Vivo Efficacy Research Articles

A nanoluciferase SFTSV for rapid screening antivirals and real-time visualization of virus infection in mice

Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging tick-borne pathogen that causes severe hemorrhagic fever in humans, but no FDA-approved specific antivirals or vaccines are available to treat or prevent SFTS. The plasmids construction and transfection were performed to generate the recombinant SFTSV harboring the nanoluciferase gene (SFTSV-Nluc). Immunostaining plaque assay was performed to measure viral titers, and DNA electrophoresis and Sanger sequencing were performed to evaluate the genetic stability. Luciferase assay and quantitative RT-PCR were performed to evaluate the efficacy of antivirals invitro. Bioluminescence imaging, titration of virus from excised organs, hematology, and histopathology and immunohistochemistry were performed to evaluate the efficacy of antivirals invivo. SFTSV-Nluc exhibited high genetic stability and replication kinetics similar to those of wild-type virus (SFTSVwt), then a rapid high-throughput screening system for identifying inhibitors to treat SFTS was developed, and a nucleoside analog, 4-FlU, was identified to effectively inhibit SFTSV invitro. SFTSV-Nluc mimicked the replication characteristics and localization of SFTSVwt in counterpart model mice. Bioluminescence imaging of SFTSV-Nluc allowed real-time visualization and quantification of SFTSV replication in the mice. 4-FlU was demonstrated to inhibit the replication of SFTSV with more efficiency than T-705 and without obvious adverse effect invivo. The high-throughput screening system based on SFTSV-Nluc for use invitro and invivo revealed that a safe and effective antiviral nucleoside analog, 4-FlU, may be a basis for the strategic treatment of SFTSV and other bunyavirus infections, paving the way for the discovery of antivirals. This work was supported by grants from the National Key Research and Development Plan of China (2021YFC2300700 to L. Zhang, 2022YFC2303300 to L. Zhang), Strategic Priority Research Program of Chinese Academy of Sciences (XDB0490000 to L. Zhang), National Natural Science Foundation of China (31970165 to L. Zhang, U22A20379 to G. Xiao), the Science and Technology Commission of Shanghai Municipality (21S11903100 to Y. Xie), Hubei Natural Science Foundation for Distinguished Young Scholars (2022CFA099 to L. Zhang).

Antitumor efficacy of a sequence-specific DNA-targeted γPNA-based c-Myc inhibitor

Targeting oncogenes at the genomic DNA level can open new avenues for precision medicine. Significant efforts are ongoing to target oncogenes using RNA-targeted and protein-targeted platforms, but no progress has been made to target genomic DNA for cancer therapy. Here, we introduce a gamma peptide nucleic acid (γPNA)-based genomic DNA-targeted platform to silence oncogenes invivo. γPNAs efficiently invade the mixed sequences of genomic DNA with high affinity and specificity. As a proof of concept, we establish that γPNA can inhibit c-Myc transcription in multiple cell lines. We evaluate the invivo efficacy and safety of genomic DNA targeting in three pre-clinical models. We also establish that anti-transcription γPNA in combination with histone deacetylase inhibitors and chemotherapeutic drugs results in robust antitumor activity in cell-line- and patient-derived xenografts. Overall, this strategy offers a unique therapeutic platform to target genomic DNA to inhibit oncogenes for cancer therapy.

Development and characterization of Tinospora cordifolia extract-loaded SLNs for the treatment of autoimmune hepatitis

Solid Lipid Nanoparticles (SLNs) have emerged as promising drug delivery systems with the potential to enhance the therapeutic efficacy of bioactive compounds. In this study, T. Cordifolia extract-loaded SLNs were developed and characterized for their application in the treatment of autoimmune hepatitis. T. Cordifolia, known for its immunomodulatory and anti-inflammatory properties, was chosen as the active ingredient. The formulations were systematically evaluated for drug-excipient compatibility, particle size, zeta potential, drug loading efficiency, drug release kinetics, encapsulation efficiency, in-vitro release, and stability. The results indicated "No Change" in drug-excipient compatibility, ensuring the formulation's stability. The SLNs exhibited nanoscale particle sizes (159.30 nm to 172.12 nm) with narrow size distributions, facilitating consistent drug delivery. Negative zeta potentials (-28.28 mV to -35.44 mV) indicated colloidal stability. High drug loading efficiencies (up to 32.23%) and controlled drug release kinetics were observed, suggesting the potential for sustained and targeted drug delivery. Encapsulation efficiencies of up to 83.41% highlighted efficient drug loading within the SLNs. In-vitro release studies revealed that SLN2 and SLN4 exhibited superior drug release profiles compared to other formulations. These findings indicate the potential of these formulations for controlled drug delivery. In-vivo efficacy studies in murine models of autoimmune hepatitis are recommended to assess the therapeutic benefits of T. Cordifolia extract-loaded SLNs. Additionally, stability studies demonstrated the maintenance of critical parameters, such as particle size, zeta potential, and drug loading efficiency, under different storage conditions.

Regulatory effects of mangiferin on LPS-induced inflammatory responses and intestinal flora imbalance during sepsis.

Studies suggest that mangiferin (MAF) has good therapeutic effects on chronic bronchitis and hepatitis. Also, it is one of the antiviral ingredients in Anemarrhena asphodeloides Bunge. However, its effect on the LPS-induced inflammation and intestinal flora during sepsis remains unclear yet. In the present study, LPS-stimulated inflammation RAW264.7 cells and LPS-induced sepsis mice were used to evaluate the efficacy of MAF invitro and invivo. 16S rDNA sequencing was performed to analyze the characteristics of intestinal flora of the sepsis mice. It has been demonstrated that MAF (12.5 and 25 μg/mL) significantly inhibited protein expressions of TLR4, MyD88, NF-κB, and TNF-α in the LPS-treated cells and reduced the supernatant TNF-α and IL-6 levels. Invivo, MAF (20 mg/kg) markedly protected the sepsis mice and reduced the serum TNF-α and IL-6 levels. Also, MAF significantly downregulated the protein expressions of TLR4, NF-κB, and MyD88 in the livers. Importantly, MAF significantly attenuated the pathological injuries of the livers and small intestines. Further, MAF significantly increased proportion of Bacteroidota and decreased the proportions of Firmicutes, Desulfobacterota, Actinobacteriota, and Proteobacteria at phylum level, and it markedly reduced the proportions of Escherichia-Shigella, Pseudoalteromonas, Staphylococcus at genus level. Moreover, MAF affects some metabolism-related pathways such as citrate cycle (TCA cycle), lipoic acid metabolism, oxidative phosphorylation, bacterial chemotaxis, fatty acid biosynthesis, and peptidoglycan biosynthesis of the intestinal flora. Thus, it can be concluded that MAF as a treatment reduces the inflammatory responses invitro and invivo by inhibiting the TLR4/ MyD88/NF-κB pathway, and corrects intestinal flora imbalance during sepsis to some degree.

Progressive Design of a Ranatuerin-2 Peptide from Amolops wuyiensis: Enhancement of Bioactivity and In Vivo Efficacy.

Antimicrobial peptides (AMPs) that exert multiple functions are considered promising candidates to combat the bacterial drug resistance crisis. Nowadays, targeted peptide modification has been widely recognised to improve biological activity and make up for deficiencies in clinical applications such as toxicity. In this study, a helix-loop peptide was isolated and identified from the skin secretion of the Wuyi torrent frog Amolops wuyiensis, namely, ranatuerin-2-AW (R2AW) (GFMDTAKNVAKNVAATLLDKLKCKITGGC). Target modifications were made to R2AW to study the structure-activity relationships and to optimise its bioactivities. Five analogues were progressively designed via residue substitution and truncation and the antibacterial and anticancer activities were evaluated. We found that the serine-substitution and cyclic-domain-deletion products showed similar antibacterial activity to the natural peptide R2AW, implying that the disulphide bridge and Rana box were dispensable for the antibacterial activity of ranatuerin-2 peptides. Notably, the cationicity- and hydrophobicity-enhanced variant, [Lys4,19, Leu20]R2AW(1-22)-NH2, exhibited significantly optimised antibacterial and anticancer activities. Additionally, it killed bacteria by membrane disruption at a highly efficient rate. Moreover, [Lys4,19, Leu20]R2AW(1-22)-NH2 exerted potential in vivo efficacy in a methicillin-resistant Staphylococcus aureus (MRSA)-infected waxworm model. Overall, this study demonstrated some rational design ideas for optimising the dual antibacterial and anticancer activities of ranatuerin-2 peptides and it proposes [Lys4,19, Leu20]R2AW(1-22)-NH2 as an appealing candidate for therapeutic development.

Development and early testing of a simple, low cost, fast sensor for maternal and neonatal group B Streptococcus

Streptococcus agalactiae, (Group B Streptococcus (GBS)), is a common colonizer of the female vagina. In women giving birth it can be transmitted to the baby and cause serious illness and even death to the child. We have developed a biosensor comprising of phospholipids and fatty acids vesicles encapsulating high concentration, self-quenched carboxyfluorescein, which is released by the lysis of the vesicle by virulence factors expressed by GBS, becoming diluted and fluorescent. The microbial specificity of the sensor was tested against a number of GBS strains and other microbes including Candida albicans, Enterococcus faecalis and Staphylococcus epidermidis and a statistically significant response to GBS measured over these other microbes. To test the invivo efficacy of the biosensor, a pilot study using donated lower vaginal swabs from non-pregnant women was conducted, where 58 female adults were recruited. Participants donated two swabs, one which was used for the vesicle test and one for the ‘gold standard’, enriched culture media (ECM) test. An overall GBS carriage rate of 17.2% was measured using the ECM test. The vesicle biosensor test took 45 min to obtain a result, and showed a sensitivity of 83.3%, specificity of 85.7% and accuracy of 85.3%. The test accuracy is in line with current novel GBS identification tests, with the advantage of being rapid, easy to use, low-cost and able to be conducted by bedside during start of labour.

Multifunctional Biotechnological Lip Moisturizer for Lip Repair and Hydration: Development, In Vivo Efficacy Assessment and Sensory Analysis

The demand for sustainable cosmetics leads to the search for natural and biotechnological ingredients. The present study reports the development of a multifunctional lip moisturizer containing levan (LEV) from Bacillus subtilis natto, sophorolipids (SOPs) from Starmerella bombicola and Citrus paradisi (OCP) essential oil, using a simplex-centroid experimental design. The formulations were evaluated physicochemically, pharmacotechnically and by DPPH assay. The optimized formulation was selected through the Response Surface Method, and the evaluation of its efficiency in lip hydration was carried out using the bioimpedance method and sensory analysis. The formulations showed pH compatibility with lips and remained stable after a centrifuge test and thermal stress. Spreadability varied between 415.3 and 1217.1 mm2, moisture retention was above 95% and antioxidant capacity was around 50% for all formulations. The optimized formulation, containing 0.4% LEV and 0.8% SOF, maintained the lip hydration already shown by the participants; 85% of them reported improvement in this aspect. For the first time, LEV and SOP were incorporated in lip moisturizers, which is an environmentally friendly product with marketing potential. Furthermore, the use of the Skin Analyzer Digital equipment, a low-cost and non-invasive technique, to evaluate the effectiveness of lip products is innovative; this methodology may help in the development of future cosmetology studies.

Fabrication of chitosan/fibrin-armored multifunctional silver nanocomposites to improve antibacterial and wound healing activities

A wound healing substitute promotes rapid tissue regeneration and protects wound sites from microbial contamination. The silver-based antiseptic frequently moist skin stains, burns and irritation, penetrates deep wounds and protects against pathogenic infections. Thus, we formulated a novel fibrin/chitosan encapsulated silver nanoparticle (CH:F:SPG-CH:SNP) composites bandage accelerating the polymicrobial wound healing. Electrospinning method was employed to form the nano-porous, inexpensive, and biocompatible smart bandages. The structural, functional, and mechanical properties were analyzed for the prepared composites. The biological capacity of prepared CH:F:SPG-CH:SNP bandage was assessed against NIH-3 T3 fibroblast and HaCaT cell lines. In vitro hemolytic assays using red blood cells were extensively studied and explored the low hemolytic effect (4.5 %). In addition, the improved drug delivery nature captured for the CH:F:SPG-CH:SNP composite bandage. Antibacterial experiments were achieved against Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus and Lactobacillus bulgaricus using zone inhibition method. Moreover, in-vivo wound healing efficacy of fabricated smart bandage was evaluated on the albino Wistar rats which revealed the significant improvement on the postoperative abdomen wounds.

Abstract B128: Preclinical characterization of ETx-22, a next-generation antibody drug conjugate (ADC) targeting nectin-4

Abstract Nectin-4 is overexpressed in a variety of cancers including urothelial, breast, cervix, lung and ovarian, with low level of expression in normal tissues. Enfortumab vedotin (EV), an anti-Nectin-4 ADC with a monomethyl auristatin (MMAE) payload, has been a significant advancement in the treatment of urothelial cancer. Yet, it is associated with important skin toxicity owing to the expression of nectin-4 in the skin, and peripheral neuropathy secondary to its toxin. We have developed a next-generation anti-nectin-4 ADC, ETx-22, which consists of a humanized IgG1, Fcg-silent antibody that is bound via maleimide-ß-glucuronide poly-sarcosine linkers to 8 molecules of the topoisomerase-I inhibitor, exatecan. Here, we describe the key pharmacological experiments, in-vivo efficacy and results from the good laboratory practice (GLP) toxicology studies. In-vitro studies have shown that the ETx-22 antibody exhibits a high affinity for tumor-expressed Nectin-4, but 10-fold lower affinity for nectin-4 expressed on skin, with significantly higher internalization in tumor cells. In-vivo efficacy was evaluated in various patient-derived xenograft models. ETx-22 was shown to be consistently superior to EV as well as chemotherapy, in a broad array of tumors, including urothelial, ovarian, cervical and triple negative breast cancer irrespective of the degree of nectin-4 expression. Antitumor activity was also observed in MMAE-resistant models. GLP studies evaluated repeated doses of ETx-22 in cynomolgus monkeys at two doses, 10 and 20 mg/kg. The treatment was administered as 30-minute intravenous infusions once every 2 weeks for a total of three cycles. The ETx-22 antibody alone was also evaluated at 20 mg/kg. Both ETx-22 doses were well tolerated and all animals survived up to scheduled euthanasia. The common observation at both doses was mild multifocal skin hyperpigmentation, which was reversible. No skin rash or pruritus was reported. Mild transient decrease in red blood cells was observed in between cycles. No gastrointestinal toxicities were observed. No signs of interstitial lung disease or eye toxicity were detected. Based on these findings, the highly non- severely toxic dose (HNSTD) is at least 20 mg/kg. The half-life of ETx-22 ranged from 3.36 – 3.6 days. In conclusion, preclinical and pharmacological shows that ETx-22 has better tolerance, longer half-life, and higher efficacy to EV. No skin rash was reported in toxicology studies. A first in human study (EXCEED) is planned to start in 2023. Citation Format: Hatem Azim, Florence Lhospice, Xavier Preville, Joanna Fares, Marc Lopez, Emerence Crompot, Emmanuelle Josselin, Remy Castellano, Maria Wehbeh, Yves Collette, Jack Elands, Daniel Olive. Preclinical characterization of ETx-22, a next-generation antibody drug conjugate (ADC) targeting nectin-4 [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B128.

Abstract A024: Preclinical profile of novel and potent small molecule inhibitors of KIF18A inhibitors in chromosomally unstable solid tumor lines

Abstract Accurate alignment of chromosomes at the equator region and equal chromosome separation during cell division is essential for maintenance of genome integrity. KIF18A belongs to kinesin superfamily of microtubule associated motor proteins and is responsible for maintenance of bipolar spindle fiber integrity during mitotic phase. Cancer cells with chromosomally unstable (CIN+) DNA show synthetic lethal phenotype with genetic ablation of KIF18A. Thus, pharmacological inhibition of KIF18A represents a potential strategy aimed at attenuating growth of CIN +ve tumors. A microtubule dependent ADP Glo kinase assay was used to identify potent KIF18A inhibitors. Mitotic arrest, manifested by an increase in phosphoH3, was determined in cells using fluorescent microscopy. Anti-proliferative activity was confirmed in CIN+ve solid tumor cells with significant selectivity over CIN-ve cancer cells. Compounds inhibited KIF18A at with potencies ranging from 50-80 nM. A dose dependent increase in mitotic index in CIN+ve OVCAR-3 cells (via fluorescence microscopy) was observed in contrast to CIN-ve HCT-116 cells (10-100X selectivity). Translation was evident in an antiproliferation assay using CIN-ve cancer cells with IC50 <10 nM. Selected KIF18A inhibitors demonstrated favorable physicochemical, ADME and PK properties. In Vivo efficacy studies are planned. Citation Format: Sukanya Patra, Manoj Pothuganti, Navnath Karche, Ganesh Gudade, Venu Sankeshi, Githavani Kummari, Srikant Vishwanadha. Preclinical profile of novel and potent small molecule inhibitors of KIF18A inhibitors in chromosomally unstable solid tumor lines [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A024.

Abstract C102: mRNA-mediated restoration of BRCA-1 tumor suppressor function prevents tumor growth and reverses resistance to PARP inhibitors in triple-negative breast cancers

Abstract BRCA1 and BRCA2 proteins are involved in the repair of DNA damage such as double-strand breaks during S and G2 phases, by intervening in homologous recombination (HR) steps. Breast cancers arising from BRCA-1 mutation carriers, up to 10% BC, are associated with a lack of expression and/or function of BRCA-1 protein, which induces genomic instability. Considering the major role of BRCA1 gene in DNA repair process, therapeutic approaches using cytotoxic agents such as the platinum and PARP inhibitors are used as treatments. However, hypersensitivity to PARPi is limited in the clinic by acquired resistance after restoration of HR. We have developed a new potent strategy combining mRNAs with a tumor selective nanocarrier, to selectively rescue BRCA-1 functions and restore PARPi sensitivity, as potential therapeutic approach in cancers. ADGN-technology is based on short amphipathic peptides that form stable neutral nanoparticles with mRNA. ADGN-901, -902 , -903 nanoparticles containing mRNA derived from BRCA-1 were evaluated on breast cancer cells, harboring different BRCA-1 deletion and mutations. Cell proliferation, cell cycle level and apoptosis activation were determined by flow cytometry and Tunel assay. BRCA-1 and RAD51 protein levels were evaluated by western blot. Sensitivity to Veliparib (PARPi) following ADGN-mRNA treatment was evaluated on PARPI resistance and PARPi sensitive cells. The recruitment of RAD51 to sites of double stranded DNA break was quantified by immunofluorescence. In-vivo efficacy of IV-administered ADGN-BRCA NPs (2.0 mg/kg) was evaluated on breast (HCC1937-BRCA-1 deficient, PARPi resistant) cancer mouse xenografts. We have selected specific mRNA sequences encoding for different domains of BRCA-1 protein. We showed than ADGN-901 mediated rescue of BRCA-1 wild type expression inhibits the proliferation of BRCA-1 deficient cell lines from 20 to 60% depending on the type of BRCA-1 alterations. ADGN-902, specifically reduces the proliferation of cells expressing N-terminal truncated BRCA-1, by 50 to-60% and ADGN-903 markedly delay (>60%) the growth of all cells harboring defect in BRCA-1. In contrast, no significant cytotoxicity related to vehicle was observed. ADGN-901 and ADGN-903 resensitize the resistant cells to PARPi Veliparib inhibition. We demonstrated that the expression of domain of BRCA-1 encoded by ADGN-903 mRNA, appears to prevent RAD51 recruitment to the damage site by reducing RAD51 protein level as well as formation of RAD51 foci following irradiation. Intravenous-administration ADGN-901 and ADGN-903 (2.0mg/kg) prevented HCC-1937 tumor growth. ADGN-901 reduced tumor growth by 95% and ADGN-903 resulted in a tumor regression of 50%. ADGN-901 and ADGN-903 treatments are well tolerated, without inducing clinical toxicity. ADGN-BRCA nanoparticles are effective in rescuing BRCA-1 functions in Breast cancer cells. Our study provides a proof-of-concept that restoration of the expression of the wild type form of BRCA-1 could be combined together with PARP inhibition for potent combinatorial cancer treatment. Citation Format: Gilles DIVITA, Audrey Grunenberger, Léa Cabrera, Nathalie Durany, Khadidja Boularache, Veronique Josserand, Neil Desai. mRNA-mediated restoration of BRCA-1 tumor suppressor function prevents tumor growth and reverses resistance to PARP inhibitors in triple-negative breast cancers [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C102.

Abstract C075: A first-in-class and highly selective SHP1 allosteric inhibitor exhibits robust anti-tumor immunity and synergizes with PD-1 blockade

Abstract Background: SHP1 is a member of the src homology 2 (SH2) domain-containing protein phosphatases. It contains two SH2 domains at the N-terminus, a catalytic domain and a C-terminal tail. SHP1 is primarily expressed in hematopoietic cells and commonly recognized as a negative regulator of multiple signaling pathways. It has been known that SHP1 interacts with proteins of the inhibitory-receptor superfamily containing immunoreceptor tyrosine-based inhibitory motifs. Methods: To screen for SHP1 allosteric inhibitors, we employed a DiFMUP high-throughput biochemical assay system. Compounds that only perturbed the activity of SHP1 was further optimized for cellular activity on target, in vitro ADME, and in vivo pharmacokinetic properties. X-ray crystallography has been introduced to determine the interaction between the inhibitor and the allosteric site of SHP1. We also evaluated the lead series in in-vitro studies with human primary immune cells and in-vivo efficacy studies in syngeneic mouse models. Results: We discovered a series of SHP1 allosteric inhibitors with IC50 values at the nanomolar level and high selectivity over the other PTP enzymes, including SHP2. Structurally, SHP1 adopts an autoinhibited conformation in its basal state, where the N-SH2 domain interacts with the PTP domain and blocks access to the catalytic site. X-ray crystallography reveals that our SHP1 inhibitors are bound to non-catalytic sites of the SHP1 inactive conformation. Among the compounds, SB6299 demonstrated potent cytokine responses in various human primary immune cell types. SB6299 significantly enhanced IL-2, IFN-γ, and TNF-α productions in human primary pan-T, NK, and monocyte cells, respectively. The cytokine release was confirmed as a result of selective SHP1 inhibition through the evaluation of SHP1 knockout in primary immune cells. Moreover, SB6299 showed dose responses in costimulatory molecules and cytokines (TNF-α and IL6) in human monocyte derived dendritic cells (DCs) and enhanced DC maturation markers such as CD80, CD86, and CD40. The anti-tumor efficacy of SB6299 was evaluated in the CT26 and MC38 syngeneic mouse models. Orally administered SB6299 showed tumor growth inhibition as a single agent, and the efficacy was further enhanced when administered in combination with an anti-PD1 antibody. Conclusions: 1. Our studies successfully demonstrate that SHP1 can be allosterically inhibited, allowing selectivity with other phosphatases, including SHP2. 2. SB6299 is the first-in-class orally available, potent, and selective SHP1 allosteric inhibitor. 3. Attenuation of SHP1 activity by SB6299 could promote cytokine secretion and cytotoxic effects in human primary immune cells. 4. Orally administered SB6299 induced tumor growth inhibition as a single agent and in combination with an anti-PD1 antibody. 5. Currently, more selective and potent inhibitors are being tested. Citation Format: Jun Gyu Kim, Ki Moon Ryu, Mi Yeon Jang, Jongho Cho, Dajeong Kim, Dae Hyeon Seong, Kyu Hwan Kim, Ik-Soo Jang, Jeong Yoon Shin, Hong Sik Han, Joon Yonug Hwang, Dae Young Lee, Chae Lim Ryu, Song Yi Lee, Tae-Dong Han, SeungMin Yang, Hyounmie Doh. A first-in-class and highly selective SHP1 allosteric inhibitor exhibits robust anti-tumor immunity and synergizes with PD-1 blockade [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C075.

T Cell Selection Is Not Helpful for Therapeutic Efficacy of CART19 in Relapsed or Refractory B-ALL Patients with Peripheral Blood Leukemia Burden at Time of Apheresis

Abstract Introduction: We have demonstrated peripheral blood (PB) blasts negatively affected chimeric antigen receptor T cells targeting CD19 (CART19) production and the clinical efficacy of CART19 therapy in patients (pts) with refractory/relapsed B cell acute lymphoblastic leukemia (r/r B-ALL) (published in Molecular Therapy: Methods & Clinical Development ,2021). Whether sorting T cells before activation might be helpful for ex-vivo CART19 quality and in-vivo treatment efficacy requires further investigation. Here, we retrospectively analyzed 40 pts with or without upfront T cell purification to evaluate these indexes. Patients and Methods: From July 2021 to June 2023, 40 r/r B-ALL with PB blasts at time of apheresis experienced CART19 therapy. After leukapheresis, all pts' PBMCs were isolated using lymphocyte separation liquid. 30 pts didn't perform T cells selection (group 1) and 10 pts performed T cells selection (group 2). All of cells were activated with CD3 and CD28 antibodies for 24h, then transduced with the lentivirus encoding anti-CD19-CD3zeta-4-1BB CAR and cultured for 5-6 days in serum-free media containing IL2, IL7, IL15, IL21. The baseline characteristics, including age, sex, previous therapy period, blasts in BM, EMD distribution, complex chromosome aberration, poor fusion genes and gene mutations, did not differ between the two groups (Table 1). Cell viability, expanding fold, percentage of CD3 +T and CD3 + CD8 +T cells, transfection efficiency, B cells were all examined pre CART19 infusion. All pts received cyclophosphamide 250 mg/m2/d X 3d, and fludarabine 30 mg/m2/d X 3d, followed by CART19 infusion with a median number of 9.7(0.17-60) X10 5cells/kg in group 1 and 10 (4-16.3) X10 5cells/kg in group 2 ( P=0.99). PB and BM blasts was determined by flow cytometry (FCM). All pts underwent bone marrow (BM) biopsy examination and FCM studies on day 15 or day 30 to determine the response and remission status. Results: At time of apheresis, group 1 and group 2 had a median leukemia cell of 18% (2%-71%) and 19.4% (7%-54.4%) in PB ( P=0.5), respectively. In group 1 and group 2, the median percent of CD3 +T and CD3 + CD8 +T cells were 97.7% (70% - 99.5%) vs. 97.7% (94.8%-99.2%) ( P=0.46) and 66% (24.3% - 88.3%) vs. 64.2% (28% - 84.4%) ( P=0.45), respectively. The median cell viability, transfection efficiency and cell expanding fold in the final products was 82.9%（44.4%-92.2%) vs. 88.6%（76.4%-95.2%）( P=0.09), 31.4%（3.1%-65.5%) vs. 23.6% (12.4%-35.5%) ( P=0.08) and 3.18-fold（0.24-13.6) vs. 2.32-fold（0.48-8.13) ( P=0.83), respectively. Both of normal B and leukemia cells were undetectable pre CART19 infusion in two groups. After CART19 infusion, the median peak CART19/ lymphocyte cells, expanding fold and absolute CART19 count in vivo was 45% (0-79.4%) vs. 57.9% (0-75%) ( P=0.86), 16.7-fold (0-397.3) vs.13.2-fold (0-406)( P=0.96) and 229 (0-4810) X10 6/L blood vs. 242 (0-3220) X10 6/L blood ( P=0.83) in two groups, respectively. In group 1, 28/30 cases were evaluable and 25/28 (89.3%) obtained complete remission (CR)/ incomplete count recovery CR (CRi) and 24/28 (85.7%) obtained minimal residual disease (MRD) - CR. 1/2 patient unevaluable died from pneumonia due to severe and persisted neutropenia. One no-responder was infused at <1 X10 5 cells/kg and his culture failed to expand in vivo, and the other no-responder was infused at 1.16 X10 5 cells/kg (with TEL-AML1 positive). In group 2, 8/10 cases were evaluable and 6/8 (75%) obtained CR/CRi with all MRD - CR (75%). The two non-responders had no detectable CART19 in PB after infusion for one month. Results were shown in Table2. Conclusion: We have demonstrated pts with PB blasts without sorting T cells had poorer ex-vivo CART19 quality and in-vivo treatment efficacy compared to pts without PB blasts (published). Sorting T cells before activation is expected to remove the adverse culture conditions in the system. However, in the present study, upfront T cell purification didn't improve ex vivo cell characteristics and in vivo cell dynamics in r/r B-ALL pts with PB blasts, showing that the negative influence of PB blasts at the time of apheresis could not be reversed by upfront T cell purification. Therefore, leukemia burden in PB is suggested to be reduced before leukapheresis. Absolutely, one of the limitations of our study is that a relatively small number of products was evaluated, and larger samples need to be included to further demonstrated.

A Novel Patient-Derived Xenograft Model IO-FIVE for Drug Discovery and Precision Medicine in Lymphoma and Leukemia Patients

Lymphoma and leukemia have been the top two hematopoietic cancers in US, leading to more than 40,000 deaths each year. Although the 5-year relative survival rates have been improved up to 60%, there is still a lot of patients showed not response to standard therapy. Patient-Derived Xenograft model (PDX model), as the classical mouse xenograft tumor model that best represents the genetic information characteristics of human tumors, can be used to predict the therapeutic effect, and develop individualized treatment for patients. Its greatest advantage is that the model retains the microenvironment of the original tumor cells, inherits all the molecular biological properties of the primary tumor, and preserves the heterogeneity of the tumor. However, due to the factors that the loss of tumor heterogeneity in hematopoietic cancers after ex vivo clonal expansion and selection, and this in vitro assay based system could not fully reflect the host response to the drugs, including a range of novel immunotherapeutic agents such as PD-1 monoclonal antibody, CD38 monoclonal antibody, etc. With the increasing clinical use of oncology immunological agents, recently we are developing a new assay, named IO-FIVE (Immuno-Oncology drugs Fast In Vivo Efficacy test) for research and therapy, by retaining an appropriate proportion of tumor-infiltrating immune cells (and stromal cells) mixed with tumor cells when digesting clinical tumor samples or patient-derived xenografts-preserved tumor samples into cell suspensions, and then injecting them into the specialized IO-FIVE device, a modified microencapsulation and hollow fiber culture system (OncoVee® MiniPDX), which is implanted subcutaneously into mice and administered systematically for immuno-drug susceptibility testing to screen for immuno-drugs or combinations suitable for individual treatment. The IO-FIVE testing cycle takes only 14 days and is performed using Celltiter Glo before and after the injection of cell suspensions into the specialized device. Celltiter Glo, flow cytometry and Omics (RNA-seq, DNA-seq) are used to measure the viability of total cell subpopulations, the relative ratio of tumor cells to immune cells and the alteration of cellular transcription levels in the device, in order to further explore the sensitivity of tumor tissues to the immunotherapeutic drugs and the potential molecular mechanism for the responders and non-responders. Immune regulatory CD38 antibody Daratumumab has been tested in more than 40 acute myeloid leukemia (AML) cases, with its overall in vivo efficacy around 25%, which is consistent with previous research. It is interesting to uncover novel pathways for drug discovery and clinic treatment since CD38 is well expressed in most AML patients, but at least half of those CD38 + AML patients are not sensitive or resistant to Daratumumab monotherapy. Expression of CD38 on AML is not enough to distinguish the responder vs non-responder of Daratumumab. IO-FIVE could potentially be a companion diagnosis on AML for the patient stratification of Daratumumab. PD1 antibody Sintilimab has been tested in all the patients (12 lymphoma, 52 leukemia), and the only two laboratory PD1-Ab non-responsive T cell lymphoma patients have been showed to be clinically non-responders as well. Immunotherapy and chemical or targeted therapy combination could enhance tumor killing ability and disease control. We will use the IO-FIVE data to further compare it with its source of real-world clinical patients to obtain more evidence-based evidence that can predict individualized treatment with immunotherapy.

Improving In Vivo Tumor Accumulation and Efficacy of Platinum Antitumor Agents by Electronic Tuning of the Kinetic Lability.

The impact of kinetic lability or reactivity on in vitro cytotoxicity, stability in plasma, in vivo tumor and tissue accumulation, and antitumor efficacy of functional platinum(II) (Pt) anticancer agents containing a O˄O β-diketonate leaving ligand remain largely unexplored. To investigate this, we synthesized Pt complexes [(NH3 )2 Pt(L1-H)]NO3 and [(DACH)Pt(L1-H)]NO3 (L1=4,4,4-trifluoro-1-ferrocenylbutane-1,3-dione, DACH=1R,2R-cyclohexane-1,2-diamine) containing an electron deficient [L1-H]- O˄O leaving ligand and [(NH3 )2 Pt(L2-H)]NO3 and [(DACH)Pt(L2-H)]NO3 (L2=1-ferrocenylbutane-1,3-dione) containing an electron-rich [L2-H]- O˄O leaving ligand. While all four complexes have comparable lipophilicity, the presence of the electron-withdrawing CF3 group was found to dramatically enhance the reactivity of these complexes toward nucleophilic biomolecules. In vitro cellular assays revealed that the more reactive complexes have higher cellular uptake and higher anticancer potency as compared to their less reactive analogs. But the scenario is opposite in vivo, where the less reactive complex showed improved tissue and tumor accumulation and better anticancer efficacy in mice bearing ovarian xenograft when compared to its more reactive analog. Finally, in addition to demonstrating the profound but contrasting impact of kinetic lability on in vitro and in vivo antitumor potencies, we also described the impact of kinetic lability on the mechanism of action of this class of promising antitumor agents.

2062. In Vivo Efficacy of CD388, a Novel Drug Fc-Conjugate (DFC), Against Seasonal Subtypes of Influenza in Prophylaxis in Immune Competent Mice, and in a Severe Immunodeficient (SCID) Mouse Model

Abstract Background Influenza remains a significant public health concern due to the limited efficacy of vaccines. Cidara has developed CD388, a multivalent conjugate of a dimeric neuraminidase inhibitor with a proprietary hIgG1 Fc domain engineered for extended half-life, currently in clinical development. Here we describe the efficacy of CD388 against a panel of geographically and temporally diverse influenza A and B subtypes in mouse prophylactic models, and against H1N1 in a SCID model. Methods Efficacy studies were conducted in BALB/c mice lethally challenged with influenza virus. CD388 was administered intramuscularly (IM), seven days prior to challenge. Animals were monitored daily for 21 days or longer for survival (< 20% BW loss). For SCID studies, mice were dosed with CD388 two hours prior to lethal challenge with influenza A H1N1. Oseltamivir and baloxavir were included as comparators. Results Across all influenza subtypes, CD388 treatment at ≤1 mg/kg resulted in 100% survival (Table 1). Daily BW measurements of treatment groups supported survival findings and showed a transient BW loss (generally between 5 and 15% of starting weight), before animals recovered weight. In SCID studies, the median time of death for vehicle treated animals was seven days. Treatment with oseltamivir (5 mg/kg, bid x5d) and baloxavir (15 mg/kg, bid x1d) at their human equivalent doses extended the median survival time to Day 10 and 16, respectively (Figure 1). In contrast, a single dose of CD388 at 1 mg/kg extended the median time of death to beyond Day 21. A dose response was evident at higher CD388 doses with the median time of death extended to Day 30 and 35 for 3 and 10 mg/kg groups, respectively. Conclusion This data demonstrated a single IM administration of CD388 fully protected animals from lethal challenge against seasonal influenza A and B. Importantly, CD388 was found to be efficacious in a severe model of immune deficiency. This data supports the prophylactic use of CD388 to prevent seasonal influenza in individuals. Disclosures James Levin, PhD, Cidara Therapeutics: Stocks/Bonds Simon Döhrmann, PhD, Cidara Therapeutics: Stocks/Bonds Jason N. Cole, Ph.D., Cidara Therapeutics: Stocks/Bonds Karin Amundson, BSc, Cidara Therapeutics: Stocks/Bonds Allen Borchardt, PhD, Cidara Therapeutics: Stocks/Bonds Thomas P. Brady, Ph.D., Cidara Therapeutics: Stocks/Bonds Thanh Lam, PhD, Cidara Therapeutics: Stocks/Bonds Joanne Fortier, BSc, Cidara Therapeutics: Stocks/Bonds Amanda Almaguer, Bachelors, Cidara Therapeutics: Stocks/Bonds Leslie W. Tari, Ph.D., Cidara Therapeutics: Stocks/Bonds

Tuning the Reactivity of Nuclear Factor Erythroid 2-Related Factor 2 (Nrf2) Activators for Optimal in Vivo Efficacy.

Dimethyl fumarate 1 is approved for the treatment of multiple sclerosis but is also associated with off-target activation of the niacin receptor. By using a tetrazolone or triazolone bioisostere approach to the fumarate and vinyl sulfone series of Nrf2 activators, we have optimized the electrophilicity of the double bond to tune the on-target Nrf2 activation with PK properties to achieve efficacy in animal models of multiple sclerosis. The study linked highly potent, highly electrophilic molecules to low plasma stability and, subsequently, limited efficacy. By contrast, a sulfonylvinyltriazolone 17 retains on-target potency but shows much weaker electrophilic potential. As a consequence, in vivo high exposures of 17 are obtained, resulting in efficacy in the EAE model similar to that observed for DMF. 17 (R079) is Ames negative, is not cytotoxic to cells, and shows little inhibition of either the niacin receptor or a panel of off-target receptors.

Comparison of five retinoids for anti-photoaging therapy: Evaluation of anti-inflammatory and anti-oxidative activities invitro and therapeutic efficacy invivo.

Over the past decades, increasing evidences have demonstrated that five retinoids, including retinol (ROL), retinol acetate (RAc), retinol propionate (RP), retinol palmitate (RPalm), and hydroxypinacolone retinoate (HPR), can be potential therapeutic agents for skin photoaging. However, therapeutic efficacies and biosafety have never been compared to these compounds. This study aimed to determine the optimal retinoid type(s) for anti-photoaging therapy both invitro and invivo. Our data demonstrated that four retinoids (RPalm, RP, HPR and ROL) but not RAc were effective for anti-photoaging treatment at 5 μg/mL invitro, with action mechanisms associated with antioxidative, anti-inflammatory and anti-skin ECM degradation activities. Notably, both RPalm and RP appeared superior to HPR and ROL for those activities. Importantly, both RPalm and RP were shown to be optimal for anti-photoaging therapy when topically applied at 5 mg/kg in a UVB-induced mice model of photoaging, which is consistent with their high anti-photoaging activities invitro. Additionally, topical application of these five retinoids showed satisfactory biosafety without causing significant apoptosis in animal organs, although RP application led to a slight decline in animal body weights. Collectively, these data have laid a good foundation for the next development of the clinical application of these retinoids for skin healthcare.

Reinforced dentin remineralization via a novel dual-affinity peptide

ObjectivesIn light of the constantly flowing saliva, anti-caries remineralization agents are inclined to be taken away. Owing to their limited residence time, the remineralization effect is not as desirable as expected. Hence, our study aimed to synthesize a novel peptide (DGP) with high affinity to both collagen fibrils and hydroxyapatite, and investigated its dentin remineralization efficacy in vitro and anti-caries capability in vivo. MethodsDGP was synthesized through Fmoc solid-phase reaction. The binding ability and interaction mechanism of DGP to demineralized dentin were investigated. Dentin specimens were demineralized, then treated with DGP and deionized water respectively. The specimens were incubated in artificial saliva and in-vitro remineralization effectiveness was analyzed after 14 days. The rat caries model was established to further scrutinize the in-vivo efficacy of caries prevention. ResultsDGP possesses an enhanced adhesion force of 12.29 ± 1.12 nN to demineralized dentin. The favorable adsorption capacity is ascribed to the stable hydrogen bonds between S2P-101 and ASP-100 of DGP and GLY33 and PRO-16 of collagen fibers. Abundant mineral deposits and remarkable tubule occlusion were observed in the DGP group. DGP-treated dentin obtained notable microhardness recovery and higher mineral content after a 14-day remineralization regimen. DGP also demonstrated potent caries prevention in vivo, with substantially fewer carious lesions and significantly lower Keyes scoring. SignificanceDGP proves to possess a high affinity to demineralized dentin regardless of saliva flowing, thus enhancing remineralization potency significantly in vitro and in vivo, potential for dental caries prevention and combatting initial dentin caries clinically.

Explaining in-vitro to in-vivo efficacy correlations in oncology pre-clinical development via a semi-mechanistic mathematical model

In-vitro to in-vivo correlations (IVIVC), relating in-vitro parameters like IC50 to in-vivo drug exposure in plasma and tumour growth, are widely used in oncology for experimental design and dose decisions. However, they lack a deeper understanding of the underlying mechanisms. Our paper therefore focuses on linking empirical IVIVC relations for small-molecule kinase inhibitors with a semi-mechanistic tumour-growth model. We develop an approach incorporating parameters like the compound’s peak-trough ratio (PTR), Hill coefficient of in-vitro dose-response curves, and xenograft-specific properties. This leads to formulas for determining efficacious doses for tumor stasis under linear pharmacokinetics equivalent to traditional empirical IVIVC relations, but enabling more systematic analysis. Our findings reveal that in-vivo xenograft-specific parameters, specifically the growth rate (g) and decay rate (d), along with the average exposure, are generally more significant determinants of tumor stasis and effective dose than the compound’s peak-trough ratio. However, as the Hill coefficient increases, the dependency of tumor stasis on the PTR becomes more pronounced, indicating that the compound is more influenced by its maximum or trough values rather than the average exposure. Furthermore, we discuss the translation of our method to predict population dose ranges in clinical studies and propose a resistance mechanism that solely relies on specific in-vivo xenograft parameters instead of IC50 exposure coverage. In summary, our study aims to provide a more mechanistic understanding of IVIVC relations, emphasizing the importance of xenograft-specific parameters and PTR on tumor stasis.