Bioconjugated Antibody‐Trojan Immune Converter Enhance Cancer Immunotherapy with Minimized Toxicity by Programmed Two‐Step Immunomodulation of Myeloid Cells

Abstract

Current immune checkpoint blockade therapy (ICBT) predominantly targets T cells to harness the antitumor effects of adaptive immune system. However, the effectiveness of ICBT is reduced by immunosuppressive innate myeloid cells in tumor microenvironments (TMEs). Toll‐like receptor 7/8 agonists (TLR7/8a) are often used to address this problem because they can reprogram myeloid‐derived suppressor cells (MDSCs) and tumor‐associated M2 macrophages, and boost dendritic cell (DC)‐based T‐cell generation; however, the systemic toxicity of TLR7/8a limits its clinical translation. Here, to address this limitation and utilize the effectiveness of TLR7/8a, this work suggests a programmed two‐step activation strategy via Antibody‐Trojan Immune Converter Conjugates (ATICC) that specifically targets myeloid cells by anti‐SIRPα followed by reactivation of transiently inactivated Trojan TLR7/8a after antibody‐mediated endocytosis. ATICC blocks the CD47‐SIRPα (“don't eat me” signal), enhances phagocytosis, reprograms M2 macrophages and MDSCs, and increases cross‐presentation by DCs, resulting in antigen‐specific CD8⁺ T‐cell generation in tumor‐draining lymph nodes and TME while minimizing systemic toxicity. The local or systemic administration of ATICC improves ICBT responsiveness through reprogramming of the immunosuppressive TME, increased infiltration of antigen‐specific CD8⁺ T cells, and antibody‐dependent cellular phagocytosis. These results highlight the programmed and target immunomodulation via ATICC could enhance cancer immunotherapy with minimized systemic toxicities.

This work develops Antibody‐Trojan Immune Converter Conjugates that enable the programmed two‐step immunomodulation of suppressive myeloid cells in tumor microenvironments through specific targeting of myeloid cells by anti‐SIRPα antibody (as a first step) and subsequent reactivation of transiently inactivated Trojan Toll‐like receptor 7/8 agonist after antibody‐mediated endocytosis (as a second step).

1. Introduction

While recent immune checkpoint blockade therapy (ICBT) trials have achieved success and become the standard of care for various cancers,^{[ 1} , ^{2 ]} a significant number of patients do not respond to treatment, or their responses are short lived. This limited responsiveness to ICBT can be attributed to various defects in the cancer‐immunity cycle.^{[ 3} , ^{4 ]} These defects include 1) insufficient development of tumor‐specific T cells within the tumor microenvironment (TME),^{[ 5} , ⁶ , ⁷ , ⁸ , ^{9 ]} 2) reduced functionality of tumor‐specific T cells caused by the immunosuppressive TME^{[ 10 ]} and 3) decreased phagocytic activity of tumor‐associated macrophages (TAMs) within the TME.^{[ 11} , ¹² , ¹³ , ¹⁴ , ^{15 ]}

Myeloid innate immune cells, such as monocytes, macrophages, and dendritic cells (DCs), are abundant in the TME. However, despite their numerical prevalence, myeloid cells undergo differentiation during tumor progression and shift toward M2‐type macrophages and myeloid‐derived suppressor cells (MDSCs), which exhibit protumoral immune responses.^{[ 16} , ¹⁷ , ¹⁸ , ^{19 ]} Additionally, the “don't eat me” signal from the CD47‐SIRPα interaction inhibits macrophage phagocytosis, thereby limiting antibody‐dependent cellular phagocytosis (ADCP).^{[ 20} , ²¹ , ²² , ²³ , ²⁴ , ^{25 ]} Consequently, the antitumor activity of myeloid innate immune cells is compromised by immunosuppressive phenotype differentiation induced by tumors and blockade of phagocytosis.^{[ 26} , ²⁷ , ²⁸ , ²⁹ , ³⁰ , ^{31 ]} Moreover, in the absence of sufficient activation signals, DC‐mediated T‐cell immunity is also restrained, leading to deficient generation of antigen‐specific T cells within the TME. The conventional perspective on ICBT is predominantly based on the notion that these therapies target T cells, thereby unleashing the antitumor potential of the adaptive immune system. However, due to the observed limitation in responsiveness to ICBT, we focused on the importance of modulating innate myeloid cells within the TME to overcome the limitations or synergize with the therapeutic effects of traditional ICBT.

Toll‐like receptor 7/8 agonists (TLR7/8a) have been traditionally considered adjuvants that can trigger the innate immune system and promote the development of antigen‐specific T‐cell immunity.^{[ 32} , ^{33 ]} In addition to its well‐established immunostimulatory functions, TLR7/8a has been highlighted in recent studies for its role in modulating immunosuppressive cells within the TME.^{[ 34} , ³⁵ , ³⁶ , ³⁷ , ^{38 ]} However, despite the versatility of TLR7/8a, nonspecific distribution through systemic leakage induces systemic toxicity, diminishing its overall effectiveness.^{[ 39} , ⁴⁰ , ⁴¹ , ^{42 ]}

To address this limitation and fully utilize TLR7/8a in modulating innate myeloid immune cells in the TME, we suggested a programmed two‐step activation strategy via Antibody‐Trojan Immune Converter Conjugates (ATICC) developed by chemically linking Trojan TLR7/8a to anti‐SIRPα antibody with a cleavable linker (Figure  1a,b).^{[ 43 ]} Trojan TLR7/8a whose active site is transiently masked with an enzyme‐specific cleavable linker can be reactivated by the interferon‐gamma (IFN‐γ)‐inducible lysosomal thiol reductase (GILT) in endolysosomes after antibody‐mediated endocytosis (Figure 1a). The programmed two‐step immunomodulation involved specific targeting of myeloid cells by help of anti‐SIRPα antibody (as a first step) and subsequent activation of transiently inactivated trojan TLR7/8a by help of GILT enzyme in endolysome (as a second step) (Figure 1b). In the tissue level, considering the elevated expression of SIRPα in TME‐resident myeloid cells, ATICC effectively targeted and localized TLR7/8a to these specific cells within the TME. Upon internalization, in the cellular‐level, ATICC selectively reactivates in a timely manner, reducing systemic toxicity while augmenting its multifaceted immunomodulatory function. ATICC, which is highly internalized by MDSCs and macrophages, successfully remodeled the immunosuppressive phenotypes of MDSCs and M2 macrophages (Figure 1c,d). By reprogramming M2 macrophages toward the M1 phenotype and blocking the “do not eat me” signal, ATICC could synergize the phagocytosis and ADCP with ICBT (Figure 1c). Additionally, DC activation was successfully augmented, leading to the generation of antigen‐specific CD8⁺ T cells in tumor‐draining lymph nodes (TDLNs) and the TME (Figure 1d).

Figure 1.

Synthesis and characterization of ATICC. a) Process of synthesizing the Trojan7/8 agonist, Antibody‐Trojan Immune Converter Conjugates (ATICC) and scheme for the endocytosis‐mediated cleavage of ATICC. b) Two‐step (cellular‐level and tissue‐level) immune‐therapeutic strategy of ATICC. The pivotal role of ATICC in orchestrating enhanced uptake by myeloid cells and their selective reactivation which reduces systemic toxicity and enhancing efficacy. c) Synergistic effects of ATICCs and immune checkpoint blockade therapies (ICBTs) on tumor‐associated macrophages. d) Effects of ATICCs in MDSCs and DCs. e) The UV absorbance of ATICC and Trojan TLR7/8a. f) Comprehensive flow cytometric analysis of SIRPα expression in myeloid cells, including bone marrow‐derived cells (BMDMs and BMDCs) and RAW 264.7 cells. g,h) Examination of the binding affinity of ATICC for SIRPα as a function of concentration (g) and time (h) in BMDMs. i) In vitro endosomal‐dependent internalization of ATICC. The internalization of ATICC in RAW 264.7 cells at 12 h after treatment was imaged through live‐cell confocal microscopy; Endolysosomes (green), nuclei (blue), and ATICC (red). j) GILT‐dependent linker cleavage of ATICC in GILT with cysteine buffer. TNF‐α production in GILT‐knockdown RAW 264.7 cells following R848 or ATICC incubation for 12 h. All the data are presented as the means ± s.d. Statistical significance was evaluated by an unpaired two‐tailed t‐test in g, j and one‐way ANOVA with Tukey's multiple comparison test in h. p values: NS, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

2. Result

2.1. Synthesis and Characterization of ATICC

For the synthesis of ATICC (anti‐SIRPα‐TLR7/8a conjugate), Trojan TLR7/8a of which active site was masked with GILT enzyme‐responsive cleavable chemical linker was synthesized (Figures S1–S4, Supporting Information). Then, anti‐SIRPα was partially reduced and reacted with the 2‐pyridyl disulfide moiety in Trojan TLR7/8a to facilitate conjugation (Figure 1a). Following the reaction, the ATICC was purified with a Zeba desalting column to remove excess unreacted reagents (Figure S5, Supporting Information). The drug‒to‐antibody ratio (DAR) was determined by measuring the antibody concentration via a BCA assay and the small molecule Trojan TLR7/8a via UV–vis spectrometry (Figure 1e; Figure S6, Supporting Information). To validate the in vitro characterization of ATICC, we checked for the presence of sufficient SIRPα expression in various in vitro cells, including RAW 264.7 cells, bone marrow‐derived macrophages (BMDMs), and bone marrow‐derived dendritic cells (BMDCs) (Figure 1f). Using SIRPα‐expressing cells, we assessed the binding affinity of ATICC for SIRPα. With respect to BMDMs and RAW 264.7 cells, we confirmed that the binding affinity of anti‐SIRPα was not compromised after ATICC was synthesized (Figure 1g; Figure S7, Supporting Information). Furthermore, we observed that the SIRPα receptor did not rebuild within 48 h after binding, suggesting that ATICC effectively blocked SIRPα (Figure 1h).

After binding to the SIRPα receptor on the cell surface, ATICC undergoes endosomal‐dependent cellular internalization (Figure 1i). Initially, the active site of Trojan TLR7/8a in ATICC is masked by a chemical linker, and subsequent investigations revealed that this site is reactivated in a GILT‐dependent manner, which is abundant in endolysosomes^{[ 44 ]} (Figure 1j). Additionally, by comparing the immunostimulatory activities of ATICC in wild‐type and GILT‐knockdown RAW 264.7 cells, we confirmed that Trojan TLR7/8a in ATICC is reactivated in a GILT‐dependent manner after internalization into the endolysosome.

2.2. In Vitro Characterization of the Role of ATICC in Reprogramming TAMs and MDSCs

TAMs and MDSCs are among the most abundant infiltrating myeloid cells with immunosuppressive effects, and they undergo significant expansion during tumor progression.^{[ 18} , ^{45 ]} Consequently, TAMs and MDSCs have emerged as promising therapeutic targets in cancer immunotherapy. Recent studies have revealed that treatment with TLR7/8a can reverse MDSC‐ and TAM‐mediated immunosuppression by driving MDSC differentiation into tumoricidal antigen‐presenting cells (APCs).^{[ 46} , ^{47 ]}

To validate the ability of ATICC to polarize MDSCs and M2 macrophages, we examined the immune phenotypes of in vitro‐generated MDSCs and M2 macrophages following treatment with ATICC. To generate MDSCs in vitro, they were isolated from the splenocytes of tumor‐bearing mice using a negative selection method with an MDSC isolation kit. Purified MDSCs treated with R848 or ATICC exhibited significant secretion of proinflammatory cytokines, such as IL‐6 and IL‐12, indicating that the MDSCs had converted into tumoricidal mature APCs (Figure  2a). Subsequently, we generated protumorigenic M2 macrophages in vitro through IL‐4 stimulation, which was verified by the upregulation of CD206 surface marker expression. After treatment with R848 or ATICC, the expression of the M1 surface marker (CD80) increased, while that of the M2 surface marker (CD206) decreased. Furthermore, the levels of the proinflammatory cytokines IL‐6 and IL‐12p70 were elevated after treatment with R848 or ATICC (Figure 2b).

Figure 2.

ATICC increased in vitro polarization of MDSC and M2 BMDM and enhanced phagocytosis. a) Quantification of IL‐6 and IL‐12p70 produced by MDSCs 12 h after PBS, R848 or ATICC treatment (n = 3). b) Percentage of M1(CD80 in CD11b⁺ F4/80⁺) or M2(CD206 in CD11b⁺ F4/80⁺) marker (left) (n = 5) and quantification of pro‐inflammatory cytokines (IL‐6 and IL‐12p70) production (right) (n = 3) after treatment with PBS, R848 or ATICC for 24 h. c) Schematic of the phagocytosis assay. d) Flow cytometry gating strategy for phagocytosis. e) Mechanisms underlying the ATICC‐mediated increase in macrophage phagocytosis. f) The percentage of phagocytosis was determined by measuring the percentage of dual‐stained cells (APC‐CD11b macrophage and CFSE‐labeled B16OVA) relative to the total number of live cells (n = 3). g) Representative confocal images showing the effect of different treatments on the phagocytosis of B16OVA in a coculture assay with macrophages (left). The percentage of cancer cells phagocytosed by macrophages was calculated by assessing the colocalization of the CFSE signal with the WGA signal using ImageJ (right). The nuclei were stained with DAPI, and the macrophages were labeled with wheat germ agglutinin (WGA). Magnified images from corresponding merged images show the phagocytosis of CFSE‐labeled cancer cells (green) by macrophages (red). Scale bar, 10 µm. All the data are presented as the means ± s.d. Statistical significance was evaluated by one‐way ANOVA with Tukey's multiple comparison test in a, b and f and an unpaired two‐tailed t‐test in g. p values: NS, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Despite the abundant presence of TAMs in the TME, their immunosuppressive function and the “do not eat me” signal from CD47‐SIRPα interaction inhibit macrophage phagocytosis, thereby constraining ADCP. Consequently, we investigated whether ATICC, which simultaneously reprograms suppressive macrophages and blocks the CD47‐SIRPα interaction, can enhance macrophage phagocytosis and ADCP. For the phagocytosis assay, we upregulated CD47 expression on B16OVA tumor cells by pretreating the cells with IFN‐γ (Figure S8, Supporting Information). Concurrently, we stimulated M1‐ or M2‐polarized BMDMs with anti‐SIRPα, R848, anti‐SIRPα + R848 or ATICC and cocultured them for 12 h (Figure 2c). We then measured the population of CD11b⁺ cells that had engulfed tumor cells (Figure 2d). An ADCP assay using an anti‐PD‐L1 antibody confirmed that treatment with anti‐SIRPα + R848 or ATICC could enhance ADCP expression in M1 and M2 BMDMs (Figure 2e,f). These results were consistent with those obtained with confocal imaging in RAW 264.7 cells (Figure 2g). Compared with PBS‐treated RAW 264.7 cells, ATICC‐treated RAW 264.7 cells exhibited four to fivefold greater phagocytosis (colocalization of WGA‐labeled BMDMs and CFSE‐labeled cancer cells).

2.3. In Vitro Characterization of ATICC in DC Activation and Its Effect on Adaptive Immunity by Cross‐Presentation to CD8⁺ T Cells

DCs play a pivotal role in the generation of antigen‐specific T‐cell immunity. Mature DCs present the following crucial signals that determine the fate of naive T cells: antigens loaded onto MHC I or II, costimulatory markers (CD80 and CD86) expressed on the surface of DCs, and secretory cytokines. In our study, we confirmed that R848 and ATICC significantly enhanced immunogenicity by upregulating the expression of costimulatory markers (CD80 and CD86) (Figure  3a) and inducing the production of proinflammatory cytokines, such as IL‐6 and IL‐12p70 (Figure 3b). Furthermore, we investigated whether ATICC could successfully induce antigen‐specific CD8⁺ T‐cell immunity using OT‐1 CD8⁺ T cells purified from the splenocytes of OT‐1 mice. BMDCs were treated with OVA protein, along with R848 or ATICC for 12 h and subsequently cocultured with OT‐1 CD8⁺ T cells for 3 days. Our observations revealed that R848 and ATICC exhibited significant effects that induced the proliferation of OT‐1 CD8⁺ T cells and promoted IFN‐γ production (Figure 3c,d). These findings suggested that ATICC‐treated DCs can effectively induce cross‐presentation to CD8⁺ T cells. Additionally, we assessed whether ATICC‐treated DCs could induce cross‐presentation by phagocytosing tumor cells. For verification, we cocultured ATICC‐treated DCs with B16OVA tumor cells and OT‐1 CD8⁺ T cells for 72 h. Consistent with the cross‐presentation analysis with OVA protein antigen, ATICC‐treated DCs were effective at cross‐presenting phagocytosed B16OVA tumor cells (Figure 3e,f). This result emphasizes the potential of ATICC to generate in situ vaccination in the TME, even without the injection of other protein antigen.

Figure 3.

In vitro BMDC activation and CD8⁺ T‐cell cross‐presentation. a,b) BMDCs were treated with PBS, R848 or ATICC for 24 h. Evaluation of activation markers (CD80 in CD11c⁺ and CD80 in CD11c⁺) (n = 5) (a) and quantification of IL‐6 and IL‐12p70 production (b) (n = 5). c,d) Analysis of cross‐presentation of the OVA protein. BMDCs were treated with OVA protein and adjuvant (R848 or ATICC) for 24 h. OT‐1 CD8⁺ T cells were subsequently cocultured for 72 h to assess cross‐presentation via analysis of T‐cell proliferation (c). Quantification of IFN‐γ produced by OT‐1 CD8⁺ T cells (n = 3) (d). e,f) Analysis of cross‐presentation of B16OVA tumor cells. BMDCs were treated with adjuvant (R848 or ATICC) for 24 h and subsequently cocultured with B16OVA tumor cells and OT‐1 CD8⁺ T cells for 72 h to assess cross‐presentation via analysis of T‐cell proliferation (e). Quantification of IFN‐γ produced by OT‐1 CD8⁺ T cells (n = 3) (f). All the data are presented as the means ± s.d.s. Statistical significance was evaluated by one‐way ANOVA with Tukey's multiple comparison test in a, b, d and f. p values: NS, not significant; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

2.4. Targeted Delivery of ATICC Reprogram Myeloid Cells in TME

TLR7/8a has attracted interest not only for its adjuvant properties in stimulating the innate immune system and generating antigen‐specific T‐cell responses but also for their immunomodulatory functions, which play crucial roles in reprogramming the immunosuppressive TME. Despite its versatility, the use of conventional TLR7/8a, R848 in vivo involves significant limitations. These constraints are primarily attributed to its poor circulation and the flu‐like symptoms it induces, which are indicative of elevated cytokine levels in the bloodstream. These characteristics not only contribute to systemic toxicity but also diminish the effectiveness of TLR7/8a. To address these issues, we developed an ATICC (anti‐SIRPα‐TLR7/8a conjugate), which allows for the targeted delivery of TLR7/8a to myeloid cells within the TME. To confirm the targeting efficacy of this approach, we assessed the expression level of SIRPα in immune cells within the TME. Our findings revealed high expression of SIRPα in macrophages (CD11b⁺ F4/80⁺ in CD45⁺), DCs (CD11c⁺ MHCII⁺ in CD45⁺) and MDSCs (CD11b⁺ Gr‐1⁺ in CD45⁺) (Figure  4a). We subsequently investigated the cellular distribution and accumulation of ATICC in the TME. As expected, compared with Cy5 (as a mimic of free TLR7/8a), ATICC‐Cy5 (anti‐SIRPα‐Cy5 conjugate) was localized primarily to DCs, macrophages, and MDSCs. Compared with Cy5, ATICC‐Cy5 exhibited 15.4‐fold greater localization in DCs (Figure 4b).

Figure 4.

Myeloid cell‐targeting ATICC causes the immunosuppressive TME to be reprogrammed and enhances myeloid cell function. a) SIRPα expression in TME‐resident immune cells, including macrophages (CD11b⁺ F4/80⁺ in CD45⁺), DCs (CD11c⁺ MHCII⁺ in CD45⁺), MDSCs (CD11c⁺ MHCII⁺ in CD45⁺), CD4⁺ T cells (CD3⁺ CD4⁺ in CD45⁺), and CD8⁺ T cells (CD3⁺ CD8⁺ in CD45⁺). b) Quantification of the cellular uptake of Cy5 (as a mimic of free TLR7/8a) or ATICC‐Cy5 (anti‐SIRPα‐Cy5 conjugate) by various immune cells within the TME. c) Population of tumor‐infiltrating MDSCs (Gr‐1⁺ in the CD45⁺ CD11b⁺ population) in the TME. d) Evaluation of the M1 (CD80⁺ in CD11b⁺ F4/80⁺) phenotype or M2 (CD206⁺ in CD11b⁺ F4/80⁺) phenotype of TAMs. e) In vivo phagocytosis index of TAMs. The percentage of phagocytosis was determined by quantifying dual‐stained cells (APC‐CD11b and CFSE‐labeled B16OVA) relative to total live cells. f) ADCP index of TAMs. The percentage of phagocytosis was determined by quantifying dual‐stained cells (APC‐CD11b and CFSE‐labeled B16OVA) relative to total live cells. g,h) Flow cytometry analysis of DC activation (CD86⁺ in CD11c⁺) (g) and (CD80⁺ in CD11c⁺) (h). All the data are presented as the means ± s.d. Statistical significance was determined by an unpaired two‐tailed t‐test in b. Statistical significance was evaluated by one‐way ANOVA with Tukey's multiple‐comparison test in c–h. p values: NS, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Reprogramming of myeloid cells within the TME was intensified by the targeted delivery of TLR7/8a to myeloid cells using ATICC. We assessed the immunomodulatory effects of ATICC in the B16OVA tumor model following three immunizations at 3‐day intervals. The results revealed a drastic reduction in the population of MDSCs within the TME upon ATICC immunization, while the combination of anti‐SIRPα and R848 (anti‐SIRPα + R848) exhibited limited effects (Figure 4c; Figure S9, Supporting Information). Furthermore, ATICC facilitated the repolarization of macrophages toward the antitumoral M1 phenotype (Figure 4d; Figure S9, Supporting Information). This activity was evident in the increased population of M1 macrophages (CD80⁺ in CD11b⁺ F4/80⁺ cells) and the decreased population of M2 macrophages (CD206⁺ in CD11b⁺ F4/80⁺ cells).

As part of the innate immune system, TAMs function as one of the first lines of defense by phagocytosing cancer cells; however, the phagocytic activities of TAMs are strongly hindered by multiple factors. Cancer cells express a “do not eat me” signal, CD47, which binds to SIRPα on macrophages and inhibits phagocytosis. Additionally, TAMs tend to adopt an M2‐like lineage with reduced phagocytic capabilities. Hence, we propose that ATICC could synergistically enhance the phagocytic potential of TAMs through its dual actions, that is, reprogramming M2 macrophages to M1 macrophages and blocking the CD47‐SIRPα interaction, as demonstrated in Figure 4d. To confirm this, we inoculated CFSE‐labeled B16OVA tumor cells into C57BL/6 mice, and after 10 days, we intratumorally injected R848, anti‐SIRPα + R848, or ATICC. We then analyzed the percentage of CFSE⁺ cells within macrophages (CD11b⁺ F4/80⁺ cells) in the TME as an indicator of phagocytosis (Figure S10, Supporting Information). Phagocytosis was slightly enhanced in the R848‐injected group compared to the PBS‐injected group, while the anti‐SIRPα + R848‐injected group exhibited even greater phagocytosis than the R848‐injected group (Figure 4e). Interestingly, the ATICC‐injected group, in which anti‐SIRPα and R848 synergistically improved the efficacy, demonstrated the highest percentage of phagocytosis. Furthermore, we assessed the percentage of phagocytosis in the presence of an anti‐PD‐L1 antibody, which is an indicator of ADCP. ATICC significantly enhanced the phagocytic potential of the anti‐PD‐L1 antibody (Figure 4f).

The enhanced targeting of DCs by anti‐SIRPα in the ATICC group resulted in robust stimulation and maturation of DCs, which was characterized by increased expression levels of CD80 and CD86 in CD11c⁺ cells (Figure 4g,h; Figure S11, Supporting Information).

2.5. ATICC Enhanced Adaptive Immunity in the TDLN and TME

As shown in Figure 3, ATICC induced robust DC maturation, resulting in the generation of antigen‐specific CD8⁺ T cells in vitro. This significant maturation of DCs by ATICC in the TME, as depicted in Figure 4g,h, promoted the development of antigen‐specific CD8⁺ T cells in the TDLN; this finding was supported by the notable secretion of cytokines that occurred upon restimulation with the SIINFEKL peptide antigen (Figure  5a–c; Figures S12 and S13, Supporting Information). Furthermore, ATICC‐induced reprogramming of the TME increased the infiltration of CD8⁺ T cells throughout the TME (Figure 5d; Figure S14, Supporting Information) and expanded the population of antigen‐specific CD8⁺ T cells in the TME (Figure 5e–g; Figure S12, Supporting Information).

Figure 5.

Antigen‐specific CD8⁺ T cells in the TDLN and TME with enhanced infiltration of CD8⁺ T cells via the ATICC. a–c) Antigen‐specific CD8⁺ T cells in the TDLN. Representative flow cytometry dot plots and percentages of TNF‐α⁺ (a), IFN‐γ⁺ (b) and Granzyme B⁺ (c) CD8⁺ T cells (CD3⁺CD8⁺) after SIINFEKL restimulation in the presence of IL‐2 for 12 h. d) Representative flow cytometry dot plots and percentage of the tumor‐infiltrated CD8⁺ T‐cell population in the TME. e–g) Antigen‐specific CD8⁺ T cells in the TME. Representative flow cytometry dot plots and percentages of TNF‐α⁺ (e), IFN‐γ⁺ (f), and Granzyme B⁺ (g) CD8⁺ T cells (CD3⁺CD8⁺) after SIINFEKL restimulation in the presence of IL‐2 for 12 h. Statistical significance was evaluated by one‐way ANOVA with Tukey's multiple comparison test for a–g. p values: NS, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

2.6. Enhanced Therapeutic Efficacy of ATICC with Reduced Systemic Toxicity

To evaluate the antitumor efficacy of ATICC, we compared the percent survival rate and tumor growth volume in mice treated with the indicated samples (Figure  6a). Compared with anti‐SIRPα, ATICC initially demonstrated enhanced antitumor efficacy, indicating the effectiveness of the antibody‐adjuvant conjugation strategy in improving the impact of TLR7/8a by targeting myeloid cells in the TME. Furthermore, the notable antitumor efficacy of ATICC may be derived from the modulation of myeloid cells in various ways, including reprogramming M2 macrophages and MDSCs, augmenting phagocytic activity in TAMs, and enhancing cross‐presentation by DCs to generate antigen‐specific CD8⁺ T cells. Additionally, ATICC effectively increased the therapeutic response rate to anti‐PD‐L1 therapy. ATICC could enhance the therapeutic efficacy of anti‐PD‐L1 therapy through reprogramming the immunosuppressive TME, increasing infiltration of antigen‐specific CD8⁺ T cells, and performing ADCP. Finally, ATICC exhibited equivalent or even superior effectiveness compared to fivefold higher amounts of R848. Even though fivefold higher amounts of R848 exhibited equivalent antitumor activity compared to that of ATICC, the intratumoral administration of high amounts of R848 led to significant increases in the serum cytokine levels and activities of alanine transaminase (ALT) and aspartate transaminase (AST), indicating systemic toxicity (Figure 6b). In contrast, ATICC, which showed substantial antitumor efficacy, did not noticeably increase the serum cytokine levels or the activities of ALT and AST. This outcome may result from the efficient targeting of myeloid cells by ATICC, which allows for effective modulation of small amounts of TLR7/8a while minimizing unwanted cell activation. Interestingly, similar toxicity profiles were observed after intravenous administration of R848, fivefold higher amounts of R848, and ATICC (Figure 6c). ATICC therapy may be effective at reducing the systemic toxicity of TLR7/8a, even when the treatment is systemically delivered. Finally, we assessed the therapeutic efficacy of intravenously administered ATICC; notably, ATICC and the combination of ATICC with anti‐PD‐L1 therapy exhibited antitumor effects, indicating that ATICC could be a promising candidate for systemic therapeutic agents for TLR7/8a (Figure 6d).

Figure 6.

Antitumor efficacy and systemic toxicity in local injection or systemic injection models. a) Survival and tumor growth curves of mice intratumorally immunized with PBS, R848, anti‐SIRPα, a combination of R848 and anti‐SIRPα, or ATICC three times at 3‐day intervals (n = 6). b) Serum cytokine (IL‐6 and TNF‐α) levels and serum ALT and AST concentrations after intratumoral immunization with R848, R848 (fivefold) or ATICC (n = 3). c) Serum cytokine (IL‐6 and TNF‐α) levels and serum ALT and AST concentrations after intravenous immunization with R848, R848 (fivefold) or ATICC (n = 3). d) Survival and tumor growth curves of mice intravenously immunized with PBS, ATICC or the combination of ATICC and an anti‐PD‐L1 antibody three times at 3‐day intervals (n = 6). All the data are presented as the means ± s.d. Statistical significance was determined by an unpaired two‐tailed t‐test. p values: NS, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

3. Conclusion

In summary, we introduced ATICC as a novel and promising programmed two‐step immuno‐therapeutic strategy for cancer immunotherapy by leveraging a targeted delivery system that combines TLR7/8a with an anti‐SIRPα antibody. This innovative approach strengthens the effectiveness of TLR7/8a while addressing the limitations of TLR7/8a, such as poor circulation and systemic toxicity, through programmed two‐step immunomodulation which involved specific targeting of myeloid cells by help of anti‐SIRPα antibody (as a first step) and subsequent activation of transiently inactivated trojan TLR7/8a by help of GILT enzyme in endolysome (as a second step). This is achieved by specifically directing the therapeutic payload to myeloid cells within the TME and ensuring their selective reactivation only after internalization. Importantly, through the implementation of a programmed two‐step immuno‐therapeutic strategy, ATICC demonstrated a favorable safety profile, minimizing systemic toxicity even with systemic delivery. In tissue level, ATICC demonstrated efficient targeting and localization to myeloid cells within the TME. Subsequently upon internalization, in cellular‐level, ATICC selectively reactivated in a timely manner by the help of GILT enzyme. This precise activation mechanism allowed for effective immunomodulation while minimizing the risk of unwanted systemic cell activation.

We also focused on leveraging the multifaceted roles of TLR7/8a, aiming not only to elucidate DC‐based innate immune responses for generating antigen‐specific T‐cell responses, but also to harness its immune modulation functions in reprogramming the immunosuppressive TME. ATICC has demonstrated multifaceted immunomodulatory effects within the TME. It effectively reprogrammed MDSCs and M2 macrophages, augmented the phagocytic activity of TAMs, and enhanced cross‐presentation by DCs, leading to the generation of antigen‐specific CD8⁺ T cells. Additionally, ATICC exhibited remarkable antitumor efficacy, surpassing the effects of anti‐SIRPα and showing comparable or superior results to higher doses of free TLR7/8a. Through conjugating TLR7/8a to ATICC, we presented a targeted and effective approach to amplify the effects of TLR7/8a at a low dose, thereby improving overall therapeutic outcomes in cancer immunotherapy. Due to this strategic balance between enhanced efficacy and reduced toxicity, ATICC is expected to be a versatile therapeutic agent in TLR7/8a‐based cancer immunotherapy. This enhanced therapeutic response could be also expanded to the combination of ATICC with anti‐PD‐L1 therapy, further highlighting the potential synergy of immune checkpoint inhibitors in innate immunity (i.e., anti‐SIRPα antibody) with adaptive immunity (i.e., anti‐PD‐L1).

4. Experimental Section

Synthesis of Trojan TLR7/8a and Fabrication of ATICC

Trojan TLR7/8a was synthesized following the chemical scheme outlined in Figures S1–S4, Supporting Information. The structural characterization of the synthesized compounds was conducted using LC‒MS (Agilent 1260–6120 system) and HPLC (Waters Acquity UPLC H‐Class instrument). Subsequently, the Antibody‐Trojan Immune Converter Conjugates (ATICC) was developed according to the chemical scheme depicted in Figure 1b. Trojan7/8a was conjugated to the anti‐SIRPα antibody. To initiate disulfide reduction, a mixture containing an anti‐SIRPα antibody, TCEP (molar ratio, antibody:TCEP = 1:2.3), borate buffer (20×), and EDTA (500×) was dissolved in PBS. The solvent was incubated for 1 h at 37 °C. Then, Trojan7/8a was added to the reduced antibody (molar ratio, 10:1), and the mixture was further incubated for 2 h at 4 °C. L‐cysteine was introduced (molar ratio, 100:1) to stop the reaction, and the purification of ATICC was achieved using a desalting column. Through this process, unbound Trojan7/8a was effectively removed from the antibody. Prior to use, the desalting column was centrifuged at 1500 ×g for 1 min. After the compound was loaded, an additional centrifugation at 1500 ×g for 2 min was performed. The quantities of Trojan7/8a were quantified using ultraviolet–visible light spectrometry (UV‐1800), while the amounts of anti‐SIRPα antibodies were determined through a BCA assay.

In Vitro ATICC Binding Affinity

The binding affinity of the ATICC was evaluated based on the percentage of SIRPα molecules on BMDMs. A total of 1 × 10⁶ BMDMs were subjected to treatment with ATICC or an anti‐SIPRα antibody. Initially, cells were treated with ATICC or an anti‐SIRPα antibody at various concentrations (100 µg, 10 µg, 1 µg, 100 ng, 10 ng, 1 ng, or 100 pg), after which the expression rate of SIRPα was assessed after 24 h. Subsequently, the expression rate of SIRPα was confirmed after BMDMs were treated with ATICC for different durations (6, 12, 24, and 48 h).

Animals, Cell Lines and Antibodies

The animal experimentation protocol involved scrutiny and received approval from the Institutional Animal Care and Use Committee (IACUC) at the Sungkyunkwan University School of Medicine (Approval Code: SKKUIACUC2022‐12‐05‐1). The aforementioned committee is endorsed by the Association for Assessment and Accreditation of Laboratory Animal Care International and adheres to the guidelines stipulated by the Institute of Laboratory Animal Resources. C57BL/6 and BALB/C mice (6‐ to 8‐week‐old females) were procured from Orient Bio (Korea), while OT‐I mice were obtained from Yong‐Soo Bae (Sungkyunkwan University, Korea). All animals were housed in individually ventilated cages, which were maintained at humidity levels between 30% and 70% and temperatures ranging from 21 to 26 °C, and subjected to a 12 h light–dark cycle. RAW 264.7 cells (macrophage line, ATCC) and murine B16OVA tumor cells (melanoma, ATCC) were cultured in Dulbecco's modified Eagle's medium (DMEM, Thermo Fisher). Prior to utilization in this study, all cell lines were scrutinized to confirm the absence of mycoplasma contamination and exclusion from the list of misidentified cell lines. The culture media for the cells were supplemented with 10% heat‐inactivated fetal bovine serum (Thermo Fisher), penicillin (50 IU mL⁻¹), and streptomycin (50 µg mL⁻¹; Thermo Fisher). Antibodies (InVivoMAb anti‐mouse CD172a (SIRPα) and InVivoMAb anti‐mouse PD‐L1 (B7‐H1)) were procured from BioXcell. Detailed information regarding the antibodies used in this study, including the fluorescent antibody type, manufacturer, clone, and catalog number, is provided in Table S1, Supporting Information.

In Vitro Culture of BMDMs and BMDCs

BMDMs and BMDCs were generated from the bone marrow of 6‐ to 8‐week‐old female C57BL/6 mice. The femurs and tibias were isolated, and bone marrow was extracted using a 26‐gauge syringe and flushed with DMEM (for BMDMs) or RPMI 1640 medium (with HEPES, Thermo Fisher Scientific) (for BMDCs). Red blood cells (RBCs) were eliminated using RBC lysis buffer (BioLegend). Following washing, the cells were resuspended in DMEM (for BMDMs) or RPMI (for BMDCs) medium (50 mL) supplemented with M‐CSF (20 ng mL⁻¹; CreaGene) for BMDMs or mGM‐CSF (20 ng mL⁻¹; CreaGene) for BMDCs and seeded at a density of 5 × 10⁵ cells/10 mL (for BMDMs) or 1 × 10⁶ cells/10 mL (for BMDCs) in a 100 mm plate. On day 3, the medium, which contained M‐CSF (20 ng mL⁻¹) for BMDMs or mGM‐CSF (20 ng mL⁻¹) for BMDCs, was replaced. On day 6, fresh medium supplemented with M‐CSF (20 ng mL⁻¹) for BMDMs or mGM‐CSF (20 ng mL⁻¹) for BMDCs was added. Immature BMDMs and BMDCs were differentiated and utilized for experimentation on day 7.

In Vitro BMDM Polarization

BMDMs were cultured at a density of 1 × 10⁶ cells per plate in a 100 mm plate. M1 macrophages were generated by stimulating the cells with lipopolysaccharide (LPS) at a concentration of 100 ng mL⁻¹ (Sigma‒Aldrich) for 24 h, while M2 macrophages were generated by treating the cells with interleukin‐4 (IL‐4) at a concentration of 20 ng mL⁻¹ (BioLegend).

Preparation of Single‐Cell Suspensions

Tumor tissues and TDLNs were mechanically disrupted and suspended in medium containing collagenase D at a concentration of 1 mg mL⁻¹ (Sigma‒Aldrich). The resulting solutions were incubated in a shaking incubator for 40 min at 37 °C. Subsequently, the cells were subjected to two washes with phosphate‐buffered saline (PBS) following filtration through 70 µm cell strainers. The tumors were then mechanically homogenized and suspended in red blood cell (RBC) lysis buffer (BioLegend) for RBC removal. The solutions were filtered through a 70 µm cell strainer, and additional medium was added. Single cells within the TME or TDLN were obtained through centrifuging the suspensions at 480 ×g for 3 min.

SIRPα Expression Analysis

For in vitro analysis, BMDMs and BMDCs were prepared. M1 BMDMs were pretreated with lipopolysaccharide (LPS) at a concentration of 100 ng mL⁻¹, while M2 BMDMs were pretreated with interleukin‐4 (IL‐4) at a concentration of 20 ng mL⁻¹. The expression of signal regulatory protein alpha (SIRPα) on bone marrow‐derived immune cells was assessed using flow cytometry. For in vivo analysis, B16OVA tumor cells were subcutaneously inoculated into the right flanks of 6‐week‐old female C57BL/6 mice at a density of 5 × 10⁵ cells per mouse. After a 10‐day period, the tumors were dissociated into single cells. Subsequently, the expression of SIRPα on immune cells within the TME was evaluated using flow cytometry.

GILT Gene Knockdown Analysis

RAW 264.7 cells were seeded at a density of 2 × 10⁵ cells per well in a 6‐well culture plate. After 24 h, the cells were replenished with fresh Dulbecco's modified Eagle's medium (DMEM). Lipofectamine RNAiMAX (Thermo Fisher, 12 µL), IFI30‐specific small interfering RNA (siRNA) (Genolotion), and Opti‐MEM (Thermo Fisher) were combined in a total volume of 300 µL, and the mixture was incubated at room temperature for 5 min. The cells were treated with this solution (40 pmol per well) and incubated for 18 h. Following induction of gamma‐interferon‐inducible lysosomal thiol reductase (GILT) gene knockdown, the cells were replenished with fresh medium and exposed to ATICC at a concentration of 100 µg mL⁻¹. After 24 h, the cell supernatants were collected, and the secretion of tumor necrosis factor alpha (TNF‐α) was quantified using an enzyme‐linked immunosorbent assay (ELISA).

GILT‐Dependent Trojan7/8a Cleavage

ATICC at a concentration of 100 µg in a volume of 20 µL was prepared in a 5 mL tube. At a concentration of 1 µM, cysteine was dissolved in PBS and added to the tube at a volume of 50 µL, with or without the inclusion of GILT at a concentration of 2.5 µg (recombinant human IFI30, RayBiotech). All the samples were incubated in a shaking incubator set at 37 °C. After 12 h, the samples were loaded onto desalting columns to remove free R848. Subsequently, the remaining R848 was quantified using ultraviolet–visible light spectrometry (UV‐1800).

In Vitro Cellular Uptake

For the in vitro assessment of the cellular uptake of ATICC, ATICC was conjugated with Cy5 dye. RAW 264.7 cells (5 × 10⁴ cells) were seeded in an ibidi µ‐slide 8‐well microscopy chamber. The cells were incubated with 100 µg mL⁻¹ Cy5‐conjugated ATICC at 37 °C for 24 h. Following washing with PBS, the endolysosomes were stained with LysoTracker (Thermo Fisher), and the nuclei were stained with Hoechst (Thermo Fisher). Cell imaging was conducted using a TCS SP8 HyVolution (Leica Microsystems CMS GmbH) instrument equipped with a 100× objective and the following filter sets (excitation (nm)/emission (nm)): Cy5 (651/670), LysoTracker (577/590) and Hoechst (380/461). For the in vivo assessment of cellular uptake, B16OVA tumor cells (5 × 10⁵ cells per mouse) were subcutaneously inoculated into the right flanks of 6‐week‐old female C57BL/6 mice. After 10 days, Cy5‐conjugated ATICC (500 µg per mouse) was administered. After 24 h, the tumors were dissociated into single cells, and the cellular uptake of each immune cell within the TME was analyzed via flow cytometry.

In Vitro Macrophage Polarization

After M2 macrophages were induced, the cells were subjected to a 24‐h treatment regimen comprising PBS, soluble R848, and ATICC. The analysis of macrophage surface marker expression was performed using a flow cytometer. Additionally, the quantification of cytokines secreted by macrophages was conducted through an ELISA.

In Vitro MDSC Polarization

To isolate MDSCs, C57BL/6 mice (6‐week‐old females) were subcutaneously injected with B16OVA tumor cells (5 × 10⁵ cells per mouse) in the right flank. After 14 days, the spleens were harvested and processed into single cells. Splenocytes were suspended in MACS buffer (PBS, 2 mM ethylenediaminetetraacetic acid, and 0.5% bovine serum albumin), and MDSCs were isolated through negative selection using an MDSC Isolation Kit (Miltenyi Biotec, Gladbach, Germany). Purified MDSCs (1 × 10⁶ cells well⁻¹) were cultured in 6‐well plates and treated with PBS, soluble R848 (1 µg mL⁻¹), or ATICC (100 µg mL⁻¹) for 24 h. Following MDSC polarization, the culture medium was collected, and the secretion of interleukin‐6 (IL‐6) and interleukin‐12 (IL‐12) was analyzed using an ELISA.

In Vitro BMDC Activation

BMDCs (1 × 10⁶) were subjected to treatment with PBS, R848 at a concentration of 5 µg mL⁻¹, or ATICC at a concentration of 100 µg mL⁻¹. After a 24‐h treatment, the expression of dendritic cell activation markers (CD80 and CD86) was assessed using a flow cytometer. Additionally, the quantification of cytokines secreted by the dendritic cells was performed using an ELISA.

Cross‐Presentation

For the cross‐presentation of the protein antigen, 2.5 × 10⁵ BMDCs were treated with PBS, R848 at a concentration of 5 µg mL⁻¹, or ATICC at a concentration of 100 µg mL⁻¹. After 24 h, ovalbumin (OVA) was added to the culture at a concentration of 10 µg mL⁻¹. Spleens were harvested from OT‐1 mice, and CD8⁺ T cells were isolated using a naive CD8⁺ T‐cell isolation kit (Miltenyi Biotec) following the manufacturer's protocol. Preincubated BMDCs were then cocultured with CD8⁺ T cells at a ratio of 1:10 in a 6‐well plate. After 3 days, the cells were labeled with CellTrace Violet (Invitrogen) to confirm the proliferation of CD8⁺ T cells, cell proliferation was analyzed using a flow cytometer, the cell culture supernatants were collected, and interferon‐gamma (IFN‐γ) secretion was measured using an ELISA. For the cross‐presentation of tumor cells, 2.5 × 10⁵ BMDCs were treated with PBS, R848 (5 µg mL⁻¹), or ATICC (100 µg mL⁻¹). After 24 h, 2.5 × 10⁵ B16OVA tumor cells were added to the culture. Spleens were harvested from OT‐1 mice, and CD8⁺ T cells were isolated using a naive CD8⁺ T‐cell isolation kit (Miltenyi Biotec) following the manufacturer's protocol. Preincubated DCs were then cocultured with CD8⁺ T cells at a ratio of 1:10 in a 6‐well plate. After 3 days, the cells were labeled with CellTrace Violet (Invitrogen) to confirm the proliferation of CD8⁺ T cells, cell proliferation was analyzed using a flow cytometer, the cell culture supernatants were collected, and IFN‐γ secretion was measured using an ELISA.

In Vitro Phagocytosis Confocal Image

In each well of an eight‐well chamber slide, 5 × 10⁴ RAW 264.7 cells were seeded. After 24 h of stimulation with PBS or ATICC, the macrophages were labeled with Alexa Fluor 488. B16OVA melanoma cells (1 × 10⁵) were labeled with CellTrace (Thermo Fisher) according to the manufacturer's protocol and subsequently added to the macrophages at a 1:2 ratio, followed by a 12‐h incubation. Cell imaging was conducted using a HyVolution (Leica Microsystems CMS GmbH) instrument equipped with a 100× objective and the following filter sets (excitation (nm)/emission (nm)): Wheat Germ Alexa Fluor 488 (493/519), CellTrace (630/661), and Hoechst (380/461).

In Vitro Phagocytosis Assay

In each well of a 6‐well plate, 1 × 10⁵ macrophages displaying either the M1 or M2 phenotype were seeded. Subsequently, the macrophages were subjected to PBS, R848, or ATICC stimulation. B16OVA melanoma cells (2 × 10⁵) pretreated with IFN‐γ for 24 h to enhance CD47 expression were labeled with CellTrace carboxyfluorescein diacetate succinimidyl ester (CFSE) according to the manufacturer's protocol and added to the macrophages at a 1:2 ratio, followed by a 12‐h incubation period. The phagocytic activity was assessed by measuring the percentage of dual‐stained cells (APC‐CD11b and CFSE‐labeled B16OVA) relative to the total live cells. The percentage of stained cells was analyzed using a flow cytometer.

In Vivo Phagocytosis Assay

CFSE‐labeled B16OVA tumor cells (5 × 10⁵ cells per mouse) were introduced subcutaneously into the right flanks of 6‐week‐old female C57BL/6 mice. After a 10‐day interval, R848 (at doses of 5 or 25 µg per mouse), the anti‐SIRPα antibody (at a dose of 500 µg per mouse), ATICC (at a dose of 500 µg per mouse), or the anti‐programmed death‐ligand 1 (anti‐PD‐L1) antibody (at a concentration of 200 µg mL⁻¹) were administered intratumorally. After a 24‐h period, the tumors were dissociated into single cells. The phagocytic activity was quantified by assessing the percentage of dual‐stained cells (APC‐CD11b and CFSE‐labeled B16OVA) relative to the total live cells. The percentage of stained cells was analyzed using flow cytometry.

In Vivo Flow Cytometry Analysis

B16OVA tumor cells (5 × 10⁵ cells per mouse) were subcutaneously injected into the right flanks of 6‐week‐old female C57BL/6 mice. 10 days later, the tumor‐bearing mice were randomly assigned to groups. Subsequently, R848 (at doses of 5 or 25 µg, equivalent to 79.5 µmol), an anti‐SIRPα antibody (at a dose of 500 µg), ATICC (at a dose of 500 µg), or an anti‐PD‐L1 antibody (at a dose of 200 µg) were administered to the respective groups. All immunizations were repeated every 3 days for a total of three administrations. The nontreated group served as the control. To analyze the TME and TDLNs, tumors and TDLNs were harvested 2 days after the last immunization. Single‐cell suspensions of tumors and TDLNs were prepared and analyzed using flow cytometry. Flow cytometry data were analyzed using a BD FACSCanto II (at the BIORP of the Korea Basic Science Institute) and quantified using FlowJo v.10. Detailed information regarding the antibodies and gating strategies employed is provided in Table S1, Supporting Information.

Analysis of Antigen Specificity of CD8⁺ T Cells

To analyze the antigen‐specific CD8⁺ T cells, single cells (5 × 10⁵ per well) were seeded in a round‐bottom 96‐well plate. Then, the cells were restimulated with OVA SIINFEKL peptide (10 µg mL⁻¹), IL‐2 (30 ng mL⁻¹; PeproTech), and GolgiPlug (a protein transport inhibitor; 0.6 µg mL⁻¹; BD Bioscience) for 6 h. Following stimulation, the cells were collected and washed twice. Surface marker antibodies were applied for a 30‐min incubation at 4 °C. For intracellular staining, the cells were washed and then suspended in fixation/permeabilization solution for 20 min at 4 °C. Fixed cells were washed twice with BD Perm/Wash buffer (BD Bioscience) and stained with antibodies for another 30 min at 4 °C. Following staining, the cells were washed twice with BD Perm/Wash buffer and suspended in staining buffer. Flow cytometry data were analyzed using a BD FACSCanto II (at the BIORP of the Korea Basic Science Institute) and quantified using FlowJo v.10. Detailed information regarding the antibodies and gating strategies employed is provided in Table S1, Supporting Information.

AST and ALT Activity Analysis

Blood samples were obtained from each mouse that had been injected with R848 at a dose of 5 or 25 µg (equivalent to 79.5 µmol) or ATICC at a dose of 500 µg. The collected blood was subjected to centrifugation at 10 000 ×g for 20 min to isolate the serum. The obtained serum was subsequently analyzed using aspartate aminotransferase (AST) or alanine aminotransferase (ALT) activity analysis kits (Sigma) to characterize AST or ALT activity.

Serum Cytokine Level Analysis

Blood samples were obtained from each mouse that had been injected with R848 at a dose of 5 or 25 µg (equivalent to 79.5 µmol) or ATICC at a dose of 500 µg. The collected blood was subjected to centrifugation at 10 000 ×g for 20 min to isolate the serum. The obtained serum was subsequently analyzed using ELISA kits (BD Bioscience) to quantify TNF‐α and IL‐6 concentrations.

In Vivo Antitumor Study

B16OVA tumor cells (5 × 10⁵ cells per mouse) were subcutaneously injected into the right flanks of 6‐week‐old female C57BL/6 mice. 10 days later, the tumor‐bearing mice were randomly assigned to groups. Subsequently, R848 (at doses of 5 or 25 µg, equivalent to 79.5 µmol), an anti‐SIRPα antibody (at a dose of 500 µg), ATICC (at a dose of 500 µg), or an anti‐PD‐L1 antibody (at a dose of 200 µg) were administered to the respective groups. All immunizations were repeated every 3 days for a total of three administrations. The nontreated group served as the control. Mouse tumor growth and survival were monitored at various time points. Tumor volume was calculated using the following formula: (long‐axis diameter) × (short‐axis diameter)²/2. The mice were killed when the tumor volume reached the maximum tumor size (1000 mm³) approved by the IACUC, Sungkyunkwan University School of Medicine.

Conflict of Interest

The authors declare no conflict of interest.

Supporting information

Supporting Information

Park S., Jin S. M., Kim S., Cho J. H., Hong J., Bae Y.‐S., Lim Y. T., Bioconjugated Antibody‐Trojan Immune Converter Enhance Cancer Immunotherapy with Minimized Toxicity by Programmed Two‐Step Immunomodulation of Myeloid Cells. Adv. Healthcare Mater. 2024, 13, 2401270. 10.1002/adhm.202401270

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.