scholarly journals Assessment of chimeric antigen receptor T (CAR-T) cytotoxicity by droplet microfluidics in vitro

2021 ◽  
Author(s):  
Kuan Un Wong ◽  
Jingxuan Shi ◽  
Peng Li ◽  
Haitao Wang ◽  
Yanwei Jia ◽  
...  
Keyword(s):  
T Cells ◽  
Chimeric Antigen Receptor ◽  
Interleukin 2 ◽  
Antigen Receptor ◽  
Droplet Microfluidics ◽  
Fluid Viscosity ◽  
Label Free ◽  
Car T ◽  
Xenograft Models

Chimeric antigen receptor T (CAR-T) cells are cytotoxic T cells engineered to specifically kill cancer cells expressing specific target receptor(s). Prior CAR-T efficacy tests include CAR expression analysis by qPCR or ELISA, in vitro measurement of interferon-gamma; (IFNgamma) or interleukin-2 (IL-2), and xenograft models. However, the in vitro measurements did not reflect CAR-T cytotoxicity, whereas xenograft models are low throughput and costly. Here we presented a robust in vitro droplet microfluidic assay for CAR-T cytotoxicity assessment. This method not only enabled assessment of CAR-T cytotoxic activity under different fluid viscosity conditions, but also facilitated measurement of CAR-T expansion and dissection of mechanism of action via phenotype analysis in vitro. Furthermore, our data suggested that label-free cytotoxicity analysis is feasible by acquiring data before and after treatment. Hence, this study presented a novel in vitro method for assessment of cellular cytotoxicity that could potentially be applied to any cell-kill-cell experiment with varying solvent composition.

scholarly journals Evaluation of chimeric antigen receptor T cell therapy in non-human primates infected with SHIV or SIV

PLoS ONE ◽  
2021 ◽  
Vol 16 (3) ◽  
pp. e0248973
Author(s):  
Nami Iwamoto ◽  
Bhavik Patel ◽  
Kaimei Song ◽  
Rosemarie Mason ◽  
Sara Bolivar-Wagers ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Responses ◽  
Chimeric Antigen Receptor ◽  
Viral Suppression ◽  
Antigen Receptor ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T

Achieving a functional cure is an important goal in the development of HIV therapy. Eliciting HIV-specific cellular immune responses has not been sufficient to achieve durable removal of HIV-infected cells due to the restriction on effective immune responses by mutation and establishment of latent reservoirs. Chimeric antigen receptor (CAR) T cells are an avenue to potentially develop more potent redirected cellular responses against infected T cells. We developed and tested a range of HIV- and SIV-specific chimeric antigen receptor (CAR) T cell reagents based on Env-binding proteins. In general, SHIV/SIV CAR T cells showed potent viral suppression in vitro, and adding additional CAR molecules in the same transduction resulted in more potent viral suppression than single CAR transduction. Importantly, the primary determinant of virus suppression potency by CAR was the accessibility to the Env epitope, and not the neutralization potency of the binding moiety. However, upon transduction of autologous T cells followed by infusion in vivo, none of these CAR T cells impacted either acquisition as a test of prevention, or viremia as a test of treatment. Our study illustrates limitations of the CAR T cells as possible antiviral therapeutics.

scholarly journals HIV-1-Specific Chimeric Antigen Receptor T Cells Fail To Recognize and Eliminate the Follicular Dendritic Cell HIV Reservoir In Vitro

2020 ◽  
Vol 94 (10) ◽  
Author(s):  
Matthew T. Ollerton ◽  
Edward A. Berger ◽  
Elizabeth Connick ◽  
Gregory F. Burton
Keyword(s):  
T Cells ◽  
T Cell ◽  
Antiretroviral Therapy ◽  
Chimeric Antigen Receptor ◽  
Antigen Receptor ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Follicular Dendritic

ABSTRACT The major obstacle to a cure for HIV infection is the persistence of replication-competent viral reservoirs during antiretroviral therapy. HIV-specific chimeric antigen receptor (CAR) T cells have been developed to target latently infected CD4+ T cells that express virus either spontaneously or after intentional latency reversal. Whether HIV-specific CAR-T cells can recognize and eliminate the follicular dendritic cell (FDC) reservoir of HIV-bound immune complexes (ICs) is unknown. We created HIV-specific CAR-T cells using human peripheral blood mononuclear cells (PBMCs) and a CAR construct that enables the expression of CD4 (domains 1 and 2) and the carbohydrate recognition domain of mannose binding lectin (MBL) to target native HIV Env (CD4-MBL CAR). We assessed CAR-T cell cytotoxicity using a carboxyfluorescein succinimidyl ester (CFSE) release assay and evaluated CAR-T cell activation through interferon gamma (IFN-γ) production and CD107a membrane accumulation by flow cytometry. CD4-MBL CAR-T cells displayed potent lytic and functional responses to Env-expressing cell lines and HIV-infected CD4+ T cells but were ineffective at targeting FDC bearing HIV-ICs. CD4-MBL CAR-T cells were unresponsive to cell-free HIV or concentrated, immobilized HIV-ICs in cell-free experiments. Blocking intercellular adhesion molecule-1 (ICAM-1) inhibited the cytolytic response of CD4-MBL CAR-T cells to Env-expressing cell lines and HIV-infected CD4+ T cells, suggesting that factors such as adhesion molecules are necessary for the stabilization of the CAR-Env interaction to elicit a cytotoxic response. Thus, CD4-MBL CAR-T cells are unable to eliminate the FDC-associated HIV reservoir, and alternative strategies to eradicate this reservoir must be sought. IMPORTANCE Efforts to cure HIV infection have focused primarily on the elimination of latently infected CD4+ T cells. Few studies have addressed the unique reservoir of infectious HIV that exists on follicular dendritic cells (FDCs), persists in vivo during antiretroviral therapy, and likely contributes to viral rebound upon cessation of antiretroviral therapy. We assessed the efficacy of a novel HIV-specific chimeric antigen receptor (CAR) T cell to target both HIV-infected CD4+ T cells and the FDC reservoir in vitro. Although CAR-T cells eliminated CD4+ T cells that express HIV, they did not respond to or eliminate FDC bound to HIV. These findings reveal a fundamental limitation to CAR-T cell therapy to eradicate HIV.

scholarly journals CRISPR-mediated insertion of a chimeric antigen receptor produces nonviral T cell products capable of inducing solid tumor regression

2021 ◽  
Author(s):  
Katherine Mueller ◽  
Nicole Piscopo ◽  
Matthew Forsberg ◽  
Louise Saraspe ◽  
Amritava Das ◽  
...  
Keyword(s):  
T Cells ◽  
Chimeric Antigen Receptor ◽  
Viral Vectors ◽  
Viral Vector ◽  
Tumor Regression ◽  
Antigen Receptor ◽  
Car T Cells ◽  
Car T

Chimeric antigen receptor (CAR) T cells traditionally harbor viral vectors that encode the CAR transgene in the genome. However, viral vector manufacturing typically is resource intensive, suffers from batch-to-batch variability, and includes several animal components, adding regulatory and supply chain pressures. Here, CAR T cells were generated within nine days using recombinant SpCas9 protein and nucleic acids, without any viral vectors or animal components. In comparison to traditional retroviral CAR T cells, nonviral CRISPR CAR T cells exhibit TRAC-targeted genomic integration of the CAR transgene, higher frequency of gene expression signatures associated with a memory phenotype, low receptor signaling prior to infusion, and potent cytotoxicity against GD2+ neuroblastoma in vitro and in vivo. This proof-of-principle study eliminating viral vectors and animal components during CAR gene transfer could enable more flexible and scalable manufacturing of clinically-relevant, high-quality CAR T cells to treat cancers, including solid tumors.

scholarly journals Characterization of novel dual tandem CD19/BCMA chimeric antigen receptor T cells to potentially treat multiple myeloma

2020 ◽  
Author(s):  
Liqing Kang ◽  
Jian Zhang ◽  
Minghao Li ◽  
Nan Xu ◽  
Wei Qi ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
Tumor Cells ◽  
Chimeric Antigen Receptor ◽  
Antigen Receptor ◽  
Cytokine Release ◽  
Car T Cells ◽  
Car T

Abstract Background: Treatment with chimeric antigen receptor (CAR)-engineered T cells directed against the B-cell maturation antigen (BCMA) promoted transient recovery from multiple myeloma (MM). However, the absence of this antigen on immature plasma cells may limit the efficacy of this modality and facilitate relapse. The purpose of this study is to characterize a novel CAR that includes both a single-chain variable fragment (scFv)-BCMA and an scFv-CD19 in tandem orientation (tan-CAR) in an attempt to target both BCMA and CD19 expression on MM cells. Method: The scFv sequences from the anti-CD19 antibody FMC63 and the anti-BCMA antibody C11D5.3 were ligated in tandem with transmembrane and T-cell signaling domains to generate the tan-CAR construct. Specificity and efficacy of activated tan-CAR T cells were analyzed using in vitro proliferation, cytokine release, and cytolysis assays. We also evaluated the in vivo efficacy with a xenograft mouse model that included target tumor cells that expressed CD19 or BCMA and compared the results to those obtained with conventional CAR T cells. Results: The in vitro studies revealed specific activation of tan-CAR T cells by K562 cells that overexpressed CD19 and/or BCMA. Cell proliferation, cytokine release, and cytolytic activity were all comparable to the responses of single scFv CAR T cells. Importantly, in vivo studies of tan-CAR T cells revealed specific inhibition of tumor growth in the mouse xenograft model that included cells expressing both CD19 and BCMA. Systemic administration of tan-CAR T cells resulted in complete tumor remission, in contrast to the reduced efficacies of BCMA-CAR T and CD19-CAR T alone in this setting. Conclusion: We report the successful design and execution of novel tan-CAR T cells that promote significant anti-tumor efficacy against both CD19 and BCMA antigen-positive tumor cells in vitro and in vivo . The data from this study reveal a novel strategy that may help to reduce the rate of relapse in the treatment with single scFv-CAR T cells.

scholarly journals In Vitro-Transcribed mRNA Chimeric Antigen Receptor T Cell (IVT mRNA CAR T) Therapy in Hematologic and Solid Tumor Management: A Preclinical Update

2020 ◽  
Vol 21 (18) ◽  
pp. 6514
Author(s):  
Thangavelu Soundara Rajan ◽  
Agnese Gugliandolo ◽  
Placido Bramanti ◽  
Emanuela Mazzon
Keyword(s):  
T Cells ◽  
T Cell ◽  
Solid Tumors ◽  
Chimeric Antigen Receptor ◽  
Solid Tumor ◽  
Antigen Receptor ◽  
Hematologic Malignancies ◽  
Car T ◽  
Tumor Management

Adoptive T cell immunotherapy has received considerable interest in the treatment of cancer. In recent years, chimeric antigen receptor T cell (CAR T) therapy has emerged as a promising therapy in cancer treatment. In CAR T therapy, T cells from the patients are collected, reprogrammed genetically against tumor antigens, and reintroduced into the patients to trigger an immense immune response against cancer cells. CAR T therapy is successful in hematologic malignancies; however, in solid tumors, CAR T therapy faces multiple challenges, including the on-target off-tumor phenomenon, as most of the tumor-associated antigens are expressed in normal cells as well. Consequently, a transient in vitro-transcribed anti-mRNA-based CAR T cell (IVT mRNA CAR T) approach has been investigated to produce controlled cytotoxicity for a limited duration to avoid any undesirable effects in patients. In vitro and in vivo studies demonstrated the therapeutic ability of mRNA-engineered T cells in solid tumors, including melanoma, neuroblastoma and ovarian cancer; however, very few clinical trials are registered. In the present review, we discuss the effect of IVT mRNA CAR T therapy in preclinical studies related to hematologic malignancies and solid tumor management. In addition, we discuss the clinical trial studies based on IVT mRNA CAR T therapy in cancer.

scholarly journals Pre-Clinical Evaluation of B7-H3-Specific Chimeric Antigen Receptor T-Cells for the Treatment of Acute Myeloid Leukemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 701-701 ◽  
Author(s):  
Eben Lichtman ◽  
Hongwei Du ◽  
Barbara Savoldo ◽  
Soldano Ferrone ◽  
Guangming Li ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
Hematopoietic Stem Cells ◽  
Chimeric Antigen Receptor ◽  
Antigen Receptor ◽  
Surface Antigens ◽  
Hematopoietic Stem ◽  
Car T ◽  
Hematopoietic Toxicity

Abstract Background: The development of safe and effective chimeric antigen receptor T-cell (CAR-T) therapy for acute myeloid leukemia (AML) remains an elusive goal. Whereas CD19-directed CAR-T therapies have shown great promise for the treatment of B-cell malignancies, the identification of AML-associated surface antigens that can be safely and effectively targeted by CAR T-cells has remained a challenge. Because most AML-associated surface antigens are also expressed on normal myeloid progenitors, the potential for unacceptable hematopoietic toxicity has been a major limitation. The identification of cell surface antigens that are absent on all normal myeloid progenitors and yet expressed on all subtypes of AML is not likely. On the other hand, it seems plausible that some antigens not detected on early myeloid lineage cells may be preferentially overexpressed in certain AML subtypes. We have identified B7-H3 as one such candidate. B7-H3 (B7-homolog 3, or CD276) is a coreceptor belonging to the B7 family of immune checkpoint molecules. B7-H3 protein expression in normal tissues is largely restricted to certain antigen-presenting cells. In multiple human cancers, however, B7-H3 protein is significantly overexpressed. This includes a substantial subset of AML, and B7-H3 expression appears to be higher in AML with a monocytic immunophenotype. Furthermore, B7-H3 on tumor cells, and on myeloid-derived suppressor cells in the tumor microenvironment, is likely to play an immunosuppressive role, and may drive immune escape in multiple cancer types. This suggests that targeting B7-H3 could also enhance anti-tumor adaptive immune responses. We therefore hypothesized that B7-H3-specific CAR-Ts (B7-H3.CARs) could be effective in eliminating B7-H3-expressing AML cells and would not cause unacceptable hematopoietic toxicity. Methods and Results: We obtained bone marrow aspirates from patients with monocytic/myelomonocytic AML (n=10) and demonstrated surface expression of B7-H3 on a median of 62.5% (range 27.8 to 98.5) of primary AML blasts. We also showed that B7-H3 is highly expressed on monocytic/myelomonocytic AML cell lines (THP1, U937, OCI-AML2, OCI-AML3), and that B7-H3 expression compares favorably to that of other previously identified candidate antigens for AML-directed CAR-T therapy. Next, we generated B7-H3.CARs via retroviral transduction of CD3/CD28-activated T-cells, followed by expansion in vitro with interleukin- (IL) 7 and IL-15. When B7-H3.CARs (n=3-5 donors) were cocultured with B7-H3-positive AML cell lines (listed above) and with primary AML blasts (n=10 patients), B7-H3.CARs proliferated, released high amounts of IL-2 and interferon-γ, and demonstrated efficient B7-H3-specific cytotoxicity. Autologous B7-H3.CARs also demonstrated significant cytotoxicity against primary AML blasts (n=4). Additionally, B7-H3.CARs controlled tumor cell proliferation and prolonged survival in xenograft mouse models of disseminated AML using OCI-AML2 (p=0.0025, n=5 mice per group) and THP1 (p<0.0001, n=10 mice per group). Next, we showed that B7-H3 is not significantly expressed on hematopoietic stem cells or myeloid progenitor cell populations in normal human bone marrow samples (n=2). We also evaluated the effects of B7-H3.CARs (n=4 donors) on normal hematopoietic stem cells via in vitro colony formation assays using umbilical cord-blood (n=4 donors) derived CD34+ cells, and showed that B7-H3.CARs did not significantly inhibit the formation of myeloid progenitor colonies. We then showed in a humanized mouse model (using fetal liver-derived hematopoietic stem cells) that B7-H3.CARs did not lead to significant reductions in the populations of circulating CD45/CD14-positive or CD45/CD33-positive cells. Conclusions: B7-H3 is expressed on a significant proportion of AML blasts from patients with monocytic AML. Adoptive transfer of B7-H3.CARs could be an effective treatment option for patients with B7-H3-positive AML, since i) we have previously demonstrated limited expression of B7-H3 in normal tissues, and ii) the present results show that B7-H3.CARs are unlikely to cause significant hematopoietic toxicity. Given variable expression of B7-H3 in AML, however, it may be necessary to develop a dual-targeting approach, combining B7-H3 with a second target AML-associated surface antigen. Disclosures Du: N/A: Patents & Royalties: Patent filed for B7-H3 chimeric antigen receptor. Ferrone:N/A: Patents & Royalties: Patent filed for B7-H3 chimeric antigen receptor. Dotti:University of North Carolina: Patents & Royalties: Patent filed for B7-H3 chimeric antigen receptor.

scholarly journals CAR T Cells Equipped With a Fully Human scFv Targeting Trop2 Can Be Used to Treat Pancreatic Cancer

2021 ◽  
Author(s):  
Hong Jia Zhu ◽  
Yujie Jia ◽  
Jingwen Tan ◽  
Xiaoyan Fang ◽  
Jing Ye ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
T Cells ◽  
Cancer Cells ◽  
Chimeric Antigen Receptor ◽  
Antigen Receptor ◽  
Car T Cells ◽  
Pancreatic Cancer Cells ◽  
Car T

Abstract Purpose: Chimeric antigen receptor (CAR) T cell therapy has demonstrated clinical success in treating haematologic malignancies but has not been effective against solid tumours thus far. Trop2 is a tumour-related antigen broadly overexpressed on a variety of tumours and has been reported as a promising target for pancreatic cancers. Our study aimed to determine whether CAR T cells designed with a fully human Trop2-specific single-chain fragment variable (scFv) can be used in the treatment of Trop2-positive pancreatic tumours.Methods: We designed Trop2-targeted chimeric antigen receptor engineered T cells with a novel human anti-Trop2 scFv (2F11) and then investigated the cytotoxicity, degranulation, and cytokine secretion profiles of the anti-Trop2 CAR T cells when they were exposed to Trop2+ cancer cells in vitro. We also studied the antitumour efficacy and toxicity of Trop2-specific CAR T cells in vivo using a BxPC-3 pancreatic xenograft model.Results: Trop2-targeted CAR T cells designed with 2F11 effectively killed Trop2-positive pancreatic cancer cells and produced high levels of cytotoxic cytokines in vitro. In addition, Trop2-targeted CAR T cells, which persistently circulate in vivo and efficiently infiltrate into tumour tissues, significantly blocked and even eliminated BxPC-3 pancreatic xenograft tumour growth without obvious deleterious effects observed after intravenous injection into NSG mice. Moreover, disease-free survival was efficiently prolonged.Conclusion: These results show that Trop2-targeted CAR T cells equipped with a fully human anti-Trop2 scFv could be a potential treatment strategy for pancreatic cancer and could be useful for clinical evaluation.

Development of chimeric antigen receptor-modified T cells for the treatment of esophageal cancer

2020 ◽  
pp. 030089162096022
Author(s):  
Feng Yu ◽  
Xiaoyan Wang ◽  
Hui Shi ◽  
Maorong Jiang ◽  
Jun Xu ◽  
...  
Keyword(s):  
T Cells ◽  
Esophageal Cancer ◽  
Cell Lines ◽  
Chimeric Antigen Receptor ◽  
Interleukin 2 ◽  
Antigen Receptor ◽  
Her2 Positive ◽  
Nude Mouse Model ◽  
Car T Cells ◽  
Car T

Background: Human epidermal growth factor receptor 2 (HER2) is an overexpressed antigen in esophageal squamous cell carcinomas (ESCCs) but with limited expression levels in normal esophageal tissues. Therefore, employing the adoptive transfer of T cells genetically modified to express chimeric antigen receptor (CAR) targeting HER2 could be a promising therapeutic strategy against ESCC. Methods: Two different second-generation CAR-T cells expressing antibodies for HER2 and CD19 antigens were developed using retroviral vector transduction. The expression of HER2 antigen in ESCC tissue and cell lines was examined by immunohistochemistry and flow cytometry, respectively. The tumor killing efficacy of the CAR-T cells in mice model and ESCC cell lines and its potential for the treatment of ESCC was evaluated by determining tumor size in mice xenograft, and by crystal violet staining, MTS assay, and cytokine release. Results: In vitro, HER2.CAR-T cells efficiently recognized and killed HER2-positive tumor cells as evidenced by the secretion of proinflammatory cytokines, interferon-γ, and interleukin 2 and by cytotoxicity assays. In vivo, intratumor injection of HER2.CAR-T cells resulted in a significant suppression of established ESCCs in a subcutaneous xenograft BALB/c nude mouse model. In contrast, the injection of CD19.CAR-T cells did not affect the tumor growth pattern. Conclusions: An effective HER2 CAR targeting ESCC was developed successfully. The HER2.CAR-T cell showed promising immunotherapeutic potential for the treatment of HER2-positive esophageal cancer.

scholarly journals Engineering T cells with hypoxia-inducible chimeric antigen receptor (HiCAR) for selective tumor killing

2020 ◽  
Vol 8 (1) ◽  
Author(s):  
Qibin Liao ◽  
Huan He ◽  
Yunyu Mao ◽  
Xiangqing Ding ◽  
Xiaoyan Zhang ◽  
...  
Keyword(s):  
T Cells ◽  
Solid Tumors ◽  
Chimeric Antigen Receptor ◽  
Antigen Receptor ◽  
Normal Tissues ◽  
Car T Cells ◽  
Car T ◽  
Tumor Killing

Abstract Chimeric antigen receptor-modified T cells (CAR-T cells) have shown good effects in the treatment of hematologic cancers; however, they may cause on-target off-tumor toxicity because of minimal expression of tumor-associated antigens (TAAs) on normal tissues, particularly in the context of treating solid tumors. Hypoxia is a common hallmark of solid tumors because of the Warburg effect. To minimize side effects, we designed a hypoxia-inducible CAR (HiCAR), which is driven by a hypoxia response element (HRE), and consists of a conventional CAR and an oxygen-dependent degradation domain (ODD) that is actively degraded under normoxia but stabilized under hypoxia. HiCAR-T cells showed enhanced cytotoxicity against tumor cells under hypoxia compared to normoxia in vitro and antitumor efficacy comparable to that of conventional CAR-T cells in vivo. Overall, our study demonstrates the potential of the HiCAR for improving the safety of CAR-T cells to promote the clinical application of CAR-T immunotherapy.

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