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International Journal of Molecular... May 2021CAR-T (chimeric antigen receptor T) cells have emerged as a milestone in the treatment of patients with refractory B-cell neoplasms. However, despite having... (Review)
Review
CAR-T (chimeric antigen receptor T) cells have emerged as a milestone in the treatment of patients with refractory B-cell neoplasms. However, despite having unprecedented efficacy against hematological malignancies, the treatment is far from flawless. Its greatest drawbacks arise from a challenging and expensive production process, strict patient eligibility criteria and serious toxicity profile. One possible solution, supported by robust research, is the replacement of T lymphocytes with NK cells for CAR expression. NK cells seem to be an attractive vehicle for CAR expression as they can be derived from multiple sources and safely infused regardless of donor-patient matching, which greatly reduces the cost of the treatment. CAR-NK cells are known to be effective against hematological malignancies, and a growing number of preclinical findings indicate that they have activity against non-hematological neoplasms. Here, we present a thorough overview of the current state of knowledge regarding the use of CAR-NK cells in treating various solid tumors.
Topics: Animals; Antigens, Neoplasm; Cell Culture Techniques; Clinical Trials as Topic; Combined Modality Therapy; Disease Models, Animal; Drug Evaluation, Preclinical; Genetic Engineering; Humans; Immunotherapy, Adoptive; Killer Cells, Natural; Neoplasms; Receptors, Antigen, T-Cell; Receptors, Chimeric Antigen; Treatment Outcome
PubMed: 34072732
DOI: 10.3390/ijms22115899 -
Nature Dec 2022The B cell antigen receptor (BCR) is composed of a membrane-bound class M, D, G, E or A immunoglobulin for antigen recognition and a disulfide-linked Igα (also known as...
The B cell antigen receptor (BCR) is composed of a membrane-bound class M, D, G, E or A immunoglobulin for antigen recognition and a disulfide-linked Igα (also known as CD79A) and Igβ (also known as CD79B) heterodimer (Igα/β) that functions as the signalling entity through intracellular immunoreceptor tyrosine-based activation motifs (ITAMs). The organizing principle of the BCR remains unknown. Here we report cryo-electron microscopy structures of mouse full-length IgM BCR and its Fab-deleted form. At the ectodomain (ECD), the Igα/β heterodimer mainly uses Igα to associate with Cµ3 and Cµ4 domains of one heavy chain (µHC) while leaving the other heavy chain (µHC') unbound. The transmembrane domain (TMD) helices of µHC and µHC' interact with those of the Igα/β heterodimer to form a tight four-helix bundle. The asymmetry at the TMD prevents the recruitment of two Igα/β heterodimers. Notably, the connecting peptide between the ECD and TMD of µHC intervenes in between those of Igα and Igβ to guide TMD assembly through charge complementarity. Weaker but distinct density for the Igβ ITAM nestles next to the TMD, suggesting potential autoinhibition of ITAM phosphorylation. Interfacial analyses suggest that all BCR classes utilize a general organizational architecture. Our studies provide a structural platform for understanding B cell signalling and designing rational therapies against BCR-mediated diseases.
Topics: Animals; Mice; B-Lymphocytes; Cryoelectron Microscopy; Receptors, Antigen, B-Cell; Signal Transduction; Immunoglobulin Fab Fragments; Protein Domains; Phosphorylation
PubMed: 36228656
DOI: 10.1038/s41586-022-05412-7 -
Journal of Translational Medicine Dec 2022Chimeric antigen receptor T (CAR-T) cells are genetically modified T cells with redirected specificity and potent T-cell-mediated cytotoxicity toward malignant cells.... (Clinical Trial)
Clinical Trial
A simple and effective method to purify and activate T cells for successful generation of chimeric antigen receptor T (CAR-T) cells from patients with high monocyte count.
BACKGROUND
Chimeric antigen receptor T (CAR-T) cells are genetically modified T cells with redirected specificity and potent T-cell-mediated cytotoxicity toward malignant cells. Despite several CAR-T products being approved and commercialized in the USA, Europe, and China, CAR-T products still require additional optimization to ensure reproducible and cost-effective manufacture. Here, we investigated the critical parameters in the CD3 T-cell isolation process that significantly impacted CAR-T manufacturing's success.
METHODS
CAR-T cells were prepared from cryopreserved peripheral blood mononuclear cells (PBMC). The thawed PBMC was rested overnight before the CD3 T cell isolation process using CTS Dynabeads CD3/CD28. Different isolation media, cell-bead co-incubation time, and cell density were examined in this study. Activated CD3 T cells were transduced with a gamma retroviral vector carrying the CD19 or BCMA CAR sequence. The CAR-T cells proliferated in a culture medium supplemented with interleukin 2 (IL-2).
RESULTS
CD14 monocytes hindered T-cell isolation when X-VIVO 15 basic medium was used as the selection buffer. The activation of T cells was blocked because monocytes actively engulfed CD3/28 beads. In contrast, when DPBS was the selection medium, the T-cell isolation and activation were no longer blocked, even in patients whose PBMC contained abnormally high CD14 monocytes and a low level of CD3 T cells.
CONCLUSIONS
In this study, we discovered that selecting CD3 T-cell isolation media is critical for improving T-cell activation, transduction, and CAR-T proliferation. Using DPBS as a CD3 T cell isolation buffer significantly improved the success rate and shortened the duration of CAR-T production. The optimized process has been successfully applied in our ongoing clinical trials. Trial registration NCT03798509: Human CD19 Targeted T Cells Injection Therapy for Relapsed and Refractory CD19-positive Leukemia. Date of registration: January 10, 2019. NCT03720457: Human CD19 Targeted T Cells Injection (CD19 CAR-T) Therapy for Relapsed and Refractory CD19-positive Lymphoma. Date of registration: October 25, 2018. NCT04003168: Human BCMA Targeted T Cells Injection Therapy for BCMA-positive Relapsed/Refractory Multiple Myeloma. Date of registration: July 1, 2019.
Topics: Humans; Antigens, CD19; B-Cell Maturation Antigen; Immunotherapy, Adoptive; Leukocytes, Mononuclear; Lymphoma; Monocytes; Receptors, Antigen, T-Cell; Receptors, Chimeric Antigen; T-Lymphocytes
PubMed: 36536403
DOI: 10.1186/s12967-022-03833-6 -
Frontiers in Immunology 2023Adoptive cell therapy (ACT) has seen a steep rise of new therapeutic approaches in its immune-oncology pipeline over the last years. This is in great part due to the... (Review)
Review
Adoptive cell therapy (ACT) has seen a steep rise of new therapeutic approaches in its immune-oncology pipeline over the last years. This is in great part due to the recent approvals of chimeric antigen receptor (CAR)-T cell therapies and their remarkable efficacy in certain soluble tumors. A big focus of ACT lies on T cells and how to genetically modify them to target and kill tumor cells. Genetically modified T cells that are currently utilized are either equipped with an engineered CAR or a T cell receptor (TCR) for this purpose. Both strategies have their advantages and limitations. While CAR-T cell therapies are already used in the clinic, these therapies face challenges when it comes to the treatment of solid tumors. New designs of next-generation CAR-T cells might be able to overcome these hurdles. Moreover, CARs are restricted to surface antigens. Genetically engineered TCR-T cells targeting intracellular antigens might provide necessary qualities for the treatment of solid tumors. In this review, we will summarize the major advancements of the CAR-T and TCR-T cell technology. Moreover, we will cover ongoing clinical trials, discuss current challenges, and provide an assessment of future directions within the field.
Topics: Humans; Receptors, Chimeric Antigen; Receptors, Antigen, T-Cell; Neoplasms; Immunotherapy, Adoptive; T-Lymphocytes
PubMed: 36949949
DOI: 10.3389/fimmu.2023.1121030 -
Immunological Reviews Jul 2019Adoptive cell transfer (ACT) using chimeric antigen receptor (CAR)-modified T cells can induce durable remissions in patients with refractory B-lymphoid cancers. By... (Review)
Review
Adoptive cell transfer (ACT) using chimeric antigen receptor (CAR)-modified T cells can induce durable remissions in patients with refractory B-lymphoid cancers. By contrast, results applying CAR-modified T cells to solid malignancies have been comparatively modest. Alternative strategies to redirect T cell specificity and cytolytic function are therefore necessary if ACT is to serve a greater role in human cancer treatments. T cell receptors (TCRs) are antigen recognition structures physiologically expressed by all T cells that have complementary, and in some cases superior, properties to CARs. Unlike CARs, TCRs confer recognition to epitopes derived from proteins residing within any subcellular compartment, including the membrane, cytoplasm and nucleus. This enables TCRs to detect a broad universe of targets, such as neoantigens, cancer germline antigens, and viral oncoproteins. Moreover, because TCRs have evolved to efficiently detect and amplify antigenic signals, these receptors respond to epitope densities many fold smaller than required for CAR-signaling. Herein, we summarize recent clinical data demonstrating that TCR-based immunotherapies can mediate regression of solid malignancies, including immune-checkpoint inhibitor refractory cancers. These trials simultaneously highlight emerging mechanisms of TCR resistance. We conclude by discussing how TCR-based immunotherapies can achieve broader dissemination through innovations in cell manufacturing and non-viral genome integration techniques.
Topics: Animals; Antigens, Neoplasm; Clinical Studies as Topic; Genetic Engineering; Humans; Immunotherapy, Adoptive; Neoplasms; Receptors, Antigen, T-Cell; Receptors, Chimeric Antigen; T-Cell Antigen Receptor Specificity; T-Lymphocytes; Treatment Outcome
PubMed: 31355495
DOI: 10.1111/imr.12772 -
Cell Aug 2022The T cell receptor (TCR) expressed by T lymphocytes initiates protective immune responses to pathogens and tumors. To explore the structural basis of how TCR signaling...
The T cell receptor (TCR) expressed by T lymphocytes initiates protective immune responses to pathogens and tumors. To explore the structural basis of how TCR signaling is initiated when the receptor binds to peptide-loaded major histocompatibility complex (pMHC) molecules, we used cryogenic electron microscopy to determine the structure of a tumor-reactive TCRαβ/CD3δγεζ complex bound to a melanoma-specific human class I pMHC at 3.08 Å resolution. The antigen-bound complex comprises 11 subunits stabilized by multivalent interactions across three structural layers, with clustered membrane-proximal cystines stabilizing the CD3-εδ and CD3-εγ heterodimers. Extra density sandwiched between transmembrane helices reveals the involvement of sterol lipids in TCR assembly. The geometry of the pMHC/TCR complex suggests that efficient TCR scanning of pMHC requires accurate pre-positioning of T cell and antigen-presenting cell membranes. Comparisons of the ligand-bound and unliganded receptors, along with molecular dynamics simulations, indicate that TCRs can be triggered in the absence of spontaneous structural rearrangements.
Topics: Humans; Major Histocompatibility Complex; Neoplasms; Peptides; Protein Binding; Receptors, Antigen, T-Cell; Receptors, Antigen, T-Cell, alpha-beta
PubMed: 35985289
DOI: 10.1016/j.cell.2022.07.010 -
Frontiers in Immunology 2022In recent years, the introduction of chimeric antigen receptor (CAR) T-cell therapies into clinics has been a breakthrough in treating relapsed or refractory... (Review)
Review
In recent years, the introduction of chimeric antigen receptor (CAR) T-cell therapies into clinics has been a breakthrough in treating relapsed or refractory malignancies in hematology and oncology. To date, Food and Drug Administration (FDA) has approved six CAR-T therapies for specific non-Hodgkin lymphomas, B-cell acute lymphoblastic leukemia, and multiple myeloma. All registered treatments and most clinical trials are based on so-called 2nd generation CARs, which consist of an extracellular antigen-binding region, one costimulatory domain, and a CD3z signaling domain. Unfortunately, despite remarkable overall treatment outcomes, a relatively high percentage of patients do not benefit from CAR-T therapy (overall response rate varies between 50 and 100%, with following relapse rates as high as 66% due to limited durability of the response). Moreover, it is associated with adverse effects such as cytokine release syndrome and neurotoxicity. Advances in immunology and molecular engineering have facilitated the construction of the next generation of CAR-T cells equipped with various molecular mechanisms. These include additional costimulatory domains (3rd generation), safety switches, immune-checkpoint modulation, cytokine expression, or knockout of therapy-interfering molecules, to name just a few. Implementation of next-generation CAR T-cells may allow overcoming current limitations of CAR-T therapies, decreasing unwanted side effects, and targeting other hematological malignancies. Accordingly, some clinical trials are currently evaluating the safety and efficacy of novel CAR-T therapies. This review describes the CAR-T cell constructs concerning the clinical application, summarizes completed and ongoing clinical trials of next-generation CAR-T therapies, and presents future perspectives.
Topics: United States; Humans; Receptors, Chimeric Antigen; Receptors, Antigen, T-Cell; T-Lymphocytes; Multiple Myeloma; Hematology
PubMed: 36389658
DOI: 10.3389/fimmu.2022.1034707 -
Methods in Cell Biology 2022The generation of chimeric antigen receptor (CAR) T cells requires the transfer of the CAR gene into primary T cells. Among various gene transfer strategies,...
The generation of chimeric antigen receptor (CAR) T cells requires the transfer of the CAR gene into primary T cells. Among various gene transfer strategies, gammaretroviral vectors have been widely used to generate CAR T cells for both preclinical and clinical settings. Here we describe the detailed method of generating CAR T cells utilizing gammaretroviral vectors. This approach consists of two parallel parts: (1) production of the gammaretroviral particles and (2) gammaretroviral transduction of activated T cells. The gammaretroviral particles are produced by co-transfecting the gammaretroviral vector with packaging plasmids into 293T cells. The manufactured viral particles then efficiently infect activated T cells where the CAR transgene is integrated into host genomic DNA, resulting in stable expression of the CAR molecule on the surface of T cells.
Topics: Genetic Vectors; Plasmids; Receptors, Antigen, T-Cell; T-Lymphocytes; Transgenes
PubMed: 35152995
DOI: 10.1016/bs.mcb.2021.06.014 -
International Journal of Biological... 2022Breast cancer rises as the most commonly diagnosed cancer in 2020. Among women, breast cancer ranks first in both cancer incidence rate and mortality. Treatment... (Review)
Review
Breast cancer rises as the most commonly diagnosed cancer in 2020. Among women, breast cancer ranks first in both cancer incidence rate and mortality. Treatment resistance developed from the current clinical therapies limits the efficacy of therapeutic outcomes, thus new treatment approaches are urgently needed. Chimeric antigen receptor (CAR) T cell therapy is a type of immunotherapy developed from adoptive T cell transfer, which typically uses patients' own immune cells to combat cancer. CAR-T cells are armed with specific antibodies to recognize antigens in self-tumor cells thus eliciting cytotoxic effects. In recent years, CAR-T cell therapy has achieved remarkable successes in treating hematologic malignancies; however, the therapeutic effects in solid tumors are not up to expectations including breast cancer. This review aims to discuss the development of CAR-T cell therapy in breast cancer from preclinical studies to ongoing clinical trials. Specifically, we summarize tumor-associated antigens in breast cancer, ongoing clinical trials, obstacles interfering with the therapeutic effects of CAR-T cell therapy, and discuss potential strategies to improve treatment efficacy. Overall, we hope our review provides a landscape view of recent progress for CAR-T cell therapy in breast cancer and ignites interest for further research directions.
Topics: Breast Neoplasms; Cell- and Tissue-Based Therapy; Female; Humans; Immunotherapy, Adoptive; Receptors, Antigen, T-Cell; Receptors, Chimeric Antigen
PubMed: 35414783
DOI: 10.7150/ijbs.70120 -
Proceedings of the National Academy of... Apr 2023Regulatory T cell (Treg) therapy is a promising approach to improve outcomes in transplantation and autoimmunity. In conventional T cell therapy, chronic stimulation can...
Regulatory T cell (Treg) therapy is a promising approach to improve outcomes in transplantation and autoimmunity. In conventional T cell therapy, chronic stimulation can result in poor in vivo function, a phenomenon termed exhaustion. Whether or not Tregs are also susceptible to exhaustion, and if so, if this would limit their therapeutic effect, was unknown. To "benchmark" exhaustion in human Tregs, we used a method known to induce exhaustion in conventional T cells: expression of a tonic-signaling chimeric antigen receptor (TS-CAR). We found that TS-CAR-expressing Tregs rapidly acquired a phenotype that resembled exhaustion and had major changes in their transcriptome, metabolism, and epigenome. Similar to conventional T cells, TS-CAR Tregs upregulated expression of inhibitory receptors and transcription factors such as PD-1, TIM3, TOX and BLIMP1, and displayed a global increase in chromatin accessibility-enriched AP-1 family transcription factor binding sites. However, they also displayed Treg-specific changes such as high expression of 4-1BB, LAP, and GARP. DNA methylation analysis and comparison to a CD8 T cell-based multipotency index showed that Tregs naturally exist in a relatively differentiated state, with further TS-CAR-induced changes. Functionally, TS-CAR Tregs remained stable and suppressive in vitro but were nonfunctional in vivo, as tested in a model of xenogeneic graft-versus-host disease. These data are the first comprehensive investigation of exhaustion in Tregs and reveal key similarities and differences with exhausted conventional T cells. The finding that human Tregs are susceptible to chronic stimulation-driven dysfunction has important implications for the design of CAR Treg adoptive immunotherapy strategies.
Topics: Humans; Receptors, Chimeric Antigen; T-Lymphocytes, Regulatory; T-Cell Exhaustion; Immunotherapy, Adoptive; Graft vs Host Disease; Receptors, Antigen, T-Cell
PubMed: 36972454
DOI: 10.1073/pnas.2219086120