific CD8⁺ T cells. Mice were administered control mRNA, Rpl18-MITD mRNA alone, or a cocktail of Rpl18-MITD, CD80, and IL-2 mRNAs once weekly for three consecutive weeks, in accordance with the administration protocol established in the OVA model (Figure 3a, Supplementary Figure 3a). One week after the final injection, the spleens were harvested and analyzed for Rpl18-specific CD8⁺ T cells using MHC class I tetramers. Mice receiving Rpl18-MITD alone exhibited modest expansion of tetramer-positive CD8⁺ T cells, whereas combination treatment induced robust expansion and differentiation into CD44^hiCD62L^low effector and effector memory phenotypes (Figure 3b–d, Supplementary Figure 3b–d). As observed in the OVA model, the total splenocyte numbers remained unchanged across groups (Supplementary Figure 3e), suggesting that the response was antigen-specific rather than nonspecific.

A ten-panel figure showing mRNA immunotherapy effects on neoantigen-specific CD8 T cell expansion and tumor control in mice. A ten-panel figure demonstrates mRNA cocktail immunotherapy effects on CD8 plus T cell responses and tumor growth in C57BL/6 mice. Panel A shows experimental timeline with three weekly intravenous injections on days 0, 7, and 14. Panel B displays flow cytometry dot plots of Rpl18-specific CD8 + T cells in spleens across control mRNA, Rpl18-MITD mRNA, and cocktail of Rpl18-MITD, CD80,IL2 , with percentages 3.87, 16.6, and 50.6 respectively. Panel C shows quantification of tetramer⁺ CD8⁺ T cells in the spleen, shown as cell numbers. Panel D shows quantification of IFN-γ⁺ CD8⁺ T cells in the spleen, shown as cell numbers. Panel E outlines tumor challenge studies with Rpl18-MC38 cells and mRNA injections on days 8, 15, and 22. Panel F displays tumor growth curves showing untreated and control mRNA tumors growing rapidly , Rpl18-MITD showing intermediate control, and Rpl18-MITD + CD80 + IL2 cocktail showing delayed tumor growth. Panel G shows Kaplan-Meier survival curves of tumor-bearing mice following treatment. Panel H experimental design for combined intravenous and intratumoral mRNA administration, with treatment given once weekly on days 8, 15, and 22. Panel i shows tumor growth curves following combined intravenous and intratumoral treatment. Panel J shows Kaplan–Meier survival curves following combined intravenous and intratumoral treatment.. — In vivo administration of an mRNA cocktail expands neoantigen-specific CD8⁺ T cells. (a) C57BL/6 mice received three weekly intravenous injections on days 0, 7, and 14 of control mRNA (15 µg), Rpl18-MITD mRNA (5 µg) supplemented with control mRNA (10 µg), or a cocktail of Rpl18-MITD, CD80, and membrane-anchored IL-2 mRNAs (5 µg each; total 15 µg). Spleens were collected 7 d after the final injection. (b) Representative dot plots of Rpl18-specific CD8⁺ T cells in the spleens. (c) Number of tetramer⁺ CD8⁺ T cells in the spleens. (d) Number of IFN-γ⁺ CD8⁺ T cells in the spleens. (e) Mice were subcutaneously inoculated with Rpl18-MC38 cells, followed by intravenous administration of the indicated mRNAs on days 8, 15, and 22. (f) Tumor growth curves. (g) Kaplan–Meier survival curves of tumor-bearing mice. (h) Experimental design for combined intravenous and intratumoral (i.v. + i.t.) treatment. Tumor-bearing mice received the indicated mRNAs once weekly on days 8, 15, and 22 via combined i.v. + i.t. administration. (i) Tumor growth curves following combined i.v. + i.t. treatment. (j) Kaplan–Meier survival curves following combined i.v. + i.t. treatment. Unless otherwise indicated, n = 5 mice per group. Data in panels c and d are presented as mean ± SEM. Data normality was assessed using the Shapiro–Wilk test. Normally distributed data were analyzed using an unpaired two-tailed Student’s t-test, whereas non-normally distributed data were analyzed using the Mann–Whitney U test. Survival differences in (g and j) were analyzed using the log-rank (Mantel–Cox) test.

To assess the therapeutic efficacy, Rpl18-MC38 tumors were subcutaneously implanted and allowed to grow to approximately 100 mm³. Following the same weekly schedule as the OVA model, mice received either control mRNA, Rpl18-MITD alone or the mRNA cocktail once per week for three weeks (Figure 3e). While Rpl18-MITD alone modestly delayed tumor growth, combination treatment resulted in significantly greater tumor suppression and extended survival (Figure 3f, g).

To determine whether combined systemic and local delivery could enhance therapeutic efficacy, we evaluated combined intravenous and intratumoral (i.v. + i.t.) administration using the same weekly treatment schedule (days 8, 15, and 22) (Figure 3h). Among the groups receiving combined i.v. + i.t. administration, the mRNA cocktail produced the greatest tumor control, with several mice achieving durable tumor suppression, and prolonged survival compared with the control mRNA and Rpl18-MITD groups (Figure 3i, j).

We next examined whether earlier treatment initiation could improve therapeutic efficacy. Intravenous administration of the mRNA cocktail beginning on day 3 after tumor implantation, before tumors reached the approximately 100 mm³ treatment threshold used in the standard therapeutic model, delayed tumor growth and prolonged survival compared with the untreated, control mRNA, and Rpl18-MITD groups (Supplementary Figure 3f–h).

These results demonstrate that in vivo administration of the mRNA cocktail promotes the expansion and functional differentiation of neoantigen-specific CD8⁺ T cells, thereby enhancing antitumor immunity and improving survival compared with antigen-only mRNA vaccination.

Prophylactic vaccination delays tumor progression and is associated with protective antitumor immune memory

We next examined whether prophylactic immunization with the mRNA cocktail could induce protective immunity against subsequent tumor challenge. Naïve C57BL/6 mice were immunized intravenously with the Rpl18-MITD+CD80+IL-2 mRNA cocktail or the indicated control formulations once weekly for three consecutive weeks on days −21, −14, and −7, followed by subcutaneous inoculation of Rpl18-MC38 cells on day 0 (Figure 4a).

A four-panel figure showing tumor growth curves and survival data from a prophylactic mRNA vaccination study in mice. A four-panel figure displays prophylactic vaccination results in C57BL/6 mice. Panel A shows the experimental timeline with mRNA injections on days minus 21, minus 14, and minus 7, followed by tumor cell transplantation on day 0. Panel B show individual tumor growth curves following tumor challenge, showing reduced tumor growth in mice that received the mRNA cocktail before tumor implantation. Panel C presents Kaplan-Meier survival curves corresponding to the tumor growth experiment. Panel D shows tumor rechallenge experiment showing individual tumor growth curves in previously treated mice compared with age-matched naïve mice. Previously treated mice included the tumor-free mouse from the prophylactic vaccination experiment and mice that achieved complete tumor regression following combined intravenous and intratumoral therapy.. — Prophylactic vaccination with an mRNA cocktail induces durable protective antitumor immunity. (a) Experimental design. Naïve C57BL/6 mice received weekly intravenous injections of control mRNA, Rpl18-MITD mRNA, or the Rpl18-MITD+CD80+IL-2 mRNA cocktail on days −21, −14, and −7, followed by subcutaneous implantation of Rpl18-MC38 cells on day 0. (b) Individual tumor growth curves after tumor challenge following prophylactic vaccination (n = 5 mice per group). (c) Kaplan–Meier survival curves corresponding to (b). (d) Tumor rechallenge experiment. The mouse that remained tumor-free after prophylactic vaccination (n = 1) and mice that achieved complete tumor regression after combined intravenous and intratumoral (i.v. + i.t.) therapy (n = 2; Figure 3h–j) were rechallenged with Rpl18-MC38 cells without additional mRNA administration. Age-matched naïve mice served as controls (n = 5). Individual tumor growth curves after rechallenge are shown. Survival differences in (c) were analyzed using the log-rank (Mantel–Cox) test.

Prophylactic vaccination with the mRNA cocktail delayed tumor growth compared with the untreated, control mRNA, and Rpl18-MITD groups (Figure 4b). Although four of the five mice receiving the mRNA cocktail eventually developed tumors, tumor progression was markedly slower than in the control groups, and one mouse remained tumor-free throughout the observation period. Consistent with these findings, all mice receiving the mRNA cocktail survived until the end of the study, whereas mice in the control groups reached humane endpoints substantially earlier (Figure 4c).

To examine whether successful tumor protection or regression was associated with durable antitumor immune memory, the tumor-free mouse from the prophylactic vaccination group and mice that had achieved complete tumor regression following combined intravenous and intratumoral (i.v. + i.t.) cocktail therapy (Figure 3h–j) were rechallenged with Rpl18-MC38 cells without additional treatment. All rechallenged mice showed marked resistance to secondary tumor challenge and substantially reduced tumor growth compared with age-matched naïve control mice (Figure 4d). These findings indicate that successful tumor protection or regression induced by the mRNA cocktail can be associated with protective antitumor immune memory.

An IL-12-integrated mRNA cocktail promotes Th1 differentiation of antigen-specific CD4⁺ T cells in vivo

To extend the functionality of the mRNA cocktail beyond CD8⁺ T cell activation, we designed constructs to promote the differentiation of antigen-specific CD4⁺ helper T cells. We generated mRNAs encoding membrane-anchored IL-12 and a fusion protein comprising the OVA peptide linked to the MHC class II molecule (I-A^b) via a flexible linker, enabling direct epitope presentation without intracellular processing (Figure 5a, Supplementary Figure 4a, b). Mice were intravenously injected with either OVA-MHC-II mRNA alone or a combination of OVA-MHC-II, CD80, and IL-12 mRNAs once weekly for two consecutive weeks. One week after the final injection, spleens were harvested and analyzed by flow cytometry using MHC class II tetramers to identify OVA-specific CD4⁺ T cells (Figure 5b). In mice treated with OVA-MHC-II mRNA alone, only minimal expansion of antigen-specific CD4⁺ T cells was observed. In contrast, mice receiving the mRNA cocktail exhibited a marked increase in the frequency of MHC-II tetramer⁺ CD4⁺ T cells (Figure 5c, d; Supplementary Figure 4c, d). Among these cells, 27% expressed T-bet, a master regulator of Th1 differentiation ²³ (Figure 5e, f; Supplementary Figure 4e). Furthermore, upon ex vivo stimulation with the OVA peptide, a substantial proportion of CD4⁺ T cells from the cocktail group produced IFN-γ, confirming functional Th1 polarization (Figure 5g; Supplementary Figure 4f). Comparable splenocyte numbers across groups (Supplementary Figure 4g) support an antigen-specific immune response. These results demonstrate that in vivo administration of the mRNA cocktail encoding an MHC-II-linked antigen and IL-12 effectively promotes the expansion and Th1 differentiation of antigen-specific CD4⁺ T cells.

Eleven panels showing mRNA constructs, Th1 differentiation, tumor growth, and survival in mice receiving mRNA cocktails. Eleven-panel immunotherapy figure showing mRNA construct design and therapeutic efficacy. Panel A: Schematic representations of the OVA-MHC-II, CD80, and membrane-anchored IL-12 mRNA constructs.. Panel B: Experimental timeline showing two weekly intravenous mRNA administrations in C57BL/6 mice, followed by spleen collection 7 days after the final dose.. Panel C: Representative flow cytometry plots showing OVA-specific CD4⁺ T cells in the spleen.. Panel D: Bar graph of tetramer+ CD4+ T cells across groups. Panel E: Flow cytometry plots measuring T-bet expression in OVA-specific CD4+ T cells . Panel F: Bar graph quantifying T-bet+ OVA-specific CD4+ T cells. Panel G: IFN-gamma+ CD4+ T cell numbers. Panel H: Therapeutic treatment schedule with four groups: untreated, MHC-I cocktail, MHC-II cocktail, and combined MHC-I and MHC-II cocktail, treatments on days 8, 11, 15, and 18. Panel I: Four tumor growth curves over 60 days showing combined treatment substantially reduces growth versus untreated and single-cocktail treatment groups. Panel J: Kaplan-Meier survival curves for the treatment groups. Panel K: Tumor rechallenge curves comparing naive mice to combined-treatment survivors, , demonstrating protective immunity. — An IL-12-integrated mRNA cocktail promotes Th1 differentiation of antigen-specific CD4⁺ T cells and enhances antitumor efficacy when combined with an MHC-I-targeted cocktail. (a) Schematic representations of the mRNA constructs encoding OVA-MHC-II, CD80, and membrane-anchored IL-12. (b) C57BL/6 mice received two weekly intravenous injections of control mRNA (15 µg), OVA-MHC-II mRNA (5 µg) supplemented with control mRNA (10 µg), or a cocktail of OVA-MHC-II, CD80, and membrane-anchored IL-12 mRNAs (5 µg each; total 15 µg). Spleens were collected 7 days after the final dose. (c) Representative dot plots of OVA-specific CD4⁺ T cells in the spleens. (d) Number of tetramer⁺ CD4⁺ T cells in the spleens. (e) Representative dot plots of T-bet expression in OVA-specific CD4⁺ T cells. (f) Number of T-bet⁺ OVA-specific CD4⁺ T cells in the spleens. (g) Number of IFN-γ⁺ CD4⁺ T cells following ex vivo stimulation with OVA peptide. (h) Experimental design for therapeutic treatment with MHC-I- and MHC-II-targeted mRNA cocktails. E.G7 tumor-bearing mice received the MHC-I-targeted cocktail (OVA-MITD+CD80+IL-2) on days 8 and 15, the MHC-II-targeted cocktail (OVA-MHC-II+CD80+IL-12) on days 8 and 15, or both cocktails according to an interdigitated schedule, with the MHC-I-targeted cocktail administered on days 8 and 15 and the MHC-II-targeted cocktail administered on days 11 and 18. (i) Individual tumor growth curves. (j) Kaplan–Meier survival curves. (k) Tumor rechallenge experiment. Tumor-free mice from the combined-treatment group (n = 2) were rechallenged with E.G7 cells without additional mRNA administration. Age-matched naïve mice served as controls (n = 5). Individual tumor growth curves after rechallenge are shown. Unless otherwise indicated, n = 5 mice per group. Data in (d, f, and g) are presented as mean ± SEM. Data normality was assessed using the Shapiro–Wilk test. Normally distributed data were analyzed using an unpaired two-tailed Student’s t-test, whereas non-normally distributed data were analyzed using the Mann–Whitney U test. Survival differences in (j) were analyzed using the log-rank (Mantel–Cox) test.

Combined administration of MHC class I- and class II-restricted mRNA cocktails enhances antitumor efficacy and establishes durable protective immunity

Given that the composition of the mRNA cocktail can be tailored to preferentially activate CD8⁺ T cell responses using CD80 and IL-2 or CD4⁺ T cell responses using CD80 and IL-12, we next investigated whether combined administration of an MHC-I-targeted cocktail (OVA-MITD+CD80+IL-2) and an MHC-II-targeted cocktail (OVA-MHC-II+CD80+IL-12) could further improve therapeutic efficacy. C57BL/6 mice bearing established E.G7 tumors were left untreated, treated with either the MHC-I-targeted or MHC-II-targeted cocktail on days 8 and 15, or treated with both cocktails according to an interdigitated schedule, with the MHC-I-targeted cocktail administered on days 8 and 15 and the MHC-II-targeted cocktail administered on days 11 and 18 (Figure 5h). Combined treatment with the MHC-I- and MHC-II-targeted cocktails resulted in greater tumor suppression than either cocktail alone, with several mice achieving complete tumor regression, and prolonged survival (Figure 5i, j). To determine whether this treatment also induced durable protective antitumor immunity, tumor-free mice from the combined-treatment group were rechallenged with E.G7 tumor cells without additional treatment. All rechallenged mice exhibited strong control of secondary tumor growth, with tumor volumes remaining below approximately 50 mm³ during the observation period, whereas age-matched naïve mice developed progressively growing tumors (Figure 5k). Collectively, these findings indicate that coordinated induction of antigen-specific CD8⁺ and CD4⁺ T cell responses through the combined MHC-I- and MHC-II-targeted cocktail regimen enhances antitumor efficacy and establishes durable protective immune memory.

In vivo evaluation of HLA-A*02:01-restricted responses induced by an mRNA cocktail

To assess the function of the mRNA cocktail in an HLA-A*02:01 transgenic model, we used HHD mice, which express a chimeric MHC class I molecule composed of the α1 and α2 domains of human HLA-A*02:01, the α3 domain of murine H-2D^b, and human β2-microglobulin. ¹⁷ This model enables the evaluation of HLA-A*02:01-restricted CD8⁺ T cell responses in vivo. As a representative tumor antigen, we selected NY-ESO-1, a cancer/testis antigen widely expressed in various malignancies and used its well-characterized HLA-A*02:01-restricted epitope (amino acids 157-165). ²⁴ Mice were administered either mRNA encoding NY-ESO-1-MITD alone or a combination of mRNAs encoding NY-ESO-1-MITD, CD80, and IL-2 (Figure 6a, Supplementary Figure 5a). NY-ESO-1-MITD alone failed to induce significant expansion of antigen-specific CD8⁺ T cells, whereas the combination treatment resulted in robust expansion and differentiation of CD44^hiCD62L^low effector/memory CD8⁺ T cells (Figure 6b–d, Supplementary Figure 5b, c). Consistent with earlier results, total splenocyte numbers remained unchanged across groups, indicating antigen-specific activation (Supplementary Figure 5d).

Seven panels showing NY-ESO-1-specific CD8+ T-cell responses and in vivo killing in HLA-A*02:01 transgenic mice. A seven-panel experimental diagram presents an mRNA immunotherapy study in HLA-A*02:01 transgenic mice. Panel A shows the timeline: intravenous mRNA injection on day 0, analysis on day 7. Panel B displays flow cytometry dot plots of NY-ESO-1-specific CD8+ T cells. Panel C bar graph shows tetramer+ CD8+ T cells. Panel D bar graph of IFNgamma+ CD8+ T cells . Panel E illustrates the in vivo killing assay, in which NY-ESO-1 peptide-pulsed CTV-labeled splenocytes and control WT1 peptide-pulsed CFSE-labeled splenocytes were transferred into treated mice, followed by analysis of target-cell killing 24 hours later. Panel F represents flow cytometry dot plots showing CTV+ and CFSE+ target cells across three treatment groups with percentages in each quadrant. Panel G bar graph displays quantification of the CTV⁺/CFSE⁺ target-cell ratio, showing enhanced elimination of NY-ESO-1 peptide-pulsed target cells following treatment with the mRNA cocktail. — An mRNA cocktail activates HLA-A*02:01-restricted NY-ESO-1-specific CD8⁺ T cells in mice. (a) HLA-A*02:01 transgenic mice received a single intravenous dose of control mRNA (15 µg), NY-ESO-1-MITD mRNA (5 µg) supplemented with control mRNA (10 µg), or a cocktail of NY-ESO-1-MITD, mouse CD80, and membrane-anchored mouse IL-2 mRNAs (5 µg each; total 15 µg). Spleens were collected 7 d later. (b) Representative dot plots of NY-ESO-1-specific CD8⁺ T cells in the spleens. (c) Number of tetramer⁺ CD8⁺ T cells in the spleens. (d) Number of IFN-γ⁺ CD8⁺ T cells in the spleens. (e) In vivo killing assay. On day 0, mice were treated as described in (a). On day 7, 1 × 10⁶ NY-ESO-1 peptide-pulsed splenocytes labeled with CellTrace Violet (CTV) and 1 × 10⁶ WT1 peptide-pulsed splenocytes labeled with CFSE were transferred intravenously. Spleens were harvested 24 h later, and target-cell killing was analyzed by flow cytometry. (f) Representative dot plots of CTV⁺ and CFSE⁺ target cells. (g) CTV⁺/CFSE⁺ target-cell ratio in the spleens. Unless otherwise indicated, n = 5 mice per group. Data in (c, d, and g) are presented as mean ± SEM. Data normality was assessed using the Shapiro–Wilk test. Normally distributed data were analyzed using an unpaired two-tailed Student’s t-test, whereas non-normally distributed data were analyzed using the Mann–Whitney U test. For panel (g), n = 4 mice per group.

To assess cytotoxic activity, we performed an in vivo killing assay using a 1:1 mixture of splenocytes pulsed with NY-ESO-1 peptide (CellTrace Violet-labeled) and control peptide (CFSE-labeled), injected into mRNA-treated mice seven days after mRNA administration (Figure 6e). NY-ESO-1 peptide-pulsed target cells were selectively eliminated in mice treated with the mRNA cocktail, but not in those receiving control mRNA or NY-ESO-1-MITD mRNA alone (Figure 6f, g). These findings demonstrate that in vivo administration of the mRNA cocktail can effectively induce functional antigen-specific cytotoxic T lymphocytes (CTLs) within an HLA-A*02:01-restricted setting.

Discussion

In this study, we developed a modular mRNA cocktail that integrates antigen presentation with co-stimulatory and cytokine signals to enhance antigen-specific T cell responses. Co-delivery of antigen-encoding mRNA with CD80 and membrane-anchored IL-2 markedly increased the expansion and effector differentiation of antigen-specific CD8⁺ T cells and improved tumor control in both OVA-expressing E.G7 and Rpl18-expressing MC38 tumor models. By replacing IL-2 with membrane-anchored IL-12 and using an MHC class II-linked antigen construct, the same platform also promoted the expansion and Th1 differentiation of endogenous antigen-specific CD4⁺ T cells. These findings demonstrate that the composition of the mRNA cocktail can be adjusted to direct distinct antigen-specific T cell responses and provide a strategy to enhance the immunogenicity of mRNA cancer vaccines.

Our results extend recent studies examining IL-12 mRNA as a vaccine adjuvant. Co-administration of IL-12 mRNA-LNPs with antigen-encoding mRNA-LNPs has been shown to enhance CD8⁺ T cell expansion and effector differentiation, whereas membrane-tethered IL-12 mRNA can promote Th1-oriented immune responses in vivo. ²⁵ ^, ²⁶ In our platform, the combination of an MHC class II-linked antigen, CD80, and membrane-anchored IL-12 induced both expansion and functional Th1 polarization of endogenous antigen-specific CD4⁺ T cells, as indicated by T-bet expression and IFN-γ production. Moreover, interdigitated administration of the MHC-I- and MHC-II-targeted cocktails improved tumor control compared with either cocktail alone. This finding is consistent with the established importance of CD4⁺ T cell help in supporting effective and durable antitumor CD8⁺ T cell responses ²⁷ and highlights the value of coordinating complementary T cell subsets through modular mRNA design.

The therapeutic studies also suggest that treatment regimen influences the efficacy of the mRNA cocktail. Earlier treatment initiation and combined intravenous and intratumoral administration were both associated with potent antitumor activity, indicating that further optimization of treatment timing and administration route may improve therapeutic outcomes. Although a three-dose weekly regimen was used in the present study, the optimal number of vaccine doses and dosing intervals were not systematically evaluated and should be determined in future studies. Prophylactic vaccination delayed tumor progression and prolonged survival, although it did not completely prevent primary tumor formation in all mice. Moreover, mice that remained tumor-free after prophylactic vaccination or achieved complete tumor regression after therapeutic treatment showed marked protection against subsequent challenge with the same tumor. Strong protection against E.G7 rechallenge was also observed in mice treated with the combined MHC-I- and MHC-II-targeted regimen. Together, these findings support the ability of the mRNA cocktail platform to induce protective antitumor immune memory in addition to primary effector responses.

The induction of NY-ESO-1-specific CD8⁺ T cell responses in HLA-A*02:01 transgenic mice further supports the translational potential of this approach. The NY-ESO-1-targeted cocktail increased the number of antigen-specific effector CD8⁺ T cells and induced selective elimination of NY-ESO-1 peptide-pulsed target cells in vivo. These results demonstrate that the platform can generate functional cytotoxic T lymphocytes against a clinically relevant human tumor antigen in an HLA-restricted setting. However, HLA-A*02:01 transgenic mice do not reproduce the full complexity of the human immune system. Evaluation using primary human antigen-presenting cells and T cells, followed by studies in more physiologically relevant models, will therefore be required to establish its applicability to human cancer immunotherapy.

Several limitations should be considered. First, we used a commercially available, non-targeted liposomal reagent and did not determine which cell populations were primarily responsible for mRNA uptake, antigen presentation, and T cell priming in vivo. Incorporation of the cocktail into delivery systems optimized for dendritic cells, including LNPs or LPX formulations, may improve delivery efficiency and reduce off-target expression. ^28-30 Second, although increased antigen-specific CD8⁺ and CD4⁺ T cell responses were associated with improved tumor control, depletion or genetic experiments will be required to define the relative contribution of each T cell subset to therapeutic efficacy. Third, the distribution, duration of expression, and systemic exposure of the membrane-anchored cytokines were not comprehensively evaluated. Membrane tethering was intended to spatially restrict cytokine activity, but detailed dose optimization, biodistribution analysis, and long-term safety studies remain necessary, particularly for IL-12-containing formulations. ³¹ ^, ³² Finally, direct comparisons of alternative administration routes and larger rechallenge studies will be needed to validate the effects of treatment route and the durability of immune memory.

The modular nature of this platform should facilitate rapid evaluation of alternative antigens, co-stimulatory molecules, and cytokines. During the initial development of this platform, we also evaluated a single multicistronic mRNA encoding the antigen, CD80, and cytokine components using 2 A peptide sequences. However, the increased transcript length reduced the reproducibility of intact mRNA synthesis and appeared to compromise the expression of the individual components. We therefore adopted a modular cocktail of separately synthesized mRNAs, which also allows the dose of each component to be adjusted independently. Nevertheless, once an optimal combination has been identified, incorporation of the selected components into a single multicistronic mRNA may simplify manufacturing and improve co-expression in individual recipient cells. Future advances in mRNA synthesis and multicistronic expression strategies may help overcome the limitations observed in the present study. The platform may also be suitable for combination with immune checkpoint blockade, which could help maintain the function of vaccine-expanded tumor-reactive T cells within an immunosuppressive tumor microenvironment. ³³ ^, ³⁴ In summary, our findings establish a modular mRNA cocktail strategy that coordinates antigen presentation, co-stimulation, and cytokine signaling to induce antigen-specific CD8⁺ and CD4⁺ T cell responses. This approach produced antitumor activity, functional HLA-A*02:01-restricted cytotoxic T cells, and durable protective immunity, providing a preclinical foundation for the further development of programmable mRNA cancer vaccines.

Supplementary Material

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Acknowledgments

We thank T. Yoshida for helpful discussions. We thank the NIH Tetramer Core Facility (NIH contract 75N93020D00005; RRID: SCR_026557) for providing H-2K^b/OVA (257-264), H-2K^b/Rpl18 (KILTFDRL), HLA-A*02:01-H2K^b/NY-ESO-1 (SLLMWITQC), and I-A^b/OVA (323-339) tetramers. ChatGPT (GPT-4, OpenAI) was used to refine language and enhance readability. The authors take full responsibility for the final manuscript.

Funding Statement

This work was supported by the World Premier International Research Center Initiative (WPI), MEXT, Japan, the World-leading Innovative & Smart Education (WISE) Program for Nano-Precision Medicine (TVL, SI), the Japan Science and Technology Agency (JST) Fusion Oriented Research for Disruptive Science and Technology (FOREST) grant no. JPMJFR2115 (TY), Practical Research for Innovative Cancer Control from the Japan Agency for Medical Research and Development (AMED) grant no. 24ck0106967h0001 (TY) and AMED grant no. JP256f0137001(TY), and Research and Development Program for Innovative Biologics from AMED grant no. 26am0521007h0003 (RH).

Disclosure of potential conflicts of interest

R.H. and T.Y. have applied for a patent (Japanese Patent Application No. 2025-076452) entitled “Method for Immune Regulation and Composition for Immunomodulation.” All other authors declare that they have no competing interests to disclose.

Data availability statement

Upon reasonable request, data can be obtained from the corresponding author.

Ethics approval statement

The experiments included in the original submission were conducted from 1 April 2024 to 26 May 2025. The additional experiments were performed separately from 27 May 2025 to 16 July 2026. All animal experiments were performed in accordance with the ARRIVE guidelines and were approved by the Animal Experimentation Committee of Kanazawa University (Kanazawa, Ishikawa, Japan; approval number: AP-204131 and AP25-046). Female C57BL/6 mice aged 6 to 8 weeks and weighing 18–22 g were used in this study. In total, 216 animals were included. Mice were group-housed (up to five per cage) under specific pathogen-free conditions, with ad libitum access to standard rodent chow and water, and maintained in a controlled environment (temperature, humidity, and 12-hour light/dark cycle). Environmental enrichment (e.g., nesting materials and shelters) was performed to minimize stress. At the end of the experiments, the animals were euthanized using CO₂ inhalation with a gradual fill rate, in accordance with the AVMA guidelines.

Supplementary material

Supplemental data for this article can be accessed at https://doi.org/10.1080/2162402X.2026.2736339.

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12. Del Prete A, Salvi V, Soriani A, Laffranchi M, Sozio F, Bosisio D, Sozzani S. Dendritic cell subsets in cancer immunity and tumor antigen sensing. Cell Mol Immunol. 2023;20(5):432–447. doi: 10.1038/s41423-023-00990-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
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20. Kreiter S, Selmi A, Diken M, Sebastian M, Osterloh P, Schild H, Huber C, Türeci O, Sahin U. Increased antigen presentation efficiency by coupling antigens to MHC class I trafficking Signals. J Immunol. 2008;180(1):309–318. doi: 10.4049/jimmunol.180.1.309. [DOI] [PubMed] [Google Scholar]
21. Gallie DR. The cap and poly(A) tail function synergistically to regulate mRNA translational efficiency. Genes Dev. 1991;5(11):2108–2116. doi: 10.1101/gad.5.11.2108. [DOI] [PubMed] [Google Scholar]
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27. Borst J, Ahrends T, Bąbała N, Melief CJM, Kastenmüller W. CD4+ T cell help in cancer immunology and immunotherapy. Nat Rev Immunol. 2018;18(10):635–647. doi: 10.1038/s41577-018-0044-0. [DOI] [PubMed] [Google Scholar]
28. Sasaki K, Sato Y, Okuda K, Iwakawa K, Harashima H. mRNA-Loaded lipid nanoparticles targeting dendritic cells for cancer immunotherapy. Pharmaceutics. 2022;14(8):1572. doi: 10.3390/pharmaceutics14081572. [DOI] [PMC free article] [PubMed] [Google Scholar]
29. Mahalingam G, Rachamalla HK, Arjunan P, Karuppusamy KV, Periyasami Y, Mohan A, Subramaniyam K, M S, Rajendran V, Moorthy M, et al. SMART-lipid nanoparticles enabled mRNA vaccine elicits cross-reactive humoral responses against the omicron sub-variants. Mol Ther. 2024;32(5):1284–1297. doi: 10.1016/j.ymthe.2024.02.028. [DOI] [PMC free article] [PubMed] [Google Scholar]
30. Sahin U, Oehm P, Derhovanessian E, Jabulowsky RA, Vormehr M, Gold M, Maurus D, Schwarck-Kokarakis D, Kuhn AN, Omokoko T, et al. An RNA vaccine drives immunity in checkpoint-inhibitor-treated melanoma. Nature. 2020;585(7823):107–112. doi: 10.1038/s41586-020-2537-9. [DOI] [PubMed] [Google Scholar]
31. Bitounis D, Jacquinet E, Rogers MA, Amiji MM. Strategies to reduce the risks of mRNA drug and vaccine toxicity. Nat Rev Drug Discov. 2024;23(4):281–300. doi: 10.1038/s41573-023-00859-3. [DOI] [PubMed] [Google Scholar]
32. Wang J, Ding Y, Chong K, Cui M, Cao Z, Tang C, Tian Z, Hu Y, Zhao Y, Jiang S. Recent advances in lipid nanoparticles and their safety concerns for mRNA delivery. Vaccines (Basel). 2024;12(10):1148. doi: 10.3390/vaccines12101148. [DOI] [PMC free article] [PubMed] [Google Scholar]
33. Sharma P, Allison JP. The future of immune checkpoint therapy. Science. 2015;348(6230):56–61. doi: 10.1126/science.aaa8172. [DOI] [PubMed] [Google Scholar]
34. Ribas A, Wolchok JD. Cancer immunotherapy using checkpoint blockade. Science. 2018;359(6382):1350–1355. doi: 10.1126/science.aar4060. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material

Author Checklist Full 0731

KONI_A_2736339_SM9504.pdf^{(448.8KB, pdf)}

Fig S4 260716.tif

KONI_A_2736339_SM4493.tif^{(16.5MB, tif)}

Supplementary_Figure_Legend_260717 Clean version.docx

KONI_A_2736339_SM4492.docx^{(18KB, docx)}

Fig S1 260716.tif

KONI_A_2736339_SM4491.tif^{(16.1MB, tif)}

Fig S2 260716.tif

KONI_A_2736339_SM4490.tif^{(15.7MB, tif)}

Fig S5 260716.tif

KONI_A_2736339_SM4489.tif^{(16.6MB, tif)}

Fig S3 260716.tif

KONI_A_2736339_SM4488.tif^{(19.3MB, tif)}

Data Availability Statement

Upon reasonable request, data can be obtained from the corresponding author.

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[cit0020] 20. Kreiter S, Selmi A, Diken M, Sebastian M, Osterloh P, Schild H, Huber C, Türeci O, Sahin U. Increased antigen presentation efficiency by coupling antigens to MHC class I trafficking Signals. J Immunol. 2008;180(1):309–318. doi: 10.4049/jimmunol.180.1.309. [DOI] [PubMed] [Google Scholar]

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[cit0030] 30. Sahin U, Oehm P, Derhovanessian E, Jabulowsky RA, Vormehr M, Gold M, Maurus D, Schwarck-Kokarakis D, Kuhn AN, Omokoko T, et al. An RNA vaccine drives immunity in checkpoint-inhibitor-treated melanoma. Nature. 2020;585(7823):107–112. doi: 10.1038/s41586-020-2537-9. [DOI] [PubMed] [Google Scholar]

[cit0031] 31. Bitounis D, Jacquinet E, Rogers MA, Amiji MM. Strategies to reduce the risks of mRNA drug and vaccine toxicity. Nat Rev Drug Discov. 2024;23(4):281–300. doi: 10.1038/s41573-023-00859-3. [DOI] [PubMed] [Google Scholar]

[cit0032] 32. Wang J, Ding Y, Chong K, Cui M, Cao Z, Tang C, Tian Z, Hu Y, Zhao Y, Jiang S. Recent advances in lipid nanoparticles and their safety concerns for mRNA delivery. Vaccines (Basel). 2024;12(10):1148. doi: 10.3390/vaccines12101148. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0033] 33. Sharma P, Allison JP. The future of immune checkpoint therapy. Science. 2015;348(6230):56–61. doi: 10.1126/science.aaa8172. [DOI] [PubMed] [Google Scholar]

[cit0034] 34. Ribas A, Wolchok JD. Cancer immunotherapy using checkpoint blockade. Science. 2018;359(6382):1350–1355. doi: 10.1126/science.aar4060. [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

Modular mRNA cocktail enables synergistic activation of antigen-specific T cells for cancer immunotherapy

Toan Van Le

Shota Imai

Iriya Fujitsuka

Makie Ueda

Sayuri Nakamae

Shusaku Mizukami

Rikinari Hanayama

Tomoyoshi Yamano

Roles

Abstract

Introduction

Materials and methods

Cell lines

Mice

mRNA construct and in vitro transcription

mRNA transfection in vitro

In vitro T-cell proliferation assay

mRNA formulation and intravenous administration

Tetramer staining

In vivo tumor models and mRNA treatment regimen

Early initiation of mRNA treatment

Combined intravenous and intratumoral administration

Prophylactic vaccination and tumor rechallenge

Combined MHC-I- and MHC-II-targeted mRNA cocktail treatment

Isolation and preparation of tumor-infiltrating lymphocytes (TILs)

In vivo killing assay

Antibodies and flow cytometry

Statistical analysis

Results

Generation of mRNA cocktail-transfected cells for activation of antigen-specific CD8⁺ T cells

Figure 1.

In vivo administration of an mRNA cocktail stimulates antigen-specific CD8⁺ T cells

Figure 2.

In vivo administration of an mRNA cocktail expands neoantigen-specific CD8⁺ T cells

Figure 3.

Prophylactic vaccination delays tumor progression and is associated with protective antitumor immune memory

Figure 4.

An IL-12-integrated mRNA cocktail promotes Th1 differentiation of antigen-specific CD4⁺ T cells in vivo

Figure 5.

Combined administration of MHC class I- and class II-restricted mRNA cocktails enhances antitumor efficacy and establishes durable protective immunity

In vivo evaluation of HLA-A*02:01-restricted responses induced by an mRNA cocktail

Figure 6.

Discussion

Supplementary Material

Acknowledgments

Funding Statement

Disclosure of potential conflicts of interest

Data availability statement

Ethics approval statement

Supplementary material

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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