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MASS CYTOMETRY PUBLICATIONS TRENDING NOW

ON DEVELOPMENT Welcome to the January issue of Trending Now, a quarterly anthology of recent impactful publications by researchers using CyTOF technology. January 2021 This month’s issue is the second in a two-part series focusing on the application of mass cytometry to vaccine development. Part one highlighted the area of infectious diseases. In this issue we focus on vaccine and development for cancer. WHAT’S NEW IN MASS CYTOMETRY

The development and testing of new treatment for cancer begins with a comprehensive understanding of tumor cell and immune cell behavior, made possible with mass cytometry.

2 What makes a cure and treatments for cancer so To improve current or develop elusive? While our understanding of the unique and new ones, more work must be done to better variable processes of cancer progression continues understand how immune checkpoint blockades to develop, we have yet to create a clear picture of affect and activate immune cells. Deep immune how cancer works and what we can do to stop it. monitoring using CyTOF® technology allows a Every causative mutation, cell surface change and comprehensive view of all cells involved in immune immune evasion process can differ with and within response and within a tumor microenvironment, a cancer type down to the individual level, making exposing cell-cell interactions and identifying new prevention, treatment and even basic understanding tumor-specific neoantigens. An in-depth look at a challenge. these tumor-specific T cells and neoantigens can Vaccines provide new possibilities for cancer reveal why these cells are inconsistent in their ability prevention and treatment by strengthening the to eradicate a tumor, and what can be done to to recognize and react to develop more reliably effective treatments. and destroy cancer cells. Immunotherapies based Here we review several publications and insightful on immune checkpoint blockades (ICB) remain abstracts from 2020 that outline new work using the most promising treatment vaccine approach mass cytometry for immune monitoring to identify to date. However, issues with specificity and predictive biomarkers, develop novel therapy tumor resistance plague ICB immunotherapies, approaches and evaluate vaccine efficacy for a resulting in uneven responses to treatment and variety of . They are among a multitude of unpredictable efficacy. publications, in addition to more than 100 clinical trials, that attest to the success of mass cytometry in vaccine research and development.

Treg CTLA-4 Immune system Cancer growth evasion and proliferation B7 TCR MHC peptide Dendritic cell Cancer cell

T cell PD-1 CD28 PD-L1

CTLA-4 PD-1 PD-L1 inhibitor inhibitor inhibitor

Figure 1. The immune checkpoint blockade effect on T cell activation, typically acting through PD-1/PD-L1

3 How CyTOF specificity helps identify predictive biomarkers Immunotherapy has received considerable attention In a multi-site study conducted through the Pacific as a treatment for cancers harboring tumor-specific Pediatric Neuro-Oncology Consortium (PNOC), neoantigens, a class of immunogens that confer researchers evaluated the safety, immunoreactivity tumor specificity and immune activation. and efficacy of a vaccine based on a specific H3 Currently, technologies that can detect tumor mutation in patients with DMGs containing the specificity of antigens stand out as a much-needed variant. The vaccine was administered to newly solution to distinguishing immune cells specifically diagnosed patients on an ongoing schedule. activated by the vaccine from other activated Immune monitoring and imaging were performed cells. The significance of this distinction lies in every three months, with immune responses the ability to improve efficacy and specificity of a assessed in PBMC using mass cytometry. vaccine. Mass cytometry holds the unique ability to identify neoantigen-specific T cells in the context of high-parameter phenotyping, as well as with This research validates the power of CyTOF high-parameter tetramer use. technology for high-dimensional immune A University of California, San Francisco clinical monitoring of immunotherapies based on research study focused efforts on vaccine CD8+ T cells. development for diffuse midline gliomas (DMGs), a fatal pediatric brain cancer (Mueller et al. NCT02960230). Even with treatment, survival The group combined the use of HLA dextramers outcomes for this type of cancer are dismal, with less with CyTOF technology to analyze multiple immune than 10% of patients surviving beyond two years. subsets to better explore associations between Recently, known mutations in genes encoding histone targeted -reactive CD8+ T cells triggered 3 variants have become a tumor-specific target by the vaccine and prolonged overall survival. for immunotherapy. Dextramers exhibit high affinity and specificity for -specific CD8+ T cell populations, allowing identification and evaluation of epitope-specific CD8+ T cell responses in cancer patients treated with peptide-based vaccines.

—CD8+ or CD4+ T cell Results indicated that the vaccine is safe for use, while the study itself demonstrated the significance —T cell receptor of immune monitoring for identification of predictive biomarkers, detectable across any location (cell —CD8 or CD4 co-receptor surface, cytoplasm, nucleus). This research validates the power of CyTOF technology for high-dimensional —MHC class I or class II immune monitoring of immunotherapies based on —Antigen CD8+ T cells.

—Metal isotope

Figure 2. Metal-tagged tetramers enable identification of antigen-specific T cells in the context of deep immune profiling with 40 or more markers using mass cytometry.

4 Using mass cytometry to support immunotherapy development Mass cytometry’s ability to deeply characterize valuable tool to guide the development of novel tumor and immune cells and their interactions immunotherapy strategies through assessment of empowers a new level of insight into how tumor- drug biological activity, definition of mechanism of specific immune cells can relate to clinical response action, and identification of biomarkers of patient to therapies, identifying infiltrating behavior and response,” the paper states. specific immune signatures. A new approach to identifying tumor-specific A joint effort byI mmunoScape, Fred Hutchinson neoantigens and investigating the role of neoantigen- Center and Genentech examined specific T cells could be a significant step for the nature and function of tumor-specific T cells, known designing promising cancer immunotherapies. to play a role in tumor rejection, aiming to support Since immune checkpoint receptors such as PD-1 the development of immunotherapy strategies. and CTLA-4 and the associated T cell checkpoint Their new study (Abel et al.) leverages the high- blockade have no inherent tumor specificity, dimensional immune profiling capabilities of CyTOF response to these treatments is variable at best. technology to identify and characterize circulating Researchers at Fred Hutchinson Cancer Research neoantigen-specific T cells in lung carcinoma Center applied combinatorial tetramer staining by patients undergoing atezolizumab treatment. CyTOF technology to study an immunotherapy- Combinatorial peptide-MHC tetramer staining resistant mouse model (Li et al.) in order to learn and high-performance dimensional analysis tools, more about how immune checkpoint blockade applied together with mass cytometry, revealed affects tumor-specific T cells and why these cells are 13 unique neoantigens eliciting T cell reactivity unsuccessful in attacking tumor cells after treatment. among responding patients. Additionally, seven The team identified a new tumor-specific neoantigen neoantigens were recognized in patients who did in Lewis lung carcinoma, activated tumor-specific not respond to treatment, and differentiated T cell CD8+ tumor-infiltrating lymphocytes (TILs). However, phenotypes were observed between the two even with cell expansion after treatment, the cells did patient groups. This broad and sensitive map of not appear to affect tumor regression. The approach T cell reactivity against MHC-class I and enabled the group to discover novel cell types and simultaneous in-depth characterization of these rare behaviors associated with both immune checkpoint antigen-specific T cells suggest that tumor-reactive blockade and neoantigen peptide , T cell signatures could be associated with clinical providing a model for studying neoantigens in tumors. response to anti-PD-L1 treatment. Further investigation into the role of neoantigen- “By providing insight into the nature and function specific T cells in ICB could have a significant impact of tumor-specific T cells, ImmunoScape’s unique on the design of future immunotherapeutic strategies. high-dimensional immune profiling platform is a

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Study participants Cryopreserved CD45 live-cell Antibody staining Data acquisition Computational PBMC barcoding analysis

Figure 3. Example workflow using mass cytometry for in-depth cell identification and characterization with sample barcoding.

5 Measuring vaccine efficacy by immune monitoring Immune cell infiltration into tumors provides evidence In an effort to find new combination therapies of the level of immune response activated by a to prolong overall survival for pancreatic ductal vaccine. Mass cytometry offers unparalleled analysis adenocarcinoma (PDA), an aggressive and of the tumor immune cell infiltrate, generating detailed lethal malignancy with poor prognosis, a team information on cell type, behavior and interactions from Johns Hopkins University attempted a from limited samples. Phase 2 study to evaluate potential vaccine efficacy (HCC) is the most common (Wu et al.). The combination therapy given after type of primary . Though curative resection frontline chemotherapy consisted of granulocyte- can be successful in tumor removal, recurrence of macrophage colony-stimulating factor-allogeneic HCC remains high. Having demonstrated safety and pancreatic tumor cells (GVAX) and ipilimumab. efficacy of a novel therapeutic that induces antigen- While the study was discontinued after interim specific cytotoxic T lymphocytes, researchers at analysis, important clinical responses and immune Yamaguchi University in Japan conducted further cell effects were observed. The study used a study of the same vaccine as a perioperative 32-marker CyTOF panel including both surface and immunotherapy option in patients with resectable intracellular markers to monitor immune response HCC (Nakajima et al.). after treatment and vaccination. The combination Patients received 10 injections of adjuvant vaccine resulted in T cell differentiation into effector immunotherapy over four months before liver memory phenotypes in all samples and increased resection surgery. Researchers used mass cytometry M1 macrophages in the tumor. to analyze liver samples and PBMC, choosing 66 Even though the therapy did not improve overall antibodies across several panels to monitor T cell survival in patients, the study demonstrates the use exhaustion, T cell activation and effector T regulatory of mass cytometry for unraveling immune response cell induction. Three response types were identified to treatment and the feasibility of testing novel based on the level of tumor immune cell infiltration combinations in the maintenance treatment of observed: “hot cellular immunity,” “intermediate metastatic PDA. humoral immunity” and “cold response.” CyTOF analysis also revealed a difference in certain T cell subtypes in the tumor specimens, not seen in the Myeloid-derived PBMC, suggesting tumor-infiltrating lymphocyte suppressor cell (MDSC) analysis is critical to understanding immune response. T cell (CD8+, CD4+) This novel immunotherapy induced sustained immune cell infiltration into tumor microenvironments, confirming the ability to convert progressive tumors into tumors containing PD-1+ lymphocytes and indicating the potential to combine the therapy with PD-1 antibodies.

Treg Dendritic cell (DC) Macrophage Cancer cells NK cell

Figure 4. Immune infiltration into tumors

6 Mass cytometry’s impact on targeted vaccine research Mass cytometry’s unique ability to a detect a large regardless of their cellular location, that indicate array (50 or more) of diverse targets in a single progression or improvement of disease. As the tube of either tumor digests or peripheral blood scientific community works tirelessly toward effective samples makes it highly efficient for obtaining a development of treatment vaccines for certain types comprehensive immune picture quickly and reliably. of cancers, mass cytometry is helping bring research It enables simultaneous screening of numerous T closer to success. cell antigen-specific epitopes as well as biomarkers,

References

Abel, B. et al. “Immuno-phenotyping of tumor-specific CD8 Nakajima, M. et al. “Immunological and histopathological tumor T cells using high-dimensional mass cytometry.” Journal of responses to a novel neoadjuvant targeting Immunology (2020): 86.7. HSP70 and GPC3 antigens in patients with resectable HCC.” Cancer Research (2020): doi:10.1158/1538-7445.AM2020-CT251. Li, S. et al. “Unsuccessful checkpoint blockade and vaccination therapy despite expansion of tumor neoantigen-specific CD8 Wu, A.A. et al. “A Phase II study of allogeneic GM-CSF– T cells in the Lewis lung carcinoma (LLC) model.” Journal of transfected pancreatic tumor vaccine (GVAX) with ipilimumab Immunology (2020): 165.13. as maintenance treatment for metastatic pancreatic cancer.” Clinical Cancer Research (2020): 5,129–5,139. Mueller, S. et al. “Mass cytometry detects H3.3K27M-specific vaccine responses in diffuse midline glioma.” Journal of Clinical Investigation (2020): 6,325–6,337.

Please see these additional references using mass cytometry in therapeutic research

Beyranvand Nejad, E. et al. “Lack of myeloid cell infiltration as Sugiyama, E. et al. “Blockade of EGFR improves responsiveness an acquired resistance strategy to immunotherapy.” Journal for to PD-1 blockade in EGFR-mutated non-small cell .” ImmunoTherapy of Cancer (2020): e001326. Science Immunology (2020): eaav3937. Crosby, E.J. et al. “Long-term survival of patients with stage III Tu, C. et al. “Exploration of the personalized immune checkpoint colon cancer treated with VRP-CEA(6D), an alphavirus vector atlas of plasma cell dyscrasias patients using high‑dimensional that increases the CD8+ effector memory T cell to Treg ratio.” single‑cell analysis.” Oncology Reports (2020): 224–240. Journal for ImmunoTherapy of Cancer (2020): e001662. Wang, J. et al. “Identification of the immune checkpoint signature Friebel, E. et al. “Single-cell mapping of human brain cancer of multiple myeloma using mass cytometry-based single-cell reveals tumor-specific instruction of tissue-invading leukocytes.” analysis.” Clinical and Translational Immunology (2020): e01132. Cell (2020): 1626–1642.e20.

7 Explore mass cytometry at go.fluidigm.com/cytof

Listen to recorded seminars on the tumor microenvironment given by Joshua Brody, Jonathan Irish and Evan Lind. Learn more about decoding immune cell heterogeneity with mass cytometry in an interview with Evan Newell.

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