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tumor cell lysis by CAR-T cells on the tumor cells that do not express the CAR target antigen/s [96,97] . These
effects include the direct tumor cell lysis from cytokines secreted as a byproduct from CAR-T cells or tumor
cell lysis caused by immune cells attracted to the tumor by these secreted cytokines. However, it is thought
that the most potent bystander effects are due to epitope spreading. Following CAR-T cell tumor cell lysis,
release/spread tumor debris containing tumor epitopes are released/spread in the TME and taken up by
antigen-presenting cells (APCs). Cross-presentation of these epitopes to lymphocytes in the lymph nodes
ultimately generates an endogenous T cell response against tumor antigens not originally targeted by the
+
[96]
CAR-T cells . There is evidence that tumor-specific CD8 T cells can mediate this process of antigen or
epitope spreading via (i) therapeutic cancer vaccines; (ii) immune checkpoint inhibition; and (iii) adoptive
T cell therapies . During a clinical trial of MSLN-targeting CAR-T cell therapy in patients with MSLN-
[82]
expressing solid malignancies, two patients (one with advanced MPM and the other with metastatic
pancreatic cancer) demonstrated clinical evidence of broad antitumor immune response consistent with
[83]
epitope spreading . Following treatment with MSLN CAR-T cells, new anti-self antibodies were seen in
these two patients , who presented with clinical antitumor activity despite not receiving lymphodepletion
[83]
[83]
therapy before the CAR-T cell infusion . Another study by the same group highlighted that clonal
expansion of endogenous T cells could be induced by anti-MSLN CAR-T cells in several patients with
[98]
advanced solid cancers, which was detected by deep sequencing of the TCR beta chain . This was not
observed in patients receiving lymphodepletion before CAR-T cell transfer . These findings suggest that
[98]
CAR-T cells elicit a ‘vaccine’ effect with potential therapeutic implications. Additionally, CAR-T cells can
induce augmentation of humoral responses, as well as epitope spreading in patients, both of which appear
to be hampered by lymphodepletion. An example of epitope spreading has also been reported with
bispecific antibody (BiAb) therapy. A bispecific T cell engager (BiTE) antibody targeting Wilms’ tumor
protein (WT1) led to the expansion of secondary T cell clones with specificity for TAAs other than WT1 in
vitro co-cultures of patient PBMCs with autologous tumor cells . This study indicates that the therapeutic
[80]
efficacy of BiTEs could be attributed partly to epitope spreading following intracellular tumor antigen
targeting.

One of the few preclinical studies on the immunologic bystander effects of CAR-T cell therapy so far was
conducted on syngeneic animal models of MM . In this study, mesothelin-targeting CAR-T cells were
[84]
unable to eliminate tumors that were < 100% positive for mesothelin, demonstrating the limitation of CAR-
T cell therapy when faced with antigen-heterogenous tumors. The authors tried to augment therapeutic
efficacy by inducing bystander effects in various ways and found that pre-treatment of mice with a low,
non-lymphodepleting dose of cyclophosphamide (CTX), followed by CAR-T cell therapy, had a curative
[84]
effect in tumors that were < 100% mesothelin positive . This effect was demonstrated to be dependent on
endogenous CD8 T cells but not on basic leucine zipper transcription factor ATF-like 3 (BATF3)-dependent
[84]
type 1 dendritic cells (DCs) . In another study by Lai et al., T cells were engineered to secrete the dendritic
cell (DC) growth factor Fms-like tyrosine kinase 3 ligand (FLT3L). FLT3L-secreting T cells expanded
intratumoral conventional type 1 dendritic cells (DCs) and substantially increased host DC and T cell
[99]
activation when combined with immune agonists poly (I:C) and anti-4-1BB . These data reveal that
augmenting endogenous DCs is a promising strategy to overcome the clinical problem of antigen-negative
[99]
tumor escape following adoptively-transferred cell therapy . A study using a syngeneic mouse melanoma
model with defined tumor antigens showed that epitope spreading after treatment with TCR-engineered T
cells targeted to the melanoma tumor antigen gp100 was minimal . However, epitope spreading mediated
[100]
by BATF3-dependent DCs and bystander effects was seen following co-transduction of T cells with a second
TCR that recognizes a bacterially-expressed antigen (ovalbumin) and treating tumors with modified Listeria
[100]
bacteria that expresses ovalbumin .