MHC-Associated Peptide Proteomics: Immunogenicity and Vaccine Design

Colorful 3D glass molecules and atoms in blue background. | Image Credit: ©Drawbot - stock.adobe.com

Immunogenicity assessment is essential in any therapeutic protein development. But there’s a key step in this process that’s both powerful and versatile: major histocompatibility complex (MHC)-associated peptide proteomics (MAPPs), a complex yet informative assay that gives detailed insights into the immunogenic potential of a biologic. The real power of MAPPS, however, lies in its versatility in being applicable to other areas of drug development like vaccine design. This article looks at how the MAPPs assay works, how it integrates with other immunogenicity assays, and just how it can be flipped to help in vaccine design.

How the MAPPs assay works

At its core, MAPPs is a method to identify peptides that bind major histocompatibility complex (MHC) molecules on antigen-presenting cells (APCs). The MAPPs assay can be broken into four main steps:

• PBMC isolation: First, the peripheral blood mononuclear cells (PBMCs) are isolated from healthy donors. Then monocytes are isolated from the PBMCs by immunomagnetic cell separation to give a highly enriched monocyte population.. The monocytes are then incubated with differentiation medium to produce monocyte-derived dendritic cells (moDCs).
• Therapeutic protein exposure: The semi-mature moDCs are then exposed to the full-length therapeutic protein. This exposure allows the immune system to process the protein naturally. The moDCs internalize the protein, degrade it into peptide fragments, and present these fragments on MHC Class II molecules.
• Purification of MHC-peptide complexes: The mature moDCs are harvested followed by the purification of MHC-peptide complexes via immunoprecipitation.
• Peptide elution and analysis: Finally, any peptides bound to MHC molecules are eluted under acidic conditions. These peptides are then analyzed using high-resolution mass spectrometry. It’s at this stage that the exact sequences of the peptides presented by the MHC molecules are obtained. By identifying these peptides, one has direct mapping of any epitopes likely to elicit an in vivo immune response.

Integration within the immunogenicity risk assessment toolbox

MAPPs works alongside other immunogenicity testing methods by offering a deeper look at peptide presentation and T-cell epitope identification (1).

MAPPs and T-cell assays. MAPPs identifies peptides that APCs present, meaning you can then use traditional T-cell assays to measure the T-cell responses to these peptides. MAPPs gives a map of naturally presented peptides, which affords us a good deal of confidence that the peptides tested in our T-cell assays are relevant in vivo.

MAPPs versus HLA-peptide binding assays. Human leukocyte antigen (HLA)-peptide binding assays predict potential T-cell epitopes based on binding affinity to HLA molecules but can miss naturally processed peptides. MAPPs identifies these naturally processed peptides and provides more accurate in vivo representation. This confirms which peptides are naturally presented and potentially immunogenic, complementing HLA binding assays.

MAPPs and in silico algorithms. In silico algorithms can predict peptide binding to HLA molecules based on known binding motifs. MAPPs can then move to validate these predictions by showing which peptides APCs present. However, many in silico algorithms are trained on datasets that only represent a small fraction of the naturally occurring peptidome,(1) leading them to overestimate risk as they include peptides that may never be processed by APCs. MAPPs circumvents this limitation by focusing on peptides actually processed and presented by human cells, which gives us a more accurate immunogenicity profile. Thankfully, MAPPs data has the potential to refine these algorithms to improve the predictive power.

Rather than being a standalone assay, MAPPs integrates with and enhances traditional immunogenicity assays, providing a comprehensive view of a biotherapeutic's immunogenicity potential. This leads to better-informed drug development decisions and effective strategies to reduce unwanted immune responses.

Identifying immunogenic risks and engineering out liabilities

Using MAPPs, the specific region(s) of a biologic that may induce anti-drug antibodies (ADAs) can be identified. These ADAs can dimmish a drug's desired effect and induce adverse immune reactions. By mapping these regions, the protein can be re-engineered to minimize these immunogenic ‘hot spots’ and increase the drug's safety and efficacy.

A good example is the re-engineering of Vatreptacog Alfa, a genetically engineered variant of recombinant factor VIIa (rFVIIa), which initially showed high immunogenicity. In a paper co-authored with FDA, the MAPPs assay's ability to predict the immunogenicity of therapeutic proteins using a recombinant FVIIa variant (VA) was evaluated (2). The results indicated that VA, which elicited stronger T cell responses than the wild-type FVIIa, had a high affinity for MHC-II alleles in 100% of patients with anti-drug antibodies. In contrast, only 44% of patients without these antibodies showed similar affinity. Two de-immunized VA variants, DI-1 and DI-2—designed to reduce MHC-II binding affinity—showed significantly lower T-cell responses. The MAPPs assay identified 10 clusters of peptides from FVIIa molecules, with mutations E296V and M298Q in VA linked to higher immunogenicity. DI-1 and DI-2 showed fewer peptides from these clusters, consistent with reduced immunogenicity.

This work demonstrated that the MAPPs assay, combined with in silico assessments and T-cell proliferation assays, was able to effectively assess the immunogenicity risk of this therapeutic proteins. MAPPs identified therapeutic protein-derived peptides presented by MHC-II, which was relevant for de-immunization, and cluster frequency analysis further supported that DI-1 and DI-2 variants had reduced immunogenicity compared to VA. Although limited by a small donor cohort, it’s clear that the MAPPs assay is a valuable tool for predicting clinical outcomes and guiding the design of safer therapeutic proteins.

Flipping MAPPs for vaccine design

In a recent study, researchers used bioinformatics and mass spectrometry to determine the SARS-CoV-2 peptide sequences in HLA-I and HLA-II peptidomes recognized by circulating CD8+ and CD4+ T-cells from COVID-19 patients. The study identified peptides derived from canonical and out-of-frame ORFs in SARS-CoV-2 nucleocapsid and spike proteins, which were not targeted by existing vaccines yet still stimulated effective T-cell responses in vivo (3).

In earlier work, a different research group used MAPPs in experiments where there intentionally pulse human dendritic cells (DCs) with the virus's spike protein (4). They isolated CD14+ monocytes from healthy human donors prior to the COVID-19 pandemic, and then differentiated these into immature DCs before treating them with the viral spike glycoprotein. Next, they isolated HLA-II molecules from the mature and lysed cells via immunoprecipitation, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) detected the HLA-II antigen-derived peptides. Their mass spectrometry analysis identified 876 peptide sequences from the SARS-CoV-2 spike protein—526 of which were unique. The dendritic cells presented peptides spanning the entire viral spike protein. Identified HLA-II peptides from 11 regions were presented by most analyzed donors.

The study highlighted a significant finding: the correlation between presented and predicted peptides was low. When comparing the experimental results to predictive algorithms, only two of the 11 observed HLA-II peptides of the SARS-CoV-2 spike protein matched predictions made by the predicative algorithm for dominant HLA-II peptides (5). This discrepancy underscores the importance of empirical validation provided by MAPPs over relying solely on computational predictions.

Abzena has conducted its own bioinformatic analysis of the SARS-CoV-2 proteome. The company first exposed recombinant viral proteins to APCs to allow for the natural processing and presentation of peptides by MHC molecules. Peptides bound to MHC complexes were then isolated and analyzed using high-resolution mass spectrometry to identify the exact peptide sequences presented on MHC molecules. Rather than just relying on one or the other, MAPPs was integrated with bioinformatics to generate a robust dataset—with MAPPs bring a critical layer of validation for the bioinformatic predictions by confirming the biological presence of these epitopes. This dataset was instrumental in identifying optimal vaccine targets.

Finally, peptides were synthesized from the identified ‘hot spots’ and tested for their ability to induce an immune response using blood samples from both naïve and COVID-19 convalescent individuals. The results were promising, so those peptides that produced a strong immune response were synthesized and validated for their potential as effective components of a COVID-19 vaccine.

The broad benefits of MAPPs

The role of MAPPs in these examples is multifaceted. It provided detailed insights into the viral proteome, clearly highlighting those regions processed and presented by human immune cells. This information guided the design of vaccine candidates so ensure they targeted only the most relevant epitopes. MAPPs also identified regions likely to induce strong and specific T-cell responses—crucial for developing a vaccine—such as those capable of providing broad-spectrum immunity against SARS-CoV-2.

The success of MAPPs in COVID-19 vaccine development should form a foundation for future efforts. Similar strategies can also be used to develop vaccines for other emerging infectious diseases. The key is the ability to accurately identify and validate T-cell epitopes—something MAPPs, alongside T cell proliferation assays, provides.

But this goes beyond infectious diseases: these can be applied to other areas like immunotherapy. For instance, cancer vaccines, which have to precisely target tumor antigens, would benefit from the detailed mapping provided by MAPPs. Being able to identify tumor-specific epitopes processed and presented by the immune system allows us to design vaccines that improve the body's ability to recognize and attack cancer cells.

Consider the broader implications here. The COVID-19 pandemic highlighted the need for rapid vaccine development, yet all too often we rely on time-consuming traditional methods that rely on trial and error. MAPPs, on the other hand, offers a more targeted approach. By focusing on naturally processed and presented peptides, the vaccine design process can be streamlined. This would not only accelerate development but also improve the efficacy and safety of the vaccines.

Future perspectives

Looking ahead, integrating MAPPs with other immunogenicity tools has the potential for even greater precision in drug development. Combining MAPPs data with in silico models and T-cell assays would help to create a comprehensive immunogenicity profile for candidate therapeutics. Furthermore, expanding the use of MAPPs to vaccine development, especially for emerging diseases, could speed up the creation of robust, broad-spectrum vaccines.

As technology advances, the precision and efficiency of MAPPs are likely to improve while it’s relative costs will decrease. Its role in de-risking biologics development, coupled with its capacity to enhance vaccine design, puts MAPPs at the forefront of immunological research and therapeutic innovation. Moreover, the continued evolution of MAPPs will likely see enhancements in its analytical capabilities. High-resolution mass spectrometry and bioinformatics tools will further refine the identification of epitopes, which will bring more precise engineering of therapeutic proteins and vaccines. Better data integration methods will also lead to a better correlation between what is seen in vitro versus in vivo outcomes, and hugely boosting the predictive power of immunogenicity assessments.

MAPPs not only enriches the immunogenicity assessment options but also drives innovation in biologics and vaccine design. With its proven success in COVID-19 vaccine development, MAPPs exemplifies how scientific ingenuity can meet urgent public health needs. As we continue to refine and improve this powerful tool, the scientific community will have the chance to address future challenges more swiftly and effectively. Integrating MAPPs with emerging technologies and methodologies will undoubtedly further its impact, solidifying its place as a cornerstone of modern immunology.

References

1. Karle, A. C.. Applying MAPPs Assays to Assess Drug Immunogenicity. Front. Immunol. 2020 11. DOI: https://doi.org/10.3389/fimmu.2020.00698

2. Jankowski, W.; Kidchob, C.; Bunce, C.; Cloake, E.; Resende, R.; Sauna, Z. E. The MHC Associated Peptide Proteomics Assay is a Useful Tool for the Non-clinical Assessment of Immunogenicity. Front. Immunol. 2023 14, 1271120. DOI: DOI: 10.3389/fimmu.2023.1271120

3. Abd El-Baky, N.; Amara, A. A.; Redwan, E. M. HLA-I and HLA-II Peptidomes of SARS-CoV-2: A Review. Vaccines (Basel) 2023 11, 548. DOI: https://doi.org/10.3390/vaccines11030548.

4. MKnierman, . D.; Lannan, M. B.; Spindler, L. J.; McMillian, C. L.; Konrad, R. J.; Siegel, R. W. The Human Leukocyte Antigen Class II Immunopeptidome of the SARS-CoV-2 Spike Glycoprotein. Cell Reports 2020 33. DOI: https://doi.org/10.1016/j.celrep.2020.108454

5. Grifoni, A.; Sidney, J.; Zhang, Y.; Scheuermann, R. H.; Peters, B.; Sette, A. A Sequence Homology and Bioinformatic Approach Can Predict Candidate Targets for Immune Responses to SARS-CoV-2. Cell Host Microbe 2020 27, 671-680.e2. DOI: https://doi.org/10.1016/j.chom.2020.03.002.

About the authors

Edward Cloake is director of Immunology at Abzena with a successful track record of delivering immunogenicity risk assessment assays for a broad range of protein biotherapeutics. Edward has over 16 years experience in the pre-clinical immunogenicity assessment of biologics. He leads a team that utilizes a range of assays to evaluate the presence of potential T cell epitopes, and subsequent mitigation strategies to de-risk candidates.

Campbell Bunce is CSO and Cambridge Site Head and leads a talented team of scientists across a diverse range of expertise and capabilities to support drug discovery, design, and developability; and cell line development. He ensures that Abzena’s strong innovation focus and depth of scientific expertise is maintained through technological developments and works in partnership with clients to design and deliver solutions that support their program needs. Campbell has over 25 years of experience working in the biotech and diagnostics sectors. Before joining Abzena in 2015, he held multiple positions of increasing responsibility in Biotech including Head of Cellular Immunology at Cantab Pharmaceuticals, Director of Programs at Piramed Pharma, and R&D Director at Immune Targeting systems. Throughout his career he has applied innovative solutions for the design, manufacture and clinical evaluation of novel products including vaccines, biologics, and small molecules in multiple therapeutic areas. These include inflammation, cancer, infectious disease, and addiction. Campbell has a Ph.D. in Immunology from the University of Manchester, UK, an Executive MBA from Judge Business School, Cambridge University, UK and has published a number of papers on cell-mediated immunity, immunotherapy and vaccines.