Potential Immunotherapeutic Agent Research Articles

Design and Synthesis of Cyclic Dinucleotide Analogues Containing Triazolyl C-Nucleosides.

Natural cyclic dinucleotide (CDN) is the secondary messenger involved in bacterial hemostasis, human innate immunity, and bacterial antiphage immunity. Synthetic CDN and its analogues are key molecular probes and potential immunotherapeutic agents. Several CDN analogues are under clinical research for antitumor immunotherapy. A myriad of synthetic methods have been developed and reported for the preparation of CDN and its analogues. However, most of the protocols require multiple steps, and only one CDN or its analogue is prepared at a time. In this study, a strategy based on a macrocyclic ribose phosphate skeleton containing a 1'-alkynyl group was designed and developed to prepare CDN analogues containing triazolyl C-nucleosides by click chemistry. Combinatorial application of click chemistry and the sulfenylation cascade to the macrocyclic skeleton expanded the diversity of the CDN analogues. This macrocyclic skeleton strategy rapidly and efficiently provides CDN analogues to facilitate research on microbiology, immunology, and immunotherapy.

The deubiquitinase USP2a promotes tumor immunosuppression by stabilizing immune checkpoint B7–H4 in lung adenocarcinoma harboring EGFR-activating mutants

B7–H4 is an immune checkpoint crucial for inhibiting CD8+ T-cell activity. A clinical trial is underway to investigate B7–H4 as a potential immunotherapeutic agent. However, the regulatory mechanism of B7–H4 degradation via the ubiquitin-proteasome pathway (UPP) remains poorly understood. In this study, we discovered that proteasome inhibitors effectively increased B7–H4 expression, while EGFR-activating mutants promoted B7–H4 expression through the UPP. We screened B7–H4 binding proteins by co-immunoprecipitation and mass spectrometry and found that USP2a acted as a deubiquitinase of B7–H4 by removing K48- and K63-linked ubiquitin chains from B7–H4, leading to a reduction in B7–H4 degradation. EGFR mutants enhanced B7–H4 stability by upregulating USP2a expression. We further investigated the role of USP2a in tumor growth in vivo. Depletion of USP2a in L858R/LLC cells inhibited tumor cell proliferation, consequently suppressing tumor growth in immune-deficient nude mice by destabilizing downstream molecules such as Cyclin D1. In an immune-competent C57BL/6 mouse tumor model, USP2a abrogation facilitated infiltration of CD95+CD8+ effector T cells and hindered infiltration of Tim-3+CD8+ and LAG-3+CD8+ exhausted T cells by destabilizing B7–H4. Clinical lung adenocarcinoma samples showed a significant correlation between B7–H4 abundance and USP2a expression, indicating the contribution of the EGFR/USP2a/B7–H4 axis to tumor immunosuppression. In summary, this study elucidates the dual effects of USP2a in tumor growth by stabilizing Cyclin D1, promoting tumor cell proliferation, and stabilizing B7–H4, contributing to tumor immunosuppression. Therefore, USP2a represents a potential target for tumor therapy.

Immunotherapy in HIV-associated Kaposi Sarcoma: Challenges and Prospects

Abstract: Kaposi Sarcoma [KS] stems from malignant endothelial cells targeting the cutaneous and lymphatic systems. The aetiological agent, human herpesvirus type 8, has been implicated in the induction of KS. Of the four variants of KS that exist, HIV/AIDS associated KS remains one of the leading cancers in people living with HIV in sub-Saharan Africa [SSA]. It is estimated that approximately 80% of KS cases were attributed to HIV in 2020. Although the introduction of anti-retroviral therapy [ART] alleviated the burden in 1981, its prevalence on the continent remains significant in comparison to the rest of the world. Traditional therapeutics such as chemotherapy continue to be the most common form of managing HIV-associated KS; however, the incidence of this global cancer continues to rise, predominantly in SSA. Furthermore, a significant number of HIV/AIDS-associated KS patients had been observed with normal CD4+ count and low viral load levels. This necessitates the development of other therapeutic strategies to collectively manage the continental crisis. Various strategies, such as immunomodulatory agents, monoclonal antibodies and therapeutic cytokines, are being investigated to be used as potential therapeutic strategies. One strategy highlights targeting the signalling pathways and growth factors involved in angiogenesis, which is an important characteristic of KS. The PD-1/PD-L1 immune checkpoint blockades are particularly interesting since cell cycle inhibitors have shown promising results as a potential immunotherapeutic agent. Predictive biomarkers and alternative vaccines will be discussed here while potential barriers which reduce the impact of immunotherapy are discussed throughout the review.

Sokotrasterol Sulfate Suppresses IFN-γ-Induced PD-L1 Expression by Inhibiting JAK Activity.

PD-1/PD-L1 monoclonal antibodies exhibit promising therapeutic effectiveness in multiple cancers. However, developing a simple and efficient non-antibody treatment strategy using the PD-1/PD-L1 signaling pathway still remains challenging. In this study, we developed a flow cytometry assay to screen bioactive compounds with PD-L1 inhibitory activity. A total of 409 marine natural products were screened, and sokotrasterol sulfate (SKS) was found to efficiently suppress the IFN-γ-induced PD-L1 expression. SKS sensitizes the tumor cells to antigen-specific T-cell killing in the T cell-tumor cell coculture system. Mechanistically, SKS directly targeted Janus kinase (JAK) to inhibit the downstream activation of signal transducer and activator of transcription (STAT) and the subsequent transcription of PDL1. Our findings highlight the immunological role of SKS that may act as a basis for a potential immunotherapeutic agent.

The Chemokine CXCL14 as a Potential Immunotherapeutic Agent for Cancer Therapy.

There is great enthusiasm toward the development of novel immunotherapies for the treatment of cancer, and given their roles in immune system regulation, chemokines stand out as promising candidates for use in new cancer therapies. Many previous studies have shown how chemokine signaling pathways could be targeted to halt cancer progression. We and others have revealed that the chemokine CXCL14 promotes antitumor immune responses, suggesting that CXCL14 may be effective for cancer immunotherapy. However, it is still unknown what mechanism governs CXCL14-mediated antitumor activity, how to deliver CXCL14, what dose to apply, and what combinations with existing therapy may boost antitumor immune responses in cancer patients. Here, we provide updates on the role of CXCL14 in cancer progression and discuss the potential development and application of CXCL14 as an immunotherapeutic agent.

Peptide-ligand conjugate based immunotherapeutic approach for targeted dismissal of non-structural protein 1 of dengue virus: A novel therapeutic solution for mild and severe dengue infections

Dengue virus infection has significantly increased, with reported cases soaring from 505,430 in 2000 to 2,809,818 in 2022, emphasizing the need for effective treatments. Among the eleven structural and non-structural proteins of DENV, Non-structural protein 1 (NS1) has emerged as a promising target due to its diverse role in modulating the immune response, inducing vascular leakage, and facilitating viral replication and assembly. Monoclonal antibodies are the sole therapeutics to target NS1, but concerns about their cross-reactivity persist. Given these concerns, our study focuses on designing a novel Peptide Ligand Conjugate (PLC) as a potential alternative immunotherapeutic agent against NS1. This PLC aims to mediate the immune elimination of soluble NS1 and NS1-presenting DENV-infected host cells by pre-existing vaccine-induced immunity. By employing the High Throughput Virtual Screening (HTVS) method, QikProp analysis, and Molecular Dynamics studies, we identified three hits from Asinex Biodesigned Ligands out of 220,177 compounds that show strong binding affinity towards the monoclonal binding site of NS1 protein. After a rigorous analysis of physicochemical characteristics, antigenicity, allergenicity, and toxicity using various servers, we selected two peptides: the minimum epitopic region of the Diphtheria and Tetanus toxins as the peptide components of the PLCs. A non-cleavable, non-reactive oxime linker connected the ligand with the peptide through oxime and amide bonds. DPT vaccine is widely used in dengue-endemic countries, and it has been reported that antibodies titer against MER of Diphtheria toxin and Tetanus toxins persist lifelong in DPT-vaccinated people. Therefore, once the rationally designed PLCs bind to NS1 through the ligands, the peptide will induce an immune response against NS1 by triggering pre-existing DPT antibodies and activating memory cells. This orchestrated immune response will destroy soluble NS1 and NS1-expressing DENV-infected cells, thereby reducing the illness of severe dengue hemorrhagic fever and the DENV infection, respectively. Given the increasing demand for new therapeutics for DENV treatment, further investigation into this novel immune-therapeutic strategy may offer a new avenue for treating mild and severe dengue infections.

NK3.3-Derived Extracellular Vesicles Penetrate and Selectively Kill Treatment-Resistant Tumor Cells.

Cancer treatments often become ineffective due to the development of tumor resistance, leading to metastasis and relapse. Treatments may also fail because of their inability to access cells deep within the tumor tissue. When this occurs, new therapeutic agents are needed. We previously reported that NK3.3EVs, extracellular vesicles (EVs) derived from the normal human natural killer (NK) cell line, NK3.3, have strong cytotoxic activity against leukemia and breast cancer cell lines, without harming normal cells. Here, we used a three-dimensional (3D) MCF7 breast cancer mammosphere model to reproduce a more physiological environment that NK3.3EVs would encounter in vivo. NK3.3EVs penetrated MCF7 mammospheres, inducing death by apoptosis. We generated an imatinib-resistant K562 chronic myeloid leukemia (CML) cell line to investigate whether NK3.3EVs were able to kill tumor cells resistant to front-line chemotherapy. NK3.3EVs were even more cytotoxic to imatinib-resistant cells than parental cells, inducing apoptosis via caspase-3/-7 activation. The small population of cancer stem cells (CSCs) within tumors also contributes to therapeutic resistance. NK3.3EVs reduced the CSC-like CD34+/CD38- subpopulation in imatinib-resistant and parental K562 cultures and decreased CSC-associated expression of tumor-promoting genes. Our results provide strong evidence that NK3.3EVs may be a potential new immunotherapeutic agent for difficult-to-treat cancers.

The potential of swine pseudorabies virus attenuated vaccine for oncolytic therapy against malignant tumors

BackgroundOncolytic viruses are now well recognized as potential immunotherapeutic agents against cancer. However, the first FDA-approved oncolytic herpes simplex virus 1 (HSV-1), T-VEC, showed limited benefits in some patients in clinical trials. Thus, the identification of novel oncolytic viruses that can strengthen oncolytic virus therapy is warranted. Here, we identified a live-attenuated swine pseudorabies virus (PRV-LAV) as a promising oncolytic agent with broad-spectrum antitumor activity in vitro and in vivo.MethodsPRV cytotoxicity against tumor cells and normal cells was tested in vitro using a CCK8 cell viability assay. A cell kinase inhibitor library was used to screen for key targets that affect the proliferation of PRV-LAV. The potential therapeutic efficacy of PRV-LAV was tested against syngeneic tumors in immunocompetent mice, and against subcutaneous xenografts of human cancer cell lines in nude mice. Cytometry by time of flight (CyTOF) and flow cytometry were used to uncover the immunological mechanism of PRV-LAV treatment in regulating the tumor immune microenvironment.ResultsThrough various tumor-specific analyses, we show that PRV-LAV infects cancer cells via the NRP1/EGFR signaling pathway, which is commonly overexpressed in cancer. Further, we show that PRV-LAV kills cancer cells by inducing endoplasmic reticulum (ER) stress. Moreover, PRV-LAV is responsible for reprogramming the tumor microenvironment from immunologically naïve (“cold”) to inflamed (“hot”), thereby increasing immune cell infiltration and restoring CD8+ T cell function against cancer. When delivered in combination with immune checkpoint inhibitors (ICIs), the anti-tumor response is augmented, suggestive of synergistic activity.ConclusionsPRV-LAV can infect cancer cells via NRP1/EGFR signaling and induce cancer cells apoptosis via ER stress. PRV-LAV treatment also restores CD8+ T cell function against cancer. The combination of PRV-LAV and immune checkpoint inhibitors has a significant synergistic effect. Overall, these findings point to PRV-LAV as a serious potential candidate for the treatment of NRP1/EGFR pathway-associated tumors.

Programmed Cell Death Protein 1 (PD-1) and Programmed Cell Death Ligand 1 (PD-L1) Immunotherapy: A Promising Breakthrough in Cancer Therapeutics.

The advent of immune checkpoint inhibitors has revolutionized cancer therapy by leveraging the body's immune system to combat malignancies effectively. Among these groundbreaking agents, programmed cell death protein 1 (PD-1) and programmed cell death ligand 1 (PD-L1) inhibitors have emerged as pivotal therapeutic approaches. PD-L1, a key proteinexpressed on the surface of various cells, including cancer cells, plays a central role in immune regulation by interacting with the programmed cell death protein 1 (PD-1) receptor on T-cellsleading to immune suppression. The substantial increase in PD-L1 expression on cancer cell surfaces has driven the exploration of PD-1/PD-L1 inhibitors as potential immunotherapeutic agents. These inhibitors are monoclonal antibodies designed to impede the PD-L1 and PD-1 interaction and disrupt the immunosuppressive signal, thereby reinvigorating the anti-tumor immune response mediated by activated T-cells. Clinical trials investigating PD-1/PD-L1 inhibitors have demonstrated remarkable efficacy in the treatment of diverse advanced or metastatic cancers, including leukemia, non-small cell lung (NSCLC), hepatocellular, melanoma, gastric, colorectal, and breast cancers, among others. Regulatory approvals have been granted for both monotherapy and combination therapy with other cancer treatments, encompassing chemotherapy and additional immune checkpoint inhibitors. While PD-1/PD-L1 inhibitors have exhibited significant success, they are not devoid of challenges. The emergence of intrinsic or acquired resistance, as well as immune-related adverse events, warrants thorough investigation and management. Consequently, researchers have embarked on combination trials to augment the therapeutic potential of PD-1/PD-L1 inhibitors and surmount resistance mechanisms.

Anti-Tumor Activity of Novel Nimotuzumab-Functionalized Gold Nanoparticles as a Potential Immunotherapeutic Agent against Skin and Lung Cancers.

The epidermal growth factor receptor (EGFR) is vital for many different types of cancer. Nimotuzumab (NmAb), an anti-EGFR monoclonal antibody (mAb), is used against some of EGFR-overexpressed cancers in various countries. It targets malignant cells and is internalized via receptor-mediated endocytosis. We hypothesized that mAb-nanoparticle conjugation would provide an enhanced therapeutic efficacy, and hence we conjugated NmAb with 27 nm spherical gold nanoparticles (AuNPs) to form AuNP-NmAb nanoconjugates. Using biophysical and spectroscopic methods, including ultraviolet-visible spectroscopy (UV-Vis), transmission electron microscopy (TEM), dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and Fourier-transform infrared spectroscopy (FTIR), the AuNP-NmAb complex was characterized. Furthermore, in vitro studies were performed using a medium-level EGFR-expressing skin cancer cell (A431, EGFRmedium) and low-level EGFR-expressing lung cancer cell (A549, EGFRlow) to evaluate anti-tumor and cellular uptake efficiency via MTT assay and single-particle inductively coupled plasma mass spectrometry (spICP-MS), respectively. In comparison to NmAb monotherapy, the AuNP-NmAb treatment drastically reduced cancer cell survivability: for A431 cells, the IC50 value of AuNP-NmAb conjugate was 142.7 µg/mL, while the IC50 value of free NmAb was 561.3 µg/mL. For A549 cells, the IC50 value of the AuNP-NmAb conjugate was 163.6 µg/mL, while the IC50 value of free NmAb was 1,082.0 µg/mL. Therefore, this study highlights the unique therapeutic potential of AuNP-NmAb in EGFR+ cancers and shows the potential to develop other mAb nanoparticle complexes for a superior therapeutic efficacy.

Abstract 5175: DX-002, a dual EP2 and EP4 antagonist, alters the tumor microenvironment to enhance tumor immunogenicity and responses to immune checkpoint inhibitors

Abstract Prostaglandin E2 (PGE2) generated by the cyclooxygenase (COX) pathways regulates both inflammation and tumor microenvironment (TME). Among the four E-type prostanoid receptors EP1-4, it has been demonstrated that PGE2 exhibits its immune suppression and tumorigenic function through engagement with EP2 and EP4. Mechanistically, tumor-derived PGE2 acts on EP2 and EP4 in immune cells (e.g., NK cells and dendritic cells) and elicits immunosuppression in TME1,2. Importantly, the deletion of both EP2 and EP4, but not deletion of either receptor, is required to reverse PGE2-mediated immune evasion. Furthermore, high levels of PGE2-EP2/EP4 associated inflammatory signature have been shown in multiple human cancer types to correlate with poor prognosis and worse response to immune checkpoint blockade in the clinic1. Therefore, PGE2-EP2/EP4 signaling likely acts as a key regulatory node linking inflammation and immunosuppression in TME, and its inhibition may lead to a less immunosuppressive TME and enhance the response to immune checkpoint inhibitors. COX inhibitors have been used to block PGE2 synthesis but cause cardiovascular and gastrointestinal risks due to the inhibition of all prostanoids synthesis3. Thus, therapeutically targeting both EP2 and EP4 is an effective and more selective approach to limit the immunosuppressive functions of PGE2 while avoiding potential side effects associated with COX inhibition. Here we describe the discovery of a potent and selective EP2/EP4 dual antagonist DX-002. At 10 nM concentration, it displayed >90% inhibition of PGE2 binding to EP2 or EP4, whereas no significant inhibition was seen towards PGE2 binding to EP1 or EP3. In human NK cells DX-002 reversed PGE2-mediated inhibition of IFN-γ production more potently than selective EP2 or EP4 antagonist. DX-002 has excellent pharmacokinetics profiles across all animal species. In the mouse tumor model, treatment of DX-002 produced superior tumor regression as a monotherapy over selective EP2 or EP4 antagonists. In addition, DX-002 also provided significant synergistical effect for tumor regression in combination with anti-PD1 in both immune checkpoint inhibitor insensitive and sensitive models, in association with increased tumor infiltration of CD8+ T cells. Based on its in vitro and in vivo profiles along with other properties, DX-002 is being profiled as a development candidate as a potential cancer immunotherapeutic agent. Citation Format: Lintong Li, Guorui Yao, Wei Zheng, Yongshuai Chai, Zhongying Huang, Guojian Ma, Linlin Nie, Dengfeng Xu, Jiuquan Yang, Nanxin Li. DX-002, a dual EP2 and EP4 antagonist, alters the tumor microenvironment to enhance tumor immunogenicity and responses to immune checkpoint inhibitors. [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 5175.

Bioinformatics Analysis of Molecular Interactions between Endoplasmic Reticulum Stress and Ferroptosis under Stress Exposure.

Stress has become a universal biological phenomenon in the body, which leads to pathophysiological changes. However, the molecular network interactions between endoplasmic reticulum (ER) stress and ferroptosis under stressful conditions are not clear. For this purpose, we screened the gene expression profile of GSE173795 for intersection with ferroptosis genes and screened 68 differentially expressed genes (DEGs) (63 up-regulated, 5 down-regulated), mainly related to lipid and atherosclerosis, autophagy-animal, mitophagy-animal, focal adhesion, DNA replication, proteasome, oocyte meiosis, toll-like receptor signaling pathway, cell cycle, etc. Immune infiltration analysis revealed that stress resulted in decreased B cells memory, T cells CD8 and T cells CD4 memory resting, monocytes, macrophages M2, and increased B cells naive, T cells follicular helper, and macrophages M1. 19 core-DEGs (ASNS, TRIB3, ATF4, EIF2S1, CEBPG, RELA, HSPA5, DDIT3, STAT3, MAP3K5, HIF1A, HNF4A, MAPK14, HMOX1, CDKN1A, KRAS, SP1, SIRT1, EGFR) were screened, all of which were up-regulated DEGs. These biological processes and pathways were mainly involved in responding to ER stress, lipid and atherosclerosis, cellular response to stress, cellular response to chemical stress, and regulation of DNA-templated transcription in response to stress, etc. Spearman analysis did not find MAPK14 to be significantly associated with immune cells. Other core-DEGs were associated with immune cells, including B cells naive, T cells follicular helper, and monocytes. Based on core-DEGs, 283 miRNAs were predicted. Among the 22 miRNAs with highly cross-linked DEGs, 11 had upstream lncRNA, mainly targeting STAT3, SP1, CDKN1A, and SIRT1, and a total of 39 lncRNA were obtained. 85 potential drugs targeting 11 core-DEGs were identified and were expected to be potential immunotherapeutic agents for stress injury. Our experiments also confirmed that Liproxstatin-1 alleviates common cross-linked proteins between ER stress and ferroptosis. In conclusion, our study explored the molecular mechanisms and network interactions among stress-ER stress-ferroptosis from a novel perspective, which provides new research ideas for studying stressful injury.

Exosomal microRNAs in cancer: Potential biomarkers and immunotherapeutic targets for immune checkpoint molecules.

Exosomes are small extracellular vesicles with a lipid bilayer structure secreted from different cell types which can be found in various body fluids including blood, pleural fluid, saliva and urine. They carry different biomolecules including proteins, metabolites, and amino acids such as microRNAs which are small non-coding RNAs that regulate gene expression and promote cell-to-cell communication. One main function of the exosomal miRNAs (exomiRs) is their role in cancer pathogenesis. Alternation in exomiRs expression could indicate disease progression and can regulate cancer growth and facilitate drug response/resistance. It can also influence the tumour microenvironment by controlling important signaling that regulating immune checkpoint molecules leading to activation of T cell anti-tumour immunity. Therefore, they can be used as potential novel cancer biomarkers and innovative immunotherapeutic agents. This review highlights the use of exomiRs as potential reliable biomarkers for cancer diagnosis, treatment response and metastasis. Finally, discuses their potential as immunotherapeutic agents to regulate immune checkpoint molecules and promote T cell anti-tumour immunity.

Neutrophil-activating therapy for the treatment of cancer.

Despite their cytotoxic capacity, neutrophils are often co-opted by cancers to promote immunosuppression, tumor growth, and metastasis. Consequently, these cells have received little attention as potential cancer immunotherapeutic agents. Here, we demonstrate in mouse models that neutrophils can be harnessed to induce eradication of tumors and reduce metastatic seeding through the combined actions of tumor necrosis factor, CD40 agonist, and tumor-binding antibody. The same combination activates human neutrophils invitro, enabling their lysis of human tumor cells. Mechanistically, this therapy induces rapid mobilization and tumor infiltration of neutrophils along with complement activation in tumors. Complement component C5a activates neutrophils to produce leukotriene B4, which stimulates reactive oxygen species production via xanthine oxidase, resulting in oxidative damage and Tcell-independent clearance of multiple tumor types. These data establish neutrophils as potent anti-tumor immune mediators and define an inflammatory pathway that can be harnessed to drive neutrophil-mediated eradication of cancer.

Reprogramming of T cell-derived small extracellular vesicles using IL2 surface engineering induces potent anti-cancer effects through miRNA delivery.

T cell-derived small extracellular vesicles (sEVs) exhibit anti-cancer effects. However, their anti-cancer potential should be reinforced to enhance clinical applicability. Herein, we generated interleukin-2-tethered sEVs (IL2-sEVs) from engineered Jurkat T cells expressing IL2 at the plasma membrane via a flexible linker to induce an autocrine effect. IL2-sEVs increased the anti-cancer ability of CD8+ T cells without affecting regulatory T (Treg ) cells and down-regulated cellular and exosomal PD-L1 expression in melanoma cells, causing their increased sensitivity to CD8+ T cell-mediated cytotoxicity. Its effect on CD8+ T and melanoma cells was mediated by several IL2-sEV-resident microRNAs (miRNAs), whose expressions were upregulated by the autocrine effects of IL2. Among the miRNAs, miR-181a-3p and miR-223-3p notably reduced the PD-L1 protein levels in melanoma cells. Interestingly, miR-181a-3p increased the activity of CD8+ T cells while suppressing Treg cell activity. IL2-sEVs inhibited tumour progression in melanoma-bearing immunocompetent mice, but not in immunodeficient mice. The combination of IL2-sEVs and existing anti-cancer drugs significantly improved anti-cancer efficacy by decreasing PD-L1 expression in vivo. Thus, IL2-sEVs are potential cancer immunotherapeutic agents that regulate both immune and cancer cells by reprogramming miRNA levels.

The IL7R-Antagonist OSE-127 Blocks Acute Lymphoblastic Leukemia Development Via a Dual Mode of Action

Acute lymphoblastic leukemia (ALL) may arise from precursor B or T cells (BCP- and T-ALL, respectively) and treatment is based on polychemotherapy. There is an urgent need for novel immunotherapy targets to reduce therapy toxicity and to target relapsed/refractory (r/r) disease, especially in T-ALL where immunotherapy approaches are scarce. The Interleukin-7-receptor (IL7R) has been shown to play a pivotal role in the development of both BCP-ALL and T-ALL (Geron et al. 2022; Silva et al. 2022). The IL7Rα-chain (CD127)-targeting antibody OSE-127 has a good safety profile in healthy volunteers (NCT03980080) and has entered phase 2 trials in Ulcerative Colitis (NCT04882007) and Sjögren's syndrome (NCT04605978). We previously reported high preclinical efficacy of OSE-127 in BCP-ALL patient derived xenograft (PDX) models of different cytogenetic backgrounds (Lenk and Baccelli et al. ASH, 2021). However, the preclinical efficacy of OSE-127 in T-ALL had not yet been investigated and the mechanism of action underlying its anti-leukemic efficacy remained unclear. First, we investigated the in vivo efficacy of OSE-127 treatment on BCP-ALL and T-ALL cells by conducting a preclinical phase 2-like PDX study. Two NSG mice per patient were injected with BCP-ALL (n=13) or T-ALL (n=9) PDX cells (including 7 r/r ALL samples), randomly assigned into treatment groups and OSE-127 therapy was initiated when 1% leukemic blasts were detected in the peripheral blood (PB). OSE-127 treated mice showed a reduction of PB blasts in 21/22 (95%) PDX samples in vivo when the corresponding control mice showed clinical signs of overt leukemia (Figure 1A). This translated into a 1.6-fold prolongation of median survival (MS) of OSE-127 treated PDX mice (P<0.0001, Figure 1B). Of note, blast reduction strongly correlated with CD127 expression levels in the samples (Pearson r=0.5372, P=0.0099). Next, we characterized the mode of action of OSE-127. As expected, OSE-127 blocked STAT5 phosphorylation in IL7-dependent ALL cell lines and PDX samples. As OSE-127 is not internalized by CD127 expressing cells (Belarif et al. 2018), we investigated whether OSE-127 might also exert immune-mediated effects. We found that OSE-127 did not promote antibody-dependent cellular cytotoxicity (ADCC), but efficiently induced macrophage-mediated antibody-dependent cellular phagocytosis (ADCP) in BCP- and T-ALL cell lines and PDX cells in vitro, in a manner dependent on CD127 expression levels (Pearson r=0.5000, P=0.0352, Figure 1C-D). In line with these observations, inactivating mutations of the Fc fraction of OSE-127 (OSE-127-LALAPG) prevented ADCP of ALL cells in vitro, while conserving its antagonistic properties on IL7R signaling. To further evaluate the contribution of ADCP to the anti-leukemic activity of OSE-127, we compared the in vivo efficacy of OSE-127 and OSE-127-LALAPG in ALL samples with dysfunctional IL7R signaling (e. g., DND41 cells showing constitutive pathway activation due to IL7Rα mutations or NALM6 cells being absent of P-STAT5 induction upon IL7 stimulation). In this context, OSE-127-LALAPG did not impact leukemic engraftment as compared to control treatment, while OSE-127 significantly reduced leukemic burden, thereby substantiating that ADCP contributes to the anti-leukemic efficacy of OSE-127 (Figure 1E). Finally, we conducted a phase2-like PDX overt leukemia study testing OSE-127 alone or in combination with Dexamethasone/Vincristine/PEG-Asparaginase chemotherapy employing PDX samples with high CD127 expression (6 BCP-ALL and 3 T-ALL-PDX samples with >50% CD127+ cells in flow cytometry analysis). OSE-127 alone significantly prolonged the survival of PDX mice in comparison to control treatment (MS 106d vs 79d, P=0.0071), which was comparable to the effect of chemotherapy (MS 106d vs 79d, P=0.0037). Importantly, the strongest effects were observed when combining OSE-127 with chemotherapy (MS 79d vs undefined, P=0.0003, Figure 1F). Overall, we propose OSE-127 as a novel potential immunotherapeutic agent for the treatment of BCP-ALL and T-ALL, particularly in r/r disease and in combination with chemotherapy. Due to its dual mode of action comprising ADCP induction and IL7R-pathway blockade, OSE-127 could be efficacious in CD127+ ALL with and without IL7R pathway alterations. L.L. and I.B. contributed equally to this work. N.P. and D.M.S. share senior authorship Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

CD4+ T-lymphocytes from asymptomatic dogs infected with Leishmania infantum are able to activate macrophages for higher leishmanicidal ability in an in vitro co-culture experiment

Dogs are the most common domestic reservoir of Leishmania infantum, making canine visceral leishmaniasis (CVL) a serious public health issue. Identifying new methodologies that can mimic lymphoid and myeloid competence in naturally infected dogs could lower costs and save time in preliminary screenings of potential immunotherapeutic agents and vaccines against CVL. For that, we established a cell-to-cell communication approach between lymphocytes and myeloid cells from healthy, asymptomatic (infected, without apparent clinical signs) and symptomatic (infected with apparent clinical signs) dogs. Peripheral blood mononuclear cells (PBMC) from these dogs were used as source of CD4+, CD8+ T lymphocytes and macrophages, that were posteriorly infected with L. infantum GFP+ promastigotes (green fluorescent protein). Macrophages co-cultured with purified lymphocytes were tested for the ability to control cellular parasitism, and their microbicidal function by producing nitric oxide (NO) and reactive oxygen species (ROS). The kind of T cell response within the co-culture was also evaluated, by assessing their ability to produce interferon-gamma (IFN-γ) and interleukin 4 (IL-4). The data suggests that T lymphocytes from symptomatic dogs are more prone to produce IL-4 than the ones from asymptomatic dogs. Macrophages from asymptomatic dogs also demonstrated a higher microbicidal potential, with increased levels of NO and ROS production, compared to symptomatic dogs, mainly in highly parasitized cells. Together, our results identify the ratio of IL-4/IFN-γ produced by CD4+ and CD8+ T cells, as well as, the ratio between parasite GFP signal/NO and ROS signal in macrophages as potential immunological biomarkers of failure and success of the screened agents. Our findings also propose a reliable methodology that can be used to follow the immune response in trials of potential drugs or vaccines targeting CVL.

Patient stratification based on urea cycle metabolism for exploration of combination immunotherapy in colon cancer

BackgroundOwing to the low ratio of patients benefitting from immunotherapy, patient stratification becomes necessary. An accurate patient stratification contributes to therapy for different tumor types. Therefore, this study aimed to subdivide colon cancer patients for improved combination immunotherapy.MethodsWe characterized the patients based on urea cycle metabolism, performed a consensus clustering analysis and constructed a risk model in the cancer genome atlas cohort. Colon cancer patients were further categorized into two tags: clusters, and risk groups, for the exploration of combination immunotherapy. In addition to external validation in the Gene Expression Omnibus datasets, several images of immunohistochemistry were used for further validation.ResultsPatient characterization based on urea cycle metabolism was related to immune infiltration. An analysis of consensus clustering and immune infiltration generated a cluster distribution and identified patients in cluster 1 with high immune infiltration levels as hot tumors for immunotherapy. A risk model of seven genes was constructed to subdivide the patients into low- and high-risk groups. Validation was performed using a cohort of 731 colon cancer patients. Patients in cluster 1 had a higher immunophenoscore (IPS) in immune checkpoint inhibitor therapy, and those other risk groups displayed varying sensitivities to potential combination immunotherapeutic agents. Finally, we subdivided the colon cancer patients into four groups to explore combination immunotherapy. Immunohistochemistry analysis showed that protein expression of two genes were upregulated while that of other two genes were downregulated or undetected in cancerous colon tissues.ConclusionUsing subdivision to combine chemotherapy with immunotherapy would not only change the dilemma of immunotherapy in not hot tumors, but also promote the proposition of more rational personalized therapy strategies in future.

Immunoinformatics-based Identification of Highly Conserved Cytotoxic T-Cell Epitopes in Polyprotein pp220 of African Swine Fever Virus

Background:High mortality rate of pigs peaked in 2020 due to the re-emergence of a deadly African swine fever virus (ASFV) which has led to transcontinental outbreaks in Europe, reportedly from 2014 to 2019, and in Asia and the Pacific from 2018–2020. Given the huge socioeconomic consequences of the disease, vaccines that will prime the immunity of swine against this pathogen is a dire necessity.Methods:Insilicoidentification and characterization of highly conserved cytotoxic T-cell (CD8+) epitopes derived from one of its structural proteins, pp220, were analyzed. Protein sequences of pp220 were retrieved and clustered to obtain highly conserved sequences. Cross-reactive epitopes were filtered out, and the remaining epitopes were docked with swine leukocyte antigen-1*0401 (SLA-1*0401). Furthermore, the epitope stability was determined by comparing binding energy, dissociation constant, and eigenvalues of the epitopes with the values of positive control, influenza-epitope complex.Results:This study showed that 20 highly conserved epitopes promiscuously bind to two or more SLAs and 9 of which epitopes (ALDLSLIGF, QIYKTLLEY, FLNKSTQAY, IADAINQEF, IINPSITEY, AINTFMYYY, SLYPTQFDY, RSNPGSFYW, and RLDRKHILM) that were validated exhibit potential immunogenicity based on the acceptable binding energy, dissociation constant, and eigenvalues.Conclusion:This study has identified epitopes that show high conservancy, reducing the chance of epitope immune evasion. It is anticipated that the identified epitopes must be further evaluated as a potential immunotherapeutic agent in developing an epitope-based vaccine against ASFV.

P598: EBI3/IL-27 DEPLETED MICROENVIRONMENT FAVOURS TUMOUR PROGRESSION IN CHRONIC LYMPHOCYTIC LEUKAEMIA

Background: Chronic Lymphocytic Leukaemia (CLL) is the most common adult leukaemia in western countries. Unfortunately, CLL remains an incurable disease, evidencing the urgency to develop novel effective treatments for this malignancy. CLL cells are known to be highly dependent on interactions with non-malignant cells in their tumour microenvironment (TME) for survival and proliferation. Such dependency highlights the potential of immunotherapy as a therapeutic approach in CLL. Recently, several pro-inflammatory cytokines have emerged as a powerful tool to reactivate the immune system in a wide range of malignancies. IL-27 is a member of the IL-12 family of heterodimeric cytokines reported to have pleiotropic functions during cancer development in different malignancies. Based on the existing literature, we wondered whether IL-27 affects the development and progression of CLL. Aims: The goal of this study is to elucidate the role of IL-27 in CLL development and progression. Methods: We used a constitutive knockout mouse model of the EBI3 subunit of IL-27 to abrogate IL-27 expression in vivo. We performed adoptive transfer (AT) of TCL1 derived CLL cells into Ebi3-/- and WT mice, as well as generated the transgenic mouse model Eµ-TCL1-Ebi3-/-.The splenic TME of these mice was characterized using flow cytometry. In addition, in vivo neutralization of IL-27 using a blocking antibody was conducted. Immunodeficient Rag2-/- mice were also injected with TCL1-derived CLL cells and either WT or Ebi3-/- CD3+ T cells to assess the role of T-cells in leukaemia control in the presence and absence of IL-27. Finally, the transcriptional differences among WT and Ebi3-/- T cells were assessed using bulk-RNA sequencing, and their impact on T cell cytotoxicity was investigated both in humans and mice using in vitro killing assays. The serum levels of IL-27 in CLL patients and sick mice were quantified via ELISA and compared to those of healthy controls. Results: A strikingly enhanced CLL development and survival reduction was observed when CLL was AT into Ebi3-/- mice, as well as in the transgenic Eµ-TCL1-Ebi3-/- mice when compared to controls (A). Consistently, flow cytometry analysis revealed an increasingly immunosuppressive splenic TME in the absence of EBI3, characterized by an enrichment of highly activated and immunosuppressive Tregs and a terminally exhausted CD8+ T cell subset. The aforementioned results were recapitulated in an IL-27 neutralization experiment (B). Using Rag2-/- mice, we demonstrated that T cells from EBI3-/- mice were less efficient in controlling CLL development (C). RNA sequencing revealed major changes in CD8+ T cells transcriptional program, in particular a decreased expression of crucial transporters (D). Finally, in vitro killing assays showed an increased cytotoxicity of both human and murine T cells in the presence of IL-27, while serum protein quantification showed a decrease in the levels of IL-27 in both CLL patients (E) and sick mice (F) when compared to their healthy counterparts. Image:Summary/Conclusion: Overall, our results demonstrate the antitumor role of IL-27 in CLL development and progression by promoting T-cell-mediated anti-tumor immunity, as well as establish this cytokine as a potential immunotherapeutic agent in CLL.