Precision Neoantigen Discovery Using Large-Scale Immunopeptidomes and Composite Modeling of MHC Peptide Presentation.

Major histocompatibility complex (MHC)-bound peptides that originate from tumor-specific genetic alterations, known as neoantigens, are an important class of anticancer therapeutic targets. Accurately predicting peptide presentation by MHC complexes is a key aspect of discovering therapeutically relevant neoantigens. Technological improvements in mass spectrometry-based immunopeptidomics and advanced modeling techniques have vastly improved MHC presentation prediction over the past 2 decades. However, improvement in the accuracy of prediction algorithms is needed for clinical applications like the development of personalized cancer vaccines, the discovery of biomarkers for response to immunotherapies, and the quantification of autoimmune risk in gene therapies. Toward this end, we generated allele-specific immunopeptidomics data using 25 monoallelic cell lines and created Systematic Human Leukocyte Antigen (HLA) Epitope Ranking Pan Algorithm (SHERPA), a pan-allelic MHC-peptide algorithm for predicting MHC-peptide binding and presentation. In contrast to previously published large-scale monoallelic data, we used an HLA-null K562 parental cell line and a stable transfection of HLA allele to better emulate native presentation. Our dataset includes five previously unprofiled alleles that expand MHC diversity in the training data and extend allelic coverage in underprofiled populations. To improve generalizability, SHERPA systematically integrates 128 monoallelic and 384 multiallelic samples with publicly available immunoproteomics data and binding assay data. Using this dataset, we developed two features that empirically estimate the propensities of genes and specific regions within gene bodies to engender immunopeptides to represent antigen processing. Using a composite model constructed with gradient boosting decision trees, multiallelic deconvolution, and 2.15 million peptides encompassing 167 alleles, we achieved a 1.44-fold improvement of positive predictive value compared with existing tools when evaluated on independent monoallelic datasets and a 1.17-fold improvement when evaluating on tumor samples. With a high degree of accuracy, SHERPA has the potential to enable precision neoantigen discovery for future clinical applications.

Precision Neoantigen Discovery Using Large-scale Immunopeptidomes and Composite Modeling of MHC Peptide Presentation.

Major histocompatibility complex (MHC)-bound peptides that originate from tumor-specific genetic alterations, known as neoantigens, are an important class of anticancer therapeutic targets. Accurately predicting peptide presentation by MHC complexes is a key aspect of discovering therapeutically relevant neoantigens. Technological improvements in mass-spectrometry-based immunopeptidomics and advanced modeling techniques have vastly improved MHC presentation prediction over the past two decades. However, improvement in the sensitivity and specificity of prediction algorithms is needed for clinical applications such as the development of personalized cancer vaccines, the discovery of biomarkers for response to checkpoint blockade, and the quantification of autoimmune risk in gene therapies. Toward this end, we generated allele-specific immunopeptidomics data using 25 monoallelic cell lines and created Systematic HLA Epitope Ranking Pan Algorithm (SHERPA), a pan-allelic MHC-peptide algorithm for predicting MHC-peptide binding and presentation. In contrast to previously published large-scale monoallelic data, we used an HLA-null K562 parental cell line and a stable transfection of HLA alleles to better emulate native presentation. Our dataset includes five previously unprofiled alleles that expand MHC-binding pocket diversity in the training data and extend allelic coverage in under profiled populations. To improve generalizability, SHERPA systematically integrates 128 monoallelic and 384 multiallelic samples with publicly available immunoproteomics data and binding assay data. Using this dataset, we developed two features that empirically estimate the propensities of genes and specific regions within gene bodies to engender immunopeptides to represent antigen processing. Using a composite model constructed with gradient boosting decision trees, multiallelic deconvolution, and 2.15 million peptides encompassing 167 alleles, we achieved a 1.44-fold improvement of positive predictive value compared with existing tools when evaluated on independent monoallelic datasets and a 1.15-fold improvement when evaluating on tumor samples. With a high degree of accuracy, SHERPA has the potential to enable precision neoantigen discovery for future clinical applications.

Versatile genetic assembly system (VEGAS) to assemble pathways for expression in S. cerevisiae.

We have developed a method for assembling genetic pathways for expression in Saccharomyces cerevisiae. Our pathway assembly method, called VEGAS (Versatile genetic assembly system), exploits the native capacity of S. cerevisiae to perform homologous recombination and efficiently join sequences with terminal homology. In the VEGAS workflow, terminal homology between adjacent pathway genes and the assembly vector is encoded by 'VEGAS adapter' (VA) sequences, which are orthogonal in sequence with respect to the yeast genome. Prior to pathway assembly by VEGAS in S. cerevisiae, each gene is assigned an appropriate pair of VAs and assembled using a previously described technique called yeast Golden Gate (yGG). Here we describe the application of yGG specifically to building transcription units for VEGAS assembly as well as the VEGAS methodology. We demonstrate the assembly of four-, five- and six-gene pathways by VEGAS to generate S. cerevisiae cells synthesizing ß-carotene and violacein. Moreover, we demonstrate the capacity of yGG coupled to VEGAS for combinatorial assembly.

qPCRTag Analysis--A High Throughput, Real Time PCR Assay for Sc2.0 Genotyping.

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic chromosome, synIII(1), and one synthetic chromosome arm, synIXR(2), have been constructed and their in vivo function validated in the absence of the corresponding wild type chromosomes. An important design feature of Sc2.0 chromosomes is the introduction of PCRTags, which are short, re-coded sequences within open reading frames (ORFs) that enable differentiation of synthetic chromosomes from their wild type counterparts. PCRTag primers anneal selectively to either synthetic or wild type chromosomes and the presence/absence of each type of DNA can be tested using a simple PCR assay. The standard readout of the PCRTag assay is to assess presence/absence of amplicons by agarose gel electrophoresis. However, with an average PCRTag amplicon density of one per 1.5 kb and a genome size of ~12 Mb, the completed Sc2.0 genome will encode roughly 8,000 PCRTags. To improve throughput, we have developed a real time PCR-based detection assay for PCRTag genotyping that we call qPCRTag analysis. The workflow specifies 500 nl reactions in a 1,536 multiwell plate, allowing us to test up to 768 PCRTags with both synthetic and wild type primer pairs in a single experiment.