Faculty Opinions recommendation of T cells expressing a LMP1-specific chimeric antigen receptor mediate antitumor effects against LMP1-positive nasopharyngeal carcinoma cells in vitro and in vivo.

Infusion of regulatory T cells (Tregs) engineered with a chimeric antigen receptor (CAR) targeting donor-derived human leukocyte antigen (HLA) is a promising strategy to promote transplant tolerance. Here, we describe an anti-HLA-A2 CAR (A2-CAR) generated by grafting the complementarity-determining regions (CDRs) of a human monoclonal anti-HLA-A2 antibody into the framework regions of the Herceptin 4D5 single-chain variable fragment and fusing it with a CD28-zeta signaling domain. The CDR-grafted A2-CAR maintained the specificity of the original antibody. We then generated HLA-A2 mono-specific human CAR Tregs either by deleting the endogenous T-cell receptor (TCR) via CRISPR/Cas9 and introducing the A2-CAR using lentiviral transduction or by directly integrating the CAR construct into the TCR alpha constant locus using homology-directed repair. These A2-CAR+TCRdeficient human Tregs maintained both Treg phenotype and function in vitro. Moreover, they selectively accumulated in HLA-A2-expressing islets transplanted from either HLA-A2 transgenic mice or deceased human donors. A2-CAR+TCRdeficient Tregs did not impair the function of these HLA-A2+ islets, whereas similarly engineered A2-CAR+TCRdeficientCD4+ conventional T cells rejected the islets in less than 2 weeks. A2-CAR+TCRdeficient Tregs delayed graft-versus-host disease only in the presence of HLA-A2, expressed either by co-transferred peripheral blood mononuclear cells or by the recipient mice. Altogether, we demonstrate that genome-engineered mono-antigen-specific A2-CAR Tregs localize to HLA-A2-expressing grafts and exhibit antigen-dependent in vivo suppression, independent of TCR expression. These approaches may be applied towards developing precision Treg cell therapies for transplant tolerance.

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CD137 Co-Stimulation Improves The Antitumor Effect Of LMP1-Specific Chimeric Antigen Receptor T Cells In Vitro And In Vivo

OncoTargets and Therapy ◽

10.2147/ott.s221040 ◽

2019 ◽

Vol Volume 12 ◽

pp. 9341-9350 ◽

Cited By ~ 1

Author(s):

Xiaojun Tang ◽

Qi Tang ◽

Yuan Mao ◽

Xiaochen Huang ◽

Lizhou Jia ◽

...

Keyword(s):

T Cells ◽

Chimeric Antigen Receptor ◽

Antitumor Effect ◽

Antigen Receptor

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

10.1101/2021.08.06.455489 ◽

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.

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Development of human NKG2D-CD3ε chimeric antigen receptor (CAR) for T-cell-mediated cancer immunotherapy.

Journal of Clinical Oncology ◽

10.1200/jco.2017.35.7_suppl.150 ◽

2017 ◽

Vol 35 (7_suppl) ◽

pp. 150-150

Author(s):

Sergei Kusmartsev ◽

Johaness Vieweg ◽

Victor Prima

Keyword(s):

T Cells ◽

T Cell ◽

Tumor Cells ◽

Chimeric Antigen Receptor ◽

Antigen Receptor ◽

Adaptor Protein ◽

T Cell Subsets ◽

Human T Cells

150 Background: NKG2D is a lectin-like type 2 transmembrane receptor that expressed by natural killer cells and some T cell subsets. Stimulation of NKG2D receptor with specific agonistic ligands produces activating signals through signaling adaptor protein DAP10 leading to the enhanced cytokine production, proliferation, and cytotoxicity against tumor cells. There is strong evidence that NKG2D ligands are expressed in many human tumors, including melanoma, leukemia, myeloma, glioma, and carcinomas of the prostate, breast, lung, and colon. Recent studies also demonstrated that T cells bearing chimeric antigen receptor (CAR) NKG2D linked to CD3ζ (zeta) chain produce marked in vitro and in vivo anti-tumor effects. The aim of current study was to determine whether human T cells bearing chimeric antigen receptor (CAR) NKGD2 linked to CD3ε (epsilon) chain could be activated by the NKG2D-specific stimulation and able to kill human cancer cells. Given the important role of CD3ε in activation and survival of T cells, we hypothesized that NKG2D-CDε-bearing T cells could exert strong in vitro and in vivo anti-tumor effects. Methods: NKG2D CAR was produced by linking human NKG2D to DAP10 and the cytoplasmic portion of the CD3ε chain. Original full-length human cDNA clones were obtained from NIH Mammalian Gene Collection (MGC). Functional domain analysis and oligonucleotide design in the in-Fusion system of DNA cloning (Clontech) was used to generate the retroviral expression constructs. Results: Human PBMC-derived T cells were retrovirally transduced with newly generated NKG2D-CD3ε CAR DNA construct. These NKG2D CAR-expressing human T cells responded to NKG2D-specific activation by producing IFN-γ and exhibited significant cellular cytotoxicity against human tumor cells in vitro. In vivo studies demonstrated that NKG2D-CD3ε-bearing cells are capable of inhibiting growth of DU-145 human prostate cancer in the immunodeficient mice. Conclusions: Collectively, our data indicate the feasibility of developing chimeric antigen receptor NKG2D-CD3ε for T cells and suggest that adoptive transfer of T cells bearing NKG2D-CD3ε CAR could be potentially effective for immunotherapy of cancer patients.

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

10.21203/rs.3.rs-18044/v2 ◽

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.

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

10.21203/rs.3.rs-1203691/v1 ◽

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.

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

Biomarker Research ◽

10.1186/s40364-020-00238-9 ◽

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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Chlorotoxin Redirects Chimeric Antigen Receptor T Cells for Specific and Effective Targeting of Glioblastoma

10.1101/2020.01.24.918888 ◽

2020 ◽

Cited By ~ 1

Author(s):

Dongrui Wang ◽

Renate Starr ◽

Wen-Chung Chang ◽

Brenda Aguilar ◽

Darya Alizadeh ◽

...

Keyword(s):

T Cells ◽

Chimeric Antigen Receptor ◽

Antigen Receptor ◽

Matrix Metalloproteinase 2 ◽

Cell Surface Expression ◽

Binding Potential ◽

Car T Cells ◽

Car T

AbstractWhile chimeric antigen receptor (CAR) T cells have demonstrated antitumor activity against glioblastoma (GBM), tumor heterogeneity remains a critical challenge. To more effectively target heterogeneous GBMs, we report the development of a novel peptide-based CAR exploiting the GBM-binding potential of chlorotoxin (CLTX). CLTX bound a greater proportion of tumor cells than GBM-associated antigens EGFR, HER2 and IL13Rα2. CAR T cells bearing CLTX as the targeting domain (CLTX-CAR), mediated potent in vitro and in vivo anti-GBM activity, and efficiently targeted tumors lacking expression of other GBM-associated antigens. Importantly, CLTX-CAR T cells exhibited no observable off-target effector activity against normal cells, or when adoptively transferred into mice. Effective targeting by CLTX-CAR T cells required cell surface expression of matrix metalloproteinase-2 (MMP-2). Our results are the first demonstration of a peptide toxin utilized as a CAR targeting domain, expanding the repertoire of tumor-selective CAR T cells with the potential to reduce antigen escape.One Sentence SummaryChimeric antigen receptors incorporating chlorotoxin as the tumor targeting domain recognize and kill glioblastoma with high specificity and potency.

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Natural Flt3Lg-Based Chimeric Antigen Receptor (Flt3-CAR) T Cells Successfully Target Flt3 on AML Cell Lines

Vaccines ◽

10.3390/vaccines9111238 ◽

2021 ◽

Vol 9 (11) ◽

pp. 1238

Author(s):

Varvara Maiorova ◽

Murad D. Mollaev ◽

Polina Vikhreva ◽

Elena Kulakovskaya ◽

Dmitry Pershin ◽

...

Keyword(s):

T Cells ◽

Chimeric Antigen Receptor ◽

Antigen Receptor ◽

Flt3 Ligand ◽

Antitumor Effects ◽

Car T Cells ◽

Car T ◽

Dependent Inhibition ◽

Functional Equivalent

Relapsed/refractory acute myeloid leukemia (AML) cannot be cured with chemotherapy alone, as the blasts survive the treatment. Chimeric antigen receptor (CAR) approaches for AML are being actively developed. CARs promote immune reactions through recognition of the target molecular epitopes at the surface of cancer cells. The recognition involves the extracellular portion of the CAR protein, which corresponds to either the antibody or the physiological binding partner of the targeted antigen. Here, we design a chimeric receptor with a full-length natural Flt3-ligand recognition module that targets Flt3 tyrosine kinase, known as an adverse marker in AML. We demonstrate specific killing of Flt3-positive THP-1 cells by Flt3-CAR T cells and the lack of cytotoxicity towards Flt3-negative U937 cells. We prove that the inherent cytolytic capacity of T cells is essential for the killing. Finally, we confirm the authenticity of targeting by its competitive dose-dependent inhibition with a soluble Flt3-ligand. The developed system can be viewed as a non-immunogenic functional equivalent of scFv-mediated targeting. The robust in vitro antitumor effects of Flt3-CAR T cells, combined with their low off-target cytotoxicity, hold promise for AML treatment.

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