Gastric Cancer Xenograft Model Research Articles

Development of a bioengineered Erwinia chrysanthemi asparaginase to enhance its anti-solid tumor potential for treating gastric cancer

Asparaginase has been traditionally applied for only treating acute lymphoblastic leukemia due to its ability to deplete asparagine. However, its ultimate anticancer potential for treating solid tumors has not yet been unleashed. In this study, we bioengineered Erwinia chrysanthemi asparaginase (ErWT), one of the US Food and Drug Administration-approved types of amino acid depleting enzymes, to achieve double amino acid depletions for treating a solid tumor. We constructed a fusion protein by joining an albumin binding domain (ABD) to ErWT via a linker (GGGGS)5 to achieve ABD-ErS5. The ABD could bind to serum albumin to form an albumin-ABD-ErS5 complex, which could avoid renal clearance and escape from anti-drug antibodies, resulting in a remarkably prolonged elimination half-life of ABD-ErS5. Meanwhile, ABD-ErS5 did not only deplete asparagine but also glutamine for ∼2 weeks. A biweekly administration of ABD-ErS5 (1.5 mg/kg) significantly suppressed tumor growth in an MKN-45 gastric cancer xenograft model, demonstrating a novel approach for treating solid tumor depleting asparagine and glutamine. Multiple administrations of ABD-ErS5 did not cause any noticeable histopathological abnormalities of key organs, suggesting the absence of acute toxicity to mice. Our results suggest ABD-ErS5 is a potential therapeutic candidate for treating gastric cancer.

CLDN18.2 BiTE Engages Effector and Regulatory T Cells for Antitumor Immune Response in Preclinical Models of Pancreatic Cancer

BiTE (bispecific T-cell engager) immune therapy has demonstrated clinical activity in multiple tumor indications, but its influence in the tumor microenvironment remains unclear. CLDN18.2 is overexpressed in solid tumors including gastric cancer (GC) and pancreatic ductal adenocarcinoma (PDAC), both of which are characterized by the presence of immunosuppressive cells, including regulatory T cells (Tregs) and few effector T cells (Teffs). We evaluated the activity of AMG 910, a CLDN18.2-targeted half-life extended (HLE) BiTE molecule, in GC and PDAC preclinical models and cocultured Tregs and Teffs in the presence of CLDN18.2-HLE-BiTE. AMG 910 induced potent, specific cytotoxicity in GC and PDAC cell lines. In GSU and SNU-620 GC xenograft models, AMG 910 engaged human CD3+ T cells with tumor cells, resulting in significant antitumor activity. AMG 910 monotherapy, in combination with a programmed death-1 (PD-1) inhibitor, suppressed tumor growth and enhanced survival in an orthotopic Panc4.14 PDAC model. Moreover, Treg infusion enhanced the antitumor efficacy of AMG 910 in the Panc4.14 model. In syngeneic KPC models of PDAC, treatment with a mouse surrogate CLDN18.2-HLE-BiTE (muCLDN18.2-HLE-BiTE) or the combination with an anti-PD-1 antibody significantly inhibited tumor growth. Tregs isolated from mice bearing KPC tumors that were treated with muCLDN18.2-HLE-BiTE showed decreased T cell suppressive activity and enhanced Teff cytotoxic activity, associated with increased production of type I cytokines and expression of Teff gene signatures. Our data suggest that BiTE molecule treatment converts Treg function from immunosuppressive to immune enhancing, leading to antitumor activity in immunologically "cold" tumors.

Abstract CT156: MCLA-158 (petosemtamab), an IgG1 bispecific antibody targeting EGFR and LGR5, in advanced gastric/esophageal adenocarcinoma (GEA)

Abstract The epidermal growth factor receptor (EGFR) is a known driver of cancer growth and the leucine-rich, repeat-containing, G-protein coupled receptor 5 (LGR5) is a transmembrane receptor expressed on cancer stem cells. Petosemtamab is a human, common light chain, IgG1 bispecific antibody with enhanced ADCC activity targeting EGFR and LGR5, which has shown potent antitumor activity in patient (pt)-derived xenograft models of gastric and esophageal cancer. The dose escalation part of an ongoing phase 1/2 study is completed (JCO 2021:39.3 Sup 62). In the expansion part, petosemtamab is being investigated at the RP2D (1500 mg Q2W, 4-week cycle) in pts with selected advanced solid tumors, including GEA. Primary objective (expansion): investigator-assessed ORR per RECIST 1.1. Secondary objectives: ORR, DOR, PFS (per investigator and central review), OS, and safety and tolerability. Key eligibility criteria: advanced/metastatic GEA, prior exposure to ≥2 lines of standard therapy, ECOG PS 0-1, measurable disease (RECIST 1.1), available baseline tumor biopsy. At the data cutoff date of 24 October 2022, 14 GEA pts were treated at the RP2D. Median age was 63 years (range 40-80), ECOG PS 0/1: 3/11 pts, and 79% of pts were male. Primary tumor locations were esophagus (57%), stomach (36%), and gastroesophageal junction (7%), and all were adenocarcinoma histology. Pts received a median of 3 lines of prior systemic therapy (range 1-4) including platinum-based chemotherapy (100%) and checkpoint inhibitors (14%). 3/14 pts had EGFR overexpression (H score ≥200). A median of 2 treatment cycles (range 1-24) was administered, with 1 pt continuing therapy at the cutoff. Among the 14 pts evaluable for efficacy (≥2 cycles and ≥1 postbaseline scan, or early progression) limited activity was seen, however 1 pt with tumor EGFR protein overexpression and gene copy number amplification (CNA) showed a confirmed sustained PR (67% tumor reduction; response ongoing after 24 cycles) and 3 pts had SD (1 with EGFR overexpression and gene CNA; 2 not evaluable for IHC), with tumor reductions of 2%, 17%, and 40%. Among 78 pts treated at the RP2D (escalation and all expansion cohorts; cutoff 28 November 2022), the most frequent AEs regardless of causality (all grades/G3-4) were rash (33%/0%), hypotension (26%/6%), dyspnea (26%/4%), nausea (26%/1%), dermatitis acneiform (24%/1%), blood magnesium decreased (19%/5%), erythema (19%/0%), diarrhea (19%/0%); IRRs (composite term) were reported in 74%/21% of pts, mostly at the first infusion, and all resolved. 5 pts (6%) discontinued treatment due to IRRs on Day 1. 1 esophageal cancer pt died due to unrelated G5 GI bleeding. Petosemtamab demonstrated promising clinical efficacy among patients with pretreated GEA having EGFR gene amplification and/or overexpression, with a manageable safety profile. Citation Format: Marc Díez García, Antoine Hollebecque, Rocio Garcia-Carbonero, Christiane Jungels, Elisabeth Smyth, Shumei Kato, Guillem Argilés, Carlos Gomez Martin, Marina Magin, Yu-Ming Shen, Renée de Leeuw, Mohamed Bekradda, Eduardo Pennella, Ernesto Wasserman, Viktoriya Stalbovskaya, Jeroen Lammerts van Bueren, Aline Engbers, Andrew Joe, Josep Tabernero. MCLA-158 (petosemtamab), an IgG1 bispecific antibody targeting EGFR and LGR5, in advanced gastric/esophageal adenocarcinoma (GEA) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT156.

Abstract 2633: Zanidatamab zovodotin (ZW49) induces hallmarks of immunogenic cell death and is active in patient-derived xenograft models of gastric cancer

Abstract Zanidatamab zovodotin (ZW49) is an antibody drug conjugate (ADC) comprised of a bispecific anti-HER2 IgG1 antibody (ZW25, zanidatamab) conjugated to a microtubule inhibitor auristatin payload (ZD02044) via a protease cleavable linker. Clinical studies of ZW49 in HER2-expressing advanced solid tumors are currently underway (NCT03821233). We have previously presented data illustrating the rapid internalization of ZW49 and the anti-tumor activity of ZW49 in multiple patient-derived xenograft models originating from breast carcinomas. Here we present ZW49’s ability to induce in vitro hallmarks of immunogenic cell death (ICD) including increased calreticulin exposure, increased high mobility group box-1 (HMGB1) exposure, and increased extracellular ATP secretion in a HER2 dependent manner. Additionally, we present the results from a study evaluating the anti-tumor activity of ZW49 in a panel of patient-derived xenografts of gastric cancer. HER2-positive SK-BR-3 and HER2-negative MDA-MB-468 tumor cell lines were treated with ZW49 and cell surface calreticulin was assessed by flow cytometry. ZW49 treatment of SKBR-3 cells induced a significantly higher percentage of cells staining positive for cell surface calreticulin compared to untreated cells. In contrast, ZW49 treatment of MDA-MB-468 cells resulted in no significant difference in the percentage of cells staining positive for calreticulin compared to untreated cells. ZW49 treated HER2-positive cancer cell lines SK-BR-3 and SK-OV-3 induced higher levels of extracellular ATP compared to untreated cells. In the HER2-negative cell line MDA-MB-468, treatment with ZW49 did not alter levels of extracellular ATP compared to untreated cells. ZW49 induced higher levels of HMGB1 in the HER2-positive cancer cell lines SKBR3 and N87 compared to untreated cells, whereas in the HER2-negative cell line MDA-MB-468, ZW49 induced similar levels of HMGB1 to untreated cells. Anti-tumor activity was observed in 5/7 (71%) PDX models of gastric cancer after a single i.v. dose of 6 mg/kg, including in models with moderate and weak HER2 expression. The strong anti-tumor activity of ZW-49 in vivo, together with its ability to induce ICD and potential adaptive immune responses, support ZW49 as a promising ADC for the treatment of HER2-expressing cancers warranting further investigation, including potential combination with checkpoint inhibitors. Citation Format: Stuart D. Barnscher, Andrea Hernández Rojas, Kevin J. Hamblett, Nichole Escalante. Zanidatamab zovodotin (ZW49) induces hallmarks of immunogenic cell death and is active in patient-derived xenograft models of gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2633.

Abstract 1540: A click chemistry-activated auristatin protodrug (TCO-MMAE) demonstrates potency and safety with antibody and intratumoral tumor-targeting agents

Abstract Click chemistry encompasses chemical reactions that are highly efficient, rapid and proceed in complex environments. The 2022 Nobel prize in Chemistry was awarded to Sharpless, Meldal and Bertozzi in recognition of the transformative effect click chemistry has had in research and drug development. Using click chemistry to precisely control the activation of drugs at tumors, Shasqi has developed the Click Activated Protodrugs Against Cancer (CAPAC™) platform. In essence, a click chemistry activator is localized to tumors, which subsequently activates protodrug(s) bearing the companion click chemistry reaction partner. Shasqi is pioneering the use of click chemistry in the clinic. The lead candidate SQ3370 consists of an intratumorally (IT) injected tetrazine (Tz)-modified biopolymer and an intravenously (IV) administered trans-cyclooctene (TCO)-modified doxorubicin (Dox) protodrug. SQ3370 provides enhanced safety and efficacy in preclinical models compared to conventional Dox and is being evaluated in a Phase 2a study in soft tissue sarcomas (NCT04106492). We will present our research on an MMAE-based protodrug (TCO-MMAE), which is stable in plasma with dramatically attenuated activity in vitro and in vivo without the Tz activator. When treated with an activator in vitro, the potent cytotoxic activity of MMAE is restored. When paired with the Tz-biopolymer (IT), testing in the Karpas299 model of human non-Hodgkin’s lymphoma led to 6 of 8 (75%) complete responses in the test group. Treatment with TCO-MMAE (IV) + Tz-biopolymer (IT) also elicited significant tumor growth inhibition (p-value <0.01) in the RENCA model of murine renal adenocarcinoma. Modest body weight loss and transient cytopenia support the tolerability of the treatment. To enable systemic delivery of the click chemistry activator, we are developing a series of antigen-targeted conjugates to pair with the TCO-MMAE protodrug. We designed and generated a Fab-Tz conjugate that binds HER2. The conjugate had an average of 2.2 Tz per Fab and efficiently bound HER2-positive NCI-N87 cells. In a xenograft model of gastric cancer (NCI-N87), the Fab-Tz was dosed IV, followed by the TCO-MMAE protodrug (IV). Treatment with HER2 Fab-Tz + TCO-MMAE induced significant tumor regression compared to vehicle while a negative control Fab-Tz + TCO-MMAE treatment did not (p-value <0.05). To date, we have demonstrated the versatility and modularity of the CAPAC platform. Anti-tumor activity was observed in multiple preclinical models for the TCO-MMAE protodrug in combination with both biopolymer and Fab targeting agents. These results highlight the power of click chemistry to activate a protodrug at the tumor site, independent of tumor biology, the molecular format or delivery method of the targeting agent. Citation Format: Jesse M. McFarland, Masa Aleckovic, George Coricor, Sangeetha Srinivasan, Leslie Priddy, Matthew Tso, Tri-Hung Nguyen, John Lee, José M. Mejía Oneto. A click chemistry-activated auristatin protodrug (TCO-MMAE) demonstrates potency and safety with antibody and intratumoral tumor-targeting agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1540.

Abstract 2904: CAT-179, an allogeneic NK cell product expressing HER2-CAR, IL-15 and TGFβ dominant negative receptor, durably regresses HER2-expressing xenograft tumors in mice

Abstract While chimeric antigen receptor (CAR)-engineered immune cell therapies have been at the forefront of cancer immunotherapy for hematological malignancies, patients with solid tumors have yet to benefit from such therapies. Engineered, off-the-shelf, allogeneic natural killer (NK) cells are particularly attractive as cell therapies for solid tumors given their clinical safety, efficacy, and multimodal recognition of tumor cells. We describe here the pre-clinical pharmacokinetics, efficacy, biodistribution and safety of CAT-179, a novel allogeneic, cryopreserved CAR-NK cell therapy, in naïve animals as well as multiple xenograft models of HER2-amplified ovarian and gastric cancer. CAT-179 cells are engineered to express an optimized HER2-directed CAR to effectively target tumor cells, a transforming growth factor b (TGFβ) dominant negative receptor (DNR) to protect against TGFβ-mediated immunosuppression, and interleukin-15 (IL-15) to enhance NK cell persistence. A single intravenous (IV) dose of CAT-179 resulted in IL-15-dependent (p<0.0001) expansion and persistence of CAT-179 cells for at least 140 days in NOD-scid IL2Rgnull (NSG) mice, peaking at approximately 15,000 cells/µL of blood (by day 60). Persisting CAT-179 NK cell levels at day 60 were functionally active and significantly reduced tumor burden when challenged intraperitoneally (IP) with HER2+ SKOV3-luc tumor cells. No significant changes in body weight or condition were observed during this study. The therapeutic anti-tumor activity of CAT-179 against established tumors was assayed using two different xenograft models. In the first model, NSG mice were given an IP dose of 1 million SKOV3-luc tumor cells (derived from ovarian cancer) followed by either 4 million CAT-179 or control NK cells on days 4, 11 and 18. CAT-179 dosed animals showed a rapid and sustained 95% decline in tumor burden (p<0.0001) and a significant extension in survival relative to animals dosed with control NK cells (p<0.0001). In the second model, 1 million HER2+ N87 cancer cells (derived from gastric cancer) were implanted subcutaneously into the right flank of NSG mice. When tumors reached 70mm3, a single IV dose of 2M CAT-179 or control NK cells was administered. CAT-179 dosed animals showed a 96% durable tumor regression and significant survival benefit relative to animals dosed with control NK cells (p<0.0001). Efficacy strongly correlated with the circulating levels of CAT-179, which significantly infiltrated the tumor xenograft. Our pre-clinical results demonstrate the potential of CAT-179 as a novel, durable, and off-the-shelf cell therapy to overcome the challenges associated with solid tumors and provide quantitative insights into pharmacokinetics, pharmacodynamics and anti-tumor activity of engineered NK cells expressing CAR, TGFb DNR and IL-15. Citation Format: Bashar Hamza, Angela Nunez, Marilyn Marques, Alexia Barandiaran, Henry Moreno, Finola Moore, Meghan Walsh, Eugene Choi, Kisha Pradhan, Krista Daniel, Jennifer Johnson, Charlotte Franco, Andres Alvarez, Karl Malakian, Keith H. Wong, Joseph Gold, Vipin Suri, Dominic Picarella. CAT-179, an allogeneic NK cell product expressing HER2-CAR, IL-15 and TGFβ dominant negative receptor, durably regresses HER2-expressing xenograft tumors in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2904.

Inhibition of gastric cancer by a new controlled releasing compound of TLR-7/8 agonist via immune activation in a gastric cancer xenograft mice model.

420 Background: TLR-7/8 agonist has been suggested to be an effective immunotherapeutic agent, possible systemic side effect has forbid its clinical usage. A new controlled-releasing compound of TLR-7/8 agonist was developed to overcome the limitations, and this study aimed to evaluate efficacy and safety of this new compound in xenograft mouse model using cell-lines derived from triple conditional (Tcon) mouse which develop gastric cancer spontaneously. Methods: A cell line was established by tumor tissue in Tcon mouse which was developed by activation of Kras and suppression of E-cadherin and P53. Tcon cell line was evaluated its growth rate, sensitivity to anticancer agents and in vivo tumorigenicity. Tcon cell line was subcutaneously injected to the flank of the syngeneic mice, and 5-FU and/or TLR-7/8 agonist were administered into 4 groups of mice (control group, 5-FU alone, TLR-7/8 agonist alone, and combination of 5-FU and TLR-7/8). 5-FU was administered i.p(intra-peritoneal) in once a week and TLR-7/8 agonist was injected i.t(intra-tumoral) 3 times a week. Tumor size and mouse weight were measured for drug efficacy and safety. Immune profile of the tumor was analyzed by fluorescence-activated cell sorting. Results: Tcon cell-line show a rapid growth with 24 hours of doubling time, and good in vivo tumorigenicity in syngeneic mice. Cell-line showed moderate sensitivity to 5-FU and paclitaxel but resistance to oxaliplatin. Tumors in 5-FU alone group showed slight decrease in size compared with that in control group, However, TLR-7/8 agonist alone and combination significantly inhibited tumor growth. Weight loss or any significant side effect was not observed in any group. In immune profiling, TLR-7/8 mono or combination group showed increased levels of infiltrating CD4, CD8 T cells, and recruited NK cell as well as significantly increase M1/M2 ratio of macrophages compared to 5-FU alone or control group. Conclusions: A new controlled releasing system of TLR-7/8 agonist with or without 5-FU showed a excellent tumor inhibition efficacy with low toxicity in gastric cancer xenograft model. The effect seemed to be through multiple immune activations including cell-mediated immune response, M1 macrophage polarization as well as innate NK cells.

The organic arsenical-derived thioredoxin and glutathione system inhibitor ACZ2 induces apoptosis and autophagy in gastric cancer via ROS-dependent ER stress

Developing novel drugs for gastric cancer (GC) is greatly needed, and a reactive oxygen species (ROS)-modulating strategy has been demonstrated to be useful for cancer treatment. However, no organic arsenical-derived ROS-modulating drug has been developed in GC. Here, we constructed ACZ2 and investigated its efficacy and potential mechanism for GC in vitro and in vivo. Our data showed that ACZ2 could inhibit GC proliferation and cause G2/M phase arrest. Moreover, ACZ2 induced ROS accumulation by depleting glutathione (GSH) and TrxR1, triggering a subsequent ER stress response by activating the PERK/EIF2/ATF4/CHOP signalling pathways, which is a crucial step for ACZ2-mediated apoptosis and autophagy. Vitally, ROS scavenger (NAC) and ER stress inhibitor (4PBA) reversed ACZ2/ROS/ER stress-mediated apoptosis and autophagy. Our in vivo results clearly demonstrated that ACZ2 suppressed tumour growth in a GC xenograft model. Collectively, our data indicated that ACZ2 is a potential agent against GC.

Interleukin-15 and chemokine ligand 19 enhance cytotoxic effects of chimeric antigen receptor T cells using zebrafish xenograft model of gastric cancer.

Chimeric antigen receptor (CAR) T cells have been proven effective for the treatment of B-cell-mediated malignancies. Currently, the development of efficient tools that supply CAR T cells for the treatment of other malignancies would have great impact. In this study, interleukin (IL)-15 and C-C motif chemokine ligand 19 (CCL19) were introduced into natural killer group 2D (NKG2D)-based CARs to generate 15×19 CAR T cells, which remarkably increased T-cell expansion and promoted the production of central memory T (Tcm) cells. 15×19 CAR T cells showed greater cytotoxicity to gastric cell lines than conventional CAR T cells and produced higher levels of IL-15 and CCL-19, which resulted in increased responder T cell chemotaxis and reduced expression of T cell exhaustion markers. A live zebrafish model was used for single-cell visualization of local cytotoxicity and metastatic cancers. Administration of 15×19 CAR T cells resulted in significant shrinking of gastric cancer xenograft tumors and expansion of 15×19 CAR T cells in zebrafish models. Taken together, these findings demonstrate that 15×19 CAR T cells are highly efficient in killing gastric cancer cells, are effective to avoid off-target effects, and migrate to local and metastatic sites for long-term surveillance of cancers.

Near-infrared-dye labeled tumor vascular-targeted dimer GEBP11 peptide for image-guided surgery in gastric cancer.

Positive resection margins occur in about 2.8%-8.2% gastric cancer surgeries and is associated with poor prognosis. Intraoperative guidance using Nearinfrared (NIR) fluorescence imaging is a promising technique for tumor detection and margin assessment. The goal of this study was to develop a tumor-specific probe for real-time intraoperative NIR fluorescence imaging guidance. The tumor vascular homing peptide specific for gastric cancer, GEBP11, was conjugated with a near-infrared fluorophore, Cy5.5. The binding specificity of the GEBP11 probes to tumor vascular endothelial cells were confirmed by immunofluorescent staining. The ability of the probe to detect tumor lesions was evaluated in two xenograft models. An orthotopic gastric cancer xenograft model was used to evaluate the efficacy of the GEBP11 NIR probes in real-time surgical guidance. In vitro assay suggested that both mono and dimeric GEBP11 NIR probes could bind specifically to tumor vascular epithelial cells, with dimeric peptides showed better affinity. In tumor xenograft mice, live imaging suggested that comparing with free Cy5.5 probe, significantly stronger NIR signals could be detected at the tumor site at 24-48h after injection of mono or dimeric GEBP11 probes. Dimeric GEBP11 probe showed prolonged and stronger NIR signals than mono GEBP11 probe. Biodistribution assay suggested that GEBP11 NIR probes were enriched in gastric cancer xenografts. Using dimeric GEBP11 NIR probes in real-time surgery, the tumor margins and peritoneal metastases could be clearly visualized. Histological examination confirmed the complete resection of the tumor. (GEBP11)2-ACP-Cy5.5 could be a potential useful probe for intraoperative florescence guidance in gastric cancer surgery.

MiRNA-223-3p regulates ECT2 to promote proliferation, invasion, and metastasis of gastric cancer through the Wnt/β-catenin signaling pathway.

Expression of the guanine nucleotide exchange factor epithelial cell transforming 2 (ECT2) is elevated in gastric cancer (GC) but its biological function in GC is poorly understood. MicroRNAs (miRNAs) have great potential as therapeutic targets for GC through their ability to modulate gene expression. In the present study, we sought to identify potential miRNA-mRNA-protein regulatory pathways that might control ECT2 expression and function in GC. ECT2 expression was examined in clinical GC specimens by immunohistochemical staining, and protein levels were correlated with clinicopathological features and prognosis. TargetScan was used to identify potential ECT2 mRNA-complementary miRNAs, and the roles of ECT2 and miRNA-223-3p (miR-223-3p) in GC cell biology and signaling pathway activation were examined by targeted knockdown (KD) or overexpression (OE) of ECT2 and miR-223-3p in GC cell lines. A murine GC xenograft model was developed to explore the impact of ECT2 OE on tumor growth in vivo. ECT2 expression was significantly elevated in GC specimens compared with normal gastric tissues and the level correlated positively with depth of invasion, ulceration, vascular tumor thrombus, neural invasion, and lymph node metastasis (p < 0.05). ECT2 was an independent prognostic factor for overall survival of GC patients (high ECT2 expression v.s. low ECT2 expression: χ2 = 29.831, p < 0.001). ECT2 KD or miR-223-3p OE markedly suppressed the proliferation, migration, and invasion of GC cells in vitro, whereas ECT2 OE had the opposite effects. ECT2 OE also promoted the growth of GC tumors in vivo. Tumor expression of Wnt2, β-catenin, and several downstream target proteins in GC cells were decreased by ECT2 KD or miR-223-3p OE but increased by ECT2 OE. miR-223-3p regulates ECT2 expression to promote tumorigenic behavior of GC via activation of the Wnt/β-catenin signaling pathway, suggesting that ECT2 and miR-223-3p as potential therapeutic targets for GC.

Antibody-Drug Conjugate Sacituzumab Govitecan Drives Efficient Tissue Penetration and Rapid Intracellular Drug Release.

Antibody-drug conjugates (ADC) are a rapidly growing class of targeted cancer treatments, but the field has experienced significant challenges from their complex design. This study examined the multiscale distribution of sacituzumab govitecan (SG; Trodelvy), a recently clinically approved ADC, to clarify the mechanism(s) of efficacy given its unique design strategy. We employed a multiscale quantitative pharmacokinetic approach, including near-infrared fluorescence imaging, single-cell flow cytometry measurements, payload distribution via γH2AX pharmacodynamic staining, and a novel dual-labeled fluorescent technique to track the ADC and payload in a high trophoblast cell-surface antigen 2 expression xenograft model of gastric cancer (NCI-N87). We found that rapid release of the SN-38 payload from the hydrolysable linker inside cells imparts more DNA damage in vitro and in vivo than an ADC with a more stable enzyme cleavable linker. With SG, little to no extracellular payload release in the tumor was observed using a dual-labeled fluorescence technique, although bystander effects were detected. The high dosing regimen allowed the clinical dose to reach the majority of cancer cells, which has been linked to improved efficacy. In addition, the impact of multiple doses (day 1 and day 8) of a 21-day cycle was found to further improve tissue penetration despite not changing tumor uptake [percent injected dose per gram (%ID/g)] of the ADC. These results show increased ADC efficacy with SG can be attributed to efficient tumor penetration and intracellular linker cleavage after ADC internalization. This quantitative approach to study multiscale delivery can be used to inform the design of next-generation ADCs and prodrugs for other targets.

Evaluating the efficacy and microenvironment changes of HER2 + gastric cancer during HLX02 and Endostar treatment using quantitative MRI

Background and objectivesTrastuzumab is an important targeted drug for HER2-positive gastric cancer. The treatment efficacy of a more cost-effective and accessible trastuzumab biosimilar, HLX02, was not well investigated, especially when combined with antiangiogenic treatment. In addition, the tumour microenvironment detected by functional MRI was still unclear during treatment. This study attempts to evaluate the therapeutic effect of antiangiogenic agents combined with HLX02 in a HER2-positive gastric cancer xenograft model and to detect microenvironmental changes using intravoxel incoherent motion diffusion-weighted imaging (IVIM-DWI).Materials and methodsWe subcutaneously injected MKN-45 human gastric cancer cells into BALB/C nude mice to establish a tumour model. Twenty-eight mice were divided into four groups and treated with saline (Group 1), Endostar (Group 2), trastuzumab biosimilar HLX02 (Group 3), or the combination of Endostar and HLX02 (Group 4). We then performed IVIM-DWI before and at different time points after treatment. HE, HER2, TUNEL, E-cadherin staining, and α-SMA and CD31 double-staining were used to confirm the pathological changes.ResultsGroup 4 demonstrated the smallest tumour volume at the end of treatment. The D value in Group 4 increased more dramatically, with the highest value on Day 20, compared with the other groups. Perfusion-related parameters (D* and f values) in Groups 2 and 4 increased initially and reversed after Day 10. Group 4 showed the lowest CD31 and HER2 and the highest TUNEL- and E-cadherin-positive staining rates. The D value was positively correlated with TUNEL but negatively correlated with HER2 staining. The D* and f values had positive correlations with CD31 and E-cadherin expression and the vessel maturity index.ConclusionsThe trastuzumab biosimilar drug HLX02 exhibited good treatment efficacy in HER2-positive gastric cancer, especially when combined with Endostar. IVIM-DWI can noninvasively monitor the process of vascular normalization and reflect the treatment effect early at the molecular level.

Chemo-immune cell therapy by intratumoral injection of adoptive NK cells with capecitabine in gastric cancer xenograft model.

Gastric cancer is one of the most commonly known malignancies and is the fifth cancer-related death globally. Whereas natural killer (NK) cells play a critical role in tumor elimination; therefore, adoptive NK cell therapy has become a promising approach in cancer cytotherapy. Hence, this study investigated the chemo-immune cell therapy in MKN-45 derived xenograft gastric cancer model. Three groups of animals have received the following treatments separately: activated NK cells, capecitabine, the combination of capecitabine and activated NK cells, and one was considered as the control group. Morphometric properties of tumor samples were evaluated at the end of the study. NK cells infiltration was evaluated by immunohistochemistry (IHC) of hCD56. Mitotic count and treatment response was assessed by hematoxylin and eosin (H&E) staining. The proliferation ratio to apoptosis was determined by IHC assessment of Ki67 and caspase 3. The results indicated that the NK cell therapy could effectively decrease the mitotic count in pathology assessment, but the tumor was not completely eradicated. In combination with metronomic chemotherapy (MC) of capecitabine, NK cell therapy demonstrated a significant difference in tumor morphometric properties compared to the control group. The proliferation ratio to apoptosis was also in line with pathology data. Although NK cell therapy could effectively decrease the mitotic count in vivo, the obtained findings indicated lesser potency than MC despite ex vivo activation. In order to enhance NK cell therapy effectiveness, suppressive features of the tumor microenvironment and inhibitory immune checkpoints blockade should be considered.

Gui Shao Tea Extracts Inhibit Gastric Cancer Growth in Vitro and in Vivo and Prolong Survival in Nude Mice.

Gui Shao Tea (GST), a long-aged tea with a Chinese herbal aroma, can treat many stubborn and malignant diseases, according to traditional Chinese medicine. This research aimed to discover and define GST, study the anti-gastric cancer effects of GST extracts and preliminarily elucidate the mechanism of action in the PI3K/Akt signaling pathway and the gut microbiota. GST was analyzed by GC/MS and HPLC. Cell proliferation, the cell cycle and apoptosis were evaluated by a CCK8 assay and flow cytometry. The effects of GST extracts on tumor inhibition and survival time were explored by a gastric cancer xenograft model in nude mice. The PI3K/Akt signaling pathway was assessed by western blotting and immunohistochemistry. Gut microbiota detection and fecal microbiota transplantation were performed to examine whether the tumor inhibition observed in mice was related to gut microbiota changes. The ingredients in GST, mostly terpenes and their derivatives, were novel and more concentrated than those in tea made from the branches and leaves of the same plant species, Camellia sinensis, picked and produced the same year, while the levels of polyphenols and alkaloids were significantly reduced. In BGC-823, MGC-803, and SGC-7901 gastric cancer cells, GST extracts significantly inhibited proliferation (p = 0.037), induced G0/G1 arrest (p < 0.001) and promoted early apoptosis (p = 0.041). In mice, gastric tumor growth was significantly inhibited in both the high-dose (HTF) and middle-dose (MTF) GST-fed groups. The inhibition rate in the HTF group was 33.77% on Day 14 (p = 0.042), and that in the MTF group was 55.21% on Day 14 (p = 0.002) and 61.6% on Day 28 (p = 0.008). The survival time of MTF group mice was significantly prolonged by 22.2% (p = 0.013). GST extracts inhibited the PI3K/AKT signaling pathway in gastric cancer cells (p = 0.016) and tissues (p = 0.029), downregulated the protein p-Rb and further downregulated E2F1, thereby affecting the cell cycle and proliferation. GST extracts altered the gut microbiota in mice, but these alterations alone were insufficient to inhibit gastric cancer growth. We confirmed the anti-gastric cancer effects of GST extracts, which might provide new approaches and methods for research and development of gastric cancer drugs.

Mono-PEGylated thermostable Bacillus caldovelox arginase mutant (BCA-M-PEG20) induces apoptosis, autophagy, cell cycle arrest and growth inhibition in gastric cancer cells

Gastric cancer is one of the most common malignant solid tumors in the world, especially in Asia with high mortality due to a lack of effective treatment. The potential usage of the newly constructed arginine-depleting enzyme—mono-PEGylated Bacillus caldovelox arginase mutant (BCA-M-PEG20), an effective drug against multiple cancer cell lines such as cervical and lung cancers, for the treatment of gastric cancer was demonstrated. Our results indicated that BCA-M-PEG20 significantly inhibited argininosuccinate synthetase (ASS)-positive gastric cancer cells, MKN-45 and BGC-823, while another arginine-depleting enzyme, arginine deiminase (ADI, currently under Phase III clinical trial), failed to suppress the growth of gastric cancer cells. In vitro studies demonstrated that BCA-M-PEG20 inhibited MKN-45 cells by inducing autophagy and cell cycle arrest at the S phase under 0.58 U/mL (IC50 values). Significant caspase-dependent apoptosis was induced in MKN-45 after the treatment with 2.32 U/mL of BCA-M-PEG20. In vivo studies showed that administrations of BCA-M-PEG20 at 250 U/mouse twice per week significantly suppressed about 50% of tumor growth in the MKN-45 gastric cancer xenograft model. Taken together, BCA-M-PEG20 demonstrated a superior potential to be an anti-gastric cancer drug.

Self-assembled amphiphilic copolymers–doxorubicin conjugated nanoparticles for gastric cancer therapy with low in vivo toxicity and high efficacy

Amphiphilic copolymers have long been utilized to turn hydrophobic anticancer drugs into nanoparticles administered to patients with cancer. A lack of stability in these monotherapies may be blamed for their poor clinical results in patients with cancer. We propose novel nanotherapies based on polymeric small prodrugs that preserve pharmacologic effectiveness while significantly reducing the toxicity of the fabricated drugs in animals to overcome this problem. Doxorubicin is attached to the end of the PLA fragments through a hydrolyzable ester bond utilizing methoxypolyethylene glycol-block-poly(d, l-lactic acid) (mPEG-PCL(2K)) with conjugates to mimic the self-assembly of colloidal nanotherapies. In a gastric cancer xenograft model, this nanotherapy displays a long-lasting suppression of tumor growth once a reasonable dosage is administered. Our findings imply that a toxic chemical and hydrophobic can be converted into therapeutic effective self-delivery nanotreatment.

Abstract 1763: Gastric cancer antibody fragment drug-conjugates (FDCs): Succeeding in solid tumors where ADCs fail

Abstract Antibody Drug Conjugates (ADCs) are failing due to 3 critical limitations: Low potency, ineffective solid-tumor penetration and poor tolerability. The industry is full of approaches where full-length Immunoglobulins have been engineered to carry defined numbers of payloads with higher-loadings of less potent payloads appearing to be well-tolerated. However, antibody fragments (e.g. single-chain Fcs-scFvs), which have many advantages including rapid tumor penetration, faster clearance, inexpensive manufacture, have been technologically challenging to apply in oncology. Our novel approach enables scFvs to have a high Drug:Antibody loading ratio (DAR) whilst retaining effective binding and other favorable biophysical properties, leading to a new product class tailored for solid tumors. Antikor has two FDC products in development for solid tumors, notably gastric: anti-HER2 FDC (ANT-043) and a second target (ANT-045) which will be disclosed during this presentation. ANT-043 has pM potencies in a range of HER2-expressing cell-lines, including trastuzumab-resistant models, excellent tumor ablation effects in breast, ovarian and gastric cancer xenograft models and superior tolerability compared to an ADC in rat toxicology studies at a dose of 1mg/kg/weekly. Quantitative payload tumor uptake and fluorescent immuno-histological studies demonstrate superior solid tumor penetration across the entire tumor and diffusion from blood vessels. In collaboration with our partners, Essex Biotechnology, Antikor is taking ANT-043 into IND-enabling studies for clinical development. ANT-045, which emerged from Antikor’s proprietary FDC ‘discovery engine’, is progressing towards IND-filing and updated data will illustrate how ANT-045 could have a broader patient benefit in gastro-intestinal cancers. ANT-045 has excellent in vitro cell-kill potency (pM IC50s) and excellent stability and superior in vivo tumor cure efficacy, compared to a leading clinical stage benchmark ADC. This presentation will focus on Antikor’s FDC discovery platform (stable high-DAR scFv-display libraries, tailored linker-payloads and design features) that has the potential to generate first-in-class products for difficult to treat solid tumors for patient benefit and promising to succeed where ADCs have failed to deliver. Citation Format: Mahendra P. Deonarain, Gokhan Yahioglu, Ioanna Stamati, Bryan Edwards, Soraya Diez-Posada, Isabel Perez-Castro, Anja Pomowski, Laura Bouche, Ashleigh Stewart, Monika Maciuszek, Sam Ness, Malcolm Ngiam, Quinn Xue. Gastric cancer antibody fragment drug-conjugates (FDCs): Succeeding in solid tumors where ADCs fail [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1763.

Abstract 328: LCB84, a TROP2-targeted ADC, for treatment of solid tumors that express TROP-2 using the hu2G10 tumor-selective anti-TROP2 monoclonal antibody, a proprietary site-directed conjugation technology and plasma-stable tumor-selective linker chemistry

Abstract LCB84 is a human Trop-2-targeting antibody drug conjugate (ADC) composed of monomethyl auristatin E (MMAE) as payload and the Hu2G10 (by Mediterranea Theranostic) humanized IgG1 antibody that selectively targets the ADAM10-activated Trop-2 protein selectively expressed in transformed cancer cells (1). LCB84 was prepared using ConjuAll࣪, a proprietary site-directed conjugation technology of LegoChem Biosciences, which incorporates a conjugation ‘handle’ joined by enzymatic prenylation to a specifically engineered recognition sequence (CaaX) on antibody light chains. This conjugation handle facilitates simple versatile chemical conjugation to the linker-payload. A proprietary plasma-stable cleavable linker that is recognized and cleaved by a cancer-associated lysosomal enzyme, β-glucuronidase, was used to enable efficient and traceless payload release in a cancer-specific manner. LCB84 has been evaluated for anti-tumor activity and showed superior anticancer efficacy in triple-negative breast cancer (TNBC), pancreatic ductal adenocarcinoma (PDAC), gastric cancer and non-small cell lung cancer (NSCLC) cell line-derived xenograft (CDX) models compared to the ADC competitors Trodelvy and DS-1062. The LCB84 treatments were well tolerated, with no changes in body weight compared to control animals, for all dosing groups. LCB84 has robust cross-reactivity against primate Trop-2, which allows rigorous toxicity studies in monkeys. Remarkably, preliminary toxicity studies using cynomolgus monkeys showed that LCB84 is well tolerated, with calculated therapeutic index (TI, MTD/MED) of ~30 for single dosing and ~40 for repeat dosing. In conclusion, LCB84 is highly effective against Trop-2-positive CDX models in mice at doses that are well tolerated in mice and in primate models. Use of this proprietary plasma-stable cancer-selective linker technology and the Hu2G10 anti-Trop-2 monoclonal antibody that targets cancer-activated Trop-2 has led to a greatly improved next generation ADC for the treatment of various Trop-2-positive solid cancers including TNBC, PDAC, NSCLC and gastric cancer. Citation Format: Hyejung Kim, Emanuela Guerra, Eunji Baek, Yeojin Jeong, Hyogeun You, Byeongjun Yu, Taeik Jang, Alberti Saverio, Chul-Woong Chung, Changsik Park. LCB84, a TROP2-targeted ADC, for treatment of solid tumors that express TROP-2 using the hu2G10 tumor-selective anti-TROP2 monoclonal antibody, a proprietary site-directed conjugation technology and plasma-stable tumor-selective linker chemistry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 328.

Berberine retarded the growth of gastric cancer xenograft tumors by targeting hepatocyte nuclear factor 4α.

BACKGROUNDGastric cancer is the third deadliest cancer in the world and ranks second in incidence and mortality of cancers in China. Despite advances in prevention, diagnosis, and therapy, the absolute number of cases is increasing every year due to aging and the growth of high-risk populations, and gastric cancer is still a leading cause of cancer-related death. Gastric cancer is a consequence of the complex interaction of microbial agents, with environmental and host factors, resulting in the dysregulation of multiple oncogenic and tumor-suppressing signaling pathways. Global efforts have been made to investigate in detail the genomic and epigenomic heterogeneity of this disease, resulting in the identification of new specific and sensitive predictive and prognostic biomarkers. Trastuzumab, a monoclonal antibody against the HER2 receptor, is approved in the first-line treatment of patients with HER2+ tumors, which accounts for 13%-23% of the gastric cancer population. Ramucirumab, a monoclonal antibody against VEGFR2, is currently recommended in patients progressing after first-line treatment. Several clinical trials have also tested novel agents for advanced gastric cancer but mostly with disappointing results, such as anti-EGFR and anti-MET monoclonal antibodies. Therefore, it is still of great significance to screen specific molecular targets for gastric cancer and drugs directed against the molecular targets.AIMTo investigate the effect and mechanism of berberine against tumor growth in gastric cancer xenograft models and to explore the role of hepatocyte nuclear factor 4α (HNF4α)-WNT5a/β-catenin pathways played in the antitumor effects of berberine.METHODSMGC803 and SGC7901 subcutaneous xenograft models were established. The control group was intragastrically administrated with normal saline, and the berberine group was administrated intragastrically with 100 mg/kg/d berberine. The body weight of nude mice during the experiment was measured to assess whether berberine has any adverse reaction. The volume of subcutaneous tumors during this experiment was recorded to evaluate the inhibitory effect of berberine on the growth of MGC803 and SGC7901 subcutaneous transplantation tumors. Polymerase chain reaction assays were conducted to evaluate the alteration of transcriptional expression of HNF4α, WNT5a and β-catenin in tumor tissues and liver tissues from the MGC803 and SGC7901 xenograft models. Western blotting and IHC were performed to assess the protein expression of HNF4α, WNT5a and β-catenin in tumor tissues and liver tissues from the MGC803 and SGC7901 xenograft models.RESULTSIn the both MGC803 and SGC7901 xenograft tumor models, berberine significantly reduced tumor volume and weight and thus retarded the growth rate of tumors. In the SGC7901 and MGC803 subcutaneously transplanted tumor models, berberine down-regulated the expression of HNF4α, WNT5a and β-catenin in tumor tissues from both transcription and protein levels. Besides, berberine also suppressed the protein expression of HNF4α, WNT5a and β-catenin in liver tissues.CONCLUSIONBerberine retarded the growth of MGC803 and SGC7901 xenograft model tumors, and the mechanism behind these anti-growth effects might be the downregulation of the expression of HNF4α-WNT5a/β-catenin signaling pathways both in tumor tissues and liver tissues of the xenograft models.