Introduction

The discovery of eukaryotic split genes, with non-coding intronic sequences interspersed within protein-coding regions, revolutionized our understanding of gene structure and regulation. This breakthrough illuminated the process of RNA splicing, where introns are excised from pre-messenger RNA (pre-mRNA) by the spliceosome, a ribonucleoprotein complex that ensures the correct ligation of exons to form mature mRNA for translation [1, 2]. Splicing, which occurs in approximately 94% of human genes, is a ubiquitous and essential process in higher eukaryotes, playing a pivotal role in gene expression. Notably, a significant portion of these genes undergoes alternative splicing, generating diverse mRNA isoforms that enable a single gene to produce multiple protein variants [3–5]. This diversification is essential for cellular differentiation, development, and homeostasis. Aberrant splicing, however, can lead to the production of dysfunctional protein isoforms, contributing to various pathological conditions, including cancer and neurodegenerative diseases [6–8]. Furthermore, alternative splicing plays a crucial role in the presentation of neoantigens, which are tumor-specific antigens (TSAs) formed due to genetic mutations and splicing alterations. These neoantigens have emerged as key targets for cancer immunotherapies, highlighting the significance of understanding splicing mechanisms in the development of novel therapeutic strategies.

In cancer biology, the concept of TSAs, or neoantigens, has emerged as a transformative approach in immunotherapy [9, 10]. Neoantigens arise from somatic mutations and are presented on tumor cell surfaces by major histocompatibility complex (MHC) molecules, enabling cytotoxic T lymphocytes (CTLs) to target and eliminate tumor cells [9, 11, 12]. The identification of neoantigens has catalyzed the development of cancer vaccines and adoptive T cell therapies, all of which amplify tumor-specific immune responses [9, 13, 14]. Notably, neoantigens are exclusive to tumor cells, minimizing off-target effects and enabling personalized therapeutic approaches.

While most neoantigen research has focused on mutations, recent studies have highlighted a novel source: aberrant RNA splicing. Dysregulated splicing can produce cryptic exons or exon-skipping events, leading to the generation of tumor-specific splicing isoforms that may serve as immunotherapeutic targets [3]. Advances in next-generation sequencing (NGS) and RNA-seq technologies have facilitated the profiling of cancer-specific splicing landscapes, uncovering new neoepitopes [15, 16]. Additionally, predictive bioinformatics tools that assess MHC-binding affinity and immunogenicity further expand the potential pool of actionable targets [17, 18].

Glioblastomas (GBMs) exemplify the need for innovative therapies. Despite multimodal treatments, including surgery, radiotherapy, and chemotherapy with temozolomide, median survival remains limited to 15 months [19, 20]. FDA-approved therapies such as bevacizumab and tumor-treating fields (TTF) offer minimal survival benefits, and immunotherapeutic agents like nivolumab and EGFRvIII vaccines have failed to achieve efficacy in phase 3 trials. GBM’s inherent heterogeneity, infiltrative growth, and immunosuppressive microenvironment contribute to its resistance to conventional treatments [21, 22].

Neoantigen-based personalized peptide vaccines offer a promising avenue for GBM treatment [9, 23]. By utilizing NGS to identify tumor-specific mutations and splicing variants, these vaccines target unique neoepitopes, generating robust and specific immune responses. Clinical trials targeting the H3K27M mutation in diffuse midline gliomas have demonstrated safety and immunogenicity, underscoring the potential of personalized immunotherapy for GBM management [24, 25]. This strategy represents a critical step in addressing the challenges posed by this devastating malignancy.

This article explores the transformative potential of neoantigen-based immunotherapy, a precision oncology approach that targets TSAs arising from genetic, transcriptomic, and proteomic alterations. It examines various therapeutic modalities, including neoantigen vaccines, tumor-infiltrating lymphocyte (TIL) therapy, T-cell receptor-engineered T cells therapy (TCR-T), and chimeric antigen receptor T cells therapy (CAR-T), all of which show promise in treating GBM. The article also highlights how advances in NGS, RNA-based platforms, and CRISPR gene-editing technologies have accelerated neoantigen discovery, facilitating the development of highly personalized treatments. However, clinical application remains challenging due to factors such as tumor heterogeneity, immune evasion, and therapeutic resistance. To overcome these obstacles, the article underscores the need for an integrated approach that combines multi-omic data analysis, AI-driven predictive modeling, and collaborative efforts between researchers and clinicians to optimize neoantigen-based immunotherapies, paving the way for more effective and durable cancer treatments.

Advances in neoantigen research: from early insights to clinical applications in targeted cancer immunotherapy

Neoantigen research has significantly advanced our understanding of tumor immunology and has been crucial in the development of targeted cancer therapies. Neoantigens are tumor-specific proteins that arise from somatic mutations in cancer cells, making them highly specific and effective targets for immune system recognition. The identification and validation of these neoantigens have revolutionized cancer immunotherapy by opening up new possibilities for personalized treatments [10, 26].

The exploration of neoantigens dates back to foundational studies in the mid-20th century, which demonstrated the immune system’s ability to recognize tumor cells as foreign [27, 28]. These early insights set the stage for further discoveries, including the recognition of T cell-mediated immune responses against neoantigens in both murine and human tumor models, validating their immunogenic potential [13, 29].

The technological breakthroughs of the early 2000s, such as NGS and flow cytometry, played a pivotal role in advancing neoantigen research. These tools allowed for high-throughput identification and validation of neoantigens, deepening our understanding of tumor immunology and T cell responses [30, 31].

Key milestones in neoantigen research are summarized in Table 1, highlighting how the field has evolved from initial discoveries to the application of personalized neoantigen vaccines and adoptive cell therapies (ACTs). The progress made in identifying and targeting TSAs demonstrates the transformative potential of these.

Recent advancements in technologies, such as NGS and RNA-based platforms, have greatly accelerated the clinical translation of neoantigen research, enhancing the specificity and efficacy of therapeutic strategies [31, 53]. Ongoing innovations continue to refine neoantigen-based immunotherapies by improving immune responses while reducing off-target effects.

Looking ahead, the integration of cutting-edge technologies like CRISPR and AI may revolutionize the discovery and validation of neoantigens. These advancements promise to further enhance the effectiveness of cancer treatments, broadening the applicability of neoantigen-based therapies across various cancer types.

Mechanisms driving neoantigen diversity: genetic, transcriptomic, and proteomic contributions

Neoantigen generation results from a multifaceted interplay of genetic, transcriptomic, and proteomic mechanisms, all of which contribute to the diversity of the antigenic landscape. These molecular alterations can profoundly affect the immune system’s ability to recognize cancer cells by presenting novel peptide sequences that are not found in normal, healthy tissues [54, 55].

At the genomic level, various mutations, such as single nucleotide variants (SNVs), insertions and deletions (INDELs), and gene fusions, disrupt the normal coding sequences of genes (Table 2). These alterations can lead to the creation of novel open reading frames (ORFs) and chimeric proteins that may possess antigenic properties, making them recognizable by the immune system [56, 57]. Additionally, the integration of viral sequences or the reactivation of endogenous retroviruses (ERVs) further contributes to the diversity of the neoantigen repertoire by introducing foreign protein-coding regions into the genome [58, 59]. These changes can be particularly impactful in cancer, where such genomic alterations can create unique targets for immune recognition.

The transcriptomic landscape also plays a crucial role in expanding neoantigen diversity. Alternative mRNA processing events, such as exon skipping, alternative splice site selection, and intron retention, can modify the protein-coding potential of genes (Table 2). These variations can lead to the translation of modified or truncated protein products, which may differ significantly from the normal protein isoform [89–91]. In addition, mechanisms like mutually exclusive exon expression and the partial retention of exitrons further contribute to the diversity of protein isoforms that can be produced from a single gene [75]. Moreover, post-transcriptional modifications, such as adenosine-to-inosine (A-to-I) RNA editing, introduce codon changes that alter the sequence of the resulting peptides without modifying the underlying DNA sequence. This adds another layer of potential neoantigen generation from modified RNA transcripts [92, 93].

At the proteomic level, the generation of neoantigens is further enhanced by variations in translation and post-translational modifications (Table 2). ORFs can emerge from untranslated regions of the genome, and small peptides derived from long non-coding RNAs (lncRNAs) may also generate unique antigenic peptides [94, 95]. Translation defects, such as premature termination, frameshift mutations, or the use of non-canonical start sites, also contribute to antigenic diversity by producing altered peptide sequences that are not present in healthy tissues [96]. Additionally, post-translational modifications, such as phosphorylation, glycosylation, and ubiquitination, can expose previously hidden epitopes or alter peptide structures in ways that make them more immunologically recognizable [97, 98].

A comprehensive summary of the genetic, transcriptomic, and proteomic mechanisms that contribute to neoantigen diversity is provided in Table 2.

Neoantigen generation and treatment modalities in cancer immunotherapy

The development of effective cancer immunotherapies relies heavily on the identification and targeting of tumor-specific neoantigens. Neoantigens, which arise from genetic and molecular alterations unique to cancer cells, play a critical role in immune recognition and the design of personalized treatments [26, 42, 99]. Understanding how various therapeutic strategies influence neoantigen generation and presentation is essential for optimizing immunotherapy efficacy.

A comprehensive overview of various treatment modalities and their roles in neoantigen generation within the context of cancer immunotherapy are discussed in Figure 1. Each therapeutic approach affects neoantigen formation through distinct biological mechanisms, with implications for immune response modulation, clinical application, and combination therapy potential.

Display full size

Figure 1. 

Treatment modalities and their role in neoantigen generation for cancer immunotherapy

Chemotherapy, particularly alkylating agents such as temozolomide and cyclophosphamide, induces DNA alkylation that causes direct strand damage, leading to point mutations and small insertions or deletions [100–102]. These genetic alterations give rise to novel antigenic peptides presented on MHC molecules, making tumor cells more immunogenic. For example, temozolomide treatment has been associated with enhanced neoantigen expression in GBM, particularly when paired with cancer vaccines [23]. However, chemotherapy poses challenges in predicting the extent of neoantigen generation and may cause immunosuppression, complicating its standalone efficacy [103, 104].

Radiation therapy also contributes significantly to neoantigen generation by causing DNA damage through direct ionization and reactive oxygen species (ROS) production. This damage induces genomic instability, leading to the formation of neoantigens from fragmented or mutated tumor DNA [105–107]. Preclinical studies in triple-negative breast cancer and non-small cell lung cancer (NSCLC) have demonstrated that radiation, when combined with immune checkpoint inhibitors such as anti-programmed death-1 (PD-1) inhibitors, enhances antitumor immunity by increasing immune cell infiltration [108, 109]. Nonetheless, radiation therapy carries the risk of localized tissue damage and immune-related adverse effects [110].

Microbial activity, specifically genotoxic microbial metabolites such as colibactin produced by Escherichia coli, can lead to tumor-specific mutations. Strains like pks+ E. coli induce double-strand breaks in host DNA, contributing to neoantigen formation in colorectal cancer [111, 112]. Similarly, Fusobacterium nucleatum and Bacteroides fragilis have been linked to mutagenic effects in colorectal and oral cancers [113, 114]. These microbial-induced neoantigens can promote tumor-specific immune responses, yet challenges remain in targeting specific microbial strains without disrupting the broader microbiome [115].

Targeted therapies, such as tyrosine kinase inhibitors (e.g., imatinib and erlotinib), interfere with signaling pathways involved in tumor growth and survival. These agents can induce mutations or unmask cryptic antigens within the tumor microenvironment (TME) [116, 117]. For instance, imatinib has been associated with increased mutation rates in chronic myelogenous leukemia, contributing to antigenic diversity [118]. However, the emergence of resistance and limited efficacy in tumors with low mutation rates restricts the widespread impact of targeted therapies on neoantigen generation [119].

Immunotherapy, particularly immune checkpoint inhibitors (e.g., pembrolizumab and nivolumab), plays a central role in restoring immune surveillance by blocking inhibitory proteins like PD-1 and CTLA-4 [120, 121]. These inhibitors amplify T-cell activity against tumor-specific neoantigens, especially in tumors with high mutational burdens. However, immune-related adverse events, such as colitis and pneumonitis [11, 122], pose clinical management challenges, necessitating careful patient selection.

Emerging therapeutic modalities, including oncolytic virus therapy and gene therapy, further expand the landscape of neoantigen generation. Oncolytic viruses like T-VEC and Reolysin selectively infect tumor cells, inducing direct lysis and the release of neoantigens [123, 124]. Additionally, viral infection can trigger immunogenic cell death, promoting immune system activation [125]. Gene therapy approaches, such as CAR-T cell therapy and oncolytic virus gene modification, enhance neoantigen presentation through engineered genetic alterations that promote novel antigen expression [126]. However, these advanced therapies face challenges like delivery efficiency, off-target effects, and high costs.

Epigenetic modifiers, including DNA methylation inhibitors and histone deacetylase inhibitors like vorinostat and azacitidine, represent another innovative strategy for neoantigen generation [127, 128]. By altering the epigenetic landscape, these agents can reactivate silenced tumor antigens and promote immune recognition [129, 130]. Nonetheless, concerns regarding off-target epigenetic changes and unpredictable long-term effects remain significant hurdles.

Advancing cancer immunotherapy: the role of neoantigen-based approaches

Neoantigen-based immunotherapies—personalized approaches and synergistic strategies in cancer treatment

Neoantigen-based immunotherapies represent a promising frontier in cancer treatment, leveraging the body’s immune system to target TSAs [131]. These therapies encompass a variety of approaches, including vaccines, TIL therapy, TCR-T therapy, and CAR-T cells, each designed to stimulate or enhance immune responses against tumor neoantigens [132] (Figure 2). The breadth of neoantigen-targeted strategies underscores the potential for personalized cancer treatments [133, 134], emphasizing the importance of tailoring interventions to the unique antigenic landscape of individual tumors.

Display full size

Figure 2. 

Comparative analysis of neoantigen-based immunotherapies: mechanisms, targeted tumors, and clinical potential

Neoantigen vaccines, for instance, aim to stimulate T cells by presenting neoantigen peptides or mRNA to the immune system, primarily targeting tumors such as melanoma, glioma, and lung cancer [131]. These vaccines offer advantages like non-toxicity and the ability to customize treatment for individual patients. However, challenges remain, including limited neoantigen identification and immune evasion by tumors. Despite these hurdles, studies have demonstrated the high therapeutic potential of neoantigen vaccines, particularly when combined with immune checkpoint inhibitors, leading to improved survival rates and tumor shrinkage [135, 136].

TIL therapy involves isolation, expansion, and reinfusion of TILs from the patient’s tumor to enhance antitumor activity. This approach has shown effectiveness in cancers with high TIL burden, such as melanoma and NSCLC [137, 138]. TIL therapy’s advantages include its ability to target multiple tumor mutations simultaneously and its direct engagement with the TME. However, invasive procedures for TIL harvesting and challenges related to tumor heterogeneity pose significant limitations [138]. Nonetheless, TIL therapy remains a powerful option, especially in immune-permissive tumors, with notable results in metastatic colorectal cancer [139, 140].

Combination therapies, including the use of neoantigen vaccines with TILs or immune checkpoint inhibitors, have gained attention for their synergistic effects in overcoming immune resistance [141]. As shown in Figure 2, combining these therapies can enhance the immune response by targeting multiple pathways simultaneously, particularly in cancers with high mutational burdens like melanoma and NSCLC [142, 143]. However, complex logistics, regulatory hurdles, and the risk of excessive immune activation remain barriers to widespread clinical adoption [144]. Still, the potential for achieving complete remission in refractory cancers underscores the promise of these multimodal strategies in advancing cancer immunotherapy.

Comparative analysis of neoantigen-based therapeutic strategies in cancer immunotherapy

Neoantigen-based therapeutic strategies represent a transformative advancement in cancer immunotherapy. These neoantigens are absent in normal tissues, allowing for precise tumor targeting with minimal risk to healthy cells [133, 145]. By focusing on these highly specific antigens, neoantigen therapies aim to harness and amplify the immune system’s natural ability to recognize and destroy malignant cells, thereby reducing the likelihood of off-target cytotoxicity and enhancing the therapeutic index [146, 147]. This precision is particularly advantageous in cancers with high mutational burdens, where the diversity of neoantigens increases the likelihood of effective immune responses [11, 148].

The principal neoantigen-based strategies include TCR-T therapy, CAR-T cell therapy, bispecific antibodies, neoantigen vaccines, TIL therapy, oncolytic virus therapies, cancer peptide vaccines, dendritic cell (DC) vaccines, and personalized multi-epitope vaccines [149]. TCR-T therapy involves the genetic engineering of autologous T cells to express TCRs capable of recognizing neoantigens presented by MHC-I molecules on the tumor cell surface [150]. CAR-T therapy modifies T cells to express synthetic antigen-binding receptors that can directly recognize tumor surface antigens without the need for MHC presentation, broadening their applicability to tumors with low MHC expression [151]. Bispecific antibodies act as immune engagers by simultaneously binding TSAs and CD3 on T cells, physically linking effector cells to tumor cells for targeted cytotoxicity [152, 153]. Neoantigen vaccines, including RNA, DNA, peptide, and DC-based platforms, work by introducing TSAs into the host to prime and expand tumor-reactive T cells [131, 154]. TIL therapy involves isolating and expanding TILs directly from the patient’s tumor, which are then reinfused into the patient to boost anti-tumor immunity [140, 155, 156]. Oncolytic virus therapies use genetically modified viruses that selectively infect and lyse tumor cells while also stimulating a systemic immune response against neoantigens released during tumor cell lysis [126]. Cancer peptide vaccines use synthetic peptides corresponding to TSAs to stimulate T cell responses, while DC vaccines involve loading patient-derived DCs with tumor neoantigens ex vivo before reinfusion [157–159]. Personalized multi-epitope vaccines combine multiple tumor neoantigens tailored to an individual’s tumor mutation profile, maximizing immune coverage [160, 161].

A comparative analysis of these neoantigen-based therapeutic strategies, evaluating critical parameters such as immune response potency, manufacturing complexity, scalability, and antigen breadth, is presented in Figure 3. TCR-T and CAR-T therapies exhibit robust immune responses but face challenges due to high manufacturing complexity and limited scalability, as they require patient-specific modifications [162, 163]. Bispecific antibodies provide a balance of moderate potency and manageable manufacturing demands, making them versatile, though still constrained by limited antigen breadth [164]. Neoantigen vaccines, while highly scalable and capable of broad antigen coverage, often elicit weaker immune responses and struggle to overcome immune suppression in advanced malignancies [146]. TIL therapy offers a potent and personalized response but relies on the availability of tumor-infiltrating cells and involves complex ex vivo expansion protocols [156]. Oncolytic viruses provide a dual mechanism of direct tumor lysis and immune activation but are often limited by pre-existing immunity against the viral vector [165, 166]. Peptide and DC vaccines offer moderate scalability and are less complex to produce but face challenges in generating sustained immune responses [167, 168]. Personalized multi-epitope vaccines maximize antigen breadth but require highly individualized tumor profiling, increasing complexity and cost [134, 141]. This comparative framework aids in selecting the optimal strategy based on clinical context and disease stage.

Display full size

Figure 3. 

Comparative evaluation of neoantigen-targeted immunotherapies: key features, advantages, and clinical feasibility

Advanced methodologies and tools for neoantigen prediction in personalized cancer immunotherapy

The role of neoantigen prediction in personalized cancer immunotherapy

In personalized cancer immunotherapy, the identification and targeting of neoantigens are critical for developing tailored therapeutic strategies [13, 193]. The process begins with mutation calling, which involves the detection of genetic alterations, such as SNVs, INDELs, gene fusions, and alternative splice variants, all of which are potential sources of neoantigens [84]. Tools like INTEGRATE-neo, PAVIS, and Cancer Genome Interpreter leverage NGS data and machine learning integration to identify these mutations with high sensitivity [194–196] (Figure 4). However, challenges such as false positives from sequencing errors or low variant allele frequencies can complicate mutation detection, highlighting the need for robust validation methods in the neoantigen discovery pipeline [197].

Display full size

Figure 4. 

Overview of computational and experimental tools for neoantigen discovery and immunogenicity assessment. HLA: human leukocyte antigen; TCR: T-cell receptor; MHC: major histocompatibility complex

Once mutations are identified, the next crucial step is human leukocyte antigen (HLA) typing, which determines the MHC alleles expressed by the individual. This is vital for predicting the potential presentation of peptides on the surface of tumor cells [198–200]. High-resolution genotyping tools like Polysolver and OptiType use sequence-based typing and Bayesian inference to accurately determine an individual’s HLA haplotype [201–203] (Figure 4). The accurate matching of peptides to specific MHCs is essential for the subsequent prediction of antigen presentation [17, 204, 205]. However, challenges arise in cases of low-coverage sequencing data, where the performance of HLA typing tools may be limited, necessitating further refinement of these methods for clinical applications.

HLA binding prediction is the next step, focusing on the prediction of peptide-MHC binding affinity and stability. The ability of peptides derived from mutations to bind to MHC molecules plays a pivotal role in eliciting a T-cell-mediated immune response [206, 207]. Tools such as NetMHCpan and pVAC-Seq employ advanced algorithms, including neural networks and position-specific scoring matrices, to predict the binding of peptides to MHC class I molecules (for CD8+ T cells) and class II molecules (for CD4+ T cells) [57, 172, 208] (Figure 4). These tools provide reliable predictions for peptide-MHC affinity, although they may overlook complex, non-linear binding patterns or peptide stability, which could affect the immune response. As a result, the integration of multiple prediction tools and experimental validation becomes necessary for optimizing neoantigen identification and ensuring effective immunogenicity [209].

To further refine neoantigen candidates, T cell recognition analysis assesses the likelihood of TCR binding to peptide-MHC complexes. Tools such as NetCTL and TCRMatch utilize structural modeling and TCR-peptide docking models to predict TCR interaction and immune activation [210, 211]. Despite their usefulness, these models are often complex and face challenges due to limited experimental validation data. Additionally, neoantigen ranking and prioritization tools like Neopepsee and MuPeXI evaluate immunogenicity and expression levels to prioritize the most promising neoantigens for therapeutic development [172, 212, 213]. Finally, the validation of computational predictions through experimental tools like ELISpot and MHC Tetramer assays is essential to confirm the immunogenicity of neoantigens [41, 214, 215]. While these experimental methods provide the gold standard for validation, they are labor-intensive and require specialized expertise and reagents. The integration of computational tools with experimental validation creates a comprehensive approach to neoantigen prediction, ultimately enabling the development of personalized cancer immunotherapies that target specific tumor mutations.

Comparative analysis of current therapies for GBM and personalized neoantigen vaccines

GBM is a highly aggressive and treatment-resistant brain cancer, presenting significant challenges in clinical management [246, 247]. Current and emerging therapeutic strategies for GBM encompass a broad spectrum of approaches, each characterized by distinct mechanisms of action, varying levels of efficacy, inherent limitations, and impacts on patient outcomes and quality of life [20, 248, 249]. A thorough evaluation of these therapies highlights the complexity of GBM treatment and underscores the urgent need for innovative and multimodal strategies to achieve meaningful improvements in patient survival and overall well-being.

Conventional treatment options, including surgical resection, temozolomide chemotherapy, and radiation therapy, remain the standard of care for GBM patients. While these modalities collectively offer modest survival benefits, with a median overall survival of approximately 15 months, their limitations are pronounced [250]. Tumor heterogeneity and the emergence of therapeutic resistance contribute to inevitable disease recurrence (Figure 5). Moreover, adverse effects, such as cognitive decline, fatigue, and immunosuppression, significantly compromise the quality of life. These challenges underscore the importance of integrating advanced therapeutic approaches to enhance efficacy and address the long-term limitations of these foundational treatments [251, 252].

Display full size

Figure 5. 

Comparative analysis of treatment approaches for glioblastoma (GBM)

Emerging therapies, such as bevacizumab, TTF, immune checkpoint inhibitors, and targeted therapies, provide novel avenues for GBM management (Figure 5) [253, 254]. Bevacizumab, an anti-angiogenic monoclonal antibody, has demonstrated promise in improving progression-free survival (PFS) in recurrent GBM cases but has not shown a significant extension of overall survival, particularly in newly diagnosed patients [255, 256]. Similarly, TTF, a non-invasive therapeutic modality that disrupts mitotic processes, achieves only limited survival benefits [257]. Immune checkpoint inhibitors and targeted therapies face significant challenges, including tumor immune evasion and heterogeneity, which limit their standalone efficacy [258, 259]. However, the potential for combination therapies to overcome these obstacles represents a critical area for further research and optimization.

Personalized neoantigen-derived peptide vaccines represent a promising advancement in precision medicine for GBM (Figure 5) [23, 260]. These vaccines leverage patient-specific tumor antigens to elicit robust T-cell responses, offering the potential for extended survival and improved quality of life [23]. Despite their promise, challenges such as the time-intensive production process and the immunosuppressive TME hinder their broad applicability [41, 103]. When combined with complementary therapies like checkpoint inhibitors, these vaccines demonstrate synergistic potential, paving the way for transformative developments in GBM treatment [261, 262].

By critically analyzing the strengths and limitations of these therapeutic modalities, researchers and clinicians can navigate the evolving landscape of GBM management more effectively and identify innovative strategies to enhance patient outcomes. The next section will explore the role of personalized neoantigen-derived peptide vaccines in greater detail.

Targeting tumor-specific neoantigens in GBM: innovations in immunotherapy, personalized vaccines, and overcoming immunosuppressive barriers

Clinical strategies in neoantigen-based immunotherapies for cancer treatment

Neoantigen-based immunotherapies have emerged as a promising approach for personalized cancer treatment, leveraging TSAs to provoke robust immune responses. Currently, a variety of therapeutic strategies—such as cancer vaccines, ACT, CAR-T therapy, and immune checkpoint blockade (ICB)—are undergoing clinical evaluation (Table 5). These therapies are being assessed for their ability to enhance immune responses, reduce tumor burden, and improve patient outcomes. Clinical trials are closely monitoring the safety, efficacy, and overall clinical impact of these therapies across various cancer types, highlighting the ongoing evolution of neoantigen-targeted immunotherapies (Table 5).

The clinical trials focus on five primary therapeutic categories: neoantigen-directed cancer vaccines, adoptive cell transfer, TIL therapy, CAR-T therapy, and ICB therapy. Each approach seeks to harness the body’s immune system to more effectively target cancer, either by stimulating immune responses against neoantigens or by genetically engineering immune cells to recognize and eliminate cancer cells (Table 5). For example, vaccines utilizing DCs, synthetic long peptides (SLPs), and RNA aim to prime the immune system to identify and attack tumors expressing neoantigens [305, 306]. ACT and TCR-engineered adoptive cell transfer (TCR-ACT) approaches involve modifying and expanding T cells to enhance their ability to recognize and kill cancer cells [150]. CAR-T therapy involves engineering T cells to express chimeric receptors that specifically target cancer cells [307], while ICB therapy aims to lift immune system inhibitory signals, enabling immune cells to attack tumor cells more effectively [308].

These clinical trials encompass a wide range of cancer types, including melanoma, GBM, NSCLC, and colorectal cancer, among others (Table 5). The studies also explore various combinations of these therapies with other treatments, such as PD-1 inhibitors, chemotherapy, radiotherapy, and ICB, to further enhance therapeutic efficacy. Clinical outcomes, such as overall response rate (ORR), disease-free survival (DFS), and PFS, are being used to evaluate the success of these therapies.

Discussion

The increasing focus on neoantigen-based cancer immunotherapies—encompassing vaccines, ACTs, and immune checkpoint inhibitors—highlights their transformative potential in revolutionizing cancer treatment [10, 103]. Neoantigens, by being tumor-specific, allow for precise targeting of malignancies while sparing healthy tissues, offering a more personalized alternative to traditional therapies [133, 145, 301]. However, while promising, several obstacles need to be addressed to maximize the efficacy and applicability of these treatments.

One primary challenge lies in the identification of immunogenic neoantigens and their corresponding TCRs [30, 309]. Current methods, such as NGS and immunopeptidomics, have made strides in identifying neoantigens [9]. Despite these advancements, the accuracy of epitope prediction remains suboptimal, necessitating the refinement of computational workflows [36, 41]. The experimental validation of these antigens is resource-intensive and time-consuming, further complicating progress. Emerging technologies, such as T-Scan, which assess T cell-mediated killing, hold promise for scaling these processes, but a more efficient approach to TCR mapping, high-throughput platforms, and enhanced algorithms is vital for advancing personalized therapies [310, 311].

The financial and time-related constraints associated with personalized neoantigen therapies represent another significant hurdle. While these therapies promise tailored treatments, the high costs and lengthy timelines associated with producing custom vaccines and identifying TCRs hinder their widespread implementation. A promising alternative lies in the development of off-the-shelf therapies targeting public neoantigens, which arise from common mutations across different tumor types [84, 312]. Although such therapies could alleviate cost and time challenges, targeting shared neoantigens across diverse patient populations and creating effective vaccines for these antigens remains a major scientific hurdle [131, 147]. Future research should focus on the development of public neoantigen-based vaccines, bispecific antibodies, and TCR-based therapies to overcome these limitations.

Combination therapies, particularly pairing neoantigen-based vaccines with ICIs, are emerging as a potential solution to improve treatment efficacy. These combination strategies, which may also include chemotherapy or radiation to boost neoantigen presentation, have shown promise in overcoming immune evasion in solid tumors, especially epithelial cancers [12, 131, 145, 313]. However, combining therapies introduces its own set of challenges, particularly with respect to balancing immune activation and managing immune-related adverse effects [122, 314, 315]. Further exploration is needed into immune-modulating agents such as interferons and TLR agonists to determine optimal dosing schedules and improve their therapeutic impact.

Despite the challenges, the future of neoantigen-based immunotherapies is promising. Future research must focus on optimizing the screening and production processes to enhance scalability and reduce costs. Advances in bioinformatics, machine learning, and AI could significantly improve the accuracy of neoantigen prediction and streamline the process of identifying optimal therapeutic targets. Moreover, novel delivery systems, such as nanoparticles and tumor-derived extracellular vesicles (EVs), hold great potential in augmenting immune responses and improving overall therapeutic outcomes [316, 317]. As clinical trials continue, the integration of neoantigen-based therapies into personalized treatment regimens for specific patient populations, such as the elderly, could further improve treatment efficacy, considering distinct disease progression patterns in these individuals.

In conclusion, neoantigen-based immunotherapies hold significant promise as a frontier in cancer treatment, yet several challenges must be addressed to fully realize their potential. Key obstacles include the efficient identification of neoantigens, cost reduction, and the scalability of therapies. Overcoming these hurdles will require continued advancements in bioinformatics, high-throughput technologies, and combination therapeutic strategies. While the future of neoantigen-based cancer immunotherapy appears promising, achieving its full impact will require overcoming technical, logistical, and financial barriers.

To unlock the potential of personalized therapies, substantial investments in infrastructure, computational tools, and collaborative efforts across disciplines are essential. Collaboration between industry partners, academic institutions, and regulatory bodies will play a pivotal role in translating research findings into clinical applications. The challenge of ensuring cost-effectiveness remains critical to providing equitable access to these therapies for all patients.

Looking forward, enhancing the precision of neoantigen identification through improved bioinformatics models and high-throughput technologies represents a promising direction. Additionally, exploring the use of combination therapies—integrating immune checkpoint inhibitors with other immune modulators—could offer a more comprehensive approach to overcoming tumor resistance mechanisms. Expanding the focus to investigate common neoantigens across various cancers could also lead to the development of more broadly applicable therapies. However, challenges related to computational prediction, resource constraints, and the complexity of immune responses must be addressed through continuous innovation and rigorous clinical testing. Ultimately, while substantial work remains, the future of neoantigen-based immunotherapy is bright, with the potential to revolutionize cancer treatment, particularly in the realm of personalized medicine.