Immunological Effects Research Articles

Characterization of structural, genotoxic, and immunological effects of methyl methanesulfonate (MMS) induced DNA modifications: Implications for inflammation-driven carcinogenesis

Genotoxic DNA damaging agents are the choice of chemicals for studying DNA repair pathways and the associated genome instability. One such preferred laboratory chemical is methyl methanesulfonate (MMS). MMS, an SN2-type alkylating agent known for its ability to alkylate adenine and guanine bases, causes strand breakage. Exploring the outcomes of MMS interaction with DNA and the associated cytotoxicity will pave the way to decipher how the cell confronts methylation-associated stress. This study focuses on an in-depth understanding of the structural instability, induced antigenicity on the DNA molecule, cross-reactive anti-DNA antibodies, and cytotoxic potential of MMS in peripheral lymphocytes and cancer cell lines. The findings are decisive in identifying the hazardous nature of MMS to alter the intricacies of DNA and morphology of the cell. Structural alterations were assessed through UV–Vis, fluorescence, liquid chromatography, and mass spectroscopy (LCMS). The thermal instability of DNA was analyzed using duplex melting temperature profiles. Scanning and transmission electron microscopy revealed gross topographical and morphological changes. MMS-modified DNA exhibited increased antigenicity in animal subjects. MMS was quite toxic for the cancer cell lines (HCT116, A549, and HeLa). This research will offer insights into the potential role of MMS in inflammatory carcinogenesis and its progression.

Photoactivated theranostic nanomaterials based on aggregation‐induced emission luminogens for cancer photoimmunotherapy

AbstractIn this study, a series of high liposoluble near‐infrared emissive aggregation‐induced emission luminogens with triphenylamine derivatives as donor units and 1‐indanone as electron acceptor units are developed. Specifically, MTTOI has promising lipid droplets targeting ability and viscosity‐response characteristics, and it can monitor the viscosity fluctuations in living cells in real time. It is found that MTTOI is able to fulfill the activation of the apoptosis‐related signaling pathways under white light, and it can also activate the ferroptosis to synergize apoptosis resulting in tumor elimination. Interestingly, the occurrence of ferroptosis is positively related to the enhancement of immunogenic cell‐death effect, thus boosting the tumor infiltration of CD8+ T cells and reducing the proportion of regulatory T cells by cooperating with dendritic cells, which can not only carry out primary antitumor treatment, but also prevent tumor metastasis through strong immunological memory effect. Moreover, in vivo experimental results confirm that MTTOI successfully effectuates fatty liver tissue imaging. MTTOI fluorescence signals can be captured at the tumor site after 3 days of administration due to its high fat‐solubility and ease of fusion with tumors. This classic example could promote the further development of phototherapeutic agents in preclinical research and clinical applications.

Tubulin alpha-1b chain was identified as a prognosis and immune biomarker in pan-cancer combing with experimental validation in breast cancer

The α-tubulin subtype, Tubulin α-1b chain (TUBA1B), has been shown to influence immune cell infiltration, cancer growth, and survival across various malignancies. However, a comprehensive study has not yet been undertaken examining the immunological and predictive effects of TUBA1B in a pan-carcinoma context. Using data from TCGA, GEO, and other databases, we analyzed TUBA1B expression across various carcinoma types using transcriptional profiling, prognostic implications, genetic and epigenetic alterations, methylation patterns, and immunological significance. To validate our findings, we conducted Western blot analysis to assess TUBA1B protein levels in matched breast cancer tissue samples and performed CCK-8 proliferation assay, flow cytometry, transwell invasion, and migration assays to comprehensively examine the functional impact of TUBA1B on breast cancer cells. Our pan-cancer analysis found TUBA1B upregulation across most tumor types, with varying expression patterns in distinct immune and molecular subtypes. High TUBA1B expression was an independent risk factor and associated with poor prognoses in several cancers, including BRCA, KICH, LGG, LUAD, and MESO. TUBA1B also demonstrates moderate to high diagnostic accuracy in most tumor types. Increased m6A methylation levels were observed in the TUBA1B gene, while its promoter region displayed low methylation levels. TUBA1B's expression impacted some cancers by elevating tumor mutation burden, microsatellite instability, neoantigen formation, immune cell infiltration, and the modulation of immune checkpoints. Functional enrichment analysis highlights TUBA1B’s involvement in important cellular processes such as the cell cycle, p53 signaling, cell senescence, programmed cell death, and the regulation of immune-related pathways. Moreover, our study reveals higher TUBA1B protein expression in breast cancer tissues compared to adjacent tissues. In vitro experiments confirm that TUBA1B deletion reduces breast cancer cell proliferation, invasion, and migration while increasing apoptosis. In conclusion, our study suggests that TUBA1B could potentially serve as a diagnostic marker for predicting cancer immunological profiles and survival outcomes and shed light on the expression and role of TUBA1B in breast cancer, providing a solid foundation for considering it as a promising therapeutic target for breast cancer patient treatment.

Induction of therapeutic immunity and cancer eradication through biofunctionalized liposome-like nanovesicles derived from irradiated-cancer cells.

Immunotherapy has revolutionized the treatment of cancer. However, its efficacy remains to be optimized. There are at least two major challenges in effectively eradicating cancer cells by immunotherapy. Firstly, cancer cells evade immune cell killing by down-regulating cell surface immune sensors. Secondly, immune cell dysfunction impairs their ability to execute anti-cancer functions. Radiotherapy, one of the cornerstones of cancer treatment, has the potential to enhance the immunogenicity of cancer cells and trigger an anti-tumor immune response. Inspired by this, we fabricate biofunctionalized liposome-like nanovesicles (BLNs) by exposing irradiated-cancer cells to ethanol, of which ethanol serves as a surfactant, inducing cancer cells pyroptosis-like cell death and facilitating nanovesicles shedding from cancer cell membrane. These BLNs are meticulously designed to disrupt both of the aforementioned mechanisms. On one hand, BLNs up-regulate the expression of calreticulin, an "eat me" signal on the surface of cancer cells, thus promoting macrophage phagocytosis of cancer cells. Additionally, BLNs are able to reprogram M2-like macrophages into an anti-cancer M1-like phenotype. Using a mouse model of malignant pleural effusion (MPE), an advanced-stage and immunotherapy-resistant cancer model, we demonstrate that BLNs significantly increase T cell infiltration and exhibit an ablative effect against MPE. When combined with PD-1 inhibitor (α-PD-1), we achieve a remarkable 63.6% cure rate (7 out of 11) among mice with MPE, while also inducing immunological memory effects. This work therefore introduces a unique strategy for overcoming immunotherapy resistance.

Immunological effects of Chinese herbal therapy for allergies

Immunological effects of Chinese herbal therapy for allergies

Prenatal risk factors of indoor environment and incidence of childhood eczema in the Japan Environment and Children's Study

The quality of indoor environment is a risk factor for early childhood eczema and atopic dermatitis; however, its influence during pregnancy on childhood eczema in Japan has not been investigated. In this study, we aimed to determine the indoor environmental factors that are associated with eczema in children up to 3 years of age, using national birth cohort data from the Japan Environment and Children's Study (JECS).Information on indoor environments and eczema symptoms until 3 years of age was collected using self-administered questionnaires to the mothers. A total of 71,883 and 58,639 mother-child pairs at 1.5- and 3-years-old, respectively, were included in the former analyses. To account for prenatal indoor risk factors, 17,568 (1.5-years-old) and 7063 (3-years-old) children without indoor mold and/or ETS exposure were included in the final analysis. A higher mold index, gas heater use, parquet flooring use, and frequent insecticide use showed significantly increased risks for childhood eczema up to 3 years of age. These associations were consistent after stratification analysis among children whose parents did not have a history of allergies. The updated WHO guidelines on indoor air quality should be implemented based on recent findings regarding the effects of prenatal exposure to indoor dampness on health effects of children further in life, including asthma, respiratory effects, eczema, and other immunological effects.

Immunological effects of radiopharmaceutical therapy.

Radiation therapy (RT) is a pillar of cancer therapy used by more than half of all cancer patients. Clinically, RT is mostly delivered as external beam radiation therapy (EBRT). However, the scope of EBRT is limited in the metastatic setting, where all sites of disease need to be irradiated. Such a limitation is attributed to radiation-induced toxicities, for example on bone marrow and hematologic toxicities, resulting from a large EBRT field. Radiopharmaceutical therapy (RPT) has emerged as an alternative to EBRT for the irradiation of all sites of metastatic disease. While RPT can reduce tumor burden, it can also impact the immune system and anti-tumor immunity. Understanding these effects is crucial for predicting and managing treatment-related hematological toxicities and optimizing their integration with other therapeutic modalities, such as immunotherapies. Here, we review the immunomodulatory effects of α- and β-particle emitter-based RPT on various immune cell lines, such as CD8+ and CD4+ T cells, natural killer (NK) cells, and regulatory T (Treg) cells. We briefly discuss Auger electron-emitter (AEE)-based RPT, and finally, we highlight the combination of RPT with immune checkpoint inhibitors, which may offer potential therapeutic synergies for patients with metastatic cancers.

Induction of Immunological Antitumor Effects by the Combination of Adenovirus-Mediated Gene Transfer of B7-1 and Anti-Programmed Cell Death-1 Antibody in a Murine Squamous Cell Carcinoma Model.

The goal of this study was to evaluate the antitumor immune effects of B7-1 gene expression in addition to immune checkpoint inhibitor against squamous cell carcinoma. A murine SCC cell line, KLN205, was infected with adenoviral vector carrying B7-1 (AdB7). Infected cells were injected subcutaneously in the flanks of DBA/2 mice. Three weeks after implantation, anti-mouse PD-1 antibody (antiPD1) was intraperitonially administrated twice a week for a total of six times. CD80 was significantly overexpressed in the AdB7-infected tumors. IFN-gamma in the T cells in the spleen was significantly increased and tumor size was significantly reduced in the mice treated with both AdB7 and antiPD1. Targeted tumors treated with both AdB7 and antiPD1 exhibited significantly increased cell densities of total immune cells as well as Ki-67+ CD8+ T cells and decreased regulatory T cells. These results suggest that the B7-1 gene transfer may enhance the antitumor effect of anti-PD1 antibody against SCC.

Retinoic Acid Treatment Mitigates PM2.5-Induced Type 2 Inflammation: Insights into Modulation of Innate Immune Responses.

Some studies have demonstrated the effects of particulate matter (PM) on chronic rhinosinusitis with nasal polyps (CRSwNP) development, as well as the therapeutic role of retinoic acid (RA) in nasal polypogenesis. However, the immunologic effect of PM in innate lymphoid cells (ILCs) and the exact mechanism of the therapeutic effect of RA remain unclear. Therefore, the present study investigated the effects of fine-dust-induced inflammation in CRSwNP and the mechanisms of the therapeutic effect of RA. PM2.5 exposure exacerbated pathological damage in the nasal mucosa of mice with nasal polyps (NP) via upregulation of type 2 inflammation. Additionally, PM2.5 exposure increased the expression of type 2 cytokines and epithelial-cell-derived cytokines (IL-33 and IL-25) significantly, as well as the ILC populations in human-NP-derived epithelial cells (HNECs). Moreover, RA supplementation significantly increased the expression of ILCreg in Lin-CD45+CD127+ cells, which in turn increased the levels of the anti-inflammatory cytokine IL-10. The findings suggest that PM2.5 exposures could aggravate the CRSwNP type 2 inflammation, and RA treatment may ameliorate fine-dust-induced inflammation by modulating the innate immune response.

Adjuvant nivolumab, capecitabine or the combination in patients with residual triple-negative breast cancer: the OXEL randomized phase II study

Chemotherapy and immune checkpoint inhibitors have a role in the post-neoadjuvant setting in patients with triple-negative breast cancer (TNBC). However, the effects of nivolumab, a checkpoint inhibitor, capecitabine, or the combination in changing peripheral immunoscore (PIS) remains unclear. This open-label randomized phase II OXEL study (NCT03487666) aimed to assess the immunologic effects of nivolumab, capecitabine, or the combination in terms of the change in PIS (primary endpoint). Secondary endpoints included the presence of ctDNA, toxicity, clinical outcomes at 2-years and association of ctDNA and PIS with clinical outcomes. Forty-five women with TNBC and residual invasive disease after standard neoadjuvant chemotherapy were randomized to nivolumab, capecitabine, or the combination. Here we show that treatment with immunotherapy containing arms (nivolumab or a combination of nivolumab plus capecitabine) leads to an increase in PIS from baseline to week 6 compared with capecitabine alone, meeting the pre-specified primary endpoint. In addition, the presence of circulating tumor DNA (ctDNA) is associated with disease recurrence, with no new safety signals in the combination arm. Our results provide efficacy and safety data on this combination in TNBC and support further development of PIS and ctDNA analyses to identify patients at high risk of recurrence.

Human Cytomegalovirus Oncoprotection across Diverse Populations, Tumor Histologies, and Age Groups: The Relevance for Prospective Vaccinal Therapy.

The oncogenicity of the human cytomegalovirus (CMV) is currently being widely debated. Most recently, mounting clinical evidence suggests an anti-cancer effect via CMV-induced T cell-mediated tumor destruction. However, the data were mostly obtained from single-center studies and in vitro experiments. Broad geographic coverage is required to offer a global perspective. Our study examined the correlation between country-specific CMV seroprevalence (across 73 countries) and the age-standardized incidence rate (of 34 invasive tumors). The populations studied were stratified according to decadal age periods as the immunologic effects of CMV seropositivity may depend upon age at initial infection. The International Agency for Research on Cancer of the World Health Organization (IARC WHO) database was used. The multivariate linear regression analysis revealed a worldwide inverse correlation between CMV seroprevalence and the incidences of 62.8% tumors. Notably, this inverse link persists for all cancers combined (Spearman's ρ = -0.732, p < 0.001; β = -0.482, p < 0.001, adjusted R2 = 0.737). An antithetical and significant correlation was also observed in particular age groups for the vast majority of tumors. Our results corroborate the conclusions of previous studies and indicate that this oncopreventive phenomenon holds true on a global scale. It applies to a wide spectrum of cancer histologies, additionally supporting the idea of a common underlying mechanism-CMV-stimulated T cell tumor targeting. Although these results further advance the notion of CMV-based therapies, in-depth investigation of host-virus interactions is still warranted.

CGA mitigates HMGB1 mediated TLR4 activated hepatic cancer in urethane primed mice

BackgroundLiver cancer is fourth leading cause of cancer deaths worldwide. Urethane presents in herbicides, pesticides and food beverages as preservatives upregulate the expression of inflammatory cytokines in liver tissues for the genesis of cancer. It also naturally formed in fermented food products and distilled beverages. Study designBalb/c mice of approx same age and body weight were selected and divided into four groups G-I (PBS treated), G-II (urethane of 500 mg/kgb.w.), G-III (CGA of 50 mg/kg b.w.) and G-IV (urethane & CGA). Treatments were given on alternate day upto eight consecutive weeks and 3-12 month of latency period. Blood sample collected was used for counting of inflammatory immune cells by automated cell counter. Total RNA and protein samples isolated from dissected liver of sacrificed mice were used for molecular analysis at transcriptional and translational level. MethodsGene expression is assessed by RT-PCR at transcriptional level and by western blot at translational level. Immunological effects are evaluated by counting inflammatory immue cells and histopathology is done to examine hepatic tissues injury by carcinogen urethane. Hypothesis and purposeInflammatory cytokines create microenvironment for tumor initiation and development which is mediated through TLR-4 pathway activation via HMGB1 upon carcinogen exposure. Here aimed to find out the role of Chlorogenic acid (CGA) a phenolic compound naturally present in plants as anti-cancerous properties to prevent and cure cancer by acting upon HMGB1 & TLR4 pathway. ResultsAt transcriptional level up-regulated expression of HMGB1, TLR-4 pathway genes, pro-inflammatory cytokines IL-1β, IL-6, TNF-α and IL-18 in urethane treated mice is observed. Western blot results showed up-regulated expression of HMGB1 and NF-κB protein at translational level. CGA treated mice liver showed down-regulated expression of these inflammatory molecules. Increased number of inflammatory immune cells and presence of hepatic tissues injury in histopathological slides is observed in urethane treated mice that is mitigated by CGA. ConclusionCGA is an important anti-cancerous bioactive compound targets inflammatory cytokines and HMGB1 at molecular level to prevent, cure and mitigate liver cancer. Down regulated expression of HMGB1 and TLR-4 activation pathway in cancerous Balb/c mice liver proved its anti-cancerous properties to become a potential future drug.

Abstract 5259: Mechanistic evaluations of the immunological and cell biological effects that distinguish eribulin from other microtubule-targeted chemotherapeutics

Abstract Microtubule-targeted agents (MTAs) are some of the most widely used and effective chemotherapeutics and they continue to find utility as the payloads of antibody-drug conjugates and in combination with targeted agents to improve response, including with immune checkpoint inhibitors. While all MTAs have shared antiproliferative effects, over the last decade it has become increasingly clear that individual drugs of this class have distinct effects on microtubule-dependent signaling and trafficking that are critical for their anticancer efficacy. We and others have demonstrated that eribulin is unique from other MTAs in its ability to promote vascular remodeling, reverse epithelial-to-mesenchymal transition (EMT), and increase immune infiltration into tumors both in animal models and in the clinic. However, the mechanism linking eribulin-mediated microtubule destabilization to these phenotypes, which are associated with improved overall survival of eribulin-treated patients, is unclear. We found that acute eribulin treatment at clinically relevant concentrations promotes the production of antitumor cytokines in both immune and cancer cells and synergizes with direct STING agonists to promote enhanced immune infiltration and improved antitumor efficacy in vivo in both breast and bladder cancer models. This effect was shown to involve activation of natural killer cells and is associated with TBK1-mediated IRF3 phosphorylation, which is regulated downstream of the microtubule associated GTPase GEF-H1. Intriguingly, GEF-H1 release from destabilized microtubules has also been shown to enhance the formation of focal adhesions and promote the differentiation of mesenchymal cells to a more epithelial phenotype. We show that eribulin is indeed sufficient to promote the formation and activation of focal adhesion complexes in mesenchymal triple-negative breast cancer cells, which was not observed when cells were subjected to treatment with microtubule stabilizing chemotherapeutics. Both genetic and pharmacological inhibitors of GEF-H1 were utilized to interrogate the relationship between eribulin-mediated microtubule destabilization and TBK1/IRF3-dependent immunological activation as well as the formation of RhoA-dependent focal adhesions and reversal of EMT-associated phenotypes. Together, our data provide a mechanistic rationale for unique effects of eribulin as compared to other classes of MTAs that could inform on the use of these widely used, but mechanistically underappreciated drugs in a more personalized manner and inform on their rational combination with targeted agents, including immunotherapy. Citation Format: Zaineb Hassouneh, Nancy Wilkinson, Leila Takahashi-Ruiz, Neelam Mukherjee, April L. Risinger. Mechanistic evaluations of the immunological and cell biological effects that distinguish eribulin from other microtubule-targeted chemotherapeutics [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 5259.

Abstract 5087: ILB-3101, a novel B7H3-targeting ADC with Eribulin as payload, demonstrates strong tumor killing activities and favorable PK/TOX profile in preclinical evaluation

Abstract B7H3, a B7 family member of immune checkpoint proteins, is overexpressed in a broad spectrum of malignant tumors. It has become an attractive target for cancer therapies in recent years. ILB-3101, a potential best-in-class ADC, was constructed by conjugating a potent mitotic inhibitor Eribulin to a proprietary anti-B7H3 mAb through a proprietary linker. This ADC molecule has been developed and preclinically evaluated as both the 1st line ADC treatment with B7H3 expressed patients in Eribulin sensitive cancer types and the later line treatment for patients whose cancer are B7H3 positive but TopoI inhibitor resistant. The humanized antibody of ILB-3101 is IgG1 type and shows high affinity to both human and monkey B7H3 proteins. It also shows better internalization than benchmark antibodies such as that of DS-7300 analog. The DAR value of ILB-3101 is 3.5. ILB-3101 has potent and dose-dependent cytotoxicity to B7H3-positive tumor cells and shows better activities than DS-7300 analog as positive control. In vivo studies based on xenograft tumor models reveal that ILB-3101 has potent anti-tumor activities in various tumor types including TNBC, sarcoma, ovarian cancer and AML etc., which are superior or comparable to DS-7300 analog. Furthermore, ILB-3101 shows strong anti-tumor activity on Dxd based ADC resistant tumors in xenograft tumor models. The PK study of ILB-3101 in cynomolgus monkeys shows dose-dependent exposure and desirable PK profile. ILB-3101 was well tolerated in cynomolgus monkeys with HNSTD at 6 mg/kg. No serious adverse events were observed, and the main safety findings were limited and reversible hematological and immunological effects, which are consistent with the safety profile of Eribulin as an approved chemotherapy. The in vitro and in vivo preclinical results above demonstrate that ILB-3101 has good efficacy and safety profile and it is a promising and potentially best-in-class B7H3 targeting ADC drug candidate. ILB-3101 is worthy of further translational research and clinical trials. Its IND application has been submitted in China, followed by US FDA filing soon. Citation Format: Yushuan Shang, Junzhuan Qiu, Yanfei Xin, Min Zhao, Qiang Liu, Zhengsheng Wang, Xiaojing Gong, Yan Xu, Yan Li, Mingde Xia, Rulei Chen. ILB-3101, a novel B7H3-targeting ADC with Eribulin as payload, demonstrates strong tumor killing activities and favorable PK/TOX profile in preclinical evaluation [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 5087.

Abstract 6397: Immune profiling for improving responsiveness to ipilimumab plus nivolumab checkpoint blockade treatment in patients with metastatic melanoma

Abstract Background:aCTLA-4 and aPD-1 combination therapy has significantly improved clinical outcomes in patients with metastatic melanoma, with 50%-60% of patients responding to treatment, but predictors of response and treatment induced immunological effects are poorly characterized. Identifying circulating immune populations as biomarkers could reveal crucial insights into the mechanisms driving treatment success. Methods:Using unbiased single-cell profiling (with cytometry by Time-Of-Flight, CyTOF), we characterized the systemic immune compartments of over 250 peripheral blood mononuclear samples from 89 patients with advanced melanoma before and during combination therapy. We evaluated the association between circulating immune populations and clinical outcome using nonparametric Mann-Whitney tests, and investigated the prognostic values of these features using survival analysis. Additionally, we assessed treatment-induced immunological effects, providing insights into the dynamic changes within the immune system during therapy. Results:We characterized circulating T cells, NK cells, B cells, and monocytes from peripheral blood. Following combination therapy, there was a shift from naïve and central memory T cells to effector populations in both CD4+ and CD8+ T cells compartments. There was also a broad activation observed in both adaptive and innate populations, characterized by an elevation of multiple co-stimulatory and co-inhibitory molecules. Lower CD8+ NK cell populations prior to combination therapy were predictive of durable clinical benefit (DCB). Pre-treatment measurements of Tregs were not predictive of outcome, but higher Tregs one month after starting therapy, as well as increases of Tregs from pre to post treatment, predicted better progression free survival (PFS) and overall survival (OS). Starting from early on treatment, higher “exhausted-like” CD8+ T cells, characterized by positive expression of at least two co-inhibitory markers, is associated with resistance. Conclusions:aCTLA-4 and aPD-1 combination therapy led to a longitudinal shift from naïve and central memory to effector phenotypes in T cells, along with a broad activation in both adaptive and innate populations. Within innate immune compartment, favorable clinical outcome was associated with lower CD8+ NK cells, capable of regulating proliferation and activation of CD4+ T cells. Interestingly, one month after the start of the therapy, the increase in Tregs and a decrease in 'exhausted-like' CD8+ T populations are associated with a better response. Collectively, these analyses suggest potential treatment-specific correlates of efficacy and may enable biomarker-selected patient populations in future. Citation Format: Joanna Baginska, Jiajia Chen, Giuseppe Tarantino, Anita Giobbie-Hurder, Jason L. Weirather, Mariano Severgnini, Michael Manos, Kelly Burke, Janice D. Russell, Martha Brainard, Elizabeth Gabriel, Ryan Brennick, Matthew Nazzaro, Emma Hathaway, Marta Holovatska, Claire Manuszak, Srinika Ranashinge, David Liu, F. Stephen Hodi. Immune profiling for improving responsiveness to ipilimumab plus nivolumab checkpoint blockade treatment in patients with metastatic melanoma [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 6397.

Abstract 2918: Potential for a novel therapeutic target: Effects of lactate &amp; lactylation in expression of key regulatory cancer proteins

Abstract The purpose of our study is to investigate the immunologic, metastatic, and gene regulation effects of lactate in the context of cancer in normoxic and hypoxic conditions to identify novel biomarkers and potential therapeutic targets. We aim to modify the in-vitro cancer microenvironment to show how lactate and lactylation can be exploited using existing cancer treatments. While hyperlactatemia is defined as lactate levels between 2 mmol/L and 4 mmol/L (Foucher et al 2023), tumor biopsies have demonstrated extracellular concentrations as high as 40 mM (Pérez-Tomás et al 2020). Through CTG viability assays we identified lactate concentrations that PANC1 cells can survive, which are in agreement with the in-vivo findings of Brizel et al (2001). We observed pancreatic cancer cells achieving over 50% viability for 5 days at 50 mM lactate concentrations while at 30 mM on day 5 they proliferated past their initial plating numbers. Furthermore, Brizel et al (2001) found that patients with high tumor lactate concentrations had a significantly higher incidence of metastatic relapse. Through our scratch assay, we show qualitative evidence of increased mobility and inferred migration potential of PANC1 at 7.5mM [lactate] compared to the absence of lactate. We have also observed increased migration at higher lactate concentrations for which we are pursuing quantitative measurements to evaluate significance. Migration experiments will be followed by the investigation of gene regulation (eg. mRNA seq, lactylation profile) involved in the observed increased mobility. Lactate has been shown to affect immune regulation, namely suppression of iNKT (Fu et al, 2020), macrophages (Yang et al, 2020), dendritic cell differentiation and antigen recognition/presentation (Wang et al, 2022), however not much is known about its effect on NK cells and their cytokine expression. Through NK92MI/PANC1 cocultures we show the effects of lactate concentration on the killing ability of NK cells (in the works). And plan on investigating the cytokine profile leading to the phenotypic killing differences. Lastly, via lactate-titrated incubation of PANC1 cells in normoxia we have demonstrated a positive correlation of HIF1a expression, pan-lactyllysine lactylation, and microenvironment lactate concentration. We speculate HIF1a inhibition post-lactate-sensitization may induce cancer killing and we will investigate this theory in normoxia and hypoxia using existing treatment options. Citation Format: Christos P. Costeas, Connor Purcell, Wafik S. El-Deiry. Potential for a novel therapeutic target: Effects of lactate &amp; lactylation in expression of key regulatory cancer proteins [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 2918.

Abstract 3864: Role of NK/TAM cross-talk on T-CD8+ recruitment and efficacy of chemoimmunotherapy and MEKi combination in "cold" tumors

Abstract In patients with non-small cell lung cancer (NSCLC), double platinum-based chemotherapy (cisplatin or carboplatin and pemetrexed) combined with the anti-PD-1 antibody has shown significant clinical benefit and has become the standard of care. In the context of so-called "cold" tumors, the immunogenic capacities of chemotherapies increase danger signals (type I IFNs, CXCL9/10) and favor both infiltration of NK/T-CD8+ cells and response to anti-PD-1. Despite the therapeutic progress represented by this new combination, some patients have limited benefit from chemo-immunotherapy, suggesting that in these patients, the immunogenic capacities of chemotherapy are limited. Recently, we were able to show that KRAS oncogene signaling restricts the ability of the cisplatin/pemetrexed doublet to induce the chemokine CXCL10, lymphocyte recruitment and sensitize to PD-L1 blockade. Thus, the addition of a MEK inhibitor (MEKi) to the chemo-immunotherapy combination restores an immunogenic signal through CXCL10, enabling T-CD8+ to infiltrate the tumor more extensively, making it more sensitive to immunotherapy. Here, we unravel the essential role of NK cells in the immunological and antitumoral effect of chemo-immunotherapy plus MEKi combination in the LLC1 preclinical lung cancer model. We showed that chemo/MEKi combination favors NK cell recruitment and activation in tumor bed through both CXCL10 and NKG2D-ligand (Rae1, Mult1). Then, IFNγ-producing NK cells trigger tumor associated macrophage switch from M2 toward M1 phenotype and amplify T-CD8+ cells recruitment through CXCL9 production by macrophages. Thus, NK cell depletion, CXCR3, CXCL9 or NKG2D-neutralization restrained chemo/MEKi therapeutic efficacy. However, IFNγ also triggers NKG2A-ligand expression (Qa-1b, HLA-E in human) which restrained NK cell activation and CD8+ T cell recruitment. Thus reducing the NKG2A pathway, by using Qa-1b-/- LLC1 cells, enhances chemo-immunotherapy/MEKi anti-tumor efficacy. Similar results were obtained in preclinical “cold” CT26 colon cancer model treated by chemo-immunotherapy. Our results underline a new role for NK cell activation to trigger TAM2/TAM1 switch, CXCL9 expression and amplify CD8 T cell recruitment in “cold” tumor. In a context of resistance to chemo-immunotherapy, MEKi or ICI targeting NK cells could be an interesting way to amplify the anti-tumor immune response by modulating both TAMs biology and the recruitment of CD8+ T cells. Citation Format: Lisa Nuttin, Charlène Latour, Solène Revy, Romain Aucagne, Guilhem Lalle, François Ghiringhelli, Emeric Limagne. Role of NK/TAM cross-talk on T-CD8+ recruitment and efficacy of chemoimmunotherapy and MEKi combination in "cold" tumors [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 3864.

Metal‐based smart nanosystems in cancer immunotherapy

AbstractMetals are an emerging topic in cancer immunotherapy that have shown great potential in modulating cancer immunity cycle and promoting antitumor immunity by activating the intrinsic immunostimulatory mechanisms which have been identified in recent years. The main challenge of metal‐assisted immunotherapy lies in the fact that the free metals as ion forms are easily cleared during circulation, and even cause systemic metal toxicity due to the off‐target effects. With the rapid development of nanomedicine, metal‐based smart nanosystems (MSNs) with unique controllable structure become one of the most promising delivery carriers to solve the issue, owing to their various endogenous/external stimuli‐responsiveness to release free metal ions for metalloimmunotherapy. In this review, the state‐of‐the‐art research progress in metal‐related immunotherapy is comprehensively summarized. First, the mainstream mechanisms of MSNs‐assisted immunotherapy will be delineated. The immunological effects of certain metals and categorization of MSNs with different characters and compositions are then provided, followed by the representative exemplar applications of MSNs in cancer treatment, and synergistic combination immunotherapy. Finally, we conclude this review with a summary of the remaining challenges associated with MSNs and provide the authors' perspective on their further advances.

Abstract 4076: Interleukin-18 engineered for resistance to IL-18 binding protein (IL-18BP) and half-life extension to enhance its therapeutic potential

Abstract Interleukin-18 (IL-18) possesses a unique combination of innate and acquired immune functions with high potential to be transformative in cancer immunotherapy of solid tumors. In clinical studies, recombinant wild-type IL-18 was limited by rapid upregulation of IL-18 binding protein (IL-18BP), which serves as a negative feedback checkpoint of the IL-18 pathway. As its name implies, IL-18BP tightly binds to IL-18, thereby blocking its ability to activate the IL-18 receptor. We have focused our protein engineering efforts to obtain variants of IL-18 that are resistant to IL-18BP suppression while retaining IL-18 biological activity. To further capitalize on the immunological effects of IL-18, the IL-18BP resistant variants were fused to half-life extension protein scaffolds for enhanced in vivo exposure. Rational protein design and combinatorial approaches were used to generate a pool of IL-18 variants that were screened for potency and resistance to IL-18BP. Several IL-18 variants showed undetectable binding to IL-18BP (up to 1 mM IL-18BP) and displayed a spectrum of potencies relative to recombinant wild-type IL-18. Importantly, these variants retained their full biological activity in the presence of super-physiological levels of IL-18BP, as tested with in vitro cell-based IFN-γ release assays. In vivo pharmacokinetic studies in mice compared several IL-18BP resistant variants fused to a half-life extension scaffold with a non-half-life enhanced IL-18BP resistant variant. The half-lives for the fusion proteins ranged from 15 - 30h, while the non-fused variant showed a half-live of 0.6h. Furthermore, the IL-18 variants displayed a more durable, Th1 immune response compared to a non-half-life enhanced variant. Thus, IL-18 variants with strong resistance to IL-18BP that are fused to a half-life extension protein scaffold displayed enhanced pharmacokinetic and pharmacodynamic properties in preclinical mouse models. These protein engineering modifications will enable us to select an appropriate development candidate with enhanced pharmaceutical properties to achieve the full therapeutic potential of IL-18 to assess in patients with cancer. Citation Format: Jean Chamoun, Clifford DiLea, Kristiana Dreaden, Pinar Gurel, Joshua Heiber, Robert G. Newman, Su-Ping Pearson, Chunhua Wang, Yanchun Zhao, Mark Whitmore. Interleukin-18 engineered for resistance to IL-18 binding protein (IL-18BP) and half-life extension to enhance its therapeutic potential [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 4076.

Abstract 6648: Low-intensity pulsed ultrasound enhances the presentation of brain-derived antigens by facilitating the migration of antigen-presenting cells from the brain to the periphery

Abstract Gliomas, the most common primary brain tumors, remain largely incurable despite advancements in surgical and adjuvant therapies. While immunotherapies offer promising avenues, their efficacy has been limited by formidable anatomical barriers, such as the blood-brain barrier (BBB), which restricts the migration of immune cells into the brain and impedes the efficient drainage of brain-derived antigens, establishing an immune-privileged environment. Consequently, neoplastic formations within the brain often escape detection by the peripheral immune system, complicating therapeutic interventions. The application of low-intensity pulsed ultrasound with microbubbles (LIPU/MB) has emerged as a potential solution, enabling the transient opening of the BBB. This facilitates drug transport and immune cell trafficking, including Chimeric Antigen Receptor (CAR) T cells, into brain tumors. However, challenges such as antigen-loss escape persist with CAR T cells. One proposed solution involves promoting antigen/epitope spreading by inducing the presentation of brain (tumor) antigens to the systemic immune system. To investigate this, we conducted intravital imaging using Colony-stimulating factor receptor 1 (CSFR1) and CX3CR1 reporter mice, visualizing antigen-presenting cell locomotion in the brain before and after LIPU/MB treatment using two-photon microscopy. Within one hour after LIPU/MB, we observed the migration of CSFR1+ and CX3CR1+ immune cells from the brain parenchyma and perivascular space into the bloodstream, a phenomenon absent prior to LIPU/MB treatment. Beyond that, flow cytometry identified CD45intermediateCD11b+-CX3CR1+-TMEM119+-P2RY12+microglia-like' cells in the blood. To further assess the immunological effects of LIPU/MB on brain antigen presentation, we utilized transgenic mice expressing a minigene encoding four immunogenic peptides under the glial fibrillary acidic protein (GFAP) promoter. LIPU/MB treatment was associated with increased proliferation and priming of adoptively transferred CNS antigen-specific naïve T cells in cervical lymph nodes, ultimately homing into the CNS, indicating enhanced antigen presentation. Furthermore, in a subset of glioblastoma (GBM) patients undergoing LIPU/MB combined with doxorubicin and anti-PD-1 blocking antibodies, CD11b+CD14+P2YR12+ microglia were detected in blood samples during cycles 3-6 of treatment. This increase was absent in blood samples from healthy donors.Collectively, our findings suggest that LIPU/MB represents a novel approach to augment brain-antigen presentation in the periphery, holding potential implications for improving the immunotherapeutic management of gliomas.[M.G., V.A.A., A.M.S., and H.O. contributed equally to this work.] Citation Format: Marco Gallus, Victor Andres Arrieta, Atsuro Saijo, Pavlina Chuntova, Akane Yamamichi, Jeffrey Haegelin, Su Phyu, Lan Phung, Shayan A. Amir-Kabirian, Senthilnath Lakshmanachetty, Li Chen, Karl Habashy, Andrew Gould, Christina Amidei, Catalina Lee Chang, Roger Stupp, Michael Canney, Hideho Okada. Low-intensity pulsed ultrasound enhances the presentation of brain-derived antigens by facilitating the migration of antigen-presenting cells from the brain to the periphery [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 6648.