Proteogenomic discovery of neoantigens for personalized T cell immunotherapy in medulloblastoma
Neoantigen discovery in pediatric brain tumors is hampered by their low mutational burden and scant tissue availability. We developed a low-input proteogenomic approach combining tumor DNA/RNA sequencing and mass spectrometry proteomics to identify tumor-restricted peptides arising from multiple genomic aberrations to generate a highly target-specific, autologous, personalized T cell immunotherapy.
Effective therapy for pediatric brain tumors requires intensive chemo- and radiation therapies, leaving survivors with significant long-term burdens including life-altering cognitive deficits. If the tumor recurs after radio-chemotherapy, there are no standard effective therapies and virtually no long-term survivors. Therefore, there is an urgent need to develop new therapeutics that can augment standard therapies to effectively prevent tumor recurrence without increasing toxicity. Immunotherapy is now widely considered to be a promising tool for the treatment of individuals with cancer. Therapies that boost the ability of endogenous T cells to destroy cancer cells have demonstrated therapeutic efficacy in a variety of adult cancers. The infusion of autologous ex vivo expanded lymphocytes is a promising strategy for pediatric brain tumors which can actively home to sites of disease across the blood brain barrier; possess exquisitely sensitive peptide antigen recognition; and mediate continued, life-long protection through generation of immune memory. The endogenous T cells are able to recognize peptide epitopes that are displayed on Major histocompatibility complexes (MHCs) on the surface of the cancer cells. Generally, the epitopes derive from two classes of antigens. The first type of antigens, tumor-associated antigens (TAA), are formed by nonmutated proteins to which T cell tolerance is incomplete because of their restricted tissue expression pattern. A second class of antigens, tumor-specific antigens (TSA), is formed by peptides that are entirely absent from the normal human cells; these are also called neoantigens. These neoantigens are created by tumor-specific DNA alterations that result in the formation of novel protein sequences. As compared with TAA, TSA have been postulated to be of particular relevance to tumor control, as the quality of the T cell pool that is available for these antigens is not affected by central T cell tolerance. Additionally, targeting antigens that are completely specific to the tumor, and expressed nowhere else in the body, will potentially decrease on-target, off-tumor toxicity. Effective T cell therapies will also need to target multiple antigens while maintaining tumor specificity in order address tumor heterogeneity.
Cancer proteogenomics is a field that aims to identify and quantify protein sequence changes associated with the cancer genome. Besides being involved in cancer development and progression, such protein variants may serve as neoantigens, which can provide a T-cell response against tumors. In this study, we developed a personalized, low-input proteogenomic approach to identify TSAs resulting from an individual tumor’s genomic aberrations and their use to manufacture T cells specific for multiple TSAs.
To identify sufficient TSA for multi-antigen targeting, it is necessary to expand their sources beyond somatic mutations alone. This is especially true for pediatric cancers which have many fewer mutations than to their adult counterparts. In order to identify tumor-specific genomic events, we obtained 46 freshly frozen medulloblastoma tumor tissues and high coverage whole genome sequencing (WGS) and RNA-seq data from the Children’s Brain Tumor Network (CBTN). Four different types of tumor-specific genomic events were identified from genomic data: gene fusions, aberrant splice junctions, small insertions/deletions and single nucleotide variants (SNVs). The tumor-specific genomic events for each tumor were translated into peptides and included in individualized databases together with the normal human proteome. We next performed high-resolution LC-MS/MS on the same panel of freshly frozen tumors for which we previously created individualized protein databases using total protein lysates partially digested with LysC. In addition to the tumors, we also performed LC-MS/MS on five healthy childhood cerebellums for comparison. LS-MS/MS spectra from MB tumors and healthy cerebellums were searched together against the individualized tumor databases described above. We effectively identified TSAs arising from four types of genomic events. To verify that our findings are not simply unannotated normal proteins, we also developed a multi-step strategy to ensure that the tumor specific peptides are not present in normal tissues. The vast majority of these tumor specific peptides were non-overlapping between the tumors. In addition, we identified aberrant splice junctions as the main source of neoantigens in medulloblastoma tumors. This approach succeeds in identifying a mean of 12 neoantigens per tumor, making multi-antigen targeting possible. Because the pipeline only requires a small amount of tissue, it is uniquely suited to brain tumors though it is applicable to all cancer types. As a proof-of-principle, we demonstrate that T cells selected and expanded in response to these peptides contain both CD4 and CD8 populations and are immunogenic, as demonstrated by cytokine profiling and robust cytotoxicity in vitro.
In summary, our workflow identifies a robust number of peptide neoantigens sourced from multiple types of tumor-specific genomic and transcriptomic events translated into de facto proteins. The pipeline uses very low tissue input and employs native immuno-proteosome processing and presentation machinery to select and expand autologous T cells for an HLA agnostic, personalized T cell therapy. Such a specific, targeted T cell product should make an ideal backbone for the addition of potentiating immunoadjuvants. The next step for this work is to employ the pipeline in support of an early phase clinical trial treating children with brain tumors (Figure 1).