Engineered T-cell Receptor Research Articles

The current landscape of optogenetics for the enhancement of adoptive T-cell therapy

Abstract Immunotherapy, medicinal modulation of a host’s immune response to better combat a pathogen or disease, has transformed cancer treatments in recent decades. T-cells, an important component of the adaptive immune system, are further paramount for therapy success. Recent immunotherapeutic modalities have therefore more frequently targeted T-cells for cancer treatments and other pathologies and are termed adoptive T-cell (ATC) therapies. ATC therapies characterise various types of immunotherapies but predominantly fall into three established techniques: Tumour infiltrating lymphocyte (TIL), Chimeric Antigen Receptor T-cell (CAR-T) and engineered T-cell receptor (eTCR) therapies. Despite promising clinical results, all ATC therapy types fall short in providing long-term sustained tumour clearance whilst being particularly ineffective against solid tumours, with substantial developments aiming to understand and prevent the typical drawbacks of ATC therapy. Optogenetics is a relatively recent development, incorporating light-sensitive protein domains into cells or tissues of interest to optically tune specific biological processes. Optogenetic manipulation of immunological functions is rapidly becoming an investigative tool in immunology, with light-sensitive systems now being used to optimise many cellular therapeutic modalities and ATC therapies. This review focuses on how optogenetic approaches are currently utilised to improve ATC therapy in clinical settings by deepening our understanding of the molecular rationale behind therapy success. Moreover, this review further critiques current immuno-optogenetic systems and speculates on the expansion of recent developments, enhancing current ATC-based therapeutic modalities to pave the way for clinical progress.

Phase separation of chimeric antigen receptor promotes immunological synapse maturation and persistent cytotoxicity.

Major challenges of chimeric antigen receptor (CAR)-T cell therapy include poor antigen sensitivity and cell persistence. Here, we report a solution to these issues by exploiting CAR phase separation. We found that incorporation of an engineered Tcell receptor CD3ε motif, EB6I, into the conventional 28Z or BBZ CAR induced self-phase separation through cation-π interactions. EB6I CAR formed a mature immunological synapse with the CD2 corolla to transduce efficient antigen and costimulatory signaling, although its tonic signaling remained low. Functionally, EB6I CAR-T cells exhibited improved signaling and cytotoxicity against low-antigen tumor cells and persistent tumor-killing function. In multiple primary and relapsed murine tumor models, EB6I CAR-T cells exerted better antitumor functions than conventional CAR-T cells against blood and solid cancers. This study thus unveils a CAR engineering strategy to improve CAR-T cell immunity by leveraging molecular condensation and signaling integration.

Exploring the potential of structure-based deep learning approaches for T cell receptor design.

Deep learning methods, trained on the increasing set of available protein 3D structures and sequences, have substantially impacted the protein modeling and design field. These advancements have facilitated the creation of novel proteins, or the optimization of existing ones designed for specific functions, such as binding a target protein. Despite the demonstrated potential of such approaches in designing general protein binders, their application in designing immunotherapeutics remains relatively underexplored. A relevant application is the design of T cell receptors (TCRs). Given the crucial role of T cells in mediating immune responses, redirecting these cells to tumor or infected target cells through the engineering of TCRs has shown promising results in treating diseases, especially cancer. However, the computational design of TCR interactions presents challenges for current physics-based methods, particularly due to the unique natural characteristics of these interfaces, such as low affinity and cross-reactivity. For this reason, in this study, we explored the potential of two structure-based deep learning protein design methods, ProteinMPNN and ESM-IF1, in designing fixed-backbone TCRs for binding target antigenic peptides presented by the MHC through different design scenarios. To evaluate TCR designs, we employed a comprehensive set of sequence- and structure-based metrics, highlighting the benefits of these methods in comparison to classical physics-based design methods and identifying deficiencies for improvement.

Cellular and immunotherapies for myelodysplastic syndromes

In this review article, we outline the current landscape of immune and cell therapy-based approaches for patients with myelodysplastic syndromes (MDS). Given the well characterized graft-versus-leukemia (GVL) effect observed with allogeneic hematopoietic cell transplantation, and the known immune escape mechanisms observed in MDS cells, significant interest exists in developing immune-based approaches to treat MDS. These attempts have included antibody-based drugs that block immune escape molecules, such as inhibitors of the PD-1/PD-L1 and TIM-3/galectin-9 axes that mediate interactions between MDS cells and T-lymphocytes, as well as antibodies that block the CD47/SIRPα interaction, which mediates macrophage phagocytosis. Unfortunately, these approaches have been largely unsuccessful. There is significant potential for T-cell engaging therapies and chimeric antigen receptor T (CAR-T) cells, but there are also several limitations to these approaches that are unique to MDS. However, many of these limitations may be overcome by the next generation of cellular therapies, including those with engineered T-cell receptors or natural killer (NK)-cell based platforms. Regardless of the approach, all these immune cells are subject to the complex bone marrow microenvironment in MDS, which harbours a variable and heterogeneous mix of pro-inflammatory cytokines and immunosuppressive elements. Understanding this interaction will be paramount to ensuring the success of immune and cellular therapies in MDS.

Strengths and limitations of web servers for the modeling of TCRpMHC complexes

Cellular immunity relies on the ability of a T-cell receptor (TCR) to recognize a peptide (p) presented by a class I major histocompatibility complex (MHC) receptor on the surface of a cell. The TCR-peptide-MHC (TCRpMHC) interaction is a crucial step in activating T-cells, and the structural characteristics of these molecules play a significant role in determining the specificity and affinity of this interaction. Hence, obtaining 3D structures of TCRpMHC complexes offers valuable insights into various aspects of cellular immunity and can facilitate the development of T-cell-based immunotherapies. Here, we aimed to compare three popular web servers for modeling the structures of TCRpMHC complexes, namely ImmuneScape (IS), TCRpMHCmodels, and TCRmodel2, to examine their strengths and limitations. Each method employs a different modeling strategy, including docking, homology modeling, and deep learning. The accuracy of each method was evaluated by reproducing the 3D structures of a dataset of 87 TCRpMHC complexes with experimentally determined crystal structures available on the Protein Data Bank (PDB). All selected structures were limited to human MHC alleles, presenting a diverse set of peptide ligands. A detailed analysis of produced models was conducted using multiple metrics, including Root Mean Square Deviation (RMSD) and standardized assessments from CAPRI and DockQ. Special attention was given to the complementarity-determining region (CDR) loops of the TCRs and to the peptide ligands, which define most of the unique features and specificity of a given TCRpMHC interaction. Our study provides an optimistic view of the current state-of-the-art for TCRpMHC modeling but highlights some remaining challenges that must be addressed in order to support the future application of these tools for TCR engineering and computer-aided design of TCR-based immunotherapies.

Pharmacotherapy of autoimmune rheumatic diseases – from monoclonal antibodies to CAR T cells: 20 years later

Autoimmunity is a pathological process associated with a violation of immunological tolerance to normal structural components of the body (autoantigens), associated with the predominance of active (adaptive) immunity and manifested by hyperproduction of autoantibodies. Systemic autoimmune rheumatic diseases (SARDs) are among the most common and severe nosological forms of this pathology associated with autoimmunity. Problems of pharmacotherapy of SARDs are the subject of intensive research. At the beginning of the 21st century, more than 20 biologic agents were developed for the treatment of rheumatoid arthritis – monoclonal antibodies (mAbs) and recombinant proteins that control inflammation associated with the overproduction of “pro-inflammatory” cytokines, the use of which has dramatically improved the results of pharmacotherapy. However, much less research has been devoted to studying the possibilities of pharmacotherapy aimed at selective suppression of the “autoimmune” component of the pathogenesis of SADRs associated with uncontrolled activation of B cells and restoration of immunological tolerance to autoantigens. In the spectrum of drugs whose mechanism of action is associated with the suppression of pathological activation of B cells, the leading place is occupied by rituximab (RTM). It is noteworthy that 20 years ago (2004), a group of researchers led by prof. J.C. Edwards first demonstrated the effectiveness of RTM in patients with RA, which was soon successfully repositioned to treat a wide range of SARDs. A major achievement in the pharmacotherapy of SARDs is associated with the use of CAR (сhimeric antigen receptor) T cell therapy, developed for the treatment of refractory hematological tumors. The main component of CART-cells is a genetically engineered T-cell receptor that recognizes the target antigen without the participation of the major histocompatibility complex. Although limited, extremely impressive data regarding high remission rates have been obtained by adapting CD19 CART-cell therapy to treat patients with severe systemic lupus erythematosus (SLE) and other SARDs refractory to standard immunosuppressive medications. The article discusses the results of the use of CART-cell therapy in SLE and other SARDs and prospects for further research.

Abstract PO4-16-05: Identification of functional HLA-A*11:01-restricted driver gene PIK3CA mutation specific T-cell receptors

Abstract The adoptive transfer of genetically engineered T-cell receptors (TCR-T) has emerged as a promising therapeutic strategy for the treatment of solid tumors. Recent clinical trials have demonstrated the safety and efficacy of TCR-T cell therapies against certain types of metastatic solid cancers. In our study, we aimed to investigate the potential of TCR-T cell therapies targeting neoantigens, which are exclusively expressed in cancer cells, with a focus on metastatic breast cancer (MBC). Specifically, we identified four driver missense mutations (PIK3CA E542K, E545K, H1047L, and H1047R) frequently observed in MBC patients and utilized computational tools to predict the generation of neoantigens. Utilizing NetMHCpan V.4.1, we performed screening of peptides spanning the mutation region and predicted a neoantigen epitope, PIK3CA_H1047L, to bind with HLA-A*11:01. To validate the presentation and stability of the neoantigen epitope, we employed TAP1-deficient K562 cells and observed stable expression of the mutant peptide-major histocompatibility complex (MHC) complex on the cell membrane. Subsequently, we isolated T cells from healthy donors possessing the HLA-A11:01 genotype, and subjected them to successive stimulations with neopeptide-pulsed dendritic cells. T cells stimulated with neopeptide were examined for neoantigen specificity using an IFN-γ ELISpot assay. Employing pMHC tetramer staining and single-cell sorting via flow cytometry, we successfully isolated five HLA-A11:01-restricted PIK3CA_H1047L-specific TCRs. Through lentivirus transduction of these TCRs into T cells, we achieved the generation of TCR-T cells that exhibited potent and specific activity exclusively against the PIK3CA_H1047L neoantigen. The functionality of the TCR-T cells was confirmed through assessments of CD137 expression, IFN-γ secretion, and cytotoxicity assays. In conclusion, our study demonstrates T cell responses towards neoantigens derived from PIK3CA, a commonly observed driver mutation in MBC. Furthermore, we successfully isolated five distinct neoantigen-specific TCRs that specifically recognized the PIK3CA_H1047L mutation and were restricted to the prevalent HLA-A*11:01 allele. The transfer of these TCRs into T cells resulted in the generation of TCR-T products exhibiting remarkable activity against the PIK3CA_H1047L neoantigen. These findings strongly encourage further investigation into TCRs targeting driver mutations in MBC, with the aim of advancing neoantigen-targeted TCR-T therapies into clinical trials. Additionally, the PIK3CA_H1047L neoantigen holds great promise as a potential candidate for the development of effective cancer vaccines. Citation Format: Meiying Shen, Xiaojian Han, Siyin Chen, Aishun Jin, Shengchun Liu. Identification of functional HLA-A*11:01-restricted driver gene PIK3CA mutation specific T-cell receptors [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-16-05.

ScRNA-seq reveals novel immune-suppressive T cells and investigates CMV-TCR-T cells cytotoxicity against GBM

BackgroundGlioblastoma (GBM) is a fatal primary brain malignancy in adults. Previous studies have shown that cytomegalovirus (CMV) is a risk factor for tumorigenesis and aggressiveness for glioblastoma. However, little is...

Abstract 1514: A novel workflow to assess the T-cell and patient-derived organoid interaction

Abstract Immunotherapy is increasingly popular as a type of cancer treatment. These therapies include the use of Chimeric Antigen Receptor-engineered T-cells (CAR T-cells), tumor-infiltrating lymphocytes (TIL), and other genetically modified T-cells to specifically target the cancer cells. Although much success has been achieved with immunotherapy for treatment of blood cancers, its efficacy remains limited in solid tumors. One of the reasons for the low success rate is attributed to the solid tumor microenvironment (TME) where suppressive cytokines limit the tumor killing ability of T-cells. Thus, understanding the role the TME plays in T-cell responses is essential for the development of effective cancer therapies. The benefits of using 3-dimensional (3D) PDOs lie in the physical and chemical cues present within the TME that cannot be mimicked in traditional 2D monolayer cultures. Studies show that PDOs show similar responses to drugs as original tumors, suggesting the value of using PDOs to improve therapeutic outcomes. Thus, PDOs can provide more relevant physiological and pathological cancer models that recapitulate the basic features of primary tumors and is more suited for assessing the effectiveness of T-cell killing than 2D cell models. Despite the benefits associated with the use of PDOs, there are significant barriers to widespread adoption of PDOs in drug discovery. Organoid production is a costly and highly labor-intensive process. Moreover, organoid culture is a skilled manual process, and thus there can be significant variability between operators. To address the challenges associated with the use of PDOs in large scale applications, a semi-automated bioprocess has been developed for the large-scale expansion of assay ready organoids. Here, we developed a method to assess the effectiveness of T-cell invasion in solid tumors using patient derive organoids (PDOs). Using bioreactor expanded patient-derived colorectal cancer organoids (CRCs), activated PBMCs (human peripheral blood mononuclear cells) stained with CellTracker were added to CRCs (stained with MitoTracker) in a 96well microtiter plate and monitored on every 4 hours for 3 days using high content imager. To quantify T-cell invasion, we developed an image analysis method to measure the distance of each T-cell to the nearest organoid (interaction distance). We find that stimulated T-cell resulted in smaller interaction distance than non-stimulated T-cells. The results demonstrate the utility of the bioreactor-expanded organoids in large scale T-cell based screens. Citation Format: Zhisong Tong, Angeline Lim, Oksana Sirenko. A novel workflow to assess the T-cell and patient-derived organoid interaction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1514.

Cas9-induced targeted integration of large DNA payloads in primary human T cells via homology-mediated end-joining DNA repair.

The reliance on viral vectors for the production of genetically engineered immune cells for adoptive cellular therapies remains a translational bottleneck. Here we report a method leveraging the DNA repair pathway homology-mediated end joining, as well as optimized reagent composition and delivery, for the Cas9-induced targeted integration of large DNA payloads into primary human T cells with low toxicity and at efficiencies nearing those of viral vectors (targeted knock-in of 1-6.7 kb payloads at rates of up to 70% at multiple targeted genomic loci and with cell viabilities of over 80%). We used the method to produce T cells with an engineered T-cell receptor or a chimaeric antigen receptor and show that the cells maintained low levels of exhaustion markers and excellent capacities for proliferation and cytokine production and that they elicited potent antitumour cytotoxicity in vitro and in mice. The method is readily adaptable to current good manufacturing practices and scale-up processes, and hence may be used as an alternative to viral vectors for the production of genetically engineered T cells for cancer immunotherapies.

Assessment and comparison of viability assays for cellular products

Background aimsAccurate assessment of cell viability is crucial in cellular product manufacturing, yet selecting the appropriate viability assay presents challenges due to various factors. This study compares and evaluates different viability assays on fresh and cryopreserved cellular products, including peripheral blood stem cell (PBSC) and peripheral blood mononuclear cell (PBMC) apheresis products, purified PBMCs and cultured chimeric antigen receptor and T-cell receptor-engineered T-cell products. MethodsViability assays, including manual Trypan Blue exclusion, flow cytometry-based assays using 7-aminoactinomycin D (7-AAD) or propidium iodide (PI) direct staining or cell surface marker staining in conjunction with 7-AAD, Cellometer (Nexcelom Bioscience LLC, Lawrence, MA, USA) Acridine Orange/PI staining and Vi-CELL BLU Cell Viability Analyzer (Beckman Coulter, Inc, Brea, CA, USA), were evaluated. A viability standard was established using live and dead cell mixtures to assess the accuracy of these assays. Furthermore, precision assessment was conducted to determine the reproducibility of the viability assays. Additionally, the viability of individual cell populations from cryopreserved PBSC and PBMC apheresis products was examined. ResultsAll methods provided accurate viability measurements and generated consistent and reproducible viability data. The assessed viability assays were demonstrated to be reliable alternatives when evaluating the viability of fresh cellular products. However, cryopreserved products exhibited variability among the tested assays. Additionally, analyzing the viability of each subset of the cryopreserved PBSC and PBMC apheresis products revealed that T cells and granulocytes were more susceptible to the freeze–thaw process, showing decreased viability. ConclusionsThe study demonstrates the importance of careful assay selection, validation and standardization, particularly for assessing the viability of cryopreserved products. Given the complexity of cellular products, choosing a fit-for-purpose viability assay is essential.

Novel Cancer Immunotherapy Using Cell-Derived Membrane Vesicles Orchestrates Multimodal Antitumor Immunity

Cancer immunotherapy has emerged as a promising therapy for hematological malignancies. Although multiple immunotherapeutics are currently available in the clinic such as chimeric antigen receptor-engineered T-cell therapy (CAR-T cell therapy) and bispecific T-cell engager (BiTE), their long-term efficacy is still insufficient in most of the cases. Simultaneous inputs of multiple signals may provide more optimal T-cell activation and overcome the immunosuppressive tumor microenvironment (TME), which results in a durable therapeutic effect. However, integrating multifaceted immunomodulatory signals into a single drug or cell is technically difficult. To overcome these limitations, we developed nanosized cell-derived membrane vesicles (MVs) that orchestrate multimodal antigen-specific antitumor immunity in endogenous T cells. We isolated MVs with 100-150 nm in size and intact membrane-bound proteins from the HLA-negative leukemia cell line K562 by mechanical homogenization followed by isolation of the plasma membrane fraction using magnetic beads and ultracentrifugation. MVs can be loaded with a desired combination of immunomodulatory molecules on the surface by preparing cells stably transduced with those proteins. First, K562 cells were genetically engineered with a membrane-bound form of the anti-CD3/CD19 BiTE and homogenized to form BiTE-expressing MVs. When co-cultured with T cells and CD19-positive leukemia cell line NALM-6, MVs loaded with anti-CD3/CD19 BiTE induced potent cytotoxic activity in nonspecific T cells in a dose-dependent manner. To provide optimal T-cell activation, we further loaded MVs with a variety of immunostimulatory molecules including costimulatory ligands (CD80 and 4-1BBL) and cytokines (IL-7 and IL-15) on the surface. The generated antitumor MVs simultaneously induced multiple immunostimulatory signals in T cells, resulting in superior proliferation, cytokine production, and cytolytic functions compared with MVs loaded with BiTE alone. Intriguingly, MVs activated T cells only in the presence of target tumor cells, which is considered to provide a safety advantage in clinical settings. Changing the antitumor mAb sequences of the BiTE enabled to generate MVs against any type of antigen including B cell maturation antigen (BCMA), EGFR, and mesothelin. To test functions of our MVs in vivo, NSG mice were transplanted with NALM-6 and then infused with human peripheral blood T cells. While infusion of T cells alone did not contribute to controlling leukemia progression, infusion of anti-CD19 MVs with CD80, 4-1BBL, IL-7, and Il-15 induced potent antitumor response. Repeated infusion of MVs further augmented their therapeutic efficacy. To overcome the immunosuppressive TME, we additionally loaded MVs with immune checkpoint inhibitors (anti-PD-1 and anti-CTLA-4 antibody), TGF-b receptors to trap TGF-b, and inflammatory cytokines (IL-12 and IL-18) that activate both T cells and myeloid cells in the TME. Immunocompetent Balb/c mice were subcutaneously inoculated with colon carcinoma cell line CT26 ectopically expressed with CD19 and treated with MVs targeting CD19. The injected MVs converted tumor-associated M2-type macrophages into an inflammatory phenotype and promoted CD8+ T cell infiltration within the TME, resulting in better control of tumor progression in in vivo solid tumor mouse models. K562 cells are highly sensitive to NK cells because they endogenously express several ligands for NK cell activation receptors such as MIC-A and MIC-B, and lack the expression of HLA class I that represses NK cell activation. K562-derived MVs induced potent cytotoxic activity in NK cells against NK-cell-resistant myeloma cell line MM1S in an antigen-specific manner. In summary, this novel technology enables to deliver desired combinations of antitumor immune signals to not only T cells but also NK cells and other types of immune cells, which can transmit superior therapeutic efficacy against the tumor cells that are resistant to the present immunotherapeutics.

Evaluation of Central Nervous System Involvement in Patients undergoing Chimeric antigen receptor-engineered T-cell Therapy by Magnetic Resonance Imaging

Chimeric antigen receptor-engineered (CAR) T-cell therapy is an effective immunotherapy for aggressive hematologic cancers. However, it can lead to complications such as immune effector cell-associated neurotoxicity syndrome (ICANS), or complications related to the immunosuppressive status of these patients. The role of imaging in this context is essential to help in ICANS diagnosis and to rule out other potential diagnosis, such as central nervous system infections. Two cases are presented to illustrate this clinical problem. Case 1describes a 38-year-old patient with diffuse large B-cell lymphoma who developed ICANS after CAR T-cell therapy. MRI revealed signs of leukoencephalopathy. Case 2 involves a 57-year-old patient with mantle-cell lymphoma who presented neurologic symptoms -clinically suggestive of ICANS- after CAR-T therapy. MRI showed signs indicative of limbic encephalitis.These two cases highlight the importance of MRI in clinical practice after CAR T-Cell Therapy underscoring the role of MRI in the diagnosis of complications in patients with neuropsychiatric symptoms.

Creation of a High-Throughput Microfluidic Platform for Single-Cell Transcriptome Sequencing of Cell-Cell Interactions.

Cell-cell interaction is one of themajor modalities for transmitting information between cells and activating the effects of functional cells. However, the construction of high-throughput analysis technologies from cell omics focusing on the impact of interactions of functional cells on targets has been relatively unexplored. Here, they propose a droplet-based microfluidicplatform for cell-cell interaction sequencing (c-c-seq) and screening in vitro to address this challenge. A class of interacting cells is pre-labeled using cell molecular tags, and additional single-cell sequencing reagents are introduced to quickly form functional droplet mixes. Lastly, gene expression analysis is used to deduce the impact of the interaction, while molecular sequence tracing identifies the type of interaction. Research into the active effect between antigen-presenting cells and T cells, one of the most common cell-to-cell interactions, is crucial for the advancement of cancer therapy, particularly T cell receptor-engineered Tcell therapy. As it allows for high throughput, this platform is superior to well plates as a research platform for cell-to-cell interactions. When combined with the next generation of sequencing, the platform may be able to more accurately evaluate interactions between epitopes and receptors and verify their functional relevance.

Innovative Breakthroughs for the Treatment of Advanced and Metastatic Synovial Sarcoma.

Synovial sarcoma (SyS) is a rare aggressive soft tissue sarcoma carrying the chromosomal translocation t(X;18), encoding the fusion transcript SS18::SSX. The fusion oncoprotein interacts with both BAF enhancer complexes and polycomb repressor complexes, resulting in genome-wide epigenetic perturbations and a unique altered genetic signature. Over 80% of the patients are initially diagnosed with localized disease and have a 5-year survival rate of 70-80%, but metastatic relapse occurs in 50% of the cases. Advanced, unresectable, or metastatic disease has a 5-year survival rate below 10%, representing a critical issue. This review summarizes the molecular mechanisms behind SyS and illustrates current treatments in front line, second line, and beyond settings. We analyze the use of immune check point inhibitors (ICI) in SyS that do not behave as an ICI-sensitive tumor, claiming the need for predictive genetic signatures and tumor immune microenvironment biomarkers. We highlight the clinical translation of innovative technologies, such as proteolysis targeting chimera (PROTAC) protein degraders or adoptive transfer of engineered immune cells. Adoptive cell transfer of engineered T-cell receptor cells targeting selected cancer/testis antigens has shown promising results against metastatic SyS in early clinical trials and further improvements are awaited from refinements involving immune cell engineering and tumor immune microenvironment enhancement.

Adoptive Cell Therapy for Nonhematologic Solid Tumors.

The long-term benefits demonstrated by immunotherapy in select tumors have failed to generalize to most nonhematologic solid tumors. Adoptive cell therapy (ACT)-a treatment on the basis of the isolation and engineering of living T cells and other immune cells-has shown early clinical advances. ACT, through tumor-infiltrating lymphocyte therapy, has shown activity in traditionally immunogenic tumors such as melanoma and cervical cancers, and has the potential to improve immune reactivity in these tumor types where traditional therapies have failed. Engineered T-cell receptor and chimeric antigen receptor T-cell therapies have also shown activity in select nonhematologic solid tumors. Through receptor engineering, and improved understanding of tumor antigens, these therapies have the potential to target poorly immunogenic tumors to deliver long-lasting responses. Additionally, non-T-cell therapies such as natural killer-cell therapy may allow for allogeneic forms of ACT. Each form of ACT has trade-offs that will likely limit their application to specific clinical settings. Key challenges with ACT include the logistical challenges of manufacturing, accurate antigen identification, and the risk of on-target, off-tumor toxicity. The successes of ACT are built on decades of advances in cancer immunology, antigen identification, and cell engineering. With continued refinements in these processes, ACT may extend the benefits of immunotherapy to more patients with advanced nonhematologic solid tumors. Herein, we review the major forms of ACT, their successes, and strategies to overcome the trade-offs of current ACTs.

TCR sequencing and cloning methods for repertoire analysis and isolation of tumor-reactive TCRs

T cell receptor (TCR) technologies, including repertoire analyses and Tcell engineering, are increasingly important in the clinical management of cellular immunity in cancer, transplantation, and other immune diseases. However, sensitive and reliable methods for repertoire analyses and TCR cloning are still lacking. Here, we report on SEQTR, a high-throughput approach to analyze human and mouse repertoires that is more sensitive, reproducible, and accurate as compared with commonly used assays, and thus more reliably captures the complexity of blood and tumor TCR repertoires. We also present a TCR cloning strategy to specifically amplify TCRs from Tcell populations. Positioned downstream of single-cell or bulk TCR sequencing, it allows time- and cost-effective discovery, cloning, screening, and engineering of tumor-specific TCRs. Together, these methods will accelerate TCR repertoire analyses in discovery, translational, and clinical settings and permit fast TCR engineering for cellular therapies.

Engineering second-generation TCR-T cells by site-specific integration of TRAF-binding motifs into the CD247 locus

BackgroundThe incorporation of co-stimulatory signaling domains into second-generation chimeric antigen receptors (CARs) significantly enhances the proliferation and persistence of CAR-T cells in vivo, leading to successful clinical outcomes.MethodsTo achieve such...

Emerging targeted and cellular therapies in the treatment of advanced and metastatic synovial sarcoma.

Synovial sarcoma is a soft tissue sarcoma accounting for approximately 1,000 cases per year in the United States. Currently, standard treatment of advanced and metastatic synovial sarcoma is anthracycline-based chemotherapy. While advanced synovial sarcoma is more responsive to chemotherapy compared to other soft tissue sarcomas, survival rates are poor, with a median survival time of less than 18 months. Enhanced understanding of tumor antigen expression and molecular mechanisms behind synovial sarcoma provide potential targets for treatment. Adoptive Cell Transfer using engineered T-cell receptors is in clinical trials for treatment of synovial sarcoma, specifically targeting New York esophageal squamous cell carcinoma-1 (NY-ESO-1), preferentially expressed antigen in melanoma (PRAME), and melanoma antigen-A4 (MAGE-A4). In this review, we explore the opportunities and challenges of these treatments. We also describe artificial adjuvant vector cells (aAVCs) and BRD9 inhibitors, two additional potential targets for treatment of advanced synovial sarcoma. This review demonstrates the progress that has been made in treatment of synovial sarcoma and highlights the future study and qualification needed to implement these technologies as standard of care.

Global analysis of HLA-A2 restricted MAGE-A3 tumor antigen epitopes and corresponding TCRs in non-small cell lung cancer.

Background: Advanced non-small cell lung cancer (NSCLC) is the most common type of lung cancer with poor prognosis. Adoptive cell therapy using engineered T-cell receptors (TCRs) targeting cancer-testis antigens, such as Melanoma-associated antigen 3 (MAGE-A3), is a potential approach for the treatment of NSCLC. However, systematic analysis of T cell immune responses to MAGE-A3 antigen and corresponding antigen-specific TCR is still lacking. Methods: In this study, we comprehensively screened HLA-A2 restricted MAGE-A3 tumor epitopes and characterized the corresponding TCRs using in vitro artificial antigen presentation cells (APC) system, single-cell transcriptome and TCR V(D)J sequencing, and machine-learning. Furthermore, the tumor-reactive TCRs with killing potency was screened and verified. Results: We identified the HLA-A2 restricted T cell epitopes from MAGE-A3 that could effectively induce the activation and cytotoxicity of CD8+ T cells using artificial APC in vitro. A cohort of HLA-A2+ NSCLC donors demonstrated that the number of epitope specific CD8+ T cells increased in NSCLC than healthy controls when measured with tetramer derived from the candidate MAGE-A3 epitopes, especially epitope Mp4 (MAGE-A3: 160-169, LVFGIELMEV). Statistical and machine-learning based analyses demonstrated that the MAGE-A3-Mp4 epitope-specific CD8+ T cell clones were mostly in effector and proliferating state. Importantly, T cells artificially expressing the MAGE-A3-Mp4 specific TCRs exhibited strong MAGE-A3+ tumor cell recognition and killing effect. Cross-reactivity risk analysis of the candidates TCRs showed high binding stability to MAGE-A3-Mp4 epitope and low risk of cross-reaction. Conclusions: This work identified candidate TCRs potentially suitable for TCR-T design targeting HLA-A2 restricted MAGE-A3 tumor antigen.