In this review, it’s provided an overview of the major mechanisms used by tumor cells to evade immune defenses and are critically exposed the most optimistic engineering strategies to make CAR-T cell therapy a solid option for solid tumors.

CAR-T cell based cell therapy is a moving field, which showed impressive results in hematopoietic cancer management and brought hope to incurable patients. Unfortunately, success in managing solid cancers was less outstanding. Assiduous research has been done to overcome unexpected roadblocks which impede CAR-T cells trafficking, infiltration, persistence or function in the unwelcoming tumor environment. Indeed, research focused on identifying target antigens and avoiding on-target-off tumor toxicity, improving CAR-T cell trafficking and entry into the tumor site, promoting better signaling, less exhaustion, and memory phenotypes in solid tumors. Preclinical models propose various engineering strategies, some of which have already advanced from bench to bedside, with encouraging preliminary results.

As reviewed herein, trafficking and infiltration have been addressed by genetically manipulating chemotaxis and tissue homing. Moreover, tumor stroma targeting emerged as a promising strategy, based either on depletion of stromal cells/immunosuppressive cells or at reprogramming strategies directed at regulating TME plasticity. To this regard, a new generation of CAR-T cells has been designed to directly target stroma components like fibroblasts and immunosuppressive cells (Tregs, TAMs or MDSCs). However, a remaining challenge for the development of both effective and safe CAR-T cell therapies is the insufficient clinical relevance of preclinical mouse models. Indeed, these models sometimes failed to predict clinical level toxicities or, on the other hand, inefficient tumor targeting when translated to the clinic. Further research is still needed to overcome this hurdle and develop advanced preclinical models able to address tumor heterogeneity and TME complexity in order to accomplish a perfect balance between efficacy and safety of CAR-T cell therapies in solid tumors.

Furthermore, exciting new opportunities emerged thanks to gene editing/gene ablation techniques based on the revolutionary, highly specific and efficient CRISPR/Cas9 tools, which have been used not only to generate immune-checkpoint knock-outs (PD-1 KO) but also to design “universal” CARs, edited for TCR and/or HLA molecules expression, which could pave the road towards cost-effective allogeneic CAR-T cells for an “off-the-shelf” ACT with a broader spectrum. This technique can even be used for multiplexed genome editing. To this regard, feasibility of targeting multiple genes in T cells by multiplex CRISPR-Cas9 has recently been proven in a small interventional study in patients with advanced, refractory cancer (NCT03399448). Further improvements of this technology are awaited as recent advances seem to insure increased precision and minimized side effects both in case of gene deletion and gene insertion. As allogeneic (allo)-CAR-T cells could offer readily available ACT sources that could expand the usage of CAR-cells based immunotherapy, other recent strategies for allo-CAR-T cells generation emerged, like the NKG2D (an NK-based activating receptor) expression. NKG2D expression in allogeneic CAR-T cells offers a non-TCR edited cellular therapy with broad solid tumor targeting, and two clinical trials are ongoing in metastatic colon cancer (NCT04991948 and NCT03692429, Celyad Oncology) with encouraging preliminary results in the second one (2/15 PR and 9/15 SD). Another allo-CAR-T cells product, the CD70-targeting ALLO-316 cells (Allogene Therapeutics) is under evaluation in a clinical trial on renal cell carcinoma (together with anti-CD52 mAb, NCT04696731).

On the other hand, a part from innovations in CAR design addressed in this review, advances in transduction techniques, cell culture and amplification conditions (like IL-7/IL-15 media) as well as identification of the most suitable stage of T cell differentiation (TCM/TSCM) to use for adoptive transfer represent additional steps towards effective CAR-T cell therapy in solid tumors. To this regard, the need for large-scale CAR-T manufacturing persists and could limit cancer patients’ accessibility to CAR-T cell-based ACT. Therefore, the already engaged transition from academic to industrial manufacturing could ensure increased availability and reproducibility as well as shorter delays thanks to Good Manufacturing Practice (GMP)-compliant automated, closed systems. Contrary to large scale, commercial in vitro manufacturing, Smith and colleagues recently described an in-vivo manufacturing technique of CAR-T cells, by programming circulating, bloodstream T cells with DNA-carrying polymer nanoparticles, which efficiently introduced leukemia-targeting CAR genes into T-cell nuclei.Accumulating knowledge on efficacy, toxicity and resistance drawn from clinical trials as well as fundamental research data on TILs interaction with the TME will allow for the identification of novel molecular targets in CAR-T cells design. To this regard, and pointing out once more the role of the hypoxia response in cancer, the VHL-HIF axis and particularly HIF’s activity, has recently been identified as a tool to potentiate tissue residency of CD8+ CTLs, as well as a potential molecular candidate to modulate CAR-T cell therapy efficacy. Genetic targeting of precise molecular or metabolic pathways critical for TILs survival in the TME emerge therefore as novel strategies to overcome insufficient amplification and persistence of CAR-T cells in solid tumors.

This review focuses less on engineering strategies aiming at enhancing tumor recognition and preventing antigen escape. Such combinatorial targeting strategies employ bispecific/dual CARs or Tandem CARs, trivalent or pool CARs and have already been reviewed by autors and others. Bispecific CARs have gained an important place in hematologic cancers management, with numerous ongoing clinical trials (NCT04662099, NCT0327115, NCT03919526, NCT03879382, NCT03881761, NCT03706547, NCT04303520, NCT04412174, NCT03825731, NCT04499573, NCT05098613, NCT04034446, NCT04007029, and NCT04215016). However, usage of bispecific CARs in solid tumors is still at its beginning (NCT03672305, NCT04483778, NCT03618381 and NCT04684459). Nonetheless, multiple antigen targeting by employing universal immune receptors CAR also gains increasing interest and a universal CAR is being tested in a clinical trial on prostate cancer patients (UniCAR02, NCT04633148). Moreover, toxicity management strategies and especially prevention of on-target off tumor effects were not thoroughly described here-in but were reviewed previously. Switchable CARs for instance emerge as valuable safety strategies, like is the case of the iCas9 safety suicide switch employed in some ongoing clinical trials (NCT04715191, NCT03721068). Moreover, orthogonal switchable CARs or dual-switch CAR-T cells capable of both regulated costimulation/inducible activation to drive CAR-T cell expansion and activity and regulated iCasp9 safety switch for CAR elimination have recently been described. Co-activated switchable CAR-T cells also advanced to clinical testing (ongoing clinical trial NCT02744287 of BPX-601 CAR-T cells expressing a PSCA specific CAR and a rimiducid-inducible MyD88/CD40 co-activation switch, Bellicum Pharmaceuticals), with encouraging preliminary results.

Besides optimization of costimulatory domains discussed here-in, modulation of scFv avidity could be another strategy to increase antigen recognition and CAR-T cell engagement. Surprisingly, lower avidity CAR-T cells (the 8F8-BBz CAR-T cells) could show greater therapeutic potential, by increased resistance to exhaustion and apoptosis in an HCC context.

Considering all the aforementioned hurdles in CAR-T cells homing, as well as the diversity and plasticity of cells composing the tumor microenvironment, the best engineering option could be based on a combination of strategies that enhance at the same time trafficking, penetration, persistence and/or CAR-T cell function. Some combinations have already been tested, like it is the case of armored CAR-T cells/TRUCKs engineered to co-express chemokine receptors and secrete vital cytokines. As monotherapeutic approaches are rarely effective, strategies targeting multiple antigens, combinations of different genetic engineering strategies or combinations based on CAR-T cells and innovative immunotherapies (like ICIs) could represent a turning point in a still ongoing revolution in solid cancer management. Nonetheless, CAR-T cells could also be combined with other therapeutic modalities, such as standard chemotherapy and/or radiation therapy, tyrosine kinase inhibitors, epigenetic modulators, other small molecule drugs or vaccines.

All in all, CAR-T cell immunotherapy stands out as a promising, evolving weapon in the fight against solid cancer.

Beside CAR-T cell based ACT, novel genetic engineering techniques, such as gene-editing and cellular reprogramming allowed for the emergence of new ACT strategies employing various innate killer cells (IKC) (like NK cells, NKT cells, and γδ T-cells (CAR-IKC), macrophages (CAR-M) and even B lymphocytes (CAR-B cells). A combination of “classic” CAR-T cells and CAR- IKC/CAR-Macrophages as bridging therapy could potentially increase efficiency in solid tumors by increasing the cross-talk between various immune cells or by TME remodeling effects. Unfortunately, CAR-NK also have some limitations, as for example high-dosage conditioned efficacy and decreased persistence. On the other hand, co-administration of cord-blood derived-NK cells (CB-NKs) proved to be a potent immunoregulatory strategy, promoting early activation and migration, enhanced fitness and increased anti-tumor efficacy of CAR-T cells. Surprisingly, chimeric receptor engineered Tregs (CAR-Tregs), which emerged as potential immune-tolerance inducers in autoimmunity or transplantation, also showed potent anti-tumor effect. Moreover, CAR-B cells, which could represent safe and controllable vehicles for local delivery of monoclonal antibodies emerged in preclinical studies as potential candidates for infectious diseases and protein deficiencies and might therefore be interesting candidates for cancer therapy as well.