Time-Prolonged Release of Tumor-Targeted Protein–MMAE Nanoconjugates from Implantable Hybrid Materials

The sustained release of small, tumor-targeted cytotoxic drugs is an unmet need in cancer therapies, which usually rely on punctual administration regimens of non-targeted drugs. Here, we have developed a novel concept of protein–drug nanoconjugates, which are packaged as slow-releasing chemically hybrid depots and sustain a prolonged secretion of the therapeutic agent. For this, we covalently attached hydrophobic molecules (including the antitumoral drug Monomethyl Auristatin E) to a protein targeting a tumoral cell surface marker abundant in several human neoplasias, namely the cytokine receptor CXCR4. By this, a controlled aggregation of the complex is achieved, resulting in mechanically stable protein–drug microparticles. These materials, which are mimetics of bacterial inclusion bodies and of mammalian secretory granules, allow the slow leakage of fully functional conjugates at the nanoscale, both in vitro and in vivo. Upon subcutaneous administration in a mouse model of human CXCR4+ lymphoma, the protein–drug depots release nanoconjugates for at least 10 days, which accumulate in the tumor with a potent antitumoral effect. The modification of scaffold cell-targeted proteins by hydrophobic drug conjugation is then shown as a novel transversal platform for the design of slow releasing protein–drug depots, with potential application in a broad spectrum of clinical settings.

Azaindole inhibits liver cancer cell proliferation in vitro and in vivo by targeting the expression of kinesin family member C1

Purpose: To investigate the effect of azaindole on proliferation of liver cancer cells, as well as the underlying mechanism. Methods: Colony forming and 3-(4,5-dimethylthiazole-2-yl)-2,5-biphenyl tetrazolium bromide (MTT) assays were used to determine the effect of azaindole on cell proliferation. A tumor model was established through subcutaneous administration of HEPG2 cells to rats. Thereafter, in vivo tumor development was measured using Vernier caliper. Results: The proliferation potential of HEPG2 and SNU-398 cells was markedly and dose-dependently suppressed by treatment with azaindole at doses of 2, 4, 8, 16 and 20 μM (p < 0.05). The expression levels of Ki67 and PCNA levels were significantly down-regulated in HEPG2 and SNU-398 cells on treatment with 20 μM azaindole. Moreover, azaindole significantly suppressed mRNA and protein expressions of KIFC1 in HEPG2 and SNU-398 cells (p < 0.05). Tumor volume in azaindole-treated rats on day 21 was greatly reduced, while KIFC1 expression in azaindole-treated rat tumor tissue was significantly down-regulated, when compared to the model group (p < 0.05). Conclusion: Azaindole targets proliferation of liver cancer cells in vitro and inhibits tumor growth in vivo through a mechanism involving down-regulation of KIFCI expression. Thus, azaindole is a potential therapeutic candidate for liver cancer.

Oral Immunogenicity of Enterotoxigenic Escherichia coli Outer Membrane Vesicles Encapsulated into Zein Nanoparticles Coated with a Gantrez® AN–Mannosamine Polymer Conjugate

Enterotoxigenic Escherichia coli (ETEC) represents a major cause of morbidity and mortality in the human population. In particular, ETEC infections affect children under the age of five from low-middle income countries. However, there is no licensed vaccine against this pathogen. ETEC vaccine development is challenging since this pathotype expresses a wide variety of antigenically diverse virulence factors whose genes can be modified due to ETEC genetic plasticity. To overcome this challenge, we propose the use of outer membrane vesicles (OMVs) isolated from two ETEC clinical strains. In these OMVs, proteomic studies revealed the presence of important immunogens, such as heat-labile toxin, colonization factors, adhesins and mucinases. Furthermore, these vesicles proved to be immunogenic after subcutaneous administration in BALB/c mice. Since ETEC is an enteropathogen, it is necessary to induce both systemic and mucosal immunity. For this purpose, the vesicles, free or encapsulated in zein nanoparticles coated with a Gantrez®–mannosamine conjugate, were administered orally. Biodistribution studies showed that the encapsulation of OMVs delayed the transit through the gut. These results were confirmed by in vivo study, in which OMV encapsulation resulted in higher levels of specific antibodies IgG2a. Further studies are needed to evaluate the protection efficacy of this vaccine approach.

The Route of Vaccine Administration Determines Whether Blood Neutrophils Undergo Long-Term Phenotypic Modifications

Innate immunity modulates adaptive immunity and defines the magnitude, quality, and longevity of antigen-specific T- and B- cell immune memory. Various vaccine and administration factors influence the immune response to vaccination, including the route of vaccine delivery. We studied the dynamics of innate cell responses in blood using a preclinical model of non-human primates immunized with a live attenuated vaccinia virus, a recombinant Modified vaccinia virus Ankara (MVA) expressing a gag-pol-nef fusion of HIV-1, and mass cytometry. We previously showed that it induces a strong, early, and transient innate response, but also late phenotypic modifications of blood myeloid cells after two months when injected subcutaneously. Here, we show that the early innate effector cell responses and plasma inflammatory cytokine profiles differ between subcutaneous and intradermal vaccine injection. Additionally, we show that the intradermal administration fails to induce more highly activated/mature neutrophils long after immunization, in contrast to subcutaneous administration. Different batches of antibodies, staining protocols and generations of mass cytometers were used to generate the two datasets. Mass cytometry data were analyzed in parallel using the same analytical pipeline based on three successive clustering steps, including SPADE, and categorical heatmaps were compared using the Manhattan distance to measure the similarity between cell cluster phenotypes. Overall, we show that the vaccine per se is not sufficient for the late phenotypic modifications of innate myeloid cells, which are evocative of innate immune training. Its route of administration is also crucial, likely by influencing the early innate response, and systemic inflammation, and vaccine biodistribution.

solPredict: Antibody apparent solubility prediction from sequence by transfer learning

There is growing interest in developing therapeutic mAbs for the route of subcutaneous administration for several reasons, including patient convenience and compliance. This requires identifying mAbs with superior solubility that are amenable for high-concentration formulation development. However, early selection of developable antibodies with optimal high-concentration attributes remains challenging. Since experimental screening is often material and labor intensive, there is significant interest in developing robust in silico tools capable of screening thousands of molecules based on sequence information alone. In this paper, we present a strategy applying protein language modeling, named solPredict, to predict the apparent solubility of mAbs in histidine (pH 6.0) buffer condition. solPredict inputs embeddings extracted from pretrained protein language model from single sequences into a shallow neutral network. A dataset of 220 diverse, in-house mAbs, with extrapolated protein solubility data obtained from PEG-induced precipitation method, were used for model training and hyperparameter tuning through five-fold cross validation. An independent test set of 40 mAbs were used for model evaluation. solPredict achieves high correlation with experimental data (Spearman correlation coefficient = 0.86, Pearson correlation coefficient = 0.84, R2 = 0.69, and RMSE = 4.40). The output from solPredict directly corresponds to experimental solubility measurements (PEG %) and enables quantitative interpretation of results. This approach eliminates the need of 3D structure modeling of mAbs, descriptor computation, and expert-crafted input features. The minimal computational expense of solPredict enables rapid, large-scale, and high-throughput screening of mAbs during early antibody discovery.

Association of subcutaneous or intravenous route of administration of casirivimab and imdevimab monoclonal antibodies with clinical outcomes in COVID-19.

Importance: Monoclonal antibody (mAb) treatment decreases hospitalization and death in outpatients with mild to moderate COVID 19; however, only intravenous administration has been evaluated in randomized clinical trials of treatment. Subcutaneous administration may expand outpatient treatment capacity and qualified staff available to administer treatment, but association with patient outcomes is understudied. Objective: To evaluate whether or not, i.) subcutaneous casirivimab and imdevimab treatment is associated with reduced 28 days hospitalization/death than non-treatment among mAb-eligible patients, and ii.) subcutaneous casirivimab and imdevimab treatment is clinically and statistically similar to intravenous casirivimab and imdevimab treatment. Design, Setting, and Participants: Prospective cohort study of outpatients in a learning health system in the United States with mild to moderate COVID 19 symptoms from July 14 to October 26, 2021 who were eligible for mAb treatment under emergency use authorization. A nontreated control group of eligible patients was also selected. Intervention: Subcutaneous injection or intravenous administration of the combined single dose of casirivimab 600mg and imdevimab 600mg. Main Outcomes and Measures: The primary outcome was the 28 day adjusted risk ratio or adjusted risk difference for hospitalization or death. Secondary outcomes included 28 day adjusted risk ratios/differences of hospitalization, death, composite endpoint of ED admission and hospitalization, and rates of adverse events. Results: Among 1,956 matched adults with mild to moderate COVID 19, patients who received casirivimab and imdevimab subcutaneously had a 28-day rate of hospitalization/death of 3.4% (n=652) compared to 7.8% (n=1,304) in nontreated controls [risk ratio 0.44 (95% confidence interval: 0.28 to 0.68, p < .001)]. Among 2,185 patients treated with subcutaneous (n=969) or intravenous (n=1,216) casirivimab and imdevimab, the 28 day rate of hospitalization/death was 2.8% vs. 1.7%, respectively which resulted in an adjusted risk difference of 1.5% (95% confidence interval: -0.5% to 3.5%, p=.14). The 28 day adjusted risk differences (subcutaneous and intravenous) for death, ICU admission, and mechanical ventilation were 0.3% or less, although the 95% confidence intervals were wide. Conclusions and Relevance: Subcutaneously administered casirivimab and imdevimab is associated with reduced risk adjusted hospitalization or death amongst outpatients with mild to moderate COVID 19 compared to no treatment and indicates low adjusted risk difference compared to patients treated intravenously.