Localization of Therapeutic Fab-CHP Conjugates to Sites of Denatured Collagen for the Treatment of Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation in synovial joints and protease-induced cartilage degradation. Current biologic treatments for RA can effectively reduce symptoms, primarily by neutralizing the proinflammatory cytokine TNFα; however, continued, indiscriminate overinhibition of inflammatory factors can significantly weaken the host immune system, leading to opportunistic infections and interrupting treatment. We hypothesize that localizing anti-TNFα therapeutics to denatured collagen (dCol) present at arthritic joints, via conjugation with collagen-hybridizing peptides (CHPs), will reduce off-site antigen binding and maintain local immunosuppression. We isolated the antigen-binding fragment of the clinically approved anti-TNFα therapeutic infliximab (iFab) and prepared iFab-CHP conjugates via lysine-based conjugation with an SMCC linker. After successful conjugation, confirmed by LC-MS, the binding affinity of iFab-CHP was characterized by ELISA-like assays, which showed comparable antigen binding relative to infliximab, comparable dCol binding relative to CHP, and the hybrid ability to bind both dCol and TNFα simultaneously. We further demonstrated localization of Fab-CHP to areas of high dCol in vivo and promising therapeutic efficacy, assessed by histological staining (Safranin-O and H&E), in a pilot mouse study.

Split-enzyme fragment as a single affinity tag that enables protein expression, purification, and functional assays.

Expressing, isolating, and characterizing recombinant proteins is crucial to many disciplines within the biological sciences. Different molecular tagging technologies have been developed to enable each individual step of protein production, from expression through purification and characterization. Monitoring the entire production process requires multiple tags or molecular interactions, because no individual tag has provided the comprehensive breadth of utility. An ideal molecular tag is small and does not interrupt expression, solubility, folding or function of the protein being purified and can be used throughout the production process. We adapted and integrated a split-luciferase system (NanoBiT®, Promega ®) to perform the range of techniques essential to protein production. We developed a simple method to monitor protein expression in real time to optimize expression conditions. We constructed a novel affinity chromatography system using the split-luciferase system to enable purification. We adapted western blot analysis, enzyme-linked immunosorbent assay, and cell-based bioassay to characterize the expressed proteins. Our results demonstrate that a single-tag can fulfill all aspects needed throughout protein production.

Imiquimod Induces Apoptosis in Human Endometrial Cancer Cells In vitro and Prevents Tumor Progression In vivo.

PURPOSE: The increasing incidence of endometrial cancer (EC), in younger age at diagnosis, calls for new tissue-sparing treatment options. This work aims to evaluate the potential of imiquimod (IQ) in the treatment of low-grade EC. METHODS: Effects of IQ on the viabilities of Ishikawa and HEC-1A cells were evaluated using MTT assay. The ability of IQ to induce apoptosis was evaluated by testing changes in caspase 3/7 levels and expression of cleaved caspase-3, using luminescence assay and western blot. Apoptosis was confirmed by flow cytometry and the expression of cleaved PARP. Western blot was used to evaluate the effect of IQ on expression levels of Bcl-2, Bcl-xL, and BAX. Finally, the in vivo efficacy of IQ was tested in an EC mouse model. RESULTS: There was a decrease in EC cell viability following IQ treatment as well as increased caspase 3/7 activities, cleaved caspase-3 expression, and Annexin-V/ 7AAD positive cell population. Western blot results showed the ability of IQ in cleaving PARP, decreasing Bcl-2 and Bcl-xL expressions, but not affecting BAX expression. In vivo study demonstrated IQ's ability to inhibit EC tumor growth and progression without significant toxicity. CONCLUSIONS: IQ induces apoptosis in low-grade EC cells in vitro, probably through its direct effect on Bcl-2 family protein expression. In, vivo, IQ attenuates EC tumor growth and progression, without an obvious toxicity. Our study provides the first building block for the potential role of IQ in the non-surgical management of low-grades EC and encouraging further investigations.

Surface-Available HER2 Levels Alone Are Not Indicative of Cell Response to HER2-Targeted Antibody-Drug Conjugate Therapies.

HER2-targeting therapies have advanced breast cancer treatment over the past decade. Clinically, eligibility for HER2 therapies is determined by assessing HER2 levels on tumor cell surfaces through immunohistochemistry or by gene regulation through fluorescence in situ hybridization. HER2 therapies are not always effective in patients with elevated levels of HER2, questioning whether the amount of HER2 is sufficiently predictive of patient outcomes. Additionally, the HER2-targeting antibody-drug conjugate (ADC) Enhertu® was recently approved for metastasized HER2-low cancers, confirming the benefits of HER2 treatment for patients with low HER2 levels. To evaluate the correlation between HER2 levels and treatment efficacy, we quantified HER2 on eight cell lines using flow cytometry while simultaneously determining the toxicity of two HER2-targeting ADCs. Both HER2-high cell lines and HER2-low cell lines had significant toxicity responses to ADCs. We quantified HER2 internalization and found no correlation between HER2 levels and the percentage of internalization. We found a useful metric suggesting that a minimum number of HER2 receptors trafficked to lysosomes is sufficient to provide effective treatment. Our results indicate that the current standards of determining eligibility for HER2 therapy could limit patients' access to effective treatment. In conclusion, HER2 levels are not wholly adequate to determine the response to ADC treatment.

Double click: dual functionalized polymeric micelles with antibodies and peptides.

Polymeric nanoparticle micelles provide a possible platform for theranostic delivery, combining the role of therapeutics and diagnostics in one vehicle. To explore dual-functional micelles, the amphiphilic copolymer of poly(d,l-lactide-co-2-methyl-2-carboxytrimethylene carbonate)-graft-poly(ethylene glycol)-X (P(LA-co-TMCC)-g-PEG-X) was self-assembled to form micelles, with X representing either azide or furan. Micelles of P(LA-co-TMCC)-g-PEG-azide and P(LA-co-TMCC)-g-PEG-furan terminal functional groups were used to conjugate dibenzylcyclooctyne and maleimide-modified probes, respectively, taking advantage of orthogonal coupling chemistry. To verify the utility of the dual-functional micelles, trastuzumab-maleimide antibodies and FLAG-dibenzylcyclooctyne peptides were covalently bound by sequential click chemistry reactions. SKOV-3luc cells that were treated with the dual-functionalized micelles showed colocalization of the antibodies and peptides by confocal imaging, demonstrating the promise of this approach.

Hyaluronic acid click hydrogels emulate the extracellular matrix.

Hydrogels are used to create 3D microenvironments with properties that direct cell function. The current study demonstrates the versatility of hyaluronic acid (HA)-based hydrogels with independent control over hydrogel properties such as mechanics, architecture, and the spatial distribution of biological factors. Hydrogels were prepared by reacting furan-modified HA with bis-maleimide-poly(ethylene glycol) in a Diels-Alder click reaction. Biomolecules were photopatterned into the hydrogel by two-photon laser processing, resulting in spatially defined growth factor gradients. The Young's modulus was controlled by either changing the hydrogel concentration or the furan substitution on the HA backbone, thereby decoupling the hydrogel concentration from mechanical properties. Porosity was controlled by cryogelation, and the pore size distribution, by the thaw temperature. The addition of galactose further influenced the porosity, pore size, and Young's modulus of the cryogels. These HA-based hydrogels offer a tunable platform with a diversity of properties for directing cell function, with applications in tissue engineering and regenerative medicine.

Therapeutic Fusion Proteins.

Therapeutic fusion proteins are a class of hybrid constructs that combine distinct biomolecules into a single platform with the additive effects of the components. The ability to fuse two unrelated proteins provides a means to localize mechanisms to better treat a range of diseases. Fusion proteins can be designed to impart diverse functions, including increasing half-life, providing targeting, and enabling sustained signaling. Of these, half-life extenders, which are fused to a therapeutic protein to increase exposure, are the most established group of fusion proteins, with many clinical successes. Rapid advances in antibody and antibody-derivative technology have enabled the fusion of targeting domains with therapeutic proteins. An emerging group of therapeutic fusion proteins has two separate active functions. Although most research for therapeutic fusion proteins focuses on cancer, prior successes provide a foundation for studies into other diseases as well. The exponential emergence of biopharmaceuticals gives precedence for increased research into therapeutic fusion proteins for a multitude of diseases.

Bioluminescence Assay of Lysine Deacylase Sirtuin Activity.

Lysine acylation can direct protein function, localization, and interactions. Sirtuins deacylate lysine towards maintaining cellular homeostasis, and their aberrant expression contributes to the pathogenesis of multiple pathological conditions, including cancer. Measuring sirtuins' activity is essential to exploring their potential as therapeutic targets, but accurate quantification is challenging. We developed 'SIRTify', a high-sensitivity assay for measuring sirtuin activity in vitro and in vivo. SIRTify is based on a split-version of the NanoLuc® luciferase consisting of a truncated, catalytically inactive N-terminal moiety (LgBiT) that complements with a high-affinity C-terminal peptide (p86) to form active luciferase. Acylation of two lysines within p86 disrupts binding to LgBiT and abates luminescence. Deacylation by sirtuins reestablishes p86 and restores binding, generating a luminescence signal proportional to sirtuin activity. Measurements accurately reflect reported sirtuin specificity for lysine acylations and confirm the effects of sirtuin modulators. SIRTify effectively quantifies lysine deacylation dynamics and may be adaptable to monitoring additional post-translational modifications.

Blood vessel hyperpermeability and pathophysiology in human tumour xenograft models of breast cancer: a comparison of ectopic and orthotopic tumours.

BACKGROUND: Human tumour xenografts in immune compromised mice are widely used as cancer models because they are easy to reproduce and simple to use in a variety of pre-clinical assessments. Developments in nanomedicine have led to the use of tumour xenografts in testing nanoscale delivery devices, such as nanoparticles and polymer-drug conjugates, for targeting and efficacy via the enhanced permeability and retention (EPR) effect. For these results to be meaningful, the hyperpermeable vasculature and reduced lymphatic drainage associated with tumour pathophysiology must be replicated in the model. In pre-clinical breast cancer xenograft models, cells are commonly introduced via injection either orthotopically (mammary fat pad, MFP) or ectopically (subcutaneous, SC), and the organ environment experienced by the tumour cells has been shown to influence their behaviour. METHODS: To evaluate xenograft models of breast cancer in the context of EPR, both orthotopic MFP and ectopic SC injections of MDA-MB-231-H2N cells were given to NOD scid gamma (NSG) mice. Animals with matched tumours in two size categories were tested by injection of a high molecular weight dextran as a model nanocarrier. Tumours were collected and sectioned to assess dextran accumulation compared to liver tissue as a positive control. To understand the cellular basis of these observations, tumour sections were also immunostained for endothelial cells, basement membranes, pericytes, and lymphatic vessels. RESULTS: SC tumours required longer development times to become size matched to MFP tumours, and also presented wide size variability and ulcerated skin lesions 6 weeks after cell injection. The 3 week MFP tumour model demonstrated greater dextran accumulation than the size matched 5 week SC tumour model (for P < 0.10). Immunostaining revealed greater vascular density and thinner basement membranes in the MFP tumour model 3 weeks after cell injection. Both the MFP and SC tumours showed evidence of insufficient lymphatic drainage, as many fluid-filled and collagen IV-lined spaces were observed, which likely contain excess interstitial fluid. CONCLUSIONS: Dextran accumulation and immunostaining results suggest that small MFP tumours best replicate the vascular permeability required to observe the EPR effect in vivo. A more predictable growth profile and the absence of ulcerated skin lesions further point to the MFP model as a strong choice for long term treatment studies that initiate after a target tumour size has been reached.

Complementation Dependent Enzyme Prodrug Therapy Enables Targeted Activation of Prodrug on HER2-Positive Cancer Cells.

Antibodies have been explored for decades for the delivery of small molecule cytotoxins directly to diseased cells. In antibody-directed enzyme prodrug therapy (ADEPT), antibodies are armed with enzymes that activate nontoxic prodrugs at tumor sites. However, this strategy failed clinically due to off-target toxicity associated with the enzyme prematurely activating prodrug systemically. We describe here the design of an antibody-fragment split enzyme platform that regains activity after binding to HER2, allowing for site-specific activation of a small molecule prodrug. We evaluated a library of fusion constructs for efficient targeting and complementation to identify the most promising split enzyme pair. The optimal pair was screened for substrate specificity among chromogenic, fluorogenic, and prodrug substrates. Evaluation of this system on HER2-positive cells revealed 7-fold higher toxicity of the activated prodrug over prodrug treatment alone. Demonstrating the potential of this strategy against a known clinical target provides the basis for a unique therapeutic platform in oncology.

Homogeneous surrogate virus neutralization assay to rapidly assess neutralization activity of anti-SARS-CoV-2 antibodies.

The COVID-19 pandemic triggered the development of numerous diagnostic tools to monitor infection and to determine immune response. Although assays to measure binding antibodies against SARS-CoV-2 are widely available, more specific tests measuring neutralization activities of antibodies are immediately needed to quantify the extent and duration of protection that results from infection or vaccination. We previously developed a 'Serological Assay based on a Tri-part split-NanoLuc® (SATiN)' to detect antibodies that bind to the spike (S) protein of SARS-CoV-2. Here, we expand on our previous work and describe a reconfigured version of the SATiN assay, called Neutralization SATiN (Neu-SATiN), which measures neutralization activity of antibodies directly from convalescent or vaccinated sera. The results obtained with our assay and other neutralization assays are comparable but with significantly shorter preparation and run time for Neu-SATiN. As the assay is modular, we further demonstrate that Neu-SATiN enables rapid assessment of the effectiveness of vaccines and level of protection against existing SARS-CoV-2 variants of concern and can therefore be readily adapted for emerging variants.

Diels-Alder Click cross-linked hyaluronic acid hydrogels for tissue engineering.

Hyaluronic acid (HA) is a naturally occurring polymer that holds considerable promise for tissue engineering applications. Current cross-linking chemistries often require a coupling agent, catalyst, or photoinitiator, which may be cytotoxic, or involve a multistep synthesis of functionalized-HA, increasing the complexity of the system. With the goal of designing a simpler one-step, aqueous-based cross-linking system, we synthesized HA hydrogels via Diels-Alder "click" chemistry. Furan-modified HA derivatives were synthesized and cross-linked via dimaleimide poly(ethylene glycol). By controlling the furan to maleimide molar ratio, both the mechanical and degradation properties of the resulting Diels-Alder cross-linked hydrogels can be tuned. Rheological and degradation studies demonstrate that the Diels-Alder click reaction is a suitable cross-linking method for HA. These HA cross-linked hydrogels were shown to be cytocompatible and may represent a promising material for soft tissue engineering.

Split-enzyme immunoassay to monitor EGFR-HER2 heterodimerization on cell surfaces.

Over 30,000 protein-protein interactions with pathological implications have been identified; yet, discovering and investigating drugs that target these specific interactions is greatly limited by the inability to monitor native protein-protein interactions (PPIs) efficiently. The two most frequently used tools to monitor PPIs, resonance-energy transfer (RET) assays and protein complementation assays (PCA), face significant limitations. RET assays have a narrow working range of 10 to 50 Å, while PCA require permanent attachment of a reporter probe to a protein of interest by chemical conjugation or genetic engineering. We developed a non-invasive assay platform to measure PPIs without modifications to the proteins of interest and is functional at a greater working range than RET assays. We demonstrate our approach by monitoring the EGFR-HER2 heterodimerization on relevant cell surfaces, utilizing various EGFR- and HER2-specific binders (e.g., Fab, DARPin, and VHH) fused with small fragments of a tri-part split-luciferase derived from NanoLuc®. Following independent binding of the binder fusions to their respective targets, the dimerization of EGFR and HER2 induces complementation of the luciferase fragments into a functional native structure, producing glow-type luminescence. We have confirmed the functionality of the platform to monitor EGFR-HER2 dimerization induction and inhibition. STATEMENT OF SIGNIFICANCE: We describe a platform technology for rapid monitoring of protein-protein interactions (PPIs). Our approach is uses a luciferase split into three parts - two short peptide "tags" and a large third fragment. Each of the short peptides can be fused to antibodies which bind to domains of a target antigens which orients the two tags and facilitates refolding of an active enzyme. To our knowledge this is the first example of a split-enzyme used to monitor PPIs without requiring any modification of the target proteins. We demonstrate our approach on the important PPI of HER2 and EGFR. Significantly, we quantify stimulation and inhibition of these partners, opening the possibility of using our approach to assess potential drugs without engineering cells.

Homogeneous Immunoassay Using a Tri-Part Split-Luciferase for Rapid Quantification of Anti-TNF Therapeutic Antibodies.

Anti-TNF therapeutics bind and sequester tumor necrosis factor (TNF) to prevent downstream signaling and are clinically important in the treatment of several autoimmune diseases. Effective treatment with these drugs requires frequent therapeutic drug monitoring (TDM). Current analytical methods, including reporter gene assay (RGA), enzyme-linked immunosorbent assay (ELISA), and mobility shift assay (MSA), can be technically rigorous, slow, and expensive. These qualities prevent the implementation of point-of-care testing and ultimately limit the frequency and utility of monitoring. An assay simple enough to be performed in the clinic would enable increased TDM frequency, more accurate dosing, and improved patient outcomes. Toward this end, we developed a homogeneous immunoassay based on a tri-part split-luciferase system for "add-and-read" detection of anti-TNF therapeutics. In our platform, two small fragments of the split-luciferase, called ß9 and ß10, are each fused to a different interacting protein. The binding of each of these proteins to anti-TNF antibodies forces the split-luciferase components into proximity where they reform the active luciferase. We identified the fusion proteins, ß9-protein A (ß9-A) and ß10-TNF, as promising binding pairs. We systematically adjusted assay conditions to optimize the signal/background (S/B) ratio, limit of detection (LOD), and percent recovery. The assay has a large dynamic range (0.5-32 µg/mL) and is sensitive enough to monitor both subtherapeutic and supratherapeutic serum concentrations of anti-TNF antibodies, as demonstrated in clinical samples.

Antibody-drug conjugate and free geldanamycin combination therapy enhances anti-cancer efficacy.

Antibody drug-conjugates (ADCs) targeting human epidermal growth factor (HER2) are a rapidly expanding class of cancer therapeutics. Such ADCs are known to suffer from inefficient trafficking to the lysosome due to HER2 endosomal recycling, leaving most bound ADCs at the cell surface or in early endosomes. This study aims to increase the maximum cytotoxicity of ADC treatment by co-delivering a small molecule inhibitor targeting the primary chaperone of HER2, heat shock protein 90 (HSP90). We hypothesized that inhibiting HSP90 could aid ADC cytotoxicity by overcoming HER2 endosomal recycling. Flow cytometric studies tracking HER2 surface expression revealed âˆ¼ 10 nM geldanamycin (GA) as the threshold for inhibiting HSP90 mediated HER2 recycling. Cytotoxicity studies in HER2 overexpressing cancer cell lines NCI-N87, MDA-MB-453, and SKOV3 demonstrated that co-administration of ADC alongside 100 nM GA significantly increased cytotoxicity compared to ADC alone. In all cases, baseline cytotoxicity was observed even in low HER2 expressing line MDA-MB-231 cells, indicating possible off-target effects. To mitigate this baseline cytotoxicity, a "pulse treatment" regime was adopted where cells are pre-loaded with T-DM1 or T-MMAE ADCs for 4 h, followed by a 4-hour pulse treatment with ADC and 100 nM GA to initiate trafficking of HER2 bound ADC to the lysosome. Afterwards, GA is removed, and ADC treatment is continued. GA pulse co-treatment decreased the amount of ADC required to achieve maximum cytotoxicity while minimizing baseline cytotoxicity. No such co-treatment regime featuring a pulse sequence has been explored before. Such co-treatments could offer a viable solution to increase ADC efficacy in hard to treat or resistant HER2-positive cancers.

SIRT5 IS A DRUGGABLE METABOLIC VULNERABILITY IN ACUTE MYELOID LEUKEMIA.

We discovered that the survival and growth of many primary acute myeloid leukemia (AML) samples and cell lines, but not normal CD34+ cells, are dependent on SIRT5, a lysine deacylase implicated in regulating multiple metabolic pathways. Dependence on SIRT5 is genotype-agnostic and extends to RAS- and p53-mutated AML. Results were comparable between SIRT5 knockdown and SIRT5 inhibition using NRD167, a potent and selective SIRT5 inhibitor. Apoptosis induced by SIRT5 disruption is preceded by reductions in oxidative phosphorylation and glutamine utilization, and an increase in mitochondrial superoxide that is attenuated by ectopic superoxide dismutase 2. These data indicate that SIRT5 controls and coordinates several key metabolic pathways in AML and implicate SIRT5 as a vulnerability in AML.

Hyaluronic acid binding to CD44S is indiscriminate of molecular weight.

The ubiquitous presence of hyaluronic acid (HA) in the extracellular matrix (ECM) of both healthy and diseased tissues underscores its importance in human physiology. Previous studies suggest that HA can be used as a probe to qualitatively monitor cell surface levels of CD44 and other important HA receptors; however, these studies use mixtures of HA at various molecular weights. Using fluorescently labeled HA, we evaluated the apparent differences of low (25 kilodalton) and high (700 kilodalton) molecular weight HA interacting with breast cancer cell lines of varying levels of CD44. Our results confirm that CD44 expression and the apparent level of HA interaction correlates with molecular weight. Importantly, we show that HA only binds a small fraction of the major CD44 isoform, CD44S, on cell surfaces and that CD44S interactions account for <50% of the total HA bound to cell surfaces. Although increased fluorescence level correlates with higher molecular weight of HA, this appears to be an artifact of chain length and not a result of multivalent binding between HA and CD44S. Accordingly, we verify that HA binding characteristics of cell surfaces is similar to previous artificial membrane models which proposed that HA anchors to CD44S and forms a non-binding corona of HA that extends beyond the surface.

Multivalent HER2-binding polymer conjugates facilitate rapid endocytosis and enhance intracellular drug delivery.

Incorporating targeting moieties that recognize cancer-specific cellular markers can enhance specificity of anticancer nanomedicines. The HER2 receptor is overexpressed on numerous cancers, making it an attractive target. However, unlike many receptors that trigger endocytosis upon ligand binding, HER2 is an internalization-resistant receptor. As most chemotherapeutics act on intracellular targets, this presents a significant challenge for exploiting HER2 overexpression for improved tumor killing. However, hyper-crosslinking of HER2 has been shown to override the receptor's native behavior and trigger internalization. This research co-opts this crosslinking-mediated internalization for efficient intracellular delivery of an anticancer nanomedicine - specifically a HPMA copolymer-based drug delivery system. This polymeric carrier was conjugated with a small (7 kDa) HER2-binding affibody peptide to produce a panel of polymer-affibody conjugates with valences from 2 to 10 peptides per polymer chain. The effect of valence on surface binding and uptake was evaluated separately. All conjugates demonstrated similar (nanomolar) binding affinity towards HER2-positive ovarian carcinoma cells, but higher-valence conjugates induced more rapid endocytosis, with over 90% of the surface-bound conjugate internalized within 4 h. Furthermore, this enhancement was sensitive to crowding - high surface loading reduced conjugates' ability to crosslink receptors. Collectively, this evidence strongly supports a crosslinking-mediated endocytosis mechanism. Lead candidates from this panel achieved high intracellular delivery even at picomolar treatment concentrations; untargeted HPMA copolymers required 1000-fold higher treatment concentrations to achieve similar levels of intracellular accumulation. This increased intracellular delivery also translated to a more potent nanomedicine against HER2-positive cells; incorporation of the chemotherapeutic paclitaxel into this targeted carrier enhanced cytotoxicity over untargeted polymer-drug conjugate.