From T-cell Engineering to CAR therapy: Progress and Prospects
Authors: Sadelain Michel 
Published: October 2019

The genetic engineering of T cells provides a means to rapidly generate anti-tumor T cells for any cancer patient. This approach is predicated on gene transfer technology that enables the expression of receptors for antigen and other gene products in primary T cells. Tumor targeting may be achieved through the transfer of a physiological receptor for antigen, which is known as the T cell receptor (TCR), or synthetic fusion receptors, which we grouped under the general term of chimeric antigen receptor (CAR).
CARs are recombinant receptors for antigen, which, in a single molecule, redirect T cell specificity and eventually enhance anti-tumor potency. Functional augmentation is achieved through the design of second generation CARs, which not only redirect cytotoxicity, but also reprogram T cell function and longevity through their costimulatory properties. The combined activating and costimulatory domains incorporated in second-generation CARs critically determine the function, differentiation, metabolism and persistence of engineered T cells. CD19 CARs that incorporate CD28 or 4-1BB signalling domains are the best known to date.
Two decades ago, we selected CD19 as the prime target for developing our CAR technology and provided the first proof-of-principle that CD19-targeted human peripheral blood T cells could eradicate a broad range of B cell malignancies in immunodeficient mice (Brentjens RJ, Riviere I, et al. Eradication of systemic B-cell tumors by genetically targeted human T lymphocytes co-stimulated by CD80 and interleukin-15. 2003;9(3):279-86). CD19 has since become the poster child for CAR therapies. Complete remissions have been reported from several centers in patients with non-Hodgkin lymphoma, chronic lymphocytic leukemia and, most dramatically, acute lymphoblastic leukemia. Two types of second generation CARs, utilizing either CD28 or 4-1BB as their costimulatory signaling components, have been used in ALL patients. Both have yielded dramatic outcomes, in adults as well as in children.
Our data indicate that CD28-based CARs direct a brisk proliferative response and boost effector functions, while 4-1BB-based CARs direct a gradual T cell accumulation that may eventually overcome lesser functional potency. These distinct kinetic features can be exploited to further develop CAR T cell therapies for a variety of cancers.
We have now modeled CD19 CAR therapy for ALL in a "stress test", wherein we purposefully lower the infused T cell doses to challenge the CAR therapy. We have compared novel CAR designs intended to recruit both CD28 and 4-1BB signaling. These quantitative analyses reveal striking disparities that hinge on subtle variations in the structural design of CARs and co-expressed costimulatory molecules. Remarkably, we find that some of the most effective engineering strategies activate and sustain the recruitment of the IFNβ pathway through the induction of IRF7, while lowering the induction of exhaustion markers relative to second generation CARs activating either CD28 or 4-1BB alone.
The field is thus poised to move beyond the CD28 vs 4-1BB debate, which will be rendered obsolete by the emergence of superior CAR designs that coopt the use of costimulatory ligands, cytokines and/or checkpoint blockade inhibitors. A new field of immunopharmacology is emerging.

Disclosures
Sadelain: Juno Therapeutics: Consultancy, Equity Ownership, Other: Co-Founder, stockholder, Patents & Royalties: Licensed patents on CARs.