Isolation and Characterization of Neutralizing Monoclonal Antibodies from a Large Panel of Murine Antibodies against RBD of the SARS-CoV-2 Spike Protein.

The COVID-19 pandemic, once a global crisis, is now largely under control, a testament to the extraordinary global efforts involving vaccination and public health measures. However, the relentless evolution of SARS-CoV-2, leading to the emergence of new variants, continues to underscore the importance of remaining vigilant and adaptable. Monoclonal antibodies (mAbs) have stood out as a powerful and immediate therapeutic response to COVID-19. Despite the success of mAbs, the evolution of SARS-CoV-2 continues to pose challenges and the available antibodies are no longer effective. New variants require the ongoing development of effective antibodies. In the present study, we describe the generation and characterization of neutralizing mAbs against the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein by combining plasmid DNA and recombinant protein vaccination. By integrating genetic immunization for rapid antibody production and the potent immune stimulation enabled by protein vaccination, we produced a rich pool of antibodies, each with unique binding and neutralizing specificities, tested with the ELISA, BLI and FACS assays and the pseudovirus assay, respectively. Here, we present a panel of mAbs effective against the SARS-CoV-2 variants up to Omicron BA.1 and BA.5, with the flexibility to target emerging variants. This approach ensures the preparedness principle is in place to address SARS-CoV-2 actual and future infections.

Immunogenicity of COVID-eVax Delivered by Electroporation Is Moderately Impacted by Temperature and Molecular Isoforms.

DNA integrity is a key issue in gene therapy and genetic vaccine approaches based on plasmid DNA. In contrast to messenger RNA that requires a controlled cold chain for efficacy, DNA molecules are considered to be more stable. In this study, we challenged this concept by characterizing the immunological response induced by a plasmid DNA vaccine delivered using electroporation. As a model, we used COVID-eVax, a plasmid DNA-based vaccine that targets the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. Increased nicked DNA was produced by using either an accelerated stability protocol or a lyophilization protocol. Surprisingly, the immune response induced in vivo was only minimally affected by the percentage of open circular DNA. This result suggests that plasmid DNA vaccines, such as COVID-eVax that have recently completed a phase I clinical trial, retain their efficacy upon storage at higher temperatures, and this feature may facilitate their use in low-/middle-income countries.

COVID-eVax, an electroporated DNA vaccine candidate encoding the SARS-CoV-2 RBD, elicits protective responses in animal models.

The COVID-19 pandemic caused by SARS-CoV-2 has made the development of safe and effective vaccines a critical priority. To date, four vaccines have been approved by European and American authorities for preventing COVID-19, but the development of additional vaccine platforms with improved supply and logistics profiles remains a pressing need. Here we report the preclinical evaluation of a novel COVID-19 vaccine candidate based on the electroporation of engineered, synthetic cDNA encoding a viral antigen in the skeletal muscle. We constructed a set of prototype DNA vaccines expressing various forms of the SARS-CoV-2 spike (S) protein and assessed their immunogenicity in animal models. Among them, COVID-eVax-a DNA plasmid encoding a secreted monomeric form of SARS-CoV-2 S protein receptor-binding domain (RBD)-induced the most potent anti-SARS-CoV-2 neutralizing antibody responses (including against the current most common variants of concern) and a robust T cell response. Upon challenge with SARS-CoV-2, immunized K18-hACE2 transgenic mice showed reduced weight loss, improved pulmonary function, and lower viral replication in the lungs and brain. COVID-eVax conferred significant protection to ferrets upon SARS-CoV-2 challenge. In summary, this study identifies COVID-eVax as an ideal COVID-19 vaccine candidate suitable for clinical development. Accordingly, a combined phase I-II trial has recently started.

The Nuts and Bolts of SARS-CoV-2 Spike Receptor-Binding Domain Heterologous Expression.

COVID-19 is a highly infectious disease caused by a newly emerged coronavirus (SARS-CoV-2) that has rapidly progressed into a pandemic. This unprecedent emergency has stressed the significance of developing effective therapeutics to fight the current and future outbreaks. The receptor-binding domain (RBD) of the SARS-CoV-2 surface Spike protein is the main target for vaccines and represents a helpful "tool" to produce neutralizing antibodies or diagnostic kits. In this work, we provide a detailed characterization of the native RBD produced in three major model systems: Escherichia coli, insect and HEK-293 cells. Circular dichroism, gel filtration chromatography and thermal denaturation experiments indicated that recombinant SARS-CoV-2 RBD proteins are stable and correctly folded. In addition, their functionality and receptor-binding ability were further evaluated through ELISA, flow cytometry assays and bio-layer interferometry.

Preclinical evaluation of the novel 7-substituted camptothecin Namitecan (ST1968) in paediatric tumour models.

PURPOSE: The present study aimed to evaluate the new water soluble camptothecin analogue Namitecan (ST1968) in preclinical paediatric tumour models of the nervous system comprehensive of neuroblastoma, primitive neuroectodermal tumours/PNET and medulloblastoma where the drug was compared to Irinotecan. METHODS: Cellular sensitivity to the drug was assessed by MTT and clonogenic assays. Propidium iodide staining was used for cell cycle perturbation studies. The genotoxic effects were quantified by Comet assay, whereas apoptosis was assessed by PARP cleavage and sub-G1 accumulation. Tumour response was investigated in xenograft models in nude mice. RESULTS: The cellular response to Namitecan was heterogeneous with IC(50) (2 h) ranging between 0.14 and 13.26 µM, whereas SN38 (the active metabolite of Irinotecan) appeared more effective (IC(50): 0.03-11.7 µM). Interestingly, prolonged drug incubation times up to 72 h enhanced Namitecan cytotoxicity, with similar colony inhibition curves between the two analogues (IC(50), nM-SN38: 0.9 ± 0.2; Namitecan: 0.7 ± 0.4). DNA damage, accumulation in late-S/G2 phases and induction of apoptosis appeared important players of Namitecan cytotoxicity in our models. In vivo, Namitecan was superior to Irinotecan in three out of five xenograft models, with reversible weight loss (10 %). In the sensitive SK-N-AS xenograft, Namitecan showed a high retention in tumours consistently with: high antitumour response, rapid drug-mediated DNA damage (60 % mean TailDNA after 1 h from drug inoculation), persistent cell cycle perturbation (60-40 % G2 accumulation after 48-72 h, respectively) and apoptosis. Studies with Namitecan and platinum agents in this model showed a significant enhancement of antitumour activity of the drugs combination versus single agents. CONCLUSIONS: Our preclinical data strongly support the interest of further investigations on the well-tolerated Namitecan either as a single agent or in combination in paediatric oncology.

The curative efficacy of namitecan (ST1968) in preclinical models of pediatric sarcoma is associated with antiangiogenic effects.

Namitecan (ST1968), a novel hydrophilic camptothecin analog of the 7-oxyiminomethyl series, was selected for clinical development on the basis of its promising preclinical efficacy. Since there is clinical evidence of efficacy of camptothecins against pediatric tumors, this study was performed to explore the antitumor and antiangiogenic activity of the camptothecin derivative in pediatric sarcoma models. With the exception of an undifferentiated rhabdomyosarcoma (A204), namitecan exhibited curative efficacy even at well-tolerated suboptimal doses in a panel of five models. The good therapeutic index of namitecan likely reflected a high and persistent drug accumulation at tumor site. The four responsive tumors were characterized by high topoisomerase I expression. In the RD/TE-671 rhabdomyosarcoma model the drug activity was associated with a marked antiangiogenic effect, which was consistent with the downregulation of proangiogenic factors, including VEGF, bFGF and the multifunctional chemokines CCL-2 and CXCL16. In agreement with this modulation, the combination of low doses of namitecan with other antiangiogenic agents, such as bevacizumab (a humanized anti-VEGF antibody) and sunitinib (a multitarget tyrosine kinase inhibitor effective against receptors implicated in the angiogenesis process), enhanced the antitumor effects. In conclusion, this preclinical study provides evidence of curative efficacy of namitecan at well-tolerated doses against pediatric sarcoma models, likely reflecting a contribution of antiangiogenic effects. Based on the promising therapeutic profile, namitecan is a good candidate for clinical evaluation in pediatric sarcomas.

New retinoid derivatives as back-ups of Adarotene.

Adarotene belongs to the so-called class of atypical retinoids. The presence of the phenolic hydroxyl group on Adarotene structure allows a rapid O-glucuronidation as a major mechanism of elimination of the drug, favoring a fast excretion of its glucuronide metabolite in the urines. A series of ether, carbamate and ester derivatives was synthesized. All of them were studied and evaluated for their stability at different pH. The cytotoxic activity in vitro on NCI-H460 non-small cell lung carcinoma and A2780 ovarian tumor cell lines was also tested. A potential back-up of Adarotene has been selected to be evaluated in tumor models.

Conjugates of a novel 7-substituted camptothecin with RGD-peptides as α(v)ß3 integrin ligands: An approach to tumor-targeted therapy.

Eight conjugates of a novel camptothecin derivative (Namitecan, NMT) with RGD peptides have been synthesized and biologically evaluated. This study focused on factors that optimize the drug linkage to the transport vector. The different linkages investigated consist of heterofunctional glycol fragments and a lysosomally cleavable peptide. The linkage length and conformation were systematically modified with the purpose to understand their effect on receptor affinity, systemic stability, cytotoxicity, and solubility of the corresponding conjugates. Among the new conjugates prepared, C6 and C7 showed high receptor affinity and tumor cell adhesion, acceptable stability in murine blood, and high cytotoxic activity (IC50 = 8 nM). The rationale, synthetic strategy, and preliminary biological results will be presented.

Novel tumor-targeted RGD peptide-camptothecin conjugates: synthesis and biological evaluation.

Five RGD peptide-camptothecin (CPT) conjugates were designed and synthesized with the purpose to improve the therapeutic index of this antitumoral drug family. New RGD cyclopeptides were selected on the basis of their high affinity to alpha(v) integrin receptors overexpressed by tumor cells and their metabolic stability. The conjugates can be divided in two groups: in the first the peptide was attached to the drug through an amide bond, in the second through a hydrazone bond. The main difference between the two spacers lies in their acid stability. Affinity to the receptors was maintained for all conjugates and their internalization into tumor cells was demonstrated. The first group conjugates showed lower in vitro and in vivo activity than the parent drug, probably due to the excessive stability of the amide bond, even inside the tumor cells. Conversely, the hydrazone conjugates exhibited in vitro tumor cell inhibition similar to the parent drug, indicating high conversion in the culture medium and/or inside the cells, but their poor solubility hampered in vivo experiments. On the basis of these results, information was acquired for additional development of derivatives with different linkers and better solubility for in vivo evaluation.

Efficacy of ST1968 (namitecan) on a topotecan-resistant squamous cell carcinoma.

ST1968 (namitecan), a novel 7-modified hydrophilic camptothecin, was found to be effective against tumor models relatively resistant to topotecan and irinotecan. Based on this observation, this study was designed to investigate the cellular and antitumor effects of ST1968 in a subline of A431, squamous cell carcinoma, selected for resistance to topotecan (A431/TPT). This model was characterized by a slow growth rate, associated with downregulation of EGFR and topoisomerase I. In contrast to other camptothecins (SN38 and gimatecan), ST1968 was able to overcome almost completely the resistance at cellular level. The cellular pharmacokinetics indicated a comparable accumulation and retention of ST1968 in sensitive and resistant cells, in spite of expression of the efflux transporter, P-glycoprotein, in resistant cells. The uptake and retention of topotecan were dramatically reduced in both tumor cell lines, but more evident in the resistant one. In contrast to topotecan, ST1968 retained an outstanding efficacy in vivo against the resistant tumor (A431/TPT). The results are consistent with the interpretation that ST1968 was able to overcome the most relevant mechanisms associated with the development of topotecan resistance (i.e., slow proliferation and target downregulation) owing to its peculiar pharmacokinetic behaviour.

Intracellular accumulation and DNA damage persistence as determinants of human squamous cell carcinoma hypersensitivity to the novel camptothecin ST1968.

ST1968, a novel hydrophilic camptothecin analogue of the 7-oxyiminomethyl series, is characterised by the formation of stable DNA-topoisomerase I cleavable complex and by a promising profile of antitumour activity. The present study was designed to extend preclinical evaluation of the novel camptothecin in human squamous cell carcinoma (SCC) models. ST1968 exhibited an impressive activity with a high cure rate in SCC models. ST1968 produced 100% of complete response without evidence of regrowth in tumours characterised by susceptibility to drug-induced apoptosis (FaDu, A431 and A2780). In contrast to irinotecan, ST1968 still showed an excellent, persisting activity in models less susceptible to apoptosis induction (KB, Caski and SiHa), in which drug treatment elicited a persistent DNA damage response, as documented by phosphorylation of p53, RPA-2 and histone H2AX, resulting in delayed apoptosis and senescence. This behaviour was associated with a marked cellular/tumour drug accumulation. In conclusion, ST1968 exhibited an outstanding antitumour activity superior to that of irinotecan against SCC. A high intracellular accumulation, resulting in fast apoptosis or DNA damage persistence, appeared to be a critical determinant of SCC sensitivity to ST1968.

Novel combretastatin analogues endowed with antitumor activity.

We studied the anticancer activity of a series of new combretastatin derivatives with B-ring modifications. The structure-activity relationship (SAR) information confirmed the importance of cis-stereochemistry and of a phenolic moiety in B-ring. We selected the benzo[b]thiophene and benzofuran combretastatin analogues 11 (ST2151) and 13 (ST2179) and their phosphate prodrugs (29 and 30) for their high antitumor activity in in vitro and in vivo models. Cell exposure to IC50 of 11, 13, and CA-4 led to the arrest of various cell types in the G2/M phase of the cell cycle and induction of apoptosis. Mainly, 11 and 13 induced the formation of multinucleated cells with abnormal chromatin distribution, with only a minimal effect on the microtubule organization, with respect to CA-4. Interestingly, both the pharmacokinetic profile of 29 and its in vivo antitumor effect and those of 30, active even after oral administration, suggest additional pharmacological differences between these compounds and CA-4P.

Development of resistance to the atypical retinoid, ST1926, in the lung carcinoma cell line H460 is associated with reduced formation of DNA strand breaks and a defective DNA damage response.

Atypical retinoids are potent inducers of apoptosis, but activation of the apoptotic pathway seems to be independent of retinoid receptors. Previous studies with a novel adamantyl retinoid, ST1926, have shown that apoptosis induction is associated with an early genotoxic stress. To better understand the relevance of these events, we have selected a subline of the H460 lung carcinoma cell line resistant to ST1926. Resistant cells exhibited cross-resistance to a related molecule, CD437, but not cross-resistance to agents with different mechanisms of action. In spite of a lack of defects in intracellular drug accumulation, induction of DNA strand breaks in resistant cells required exposure to a substantially higher concentration, which was consistent with the degree of resistance. At drug concentrations causing a similar antiproliferative effect (IC80) and a comparable extent of DNA lesions in sensitive and resistant cells, the apoptotic response was a delayed and less marked event in resistant cells, thus indicating a reduced susceptibility to apoptosis. In spite of recognition of DNA lesions in resistant cells, as supported by phosphorylation of p53 and histone H2AX, resistant cells exhibited no activation of the mitochondrial pathways of apoptosis. Following exposure to equitoxic drug concentrations, only sensitive cells exhibited a typical stress/DNA damage response, with activation of the S-phase checkpoint. The cellular resistance to ST1926 reflects alterations responsible for a reduced generation of DNA lesions and for an enhanced tolerance of the genotoxic stress, resulting in lack of activation of the intrinsic pathway of apoptosis. The defective DNA damage response, accompanied by a reduced susceptibility to apoptosis in resistant cells, provides further support to the involvement of genotoxic stress as a critical event in mediating apoptosis induction by ST1926.

Novel camptothecin analogue (gimatecan)-containing liposomes prepared by the ethanol injection method.

Small-sized liposomes have several advantages as drug delivery systems, and the ethanol injection method is a suitable technique to obtain the spontaneous formation of liposomes having a small average radius. In this paper, we show that liposomal drug formulations can be prepared in situ, by simply injecting a drug-containing lipid(s) organic solution into an aqueous solution. Several parameters should be optimized in order to obtain a final suitable formulation, and this paper is devoted to such an investigation. Firstly, we study the liposome size distributions determined by dynamic light scattering (DLS), as function of the lipid concentration and composition, as well as the organic and aqueous phases content. This was carried out, firstly, by focusing on POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) then on the novel L-carnitine derivative PUCE (palmitoyl-(R)-carnitine undecyl ester chloride), showing that it is possible to obtain monomodal size distributions of rather small vesicles. In particular, depending on the conditions, it was possible to achieve a population of liposomes with a mean size of 100 nm, when a 50 mM POPC ethanol solution was injected in pure water; in the case of 50 mM PUCE the mean size was around 30 nm, when injected in saline (0.9% NaCl). The novel anticancer drug Gimatecan, a camptothecin derivative, was used as an example of lipophilic drug loading by the injection method. Conditions could be found, under which the resultant liposome size distributions were not affected by the presence of Gimatecan, in the case of POPC as well as in the case of PUCE. To increase the overall camptothecin concentration in the final liposomal dispersion, the novel technique of "multiple injection method" was used, and up to a final 5 times larger amount of liposomal drug could be reached by maintaining approximately the same size distribution. Once prepared, the physical and chemical stability of the liposome formulations was satisfactory within 24, as judged by DLS analysis and HPLC quantitation of lipids and drug. The Gimatecan-containing liposomes formulations were also tested for in vitro and in vivo activity, against the human nonsmall cell lung carcinoma NCI-H460 and a murine Lewis lung carcinoma 3 LL cell lines. In the in vitro tests, we did not observe any improvement or reduction of the Gimatecan pharmacological effect by the liposomal delivery system. More interestingly, in the in vivo Lewis lung carcinoma model, the intravenously administration of liposomal Gimatecan formulation showed a mild but significant increase of Tumor Volume Inhibition with respect to the oral no-liposomal formulation (92% vs. 86 %, respectively; p < 0.05). Finally, our study showed that the liposomal formulation was able to realize a delivery system of a water-insoluble drug, providing a Gimatecan formulation for intravenous administration with a preserved antitumoral activity.

Cellular bases of the antitumor activity of a 7-substituted camptothecin in hormone-refractory human prostate carcinoma models.

ST1481, a lead compound of a novel potent 7-substituted lipophilic camptothecin series, is able to overcome several mechanisms of drug resistance and was selected for clinical development. This study was designed to examine the antitumor activity of ST1481 in the treatment of preclinical models of human p53-defective hormone-refractory prostate carcinoma (DU145, PC3, and JCA-1) and to explore the cellular bases of the efficacy of camptothecins. A cellular pharmacology study (cytotoxicity, apoptosis, cellular drug accumulation, DNA damage, and cell cycle perturbation) was performed in DU145 and PC3 cells, characterized by a different cell cycle checkpoint status. The introduction of wild-type p53 in PC3 cells appreciably decreased the drug sensitivity. The 7-substituted camptothecins exhibited a high cytotoxic potency that paralleled their relative ability to induce DNA damage and a substantially increased cellular accumulation as compared to topotecan. The cytotoxic effect of camptothecins in DU145 cells was associated with arrest in S phase and early activation of apoptosis, whereas PC3 cells responded to drugs by a persistent block in G2 phase with a cytostatic effect and a late apoptosis. The efficiency of S phase checkpoint in DU145 cells was supported by a time-dependent decrease of DNA synthesis following treatment. In spite of an apparent cytostatic response and apoptosis resistance, the PC3 tumor was more responsive to in vivo treatment with camptothecins than the DU145 model. Indeed, the therapeutic outcome did not reflect the cell susceptibility to early activation of apoptosis. We suggest that cell death in PC3 cells is a delayed event consequent to persistent arrest in G2 and insufficient repair of DNA damage. ST1481 was appreciably more effective than topotecan in all tested tumors. In conclusion, the results indicated a relevant efficacy of camptothecins against human prostate carcinoma models, in spite of p53 alterations. Although p53 status could influence DNA damage and cell cycle checkpoints, p53 mutation was not a determinant of resistance. The results support that, in addition to the extent and persistence of topoisomerase I-mediated DNA damage, cell cycle checkpoints and DNA damage signaling pathways are critical determinants of tumor responsiveness to camptothecins. A role of cell cycle checkpoints activated by DNA damage in cell response is supported by the modulation of transcriptional profile.