Advances in mRNA vaccine therapy for breast cancer research

Abstract

Breast cancer represents the most prevalent cancer among women globally, constituting approximately 30% of newly diagnosed female malignancies and serving as the second leading cause of cancer-related mortality, accounting for 11.6% of deaths. Despite notable advancements in survival rates and quality of life for breast cancer patients over recent decades—achieved through interventions such as surgery, chemotherapy, radiotherapy, and endocrine therapy—there remains an urgent need for novel therapeutic strategies. This necessity arises from challenges associated with recurrence, metastasis, and drug resistance. The COVID-19 pandemic has accelerated the development of Messenger RNA (mRNA) vaccines at an unprecedented pace, and as a novel form of precision immunotherapy, mRNA vaccines are increasingly being recognized for their potential in cancer treatment. mRNA vaccines efficiently produce antigens within the cytoplasm, specifically activating the immune system to target tumor cells while minimizing the risk of T-cell tolerance. Therefore, mRNA vaccines have emerged as a promising approach in cancer immunotherapy. This review systematically examines the principles, mechanisms, advantages, key targets, and recent progress in mRNA vaccine therapy for breast cancer. Furthermore, it discusses current challenges and suggests potential directions for future research.

Introduction

According to the latest statistics from the International Agency for Research on Cancer of the World Health Organization, in 2022, there were 20 million new cancer cases and 9.7 million cancer-related deaths worldwide. Approximately 1 in 5 individuals will be diagnosed with cancer during their lifetime, with around 1 in 9 men and 1 in 12 women dying from the disease [1]. Breast cancer is the most frequently diagnosed cancer and the leading cause of cancer-related deaths among women. By 2022, it accounted for an estimated 670,000 deaths, surpassing gastric cancer to become the fourth leading cause of cancer mortality worldwide. This number far exceeds the mortality rates of other gynecological cancers, such as uterine and ovarian cancers [1]. According to the Breast Cancer Statistics 2024, the lifetime risk of developing invasive breast cancer for women in the United States is estimated to be 13%. Furthermore, approximately 2% of women diagnosed with the disease succumb to it. The overall mortality rate associated with breast cancer is reported to be 19.3 per 100,000 individuals [2]. Breast cancer mortality is partly attributed to inadequate early-stage diagnosis, leading to tumor metastasis in advanced stages. The development of highly sensitive diagnostic tools for early-stage breast cancer and novel targeted molecular imaging agents can effectively reduce mortality. For instance, molecular imaging techniques such as single-photon emission computed tomography (SPECT) and positron emission tomography (PET) utilize peptide-based radiopharmaceuticals to target specific receptors overexpressed on breast cancer cells (e.g., HER2, EGFR, SSTR, Galectin-3), enabling early diagnosis and precise therapeutic interventions [3]. In recent decades, advancements in surgery, radiotherapy, chemotherapy, endocrine therapy, targeted therapy, and immunotherapy have significantly improved the survival rates and quality of life for breast cancer patients [4, 5]. Immune checkpoint inhibitors (ICBs) and other passive immunotherapies have also shown success in treating triple-negative breast cancer (TNBC) [6]. However, the monotherapy efficacy of ICBs remains suboptimal [7], and strategies to overcome resistance in advanced-stage immunotherapy are still under investigation, as these approaches have not yet fundamentally transformed treatment paradigms.

With the increasing diversity of immunotherapeutic agents, there is also an escalation in immune-related adverse events. Tumor vaccines, as a form of active immunotherapy, represent a potential approach to stimulating the host's immune system. Current research suggests tumor vaccines may hold particular therapeutic value for treating solid tumors such as breast cancer, which is often associated with low mutational burden [8], generally low programmed cell death-ligand 1 (PD-L1) expression [9, 10], potential impairments in antigen processing/presentation, and the frequent presence of an immunosuppressive tumor microenvironment (TME) [11]. The primary objective of tumor vaccines is to induce immune responses with potential durability for targeting tumor cells. Preliminary studies suggest that mRNA vaccines, compared to traditional tumor vaccine platforms, exhibit certain advantages such as enhanced immune response durability, reduced production costs, and improved scalability. These features highlight their potential as a therapeutic strategy under investigation for breast cancer treatment. Breast cancer cells often evade immune surveillance through various mechanisms, such as TGF-β-mediated immunosuppression, upregulation of cytotoxic T-lymphocyte-associated protein 4 (CTLA4), and activation of the programmed cell death-1 (PD-1) / PD-L1 pathway [12]. mRNA vaccines can break this immune escape by activating T- and B-cell immune responses. Studies have shown that mRNA vaccines can induce effective anti-tumor immune responses in breast cancer models. For example, mRNA vaccines against HER2-positive breast cancer have shown potential in trials [13]. Compared to current emerging therapies for breast cancer, such as the antibody-coupled drug ARX788, which is highly target-dependent, has complex toxicity management, and is prone to adverse effects such as interstitial lung disease (incidence 32.7%) and ocular reactions [14], mRNA tumor vaccines can be personalized, establish a long-lasting immune memory, and have a controllable immunogenicity, with the common side-effects of the mRNA vaccine being injection site reactions and transient fever. Furthermore, the inherently limited duration of action of mRNA vaccines reduces the likelihood of irreversible side effects. Therefore, we anticipate that mRNA vaccination will play an increasingly important role in the treatment of breast cancer.

mRNA cancer vaccine principles and mechanisms

mRNA is a negatively charged single-stranded RNA molecule that carries genetic information transcribed from DNA and is translated into proteins by ribosomes to perform specific biological functions. mRNA vaccines can be categorized into three distinct types: non-replicating mRNA vaccines, self-amplifying mRNA vaccines, and circular RNA (circRNA) vaccines, according to their specific characteristics [15]. Traditional non-replicating mRNA molecules are composed of several key structural elements: a 5′cap structure, a 5′untranslated region, an open reading frame that encodes the target protein, a 3′UTR, and a poly(A) tail [16]. While the ORF encodes the protein of interest, the other components play critical roles in enhancing mRNA stability and translation efficiency.

One notable advantage of mRNA in vaccines is its inherent immunogenicity, which allows it to act as an adjuvant. These vaccines are synthesized using in vitro transcription, where linearized plasmid DNA serves as a template, and nucleoside triphosphates provide the raw materials. RNA polymerases, such as T7, T3, or Sp6, facilitate transcription, incorporating a 5′ cap structure and a 3′ poly(A) tail. Unlike cellular-based methods, this approach eliminates the need for amplification, enabling rapid, large-scale production [17]. To ensure the safety and efficacy of mRNA products for clinical application, it is essential to establish a balance between immune activation and the expression of the target antigen.

The immune response generated by mRNA vaccines closely resembles that triggered by viral infections, activating both cellular and humoral immunity to provide protective effects. The foreign mRNA is introduced into the body and recognized by receptors on the cell surface, endosomes, and cytoplasm, triggering an immune response. mRNA is translated into protein in the cytoplasm, and the proteasome then breaks it down into antigenic peptides. Major Histocompatibility Complex (MHC) class I molecules deliver these peptides to CD8 + T cells, triggering a cytotoxic T-cell response [18, 19]. Additionally, the protein encoded by mRNA can be secreted into the extracellular space, where it is taken up by antigen-presenting cells (APCs). These antigenic peptides are presented to CD4 + T cells via MHC class II molecules, inducing cytokine secretion and activating antigen-specific B cells that differentiate into plasma cells to produce specific antibodies, thus promoting humoral immunity [18, 19](Fig. 1). By encoding altered or defective tumor suppressor proteins, mRNA vaccines can modulate the tumor immune microenvironment to achieve cancer immunotherapy. Current research mainly focuses on using mRNA as a therapeutic vaccine to train the immune system to target tumor-associated antigens (TAAs) and tumor-specific antigens (TSAs), enhancing the immune system's ability to recognize and attack tumor cells [20].

Fig. 1.

The mechanism of action of mRNA tumor vaccine. The mRNA tumor vaccine activates cell-mediated immune responses by delivering mRNA encoding TSA to the patient, which are translated into proteins by ribosomes in the cytoplasm of the host cell and degraded by the proteasome to antigenic peptides, which are presented to the CD8 + T cells via the MHC. In addition, proteins based on mRNA translation can be secreted into the extracellular environment and thus enter the circulatory system for uptake by APCs, and antigenic peptides are presented as exogenous antigens to CD4 + T cells as MHC class II molecules, exerting humoral immunity

Cancer vaccines are generally categorized into preventive and therapeutic types. Preventive vaccines, such as the Human Papillomavirus (HPV) vaccine for cervical cancer, target viruses involved in malignant transformation. In contrast, therapeutic vaccines offer key advantages over other immunotherapies, such as generating highly specific adaptive immune responses and establishing immunological memory, potentially controlling or eliminating residual disease. Preventive vaccines have broader applications and more ideal outcomes compared to therapeutic vaccines, and they can be mass-produced. Identifying early-stage antigens that are expressed in breast cancer and stimulating the immune system prior to the onset of the disease to eliminate cells that express aberrant proteins represents a viable approach to primary prevention. Additionally, intercepting high-risk lesions serves as another strategy to impede the progression of breast cancer. One effective method for preventing pre-invasive lesions, such as ductal carcinoma in situ (DCIS) and lobular carcinoma in situ, from advancing to invasive cancers is through their surgical removal [21]. This method represents a therapeutic vaccine targeting high-risk lesions. For example, HER2 protein is expressed in a small proportion of DCIS cases and is associated with tumor invasion, metastasis, recurrence, and drug resistance risks. A dendritic cell (DC)-based HER-2/neu vaccine strategy was designed for patients with HER-2/neu overexpressing DCIS in a phase II clinical trial. The trial enrolled 27 patients in which a DC vaccine pulsed with HER-2/neu peptide was injected into the inguinal lymph nodes by ultrasound guidance once a week for four times prior to surgery. Postoperative pathologic examination revealed no evidence of residual disease in 18.5% (5/27) of patients and complete resolution of HER-2/neu expression in 50% (11/22) of the remaining 22 patients [22]. This study demonstrates the potential application of HER-2/neu vaccine in the treatment of early breast cancer. mRNA-based therapeutic cancer vaccines function by delivering mRNA fragments encoding exogenous antigens into human cells. Upon internalization by host cells, these mRNA transcripts are translated into TAAs or tumor-TSAs. The MHC class I and II pathways present these antigens, thereby activating immune cells such as helper T cells and cytotoxic T lymphocytes (CTLs). The primary objective is to target and eliminate tumor cells to achieve antitumor effects by overcoming tumor-induced immunosuppression, enhancing the immunogenicity of tumor cells, and augmenting the patient's immune response [23]. Prophylactic tumor vaccines reduce the risk of cancer development or limit the progression of these tumors to invasive cancers by targeting pathogens associated with tumorigenesis or intercepting high-risk lesions in tumors, while therapeutic tumor vaccines treat established tumors by stimulating the immune system to recognize and eliminate cancer cells. Compared with preventive vaccines, which require large-scale clinical trials for validation, therapeutic vaccines can rapidly develop personalized vaccines based on the tumor mutation characteristics of patients, designed to target patient's tumor-specific mutations (e.g., neoantigens), and stimulate the patient's own immune system from the forehead to recognize and attack tumor cells, which is more targeted [24]. Moreover, therapeutic tumor vaccines can attack clinically undetected occult lesions by activating the patient's own immune system, which offers the possibility of treating advanced breast cancer. Therefore, this paper focuses on therapeutic tumor vaccines (Table 1).

Table 1.

Clinical trials mRNA vaccine against breast cancer

Trial numbers (clinical phase)	Vaccine type	Target antigen or agonist	Combination therapy	Route of administration	Condition	Results or recruitment status	Sponsor/collaborators
NCT01526473(I)	SAM vaccine (AVX901)	HER2	NA	i.m	HER2 + Breast Cancer	Completed, Safety and Toxicities	H. Kim Lyerly Susan G. Komen Breast Cancer Foundation Duke University
NCT03313778(I)	Personalized neoantigen tumor vaccine	Neo-antigen	pembrolizumab	i.m	Solid Tumors	Recruiting	ModernaTX, Inc
NCT00529984(I/II)	CEA(6D) VRP Vaccine	TAA, CEA	NA	NA	Colorectal Cancer Breast Cancer Lung Cancer Pancreatic Cancer	Completed	AlphaVax, Inc
NCT06497010 (I)	mRNA Cancer Vaccine	Neo-antigen	Tislelizumab	i.m	Advanced Solid Tumor	Recruiting	Shengfa Su, The Affiliated Hospital of Guizhou Medical University
NCT03632941(II)	SAM vaccine (AVX901)	HER2	Pembrolizumab	i.m. + i.v	HER2 + Breast Cancer	Recruiting	Herbert Lyerly Merck Sharp & Dohme Corp Duke University
NCT00978913 (I)	DC vaccine	Survivin, hTERT, and p53	Cyclophosphamide	i.d	Breast Cancer Malignant Melanoma	Complete, safety, and toxicities	Inge Marie Svane Herlev Hospital
NCT00004604 (I)	DC vaccine	CEA	NA	i.v	Metastatic Cancer with CEA expression (Breast Cancer, et al.)	Completed, Safety and Toxicities	Duke University National Cancer Institute (NCI)
NCT05942378(I)	mRNA cancer vaccines	neoantigen	Adebrelimab	NA	Advanced Solid Tumors	Not yet recruiting	Jian Zhang, MD, Fudan University
NCT03289962(I)	Lipoplex vaccine	TSA, poly-neoepitope	Atezolizumab	i.v	Advanced or Metastatic Tumors (Triple Negative Breast Cancer, et al.)	Active, not recruiting	Genentech, Inc
NCT05359354(I)	Personalized neoantigen tumor vaccine	neoantigen	PD-1	NA	Solid Tumor	Recruiting	YueJuan Cheng, Peking Union Medical College Hospital
NCT00004604(I)	DC vaccine	TAA, CEA	NA	i.v	Breast Cancer Colorectal Cancer Extrahepatic Bile Duct Cancer Gallbladder Cancer	Completed	Duke University
NCT06530082(I)	DC vaccine	CircFAM53B	camrelizumab	i.d	Breast Neoplasms	Not yet recruiting	Erwei Song, M.D., Ph.D., Sun Yat-Sen Memorial Hospital of Sun Yat-Sen University
NCT03739931 (I)	ISV (LNP encapsulated)	mRNA-2752 (Human OX40L, IL-23, and IL-36γ)	Durvalumab	i.t. + i.v	Triple Negative Breast Cancer Head and Neck Squamous Cell Carcinoma	Recruiting	ModernaTX, Inc AstraZeneca
NCT05198752(I)	Neoantigen mRNA Personalised Cancer	Neoantigen	NA	i.h	Solid Tumor	Unknown status	Stemirna Therapeutics
NCT01291420 (I/II)	DC vaccine	WT1	NA	i.d	Solid Tumors (Breast Cancer, et al.)	Phase I study: vaccination with DC will be well tolerated and will increase WT1-specifc CD8 + T-cell responses	BioNTech SE Seventh Framework Programme
NCT02316457 (I)	LNP mRNA vaccine	Neoantigens + 4 TAAs (2–3 variant RNAs + p53 RNA)	NA	i.m	Triple Negative Breast Cancer	Active, not recruiting	BioNTech SE Seventh Framework Programme
NCT03788083 (I)	ISV	TriMix (caTLR4, CD40L and CD70)	NA	i.t	Early-stage Breast Cancer	Recruiting	Universitair Zieken huis Brussel eTheRNA immuno therapies
NCT06195384(I)	Neoantigen mRNA Vaccine	neoantigen	PD1/PDL1/CTLA4 antibodies	i.d	Solid Tumor, Adult	Recruiting	Second Affiliated Hospital of Guangzhou Medical University

Advantages of mRNA cancer vaccines

Both mRNA vaccines and traditional vaccines aim to activate the immune system to generate targeted immune responses against specific pathogens or antigens. The distinguishing advantage of mRNA vaccines lies in their enhanced safety profile. Unlike traditional vaccines that utilize inactivated/attenuated pathogens, pathogen-derived proteins, or subunit components, mRNA vaccines do not require introducing live biological entities into the body. Instead, they leverage the body's own cellular machinery to produce specific viral or pathogenic proteins, thereby initiating immune responses. This mechanism reduces the inherent risks of infection or adverse reactions associated with live-attenuated vaccine platforms [25]. Moreover, mRNA vaccines can be produced rapidly by synthesizing only mRNA sequences without culturing pathogens or proteins. In addition, the in vitro preparation process avoids immunogenicity and cytotoxicity caused by contaminants of viral origin [26]. While traditional vaccine immune responses rely on repeated vaccination to enhance memory B cells, but T cell memory is limited, e.g., influenza vaccines need to be renewed annually, mRNA vaccines induce stronger immune responses, especially T cell responses, through dual signaling activation (antigenic signaling + adjuvant effect), e.g., the mRNA-4157 vaccine, as mentioned below, was shown to be effective in melanoma patients, with peripheral blood still able to neoantigen-specific T cells were detected [27].

Other advantages of mRNA vaccines include: 1. Cellular mechanisms govern the degradation of mRNA [28], thereby enabling the establishment of a controlled half-life in vivo, which ensures safety and prevents integration into the host DNA. 2. The immune response induced by mRNA vaccines is similar to that of viral infections. Upon entering cells via endocytosis, mRNA vaccines express and accumulate antigens, which are processed into peptides and loaded onto MHC-I molecules. On the other hand, secreted proteins encoded by mRNA can activate the MHC-II pathway or directly deliver antigens from the cytoplasm to lysosomes, stimulating both cellular and humoral immunity, especially by inducing CD8 + T cell responses for effective protection [29]. 3. Unlike protein-based vaccines, mRNA vaccines have the capability to encode entire antigen proteins, thereby presenting multiple epitopes without the limitations associated with specific human leukocyte antigen (HLA) epitopes, which is a characteristic of peptide-based vaccines. Additionally, the use of multiple mRNA strands or a single mRNA strand that expresses multiple antigens can elicit broader immune responses. This feature is particularly advantageous for targeting tumors that produce a diverse array of novel antigens. 4. mRNA can be synthesized in a cell-free environment, which contributes to a reduction in production costs. Furthermore, the established production processes and platform technologies for mRNA vaccines facilitate rapid and scalable manufacturing [25]. These principles and characteristics provide new insights into potential therapeutic strategies for breast cancer.

Although extensive preclinical and clinical trials have been conducted to ensure safety, mRNA tumor vaccines, as an emerging means of individualized immunotherapy, have been evaluated as therapeutic vaccines usually require repeated administration, and ongoing monitoring and evaluation is necessary to monitor any long-term effects or rare adverse events. mRNA its potential side effects include inflammatory reactions, toxic reactions, etc., for example, mRNA vaccines have an inherent immunostimulatory properties, capable of activating the innate immune system through multiple pathways, including Toll-like receptors (TLR3, TLR7, TLR8) and cytoplasmic proteins (e.g., PKR, OAS, RIG-I, MDA5). These immune activations may bring about local and systemic inflammation, as well as more autoimmune reactions, and reduced stability of mRNAs, decreased translation efficiency, and even affect the type of immune response [30]. Therefore, further optimization and improvement are still needed to reduce adverse reactions and improve vaccine efficacy.

mRNA cancer vaccine targets

Designing and manufacturing mRNA-based breast cancer vaccines requires choosing one or more targets for active immunotherapy, informed by the unique biological characteristics of the various molecular subtypes of the disease. TAAs, TSAs, and tumor TME antigens are among the many target possibilities available in mRNA-based tumor vaccines. These vaccines allow the production of target antigens or proteins to prevent tumor growth by introducing mRNA encoding these proteins into the cytoplasm of host cells. Alternatively, these antigens can be presented on the cell surface via MHC molecules, thereby activating adaptive immunity to selectively eliminate tumor cells. Furthermore, the induction of memory T and B cells enables the establishment of long-term therapeutic effects.

mRNA vaccines targeting TAAs

TAAs, non-mutated proteins that are highly expressed in tumors but low or absent in normal tissues [31], are targets of the immune system to kill tumor cells. These antigens, which may include growth-related factors or those resulting from somatic mutations specific to malignant cells, are essential for the advancement of cancer vaccine development [32]. Common TAAs, including HER2, CD30, and CA125, are frequently utilized in clinical diagnostics and tumor classification. Compared to traditional vaccines, mRNA vaccines can deliver multiple TAAs simultaneously, enhancing immunogenic efficacy. Furthermore, the encoded full-length tumor antigens facilitate the activation of both HLA class I and class II molecules by APCs, thereby eliciting a more vigorous T-cell immune response [33].

Currently, TAA-targeted immunotherapy represents a significant area of research, particularly in the treatment of solid tumors, where TAA targets are of primary interest. Antibodies directed against TAAs not only facilitate direct tumor cell destruction through antibody-dependent cellular cytotoxicity but also function as diagnostic markers and enhance the specificity of conventional cancer therapies. Therapeutic antibodies, exemplified by antibody–drug conjugates (ADCs), have emerged as pivotal tools in cancer treatment. These ADCs primarily target TAAs such as HER2 and CD30, demonstrating their significant role in advancing tumor-targeted therapies.

A preclinical study to develop potentially relevant antigens for anti-breast cancer mRNA vaccines and populations suitable for mRNA vaccination by integrating multi-omics data (TCGA, METABRIC, single-cell sequencing, etc.) found that three TAAs ( CD74, IRF1, and PSME2) were associated with improved prognosis of the patients and infiltration of APCs (e.g., B-cells, DCs) significantly correlated [34].

However, a common problem with TAAs as tumor vaccine targets is that they are also expressed to some extent in non-malignant tissues, and thus may fail to trigger an anti-tumor immune response due to self-tolerance mechanisms, or produce off-target effects leading to autoimmune toxicity [35, 36].

mRNA vaccines encoding TSA

TSAs include proteins derived from oncogenic viruses expressed by transformed cells (e.g., HPV) and specific mutated proteins generated by somatic mutations or frameshift mutations. TSAs are absent in normal cells and can be recognized by the host immune system as “non-self” entities [37]. These antigens exhibit strong tumor specificity and immunogenicity, with weaker central tolerance [36], making them a major focus of cancer vaccine research in recent years. Personalized neoantigen-based cancer vaccines can serve as tumor-specific therapies. Unlike TAAs, neoantigen-specific T cells may survive during immune self-tolerance progression, thereby effectively stimulating robust T cell responses [24]. Several clinical trials are currently being conducted to evaluate the safety and efficacy of mRNA vaccines that encode neoantigens [20, 38].

mRNA-4157 is an individualized neoantigen mRNA vaccine. mRNA-4157 can integrate sequences encoding up to 34 neoantigens onto a single synthetic mRNA molecule. 3-year follow-up clinical trial (NCT03897881), published in December 2023 showed that a total of 157 patients with completely resected, high-risk cutaneous melanoma (stages IIIB/C/D and IV) were enrolled in the current study, and the results after 18 months of treatment demonstrated that compared to Keytruda monotherapy, the mRNA-4157 in combination with Keytruda treatment significantly improved recurrence-free survival (RFS), which resulted in a 49% reduction in the risk of recurrence or death, and distant metastasis-free survival, which resulted in a 62% reduction in the patient's risk of distant metastasis or death. The combination therapy of this mRNA personalized cancer vaccine, mRNA-4157, and PD-1 monoclonal antibody (Keytruda) has advanced to Phase 3 clinical trials, making it the first mRNA cancer vaccine in the world to enter Phase 3 clinical trials [27].

The safety and initial effectiveness of autogene cevumeran, an individualized neoantigenic vaccine based on uridine mRNA, in patients with advanced solid tumors are being assessed in a Phase I study (NCT03289962), both alone and in conjunction with atezolizumab, an anti-PD-L1 checkpoint inhibitor. The study, which involved 213 patients, showed that 71% of patients had poly-epitopic neoantigen-specific T cell responses to autogene cevumeran, with responses being detectable up to 23 months after the start of treatment. Furthermore, clinical activity was noted, including one objective response in the dose escalation cohort for monotherapy and disease control in two patients in the combination cohort who had unfavorable characteristics for immunotherapy response [39]. These studies reveal that T cells recognizing neoantigens exist in most cancers, providing highly immunogenic and specific targets for personalized vaccination.

Similarly, a phase I clinical trial (NCT04161755) evaluated the immunogenicity and clinical efficacy of autogene cevumeran in combination with anti-PD-L1 (atezolizumab) and chemotherapy (mFOLFIRINOX) in patients with postoperative pancreatic ductal adenocarcinoma (PDAC). The study showed that over a median follow-up period of 3.2 years, the median recurrence-free survival (RFS) of non-responders (n = 8) who did not generate a vaccine-induced T-cell response was 13.4 months (HR = 0.14, 95% CI 0.03–0.59, p = 0.007); those who did generate a vaccine-induced T-cell response responders (n = 8) had a significantly prolonged RFS (median value not yet reached at the time of publication of this paper), and 6 of these patients were relapse-free. Vaccine-induced CD8 + T-cell clones had a median lifespan of 7.7 years, with 86% of clones persisting 3 years after vaccination and exhibiting cytotoxicity and tissue-resident memory T-cell (TRM-like) phenotypes, as revealed by T cell receptor (TCR) sequencing and single-cell transcriptome analysis. In addition, relapsed patients (2/8) had significantly reduced numbers of vaccine-induced T cells and immune clearance of vaccine-targeted tumor clones in relapsed lesions [40]. It was demonstrated that individualized mRNA vaccines can delay PDAC recurrence by activating long-acting, high-affinity neoantigen-specific T cells through a mechanism closely related to the induction of TRM-like effector cell phenotypes and clonal persistence, which provides a new strategy for immunotherapy of low mutation load tumors.

Despite theoretical validation, obstacles related to design, manufacturing, and cost remain. For example, tumor mutations produce a rich variety of antigens, but during the escape phase of the immune editing process, tumor cells escape the surveillance of the immune system through mechanisms such as loss of tumor antigen expression, and undergo proliferation and invasion [41]. The presentation of neoantigens, as well as their persistence, etc., require more advanced predictive tools as well as a large number of clinical trials to optimize and accelerate the translation of this innovative approach from theory to the clinic, ultimately benefiting cancer patients [42].

mRNA vaccines encoding TME antigens

The TME consists of both cellular and non-cellular components that surround tumors, including blood and lymphatic vessels, immune cells, fibroblasts, signaling molecules, and the extracellular matrix. As a critical external factor in tumor immunotherapy, the role of the TME has garnered considerable attention in recent research. The TME is shaped by the interactions between tumor cells and the host's defense system. Tumor cells secrete extracellular signals that facilitate the recruitment of cells and nutrients necessary for their proliferation and migration, while the immune system mobilizes immune cells to eliminate malignant cells, thereby adding complexity to the TME.

An immunosuppressive TME—comprising myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages, and regulatory T cells —inhibits T cell function [43]. This state allows malignant tumors to evade immune surveillance, promoting rapid growth and metastasis [44]. Evidence suggests a complex interplay between the TME and vaccine efficacy: while the TME can influence vaccine outcomes, vaccines can reshape the TME, enhancing their effectiveness.

Using TME antigens instead of tumor cell antigens represents a novel approach to cancer vaccination. mRNA vaccines can target multiple antigens to manage disease burden, and TME antigen-directed vaccines or in situ vaccination may address TME-related challenges. Therapeutic cancer vaccines that activate local immune TMEs have demonstrated enhanced tumor cell death induction, underscoring the TME’s critical role in vaccine efficacy [45]. Therefore, the TME is increasingly viewed as a promising therapeutic target, driving the development of TME-focused cancer vaccines. A preclinical study was reported that ICB and a TRP2(TRP2 is a melanoma antigen peptide) mRNA vaccination were shown to downregulate PD-L1 in DCs, which inhibited tumor growth in a melanoma animal model [46]. Likewise, the immune microenvironment of B16 melanomas was enhanced by a combination of anti-PD-L1 treatment and Trp2 peptide vaccines, suggesting a possibly favorable prognosis [47].

TME antigens’ genomic stability prevents immune escape caused by antigen mutation, enhancing immunotherapy effectiveness. Combining mRNA vaccines with TME-modulating technologies can address two key features of breast cancer: pathological complexity [48] and a highly immunosuppressive TME [49, 50]. This combination also prevents immune escape pathways and strengthens antitumor immune responses. A novel mRNA cancer vaccine, based on “onion-like” multilayered RNA-lipid particle aggregates, reprograms the TME into a “hot” immune environment through stromal cell activation of innate immunity. In preclinical trials, spontaneous glioma canines showed rapid inflammation of the TME after a single infusion, extending median survival from 35 to 139 days. And in a first-in-human trial in four adult patients for the treatment of glioblastoma, the mRNA cancer vaccine rapidly reprogrammed the patient's immune system after injection, triggering cytokine/chemokine release, immune activation/transport, and specific immune responses in glioblastoma patients [51]. These studies support the novel mRNA cancer vaccine as a new technology that simultaneously reprograms the TME and triggers rapid and durable cancer immunotherapy.

mRNA vaccines encoding chimeric antigen receptors (CARs)

CAR-T combined with mRNA technology has made progress in tumor therapy, especially in the field of solid tumor therapy. CAR-T in solid tumors faces many challenges, such as lack of highly specific targets, highly immunosuppressive TME, ineffective T-cell trafficking and tumor infiltration [52, 53]. The use of mRNA encoding CARS, thus enabling a synergistic approach of CAR-T technology and mRNA tumor vaccines to target and kill tumor cells, thereby enhancing anti-tumor efficacy. Traditional retroviral-based CAR-T approaches carry risks of off-target toxicity. In contrast, mRNA vaccine technology employs mRNA-transfected T cells, avoiding these risks. Additionally, CAR-encoding mRNA directly delivered to T cells enables efficient, cost-effective CAR-T cell production in vivo, eliminating the need for complex ex vivo cell manipulation and expansion. Gene-edited allogeneic T cells offer the potential as universal donor cells, bypassing the complexities and high costs associated with autologous T cells [54]. Introducing mRNA-encoded CARs into these genetically engineered allogeneic T cells reprograms circulating T cells to recognize tumor antigens. A preclinical study aimed at developing an injectable nanocarrier to directly program T cells in vivo to express CARs or TCRs targeting tumor or viral antigens by delivering ex vivo transcribed mRNAs has yielded sufficient host T cells expressing tumor-specific CARs or viral-specific TCRs to cause comparable disease regression to infusion in mouse models of prostate cancer, hepatocellular carcinoma and leukemia. CAR-T cells equivalent to disease regression [55].

A phase I/II clinical trial (BNT211-01) (NCT04503278), using a dose-escalation design to assess the safety and tolerability of CAR-T cells alone or in combination with an mRNA vaccine, enrolled 22 patients with relapsed or refractory CLDN6-positive solid tumors. No significant targeted/non-tumor toxicity was observed in the study, with 10 of 22 patients (46%) developing cytokine release syndrome, including one grade 3 event, and one patient developing grade 1 immune effector cell-associated neurotoxicity syndrome. Dose-limiting toxicity occurred in two patients who received higher doses, but neither left sequelae. Of the 21 evaluable patients, the unconfirmed ORR was 33% (7/21), including one complete remission. Disease control was 67% (14/21), including 7 patients with stable disease, demonstrating significant antitumor activity [56].

A safety and efficacy clinical trial (NCT01837602) of CAR T-cell injections in metastatic breast cancer evaluated the safety and feasibility of intratumoral treatment of metastatic breast cancer with mRNA-transfected c-MET-CAR-T-cells. c-MET is a tyrosine kinase receptor that is upregulated in a variety of solid tumors, particularly in about 50% of breast cancers. Results showed that CAR-T mRNA was detected in peripheral blood and tumor tissue after intratumoral injection in 2 and 4 patients, respectively. mRNAc-Met-CAR-T cell injection was well tolerated, as no patient had drug-related adverse reactions exceeding grade 1. Excision of tumors treated with intratumoral injection of mRNAc-Met-car-T cells and analysis by immunohistochemistry revealed extensive tumor necrosis at the injection site, cellular debris, loss of c-Met immunoreactivity, and macrophages surrounding both the margins and within the necrotic areas. This result suggests that intratumor injection of mRNAc-Met-CAR-T cells was well tolerated and triggered an inflammatory response within the tumor [57].

mRNA vaccines for breast cancer treatment

Breast cancer is classified as a "cold tumor," characterized by its limited responsiveness to immunotherapy. Vaccination with mRNA presents a promising strategy to overcome the adverse clinical trial variables associated with breast cancer vaccines. Currently, breast cancer is categorized into four main molecular subtypes based on immunohistochemical phenotypes: luminal A (HR + /HER2 −), luminal B (HR + /HER2 +), HER2-positive, and TNBC (HR − /HER2 −) [58]. Among these, TNBC and HER2-positive breast cancers exhibit higher immunogenicity [59], making these subtypes more amenable to tumor vaccine-mediated immune activation. The following table lists current clinical trials of mRNA vaccines in breast cancer (Table 1).

mRNA antitumor vaccines for HER2-positive breast cancer

HER2 (human epidermal growth factor receptor 2) amplification and/or overexpression occurs in approximately 20–30% of breast cancers. This overexpression correlates with enhanced tumor growth, reduced responses to conventional chemotherapy, and decreased overall survival [60]. HER2/neu proteins circulating in the bloodstream of breast cancer patients serve as TAAs, making them ideal targets for tumor vaccines. Recent preclinical studies demonstrate that mRNA vaccines elicit stronger immune-stimulatory antigen presentation compared to HER2-positive tumor cell vaccines or peptide-loaded dendritic cells (DCs). These vaccines efficiently induce HER2-specific CTLs, leading to potent tumor cell lysis [61]. For instance, a preclinical study suggested that a HER2 mRNA vaccine (VRP-HER2) stimulated HER2-specific T cell responses in mice, which was associated with tumor growth suppression. This effect appeared more pronounced in a subset of memory CD8 + T cells expressing perforin, and preliminary data indicated a potential correlation with progression-free survival (PFS) trends in breast cancer patients [13]. Additionally, sera from VRP-HER2 vaccinated mice exhibited higher levels of HER2-specific antibodies with diverse functional capacities compared to antibodies induced by monoclonal antibody therapies or protein vaccines. These polyclonal antibodies were more effective in mediating HER2 internalization, degradation, and reduced signaling [62], ultimately enhancing clinical outcomes in HER2-resistant diseases [63, 64].

A clinical trial evaluating clinical outcomes by administering the HER-2/neu vaccine prior to surgical resection and performing pre-vaccination and post-vaccination analyses demonstrated that vaccination against HER-2/neu was safe and well-tolerated and induced a decrease and/or eradication of HER-2/neu expression. This clinical trial demonstrates that vaccination in this early stage disease is safe and effective in inducing a long-term stable immune response and eradicating HER-2/neu-expressing tumor cells in residual DCIS lesions [22], and further suggests a possible new therapeutic option for patients with phenotypes that are underserved by currently available therapies.

mRNA antitumor vaccines for TNBC

TNBC is biologically and clinically more aggressive than other breast cancer subtypes. Its highly proliferative tumor cells, often classified as intermediate- or high-grade cancers, are associated with poor prognosis and limited treatment options [65]. mRNA vaccines are a potential treatment option for TNBC because they reshape the host immune system to eliminate tumor cells. However, the lack of common tumor antigens in TNBC complicates the identification of appropriate vaccination targets. Preclinical and clinical studies have explored MUC1, p53, and neoantigens as promising targets for TNBC, demonstrating effective antitumor activity. Combining mRNA vaccines with ICBs can improve tumor antigen recognition (via vaccinations) while also increasing anticancer immune responses (by checkpoint blockage) [66, 67]. A preclinical study to develop a nanoparticle-based mRNA vaccine for delivery to DCs in lymph nodes to activate and expand tumor-specific T cells. The combined therapeutic effects of the vaccine and anti-CTLA-4 antibody were evaluated in a mouse TNBC model, including tumor growth inhibition, CTL responses, interferon-γ production, and analysis of tumor-infiltrating T cells. The results showed that MUC1 mRNA vaccine was successfully expressed in DCs of lymph nodes and induced strong antigen-specific CTL responses. The combination therapy significantly increased the number of tumor-infiltrating CD8 + T cells and enhanced IFN-γ production, which inhibited the growth of TNBC tumors, followed by toxicity studies that showed that the treatment had no significant adverse effects on organ function and blood indices in mice [68].

Due to low tumor-infiltrating lymphocyte (TIL) levels, monotherapy is often less effective for TNBC. A clinical retrospective cohort trial of nearly 16,000 patients across five trials found that TIL levels were prognostic for both TNBC and HER2 + breast cancer. Among 821 TNBC patients from three trials, a 10% increase in stromal TILs correlated with approximately 15–20% improvement in disease-free survival (DFS; P = 0.005–0.035). Similarly, in 387 HER2 + breast cancer patients, a 1% increase in stromal TILs reduced recurrence rates by 3% (P = 0.002). mRNA vaccines effectively induce humoral and cellular immunity [25, 69], particularly enhancing CD8 + T cell responses [70], which improves TIL levels, extends DFS, and reduces recurrence rates in breast cancer.

Current clinical strategies for mRNA breast cancer vaccines

Natural mRNA molecules are unstable because of their linear shape, which makes them extremely vulnerable to RNase breakdown. A new synthesized RNA molecule with a circular shape, circRNA, is very stable and resistant to RNase [71–73]. Circular messenger RNAs are single-stranded RNAs with a covalently closed circular structure, generated through back-splicing processes [74]. CircRNAs lack the terminal motifs required for degradation by exonucleases, rendering them more stable than linear mRNAs [75, 76]. Compared to single-stranded mRNAs, circRNAs exhibit enhanced resistance to intracellular enzymatic degradation, thereby extending their functional duration. This stability enhances the translation efficiency of synthetic mRNAs, reduces intrinsic immunogenicity, and makes them more suitable for therapeutic applications by facilitating the development of long-term antitumor immunological memory and prolonged antigenic stimulation.

Emerging research indicates that circRNAs hold significant promise for therapeutic applications. A circRNA-based vaccine against SARS-CoV-2 has been successfully developed, providing a proof-of-concept for the potential applicability of this technology in antitumor strategies [77]. In a preclinical study, circRNAs supplied using lipid nanoparticle (LNP) packing outperformed linear counterparts in terms of stability and protein expression in vitro. CircRNA-LNPs elicited robust innate immune responses and antigen-specific cytotoxic T cell activation, effectively eradicating refractory tumors in murine models. Furthermore, combining circRNA-LNPs with adoptive cell transfer therapy exhibited potent antitumor efficacy in advanced tumor-bearing mice [78]. Additionally, a preclinical study injecting a mixture of circular mRNAs encoding four cytokines into tumors successfully modulated intratumoral and systemic antitumor immune responses and augmented tumor suppression induced by an anti-PD-1 antibody [79].

Neoepitope-targeted immunotherapies are restricted in their efficacy due to the limited number of cancer-associated mutations that can trigger strong immune responses [80, 81]. To overcome this issue, mRNA vaccines should concurrently target numerous TAAs or TSAs to improve effectiveness. Personalized multi-neoepitope vaccination techniques can attract a larger range of immune effectors, lowering the chance of tumor escape owing to loss of single neoantigens. Unlike peptide-based vaccinations, mRNAs may encode full-length antigens, ensuring the presentation of various epitopes without regard to HLA type. Furthermore, mRNAs can be engineered to express multiple neoantigens, either as individual molecules or as tandem sequences. Certain tumor forms may produce hundreds of neoantigens, and in terms of producing a thorough immune response, the expression of many epitopes capable of activating T cells is favorable [82].

A bioinformatics analysis study suggests that multi-epitope vaccines designed to elicit more robust anti-tumor immune responses can be used to target TNBC alone or in combination with other therapies. Comprehensive experimental validation is necessary to determine the effectiveness of such vaccinations [83].

Preclinical studies have found that in animal models of breast cancer transplantation, mouse tumor tissues express a certain percentage of gene mutations that are immunogenic (21–45%), and that multi-epitope mRNA vaccination is effective in controlling advanced tumors in mice [84]. To maximize the effectiveness of mRNA breast cancer vaccines, numerous TAAs must be targeted in relevant individuals. The clinical feasibility, safety, and anticancer effectiveness of multi-epitope mRNA vaccines targeting specific cancer mutations have been proven, giving data to encourage increased access to customized medicines for breast cancer patients.

To provide tailored neoantigen vaccines to clinical patients, it is first important to discover and confirm patient-specific immunogenic mutations expressed within malignancies [85, 86]. Subsequent steps involve screening, analyzing, and prioritizing mutations with the highest immunogenic potential. Based on in vitro binding assays, a ranked list of candidate antigens is generated, TSAs are manufactured, and the vaccine is administered to the patient [87]. Patients then undergo clinical and immunological monitoring to evaluate the therapeutic efficacy and immunogenicity of the neoantigens included in the vaccine. Currently, this process takes approximately 8 to 12 weeks [88], thus developing more convenient kits or optimizing the assay technique to save time is critical for the next steps in development. Personalized mRNA vaccines are better suited for newly diagnosed patients than progressive patients because nonantigenic vaccines are drugs that are individualized and tailored to a patient's mutation status and can be produced before the clinical course of the disease, and the time required to produce a customized vaccine does not meet the urgent need to treat progressive or recurrent disease.

The combination of mRNA vaccines with antitumor drug therapy could address various problems in antitumor therapy for breast cancer at this stage. first, the combination therapy could be used to overcome multidrug resistance (MDR), which is a considerable challenge in cancer therapy. Second, combination therapies can improve overall therapeutic efficacy and overcome the limitations of monotherapy [89]. Combination immunotherapy, cancer vaccines that encourage the patient's immune system to recognize and target tumor cells, and immunotherapy to boost the immune response and counteract immunosuppressive mechanisms in the TME are all examples. Combining certain chemotherapeutic agents can eliminate the effects of intratumoral treg and MDSC [90]. Third, because high doses are frequently needed to produce therapeutic effects when anticancer medications are taken alone, combination therapy also lessens the toxicity associated with cancer monotherapy. Fourth, mRNA vaccines by themselves cannot kill larger or advanced tumors. Immunocheckpoint inhibitors, chemotherapy, radiation, targeted therapy, and lysosomal virus therapy are examples of combination medicines that offer a tremendous chance to address this problem and increase therapeutic efficacy [91–94]. Combination trials will dominate the field of clinical trials in immuno-oncology in the coming years, the vast majority of them evaluating an investigational drug on the basis of an ICB [95].

To sum up, a scientifically sound approach is required to enable the effective combination of standard breast cancer therapy and mRNA vaccines. For example, combining medications that target the biology of breast cancer (endocrine therapy and trastuzumab) or that alter the immune milieu (cyclophosphamide and paclitaxel) can be used to increase the activity of mRNA vaccines for the treatment of advanced breast cancer. This will reduce the tumor load, balance the severity of the disease and the immune response, and lessen the effects of immune tolerance and suppression. This will give patients more effective, individualized treatment options and provide a promising avenue for future research on breast cancer as well as other diseases. Numerous benefits can be obtained from this strategy, including the enhanced effectiveness of combination therapy techniques and the enhanced quality of life linked to decreased medication toxicity.

When used with traditional chemotherapeutic medicines, mRNA therapy can increase patient responsiveness to treatment and increase efficacy through synergistic therapeutic effects. Several chemotherapeutic agents have been used in combination cancer therapy, including benzethonium chloride (BZN), paclitaxel, cabazitaxel, adriamycin, and orlistat. BZN has been used in nanocomplexes formulations with bcl -2-targeted siRNAs and has demonstrated therapeutic efficacy in vivo [96]. In preclinical models, mRNAs have been utilized to reintroduce tumor suppressors like TP53 and PTEN into tumors to slow their growth and make them more sensitive to chemotherapeutic treatments. Additionally, research has demonstrated that TIL levels are lower in pre-chemotherapy samples than in post-chemotherapy samples, corroborating the idea that chemotherapy can trigger an immune response against the tumor. When neoadjuvant chemotherapy was used, an increase in TIL was associated with a higher pCR, regardless of the type and duration of neoadjuvant chemotherapy treatment. Cyclophosphamide and temozolomide deplete Treg and create a more receptive tumor environment [97–99]. Doxorubicin, used in the treatment of breast cancer among others, enhances antigen presentation and enhances immune recognition of tumor cells [100]. Gemcitabine targets and reduces MDSCs [101]. Numerous preclinical and clinical investigations have demonstrated that mRNA and chemotherapy can work in concert to lower the dosage of chemotherapy and provide novel treatment options for patients who are unable to tolerate it.

Advances in mRNA delivery systems

In the past, the development of mRNA drugs faced challenges due to the inherent instability of mRNA single-strand structure, its immunogenicity in vivo, and limitations in delivery efficiency [102]. With advancements in stabilization techniques, sequence optimization, and lipid nanoparticle delivery systems, mRNA-based approaches are now being explored for both preventive and therapeutic applications, offering promising avenues for population-level and individualized medical interventions. Installing cap motifs, altering nucleotide structures, and optimizing nucleotide sequences have all improved the shelf and in vivo stability of mRNA therapeutic compounds. The 2023 Nobel Prize in Physiology or Medicine recognized Katalin Karikó and Drew Weissman for their contributions to mRNA modification, which provided a platform for the rapid development of mRNA vaccines during the global COVID-19 crisis.

The delivery system is one of the most important aspects of mRNA drug development. mRNAs face numerous obstacles during intracellular delivery: naked mRNAs are unstable in vivo and susceptible to degradation by ribonuclease (RNase) [103] Negatively charged mRNA macromolecules have difficulty crossing the host cell membrane, which is also negatively charged [104], resulting in inefficient cell permeation. Exogenous mRNAs' potent immunogenicity may cause the body to mount an immune response, which could enhance cytotoxicity and hinder the translation of therapeutic proteins [105]. Therefore, the development of efficient and safe delivery systems is an important research direction for current mRNA therapy.

Currently, viral vectors and non-viral vectors are commonly used. The commonly used viral vectors are retroviruses, adenoviruses, adeno-associated viruses and lentiviruses. Although they have benefits including a high rate of transfection, the toxicity, immunogenicity, and bulk of viral vectors are barriers to their continued advancement [106]. Various non-viral nanocarriers, such as LNP, polymer nanoparticles, and lipid-polymer hybrid nanoparticles, have received greater attention [107–109]. LNPs are currently the most studied and clinically advanced non-viral delivery vehicles for mRNA [19]. To improve mRNA loading capacity and delivery effectiveness, LNPs can be altered through straightforward and controlled chemical production. Currently widely used are LNPs with cationic or ionizable lipids [110, 111], which usually contain cationic or ionizable lipids, cholesterol, co-lipids and polyethylene glycol (PEG) [112]. Ionizable lipids are the most important components responsible for nucleic acid encapsulation during preparation and lysosomal escape after cellular uptake. Ionizable lipids are positively charged under acidic pH conditions and interact electrostatically with negatively charged mRNA and increase the encapsulation efficiency, which in turn forms LNP-mRNA through interactions with other lipid components. LNP-mRNA is electrically neutral at physiological pH and is unable to interact with anions on the cellular membrane, which reduces cytotoxicity [113]. After the cell absorbs LNP-mRNA, the pH in the endosome drops, which causes ionizable lipids to get protonated. These lipids then interact with the endosomal membrane, disrupting it and allowing mRNA to escape into the cytoplasm to synthesis target proteins there [114, 115]. As a class of amphiphilic lipids, PEG lipids are essential for controlling the stability, half-life, and clearance of LNPs. plays a vital part. The accurate delivery of LNP-mRNA has been made possible by research on PEG length, in vivo shedding kinetics, and adsorbed protein crowns. Phospholipids and cholesterol are essential for enhancing lipid bilayer stability, facilitating membrane fusion, and facilitating endosomal escape [113, 116]. SLN are spherical nanoparticles with an inner core of solid lipids used to encapsulate the drug, and an outer bilayer composed of surfactants. NLC are mixtures containing solid and liquid lipids, which is a mixture that replaces some of the solid lipids of SLN with liquid lipids, which creates a larger space and increases the drug-carrying capacity after substitution.

The good modifiability, biocompatibility, and biodegradability of polymeric nanoparticles, which are often made of biodegradable amine-containing polymers that can self-assemble with RNA, make them promise as delivery systems even though they are not as well-known as LNPs, which are currently the most studied. PNPs can also be made with adjuvant phospholipids, cholesterol, and peg-lipids, depending on the need. Cell-specific targeting can be facilitated by further modifying both LNPs and PNPs with certain ligands [82].

In real-world applications, limitations of delivery mechanisms for mRNA tumor vaccines still exist, for example, the most used LNPs are still less stable in complex in vivo environments and are prone to rapid metabolism [117]. Some mRNA vaccines have not yet achieved specific targeting of the immune system, resulting in vaccines being retained at the injection site or in liver tissue [118]. In addition, tumor vaccine therapy requires not only protection through humoral immunity, but more importantly, robust cellular immunity mediated by cytotoxic CD8 + T cells to eradicate malignant cells [82]. These limitations suggest that despite the broad potential of mRNA vaccines, there are still many scientific and technical challenges that need to be addressed for their practical application.

For LNP-loaded mRNA delivery to increase the effectiveness of mRNA tumor vaccines and broaden the region of immune stimulation, appropriate methods of administration are essential. Conventional delivery systems include intradermal, intramuscular, and subcutaneous administration [25, 119] Unconventional delivery systems include intralipid (intranodal, intravenous, intrasplenic, or intra-tumoral). Among these, intranodal injection is the injection of mRNA into secondary lymphoid tissues to target antigen-presenting cells at the site of T-cell activation, avoiding the need for DC migration [120].Kreiter et al. observed that intranodal injection stimulated robust T-cell immunity compared to other routes of administration (i.d./s.c./n.n.) [121]. Intranodal injections in combination with systemic drug delivery methods (e.g., ICBs) provide a more comprehensive approach to cancer treatment [122]. In order to facilitate antigen presentation in difficult-to-reach central nervous system cells, intratumoral injections are recommended for patients with brain metastases from breast cancer. Because the liver is full of porous capillaries, mRNA tumor vaccine antigens can target certain organs by changing the constitution and makeup of LNP when injected and supplied intravenously [123] for the treatment of liver metastases from breast cancer. This passive targeting LNP was named Selective organ targeting nanoparticles. Furthermore, cell-targeted LNPs were created to achieve a more precise mRNA delivery method. These LNPs were made to be absorbed by particular cells, which in turn caused protein expression in those cells. Although ligand-receptor interactions often facilitate this cell-specific uptake, new research has shown that changing the lipid structure in the LNP can also have this effect.

Discussion

As an emerging strategy for breast cancer treatment, mRNA vaccines are of research value due to their safety, efficacy, rapidity, and mass production. mRNA tumor vaccine technology has progressed in various aspects over the past few years, with various preclinical and clinical trials. Future research directions will revolve around the core areas of target optimization, delivery system upgrades, and combination therapies. Considering the current research progress and challenges, the following are the feasible future research directions and perspectives.

The design of mRNA tumor vaccines usually samples only a small portion of samples, which cannot provide complete information about the tumor genetic profile. Moreover, the tumor heterogeneity caused by the dynamic evolution during breast cancer progression and the subcloned composition of different genetic backgrounds limits the efficacy of mRNA vaccines, which is a major challenge for their clinical application. Therefore, it is necessary to develop algorithms to accurately predict neoantigens with high immunogenicity, such as TNBC, which lacks a clear target and needs to be screened for neoantigens with high mutation loads by whole-exome sequencing. And by taking advantage of the fact that mRNAs can encode multiple antigens, vaccines containing multiple neoantigens should be designed to overcome tumor heterogeneity and immune escape, e.g., exploring the combined application of TAAs, TSAs, etc. (e.g., HER2, MUC1).

Since the major drawbacks limiting the clinical feasibility of mRNA vaccines are their low stability and limited transfection efficiency, the concept of nanotechnology was introduced. Encapsulation of mRNA vaccines in novel nanocarriers has facilitated the development of mRNA vaccines from conceptual propositions to clinically viable universal solutions. Thus, the future can look forward to the development of ionizable lipids to improve the intracellular delivery efficiency of mRNA and to explore ligand modifications targeting breast tissues to enhance the local enrichment of the TME.

Furthermore, even if mRNA-produced antigens can induce a cellular immune response, the suppressive TME can prevent T-cells from infiltrating into the tumor and may lead to T-cell exhaustion. Therefore, therapeutic tumor vaccines need to be administered in combination with another therapy that overcomes the suppressive microenvironment (e.g., TCBs) [124], e.g., mRNA vaccines in combination with anti-PD-1/PD-L1 drugs can reverse immunosuppression in TME, and the combination of mRNA-4157 with pembrolizumab as mentioned above reduced the risk of recurrence or death in melanoma, and a similar strategy could be extended to breast cancer.

With the expanding portfolio of therapeutic targets and the innovative approaches being developed to personalize cancer drugs with mRNA vaccines. The integration of next-generation molecular sequencing technologies enhanced artificial intelligence algorithms, and advancements in vaccine preparation engineering has the potential to significantly transform tumor vaccine research. However, further studies are needed to fully realize the impact of these technologies and address existing challenges [125–127].

Abbreviations

ADCs

Antibody–drug conjugates

APCs

Antigen-presenting cells

BZN

Benzethonium chloride

CAR

Chimeric Antigen Receptor

CAR-T

Chimeric Antigen Receptor T-Cell

Circ RNA

Circular RNA

CTLs

Cytotoxic T lymphocytes

DC

Dendritic cell

DCIS

Ductal carcinoma in situ

TAAs

Tumor-associated antigens

TSAs

Tumor-specific antigens

HPV

Human Papillomavirus

HLA

Human leukocyte antigen

ICB

Immune checkpoint inhibitor

LNP

Lipid nanoparticle

MDSCs

Myeloid-derived suppressor cells

MHC

Major histocompatibility complex

mRNA

Messenger RNA

PDAC

Pancreatic ductal adenocarcinoma

PD-1

Programmed cell death protein 1

PD-L1

Programmed cell death-ligand 1

PEG

Polyethylene glycol

TCR

T cell receptor

TIL

Tumor-infiltrating lymphocyte

TME

Tumor microenvironment

TNBC

Triple-negative breast cancer

Author contributions

Jia-ying Li: conceptualized the study, wrote original draf, developed methodology. Rui-yuan Jiang: developed methodology, performed formal analysis. Jia Wang: developed software, curateddata. Xiao-jia Wang: supervised the study, administered the project. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Zhejiang Provincial Traditional Chinese Medicine Science and Technology Program Project (2022ZB050);Natural Science Foundation of Zhejiang Province, China (Grant Number: TGD23H160004).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

The manuscript is approved by all authors for publication.

Competing interests

The authors declare no competing interests.