Human Xenograft Mouse Model Research Articles

Multifunctional O-phenanthroline silver(I) complexes for antitumor activity against colorectal adenocarcinoma cells and antimicrobial properties by multiple mechanisms

A series of O-phenanthroline silver(I) complexes were synthesized and characterized by infrared (IR) spectroscopy, mass spectrometry (MS), 1H nuclear magnetic resonance (NMR) spectroscopy and single-crystal X-ray crystallography. The cytotoxicity of the silver(I) complex (P-131) was evaluated in the cancer cell lines HCT-116, HeLa, and MDA-MB-231 and the normal cell line LO2 via MTT assays. The 50% inhibition concentration (IC50) of P-131 on HCT116 cell line is 0.86 ± 0.03 μM. It is far lower than the IC50 value of cisplatin (9.08 ± 1.10 μM), the IC50 value of normal cell LO2 (76.20 ± 0.48 μM) is much higher than that of cisplatin (3.99 ± 0.74 μM), indicating that its anticancer effect is stronger than that of cisplatin, and its biological safety is greater than that of cisplatin. Furthermore, anticancer mechanistic studies showed that P-131 inhibited cell proliferation by blocking DNA synthesis and acted temporally on the nucleus in dividing HCT-116 cells. Moreover, P-131 increased intracellular reactive oxygen species (ROS) levels in a dose-dependent manner. Notably, 10 mg/kg P-131 showed better antitumor effects than oxaliplatin in an HCT116 human colorectal xenograft mouse model without inducing toxicity. Moreover, the microdilution broth method was used to evaluate the antimicrobial properties of P-131 against Pseudomonas aeruginosa and Candida albicans. A biofilm eradication study was also performed using the crystal violet method and confocal laser scanning microscopy.

A chimeric human/dog-DNA vaccine against CSPG4 induces immunity with therapeutic potential in comparative preclinical models of osteosarcoma

The high mortality rate of osteosarcoma (OSA) patients highlights the requirement of alternative strategies. The young age of patients, as well as the rarity and aggressiveness of the disease, limits opportunities for the robust testing of novel therapies, suggesting the need for valuable preclinical systems. Having previously shown the overexpression of the chondroitin sulfate proteoglycan (CSPG)4 in OSA, herein the functional consequences of its downmodulation in human OSA cells were evaluated invitro, with a significant impairment of cell proliferation, migration, and osteosphere generation. The potential of a chimeric human/dog (HuDo)-CSPG4 DNA vaccine was explored in translational comparative OSA models, including human xenograft mouse models and canine patients affected by spontaneous OSA. The adoptive transfer of HuDo-CSPG4 vaccine-induced CD8+ Tcells and sera in immunodeficient human OSA-bearing mice delayed tumor growth and metastasis development. HuDo-CSPG4 vaccination resulted safe and effective in inducing anti-CSPG4 immunity in OSA-affected dogs, which displayed prolonged survival as compared to controls. Finally, HuDo-CSPG4 was also able to induce a cytotoxic response in a human surrogate setting invitro. On the basis of these results and the high predictive value of spontaneous OSA in dogs, this study paves the way for a possible translation of this approach to humans.

Design, Synthesis, and Biological Evaluation of Potent and Selective Inhibitors of Ataxia Telangiectasia Mutated and Rad3-Related (ATR) Kinase for the Efficient Treatment of Cancer.

Ataxia telangiectasia mutated and Rad3-related (ATR), a vital member of the phosphatidylinositol 3-kinase-related kinase (PIKK) family, plays a critical role in the DNA damage response (DDR). Tumor cells with a loss of DDR function or defects in the ataxia telangiectasia mutated (ATM) gene are generally more dependent on ATR for survival, suggesting that ATR is an attractive anticancer drug target based on its synthetic lethality. Herein, we present a potent and highly selective ATR inhibitor, ZH-12 (IC50 = 0.0068 μM). It showed potent antitumor activity as a single agent or in combination with cisplatin in the human colorectal adenocarcinoma LoVo tumor xenograft mouse model. Overall, ZH-12 may be a promising ATR inhibitor based on the principle of synthetic lethality and deserves further in-depth study.

AFM13 enhances the anti‐tumor activity of AB‐101 towards CD30+ tumors, conferring tumor growth control in vivo

Introduction: Bispecific antibodies targeting CD16A and a tumor antigen have the potential to specifically redirect the cytotoxic potential of NK cells towards cancer cells through antibody-dependent cellular cytotoxicity (ADCC). It has been demonstrated that the bispecific CD16AxCD30 innate cell engager AFM13 significantly enhances the cytotoxic activity of CD16A+ NK cells towards CD30+ tumor cells. Combination of AFM13 with adoptive NK cell transfer achieved unprecedented clinical response rates in patients with relapsed/refractory (R/R) Hodgkin lymphoma (ORR 94.2% for patients treated with RP2D), while maintaining a favorable toxicity profile. AB-101 is a non-engineered, allogeneic, off-the-shelf, cryopreserved cord blood-derived NK cell product, currently being tested in a Phase 1/2 clinical trial as monotherapy and in combination with rituximab in patients with R/R B cell NHL. AB-101 is optimized for ADCC through pre-selection for the KIR-B haplotype and the natural high-affinity variant of CD16A (158V/V). In the present study, we investigated whether the combination of AFM13 with AB-101 leads to enhanced anti-tumor activity in vitro and in vivo. Methods: Cellular cytotoxic activity was assessed in 4-h calcein-release cytotoxicity assays. Cell viability, degranulation/CD107a expression and IFN-γ production were measured by flow cytometry. Tumor growth control in vivo was determined in a hematologic (Karpas-299 CD30+ T-cell lymphoma) human IL-15 NOG mouse xenograft model by whole body bioluminescence imaging. Results: We demonstrate robust, homogenous binding of AFM13 to AB-101 as assessed by flow cytometry when AB-101 cells were combined with AFM13 after thawing. Overall, the binding pattern of AFM13 was congruent to the binding pattern of high CD16A expression on AB-101, suggesting saturated loading of CD16A molecules by AFM13 on the surface of AB-101. Combination with AFM13 enhanced the cytotoxic activity of AB-101 towards CD30+ Karpas-299 tumor cells. AFM13-induced cytotoxic activity was associated with an increased functional activation status of AB-101, reflected by increased degranulation/CD107a expression and IFN-γ production in response to Karpas-299 tumor cells. Of note, viability of AB-101 cells was maintained upon exposure to AFM13 in the absence or presence of CD30+ tumor cells. Importantly, adoptive transfer of AB-101 co-administered with AFM13 conferred tumor growth control in vivo in a human IL-15 NOG mouse xenograft model. Conclusion: This study demonstrates synergistical anti-tumor activity in vivo for the combination of AFM13 and AB-101. Building on our clinical data with fresh cord blood-derived stimulated/expanded NK cells combined with AFM13 (NCT04074746), co-administration of cryopreserved AB-101 with AFM13 offers a promising highly scalable off-the-shelf immunotherapeutic treatment for patients with CD30+ malignancies. The research was funded by Affimed GmbH and Artiva Biotherapeutics Keywords: Cellular therapies, Combination Therapies, Immunotherapy Conflicts of interests pertinent to the abstract. J. Pahl Employment or leadership position: Affimed GmbH Stock ownership: Affimed GmbH T. Haneke Employment or leadership position: Affimed GmbH Stock ownership: Affimed GmbH L. Guerrettaz Employment or leadership position: Artiva Biotherapeutics S. Somanchi Employment or leadership position: Artiva Biotherapeutics H. Raymon Employment or leadership position: Artiva Biotherapeutics S. Pinto Employment or leadership position: Affimed GmbH Stock ownership: Affimed GmbH P. Flynn Employment or leadership position: Artiva Biotherapeutics J. Koch Employment or leadership position: Affimed GmbH Stock ownership: Affimed GmbH

AFNT-211: An FAS-41BB–enhanced TCR-T cell therapy with stem-like properties targeting KRAS G12V-expressing solid tumors.

2543 Background: KRAS is the most common oncogenic driver mutation in solid tumors, promoting the initiation and progression of many uncurable cancers, including colorectal, pancreatic and lung cancer. While small molecule inhibitors to KRASG12C mutations have been approved, there are no targeted therapies available for patients with highly prevalent KRASG12V mutations. TCR-T cell therapies have demonstrated remarkable responses in clinical trials, but their durability has been limited by the immunosuppressive tumor microenvironment (TME). AFNT-211 is an autologous T cell therapy engineered to express an HLA-A*11:01 KRASG12V-specific TCR, further enhanced with CD8α/β coreceptor and a FAS-41BB switch receptor to drive T cell persistence and durable clinical responses. CD8α/β coreceptor enables a coordinated CD4+/CD8+ T cell response and FAS-41BB converts the FAS ligand (FASL) TME death signal into a costimulatory signal through 41BB activation. Methods: AFNT-211 was assessed for efficacy in vitro against a panel of KRASG12V-expressing tumor cell lines and in vivo using human xenograft mouse models. In vitro safety studies were performed to assess potential cross-reactivity, alloreactivity, and cytokine-independent growth. The clinical manufacturing process consists of autologous CD4+/CD8+ T cells transduced with lentivirus and expanded using culture conditions that drive robust expansion while preserving stem-like properties. Phenotypic and functional analyses of AFNT-211 were performed using flow cytometry and cell-based assays. Results: Coculture of AFNT-211 with a panel of KRASG12V-expressing tumor cell lines led to significant effector cytokine secretion, T cell proliferation, and tumor cell killing. The CD8α/β coreceptor enabled CD4+ T cell recognition of KRASG12V and greater overall cytotoxicity. The FAS-41BB switch receptor dramatically augmented the magnitude and durability of the anti-tumor response against FASL-expressing tumor cells. XScan mutagenesis and potential off-target peptide testing revealed no significant cross-reactivities. No alloreactivity was observed against a panel of lymphoblastoid cell lines presenting the most frequent HLA types in the US population. Potent anti-tumor response was observed in vivo in a mouse xenograft model. Affini-T’s Thrive manufacturing platform used for the production of AFNT-211 consistently delivers >30-40e9 TCR-engineered T cells with a high frequency of naïve and central memory T cells expressing negligible markers of exhaustion. Conclusions: The AFNT-211 manufacturing process generates a large number of TCR-T cells with desirable stem-like properties optimized for clinical dosing. Preclinical data demonstrated a potent and safe profile for AFNT-211 that supports clinical development in HLA-A*11:01 patients with advanced/metastatic solid tumors harboring a KRAS G12V mutation.

The safety and efficacy of SNK01 (autologous natural killer cells) in combination with cytotoxic chemotherapy after failure of prior tyrosine kinase inhibitor in non-small cell lung cancer: Preclinical mouse model and phase I/IIa clinical study.

9057 Background: It is challenging to choose subsequent treatment option in patients with Osimertinib resistance in epidermal growth factor receptor (EGFR)-mutated non-small cell lung cancer (NSCLC). Meanwhile, antitumor effect of the antibody-dependent cellular cytotoxicity of natural killer (NK) cells mediated by cetuximab –anti-EGFR monoclonal antibody– has been reported. Therefore, we aimed to evaluate the safety and efficacy of SNK01 (non-genetically modified autologous NK cell) in combination with cytotoxic chemotherapy including cetuximab in NSCLC after failure to prior tyrosine kinase inhibitor (TKI) in a preclinical humanized cell line-derived xenograft (CDX) mouse model and phase I/IIa clinical study. Methods: We established a humanized CDX mouse model using Osimertinib-resistant lung cancer cell line. Reconstruction of the humanized mouse, CDX-related skills, and analytic data were made according to C&SR Inc. manufacturing technique & SOP protocol. The mice were divided into 4 groups based on treatment (no treatment [n = 2]; Cetuximab only [n = 3]; SNK01 only [n = 4]; SNK01 plus Cetuximab [n = 4]) and treated weekly for 5 weeks (SNK01, 1x107 cells/dose; Cetuximab, 20 ug/dose). In the clinical study, 12 patients with EGFR-mutated NSCLC who failed prior TKI treatment were finally enrolled. They received weekly SNK01 in combination with Gemcitabine/Carboplatin (n = 6) or Gemcitabine/Carboplatin/Cetuximab (n = 6), and dose escalation of SNK01 following “3+3” design (4×109 cells/dose or 6×109 cells/dose). The primary endpoint was safety, and secondary endpoint was efficacy. Results: In the preclinical study, flow cytometry analysis showed that NK cells (CD45+/CD56+/CD3-) were significantly increased in the groups administrated SNK01. The volume of tumor extracted after completion of treatment was the smallest in SNK01 plus cetuximab group. In the clinical study, the median age of patients was 61 years, 33.3% were male, and all patients had adenocarcinoma. The enrolled patients received weekly infusions of SNK01 for 7 to 8 weeks (4×109 cells/dose [n = 6]; 6×109 cells/dose [n = 6]). Since dose limiting toxicity was not observed, maximum tolerated dose of SNK01 was determined to be 6×109 cells/dose. SNK01-related adverse event ≥grade 3 was also not observed. In the efficacy analysis, objective response rate was 25%, disease control rate was 100% (partial response, n = 3/12; stable disease, n = 9/12), and median progression free survival (PFS) was 143 days. PFS will be updated as some patients are still being followed. Conclusions: SNK01 in combination with cytotoxic chemotherapy, including cetuximab in EGFR-mutated NSCLC with resistance to TKI was safe and showed a potential antitumor effect in this preclinical study and early phase I/IIa clinical trial. Clinical trial information: NCT04872634 .

Exploration of Imaging Biomarkers for Metabolically-Targeted Osteosarcoma Therapy in a Murine Xenograft Model.

Background: Osteosarcoma (OS) is an aggressive pediatric cancer with unmet therapeutic needs. Glutaminase 1 (GLS1) inhibition, alone and in combination with metformin, disrupts the bioenergetic demands of tumor progression and metastasis, showing promise for clinical translation. Materials and Methods: Three positron emission tomography (PET) clinical imaging agents, [18F]fluoro-2-deoxy-2-D-glucose ([18F]FDG), 3'-[18F]fluoro-3'-deoxythymidine ([18F]FLT), and (2S, 4R)-4-[18F]fluoroglutamine ([18F]GLN), were evaluated in the MG63.3 human OS xenograft mouse model, as companion imaging biomarkers after treatment for 7 d with a selective GLS1 inhibitor (CB-839, telaglenastat) and metformin, alone and in combination. Imaging and biodistribution data were collected from tumors and reference tissues before and after treatment. Results: Drug treatment altered tumor uptake of all three PET agents. Relative [18F]FDG uptake decreased significantly after telaglenastat treatment, but not within control and metformin-only groups. [18F]FLT tumor uptake appears to be negatively affected by tumor size. Evidence of a flare effect was seen with [18F]FLT imaging after treatment. Telaglenastat had a broad influence on [18F]GLN uptake in tumor and normal tissues. Conclusions: Image-based tumor volume quantification is recommended for this paratibial tumor model. The performance of [18F]FLT and [18F]GLN was affected by tumor size. [18F]FDG may be useful in detecting telaglenastat's impact on glycolysis. Exploration of kinetic tracer uptake protocols is needed to define clinically relevant patterns of [18F]GLN uptake in patients receiving telaglenastat.

From a drug repositioning to a structure-based drug design approach to tackle acute lymphoblastic leukemia

Cancer cells utilize the main de novo pathway and the alternative salvage pathway for deoxyribonucleotide biosynthesis to achieve adequate nucleotide pools. Deoxycytidine kinase is the rate-limiting enzyme of the salvage pathway and it has recently emerged as a target for anti-proliferative therapies for cancers where it is essential. Here, we present the development of a potent inhibitor applying an iterative multidisciplinary approach, which relies on computational design coupled with experimental evaluations. This strategy allows an acceleration of the hit-to-lead process by gradually implementing key chemical modifications to increase affinity and activity. Our lead compound, OR0642, is more than 1000 times more potent than its initial parent compound, masitinib, previously identified from a drug repositioning approach. OR0642 in combination with a physiological inhibitor of the de novo pathway doubled the survival rate in a human T-cell acute lymphoblastic leukemia patient-derived xenograft mouse model, demonstrating the proof-of-concept of this drug design strategy.

Improved synthesis and anticancer activity of a potent neuronal nitric oxide synthase inhibitor

An improved synthesis of 4-methyl-7-(3-((methylamino)methyl)phenethyl)quinolin-2-amine (1) is reported. A scalable, rapid, and efficient methodology was developed to access this compound with an overall yield of 35%, which is 5.9-fold higher than the previous report. The key differences in the improved synthesis are a high yielding quinoline synthesis by a Knorr reaction, a copper-mediated Sonogashira coupling to the internal alkyne in excellent yield, and a crucial deprotection of the N-acetyl and N-Boc groups achieved under acidic conditions in a single step rather than a poor yielding quinoline N-oxide strategy, basic deprotection conditions, and low yielding copper-free conditions that were reported in the previous report. Compound 1, which previously was shown to inhibit IFN-γ-induced tumor growth in a human melanoma xenograft mouse model, was found to inhibit the growth of metastatic melanoma, glioblastoma, and hepatocellular carcinoma in vitro.

Post-translational covalent assembly of CAR and synNotch receptors for programmable antigen targeting

Chimeric antigen receptors (CARs) and synthetic Notch (synNotch) receptors are engineered cell-surface receptors that sense a target antigen and respond by activating T cell receptor signaling or a customized gene program, respectively. Here, to expand the targeting capabilities of these receptors, we develop “universal” receptor systems for which receptor specificity can be directed post-translationally via covalent attachment of a co-administered antibody bearing a benzylguanine (BG) motif. A SNAPtag self-labeling enzyme is genetically fused to the receptor and reacts with BG-conjugated antibodies for covalent assembly, programming antigen recognition. We demonstrate that activation of SNAP-CAR and SNAP-synNotch receptors can be successfully targeted by clinically relevant BG-conjugated antibodies, including anti-tumor activity of SNAP-CAR T cells in vivo in a human tumor xenograft mouse model. Finally, we develop a mathematical model to better define the parameters affecting universal receptor signaling. SNAP receptors provide a powerful strategy to post-translationally reprogram the targeting specificity of engineered cells.

Combinations of ivermectin with proteasome inhibitors induce synergistic lethality in multiple myeloma

Multiple myeloma (MM) is an incurable malignancy of plasma cells. Ivermectin is a US Food and Drug Administration-approved drug for antiparasitic use. Here, we showed that ivermectin exerted anti-MM effects and significantly synergized with proteasome inhibitors in vitro and in vivo. Ivermectin alone exhibited mild anti-MM activity in vitro. Further investigation suggested that ivermectin inhibited proteasome activity in the nucleus by repressing the nuclear import of proteasome subunits, such as PSMB5-7 and PSMA3-4. Therefore, ivermectin treatment caused the accumulation of ubiquitylated proteins and the activation of the UPR pathway in MM cells. Furthermore, ivermectin treatment caused DNA damage and DNA damage response (DDR) signaling pathway activation in MM cells. Ivermectin and bortezomib exhibited synergized anti-MM activity in vitro. The dual-drug treatment resulted in synergistic inhibition of proteasome activity and increased DNA damage. An in vivo study using a human MM cell line xenograft mouse model showed that ivermectin and bortezomib efficiently repressed MM tumor growth in vivo, while the dual-drug treatment was well tolerated by experimental animals. Overall, our results demonstrated that ivermectin alone or cotreated with bortezomib might be promising in MM treatment.

Abstract 4083: In vivo efficacy of LGR5-targeting CAR-T cell therapies developed for the treatment of colorectal cancer

Abstract While CAR-T cells have demonstrated significant efficacy in the treatment of haematological malignancies, there are many challenges to overcome in developing CAR-T cell therapies against solid tumours. In particular, the scarcity of recognised effective target antigens in solid tumours remains a significant hurdle. Leucine-rich repeat-containing G-protein coupled receptor (LGR5) is a cancer stem cell marker, which mediates important roles in tumor initiation and metastasis. Upregulated LGR5 expression in malignant cell contexts highlights this surface receptor as a promising target for CAR-T cell therapy. In this study, we evaluated the efficacy of LGR5-targeting CAR-T cells against a range of human cancers. We demonstrate that LGR5 is expressed, both in vitro and in vivo, by numerous human cancers, including colorectal, ovarian, hepatic, brain, and pancreatic cancer. LGR5-targeting CAR-T cells effectively kill LGR5-expressing cancer cells in in vitro cytotoxicity assays and display significant anti-tumor activity in vivo when administered at 1, 2 or 3 weeks post-tumor inoculation in a subcutaneous human colorectal cancer xenograft mouse model. Furthermore, LGR5-targeting CAR-T cells that induce primary tumor rejection confer complete protection against a secondary tumor challenge 5 weeks post-T cell transfer. In vivo spatiotemporal distribution and repeated dosing studies demonstrate that LGR5-targeting CAR-T cells accumulate at the tumor site until tumor elimination, do not persist in off-target organs by d28 post-tumor inoculation and are safe and well-tolerated under the conditions tested. Together, these results provide the foundation for furthering the development of LGR5-targeting CAR-T cells for use in the clinic against colorectal cancers and other solid tumours. Citation Format: Dylan McPeake, Timona Tyllis, Caitlin Abbott, Jade Foeng, Veronika Bandara, Batjargal Gundsambuu, Elaheh Rohani-Rad, Silvana Napoli, Timothy Sadlon, Simon Barry, Shaun McColl. In vivo efficacy of LGR5-targeting CAR-T cell therapies developed for the treatment of colorectal cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4083.

Abstract 1862: Synergistic and long-lasing anti-tumor efficacy of CBA-1205, a novel glycoengineered humanized antibody targeting DLK-1, in combination with lenvatinib in human HCC xenograft models

Abstract Delta-like 1 homolog (DLK-1), a non-canonical notch ligand, is a membrane protein with 6 tandem EGF-like motifs in extracellular region. While the expression of DLK-1 is restricted in some neuroendocrine tissues such as adrenal gland and hypophysis in normal adult tissues, its over-expression in cancers have been reported in a variety of cancer types, such as hepatocellular carcinoma (HCC), small cell lung carcinoma, ovarian carcinoma, adrenal cortical carcinoma, pancreatic carcinoma, gastrointestinal stromal tumor, etc. Interestingly, it has been suggested that DLK-1 is a marker of cancer stem cells in HCC. CBA-1205 is a humanized antibody (IgG1/κ) targeting DLK-1, which is glycoengineered by GlymaxX® to potentiate antibody-dependent cellular cytotoxicity (ADCC) activity. CBA-1205 showed no toxicology issues in cynomolgus monkey GLP studies and the Phase 1 clinical trial in patients with solid tumors is under way (JapicCTI-205384). Previously, we demonstrated potent anti-tumor efficacy of CBA-1205 as a monotherapy in multiple human DLK-1 positive cancer xenograft mouse models including HCC (AACR 2019 annual meeting). Lenvatinib is an oral multi-kinase inhibitor which has been used for 1st line therapy of unresectable advanced HCC globally. The purpose of present study is to evaluate the combinatorial efficacy of CBA-1205 with lenvatinib in two HCC xenograft models (Hep3B and HepG2). Hep3B model was sensitive to lenvatinib monotherapy; oral daily dosing of lenvatinib at 3 mg/kg treatment showed 50% tumor growth inhibition (TGI) and at 10 mg/kg treatment showed more than 80% TGI. In contrast HepG2 model was less sensitive to Lenvatinib; TGI at 3 mg/kg treatment group was 20 % and that for 10 mg/kg treatment group was 50 %. When CBA-1205 (1 mg/kg, twice a week, total 4 times) was intraperitoneally administered in combination with 3 mg/kg of lenvatinib (daily, total 10 times) in Hep3B xenograft mice, synergistic and long-lasting anti-tumor efficacy was observed compared either with CBA-1205 treatment alone or lenvatinib treatment alone. Tumor growth of all mice (N=8) treated with CBA-1205 and lenvatinib showed almost complete inhibition even 2 weeks after the last dosing. More importantly, 4 out of 8 mice in CBA-1205 and lenvatinib treatment group showed tumor regression. Similar results were obtained with CBA-1205 at 1 mg/kg dosage in combination with lenvatinib at 10 mg/kg dosage in HepG2 xenograft model. In conclusion, we demonstrated synergistic and long-lasting anti-tumor efficacy of CBA-1205 in combination with lenvatinib in two different HCC pre-clinical models suggesting that the combination could be a new treatment option for unresectable advanced HCC. Citation Format: Koji Nakamura, Izumi Sakaguchi, Kota Takahashi. Synergistic and long-lasing anti-tumor efficacy of CBA-1205, a novel glycoengineered humanized antibody targeting DLK-1, in combination with lenvatinib in human HCC xenograft models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1862.

Abstract 1149: Universal redirection of chimeric antigen receptor T cells against solid tumors via tumor cell membrane-inserting CAR ligands

Abstract Chimeric antigen receptor (CAR) T cell therapy is effective in treating certain hematological malignancies, but its translation to solid tumors has been challenging. Selecting an effective target antigen is hampered by heterogeneous tumor antigen expression and target antigen expression in healthy tissues. We describe a strategy to promote CAR T-mediated destruction of any tumor via intratumoral administration of a membrane-inserting fluorescein isothiocyanate lipid amphiphile molecule (“amph-FITC”) recognizable by a FITC-specific CAR. Firstly, we show labeling of tumor cells with amph-FITC in vitro, mediating cytotoxicity by FITC CAR T cells in coculture. Then, we demonstrate efficient tagging of tumor cells in vivo upon intratumoral injection with minimal labeling of non-tumor tissues, driving FITC CAR T cell proliferation and accumulation in tumors. Subsequently, we determine that this strategy stimulates an anti-tumor response that facilitates tumor regression in several syngeneic and human xenograft mouse models bearing different tissues of origin. In syngeneic models, this strategy induced the infiltration of host T cells and elicited endogenous tumor-specific T cell priming, leading to protection against tumor rechallenge. These results demonstrate the capacity to treat tumors independent of antigen expression or tissue of origin, addressing a major bottleneck in adoptive T cell therapy. Citation Format: Angela Zhang. Universal redirection of chimeric antigen receptor T cells against solid tumors via tumor cell membrane-inserting CAR ligands [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1149.

In vivo near-infrared fluorescence imaging of gastric cancer in an MKN-45 gastric cancer xenograft mouse model using intraoperative ureteral identification agent ASP5354.

Accurate intraoperative identification of gastric cancer lesions and determination of the extent of resection are important for curability and function preservation. This study aimed to investigate the potential of the near-infrared fluorescence (NIRF) imaging agent ASP5354 for in vivo fluorescence imaging of gastric cancer. The capability of ASP5354 was evaluated using an MKN-45 human gastric cancer xenograft mouse model. A single dose of ASP5354 was intravenously administered to the mice at a concentration of 120nmol (0.37mg)/kg body weight. In vivo NIRF images of the mouse backs were obtained using an NIRF camera system. Moreover, the cancer tissues were dissected, and the NIRF intensity in the tissue sections was measured using the NIRF camera system. ASP5354 uptake in MKN-45 cells was assessed in vitro using the NIRF microscope. The NIRF signal of ASP5354 was selectively detected in gastric cancer tissues immediately after the intravenous administration of ASP5354. The cancer tissues emitted stronger NIRF signals than adjacent normal tissues. The difference in the NIRF intensity between the normal and cancer tissues was clearly observed at the boundary between them in the macrolevel NIRF images. Cancer tissues can be distinguished from normal tissues based on the measurement of the NIRF of ASP5354, using an NIRF camera system. ASP5354 is a promising agent for NIRF imaging of gastric cancer tissues.

Preclinical Activity of 4-Demethyl-4-cholesteryloxycarbonylpenclomedine in Melanoma.

Temozolomide plays a role in treating melanoma refractory to immunomodulatory and mitogen-activated protein kinase-targeted approaches, but its efficacy is limited. 4-Demethyl-4-cholesteryloxycarbonylpenclomedine (DM-CHOC-PEN) is a polychlorinated pyridine cholesteryl carbonate. Its mechanism of action is considered to be via alkylation/adduct formation with N7-guanine. It demonstrated activity in intracranial implanted human glioma and breast cancer xenograft mouse models. The activity of DM-CHOC-PEN in melanoma models was assessed. B-16 melanoma cells were exposed to DM-CHOC-PEN at different concentrations to assess proliferation and survival. B-16 cells were implanted subcutaneously into the flank of adult female C57BL mice which were then were treated with 200 mg/kg DM-CHOC-PEN intraperitoneally daily for 5 days in the setting of palpable subcutaneous tumor. Survival was compared to mice treated with temozolomide or saline. Five mice were treated per group. In vitro, the respective half-maximal inhibitory concentrations of DM-CHOC-PEN and temozolomide were 0.5 and ≥3.0 μg/ml. Floating, heavily melanotic cells formed and these cells were separated, analyzed, and contained 10-90 ng DM-CHOC-PEN per 105 cells. The improvement in survival of mice treated with DM-CHOC-PEN or temozolomide relative to saline controls was 142% and 78%, respectively. Longer survival was seen with DM-CHOC-PEN in a C57BL murine model relative to temozolomide and saline-treated controls, supporting the development of clinical trials assessing the efficacy of DM-CHOC-PEN as treatment for metastatic melanoma.

An integrated approach of network pharmacology, molecular docking, and experimental verification uncovers kaempferol as the effective modulator of HSD17B1 for treatment of endometrial cancer

BackgroundEndometrial cancer (EC) is one of the most common gynecological malignancies globally, and the development of innovative, effective drugs against EC remains a key issue. Phytoestrogen kaempferol exhibits anti-cancer effects, but the action mechanisms are still unclear.MethodMTT assays, colony-forming assays, flow cytometry, scratch healing, and transwell assays were used to evaluate the proliferation, apoptosis, cell cycle, migration, and invasion of both ER-subtype EC cells. Xenograft experiments were used to assess the effects of kaempferol inhibition on tumor growth. Next-generation RNA sequencing was used to compare the gene expression levels in vehicle-treated versus kaempferol-treated Ishikawa and HEC-1-A cells. A network pharmacology and molecular docking technique were applied to identify the anti-cancer mechanism of kaempferol, including the building of target-pathway network. GO analysis and KEGG pathway enrichment analysis were used to identify cancer-related targets. Finally, the study validated the mRNA and protein expression using real-time quantitative PCR, western blotting, and immunohistochemical analysis.ResultsKaempferol was found to suppress the proliferation, promote apoptosis, and limit the tumor-forming, scratch healing, invasion, and migration capacities of EC cells. Kaempferol inhibited tumor growth and promotes apoptosis in a human endometrial cancer xenograft mouse model. No significant toxicity of kaempferol was found in human monocytes and normal cell lines at non-cytotoxic concentrations. No adverse effects or significant changes in body weight or organ coefficients were observed in 3–7 weeks’ kaempferol-treated animals. The RNA sequencing, network pharmacology, and molecular docking approaches identified the overall survival-related differentially expressed gene HSD17B1. Interestingly, kaempferol upregulated HSD17B1 expression and sensitivity in ER-negative EC cells. Kaempferol differentially regulated PPARG expression in EC cells of different ER subtypes, independent of its effect on ESR1. HSD17B1 and HSD17B1-associated genes, such as ESR1, ESRRA, PPARG, AKT1, and AKR1C1\\2\\3, were involved in several estrogen metabolism pathways, such as steroid binding, 17-beta-hydroxysteroid dehydrogenase (NADP+) activity, steroid hormone biosynthesis, and regulation of hormone levels. The molecular basis of the effects of kaempferol treatment was evaluated.ConclusionsKaempferol is a novel therapeutic candidate for EC via HSD17B1-related estrogen metabolism pathways. These results provide new insights into the efficiency of the medical translation of phytoestrogens.

177Lu]Lu-PSMA-617 theranostic probe for hepatocellular carcinoma imaging and therapy.

This study aimed to explore the feasibility of using [177Lu]Lu-prostate-specific membrane antigen (PSMA)-617 and [177Lu]Lu-Evans blue (EB)-PSMA-617 for in vivo radioligand therapy by single-dose administration in a PSMA-positive hepatocellular carcinoma (HCC) xenograft mouse model. [177Lu]Lu-PSMA-617 and [177Lu]Lu-EB-PSMA-617 were prepared, and labelling efficiency and radiochemical purity were determined. A HepG2 human HCC subcutaneous xenograft mouse model was established. After intravenous injection of [177Lu]Lu-PSMA-617 or [177Lu]Lu-EB-PSMA-617 (37MBq) into the mouse model, single-photon emission computed tomography/computed tomography (SPECT/CT) was performed. Biodistribution studies were conducted to verify targeting specificity and pharmacokinetics. In the radioligand therapy study, mice were randomized into 4 groups: 37MBq [177Lu]Lu-PSMA-617, 18.5MBq [177Lu]Lu-PSMA-617, 7.4MBq [177Lu]Lu-EB-PSMA-617, and saline (control). A single-dose administration was applied at the beginning of therapy studies. Tumor volume, body weight, and survival were monitored every 2days. After the end of therapy, mice were euthanized. Tumors were then weighed, and systemic toxicity was evaluated via blood testing and histological examination of healthy organs. [177Lu]Lu-PSMA-617 and [177Lu]Lu-EB-PSMA-617 were successfully prepared with high purity and stability. SPECT/CT and biodistribution showed that tumor uptake was higher and persisted longer for [177Lu]Lu-EB-PSMA-617 compared with [177Lu]Lu-PSMA-617. [177Lu]Lu-PSMA-617 was rapidly cleared from the blood, while [177Lu]Lu-EB-PSMA-617 persisted for significantly longer. In radioligand therapy studies, tumor growth was significantly suppressed in the 37MBq [177Lu]Lu-PSMA-617, 18.5MBq [177Lu]Lu-PSMA-617, and 7.4MBq [177Lu]Lu-EB-PSMA-617 groups compared to the saline group. Median survival was 40, 44, 43, and 30days, respectively. No healthy organ toxicity was observed in safety and tolerability evaluation. Radioligand therapy using [177Lu]Lu-PSMA-617 and [177Lu]Lu-EB-PSMA-617 significantly suppressed tumor growth and prolonged survival time in PSMA-positive HCC xenograft mice without obvious toxicity. These radioligands appear promising for clinical use in humans, and future studies are warranted.

Bafilomycin A1 inhibits SARS-CoV-2 infection in a human lung xenograft mouse model

Coronavirus disease 2019 is a global pandemic caused by SARS-CoV-2. The emergence of its variant strains has posed a considerable challenge to clinical treatment. Therefore, drugs capable of inhibiting SARS-CoV-2 infection, regardless of virus variations, are in urgently need. Our results showed that the endosomal acidification inhibitor, Bafilomycin A1 (Baf-A1), had an inhibitory effect on the viral RNA synthesis of SARS-CoV-2, and its Beta and Delta variants at the concentration of 500 nM. Moreover, the human lung xenograft mouse model was used to investigate the anti-SARS-CoV-2 effect of Baf-A1. It was found that Baf-A1 significantly inhibited SARS-CoV-2 replication in the human lung xenografts by in situ hybridization and RT-PCR assays. Histopathological examination showed that Baf-A1 alleviated SARS-CoV-2-induced diffuse inflammatory infiltration of granulocytes and macrophages and alveolar endothelial cell death in human lung xenografts. In addition, immunohistochemistry analysis indicated that Baf-A1 decreased inflammatory exudation and infiltration in SARS-CoV-2-infected human lung xenografts. Therefore, Baf-A1 may be a candidate drug for SARS-CoV-2 treatment.

The CNS-penetrating taxane drug TPI 287 potentiates antiglioma activity of the AURKA inhibitor alisertib in vivo.

Glioblastoma (GBM) has a very poor prognosis despite current treatment. We previously found cytotoxic synergy between the AURKA inhibitor alisertib and the CNS-penetrating taxane TPI 287 against GBM tumor cells in vitro. We used an orthotopic human GBM xenograft mouse model to test if TPI 287 potentiates alisertib in vivo. Western blotting, immunohistochemistry, siRNA knockdown, annexin V binding, and 3-dimensional Matrigel invasion assays were used to investigate potential mechanisms of alisertib and TPI 287 treatment interactions. Alisertib + TPI 287 combination therapy significantly prolonged animal survival compared to vehicle (p = 0.011), but only marginally compared to alisertib alone. Alisertib, TPI 287, and combined alisertib + TPI 287 reduced animal tumor volume compared to vehicle-treated controls. This was statistically significant for the combination therapy at 4 weeks (p < 0.0001). Alisertib + TPI 287 treatment decreased anti-apoptotic Bcl-2 protein levels in vivo and in vitro. Expression of the pro-apoptotic protein Bak was significantly increased by combination treatment (p < 0.0001). Pro-apoptotic Bim and Bak knockdown by siRNA decreased apoptosis by alisertib + TPI 287 in GB9, GB30, and U87 cells (p = 0.0005 to 0.0381). Although alisertib and TPI 287 significantly reduced GBM cell invasion (p < 0.0001), their combination was no more effective than TPI 287 alone. Results suggest that apoptosis is the dominant mechanism of potentiation of GBM growth inhibition by alisertib + TPI 287, in part through effects on Bcl-2 family proteins, providing a rationale for further laboratory testing of an AURKA inhibitor plus TPI 287 as a potential therapy against GBM.