Antigen Identification for Orphan T Cell Receptors Expressed on Tumor-Infiltrating Lymphocytes.

The immune system can mount T cell responses against tumors; however, the antigen specificities of tumor-infiltrating lymphocytes (TILs) are not well understood. We used yeast-display libraries of peptide-human leukocyte antigen (pHLA) to screen for antigens of "orphan" T cell receptors (TCRs) expressed on TILs from human colorectal adenocarcinoma. Four TIL-derived TCRs exhibited strong selection for peptides presented in a highly diverse pHLA-A∗02:01 library. Three of the TIL TCRs were specific for non-mutated self-antigens, two of which were present in separate patient tumors, and shared specificity for a non-mutated self-antigen derived from U2AF2. These results show that the exposed recognition surface of MHC-bound peptides accessible to the TCR contains sufficient structural information to enable the reconstruction of sequences of peptide targets for pathogenic TCRs of unknown specificity. This finding underscores the surprising specificity of TCRs for their cognate antigens and enables the facile indentification of tumor antigens through unbiased screening.

Non-viral precision T cell receptor replacement for personalized cell therapy.

T cell receptors (TCRs) enable T cells to specifically recognize mutations in cancer cells1-3. Here we developed a clinical-grade approach based on CRISPR-Cas9 non-viral precision genome-editing to simultaneously knockout the two endogenous TCR genes TRAC (which encodes TCRα) and TRBC (which encodes TCRß). We also inserted into the TRAC locus two chains of a neoantigen-specific TCR (neoTCR) isolated from circulating T cells of patients. The neoTCRs were isolated using a personalized library of soluble predicted neoantigen-HLA capture reagents. Sixteen patients with different refractory solid cancers received up to three distinct neoTCR transgenic cell products. Each product expressed a patient-specific neoTCR and was administered in a cell-dose-escalation, first-in-human phase I clinical trial ( NCT03970382 ). One patient had grade 1 cytokine release syndrome and one patient had grade 3 encephalitis. All participants had the expected side effects from the lymphodepleting chemotherapy. Five patients had stable disease and the other eleven had disease progression as the best response on the therapy. neoTCR transgenic T cells were detected in tumour biopsy samples after infusion at frequencies higher than the native TCRs before infusion. This study demonstrates the feasibility of isolating and cloning multiple TCRs that recognize mutational neoantigens. Moreover, simultaneous knockout of the endogenous TCR and knock-in of neoTCRs using single-step, non-viral precision genome-editing are achieved. The manufacture of neoTCR engineered T cells at clinical grade, the safety of infusing up to three gene-edited neoTCR T cell products and the ability of the transgenic T cells to traffic to the tumours of patients are also demonstrated.

Neoantigen-targeted CD8+ T cell responses with PD-1 blockade therapy.

Neoantigens are peptides derived from non-synonymous mutations presented by human leukocyte antigens (HLAs), which are recognized by antitumour T cells1-14. The large HLA allele diversity and limiting clinical samples have restricted the study of the landscape of neoantigen-targeted T cell responses in patients over their treatment course. Here we applied recently developed technologies15-17 to capture neoantigen-specific T cells from blood and tumours from patients with metastatic melanoma with or without response to anti-programmed death receptor 1 (PD-1) immunotherapy. We generated personalized libraries of neoantigen-HLA capture reagents to single-cell isolate the T cells and clone their T cell receptors (neoTCRs). Multiple T cells with different neoTCR sequences (T cell clonotypes) recognized a limited number of mutations in samples from seven patients with long-lasting clinical responses. These neoTCR clonotypes were recurrently detected over time in the blood and tumour. Samples from four patients with no response to anti-PD-1 also demonstrated neoantigen-specific T cell responses in the blood and tumour to a restricted number of mutations with lower TCR polyclonality and were not recurrently detected in sequential samples. Reconstitution of the neoTCRs in donor T cells using non-viral CRISPR-Cas9 gene editing demonstrated specific recognition and cytotoxicity to patient-matched melanoma cell lines. Thus, effective anti-PD-1 immunotherapy is associated with the presence of polyclonal CD8+ T cells in the tumour and blood specific for a limited number of immunodominant mutations, which are recurrently recognized over time.

T cell antigen discovery via signaling and antigen-presenting bifunctional receptors.

CD8+ T cells recognize and eliminate tumors in an antigen-specific manner. Despite progress in characterizing the antitumor T cell repertoire and function, the identification of target antigens remains a challenge. Here we describe the use of chimeric receptors called signaling and antigen-presenting bifunctional receptors (SABRs) in a cell-based platform for T cell receptor (TCR) antigen discovery. SABRs present an extracellular complex comprising a peptide and major histocompatibility complex (MHC), and induce intracellular signaling via a TCR-like signal after binding with a cognate TCR. We devised a strategy for antigen discovery using SABR libraries to screen thousands of antigenic epitopes. We validated this platform by identifying the targets recognized by public TCRs of known specificities. Moreover, we extended this approach for personalized neoantigen discovery.

T cell antigen discovery via trogocytosis.

T cell receptor (TCR) ligand discovery is essential for understanding and manipulating immune responses to tumors. We developed a cell-based selection platform for TCR ligand discovery that exploits a membrane transfer phenomenon called trogocytosis. We discovered that T cell membrane proteins are transferred specifically to target cells that present cognate peptide-major histocompatibility complex (MHC) molecules. Co-incubation of T cells expressing an orphan TCR with target cells collectively presenting a library of peptide-MHCs led to specific labeling of cognate target cells, enabling isolation of these target cells and sequencing of the cognate TCR ligand. We validated this method for two clinically employed TCRs and further used the platform to identify the cognate neoepitope for a subject-derived neoantigen-specific TCR. Thus, target cell trogocytosis is a robust tool for TCR ligand discovery that will be useful for studying basic tumor immunology and identifying new targets for immunotherapy.

Isolation and characterization of NY-ESO-1-specific T cell receptors restricted on various MHC molecules.

Tumor-specific T cell receptor (TCR) gene transfer enables specific and potent immune targeting of tumor antigens. Due to the prevalence of the HLA-A2 MHC class I supertype in most human populations, the majority of TCR gene therapy trials targeting public antigens have employed HLA-A2-restricted TCRs, limiting this approach to those patients expressing this allele. For these patients, TCR gene therapy trials have resulted in both tantalizing successes and lethal adverse events, underscoring the need for careful selection of antigenic targets. Broad and safe application of public antigen-targeted TCR gene therapies will require (i) selecting public antigens that are highly tumor-specific and (ii) targeting multiple epitopes derived from these antigens by obtaining an assortment of TCRs restricted by multiple common MHC alleles. The canonical cancer-testis antigen, NY-ESO-1, is not expressed in normal tissues but is aberrantly expressed across a broad array of cancer types. It has also been targeted with A2-restricted TCR gene therapy without adverse events or notable side effects. To enable the targeting of NY-ESO-1 in a broader array of HLA haplotypes, we isolated TCRs specific for NY-ESO-1 epitopes presented by four MHC molecules: HLA-A2, -B07, -B18, and -C03. Using these TCRs, we pilot an approach to extend TCR gene therapies targeting NY-ESO-1 to patient populations beyond those expressing HLA-A2.

T cell receptors for the HIV KK10 epitope from patients with differential immunologic control are functionally indistinguishable.

HIV controllers (HCs) are individuals who can naturally control HIV infection, partially due to potent HIV-specific CD8+ T cell responses. Here, we examined the hypothesis that superior function of CD8+ T cells from HCs is encoded by their T cell receptors (TCRs). We compared the functional properties of immunodominant HIV-specific TCRs obtained from HLA-B*2705 HCs and chronic progressors (CPs) following expression in primary T cells. T cells transduced with TCRs from HCs and CPs showed equivalent induction of epitope-specific cytotoxicity, cytokine secretion, and antigen-binding properties. Transduced T cells comparably, albeit modestly, also suppressed HIV infection in vitro and in humanized mice. We also performed extensive molecular dynamics simulations that provided a structural basis for similarities in cytotoxicity and epitope cross-reactivity. These results demonstrate that the differential abilities of HIV-specific CD8+ T cells from HCs and CPs are not genetically encoded in the TCRs alone and must depend on additional factors.

Generation of mature T cells from human hematopoietic stem and progenitor cells in artificial thymic organoids.

Studies of human T cell development require robust model systems that recapitulate the full span of thymopoiesis, from hematopoietic stem and progenitor cells (HSPCs) through to mature T cells. Existing in vitro models induce T cell commitment from human HSPCs; however, differentiation into mature CD3+TCR-αß+ single-positive CD8+ or CD4+ cells is limited. We describe here a serum-free, artificial thymic organoid (ATO) system that supports efficient and reproducible in vitro differentiation and positive selection of conventional human T cells from all sources of HSPCs. ATO-derived T cells exhibited mature naive phenotypes, a diverse T cell receptor (TCR) repertoire and TCR-dependent function. ATOs initiated with TCR-engineered HSPCs produced T cells with antigen-specific cytotoxicity and near-complete lack of endogenous TCR Vß expression, consistent with allelic exclusion of Vß-encoding loci. ATOs provide a robust tool for studying human T cell differentiation and for the future development of stem-cell-based engineered T cell therapies.

Parallels between pathogens and gluten peptides in celiac sprue.

Pathogens are exogenous agents capable of causing disease in susceptible organisms. In celiac sprue, a disease triggered by partially hydrolyzed gluten peptides in the small intestine, the offending immunotoxins cannot replicate, but otherwise have many hallmarks of classical pathogens. First, dietary gluten and its peptide metabolites are ubiquitous components of the modern diet, yet only a small, genetically susceptible fraction of the human population contracts celiac sprue. Second, immunotoxic gluten peptides have certain unusual structural features that allow them to survive the harsh proteolytic conditions of the gastrointestinal tract and thereby interact extensively with the mucosal lining of the small intestine. Third, they invade across epithelial barriers intact to access the underlying gut-associated lymphoid tissue. Fourth, they possess recognition sequences for selective modification by an endogenous enzyme, transglutaminase 2, allowing for in situ activation to a more immunotoxic form via host subversion. Fifth, they precipitate a T cell-mediated immune reaction comprising both innate and adaptive responses that causes chronic inflammation of the small intestine. Sixth, complete elimination of immunotoxic gluten peptides from the celiac diet results in remission, whereas reintroduction of gluten in the diet causes relapse. Therefore, in analogy with antibiotics, orally administered proteases that reduce the host's exposure to the immunotoxin by accelerating gluten peptide destruction have considerable therapeutic potential. Last but not least, notwithstanding the power of in vitro methods to reconstitute the essence of the immune response to gluten in a celiac patient, animal models for the disease, while elusive, are likely to yield fundamentally new systems-level insights.

Interferon-gamma released by gluten-stimulated celiac disease-specific intestinal T cells enhances the transepithelial flux of gluten peptides.

Celiac sprue is a T-cell-mediated enteropathy elicited in genetically susceptible individuals by dietary gluten proteins. To initiate and propagate inflammation, proteolytically resistant gluten peptides must be translocated across the small intestinal epithelium and presented to DQ2-restricted T cells, but the effectors enabling this translocation under normal and inflammatory conditions are not well understood. We demonstrate that a fluorescently labeled antigenic 33-mer gluten peptide is translocated intact across a T84 cultured epithelial cell monolayer and that preincubation of the monolayer with media from gluten-stimulated, celiac patient-derived intestinal T cells enhances the apical-to-basolateral flux of this peptide in a dose-dependent, saturable manner. The permeability-enhancing activity of activated T-cell media is inhibited by blocking antibodies against either interferon-gamma or its receptor and is recapitulated using recombinant interferon-gamma. At saturating levels of interferon-gamma, activated T-cell media does not further increase transepithelial peptide flux, indicating the primacy of interferon-gamma as an effector of increased epithelial permeability during inflammation. Reducing the assay temperature to 4 degrees C reverses the effect of interferon-gamma but does not reduce basal peptide flux occurring in the absence of interferon-gamma, suggesting active transcellular transport of intact peptides is increased during inflammation. A panel of disease-relevant gluten peptides exhibited an inverse correlation between size and transepithelial flux but no apparent sequence constraints. Anti-interferon-gamma therapy may mitigate the vicious cycle of gluten-induced interferon-gamma secretion and interferon-gamma-mediated enhancement of gluten peptide flux but is unlikely to prevent translocation of gluten peptides in the absence of inflammatory conditions.

MATE-Seq: microfluidic antigen-TCR engagement sequencing.

Adaptive immunity is based on peptide antigen recognition. Our ability to harness the immune system for therapeutic gain relies on the discovery of the T cell receptor (TCR) genes that selectively target antigens from infections, mutated proteins, and foreign agents. Here we present a method that selectively labels peptide antigen-specific CD8+ T cells using magnetic nanoparticles functionalized with peptide-MHC tetramers, isolates these specific cells within an integrated microfluidic device, and directly amplifies the TCR genes for sequencing. Critically, the identity of the peptide recognized by the TCR is preserved, providing the link between peptide and gene. The platform requires inputs on the order of just 100 000 CD8+ T cells, can be multiplexed for simultaneous analysis of multiple peptides, and performs sorting and isolation on chip. We demonstrate 1000-fold sensitivity enhancement of detecting antigen-specific TCRs relative to bulk analysis and simultaneous capture of two virus antigen-specific TCRs from a population of T cells.

Sensitive Detection and Analysis of Neoantigen-Specific T Cell Populations from Tumors and Blood.

Neoantigen-specific T cells are increasingly viewed as important immunotherapy effectors, but physically isolating these rare cell populations is challenging. Here, we describe a sensitive method for the enumeration and isolation of neoantigen-specific CD8+ T cells from small samples of patient tumor or blood. The method relies on magnetic nanoparticles that present neoantigen-loaded major histocompatibility complex (MHC) tetramers at high avidity by barcoded DNA linkers. The magnetic particles provide a convenient handle to isolate the desired cell populations, and the barcoded DNA enables multiplexed analysis. The method exhibits superior recovery of antigen-specific T cell populations relative to literature approaches. We applied the method to profile neoantigen-specific T cell populations in the tumor and blood of patients with metastatic melanoma over the course of anti-PD1 checkpoint inhibitor therapy. We show that the method has value for monitoring clinical responses to cancer immunotherapy and might help guide the development of personalized mutational neoantigen-specific T cell therapies and cancer vaccines.

A kinetic investigation of interacting, stimulated T cells identifies conditions for rapid functional enhancement, minimal phenotype differentiation, and improved adoptive cell transfer tumor eradication.

For adoptive cell transfer (ACT) immunotherapy of tumor-reactive T cells, an effective therapeutic outcome depends upon cell dose, cell expansion in vivo through a minimally differentiated phenotype, long term persistence, and strong cytolytic effector function. An incomplete understanding of the biological coupling between T cell expansion, differentiation, and response to stimulation hinders the co-optimization of these factors. We report on a biophysical investigation of how the short-term kinetics of T cell functional activation, through molecular stimulation and cell-cell interactions, competes with phenotype differentiation. T cells receive molecular stimulation for a few minutes to a few hours in bulk culture. Following this priming period, the cells are then analyzed at the transcriptional level, or isolated as single cells, with continuing molecular stimulation, within microchambers for analysis via 11-plex secreted protein assays. We resolve a rapid feedback mechanism, promoted by T cell-T cell contact interactions, which strongly amplifies T cell functional performance while yielding only minimal phenotype differentiation. When tested in mouse models of ACT, optimally primed T cells lead to complete tumor eradication. A similar kinetic process is identified in CD8+ and CD4+ T cells collected from a patient with metastatic melanoma.

Heterologous expression, purification, refolding, and structural-functional characterization of EP-B2, a self-activating barley cysteine endoprotease.

We describe the heterologous expression in Escherichia coli of the proenzyme precursor to EP-B2, a cysteine endoprotease from germinating barley seeds. High yields (50 mg/l) of recombinant proEP-B2 were obtained from E. coli inclusion bodies in shake flask cultures following purification and refolding. The zymogen was rapidly autoactivated to its mature form under acidic conditions at a rate independent of proEP-B2 concentration, suggesting a cis mechanism of autoactivation. Mature EP-B2 was stable and active over a wide pH range and efficiently hydrolyzed a recombinant wheat gluten protein, alpha2-gliadin, at sequences with known immunotoxicity in celiac sprue patients. The X-ray crystal structure of mature EP-B2 bound to leupeptin was solved to 2.2 A resolution and provided atomic insights into the observed subsite specificity of the endoprotease. Our findings suggest that orally administered proEP-B2 may be especially well suited for treatment of celiac sprue.

Rational design of combination enzyme therapy for celiac sprue.

Celiac sprue (also known as celiac disease) is an inheritable, gluten-induced enteropathy of the upper small intestine with an estimated prevalence of 0.5%-1% in most parts of the world. The ubiquitous nature of food gluten, coupled with inadequate labeling regulations in most countries, constantly poses a threat of disease exacerbation and relapse for patients. Here, we demonstrate that a two-enzyme cocktail comprised of a glutamine-specific cysteine protease (EP-B2) that functions under gastric conditions and a PEP, which acts in concert with pancreatic proteases under duodenal conditions, is a particularly potent candidate for celiac sprue therapy. At a gluten:EP-B2:PEP weight ratio of 75:3:1, grocery store gluten is fully detoxified within 10 min of simulated duodenal conditions, as judged by chromatographic analysis, biopsy-derived T cell proliferation assays, and a commercial antigluten antibody test.

Personalized T cell-mediated cancer immunotherapy: progress and challenges.

Immunotherapies are yielding effective treatments for several previously untreatable cancers. Until recently, vaccines and adoptive cell therapies have been designed to target public tumor antigens common to multiple patients rather than private antigens specific to a single patient. Due to the difficulty of identifying public antigens that are expressed exclusively on tumor cells, these studies have yielded both clinical successes and serious immune-related adverse events. Multiple avenues of research now underscore the centrality of tumor-specific mutated private antigens to endogenous anti-tumor immunity. Immunotherapies that target these neoantigens may enable safer and more durable tumor regression, but personalized targeting presents a number of challenges. Foremost among these is to develop processes that accelerate advancement from neoantigen discovery to use of these neoantigens as vaccines or as targets for adoptive cell therapies. Exome sequencing has facilitated discovery of neoantigens for melanoma and other highly mutated cancers. New technologies - possibly proceeding from T cell receptor repertoire sequencing - are needed to identify antigens for cancers with low mutational burden and few neoantigens. In this review, we discuss progress toward personalizing T cell-mediated immunotherapy for cancer as well as challenges going forward.

Preparation of peptide-MHC and T-cell receptor dextramers by biotinylated dextran doping.

Peptide-major histocompatibility complex (pMHC) multimers enable the detection, characterization, and isolation of antigen-specific T-cell subsets at the single-cell level via flow cytometry and fluorescence microscopy. These labeling reagents exploit a multivalent scaffold to increase the avidity of individually weak T-cell receptor (TCR)-pMHC interactions. Dextramers are an improvement over the original streptavidin-based tetramer technology because they are more multivalent, improving sensitivity for rare, low-avidity T cells, including self/tumor-reactive clones. However, commercial pMHC dextramers are expensive, and in-house production is very involved for a typical biology research laboratory. Here, we present a simple, inexpensive protocol for preparing pMHC dextramers by doping in biotinylated dextran during conventional tetramer preparation. We use these pMHC dextramers to identify patient-derived, tumor-reactive T cells. We apply the same dextran doping technique to prepare TCR dextramers and use these novel reagents to yield new insight into MHC I-mediated antigen presentation.

Domain-swapped T cell receptors improve the safety of TCR gene therapy.

T cells engineered to express a tumor-specific αß T cell receptor (TCR) mediate anti-tumor immunity. However, mispairing of the therapeutic αß chains with endogenous αß chains reduces therapeutic TCR surface expression and generates self-reactive TCRs. We report a general strategy to prevent TCR mispairing: swapping constant domains between the α and ß chains of a therapeutic TCR. When paired, domain-swapped (ds)TCRs assemble with CD3, express on the cell surface, and mediate antigen-specific T cell responses. By contrast, dsTCR chains mispaired with endogenous chains cannot properly assemble with CD3 or signal, preventing autoimmunity. We validate this approach in cell-based assays and in a mouse model of TCR gene transfer-induced graft-versus-host disease. We also validate a related approach whereby replacement of αß TCR domains with corresponding γδ TCR domains yields a functional TCR that does not mispair. This work enables the design of safer TCR gene therapies for cancer immunotherapy.

Oral enzyme therapy for celiac sprue.

Celiac sprue is an inflammatory disease of the small intestine caused by dietary gluten and treated by adherence to a life-long gluten-free diet. The recent identification of immunodominant gluten peptides, the discovery of their cogent properties, and the elucidation of the mechanisms by which they engender immunopathology in genetically susceptible individuals have advanced our understanding of the molecular pathogenesis of this complex disease, enabling the rational design of new therapeutic strategies. The most clinically advanced of these is oral enzyme therapy, in which enzymes capable of proteolyzing gluten (i.e., glutenases) are delivered to the alimentary tract of a celiac sprue patient to detoxify ingested gluten in situ. In this chapter, we discuss the key challenges for discovery and preclinical development of oral enzyme therapies for celiac sprue. Methods for lead identification, assay development, gram-scale production and formulation, and lead optimization for next-generation proteases are described and critically assessed.

Visualization of transepithelial passage of the immunogenic 33-residue peptide from alpha-2 gliadin in gluten-sensitive macaques.

BACKGROUND: Based on clinical, histopathological and serological similarities to human celiac disease (CD), we recently established the rhesus macaque model of gluten sensitivity. In this study, we further characterized this condition based on presence of anti-tissue transglutaminase 2 (TG2) antibodies, increased intestinal permeability and transepithelial transport of a proteolytically resistant, immunotoxic, 33-residue peptide from alpha(2)-gliadin in the distal duodenum of gluten-sensitive macaques. METHODOLOGY/PRINCIPAL FINDINGS: Six rhesus macaques were selected for study from a pool of 500, including two healthy controls and four gluten-sensitive animals with elevated anti-gliadin or anti-TG2 antibodies as well as history of non-infectious chronic diarrhea. Pediatric endoscope-guided pinch biopsies were collected from each animal's distal duodenum following administration of a gluten-containing diet (GD) and again after remission by gluten-free diet (GFD). Control biopsies always showed normal villous architecture, whereas gluten-sensitive animals on GD exhibited histopathology ranging from mild lymphocytic infiltration to villous atrophy, typical of human CD. Immunofluorescent microscopic analysis of biopsies revealed IgG+ and IgA+ plasma-like cells producing antibodies that colocalized with TG2 in gluten-sensitive macaques only. Following instillation in vivo, the Cy-3-labeled 33-residue gluten peptide colocalized with the brush border protein villin in all animals. In a substantially enteropathic macaque with "leaky" duodenum, the peptide penetrated beneath the epithelium into the lamina propria. CONCLUSIONS/SIGNIFICANCE: The rhesus macaque model of gluten sensitivity not only resembles the histopathology of CD but it also may provide a model for studying intestinal permeability in states of epithelial integrity and disrepair.