RESUMO
Immunotherapy using TCR and especially CAR transgenic T cells is a rapidly advancing field with the potential to become standard of care for the treatment of multiple diseases. While all current FDA approved CAR T cell products are generated using lentiviral gene transfer, extensive work is put into CRISPR/Cas mediated gene delivery to develop the next generation of safer and more potent cell products. One limitation of all editing systems is the size restriction of the knock-in cargo. Targeted integration under control of an endogenous promotor and/or signaling cascades opens the possibility to reduce CAR gene size to absolute minimum. Here we demonstrate that a first-generation CAR payload can be reduced to its minimum component - the antigen-binding domain - by targeted integration under control of the CD3ε promoter generating a CAR-CD3ε fusion protein that exploits the endogenous TCR signaling cascade. Miniaturizing CAR payload in this way results in potent CAR activity while simultaneously retaining the primary antigen recognition function of the TCR. Introducing CAR-specificity using a CAR binder only while maintaining endogenous TCR function may be an appealing design for future autologous CAR T cell therapies.
Assuntos
Imunoterapia Adotiva , Linfócitos T , Imunoterapia Adotiva/métodos , Imunoterapia , Receptores de Antígenos de Linfócitos TRESUMO
CAR T cell therapy is a rapidly growing area of oncological treatments having a potential of becoming standard care for multiple indications. Coincidently, CRISPR/Cas gene-editing technology is entering next-generation CAR T cell product manufacturing with the promise of more precise and more controllable cell modification methodology. The intersection of these medical and molecular advancements creates an opportunity for completely new ways of designing engineered cells to help overcome current limitations of cell therapy. In this manuscript we present proof-of-concept data for an engineered feedback loop. We manufactured activation-inducible CAR T cells with the help of CRISPR-mediated targeted integration. This new type of engineered T cells expresses the CAR gene dependent on their activation status. This artifice opens new possibilities to regulate CAR T cell function both in vitro and in vivo. We believe that such a physiological control system can be a powerful addition to the currently available toolbox of next-generation CAR constructs.
Assuntos
Receptores de Antígenos Quiméricos , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/metabolismo , Sistemas CRISPR-Cas , Edição de Genes/métodos , Imunoterapia Adotiva/métodos , Linfócitos T/metabolismoRESUMO
Large-scale target cell isolation from patient blood preparations is one of the critical operations during drug product manufacturing for personalized cell therapy in immuno-oncology. Use of high-affinity murine antibody coated magnetic nanoparticles that remain on isolated cells is the current standard applied for this purpose. Here, we present the transformation of previously described technology - non-magnetic immunoaffinity column chromatography-based cell selection with reversible reagents into a new clinical-grade cell isolation platform called Automated Traceless Cell affinity chromatography (ATC). ATC is a fully closed and GMP-compliant cell selection and manufacturing system. Reversibility of reagents enables (sequential) positive cell selection, optionally in combination with depletion columns, enabling capture of highly specific cell subsets. Moreover, synergy with other Streptamer-based technologies allows novel uses beyond cell isolation including integrated and automated on-column target cell activation. In conclusion, ATC technology is an innovative as well as versatile platform to select, stimulate and modify cells for clinical manufacturing and downstream therapies.
Assuntos
Cromatografia , Animais , Separação Celular/métodos , Humanos , CamundongosRESUMO
The avidity of TCRs for peptide-major histocompatibility complexes (pMHCs) is a governing factor in how T cells respond to antigen. TCR avidity is generally linked to T-cell functionality and there is growing evidence for distinct roles of low and high avidity T cells in different phases of immune responses. While physiological immune responses and many therapeutic T-cell products targeting infections or cancers consist of polyclonal T-cell populations with a wide range of individual avidities, the role of T-cell avidity is usually investigated only in monoclonal experimental settings. In this report, we induced polyclonal T-cell responses with a wide range of avidities toward a model epitope by altered peptide ligands, and benchmarked global avidity of physiological polyclonal populations by investigation of TCR-pMHC koff -rates. We then investigated how varying sizes and avidities of monoclonal subpopulations translate into global koff -rates. Global koff -rates integrate subclonal avidities in a predictably weighted manner and robustly correlate with the functionality of murine polyclonal T-cell populations in vitro and in vivo. Surveying the full avidity spectrum is essential to accurately assess polyclonal immune responses and inform the design of polyclonal T-cell therapeutics.
Assuntos
Receptores de Antígenos de Linfócitos T , Linfócitos T , Animais , Antígenos , Complexo Principal de Histocompatibilidade , Camundongos , Peptídeos , Receptores de Antígenos de Linfócitos T/genéticaRESUMO
BACKGROUND: Neoantigens derived from somatic mutations correlate with therapeutic responses mediated by treatment with immune checkpoint inhibitors. Neoantigens are therefore highly attractive targets for the development of therapeutic approaches in personalized medicine, although many aspects of their quality and associated immune responses are not yet well understood. In a case study of metastatic malignant melanoma, we aimed to perform an in-depth characterization of neoantigens and respective T-cell responses in the context of immune checkpoint modulation. METHODS: Three neoantigens, which we identified either by immunopeptidomics or in silico prediction, were investigated using binding affinity analyses and structural simulations. We isolated seven T-cell receptors (TCRs) from the patient's immune repertoire recognizing these antigens. TCRs were compared in vitro by multiparametric analyses including functional avidity, multicytokine secretion, and cross-reactivity screenings. A xenograft mouse model served to study in vivo functionality of selected TCRs. We investigated the patient's TCR repertoire in blood and different tumor-related tissues over 3 years using TCR beta deep sequencing. RESULTS: Selected mutated peptide ligands with proven immunogenicity showed similar binding affinities to the human leukocyte antigen complex and comparable disparity to their wild-type counterparts in molecular dynamic simulations. Nevertheless, isolated TCRs recognizing these antigens demonstrated distinct patterns in functionality and frequency. TCRs with lower functional avidity showed at least equal antitumor immune responses in vivo. Moreover, they occurred at high frequencies and particularly demonstrated long-term persistence within tumor tissues, lymph nodes and various blood samples associated with a reduced activation pattern on primary in vitro stimulation. CONCLUSIONS: We performed a so far unique fine characterization of neoantigen-specific T-cell responses revealing defined reactivity patterns of neoantigen-specific TCRs. Our data highlight qualitative differences of these TCRs associated with function and longevity of respective T cells. Such features need to be considered for further optimization of neoantigen targeting including adoptive T-cell therapies using TCR-transgenic T cells.
Assuntos
Antígenos de Neoplasias/imunologia , Imunoterapia/métodos , Melanoma/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Animais , Humanos , CamundongosRESUMO
Adoptive transfer of T cells expressing a transgenic T cell receptor (TCR) has the potential to revolutionize immunotherapy of infectious diseases and cancer. However, the generation of defined TCR-transgenic T cell medicinal products with predictable in vivo function still poses a major challenge and limits broader and more successful application of this "living drug." Here, by studying 51 different TCRs, we show that conventional genetic engineering by viral transduction leads to variable TCR expression and functionality as a result of variable transgene copy numbers and untargeted transgene integration. In contrast, CRISPR/Cas9-mediated TCR replacement enables defined, targeted TCR transgene insertion into the TCR gene locus. Thereby, T cell products display more homogeneous TCR expression similar to physiological T cells. Importantly, increased T cell product homogeneity after targeted TCR gene editing correlates with predictable in vivo T cell responses, which represents a crucial aspect for clinical application in adoptive T cell immunotherapy.
Assuntos
Edição de Genes , Genes Codificadores dos Receptores de Linfócitos T , Imunoterapia , Linfócitos T/imunologia , Animais , Linhagem Celular , Membrana Celular/metabolismo , Feminino , Humanos , Masculino , Camundongos Endogâmicos NOD , Transcrição GênicaRESUMO
T cell activation is a cornerstone in manufacturing of T cell-based therapies, and precise control over T cell activation is important in the development of the next generation T-cell based therapeutics. This need cannot be fulfilled by currently available methods for T cell stimulation, in particular not in a time dependent manner. Here, we describe a modular activation reagent called Expamers, which addresses these limitations. Expamers are versatile stimuli that are intended for research and clinical use. They are readily soluble and can be rapidly bound and removed from the cell surface, allowing nearly instantaneous initiation and termination of activation signal, respectively. Hence, Expamers enable precise regulation of T cell stimulation duration and provide promise of control over T cell profiles in future products. Expamers can be easily adopted to different T cell production formats and have the potential to increase efficacy of T cell immunotherapeutics.
Assuntos
Indicadores e Reagentes/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Animais , Proliferação de Células , Perfilação da Expressão Gênica , Humanos , Imunoterapia Adotiva , Camundongos , Linfócitos T/citologia , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
Adoptive transfer of T cells transgenic for tumor-reactive T-cell receptors (TCR) is an attractive immunotherapeutic approach. However, clinical translation is so far limited due to challenges in the identification of suitable target antigens as well as TCRs that are concurrent safe and efficient. Definition of key characteristics relevant for effective and specific tumor rejection is essential to improve current TCR-based adoptive T-cell immunotherapies. We here characterized in-depth two TCRs derived from the human leukocyte antigen (HLA)-mismatched allogeneic repertoire targeting two different myeloperoxidase (MPO)-derived peptides presented by the same HLA-restriction element side by side comprising state of the art biochemical and cellular in vitro, in vivo, and in silico experiments. In vitro experiments reveal comparable functional avidities, off-rates, and cytotoxic activities for both TCRs. However, we observed differences especially with respect to cytokine secretion and cross-reactivity as well as in vivo activity. Biochemical and in silico analyses demonstrate different binding qualities of MPO-peptides to the HLA-complex determining TCR qualities. We conclude from our biochemical and in silico analyses of peptide-HLA-binding that rigid and high-affinity binding of peptides is one of the most important factors for isolation of TCRs with high specificity and tumor rejection capacity from the MHC-mismatched repertoire. Based on our results, we developed a workflow for selection of such TCRs with high potency and safety profile suitable for clinical translation.
Assuntos
Antígenos HLA/imunologia , Peptídeos/imunologia , Peroxidase/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Animais , Linhagem Celular , Citocinas/imunologia , Humanos , Complexo Principal de Histocompatibilidade/imunologia , Camundongos Transgênicos , Modelos Moleculares , Neoplasias/imunologia , Neoplasias/terapiaRESUMO
Therapeutic T cells with desired specificity can be engineered by introducing T-cell receptors (TCRs) specific for antigens of interest, such as those from pathogens or tumour cells. However, TCR engineering is challenging, owing to the complex heterodimeric structure of the receptor and to competition and mispairing between endogenous and transgenic receptors. Additionally, conventional TCR insertion disrupts the regulation of TCR dynamics, with consequences for T-cell function. Here, we report the outcomes and validation, using five different TCRs, of the use of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) with non-virally delivered template DNA for the elimination of endogenous TCR chains and for the orthotopic placement of TCRs in human T cells. We show that, whereas the editing of a single receptor chain results in chain mispairing, simultaneous editing of α- and ß-chains combined with orthotopic TCR placement leads to accurate αß-pairing and results in TCR regulation similar to that of physiological T cells.
Assuntos
Receptores de Antígenos de Linfócitos T alfa-beta/química , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Antígenos de Neoplasias/imunologia , Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Edição de Genes , Técnicas de Inativação de Genes , Genes Codificadores dos Receptores de Linfócitos T/genética , Vetores Genéticos/genética , Humanos , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Retroviridae/genética , Transdução Genética , TransgenesRESUMO
Immunotherapy can revolutionize anti-cancer therapy if specific targets are available. Immunogenic peptides encoded by cancer-specific genes (CSGs) may enable targeted immunotherapy, even of oligo-mutated cancers, which lack neo-antigens generated by protein-coding missense mutations. Here, we describe an algorithm and user-friendly software named RAVEN (Rich Analysis of Variable gene Expressions in Numerous tissues) that automatizes the systematic and fast identification of CSG-encoded peptides highly affine to Major Histocompatibility Complexes (MHC) starting from transcriptome data. We applied RAVEN to a dataset assembled from 2,678 simultaneously normalized gene expression microarrays comprising 50 tumor entities, with a focus on oligo-mutated pediatric cancers, and 71 normal tissue types. RAVEN performed a transcriptome-wide scan in each cancer entity for gender-specific CSGs, and identified several established CSGs, but also many novel candidates potentially suitable for targeting multiple cancer types. The specific expression of the most promising CSGs was validated in cancer cell lines and in a comprehensive tissue-microarray. Subsequently, RAVEN identified likely immunogenic CSG-encoded peptides by predicting their affinity to MHCs and excluded sequence identity to abundantly expressed proteins by interrogating the UniProt protein-database. The predicted affinity of selected peptides was validated in T2-cell peptide-binding assays in which many showed binding-kinetics like a very immunogenic influenza control peptide. Collectively, we provide an exquisitely curated catalogue of cancer-specific and highly MHC-affine peptides across 50 cancer types, and a freely available software (https://github.com/JSGerke/RAVENsoftware) to easily apply our algorithm to any gene expression dataset. We anticipate that our peptide libraries and software constitute a rich resource to advance anti-cancer immunotherapy.
RESUMO
BACKGROUND: Enteral nutrition is used to treat a subset of patients with inflammatory bowel diseases. Because dietary factors may contribute to an aggressive immune response toward the intestinal microbiota in the disease susceptible host, we used TNFΔARE/WT mice to study the therapeutic effect of a semisynthetic experimental diet in the pathogenesis of Crohn's disease (CD)-like inflammation in the ileum. METHODS: TNFΔARE/WT mice were fed chow and experimental diets partially fortified with gluten in a dose and time-dependent manner. Histopathology, markers of inflammation, intraepithelial lymphocytes phenotypes, and antigen-specific reactivation of CD4⺠T cells were determined. RESULTS: TNFΔARE/WT mice being transferred to an experimental diet with 7 but not with 10 or 14 weeks of age were protected from development of Crohn's disease-like ileitis. Although disease-related CD8αß⺠intraepithelial lymphocytes were increased irrespective of dietary intervention, the protective effect of experimental diet was associated with decreased expression of inflammation markers in ileal tissues. In addition, CD4⺠T-cell reactivation in bacterial antigen-primed dendritic cell cocultures was not altered between semisynthetic and chow diet-fed TNFΔARE/WT mice, suggesting bacteria-independent mechanisms. Most importantly, gluten-fortified experimental diet induced chronic ileitis in TNFΔARE/WT mice, despite the fact that gluten-derived peptides failed to induce CD4⺠T-cell activation. Reduced occludin expression levels suggest a negative role of gluten-fortified experimental diet on intestinal barrier integrity. CONCLUSIONS: Crohn's disease-like ileitis can be prevented at early stages of disease development using a semisynthetic experimental diet. Gluten was identified as antigen-independent dietary factor relevant for the induction of chronic inflammation in the small intestine of TNFΔARE/WT mice.