Preclinical Immuno-recognition and Neutralization of Lethality Assessment of a New Polyvalent Antivenom, VINS Snake Venom Antiserum – African IHS®, against Envenomation of Ten African Viperid and Elapid Snakes

Snakebite envenomation is a major health concern in developing countries causing significant mortality and morbidity. With over 1.2 million cases annually caused by medically important snake species belonging to the two families Viperidae (Echis spp. and Bitis spp.) and Elapidae (Naja spp. and Dendroaspis spp.). Several antivenoms are being produced and distributed to western sub-Saharan Africa for treatment of envenomation with the absence of preclinical efficacy studies. The present study evaluated the preclinical efficacy of venoms from Echis leucogaster, Echis ocellatus, Bitis arietans, Bitis gabonica, Naja haje, Naja melanoleuca, Naja nigricollis, Dendroaspis jamesoni, Dendroaspis polylepis and Dendroaspis viridis against a polyvalent Snake Venom Antiserum - African IHS (lyophilised), manufactured by VINS Bioproducts Limited (Telangana, India). Our in vitro results showed that, the SVA- AIHS contains antibodies that are capable of recognizing and binding majority of protein components representative of all eight major protein families of venoms of the snake species tested by double immunodiffusion assay and confirmed by western blot. The venom antiserum exhibited high neutralization efficacy against all the viperid and elapid snake species venoms in in vivo studies and confirmed the manufacturer’s recommended neutralization capacity. This is clear evidence that the VINS polyvalent SVA-AIHS batch tested has strong neutralizing capacity and will be useful in treating envenoming by most African viperid and some elapid snake species.

A Study on Preclinical Efficacy, Underlying Mechanisms, and Sensitivity Markers of a Novel Hypomethylating Agent Ntx-301 in Acute Myeloid Leukemia

Introduction: While intensive induction chemotherapy has been standard-or-care for patients with acute myeloid leukemia (AML), intensive regimens have often been limited in elderly patients with comorbidities. As an alternative option, less intensive hypomethylating agents (HMAs), decitabine (DAC) and azacitidine (AZA), are currently being used in these unfit patients. However, their low response rates and adverse events when administered alone suggest a need to develop novel HMAs that elicit enhanced efficacy and reduced mortality. A recent study demonstrated pharmacological improvements of a novel 4′-thio-modified analog, 5-aza-4′-thio-2′-deoxycytidine (hereafter NTX-301), including enhanced chemical stability and incorporation into DNA and improved preclinical efficacy (Thottassery, 2014). However, the mechanism of action (MoA) of NTX-301 has not yet been understood. Herein, we aim to thoroughly investigate the preclinical efficacy and MoAs of NTX-301 in AML through comparative analysis with DAC and AZA. To this end, we used in vitro and in vivo preclinical models of AML and performed multiomics-based analyses. Results: We comprehensively examined viability of 200 cancer cell lines (CCLs) upon treatment with NTX-301. Consistent with the current use of HMAs as therapeutics for hematologic malignancies, this sensitivity profiling displayed the most remarkable potency of NTX-301 toward blood CCLs (OR=3.97, p=0.0003). In addition, phenotypic assays revealed that the anti-leukemic activity of NTX-301 was superior to that of DAC, which is attributed to more effective actions in inducing apoptosis, cell cycle arrest, and differentiation. Supporting the in vitro results, orally administrated NTX-301 led to more potent tumor regression, better tolerability, and survival benefits over DAC and AZA in both systemic (1.5-2.0 mg/kg, qdx5 then 2 days off, 5 days on, and 9 days off, for 3 cycles) and subcutaneous (0.2-1.5 mg/kg) xenograft models. To assess the underlying MoAs, we interrogated global alterations at the transcriptome and methylome levels upon treatment with NTX-301 in three AML cell lines using RNA sequencing and methylation array. Methylome analyses revealed that NTX-301-induced demethylation patterns were distinguished from DAC; DAC triggered stronger global demethylation than NTX-301 did, whereas NTX-301 derived rather selective demethylation, preferentially in early-replicating regions, H3K27ac-marked regions, and non-CpG islands. In transcriptome analyses compared with DAC, NTX-301 more markedly elicited a transcriptional reversal toward a normal myeloid-like signature by increasing a differentiation signature and suppressing a leukemic stem cell signature. NTX-301 also mediated more pronounced activation of DNA damage response and the p53 pathway, which are characterized by marked induction of pH2AX and pChk1 and increased stability of p53, respectively. Given the synthetic lethality of p53 activation and BCL2 inhibition (Rongqing, 2017), stronger p53 stabilization by NTX-301 may confer more benefits in combination with venetoclax. Indeed, the combination of NTX-301 + venetoclax produced a more synergistic combination index compared with DAC + venetoclax. Strikingly, the combined NTX-301 (0.5-2.0 mg/kg) + venetoclax (50 mg/kg) achieved complete tumor remission, no notable toxicity, and prolonged survival benefits over AZA (2.5 mg/kg) + venetoclax (50 mg/kg) in preclinical models of AML. By integrating sensitivity profiles and multiomics data of 200 CCLs, we interrogated molecular determinants associated with sensitivity to NTX-301. Intriguingly, when comparing methylomes between sensitive and resistant CCLs, we found a significantly biased global hypermethylation trend toward sensitive CCLs. A combinatorial set of the most significantly biased 352 differentially methylated regions (FDR<0.05) showed potential as a predictive sensitivity marker for NTX-301, exhibiting a significant correlation (r=0.69, p<0.0001) with sensitivity to NTX-301. Conclusions: Our study demonstrated an improved therapeutic index of NTX-301 over traditional HMAs, providing a rationale for further clinical development of the agent as a single-agent or in combination with other agents. We also believe that our study for MoAs and biomarkers will improve our understanding of NTX-301. Disclosures Lim: Pinotbio: Research Funding. Yoo: Pinotbio: Research Funding. Cho: Pinotbio: Research Funding. Choi: Pinotbio: Research Funding. Jung: Pinotbio: Research Funding. Jung: Pinotbio: Current Employment. Lee: Pinotbio: Current Employment. Chun: Pinotbio: Current Employment. Go: Pinotbio: Current Employment. Lee: Pinotbio: Current Employment. Choi: Pinotbio: Research Funding.

230 Preclinical efficacy of CLEC-1 antagonist as novel myeloid immune checkpoint therapy for oncology

BackgroundC-type lectin receptors (CLRs) are powerful pattern recognition receptors shaping immune cell-mediated tissue damage by positively or negatively regulating myeloid cell functions and hence tumor elimination or evasion. We previously reported that the orphan CLR CLEC-1 expressed by dendritic cells (DCs) tempers T cell’s responses in vivo by limiting antigen cross-presentation by cDC1. Furthermore, we observed that CLEC-1 is highly expressed by myeloid cells purified from human tumor microenvironment, in particular tumor-associated macrophages.MethodsMacrophages were generated from monocytes of healthy volunteers for phagocytosis assays. MC38 and Hepa 1.6 murine tumor cells were implanted in Clec1a KO or KI mice for immunotherapeutic treatment evaluation.ResultsUsing newly developed anti-human CLEC-1 monoclonal antibodies (mAbs), we found that antagonist anti-CLEC-1 mAbs with the capacity to block CLEC-1/CLEC-1Ligand interaction, as opposed to non-antagonist CLEC-1 mAbs, increase the phagocytosis of CLEC-1Ligand-positive human tumor cells by human macrophages, in particular when opsonized by tumor-associated antigen mAbs (Rituximab, Cetuximab, Trastuzumab) or with anti-CD47 mAb (Magrolimab). In-vivo, CLEC-1 knock-out (KO) mice (n=19) display significant prolonged survival in monotherapy as compared to wild-type littermates (n=12) in an orthotopic hepatocellular carcinoma (HCC) model and anti-tumor memory responses was demonstrated by tumor rechallenge in cured mice. CLEC1 KO mice also illustrate significant eradication of MC38 colorectal tumors in combination with chemotherapy promoting CLEC-1Ligand expression by tumor cells (n=16 with Gemcitabine or n=11 with Cyclophosphamide). HCC tumor microenvironment analysis after 2 weeks of tumor implantation shows significantly higher number of CD8+ and memory CD8+ T cells with reduced PD1 expression in CLEC1 KO animals (n=16 versus n=12 for KO vs WT mice respectively). Finally, we recently generated human CLEC-1 knock-in mice expressing the extracellular human CLEC1 domain fused to the intracellular mouse CLEC1 tail and confirmed preclinical efficacy in vivo with anti-human CLEC1 antagonist mAb in monotherapy in the orthotopic HCC model.ConclusionsThese data illustrate that CLEC-1 inhibition represents a novel therapeutic target for immuno-oncology modifying T cell immune responses and tumor cell phagocytosis by macrophages.

The adenosine analogue prodrug ATV006 is orally bioavailable and has potent preclinical efficacy against SARS-CoV-2 and its variants

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes the COVID-19 pandemic, is rapidly evolving. Due to the limited efficacy of vaccination in prevention of SARS-CoV-2 transmission and continuous emergence of variants of concern (VOC), including the currently most prevalent Delta variant, orally bioavailable and broadly efficacious antiviral drugs are urgently needed. Previously we showed that adenosine analogue 69-0 (also known as GS-441524), possesses potent anti-SARS-CoV-2 activity. Herein, we report that esterification of the 5-hydroxyl moieties of 69-0 markedly improved the antiviral potency. The 5-hydroxyl -isobutyryl prodrug, ATV006, showed excellent oral bioavailability in rats and cynomolgus monkeys and potent antiviral efficacy against different VOCs of SARS-CoV-2 in cell culture and three mouse models. Oral administration of ATV006 significantly reduced viral loads, alleviated lung damage and rescued mice from death in the K18-hACE2 mouse model challenged with the Delta variant. Moreover, ATV006 showed broad antiviral efficacy against different mammal-infecting coronaviruses. These indicate that ATV006 represents a promising oral drug candidate against SARS-CoV-2 VOCs and other coronaviruses.