Gene-edited healthy donor CAR T cells show superior anti-tumour activity compared to CAR T cells derived from patients with lymphoma in an in vivo model of high-grade lymphoma.
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ABSTRACT:
SUBMITTER: Graham CE
PROVIDER: S-EPMC8632681 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Mitochondrial failure is recognized to play an important role in a variety of diseases. We previously showed hibernating species to have cell-autonomous protective mechanisms to resist cellular stress and sustain mitochondrial function. Here, we set out to detail these mitochondrial features of hibernators. We compared two hibernator-derived cell lines (HaK and DDT1MF2) with two non-hibernating cell lines (HEK293 and NRK) during hypothermia (4 °C) and rewarming (37 °C). Although all cell lines showed a strong decrease in oxygen consumption upon cooling, hibernator cells maintained functional mitochondria during hypothermia, without mitochondrial permeability transition pore (mPTP) opening, mitochondrial membrane potential decline or decreased adenosine triphosphate (ATP) levels, which were all observed in both non-hibernator cell lines. In addition, hibernator cells survived hypothermia in the absence of extracellular energy sources, suggesting their use of an endogenous substrate to maintain ATP levels. Moreover, hibernator-derived cells did not accumulate reactive oxygen species (ROS) damage and showed normal cell viability even after 48 h of cold-exposure. In contrast, non-hibernator cells accumulated ROS and showed extensive cell death through ferroptosis. Understanding the mechanisms that hibernators use to sustain mitochondrial activity and counteract damage in hypothermic circumstances may help to define novel preservation techniques with relevance to a variety of fields, such as organ transplantation and cardiac arrest.
| S-EPMC7084219 | biostudies-literature
Project description:The potential of adoptive cell therapy can be extended when combined with genome editing. However, variation in the quality of the starting material and the different manufacturing steps are associated with production failure and product contamination. Here, we present an automated T cell engineering process to produce off-the-shelf chimeric antigen receptor (CAR) T cells on an extended CliniMACS Prodigy platform containing an in-line electroporation unit. This setup was used to combine lentiviral delivery of a CD19-targeting CAR with transfer of mRNA encoding a TRAC locus-targeting transcription activator-like effector nuclease (TALEN). In three runs at clinical scale, the T cell receptor (TCR) alpha chain encoding TRAC locus was disrupted in >35% of cells with high cell viability (>90%) and no detectable off-target activity. A final negative selection step allowed the generation of TCRα/β-free CAR T cells with >99.5% purity. These CAR T cells proliferated well, maintained a T cell memory phenotype, eliminated CD19-positive tumor cells, and released the expected cytokines when exposed to B cell leukemia cells. In conclusion, we established an automated, good manufacturing practice (GMP)-compliant process that integrates lentiviral transduction with electroporation of TALEN mRNA to produce functional TCRα/β-free CAR19 T cells at clinical scale.
| S-EPMC7848723 | biostudies-literature
Project description:Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potentially curative therapy for hematological malignancies. However, graft-versus-host disease (GVHD) and relapse after allo-HSCT remain major impediments to the success of allo-HSCT. Chimeric antigen receptors (CARs) direct tumor cell recognition of adoptively transferred T cells. CD19 is an attractive CAR target, which is expressed in most B cell malignancies, as well as in healthy B cells. Clinical trials using autologous CD19-targeted T cells have shown remarkable promise in various B cell malignancies. However, the use of allogeneic CAR T cells poses a concern in that it may increase risk of the occurrence of GVHD, although this has not been reported in selected patients infused with donor-derived CD19 CAR T cells after allo-HSCT. To understand the mechanism whereby allogeneic CD19 CAR T cells may mediate anti-lymphoma activity without causing a significant increase in the incidence of GVHD, we studied donor-derived CD19 CAR T cells in allo-HSCT and lymphoma models in mice. We demonstrate that alloreactive T cells expressing CD28-costimulated CD19 CARs experience enhanced stimulation, resulting in the progressive loss of both their effector function and proliferative potential, clonal deletion, and significantly decreased occurrence of GVHD. Concurrently, the other CAR T cells that were present in bulk donor T cell populations retained their anti-lymphoma activity in accordance with the requirement that both the T cell receptor (TCR) and CAR be engaged to accelerate T cell exhaustion. In contrast, first-generation and 4-1BB-costimulated CAR T cells increased the occurrence of GVHD. These findings could explain the reduced risk of GVHD occurring with cumulative TCR and CAR signaling.
| S-EPMC5528161 | biostudies-literature
Project description:Targeting T cell malignancies using chimeric antigen receptor (CAR) T cells is hindered by 'T v T' fratricide against shared antigens such as CD3 and CD7. Base editing offers the possibility of seamless disruption of gene expression of problematic antigens through creation of stop codons or elimination of splice sites. We describe the generation of fratricide-resistant T cells by orderly removal of TCR/CD3 and CD7 ahead of lentiviral-mediated expression of CARs specific for CD3 or CD7. Molecular interrogation of base-edited cells confirmed elimination of chromosomal translocations detected in conventional Cas9 treated cells. Interestingly, 3CAR/7CAR co-culture resulted in 'self-enrichment' yielding populations 99.6% TCR-/CD3-/CD7-. 3CAR or 7CAR cells were able to exert specific cytotoxicity against leukaemia lines with defined CD3 and/or CD7 expression as well as primary T-ALL cells. Co-cultured 3CAR/7CAR cells exhibited highest cytotoxicity against CD3 + CD7 + T-ALL targets in vitro and an in vivo human:murine chimeric model. While APOBEC editors can reportedly exhibit guide-independent deamination of both DNA and RNA, we found no problematic 'off-target' activity or promiscuous base conversion affecting CAR antigen-specific binding regions, which may otherwise redirect T cell specificity. Combinational infusion of fratricide-resistant anti-T CAR T cells may enable enhanced molecular remission ahead of allo-HSCT for T cell malignancies.
| S-EPMC8632682 | biostudies-literature
Project description:Adoptive cell therapy using patient-derived chimeric receptor antigen (CAR) T cells redirected against tumor cells has shown remarkable success in treating hematologic cancers. However, wider accessibility of cellular therapies for all patients is needed. Manufacture of patient-derived CAR T cells is limited by prolonged lymphopenia in heavily pre-treated patients and risk of contamination with tumor cells when isolating T cells from patient blood rich in malignant blasts. Donor T cells provide a good source of immune cells for adoptive immunotherapy and can be used to generate universal off-the-shelf CAR T cells that are readily available for administration into patients as required. Genome editing tools such as TALENs and CRISPR-Cas9 and non-gene editing methods such as short hairpin RNA and blockade of protein expression are currently used to enhance CAR T cell safety and efficacy by abrogating non-specific toxicity in the form of graft versus host disease (GVHD) and preventing CAR T cell rejection by the host.
| S-EPMC8973942 | biostudies-literature
Project description:Autologous T cells engineered to express a chimeric antigen receptor (CAR) against the CD19 antigen are in the frontline of contemporary hemato-oncology therapies, leading to high remission rates in B-cell malignancies. Although effective, major obstacles involve the complex and costly individualized manufacturing process, and CD19 target antigen loss or modulation leading to resistant and relapse following CAR therapy. A potential solution for these limitations is the use of donor-derived ??T cells as a CAR backbone. ??T cells lack allogenecity and are safely used in haploidentical transplants. Moreover, ??T cells are known to mediate natural anti-tumor responses. Here, we describe a 14-day production process initiated from peripheral-blood mononuclear cells, leading to a median 185-fold expansion of ?? T cells with high purity (>98% CD3+ and >99% ??TCR+). CAR transduction efficacy of ?? T cells was equally high when compared to standard CAR-T cells (60.5 ± 13.2 and 65.3 ± 18.3%, respectively). CD19-directed ??CAR-T cells were effective against CD19+ cell lines in vitro and in vivo, showing cytokine production, direct target killing, and clearance of bone marrow leukemic cells in an NSG model. Multiple injections of ??CAR-T cells and priming of mice with zoledronate lead to enhanced tumor reduction in vivo. Unlike standard CD19 CAR-T cells, ??CAR-T cells were able to target CD19 antigen negative leukemia cells, an effect that was enhanced after priming the cells with zoledronate. In conclusion, ??CAR-T cell production is feasible and leads to highly pure and efficient effector cells. ??CAR-T cell may provide a promising platform in the allogeneic setting, and may target leukemic cells also after antigen loss.
| S-EPMC7343910 | biostudies-literature
Project description:The current treatment for pediatric acute lymphoblastic leukemia (ALL) is highly successful with high cure rate. However, the treatment of adult ALL remains a challenge, particularly for refractory and/or relapsed (R/R) ALL. The advent of new targeted agents, blinatumomab, inotuzumab ozogamycin, and chimeric antigen receptor (CAR) T cells, are changing the treatment paradigm for ALL. Tisagenlecleucel (kymriah, Novartis) is an autologous CD19-targeted CAR T cell product approved for treatment of R/R B cell ALL and lymphoma. In an attempt to reduce the relapse rate and treat those relapsed patients with antigen loss, donor-derived CAR T cells and CD19/CD22 dual-target CAR T cells are in clinical trials. Gene-edited "off-the-shelf" universal CAR T cells are also undergoing active clinical development. This review summarized new clinical trials and latest updates at the 2018 ASH Annual Meeting on CAR T therapy for ALL with a focus on dual-target CAR T and universal CAR T cell trials.
| S-EPMC6376657 | biostudies-literature
Project description:Chimeric antigen receptor-modified (CAR-modified) T cells have shown promising therapeutic effects for hematological malignancies, yet limited and inconsistent efficacy against solid tumors. The refinement of CAR therapy requires an understanding of the optimal characteristics of the cellular products, including the appropriate composition of CD4+ and CD8+ subsets. Here, we investigated the differential antitumor effect of CD4+ and CD8+ CAR T cells targeting glioblastoma-associated (GBM-associated) antigen IL-13 receptor ?2 (IL13R?2). Upon stimulation with IL13R?2+ GBM cells, the CD8+ CAR T cells exhibited robust short-term effector function but became rapidly exhausted. By comparison, the CD4+ CAR T cells persisted after tumor challenge and sustained their effector potency. Mixing with CD4+ CAR T cells failed to ameliorate the effector dysfunction of CD8+ CAR T cells, while surprisingly, CD4+ CAR T cell effector potency was impaired when coapplied with CD8+ T cells. In orthotopic GBM models, CD4+ outperformed CD8+ CAR T cells, especially for long-term antitumor response. Further, maintenance of the CD4+ subset was positively correlated with the recursive killing ability of CAR T cell products derived from GBM patients. These findings identify CD4+ CAR T cells as a highly potent and clinically important T cell subset for effective CAR therapy.
| S-EPMC6012522 | biostudies-literature
Project description:Vδ2+ T cells can recognize malignantly transformed cells as well as those infected with mycobacteria. This cross-reactivity supports the idea of using mycobacteria to manipulate Vδ2+ T cells in cancer immunotherapy. To date, therapeutic interventions using Vδ2+ T cells in cancer have involved expanding these cells in or ex vivo using zoledronic acid (ZA). Here, we show that the mycobacterium Bacillus Calmette-Guérin (BCG) also causes Vδ2+ T-cell expansion in vitro and that resulting Vδ2+ cell populations are cytotoxic toward tumour cell lines. We show that both ZA and BCG-expanded Vδ2+ cells effectively killed both Daudi and THP-1 cells. THP-1 cell killing by both ZA and BCG-expanded Vδ2+ cells was enhanced by treatment of targets cells with ZA. Although no difference in cytotoxic activity between ZA- and BCG-expanded Vδ2+ cells was observed, BCG-expanded cells degranulated more and produced a more diverse range of cytokines upon tumour cell recognition compared to ZA-expanded cells. ZA-expanded Vδ2+ cells were shown to upregulate exhaustion marker CD57 to a greater extent than BCG-expanded Vδ2+ cells. Furthermore, ZA expansion was associated with upregulation of inhibitory markers PD-1 and TIM3 in a dose-dependent manner whereas PD-1 expression was not increased following expansion using BCG. Intradermal BCG vaccination of rhesus macaques caused in vivo expansion of Vδ2+ cells. In combination with the aforementioned in vitro data, this finding suggests that BCG treatment could induce expansion of Vδ2+ T cells with enhanced anti-tumour potential compared to ZA treatment and that either ZA or BCG could be used intratumourally as a means to potentiate stronger anti-tumour Vδ2+ T-cell responses.
| S-EPMC9226146 | biostudies-literature