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surface of T cells affects the CAR-T cell activation; lower levels of CAR expression results in sub-activation
of the CAR-T cells, while the overexpression of CARs can induce antigen-independent activation,
[68]
accelerating cell differentiation and exhaustion, or apoptosis . Avidity or functional avidity generally refers
to the interaction between the receptor and the antigen. Avidity is the multimeric interaction between the
tumor cell and the T-cell, whereas functional avidity is the functional response of a T cell (e.g., cytokine
[69]
secretion or cytotoxicity) . An overall increase in the affinity or avidity of CAR-T cells typically induces a
more robust response . Furthermore, tumor antigen-mediated downregulation of CARs also affects CAR
[70]
density on the cell surface . This downregulation of CAR on the cell surface is independent of CAR affinity
[71]
and associated with T cell CAR density and TAA density . CAR scFvs, in general, have a higher affinity to
[72]
the target antigen compared to the affinity of TCRs. Considering the majority of the TAAs are self-antigens
that are overexpressed on tumor cells and are weakly expressed on normal cells, there is a tendency for the
high-affinity scFv CARs to attack normal cells . This on-target, off-tumor toxicity may raise safety
[73]
concerns and is one of the inevitable and common adverse effects of CAR-T cell therapy. The new
generation of the CARs has modified scFvs with optimized affinity, enabling CAR-T cells to favorably attack
[74]
tumor cells with overexpressed TAAs . Remarkably, it is now possible to generate a large pool of
antibodies with a wide range of affinity, each targeting the same epitope with a different affinity performed
by light chain exchange technology. This approach provides a feasible option to screen a number of scFvs
simultaneously and enables determining the best scFv affinity required for the activation of the CAR-T
cell . Therefore, understanding the affinity and interaction between the TAA density on tumor cells and
[75]
the scFv density on the CAR-T cells will contribute to developing strategies for better CAR-T cell therapies.

Tumor antigen escape
TAA heterogeneity and/or high mutation rates are linked to the tumor antigen escape from immune
recognition, which occurs by modulation of targeted TAA expression. In solid tumors, loss of target antigen
and/or mutations in the target antigen are frequent observations post CAR-T cell infusion ; in such cases,
[76]
employing an additional target is one possible solution. Even in hematological cancers, patients have
exhibited resistance to CAR-T cell therapy and the emergence of tumor antigen escape variants is a primary
concern, accounting for about 7%-25% of relapsed patients post therapy with CD19-targeting CAR-T
[59]
cells . The most common mechanisms of this include antigen loss due to mutations, splice variants of the
[77]
gene, and lineage switching from CD19+ to CD19- (lymphoid to myeloid malignancy) . In the case of solid
tumors with an inherent heterogeneous expression of the tumor antigens, antigen escape is further
[60]
exacerbated . One of the studies in this field found that heterogeneous expression of TAA EGFRvIII and
the inhibitory TME, which becomes even more immunosuppressive after CAR-T cell infusion, were
significant barriers to the clinical efficacy of CAR-T cell therapy . While the heterogeneous expression of
[76]
TAAs could be overcome by CARs with dual or triple antigen targeting, the TME would require drug
targeting against its immunosuppressive components. In a recent study by Larson et al., a genome-wide
CRISPR knockout screen was conducted in glioblastoma, in which CAR-T cell therapy has limited efficacy,
and it was found that the loss of interferon-γ receptor (IFNγR) signaling pathway genes, such as JAK1 or
JAK2 and IFNGR1, rendered glioblastoma and other solid tumors more resistant to killing by CAR-T cells
both in vitro and in vivo . This study further reported that the defect in IFNGR1 signaling is one of the
[78]
important mechanisms of tumor cell-intrinsic resistance to CAR-T cells, which may require improved
binding interactions between the CAR-T cells and the tumor cells for better outcomes in solid tumors .
[78]

APPROACHES TO OVERCOME TAA HETEROGENEITY
Several modifications to the CAR design and translational strategies have aided in overcoming TAA
heterogeneity to some extent in recent years. First is the choice of the target antigen, which can be focused
on the ones associated with tumor invasion or metastasis, so the CAR-T cells generated are directed to more
aggressive cancer cells. Another strategy is targeting multiple antigens or having the CAR deliver “payloads”