Introduction
Immunotherapies such as adoptive cell therapy (ACT) and immune checkpoint blockade (ICB) have revolutionized cancer treatment, especially for patients with diseases that were previously considered incurable, such as melanoma. ACTs have made an incredible impact towards the treatment of cancer, particularly for hematological malignancies, by utilizing genetically engineered T cells, notably the chimeric antigen receptor-engineered T (CAR-T) cells. Unlike traditional immune responses, CAR-T cells bypass antigen presentation and T-cell priming, thereby directly recognizing, and attacking cancer cells upon administration. This mechanism has led to remarkable efficacy in hematological cancers, yielding durable clinical responses despite challenges like resistance within the tumor microenvironment. On the other hand, ICBs have transformed cancer treatment by targeting immune checkpoints, which are critical pathways exploited by cancer cells to suppress anti-tumor immunity. ICB drugs, such as monoclonal antibodies, block checkpoint proteins, including programmed cell death 1 (PD1), PD1 ligand 1 (PD-L1), and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), thus preventing the “off” signal and allowing T cells to unleash their cytotoxic potential against cancer cells. This approach has led to long-term remission and survival in metastatic cancers, where conventional treatments often fell short, therefore, marking a significant clinical impact.
The degree of immune cell penetration into tumors can differ based on the tumor’s stage and histological characteristics. However, studies have shown that most metastatic human tumors continue to exhibit immune recognition of tumor antigens and epitopes, or neoantigens, indicating that there’s an active immune response against the tumor, even in cancers that are typically considered immunologically ‘cold’ or unresponsive to immune activity. Tumor-infiltrating lymphocytes (TILs), which are obtained from surgically removed tumors, serve as a rich source for isolating and identifying these antitumor T cells. When cultured in vitro, these TILs can be evaluated for their ability to recognize tumor-specific antigens. Furthermore, the T-cell receptors (TCRs), which are essential for antigen recognition, can be isolated and sequenced to provide insight into the anti-tumor activity of TILs. However, TCRs are often non-specific, and can often be expressed on TILs that are irrelevant to the tumor, which complicates the accurate identification of antitumor TILs. This is particularly problematic as these tumor-irrelevant TILs can also exhibit functional impairment. Single-cell transcriptomic sequencing (scRNA-seq) based profiling of neoantigen-specific TILs have provided insights into the phenotypes, differentiation states, and TCR repertoire of tumor-reactive TILs. Through scRNA-seq, studies have shown that it’s feasible to identify antitumor TCRs solely based on the transcriptomic states of TILs. However, this analysis requires surgical resection of tumors from patients, followed by the separation of TILs from tumors prior to analysis. As such, there is considerable interest in the scientific community and the pharmaceutical industry towards the use of circulating blood as a source of tumor-specific T cells and their TCRs. Towards this endeavor, recent murine studies have demonstrated the presence of a clonal and phenotypic overlap between antitumor T cells in circulating blood and TILs. However, the clinical relevance of these findings in the context of metastatic human cancers remains unclear.
In a recent publication in Cancer Cell, Yossef et al. (2023) utilize single cell sequencing to provide the first in-human deep transcriptional profiling of neoantigen-specific anti-tumor CD8+ T cells in the peripheral blood lymphocytes (PBL) that support findings from murine T cell phenotyping efforts.
Transcriptional profiling of PBL derived neoantigen-specific CD8+ T-cells (NeoTCRPBL) Enables Capturing of Circulating Antitumor TCRs
The circulating neoantigen reactive CD8+ T-cells (NeoTCRPBL) in patients with metastatic cancer provide a glimpse into the broader antitumor immune response. Dissecting the transcriptional and cell surface signatures of NeoTCRPBL cells sheds light on the landscape of antitumor immunity beyond the tumor microenvironment. To characterize NeoTCRPBL cells, Yossef et al. (2023) isolated known neoantigen-specific CD8+ clonotypes from TILs, followed by identification of the same clonotypes within the PBLs using tetramer enrichment via fluorescent-activated cell sorting (FACS). Subsequently, they reintroduced the known proportion of neoantigen tetramer-positive PBL T cells into CD8+ tetramer-negative PBL T cells. Finally, Yossef et al. (2023) performed scRNA-seq and single cell T cell receptor (scTCR-seq) sequencing to dissect the phenotypic diversity of antitumor NeoTCRPBL T cells.
Dimensionality reduction and unsupervised clustering, followed by uniform manifold approximation and projection (UMAP) of the PBL CD8+ T cells identified 24 distinct cell states. The NeoTCRPBL T-cells were found to be less dysfunctional than their TIL counterparts, and displayed memory phenotypes thereby suggesting a poised readiness for immune surveillance. Cellular Indexing of Transcriptomes and Epitopes by sequencing (CITE-seq) of the neoantigen specific CD8+ PBLs indicated relatively high levels of CD45RO, HLA-DRA activation marker, as well as the tissue residency marker CD103, and TIGIT, PD-1, and CD39. These markers are known to be enriched in neoantigen specific TILs. Furthermore, anti-timor TCR clonotypes could be derived from PBLs by analyzing the transcriptional signatures of NeoTCRPBL T cells. Overall, these findings indicate that the circulating NeoTCRPBL T-cells possess a unique cell-surface marker profile, target the same neoantigens as TILs, and provide a non-invasive source for antitumor TCRs.
Challenges and future directions for the use of NeoTCRPBL in the clinic
The single cell transcriptional profiling of PBLs to characterize NeoTCRPBL in patients with metastatic cancer affords the promise of identifying anti-tumor TCRs that target the same tumor-relevant neoantigens as the TIL compartment. Therefore, the NeoTCRPBL gene signature could be instrumental in identifying tumor-relevant TCRs for the development of PBL-derived TCR-engineered cell therapies against metastatic tumors. However, the NeoTCRPBLwere found to be of low frequency, comprising less than 0.002 % of all cells. Overcoming this limitation by the selective stimulation of the NeoTCRPBL T cells using novel chemotherapeutics may provide a diverse selection of anti-tumor TCRs, while simultaneously increasing the tumor-killing potential of PBLs.
The integration of single-cell transcriptomics to identify anti-tumor TCRs in metastatic cancer patients marks a potential paradigm shift towards personalized medicine. However, extending these findings to solid tumors demands careful exploration due to their heightened complexity. Solid tumors present intricate microenvironments and cellular compositions, posing challenges in deciphering immune responses accurately. However, by understanding these complexities, future research can uncover valuable insights into tumor-immune interactions, potentially leading to the development of targeted immunotherapies tailored to individual tumor characteristics. Overcoming technical hurdles and validating findings across diverse patient cohorts and tumor types are crucial steps towards realizing the transformative nature of this therapy.
How Bridge Informatics Can Help
At Bridge Informatics, we are passionate about empowering life science companies with cutting-edge technologies. We believe the ability to find neoantigen specific TCR sequences from peripheral blood a significant advancement in the field of personalized immunotherapy. Our team of bioinformatics experts can assist your organization in:
- Understanding the implications of this research for your specific drug development programs.
- Developing strategies to incorporate this technology into your research and development pipeline.
- Leveraging our bioinformatics expertise to design and optimize experiments for TCR extraction and analysis.
By embracing innovative solutions like the identification of neoantigen specific TCR sequences from peripheral blood, we can collectively accelerate progress towards developing safer and more effective therapies for patients in need. Bridge Informatics’ bioinformaticians are trained bench biologists, so they understand the biological questions driving your computational analysis. Click here to schedule a free introductory call with a member of our team.
Haider M. Hassan, Data Scientist, Bridge Informatics
Haider is one of our premier data scientists. He provides bioinformatic services to clients, including high throughput sequencing, data pre-processing, analysis, and custom pipeline development. Drawing on his rich experience with a variety of high-throughput sequencing technologies, Haider analyzes transcriptional (spatial and single-cell), epigenetic, and genetic landscapes.
Before joining Bridge Informatics, Haider was a Postdoctoral Associate at the London Regional Cancer Centre in Ontario, Canada. During his postdoc, he investigated the epigenetics of late-onset liver cancer using murine and human models. Haider holds a Ph.D. in biochemistry from Western University, where he studied the molecular mechanisms behind oncogenesis. Haider still lives in Ontario and enjoys spending his spare time visiting local parks. If you’re interested in reaching out, please email [email protected] or [email protected]
