Viral Engineering of Chimeric Antigen Receptor Expression on Murine and Human T Lymphocytes

Hammill, Joanne A.; Afsahi, Arya; Bramson, Jonathan L.; Helsen, Christopher W.

doi:10.1007/978-1-4939-3801-8_11

Cited by 9 publications

(12 citation statements)

References 18 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Self-inactivating, non-replicative lentivirus produced using a third-generation system has been previously discussed 46 , 49 . Briefly, 8 × 10 6 HEK293T cells cultured on 15 cm diameter tissue culture-treated dishes (NUNC; Thermo Fisher Scientific) were transfected with the packaging plasmids pRSV-Rev (6.25 μg), pMD2.G (9 μg), pMDLg-pRRE (12.5 μg) and the transfer plasmid pCCL containing the transgene (32 μg) using Opti-MEM (Gibco; Thermo Fisher Scientific) and Lipofectamine 2000 (Thermo Fisher Scientific).…”

Section: Methodsmentioning

confidence: 99%

“…This research was approved by the McMaster Health Sciences Research Ethics Board and all donors in this study provided informed written consent. Receptor-engineered human T cells were generated as previously described 49 . Human peripheral blood mononuclear cells (PBMC) from healthy donors (McMaster Adult Cohort (MAC) donor) or commercial leukapheresis products (LEUK donor) (HemaCare, Van Nuys, CA) were isolated by Ficoll-Paque-Plus gradient centrifugation (GE Healthcare, Baie d’Urfe, QC) and cryopreserved in inactivated human AB serum (Corning, Corning, NY) containing 10% DMSO (Sigma-Aldrich Canada Co.).…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

The chimeric TAC receptor co-opts the T cell receptor yielding robust anti-tumor activity without toxicity

et al. 2018

Self Cite

View full text Add to dashboard Cite

Engineering T cells with chimeric antigen receptors (CARs) is an effective method for directing T cells to attack tumors, but may cause adverse side effects such as the potentially lethal cytokine release syndrome. Here the authors show that the T cell antigen coupler (TAC), a chimeric receptor that co-opts the endogenous TCR, induces more efficient anti-tumor responses and reduced toxicity when compared with past-generation CARs. TAC-engineered T cells induce robust and antigen-specific cytokine production and cytotoxicity in vitro, and strong anti-tumor activity in a variety of xenograft models including solid and liquid tumors. In a solid tumor model, TAC-T cells outperform CD28-based CAR-T cells with increased anti-tumor efficacy, reduced toxicity, and faster tumor infiltration. Intratumoral TAC-T cells are enriched for Ki-67+ CD8+ T cells, demonstrating local expansion. These results indicate that TAC-T cells may have a superior therapeutic index relative to CAR-T cells.

show abstract

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

The chimeric TAC receptor co-opts the T cell receptor yielding robust anti-tumor activity without toxicity

et al. 2018

Self Cite

View full text Add to dashboard Cite

show abstract

“…Third generation, self-inactivating and non-replicative lentivirus was produced as previously described (Hammill et al, 2016;Helsen et al, 2018). Briefly, 9 3 10 6 HEK 293T cells were first cultured on 15 cm diameter tissue culture-treated plates (NUNC; Thermo Fisher) until 70% confluency.…”

Section: Limitations Of the Studymentioning

confidence: 99%

“…Engineered T cells were generated from cryopreserved PBMCs of healthy donors as previously described (Hammill et al, 2016). Briefly, 1 3 10 5 human T cells were activated from cryopreserved PBMCs using anti-CD3/CD28 beads at a 0.8:1 bead-to-cell ratio (Dynabeads, Thermo Fisher) in a U-bottom 96-well plate with T cell media (RPMI media supplemented with 10% FBS, 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 13 non-essential amino acids, 55 mM 2-Mercaptoethanol, and 100 U/mL penicillin +100 ug/mL streptomycin) containing 1.5 ng/mL rhIL-2 and 10 ng/mL rhIL-7 at 37 C. Twenty-four hours after activation T cells were transduced with thawed lentivirus at a MOI of 5.…”

Section: Transduction Of Human T Cellsmentioning

confidence: 99%

Expanded human NK cells armed with CAR uncouple potent anti-tumor activity from off-tumor toxicity against solid tumors

et al. 2021

Self Cite

View full text Add to dashboard Cite

Despite the remarkable success of chimeric antigen receptor (CAR)-T cells against hematologic malignancies, severe off-tumor effects have constrained their use against solid tumors. Recently, CAR-engineered natural killer (NK) cells have emerged as an effective and safe alternative. Here, we demonstrate that HER2 CAR-expression in NK cells from healthy donors and patients with breast cancer potently enhances their anti-tumor functions against various HER2-expressing cancer cells, regardless of MHC class I expression. Moreover, HER2 CAR-NK cells exert higher cytotoxicity than donor-matched HER2 CAR-T cells against tumor targets. Importantly, unlike CAR-T cells, HER2 CAR-NK cells do not elicit enhanced cytotoxicity or inflammatory cytokine production against non-malignant human lung epithelial cells with basal HER2 expression. Further, HER2 CAR-NK cells maintain high cytotoxic function in the presence of immunosuppressive factors enriched in solid tumors. These results show that CAR-NK cells may be a highly potent and safe source of immunotherapy in the context of solid tumors.

show abstract

“…Self-inactivating, non-replicative lentivirus was produced using a third-generation system, which has been previously discussed. 42,43 Briefly, 8 Â 10 6 HEK293T cells cultured on 15-cm-diameter tissueculture-treated dishes (NUNC; Thermo Fisher Scientific) were transfected with the packaging plasmids pRSV-Rev (6.25 mg), pMD2.G (9 mg), pMDLg-pRRE (12.5 mg), and the desired pCCL transfer plasmid (described earlier; 32 mg) using Opti-MEM (GIBCO; Thermo Fisher Scientific) and Lipofectamine 2000 (Thermo Fisher Scientific). Twelve to 16 h after transfection, media were replaced; fresh medium was supplemented with sodium butyrate (1 mM; Sigma-Aldrich).…”

Section: Lentivirus Productionmentioning

confidence: 99%

A Cross-Reactive Small Protein Binding Domain Provides a Model to Study Off-Tumor CAR-T Cell Toxicity

Hammill

Kwiecień

Dvorkin‐Gheva

et al. 2020

Molecular Therapy - Oncolytics

Self Cite

View full text Add to dashboard Cite

Tumor-targeted chimeric antigen receptor (CAR)-engineered T lymphocytes (CAR-T cells) have demonstrated striking clinical success, but their use has been associated with a constellation of toxicities. A better understanding of the pathogenesis of these toxicities is required to improve the safety profile of CAR-T cells. Herein, we describe a xenograft model of off-tumor CAR-T cell-associated toxicity. Human CAR-T cells targeted against HER2 using a small-protein binding domain induced acute, dose-dependent toxicities in mice. The inclusion of a CD28 or 4-1BB co-stimulatory domain in the CAR was required to produce toxicity; however, co-stimulation through CD28 was most toxic on a per-cell basis. CAR-T cell activation in the lungs and heart was associated with a systemic cytokine storm. The severity of observed toxicities was dependent upon the peripheral blood mononuclear cell (PBMC) donor used as a T cell source and paralleled the CD4 + -to-CD8 + T cell ratio in the adoptive transfer product. CD4 + CAR-T cells were determined to be the primary contributors to CAR-T cell-associated toxicity. However, donor-specific differences persisted after infusion of a purified CD4 + CAR-T cell product, indicating a role for additional variables. This work highlights the contributions of CAR-T cell-intrinsic variables to the pathogenesis of off-tumor toxicity.

show abstract

Viral Engineering of Chimeric Antigen Receptor Expression on Murine and Human T Lymphocytes

Cited by 9 publications

References 18 publications

The chimeric TAC receptor co-opts the T cell receptor yielding robust anti-tumor activity without toxicity

The chimeric TAC receptor co-opts the T cell receptor yielding robust anti-tumor activity without toxicity

Expanded human NK cells armed with CAR uncouple potent anti-tumor activity from off-tumor toxicity against solid tumors

A Cross-Reactive Small Protein Binding Domain Provides a Model to Study Off-Tumor CAR-T Cell Toxicity

Contact Info

Product

Resources

About