ABSTRACT
Novel chimeric antigen receptor (CAR) T-cell approaches are needed to improve therapeutic efficacy in solid tumors. High-risk neuroblastoma is an aggressive pediatric solid tumor that expresses cell-surface GPC2 and GD2 with a tumor microenvironment infiltrated by CD16a-expressing innate immune cells. Here we engineer T-cells to express a GPC2-directed CAR and simultaneously secrete a bispecific innate immune cell engager (BiCE) targeting both GD2 and CD16a. In vitro, GPC2.CAR-GD2.BiCE T-cells induce GPC2-dependent cytotoxicity and secrete GD2.BiCE that promotes GD2-dependent activation of antitumor innate immunity. In vivo, GPC2.CAR-GD2.BiCE T-cells locally deliver GD2.BiCE and increase intratumor retention of NK-cells. In mice bearing neuroblastoma patient-derived xenografts and reconstituted with human CD16a-expressing immune cells, GD2.BiCEs enhance GPC2.CAR antitumor efficacy. A CAR.BiCE strategy should be considered for tumor histologies where antigen escape limits CAR efficacy, especially for solid tumors like neuroblastoma that are infiltrated by innate immune cells.
Subject(s)
Gangliosides , Immunity, Innate , Immunotherapy, Adoptive , Killer Cells, Natural , Neuroblastoma , Receptors, Chimeric Antigen , T-Lymphocytes , Neuroblastoma/immunology , Neuroblastoma/therapy , Neuroblastoma/pathology , Animals , Humans , Receptors, Chimeric Antigen/immunology , Receptors, Chimeric Antigen/metabolism , Mice , Gangliosides/immunology , Cell Line, Tumor , Immunotherapy, Adoptive/methods , Killer Cells, Natural/immunology , T-Lymphocytes/immunology , Xenograft Model Antitumor Assays , Glypicans/immunology , Glypicans/metabolism , Tumor Microenvironment/immunology , FemaleABSTRACT
The application of CAR-T cells in solid tumors poses several challenges, including poor T cell homing ability, limited infiltration of T cells and an immunosuppressive tumor environment. In this study, we developed a novel approach to address these obstacles by designing GPC3-specific CAR-T cell that co-express IL-21 and CXCL9 (21 × 9 GPC3 CAR-T cells) and blocking the PD-1 expression on it. The proliferation, cell phenotype, cytokine secretion and cell migration of indicated CAR-T cells were evaluated in vitro. The cytotoxic activities of genetically engineered CAR-T cells were accessed in vitro and in vivo. Compared to conventional GPC3 CAR-T cells, the 21 × 9 GPC3 CAR-T cells demonstrated superior proliferation, cytokine secretion and chemotaxis capabilities in vitro. Furthermore, when combined with PD-1 blockade, the 21 × 9 GPC3 CAR-T cells exhibited enhanced proliferation, cytokine secretion and enrichment of effector T cells such as CTL, NKT and TEM cells. In xenograft tumor models, the PD-1 blocked 21 × 9 GPC3 CAR-T cells effectively suppressed HCC xenograft growth and increased T cell infiltration. Overall, our study successfully generated GPC3 CAR-T cells expressing both IL-21 and CXCL9, demonstrated that combining PD-1 blockade can further enhance CAR-T cell function by promoting proliferation, cytokine secretion, chemotaxis and antitumor activity. These findings present a hopeful and potentially effective strategy for GPC3-positive HCC patients.
Subject(s)
Carcinoma, Hepatocellular , Chemokine CXCL9 , Glypicans , Immunotherapy, Adoptive , Interleukins , Liver Neoplasms , Programmed Cell Death 1 Receptor , Receptors, Chimeric Antigen , Glypicans/immunology , Glypicans/metabolism , Glypicans/antagonists & inhibitors , Glypicans/genetics , Interleukins/metabolism , Interleukins/genetics , Carcinoma, Hepatocellular/therapy , Carcinoma, Hepatocellular/pathology , Carcinoma, Hepatocellular/immunology , Carcinoma, Hepatocellular/drug therapy , Animals , Humans , Immunotherapy, Adoptive/methods , Liver Neoplasms/therapy , Liver Neoplasms/pathology , Liver Neoplasms/immunology , Liver Neoplasms/drug therapy , Programmed Cell Death 1 Receptor/metabolism , Programmed Cell Death 1 Receptor/antagonists & inhibitors , Mice , Chemokine CXCL9/metabolism , Chemokine CXCL9/genetics , Receptors, Chimeric Antigen/immunology , Receptors, Chimeric Antigen/metabolism , Xenograft Model Antitumor Assays , Cell Proliferation , Cell Line, TumorABSTRACT
This study aims to prepare bacterial outer membrane vesicles (OMVs) with anti-glypican-3 (GPC3) single-chain antibody and analyze their targeting effects on Hep G2 hepatocellular carcinoma (HCC) cells and tissue. The recombinant plasmid pET28a-Hbp-hGC 33-scFv was constructed by ligating Hbp-hGC 33-scFv to pET28a. Western blotting was employed to determine the prokaryotic expression of the fusion protein Hbp-hGC 33-scFv, on the basis of which the optimal induction conditions were determined. Hbp-hGC 33-OMVs secreted from the recombinant expressing strains were collected by ultrafiltration concentration and then characterized. The localization of Hbp-hGC 33-scFv in bacteria and Hbp-hGC 33-OMVs was analyzed by immune electron microscopy. The binding of Hbp-hGC 33-scFv to Hep G2 cells was observed by immunofluorescence. The Hep G2 tumor-bearing mouse model was established, and the targeted retention of Hbp-hGC 33-OMVs in the tumor site of mice was observed by a fluorescence imaging system in vivo. The results showed that the actual molecular weight of the fusion protein was 175.3 kDa, and the optimal induction conditions were as follows: OD600=0.5, IPTG added at a final concentration of 0.5 mmol/L, and overnight induction at 16 â. The prepared Hbp-hGC 33-OMVs were irregular spherical structures with an average particle size of (112.3±4.6) nm, expressing OmpC, OmpA, and the fusion protein Hbp-hGC 33-scFv. The Hbp-hGC 33-OMVs prepared in this study demonstrated stronger ability of binding to Hep G2 cells than the wild-type OMVs (P=0.008). All the data indicated that Hbp-hGC 33-OMVs with anti-GPC3 single-chain antibody were successfully prepared and could be used for research on the targeted therapy of hepatocellular carcinoma.
Subject(s)
Bacterial Outer Membrane , Carcinoma, Hepatocellular , Glypicans , Liver Neoplasms , Single-Chain Antibodies , Single-Chain Antibodies/immunology , Single-Chain Antibodies/genetics , Single-Chain Antibodies/chemistry , Animals , Mice , Humans , Liver Neoplasms/immunology , Liver Neoplasms/metabolism , Bacterial Outer Membrane/metabolism , Bacterial Outer Membrane/immunology , Hep G2 Cells , Glypicans/immunology , Glypicans/metabolism , Glypicans/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Recombinant Fusion Proteins/biosynthesis , Escherichia coli/genetics , Escherichia coli/metabolism , Drug Delivery Systems , Mice, NudeABSTRACT
Liver cancer, primarily hepatocellular carcinoma (HCC), is the second leading cause of cancer-related deaths globally. It is typically characterized by rapid progression, poor prognosis, and high mortality rates. Given these challenges, the search for molecular targets aiding early diagnosis and targeted therapy remains imperative. Glypican 3 (GPC3), a cell-surface glycoprotein, emerges as a promising candidate for addressing HCC Overexpressed in HCC tissues; GPC3 is a credible immunohistochemical marker for liver cancer diagnosis and a potential marker for liquid biopsy through soluble GPC3 in serum. Various immunotherapies targeting GPC3 have been developed, including vaccines, anti-GPC3 immunotoxins, and chimeric antigen receptor-modified cells. This review comprehensively covers the structure, physicochemical properties, biological functions, and clinical applications of GPC3. It explores diagnostic and treatment strategies centered around GPC3, offering hope for improved early detection and targeted therapies in the challenging landscape of HCC.
Subject(s)
Biomarkers, Tumor , Carcinoma, Hepatocellular , Glypicans , Immunotherapy , Liver Neoplasms , Glypicans/immunology , Glypicans/metabolism , Humans , Carcinoma, Hepatocellular/immunology , Carcinoma, Hepatocellular/diagnosis , Carcinoma, Hepatocellular/therapy , Liver Neoplasms/immunology , Liver Neoplasms/therapy , Liver Neoplasms/diagnosis , Liver Neoplasms/metabolism , Immunotherapy/methods , Biomarkers, Tumor/metabolism , Biomarkers, Tumor/immunology , Biomarkers, Tumor/blood , Molecular Targeted Therapy/methods , Precision Medicine/methods , Cancer Vaccines/immunology , Cancer Vaccines/therapeutic useABSTRACT
CD39 serves as a crucial biomarker for neoantigen-specific CD8+ T cells and is associated with antitumor activity and exhaustion. However, the relationship between CD39 expression levels and the function of chimeric antigen receptor T (CAR-T) cells remains controversial. This study aimed to investigate the role of CD39 in the functional performance of CAR-T cells against hepatocellular carcinoma (HCC) and explore the therapeutic potential of CD39 modulators, such as mitochondrial division inhibitor-1 (mdivi-1), or knockdown CD39 through short hairpin RNA. Our findings demonstrated that glypican-3-CAR-T cells with moderate CD39 expression exhibited a strong antitumor activity, while high and low levels of CD39 led to an impaired cellular function. Methods modulating the proportion of CD39 intermediate (CD39int)-phenotype CAR-T cells such as mdivi-1 and CD39 knockdown enhanced and impaired T cell function, respectively. The combination of mdivi-1 and CD39 knockdown in CAR-T cells yielded the highest proportion of infiltrated CD39int CAR-T cells and demonstrated a robust antitumor activity in vivo. In conclusion, this study revealed the crucial role of CD39 in CAR-T cell function, demonstrated the potential therapeutic efficacy of combining mdivi-1 with CD39 knockdown in HCC, and provided a novel treatment strategy for HCC patients in the field of cellular immunotherapy.
Subject(s)
Apyrase , Carcinoma, Hepatocellular , Glypicans , Immunotherapy, Adoptive , Liver Neoplasms , Receptors, Chimeric Antigen , Carcinoma, Hepatocellular/immunology , Carcinoma, Hepatocellular/therapy , Carcinoma, Hepatocellular/genetics , Glypicans/immunology , Glypicans/genetics , Glypicans/metabolism , Liver Neoplasms/immunology , Liver Neoplasms/therapy , Liver Neoplasms/genetics , Apyrase/metabolism , Apyrase/genetics , Humans , Animals , Mice , Immunotherapy, Adoptive/methods , Receptors, Chimeric Antigen/immunology , Receptors, Chimeric Antigen/genetics , Cell Line, Tumor , Antigens, CD/genetics , Antigens, CD/metabolism , Xenograft Model Antitumor AssaysABSTRACT
PURPOSE: Retinoblastoma is the most common intraocular malignancy in children. Although new chemotherapeutic approaches have improved ocular salvage rates, novel therapies are required for patients with refractory intraocular and metastatic disease. Chimeric antigen receptor (CAR) T cells targeting glypican-2 (GPC2) are a potential new therapeutic strategy. EXPERIMENTAL DESIGN: GPC2 expression and its regulation by the E2F1 transcription factor were studied in retinoblastoma patient samples and cellular models. In vitro, we performed functional studies comparing GPC2 CAR T cells with different costimulatory domains (4-1BB and CD28). In vivo, the efficacy of local and systemic administration of GPC2 CAR T cells was evaluated in intraocular and leptomeningeal human retinoblastoma xenograft models. RESULTS: Retinoblastoma tumors, but not healthy retinal tissues, expressed cell surface GPC2, and this tumor-specific expression was driven by E2F1. GPC2-directed CARs with 4-1BB costimulation (GPC2.BBz) were superior to CARs with CD28 stimulatory domains (GPC2.28z), efficiently inducing retinoblastoma cell cytotoxicity and enhancing T-cell proliferation and polyfunctionality. In vivo, GPC2.BBz CARs had enhanced persistence, which led to significant tumor regression compared with either control CD19 or GPC2.28z CARs. In intraocular models, GPC2.BBz CAR T cells efficiently trafficked to tumor-bearing eyes after intravitreal or systemic infusions, significantly prolonging ocular survival. In central nervous system (CNS) retinoblastoma models, intraventricular or systemically administered GPC2.BBz CAR T cells were activated in retinoblastoma-involved CNS tissues, resulting in robust tumor regression with substantially extended overall mouse survival. CONCLUSIONS: GPC2-directed CAR T cells are effective against intraocular and CNS metastatic retinoblastomas.
Subject(s)
Glypicans , Immunotherapy, Adoptive , Receptors, Chimeric Antigen , Retinoblastoma , T-Lymphocytes , Xenograft Model Antitumor Assays , Humans , Animals , Retinoblastoma/immunology , Retinoblastoma/pathology , Retinoblastoma/therapy , Mice , Receptors, Chimeric Antigen/immunology , Glypicans/immunology , Glypicans/antagonists & inhibitors , Immunotherapy, Adoptive/methods , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Cell Line, Tumor , Central Nervous System Neoplasms/therapy , Central Nervous System Neoplasms/immunology , Central Nervous System Neoplasms/secondary , Central Nervous System Neoplasms/pathology , Disease Models, Animal , FemaleABSTRACT
Chimeric antigen receptor-expressing T (CAR-T) cells induce robust antitumor responses in patients with hematologic malignancies. However, CAR-T cells exhibit only limited efficacy against solid tumors such as hepatocellular carcinoma (HCC), partially due to their limited expansion and persistence. CD8+ T cells, as key components of the adaptive immune response, play a central role in antitumor immunity. Aerobic glycolysis is the main metabolic feature of activated CD8+ T cells. In the tumor microenvironment, however, the uptake of large amounts of glucose by tumor cells and other immunosuppressive cells can impair the activation of T cells. Only when tumor-infiltrating lymphocytes (TILs) in the tumor microenvironment have a glycolytic advantage might the effector function of T cells be activated. Glucose transporter type 1 (GLUT1) and acylglycerol kinase (AGK) can boost glycolytic metabolism and activate the effector function of CD8+ T cells, respectively. In this study, we generated GPC3-targeted CAR-T cells overexpressing GLUT1 or AGK for the treatment of HCC. GPC3-targeted CAR-T cells overexpressing GLUT1 or AGK specifically and effectively lysed GPC3-positive tumor cells in vitro in an antigen-dependent manner. Furthermore, GLUT1 or AGK overexpression protected CAR-T cells from apoptosis during repeated exposures to tumor cells. Compared with second-generation CAR-T cells, GPC3-targeted CAR-T cells overexpressing GLUT1 or AGK exhibited greater CD8+ T-cell persistence in vivo and better antitumor effects in HCC allograft mouse models. Finally, we revealed that GLUT1 or AGK maintained anti-apoptosis ability in CD8+ T cells via activation of the PI3K/Akt pathway. This finding might identify a therapeutic strategy for advanced HCC.
Subject(s)
Carcinoma, Hepatocellular , Glucose Transporter Type 1 , Glypicans , Liver Neoplasms , Carcinoma, Hepatocellular/therapy , Carcinoma, Hepatocellular/immunology , Carcinoma, Hepatocellular/metabolism , Animals , Liver Neoplasms/immunology , Liver Neoplasms/therapy , Liver Neoplasms/pathology , Glucose Transporter Type 1/metabolism , Humans , Mice , Glypicans/metabolism , Glypicans/immunology , Immunotherapy, Adoptive/methods , Cell Line, Tumor , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Receptors, Chimeric Antigen/immunology , Receptors, Chimeric Antigen/metabolism , Tumor Microenvironment , ApoptosisABSTRACT
BACKGROUND: Although most patients with newly diagnosed high-risk neuroblastoma (NB) achieve remission after initial therapy, more than 50% experience late relapses caused by minimal residual disease (MRD) and succumb to their cancer. Therapeutic strategies to target MRD may benefit these children. We developed a new chimeric antigen receptor (CAR) targeting glypican-2 (GPC2) and conducted iterative preclinical engineering of the CAR structure to maximize its anti-tumor efficacy before clinical translation. METHODS: We evaluated different GPC2-CAR constructs by measuring the CAR activity in vitro. NOD-SCID mice engrafted orthotopically with human NB cell lines or patient-derived xenografts and treated with human CAR T cells served as in vivo models. Mechanistic studies were performed using single-cell RNA-sequencing. RESULTS: Applying stringent in vitro assays and orthotopic in vivo NB models, we demonstrated that our single-chain variable fragment, CT3, integrated into a CAR vector with a CD28 hinge, CD28 transmembrane, and 4-1BB co-stimulatory domain (CT3.28H.BBζ) elicits the best preclinical anti-NB activity compared with other tested CAR constructs. This enhanced activity was associated with an enrichment of CD8+ effector T cells in the tumor-microenvironment and upregulation of several effector molecules such as GNLY, GZMB, ZNF683, and HMGN2. Finally, we also showed that the CT3.28H.BBζ CAR we developed was more potent than a recently clinically tested GD2-targeted CAR to control NB growth in vivo. CONCLUSION: Given the robust preclinical activity of CT3.28H.BBζ, these results form a promising basis for further clinical testing in children with NB.
Subject(s)
Glypicans , Neuroblastoma , Receptors, Chimeric Antigen , Animals , Child , Humans , Mice , CD28 Antigens , Gangliosides , Glypicans/immunology , Glypicans/therapeutic use , Immunotherapy, Adoptive/methods , Mice, Inbred NOD , Mice, SCID , Neuroblastoma/metabolism , Neuroblastoma/therapy , Receptors, Chimeric Antigen/geneticsABSTRACT
Currently, ERY974, a humanized IgG4 bispecific T cell-redirecting antibody recognizing glypican-3 and CD3, is in phase I clinical trials. After a first-in-human clinical trial of an anti-CD28 agonist monoclonal antibody resulting in severe life-threatening adverse events, the minimal anticipated biological effect level approach has been considered for determining the first-in-human dose of high-risk drugs. Accordingly, we aimed to determine the first-in-human dose of ERY974 using both the minimal anticipated biological effect level and no observed adverse effect level approaches. In the former, we used the 10% effective concentration value from a cytotoxicity assay using the huH-1 cell line with the highest sensitivity to ERY974 to calculate the first-in-human dose of 4.9 ng/kg, at which maximum drug concentration after 4 h of intravenous ERY974 infusion was equal to the 10% effective concentration value. To determine the no observed adverse effect level, we conducted a single-dose study in cynomolgus monkeys that were intravenously infused with ERY974 (0.1, 1, and 10 µg/kg). The lowest dose of 0.1 µg/kg was determined as the no observed adverse effect level, and the first-in-human dose of 3.2 ng/kg was calculated, considering body surface area and species difference. For the phase I clinical trial, we selected 3.0 ng/kg as a starting dose, which was lower than the first-in-human dose calculated from both the no observed adverse effect level and minimal anticipated biological effect level. Combining these two methods to determine the first-in-human dose of strong immune modulators such as T cell-redirecting antibodies would be a suitable approach from safety and efficacy perspectives.Clinical trial registration: JapicCTI-194805/NCT05022927.
Subject(s)
Antibodies, Bispecific , Glypicans , T-Lymphocytes , Antibodies, Bispecific/administration & dosage , Dose-Response Relationship, Immunologic , Glypicans/immunology , Humans , T-Lymphocytes/immunologyABSTRACT
Pediatric cancers often mimic fetal tissues and express proteins normally silenced postnatally that could serve as immune targets. We developed T cells expressing chimeric antigen receptors (CARs) targeting glypican-2 (GPC2), a fetal antigen expressed on neuroblastoma (NB) and several other solid tumors. CARs engineered using standard designs control NBs with transgenic GPC2 overexpression, but not those expressing clinically relevant GPC2 site density (â¼5,000 molecules/cell, range 1-6 × 103). Iterative engineering of transmembrane (TM) and co-stimulatory domains plus overexpression of c-Jun lowered the GPC2-CAR antigen density threshold, enabling potent and durable eradication of NBs expressing clinically relevant GPC2 antigen density, without toxicity. These studies highlight the critical interplay between CAR design and antigen density threshold, demonstrate potent efficacy and safety of a lead GPC2-CAR candidate suitable for clinical testing, and credential oncofetal antigens as a promising class of targets for CAR T cell therapy of solid tumors.
Subject(s)
Glypicans/immunology , Immunotherapy, Adoptive , Neuroblastoma/drug therapy , Receptors, Antigen, T-Cell/metabolism , Animals , Cell Line, Tumor , Glypicans/metabolism , Humans , Immunotherapy/methods , Neuroblastoma/pathology , Receptors, Antigen, T-Cell/immunology , Receptors, Chimeric Antigen/immunology , T-Lymphocytes/drug effects , T-Lymphocytes/immunology , Xenograft Model Antitumor Assays/methodsABSTRACT
BACKGROUND: Glypican-3 (GPC-3) is an oncofetal protein that is highly expressed in various solid tumors, but rarely expressed in healthy adult tissues and represents a rational target of particular relevance in hepatocellular carcinoma (HCC). Autologous chimeric antigen receptor (CAR) αß T cell therapies have established significant clinical benefit in hematologic malignancies, although efficacy in solid tumors has been limited due to several challenges including T cell homing, target antigen heterogeneity, and immunosuppressive tumor microenvironments. Gamma delta (γδ) T cells are highly cytolytic effectors that can recognize and kill tumor cells through major histocompatibility complex (MHC)-independent antigens upregulated under stress. The Vδ1 subset is preferentially localized in peripheral tissue and engineering with CARs to further enhance intrinsic antitumor activity represents an attractive approach to overcome challenges for conventional T cell therapies in solid tumors. Allogeneic Vδ1 CAR T cell therapy may also overcome other hurdles faced by allogeneic αß T cell therapy, including graft-versus-host disease (GvHD). METHODS: We developed the first example of allogeneic CAR Vδ1 T cells that have been expanded from peripheral blood mononuclear cells (PBMCs) and genetically modified to express a 4-1BB/CD3z CAR against GPC-3. The CAR construct (GPC-3.CAR/secreted interleukin-15 (sIL)-15) additionally encodes a constitutively-secreted form of IL-15, which we hypothesized could sustain proliferation and antitumor activity of intratumoral Vδ1 T cells expressing GPC-3.CAR. RESULTS: GPC-3.CAR/sIL-15 Vδ1 T cells expanded from PBMCs on average 20,000-fold and routinely reached >80% purity. Expanded Vδ1 T cells showed a primarily naïve-like memory phenotype with limited exhaustion marker expression and displayed robust in vitro proliferation, cytokine production, and cytotoxic activity against HCC cell lines expressing low (PLC/PRF/5) and high (HepG2) GPC-3 levels. In a subcutaneous HepG2 mouse model in immunodeficient NSG mice, GPC-3.CAR/sIL-15 Vδ1 T cells primarily accumulated and proliferated in the tumor, and a single dose efficiently controlled tumor growth without evidence of xenogeneic GvHD. Importantly, compared with GPC-3.CAR Vδ1 T cells lacking sIL-15, GPC-3.CAR/sIL-15 Vδ1 T cells displayed greater proliferation and resulted in enhanced therapeutic activity. CONCLUSIONS: Expanded Vδ1 T cells engineered with a GPC-3 CAR and sIL-15 represent a promising platform warranting further clinical evaluation as an off-the-shelf treatment of HCC and potentially other GPC-3-expressing solid tumors.
Subject(s)
Carcinoma, Hepatocellular/therapy , Glypicans/immunology , Immunotherapy, Adoptive/methods , Interleukin-15/immunology , Liver Neoplasms/therapy , Receptors, Antigen, T-Cell, gamma-delta/immunology , Receptors, Chimeric Antigen/immunology , Animals , Apoptosis , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/immunology , Carcinoma, Hepatocellular/pathology , Cell Proliferation , Female , Humans , Leukocytes, Mononuclear , Liver Neoplasms/genetics , Liver Neoplasms/immunology , Liver Neoplasms/pathology , Mice , Mice, Inbred NOD , Mice, SCID , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
Glypican 2 (GPC2) is a MYCN-regulated, differentially expressed cell-surface oncoprotein and target for immune-based therapies in neuroblastoma. Here, we build on GPC2's immunotherapeutic attributes by finding that it is also a highly expressed, MYCN-driven oncoprotein on small-cell lung cancers (SCLCs), with significantly enriched expression in both the SCLC and neuroblastoma stem cell compartment.By solving the crystal structure of the D3-GPC2-Fab/GPC2 complex at 3.3 Å resolution, we further illustrate that the GPC2-directed antibody-drug conjugate (ADC; D3-GPC2-PBD), that links a human GPC2 antibody (D3) to DNA-damaging pyrrolobenzodiazepine (PBD) dimers, binds a tumor-specific, conformation-dependent epitope of the core GPC2 extracellular domain. We then show that this ADC induces durable neuroblastoma and SCLC tumor regression via induction of DNA damage, apoptosis, and bystander cell killing, notably with no signs of ADC-induced in vivo toxicity. These studies provide preclinical data to support the clinical translation of ADCs targeting GPC2.
Subject(s)
Epitopes/chemistry , Epitopes/metabolism , Glypicans/immunology , Immunoconjugates/pharmacology , Lung Neoplasms/pathology , Neuroblastoma/pathology , Small Cell Lung Carcinoma/pathology , Animals , Bystander Effect/drug effects , Cell Compartmentation , Cell Death/drug effects , Cell Membrane/metabolism , DNA Damage , Female , Humans , Mice, Inbred C57BL , Mice, SCID , N-Myc Proto-Oncogene Protein/metabolism , Oncogene Proteins/metabolism , Protein ConformationABSTRACT
Although chimeric antigen receptor (CAR)-engineered T cells have shown great success in the treatment of B cell malignancies, this strategy has limited efficacy in patients with solid tumors. In mouse CAR-T cells, IL-7 and CCL19 expression have been demonstrated to improve T cell infiltration and CAR-T cell survival in mouse tumors. Therefore, in the current study, we engineered human CAR-T cells to secrete human IL-7 and CCL19 (7 × 19) and found that these 7 × 19 CAR-T cells showed enhanced capacities of expansion and migration in vitro. Furthermore, 7 × 19 CAR-T cells showed superior tumor suppression ability compared to conventional CAR-T cells in xenografts of hepatocellular carcinoma (HCC) cell lines, primary HCC tissue samples and pancreatic carcinoma (PC) cell lines. We then initiated a phase 1 clinical trial in advanced HCC/PC/ovarian carcinoma (OC) patients with glypican-3 (GPC3) or mesothelin (MSLN) expression. In a patient with advanced HCC, anti-GPC3-7 × 19 CAR-T treatment resulted in complete tumor disappearance 30 days post intratumor injection. In a patient with advanced PC, anti-MSLN-7 × 19 CAR-T treatment resulted in almost complete tumor disappearance 240 days post-intravenous infusion. Our results demonstrated that the incorporation of 7 × 19 into CAR-T cells significantly enhanced the antitumor activity against human solid tumor. Trial registration: NCT03198546. Registered 26 June 2017, https://clinicaltrials.gov/ct2/show/NCT03198546?term=NCT03198546&draw=2&rank=1.
Subject(s)
Chemokine CCL19/immunology , GPI-Linked Proteins/analysis , Glypicans/analysis , Immunotherapy, Adoptive/methods , Interleukin-7/immunology , Neoplasms/therapy , Animals , Carcinoma, Hepatocellular/immunology , Carcinoma, Hepatocellular/therapy , Female , GPI-Linked Proteins/immunology , Glypicans/immunology , Hep G2 Cells , Humans , Liver Neoplasms/immunology , Liver Neoplasms/therapy , Mesothelin , Mice , Neoplasms/immunology , Neoplasms/pathology , Ovarian Neoplasms/immunology , Ovarian Neoplasms/therapy , Pancreatic Neoplasms/immunology , Pancreatic Neoplasms/therapy , T-Lymphocytes/immunology , Treatment OutcomeABSTRACT
Targeting solid tumors must overcome several major obstacles, in particular, the identification of elusive tumor-specific antigens. Here, we devise a strategy to help identify tumor-specific epitopes. Glypican 2 (GPC2) is overexpressed in neuroblastoma. Using RNA sequencing (RNA-seq) analysis, we show that exon 3 and exons 7-10 of GPC2 are expressed in cancer but are minimally expressed in normal tissues. Accordingly, we discover a monoclonal antibody (CT3) that binds exons 3 and 10 and visualize the complex structure of CT3 and GPC2 by electron microscopy. The potential of this approach is exemplified by designing CT3-derived chimeric antigen receptor (CAR) T cells that regress neuroblastoma in mice. Genomic sequencing of T cells recovered from mice reveals the CAR integration sites that may contribute to CAR T cell proliferation and persistence. These studies demonstrate how RNA-seq data can be exploited to help identify tumor-associated exons that can be targeted by CAR T cell therapies.
Subject(s)
Antibodies, Monoclonal/pharmacology , Glypicans/genetics , Nervous System Neoplasms/therapy , Neuroblastoma/therapy , Receptors, Antigen, T-Cell/genetics , Receptors, Chimeric Antigen/genetics , Animals , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/metabolism , Cell Line, Tumor , Cell Proliferation , Exons , Female , Gene Expression , Glypicans/antagonists & inhibitors , Glypicans/chemistry , Glypicans/immunology , Humans , Immunotherapy, Adoptive/methods , Mice , Mice, Nude , Models, Molecular , Nervous System Neoplasms/genetics , Nervous System Neoplasms/mortality , Nervous System Neoplasms/pathology , Neuroblastoma/genetics , Neuroblastoma/mortality , Neuroblastoma/pathology , Protein Binding , Protein Conformation , Receptors, Antigen, T-Cell/immunology , Receptors, Chimeric Antigen/immunology , Sequence Analysis, RNA , Survival Analysis , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Tumor Burden , Xenograft Model Antitumor AssaysABSTRACT
Cholangiocarcinoma is a highly malignant cancer. Many patients need systemic chemotherapy to prevent tumor development and recurrence; however, their prognosis is poor due to the lack of effective therapy. Therefore, a new treatment option is urgently required. We recently identified glypican-1 (GPC1) as a novel cancer antigen of esophageal squamous cell carcinoma. We also demonstrated the efficacy and safety of GPC1-targeted ADC (GPC1-ADC) conjugating anti-GPC1 mAb possessing high internalization activity with monomethyl auristatin F (MMAF), which is a potent tubulin polymerizing inhibitor. In this study, we confirmed that GPC1 was highly expressed in cholangiocarcinoma cells and tissues. IHC analysis of 49 extrahepatic cholangiocarcinoma patient tumor specimens revealed high expression of GPC1 in 47% of patients. These patients demonstrated significantly poorer prognosis compared with the low-expression group in terms of disease-free survival and overall survival (P < 0.05). GPC1 was also expressed in tumor vessels of cholangiocarcinoma, but not on the vessels of nontumor tissues. MMAF-conjugated GPC1-ADC showed potent tumor growth inhibition against GPC1-positive cholangiocarcinoma cells in vitro and in vivo In a GPC1 knockout xenograft model, GPC1-ADC partially inhibited tumor growth. Vascular endothelial cells in tumor tissues of GPC1-negative xenograft mice expressed GPC1 and were arrested in the G2-M phase of cell cycle by GPC1-ADC. GPC1-ADC exhibits direct as well as indirect antitumor effects via inhibition of tumor angiogenesis. Our preclinical data highlight GPC1-ADC as a promising therapy for GPC1-positive cholangiocarcinoma.
Subject(s)
Bile Duct Neoplasms/drug therapy , Cholangiocarcinoma/drug therapy , Glypicans/antagonists & inhibitors , Immunoconjugates/pharmacology , Neovascularization, Pathologic/prevention & control , Animals , Apoptosis , Bile Duct Neoplasms/blood supply , Bile Duct Neoplasms/metabolism , Bile Duct Neoplasms/pathology , Cell Proliferation , Cholangiocarcinoma/blood supply , Cholangiocarcinoma/metabolism , Cholangiocarcinoma/pathology , Female , Glypicans/immunology , Humans , Mice , Mice, SCID , Neovascularization, Pathologic/immunology , Neovascularization, Pathologic/metabolism , Neovascularization, Pathologic/pathology , Prognosis , Survival Rate , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
BACKGROUND: Glypican-3 (GPC3), a cell surface glycoprotein that is pathologically highly expressed in hepatocellular carcinoma (HCC), is an attractive target for immunotherapies, including chimeric antigen receptor (CAR) T cells. The serum GPC3 is frequently elevated in HCC patients due to the shedding effect of cell surface GPC3. The shed GPC3 (sGPC3) is reported to block the function of cell-surface GPC3 as a negative regulator. Therefore, it would be worth investigating the potential influence of antigen shedding in anti-GPC3 CAR-T therapy for HCC. METHODS: In this study, we constructed two types of CAR-T cells targeting distinct epitopes of GPC3 to examine how sGPC3 influences the activation and cytotoxicity of CAR-T cells in vitro and in vivo by introducing sGPC3 positive patient serum or recombinant sGPC3 proteins into HCC cells or by using sGPC3-overexpressing HCC cell lines. RESULTS: Both humanized YP7 CAR-T cells and 32A9 CAR-T cells showed GPC3-specific antitumor functions in vitro and in vivo. The existence of sGPC3 significantly inhibited the release of cytokines and the cytotoxicity of anti-GPC3 CAR-T cells in vitro. In animal models, mice carrying Hep3B xenograft tumors expressing sGPC3 exhibited a worse response to the treatment with CAR-T cells under both a low and high tumor burden. sGPC3 bound to CAR-T cells but failed to induce the effective activation of CAR-T cells. Therefore, sGPC3 acted as dominant negative regulators when competed with cell surface GPC3 to bind anti-GPC3 CAR-T cells, leading to an inhibitory effect on CAR-T cells in HCC. CONCLUSIONS: We provide a proof-of-concept study demonstrating that GPC3 shedding might cause worse response to CAR-T cell treatment by competing with cell surface GPC3 for CAR-T cell binding, which revealed a new mechanism of tumor immune escape in HCC, providing a novel biomarker for patient enrolment in future clinical trials and/or treatments with GPC3-targeted CAR-T cells.
Subject(s)
Biomarkers, Tumor/antagonists & inhibitors , Carcinoma, Hepatocellular/therapy , Glypicans/antagonists & inhibitors , Immunotherapy, Adoptive , Liver Neoplasms/therapy , Receptors, Chimeric Antigen/genetics , T-Lymphocytes/transplantation , Animals , Binding, Competitive , Biomarkers, Tumor/blood , Biomarkers, Tumor/immunology , Carcinoma, Hepatocellular/blood , Carcinoma, Hepatocellular/immunology , Carcinoma, Hepatocellular/pathology , Cell Line, Tumor , Cytokines/metabolism , Cytotoxicity, Immunologic , Female , Glypicans/blood , Glypicans/immunology , Liver Neoplasms/blood , Liver Neoplasms/immunology , Liver Neoplasms/pathology , Lymphocyte Activation , Mice, Inbred BALB C , Mice, Inbred NOD , Mice, Nude , Proof of Concept Study , Protein Binding , Receptors, Chimeric Antigen/immunology , Receptors, Chimeric Antigen/metabolism , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Tumor Burden , Xenograft Model Antitumor AssaysABSTRACT
Aim: Two peptide cocktail vaccines using glypican-3, WD-repeat-containing protein up-regulated in hepatocellular carcinoma (HCC) and nei endonuclease VIII-like three epitopes were evaluated in advanced HCC in two Phase I studies. Patients & methods: Study 1 evaluated dose-limiting toxicities (DLTs) of peptides 1-3 (HLA-A24-restricted) and study 2 evaluated DLTs of peptides 1-6 (HLA-A24 or A02-restricted). Results: Overall, 18 and 14 patients were enrolled in studies 1 and 2, respectively. No DLTs were observed up to 7.1 mg of the vaccine cocktail. No complete response/partial response was observed. Stable disease was reported in nine and five patients with a disease control rate of 52.9% and 35.7% in studies 1 and 2, respectively. Conclusion: Both vaccines showed good tolerability and potential usefulness against HCC. Clinical trial registration: JapicCTI-121933; JapicCTI-142477.
Subject(s)
Cancer Vaccines/immunology , Carcinoma, Hepatocellular/drug therapy , Carrier Proteins/immunology , Cilia/immunology , Glypicans/immunology , Liver Neoplasms/drug therapy , N-Glycosyl Hydrolases/immunology , Adult , Aged , Antigens, Neoplasm/immunology , Cancer Vaccines/administration & dosage , Cancer Vaccines/adverse effects , Carcinoma, Hepatocellular/immunology , Carcinoma, Hepatocellular/pathology , Endpoint Determination , Epitopes/administration & dosage , Epitopes/adverse effects , Epitopes/immunology , Female , HLA-A Antigens/immunology , Humans , Liver Neoplasms/immunology , Liver Neoplasms/pathology , Male , Middle Aged , T-Lymphocytes, Cytotoxic/immunology , Treatment Outcome , Vaccines, Subunit/administration & dosage , Vaccines, Subunit/adverse effects , Vaccines, Subunit/immunologyABSTRACT
Glypican-3 (GPC3) is a tumor associated antigen expressed by hepatocellular carcinoma (HCC) cells. This preclinical study evaluated the efficacy of a theranostic platform using a GPC3-targeting antibody αGPC3 conjugated to zirconium-89 (89Zr) and yttrium-90 (90Y) to identify, treat, and assess treatment response in a murine model of HCC. A murine orthotopic xenograft model of HCC was generated. Animals were injected with 89Zr-labeled αGPC3 and imaged with a small-animal positron emission/computerized tomography (PET/CT) imaging system (immuno-PET) before and 30 days after radioimmunotherapy (RIT) with 90Y-labeled αGPC3. Serum alpha fetoprotein (AFP), a marker of tumor burden, was measured. Gross tumor volume (GTV) and SUVmax by immuno-PET was measured using fixed intensity threshold and manual segmentation methods. Immuno-PET GTV measurements reliably quantified tumor burden prior to RIT, strongly correlating with serum AFP (R2 = 0.90). Serum AFP was significantly lower 30 days after RIT in 90Y-αGPC3 treated animals compared to those untreated (p = 0.01) or treated with non-radiolabeled αGPC3 (p = 0.02). Immuno-PET GTV measurements strongly correlated with tumor burden after RIT (R2 = 0.87), and GTV of animals treated with 90Y-αGPC3 was lower than in animals who did not receive treatment or were treated with non-radiolabeled αGPC3, although this only trended toward statistical significance. A theranostic platform utilizing GPC3 targeted 89Zr and 90Y effectively imaged, treated, and assessed response after radioimmunotherapy in a GPC3-expressing HCC xenograft model.
Subject(s)
Carcinoma, Hepatocellular/therapy , Drug Delivery Systems/methods , Glypicans/immunology , Animals , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Cell Line, Tumor , Glypicans/metabolism , Humans , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Liver Neoplasms/therapy , Mice , Mice, Nude , Positron-Emission Tomography/methods , Precision Medicine/methods , Radioimmunotherapy , Radioisotopes/pharmacology , Radiopharmaceuticals , Tissue Distribution , Xenograft Model Antitumor Assays , Yttrium Radioisotopes/pharmacology , Zirconium/pharmacologyABSTRACT
Glypican-3 (GPC3) is a well-characterized hepatocellular carcinoma (HCC)-associated antigen, yet anti-GPC3 therapies have achieved only minimal clinical progress. CD47 is a ubiquitously expressed innate immune checkpoint that promotes evasion of tumors from immune surveillance. Given both the specific expression of GPC3 in HCC and the known phagocytosis inhibitory effect of CD47 in liver cancer, we hypothesized that a bispecific antibody (BsAb) that co-engages with GPC3 and CD47 may offer excellent antitumor efficacy with minimal toxicity. Here, we generated a novel BsAb: GPC3/CD47 biAb. With the use of both in vitro and in vivo assays, we found that GPC3/CD47 biAb exerts strong antitumor activity preferentially against dual antigen-expressing tumor cells. In hCD47/human signal regulatory protein alpha (hCD47/hSIRPα) humanized mice, GPC3/CD47 biAb had an extended serum half-life without causing systemic toxicity. Importantly, GPC3/CD47 biAb induced enhanced Fc-mediated effector functions to dual antigen-expressing HCC cells in vitro, and both macrophages and neutrophils are required for its strong efficacy against xenograft HCC tumors. Notably, GPC3/CD47 biAb outperformed monotherapies and a combination therapy with anti-CD47 and anti-GPC3 monoclonal antibodies (mAbs) in a xenograft HCC model. Our study illustrates a strategy for improving HCC treatment by boosting innate immune responses and presents new insights to inform antibody design for the future development of innovative immune therapies.
Subject(s)
CD47 Antigen/genetics , Carcinoma, Hepatocellular/drug therapy , Glypicans/genetics , Liver Neoplasms/drug therapy , Animals , Antibodies, Bispecific/pharmacology , Antibodies, Monoclonal/pharmacology , CD47 Antigen/antagonists & inhibitors , CD47 Antigen/immunology , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/immunology , Carcinoma, Hepatocellular/pathology , Cell Line, Tumor , Glypicans/antagonists & inhibitors , Glypicans/immunology , Humans , Immunity, Innate/drug effects , Immunity, Innate/immunology , Liver Neoplasms/genetics , Liver Neoplasms/immunology , Liver Neoplasms/pathology , Mice , Xenograft Model Antitumor AssaysABSTRACT
Glypican-3 (GPC3) is a highly specific diagnostic marker for hepatocellular carcinoma (HCC) diagnosis and a potential target in HCC therapy. Nanobodies (Nbs) are promising targeting molecules due to their high specificity and strong affinities to antigens, high stability, deep tissue penetration, and low immunogenicity. In this study, we isolated Nbs against GPC3 marker protein from a synthetic Nb library by phage display. To characterize these Nbs, we performed enzyme-linked immunosorbent assay, immunoprecipitation assay, and immunofluorescent assay to demonstrate that four (G8, G10, G11, and G64) of them bound specifically to recombinant as well as endogenous GPC3, and epitope mapping showed they all bound to N-terminal subunit of GPC3. Furthermore, we found that G64 exhibited high protein stability and GPC3 binding activity in serum at 37â for at least 96 h, and G64 did not affect the proliferation of HEK293T cells and HCC cell line HepG2. Our study provides four anti-GPC3 Nbs as promising targeting molecules for HCC diagnostic and therapeutic drugs.