Optimized NGFR-derived hinges for rapid and efficient enrichment and detection of CAR T cells in vitro and in vivo

Bister, A; Ibach, Tabea C.; Haist, C; Gerhorst, G; Smorra, Denise; Soldierer, Maren; Roellecke, Katharina; Wagenmann, Martin; Scheckenbach, Kathrin; Gattermann, Norbert; Wiek, Constanze

doi:10.1016/j.omto.2022.05.012

Cited by 5 publications

(3 citation statements)

References 63 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…To enable enrichment of the desired CAR + knock-in population we optimized CEMENT based selection by comparing three different clinically relevant enrichment markers: Dihydrofolate Reductase L22F/F31S (DHFR-FS), truncated Epidermal Growth Factor Receptor (tEGFR), and truncated Nerve Growth Factor Receptor (tNGFR) [ 9 , 17 , 39 ]. All three enrichment markers were successfully co-inserted along with the GD2-CAR resulting in total transgene sizes of ˜2.5-3 kb.…”

Section: Methodsmentioning

confidence: 99%

“…To enhance purity of HITI GD2 knock-in CAR-T cells, we optimized CEMENT by comparing three enrichment systems compatible with clinical application, which incorporate human proteins to diminish the risk of immunogenicity, and utilize GMP grade reagents or clinically approved drugs. We tested immunomagnetic enrichment targeting tEGFR and tNGFR as previously described [ 9 , 39 ], and drug based selection of cells expressing DHFR-FS, which confers resistance to the approved drug methotrexate (MTX) [ 49 , 50 ]. To optimize conditions for surface marker-based selection we compared column-based versus column-free magnetic selection and optimized the timing of enrichment.…”

Section: Optimization Of Cement Using Clinical Grade Enrichmentmentioning

confidence: 99%

See 1 more Smart Citation

Homology-independent targeted insertion (HITI) enables guided CAR knock-in and efficient clinical scale CAR-T cell manufacturing

et al. 2023

View full text Add to dashboard Cite

Background Chimeric Antigen Receptor (CAR) T cells are now standard of care (SOC) for some patients with B cell and plasma cell malignancies and could disrupt the therapeutic landscape of solid tumors. However, access to CAR-T cells is not adequate to meet clinical needs, in part due to high cost and long lead times for manufacturing clinical grade virus. Non-viral site directed CAR integration can be accomplished using CRISPR/Cas9 and double-stranded DNA (dsDNA) or single-stranded DNA (ssDNA) via homology-directed repair (HDR), however yields with this approach have been limiting for clinical application (dsDNA) or access to large yields sufficient to meet the manufacturing demands outside early phase clinical trials is limited (ssDNA). Methods We applied homology-independent targeted insertion (HITI) or HDR using CRISPR/Cas9 and nanoplasmid DNA to insert an anti-GD2 CAR into the T cell receptor alpha constant (TRAC) locus and compared both targeted insertion strategies in our system. Next, we optimized post-HITI CRISPR EnrichMENT (CEMENT) to seamlessly integrate it into a 14-day process and compared our knock-in with viral transduced anti-GD2 CAR-T cells. Finally, we explored the off-target genomic toxicity of our genomic engineering approach. Results Here, we show that site directed CAR integration utilizing nanoplasmid DNA delivered via HITI provides high cell yields and highly functional cells. CEMENT enriched CAR T cells to approximately 80% purity, resulting in therapeutically relevant dose ranges of 5.5 × 108–3.6 × 109 CAR + T cells. CRISPR knock-in CAR-T cells were functionally comparable with viral transduced anti-GD2 CAR-T cells and did not show any evidence of off-target genomic toxicity. Conclusions Our work provides a novel platform to perform guided CAR insertion into primary human T-cells using nanoplasmid DNA and holds the potential to increase access to CAR-T cell therapies.

show abstract

Section: Methodsmentioning

confidence: 99%

Section: Optimization Of Cement Using Clinical Grade Enrichmentmentioning

confidence: 99%

Homology-independent targeted insertion (HITI) enables guided CAR knock-in and efficient clinical scale CAR-T cell manufacturing

et al. 2023

View full text Add to dashboard Cite

show abstract

“…Directly targeting PDL1 using CAR-T cells can lead to severe and systemic toxicity because of the widespread expression of PDL1 on hematopoietic and parenchymal cells [ 25 ]. The hinge region (HD) of the CAR provides the necessary flexibility and length to access the target antigen while overcoming steric hindrance [ 26 , 27 ]. In our previous study, we demonstrated that reducing the flexibility of the hinge region can weaken the antigen-mediated activation of CAR-T cells [ 28 ].…”

Section: Introductionmentioning

confidence: 99%

Targeting and cytotoxicity of chimeric antigen receptor T cells grafted with PD1 extramembrane domain

Zhang,

Wang,

Sun

et al. 2023

Exp Hematol Oncol

View full text Add to dashboard Cite

Background Immunosuppression induced by programmed cell death protein 1 (PD1) presents a significant constraint on the effectiveness of chimeric antigen receptor (CAR)-T therapy. The potential of combining PD1/PDL1 (Programmed cell death 1 ligand 1) axis blockade with CAR-T cell therapy is promising. However, developing a highly efficient and minimally toxic approach requires further exploration. Our attempt to devise a novel CAR structure capable of recognizing both tumor antigens and PDL1 encountered challenges since direct targeting of PDL1 resulted in systemic adverse effects. Methods In this research, we innovatively engineered novel CARs by grafting the PD1 domain into a conventional second-generation (2G) CAR specifically targeting CD19. These CARs exist in two distinct forms: one with PD1 extramembrane domain (EMD) directly linked to a transmembrane domain (TMD), referred to as PE CAR, and the other with PD1 EMD connected to a TMD via a CD8 hinge domain (HD), known as PE8HT CAR. To evaluate their efficacy, we conducted comprehensive assessments of their cytotoxicity, cytokine release, and potential off-target effects both in vitro and in vivo using tumor models that overexpress CD19/PDL1. Results The findings of our study indicate that PE CAR demonstrates enhanced cytotoxicity and reduced cytokine release specifically towards CD19 + PDL1 + tumor cells, without off-target effects to CD19-PDL1 + tumor cells, in contrast to 2G CAR-T cells. Additionally, PE CAR showed ameliorative differentiation, exhaustion, and apoptosis phenotypes as assessed by flow cytometry, RNA-sequencing, and metabolic parameter analysis, after encountering CD19 + PDL1 + tumor cells. Conclusion Our results revealed that CAR grafted with PD1 exhibits enhanced antitumor activity with lower cytokine release and no PD1-related off-target toxicity in tumor models that overexpress CD19 and PDL1. These findings suggest that our CAR design holds the potential for effectively addressing the PD1 signal.

show abstract

Generation of Human 293-F Suspension NGFR Knockout Cells Using CRISPR/Cas9 Coupled to Fluorescent Protein Expression

Schatz,

van Dijk,

Dubiel

et al. 2023

Methods in Molecular Biology

View full text Add to dashboard Cite

Optimized NGFR-derived hinges for rapid and efficient enrichment and detection of CAR T cells in vitro and in vivo

Cited by 5 publications

References 63 publications

Homology-independent targeted insertion (HITI) enables guided CAR knock-in and efficient clinical scale CAR-T cell manufacturing

Homology-independent targeted insertion (HITI) enables guided CAR knock-in and efficient clinical scale CAR-T cell manufacturing

Targeting and cytotoxicity of chimeric antigen receptor T cells grafted with PD1 extramembrane domain

Generation of Human 293-F Suspension NGFR Knockout Cells Using CRISPR/Cas9 Coupled to Fluorescent Protein Expression

Contact Info

Product

Resources

About