Generation of CAR-T Cells for Cancer Immunotherapy

Xu, Qumiao; Harto, Hizkia; Berahovich, Robert; Xu, Shuning; Zhou, Hua; Golubovskaya, Vita M.; Wu, Lijun

doi:10.1007/978-1-4939-8885-3_24

Cited by 13 publications

(12 citation statements)

References 25 publications

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“…These data support our hypothesis that the TSC population can be targeted using a DCLK1-specific CAR-T. Here, we report that, in collaboration with ProMab Inc., we have developed a novel CAR-T based on the DCLK1 ScFv containing a CD28 transmembrane and co-stimulatory domain and CD3ζ activation domain [39][40][41][42][43][44][45][46]. CAR-T cells generated using DCLK1 ScFv (CBT-511) demonstrated~20% CAR expression and significantly induced CRC cell cytotoxicity.…”

Section: Introductionsupporting

confidence: 72%

DCLK1 Monoclonal Antibody-Based CAR-T Cells as a Novel Treatment Strategy against Human Colorectal Cancers

Sureban

Berahovich

Zhou

et al. 2019

Cancers

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CAR-T (chimeric antigen receptor T cells) immunotherapy is effective in many hematological cancers; however, efficacy in solid tumors is disappointing. Doublecortin-like kinase 1 (DCLK1) labels tumor stem cells (TSCs) in genetic mouse models of colorectal cancer (CRC). Here, we describe a novel CAR-T targeting DCLK1 (CBT-511; with our proprietary DCLK1 single-chain antibody variable fragment) as a treatment strategy to eradicate CRC TSCs. The cell surface expression of DCLK1 and cytotoxicity of CBT-511 were assessed in CRC cells (HT29, HCT116, and LoVo). LoVo-derived tumor xenografts in NOD Scid gamma (NSG™) mice were treated with CBT-511 or mock CAR-T cells. Adherent CRC cells express surface DCLK1 (two-dimensional, 2D). A 4.5-fold increase in surface DCLK1 was observed when HT29 cells were grown as spheroids (three-dimensional, 3D). CBT-511 induced cytotoxicity (2D; p < 0.0001), and increased Interferon gamma (IFN-γ) release in CRC cells (2D) compared to mock CAR-T (p < 0.0001). Moreover, an even greater increase in IFN-γ release was observed when cells were grown in 3D. CBT-511 reduced tumor growth by approximately 50 percent compared to mock CAR-T. These data suggest that CRC cells with increased clonogenic capacity express increased surface DCLK1. A DCLK1-targeted CAR-T can induce cytotoxicity in vitro and inhibit xenograft growth in vivo.

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Section: Introductionsupporting

confidence: 72%

DCLK1 Monoclonal Antibody-Based CAR-T Cells as a Novel Treatment Strategy against Human Colorectal Cancers

Sureban

Berahovich

Zhou

et al. 2019

Cancers

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“…The medium was cleared of cell debris by centrifugation at 2100× g for 30 min. The virus particles were collected by centrifugation at 110,000× g for 100 min, suspended in AIM V medium (Thermo Fisher), aliquoted and frozen at −80 °C, as described [16,37,38,39].…”

Section: Methodsmentioning

confidence: 99%

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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“…Thawing glioblastoma organoids (Timing 1 h) 40. Locate cryogenic vials from liquid nitrogen storage and place them on dry ice.…”

Section: Cryopreservation Of Glioblastoma Organoids (Timing 2-3 H)mentioning

confidence: 99%

“…CRITICAL CAR-T cells are generated by transducing purified peripheral blood mononuclear cells (PBMCs) with integrating viruses harboring the CAR sequence. Not included in this procedure are detailed protocols for generating CAR-T cells as this is beyond our scope and already described in the literature 40 .…”

Section: Preparation Of Car-t Cells (Timing 3 Days)mentioning

confidence: 99%

Generation and biobanking of patient-derived glioblastoma organoids and their application in CAR-T cell testing

Jacob

Ming

Song

2020

Preprint

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Short Summary: Detailed procedures for generating and biobanking glioblastoma organoids from resected patient tumor tissue and testing CAR-T cell efficacy by co-culture. Additional procedures for tissue processing, immunohistology, and detecting hypoxia gradients and actively proliferating cells. AbstractGlioblastoma tumors exhibit extensive inter-and intra-tumoral heterogeneity, which has contributed to the poor outcomes of numerous clinical trials and continues to complicate the development of effective therapeutic strategies. Current in vitro models do not preserve the cellular and mutational diversity of parent tumors and often require a lengthy generation time with variable efficiency. Here, we describe detailed procedures for generating glioblastoma organoids (GBOs) from surgically resected patient tumor tissue using a chemically defined medium without cell dissociation. By preserving cell-cell interactions and minimizing clonal selection, GBOs maintain the cellular heterogeneity of parent tumors. We include methods for passaging and cryopreserving GBOs for continued use, biobanking, and long-term recovery. We further describe procedures for investigating patient-specific responses to immunotherapies by co-culturing GBOs with chimeric antigen receptor (CAR) T cells. This protocol takes approximately 2-4 weeks to generate GBOs and 5-7 days to perform CAR-T cell co-culture. Competence with human cell culture, tissue processing, and immunohistology is required for optimal results.An ideal in vitro model for glioblastoma would maintain cellular diversity, preserve native cell-cell interactions, recapitulate defining histological characteristics, and maintain the transcriptomes and mutation profiles of parent tumors. To be useful for personalized medicine approaches, the model would need a quick generation time with high fidelity for success and be scalable to test potential therapeutics. This would require maintaining patient-derived glioblastoma tissue in culture with minimal perturbation. Towards this goal, we opted to directly culture micro-dissected tumor pieces without cell dissociation to preserve cell-cell interactions and minimize clonal selection. We start from resected patient tumor tissue that can be sampled from different tumor foci or subdivided into regional samples to analyze intra-tumoral heterogeneity. Through testing of many medium formulations, we learned that tumor pieces did not require basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), serum, or added extracellular matrix such as Matrigel to survive, form organoids, and proliferate at rates similar to parent tumors 11 . We optimized a chemically defined medium that contains basic nutrients and components needed to support glial cell health with few exogenous factors to minimize clonal selection induced by growth factors and decrease potential treatment confounders. This resulted in an optimized version of our medium used to maintain human brain organoid cultures 12,13 . We culture tumor pieces on an orbital shaker to inc...

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Generation of CAR-T Cells for Cancer Immunotherapy

Cited by 13 publications

References 25 publications

DCLK1 Monoclonal Antibody-Based CAR-T Cells as a Novel Treatment Strategy against Human Colorectal Cancers

DCLK1 Monoclonal Antibody-Based CAR-T Cells as a Novel Treatment Strategy against Human Colorectal Cancers

Hypoxia Selectively Impairs CAR-T Cells In Vitro

Generation and biobanking of patient-derived glioblastoma organoids and their application in CAR-T cell testing

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