Complete Eradication Research Articles

Efficacy of LCMV-based cancer immunotherapies is unleashed by intratumoral injections of polyI:C

BackgroundLymphocytic choriomeningitis virus (LCMV) belongs to the Arenavirus family known for inducing strong cytotoxic T-cell responses in both mice and humans. LCMV has been engineered for the development of cancer...

Genome mining of Mycobacterium tuberculosis: targeting SufD as a novel drug candidate through in silico characterization and inhibitor screening.

Tuberculosis (TB) stands as the second most fatal infectious disease globally, causing 1.3 million deaths in 2022. The resurgence of TB and the alarming rise of antibiotic resistance demand urgent call to develop novel antituberculosis drugs. Despite concerted efforts to control TB, the disease persists and spreads rapidly on a global scale. Targeting stress response pathways in Mycobacterium tuberculosis (Mtb) has become imperative to achieve complete eradication. This study employs subtractive genomics to identify and prioritize potential drug targets among the hypothetical proteins of Mtb, focusing on indispensable pathways. Amongst 177 essential hypothetical proteins, 152 were nonhomologous to human. These proteins participated in 34 pathways, and a 20-fold enrichment of SUF pathway genes led to its selection as a target pathway. Fe-S clusters are fundamental, widely distributed protein cofactors involved in vital cellular processes. The survival of Mtb in a hypoxic environment relies on the iron-sulfur (Fe-S) cluster biogenesis pathway for the repair of damaged Fe-S clusters. It also protects pathogen against drugs, ensuring controlled iron utilization and contributing to drug resistance. In Mtb, six proteins of Fe-S cluster assembly pathway are encoded by the suf operon. The present study was focused on SufD because of its role in iron acquisition and prevention of Fenton reaction. The research further delves into the in silico characterization of SufD, utilizing bioinformatics tools for sequence and structure based analysis. The protein's structural features, including the identification of conserved regions, motifs, and 3D structure prediction enhanced functional annotation. Target based virtual screening of compounds from the ChEMBL database resulted in 12 inhibitors with best binding affinities. Drug likeness and ADMET profiling of potential inhibitors identified promising compounds with favorable drug-like properties. The study also involved cloning in SUMO-pRSF-Duet1 expression vector, overexpression, and purification of recombinant SufD from E. coli BL21 (DE3) cells. Optimization of expression conditions resulted in soluble production, and subsequent purification highlighting the efficacy of the SUMO fusion system for challenging Mtb proteins in E. coli. These findings provide valuable insights into pharmacological targets for future experimental studies, holding promise for the development of targeted therapy against Mtb.

Hepatitis B virus-specific human stem cell memory T cells differentiate into cytotoxic T cells and eradicate HBV-infected hepatocytes in mice.

Chronic infection with the hepatitis B virus (HBV) induces progressive hepatic impairment. Achieving complete eradication of the virus remains a formidable challenge. Cytotoxic T lymphocytes, specific to viral antigens, either exhibit a numerical deficiency or succumb to an exhausted state in individuals chronically afflicted with HBV. The comprehension of the genesis and dissemination of stem cell memory T cells (TSCMs) targeting HBV remains inadequately elucidated. We identified TSCMs in subjects with chronic HBV infection and scrutinized their efficacy in a murine model with human hepatocyte transplants, specifically the TK-NOG mice. TSCMs were discerned in all subjects under examination. Introduction of TSCMs into the HBV mouse model precipitated a severe necro-inflammatory response, resulting in the elimination of human hepatocytes. TSCMs may constitute a valuable tool in the pursuit of a remedial therapy for HBV infection.

Introducing Copi as a Positive Path Toward Combatting Invasive Carps in North America

A group of loosely related, large‐bodied fishes collectively called carps have had a complex relationship with North Americans. Despite lessons learned about invasive Common Carp Cyprinus carpio in the early 1900s, Bighead Carp Hypophthalmichthys nobilis, Black Carp Mylopharyngodon piceus, Grass Carp Ctenopharyngodon idella, and Silver Carp H. molitrix were introduced to the United States more than 50 years ago and are expanding throughout the Mississippi River basin. Increased economic value in the North American seafood market could aid management. Complete eradication through harvest is unlikely, but controlling densities and containing dispersal may be possible. Improving perceptions of nutrition, palatability, and safety of wild‐caught carps should increase consumer demand. A branding and marketing effort launched in June 2022 renamed the foodstuff produced from the four species as the trademarked brand Copi. The “Choose Copi: Eat Well. Do Good.” campaign allows consumers to know that these fishes are an environmentally sound and responsible alternative to other seafood choices. The Copi brand has gained interest nationwide, with food processors and distributors engaged, although the contribution of Copi to harvest removal from rivers and resulting population dynamics is yet to be quantified. Developing a regional fishing industry for Copi, while also aiding fisheries and aquaculture for native species, remains an economic and logistical challenge within the vast river network.

Engineering resveratrol-loaded chitosan nanoparticles for potential use against Helicobacter pylori infection

Helicobacter pylori (H. pylori) is a microorganism directly linked to severe clinical conditions affecting the stomach. The virulence factors and its ability to form biofilms increase resistance to conventional antibiotics, growing the need for new substances and strategies for the treatment of H. pylori infection. The trans-resveratrol (RESV), a bioactive polyphenol from natural sources, has a potential activity against this gastric pathogen. Here, Chitosan nanoparticles (NP) containing RESV (RESV-NP) were developed for H. pylori management. The RESV-NP were prepared using the ionic gelation method and characterized by Dynamic Light Scattering (DLS), Nanoparticle Tracking Analysis (NTA) and, Cryogenic Transmission Electron Microscopy (Cryo – TEM). The encapsulation efficiency (EE) and in vitro release rate of RESV were quantified using high-performance liquid chromatography (HPLC). RESV-NP performance against H. pylori was evaluated by the quantification of the minimum inhibitory/bactericidal concentrations (MIC/MBC), time to kill, alterations in H. pylori morphology in its planktonic form, effects against H. pylori biofilm and in an in vitro infection model. RESV-NP cytotoxicity was evaluated against AGS and MKN-74 cell lines and by hemolysis assay. Acute toxicity was tested using Galleria mellonella model assays. RESV-NP showed a spherical shape, size of 145.3 ± 24.7 nm, polydispersity index (PDI) of 0.28 ± 0.008, and zeta potential (ZP) of + 16.9 ± 1.81 mV in DLS, while particle concentration was 3.12 x 1011 NP/mL (NTA). RESV-NP EE was 72 %, with full release within the first 5 min. In microbiological assays, RESV-NP presented a MIC/MBC of 3.9 µg/mL, a time to kill of 24 h for complete eradication of H. pylori. At a concentration of 2xMIC (7.8 µg/mL), RESV-NP completely eradicated the H. pylori biofilm, and in an in vitro infection model, RESV-NP (4xMIC – 15.6 µg/mL) showed a significant decrease in bacterial load (1 Log10CFU/mL) when compared to the H. pylori J99 control. In addition, they did not demonstrate a toxic character at MIC concentration for both cell lines. The use of the RESV-NP with mucoadhesion profile is an interesting strategy for oral administration of substances targeting gastric disorders, linked to H. pylori infections.

Abstract NG05: Epitope editing enables targeted immunotherapy of acute myeloid leukemia

Abstract NG05: Epitope editing enables targeted immunotherapy of acute myeloid leukemia

Susceptibility of Salmonella enterica Typhimurium dry surface biofilms to disinfection

AbstractIn food preparation and manufacturing environments, surfaces contaminated with Salmonella can lead to outbreaks of Salmonellosis. We hypothesise that Salmonella resides on dry surfaces in a biofilm form leading to potential environmental persistence and transfer following contact. This is the first study reporting that Salmonella Typhimurium can form dry surface biofilm (DSB). Six disinfectants commonly used in the food industry were evaluated for their efficacy against the DSB. The two most efficacious formulations reduced bacterial viability in DSB by >99.99% when combined with mechanical removal (5 sec wiping; 300 g weight). Five out of six formulations significantly reduced bacterial transfer when combined with wiping. Complete eradication of Salmonella Typhimurium DSB was challenging, and mechanical removal was essential to produce a >99.99% reduction in bacterial viability within DSB. This study highlights a potential mode of survival of Salmonella Typhimurium on food‐contact surfaces and DSB challenges for disinfection.

Apoptosis and Autophagy: Therapeutic Implications in Cancer

Despite the advances in the medical field so far, cancer remains a global health priority even now. Considering the drug resistance and the failure of cancer therapies to achieve complete eradication of cancer cells in certain populations, developing molecules that induce programmed cell death or apoptosis has been the focus of cancer research for several decades. Apoptosis evasion is one of the hallmarks of cancer cells, and efforts continue to achieve complete annihilation of cancer cells through selective killing. On the other hand, autophagy, a mode of cell degradation, is considered a double-edged sword. Recent studies show that autophagy also can be manipulated to selectively target cancer cells based on the tumor microenvironment and cellular context. Studies show that autophagy is an evolutionarily conserved process initiated during stress response and has enormous importance in maintaining physiological balance. Most importantly, the dynamic equilibrium between apoptosis and autophagy is crucial in maintaining cellular homeostasis. Although a ‘cell eating’ process, the fate of autophagic cells depends entirely on the nature of stress and the extent of crosstalk between autophagy. This understanding is of immense significance when designing therapeutic interventions targeting apoptosis and autophagy. Currently, several studies are ongoing to gain insights into the role of autophagy in cancer initiation, invasion, progression, angiogenesis, and metastasis. This review focuses on the two major cell death mechanisms, apoptosis and autophagy, in the context of cancer, their crosstalk, and the therapeutic interventions targeting both modes of cell death.

Femtosecond pulsed laser photodynamic therapy activates melanin and eradicates malignant melanoma

Photodynamic therapy (PDT) relies on a series of photophysical and photochemical reactions leading to cell death. While effective for various cancers, PDT has been less successful in treating pigmented melanoma due to high light absorption by melanin. Here, this limitation is addressed by 2-photon excitation of the photosensitizer (2p-PDT) using ~100 fs pulses of near-infrared laser light. A critical role of melanin in enabling rather than hindering 2p-PDT is elucidated using pigmented and non-pigmented murine melanoma clonal cell lines in vitro. The photocytotoxicities were compared between a clinical photosensitizer (Visudyne) and a porphyrin dimer (Oxdime) with ~600-fold higher σ2p value. Unexpectedly, while the 1p-PDT responses are similar in both cell lines, 2p activation is much more effective in killing pigmented than non-pigmented cells, suggesting a dominant role of melanin 2p-PDT. The potential for clinical translational is demonstrated in a conjunctival melanoma model in vivo, where complete eradication of small tumors was achieved. This work elucidates the melanin contribution in multi-photon PDT enabling significant advancement of light-based treatments that have previously been considered unsuitable in pigmented tumors.

Endoscopic treatment of scarred polyps with a non-thermal device (Endorotor): A review of the literature‎.

Endoscopic resection (ER) of colorectal polyps has become a daily practice in most endoscopic units providing a colorectal cancer screening program and requires the availability of local experts and high-end endoscopic devices. ER procedures have evolved over the past few years from endoscopic mucosal resection (EMR) to more advanced techniques, such as endoscopic submucosal dissection and endo-scopic full-thickness resection. Complete resection and disease eradication are the ultimate goals of ER-based techniques, and novel devices have been developed to achieve these goals. The EndoRotor® Endoscopic Powered Resection System (Interscope Medical, Inc., Northbridge, Massachusetts, United States) is one such device. The EndoRotor is a powered resection tool for the removal of alimentary tract mucosa, including post-EMR persistent lesions with scarring, and has both CE Mark and FDA clearance. This review covers available published evidence documenting the usefulness of EndoRotor for the management of recurrent colorectal polyps.

MICROBIOLOGICAL CHARACTERISTICS OF THE EFFECTIVENESS OF THE TREATMENT OF RATS WITH AN EXPERIMENTAL INFECTED WOUND USING AN ANTISEPTIC AND AN IMMUNOMODULATOR

Despite the outstanding achievements in treating wounds, the question of the development and search of new medicines and new medicinal antimicrobial compositions, which would provide an opportunity to improve the treatment of infected and purulent wounds in modern conditions, is still relevant.Material and methods. On the basis of the Department of Microbiology and the veterinary medical clinic of National Pyrogov Memorial Medical University (Vinnytsya, Ukraine), an experimental study was conducted, which consisted of the microbiological evaluation of the use of the immunomodulator "Blastomunil" in the treatment of infected wounds in rats. Under anesthesia, a wound of 1.5x1.5 cm was made in the interscapular area of the animals. It was infected, and then it was treated by intramuscular injection of Blastomunyl, or its local application as monotherapy or in combination with Decamethoxine. The collection of material from the wounds for microbiological research was carried out for 3, 7, 10 and 14 days, followed by a microbiological study of the quantitative content of microorganisms in the wound and their species identification. Numerical values of the number of microorganisms in wounds were expressed as the decimal logarithm of colony-forming units per ml (lg CFU/ml).Research results. Indicators of S.aureus wound contamination on the third day decreased from the 3rd to the 6th group by 1.35, 1.32, 1.46, 1.49, 1.26. and 1.29 times, respectively, with the most significant indicators in 5 (3.74±0.14 lg CFU/ml) and 6 (2.48±0.45 lg CFU/ml) groups, which were significantly different from the control (p<0.05). Regarding P.aeruginosa, a significant logarithmic decrease in microbial contamination was observed in groups 3 (3.78±0.26 lg CFU/ml), 5 (3.12±0.17 lg CFU/ml), 6 (2.48±0.45 lg CFU/ml), and the reduction was respectively, 1.37, 1.66 and 2.09 times, and for the 6th group was reliably the most significant (p<0.05). On the 7th day, a decrease in the number of S.aureus and P.aeruginosa CFU was observed in all groups compared to the control. The most significant decrease in the level of microbial colonization of S.aureus wounds was found in group 5 (2.96±0.08 lg CFU/ml), and its multiplicity was 1.99 times, respectively, compared to the control (p<0.05). A significant reduction in the colonization of wounds by the opportunistic gram-negative bacillus P.aeruginosa was established on the 7th day in groups 5 (2.32±0.36 lg CFU/ml) and 6 (1.43±0.65 lg CFU/ml), the multiplicity of logarithmic reduction of contamination was significant and reliable ( p<0.05) and was 2.0 and 3.26, respectively. On the 10th day, the load of S.aureus in wounds in groups 3-6 significantly decreased. Multiplicity compared to the control was determined in 1.4, 1.54, 2.24, 2.2 times, respectively, with the best result in 5 (2.06±0.04 lg CFU/ml) and 6 (1.9±0.52 lg CFU/ml) groups. For P.aeruginosa, a reliable logarithmic decrease in microbial colonization was determined for groups 2-6, and the multiplicity compared to the control was 1.46, 1.38, 1.91, 1.86, 1.38 times with the best indicators in groups 5 (1.85±0.66 lg CFU/ml) and 6 (1.9±0.52 lg CFU/ml) (p<0.05). On the 14th day, complete eradication of S.aureus and P.aeruginosa was established in wounds of groups 3-6. In the second group, the level of contamination still remained at an insignificant level and was 1.55±0.49 lg CFU/ml S.aureus and 1.04±0.2 lg CFU/ml P.aeruginosa, which is 4.2 and 5 times less than in the control group.Conclusion. Blastomunyl indirectly, apparently due to its immunomodulatory effect, exhibits significant antimicrobial properties, both when administered intramuscularly and when used locally as monotherapy or in combination with Decasan.

Current application and future perspectives of antimicrobial degradable bone substitutes for chronic osteomyelitis.

Chronic osteomyelitis remains a persistent challenge for the surgeons due to its refractory nature. Generally, treatment involves extensive debridement of necrotic bone, filling of dead space, adequate antimicrobial therapy, bone reconstruction, and rehabilitation. However, the optimal choice of bone substitute to manage the bone defect remains debatable. This paper reviewed the clinical evidence for antimicrobial biodegradable bone substitutes in the treatment of osteomyelitis in recent years. Indeed, this combination was proved to eradicate infection and facilitate bone reconstruction, which might reduce the cost and hospital stay. Handling was associated with increased risk of unwanted side effect to affect bone healing. The study provides some valuable insights into the clinical evaluation of treatment outcomes in the aspects of infection eradication, bone reconstruction, and complications caused by materials. However, achieving complete infection eradication and subsequently perfect bone reconstruction remains challenging in compromised conditions, hence advanced innovative bone substitutes are imperative. In this review, we mainly focus on the desired functional effects of advanced bone substitutes on infection eradication and bone reconstruction from the future perspective. Handling property was optimized to simplify surgery process. It is expected that this review will provide an important opportunity to enhance the understanding of the design and application of innovative biomaterials to synergistically eradicate infection and restore integrity and function of bone.

A Review of FDA-Approved Anti-HIV-1 Drugs, Anti-Gag Compounds, and Potential Strategies for HIV-1 Eradication.

Acquired immunodeficiency syndrome (AIDS) is an enormous global health threat stemming from human immunodeficiency virus (HIV-1) infection. Up to now, the tremendous advances in combination antiretroviral therapy (cART) have shifted HIV-1 infection from a fatal illness into a manageable chronic disorder. However, the presence of latent reservoirs, the multifaceted nature of HIV-1, drug resistance, severe off-target effects, poor adherence, and high cost restrict the efficacy of current cART targeting the distinct stages of the virus life cycle. Therefore, there is an unmet need for the discovery of new therapeutics that not only bypass the limitations of the current therapy but also protect the body's health at the same time. The main goal for complete HIV-1 eradication is purging latently infected cells from patients' bodies. A potential strategy called "lock-in and apoptosis" targets the budding phase of the life cycle of the virus and leads to susceptibility to apoptosis of HIV-1 infected cells for the elimination of HIV-1 reservoirs and, ultimately, for complete eradication. The current work intends to present the main advantages and disadvantages of United States Food and Drug Administration (FDA)-approved anti-HIV-1 drugs as well as plausible strategies for the design and development of more anti-HIV-1 compounds with better potency, favorable pharmacokinetic profiles, and improved safety issues.

CURRENT STATE AND PROSPECTS FOR THE DEVELOPMENT OF COUNTERING THE IDEOLOGY OF EXTREMISM IN THE RUSSIAN FEDERATION

Extremism is a complex and multifaceted phenomenon of a destructive nature that exists in almost all states. The natural nature of extremism is associated with the public reaction to processes affecting society. The complete eradication of extremism seems impossible, and therefore, at present, the most urgent thing is to prevent the large-scale spread of extremist behavior in society, which can create threats to the stable functioning of the state. The purpose of the presented research is to study current issues of countering the spread of the ideology of extremism in the Russian Federation, since it is the presence of an appropriate ideology that determines the dynamic development of this illegal phenomenon. The authors come to the conclusion that currently the ideology of extremism is being transformed under the conditions of a number of factors related to the foreign and domestic policies of the state. As the main directions of countering the spread of this ideology, not only preventive and preventive activities of a direct anti-extremist orientation are highlighted, but also the improvement of the ideological views of the population as a whole is of paramount importance. Attention is focused on the need to develop intolerance among the Russian population towards violent methods of conflict resolution and to demonstrate successful forms of self-realization of people within the legal framework.

Abstract 1961: Discovery of a highly potent and brain penetrant fourth generation EGFR tyrosine kinase inhibitor

Abstract Third generation EGFR tyrosine kinase inhibitors (TKIs) such as Osimertinib have emerged as first-line standard of care therapy to treat non-small cell lung cancer (NSCLC) patients with activating EGFR mutations (del19, L858R or T790M). However, within 12 to 18 months following treatment, 15-20% of the patients develop C797S mutation, which drives resistance to these drugs; with the progression of disease brain metastasis is observed in 40-70% of the EGFRm+ NSCLC patients. Here, we report a highly potent and brain penetrant fourth generation EGFR-TKI ACE-34237307, designed to overcome C797S mutation (del19/C797S and L858R/C797S), and common single point mutations (del19 and L858R). ACE-34237307 is also designed to potently inhibit the more prevalent non-canonical EGFR mutations such as G719S and L861Q. ACE-34237307 showed robust anti-proliferative activity against EGFR double mutations in Ba/F3 (EGFR-del19/C797S and EGFR-L858R/C797S) cell lines with IC50 values of 0.1 and 0.23 nM, respectively. In phospho-EGFR assay, ACE-34237307 demonstrates over 79-fold selectivity against A431 (EGFR-WT) cell line when compared to PC-9 (EGFR-del 19) cell line. ACE-34237307 is highly brain penetrant with B/P ratio of 2.3 in rat and 1.2 in mouse. It has an excellent pharmacokinetic profile in rat, mouse and dog with good bioavailability, a flat PK curve and reduced Cmax, which will translate to a better safety profile. The in vivo anti-tumor efficacy was evaluated in Osimertinib resistant double mutant Ba/F3-EGFR-del19/C797S and L858R/C797S as well as del19 and L858R single mutant xenograft models. It has been shown that ACE-34237307 at doses of ≥ 0.6 mg/kg induces complete tumor regression or eradication in all tumor models tested. In brain tumor models with Ba/F3 (EGFR-del19/C797S) and PC-9 (EGFR-del19), ACE-34237307 showed complete inhibition or eradication of brain tumors and drastically extended animal survival at ≥ 0.6 mg/kg. In summary, ACE-34237307 is a potent fourth generation EGFR-TKI with high WT selectivity, excellent brain penetration, highly favorable DMPK profile, outstanding in vivo efficacy in subcutaneous and brain tumor models and low projected human dose amenable to QD dosing. Citation Format: sanjeev kumar, junmei wang, wenqian li, weitao pan, zhiming wen, Kuo-Long yu, Genshi Zhao, tinggui yin. Discovery of a highly potent and brain penetrant fourth generation EGFR tyrosine kinase inhibitor [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 1961.

Abstract 6646: Investigating the combination of radiation and subsequent immune checkpoint inhibitor treatment in head and neck squamous cell carcinoma models

Abstract Purpose: Head and neck squamous cell carcinoma (HNSCC) is often marked by an immunosuppressive tumor microenvironment, which contributes to the dismal 10-20% response rate to immune checkpoint inhibitor (ICI) therapy in HNSCC patients. Radiotherapy (RT) is a currently standard of care and frontline therapy for HNSCC, yet resistance is common. We sought to examine the combination therapy of RT followed by ICI therapy to test if RT induces an in situ vaccination which enhances the efficacy of subsequent ICI. Methods: We utilized SCC cell lines derived from K15.KrasG12D/Smad4−/− SCCs, which mimic the progression of HPV-unrelated HNSCC, and transplanted SCC cells into immune-competent C57BL/6J mouse recipients. Mice were subjected to RT followed by anti-PD-L1/anti-TGFb therapy. CD8+ splenic T cells of responder or non-responder mice were examined by paired single-cell VDJ sequencing and single cell RNA sequencing (TCR/sc-RNAseq) to look for adaptations of the cytotoxic T cell compartment following combination therapy. Responder and non-responder tumor lines or tumors were treated with RT alone in vitro and in vivo, respectively, to examine major histocompatibility complex I (MHC-I) levels on tumor cells. Results: Responders to this combination therapy exhibited complete tumor eradication while non-responders displayed rapid tumor progression. When re-challenged with same HNSCCs cells, responders rejected implanted tumor cells while naïve recipients rapidly developed HNSCCs. Single-cell TCR sequencing of splenic CD8+ T cells revealed that responders, but not naïve recipients exhibited multiclonal CD8+ T cell expansion specifically of memory cell populations. Responder tumor cells exhibited greater levels of MHC-I protein level presentation than non-responders when stimulated with RT. Conclusions: Our data suggest that 1) elevated MHC-I by SCC tumor cells presentation driven by RT and ICI combination therapy facilitate a systemic expansion of memory CD8+ T cell clonotypes which drive anti-tumor immunity and 2) MHC-I expression at baseline or when induced by RT may serve as a predictive marker for therapeutic response to RT and ICI combination therapy in HNSCCs. Citation Format: Hanne Lind, Alexander Strait, Spencer Hall, Jack B. Goon, John D. Aleman, Samantha Chen, Philip Owens, Jing H. Wang, Christian Young, Xiao-Jing Wang. Investigating the combination of radiation and subsequent immune checkpoint inhibitor treatment in head and neck squamous cell carcinoma 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 6646.

Abstract 7239: Eradication of B cell lymphoma in a mouse model through in vivo transduction of T cells with an aCD19-CAR utilizing a CD3-targeted lentiviral vector

Abstract One of the challenges in CAR-T cell therapies is the need for ex vivo processing of autologous T cells. The generation of CAR-T cells directly within the patient has therefore become an attractive alternative. Here, we pseudotyped lentiviral vectors with a VSV-G protein engineered to de-target the LDL receptor and to bind to CD3 via a displayed CD3-specific single-chain antibody (G-CD3), allowing for targeted recognition of T cells. In ex vivo experiments, we demonstrated that engineered G-CD3-LV specifically recognizes and transduces T cells in PBMC and whole blood without apparent off-target transduction of other cells. Furthermore, unlike G-WT-LV, which necessitates T cell activation for transduction, G-CD3-LV does not require pre-activation or the presence of cytokines for successful T cell transduction. The presence of CD3 antibodies on the surface of G-CD3-LV alone was sufficient to activate T cells, as evidenced by CD25 expression and the induction of T cell proliferation. For in vivo evaluation of retargeted LV efficiency NSG mice implanted with NALM6 B-lymphoma were treated with G-CD3 LV encoding αCD19-CAR/GFP (G-CD3/LV-CAR). The tumor was established by tail vein injection of NALM6/F-Luc cells, and 3 or 10 days later animals were humanized via intraperitoneal (IP) injection of human PBMC. Four hours after PBMC transfer, G-CD3/LV-CAR was delivered by IP route. Blood was drawn every week to monitor the generation of CAR-T cells, cytokine levels, and vector copy numbers. Tumor burden was monitored by bioluminescence imaging. Treatment with G-CD3/LV-CAR at both time points resulted in complete tumor eradication. Subsequently, significant levels of CAR-T cells were observed in the blood through Flow analysis, peaking at two weeks after LV injection. Importantly, mice treated on day 10 after tumor establishment reached higher levels of CAR-T cells compared to those treated on day 3 (CAR-T levels were 38±6.5% vs. 10±2.38% of hCD45+ cells, respectively). These results suggest that CAR-T cells proliferated proportionally in response to the level of tumor antigen. With the eradication of the tumor, CAR-T levels in the blood diminished. We also demonstrated that the levels of CAR-T cells correlated well with vector copy numbers. Throughout the treatment, mice did not exhibit any signs of toxicity or body weight loss. Additionally, by measuring cytokines in the blood produced by human cells, we found only a moderate increase in hIFNγ (0.852±0.681 ng/ml) at the peak of CAR-T cell levels in the blood, while the levels of hIL-2 and hTNFα were below the detection limit. In summary, these data highlight the efficiency and potential applicability of fully retargeted, serum stable LV vectors for in vivo CAR-T generation, offering promising prospects to optimize CAR-T cells therapy applications. Citation Format: Karina Krotova, Gopal Naik Nenavath, Nandakumar Packiriswamy, Rianna Vandergaast, Yumeng McDaniel, Melissa Moy, Zachary Larson, Luke Schnebeck, Chia-Hsuan Chin, Kyle Gromer, Kah-Whye Peng, Stephen J. Russell. Eradication of B cell lymphoma in a mouse model through in vivo transduction of T cells with an aCD19-CAR utilizing a CD3-targeted lentiviral vector [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 7239.

Abstract 3184: Development of an innovative bifunctional CPL976-MMAE conjugate based on a bispecific anti-PD-L1/ant-AXL antibody with a dual mechanism of action

Abstract Background: Tumor immunotherapy, especially the combination of PD-1/PD-L1 inhibitors and chemotherapy, has developed rapidly. However, the systemic side effects induced by chemotherapy remain a crucial problem that needs to be addressed. Antibody-drug conjugates (ADCs) are exceptional as a target-specific prodrugs that greatly improve the therapeutic window of chemotherapy drugs. Therefore, we designed the ADC based on a bispecific anti-PD-L1/anti-AXL antibody with toxic MMAE (Monomethyl auristatin E) for precise targeting of cancer cells and reduction of the side effects. Materials and Methods: The CPL976-MMAE conjugate was prepared with site-specific conjugation technology of Fc-glycan remodeling and click-chemistry. The binding to the extracellular domain of AXL and PD-L1 was confirmed with use of the Surface Plasmon Resonance (SPR). After the conjugation with MMAE, cytotoxicity and selectivity of the ADC were evaluated on the human cancer cell lines with different expression levels of AXL and PD-L1. The ADC's ability to force internalization of targets was shown on a panel of cell lines. The effectiveness of ADC was evaluated based on changes in the cell cycle and of treated cells. The high efficacy of the conjugates was confirmed in a mouse model of MDA-MB-231 xenografts in bi-weekly intravenous injection of 6 doses. Results: CPL976-MMAE showed improved cellular uptake, selectivity, and anticancer activity, compared to unconjugated MMAE in the cancer cell lines with different expression of the targets AXL/PD-L1. We confirmed ADCs efficacy using cells expressing high levels of AXL and PDL-1 shown as arrest in G2-M phase of the cell cycle and dose-depended apoptosis. The in vivo experiment on a mouse xenograft model confirmed complete tumor reduction (8/8) after ADC treatment, despite the application of an 11 times lower dose in comparison to the free-MMAE control group and was well-tolerated by mice. Importantly, even after the withdrawal of 976-MMAE for 3 weeks, no tumor regrowth was observed. Conclusions: We presented a new, ADC based on bispecific anti-PD-L1/anti-AXL antibody conjugated with MMAE, that combines the effects of cancer growth inhibition by interaction with AXL and immunotherapy by disruption of the PD-1/PD-L1 axis, with selective delivery of MMAE to cancer cells. The simultaneous action of delivering the toxin and acting on both therapeutic targets provides a potent synergistic anti-tumor effect, that can lead to complete tumor eradication and potentially overcome the primary and secondary resistance to PD-1/PD-L1 targeted therapies. Citation Format: Krysztof Lacek, Anna Jablonska, Olga Abramczyk, Martyna Janowska, Filip Mitula, Aleksandra Sowinska, Agnieszka Bojko-Matuszek, Tomasz Kornatowski, Damian Kolakowski, Magdalena Bojko, Monika Skupinska, Wiktor Pyra, Michal Gorka, Joanna Hucz-Kalitowska, Emilia Mroz, Malgorzata Choros, Jerzy Pieczykoland, Maciej Wieczorek, Delfina Popiel. Development of an innovative bifunctional CPL976-MMAE conjugate based on a bispecific anti-PD-L1/ant-AXL antibody with a dual mechanism of action [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 3184.

Abstract 6653: Oncolytic virotherapy induces a long-term immune antitumoral response leading to glioma eradication

Abstract Oncolytic herpes simplex viruses (oHSVs) have shown promise as effective treatments against various cancers. Our study evaluated the efficacy of R-115, an oHSV retargeted to human Her2 (hHer2), fully virulent in its target cells and expressing murine interleukin 12 (mIL12), in a high-grade, immunocompetent murine glioma model. This model is based on orthotopically transplant of cells derived from PDGFB-induced primary gliomas in syngeneic mice, engineered to express hHer2. Our experimental design compared the impact of R-115 on gliomas where all cells expressed hHer2 against a setup where only about 50% of cells expressed hHer2. The latter aimed to replicate the heterogeneity of patient’s high-grade gliomas, where not all cells express the target molecule. Strikingly, results in terms of overall survival and tumor eradication were similar in both setups, with about a quarter of animals showing complete glioma eradication. This suggests that mIL12-armed oHSV's impact is less about direct viral-induced cell death and more about the immune response elicited by the mIL12-expressing virus. This response effectively targets cells lacking the viral-targeted protein but likely sharing other neoantigens with hHer2-positive cells. Mice cured in both experimental setups were rechallenged with purely hHer2-negative cells. Almost all did not develop gliomas, indicating a strong immune response capable of rejecting the tumor. Plasma analysis revealed that R-115-treated mice from both setups were more immunoreactive against both hHer2-positive and negative cells compared to untreated mice. Further, co-culturing long-surviving mice's splenocytes with glioma cells showed increased cell division in CD8, CD4 and CD19 populations, and elevated IFN-γ, granzyme B, and TNF-α production. These findings underscore R-115's profound anti-cancer potential, especially its capacity to elicit an immune response effective against glioma cells, irrespective of their expression of the target molecule. This outcome, coupled with the results of the rechallenge experiments, is pivotal. It suggests that in a clinical context, an interleukin-12-expressing oHSV could effectively target not only residual cells post-surgical resection but also infiltrating cells that are distant from the site of virus administration. Encouraged by these promising findings, we are currently advancing our research with a novel oHSV that expresses mIL12 and targets EGFRvIII, a mutation found exclusively in tumor cells. This is aimed at enhancing the safety and translatability of this therapeutic approach. Citation Format: Francesca Piaggio, Francesco Alessandrini, Chiara Riviera, Irene Appolloni, Davide Ceresa, Daniela Marubbi, Tatiana Gianni, Gabriella Campadelli-Fiume, Paolo Malatesta. Oncolytic virotherapy induces a long-term immune antitumoral response leading to glioma eradication [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 6653.

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.