Therapeutic Discovery

Neoantigen Immunopeptidome Profiling and Production for Personalized Immuno-oncology Therapeutic Discovery

This Nature Research Webinar, hosted by GenScript and Cayman Chemical, focuses on neoantigen identification and production services. In partnership with Cayman Chemical, GenScript is leveraging its expertise in peptide synthesis to support immunopeptidome profiling and neoantigen discovery.

Neoantigens are “new” protein sequences that arise in the process of tumorigenesis and are otherwise absent from normal tissues. Because tumor-specific neoantigens are truly foreign, they are recognized by the adaptive immune system and are highly immunogenic. Therefore, neoantigen peptides have become highly sought-after tools for personalized immunotherapy. Two main modalities of neoantigen based immunotherapy include personalized CAR T-cell therapy and cancer vaccines.

Neoantigen identification and neoantigen peptide synthesis are two main challenges for the development of personalized immunotherapies. To identify targetable neoantigens, a series of approaches may be followed including whole genome sequencing, whole exome sequencing, RNAseq, in silico prediction, and immunopeptidome profiling. Once neoantigen candidates are identified, validation may be implemented through the use of neoantigen synthetic peptides in T cell activation assays (e.g., ELISPOT, ELISA, or Flow cytometry).

Immunopeptidome Profiling Service at Cayman Chemical (Dr. Julie Rumble, Manager of Immunology Contract Services, Cayman Chemical)

Immunopeptidome profiling involves the identification through sequencing of major histocompatibility complex (MHC) bound peptides presented on the surface of cancer cells. Because cancer cells are characterized by genomic instability which leads to accumulated mutations, this approach provides the opportunity to identify novel and tumor-specific peptides with the potential to activate T-cell immune responses.

In the immunopeptidome profiling workflow, MHC complexes are enriched through immunoaffinity chromatography from tumor cells or tissues, and eluted peptides are analyzed by LC-MS/MS.

LEARN More About Cayman Chemical’s Immunopeptidome Service

Neoantigen Target Validation

Neoantigen candidates must be validate before their use in downstream applications. Validation of MHC I peptides, which are derived from endogenous proteins, may rely on expression systems where the candidate neoantigen peptide is expressed as part of the sequence of origin or as tandem minigenes. Alternatively, soluble synthetic peptides may be incubated with cells in culture. MHC II peptides, which are derived from phagocytosed proteins, may require strategies to promote phagocytosis such as the use of protein coated latex beads. Finally, another tool that may be leveraged for neoantigen validation is the use of monoallelic MHC cell lines.

Optimizing Immunopeptidome Profiling Workflows

Some key considerations for immunopeptidome profiling workflow optimization include the amount of starting tumor material, the choice of antibody for immunoprecipitation of MHC complexes, and the impact of tumor treatments on MHC expression and peptide number. Additionally, knowledge of the expected experimental output is critical for implementing the most effective data analysis strategy. For example, identification of neoantigens carrying expansive mutations such as indels, splice variations, or translocations would necessitate mutated sequence information, derived from transcriptome or exome sequencing analysis. Ultimately, neoantigen identification requires the use of relevant databases and depends in great part on the goals of each study.

Neoantigen Peptide Synthesis: Producing Difficult Neoantigen Peptides(Raymond Miller, Senior Global Product Manager, GenScript)

Neoantigen peptide synthesis presents a significant challenge for the validation and ultimately implementation of life-saving immunotherapy strategies. A combination of factors complicate neoantigen peptide synthesis including their size, high hydrophobicity, and purity requirements.

GenScript is building technology and infrastructure in order to support its platform for neoantigen peptide synthesis services, with the goal of achieving 95% success rate in the synthesis of difficult peptides. For example, use of the bioinformatics tool NeoPre™ algorithm is at the forefront of the neoatingen peptide synthesis workflow. This proprietary algorithm provides an initial assessment of synthesis difficulty, based on neoantigen sequence, length, hydrophobicity, and aggregation properties. Additionally, based on the initial assessment and calculated score, NeoPre™ instructs synthesis methods that are suited for simple, middle or hard to synthetize peptides. By implementing the NeoPre™ algorithm to instruct synthesis processes for ~2,300 neoantigen peptides, GenScript’s platform has achieved ~95% synthesis success rate.