Graphical abstract

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Highlights

     * •
       Immunocyte-tropic LNP-delivered circRNAs form in vivo panCAR
       (CAR-T/NK/Mφ) cells
     * •
       In vivo panCAR effectively inhibits tumor growth and reshapes the
       TME in mice
     * •
       CircRNA vaccines synergistically boost the anti-tumor efficacy of
       in vivo panCAR
     * •
       Depleting NK cells or macrophages impairs the efficacy of in vivo
       panCAR-VAC
     __________________________________________________________________

   Wang et al. establish circRNA-based in vivo panCAR (CAR-T/NK/Mφ)
   immunotherapy, effectively inhibiting tumor growth and reprogramming
   the tumor microenvironment in mice. In combination with vaccination, in
   vivo panCAR demonstrates a synergistic enhancement of anti-tumor
   immunity across various mouse models, thereby establishing a framework
   for the synergistic in vivo panCAR-VAC immunotherapy.

Introduction

   Chimeric antigen receptor (CAR) T cell therapy is an important method
   of cancer immunotherapy that has successfully treated hematologic
   malignancies, particularly B lymphomas.[58]^1 However, CAR-T cell
   therapy still faces several challenges, including high costs, a
   time-consuming manufacturing process, the necessity of lymphodepletion,
   and side effects such as cytokine storm and on-target, off-tumor
   killing.[59]^2^,[60]^3^,[61]^4^,[62]^5 It is important to solve these
   issues and improve the effectiveness of adoptive CAR-T cell therapy.

   To address the limitations of conventional adoptive CAR-T cell therapy,
   shorten preparation and processing times, and decrease treatment
   costs,[63]^6 researchers recently reported that in vivo delivery of
   CAR-encoding mRNAs to T cells or macrophages could generate functional
   CAR-T or CAR-macrophage (CAR-M) to exert tumor-killing
   function.[64]^7^,[65]^8^,[66]^9^,[67]^10^,[68]^11 This innovative
   approach has demonstrated efficacy in treating hematologic malignancies
   and myocarditis in murine models, providing an off-the-shelf
   immunotherapy strategy with significant potential for therapeutic
   applications.[69]^7^,[70]^8^,[71]^9^,[72]^10^,[73]^11 Compared to DNA
   transposase-mediated[74]^7 or lentivirus-based[75]^12^,[76]^13
   delivery, mRNA technology provides a significant safety advantage
   without integrating into the genome.

   Similar to mRNAs, circular RNAs (circRNAs) are also not integrated into
   the genome. However, unlike the linear conformation of mRNA, circRNAs
   are covalently closed circular RNA molecules that are more stable than
   linear mRNAs in mammalian cells.[77]^14^,[78]^15^,[79]^16 Therefore,
   circRNA-based in vivo CAR might enable higher and more durable
   expression of CAR proteins on the cell membrane of functional immune
   cells than mRNAs, holding the potential to generate more effective
   killing effects.

   Recent studies have also shown that the combination therapy of adoptive
   CAR-T therapy and vaccination could enhance anti-tumor immunity, which
   might occur through further in vivo activation of reinfused CAR-T cells
   via the interaction between antigen-presenting cells expressing
   transmembrane tumor antigens and the reinfused CAR-T cells expressing
   CAR molecules against the corresponding
   antigens.[80]^17^,[81]^18^,[82]^19^,[83]^20 However, while previous
   studies have explored combining adoptive CAR-T cell therapy with
   vaccines, the potential effects of in vivo CAR immunotherapy on cancer
   vaccines still remain unclear. Moreover, the anti-tumor activity of
   vaccination-elicited antibodies, especially the antibody-dependent
   cellular cytotoxicity (ADCC) or antibody-dependent cellular
   phagocytosis (ADCP) effects, still remains to be clarified, while the
   therapeutic antibody drugs were widely used in treating cancers, such
   as anti-programmed cell death protein 1 (PD-1)/programmed cell death
   ligand 1 (PD-L1) antibodies and anti-human epidermal growth factor
   receptor 2 (HER2) antibodies.[84]^21^,[85]^22 Therefore, it is tempting
   to explore whether circRNA vaccines could synergistically boost the
   anti-tumor immunity of circRNA-based in vivo CAR. Given the potential
   superiority of circRNAs in generating CAR molecules and producing
   transmembrane tumor antigens in vivo, we attempted to develop the
   combination immunotherapy between circRNA-based in vivo CAR and cancer
   vaccines, aiming to synergistically enhance the anti-tumor activity of
   in vivo CAR via simultaneously mobilizing both the adaptive and innate
   immune responses.

Results

CircRNA^CAR efficiently expressed CAR proteins in both human primary T cells
and macrophages

   We employed the group I intron self-splicing strategy to produce
   circRNAs that encode anti-HER2 CAR transmembrane proteins, designated
   as circRNA^CAR ([86]Figure 1A; [87]Table S1).[88]^16^,[89]^23 We also
   generated control circRNA, termed circRNA^Ctrl, which was an empty
   vector comprising circRNA circularization elements without the
   protein-encoding sequence. To assess the translation efficacy of
   circRNA^CAR, we transfected the prepared circRNA^CAR into HEK293T
   cells. Western blot revealed clear CAR protein expression ([90]Figure
   1B), and flow cytometry analysis further confirmed the expression of
   CAR proteins ([91]Figure 1C). Prolonged CAR protein expression is
   crucial for in vivo CAR-mediated functionality. To compare circRNA^CAR
   and its linear counterpart mRNA^CAR, we also generated
   1-methylpseudouridine-modified mRNA (1mΨ-mRNA) and unmodified mRNA
   through in vitro transcription. The flow cytometry result showed that
   circRNA^CAR exhibited significantly higher and more durable CAR
   expression ([92]Figure 1D). The elevated levels and prolonged presence
   of CAR proteins produced by circRNA^CAR suggest the potential
   superiority of circRNA-based in vivo CAR immunotherapy.

Figure 1.

   [93]Figure 1
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   CircRNA^CAR efficiently expressed CAR proteins and mediated remarkable
   tumor killing

   (A) Schematic representation of circRNA^Anti-HER2-CAR circularization
   via group I intron autocatalysis.

   (B and C) Detecting the expression of CAR proteins in HEK293T cells
   after circRNA^Anti-HER2-CAR transfection via western blot (B) and flow
   cytometry (C).

   (D) Comparative analysis of CAR expression levels from
   circRNA^Anti-HER2-CAR, 1mΨ-mRNA^Anti-HER2-CAR, and unmodified
   mRNA^Anti-HER2-CAR in HEK293T cells.

   (E–G) Optimization of circRNA^Anti-HER2-CAR encoding CAR in Jurkat (E),
   THP-1 (F), and J774A.1 (G).

   (H) Detection of CAR expression in primary T cells using flow
   cytometry.

   (I–K) Cytotoxic effects of primary T cells transfected with
   circRNA^Anti-HER2-CAR on SK-OV-3 (I), B16F10-HER2 (J), and 4T1-HER2 (K)
   tumor cells.

   (L–N) Cytotoxic effects of Jurkat cells transfected with
   circRNA^Anti-HER2-CAR on SK-OV-3 (L), B16F10-HER2 (M), and 4T1-HER2 (N)
   tumor cells.

   (O–Q) Cytotoxic effects of THP-1 cells transfected with
   circRNA^Anti-HER2-CAR on SK-OV-3 (O), B16F10-HER2 (P), and 4T1-HER2 (Q)
   tumor cells.

   In (C)–(Q), data were presented as mean ± SEM (n = 3). An unpaired
   two-sided Student’s t test was performed for comparison; ∗p < 0.05; ∗∗p
   < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001; ns, not significant. See also
   [95]Figure S1; [96]Table S1.

   However, while circRNA^CAR expressed high-level CAR proteins in HEK293T
   cells ([97]Figure 1D), it produced CAR proteins at much lower levels in
   both T cells and macrophages ([98]Figures S1A and S1B). Therefore, we
   further optimized the protein-encoding sequence of circRNA^CAR based on
   the degeneracy of codons in mammalian cells, aiming to increase its
   expression in immune cells. We screened out several circRNAs that
   enable higher expression of CAR proteins in both T cells and
   macrophages ([99]Figures 1E–1G and [100]S1C). Among these optimized
   circRNAs, the circRNA^CAR-opt-3 exhibits the most efficient expression
   of CAR proteins in Jurkat cells, THP-1 cells, J774A.1 cells, and human
   primary T cells ([101]Figures 1E–1H). Therefore, we used
   circRNA^CAR-opt-3 to encode anti-HER2-CAR proteins for the following
   research in this study.

CircRNA^CAR mediated effective tumor killing in both human primary T cells
and macrophages

   Next, we investigated whether T cells or macrophages transfected with
   circRNA^CAR exhibited cytotoxic effects on the targeted tumor cells.
   The human primary T cells, Jurkat cells, THP-1 cells, or J774A.1 were
   transfected with circRNA^Anti-HER2-CAR and then co-cultured with
   various targeted tumor cells, including SK-OV-3 cells, B16F10-HER2
   cells, 4T1-HER2 cells, MC38-HER2 cells, and CT26-HER2 cells,
   respectively. The results showed that the human primary T cells
   transfected with circRNA^CAR could significantly kill those tumor cells
   in an effector:target (E:T) ratio-dependent manner ([102]Figures 1I–1K,
   [103]S1D, and S1E). Similarly, circRNA^CAR also mediated efficient
   tumor killing in immortalized Jurkat cells, THP-1 cells, and J774A.1
   cells in an E:T ratio-dependent manner ([104]Figures 1L–1Q and
   [105]S1F–S1J).

Macrophages exhibited efficient tumor phagocytosis and pro-inflammatory
polarization induced by circRNA^CAR

   As a unique mechanism of macrophage, the immune defense mechanism of
   macrophages plays a pivotal role in phagocytosis, a process crucial for
   combating various threats, including tumors.[106]^24 Previous studies
   have illuminated the role of adenovirally transduced CAR-macrophages in
   mediating tumor phagocytosis.[107]^24 To explore the potential of
   circRNA^CAR in orchestrating tumor phagocytosis by macrophages, the
   THP-1 or J774A.1 cells were transfected with circRNA^CAR and then
   co-cultured with various targeted tumor cells, respectively. The
   results unveiled that circRNA^CAR mediated significant phagocytosis of
   diverse cancer cells in macrophage in an E:T ratio-dependent manner
   ([108]Figures 2A–2C and [109]S1K–S1O).

Figure 2.

   [110]Figure 2
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   Macrophages exhibited efficient tumor phagocytosis and pro-inflammatory
   polarization induced by circRNA^CAR

   (A and B) Phagocytosis of THP-1 cells transfected with
   circRNA^Anti-HER2-CAR against SK-OV-3 (A) and MC38-HER2 (B) cells.

   (C) Phagocytosis of J774A.1 cells transfected with
   circRNA^Anti-HER2-CAR against CT26-HER2 cells.

   (D and E) Effects of circRNA^Anti-HER2-CAR on iNOS (D) and CD206 (E)
   expression in J774A.1.

   (F and G) Effects of circRNA^Anti-HER2-CAR on iNOS (F) and CD206 (G)
   expression in THP-1 cells.

   (H) Volcano plot illustrating differentially expressed genes in THP-1
   cells.

   (I) Heatmap depicting gene expression patterns in THP-1 cells (n = 2).

   (J) Bubble chart of relevant biological processes via Gene Ontology
   (GO) analysis (n = 2). Bubble size represents the number of genes.

   In (A)–(G), data were presented as mean ± SEM (n = 3). An unpaired
   two-sided Student’s t test was conducted for comparison; ∗p < 0.05; ∗∗p
   < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001; ns, not significant. See also
   [112]Figure S1.

   In the tumor microenvironment (TME), macrophages could be polarized to
   two opposite states, pro-inflammatory state (M1) or anti-inflammatory
   state (M2).[113]^25 Subsequently, we tested whether circRNA^CAR could
   affect the polarization of macrophages. Flow cytometry analysis results
   showed that circRNA^CAR-transfected J774A.1 cells demonstrated elevated
   levels of inducible nitric oxide synthase (iNOS) ([114]Figure 2D).
   Notably, there was only a very slight increase in CD206 ([115]Figure
   2E). Similarly, circRNA^CAR-transfected THP-1 cells also exhibited the
   pro-inflammatory M1 phenotype but not the anti-inflammatory M2
   phenotype ([116]Figures 2F and 2G).

   To further verify the impact of circRNA^CAR on the polarization of
   macrophages, we collected these transfected THP-1 cells for
   transcriptome-wide RNA sequencing (RNA-seq) analysis. The RNA-seq
   results showed that circRNA^CAR group exhibited upregulation of a
   series of genes related pro-inflammatory function, phagocytic
   capability, metabolism, and other immune responses of macrophages in
   comparison with circRNA^Ctrl or untreated group ([117]Figures 2H, 2I,
   [118]S1P, and S1Q). The pathway enrichment analysis showed that
   circRNA^CAR exhibited enhanced intracellular signal transduction and
   several enriched inflammatory-associated pathways such as tumor
   necrosis factor (TNF) production, interferon-gamma (IFN-γ) production,
   nuclear factor κB signaling, and mitogen-activated protein kinase
   signaling ([119]Figures 2J and [120]S1R). Collectively, these results
   suggested that circRNA^CAR held the potential to induce a
   pro-inflammatory M1 polarization in macrophages, which provided
   potential insights into the immunomodulatory capabilities of
   circRNA^CAR.

Immunocyte-tropic lipid nanoparticles efficiently delivered circRNAs into
immune cells in mice

   It is crucial to deliver circRNA^CAR into T cells and macrophages in
   vivo. The spleen is an important immune organ for the targeted delivery
   of circRNA^CAR using lipid nanoparticles
   (LNPs).[121]^26^,[122]^27^,[123]^28^,[124]^29^,[125]^30 Therefore, we
   synthesized various cationic lipids for LNPs by altering the head and
   tail groups. We screened out an LNP composition that contains four key
   components: the specific cationic lipids, cholesterol,
   1,2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC), and
   1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000
   (DMG-PEG2000), which efficiently targeted the spleen ([126]Figure 3A).
   Then, the circRNA^Luciferase was encapsulated within this LNP. The
   average size of LNP-circRNA^Luciferase formulation was approximately
   110 nm, and the zeta potential was approximately 8 mV ([127]Figures 3B
   and 3C). To investigate the in vivo targeting effects, the
   LNP-circRNA^Luciferase was intravenously injected into BALB/c mice. The
   in vivo and ex vivo luciferase imaging results showed that
   LNP-circRNA^Luciferase injected intravenously was mainly concentrated
   in the spleen, indicating the immunocyte-tropic feature of this LNP
   ([128]Figures 3D and 3E). In addition, the TME contained substantial
   tumor-associated macrophages, T cells, and dendritic cells.[129]^31 The
   LNP-circRNA^Luciferase was subsequently injected into the tumors of
   subcutaneous tumor-bearing mice. In vivo luciferase imaging results
   showed that luciferase was mainly concentrated within the local tumor
   region ([130]Figure S2A).

Figure 3.

   [131]Figure 3
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   Screening for immunocyte-tropic LNPs that efficiently delivered
   circRNAs into immune cells in mice

   (A) Schematic representation of the LNP-circRNA complex.

   (B and C) The size distribution (B) and zeta potential (C) of the
   LNP-circRNA^Luciferase complex.

   (D and E) Bioluminescence imaging in vivo (D) or ex vivo (E) of BALB/c
   mice intravenously injected with PBS or LNP-circRNA^Luciferase.

   (F and G) Bioluminescence imaging in vivo (F) or ex vivo (G) of BALB/c
   mice intravenously injected with PBS or SORT-circRNA^Luciferase.

   (H) Evaluation of targeting efficiency of SORT-circRNA^Cre in the
   spleen of reporter mice.

   (I) Detection of anti-HER2-CAR expression at different time points in
   various immune cells of mouse spleen.

   In (H) and (I), data were presented as mean ± SEM, an unpaired
   two-sided Student’s t test was performed for comparison; ∗p < 0.05; ∗∗p
   < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001; ns, not significant. See also
   [133]Figure S2.

   Besides, we also tested the selective organ targeting (SORT) approach,
   a previously reported method where LNPs are engineered with
   complementary lipids for targeting extrahepatic tissues.[134]^26 This
   technique employs negatively charged SORT-LNPs for targeted spleen
   delivery, and Imaging results revealed a predominant luciferase
   expression in the spleen via intravenous injection of
   SORT-circRNA^Luciferase, indicating the immunocyte-tropic specificity
   of negatively charged SORT-LNPs ([135]Figures 3F and 3G). We also
   tested the intratumoral injection of SORT-circRNA^Luciferase in
   tumor-bearing BALB/C mice, the imaging results elucidated a localized
   high concentration in the tumor region ([136]Figure S2B), which was
   consistent with the above LNP we initially prepared ([137]Figure S2A).
   Next, we further utilized flow cytometry analysis to detect the
   cell-targeting specificity of SORT-circRNA using reporter mice,
   harboring a CAG-loxp-zsGreen-stop-loxp-tdTomato-PolyA cassette
   integrated into the mouse genome. PBS or SORT-circRNA^Cre was
   intravenously administered into the reporter mice, and then the spleens
   and lymph nodes were collected for flow cytometry analysis. The results
   revealed that SORT-circRNA^Cre enables effective delivery of circRNA in
   T cells, natural killer (NK) cells, macrophages, and dendritic cells
   ([138]Figures 3H and [139]S2C).

   Moreover, to further assess the expression of circRNA-encoded
   anti-HER2-CAR proteins at different time points in immune cells of
   mice, BALB/c mice were intravenously injected with PBS,
   SORT-circRNA^Ctrl, or SORT-circRNA^CAR, respectively. The flow
   cytometry analysis results demonstrated that SORT-circRNA^CAR
   effectively expressed anti-HER2-CAR proteins in T cells, NK cells, and
   macrophages in the spleens and lymph nodes, forming in vivo panCAR
   (CAR-T, CAR-NK, and CAR-Macrophage) cells ([140]Figures 3I and
   [141]S2D). A single dose of circRNA-based in vivo CAR could produce in
   vivo panCAR lasting more than 72 h in the spleens and lymph nodes
   ([142]Figures 3I and [143]S2D). These results indicated that the
   circRNA-based in vivo panCAR could simultaneously utilize both adaptive
   and innate immune responses to exert targeted anti-tumor immunity.

CircRNA^CAR efficiently inhibited tumor growth and improved survival time in
mice

   To test whether circRNA^CAR can inhibit tumor growth in vivo, we
   firstly used immune-competent BALB/c mice with CT26-HER2 colorectal
   carcinomas ([144]Figure 4A). When the subcutaneous CT26-HER2 tumor
   volume reached approximately 50–80 mm^3, the tumor-bearing mice were
   randomly grouped and intravenously administered three times with PBS,
   15 μg of SORT-circRNA^Ctrl, or 15 μg of SORT-circRNA^CAR ([145]Figure
   4A). The result showed that SORT-circRNA^CAR significantly inhibited
   tumor growth, in comparison with PBS or SORT-circRNA^Ctrl ([146]Figure
   4B).

Figure 4.

   [147]Figure 4
   [148]Open in a new tab

   CircRNA^CAR efficiently inhibited tumor growth and improved survival
   time in mice

   (A) Schematic illustration of intravenous PBS, LNP-circRNA^Ctrl, or
   SORT-circRNA^Anti-HER2-CAR treatment in the CT26-HER2 tumor model.

   (B) Tumor growth curves for CT26-HER2 tumor-bearing mice treated as
   indicated in (A).

   (C) Schematic illustration of intratumoral PBS, LNP-circRNA^Ctrl, or
   LNP-circRNA^Anti-HER2-CAR treatment in the CT26-HER2 tumor model.

   (D and E) Tumor growth curves (D) and survival curves (E) of CT26-HER2
   tumor-bearing mice treated as indicated in (C).

   (F) In vivo bioluminescence imaging of CT26-HER2 tumor-bearing mice
   treated as indicated in (C).

   (G) The quantified signal intensity of bioluminescence imaging in (F).

   (H) Schematic illustration of intratumoral PBS, LNP-circRNA^Ctrl, or
   LNP-circRNA^Anti-HER2-CAR treatment in the 4T1-HER2 tumor model.

   (I) Tumor growth curves of 4T1-HER2 tumor-bearing mice treated as
   indicated in (H).

   (J) Tumor growth curves of individual 4T1-HER2 tumor-bearing mice
   treated as indicated in (H).

   (K) Schematic illustration of intratumoral PBS, LNP-circRNA^Ctrl, or
   LNP-circRNA^CAR treatment in the MC38-HER2 tumor model.

   (L and M) Tumor growth curves (L) and survival curves (M) of MC38-HER2
   tumor-bearing mice treated as indicated in (K).

   In (B), (D), (I), and (L), data were represented as the mean ± SEM, the
   tumor growth curves were calculated by two-way ANOVA analysis (n = 6).
   In (E) and (M), data were represented as the mean ± SEM, the survival
   curves were calculated by Kaplan-Meier simple survival analysis (n =
   6). In (G), data were represented as the mean ± SEM, an unpaired
   two-sided Student’s t test was conducted for comparison. ∗p < 0.05; ∗∗p
   < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. See also [149]Figure S2.

   Apart from the intravenous administration, we also tested the
   intratumoral administration in mice with subcutaneous CT26-HER2 tumors
   ([150]Figure 4C). Similarly, when the tumor volume reached
   approximately 50–80 mm^3, the mice were randomly grouped and
   intratumorally injected four times with PBS, LNP-circRNA^Ctrl, or
   LNP-circRNA^CAR ([151]Figure 4C). We found that LNP-circRNA^CAR could
   also significantly reduce tumor burden and prolong survival time, in
   comparison with PBS or LNP-circRNA^Ctrl ([152]Figures 4D–4G and
   [153]S2E). Similarly, in 4T1 breast carcinoma-bearing mouse models
   ([154]Figure 4H), the intratumoral administration of LNP-circRNA^CAR
   significantly inhibited tumor growth and even achieved tumor regression
   in comparison with PBS or LNP-circRNA^Ctrl ([155]Figures 4I and 4J). As
   for the subcutaneous MC38-HER2 tumors ([156]Figure 4K), the
   intratumoral administration of LNP-circRNA^CAR also significantly
   inhibited tumor growth and increased the survival time of tumor-bearing
   mice in comparison with PBS or LNP-circRNA^Ctrl ([157]Figures 4L, 4M,
   and [158]S2F). Collectively, these results demonstrated that the
   circRNA-based in vivo CAR exerted potent efficacy in treating tumors.

CircRNA^CAR reshaped the TME to a pro-inflammatory state in mice

   Solid tumors are often refractory partly due to the immunosuppressive
   TME.[159]^32 Therefore, we further investigated the effects of
   circRNA^CAR on the TME. At the end of experiments via intravenous
   administration of PBS, circRNA^Ctrl, or circRNA^CAR, the mouse spleens
   and tumor tissues were collected for detecting the infiltration of
   immune cells via flow cytometry analysis ([160]Figures S3A–S3C). The
   results showed that circRNA^CAR exhibited a significant increase in the
   proportion of CD8^+ T cells, CD8^+ effector memory T cells (Tem), CD8^+
   central memory T cells (Tcm), and CD4^+ T cells but a significant
   decrease in the proportion of regulatory T (Treg) cells in the spleen,
   compared to the PBS or circRNA^Ctrl ([161]Figures 5A and [162]S3D). As
   for tumor tissues, we also found that circRNA^CAR exhibited increased
   proportions of tumor-infiltrating CD45^+ cells, CD8^+ T cells, and
   CD4^+ T cells but a decreased proportion of tumor-infiltrating Treg
   cells, compared to the PBS or circRNA^Ctrl ([163]Figures 5B and
   [164]S3E). The flow cytometry results also showed that circRNA^CAR
   exhibited a significantly increased proportion of pro-inflammatory
   M1-polarized macrophages (major histocompatibility complex [MHC] II^+
   macrophages) in the TME, in comparison with PBS or circRNA^Ctrl
   ([165]Figures 5B and [166]S3E).

Figure 5.

   [167]Figure 5
   [168]Open in a new tab

   In vivo panCAR reshaped the tumor microenvironment to a
   pro-inflammatory state

   (A) Changes in the proportion of immune cells of spleen via flow
   cytometry. CD8^+ T cells, CD8^+ Tem cells, CD8^+ Tcm cells, or CD4^+ T
   cells were gated from CD45^+ cell population, and Treg cells were gated
   from CD4^+ T cell population.

   (B) Flow cytometric analysis of changes in the proportion of
   infiltrating immune cells in tumors. CD8^+ T cells, CD4^+ T cells, or
   MHC II^+ macrophages were gated from CD45^+ cell population, and Treg
   cells were gated from CD4^+ T cell population.

   (C and D) H&E staining (C) or IHC staining (D) of tumor tissue sections
   obtained from CT26-HER2 tumor-bearing mice after PBS,
   SORT-LNP-circRNA^Ctrl, or SORT-LNP-circRNA^Anti-HER2-CAR treatment. The
   integrated density of IHC staining was quantified using ImageJ.

   (E) Heatmap of gene expression patterns of immune cells extracted from
   tumor tissues (n = 2).

   (F) Bubble chart of relevant biological processes through GO analysis
   (n = 2). The size of the bubbles represented the number of genes.

   (G) Gene set enrichment analysis (GSEA) showing enriched pathways in
   immune cells extracted from tumor tissues (n = 2).

   In (A), (B), and (D), data were represented as the mean ± SEM; an
   unpaired two-sided Student’s t test was conducted for comparison; ∗p <
   0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001; ns, not significant.
   Each symbol represents an individual mouse. See also [169]Figures S3
   and [170]S4.

   Besides, we also harvested tumor tissue specimens from treated mice for
   hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC)
   staining. H&E staining showed that circRNA^CAR exhibited a significant
   increase in tumor cell death with enlargement of tumor cell gaps, pale
   cytoplasm, cell disintegration, and loss of structure, while PBS or
   circRNA^Ctrl exhibited diffuse growth and dense arrangement in both the
   CT26-HER2 and MC38-HER2 colorectal carcinoma-bearing mice ([171]Figures
   5C, [172]S4A, and S4B). IHC staining demonstrated increased
   infiltration of both CD8^+ T cells and CD86^+ cells in the circRNA^CAR
   group, compared to the PBS or circRNA^Ctrl group in both MC38-HER2 and
   CT26-HER2 tumor-bearing mice ([173]Figures 5D, [174]S4C, and S4D).

   To further explore the impact of circRNA^CAR on the TME, we utilized
   Percoll to isolate immune cells from tumor tissues via density gradient
   centrifugation and performed RNA-seq analysis. The differential gene
   expression analysis showed that circRNA^CAR exhibited upregulation of
   genes related to pro-inflammatory cytokines, chemokines, and chemokine
   receptors, in comparison with circRNA^Ctrl or PBS, suggesting the
   infiltration of immune cells in the TME ([175]Figures 5E and [176]S4E).
   The pathway enrichment analysis showed that circRNA^CAR exhibited
   several enriched pathways related to T cell proliferation, dendritic
   cell migration, phagocytosis, and pro-inflammatory responses such as
   IFN-γ signaling, TNF alpha (TNF-α) signaling, and interleukin
   (IL)-6-JAK-STAT3 signaling, in comparison with PBS or circRNA^Ctrl
   ([177]Figures 5F, 5G, [178]S4F, and S4G). We found that multiple
   pathways related to B cell immunity, such as B cell activation, B cell
   proliferation, immunoglobulin-mediated immune response, immunoglobulin
   production, and complement activation, were significantly enriched in
   the circRNA^CAR group ([179]Figures 5F and [180]S4F). This finding
   suggested that the antibodies might play an important role in the
   anti-tumor immunity of in vivo panCAR immunotherapy, warranting further
   investigation. Collectively, these results demonstrated that
   circRNA^CAR could markedly reshape TME to a pro-inflammatory state in
   tumor mouse models, which tended to sensitize immunotherapy.

CircRNA vaccine synergistically boosted the anti-tumor activity of in vivo
panCAR

   Recent reports proposed that vaccines might further enhance the
   therapeutic efficacy of CAR-T adoptive cell therapy in mouse tumor
   models.[181]^17^,[182]^18^,[183]^19^,[184]^20 Notably, the targeted
   proteins of the CAR molecules align with the antigens of the cancer
   vaccine,[185]^19^,[186]^20 which were designed to induce antigen
   spreading through the reactivation of infused CAR-T cells by the
   corresponding antigens of cancer vaccine.[187]^17 The aforementioned
   RNA-seq results suggested that the anti-tumor effects of circRNA^CAR
   therapy might be related to B cell responses ([188]Figures 5F and
   [189]S4F). Additionally, antibodies could promote phagocytosis or
   killing effects of macrophages or NK cells through ADCC and
   ADCP.[190]^21^,[191]^22 Therefore, we aimed to investigate whether
   circRNA vaccines could synergistically boost the anti-tumor immunity of
   the circRNA-based in vivo panCAR, in the manner of ADCC or ADCP.

   As the aforementioned circRNA^CAR encoded anti-HER2 CAR molecules that
   target human HER2 proteins, we developed a circRNA vaccine
   (circRNA^VAC) that encoded a human HER2 fusion protein, in which the
   endocytosis prevention motif (EPM) and ESCRT- and ALIX-binding region
   (EABR) domain were fused at the C-terminal instead of the original
   intracellular domain of human HER2 protein, aiming to help elicit
   higher level of HER2-specific antibodies ([192]Figure 6A; [193]Table
   S1).[194]^33 Therefore, we tested the combination immunotherapy between
   the circRNA-based in vivo CAR and circRNA-based cancer vaccines (in
   vivo panCAR-VAC). In HEK293T cells, circRNA^VAC exhibited efficient
   expression of HER2-EPM-EABR fusion protein ([195]Figure 6B),
   concomitant with the efficient secretion of vesicles in the supernatant
   ([196]Figure 6C). Next, we tested whether circRNA^VAC could induce
   HER2-specific antibodies. The BALB/c mice were immunized with
   LNP-circRNA^VAC through intramuscular injection twice at a 3-week
   interval, and the sera of immunized mice were collected at 1 week after
   the boost. The ELISA result showed that LNP-circRNA^VAC could elicit
   HER2-specific IgG antibodies, and the geometric mean titer was ∼4.9 ×
   10^6 ([197]Figure 6D).

Figure 6.

   [198]Figure 6
   [199]Open in a new tab

   CircRNA vaccine synergistically boosted the anti-tumor activity of in
   vivo panCAR

   (A) Schematic illustration of circRNA^VAC design. EPM, the endocytosis
   prevention motif; EABR, the ESCRT- and ALIX-binding region domain.

   (B) Flow cytometric analysis detecting the translation of circRNA^HER2
   in HEK293T cells (n = 3).

   (C) Electron microscopy showing the vesicles secreted by HEK293T cells
   transfected with circRNA^HER2-EPM-EABR.

   (D) Measurement of the endpoint titer of HER2-specific IgG with ELISA.

   (E) Schematic illustration of 4T1-HER2 tumor-bearing mice receiving PBS
   (intravenously), circRNA^CAR (intravenously), circRNA^VAC
   (intramuscularly), or circRNA^CAR (intravenously) plus circRNA^VAC
   (intramuscularly) combined therapy (n = 5).

   (F) Tumor growth curves of overall mice treated as indicated in (E).

   (G) Tumor growth curves of individual mouse treated as indicated in
   (E).

   (H) Schematic illustration of B16F10-HER2 tumor-bearing mice receiving
   PBS (intravenously), circRNA^CAR (intravenously), circRNA^VAC
   (intramuscularly), or circRNA^CAR (intravenously) plus circRNA^VAC
   (intramuscularly) combined therapy (n = 5).

   (I) Tumor growth curves of overall mice treated as indicated in (H).

   (J) Tumor growth curves of individual mouse treated as indicated in
   (H).

   (K) Survival curves of B16F10-HER2 tumor-bearing mice treated as
   indicated in (H).

   In (B), data were represented as the mean ± SEM; an unpaired two-sided
   Student’s t test was performed for comparison. In (D), data were
   represented as the geometric mean ± geometric SD; an unpaired two-sided
   Student’s t test was performed for comparison. In (F) and (I), data
   were represented as the mean ± SEM, and the tumor growth curves were
   calculated by two-way ANOVA analysis. In (K), the survival curves were
   calculated by Kaplan-Meier simple survival analysis. ∗p < 0.05; ∗∗p <
   0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001; ns, not significant. See also
   [200]Figures S5−S7; [201]Table S1.

   To test the effects of in vivo panCAR-VAC therapy, the BALB/c mice with
   subcutaneous 4T1-HER2 tumors were injected with PBS (intravenously),
   circRNA^CAR (intravenously), circRNA^VAC (intramuscularly), or
   circRNA^CAR (intravenously) plus circRNA^VAC (intramuscularly) at 6,
   10, and 18 days post tumor engraftment ([202]Figure 6E). The results
   showed that the combination of circRNA^CAR and circRNA^VAC showed
   improved tumor inhibition, in comparison with PBS, individual
   circRNA^CAR, or circRNA^VAC ([203]Figures 6F and 6G). To further
   confirm the enhanced effect of in vivo panCAR-VAC on tumor inhibition
   in mice models, we used additional tumor models, including B16F10-HER2
   and MC38-HER2 tumor models ([204]Figures 6H and [205]S5A). The results
   showed that both circRNA^CAR and circRNA^VAC individually inhibited
   tumor growth compared to the PBS group, and the combination of
   circRNA^CAR and circRNA^VAC showed improved tumor inhibition and
   significantly enhanced survival rate ([206]Figures 6I–6K, [207]S5B, and
   S5C).

   In addition to delivering circRNA^CAR via SORT-LNP, we also assessed
   the impact of delivering circRNA^CAR using the LNP we screened out
   ([208]Figures 3A–3D) on tumor growth. As before, CT26-HER2 tumors were
   subcutaneously implanted in BALB/c mice, and when the tumor volume
   reached 50–80 mm^3, the mice were randomly divided and injected with
   PBS, circRNA^CAR, circRNA^VAC, or the combination group of circRNA^CAR
   and circRNA^VAC ([209]Figure S5D). The results showed that compared to
   PBS, the combination group had a significant effect on inhibiting tumor
   growth and improving survival rates in mice ([210]Figures S5E–S5G).
   Additionally, we also investigated the efficacy of intratumoral
   administration, as an alternative to the intravenous administration,
   for the delivery of circRNA^CAR ([211]Figure S5H). Impressively, the
   combination of intratumorally delivered circRNA^CAR and circRNA^VAC
   vaccine (intramuscularly) markedly impeded tumor growth, resulting in a
   noteworthy enhancement in the survival rate of tumor-bearing mice
   ([212]Figures S5I–S5K).

   Collectively, these results demonstrated that the circRNA vaccines
   could synergistically boost the anti-tumor immunity of circRNA-based in
   vivo panCAR administered either intratumorally or intravenously, which
   holds the potential for an upgraded off-the-shelf in vivo panCAR-VAC
   immunotherapy.

   To further investigate the effects of in vivo panCAR-VAC immunotherapy
   on the TME, we euthanized MC38-HER2 tumor-bearing mice at 1 week after
   the final injection and collected the tumor tissues for analysis. The
   flow cytometry results showed that the in vivo panCAR-VAC-treated group
   significantly enhanced immune cell infiltration in the TME ([213]Figure
   S6A). Specifically, the panCAR-VAC group exhibited a significantly
   increased proportion of pro-inflammatory M1-polarized macrophages and a
   reduced proportion of anti-inflammatory M2-polarized macrophages
   compared to PBS, individual circRNA^CAR, or individual circRNA^VAC
   ([214]Figures S6B–S6D), while the proportions of exhausted T cells,
   exhausted NK cells, regulatory T cells, and myeloid-derived suppressor
   cells were reduced in the in vivo panCAR-VAC-treated group
   ([215]Figures S6E–S6J). H&E staining revealed that tumor tissues in the
   PBS group were tightly structured with deeper nuclear staining, while
   the individual circRNA^CAR or circRNA^VAC groups disrupted the tumor
   cell structure, and the in vivo panCAR-VAC combination group exhibited
   a significant increase in tumor cell death, characterized by enlarged
   tumor cell gaps and cell disintegration ([216]Figure S6K). IHC staining
   demonstrated that in vivo panCAR-VAC combination therapy promoted the
   infiltration of CD8^+ cells, CD11b^+ cells, and CD86^+ cells while
   reducing the infiltration of immunosuppressive Foxp3^+ cells
   ([217]Figures S6L and S6M). Collectively, these results demonstrated
   that in vivo panCAR-VAC combination therapy could markedly reprogram
   the TME to a pro-inflammatory state.

   To assess the safety of in vivo panCAR and panCAR-VAC therapies in
   mice, the BALB/c mice were treated with PBS, circRNA^CAR, circRNA^VAC,
   or the combination of circRNA^CAR and circRNA^VAC for short-term and
   long-term observations. The ELISA results indicated that neither the
   individual nor the combined groups exhibited increased levels of
   cytokines, including TNF-α, IL-1β, and IL-6, as well as alanine
   aminotransferase, aspartate aminotransferase, and alkaline phosphatase
   in both the short-term and long-term observations ([218]Figures
   S7A–S7C). Additionally, H&E staining revealed that in vivo panCAR and
   in vivo panCAR-VAC therapies resulted in no significant organ damage or
   major abnormalities in the important organs (heart, liver, spleen,
   lung, and kidney) of treated mice ([219]Figure S7D).

CircRNA^CAR boosted the level of circRNA vaccine-elicited antibodies
enhancing the anti-tumor activity via antibody-mediated cellular cytotoxicity

   Inspired by the finding of potential antibody-mediated anti-tumor
   immunity of in vivo panCAR therapy ([220]Figures 5F and [221]S4F), we
   further collected the antibodies of serum from mice treated with PBS,
   circRNA^CAR, circRNA^VAC, or circRNA^CAR and circRNA^VAC combined
   therapy. The ELISA results showed that the circRNA^CAR and circRNA^VAC
   combined group elicited higher endpoint titer of total IgG, IgG1,
   IgG2A, IgG2B, and IgG2C subtype than individual circRNA^VAC,
   circRNA^CAR, or PBS, while the endpoint titer of IgG3 and unmatured IgM
   showed no obvious difference ([222]Figures 7A and [223]S7E–S7G),
   indicating that the in vivo panCAR therapy might boost the level of
   circRNA vaccine-elicited functional antibodies.

Figure 7.

   [224]Figure 7
   [225]Open in a new tab

   In vivo panCAR enhanced the anti-tumor activity via antibody-mediated
   cellular cytotoxicity

   (A) Measurement of HER2-specific IgG, IgG1, IgG2A, IgG2B, or
   IgG2C-binding antibodies with ELISA (n = 5 or 6).

   (B and C) HER2-specific antibodies in (A) mediated cellular
   cytotoxicity against SK-OV-3 (B) and MC38-HER2 (C) tumor cells in
   J774A.1.

   (D and E) HER2-specific antibodies in (A) mediated cellular
   cytotoxicity against SK-OV-3 (D) and MC38-HER2 (E) tumor cells in RAW
   264.7.

   (F) Tumor growth curves of overall CT26 tumor-bearing mice treated with
   PBS, in vivo panCAR-VAC, or in vivo panCAR-VAC plus anti-NK1.1
   antibodies to deplete NK cells (n = 5).

   (G) Tumor growth curves of individual mouse treated as indicated in
   (F).

   (H) Survival curves of mice treated as indicated in (F).

   (I) Tumor growth curves of overall CT26 tumor-bearing mice treated with
   PBS, in vivo panCAR-VAC, or in vivo panCAR-VAC plus anti-CSF1R antibody
   to deplete macrophages (n = 5).

   (J) Tumor growth curves of individual mouse treated as indicated in
   (I).

   (K) Survival curves of mice treated as indicated in (I).

   (L) The potential mechanism diagram of synergistic in vivo panCAR-VAC
   immunotherapy.

   In (A), data are shown as the mean ± SEM; In (B)–(E), data were
   represented as the mean ± SEM; an unpaired two-sided Student’s t test
   was performed for comparison. In (F) and (I), tumor growth curves were
   calculated by two-way ANOVA analysis (n = 5). In (H) and (K), survival
   curves were calculated by Kaplan-Meier simple survival analysis (n =
   5). ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001; ns, not
   significant. See also [226]Figure S7.

   Next, we further explored whether the HER2-specific antibodies could
   exercise anti-tumor function, such as ADCC or ADCP activity. The in
   vitro tumor-killing results showed that the HER2-specific antibodies
   elicited by circRNA^CAR and circRNA^VAC combined therapy mediated
   significantly higher killing effects in both J774A.1 and RAW 264.7
   macrophages, in comparison with PBS, circRNA^CAR, or circRNA^VAC
   ([227]Figures 7B–7E).

   Next, to further verify the function of NK cells or macrophages in in
   vivo panCAR-VAC-mediated tumor inhibition, we employed an anti-NK1.1
   antibody or anti-CSF1R antibody to deplete NK cells or macrophages in
   tumor-bearing mice treated with in vivo panCAR-VAC, respectively. The
   results demonstrated that depleting NK cells or macrophages
   significantly reduced the efficacy of in vivo panCAR-VAC-mediated tumor
   inhibition and the survival of mice ([228]Figures 7F–7K), indicating
   that both NK cells and macrophages played crucial roles in the
   anti-tumor immunity mediated by in vivo panCAR-VAC.

   Traditional strategies of cancer vaccines have been mainly focusing on
   the cellular immunity but not the humoral immunity, whereas in this
   study, we found that B cell-mediated immune responses played an
   important role in tumor inhibition. On one hand, circRNA
   vaccine-elicited tumor-specific antibodies might boost the in vivo
   circRNA^CAR-mediated tumor killing via ADCC and ADCP effects, beyond
   the basic CAR-mediated phagocytosis or killing effects in the TME
   ([229]Figure 7L). On the other hand, in return, the elevated exposure
   of tumor antigens might further boost the humoral immunity to produce
   potent tumor-specific antibodies ([230]Figure 7L). This potential
   positive feedback loop between antibodies and antigens might partly
   explain the synergically enhanced anti-tumor immunity of in vivo
   panCAR-VAC immunotherapy.

   In summary, we provided the proof-of-concept study, which established
   the combined immunotherapy of circRNA-based in vivo panCAR and cancer
   vaccine, termed in vivo panCAR-VAC, achieving synergically enhanced
   anti-tumor immunity via simultaneously mobilizing both the adaptive and
   innate immune responses. Nevertheless, the potential of
   antibody-mediated anti-tumor immunity elicited by cancer vaccines still
   awaits further investigation.

Discussion

   CircRNA, a covalently closed circular RNA molecule, is highly stable
   and has been engineered for RNA editing, RNA vaccines against
   infectious diseases, cancer vaccines, and gene
   therapy.[231]^34^,[232]^35^,[233]^36^,[234]^37^,[235]^38^,[236]^39
   Recent research reported the circRNA-based T cell receptor-T adoptive
   cell therapy for treating cytomegalovirus infection.[237]^40 Unlike DNA
   vector platforms, such as DNA transposase,[238]^7 adeno-associated
   virus vectors,[239]^41^,[240]^42 and lentivirus-based
   vectors,[241]^12^,[242]^13 the expression of circRNA^CAR was transient
   and reversible without integrating into the genomic DNA. In this study,
   we generated circRNA^CAR, which encoded anti-HER2 CAR proteins for in
   vivo CAR immunotherapy to treat tumors in mice. We demonstrated that
   circRNA^CAR exhibited higher and more durable expression of CAR
   proteins compared with mRNA^CAR, indicating the potential superiority
   of circRNA-based in vivo CAR immunotherapy ([243]Figure 1D).

   For in vivo study, using the immunocyte-tropic LNPs ([244]Figure 3),
   circRNA^CAR was delivered into tumor-bearing mice and effectively
   suppressed tumor growth and markedly improved the survival rate in
   multiple mouse models ([245]Figure 4). Moreover, we also found that
   circRNA^CAR could reshape the TME to a highly pro-inflammatory state
   ([246]Figures 5, [247]S3, and [248]S4). Therefore, this circRNA-based
   in vivo CAR therapy held the potential to sensitize the anti-tumor
   immunity via combination with other cancer therapies, such as the
   targeted therapy or immune checkpoint blockade therapies (e.g.,
   anti-PD-1/PD-L1 antibodies). In this study, we observed that
   circRNA^Ctrl also exhibited partial anti-tumor effects ([249]Figure 4),
   which might result from the innate immune responses of in vitro
   transcription-produced circRNAs[250]^16^,[251]^43^,[252]^44^,[253]^45
   or the adjuvant activity of LNP.[254]^46^,[255]^47^,[256]^48^,[257]^49
   In comparison with the adoptive CAR-T cell therapy, this off-the-shelf
   in vivo circRNA^CAR therapy potentially had multiple advantages such as
   (1) lower cost, (2) off-the-shelf therapy, (3) avoiding the risk of
   genomic integration, (4) no need for lymphodepletion, and (5)
   repeatable dosing.

   It was worth mentioning that in this study, circRNA^CAR was delivered
   to a variety of immune cells in the spleen and lymph nodes, including T
   cells, macrophages, and NK cells, through immunocyte-tropic LNPs,
   forming panCAR (CAR-T, CAR-M, and CAR-NK) immune effector cells
   ([258]Figure 3), thereby exerting efficient targeted killing function.
   Compared with CAR-T cell therapy[259]^50^,[260]^51 or T cell engager
   therapy,[261]^52^,[262]^53 in vivo panCAR therapy simultaneously
   mobilized adaptive (T cell) and innate (NK cells and macrophages)
   immunity to exert targeted anti-tumor function and would have higher
   universality in disease treatment applications, especially for people
   with T cell immunodeficiency or T cell immune dysfunction. In addition,
   T cell exhaustion was the main difficulty in treating solid
   tumors.[263]^54 The in vivo panCAR strategy also mobilized innate
   immune cells such as NK cells and macrophages to exert anti-tumor
   immunity, which was an important supplement to adaptive immunity to
   eliminate tumors.

   Previous research reported that mRNA vaccines encoding tumor-associated
   antigens could help enhance the anti-tumor effects of adoptive CAR-T
   cell therapy.[264]^19^,[265]^20 Up to now, there are no reports about
   the combined immunotherapy between in vivo CAR therapy and RNA
   vaccines. More importantly, antibodies play an important role in
   macrophages or NK cell-mediated phagocytosis or killing via ADCC or
   ADCP effects.[266]^21^,[267]^22^,[268]^55^,[269]^56 Current studies on
   the combined therapy of CAR-T adoptive cell therapy and vaccines have
   only reported on the interactions between CAR-T cells and
   antigen-presenting cells,[270]^17 while the functions of
   vaccination-elicited antibodies still remain to be clarified. In this
   study, we formulated a circRNA-based cancer vaccine encoding human HER2
   antigens with the fused intracellular EPM-EABR motif to enhance the
   level of HER2-specific antibodies.[271]^33 We further demonstrated that
   this circRNA vaccine could synergistically boost the anti-tumor
   immunity of circRNA-based in vivo panCAR ([272]Figures 6 and [273]S5).
   More importantly, vaccination-induced endogenous antibodies had a
   complete structure and could effectively mediate ADCC and ADCP effects,
   which were an important bridge connecting innate immune responses and
   adaptive immune responses. We also demonstrated that depleting NK cells
   or macrophages significantly reduced the effects of in vivo
   panCAR-VAC-mediated tumor inhibition and the survival rate of mice
   ([274]Figures 7F–7K). These results indicated that the anti-HER2
   antibody promoted tumor killing, possibly via ADCC or ADCP, but likely
   did not interfere with CAR binding. Cancer immunotherapy relied on
   systemic immune responses. The in vivo panCAR-VAC strategy could
   effectively mobilize adaptive and innate immune cells to
   synergistically exert anti-tumor immunity.

   Mechanistically, it was possible that the immune cells expressing CAR
   molecules, such as T cells, macrophages, and NK cells, could
   specifically kill tumor cells, release inflammatory cytokines, reshape
   the immunosuppressive TME, and promote the infiltration of
   pro-inflammatory immune cells. Beyond the CAR-mediated tumor killing,
   the corresponding circRNA vaccine could concurrently elicit strong B
   cell responses and produce high-level tumor-specific antibodies to
   boost the in vivo circRNA^CAR-mediated tumor killing via ADCC and ADCP.
   On the other hand, the elevated tumor killing generated more exposure
   of tumor antigens to further boost humoral immunity producing potent
   tumor-specific antibodies in the germinal centers of tumor-draining
   lymph node (TDLN) or spleen, forming the positive feedback loop of
   antibody-antigen ([275]Figure 7L). This proof-of-concept study
   established a potent combined immunotherapy between in vivo panCAR and
   cancer vaccines, which achieved synergistically enhanced anti-tumor
   effects in multiple mouse models. Notably, this study found that the
   vaccination-elicited antibodies held an important role in anti-tumor
   immunity, providing a concept for the synergistic in vivo panCAR-VAC
   immunotherapy.

Limitations of the study

   In this study, the intravenous administration of circRNA^Anti-HER2-CAR
   exhibited slightly inferior anti-tumor activity, in comparison with the
   intratumoral administration of circRNA^Anti-HER2-CAR, which may be
   related to the targeting efficiency of LNPs. In the future, using
   optimized LNPs with higher targeting efficiency, the intravenously
   delivered circRNA^Anti-HER2-CAR might achieve remarkable anti-tumor
   effects. Besides, this study also lacked detailed research about the
   mechanism of synergistically enhanced anti-tumor immunity of in vivo
   panCAR-VAC therapy, and the further analysis of the TME with
   single-cell RNA-seq method and immunological experiments with gene
   knockout mouse models might help further clarify the synergistic
   mechanism of in vivo panCAR-VAC immunotherapy.

Resource availability

Lead contact

   Further information and requests for resources and reagents should be
   directed to and will be fulfilled by the lead contact, Liang Qu
   (quliang@fudan.edu.cn).

Materials availability

   All unique reagents generated in this study are available from the
   [276]lead contact with a completed material transfer agreement.

Data and code availability

   The data supporting the findings of this study are available from the
   [277]lead contact upon a reasonable request under a completed material
   transfer agreement. This study did not generate any unique code. Any
   additional information required to re-analyze the data reported in this
   study is available from the [278]lead contact upon request. The
   accession code of the transcriptome-wide RNA-seq data in this study was
   GEO: [279]GSE268105
   of NCBI Gene Expression Omnibus database.

Acknowledgments