Gastric Cancer Xenograft Model Research Articles

Preparation and identification of a fluorescent probe with CsPbBr3perovskite quantum dots and CD44v6 specific peptide for gastric cancer imaging.

Since the sensitivity and accuracy of traditional detection for early gastric cancer diagnosis are still insufficient, it is significant to continuously optimize the optical molecular imaging detection technology based on an endoscopic platform. The signal intensity and stability of traditional chemical fluorescent dyes are low, which hinders the clinical application of molecular imaging detection technology. This work developed a probe based on perovskite quantum dots (PQDs) and peptide ligands. By utilizing CsPbBr3perovskite PQDs modified by azithromycin (AZI), combined with the specific polypeptide ligand of CD44v6, a gastric cancer biomarker, the perovskite-based probe (AZI-PQDs probe) which can specifically identify gastric cancer tumor was prepared. Owing to the high photoluminescence quantum yield of CsPbBr3PQDs, the naked eye can observe the imaging under the excitation of the hand-held ultraviolet light source. AZI-PQDs probe can accurately identify gastric cancer cells, tissues, and xenograft models with experiments ofex vivoandin vivofluorescence imaging detection. It also exhibited low toxicity and immunogenicity, indicating the safety of the probe. This work provides a probe combined with cancer specificity and a reliable fluorescent signal that has the potential for application in gastric cancer optical molecular imaging.

Effects of phospholipase D1-inhibitory peptide on the growth and metastasis of gastric cancer cells

Phospholipase D1 (PLD1) contributes to cancer development and progression through its effects on cell proliferation, survival, invasion, metastasis, angiogenesis, drug resistance, and modulation of the tumor microenvironment. Its central role in these processes makes it a promising target for novel cancer treatments aimed at inhibiting its activity and disrupting the signaling pathways it regulates. In this study, we aimed to investigate the effect of PLD1 inhibition on gastric cancer cell growth using a novel peptide inhibitor, TAT-TVTSP. PLD1, which plays a role in cancer progression, catalyzes the conversion of phosphatidylcholine into choline and phosphatidic acid through hydrolysis. To effectively target PLD1 in cells, we engineered TAT-TVTSP by fusing a PLD1-inhibitory peptide (TVTSP) with a cell-penetrating peptide (TAT). We observed that TAT-TVTSP effectively inhibited PLD1 activity in AGS gastric cancer cells. Moreover, TAT-TVTSP significantly inhibited the mammalian target of the rapamycin signaling pathway, including the phosphorylation of key downstream targets such as S6K1, AKT, S473, glycogen synthase kinase-3b, and forkhead box O1. TAT-TVTSP did not induce cell death, but it triggered cell cycle arrest by activating p21 and p27 via AKT phosphorylation. Functional assays revealed that TAT-TVTSP significantly impaired the colony-forming ability of AGS cells, thus inhibiting cell proliferation. Transwell and wound-healing assays revealed that this peptide disrupted the cellular behaviors critical to cancer progression, such as migration and invasion. In vivo, TAT-TVTSP significantly reduced tumor growth in the xenograft model of gastric cancer without any toxicity. Overall, our results suggest that TAT-TVTSP is a novel therapeutic agent for PLD1-mediated cancers.

Enhanced Efficacy of Gastric Cancer Treatment through Targeted Exosome Delivery of 17-DMAG Anticancer Agent.

In this study, we explored the potential of genetically engineered exosomes as vehicles for precise drug delivery in gastric cancer therapy. A novel antitumor strategy using biocompatible exosomes (Ex) was devised by genetically engineering adipose-derived stem cells to express an MKN45-binding peptide (DE532) on their surfaces. 17-(Dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG) was encapsulated in engineered exosomes, resulting in 17-DMAG-loaded DE532 exosomes. In both in vitro and in vivo experiments using mouse gastric cancer xenograft models, we demonstrated that 17-DMAG-loaded DE532 Ex exhibited superior targetability over DE532 Ex, 17-DMAG-loaded Ex, and Ex. Administration of the 17-DMAG-loaded DE532 Ex yielded remarkable antitumor effects, as evidenced by the smallest tumor size, lowest tumor growth rate, and lowest excised tumor weight. Further mechanistic examinations revealed that the 17-DMAG-loaded DE532 Ex induced the highest upregulation of the pro-apoptotic marker B-cell lymphoma-2-like protein 11 and the lowest downregulation of the anti-apoptotic marker B-cell lymphoma-extra large. Concurrently, the 17-DMAG-loaded DE532 Ex demonstrated the lowest suppression of antioxidant enzymes, such as superoxide dismutase 2 and catalase, within tumor tissues. These findings underscore the potential of 17-DMAG-loaded DE532 exosomes as a potent therapeutic strategy for gastric cancer, characterized by precise targetability and the potential to minimize adverse effects.

Abstract LB045: Exploiting biparatopic binding to deliver novel, differentiated ALPP/ALPPL2 targeting protein drug conjugates

Abstract The placental alkaline phosphatases ALPP and ALPPL2 are attractive targets for antibody and protein-drug conjugate (ADC and PDC) approaches. These GPI-anchored cell-surface proteins, which share very high sequence identity, are over-expressed in a number of different solid tumor indications including testicular, ovarian, endometrial, gastric, pancreatic and non-small cell lung cancers but have very little expression on normal adult tissues. Consequently, there is significant interest in exploiting ALPP/ALPPL2 for the development of new targeted oncology therapeutics, and an ALPP/ALPPL2 ADC, utilizing a classical full-length IgG antibody, is currently in Phase I clinical trials. The exploitation of biparatopic binding is an attractive approach for improving the therapeutic index of ADCs. This mode of target engagement increases the effective number of binding sites on the tumor cells and can promote internalisation and trafficking, thereby delivering increased efficacy for the same payload bolus and opening up patient populations with lower target expression. This can lead to a widening of the therapeutic window, by improving the efficacy achievable for a given amount of payload administered (often defined as the “platform” toxicity). For the development of biparatopic conjugates, the use of small protein domain binders offers advantages over the use of full-length antibodies, as they can readily be reformatted into a variety of biparatopic architectures to deliver homogeneous drug conjugates in high yield. Additionally, smaller protein formats have the capacity to penetrate deeper into solid tumors than large antibodies. We have identified a series of highly selective, high affinity ALPP/ALPPL2 single domain VHH binders, which target distinct epitopes on the protein and which, importantly, show no binding to the closely related ALPI or ALPL isoforms (which have high normal tissue expression). Using protein engineering approaches, a series of homogenous site-specifically labelled biparatopic PDCs have been developed employing the clinically validated vcMMAE (vedotin) payload. In pre-clinical studies, these PDC agents demonstrate excellent anti-tumor efficacy in vivo in ALPP/ALPPL2 positive cell-line derived xenograft models of gastric, pancreatic and ovarian cancer displaying a range of target expression. In benchmarking studies, lead biparatopic PDCs show increased internalisation and target-mediated in vitro cell kill potency compared to an ALPP/ALPPL2 targeting ADC. Additionally, in multiple CDX models, lead biparatopic PDCs demonstrate improved efficacy over an ALPP/ALPPL2 ADC both on a mg/kg and on a payload equivalents basis, in both single dose and multi-dose studies, providing complete regressions. Based on this excellent efficacy profile and favourable developability characteristics, preclinical development activities have been initiated. Citation Format: Graham Cotton, Estelle G. McLean, Paul Trumper, Mark Wappett, Stephanie Gatdula, Stacey Bell, Greg Papadakos, Kieron Lucas, Stephanie Burton, Chiara Saladino, Georgiana Parau, Aidan McCann, Jennifer Thom, Aaron N. Cranston, Tim Harrison. Exploiting biparatopic binding to deliver novel, differentiated ALPP/ALPPL2 targeting protein drug conjugates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB045.

DR30318, a novel tri-specific T cell engager for Claudin 18.2 positive cancers immunotherapy

BackgroundClaudin 18.2 (CLDN18.2) is a highly anticipated target for solid tumor therapy, especially in advanced gastric carcinoma and pancreatic carcinoma. The T cell engager targeting CLDN18.2 represents a compelling strategy for enhancing anti-cancer efficacy.MethodsBased on the in-house screened anti-CLDN18.2 VHH, we have developed a novel tri-specific T cell engager targeting CLDN18.2 for gastric and pancreatic cancer immunotherapy. This tri-specific antibody was designed with binding to CLDN18.2, human serum albumin (HSA) and CD3 on T cells.ResultsThe DR30318 demonstrated binding affinity to CLDN18.2, HSA and CD3, and exhibited T cell-dependent cellular cytotoxicity (TDCC) activity in vitro. Pharmacokinetic analysis revealed a half-life of 22.2–28.6 h in rodents and 41.8 h in cynomolgus monkeys, respectively. The administration of DR30318 resulted in a slight increase in the levels of IL-6 and C-reactive protein (CRP) in cynomolgus monkeys. Furthermore, after incubation with human PBMCs and CLDN18.2 expressing cells, DR30318 induced TDCC activity and the production of interleukin-6 (IL-6) and interferon-gamma (IFN-γ). Notably, DR30318 demonstrated significant tumor suppression effects on gastric cancer xenograft models NUGC4/hCLDN18.2 and pancreatic cancer xenograft model BxPC3/hCLDN18.2 without affecting the body weight of mice.

Abstract 5618: Evaluating the combination of datopotamab deruxtecan (Dato-DXd) with AZD5305, a highly potent, PARP1-selective inhibitor, -in preclinical models

Abstract Background: Dato-DXd is a TROP2-directed antibody drug conjugate comprised of a topoisomerase I inhibitor (an exatecan derivative, DXd) conjugated to datopotamab, a humanized anti-TROP2 IgG1 antibody, via a cleavable plasma-stable tetrapeptide-based linker. Topoisomerase I inhibitors stabilize DNA-topoisomerase cleavage complexes (Topo1cc) that lead to double-strand breaks and activation of DNA damage response (DDR). Poly(ADP-ribose) polymerase 1 (PARP1) mediates signal transduction in the DDR as an important regulator. AZD5305 is a highly potent and selective inhibitor of PARP1. Since PARP1 is a key component driving the repair of trapped Topo1cc, we aimed to investigate if combinations of Dato-DXd with AZ5305 lead to synergistic anti-tumor effects in preclinical models. Methods: We evaluated the cytotoxic effect of the combination of Dato-DXd with AZD5305 in a panel of six non-small cell lung cancer (NSCLC) and five triple negative breast cancer (TNBC) cell lines in a 7-day viability assay. We evaluated PARylation inhibition and gH2AX response by Western blotting. Topo1cc accumulation in response to combination treatment was evaluated by immunofluorescence. This combination was evaluated in vivo in TROP2+, HRD-negative gastric cancer (GC) N87 cell line xenograft at varying concentrations of AZD5305 and in a TROP2+, PARPi-resistant Ovarian cancer patient derived xenograft (PDX) model (CTG-3718). Results: The in vitro combination screen demonstrated enhanced cell killing by the Dato-DXd and AZD5305 combination over either single agent in both NSCLC and TNBC cell lines. Combination benefit was observed at all doses regardless of homologous recombination deficiency (HRD) status. We observed dose-dependent increase in PARylation inhibition and enhanced induction of gH2AX in the combination treatment. In a N87 tumor model, while Dato-DXd and AZD5305 provided 84% and 31% tumor growth inhibition (TGI) as monotherapies respectively, the addition of AZD5305 to Dato-DXd resulted in 95% TGI. This effect was observed with multiple dose levels of AZD5305. In a TROP2+, ovarian cancer PDX model, AZD5305 demonstrated limited monotherapy activity (<10% TGI) and Dato-DXd demonstrated robust monotherapy activity with 72% TGI. Additional anti-tumor activity was observed for the combination treatment compared to either monotherapy with 88% TGI. All treatment groups were well tolerated in vivo. Conclusion: Dato-DXd combination with AZD5305 increased cytotoxic activity and pharmacodynamic response in vitro. This combination led to superior tumor growth inhibition in a GC xenograft model and a PARP1i-resistant Ovarian PDX model in vivo. These pre-clinical findings support the ongoing clinical evaluation of Dato-DXd as a monotherapy and in combination with AZD5305 in patients with various advanced solid tumor types (NCT05489211, NCT04644068). Citation Format: Gopi Patel, Joe’l Owusu, Chetan Rane, Alan Rosen, Matthew Griffin, Matthew Sung. Evaluating the combination of datopotamab deruxtecan (Dato-DXd) with AZD5305, a highly potent, PARP1-selective inhibitor, -in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5618.

Abstract 3317: KRASmulti inhibitor BI 3706674 shows efficacy in KRAS-driven preclinical models of cancer that supports clinical testing in patients with tumors harbouring KRASG12V mutations and KRAS wild-type amplifications

Abstract Alterations in KRAS are a main cancer driver. Amplifications of the KRAS wild-type (wt) allele account for ~7% of all KRAS-driven cancers and are frequent in gastric (~5%), esophageal (~13%) and gastroesophageal junction cancers (~12%). KRASG12V mutations account for ~28% of pancreatic cancers, 9% of colorectal cancers and 6% of lung adenocarcinoma. There is an urgent medical need to identify targeted therapies for these frequent KRAS alterations. Herein, we describe BI 3706674, a novel, potent and orally bioavailable small molecule inhibitor of the KRAS oncogene for clinical testing in patients with tumors harboring KRASG12V mutations and KRAS wild-type amplifications. BI 3706674 binds non-covalently to multiple KRAS mutant alleles, including the KRAS wt allele, in the GDP-bound state and thereby disrupts oncogenic signaling. Importantly, BI 3706674 is highly selective for KRAS vs HRAS or NRAS, and it is therefore expected to be well-tolerated in normal tissues. In two large cancer cell line panels (PRISM and Horizon), BI 3706674 shows sensitivity across a wide range of KRAS alleles (KRAS wt amplifications and KRAS mutations e.g., G12A/C/D/V, G13D and Q61H). The strongest sensitivity was observed for cell lines with KRAS wt amplifications (relative copy number (CN) of > 10) followed by cell lines with KRASG12V mutations along with significant pharmacodynamic (PD) biomarker modulation (e.g., down-regulation of DUSP6 mRNA). In vivo, BI 3706674 was well-tolerated and showed dose-dependent efficacy in cell line-derived and patient-derived xenograft models of human gastric cancer with KRAS wt CN > 10 and diverse tumor types with KRASG12V mutations. A twice daily oral dose of 30 mg/kg induced significant tumor regression and PD biomarker modulation. Combination of BI 3706674 with standard of care therapies and novel agents are being tested in preclinical models to guide clinical development. Results of the ongoing pre-clinical analysis will be shared. A Phase I clinical trial is in preparation in patients with advanced solid cancers harboring KRASG12V mutations and KRAS wt amplifications to evaluate safety, tolerability, pharmacokinetic and pharmacodynamic properties, and efficacy of BI 3706674. Citation Format: Antonio Tedeschi, David H. Peng, Fiorella Schischlik, Lorenz Herdeis, Otmar Schaaf, Valeria Santoro, Daniel Gerlach, Fabio Savarese, Jesse Lipp, Christian Haslinger, Francesca Rocchetti, Johannes Popow, Heinrich J. Huber, Birgit Wilding, Matthias Treu, Julian Fuchs, Joachim Broeker, Tobias Wunber, Michael Gmachl, Klaus Rumpel, Matthew Rees, Melissa Ron, Jennifer Roth, Mariah Williams, Charles Deckard, Vandhana Ramamoorthy, Joseph R. Daniele, Jaffer A. Ajani, Funda Meric-Bernstam, Scott Kopetz, Michael Kim, Don L. Gibbons, Christopher P. Vellano, Joseph R. Marszalek, Timothy P. Heffernan, Darryl McConnell, Mark Pearson, Norbert Kraut, Dorothea Rudolph. KRASmulti inhibitor BI 3706674 shows efficacy in KRAS-driven preclinical models of cancer that supports clinical testing in patients with tumors harbouring KRASG12V mutations and KRAS wild-type amplifications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3317.

Abstract 2120: Asparaginases-Treatment potential for the biomarker selected hepatocellular carcinoma (HCC) population

Abstract Asparaginase is a key component of the therapeutic regimen used to treat patients with acute lymphoblastic leukemia (ALL). Calaspargase pegol (Asparlas) is composed of E. coli L-asparaginase and is PEGylated with approximately 5000 Da polyethylene glycol (PEG). Asparlas depletes asparagine (Asn) by deamination into aspartic acid. Asn synthetase (ASNS) is the key enzyme responsible for de novo Asn synthesis. In the absence of Asn in their direct environment, normal cells having adequate ASNS activity can survive with their own Asn supply while cancer cells lacking ASNS activity undergo apoptosis as they depend on extracellular sources of Asn. The ASNS gene has a typical CpG island in its promoter and its methylation status in tumor cells has been reported to be associated with asparaginase sensitivity clinically in T-ALL and in solid tumors preclinically. In this study we have investigated ASNS promoter hypermethylation as a biomarker for Asparlas activity in solid tumors and explored the feasibility of translating these results into the clinic. Analyses of the TCGA datasets indicated that a relatively high fraction of hepatocellular carcinoma (HCC) and gastric cancers are hypermethylated at the ASNS promoter. Testing Asparlas across a panel of HCC cell lines demonstrated that ASNS promoter-hypermethylated cell lines with lowest ASNS mRNA expression are the most responsive to Asparlas treatment in vitro. In vivo efficacy studies in mice bearing human ASNS promoter-hypermethylated xenograft models of HCC and gastric cancer showed that Asparlas treatment inhibited tumor growth in a dose-dependent manner and resulted in tumor rejections at the highest doses. Data from the HCC model was used to build a PK/PD efficacy model to project efficacious concentrations in patients. A panel of HCC PDX models with different levels of ASNS promoter methylation was evaluated to better understand the relationship between this biomarker and response to Asparlas. Treatment with Asparlas resulted in complete depletion of Asn in plasma and a partial depletion of Asn in the tumor for all models tested and strong anti-tumor activity against a model that had high methylation of the ASNS promoter. This activity was lost in models with lesser degrees of methylation indicating a strict threshold of promoter methylation for efficacy. These data confirm and extend previous findings that hypermethylation of the ASNS promoter has a synthetic lethal relationship with Asn-depleting therapies including Asparlas and highlight the need to carefully define the minimum threshold of hypermethylation to select HCC patients who could potentially benefit from Asparlas treatment. Citation Format: Ethika Tyagi, Magdalena Pohorecka, Valérie Duvivier, Aurelie Studeny, Damien Valour, Natacha Moulharat, Hélène Darville, Nicolas Provost, Pierre Barbier Saint Hilaire, Eef Hoeben, Donald Simons. Asparaginases-Treatment potential for the biomarker selected hepatocellular carcinoma (HCC) population [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2120.

Abstract 3137: Development of AT2604, a highly efficacious ADC targeting alkaline phosphatases ALPP and ALPPL2​

Abstract Placental alkaline phosphatases, ALPP and ALPPL2, are highly homologous, membrane-bound proteins involved in fetal development. Despite having limited expression in normal tissues, they are highly differentiated in tumor cells making them ideal targets for antibody drug conjugate (ADC) development. AT2604 is an ADC that consists of a humanized IgG1 anti-ALPP/ALPPL2 antibody that displays specificity over other human alkaline phosphatases and is cross reactive to NHP ALPPL2 but not to murine ALPPL2. The anti-ALPP/ALPPL2 antibody shows high target binding affinity and internalization into ALPP and ALPPL2 positive cells.​ AT2604 utilizes AxcynCYSTM technology for site-specific conjugation to achieve a highly homogeneous (>97%) DAR4 ADC product. The payload is the clinically validated microtubule disrupting agent monomethyl auristatin E (MMAE) conjugated to the antibody via a protease-cleavable peptide linker approved for ADC use. When evaluated in mouse xenograft models of gastric (NCI-N87) and pancreatic (HPAC) cancer, AT2604 displayed strong tumor growth inhibition (>90%) at 1 mg/kg (QW3 × 2 dosing) in both models. Furthermore, a pre-tox study in non-human primates concluded AT2604 has a HNSTD of 10 mg/kg (QW3 × 3 dosing) and did not exhibit any non-reversible adverse effects; implying that AT2604 possesses an improved safety profile over other vedotin-based ADCs. Taken together, the data supports the continued development of AT2604 towards evaluation in human trials.​ Citation Format: Bryan K. Yeung, Xiao Hui Koh, Wei Li Ling, Weiqi Tan, You Xue Wu, Xi Yun Zhang, Hong Yee Tan, Shao Jun Liu, Chen Zhong, Bin Zou. Development of AT2604, a highly efficacious ADC targeting alkaline phosphatases ALPP and ALPPL2​ [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3137.

Abstract 7216: Improving anti-tumor efficacy by modulating a separate HER2 activator and an MMAE payload and reuniting both through click chemistry

Abstract Shasqi is advancing the Click Activated Protodrugs Against Cancer (CAPAC®) platform based on click chemistry, a Nobel Prize winning technology. The platform is modular and comprises 1) an activator that target specific antigens, and 2) inert cancer drugs, protodrugs, which are selectively activated at tumors via click chemistry. The CAPAC technology separates the tumor targeting function from the payload and reunites them at the tumor, creating the flexibility to optimize activity while limiting toxicity during preclinical and clinical development. The modularity of the platform enables the rapid development of new therapies as well as unlocking unique treatment benefits such as tunable combinations and payload cycling. We envision that CAPAC will expand the scope of potential targets and widen the therapeutic index of antibody drug conjugates (ADCs). Here we present proof of concept for a HER2-targeting activator with a monomethyl auristatin E (MMAE) payload. The activator SQT01 is composed of a HER2-binding Fab conjugated with tetrazines (Tz), a click chemistry component. The protodrug SQP22 is formed by coupling trans-cyclooctene (TCO), another click chemistry component, to MMAE, a cytotoxic spindle poison. SQT01 binding was confirmed via flow cytometry, while SQP22 was evaluated for stability and activation via the reaction of Tz and TCO moieties activating MMAE at the tumor site. In vivo activity was evaluated in a HER2+ gastric cancer (NCI-N87) xenograft model. SQP22 was stable in plasma and had minimal in vivo anti-tumor activity without the activator. When combined with SQT01 in tumor-bearing mice, SQP22 was rapidly activated and led to active MMAE at the tumor site within 15 minutes. In efficacy studies, this activation resulted in significant tumor growth inhibition compared to an isotype control or disitamab vedotin, an ADC that targets HER2 and carries an MMAE payload. Treatment with SQT01 and 3 daily doses of SQP22 resulted in 4/6 complete responses with no body weight loss. This response can be differentiated from the activity of the isotype control by modulating the dose levels of SQT01 or SQP22 and the time interval between the two. In a dose escalation study, increasing dose level combinations were evaluated. In another efficacy study the optimal timing between the administration of SQT01 and SQP22 was investigated. It was found that anti-tumor efficacy is maintained over a 4 to 24 hours dose gap, but non-specific activity is decreased by dosing SQP22 8 - 24 hours after SQT01, corresponding to increased safety. Additional toxicology studies are ongoing. This data indicates that the CAPAC platform, which separates the targeting agent from the payload, can be optimized for antigen specific efficacy and safety by modulating dose amounts and time intervals between both components. Citation Format: Stefanie Wagner, Maša Aleckovic, George Coricor, Sangeetha Srinivasan, Jesse McFarland, Tri-Hung Nguyen, Jose M. Mejía Oneto. Improving anti-tumor efficacy by modulating a separate HER2 activator and an MMAE payload and reuniting both through click chemistry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7216.

Abstract 7213: Tumor targeted activation of an attenuated exatecan protodrug through Click Chemistry demonstrates efficacy in murine tumor studies

Abstract Shasqi is advancing the Click Activated Protodrugs Against Cancer (CAPAC®) platform based on click chemistry, a Nobel Prize winning technology. The platform is modular and comprises 1) an activator that target specific antigens, and 2) inert cancer drugs, protodrugs, which are selectively activated at tumors via click chemistry. The CAPAC technology separates the tumor targeting function from the payload and re-unites them at the tumor creating the flexibility to optimize activity while limiting toxicity during preclinical and clinical development. The modularity of the platform enables the rapid development of new therapies as well as unlocking unique treatment benefits such as tunable combinations and payload cycling. We envision that CAPAC will expand the scope of potential targets, widen the therapeutic window, and enhance the safety of locally activated cancer therapeutics. Here, we demonstrate the efficacy and safety of our exatecan protodrug (SQP07) used with intratumorally injected activators. The activators, tetrazine-modified sodium hyaluronate biopolymer(s), are injected at the tumor site and followed by a systemic dose of SQP07, a trans-cyclooctene (TCO)-modified protodrug of exatecan (Exa). An efficient covalent reaction between tetrazine and TCO moieties releases active Exa at the tumor. In vitro cytotoxicity studies showed that SQP07 activated with tetrazine was effective against mouse and human tumor cell lines (MC38, CT26, EMT6 and NCI-N87); SQP07 without activator had potency attenuated by 60-133 fold. In vivo tumor distribution and activity of SQP07 with the intratumoral biopolymer activators was evaluated in an NCI-N87 gastric cancer xenograft model. Female C.B-17 SCID mice received a single intravenous dose of vehicle (10% HPCD), Exa (50 mg/kg) or biopolymer (40 or 100 μL intratumorally) followed by SQP07 or SQP07 alone (50 mg/kg Exa mol equivalent). 1 hour after biopolymer + SQP07 treatment, activated Exa was highly concentrated in the tumor and stroma. This led to tumor regression in animals up to 40 days, performing significantly better than the free drug cytotoxic, Exa alone. Similarly, median survival with biopolymer + SQP07 was higher (>81 days) compared to Exa (68.5 days). Toxicity, measured as body weight loss, was comparable (~5%) and transient between Exa and biopolymer + SQP07. SQP07 alone treatment without the biopolymer was not differentiated from the vehicle control. These data show that the CAPAC platform, which separates the targeting agent from the payload, is an effective way to administer cytotoxic therapies systemically. Additional work with other tumor models, different biopolymers and the SQP07 protodrug is ongoing. Citation Format: Sangeetha Srinivasan, Stefanie Wagner, George Coricor, Tri-Hung Nguyen, Jesse M. McFarland, José M. Mejía Oneto. Tumor targeted activation of an attenuated exatecan protodrug through Click Chemistry demonstrates efficacy in murine tumor studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7213.

Abstract 7215: Nectin-4 targeted therapy with MMAE protodrug results in anti-tumor efficacy mediated by click chemistry

Abstract Shasqi is advancing the Click Activated Protodrugs Against Cancer (CAPAC®) platform based on click chemistry, a Nobel Prize winning technology. The platform is modular and comprises 1) an activator that target specific antigens, and 2) inert cancer drugs, protodrugs, which are selectively activated at tumors via click chemistry. The CAPAC technology separates the tumor targeting function from the payload and reunite them at the tumor creating the flexibility to optimize activity while limiting toxicity during preclinical and clinical development. The modularity of the platform enables the rapid development of new therapies as well as unlocking unique treatment benefits such as tunable combinations and payload cycling. We envision that CAPAC will expand the scope of potential targets, widen the therapeutic index of antibody drug conjugates (ADCs). Here we present a Nectin-4-targeting activator with a monomethyl auristatin E (MMAE) payload. Nectin-4 is a clinically validated tumor target expressed in a wide range of solid tumors, making Nectin-4 an attractive target for developing new therapeutic approaches for cancer patients. Substantial numbers of Nectin-4 targeting drugs are being developed but exhibit high rates of drug toxicities and highlight the need for alternative approaches that are efficacious and safe. We have developed a Nectin-4 Fab binder conjugated with tetrazine (~2.3 tetrazines per protein), SQT03, and tested it in combination with SQP22, an attenuated protodrug of MMAE conjugated to trans-cyclooctene (TCO). The reaction between tetrazine and TCO moieties releases MMAE at the tumor site. We have previously demonstrated that SQP22 is highly attenuated in vivo at doses that are > 20x the toxic dose of free MMAE, and in combination with a HER2 targeting activator, led to significant tumor growth inhibition in NCI-N87 gastric cancer xenograft model. The separation of an antigen-targeting activator from the payload by CAPAC technology allowed the rapid development and subsequent testing of SQT03 in combination with SQP22 in vitro and in vivo. SQT03 displayed <10nM binding affinity to Nectin-4 positive cells by flow cytometry analysis. In vivo, dosing of SQT03 occurs 8-24hrs prior to SQP22 to reduce systemic activation and to enable specific release of the active cytotoxic payload at the tumor site. In the T47D breast cancer xenograft model, a single dose of SQP22 at 8-24hrs after SQT03 led to tumor regression without body weight loss, in contrast to the 5% of body weight loss presented by two doses of Enfortumab-vedotin required to lead to similar activity. The presented data illustrates how the CAPAC platform can be used to test various therapeutic combinations, accelerating development, and reducing the time it takes to bring treatments to the clinic. The results support further development and testing of MMAE protodrug with Nectin-4 targeting agent in clinical settings. Citation Format: George Coricor, Jesse M. McFarland, Sangeetha Srinivasan, Stefanie Wagner, Nathan Yee, Tri-Hung Nguyen, Jose M. Mejia Oneto. Nectin-4 targeted therapy with MMAE protodrug results in anti-tumor efficacy mediated by click chemistry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7215.

Abstract 4113: AST-301,a pDNA-based cancer vaccine encoding HER2-ICD, enhances anti-tumor effect of HER2-ADC in a HER2-expressed gastric cancer xenograft model

Abstract Background: In gastric cancer, the HER2 overexpression (6% to 29.5% of gastric cancers) is considered to increase proliferation activity and suppress apoptosis of the cancer cells. However, HER2-targeting therapy in gastric cancer has not been fully evaluated. Currently, new approaches with HER2-tageted antibody-drug conjugates (ADCs) have led to promising results in HER2-related solid tumors. ADCs may have immune-modulating properties and that the payloads delivered by ADCs may induce immunogenic cell death. Based on this rationale, ADC and cancer vaccine combination would be potentially synergistic to improve clinical outcomes. This study was conducted to evaluate an synergistic effect and immune response of the combination of AST-301 and HER2-ADC in HER2-expressed gastric cancer xenograft model. AST-301 phase 1 study (PN109) was completed and phase 2 randomized-control trials (CornerStone-001, CornerStone-003) have been conducted in breast cancer and gastric cancer. Methods: NCI-N87 cells were mixed with the same amount of matrigel at 1x107 cells to formation tumors in the flanks of athymic BALB/c nude mice. After the tumor size of mice reached 100-200 mm3, all of them were randomly classified into four groups (n=7/group) and drug administration was initiated. AST-301 (100 μg/animal, intradermal injection, mixed with rhuGM-CSF as an immune adjuvant) was administered once a week to a total of fourth. LCB01 (1 mg/kg, intravenous injection) was administered to once. Enhertu (1 or 3 mg/kg, intravenous injection) was administered to twice for four weeks. The relative tumor size and TGI rate at day 25 were key endpoints of efficacy. To evaluate the mechanism underlying the enhanced anti-tumor effect due to the combined administration of AST-301 and HER2-ADCs, immune-cell profiling was also conducted using FACS analysis. Results: Our previous study has shown that AST-301 has anti-tumor effects in human gastric cancer xenograft athymic BALB/c nude mouse model. It was showed that adding AST-301 to LCB01 or Enhertu inhibited tumor growth based on a TGI rate at days 25; AST-301 combining with LCB01 vs LCB01 mono (52 % vs 43 %), AST-301 combining with Enhertu vs Enhertu mono (42 % vs 24 %).As an explorative endpoint, it was observed that myeloid-derived suppressor cells (MDSCs) elevation was modestly inhibited in tumor site in AST-301 mono or certain combination regimen adding AST-301. It was remarkable (or observed or captured) that MDSCs elevation was inhibited in tumor site in the combination groups of all ADCs with AST-301 compared to normal saline groups. Conclusion: With combining AST-301 and HER2 ADCs, it was demonstrated that anti-tumor effect was likely synergistic in the athymic mouse model. Phase 2 study of AST-301 in HER2-expressed gastric cancer has been on going (CornerStone-003, NCT 05771584). Citation Format: Jinho Kang, Hyo-Hyun Park, Jee Hyun Choi, Jinback Lim, Seong-Yong Jang, Min-Ah Kim, Myeong-Kyu Park, Yun-Hee Park, Soyeon Lim, Chul-Woong Chung, Jin Kyeong Choi, Eunkyo Joung, Ashley Yongmin Kim, Hun Jung. AST-301,a pDNA-based cancer vaccine encoding HER2-ICD, enhances anti-tumor effect of HER2-ADC in a HER2-expressed gastric cancer xenograft model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4113.

Abstract 721: Novel and distinctive anti-HLA-G antibody, IMB-201, inhibits tumor progression by breaking immune escape mechanism and enhancing cytotoxicity

Abstract Human leukocyte antigen-G (HLA-G), a type of MHC class I, is known to be expressed only in the placenta and acts as an immune suppressor to protect the fetus from maternal immunity. Recent studies have revealed that HLA-G suppresses various tumor-infiltrated immune cells, such as T cells, NK cells, and macrophages, through interaction with inhibitory receptors Ig-like transcript 2 (ILT2). HLA-G has recently emerged as an attractive anticancer target because HLA-G is a highly specific tumor antigen and has a role in immune evasion of cancer cells. Additionally, the expression pattern of ILT2 differs from that of PD-1 in tumor-infiltrated CD8+ T cells. Therefore, an HLA-G targeting strategy is expected to be an alternative to address unmet medical needs for those not responding to anti-PD(L)1 therapies. In this study, we screened the novel anti-HLA-G antibody, IMB-201, using phage display technique and confirmed that IMB-201 selectively bound to the membrane HLA-G of cancer cells. It exhibited superior activities in ILT2 blockade, NK cell activation, and antibody-dependent cellular cytotoxicity (ADCC) compared to the reference antibody. Furthermore, IMB-201 demonstrated more efficient suppression of tumor growth compared to the reference antibody in subcutaneous gastric cancer xenograft model. Notably, the group administered IMB-201 with NK92MI cells (CD16-negative NK cells) in the ovarian cancer xenograft model showed stronger tumor growth inhibition compared to the group treated only IMB-201, indicating that NK92MI cells were activated by IMB-201. This result indicates that one of the mechanisms of action (MoA) of IMB-201 worked effectively in vivo. In addition, to maximize the efficacy of IMB-201 and differentiate it from competitors, various efficacy enhancing modality could be applicable. We aim to develop biopharmaceuticals by selecting the optimized modality. Based on these results, IMB-201 could overcome the limitations of existing cancer immunotherapy, potentially becoming highly influential in cancer treatment as a novel immune checkpoint inhibitor. It is also expected to exhibit higher anticancer efficacy in combination therapy with immune checkpoint inhibitors. Citation Format: Hyeonju Kang, Seungil Baek, Inyoung Lee, Manseok Ju, Seonggu Han, Eunjung Han, Sungmuk Kang, Jihye Koo, Yoojin Kim, Suho Park, Eunyoung Cho, Chungmin Lee, Gyongsik Ha. Novel and distinctive anti-HLA-G antibody, IMB-201, inhibits tumor progression by breaking immune escape mechanism and enhancing cytotoxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 721.

Pre-clinical Efficacy and Safety Pharmacology of PEGylated Recombinant Human Endostatin.

This study aimed to outline the pre-clinical efficacy and safety pharmacology of PEGylated recombinant human endostatin (M2ES) according to the requirements of new drug application. The purity of M2ES was evaluated by using silver staining. Transwell migration assay was applied to detect the bioactivity of M2ES in vitro. The antitumor efficacy of M2ES was evaluated in an athymic nude mouse xenograft model of pancreatic cancer (Panc-1) and gastric cancer (MNK45). BALB/C mice were treated with different doses of M2ES (6, 12 and 24 mg/kg) intravenously, both autonomic activity and cooperative sleep were monitored before and after drug administration. The apparent molecular weight of M2ES was about 50 kDa, and the purity was greater than 98%. Compared with the control group, M2ES significantly inhibits human micro-vascular endothelial cells (HMECs) migration in vitro. Notably, weekly administration of M2ES showed a significant antitumor efficacy when compared with the control group. Treatment of M2ES (24mg/kg or below) showed no obvious effect on both autonomic activity and hypnosis. On the basis of the pre-clinical efficacy and safety pharmacology data of M2ES, M2ES can be authorized to carry out further clinical studies.

High Engraftment and Metastatic Rates in Orthotopic Xenograft Models of Gastric Cancer via Direct Implantation of Tumor Cell Suspensions.

Although the implantation of intact tumor fragments is a common practice to generate orthotopic xenografts to study tumor invasion and metastasis, the direct implantation of tumor cell suspensions is necessary when prior manipulations of tumor cells are required. However, the establishment of orthotopic xenografts using tumor cell suspensions is not mature, and a comparative study directly comparing their engraftment and metastatic capabilities is lacking. It is unclear whether tumor fragments are superior to cell suspensions for successful engraftment and metastasis. In this study, we employed three GC cell lines with varying metastatic capacities to stably express firefly luciferase for monitoring tumor progression in real time. We successfully minimized the risk of cell leakage during the orthotopic injection of tumor cell suspensions without Corning Matrigel by systematically optimizing the surgical procedure, injection volume, and needle size options. Comparable high engraftment and metastatic rates between these two methods were demonstrated using MKN-45 cells with a strong metastatic ability. Importantly, our approach can adjust the rate of tumor progression flexibly and cuts the experimental timeline from 10-12 weeks (for tumor fragments) to 4-5 weeks. Collectively, we provided a highly reproducible procedure with a shortened experimental timeline and low cost for establishing orthotopic GC xenografts via the direct implantation of tumor cell suspensions.

Development and validation of cyclic peptide probe for gastric cancer based on phage display technique

AbstractCancer‐targeting diagnostics should have a sensitive and specific binding affinity. To achieve this, biomarker development is critical. This study aimed to develop and validate a 7‐mer cyclic peptide probe that can target gastric cancer. We developed this probe based on LGR5 (leucine‐rich repeat‐containing G‐protein coupled receptor 5)‐specific targeting, which is a marker for gastric cancer stem cells. An LGR5 targeting peptide sequence that was developed using phage display technology resulted in a cyclic peptide, C‐YLASRVH‐C (named YLA). We conjugated this peptide with fluorescent probes to validate its specific targeting ability for gastric cancer. The fluorescence‐labeled YLA peptide exhibited 3.0‐fold higher fluorescence intensity in a gastric cancer cell line (MKN45) than it did in a normal cell line (CCD841 cells). In contrast, pancreatic and colorectal cancer cells did not show significant fluorescence intensity with the YLA peptide. To verify its tumor‐targeting affinity, we developed a control peptide, C‐YLASAVH‐C (named YLASA) using an ALA scanning experiment. Whole‐body imaging of a gastric cancer xenograft model showed higher fluorescence intensity of tumors in the YLA peptide group than in the control peptide group. Moreover, ex vivo imaging of tumor tissues exhibited 6.8‐fold higher fluorescence intensity in the YLA peptide group compared to that in the YLASA control peptide group. In conclusion, we confirmed that the YLA peptide probe functions as a specific diagnostic probe for gastric cancer. We anticipate that it will play a theranostic role through further development.

ESM1 facilitates the EGFR/HER3-triggered epithelial-to-mesenchymal transition and progression of gastric cancer via modulating interplay between Akt and angiopoietin-2 signaling.

Gastric cancer (GC) poses global challenges due to its difficult early diagnosis and drug resistance, necessitating the identification of early detection markers and understanding of oncogenic pathways for effective GC therapy. Endothelial cell-specific molecule 1 (ESM1), a secreted glycoprotein, is elevated in various cancers, but its role in GC remains controversial. In our study, ESM1 was elevated in GC tissues, and its concentration was correlated with progression and poorer patient prognosis in independent cohorts. Functionally, ESM1 expression promoted proliferation, anoikis resistance, and motility of GC cells, as well as tumor growth in PDOs and in GC xenograft models. Mechanistically, ESM1 expression triggered the epithelial-to-mesenchymal transition (EMT) of GC cells by enhancing epidermal growth factor receptor (EGFR)/human EGFR 3 (HER3) association and activating the EGFR/HER3-Akt pathway. Additionally, angiopoietin-2 (ANGPT2) was found to be highly correlated with ESM1 and interplayed with Akt to induce the EMT and cancer progression. Use of a signal peptide deletion mutant (ESM1-19del) showed that the secreted form of ESM1 is crucial for its protumorigenic effects by activating the EGFR/HER3-Akt/ANGPT2 pathway to promote the EMT. Patients with high levels of both ESM1 and ANGPT2 had the poorest prognoses. Furthermore, therapeutic peptides successfully inhibited ESM1's induction of the aforementioned signals and motility of GC cells. ESM1's oncogenic role in GC involves activating the EGFR/HER3-Akt/ANGPT2 pathway, presenting a potential therapeutic target for GC.

Abstract A085: BI KRASmulti, a first-in-class, orally bioavailable and direct inhibitor of diverse oncogenic KRAS variants drives tumor regression in preclinical models and validates wild-type amplified KRAS as a therapeutic target

Abstract Alterations of KRAS are found in approximately one in seven of all human cancers, making KRAS one of the main oncogenic drivers in cancer. Gain-of-function missense mutations in KRAS are found in ~90% of pancreatic cancer, ~40% of colorectal cancer and ~30% of lung adenocarcinoma. In addition, focal high-level amplification of the KRAS wild-type (wt) allele, an alternative means of activating this oncoprotein, is observed in ~7% of cancers and lacks effective targeted therapies. Thus, there is an urgent need to identify effective therapies to address KRAS-driven tumors. Recently, we have reported the identification of compounds active against a broad range of oncogenic KRAS variants (KRASmulti) that selectively inactivate downstream signaling and tumor growth (Kim et al., Nature 2023). Herein we describe BI 3706674, a novel, potent and orally available small molecule inhibitor of the KRAS oncogene. BI 3706674 binds non-covalently to multiple KRAS mutant alleles, including the KRAS wt allele, in the GDP-bound state and thereby disrupts oncogenic signaling. Using several in vitro assays, we show that BI 3706674 is a) highly selective for KRAS vs HRAS or NRAS; b) blocks the interaction between KRAS mutant/wt and its guanidine exchange factor (GEF) SOS1 and c) shows potent antiproliferative activity in isogeneic cell lines that are dependent on various KRAS mutant alleles. In two large cancer cell line panels, BI 3706674 potently and selectively inhibits proliferation of KRAS mutant cancer cell lines as well as cancer cell lines with an amplification of the KRAS wt allele. Significant pharmacodynamic (PD) biomarker modulation (including inhibition of ERK1/2 phosphorylation and DUSP6 mRNA down-regulation) was observed in cell lines with a KRAS wt relative copy number (CN) > 10, treated with BI 3706674. Amplification of the KRAS wt allele is most frequently observed in gastric (~5%), esophageal (~13%) and gastroesophageal junction cancers (~12%). BI 3706674 was well-tolerated and showed dose-dependent efficacy in cell line-derived and patient-derived xenograft models (CDX and PDX) of human gastric cancer with a KRAS wt CN > 10. A twice daily oral dose of 30 mg/kg was sufficient to induce significant tumor regression. BI 3706674 treatment induced PD biomarker modulation (including inhibition of ERK1/2 phosphorylation, DUSP6 down-regulation, repression of the MAPK Pathway Activity Score (MPAS) signature and Ki67 reduction) in two KRAS wt amplified gastric cancer xenograft models. The depth and duration of PD biomarker responses following treatment correlated well with the doses used in the respective efficacy studies. Finally, these results support the clinical use of biomarkers such as the MPAS signature score and Ki67 to investigate treatment effects elicited by BI 3706674 in tumor biopsies. The KRASmulti inhibitor BI 3706674 is undergoing IND enabling studies and is intended to target tumors driven by KRAS wt amplifications as well as oncogenic KRAS missense mutations. Citation Format: Antonio Tedeschi, Lorenz Herdeis, Valeria Santoro, Fabio Savarese, Birgit Wilding, Matthias Treu, Julian Fuchs, Joachim Bröker, Tobias Wunberg, Michael Gmachl, Rumpel Klaus, Fiorella Schischlik, Jesse Lipp, David H. Peng, Mariah Williams, Charles Deckard, Vandhana Ramamoorthy, Joseph R. Daniele, Jaffer A. Ajani, Funda Meric-Bernstam, Christopher P. Vellano, Joseph R. Marszalek, Timothy P. Heffernan, Darryl McConnell, Mark Pearson, Norbert Kraut, Dorothea Rudolph. BI KRASmulti, a first-in-class, orally bioavailable and direct inhibitor of diverse oncogenic KRAS variants drives tumor regression in preclinical models and validates wild-type amplified KRAS as a therapeutic target [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A085.

Regulation of the Hippo/YAP axis by CXCR7 in the tumorigenesis of gastric cancer

BackgroundThe Hippo pathway is crucial in organ size control and tumorigenesis. Dysregulation of the Hippo/YAP axis is commonly observed in gastric cancer, while effective therapeutic targets for the Hippo/YAP axis are lacking. Identification of reliable drug targets and the underlying mechanisms that could inhibit the activity of the Hippo/YAP axis and gastric cancer progression is urgently needed.MethodsWe used several gastric cancer cell lines and xenograft models and performed immunoblotting, qPCR, and in vivo studies to investigate the function of CXCR7 in gastric cancer progression.ResultsIn our current study, we demonstrate that the membrane receptor CXCR7 (C-X-C chemokine receptor 7) is an important modulator of the Hippo/YAP axis. The activation of CXCR7 could stimulate gastric cancer cell progression through the Hippo/YAP axis in vitro and in vivo, while pharmaceutical inhibition of CXCR7 via ACT-1004–1239 could block tumorigenesis in gastric cancer. Molecular studies revealed that the activation of CXCR7 could dephosphorylate YAP and facilitate YAP nuclear accumulation and transcriptional activation in gastric cancer. CXCR7 functions via G-protein Gαq/11 and Rho GTPase to activate YAP activity. Interestingly, ChIP assays showed that YAP could bind to the promoter region of CXCR7 and facilitate its gene transcription, which indicates that CXCR7 is both the upstream signalling and downstream target of the Hippo/YAP axis in gastric cancer.ConclusionIn general, we identified a novel positive feedback loop between CXCR7 and the Hippo/YAP axis, and blockade of CXCR7 could be a plausible strategy for gastric cancer.