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Summary:CD19- and B-cell maturation antigen (BCMA)–directed chimeric antigen receptor (CAR) T cells have enabled unprecedented responses in a subset of refractory patients with B-cell and plasma cell malignancies, leading to their approval by the FDA for the treatment of leukemia, lymphoma, and myeloma. These “living drugs” can become part of a synthetic immune system, persisting at least a decade in some patients. However, despite this tremendous impact, significant unmet treatment needs remain for patients with hematologic malignancies and solid cancers. In this perspective, we highlight recent innovations that advance the field toward production of a more potent and universal cellular immunotherapy of the future. Next-generation CAR T cells will incorporate advances in gene engineering and synthetic biology to enhance functionality and persistence, and reduce treatment-associated toxicities. The combination of autologous CAR T cells with various allogeneic cell treatment strategies designed to target the immunosuppressive tumor microenvironment will broaden the impact of future CAR T-cell therapies.

T cells engineered to express transgenes such as chimeric antigen receptors (CAR) or modified T cell receptors (TCR) represent a new pillar of cancer therapy. Use of CRISPR/Cas gene-editing tools now allows even stronger and more precise control over the fate and function of engineered T cell therapies, including multiplex genome editing to facilitate use of off-the-shelf allogeneic T cells and novel approaches which have the potential to overcome some of the limitations of canonical Cas9-mediated DNA cleavage. This review summarizes the CRISPR/Cas techniques that have been used in preclinical research and outlines those that currently being tested in clinical trials.

In the 21st century, several emergent viruses have posed a global threat. Each of these pathogens has emphasized the value of rapid and scalable vaccine development programs. The ongoing SARS-CoV-2 pandemic has made the importance of such efforts especially clear. In particular, new biotechnological advances in vaccine design allowed for new advances in vaccines that provide only the nucleic acid building blocks of an antigen, eliminating many safety concerns. During the COVID-19 pandemic, these DNA and RNA vaccines have facilitated the development and deployment of vaccines at an unprecedented pace. This success was attributable at least in part to broader shifts in scientific research relative to prior epidemics. For example, the genome of SARS-CoV-2 was available as early as January 2020, allowing for global efforts in the development of DNA and RNA vaccines to begin within two weeks of the international community becoming aware of the new viral threat. Additionally, these technologies that were previously only theoretical were found to be not only safe but also to have high vaccine efficacy. Although historically a slow process, the rapid development of vaccines during the COVID-19 public health crisis reveals a major shift in vaccine technologies. In this review, we provide historical context for the emergence of these paradigm-shifting vaccines. Further context is provided in a companion review exploring more established vaccines platforms. We describe several DNA and RNA vaccines and in terms of their efficacy, safety, and approval status. We also discuss patterns in worldwide distribution. The advances made since early 2020 provide an exceptional illustration of how rapidly vaccine development technology has advanced in the last two decades in particular, and suggest a new era in vaccines against emerging pathogens.

Over the past 150 years, vaccines have revolutionized the relationship between people and disease. During the COVID-19 pandemic, technologies such as mRNA vaccines have received significant attention due to their novelty and successes. However, more traditional vaccine development platforms have also been applied against SARS-CoV-2, yielding important tools in the worldwide fight against the virus. A variety of approaches have been used to develop COVID-19 vaccines that are now authorized for use in countries around the world. In this review, we highlight strategies that focus on the viral capsid outwards, rather than on the nucleic acids inside. Such approaches broadly fall into two categories: whole-virus vaccines and subunit vaccines. Whole-virus vaccine approaches use the virus itself, either in an inactivated or attenuated state. Subunit vaccines isolate an immunogenic component of the virus using various strategies that is then introduced through vaccination. We highlight specific vaccine candidates that utilize these approaches in different ways. In a companion manuscript, we review the more recent and novel development of nucleic-acid based vaccine technologies. We further consider the role that these COVID-19 vaccine development programs have played in providing immunity to people around the world. Well-established vaccine technologies have proved especially important because of the significant role they have played in COVID-19 vaccine access at the global scale. Vaccine development programs that use established platforms have been undertaken in a much wider range of countries than those using nucleic-acid-based technologies, which have been led by wealthy Western countries. Therefore, these vaccine platforms, while less cutting-edge on the biotechnology side, have proven to be extremely important to the management of SARS-CoV-2.

The COVID-19 pandemic has presented many challenges that have spurred biotechnological research to address specific problems. Diagnostics is one area where biotechnology has been critical. Diagnostic tests play a vital role in managing a viral threat by facilitating the detection of infected and/or recovered individuals. From the perspective of what information is provided, these tests fall into two major categories, molecular and serological. Molecular diagnostic techniques assay whether a virus is present in a biological sample, thus making it possible to identify individuals who are currently infected. Additionally, when the immune system is exposed to a virus, it responds by producing antibodies specific to the virus. Serological tests make it possible to identify individuals who have mounted an immune response to a virus of interest and therefore facilitate the identification of individuals who have previously encountered the virus. These two categories of tests provide different perspectives valuable to understanding the spread of SARS-CoV-2. Within these categories, different biotechnological approaches offer specific advantages and disadvantages. Here we review the categories of tests developed for the detection of the SARS-CoV-2 virus or antibodies against SARS-CoV-2 and discuss the role of diagnostics in the COVID-19 pandemic.

Adoptive cell therapies using chimeric antigen receptor T cells (CAR-T cells) have demonstrated remarkable clinical efficacy in patients with hematological malignancies and are currently being investigated for various solid tumors. CAR-T cells are generated by removing T cells from a patient's blood and engineering them to express a synthetic immune receptor that redirects the T-cells to recognize and eliminate target tumor cells. Gene editing of CAR-T cells has the potential to improve safety of current CAR-T cell therapies and further increase the efficacy of CAR-T cells. Here, we describe methods for the activation, expansion, and characterization of human CRISPR-engineered CD19 directed CAR-T cells. This comprises transduction of the CAR lentiviral vector and use of single guide RNA (sgRNA) and Cas9 endonuclease to target genes of interest in T cells. The methods described in this protocol can be universally applied to other CAR constructs and target genes beyond the ones used for this study. Furthermore, this protocol discusses strategies for gRNA design, lead gRNA selection and target gene knockout validation to reproducibly achieve high-efficiency, multiplex CRISPR-Cas9 engineering of clinical grade human T cells.

Chimeric antigen receptor (CAR) T cell immunotherapy is FDA approved for the treatment of a subset of B cell malignancies but has shown limited clinical success in solid tumor therapy. T cell exhaustion is an important factor involved in treatment failure, and can, in part, result from continuous stimulation of the CAR by tumor cells expressing the cognate antigen. To gain deeper understanding of CAR T cell exhaustion induced by chronic antigen exposure (CAE), we developed and validated a robust in vitro model, in which mesothelin-redirected CAR T cells (M5CAR) were continuously stimulated with mesothelin-expressing AsPC-1 pancreatic tumor cells such that tumor cells were never cleared, and we characterized these CAE CAR T cells by gene expression at population and single-cell levels, and by epigenetic analyses. CAE M5CAR T cells recapitulate the hallmarks of T cell exhaustion, including reduced proliferation, down-modulation of surface CAR, decreased cytokine production, and reduced cytotoxicity. In addition, CAR T cells undergoing CAE have a transcriptional signature and an epigenetic landscape consistent with exhaustion. Further, transcriptomic analysis revealed that CAE M5CAR T cells undergo a transition from T cells to a post-thymic NK-like T cell phenotype. This plasticity was confirmed by TCR lineage tracing and was also detected in CD19 CAR T cells analyzed from post-infusion blood from DLBCL patients and in M5CAR T cells infiltrating relapsed tumors derived from a xenograft NSG/AsPC-1 mouse model. The dysfunctional signature and the NK phenotype were further detected in vivo by transcriptomic analysis in Ly95 TCR-specific TILs infiltrating NY-ESO-1 tumors. Among the genes included in the dysfunctional signature, the transcription factors SOX4 and ID3 were identified as potential regulators of dysfunction by differential gene expression and pathway analyses. To determine their role in the establishment of a dysfunctional phenotype, we generated ID3KO.M5 and SOX4KO.M5 CAR T cells using CRISPR technology in primary human lymphocytes. The cytotoxic potential of the M5 CAR T cell product generated was not modified by the genetic disruption of the transcription factors. However, when CAR T cells were challenged with chronic antigen stimulation, KO CAR T cells showed a significant reduction of the dysfunctional signature and the NK-like T cell gene expression. Importantly, CAE KO CAR T cells exhibit improved cytotoxicity as compared to Mock.M5CAR T cells. In summary, we have developed a robust in vitro model that recapitulates the hallmarks of T cell exhaustion and that facilitated the identification of a gene signature defining CAR dysregulation, a T-to-NK-like-T cell transition as a novel feature of CAR T cell dysfunction and the transcription factors SOX4 and ID3 as key regulators of CAR T cell exhaustion. Citation Format: M. Angela Aznar, Charly R. Good, Shunichiro Kuramitsu, Parisa Samareh, Sangya Agarwal, Greg Donahue, Kenichi Ishiyama, Nils Wellhausen, Austin K. Rennels, Yujie Ma, Lifeng Tian, Sonia Guedan, Katherine A. Alexander, Zhen Zhang, Philipp C. Rommel, Nathan Singh, Karl M. Glastad, Max W. Richardson, Keisuke Watanabe, Janos L. Tanyi, Mark H. O’Hara, Marco Ruella, Simon F. Lacey, Edmund K. Moon, Stephen J. Schuster, Steven M. Albelda, Lewis L. Lanier, Regina M. Young, Shelley L. Berger, Carl H. June. Mechanisms of CAR T cell dysfunction and identification of transcription factors that drive the exhaustion phenotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3602.

Despite the high tumor mutational burden (TMB), immune check point blockade (ICB) still fails in about half of microsatellite instability-high (MSI-H) cancer patients, suggesting underlying immune dysregulation. Immune profiling of peripheral blood from chemotherapy-resistant MSI-H uterine cancer patients treated with nivolumab identified a rapid proliferative CD8 T cell response 2-4 weeks post PD-1 blockade (N = 32). This immunological response, however, did not correlate with clinical response, suggesting additional parameters may be relevant. We focused on DNA damage and repair (DDR) in T cells as potential novel parameters. DDR has been extensively studied in the context of inducing cell death in highly-proliferating tumor cells. However, despite induction of rapid proliferation of T cells upon ICB, whether T cell-intrinsic DDR impacts T cell function, and how the coordination of DDR affects immunological and clinical response to ICBs have been largely unexplored. We hypothesized that the T celI-intrinsic DDR responses to proliferative and genotoxic stress might contribute to the disparity between immunological and clinical response. To test the hypothesis, we developed a novel high-dimensional cytometry platform. This DDR-Immune platform enables simultaneous analysis of T cell differentiation state with changes in major DDR pathways at single-cell resolution. The DDR-Immune platform revealed consistent T cell subset specific patterns of DDR, as well as specific DDR pathways induced by different types of DNA damage, such as γ-irradiation (IR), UV irradiation (UV) or proliferative stress (i.e. anti-CD3/CD28 stimulation). For example, terminally differentiated effector T cells accumulated higher DNA damage and cell death. In contrast, stem cell memory (TSCM) and regulatory T cells (Treg) displayed high DDR with less cell death, suggesting that effective cell-intrinsic DDR against genotoxic stress in T cells confers a survival advantage. We applied the platform to MSI-H uterine cancer cohort to test if T cell-intrinsic DDR distinguish the clinical response. Indeed, we found that in clinical responders but not clinical non-responders, Ki67+ CD8 T cells responding to PD-1 blockade exhibited rapid induction of DDR represented as a spike increase of phosphorylated-ATM (pATM). This likely indicates the T cell ‘fitness’ response to proliferative stress induced by PD-1 blockade. Furthermore, patients with higher induction of pATM in responding CD8 T cells at the peak of the immunological response to PD-1 blockade had longer progression-free survival (PFS). Collectively, the new platform reveals previously unrecognized roles for T cell-intrinsic DDR as a novel determinant of immune responsiveness and clinical outcome to ICB and has potential application to other cancer therapies including chemotherapy and radiotherapy. Citation Format: Yuki Muroyama, Sasikanth Manne, Derek A. Oldridge, Nils Wellhausen, Allison R. Greenplate, Lakshmi Chilukuri, Divij Mathew, Caiyue Xu, Ramin S. Herati, Alexander C. Huang, Dmitriy Zamarin, Claire F. Friedman, E. John Wherry. T cell intrinsic DNA damage and repair response as a novel marker associated with clinical response to PD-1 blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3579.

Summary Chimeric antigen receptor (CAR) T cell therapy has achieved remarkable success in hematological malignancies but remains ineffective in solid tumors, due in part to CAR T cell exhaustion in the solid tumor microenvironment. To study dysfunction of mesothelin-redirected CAR T cells in pancreatic cancer, we establish a robust model of continuous antigen exposure that recapitulates hallmark features of T cell exhaustion and discover, both in vitro and in CAR T cell patients, that CAR dysregulation is associated with a CD8+ T-to-NK-like T cell transition. Furthermore, we identify a gene signature defining CAR and TCR dysregulation and transcription factors, including SOX4 and ID3 as key regulators of CAR T cell exhaustion. Our findings shed light on the plasticity of human CAR T cells and demonstrate that genetic downmodulation of ID3 and SOX4 expression can improve the efficacy of CAR T cell therapy in solid tumors by preventing or delaying CAR T cell dysfunction.

Chimeric antigen receptor (CAR)-engineered T cell therapy for solid tumors is limited by the lack of both tumor-restricted and homogeneously expressed tumor antigens. Therefore, we engineered an oncolytic virus to express a non-signaling, truncated CD19 (CD19t) protein for tumor-selective delivery, enabling targeting by CD19-CAR T cells. Infecting tumor cells with an oncolytic vaccinia virus coding for CD19t (OV19t) produced de novo CD19 at the cell surface prior to virus-mediated tumor lysis. Co-cultured CD19-CAR T cells secreted cytokines and exhibited potent cytolytic activity against infected tumors. Using several mouse tumor models, delivery of OV19t promoted tumor control following CD19-CAR T cell administration. Importantly, OV19t induced local immunity characterized by tumor infiltration of endogenous and adoptively-transferred T cells. CAR T cell–mediated tumor killing also induced release of virus from dying tumor cells, which propagated tumor expression of CD19t. Our study features a combination immunotherapy approach using oncolytic viruses to promote de novo CAR T cell targeting of solid tumors.

Chimeric antigen receptor (CAR) T cell therapy has achieved remarkable success in hematological malignancies but remains largely ineffective in solid tumors. A major factor leading to the reduced efficacy of CAR T cell therapy is T cell dysfunction, and the mechanisms mediating this dysfunction are under investigation. Here we establish a robust model to study dysfunction of mesothelin-redirected CAR T cells in pancreatic cancer. Continuous antigen exposure results in hallmark features of exhaustion including reduced proliferation capacity and cytotoxicity, and severe defects in cytokine production. Here we identified a transcriptional signature at both population and single-cell levels in CAR T cells after continuous antigen exposure. In addition, TCR lineage tracing revealed a CD8+ T-to-NK-like T cell plasticity that results in reduced tumor cell killing. The transcription factors SOX4 and ID3 are specifically expressed in the dysfunctional CAR NK-like T cells and are predicted to be master regulators of the dysfunction gene expression signature and the post-thymic acquisition of an NK-like T cell fate. Finally, we identified the emergence of NK-like CAR T cells in a subset of patients after infusion of CAR T cells. The findings gleaned from this study shed light on the plasticity of human CAR T cells and suggest new approaches to improve the efficacy of CAR T cell therapy in solid tumors by preventing or revitalizing CAR T cell dysfunction. Citation Format: Charly R. Good, Shunichiro Kuramitsu, Parisa Samareh, Greg Donahue, Kenichi Ishiyama, Yujie Ma, Nils Wellhausen, Lifeng Tian, Sangya Agarwal, Sonia Guedan, M. Angela Aznar, Katherine A. Alexander, Zhen Zhang, Nathan Singh, Max W. Richardson, Keisuke Watanabe, Janos L. Tanyi, Mark H. O'Hara, Marco Ruella, Simon F. Lacey, Edmund K. Moon, Stephen J. Schuster, Steven M. Albelda, Lewis L. Lanier, Regina M. Young, Shelley L. Berger, Carl H. June. Induction of T cell dysfunction and NK-like T cell differentiation in vitro and in patients after CAR T cell treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 60.