What are neoantigens?

Cancer is driven by genetic changes which alter the survival and proliferation of cells. The mutations that are introduced into the DNA sequences of cancer cells have the potential to generate altered or entirely new protein products that can be recognized by the immune system as ‘non-self’. These altered or new proteins are antigenically novel for the host and are, therefore, referred to as neoantigens. Typically, one distinguishes between tumor associated antigens (TAAs), which are also present on other tissue and tumor-specific antigens (TSAs), which are exclusively presented on tumor cells Katsikis, et al (2023)

Neoantigens can arise through a variety of mutational events, with the numbers and types of mutation varying by cancer type. These events include point mutations, insertions or deletions (indels) and gene fusions. Tumors with a high mutational burden are likely to have more neoantigens vaccine candidates than tumors with low mutational burden Blass, et al (2021)

Genuine TSAs/neoantigens develop through non-synonymous mutations that result in aberrant proteins whose degradation results in HLA-binding neopeptides. Being the most abundant and simple form of mutations, non-synonymous point mutations are currently the best studied mutation-derived neoantigen precursors. Single amino acid changes may either alter the immunogenicity of an HLA-binding peptide or, if they occur in anchor positions, turn a non-binding sequence into an HLA-binding one. Alternatively, a mutated amino acid could give rise to a new proteasomal cleavage site, thus allowing peptide processing and HLA loading. However, the majority of discovered neoantigens exhibit point mutations at the TCR-exposed region of the neopeptide Peri, et al(2023)

In contrast, recurrent or public neoantigens derived from both point mutations and larger genetic aberrations, although scarce, have also been identified. Therapies against recurrent mutations can benefit many patients with the same tumor type, but also patients with different cancer types harboring the same recurrent mutation. Among the known neoantigens derived from recurrent mutations are CDK4.R24C47–50, KRAS.G12V/C/D51–53, EGFR54–59 and PIK3CA.H1047L60 Peri, et al(2023)

Neoantigens are generally not germ line encoded and therefore the host theoretically harbors no central tolerance towards these antigens. Importantly, owing to negative selection of T cells against self-antigens in the thymus, neoantigen-specific T cell receptors (TCRs) tend to be of lower affinity. This does not, however, alleviate the concern for cross-reactivity toward the wild-type variant, given their similarity, when targeting neoantigens. Both aspects must be considered when choosing antigens for vaccine development (Katsikis, et al (2023); Saxena, et al (2021)

For neoantigens to mount a TH1 and or CTL response against the tumor, it needs to display suitable characteristics Saxena, et al (2021):

High foreignness	The higher the similarity to wild-type peptide, the higher the risk of the respective T-cell being deleted in the Thymus
Clonal distribution	If the mutation is only found in subclones of the tumor, the T-cell response is restricted to those
Targeting Driver mutations	Passenger mutations are subject to loss of expression through tumor evolution or immune resistance. Driver mutations are more conserved as these serve critical survival functions
MHC presentation	Neoantigen presentation on MHC class I and/or MHC class II molecules increases probabilities of cross-presentation
TCR avidity	High TCR avidity of neoantigens induces a strong CTL-driven response

 

Taken together, vaccines based on neoantigens (TSA) have several advantages over traditionally used TAAs. First, neoantigens are exclusively expressed by tumor cells and can elicit truly tumor- specific T cell responses, preventing ‘off- target’ damage to nonmalignant tissues. Second, neoantigens are de novo epitopes derived from somatic mutations, which presents the possibility to circumvent T cell central tolerance of self- epitopes and thus induce immune responses to tumors. As such, personalized neoantigen- based vaccines therefore afford the opportunity to boost tumor- specific immune responses. Finally, the vaccine- boosted neoantigen- specific T cell responses may persist and provide post- treatment immunological memory, presenting the possibility of long- term protection against disease recurrence Blass, et al (2021)

How are neoantigens identified?

While the sequence and particular methods used do vary, the procedure of neoantigen identification generally is as follows:

1. Peripheral blood, tumor biopsies and control tissues are taken from patients.
2. Using the obtained tissues and cells, cancer-specific mutations are mapped using Whole Exome Sequencing (WES).
3. Expression of these mutations in cancer cells is determined by RNA sequencing (RNA transcriptome)
4. HLA typing
5. In silico prediction of antigens
6. Vaccine production

Some groups choose to validate the in-silico predictions using immune-peptidomics. To identify neoantigen-derived peptides presented on the surface of cancer cells by MHC class I molecules, mass spectrometry-based detection of antigenic peptides is performed after MHC immunoprecipitation and peptide purification from cancer and control material (Nelde, et al (2021) ; Chong, et al (2022)).

For more information also refer to Arnaud, et al (2022) , describing their NeoDisc pipeline for prioritization of neoantigens in PDAC for the design of optimally long peptides for vaccination and Mueller (2023) for the incorporation of machine learning into neoantigen identification.

What delivery platforms for neoantigen cancer vaccines have been established?

As there are multiple ways to identify neoantigens and choose suitable antigens for vaccination, there are several platforms to activate the immune system to target tumor cells.

DNA vaccines

DNA vaccines are easy to manufacture, carry built-in adjuvants, represent a concentrated form of tumor-associated antigens but require transcription and translation before cross-presentation by dendritic cells (DCs). DNA vaccines are most effective in driving sufficient antigen processing and presentation for induction of CD4+ T cell and CD8+ T cell responses when administered at relatively high doses via intramuscular injection in combination with electroporation Stephens, et al (2021)

mRNA vaccines

Like DNA vaccines, RNA vaccines are relatively straightforward to manufacture and have built-in adjuvants. However, unlike DNA vaccines, RNA vaccines do not require transcription and are thus closer to protein antigen expression and processing and presentation on MHC molecules. Boosted by the research on Covid19, the formulation of mRNA vaccines with LNPs has led to personalized mRNA vaccines being currently assessed for efficacy in clinical trials. At this stage mRNA vaccines still pose the challenge of low temperature logistics Stephens, et al (2021)

Dendritic cell vaccines

DCs that have been isolated or cultured from blood, adjuvant activated and loaded with antigens have been tested in several trials. They can be pulsed with TAA/TSA peptides, transduced with mRNA or leni viruses, fused with tumor cells or incubated with whole tumor lysate. After Transduction they are injected intracutaneously or subcutaneously or even intravenously. The dendric cell vaccine DCVax-L is close to FDA approval for application in glioblastoma Stephens, et al (2021)

Peptide vaccines

During the recent decades, peptides have become the mainstay of vaccine development and are being used to address infectious and even chronic diseases. Synthetic peptides offer a plethora of advantages over other vaccine platforms (Purcell, et al (2007); Malonis, et al (2019)):

• Highly modifiable – from amino acid sequence to sidechain modifications. Lipid, carbohydrate, and phosphate groups can be readily introduced in a controlled manner to improve immunogenicity, stability and solubility.
• Peptide-based vaccines can be designed to include multiple determinants from several pathogens, or multiple epitopes from the same pathogen.
• Peptides are easily characterized and analyzed for purity using well-established analytical techniques such as liquid chromatography and mass spectrometry. This facilitates quality control and ultimately approval by the regulatory authorities.
• The production of chemically defined peptides can be carried out economically on a large scale.
• Peptide preparations can be stored freeze-dried, which avoids the need to maintain a 'cold-chain' facility in storage, transport and distribution.
• There is no risk of genetic integration or recombination, which can be a problem with DNA or mRNA vaccines.

Currently a multitude of clinical trials investigates the safety and efficacy of different neoantigen peptide cancer vaccine approaches.

How can neoantigen peptide cancer vaccines be translated into the clinic?

Three different study and treatment strategies have been formulated for the clinical application of neoantigen peptide cancer vaccines: stratification, warehouse-based personalization, and full individualization Nelde, et al (2021)

Stratification: This strategy selects suitable patients for a specific therapy based on predefined tumor-associated criteria. Only patients with the respective tumor feature receive the therapy. Stratification is a standard practice in clinical routine, such as biomarker-based targeted therapies for specific mutations or antibody-based immunotherapies. It is relatively straightforward and cost-effective but excludes a substantial proportion of patients Nelde, et al (2021)

Warehouse-based personalization: This strategy uses a predefined set of high-frequency tumor-associated peptides for specified HLA allotypes. It allows the personalization of vaccination within these parameters for each patient. For example, a hypothetical warehouse peptide vaccination trial might cover eight different HLA allotypes and five peptides each, resulting in only 40 different peptides to be produced. This concept enables the individualization of vaccine cocktails in a time- and cost-saving manner Nelde, et al (2021)

Personalization: This strategy involves the selection and on-demand production of single patient-specific drug products. It aims to design patient-specific vaccines tailored to the patient's HLA allotypes, rare mutations, and tumor-presented peptides. Personalized peptide vaccines can include patient-tailored neoepitopes based on individual sequencing data and/or non-mutated tumor-associated peptides. This approach maximizes the drug benefit for each cancer patient Nelde, et al (2021)

How are neoantigen peptide cancer vaccines formulated?

Neoantigen peptide cancer vaccines require CD8+ epitopes for antigen cross-presentation and CTL activation, as well as CD4+ epitopes for T-helper cell activation. The length of the peptides is crucial for a robust immune response. Peptides that are too short may bind to non-professional antigen-presenting cells, resulting in incomplete T cell activation and a weak immune response or immune tolerance. Short peptides are also limited by HLA-type restriction and susceptibility to enzymatic digestion and rapid elimination. In contrast, synthetic long peptides (SLPs) offer broader HLA-type coverage, allow for multi-epitope inclusion to enhance CD4+ and CD8+ response, and enable the incorporation of binding motifs to enhance immunogenicity with promising pre-clinical efficacy Stephens, et al (2021)

Recombinant overlapping peptides (ROPs) are another design strategy for peptide vaccines that have shown promising pre-clinical efficacy. They consist of sequential overlapping long peptide sequences covering the entire sequence of a target, with Cathepsin S protease-sensitive linkers between the peptide sequence overlaps. The overlapping region allows for diversity in epitope, especially with MHC-II molecules, which have shown different but overlapping recognition of CD4+ epitopes between HLA haplotypes Stephens, et al (2021)

Although pre-clinical studies have shown promising immunogenicity with peptide-based cancer vaccines, achieving strong clinical efficacy remains challenging. Current barriers include inappropriate adjuvants, tumor heterogeneity, tumor antigen loss, decreased MHC expression, limited infiltration of T cells in tumor tissue, and T cell dysfunction-mediated immune suppression [Stephens]. The focus of peptide vaccination is to induce a type 1–polarized, cell-mediated immunity rather than a type 2–polarized and humoral response. Neopeptides derived from tumor mutations are often similar to self-peptides and have lower TCR affinity, making them generally lacking sufficient immunogenicity in the absence of adjuvants. To maximize effective T cell responses, the choice of optimal, strong adjuvants or immunostimulators is of paramount importance Nelde, et al (2021)

While plasmid DNA and mRNA vaccines can act as their own adjuvants, neoantigen peptide cancer vaccines require exogenous adjuvants to stimulate immunity. Classical adjuvants like alum, MF59, AS01, AS04, AS03, and CpG 1018 were primarily designed to induce a humoral response and have a lower ability to induce CD8+ T cell responses. Intense efforts are being made to generate suitable adjuvants for peptide vaccinations. Promising candidates include CpG ODNs (humoral and cellular response), STING agonists, the combination of K3 CpG-ODNs and STING, or Montanide with poly-ICLC [katsekis]. Recently, the novel water-soluble adjuvant XS15, a synthetic TLR1/2-binding Pam3-Cys-derivate covalently linked to a single synthetic—nonvaccine—peptide (GDPKHPKSF), was described as an effective vaccine adjuvant inducing unprecedented strong and long-lasting CD8+ and CD4+ T cell responses in first-in-man proof-of-concept experiments (Katsikis, et al (2023), Nelde, et al (2021))

Finally, non-cellular nanoparticles, which are smaller than DCs (size range 20–100 nm), are being explored as carriers to improve non-cellular protein or peptide-based neoantigen cancer vaccine platforms that may not efficiently travel to or target lymph node DCs. Such approaches include synthetic particle delivery by lipoplexes, amphiphile vaccines, liposomes, and the novel self-assembling nanoparticles comprising TLR7/8-SLP neoantigen conjugate (SNP-7/8a) Saxena, et al (2021)

How Intavis Peptide Services supports your personalized neoantigen peptide cancer vaccine trial

When it comes to the manufacturing of neoantigen personalized peptide cancer vaccines, we understand that expertise, reliability, and speed are crucial. Consequently, we offer exactly what investigators look for in a peptide manufacturer:

1. 1. One-stop-shop – GMP peptide drug substance and fill and finish
   2. High Quality - Peptide expertise, experience and the proper certifications
   3. Dedicated Project Team – a safe pair of hands to manage your project as top priority
   4. Traceability – transparent and reproducible
   5. IND/IMPD Support – we know what regulators want
   6. Fast Turn-around – two months from sequence to drug product or you get your money back

Our neoantigen GMP peptide manufacturing site is situated within the peptide immunology hub of Tuebingen, Germany. We are specialized in small scale parallel synthesis, with 30 years’ experience in the peptide world, and can therefore provide the GMP grade peptide drug substance for your personalized neoantigen peptide cancer vaccine trial, quickly with high quality. As operational excellence is in our DNA, a dedicated project team will be at your service from project inception to end. We have experts on peptide chemistry, immunology, oncology and QC/QA at your disposal.

Furthermore, we offer to create the peptide pools following your specifications and, of course, all the analytics necessary to meet regulatory demands. In addition, you can have your peptide pools filled, labelled, packaged, and shipped to the clinical site of your desire. Intavis Peptide Services, a true one-stop-shop for neoantigen personalized peptide cancer vaccines.

Analytical offering

Individual peptides

• Identity
• Purity
• Peptide Content
• Amount

Peptide pools

• Identity
• Amino Acid Sequence
• Assay
• Total Product Related Impurities
• Water Content
• Residual Solvents
• Trifluoroacetic Acid Content
• Bacterial Endotoxins
• Sterility
• Container Closure Integrity

But our offering does not end here. During your trial, you are probably monitoring immune responses using ELISpot or other techniques which require high purity peptide libraries. We can also provide those for competitive prices.

We understand that GMP grade peptides are just the basic needs for your trial. Time-to-needle is crucial as your patients often are terminally ill. In cooperation with HB technology, a leading developer of healthcare software and automation, we have created automated peptide printers which allow us to beat turn around times of other neoantigen personalized peptide cancer vaccine manufacturers. From order to fill and finish, we can do it in two months.

 

Bibliography

Arnaud, et al (2022)  "Sensitive identification of neoantigens and cognate TCRs in human solid tumors." Nature Biotechnology 40.5: 656-660.

Blass, et al (2021)"Advances in the development of personalized neoantigen-based therapeutic cancer vaccines." Nature Reviews Clinical Oncology 18.4: 215-229.

Chong, et al (2022) "Identification of tumor antigens with immunopeptidomics." Nature biotechnology 40.2: 175-188.

Katsikis, et al (2023)"Challenges in developing personalized neoantigen cancer vaccines." Nature Reviews Immunology: 1-15.

Malonis, et al (2019) "Peptide-based vaccines: current progress and future challenges." Chemical reviews 120.6 (2019): 3210-3229.

Müller, et al (2023)"Machine learning methods and harmonized datasets improve immunogenic neoantigen prediction." Immunity 56.11 (2023): 2650-2663.

Nelde, et al (2021) "The peptide vaccine of the future." Molecular & Cellular Proteomics 20 (2021).

Peri, et al(2023)"The landscape of T cell antigens for cancer immunotherapy." Nature cancer 4.7 (2023): 937-954.

Purcell, et al (2007)"More than one reason to rethink the use of peptides in vaccine design." Nature reviews Drug discovery 6.5: 404-414.

Saxena, et al (2021)"Therapeutic cancer vaccines." Nature Reviews Cancer 21.6: 360-378.

Stephens, et al (2021) "Beyond just peptide antigens: the complex world of peptide-based cancer vaccines." Frontiers in Immunology 12: 696791.

Zitvogel & Kroemer (2018)" Oncoimmunology: A Practical Guide for Cancer Immunotherapy: 1-2.