Potent Antitumor Efficacy Research Articles

Abstract 4256: YH012, a novel bispecific anti-HER2 and TROP2 antibody-drug conjugate, exhibits potent antitumor efficacy

Abstract Bispecific antibodies (BsAbs) and antibody-drug conjugates (ADCs) are among the most promising and fastest growing classes of next-generation antibody therapeutics for cancer therapy. The strategy of targeting dual tumor-associated antigens (TAAs) with a BsAb offers several advantages, such as improved efficacy due to synergistic effects, increased target cross-linking and internalization, increased tumor cell specificity and reduced side effects in normal tissues. HER2 is an established therapeutic target for approximately 20% of breast cancers, and its overexpression has been observed in a variety of other solid tumors. Trop-2 is another ideal tumor-associated target overexpressed on a wide variety of solid tumors. Co-expression of HER2 and TROP2 has been found in a number of tumors including gastric, colorectal, bladder and breast cancers. Therefore, co-targeting these TAAs using bispecific antibodies has clinical potential. YH012 is a novel first-in-class bispecific antibody-drug conjugate targeting HER2-TROP2 (HER2-TROP2-ADC), which contains a novel fully human bispecific anti-HER2/TROP2 antibody conjugated with Monomethyl auristatin E (MMAE) via a protease-cleavable linker. The anti-HER2/TROP2 BsAb showed increased endocytosis activity compared with its parental mAbs in tumor cells co-expressing HER2/TROP2, whereas the monovalent HER2 or TROP2 antibodies showed reduced internalization. As expected, YH012 showed increased in vitro potency of cell killing compared to the monovalent HER2/TROP2-ADC. These results suggest that YH012 increases the selectivity of payload delivery demonstrating the beneficial effects of targeting dual TAAs using BsAb-ADC. Moreover, YH012 showed robust anti-tumor efficacy in multiple cell line-derived none-small cell lung cancer (NSCLC) and gastric xenografts. Treatment with YH012 showed sustained tumor growth inhibition, which is more pronounced than the effects of the parental mAb-ADCs and monovalent ADCs. These preclinical data suggest that YH012 could potentially benefit patients with tumors co-expressing HER2 and TROP2 through improved drug selectivity and efficacy. Further investigations on the antitumor activity and safety profile of YH012 are ongoing in dogs. Citation Format: Chengzhang Shang, Liu Yang, Jiyong Zhang, Yanfei Han, Zhuolin Li, Zhenyan Han, Jun Li, Ying Meng, Gao An, Hao Yang, Wenqian An, Lei Chen, John Charpentier. YH012, a novel bispecific anti-HER2 and TROP2 antibody-drug conjugate, exhibits potent antitumor efficacy [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 4256.

Abstract 4032: Identification of Muc5B mutation as a positive predictive biomarker for mTORC1/2 inhibition by ATG-008 in lung cancer

Abstract Background: mTOR is a serine/threonine kinase that regulates cell growth, metabolism, proliferation, and survival. mTOR complex-1 (mTORC1) and mTOR complex-2 (mTORC2) are critical mediators of the PI3K-AKT pathway. Signaling by the PI3K/AKT/mTOR pathway is frequently deregulated in solid tumors and hematological malignancies. Inhibition of mTOR by small molecule inhibitors has therefore attracted great attention as an anti-cancer therapy. However, due to relatively modest clinical efficacy, no mTOR inhibitor has been approved for the treatment of lung cancer. The research of potentially predictive biomarkers is a key aspect of all anti-tumor treatment strategies to improve the proportion of patients benefiting from therapy. Multiple genetic alternations have been reported to be correlated with better anti-tumor efficacy of mTOR inhibition in animal models or patients, such as NFE2L2 mutation, STK11 mutation, or RICTOR amplification. Here, we report Muc5B mutation as a positive predictive biomarker for mTORC1/2 inhibition by ATG-008 (Onatasertib), a dual mTORC1/2 kinase inhibitor, in preclinical lung cancer models. Methods: 31 different lung cancer cell lines were treated with ATG-008 in vitro to determine the dose-response curves and the Area Under Curve (AUC) values. According to the AUC, the cell lines were classified as a sensitive group, medium group, and insensitive group. Unbiased genomic analysis of the tested cell lines was used to correlate the gene mutation, amplification, and expression with the sensitivity to ATG-008. The potential positive predictive biomarker, MUC5B mutation, was further validated in lung cancer CDX mouse models. Mouse bearing lung cancer cell lines with or without MUC5B mutation were orally treated with ATG-008 to determine the in vivo tumor growth inhibition. Results: ATG-008 inhibits lung cancer cell growth in vitro, with IC50 values ranging from 0.36µM to >10µM. By genomic analysis, we identified 23 genes with a significant difference in AUC values between mutant and wild type cell lines, among which MUC5B mutation was found in 7/10 sensitive cell lines and only 2/10 in insensitive cell lines. Cell lines that carried MUC5B mutation had significantly lower AUC compared with MUC5B wild type cell lines (p=0.0084). Oral dosing of ATG-008, 10mg/kg, QD, induced potent tumor growth inhibition(TGI) in MUC5B mutant CDX models, with 53.2% TGI in NCI-H82 model and 81.9% TGI in SK-MES-1 model. However, in MUC5B wild type models, the TGI is only 20.5% in NCI-H209 model and 36% in NCI-H526. Conclusion: These results suggest that the presence of MUC5B mutation correlates with more potent anti-tumor efficacy of ATG-008, potentially serving as a positive predictive biomarker for patient enrichment that warrants further investigation in the clinic. Citation Format: Bing Hou, Hui Xie, Shengyue Piao, Zhi Guo, Bo Shan, Jay Mei. Identification of Muc5B mutation as a positive predictive biomarker for mTORC1/2 inhibition by ATG-008 in lung cancer [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 4032.

Abstract 3299: Development of ONCR-788, a synthetic oncolytic virus based on Seneca Valley virus for the treatment of neuroendocrine tumors

Abstract Oncolytic viruses (OVs) are an emerging therapeutic approach for the treatment of cancer. Clinical benefit has been demonstrated for intratumoral administration. Still, the therapeutic effectiveness of intravenous delivery has been limited by neutralizing antibody responses against the virus. To circumvent this limitation, we developed Synthetic RNA viruses, a novel approach for intravenous and repeated administration of OVs, consisting of a viral RNA genome (vRNA) formulated within lipid nanoparticles. Systemic administration of ONCR-788, the Synthetic RNA virus drug candidate encoding the vRNA of Seneca Valley virus (SVV), led to potent anti-tumor efficacy, even in the presence of OV neutralizing antibodies in the bloodstream. After a single intravenous administration of ONCR-788, we demonstrated vRNA delivery, viral replication, spread, and lysis of tumor cells. Robust anti-tumor efficacy was observed across diverse neuroendocrine tumor models, including tumor CDX and PDX xenografts, lung orthotopic, and GEMM-derived models. In a syngeneic model, ONCR-788 stimulated both tumor lysis and modulation of the tumor microenvironment. Enhanced T cell recruitment and activation, increased expression of PD-L1 on tumor cells and myeloid cells, and M2 to M1 macrophage conversion were observed. Combination of ONCR-788 with anti-PD-1 resulted in improved anti-tumor activity versus the monotherapy arms. Altogether, ONCR-788 provides an innovative approach that enables repeat intravenous administration of viral immunotherapy. Citation Format: Edward M. Kennedy, Agnieszka Denslow, Jacqueline Hewett, Lingxin Kong, Ana De Almeida, Jeffrey Bryant, Jennifer S. Lee, Judy Jacques, Sonia Feau, Hayes Hayes, Elizabeth L. McMichael, Daniel Wambua, Sanmit Adhikari, Jessica Deterling, Matthew Scott, Brian H. Haines, Mitchel H. Finer, Ted Ashburn, Christophe Quéva, Lorena Lerner. Development of ONCR-788, a synthetic oncolytic virus based on Seneca Valley virus for the treatment of neuroendocrine tumors [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 3299.

Abstract 314: Amplifying STING activation by cyclic dinucleotide-manganese particles for systemic cancer immunotherapy

Abstract Background: Innate immune pathway of stimulator of interferon genes (STING) orchestrates antitumor immune responses and can elicit potent antitumor efficacy. However, clinical development of cyclic-dinucleotide (CDN) STING agonists is mostly limited to intratumoral administration because of the undesirable pharmacological properties of CDN. This limits their applications in treating metastasis, hematologic malignancies, and hard-to-reach solid tumor. Here, we aim to develop a pharmacological formulation that facilitate systemic delivery of CDN STING agonist to broaden its applications for cancer treatment. Methods: Nutritional metal ions physiologically mediate various immune processes, which could potentially be utilized for modulating the STING pathway. By screening different nutritional metal ions, we discovered manganese ion (Mn2+) and cobalt ion (Co2+) synergize with CDN STING agonists and significantly enhance type-I interferon (IFN-I) responses. As Mn2+ has been used in FDA approved drugs, we further developed the formulation to enable systemic codelivery of Mn2+ and CDN. Results: Mn2+ could significantly potentiate the STING activation by up to 77-fold. In addition, Mn2+ interact with CDNs and self-assemble into nanocrystals that could be stabilized as uniform nanoparticles (~120 nm) through lipid layer coating (CDN-Mn Particle, CMP). After either intratumoral or intravenous administrations, CMP effectively elevate the serum levels of inflammatory cytokines (IFN-β, TNF-α, CXCL-9, and CXCL-10), mitigates the suppressive functions of myeloid cells, relieve the immunosuppressive tumor microenvironment (TME), and induce antigen-specific CD8+ T cells expansion. Minute dose of CMP given intravenously exert remarkable antitumor efficacy in CT26, B16F10, and immune checkpoint blocker (ICB)-resistance tobacco-associated NOOC1 murine tumor models, leading to efficient tumor regression. Furthermore, the systemic biodistribution of CMP could be modulated by changing the composition of the lipid layer coating, allowing for organ-specific delivery of CMP to target tumor malignancies in certain organs. Conclusions: CMP provides a novel and versatile platform for local and systemic cancer treatment. More importantly, it highlights the potential of harnessing metal ions in treating various immune-mediated diseases. Citation Format: Xingwu Zhou, Xiaoqi Sun, Wang Gong, Yu Leo Lei, James J. Moon. Amplifying STING activation by cyclic dinucleotide-manganese particles for systemic cancer immunotherapy [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 314.

Charge-Reversible Pro-Ribonuclease Enveloped in Virus-like Synthetic Nanocapsules for Systemic Treatment of Intractable Glioma.

We have tailored multifaceted chemistries into the manufacture of artificial virus-like delivery vehicles mimicking viral "intelligent" transportation pathways through sequential biological barriers; these vehicles can acquire the ability to dynamically "program transfer" to their target sites. To accomplish this, we created anionic pro-proteins, which facilitate charge reversal when subject to acidic endosomal pH; in this way, carboxylation reactions are performed on proteins with amine-reactive cis-aconitic anhydride. Electrostatic associations then initiate the envelopment of these pro-proteins into multilayered nanoarchitectural vehicles composed of multiple-segmental block copolycationic cyclic Arg-Gly-Asp (RGD)-poly(ethylene glycol)(PEG)-GPLGVRG-polylysine(thiol). Therefore, upon the pro-proteins' initial binding to the tumors via the protruding RGD ligands, the bio-inert PEG surroundings are detached through the enzymolysis of the intermediate GPLGVRG linkage by tumor-enriched matrix metalloproteinases, unveiling the cationic polylysine palisade and imparting intimate affinities to the anionic cytomembranes of the targeted tumors. Essentially, through their active endocytosis into the subcellular endosomal compartments, the pro-proteins are made capable of retrieving the original amine groups through a charge reversal decarboxylation process, consequently eliciting augmented charge densities (charge nonstoichiometric protein@polylysine(disulfide)) to disrupt the anionic endosomal membranes to facilitate translocation into the cytosol. Eventually, the active protein payloads can be liberated from nonstoichiometric protein@polylysine(thiol) by the disassembly of polylysine palisade upon the cleavage of disulfide crosslinking in response to the very high level of glutathione in the cytosol, thereby contributing toward extreme cytotoxic potency. Hence, our elaborated virus-mimicking platform has demonstrated potent antitumor efficacy through the systemic administration of ribonucleases, which will consequently lead to an innovative new therapeutic method by which proteins could reach intracellular targets.

Multicenter phase II study of abemaciclib and ramucirumab in metastatic esophageal/gastroesophageal junction carcinoma.

TPS4169 Background: Cyclin dependent kinases (CDKs) are serine/threonine kinases that are responsible for phosphorylating the intracellular proteins that coordinate cell-cycle progression. The Cancer Genome Atlas (TCGA) molecular analysis of esophageal/gastroesophageal junction (E/GEJ) carcinomas has highlighted significant dysregulation of CDKN2A, the gene coding for the tumor suppressor p16, through deletion, mutation or epigenetic silencing in up to 76% of cases and the associated significant upregulation of the CDK4/6-Cyclin D-axis (The Cancer Genome Atlas Research Network. Integrated genomic characterization of oesophageal carcinoma. Nature 2017). Preclinical experiments demonstrated that single agent abemaciclib has potent antitumor efficacy both in vitro and in vivo in esophageal adenocarcinoma with direct pathway inhibition (Kosovec J et al. CDK4/6 dual inhibitor abemaciclib demonstrates compelling preclinical activity against esophageal adenocarcinoma: a novel therapeutic options for a deadly disease. Oncotarget 2017 Nov 1;8[59]). Significant activity was seen in the OE33 (p=0.048) and FLO1 (p=0.043) esophageal cancer cell lines while western blot revealed downregulation of Cyclin D, phospho-pRb, E2F1, and Cyclin A2 (Kosovec J et al, 2017). In vivo experiments , utilizing the Levrat model of end-to-side esophagojejunostomy performed on Sprague-Dawley rats demonstrated that 78.9% of animals given abemaciclib demonstrated >20% tumor volume decrease (placebo 0%). Post treatment comparison between placebo and abemaciclib of qRT-PCR gene expression demonstrated downregulation of pathway correlates, including CDK4 (p=0.023), CDK6 (p=0.068), E2F1 (p=0.005), Rb1 (p=0.067), Cyclin D (p=0.031), and Cyclin A (p=0.039) (Kosovec J et al, 2017). The JPBJ phase 1b study has previously demonstrated the safety of combining abemaciclib with ramucirumab in NSCLC. Methods: Subjects with previously treated metastatic esophageal or gastroesophageal junction adenocarcinomas are eligible for this study which is currently open and enrolling patients. Squamous cell and mixed histology with neuroendocrine features are excluded. A total of 30 patients will be enrolled across 3 sites at Baylor University Medical Center in Dallas, The Allegheny Health Network in Pittsburgh and at Johns Hopkins Hospital in Baltimore. The primary objective is to describe the safety profile of abemaciclib (150mg po bid) and ramucirumab (8mg/kg iv every 2 weeks) in this patient population as assessed according to Common Terminology Criteria for Adverse Events version 4.0. Secondary objectives are to assess response rate, progression free and overall survival. In addition, we will determine the rate of stable disease at 3 months post targeted therapy. Correlative studies investigating changes in the expression of selected serum/tissue genomic markers of interest will be performed. Clinical trial information: NCT04921904.

WX-0593 combined with an epithelial growth factor receptor (EGFR) monoclonal antibody in the treatment of xenograft tumors carrying triple EGFR mutations.

BackgroundTo evaluate the safety and therapeutic efficacy of WX-0593, a newly developed potent anaplastic lymphoma kinase (ALK) inhibitor, in combination with an epithelial growth factor receptor (EGFR) monoclonal antibody (QL1203 or Vectibix) for the treatment of xenograft tumors carrying mutant EGFR and osimertinib-resistant mutations (EGFR/T790M/C797S).MethodsThe inhibition of tumor cell proliferation by WX-0593 and Vectibix alone or combined was evaluated in four EGFR triple-mutant cell lines: PC9 (EGFR Del19/T790M/C797S), NCI-H1975 (EGFR L858R/T790M/C797S), Ba/F3 (EGFR L858R/T790M/C797S and EGFR Del19/T790M/C797S). The in vivo antitumor efficacy of WX-0593 alone or combined with QL1203 or Vectibix was evaluated in xenograft tumor models of BALB/c nude mice developed from H1975 (EGFR-Del19/T790M/C797S) and Ba/F3 (EGFR-L858R/T790M/C797S) cell lines. Mice were randomized into groups and treated with or without WX-0593, QL1203, Vectibix, or their combination. The tumor volume, mouse body weight, and therapeutic side effects were monitored routinely. Blood samples were obtained from all mice at different time points after the last dosage of treatment to evaluate the pharmacokinetic parameters of the drugs.ResultsWX-0593 and Vectibix showed a strong synergistic inhibitory effect on the proliferation of two EGFR triple-mutant Ba/F3 cell lines (EGFR L858R/T790M/C797S and Del19/T790M/C797S), but little synergistic inhibitory effect on the proliferation of NCI-H1975 (EGFR L858R/T790M/C797S) and PC9 (EGFR Del19/T790M/C797S). In vivo, WX-0593 (25 mg/kg) showed a modest therapeutic effect when combined with QL1203 or Vectibix, but had no effect on tumor growth as a monotherapy at this dosage. WX-0593 (75 mg/kg) exhibited modest antitumor efficacy that was further enhanced in combination with QL1203 or Vectibix in both tumor models (H1975 and Ba/F3). No significant body weight alteration, any other side effect, or deaths were observed during treatment. Pharmacokinetic analysis showed that the serum level of QL1203 or Vectibix was significantly increased and lasted longer when combined with WX-0593.ConclusionsWX-0593 exhibited a synergetic effect with an EGFR monoclonal antibody on osimertinib-resistant EGFR-mutant non-small cell lung cancer (NSCLC) both in vitro and in vivo. Their combination showed potent antitumor efficacy and an acceptable safety profile, which may be a promising strategy for the treatment of patients with EGFR triple-mutant NSCLC resistant to osimertinib.

A phase II study of AK112 (PD-1/VEGF bispecific) in combination with chemotherapy in patients with advanced non-small cell lung cancer.

9019 Background: Based on IMPOWER 150, atezolizumab in combination with bevacizumab and platinum-based chemotherapy has been the first-line treatment for advanced non-squamous NSCLC with negative driver genes. AK112 is a global first-in-class anti-PD-1/VEGF bi-specific antibody developed by Akesobio. Preclinical and clinical studies have indicated that AK112 possess potential anti-tumor efficacy in solid tumors. Therefore, we aimed to assess the efficacy and safety of AK112 in combination with chemotherapy for patients with advanced NSCLC. Methods: This was an open-label, multi-center phase II study evaluating the efficacy and safety of AK112 in combination with chemotherapy in pts with advanced NSCLC. Enrolled pts were divided into three cohorts: Previously untreated advanced NSCLC pts with wide-type EGFR/ALK (Cohort 1), pts with EGFR mutations who had failed prior EGFR-TKI therapies without T790M mutation or failed osimertinib treatment (Cohort 2), and pts who progressed after anti-PD-1/L1 and platinum-based chemotherapy (Cohort 3). Pts were treated with 10mg/kg or 20mg/kg AK112 once every 3 weeks in combination with carboplatin and pemetrexed or carboplatin and paclitaxel for Cohort 1/2, and docetaxel for Cohort 3. Primary endpoint was ORR as assessed by investigator per RECIST v1.1. Results: 133 pts were enrolled from Feb 03, 2021 to Dec 31, 2021 and received AK112 plus chemotherapy (44 received AK112 10 mg/kg and 89 received AK112 20 mg/kg). As of Dec 31, 2021, in cohort-1, among 26 evaluable pts with squamous cell carcinoma, 20 partial response and 6 stable disease were observed for a 76.9% ORR and a 100.0% DCR, median DOR and median PFS was not reached while 6-month PFS rate was 86.2%. In Cohort-2, among 19 evaluable pts, 13 partial response and 5 stable disease were observed for a 68.4% ORR and a 94.7% DCR while median DOR was 5.5 months, and median PFS was 8.3 months. In Cohort-3, among 20 evaluable pts, 8 partial response and 8 stable disease were observed for a 40.0% ORR and a 80.0% DCR, median DOR and median PFS was not reached while 6-month PFS rate was 71.1%. Treatment emergent adverse events (TEAE) occurred in 86.5% (115/133) of the pts, and grade ≥3 AEs occurred in 28.6% (38/133) of pts including two deaths. Most common AE (incidence ≥ 5%) included alanine/aspartate aminotransferase increased, epistaxis, anemia, vomiting, nausea, rash, leukopenia, thrombocytopenia, and neutropenia. Treatment discontinuation due to AE occurred in 3.0% (4/133) of the pts. Conclusions: AK112 plus chemotherapy has shown a promising anti-tumor efficacy in each cohort and warrants potentially superior safety in comparison to anti-PD-(L)1 and anti-VEGF combination therapies in advanced NSCLC. PFS and ORR were also significantly improved with AK112 plus chemotherapy. Therefore, phase III registration studies of AK112 plus chemotherapy in advanced NSCLC will be initiated in 2022. Clinical trial information: NCT04736823.

Targeted Drug/Gene/Photodynamic Therapy via a Stimuli-Responsive Dendritic-Polymer-Based Nanococktail for Treatment of EGFR-TKI-Resistant Non-Small-Cell Lung Cancer.

Yes-associated protein (YAP) has been identified as a key driver for epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) resistance. Inhibition of YAP expression could be a potential therapeutic option for treating non-small-cell lung cancer (NSCLC). Herein, a nanococktail therapeutic strategy is proposed by employing amphiphilic and block-dendritic-polymer-based nanoparticles (NPs) for targeted co-delivery of EGFR-TKI gefitinib (Gef) and YAP-siRNA to achieve a targeted drug/gene/photodynamic therapy. The resulting NPs are effectively internalized into Gef-resistant NSCLC cells, successfully escape from late endosomes/lysosomes, and responsively release Gef and YAP-siRNA in an intracellular reductive environment. They preferentially accumulate at the tumor site after intravenous injection in both cell-line-derived xenograft (CDX) and patient-derived xenograft (PDX) models of Gef-resistant NSCLC, resulting in potent antitumor efficacy without distinct toxicity after laser irradiation. Mechanism studies reveal that the cocktail therapy could block the EGFR signaling pathway with Gef, inhibit activation of the EGFR bypass signaling pathway via YAP-siRNA, and induce tumor cell apoptosis through photodynamic therapy (PDT). Furthermore, this combination nanomedicine can sensitize PDT and impair glycolysis by downregulating HIF-1α. These results suggest that this stimuli-responsive dendritic-polymer-based nanococktail therapy may provide a promising approach for the treatment of EGFR-TKI resistant NSCLC.

A Stimuli‐Responsive Small‐Molecule Metal‐Carrying Prochelator: A Novel Prodrug Design Strategy for Metal Complexes

AbstractSelective activation of prodrugs is an important approach to reduce the side effects of disease treatment. We report a prodrug design concept for metal complexes, termed “metal‐carrying prochelator”, which can co‐carry a metal ion and chelator within a single small‐molecule compound and remain inert until it undergoes a specifically triggered intramolecular chelation to synthesize a bioactive metal complex in situ for targeted therapy. As a proof‐of‐concept, we designed a H2O2‐responsive small‐molecule prochelator, DPBD, based on the strong chelator diethyldithiocarbamate (DTC) and copper. DPBD can carry Cu2+ (DPBD‐Cu) and respond to elevated H2O2 levels in tumor cells by releasing DTC, which rapidly chelates Cu2+ from DPBD‐Cu affording a DTC–copper complex with high cytotoxicity, realizing potent antitumor efficacy with low systemic toxicity. Thus, with its unique intramolecularly triggered activation mechanism, this concept based on a small‐molecule metal‐carrying prochelator can help in the prodrug design of metal complexes.

A Stimuli-Responsive Small-Molecule Metal-Carrying Prochelator: A Novel Prodrug Design Strategy for Metal Complexes.

Selective activation of prodrugs is an important approach to reduce the side effects of disease treatment. We report a prodrug design concept for metal complexes, termed "metal-carrying prochelator", which can co-carry a metal ion and chelator within a single small-molecule compound and remain inert until it undergoes a specifically triggered intramolecular chelation to synthesize a bioactive metal complex in situ for targeted therapy. As a proof-of-concept, we designed a H2 O2 -responsive small-molecule prochelator, DPBD, based on the strong chelator diethyldithiocarbamate (DTC) and copper. DPBD can carry Cu2+ (DPBD-Cu) and respond to elevated H2 O2 levels in tumor cells by releasing DTC, which rapidly chelates Cu2+ from DPBD-Cu affording a DTC-copper complex with high cytotoxicity, realizing potent antitumor efficacy with low systemic toxicity. Thus, with its unique intramolecularly triggered activation mechanism, this concept based on a small-molecule metal-carrying prochelator can help in the prodrug design of metal complexes.

A Dimeric FAP-Targeting Small-Molecule Radioconjugate with High and Prolonged Tumor Uptake.

Imaging procedures based on small-molecule radioconjugates targeting fibroblast activation protein (FAP) have recently emerged as a powerful tool for the diagnosis of a wide variety of tumors. However, the therapeutic potential of radiolabeled FAP-targeting agents is limited by their short residence time in neoplastic lesions. In this work, we present the development and invivo characterization of BiOncoFAP, a new dimeric FAP-binding motif with an extended tumor residence time and favorable tumor-to-organ ratio. Methods: The binding properties of BiOncoFAP and its monovalent OncoFAP analog were assayed against recombinant human FAP. Preclinical experiments with 177Lu-OncoFAP-DOTAGA (177Lu-OncoFAP) and 177Lu-BiOncoFAP-DOTAGA (177Lu-BiOncoFAP) were performed on mice bearing FAP-positive HT-1080 tumors. Results: OncoFAP and BiOncoFAP displayed comparable subnanomolar dissociation constants toward recombinant human FAP in solution, but the bivalent BiOncoFAP bound more avidly to the target immobilized on solid supports. In a comparative biodistribution study, 177Lu-BiOncoFAP exhibited a more stable and prolonged tumor uptake than 177Lu-OncoFAP (∼20 vs. ∼4 percentage injected dose/g, respectively, at 24 h after injection). Notably, 177Lu-BiOncoFAP showed favorable tumor-to-organ ratios with low kidney uptake. Both 177Lu-OncoFAP and 177Lu-BiOncoFAP displayed potent antitumor efficacy when administered at therapeutic doses to tumor-bearing mice. Conclusion: 177Lu-BiOncoFAP is a promising candidate for radioligand therapy of cancer, with favorable invivo tumor-to-organ ratios, a long tumor residence time, and potent anticancer efficacy.

LAG-3xPD-L1 bispecific antibody potentiates antitumor responses of T cells through dendritic cell activation

Several preclinical studies demonstrate that antitumor efficacy of programmed cell death-1 (PD-1)/programmed death-ligand 1 (PD-L1) blockade can be improved by combination with other checkpoint inhibitors. Lymphocyte-activation gene 3 (LAG-3) is an inhibitory checkpoint receptor involved in Tcell exhaustion and tumor immune escape. Here, we describe ABL501, a bispecific antibody targeting LAG-3 and PD-L1 in modulating immune cell responses against tumors. ABL501 that efficiently inhibits both LAG-3 and PD-L1 pathways enhances the activation of effector CD4+ and CD8+ Tcells with a higher degree than a combination of single anti-LAG-3 and anti-PD-L1. The augmented effector Tcell responses by ABL501 resulted in mitigating regulatory-T-cell-mediated immunosuppression. Mechanistically, the simultaneous binding of ABL501 to LAG-3 and PD-L1 promotes dendritic cell (DC) activation and tumor cell conjugation with Tcells thatsubsequently mounts effective CD8+ Tcell responses. ABL501 demonstrates its potent invivo antitumor efficacy ina humanized xenograft model and with knockin mice expressing human orthologs. The immune profiling analysis of peripheral blood reveals an increased abundance of LAG-3hiPD-1hi memory CD4+ Tcell subset in relapsed cholangiocarcinoma patients after gemcitabine plus cisplatin therapy, which are more responsive to ABL501. This study supports the clinical evaluation of ABL501 as a novel cancer immunotherapeutic, and a first-in-human trial has started (NCT05101109).

A novel SRSF3 inhibitor, SFI003, exerts anticancer activity against colorectal cancer by modulating the SRSF3/DHCR24/ROS axis

As the modulation of serine/arginine-rich splicing factor 3 (SRSF3) may be therapeutically beneficial to colorectal cancer (CRC) treatment, the identification of novel SRSF3 inhibitors is highly anticipated. However, pharmaceutical agents targeting SRSF3 have not yet been discovered. Here, we propose a functional SRSF3 inhibitor for CRC therapy and elucidate its antitumor mechanisms. We found high expression of SRSF3 in 70.6% CRC tissues. Silencing SRSF3 markedly inhibits the proliferation and migration of CRC cells through suppression of its target gene 24-dehydrocholesterol reductase (DHCR24). This is evidenced by the links between SRSF3 and DHCR24 in CRC tissues. The novel SRSF3 inhibitor SFI003 exhibits potent antitumor efficacy in vitro and in vivo, which drives apoptosis of CRC cells via the SRSF3/DHCR24/reactive oxygen species (ROS) axis. Moreover, SFI003 is druggable with suitable pharmacokinetic properties, bioavailability, and tumor distribution. Thus, SRSF3 is a novel potential therapeutic target for CRC. Its inhibitor SFI003 may be developed as an anticancer therapeutic.

Cell membrane-anchored and tumor-targeted IL-12 (attIL12)-T cell therapy for eliminating large and heterogeneous solid tumors

Abstract Background Adoptive T-cell transfer has become an attractive therapeutic approach for hematologic malignancies but shows poor activity against large and heterogeneous solid tumors. Interleukin-12 (IL-12) potent antitumor efficacy against solid tumors, but its clinical application has been stalled because of toxicity. Here, we aimed to develop a safe approach to IL-12 T-cell therapy for eliminating large solid tumors. Methods We generated a cell membrane-anchored IL-12 (aIL12), a tumor-targeted IL-12 (ttIL12), and a cell membrane-anchored and tumor-targeted IL-12 (attIL12) armed T cells, CAR-T cells, and TCR-T cells with each. We compared the antitumor efficacy of these armed T cells in treating osteosarcoma PDX tumors and mouse melanoma tumors, and addressed the safety concerns of IL-12 expressing T cell therapy after intravenous infusions of the armed T cells. Results attIL12-T cell infusion showed remarkable antitumor efficacy in human and mouse large solid tumor models. Mechanistically, attIL12-T cells target tumor cells that express cell-surface vimentin (CSV) and accumulate in tumor sites. Such CSV-attIL12 interaction stimulates effector T cell to produce high levels of inflammatory cytokines (IFNγ, Flt3L, GM-CSF and etc.) only in tumors, which in turn triggers dendritic cell maturation for activating secondary T-cell responses and tumor antigen spreading. Both attIL12- and aIL12-T-cell transfer eliminated peripheral cytokine release and the associated toxic effects due to tumor-specific T cell distribution and compartment-dependent cytokine secretion. Conclusions This novel approach sheds light on the safe application of IL-12–based T-cell therapy for large and heterogeneous solid tumors. Supported by R01 CA200574

Novel MDM2 Inhibitor XR-2 Exerts Potent Anti-Tumor Efficacy and Overcomes Enzalutamide Resistance in Prostate Cancer

Background: The inactivation of tumor-suppressor p53 plays an important role in second generation anti-androgens (SGAs) drug resistance and neuroendocrine differentiation in castration-resistant prostate cancer (CRPC). The reactivation of p53 by blocking the MDM2–p53 interaction represents an attractive therapeutic remedy in cancers with wild-type or functional p53. Whether MDM2-p53 inhibitor could overcome SGAs drug resistance in CRPC is still needed further research. Here, we investigated the anti-tumor efficacy and mechanisms of a novel MDM2-p53 inhibitor XR-2 in CRPC. Methods: To investigate the functions and mechanisms of XR-2 in prostate cancer, in vitro and in vivo biofunctional assays were performed. Western blot and qRT-PCR assay were performed to detect the protein and mRNA expression levels of indicated genes. CCK8, colony formation, flow cytometry and senescence assays were performed for cell function identifications. RNA-sequencing and bioinformatics analysis were mainly used to identify the influence of XR-2 on prostate cancer cells transcriptome. Subcutaneous 22Rv1 derived xenografts mice model was used to investigate the in vivo anti-tumor activity of XR-2. In addition, the broad-spectrum anti-tumor activities in vivo of XR-2 were evaluated by different xenografts mice models. Results: XR-2 could directly bind to MDM2, potently reactivate the p53 pathway and thus induce cell cycle arrest and apoptosis in wild-type p53 CRPC cell lines. XR-2 also suppresses the AR pathway as p53 regulates AR transcription inhibition and MDM2 participates in AR degradation. As a result, XR-2 efficiently inhibited CRPC cell viability, showed a synergistic effect with enzalutamide and overcame enzalutamide resistance both in vitro and in vivo. Moreover, results illustrated that XR-2 possesses broad-spectrum anti-tumor activities in vivo with favourable safety. Conclusion: MDM2-p53 inhibitor (XR-2) possesses potently prostate cancer progresses inhibition activity both in vitro and in vivo. XR-2 shows a synergistic effect with enzalutamide and overcomes enzalutamide resistance.

Chimeric antigen receptor clustering via cysteines enhances T-cell efficacy against tumor.

Chimeric antigen receptor (CAR) T-cell therapy achieves great success for hematological malignancies. However, clinical trials have revealed some limitations in both improving the efficacy and reducing the relapse, which calls for innovative strategies to engineer more powerful CAR-T cells. Promoting the formation of CAR clusters provides an alternative approach and potentially improves current CAR T-cell therapy against cancers. Here, we generated CARCys-T cells using a 4-1BB-derived hinge region including 11 cysteines residues. The cysteines in the hinge were found to facilitate CARCys clustering upon antigen stimulation and promote the antitumor activity of CAR-T cells. Compared with most conventionally used CAR-T cells with CD8α-derived hinge (CARconv-T cells), CARCys-T cells exhibited larger diameter of CAR clusters and enhanced antigen-specific tumor lysis both in vitro and in vivo. In addition, the CARCys-mediated enhancement could be applied to HER2, CD19 as well as GPC3-targeted CAR-T cells. More importantly, CARCys-T cells showed potent antitumor efficacy in clinically relevant patient-derived primary tumor cells and organoids. Thus, the novel hinge containing 11 cysteines provides a promising strategy to facilitate CAR clustering and maximize anti-tumor activity of CAR-T cells, which emphasizes the importance of CAR clustering to improve CAR T-cell therapy in the clinic.

Targeting the tumor microenvironment by an enzyme-responsive prodrug of tubulin destabilizer for triple-negative breast cancer therapy with high safety

In response to the long-term potential toxicity concerns of tubulin destabilizer, an enzyme-responsive prodrug therapy for triple-negative breast cancer was developed based on the different β-glucuronidase levels between tumor and normal tissues in this study. All the prodrugs synthesized herein showed remarkable stability in phosphate buffer and bovine serum solution, among which 17a was found to be more susceptible to enzymatic cleavage. 17a exhibited excellent selectivity between the in vitro antiproliferative activities against β-glucuronidase-pretreated and -untreated cancer cells (IC50 (+Enz) = 8.9–15.7 nM, IC50 (-Enz) > 50 μM), along with favorable liver microsomal metabolic stability and improved aqueous solubility. Furthermore, as a candidate prodrug 17a showed potent antitumor efficacy in MDA-MB-231 xenograft mouse model without causing perceptible injury to organs. Importantly, 17a exhibited superior safety profiles with higher LD50 value and no perceivable cardiotoxicity, which was a major dose-limiting adverse effect for the parent compound 1. These salient toxicity-reduced effects of 17a would merit further in-depth assessment of this compound for preclinical therapeutic usages.

Emerging Albumin-Binding Anticancer Drugs for Tumor-Targeted Drug Delivery: Current Understandings and Clinical Translation.

Albumin has shown remarkable promise as a natural drug carrier by improving pharmacokinetic (PK) profiles of anticancer drugs for tumor-targeted delivery. The exogenous or endogenous albumin enhances the circulatory half-lives of anticancer drugs and passively target the tumors by the enhanced permeability and retention (EPR) effect. Thus, the albumin-based drug delivery leads to a potent antitumor efficacy in various preclinical models, and several candidates have been evaluated clinically. The most successful example is Abraxane, an exogenous human serum albumin (HSA)-bound paclitaxel formulation approved by the FDA and used to treat locally advanced or metastatic tumors. However, additional clinical translation of exogenous albumin formulations has not been approved to date because of their unexpectedly low delivery efficiency, which can increase the risk of systemic toxicity. To overcome these limitations, several prodrugs binding endogenous albumin covalently have been investigated owing to distinct advantages for a safe and more effective drug delivery. In this review, we give account of the different albumin-based drug delivery systems, from laboratory investigations to clinical applications, and their potential challenges, and the outlook for clinical translation is discussed. In addition, recent advances and progress of albumin-binding drugs to move more closely to the clinical settings are outlined.

In vivo imaging with two-photon microscopy to assess the tumor-selective binding of an anti-CD137 switch antibody

STA551, a novel anti-CD137 switch antibody, binds to CD137 in an extracellular ATP concentration-dependent manner. Although STA551 is assumed to show higher target binding in tumor tissues than in normal tissues, quantitative detection of the target binding of the switch antibody in vivo is technically challenging. In this study, we investigated the target binding of STA551 in vivo using intravital imaging with two-photon microscopy. Tumor-bearing human CD137 knock-in mice were intravenously administered fluorescently labeled antibodies. Flow cytometry analysis of antibody-binding cells and intravital imaging using two-photon microscopy were conducted. Higher CD137 expression in tumor than in spleen tissues was detected by flow cytometry analysis, and T cells and NK cells were the major CD137-expressing cells. In the intravital imaging experiment, conventional and switch anti-CD137 antibodies showed binding in tumors. However, in the spleen, the fluorescence of the switch antibody was much weaker than that of the conventional anti-CD137 antibody and comparable with that of the isotype control. In conclusion, we were able to assess switch antibody biodistribution in vivo through intravital imaging with two-photon microscopy. These results suggest that the tumor-selective binding of STA551 leads to a wide therapeutic window and potent antitumor efficacy without systemic immune activation.