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Abstract Background Basket clinical trials targeting the KRASG12C-mutation in solid tumors have shown initial promise, including in orphan KRASG12C head and neck cancer (HNC). However, development of resistance to KRASG12C-mutant-specific inhibitors (KRASG12Ci) remains a major obstacle. Here, we investigated the intrinsic (tumor-cell autonomus) and tumor-microenvironment (TME) mechanisms of resistance to the KRASG12Ci—MRTX849 and AMG510 in a unique syngenic murine KRASG12C-mutated HNC cell line. Methods Western-blotting was used for protein abundance and activation, overexpression, and ligand activation studies to verify the intrinsic mechanism of resistance to KRASG12Ci in KRASG12C-mutated HNC cell line, 4NQO-L. In vitro KRASG12C-acquired-resistant cells were developed from 4NQO-L (4NQO-L-AcR). MRTX849/lapatinib combination efficacy, and CD8+ T-cells depletion, were assessed in C57BL/6 J mice and supplementation of anti-PD-1 (αPD-1) to MRTX849/lapatinib was also performed in 4NQO-L– KRASG12Ci-senisitve and 4NQO-L-AcR tumors. Immunohistochemistry (IHC) and Immunoflourescence (IF) analyses were performed to profile the TME and programmed death-ligand 1 (PD-L1) expression in tumors. Results Activation and upregulation of EGFR and HER2/3 (pan-HERs) are the intrinsic mechanism of resistance to KRASG12Ci in 4NQO-L cells, and blocking pan-HERs signaling with lapatinib enhanced MRTX849 efficacy in vitro by inhibiting the MAPK and AKT/mTOR pathways. 4NQO-L-AcR upregulated the expression of pan-HERs, and lapatinib treatment re-sensitized 4NQO-L-AcR to MRTX849. In mice, MRTX849 showed a slight anti-tumor effect, but in combination with lapatinib a significant tumor growth delay was observed, but all tumors progressed over time. Histopathology analysis of the TME revealed infiltration of CD8+ T-cells after treatment combination, and these CD8+ T-cells play a key role in MRTX849/lapatinib efficacy. MRTX849/lapatinib treatment upregulated PD-L1 overexpression in both stromal and tumor cells, which presumably suppressed CD8+ T-cells and enabled immune escape and tumor progression. Supplementation of αPD-1 prolonged the progression-free survival of 4NQO-L-bearing mice treated with MRTX849/lapatinib. MRTX849/lapatinib treatment delayed tumor growth of 4NQO-L-AcR in mice; however, the percentages of CD8+ T-cells in 4NQO-L-AcR were low, and supplementation of MRTX849/lapatinib with αPD-1 did not improve the outcome. Conclusions Our study highlights the critical need for blocking both intrinsic and extrinsic mechanisms of resistance for the prolonged response and shows that such treatment is ineffective in KRASG12Ci-AcR tumors.

Abstract Cancer immunotherapies are highly potent and are gaining wide clinical usage. However, severe side effects require focusing effector immune cell activities on the tumor microenvironment (TME). We recently developed a chimeric antigen receptor tumor-induced vector (CARTIV), a synthetic promoter activated by TME factors. To improve CARTIV functions including background, activation levels, and synergism, we screened a library of promoters with variations in key positions. Here, we present a screening method involving turning ON/OFF stimulating TNFα and IFNγ cytokines, followed by sequential cell sorting. Sequencing of enriched promoters identified seventeen candidates, which were cloned and whose activities were then validated, leading to the identification of two CARTIVs with lower background and higher induction. We further combined a third hypoxia element with the two-factor CARTIV, demonstrating additional modular improvement. Our study presents a method of fine-tuning synthetic promoters for desired immunotherapy needs.

Crustacean cell line immortalization has gained a great deal of attention in recent decades for both scientific and applied reasons. Our goal in this study was to advance the state of art towards establishing an immortalized cell line by improving the proliferation rates of primary cells isolated from embryos of the giant freshwater prawn Macrobrachium rosenbergii by using a lentivirus expressing the Ras oncogene. The choice of Ras derived from its involvement in various cellular pathways, such as cell growth, differentiation, and survival, and its use as a tool for in-vitro immortalization, e.g., a specific mutated Ras (RasV12) was used to generate an arthropod cell line. Complementarily, in-silico screening of M. rosenbergii transcriptomic libraries for Ras expression indicated that Ras is already expressed at very early stages of embryo development. In the current study, we transduced primary M. rosenbergii embryonic cells with a lentivirus expressing RasV12 by using the white spot syndrome virus (WSSV IE1) promoter. Expression and sequencing (as followed by sequencing cDNA, confocal microscopy and FACS analysis) of the mutated Ras in the transduced cells confirmed that the lentivirus was successfully integrated into the genome. The lenti-MrRas transduction rate was 23% in the total primary cell population and more than 80% in a sub-population of cells with high granularity. Proliferation of lenti-MrRas transfected cells was enhanced to almost 1200% of the seeding density by the end of our experiment (18 days), which was double that of the control. We were thus successful in enhancing the longevity of embryonic primary cell cultures by ectopic expression of the mutated Ras protein, but the improvement was not sufficient for immortalization.

Abstract IL-2 is the master-regulator cytokine for T cell dependent responses and is crucial for proliferation and survival of T cells. However, IL-2-based treatments remained marginal, in part due to short half-life. Thus, we aimed to extend IL-2 half-life by flanking the IL-2 core with sequences derived from the extensively glycosylated hinge region of the NCR2 receptor. We termed this modified IL-2: “S2A”. Importantly, S2A blood half-life was extended 14-fold compared to the clinical grade IL-2, Proleukin. Low doses inoculation of S2A significantly enhanced induction of Tregs (CD4+ Regulatory T cells) in vivo, as compared to Proleukin, while both S2A and Proleukin induced low levels of CD8+ T cells. In a B16 metastatic melanoma model, S2A treatment was unable to reduce the metastatic capacity of B16 melanoma, while enhancing induction and recruitment of Tregs, compared to Proleukin. Conversely, in two autoimmune models, rheumatoid arthritis and DSS-induced colitis, S2A treatment significantly reduced the progression of disease compared to Proleukin. Our results suggest new avenues for generating long-acting IL-2 for long-standing treatment and a new technique for manipulating short-life proteins for clinical and research uses.

Greenshpan et al. explore the use of synthetic promoters based on IFNgamma, TNF alpha and hypoxia responsive elements to control gene expression. The authors describe this technology in the context of chimeric antigen expression (CAR) and propose that this method can circumvent off-target toxic effects via specific expression of the CAR in the tumor microenvironment.

The current Covid-19 pandemic has a profound impact on all aspects of our lives. Aside from contagion by aerosols, the presence of the SARS-CoV-2 is ubiquitous on surfaces that millions of people handle daily. Therefore, controlling this pandemic involves the reduction of potential infections via contaminated surfaces. We developed antiviral surfaces by preparing suspensions of copper and cupric oxide nanoparticles in two different polymer matrices, poly(methyl methacrylate) and polyepoxide. For total copper contents as low as 5%, the composite material showed remarkable antiviral properties against the HCoV-OC43 human coronavirus and against a model lentivirus and proved well-resistant to accelerated aging conditions. Importantly, we showed that the Cu/CuO mixture showed optimal performances. This product can be implemented to produce a simple and inexpensive coating with long-term antiviral properties and will open the way to developing surface coatings against a broad spectrum of pathogens including SARS-CoV-2.

Novel monosex biotechnologies in crustacean aquaculture involve the induction of sex reversal through manipulations of the androgenic gland (AG), and its most prominent factor, the insulin-like androgenic gland (IAG) hormone, during early developmental stages. In the giant freshwater prawn Macrobrachium rosenbergii, all-female populations are desirable for aquaculture, since the females can be stocked at higher densities and exhibit more uniform growth than males. Recently, a novel biotechnology for all-female aquaculture was developed based on injection into post-larvae of suspended primary AG cells from mature M. rosenbergii males. However, this biotechnology depends on the availability of appropriate male donors and it requires delicate surgical procedures on the small endocrine AG to produce appropriate quantities of cells for the sex manipulation. We therefore established a new platform for the production of M. rosenbergii IAG (Mr-IAG) in hematopoietic cells. The method rests on the induction of ectopic Mr-IAG expression under the white spot syndrome virus (WSSV) immediate-early 1 (IE1) promoter via a novel lentiviral vector. Our results demonstrate that M. rosenbergii primary cells infected with the Mr-IAG lentiviruses are capable of transcription, translation, and secretion of Mr-IAG in culture. Our new platform, which produces easy-to-harvest cells in abundance, could replace the AG cells used in the first step of the above-mentioned biotechnology for all-female aquaculture and, importantly, pave the way for producing monosex populations in other edible crustacean aquaculture species. In addition, a lentiviral system for crustacean cells provides a useful tool for basic and applied research in crustacean species.

The Ebola Virus (EBOV) glycoprotein (GP) sterically shields cell-membrane ligands to immune receptors such as human leukocyte antigen class-1 (HLA-I) and MHC class I polypeptide-related sequence A (MICA), thus mediating immunity evasion. It was suggested that the abundant N-glycosylation of the EBOV-GP is involved in this steric shielding. We aimed to characterize (i) the GP N-glycosylation sites contributing to the shielding, and (ii) the effect of mutating these sites on immune subversion by the EBOV-GP. The two highly glycosylated domains of GP are the mucin-like domain (MLD) and the glycan cap domain (GCD) with three and six N-glycosylation sites, respectively. We mutated the N-glycosylation sites either in MLD or in GCD or in both domains. We showed that the glycosylation sites in both the MLD and GCD domains contribute to the steric shielding. This was shown for the steric shielding of either HLA-I or MICA. We then employed the fluorescence resonance energy transfer (FRET) method to measure the effect of N-glycosylation site removal on the distance in the cell membrane between the EBOV-GP and HLA-I (HLA.A*0201 allele). We recorded high FRET values for the interaction of CFP-fused HLA.A*0201 and YFP-fused EBOV-GP, demonstrating the very close distance (

Receptor tyrosine kinases (RTKs) are major players in signal transduction, regulating cellular activities in both normal regeneration and malignancy. Thus, many RTKs, c-Kit among them, play key roles in the function of both normal and neoplastic cells, and as such constitute attractive targets for therapeutic intervention. We thus sought to manipulate the self-association of stem cell factor (SCF), the cognate ligand of c-Kit, and hence its suboptimal affinity and activation potency for c-Kit. To this end, we used directed evolution to engineer SCF variants having different c-Kit activation potencies. Our yeast-displayed SCF mutant (SCFM) library screens identified altered dimerization potential and increased affinity for c-Kit by specific SCF-variants. We demonstrated the delicate balance between SCF homo-dimerization, c-Kit binding, and agonistic potencies by structural studies, in vitro binding assays and a functional angiogenesis assay. Importantly, our findings showed that a monomeric SCF variant exhibited superior agonistic potency vs. the wild-type SCF protein and vs. other high-affinity dimeric SCF variants. Our data showed that action of the monomeric ligands in binding to the RTK monomers and inducing receptor dimerization and hence activation was superior to that of the wild-type dimeric ligand, which has a higher affinity to RTK dimers but a lower activation potential. The findings of this study on the binding and c-Kit activation of engineered SCF variants thus provides insights into the structure–function dynamics of ligands and RTKs.