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Most investigators have been using γ retroviruses these are relatively easy to produce, they efficiently and permanently transduce T cells, and they have preliminarily proven safe from an integration standpoint in primary human T cells. This system 13 is now entering clinical trials as an approach to engineer T cells. 10, 11 Sleeping Beauty was shown to provide efficient stable gene transfer and sustained transgene expression in multiple cell types, including T cells, 12 but the culture time remains relatively prolonged. Transposon-based systems can integrate transgenes more efficiently than plasmids that do not contain an integrating element. The long-term culture may be detrimental to the activity and persistence of the infused cells, and the antibiotic-resistance gene products may render them immunogenic. This method requires long-term culture and antibiotic selection due to the relative inefficiency of gene transfer. Non–viral-based DNA transfection was initially used because of cost and the low risk of insertional mutagenesis. 9 Each has advantages and disadvantages with regard to cost, safety, and level of expression.
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Investigators in the field are using a variety of methods to introduce their CAR constructs into T cells. 6-8 A major challenge to the field is that it is currently necessary to empirically test these design variables as there are no general rules guiding CAR design for target molecules. 5 The length, flexibility, and origin of the hinge domain is also an important variable in the design of CARs. Preclinical data suggest that the spatial location of epitope binding has a bigger effect on CAR activity than variation in affinity.
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4 Unlike TCRs, where a narrow range of affinity dictates the activation and specificity of the T cell, CARs typically have a much higher and perhaps broader range of affinities that will engage the target without necessarily encountering cross-reactivity issues. 3 Individual scFvs targeting a surface molecule are either derived from murine or humanized antibodies or synthesized and screened via phage display libraries. CARs typically engage the target via a single-chain variable fragment (scFv) derived from an antibody, although natural ligands have also been used. 2 Unlike TCRs, CARs engage molecular structures independent of antigen processing by the target cell and independent of MHC. Anatomy of CARs and CAR T-cell productsĬARs are synthetic, engineered receptors that can target surface molecules in their native conformation. We limit our discussion to CAR T cells in hematologic malignancies and will not cover CARs that have been tested in solid tumors or engineered T-cell receptors (TCRs) that have been tested in any setting. We will also discuss the emerging toxicity profiles and management strategies and future outlook of CAR T-cell therapies.
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Here, we will introduce the various CAR designs that have been tested clinically, the results from a series of clinical trials testing CAR T cells, and an overview and comparison of the manufacturing processes used. It is not surprising that CAR T cells directed to hematologic malignancies have been the first ones tested, given the extent of the known surface antigens expressed on hematologic cells, the relative ease of sampling tumor, and the natural preference of T-cell homing to hematologic organs such as the blood, bone marrow, and lymph nodes. In the last 5 years, chimeric antigen receptor (CAR)-redirected T cells have emerged from the bench and made splashy headlines in the clinical setting at a number of academic institutions. 1 The main barriers to this approach have been the difficulty in culturing and manufacturing of tumor-infiltrating lymphocytes, immune tolerance to self-antigens, and the requirement for major histocompatibility complex (MHC) presentation of antigens ( Figure 1). Adoptive T-cell therapy also offers this possibility but has thus far been limited in application to those patients with melanoma who have adequate culture and expansion of isolated tumor-infiltrating lymphocytes. This is the basis for the US Food and Drug administration (FDA) approval of interleukin-2 (IL-2) in melanoma more recent immune therapies that are FDA-approved treatments for cancer involve checkpoint blockade, which is a form of releasing the brakes on tumor-specific T cells and allowing them to persist and expand in vivo, leading to control or regression of cancer. Immune-based therapies for cancer have the tantalizing possibility of effecting long-term durable remissions and perhaps even offering the possibility of a cure.