Xenogeneic Tumor Research Articles

The expression of PD-L1 on tumor-derived exosomes enhances infiltration and anti-tumor activity of αCD3 × αPD-L1 bispecific antibody-armed T cells

Anti-cluster of differentiation (CD) 3 × α programmed death-ligand 1 (PD-L1) bispecific T-cell engager (BsTE)-bound T-cells (BsTE:T) are a promising new cancer treatment agent. However, the mechanisms of action of bispecific antibody-armed activated T-cells are poorly understood. Therefore, this study aimed to investigate the anti-tumor mechanism and efficacy of BsTE:T. The BsTE:T migration was assessed in vivo and in vitro using syngeneic and xenogeneic tumor models, flow cytometry, immunofluorescence staining, transwell migration assays, microfluidic chips, Exo View R100, western blotting, and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 technology. In murine B16 melanoma, MC38 colon cancer, and human multiple myeloma cells, BsTE:T exhibited superior tumor elimination relative to that of T-cells or BsTE alone. Moreover, BsTE:T migration into tumors was significantly enhanced owing to the presence of PD-L1 in tumor cells and secretion of PD-L1-containing exosomes. Furthermore, increased infiltration of CD44highCD62Llow effector memory CD8+ T-cells into tumors was closely associated with the anti-tumor effect of BsTE:T. Therefore, BsTE:T is an innovative potential anti-tumor therapy, and exosomal PD-L1 plays a crucial role both in vitro and in vivo in the anti-tumor activity of BsTE:T.Graphical abstract

GT-00AxIL15, a Novel Tumor-Targeted IL-15-Based Immunocytokine for the Treatment of TA-MUC1-Positive Solid Tumors: Preclinical In Vitro and In Vivo Pharmacodynamics and Biodistribution Studies.

GT-00AxIL15 is a novel interleukin-15-based immunocytokine targeting a tumor-specific, glycosylated epitope of MUC1 (TA-MUC1). We characterized mode of action, pharmacokinetic (PK) and pharmacodynamic (PD) properties and investigated the relevance of TA-MUC1 binding for the concept of delivering IL-15 to solid tumors. In vitro pharmacology was analyzed in binding and cell-based assays. The in vivo PK profile and IL-15-mediated PD effects of GT-00AxIL15 were investigated in tumor-free mice. Tumor accumulation, immune infiltration and anti-tumor activity were assessed in TA-MUC1+ syngeneic and xenogeneic murine tumor models. GT-00AxIL15 was shown to specifically bind TA-MUC1 on tumor cells via its mAb moiety, to IL-15 receptors on immune cells via its IL-15 fusion modules and to FcγRs via its functional Fc-part. In vitro, NK, NKT and CD8+ T cells were activated and proliferated, leading to anti-tumor cytotoxicity and synergism with antibody-dependent cellular cytotoxicity (ADCC)-mediating mAbs. In vivo, GT-00AxIL15 exhibited favorable PK characteristics with a serum half-life of 13 days and specifically accumulated in TA-MUC1+ tumors. In the tumor microenvironment, GT-00AxIL15 induced robust immune activation and expansion and mediated anti-metastatic and anti-tumor effects in syngeneic and xenograft tumor models. These results support the rationale to improve PK and anti-tumor efficacy of IL-15 by increasing local concentrations at the tumor site via conjugation to a TA-MUC1 binding mAb. The tumor-selective expression pattern of TA-MUC1, powerful immune activation and anti-tumor cytotoxicity, long serum half-life and tumor targeting properties, render GT-00AxIL15 a promising candidate for treatment of solid tumors with high medical need, e.g., ovarian, lung and breast cancer.

Characteristics of adaptive immune effectors involved in the secondary xenogeneic immune response to human melanoma cells

Introduction. Current approaches are being developed for adoptive cancer therapy using T-cells genetically modified with T-cell receptors (TCRs) with specificity for tumor antigens. The complexities of identifying antigen-specific TCRs in a patient’s repertoire and selecting therapeutic receptors necessitate the development of experimental strategies for generating tumor-specific T cells. One of such approaches could be the xenogeneic immunization of mice with human tumor cells. It seems plausible that the T cell repertoire stimulated by xenogeneic vaccination could be a source of TCRs suitable for adoptive cancer immunotherapy.Aim. To assess the prospects for using xenogeneic immunizations to generate tumor-specific memory T cells and identify their TCRs suitable for adoptive immunotherapy, we studied the dynamics of the secondary xenogeneic response in a model of induction of an immune response in mice to human melanoma cells.Materials and methods. Mice were immunized with human melanoma cells, and 45 days later, they were re-challenged with the immunizing tumor. The dynamics of the development of the secondary immune response in vivo and the composition of the involved effectors of adaptive immunity were analyzed by flow cytometry. The proliferation of lymphocytes from immune mice in response to human melanoma cells was evaluated in in vitro culture.Results. The secondary xenogeneic response was characterized by a more intense accumulation of T cells and the rapid development of the effector phase at the injection site of human melanoma. This correlated with an enhanced in vitro proliferative response of lymphocytes from immune animals to xenoantigens of the immunizing tumor. CD4+ and CD8+ memory T cells contributed equally to the development of a secondary response to human melanoma cells expressing HLA class I and II molecules. When only HLA class I was expressed on the cells of the immunizing xenogeneic tumor, CD8+ memory cells were formed, which dominated the secondary immune response.Conclusion. Our findings confirmed the formation of a specific immunological memory for xenoantigens during xenogeneic immunization. This suggests the possibility of generating xenogeneic TCRs specific for human tumor antigens, which opens up opportunities to developing approaches for screening among them for receptor variants suited for adoptive immunotherapy of human cancers.

Cirsiliol induces autophagy and mitochondrial apoptosis through the AKT/FOXO1 axis and influences methotrexate resistance in osteosarcoma

BackgroundOsteosarcoma (OS) is the most common primary malignant bone tumor in children and adolescents, with poor outcomes for patients with metastatic disease or chemotherapy resistance. Cirsiliol is a recently found flavonoid with anti-tumor effects in various tumors. However, the effects of cirsiliol in the regulation of aggressive behaviors of OS remain unknown.MethodsThe effect of cirsiliol on the proliferation of OS cells was detected using a cell counting kit-8 (CCK-8) assay and 5-ethynyl-2′-deoxyuridine (EdU) staining, while cell apoptosis was detected using flow cytometry. Immunofluorescence was applied to visualize the expression level of the mitochondria, lysosomes and microtubule-associated protein light chain 3 (LC3). A computational molecular docking technique was used to predict the interaction between cirsiliol and the AKT protein. The impact of cirsiliol on resistance was investigated by comparing it between a methotrexate (MTX)-sensitive OS cell line, U2OS, and a MTX-resistant OS cell line, U2OS/MTX. Finally, in situ xenogeneic tumor models were used to validate the anti-tumor effect of cirsiliol in OS.ResultsCirsiliol inhibited cell proliferation and induced apoptosis in both U2OS and U2OS/MTX300 OS cells. In addition, treatment with cirsiliol resulted in G2 phase arrest in U2OS/MTX300 and U2OS cells. Cell fluorescence probe staining results showed impaired mitochondria and increased autophagy in OS cells after treatment with cirsiliol. Mechanistically, it was found that cirsiliol targeted AKT by reducing the phosphorylation of AKT, which further activated the transcriptional activity of forkhead Box O transcription factor 1 (FOXO1), ultimately affecting the function of OS cells. Moreover, in situ tumorigenesis experiments showed that cirsiliol inhibited the tumorigenesis and progression of OS in vivo.ConclusionsCirsiliol inhibits OS cell growth and induces cell apoptosis by reducing AKT phosphorylation and further promotes FOXO1 expression. These phenomena indicate that cirsiliol is a promising treatment option for OS.

Aurora Kinase a Inhibition for Gvhd and Relapse Prevention after Allogeneic HCT: Phase I Trial in Combination with Ptcy/Sirolimus

Introduction: With the wider use of post-transplant cyclophosphamide (PTCy) graft-versus-host disease (GVHD) prophylaxis, there is interest in replacing tacrolimus (TAC) with sirolimus (SIR, mTOR inhibitor) to avoid calcineurin inhibitor toxicity, maintain excellent GVHD control, and potentially allow for a greater graft-versus-tumor effect. mTOR and Aurora kinase A (AURKA) mediate CD28 costimulation in alloreactive T cells. SIR can only partially block CD28 costimulation. Adding selective AURKA inhibition to SIR following PTCy fully suppresses CD28 signal transduction, with synergistic GVHD prevention in mice (see ASH 2023 abstract ID 173376). AURKA inhibitors also have direct anti-leukemia activity. Here, we present the phase 1 dose escalation portion of a first-in-human clinical trial of PTCy plus SIR and VIC-1911 (VIC), a selective oral AURKA inhibitor, for GVHD and relapse prophylaxis after myeloablative allogeneic hematopoietic cell transplantation (alloHCT). Methods: This single-arm phase I dose escalation trial (NCT05120570) is designed to evaluate the pharmacodynamic potency of AURKA pathway suppression and the safety of PTCy/SIR/VIC. The primary endpoint of phase I is to identify the lowest biologically active dose of VIC, defined as <54% pH3ser10+ CD4+ T cells at day +21 (the lower confidence interval of the normal, pre-transplant frequency of pH3ser10+ CD4+ T cells - a marker of AURKA activity), that is safe with PTCy/SIR. Patients aged 18-60 years receive myeloablative conditioning of TBI alone at a dose of 1320 cGy. PTCy is given as 50 mg/kg on days +3 and +4, SIR on day +5 with levels targeting 8-12 ng/ml and tapered after day +100, and VIC given on days +5 to +45 at doses of 25, 50, or 75 mg BID. Patients undergoing myeloablative TBI-based alloHCT with PTCy/SIR plus mycophenolate mofetil (MMF), without VIC on a separate protocol, served as a control. Results: Proof-of-concept experiments with PTCy/SIR/alisertib (AURKA inhibitor) demonstrate synergistic protection against xenogeneic GVHD and tumor (Raji cells) in mice, compared to PTCy/TAC (median survival: 45 days v undefined, 9 mice/group, 2 experiments, P<0.0001). Unlike alisertib, VIC-1911 is not myelosuppressive, providing rationale for this trial. In vitro studies show killing of multiple leukemia cells lines at physiologically achievable VIC-1911 concentrations. VIC 75 mg BID is the lowest biologically active dose suppressing AURKA activity below the primary endpoint target of <54%, with reduced mean frequency of day +21 pH3ser10+ CD4+ T cells at 23.7% compared to 58.5% with PTCy/SIR/MMF (Figure 1A, P=0.0091). PTCy/SIR/VIC also suppresses mTOR activity in donor T cells (3.9% v 16.6% pS6+ CD4+ T cells, P=NS; pS6 frequency in healthy donor T cells is ~30-40%), concurrently ablating CD28 signal transduction. Data trends show greater Tregs at day +21 with PTCy/SIR/VIC, compared to PTCy/SIR/MMF controls (40.2% v 10.5%, P=NS). Patient characteristics are detailed in Figure 1B. Neutrophil engraftment occurred in 9/9 evaluable patients at a median of 18 days. Platelet engraftment occurred in 8/9 evaluable patients at a median of 21 days; one patient is awaiting platelet engraftment. No dose-limiting toxicities have been observed during dose escalation. Patient 1 died of non-COVID coronavirus during initial hospitalization; he received high-dose methylprednisolone for ARDS beginning on day +17, which made him unevaluable for the primary pharmacodynamic endpoint. Conclusions: A VIC-1911 dose of 75 mg BID from day +5 to day +45 effectively suppresses AURKA activity as determined by a low frequency of pH3ser10+ CD4+ T cells, ablating CD28 T cell costimulation when combined with sirolimus, resulting in no dose-limiting toxicities. VIC 75 mg BID will be studied further in an expanded phase I cohort to obtain estimates of efficacy in preventing both GVHD and relapse in PTCy-based myeloablative alloHCT.

Dual blockade of CD47 and CD24 signaling using a novel bispecific antibody fusion protein enhances macrophage immunotherapy

CD47 and its receptor SIRPα act as a dominant anti-phagocytic, ‘don’t eat me’ signal. Recent studies reveal CD24 as a novel target for cancer immunotherapy by macrophages in ovarian cancer and breast cancer. However, whether simultaneous blockade of CD47 and CD24 by bispecific antibody may result in a potential synergy is still unclear. In the present study, we for the first time designed and developed a bispecific antibody fusion protein, PPAB001 for co-targeting CD47 and CD24. Data demonstrate that simultaneous blockade of CD47/SIRPα and CD24/Siglec-10 signaling by PPAB001 potently promoted macrophage phagocytosis of tumor cells. Compared to single CD47 or CD24-targeting agent, PPAB001 was more effective in inhibiting tumor growth in both mouse 4T-1 syngeneic and human SK-OV-3 xenogeneic tumor models. Mechanistically, we found that PPAB001 therapy markedly increased the proportion of tumor-infiltrating macrophages and upregulated IL-6 and TNF-α levels that were representative macrophage inflammatory cytokines. Notably, an increased ratio of M1/M2 in tumor-infiltrating macrophages in the mice treated with PPAB001 suggested that the dual blockade may promote the transition of macrophages from M2 to M1. Taken together, our data supported the development of PPAB001 as a novel immunotherapeutic in the treatment of CD47 and CD24 double positive cancers.

Luteolin inhibits A549 cells proliferation and migration by down-regulating androgen receptors

BackgroundYi Fei Qing Hua Granules (YQG) is a traditional Chinese herbal medicine with the effects of inhibiting the proliferation of lung cancer cells. Luteolin is one of the active compounds of YQG. Luteolin is a common flavonoid extracted from natural herbs and it can promote cancer cells apoptosis has been reported. However, the underlying molecular mechanism and effects of luteolin on human lung cancer needs to be validated.MethodsMolecular docking, network pharmacology methods and quantitative structure–activity relationship (QSAR) model were used to identify the active components of YQG and their possible mechanisms of action. Western blot analysis was used to measure AR expression in A549 cells. Cell migration assays were used to detect A549 cells proliferation transfected by AR plasmid and AR mutation plasmid, respectively.ResultsTCMSP search results revealed that there are 182 active compounds in YQG, which correspond to 232 target genes. Sixty-one genes were overlapping genes in the 2 datasets of TCMSP and GeneCards. Through bioinformatics tagging of these overlapping genes, a total of 1,951 GO functional tagging analysis and 133 KEGG pathways were obtained. Through molecular docking technology and QSAR model verification, the multi-target active compound luteolin was screened out as one of the active components of YQG for in vitro verification. Androgen receptor (AR) was the hub protein with the highest docking score of luteolin. Western blot showed that luteolin could inhibit AR protein expression in lung cancer cell line A549. After the phosphorylation site of AR protein 877 was inactivated, the ability of luteolin to inhibit the proliferation of lung cancer cells was weakened. Luteolin significantly inhibited the growth of A549 xenogeneic tumors at day 25 and 28 and inhibited the expression of AR.ConclusionIn this study, we have explored luteolin as one of the active components of YQG, and may inhibit the proliferation and migration of A549 cells by decreasing the expression of AR and the regulation of phosphorylation at AR-binding sites.

Bispecific antibody targeting both B7-H3 and PD-L1 exhibits superior antitumor activities.

Clinical application of PD-1andPD-L1 monoclonal antibodies (mAbs) is hindered by their relatively low response rates and the occurrence of drug resistance. Co-expression of B7-H3 with PD-L1 has been found in various solid tumors, and combination therapies that target both PD-1/PD-L1 and B7-H3 pathways mayprovide additional therapeutic benefits. Up to today, however, no bispecific antibodies targeting both PD-1 and B7-H3 have reached the clinical development stage. In this study, we generated a stable B7-H3×PD-L1 bispecific antibody (BsAb) in IgG1-VHH format by coupling a humanized IgG1 mAb against PD-L1 with a humanized camelus variable domain of the heavy-chain of heavy-chain antibody (VHH) against human B7-H3. The BsAb exhibited favorable thermostability, efficient T cell activation, IFN-γ production, and antibody-dependent cell-mediated cytotoxicity (ADCC). In a PBMC humanized A375 xenogeneic tumor model, treatment with BsAb (10 mg/kg, i.p., twice a week for 6 weeks) showed enhanced antitumor activities compared to monotherapies and, to some degree, combination therapies. Our results suggest that targeting both PD-1 and B7-H3 with BsAbs increases their specificities to B7-H3 and PD-L1 double-positive tumors and induces a synergetic effect. We conclude that B7-H3×PD-L1 BsAb is favored over mAbs and possibly combination therapies in treating B7-H3 and PD-L1 double-positive tumors.

Human CAR19 CD8 +iTreg exceed CAR19 CD8 CTLs both in suppressing GVHD lethality and eliminating CD19 +Nalm-6 lymphoma cells in vivo

Abstract Simultaneously achieving a graft versus leukemia (GVL) effect while suppressing graft versus host disease (GVHD) would facilitate the development of immunotherapies for hematopoietic stem cell transplantation. Here, we show that human peripheral blood CD8+CD25− T-cells cultured with IL-2, TGF-β and rapamycin generates CD8 induced Tregs (iTregs). CD8 iTregs expressed FoxP3, CD25 and higher CD103 and CD39, but lower Helios, CTLA-4, and TIGIT than CD4 Tregs. While in vitro suppressor function was modestly reduced with CD8 iTreg, in vivo suppression of GVHD lethality was comparable to that of CD4 Treg. In vitro, cytolytic activity with an αCD3×αCD19 bispecific engager against CD19+ Nalm-6 lymphoma cells was comparable to CD8 CTLs. We subsequently generated CD8 iTreg and CTLs expressing αCD19scFv (CAR19). Following CAR19-EGFR sort purification, CAR19 CD8 iTreg and CAR19 CTL had similar expression of perforin, IFNγ and TNFα. In a 48h Incucyte killing assay against CD19+Nalm-6 targets, using CAR19 CD8 iTreg and CAR19 CTLs generated from the same donors, no significant differences were noted; CD8 iTreg killing was perforin-dependent and granzyme-independent. No killing was observed against CD19 koNalm-6 targets. Unexpectedly, CAR19 CD8 iTreg were found to be significantly more effective at limiting tumor burden in vivo and significantly delayed tumor-related mortality in a xenogeneic Nalm-6 tumor model compared to CAR19 CTLs (p = 0.0018). Mechanistic studies are ongoing. Together, these findings highlight distinct advantages of CD8 iTreg as a concurrent immunotherapy and provide a rationale for CD8 iTreg based CAR therapies to prevent GVHD and promote GVL. Supported by grants from NIH (R01 HL11879-07 and 2P01 CA065493-25) as well as funding from the Childrens' Cancer Research Fund

Testing Cancer Immunotherapeutics in a Humanized Mouse Model Bearing Human Tumors.

Reversing the immunosuppressive nature of the tumor microenvironment is critical for the successful treatment of cancers with immunotherapy drugs. Murine cancer models are extremely limited in their diversity and suffer from poor translation to the clinic. To serve as a more physiological preclinical model for immunotherapy studies, this protocol has been developed to evaluate the treatment of human tumors in a mouse reconstituted with a human immune system. This unique protocol demonstrates the development of human immune system (HIS, "humanized") mice, followed by implantation of a human tumor, either a cell-line derived xenograft (CDX) or a patient derived xenograft (PDX). HIS mice are generated by injecting CD34+ human hematopoietic stem cells isolated from umbilical cord blood into neonatal BRGS (BALB/c Rag2-/- IL2RγC-/- NODSIRPα) highly immunodeficient mice that are also capable of accepting a xenogeneic tumor. The importance of the kinetics and characteristics of the human immune system development and tumor implantation is emphasized. Finally, an in-depth evaluation of the tumor microenvironment using flow cytometry is described. In numerous studies using this protocol, it was found that the tumor microenvironment of individual tumors is recapitulated in HIS-PDX mice; "hot" tumors exhibit large immune infiltration while "cold" tumors do not. This model serves as a testing ground for combination immunotherapies for a wide range of human tumors and represents an important tool in the quest for personalized medicine.

BCMA CAR-T Cell Phenotype and Functionality Is Affected By Disease Stage of Multiple Myeloma Patients

Background: Refractory or relapsed (R/R) multiple myeloma (MM) is nowadays an incurable disease. CAR-T immunotherapy against BCMA has proven its efficacy with complete response rates of 83-85%. However, despite the initial favorable response, most patients relapse with a median progression free survival of 8-24 months. This lack of long-term response could be related to the fitness and the functionality of the CAR-T cells. Thus, we hypothesize that CAR-T cells generated from patients at early stages of the disease would present improved phenotype and functionality. In this work we performed phenotypic and functional characterization of CAR-T generated from healthy donors and MM patients at different disease stage. Methodology: Second generation CAR-T cells targeting BCMA and co-expressing BFP as a reporter gene (modified from ARI0002h CAR-T sequence1), were generated by lentiviral transduction of aCD3/aCD28 activated T cells, from each group of donor/patients, and expanded in the presence of IL-7/IL-15. Phenotypic analyses were performed by flow cytometry. Cytotoxic activity was measured by standard luciferase-based procedures and cytokine production was quantified by ELISA and Luminex. In vivo antitumoral efficacy was evaluated in xenogeneic tumor models in NSG mice. RNA-seq analysis was performed following stablished protocols2. Results: We were able to generate CAR-T cells from young (n=5) and senior healthy donors (n=5), as well as patients with MGUS (n=5), SMM (n=5), MM at diagnosis (n=3), MM after 1st line of treatment (n=5) and MM after >2 lines of treatment (n=4). CAR-T cells presented increasing variability in CAR transduction and cell expansion along disease progression. Phenotypic analysis revealed a decreasing memory phenotype, of both stem cell memory and central memory CAR-T cells, with significantly increasing number of effector cells during disease progression. This phenotype was already observed in T cells used for CAR-T cell generation, suggesting that initial phenotype strongly affects final CAR-T cell features. However, no significant differences were observed in activation (HLA-DR, CD137) and exhaustion markers (PD1, LAG3, TIM3, TIGIT). Functional in vitro characterization of CAR-T cells revealed a reduced cytotoxicity against MM cell lines once MM is diagnosed, that was associated with lower production of effector cytokines (IFNg, Granzyme A, MIP1a), especially in CAR-T cells form MM patients with >2 lines of treatment. Moreover, after continuous exposure to tumoral cells, CAR-T cells from these MM patients presented a significant upregulation of exhaustion markers (PD1, LAG3, TIM3, TIGIT), suggesting that long-term functionality could be compromised. In contrast, these phenotypic and functional differences observed in vitro were not reflected in the in vivo antitumoral response, that showed a similar efficacy between CAR-T cells from the different groups. This lack of differences could be probably due to the xenogeneic tumor model in NSG mice, that would require stringent conditions to observe differences in antitumor efficacy. Transcriptomic analyses revealed that CAR-T cells did not cluster according to disease stage due to the high variability between patients. We focus our analysis on CAR-T cells from MM patients at diagnosis (MMd) and after >2 lines of treatment (MM>2Lines), using age-matched senior healthy donors (SHD) as control. Major differences were observed between MM>2Lines and MMd/SHD, with few differences between these last groups. Gene ontology analysis of differentially expressed genes in MM>2Lines group revealed enrichment in pathways related to T cell activation, cell-cell adhesion, and lymphocyte differentiation. Moreover, deeper characterization of CAR-T cells from MM>2Lines patients showed decreased score of proliferation and polyfunctional gene sets, with enrichment in genes related to terminally effector features, corroborating functional results previously observed. Conclusions: Our results would indicate that CAR-T generated from patients at later stages of the disease present a more differentiated phenotype, with reduced functionality and increased exhaustion features, that could compromise antitumor efficacy. Transcriptomic analysis allows the identification of molecular mechanisms that could be modulated for the development of improved therapies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Oxaliplatin inhibits colorectal cancer progression by inhibiting CXCL11 secreted by cancer-associated fibroblasts and the CXCR3/PI3K/AKT pathway.

Colorectal cancer (CRC) is a malignant tumor. Oxaliplatin (OXA) can inhibit cancer-associated fibroblasts (CAFs)-induced cancer progression. This study sought to explore the mechanism of OXA in CAFs-induced CRC development. CRC cell lines (Caco-2, SW620), normal fibroblasts (NFs), and CAFs were treated with OXA. NFs and CAFs were cultured. CAFs were treated with/without OXA (0.4mM), and the supernatant was extracted as the conditioned medium (CM) to culture CRC cells. Cell malignant episodes, E-cadherin and Vimentin levels, CXCL1, CXCL2, CXCL3, CXCL8, and CXCL11 mRNA levels, CXCL11 protein level, and extracellular release were assessed. CAFs were transfected with interfering RNA sh-CXCL11 to silence CXCL11 or transfected with CXCL11 overexpression plasmids and treated with OXA to explore the role of CXCL11 in OXA-mediated CRC cells through CAFs. CXCL11 receptor CXCR3 levels in CRC cells and the PI3K/AKT pathway changes were examined. The xenogeneic tumor was transplanted in nude mice. CXCL11 and CXCR3 levels in tumor tissues, tumor volume, shape, size, weight, and Ki67 positive expressions were assessed. CRC cell growths and epithelial-mesenchymal transformation were stimulated after culture with CAFs-CM, while OXA averted these trends. CXCL11 mRNA level was elevated most significantly, and its protein and extracellular secretion levels were raised, while OXA diminished the levels. CXCL11 silencing weakened the effects of CAFs-CM on promoting CRC proliferation and malignant episodes and CXCL11 overexpression averted OXA property on inhibiting CAFs-promoted CRC cell growth. CXCR3 and PI3K and AKT1 phosphorylation levels were raised in the CAFs-CM group but diminished by OXA. CXCL11 overexpression in CAFs averted OXA property on inhibiting CAFs-activated CXCR3/PI3K/AKT in CRC cells. OXA also inhibited the progression of xenograft tumors by limiting CAFs-secreted CXCL11. OXA repressed CRC progression by inhibiting CAFs-secreted CXCL11 and the CXCR3/PI3K/AKT pathway.

Abstract 2884: Optimized CD28 bispecific antibodies for targeted activation of T cells within the tumor microenvironment

Abstract Tumor-targeted CD28 bispecific antibodies (bsAbs) are designed to co-stimulate T cells specifically within the tumor microenvironment. By bridging T cells to malignant cells expressing a selected tumor-associated antigen (TAA), CD28 bsAbs deliver the so-called signal 2 to T cells unleashing their full cytotoxic potential. Contrary to bivalent CD28 monoclonal antibody (mAb) superagonists, the activation of CD28 with monovalent CD28 bispecifics requires the binding of a TAA expressed by the cancer cells to induce CD28 clustering. Preclinical studies have shown the benefit of adding costimulatory CD28 bsAbs for the treatment of solid tumors, boosting the efficacy of bispecific T cell engagers (CD3 bsAbs) or PD-(L)1 checkpoint inhibitors. Using our proprietary fully human κλ body antibody platform, which relies on a common heavy chain and on two distinct light chains driving specificity and affinity (i.e., one kappa and one lambda), we have identified a panel of agonist anti-CD28 kappa arms. Using our existing lambda anti-TAA arms (e.g., targeting CD19, MSLN, HER2, EGFR), an array of TAA-CD28 κλ bodies targeting different tumors were generated. The capacity of these CD28 κλ bodies to provide signal 2 to T cells was assessed both in vitro and in vivo in combination with CD3 bsAbs. In the presence of cancer cells and corresponding TAA-CD3 bsAbs, the CD28 κλ bodies enabled the activation of primary human CD4 and CD8 T cells, leading to T cell proliferation and secretion of cytokines such as IL-2 and IFNγ, and granzyme B. Importantly, when used alone or in combination with an untargeted CD3 bsAb (that is, in the absence of signal 1), CD28 κλ bodies showed no sign of T cell activation, potentially limiting peripheral toxicity. The resulting change in the T cell activation status led to a potent in vitro T cell-dependent cellular cytotoxicity (TDCC) against TAA-expressing cancer cell lines. CD28 κλ bodies targeting gastrointestinal tumors were tested in combination with CD3 bsAbs in xenogeneic tumor models and synergistically improved the antitumoral activity of T cell engagers, leading to an enhanced tumor control. Thus, via the identification of optimal agonist anti CD28 arms, CD28 bsAbs targeting multiple TAAs were designed for tumor-specific activation of the immune system. The resulting TAA-CD28 κλ bodies can enhance the antitumor response induced by CD3-retargeting bsAbs via the induction of T cell proliferation and activation, increased cytokine secretion and boosted anti-tumoral cytotoxicity. Other anti-TAA arms are being explored, to expand the panel of TAAs that could be easily paired with our anti-CD28 platform arms. Citation Format: Sara Majocchi, Pauline Lloveras, Coline Burnet-Merlin, Lise Nouveau, Nicolas Pleche, Pauline Malinge, Claudia Batista, Christelle Daviet, Maud Charreton, Guillemette Pontini, Franck Gueneau, Valery Moine, Giovanni Magistrelli, Limin Shang, Walter Ferlin, Krzysztof Masternak. Optimized CD28 bispecific antibodies for targeted activation of T cells within the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2884.

Abstract 1654: Mouse models of hepatocellular carcinoma: A comprehensive and functional preclinical platform for immunotherapy research

Abstract Hepatocellular carcinoma (HCC) is the fourth leading cause of cancer-related death and accounts for over 80% of primary liver cancer worldwide. Early stage HCC can be treated by local ablation, surgical resection or liver transplantation. Systemic pharmacological options are limited (kinase and immune-checkpoint inhibitors). Most cases of liver cancer occur in the setting of chronic liver diseases. Risk factors include chronic Hepatitis B and C, alcohol addiction and metabolic diseases. HCC is a multistep process comprising chronic liver injury, inflammation, fibrosis/cirrhosis and cancer formation. Therefore, providing palliative and curative options remains a high medical need. And with the recent success of immunotherapies in HCC, mouse models that better recapitulate the human disease and antitumor immune response are needed. In order to better evaluate new preventive and curative treatments of liver cancers we developed complementary and integrated strategies to mimic the liver cancer initiation and progression steps in mouse models. These models involve chemotoxic agents, diet-induced disorders and syngeneic or xenogeneic tumor implantation strategies. We established an orthotopic syngeneic model using Hepa1.6 mouse liver hepatoma cells, characterized through liver index, alpha-fetoprotein measurement in serum and liver, and MRI. The response to chemotherapy (sorafenib) and immunotherapy (anti PD-1, anti-TLR) was also assessed and show moderate to high efficiency. Moreover, a panel of xenograft models including Patient-Derived Xenograft models (PDXs) are available to assess new treatment options in human HCC with regards of the genetic mutations and the variety of etiologies seen in human. But as xenograft models are not completely mimicking the human situation of both immune and liver microenvironment, we have recently initiated the development of a double humanized (immune and liver) transgenic mouse as a better host for human tumor engraftment. During the course of HCC formation, the liver undergoes cycles of inflammation, necrosis with regeneration, fibrosis, cell dysplasia and ultimately HCC. Thus, we developed a model induced by a low dose of streptozotocin and high fat diet regimen. In this model, mice develop metabolic syndrome NASH and fibrosis within 12 weeks and HCC within 16 weeks. Of interest, 100% of male mice develop HCC within 16 weeks, in accordance with studies showing that men had a 2-to 7-fold higher risk of developing HCC in human. Treatment of mice with lenvatinib alone or in combination with anti-PD1 increases survival and reduces tumor burden as shown with reduced liver weight/body weight ratio at 16 weeks. Altogether, these results demonstrate the usefulness of this comprehensive platform of preclinical in vivo HCC models to discover and identify novel therapeutic strategies that could circumvent the progression of liver cancers. Citation Format: Olivier Duchamp, Gael Krysa, Loic Morgand, Hugo Quillery, Robin Artus, Maxime Ramelet, Jeremy Odillard, Peggy Provent, Sylvie Maubant, Caroline Mignard, Fabrice Viviani, Samira Benhamouche-Trouillet. Mouse models of hepatocellular carcinoma: A comprehensive and functional preclinical platform for immunotherapy research [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1654.

Niclosamide induces miR-148a to inhibit PXR and sensitize colon cancer stem cells to chemotherapy.

SummaryTumor recurrence is often attributed to cancer stem cells (CSCs). We previously demonstrated that down-regulation of Pregnane X Receptor (PXR) decreases the chemoresistance of CSCs and prevents colorectal cancer recurrence. Currently, no PXR inhibitor is usable in clinic. Here, we identify miR-148a as a targetable element upstream of PXR signaling in CSCs, which when over-expressed decreases PXR expression and impairs tumor relapse after chemotherapy in mouse tumor xenografts. We then develop a fluorescent reporter screen for miR-148a activators and identify the anti-helminthic drug niclosamide as an inducer of miR-148a expression. Consequently, niclosamide decreased PXR expression and CSC numbers in colorectal cancer patient-derived cell lines and synergized with chemotherapeutic agents to prevent CSC chemoresistance and tumor recurrence in vivo. Our study suggests that endogenous miRNA inducers is a viable strategy to down-regulate PXR and illuminates niclosamide as a neoadjuvant repurposing strategy to prevent tumor relapse in colon cancer.

Pharmacological inhibition of Ref-1 enhances the therapeutic sensitivity of papillary thyroid carcinoma to vemurafenib

The use of the BRAF inhibitor vemurafenib exhibits drug resistance in the treatment of thyroid cancer (TC), and finding more effective multitarget combination therapies may be an important solution. In the present study, we found strong correlations between Ref-1 high expression and BRAF mutation, lymph node metastasis, and TNM stage. The oxidative stress environment induced by structural activation of BRAF upregulates the expression of Ref-1, which caused intrinsic resistance of PTC to vemurafenib. Combination inhibition of the Ref-1 redox function and BRAF could enhance the antitumor effects of vemurafenib, which was achieved by blocking the action of Ref-1 on BRAF proteins. Furthermore, combination treatment could cause an overload of autophagic flux via excessive AMPK protein activation, causing cell senescence and cell death in vitro. And combined administration of Ref-1 and vemurafenib in vivo suppressed PTC cell growth and metastasis in a cell-based lung metastatic tumor model and xenogeneic subcutaneous tumor model. Collectively, our study provides evidence that Ref-1 upregulation via constitutive activation of BRAF in PTC contributes to intrinsic resistance to vemurafenib. Combined treatment with a Ref-1 redox inhibitor and a BRAF inhibitor could make PTC more sensitive to vemurafenib and enhance the antitumor effects of vemurafenib by further inhibiting the MAPK pathway and activating the excessive autophagy and related senescence process.

CAR Density Influences Antitumoral Efficacy of BCMA CAR-T Cells and Correlates with Clinical Outcome

Background: Chimeric Antigen Receptor-modified T cell (CAR-T) therapies have revolutionized cancer immunotherapy, especially in hematological malignancies. Although great results have been achieved during the last years, long-term efficacy is still compromised in some cases and factors behind CAR-T cell disfunction are not fully understood. Recent studies have shown that the control of CAR expression influences CAR-T fitness and antitumoral efficacy 1. Therefore, we hypothesized that CAR density on the membrane of CAR-T cells could directly affect CAR-T cell function. In this study we perform a functional and genomic analysis of FACS-isolated subpopulations of CAR-T cells with different CAR densities (CAR High and CAR Low).Methodology: Second generation CAR-T cells with 4-1BB costimulatory domain targeting BCMA were generated by lentiviral transduction of αCD3/αCD28 activated T cells that were expanded for 12-14 days in the presence of IL-7/IL-15. Phenotypic analyses were performed by flow cytometry before and after coculture with MM cells. Cytotoxic activity and cytokine production were measured by standard procedures. In vivo antitumoral efficacy was evaluated in xenogeneic tumor models in NSG mice. Transcriptomic (RNA-seq) and epigenetic (ATAC-seq) analysis were performed following stablished protocols 2. Single cell analysis was performed using the Chromium Single Cell Immune Profiling solution from 10x Genomic that allows simultaneous analysis of gene expression and paired T-cell receptors from a single cell. Gene Regulatory Network (GRN) analysis was performed using SimiC, a novel computational method that infers regulatory dissimilarities 3.Results: RNA-seq and ATAC-seq analysis revealed completely different profiles between CAR High- and CAR Low-T cells in both CD4 +and CD8 + cell subsets, with >3500 differentially expressed genes (2086 for CD4 + and 1553 for CD8 +) that were related with increased tonic signaling, T cell activation and proliferation in CAR High-T cells. Functional studies at resting state (before antigen encounter) corroborated that CAR High-T cells presented increased tonic signaling, that lead to a higher basal activation and a more differentiated phenotype with skewed presence of CCR7 +/CD45RA +/CXCR3 + T SCM cells. After antigen-driven activation, increased cytotoxicity and cytokine production was observed in CAR High-T cells, that also presented higher percentage of terminally differentiated effector cells (CCR7 -/CD45RA +), along with increased exhaustion (PD1 +/LAG3 +/TIGIT +). This effect was also observed in the infusion products of CARTBCMA-HCB-01 clinical trial for patients with R/R MM (NCT04309981), where products enriched in CAR High-T cells presented increased cytotoxic activity. Although no significant differences were observed in the antitumoral efficacy in vivo, CAR Low-T cells presented increased persistence, suggesting that higher CAR levels could reduce long-term efficacy.Further characterization of CAR-T cells at single cell level (scRNA-seq) showed enrichment of CAR High-T cells in activated CD4 + and exhausted CD8 + cell clusters. The analysis of regulatory dissimilarities driven by different CAR densities with SimiC revealed an increased activity of the regulon associated to NR4A1 transcription factor (a well-known TF driving T cell exhaustion 4) in CAR High-T cells, providing mechanistic insights of the regulatory networks behind differential functionality of CAR High-T cells.Finally, to evaluate the impact of CAR density in the clinical outcome of CAR-T therapies, we developed a gene signature associated to increased CAR density, that was applied to transcriptomic data available from public studies 5. We score the infusion products of several clinical trials testing CTL019 (NCT01029366, NCT01747486 and NCT02640209) and we observed an enrichment on CAR High signature in the products from non-responder patients.Conclusions: Our data demonstrate that CAR density on the membrane of engineered T cells plays important roles in CAR-T activity with a significant impact on clinical outcome. Moreover, the comprehension of regulatory mechanisms driven by CAR densities at the single cell level offer an important tool for the identification of key regulatory factors that could be modulated for the development of improved therapies. [Display omitted] DisclosuresRodríguez-Otero: Oncopeptides: Honoraria, Membership on an entity's Board of Directors or advisory committees; Kite: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Regeneron: Membership on an entity's Board of Directors or advisory committees; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS/Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel and other expenses. Paiva: Bristol-Myers Squibb-Celgene, Janssen, and Sanofi: Consultancy; Adaptive, Amgen, Bristol-Myers Squibb-Celgene, Janssen, Kite Pharma, Sanofi and Takeda: Honoraria; Celgene, EngMab, Roche, Sanofi, Takeda: Research Funding. San-Miguel: AbbVie, Amgen, Bristol-Myers Squibb, Celgene, GlaxoSmithKline, Janssen, Karyopharm, Merck Sharpe & Dohme, Novartis, Regeneron, Roche, Sanofi, SecuraBio, Takeda: Consultancy, Other: Advisory board. Prósper: Oryzon: Honoraria; Janssen: Honoraria; BMS-Celgene: Honoraria, Research Funding.

F-circEA1 regulates cell proliferation and apoptosis through ALK downstream signaling pathway in non-small cell lung cancer

Studies have confirmed that circular RNA (circRNA) has a stable closed structure, which plays an important role in the progression of tumors. Cancers with positive fusion genes can produce associated fusion circRNA (F-cirRNA). However, there are no reports concerning a role for F-circRNA of the echinoderm microtubule associated-protein like 4-anaplastic lymphoma kinase variant 1 (EML4-ALK1) in non-small cell lung cancer (NSCLC). Our study confirmed the existence of fusion circEA1 (F-circEA1) in NCI-H3122 cells (carrying the EML4-ALK1 gene), F-circEA1 was expressed both in the cytoplasm and nucleus as determined by fluorescence in situ hybridization (FISH) and Sanger sequencing. CCK8 and transwell assays showed that F-circEA1 was beneficial to cell proliferation, metastasis, and invasion. Overexpression of F-circEA1 can also promote cell proliferation, migration and invasion in A549 and SPCA1 cells (non-small cell lung cancer cell line not carrying the EML4-ALK1 gene). Interference with F-circEA1, induced cell cycle arrest and promoted apoptosis as determined by flow cytometry, and increased drug sensitivity to crizotinib in H3122 cells. F-circEA1 directly affected the expression of parental gene EML4-ALK1. Further research found that F-circEA1 can affect the downstream signaling pathway of ALK. In vivo, the growth rate of xenogeneic tumors was reduced and the protein expression level of EML4-ALK1 was significantly decreased in transplanted tumors measured by immunohistochemistry (IHC) after interference with F-circEA1. In conclusion, F-circEA1 can be considered as a proto-oncogene that regulates cell proliferation and apoptosis by affecting the expression of the parental gene EML4-ALK1 and its ALK downstream signaling pathway in non-small cell lung cancer.

MicroRNA-5100 Modulates Lung Cancer Cell Proliferation and Apoptosis via Inhibiting X-Linked Inhibitor of Apoptosis Protein (XIAP) Expression

This study assesses the miR-5100 expression and its function in human lung cancer. The expression of miR-5100 was analyzed by miScript miRNA method. Cancer cells were transfected with miR-5100 mimics (miR-5100), miR-5100 inhibitors (ASO-miR-5100), XIAP inhibitors (si-XIAP), negative controls (NC) followed by analysis of cell proliferation by MTT and apoptosis by flow cytometry, the expression of XIAP related proteins by Western blot. miR-5100’ target was predicted by bioinformatics website and verified by dual luciferase assay. Finally, a xenogeneic tumor inhibition model was established to detect tumor progression after treatments. Lung cancer cells and tissues exhibited significantly reduced miR-5100 level. Dual luciferase assay showed that miR-5100 bound XIAP 3′-UTR and reduced XIAP mRNA and protein level. Further, miR-5100 inhibited cell proliferation, increased apoptosis and the expression of cleaved-capsase-3 and cleaved-capsase-9, the XIAP downstream factor. Finally, miR-5100 inhibited tumor growth, decreased cellular proliferation and promoted apoptosis, accompanied by reduced XIAP expression in vivo. miR-5100 inhibits lung cancer cell proliferation and enhances apoptosis through inhibiting XIAP expression in vitro and in vivo.

Alternol Sensitizes Renal Carcinoma Cells to TRAIL-Induced Apoptosis.

Purpose: Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL), a member of the TNF family, can selectively induce cancer cell death while sparing normal cells. However, the application of TRAIL-based antitumor therapies has been hindered due to drug resistance. Alternol is a new compound isolated from microbial fermentation that possesses antitumor activity in different tumors. In our research, we discovered that alternol can sensitize TRAIL-induced apoptosis in renal carcinoma cells (RCCs). Materials and Methods: Cytotoxic activity was measured by MTT assay. Apoptosis was probed using the PI/annexin V method. Real-time PCR and western blot were used to test the levels of mRNA and protein, respectively. Luciferase assay was used to investigate whether CHOP regulated the expression of death receptor (DR) 5 through transcription. A xenogeneic tumor transplantation model was used to evaluate the anticancer effects of alternol/TRAIL in vivo. Results: When the mechanisms were investigated, we discovered that alternol increased DR5 expression. DR5 knockdown by siRNA eliminated the enhanced effect of alternol on TRAIL-mediated apoptosis. Alternol reduced the expression of antiapoptotic proteins and increased the levels of proapoptotic proteins. Moreover, alternol increased the level of CHOP, which is necessary for the enhancing effect of alternol on TRAIL-induced apoptosis, given that downregulation of CHOP abrogated the synergistic effect. DR5 upregulation induced by alternol required the production of reactive oxygen species (ROS). Removing ROS inhibited the induction of DR5 and blocked the antiapoptotic proteins induced by alternol. Conclusion: Taken together, our research suggested that alternol increased TRAIL-mediated apoptosis via inhibiting antiapoptotic proteins and upregulating DR5 levels via ROS generation and the CHOP pathway.