Complete Rejection Research Articles

Abstract PR-07: Optimally designed mRNA vaccine encoding tumor-specific antigens identified in a colorectal cancer model leads to complete tumor rejection in mice

Abstract mRNA vaccines can be designed to encode multiple epitopes of choice, which is a crucial advantage when aiming to include a broad coverage immune response. Upon translation, the cell machinery must process the vaccine-encoded poly-epitope protein to release individual peptides. This processing step is key in inducing an immune response toward selected epitopes, as efficient processing yields a higher amount of peptide available for MHC-I presentation. The efficiency of peptide processing is greatly influenced by the peptide surrounding context (i.e. the flanking amino acids in N-and C-terminal). Aberrantly expressed tumor-specific antigens (aeTSAs) are MHC-I-associated peptides (MAPs) resulting from cancer-specific epigenetic changes and splicing aberrations. In contrast to TSAs derived from mutated protein-coding exons, aeTSAs are highly shared by different tumors. Moreover, unlike commonly used tumor-associated antigens (TAAs), they are not expressed by normal healthy cells, which is pivotal for inducing strong CD8+ T cell responses. The present study aimed to determine whether RNA vaccines encoding aeTSAs would elicit protective anti-tumor responses against a colorectal cancer cell line model (MC38). We hypothesized that multiepitope mRNA vaccine efficacy would be improved if the mRNA construct was composed of minimal epitopes (here, cancer antigens) bordered by optimal (rather than natural) flanking sequences. Thus, we designed 2 vaccine constructs encoding 5 aeTSAs identified by mass spectrometry in MC38 tumor cells. The 2 constructs differ in the identity of the amino acids flanking the antigens (natural vs. substituted antigen flanking sequences). Mice were injected subcutaneously with MC38 cells on Day 0. Three mRNA vaccine doses were administered intravenously one week apart, starting at Day 4. Vaccination with our improved mRNA vaccine design showed a clear therapeutic effect. As opposed to the natural flanking sequences design resulting in delayed tumor growth, vaccination with the substituted flanking sequences design led to the complete elimination of tumors and the survival of all mice. Elispot and dextramer stainings revealed that at least 3 of the 5 MC38 aeTSAs were immunogenic and that the amplitude of antigen-specific CD8+ T cell responses was significantly higher in mice vaccinated with the substituted flanking sequences design. Our results confirm our hypothesis that highly proficient flanking regions have intrinsic benefits that can be carried over to different antigens. From a translational perspective, our work provides new insights into the therapeutic potential of optimally designed aeTSA-encoding mRNA vaccines for treating cancers. Citation Format: Marie-Pierre Hardy, Krystel Vincent, Gabriel Ouellet-Lavallée, Chantal Durette, Isabelle Caron, Joel Lanoix, Mathieu Courcelles, Jean-Philippe Laverdure, Pierre Thibault, Claude Perreault. Optimally designed mRNA vaccine encoding tumor-specific antigens identified in a colorectal cancer model leads to complete tumor rejection in mice [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2024 Oct 18-21; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2024;12(10 Suppl):Abstract nr PR-07.

The Essence of the Ex aequo et bono Method and the Rules of its Application for the Resolution of International Disputes

One of the most important functions of law is the resolution of disputes. However, legal means of dispute resolution, such as judicial settlement and arbitration, are often not chosen by the parties involved in international disputes. This can be explained by several reasons. In legal doctrine, it is a common view that parties avoid legal means due to the binding nature of the decisions rendered, yet this is far from the only reason why parties refuse these methods. In particular, one of the main reasons they avoid legal means is the uncertainty regarding whether the decision based on law will be equitable. There are many grounds for this, as legal norms contained in international legal documents, adopted several decades ago, may not correspond to modern realities. Therefore, the application of such norms can quite reasonably raise concerns among the parties about the potential for an inequitable decision based on them. In such cases, the method of ex aequo et bono may prove to be a practical tool for excluding the possibility of an inequitable decision based on legal norms. Contrary to popular misconception, the use of the ex aequo et bono method does not imply a complete rejection of legal norms. When applying this method, basic legal norms are often used to provide a legal foundation for decisions and to enhance their overall objectivity. However, in the application of the method ex aequo et bono, those legal norms that may lead to an inequitable decision are not applied. In this case, an equitable decision is often determined based on an analysis of the goals and principles of legal documents regulating the relevant issues, including an analysis of the document containing the legal norm whose application could have led to an inequitable decision. Thus, the ex aequo et bono method is a rather flexible peaceful means, combining valuable features of both equity and law. There is a deficit of information regarding the ex aequo et bono method in international legal documents. In particular, the lack of a clear definition, conditions, and rules for applying the ex aequo et bono method presents a significant obstacle to its extensive use in international practice. Therefore, appropriate lawmaking work should be undertaken in this area to fill legal gaps. The goal of the scientific article is to study the legal aspects of the ex aequo et bono method and identify the main problems negatively affecting the practice of its application. The results of the research showed the high practical value of the ex aequo et bono method in dispute resolution, especially in modern conditions when the use of flexible means is becoming increasingly relevant. The article aimed to condense, specify and expand legal knowledge about the ex aequo et bono method and may present significant scientific value for lawyers interested in judicial and arbitration practices; legal scholars; practicing judges and arbitrators; parties with unresolved disputes; as well as philosophers researching the relationship between equity and law.

An oncolytic HSV-1 vector induces a therapeutic adaptive immune response against glioblastoma

BackgroundGlioblastoma (GBM) is the most frequent and aggressive brain tumor in adults with the lowest survival rates five years post-diagnosis. Oncolytic viruses (OVs) selectively target and damage cancer cells, and for this reason they are being investigated as new therapeutic tools also against GBM.MethodsAn oncolytic herpes simplex virus type 1 (oHSV-1) with deletions in the γ34.5 neurovirulence gene and the US12 gene, expressing enhanced green fluorescent protein (EGFP-oHSV-1) as reporter gene was generated and tested for its capacity to infect and kill the murine GL261 glioblastoma (GBM) cell line. Syngeneic mice were orthotopically injected with GL261cells. Seven days post-implantation, EGFP-oHSV-1 was administered intratumorally. Twenty-one days after parental tumor challenge in the opposite brain hemisphere, mice were sacrified and their brains were analysed by immunohistochemistry to assess tumor presence and cell infiltrate.ResultsoHSV-1 replicates and induces cell death of GL261 cells in vitro. A single intracranial injection of EGFP-oHSV-1 in established GL261 tumors significantly prolongs survival in all treated mice compared to placebo treatment. Notably, 45% of treated mice became long-term survivors, and rejected GL261 cells upon rechallenge in the contralateral brain hemisphere, indicating an anamnestic antitumoral immune response. Post-mortem analysis revealed a profound modification of the tumor microenvironment with increased infiltration of CD4 + and CD8 + T lymphocytes, intertumoral vascular collapse and activation and redistribution of macrophage, microglia, and astroglia in the tumor area, with the formation of intense fibrotic tissue suggestive of complete rejection in long-term survivor mice.ConclusionsEGFP-oHSV1 demonstrates potent antitumoral activity in an immunocompetent GBM model as a monotherapy, resulting from direct cell killing combined with the stimulation of a protective adaptive immune response. These results open the way to possible application of our strategy in clinical setting.

Understanding Human-Robot Interactions with a Humanoid Robot

Human responses towards robots vary from excitement and acceptance to complete rejection, and as robots become more prevalent in personal and public spaces, understanding the factors that elicit those responses is important. This research explores the factors influencing these responses, specifically in the context of human-robot collaborative tasks. Under mechatronics and robotics, the study focuses on identifying behavioural features (like triggers or positive/negative eliciting actions) and human-designed robot algorithms that elicit different human reactions. It also examines how these features interact to shape human behaviour and emotions in stressful and calm situations. I developed a study in a simulated medical setting where participants are tasked with developing dialogue scripts for a social robot. The study utilizes the NAO robot and a programmable platform called the Robot Management System developed by RobotLab. Participants will program NAO to interact with a patient, perform a checkup, and decide on actions like writing a prescription or referring the patient to medical professionals. The experiment is designed to observe how the participant interacts with the robot to complete tasks and how the robot’s design impacts the participants' perception and interaction following and during tasks. While no experimental data has been collected due to pending ethics approval, the research will provide insights into human-robot interaction, including algorithm design. This study aims to enhance our understanding of human-robot relationships, contributing to developing robotic systems that can be effectively integrated into social environments, such as healthcare settings or similar environments. These insights may inform the design and deployment of robots to ensure they support rather than disrupt social dynamics.

IgG-BSA separation and purification by internally staged ultrafiltration

Purification of IgG from residual host cell proteins (HCPs) in post-Protein A chromatography is important since some HCPs bind with Protein A and elute with the monoclonal antibody (mAb); removal of HCPs from CHO cell lines is essential. To that end, an advanced separation and purification technique in biopharmaceutical manufacturing, namely, internally staged ultrafiltration (ISUF), is investigated here. Choosing BSA as a model for HCPs in post-protein A eluate, separation of a binary mixture of IgG and BSA containing 1.0 mg/ml IgG and 0.1 mg/ml BSA is successfully demonstrated here using a modified ISUF technique: two Omega 100 kDa membranes on top followed by one Omega 70 kDa membrane at the bottom. This modified configuration demonstrated exceptional performance with almost complete rejection, 99 % purity, and 99.5 % retention of IgG, along with 96.5 % recovery of BSA over 10 diavolumes. This modified membrane stacking resulted from strategic considerations of membrane stacking and careful selection of molecular weight cutoffs and materials, and performance analysis of different membranes and stacking configurations using rejection behaviors, purity levels, and recovery rates under varying diavolume and pressure differential. The approach adopted here enhances flexibility in membrane choices in ISUF and provides valuable insights for optimizing membrane-based biopharmaceutical separation techniques.

مآلات فتاوى العلامة البوطي في مواجهة الربيع العربي دراسة تحليلية في أحداث الجمهورية العربية السورية

The study aims to clarify the consequences of the fatwas of Sheikh al-Buti in the Syrian events. The researcher focused on the concept of legal consequences to highlight the extent to which fatwas and their consequences are governed by Islamic law. The researcher followed an inductive and analytical approach to examine the fatwas, collect texts, and opinions of scholars related to these fatwas from reliable sources. The study analyzed these opinions and examined their consequences from the perspective of Islamic law. The study concluded with several findings, including:Al-Buti followed the approach of early scholars in tracing the consequences of rulings.Al-Buti's stance on the uprising and rebellion against the ruler is complete rejection, adhering to the principles of Islamic law. Al-Buti believes that rebellion against the ruler is permissible if the ruler openly commits kufr (disbelief). He believes that rebellion against the ruler is not permissible when the rebels do not rely on a legitimate Islamic basis. Demonstrations and marches are forbidden because they are a path and a pretext for opening a confrontation, a path to killing and committing forbidden acts, facilitating the way for external aggression to control the country, and blocking the means is a religious obligation.

Enhancing the permeability and selectivity of graphene oxide membrane through polyhedral oligomeric silsesquioxane intercalation for textile wastewater treatment

Graphene oxide-based loose nanofiltration membranes have shown significant potential in nanofiltration processes due to their unique 2D mass transfer nanochannels. However, they still suffer from poor structural stability and overly compact laminar stacking. To address these issues, a novel AP-POSS was intercalated into GO-based membrane to construct a tuned interlayer structure and mass transfer characteristics. The aromatic ring structure of aminophenyl groups on AP-POSS act as hydrophobic side walls of the mass transfer channels between adjacent GO nanosheets, reducing the interaction between water molecules and channel structure. With an optimal amount of AP-POSS as an intercalator, the permeation flux of the prepared POSS-GO membrane could be improved by 30 wt% while maintaining nearly complete rejection of Congo Red (CR). Moreover, the prepared membrane exhibited enhanced structural stability, long-term stability, and a separation factor above 91.0 for CR/NaCl in binary solutions. Our study highlights the excellent performance of the POSS-GO membrane and demonstrates its potential application in dye/salt wastewater treatment.

Recovery of organic nutrients from biogas slurry using decolorized ultra filtration membrane compared with nanofiltration membrane

Membrane separation technology has played a crucial role in concentrating biogas slurry (BS) for efficient water discharge. However, a membrane that selectively separates organic molecules and inorganic ions for the recovery of organic nutrients from BS for its valorization has not yet been reported. In this study, we investigated the use of decolorized ultra filtration membrane (DUFM) for the separation of organic molecules and inorganic ions from BS, compared it with the commonly used commercial nanofiltration (NF). The results showed that DUFM concentrated approximately 63 % of the COD content, which was 24 % lower than that achieved by NF. However, the concentrated BS using DUFM exhibited higher levels of protein-like substances (9 %), aromatic humic-like substances (14 %), and pigments (8 %) compared to NF, as indicated by absorbance of λ254, λ420 and λ560. In terms of inorganic ions, NH4+, Ca2+, HCO3-, and PO43- constituated approximately 88 %, 90 %, 80 %, and 70 % in the permeate side of DUFM, respectively. In constrast, NF allowed only about 70 % of NH4+ and 67 % of HCO3- to pass through, with almost complete rejection of PO43- and Ca2+. The mix-charged DUFM (500 Da) demonstrated superior separation of organic molecules and inorganic salts with low energy consumption compared to NF (150 Da) with negative charges. The DUFM with mix-charged performance increases organic molecules rejection by Donnan effect via electrostatic exclusion and with 500 Da reinforces inorganic ions’ permeation by size exclusion. Additionally, DUFM exhibited tolerance for large molecular dissolved organic matter in BS due to its membrane’s high surface hydrophilicity and smoothness. In conclusion, this study suggests DUFM, with its mix-charged characteristics, could serve as a promising membrane for the selective separation of organic molecules and inorganic ions, facilitating the recovery of organic nutrients for BS valorization.

Wytwarzanie ochronnych struktur kompozytowych za pomocą technologii infuzji w celu zapewnienia odporności na fale uderzeniowe

The paper presents the results of experiments on the use of infusion of polymer binders into a pre-constructed skeleton of reinforcing structures. Currently, three-layer structures with an average layer of aluminum honeycombs are considered to be one of the effective structural elements that ensure the damping of the blast wave. As an alternative middle layer, it is possible to offer elements of various shapes (cylinders, cubes, etc.) and geometries made of modern composites that are well-proven under the impact of a shock wave. These are, as is known, metal-polymer composites, i.e. materials containing, in addition to polymer binder and fabric filler, thin layers of metal. In order to form a solid structure that perceives an explosive load, an attempt was made to use RFI technology to obtain individual elements with which two steel sheets are connected. Aramid, glass fabrics and aluminum thin (0.5, 0.15, 0.05 mm) plates were used in the experiments. For the implementation of the infusion process, an epoxy resin was used, suitable in its technological parameters for this process. The use of this resin leads to the minimization of non-nourished areas and pores. In this case, the uniformity of the product obtained from the composite material is achieved. It should be added that RFI technology differs from other polymer processing technologies in the following significant advantages: the possibility of abandoning expensive equipment, reducing energy costs for equipment and its maintenance, and complete rejection of the use of prepregs. However, there are a number of difficulties that are associated with the technological process of infusion, these primarily include the rheological requirements of the binder: at room temperature, the resin should have a low viscosity, and in the practice of fillers, the viscosity should have rather low values. Below are some preliminary plans for the development of this technology in order to obtain a product that provides attenuation of the blast wave.

SYNTHESIS OF NATIONAL AND WESTERN TRADITION IN EARLY EXAMPLES OF JAPANESE MODERNISM (THE CASE OF WORLD PEACE MEMORIAL CATHEDRAL IN HIROSHIMA)

The paper is devoted to the analysis of the existing influences of traditional Japanese architecture in the modernist World Peace Memorial Cathedral in Hiroshima built after the atomic bombing in Hiroshima on the site of the Noboricho Catholic Church destroyed by the explosion, designed by the architect Togo Murano (using some of the competition ideas of the Kenzo Tange project). According to the results of the study, it was found that the architecture of the World Peace Memorial Cathedral in Hiroshima in Hiroshima represents a synthesis of the traditions of European Romanesque, the Japanese tradition of temple building, and the actual style of modernism, transformed through the prism of the creative manner of the talented architect of Japan of his time, Togo Murano, who had a huge experience in creating architecture with reminiscences of various , both Japanese and European styles of the past and present. In particular, it was established that the general planning and composition of the cathedral is close to the style of basilica churches of European Romanesque, taking into account the simplified global forms of modernism. At the same time, the appearance on the facade of the cathedral of its frame structure and a number of decoration elements refers to the tradition of ancient Japanese architecture, which is especially noticeable in the decoration of the main facade in the form of Japanese carved partitions, which, however, in accordance with the ideas of modernism, are made of concrete. From the results of the work, it is generally clear that Japanese modernism, from the very beginning of its spread in Japan, fundamentally differed from Europe, as it did not accept the idea manifested by European modernism of a complete rejection of traditional form-making. On the contrary, modernism in Japan actively used the national tradition as a basis for ideas.

CRISPR-Cas9 immune-evasive hESCs are rejected following transplantation into immunocompetent mice.

Although current stem cell therapies exhibit promising potential, the extended process of employing autologous cells and the necessity for donor-host matching to avert the rejection of transplanted cells significantly limit the widespread applicability of these treatments. It would be highly advantageous to generate a pluripotent universal donor stem cell line that is immune-evasive and, therefore, not restricted by the individual's immune system, enabling unlimited application within cell replacement therapies. Before such immune-evasive stem cells can be moved forward to clinical trials, in vivo testing via transplantation experiments in immune-competent animals would be a favorable approach preceding preclinical testing. By using human stem cells in immune competent animals, results will be more translatable to a clinical setting, as no parts of the immune system have been altered, although in a xenogeneic setting. In this way, immune evasiveness, cell survival, and unwanted proliferative effects can be assessed before clinical trials in humans. The current study presents the generation and characterization of three human embryonic stem cell lines (hESCs) for xenogeneic transplantation in immune-competent mice. The major histocompatibility complexes I- and II-encoding genes, B2M and CIITA, have been deleted from the hESCs using CRISPR-Cas9-targeted gene replacement strategies and knockout. B2M was knocked out by the insertion of murine CD47. Human-secreted embryonic alkaline phosphatase (hSEAP) was inserted in a safe harbor site to track cells in vivo. The edited hESCs maintained their pluripotency, karyotypic normality, and stable expression of murine CD47 and hSEAP in vitro. In vivo transplantation of hESCs into immune-competent BALB/c mice was successfully monitored by measuring hSEAP in blood samples. Nevertheless, transplantation of immune-evasive hESCs resulted in complete rejection within 11days, with clear immune infiltration of T-cells on day 8. Our results reveal that knockout of B2M and CIITA together with species-specific expression of CD47 are insufficient to prevent rejection in an immune-competent and xenogeneic context.

Practical design of an optical filter for thermal management of photovoltaic modules

AbstractThis work presents a practical approach to designing an optical filter for thermal management for photovoltaic modules. The approach emphasizes the practicality of manufacturing over optical performance. Simulation work demonstrates that, for an interdigitated back contact solar cell architecture, complete rejection of infrared radiation offers limited thermal benefits requiring highly complex optical filter designs. An alternative approach consists of reducing thermalization losses by providing reflectance at lower wavelength values. An optical filter design that fulfills this requirement is possible using simple structures based on two materials and taking advantage of the harmonics present in quarter wavelength optical thickness designs. The filter is later optimized for angular performance via second‐order algorithms, resulting in a device consisting of only 15 thin‐film layers. Performance simulations on two locations, Delft (the Netherlands) and Singapore, estimate a temperature reduction of 2.20°C and 2.45°C, respectively. In a single year, the optical loss produced by the filter is not compensated via temperature reduction. However, improvements in the annual degradation rate show that in Singapore, the overall effect of the filter on the lifetime DC energy yield is positive.

Treatment organization of disabled veterans with mental illnesses in the post-war years (1945–1950) (based on the materials of the Tambov Psychiatric Hospital)

Importance. Studying the treatment features of participants and disabled veterans of the Great Patriotic War of 1941–1945, who were in regional psychiatric hospitals, allows not only to clarify the levels of medical care for such military veterans, but also to evaluate the results of public health organization in the postwar years through the prism of Soviet psychiatry development in the 20th century, as well as the results of medical care for war veterans. A detailed study of the situation of disabled veterans in psychiatric clinics in the post-war period is very useful, which will clarify many little-studied aspects not only of the history of medicine, but also of the development of interpersonal, group, and regional relations in socialist society.Research Methods. The methodological features of the study of the stated problems are based on the author’s concept of involving the works of contemporaries, primarily specialists in the field of psychiatry, practitioners of the Tambov region, as well as the actual case histories and anamnesis of mental illness. Materials from state and party archives of the Tambov region and memoirs of contemporaries were used, many of which were introduced into scientific circulation for the first time.Results and Discussion. In fact, patients of psychiatric hospitals in the post-war period received almost the entire necessary complex of therapeutic and preventive procedures and rehabilitation measures. At the same time, it must be borne in mind that the post-war devastation, lack of food, clothing, and a number of medicines seriously complicated the capabilities of medical organizations, which often did not have the necessary resources to fully organize the treatment of war veterans suffering from mental illness. In addition, there are often problems with heating, the supply of necessary equipment and supplies for the psychiatric hospital, which left an imprint on practical health care at the regional level. Nevertheless, Tambov psychiatrists used the full range of medical procedures and medicines available to them, both traditional and new, which gave a tangible therapeutic effect. It is important to note that in the post-war years there is a complete rejection of the elements of “punitive” psychiatry due to the cessation of placing patients in isolation, as well as wet wraps. In addition, the use of intensive treatment methods required coordination with the chief physician.Conclusion. The results of the study of the problem made it possible to assess these processes, viewed through the prism of the development of regional health care, to identify trends in the development of Soviet psychiatry in the first post-war decade. The development of an outpatient network for the provision of medical care to war invalids suffering from mental disorders and diseases is clarified. The assessment of the placement of military veterans in labor unions, enterprises and homes for the disabled is given. It is concluded that, in general, regional psychiatry has shown its capacity and effectiveness in terms of treatment and rehabilitation of military invalids who had mental illnesses.

Accurate prediction of solvent flux in sub-1-nm slit-pore nanosheet membranes.

Nanosheet-based membranes have shown enormous potential for energy-efficient molecular transport and separation applications, but designing these membranes for specific separations remains a great challenge due to the lack of good understanding of fluid transport mechanisms in complex nanochannels. We synthesized reduced MXene/graphene hetero-channel membranes with sub-1-nm pores for experimental measurements and theoretical modeling of their structures and fluid transport rates. Our experiments showed that upon complete rejection of salt and organic dyes, these membranes with subnanometer channels exhibit remarkably high solvent fluxes, and their solvent transport behavior is very different from their homo-structured counterparts. We proposed a subcontinuum flow model that enables accurate prediction of solvent flux in sub-1-nm slit-pore membranes by building a direct relationship between the solvent molecule-channel wall interaction and flux from the confined physical properties of a liquid and the structural parameters of the membranes. This work provides a basis for the rational design of nanosheet-based membranes for advanced separation and emerging nanofluidics.

Novel MXene/PVDF nanocomposite ultrafiltration membranes for optimized Eriochrome black T (azo dye) removal

In the context of recycling and reusing textile wastewater, the quest for a membrane possessing superior efficiency has garnered considerable attention. This research endeavors to advance this objective by introducing a novel asymmetric ultrafiltration membrane constructed from PVDF (Polyvinylidene Fluoride) modified with MXene through bulk modification, thereby augmenting its resistance to fouling for textile wastewater treatment applications. The investigation employed a comprehensive array of characterization techniques. The proposed interaction mechanism between the contents of PVDF/MXene and the interaction mechanism of composite membrane with water molecules were also presented. Comparative analysis with pure membranes revealed that the incorporation of MXene resulted in enhanced hydrophilicity and porosity, concurrently reducing its surface roughness when compared to a pristine membrane. The lowest water contact angle about 38.5 (⁰) was observed in the mixed matrix membrane containing 0.5 wt% MXene. The developed membranes exhibited a maximum pure water flux of 538 LMH and a flux of 467.8 LMH for EBT dye solution. Furthermore, optimization of the nanocomposite membrane composition led to nearly complete rejection of Eriochrome black T (EBT). A continuous improvement in the flux recovery ratio (FRR%) culminating in a peak value of 85.6% for the MMMs prepared from 0.5 wt% MXene.

Abstract 3251: QTOLIMOD: A specific TLR9 agonist nanomedicine with high anti-solid tumor activity

Abstract Background Toll-like receptor 9 (TLR9) agonists are extensively studied for cancer treatment, but face challenges in delivery, clearance, and side effects. Our study showed that QTOLIMOD, a 26nt CpG-ODN delivered via QTsomeTM lipid nanoparticle, significantly inhibited tumor growth in MC38-bearing mice (TGI%=99.06%, p<0.001). Here, we report QTOLIMOD's potential for complete tumor rejection, prevention of recurrence, and advantages in safety and immune-cell induction. Methods C57BL/6 mice were subcutaneously inoculated with 1 × 106 MC38 cells. To assess drug tissue distribution, Cy5-labeled naked CpG-ODN or QTOLIMOD were intratumorally injected and in vivo imaging was conducted. ELISA was utilized to determine the levels of cytokines in serum and tumor samples. Once the tumor volume reached an average size of 80-100 mm3, mice were grouped and intratumorally injected with saline, vehicle, or QTOLIMOD. Tumor volume was calculated using the formula: length × width2 × 1/2. Results Following intratumoral injection of QTOLIMOD or naked CpG-ODN, free CpG-ODN were significantly reduced within 24 hours, while QTOLIMOD exhibited extended duration of action up to 7 days. Anatomical examination revealed that a dose of 5 mg/kg naked CpG-ODN caused liver and spleen enlargement in mice, whereas QTOLIMOD at the same dose did not cause the adverse events. Immunohistochemical assay indicated a significant increase in the infiltration of macrophages, dendritic cells, and CD8+ T cells at the tumor site after dosing. IL-10, IFN-γ, and IL-12 were significantly upregulated in the tumor samples, but not in the serum. In a dose escalation experiment, tumor suppression were observed with intratumoral injection of 0.5, 1, 1.5, or 2 mg/kg (every 3 days, 5 doses), resulting in tumor growth inhibition rates ranging from 92.5% to 98.8%. In the QTOLIMOD treated groups, complete tumor rejection was achieved in 4 out of 6 animals in the 0.5 mg/kg group and in all animals in the 1-2 mg/kg groups within 56 days. To investigate the effect of QTOLIMOD on tumor recurrence, cured mice were re-inoculated with MC38 tumor cells at a distant site. All cured mice rejected newly inoculated tumors without additional injection, resulting in 100% survival for up to 60 days. In a dosing frequency experiment, mice were administered with QTOLIMOD at 1 mg/kg once every 3, 5, 7, or 14 days for a total of 5 doses. The results suggested that fortnightly dosing of QTOLIMOD is more effective than more frequent dosing. Conclusions QTOLIMOD exhibits a high intratumoral retention rate, resulting in increased safety, while effectively promoting the expansion and infiltration of immune cells associated with tumors. Moreover, QTOLIMOD demonstrates potent efficacy in inhibiting tumor growth, potentially leading to complete eradication. Furthermore, treatment with QTOLIMOD effectively prevents tumor from recurrence, offering promising long-term effect. Citation Format: Fei Su, Jun Bai, Chen Li, Yujing Wu, Jing Li, Xiaobin Zhao, Yongsheng Yang, Robert J. Lee. QTOLIMOD: A specific TLR9 agonist nanomedicine with high anti-solid tumor activity [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 3251.

Abstract 2367: STING agonist ADU-S100 improves the antitumor response of CLDN18.2/CD3 BiTEs by enhancing stem-like tumor-reactive CD8+ T cells in pancreatic adenocarcinoma

Abstract Background: Immunotherapy with CLDN18.2/CD3 BiTEs (bispecific T-cell engagers) has shown clinical activity in pancreatic adenocarcinoma. However, only few patients benefit from the treatment, and most responders develop acquired resistance. STING agonists may promote the development of stem-like central memory CD8+ T cells and enhance antitumor responses. This study aimed to investigate the synergistic effect and related mechanism of STING agonist plus CLDN18.2/CD3 BiTEs regimen. Methods: Single-cell RNA sequencing and flow cytometry were used to examine the tumor immune landscape in humanized PDX mouse models. By qPCR, western blot and flow cytometry, an orthotopic transplantation mouse model of pancreatic cancer and an in vitro tumor-PBMC coculture system were established to reveal the potential molecular mechanism of STING agonist ADU-S100 in optimizing the effects of CLDN18.2/CD3 BiTEs (IBI389). Results: In PDAC organoids, IBI389 induced potent, specific cytotoxicity, the CLDN18.2 BiTEs significantly engaged human CD3+ T cells with tumor cells in an organoid-PBMC coculture system in vitro, resulting in the formation of the immunological synapse (IS). IBI389 monotherapy significantly suppressed tumor growth and improved survival in vivo in hCD3e C57BL/6 and humanized hCD34+ PDX mouse models. Further, single-cell RNA sequencing and flow cytometry analysis revealed that the intertumoral TCF1+PD-1+ stem-like T cells and STING signaling activity of T cells were enriched in the early stage of tumor development and sharply decreased in the late stage during CLDN18.2 BiTEs administration. Furthermore, we discovered that the STING agonist ADU-S100 could significantly induce the enrichment of TCF1+PD-1+ stem-like CD8+ T cells in vitro and in vivo, increasing IBI389’s therapeutic effects. Furthermore, humanized PDX mice that were given the combination of IBI389 and ADU-S100 and survived after complete tumor rejection were rechallenged with PDX tumors. Humanized PDX tumors and hCD3e KPC orthotopic tumors from mice treated with the combination contained increased numbers of TCF1+PD-1+ stem-like CD8+ T cells, CD4+CD127+ memory T cells, CD8+CD127+ memory T cells, and fewer numbers of CD4+PD-1+Tim-3+ exhausted T cells, CD8+PD-1+Tim-3+ exhausted T cells and CD4+CD25+ Treg cells. Conclusions: Pancreatic tumors appear to evade immune response by inducing immune-suppressive tumor microenvironment development. In mice, the combination of STING agonist ADU-S100 and CLDN18.2 BiTEs increased the proportion of memory and stem-like CD4+ and CD8+ T cells while decreasing the proportion of regulatory and exhausted T cells in pancreatic tumors, eradicating all detectable tumors. This data may be utilized to formulate immune-based combination therapies for PDAC. Citation Format: Tianxing Zhou, Jingrui Yan, Bin Wang, Yongjie Xie, Chao Yang, Jun Yu, Jihui Hao. STING agonist ADU-S100 improves the antitumor response of CLDN18.2/CD3 BiTEs by enhancing stem-like tumor-reactive CD8+ T cells in pancreatic adenocarcinoma [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 2367.

Abstract 3979: PD-1 signals regulate NKG7 that is required for CD8+ T cells to sustain cytotoxicity and durable antitumor memory responses

Abstract Cytotoxic CD8+ T cells play a key role in response to anti-PD-1/PD-L1 immune checkpoint inhibitors (ICI) therapy. However, the exact effector molecules that are regulated by PD-1 signals in cytotoxic CD8+ T cells have not been completely defined. Lack of this knowledge is a crucial problem in addressing how to overcome the low clinical response rate to ICI therapy. When exploring why certain patients not responding to ICI therapy, we found non-responders did not have an elevated NKG7 expression in CD8+ T cells as responders to ICI therapy. The difference in NKG7 expression in CD8+ T cells between non-responders and responders to anti-PD-1 therapy prompted us to investigate the potential role of PD-1 signaling in regulating NKG7 expression. To that end, we generated a CD8+ T cell-specific Pdcd1 conditional knockout mice (CD8 E8ICre-Pdcd1fl/fl, also known as CD8-PD-1 cKO) where Pdcd1 is deleted after thymic selection of single positive CD8+ T cells. We observed a delayed tumor growth and prolonged survival in CD8-PD-1 cKO mice compared to control mice after B16-OVA melanoma tumor challenge. On Day 12, when the CD8-PD-1 cKO mice demonstrated their ability to control tumor growth, we conducted single cell RNA sequencing on isolated tumor infiltrating CD8+ T cells, revealing a significant increase of Nkg7 expression in effector CD8+ T cells in the Pdcd1 deficient CD8+ T cells compared to the control wild type T cells. Pdcd1-deficient effector CD8+ T cells also had increased expression of genes linked to NFAT/Calcineurin signaling pathway including Nfatc1, S100a4, Tbet and Tox, suggesting Nkg7 could be regulated by Pdcd1 via the NFAT pathway. To determine the impact of increased NKG7 expression in CD8+ T cells, we used OVA protein as a surrogate tumor antigen in vaccination. We found a complete B16-OVA tumor rejection in CD8-PD-1 cKO mice compared to control mice when tumor cells were injected on day 7 after vaccination with OVA protein and poly (I:C) (as an adjuvant), but only a partial rejection on day 21. The CD8-PD-1 cKO mice that were tumor-free for 30-40 days rejected 2nd tumor challenge with B16-OVA tumors. This long-term protection is tumor antigen (OVA) specific because these mice did not reject B16F10 tumor cells that do not express OVA antigen. We also found effector memory CD8+ T cells with high cytotoxicity increased in the spleen of CD8-PD-1 cKO mice after OVA antigen immunization compared to control. In conclusion, we found NKG7 is one of the effector molecules in cytotoxic CD8+ T cells, which can be regulated by PD-1 signals through NFAT signaling. Targeting the PD-1-NFAT pathway may be a new option to overcome low responses of T cells to ICI therapy in treatment of refractory cancer. Citation Format: Zhiming Mao, Joanina Gicobi, Jacob Hirdler, Xin Liu, Michelle Hsu, Emilia Dellacecca, Wenjing Zhang, Fabrice Lucien-Matteoni, Hai dong. PD-1 signals regulate NKG7 that is required for CD8+ T cells to sustain cytotoxicity and durable antitumor memory responses [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 3979.

Abstract 4102: Treatment with androgen receptor targeting vaccines provides an overall survival benefit in AR-V7 expressing tumor models

Abstract Background: Androgen receptor (AR) signaling inhibition is part of the mechanism behind many effective prostate cancer therapies, including androgen deprivation and next generation anti-androgens. However in castration resistant prostate cancer up to 50% of treatment resistance has been linked to AR amplification or expression of the constitutively active RNA splice variant, AR-V7. Although the use of immune checkpoint blockade has failed to demonstrate clinical success, immunotherapeutic interventions remain attractive as prostate cancer’s relatively slow progression provides time for anti-tumor immune responses to develop. Cancer specific vaccines have the potential to enable and enhance tumor specific immune responses by stimulating and directing T cell immunity to important oncologic targets. We hypothesized that a cancer vaccine designed to induce a T cell response against cells expressing high levels of AR would result in an anti-tumor effect and potentially prevent or delay the development of therapeutic resistance. Methods: Adenoviral vaccines against AR (Ad-AR) or AR splice variant 7 (Ad-AR-V7) were used to evaluate the potential of AR targeting vaccination in mouse models. We evaluated AR and AR variant specific adaptive responses produced by our vaccines with naïve and tumor-bearing mice by ex vivo peptide restimulation. We challenged vaccinated mice with multiple syngeneic subcutaneous tumor models that we engineered to express AR-V7, including a PTEN-/- p53-/- prostate tumor cell line. Finally we tested our vaccines in a therapeutic regimen, vaccinating mice after implanting our subcutaneous AR-V7 expressing models. Results: Across multiple strains of mice, AR or AR-V7 targeting adenoviral vaccination induced robust adaptive immune responses against the N-terminal domain of AR. Vaccination with either Ad-AR or Ad-AR-V7 resulted in significant anti-tumor responses, providing a significant overall survival benefit and complete tumor rejection by the majority of mice vaccinated against AR or AR-V7. Similarly tumor bearing mice treated with Ad-AR or Ad-AR-V7 vaccination displayed tumor growth inhibition or complete tumor regression resulting in an overall survival benefit. Additionally, we found that vaccine responses were unaffected by the use of the approved antiandrogen drug enzalutamide, suggesting that vaccinations could be deployed in tandem with standard-of-care therapies. Conclusions: Vaccination against AR and AR splice variants can induce AR specific adaptive immune responses capable of inhibiting the growth of AR-V7 expressing tumor models, illustrating the potential of AR and its splice variants as immunologic targets in prostate cancer. Citation Format: Robert D. Marek, Junping Wei, Tao Wang, Xiao-Yi Yang, Gangjun Lei, Zachary C. Hartman. Treatment with androgen receptor targeting vaccines provides an overall survival benefit in AR-V7 expressing 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 4102.

Abstract 2652: NDI-101150 is a potent and highly selective HPK1 inhibitor that both synergizes with and differentiates from anti-PD1 immune checkpoint blockade

Abstract Introduction HPK1 is a member of the MAP4K family of protein serine/threonine kinases that negatively regulates activation signals in multiple immune cells and is an attractive therapeutic target for many cancers. Using structure-based drug design, we developed a highly selective HPK1 inhibitor, NDI-101150, with nanomolar potency and physiochemical properties suitable for once daily oral administration. Methods & Results HPK1 inhibition with NDI-101150 has a differentiated pharmacology profile when compared directly to PD1 blockade. NDI-101150 was able to overcome the immunosuppressive effects of prostaglandin E2, adenosine, and TGF-β on human T cell activation. HPK1 inhibition also directly limited the suppressive capacity of T regulatory cells. In contrast, anti-PD1 treatment was not able to overcome the immunosuppressive activity associated with any of these mechanisms. In vivo, HPK1 inhibition significantly enhanced the production of antigen-specific antibodies in response to immunization with both T-dependent and T-independent antigens, while anti-PD1 treatment had minimal to no effects on antibody production. In the EMT-6 syngeneic model, 7/10 mice receiving NDI-101150 exhibited complete tumor regressions, compared to only 1/10 from an anti-PD1 cohort. Furthermore, NDI-101150 established a robust and durable immune memory response as 100% of treated mice showed complete rejection of tumor growth upon subsequent re-challenge. HPK1 inhibition with NDI-101150 also shows robust synergy with PD1 blockade in vitro and in vivo. Both NDI-101150 and anti-PD1 were able to reinvigorate exhausted human T cells, restoring cytokine production and proliferative effects in mixed lymphocyte reactions. Further, in combination HPK1 inhibition and PD1 blockade synergized to enhance the activity of exhausted human T cells to an extent exceeding that observed with naïve human T cells in similar experiments. In vivo, while NDI-101150 or anti-PD1 treatments alone induced tumor growth inhibition in the murine CT-26 syngeneic tumor model, NDI-101150 and anti-PD1 in combination mediated complete tumor regressions in several mice. Furthermore, NDI-101150 seemed to induce a durable immune memory response as evidenced by the combination-treated animals showing complete rejection of tumor growth upon subsequent re-challenge, without any further dosing of NDI-101150. Conclusions Pharmacological inhibition of HPK1 with NDI-101150 represents a powerful system-wide immunomodulatory approach with the potential to enhance anti-tumor immunity in patients failing to respond to currently approved immune checkpoint therapies or in combination with immune checkpoint blockade therapies. NDI-101150 is currently being tested in a Phase 1/2 trial (NCT05128487) in patients with advanced solid tumors. Citation Format: David Ciccone, Fu-Shan Kuo, Scott Daigle, Neelu Kaila, Gene Yau, Mark Carlson, Denise Levasseur, Bhaskar Srivastava, Frank G. Basile, Christine Loh. NDI-101150 is a potent and highly selective HPK1 inhibitor that both synergizes with and differentiates from anti-PD1 immune checkpoint blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2652.