---
title: "53L10 T‑cell engager tribody for immunotherapy of multiple solid tumors — Frontiers in Immunology"
id: "frontiers-in-immunology-11-53l10-t-cell-engager-tribody-for-immunotherapy-of-multiple-solid-tumors"
canonical_url: "https://medichelpline.com/clinical-feed/frontiers-in-immunology-11-53l10-t-cell-engager-tribody-for-immunotherapy-of-multiple-solid-tumors"
content_type: "clinical_feed_article"
specialty: "Oncology"
source_name: "Frontiers in Immunology"
source_url: "https://www.frontiersin.org/articles/10.3389/fimmu.2026.1934039"
published_at: "2026-10-06T00:00:00.000Z"
evidence_level: "Journal Feed"
license: "CC-BY-NC-4.0 / Informational Use"
---
# 53L10 T‑cell engager tribody for immunotherapy of multiple solid tumors — Frontiers in Immunology
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- **Specialty:** [Oncology](https://medichelpline.com/clinical-feed/oncology.md)
- **Primary Source:** Frontiers in Immunology
- **Source URL:** [Original Journal Publication](https://www.frontiersin.org/articles/10.3389/fimmu.2026.1934039)
- **Published At:** 2026-10-06T00:00:00.000Z
- **Evidence Rating:** Journal Feed
## Executive GIST (TL;DR)
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## Clinical Analysis & Structured Key Points
Frontiers | 53L10 T-cell engager tribody for immunotherapy of multiple solid tumors BRIEF RESEARCH REPORT article Front. Immunol. , 06 October 2026 Sec. Cancer Immunity and Immunotherapy Volume 17 - 2026 | https://doi.org/10.3389/fimmu.2026.1934039 Published in Frontiers in Immunology Cancer Immunity and Immunotherapy 7 impact factor 11.3 citescore Editor & Reviewers Edited by M F Massimo Fantini Reviewed by D T Dianzhe Tian Q G Qintao Ge Outline Figures and Tables Figure 1 View in article Figure 2 View in article Figure 3 View in article Figure 4 View in article Table 1 Cell surface expression of 5T4 and PD-L1 and EC50 values for 53L10 effect on tumor cells. The values were calculated by fitting the dose–response curves reported in Figure 2 after treatment of the cells with hPBMCs from three different donors and performed in triplicates. View in article Table 2 Comparison of the antibody constructs, tumor models, PBMC conditions, treatment schedules, endpoints, and principal findings of the previous and current studies. View in article BRIEF RESEARCH REPORT article Front. Immunol. , 06 October 2026 Sec. Cancer Immunity and Immunotherapy Volume 17 - 2026 | https://doi.org/10.3389/fimmu.2026.1934039 53L10 T-cell engager tribody for immunotherapy of multiple solid tumors M P Margherita Passariello 1,2 † M B Martina Belardo 1,2 † T I Toshikazu Inoue 3 K K Kentaro Kajiwara 3 R R Rosa Rapuano Lembo 2,4 S H Shu-ichi Hashimoto 3 * C D Claudia De Lorenzo 1,2 * 1. Department of Molecular Medicine and Medical Biotechnologies, University of Naples “Federico II”, Naples, Italy 2. Ceinge-Biotecnologie Avanzate s.c.a.r.l., Naples, Italy 3. Chiome Bioscience Inc., Tokyo, Japan 4. European School of Molecular Medicine, University of Milan, Milan, Italy See more Article metrics View details Abstract Cancer immunotherapy with immune checkpoint inhibitors (ICIs), i.e., antibodies targeting inhibitory ICs, such as PD-1, PD-L1, and CTLA-4, have successfully revolutionized cancer treatment. One of the most promising cancer immunotherapies is based on bi-specific T-cell engagers that simultaneously bind with one arm to a tumor-associated antigen on tumor cells and with the other one to the CD3 complex on T cells to form a TCR-MHC-independent immune synapse. We previously generated four novel tribodies, tri-specific antibody derivative constructs, made up of a Fab targeting 5T4, an oncofetal tumor antigen expressed on several types of tumors, an scFv targeting CD3 on T cells, and an additional scFv specific for an immune checkpoint. In particular, the 53L10 tribody, targeting PD-L1, displayed the best binding properties and stronger anti-tumor activity in vitro and in vivo in mouse models. Here, we further investigated the anti-tumor potential of 53L10 by testing its effects on a panel of tumor cell types with a more aggressive profile, including gastric, pancreatic, prostate, ovarian, and epidermoid tumor cells. We found that 53L10 was very effective on these cells by inducing stronger T-cell activation and tumor cell lysis than the parental bi-specific 53P lacking an ICI. To further test its therapeutic potential, we also analyzed its stability and the effects on tumor T-cell infiltration in vivo on a mouse model. 53L10 was found to be stable after incubation in serum at 37 °C for 3 days and induced a more efficient and earlier increase in tumor tissues of both CD4 and CD8 T cells than the parental bi-specific 53P. These findings support further preclinical evaluation of 53L10 as a new tool for multiple solid tumor-targeted therapeutic approaches. 1 Introduction Immunotherapy based on ICIs (immune checkpoint inhibitors) has gained increasing success in the last two decades. Several monoclonal antibodies (mAbs) targeting immune checkpoints (ICs) are already in clinical use for the treatment of many types of cancer, and the most common examples of checkpoint blockade strategies are those targeting PD-1/PD-L1 and CTLA-4 ( 1 ). Some mAbs, such as the anti-PD-1 nivolumab and pembrolizumab or the anti-PD-L1 atezolizumab, have already been approved by the Food and Drug Administration (FDA) for the therapy of melanoma, non-small cell lung cancer (NSCLC), head and neck squamous cell cancer (HNSCC), urothelial cancer, classical Hodgkin lymphoma, and renal cell cancer ( 2 ). However, mAbs binding to single targets still fail in achieving long-term therapeutic response and are effective only in a reduced percentage of patients. Thus, novel approaches led to a switch from standard mAbs to multi-functional molecules that simultaneously hit different targets with the aim of simultaneously “releasing the brakes” and “pushing the accelerator” at the same time ( 3 ). In particular, the clinical development of bi-specific antibodies that can act as T-cell engagers, targeting different tumor-associated antigens (TAAs) or ICs, has led to the FDA approval of 11 new drugs in the last 4 years ( 4 ). Most of them are T-cell engagers, such as glofitamab (CD20 × CD3), teclistamab (BCMA × CD3), and elranatamab (BCMA × CD3), which, in clinical use, show high overall response rates and efficacy especially for the therapy of lymphoma and multiple myeloma. More recently, even tri-specific molecules containing an additional arm to potentiate the anti-tumor effects are undergoing evaluation in clinical phases. Examples are represented by ISB2001 in phase 1, a tri-specific antibody for relapsed or refractory multiple myeloma, which targets two TAAs on myeloma cells (CD38 and B-cell maturation antigen) and CD3 on T cells ( 5 , NCT05862012); DR30206 in phase 2, which is a tri-specific antibody that simultaneously targets PD-L1, VEGF, and TGF-β in metastatic tumors ( 6 ; NCT06132828); and GB268 in phase 1, a tri-specific antibody simultaneously binding PD-L1, CTLA-4, and VEGF ( 7 ; NCT06934616). In this field, we recently designed and constructed novel immunomodulatory bi-specific and tri-specific antibody derivative constructs, starting from the parental bi-specific tribody Tb535H, targeting CD3 on T cells and 5T4 on tumor cells. The novel tri-specific tribodies not only retain the ability of the parental tribody to bind 5T4-TAA and CD3 on T cells, but also can potentially overcome the immunosuppressive environment of solid tumors by an additional scFv derived from an antibody targeting a specific IC, such as anti-PD-L1, anti-PD-1, or anti-LAG-3 mAbs. Among these, the tribody named 53L10, containing the anti-PD-L1 scFv, demonstrated the strongest anti-tumor efficacy in vitro and total tumor regression in vivo in previous studies on mouse models bearing lung xenografts ( 8 ). Here, we first evaluated the anti-tumor efficacy of 53L10 on a different panel of tumor cells with a more aggressive phenotype, such as gastric, pancreatic, prostate, ovarian, and epidermoid ( 9 ) not tested before, and then we investigated its in vivo mechanism of action and its stability in physiological conditions. 2 Materials and methods 2.1 Antibodies The antibodies used were as follows: the commercial human anti-PD-L1 (MAB0784, G&P Biosciences, Santa Clara, CA, USA), horseradish peroxidase (HRP)-conjugated anti-mouse IgG secondary antibody (62-6520, Invitrogen Thermo Fisher Scientific, Rockford, IL, USA), HRP-conjugated goat anti-human IgG (Fab′)2 mAb (ab87422, Abcam, Biomedical Campus, Cambridge, UK), HRP-conjugated His-Antibody (130-092-785, Miltenyi Biotec, Bergisch Gladbach, Germany), and HRP-conjugated goat anti-Rabbit IgG (H+L) secondary antibody (31460, Invitrogen, Thermo Fisher Scientific). The human Tb535H, 53L10, and 53P tribodies were generated and produced as previously described ( 8 , 10 , 11 ). 2.2 Tumor cell lines The human tumor cell lines used were as follows: triple-negative breast cancer (TNBC) MDA-MB-231 and BT-549 cells, HER2-negative breast cancer MCF-7 cells, gastric cancer NCI-N87 cells, pancreatic cancer PANC-1 and BxPC-3 cells, ovarian cancer SKOV-3 cells, NSCLC A-549 cells, epidermoid cancer A-431 cells, and prostate cancer DU145 cells, from the American Type Culture Collection (ATCC) (Virginia, USA). MDA-MB-231 and MCF-7 cells were cultured in Dulbecco’s Modified Eagle’s Medium (11574486, DMEM Gibco, Life Technologies, Paisley, Scotland UK) and Modified Eagle’s Medium (32561029, MEM, Gibco, Life Technologies, Grand Island, USA), respectively. A-549 cells were cultured in Kaighn’s Modification of Ham’s F-12 Medium (10472692, F-12 K Gibco, Life Technologies, Paisley, Scotland UK). NCI-N87, PANC-1, and SKOV-3 cell lines were cultured in Roswell Park Memorial Institute 1640 Medium (12633012, RPMI 1640, Gibco, Life Technologies, Paisley, UK). The media were supplemented with 10% (vol/vol) heat-inactivated fetal bovine serum (50-165-7328, FBS, Sigma-Aldrich, St Louis, MO, USA), 50 U/mL penicillin, 50 μg/mL streptomycin, and 2 mM L-glutamine (all from Gibco Life Technologies, Paisley, Scotland UK). BxPC-3 cells were cultured in RPMI 1640 Medium (A1049101, ATCC modified, Thermo Fisher Scientifc). A431 cells were cultured in DMEM (08458045, Nacalai Tesque, Kyoto, Japan), and DU145 cells were cultured in MEM (21443-15, Nacalai Tesque, Kyoto, Japan). The media were supplemented with 10% (v/v) FBS (SH30088.03, Hyclone, Cytiva), 100 U/mL penicillin, and 100 μg/mL streptomycin (Nacalai Tesque, Kyoto, Japan). Mycoplasma contamination was assessed using the MycoAlert™ Mycoplasma Detection Kit (Lonza, LT07-218) according to the manufacturer’s instructions. Human peripheral blood mononuclear cells (hPBMCs) were purchased from ATCC (PCS-800-011). All tissues used for isolation by ATCC conform to the privacy rule guidance and were obtained under informed consent and conform to HIPAA regulations to protect the privacy of the donor’s Personally Identifiable Information. hPBMCs used in the experiments were derived from five different donors. hPBMCs were thawed out by using a medium composed of RPMI 1640 with 1% L-glutamine, 1% CTLWash™ (CTLW-010, Immunospot by Cellular Technology Limited, Shaker Heights, OH, USA), and 100 U/mL Benzonase (K501 45697 818, Merck Millipore, Darmstadt, Germany) and cultured at 37 °C in R10 medium (RPMI 1640 supplemented with 10% inactivated FBS, 1% L-glutamine, 50 U/mL penicillin, 50 μg/mL streptomycin, and 1% HEPES; 15630-056, Gibco, Life Technologies). 2.3 ELISAs Enzyme-linked immunosorbent assays (ELISAs) were performed to analyze the functional binding of 53L10, after incubation in 100% FBS at 37 °C for 24, 48, and 72 h. Human PD-L1/Fc recombinant protein was immobilized at a concentration of 5 μg/mL in a Nunc 96-well plate for 72 h at 4 °C. The plate was blocked with phosphate-buffered saline (PBS)/milk 5% for 1 h at 37 °C. The untreated or treated tribody 53L10 was incubated at concentrations of 30 and 100 nM for 1 h 30 min at room temperature (RT). FBS or anti-PD-L1 commercial Ab was used as negative or positive control, respectively. After extensive washes, an HRP-conjugated anti-His tag antibody was added for 1 h at RT. The detection was carried out by incubating with 3,3′,5,5′-tetramethylbenzidine (T8665, TMB, Sigma-Aldrich), the reaction was stopped with 1 N HCl, and absorbance was measured at 450 nm with an Envision plate reader (PerkinElmer, San Diego, CA, USA), as previously described ( 12 ). Cell ELISAs were performed to check the expression levels of 5T4 and PD-L1 on breast MDA-MB-231 and MCF-7, lung A-549, gastric NCI-N87, pancreatic PANC-1, and ovarian SKOV-3 cancer cell by using the anti-PD-L1 commercial Ab or the parental bivalent anti-5T4 tribody at a concentration of 50 nM. Tumor cells were plated in triplicate into round-bottom 96-well plates (2 × 10 5 cells/well), blocked with PBS/BSA 6% for 30 min, and then incubated with the antibodies in PBS/BSA 3% for 1 h 30 min at RT under gentle agitation. After extensive washes, the secondary goat anti-human IgG (Fab′)2 mAb and anti-human HRP-conjugated IgG (Fab′), respectively, were added for 1 h, and detection was carried out by reading the absorbance at 450 nm, as previously described ( 13 ). 2.4 LDH cytotoxic assays and morphology analyses To test the cytotoxic effects of 53L10, target tumor cells were co-cultured with hPBMCs [effector-to-target cell (E/T) ratio 5:1] in 96-well flat-bottom plates and incubated for 48 h at 37 °C in the presence of 53L10 used at increasing concentrations (0.1–1 nM). After treatments, the supernatants of the co-cultures were collected by centrifugation at 1,200 rpm for 10 min and the supernatant was tested for LDH release in co-cultures by using the LDH Detection Kit (C20300, Thermo Fisher Scientific). Tumor cell lysis was analyzed by measuring the fold increase of LDH in the presence of each treatment, with respect to the amount present in the supernatant of untreated co-cultures (spontaneous LDH activity) and expressed as percentage with respect to the maximum lysis represented by the cells treated with 10% Triton X-100 as previously described ( 14 ). The formula used for calculating the % of cytotoxicity is the following: In parallel assays, spontaneous effector cell release, represented by hPBMCs cultured alone, and spontaneous target cell release, represented by tumor cells alone, were measured as well. Images of NCI-N87, PANC-1, and SKOV-3 cells, untreated or treated with 53L10 in the presence of hPBMCs, were acquired and processed by using digital microscopy from Zen 3.2 Cell Discover. Cells co-cultured with hPBMCs and treated with an unrelated human IgG1 were used as negative control. The cytotoxic activity of 53L10 was also evaluated in co-cultures of various cancer cell lines or CHO-K1 cell lines expressing varying levels of 5T4 or PD-L1 and hPBMCs pre-activated with anti-CD3/anti-CD28 beads. Cells were mixed at an E/T ratio of 5:1 or 10:1. The tribody 53L10 was added at a final concentration of 10 5 to 10 −1 pM for 72 h. The LDH released in the culture supernatant was measured with LDH-Glo™ Cytotoxicity Assay (J2380, Promega, Milano, Italy) following the manufacturer’s instructions. 2.5 Cytokine secretion assays To evaluate the effects of the antibodies on immune cell activation, the secretion of human IL-2 and IFNγ in supernatants of co-cultures of tumor cells with hPBMCs was assessed by using the DuoSet ELISA kit assays (DY007B, Bio-Thecne R&D Systems, Inc., Northeast, Minneapolis, USA). The culture supernatants were collected after treatments and the cytokines were quantified by using the IL-2 (DY202) and IFNγ (DY285B) kits, according to the manufacturer’s recommendations. Absorbance values were converted in pg/mL by plotting the mean absorbance for each sample in comparison with the standard curve constructed within the experiment. 2.6 TIL analysis of PBMC-humanized xenograft model hPBMCs from healthy donors (CTL Laboratories, ShenZhen China) cultured in ImmunoCult-XF T Cell Expansion Medium (10981, STEMCELL Technologies, Vancouver, Canada) in the presence of 30 U/mL of recombinant hIL-2 (200-02-50UG, Thermo Fisher Scientific) were activated by Dynabeads Human T-Activator CD3/CD28 (11131D, Thermo Fisher Scientific) for 3 days. After removing magnetic beads, hPBMCs were further cultured in the medium containing 30 U/mL of rhIL-2, and then used for transplantation. A-549 tumor cells (5 × 10 6 cells) (ATCC) were transplanted into the right flank of 5-week-old female NOD.CB17-Prkdcscid/J mice (Jackson Laboratories Japan, Bar Harbor, ME). On the day the average tumor volume reached 100 mm³, mice were treated with anti-asialo GM1 pAb (014-09801, Fuji Film Wako, Osaka, Japan). In the following day (day 0), 3 × 10 7 activated hPBMCs were introduced intraperitoneally, and doses of the tribodies were subsequently administered intravenously once every 2 days. Mice were sacrificed and tumors were resected on days 6 and 10 for analyses of T-cell infiltration. Tumor tissues were harvested from three mice per group and pooled for analysis. Tumor tissues were mechanically and enzymatically dissociated into single-cell suspensions using the gentleMACS Octo Dissociator and the Tumor Dissociation Kit (130-095-929, Miltenyi Biotec) according to the manufacturer’s instructions. Briefly, tumor samples were minced into small fragments and processed using the gentleMACS program with enzyme mixtures provided by the kit. The resulting cell suspensions were filtered through a 100-µm cell strainer to remove debris and washed with cold PBS containing 1% FBS. Cells were then counted and washed twice with cold PBS containing 1% FBS. For surface staining, cells (1.6 × 10 6 cells per sample) were incubated with Human Trustain FcX (422305, BioLegend, San Diego, CA, USA) and mAbs specific for CD45 (clone 2D1, FITC-conjugated, 368508, BioLegend; 1 μg/mL), CD4 (clone RPA-T4, Brilliant Violet 605-conjugated, 562658, BD Biosciences; 1 μg/mL), and CD8 (clone SK1, PerCP/Cyanine5.5-conjugated, 344710 BioLegend; 0.5 μg/mL) in Brilliant Stain Buffer (563794, BD Biosciences) for 30 min at 4 °C in the dark. After staining, cells were washed twice with PBS containing 1% FBS and resuspended in PBS containing 1% FBS for analysis. Appropriate isotype and unstained controls were included for gating. Dead cells were excluded by using Fixable Viability Stain 780 (BD Biosciences). Data acquisition was performed using a flow cytometer FACSLyric (BD Biosciences, San Jose, CA, USA), and at least 300,000 events were collected per sample. Data were analyzed using FlowJo software (version 10.10, BD Biosciences). Gating was performed sequentially to exclude debris based on forward scatter (FSC) and side scatter (SSC), followed by selection of single cells using FSC-A versus FSC-H. Live cells were then gated by excluding Fixable Viability Stain 780-positive dead cells. Among live cells, CD45 + leukocytes were selected, and subsequent analysis of CD4 + and CD8 + T-cell populations was performed. 2.7 Quantitative flow cytometry analysis Cell surface antigen density was quantified by quantitative flow cytometry using QIFIKIT ® (K007811-8; Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer’s instructions. Anti-human 5T4 mouse mAb (Cat. No. MAB49751; R&D Systems, Minneapolis, MN, USA) and anti-human PD-L1 mouse mAb (clone MIH1, purified NA/LE; Cat. No. 565959; BD Pharmingen, BD Biosciences, San Jose, CA, USA) were used as antigen-specific antibodies, and mouse IgG1 isotype control antibody (clone 2E12; Code No. M075-3; Medical & Biological Laboratories, Nagoya, Japan) was used as isotype control antibody. Expression values were calculated by subtracting the ABC value obtained with the negative control antibody from that obtained with the antigen-specific antibody. 2.8 Stability analyses To test the integrity and stability of 53L10 after incubation in serum for increasing time intervals at 37 °C, aliquots were collected after 24, 48, and 72 h and were analyzed by Western blotting under non-reducing conditions. The nitrocellulose membrane was blocked with 0.1% tween-PBS/milk 5% for 1 h at 37 °C and then incubated with an HRP-conjugated anti-His antibody for 1 h at RT. The signals were detected by using a ChemiDoc imaging system (Bio-Rad), and the intensity of the bands was visualized by using Image Lab software (version 6.0.1). 2.9 Statistical analyses Error bars were calculated by considering the results obtained by at least three determinations obtained in three independent experiments. Error bars depict means ± SD. Statistical analyses were assessed by Student’s t -test for comparisons between two groups, or one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test for comparisons among three or more groups, based on data from at least three independent biological experiments. Statistical significance was established as *** p ≤ 0.001; ** p < 0.01; * p < 0.05, as reported in Figure 1 . Figure 1 Expression of 5T4 and PD-L1 and anti-tumor effects of 53L10 on a panel of tumor cells. (A) The expression levels of 5T4 and PD-L1 on breast MDA-MB-231, lung A-54
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