Potent Antitumor Efficacy Research Articles

Red blood cell membrane-coated FLT3 inhibitor nanoparticles to enhance FLT3-ITD acute myeloid leukemia treatment

FMS-like tyrosine kinase 3 (FLT3) is a viable and important therapeutic target for acute myeloid leukemia (AML). FLT3 internal tandem duplication (FLT3-ITD) mutations have been identified in approximately 30% of AML patients, and are associated with unfavorable prognosis, higher risk of relapse, drug resistance, and poor clinical outcome. Even FLT3 inhibitors have demonstrated promising efficacy, they cannot cure AML or even significantly extend the lives of patients with FLT3-ITD mutations. This is partly because of poor water solubility, insufficient membrane penetration and short half-life of small molecule inhibitors. Besides, the presence of enzymes like CYP3A4 in bone marrow accelerate the elimination and metabolism of FLT3 inhibitors, resulting in low plasma concentrations and side effects. Here we report the erythrocyte membrane-camouflaged FLT3 inhibitor nanoparticles to enhance FLT3-ITD AML treatment. Briefly, we physically coextruded red blood cell (RBC) membrane vesicles with nanoparticles derived from FLT3 inhibitor F30 to obtain F30@RBC-M, which exhibited comparable potent FLT3-ITD inhibitory effects compared to free F30in vitro, while displaying a higher potent antitumor efficacy in xenograft models due to the prolonged circulation properties. Furthermore, administration of F30@RBC-M significantly extended the survival of mice in a transplanted mouse model than F30 free drug. These findings suggest that RBC membrane-coated nanoparticles derived from FLT3 inhibitors hold promise as a tool to enhance the therapeutic efficacy to treat FLT3-ITD AML.

Synthesis, crystal structure, DFT calculation, Hirshfeld suface analysis and antitumor activity of 4-(tert-butyl)-5-(1H-1,2,4-triazole-1-yl)thiazole derivatives containing benzoxazinone

Seven novel 4-(tert-butyl)-5-(1H-1,2,4-triazole-1-yl)thiazole derivatives containing benzoxazinone (B1-B7) were obtained by cyclization of 4-(tert-butyl)-5-(1H-1,2,4-triazol-1-yl)-N-(2-hydroxy-benzyl)thiazol-2-amine. The chemical structures of the target compounds were confirmed by 1H NMR and 13C NMR. The single crystal X-ray diffraction revealed that B1 crystallizes in a triclinic system with the space group P1¯. Hirshfeld surfaces and two dimensional (2D) fingerprint plots were employed to quantitatively characterize the influence of intermolecular interactions within the crystal lattice of B1. And the molecular electrostatic potential (MEP) surface was calculated employing Becke-3-Lee Yang Parr (B3LYP)/6-311++G (d,p) theoretical approach. Meanwhile, the Frontier-molecular orbitals of B1 and B2 were calculated using density functional theory (DFT) B3LYP technique with the def2-SVP basis set in the ground state. Preliminary biological tests revealed that the title compounds had certain antitumor activities against three cell lines MCF-7, Hela and A549, and compound B2 presented the most potent antitumor efficacy against A549.

Oncolytic varicella-zoster virus engineered with ORF8 deletion and armed with drug-controllable interleukin-12

BackgroundThe varicella-zoster virus (VZV), belonging to the group of human α-herpesviruses, has yet to be developed as a platform for oncolytic virotherapy, despite indications from clinical case reports suggesting a...

Sialic Acid Conjugate-Modified Cationic Liposomal Paclitaxel for Targeted Therapy of Lung Metastasis in Breast Cancer: What a Difference the Cation Content Makes.

Cationic lipids play a pivotal role in developing novel drug delivery systems for diverse biomedical applications, owing to the success of mRNA vaccines against COVID-19 and the Phase III antitumor agent EndoTAG-1. However, the therapeutic potential of these positively charged liposomes is limited by dose-dependent toxicity. While an increased content of cationic lipids in the formulation can enhance the uptake and cytotoxicity toward tumor-associated cells, it is crucial to balance these advantages with the associated toxic side effects. In this work, we synthesized the cationic lipid HC-Y-2 and incorporated it into sialic acid (SA)-modified cationic liposomes loaded with paclitaxel to target tumor-associated immune cells efficiently. The SA-modified cationic liposomes exhibited enhanced binding affinity toward both RAW264.7 cells and 4T1 tumor cells in vitro due to the increased ratios of cationic HC-Y-2 content while effectively inhibiting 4T1 cell lung metastasis in vivo. By leveraging electrostatic forces and ligand-receptor interactions, the SA-modified cationic liposomes specifically target malignant tumor-associated immune cells such as tumor-associated macrophages (TAMs), reduce the proportion of cationic lipids in the formulation, and achieve dual objectives: high cellular uptake and potent antitumor efficacy. These findings highlight the potential advantages of this innovative approach utilizing cationic liposomes.

High-Specificity CRISPR-Mediated Genome Engineering in Anti-BCMA Allogeneic CAR T Cells Suppresses Allograft Rejection in Preclinical Models.

Allogeneic chimeric antigen receptor (CAR) T cell therapies hold the potential to overcome many of the challenges associated with patient-derived (autologous) CAR T cells. Key considerations in the development of allogeneic CAR T cell therapies include prevention of graft-vs-host disease (GvHD) and suppression of allograft rejection. Here, we describe preclinical data supporting the ongoing first-in-human clinical study, the CaMMouflage trial (NCT05722418), evaluating CB-011 in patients with relapsed/refractory multiple myeloma. CB-011 is a hypoimmunogenic, allogeneic anti-B-cell maturation antigen (BCMA) CAR T cell therapy candidate. CB-011 cells feature 4 genomic alterations and were engineered from healthy donor-derived T cells using a Cas12a CRISPR hybrid RNA-DNA (chRDNA) genome-editing technology platform. To address allograft rejection, CAR T cells were engineered to prevent endogenous HLA class I complex expression and overexpress a single-chain polyprotein complex composed of beta-2 microglobulin (B2M) tethered to HLA-E. In addition, T-cell receptor (TCR) expression was disrupted at the TCR alpha constant locus in combination with the site-specific insertion of a humanized BCMA-specific CAR. CB-011 cells exhibited robust plasmablast cytotoxicity in vitro in a mixed lymphocyte reaction in cell cocultures derived from patients with multiple myeloma. In addition, CB-011 cells demonstrated suppressed recognition by and cytotoxicity from HLA-mismatched T cells. CB-011 cells were protected from natural killer cell-mediated cytotoxicity in vitro and in vivo due to endogenous promoter-driven expression of B2M-HLA-E. Potent antitumor efficacy, when combined with an immune-cloaking armoring strategy to dampen allograft rejection, offers optimized therapeutic potential in multiple myeloma. See related Spotlight by Caimi and Melenhorst, p. 385.

Personalized neoantigen vaccine enhances the therapeutic efficacy of bevacizumab and anti-PD-1 antibody in advanced non-small cell lung cancer

Clinically, a considerable number of non-small cell lung cancer (NSCLC) patients are unable to receive or resist chemotherapy, and the efficacy of non-chemotherapy treatment strategies based on anti-angiogenic agents combined with immune checkpoint blockade is still unsatisfactory. Neoantigen vaccine, based on personalized tumor DNA mutations, could elicit tumor specific T cell infiltration into the tumor site, exerting potent anti-tumor efficacy. Here, we evaluated the feasibility and safety of a new antitumor strategy by adding neoantigen vaccine to the regimen of bevacizumab and anti-PD-1 antibody. Firstly, 7 novel immunogenic neoantigen peptides were identified and developed for neoantigen vaccine (LLCvac), which can elicit strong antitumor immune response in vivo. Then, in orthotopic lung cancer model, LLCvac further combining with bevacizumab and anti-PD-1 antibody exerted a stronger antitumor effect, exhibiting significant decrease of tumor volume without obvious toxicity. Furthermore, tumor immune microenvironment assessment also showed that the proportion of neoantigen-specific T cells in blood could be induced dramatically by the combined therapy. And a large amount of neoantigen-specific Ki67-positive CD8+ T cells were found in tumor tissues, which infiltrated tumor tissues effectively to kill tumor cells expressing identified neoantigens. Overall, these results suggested that this combined therapy could safely induce robust antitumor efficacy, serving as an effective chemotherapy-free strategy for NSCLC treatment.

Heterostructure nanozymes with sustainable tumor microenvironment regulation and reinforced hyperthermia-responsive catalytic activities for orchestrated osteosarcoma catalytic therapy

Catalytic therapy employing nanozymes has evoked extensive exploration as a promising regimen for cancer theranostics, with specific reactivity in response to tumor microenvironment (TME). However, inherently deficient catalytic efficiency and instability compromise their anticancer efficacy, thus limiting biomedical applications. Herein, we report a novel gold-based heterostructure nanozyme (ATP@IR) with dual enzymes-mimicking activities and illustrious photothermal effect for bone tumor catalytic treatment, aiming at obtaining balanced synergism of catalytic therapy and hyperthermia. With the advantage of its unique tipping-deposition heterostructure structure, ATP@IR nanozymes expedite the electron-hole pair separation and produce massive hot electron, leading to illustrious photothermal effect for tumor hyperthermia treatment. Intriguingly, ATP@IR nanozymes display both intrinsic peroxidase (POD)-mimicking (Km: 39.56 mM) and catalase (CAT)-mimicking activities under weakly acidic TME, which could breakdown endogenous H2O2 into deleterious hydroxyl radicals (·OH) and simultaneously replenish oxygen, realizing efficient tumor ablation and hypoxia alleviation. Meanwhile, elevated oxygen level is leveraged to synergistically produce more reactive oxygen species by reacting with ICG for enhanced photodynamic efficacy. Benefiting from phenomenal thermal conversion efficiency (η = 73.6 %), the dual enzyme-mimicking activities of nanozymes are dramatically improved upon mild hyperthermia when exposure to laser, which actualize hyperthermia-augmented catalytic treatment. Impressively, the potent antitumor efficacy is demonstrated by in vivo xenograft osteosarcoma models without causing lung metastasis. Accordingly, this work provides a reasonable strategy by integrating nanocatalytic treatment, hyperthermia treatment, and TME modulation for efficient treatment of osteosarcoma.

Fc receptor-like 5 (FCRL5)-directed CAR-T cells exhibit antitumor activity against multiple myeloma

Multiple myeloma (MM) remains a challenging hematologic malignancy despite advancements in chimeric antigen receptor T-cell (CAR-T) therapy. Current targets of CAR-T cells used in MM immunotherapy have limitations, with a subset of patients experiencing antigen loss resulting in relapse. Therefore, novel targets for enhancing CAR-T cell therapy in MM remain needed. Fc receptor-like 5 (FCRL5) is a protein marker with considerably upregulated expression in MM and has emerged as a promising target for CAR-T cell therapeutic interventions, offering an alternative treatment for MM. To further explore this option, we designed FCRL5-directed CAR-T cells and assessed their cytotoxicity in vitro using a co-culture system and in vivo using MM cell-derived xenograft models, specifically focusing on MM with gain of chromosome 1q21. Given the challenges in CAR-T therapies arising from limited T cell persistence, our approach incorporates interleukin-15 (IL-15), which enhances the functionality of central memory T (TCM) cells, into the design of FCRL5-directed CAR-T cells, to improve cytotoxicity and reduce T-cell dysfunction, thereby promoting greater CAR-T cell survival and efficacy. Both in vitro and xenograft models displayed that FCRL5 CAR-T cells incorporating IL-15 exhibited potent antitumor efficacy, effectively inhibiting the proliferation of MM cells and leading to remarkable tumor suppression. Our results highlight the capacity of FCRL5-specific CAR-T cells with the integration of IL-15 to improve the therapeutic potency, suggesting a potential novel immunotherapeutic strategy for MM treatment.

Preliminary Study on Pharmacokinetics and Antitumor Pharmacodynamics of Folic Acid Modified Crebanine Polyethyleneglycol-Polylactic Acid Hydroxyacetic Acid Copolymer Nanoparticles.

Liver cancer is associated significantly with morbidity and mortality. The combination of low-intensity ultrasound with nanomedicine delivery systems holds promise as an alternative for the treatment for liver cancer. This study focuses on the utilization of folic acid (FA) modified nanoparticles, which are loaded with fluorescent dye DiR and liquid fluorocarbon (PFP). These nanoparticles have the potential to enhance liver cancer targeting under ultrasound stimulation and future applications in vivo. The pharmacokinetics and tissue distribution of folic acid-modified Crebanine polyethylene glycol-polylactic acid copolymer nanoparticles (FA-Cre@PEG-PLGA NPs) were investigated. The pharmacokinetic parameters, liver targeting, and in vivo distribution were assessed. Additionally, the inhibitory impacts of FA-Cre@PEG-PLGA NPs in combination with ultrasonic irradiation on the proliferation and acute toxicity of H22 cells of mouse hepatoma were investigated in vitro. The tumor targeting and anti-tumor efficacy of FA-Cre@PEG-PLGA NPs were assessed utilizing a small animal in vivo imaging system and an in situ hepatocellular carcinoma transplantation model, respectively. The pharmacokinetic studies and tissue distribution tests demonstrated that FA-Cre@PEG-PLGA NPs conspicuously prolonged the half-life and retention time of the drug in rats, and the liver targeting effect was pronounced. Additionally, the in vivo acute toxicity test indicated that FA-Cre@PEG-PLGA NPs had minimal adverse reactions and could fulfill the aim of attenuating the drug. The outcomes of the animal experiments further substantiated that FA-Cre@PEG-PLGA NPs had a longer retention time at the tumor site, a superior anti-tumor effect, and less damage to liver and kidney tissue. The integration of FA-Cre@PEG-PLGA NPs with ultrasound irradiation demonstrated exceptional safety and potent anti-tumor efficacy in vivo, presenting a promising therapeutic strategy for the treatment of liver cancer through the combination of ultrasound technology with a nanomedicine delivery system.

Synthesis of a celastrol derivative as a cancer stem cell inhibitor through regulation of the STAT3 pathway for treatment of ovarian cancer.

Accumulating evidence suggests that the root of drug chemoresistance in ovarian cancer is tightly associated with subpopulations of cancer stem cells (CSCs), whose activation is largely associated with signal transducer and activator of transcription 3 (STAT3) signaling. Recently, celastrol has shown a significant anti-cancer effect on ovarian cancer, but its clinical translation is very challenging due to its oral bioavailability and high organ toxicity. In this study, a celastrol derivative (Cel-N) was synthesized to augment the overall efficacy, and its underlying mechanisms were also explored. Different ovarian cancer cells, SKOV3 and A2780, were used to evaluate and compare the anticancer effects. Cel-N displayed potent activities against all the tested ovarian cancer cells, with the lowest IC50 value of 0.14-0.25 μM. Further studies showed that Cel-N effectively suppressed the colony formation and sphere formation ability, decreased the percentage of CD44+CD24- and ALDH+ cells, and induced ROS production. Furthermore, western blot analysis indicated that Cel-N significantly inhibited both Tyr705 and Ser727 phosphorylation and reduced the protein expression of STAT3. In addition, Cel-N could dramatically induce apoptosis and cell cycle arrest, and inhibit migration and invasion. Importantly, Cel-N showed a potent antitumor efficacy with no or limited systemic toxicity in mice xenograft models. The anticancer effect of Cel-N is stronger than celastrol. Cel-N attenuates cancer cell stemness, inhibits the STAT3 pathway, and exerts anti-ovarian cancer effects in cell and mouse models. Our data support that Cel-N is a potent drug candidate for ovarian cancer.

Engineering macrophage membrane-camouflaged nanoplatforms with enhanced macrophage function for mediating sonodynamic therapy of ovarian cancer.

Cancer immunotherapy has demonstrated remarkable efficacy in the treatment of cancer, and it has been successfully applied in the treatment of various solid tumors. However, the response rates to immunotherapy in patients with ovarian cancer remain modest because of the immunosuppressive tumor microenvironment (TME). Tumor-associated macrophages (TAMs) represent the predominant myeloid cell population within the TME, which adopt the protumorigenic M2 phenotype and are blinded by the "don't eat me" signals from tumor cells. These characteristics of TAMs result in insufficient phagocytic activation. In this study, we constructed a SIM@TR-NP-mediated combination therapy of sonodynamic and immunotherapy. SIM@TR-NPs were modified by engineered macrophage membranes with overexpressed sialic acid-binding Ig-like lectin 10 (Siglec-10), and were internally loaded with sonosensitizer 4,4',4'',4'''-(porphine-5,10,15,20-tetrayl)tetrakis(benzoic acid) and immune adjuvant resiquimod. SIM@TR-NPs can block "don't eat me" signals to enhance macrophage phagocytosis and trigger the polarization of TAMs toward the M1 phenotype, thereby improving the immunosuppressive TME. Simultaneously, upon ultrasound irradiation, SIM@TR-NP-mediated sonodynamic therapy (SDT) triggered immunogenic cell death in tumor cells, in combination with TAM-based immunotherapy, transforming the "immune cold tumor" into an "immune hot tumor". SIM@TR-NP-mediated sonodynamic immunotherapy exhibited potent antitumor efficacy in ovarian cancer and exhibited substantial potential for improving the immunosuppressive TME. This study presents an emerging therapeutic regimen for ovarian cancer that synergizes TAM-based antitumor immunotherapy and SDT.

Tackling Esophageal Squamous Cell Carcinoma with ITFn-Pt(IV): A Novel Fusion of PD-L1 Blockade, Chemotherapy, and T-cell Activation.

PD-1/PD-L1 blockade immunotherapy has gained approval for the treatment of a diverse range of tumors; however, its efficacy is constrained by the insufficient infiltration of T lymphocytes into the tumor microenvironment, resulting in suboptimal patient responses. Here, a pioneering immunotherapy ferritin nanodrug delivery system denoted as ITFn-Pt(IV) is introduced. This system orchestrates a synergistic fusion of PD-L1 blockade, chemotherapy, and T-cell activation, aiming to augment the efficacy of tumor immunotherapy. Leveraging genetic engineering approach and temperature-regulated channel-based drug loading techniques, the architecture of this intelligent responsive system is refined. It is adept at facilitating the precise release of T-cell activating peptide Tα1 in the tumor milieu, leading to an elevation in T-cell proliferation and activation. The integration of PD-L1 nanobody KN035 ensures targeted engagement with tumor cells and mediates the intracellular delivery of the encapsulated Pt(IV) drugs, culminating in immunogenic cell death and the subsequent dendritic cell maturation. Employing esophageal squamous cell carcinoma (ESCC) as tumor model, the potent antitumor efficacy of ITFn-Pt(IV) is elucidated, underscored by augmented T-cell infiltration devoid of systemic adverse effects. These findings accentuate the potential of ITFn-Pt(IV) for ESCC treatment and its applicability to other malignancies resistant to established PD-1/PD-L1 blockade therapies.

Structure-activity relationship study of 1,6-naphthyridinone derivatives as selective type II AXL inhibitors with potent antitumor efficacy

The role of AXL in various oncogenic processes has made it an attractive target for cancer therapy. Currently, kinase selectivity profiles, especially circumventing MET inhibition, remain a scientific issue of great interest in the discovery of selective type II AXL inhibitors. Starting from a dual MET/AXL-targeted lead structure from our previous work, we optimized a 1,6-naphthyridinone series using molecular modeling-assisted compound design to improve AXL potency and selectivity over MET, resulting in the potent and selective type II AXL-targeted compound 25c. This showed excellent AXL inhibitory activity (IC50 = 1.1 nM) and 343-fold selectivity over the highly homologous kinase MET in biochemical assays. Moreover, compound 25c significantly inhibited AXL-driven cell proliferation, dose-dependently suppressed 4T1 cell migration and invasion, and induced apoptosis. Compound 25c also showed noticeable antitumor efficacy in a BaF3/TEL-AXL xenograft model at well-tolerated doses. Overall, this study presented a potent and selective type II AXL-targeted lead compound for further drug discovery.

Abstract B127: Novel anti-CD147 antibody DS-1471a exerts antitumor effect in hepatocellular carcinoma Patient Derived Xenograft models and its efficacy correlates with the expression of CD147

Abstract Background: CD147 is an immunoglobulin-like receptor that is highly expressed in various cancers and involved in the growth, metastasis, and activation of inflammatory pathways via interactions with various functional molecules, such as integrins, CD44, and monocarboxylate transporters. Through screening of CD147-targeting antibodies with antitumor efficacy, we developed a novel humanized IgG4-formatted monoclonal anti-CD147 antibody, DS-1471a. Our previous studies revealed this antibody showed potent antitumor efficacy superior to standard of care drugs in various cell line xenograft mouse models such as hepatocellular carcinoma (HCC), pancreatic invasive ductal adenocarcinoma, and chronic myelogenous leukemia. In this study, we further examined antitumor efficacy and biomarker expression in 18 patient derived xenograft (PDX) models established from HCC patients. Methods: DS-1471a or vehicle was administered intravenously once per week at 10 mg/kg doses in HCC-PDX models and tumor volumes were measured every 2 or 3 days. After tumors were harvested, samples were analyzed by simple western blotting (sWB, capillary based western blotting system) and immunohistochemistry (IHC). H-scores were calculated from IHC results and correlation between H-score, protein expression by sWB, and tumor growth inhibition (TGI) ratio were examined. Results: Seven out of 18 PDX models showed over 50% TGI with DS-1471a treatment in comparison to vehicle, regardless of gender, tumor grade, and hepatitis virus type. Protein expression analysis by sWB showed good correlation of several biomarker candidates with efficacy of DS-1471a. Among the biomarker candidates tested, CD147, the DS-1471a target molecule, showed the strongest correlation with efficacy (r = 0.78). SMAD4, which regulates the downstream signaling of the CD147 complex, and FBXO22, reported to contribute to CD147 degradation, also showed good correlation with DS-1471a efficacy (r = 0.66 and r = 0.77, respectively). By both sWB and IHC analysis, all PDX models showed negative or weak KLF5 expression, another biomarker candidate for DS-1471a that is known to be a suppressor for SMAD signaling pathway. A clear correlation between CD147 H-score and TGI was observed (r= 0.74) in these patient derived PDX models. Correlation between CD147 H-score and protein expression levels by sWB was also observed (r = 0.71). Of note, all PDX models showed moderate to high expression of CD147. Conclusion: These pre-clinical study results suggest DS-1471a may be potentially beneficial for HCC treatment. In addition, our results show correlation of DS-1471a efficacy with expression of CD147. Further studies are needed to further understand the full potential benefit of DS-1471a in clinical trials. Citation Format: Hiroshi Yuita, Ryuichi Nakamura, Jun Tsukada, Hideo Yukinaga, Huynh The Hung, Keisuke Fukuchi. Novel anti-CD147 antibody DS-1471a exerts antitumor effect in hepatocellular carcinoma Patient Derived Xenograft models and its efficacy correlates with the expression of CD147 [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 B127.

ZGGS15, a Novel IgG4 Bispecific Monoclonal Antibody Targeting Both LAG-3 and Tigit, Demonstrates Outstanding Anti-Tumor Activity and Can be Used As Monotherapy or in Combination with Anti-PD-1 Antibodies for Cancer Immunotherapy

Background This study aims to evaluate the anti-tumor efficacy of ZGGS15, a novel IgG4 bispecific monoclonal antibody (BsAb) targeting TIGIT and LAG-3, for cancer immunotherapy. Methods: ZGGS15 was generated in-house with the standard recombinant BsAb technology. ZGGS15 proteins were purified with protein A affinity column chromatography and evaluated for purity by size-exclusion high-performance liquid chromatography. The ZGGS15 affinity was assessed using a biolayer interferometry (BLI) assay. The EC50s of ZGGS15 binding to TIGIT and LAG-3 were determined using the ELISA technique. The EC50s of ZGGS15 binding to REH, Hela, MCF-7, and HCC1954 cells were measured by flow cytometry. Antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) activities were also evaluated by flow cytometry. BALB/c-hPD-1/hLAG-3 and BALB/c-hPD-1/hTIGIT subcutaneously transplanted with CT26.WT tumor mouse models, and peripheral blood mononuclear cells (PBMC) -humanized NOG bearing A375 tumor mouse model were used for in vivo anti-tumor efficacy evaluation. Results ZGGS15 binds specifically to activated CD4 + and CD8 + T cells and human LAG-3 and TIGIT proteins expressed by CHO-K1 cells identically with a dose-dependent nanomolar affinity of 0.69 nM and 1.87 nM for human LAG-3 and TIGIT, respectively. In vitro kinetic analysis BLI results revealed ZGGS15 has high affinity for human LAG-3 and human TIGIT proteins, with KDs of 3.05 nM and 2.65 nM, respectively. ZGGS15 binds to human LAG-3 and human TIGIT simultaneously with similar affinity. Furthermore, ZGGS15 competitively inhibited the binding of LAG-3 to MHC-II (IC50 = 0.77 nM) and the binding of TIGIT to CD155 (IC50=0.24 nM). ZGGS15 induced better T cell activation than single anti-LAG3 or anti-TIGIT mAb agent or combined with different stimuli with or without cancer cells. When combined with nivolumab, ZGGS15 can further enhance the T cell activation in a dose-dependent manner. ADCC/CDC assay results showed that ZGGS15 binding to target cells does not induce ADCC or CDC. ZGGS15 interacts with the human neonatal Fc receptor in a pH-dependent manner, which can promote recirculation of ZGGS15 with a prolonged half-life in plasma. Besides, ZGGS15 interacts with human Fc-γ receptors and the human complement component, C1q, with low binding affinities. However, ZGGS15 does not induce obvious cytokine production from PBMCs, including IFN-γ, IL-2, IL-6, IL-10, and TNF-α. In vivo anti-tumor efficacy for ZGG15 in PBMC humanized NOG mice bearing A375 tumors showed that ZGGS15 had significant anti-tumor effects in the subcutaneous A375 xenograft model. ZGGS15 showed a better anti-tumor inhibition than single anti-LAG-3 or anti-TIGIT agent and a synergetic effect when combined with nivolumab. On day 18 post-treatment, the ZSAB015 plus nivolumab treatment demonstrated a significantly tumor growth inhibition (TGI) of 95.80% ( p =0.001), compared with the vehicle group. In vivo efficacy tests in BALB/c-hPD-1/hLAG-3 mice subcutaneously transplanted with CT26.WT tumor model showed that both high and low doses of ZGGS15 could inhibit the growth of CT26.WT colon cancer. The combination of high-dose ZGGS15 plus nivolumab has better efficacy than high-dose ZGGS15 and is slightly better than nivolumab alone. The tumor volume inhibition rate (TGITV) for ZGGS15 at 2 mg/kg was 69.70%, and for ZGGS15 at 5 mg/kg plus nivolumab at 1 mg/kg, it was 94.03% ( p < 0.001). During the experiment, the tumor-bearing mice tolerated it well, and no adverse reactions were observed. In vivo efficacy tests of ZGG15 in BALB/c-hPD-1/hTIGIT mice subcutaneously transplanted with CT26.WT tumor model showed that ZGGS15 at a low dose (1.5 mg/kg) demonstrated a significant tumor inhibitory effect, with the TGITV of 51.42% ( p < 0.01). The combination of ZGGS15 high-dose with nivolumab (5 mg/kg + 1 mg/kg) showed a better tumor inhibitory effect compared with ZGGS15 high-dose (5 mg/kg), with the TGITV of 81.15% and 41.54%, respectively ( p < 0.05). Conclusions ZGGS15 has excellent anti-tumor activity by inhibiting LAG-3 and TIGIT as a new IgG4 BsAb. ZGGS15 also exhibits potent anti-tumor efficacy in transgenic mouse models combined with nivolumab. Additionally, ZGGS15 does not elicit ADCC or CDC, therefore avoiding the cross-reaction of non-target binding. Finally, ZGGS15 does not induce cytokine production, mitigating any potential adverse effects of CRS.

CDC7 inhibition induces replication stress-mediated aneuploid cells with an inflammatory phenotype sensitizing tumors to immune checkpoint blockade

Serine/threonine kinase, cell division cycle 7 (CDC7) is critical for initiating DNA replication. TAK-931 is a specific CDC7 inhibitor, which is a next-generation replication stress (RS) inducer. This study preclinically investigates TAK-931 antitumor efficacy and immunity regulation. TAK-931 induce RS, generating senescence-like aneuploid cells, which highly expressed inflammatory cytokines and chemokines (senescence-associated secretory phenotype, SASP). In vivo multilayer-omics analyses in gene expression panel, immune panel, immunohistochemistry, RNA sequencing, and single-cell RNA sequencing reveal that the RS-mediated aneuploid cells generated by TAK-931 intensively activate inflammatory-related and senescence-associated pathways, resulting in accumulation of tumor-infiltrating immune cells and potent antitumor immunity and efficacy. Finally, the combination of TAK-931 and immune checkpoint inhibitors profoundly enhance antiproliferative activities. These findings suggest that TAK-931 has therapeutic antitumor properties and improved clinical benefits in combination with conventional immunotherapy.

Tumor Regression upon Intratumoral and Subcutaneous Dosing of the STING Agonist ALG-031048 in Mouse Efficacy Models.

Stimulator of interferon genes (STING) agonists have shown potent anti-tumor efficacy in various mouse tumor models and have the potential to overcome resistance to immune checkpoint inhibitors (ICI) by linking the innate and acquired immune systems. First-generation STING agonists are administered intratumorally; however, a systemic delivery route would greatly expand the clinical use of STING agonists. Biochemical and cell-based experiments, as well as syngeneic mouse efficacy models, were used to demonstrate the anti-tumoral activity of ALG-031048, a novel STING agonist. In vitro, ALG-031048 is highly stable in plasma and liver microsomes and is resistant to degradation via phosphodiesterases. The high stability in biological matrices translated to good cellular potency in a HEK 293 STING R232 reporter assay, efficient activation and maturation of primary human dendritic cells and monocytes, as well as long-lasting, antigen-specific anti-tumor activity in up to 90% of animals in the CT26 mouse colon carcinoma model. Significant reductions in tumor growth were observed in two syngeneic mouse tumor models following subcutaneous administration. Combinations of ALG-031048 and ICIs further enhanced the in vivo anti-tumor activity. This initial demonstration of anti-tumor activity after systemic administration of ALG-031048 warrants further investigation, while the combination of systemically administered ALG-031048 with ICIs offers an attractive approach to overcome key limitations of ICIs in the clinic.

Multispecific Hybrid T Cell Receptors for Sensitive Targeting of Cancer

CAR T cells represent a promising therapy for a growing list of tumors. Despite high initial response rates, 50-60% of patients relapse within 1 year after CAR T cell infusion. Heterogeneity of target antigen expression can lead to outgrowth of tumor cells with low or negative antigen levels. Conventional CARs require high antigen levels for efficient T cell activation and tumor elimination. By contrast, T cell receptors (TCRs) are 10-100 times more sensitive than CARs. To achieve greater antigen sensitivity, we and others designed synthetic hybrid T cell receptors by fusing antibody-based recognition domains to TCRa and TCRb constant chains. Here we extend the initial monospecific design to incorporate two antigen recognition domains. We evaluated 2 monospecific hybrid receptors, one with VH and VL domains fused to TCRa and TCRb constant chains respectively (Split TCR/CAR) and a second where the entire scFv (VL-linker-VH) is fused only to the TCRa chain (Full TCR/CAR) leaving TCRb available for a second scFv. We used based editing to knock out endogenous TCRa and b chains to avoid mispairing. Using CD19 as a model target we found that both TCR/CAR designs lacked tonic signaling, induced TCR-like changes in Ca+ flux, more efficiently phosphorylated LAT, and exhibited superior recognition of Nalm-6 clones expressing a range of CD19 levels compared to conventional CD19 28z or 4-1BBz CARs. Interestingly, the Full TCR/CAR outperformed the Split format in these assays. Using soluble bilayer and TIRF microscopy, we demonstrated that unlike CARs, TCR/CAR T cells recapitulate a classical TCR-like immunological synapse. I n vivo studies using NSG mice engrafted with CD19+ Raji cells demonstrated potent anti-tumor efficacy, and the Full format outperformed the Split TCR/CAR. Capitalizing on the superiority of the Full TCR/CAR format, we next designed bispecific hybrid receptors composed of two scFvs of different specificities fused to the TCRa and TCRb chains. Multiple Myeloma is particularly suitable for bispecific targeting as relapse following CAR T cell therapy is common and multiple target antigens have been described. We designed bispecific Full TCR/CARs specific for pairs of myeloma antigens (BCMA/SLAMF7 and BCMA/GPRC5D) and expressed them in primary T cells. We used base editing to knock out SLAMF-7 which can be expressed in T cells and could cause fratricide. Bispecific TCR/CAR receptors conferred sensitive T cell recognition of target cells expressing BCMA and SLAMF-7 as measured by cytokine production, proliferation, and cytotoxicity. T cells equipped with these novel bispecific receptors maintained function when exposed to L363 cells BCMA ko or SLAMF-7 ko demonstrating that both antigen binding domains can trigger T cell functions against target cells. In vivo studies using NSG mice implanted with a mixture composed of 33% of Nalm-6 BCMA+, Nalm-6 SLAMF-7+ and Nalm-6 BCMA+SLAMF-7+ cells to mimic target antigen expression heterogeneity and antigen loss showed superior anti-tumor efficacy for bispecific TCR/CAR cells compared to conventional BCMA and SLAMF-7 BBz CARs. Our hybrid bispecific TCR/CAR also demonstrated superior anti-tumor efficacy compared to a previously described, and systematically optimized bispecific CAR format (Zah et al., 2020). Bispecific hybrid T cell receptors embedded with natural TCR signaling machinery represent a promising therapeutic option to address antigen downmodulation/loss and antigen heterogeneity for treatment of cancer.

Mono- and multimeric PSMA-targeting small molecule-thorium-227 conjugates for optimized efficacy and biodistribution in preclinical models

PurposePSMA (prostate-specific membrane antigen) is highly expressed on prostate cancer (PrCa) cells and extensively used as a homing target for PrCa treatment. Most prominently, PSMA-targeting conjugate PSMA-617, carrying a DOTA chelator and labeled with therapeutic radionuclides like beta-emitting lutetium-177 or alpha-emitting actinium-225, has shown clinical activity in PrCa patients. We sought to develop PSMA-targeting small molecule (SMOL) conjugates that show high uptake in PSMA-expressing tumors and fast clearance, and can easily be labeled with the alpha emitter thorium-227 (half-life 18.7 days).MethodsA novel linker motif with improved competition against 3H-PSMA-617 on PSMA-expressing LNCaP cells was identified. A 2,3-hydroxypyridinone chelator modified with carboxyl groups (carboxy-HOPO) with increased hydrophilicity and robust labeling with thorium-227 was developed and allowed the synthesis of mono-, di-, tri-, and tetrameric conjugates. The resulting monomeric and multimeric PSMA SMOL-TTCs (targeted thorium conjugate) were evaluated for cellular binding, internalization, and antiproliferative activity. The in vivo antitumor efficacy of the PSMA SMOL-TTCs was determined in ST1273 and KUCaP-1 PrCa models in mice, and their biodistribution was assessed in cynomolgus monkeys, minipigs, and mice.ResultsThe monomeric and multimeric PSMA SMOL conjugates were readily labeled with thorium-227 at room temperature and possessed high stability and good binding, internalization, and antiproliferative activity in vitro. In vivo, the monomeric, dimeric, and trimeric PSMA SMOL-TTCs showed fast clearance, potent antitumor efficacy, and high uptake and retention in prostate tumors in mice. No major uptake or retention in other organs was observed beyond kidneys. Low uptake of free thorium-227 into bone confirmed high complex stability in vivo. Salivary gland uptake remained inconclusive as mini pigs were devalidated as a relevant model and imaging controls failed in cynomolgus monkeys.ConclusionMonomeric and multimeric PSMA SMOL-TTCs show high tumor uptake and fast clearance in preclinical models and warrant further therapeutic exploration.

HIMB1636-MMAE, a Novel TROP2-Targeting Antibody-Drug Conjugate Exerting Potent Antitumor Efficacy in Pancreatic Cancer.

Herein, we first prepared a novel anti-TROP2 antibody-drug conjugate (ADC) hIMB1636-MMAE using hIMB1636 antibody chemically coupled to monomethyl auristatin E (MMAE) via a Valine-Citrulline linker and then reported its characteristics and antitumor activity. With a DAR of 3.92, it binds specifically to both recombinant antigen (KD ∼ 0.687 nM) and cancer cells and could be internalized by target cells and selectively kill them with IC50 values at nanomolar/subnanomolar levels by inducing apoptosis and G2/M phase arrest. hIMB1636-MMAE also inhibited cell migration, induced ADCC effects, and had bystander effects. It displayed significant tumor-targeting ability and excellent tumor-suppressive effects in vivo, resulting in 5/8 tumor elimination at 12 mg/kg in the T3M4 xenograft model or complete tumor disappearance at 10 mg/kg in BxPc-3 xenografts in nude mice. Its half-life in mice was about 87 h. These data suggested that hIMB1636-MMAE was a promising candidate for the treatment of pancreatic cancer with TROP2 overexpression.