Neoantigen mRNA vaccines and A₂A receptor antagonism: A strategy to enhance T cell immunity

ABSTRACT

Although neo-antigen mRNA vaccines are promising for personalized cancer therapy, their effectiveness is often limited by the immunosuppressive tumor microenvironment (TME). The adenosine A₂A receptor (A₂AR) inhibits dendritic cell (DC) function and weakens antitumor T cell responses through hypoxia-driven mechanisms within the TME. This review explores a novel strategy combining neo-antigen mRNA vaccines with A₂AR antagonists (A₂ARi). By targeting A₂AR, this approach reduces TME-induced immunosuppression, enhances DC activation, and improves neo-antigen presentation. The review also discusses lipid nanoparticles (LNPs) to co-deliver A₂ARi and mRNA vaccines, optimizing their effectiveness. The integration of neo-antigen mRNA-LNPs with A₂ARi modulation offers a promising strategy to overcome immunosuppression, stimulate DC activation, and achieve precise anti-tumor responses with minimal off-target effects. This synergy represents significant progress in cancer immunotherapy, advancing the potential for personalized neoantigen therapies.

Graphical abstract

Introduction

Developing neo-antigen messenger RNA (mRNA) vaccines represents a significant advancement in personalized neoantigen therapy.^1,2 These vaccines encode tumor-specific antigens, facilitating targeted immune responses against cancer cells with precision.^2,3 Their safety, efficacy, and scalability enable rapid clinical implementation and cost-efficient production, positioning neo-antigen mRNA vaccines as a compelling alternative to conventional therapeutic strategies.^2–4

Within the tumor microenvironment (TME), adenosine (AD) emerges as a pivotal regulator of the immune response.^5,6 Its interaction with G-protein-coupled adenosine receptors (ARs), particularly adenosine A₂A receptors (A₂AR), which influences immune dynamics.⁶ A₂AR, found in various immune cell types, including regulatory T cells, cytotoxic T cells, and macrophages, plays a central role in immune tolerance maintenance.^7,8 Activation of the A₂AR pathway triggers intricate signaling cascades, particularly the cAMP/PKA/CREB pathway.^9,10 Recent research has highlighted FDA-approved small-molecule A₂AR antagonists (A₂ARi) for cancer therapy, including AZD4635 (AstraZeneca),¹¹ Ciforadenant (Corvus Pharm),¹² EOS100850 (iTeos Therapeutics),¹³ Etrumadenant (Arcus Biosciences),¹⁴ JNJ-40255293 (Johnson & Johnson),¹⁵ Taminadenant (Pablobio/Novartis),¹⁶ and AB928 (Arcus Biosciences).¹⁷ These compounds are being evaluated for their potential effectiveness as monotherapies or in combination with other anticancer agents.¹⁸ A₂ARi can significantly enhance the expression of targeted genes across various diseases, including neurodegenerative diseases,¹⁹ cellular hypoxia,²⁰ inflammatory disorders,²¹ and cancer.^18,22 For example, experiments conducted with rat retinal Müller cells exposed to hypoxic conditions showed a notable increase in the expression of Kir 2.1 and Kir 4.1 channels, which could be beneficial for treating retinal diseases.²³ These findings suggest that A₂ARi holds promise as an effective therapeutic compound beyond oncology, offering potential applications in a wide range of clinical conditions. The A₂ARi has become a key immunosuppressive factor within the TME, and its blockade can restore anti-tumor immunity, resulting in improved overall survival for patients.^24,25

This review perspective explores the innovative synergy between A₂ARi and neo-antigen mRNA vaccines in cancer immunotherapy, particularly emphasizing the role of dendritic cells (DCs).^7,26 By co-administering neo-antigen mRNA encapsulated in LNPs alongside A₂ARi, we aim to enhance the anti-tumor immune response through complementary pathways. The review highlights the necessity of optimal encapsulation for both components within LNPs and proposes strategies to refine LNP delivery systems for seamless integration. This combined approach marks a significant advancement in the development of neo-antigen mRNA vaccines and holds great promise for INT.

Body

ARs expression across immune cell subsets

Firstly, we conducted a comprehensive analysis of The Cancer Genome Atlas (TCGA) dataset to examine the expression patterns of ARs- specifically A₁R, A₂AR, A₂BR, and A₃R-across various immune cell subsets in the context of acute myeloid leukemia (AML).^8,27 AML was chosen for this study due to its pronounced heterogeneity and the high expression of AR family members within the TME, making it an ideal model for investigating the role of ARs in immune modulation. This analysis aimed to identify potential correlations between AR expression and immune cell populations, providing valuable insights into the therapeutic relevance of ARs in regulating immune responses and their potential for targeted therapies in AML. Figure 1 shows the expression of ARs (A₁R, A₂AR, A₂BR, A₃R) in immune cell subsets of AML, showing that A₂AR is widely expressed in immune populations, including DCs, macrophages, T cells, natural killer (NK) cells, and tumor-associated macrophages (TAMs), playing a significant role in modulating immune responses within the TME.⁸ Both CD4⁺ and CD8⁺ T cells express A₂AR, indicating its role in regulating T cell functions. Additionally, activated regulatory T cells (aTreg), memory regulatory T cells (mTreg), and naive regulatory T cells (nTreg) also show substantial A₂AR expression, underscoring their importance in regulatory T cell-mediated immune responses.^28,29

Figure 1.

Expression profile of main ARs across immune cell subsets in TME. This heatmap depicts the differential expression levels of A₁R, A₂AR, A₂BR, and A₃R across various immune cell populations implicated in the TME. Immune cell types include DCs, Th cells, macrophages, NK cells, granulocytes, mast cells, eosinophils, basophils, monocytes, platelets, and ILCs. The data suggest prominent expression of A₂AR on DCs, Th cells, and macrophages, highlighting its potential role in modulating immune responses within the TME. This comprehensive analysis provides insights into the distribution of A₂AR across immune cell subsets, underscoring its significance as a potential target for cancer immunotherapy. The data presented in this figure are derived from extensive genomic analyses, incorporating datasets from the cancer genome atlas (TCGA).²⁷

The expression of A₂AR in DCs suggests its involvement in modulating DC-mediated immune responses, potentially influencing antigen presentation and T-cell activation.³⁰ A₁R and A₃R are also expressed in various immune cells, including T cells, macrophages, and DCs, albeit to a lesser extent compared to A₂AR. While A₁R and A₃R may not be as abundantly expressed as A₂AR, their activation modulates intracellular signaling pathways by specific mechanisms, such as altering cAMP levels or activating specific kinases. This, in turn, influences immune cell functions such as cytokine production, cell migration, or cytotoxic activity within the TME.³¹ A₂BR, expressed in various immune cells including macrophages and DCs, influences their maturation and antigen-presenting capabilities, affecting T-cell priming and anti-tumor immunity. The robust expression of A₂AR on both M1 and M2 macrophages suggests its involvement in modulating macrophage polarization and function within the TME, especially via an M2-like macrophage.

In Supplementary Figure S1, we present the 3D structural diversity of ARs, highlighting the heterodimeric complexes of A₁R-A₃R and A₂AR-A₂BR. The figure showcases the 3D conformation and key features of A₁R-A₃R and A₂AR-A₂BR, including their transmembrane helices, binding pockets, and the conformational changes associated with ligand binding and receptor activation. The A₂AR-A₂BR heterodimers in the TME suppress immune responses by affecting three main immune cell types, cytotoxic T lymphocytes (CTLs), NK cells, and macrophages.³² These heterodimers profoundly influence immune function, particularly impacting CD4⁺ T cells essential for cellular immunity.³³ Additionally, A₂AR-A₂BR heterodimers enhance the generation of FoxP3⁺ Tregs, which suppress anti-tumor responses while maintaining immune balance.^25,34,35 Inhibiting A₂AR-A₂BR heterodimers enhances the infiltration and function of CD8⁺ T cells within tumors, significantly impacting tumor growth and metastasis in various cancer models.³⁶ The sensitivity of CD8⁺ T cells to AD varies with their differentiation stages, affecting cytokine secretion and migratory capabilities, which can be partially restored by targeting specific potassium channels. Furthermore, A₂AR-A₂BR heterodimers affect metabolic pathways like PKA and mTORC1, impairing the metabolic fitness crucial for sustained anti-tumor responses.^37,38 As Supplement Figure S1 displays the structures of ARs and their homology. It compares the structures of A₁R-A₃R and A₂AR-A₂BR, highlighting their similarities and differences. Similarly, A₂AR-A₂BR heterodimers restrict NK cell development and suppress NK cell maturation and proliferation.⁸ They reduce the expression of key pro-inflammatory factors such as IFN-γ, IL-2, GM-GSF, MIP-1α, and TNF-α, and diminish NK cell cytotoxicity by lowering perforin levels and impairing the FASL-dependent pathway. Moreover, A₂AR-A₂BR heterodimers skew macrophages toward an M2 phenotype by increasing IL-10 and decreasing pro-inflammatory factors, impairing their ability to perform antibody-mediated cellular phagocytosis, crucial for anti-tumor immunity.³⁹

A₂AR in immune function and antigen presentation

Upregulation of A₂AR signaling profoundly affects various immune cells, contributing to tumor progression by impairing the immune system’s ability to target and eliminate cancer cells (Figure 2). A₂AR signaling significantly affects the functions of various immune cells, including CD8⁺ T cells, NK cells (both CD56^dimCD16⁻ and CD56^brightCD16⁻), DCs (cDC1s, cDC2s, and pDCs), macrophages (M1 and M2), and MDSCs (PMN-MDSCs and M-MDSCs).^40,41

Figure 2.

The A₂AR signaling within the TME. The generation and modulation of extracellular adenosine accentuate its multifaceted role in shaping the TME. This intricate interplay, orchestrated by the A₂AR signaling, involves diverse immune cell subsets within the TME, including dendritic cells (DCs), regulatory T cells (Tregs), effector T cells (Teffs), natural killer (NK) cells, tumor-associated macrophages (TAMs), and myeloid-derived suppressor cells (MDSCs). This signaling axis shapes the dynamics of tumor immune evasion and progression at both cellular and molecular levels. Furthermore, A₂AR activation extends its influence beyond immune cells to nonimmune components of the TME, including tumor cells and stromal cells, impacting processes like epithelial-mesenchymal transition and contributing to tumor progression.

Upon A₂AR activation, DCs exhibit impaired maturation and antigen-presenting capacity, compromising their ability to initiate effective anti-tumor immune responses. This leads to the expansion and dominance of Tregs, suppressing effector T cells (Teffs) and upregulating inhibitory receptors like PD-1.⁴² Consequently, the immunosuppressive milieu within the TME is amplified, impeding anti-tumor immunity. NK cells, known for their cytotoxic activity against tumors, also experience diminished activity following A₂AR activation, reducing their ability to recognize and eliminate tumor cells. Similarly, TAMs polarize toward an immunosuppressive M2-like phenotype in response to A₂AR signaling, fostering an environment conducive to tumor progression.⁴³

Moreover, MDSCs expand and enhance their suppressive capabilities under A₂AR activation, hindering effector T-cell function and promoting Treg proliferation, thus exacerbating immunosuppression within the TME.⁴¹ A₂AR activation also initiates intracellular events, including the cAMP/PKA/CREB pathway, suppressing pro-inflammatory signaling pathways like NFκB and JAK/STAT, further fortifying immunosuppressive barriers to tumor progression.⁴⁴ Similarly, it undermines the maturation and antigen-presenting capabilities of DCs, hindering effective antigen presentation and bolstering immune evasion. Additionally, A₂AR activation suppresses NK cell cytotoxicity and promotes Treg expansion, fostering an immunosuppressive milieu within the TME. Within TAMs, A₂AR signaling orchestrates a transformation toward an immunosuppressive phenotype.²⁴

A₂AR activation suppresses the expression of MHC-II and co-stimulatory molecules like CD80 and CD86 on DCs, compromising their ability to present tumor antigens to CD4⁺ and CD8⁺ T cells and initiate anti-tumor responses.⁴⁵ In mature DCs, A₂AR activation reduces MHC-II antigen presentation, impeding T-cell activation and proliferation within the TME. A₂AR signaling also disrupts the maturation process of nascent DCs (nDCs), altering cytokine secretion profiles toward an anti-inflammatory state dominated by IL-10 and TGF-β.⁴⁶ Conventional DCs (cDCs), including cDC1s and cDC2s, experience reduced expression of MHC molecules, affecting their ability to present MHC class I- and II-restricted neoantigens to T cells. This suppression significantly impairs the activation of both CD8⁺ and CD4⁺ T cells, further undermining anti-tumor immune responses.⁴⁷

Plasmacytoid DCs (pDCs), though primarily involved in antiviral responses, may also exhibit decreased neoantigen presentation under A₂AR activation (Figure 2). A₂AR signaling in pDCs alters their cytokine secretion, reducing pro-inflammatory cytokines and enhancing the production of anti-inflammatory cytokines such as IL-10 and TGF-β, which contributes to immune tolerance and the expansion of Tregs.⁴⁸ Similarly, A₂AR activation in macrophages downregulates MHC-II expression and co-stimulatory molecules, impairing their antigen-presenting capacity and weakening the adaptive immune response to tumors.⁴⁹

Neo-antigen mRNA vaccines: advances and challenges

Personalized neo-antigen mRNA vaccines offer a groundbreaking approach to personalized neoantigen therapy by targeting tumor-specific neo-antigens. These vaccines utilize non-replicating mRNA (NRM) and self-amplifying mRNA (SAM) techniques, each with distinct advantages affecting stability, immunogenicity, and translatability, as outlined in Table 1. Table 1 compares the different neo-antigen mRNA platforms, including In vitro transcribed mRNA (IVT-mRNA), Chemically synthesized minimal mRNA (CmRNA), and Circular RNA (circRNA), in terms of stability, immunogenicity, and translational efficiency. By delivering neo-antigen mRNA-LNPs encoding tumor-specific antigens directly to DCs, they trigger precise and effective immune responses against cancer cells while avoiding integration with host DNA, thus minimizing genetic alteration risks.⁵⁰ Structural modifications in the mRNA further enhance vaccine stability and processing by DCs, while streamlined production in a cell-free environment enables rapid, scalable manufacturing.⁵¹

Table 1.

Comparison of neo-antigen mRNA technologies: ivt-mRNA, CmRNA, and circRNA.

	Non-replicating mRNA (NRM)		Self-amplification mRNA (SAM)
Type	ssRNA	ssRNA	ss-circRNA
Length (bp)	20–100	900–3000	500–50000
Potency	High protein levels with enhanced expression	Low levels of proteins	High protein levels with enhanced expression
Stability	High, engineered for stability with chemical modifications	Moderate, enhanced with UTRs modifications	High, naturally stable due to its circular structure
Translatability	High, enhanced by chemical modifications and specific nucleotide arrangements	Moderate, optimized with tailored sequences	Low, limited by circular structure’s impact on translation
Concentration/dose	0.5–2 ug/dose	50–200 ug/dose	1 ug/dose
Delivery	Not specified	LNPs, electroporation, direct injection	Viral vectors, LNPs, exosome
Immunity	Innate and adaptive – Mϕ, DCs, NK	Mainly adaptive; T Cells, B Cells	Mainly innate; Mϕ, NK
Therapeutical risk	No therapeutical risk	No therapeutical risk	Anti – vector effects due to interactions with host factors and encoded proteins

Abbreviations: ssRNA, single-stranded RNA; ss-circRNA, single-stranded circular RNA; bp, base pairs; UTRs, untranslated regions; ug, micrograms; LNPs, lipid nanoparticles; Mϕ, macrophages; DCs, dendritic cells; NK, natural killer cells; T Cells, T lymphocytes; B Cells, B lymphocytes.

However, the development of these vaccines presents challenges. One key issue is generating potent and lasting immune responses against tumor-specific antigens without causing off-target effects or adverse reactions. Additionally, the rapid identification of patient-specific neo-antigens and the natural instability of mRNA present obstacles to effective vaccine delivery.^52–54 Improving the stability and immune response of mRNA vaccines is being addressed through tailored lipid nanoparticles (LNPs) and advanced encapsulation methods.⁵⁰ Accurate targeting of antigen-presenting cells, particularly different subsets of DCs like plasmacytoid DCs (pDCs), conventional type 1 DCs (cDC1s), and conventional type 2 DCs (cDC2s), is crucial for maximizing vaccine efficacy while minimizing off-target effects.⁵⁵ Moreover, ensuring scalability, reproducibility, and meeting regulatory standards in vaccine manufacturing remain significant hurdles.^56,57 Enhancing DC uptake, activation, and MHC class I and II expression could further improve vaccine effectiveness.⁵⁸

The A₂ARi in neo-antigen mRNA vaccines

Figure 3 shows the structure of neo-antigen mRNA-loaded LNPs with A₂ARi, highlighting their lipid components and design for targeted delivery to A₂AR-expressing DCs. Incorporating A₂ARi into neo-antigen mRNA vaccine formulations represents a pivotal advancement in cancer immunotherapy.⁵⁰ By disrupting the immunosuppressive AD pathway, these antagonists unleash the full immunostimulatory potential of neo-antigen mRNA vaccines, aiming for more robust and enduring anti-tumor immune responses.

Figure 3.

Schematic of neo-antigen mRNA-LNPs+A₂ARi vaccines architecture. This diagram shows neo-antigen mRNA-loaded lipid nanoparticles (LNPs) conjugated with A₂A receptor inhibitors (A₂ARi). it highlights how A₂ARi is attached to the LNPs for targeted delivery to dendritic cells expressing A₂AR. The lipid-based core of the LNPs encapsulates the neo-antigen mRNA payload, and various A₂ARi types are compatible with this delivery system.

Many ongoing clinical trials are being conducted in different phases to study the use of small-molecule A₂ARi for cancer therapy.^{11,14–16,17} Among the leading candidates, AZD4635 is being investigated in several Phase I and Phase II clinical trials, where its potential to enhance immune responses is being examined. This A₂ARi has been studied both as a monotherapy and in combination with other treatments, such as Durvalumab, a PD-L1 checkpoint inhibitor, in patients with advanced solid tumors.^11,59 Initial findings suggest that AZD4635 may help counteract tumor-induced immune evasion by restoring T-cell function, a key mechanism that could enhance antitumor immunity.¹¹

Additionally, Ciforadenant (CPI-444), another potent A₂ARi, has been extensively studied in Phase I/II trials for malignancies like renal cell carcinoma.¹² These trials have highlighted Ciforadenant’s ability to modulate the TME, with results indicating that it can improve immune activity when combined with checkpoint inhibitors like Atezolizumab.⁶⁰ Similarly, EOS100850, developed by iTeos Therapeutics, is a promising A₂ARi currently under clinical evaluation.¹³ This compound is designed to block the suppressive effects of AD within the TME, enhancing T-cell-mediated immune responses. Early findings from trials supported by the National Cancer Institute suggest that EOS100850 can disrupt AD-induced immune suppression, potentially leading to better outcomes in patients with solid tumors.¹⁸

Another important agent, Etrumadenant, developed by Arcus Biosciences, is undergoing clinical trials to assess its efficacy both as a monotherapy and in combination with PD-1 inhibitors.¹⁴ These trials aim to evaluate how Etrumadenant can enhance immune responses and reinvigorate T cells to combat tumor progression.¹⁴ JNJ-40255293, developed by Johnson & Johnson, is also being studied for its potential to enhance immune activation in cancer. Currently undergoing Phase I trials, this compound is being tested both as a monotherapy and in combination with other cancer therapies. Early data suggest that JNJ-40255293 may significantly augment the immune system’s ability to recognize and attack cancer cells, especially when paired with immune checkpoint inhibitors.

Similarly, Taminadenant, a compound being studied by PabloBio/Novartis, targets AD pathways that suppress immune activity in the TME.¹⁶ Ongoing clinical trials are evaluating Taminadenant both as a monotherapy and in combination with immune checkpoint inhibitors like Pembrolizumab to determine its effectiveness in restoring immune function.¹⁸ Lastly, AB928, developed by Arcus Biosciences, is a unique dual A₂A/A₂B receptor antagonist that is distinct from other A₂AR-specific agents. It is currently being investigated in clinical trials for its ability to enhance immune responses against cancer by targeting both the A₂A and A₂B receptors. These trials aim to assess whether AB928 can provide a more comprehensive solution for overcoming immune suppression in the TME. AB928 is being tested both as a monotherapy and in combination with other immune-modulating therapies to maximize its potential in treating a range of cancers.¹⁷

As clinical trials progress, these A₂ARi play a pivotal role in improving cancer treatment outcomes, offering new hope for patients with advanced malignancies. The results from these trials will help define the future direction of cancer immunotherapy and solidify the potential of A₂ARi as a transformative therapy.

The integration of A₂ARi into LNP-neo-antigen mRNA vaccines opens promising avenues in personalized neo-antigen therapies. Key candidates such as SCH 58,261⁶¹ and KW-6002⁶² exhibit selective antagonistic activity against A₂AR, known for their anti-inflammatory properties.⁶² These antagonists block A₂AR on DCs and Th cells, effectively alleviating AD-mediated immunosuppression within the TME. This mechanism holds the potential to significantly enhance the efficacy of neo-antigen mRNA vaccines against cancer cells.⁶³

Furthermore, compounds like SYN115 (Tozadenant)⁶⁴ and SCH 420,814 (Preladenant),⁶⁵ initially explored for Parkinson’s disease, show promise in cancer therapy through A₂AR inhibition. By disrupting AD signaling pathways on DCs and Th cells, these A₂ARi aim to invigorate the immune landscape within the TME, thereby amplifying the effectiveness of mRNA vaccines against cancer.^66,67

Synthetic A₂ARi such as ZM 241385^68,69 SCH 58,261,⁶¹ and BG9928⁷⁰ effectively counteract AD-mediated immune suppression by targeting A₂AR on DCs, allowing γδ T cells to sequester AD and mitigate its suppressive effects.⁷¹ This section provides a detailed analysis of the structural considerations for neo-antigen mRNA-LNPs+ A₂ARi, including the selection of suitable antagonists potentially linked to LNPs via appropriate linkers.^56,72 The studies published by Zhang et al. and Leone et al. provide insight into mechanisms by which these combinations can facilitate targeted delivery to A₂AR-expressing DCs within the TME.^57,73 Among the six A₂ARi discussed, SCH 58,261 and KW-6002 emerge as promising candidates for integration into the neo-antigen mRNA-LNPs+A₂ARi vaccines.⁷⁴ SCH 58,261 exhibits a high affinity for A₂AR and a relatively long half-life of 20 hours, showing promising results in various cancers.⁷⁵ Conversely, KW-6002 demonstrates a high affinity for A₂AR and enhances anti-tumor immune responses, making it suitable for different cancer types.⁵⁸ Their high affinity for A₂AR and demonstrated enhancement of anti-tumor immune responses make them viable options for inclusion in neo-antigen mRNA vaccine strategies across various cancer types.

Table 2 complements this discussion by providing a comprehensive overview of each A₂ARi, detailing their chemical structures, mechanisms of action, pharmacological profiles, clinical applications, binding activities, and modes of action. This detailed information facilitates informed decisions regarding the incorporation of these compounds into neo-antigen mRNA vaccine strategies.

Table 2.

Candidate A₂ARi for neo-antigen mRNA vaccines.

Antagonist	Structure	Kd (nM)	Half-life(h)	Applications	Functions	Side Effects	Cancers	Trials	Refer.
SCH 58,261		High-3.5	20	Enhances immune response, anti-inflammation, TME modulation, mRNA vaccine efficacy	T cell activation, proliferation; Regulatory T cell suppression	Nausea, dyskinesia, headache	Breast, Lung, Melanoma	Phase 3, NCT01227655	61
KW-6002		High-2.2	16–20	Parkinson’s disease, anti-tumor immune enhancement	NK cell cytotoxicity; Th1 cytokine production	Dyskinesia, insomnia, nausea	Breast, Colorectal, Renal	Phase 2, NCT03465618	62
SYN115		Moderate-21	6	Adenosine reduction, TME modulation, immune enhancement	Dendritic cell maturation; TAM polarization inhibition	Dyskinesia, hallucinations, headache	Breast, Lung, Pancreatic	Phase 2, NCT03593018	64
SCH 420,814		High-1.1	9–12	Dopamine increase,Th cell enhancement	CD4+ T cell proliferation	Nausea, headache, dizziness	Lung, Prostate, Renal	Phase 1, NCT02071095	65
ZM 241,385		High-1.2	3	Adenosine blockage, anti-tumor immunity	Angiogenesis inhibition; Tumor cell proliferation inhibition	Hypotension, tachycardia, dizziness	Breast, Colorectal, Melanoma	Phase 1, NCT02289151	68
BG9928		High-1.3	16–20	Active immunity enhancement, mRNA vaccine efficacy	CD8+ T cell activation	Fatigue, diarrhea, nausea	Colorectal, Lung, Melanoma	Phase 2, NCT02655822	70

Abbreviations: Kd, binding affinity; Th, T helper; TME, tumor microenvironment; TAM, tumor-associated macrophage; CD, cluster of differentiation.

Synergy of neo-antigens mRNA-LNPs+A₂ARi vaccines

Figure 4 illustrates the concept of engineering neo-antigen mRNA with A₂ARi in LNP-encapsulated neo-antigen mRNA vaccines, emphasizing their dual functionality. This targeted delivery approach holds promise in enhancing mRNA vaccine effectiveness for cancer immunotherapy by overcoming immunosuppressive barriers and stimulating potent anti-tumor immune responses. Engineering mRNA neo-antigens with A₂AR ligands aims to boost neo-antigen presentation, enhancing the anti-tumor immune response and overcoming immunosuppression within the TME, thereby maximizing the potential of mRNA vaccines in personalized neoantigen therapy.^76,77

Figure 4.

Enhanced cancer immunotherapy through neo-antigen mRNA-LNPs+A₂ARi. A₂ARi, linked to LNPs, enables targeted delivery to A₂AR-expressing dendritic cells (DCs) and reduces the DCs migrating in TME. This delivery enhances neo-antigen presentation on MHC class I molecules, priming cytotoxic T lymphocytes (CTLs) for cancer cell destruction. Additionally, A₂ARi on DCs alleviate ad-mediated immunosuppression, promoting CTL activation. Enhanced mRNA neo-antigens with A₂AR ligands enhance uptake by other immune cells, such as macrophages and natural killer cells, amplifying the anti-tumor immune response. mRNA-LNPs+A₂ARi vaccines facilitate robust endogenous neo-antigen presentation and CTL activation through this dual mechanism.

Optimizing the effects of A₂ARi during the vaccination period enhances multiple facets of the immune response, including T cell activation, expansion, and effector function. Preclinical studies in A₂AR-deficient mice have demonstrated enhanced survival and tumor rejection compared to wild-type mice.⁷⁸ For example, Waickman et al. showed increased rejection of EL4 lymphoma cells and enhanced OVA-specific CD8⁺ T cell responses in A₂AR knockout mice.⁷⁹ Mechanistically, success has been demonstrated in murine models, where combining CD73 blockade with doxorubicin chemotherapy inhibited AD production and improved outcomes in breast cancer⁸⁰ and melanoma.⁷⁹

A₂ARi has shown potential therapeutic applications in combination with various cancer treatments such as chemotherapy, immunotherapy, checkpoint therapy, and even adoptive T-cell therapy. Several chemotherapeutic agents, including anthracyclines, oxaliplatin, cyclophosphamide, gemcitabine, and bortezomib, appear to induce in situ vaccination effects due to their initial cytotoxic impact when combined with A₂ARi.⁸¹ For instance, Allard et al. demonstrated success in inhibiting AD production (upstream of A₂AR) with CD73 blockade in combination with doxorubicin chemotherapy in a murine breast cancer model.⁸²

Additionally, A₂AR pathway blockade shows additive effects when combined with targeting other checkpoint pathways, with mechanistic and clinical implications. Clinically, combining A₂ARi with CTLA-4 and/or PD-1 blockade reported an initial overall response rate of 42%- significantly higher than either agent used alone.⁸³

Combining A₂ARi with adoptive T-cell therapy enhances T-cell function and extends the duration of cytotoxic responses.⁸¹ Recently, Chen et al. demonstrated that Liproxstatin-1, which promotes iron-dependent lipid peroxidation, enhances mitochondrial function in activated CD8+ T cells during in vivo adoptive T cell therapy.⁸⁴ The potential for concurrent genetic targeting of Tim3 and A₂AR could augment the efficacy of CAR T cell therapy in solid tumors.⁸¹

Effective targeting of A₂AR-expressing cells requires careful selection of linkers between A₂ARi and the LNP carrier for optimal delivery and interaction with DCs in the TME. The linker must maintain stability under physiological conditions to ensure sustained conjugation with the LNP carrier during circulation and cellular uptake. Flexibility is essential for optimal A₂ARi positioning on the LNP surface, facilitating efficient binding to cDC1s and subsequent internalization.⁸⁵ Specificity is crucial for the selective binding of A₂ARi to A₂AR-expressing DCs, minimizing off-target effects and maximizing the therapeutic impact on anti-tumor immune responses.⁸⁶ Biocompatibility is necessary to ensure safe in vivo administration without adverse reactions. Lastly, the linker should be minimally immunogenic to avoid unwanted immune responses that could compromise neo-antigen mRNA vaccine efficacy.

Integrating A₂ARi into mRNA-LNP neo-antigen vaccines represents a significant advancement by enhancing DC activation and creating a favorable TME.^24,87 This synergy promotes neo-antigen delivery and initiates potent immune responses by disrupting AD pathways and bolstering effector functions of T cells, particularly CD8⁺ CTLs.⁸⁷ By counteracting the inhibitory influence of A₂AR on cDC1s activation, these vaccines create an environment conducive to heightened DC activity, facilitating efficient neo-antigen delivery and initiating a potent immune response.⁸⁸ Simultaneously, A₂ARi disrupts AD pathways within the TME, impeding suppressive signals and establishing an immune-permissive landscape.⁹ Key steps in the immune response triggered by mRNA-LNPs combined with A₂ARi include the binding of neoantigen-loaded MHC molecules to TCRs on CD8+ CTLs.^86,89 Co-stimulatory signals from molecules like CD40 on DCs and CD137 on T cells enhance their functions, while CD4+ Th cells, especially the Th1 subset, boost CTL activity by producing IFN-γ.^86,90

Conclusion

In summary, the combination of neo-antigen mRNA-LNPs and A₂ARi vaccines presents a promising approach to cancer immunotherapy. This strategy may offer potent anti-tumor responses with minimal off-target effects by potentially counteracting A₂AR-mediated immunosuppression and enhancing pDCs and cDC1s activation. Ongoing studies and future randomized controlled trials will be crucial in determining the potential of this combination therapy to improve outcomes and quality of life for cancer patients.

Expert opinion

The integration of A₂ARi with neo-antigen mRNA vaccines holds considerable promise for advancing cancer immunotherapy by overcoming some of the critical challenges associated with TME. This approach combines the specificity of neo-antigen vaccines with the immunomodulatory potential of A₂ARi, particularly within the context of DCs and CTLs. However, to fully realize the therapeutic potential of this combination, several key challenges and future research directions must be addressed.

One of the most promising avenues of this combination therapy lies in enhancing the immune system’s ability to recognize and target cancer cells more effectively. Neo-antigen mRNA vaccines are designed to encode tumor-specific antigens, which are delivered through LNPs to APCs, especially DCs. The vaccines aim to activate CTLs, which are essential for recognizing and destroying tumor cells. However, the efficacy of this process is often limited by the immunosuppressive properties of the TME. Here, A₂ARi plays a crucial role in counteracting the suppressive effects of AD on T cells. By inhibiting the A₂AR, A₂ARi helps sustain T cell activation, thereby allowing the immune system to mount a more robust response against tumors. This combination, therefore, holds the potential to increase vaccine efficacy by disrupting the inhibitory pathways in the TME.

The clinical implementation of this combined approach, however, is not without hurdles. One key challenge is the precise delivery and targeting of both mRNA vaccines and A₂ARi to the desired cellular populations. In particular, achieving selective uptake by specific subsets of DCs, such as pDCs and cDC1s, is crucial for ensuring an effective immune response. Ligand-conjugation and surface modification techniques are being explored to enhance the specificity of targeting, but more research is needed to refine these methods and reduce potential off-target effects. The stability of mRNA in the bloodstream, as well as the efficiency of its encapsulation in LNPs, also represents a technological challenge. Further advancements in LNP technology are required to improve the delivery of mRNA and A₂ARi to DCs within the TME, thereby optimizing antigen presentation and subsequent immune activation.

Another significant barrier to clinical application is the variability of immune responses among patients. Personalized neo-antigen mRNA vaccines, by their very nature, require the rapid and cost-effective identification of patient-specific neo-antigens. This process involves extensive genomic analysis, which is currently both time- and resource-intensive, potentially limiting the widespread adoption of these vaccines in clinical practice. In this context, there is an urgent need to develop more efficient bioinformatics pipelines and scalable production processes for mRNA vaccines. Streamlining the identification of neo-antigens and ensuring the timely production of personalized vaccines will be crucial for translating these therapies from the research setting into routine clinical care.

From a research perspective, the synergy between neo-antigen mRNA vaccines and A₂ARi opens new opportunities for improving the treatment landscape. Current research is already exploring the combination of neo-antigen vaccines with other immune checkpoint inhibitors, such as PD-1/PD-L1 inhibitors. Combining A₂ARi with PD-1 inhibitors could offer a more comprehensive approach to overcoming immune suppression within the TME. Furthermore, exploring different dosing regimens and identifying biomarkers that predict patient response to these therapies will be essential for personalizing treatment plans and maximizing therapeutic efficacy.

The potential for future research in this area is substantial. Investigating how different subsets of DCs respond to combined mRNA vaccines and A₂ARi will yield valuable insights into optimizing immune responses. Moreover, understanding the precise mechanisms by which A₂AR signaling influences both the activation and suppression of immune cells within the TME could pave the way for the development of next-generation immunotherapies. As more data emerge, particularly from ongoing clinical trials, we can expect to see a shift toward more refined and effective strategies that integrate mRNA vaccines with a broad array of immunomodulatory agents. Over the next 5 to 10 years, we will witness the evolution of cancer immunotherapy into a more personalized and multi-modal treatment approach. Advances in mRNA delivery systems, combined with an enhanced understanding of the TME, will likely lead to therapies that are not only more effective but also more accessible. As these therapies become more widely adopted, the standard of care for cancer patients will likely shift toward more individualized, immune-based treatments that can adapt to the unique characteristics of each patient’s tumor and immune environment.

Article highlights

• Combining adenosine A₂A receptor antagonist (A₂ARi) with neo-antigen mRNA vaccines enhances innate and adaptive immune responses, boosting vaccine efficacy.

• A₂ARi coupled with neo-antigen mRNA vaccines increases antitumor T cell responses by modulating the hypoxia-adenosinergic signaling, improving dendritic cell activation, and increasing MHC class II-restricted neoantigen presentation.

• Lipid nanoparticles (LNPs) for co-delivery of mRNA and A₂ARi ensure more precise targeting, reducing systemic toxicity and off-target immune activation.

• The precision-guided mRNA-LNPs+A₂ARi strategy minimizes off-target effects, providing tailored cancer treatment options.

Biographies

Saber Imani, Ph.D., is an Assistant Professor at Shulan International Medical College, Zhejiang Shuren University, in Hangzhou, China. His research lab focuses on “mRNA Cancer Vaccines” and “Translational Oncogenesis,” aiming to synthesize and deliver innovative cancer vaccines and biopharmaceuticals. Dr. Imani prioritizes mRNA-based approaches to develop novel antigen-encoding vaccines for personalized cancer therapy, leveraging mRNA’s potential to elicit targeted immune responses against cancer cells. Additionally, his lab investigates translational oncogenesis to understand abnormal protein synthesis in cancer development and to explore therapeutic targets. With a robust background in molecular biology and translational oncology. He has successfully secured several competitive research grants and led numerous projects in cancer therapeutics across China and Europe. His expertise in advanced sequencing technologies, combined with extensive project management and collaborative skills, aligns well with the current research landscape in translational oncology and mRNA cancer vaccines.

Mazaher Maghsoudloo, Ph.D. in Bioinformatics, graduated from Tehran University in 2020. With a background in Computer Science, he integrates computational expertise with a deep understanding of biological systems. Since 2023, he has been a Research Associate at Southwest Medical University, working in the Key Laboratory of Epigenetics and Oncology within the Research Center for Preclinical Medicine. His research focuses on cancer biology, particularly biomarker detection, mRNA cancer vaccines, immunology, immunotherapy, and the reconstruction of disease networks, with a special emphasis on cancer-related applications.

Funding Statement

This work was supported by the Talent Scientific Research Project of Zhejiang Shuren University under Grant [numbers KXJ1723104, 2021].

Disclosure statement

No potential conflict of interest was reported by the author(s).

Author contributions

CRediT authorship contribution statement. Investigation: Ali Babaeizad and Mazaher Maghsoudloo; Validation: Ali Babaeizad and Saber Imani. Writing-original draft: Saber Imani and Parham Jabbarzadeh Kaboli. Writing-review & editing: Ali Babaeizad, Mazaher Maghsoudloo, and Parham Jabbarzadeh Kaboli. Supervision: Saber Imani and Mazaher Maghsoudloo. All authors have read and edited the published version of the manuscript.

Data availability statement

All data associated with this study are in the paper or the Supplementary Materials.

Ethical approval

This study does not involve human participants; therefore, ethical approval is not applicable.

Supplementary material

Supplemental data for this article can be accessed on the publisher’s website at https://doi.org/10.1080/21645515.2025.2458936