Colon Tumors In Mice Research Articles

Tertiary lymphoid structure formation induced by LIGHT-engineered and photosensitive nanoparticles-decorated bacteria enhances immune response against colorectal cancer

Tertiary lymphoid structures (TLSs) are known to enhance the prognosis of patients with colorectal cancer (CRC) by fostering an immunologically active tumor microenvironment (TME). Inducing TLS formation therapeutically holds promise for treating immunologically cold CRC, though it poses technical challenges. Here, we design and fabricate a photosensitive bacterial system named E@L-P/ICG. This system is engineered bacteria internally loaded with the cytokine LIGHT and surface-modified with PLGA/ICG nanoparticles (P/ICG NPs). Once accumulated in orthotopic colonic tumors in mice, E@L-P/ICG generates a mild photothermal effect under laser irradiation due to the photosensitive P/ICG NPs. This photothermal effect triggers the self-rupture of E@L-P/ICG and the death of surrounding tumor cells to release adjuvants and antigens, respectively, which in turn synergistically activate the adaptive immune responses. Furthermore, the cytokine LIGHT released from ruptured E@L-P/ICG stimulates the generation of high endothelial vessels (HEVs), promoting lymphocyte recruitment within the TME. These mechanisms lead to the TLS formation in CRC, which further boosts adaptive immune responses through effective infiltration of T cells and B cells, resulting in effectively inhibited tumor growth and extended survival of mice. Our study shows the potential of the E@L-P/ICG system in photosensitively inducing the TLS formation to treat CRC in clinic.

Interleukin-17F suppressed colon cancer by enhancing caspase 4 mediated pyroptosis of endothelial cells

The combination of anti-angiogenic treatment and immunotherapy presents a promising strategy against colon cancer. Interleukin-17F (IL-17F) emerges as a critical immune cell cytokine expressed in colonic epithelial cells, demonstrating potential in inhibiting angiogenesis. In order to clarify the roles of IL-17F in the colon cancer microenvironment and elucidate its mechanism, we established a mouse colon carcinoma cell line CT26 overexpressing IL-17F and transplanted it subcutaneously into syngeneic BALB/c mice. We also analyzed induced colon tumor in IL-17F knockout and wild type mice. Our results demonstrated that IL-17F could suppress colon tumor growth in vivo with inhibited angiogenesis and enhanced recruitment of cysteine-cysteine motif chemokine receptor 6 (CCR6) positive immune cells. Additionally, IL-17F suppressed the tube formation, cell growth and migration of endothelial cells EOMA in vitro. Comprehensive bioinformatics analysis of transcriptome profiles between EOMA cells and those treated with three different concentrations of IL-17F identified 109 differentially expressed genes. Notably, a potential new target, Caspase 4, showed increased expressions after IL-17F treatment in endothelial cells. Further molecular validation revealed a novel downstream signaling for IL-17F: IL-17F enhanced Caspase 4/GSDMD signaling of endothelial cells, CT26 cells and CT26 transplanted tumors, while IL-17F knockout colon tumors exhibited decreased Caspase 4/GSDMD signaling. The heightened expression of the GSDMD N-terminus, coupled with increased cellular propidium iodide (PI) uptake and lactate dehydrogenase (LDH) release, revealed that IL-17F promoted pyroptosis of endothelial cells. Altogether, IL-17F could modulate the colon tumor microenvironment with inhibited angiogenesis, underscoring its potential as a therapeutic target for colon cancer.

Changes in the Expression of Genes Regulating the Response to Hypoxia, Inflammation, Cell Cycle, Apoptosis, and Epithelial Barrier Functioning during Colitis-Associated Colorectal Cancer Depend on Individual Hypoxia Tolerance.

One of the factors contributing to colorectal cancer (CRC) development is inflammation, which is mostly hypoxia-associated. This study aimed to characterize the morphological and molecular biological features of colon tumors in mice that were tolerant and susceptible to hypoxia based on colitis-associated CRC (CAC). Hypoxia tolerance was assessed through a gasping time evaluation in a decompression chamber. One month later, the animals were experimentally modeled for colitis-associated CRC by intraperitoneal azoxymethane administration and three dextran sulfate sodium consumption cycles. The incidence of tumor development in the distal colon in the susceptible to hypoxia mice was two times higher and all tumors (100%) were represented by adenocarcinomas, while in the tolerant mice, only 14% were adenocarcinomas and 86% were glandular intraepithelial neoplasia. The tumor area assessed on serially stepped sections was statistically significantly higher in the susceptible animals. The number of macrophages, CD3-CD19+, CD3+CD4+, and NK cells in tumors did not differ between animals; however, the number of CD3+CD8+ and vimentin+ cells was higher in the susceptible mice. Changes in the expression of genes regulating the response to hypoxia, inflammation, cell cycle, apoptosis, and epithelial barrier functioning in tumors and the peritumoral area depended on the initial mouse's hypoxia tolerance, which should be taken into account for new CAC diagnostics and treatment approaches development.

Anti-TNFR2 Antibody-Conjugated PLGA Nanoparticles for Targeted Delivery of Adriamycin in Mouse Colon Cancer.

High levels of tumor necrosis factor receptor type II (TNFR2) are preferentially expressed by immunosuppressive CD4+Foxp3+ regulatory T cells (Tregs), especially those present in the tumor microenvironment, as initially reported by us. There is compelling evidence that targeting TNFR2 markedly enhances antitumor immune responses. Furthermore, a broad spectrum of human cancers also expresses TNFR2, while its expression by normal tissue is very limited. We thus hypothesized that TNFR2 may be harnessed for tumor-targeted delivery of chemotherapeutic agents. In this study, we performed a proof-of-concept study by constructing a TNFR2-targeted PEGylated poly(dl-lactic-co-glycolic acid) (PLGA-PEG) nanodrug delivery system [designated as TNFR2-PLGA-ADR (Adriamycin)]. The results of invitro study showed that this TNFR2-targeted delivery system had the properties in cellular binding and cytotoxicity toward mouse colon cancer cells. Further, upon intravenous injection, TNFR2-PLGA-ADR could efficiently accumulate in MC38 and CT26 mouse colon tumor tissues and preferentially bind with tumor-infiltrating Tregs. Compared with ADR and ISO-PLGA-ADR, the invivo antitumor effect of TNFR2-PLGA-ADR was markedly enhanced, which was associated with a decrease of TNFR2+ Tregs and an increase of IFNγ+CD8+ cytotoxic T lymphocytes in the tumor tissue. Therefore, our results clearly show that targeting TNFR2 is a promising strategy for designing tumor-specific chemoimmunotherapeutic agent delivery system.

A Tumor Environment-Activated Photosensitized Biomimetic Nanoplatform for Precise Photodynamic Immunotherapy of Colon Cancer.

Aggressive nature of colon cancer and current imprecise therapeutic scenarios simulate the development of precise and effective treatment strategies. To achieve this, a tumor environment-activated photosensitized biomimetic nanoplatform (PEG2000-SiNcTI-Ph/CpG-ZIF-8@CM) is fabricated by encapsulating metal-organic framework loaded with developed photosensitizer PEG2000-SiNcTI-Ph and immunoadjuvant CpG oligodeoxynucleotide within fusion cell membrane expressing programmed death protein 1 (PD-1) and cluster of differentiation 47 (CD47). By stumbling across, systematic evaluation, and deciphering with quantum chemical calculations, a unique attribute of tumor environment (low pH plus high concentrations of adenosine 5'-triphosphate (ATP))-activated photodynamic effect sensitized by long-wavelength photons is validated for PEG2000-SiNcTI-Ph/CpG-ZIF-8@CM, advancing the precision of cancer therapy. Moreover, PEG2000-SiNcTI-Ph/CpG-ZIF-8@CM evades immune surveillance to target CT26 colon tumors in mice mediated by CD47/signal regulatory proteins α (SIRPα) interaction and PD-1/programmed death ligand 1 (PD-L1) interaction, respectively. Tumor environment-activated photodynamic therapy realized by PEG2000-SiNcTI-Ph/CpG-ZIF-8@CM induces immunogenic cell death (ICD) to elicit anti-tumor immune response, which is empowered by enhanced dendritic cells (DC) uptake of CpG and PD-L1 blockade contributed by the nanoplatform. The photodynamic immunotherapy efficiently combats primary and distant CT26 tumors, and additionally generates immune memory to inhibit tumor recurrence and metastasis. The nanoplatform developed here provides insights for the development of precise cancer therapeutic strategies.

Morphological Characteristics of Colon Tumors in Mice with Different Tolerance to Hypoxia.

In adult male C57BL/6 mice with high (HR) and low (LR) resistance to hypoxia, morphological features of colon tumors and blood parameters were evaluated 70 days after intraperitoneal injection of azoxymethane and subsequent consumption of 3 cycles of dextran sulfate sodium. On macroscopic analysis, tumors were found in the distal colon in 35% (7 of 20 animals) of HR and 31% (4 of 13 animals) of LR animals. Microscopic analysis of the distal colon revealed tumors in 75% (15 of 20 animals) of HR and 69% (9 of 13 animals) of LR mice. The tumors were presented by areas of glandular intraepithelial neoplasia and adenocarcinomas; the incidence and the area of the tumors did not differ in groups of HR and LR mice. The number of neuroendocrine and goblet cells in the distal colon mucosa in the areas of tumors was similar in the compared groups. However, in both HR and LR mice of the experimental groups, the content of goblet cells in tumors was lower and the content of endocrine cells was higher than in the corresponding control groups. In the peripheral blood, the erythrocyte count and hemoglobin content decreased in HR and LR mice of the experimental groups; the relative number of monocytes increased only in HR mice and the absolute number of lymphocytes and monocytes decreased in LR mice. Thus, 70 days after azoxymethane administration and dextran sulfate sodium consumption, the tumors in mice were presented by glandular intraepithelial neoplasia and adenocarcinomas, and their incidence and area did not differ between animals with different tolerance to hypoxia.

Abstract 693: The effect of butyrophilin 1A1 blockade in combination with radiation treatment in tumor models

Abstract Expression of radiation-induced immune escape molecules, including PD-L1, contributes to the efficacy of cancer immunotherapy. We used an unbiased in vivo gene knockdown screen in naïve T-cells and introduced them into irradiated host tumors to identify novel immunosuppressive genes activated during radiation. From the screen, we found that butyrophilin 1A1 (BTN1A1) knockdown causes T-cell expansion in irradiated tumors. This points to the potential immune checkpoint role of BTN1A1. We validated that BTN1A1 expression is inducible in activated T-cells and that blocking BTN1A1 contributes to T-cell activation. We tested an anti-mouse BTN1A1 antibody (STC109) in mouse colon tumor models (CT26 tumors), we observed partial anti-tumor efficacy using single agent STC109, which was enhanced by the combination of STC109 with radiation. We also got similar result in in mouse lung tumor models (LLC tumors) using anti-mouse BTN1A1 antibody, and the combination treatment showed an improved suppression of tumor growth and lung metastasis compared to single agent. Moreover, our data shown that knockout of BTN1A1 in 4T1 tumor models shown dramatic defect of tumor growth, and the BTN1A1 ko tumors are more sensitive to radiotherapy. These findings indicate the role of BTN1A1 in tumor immune response from radiation stress, and that anti-BTN1A1 has strong potential to enhance efficacy of radiotherapy for cancer treatment. Citation Format: Nan Li, Chen Braun, Young-Seung Kim, Andrew Park, Stephen S Yoo, Steven H. Lin. The effect of butyrophilin 1A1 blockade in combination with radiation treatment in tumor models [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 693.

Jiedu Xiaozheng Yin Inhibits the Progression of Colitis Associated Colorectal Cancer by Stimulating Macrophage Polarization Towards an M1 Phenotype via the TLR4 Pathway.

To investigate the effect of Jiedu Xiaozheng Yin (JXY) on the polarization of macrophages in colitis-associated colon cancer (CAC). An orthotopic model of CAC was established to monitor changes in the pathological state of mice. Colon length, number of colon tumors were recorded, and indices for liver, spleen, and thymus were calculated. Hematoxylin and eosin (H&E) staining was employed to observe intestinal mucosal injury and tumor formation. Immunohistochemistry (IHC) staining was utilized to investigate the effect of JXY on M1 and M2 polarization of macrophages in the colonic mucosa of CAC mice. For in vitro experiments, RT-qPCR (Reverse Transcription-quantitative PCR) and flow cytometry were used to observe the effect of JXY on various M1-related molecules such as IL-1β, TNF-α, iNOS, CD80, CD86, and its phagocytic function as well as M2-related molecules including Arg-1, CD206, and IL-10. Subsequently, after antagonizing the TLR4 pathway with antagonists (TAK242, PDTC, KG501, SR11302, LY294002), the expression of IL-6, TNF-α, iNOS, and IL-1β mRNA were detected by RT-qPCR. In vivo experiments, the results showed that JXY improved the pathological condition of mice in general. And JXY treatment decreased the shortening of colon length and number of tumors as compared to non-treated CAC mice. Additionally, JXY treatment improved the lesions in the colonic tissue and induced a polarization of intestinal mucosal macrophages towards the M1 phenotype, while inhibiting polarization towards the M2 phenotype. In vitro experiments further confirmed that JXY treatment promoted the activation of macrophages towards the M1 phenotype, leading to increased expression of IL-1β, TNF-α, iNOS, CD80, CD86, as well as enhanced phagocytic function. JXY treatment concomitantly inhibited the expression of M2-phenotype related molecules Arginase-1 (Arg-1), CD206, and IL-10. Furthermore, JXY inhibited M1-related molecules such as IL-6, TNF-α, iNOS, and IL-1β after antagonizing the TLR4 pathway. Obviously, JXY could exhibit inhibitory effects on the development of colon tumors in mice with CAC by promoting M1 polarization through TLR4-mediated signaling and impeding M2 polarization of macrophages.

Interleukin-17 directly stimulates tumor infiltrating Tregs to prevent cancer development.

Interleukin-17 (IL-17) family cytokines promote protective inflammation for pathogen resistance, but also facilitate autoimmunity and tumor development. A direct signal of IL-17 to regulatory T cells (Tregs) has not been reported and may help explain these dichotomous responses. We generated a conditional knockout of Il17ra in Tregs by crossing Foxp3-YFP-Cre mice to Il17ra-flox mice (Il17ra ΔTreg mice). Subsequently, we adoptively transferred bone marrow cells from Il17ra ΔTreg mice to a mouse model of sporadic colorectal cancer (Cdx2-Cre +/Apc F/+), to selectively ablate IL-17 direct signaling on Tregs in colorectal cancer. Single cell RNA sequencing and bulk RNA sequencing were performed on purified Tregs from mouse colorectal tumors, and compared to those of human tumor infiltrating Treg cells. IL-17 Receptor A (IL-17RA) is expressed in Tregs that reside in mouse mesenteric lymph nodes and colon tumors. Ablation of IL-17RA, specifically in Tregs, resulted in increased Th17 cells, and exacerbated tumor development. Mechanistically, tumor-infiltrating Tregs exhibit a unique gene signature that is linked to their activation, maturation, and suppression function, and this signature is in part supported by the direct signaling of IL-17 to Tregs. To study pathways of Treg programming, we found that loss of IL-17RA in tumor Tregs resulted in reduced RNA splicing, and downregulation of several RNA binding proteins that are known to regulate alternative splicing and promote Treg function. IL-17 directly signals to Tregs and promotes their maturation and function. This signaling pathway constitutes a negative feedback loop that controls cancer-promoting inflammation in CRC.

The microbial genotoxin colibactin exacerbates mismatch repair mutations in colorectal tumors

Certain Enterobacteriaceae strains contain a 54-kb biosynthetic gene cluster referred to as “pks” encoding the biosynthesis of a secondary metabolite, colibactin. Colibactin-producing E. coli promote colorectal cancer (CRC) in preclinical models, and in vitro induce a specific mutational signature that is also detected in human CRC genomes. Yet, how colibactin exposure affects the mutational landscape of CRC in vivo remains unclear. Here we show that colibactin-producing E. coli-driven colonic tumors in mice have a significantly higher SBS burden and a larger percentage of these mutations can be attributed to a signature associated with mismatch repair deficiency (MMRd; SBS15), compared to tumors developed in the presence of colibactin-deficient E. coli. We found that the synthetic colibactin 742 but not an inactive analog 746 causes DNA damage and induces transcriptional activation of p53 and senescence signaling pathways in non-transformed human colonic epithelial cells. In MMRd colon cancer cells (HCT 116), chronic exposure to 742 resulted in the upregulation of BRCA1, Fanconi anemia, and MMR signaling pathways as revealed by global transcriptomic analysis. This was accompanied by increased T>N single-base substitutions (SBS) attributed to the proposed pks+E. coli signature (SBS88), reactive oxygen species (SBS17), and mismatch-repair deficiency (SBS44). A significant co-occurrence between MMRd SBS44 and pks-associated SBS88 signature was observed in a large cohort of human CRC patients (n=2,945), and significantly more SBS44 mutations were found when SBS88 was also detected. Collectively, these findings reveal the host response mechanisms underlying colibactin genotoxic activity and suggest that colibactin may exacerbate MMRd-associated mutations.

Abstract 6371: Identification of a lymphocyte activation gene-3-binding peptides using phage displayed-peptide libraries for cancer immunotherapy

Abstract Treatment with immune checkpoint blockades against CTLA-4, PD-1, and PD-L1 is currently giving improved results to many cancer patients. Nevertheless, a significant proportion of cancer patients treated with immune checkpoint blockades are still largely unsatisfied with the benefits of these drugs. One of the reasons is that cancer cells also use other immune checkpoints such as lymphocyte activation gene-3 (LAG-3), T cell immunoreceptor with Ig and ITIM domains, and T-cell immunoglobulin and mucin-domain containing-3. In this study, we screened phage displayed-peptide libraries for peptides that selectively bind to LAG-3. After five rounds of screening, we selected a candidate peptide and named it LAG3Pep. LAG3Pep preferentially bound to human LAG-3-transfected HEK 293T cells over mock-transfected cells and phorbol myristate acetate/ionomycin/chloroquine-stimulated Jurkat T cells over unstimulated cells. LAG3Pep also bound to splenocytes isolated from tumor-bearing mice, suggesting the reactivity across human and mouse LAG-3. Pull-down assay of human recombinant LAG-3 protein using biotin-labeled LAG3Pep yielded a protein band of LAG-3. LAG3Pep showed a high-affinity binding to the LAG-3 protein in SPR assays. Pre-treatment of a LAG-3-blocking antibody competed the binding of LAG3Pep to stimulated Jurkat T cells. LAG3Pep inhibited the binding of the LAG-3 protein to THP-1 cells expressing HLA-DR, a well-known LAG-3 ligand. In addition, treatment of T-cells with LAG3Pep in combination with a PD-L1-blocking antibody recovered the secretion of IL-2 by T-cells which was suppressed by FGL-1, another ligand of LAG-3, secreted from HepG2 tumor cells in co-culture. Intravenous administration of LAG3Pep in combination with a PD-L1-blocking peptide showed an anti-tumor growth activity in the MC38 syngeneic mouse colon tumor model. These results show that LAG3Pep acts as LAG-3 blockade and, in combination with a PD-L1-blockade, could be a promising tool for cancer immunotherapy. Citation Format: Seok-Min Lee, Byungheon Lee. Identification of a lymphocyte activation gene-3-binding peptides using phage displayed-peptide libraries for cancer immunotherapy. [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 6371.

Abstract 462: A novel and potent Cblb inhibitor demonstrates robust immunological profile and anti-tumor efficacy

Abstract Background: Casitas B-lineage lymphoma b (Cbl-b), a RING finger E3 ligase, is a negative regulator of immune cell activation1. Genetic deletion or pharmacological inhibition of Cbl-b resulted in hyper-reactive and co-stimulation independent T cell activation and cytokine production1. In syngeneic tumor models, CD8 T-cell and NK-cell mediated rejection of tumours were observed1. These findings point to Cbl-b as a therapeutic target in cancer immunotherapy. Inhibition of Cbl-b also demonstrated the potential to enhance the efficacy of check-point blockers like anti-PD-1 antibody, an unmet need in the clinic. Methods: Using intuitive medicinal chemistry design supported by computational approaches, we identified a lead Cbl-b inhibitor. SAR was developed using a battery of biochemical assays, functional read-outs and primary human in vitro T-cell activation and exhaustion assays. In vivo efficacy was demonstrated in syngeneic mouse colon tumor model. Results: Our lead Cbl-b inhibitor demonstrated potent binding to Cbl-b, robust anti-tumor cytokine secretion in human and mouse T cells, whole blood and potent reversal of T cell exhaustion. A strong tumor growth inhibition was demonstrated by the lead compound in a mouse colon tumor model. Compared to single agent, a combination of the lead compound with anti-PD-1 antibody induced enhanced complete tumor rejections. Conclusions: We have identified a novel, potent and orally bioavailable Cbl-b inhibitor that demonstrated robust in vitro and in vivo anti-tumor profiles. Acknowledgements: We thank Sanjib Das, Ajit Patil, Gauri Gawas, Savita Pandita, Priya Yadav, Sneha Pusadkar, Mayura Behere, Subhadip Das, Shravankumar Kolli and Radheshyam Yadav for their contributions to the project

Abstract 6327: Intratumoral administration of ultra high-concentration nitric oxide (UNO) is more efficacious than anti-PD1 therapy in 4T1 tumor-bearing mice

Abstract Background: For the majority of metastatic triple-negative breast cancer (mTNBC) patients, chemotherapy is the standard first-line treatment, however, outcomes in this population are poor. A monoclonal antibody against the immune checkpoint inhibitor anti-programmed death 1 (PD-1) also has antitumor activity in patients with mTNBC. Previously, we reported that delivering ultra-high concentrations of nitric oxide (UNO) to mouse colon tumors stimulated innate and adaptive immune responses leading to the rejection of secondarily-induced tumors. Moreover, adding an anti-PD-1 antibody to UNO treatment resulted in primary tumor regression in 53% of the mice, the rejection of a distant tumors in all mice, and prolonged survival compared to control and anti-PD-1 arms. In this in vivo study, we investigated the ability of UNO to improve the efficacy of anti-PD-1 antibody treatment in the aggressive mouse breast cancer model, 4T1. Methods: We tested two treatment protocols: 4T1 mice tumors were treated with either a single dose of UNO and anti-PD-1 or a repeated dose of UNO and anti-PD-1. In the single dose design, 4T1 tumors were treated with a single administration of UNO combined with Anti-PD1 administration starting one day after UNO treatment (n=9-10 mice per group). In the repeated dosing design, 4T1 tumors were treated with UNO twice combined with Anti-PD-1 administration starting on the first UNO treatment day (n=8 mice per group). • UNO treatment regimen: 50,000 ppm, 10 minutes, flow rate ~0.2 liters per minute • Anti-PD1 treatment regimen: 10 mg/kg antibody was administered every 3 days for a total of 5 treatments. Results (UNO Single Dose): The primary tumors of 4T1 tumor-bearing mice were 13.2% smaller in the combined single-dose UNO group compared to anti-PD-1 alone 15 days post-UNO treatment. Results (UNO Repeated Dosing): The primary tumors of 4T1 tumor-bearing mice were 25.2% smaller in the combined repeated dose UNO group compared to the anti-PD-1 alone group 16 days after the first UNO treatment. Conclusion: Treating 4T1 tumors with single and repeated doses of UNO improved outcomes compared to anti-PD-1 alone. This suggests that local short-term treatment with UNO can serve as a treatment option for cancer patients with tumors that are not amenable to immune checkpoint inhibitor treatment. Citation Format: Yana Epshtein, Matan Goldshtein, Selena Chaisson, Jedidiah Monson, Matt Johnson, Amir Avniel, Steve Lisi, Hila Confino. Intratumoral administration of ultra high-concentration nitric oxide (UNO) is more efficacious than anti-PD1 therapy in 4T1 tumor-bearing mice [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 6327.

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

Modular-designed engineered bacteria for precision tumor immunotherapy via spatiotemporal manipulation by magnetic field

Micro-nano biorobots based on bacteria have demonstrated great potential for tumor diagnosis and treatment. The bacterial gene expression and drug release should be spatiotemporally controlled to avoid drug release in healthy tissues and undesired toxicity. Herein, we describe an alternating magnetic field-manipulated tumor-homing bacteria developed by genetically modifying engineered Escherichia coli with Fe3O4@lipid nanocomposites. After accumulating in orthotopic colon tumors in female mice, the paramagnetic Fe3O4 nanoparticles enable the engineered bacteria to receive and convert magnetic signals into heat, thereby initiating expression of lysis proteins under the control of a heat-sensitive promoter. The engineered bacteria then lyse, releasing its anti-CD47 nanobody cargo, that is pre-expressed and within the bacteria. The robust immunogenicity of bacterial lysate cooperates with anti-CD47 nanobody to activate both innate and adaptive immune responses, generating robust antitumor effects against not only orthotopic colon tumors but also distal tumors in female mice. The magnetically engineered bacteria also enable the constant magnetic field-controlled motion for enhanced tumor targeting and increased therapeutic efficacy. Thus, the gene expression and drug release behavior of tumor-homing bacteria can be spatiotemporally manipulated in vivo by a magnetic field, achieving tumor-specific CD47 blockage and precision tumor immunotherapy.

Constitutive Expression of a Cytotoxic Anticancer Protein in Tumor-Colonizing Bacteria.

Bacterial cancer therapy is a promising next-generation modality to treat cancer that often uses tumor-colonizing bacteria to deliver cytotoxic anticancer proteins. However, the expression of cytotoxic anticancer proteins in bacteria that accumulate in the nontumoral reticuloendothelial system (RES), mainly the liver and spleen, is considered detrimental. This study examined the fate of the Escherichia coli strain MG1655 and an attenuated strain of Salmonella enterica serovar Gallinarum (S. Gallinarum) with defective ppGpp synthesis after intravenous injection into tumor-bearing mice (~108 colony forming units/animal). Approximately 10% of the injected bacteria were detected initially in the RES, whereas approximately 0.01% were in tumor tissues. The bacteria in the tumor tissue proliferated vigorously to up to 109 colony forming units/g tissue, whereas those in the RES died off. RNA analysis revealed that tumor-associated E. coli activated rrnB operon genes encoding the rRNA building block of ribosome needed most during the exponential stage of growth, whereas those in the RES expressed substantially decreased levels of this gene and were cleared soon presumably by innate immune systems. Based on this finding, we engineered ΔppGpp S. Gallinarum to express constitutively a recombinant immunotoxin comprising TGFα and the Pseudomonas exotoxin A (PE38) using a constitutive exponential phase promoter, the ribosomal RNA promoter rrnB P1. The construct exerted anticancer effects on mice grafted with mouse colon (CT26) or breast (4T1) tumor cells without any notable adverse effects, suggesting that constitutive expression of cytotoxic anticancer protein from rrnB P1 occurred only in tumor tissue.

TCP-1, a novel peptide to diagnose early colon cancer

A nine cyclic peptide (TCP-1) showed excellent specificity for colon cancer. TCP-1 binds with human tumor tissues at early stages and mice tumor with diameters of 1-4 mm, suggesting that TCP-1 may be used for early diagnosis of colon cancer. The mechanism of the targeted binding of TCP-1 to colon cancer was also studied using immunoprecipitation, LC-MS and bioinformatics. After screening and identifying of the possible binding target proteins of TCP-1, keratin, type II cytoskeletal 5 was speculated to be the specific binding target protein of TCP-1 in human tumor tissue. Pharmacokinetics studies were conducted to investigate the target-mediated drug disposition of the new tumor-specific peptide by LC-MS/MS. The tissue distribution study showed that TCP-1 was found only in colon tumors (the target site) in tumor mice did not bind to any other tissues. Conjugating TCP-1 to tumor markedly increased its removal rate from blood circulation but mildly extended its staying time in vivo. In tumor mice, a lower AUC of TCP-1 (reduced by almost 35%) and 2-fold higher clearance were found compared to that of normal mice. The proposed metabolic pathway of TCP-1 in the kidney was also determined using LC-MSn-IT-TOF. The high specificity and low toxicity of the peptide may be caused by its extremely tight binding to the targets. Potential applications for future clinical use, including MRI and PET/CT were also explored, and this research may promote the development of colon cancer diagnostic technology research and provide new ideas and technical routes for tumor diagnostic technology.

Manganese‐CpG Nanocomposite Integrates ROS‐Induced Cell Apoptosis and STING‐Activated and Adjuvant‐Augmented Immune Responses for Tumor Elimination and Prevention

AbstractChemodynamic therapy (CDT) based on Fenton‐like reaction possesses great potential for anticancer therapy with high efficiency and specificity. However, CDT still suffers low therapeutic efficiency due to intratumoral insufficient H2O2 or unfavorable tumor microenvironment. To enhance tumor treatment and in particular prevent tumor recurrence, the combined strategies with CDT are essentially needed. Herein, a nanocomposite based on Mn2+ and cytosine‐phosphate‐guanine oligonucleotides (CpG ODNs) (MnCpGPNCs) is constructed to combine CDT with immunostimulating responses by exploiting Mn2+‐induced CDT and Mn2+/CpG ODNs‐coinduced immunotherapy against mouse colon tumor cells. Specifically, MnCpGPNCs efficiently deliver and sensitively release Mn2+ in tumor cells to trigger Fenton‐like reaction, causing cell apoptosis. Importantly, Mn2+ also promotes the stimulator of interferon genes pathway to increase the production of type I interferons, thus increasing cytotoxic T lymphocyte infiltration, dendritic cell maturation, and proinflammatory cytokine secretion. Furthermore, CpG ODNs serve as immune adjuvant enhanced the antigen presentation and immune‐eliciting potency. The combination of Mn2+‐based CDT with augmented immunotherapy efficiently inhibits the primary/distance tumors and prevents tumorigenesis. Overall, this newly developed metal‐CpG system demonstrates a paradigm to expand chemodynamic agents as immunotherapy boosters to complement current treatments for tumor elimination and prevention.

Blocking DCIR mitigates colitis and prevents colorectal tumors by enhancing the GM-CSF-STAT5 pathway.

Dendritic cell immunoreceptor (DCIR; Clec4a2), a member of the C-type lectin receptor family, plays important roles in homeostasis of the immune and bone systems. However, the intestinal role of this molecule is unclear. Here, we show that dextran sodium sulfate (DSS)-induced colitis and azoxymethane-DSS-induced intestinal tumors are reduced in Clec4a2-/- mice independently of intestinal microbiota. STAT5 phosphorylation and expression of Csf2 and tight junction genes are enhanced, while Il17a and Cxcl2 are suppressed in the Clec4a2-/- mouse colon, which exhibits reduced infiltration of neutrophils and myeloid-derived suppressor cells. Granulocyte-macrophage colony-stimulating factor (GM-CSF) administration ameliorates DSS colitis associated with reduced Il17a and enhanced tight junction gene expression, whereas anti-GM-CSF exacerbates symptoms. Furthermore, anti-NA2, a ligand for DCIR, ameliorates colitis and prevents colorectal tumors. These observations indicate that blocking DCIR signaling ameliorates colitis and suppresses colonic tumors, suggesting DCIR as a possible target for the treatment of these diseases.

Improved Tumor Responses with Sequential Targeted α-Particles Followed by Interleukin 2 Immunocytokine Therapies in Treatment of CEA-Positive Breast and Colon Tumors in CEA Transgenic Mice.

Targeted α-therapy (TAT) delivers high-linear-transfer-energy α-particles to tumors with the potential to generate tumor immune responses that may be augmented by antigen-targeted immunotherapy. Methods: This concept was evaluated in immunocompetent carcinoembryonic antigen (CEA) transgenic mice bearing CEA-positive mammary or colon tumors. Tumors were targeted with humanized anti-CEA antibody M5A labeled with 225Ac for its 10-d half-life and emission of 4 α-particles, as well as being targeted with the immunocytokine M5A-interleukin 2. Results: A dose response (3.7, 7.4, and 11.1 kBq) to TAT only, for orthotopic CEA-positive mammary tumors, was observed, with a tumor growth delay of 30 d and an increase in median survival from 20 to 36 d at the highest dose. Immunocytokine (4 times daily) monotherapy gave a tumor growth delay of 20 d that was not improved by addition of 7.4 kBq of TAT 5 d after the start of immunocytokine. However, TAT (7.4 kBq) followed by immunocytokine 10 d later led to a tumor growth delay of 38 d, with an increase in median survival to 45 d. Similar results were seen for TAT followed by immunocytokine at 5 versus 10 d. When a similar study was performed with subcutaneously implanted CEA-positive MC38 colon tumors, TAT (7.4 kBq) monotherapy gave an increase in median survival from 29 to 42 d. The addition of immunocytokine 10 d after 7.4 kBq of TAT increased median survival to 57 d. Immunophenotyping showed increased tumor-infiltrating interferon-γ-positive, CD8-positive T cells and an increased ratio of these cells to Foxp3-positive, CD4-positive regulatory T cells with sequential therapy. Immunohistochemistry confirmed there was an increase in tumor-infiltrating CD8-positive T cells in the sequential therapy group, strongly suggesting that immunocytokine augmented TAT can lead to an immune response that improves tumor therapy. Conclusion: Low-dose (7.4 kBq) TAT followed by a 4-dose immunocytokine regimen 5 or 10 d later gave superior tumor reductions and survival curves compared with either monotherapy in breast and colon cancer tumor models. Reversing the order of therapy to immunocytokine followed by TAT 5 d later was equivalent to either monotherapy in the breast cancer model.