Nonglycosylated, Legumain-Cleavable ISACs Drive Potent Antitumor Immunotherapy via a Bystander Effect

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is the most lethal form of pancreatic cancer, with poor prognosis driven by late diagnosis, therapeutic resistance, and an immunosuppressive tumor microenvironment. Interactions between tumor cells and immune cells promote immune evasion and tumor progression, limiting the efficacy of immune checkpoint blockade and other immunotherapies. Given the high expression of trophoblast cell surface antigen 2 (TROP2) in PDAC, we developed a TROP2-targeted Toll-like receptor 7 (TLR7) agonist (E104) designed for selective accumulation within the tumor microenvironment to activate antitumor immunity. Although antibody bioactivity is traditionally linked to Fcγ receptor (FcγR) engagement and recruitment of effector cells, our legumain-cleavable, nonglycosylated immune-stimulating antibody conjugates (NG-ISAC) induce robust myeloid activation, cytokine release, and tumor regression without FcγR-mediated functions of NK cells or macrophages. Rather, NG-anti-TROP2-E104-ISACs depend on tumor antigen recognition and TLR7 activation, not FcγR-driven antibody-dependent cellular cytotoxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP), to elicit tumor regression and adaptive immunity as evidenced by anti-TROP2 antibody generation in a syngeneic model. By incorporating the cell-permeable E104 payload with bystander activity, NG-ISACs can activate immune responses independently of FcγR binding. In vitro, NG-anti-TROP2-E104-ISACs bypass FcγRIIa-mediated ADCC and FcγRIIIa-mediated ADCP while maintaining potency in cocultures of TROP2-positive tumor and effector cells. Moreover, NG-anti-TROP2-E104-ISACs display reduced acute toxicity compared with glycosylated counterparts. Together, these findings delineate the bystander mechanism underlying FcγR-independent immune stimulation and establish a framework for designing ISACs with improved safety.

Introduction

Pancreatic cancer is one of the most lethal malignancies (1), accounting for 85% of all cases worldwide, with a 5-year survival rate of approximately 10% in the United States (2). It is projected to become the second leading cause of cancer-related deaths by 2030 (3, 4). Pancreatic ductal adenocarcinoma (PDAC) accounts for more than 90% of pancreatic tumors, with poor survival outcomes (2, 5) because of rapid disease progression and late-stage diagnosis, often attributed to the absence of early symptoms (3). The tumor microenvironment (TME) of PDAC plays a critical role in both tumor growth and resistance to therapy, posing a significant challenge to the development of effective treatments (6).

Efforts to enhance antitumor immunity have focused on immune checkpoint inhibitors (3), which have shown success in several malignancies (7). However, their application in PDAC has been largely unsuccessful because of the highly immunosuppressive TME (2). This environment is enriched with myeloid-derived suppressor cells, regulatory T cells, M2 macrophages, and cancer-associated fibroblasts, all of which contribute to immune evasion and tumor progression (8). Given the limitations of immune checkpoint blockade, alternative immunotherapeutic strategies targeting innate immune pathways have gained significant attention.

Toll-like receptors (TLR), a class of pattern recognition receptors (PRR), are central mediators of antitumor immune responses (9, 10). TLR agonists (TLRa) have emerged as promising immunomodulators, capable of activating cytokine signaling cascades that drive immune-mediated tumor clearance (11). Among these, TLR7—an endosomal receptor—activates NF-κB and IFN regulatory factor (IRF) pathways upon stimulation, thereby promoting both innate and adaptive immune responses (12). TLRa can repolarize tumor-associated macrophages (TAM) from an immunosuppressive M2 phenotype toward an M1-like phenotype (13), thereby enhancing proinflammatory cytokine production (14). This shift not only alters macrophage function but also triggers a broader proinflammatory response across other immune cells within the TME, leading to robust tumor suppression (15). Furthermore, TLRa can enhance T cell–mediated antitumor immunity, thereby promoting both immediate and durable responses (16). However, systemic administration of TLRa is limited by dose-dependent toxicity resulting from widespread immune activation (17). Thus, strategies that enhance tumor selectivity while limiting systemic exposure are essential for clinical translation.

Immune-stimulating antibody conjugates (ISAC) represent a promising strategy to achieve localized delivery of TLRa, thereby reducing systemic toxicity (18). By conjugating TLR7 agonists (e.g., E104) to tumor-targeting antibodies, ISACs enable selective immune activation within the TME while minimizing off-target effects (19). Trophoblast cell surface antigen 2 (TROP2), a transmembrane glycoprotein overexpressed in approximately 55% of pancreatic cancers (20), serves as an attractive target for ISAC-based therapies (21–23). Preclinical studies have demonstrated several advantages of ISACs over conventional antibody–drug conjugates that deliver cytotoxic payloads (10, 24). First, ISAC-mediated antitumor responses can target a broad range of tumor-associated damage-associated molecular patterns. Second, ISACs activate antigen-presenting cells (APC) and can further stimulate other tumor-infiltrating immune cells, including T cells, NK cells, and dendritic cells (Fig. 1A). Third, ISACs can elicit immunologic memory through adaptive immune activation, contributing to durable antitumor responses and lowering the risk of recurrence.

Figure 1.

A, Schematic illustration of NG-ISAC mechanism of action. NG-ISACs bind to TROP2 on tumor cells and are internalized, releasing the conjugated TLR7 agonist E104 intracellularly. The released E104 diffuses within the TME, triggering bystander activation of immune-suppressive cells such as regulatory T cells (Treg), myeloid-derived suppressor cells (MDSC), and TAMs. This activation promotes proinflammatory cytokine production, recruits immune effector cells, including T cells, NK cells, dendritic cells, and B cells, and reduces immunosuppressive myeloid populations, collectively shifting the TME toward an immunostimulatory phenotype. B, Schematic overview of the three-step conjugation process for NG-ISACs. C, Representative chemical structure of E104-ISACs.

Mechanistically, ISACs are typically thought to achieve tumor suppression through opsonization of tumor cells, followed by Fc-mediated endocytosis (or trogocytosis) by APCs (25), and subsequent activation of intracellular TLRs or other PRRs (10). Upon uptake in APCs, ISACs release their conjugated agonists, thus synergistically driving proinflammatory cytokine production, enhancing antigen presentation, and attracting additional immune cells to the tumor site (26).

Most ISACs to date utilize conventional IgG backbones that retain an N-linked glycan at Asn297, a feature that is widely reported to facilitate interactions with Fcγ receptors (FcγR) and complement component C1q (27). These interactions, in turn, drive uptake into APCs and contribute to tumor regression (25). In humans, three activating type I FcγRs—FcγRI, FcγRIIa, and FcγRIIIa—are broadly expressed on leukocytes and mediate antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP; refs. 28, 29).

In this study, we investigated whether ISAC efficacy requires Fc effector functions. Rather than removing the Fc segment, which is known to markedly shorten antibody half-life in vivo (30), we developed nonglycosylated (NG) ISACs using an NG-anti-TROP2 monoclonal antibody (mAb) (31), which significantly reduces FcγR binding and Fc-mediated uptake and effector functions while preserving tumor-targeted immune activation. Importantly, Fc glycosylation is not required for binding to the neonatal Fc receptor (FcRn; refs. 25, 32), which supports FcRn-mediated recycling and favorable pharmacokinetics (PK; ref. 33). As Fcγ-mediated uptake of ISACs by APCs may also contribute to the formation of antidrug antibodies (ADA; ref. 34), this design may enhance ISAC safety by reducing immunogenicity. The resulting NG-ISACs consist of an NG-anti-TROP2 antibody conjugated to a potent TLR7 agonist (E104) via a cleavable linker.

We demonstrate that NG-anti-TROP2-E104-ISACs elicit potent antitumor immunity independent of FcγR-driven ADCC or ADCP. NG-ISACs induce a bystander effect within the TME, reprogramming M2 macrophages toward an M1-like phenotype and promoting antitumor cytokine production in the presence of TROP2-expressing PDAC cells. In a syngeneic mouse model, systemic administration of NG-ISACs triggers robust innate and adaptive immune responses, resulting in significant tumor regression while minimizing systemic toxicity. These findings highlight the potential of NG-ISACs as a novel targeted immunotherapeutic strategy for PDAC, capable of remodeling the immunosuppressive TME and enhancing antitumor immunity with an improved safety profile.

Materials and Methods

General

All synthetic reagents were purchased from commercial suppliers and used without further purification. All compounds used in this study exhibited >90% purity as determined by the Waters SQD2 LC/MS system (RRID: SCR_022217) and ¹H nuclear magnetic resonance (NMR) analyses. ¹H NMR spectra were recorded on a Bruker Avance III HD 400 MHz spectrometer (RRID: SCR_027378). Compound identity and purity were confirmed by both LC/MS and ¹H NMR. The general reaction scheme (Supplementary Scheme S1) and synthetic methods (Spectra S1–S16) are described in the Supplementary Materials.

Antibody conjugation and characterization

Sacituzumab (Saci) is a clinically validated humanized IgG1 antibody targeting human TROP2 (hTROP2), whereas palivizumab (Pali) is a humanized IgG1 antibody that binds a viral capsid protein of respiratory syncytial virus and was used here as an isotype control. Biosimilar Saci and Pali (CHO-expressed biosimilars with amino acid sequences identical to the commercial products) were obtained from Syd Labs (RRID: SCR_013614). NG-ISACs were prepared using a three-step conjugation method (Fig. 1A and B) adapted from previously published protocols (31). Briefly, mAbs Saci and Pali were treated with PNGase F to remove N-linked glycans prior to conjugation. The resulting deglycosylated antibodies (NG-mAbs) were then reduced with tris(2-carboxyethyl)phosphine (TCEP) to generate free thiol groups. For conjugation, linker–payloads were reacted with the reduced antibodies at room temperature for 2 hours. In parallel, conventional ISACs were prepared using a two-step protocol in which mAbs were reduced with TCEP and subsequently conjugated with the same linker–payloads. All resulting immunoconjugates were buffer-exchanged into PBS using Vivaspin 500 centrifugal concentrators (10 kDa MWCO, Sartorius). Protein concentrations were determined by absorbance at 280 nm using a Thermo Fisher Scientific NanoDrop One/OneC Microvolume UV-Vis Spectrophotometer (RRID: SCR_023005). Yields exceeded 75% based on recovered protein. Size-exclusion chromatography (SEC) analysis performed on an Agilent Infinity 1260 Liquid Chromatography System (RRID: SCR_019511) showed minimal aggregation for all conjugates, except for NG-Saci-ValCitPABC-E104. Drug-to-antibody ratios (DAR) were quantified by LC/MS (Supplementary Table S1, Spectra S17–S37). Final products were sterile filtered using 0.2-µm filters and stored at −20°C until use.

Cell culture

Unless otherwise specified, all cell culture experiments were performed using complete medium composed of commercially available basal medium supplemented with 10% heat-inactivated FBS and 1% penicillin/streptomycin (Corning) and maintained at 37°C in a humidified atmosphere with 5% CO₂. Cell lines were obtained from ATCC, Sigma-Aldrich, InvivoGen, or other vendors as outlined below and were used within 20 passages. All cell lines were authenticated by the suppliers and confirmed to be free of Mycoplasma contamination prior to shipment. Cell lines were not reauthenticated after arrival. Ramos-Blue reporter cells (2024, InvivoGen, cat. #rms-sp, RRID: CVCL_X591), human peripheral blood mononuclear cells (PBMC; male; iXCells Biotechnologies, November of 2024), mouse BMDM-M2 and splenocytes (March 2024; iQ Biosciences; female, IQB-MSP202), BxPC-3 cells (2023, ATCC, cat. #CRL-1687, RRID: CVCL_0186), RAW-Dual reporter cells (May 2019, InvivoGen, cat. #rawd-ismip, RRID: CVCL_A7ZK), MC38-hTROP2 (April 2024, Kyinno Biotechnology, cat. #KC-1698), Jurkat-Lucia NFAT-CD16 (June 2024, InvivoGen, cat. #jkt-nfat-cd16, RRID: CVCL_A7ZT), and NFAT-CD32 (August 2024, InvivoGen, cat. #jkt-nfat-cd32, RRID: CVCL_A7ZU) were also obtained.

Differentiation and polarization of M2-like macrophages

Human PBMCs (male) were cultured as described previously (14). Briefly, PBMCs were plated in monocyte attachment medium (PromoCell) and incubated for 2 hours to allow monocyte adherence. The attached monocytes were then differentiated into M0-like macrophages by culturing in medium containing 20 ng/mL human macrophage colony-stimulating factor (M-CSF) for 7 days (35). To induce M2-like polarization, M0 macrophages were subsequently incubated for 2 days in tumor cell–conditioned medium collected from CFPAC-1 cells (ATCC, cat. #CRL-1918, RRID: CVCL_1119).

Mouse bone marrow–derived macrophages (BMDM; mixed gender) were prepared following the STAR protocol with minor modifications (36). Briefly, bone marrow was harvested from the femurs and tibias of mice, centrifuged at 4,000 g for 1.5 minutes, and resuspended in DMEM containing 10% FBS and 10 ng/mL mouse M-CSF. Cells were cultured for 7 days to allow differentiation into macrophages. To induce M2 polarization, BMDMs were seeded into 96-well plates at a density of 10,000 cells/well and incubated for 24 hours in MC38 (Sigma-Aldrich, cat. #SCC172, RRID: CVCL_B288) tumor cell–conditioned medium.

Cellular activation assays

PBMCs and PBMC-derived M2 macrophages (PBMC-M2) were seeded into flat-bottom 96-well plates at densities of 1 × 10⁵ and 5 × 10⁴ cells per well, respectively, in RPMI-1640 complete medium. Mouse BMDM-M2 and splenocytes were seeded at 1 × 10⁵ cells per well in DMEM complete medium. Cells were then treated with immunostimulatory agents or control solutions and incubated for 24 hours.

For coculture experiments, PBMC-M2 cells were incubated with CFPAC-1 tumor cells at a 3:1 ratio. Mouse BMDM-M2 cells were cocultured with BxPC-3 tumor cells at a 5:1 ratio. Mouse splenocytes were cocultured with either CFPAC-1 or BxPC-3 cells at a 10:1 ratio. All cultures were treated with immunostimulatory agents or control solutions in fresh medium and incubated for 24 hours.

Cellular activation was assessed by measuring cytokine production in the supernatants using ELISA. The following kits were used: ELISA MAX Deluxe Set Human TNFα, Human IL6, and Mouse IL6 (BioLegend).

Reporter cell assays

Ramos-Blue reporter cells, which stably express an NF-κB–/AP-1–inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene, were used to assess human NF-κB pathway activation. Cells were maintained according to the manufacturer’s protocol in complete Iscove’s Modified Dulbecco’s Medium (IMDM) supplemented with 2 mmol/L L-glutamine, normocin, and zeocin. For coculture experiments, Ramos-Blue cells were seeded in 96-well plates and cocultured with BxPC-3 tumor cells at a 1:1 ratio. After overnight incubation, cells were treated with test agents for 24 hours. Following treatment, culture supernatants were collected, and NF-κB pathway activation was quantified using the QUANTI-Blue assay (InvivoGen), according to the manufacturer’s instructions.

RAW-Dual reporter cells, murine macrophages engineered to co-express SEAP under an NF-κB/AP-1 promoter and Lucia luciferase under an IRF-inducible promoter, were used to evaluate both NF-κB and IRF pathway activation. Cells were cultured in complete DMEM supplemented with normocin and zeocin. Upon reaching 80% to 85% confluency, cells were seeded at 1 × 10⁵ cells per well in 96-well plates and cocultured with BxPC-3, CFPAC-1, or MC38-hTROP2 tumor cells at ratios of 1:1, 5:4, and 5:1, respectively. After overnight coculture, cells were treated for 24 hours. Supernatants were collected, and NF-κB and IRF pathway activation was assessed using the QUANTI-Blue and QUANTI-Luc assays (InvivoGen), respectively, following the manufacturer’s protocols.

Jurkat-Lucia NFAT-CD16 and NFAT-CD32 reporter cells, which stably express the FcγRs CD16A (FcγRIIIA) or CD32A (FcγRIIA), respectively, along with an NFAT-inducible Lucia luciferase reporter gene, were used to evaluate ADCC and ADCP potential. Both cell lines were cultured in complete IMDM supplemented with normocin, blasticidin, and zeocin. For coculture experiments, reporter cells were seeded in 96-well plates and incubated with CFPAC-1 tumor cells at a 2:1 effector-to-target ratio. CFPAC-1 cells were pretreated for 1 hour before addition of Jurkat-Lucia reporter cells, followed by a 6-hour co-incubation. Following treatment, supernatants were collected, and NFAT pathway activation was measured using the QUANTI-Luc assay (InvivoGen), following the manufacturer’s instructions.

Payload release assays

BxPC-3 tumor cells or RAW-Dual murine macrophages were seeded in 96-well plates at 1.5 × 10⁴ cells per well and treated with NG-ISACs for 4 days. Following incubation, supernatants were collected, and the release of E104 was quantified by LC/MS.

OpenSPR binding analysis

Histidine-tagged FcγR proteins were obtained from commercial suppliers: CD64 (FcγRI, Sino Biological, cat. #10256-H08S-100), CD16a (FcγRIIIa, Invitrogen, cat. #RP87975), and CD32a (FcγRIIa, Sino Biological, cat. #10374-H08H1-100). Binding assays were performed using a two-channel OpenSPR (or OpenSPR-XT) instrument (Nicoya Lifesciences) with a carboxyl-functionalized sensor chip (Carboxyl Chip, Nicoya Lifesciences, cat. #SEN-AU-100-10-HC-COOH) in PBS running buffer. Anti-His antibody (GeneTex, cat. #GTX40628, RRID: AB_11172041) was immobilized onto the sensor chip surface to capture His-tagged FcγRs. NG-ISACs, conventional ISACs, or unconjugated mAbs were then injected over the flow cells containing the captured receptors. Binding data were double-referenced using a blank anti-His surface and buffer-only injections. A kinetic titration approach was used for analyte injections, and sensor surfaces were regenerated between cycles using 10 mmol/L glycine-HCl (pH 1.5). Data were analyzed using TraceDrawer software (Nicoya Lifesciences). Relative binding affinities were normalized to those of Saci.

Stability evaluations of NG-ISACs

Hepatic metabolism

The in vitro clearance of E104 was evaluated in human liver microsomes (XenoTech, LLC) in the presence of a RapidStart NADPH Regenerating System (XenoTech, LLC), following the vendor’s protocol. R848 (VWR, cat. #76810-408) and 7-ethoxycoumarin were included as reference compounds (37, 38).

Lysosomal stability assay

Pooled Sprague–Dawley rat liver tritosomes (mixed gender, XenoTech, LLC) were used to evaluate the lysosomal stability of the linker–payloads. To prevent maleimide-mediated enzyme deactivation, the maleimide (Mc) group on the linker was quenched using cysteine, yielding cys-Mc-AsnAsn-E104 and cys-Mc-ValCitPABC-E104. Enzymatic activation of the linkers was performed according to the vendor’s protocol. After activation, 10 µL of the quenched linker–payload (5 mmol/L) was diluted in 50 µL of sodium acetate buffer (pH 4.7), followed by the addition of 40 µL of activated tritosome preparation. The reaction mixtures were incubated at 37°C, and 10 µL aliquots were collected at 1, 5, 15, 30, 60, 120, 240, and 360 minutes. Each aliquot was quenched with 90 µL of acetonitrile (ACN) to precipitate proteins. Analyses were performed using a Xevo TQD LC/MS system.

Aggregation assessment

NG-ISACs were diluted to a concentration of 0.5 mg/mL and incubated at 45°C for 4 weeks to evaluate thermal stability. Aggregation was assessed by SEC.

Serum stability of NG-ISACs

To assess serum stability, NG-ISACs were diluted to 0.2 mg/mL in a sterile solution composed of 70% mouse or human serum (GeminiBio) and 30% PBS. Samples were incubated at 37°C with 5% CO₂. On days 0, 1, 4, and 7, 50 μL aliquots were collected. Plasma proteins were precipitated by adding three volumes of ACN, and the supernatants were analyzed by LC/MS to determine the extent of E104 release.

Flow cytometric analysis

Cells were stained on ice for 30 minutes with Zombie NIR Fixable Viability Dye (BioLegend, cat. #423101) and a synthesized Cy5-labeled anti-TROP2 antibody. Following staining, samples were washed twice and resuspended in cell staining buffer (BioLegend, cat. #420201). Flow cytometric analysis was performed using a Bio-Rad ZE5 Cell Analyzer (RRID: SCR_019712), and data were processed using BD Accuri C6 Plus (RRID: SCR_014422).

Animal studies

All animal procedures were conducted in accordance with protocols approved by the Institutional Animal Care and Use Committee (IACUC).

Xenograft tumor model experimental procedure

The human pancreatic cancer cell line CFPAC-1 (ATCC) was cultured according to the manufacturer’s instructions. Cells were harvested at 80% to 90% confluency, washed with PBS, and resuspended at a concentration of 5 × 10⁷ cells/mL in PBS. The suspension was kept on ice for no longer than 2 hours. A volume of 100 μL (5 × 10⁶ cells) was injected subcutaneously into the right flank of 6- to 8-week-old female Nu/J mice (RRID: IMSR_JAX:002019). Tumor growth was monitored every other day using caliper measurements, and volume was calculated using the formula (length × width²)/2. Treatment was initiated when tumor volumes reached ∼100 mm³, typically within 2 weeks after implantation. Mice were randomized into treatment groups, and treatments were initiated when tumors were of required size. DAR8 NG-ISACs were formulated in PBS at 1.2 mg/mL and administered intraperitoneally at a dose of 6 mg/kg. Treatments, including NG-Saci or vehicle controls, were given at a dosage of 6 mg/kg every 4 days for a total of three dosages, as detailed in the corresponding figure legends. Study blinding was maintained until treatment completion. Mice were humanely euthanized if tumor volumes exceeded 1,000 mm³ or if ulcerations or distress was observed.

Syngeneic tumor model experimental procedure

MC38-hTROP2 cells (Kyinno Biotechnology) were cultured in RPMI-1640 medium supplemented with 10% FBS and 5 μg/mL puromycin at 37°C in a humidified incubator with 5% CO₂. Cells at 80% to 90% confluency were detached with trypsin, resuspended in PBS at 5 × 10⁷ cells/mL, and maintained on ice prior to injection. A 100 μL suspension (5 × 10⁶ cells) was injected subcutaneously into 6- to 8-week-old female C57BL/6J mice (RRID: IMSR_JAX:000664). Tumor size was measured three times weekly, and volume was calculated using the formula above. Treatments were initiated at ∼100 mm³ tumor volume (typically reached 1–2 weeks after implantation), and mice were randomized into treatment groups. DAR4 NG-ISACs or control agents were administered intraperitoneally at 6 mg/kg every 4 days for a total of three dosages, following the schedule detailed in the figure legends. Mice were euthanized following IACUC protocols if tumor volumes exceeded 1,000 mm³ or if signs of distress were present.

For immunogenicity assessment, blood samples were collected via submandibular bleeding at multiple time points (days 4–30 after dose; n = 4 mice per time point) into serum collection tubes (Sarstedt Microvette). Samples were centrifuged at 4,000 g for 4 minutes at room temperature, and serum was stored at −80°C. Detection of mouse anti-hTROP2 antibodies was performed using the PIXI Automated Immunoassay Platform (Correlia Biosystems).

Serum PK and toxicity assessment

For PK evaluation, ISACs were administered either intraperitoneally or intravenously via tail vein injection, with dose volumes adjusted to 80 to 120 μL based on body weight. Injections were completed within 5 seconds. Mice were closely monitored for 1 hour after injection and assessed for toxicity at 3, 6, and 24 hours. Blood samples were collected at defined intervals (15 minutes to 14 days after dose; n = 3 mice per time point) via submandibular bleeding. Serum was prepared as described above and stored at −80°C until analysis. ISAC serum concentrations were quantified using the PIXI Automated Immunoassay Platform (Correlia Biosystems) or the MESO QuickPlex SQ 120 (RRID: SCR_020304).

For total human IgG quantitation, antibody and ISAC concentrations were measured on the Correlia PIXI Automated ELISA system using the Human IgG Detection Kit (Correlia, KPX1-PCEA101-001). For MSD assays, rabbit anti–human IgG (H + L) secondary antibody (Thermo Fisher Scientific, cat. #31143, RRID: AB_228410) was used as the capture antibody. The assay was performed on 96-well 1-Spot SECTOR Plates (MSD, L15XA-3) with MSD Blocker A Kit (R93AA-2), SULFO-TAG anti–human IgG antibody (D21ADF-3), and MSD Gold Read Buffer B (R60AM-3). The data were analyzed using noncompartmental analysis conducted with Phoenix WinNonlin software (version 8.40, RRID: SCR_024504). ADA detection was performed using synthesized MSD TAG–conjugated Saci and biotinylated Saci, with MSD GOLD 96-well Streptavidin Plates (MSD, L45SA), following the manufacturer’s instructions.

Statistics and reproducibility

The number of biological and technical replicates for each experiment is indicated in the corresponding figure legends. In vitro experiments were not randomized, and investigators were not blinded to sample allocation or outcome assessment during these studies. For all in vivo experiments, the exact number of mice per treatment group is provided in the figure legends. Animals were randomized into groups based on tumor size prior to treatment initiation. Investigators conducting in vivo studies were blinded to group allocation during both treatment and data collection.

Sample sizes for in vitro studies were selected to ensure sufficient statistical power, with a minimum of three independent biological replicates included in most assays. Efforts were made to minimize technical variation and ensure reproducibility across experiments.

All statistical analyses were performed using Microsoft Excel (RRID: SCR_016137) or GraphPad Prism (RRID: SCR_002798). Comparisons between two groups were conducted using either paired or unpaired two-tailed Student t tests, as appropriate. For comparisons involving more than two groups, one-way or two-way ANOVA was applied, followed by post hoc multiple comparisons using Tukey correction. Statistical significance was defined based on the highest P value obtained when comparing each treatment group with all others, unless specified otherwise in the figure legends. A P value of less than 0.05 was considered statistically significant.

Results

Design and preparation of NG-ISACs that function via a bystander mechanism

In 2022, we reported a HER2-targeted ISAC design using a cleavable mc-Val-Cit-PABC linker conjugated to the selective TLR7 agonist E104 (Fig. 1C). Earlier studies suggested that FcγR binding was required for ISAC efficacy (19), but our design employed an NG IgG1 backbone lacking FcγR affinity. We hypothesized that NG-ISACs could act through a bystander mechanism, in which antigen-dependent uptake by tumor cells releases E104, which diffuses to TAMs and promotes M2-to-M1 polarization (Fig. 1A). To rigorously test FcγR involvement, we generated matched glycosylated and NG-ISACs. NG-ISACs were produced by enzymatic removal of Fc N-linked glycans to abolish FcγR binding while preserving FcRn interactions (31). This modification preserves FcRn binding while eliminating engagement with Fc receptors (32, 33). E104 was conjugated to NG-Saci, and isotype controls were prepared with NG-Pali. Conjugation used TCEP-mediated reduction of interchain disulfides, followed by coupling to maleimide-containing E104 linkers (Fig. 1B). Conjugation efficiency and DAR were confirmed by LC/MS (Supplementary Table S1), and stability was assessed by SEC and hydrophobic interaction chromatography.

Because mc-Val-Cit-PABC linkers displayed hydrophobicity, aggregation, and premature cleavage, four alternative linkers were synthesized (Supplementary Scheme S1): (i) mcValCitGlyPro-E104 (4a), with a polar GlyPro spacer (39), (ii) PEG12-ValCitPABC-E104 (4b), a PEGylated construct, and two legumain-sensitive linkers, (iii) mcAsn-E104 (4c) and (iv) mcAsnAsn-E104 (4d), building on our prior findings that Asn-containing motifs are rapidly cleaved by lysosomal legumain (40, 41). Each was synthesized using Fmoc peptide chemistry, fitted with maleimide handles, and conjugated to NG-Saci and NG-Pali. Complete disulfide reduction yielded DAR ∼8, although in selected cases, constructs with DAR ∼4 were prepared to mitigate aggregation and improve PK exposure (10).

Analysis of FcγR-mediated ADCC and ADCP

To evaluate FcγR engagement, we measured binding affinities of ISACs and NG-ISACs to FcγRI, FcγRIIa, and FcγRIIIa using surface plasmon resonance (SPR), with Saci as the reference. Compared with their glycosylated counterparts (Saci-ISACs), NG-Saci-ISACs exhibited markedly reduced or undetectable binding to FcγRIIa and FcγRIIIa (Supplementary Table S2) and showed diminished FcγRI binding (Supplementary Fig. S1A). As FcγRI is typically saturated by monomeric IgG on circulating cells in vivo (42, 43), this reduction is consistent with loss of Fc-mediated effector potential.

We next examined functional FcγR engagement using engineered Jurkat-Lucia NFAT reporter cells expressing either FcγRIIa (CD32) or FcγRIIIa (CD16), cocultured with CFPAC-1‒TROP2^medium tumor cells (Fig. 2A; Supplementary Fig. S1B). In these assays, NFAT-driven luciferase expression serves as a readout of FcγR activation. As expected, NG-ISACs failed to induce NFAT activation, whereas glycosylated ISACs retained FcγR binding and triggered robust reporter responses, confirming that NG-ISACs function independently of FcγR-mediated ADCC and ADCP.

Figure 2.

NG-ISACs activate myeloid and lymphoid cells independent of ADCC and ADCP. A, FcγR-mediated activation was assessed using Jurkat-Lucia NFAT-CD16 and NFAT-CD32 reporter cells cocultured with CFPAC-1 tumor cells. ISAC-induced NFAT signaling was measured by Lucia luciferase activity 6 hours after incubation. Glycosylated ISACs are shown with dashed lines whereas NG-ISACs are shown with solid lines. B and C, Mouse BMDM-M2 (B) and splenocytes (C) were stimulated with E104 for 24 hours, followed by analysis of IL6 production using ELISA. D and E, RAW-Dual reporter macrophages were cocultured with BxPC-3‒TROP2^high (D) or CFPAC-1‒TROP2^medium tumor cells (E) in the presence of NG-ISACs or controls. Activation of NF-κB signaling pathways was measured. H, Mouse BMDM-M2 cells were cocultured with BxPC-3‒TROP2^high tumor cells and stimulated with NG-ISACs for 24 hours. IL6 production was quantified by ELISA. I, Mouse splenocytes were cocultured with hTROP2-expressing tumor cells and stimulated with NG-ISACs for 24 hours. IL6 production was quantified by ELISA. Data are presented as the mean ± SD (n = 3), and EC₅₀ values are from three independent experiments. OD, optical density; RLU, relative light units.

NG-ISACs activated myeloid and lymphoid cells independent of ADCC and ADCP

E104 engages TLR7 at low nanomolar concentrations with high cell permeability (44). In mouse BMDM-M2 macrophages and splenocytes, E104 induced strong activation (Fig. 2B and C), consistent with its known mechanism. In human liver microsomes, E104 cleared faster than R848 (Supplementary Fig. S2A; Supplementary Table S3), suggesting reduced systemic exposure from free payload (45).

RAW-Dual macrophages cocultured with BxPC-3‒TROP2^high tumor cells showed robust NF-κB activation with NG-ISACs (Fig. 2D). NG-Saci-AsnAsn-E104 and NG-Saci-PEG12ValCitPABC-E104 displayed EC₅₀ ∼1.0 μg/mL, whereas NG-Saci-Asn-E104 was less potent (EC₅₀ = 1.9 μg/mL). Isotype controls generally showed minimal activity, although Pali-ValCitGlyPro-E104 and Pali-Asn-E104 triggered responses, suggesting linker instability (Supplementary Fig. S2C). In CFPAC-1‒TROP2^medium cocultures, NF-κB activation was weaker than in BxPC-3‒TROP2^high cells (Fig. 2E; Supplementary Fig. S2D), supporting antigen-dependent uptake.

To extend our findings to a murine system, we performed coculture assays using BMDM-M2 macrophages with hTROP2-expressing tumor cells. Cocultures of BMDM-M2 with BxPC-3‒TROP2^high tumor cells (Fig. 2F) showed robust activation by NG-ISACs compared with both the isotype control (Supplementary Fig. S3A) and the unconjugated NG-Saci antibody. Among the constructs tested, NG-Saci-ValCitPABC-E104 exhibited an EC₅₀ of 2.0 μg/mL, whereas NG-Saci-AsnAsn-E104 showed slightly lower potency with an EC₅₀ of 4.7 μg/mL. Notably, when BMDM-M2 cells were incubated with ISACs in the absence of tumor cells, glycosylated ISACs retained activity (Supplementary Fig. S3B), but NG-ISACs showed markedly reduced responses (Supplementary Fig. S3C), suggesting that removal of Fc glycosylation may lessen off-target immune activation mediated by FcγR binding. In cocultures of mouse splenocytes, which contain abundant T, B, and NK cells, with TROP2-expressing tumor cells, NG-ISACs induced marked IL6 production, whereas the isotype control and naked antibody elicited no measurable response (Fig. 2G; Supplementary Fig. S3D and S3E).

NG-ISACs activate human macrophages and lymphocytes via a bystander effect

Macrophages are key components of the TME and can constitute up to 50% of tumor mass (46). In both PBMCs and PBMC-derived M2 macrophages, the TLR7 agonist E104 induced strong activation (Fig. 3A and B), consistent with its known mechanism. To assess lymphocyte activation (47), Ramos-Blue NF-κB reporter B cells were cocultured with BxPC-3‒TROP2^high cells (Fig. 3C). NG-Saci-E104 ISACs triggered NF-κB activation in B lymphocytes, with NG-Saci-AsnAsn-E104 showing the highest potency (EC₅₀ = 3.6 μg/mL). No activation was observed with unconjugated antibodies or isotype controls (Fig. 3D).

Figure 3.

NG-ISACs activate human macrophages and lymphocytes via a bystander effect. A and B, Human PBMCs (A) and human PBMC-M2 (B) were stimulated with E104 for 24 hours, followed by analysis of TNFα and IL6 production using ELISA. C and D, NF-κB–induced SEAP activity in Ramos-Blue reporter cells after incubation with NG-ISACs (C) or controls (D) in the presence of BxPC-3‒TROP2^high cells. E, Human PBMC-M2 were cocultured with NG-ISACs in the presence of CFPAC-1‒TROP2^medium tumor cells and analyzed for TNFα production. F, Human PBMCs were cultured with NG-ISACs in the absence of TROP2-expressing cells and analyzed for TNFα and IL6 production. Data are shown as the mean ± SD (n = 3). Data are presented as the mean ± SD, and EC₅₀ values are from n = 3 independent experiments.

High loading on antibodies (DAR) can limit payload delivery efficiency because of rapid clearance of hydrophobic, high-DAR species (10). Because NG-Saci-AsnAsn-E104 induced robust stimulation in the coculture with TROP2-expressing pancreatic tumor cells, we generated a reduced-DAR (∼4) version of this construct, along with an isotype control (NG-Pali-AsnAsn-E104, DAR ∼4). To evaluate the ability of NG-ISACs to reprogram human macrophages and promote proinflammatory cytokine release independent of Fc effector function, coculture experiments were conducted using human PBMC-derived M2 macrophages. NG-Saci-AsnAsn-E104 (DAR4) induced dose-dependent TNFα secretion in cocultures of human M2 macrophages and CFPAC-1‒TROP2^medium tumor cells (Fig. 3E). This activation was absent with isotype control (NG-Pali-AsnAsn-E104), unconjugated Saci, and NG-Saci. NG-Saci-AsnAsn-E104 (DAR4) also displayed minimal off-target activity in TROP2-negative PBMCs, presumably because of ablated FcγR-mediated uptake (Fig. 3F).

Together, these findings demonstrate that NG-ISACs stimulate activation of both myeloid cells and lymphocytes via TLR7 engagement in the presence of TROP2-positive tumor cells, supporting a bystander mechanism of action.

Linker stability and E104 release profiles

We compared NG-Saci-AsnAsn-E104 and NG-Saci-ValCitPABC-E104, which showed the strongest activity in vitro. NG-Saci-AsnAsn-E104 exhibited high stability, with only 12.1% aggregation after 28 days at 45°C, which was comparable with unconjugated NG-Saci (7.3%). NG-Saci-ValCitPABC-E104 aggregated more substantially, increasing from 18.6% to 46.9% (Supplementary Table S4). In serum, NG-Saci-AsnAsn-E104 released significantly less E104 than ValCitPABC-linked ISACs (Fig. 4A). PEG12-ValCitPABC showed especially high release in mouse serum, raising concerns about species-specific instability. In tritosomal release assays, NG-Saci-AsnAsn-E104 released E104 more slowly than ValCitPABC constructs (Fig. 4B).

Figure 4.

E104 release profiles of NG-ISACs. A and B, E104 release from NG-ISACs was evaluated in human and mouse serum (A) or rat liver tritosomes (B). Data are presented as mean ± SD (n = 3). C, E104 release in the presence of BxPC-3 tumor cells or RAW-Dual cells was measured to assess tumor-selective payload liberation. Data are presented as mean ± SD (n = 6).

In cell-based assays, NG-ISACs released more E104 into media when incubated with BxPC-3‒TROP2^high tumor cells as compared with RAW-Dual macrophages (Fig. 4C), consistent with tumor-targeted delivery. Isotype control ISACs showed negligible release (Supplementary Fig. S4).

Systemically administered NG-ISAC eliminates tumors in the absence of T-cell, B-cell, or NK-cell activity in a human xenograft tumor model

In CFPAC-1‒TROP2^medium xenografts, tumor-bearing mice received intraperitoneal injections of NG-Saci, NG-Saci-ValCitPABC-E104, NG-Saci-PEG12ValCitPABC-E104, NG-Saci-AsnAsn-E104, or PBS vehicle control. NG-Saci-AsnAsn-E104 showed the most potent antitumor response, resulting in complete tumor regression in a subset of treated animals (Fig. 5A; Supplementary Fig. S5A–S5E). By contrast, ValCitPABC-based NG-ISACs significantly delayed tumor growth, comparable with NG-Saci, but did not induce regression.

Figure 5.

NG-ISACs elicit robust antitumor immunity through targeted E104 release in the CFPAC-1‒TROP2^medium human pancreatic tumor xenograft model. Nu/J mice bearing subcutaneous CFPAC-1 tumors (∼100 mm³) were randomized and treated intraperitoneally with NG-ISACs, NG-Saci, or vehicle control at 6 mg/kg every 4 days for three dosages (n = 5 per group). A, Tumor volume (left) and body weight change (right) are shown as mean ± SD. B, Kaplan–Meier survival analysis of treated cohorts; mice surviving 30 days after treatment initiation are indicated in the legend.

Importantly, all NG-ISAC formulations were well tolerated, with no observable body weight loss during the treatment course (Fig. 5A; Supplementary Fig. S5F). Survival analysis further confirmed the superior efficacy of NG-Saci-AsnAsn-E104, with treated mice exhibiting significantly extended survival compared with vehicle-treated controls (Fig. 5B).

NG-ISACs elicit tumor regression in a syngeneic mouse colon tumor model

To explore the immunomodulatory potential of NG-ISACs in a fully functional immune system, we employed a syngeneic mouse model using MC38 colon cancer cells engineered to express hTROP2 (MC38-hTROP2, Supplementary Fig. S6A). In cocultures of MC38-hTROP2 cells and RAW-Dual macrophages, NG-Saci-AsnAsn-E104 and NG-Saci-ValCitPABC-E104 induced strong NF-κB and IRF pathway activation, whereas control constructs showed no response (Fig. 6A and B; Supplementary Fig. S6B and S6C).

Figure 6.

NG-ISACs elicit potent antitumor responses in the MC38-hTROP2 syngeneic tumor model. A and B, RAW-Dual reporter macrophages were cocultured with MC38-hTROP2 tumor cells and treated with NG-ISACs for 24 hours. Activation of NF-κB (A) and IRF (B) signaling pathways was quantified. Data are presented as mean ± SD (n = 3). C, BMDM-M2 cells were cocultured with BxPC-3 tumor cells and stimulated with NG-ISACs (DAR4). IL6 production was measured by ELISA. Data are presented as mean ± SD (n = 3). D and E, C57BL/6J mice bearing subcutaneous MC38-hTROP2 tumors (∼100 mm³) were randomized and treated intraperitoneally with NG-ISACs or control agents at 6 mg/kg every 4 days for three dosages (n = 4 per group). D, Tumor volume (left) and body weight change (right) are shown as mean ± SD. E, Kaplan–Meier survival analysis of each treatment group. F, Serum anti-hTROP2 antibody titers were measured using the PIXI Automated Immunoassay Platform at specified time points after treatment. Data are shown as mean ± SD. G, Serum concentration–time profiles of NG-ISACs following intraperitoneal administration in C57BL/6J mice. Data are shown as mean ± SD (n = 3 mice per group). Concentrations were quantified using the PIXI Automated Immunoassay Platform. Note that measured values after the second dose were near the limit of quantitation. DPBS, Dulbecco’s PBS.

NG-Saci-AsnAsn-E104 (DAR4) was selected for in vivo evaluation in an immunocompetent mouse model instead of the DAR8 construct because of its improved PK exposure (10), alongside an isotype control (NG-Pali-AsnAsn-E104). In cocultures of BMDM-M2 macrophages with BxPC-3‒TROP2^high tumor cells, NG-Saci-AsnAsn-E104 (DAR4) elicited significant IL6 secretion, whereas the isotype control had no measurable effect (Fig. 6C).

In vivo, the administration of NG-Saci-AsnAsn-E104 (DAR4) in C57BL/6J mice bearing MC38-hTROP2 tumors led to complete tumor regression in most animals and was well tolerated, with no evidence of systemic toxicity or weight loss (Fig. 6D; Supplementary Fig. S6D–S6H). In contrast, NG-Pali-AsnAsn-E104 and unconjugated antibodies failed to suppress tumor growth, underscoring the necessity of TROP2 targeting and TLR7 agonist delivery. Survival analysis confirmed the durable benefit of NG-Saci-AsnAsn-E104 (DAR4), with significantly prolonged survival compared with control groups (Fig. 6E).

To test adaptive immunity, we measured serum levels of mouse anti-hTROP2 antibodies recognizing the hTROP2 antigen expressed on MC38-hTROP2 tumor cells in mice treated with NG-Saci-AsnAsn-E104 (DAR4). Mice receiving NG-Saci-AsnAsn-E104 (DAR4) developed significantly elevated anti-hTROP2 titers by day 4, which remained elevated at 1 month (Fig. 6F). In contrast, mice treated with NG-Saci or vehicle showed no appreciable anti-hTROP2 antibody production.

Serum PK and toxicity assessment of NG-ISACs

PK studies were performed to evaluate systemic exposure and clearance. The multi-dose exposure of NG-Saci-AsnAsn-E104 (DAR ∼4) was evaluated following both intraperitoneal and intravenous dosing (Fig. 6G; Supplementary Fig. S7A). Following the first intraperitoneal dose, NG-Saci-AsnAsn-E104 (DAR4) exhibited a C_max of 8.9 μg/mL and an AUC_last of 580.1 hours × μg/mL. Exposure after the second dose of NG-Saci-AsnAsn-E104 (DAR4) was near the limit of quantitation for both intraperitoneal and intravenous routes, with AUC_0-last values estimated at 30.8 and 19.5 hours × μg/mL, respectively. This dramatically lower exposure following the second dose of NG-ISAC strongly suggests that clearance is influenced by the adaptive immune system. Consistent with this hypothesis, NG-Saci-AsnAsn-E104 (DAR4) exhibited higher serum concentrations at 4 days in C57BL/6J mice as compared with glycosylated Saci-AsnAsn-E104 (Supplementary Fig. S7B). DAR reduction improved PK characteristics.

In Nu/J mice, the bioavailability of NG-ISACs (DAR8) after intraperitoneal administration was assessed (Supplementary Fig. S7C; Supplementary Table S5). The AUC_last for NG-Saci-AsnAsn-E104 and NG-Saci-ValCitPABC was 1,040 hours × μg/mL and 1,670 hours × μg/mL, respectively, both significantly lower than the exposure of the naked mAb (5,570 hours × μg/mL).

ADA formation was minimal after the first dose but more pronounced after the second (Supplementary Fig. S7D and S7E). NG-ISACs produced slightly less ADA than glycosylated ISACs, suggesting reduced immune complex clearance. Toxicity assessments showed that NG-Saci and NG-Saci-AsnAsn-E104 (DAR4) were well tolerated with minimal adverse effects. In contrast, glycosylated ISACs produced higher clinical scores and acute immune-related toxicity (Supplementary Fig. S7F; Supplementary Table S6).

Together, these data demonstrate that legumain-cleavable NG-ISACs mediate tumor-selective immune activation via TROP2 internalization and TLR7 release, while minimizing FcγR-driven off-target effects (48, 49).

Discussion

This study introduces a next-generation strategy for ISACs that eliminates the requirement for Fc receptor engagement while preserving tumor-specific immune activation. Traditional ISACs rely on Fc-mediated mechanisms such as ADCC and ADCP to engage effector cells, including macrophages and NK cells. However, these mechanisms are not universally required for therapeutic efficacy. Notably, when ISACs incorporate cell-permeable payloads capable of inducing a bystander effect, immune activation may occur through mechanisms that do not involve Fc receptor binding. Although FcγR-mediated activities can be assessed using hydrophilic payloads that do not induce bystander effects, evaluating bystander activity in isolation without concurrent ADCC or ADCP requires separation from FcγR engagement. To achieve this, we developed NG-ISACs that bypass FcγRIIa-mediated ADCC and FcγRIIIa-mediated ADCP and instead rely on antigen-targeted delivery and intracellular release of TLR7 agonists for immune activation.

The mechanism of action for NG-ISACs involves a three-step process. First, the antibody binds to and is internalized into tumor cells expressing TROP2. Second, the cleavable linker is processed within the endolysosomal compartment, releasing the TLR7 agonist. Third, the released agonist diffuses into the TME and activates surrounding immune cells through a bystander effect. This approach eliminates the need for ADCC and ADCP and instead depends on targeted delivery and controlled intracellular payload release.

NG-ISACs were constructed by conjugating the synthetic TLR7 agonist E104 to an NG anti-TROP2 mAb (NG-Saci) using a panel of cleavable linkers, including cathepsin- and legumain-sensitive motifs. Enzymatic removal of N-linked glycans from the Fc region abolished FcγR binding while potentially preserving FcRn interactions. This design aimed to separate FcγR-mediated signaling from the PK and biodistribution benefits of the IgG scaffold. NG-ISACs were synthesized using cysteine–maleimide chemistry, and linker selection was optimized for cleavage specificity and plasma stability. Linker chemistry was a critical determinant of ISAC performance. The AsnAsn linker, designed to be cleaved by the endo/lysosomal protease legumain, demonstrated superior intracellular release kinetics and enhanced plasma stability compared with the traditional ValCitPABC linker.

SPR assays demonstrated that NG-ISACs had minimal-to-undetectable binding affinity for FcγRIIa and FcγRIIIa, whereas glycosylated ISACs retained strong receptor binding. Functional assays using FcγR-dependent Jurkat-Lucia NFAT reporter cells cocultured with CFPAC-1 tumor cells showed that NG-ISACs failed to activate NFAT signaling, confirming successful bypass of ADCC and ADCP pathways. In coculture experiments with RAW-Dual macrophages and hTROP2-expressing tumor cells, NG-ISACs triggered robust activation of the NF-ĸB pathway. Among the evaluated constructs, NG-Saci-AsnAsn-E104 demonstrated the highest potency. Neither the isotype control nor unconjugated antibodies induced measurable immune activation, highlighting the necessity of both antigen targeting and intracellular delivery of the immunostimulatory payload. Moreover, NG-ISAC activity correlated with surface antigen density, inducing stronger responses in BxPC-3‒TROP2^high than in CFPAC-1‒TROP2^medium cocultures, supporting the importance of antigen-mediated internalization. In murine cocultures, NG-ISACs activated BMDM-M2 macrophages and splenocytes in the presence of tumor cells. In the absence of tumor cells, glycosylated ISACs retained Fc-mediated activation of BMDM-M2 macrophages, whereas NG-ISACs did not, demonstrating that NG-ISACs promote myeloid activation through targeted TLR7 delivery and bystander signaling without requiring ADCC or ADCP.

The AsnAsn linker released E104 more slowly in tritosome digestion studies than ValCitPABC, suggesting prolonged intracellular exposure. Stability assays in mouse and human serum revealed significantly reduced spontaneous E104 release from NG-Saci-AsnAsn-E104 compared with NG-Saci-ValCitPABC-E104. The influence of linker chemistry was further validated in in vivo efficacy studies. In CFPAC-1 xenograft models, NG-Saci-AsnAsn-E104 induced complete tumor regression in several animals, whereas ValCitPABC-based constructs only delayed tumor progression with efficacy on par with unconjugated NG-Saci. Although the reason for this improved activity of the AsnAsn-E104 ISAC was not established with certainty, we hypothesize that it is likely due to either improved exposure (due to increased polarity of the linker) or increased stability of the linker. Importantly, these results were observed in immunodeficient mice lacking B, T, and NK cells, indicating that innate immune activation alone was sufficient for tumor control. The membrane-permeable nature of E104 allowed for bystander activation of TAMs, which enabled effective immune responses in the absence of adaptive immunity.

To investigate the role of adaptive immunity, we utilized an immunocompetent syngeneic mouse model bearing MC38 tumors engineered to express hTROP2. NG-ISAC–induced lymphocyte activation was assessed in cocultures with mouse splenocytes, which contain B, T, and NK cells. NG-ISACs activated splenocytes in the presence of TROP2-positive tumor cells, whereas isotype controls and unconjugated antibodies showed no measurable activation. Systemic administration of NG-Saci-AsnAsn-E104 (DAR4) in immune-competent C57BL6/J tumor-bearing mice led to complete tumor elimination and significantly extended survival. Moreover, mice treated with NG-ISACs developed sustained levels of anti-hTROP2 antibodies, indicating that localized TLR7 activation can overcome immune tolerance and promote antigen-specific humoral immunity. Control constructs, including isotype antibodies and free E104, did not elicit this response, emphasizing the importance of targeted antigen engagement and intracellular delivery of the immunostimulatory payload.

PK analysis revealed that NG-ISACs achieved higher C_max and AUC_last values in Nu/J mice than in C57BL/6J mice, likely because of adaptive immune–mediated clearance. After the second dose, serum levels declined rapidly in C57BL/6J mice following both intraperitoneal and intravenous administration, consistent with ADA-driven clearance. ADA formation was minimal after the first dose but increased dramatically after the second dose. NG-ISACs induced slightly lower ADA levels than glycosylated ISACs, consistent with reduced FcγR binding–associated uptake into APCs. Encouragingly, toxicity assessments confirmed that NG-ISACs were well tolerated. No signs of systemic inflammation, weight loss, or adverse immune reactions were observed, even at therapeutic doses. In contrast, glycosylated ISACs caused greater levels of inflammation and toxicity, likely because of Fc effector–mediated immune stimulation. These findings support the safety benefit of eliminating FcγR interactions from the ISAC design.

Finally, to evaluate NG-ISAC activity in human immune cells, we conducted coculture experiments with human PBMC-derived M2 macrophages. NG-Saci-AsnAsn-E104 induced dose-dependent secretion of TNFα when cocultured with CFPAC-1 tumor cells. This response was not observed with the isotype control, unconjugated Saci, or NG-Saci alone. Additionally, minimal activation occurred in TROP2-negative PBMC cultures, indicating reduced off-target uptake resulting from the absence of FcγR binding. In parallel, we examined NG-ISAC activity in Ramos-Blue reporter B cells cocultured with BxPC-3 tumor cells. NG-Saci-E104 constructs induced NF-ĸB activation, with NG-Saci-AsnAsn-E104 (DAR4) showing the greatest potency. No activation was observed with unconjugated or isotype control antibodies. These results suggest that NG-ISACs can stimulate both macrophages and lymphocytes within the TME, thereby supporting activation of both innate and adaptive immune responses.

In conclusion, this study establishes NG-ISACs as a novel antibody conjugate platform capable of eliciting tumor-specific immune activation independently of ADCC and ADCP. Key innovations include the use of NG antibodies for precise antigen targeting, legumain-sensitive linkers for controlled intracellular release, and highly permeable small-molecule agonists that exhibit strong bystander activity. These combined features enable selective immune stimulation while reducing systemic toxicity. By moving beyond traditional ADCC-/ADCP-dependent mechanisms, NG-ISACs expand the therapeutic scope of antibody conjugates for solid tumors and immune-mediated diseases. This is particularly relevant for the delivery of potent innate immune ligands such as TLR7 agonists, which may cause systemic inflammation if not properly localized. The NG-ISAC platform offers a controllable and tumor-focused approach to immune stimulation and may have applications in solid tumors and immune-mediated diseases. Overall, our findings highlight the therapeutic potential of NG-ISACs and provide a foundation for the rational design of antibody-based immunotherapies with improved safety and efficacy. Future studies will focus on optimizing antibody engineering and linker chemistry, exploring alternative payloads, and evaluating NG-ISACs in additional tumor models and disease settings, including patient populations with low-affinity FcγR polymorphisms that are less responsive to traditional mAb therapies.

Supplementary Material

Supplemental Figure S2

Hepatic metabolism of E104 and co-culture assays using RAW-Dual cells

Supplemental Figure S3

Experiments with mouse primary immune cells

Supplemental Figure S4

E104 release profile.

Supplemental Figure S5

NG-ISACs exhibit efficacy in CFPAC-1 tumor model

Supplemental Figure S6

Therapeutic efficacy of NG-ISACs in MC38-hTROP2 syngeneic tumor cell line model

Supplemental Figure S7

Serum pharmacokinetics and toxicity evaluation of ISACs and NG-ISACs

Supplementary Data

Supporting spectra and methods

Supplemental Figure S1

FcγR engagement profile of ISACs and NG-ISACs

Data Availability

The data generated in this study are available within the article and its supplementary data files.

Authors’ Disclosures

L.N. Tumey reports grants from NIH and Heidelberg Pharma during the conduct of the study; grants from BigHat Biosciences outside the submitted work; and a patent for WO 2025/212999 pending, a patent for WO2024238588 pending, and a patent for WO2022187809 pending. No disclosures were reported by the other authors.

Authors’ Contributions

A. Zhang: Investigation, methodology, writing–original draft, writing–review and editing. M.R. Mullapudi: Investigation, methodology, writing–original draft. L. Wu: Investigation, methodology. M. Yi: Investigation, methodology. M.A. Rahman: Investigation, methodology. A.A. Yaseen: Investigation, methodology. H.K. Zhong: Investigation, methodology. T.A. Brooks: Conceptualization, supervision, funding acquisition, investigation, writing–review and editing. L.N. Tumey: Conceptualization, resources, supervision, funding acquisition, methodology, writing–review and editing.