Browsing by Author "Chilkoti, Ashutosh"

The primary barriers to multiplexed point of care immunoassays are: (1) cost; (2) response time; and (3) sample handling. Described here is a self-contained, multiplexed immunoassay platform for point of care detection that leverages a number of enabling technologies to address these barriers. This platform is referred to as the "D4" assay, as it is composed of the following four sequential, concerted events (Figure 1): (1) Dispense (droplet of blood); (2) Dissolve (printed reagents on chip); (3) Diffuse across surface; (4) Detect binding event.

The D4 assay process begins when a finger-stick is administered and the resulting droplet of blood is applied to the surface of a detector chip. Hydrophobic ink printed onto the surface of the chip confines the blood droplet to a non-fouling region containing soluble, labile spots of detection antibodies and insoluble, non-labile spots of capture antibodies. As the soluble detection antibodies are dissolved from their printed spots by the droplet of blood, three serial events occur to generate signal (Figure 2): (1) the first half of the detection complex is formed by the binding of analytes present in blood to the stable capture agent spots; (2) diffusion of the blood laterally through the polymer brush, resulting in the dissolution and diffusion of soluble detection antibody spots; (3) solubilized detection antibodies bind to their respective analyte-capture agent spots, completing the detection complex and resulting in signal generation at the position of the non-labile capture antibody spots.

This assay relies upon the ability of labeled detection antibodies, printed into a nonfouling brush as "labile spots", to be carried by blood flow to adjacent rows of stably immobilized capture antibodies by diffusion of the analyte solution (Figure 2). Generation of signal at a given capture spot location provides identification of individual analytes (positives). Quantification of the concentration of the different analytes is carried out identically to a conventional fluorescence immunoassay by pre-calibration of the system using a dilution series of the analyte spiked into whole blood.

The D4 assay addresses several critical needs in point of care testing as follows: First, the cost of testing is reduced through miniaturization, multiplexing and one-step, on-site processing of undiluted whole blood obtained from a finger stick. Second, in order to simplify the immunoassay process, the D4 relies on diffusion to bring spatially localized reagents together to create a functional assay and thereby eliminate the need for liquid transfer steps, microfluidic manipulation of sample or reagents, and wash steps. Third, this multiplexed platform is capable of screening for a panel of markers in a single drop of blood with no sample preprocessing. Fourth, the assay is fast, which alleviates the difficulties often associated with communicating the outcome of diagnostic tests. A prototype of the D4 assay is shown in Figure 3 below.

Laboratory enabled disease diagnosis is one of the cornerstones in patient care and greatly relies in immunological techniques to detect pathogens and quantify biomarkers related to a myriad of clinical conditions. The performance of immunoassays is directly dependent on the binding affinity of the molecules that enable capture and detection of analytical targets of interest and especially on the surfaces that interact with these sensing molecules and the complex elements present in biological samples. The lack of high-quality antibodies and nonspecific protein absorption (NSPA) on test substrates have historically hindered assay sensitivity and overall performance. While antibody development has witnessed a significant evolution over time, mostly driven by the interest in antibody-based drugs, major challenges still exist in the IVD reagent generation process. Regarding NSPA, only recently protein resistant surfaces found their way into immunoassay development applications with extremely promising results.

In this dissertation, we aimed to better understand the surface properties required to deliver a new generation of immunodiagnostics with high sensitivity and broad dynamic range. Our studies demonstrated that there are very specific physicochemical requirements a surface must present to enable inkjet based, simple fabrication of antigen detection tests. Interestingly, we determined that POEGMA based brushes, our prototypical surface, naturally presents a fine balance between protein resistance and hydrophilicity that enables their non-covalent biofunctionalization and use for IVD applications. Once we confirmed POEGMA as the ideal coating for the fabrication of antigen detection tests, our work evolved to address several of the challenges related to antibody generation for diagnostic test development. Towards this end, our work entailed the development of a new antibody pair that targets non-overlapping epitopes of ebolavirus secreted glycoprotein, a truncated version of the structural glycoprotein that is actively secreted from infected cells in early stages of the infection. The generation of these antibodies was achieved by associating scFv phage-display technology with the transient expression of promising scFv candidates as Fc fusions in mammalian cells followed by their seamless purification with an IsoTag based chromatography-free system. These elements when combined with a novel antibody pairing strategy, that leverages the D4 Assay’s multiplexing capabilities and ease of fabrication, warranted the rapid identification of optimal capture and detection reagents. Finally, employing these reagents, we developed and validated an ultra-sensitive ebolavirus detection test based on the D4 Assay test format, which was able to not only match but outperform the sensitivity of qRT-PCR. This exceptional sensitivity which can enable the deployment of life-saving treatment and containment efforts was demonstrated in two independent nonhuman primate models of the disease and attests to the success of this new IVD test development work-flow.

Serological testing—detection of antibodies—plays a key role in the diagnosis, management, and surveillance of infectious diseases. Serological assays can detect both active and past infections which is essential in understanding epidemiological variables such as incidence and fatality rates. Another important metric, antibody avidity, provides additional insight into recency of infection, can be used to discriminate between closely related infectious species, assess vaccine efficacy and provides estimates of who is and who is not immune to certain infections. However, conventional methods of measuring antibody avidity are costly, time consuming, and utilize harsh denaturing reagents that negatively impact automated immuno-ELISA equipment. These challenges have deterred the development of point of care tests for antibody avidity. In this thesis, we investigated the performance of four assay formats for antibody detection developed by inkjet-printing assay reagents on glass surfaces coated with a non-fouling polymer brush. We then adopted the antibody detection formats to determine antibody avidity by measuring resistance of the antibody-antigen bonds to chaotropic agents. We further developed a new technique of measuring antibody avidity by reducing the concentration of capture antigen (cAg) on the immunoassay platform. In this new technique, avidity index was determined as the ratio of fluorescence intensity measured at a lower cAg concentration to intensity measured at a higher cAg concentration. This technique showed strong correlation (R > 0.8) with the conventional method of antibody avidity measurement (resistance to chaotropic agent) in three antibody-antigen systems. Additionally, we showed that the proposed platform can detect key biomarkers for identifying recent HIV1 infections. The targeted biomarkers were based on measuring titers and avidity of antibodies secreted against specific clades of HIV envelope proteins. They included clade C GP140 IgG3, transmitted/founder clade C GP140 IgG4 avidity, clade B GP140 IgG4 avidity, and GP41 immunodominant region (GP41-ID) IgG avidity. The proposed assay detected all four biomarkers with wide dynamic ranges (>103.6) and high sensitivity in diluted pooled human serum. The proposed platform for antibody avidity testing is rapid, easy to use and has high correlation with chaotropic resistance. It therefore has potential to enable measurement of antibody avidity at the point of care for clinical applications.

Detection and quantification of biomarkers directly from complex clinical specimens is desired and often required by healthcare professionals for the effective diagnosis and screening of disease, and for general patient care. Current methodologies to accomplish this task have critical shortcomings. Laboratory immunoassays, most notably enzyme-linked immunosorbent assay (ELISA) require extensive clinical infrastructure and complex user intervention steps to generate results and often are accompanied by a lengthy time-to-result. Conversely, available point-of-care (POC) diagnostic solutions, most notably available lateral flow immunoassays (LFIAs), often struggle with sensitivity and specificity in complex fluids, lack quantitative output and are not easily multiplexed. In this dissertation I will discuss the design, fabrication, testing, and refinement of an all-in-one fluorescence microarray integrated into a passive microfluidic fluid handling system to create a versatile and automated POC platform that can detect biomarkers from complex samples for disease management with the relative ease-of-use of an LFIA and the performance of a laboratory-grade test. The platform is driven by capillary and gravitational forces and automates all intervention steps after the addition of the sample and running buffer at the start of testing. The microfluidic cassette is built on a (poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA) polymer brush which imparts two key functionalities, (1) it eliminates cellular and protein binding, and when combined with the vertical orientation of the microfluidic cassette prevents settling of debris during all assay steps. This allows for impressive sensitivities and specificities to be obtained from samples as complex as undiluted whole blood even when relying on gentle capillary and hydrostatic pressures for cassette operation. (2) Paradoxically, printed biorecognition elements can be stably and non-covalently immobilized into the POEGMA allowing for all reagents needed to conduct a sandwich immunoassay in a single step to be easily inkjet printed as spatially discrete spots into the POEGMA brush, which also stabilizes them at room temperature. Additionally, the microfluidic cassette is compatible with the “D4Scope” a handheld fluorescence detector that can quantify the output of the microfluidic cassette in seconds at the POC and is the only piece of auxiliary equipment required to operate the test.

This dissertation discusses early cassette prototypes and characterizes the performance of major device iterations (Chapter 2) before moving into three clinical applications of the cassette. First, a multiplexed serological test to detect antibodies against different proteins of the SARS-CoV-2 virus was developed (Chapter 3). Second, a multiplexed COVID-19 diagnostic test that simultaneously differentiates which variant you are infected with was developed (Chapter 4). Third, a sensitive fungal infection test for the diagnosis of talaromycosis was developed (Chapter 5). Finally, a rapidly iterative yet highly scalable injection molding fabrication process flow was created and characterized to improve performance and translatability of the cassette (Chapter 6).

Bioanalytical techniques such as immunoassays are ubiquitous in clinical and basic research laboratories and have transformed how we diagnose patients, monitor health, and study disease. Immunoassays typically use capture reagents, such as antibodies or antigens, to detect and quantify a biomarker of interest from a clinical specimen based on highly sensitive and specific binding interactions. While laboratory-based assays, such as enzyme-linked immunosorbent assay (ELISA), are the workhorses of clinical laboratories, they have several shortcomings that limit their overall utility, especially in low resource settings. Of note, ELISA requires multiple timed incubation steps, trained personnel, expensive equipment, and suffers from long times to result. To democratize access to clinical-grade tests, researchers have sought out different methods for point-of-care (POC) testing that are easy to perform and maintain high sensitivity and specificity. This dissertation describes the use of a “zero-background” polymer coating—poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA)—as a substrate for highly sensitive and specific POC diagnostic tests. The POEGMA coating eliminates nearly all non-specific protein adsorption and cellular adhesion, thus leading to high signal-to-noise ratios, even from complex biological samples, such as whole blood. In addition, the POEGMA brush contains all biomolecules necessary to complete an assay after addition of a liquid sample, thus allowing assays to be conducted in a single step. Further, the POEGMA coating stabilizes biomolecules on the surface, which allows tests to be stored at ambient conditions without refrigeration. Assays are read out using a fluorescence detector which quantifies the concentration for a given biomarker of interest. By inkjet printing capture biomolecules at discrete spatial addresses on the POEGMA-slides, multiplexing can be accomplished using a single fluorophore which greatly reduces the complexity and costs for assay readout. This dissertation focuses on adapting and applying this platform to several clinically relevant applications. First, we developed a test for molecular and cellular credentialing of breast cancer tissue at the POC (Chapter 2). With the onset of the coronavirus 2019 (COVID-19) pandemic, we adapted the platform to detect several different relevant biomarkers for COVID-19, including total antibody concentration against several viral proteins (Chapter 3), neutralizing antibodies (Chapter 4), and viral proteins (Chapter 5). All the tests developed for COVID-19 use multiplexed sensing strategies and can be conducted with minimal/no user intervention or clinical infrastructure. Taken together, these studies highlight the great potential for bioanalytical assays built upon POEGMA-coated substrates to be used for clinical applications in disease diagnosis, surveillance, and management.

Antivascular targeting is a promising strategy for tumor therapy. This strategy overcomes many of the transport barriers and has shown efficacy in many preclinical models, but targeting epitopes on tumor vasculature can also promote accumulation in healthy tissues. We used Elastin-like Polypeptide (ELP) to form block copolymers (BCs) consisting of two separate ELP blocks seamlessly fused at the genetic level. ELPBCs self-assemble into spherical micelles at a critical micelle temperature (CMT), allowing external control over monovalent unimer and multivalent micelle forms. We hypothesized that thermal self-assembly could trigger specific binding of ligand-ELPBC to target receptors via the multivalency effect as a method to spatially restrict high-avidity interactions. We termed this approach Dynamic Affinity Modulation (DAM). The objectives of this study were to design, identify, and evaluate protein-based drug carriers that specifically bind to target receptors through static or dynamic multivalent ligand presentation.

ELPBCs were modified to include a low-affinity GRGDS or GNGRG ligand and a unique conjugation site for hydrophobic compounds. This addition did not disrupt micelle self-assembly and facilitated thermally-controlled multivalency. The ability of ligand-ELPBC to specifically interact with isolated AvB3 or CD13 was tested using an in vitro binding assay incorporating an engineered cell line. RGD-ELPBC promoted specific receptor binding in response to multivalent presentation but NGR-ELPBC did not. Enhanced binding with multivalent presentation was also observed only with constructs exhibiting CMT < body temperature. This study establishes proof-of-principle of DAM, but ELPBC requires thermal optimization for use with applied hyperthermia. Static affinity targeting of fluorescent ligand-ELPBC was then analyzed in vivo using intravital microscopy (IM), immunohistochemistry (IHC), and custom image processing algorithms. IM showed increased accumulation of NGR-ELPBC in tumor tissue relative to normal tissue while RGD-ELPBC and non-ligand ELPBC did not, and IHC verified these observations. This study shows (1) multivalent NGR presentation is suitable for static multivalent targeting of tumors and tumor vasculature, (2) multivalent RGD presentation may be suitable for DAM with thermal optimization, and (3) ELPBC micelles may selectively target proteins at the tumor margin.

Phase separation of macromolecules is a critical phenomenon for the human condition. This phenomenon has also been exploited for biotechnological development to improve human morbidity and mortality. However, there is still much more to learn regarding how this behavior is encoded within a protein sequence. Thus, this thesis seeks to 1) further explore the sequence space to understand how phase separation is encoded, with an emphasis on polypeptides with upper critical solution temperature (UCST) transitions and 2) use this phase separation to control availability of macromolecules at various length scales.

Using traditional molecular biology techniques, we will recombinantly express and purify a large number of polypeptides with variable sequence composition and sequence architecture. Then, using traditional polymer science and material science techniques combined with microscopic techniques that span the macro-scale and nano-scale, we will characterize their phase separation behavior and the interaction of these materials with biological systems.

We developed a practical mutation strategy that allows for complete control of the UCST binodal line in physiologic conditions that is useful for de novo design of artificial IDPs with UCST phase behavior. We evaluated the interaction of these polypeptides and their phase separation in the presence of bacterial, eukaryotic cells and in mice demonstrating how this binodal line fused to biological active partners can control biologic functions.

In bacteria, we made artificial phase separated puncta, akin to naturally occurring phase separated droplets, that have non-canonical function, demonstrating how primary features of the polypeptide chain affect enzymatic function. We created block co-polypeptides comprised of UCST and LCST protein sequences that exhibit remarkably tunable and robust nanoscale self-assembly into spherical micelles, worm-like micelles and vesicular structures capable of displaying large targeting domains on their surface. In the presence of eukaryotic cells, these nanomaterials can dramatically increase polypeptide uptake, increasing the avidity of the targeting molecule by over 1000-fold.

Finally, we demonstrated that phase separated polypeptides can sequester an active peptide GLP-1 from systemic circulation, controlling the peptide’s bioactivity through control of the phase diagram. Taken together, we demonstrate the universal power of the phase diagram, across many length scales, where the transducing agent for controlling biological activity is an engineered, repetitive polypeptide sequence.

Traditional small molecule chemotherapeutics show limited effectiveness in the clinic as their poor pharmacokinetics lead to rapid clearance from circulation and their exposure to off-target tissues results in dose-limiting toxicity. The objective of this dissertation is to exploit a class of recombinant chimeric polypeptides (CPs) to actively target drugs to tumors as conjugation to macromolecular carriers has demonstrated improved efficacy by increasing plasma retention time, reducing uptake by healthy tissues, and enhancing tumor accumulation by exploiting the leaky vasculature and impaired lymphatic drainage characteristic of solid tumors. CPs consist of two principal components: (1) a thermally responsive elastin-like polypeptide (ELP) that displays a soluble-to-aggregate phase transition above a characteristic transition temperature (Tt); and (2) a cysteine-rich peptide fused to one end of the ELP to which small molecule therapeutics can be covalently attached (the conjugation domain). This work describes the development of CP drug-loaded nanoparticles that can be targeted to solid tumors by the external application of mild regional hyperthermia (39-43°C).

Highly repetitive ELP polymers were assembled by Plasmid Reconstruction Recursive Directional Ligation (PRe-RDL), in which two halves of a parent plasmid, each containing a copy of an oligomer, were ligated together to dimerize the oligomer and reconstitute the functional plasmid. Chimeric polypeptides were constructed by fusing the ELP sequence to a (CGG)8 conjugation domain, expressed in Escherichia coli, and loaded with small molecule hydrophobes through site specific attachment to the conjugation domain. Drug attachment induced the assembly of nanoparticles that retained the thermal responsiveness of the parent ELP in that they experienced a phase transition from soluble nanoparticles to an aggregated phase above their Tt. Importantly, the Tt of these nanoparticles was near-independent of the CP concentration and the structure of the conjugated molecule as long as it displayed an octanol-water distribution coefficient (LogD) > 1.5.

A series of CP nanoparticles with varying ratios of alanine and valine in the guest residue position was used to develop a quantitative model that described the CP transition temperature in terms of three variables - sequence, chain length, and concentration - and the model was used to identify CPs of varying molecular weights that displayed transition temperatures between 39°C and 43°C. A murine dorsal skin fold window chamber model using a human tumor xenograft was used to validate that only the thermoresponsive CP nanoparticles (and not the controls) exhibited a micelle-to-aggregate phase transition between 39-43°C in vivo. Furthermore, quantitative analysis of the biodistribution profile demonstrated that accumulation of these thermoresponsive CP nanoparticles was significantly enhanced by applying heat in a cyclical manner. It is hoped that this work will provide a helpful resource for the use of thermoresponsive CP nanoparticles in a variety of biomedical applications.

Recombinant technology has given us the powerful ability to imagine and create novel biological entities, from potent therapeutics to functionally active materials. By harnessing nature’s building blocks and reconfiguring these components, recombinant engineering unlocks the potential to tailor drug specificity and pharmacokinetics, rationally design biomaterials, understand and define protein structure, and probe cellular function with molecular precision. These technological feats are made possible with a few simple biological ingredients: nucleotides, sugars, and amino acids. These components, exquisitely crafted by evolution, are individually combined in useful ratios and precise sequences in living systems to synthesize DNA, RNA, polysaccharides, and proteins. These macromolecules collectively support organismal structure and function and give rise to the incredible diversity in Charles Darwin’s “great tree” of life. However, the seemingly infinite potential for new materials built from these components is, in fact, limited. The chemical identity of these building blocks – with a particular focus herein on the twenty naturally-occurring amino acids – limits the scope and functionality of the recombinant materials we can produce. In order to functionalize these products, to fundamentally change their chemical identity while preserving their biological functionality, we require the finesse of bioorthogonal chemistries and modification techniques.

Bioorthogonal reactions modify biological materials within living systems without perturbing function, much as two orthogonal lines can extend in different directions and intersect only at a single point. That point of intersection can be precisely defined through recombinant technology and gives us access to new classes of biomaterials. The term “bioorthogonal”, coined by Carolyn Bertozzi, importantly defines these unique chemistries, which inertly co-exist with biology until the exact moment when the desired reactions are initiated, to enhance – and even transform – biological systems.

Bioorthogonal modification of proteins will, by definition, require expansion of the biochemical toolbox; there are a variety of techniques used to achieve this goal. In these studies, we explore the use of genetic code expansion for incorporation of unnatural amino acids. This technology permits co-translational incorporation of amino acids with unique and non-canonical R-groups directly into the polypeptide backbone of a protein or biopolymer. These residues introduce unique chemical reactivity for further functionalization with desired moieties or chemical transformation.

We have used this technology to develop novel therapeutic and material platforms comprised of a unique biopolymer, elastin-like polypeptide (ELP). This thermally responsive biopolymer is easily recombinantly synthesized, though more biochemically complex ELPs require successful bioorthogonal modification. We designed the unnatural amino acid-containing ELPs necessary to enable our strategies for developing three distinct biomaterial platforms: 1) photoreactive ELPs which can generate stable hydrogel particles spanning four orders of magnitude in size; 2) a universal strategy for drug-loaded, targeted ELP nanoparticles by incorporation of a unique site for drug attachment; 3) a sustained-release therapeutic for treatment of brain tumors combining proteins of distinct cellular origin.

We have combined existing tools, technologies, and materials to generate these novel platforms with utility in biomaterials, drug delivery, and cancer therapeutics. The optimizations performed in developing each of these systems will inform future studies with similar goals; similarly, the reactions and strategies employed will contribute to furthering our understanding of the full potential of these important bioorthogonal chemistries.

Targeted drug delivery to solid tumors aims to increase the accumulation of drug at the site of disease while limiting accumulation in healthy tissues. Thus, targeted delivery serves to enhance therapeutic efficacy while minimizing off-target side effects. Targeting drug to the site of disease is especially important for many current anti-cancer therapeutics whose cytotoxic effects are not exclusive to cancer cells. Drug carriers can improve tumor targeting of drug cargo by either passive or active mechanisms. Passive targeting of drug carriers occurs by the enhanced permeability and retention effect, whereby long circulating drug carriers can accumulate in the tumor by extravasation from the tumor's leaky vasculature and be retained in the tumor due to the lack of an organized tumor lymphatic system. Alternatively, active targeting can improve drug delivery to the tumor by means of functionalizing a drug carrier such that it interacts specifically with the tumor tissue. Traditionally, actively targeted drug carriers rely on intrinsic features of the tumor such as upregulated cell receptors, overexpressed extracellular enzymes, or depressed tissue pH. These intrinsic targets, however, are heterogeneous across cancer classes and between patients with a single tumor type. Therefore traditional active targeting cannot be applied to a breadth of cancers or patients without prior knowledge of the cancer phenotype.

Active targeting can alternatively be achieved by an extrinsic trigger, independent of the characteristics of the tumor. This approach could thereby achieve targeted drug delivery in a breadth of tumor types and cancer patients. This dissertation describes one such approach that exploits cell-penetrating peptides (CPPs) to achieve receptor-independent and non-specific uptake in a variety of cancer cells. The function of this non-specific CPP is controlled by an extrinsic trigger by means of the modulation of its local interfacial density with temperature-triggered micelle assembly. Elastin-like polypeptide diblock copolymers (ELP_BCs) were used as the drug carrier platform, as their lower critical solution temperature phase transition behavior permits their controlled self-assembly from unimer to micelle in response to a thermal stimulus. CPP-ELP_BCs were recombinantly synthesized in E. coli with CPP-functionalization at their hydrophilic terminus, such that temperature-triggered micelle assembly would result in the decoration of CPP on the micelle corona. The CPP-ELP_BC design was carefully optimized to permit micelle self-assembly in response to the clinically relevant trigger of mild hyperthermia.

Temperature-triggered micelle assembly of CPP-ELP_BCs achieved controlled cellular uptake in vitro by means of their CPP density modulation, such that cellular uptake was minimized at physiologic temperature and was greatly enhanced at conditions of mild hyperthermia. This effect was achieved in multiple cell lines, albeit with variable magnitude. Controlled uptake of the CPP-ELP_BC carrier could control the intracellular delivery of appended drug cargo. This controlled intracellular delivery was translated to controlled therapeutic effect when the CPP-ELP_BC was genetically appended to a proapoptotic peptide drug cargo. These drug-loaded CPP-ELP_BCs achieved controlled cytotoxicity in cancer cells, whereby significant cell death was induced at conditions of mild hyperthermia, but cells at physiologic temperature were spared.

For use as targeted drug carriers in vivo, CPP-ELP_BCs would be systemically administered and circulate throughout the body in their soluble state. It would only be at the site of the solid tumor that local mild hyperthermia would be applied and induce the self-assembly of CPP-ELP_BC micelles that could induce internalization into cancer cells. Translation of CPP-ELP_BC function from in vitro to in vivo environments proved to be quite challenging. Issues such as perturbation of temperature-triggered assembly in serum and interference of CPP-ELP_BC internalization by serum proteins likely played a role in preventing the extrinsically targeted accumulation of CPP-ELP_BCs in hyperthermia treated tumors, as investigated by intravital tumor microscopy and biodistribution studies. Further optimization of the CPP-ELP_BC platform is thus required to achieve extrinsically targeted drug carrier delivery in vivo.

Over 30 million people in the United States suffer from type 2 diabetes (T2D), and this figure is rapidly increasing. Currently available glucose-lowering drugs largely treat the symptoms of diabetes and not the underlying pathology, leaving one third of diabetes patients with improperly managed disease. Thus, there exists an urgent need for novel drugs that slow T2D progression while posing a minimal burden on the patient.

The metabolic regulatory factor fibroblast growth factor 21 (FGF21) is under investigation as a T2D therapeutic due to its favorable effects on glycemic control and body weight. However, the feasibility of native FGF21 as a drug candidate is impeded by its rapid in vivo clearance and by costly production methods associated with poor protein solubility. To address these issues, FGF21 was recombinantly expressed in E. coli as a fusion with an elastin-like polypeptide (ELP), a repetitive peptide polymer with reversible thermal phase behavior. Below their transition temperature (Tt), ELPs exist as soluble unimers, while above their Tt, they aggregate into an insoluble coacervate. The thermal responsiveness of the ELP was retained when genetically fused to FGF21, with several notably positive impacts for the synthesis and efficacy of this protein drug. First, the ELP fusion partner acted as a solubility enhancer, yielding 50 mg/L of active FGF21 protein from the soluble cell lysate fraction in shaker flask culture, and eliminating the need for protein refolding. Second, the phase transition behavior of the ELP was exploited for chromatography-free FGF21 purification. Third, the Tt of the ELP was tuned to below body temperature, such that the phase transition was initiated solely by body heat. Indeed, in vivo injection of the fusion resulted in an immiscible viscous phase in the subcutaneous (s.c.) space that dissolved at a steady rate, temporally releasing fusion unimers into circulation. The injectable FGF21 drug depot was tested in diabetic ob/ob mice, and conferred dose-dependent glucose-and weight-lowering effects that were sustained out to 5 days following a single s.c. injection.

Once an optimized ELP-based FGF21 delivery strategy was established, the fusion concept was applied to a combination therapy to afford even greater metabolic benefits, while providing controlled release properties exclusive to the ELP platform. Recent evidence supports the development of combination drug treatments that incorporate complementary mechanisms of action to more effectively treat T2D. Thus, we developed a unimolecular dual agonist by combining the incretin glucagon-like peptide-1 (GLP1) with FGF21, hypothesizing that this agent would merge the insulinotropic and anorectic effects of GLP1 with the enhanced insulin sensitivity and energy expenditure associated with FGF21 signaling. The dual agonist was designed as a single polypeptide fusion, with GLP1 located at the N terminus and FGF21 at the C terminus. This orientation allowed each peptide to activate its endogenous receptor, while the linear architecture enabled facile synthesis in a bacterial expression system. An ELP was fused between GLP1 and FGF21 to serve as both a flexible linker and a depot-forming delivery scaffold. Indeed, a single s.c. injection of GLP1-ELP-FGF21 into diabetic db/db mice resulted in potent metabolic effects that were sustained at least 7 days, indicating formation of an ELP depot with a highly controlled rate of drug release. Furthermore, dual agonist treatment outperformed a long-acting GLP1 analog in restoring glycemic control and inducing weight loss, supporting the rationale for a GLP1/FGF21 combination therapy.

With a significant proportion of T2D patients failing to properly manage their disease, there is an urgent need for novel drug and drug combinations that effectively target disease pathophysiology, while posing a minimal burden on the patient. Meanwhile, the vast – and global – prevalence of metabolic disease argues for cost-effective and scalable manufacturing methods for new drugs. An ELP-based approach to therapeutics precisely addresses these needs by providing a streamlined method for production, as well as an innovative strategy for drug delivery to reduce the frequency of administration and thereby promote patient compliance. Furthermore, the ELP platform can be utilized to unite distinct drugs into one multi-functioning molecule to more effectively treat diabetes, altogether simplifying and improving metabolic disease management.

Monoclonal antibodies have been successfully developed as PD-L1 antagonists, showing unprecedented anti-cancer immune response and efficacy with their high affinity and exquisite specificity. Despite its substantial success, the development of antibody drugs is approaching an asymptote because their structural inflexibility limits the ability to tune their valency, receptor accessibility, and blood circulation duration. Their efficacy in treating solid tumors is also limited by low tumor penetration due to their large size and structural inflexibility. These inherent challenges to utilizing antibodies as therapeutics confine their further improvements. To address these limitations, we have developed a modular protein therapeutic with rationally tunable valency, affinity, pharmacokinetics, and tumor penetration. In modular protein therapeutics, we can independently tune their affinity and valency for any given target, as well as modulate their pharmacokinetics and tumor penetration. To create a modular PD-L1 antagonist, we chose the human tenth fibronectin type III domain (FN3) as the “affinity module” because it is a small (~10 kDa), structurally robust protein domain that has six disordered loops that are similar to the complementarity-determining regions (CDRs) of antibodies. We then oligomerized the affinity module to enhance its binding to PDL1 via the avidity. To optimize pharmacokinetics, we fused the oligomerized affinity module with a “half-life module”: 1) an elastin-like polypeptide (ELP) that is injectable as a solution at room temperature but forms a gel-like depot at 37 °C and provides sustained, the zero-order release of the fusion; or 2) an albumin-binding domain that binds to and exploits the endogenous albumin to significantly extend the plasma half-life. The fusion with a “half-life module” would enable our modular protein therapeutics to rival the pharmacokinetics of antibodies. In this thesis, we discovered PD-L1-binding FN3 proteins (aPDL1-FN3) using phage display and modulated their valency and affinity for their equilibrium dissociation constants (KD) in the picomolar range. Unlike bivalent antibodies, the multivalency of FN3 is not restricted, and tetra-valency was chosen because (aPDL1-FN3)4 reached a plateau in terms of binding ability to PD-L1, measured by surface plasmon resonance (SPR), in vitro PD-L1 neutralization assay, and flow cytometry. To overcome glomerular filtration cutoff (~50 kDa) and improve pharmacokinetics, we genetically fused the (aPDL1-FN3)4 protein with either elastin-like polypeptides (ELPs) or albumin-binding proteins (ABDs). Using these fusions, we studied pharmacokinetics, biodistribution, and tumor uptake as compared to anti-PD-L1 antibodies. Also, we validated in vivo preclinical efficacy using three different immunocompetent mice models: 1) B16.F10 melanoma model; 2) CT26 colon cancer model; and 3) MC38 colon cancer model. These results demonstrated that our modular protein therapeutics successfully mimic antibodies as alternative therapeutics and have the potential to outperform antibodies regarding multivalency and cellular and tumor penetration. We believe that this research project serves as a proof-of-concept for modular protein therapeutics where tunable efficacy and pharmacokinetics can lead to a clinical utility that can eventually overcome the hurdles of traditional antibody-based therapy.

The clinical utility of many peptide, protein and small molecule drugs is limited by their short in-vivo ¬half-life. To address this limitation, we report a new class of biomaterials that have a long plasma circulation time. In particular, taking inspiration and cues from natural proteins and synthetic polymers, we have worked to create polypeptide-based drug carriers that are biocompatible and biodegradable. These peptide polymers or polypeptides can be attached to therapeutics with molecular precision as they are designed from the gene level.

In the first part of this thesis (Chapter 3-4), we report on the development of a new class of biomaterials called zwitterionic polypeptides (ZIPPs) that exhibit “stealth” behavior, and when fused to therapeutics, improve their pharmacological efficacy. To identify an optimal polypeptide design, we first synthesized a library of ZIPPs by incorporating various oppositely charged amino acids within an intrinsically disordered polypeptide motif, (VPX1X2G)n, where X1 and X2 are cationic and anionic amino acids, respectively, and n is the number of repeats. The (VPX1X2G)n motif is derived from the disordered region of human tropo-elastin. By systematically varying the identity of the charged amino acids and the chain length of the polypeptide, we determined the optimal polypeptide sequence that maximizes the pharmacokinetics for intravenous and subcutaneous routes of administration. We show that a combination of lysine and glutamic acid in the ZIPP confer superior pharmacokinetics, for both intravenous and subcutaneous administration, compared to uncharged control polypeptides. We report detailed physicochemical characterization of this new class of polypeptide-based drug carriers and show its clinical utility for drug delivery by using it to deliver a peptide drug. The peptide drug used is Glucagon like peptide 1 (GLP1) – a therapeutic peptide that is approved for treatment of type 2 diabetes but has seen limited clinical utility because of its short two-minutes half-life. We find that the GLP1-ZIPP conjugate reduced blood glucose level for up to 3 days in a diet induced obesity model of type-2 diabetes in mice after a single s.c. injection. This is a 70-fold improvement over the injection of the unmodified drug and a 1.5-fold improvement over an uncharged polypeptide control.

To further demonstrate the clinical utility of ZIPPs, in the second part of this thesis (Chapter 5), we used ZIPPs to create a nanoparticle system that can package and deliver hydrophobic chemotherapeutic drugs to the tumor with higher efficacy and lower toxicity. Such nanoparticle drug carriers are attractive for systemic delivery of chemotherapeutics because they improve the half-life of the drug, protect the drug from early degradation, and increase selective accumulation of drugs in tumors via the enhanced permeation and retention effect (EPR). The EPR effect is a consequence of the leaky vasculature and poorly developed lymphatic drainage system present in the tumors. These attributes of nanoparticles are significant and desirable because drug delivery systems that can improve circulation time and tumor accumulation of chemotherapeutics have the ability to improve the patient prognosis and survival by controlling the tumors at their local sites. To that end, we conjugated paclitaxel a chemotherapy drug that is used to treat different types of cancer to ZIPPs and showed that it imparts sufficient amphiphilicity to the polypeptide chain to drive its self-assembly into sub-100 nm nanoparticles. We report that ZIPPs can increase the systemic exposure of paclitaxel by 17-fold compared to the free drug and 1.6-fold compared to uncharged recombinant control. Treatment of mice bearing highly aggressive prostate cancer or colon cancer with a single dose of ZIPP-Paclitaxel nanoparticles leads to a near complete-eradication of the tumors (5 out of 7 cures in prostate cancer) and (2 out of 7 cures in colon cancer) and it outperforms Abraxane, which is an FDA approved taxane nanoformulation and current gold standard for paclitaxel delivery.

In summary, this doctoral research is multidisciplinary, which integrates the field of protein engineering, molecular biology, bioconjugate chemistry, soft matter physics and cancer biology for rational design of biomaterials for drug delivery.

Brachytherapy has emerged as one of the pre-eminent radiotherapy modalities for the treatment of prostate cancer. Current clinical methods utilize titanium encased radioactive seeds that are fixated within the prostate and permanently implanted. A novel brachytherapy alternative that has been developed to improve the delivery of radionuclide intratumorally is the synthetically designed elastin-like polypeptide (ELP). ELP can be injected in fluid form and undergoes an inverse phase transition to a biocompatible coascervate capable of serving as a biocompatible, intratumoral depot. Utilizing a previously developed ELP with a 7 tyrosine C-terminus tail, the therapeutic efficacy of ELP as a radioactive depot for treating prostate cancer was examined in a preclinical, orthotopic model. The orthotopic prostate model was first established by xenografting Bioware® PC-3M-luc-C6 cells into immunoincompetent, Balb/c nude mice. A non-invasive method for tracking tumor progression in vivo was developed using a correlation model comparing quantitative luminescent flux emitted from the cell line against the actual tumor size. The correlation between flux and tumor volume was determined to as Volume = 7.234x10-9x - 18.54, (±21.7%), where x is the supine photon flux measured from a 10 second exposure taken 18 minutes after D-luciferin injection. Radionuclide conjugation of 131I to ELP was conducted using the established IODO-GEN reaction methodology and mice were administered a therapeutic dose of 2mCi / 40µl ELP / 150 mm3 prostate tumor. Intratumoral deposition resulted in tumor regression in 90.9% of treated mice (n=11); 63.6% of which achieved tumor size reduction by over 60%. Radioactivity measurements demonstrate an 89.9% ELP depot retention over 2 weeks. Survival rates of the test group (64%) compared with controls (100%, n=14) indicate further testing is required to optimize radionuclide dosimetry.