HYBRID TUMOR PENETRATING FERRITIN NANOCAGES AS A HYPOXIA TARGETING NANOCARRIER FOR CANCER TREATMENT

Background: Tumor hypoxia plays a key role in promoting tumor progression and metastasis, and thus serves as a critical therapeutic target. However, hypoxic regions within solid tumors are hard to be reached by systemically administered therapeutics due to the poor vascularization and tumor extracellular matrix (ECM) barrier. In reality, hypoxia-tropic drug delivery platforms have been lacking. Human ferritin nanocage (FTn) possesses intrinsic affinity to human transferrin receptor 1 (TfR1) and rodent T-cell immunoglobulin mucin 2 (TIM-2) which are upregulated in hypoxic tumor cells, and thus FTn can be potentially used to deliver therapeutic payloads to tumor hypoxia. Methods: Protein disassembly and reassembly process was used to engineer hybrid FTn that possessed varying degrees of surface polyethylene glycol (PEG) coatings, while retaining the TfR1/TIM-2 target ability. The resulting hybrid PEG-FTn were characterized by transmission electron microscopy, size exclusion chromatography and dynamic light scattering. The tissue penetration and cellular uptake of hybrid PEG-FTn were evaluated and compared using 3D multicellular spheroids in vitro or flank tumor-bearing mice in vivo, both based on 3LL tumor cells. A hypoxia-inducible factor 1 inhibitor, acriflavine (AF), was loaded into the nanocages as a model drug and the pharmacokinetic profile was compared with free AF. Results: Hybrid PEG-FTn with varying PEGylation degrees were synthesized and extensively screened based on physicochemical properties and in vitro behaviors. We identified a lead hybrid PEG-FTn of which surface PEG modification promoted penetration of the nanocages through 3LL-based tumor tissues both in vitro and in vivo without compromising their ability to accumulate in tumor hypoxia via TfR1/TIM-2. The lead hybrid PEG-FTn, following systemic administration, provided prolonged circulation and enhanced accumulation of the payloads (i.e. AF) compared to the identically administered, dose-matched free AF. Conclusion: The hybrid PEG-FTn is an excellent drug delivery system for the delivery of therapeutics, particularly hypoxia-modulating agents, to hypoxic regions within solid tumors

HYBRID TUMOR PENETRATING FERRITIN NANOCAGES AS A HYPOXIA TARGETING NANOCARRIER FOR CANCER TREATMENT

Background: Tumor hypoxia plays a key role in promoting tumor progression and metastasis, and thus serves as a critical therapeutic target. However, hypoxic regions within solid tumors are hard to be reached by systemically administered therapeutics due to the poor vascularization and tumor extracellular matrix (ECM) barrier. In reality, hypoxia-tropic drug delivery platforms have been lacking. Human ferritin nanocage (FTn) possesses intrinsic affinity to human transferrin receptor 1 (TfR1) and rodent T-cell immunoglobulin mucin 2 (TIM-2) which are upregulated in hypoxic tumor cells, and thus FTn can be potentially used to deliver therapeutic payloads to tumor hypoxia. Methods: Protein disassembly and reassembly process was used to engineer hybrid FTn that possessed varying degrees of surface polyethylene glycol (PEG) coatings, while retaining the TfR1/TIM-2 target ability. The resulting hybrid PEG-FTn were characterized by transmission electron microscopy, size exclusion chromatography and dynamic light scattering. The tissue penetration and cellular uptake of hybrid PEG-FTn were evaluated and compared using 3D multicellular spheroids in vitro or flank tumor-bearing mice in vivo, both based on 3LL tumor cells. A hypoxia-inducible factor 1 inhibitor, acriflavine (AF), was loaded into the nanocages as a model drug and the pharmacokinetic profile was compared with free AF. Results: Hybrid PEG-FTn with varying PEGylation degrees were synthesized and extensively screened based on physicochemical properties and in vitro behaviors. We identified a lead hybrid PEG-FTn of which surface PEG modification promoted penetration of the nanocages through 3LL-based tumor tissues both in vitro and in vivo without compromising their ability to accumulate in tumor hypoxia via TfR1/TIM-2. The lead hybrid PEG-FTn, following systemic administration, provided prolonged circulation and enhanced accumulation of the payloads (i.e. AF) compared to the identically administered, dose-matched free AF. Conclusion: The hybrid PEG-FTn is an excellent drug delivery system for the delivery of therapeutics, particularly hypoxia-modulating agents, to hypoxic regions within solid tumors

De novo design of protein binders as functional therapeutics

Thesis (Ph.D.)--University of Washington, 2023De novo design of protein binding proteins (minibinders) with target structure information alone remains a grand challenge. A general computational design framework includes (1) generation of binder backbones, (2) sequence design and side-chain refinement, (3) resampling, and (4) prediction of binding and evaluation of the minibinders as a monomer. In Chapter 1, I review the improved computational minibinder design method I have contributed to develop. With these cutting-edge pipelines, I describe two strategies of applying designed minibinders as novel functional therapeutics: in Chapter 2, I report the design of minibinder antagonists as immune modulator for cytokine storm; in Chapter 3, I report the design of endocytosis ligands for target degradation and signaling amplification. Overall, the minibinder is a brand-new drug modality/platform with advantages of ultra-stability, high-specificity, robust production, and modularity. The work described indicate the great potential of the minibinder to bridge the gap of existing therapeutics and revolutionize the future of protein drug development

Improving de novo protein binder design with deep learning

Abstract Recently it has become possible to de novo design high affinity protein binding proteins from target structural information alone. There is, however, considerable room for improvement as the overall design success rate is low. Here, we explore the augmentation of energy-based protein binder design using deep learning. We find that using AlphaFold2 or RoseTTAFold to assess the probability that a designed sequence adopts the designed monomer structure, and the probability that this structure binds the target as designed, increases design success rates nearly 10-fold. We find further that sequence design using ProteinMPNN rather than Rosetta considerably increases computational efficiency

Stretchable, dynamic covalent polymers for soft, long-lived bioresorbable electronic stimulators designed to facilitate neuromuscular regeneration.

Bioresorbable electronic stimulators are of rapidly growing interest as unusual therapeutic platforms, i.e., bioelectronic medicines, for treating disease states, accelerating wound healing processes and eliminating infections. Here, we present advanced materials that support operation in these systems over clinically relevant timeframes, ultimately bioresorbing harmlessly to benign products without residues, to eliminate the need for surgical extraction. Our findings overcome key challenges of bioresorbable electronic devices by realizing lifetimes that match clinical needs. The devices exploit a bioresorbable dynamic covalent polymer that facilitates tight bonding to itself and other surfaces, as a soft, elastic substrate and encapsulation coating for wireless electronic components. We describe the underlying features and chemical design considerations for this polymer, and the biocompatibility of its constituent materials. In devices with optimized, wireless designs, these polymers enable stable, long-lived operation as distal stimulators in a rat model of peripheral nerve injuries, thereby demonstrating the potential of programmable long-term electrical stimulation for maintaining muscle receptivity and enhancing functional recovery

Design of protein-binding proteins from the target structure alone

The design of proteinsthat bind to a specific site on the surface of a target protein using no information other than the three-dimensional structure of the target remains a challenge' 5 . Here we describe a general solution to this problem that starts with a broad exploration of the vast space of possible binding modes to a selected region of a protein surface, and then intensifiesthe search in the vicinity of the most promising binding modes. We demonstrate the broad applicability of this approach through the de novo design of binding proteins to 12 diverse protein targets with different shapes and surface properties. Biophysical characterization showsthat the binders, which are all smaller than 65 amino acids, are hyperstable and, following experimental optimization, bind their targets with nanomolar to picomolar affinities. We succeeded in solving crystal structures of five of the binder-target complexes, and all five closely match the corresponding computational design models. Experimental data on nearly half a million computational designs and hundreds of thousands of point mutants provide detailed feedback on the strengths and limitations of the method and of our current understanding of protein-protein interactions, and should guide improvements of both. Our approach enables the targeted design of bindersto sites of interest on a wide variety of proteins for therapeutic and diagnostic applications

Designed endocytosis-inducing proteins degrade targets and amplify signals.

Endocytosis and lysosomal trafficking of cell surface receptors can be triggered by endogenous ligands. Therapeutic approaches such as lysosome-targeting chimaeras1,2 (LYTACs) and cytokine receptor-targeting chimeras3 (KineTACs) have used this to target specific proteins for degradation by fusing modified native ligands to target binding proteins. Although powerful, these approaches can be limited by competition with native ligands and requirements for chemical modification that limit genetic encodability and can complicate manufacturing, and, more generally, there may be no native ligands that stimulate endocytosis through a given receptor. Here we describe computational design approaches for endocytosis-triggering binding proteins (EndoTags) that overcome these challenges. We present EndoTags for insulin-like growth factor 2 receptor (IGF2R) and asialoglycoprotein receptor (ASGPR), sortilin and transferrin receptors, and show that fusing these tags to soluble or transmembrane target protein binders leads to lysosomal trafficking and target degradation. As these receptors have different tissue distributions, the different EndoTags could enable targeting of degradation to different tissues. EndoTag fusion to a PD-L1 antibody considerably increases efficacy in a mouse tumour model compared to antibody alone. The modularity and genetic encodability of EndoTags enables AND gate control for higher-specificity targeted degradation, and the localized secretion of degraders from engineered cells. By promoting endocytosis, EndoTag fusion increases signalling through an engineered ligand-receptor system by nearly 100-fold. EndoTags have considerable therapeutic potential as targeted degradation inducers, signalling activators for endocytosis-dependent pathways, and cellular uptake inducers for targeted antibody-drug and antibody-RNA conjugates