Table 2.

CAR-T cell therapy main challenges and potential solutions

Main challenges of CAR-T cell therapy	Potential solutions
Choosing tumor-specific antigen
Diverse expression of TAA in cancer cells Variable and changing levels of antigen expression in different tumor sites Presence of TAA in healthy tissues resulting in cross reactions with regional non-tumor cells	Co-administration of different CAR-T cell products Combining vectors for two separate CARs Bispecific CARs (Dual CAR, tanCAR, iCAR) Trivalent CAR-T cells TRUCKS-synNotch system Nanobody-based antigen recognition domain Targeting cancer stem cells
CAR-T cell trafficking and tumor penetration
Tight connections with tumor-surrounding cells, presence of blood vessels, fibroblasts, and ECM proteins, signaling molecules and decreased levels of oxygen / nutrients Presence of dense fibrotic matrix in the tumor site Mismatching of endogenous T-cell chemokine receptors with tumor-secreted chemokines	Local administration of CAR-T cells in the tumor site Implantable biopolymer devices for direct delivery Transgenic expression of chemokine receptors on CAR-T cells Combination of CAR-T cells with oncolytic viruses Heparinase-secreting CAR-T cells FAP-specific CAR-T cells Co-administration of anti-VEGF antibodies
Immunosuppressive tumor microenvironment
Presence of immune suppressor cells (Tregs, MDSCs, TAMs) Secretion of cytokines, growth factors and chemokines (IL-4, IL-10 and TGF-β) Presence of immune checkpoint molecules / inhibitory pathways (PD-1 or CTLA-4) Increased levels of adenosine and reactive oxygen species	Combination of CAR-T cells and immune checkpoint inhibitors Engineering PD-1 deficient CAR-T cells Depletion of Tregs and/or MDSCs CAR-T cells expressing dominant negative TGF-beta type II receptor Manufacturing CAR-T cells that secrete anti-cancer cytokines (IL-12, IL-15) TRUCKS-synNotch system

CAR chimeric antigen receptor, TAA tumor-associated antigen, tanCAR tandem CAR, iCAR inhibitory CAR, TRUCKS T cells redirected for antigen-unrestricted cytokine-initiated killing, synNotch synthetic notch, FAP fibroblast activation protein, VEGF vascular endothelial growth factor, Tregs regulatory T cells, MDSCs myeloid-derived suppressor cells, TAMs tumor-associated macrophages, TGF-β transforming growth factor-β, PD-1 programmed cell death protein 1, CTLA-4 cytotoxic T-lymphocyte-associated protein 4, IL interleukin