kDa PEG Research Articles

Functionalized Polysaccharides Improve Sensitivity of Tyramide/Peroxidase Proximity Labeling Assays through Electrostatic Interactions.

High-throughput assays that efficiently link genotype and phenotype with high fidelity are key to successful enzyme engineering campaigns. Among these assays, the tyramide/peroxidase proximity labeling method converts the product of an enzymatic reaction of a surface expressed enzyme to a highly reactive fluorescent radical, which labels the cell surface. In this context, maintaining the proximity of the readout reagents to the cell surface is crucial to prevent crosstalk and ensure that short-lived radical species react before diffusing away. Here, we investigated improvements in tyramide/peroxidase proximity labeling for enzyme screening. We modified chitosan (Cs) chains with horseradish peroxidase (HRP) and evaluated the effects of these conjugates on the efficiency of proximity labeling reactions on yeast cells displaying d-amino acid oxidase. By tethering HRP to chitosan through different chemical approaches, we localized the auxiliary enzyme close to the cell surface and enhanced the sensitivity of tyramide-peroxidase labeling reactions. We found that immobilizing HRP onto chitosan through a 5 kDa PEG linker improved labeling sensitivity by over 3.5-fold for substrates processed with a low turnover rate (e.g., d-lysine), while the sensitivity of the labeling for high activity substrates (e.g., d-alanine) was enhanced by over 0.6-fold. Such improvements in labeling efficiency broaden the range of enzymes and conditions that can be studied and screened by tyramide/peroxidase proximity labeling.

PLX038A, a long-acting SN-38, penetrates the blood-tumor-brain-barrier, accumulates and releases SN-38 in brain tumors to increase survival of tumor bearing mice

Central nervous system tumors have resisted effective chemotherapy because most therapeutics do not penetrate the blood-tumor-brain-barrier. Nanomedicines between ~ 10 and 100 nm accumulate in many solid tumors by the enhanced permeability and retention effect, but it is controversial whether the effect can be exploited for treatment of brain tumors. PLX038A is a long-acting prodrug of the topoisomerase 1 inhibitor SN-38. It is composed of a 15 nm 4-arm 40 kDa PEG tethered to four SN-38 moieties by linkers that slowly cleave to release the SN-38. The prodrug was remarkably effective at suppressing growth of intracranial breast cancer and glioblastoma (GBM), significantly increasing the life span of mice harboring them. We addressed the important issue of whether the prodrug releases SN-38 systemically and then penetrates the brain to exert anti-tumor effects, or whether it directly penetrates the blood-tumor-brain-barrier and releases the SN-38 cargo within the tumor. We argue that the amount of SN-38 formed systemically is insufficient to inhibit the tumors, and show by PET imaging that a close surrogate of the 40 kDa PEG carrier in PLX038A accumulates and is retained in the GBM. We conclude that the prodrug penetrates the blood-tumor-brain-barrier, accumulates in the tumor microenvironment and releases its SN-38 cargo from within. Based on our results, we pose the provocative question as to whether the 40 kDa nanomolecule PEG carrier might serve as a “Trojan horse” to carry other drugs past the blood-tumor-brain-barrier and release them into brain tumors.

Evaluation of cell‐laden three‐dimensional bioprinted polymer composite scaffolds based on synthesized photocrosslinkable poly(ethylene glycol) dimethacrylate with different molecular weights

AbstractThis manuscript aims to three‐dimensional bioprint and evaluate new polymer composite scaffolds based on synthesized poly(ethylene glycol) dimethacrylate (PEGDMA) as well as methyl cellulose and gelatin. The PEGDMA was synthesized by a simple microwave‐assisted method using three distinct molecular weights (MWs) of poly(ethylene glycol) (PEG), 3, 6, and 12 kDa, and methacrylic anhydride. The percent functionalization of the PEGDMA was analyzed using the nuclear magnetic resonance spectrum, and the theoretical calculations indicated that over 50% of methacrylation was achieved in all samples, with the PEGDMA synthesized from 6 kDa PEG surpassing 66% methacrylation. These three PEGDMA‐based bioinks were investigated for their suitability for bioprinting scaffolds. It was observed that lower MW PEGDMA resulted in a higher degree of crosslinking, leading to more stable composite scaffolds. However, higher crosslinking degree did not support long‐term cell viability when encapsulated with cells. Higher MW PEGDMA showed higher cell viability over time though overall stability was lower. Synthesized PEGDMA with 6 kDa PEG showed both stability and long‐term cell viability after postprinting. Over 80% of cell viability was maintained for a 7‐day study period, showing potential use in tissue engineering applications as a cell delivery vehicle.

Surface modulation of extracellular vesicles with cell-penetrating peptide-conjugated lipids for improvement of intracellular delivery to endothelial cells.

Exosomes (diameter 30-200nm) are a subtype of extracellular vesicles secreted by cells containing DNA, microRNA (miRNA), and proteins. Exosomes are expected to be valuable as a means of delivering drugs or functional miRNAs in treatment of diseases. However, the delivery of exosomes is not sufficiently effective, even though exosomes have intrinsic delivery functions. Cell-penetrating peptides (CPPs) are short peptide families that facilitate cellular intake of molecules and vesicles. We previously reported that the modification of cells, and liposomes with CPP-conjugated-lipids, CPPs conjugated with poly (ethylene glycol)-conjugated phospholipids (PEG-lipid), that induce adhesion by CPPs, can be useful for cell-based assays and harvesting liposomes. In this study, we aimed to modulate the exosome surface using Tat peptide (YGRKKRRQRRR)-PEG-lipids to improve intracellular delivery to endothelial cells. We isolated and characterized exosomes from the medium of HEK 293T cell cultures. Tat conjugated PEG-lipids with different spacer molecular weights and lipid types were incorporated into exosomes using fluorescein isothiocyanate labeling to optimize the number of Tat-PEG-lipids immobilized on the exosome surface. The exosomes modified with Tat-PEG-lipids were incubated with human umbilical vein endothelial cells (HUVECs) to study the interaction. Tat conjugated with 5kDa PEG and C16 lipids incorporated on the exosome surface were highly detected inside HUVECs by flow cytometry. Fluorescence was negligible in HUVECs for control groups. Thus, Tat-PEG-lipids can be modified on the exosome surface, improving the intracellular delivery of exosomes.

Engineering endosomolytic nanocarriers of diverse morphologies using confined impingement jet mixing.

The clinical translation of many biomolecular therapeutics has been hindered by undesirable pharmacokinetic (PK) properties, inadequate membrane permeability, poor endosomal escape and cytosolic delivery, and/or susceptibility to degradation. Overcoming these challenges merits the development of nanoscale drug carriers (nanocarriers) to improve the delivery of therapeutic cargo. Herein, we implement a flash nanoprecipitation (FNP) approach to produce nanocarriers of diverse vesicular morphologies by using various molecular weight PEG-bl-DEAEMA-co-BMA (PEG-DB) polymers. We demonstrated that FNP can produce uniform (PDI < 0.1) particles after 5 impingements, and that by varying the copolymer hydrophilic mass fraction, FNP enables access to a diverse variety of nanoarchitectures including micelles, unilamellar vesicles (polymersomes), and multi-compartment vesicles (MCVs). We synthesized a library of 2 kDa PEG block copolymers, with DEAEMA-co-BMA second block molecular weights of 3, 6, 12, 15, 20, and 30 kDa. All formulations were both pH responsive, endosomolytic, and capable of loading and cytosolically delivering small negatively charged molecules - albeit to different degrees. Using a B16.F10 melanoma model, we showcased the therapeutic potential of a lead FNP formulated PEG-DB nanocarrier, encapsulating the cyclic dinucleotide (CDN) cGAMP to activate the stimulator of interferon genes (STING) pathway in a therapeutically relevant context. Collectively, these data demonstrate that an FNP process can be used to formulate pH-responsive nanocarriers of diverse morphologies using a PEG-DB polymer system. As FNP is an industrially scalable process, these data address the critical translational challenge of producing PEG-DB nanoparticles at scale. Furthermore, the diverse morphologies produced may specialize in the delivery of distinct biomolecular cargos for other therapeutic applications, implicating the therapeutic potential of this platform in an array of disease applications.

Efficient Magneto-Luminescent Nanosystems based on Rhodamine-Loaded Magnetite Nanoparticles with Optimized Heating Power and Ideal Thermosensitive Fluorescence.

Nanosystems that simultaneously contain fluorescent and magnetic modules can offer decisive advantages in the development of new biomedical approaches. A biomaterial that enables multimodal imaging and contains highly efficient nanoheaters together with an intrinsic temperature sensor would become an archetypical theranostic agent. In this work, we have designed a magneto-luminescent system based on Fe3O4 NPs with large heating power and thermosensitive rhodamine (Rh) fluorophores that exhibits the ability to self-monitor the hyperthermia degree. Three samples composed of highly homogeneous Fe3O4 NPs of ∼25 nm and different morphologies (cuboctahedrons, octahedrons, and irregular truncated-octahedrons) have been finely synthesized. These NPs have been thoroughly studied in order to choose the most efficient inorganic core for magnetic hyperthermia under clinically safe radiofrequency. Surface functionalization of selected Fe3O4 NPs has been carried out using fluorescent copolymers composed of PMAO, PEG and Rh. Copolymers with distinct PEG tail lengths (5-20 kDa) and different Rh percentages (5, 10, and 25%) have been synthesized, finding out that the copolymer with 20 kDa PEG and 10% Rh provides the best coating for an efficient fluorescence with minimal aggregation effects. The optimized Fe3O4@Rh system offers very suitable fluorescence thermosensitivity in the therapeutic hyperthermia range. Additionally, this sample presents good biocompatibility and displays an excellent heating capacity within the clinical safety limits of the AC field (≈ 1000 W/g at 142 kHz and 44 mT), which has been confirmed by both calorimetry and AC magnetometry. Thus, the current work opens up promising avenues toward next-generation medical technologies.

Surface Modification of Biodegradable Microparticles with the Novel Host-Derived Immunostimulant CPDI-02 Significantly Increases Short-Term and Long-Term Mucosal and Systemic Antibodies against Encapsulated Protein Antigen in Young Naïve Mice after Respiratory Immunization

Generating long-lived mucosal and systemic antibodies through respiratory immunization with protective antigens encapsulated in nanoscale biodegradable particles could potentially decrease or eliminate the incidence of many infectious diseases, but requires the incorporation of a suitable mucosal immunostimulant. We previously found that respiratory immunization with a model protein antigen (LPS-free OVA) encapsulated in PLGA 50:50 nanoparticles (~380 nm diameter) surface-modified with complement peptide-derived immunostimulant 02 (CPDI-02; formerly EP67) through 2 kDa PEG linkers increases mucosal and systemic OVA-specific memory T-cells with long-lived surface phenotypes in young, naïve female C57BL/6 mice. Here, we determined if respiratory immunization with LPS-free OVA encapsulated in similar PLGA 50:50 microparticles (~1 μm diameter) surface-modified with CPDI-02 (CPDI-02-MP) increases long-term OVA-specific mucosal and systemic antibodies. We found that, compared to MP surface-modified with inactive, scrambled scCPDI-02 (scCPDI-02-MP), intranasal administration of CPDI-02-MP in 50 μL sterile PBS greatly increased titers of short-term (14 days post-immunization) and long-term (90 days post-immunization) antibodies against encapsulated LPS-free OVA in nasal lavage fluids, bronchoalveolar lavage fluids, and sera of young, naïve female C57BL/6 mice with minimal lung inflammation. Thus, surface modification of ~1 μm biodegradable microparticles with CPDI-02 is likely to increase long-term mucosal and systemic antibodies against encapsulated protein antigen after respiratory and possibly other routes of mucosal immunization.

Abstract 4224: BPT-143: a fully synthetic alpha-dead IL-2 with a best-in-class preclinical pharmacodynamic and efficacy profile supporting first-in-human clinical development

Abstract High-dose recombinant IL-2 (aldesleukin) is approved for treatment of advanced melanoma and renal cell carcinoma, however, major limitations restrict its therapeutic use. Wild-type IL-2 acts via binding to the medium-affinity IL-2 receptor βγ (IL2Rβγ) expressed in CD8+ T effector cells and NK cells. At the same time, its efficacy is dampened due to strong activation of regulatory T cells (Treg) expressing the high-affinity IL-2 receptor αβγ (IL2Rαβγ). Furthermore, binding to CD25/IL2Rα on endothelial cells and type 2 innate lymphoid cells is thought to be involved in the induction of severe toxicity including vascular leak syndrome (VLS). In addition, aldesleukin exhibits a very short half-life that, combined with its safety risks, requires a burdensome inpatient treatment schedule. We set out to rationally design a variant of human IL-2 that addresses and overcomes the major limitations of aldesleukin. Using our chemical protein synthesis technology, we introduced select modified amino acids including site-specific chemical conjugation handles to optimize the properties of IL-2 for cancer therapy while maintaining high homology to the wild-type IL-2 sequence. Bright Peak’s enhanced cytokine shows increased binding to CD122/IL2Rβ and does not interact with CD25/IL2Rα to improve safety and prevent the preferential activation of Tregs compared to CD8+ T effector cells. Site-specific conjugation to a 30 kDa PEG for half-life extension resulted in the generation of BPT-143, which is equipotent to aldesleukin in activating CD8+ T cells in vitro. In mice, BPT-143 induces a strong expansion of CD8+ T cells with only transient and minor effects on Tregs in vivo and exhibits improved PK properties allowing for a convenient dosing schedule. In the syngeneic CT26 tumor model, BPT-143 showed strong anti-tumor efficacy as a single agent as well as enhanced efficacy in combination with an anti-PD-1 antibody. BPT-143 induced a 55% complete response rate and, upon tumor re-challenge, all cured animals rejected CT26 tumor cells indicating the development of immunologic memory. In multiple-dose PK/PD studies in non-human primates (NHP), BPT-143 was well tolerated and induced robust and repeated expansion of CD8+ T cells, CD4+ conventional T cells and NK cells while showing only negligible effects on Tregs and eosinophils. Both in vivo efficacy studies in murine tumor models as well as PD effects observed in NHP indicate that BPT-143 has a best-in-class profile among “not-alpha” IL-2 compounds currently in development. IND-enabling studies are ongoing and a first in human trial is planned to start in 2022. Citation Format: Jean-Philippe Carralot, Rubén Alvarez Sanchez, Matilde Arévalo Ruiz, Magali Muller, Vijaya R. Pattabiraman, Bertolt Kreft. BPT-143: a fully synthetic alpha-dead IL-2 with a best-in-class preclinical pharmacodynamic and efficacy profile supporting first-in-human clinical development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4224.

Abstract 2138: Creating next-generation biologics using a novel chemistry platform technology

Abstract Bright Peak Therapeutics is developing a portfolio of differentiated biotherapeutics using chemistry for applications in immuno-oncology and autoimmune diseases. Our unique chemical protein synthesis and engineering platform allows us to fine-tune cytokines and other proteins to interrogate and modulate biological functions by incorporating new functional modifications. Standard recombinant bacterial or cellular expression systems used to produce proteins are largely restricted to using canonical amino acids, which limits access to diverse modifications that can bestow additional functional properties. With chemical protein synthesis technology, canonical and non-canonical modifications including conjugation handles can be easily introduced, ultimately enabling a medicinal chemistry approach for engineering cytokine structures. Enhanced cytokines with differentiated biology developed using this approach can be further elaborated by conjugating to a diverse array of molecules. We first applied our technology platform to identify BPT-143, a rationally designed enhanced IL-2 variant currently in IND-enabling studies. BPT-143 is engineered to have enhanced binding to IL2Rβ and no binding to IL2Rα for improved efficacy and safety independent of the conjugation to a half-life extending 30 kDa PEG. The chemical synthesis technology is robust, reproducible, and scalable. We are applying our platform to enhance a number of other cytokines for use in immuno-oncology and autoimmune diseases. Additionally, our synthetically engineered cytokines can be easily conjugated to monoclonal antibodies as ‘payloads’ using a distinct chemical conjugation technology. A rapid and simple chemical process allows site-selective conjugation of our engineered cytokines to existing antibodies ‘as-is’ to generate novel immunocytokines (IC). This ‘off-the-shelf’ approach is orthogonal to recombinant fusion methods to create ICs and does not require complex recombinant protein expression optimization and lengthy cell-line development. Moreover, it allows rapid screening of cytokine payloads in a structure-activity relationship (SAR) format to identify dual-targeting ICs with precisely tailored properties to generate the desired biological effect. We have prepared a number of ICs including anti-PD-1/IL-2 ICs with various drug-antibody ratio (DAR) and conjugation sites within the antibody. We will provide an overview of the platform technology and present highlights of its application for discovery and development of designer therapeutic cytokines and ICs. Citation Format: Vijaya R. Pattabiraman, Matilde Arévalo Ruiz, Régis Boehringer, Benoit Hornsperger, Roy Meoded, Robert C. Tam, Bertolt Kreft. Creating next-generation biologics using a novel chemistry platform technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2138.

OP0008 A NOVEL SITE-SPECIFIC PEGYLATED IL-2 WITH POTENT AND TREG-SELECTIVE ACTIVITY IN VIVO

BackgroundDecreased regulatory T cells (Tregs) and Treg dysfunction are hallmarks of a various autoimmune and inflammatory diseases. While low-dose IL-2 therapy induces Treg expansion in vivo and has clinical benefits in some diseases (e.g., SLE and chronic graft-versus-host disease [GvHD]), there are many concerns about adverse events due to low Treg-selectivity. Furthermore, frequent dosing is needed due to the short half-life.ObjectivesWe discovered a novel site-specific PEGylated IL-2 variant, KKC80, with high Treg selectivity and a long half-life in vivo, which overcomes the issues of low-dose IL-2 therapy.MethodsBased on the co-crystal structure of wild-type IL-2 and its heterotrimeric receptor (PBD ID: 2ERJ), amino acid residues that were to be PEGylation sites were substituted with oAzZLys, an azide-containing lysine derivative. The PEG molecule was site-specifically attached to oAzZLys-incorporated IL-2 by copper-free click chemistry. The binding property to the IL-2 receptors were measured by surface plasmon resonance (SPR). In vitro, Treg selectivity was evaluated by the IL-2-dependent proliferation activity of Tregs and NK cells from human peripheral blood mononuclear cells (PBMCs). In vivo pharmacological activity after the single subcutaneous administration in cynomolgus monkeys was measured by changes in Treg count and Treg activation status in peripheral blood by flow cytometry. Pharmacokinetic parameters were calculated according to serum PEGylated IL-2 concentration. Efficacy in mouse xenogeneic GvHD model using human PBMC-transplanted NOG mice and in monkey DTH model were evaluated.ResultsA novel PEGylated IL-2, KKC80 (human IL-2 desA1/C125S /I129oAzZLys_W-shaped 80 kDa PEG) was discovered by optimizing the PEGylation site and PEG structure based on Treg selectivity and PK. SPR analysis showed that the binding affinity of KKC80 to CD25 was moderately decreased from wild-type IL-2, while binding affinity of KKC80 to IL-2Rβγ was remarkably decreased due to a significant change of the association rate constant. In vitro, wild-type IL-2 activated both Tregs and NK cells in the same concentration range, whereas KKC80 selectively activated Tregs. The Treg selectivity of KKC80 was comparable to another IL-2 mutein, Fc.IL-2 V91K. KKC80, but not Fc.IL-2 V91K, retained its biological activity, even in the presence of a large amount of recombinant soluble CD25, which mimicked the endogenous decoy receptor for IL-2. In monkeys, KKC80 selectively increased peripheral blood Tregs in a dose-dependent manner; the average maximum rate of increase of Treg count in animals treated with 0.01, 0.03, 0.1, 0.3 and 1 mg/kg was 1.5, 3.5, 28, 50 and 154-fold, respectively. In contrast to Tregs, the rates of increase of conventional CD4+ T, CD8+ T and NK cells were low. The Treg increase peaked on day 8 or 11 and lasted for over day 29. KKC80 showed a more sustained upregulation of functional Treg markers (e.g., Foxp3 and CD25) in comparison to Fc.IL-2 V91K. The half-life of KKC80 was calculated as 83.5 to 150 h. At high doses, inflammation-related adverse effects, including increased CRP (≥0.3 mg/kg) and deterioration of general conditions (1 mg/kg) were observed. In the mouse xenogenic GvHD model, KKC80 ameliorated GvHD symptoms and suppressed multiple tissue inflammation markers. Decreased soluble CD25 and IFN-γ were also confirmed, suggesting Treg-mediated anti-inflammatory effect by KKC80 administration were exerted in vivo. In the monkey DTH model, KKC80 suppressed skin inflammation and antibody production.ConclusionAmong next-generation IL-2 variants, KKC80 showed a best-in-class biological profile for Treg activation. A drastic and sustained increase of Tregs with high Treg-selectivity and anti-inflammatory effects were observed in vivo. These data suggest that in comparison to current IL-2 therapy, KKC80 provides superior therapeutic index and efficacy in patients with autoimmune and inflammatory diseases.Figure 1.Disclosure of InterestsMasahiro Ikeda Employee of: Kyowa Kirin Co., Ltd., Shinpei Yamaguchi Employee of: Kyowa Kirin Co., Ltd., Masumi Murakami Employee of: Kyowa Kirin Co., Ltd., Shigeki Takaoka Employee of: Kyowa Kirin Co., Ltd., Yasuko Sakaguchi Employee of: Kyowa Kirin Co., Ltd., Shunki Yasui Employee of: Kyowa Kirin Co., Ltd., Kousuke Iijima Employee of: Kyowa Kirin Co., Ltd., Kenichiro Nanya Employee of: Kyowa Kirin Co., Ltd., Hideyuki Onodera Employee of: Kyowa Kirin Co., Ltd., Toru Amano Employee of: Kyowa Kirin Co., Ltd.

Injectable gelatin-poly(ethylene glycol) adhesive hydrogels with highly hemostatic and wound healing capabilities

Although numerous poly(ethylene glycol) (PEG)-based bioadhesives have been developed and commercialized for various biomedical applications, their biocompatibility and bioactivity performances are generally lacking. In addition, the development of adhesive hydrogels with rapid gelation and high tissue adhesion are desirable to promote the surgical procedures and enhance patient compliance. To overcome these problems, we designed a series of chemically crosslinked gelatin-PEG adhesive hydrogels (GP) through the enzymatic crosslinking reaction of horseradish peroxidase (HRP). The resultant GP hydrogels were rapidly formed in situ within seconds to minutes after injection, and their mechanical as well as adhesive properties were adjustable in a wide range by changing the molecular weight and content of PEG. Notably, the hybrid hydrogels composed of 20 kDa PEG with ratio of 2.5/7.5 gelatin/PEG (wt/wt) showed 11 times greater tissue adhesiveness than commercially available fibrin glues. From in vitro cell studies, the hybrid adhesive hydrogels are nontoxic and improve the cell proliferation. Importantly, the hydrogels exhibited excellent hemostatic capability and accelerated the wound healing in vivo, compared to fibrin glue. These injectable GP hydrogels effectively improved the tissue adhesiveness and bioactivity of PEG-based adhesives, and hold great potential for hemostatic and wound healing treatments.

Multiscale imaging of therapeutic anti-PD-L1 antibody localization using molecularly defined imaging agents

BackgroundWhile immune checkpoint inhibitors such as anti-PD-L1 antibodies have revolutionized cancer treatment, only subgroups of patients show durable responses. Insight in the relation between clinical response, PD-L1 expression and intratumoral localization of PD-L1 therapeutics could improve patient stratification. Therefore, we present the modular synthesis of multimodal antibody-based imaging tools for multiscale imaging of PD-L1 to study intratumoral distribution of PD-L1 therapeutics.ResultsTo introduce imaging modalities, a peptide containing a near-infrared dye (sulfo-Cy5), a chelator (DTPA), an azide, and a sortase-recognition motif was synthesized. This peptide and a non-fluorescent intermediate were used for site-specific functionalization of c-terminally sortaggable mouse IgG1 (mIgG1) and Fab anti-PD-L1. To increase the half-life of the Fab fragment, a 20 kDa PEG chain was attached via strain-promoted azide-alkyne cycloaddition (SPAAC). Biodistribution and imaging studies were performed with 111In-labeled constructs in 4T1 tumor-bearing mice. Comparing our site-specific antibody-conjugates with randomly conjugated antibodies, we found that antibody clone, isotype and method of DTPA conjugation did not change tumor uptake. Furthermore, addition of sulfo-Cy5 did not affect the biodistribution. PEGylated Fab fragment displayed a significantly longer half-life compared to unPEGylated Fab and demonstrated the highest overall tumor uptake of all constructs. PD-L1 in tumors was clearly visualized by SPECT/CT, as well as whole body fluorescence imaging. Immunohistochemistry staining of tumor sections demonstrated that PD-L1 co-localized with the fluorescent and autoradiographic signal. Intratumoral localization of the imaging agent could be determined with cellular resolution using fluorescent microscopy.ConclusionsA set of molecularly defined multimodal antibody-based PD-L1 imaging agents were synthesized and validated for multiscale monitoring of PD-L1 expression and localization. Our modular approach for site-specific functionalization could easily be adapted to other targets.Graphical

Multichromatic near-infrared imaging to assess interstitial lymphatic and venous uptake in vivo.

.Significance: Changes in interstitial fluid clearance are implicated in many diseases. Using near-infrared (NIR) imaging with properly sized tracers could enhance our understanding of how venous and lymphatic drainage are involved in disease progression or enhance drug delivery strategies.Aim: We investigated multichromatic NIR imaging with multiple tracers to assess in vivo microvascular clearance kinetics and pathways in different tissue spaces.Approach: We used a chemically inert IR Dye 800CW (D800) to target venous capillaries and a purified conjugate of IR dye 680RD with 40 kDa PEG (P40D680) to target lymphatic capillaries in vivo. Optical imaging settings were validated and tuned in vitro using tissue phantoms. We investigated multichromatic NIR imaging’s utility in two in vivo tissue beds: the mouse tail and rat knee joint. We then tested the ability of the approach to detect interstitial fluid perturbations due to exercise.Results: In an in vitro simulated tissue environment, free dye and PEG mixture allowed for simultaneous detection without interference. In the mouse tail, co-injected NIR tracers cleared from the interstitial space via distinct routes, suggestive of lymphatic and venous uptake mechanisms. In the rat knee, we determined that exercise after injection transiently increased lymphatic drainage as measured by lower normalized intensity immediately after exercise, whereas exercise pre-injection exhibited a transient delay in clearance from the joint.Conclusions: NIR imaging enables simultaneous imaging of lymphatic and venous-mediated fluid clearance with great sensitivity and can be used to measure temporal changes in clearance rates and pathways.

The mode of dexamethasone decoration influences avidin-nucleic-acid-nano-assembly organ biodistribution and in vivo drug persistence

Avidin-Nucleic-Acid-NanoASsemblies (ANANAS) possess natural tropism for the liver and, when loaded with dexamethasone, reduce clinical progression in an autoimmune hepatitis murine model. Here, we investigated the linker chemistry (hydrazide-hydrazone, Hz-Hz, or carbamate hydrazide-hydrazone, Cb-Hz bond) and length (long, 5 kDa PEG, or short, 5-6 carbons) in biotin-dexamethasone conjugates used for nanoparticle decoration through in vitro and in vivo studies. All four newly synthesized conjugates released the drug at acidic pH only. In vitro, the Hz-Hz and the PEG derivatives were less stable than the Cb-Hz and the short chain ones, respectively. Once injected in healthy mice, dexamethasone location in the PEGylated ANANAS outer layer favors liver penetration and resident macrophages uptake, while drug Hz-Hz, but not Cb-Hz, short spacing prolongs drug availability. In conclusion, the tight modulation of ANANAS decoration can significantly influence the host interaction, paving the way for the development of steroid nanoformulations suitable for different pharmacokinetic profiles.

Production of PEGylated GCSF from Non-classical Inclusion Bodies Expressed in Escherichia coli

The recombinant human granulocyte colony stimulating factor conjugated with polyethylene glycol (PEGylated GCSF) has currently been used as an efficient drug for the treatment of neutropenia caused by chemotherapy due to its long circulating half-life. Previous studies showed that Granulocyte Colony Stimulating Factor (GCSF) could be expressed as non-classical Inclusion Bodies (ncIBs), which contained likely correctly folded GCSF inside at low temperature. Therefore, in this study, a simple process was developed to produce PEGylated GCSF from ncIBs. BL21 (DE3)/pET-GCSF cells were cultured in the LiFlus GX 1.5 L bioreactor and the expression of GCSF was induced by adding 0.5 mM IPTG. After 24 hr of fermentation, cells were collected, resuspended, and disrupted. The insoluble fraction was obtained from cell lysates and dissolved in 0.1% N-lauroylsarcosine solution. The presence and structure of dissolved GCSF were verified using SDS-PAGE, Native-PAGE, and RP-HPLC analyses. The dissolved GCSF was directly used for the conjugation with 5 kDa PEG. The PEGylated GCSF was purified using two purification steps, including anion exchange chromatography and gel filtration chromatography. PEGylated GCSF was obtained with high purity (∼97%) and was finally demonstrated as a form containing one GCSF molecule and one 5 kDa PEG molecule (monoPEG-GCSF). These results clearly indicate that the process developed in this study might be a potential and practical approach to produce PEGylated GCSF from ncIBs expressed in Escherichia coli (E. coli).

3D printing of sacrificial thioester elastomers using digital light processing for templating 3D organoid structures in soft biomatrices

Biofabrication allows for the templating of structural features in materials on cellularly-relevant size scales, enabling the generation of tissue-like structures with controlled form and function. This is particularly relevant for growing organoids, where the application of biochemical and biomechanical stimuli can be used to guide the assembly and differentiation of stem cells and form architectures similar to the parent tissue or organ. Recently, ablative laser-scanning techniques was used to create 3D overhang features in collagen hydrogels at size scales of 10–100 µm and supported the crypt-villus architecture in intestinal organoids. As a complementary method, providing advantages for high-throughput patterning, we printed thioester functionalized poly(ethylene glycol) (PEG) elastomers using digital light processing (DLP) and created sacrificial, 3D shapes that could be molded into soft (G′ < 1000 Pa) hydrogel substrates. Specifically, three-arm 1.3 kDa PEG thiol and three-arm 1.6 kDa PEG norbornene, containing internal thioester groups, were photopolymerized to yield degradable elastomers. When incubated in a solution of 300 mM 2-mercaptoethanol (pH 9.0), 1 mm thick 10 mm diameter elastomer discs degraded in <2 h. Using DLP, arrays of features with critical dimensions of 37 ± 4 µm, resolutions of 22 ± 5 µm, and overhang structures as small as 50 µm, were printed on the order of minutes. These sacrificial thioester molds with physiologically relevant features were cast-molded into Matrigel and subsequently degraded to create patterned void spaces with high fidelity. Intestinal stem cells (ISCs) cultured on the patterned Matrigel matrices formed confluent monolayers that conformed to the underlying pattern. DLP printed sacrificial thioester elastomer constructs provide a robust and rapid method to fabricate arrays of 3D organoid-sized features in soft tissue culture substrates and should enable investigations into the effect of epithelial geometry and spacing on the growth and differentiation of ISCs.

Molecular Design and Characterization of a Novel, Vwf Independent Coagulation Factor VIII (LAFATE)

We have known for long that most FVIII is carried by vWF in the blood stream, and began to aware that vWF determines the fate of FVIII whatever modification we make on it for its prolonged circulation. Thus, the consideration that vWF binding property of FVIII is to be conserved is now facing change.The aim of this study is to find out a way to unbind FVIII from endogenous vWF but to stabilize it to generate a novel longer-acting coagulation FVIII. We took a way to shield three major LRP1 binding sites by conjugating PEG and at the same time vWF binding sites by connecting D'D3 domains, the FVIII binding domains of vWF, or its variants using a flexible amino acid linker. We constructed a stable single chain form of FVIII (scFVIII) and employed it as a basic FVIII backbone structure for doing so.We generated FVIII variants according to this strategy, and evaluated their pharmacokinetic property in hemophilia A mice. Unmodified rFVIII (Advate®) was used as the reference. The mice were intravenously injected with the reference and test articles at a dose of 125 IU/kg rFVIII, respectively, and blood was sampled 5 min to 72 h or further after administration. Citrated plasma was prepared and analyzed for FVIII activity (Coamatic®, Chromogenix). PK parameters were determined using non-compartmental modeling with Phoenix WinNonlin 6.4 (Pharsight).We first generated three different types of FVIII variants containing vWF D'D3 domains (D'D3 monomer, D'D3 dimer, D'D3-Fc dimer linked to C-terminus of C2 domain or N-terminus of A1 domain of FVIII), and then conjugated a 40kDa PEG to the B-domain of each variant site-specifically. In the subsequent pharmacokinetic study in hemophilia A mice, we observed significant prolongation of half-lives from 1.8- to 3.6-fold comparing with Advate®. The D'D3 monomer linked FVIII exhibited most prolonged half-life, suggesting that a D'D3 monomer is active enough to substantially attenuate binding of endogenous vWF to the chimera.Recently published electron microscopy and hydrogen-deuterium exchange analyses established the C1 domain as the major binding site for the vWF D'D3 domains on FVIII, and noted additional sites located within the a3 acidic peptide and the A3 and C2 domains of FVIII as being implicated in the FVIII-vWF interaction. In the following experiment, we aimed to find out optimal length of the amino acids linker that would secure easy access of D'D3 domains to A3 and C2 domains of FVIII. Considering the distance between these two domains measurable from the structural information of FVIII, we generated D'D3 monomer connected with 60, 120 and 300 amino acids linkers to scFVIII, respectively, PEGylated them and evaluated their pharmacokinetic properties in hemophilia A mice again. These molecules showed 2.9-, 3.5-, and 2.5-fold more prolonged half-lives than Advate®, respectively, and the 120 amino acids linker seemed optimal.Finally we conjugated several PEGs of different type and size, respectively, to the FVIII-D'D3 connected with this optimal linker, and evaluated their PK properties in hemophilia A mice in the next experiment. Consequently, we identified four different PEGs that support best performance, and named these compounds as LAFATE. LAFATE corrected impaired thrombin generation of the FVIII deficient plasma in a concentration-dependent manner, and stopped acute bleeding as effectively as Advate®, evaluated by the amount of hemoglobin loss for a given time period in a tail-clip bleeding model of hemophilia A mice. LAFATE has a good potential to bring in possible administration of a hemophilia A drug on weekly bases and therefore, warrants further investigation.Related Articles1. The tertiary structure and domain organization of coagulation factor VIII. Betty W. Shen et al., Blood, 20082. A von Willebrand factor fragment containing the D'D3 domains is sufficient to stabilize coagulation factor VIII in mice. Andrew Yee et al., Blood, 20143. von Willebrand factor biosynthesis, secretion, and clearance: connecting the far ends. Peter J. Lenting et al., Blood, 20154. Mapping the interaction between factor VIII and von Willebrand factor by electron microscopy and mass spectrometry. Po-Lin Chiu et al., Blood, 20155. Life in the shadow of a dominant partner: the FVIII-VWF association and its clinical implications for hemophilia A. Steven W. Pipe et al., Blood, 2016 DisclosuresJo:Mogam Institute for Biomedical Research: Employment. Oh:Mogam Institute for Biomedical Research: Employment. Lee:Mogam Institute for Biomedical Research: Employment. Oh:Green Cross Corporation: Employment. Ryu:Mogam Institute for Biomedical Research: Employment. Kim:Green Cross Corporation: Employment, Equity Ownership. Park:Green Cross Corporation: Employment. Kim:Green Cross Corporation: Employment. Jung:Green Cross Corporation: Employment, Equity Ownership.

PEG Linker Improves Antitumor Efficacy and Safety of Affibody-Based Drug Conjugates.

Antibody drug conjugates (ADCs) have become an important modality of clinical cancer treatment. However, traditional ADCs have some limitations, such as reduced permeability in solid tumors due to the high molecular weight of monoclonal antibodies, difficulty in preparation and heterogeneity of products due to the high drug/antibody ratio (4–8 small molecules per antibody). Miniaturized ADCs may be a potential solution, although their short circulation half-life may lead to new problems. In this study, we propose a novel design strategy for miniaturized ADCs in which drug molecules and small ligand proteins are site-specifically coupled via a bifunctional poly(ethylene glycol) (PEG) chain. The results showed that the inserted PEG chains significantly prolonged the circulation half-life but also obviously reduced the cytotoxicity of the conjugates. Compared with the conjugate ZHER2-SMCC-MMAE (HM), which has no PEG insertion, ZHER2-PEG4K-MMAE (HP4KM) and ZHER2-PEG10K-MMAE (HP10KM) with 4 or 10 kDa PEG insertions have 2.5- and 11.2-fold half-life extensions and 4.5- and 22-fold in vitro cytotoxicity reductions, respectively. The combined effect leads to HP10KM having the most ideal tumor therapeutic ability at the same dosages in the animal model, and its off-target toxicity was also reduced by more than 4 times compared with that of HM. These results may indicate that prolonging the half-life is very helpful in improving the therapeutic capacity of miniaturized ADCs. In the future, the design of better strategies that can prolong half-life without affecting cytotoxicity may be useful for further improving the therapeutic potential of these molecules.

Development of Site-Specific PEGylated Granulocyte Colony Stimulating Factor With Prolonged Biological Activity.

Currently, amino-terminal PEGylated human granulocyte colony stimulating factor (huG-CSF) is used to prevent and treat neutropenia. Although huG-CSF has been used as a drug for more than 20 years, it has three significant drawbacks: (i) it relies on PEG aldehyde for PEGylation of the alpha-amino group of the first amino acid, and this leads to non-specific PEGylation of the epsilon amino group of lysine residues within the G-CSF; (ii) longer-acting G-CSF variants are desirable to reduce the risk of chemotherapy-associated neutropenia; and (iii) G-CSF cannot be administered on the day of chemotherapy. In an attempt to overcome the above drawbacks, we engineered cysteine variants of G-CSF to facilitate the maleimide PEG-based site-specific PEGylation that leads to a highly homogenous PEGylated product. Importantly, we have demonstrated that 20 kDa thiol-reactive PEG conjugated by maleimide chemistry to the Cys2 G-CSF variant exhibits leukocyte proliferative activity similar to that of the commercially available G-CSF conjugated with aldehyde PEG in a neutropenia mice model. Moreover, we have demonstrated that PEGylation of the cysteine variant of huG-CSF with higher molecular weight PEGs, such as 30 kDa PEG and 40 kDa PEG, leads to significantly prolonged leukocyte proliferation activity compared to the variant conjugated with 20 kDa PEG. Importantly, even a half-dose of the engineered variant conjugated with 40 kDa PEG exhibited significantly longer biological activity than the commercially available 20 kDa PEGylated huG-CSF. Finally, we have demonstrated that administration of the engineered variant conjugated with 40 kDa PEG on the day of administration of cyclophosphamide for inducing neutropenia in mice can alleviate neutropenia through leukocyte proliferation. In summary, this study provides the design of site-specific PEGylated huG-CSF variants with improved therapeutic potential. It opens the possibility of long-acting and same-day prophylactic administration of G-CSF after chemotherapy drug regimens. These results may pave the way for the development of potential G-CSF derivatives possessing longer half-lives and favorable clinical attributes.

Improvement of Certolizumab Fab\u2032 properties by PASylation technology

Certolizumab pegol is a Fab′ antibody fragment for treatment of rheumatoid arthritis and Crohn’s disease which is conjugated to a 40 kDa PEG molecule in order to increase the protein half-life. PEGylation may have disadvantages including immunogenicity, hypersensitivity, vacuolation, decreased binding affinity and biological activity of the protein. To overcome these problems, PASylation has been developed as a new approach. The nucleotide sequence encoding 400 amino acid PAS residues was genetically fused to the corresponding nucleotide sequences of both chains of certolizumab. Then, the bioactivity as well as physicochemical and pharmacokinetic properties of the recombinant PASylated expressed protein was assayed. Circular dichroism spectroscopy demonstrated that the random coil structure of PAS sequences did not change the secondary structure of the PASylated Fab′ molecule. It was observed that PASylation influenced the properties of the Fab′ molecule by which the hydrodynamic radius and neutralization activity were increased. Also, the antigen binding and binding kinetic parameters improved in comparison to the PEGylated Fab′ antibody. Pharmacokinetic studies also showed prolonged terminal half-life and improved pharmacokinetic parameters in PASylated recombinant protein in comparison to the PEGylated and Fab′ control molecules. The results reconfirmed the efficiency of PASylation approach as a potential alternative method in increasing the half-life of pharmaceutical proteins.