CT26 Tumor Model Research Articles

Leveraging long-acting IL-15 agonists for intratumoral delivery and enhanced antimetastatic activity

IntroductionIL-15 agonists hold promise as immunotherapeutics due to their ability to induce the proliferation and expansion of cytotoxic immune cells including natural killer (NK) and CD8+ T cells. However, they generally have short half-lives that necessitate frequent administration to achieve efficacy. To address this limitation, we have developed a half-life extension technology using hydrogel microspheres (MS). Here, the therapeutic is tethered to MSs by a releasable linker with pre-programed cleavage rates. We previously showed the MS conjugate of single-chain IL-15, MS~IL-15, effectively increased the half-life of IL-15 to approximately 1 week and enhanced the pharmacodynamics. We sought to determine whether the same would be true with a MS conjugate of the IL-15 agonist, receptor-linker IL-15 (RLI).MethodsWe prepared a long acting MS conjugate of RLI, MS~RLI. The pharmacokinetics and pharmacodynamics of MS~RLI were measured in C57BL/6J mice and compared to MS~IL-15. The antitumor efficacy of MS~RLI was measured when delivered subcutaneously or intratumorally in the CT26 tumor model and intratumorally in the orthotopic EO771 tumor model.ResultsMS~RLI exhibited a half-life of 30 h, longer than most IL-15 agonists but shorter than MS~IL-15. The shorter than expected half-life of MS~RLI was shown to be due to target-mediated-disposition caused by an IL-15 induced cytokine sink. MS~RLI resulted in very potent stimulation of NK and CD44hiCD8+ T cells, but also caused significant injection-site toxicity that may preclude subcutaneous administration. We thus pivoted our efforts toward studying the MS~RLI for long-acting intra-tumoral therapy, where some degree of necrosis might be beneficial. When delivered intra- tumorally, both MS~IL-15 and MS~RLI had modest anti-tumor efficacy, but high anti- metastatic activity.ConclusionIntra-tumoral MS~RLI and MS~RLI combined with systemic treatment with other agents could provide beneficial antitumor and anti-metastatic effects without the toxic effects of systemic IL-15 agonists. Our findings demonstrate that intra-tumorally administered long-acting IL-15 agonists counter two criticisms of loco-regional therapy: the necessity for frequent injections and the challenge of managing metastases.

Blocking S100A9-signaling is detrimental to the initiation of anti-tumor immunity.

S100A9, a multifunctional protein mainly expressed by neutrophils and monocytes, poses an immunological paradox. In virus infections or sterile inflammation, it functions as an alarmin attracting innate immune cells, as well as mediating proinflammatory effects through TLR4 signaling. However, in cancer, S100A9 levels have been shown to associate with poor prognosis and lack of response to immunotherapy. Its expression by myeloid cells has been related to an immune suppressive phenotype, the so-called myeloid derived suppressor cells (MDSCs). Targeting S100A9 in cancer has therefore been proposed as a potential way to relieve myeloid-mediated immune suppression. Surprisingly, we found that blocking the extracellular TLR4 signaling from S100A9 using the inhibitor Paquinimod, resulted in increased tumor growth and a detrimental effect on anti-PD-L1 efficacy in the CT26 tumor model. This effect was caused by a reduction in the tumor immune infiltration to about half of untreated controls, and the reduction was made up of a 5-fold decrease in Ly6Chigh monocytic cells. The suppressive Ly6G+ myeloid cells compartment was not reduced by Paquinimod treatment, suggesting alternative mechanisms by which S100A9 contributes to myeloid-mediated suppression. Intratumoral injection of recombinant S100A9 early after mice inoculation with CT26 cells had an anti-tumor effect. These findings indicate an important yet understudied role of S100A9 as an alarmin and immune stimulatory signal in cancer settings, and highlight the potential to exploit such signals to promote beneficial anti-tumor responses.

Sequential responsive nano-PROTACs for precise intracellular delivery and enhanced degradation efficacy in colorectal cancer therapy

PROteolysis TArgeting Chimeras (PROTACs) have been considered the next blockbuster therapies. However, due to their inherent limitations, the efficacy of PROTACs is frequently impaired by limited tissue penetration and particularly insufficient cellular internalization into their action sites. Herein, based on the ultra-pH-sensitive and enzyme-sensitive nanotechnology, a type of polymer PROTAC conjugated and pH/cathepsin B sequential responsive nanoparticles (PSRNs) are deliberately designed, following the construction of the PROTAC for Cyclin-dependent kinase 4 and 6 (CDK4/6). Colorectal cancer (CRC) which hardly responds to many treatments even immune checkpoint blockades was selected as the tumor model in this study. As a result, PSRNs were found to maintain nanostructure (40 nm) in circulation and efficiently accumulated in tumors via enhanced permeation and retention effect. Then, they were dissociated into unimers (<10 nm) in response to an acidic tumor microenvironment, facilitating tumor penetration and cellular internalization. Eventually, the CDK4/6 degrading PROTACs were released intracellularly following the cleavage of cathepsin B. Importantly, PSRNs led to the enhanced degradation of target protein in vitro and in vivo. The degradation of CDK4/6 also augmented the efficacy of immune checkpoint blockades, through the upregulation of programmed cell death-ligand 1 (PD-L1) expression in cancer cells and the suppression of regulatory T cells cell proliferation in tumor microenvironment. By combination with α-PD-1, an enhanced anti-tumor outcome is well achieved in CT26 tumor model. Overall, our study verifies the significance of precise intracellular delivery of PROTACs and introduces a promising therapeutic strategy for the targeted combination treatment of CRC.

PAMAM-Based Polymeric Immunogenic Cell Death Inducer To Potentiate Cancer Immunotherapy.

Immunogenic cell death (ICD) has been widely employed to potentiate cancer immunotherapy due to its capability to activate the anticancer immune response. Although various ICD inducers have been described, the development of synthetic materials with intrinsic ICD-inducing competency has rarely been reported. Herein, we identify a derivative of the fourth generation polyamidoamine (PAMAM) modified with multiple seven-membered heterocyclic rings, G4P-C7A, as a robust ICD inducer. G4P-C7A evokes characteristic release of damage-associated molecular patterns in tumor cells and induces efficient dendritic cell maturation. Mechanistic studies suggest that G4P-C7A can selectively accumulate in the endoplasmic reticulum and mitochondria to generate reactive oxygen species. G4P-C7A-treated tumor cells can work as potent vaccines to protect against secondary tumor implantation. Either local or systemic injection of G4P-C7A alone can effectively inhibit tumor growth by eliciting robust antitumor immune response. The combination of G4P-C7A with immunotherapeutic antibodies such as anti-PD1 (aPD-1) and anti-CD47 (aCD47) further potentiates the antitumor effect in either CT26 or 4T1 tumor model. This study offers a simple but effective strategy to induce ICD to boost cancer immunotherapy.

Towards reliable hyperspectral imaging biomarkers of CT26 murine tumor model

The non-invasive monitoring of tumor growth can offer invaluable diagnostic insights and enhance our understanding of tumors and their microenvironment. Integrating hyperspectral imaging (HSI) with three-dimensional optical profilometry (3D OP) makes contactless and non-invasive tumor diagnosis possible by utilizing the inherent tissue contrast provided by visible (VIS) and near-infrared (NIR) light. Consequently, valuable information regarding tumors and healthy tissues can be extracted from the acquired hyperspectral images. Until now, very few methods have been used to monitor tumor models in vivo daily and non-invasively. In this research, we conducted a 14-day study monitoring BALB/c mice with subcutaneously grown CT26 murine colon carcinomas in vivo, commencing on the day of tumor cell injection. We extracted physiological properties such as total hemoglobin (THB) and tissue oxygenation (StO2) using the inverse adding-doubling (IAD) algorithm and manually segmented the tissues. We then selected the ten most relevant features describing tumors using the Max-Relevance Min-Redundancy (MRMR) algorithm and utilized 30 classic and advanced machine learning (ML) algorithms to discriminate tumors from healthy tissues. Finally, we tested the robustness of feature selection and model performance by smoothing tissue parameter maps extracted by IAD with a variable kernel and omitting selected training data. We could discriminate CT26 tumor models from surrounding healthy tissues with an area under the curve (AUC) of up to 1 for models based on the gradient boosting method, linear discriminant analysis, and random forests. Our findings help pave the way for precise and robust imaging biomarkers that could aid tumor diagnosis and advance clinical practice.

ALK5/VEGFR2 dual inhibitor TU2218 alone or in combination with immune checkpoint inhibitors enhances immune-mediated antitumor effects

Transforming growth factor β (TGFβ) is present in blood of patients who do not respond to anti-programmed cell death (ligand) 1 [PD-(L)1] treatment, and through synergy with vascular endothelial growth factor (VEGF), it helps to create an environment that promotes tumor immune evasion and immune tolerance. Therefore, simultaneous inhibition of TGFβ/VEGF is more effective than targeting TGFβ alone. In this study, the dual inhibitory mechanism of TU2218 was identified through in vitro analysis mimicking the tumor microenvironment, and its antitumor effects were analyzed using mouse syngeneic tumor models. TU2218 directly restored the activity of damaged cytotoxic T lymphocytes (CTLs) and natural killer cells inhibited by TGFβ and suppressed the activity and viability of regulatory T cells. The inactivation of endothelial cells induced by VEGF stimulation was completely ameliorated by TU2218, an effect not observed with vactosertib, which inhibits only TGFβ signaling. The combination of TU2218 and anti-PD1 therapy had a significantly greater antitumor effect than either drug alone in the poorly immunogenic B16F10 syngeneic tumor model. The mechanism of tumor reduction was confirmed by flow cytometry, which showed upregulated VCAM-1 expression in vascular cells and increased influx of CD8 + CTLs into the tumor. As another strategy, combination of anti-CTLA4 therapy and TU2218 resulted in high complete regression (CR) rates in CT26 and WEHI-164 tumor models. In particular, immunological memory generated by the combination of anti-CTLA4 and TU2218 in the CT26 model prevented the development of tumors after additional tumor cell transplantation, suggesting that the TU2218-based combination has therapeutic potential in immunotherapy.

Naked mesoporous rhodium nanospheres with glutathione depletion and photothermal capabilities for tumor therapy

Pancreatic and colon cancer are malignant tumors of the digestive system that currently lack effective treatments. In cancer cells, a high level of glutathione (GSH) is indispensable to scavenge excessive reactive oxygen species (ROS) and detoxify xenobiotics, which make it a potential target for cancer therapy. GSH depletion has been proved to improve the therapeutic efficacy of photodynamic therapy. Here, we reported that naked mesoporous rhodium nanospheres (Rh MNs), prepared by soft template redox method, can act as GSH depletion agent and photothermal conversion agent to achieve synergistic therapy respectively. Different from conventional nanoagents, Rh MNs with the characteristics of easy synthesis, simple structure and multiple functions can decrease the GSH level in tumor and depict excellent photothermal ability with a high photothermal conversion efficiency (PTCE) up to 39%. Notably, multiple anti-tumor mechanisms in CT26 and BxPC-3 tumor models, include inhibited anti-apoptosis, DNA replication repair, and GSH synthesis are revealed, and the pancreatic tumor cure rate of the cooperative treatment group is 80%. Collectively, we developed Rh MNs to combine GSH depletion with photothermal therapy for cancer treatment.

Impact of in vivo fate of STING agonist-loaded lipid nanoparticles on antitumor immunity

Therapeutically manipulating the stimulator of interferon genes (STING) pathway has promising potential for enhancing antitumor immunity. Agonists of this pathway (STING agonists) are being evaluated in clinical trials. Loading the STING agonists into lipid nanoparticles (LNPs) increases their safety and efficacy. We previously developed STING agonists loaded LNPs consisting of the ionizable lipid YSK12-C4 (YSK12-LNPs), which showed significant antitumor effects. However, it is largely unclear how the in vivo fate of STING agonists loaded LNPs affects the antitumor immune responses. In this study, we compared the YSK12-LNPs with LNPs composed of DLin-MC3-DMA (MC3-LNPs) showing different in vivo fates. Biodistribution and flow cytometry analyses of mouse tissues revealed that the MC3-LNPs delivered higher amounts of STING agonists to the liver than the YSK12-LNPs. Additionally, significantly more liver leukocytes internalized the MC3-LNPs than the YSK12-LNPs. In contrast, the YSK12-LNPs delivered higher amounts of STING agonists to the liver leukocytes than the MC3-LNPs, leading to the effective induction of innate immunity and inflammation in the tumors. However, the antitumor effects in the B16-F10 lung metastasis and CT26 tumor models were comparable. Interestingly, flow cytometry analyses suggested that the YSK12-LNPs were more likely to activate natural killer cells and M1 macrophages, while the MC3-LNPs were more likely to activate CD8+ T cells. Our data suggest that different antitumor immune response mechanisms may operate depending on the characteristics and distribution of the LNPs.

Non-viral-mediated gene transfer of OX40 ligand for tumor immunotherapy.

Immune checkpoint blockade (ICB) is rapidly becoming a standard of care in the treatment of many cancer types. However, the subset of patients who respond to this type of therapy is limited. Another way to promote antitumoral immunity is the use of immunostimulatory molecules, such as cytokines or T cell co-stimulators. The systemic administration of immunotherapeutics leads to significant immune-related adverse events (irAEs), therefore, the localized antitumoral action is needed. One way to achieve this is intratumoral non-viral gene-immune therapy, which allows for prolonged and localized gene expression, and multiple drug administration. In this study, we combined the previously described non-viral gene delivery system, PEG-PEI-TAT copolymer, PPT, with murine OX40L-encoding plasmid DNA. The resulting OX40L/PPT nanoparticles were characterized via gel mobility assay, dynamic light scattering analysis and in vitro transfection efficiency evaluation. The antitumoral efficacy of intratumorally (i.t.) administered nanoparticles was estimated using subcutaneously (s.c.) implanted CT26 (colon cancer), B16F0 (melanoma) and 4T1 (breast cancer) tumor models. The dynamics of stromal immune cell populations was analyzed using flow cytometry. Weight loss and cachexia were used as irAE indicators. The effect of combination of i.t. OX40L/PPT with intraperitoneal PD-1 ICB was estimated in s.c. CT26 tumor model. The obtained OX40L/PPT nanoparticles had properties applicable for cell transfection and provided OX40L protein expression in vitro in all three investigated cancer models. We observed that OX40L/PPT treatment successfully inhibited tumor growth in B16F0 and CT26 tumor models and showed a tendency to inhibit 4T1 tumor growth. In B16F0 tumor model, OX40L/PPT treatment led to the increase in antitumoral effector NK and T killer cells and to the decrease in pro-tumoral myeloid cells populations within tumor stroma. No irAE signs were observed in all 3 tumor models, which indicates good treatment tolerability in mice. Combining OX40L/PPT with PD-1 ICB significantly improved treatment efficacy in the CT26 subcutaneous colon cancer model, providing protective immunity against CT26 colon cancer cells. Overall, the anti-tumor efficacy observed with OX40L non-viral gene therapy, whether administered alone or in combination with ICB, highlights its potential to revolutionize cancer gene therapy, thus paving the way for unprecedented advancements in the cancer therapy field.

Discovery of Small and Bifunctional Molecules Targeting PD-L1/CD73 for Cancer Dual Immunotherapy.

In this work, a series of bifunctional PD-L1/CD73 (cluster of differentiation 73) small-molecule inhibitors were designed and synthesized. Among them, CC-5 showed the strongest PD-L1 inhibitory effects with an IC50 of 6 nM and potent anti-CD73 activity with an IC50 of 0.773 μM. The high PD-L1/CD73 inhibitory activity of CC-5 was further confirmed by SPR assays with KD of 182 nM for human PD-L1 and 101 nM for CD73, respectively. Importantly, CC-5 significantly suppressed tumor growth in a CT26 and B16-F10 tumor model with TGI of 64.3% and 39.6%, respectively. Immunohistochemical (IHC) and flow cytometry analysis of tumor-infiltrating lymphocytes (TILs) indicated that CC-5 exerted anticancer effects via activating the tumor immune microenvironment. Collectively, CC-5 represents the first dual PD-L1/CD73 inhibitor worthy of further research as a bifunctional immunotherapeutic agent.

Abstract 3720: Utility of oncolytic virus as an immunostimulants and potential for combination therapy with ICIs

Abstract Background: Anti-PD-1 antibodies have been approved for a variety of carcinomas, but their efficacy in gastrointestinal cancers is limited. CD8-positive T cells (CD8) of tumor-infiltrating lymphocytes (TILs) and resident memory T cells (TRMs) with long-term anti-tumor immune function have been reported to be associated with the therapeutic efficacy of anti-PD-1 antibodies. Among our oncolytic adenoviruses (OVs), OBP-301, in which the human telomerase reverse transcriptase (hTERT) promoter element drives the expression of the viral E1A and E1B genes, is currently undergoing clinical trials. In the present study, we investigated the usefulness of OVs as immunostimulants and their potential efficacy in combination with anti-PD-1 antibodies. Methods: The cell lines used were the mouse colon cancer cell line CT26 and the mouse pancreatic cancer cell line PAN02. In vitro, the cytotoxic activity of the OVs and extracellular ATP in the culture supernatant were determined. OBP-502, an OBP-301 variant with the gene cassette of the RGD peptide, and anti-PD-1 antibody were used to examine the antitumor effect on subcutaneous tumors. OBP-702, another OBP-301 variant with the gene cassette of the wild-type p53 tumor suppressor gene, was used to examine the effect of long-term anti-tumor immunity. The effects on memory T-precursor cells (MPEC), CD8 and TRM were immunologically assessed in a rechallenge study. Results: OBP-502 and OBP-702 showed cytotoxic activity against CT26 and PAN02 cells in vitro, and significantly increased ATP in the culture supernatant. In vivo, the anti-PD-1 antibody/OBP-502 combination therapy significantly increased intratumor CD8 in CT26 and PAN02 subcutaneous tumors compared to each monotherapy. Complete response was achieved in 4 out of 12 mice by the combination therapy against CT26 tumors. When CT26 cells was re-seeded into tumor-free mice cured by the combination therapy, 2 out of 4 mice remained tumor-free. In the CT26 and PAN02 subcutaneous tumor model, OBP-702 increased CD44-positive CD62L-negative MPECs that differentiated into TRMs in the spleen, and this effect was blocked by ATP receptor inhibition. In CT26 and PAN02 bilateral subcutaneous tumor models, OBP-702 had a significant antitumor effect on both OBP-702-treated and -non-treated sides, and TIL analysis showed a significant increase in CD8 and TRM. In a rechallenge study, OBP-702-pretreatment showed significant growth inhibition of re-inoculated tumors. Conclusion: The OVs showed potent and long-term immune activation by inducing CD8 and TRM in tumors as well as direct anti-tumor effects, and synergistic effects with ICI. Citation Format: Daisuke Kadowaki, Shinji Kuroda, Masashi Hashimoto, Nobuhiko Kanaya, Yoshihiko Kakiuchi, Satoru Kikuchi, Kazuhiro Noma, Hiroshi Tazawa, Shunsuke Kagawa, Yasuo Urata, Toshiyoshi Fujiwara. Utility of oncolytic virus as an immunostimulants and potential for combination therapy with ICIs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3720.

Abstract 2460: Receptor antibody conjugated engager (RACE): A novel bispecific 4-1BB engager platform for enhanced immuno-oncology therapy

Abstract Background: Traditional immuno-oncology strategies, such as checkpoint blockade therapy, are evolving to immune cell engagers as a smarter strategy. The early successes of Blincyto, a CD3 engager, demonstrated its capacity to activate host pan-T cells irrespective of their TCR-pMHC restriction status. However, this broad activation has been linked to off-target immunotoxicities, thus limiting its therapeutic applicability. In response, targeting immune co-stimulatory receptors like CD28 or 4-1BB has gained attention by their expression being induced on activated immune effector cells, presenting a more targeted approach to cancer therapy. Methods: We developed RACE, a novel bispecific 4-1BB engager. This platform fuses a 4-1BB ligand (4-1BBL) trimer with antibody F(ab’)2 fragments, excluding Fc domains. A Key and lock design on the 4-1BBL trimer interface facilitates asymmetric assembly of 4-1BBL dimer and monomer in the endoplasmic reticulum, achieving over 95% assembly efficiency in mammalian systems. Results: RACE differs from conventional 4-1BB agonistic antibodies in its ability to form natural hexagonal hyper-clusters of 4-1BB receptors, enhancing NF-κB signal transduction. Unlike first-generation 4-1BB antibodies, which incurred off-tumor hepatotoxicity, RACE selectively activates 4-1BB receptors only in the presence of tumor antigens. Enhanced cytotoxicity requires successful TCR signaling complex formation between T cells and cancer cells. In CT26 and MC38 tumor models, RACE not only induced strong anti-tumor effects without hepatic or systemic toxicities but showed synergistic efficacy with anti-PD-1 antibodies as well. Conclusion: The novel RACE platform represents a potent as well as safe immuno-oncology strategy, demonstrating significant potential for clinical application. Citation Format: DaeHee Lee, Jihye Yoon, Jonggwan Jeong, Jeonghyeon Bak, KkaBi Son, Heechan Kim, Seil Jang, Minseok S. Kim. Receptor antibody conjugated engager (RACE): A novel bispecific 4-1BB engager platform for enhanced immuno-oncology therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2460.

Abstract 4042: Cbl inhibition increases the response of cytotoxic T cells to sub-optimal stimulation and drives enhanced anti-tumor responses alone or in combination with PD-1 blockade

Abstract INTRODUCTION: Herein, we describe a series of experiments to define the unique biology of dual Cbl-b/c-Cbl (Cbl) inhibition in amplifying T cell activation and anti-tumor responses. Through their E3 ubiquitin ligase activity, Cbl-b and c-Cbl have been shown to negatively regulate multiple signaling proteins downstream of the T cell receptor (TCR), suggesting that inhibition may yield enhanced anti-tumor T cell activity. Indeed, using a dual Cbl-b/c-Cbl small molecule inhibitor, we demonstrate that Cbl blockade enhances T cell activity under standard and sub-optimal stimulation conditions. Moreover, Cbl inhibition increases mouse syngeneic tumor control alone or in combination with α-PD-1, as described below. METHODS: Human CD8+ T cells were isolated from healthy human blood and activated using α-CD3 or α-CD3/CD28 stimulation. Mouse OT-I splenocytes were stimulated with SIINFEKL or lower affinity ovalbumin peptides and cytokine production was assessed. For tumor studies, mice were inoculated with syngeneic cancer cells and were treated with Cbl inhibitor (30 mg/kg, QD) +/- α-PD-1 (10 mg/kg, Q5D) starting at a tumor volume of 75 mm3. RESULTS: Human CD8+ T cells, stimulated in the presence of a Cbl inhibitor, demonstrated a concentration-dependent increase in activation hallmarks including cytokine secretion (IL-2, IFN-γ and granzyme B), proliferation, and the presence of highly polyfunctional cells. Furthermore, using serially stimulated CD8+ T cells with reduced overall activity, Cbl inhibition showed a marked enhancement of IFN-γ production and cytotoxicity compared to controls upon re-stimulation. We then used an antigen-specific T cell assay where OT-I splenocytes were stimulated by peptides with optimal or reduced TCR affinities, Cbl inhibition produced significantly higher IFN-γ levels with all peptides relative to controls demonstrating that Cbl inhibition can increase the response of T cells to sub-optimal stimulation. Using mouse CT26 and MC38 tumor models, Cbl inhibition was able to significantly enhance tumor control in combination with α-PD-1 and as a monotherapy in CT26 tumors. Notably, a greater number of tumor antigen-specific cytotoxic T cells were found in Cbl inhibitor-treated tumors. Higher expression levels of the co-stimulatory molecule CD226 and the TCR in these anti-tumor cells were also observed, demonstrating the unique biology of Cbl inhibition in enhancing anti-tumor responses. Finally, Cbl inhibition had no effect on the growth or survival of cancer cells in vitro consistent with the notion that enhanced tumor control was driven by immune activity. CONCLUSIONS: Altogether, these data demonstrate that pharmacologic Cbl inhibition robustly enhances T cell activity in vitro and in vivo and provide a mechanistic rationale for targeting Cbl-b/c-Cbl to amplify anti-tumor immune responses. Citation Format: Livia Yamashiro, Sachie Marubayashi, Rameshwari Rayaji, Anudari Battsooj, Karim Mrouj, Hema Singh, Yue Tong Lee, Ada Chen, Lunda Shen, Artur Mailyan, Kai Yu, Masha Podunavac, Julie Yu, Dongdong Liu, Clayton Hardman, Johnny Yin, Srikanth Gangam, Ken Lawson, Manmohan Leleti, Matthew Walters, Daniel DiRenzo. Cbl inhibition increases the response of cytotoxic T cells to sub-optimal stimulation and drives enhanced anti-tumor responses alone or in combination with PD-1 blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4042.

Abstract 4065: Entinostat synergizes with the IL-15 superagonist N-803 and anti-PD-1 to engage cDC1 and stem-like TCF-1+ CD8+ T and NK cells to overcome colorectal tumors refractory to anti-PD-1 therapy

Abstract Background: Immune checkpoint blockade (ICB) has limited efficacy in colorectal carcinoma (CRC), where ~85% of patients harbor microsatellite stability (MSS). A minor subset of CRC patients (~15%) harbor microsatellite instability (MSI) and demonstrate improved responses to ICB ranging from 30-50%. Combination therapy has the potential to increase the response rate. Entinostat, a class I HDAC inhibitor, has demonstrated potential to enhance ICB efficacy, such as αPD-1 and immunostimulatory cytokines. N-803, a heterodimeric IL-15 superagonist that enhances proliferation and activation of CD8+ and NK cells has demonstrated to enhance the clinical benefit of Nivolumab in non-small cell lung cancer patients that were relapsed or refractory to αPD-1 monotherapy. Here, we investigated the antitumor efficacy and mode of action of combination therapy comprising αPD1, Entinostat, and N-803 in αPD1-refractory CT26 and Jak1-deleted MC38 CRC tumor models. Methods: Entinostat, N-803, and αPD1 alone or in combination were administered to mice bearing established CT26 or Jak1-deleted MC38 tumors. Antitumor efficacy, survival, and protective memory upon tumor rechallenge were monitored. Comprehensive flow cytometric profiling of the tumor microenvironment (TME), tumor-draining lymph node (tdLN), and spleen cell populations was conducted. CT26 tumor and tdLN immunomes were examined by single-cell transcriptomics. Cytokines and chemokines were examined in the TME and sera. Antigen-specific IFNg T cell responses were evaluated by ELISpot. Results: We demonstrated that combination therapy with Entinostat, N-803 and αPD1 elicits potent suppression of CT26 and Jak1-deleted MC38 tumors, with median overall survival increasing 33% to not reached. These effects were associated with increased activation, infiltration, cytokine production, and cytolytic function of tumor CD8+ T and NK cells, and increased sera IFNg and TNFα. Tumor resolution and protective memory were associated with development of p15e-specific T cell responses. Increased migration of CD8+ T cells and cross-presenting dendritic cells (cDC1) to the tdLN, as well as significant enrichment of cross-presentation genes in tumor cDC1s was observed. Transcriptomic and phenotypic analyses demonstrated increased stem-like TCF-1+ CD8+ T cell accumulation in the tdLNs and TME of mice treated with triple combination. Conclusions: Combination therapy with Entinostat plus N-803 and αPD1 synergized to promote a sustained antitumor response and prolonged survival. We hypothesize the antitumor effects were due to collaborative efforts of CD8+ T and NK cells, and cDC1 cells in tdLN and TME. These results provide a rationale for the clinical testing of αPD1, Entinostat and N-803 combination for patients with CRC, including those with acquired resistance to ICB. Citation Format: Christine M. Minnar, Masaya Miyamoto, Asma S. Khelifa, Grace Lui, Katherine L. Lothstein, Paul L. Chariou, Thomas J. Meyer, Alexei Lobanov, Margaret Cam, Jeffrey Schlom, Sofia R. Gameiro. Entinostat synergizes with the IL-15 superagonist N-803 and anti-PD-1 to engage cDC1 and stem-like TCF-1+ CD8+ T and NK cells to overcome colorectal tumors refractory to anti-PD-1 therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4065.

Introducing urea into tirapazamine derivatives to enhance anticancer therapy.

Tirapazamine (TPZ) has been approved for multiple clinical trials relying on its excellent anticancer potential. However, as a typical hypoxia-activated prodrug (HAP), TPZ did not exhibit survival advantages in Phase III clinical trials when used in combination therapy due to the insufficient hypoxia levels in patients' tumors. In this study, to improve the therapeutic effects of TPZ, we first introduced urea to synthesize a series of urea-containing derivatives of TPZ. All urea-containing TPZ derivatives showed increased hypoxic cytotoxicity (9.51-30.85-fold) compared with TPZ, while maintaining hypoxic selectivity. TPZP, one of these derivatives, showed 20-fold higher cytotoxicity than TPZ while maintaining a similar hypoxic cytotoxicity ratio. To highly efficiently deliver TPZP to the tumors and reduce its side effects on healthy tissues, we further prepared TPZP into a nanodrug with fibrin-targeting ability: FT11-TPZP-NPs. CA4-NPs, a vascular disrupting agent, was used to increase the fibrin level within tumors and exacerbate tumor hypoxia. By being combined with CA4-NPs, FT11-TPZP-NPs can accumulate in the hypoxia-aggravated tumors and activate sufficiently to kill tumor cells. After a single-dose treatment, FT11-TPZP-NPs+CA4-NPs showed a high inhibition rate of 98.1% against CT26 tumor models with an initial volume of ∼480 mm3 and four out of six tumors were completely eliminated; it thereby exerted a significant antitumor effect. This study provides a new strategy for improving the therapeutic effect of TPZ and other HAPs in anticancer therapy.

Hemin blocks TIGIT/PVR interaction and induces ferroptosis to elicit synergistic effects of cancer immunotherapy.

The immune checkpoint TIGIT/PVR blockade exhibits significant antitumor effects through activation of NK and CD8+ T cell-mediated cytotoxicity. Immune checkpoint blockade (ICB) could induce tumor ferroptosis through IFN-γ released by immune cells, indicating the synergetic effects of ICB with ferroptosis in inhibiting tumor growth. However, the development of TIGIT/PVR inhibitors with ferroptosis-inducing effects has not been explored yet. In this study, the small molecule Hemin that could bind with TIGIT to block TIGIT/PVR interaction was screened by virtual molecular docking and cell-based blocking assay. Hemin could effectively restore the IL-2 secretion from Jurkat-hTIGIT cells. Hemin reinvigorated the function of CD8+ T cells to secrete IFN-γ and the elevated IFN-γ could synergize with Hemin to induce ferroptosis in tumor cells. Hemin inhibited tumor growth by boosting CD8+ T cell immune response and inducing ferroptosis in CT26 tumor model. More importantly, Hemin in combination with PD-1/PD-L1 blockade exhibited more effective antitumor efficacy in anti-PD-1 resistant B16 tumor model. In summary, our finding indicated that Hemin blocked TIGIT/PVR interaction and induced tumor cell ferroptosis, which provided a new therapeutic strategy to combine immunotherapy and ferroptosis for cancer treatment.

Supramolecular assembly of isomeric SN-38 prodrugs regulated by conjugation sites.

Supramolecular polymers (SPs) are an emerging class of drug transporters employed to improve drug therapy. Through the rational design of self-assembling monomers, one can optimize the properties of the resulting supramolecular nanostructures, such as size, shape, surface chemistry, release, and, therefore, biological fates. This study highlights the design of isomeric SN38 prodrugs through the conjugation of hydrophilic oligo(ethylene glycol) (OEG) with hydroxyls at positions 10 and 20 on hydrophobic SN-38. Self-assembling prodrug (SAPD) isomers 10-OEG-SN38 and 20-OEG-SN38 can self-assemble into giant nanotubes and filamentous assemblies, respectively, via aromatic associations that dominate self-assembly. Our study reveales the influence of modification sites on the assembly behavior and ability of the SN38 SAPDs, as well as drug release and subsequent in vitro and in vivo antitumor effects. The SAPD modified at position 20 exhibits stronger π-π interactions among SN38 units, leading to more compact packing and enhanced assembly capability, whereas OEG at position 10 poses steric hindrance for aromatic associations. Importantly, owing to its higher chemical and supramolecular stability, 20-OEG-SN38 outperforms 10-OEG-SN38 and irinotecan, a clinically used prodrug of SN38, in a CT26 tumor model, demonstrating enhanced tumor growth inhibition and prolonged animal survival. This study presents a new strategy of using interactions among drug molecules as dominating features to create supramolecular assemblies. It also brings some insights into creating effective supramolecular drug assemblies via the engineering of self-assembling building blocks, which could contribute to the optimization of design principles for supramolecular drug delivery systems.

Abstract A005: Synergistically Engaging a b-Selective IL-2 Agonist with PD1/PDL-1 Blockade in a Bifunctional Superkine, MDNA223

Abstract Introduction: Combination treatment with a long-acting IL-2 superkine (MDNA109FEAA-Fc; aka MDNA19) and anti-PD1 or anti-CTLA4 has shown clear evidence of synergy in blunting tumor growth. To further leverage this synergism, we designed MDNA223, a BiSKIT (Bifunctional SuperKine for ImmunoTherapy) comprising MDNA109FEAA with enhanced affinity for IL-2Rb and no binding to IL-2Ra, fused to either human or mouse anti-PD1. We present in vitro and in vivo characterization of MDNA223 and demonstrate its enhanced IL-2R agonism and effective PD1/PDL-1 blockade in multiple “hot” and “cold” syngeneic tumor models. Experimental Procedure: In vitro studies included human PBMCs and cell-based reporter assays. In vivo studies were conducted in CT26, B16F10, E0771 and TRAMP-C1 tumor models by intraperiotonneal (IP) administration. Intratumoral treatment was performed in a CT26 bilateral tumor model with or without a STING agonist. Analysis of tumor infiltrating lymphocytes (TILs) was performed using flow cytometry. Results: In studies with human PBMCs, MDNA223 showed increased p-STAT5 signaling in CD8+ T cells and reduced activity in Tregs compared to rhIL-2, consistent with its enhanced receptor selectivity. MDNA223 and anti-PD1 were similarly potent at PD1/PDL-1 blockade in a cell-based reporter assay. In mice, MDNA223 induced proliferation (based on Ki67 expression) and expansion of peripheral CD8+ T cells for over 7 days. IP treatment with MDNA223 resulted in significantly greater inhibition of CT26, B16F10, E0771 and TRAMP-C1 tumor growth when compared to combination of MDNA19 with anti-PD1 at equimolar dosage. This observation is consistent with the proposed cis-binding mechanism of MDNA223 to both, IL-2R and PD1 on the same effector immune cell. In a bilateral CT26 tumor model, IT treatment with MDNA223 not only inhibited the treated tumor but also exhibited an abscopal effect by slowing growth of the untreated tumor implanted on the opposite flank. TILs analysis of B16F10 melanoma following a single dose of MDNA223 showed increased infiltration of CD8+ T cells over Tregs compared to single agent treatment with MDNA19 or anti-PD1 as well as their combination. Treatment with MDNA223 also resulted in a larger population of functional effector CD8+ T cells expressing low levels of the Tim-3 exhaustion marker and high levels of the cytotoxic Granzyme-B protease. Conclusion: MDNA223 demonstrated superior therapeutic activity over the combination of MDNA19 and anti-PD1 in both immunologically ‘hot’ (CT26) and ‘cold’ (B16F10, E0771 and TRAMP-C1) tumor models. Additional studies with variants of MDNA223 capable of tumor selective targeting and/or conditional activation within the tumor micro-environment are in progress to maximize activity at the tumor site with tunable peripheral stimulation. Citation Format: Aanchal Sharma, Minh D To, Hardeep Kataria, Qian Liu, Rosemina Merchant, Fahar Merchant. Synergistically Engaging a b-Selective IL-2 Agonist with PD1/PDL-1 Blockade in a Bifunctional Superkine, MDNA223 [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr A005.

Vasopressin Analog [V4Q5]dDAVP Exerts Cooperative Anticancer Effects in Combination With Low-Dose 5-Fluorouracil on Aggressive Colorectal Cancer Models.

Colorectal cancer (CRC) is a leading cause of cancer-associated mortality worldwide. Despite being an essential component of systemic chemotherapy for advanced CRC, 5-fluorouracil (5-FU) clinical use has severe limitations, such as high toxicity, low selectivity and drug resistance. [V4Q5]dDAVP (1-deamino-4-valine-5-glutamine-8-D-arginine vasopressin) is a peptide vasopressin analog and a selective agonist of the arginine vasopressin type 2 membrane receptor (AVPR2), expressed in microvascular and tumor tissue. This synthetic compound has well-proven antitumor and antimetastatic activity in different tumor types, including metastatic CRC. The objective of this work was to assess the potential combinational benefits in preclinical CRC models after [V4Q5]dDAVP addition to 5-FU. Effects on cellular viability, cell cycle progression, apoptosis and molecular mechanisms associated to [V4Q5]dDAVP treatment in combination with 5-FU were evaluated in murine CT-26 and human COLO-205 cell lines. In vivo, impact of dual therapy was explored on CRC tumor growth and metastatic spread. In CRC cells, [V4Q5]dDAVP (1 µM) addition to sub-IC50 5-FU concentrations resulted in the enhancement of cytostatic effects induced by chemotherapy. Reduction of cell viability after combined treatment was associated with cell cycle arrest in the G0/G1 phase, induction of apoptosis and increased gene expression of the cyclin-dependent kinase inhibitor p21 (CDKN1A) and the tumor suppressor p53 (TP53) in malignant cells, as assessed by flow cytometry, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL), and quantitative reverse transcription polymerase chain reaction (qRT-PCR), respectively. In vivo, intravenous administration of [V4Q5]dDAVP (0.3 µg/kg) in combination with safe low doses of 5-FU (50 or 80 mg/kg for CT-26 or COLO-205 tumor models, respectively) effectively abrogated CRC growth, reducing aggressiveness of primary lesions and increasing survival of tumor-bearing mice. In addition, concomitant administration of [V4Q5]dDAVP and 5-FU inhibited pulmonary metastasis formation by CT-26 cells in immunocompetent mice, especially reducing macrometastatic disease. [V4Q5]dDAVP seems to enhance the efficacy of 5-FU-based chemotherapy in CRC by modulating tumor progression, as well as metastatic dissemination, suggesting its potential role as a safe and cost-effective co-adjuvant agent for the management of advanced CRC.

Abstract C065: Hematopoietic Progenitor Kinase 1 (HPK1) inhibition enhances antibody secretion, pro-inflammatory cytokine production and proliferation of primary human B cells

Abstract Introduction Immune checkpoint blockade (ICB) therapies have revolutionized cancer treatment, although most approaches have focused solely on enhancing T cell responses. In recent years, it has become clear that augmentation of other immune cell subsets is critical to the overall success of cancer immunotherapy. Recent data show that tumors from patients responding to ICB are infiltrated by B cells and strongly correlated with the formation of tertiary lymphoid structures, highlighting the importance of B cells in promoting and coordinating immunotherapy responses [1,2]. Here, we demonstrate that NDI-101150, a potent and highly selective HPK1 small molecule inhibitor, can enhance B cell activation with the tumor. Experimental Procedures Biochemical and cell-based assays were used to characterize the potency and selectivity of NDI-101150, respectively. Mouse splenocytes and/or human blood collections were used to characterize the effects of NDI-101150 on antibody secretion, cytokine production, and proliferation in purified B cells. CT-26 and EMT-6 syngeneic tumor models were used to study tumor-associated B cell responses in vivo, as well as immunization response to Keyhole Limpet Hemocyanin (KLH) in mice receiving daily oral dosing of NDI-101150. Results In CD19+ human B cells and donor PBMCs (peripheral blood mononuclear cells), NDI-101150 treatment inhibited (&amp;gt;90%) phosphorylation of BLNK, a B cell adapter protein phosphorylated by HPK1 downstream of B cell receptor signaling. In vitro NDI-101150 treatment dose-dependently activated primary human and mouse B cells as evidenced by increased pro-inflammatory cytokine production, enhanced IgG antibody secretion, and increased proliferation. Once daily oral administration of NDI-101150 significantly increased KLH antigen-specific IgM and IgG antibody production upon KLH immunization, increased total circulating antibody levels, and induced significant tumor growth inhibition (TGI) in CT-26 and EMT-6 murine syngeneic models (50% and 85% TGI, respectively). Immunophenotyping of tumor bearing animals treated with NDI-101150 revealed an increase in infiltrating CD19+ B cells and an increased tumor B cell transcriptional signature. Conclusions Pharmacological inhibition of HPK1 with NDI-101150 represents a novel immunomodulatory approach for engaging and enhancing B cell activation within the tumor microenvironment. NDI-101150 is currently being tested in a Phase 1 trial in patients with advanced recurrent or metastatic solid tumors. References Helmink, B.A., et al., Nature 2020. v577: 549-55. Cabrita, R., et al., Nature 2020. v577: 561-5. Citation Format: David Ciccone. Hematopoietic Progenitor Kinase 1 (HPK1) inhibition enhances antibody secretion, pro-inflammatory cytokine production and proliferation of primary human B cells [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C065.