Chemo-enzymatic Functionalization of Bovine Milk Exosomes with an EGFR Nanobody for Target-specific Drug Delivery.

Bovine milk exosomes (BmExo) have been identified as versatile nanovesicles for anti-cancer drugs delivery due to their natural availability and biocompatibility. However, tumor-specific delivery based on BmExo often requires post-isolation modifications of the membrane surface with active-targeting ligands. In this study, we report an alternative approach to functionalize BmExo with nanobody using Sortase A-mediated site-specific ligation for drug delivery. The BmExo membrane was first coated with a diglycine-containing amphiphile molecule, NH2-GG-PEG2000-DSPE, through hydrophobic insertion, following by ligation with EGFR nanobody (7D12) by Sortase A (SrtA). The successful construction of BmExo-7D12 was confirmed by Western blotting analysis, electron microscopy, and dynamic light scattering (DLS). As a demonstration model, BmExo-7D12 loaded with the chemotherapeutic drug doxorubicin (Dox) was shown to be able to deliver Dox to cancer cells in response to the expression of EGFR as manifested by immunocytochemistry and flow cytometry analysis. Finally, the cytotoxicity assay showed that BmExo-7D12-Dox was more effective in killing tumor cells with high EGFR expression while significantly reduced the non-specific toxicity to EGFR negative cells. This study developed an effective approach to functionalize BmExo with nanobody for target-specific drug delivery. This approach should prove to be versatile and efficient for the generation of protein-ligands modified BmExo.

Chemoenzymatic Synthesis of a Rhamnose-Functionalized Bispecific Nanobody as a Bispecific Antibody Mimic for Cancer Immunotherapy.

Bispecific antibodies (BsAbs) are next-generation therapeutics for complex cancer treatment. Herein, we developed a dual-targeting non-IgG format of bsAbs by using a bispecific nanobody (bsNb) that can simultaneously target EGFR and HER2 on tumor cells. Site-specific modification of the anti-EGFR-HER2 bsNb was conducted using the rhamnose (Rha) hapten via sortase A-mediated ligation to reconstitute the missing crystallizable fragment (Fc) effector biological functions. Functionally similar to bsAbs, bsNb-Rha conjugates retained dual-targeting activity and exerted potent anticancer effects via the Fc-domain-mediated engagement of endogenous anti-Rha antibodies. Further, an optimized bsNb-Rha conjugate exhibited markedly improved pharmacokinetics and efficient inhibitory effects against xenograft tumor growth in vivo. Our strategy provides a general and cost-effective platform to generate a new bsAb format for cancer immunotherapy.

Nanobody-Engineered Natural Killer Cell Conjugates for Solid Tumor Adoptive Immunotherapy.

Cancer immunotherapy based on natural killer (NK) cells is demonstrated to be a promising strategy. However, NK cells are deficient in ligands that target specific tumors, resulting in limited antitumor efficacy. Here, a glycoengineering approach to imitate the chimeric antigen receptor strategy and decorate NK cells with nanobodies to promote NK-based immunotherapy in solid tumors is proposed. Nanobody 7D12, which specifically recognizes the human epidermal growth factor receptor (EGFR) that is overexpressed on many solid tumors, is coupled to the chemically synthesized DBCO-PEG4 -GGG-NH2 by sortase A-mediated ligation to generate DBCO-7D12. The NK92MI cells bearing azide groups are then equipped with DBCO-7D12 via bioorthogonal click chemistry. The resultant 7D12-NK92MI cells exhibit high specificity and affinity for EGFR-overexpressing tumor cells in vitro and in vivo by the 7D12-EGFR interaction, causing increased cytokine secretion to more effectively kill EGFR-positive tumor cells, but not EGFR-negative cancer cells. Importantly, the 7D12-NK92MI cells also show a wide anticancer spectrum and extensive tumor penetration. Furthermore, mouse experiments reveal that 7D12-NK92MI treatment achieves excellent therapeutic efficacy and outstanding safety. The authors' works provide a cell modification strategy using specific protein ligands without genetic manipulation and present a potential novel method for cancer-targeted immunotherapy by NK cells.

Enhanced acid-stress tolerance in Lactococcus lactis NZ9000 by overexpression of ABC transporters.

BACKGROUND: Microbial cell factories are widely used in the production of acidic products such as organic acids and amino acids. However, the metabolic activity of microbial cells and their production efficiency are severely inhibited with the accumulation of intracellular acidic metabolites. Therefore, it remains a key issue to enhance the acid tolerance of microbial cells. In this study, we investigated the effects of four ATP-binding cassette (ABC) transporters on acid stress tolerance in Lactococcus lactis. RESULTS: Overexpressing the rbsA, rbsB, msmK, and dppA genes exhibited 5.8-, 12.2-, 213.7-, and 5.2-fold higher survival rates than the control strain, respectively, after acid shock for 3 h at pH 4.0. Subsequently, transcriptional profile alterations in recombinant strains were analyzed during acid stress. The differentially expressed genes associated with cold-shock proteins (csp), fatty acid biosynthesis (fabH), and coenzyme A biosynthesis (coaD) were up-regulated in the four recombinant strains during acid stress. Additionally, some genes were differentially expressed in specific recombinant strains. For example, in L. lactis (RbsB), genes involved in the pyrimidine biosynthetic pathway (pyrCBDEK) and glycine or betaine transport process (busAA and busAB) were up-regulated during acid stress, and the argG genes showed up-regulations in L. lactis (MsmK). Finally, we found that overexpression of the ABC transporters RbsB and MsmK increased intracellular ATP concentrations to protect cells against acidic damage in the initial stage of acid stress. Furthermore, L. lactis (MsmK) consistently maintained elevated ATP concentrations under acid stress. CONCLUSIONS: This study elucidates the common and specific mechanisms underlying improved acid tolerance by manipulating ABC transporters and provides a further understanding of the role of ABC transporters in acid-stress tolerance.

Design and synthesis of novel dual-cyclic RGD peptides for αvß3 integrin targeting.

The specific binding of RGD cyclic peptide with integrin αvß3 attracts great research interest for tumor-targeting drug delivery. Herein, we designed and synthesized a series of dual-ring RGD-peptide derivatives as a drug carrier for αvß3 targeting. Three novel peptides showed excellent cell adhesion inhibition effect, in which, P3 exhibited 7-fold enhancement in IC50 compared with cyclo(RGDfK). Drug-loaded cytotoxicity experiment and imaging experiment indicated that such dual-cyclic RGD peptides have good tumor targeting effects. This work provides a new strategy for the design of novel RGD peptides.

One-step immobilization and purification of genetic engineering CBD fusion EndoS on cellulose for antibodies Fc-glycan remodeling.

The endoglycosidase (EndoS and its glycosynthase mutants D233A, D233Q) gene was fused with cellulose binding domain (CBD) using pET-35b vector and the fusion enzymes were successfully expressed in Escherichia coli. Then a simplified approach for one-step immobilization and purification of EndoS enzymes using cellulose as matrices were developed and excellent loading efficiency (81-90%) was achieved in optimal condition. The cellulose immobilized CBD-EndoS and the glycosynthase mutants presented high catalytic activity and were successfully applied in a two-step antibody Fc N-glycan remodeling, generating a therapeutic antibody with homogeneous glycoform in high efficiency. The cellulose immobilized CBD-EndoS and its mutants (D233A and D233Q) displayed excellent storage stability when stored at 4 degrees for one month. Reusability studies demonstrated that the cellulose immobilized CBD-EndoS and its mutants could be recycled for five times without obvious activity loss.

Systemic understanding of Lactococcus lactis response to acid stress using transcriptomics approaches.

During fermentation, acid stress caused by the accumulation of acidic metabolites seriously affects the metabolic activity and production capacity of microbial cells. To elucidate the acid stress-tolerance mechanisms of microbial cells, we performed genome mutagenesis combined with high-throughput technologies to screen acid stress-tolerant strains. Mutant strain Lactococcus lactis WH101 showed a 16,000-fold higher survival rate than that of the parent strain after 5 h of acid shock at pH 4.0 and maintained higher ATP, NH4+, and intracellular pH (pHi) levels during acid stress. Additionally, comparative transcriptomics analysis revealed enhanced regulation of carbohydrate metabolism and sugar transport to provide additional energy, amino acid metabolism and transport to maintain pHi homeostasis and ATP generation, and fatty acid metabolism to enhance cellular acid tolerance. Moreover, overexpression of identified components resulted in 12.6- and 12.9-fold higher survival rates after acid shock for 3 h at pH 4.0 in L. lactis (ArcB) and L. lactis (MalQ) compared to the control strain, respectively. These findings provide valuable insight into the acid stress-response mechanisms of L. lactis and promote the further development of robust industrial strains.

ATF6a, a Runx2-activable transcription factor, is a new regulator of chondrocyte hypertrophy.

Our previous research has shown that the spliced isoform of XBP1 (XBP1s) is an important downstream mediator of BMP2 and is involved in BMP2-stimulated chondrocyte differentiation. Herein, we report that ATF6 and its cleaved N-terminal cytoplasmic domain (known as ATF6a) are expressed in growth plate chondrocytes. We find that these proteins are differentially induced during BMP2-triggered chondrocyte differentiation. This differential expression probably results from the activation of the ATF6 gene by Runx2 and its repression by the Sox6 transcription factor. Runx2 and Sox6 act through their respective binding elements on the ATF6 gene. When overexpressed, ATF6 and ATF6a intensify chondrogenesis; our studies demonstrate that under the stimulation of ATF6 and ATF6a, chondrocytes tend to be hypertrophied and mineralized, a process leading to bone formation. By contrast, lowering expression of ATF6a by use of its specific siRNA suppresses chondrocyte differentiation. Moreover, ATF6a interacts with Runx2 and augments the Runx2-mediated hypertrophication of chondrocytes. Importantly, overexpression and knockdown of ATF6a during the chondrocyte hypertrophy process also led to altered expressions of IHH and PTHrP (also known as PTHLH). Taken together, these findings indicate that ATF6a favorably controls chondrogenesis and bone formation (1) by acting as a co-factor of Runx2 and enhancing Runx2-incited hypertrophic chondrocyte differentiation, and (2) by affecting IHH and PTHrP signaling.

Enzymatic On-Resin Peptide Cleavage and in Situ Cyclization One-Pot Strategy for the Synthesis of Cyclopeptide and Cyclotide.

A one-pot strategy combining sortase A mediated on-resin peptide cleavage and in situ cyclization was developed for the synthesis of cyclic peptides. This strategy was applied to synthesize head-to-tail cyclic antibacterial bovine lactoferricin peptide LFcinB20-35 in a yield of 67%. The one-pot strategy was compatible with an oxidative folding reaction, and complex cyclotides containing one or two disulfide bonds, such as sunflower trypsin inhibitors-1 and α-conotoxin MII, were successfully synthesized in one pot in a yield of 77% and 61%, respectively.

Evolutionary engineering of industrial microorganisms-strategies and applications.

Microbial cells have been widely used in the industry to obtain various biochemical products, and evolutionary engineering is a common method in biological research to improve their traits, such as high environmental tolerance and improvement of product yield. To obtain better integrate functions of microbial cells, evolutionary engineering combined with other biotechnologies have attracted more attention in recent years. Classical laboratory evolution has been proven effective to letting more beneficial mutations occur in different genes but also has some inherent limitations such as a long evolutionary period and uncontrolled mutation frequencies. However, recent studies showed that some new strategies may gradually overcome these limitations. In this review, we summarize the evolutionary strategies commonly used in industrial microorganisms and discuss the combination of evolutionary engineering with other biotechnologies such as systems biology and inverse metabolic engineering. Finally, we prospect the importance and application prospect of evolutionary engineering as a powerful tool especially in optimization of industrial microbial cell factories.

Improved acid-stress tolerance of Lactococcus lactis NZ9000 and Escherichia coli BL21 by overexpression of the anti-acid component recT.

Acid accumulation caused by carbon metabolism severely affects the fermentation performance of microbial cells. Here, different sources of the recT gene involved in homologous recombination were functionally overexpressed in Lactococcus lactis NZ9000 and Escherichia coli BL21, and their acid-stress tolerances were investigated. Our results showed that L. lactis NZ9000 (ERecT and LRecT) strains showed 1.4- and 10.4-fold higher survival rates against lactic acid (pH 4.0), respectively, and that E. coli BL21 (ERecT) showed 16.7- and 9.4-fold higher survival rates than the control strain against lactic acid (pH 3.8) for 40 and 60 min, respectively. Additionally, we found that recT overexpression in L. lactis NZ9000 improved their growth under acid-stress conditions, as well as increased salt- and ethanol-stress tolerance and intracellular ATP concentrations in L. lactis NZ9000. These findings demonstrated the efficacy of recT overexpression for enhancing acid-stress tolerance and provided a promising strategy for insertion of anti-acid components in different hosts.

Immobilization of Staphylococcus aureus Sortase A on Chitosan Particles and Its Applications in Peptide-to-Peptide Ligation and Peptide Cyclization.

Chitosan macro-particles prepared by the neutralization method were applied to Sortase A (SrtA) immobilization using glutaraldehyde as a crosslinking agent. The particles were characterized by Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). Response surface methodology (RSM) was employed to optimize the immobilization process. An average specific activity of 3142 U (mg protein)-1 was obtained under optimized immobilization conditions (chitosan concentration 3%, SrtA concentration 0.5 mg·mL-1, glutaraldehyde concentration 0.5%, crosslinking and immobilization at 20 °C, crosslinking for 3 h, and an immobilization time of 8 h). The transpeptidase activity of immobilized SrtA was proved by a peptide-to-peptide ligation with a conversion yield approximately at 80%, and the immobilized catalyst was successfully reused for five cycles without obvious activity loss. Moreover, the scale-up capability of using immobilized SrtA to catalyze a head-to-tail peptide cyclization was investigated in a batch reaction and the conversion yield was more than 95% when using 20 mg of peptide as a substrate.

Efficient extracellular expression of transpeptidase sortase A in Pichia pastoris.

In order to achieve efficient extracellular expression of Sortase A (SrtA), various strategies in Pichia pastoris system were applied in this study. Among different constructed recombinant strains, the SMD1168 strain integrated 5.7 copies of srtA gene under control of AOX1 promoter was proved to be the best strain for the extracellular SrtA expression. After the optimization of fermentation conditions (induction 72 h at 28 °C, initial pH 6.0, supplemented with 1.5% methanol), the highest yield and activity of extracellular SrtA reached 97.8 mg/L and 131.9 U/mL at the shake-flask level, respectively. This is the first report on the efficient secretory expression of SrtA in P. pastoris and the yield of SrtA is the maximum compared with previous reports. In addition, the transpeptidation activity of extracellular SrtA was confirmed by the successful immobilization of enhanced green fluorescent protein (EGFP) onto Gly3-polystyrene beads.

New potent and selective αvß3 integrin ligands: Macrocyclic peptides containing RGD motif synthesized by sortase A-mediated ligation.

Three 14-mer macrocyclic peptides 1-3 containing mono-, di- and tri-RGD structure motif were designed and synthesized by sortase A-mediated ligation in good yields. The results of in intro cell-based biological assays indicated that linear peptide 5 and macrocyclic peptide 1, containing di-RGD and mono-RGD motif respectively, showed remarkable potency and selectivity to αvß3 integrin.

New Antibiotic Candidates against Helicobacter pylori.

Helicobacter pylori is a Gram-negative bacterium that colonizes the gut of over 50% of the world's population. It is responsible for most peptic ulcers and is an important risk factor for gastric cancer. Antibiotic treatment for H. pylori infections is challenging as drug resistance has developed to antibiotics with traditional mechanisms of action. H. pylori uses an unusual pathway for menaquinone biosynthesis with 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) catalyzing an essential step. We validated MTAN as a target with a transition-state analogue of the enzyme [Wang, S.; Haapalainen, A. M.; Yan, F.; et al. Biochemistry 2012, 51, 6892-6894]. MTAN inhibitors will only be useful drug candidates if they can both include tight binding to the MTAN target and have the ability to penetrate the complex cell membrane found in Gram-negative H. pylori. Here we explore structural scaffolds for MTAN inhibition and for growth inhibition of cultured H. pylori. Sixteen analogues reported here are transition-state analogues of H. pylori MTAN with dissociation constants of 50 pM or below. Ten of these prevent growth of the H. pylori with IC90 values below 0.01 µg/mL. These remarkable compounds meet the criteria for potent inhibition and cell penetration. As a consequence, 10 new H. pylori antibiotic candidates are identified, all of which prevent H. pylori growth at concentrations 16-2000-fold lower than the five antibiotics, amoxicillin, metronidazole, levofloxacin, tetracyclin, and clarithromycin, commonly used to treat H. pylori infections. X-ray crystal structures of MTAN cocrystallized with several inhibitors show them to bind in the active site making interactions consistent with transition-state analogues.

Rhamnose modified antibodies show improved immune killing towards EGFR-positive solid tumor cells.

Therapeutic monoclonal antibodies (mAbs) against the epidermal growth factor receptor (EGFR) have shown clinical efficacy in colorectal cancer and other solid cancers. Enhancing the effector functions of these anti-EGFR mAbs is believed to be a valuable approach to achieve improved efficacy in clinical setting. Here, we report the development of an effector function-enhanced antibody by rhamnose (Rha) functionalization. Cetuximab, a human/mouse chimeric anti-EGFR mAb, was selected and site-specifically conjugated with Rha haptens. The obtained cetuximab-Rha conjugate was shown to be able to selectively redirect amounts of endogenous anti-Rha antibodies onto EGFR-positive solid tumor cells and thereby provide more Fc domains to achieve enhancement of effector functions including complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated phagocytosis (ADCP). Particularly, CDC, one powerful cell killing mechanism which is inactive in cetuximab, was dramatically improved. This study demonstrates the potential of rhamnose-modified antibody for EGFR-positive solid tumor immunotherapy.

Causal associations between dietary factors and colorectal cancer risk: a Mendelian randomization study.

Background: Previous epidemiological studies have found a link between colorectal cancer (CRC) and human dietary habits. However, the inherent limitations and inevitable confounding factors of the observational studies may lead to the inaccurate and doubtful results. The causality of dietary factors to CRC remains elusive. Methods: We conducted two-sample Mendelian randomization (MR) analyses utilizing the data sets from the IEU Open GWAS project. The exposure datasets included alcoholic drinks per week, processed meat intake, beef intake, poultry intake, oily fish intake, non-oily fish intake, lamb/mutton intake, pork intake, cheese intake, bread intake, tea intake, coffee intake, cooked vegetable intake, cereal intake, fresh fruit intake, salad/raw vegetable intake, and dried fruit intake. In our MR analyses, the inverse variance weighted (IVW) method was employed as the primary analytical approach. The weighted median, MR-Egger, weighted mode, and simple mode were also applied to quality control. Heterogeneity and pleiotropic analyses were implemented to replenish the accuracy of the results. Results: MR consequences revealed that alcoholic drinks per week [odds ratio (OR): 1.565, 95% confidence interval (CI): 1.068-2.293, p = 0.022], non-oily fish intake (OR: 0.286; 95% CI: 0.095-0.860; p = 0.026), fresh fruit intake (OR: 0.513; 95% CI: 0.273-0.964; p = 0.038), cereal intake (OR: 0.435; 95% CI: 0.253-0.476; p = 0.003) and dried fruit intake (OR: 0.522; 95% CI: 0.311-0.875; p = 0.014) was causally correlated with the risk of CRC. No other significant relationships were obtained. The sensitivity analyses proposed the absence of heterogeneity or pleiotropy, demonstrating the reliability of the MR results. Conclusion: This study indicated that alcoholic drinks were associated with an increased risk of CRC, while non-oily fish intake, fresh fruit intake, cereal intake, and dried fruit were associated with a decreased risk of CRC. This study also indicated that other dietary factors included in this research were not associated with CRC. The current study is the first to establish the link between comprehensive diet-related factors and CRC at the genetic level, offering novel clues for interpreting the genetic etiology of CRC and replenishing new perspectives for the clinical practice of gastrointestinal disease prevention.

Multivalent Rhamnose-Modified EGFR-Targeting Nanobody Gains Enhanced Innate Fc Effector Immunity and Overcomes Cetuximab Resistance via Recruitment of Endogenous Antibodies.

Cetuximab resistance is a significant challenge in cancer treatment, requiring the development of novel therapeutic strategies. In this study, a series of multivalent rhamnose (Rha)-modified nanobody conjugates are synthesized and their antitumor activities and their potential to overcome cetuximab resistance are investigated. Structure-activity relationship studies reveal that the multivalent conjugate D5, bearing sixteen Rha haptens, elicits the most potent innate fragment crystallizable (Fc) effector immunity in vitro and exhibits an excellent in vivo pharmacokinetics by recruiting endogenous antibodies. Notably, it is found that the optimal conjugate D5 represents a novel entity capable of reversing cetuximab-resistance induced by serine protease (PRSS). Moreover, in a xenograft mouse model, conjugate D5 exhibits significantly improved antitumor efficacy compared to unmodified nanobodies and cetuximab. The findings suggest that Rha-Nanobody (Nb) conjugates hold promise as a novel therapeutic strategy for the treatment of cetuximab-resistant tumors by enhancing the innate Fc effector immunity and enhancing the recruitment of endogenous antibodies to promote cancer cell clearance by innate immune cells.

Chiral Nanomaterials for Cancer Vaccines.

Chirality is a fundamental characteristic of living organisms and is commonly observed at the biomolecule, cellular, and tissue levels. Chiral nanomaterials play an irreplaceable role in nanomedicine and nanobiology because of their unique enantioselectivity with biological components. Here, research progress relating to chiral nanomaterials in the field of vaccines is reviewed, including antigen presenting systems, immune adjuvants, and cancer vaccines. First, the common synthesis methods are outlined for different types of chiral nanomaterials, as well as their chiral sources, optical properties, and potential biological applications. Then, the application of chiral nanomaterials are discussed in the field of vaccines with reference to the promotion of antigen presentation and activation of the immune system for tumor immunotherapy. Finally, the current obstacles and future research directions of chiral nanomaterials are revealed with regard to regulating the immune system.

Reprogramming tumor-associated macrophages by a dually targeted milk exosome system as a potent monotherapy for cancer.

Tumor-associated macrophages (TAMs) play a key role in inducing an immunosuppressive tumor microenvironment (TME) and cancer immune escape. We previously revealed that PDL1 (a key immune checkpoint) was upregulated in TAMs and induced M2 polarization, highlighting PDL1 in TAMs as a promising cancer therapeutic target. In this study, we developed an engineered milk exosome (mExo) system decorated with M2pep (an M2 macrophage binding peptide) and 7D12 (an anti-EGFR nanobody) (7D12-mExo-M2pep-siPDL1) to specifically deliver siPDL1 into M2 TAMs. A series of in vitro and in vivo assays showed that the dually targeted engineered mExos efficiently delivered siPDL1 into M2 TAMs and repolarized them into M1 macrophages, restoring CD8+ T cell immune activity and remodeling TME. Importantly, systemically administered 7D12-mExo-M2pep-siPDL1 showed efficient single-agent antitumor activity, resulting in nearly 90% tumor growth inhibition in a mouse model of orthotopic epidermal growth factor receptor (EGFR) cancer. Collectively, our study indicates that PDL1 is a promising target for TAM-based cancer immunotherapy, and our engineered mExo-based nanomedicine represents a novel tool for specifically targeting M2 TAMs, distinguishing this novel therapeutic method from other TAM-targeting therapies and highlighting its promising clinical potential.