What Are Neoantigen Peptides?

The accumulation of somatic mutations, such as single nucleotide variations, frameshift insertions or deletions, and gene fusions, is a central feature in the tumorigenesis process. The resulting mutated genes may give rise to neoantigens or novel protein sequences within the tumor not present in otherwise healthy tissue. Once neoantigens are processed by the cellular machinery of tumor cells or antigen-presenting cells, neoantigen peptides may be uncovered and presented to immune cells.

However, not all mutations result in targets that can be leveraged to develop neoantigen-based therapies.

For these tumor-specific antigens (TSAs) to be useful in cancer vaccine development they must meet three criteria. First, they should give rise to neoepitopes that are presented by the major histocompatibility complex (MHC). Second, neoepitopes must be recognized by immune cells. Lastly, neoepitopes need to trigger an immune response.

Only neoantigens that are recognized and able to induce a strong T-cell response are considered good candidates for cancer vaccine development, as they are expected to activate efficient anti-tumor responses¹.

Neoantigen Peptide Identification

The discovery of neoantigens starts at the tumor site. Advances in sequencing technologies, such as whole exome sequencing and RNA-seq, have made genomic sequencing for cancer treatment a reality by enabling the full identification of tumor genomes and the underlying mutations driving tumorigenesis.

The use of high-throughput sequencing, bioinformatics, and new computational methods has helped predict neoepitope presentation. Additionally, improvements in the prediction of neoepitope binding affinities and T-cell activation potential have made it easier to identify important tumor-specific antigens.

Lastly, validation of predicted neoantigens via immunopeptidomics and in vitro immunological assays is essential in this complex workflow to ensure the selection of the best actionable candidates².

Neoantigen Identification Workflow for Cancer Immunotherapy Development

Neoantigen identification and validation for personalized vaccine and T-cell adoptive cell transfer. “Patient samples, encompassing both tumor and normal cells, are gathered and subjected to whole exome DNA sequencing or RNA sequencing. Subsequently, a variants calling procedure is executed to identify the alterations within protein-encoding regions, followed with neoantigens computational predictions and prioritization. The validation process involves a range of methods such as tetramer assays, Enzyme-linked immunosorbent spot (ELISPOT) assays, and T-cell activation assays. The identified neoantigens can be harnessed for the development of immunotherapies, often taking the form of cancer vaccines or ACT. In the realm of cancer vaccines, neoantigens can be administered in diverse formats, including DNA, mRNA, or peptide-based constructs. In the context of ACT, T cells are extracted from either tumor tissues or peripheral blood. Through coculture with antigen-presenting cells (APCs) primed with neoantigens, neoantigen-specific T cells are identified. These cells then undergo an expansion process facilitated by rapid expansion protocols. Upon achieving a substantial quantity, the expanded T cells are reintroduced into the patient’s system, instigating a concerted effort to suppress the tumor.” Figure 1 and legend retrieved from Zhang et al. 2023.² https://www.mdpi.com/openaccess.

Role of Neoantigens Peptides in Cancer Vaccines

Once identified, neoantigen peptides can be leveraged in vaccine development. Cancer peptide vaccines are usually highly personalized to target specific mutations in the patient’s tumor. Moreover, because tumors commonly accumulate a range of mutations, the composition of vaccines is also heterogeneous and includes a variety of neoantigen peptides. Often neoantigen peptide vaccines may contain up to 20 different peptides³.

How therapeutic cancer vaccines work. “The mechanism of cancer vaccine in vivo. After the tumor antigens migrate into the body in different forms, they are phagocytosed, intracellularly expressed, and efficiently processed by specialized antigen-presenting cells (APCs). The major histocompatibility complex (MHC) of dendritic cells presents antigens to their surface, and the MHC complexes activate antigen-specific T-cells by binding to T-cell receptors (TCR) on the surface of T-cells, therefore safely, persistently, and specifically destroying tumor cells and inhibiting tumor growth.” Figure 1 and legend retrieved from Fan et al. 2023⁴. http://creativecommons.org/licenses/by/4.0/

Mechanistically, peptide vaccines are designed to induce humoral and cell-mediated anti-tumor immune responses. Vaccines may consist of neoantigens in the form of short peptides, which directly bind to MHC-I or MHC-II molecules and induce T-cell responses. In contrast, long peptide immunogens require processing by antigen-presenting cells, which determines how they are presented by MHC molecules to tumor-specific CD4+ or CD8+ T cells⁵.

Significantly, the length of neoantigen peptides plays a critical role in the type of immune response elicited. Long peptide-based vaccines are often more immunogenic by virtue of their expanded capacity to present multiple neoepitopes⁵.

Challenges in Antigen Screening, Identification, and Peptide Production

In the field of cancer immunotherapy, personalized neoantigen peptide vaccines have shown great potential and are expected to bring more precise and effective treatment options to cancer patients. However, investigators and vaccine developers face many serious challenges in moving from research and development to the production of neoantigen peptides.

The Complexity of Neoantigen Screening and Identification

Individual differences challenge: Because each patient's tumor cells have a unique genetic mutation profile, each patient’s tumor needs to be screened and analyzed for neoantigens individually. Whole-exome sequencing and bioinformatics analysis are commonly used methods, but the quality of sequencing data varies from patient to patient, and the analysis process also requires highly specialized skills and experience to accurately identify neoantigens.

Immunogenicity prediction dilemma: Even when a neoantigen is identified, accurately predicting its immunogenicity remains a challenge. Current prediction algorithms, although constantly evolving, still do not provide complete precision for determining which antigens can trigger an effective immune response in the patient. This can lead to the inclusion of poorly immunogenic antigens in vaccine production with low to no effect on tumor growth, wasting production resources and delaying patient treatment.

The High Requirements of the Neoantigen Peptide Production Process

Peptide synthesis technology bottleneck: Personalized neoantigen peptide vaccines often contain a variety of peptides with different sequences, and the synthesis process needs to be highly precise. Current large-scale peptide synthesis technology platforms present several challenges in meeting the requirements of neoantigen peptide production, including low efficiency, high cost, and variable quality when synthesizing complex peptide sequences. For example, the synthesis of long-chain polypeptides often used in personalized neoantigen vaccines is difficult and prone to amino acid mismatch and deletion, which can affect the quality and safety of vaccines.

Quality control challenges: Ensuring the purity, stability, and potency of each personalized peptide is technically demanding. Each batch requires rigorous testing and approval, increasing complexity in comparison to large-scale standardized vaccines. Because neoantigen peptide vaccines are highly individualized, it is difficult to establish unified quality standards and testing methods, which increases the difficulty and cost of quality control.

High Costs and Lack of Optimized Production Facilities

High R&D and production costs: From patient sample collection, gene sequencing, antigen screening, and peptide synthesis, each step in the neoantigen vaccine manufacturing process requires a lot of manpower, material resources, and time. The personalized nature and required customization make it impossible to allocate the cost through large-scale production, resulting in the high price of a single dose of vaccine, which limits its clinical application and promotion.

Lack of optimized production facilities and technology: Most of the existing vaccine production facilities are designed for traditional vaccines, which are not equipped to meet the needs of personalized neoantigen peptide vaccine manufacturing. Large-scale peptide synthesis platforms are unable to address the complexities of producing individualized peptide batches often consisting of 25 to 40 unique peptide sequences at a much smaller scale ranging from 50 to 60 mgs⁶.

Personalized peptide vaccines require small-scale and adaptable GMP manufacturing capabilities, making it difficult to find a matching production supplier in the current market. Moreover, the development of specialized production facilities and technologies requires huge capital investment and technology research and development, and the investment risk is large in the case of immature technology.

The Uncertainty of Regulations and Oversight

Regulatory gaps and lags: As an emerging product, personalized neoantigen peptide vaccines are not yet perfect. The lack of clear guidelines and standards for vaccine approval, clinical trials, and manufacturing practices has brought great uncertainty to enterprises' research, development, and production, increasing their compliance costs and risks.

The approval process is complex and lengthy: Even with regulations, the approval process for neoantigen vaccines can be more complex and lengthier than that for traditional vaccines due to their personalized and innovative nature. This not only prolongs the time to market for vaccines but also increases companies' research and development costs and patients' waiting times for treatment.

Future Outlook for Personalized Neoantigen Peptide Vaccines

The production of personalized neoantigen peptide vaccines faces challenges ranging from antigen screening to regulation. However, with the continuous advancement of technology and the deepening of research, these challenges are expected to be gradually solved, bringing more hope to cancer patients.

Emerging Solutions & Innovations

Several advances in the production of neoantigen peptide vaccines and regulatory frameworks are helping these immunotherapies reach their full potential.

• Modular Manufacturing: Facilities using closed, automated systems.
• AI Acceleration: Platforms like NVIDIA CLARA for rapid neoantigen prediction and vaccine design.
• Shared Neoantigen Libraries: Off-the-shelf vaccines targeting common mutations.
• Regulatory Advocacy: Collaborations to develop adaptive approval frameworks

GenScript Neoantigen Peptide Synthesis Capabilities

Comprehensive Support for Personalized Cancer Vaccine Development

• One-Stop Solution: GenScript streamlines personalized cancer vaccine development from design to clinical trials, offering fast and reliable results.
• Design:Tackles synthesis challenges of neoantigen peptides with the NeoPre algorithm, achieving a 98% success rate and optimizing sequences with library design tools.
• Synthesis: Produces high-quality neoantigen peptides up to 35 amino acids with >95% purity, meeting GMP requirements for clinical success.
• Screening:Utilizes MS/MS, ELISPOT, and in vitro/in vivo studies for effective candidate screening.
• Clinical: Provides process development, regulatory support, and sterile filtration for seamless clinical transitions.

Relevant Resources

Reference

• 1. Lang, F., et al. (2022). Identification of neoantigens for individualized therapeutic cancer vaccines. Nat Rev Drug Discov.
• 2. Zhang, T., et al. (2023). Neoantigens: The Novel Precision Cancer Immunotherapy. Biologics.
• 3. Aziz, N., Poh C.L., (2022). Development of Peptide-Based Vaccines for Cancer. J Oncol.
• 4. Fan, T., et al. (2023). Therapeutic cancer vaccines: advancements, challenges and prospects. Sig Transduct Target Ther.
• 5. Liu, Q., et al. (2024) Old concepts, new tricks: How peptide vaccines are reshaping cancer immunotherapy? Int J Biol Macromol.
• 6. Pennington, M. P., et al. (2021). Commercial manufacturing of current good manufacturing practice peptides spanning the gamut from neoantigen to commercial large-scale products,Medicine in Drug Discovery.