Cancer development is shaped by host-microbe interactions, including viral infections. While several viruses are established oncogenic drivers, their potential protective roles in cancer remain unclear. Here we identify a dominant antibody response to CE1, a consensus epitope of enterovirus and rhinovirus, that is associated with reduced hepatocellular carcinoma (HCC) incidence and mortality. Anti-CE1 antibodies selectively recognize HCC cells and mediate anti-tumor activity through NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC). Mechanistically, anti-CE1 antibodies cross-react with aspartate β-hydroxylase (ASPH), with CE1-ASPH sequence homology underpinning tumor recognition and cytotoxicity. Clinically, ASPH is aberrantly upregulated in HCC and correlates with inferred NK cell-associated ADCC activity and improved survival in CE1-seropositive patients. Collectively, these findings reveal a mechanism by which antiviral humoral immunity confers cancer protection through molecular mimicry and highlight anti-CE1 immunity as a potential therapeutic strategy in HCC.

B7-H3 is a cell surface protein overexpressed in many solid tumors and an attractive target for chimeric antigen receptor (CAR) T cell therapy. The most clinically advanced B7-H3 CARs derive from murine monoclonal antibodies (mAbs) 376.96 and MGA271 and are now in phase 1/2 trials. However, non-human mAb sequences can provoke immune responses, leading to CAR T cell rejection and therapeutic failure. Although single-chain variable fragment (scFv) humanization reduces this risk, residual foreign residues within variable domains remain. To overcome this limitation, here we use in vitro phage display to generate fully human B7-H3-specific scFvs for CAR design. In pancreatic cancer, neuroblastoma, and glioblastoma xenograft models, CAR T cells incorporating the lead human binder Y111 are well tolerated and demonstrate superior antitumor activity compared with 376.96- and MGA271-based CARs. Y111 CAR treatment induces complete responses, tumor rejection, and significant survival benefits, identifying Y111 as a promising fully human B7-H3 CAR for solid tumors.

Chimeric antigen receptor (CAR) T-cell therapy has shown limited efficacy in solid tumors, in part due to variability in autologous T cells derived from heavily pretreated patients with advanced disease. To address these constraints, we developed an off-the-shelf allogeneic CAR-T platform using CRISPR-Cas9-mediated genome editing in T cells from healthy donors to enable targeted CAR insertion at the TRAC locus with concurrent disruption of B2M. Using adeno-associated virus (AAV) delivery, we designed CAR-T cells targeting glypican-2 (GPC2) and glypican-3 (GPC3), emerging antigens expressed in pediatric and adult solid tumors. Genome-edited allogeneic CAR-T cells exhibited potent, antigen-specific cytotoxicity across multiple tumor models. GPC2-directed allogeneic CAR-T cells demonstrated enhanced or comparable activity relative to conventional lentiviral CAR-T cells in neuroblastoma models and mediated tumor regression with prolonged survival in preclinical models. Notably, repeated dosing augmented antitumor efficacy without evidence of toxicity, supporting multi-dose regimens for solid tumors. Similarly, GPC3-targeted allogeneic CAR-T cells based on a single-domain antibody showed robust activity against hepatocellular carcinoma cells in vitro and in vivo. These findings establish a scalable, genome-engineered allogeneic CAR-T strategy with strong therapeutic potential and support the clinical development of off-the-shelf cell therapies for pediatric and adult solid tumors.

Glypican-3 (GPC3) is a heparan sulfate proteoglycan that is highly expressed in hepatocellular carcinoma and promotes tumor progression through Wnt3a/β-catenin signaling. However, how the nanoscale organization of GPC3 at the cell surface controls signaling remains unclear. Here, we combined nano-resolution MINFLUX imaging, single-molecule tracking, and functional assays to define the spatial architecture and dynamics of GPC3 on hepatoma cells. We found that GPC3 exists as both single molecules and nanoscale clusters and switches between confined and free diffusions on the plasma membrane. Heparan sulfate (HS) chains create nanoscale corrals that limit GPC3 movement, whereas removal of HS increases diffusive heterogeneity and disrupts confinement. Wnt3a stimulation induces the formation of higher-order GPC3 assemblies and enhances β-catenin signaling, while loss of HS markedly reduces this response. MINFLUX DNA-PAINT further revealed that HS chains orchestrate the spatial distribution of Wnt3a and promote its association with the Wnt receptor, Frizzled-1, an essential step for pathway activation. Collectively, these findings reveal that HS controls the nanoscale organization and dynamics of GPC3 to promote Wnt receptor assembly and efficient β-catenin signaling in hepatoma cells.

BACKGROUND:Chimeric antigen receptor (CAR) T cell therapy has shown promise in treating hematological malignancies. However, the complex tumor microenvironment of cholangiocarcinoma (CCA) poses significant challenges, particularly due to the lack of clinically validated targets and the presence of fibrosis, which hinders T cell infiltration into tumor sites. Mesothelin (MSLN) is highly expressed in CCA. In this study, we aimed to develop CAR T therapy based on a panel of humanized rabbit monoclonal antibodies targeting various epitopes of MSLN, ranging from the N- to the C-terminus, for CCA. METHODS:MSLN expression was assessed in CCA tissue samples obtained from Thai patients. CAR T cells were generated using various single-chain variable fragment (scFv) constructs, engineered in either VH-linker-VL or VL-linker-VH orientation, targeting non-overlapping epitopes of membrane-bound MSLN: hYP218 (proximal region), hYP223 and hYP3 (middle region), and hYP158 (distal region). The cytotoxicity of MSLN-specific CAR T cells was evaluated in 3 preclinical CCA mouse models: Mz-ChA-1, KMCH, and KMBC. RESULTS:MSLN was strongly expressed in 79% of the CCA specimens. Among the CAR constructs, hYP218-based CAR T cells-with the VL-linker-VH orientation and CD28-derived hinge and transmembrane domains (CD28HTM)-completely eradicated CCA tumors in all 3 CCA xenograft mouse models (Mz-ChA-1, KMCH, and KMBC). Furthermore, hYP218 VLVH CD28HTM CAR T cells showed strong persistence in mice, with low PD-1 expression (a marker of T cell exhaustion) and minimal adverse effects. CONCLUSIONS:Our findings suggest that MSLN is a promising target for CAR T cell therapy in CCA. CAR T cells engineered with hYP218 VLVH CD28HTM, which targets the membrane-proximal epitope of MSLN, may represent a novel therapeutic strategy for the clinical treatment of CCA.

Background & Aims:Liver transplantation improves outcomes in hepatocellular carcinoma (HCC), yet treatment options for patients with tumor recurrence remain limited to tyrosine kinase inhibitors. Glypican-3 (GPC3)-targeted CAR T cells offer a tumor-directed immune-based therapeutic strategy, but their efficacy may be limited by post-transplant immunosuppression. We developed a CAR T cell platform combining CRISPR/Cas9-mediated FKBP1A disruption to confer resistance to FKBP12-dependent immunosuppressive agents, including tacrolimus, everolimus, and sirolimus, with TRAC knockout to eliminate endogenous T cell receptor expression and reduce alloreactivity. Methods:Human T cells were edited using Cas9 ribonucleoprotein complexes targeting FKBP1A and TRAC, expanded, and transduced with an anti-GPC3 CAR construct. Cytokine production and cytotoxicity were assessed in vitro. Antitumor activity under tacrolimus treatment was evaluated in a Hep G2 xenograft model, and xenoreactivity was assessed in a graft-versus-host disease model. FKBP1A/TRAC double-knockout T cells were enriched using mTOR inhibitor selection combined with CD3-based MACS depletion. PBMCs from liver transplant recipients were used to evaluate feasibility for clinical translation during the early post-transplant period. Results:Tacrolimus suppressed wild-type CAR T cell function but not FKBP1A/TRAC double-knockout CAR T cells, which retained cytokine production, cytotoxicity, and in vivo antitumor activity. Cyclosporine A remained suppressive, enabling its potential use as a pharmacologic control strategy. TRAC disruption reduced xenoreactivity. CD3-based MACS depletion and mTOR inhibition achieved functional double-knockout efficiencies greater than 98%, without compromising cell viability. Functional FKBP1A/TRAC knockout CAR T cells were generated from patient PBMC samples 30 days post-transplant. Conclusions:Dual-edited GPC3 CAR T cells resist tacrolimus-based immunosuppression while limiting alloreactivity, supporting their use for recurrent HCC after liver transplantation. Sequential, high-viability selection in a modular cellular engineering framework enables adaptation to alternative tumor targets and next-generation CAR T cell designs.

Abstract Antigen mutation and heterogeneous expression remain major barriers to effective antibody- and chimeric antigen receptor (CAR)-based immunotherapies for hepatocellular carcinoma (HCC). Glypican-3 (GPC3) exhibits variable surface density and epitope alteration in advanced or recurrent HCC tumors. To address these clinically relevant obstacles, we developed a modular antibody-based γ/δ T-cell receptor (γδAbTCR) platform that integrates antibody specificity with intrinsic TCR-CD3 signaling to enhance tumor recognition under conditions of antigen variation. Using two GPC3 antibodies recognizing distinct epitopes (hYP7 and HN3), we generated four γδAbTCR constructs—hYP7-hYP7, hYP7-HN3, HN3-hYP7, and HN3-HN3—and evaluated their functional properties in primary human T cells. Among these, hYP7-hYP7 γδAbTCR-T cells demonstrated the strongest antigen binding and cytotoxic activity against Hep3B and other GPC3+ HCC models, particularly those with low or heterogeneous antigen density. In vivo, hYP7-hYP7 T cells showed superior tumor infiltration, persistence, and control of large, established xenografts compared with other configurations. Mechanistic studies revealed that the hYP7-hYP7 γδAbTCR architecture couples enhanced antigen-binding avidity with coordinated TCR-CD3 and CD30 signaling, leading to potent NF-κB and NFAT activation and rapid induction of caspase-mediated apoptosis in tumor cells. These findings establish hYP7-hYP7 as a lead γδAbTCR design that overcomes the limitations of antigen heterogeneity and mutation in liver cancer, providing a promising framework for next-generation T-cell therapies targeting liver cancer. Citation Format: Dan Li, Tianyuzhou Liang, Hsi-En Tsao, Zhijian Duan, Laura E. Hutchins, Madilyn Gaydos, Iris Yang, Elijah Edmondson, Xiaoshan Wang, Rui Zheng, Jing Zhou, Chin-Hsien (Emily) Tai, Jing Bian, Maggie Cam, Hongbing Zhang, Cheng Liu, Mitchell Ho. The GPC3-targeting hYP7 antibody-based gamma/delta TCR-T cell therapy for overcoming antigen mutation and heterogeneity in liver cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 5621.

Abstract Glypican-1 (GPC1) is a heparan sulfate proteoglycan that is overexpressed in pancreatic ductal adenocarcinoma (PDAC). We previously developed GPC1 CARs using the dromedary VHH nanobody D4 and the mouse monoclonal IgG antibody HM2, which recognize a membrane-distal epitope and a membrane-proximal epitope, respectively. Here, we integrate structural modeling with CAR functional data and animal experiments to define how epitope position and CAR geometry jointly determine GPC1 CAR efficacy. Modeling of CAR ectodomains incorporating CD8 or IgG4 hinges and CD8 or CD28 transmembrane (TM) domains indicated that only specific combinations yield an intermembrane spacing comparable to that of the TCR-pMHC immune synapse for a given epitope. These geometric predictions aligned with functional data in which D4-IgG4H-CD28TM CAR T cells rapidly regressed tumor bioluminescent in a T3M4 intraperitoneal PDAC model. Together, these results support a model in which epitope spatial location on GPC1 and CAR hinge/TM architecture are key design parameters for GPC1-targeting CAR T cells. To facilitate clinical development of D4 CAR T cells, humanization of the VHH D4 is highly desirable, although humanization of nanobodies such as VHHs is not well established. In addition to the CDR grafting to the nearest germline framework as we described previously for humanization of rabbit and mouse antibodies, we used AI-predicted structural models from multiple platforms to compare both the spatial geometry and sequence similarity of predicted D4 framework and CDR regions with available antibody and nanobody structures. Humanized constructs with the highest humanness scores, geometry, and similarity scores were back-mutated to preserve critical residues in the nanobody framework sequences and were then evaluated for GPC1 binding affinity and cell-surface binding. Using this AI-assisted, structure-guided strategy, we aim to generate clinically suitable humanized VHH nanobody scaffolds for GPC1 CAR T therapy and other nanobody-based clinical applications. Citation Format: Hsi En Tsao, Mitchell Ho, . Structure-guided protein engineering and humanization of GPC1-targeted nanobody CAR T cells for treating pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4269.

Abstract Mesothelin (MSLN) is a cell surface protein that is overexpressed in various cancers, including mesothelioma, pancreatic, and ovarian cancer. Its expression in normal tissue is limited to the mesothelial cells lining the pleura, peritoneum and pericardium; make it an attractive target for antibody-based therapeutics. Many efforts have been dedicated towards the development of these antibody-based approaches but proteases in the tumor microenvironment promote the cleavage of MSLN from cancer cells. High concentrations of shed MSLN in the tumor microenvironment bind to the antibody inhibiting its activity and preventing the death of cancer cells. To address the presence of shed MSLN, an antibody called 15B6 was designed. It binds the membrane-proximal, protease sensitive region of MSLN that is not shed in the tumor microenvironment. This study investigates the ability of 15B6-targeted bispecific antibodies to eliminate MSLN expressing cancers. We designed humanized and murine versions of a CD3x15B6 bispecific antibody, which binds the CD3 on T cells and the MSLN (15B6) epitope on cancer cells serving as a bridge to promote the activation of T cells to kill MSLN positive cancer cells. These 15B6-based antibodies were compared to SS1-based antibodies that target the distal N-terminal domain of MSLN that is shed by proteases. In-vitro, the SS1 and 15B6 antibodies hold similar cytotoxic activity, but when cocultured with MSLN 296-591, a recombinant protein mimicking shed MSLN, the SS1 antibodies activity is inhibited. In-vivo, in a human mesothelioma model in immunodeficient mice, tumor shrinkage and growth inhibition were observed when treated with the 15B6-based antibody but not those treated with the SS1-based antibody. In an immunocompetent mouse model, the complete regression of colon and breast tumors was observed when treated with the 15B6-based antibody compared to the SS1 antibody and control samples. Transcriptional analysis revealed that 15B6-treated mice had higher levels of activation of both innate and adaptive immune cells, along with significant upregulation of cytokine and STAT5 signaling pathways. Through in-vitro and in-vivo studies, we have demonstrated that this 15B6-targeted antibody binds to the protease-sensitive region, is highly active against MSLN-expressing cancer cell lines in vitro, is not inhibited by shed MSLN, and promotes upregulation of immune cell populations and signaling pathways that support robust anti-tumor activity. Citation Format: Eber Antonio Guzman-Cruz, Masanori Onda, Xiufen Liu, Tara O’Shea, Qi Zhou, Wenlong Liu, Jing Bian, Chin-Hsien Tai, Ira Pastan, Mitchell Ho, . A T-cell engager antibody targeting the non-shed site of mesothelin in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2633.

Chimeric antigen receptor (CAR) efficacy depends on the presence of surface antigen. While antigen levels in leukemias can be measured in the blood or bone marrow, expression in solid tumors is often inferred from archival tissues or remains undetermined. Neuroblastoma (NB) is an aggressive pediatric solid tumor for which we have co-developed a CAR targeting glypican 2 (GPC2). Here, we developed a radiotheranostic platform comprising an antibody-based positron emission tomography (immunoPET) agent, [89Zr]Zr-DFO-mCT3 (89Zr-mCT3), for non-invasive detection of GPC2 and [225Ac]Ac-macropa-mCT3 (225Ac-mCT3) for alpha-particle therapy. In orthotopic NB models with variable GPC2 levels, immunoPET accurately detected tumors with positive versus low/negative GPC2 status, which correlated with in vivo response to CAR. As a therapeutic agent, 225Ac-mCT3 induced tumor regression in a dose-dependent manner, with the maximal effect at 2.96 kBq (80 nCi). Because 225Ac-mCT3 does not downregulate GPC2, we assessed GPC2-CAR therapy with 225Ac-mCT3 (1.48 kBq [40 nCi]) and showed improved survival in mice with GPC2med but not GPC2high tumors. These findings establish 89Zr-mCT3 as a novel radiotracer for GPC2 monitoring and a predictive tool for CAR responses. 225Ac-mCT3 enhances GPC2-CAR therapy in tumors with medium antigen levels, offering a rationale for clinical development in NB and other pediatric tumors.

Adapting the success of chimeric antigen receptor T cell therapy from hematologic malignancies to solid tumors has become a major focus of ongoing research activities. However, the unique challenges posed by solid tumors, such as limited immune cell infiltration, reduced T cell persistence, and antigen loss, have led to only limited success in early clinical trials. Recently, combinatorial strategies incorporating next-generation armored CAR T cells along with various alternative immune cell types have rekindled optimism in the field. Hepatocellular carcinoma represents a distinct entity due to the unique characteristics of the liver microenvironment, including the influence of lipid metabolism, bile acids, and microbial compounds from the gut-liver axis. Furthermore, HCC is characterized by a variety of tumor-specific and tumor-associated antigens, enabling targeted approaches with minimal risk of on-target/off-tumor effects. The unique complexity of HCC, along with the underlying liver diseases that give rise to these tumors, presents both challenges and opportunities for cellular therapies. In this review, we examine the current landscape of CAR T cell therapy for HCC, highlighting recent clinical and preclinical developments. Furthermore, we discuss why HCC may be especially well-suited for tailored CAR-based strategies, given the liver's specific anatomical and immunological properties.

Abstract Background and Significance: Chimeric antigen receptor (CAR) T cell therapy has revolutionized the treatment of hematologic cancers but remains less effective against solid tumors, where heterogeneous antigen expression poses a major barrier to efficacy. Neuroblastoma, a pediatric solid tumor with poor survival rates in high-risk patients, often expresses glypican-2 (GPC2), a promising yet variably expressed surface antigen. To overcome these challenges, we engineered antibody-T cell receptors (AbTCRs) that combine antibody-based antigen recognition with the signaling machinery of γ/δ T cell receptors, aiming to promote more physiological activation and sustained antitumor activity in GPC2-positive neuroblastoma. Methods: We developed AbTCRs containing anti-GPC2 Fab fragments (humanized CT3 or murine CT3) fused to γ/δ TCR constant regions and a co-stimulatory domain, CD30. Primary human T cells were transduced with these constructs and tested through in vitro tumor-killing assays, repeated cytotoxicity tests, western blotting, and in vivo xenograft models bearing GPC2 neuroblastoma (IMR5, NBEB, LAN1, and SH-SY5Y) tumors. The LAN1 and SH-SY5Y models have low GPC2 antigen density, enabling evaluation of efficacy under stringent antigen conditions. Results: We found strong anti-tumor effects in humanized CT3 AbTCR-T cells, including significant tumor reduction and complete responses in multiple xenograft models. Tumors from mice administered with humanized CT3 AbTCR-T cells showed notably augmented infiltration of CD8+ and CD8+CD27+ T cells, consistent with enhanced effector persistence. Humanized CT3 AbTCR-T cells demonstrated downregulation of exhaustion markers PD1, LAG3, and TIM3 and an enriched less-differentiated Tscm subset, as assessed by peripheral blood testing. These cells maintained lower PD1 levels after prolonged coculture with tumor cells, and retained cytotoxic activity in second- and third-round killing assays. Mechanistically, upon tumor engagement, hCT3 AbTCRs elicited more robust TCR signaling, evidenced by higher NFAT nuclear translocation and greater phosphorylation of key TCR pathway proteins. Conclusion: Humanized CT3 AbTCR T cells exhibit durable and potent antitumor effects against low-GPC2 neuroblastoma by combining antibody specificity with physiological TCR signaling. Their enhanced TCR signaling, reduced exhaustion, and sustained cytotoxic ability suggest that AbTCR T cells represent a promising next-generation cellular therapy for solid tumors with heterogeneous or low antigen expression. Citation Format: Mingyu Huo, Alex Quan, Dan Li, Laura E. Hutchins, Constanza Rodriguez, Jangsuk Oh, Hsi-En Tsao, Madeline Spetz, Elijah Edmondson, Dana Ashworth, Rui Zheng, Jing Zhou, Jinyun Chen, Jingbao Liu, Guangyan Xiong, Hongbing Zhang, Cheng Liu, Rosa Nguyen, Nan Li, Mitchell Ho. Humanized antibody Fab-TCR T cells targeting GPC2 effectively regress neuroblastoma via enhanced TCR signaling and sustained cytotoxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 5617.

Hypoxia is a hallmark of the solid tumor microenvironment and is known to impair CAR T-cell function. In order to condition CAR T-cells to this microenvironment during manufacturing, we evaluated whether varying oxygen tension during the manufacturing of GPC2-CAR T-cells improves cellular characteristics and function against neuroblastoma. Cell products were produced over 8-9 days under normoxic (21% oxygen), transitional (21%, then transitioned to 1% on Day 2), or maintained hypoxic conditions (1% from Day 0 onward). RNA-sequencing, metabolic assays, phenotyping, and functional studies were performed. Sustained hypoxia induced universal changes in CAR T-cells, including increased surface CAR expression, enrichment of TN/TSCM subsets, and a shift toward glycolysis. However, manufacturing fully in hypoxic conditions yielded markedly reduced cell expansion relative to normoxic and transitional conditions. Normoxia-produced CAR T-cells were most potent in eliminating neuroblastoma tumor cells in vitro and in vivo. In parallel, hypoxia also altered neuroblastoma biology, including downregulation of GPC2 expression and induction of transcriptional programs associated mesenchymal transition and hypoproliferation. Together, these findings indicate that oxygen tension substantially alters both CAR T-cell products and tumor cell state. Sustained hypoxic manufacturing does not improve GPC2-CAR T-cell efficacy and instead compromises yield and antitumor function. Thus, for this GPC2-CAR T-cell product, normoxic manufacturing remains the most favorable approach for clinical translation.

Spearman’s plot suggesting correlation between SUVmean (A) and SUVmax (B) intensity and GPC3 staining of HepG2, Hep3B, and Huh7 tumors (n = 2).

Studies on the dynamic changes occurring in the tumor microenvironment (TME) following CAR-T cell therapy have been confounded by host lymphodepletion, multiple dosing and immunodeficient models. Here, a nanobody-based, mouse mesothelin-targeting CAR-T cell (A101) was developed, achieving effective primary tumor suppression, metastasis reduction, and improved survival after a single dose in immunocompetent, syngeneic mouse models without lymphodepletion. Temporal tumor profiling using RNA sequencing revealed initial downregulation of cell proliferation genes followed by upregulation of inflammation, epithelial-to-mesenchymal-transition (EMT) and extracellular matrix (ECM) modification genes in the CAR-T-treated tumors relative to mock-T-treated controls. This phenotype was reversed at a later timepoint which coincided with downregulation of immunosuppressive Cd274 + Lcn2 + neutrophils and upregulation of anti-tumor P2rx1 + Nrf2 - neutrophils. At the same time, upregulation of Ccl2 + in fibroblasts and a more immunomodulatory macrophage phenotype was observed in CAR-T-treated tumors, indicating a tumor adaptation mechanism. This study demonstrates complex dynamic changes in the TME, and highlights time-dependent responses of solid tumors to CAR-T cell therapy. It further highlights Lcn2 + neutrophils and Ccl2 + fibroblasts as potential therapeutic targets for improving CAR-T cell anti-tumor efficacy.

The tumor-associated antigen mesothelin is highly expressed in many human cancers, while its expression in normal tissues is limited. Its interaction with the cancer antigen 125 promotes heterotypic cell adhesion and tumor metastasis. Mesothelin-targeted immunotherapies are being intensively investigated, which is aided by growing structural knowledge of the protein and its interactions with antibodies. Recent studies have produced a complete atomic model showing mesothelin as a compact, right-handed, conformationally flexible solenoid composed of nine layers of helices, with glycans attached at all three predicted N-glycosylation sites. Structural analyses reveal that most therapeutic antibodies target the rigid and immunogenic N-terminal domain, while a few bind to middle domain or C-terminal linear tail, revealing correlation between immunogenicity and structural stability. Crystallographic studies have also extended to the interactions between mesothelin and CA-125. These structural advances offer insights into the potential function of mesothelin and guidance for further development of therapeutic antibodies.

Chimeric antigen receptor (CAR) T cell therapy is a promising approach for cancer treatment; however, its effectiveness in solid tumors is hindered by challenges such as antigen escape due to mutations, heterogenous expression, and immunosuppressive tumor microenvironment. Previously, we developed CAR-T cells targeting glypican-3 (GPC3) for liver cancer using a humanized GPC3-specific monoclonal antibody (hYP7) and human single-domain antibody (HN3). The hYP7 CAR-T cells demonstrate tumor regression in preclinical mouse models and are being used to treat liver cancer patients in a clinical trial at the NIH. In this study, we analyzed clinically-relevant mutations in GPC3 transcripts observed in hepatocellular carcinoma (HCC) patients through RNAseq analysis. Produced the GPC3 variant proteins, and tested whether these mutations may functionally reduce the effectiveness of CAR-T therapies targeting GPC3. We also developed an engineered antibody-gamma/delta TCR (AbTCR) targeting GPC3, incorporating hYP7 and HN3 antibodies against two distinct epitopes on C and N lobes of GPC3, respectively. In an HCC mouse model with low GPC3 antigen density, the GPC3-targeting AbTCR demonstrated superior tumor control and improved overall survival compared to CAR-T cells, attributable to its robust antigen-binding capacity and enhanced tumor infiltrations. Our RNA sequencing analysis revealed a unique set of upregulated genes in tumor-infiltrating AbTCR-T cells compared to CAR-T cells. These genes were associated with T cell functionality and persistence, particularly pathways involving NF-κB and NFAT signaling. The functional analysis data highlight the GPC3-targeting AbTCR as a new potent therapeutic approach for HCC. Dan Li, Tianyuzhou Liang, Hsi-En Tsao, Laura Hutchins, Madilyn Gaydos, Chin-Hsien Tai, Jing Bian, Maggie Cam, Hongbing Zhang, Cheng Liu, Mitchell Ho. A chimeric antibody-based gamma/delta TCR targeting GPC3 overcomes low antigen expression in liver cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3497.

Identifying tumor selective targets is critical for the development of precision diagnostic and therapeutic agents in oncology. Despite advances in precision oncology elsewhere, there are no FDA-approved hepatocellular carcinoma (HCC)-selective treatments. HCC is the most common type of liver cancer and accounts for significant morbidity and mortality worldwide. Here, we sought to integrate bulk (371 cases) and single cell RNA sequencing (scRNAseq, n =2 datasets, 34 cases, 102,956 cells) of patient samples to enrich for molecules that are overexpressed in HCC, which could serve as HCC-selective targets. To guide definitions of tumor and normal cell clusters with higher fidelity, we also imported a normal liver scRNAseq dataset. Using this integrated approach, we identified several HCC-selective plasma membrane molecules. To validate these targets, we performed immunohistochemical staining of HCC and normal tissue microarrays and confirmed HCC-selective staining of identified targets. Next, we verified the presence of these targets in several commercially available HCC cell lines by flow cytometry and western blot. Finally, we designed, engineered, and tested novel antibody-based positron emission tomography (immunoPET) agents to these targets in various murine models of liver cancer. Our findings confirm that we can leverage this multimodal approach to identify and validate of HCC-selective targets, which can be used to develop tumor-selective diagnostic and therapeutic radiopharmaceuticals, or radiotheranostics, and other precision oncology agents. One Sentence Summary:A multimodal pipeline defines and validates tumor-selective surface targets for radiotheranostic use in hepatocellular carcinoma.

Chimeric antigen receptor (CAR) T cells have shown promise in hematological cancers but face challenges in solid tumors, partly due to heterogeneous antigen density. Glypican-2 (GPC2) is an oncofetal antigen highly expressed in neuroblastoma and under evaluation in phase 1 clinical trials. Here, we engineer T cells with antibody-T cell receptors (AbTCRs) targeting GPC2. We generate autologous AbTCR T cells using CT3 or humanized CT3 (hCT3) antigen-binding fragments (Fab) linked to γ/δ T cell receptors (TCRs), along with a CD30 co-stimulatory domain. Both CT3 and hCT3 AbTCR T cells show superior antitumor efficacy compared to CT3 CAR T cells, with hCT3 AbTCR T cells inducing significant regression in neuroblastoma with low GPC2 antigen density. Enhanced efficacy is associated with stronger TCR signaling, expansion of stem cell-like memory T cells, and improved CD8+ T cell infiltration. These results highlight the potential of hCT3 AbTCR T cells for neuroblastoma and indicate broad application of AbTCR T cells in solid tumors.