Preclinical Results of an Allogeneic, Universal CD19/CD7-Targeting CAR-T Cell Therapy (GC502) for B Cell Malignancies

Background Autologous CD19 CAR-T therapies show very promising clinical efficacy, but are limited in their applicability by several factors including cost, time to manufacture, and other factors involving patients own T-cell qualities. GC027, a CD7 targeting allogeneic, universal CAR-T (UCAR-T) currently in development for the treatment of T-cell acute lymphoblastic leukemia (T-ALL) has demonstrated robust expansion and anti-leukemia efficacy with a manageable safety profile in an investigator-initiated trial in China. These data suggest that, a single CD7 targeting CAR-T therapy is able to generate a therapeutic window by suppressing host vs graft (HvG) rejection of UCAR-T cells by patients' own NK and T cells, and achieve efficacy in patients with T-ALL. Based on these findings we developed GC502, a CD19/CD7 dual-targeting, allogeneic CAR-T therapy for B-cell malignancies, in which the CD19 CAR moiety targets malignant cells while CD7 CAR moiety suppresses HvG in variety of preclinical models. Methods GC502 was manufactured using leukopaks from HLA-unmatched healthy donors. It contains a 4-1BB based 2 nd-generation dual targeting CAR comprising an anti-CD19 and an anti-CD7 single-chain variable fragments (scFvs). TRAC and CD7 loci were disrupted to avoid graft vs host disease and fratricide, respectively. To select the leading CAR candidate, CAR expression and functionalities of CAR constructs with different heavy-light (H-L) chain orientations of the dual CAR were analyzed via in vitro assays and mouse xenograft models, in comparing to single CD19 CAR and CD7 CAR products. To achieve optimal anti-tumor efficacy, a T-cell enhancer was included in the CAR construct. Result Gene editing and dual CAR orientation selection TRAC and CD7 double knockout efficiencies were constantly above 97% across multiple donor pan T cells. Although CD19/CD7 CAR expression levels in different H-L chain orientations were similar, in the final CAR-T product as measured by flow cytometry (FCM) analysis, significant difference was observed in their cytotoxicity and in vitro expansion under repeated antigen stimulations by CD19+ B-cell acute lymphoblastic leukemia (B-ALL) cell line Nalm6 and CD7+ T-cell line CCRF-CEM. CAR candidates mediated the strongest cytotoxicity and most durable response were selected for further optimizations. CAR construct optimizations For the leading candidates, we first assessed the dual CAR efficacy after incorporation of an enhancer. While the IL-2, TNFα and IFNγ secretion levels were comparable, enhancer addition significantly improved tumor killing and CAR-T cell expansion under repeated stimulations by either CD19+ or CD7+ target cells. Anti-leukemia response under sub-optimal CAR-T cell dosages were also greatly enhanced as assessed by both B-ALL and T-ALL mouse xenograft models. GC502 CAR functionality comparison to single CAR products with proven clinical efficacies GC502 and GC027 were compared for their CD7 CAR function to assess their anti-HvG activities. GC502 and GC027 exhibited comparable toxicities towards pan T cells and similar efficacies in a highly malignant T-ALL mouse model. The CD19 CAR functionality of GC502 were evaluated and compared to a 2 nd generation CD19 CAR product comprising a FMC63 scFv and a 4-1BB-CD3ȥ signaling domain. In a Raji based B-ALL mouse xenograft model, both products rapidly eliminated cancer cells. While CD19 CAR treated mice showed signs of relapse at 2 weeks post CAR-T infusion, GC502 treatment group maintained "leukemia free" status till the end of study (Day28). Conclusion GC502 was optimized for CD19/CD7 dual CAR functionality and in vivo durability. It demonstrated robust anti-tumor efficacy and promising potentials to suppress HvG. This report presents an example that the dual CAR design of GC502 may serve as a novel "off-the-shelf" CAR-T technology. Disclosures Ge: GracellBiotechnologies Ltd: Current Employment, Current equity holder in publicly-traded company. Yang: GracellBiotechnologies Inc: Current Employment, Current equity holder in publicly-traded company. Sun: GracellBiotechnologies Inc: Current Employment, Current equity holder in publicly-traded company. Chen: GracellBiotechnologies Inc: Current Employment, Current equity holder in publicly-traded company. Qiu: GracellBiotechnologies Inc: Current Employment, Current equity holder in publicly-traded company. Yin: GracellBiotechnologies Inc: Current Employment, Current equity holder in publicly-traded company. Shen: GracellBiotechnologies Inc: Current Employment, Current equity holder in publicly-traded company. Sersch: GracellBiotechnologies Inc: Current Employment, Current equity holder in publicly-traded company. Cao: GracellBiotechnologies Inc: Current Employment, Current equity holder in publicly-traded company. Wang: GracellBiotechnologies Inc: Current Employment, Current equity holder in publicly-traded company.

Human Glioma Nude Mouse Xenograft Model in situ

Backgrounds: Surgery and chemotherapy are difficult because of the specific location of the glioma. The establishment of a suitable in situ model of glioma is the premise of the treatment of glioma. 8 week-old female BALB/c nude mice were chose to establish the glioma model. Methods: For the orthotopic glioma mice model, 1 × 105cells/5 μL U87-MG-Luc or U87-MG cells which were trypsinized and resuspended in sterile PBS were slowly injected into the right corpus striatum (1.8 mm lateral, 0.6 mm anterior to the bregma and 3.0 mm in depth) by a stereotactic fixation device using a mouse adaptor. Results: The othotopic U87 glioma mice model identified by imaging on IVIS Spectrum and magnetic resonance imaging after 2 weeks from surgery. H&E-stained tumor sections in brain of the mice model were also observed. Conclusions: After identification, the glioma mouse xenograft in situ model obtained could be used in the evaluation system of therapeutic drugs or methods.

Cancer-associated fibroblasts-derived HAPLN1 promotes tumour invasion through extracellular matrix remodeling in gastric cancer

Background Cancer-associated fibroblasts (CAFs) are the most principal cells of depositing and remodeling extracellular matrix (ECM) within solid tumours. Both CAFs and ECM have been demonstrated to play critical roles in tumour development. However, the functional roles of CAFs-associated ECM or ECM remodeling in the pathogenesis of gastric cancer remain unclear. Methods Bioinformatics analysis of the differentially expressed genes between CAFs and corresponding normal fibroblasts (NFs) in gastric cancer was performed. The clinical relevance of hyaluronan and proteoglycan link protein 1 (HAPLN1) was investigated using TCGA data and human gastric cancer specimens. Spheroid cell invasion assay and nude mouse xenograft model were introduced to assay cell invasion. Second harmonic generation (SHG) was used to image and analyze the changes of collagen fibers in ECM. Results HAPLN1 was identified as the most significantly up-regulated gene in CAFs of gastric cancer, and higher HAPLN1 levels were associated with shorter overall survival. HAPLN1 was prominently produced by CAFs, and its levels were correlated positively with tumor T staging (P < 0.0001), lymph node metastasis (P = 0.0006) and TNM stage (P = 0.0063). Mechanically, gastric cancer cells activate fibroblasts to up-regulate HAPLN1 expression via activation of TGF-β1/Smad2/3 signaling, which in turn promotes tumour migration and invasion. Importantly, SHG assays with mouse xenograft models and human samples further demonstrated CAFs-derived HAPLN1 increased tumour invasiveness through ECM remodeling. Conclusions This study sheds light on the role of CAFs-derived HAPLN1 in the pathogenesis of gastric cancer, and provides insights for the development of novel strategies for prevention and treatment of gastric carcinoma.

Anti-tumor effects of rivoceranib against canine melanoma and mammary gland tumour in vitro and in vivo mouse xenograft models

Background Rivoceranib, a novel tyrosine kinase inhibitor, exhibits anti-tumour effects by selectively blocking vascular endothelial growth factor receptor-2 (VEGFR2) in cancer cells. Recently, the therapeutic effects of rivoceranib on solid tumours have been elucidated in human patients. However, the anti-tumour effects of rivoceranib against canine cancer remain unclear. Here, we investigated the anti-tumour effects of rivoceranib using in vitro and in vivo mouse xenograft models. Methods We performed cell proliferation, cell cycle, and migration assays to determine the effects of rivoceranib on canine solid tumour cell lines in vitro. Furthermore, apoptosis and angiogenesis in tumour tissues were examined using a TUNEL assay and immunohistochemistry methods with an anti-cluster of differentiation-31 antibody, respectively. Additionally, the expression levels of cyclin-D1 and VEGFR2 activity were determined using western blot analysis. Results Rivoceranib treatment showed anti-proliferative effects and mediated cell cycle arrest in the canine melanoma cell line (LMeC) and the mammary gland tumour (MGT) cell line (CHMp). In animal experiments, rivoceranib decreased the average volume of LMeC cells compared to that following control treatment, and similar results were observed in CHMp cells. Histologically, rivoceranib induced apoptosis and exerted an anti-angiogenic effect in tumour tissues. It also downregulated the expression of cyclin-D1 and inhibited VEGFR2 activity. Conclusion Our results show that rivoceranib inhibits proliferation and migration of tumour cells. These findings support the potential application of rivoceranib as a novel chemotherapeutic strategy for canine melanoma and MGTs.

KIF2C Is a Novel Prognostic Biomarker and Correlated with Immune Infiltration in Endometrial Cancer

Endometrial cancer (EC) is commonly diagnosed cancer in women, and the prognosis of advanced types of EC is extremely poor. Kinesin family member 2C (KIF2C) has been reported as an oncogene in cancers. However, its pathophysiological roles and the correlation with tumor-infiltrating lymphocytes in EC remain unclear. The mRNA and protein levels of KIF2C in EC tissues were detected by qRT-PCR, Western blot (WB), and IHC. CCK8, Transwell, and colony formation assay were applied to assess the effects of KIF2C on cell proliferation, migration, and invasion. Cell apoptosis and cell cycle were analyzed by flow cytometry. The antitumor effect was further validated in the nude mouse xenograft cancer model and humanized mouse model. KIF2C expression was higher in EC. Knockdown of KIF2C prolonged the G1 phases and inhibited EC cell proliferation, migration, and invasion in vitro. Bioinformatics analysis indicated that KIF2C is negatively correlated with the infiltration level of CD8+ T cells but positively with the poor prognosis of EC patients. The apoptosis of CD8+ T cell was inhibited after the knockdown of KIF2C and was further inhibited when it is combined with anti-PD1. Conversely, compared to the knockdown of KIF2C expression alone, the combination of anti-PD1 further promoted the apoptosis of Ishikawa and RL95-2 cells. Moreover, the knockdown of KIF2C inhibited the expression of Ki-67 and the growth of tumors in the nude mouse xenograft cancer model. Our study found that the antitumor efficacy was further evaluated by the combination of anti-PD1 and KIF2C knockdown in a humanized mouse model. This study indicated that KIF2C is a novel prognostic biomarker that determines cancer progression and also a target for the therapy of EC and correlated with tumor immune cells infiltration in EC.

Hydroxygenkwanin Increases the Sensitivity of Liver Cancer Cells to Chemotherapy by Inhibiting DNA Damage Response in Mouse Xenograft Models

Molecules involved in DNA damage response (DDR) are often overexpressed in cancer cells, resulting in poor responses to chemotherapy and radiotherapy. Although treatment efficacy can be improved with the concomitant use of DNA repair inhibitors, the accompanying side effects can compromise the quality of life of patients. Therefore, in this study, we identified a natural compound that could inhibit DDR, using the single-strand annealing yeast-cell analysis system, and explored its mechanisms of action and potential as a chemotherapy adjuvant in hepatocellular carcinoma (HCC) cell lines using comet assay, flow cytometry, Western blotting, immunofluorescence staining, and functional analyses. We developed a mouse model to verify the in vitro findings. We found that hydroxygenkwanin (HGK) inhibited the expression of RAD51 and progression of homologous recombination, thereby suppressing the ability of the HCC cell lines to repair DNA damage and enhancing their sensitivity to doxorubicin. HGK inhibited the phosphorylation of DNA damage checkpoint proteins, leading to apoptosis in the HCC cell lines. In the mouse xenograft model, HGK enhanced the sensitivity of liver cancer cells to doxorubicin without any physiological toxicity. Thus, HGK can inhibit DDR in liver cancer cells and mouse models, making it suitable for use as a chemotherapy adjuvant.

Long Noncoding RNA AC007639.1 Promotes the Pathogenesis and Progression of Hepatocellular Carcinoma Through Inhibiting Apoptosis and Stimulating Chemotherapeutic Resistance

BackgroundHepatocellular carcinoma (HCC) is known for its poor prognosis. Long noncoding RNAs (lncRNAs) are critical in the pathogenesis of various types of cancers. We tried to explore the role of lncRNA in the development of HCC.MethodsWe identified the role of lncRNA AC007639.1 in the pathogenesis of HCC through bioinformatics and biological experiments in HepG2, Hep3B, and SMMC-7721 cells as well as the nude mice xenograft model.ResultsWe found that lncRNA AC007639.1 was overexpressed in hepatocellular carcinoma. Knocking down of lncRNA AC007639.1 by specific siRNAs or shRNAs promoted cancer cell death. The growth of mouse xenograft tumor created using lncRNA AC007639.1 deficient HepG2 cells was significantly slowed down. Furthermore, the knockdown of lncRNA AC007639.1 in HCC cells led to the increased expression of p53 and decreased expression of angiopoietin-like 4.ConclusionLncRNA AC007639.1 was involved in the pathogenesis and progression of hepatocellular carcinoma by inhibition of apoptosis and increasing HCC resistance to chemotherapy.