A Molecular Spotlight on CAR-T Cells

Introduction

Chimeric antigen receptor-redirected T cells (CAR-T cells) have become a promising tool in the treatment of hematopoietic malignancies as well as solid tumors. CARs expressed on T cells target surface antigens in their native conformation, independent of the major histocompatibility complex. In CAR-T cell therapies the collection of patient’s T cells is followed by a genetic modification of these immune cells to obtain the expression of chimeric antigen receptors specific for the tumor/cancer cell of interest. After the characterization and expansion ex vivo, they are re-infused to the patient to fight specifically against the patient’s tumor cells.

CAR-T cell therapy procedure

The increasing number of clinical trials in recent years reflects the potential of CARs and hope of researchers worldwide to fight cancer with this individualized and therefore regulatory- and cost-intensive immunotherapy.

The molecular background of CARs

CARs consist of a single-chain variable fragment (scFv) from an antibody as the ectodomain, which provides antigen-specificity, a hinge domain that varies in length, flexibility and origin, as well as the transmembrane domain and the endodomain, which is the signaling domain of the receptor.As of today, four different generations of CARs have been investigated, mainly differing in their signaling domain:

Figure 2: Chimeric antigen receptors. scFv: single-chain variable fragment, Co-Stimulation 1: CD28, 4-1BB/CD137), Co-Stimulation 2: CD27, CD28, ICOS, 4-1BB, OX40 VH

Adapted from Nature Reviews Clinical Oncology 13, 25-40 (2016)

CARs of the first generation function by binding the target to the single chain fragment of the antibody’s variable region. T cell activation is mediated by the TCR CD3ζ pathway. This CAR molecule was already described in 1998 by Zelig Eshhar. By adding a co-stimulatory element to the signaling domain, it was possible to augment and prolong T cell activation, leading to the development of the second generation of CARs. The third generation combines two co-stimulatory domains.

The newest generation of CARs, the so-called TRUCKs (T cell redirected universal cytokine killing), carries a cytokine transgene cassette and expresses cytokines once the CAR is activated in the target tissue. This initiates an immune response that hits those cancer cells that might be unrecognizable to the CAR-T cell.

Target antigens depend on the target disease. B-lineage antigens, such as CD19, have been extensively investigated. However, more and more antigens are being tested in clinical trials. The table below shows examples of on-going clinical trials for CAR-T cell therapies^1,2.

Acute Lymphoblastic Leukemia (ALL)

CD19, CD19EGFRt

Chronic Lymphocytic Leukemia (CLL)

CD19

Non-Hodgkin Lymphoma

CD19, CD30

Hodgkin lymphoma

CD30

B-cell malignancies

CD19

CD33+ Acute Myeloid Leukemia (AML)

CD33

CD20+ Leukemia and Lymphoma

CD20

Neuroblastoma

GD2

Multiple myeloma

CD138

AML, Myelodysplastic Syndromes, Multiple Myeloma

Lewis-Y

Manufacture of CAR-T cells

The manufacturing process of gene-modified CAR-T cells requires further optimization in order to result in safe, clinically effective and robustly reproducible products regardless of the inter-patient variability. Furthermore, to ensure the availability of CAR-T cells in time for all patients that require a treatment, the process should be time- and cost-effective³. Especially for aggressive lymphomas the time aspect is a matter of life and death. Popular genomic-editing procedures for modification of T cells are viral transduction and electroporation - both having advantages and disadvantages.

With viral transduction, an integration into the host genome and low intrinsic immunogenicity can be achieved. Many groups are working with gamma-retroviral vectors. These, however, can only integrate into dividing cells, are susceptible to silencing by the host, and are limited in DNA size. An alternative to overcome these restrictions are lentiviral vectors, which are shown to have no random integration sites and do not favor proto-oncogene or tumor suppressor gene loci. Furthermore, the use of adenovirus and adenovirus-associated viruses for long-term episomal transgene expression is possible⁴. Followed by viral transduction, the CAR-T cells need to be expanded and stimulated by antibodies and/or cytokines.

Electroporation can minimize expenses and manufacturing difficulties associated with recombinant viral vectors. However, in order to achieve sufficient delivery and keep the functionality of the T cells, improved electroporation technologies specialized on primary cells (e.g. Nucleofector^TM Technology) could be advantageous.

Transposon system-based manufacture of CAR-T cells via electroporation

By using transposon systems like the Sleeping Beauty (SB)^5,6 system or piggyBac⁷ combined with culturing on artificial Antigen Presenting Cells (aAPCs), it is possible to achieve stable integration and the selective proliferation of CAR-T cells.

As a brief overview of the procedure, T cells are collected and transfected using electroporation, with a DNA plasmid carrying an IR/DR flanked CAR transposon as well as a DNA plasmid encoding a transposase. The mixed population of untransfected T cells as well as successfully transfected CAR-T cells with the CAR expression cassette integrated is then cultured on APCs (e.g. K562 cells that express endogenous molecules for T cell activation and proliferation). Thus, only the CAR expressing T cells are selected for re-infusion into the patient’s body.

Alternatively, electroporation of CAR mRNA has been investigated. This method enables a high level of CAR expression for a limited time of 3-5 days. This ensures an important safety feature for patients by “testing” for severe side effects before treating long-term.

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. There is still a lot of research needed to see CAR-T cell immunotherapy to its full potential. Lonza is committed to supporting your CAR-T cell research. That’s why we offer the Nucleofector^TM Technology and corresponding T cell Nucleofector^TM Kits for efficient T cells transfection. In addition, we provide human PBMCs as well as purified CD4+ T cells from a variety of different donors. Please contact Scientific Support for more information.

Selected References

Antibody-modified T cells: CARs take the front seat for hematologic malignancies
https://www.ncbi.nlm.nih.gov/pubmed/24578504
Genetically modified T cells in cancer therapy: opportunities and challenges
https://www.ncbi.nlm.nih.gov/pubmed/26035842
Towards a commercial process for the manufacture of genetically modified T cells for therapy
https://www.ncbi.nlm.nih.gov/pubmed/25613483
Going viral: chimeric antigen receptor T cell therapy for hematological malignancies
http://www.ncbi.nlm.nih.gov/pubmed/25510272
A new approach to gene therapy using Sleeping Beauty to genetically modify clinical-grade T cells to target CD19
https://www.ncbi.nlm.nih.gov/pubmed/24329797
Manufacture of clinical-grade CD19-specific T cells stably expressing chimeric antigen receptor using Sleeping Beauty System and artificial antigen presenting cells
https://www.ncbi.nlm.nih.gov/pubmed/23741305
Anti-proliferative effects of T cells expressing a ligand-based chimeric antigen receptor against CD116 on CD34⁺ cells of juvenile myelomonocytic leukemia
https://www.ncbi.nlm.nih.gov/pubmed/26983639

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