FLAG-tagged CD19-specific CAR-T cells eliminate CD19-bearing solid tumor cells  i in vitro  i  and  i in vivo  i

Berahovich, Robert; Xu, Shuning; Zhou, Hua; Harto, Hizkia; Xu, Qumiao; Garcı́a, Andrés J.; Liu, Fenyong; Golubovskaya, Vita M.; Wu, Lijun

doi:10.2741/4563

Cited by 15 publications

(12 citation statements)

References 3 publications

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“…CHO-CD22 and CHO-BCMA cells were purchased from BPS Bioscience (San Diego, CA, USA) and cultured in Ham’s F-12K medium (Thermo Fisher) containing 10% FBS and 1 mg/mL geneticin (Thermo Fisher). HeLa-CD19 cells were generated in our laboratory [16] and cultured in DMEM containing 10% FBS and 1 uM puromycin (Thermo Fisher). Human peripheral blood mononuclear cells (PBMC) were isolated from LRS chambers (Stanford Blood Center, Palo Alto, CA, USA) by density sedimentation over Ficoll-Paque (GE Healthcare).…”

Section: Methodsmentioning

confidence: 99%

“…Hypoxia is also present in bone marrow hematopoietic niches where B lineage cells reside [15]. Therefore, in this study, we analyzed the effects of hypoxia on CD19 CAR-T cells [16,17] and B cell maturation antigen (BCMA) CAR-T cells in vitro [18]. We generated CD19 and BCMA CAR-T cells at atmospheric (18%) and hypoxic (1%) oxygen levels, and characterized the cells for expansion, CAR expression, CD4:CD8 ratio and differentiation status.…”

Section: Introductionmentioning

confidence: 99%

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Hypoxia Selectively Impairs CAR-T Cells In Vitro

Berahovich

Liu

Zhou

et al. 2019

Cancers

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Hypoxia is a major characteristic of the solid tumor microenvironment. To understand how chimeric antigen receptor-T cells (CAR-T cells) function in hypoxic conditions, we characterized CD19-specific and BCMA-specific human CAR-T cells generated in atmospheric (18% oxygen) and hypoxic (1% oxygen) culture for expansion, differentiation status, and CD4:CD8 ratio. CAR-T cells expanded to a much lower extent in 1% oxygen than in 18% oxygen. Hypoxic CAR-T cells also had a less differentiated phenotype and a higher CD4:CD8 ratio than atmospheric CAR-T cells. CAR-T cells were then added to antigen-positive and antigen-negative tumor cell lines at the same or lower oxygen level and characterized for cytotoxicity, cytokine and granzyme B secretion, and PD-1 upregulation. Atmospheric and hypoxic CAR-T cells exhibited comparable cytolytic activity and PD-1 upregulation; however, cytokine production and granzyme B release were greatly decreased in 1% oxygen, even when the CAR-T cells were generated in atmospheric culture. Together, these data show that at solid tumor oxygen levels, CAR-T cells are impaired in expansion, differentiation and cytokine production. These effects may contribute to the inability of CAR-T cells to eradicate solid tumors seen in many patients.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Hypoxia Selectively Impairs CAR-T Cells In Vitro

Berahovich

Liu

Zhou

et al. 2019

Cancers

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“…Hela cells were purchased from the ATCC (Manassas, VA, USA) and cultured in DMEM with 10% FBS and 1% penicillin/streptomycin. Hela-CD19 cells with stable expression of CD19 were maintained in DMEM with 10% FBS, puromycin and penicillin/streptomycin.as described in [ 33 ]. Human peripheral blood mononuclear cells (PBMC) were isolated from whole blood obtained in the Stanford Hospital Blood Center (Stanford, CA, USA) according to IRB-approved protocol using Ficoll-Paque solution (GE Healthcare, Chicago, IL, USA).…”

Section: Methodsmentioning

confidence: 99%

“…The lentiviral CARs were used for generation of lentivirus using 293 FT cells, Lentivirus Packaging Mix and transfection agent (Alstem, Richmond, CA, USA) as described [ 33 ]. The virus titers were determined by quantitative RT-PCR using the Lenti-X qRT-PCR kit (Takara Bio, Mountain View, CA, USA) according to the manufacturer’s protocol and the 7900HT thermal cycler (Thermo Fisher Scientific, South San Francisco, CA, USA).…”

Section: Methodsmentioning

confidence: 99%

CD47-CAR-T Cells Effectively Kill Target Cancer Cells and Block Pancreatic Tumor Growth

Golubovskaya

Berahovich

Zhou

et al. 2017

Cancers

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CD47 is a glycoprotein of the immunoglobulin superfamily that is often overexpressed in different types of hematological and solid cancer tumors and plays important role in blocking phagocytosis, increased tumor survival, metastasis and angiogenesis. In the present report, we designed CAR (chimeric antigen receptor)-T cells that bind CD47 antigen. We used ScFv (single chain variable fragment) from mouse CD47 antibody to generate CD47-CAR-T cells for targeting different cancer cell lines. CD47-CAR-T cells effectively killed ovarian, pancreatic and other cancer cells and produced high level of cytokines that correlated with expression of CD47 antigen. In addition, CD47-CAR-T cells significantly blocked BxPC3 pancreatic xenograft tumor growth after intratumoral injection into NSG mice. Moreover, we humanized mouse CD47 ScFv and showed that it effectively bound CD47 antigen. The humanized CD47-CAR-T cells also specifically killed ovarian, pancreatic, and cervical cancer cell lines and produced IL-2 that correlated with expression of CD47. Thus, CD47-CAR-T cells can be used as a novel cellular therapeutic agent for treating different types of cancer.

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“…5 The FDA has approved two types of CAR-T cell products for the treatment of hematological tumors. 6 Although CAR-T therapy performs well in hematological tumors, they have not shown similar efficacy in the treatment of other cancers. 7 Due to the suppressive tumor microenvironment, good infiltration and existence of CAR-T cells in solid tumors is a challenge, and on the other hand, the antitumor T cell function is insufficient activated.…”

Section: Introductionmentioning

confidence: 99%

<p>JAK-STAT Domain Enhanced MUC1-CAR-T Cells Induced Esophageal Cancer Elimination</p>

Zhang¹,

Zhao²,

He³

et al. 2020

CMAR

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Purpose: Chimeric antigen receptor (CAR)-T cells have shown to play a vital role in antitumor functions in hematological malignancies, but have poor efficacy in solid tumors. To improve the activation and proliferation of CART cell in solid tumors, we constructed an enhanced CART cells to increase the survival of esophageal cancer. Materials and Methods: To construct enhanced CART cells, we chose MUC1 as the target of CART cells. Long-term co-culture of target cells and effector cells was applied to verify the antitumor activity of these enhanced MUC1-CART cells in vitro. Moreover, a mouse xenograft model was established to investigate the effects of enhanced MUC1-CART cells on tumor elimination in vivo. Results: In vitro studies showed that enhanced MUC1-CART cells have long-lasting tumor killing and proliferative capabilities. Moreover, animal experiments verified that enhanced MUC1-CART cells had significant antitumor function and a prolonged half-life by subcutaneous transplantation models of esophageal cancer and PDX models of esophageal cancer, in vivo. Conclusion: These results indicated that enhanced MUC1-CART cells have a significant cytotoxic effect on esophageal cancer, and may likely to provide a novel strategy for the treatment of esophageal cancer.

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FLAG-tagged CD19-specific CAR-T cells eliminate CD19-bearing solid tumor cells i in vitro i and i in vivo i

Cited by 15 publications

References 3 publications

Hypoxia Selectively Impairs CAR-T Cells In Vitro

Hypoxia Selectively Impairs CAR-T Cells In Vitro

CD47-CAR-T Cells Effectively Kill Target Cancer Cells and Block Pancreatic Tumor Growth

<p>JAK-STAT Domain Enhanced MUC1-CAR-T Cells Induced Esophageal Cancer Elimination</p>

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