Ruxolitinib for Refractory PL-12 Antisynthetase Syndrome-Associated Angioedema-Like Panniculitis With Clonal T-Cell Receptor Gene Rearrangement.

This case report describes a woman in her 30s who presented with a 3-year history of anti­PL-12 antisynthetase syndrome characterized by interstitial lung disease, arthritis, and myositis and was diagnosed with antisynthetase syndrome­associated panniculitis.

Safety and Efficacy of NY-ESO-1 Antigen-Specific T-Cell Receptor Gene-Transduced T Lymphocytes in Patients with Synovial Sarcoma: A Phase I/II Clinical Trial.

PURPOSE: To determine, for patients with advanced or recurrent synovial sarcoma (SS) not suitable for surgical resection and resistant to anthracycline, the safety and efficacy of the infusion of autologous T lymphocytes expressing NY-ESO-1 antigen-specific T-cell receptor (TCR) gene and siRNA to inhibit the expression of endogenous TCR (product code: TBI-1301). PATIENTS AND METHODS: Eligible Japanese patients (HLA-A*02:01 or *02:06, NY-ESO-1-positive tumor expression) received cyclophosphamide 750 mg/m2 on days -3 and -2 (induction period) followed by a single dose of 5×109 (±30%) TBI-1301 cells as a divided infusion on days 0 and 1 (treatment period). Primary endpoints were safety-related (phase I) and efficacy-related [objective response rate (ORR) by RECIST v1.1/immune-related RECIST (irRECIST); phase II]. Safety- and efficacy-related secondary endpoints were considered in both phase I/II parts. RESULTS: For the full analysis set (N = 8; phase I, n = 3; phase II, n = 5), the ORR was 50.0% (95% confidence interval, 15.7-84.3) with best overall partial response in four of eight patients according to RECIST v1.1/irRECIST. All patients experienced adverse events and seven of eight patients (87.5%) had adverse drug reactions, but no deaths were attributed to adverse events. Cytokine release syndrome occurred in four of eight patients (50.0%), but all cases recovered with prespecified treatment. Immune effector cell-associated neurotoxicity syndrome, replication-competent retrovirus, and lymphocyte clonality were absent. CONCLUSIONS: Adoptive immunotherapy with TBI-1301 to selectively target NY-ESO-1-positive tumor cells appears to be a promising strategy for the treatment of advanced or recurrent SS with acceptable toxicity.

T cell receptor gene-modified allogeneic T cells with siRNA for endogenous T cell receptor induce efficient tumor regression without graft-versus-host disease.

Adoptive immunotherapy using genetically engineered patient-derived lymphocytes to express tumor-reactive receptors is a promising treatment for malignancy. However, utilization of autologous T cells in this therapy limits the quality of gene-engineered T cells, thereby inhibiting the timely infusion of the cells into patients. In this study, we evaluated the anti-tumor efficacy and the potential to induce graft-versus-host disease (GVHD) in T cell receptor (TCR) gene-engineered allogeneic T cells that downregulate the endogenous TCR and HLA class I molecules with the aim of developing an "off-the-shelf" cell product with expanded application of genetically engineered T cells. We transduced human lymphocytes with a high-affinity TCR specific to the cancer/testis antigen NY-ESO-1 using a novel retrovirus vector with siRNAs specific to the endogenous TCR (siTCR vector). These T cells showed reduced expression of endogenous TCR and minimized reactivity to allogeneic cells in vitro. In non-obese diabetic/SCID/γcnull mice, TCR gene-transduced T cells induced tumor regression without development of GVHD. A lentivirus-based CRISPR/Cas9 system targeting ß-2 microglobulin in TCR gene-modified T cells silenced the HLA class I expression and prevented allogeneic CD8+ T cell stimulation without disrupting their anti-tumor capacity. This report is the first demonstration that siTCR technology is effective in preventing GVHD. Adoptive cell therapy with allogeneic T cells engineered with siTCR vector may be useful in developing an "off-the-shelf" therapy for patients with malignancy.

Modifications outside CDR1, 2 and 3 of the TCR variable ß domain increase TCR expression and antigen-specific function.

T cell receptor (TCR) gene modified T cells are a promising form of adoptive cellular therapy against human malignancies and viral infections. Since the first human clinical trial was carried out in 2006, several strategies have been developed to improve the efficacy and safety of TCR engineered T cells by enhancing the surface expression of the introduced therapeutic TCRs whilst reducing the mis-pairing with endogenous TCR chains. In this study, we explored how modifications of framework residues in the TCR variable domains affect TCR expression and function. We used bioinformatic and protein structural analyses to identify candidate amino acid residues in the framework of the variable ß domain predicted to drive high TCR surface expression. Changes of these residues in poorly expressed TCRs resulted in improved surface expression and boosted target cell specific killing by engineered T cells expressing the modified TCRs. Overall, these results indicate that small changes in the framework of the TCR variable domains can result in improved expression and functionality, while at the same time reducing the risk of toxicity associated with TCR mis-pairing.

[Development of "Off the Shelf" T Cells for Cancer Immunotherapy].

In the area of cancer immunotherapy, the efficacy of strategies in which patient derived T cells are genetically modified ex vivo and administered to patients has been demonstrated. However, some issues have remained to be addressed; the method using autologous T cells is costly and time consuming, and their quality is unstable. The time consuming problem can be solved by preparing allogeneic T cells in advance. Peripheral blood is being considered as a source of allogeneic T cells, and methods are being explored to avoid the risk of rejection or GVHD, but even so the issues of cost and quality stability still remain. On the other hand, use of pluripotent stem cells such as iPS cells or ES cells as material of T cells may solve the cost issue and achieve homogeneity of products. The authors group has been developing a method to generate T cells from iPS cells transduced with a certain T cell receptor gene, and is currently preparing for clinical trials. We believe that, when this strategy is realized, it becomes possible to deliver a universal and homogeneous T cell preparation immediately when needed.

Archaic humans have contributed to large-scale variation in modern human T cell receptor genes.

Human T cell receptors (TCRs) are critical for mediating immune responses to pathogens and tumors and regulating self-antigen recognition. Yet, variations in the genes encoding TCRs remain insufficiently defined. Detailed analysis of expressed TCR alpha, beta, gamma, and delta genes in 45 donors from four human populations-African, East Asian, South Asian, and European-revealed 175 additional TCR variable and junctional alleles. Most of these contained coding changes and were present at widely differing frequencies in the populations, a finding confirmed using DNA samples from the 1000 Genomes Project. Importantly, we identified three Neanderthal-derived, introgressed TCR regions including a highly divergent TRGV4 variant, which mediated altered butyrophilin-like molecule 3 (BTNL3) ligand reactivity and was frequent in all modern Eurasian population groups. Our results demonstrate remarkable variation in TCR genes in both individuals and populations, providing a strong incentive for including allelic variation in studies of TCR function in human biology.

Analysis of genomic and immune intratumor heterogeneity in linitis plastica via multiregional exome and T-cell receptor sequencing.

The molecular landscape and the intratumor heterogeneity (ITH) architecture of gastric linitis plastica (LP) are poorly understood. We performed whole-exome sequencing (WES) and T-cell receptor (TCR) sequencing on 40 tumor regions from four LP patients. The landscape and ITH at the genomic and immunological levels in LP tumors were compared with multiple cancers that have previously been reported. The lymphocyte infiltration was further assessed by immunohistochemistry (IHC) in LP tumors. In total, we identified 6339 non-silent mutations from multi-samples, with a median tumor mutation burden (TMB) of 3.30 mutations per Mb, comparable to gastric adenocarcinoma from the Cancer Genome Atlas (TCGA) cohort (P = 0.53). An extremely high level of genomic ITH was observed, with only 12.42%, 5.37%, 5.35%, and 30.67% of mutations detectable across 10 regions within the same tumors of each patient, respectively. TCR sequencing revealed that TCR clonality was substantially lower in LP than in multi-cancers. IHC using antibodies against CD4, CD8, and PD-L1 demonstrated scant T-cell infiltration in the four LP tumors. Furthermore, profound TCR ITH was observed in all LP tumors, with no T-cell clones shared across tumor regions in any of the patients, while over 94% of T-cell clones were restricted to individual tumor regions. The Morisita overlap index (MOI) ranged from 0.21 to 0.66 among multi-regions within the same tumors, significantly lower than that of lung cancer (P = 0.002). Our results show that LP harbored extremely high genomic and TCR ITH and suppressed T-cell infiltration, suggesting a potential contribution to the frequent recurrence and poor therapeutic response of this adenocarcinoma.

TCR gene segment usage and HLA alleles that are associated with cancer survival rates also represent racial disparities.

Understanding racial disparities in cancer outcomes continues to be a challenge, with likely many factors at play, including socioeconomic factors and genetic polymorphisms impacting basic cellular and molecular functions. Additionally, it is possible that specific combinations of environment and genetics have specific impacts. T-cell receptor (TCR) gene segment usage, HLA allele combinations have been associated with autoimmune and infectious disease courses, and more recently, TCR gene segment usage, HLA allele combinations have been associated with distinct survival outcomes in cancer as well. We examined several such, previously reported cancer-related TCR gene segment usage, HLA allele combinations for evidence of racial disparities, with regard to the prevalence of the combination in different racial groups. Results indicated that TCR gene segment usage, potentially reflecting environmental factors related to previous pathogen exposure, in combination with certain HLA alleles or independently, may represent a novel explanation for racial disparities in cancer outcomes. Overall, at this point, a genetic connection to racial disparities in cancer outcomes is detectable but remains modest, suggesting that other factors, such as socioeconomic factors, remain as important considerations.

T-Cell Repertoire Characterization.

T-cell repertoire characterization is a methodology that enables the identification of T-cell receptor (TCR) gene sequences in a T-cell population. TCR genes are composed of modular gene segments V (D) J that undergo somatic recombination, resulting in unique and unpredictable sequences that can be utilized to identify each T-cell clone. The analysis of the TCR composition in a T-cell population can give information on the biological phenomenon such as antigen-driven expansion and heterogeneity of T-cell responses. Bulk TCR analysis can give useful information on the clonality and can help track a specific clonotype over time or in different compartments, although the information about pairing cannot be provided. Single-cell TCR sequencing, on the other hand, can provide pairing information that are necessary to reconstruct the TCR and confirm antigen specificity.Here we describe common methods to characterize T-cell repertoires based on both bulk and single-cell next-generation sequencing.

A High-Throughput Strategy for T-Cell Receptor Cloning and Expression.

Expression of T-cell receptor (TCR) genes is a critical step for TCR characterization and epitope identification. The recent interest in using specific TCRs for cancer immunotherapy has further increased the demand for practical and robust methods to rapidly clone and express TCRs. We show that a recombination-based cloning protocol facilitates simple and rapid transfer of the TCR transgene into different expression systems. In this protocol, we first constructed all the human TRAV and TRBV genes into individual plasmid. To clone any TCR, we only need to ligate a short CDR3 fragment to its corresponding V gene plasmid using Golden Gate cloning. This strategy significantly improves the efficiency of individual TCR cloning and mutagenesis, providing a flexible high-throughput method for TCR analysis and TCR-mediated therapeutics.

Adoptive Cellular Therapy with Autologous Tumor-Infiltrating Lymphocytes and T-cell Receptor-Engineered T Cells Targeting Common p53 Neoantigens in Human Solid Tumors.

Adoptive cellular therapy (ACT) targeting neoantigens can achieve durable clinical responses in patients with cancer. Most neoantigens arise from patient-specific mutations, requiring highly individualized treatments. To broaden the applicability of ACT targeting neoantigens, we focused on TP53 mutations commonly shared across different cancer types. We performed whole-exome sequencing on 163 patients with metastatic solid cancers, identified 78 who had TP53 missense mutations, and through immunologic screening, identified 21 unique T-cell reactivities. Here, we report a library of 39 T-cell receptors (TCR) targeting TP53 mutations shared among 7.3% of patients with solid tumors. These TCRs recognized tumor cells in a TP53 mutation- and human leucocyte antigen (HLA)-specific manner in vitro and in vivo. Twelve patients with chemorefractory epithelial cancers were treated with ex vivo-expanded autologous tumor-infiltrating lymphocytes (TIL) that were naturally reactive against TP53 mutations. However, limited clinical responses (2 partial responses among 12 patients) were seen. These infusions contained low frequencies of mutant p53-reactive TILs that had exhausted phenotypes and showed poor persistence. We also treated one patient who had chemorefractory breast cancer with ACT comprising autologous peripheral blood lymphocytes transduced with an allogeneic HLA-A*02-restricted TCR specific for p53R175H. The infused cells exhibited an improved immunophenotype and prolonged persistence compared with TIL ACT and the patient experienced an objective tumor regression (-55%) that lasted 6 months. Collectively, these proof-of-concept data suggest that the library of TCRs targeting shared p53 neoantigens should be further evaluated for the treatment of patients with advanced human cancers. See related Spotlight by Klebanoff, p. 919.

T-cell Receptor Gene Therapy Clinically Targeting a TP53 Public Neoantigen.

T-cell receptors (TCR) are an antigen receptor class that can uniquely respond to epitopes resulting from cytosolic and intranuclear proteins. In this issue, Kim and colleagues report the first successful application of TCR gene therapy targeting a shared, or public, neoantigen resulting from a TP53 hotspot mutation. These results establish clinical proof of concept that an off-the-shelf TCR targeting a recurrent mutation in a molecular driver of oncogenesis can benefit patients with metastatic cancer. See related article by Kim et al., p. 932 (4) .

Autoreactive CD8 T cells in NOD mice exhibit phenotypic heterogeneity but restricted TCR gene usage.

Type 1 diabetes (T1D) is an autoimmune disorder defined by CD8 T cell-mediated destruction of pancreatic ß cells. We have previously shown that diabetogenic CD8 T cells in the islets of non-obese diabetic mice are phenotypically heterogeneous, but clonal heterogeneity remains relatively unexplored. Here, we use paired single-cell RNA and T-cell receptor sequencing (scRNA-seq and scTCR-seq) to characterize autoreactive CD8 T cells from the islets and spleens of non-obese diabetic mice. scTCR-seq demonstrates that CD8 T cells targeting the immunodominant ß-cell epitope IGRP206-214 exhibit restricted TCR gene usage. scRNA-seq identifies six clusters of autoreactive CD8 T cells in the islets and six in the spleen, including memory and exhausted cells. Clonal overlap between IGRP206-214-reactive CD8 T cells in the islets and spleen suggests these cells may circulate between the islets and periphery. Finally, we identify correlations between TCR genes and T-cell clonal expansion and effector fate. Collectively, our work demonstrates that IGRP206-214-specific CD8 T cells are phenotypically heterogeneous but clonally restricted, raising the possibility of selectively targeting these TCR structures for therapeutic benefit.

Neoantigen T-Cell Receptor Gene Therapy in Pancreatic Cancer.

A patient with progressive metastatic pancreatic cancer was treated with a single infusion of 16.2×109 autologous T cells that had been genetically engineered to clonally express two allogeneic HLA-C*08:02-restricted T-cell receptors (TCRs) targeting mutant KRAS G12D expressed by the tumors. The patient had regression of visceral metastases (overall partial response of 72% according to the Response Evaluation Criteria in Solid Tumors, version 1.1); the response was ongoing at 6 months. The engineered T cells constituted more than 2% of all the circulating peripheral-blood T cells 6 months after the cell transfer. In this patient, TCR gene therapy targeting the KRAS G12D driver mutation mediated the objective regression of metastatic pancreatic cancer. (Funded by the Providence Portland Medical Foundation.).

One-Step Next-Generation Sequencing of Immunoglobulin and T-Cell Receptor Gene Recombinations for MRD Marker Identification in Acute Lymphoblastic Leukemia.

Within the EuroClonality-NGS group, immune repertoire analysis for target identification in lymphoid malignancies was initially developed using two-stage amplicon approaches, essentially as a progressive modification of preceding methods developed for Sanger sequencing. This approach has, however, limitations with respect to sample handling, adaptation to automation, and risk of contamination by amplicon products. We therefore developed one-step PCR amplicon methods with individual barcoding for batched analysis for IGH, IGK, TRD, TRG, and TRB rearrangements, followed by Vidjil-based data analysis.

Immunoglobulin/T-Cell Receptor Gene Rearrangement Analysis Using RNA-Seq.

Identification of immunoglobulin (IG) and T-cell receptor (TR) gene rearrangements in acute lymphoblastic leukemia (ALL) patients at initial presentation are crucial for monitoring of minimal residual disease (MRD) during subsequent follow-up and thereby for appropriate risk-group stratification. Here we describe how RNA-Seq data can be generated and subsequently analyzed with ARResT/Interrogate to identify possible MRD markers. In addition to the procedures, possible pitfalls will be discussed. Similar strategies can be employed for other lymphoid malignancies, such as lymphoma and myeloma.

Minimal Residual Disease Analysis by Monitoring Immunoglobulin and T-Cell Receptor Gene Rearrangements by Quantitative PCR and Droplet Digital PCR.

Analysis of immunoglobulin and T-cell receptor gene rearrangements by real-time quantitative polymerase chain reaction (RQ-PCR) is the gold standard for sensitive and accurate minimal residual disease (MRD) monitoring; it has been extensively standardized and guidelines have been developed within the EuroMRD consortium ( www.euromrd.org ). However, new generations of PCR-based methods are standing out as potential alternatives to RQ-PCR, such as digital PCR technology (dPCR), the third-generation implementation of conventional PCR, which has the potential to overcome some of the limitations of RQ-PCR such as allowing the absolute quantification of nucleic acid targets without the need for a calibration curve. During the last years, droplet digital PCR (ddPCR) technology has been compared to RQ-PCR in several hematologic malignancies showing its proficiency for MRD analysis. So far, no established guidelines for ddPCR MRD analysis and data interpretation have been defined and its potential is still under investigation. However, a major standardization effort is underway within the EuroMRD consortium ( www.euromrd.org ) for future application of ddPCR in standard clinical practice.

Experiencia en el uso de los protocolos Biomed-2 para el estudio de reordenamientos de TCR e inmunoglobulinas en proliferaciones linfoides en el Instituto Nacional de Cancerología, Colombia / Experience with the BIOMED-2 standardized polymerase chain reaction protocol for immunoglobulin and T- cell receptor clonality analysis in the Instituto Nacional de Cancerología, Colombia

Introducción. El consorcio europeo BIOMED-2 se creó para determinar si una población linfoide de difícil clasificación patológica es clonal. En Colombia, la implementación de estas pruebas comenzó en el 2015 en el Instituto Nacional de Cancerología E.S.E. (Bogotá). Objetivos. Determinar el comportamiento de las pruebas de reordenamiento clonal o clonalidad linfoide. y determinar las dificultades de su uso en nuestro medio verificando su adaptación local y los resultados en una serie retrospectiva de casos y consecutiva de proliferaciones linfoides sometidas a los protocolos BIOMED-2. Materiales y métodos. A partir de las historias clínicas, se recolectaron los datos clínicos e histológicos y los resultados de los análisis de los reordenamientos en todos los casos de proliferaciones linfoides sometidas a los protocolos BIOMED-2, entre febrero de 2015 y mayo de 2019. Resultados. Se hallaron 132 casos, de los cuales 47 se clasificaron mediante los protocolos de Biomed-2 como hiperplasias linfoides reactivas, 62 como linfomas T, 19 como linfomas B y 3 como neoplasias linfoides de linaje no establecido. Solo en un caso falló la extracción de ADN. Según estos resultados, la mayor dificultad diagnóstica para el patólogo fue el análisis de los infiltrados linfoides T, la mayoría (44) de los cuales correspondía a lesiones cutáneas. Conclusiones. Las pruebas de clonalidad pueden usarse en tejidos de diversa calidad en nuestro medio como ayuda en el diagnóstico de proliferaciones linfoides de difícil clasificación. Es importante hacerlas e interpretarlas de manera multidisciplinaria y considerar cada caso por separado.

Introduction: The European BIOMED-2 consortium was created to evaluate clonality in lymphoproliferations that are difficult to diagnose. In Colombia, the implementation of these tests began in 2015 at the Instituto Nacional de Cancerología E.S.E., Bogotá. Objectives: To determine the behavior of the rearrangement tests for lymphoid clonality and the difficulties of its implementation in our field through a series of retrospective and consecutive cases of lymphoid proliferation subjected to the BIOMED-2 protocols. Materials and methods: Clinical and histological data and the results of the rearrangement analysis of all cases of lymphoid proliferation subjected to the BIOMED-2 protocols between February 2015 and May 2019 were collected from clinical histories. Results: We recovered 132 samples from which 47 corresponded to reactive lymphoid hyperplasias, 62 to T lymphomas, 19 to B lymphomas, and three to lymphoid neoplasms of unestablished lineage. Only in one case did DNA extraction fail. According to these results, the greatest diagnostic difficulty for the pathologist was the analysis of T lymphoid infiltrates, most of which (44) were skin lesions. Conclusions: Clonality tests can be used in tissues of different quality to help in the diagnosis of lymphoid proliferations that are difficult to classify. It is important to implement and interpret them in an interdisciplinary way considering each case separately.