CAR-T cells: the long and winding road to solid tumors

Abstract

Adoptive cell therapy of solid tumors with reprogrammed T cells can be considered the "next generation" of cancer hallmarks. CAR-T cells fail to be as effective as in liquid tumors for the inability to reach and survive in the microenvironment surrounding the neoplastic foci. The intricate net of cross-interactions occurring between tumor components, stromal and immune cells leads to an ineffective anergic status favoring the evasion from the host's defenses. Our goal is hereby to trace the road imposed by solid tumors to CAR-T cells, highlighting pitfalls and strategies to be developed and refined to possibly overcome these hurdles.

Fig. 1. Schematic representation of the chimeric antigen receptors for adoptive cell therapy.

CARs comprise an extracellular domain with a tumor-binding moiety, typically a single-chain variable fragment (scFv), followed by a hinge/spacer of varying length and flexibility, a transmembrane (TM) region, and one or more signaling domains associated with the T-cell signaling. The 1^st CARs generation is equipped with the stimulatory domain of the ζ-chain; in the 2^nd CARs generation the presence of costimulatory domains (CD28) provides additional signals to ensure full activation; in the 3^rd generation an additional transducer domain (CD27, 41-BB or OX40) is added to the ζ-chain and CD28 to maximize strength, potency, and duration of the delivered signals; the 4^th generation includes armored CARs, engineered to synthetize and deliver interleukins (green ovals)

Fig. 2. Simultaneous targeting of two antigens may serve to enhances (Tandem CAR) or cut down (iCAR) the activity of the CAR-T cells.

Tandem CARs (TanCAR) mediate bispecific activation of T cells through the engagement of two chimeric receptors designed to deliver stimulatory or costimulatory signals in response to an independent engagement of two different tumor associated antigens (TAAs). iCARs use the dual antigen targeting to shout down the activation of an active CAR through the engagement of a second suppressive receptor equipped with inhibitory signaling domains

Fig. 3. The Transposon systems of Sleeping Beauty (SB) and PiggyBac (PB) for gene delivery.

Transposition is possible through a dual vector system that comprises the transposon containing the transgene flanked by two inverted terminal repeats, and a transposase that mobilizes the transposon. The CAR is integrated into the genome through a cut-and-paste mechanism SB transposon vectors are characterized by the presence of specular IR/DR sequences, target for the transposase. The SB vector contains the gene of interest (CAR). The SB transposase (SB-100×) binds to the IR/DR sequences and cuts the vector to release the transposable portion of DNA. TA sequences in the host DNA act as acceptors of the transposed element. The PB transposon is a mobile genetic element that transposes the gene of interest (CAR) from the vector to the host DNA. The l’hyperactive PiggyBac (Hy7 PB) transposase recognizes the transposon-specific “inverted terminal repeats” sequences (ITRs) located at the ends of the gene of interest. Transposition occurs between two TTAA acceptor sites located in the host DNA

Fig. 4. Comparison of viral versus transposon-based gene delivery systems.

Plasmid-based transposon systems combine the advantages of integrating viral vectors with those of non-viral delivery systems

Fig. 5. In the CD16-CR, the chimeric receptor extracellular portion is engineered to express an FcγR domain able to complex virtually any mAb directed against TAAs expressed by malignant cells.

The FcγR module is combined to a hinge region, a transmembrane domain, and to the intracellular signaling domains of the ζ-chain and CD28. Advantages and disadvantages of the CD16-CR are illustrated