LowTempGAL: a highly responsive low temperature-inducible GAL system in Saccharomyces cerevisiae.

Temperature is an important control factor for biologics biomanufacturing in precision fermentation. Here, we explored a highly responsive low temperature-inducible genetic system (LowTempGAL) in the model yeast Saccharomyces cerevisiae. Two temperature biosensors, a heat-inducible degron and a heat-inducible protein aggregation domain, were used to regulate the GAL activator Gal4p, rendering the leaky LowTempGAL systems. Boolean-type induction was achieved by implementing a second-layer control through low-temperature-mediated repression on GAL repressor gene GAL80, but suffered delayed response to low-temperature triggers and a weak response at 30°C. Application potentials were validated for protein and small molecule production. Proteomics analysis suggested that residual Gal80p and Gal4p insufficiency caused suboptimal induction. 'Turbo' mechanisms were engineered through incorporating a basal Gal4p expression and a galactose-independent Gal80p-supressing Gal3p mutant (Gal3Cp). Varying Gal3Cp configurations, we deployed the LowTempGAL systems capable for a rapid stringent high-level induction upon the shift from a high temperature (37-33°C) to a low temperature (≤30°C). Overall, we present a synthetic biology procedure that leverages 'leaky' biosensors to deploy highly responsive Boolean-type genetic circuits. The key lies in optimisation of the intricate layout of the multi-factor system. The LowTempGAL systems may be applicable in non-conventional yeast platforms for precision biomanufacturing.

Recent advances in the production of recombinant factor IX: bioprocessing and cell engineering.

Appropriate treatment of Hemophilia B is vital for patients' quality of life. Historically, the treatment used was the administration of coagulation Factor IX derived from human plasma. Advancements in recombinant technologies allowed Factor IX to be produced recombinantly. Successful recombinant production has triggered a gradual shift from the plasma derived origins of Factor IX, as it provides extended half-life and expanded production capacity. However, the complex post-translational modifications of Factor IX have made recombinant production at scale difficult. Considerable research has therefore been invested into understanding and optimizing the recombinant production of Factor IX. Here, we review the evolution of recombinant Factor IX production, focusing on recent developments in bioprocessing and cell engineering to control its post-translational modifications in its expression from Chinese Hamster Ovary (CHO) cells.

Pharmacokinetics and Biodistribution of 89Zr-Miltuximab and Its Antibody Fragments as Glypican-1 Targeting Immuno-PET Agents in Glioblastoma.

Glioblastoma (GBM) is the most aggressive form of primary brain cancer, accounting for about 85% of all primary central nervous system (CNS) tumors. With standard treatment strategies like surgery, radiation, and chemotherapy, the median survival time of patients with GBM is only 12-15 months from diagnosis. The poor prognosis of GBM is due to a very high tumor recurrence rate following initial treatment, indicating a dire need for improved diagnostic and therapeutic alternatives for this disease. Antibody-based immunotheranostics holds great promise in treating GBM, combining the theranostic applications of radioisotopes and target-specificity of antibodies. In this study, we developed and validated antibody-based positron emission tomography (PET) tracers targeting the heparan sulfate proteoglycan, glypican-1 (GPC-1), for noninvasive detection of disease using diagnostic molecular imaging. GPC-1 is overexpressed in multiple solid tumor types, including GBM, and is a promising biomarker for novel immunotheranostics. Here, we investigate zirconium-89 (89Zr)-conjugated Miltuximab (a clinical stage anti-GPC-1 monoclonal antibody developed by GlyTherix, Ltd.) and engineered fragments for their potential as immuno-PET tracers to detect GPC-1positive GBM tumors in preclinical models. We explore the effects of molecular size, avidity, and Fc-domain on the pharmacokinetics and biodistribution in vivo, by comparing in parallel the full-length antibody (Miltuximab), Fab'2, Fab, and single-chain variable fragment (scFv) formats. High radiolabeling efficiency (>95%) was demonstrated by all the formats and the stability post-radiolabeling was higher for larger constructs of Miltuximab and the Fab. Receptor-mediated internalization of all 89Zr-labeled formats was observed in a human GBM cell line in vitro, while full-length Miltuximab demonstrated the highest tumor retention (5.7 ± 0.94% ID/g, day-9 postinjection (p.i.)) and overall better tumor-to-background ratios than the smaller Fc-less formats. Results from in vivo PET image quantification and ex vivo scintillation counting were highly correlated. Altogether, 89Zr-DFO-Miltuximab appears to be an effective immuno-PET imaging agent for detecting GPC-1positive tumors such as GBM and the current results support utility of the Fc containing whole mAb format over smaller antibody fragments for this target.

Detection of Dengue Virus 2 with Single Infected Mosquito Resolution Using Yeast Affinity Bionanofragments and Plasmonic SERS Nanoboxes.

Dengue disease is an emerging global threat triggered by dengue virus (DENV) transmission, primarily by the mosquito Aedes aegypti. The accurate surveillance and sensitive detection of DENV in mosquito populations are critical for the protection of human populations worldwide that are in the habitat of these mosquito species. There are four DENV serotypes with DENV2 reported to cause the most severe complications. There are limited ultrasensitive methods to early detect DENV2 mosquito infection and prevent human infection. Herein, we report an innovative nanobased immunoassay platform for early, specific, and ultrasensitive detection of DENV2-secreted nonstructural 1 (NS1) protein biomarker in single infected mosquitoes with the limit of detection of 500 fg of recombinant DENV2 NS1. The high sensitivity and DENV2 serotype specificity of the platform are the result of using nanomixing, plasmonic SERS nanoboxes, and yeast affinity bionanofragments displaying single-chain variable fragments (nanoyeast scFvs). Nanoyeast scFvs used for high affinity capture of DENV2 NS1 provided an innovative and cost-efficient alternative to monoclonal antibodies and differentiated DENV2 NS1 from other DENV serotypes and Zika virus NS1. The platform used electrohydrodynamically driven nanomixing to enhance NS1 capture by the nanoyeast scFvs while reducing nonspecific interactions. High sensitivity detection of captured DENV2 NS1 was achieved using NS1-specific surface-enhanced Raman scattering (SERS) nanotags. These nanotechnologies provide a significant innovation for early DENV2 detection in single infected mosquitoes, improving the accurate surveillance of mosquito habitats and preventing infection and severe disease arising from DENV2 transmission.

Perfusion culture of Chinese Hamster Ovary cells for bioprocessing applications.

Much of the biopharmaceutical industry's success over the past 30 years has relied on products derived from Chinese Hamster Ovary (CHO) cell lines. During this time, improvements in mammalian cell cultures have come from cell line development and process optimization suited for large-scale fed-batch processes. Originally developed for high cell densities and sensitive products, perfusion processes have a long history. Driven by high volumetric titers and a small footprint, perfusion-based bioprocess research has regained an interest from academia and industry. The recent pandemic has further highlighted the need for such intensified biomanufacturing options. In this review, we outline the technical history of research in this field as it applies to biologics production in CHO cells. We demonstrate a number of emerging trends in the literature and corroborate these with underlying drivers in the commercial space. From these trends, we speculate that the future of perfusion bioprocesses is bright and that the fields of media optimization, continuous processing, and cell line engineering hold the greatest potential. Aligning in its continuous setup with the demands for Industry 4.0, perfusion biomanufacturing is likely to be a hot topic in the years to come.

Antibody-Based Formats to Target Glioblastoma: Overcoming Barriers to Protein Drug Delivery.

Glioblastoma (GB) is recognized as the most aggressive form of primary brain cancer. Despite advances in treatment strategies that include surgery, radiation, and chemotherapy, the median survival time (∼15 months) of patients with GB has not significantly improved. The poor prognosis of GB is also associated with a very high chance of tumor recurrence (∼90%), and current treatment measures have failed to address the complications associated with this disease. However, targeted therapies enabled through antibody engineering have shown promise in countering GB when used in combination with conventional approaches. Here, we discuss the challenges in conventional as well as future GB therapeutics and highlight some of the known advantages of using targeted biologics to overcome these impediments. We also review a broad range of potential alternative routes that could be used clinically to administer anti-GB biologics to the brain through evasion of its natural barriers.

Amplification-Free SARS-CoV-2 Detection Using Nanoyeast-scFv and Ultrasensitive Plasmonic Nanobox-Integrated Nanomixing Microassay.

The implementation of accurate and sensitive molecular detection for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is paramount to effectively control the ongoing coronavirus disease 2019 (COVID-19) pandemic. In this regard, we herein propose the specific and highly sensitive SARS-CoV-2 detection based on nanoyeast single-chain-variable fragment (scFv) and ultrasensitive plasmonic nanobox-integrated nanomixing microassay. Importantly, this designed platform showcases the utility of nanoyeast-scFvs as specific capture reagents targeting the receptor-binding domain (RBD) of the virus and as monoclonal antibody alternatives suitable for cost-effective mass production and frequent testing. By capitalizing on single-particle active nanoboxes as plasmonic nanostructures for surface-enhanced Raman scattering (SERS), the microassay utilizes highly sensitive Raman signals to indicate virus infection. The developed microassay further integrated nanomixing for accelerating molecular collisions. Through the synergistic working of nanoyeast-scFv, plasmonic nanoboxes, and nanomixing, the highly specific and sensitive SARS-CoV-2 detection is achieved as low as 17 virus/µL without any molecular amplification. We successfully demonstrate SARS-CoV-2 detection in saliva samples of simulated patients at clinically relevant viral loads, suggesting the possibility of this platform for accurate and noninvasive patient screening.

Enhanced uptake of potassium or glycine betaine or export of cyclic-di-AMP restores osmoresistance in a high cyclic-di-AMP Lactococcus lactis mutant.

The broadly conserved bacterial signalling molecule cyclic-di-adenosine monophosphate (c-di-AMP) controls osmoresistance via its regulation of potassium (K+) and compatible solute uptake. High levels of c-di-AMP resulting from inactivation of c-di-AMP phosphodiesterase activity leads to poor growth of bacteria under high osmotic conditions. To better understand how bacteria can adjust in response to excessive c-di-AMP levels and to identify signals that feed into the c-di-AMP network, we characterised genes identified in a screen for osmoresistant suppressor mutants of the high c-di-AMP Lactococcus ΔgdpP strain. Mutations were identified which increased the uptake of osmoprotectants, including gain-of-function mutations in a Kup family K+ importer (KupB) and inactivation of the glycine betaine transporter transcriptional repressor BusR. The KupB mutations increased the intracellular K+ level while BusR inactivation increased the glycine betaine level. In addition, BusR was found to directly bind c-di-AMP and repress expression of the glycine betaine transporter in response to elevated c-di-AMP. Interestingly, overactive KupB activity or loss of BusR triggered c-di-AMP accumulation, suggesting turgor pressure changes act as a signal for this second messenger. In another group of suppressors, overexpression of an operon encoding an EmrB family multidrug resistance protein allowed cells to lower their intracellular level of c-di-AMP through active export. Lastly evidence is provided that c-di-AMP levels in several bacteria are rapidly responsive to environmental osmolarity changes. Taken together, this work provides evidence for a model in which high c-di-AMP containing cells are dehydrated due to lower K+ and compatible solute levels and that this osmoregulation system is able to sense and respond to cellular water stress.

S-Trap Eliminates Cell Culture Media Polymeric Surfactants for Effective Proteomic Analysis of Mammalian Cell Bioreactor Supernatants.

Proteomic analysis of bioreactor supernatants can inform on cellular metabolic status, viability, and productivity, as well as product quality, which can in turn help optimize bioreactor operation. Incubating mammalian cells in bioreactors requires the addition of polymeric surfactants such as Pluronic F68, which reduce the sheer stress caused by agitation. However, these surfactants are incompatible with mass spectrometry proteomics and must be eliminated during sample preparation. Here, we compared four different sample preparation methods to eliminate polymeric surfactants from filtered bioreactor supernatant samples: organic solvent precipitation; filter-assisted sample preparation (FASP); S-Trap; and single-pot, solid-phase, sample preparation (SP3). We found that SP3 and S-Trap substantially reduced or eliminated the polymer(s), but S-Trap provided the most robust cleanup and highest quality data. Additionally, we observed that SP3 sample preparation of our samples and in other published data sets was associated with partial alkylation of cysteines, which could impact the confidence and robustness of protein identification and quantification. Finally, we observed that several commercial mammalian cell culture media and media supplements also contained polymers with similar mass spectrometry profiles, and we suggest that proteomic analyses in these media will also benefit from the use of S-Trap sample preparation.

A bispecific T cell engager targeting Glypican-1 redirects T cell cytolytic activity to kill prostate cancer cells.

BACKGROUND: Glypican-1 is a heparan sulfate proteoglycan that is overexpressed in prostate cancer (PCa), and a variety of solid tumors. Importantly, expression is restricted in normal tissue, making it an ideal tumor targeting antigen. Since there is clinical and preclinical evidence of the efficacy of Bispecific T cell Engager (BiTE) therapy in PCa, we sought to produce and test the efficacy of a GPC-1 targeted BiTE construct based on the Miltuximab® sequence. Miltuximab® is a clinical stage anti-GPC-1 antibody that has proven safe in first in human trials. METHODS: The single chain variable fragment (scFv) of Miltuximab® and the CD3 binding sequence of Blinatumomab were combined in a standard BiTE format. Binding of the construct to immobilised recombinant CD3 and GPC-1 antigens was assessed by ELISA and BiaCore, and binding to cell surface-expressed antigens was measured by flow cytometry. The ability of MIL-38-CD3 to activate T cells was assessed using in vitro co-culture assays with tumour cell lines of varying GPC-1 expression by measurement of CD69 and CD25 expression, before cytolytic activity was assessed in a similar co-culture. The release of inflammatory cytokines from T cells was measured by ELISA and expression of PD-1 on the T cell surface was measured by flow cytometry. RESULTS: Binding activity of MIL-38-CD3 to both cell surface-expressed and immobilised recombinant GPC-1 and CD3 was retained. MIL-38-CD3 was able to mediate the activation of peripheral blood T cells from healthy individuals, resulting in the release of inflammatory cytokines TNF and IFN-g. Activation was reliant on GPC-1 expression as MIL-38-CD3 mediated only low level T cell activation in the presence of C3 cells (constitutively low GPC-1 expression). Activated T cells were redirected to lyse PCa cell lines PC3 and DU-145 (GPC-1 moderate or high expression, respectively) but could not kill GPC-1 negative Raji cells. The expression of PD-1 was up-regulated on the surface of MIL-38-CD3 activated T cells, suggesting potential for synergy with checkpoint inhibition. CONCLUSIONS: This study reports preclinical findings into the efficacy of targeting GPC-1 in PCa with BiTE construct MIL-38-CD3. We show the specificity and efficacy of the construct, supporting its further preclinical development.

Glycoproteomic measurement of site-specific polysialylation.

Polysialylation is the enzymatic addition of a highly negatively charged sialic acid polymer to the non-reducing termini of glycans. Polysialylation plays an important role in development, and is involved in neurological diseases, neural tissue regeneration, and cancer. Polysialic acid (PSA) is also a biodegradable and non-immunogenic conjugate to therapeutic drugs to improve their pharmacokinetics. PSA chains vary in length, composition, and linkages, while the specific sites of polysialylation are important determinants of protein function. However, PSA is difficult to analyse by mass spectrometry (MS) due to its high negative charge and size. Most analytical approaches for analysis of PSA measure its degree of polymerization and monosaccharide composition, but do not address the key questions of site specificity and occupancy. Here, we developed a high-throughput LC-ESI-MS/MS glycoproteomics method to measure site-specific polysialylation of glycoproteins. This method measures site-specific PSA modification by using mild acid hydrolysis to eliminate PSA and sialic acids while leaving the glycan backbone intact, together with protease digestion followed by LC-ESI-MS/MS glycopeptide detection. PSA-modified glycopeptides are not detectable by LC-ESI-MS/MS, but become detectable after desialylation, allowing measurement of site-specific PSA occupancy. This method is an efficient analytical workflow for the study of glycoprotein polysialylation in biological and therapeutic settings.

Identification of novel glycosylation events on human serum-derived factor IX.

Human Factor IX is a highly post-translationally modified protein that is an important clotting factor in the blood coagulation cascade. Functional deficiencies in Factor IX result in the bleeding disorder haemophilia B, which is treated with plasma-derived or recombinant Factor IX concentrates. Here, we investigated the post-translational modifications of human serum-derived Factor IX and report previously undescribed O-linked monosaccharide compositions at serine 141 and a novel site of glycosylation. At serine 141 we observed two monosaccharide compositions, with HexNAc1Hex1NeuAc2 dominant and a low level of HexNAc1Hex1NeuAc1. This O-linked site lies N-terminal to the first cleavage site for the activation peptide, an important region of the protein that is removed to activate Factor IX. The novel site is an N-linked site in the serine protease domain with low occupancy in a non-canonical consensus motif at asparagine 258, observed with a HexNAc4Hex5NeuAc2 monosaccharide composition attached. This is the first reported instance of a site of modification in the serine protease domain. The description of these glycosylation events provides a basis for future functional studies and contributes to structural characterisation of native Factor IX for the production of effective therapeutic biosimilars and biobetters.

Effect of Chain-End Chemistries on the Efficiency of Coupling Antibodies to Polymers Using Unnatural Amino Acids.

Novel conjugates that incorporate strategies for increasing the therapeutic payload, such as targeted polymeric delivery vehicles, have great potential in overcoming limitations of conventional antibody therapies that often exhibit immunogenicity and limited drug loading. Click chemistry has significantly expanded the toolbox of effective strategies for developing hybrid polymer-biomolecule conjugates, however, effective systems require orthogonality between the polymer and biomolecule chemistries to achieve efficient coupling. Here, three cycloaddition-based strategies for antibody conjugation to polymeric carriers are explored and show that a purely radical-based method for polymer synthesis and subsequent biomolecule attachment has a trade-off between coupling efficiency of the antibody and the ability to synthesize polymers with controlled chemical properties. It is shown that careful consideration of both coupling chemistries as well as the potential effect of how this modulates the chemical properties of the polymer nanocarrier should be considered during the development of such systems. The strategies described offer insight into improving conjugate development for therapeutic and theranostic applications. In this system, polymerization using conventional and established reversible addition fragmentation chain transfer (RAFT) agents, followed by multiple post-modification steps, always leads to systems with more defined chemical architectures compared to strategies that utilize alkyne-functional RAFT agents.

Wavelength-Dependent Fluorescent Immunosensors via Incorporation of Polarity Indicators near the Binding Interface of Antibody Fragments.

Herein, we describe a fluorescent immunosensor designed by incorporating an unnatural amino acid fluorophore into the binding site of an EGFR-specific antibody fragment, resulting in quantifiable EGFR-dependent changes in peak fluorescence emission wavelength. To date, immunosensor design strategies have relied on binding-induced changes in fluorescence intensity that are prone to excitation source fluctuations and sample-dependent noise. In this study, we used a rational design approach to incorporate a polarity indicator (Anap) into specific positions of an anti-EGFR single chain antibody to generate an emission wavelength-dependent immunosensor. We found that when incorporated within the topological neighborhood of the antigen binding interface, the Anap emission wavelength is blue-shifted by EGFR-binding in a titratable manner, up to 20 nm, with nanomolar detection limits. This approach could be applicable to other antibody/antigen combinations for integration into a wide range of assay platforms (including homogeneous, solid-phase assay, or microfluidic assays) for one-step protein quantification.

Single droplet detection of immune checkpoints on a multiplexed electrohydrodynamic biosensor.

Monitoring soluble immune checkpoints in circulating fluids has the potential for minimally-invasive diagnostics and personalised therapy in precision medicine. Yet, the sensitive detection of multiple immune checkpoints from small volumes of liquid biopsy samples is challenging. In this study, we develop a multiplexed immune checkpoint biosensor (MICB) for parallel detection of soluble immune checkpoints PD-1, PD-L1, and LAG-3. MICB integrates a microfluidic sandwich immunoassay using engineered single chain variable fragments and alternating current electrohydrodynamic in situ nanofluidic mixing for promoting biosensor-target interaction and reducing non-specific non-target binding. MICB provides advantages of simultaneous analysis of up to 28 samples in <2 h, requires as little as a single sample drop (i.e., 20 µL) per target immune checkpoint, and applies high-affinity yeast cell-derived single chain variable fragments as a cost-effective alternative to monoclonal antibodies. We investigate the assay performance of MICB and demonstrate its capability for accurate immune checkpoint detection in simulated patient serum samples at clinically-relevant levels. MICB provides a dynamic range of 5 to 200 pg mL-1 for PD-1 and PD-L1, and 50 to 1000 pg mL-1 for LAG-3 with a coefficient of variation <13.8%. Sensitive immune checkpoint detection was achieved with limits of detection values of 5 pg mL-1 for PD-1, 5 pg mL-1 for PD-L1, and 50 pg mL-1 for LAG-3. The multiplexing capability, sensitivity, and relative assay simplicity of MICB make it capable of serving as a bioanalytical tool for immune checkpoint therapy monitoring.

Bispecific Antibody-Functionalized Upconversion Nanoprobe.

Upconversion nanoparticles (UCNPs) are new optical probes for biological applications. For specific biomolecular recognition to be realized for diagnosis and imaging, the key lies in developing a stable and easy-to-use bioconjugation method for antibody modification. Current methods are not yet satisfactory regarding conjugation time, stability, and binding efficiency. Here, we report a facile and high-yield approach based on a bispecific antibody (BsAb) free of chemical reaction steps. One end of the BsAb is designed to recognize methoxy polyethylene glycol-coated UCNPs, and the other end of the BsAb is designed to recognize the cancer antigen biomarker. Through simple vortexing, BsAb-UCNP nanoprobes form within 30 min and show higher (up to 54%) association to the target than that of the traditional UCNP nanoprobes in the ELISA-like assay. We further demonstrate its successful binding to the cancer cells with high efficiency and specificity for background-free fluorescence imaging under near-infrared excitation. This method suggests a general approach broadly suitable for functionalizing a range of nanoparticles to specifically target biomolecules.

Multiplexed SERS Detection of Soluble Cancer Protein Biomarkers with Gold-Silver Alloy Nanoboxes and Nanoyeast Single-Chain Variable Fragments.

Highly sensitive, multiplexed detection of soluble cancer protein biomarkers can facilitate early cancer screening as well as enable real-time monitoring of patients' sensitivity and resistance to therapy. Current technologies for detection of soluble cancer protein biomarkers, e.g., enzyme-linked immunosorbent assay, however, suffer from limited sensitivity, as well as the requirement of expensive monoclonal antibodies, which undergo the quality variability. Herein, we propose a sensitive, cheap, and robust surface-enhanced Raman scattering technology to detect a panel of soluble cancer protein biomarkers, including soluble programmed death 1 (sPD-1), soluble programmed death-ligand 1 (sPD-L1) and soluble epithermal growth factor receptor (sEGFR), which are related to disease progression and treatment efficacy. In this assay, gold-silver alloy nanoboxes that have strong Raman signal enhancement capability were used as plasmonic nanostructures to facilitate highly sensitive detection. In addition, nanoyeast single-chain variable fragments were utilized as mAb alternatives to allow specific and stable protein capture performance. We successfully detected sPD-1, sPD-L1, and sEGFR with a limit of detection of 6.17 pg/mL, 0.68 pg/mL, and 69.86 pg/mL, respectively. We further tested the detection of these three soluble cancer protein biomarkers in human serum and achieved recovery rates between 82.99% and 101.67%. We believe our novel platform that achieves sensitive, multiplexed, and specific detection of soluble cancer protein biomarkers could greatly benefit cancer treatment and improve patient outcome.

Insights into the interfacial structure-function of poly(ethylene glycol)-decorated peptide-stabilised nanoscale emulsions.

The interfacial properties of nanoscale materials have profound influence on biodistribution and stability as well as the effectiveness of sophisticated surface-encoded properties such as active targeting to cell surface receptors. Tailorable nanocarrier emulsions (TNEs) are a novel class of oil-in-water emulsions stabilised by molecularly-engineered biosurfactants that permit single-pot stepwise surface modification with related polypeptides that may be chemically conjugated or genetically fused to biofunctional moieties. We have probed the structure and function of poly(ethylene glycol) (PEG) used to decorate TNEs in this way. The molecular weight of PEG decorating TNEs has considerable impact on the ζ-potential of the emulsion particles, related to differential interfacial thickness of the PEG layer as determined by X-ray reflectometry. By co-modifying TNEs with an antibody fragment, we show that the molecular weight and density of PEG governs the competing parameters of accessibility of the targeting moiety and of shielding the interface from non-specific interactions with the environment. The fundamental understanding of the molecular details of the PEG layer that we present provides valuable insights into the structure-function relationship for soft nanomaterial interfaces. This work therefore paves the way for further rational design of TNEs and other nanocarriers that must interact with their environment in controlled and predictable ways.

EGFR Targeting of Liposomal Doxorubicin Improves Recognition and Suppression of Non-Small Cell Lung Cancer.

Introduction: Despite improvements in chemotherapy and molecularly targeted therapies, the life expectancy of patients with advanced non-small cell lung cancer (NSCLC) remains less than 1 year. There is thus a major global need to advance new treatment strategies that are more effective for NSCLC. Drug delivery using liposomal particles has shown success at improving the biodistribution and bioavailability of chemotherapy. Nevertheless, liposomal drugs lack selectivity for the cancer cells and have a limited ability to penetrate the tumor site, which severely limits their therapeutic potential. Epidermal growth factor receptor (EGFR) is overexpressed in NSCLC tumors in about 80% of patients, thus representing a promising NSCLC-specific target for redirecting liposome-embedded chemotherapy to the tumor site. Methods: Herein, we investigated the targeting of PEGylated liposomal doxorubicin (Caelyx), a powerful off-the-shelf antitumoral liposomal drug, to EGFR as a therapeutic strategy to improve the specific delivery and intratumoral accumulation of chemotherapy in NSCLC. EGFR-targeting of Caelyx was enabled through its complexing with a polyethylene glycol (PEG)/EGFR bispecific antibody fragment. Tumor targeting and therapeutic potency of our treatment approach were investigated in vitro using a panel of NSCLC cell lines and 3D tumoroid models, and in vivo in a cell line-derived tumor xenograft model. Results: Combining Caelyx with our bispecific antibody generated uniform EGFR-targeted particles with improved binding and cytotoxic efficacy toward NSCLC cells. Effects were exclusive to cancer cells expressing EGFR, and increments in efficacy positively correlated with EGFR density on the cancer cell surface. The approach demonstrated increased penetration within 3D spheroids and was effective at targeting and suppressing the growth of NSCLC tumors in vivo while reducing drug delivery to the heart. Conclusion: EGFR targeting represents a successful approach to enhance the selectivity and therapeutic potency of liposomal chemotherapy toward NSCLC.

Targeted delivery of polo-like kinase 1 siRNA nanoparticles using an EGFR-PEG bispecific antibody inhibits proliferation of high-risk neuroblastoma.

High-risk neuroblastoma has poor survival due to treatment failure and off-target side effects of therapy. Small molecule inhibitors have shown therapeutic efficacy at targeting oncogenic cell cycle dysregulators, such as polo-like kinase 1 (PLK1). However, their clinical success is limited by a lack of efficacy and specificity, causing off-target toxicity. Herein, we investigate a new treatment strategy whereby a bispecific antibody (BsAb) with dual recognition of methoxy polyethylene glycol (PEG) and a neuroblastoma cell-surface receptor, epidermal growth factor receptor (EGFR), is combined with a PEGylated small interfering RNA (siRNA) lipid nanoparticle, forming BsAb-nanoparticle RNA-interference complexes for targeted PLK1 inhibition against high-risk neuroblastoma. Therapeutic efficacy of this strategy was explored in neuroblastoma cell lines and a tumor xenograft model. Using ionizable lipid-based nanoparticles as a low-toxicity and clinically safe approach for siRNA delivery, we identified that their complexing with EGFR-PEG BsAb resulted in increases in cell targeting (1.2 to >4.5-fold) and PLK1 gene silencing (>2-fold) against EGFR+ high-risk neuroblastoma cells, and enhancements correlated with EGFR expression on the cells (r > 0.94). Through formulating nanoparticles with PEG-lipids ranging in diffusivity, we further identified a highly diffusible PEG-lipid which provided the most pronounced neuroblastoma cell binding, PLK1 silencing, and significantly reduced cancer growth in vitro in high-risk neuroblastoma cell cultures and in vivo in a tumor-xenograft mouse model of the disease. Together, this work provides an insight on the role of PEG-lipid diffusivity and EGFR targeting as potentially relevant variables influencing the therapeutic efficacy of siRNA nanoparticles in high-risk neuroblastoma.