Targeted Radionuclide Therapy (TRT) enables selective delivery of radionuclides for cancer treatment. Alpha particle emitters such as 212Pb are emerging as potential gamechangers, representing highly potent payloads for precision therapy and refractory cancer treatment. While small-molecule carriers are widely explored due to favorable pharmacokinetics, nanoparticle-based TRT remains less studied due to perceived non-ideal pharmacokinetics. We report a unique nanoparticle-TRT pretargeting approach using bispecific antibodies (BsAbs) which "prime" tumor surfaces, enhancing tumor-specific delivery and minimizing off-target deposition of 212Pb. We developed a poly(ethylene glycol) (PEG)-based nanomedicine platform to carry 212Pb, and employed in-house designed and manufactured BsAb (α-epidermal growth factor receptor (EGFR)/α-PEG) to prime tumors to receive the nanocarrier. Sequential administration of each component to EGFR-expressing cells produced enhanced receptor-mediated internalization of the BsAb upon nanomedicine binding, resulting in improved radionuclide delivery and efficacy in a series of in vitro assays. The pretargeting approach more than tripled tumor retention of the 212Pb-nanomedicine compared to the untargeted nanomaterial in a murine EGFR+ breast cancer xenograft model, evidenced by single-photon emission computed tomography (SPECT) imaging of 212Pb-loaded nanomaterials and gamma analysis of the excised organs. Therapeutic studies demonstrated the 212Pb-nanomedicine to produce well-tolerated and statistically-enhanced therapeutic outcomes for the pretargeting versus conventional 212Pb-nanomedicine, with no observed long term hematological effects. This work establishes a modular strategy for targeted TRT nanomedicine delivery. The platform has potential for broad applicability, including simultaneous delivery of diverse or synergistic payloads. These findings represent an important advance toward precision nanomedicine approaches in radionuclide therapy.

Dynamic downregulation of the endogenous farnesyl pyrophosphate (FPP) synthase (Erg20p) is crucial to engineer heterologous monoterpene production in the yeast Saccharomyces cerevisiae. FPP downstream metabolite geranylgeranyl pyrophosphate (GGPP) can induce the degradation of 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase 2 (Hmg2p) through its N-terminal GGPP-sensing endoplasmic reticulum transmembrane domain (Hmg2pN) in S. cerevisiae. Here, we investigate the use of Hmg2pN to regulate Erg20p, aiming to restrict FPP synthesis and redirect metabolic flux to monoterpene production. While using the ERG1 promoter to regulate ERG20 transcription improved monoterpene limonene by ~10-fold, combinatory fusion of Hmg2pN to Erg20p N-terminus further improved limonene production by 40% to 0.52 g L-1 in synthetic minimal media. This approach yielded 0.5 g L-1 geraniol in batch cultivation, comparable to levels achieved using the N-end-rule degron K3K15 or an auxin-inducible degron to regulate Erg20p. In rich complex media, this approach was superior, leading to 2.1 g L-1 geraniol production in semi-fed batch cultivation. In summary, the Hmg2pN domain is an efficient tool to constrain FPP synthesis for improved monoterpene production in S. cerevisiae.

Immune-modulating peptides have shown potential as novel immune-stimulating agents which enhance the secretion of anticancer cytokines in vitro. However, fast clearance from blood hampers the ability of such peptides to accumulate in the tumour and results in limited therapeutic efficacy in animal studies. To address the fast blood clearance, this work reports the development and validation of a novel polymeric nanoparticle delivery system for the efficient localization of an immunomodulating peptide in the tumour microenvironment (TME). To identify the optimal polymeric nanoparticle for this study, two types of nanoparticles were developed as either branched polymers or micelles that have similar chemical functionality but different sizes. The effect of targeting the nanomedicine to the tumour-specific antigen, glycoprotein GPC-1, was explored using a bispecific antibody (BsAb) that shows an affinity for the cell protein (GPC-1) and the nanoparticle. These systems were evaluated for targeting efficiency and tumour penetration using tumour spheroids of Lewis Lung Cancer (LLC) cells and it was shown that the targeted system significantly enhanced cell association compared to the untargeted control with minor differences in penetration. The lead micelle-peptide conjugates were identified and using in vivo allograft models they were demonstrated to have high delivery efficiency of the peptide to tumours, prolonged blood circulation, enhanced tumour accumulation and tumour suppression that was associated with immune cell recruitment to the tumour.

Recent examples of immune responses directed against the synthetic polymer poly(ethylene glycol) (PEG) have led to the development of biocompatible polymers, which are viewed as promising candidates to act as surrogate materials for use in biological applications, such as hydrophilic poly(2-oxazoline)s (POx). Despite this, the characterization of critical aspects of the immune response against these emerging materials is sparse, in part because no known monoclonal antibodies (mAbs) against this family of synthetic material have been reported. To advance the understanding of such responses, we report the successful isolation and characterization of hybridoma-derived mAbs with excellent specificity for different POx species and notable selectivity for highly branched polymer architectures over linear systems. In conjunction with established mAbs targeted against PEG, we show that these antibodies can be employed for sensitive in vivo multiplex-detection of label-free polymer therapeutics based on the specificity of the polymer-antibody binding. This approach enables scalable therapeutic drug monitoring of multiple polymer therapeutics within a single animal, simultaneously.

Efficient delivery of mRNA-lipid nanoparticles (LNPs) to specific cell types remains a major challenge for mRNA therapeutics. Conventional targeting approaches involve modifying the lipid composition or functionalizing the surface of LNPs, which complicates manufacturing and alters nanoparticle size, charge, and stealth, impacting their delivery and immunogenicity. Here, we present a generalizable method for targeted mRNA-LNP delivery that uses bispecific antibodies (BsAbs) to form a bridge between LNPs and cell surface markers. BsAbs can be combined with LNPs or administered first, binding to surface proteins on target cells and later retaining unmodified LNPs in affected tissues. We demonstrate the efficient and cell-type-specific delivery of mRNA-LNPs beyond the liver, targeting epidermal growth factor receptor (EGFR)- and folate hydrolase 1 (PSMA)-positive cells in vitro and in vivo. The flexibility of this technology, achieved by substituting the cell-targeting region of the BsAbs, enables the rapid development of next-generation targeted mRNA drugs.

Surface-enhanced Raman scattering (SERS) biosensing platforms offer exceptional sensitivity and multiplexing capabilities for biomarker detection, but their performance is often limited by uncontrolled ligand orientation on nanotags. To address this, we developed a SERS nanotag incorporating a bispecific antibody fragment (BsAb) engineered to bind the SARS-CoV-2 receptor-binding domain (RBD) and methoxy polyethylene glycol (mPEG) simultaneously, enabling orientation-controlled immobilization onto mPEG-grafted plasmonic nanostructures. We systematically evaluated how immobilization conditions─particularly pH relative to the BsAb's isoelectric point (pI)─influence surface density, antigen accessibility, and overall assay performance using a combination of orthogonal physicochemical and functional assays. Our results show that immobilization at pH above the BsAb's pI improves antigen accessibility and nanotag stability, despite reduced immobilization density. When integrated into a digital SERS biosensing platform, the BsAb-functionalized SERS nanotags enabled highly specific digital detection of RBD in plasma, achieving a low limit of detection (3.97 ng/mL) and outperforming enzyme-linked immunosorbent assay in assay time, reagent consumption, and multiplexing potential. This study establishes a robust strategy for designing and optimizing antibody fragment-based SERS nanotags, advancing their application in next-generation diagnostic platforms.

Surface-enhanced Raman scattering (SERS) immunoassays have emerged as highly sensitive, multiplexed analytical techniques for detecting protein biomarkers. Traditional SERS immunoassays typically rely on antibody-based SERS probes for target protein detection; however, it is challenging to obtain antibodies that are both highly effective at identifying natural proteins and suitable for SERS probe conjugation. Herein, we engineer a MultiValent Probe (MVP), consisting of multivalent nanobodies as the protein-targeting ligand to provide improved binding avidity and Raman reporter-coated gold-silver alloy nanoboxes for single-particle signal readouts. The multivalent nanobodies exhibit precise antigen recognition and exceptional affinity, and are expressed in a mammalian system for cost-effective and large-scale production. We thoroughly characterize the MVP via nanoparticle tracking analysis, nanoflow cytometry, and differential centrifugal sedimentation. To further enhance assay performance, we integrate MVP with a nanomixing-enhanced microfluidic chip to develop an MVP-based SERS-microfluidic immunoassay. As a proof of concept, we demonstrate the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike proteins and virions from multiple strains in clinical nasopharyngeal samples (39 healthy and 39 infected), showing 84.6% concordance with RT-qPCR. This work highlights the potential of MVP-incorporated SERS-microfluidic immunoassays for diagnostics of pandemic diseases and broader applications in detecting a wide range of viral pathogens.

Laboratory-based diagnostics such as plaque reduction neutralisation tests (PRNT) and ELISA are commonly used to detect seroconversion to flavivirus infections. However, faster, qualitative screening methods are essential for quicker diagnosis and improved patient outcomes. Lateral flow assays (LFAs) offer rapid results (5-15 mins) at the point-of-care, but few commercial flavivirus antibody detection LFAs are available. We developed an LFA using novel chimeric viral antigens produced by genetically modifying the mosquito restricted Binjari virus (BinJV) to display the outer virion proteins of pathogenic viruses like West Nile virus (WNV). The BinJV chimeric platform offers several advantages for diagnostic assay development, including rapid construction of new chimeras in response to emerging viral variants, safe, scalable antigen manufacturing, and structural indistinguishability to the wild-type pathogenic virion. To demonstrate feasibility, we applied the chimeric WNV (BinJV/WNV) antigen to LFA as the capture/test line reagent for detecting seroconversion in crocodilians to WNV - a virus affecting crocodilians across multiple continents. We confirmed the antigenic conservation of the chimera on the LFA detection surface using monoclonal antibodies. Utilising well-characterised sera (n=60) from WNV-seropositive or flavivirus-naive Australian saltwater crocodiles (Crocodylus porosus), the assay exhibited 98.8 % sensitivity and 100 % specificity, with results obtained in under 15 minutes. The LFA also accurately detected seroconversion in animals experimentally infected with WNV. This qualitative screening method can be performed both inside and outside of a laboratory, and the assay design will guide the optimisation of similar tests for detecting vector-borne viral infections in humans and other animals.

The identification of adjuvants to improve vaccination efficacy is a major unmet need. One approach is to augment the functionality of dendritic cells (DCs) by using Toll-like receptor-9 (TLR9) agonists such as cytosine-phosphate-guanine oligodeoxynucleotides (CpG ODNs) as adjuvants. Another approach is adjuvant selection based on production of bioactive interleukin-12 (IL-12). We report a D-peptide isomer, designated D-15800, that induces monocyte differentiation to the DC phenotype in vitro and more effectively stimulates IL-12p70 production upon T cell receptor (TCR) activation than the L-isomer. In the absence of TCR activation and either IL-12p70 or interleukin-2 production, only D-15800 activates CD4+ T and natural killer cells. In the presence of CpG ODN, D-15800 synergistically enhances production of interferon-alpha (IFN-α). Taken together with its biostability in human serum and depot retention upon injection, co-delivery of D-15800 with TLR9 agonists could serve to improve vaccine efficacy.

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.

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.

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.

Protein functionalisation for the development of imaging agents and antibody drug conjugates still often relies on statistical amidation of the protein through accessible lysine and cysteine residues, requiring protein to protein conjugation optimisation and can potentially impact the overall function. To combat this, focus has turned to developing proteins that have noncanonical amino acids incorporated into their structure, allowing for site‐specific labelling and functionalisation. Herein we showcase the incorporation of non‐canonical amino acids bearing a tetrazine or azide orthogonal coupling modality into biologics targeted to the prostate‐specific membrane antigen and epidermal growth factor receptor respectively. The placement of these bioorthogonal residues into nanobody or single chain variable fragments (scFvs) is introduced by site‐directed mutagenesis of the protein‐coding DNA that allows for controlled insertion when these proteins are expressed. We show that bioorthogonal coupling of model compounds such as fluorophore or polymeric materials onto the protein does not significantly change the binding affinity, making these protein conjugation methods a powerful tool for development of simple customisable personalised targeted antibody‐drug conjugates where affinity is retained.

Despite the significant potential of protein biosensors, their construction remains a trial-and-error process. The most obvious approach for addressing this is to utilize modular biosensor architectures where specificity-conferring modalities can be readily generated to recognize new targets. Toward this goal, we established a workflow that uses mRNA display-based selection of hyper-stable monobody domains for the target of choice or ribosome display to select equally stable DARPins. These binders were integrated into a two-component allosteric biosensor architecture based on a calmodulin-reporter chimera. This workflow was tested by developing biosensors for liver toxicity markers such as cytosolic aspartate aminotransferase, mitochondrial aspartate aminotransferase, and alanine aminotransferase 1. We demonstrate that our pipeline consistently produced >10(3) unique binders for each target within a week. Our analysis revealed that the affinity of the binders for their targets was not a direct predictor of the binder's performance in a biosensor context. The interactions between the binding domains and the reporter module affect the biosensor activity and the dynamic range. We conclude that following binding domain selection, the multiplexed biosensor assembly and prototyping appear to be the most promising approach for identifying biosensors with the desired properties.

Hexokinase II (Hxk2) is a master protein in glucose-mediated transcriptional repression signaling pathway. Degrading Hxk2 through an auxin-inducible protein degradation previously doubled sesquiterpene (nerolidol) production at gram-per-liter levels in Saccharomyces cerevisiae. Global transcriptomics/proteomics profiles in Hxk2-deficient background are important to understanding genetic and molecular mechanisms for improved nerolidol production and guiding further strain optimization. Here, proteomic responses to Hxk2 depletion are investigated in the yeast strains harboring a GAL promoters-controlled nerolidol synthetic pathway, at the exponential and ethanol growth phases and in GAL80-wildtype and gal80Δ backgrounds. Carbon metabolic pathways and amino acid metabolic pathways show diversified responses to Hxk2 depletion and growth on ethanol, including upregulation of alternative carbon catabolism and respiration as well as downregulation of amino acid synthesis. De-repression of GAL genes may contribute to improved nerolidol production in Hxk2-depleted strains. Seventeen transcription factors associated with upregulated genes are enriched. Validating Ash1-mediated repression on the RIM4 promoter shows the variation on the regulatory effects of different Ash1-binding sites and the synergistic effect of Ash1 and Hxk2-mediated repression. Further validation of individual promoters shows that HXT1 promoter activities are glucose-dependent in hxk2Δ background, but much weaker than those in HXK2-wildtype background. In summary, inactivating HXK2 may relieve glucose repression on respiration and GAL promoters for improved bioproduction under aerobic conditions in S. cerevisiae. The proteomics profiles provide a better genetics overview for a better metabolic engineering design in Hxk2-deficient backgrounds.

Cancers and autoimmune diseases commonly co-exist and immune checkpoint inhibitor therapy (ICI) exacerbates autoimmune pathologies. We recently described a lipidic peptide, designated IK14004, that promotes expansion of immunosuppressive T regulatory (Treg) cells and uncouples interleukin-2 from interferon-gamma production while activating CD8+ T cells. Herein, we report IK14004-mediated inhibition of Lewis lung cancer (LLC) growth and re-invigoration of splenocyte-derived exhausted CD4+ T cells. In human immune cells from healthy donors, IK14004 modulates expression of the T cell receptor α/β subunits, induces Type I IFN expression, stimulates natural killer (NK) cells to express NKG2D/NKp44 receptors and enhances K562 cytotoxicity. In both T and NK cells, IK14004 alters the IL-12 receptor β1/β2 chain ratio to favour IL-12p70 binding. Taken together, this novel peptide offers an opportunity to gain further insight into the complexity of ICI immunotherapy so that autoimmune responses may be minimised without promoting tumour evasion from the immune system.

High-risk childhood leukemia has a poor prognosis because of treatment failure and toxic side effects of therapy. Drug encapsulation into liposomal nanocarriers has shown clinical success at improving biodistribution and tolerability of chemotherapy. However, enhancements in drug efficacy have been limited because of a lack of selectivity of the liposomal formulations for the cancer cells. Here, we report on the generation of bispecific antibodies (BsAbs) with dual binding to a leukemic cell receptor, such as CD19, CD20, CD22, or CD38, and methoxy polyethylene glycol (PEG) for the targeted delivery of PEGylated liposomal drugs to leukemia cells. This liposome targeting system follows a “mix-and-match” principle where BsAbs were selected on the specific receptors expressed on leukemia cells. BsAbs improved the targeting and cytotoxic activity of a clinically approved and low-toxic PEGylated liposomal formulation of doxorubicin (Caelyx) toward leukemia cell lines and patient-derived samples that are immunophenotypically heterogeneous and representative of high-risk subtypes of childhood leukemia. BsAb-assisted improvements in leukemia cell targeting and cytotoxic potency of Caelyx correlated with receptor expression and were minimally detrimental in vitro and in vivo toward expansion and functionality of normal peripheral blood mononuclear cells and hematopoietic progenitors. Targeted delivery of Caelyx using BsAbs further enhanced leukemia suppression while reducing drug accumulation in the heart and kidneys and extended overall survival in patient-derived xenograft models of high-risk childhood leukemia. Our methodology using BsAbs therefore represents an attractive targeting platform to potentiate the therapeutic efficacy and safety of liposomal drugs for improved treatment of high-risk leukemia.

Accurate and early detection of biomarkers provides the molecular evidence for disease management, allowing prompt actions and timely treatments to save lives. Multivalent biomolecular interactions between the probe and biomarker as well as controlled probe orientation on material surfaces are keys for highly sensitive detection. Here we report the bioengineering of programmable and multifunctional nanoprobes, which can provide rapid, specific and highly sensitive detection of emerging diseases in a range of widely used diagnostic systems. These nanoprobes composed of nanosized cell wall fragments, termed as synthetic bionanofragments (SynBioNFs), are generated by the fragmentation of genetically programmed yeast cells. SynBioNFs display multiple copies of biomolecules for high-affinity target binding and molecular handles for the precisely orientated attachment on surfaces used in diagnostic platforms. SynBioNFs are demonstrated for the capture and detection of SARS-CoV-2 virions using multiple diagnostic platforms, including surface-enhanced Raman scattering, fluorescence, electrochemical and colorimetric-based lateral flow systems with sensitivity comparable with the gold-standard reverse-transcription quantitative polymerase chain reaction.

Ultraviolet radiation (UVR) induces immunosuppression and DNA damage, both of which contribute to the rising global incidence of skin cancer including melanoma. Nucleotide excision repair, which is activated upon UVR-induced DNA damage, is linked to expression of interleukin-12 (IL-12) which serves to limit immunosuppression and augment the DNA repair process. Herein, we report an immunomodulating peptide, designated IK14800, that not only elicits secretion of IL-12, interleukin-2 (IL-2) and interferon-gamma (IFN-γ) but also reduces DNA damage in the skin following exposure to UVR. Combined with re-invigoration of exhausted CD4+ T cells, inhibition of UVR-induced MMP-1 release and suppression of B16F10 melanoma metastases, IK14800 offers an opportunity to gain further insight into mechanisms underlying the development and progression of skin cancers.