CT26 Tumor Model Research Articles

Comparative evaluation of antitumor effects of TNF superfamily costimulatory ligands delivered by mesenchymal stem cells

Stimulation of costimulatory receptors serves as an alternative immunotherapeutic strategy other than checkpoint inhibition. However, systemic administration of the agonistic antibodies is associated with severe toxicities, which is one of the major obstacles for their clinical application. This study aimed to develop a mesenchymal stem cell (MSC)-based system for tumor-targeted delivery of TNF superfamily ligands and assess their potential in enhancing antitumor immunity. Here we established an MSC-based system for tumor-targeted delivery of TNF superfamily ligands, including TNFSF4, 9 and 18. The TNFSF receptors (TNFRSFs) were evaluated in mouse models and patient samples for lung and colorectal cancers. TNFRSFs were all expressed at various levels on tumor-infiltrated lymphocytes, with TNFRSF18 being the most prevalent receptor. Human umbilical cord-derived MSCs expressing these costimulatory ligands (MSC-TNFSFs) effectively activated lymphocytes in vitro and elicited antitumor immunity in mice. TNFSF4 showed the least antitumor efficacy in both LLC1 and CT26 tumor models. MSC-TNFSF9 showed the most potent tumor-inhibiting effect in the LLC1 tumor model, while MSCs expressing TNFSF18 in combination with CXCL9 most significantly repressed CT26 tumor growth. Overall, TNFSF9 and TNFSF18 exhibited stronger lymphocyte-stimulating and antitumor activities than TNFSF4. Our study provides evidence that antitumor effects of agonism of different costimulatory receptors may vary in different tumor types and presents a promising approach for targeted delivery of TNF superfamily costimulatory ligands to avoid the systemic toxicities and side effects associated with immune agonist antibodies.

Methylglyoxal from gut microbes boosts radiosensitivity and radioimmunotherapy in rectal cancer by triggering endoplasmic reticulum stress and cGAS-STING activation

BackgroundPreoperative radiation therapy (preRT) is a fundamental aspect of neoadjuvant treatment for rectal cancer (RC), but the response to this treatment remains unsatisfactory. The combination of radiation therapy (RT) and...

Inflammation-Related Immune-Modulatory SLURP1 Prevents the Proliferation of Human Colon Cancer Cells, and Its Delivery by Salmonella Demonstrates Cross-Species Efficacy against Murine Colon Cancer.

This study investigates the anticancer properties of the α7-nAChR antagonist SLURP1 with a specific focus on its effect as an inflammation modulator on human colorectal cancer cell lines Caco2, Colo320DM, and H508 cells. The investigation includes the evaluation of cell cycle arrest, cell migration arrest, endogenous expression of SLURP1 and related proteins, calcium influx, and inflammatory responses. The results demonstrate that SLURP1 not only inhibits cell proliferation but also has the potential to arrest the cell cycle at the G1/S interface. The impact of SLURP1 on cell cycle regulation varied among cell lines, with H508 cells displaying the strongest response to exogenous SLURP1. Additionally, SLURP1 affects the nuclear factor kappa B expression and effectively reverses inflammatory responses elicited by purified lipopolysaccharides in H508 and Caco2 cells. This study further confirmed the expression of human SLURP1 by Salmonella, under Ptrc promoter, through Western blot analysis. Moreover, Salmonella secreting SLURP1 revealed a significant tumor regression in a mouse CT26 tumor model, suggesting the cross-species anticancer potential of human SLURP1. However, further investigations are required to fully understand the mechanisms underlying SLURP1's ability to prevent cancer proliferation and its protective function in humans.

Antibacterial Fusobacterium Nucleatum-Mimicking Nanomedicine to Selectively Eliminate Tumor-colonized Bacteria And Enhance Immunotherapy Against Colorectal Cancer.

Clinical evidence has indicated that tumor-colonizing bacteria would be closely related to the tumor development and therapeutic responses. Selectively eliminating bacteria within tumors may be an attractive approach to enhance cancer treatment without additional side effects. Herein, we found that owing to the high affinity between the membrane protein Fap-2 on Fusobacterium nucleatum (F. nucleatum) and D-galactose-β (1-3)-N-acetyl-D-galactosamine (Gal-GalNAc) overexpressed on colorectal tumor cells, F. nucleatum would colonize in colorectal tumors, as evidenced by both clinical samples and animal tumor models. Notably, F. nucleatum colonized in colorectal tumors would lead to immunosuppressive tumor microenvironment, greatly reducing their responses to immune checkpoint blockade (ICB) therapy. Inspired by this finding, we designed a F. nucleatum-mimetic nanomedicine by fusing F. nucleatum cytoplasmic membrane (FM) with Colistin loaded liposomes to achieve selectively killing tumor-colonizing F. nucleatum without affecting gut microbes. As the results, the therapeutic responses of F. nucleatum-colonized tumors to ICB therapies could be successfully restored, as demonstrated in F. nucleatum-infected subcutaneous CT-26 tumor model, chemical-induced spontaneous colorectal cancer models and MC-38 tumor model. In summary, our work presented a F. nucleatum-Mimicking nanomedicine which could selectively eliminate tumor-colonized bacteria, promising for enhancing the responses of cancer immunotherapy against F. nucleatum-colonized colorectal cancer. This article is protected by copyright. All rights reserved.

A sequential scheme including PTT and 2′3′-cGAMP/CQ-LP reveals the antitumor immune function of PTT through the type I interferon pathway

Photothermal therapy (PTT) is a promising antitumor treatment that is easy to implement, minimally invasive, and precisely controllable, and evokes strong antitumor immunity. We believe that a thorough elucidation of its underlying antitumor immune mechanisms would contribute to the rational design of combination treatments with other antitumor strategies and consequently potentiate clinical use. In this study, PTT using indocyanine green (ICG) induced STING-dependent type I interferon (IFN) production in macrophages (RAW264.7 and bone marrow-derived macrophages (BMDMs)), as proven by the use of a STING inhibitor (C178), and triggered STING-independent type I IFN generation in tumor cells (CT26 and 4T1), which was inhibited by DNase pretreatment. A novel liposome coloaded with the STING agonist 2′3′-cGAMP (cGAMP) and chloroquine (CQ) was constructed to achieve synergistic effect with PTT, in which CQ increased cGAMP entrapment efficiency and prevented STING degradation after IFN signaling activation. The sequential combination treatment caused a significant increase in tumor cell apoptosis, probably due to interferon stimulating gene products 15 and 54 (ISG15 and ISG 54), and achieved a more striking antitumor inhibition effect in the CT26 tumor model than the 4T1 model, likely due to higher STAT1 expression and consequently more intense IFN signal transduction. In the tumor microenvironment, the combination treatment increased infiltrating CD8+T cells (4-fold) and M1-like TAMs (10-fold), and decreased M-MDSCs (over 2-fold) and M2-like TAMs (over 4-fold). Above all, in-depth exploration of the antitumor mechanism of PTT provides guidance for selecting sensitive tumor models and designing reasonable clinical schemes.

Cordycepin synergizes with CTLA-4 blockade to remodel the tumor microenvironment for enhanced cancer immunotherapy

The strategy of using immune checkpoint inhibitors (ICIs) has revolutionized cancer treatment, leading to remarkable clinical outcomes. However, certain cancer types and patient demographics continue to face unique challenges. As a result, it is vital to investigate combination therapies that involve ICIs to boost therapeutic efficacy. Cordycepin, an adenosine derivative composed of adenine and pentose, holds immense promise for treating inflammation and cancer. Our recent research has demonstrated that the combined treatment of cordycepin and the anti-CD47 antibody significantly curtails tumor growth and extends the lifespan of tumor-bearing mice. In the current study, we showed that the combination of cordycepin and CTLA-4 blockade had a profound impact on suppressing tumor growth. We utilized the MC38 and CT26 tumor models to evaluate the therapeutic effect of cordycepin, CTLA-4 blockade, and their combined approach. Flow cytometry results unveiled that cordycepin, when combined with CTLA-4 blockade, considerably augmented the presence of tumor-infiltrating CD8+T cells and diminished the population of Foxp3+Tregs within the tumor microenvironment (TME). Additionally, we employed single-cell analysis to examine the TME’s reconfiguration upon the combined treatment of anti-CTLA-4 and cordycepin. We observed a significant impact on inhibiting tumor growth and substantially extended survival in tumor-bearing mice. Our data also demonstrated an increased proportion of effector CD8+T cells in the combined treatment group compared to all other groups, while exhausted CD8+T cells diminished in the combined group compared to the anti-CTLA-4 treatment alone. In conclusion, our findings supported the idea that combining cordycepin and CTLA-4 blockade could modify the effector and exhaustion status of CD8+T cells, thereby bolstering CD8+T-cell-mediated anti-tumor immunity in the TME. Collectively, our current study successfully established a combination therapeutic strategy utilizing cordycepin and CTLA-4 blockade. This strategy demonstrated a significant synergistic effect against cancer, highlighting its importance in cancer treatment.

Engineered E. coli Nissle 1917 for delivery of bioactive IL-2 for cancer immunotherapy

In this study we performed a step-wise optimization of biologically active IL-2 for delivery using E. coli Nissle 1917. Engineering of the strain was coupled with an in vitro cell assay to measure the biological activity of microbially produced IL-2 (mi-IL2). Next, we assessed the immune modulatory potential of mi-IL2 using a 3D tumor spheroid model demonstrating a strong effect on immune cell activation. Finally, we evaluated the anticancer properties of the engineered strain in a murine CT26 tumor model. The engineered strain was injected intravenously and selectively colonized tumors. The treatment was well-tolerated, and tumors of treated mice showed a modest reduction in tumor growth rate, as well as significantly elevated levels of IL-2 in the tumor. This work demonstrates a workflow for researchers interested in engineering E. coli Nissle for a new class of microbial therapy against cancer.

Effect of FAPα-expressing tumor cell vaccine with PD-1 mAb on its efficacy in mice colorectal cancer model.

e14615 Background: Colorectal cancer (CRC) is the third prevalent malignant tumor all over the world, and many cases are metastatic at diagnosis. Immunotherapy emerges a promising treatment in CRC. However, anti-PD-1 monotherapy alone has not yielded a clinical benefit in majority of these patients. Therefore, combination therapy is necessary to overcome this limitation. Previously, we have developed a FAPα-expressing tumor vaccine and demonstrated its anti-tumor effects by reversing of immunosuppressive tumor microenvironment (TME). In the current study, we hypothesized that combination therapy could enhance the effect of anti-PD1 therapy. Methods: FAPα-expressing CT26 cells were produced as previously described. To establish xenograft tumor model, CT26 cells were injected into left flank of BALB/c mice. Anti-PD-1 monoclonal antibodies(mAb) were injected intraperitoneally twice weekly up to 5 injections. FAPα-vaccines were injected subcutaneously around tumors weekly up to 3 injections. Histological sections of tumours were analysed by H&E staining and immunofluorescence. Flow cytometric analysis was done using an FACSCalibur (BD) and analyzed using FlowJo software (TreeStar). Gene expression profile in TME was analyzed by bulk RNA sequencing. Results: In mice xenograft CT26 tumor model, either anti-PD-1 immunotherapy or FAPα tumor cell vaccine produced suppression of CT26 tumor growth compared with the control group. However, combining anti-PD-1 mAb and FAPα-expressing tumor cell vaccine elicited the most remarkable anti-tumor effect and induced the most pronounced tumor growth suppression among all groups. In combination therapy group of the vaccine and PD-1 mAb, there were significantly more infiltrated lymphocytes and less FAPα+ fibroblasts. Flow cytometry demonstrated that the combination therapy promoted M1 macrophage polarization with a reduction of M2-like tumor associated macrophages (TAMs) and an increase of M1-like TAMs. This polarization was in consistent with the reduced level of IL-10 that is predominant cytokine secreted by M2 macrophages. A reduction of myeloid-derived suppressor cells (MDSCs) in the TME were also observed. Using bulk RNAseq we observed a set of immune related genes upregulated following combination therapy, including innate immune response, antigen presentation, lymphatic cells and recruitment and T cells activation. Decreased secretion of immune inhibitory chemokines (eg, CXCL12, IL-10, TGFβ) was also found by ELISA in tumors from mice treated with combination therapy. Conclusions: Our preliminary results suggested that combination therapy of FAPα-expressing tumor vaccine and anti-PD-1 therapy is a promising way to improve the efficacy of immunotherapy through modulation of immunosuppressive TME. In the future, more research is needed to further elucidate mechanism and clinical value of this combination approach.

DNA based neoepitope vaccination induces tumor control in syngeneic mouse models

Recent findings have positioned tumor mutation-derived neoepitopes as attractive targets for cancer immunotherapy. Cancer vaccines that deliver neoepitopes via various vaccine formulations have demonstrated promising preliminary results in patients and animal models. In the presented work, we assessed the ability of plasmid DNA to confer neoepitope immunogenicity and anti-tumor effect in two murine syngeneic cancer models. We demonstrated that neoepitope DNA vaccination led to anti-tumor immunity in the CT26 and B16F10 tumor models, with the long-lasting presence of neoepitope-specific T-cell responses in blood, spleen, and tumors after immunization. We further observed that engagement of both the CD4+ and CD8+ T cell compartments was essential to hamper tumor growth. Additionally, combination therapy with immune checkpoint inhibition provided an additive effect, superior to either monotherapy. DNA vaccination offers a versatile platform that allows the encoding of multiple neoepitopes in a single formulation and is thus a feasible strategy for personalized immunotherapy via neoepitope vaccination.

Carbohydrate Strengthens the Immunotherapeutic Effect of Small-Molecule PD-L1 Inhibitors.

PD-1/PD-L1 checkpoint blockade has demonstrated great success in cancer immunotherapy. Small-molecule PD-L1 inhibitors also attract significant research interests but remain challenging in the efficacy and safety. Carbohydrate moiety and carbohydrate-binding proteins (lectins) play important roles in immune modulation including antigen recognition and presenting. Herein, we reported a novel strategy to strengthen the immunotherapeutic effect of small-molecule PD-L1 inhibitors by introducing sugar motifs, which may utilize the carbohydrate-mediated immune enhancement for cancer treatment. The data revealed that glycoside compounds containing mannose or N-acetylglucosamine exhibited the best results in IFN-γ secretion. Moreover, compared to the nonglycosylated compounds, glycosides C3 and C15 demonstrated significant lower cytotoxicity and effective in vivo antitumor potency in the CT26 and melanoma B16-F10 tumor models with good tolerance. Notably, tumor-infiltrating lymphocyte (TIL) analysis validated increased CD3+, CD4+, CD8+, and granzyme B+ T cells after glycoside treatments. This work presents a new concept to improve the immunotherapy.

External cold atmospheric plasma-responsive on-site hydrogel for remodeling tumor immune microenvironment

Although immunotherapy has recently emerged as a promising anti-tumor approach, it remains limited by the immunosuppressive tumor microenvironment. Cold atmospheric plasma irradiation can generate reactive oxygen species and trigger the presentation of tumor-associated antigens. Here, we exploited cold atmospheric plasma for on-site hydrogel application in the tumor environment, aiming to facilitate the sustainable uptake of tumor-associated antigens and nanoadjuvants by dendritic cells. Hyaluronic acid-tyramine conjugate was intratumorally injected as a liquid and formed an on-site hydrogel under irradiation with cold atmospheric plasma. Intratumoral delivery of hyaluronic acid-tyramine conjugate with transforming growth factor β-blocking nanoadjuvant (TLN) followed by cold atmospheric plasma irradiation yielded a micro-network of TLN-loaded hydrogel (TLN@CHG). In vivo intratumoral injection of TLN@CHG promoted the activation of dendritic cells and more effectively increased the proportion of CD4 T cells and CD8 T cells in the tumor microenvironment, compared to the groups receiving TLN or hydrogel alone. Moreover, in CT26 tumor model mice, cold atmospheric plasma-induced TLN@CHG therapy ablated the primary tumor and provided 100% survival among mice rechallenged with CT26 cells. Taken together, our findings suggest that an on-site hydrogel-based micro-network of TLN has the potential to remodel the tumor immune microenvironment. Although we used TLN in this study, the concept could be extended to support the sustained action of other nanoadjuvants in a hydrogel micro-network.

Design of a novel chimeric peptide via dual blockade of CD47/SIRPα and PD-1/PD-L1 for cancer immunotherapy.

Although immune checkpoint inhibition has been shown to effectively activate antitumor immunity in various tumor types, only a small subset of patients can benefit from PD-1/PD-L1 blockade. CD47 expressed on tumor cells protects them from phagocytosis through interaction with SIRPα on macrophages, while PD-L1 dampens T cell-mediated tumor killing. Therefore, dual targeting PD-L1 and CD47 may improve the efficacy of cancer immunotherapy. A chimeric peptide Pal-DMPOP was designed by conjugating the double mutation of CD47/SIRPα blocking peptide (DMP) with the truncation of PD-1/PD-L1 blocking peptide OPBP-1(8-12) and was modified by a palmitic acid tail. Pal-DMPOP can significantly enhance macrophage-mediated phagocytosis of tumor cells and activate primary T cells to secret IFN-γ in vitro. Due to its superior hydrolysis-resistant activity as well as tumor tissue and lymph node targeting properties, Pal-DMPOP elicited stronger anti-tumor potency than Pal-DMP or OPBP-1(8-12) in immune-competent MC38 tumor-bearing mice. The in vivo anti-tumor activity was further validated in the colorectal CT26 tumor model. Furthermore, Pal-DMPOP mobilized macrophage and T-cell anti-tumor responses with minimal toxicity. Overall, the first bispecific CD47/SIRPα and PD-1/PD-L1 dual-blockade chimeric peptide was designed and exhibited synergistic anti-tumor efficacy via CD8+ T cell activation and macrophage-mediated immune response. The strategy could pave the way for designing effective therapeutic agents for cancer immunotherapy.

Abstract LB337: Inhibition of the E3 ligase CBL-B enhances the effector function and proliferation of natural killer cells

Abstract The E3 ubiquitin ligase CBL-B is a master regulator of effector T cells through its downstream regulation of T-cell receptor and co-stimulatory stimulation, but it has also been shown to play a negative feedback role in NK cells. CBL-B depletion enhances the production of IFNγ and the cytotoxicity of activated NK cells. Similar to the effect of depleting CD8 T cells in mice, depletion of NK cells abolishes the tumor growth delay observed in Cbl-b-/- mice. With emerging data supporting the potential role of exogenous NK cells in anti-tumor immunity, inhibition of CBL-B represents a promising approach to enhance both adaptive and innate immunity for cancer immunotherapy. Using one of our small molecules, allosteric CBL-B inhibitors, HOT-A, we demonstrated that CBL-B inhibition enhanced NK activation, IFNγ production and granzyme B secretion in response to the various stimulation conditions, including cytokines (such as IL-15 and IL12 + IL-18) and stimulatory receptors (such as NKp30 and NKG2D + CD244). NK cell proliferation was also enhanced in the presence of HOT-A. Single cell analysis further suggested that HOT-A increased NK polyfunctional cytokine expression during IL-12 + IL-18 activation. In a coculture system of NK cells and K562 target cells, HOT-A dose-dependently promoted primary human NK cell activation, cytokine production and cytotoxicity in the K562 cells. In the in vivo CT-26 tumor model, granzyme B+ NK cells in the TIL were significantly increased in the HOT-A treated tumor bearing mice and NK function gene signature was also increased by Nanostring analysis, suggesting an enhanced NK function in the tumor microenvironment. Taken together, the pre-clinical data presented here demonstrate that our CBL-B inhibitor enhances NK cell function in vitro and in vivo, suggesting that its enhancement of anti-tumor immunity may be driven through both effector T cell and NK cell-driven biology. Citation Format: Yilin Qi, Jun Kuai, Yingzhi Bi, David Greco, Samira Jaeger, Ken Carson, Hadi Danaee, Timothy Reilly, Geraldine Harriman, Fang Wang. Inhibition of the E3 ligase CBL-B enhances the effector function and proliferation of natural killer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB337.

Abstract 463: GRC 54276, a novel small molecule inhibitor of HPK1 has entered phase 1/2 clinical trial insolid malignancies and Hodgkin’s/non Hodgkin’s lymphoma

Abstract Background: Pre-clinical profile of GRC 54276, a clinical candidate with Phase 1/2 clinical trial ongoing, is presented here. GRC 54276 is a novel small molecule inhibitor of Hematopoietic progenitor kinase 1 (HPK1), a serine/threonine kinase1,2 that negatively regulates T and B cell receptor signaling3. Inhibition of HPK1 is an attractive therapeutic strategy for immuno-oncology based treatment of solid tumors3. Methods: GRC 54276 was designed and developed using SAR based medicinal chemistry design supported by computational approaches. In vitro profiling was done using a battery of biochemical assays, functional read-outs and primary human in vitro T-cell activation assays. In vivo efficacy was demonstrated in mouse colon tumor models of CT26 and MC38-hPD-L1. In vivo inhibition of biomarker pSLP76 Ser(376) by GRC 54276 was determined using CT26 tumor model. Detailed ADME-PK studies have been performed along with safety tolerability studies conducted in mice and monkeys. Results: GRC 54276, demonstrated excellent in vitro immune profile of target engagement and anti-tumor immune response activity in both human and mouse systems. As a single agent, GRC 54276 demonstrated strong inhibition of tumor growth and biomarker pSLP76 Ser(376) in the CT26 tumor model. Enhanced efficacy was demonstrated by combining GRC 54276 with check-point blocking antibodies anti-CTLA4 and Atezolizumab in the CT26 and MC38-hPD-L1 models, respectively. GRC 54276 robustly enhanced complete tumor rejections when combined with Atezolizumab in the MC38-hPD-L1 model, correlating with increased immune effector memory T cells. Pharmacokinetic profile of GRC 54276 is characterized by high permeability, rapid absorption and moderate oral bioavailable across species. GRC 54276 is non-gentoxic with no observed adverse effects in mice and no treatment related cardiovascular or respiratory effects in repeat dose toxicity study in monkeys. Conclusions: GRC 54276 is a novel HPK1 inhibtitor with acceptable pre-clincial profile and is currently undergoing a Phase 1/2 clinical trial. Acknowledgements: We thank Pooja S, Shital M, Rahul B, Ajit J, Sanjay G, Somesh K, Pramod S for their contributions to the project

Abstract 5107: A novel AhR inhibitor ‘DA-4505’ improved the anti-cancer efficacy of surgical and chemotherapy via synergistic anti-tumor effects of aPD-1

Abstract Background: The Aryl hydrocarbon receptor (AhR) is one of the most predominant regulators of cancer metabolism. The AhR exerts important immunosuppressive functions by activating Treg cells and myeloid-derived suppressor cells and repressing CD8+ effector T cells. Here, we propose that a best-in-class AhR inhibitor, DA-4505, improves anti-tumor efficacy via modulation of tumor immune surveillance compared to BAY2416964, an AHR antagonist drug candidate being studied in the clinical phase. Methods: To evaluate anti-tumor effects of DA-4505 and BAY2416964, the two AhR inhibitors were dosed at 10 mg/kg once daily alone or in combination with aPD-1 (10 mg/kg) in surgical and chemotherapy models, and a PDX model (YHIM2004). Tumor volume, relapse, and survival were evaluated, and immune profiles were analyzed with IHC, flow cytometry, and scRNAseq. Results: A significant tumor reduction appeared in the CT26 and 4T1 tumor models after the DA-4505 treatment compared to vehicle group (P<0.05). In contrast, DA-4505 treatment did not induce significant tumor regression compared to vehicle group in tumor-bearing NOG mice, suggesting that anti-tumor effects of DA-4505 were driven by immunologic mechanisms. To evaluate the role of DA-4505 in conjunction with surgery, DA-4505 alone or in combination with anti-PD-1 was given prior to and following resection of the tumors in 4T1 tumor-bearing mice. Survival of mice treated with DA-4505 alone or DA-4505 combined with anti-PD-1 was significantly prolonged after resection compared to aPD-1 treatment group (P<0.05). In addition, there were four mice that did not have a relapse by treating DA-4505 with or without aPD-1 after surgery (4/5). A tumor regression also appeared in the YHIM2004-engrafted humanized mouse study. A tumor reduction was shown by treating DA-4505 alone or in combination with pembrolizumab compared to vehicle group (P<0.05). Next, we co-administered an AhR inhibitor and aPD-1 as a partner to improve the antitumor effects of chemotherapy. The DA-4505 add-on group showed tumor regression when compared with the combination therapy group treated with aPD-1 and chemotherapy (P<0.0001). In addition, a significant increase in survival rate was shown in the group treated with a DA-4505 add-on compared to vehicle group (P<0.001). Analysis of scRNAseq showed that M1 macrophage expressing CCL7 and CCL8 were increased in DA-4505 treated group compared to the vehicle and aPD-1 groups. This suggests that immune modulatory effect of DA-4505 may be due to enhanced recruitment of immune cells into the tumor site by macrophages with high chemotactic activity. Conclusion: The AhR inhibitor DA-4505 demonstrated an improvement in anti-tumor efficacy. In addition, it has shown a synergistic effect when combined with aPD-1. Discoveries from this study provide a preclinical rationale for future clinical implications in solid tumor. Citation Format: DongKwon Kim, Sujeong Baek, Seung Min Yang, Yu Jin Han, Seong-san Kang, Chun-Bong Synn, Mi Hyun Kim, Heekyung Han, Kwangmin Na, Young Taek Kim, Sungwoo Lee, Taedong Han, Hyounmie Doh, Jongho Cho, Dajeong Kim, Daewon Cha, Jae Hwan Kim, Youngseon Byeon, Young Seob Kim, Mi Ran Yun, Ji Yun Lee, Jii Bum Lee, Chang Gon Kim, Min Hee Hong, Sun Min Lim, Byoung Chul Cho, Kyoung-Ho Pyo. A novel AhR inhibitor ‘DA-4505’ improved the anti-cancer efficacy of surgical and chemotherapy via synergistic anti-tumor effects of aPD-1. [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 5107.

Local sustained release of PD-1 monoclonal antibody and lenvatinib by thermo-sensitive hydrogel for improving tumor immunotherapy

In clinic, the combination of intravenous pembrolizumab (PD-1 monoclonal antibody) with oral Lenvatinib (LEN) exhibited an enhanced synergistic benefit for cancer therapy. However, the clinical outcomes were always limited by the problems of inconsistent pharmacokinetic profiles of two drugs, lower drug accumulation in tumor and obvious side effects during the combination therapy. Here, in situ-forming thermosensitive hydrogels based on PLGA-PEG-PLGA triblock copolymers were prepared for local administration of anti-PD1 and LEN (P&L@Gel) to improve therapeutic efficacy and safety. After peritumoral or surgical resection site injection, the significant increased concentrations of both drugs in tumor were observed with the local sustained release of P&L@Gel. In comparison with the group of intraperitoneal anti-PD1 plus oral LEN (P-ip&L-po), significantly higher tumor inhibition efficiency on CT26 tumor models could be obtained in P&L@Gel group, even at the dose of one-eighth of the former, same tumor-inhibition effects could be achieved. The enhanced antitumor efficacy of P&L@Gel group was probably associated with the 2.2 folds of increased level of CD8+ T cells and the polarization of tumor associated macrophage from M2 to M1 along with the increased drug accumulation. Moreover, compared with the obvious side effects of P-ip&L-po group, no significant changes of PLT, ALT and UA in blood, as well as IL-1α and IL-1β in mice paws were observed between P&L@Gel group and untreated group. These results suggested that local administration of anti-PD1 and LEN with thermosensitive hydrogel could offer a potential strategy for tumors or tumor postoperative adjuvant treatment.

A bispecific peptide-polymer conjugate bridging target-effector cells to enhance immunotherapy.

Peptide-based immune checkpoint inhibitors exhibited remarkable therapeutic benefits although their application was hindered by quick blood clearance and low affinity with receptors. The modification of the peptides into artificial antibodies was an ideal platform to solve these problems, and one of the optional pathways was the conjugation of peptides with a polymer. More importantly, the bridging effect, mediated by bispecific artificial antibodies, could promote the interaction of cancer cells and T cells, which will benefit cancer immunotherapy. Herein, we prepared a bispecific peptide-polymer conjugate (octa PEG-PD1-PDL1) by simultaneously conjugating PD1-binding and PDL1-binding peptides onto 8-arm-PEG. octa PEG-PD1-PDL1 bridged T cells and cancer cells and thus enhanced T cell-mediated cytotoxicity against cancer cells. Meanwhile, the tumor-targeting octa PEG-PD1-PDL1 increased the infiltration of cytotoxic T lymphocytes in tumors and reduced their exhaustion. It effectively activated the tumor immune microenvironment and exerted a potent antitumor effect against CT26 tumor models with a tumor inhibition rate of 88.9%. This work provided a novel strategy to enhance tumor immunotherapy through conjugating bispecific peptides onto a hyperbranched polymer to effectively engage target-effector cells. This article is protected by copyright. All rights reserved.

CEACAM1 Marks Highly Suppressive Intratumoral Regulatory T Cells for Targeted Depletion Therapy.

Regulatory T cells (Tregs) exert immunosuppressive functions and hamper antitumor immune responses in the tumor microenvironment. Understanding the heterogeneity of intratumoral Tregs, and how it changes with tumor progression, will provide clues regarding novel target molecules of Treg-directed therapies. From 42 patients with renal cell carcinoma and 5 patients with ovarian cancer, immune cells from tumor and peripheral blood were isolated. We performed multicolor flow cytometry and RNA-sequencing to characterize the phenotypes and heterogeneity of intratumoral Tregs. In vitro functional assays were performed to evaluate suppressive capacity of Tregs and effect of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1)-mediated depletion. The CT26 tumor model was used to evaluate the association between intratumoral Tregs and tumor growth, and examine the in vivo role of CEACAM1+ intratumoral Tregs on antitumor immunity. We found that CEACAM1 was selectively expressed on intratumoral Tregs, whereas its expression on peripheral Tregs or other immune cells was low. The CEACAM1+ intratumoral Tregs accumulated with tumor progression, whereas the CEACAM1- subset did not. Notably, we found that CEACAM1 marked intratumoral Tregs that exhibited highly suppressive and activated phenotypes with substantial clonal expansion. Depletion of CEACAM1-expressing cells from tumor-infiltrating leukocytes led to increased effector functions of tumor-infiltrating T cells. Moreover, CEACAM1+ cell depletion further enhanced anti-PD-1-mediated reinvigoration of exhausted CD8+ T cells. CEACAM1 marks highly suppressive subset of intratumoral Tregs, and can be a target for selective depletion of intratumoral Tregs. These results may inform future studies on CEACAM1-mediated depletion in patients with cancer.

Mitochondria-Targeting and Multiresponsive Nanoplatform Based on AIEgens for Synergistic Chemo-Photodynamic Therapy and Enhanced Immunotherapy.

Although photodynamic therapy (PDT) has become an attractive strategy for cancer treatment, its clinical application still suffers from some limitations, including insufficient delivery of photosensitizers, hypoxic tumor environment, and the development of PDT resistance. To address these limitations, a new class of mitochondria-targeting and fluorinated polymer with aggregation-induced emission characteristics was fabricated to sensitize PDT and co-deliver chemotherapeutic drugs. The amphiphilic fluoropolymer was able to efficiently carry oxygen and SN-38 (the active metabolite of irinotecan) and self-assemble into multifunctional micellar nanoparticles (SN-38-TTCF@O2 NPs). Upon internalization into tumor cells, these NPs could successfully escape lysosomes, selectively target mitochondria, efficiently produce reactive oxygen species (ROS) under light irradiation, and release drugs in response to ROS. In the HCT116 tumor xenograft model, they preferentially accumulated in tumor tissue and significantly alleviated tumor hypoxia, resulting in synergistic chemo-PDT efficacy without distinct toxicity. Furthermore, the nanoscale chemo-PDT induced immunogenic cell death, promoted the recruitment and activation of cytotoxic T lymphocytes, and ultimately augmented the anti-tumor efficacy of anti-PD-1 antibody in the murine CT26 tumor model. These results may provide novel insights into the development of efficient chemo-PDT nanomedicine to improve the outcome of immunotherapy.

Bioinspired nanovehicle of furoxans-oxaliplatin improves tumoral distribution for chemo-radiotherapy.

The spatiotemporal distribution of therapeutic agents in tumors remains an essential challenge of radiation-mediated therapy. Herein, we rationally designed a macrophage microvesicle-inspired nanovehicle of nitric oxide donor-oxaliplatin (FO) conjugate (M-PFO), aiming to promote intratumor permeation and distribution profiles for chemo-radiotherapy. FO was responsively released from M-PFO in intracellular acidic environments, and then be activated by glutathione (GSH) into active oxaliplatin and NO molecules in a programmed manner. M-PFO exhibited notable accumulation, permeation and cancer cell accessibility in tumor tissues. Upon radiation, the reactive peroxynitrite species (ONOO-) were largely produced, which could diffuse into regions over 400μm away from the tumor vessels and be detectable after 24h of radiation, thereby exhibiting superior efficacy in improving the spatiotemporal distribution in tumors versus common reactive oxygen species (ROS). Moreover, M-PFO mediated chemo-radiotherapy caused notable inhibition of tumor growth, with an 89.45% inhibition in HT-29 tumor models and a 92.69% suppression in CT-26 tumor models. Therefore, this bioinspired design provides an encouraging platform to improve intratumor spatiotemporal distribution to synergize chemo-radiotherapy.