A Molecular Spotlight on CAR-T Cells

CAR-T cells in immunotherapy

In order to overcome immunosuppressive effects of cancer cells, researchers have genetically modified T cells ex vivo to re-target them towards cancer cells. Such re-targeting can be achieved by generating a T cell that expresses a cancer antigen specific T cell receptor (TCR) or a chimeric antigen receptor (CAR). CARs combine the signaling pathway of a TCR with the specificity and the binding properties of a monoclonal antibody. This enables the resulting T cells, the CAR-T cells, to recognize tumor antigens in their native conformation, independent of their presentation via the major histocompatibility complex (MHC) which might be mutated on cancer cells. This provides an advantage over TCR, because antigen recognition by TCR relies on MHC presentation of the cancer antigen. Furthermore, the use of CAR expands the range of potential targets, as a CAR can bind not only to proteins but also to carbohydrate, lipids and heavily glycosylated proteins.

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Because of these features, CAR-T cells are representing a promising addition to the cancer immunotherapy toolbox for the treatment of hematopoietic malignancies as well as solid tumors.

Researchers follow two different approaches when developing CAR-T cell immunotherapies. Most current strategies are autologous. For this patient-specific therapy, T cells are isolated from an individual patient, genetically engineered by introducing the CAR, expanded, characterized and then re-infused into the donating patient. With an allogeneic approach, T cells are isolated from a healthy donor, genetically engineered, expanded and then used to treat many patients. However, in this case the engineering process requires two steps: the introduction of the CAR and the knock-out of the TCR to minimize graft-versus-host disease. Researchers are also looking into deriving allogeneic T cells from induced pluripotent stem cells.

In the past years, research on CAR-T cells for cancer immunotherapy has gained a significant focus and resulted in over 100 ongoing clinical trials (Hay & Turtle 2017). CAR-T cells targeting the antigens CD19 or BCMA which are up-regulated in many hematological malignancies, have experienced much clinical success, leading to approval of several CAR-T cell therapies, e.g. Kymriah® (Novartis), Yescarta® (Gilead), Abecma® (Bluebird) and Carvykti® (J&J and Legend). More recently the first CRISPR-based CAR-T therapy against sickle cell disease or beta-thalassemia has been approved (Casgevy™ from Vertex).

Non-viral methods for ex vivo cell and gene therapy: is the future non-viral?

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Manufacturing of CAR-T cells

As explained above, the manufacturing of CAR-T cells involves an ex vivo modification step to transfer the CAR expressing transgene into T cells isolated from a donor. Such ex vivo delivery of the CAR transgene into human T cells and its insertion into the T cell genome can either be achieved by viral transduction or by non-viral transfection in combination with genome editing tools or mRNA.

Viruses, like gamma-retrovirus, lentivirus, adeno or adeno-associated virus, are widely used because of their high transduction/insertion efficiency. However, depending on the virus type, there might be some limitations with regard to safety concerns (immunogenicity, insertional mutagenesis), production costs and lead times, and DNA insert size.

Alternatively, non-viral transfection methods are under exploration for transferring the CAR to overcome some of the limitations affiliated to viruses. Non-viral methods are supposed to be more cost- and time-effective and offer more flexibility in terms of insert size and with regards to the cargo type used. When aiming for stable insertion into the T cell genome, genome editing tools like transposition systems (e.g. Sleeping Beauty®* or piggyBac®* transposon/transposase) or CRISPR are utilized. Furthermore, CAR mRNA can be used for transient CAR expression for a limited time of 3-5 days and more dosage control and also circumventing DNA-related toxicity as well as uncontrolled integration into the genome.

Automated production of gene-modified chimeric antigen receptor T cells using the Cocoon Platform

Trainor et.al. Cytotherapy 2023

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Typically, primary T cells are considered being hard-to-transfect by non-viral methods, especially due to the need for co-transfection of multiple cargo when using genome editing tools, leading to low expression of the CAR gene. However, advanced electroporation technologies, like the Nucleofector® Technology, allow for efficient transfection of pre-stimulated, or even resting primary human T cells .

Regardless of whether viral transduction or electroporation is used, adoptive immunotherapy via CAR-T cells is a huge focus area and may lead to a powerful tool to fight not only cancer, but also infectious diseases.

Overview on Lonza products supporting the CAR-T cell process

	T cell activation / culture	T cell non-viral modification	T cell expansion
Research	Primary PBMCs or purified T cells X-VIVO 15 Media - For efficient T cell culture	4D-Nucleofector® X Unit for efficient transfection in small scale	X-VIVO 15 Media - For efficient T cell culture
Process development	Primary PBMCs or purified T cells - For PD and training purposes TheraPEAK® T-VIVO Media - For serum-free culture	4D-Nucleofector® LV Unit with research-grade consumables - For scaling up	TheraPEAK® T-Vivo Media - For serum-free culture
Manufacturing	TheraPEAK® T-VIVO Media - For serum-free culture	4D-Nucleofector® LV Unit with TheraPEAK® Consumables	TheraPEAK® T-VIVO Media - For serum-free culture
	Cocoon® Platform - For closed automated manufacturing
	MODA® Platform – For combining Manufacturing and laboratory Data into a single source to expedite product release
	CDMO Services – For tailored process and analytical development, cGMP manufacturing, and regulatory services

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References

Glaser V, Flugel C, Kath J, Du W, Drosdek V, Franke C, Stein M, Pruß A, Schmueck-Henneresse M, Volk HD, Reinke P, Wagner DL. Combining different CRISPR nucleases for simultaneous knock-in and base editing prevents translocations in multiplex-edited CAR T cells. Genome Biol. 2023 Apr 24;24(1):89

Kath J, Du W, Pruene A, Braun T, Thommandru B, Turk R, Sturgeon ML, Kurgan GL, Amini L, Stein M, Zittel T, Martini S, Ostendorf L, Wilhelm A, Akyüz L, Rehm A, Höpken UE, Pruß A, Künkele A, Jacobi AM, Volk HD, Schmueck-Henneresse M, Stripecke R, Reinke P, Wagner DL. Pharmacological interventions enhance virus-free generation of TRAC-replaced CAR T cells. Mol Ther Methods Clin Dev. 2022 Apr 12:25:311-330. doi: 10.1016/j.omtm.2022.03.018. eCollection 2022 Jun 9

Mueller KP, Piscopo NJ, Forsberg MH, Saraspe LA, Das A, Russell B, Smerchansky M, Cappabianca D, Shi L, Shankar K, Sarko L, Khajanchi M, La Vonne Denne N, Ramamurthy A, Ali A, Lazzarotto CR, Tsai SQ, Capitini CM, Saha K. Production and characterization of virus-free, CRISPR-CAR T cells capable of inducing solid tumor regression. J Immunother Cancer. 2022 Sep;10(9):e004446

Oh SA, Senger K, Madireddi S, Akhmetzyanova I, Ishizuka IE, Tarighat S, Lo JH, Shaw D, Haley B, Rutz S. High-efficiency nonviral CRISPR/Cas9-mediated gene editing of human T cells using plasmid donor DNA. J Exp Med. 2022 May 2;219(5):e20211530. doi: 10.1084/jem.20211530. Epub 2022 Apr 22

Ottaviano G, Georgiadis C, Gkazi SA, Syed F, Zhan H, Etuk A, Preece R, Chu J, Kubat A, Adams S, Veys P, Vora A, Rao K, Qasim W. Phase 1 clinical trial of CRISPR-engineered CAR19 universal T cells for treatment of children with refractory B cell leukemia. Clinical Trial Sci Transl Med. 2022 Oct 26;14(668):eabq3010. doi: 10.1126/scitranslmed.abq3010. Epub 2022 Oct 26

Prommersberger S, Reiser M, Beckmann J, Danhof JS, Amberger M, Quade-Lyssy P, Einsele H, Hudecek M, Bonig H, Ivics Z. CARAMBA: a first-in-human clinical trial with SLAMF7 CAR-T cells prepared by virus-free Sleeping Beauty gene transfer to treat multiple myeloma. Clinical Trial Gene Ther. 2021 Sep;28(9):560-571. doi: 10.1038/s41434-021-00254-w. Epub 2021 Apr 13

von Auw N, Serfling R, Kitte R, Hilger N, Zhang C, Gebhardt C, Duenkel A, Franz P, Koehl U, Fricke S, Tretbar US. Comparison of two lab-scale protocols for enhanced mRNA-based CAR-T cell generation and functionality. Sci Rep. 2023 Oct 24;13(1):18160

Zhao Z, Chen Y, Francisco NM, Zhand Y, Wu M. The application of CAR-T cell therapy in hematological malignancies: ad-vantages and challenges. Acta Pharmaceutica Sinica B, 2018 Jul; 8(4): 539–551
Grens K. The Next Frontier of CAR T-Cell Therapy: Solid Tumors. The Scientist. 2019 Apr

* piggyBac® is a registered trademark of Poseida Therapeutics. Sleeping Beauty® is a registered trademark of Regents of the University of Minnesota