Early rhombic lip Protogenin+ve stem cells in a human-specific neurovascular niche initiate and maintain group 3 medulloblastoma.

We identify a population of Protogenin-positive (PRTG+ve) MYChigh NESTINlow stem cells in the four-week-old human embryonic hindbrain that subsequently localizes to the ventricular zone of the rhombic lip (RLVZ). Oncogenic transformation of early Prtg+ve rhombic lip stem cells initiates group 3 medulloblastoma (Gr3-MB)-like tumors. PRTG+ve stem cells grow adjacent to a human-specific interposed vascular plexus in the RLVZ, a phenotype that is recapitulated in Gr3-MB but not in other types of medulloblastoma. Co-culture of Gr3-MB with endothelial cells promotes tumor stem cell growth, with the endothelial cells adopting an immature phenotype. Targeting the PRTGhigh compartment of Gr3-MB in vivo using either the diphtheria toxin system or chimeric antigen receptor T cells constitutes effective therapy. Human Gr3-MBs likely arise from early embryonic RLVZ PRTG+ve stem cells inhabiting a specific perivascular niche. Targeting the PRTGhigh compartment and/or the perivascular niche represents an approach to treat children with Gr3-MB.

Characteristics and Functions of Infection-enhancing Antibodies to the N-terminal Domain of SARS-CoV-2.

Background: Fcγ-receptor (FcγR)-independent enhancement of SARS-CoV-2 infection mediated by N-terminal domain (NTD)-binding monoclonal antibodies (mAbs) has been observed in vitro, but the functional significance of these antibodies in vivo is less clear. Methods: We characterized 1,213 SARS-CoV-2 spike (S)-binding mAbs derived from COVID-19 convalescent patients for binding specificity to the SARS-CoV-2 S protein, VH germ-line usage, and affinity maturation. Infection enhancement in a vesicular stomatitis virus (VSV)-SARS-CoV-2 S pseudovirus (PV) assay was characterized in respiratory and intestinal epithelial cell lines, and against SARS-CoV-2 variants of concern (VOC). Proteomic deconvolution of the serum antibody repertoire was used to determine functional attributes of secreted NTD-binding mAbs. Results: We identified 72/1213 (5.9%) mAbs that enhanced SARS-CoV-2 infection in a PV assay. The majority (68%) of these mAbs recognized the NTD, were identified in patients with mild and severe disease, and persisted for at least 5 months post-infection. Infection enhancement by NTD-binding mAbs was not observed in intestinal and respiratory epithelial cell lines and was diminished or lost against SARS-CoV-2 VOC. Proteomic deconvolution of the serum antibody repertoire from 2 of the convalescent patients identified, for the first time, NTD-binding, infection-enhancing mAbs among the circulating immunoglobulins directly isolated from serum. Functional analysis of these mAbs demonstrated robust activation of FcγRIIIa associated with antibody binding to recombinant S proteins. Conclusions: Functionally active NTD-specific mAbs arise frequently during natural infection and can last as major serum clonotypes during convalescence. These antibodies display functional attributes that include FcγR activation, and may be selected against by mutations in NTD associated with SARS-CoV-2 VOC.

Identification of a Fully Human Antibody VH Domain Targeting Anaplastic Lymphoma Kinase (ALK) with Applications in ALK-Positive Solid Tumor Immunotherapy.

The anaplastic lymphoma kinase (ALK, CD247) is a potential target for antibody-based therapy. However, no antibody-based therapeutics targeting ALK have entered clinical trials, necessitating the development of novel antibodies with unique therapeutic merits. Single-domain antibodies (sdAb) bear therapeutic advantages compared to the full-length antibody including deeper tumor penetration, cost-effective production and fast washout from normal tissues. In this study, we identified a human immunoglobulin heavy chain variable domain (VH domain) (VH20) from an in-house phage library. VH20 exhibits good developability and high specificity with no off-target binding to ~6000 human membrane proteins. VH20 efficiently bound to the glycine-rich region of ALK with an EC50 of 0.4 nM and a KD of 6.54 nM. Both VH20-based bispecific T cell engager (TCE) and chimeric antigen receptor T cells (CAR Ts) exhibited potent cytolytic activity to ALK-expressing tumor cells in an ALK-dependent manner. VH20 CAR Ts specifically secreted proinflammatory cytokines including IL-2, TNFα and IFNγ after incubation with ALK-positive cells. To our knowledge, this is the first reported human single-domain antibody against ALK. Our in vitro characterization data indicate that VH20 could be a promising ALK-targeting sdAb with potential applications in ALK-expressing tumors, including neuroblastoma (NBL) and non-small cell lung cancer.

Abnormal Silicon Etching Behaviors in Nanometer-Sized Channels.

Modern semiconductor fabrication is challenged by difficulties in overcoming physical and chemical constraints. A major challenge is the wet etching of dummy gate silicon, which involves the removal of materials inside confined spaces of a few nanometers. These chemical processes are significantly different in the nanoscale and bulk. Previously, electrical double-layer formation, bubble entrapment, poor wettability, and insoluble intermediate precipitation have been proposed. However, the exact suppression mechanisms remain unclear due to the lack of direct observation methods. Herein, we investigate limiting factors for the etching kinetics of silicon with tetramethylammonium hydroxide at the nanoscale by using liquid-phase transmission electron microscopy, three-dimensional electron tomography, and first-principles calculations. We reveal suppressed chemical reactions, unstripping phenomena, and stochastic etching behaviors that have never been observed on a macroscopic scale. We expect that solutions can be suggested from this comprehensive insight into the scale-dependent limiting factors of fabrication.

Induction of Fatty Acid Oxidation Underlies DNA Damage-Induced Cell Death and Ameliorates Obesity-Driven Chemoresistance.

The DNA damage response is essential for preserving genome integrity and eliminating damaged cells. Although cellular metabolism plays a central role in cell fate decision between proliferation, survival, or death, the metabolic response to DNA damage remains largely obscure. Here, this work shows that DNA damage induces fatty acid oxidation (FAO), which is required for DNA damage-induced cell death. Mechanistically, FAO induction increases cellular acetyl-CoA levels and promotes N-alpha-acetylation of caspase-2, leading to cell death. Whereas chemotherapy increases FAO related genes through peroxisome proliferator-activated receptor α (PPARα), accelerated hypoxia-inducible factor-1α stabilization by tumor cells in obese mice impedes the upregulation of FAO, which contributes to its chemoresistance. Finally, this work finds that improving FAO by PPARα activation ameliorates obesity-driven chemoresistance and enhances the outcomes of chemotherapy in obese mice. These findings reveal the shift toward FAO induction is an important metabolic response to DNA damage and may provide effective therapeutic strategies for cancer patients with obesity.

Characteristics and functions of infection-enhancing antibodies to the N-terminal domain of SARS-CoV-2.

Characterization of functional antibody responses to the N-terminal domain (NTD) of the SARS-CoV-2 spike (S) protein has included identification of both potent neutralizing activity and putative enhancement of infection. Fcγ-receptor (FcγR)-independent enhancement of SARS-CoV-2 infection mediated by NTD-binding monoclonal antibodies (mAbs) has been observed in vitro , but the functional significance of these antibodies in vivo is not clear. Here we studied 1,213 S-binding mAbs derived from longitudinal sampling of B-cells collected from eight COVID-19 convalescent patients and identified 72 (5.9%) mAbs that enhanced infection in a VSV-SARS-CoV-2-S-Wuhan pseudovirus (PV) assay. The majority (68%) of these mAbs recognized the NTD, were identified in patients with mild and severe disease, and persisted for at least five months post-infection. Enhancement of PV infection by NTD-binding mAbs was not observed using intestinal (Caco-2) and respiratory (Calu-3) epithelial cells as infection targets and was diminished or lost against SARS-CoV-2 variants of concern (VOC). Proteomic deconvolution of the serum antibody repertoire from two of the convalescent subjects identified, for the first time, NTD-binding, infection-enhancing mAbs among the circulating immunoglobulins directly isolated from serum ( i.e ., functionally secreted antibody). Functional analysis of these mAbs demonstrated robust activation of FcγRIIIa associated with antibody binding to recombinant S proteins. Taken together, these findings suggest functionally active NTD-specific mAbs arise frequently during natural infection and can last as major serum clonotypes during convalescence. These antibodies display diverse attributes that include FcγR activation, and may be selected against by mutations in NTD associated with SARS-CoV-2 VOC.

Characterization of SARS-CoV-2 Convalescent Patients' Serological Repertoire Reveals High Prevalence of Iso-RBD Antibodies.

While our understanding of SARS-CoV-2 pathogenesis and antibody responses following infection and vaccination has improved tremendously since the outbreak in 2019, the sequence identities and relative abundances of the individual constituent antibody molecules in circulation remain understudied. Using Ig-Seq, we proteomically profiled the serological repertoire specific to the whole ectodomain of SARS-CoV-2 prefusion-stabilized spike (S) as well as to the receptor binding domain (RBD) over a 6-month period in four subjects following SARS-CoV-2 infection before SARS-CoV-2 vaccines were available. In each individual, we identified between 59 and 167 unique IgG clonotypes in serum. To our surprise, we discovered that ∼50% of serum IgG specific for RBD did not recognize prefusion-stabilized S (referred to as iso-RBD antibodies), suggesting that a significant fraction of serum IgG targets epitopes on RBD inaccessible on the prefusion-stabilized conformation of S. On the other hand, the abundance of iso-RBD antibodies in nine individuals who received mRNA-based COVID-19 vaccines encoding prefusion-stabilized S was significantly lower (∼8%). We expressed a panel of 12 monoclonal antibodies (mAbs) that were abundantly present in serum from two SARS-CoV-2 infected individuals, and their binding specificities to prefusion-stabilized S and RBD were all in agreement with the binding specificities assigned based on the proteomics data, including 1 iso-RBD mAb which bound to RBD but not to prefusion-stabilized S. 2 of 12 mAbs demonstrated neutralizing activity, while other mAbs were non-neutralizing. 11 of 12 mAbs also bound to S (B.1.351), but only 1 maintained binding to S (B.1.1.529). This particular mAb binding to S (B.1.1.529) 1) represented an antibody lineage that comprised 43% of the individual's total S-reactive serum IgG binding titer 6 months post-infection, 2) bound to the S from a related human coronavirus, HKU1, and 3) had a high somatic hypermutation level (10.9%), suggesting that this antibody lineage likely had been elicited previously by pre-pandemic coronavirus and was re-activated following the SARS-CoV-2 infection. All 12 mAbs demonstrated their ability to engage in Fc-mediated effector function activities. Collectively, our study provides a quantitative overview of the serological repertoire following SARS-CoV-2 infection and the significant contribution of iso-RBD antibodies, demonstrating how vaccination strategies involving prefusion-stabilized S may have reduced the elicitation of iso-RBD serum antibodies which are unlikely to contribute to protection.

Deep Learning Model for Predicting Airway Organoid Differentiation.

BACKGROUND: Organoids are self-organized three-dimensional culture systems and have the advantages of both in vitro and in vivo experiments. However, each organoid has a different degree of self-organization, and methods such as immunofluorescence staining are required for confirmation. Therefore, we established a system to select organoids with high tissue-specific similarity using deep learning without relying on staining by acquiring bright-field images in a non-destructive manner. METHODS: We identified four biomarkers in RNA extracted from airway organoids. We also predicted biomarker expression by image-based analysis of organoids by convolution neural network, a deep learning method. RESULTS: We predicted airway organoid-specific marker expression from bright-field images of organoids. Organoid differentiation was verified by immunofluorescence staining of the same organoid after predicting biomarker expression in bright-field images. CONCLUSION: Our study demonstrates the potential of imaging and deep learning to distinguish organoids with high human tissue similarity in disease research and drug screening.

YAP governs cellular adaptation to perturbation of glutamine metabolism by regulating ATF4-mediated stress response.

Proliferating cells have metabolic dependence on glutamine to fuel anabolic pathways and to refill the mitochondrial carbon pool. The Hippo pathway is essential for coordinating cell survival and growth with nutrient availability, but no molecular connection to glutamine deprivation has been reported. Here, we identify a non-canonical role of YAP, a key effector of the Hippo pathway, in cellular adaptation to perturbation of glutamine metabolism. Whereas YAP is inhibited by nutrient scarcity, enabling cells to restrain proliferation and to maintain energy homeostasis, glutamine shortage induces a rapid YAP dephosphorylation and activation. Upon glutaminolysis inhibition, an increased reactive oxygen species production inhibits LATS kinase via RhoA, leading to YAP dephosphorylation. Activated YAP promotes transcriptional induction of ATF4 to induce the expression of genes involved in amino acid homeostasis, including Sestrin2. We found that YAP-mediated Sestrin2 induction is crucial for cell viability during glutamine deprivation by suppressing mTORC1. Thus, a critical relationship between YAP, ATF4, and mTORC1 is uncovered by our findings. Finally, our data indicate that targeting the Hippo-YAP pathway in combination with glutaminolysis inhibition may provide potential therapeutic approaches to treat tumors.

Fatty acid oxidation facilitates DNA double-strand break repair by promoting PARP1 acetylation.

DNA repair is a tightly coordinated stress response to DNA damage, which is critical for preserving genome integrity. Accruing evidence suggests that metabolic pathways have been correlated with cellular response to DNA damage. Here, we show that fatty acid oxidation (FAO) is a crucial regulator of DNA double-strand break repair, particularly homologous recombination repair. Mechanistically, FAO contributes to DNA repair by activating poly(ADP-ribose) polymerase 1 (PARP1), an enzyme that detects DNA breaks and promotes DNA repair pathway. Upon DNA damage, FAO facilitates PARP1 acetylation by providing acetyl-CoA, which is required for proper PARP1 activity. Indeed, cells reconstituted with PARP1 acetylation mutants display impaired DNA repair and enhanced sensitivity to DNA damage. Consequently, FAO inhibition reduces PARP1 activity, leading to increased genomic instability and decreased cell viability upon DNA damage. Finally, our data indicate that FAO serves as an important participant of cellular response to DNA damage, supporting DNA repair and genome stability.

Fatty acid oxidation supports melanoma cell migration through autophagy regulation.

Metastasis is one of the most malignant characteristics of cancer cells, in which metabolic reprogramming is crucial for promoting and sustaining multi-steps of metastasis, including invasion, migration and infiltration. Recently, it has been shown that melanoma cells undergo a metabolic switching toward the upregulation of fatty acid oxidation (FAO) during metastasis. However, the underlying mechanisms by which FAO contributes to metastasis of melanoma cells remain obscure. Here, we report that FAO contributes to melanoma cell migration and invasion by regulating the formation of autophagosomes. Pharmacological or genetic inhibition of FAO impairs migration of melanoma cells, which seems not to be linked to energy production or redox homeostasis. Importantly, we reveal that acetyl-CoA production by FAO contributes to melanoma cell migration through autophagy regulation. Mechanistically, FAO inhibition results in increased autophagosome formation, which suppresses migration and invasion properties of melanoma cells. Our results underscore the crucial role of FAO in melanoma cell migration and support the potential therapeutic relevance of modulating cellular acetyl-CoA levels to inhibit cancer metastasis.

Suppression of fatty acid oxidation supports pancreatic cancer growth and survival under hypoxic conditions through autophagy induction.

Hypoxia, one of the key features of solid tumors, induces autophagy, which acts as an important adaptive mechanism for tumor progression under hypoxic environment. Cellular metabolic reprogramming has been correlated with hypoxia, but the molecular connection to the induction of autophagy remains obscure. Here, we show that suppression of fatty acid oxidation (FAO) by hypoxia induces autophagy in human pancreatic ductal adenocarcinoma (PDAC) cells that is required for their growth and survival. Reduced cellular acetyl-CoA levels caused by FAO inhibition decreases LC3 acetylation, resulting in autophagosome formation. Importantly, PDAC cells are significantly dependent on this metabolic reprogramming, as improving FAO leads to a reduction in hypoxia-induced autophagy and an increase in cell death after chemotherapy. Thus, our study supports that suppression of FAO is an important metabolic response to hypoxia and indicates that targeting this pathway in PDAC may be an effective therapeutic approach.

Infant Antibody Repertoires during the First Two Years of Influenza Vaccination.

The first encounter with influenza virus biases later immune responses. This "immune imprinting," formerly from infection within a few years of birth, is in the United States now largely from immunization with a quadrivalent, split vaccine (IIV4 [quadrivalent inactivated influenza vaccine]). In a pilot study of IIV4 imprinting, we used single-cell cultures, next-generation sequencing, and plasma antibody proteomics to characterize the primary antibody responses to influenza in two infants during their first 2 years of seasonal influenza vaccination. One infant, who received only a single vaccination in year 1, contracted an influenza B virus (IBV) infection between the 2 years, allowing us to compare imprinting by infection and vaccination. That infant had a shift in hemagglutinin (HA)-reactive B cell specificity from largely influenza A virus (IAV) specific in year 1 to IBV specific in year 2, both before and after the year 2 vaccination. HA-reactive B cells from the other infant maintained a more evenly distributed specificity. In year 2, class-switched HA-specific B cell IGHV somatic hypermutation (SHM) levels reached the average levels seen in adults. The HA-reactive plasma antibody repertoires of both infants comprised a relatively small number of antibody clonotypes, with one or two very abundant clonotypes. Thus, after the year 2 boost, both infants had overall B cell profiles that resembled those of adult controls. IMPORTANCE Influenza virus is a moving target for the immune system. Variants emerge that escape protection from antibodies elicited by a previously circulating variant ("antigenic drift"). The immune system usually responds to a drifted influenza virus by mutating existing antibodies rather than by producing entirely new ones. Thus, immune memory of the earliest influenza virus exposure has a major influence on later responses to infection or vaccination ("immune imprinting"). In the many studies of influenza immunity in adult subjects, imprinting has been from an early infection, since only in the past 2 decades have infants received influenza immunizations. The work reported in this paper is a pilot study of imprinting by the flu vaccine in two infants, who received the vaccine before experiencing an influenza virus infection. The results suggest that a quadrivalent (four-subtype) vaccine may provide an immune imprint less dominated by one subtype than does a monovalent infection.

Combination Therapy of the Active KRAS-Targeting Antibody inRas37 and a PI3K Inhibitor in Pancreatic Cancer.

KRAS activating mutations, which are present in more than 90% of pancreatic cancers, drive tumor dependency on the RAS/mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K)/AKT signaling pathways. Therefore, combined targeting of RAS/MAPK and PI3K/AKT signaling pathways may be required for optimal therapeutic effect in pancreatic cancer. However, the therapeutic efficacy of combined MAPK and PI3K/AKT signaling target inhibitors is unsatisfactory in pancreatic cancer treatment, because it is often accompanied by MAPK pathway reactivation by PI3K/AKT inhibitor. Therefore, we developed an inRas37 antibody, which directly targets the intra-cellularly activated GTP-bound form of oncogenic RAS mutation and investigated its synergistic effect in the presence of the PI3K inhibitor BEZ-235 in pancreatic cancer. In this study, inRas37 remarkably increased the drug response of BEZ-235 to pancreatic cancer cells by inhibiting MAPK reactivation. Moreover, the co-treatment synergistically inhibited cell proliferation, migration, and invasion and exhibited synergistic anticancer activity by inhibiting the MAPK and PI3K pathways. The combined administration of inRas37and BEZ-235 significantly inhibited tumor growth in mouse models. Our results demonstrated that inRas37 synergistically increased the antitumor activity of BEZ-235 by inhibiting MAPK reactivation, suggesting that inRas37 and BEZ-235 co-treatment could be a potential treatment approach for pancreatic cancer patients with KRAS mutations.

One-Step Etching Characteristics of ITO/Ag/ITO Multilayered Electrode in High-Density and High-Electron-Temperature Plasma.

This paper presents the dry etching characteristics of indium tin oxide (ITO)/Ag/ITO multilayered thin film, used as a pixel electrode in a high-resolution active-matrix organic light-emitting diode (AMOLED) device. Dry etching was performed using a combination of H2 and HCl gases in a reactive ion etching system with a remote electron cyclotron resonance (ECR) plasma source, in order to achieve high electron temperature. The effect of the gas ratio (H2/HCl) was closely observed, in order to achieve an optimal etch profile and an effective etch process, while other parameters-such as the radio frequency (RF) power, ECR power, chamber pressure, and temperature-were fixed. The optimized process, with an appropriate gas ratio, constitutes a one-step serial dry etch solution for ITO and Ag multilayered thin films.

Intracellular KRAS-specific antibody enhances the anti-tumor efficacy of gemcitabine in pancreatic cancer by inducing endosomal escape.

KRAS mutation is associated with the progression and growth of pancreatic cancer and contributes to chemo-resistance, which poses a significant clinical challenge in pancreatic cancer. Here, we developed a RT22-ep59 antibody (Ab) that directly targets the intracellularly activated GTP-bound form of oncogenic KRAS mutants after it is internalized into cytosol by endocytosis through tumor-associated receptor of extracellular epithelial cell adhesion molecule (EpCAM) and investigated its synergistic anticancer effects in the presence of gemcitabine in pancreatic cancer. We first observed that RT22-ep59 specifically recognized tumor-associated EpCAM and reached the cytosol by endosomal escape. In addition, the anticancer effect of RT22-ep59 was observed in the high-EpCAM-expressing pancreatic cancer cells and gemcitabine-resistant pancreatic cancer cells, but it had little effect on the low-EpCAM-expressing pancreatic cancer cells. Additionally, co-treatment with RT22-ep59 and gemcitabine synergistically inhibited cell viability, migration, and invasion in 3D-cultures and exhibited synergistic anticancer activity by inhibiting the RAF/ERK or PI3K/AKT pathways in cells with high-EpCAM expression. In an orthotopic mouse model, combined administration of RT22-ep59 and gemcitabine significantly inhibited tumor growth. Furthermore, the co-treatment suppressed cancer metastasis by blocking EMT signaling in vitro and in vivo. Our results demonstrated that RT22-ep59 synergistically increased the antitumor activity of gemcitabine by inhibiting RAS signaling by specifically targeting KRAS. This indicates that co-treatment with RT22-ep59 and gemcitabine might be considered a potential therapeutic strategy for pancreatic cancer patients harboring KRAS mutation.

Prevalence of metabolic syndrome in the Gulf Cooperation Council countries: meta-analysis of cross-sectional studies.

Metabolic syndrome is a collection of health-related conditions that lead to serious health condition. An individual with metabolic syndrome may acquire greater risk for adult-onset complications such as cardiovascular or neurological disorders. The aim of this study was to review and provide the prevalence of metabolic syndrome in Gulf Cooperation Council countries (GCC). Literature searches were performed on PubMed, Google scholar, and Web of Science Core Collection for English-language articles along with national studies. The following search terms were used during search: "prevalence of metabolic syndrome in GCC," "prevalence of metabolic syndrome in middle east," "prevalence of metabolic syndrome in Arab," and "prevalence of metabolic syndrome name of country." Thirty-seven studies were selected for final review out of 132 studies. The weighted pooled prevalence of metabolic syndrome was 27.3% with high heterogeneity (I 2=98.94%; Cochran Q-test P<0.01). The results showed comparatively high and rising rate of metabolic syndrome in the GCC area. Preventative strategy should be considered to reduce the risk of morbidity or mortality related to metabolic syndrome.

Direct targeting of oncogenic RAS mutants with a tumor-specific cytosol-penetrating antibody inhibits RAS mutant-driven tumor growth.

Oncogenic RAS mutant (RASMUT) proteins have been considered undruggable via conventional antibody regimens owing to the intracellular location restricting conventional-antibody accessibility. Here, we report a pan-RAS-targeting IgG antibody, inRas37, which directly targets the intracellularly activated form of various RASMUT subtypes after tumor cell-specific internalization into the cytosol to block the interactions with effector proteins, thereby suppressing the downstream signaling. Systemic administration of inRas37 exerted a potent antitumor activity in a subset of RASMUT tumor xenografts in mice, but little efficacy in RASMUT tumors with concurrent downstream PI3K mutations, which were overcome by combination with a PI3K inhibitor. The YAP1 protein was up-regulated as an adaptive resistance-inducing response to inRas37 in RASMUT-dependent colorectal tumors; accordingly, a combination of inRas37 with a YAP1 inhibitor manifested synergistic antitumor effects in vitro and in vivo. Our study offers a promising pan-RAS-targeting antibody and the corresponding therapeutic strategy against RASMUT tumors.

KRAS targeting antibody synergizes anti-cancer activity of gemcitabine against pancreatic cancer.

Pancreatic cancer exhibits an oncogenic KRAS mutation rate of ∼90%. Despite research and drug development efforts focused on KRAS, no targeted therapy has been clinically approved for the treatment of pancreatic cancer with KRAS mutation. Also, the efficacy of gemcitabine is poor due to rapidly acquired resistance. We developed RT11-i antibody, which directly targets the intracellularly activated GTP-bound form of oncogenic RAS mutants. Here, we investigated the combined effects of RT11-i and gemcitabine in vitro and in vivo, and the mechanism involved. RT11-i significantly sensitized pancreatic cancer cells to gemcitabine. Also, the co-treatment synergistically inhibited angiogenesis, migration, and invasion, and showed synergistic anticancer activity by inhibiting the RAF/MEK/ERK or PI3K/AKT pathways. Furthermore, co-treatment inhibited endothelial barrier disruption in tumor vessels, which is a critical step in vascular leakiness of metastasis, and improved vessel structural stability. Importantly, co-treatment significantly suppressed tumor growth in an orthotopic tumor model. Taken together, our findings show that RT11-i synergistically increased the antitumor activity of gemcitabine by inhibiting RAS downstream signaling, which suggests RT11-i and gemcitabine be viewed a potential combination treatment option for pancreatic cancer patients with KRAS mutation.

Engineering of a tumor cell-specific, cytosol-penetrating antibody with high endosomal escape efficacy.

The main obstacles for practical uses of cytosol-penetrating peptides and proteins include their lack of cell- or tissue-specific targeting and limited cytosolic access owing to the poor endosomal escape ability. We have previously reported a cytosol-penetrating, human IgG1 antibody TMab4-WYW, generally referred to as a cytotransmab (CT), which reaches the cytosol of living cells but nonspecifically because it is endocytosed via a ubiquitously expressed receptor called heparan sulfate proteoglycan (HSPG). Here, our aim was to construct a next-generation CT with tumor cell specificity and improved endosomal escape efficiency. We first substantially reduced the HSPG-binding activity of TMab4-WYW and then fused a cyclic peptide specifically recognizing tumor-associated epithelial cell adhesion molecule (EpCAM) to the N terminus of the light chain for EpCAM-mediated endocytosis, while maintaining the endosomal escape ability in the light chain variable domain (VL), thus generating epCT05. Then, we separately engineered another CT, dubbed epCT65-AAA, with an endosomal escape ability only in the heavy chain variable domain (VH) but not in VL, by functional grafting of the endosomal escape motif of epCT05 VL to the VH. We finally combined the heavy chain of epCT65-AAA and the light chain of epCT05 to create epCT65 with endosomal escape capacity in both the VH and VL. epCT65 effectively localized to the cytosol of only EpCAM-expressing tumor cells and showed approximately twofold improved endosomal escape efficiency, as compared with CTs with endosomal escape motifs in either VH or VL. The full-IgG format CT, epCT65, with a tumor cell-specific cytosol-penetrating activity, has a great potential for practical medical applications, e.g., as a carrier for cytosolic delivery of payloads.