ImmunoPET as Stoichiometric Sensor for Glypican-3 in Models of Hepatocellular Carcinoma

BackgroundHepatocellular carcinoma (HCC) is the fifth most common cancer worldwide. While conventional imaging approaches like ultrasound, CT, and MRI play critical roles in the diagnosis and surveillance of HCC, improved methods for detection and assessment of treatment response are needed. One promising approach is the use of radiolabeled antibodies for positron emission tomography (immunoPET) imaging. Glypican-3 (GPC3) is a proteoglycan that is highly expressed in the majority of HCC tumors. GPC3-specific antibodies are used to diagnose HCC histopathologically, and have been proposed as a treatment of HCC. Here, we design, synthesize and demonstrate that our humanized immunoPET agent, [89Zr]Zr-DFO-TAB-H14, can stoichiometrically bind to models of human liver cancer with varied GPC3 expression. Methods: The GPC3-specific monoclonal humanized IgG1, TAB-H14, was used as a scaffold for engineering our immunoPET agent. Fluorescent and deferroxamine (DFO) chelate conjugates of TAB-H14 were characterized using mass spectrometry. Binding affinity of TAB-H14 and conjugates for GPC3 was determined in cell-free biolayer interferometry, and cell-based radioimmunoassays. GPC3-expression was assessed by flow cytometry and immunofluorescence using commercially available anti-GPC3 antibodies and TAB-H14 in GPC3−(A431) and GPC3+ cell lines including an engineered line (A431-GPC3+, G1) and liver cancer lines (HepG2, Hep3B, and Huh7). DFO-TAB-H14, was radiolabeled with Zr-89. Mice were subcutaneously engrafted with the aforementioned cell lines and in vivo target engagement of the immunoPET agent [89Zr]Zr-DFO-TAB-H14 was determined using PET/CT, quantitative biodistribution, and autoradiography. Results: TAB-H14 demonstrated subnanomolar to nanomolar affinity for human GPC3. Fluorescently tagged TAB-H14 was able to bind to GPC3 on cell membranes of GPC3-expressing lines by flow cytometry. These results were confirmed by immunofluorescence staining of A431, G1 HepG2, Hep3B, and Huh7 tumor sections. ImmunoPET imaging with [89Zr]Zr-DFO-TAB-H14 showed stoichiometric tumor uptake corresponding to the cell surface expression levels. Autoradiography and immunostaining confirmed in vivo findings. Conclusion: We systematically demonstrate that the humanized immnoPET agent [89Zr]Zr-DFO-TAB-H14 specifically and stoichiometrically binds to GPC3 in several models of human liver cancer, serving as a promising in vivo GPC3 sensor. This agent may provide utility in HCC diagnosis and surveillance, and the selection of candidates for GPC3-directed therapies.

Intratumoral reciprocal expression of monocarboxylate transporter 4 and glypican-3 in hepatocellular carcinomas

Objective We previously reported the identification of monocarboxylate transporter 4 (MCT4) and glypican-3 (GPC3) as prognostic factors for hepatocellular carcinoma (HCC), which are now considered significant poor prognostic factors for the disease. This study aimed to clarify the detailed interaction of these two factors in HCC to improve our understanding of aggressive HCC phenotypes. A total of 225 Japanese patients with HCC from our previous study were subjected to immunohistochemical analyses. Results The number of MCT4-positive (MCT4+) HCC cases was 47 (21%), and most MCT4+ HCC showed high GPC3 expression (94%, 44/47 cases). In 44 MCT4+/GPC3+ HCC cases, intratumoral heterogeneity of GPC3 or MCT4 expression was further evaluated. We observed reciprocal (inverse), synergistic, mixed reciprocal and synergistic, or irrelevant interaction of MCT4 and GPC3 expression in 29 (66%), 5 (11%), 1 (2%), and 9 cases (21%), respectively. The cases exhibiting reciprocal expression of both markers tended to have cirrhosis without a history of neoadjuvant therapy. In summary, although MCT4+ HCC cases are mostly GPC3+, intratumoral expression patterns of MCT4 and GPC3 are frequently reciprocal each other, suggesting that dual targeting of MCT4 and GPC3 may achieve a better antitumor effect for MCT4+ HCC cases.

Intratumoral reciprocal expression of monocarboxylate transporter 4 and glypican-3 in hepatocellular carcinomas

Objective: We previously reported the identification of monocarboxylate transporter 4 (MCT4) and glypican-3 (GPC3) as prognostic factors for hepatocellular carcinoma (HCC), which are now considered significant poor prognostic factors for the disease. This study aimed to clarify the detailed interaction of these two factors in HCC t o improve our understanding of aggressive HCC phenotype s. A total of 225 Japanese patients with HCC from our previous study were subjected to immunohistochemical analyses. Results: The number of MCT4-positive (MCT4+) HCC cases was 47 (21%), and most MCT4+ HCC showed high GPC3 expression (94%, 44/47 cases). In 44 MCT4+/GPC3+ HCC cases, intratumoral heterogeneity of GPC3 or MCT4 expression was further evaluated. We observed reciprocal (inverse), synergistic, mixed reciprocal and synergistic, or irrelevant interaction of MCT4 and GPC3 expression in 29 (66%), 5 (11%), 1 (2%), and 9 cases (21%), respectively. The cases exhibiting reciprocal expression of both markers tended to have cirrhosis without a history of neoadjuvant therapy. In summary, although MCT4+ HCC cases are mostly GPC3+, intratumoral expression patterns of MCT4 and GPC3 are frequently reciprocal each other, suggesting that dual targeting of MCT4 and GPC3 may achieve a better antitumor effect for MCT4+ HCC cases.

Intratumoral reciprocal expression of monocarboxylate transporter 4 and glypican-3 in hepatocellular carcinomas

Objective: We previously reported the identification of monocarboxylate transporter 4 (MCT4) and glypican-3 (GPC3) as prognostic factors for hepatocellular carcinoma (HCC), which are now considered significant poor prognostic factors for the disease. This study aimed to clarify the detailed interaction of these two factors in HCC t o improve our understanding of aggressive HCC phenotype s. A total of 225 Japanese patients with HCC from our previous study were subjected to immunohistochemical analyses. Results: The number of MCT4-positive (MCT4+) HCC cases was 47 (21%), and most MCT4+ HCC showed high GPC3 expression (94%, 44/47 cases). In 44 MCT4+/GPC3+ HCC cases, intratumoral heterogeneity of GPC3 or MCT4 expression was further evaluated. We observed reciprocal (inverse), synergistic, mixed reciprocal and synergistic, or irrelevant interaction of MCT4 and GPC3 expression in 29 (66%), 5 (11%), 1 (2%), and 9 cases (21%), respectively. The cases exhibiting reciprocal expression of both markers tended to have cirrhosis without a history of neoadjuvant therapy. In summary, although MCT4+ HCC cases are mostly GPC3+, intratumoral expression patterns of MCT4 and GPC3 are frequently reciprocal each other, suggesting that dual targeting of MCT4 and GPC3 may achieve a better antitumor effect for MCT4+ HCC cases.

Expression of Glypican 3 Is an Independent Prognostic Biomarker in Primary Gastro-Esophageal Adenocarcinoma and Corresponding Serum Exosomes

Exosomes are nano-sized membranous vesicles of endosomal origin that carry nucleic acids, lipids and proteins. The cargo of exosomes is cell origin specific and the release of these exosomes and uptake by an acceptor cell is seen as a vital element of cell-cell communication. Here, we sought to investigate the diagnostic and prognostic value of the expression of glypican 3 (GPC3) on primary gastro-esophageal adenocarcinoma (GEA) tissue (tGPC3) and corresponding serum exosomes (eGPC3). Circulating exosomes were extracted from serum samples of 49 patients with GEA and 56 controls. Extracted exosomes were subjected to flow cytometry for the expression of eGPC3 and GPC3 expression on primary GEA tissue samples was determined by immunohistochemistry and correlated to clinicopathological parameters. We found decreased eGPC3 levels in GEA patients compared to healthy controls (p < 0.0001) and high tGPC3 expression. This was significantly associated with poor overall survival (high vs. low eGPC3: 87.40 vs. 60.93 months, p = 0.041, high vs. low tGPC3: 58.03 vs. 84.70 months, p = 0.044). Cox regressional analysis confirmed tGPC3 as an independent prognostic biomarker for GEA (p = 0.02) and tGPC3 expression was validated in two independent cohorts. Our findings demonstrate that eGPC3 and tGPC3 can be used as potential diagnostic and prognostic biomarkers for GEA.

Glypican 3 (GPC3) expression in a lethal subgroup of refractory cisplatin-resistant testicular germ cell tumor (TGCT).

559 Background: The oncofetal protein GPC3 has been tested as a therapy target. Early phase clinical trials established the safety and efficacy of anti-GPC3 chimeric antigen receptor modified T cells in patients with refractory or relapsed GPC3+ hepatocellular carcinoma. In TGCT, GPC3 is preferentially expressed at presentation in certain germ cell histological phenotypes (yolk sac tumor [YST], choriocarcinoma), but not in others (embryonal carcinoma, teratoma, seminoma). The aim of this study is to evaluate GPC3 expression in a lethal subgroup of refractory cisplatin-resistant TGCT. Methods: We retrospectively evaluated 615 patients diagnosed with TGCT from January 2000 to December 2010. We identified patients who died from their TGCT. The histologic makeup of primary tumors, next-generation sequencing data and the clinical course of disease were determined for each patient. We prospectively evaluated GPC3 expression by immunohistochemistry (IHC) using a mouse monoclonal antibody (YP7), on tumor tissue from these patients with lethal, heavily pretreated, cisplatin-resistant TGCT. Results: We identified seven patients with fatal and cisplatin-resistant TGCT, with a median age of 30 (28-50) years and a median number of prior therapies of 5 (2-8), including 3 patients who received high-dose chemotherapy with autologous stem-cell transplant. The prospectively collected samples were from different sites of metastasis: lymph nodes (n = 4), peritoneal carcinomatosis (n = 2), and lung (n = 1). The tissue histology comprised YST (n = 2), YST + choriocarcinoma (n = 1), YST + teratoma (n = 1), teratoma (n = 1), and somatic transformation (n = 2). No consistent genetic aberration was detected. The viable germ cell tumor components comprising YST and choriocarcinoma (n = 4) stained strongly positive in a membranous distribution for GPC3. While samples with teratoma and somatic transformation histology (n = 3) did not stain for GPC3. Conclusions: Potentially lethal, heavily pretreated, cisplatin-resistant viable germ cell tumors have enhanced expression of GPC3. Targeted therapy directed against GPC3 could benefit patients harboring such tumors and further investigation is of value.

An anti–glypican 3/CD3 bispecific T cell–redirecting antibody for treatment of solid tumors

Cancer care is being revolutionized by immunotherapies such as immune checkpoint inhibitors, engineered T cell transfer, and cell vaccines. The bispecific T cell–redirecting antibody (TRAB) is one such promising immunotherapy, which can redirect T cells to tumor cells by engaging CD3 on a T cell and an antigen on a tumor cell. Because T cells can be redirected to tumor cells regardless of the specificity of T cell receptors, TRAB is considered efficacious for less immunogenic tumors lacking enough neoantigens. Its clinical efficacy has been exemplified by blinatumomab, a bispecific T cell engager targeting CD19 and CD3, which has shown marked clinical responses against hematological malignancies. However, the success of TRAB in solid tumors has been hampered by the lack of a target molecule with sufficient tumor selectivity to avoid “on-target off-tumor” toxicity. Glypican 3 (GPC3) is a highly tumor-specific antigen that is expressed during fetal development but is strictly suppressed in normal adult tissues. We developed ERY974, a whole humanized immunoglobulin G–structured TRAB harboring a common light chain, which bispecifically binds to GPC3 and CD3. Using a mouse model with reconstituted human immune cells, we revealed that ERY974 is highly effective in killing various types of tumors that have GPC3 expression comparable to that in clinical tumors. ERY974 also induced a robust antitumor efficacy even against tumors with nonimmunogenic features, which are difficult to treat by inhibiting immune checkpoints such as PD-1 (programmed cell death protein–1) and CTLA-4 (cytotoxic T lymphocyte–associated protein–4). Immune monitoring revealed that ERY974 converted the poorly inflamed tumor microenvironment to a highly inflamed microenvironment. Toxicology studies in cynomolgus monkeys showed transient cytokine elevation, but this was manageable and reversible. No organ toxicity was evident. These data provide a rationale for clinical testing of ERY974 for the treatment of patients with GPC3-positive solid tumors.