Development and therapeutic assessment of multivalent protein polymers for cancer and eye diseases

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Content i DEVELOPMENT AND THERAPEUTIC ASSESSMENT OF MULTIVALENT PROTEIN POLYMERS FOR CANCER AND EYE DISEASES by Changrim Lee A Dissertation Presented to the FACULTY OF THE USC GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY PHARMACEUTICAL SCIENCES DECEMBER 2020 Copyright 2020 Changrim Lee ii Dedication Sola Scriptura Sola Fide Sola Gratia Solus Christus Soli Deo Gloria To my wife, Audrey JungJu Cho, for her uncountable sacrifices To my family and the family of Audrey, for their ceaseless prayers and supports To my uncle, Neungku Kwon, an inspirer with unabating courage iii Acknowledgments I would like to thank my mentors Dr. J. Andrew MacKay and Dr. Sarah F. Hamm- Alvarez for their guidance, support, and patience. I would also like to thank Dr. Jianming Xie for his service as a dissertation committee member. Dr. Alan L. Epstein at the University of Southern California, Dr. Honggang Cui at Johns Hopkins University, and Dr. Gordon W. Laurie at the University of Virginia have added depth to the research presented in this manuscript. Thanks to previous and present lab members as well as friends for their supports. This work was supported by the University of Southern California (USC), USC Provost Fellowship, USC School of Pharmacy, Gavin S. Herbert Professorship, USC Stevens Technology Advancement Grant (TAG), USC Ming Hsieh Institute, USC L.K. Whittier Foundation, NIH R01EY026635, R01GM114839, RO1EY011386 P30EY029220, P30CA014089, P30DK048522, and unrestricted departmental grant from Research to Prevent Blindness (RPB). iv Table of Contents Dedication ..................................................................................................... ii Acknowledgements ...................................................................................... iii Table of Contents ......................................................................................... iv List of Tables ................................................................................................. viii List of Figures ............................................................................................... ix Abstract ......................................................................................................... xii Chapter 1. Live Long and Active: Polypeptide-Mediated Assembly of Antibodies 1 1.1. Introduction ............................................................................................................ 1 1.2. Antibodies and engineered antibody fragments ..................................................... 4 1.2.1. Engineering Fab region and binding site barrier ............................................ 4 1.2.2. Engineered antibody Fab mimetic polypeptides ............................................ 10 1.2.3. Function of Fc region and Fc engineering ..................................................... 13 1.3. Human proteins that mediate antibody self-assembly ........................................... 19 1.3.1. Bioinspired recombinant polypeptides ........................................................... 19 1.3.1.1. Elastin-like polypeptides (ELPs) ........................................................... 19 1.3.1.2. Collagen-like polypeptides (CLPs) ........................................................ 22 1.3.2. Human serum albumin (HSA) ........................................................................ 26 1.3.3. Transmembrane proteins for exosome retargeting ....................................... 29 1.4. Non-human proteins as fusion platforms for antibody assembly ........................... 32 1.4.1. Leucine zippers in yeast protein GCN4 ......................................................... 32 1.4.2. Fibroin - a silk protein .................................................................................... 35 1.4.3. Viruses for drug discovery ............................................................................. 36 1.4.4. Viruses for drug delivery ................................................................................ 40 1.5. Concluding discussion ........................................................................................... 44 Chapter 2. Real-time Receptor Clustering at the Single-cell Level Using Antibody Nanoworms Designed for Lymphoma ...................................................... 48 v 2.1. Introduction ............................................................................................................ 48 2.2. Materials and methods ........................................................................................... 53 2.2.1. Synthesis, Expression, and Purification of Nanoworms ................................ 53 2.2.2. Labeling and Refolding of Nanoworms .......................................................... 54 2.2.3. Biophysical Characterization of Nanoworms ................................................. 55 2.2.4. Cryo-TEM imaging ......................................................................................... 57 2.2.5. Cell cultures and time-lapse live cell imaging ................................................ 58 2.2.6. Target specificity of Nanoworms ................................................................... 59 2.2.7. Apoptosis, cell cycle distribution, and cell activation upon Nanoworm treatment ...................................................................................................... 60 2.3. Results ................................................................................................................... 63 2.3.1. scFv-ELP fusions form colloidally stable worm-like nanostructures .............. 63 2.3.2. Nanoworms cluster bound receptors on the immune cell-surface ................. 68 2.3.3. Nanoworm-mediated receptor clustering on the immune cell-surface activates intracellular signaling ................................................................................... 71 2.3.4. Generation of CD20 targeting Nanoworm variants ........................................ 78 2.3.5. Nanoworms’ multivalency and thermo-sensitivity coordinate receptor clustering on the cell surface ....................................................................... 81 2.3.6. Enthalpy and entropy of Nanoworm phase separation is correlated to the hydrodynamic radii of the Nanoworms ........................................................ 83 2.4. Discussion .............................................................................................................. 87 2.5. Conclusion ............................................................................................................. 89 Chapter 3. Berunda polypeptides: Bi-headed rapamycin carriers for subcutaneous treatment of autoimmune dry eye disease ...................................... 91 3.1. Introduction ............................................................................................................ 91 3.2. Materials and Methods ........................................................................................... 96 3.2.1. Synthesis, Expression and Purification of FAF .............................................. 96 3.2.2. Biophysical Characterization of FAF ............................................................. 96 3.2.3. Encapsulation of Rapa with FAF ................................................................... 97 3.2.4. Pharmacokinetic study of FAF ....................................................................... 98 3.2.5. Plasma and whole blood levels of Rapa ........................................................ 100 3.2.6. Therapeutic study in male non-obese diabetic (NOD) mice, a model of SS . 101 3.2.7. Quantitative real-time PCR ............................................................................ 102 vi 3.2.8. Stimulated tear collection and Cathepsin S activity analysis in tears and LG lysates .......................................................................................................... 103 3.2.9. Histopathology analysis and serum chemistry .............................................. 104 3.2.10. Thread wetting tests, corneal fluorescein staining analysis ......................... 105 3.2.11. Statistics ...................................................................................................... 106 3.3. Results ................................................................................................................... 107 3.3.1. Biophysical characterization of FAF, a FKBP-ELP fusion protein polymer that carries Rapa .................................................................................................. 107 3.3.2. SC administration significantly improves the pharmacokinetic profiles of FAF 110 3.3.3. Rapa significantly suppresses lymphocytic infiltration, inflammatory gene expression, and CTSS activity in the LG ....................................................... 115 3.3.4. SC delivery of Rapa has minimal effect towards ocular surface health in a two- week period ................................................................................................... 121 3.3.5. Histopathological evaluation reveals FAF-Rapa as a biocompatible system for SC delivery of Rapa ...................................................................................... 124 3.3.6. Termination of Rapa treatment resolves hyperglycemia ................................. 131 3.4. Discussion .............................................................................................................. 134 3.5. Conclusion ............................................................................................................. 137 Chapter 4. Biosynthesized Multivalent Lacritin Peptides Stimulate Exosome Production via Syndecan-1 in human corneal epithelium ...................................... 139 4.1. Introduction ............................................................................................................ 139 4.2. Materials and methods ........................................................................................... 142 4.2.1. Synthesis, Expression and Purification of LP-A96 .......................................... 142 4.2.2. Biophysical Characterization of A96 and LP-A96 ........................................... 142 4.2.3. Cell culture, mitogenic activity, and cytotoxicity .............................................. 144 4.2.4. Confocal fluorescence imaging ....................................................................... 145 4.2.5. Exosome purification and analysis .................................................................. 146 4.2.6. Adsorption and release kinetics of LP-A96 from contact lenses ..................... 148 4.2.7. Secretagogue activity in primary rabbit lacrimal gland acinar cells ................. 149 4.3. Results ................................................................................................................... 150 4.3.1. Biosynthesized LP-A96 fusion proteins self-assemble into multivalent nanoparticles ................................................................................................. 150 vii 4.3.2. Multivalent Lacripep induces Ca 2+ -dependent mitogenesis and exosome- biogenesis in corneal epithelial cells ............................................................. 155 4.3.3. Multivalent Lacripep is internalized via dynamin-mediated endocytosis and colocalizes with syndecan-1 in corneal epithelial cells .................................. 166 4.3.4. LP-A96 delivered by contact lens maintain mitogenic activity and induce exosome biogenesis in corneal epithelial cells .............................................. 168 4.4. Discussion .............................................................................................................. 172 References 179 viii List of Tables Table 1: Fragment-based antibody therapeutics approved by USFDA or in clinical trials ............................................................................................................... 7 Table 2: Fc fusion therapeutics approved by USFDA or in clinical development ......... 66 Table 3: Molecular information of scFv-A192 fusions ................................................... 66 Table 4: Molecular information of anti-CD20-ELP fusions. .......................................... 80 Table 5: Biophysical characteristics of ELP, ELP fusions, and an antibody 86 Table 6. Biophysical characteristics of FAF, FAF-Rapa, and rhodamine-labeled FAF- Rapa. ............................................................................................................ 109 Table 7. Pharmacokinetic parameters of IV- or SC-delivered FAF-Rapa analyzed using compartmental analysis and non-compartmental analysis .................. 114 Table 8. Histopathological observations in tissues of male NOD mice after 2 weeks of treatment with FAF-Rapa or other control groups ......................................... 128 Table 9. Toxicity assessment of FAF-Rapa via serum chemistry and organ/body weight measurements ................................................................................... 129 Table 10. Molecular information of LP-A96, A96, and Lacripep ................................... 154 Table 11. Thermo-sensitivity of LP-A96 and A96 ......................................................... 154 Table 12. Adsorption and release kinetics of LP-A96 in contact lenses ....................... 171 ix List of Figures Figure 1: Peptide-based biopolymers mediate self-assembly of antibodies ................ 3 Figure 2: scFv-ELP fusions self-assemble into Nanoworms that cluster and activate cell surface receptors ................................................................................... 52 Figure 3: Design, identity, and thermo-responsiveness of scFv-A192 fusions against a panel of immune receptors. ......................................................................... 65 Figure 4: ScFv-ELP fusions self-assemble Nanoworms. ............................................. 67 Figure 5: Nanoworms spontaneously cluster cell surface receptors below physiological temperature ................................................................................................. 69 Figure 6: Receptor clustering induces cell membrane reformation (blebbing) and activates lymphoid cells ............................................................................... 76 Figure 7: CD3 clustering induces either activation, apoptosis, or activation-induced cell death (AICD) on T cell lines of NHL or leukemia ......................................... 77 Figure 8: Design, identity, and biophysical characteristics of anti-CD20-ELP fusions with a range of thermal sensitivity ............................................................... 79 Figure 9: Anti-CD20-ELP Nanoworms cluster on the cell-surface below physiological temperatures ............................................................................................... 82 Figure 10: Estimated enthalpy (ΔH) and entropy and (ΔS) upon Nanoworm phase separation show significant correlation to the hydrodynamic radii of Nanoworms ................................................................................................ 84 Figure 11: Thermo-sensitivity and size of A192 ........................................................... 85 Figure 12: FAF promotes subcutaneous delivery of Rapa to alleviate dacryoadenitis 94 Figure 13: Berunda polypeptides are humanized fusions of the FKBP12 protein that promote solvent-free, burst-free subcutaneous (SC) administration of Rapa to a murine model of autoimmune dacryoadenitis. .................................... 95 x Figure 14: High molecular weight FAF-Rapa has the purity, size, and concentration- temperature phase behavior necessary for stability at body temperature ... 108 Figure 15: Pharmacokinetic analysis reveals that SC administration of FAF-Rapa improves pharmacokinetic properties of Rapa ........................................... 113 Figure 16: Rapa reduces lymphocytic infiltration in the LG of male NOD mice ........... 118 Figure 17: Gene expression profile of proteins involved in inflammation, antigen presentation and autophagy in LG of male NOD mice treated with subcutaneous Rapa ................................................................................... 119 Figure 18: Rapa suppresses proteolytic CTSS activity in the LG but not in the tears 120 Figure 19: Subcutaneously delivered FAF-Rapa has a minimal effect on tear production and ocular surface integrity over a 2-week period .................... 123 Figure 20: Histopathology of mouse organs reveals no systemic toxicity of FAF-Rapa at a therapeutic dose .................................................................................. 126 Figure 21: Rapamycin-mediated injection site toxicity is mitigated when delivered via FAF ............................................................................................................ 127 Figure 22: FAF-Rapa does not induce hepatotoxicity .................................................. 130 Figure 23: FAF-Rapa induces temporary hyperglycemia in NOD mice that resolves after termination of the treatment ............................................................... 133 Figure 24: FAF-Rapa shows superior bioavailability and equivalent therapeutic efficacy compared to free Rapa upon mTOR inhibition ............................. 138 Figure 25: An LP-A96 fusion self-assembles into stable multivalent spherical nanoparticles .............................................................................................. 152 Figure 26: Thermo-sensitivity of LP-A96 and A96 ...................................................... 153 Figure 27: LP-A96 induces Ca 2+ influx and mitogenesis in corneal epithelial cells ..... 156 Figure 28: LP-A96 activates exosome biogenesis in corneal epithelial cells .............. 159 xi Figure 29: Western blot-based densitometric analysis for residual LP-A96 in purified EV samples ................................................................................................ 160 Figure 30: LP-A96 induced exosomes are properly loaded with exosomal RNAs ...... 162 Figure 31: LP-A96-mediated exosome biogenesis does not affect cell viability in corneal epithelial cells ................................................................................ 164 Figure 32: LP-A96-mediated exosome biogenesis is less prominent when treated cells are at a high confluency ..................................................................... 165 Figure 33: LP-A96 is internalized via dynamin-dependent endocytosis and colocalized with syndecan-1 in corneal epithelial cells ................................................. 167 Figure 34: Contact lens-delivered LP-A96 maintains its activity in corneal epithelial cells ............................................................................................................ 170 Figure 35: Secretagogue activity of LP-A96, 3LP-A96, and Lacripep ......................... 176 Figure 36: Mitogenicity and Ca 2+ influx induced by 3LP-A96 ...................................... 177 Figure 37: Multivalent Lacritin peptide, LP-A96, is capable of inducing both mitogenesis and exosome biogenesis in corneal epithelium ..................... 178 xii Abstract Multivalent constructs are widely explored as molecular tools and therapeutics owing to their ability to engage multiple targets via multiple ligands. For examples, drug loaded nanoparticles have a higher chance to deliver their payloads to the target if equipped with multiple targeting ligands that allow tighter binding to the target (Koshkaryev et al., 2013). Moreover, multivalent therapeutic antibodies are shown to be better in tumor penetration and therapeutic efficacy compared to monovalent counterpart (Blanco-Toribio et al., 2013). Not only for drug targeting, multivalency-mediated supramolecular polymer assembly recapitulates complex biological systems (Ooi et al., 2020), which adds depth to drug delivery research. Among myriads of biomaterial scaffolds that are able to build multivalent modalities, bioinspired recombinant polypeptides are favored due to the ease of genetic engineering, low- immunognicity, high biocompatibility, and biodegradability. One of a such material is elastin-like polypeptides (ELPs). Comprised of pentameric repeats, (Val-Pro-Gly-Xaa-Gly)n, ELPs have been recognized as a favorable polypeptide fusion scaffold for drug delivery (Despanie et al., 2016). This manuscript discusses widely utilized bioinspired recombinant polypeptide-based protein-polymers that are able to build multivalent constructs and presents three examples of distinct multivalent modalities generated using ELPs. Especially, their biophysical properties and therapeutic implications are discussed in regard to cancer and eye diseases. The Chapter 1 introduces the recombinant polypeptide-based protein-polymers utilized in the field of drug discovery and delivery, especially those of which adopted as building blocks to self-assemble antibody fragments. Antibodies have multiple biologically relevant features that can be reengineered into new therapeutics formats. These include the adaptable specificity of their variable (Fab) region or recruitment of the immune system and mechanisms to promote long-circulation through their crystallizable (Fc) region. Since the invention of the single chain variable fragment (scFv) in 1988, the variable regions of antibody have been re-engineered and rebuilt into a wide variety of multifunctional nanostructures. Among the strategies, peptide- xiii mediated self-assembly of variable regions through heterologous expression has become a powerful way to produce homogenous and functional antibody fusion biomaterials and is an alternative to chemical bioconjugation. This chapter discusses the functions of antibody fragments, status of engineered therapeutic antibody fragments in the clinic, and challenges to overcome. Moreover, a selected list of biomaterials that mediate self-assembly of antibody variable region through biosynthesis are explored, which include elastin-like polypeptides (ELPs), collagen-like polypeptides (CLPs), albumin, transmembrane proteins, leucine zippers, silk protein, and viruses. Among the modalities introduced in Chapter 1, the Chapter 2 further discusses biophysical properties of antibody-ELP fusions, their mechanism of action, and therapeutic applications towards non-Hodgkin lymphoma (NHL). Despite advancements in antibody-based therapies for NHL, at least two major therapeutic needs remain unmet: i) heterogenous activation of host immunity towards B cell NHL; and ii) lack of antibody-based therapeutics for T cell NHL. This study explores the molecular characteristics of an adaptable modality called antibody Nanoworms and demonstrates their receptor clustering activity as a means to overcome and address abovementioned needs. To test this, four selected therapeutic receptors of B cell (CD19, CD20, HLA-DR10) and T cell (CD3) NHL were targeted by Nanoworms. Regardless of the target or the cell type, Nanoworms inherently clustered bound receptors on the cell-surface through their multivalency and activated intracellular signaling without any secondary crosslinker. As a sole agent, Nanoworms induced apoptosis by clustering CD20 or HLA-DR10, and arrested the cell cycle upon CD19 clustering. Interestingly, CD3 clustering was particularly advantageous in inducing activation-induced cell death (AICD) in an aggressive form of T cell NHL named Sézary syndrome that is fatal, limited in antibody-based therapeutics, and has poor outcomes to traditional chemotherapy. As Nanoworms can be easily designed to target any receptor for which a scFv is available, they may provide solutions and add therapeutic novelty to underserved diseases. xiv The Chapter 3 discusses bivalent small molecule carrier called ‘FAF’ that carries clinically available but highly toxic immunosuppressant Rapamycin and how FAF can improve pharmacokinetic and toxicity profile of Rapamycin observed in the clinic while exhibiting therapeutic equipotency. The USFDA-approved immunosuppressive drug Rapamycin (Rapa), despite its potency, is limited by poor bioavailability and a narrow therapeutic index. In this study, we sought to improve bioavailability of Rapa under subcutaneous (SC) administration and to test its therapeutic feasibility and practicality towards Sjögren’s Syndrome (SS), which is a systemic autoimmune disease and has no approved therapies. To improve its therapeutic index, we formulated Rapa with a carrier termed FAF, a fusion of the human cytosolic FK506- binding protein 12 (FKBP12) and an Elastin-like polypeptide (ELP). The resulting 97 kDa FAF: i) has minimal burst release; ii) is ‘humanized’; iii) is biodegradable; iv) solubilizes two Rapa per FAF; and v) avoids organic solvents or amphiphilic carriers. Demonstrating high stability, FAF remained soluble and monodisperse with a hydrodynamic radius of 8 nm at physiological temperature. A complete pharmacokinetic (PK) analysis of FAF revealed that the bioavailability of subcutaneous (SC) FAF was 60% with increased the mean residence time (MRT) up to 20.3 hrs compared to 10.7 hrs for IV administration. After 24 hrs injection, the plasma concentration of Rapa delivered by FAF was 8 times higher with significantly increased plasma-to-whole blood ratio relative to free Rapa. To evaluate therapeutic effects, FAF-Rapa was administered SC every other day for 2-weeks to non-obese diabetic (NOD) mice that develop lacrimal gland (LG) inflammation with mechanisms comparable to Sjögren’s Syndrome (SS). Both FAF-Rapa and free Rapa exhibited immunomodulatory effects by significantly suppressing lymphocytic infiltration, gene expressions of IFN-γ, MHC II, type I collagen and IL-12a, and cathepsin S (CTSS) activity in the LG compared to controls. Serum chemistry and histopathological analyses in major organs revealed no apparent toxicity of FAF-Rapa. Given its improved PK and equipotent therapeutic efficacy compared to free Rapa, FAF-Rapa may be of further interest for systemic treatments for autoimmune diseases like SS. xv The Chapter 4 introduces ELPs that is formulated in fusion with a therapeutic peptide called Lacripep. Lacripep is a chemically synthesized therapeutic peptide derived from the human tear protein, Lacritin. Lacripep interacts with the cell surface proteoglycan, syndecan-1, and induces mitogenesis upon removal of heparan sulfates (HS) that are attached at the extracellular domain of syndecan-1. The presence of HS is a prerequisite for the syndecan-1 clustering that stimulates exosome biogenesis and release. Therefore, downstream activities associated with HS cleavage and syndecan-1-mediated mitogenesis versus HS retention and exosome biogenesis are assumed to be mutually exclusive. This study introduces a biosynthesized multivalent Lacripep named LP-A96, and evaluates its activity on syndecan-1- mediated mitogenesis versus exosome biogenesis. LP-A96 activates both downstream pathways associated with syndecan-1 in a dose-dependent manner. HCE-T cells at high confluence treated with 1 μM LP-A96 induced mitogenic activity equipotent to Lacripep. However, cells at low density treated with 1 μM LP-A96 generated a 200-fold higher number of exosomes compared to those treated at low density with Lacripep. As monovalent Lacripep is capable of inducing mitogenicity but not exosome biogenesis, activation of exosome biogenesis by LP-A96 not only suggests its utility as a novel molecular tool to study the Lacritin-syndecan-1 axis in corneal epithelium but also implies activity as a potential therapeutic peptide that can further improve ocular surface health through exosome biology. 1 Chapter 1 Live Long and Active: Polypeptide-Mediated Assembly of Antibodies 1.1. Introduction Under the rationale that therapeutic molecules could become better drugs if the off- target exposures are minimized, many concepts have been explored to achieve targeted drug delivery. In contrast to the non-targeted molecules, which rely on enhanced permeability and retention (EPR) effect, the ability of antibody or antibody fragments to exponentially enhance the specificity and curb the off-target side effects has created new avenues in drug development. Along with advancements in antibody engineering, discoveries in biocompatible materials that are inspired by natural polypeptides, adopted from human endogenous proteins or non-human sources, or de novo chemical synthesis have changed these ideas into reality. These biomaterials include: i) Bioinspired recombinant polypeptides, such as Elastin-like polypeptides (ELPs), Silk-like polypeptides (SLPs), or Collagen-like polypeptides (CLPs); ii) Hybrid block copolymers made up of poly-amino acids that produce liposomal, micellar or tubular structures; iii) Endogenous proteins identified in humans or in non-human organisms, such as human serum albumin, transmembrane proteins or heterodimeric coiled-coil domains; iv) Exogenous sources such as viral proteins (Stayton et al., 2000). Although these biomaterials vary in shape, size, conformation, external stimuli responsiveness, and pharmacology, they all possess some common features: i) Components can be produced with high fidelity by ribosomal biosynthetic pathways; ii) Peptides are biodegradable via endogenous proteases; iii) Tertiary and quaternary structures can be stabilized via supramolecular assembly; iv) Biophysical properties can be fine-tuned upon grafting divergent functional moieties (Rabotyagova et al., 2011). To maximize the therapeutic potentials of abovementioned materials, antibody conjugated versions were formulated and came into play as a powerful delivery system. Further 2 advancements in antibody conjugate manufacture strategies and their broad applicability gradually will allow their progression into the clinic (Mitragotri et al., 2014). In line with the continuing efforts to functionalize biomaterials with engineered antibody fragments, this review provides insights to readership in two ways. First, general characteristics of antibodies, their functional derivatives, and the exclusive list of fragment-based therapeutic antibodies currently in the clinical trials are discussed to better understand available technologies in antibody engineering. Second, understanding the physico-chemical and biological features of biomaterials that have the ability to self-assemble antibody fragments may further improve modalities under investigation, diversify their biomedical applications, and benefit health care by complementing or overcoming known drawbacks of current antibody- based therapeutics. For this, a panel of polypeptide/protein-based antibody self-assembly platforms are examined ranging from the proof-of-concept stage to early phases of human studies. The list includes recombinant polypeptides that are inspired by consensus motifs identified in human endogenous proteins, natural proteins in human, yeast, or silk worms with favorable properties that could be directly utilized as antibody assembly platforms, viruses where infectivity and cytotoxicity can be redirected to the tumors, and bacteriophages that are extensively exploited for novel antibody discovery towards various diseases, including SARS, MERS, and more recently, COVID-19 (Figure 1). 3 Figure 1. Peptide-based biopolymers mediate self-assembly of antibodies. Human-derived recombinant polypeptides, endogenous human proteins, or non-human polypeptides mediate antibody self-assembly. Antibody Self-assembly Platforms Elastin-like Polypeptides (ELPs) (Monoblock or Di-block amphiphiles) Virus Bacteriophage TARGET Albumin Transmembrane proteins Human Endogenous Proteins Non-Human Peptides or Proteins Exosome Leucine Zipper (two α-helical peptides) Collagen trimerization domain or Collagen-like polypeptides (CLPs) Trimerization Human-derived Recombinant Polypeptides 4 1.2. Antibodies and engineered antibody fragments 1.2.1. Engineering Fab region and binding site barrier Antibodies, a class of proteins expressed by plasma B cells, are utilized by the innate immune system to identify and neutralize evading exogenous entities. There are five naturally occurring antibody classes: monomeric IgD, IgE, IgG, dimeric IgA, and pentameric IgM, from which IgG class is the most abundantly found (>80%) in the systemic circulation (Schroeder and Cavacini, 2010). IgG class antibodies are composed of four polypeptides, two heavy (H) chains and two light (L) chains, and each of the H and L chains are comprised of constant (C) and variable (V) regions. H chain has 4 domains, (NH3)-VH-CH1-CH2-CH3-(COOH), whereas L chain is comprised of 2 domains, (NH3)-VL-CL-(COOH). VL (Variable Light) and CL (Constant Light) domains of L chain pairs with VH (Variable Heavy) and CH1 (Constant Heavy 1) domains of H chain, respectively, which is known as a Fab (fragment antigen binding) region. Connection and stabilization of Fab region is achieved by a disulfide bond between cysteine residues on the L chain and H chain, of which the cysteine residues used are slightly differently between IgG subtypes. In the IgG1 molecule, the last cysteine residue of L chain and fifth cysteine residue of H chain forms a disulfide linkage, whereas in IgG 2, IgG3, and IgG4 molecules, disulfide bond is formed between the last cysteine residue of the L chain and the third cysteine residue of the H chain. Paired H and L chains interact with an identical pair of H and L chains through the CH2- CH3 domain on each H chain (homodimerization), eventually forming a Y-shaped protein. Connection and stabilization of the Fc region relies on a different set of disulfide bonds that are formed between hinge regions of each heavy chain, although the number of disulfide bonds varies among subtypes: 2 for IgG1 and IgG4, 4 for IgG2, and 11 for IgG3 (Liu and May, 2012). Various derivatives that maintain the parent IgG’s specificity have been developed and exploited as a therapeutic mean to overcome limitations found on intact antibodies and their nano-formulations. These include monovalent Fab (CH1-VH paired with CL-VL) or scFv (single chain variable fragment, VH paired with VL via short polypeptide linker) as well as bivalent, 5 trivalent and tetravalent derivatives, such as Fab2 (bispecific), bis-scFv (bispecific), diabody (bispecific), Fab3 (trispecific), minibody (bivalent), triabody (trivalent) and tetrabody (tetravalent), which engage multiple receptors (Table 1) (Holliger and Hudson, 2005). Distinct classes of antibodies that have been discovered from cartilaginous fishes and camels, such as Ig-NAR (immunoglobulin new antigen receptor, found in sharks) and hcIgG (heavy chain IgG, found in camels, llamas, and alpacas), were also subjected to engineering, owing to their simple structure yet equivalent affinity compared to human scFv (Jain et al., 2007; Muyldermans, 2013). A key determinant of an antibody activity is the affinity against the target antigen. Since antibodies sometimes have to compete with the natural ligand for binding, affinity is an essential parameter in therapeutic antibody development. However, an increase in affinity does not always translate into optimal therapeutic efficacy. The ‘binding-site barrier’ effect, proposed by Weinstein et al., explains why high antibody affinity can lead to poor tumor penetrance and mitigate therapeutic efficacy (Fujimori et al., 1990). In the context of solid tumors, once antibodies escape from vasculature, high tumor interstitial pressure is the first barrier they encounter. To reach the target antigens buried deep inside the tumor, antibodies go through a series of association and dissociation against targets to gradually move forward. Under this circumstance, high affinity with low dissociation constant decreases the availability of free antibodies to diffuse farther inside the tumor core, which leads to significant accumulation at the tumor margins. Heterogeneous distribution of high affinity antibodies was also confirmed using bioimaging techniques, which clearly showed a dense localization of those on outer cell layers of the tumor (or close to blood vessels). Using Affinity variants of anti-HER2 scFv in a mice model, Adams and colleagues reconfirmed that the deep penetration and uniform distribution can be achieved at the expense of slightly lower antibody affinity (Adams et al., 2001). More uniform distribution was achieved by comparably low affinity anti-HER2 scFv, while the high affinity anti-HER2 scFv showed discrete uneven tumor distribution. Increasing the dose can 6 partially solve the uneven tumor distribution of high affinity molecules; however, it may saturate the antibody binding sites at the tumor entry port, lead to undesired off-target effects (decreased tumor-to-non tumor ratio), and more severely damage healthy tissues (increased toxicity). Therefore, affinity modification should be performed rationally. If the delivery of anti-tumor agents deep into the tumors is crucial, use of comparably lower affinity antibody should be considered; on the other hand, if binding itself is the purpose, such as induction of apoptosis upon binding to tumor cells, then the higher affinity antibody may be favorable (Saga et al., 1995). Multiple factors, such as tumor microenvironments, density of target antigen, vascular permeability, and antibody’s affinity, should be precisely assessed in conjunction with one another to achieve optimal therapeutic index. 7 Table 1. Fragment-based antibody therapeutics approved by USFDA or in clinical trials. (as of March 2020) INN (trade name) Target Fragment type Primary indication Approval Year/Phase (NCT#) Brolucizumab (Beovuâ) VEGF-A scFv Wet Age-related Macular Degeneration 2019 Caplacizumab-yhdp (Cabliviâ) Von-Willebrand Factor Nanobody Acquired Thrombotic Thrombocytopenic Purpura 2019 Moxetumomab pasudotox-tdfk CD22 scFv- Pseudomonas Exotoxin PE38 Relapsed/Refractory Hairy Cell Leukemia 2018 Idarucizumab (Praxbindâ) Dabigatran Fab Reversal of Dabigatran(anticoagulant) 2015 Blinatumomab (Blincytoâ) CD 19, CD 3 BiTE B Cell precursor Acute Lymphoblastic Leukemia (ALL) 2014 Certolizumab pegol (Cimizaâ) TNFa Fab Crohn’s Disease 2008 Ranibizumab (Lucentisâ) VEGF-A Fab Wet Age-relate Macular Degeneration 2006 Abciximab (ReoProâ) GP IIb/IIIa Fab Unstable Angina 1994 Ozoralizumab TNF Nanobody Rheumatoid Arthritis III (NCT04077567) Vicinium/ Oportuzumab monatox EpCAM scFv- P.Aeruginosa exotoxin A Non muscle invasive bladder cancer III (NCT02449239) Daromun (Darleukin, Fibromun Comb.) Extra-domain B of Fibronectin (L19) scFv-IL2, scFv- TNFa Stage IIIb/c Melanoma III (NCT03567889/ NCT02938299) Lampalizumab Complement Factor D Fab Geographic Atrophy secondary to Age-related Macular Degeneration III (NCT02247531/ NCT02247479) Dapirolizumab-pegol CD 40L Fab-peg Systemic Lupus Erythematosus III (NCT04294667) MGD013 PD-1, LAG 3 DART Unresectable or Metastatic Neoplasm III/II (NCT04082364) I (NCT03219268) Naptumomab estafenatox 5T4 Tumor Antigen Fab- Staphylococcal enterotoxin A Advanced Renal Cell Carcinoma, Advanced or Metastatic Solid Tumor III/II (NCT0042088) I (NCT03983954) M1095/ALX-0761 IL-17a,f Bispecific Nanobody Psoriasis IIb (NCT03384745) Vobarilizumab IL-6r Nanobody Rheumatoid Arthritis IIb (NCT02287922) V565 TNFa Nanobody Crohn’s Disease II (NCT02976129) V HH batch 203027 Rotavirus Nanobody Rotavirus Diarrhea II (NCT01259765) 8 68GaNOTA-Anti- HER2 V HH HER-2 Nanobody Breast Carcinoma, Brain Metastasis of Breast Carcinoma II (NCT03924466/ NCT03331601)) Flotetuzumab /MGD006 CD 123, CD 3 DART Acute Myeloid Leukemia (AML) II (NCT03739606) I/II (NCT02152956) AFM 13 CD 30, CD 16A Tetravalent bsAb CD30 Positive T-cell Lymphoma, Hodgkin’s Lymphoma (HL) II (NCT04101331) Istiratumab/MM-141 IGF-1Rb, HER- 3 Tetravalent bsAb Metastatic Pancreatic Cancer II (NCT02399137) MM- 111 HER-2, HER-3 Bispecific scFv HER-2 Positive Carcinomas of Distal Esophagus, Gastroesophageal Junction and Stomach II (NCT01774851) Gremubamab /MEDI- 3902 PcrV, Psl Fab 2-scFv-Fc Nosocomial Pneumonia IIb (NCT02696902) MT-3724 CD 20 scFv with Shiga- like toxin 1A Relapsed/Refractory Diffuse Large B Cell Non- Hodgkin’s Lymphoma (NHL) II (NCT03488251/ NCT02361346) IMCgp100 Gp100, CD 3 ImmTAC Advanced Uveal Melanoma II (NCT03070392) IMCnyeso NY-ESO-1, CD 3 ImmTAC NY-ESO-1 and/or LARGE-1A Positive Cancer II/I (NCT03515551) OXS-3550/ GTB- 3550/ 161533 CD 16, CD 33 TriKE, scFv-IL 16- scFv High Risk Myelodysplastic Syndrome, Refractory/Relapsed AML, Advanced Systemic Mastocytosis II/I (NCT03214666) OXS-1550/ DT2219ARL CD 19, CD 22 BiKE, scFv-scFv- Diphtheria Toxin Relapsed/Refractory B- Lineage Leukemia, Lymphoma II/I (NCT02370160) CD19/20 CAR-T cell CD 19, CD20 Bispecific Nanobody derived CAR-T cell B-cell Lymphoma I (NCT03881761) M6495 ADAMTS-5 Nanobody Knee Osteoarthritis I (NCT03583346) ALX-0651 CXCR4 Nanobody Hematopoietic Stem Cell Transplant I (NCT01374503) PF-05230905 TNFa Nanobody Rheumatoid Arthritis I (NCT01284036) BI 836880 VEGF, ANG2 Bispecific Nanobody Wet Age-related Macular Degeneration I (NCT03861234, NCT03468426) Solitomab/ MT110/ AMG 110 EpCAM, CD 3 BiTE Lung Cancer, Gastric Cancer, Adenocarcinoma of Gastro-esophageal Junction, Colorectal Cancer, Breast Cancer, Hormone-Refractory Prostate Cancer, and Ovarian Cancer I (NCT00635596) 9 AMG 160 PSMA, CD 3 BiTE Metastatic Castration- resistant Prostate Cancer I (NCT03792841) AMG 199 MU-17 CD 3 BiTE Metastatic Gastric and Gastroesophageal Junction Cancer I (NCT04117958) MEDI-565 /AMG 211 Human CEA, CD 3 BiTE Gastrointestinal Adenocarcinoma I (NCT02760199) Pasotuxizumab/ BAY 2010112/AMG 212 PSMA, CD 3 BiTE Prostate Cancer 1 (NCT01723475) AMG 330 CD 33, CD 3 BiTE Relapsed/Refractory AML I (NCT02520427) AMG 420M BCMA, CD 3 BiTE Relapsed/Refractory Multiple Myeloma I (NCT03836053) AMG 562 CD 19, CD 3 BiTE Diffuse Large B-cell Lymphoma, Mantle Cell Lymphoma, Follicular Lymphoma I (NCT03571828) AMG 596 EFGRvIII, CD 3 BiTE Glioblastoma I (NCT03296696) AMG 673 CD 33, CD 3 BiTE Relapsed/Refractory AML I (NCT03224819) AMG 701 BCMA, CD 3 BiTE Multiple Myeloma I (NCT03287908) AMG 757 DLL3, CD 3 BiTE Small Cell Lung Cancer I (NCT03319940) BFCR 4350A FCRH5, CD 3 BiTE Relapsed/Refractory Multiple Myeloma I (NCT03275103) AFM 11 CD 19, CD 3 Tetravalent bsAb Relapsed/Refractory NHL I (NCT02106091) MGD 007 gpA33, CD 3 DART Relapsed/Refractory Metastatic Colorectal Carcinoma I (NCT02248805) MGD 009 B7-H3, CD 3 DART Relapsed/Refractory B7- H3 expressing tumor I (NCT03406949) MGD 010/PRV 3279 CD 32B, CD 79B DART B Cell Mediated Autoimmune Disorder Ib (NCT03955666) Duvortuxizumab /MGD 011 CD 19, CD 3 DART Relapsed/Refractory B Cell Malignancy I (NCT02454270) PF-06671008 P-Cadherin, CD 3 DART P Cadherin expressing TNBC, CRC, or NSCLC I (NCT02659631) AMV 564 CD33, CD 3 TandAb Relapsed/Refractory AML I (NCT03144245) Information retrieved from www.fda.gov and www.clinicaltrials.gov. BiKE: Bi-specific Killer Engager; BiTE: Bispecific T Cell Engager; DART = Dual Affinity Re- Targeting (Di-scFv); ImmTAC: T Cell Receptor (TCR)-scFv fusion; Nanobody = VHH; TandAb: Tandem Diabody; TriKE; Tri-specific Killer Engager 10 1.2.2. Engineered antibody Fab mimetic polypeptides Polypeptide scaffolds that are simple in structure, easy to engineer, and contain equivalent affinity and specificity compared to conventional antibodies have matured as a source for alternative targeting moieties. So called antibody-mimetic or antibody-like scaffolds, these are derived from naturally occurring endogenous proteins and possess certain region (α-helix, β- sheet, or variable loop) that can be engineered as targeting domains without substantially altering the original protein fold. This section briefly introduces the most advanced antibody Fab mimetic polypeptides that are approved for clinical use or are in the late stage of clinical evaluations, as these become alternative therapeutic options to fragment-based antibody therapeutics. Kunitz domains are 7 kDa peptides derived from the active motif of Kunitz-type protease inhibitors (Simeon and Chen, 2018). Found in yeast Pichia pastoris, the core of the Kunitz domain comprised of a twisted two-stranded antiparallel β-sheet and two α-helices, stabilized by three pairs of disulfide bonds. The three loops that connect two α-helices and two β-sheets are subjected to engineering for targeting. (Stolz and Horn, 2010). Ecallantide (trade name Kalbitor â , approved by USFDA in 2012) is a selective and reversible human plasma kallikrein inhibitor (7 kDa, Kd=25 pM) derived from the Kunitz domain of lipoprotein-associated coagulation inhibitor produced in yeast Pichia pastoris (Gelse et al., 2003). Patients with hereditary angioedema (HAE) are deficient or defective in gene encoding C1-inhibitor, by which activates the protease kallikrein. Kallikrein releases bradykinin from the its precursor kininogen, which leads to edema of the tissues. Ecallantide decreases the free bradykinin by inhibiting the kallikrein and prevent blood loss in cardiothoracic surgery (Cicardi et al., 2010). Designed Ankyrin Repeat Proteins (DARPin) are bioinspired by leucine-rich repeat proteins in the jawless vertebrates’ adaptive immune system (Pluckthun, 2015). 4~6 repeats of an α- helix that are engineered to bind a target consists the core, which are flanked by stable capping units at both the N and C-termini (Sharma et al., 2020). Abicipar pegol is an anti-VEGF DARPin 11 (34 kDa) molecule produced from E. coli that inhibits angiogenesis by binding to and thus neutralizing the active form of VEGF-A (Kd=0.5 pM) in the eyes (Binz et al., 2003; Rodrigues et al., 2018; Sharma et al., 2020). Abicipar pegol recently completed two Phase III clinical trials (NCT02462486, NCT02462928) against wetAMD. Given that Abicipar pegol is administered intravitreally, its extended intraocular half-life (>13 days vs. 7.2 days for ranibizumab) greatly decreases the treatment burden for the patient, from 4-weekly ranibizumab to 8~12-weekly Abicipar pegol (Meyer, 2019). An affibody is a 6 kDa protein derived from the B-domain of staphylococcal protein A (SPA), which is found in gram-positive bacterium Staphylococcus aureus (Frejd and Kim, 2017). The characteristic three α-helices that bind the Fc region of IgG can be further engineered to target various disease-related molecules. Affibody molecules can be recombinantly produced in either bacteria, yeast, mammalian, or insect cells as single domains or as fusions (Stahl et al., 2017). Currently, HER2 targeting affibody [ 68 Ga]ABY-025 (7 kDa, Kd=22 pM) is being evaluated as a non-invasive PET imaging agent to quantify HER2-expression in advanced breast cancer patients (NCT03655353), and IL-17a targeting ABY-035 (18 kDa, Kd=0.3 pM) is being tested as a therapeutic agent against moderate-to-severe plaque psoriasis (NCT03591887) as elevated IL-17a level is associated with this disease (Chopra, 2004; Stahl et al., 2017). Adnectins, also known as a monobody, are 10 kDa proteins derived from the extracellular domain of human fibronectin type III protein (Fn3) (Lipovsek, 2011). The 94 amino acids within the tenth extracellular domain of Fn3 ( 10 Fn3) that forms β-sandwich structure with six hypervariable loops exhibits structural similarity to variable loops and CDR regions of an antibody Fab region. Further engineering and affinity maturation on these loops enabled Adnectin to have high affinity to the target. Pegdinetanib, an adnectin molecule (40 kDa) produced from E. coli, reached phase II clinical trials against glioblastoma, non-small cell lung cancer, and colorectal cancer (Mamluk et al., 2010). Pegdinetanib antagonizes VEGF receptor 12 2 (Kd=11 nM), which is associated with blood retinal barrier breakdown, pathological ocular neovascularization, and progression of wetAMD (Boudko et al., 2009). A few examples of antibody Fab mimetic polypeptides that are approved for clinical use or are in the late stage of clinical evaluations were briefly discussed; however, many other technologies are maturing. Other technologies include Nanofitin ® (Company: Affilogic), Affimer ® (Avacta Life Science, Ltd), Atrimer™ (Anaphore Inc.), Anticalin ® (Pieris Pharmaceuticals), Fynomers ® (Creative Biolabs), Knottins (Gracy and Chiche, 2011), Armadillo repeat proteins (Hansen et al., 2017), Centyrin™ (Aro Biotherapeutics), Alphabody (Complix), Avimer™ (Amgen), Repebody (Lee et al., 2012). More extensive lists of antibody mimetic polypeptides, their structural characteristics, affinity and specificity of engineered derivative, and the status of clinical developments are summarized in detail by Vazquez-Lombardi et al. (Vazquez-Lombardi et al., 2015) and Yu et al. (Yu et al., 2017). 13 1.2.3. Function of Fc region and Fc engineering By recruiting various downstream effector molecules, the antibody Fc region is essential for initiating several cellular and immunological responses: i) The Fc region interacts with the classical complement C1q protein, which initiates a classical complement cascade. As a consequence, the membrane attack complex (MAC) is formed, which binds target cell membranes and leads to cell lysis; ii) The classical complement cascade simultaneously generates C3b proteins that bind to the target cell, a major component of the process called opsonization. C3b-opsonized cells become visible to complement receptors on phagocytes (neutrophils, dendritic cells, macrophages) and eventually undergo complement dependent cytotoxicity (CDC); iii) IgG also act as opsonins. These recruit Fc receptor (FcR) baring phagocytes to initiate antibody-dependent cellular phagocytosis (ADCP); iv) Opsonization by IgG also recruits natural killer cells (NK cells) via Fc-FcR interaction between immunoglobulin and NK cells and mediates tumor cell degranulation and lysis (antibody-dependent cell cytotoxicity, ADCC) (Oflazoglu and Audoly, 2010; Shan et al., 2000; Smith, 2003; Stamataki et al., 2011). Proteins that bind to their targets and make them visible to endogenous clearance machineries are generally called opsonins. Besides IgG, several complement system proteins (C3, C4, and C5), and circulating serum proteins (mannose-binding lectin, laminin, fibronectin, and C-reactive protein) are involved in three distinct complement activation pathways: the classical pathway, the alternative pathway, and the lectin pathway (Jha et al., 2007; Owens and Peppas, 2006). IgG and C3b are known to be the most crucial opsonins among them. The rationale for Fc engineering of an antibody is often to modulate pharmacokinetics (circulation half-life) or pharmacodynamics (enhance/decrease recruitment of FcγR-bearing immune effector units) (Table 2). Favorable pharmacokinetics can be achieved by amino acid substitutions that results in tighter binding to FcRn at low pH (Arnold et al., 2007). Zalevshy et al. described the relationship between increase in binding affinity of Fc region to FcRn and enhanced anti-tumor activity (Zalevsky et al., 2010). Double mutation of M428L/N434S led to11- 14 fold increase in FcRn affinity at pH 6.0 and elongated half-life in mice (5-fold) and cynomolgus monkeys (3-fold). Dall’Acqua et al. also developed a triple mutation of M252Y/S254T/T256E in a humanized anti-respiratory syncytial virus monoclonal antibody MEDI-524, which resulted in a 10-fold increase in binding affinity to FcRn at pH 6.0 but not at pH 7.4. Increased FcRn binding affinity lead to a 4-fold increase in plasma half-life and 4-fold increase in relative bioavailability in the lungs of cynomolgus monkeys (Dall'Acqua et al., 2006). On the contrary, amino acid substitutions to avoid FcRn binding are also performed to achieve desired pharmacokinetics. A bispecific antibody Faricimab is under clinical development to treat neovascular eye diseases, such as age-related macular degeneration (wetAMD) and macular edema. Upon intravitreal administration, Faricimab targets and neutralizes both VEGF-A and ANG-2 in the vitreous and subretinal regions, which suppresses subretinal neovascularization in human eyes (Chakravarthy et al., 2017; Sahni et al., 2019). As this neutralization activity should be confined to the eyes, three mutations (I253A, H310A, H435A) were introduced to ablate FcRn binding. This allowed therapeutic effect localized to the eyes as Faricimab experienced more rapid clearance after reaching the systemic circulation (Regula et al., 2016). Modulation of pharmacodynamics in the context of Fc engineering can be performed in two ways: amino acid substitution or glycoengineering. Faricimab is also a good example of modulated pharmacodynamics via amino acid substitution. As its mode of action is only to bind and neutralize the target molecules but not to activate local immunity, three additional mutations (P329G, L234A, L235A) were introduced within the Fc region to blunt binding to FcγRI, II, III. Introduction of these mutations prevented Fc-FcγR interaction-mediated activation of immune system in the eyes. Total of six amino acid substitutions in the Fc region of Faricimab ablated its binding to four different FcR present in the body, resulted in modulated pharmacokinetics and pharmacodynamics. Glycoengineering is another strategy to modulate pharmacodynamics given that glycoforms on Fc regions affect Fc-FcγR interaction (Arnold et al., 2007). Glycosylation occurs at the N297 on CH2 domain of the Fc region (Jefferis, 2005; Krapp et al., 15 2003). Among these N-linked carbohydrates, the presence of fucose is known to reduce Fc- FcγRIIIa binding affinity, which diminishes Fc-mediated ADCC. Therefore, modified CHO cell lines have been developed to express defucosylated Fc regions (Beck and Reichert, 2012). For example, the humanized monoclonal antibody mogamulizumab, which targets CC chemokine receptor 4 (CCR4), is engineered with a defucosylated glycoform that induces ADCC and CDC at lower antigen density compared to their fucosylated controls (Beck and Reichert, 2012; Niwa et al., 2005). Fc region glycoengineering has also been explored to modulate effector functions of CD20 targeting antibodies. A distinct class of CD20-targeting antibodies have been developed with different binding properties, downstream effects, and post-translational modifications compared to type I antibody rituximab (Cragg and Glennie, 2004). These type II antibodies include, tositumomab (trade name Bexxar ® , approved in 2003 and withdrawn in 2014 for decline in sales) and obinutuzumab (trade name Gazyva ® , approved in 2013). In contrast to a type I antibody, type II antibodies do not induce CD20 relocation into the lipid raft, receptor internalization, and complement-dependent cytotoxicity (Beers et al., 2008). The glycoengineered Fc region of the type II antibodies minimizes C1q binding and Fc-FcγRIIb interactions, which induce antibody-receptor complex internalization at lipid-rafts. In contrast, type II antibodies show enhanced Fc-FcγRIII interactions that promotes ADCC and ADCP, while abrogating CDC and premature lipid-raft dependent CD20 internalization (Goede et al., 2015). Due to these advantages, Type II antibodies have become an alternative treatment option especially for patients with impaired Fc-FcγRIIb interactions. The United States Food and Drug Administration (USFDA) approved its use for the treatment of naïve chronic lymphocytic leukemia as well as rituximab refractory tumors such as diffuse large B-cell lymphoma and follicular lymphoma (Bologna et al., 2011). To generate the glycosylation pattern that activates downstream cellular and humoral immune responses at a desired level, the choice of the host cell that can produce the glycoform of interest is crucial. Currently, USFDA approved antibodies or Fc-fusions with nonimmunogenic 16 glycoforms are produced using human embryonic kidney (HEK), NSO hybridoma (cell line derived from non-secreting murine myeloma), Sp2/0 (hybrid cell line of BALB/c mouse spleen cell and mouse myeloma P3X63AG8), or Chinese hamster ovary (CHO) cell lines (cite). These cell lines produce slightly different glycoforms that result in different pharmacodynamics. 17 Table 2. Fc-fusion therapeutics approved by USFDA or in clinical development. (as of March 2020) INN (Trade name) Target Therapeutic construct Primary Indication Approval Year/NCT # Luspatercept-aamt (Rebrozylâ)_ TFG-b ligand Human activin receptor type IIB-IgG1Fc Anemia with beta thalassemia 2019 Asfotase alfa (Strensiqâ) Factor substitute TNSALP-Fc fusion Hypophospatasia 2015 Dulaglutide (Trulicityâ) GLP1R GLP1 peptide analog- IgG4Fc Type II diabetes 2014 Antihemophilic Factor Recombinant FcFusion Protein (Eloctateâ) Coagulation FVIII-IgG1Fc Hemophilia A 2014 Alprolix (Alprolixâ) Coagulation FIX-IgG1Fc Hemophilia B 2014 Ziv-aflibercept (Zaltrapâ) VEGF-A, VEGF- B, PIGF VEGFR1-VEGFR2- IgG1Fc Colorectal cancer 2012 Aflibercept (Eyleaâ) VEGF-A, VEGF- B, PIGF VEGFR1-VEGFR2- IgG1Fc Wet age-related macular degeneration 2011 Belatacept (Nulojixâ) CD80, CD86 CTLA-4-IgG1Fc (Two amino acid substitutions, A29Y and L104E, on CTLA-4 from abatacept) Transplant rejection 2011 Rilonacept (Arcalystâ) IL-1a,b,RA IL-1R-IgG1Fc Cryopyrin- associated periodic syndrome (CAPS) 2008 Romiplostim (NPlateâ) Thrombopoietin receptor (CD110) Thrombopoietin binding peptide-IgG1Fc Refractory immune Thrombocytopenia 2008 Abatacept (Orenciaâ) CD 80/CD 86 CTLA-4-IgG1Fc Rheumatoid Arthritis 2005 Alefacept (Ameviveâ) CD 2 CD58 (LFA-3)-IgG1Fc Plaque psoriasis Transplant rejection 2003 (Withdrawn from market on 2011) Etanercept (Enbrelâ) TNFa (Both soluble and membrane bound) TNFR2-IgG1Fc Rheumatoid Arthritis Plaque psoriasis 1998 Trebananib/AMG 386 Ang1, Ang2 TIE2 mimetic peptide- IgG1Fc III (NCT01204749, NCT01281254, NCT01493505) Efgartimimod/ ARGX- 113 FcRn IgG1 Fc Myasthenia Gravis III (NCT03770403) Blisibimod/AMG 623 BAFF BAFF-IgG1Fc Systemic Lupus Erythematosus III (NCT02514967, NCT01395745) II/III (NCT02062684) 18 Information retrieved from www.fda.gov and www.clinicaltrials.gov. Atacicept B Lymphocyte, stimulator (BLys), A proliferation- inducing ligand (APRIL) BLys-APRIL-IgG1Fc Systemic Lupus Erythematosus II/III (NCT00624338) ALT-803 IL-15 IL-15-IgG1Fc Acute Myeloid Leukemia II (NCT03050216) (35 other Phase II Studies) Asunercept/APG101 CD95 ligand CD95-IgG1Fc Glioblastoma Multiforme, Myelodysplastic Syndrome Orphan Drug status, II (NCT01071837) SBI-087 CD 20 Small modular immunopharmaceutical (SMIP) CD20 scFv-Fc Rheumatoid Arthritis II (NCT01008852) TRU-015 CD 20 SMIP CD20 scFv-Fc Rheumatoid Arthritis II (NCT00634933) Otlertuzumab/ TRU- 016 CD 37 CD 37 scFv-IgG1 Fc Relapsed Indolent Lymphoma II (NCT01317901) AMG 171 GDF 15 GDF15-IgG1Fc Obesity I (NCT04199351) Efavaleukin alfa/ AMG 592 IL 2 IL 2 Fc Lupus Erythematosus I (NCT03451422) CNTO 528 Erythropoietin receptor Erythropoietin mimetic peptide(EMP1)-IgG1Fc Anemia I CNTO530 Erythropoietin receptor Erythropoietin mimetic peptide(EMP1)-IgG4Fc b-thalassemia, sickle cell anemia I SBI-087 CD 20 Erythropoietin mimetic peptide(EMP1)-IgG4Fc Systemic Lupus Erythematosus, Rheumatoid Arthritis I (NCT00714116, NCT00641225) 19 1.3. Human proteins that mediate antibody self-assembly 1.3.1. Bioinspired recombinant polypeptides 1.3.1.1. Elastin-like polypeptides (ELPs) Elastin-Like Polypeptides (ELPs) are an emerging class of protein polymer whose sequence is derived from tropoelastin, the precursor of elastin (Yeo et al., 2011). Tropoelastin is a soluble monomeric protein that is encoded by a human gene ELN and makes an insoluble fibrous elastin structure once it is crosslinked via its lysine residues. Inspired by a hydrophobic repeat motif in tropoelastin, ELPs are recombinant polypeptides of pentapeptide repeats (Val- Pro-Gly-X-Gly)n, where X can be any amino acid residue (guest residue) and n is number of repeats. Since the sequence of ELP polymers are similar to that of the naturally occurring tropoelastin, they appear to be biodegradable, biocompatible, and non-immunogenic (Cho et al., 2016; Nouri et al., 2015). One of the most unique characteristics of ELPs is their thermo- responsive phase separation (Andrew Mackay and Chilkoti, 2008). ELPs phase separate above a transition temperature (Tt), while remaining highly soluble below Tt. The Tt can be precisely controlled by various factors, including molecular weight (n), hydrophilicity of the guest residue (X), ELP concentration, pH, and co-solutes. There are multiple ways to utilize their thermo- responsiveness, one of which is as a purification strategy (Hassouneh et al., 2010). Phase separated ELPs can be collected by centrifugation at above its Tt (hot centrifugation), which promotes protein pellet formation at the bottom of the tube (MacEwan and Chilkoti, 2010b). After discarding soluble impurities (supernatant), pelleted coacervates are resuspended in a cold buffer and insoluble impurities are cleared by another centrifugation below the Tt (cold centrifugation). Repeating 2~6 rounds of hot and cold centrifugation steps usually yields highly pure ELPs (>95% purity based on SDS-PAGE) (Janib et al., 2014b). This method, called inverse temperature cycling, is an advantage compared to conventional methods that involve affinity chromatography. Cost-effective expression from bacterial fermentation and ease of genetic modification are other advantages. ELP can be expressed in Escherichia coli, yeast, or 20 plant cells. As peptides, these can be co-expressed in fusion with other biological and therapeutic proteins or peptides, which avoids the need to optimize post-purification bioconjugation strategies. Unconstrained genetic level modification has made ELPs a favorable fusion platform. Examples of functional units expressed and tested in fusion with ELPs include adenovirus knob domain (Sun et al., 2011), antibody (Lim et al., 2017; Zhao et al., 2017), peptide (Bidwell and Raucher, 2005), protein (Guise et al., 2019), receptor antagonist (Shamji et al., 2008), non-invasive imaging tool (Janib et al., 2013), and intracellular molecules (Pastuszka and Mackay, 2010). While useful as a purification tag, the ELP fusions also gain thermo-responsiveness with potential uses in drug delivery. This property was exploited by Topcic et al. in developing a strategy to overcome the barriers with current antiplatelet drug that can cause bleeding problem during cardiac surgery (Topcic et al., 2011). During cardiac surgery, long-lasting neurosurgical procedures, or out-of-hospital cardiac arrest, therapeutic hypothermia (deep: <20°C; moderate: 25~30 °C; mild: 31~34 °C) is often incorporated into surgical procedures to downregulate oxygen consumption and to protect organs from ischemia. However, applying therapeutic hypothermia can induce platelet activation, which leads to thrombocytopenia and possible severe blood loss. The currently approved drug that prevents platelet activation during surgery shows nonspecific binding to GPIIb/IIIa receptor and exhibit prolonged antiplatelet activity even after rewarming, which in turn, causes major post-operative bleeding complications. This group engineered a thermally sensitive scFv-EMP (Elastin-mimetic peptide, a derivative of ELPs) fusion that only binds to activated forms of GPIIb/IIIa under specific surgical hypothermic conditions, reducing the prolonged bleeding complications that are associated with the clinically available drug. While β-spiral structure of an EMP is located close to RXD motif of CDR3 region of fused scFv heavy chain and blocks recognition of platelet epitope at 37 °C, region that forms β-spiral structure becomes unordered and epitope recognition becomes available by CDR3 of scFv under specific hypothermic conditions. Due to this thermal responsiveness, scFv-EMP 21 fusions showed: i) enhanced affinity towards activated platelets; ii) blockage of fibrinogen binding to activated platelets; iii) inhibition of ADP-induced platelet aggregation; iv) prolonged arterial occlusion time at hypothermic conditions (below 28 °C) but not at physiological temperature (37 °C) compared to commercially available drugs, such as eptifibatide, tirofiban, or abciximab. The temperature responsiveness and tunability of Tt has made ELP-modification a favorable tool to modulate therapeutic entities. Antibody-ELP fusions sometimes self-assemble into a defined structure. Using a monoblock ELP, our group reported a scFv-ELP fusion that self-assembles into worm-like nanoparticles that are 80 nm in length 10 nm in width. These ‘Nanoworms’, in which a single- chain variable fragment (scFv) derived from anti-CD20 monoclonal antibody rituximab fused to a 192 repeats of VPGAG, significantly enhanced apoptosis in CD20 positive Raji and SU-DHL-7 cell lines, which was comparable to that of the rituximab crosslinked with GAH (goat anti-human Fc antibody) (Aluri et al., 2014b). Considering that the CD20 receptor clustering into the lipid raft on the surface of lymphoma cells enhances downstream apoptotic signaling cascade, it was evident that engaging multiple receptors with multivalent Nanoworms, induced receptor clustering on the cell surface and potentiated downstream apoptotic signaling, better than bivalent monoclonal antibody. In vivo studies showed the regression of tumor growth was pronounced in scFv-A192 treated group with an increased survival compared to the rituximab- treated group in a Raji xenograft mouse model. As opposed to self-assembly to worm-like or extended chain-like structures using monoblock ELPs, diblock ELPs form micellar structures. In this case a functional antibody fragment is fused to a hydrophilic block for proper presentation at the corona of micelles. Using diblock ELPs, Zhao et al. recently reported micellar scFv-ELP fusion nanoparticles that ablate the PD-1 immune checkpoint, in vitro and in vivo (Zhao et al., 2017). The amphiphilic immune- Tolerant Elastin-like Polypeptides (iTEP), NH2-(GAGVPG)70-(GVLPGVG)56–(GC)4, that they used to produce micellar structure is made up of a hydrophilic domain (GAGVPG)70, a 22 hydrophobic domain (GVLPGVG)56, and multiple cysteine residues at the C-terminus to stabilize the resulting micellar nanoparticles through disulfide bonds. An anti-PD-1 treatment worsens diabetes by ablating the immune checkpoint. Using this endpoint, this group tested the efficacy of anti-PD-1-iTEP fusion in non-obese diabetic mice (NOD/ShiLtj) and showed equivalent efficacy compared to an anti-PD-1 IgG control in exacerbating diabetes. While E. coli is a commonly exploited expression system for ELP or ELP fusions, Conrad and coworkers have pioneered the use of plant expression system, transgenic tobacco plant Nicotiana tabacum, to express functional antibody-ELP fusions (Conrad et al., 2011). They fused an anti-human tumor necrosis factor nanobody (anti-TNF-VHH) to 100 repeats of VPGXG (X: Gly, Val, and Ala with different ratios) to extend the systemic circulation half-life. By fusing ELPs, the serum half-life of anti-TNF-VHH was extended by 24-fold, from 0.5 hr to 11.4 hrs compared to a non-fusion control. This fusion protein was functional towards blocking the LPS/D-gal induced lethal septic shock in an animal model, while maintaining comparable survival rate to anti-TNF-VHH treated control animals. Although antibody-ELP fusions developed in pre-clinical studies seem to require further optimization to be evaluated in the clinic, ELPs do seem poised to allow new therapeutic opportunities in the near future. Currently, a long-acting vasoactive intestinal peptide (VIP) analogue in fusion with ELP (PB1046) is in a Phase 2b trial in pulmonary arterial hypertension (PAH) patients (NCT03556020). 1.3.1.2. Collagen-like polypeptides (CLPs) and collagen trimerization domain Collagen is the most abundant fibrous protein found in the extracellular matrix. Although there are many types of collagens with different roles in different tissues, a distinctive triple helix conformation characterizes the fibrotic nature of all collagens (Okuyama, 2008). Due to their mechanical properties, collagens are marketed predominantly for tissue engineering (Meyer, 2019) and their derivatives are explored in fusion with other recombinant polypeptides as a 23 tissue engineering material (Gurumurthy et al., 2018). The triple helical region contains repeats of the amino acid motif (Gly-X-Y)n, where X is mostly proline (Pro, P) and Y is mostly hydroxyproline (Hyp, O) or to a lesser extent, hydroxylysine (Hyl). About 50 % of GXY motif is comprised of GPO, largely owing to its high thermal stability (Persikov et al., 2000). The high imino acid content (compound that contains amide group bonded to the alpha carbon on the peptide backbone) and the presence of hydroxyproline significantly stabilizes the triple helix and collagen fibrils as a whole via π-π stacking and hydrogen bonding (Gelse et al., 2003). Although there are ways to process them to a medical grade material, collagen produced from various biological sources can sometimes cause heterogeneity as well as immunogenicity. As an alternative, collagen-like polypeptides (CLPs) with a signature motif (GPO)n have gained traction due to their well-defined hierarchical structure, ease of genetic engineering through recombinant technology, and superior resistance to enzymatic degradation. Several research groups recognized the triple helical nature of collagen as a platform to produce multivalent multi-specific antibodies. Fan et al. expressed anti-EGFR scFv-CLP fusion proteins in mammalian cells under the presence of 4-hydroxylase that hydroxylates prolines (Fan et al., 2008). The incorporation efficiency of hydroxyproline in GXY motif was about 60 % (maximum is 10 residues in (GPO)10). Expressed scFv-CLP fusions self-assembled into trivalent ‘collabody’ via CLP-mediated trimerization. Due to multivalency, the collabody showed a remarkable improvement in equilibrium dissociation constant (KD). The KD for the trivalent scFv- CLP was 4.8 nM, which was 1000-fold and 22-fold lower than monovalent scFv (4,960 nM) and bivalent scFv-Fc fusion (104 nM), respectively. The serum stability of scFv-CLP was better than scFv-Fc fusion or scFv alone that 60 % of its binding activity was retained after 72 hrs incubation at physiological temperature. In the same study, trivalent anti-CD3 scFv-CLP fusion was also generated, which showed a 3-fold reduction in IC50 compared to its parent monocloncal antibody, OKT3. This construct had better target binding and internalization capacity compared to its parent monoclonal antibody OKT3. 24 The trimerization domain found in non-collagenous (NC) segments in several collagen subtypes was also used to assemble functional antibody fragments. From early studies, chain selection and initiation of collagen trimerization rely on NC1 domain present at the C-terminus of the core triple helix domain (Khoshnoodi et al., 2006). Having no conserved or repeat sequences such as (GPO)n, the NC1 is stabilized mainly by hydrophobic interactions and hydrogen bonds. This particular role of the NC1 segment is found in all collagen types except two transmembrane collagens, XIII and XVII (Boudko et al., 2009). The NC1 segment in collagen XVIII was proposed as an antibody assembly platform for its relatively short residue (54 amino acids) compared to that of the other collagens. Álvarez-Vallina and co-workers pioneered the use of this NC1 segment to display antibody fragments. The resultant antibody fusion named ‘trimerbody’ formed a non-covalently oligomerized trivalent antibody. On one study, they constructed two types of monospecific trivalent trimerbodies and one bispecific hexavalent trimerbody, which the latter is a composite of two former trimerbodies (Blanco-Toribio et al., 2013). For the monospecific trimerbody, scFv was either fused to N- or C-term of NC1 segment, generating trivalent construct that displays three scFvs at the N- or C-term of triples helix. Either anti-laminin scFv or anti-CD3 scFv was fused to a N-term or C-term of a NC1 segment of human collagen XVIII, respectively. To generate the a bispecific hexavalent trimerbody, two different scFvs were fused to N- and C- term of NC1 segment, respectively. Anti-laminin scFv and anti-CD3 scFv were fused to N- and C-term of NC1, generating three anti-laminin scFvs displayed at the N-term of triple helix and three anti-CD3 scFv assembled at its C-term. Purified trimerized molecules showed high stability in serum. To test specificity and function, they incubated bispecific hexavalent trimerbodies on laminin coated plates and then attempted to target and activate T cells. By showing that laminin-immobilized bispecific hexavalent trimerbodies were better at T cell activation compared to immobilized OKT3 monoclonal antibody, they demonstrated the feasibility of collagen NC1 segment as a platform to assemble multispecific antibody fusions. 25 Following this success, the same group suggested two different constructs for cancer immunotherapy. The first construct that they proposed was named IMTXTRICEAaS, which contained three anti-carcinoembryonic antigen (CEA) scFv and three immunotoxin a-sarcin at the N- and C- terminus of the trimerization domain. Functional studies in CEA positive cells and colorectal cancer xenograft model suggested that the IMTXTRICEAaS had a more promising effect on cell survival and tumor suppression than its monomeric version (Lazaro-Gorines et al., 2019). The second construct was the anti-EGFR + anti-CD3 scFv bispecific construct called Asymmetric Tandem Trimerbody for T cell Activation and Cancer Killing (ATTACK). The difference between ATTACK and other modalities introduced in this section is the trimerization strategy. While other constructs were generated upon homodimerization via trimerization domains, the ATTACK was generated via intra-trimerization. In one DNA vector, three identical cDNAs that encode anti-EGFR nanobodies and the trimerization domain were consecutively cloned followed by the cDNA encoding an anti-CD3 scFv. This resulted in an intra-trimerized tetravalent molecule that is trivalent to EGFR and monovalent to CD3. In a flow cytometry experiment, ATTACK showed better binding to the EGFR receptor and similar binding to CD3 compared to the control molecules, which consists of a single anti-EGFR nanobody linked to a single anti-CD3 scFv (Harwood et al., 2017). ATTACK was able to activate T cells and redirect them to exhibit cytotoxicity towards EGFR positive HeLa cells, which was 10~20-fold more potent than the control molecules. Moreover, ATTACK was more potent in inhibiting the growth of EGFR positive A431 cells than the USFDA approved anti-EGFR antibody cetuximab. EGFR phosphorylation was decreased by 36 % upon cetuximab treatment, while it was 75 % for ATTACK. These two constructs, IMTXTRICEAaS and ATTACK, show not only improved pharmacokinetic properties, but also encouraging in vivo data that hopefully translates into human clinical data. 26 1.3.2. Human serum albumin (HSA) Human serum albumin (HSA, 66.5kDa) is the most abundant protein in the human blood that comprises approximately half of serum protein. Produced from the liver, it has a simple yet highly stable structure, regulates osmotic pressure of the plasma, and transports various metabolites such as long chain fatty acids, bilirubin, steroid hormones, tryptophan, calcium. Due to its long half-life (20 days) (Dennis et al., 2002) and tendency to accumulate in tumor cells (Wang et al., 1994), several therapeutic studies have explored albumin as a drug delivery vehicle to modulate the pharmacokinetics of short-lived pharmaceuticals. Interaction between albumin and neonatal Fc receptor (FcRn) on the surface of endothelial cells is the mechanism by which albumin maintains prolonged serum half-life. Its binding occurs in a pH dependent manner. Albumin binds tightly to the FcRn at a slightly acidic condition (pH 6.0) but dissociates from it at pH 7.4. This characteristic allows pinocytosed albumin tightly associated with FcRn upon endosomal acidification but readily released to the extracellular space, thereby escaping from a late endosome-lysosomal degradation pathway. Albumin or other molecules that contain the Fc region, such as IgG, maintain their half-life up to 2-3 weeks through this mechanism (Chaudhury et al., 2006). Blood flow is irregular in the tumor microenvironment, which is the major barrier for systemic delivery and homogenous distribution of the therapeutic compounds inside the tumor. While therapeutic entities that rely on passive diffusion to the tumor through the leaky vasculature may suffer from irregular blood flow, albumin has the ability to overcome this limitation. Albumin, albumin fusions, or albumin-drug conjugates traverse blood vessels (capillary lumen) through transcytosis by binding to albondin (gp60), which is selectively present on the surface of plasma membrane of continuous endothelium. It has been reported that up to 50% of albumin binds to albondin and transcytose, while the remainder traverse via intercellular junctions or fluid-phase endocytosis. Binding to albondin and subsequent internalization of the whole complex through caveolin-dependent endocytosis guarantees evasion of lysosomal 27 degradation and successful translocation to tumor interstitium (Elsadek and Kratz, 2012; Merlot et al., 2014). At the tumor interstitium, albumins or albumin fusions travel further by diffusion and convection with the aid of SPARC (Secreted Protein Acidic and Rich in Cysteine), which is overexpressed in tumor cells to facilitate extracellular matrix proliferation and tumor cell migration. SPARC captures the albumin-gp60 complex through a high-affinity interaction with albumin and facilitates tumor accumulation (Podhajcer et al., 2008). SPARC-albumin interaction enables homogenous intratumoral distribution of albumin or its fusions, which is often limited in untargeted therapeutic formulations or conventional monoclonal antibody. This distinct transportation mechanism of albumin was extensively utilized to improve the therapeutic index of short-lived antibody fragments. McDonagh et al. focused on HER2-HER3 dimerization in HER2-overexpressing tumors, such as breast and gastric carcinoma (McDonagh et al., 2012). In tumor cells, cell surface receptor HER2 and HER3 preferentially dimerize to activate the AKT pathway and maintain a strong oncogenic signal. Heregulin binding to HER3 with very high affinity induces tumor growth without inducing overexpression of HER3, mediates the resistance to anticancer agents targeting HER receptor family, and leads to poor prognosis. Due to the lack of an active kinase domain and significant overexpression of HER3, HER2 has been the primary therapeutic target for interrupting the HER2-HER3 heterodimeric complex. Currently available HER2 targeting drugs, trastuzumab and lapatinib, have greatly improved treatment outcome of the HER2 overexpressing patients (~30%) but still significant proportion of patients (~70%) do not benefit from these drugs due to the lack of HER3 inhibition. To address these limitations, they developed a novel bispecific single polypeptide albumin fusion (MM-111) protein in which one arm binds to HER2, and the other arm binds to HER3. MM-111 showed better inhibition of ligand-induced oncogenic signaling when compared to trastuzumab or lapatinib treatment alone. Furthermore, cotreatment of MM-11 with trastuzumab or lapatinib significantly inhibited tumor growth by suppressing p-AKT signaling. MM-111 also extended in vivo half-life. Serum half-life of the MM-111 in the murine model was 16.2~17.5 hours, a 28 significant improvement from ~5 hours observed for scFv alone control. MM-111 even achieved circulating serum half-life of 99 hours in cynomolgus monkeys, making it a promising agent as a second-line treatment for HER2-overexpressing tumors. However, despite successful completion of a Phase I clinical trial, the further development of MM-111 as a second-line treatment for metastatic HER2-positive gastric and gastroesophageal junction (gastric/GEJ) cancers was terminated due to inferior progression-free survival in the subjects. Yazaki et al. engineered an scFv-albumin fusion as an imaging agent. They made genetic fusion of a murine anti-CEA monoclonal antibody T84.66 scFv to a truncated HSA that referred to as ‘immunobumin’ (Yazaki et al., 2008). Two different radiolabeled immunobumins, [ 125 I]-T84.66 immunobumin and [ 111 I]-DOTA-T84.66 immunobumin, were used to study biodistribution in human colon carcinoma LS-174T xenografts. [ 125 I]-T84.66 immunobumin showed significant increase in tumor uptake of 22.7% ID/g at 18h, compared to the 4.9% ID/g for scFv alone. Tumor uptake was even more prominent for [ 111 I]-DOTA-T84.66 immunobumin, which was marked at 37.2% ID/g at 18h. Remaining at the tumor site after 72h was 27.3% ID/g with a tumor-to-blood ratio of almost 19:1. By showing a higher tumor uptake and blood half-life for immunobumins compared to scFv alone control, they successfully demonstrated the potential of immunobumins as an alternative imaging tool. One of the challenges in producing antibody-albumin fusions is the deterioration of albumin’s binding to its cognate receptor. Anderson et al. recently investigated binding capacities of various albumin fusion proteins to their cognate receptor FcRn. They fused a short peptide or scFv to the N- or C-term of albumin and tested FcRn binding to examine the binding capacity of generated fusions (Andersen et al., 2013). Fusion of the small peptide and scFv at the N-terminus of albumin did not result in a significant reduction in binding (although there was a trend in reduction) whereas fusion to its C-terminus resulted in a 2-fold decrease in KD. While a small difference in KD may seem negligible, it is possible that the difference will be amplified in an in vivo environment, where the albumin fusions have to compete with 40 mg/ml of intact 29 albumins in the blood for FcRn binding. To be used as an efficacious fusion platform, extensive biochemical engineering to improve FcRn binding is required for albumin fusions. To circumvent the abovementioned issue, current efforts in utilizing albumin for antibody delivery include incorporation of albumin binding domains within a therapeutic antibody. The three most successful platforms are Albumod ™ (MedImmune, now licensed to Affibody), AlbudAb ™ (GSK) and Albumin binding Nanobodies ® (Ablynx). Albumod ™ platform uses albumin binding domain (ABD) identified in protein G of Streptococcus strain G148. ABD is a 46 amino acids polypeptide that forms three-helix bundle with affinity (KD) of ~4 nM to HAS (Stork et al., 2007). The ABY- 035, an Affibodies’ multivalent bispecific molecule that has been tested in a Phase II clinical trial, is an 18 kDa fusion of Affibody ® and Albumod ™ that exhibits antibody-like half-life (www.affibody.se). AlbudAb ™ platform uses anti-albumin single domain antibody (variable heavy chain or variable kappa chain) selected from a phage display (Holt et al., 2008). GSK2374697, a fusion of Exendin-4 and anti-albumin variable kappa chain (Vk) showed an increased half-life of Exendin-4 from ~2.5 hrs to 6 days in humans (O'Connor-Semmes et al., 2014). GSK3128349, a fusion of 89 Zr and AlbudAb ™ , was also tested in the clinical trial using PET imaging (NCT02829307). Ablynx uses albumin binding Nanobodies ® for half-life extension. Ozorulizumab (trivalent, bispecific antibody), M1095/ALX-0761 (trivalent, trispecific antibody), and Vobarilizumab (bivalent, monospecific antibody) are examples of albumin binding nanobodies that have entered clinical trials (Table 1). 1.3.3. Transmembrane proteins for exosome retargeting Exosomes are naturally occurring lipid-bilayer vesicles with 30-150 nm in hydrodynamic diameter that are secreted by most cell types as intercellular signals. Inspired by their conventional role in delivering nucleic acids, amino acids, and proteins for intercellular communications (Kalluri and LeBleu, 2020), therapeutic exosomes are gradually expanding the list of cargos to siRNA and miRNA for gene therapy, chemotherapeutic compound such as 30 paclitaxel and doxorubicin, cell growth inhibitors, viral vectors, and bioimaging agents (Susa et al., 2019). To be used as therapeutics, exosomes have been engineered to have active targeting properties. Generating targeted exosomes necessitates an in-depth understanding on the complexity of the exosome lipid bilayer composition, which is different from widely used lipid-based nanoparticles, such as liposomes or polymeric micelles (Kooijmans et al., 2012). It requires overexpression of the fusion polypeptide in the donor cells, high enrichment of that particular fusion material throughout the exosome biogenesis pathway, and eventual secretion as a part of the exosomes. On top of that, overexpressed targeting moieties should decorate the corona of the exosome bilayer (Johnsen et al., 2014). Incorporation of targeting moieties, such as RVG (targeting acetylcholine receptor) (Alvarez-Erviti et al., 2011), folate receptor α (FRα, for crossing blood brain barrier) (Grapp et al., 2013) or GE11 (EGFR targeting peptide) (Ohno et al., 2013) have been investigated to achieve targeted delivery of exosomes. Among several exosomal proteins, membrane proteins that are highly enriched in exosomes were explored as a fusion platform to self-assemble and correctly display antibody fragments on exosome surfaces. Kooijimans et al. produced nanobody displaying exosomes from Neuro2A cells transfected with the vector that encodes anti-EGFR nanobody followed by 37 amino acids of human decay-accelerating factor (DAF). DAF (or CD55) is a 70 kDa protein that interacts with cell membrane bilayer via glycosylphosphatidylinositol (GPI) anchor, and known to be selectively sorted into secreted exosomes during reticulocyte maturation (Rabesandratana et al., 1998). The expressed nanobody-DAF fusion gets inserted into a lipid bilayer after the DAF peptide is replaced with a GPI anchor through post-translational modification. Purified exosomes were enriched with conventional exosome markers, showed identical size distribution to the unmodified exosomes, and retained ‘cup-shaped’ morphology that is a hallmark of exosomes. Nanobody-decorated exosomes successfully targeted EGFR expressing tumor cells under static cell cultures as well as flow conditions (shear rate: 82.5 s- 1 ), which was selected to 31 mimic the typical flow rate of vein (shear rate: 20-200 s- 1 ). However, their modality had limitations with regard to suboptimal cellular uptake attributed to both a low expression level and the GPI anchor-mediated segregation of nanobodies into the lipid-rafts on the host cell surface. This resulted in non-reproducible decoration of nanobodies on exosomes with low surface density. However, genetic modificaiton may be better in maintaining the functionality of the exosomal proteins compared to the chemical post-conjugation, which oftentimes transforms functional proteins non-functional. Limoni et al. achieved siRNA delivery to HER2 positive breast cancer cells by engineering exosomes with antibody-mimetic peptide DARPin (Limoni et al., 2019). By fusing anti-HER2 DARPin between the signal peptide and the mature gene segment of a membrane protein Lamp2b in lentiviral vector, they produced DARPin decorated exosomes that contain 75 kDa Lamp-DARPin chimeric proteins. This anti-HER2 DARPin exosomes, loaded with siRNA, successfully targeted HER2/neu positive cells, internalized, and suppressed gene expression of tumor protein D52 (TPD52) by 70 %. In another study, the DARPin-decorated exosomes were used for tumor imaging (Molavipordanjani et al., 2020). In a SKOV03 ovarian cancer xenograft model, they showed significantly higher accumulation of 99m Tc-DARPin-exosomes compared to trastuzumab pre-treated (saturates HER2 receptors) animals. This demonstrates possible therapeutic and imaging applications of targeted exosomes. Shi et al. developed an exosome platform termed synthetic multivalent antibodies retargeted exosome (SMART-Exo), which displayed both anti-human epidermal growth factor 2 (HER2) and anti-CD3 scFv on the exosome surface (Shi et al., 2020). Their aCD3+aHER2 bispecific SMART-Exo utilized the human platelet-derived growth factor receptor (PDGF-R) transmembrane domain for self-assembly and display of scFvs on the exosome surface. Generated SMART-Exo showed a dose dependent T cell activation in the presence of HER2 positive HCC 1954 cells, which confirmed its ability to recruit CD3 positive T cells to HER2 positive cells. Under co-incubation of non-activated peripheral blood mononuclear cells 32 (PBMC)+SMART-Exo mixture and HER2 positive cells (SK-BR-3 and HCC 1954) at a 10:1 ratio, SMART-Exo exhibited cytotoxicity with EC50 of 0.85±0.23 ng/ml and 50.2±7.67 ng/ml (concentration of total exosome protein) in SK-BR-3 and HCC 1954 cells, respectively. Under the same condition, SMART-Exo had no effect on HER2 negative MDA-MB-468 cells, and PBMC depleted with CD3 positive cells had no effect towards cell viability of HER2 positive cells. Intravenous SMART-Exo showed a significant tumor suppression in immunodeficient NSG mice that were subcutaneously implanted with HCC1954 cells. No toxicities were observed as the body weight, creatine level (kidney damage marker), and alanine aminotransferase (liver damage marker) was similar to that of control animals. Their data demonstrates the encouraging potential for bispecific SMART-Exo in the immunomodulation of breast cancer. 1.4. Non-human proteins as fusion platforms for antibody assembly 1.4.1. Leucine zippers in yeast protein GCN4 Bispecific or multi-specific antibodies are attractive strategies to enhance therapeutic outcomes (Labrijn et al., 2019). With the maturation of technology to engineer functional Fabs and scFvs, other methods, beyond the Fc domain, have been explored to control their degree of multivalency. In particular, dimerization or oligomerization domains within naturally occurring proteins have been evaluated to generate multispecific, multivalent therapeutics. Similar to the use of CLPs or the collagen trimerization domain to self-assemble antibodies, a leucine zipper found in the yeast transcription factor GCN4 has been utilized for antibody assembly. Leucine zipper is a coiled-coil structure of two α-helices. Its homodimerization is processed through a hydrophobic interaction between leucine residues that are present at every 7 th residue within an α-helix forming peptide, such as RMKQLEDKVEELLSKNYHLENEVARLKKLVGER (leucine residues are bold and underlined) (O'Shea et al., 1991). This homodimerization can be utilized to generate self-assembled multivalent antibody fusions. For example, two α-helix leucine zipper forming peptides, each 33 peptide fused with an antibody fragment either at its C- or N-terminus, become bivalent antibody complexes upon expression and homodimerization (Pluckthun and Pack, 1997). Since Pack et al. reported successful expression of scFv-leucine zipper fusion (scFvZIP) in E. coli and validated its binding activity through ELISA (Pack et al., 1993), leucine zipper and its variants were further investigated as an antibody fusion platform. One of the strategies selected for further development was amino acid substitutions within the α-helix forming peptide that produced multimeric leucine zippers. It was identified that the hydrophobic residue substitutions within the heptad repeat (a and d residues in abcdefg heptad repeat within α-helix) affects oligomeric state of the leucine zipper, from dimeric to trimeric, and tetrameric (Harbury et al., 1993). Hydrophobic residues (valine, leucine, and isoleucine) present at a and d positions that are buried in the coiled-coil structure is known to drive dimerization of leucine zippers. During point mutation analyses, incorporation of isoleucine at d position in every heptad repeat relaxed the tight ‘knobs-into-holes’ configuration formed by interactions between a and d amino acids, and results in trimeric leucine zippers. Generated trimeric leucine zippers was further modified to become a tetrameric by incorporating leucine at a position in every heptad repeat. Having leucine and isoleucine at every a and d positions transformed dimeric to tetrameric leucine zippers. Based on these molecular characteristics, Klement et al. generated scFv-ZIP (dimeric), scFv-TriZIP (trimeric), and scFv-TetraZIP (tetrameric) that target cell-surface podocalaxin like protein-1 (Klement et al., 2017). The rationale for developing multivalent scFv-ZIPs would be to achieve better tissue penetration as well as improved retention compared to the parent monoclonal antibody IgM84. IgM84 shows cytotoxicity towards undifferentiated human embryonic stem cells (hESC) by clustering podocalaxin like protein-1, which is known to induce cell death similar to oncosis (cell death with swelling); however, it shows inferior tissue penetration due to its size, thus limiting its use in removing undifferentiated hESC from differentiated cells in vitro prior to transplant into the patient. To address this issue, scFv-ZIPs were tested for their cytotoxicity and tissue 34 penetrability compared to IgM84. In terms of cytotoxicity, all scFv-ZIPs were less potent than mAb84, while scFv-TetraZIP was better tolerated than both scFv-ZIP and scFv-TriZIP due to its high valency. On the contrary, scFv-ZIPs were better for tissue penetration than mAb84. More than 80 % of the given dose was found in the hESC–derived embryoid bodies (EB) for all scFv- ZIPs, which was remarkably higher than that of the mAb84 (25 %). Although all scFv-ZIPs showed better penetrability than IgM84, the ability to penetrate to the core of the tissue was slightly different among scFv-ZIPs. A significant fraction of scFv-ZIP or scFv-TriZIP was found in the core of the EB, while scFv-TetraZIP was scarce at the center of the tissue, possibly due to its high MW (127 kDa) and valency (4). Based on cytotoxicity and tissue penetrability, it was concluded that the scFv-TriZIP may be a better candidate over scFv-ZIP or scFv-TetraZIP for further studies. This conclusion also conforms to our earlier discussion in this review on antibody affinity (or avidity) and tissue penetration. While the leucine zipper from GCN4 remains the most extensively studied platform for α- helical peptide assembly, other human homologs have been explored for antibody assembly. Ojima-Kato et al. tested two kinds of leucine zippers, c-Fos/c-Jun leucine zipper pair identified in humans and artificially designed leucine zipper pair LZA/LZB, to correctly pair the Fab that targets O157, one of the O-antigens, on the surface of E. coli. (Ojima-Kato et al., 2016). It should be noted that the purpose of the study presented by Ojima-Kato et al. is to use leucine zippers to correctly fold the heavy and light chain of Fab, whereas Klement et al. utilized leucine zippers to produce multivalent antibody formulations. Fab domains contain multiple disulfide bonds that require correct folding. Fab fragments expressed under the oxidizing conditions, i.e., cytoplasm of E. coli, are usually recovered from inclusion bodies as these conditions result in incorrectly or partially folded non-functional material. To determine if leucine zippers enhance the correct folding and solubility of Fab, heavy chain and light chain of Fab was fused to Fos and Jun (or LZA and LZB), respectively, and their pairing was mediated by leucine zipper heterodimerization. The generated two types of Fab fusions, Fab-Fos/Jun and Fab-LZA/LZB 35 (named Zipbody) bound specifically to E. coli strain that expresses O157 but not to other strains. Fab domains expressed without a leucine zipper failed to bind their targets. This Zipbody platform was further optimized for the antibody screening process to rapidly develop monoclonal antibodies via cell-free protein synthesis (Ojima-Kato et al., 2018). 1.4.2. Fibroin - a silk protein About 150,000 species including spiders and insects can produce silks (Pham and Tiyaboonchai, 2020). Among all different kinds of silks, mulberry silk produced by silkworm Bombyx mori is mostly use in textile industries (Pham and Tiyaboonchai, 2020). A silk fiber produced by B. mori is composed of fibroin and sericin. Sericin is a glycoprotein that surrounds the core protein, fibroin. Fibroin is composed of three parts: heavy chain, light chain, and glycoprotein P25 (Qi et al., 2017). The 12 hydrophobic anti-parallel β-sheets stabilized by hydrogen bonds and van der Waals forces make the core of the heavy chain. Within this core, an extended segments of amino acids (GAGA)nG(Y/S) were found to be responsible for the physico-chemical properties and crystallinity of silk films and silk fibers. The hexapeptide repeats (GAGAGX)n derived from this motif has been recombinantly expressed and named as silk-like polypeptides (SLP) (Cappello et al., 1990; Wang et al., 2009). Due to their fibrous nature, both fibroin and SLPs are widely used in tissue engineering (Huang et al., 2015). Even though the heavy chain of fibroin or its recombinant derivative SLPs are widely used, it may not be a suitable platform for antibody assembly due to their tendency to form crystalline domains. Instead, a more hydrophilic segment of fibroin was explored as a fusion site (Hino et al., 2006). The hydrophilic nature of the fibroin light chain produces elastic semi- crystalline protein instead of insoluble fibers (Wadbua et al., 2010). The light chain of the fibroin is different in its amino acid composition and solubility. Certain amino acids (aspartic acid, alanine, glycine, and serine) that are relatively hydrophilic comprise approximately 50% of light chain; however, there is no consensus motif. One group in Japan pioneered the use of this light 36 chain in antibody assembly using transgenic silkworms (Sato et al., 2012). The scFv-fibroin light chain fusions (scFv-FibL) that were purified from the cocoons of the transgenic silkworms retained their binding activity towards Wiskott-Aldrich syndrome protein (WASP) after they were freeze-dried to powder. Compared to its parent molecule anti-WASP mAb, scFv-FibL showed equivalent immunoprecipitation capability towards recombinant WASP and native WASP extracted from cell lysates. After this initial success, they expanded to target CEA and validated its function using ELISA (Sato et al., 2017; Sato et al., 2014). Interesting feature across their reports is that scFv-FibL maintained binding activity regardless of its formulation: powder, thin film, and aqueous solution. Although they showed a novel approach to self-assemble antibody fragments in silk proteins, low expression was reported as a limitation. The expression level of scFv-FibL was about 10~25 % of total light chain proteins or 1~2 % of total silk proteins extracted from cocoons (Inoue et al., 2000). 1.4.3. Viruses for drug discovery Phage display is a technology that enables natural or synthetic nucleotide sequences expressed at the surface of the bacteriophage (virus that infect bacteria). Since its first introduction in 1985 (Smith, 1985), phage display has been employed in epitope discovery, novel antibody sequence identification, affinity maturation, ligand affinity enhancement, and protein-protein or protein-ligand interaction discovery (Pande et al., 2010). Filamentous bacteriophages (M13, f1, fd phage), bacteriophage λ (lambda phage), and other lytic bacteriophages (T4 or T7) are commonly used for phage display (Beghetto and Gargano, 2011; Tikunova and Morozova, 2009). Filamentous phages are often preferred compared to others in several aspects. First, genetic modification on non-essential gene segments minimizes disturbance on phage packaging. Second, high yield can be achieved due to non-lytic propagation and easy isolation of phage genome. Third, filamentous phage assembly happens in the periplasmic space, which has fewer effects on host cell viability. However, their assembly 37 in the periplasmic space limit its application to a subset of peptides that can tolerate oxidizing condition of periplasmic space (Tikunova and Morozova, 2009). Lambda phage or lytic bacteriophages are used to overcome this limitation because they assemble their capsid proteins under reducing conditions in the cytoplasm and secrete viral particles upon host cell lysis (Archer and Liu, 2009; Beghetto and Gargano, 2011). Oligonucleotide sequence incorporation to filamentous phage vector is conducted on the genes that encode surface coat proteins, usually at the N-term of pIII, pVII, pVIII, pIX genes or C-term of pVI gene (Løset and Sandlie, 2012). One of the most widely used fusion sites is between a signal peptide that directs bacteriophage to the periplasm and the N-terminus of the actual gene encoding pIII, mainly due to the low copy number of pIII (5 copies). This low expression level is an advantage that it allows identification of highest affinity antibody sequences to their targets. In case of lambda phage, the gene of interest is usually fused to the N- or C-terminus of the gene that encodes major capsid protein gpD. Fusion at this position does not affect the replicability or infectivity of the host phage (Maruyama et al., 1994; Sternberg and Hoess, 1995). When gpD gene (405-420 copies per phage) is used as a fusion site, both wild-type gpD and fusion gpD genes are co-expressed to make the fusion product less crowded on the capsid surface (Castagnoli et al., 2001). For lytic phage T4, genes that encode capsid protein Soc or Hoc are used as fusion sites (Wu et al., 2007). Since these two fusion genes can be simultaneously expressed on one virion, these two sites are used to produce phages with high copy fusion numbers. For lytic phage T7, genes that encode capsid protein gp10A or gp10B are oftentimes used as fusion sites (Bratkovic, 2010). As fusion of large proteins (<1,200 amino aicds) into these genes produce low copy numbers on the capsid, these sites become popular targets for large protein display. To identify desired amino acid sequences, the expressed bacteriophage fusions (referred to as phage library, approximately 10 6 ~10 11 variants) are subjected to biopanning, which is based on sequential affinity selection of peptides against an immobilized target 38 molecule. After removing unbound phages, those that are tightly bound to the target are eluted. Owing to phenotype-genotype linkage (the gene inside the phage contains specific DNA sequence for the displayed fusion partner), DNAs from the eluted phages are further amplified. Usually, 3~5 rounds of biopanning elutes phages with the highest affinity peptides towards the target of interest (Ellis et al., 2012). After target enrichment steps, additional steps are often incorporated, such as negative selection or phage competition, to remove selection bias (Vodnik et al., 2011). Many clinically approved vaccines and therapeutic agents were developed on various phage display platforms, which differ in phages and technology (Nixon et al., 2014). Among the different phages and technologies used, filamentous phage to develop therapeutic antibodies were discussed as examples. Use of other phages for drug development is elaborated elsewhere (Sokullu et al., 2019). Adalimumab (Humira ® ) was the first phage display derived antibody approved by the USFDA in 2002 for the treatment of rheumatoid arthritis, juvenile idiopathic arthritis, psoriatic arthritis, ankylosing spondylitis, Crohn’s disease, ulcerative colitis, and plaque psoriasis (Bain and Brazil, 2003). This particular antibody was generated through ‘guided selection’ against human tumor necrosis factor alpha (hTNFα) (Osbourn et al., 2005). The library of heavy chain genes of rodent hTNFα antibody (mouse VH-human Cμ1) was expressed and secreted from bacteria, while the library of light chain genes of rodent hTNFα antibody (mouse Vκ-human Cκ) was fused to phage coat protein pIII, expressed and secreted. Then the secreted heavy chains and phage-bound light chains were randomly paired and subjected to biopanning against immobilized hTNFα. Rodent heavy chain sequences identified from target-bound phages were then randomly combined with a library of human light chains and screened again to identify the best human light chain that targets hTNFα. After that, human heavy chains were randomly paired with selected human light chains and screened again, to achieve a fully human sequence. After affinity maturation (Kd=10 -8 M or less), clone D2E7 was patented and became 39 available in the clinic under the trade name Humira ® (Bain and Brazil, 2003). It became the third drug that targets hTNFα after infliximab (human-murine chimeric antibody) and etanercept (TNFR-IgG Fc fusion protein), but it was the first fully human antibody among the three. Due to its high affinity and potency, it is one of the top 5 therapeutic antibodies with annual sales of more than 20 billion dollars (Schumock et al., 2019). Belimumab is the first approved antibody for the treatment of systemic lupus erythematosus (SLE), which relied on non-specific immunosuppressants for more than 50 years (Sanz et al., 2011). Despite the lack of understanding of the pathogenesis, SLE patients usually show increased levels of B-lymphocyte stimulator (BLyS), thus making it a favorable target for SLE treatment. BLyS is present in our body as two forms, membrane-bound inactive form and free soluble active form (Moore et al., 1999). Soluble form of BLyS interacts with three kinds of B cell receptors: a transmembrane activator and calcium modulator BLyS receptor 3 (BR3); the cyclophilin ligand interactor (TACI); and B cell maturation antigen (BCMA). BLyS-BR3 interactions are known to be the strongest among the three interactions, by which helps autoantibody-producing B cells to evade apoptosis and facilitate proliferation. Belimumab binds to the soluble form of BLyS and hinders its association with B cell receptors. To identify specific sequence that targets BLyS, naïve VH and VL repertoires were constructed from amplifying human VH gene segments and human kappa and lambda VL gene segments isolated from B- cells of 43 non-immunized donors. Through two-fragment PCR assembly, individual VH and linker-VL DNA fragments were amplified, assembled (VH-linker-VL), and expressed as a pIII fusion, achieving scFv repertoire of approximately 1.4 x 10 10 recombinants. Candidate scFv- phages that tightly bind to immobilized BLyS were identified after 3 rounds of biopanning. Their affinity was further enhanced by introducing mutations on the CDR3 region of the heavy chain, which was then subjected to another round of biopanning followed by 12 rounds of selection process using various concentrations of BLyS. The identified candidate, LymphoStat-B (later named belimumab, trade name: Benlysta ® ), exhibited an EC50 value of 0.024 nM on solid-phase 40 capture and IC50 value of 8.5 nM against soluble BLyS (Baker et al., 2003; Vaughan et al., 1996). The USFDA approved the phage display derived antibody called raxibacumab (Abthrax ® ) on 2012 for the prophylaxis and treatment of inhaled anthrax (Kummerfeldt, 2014). Development of this particular antibody was supported by ‘The Project Bioshield Act’ signed into law in 2004, aimed at overcoming the limitations of available therapies against prophylaxis after the devastating terrorism attack in New York City (USA, 2001) (Inglesby et al., 2002; Meadows, 2004). Raxibacumab binds to the domain IV epitope of Bacillius anthracis protective antigen (PA) and prevents its binding to the cell surface receptors ANTXR1/2 with an IC50 value of 503 pM, thus indirectly deterring binding of lethal factor (LF) and edema factor (EF) internalization via PA heptamer pore. Raxibacumab provides complete protection in rabbits and 90% protection in monkeys during prophylactic studies. Post-exposure survival rates are 44 % and 64 % on rabbits and monkeys, respectively (Migone et al., 2009). The phage display technology has become a critical component in drug discovery to protect the general public from biological threats. After SARS (Severe Acute Respiratory Syndrome) and MERS (Middle East Respiratory Syndrome) incidents, Feng and coworkers at the United States National Cancer Institute (NCI) developed a phage display library based on VNAR to identify lead therapeutic candidates to neutralize these viruses (English et al., 2020; Feng et al., 2019). It is currently extensively employed by various pharmaceutical companies to find a lead compound for the recent COVID-19 pandemic. 1.4.4. Viruses for drug delivery Despite their potential for immunogenicity, the superb specificity and infectivity of viruses have shed light on viral surface engineering to redirect them to particular tumor tissues. Surface engineering can be performed in several ways (Waehler et al., 2007). Pseudotyping is a method to transplant viral glycoproteins from other viruses, either from the same family or between 41 families, to manipulate the original tropism (affinity to bind a specific target) and expand the host range of the virion (Cronin et al., 2005a). Human immunodeficiency virus type 1 (HIV-1) has shown to incorporate glycoproteins from other viruses through phenotypic mixing (pseudotyping) to expand its host range (Cronin et al., 2005b). It can be applied to both enveloped viruses (retrovirus or lentivirus) and non-enveloped viruses (adenovirus or adeno- associated virus). Compared to adaptor systems, genetic engineering, or polymer coating, pseudotyping is less technologically challenging; however, the limited availability of pseudotypes have restricted their applications (DiPaolo et al., 2006; Rebuffat et al., 2010). Alternatively, adaptor systems can be employed to modify intact viral particles. These include: i) utilizing a peptide that binds to cognate virus receptor (e.g CAR: coxsackievirus and adenovirus receptor) to graft molecules on the surface of the virus (Pereboev et al., 2004); ii) bispecific antibodies where one variable domain binds the virus and the other end targets a receptor of interest (Würdinger et al., 2005); iii) chemical linkages that covalently link a targeting peptide to the viral surface (Stephanopoulos et al., 2010); iv) avidin-biotin interactions whereby a biotinylated virus interacts with an avidin-bound targeting moiety (Purow and Staveley- O'Carroll, 2005); and v) an antibody that functions as a targeting moiety attached to virus via immunoglobulin (Ig) binding domain expressed on viral surface (Konno et al., 2011). Adaptor systems have little effect towards the original viral structure, allowing it to be explored with different targeting molecules with very simple procedures. One limitation of this method is that the incorporated adaptors add additional regulatory burdens during clinical evaluations as these are considered a separate therapeutic entity. Third approach is a genetic modification. Several examples include: i) serotype exchange that produces chimeric virus decorated with a donor serotype (Kaufmann and Nettelbeck, 2012; Särkioja et al., 2008); ii) fusion of a targeting peptide (Shi and Bartlett, 2003); iii) scFv expressed on the outer surface of the virus particle (Russell et al., 1993); iv) two or more distinct viral attachment proteins expressed on one virus particle (mosaicism) (Gigout et 42 al., 2005); and v) gene mutations that ablates original viral tropism to reduce viral transduction into off-targets (Yun et al., 2005). Genetic fusion enables homogenous production, however, technical barriers still exist in terms of deteriorating or changing original structure and function of the virus, such as replication and encapsulation. A fourth strategy is the incorporation of a polymer coating to evade opsonization and neutralization. Polyethylene glycol (PEG) (Wortmann et al., 2008) and poly-N-(2-hydroxypropyl) methacrylamide (poly-HPMA) (Carlisle et al., 2008) are widely used polymers to coat viral particles, which helps them escape clearance by the mononuclear phagocyte system (MPS). Among the introduced strategies, genetic modifications on the genes that encode viral capsid proteins or envelop proteins have been extensively investigated for antibody fusion after a landmark report by Russell et al. in 1993 (Russell et al., 1993). Despite challenges, technological advancements have enabled a successful assembly of antibody fragments on the viruses with high titer and exceptional specificity. Chowdhury and coworkers fused anti-CEA scFv to the N-terminus of the murine leukemia virus surface subunit A, allowing the scFvs to assemble on the surface of retrovirus envelop (Chowdhury et al., 2004). After scFv-retrovirus binds to CEA, viral particles transduce into tumor cells via interaction between its surface unit gp70 and Pit-2 on the target cell. Attributed to specificity and transduction efficiency, CEA positive tumor xenografts (HT29 and Mawi) were highly transduced, while CEA negative tumors were not (A375 and HT1080). They claimed that it was 10 4 times less likely for scFv-retrovirus to infect host organs, as proviral DNA was not detected in spleen, liver, and kidney, in CEA positive tumor xenografts. Achievement of high titer and maintenance of in vivo transduction efficiency demonstrated the feasibility of scFv fused virus as targeted therapeutics. Nakamura and coworkers tested two different retargeted oncolytic measles viruses (MV) by scFv fusion at the C-terminus of the H (hemagglutinin) gene. To precisely evaluate the scFv- mediated targeting ability, the H gene was mutated to blind MV from its natural receptors CD46 and SLAM (Nakamura et al., 2005). scFv-MVs that target either CD38 or EGFR had specificity 43 against EGFR positive cell line (SKOV3ip.l) or CD38 positive cell lines (Ramos and Raji), respectively, with no residual infectivity on target negative control cells. When these were injected intratumorally, both anti-EGFR-MV and anti-CD38-MV showed a remarkable anti-tumor activity in their respective subcutaneous xenograft models. Upon intravenous administration, anti-CD38-MV treated metastatic Raji xenografts showed the best survival rate compared to animals treated with unmodified MVs or anti-EGFR-MVs. scFv-MV-H fusion was further characterized by Ayala-Breton et al. (Ayala-Breton et al., 2012). They pseudotyped VSV (an oncolytic vesicular stomatitis virus) with both scFv-MV-H and scFv-MV-F (hemagglutinin (H) and fusion (F) envelope glycoproteins) to target either EGFR, alpha-folate receptor (αFR), or prostate-specific membrane antigen (PSMA). In these fusions, they ablated the original tropism of VSV by deleting G gene from its genome (VSVΔG). Therefore, reconstituted scFv-MV-H/F pseudotyped VSVΔGs were neutralized only by anti- measles serum, but not by anti-VSV serum. Cells expressing either EGFR, αFR, or PSMA were efficiently targeted by pseudotyped VSVΔGs, while cells expressing CD46 or SLAM were not. This confirmed infectivity was dependent on the pseudotyped scFv-MV-H/F proteins onto VSVΔG. The in vitro specificity was also fully maintained in in vivo. The gene expressions of green fluorescent protein were only restricted to the receptor positive tumors in SCID or athymic xenografts engrafted with various cell lines: KAS 6/1 (EGFR-, αFR-, PSMA-), SKOV3ip.1 (EGFR+, αFR+), PC3 (PSMA-), and PC3-PSMA (PSMA+). Antibody-decorated ‘onco-lytic’ viruses, that lyse the oncogenic tissues, have been explored for the treatment of cancer. Using oncolytic viruses has proven challenging for multiple reasons. First, viral producer cells tend to die before substantial viral particles are expressed. Moreover, the spread of oncolytic virus inside the tumor is insufficient, which seems to lead to suboptimal therapy. However, due to their exceptional infectivity, they may still possess therapeutic potential. To address these challenges, Fernández-Ulibarri and coworkers genetically linked a peptide that is a fusion of anti-EGFR scFv and onconase (ONCEGFR) to the 44 capsid of oncolytic adenovirus (Fernández-Ulibarri et al., 2015). Onconase (sometimes referred to as immunoRNase) is an RNase from the Northern leopard frog Rana pipiens that resists the human cytosolic RNase inhibitor and its expression in the cytosol degrades endogenous RNAs and blocks proliferation of human cancer cells (Lee, 2008; Rybak et al., 2009). On top of ONCEGFR fusion, additional modifications were explored to eliminate EGFR positive cells that were not initially infected: i) viral expression was optimized to maintain full replication by shifting Onconase expression until the late phase of replication; ii) deletion mutation in pRb interacting domain of E1A (E1AΔ24) for cancer selective replication of the virus; iii) supplemented with adenovirus serotype 3 knob, thus making chimeric serotype 5/3, for better infection. Produced viral particles, ONCEGFR-adenovirus, showed strong binding and specificity to EGFR and induced cytotoxicity in all EGFR positive cells A431, Cal27, Panc-1, and primary HNO210 cells. Especially, they observed more than 100-fold improvement in IC50 on A431 cells compared to control virus treated A431 cells. ONCEGFR-adenovirus had no effect in the receptor negative Mel624 melanoma cell line. The ONCEGFR-adenovirus was especially effective in overcoming the antibody resistance. A non-small cell lung cancer (NSCLC) cell line H460, in which the cytotoxic effect of an anti-EGFR monoclonal antibody cetuximab was minimal, showed a significant decrease in its viability upon ONCEGFR-adenovirus treatment. This superior cytotoxicity was successfully replicated in subcutaneous A431 xenografts. Single intratumoral injection of ONCEGFR-adenovirus virotherapy showed a significant tumor regression as well as exceptionally high onconase expression in tumor cells compared to a non-targeted virotherapy. 1.5. Concluding discussion Engineering conventional antibodies found in humans as well as identification of single domain antibodies (VHH or VNAR) from non-human sources accelerated invention of the smallest functional units and their advancements into the clinics. Biomaterials that mediate their self- assembly and delivery added novelty to their biomedical applications. The aim of this review is 45 to promote a critical assessment of peptide-based biomaterial platforms that mediate antibody assembly and provide insights for their further improvements to drive efficacy in humans. Despite the promising theory and preliminary in vitro studies, in vivo applications are not always successful. The limitations may arise from sub-optimal PK. The key factors governing PK of the antibody conjugated biomaterials are size, charge, morphology, and antibody valency. Under pathologic conditions, therapeutic molecules can readily cross or escape the abnormal vascular endothelium and reach the tumor microenvironment (Hobbs et al., 1998). However, unlike the tumor periphery, blood flow is scarce in the tumor core, where many prominent target antigens reside on populations of dormant, resistant tumor cells. Therefore, whether they are molecularly targeted or non-targeted, it takes substantial time for therapeutic molecules to accumulate deep inside the tumor. Although smaller proteins penetrate better to the tumor core with their higher diffusion constants, they suffer from rapid systemic clearance. Therefore, the size of the molecule has to be precisely determined to guarantee both the elongated residence in blood and the tumor penetration (Bertrand et al., 2014). Molecules that are less than 10 nm in hydrodynamic radius are eliminated by the kidney (renal clearance), whereas those ranging from 150~300 nm are cleared by the liver and spleen (reticuloendothelial system) (Longmire et al., 2008). Therefore, molecules with size between 70~200 nm are suggested to have a higher chance to reach the target site with prolonged circulation in the bloodstream with minimal association with glomerular filtration or the reticuloendothelial system (Gaumet et al., 2008; Yokoyama, 2014). The net surface charge of the molecule also determines their longevity. In vivo toxicity study revealed that positively charged nanoparticles increased liver enzyme release, body weight loss, and interferon type I response. They also induced 15~25-fold higher mRNAs of interferon responsive genes, and 10~75-fold higher expression of pro-inflammatory cytokines compared to negatively-charged or neutral nanoparticles (Kedmi et al., 2010). Due to this reason, positively charged nanoparticles are immediately removed by macrophages through 46 opsonization. Negatively charged or charge neutral nanoparticles are better than positively charged nanoparticles in terms of systemic half-life and several reports indicate that net negative charge may be slightly better over charge neutral in preventing non-specific cellular uptake (Alexis et al., 2008); however, some of the negatively charged nanoparticles can still activate complement pathways, get opsonized, and cleared by immune system, leading to shortened systemic half-life (Inglut et al., 2020). The overall shape may be another factor governing longevity. Single-walled carbon nanotubes that are 200~300 nm in length and 350~500 kDa in molecular weight experienced rapid renal clearance with half-life of only 6 minutes. Although it was 10~20 times higher in mass for the glomerular filtration cut-off (60 kDa), their narrow and elongated tubular structure negatively affected its residence in the systemic circulation (Ruggiero et al., 2010). Advancements in antibody engineering towards generating multivalent antibodies introduced avidity into consideration. As we discussed the negative effect of high affinity antibodies in tumor penetration (binding site barrier), avidity-mediated tighter binding may not always give superior results. Cuesta et al. postulated the ‘tumor target zone’, which suggests optimal biophysical conditions for the multivalent antibody constructs in tumor targeting. The monovalent antibodies, such as scFv, have higher tumor penetration, but they experience fast off-rates and rapid systemic clearance due to their smaller size and weight. In contrast, the multivalent antibodies overcome the abovementioned setbacks leading to a tighter engagement to the targets (longer tumor retention) and longer half-life in the blood through their avidity. However, they suffer from inferior tumor penetrability due to avidity. The molecular weights between 70-120 kDa with bi- or trivalency was suggested to achieve optimal tumor targeting, tumor uptake, and low systemic clearance, which may include diabodies (bivalent), minibodies (bivalent), and trimerbodies (trivalent) (Cuesta et al., 2010). Given that antibody-biopolymer fusion modalities are scarce in clinics, the platforms discussed above likely experienced obstacles to move beyond the pre-clinical stage due to 47 unsatisfactory performance. To overcome these hurdles, research into their biophysical and pharmacological properties must continue until these modalities can complement limitations of conventional antibody therapy and ultimately become better therapeutic alternatives to patients. 48 Chapter 2 Real-time Receptor Clustering at the Single-cell Level Using Antibody Nanoworms Designed for Lymphoma 2.1. Introduction Defined by the United States National Cancer Institute (NCI, www.cancer.gov/types/lymphoma), lymphoma arises from malignant lymphocytes that can be divided into two categories, Hodgkin lymphoma and non-Hodgkin lymphoma. Hodgkin lymphoma can be often cured by standard therapy whereas non-Hodgkin lymphoma (NHL) is more progressive and has a 5-year survival rate around 71%. Estimated by the America Cancer Society (www.cancer.org), the overall chance of a man will develop NHL in his lifetime is about 1 in 41; that for a woman is about 1 in 52. The NHL can be sub-categorized into two types: B cell NHL and T/NK (natural killer) cell NHL (Campo et al., 2011). The B cell NHL makes up 85~90% of total NHL, while T cell NHL makes up less than 15%. Because of the disproportionate incidence rates between the two types, the majority of research efforts have been put to B cell NHL. This is more apparent when available antibody-based therapies are compared. Total of 23 antibody-based immunotherapies that target 13 different B cell surface receptors have been approved by USFDA for B cell NHL (Crisci et al., 2019), whereas brentuximab vedotin (anti-CD30 monoclonal antibody conjugated with monomethyl auristatin E) and mogamulizumab (anti-CCR4 monoclonal antibody) are the only two therapeutic antibodies approved for T cell NHL (Welborn and Duvic, 2019). Although addition of therapeutic antibodies, thus activating the host immune system, to the existing chemotherapy significantly improved the prognosis of B cell NHL (Borghaei et al., 2009; Wilson, 2000); sub-optimal therapeutic responses or fatal side-effects due to the heterogenous involvement of host immune system still remains as a challenge (Atmar, 2010; 49 Burkhardt et al., 2016; Gutierrez et al., 2006; Shimabukuro-Vornhagen et al., 2018; Zhuang et al., 2010). On top of that, the necessity of better antibody-based therapeutic options for T cell NHL is needless to say counting its aggressiveness and fatality. This study attempts to visit abovementioned therapeutic needs using the antibody-based therapeutic modality, which mechanism of action and potency are equivalent to that of the therapeutic antibodies. The therapeutic modality is built upon a well characterized protein polymer called Elastin-Like Polypeptides (ELPs) (Despanie et al., 2016), which streamlined the genetic fusion of single chain antibody fragment, production of homogenous population, and the purification of the functional nanomaterials. The generated antibody-ELP fusions exhibit high degree multivalency via self-assembly and have a distinct worm-like morphology (hence, Nanoworms), which together enable lateral engagement of multiple cell surface receptors and subsquent clustering without any secondary crosslinkers. This study tests, 4 different antibody Nanoworms that target therapeutically relevant cell surface receptors on B cells (CD20, HLA-DR10, and CD19) and T cells (CD3). Three aspects were studied by targeting these receptors. First, CD20 and HLA-DR10 have been extensively studied as therapeutic targets in the context of Fc-FcR mediated host immune system activation and its therapeutic effect. Because Nanoworm modality lacks the Fc domain, these are the most suitable candidates to test whether the Nanoworms can achieve therapeutic equipotency only by clustering cell surface receptor in B cell NHL without Fc-FcR interaction-mediated host immune activation. Second, CD19 became a prominent therapeutic target with the advent of BiTE (Hoffman and Gore, 2014) and CAR-T therapy (Magee and Snook, 2014); however, its therapeutic effect upon clustering had acquired less attention. Therefore, CD19 clustering and its therapeutic implications towards B cell NHL was explored. Third, it was tested whether the strategy tested on B cell NHL can be applied to other NHL models that needs better antibody- based therapeutic options. For this, the activity on the cell surface and its therapeutic effect of CD3 clustering Nanoworm was observed in the Sézary syndrome model (Bunn and Foss, 50 1996), an aggressive form of cutaneous T cell NHL. The Sézary cells are known to be resistant to activation-induced cell death (AICD) and one of the mechanisms responsible for this resistance is the lack of T cell receptor (TCR) signaling (Klemke et al., 2009). Therefore, particular attention was given to this disease in the context of CD3 clustering to test whether CD3 clustering Nanoworm can overcome the resistance and induce AICD. The dynamic clustering of cell surface receptors induced by antibody Nanoworms was observed in real-time using the experimental method that our lab has pioneered to detect the rapid and reversible segregation of the cytoplasmic molecule (Pastuszka et al., 2014) or membrane-bound receptor (Li et al., 2018d) in cell cultures and in zebrafish (Li et al., 2018c) relative to the applied thermal energy. Using this powerful technique, this study presents the real-time visual evidence of Nanoworm-mediated clustering of cell surface receptors, exact temperatures that occurs, and simultaneous induction of membrane blebbing, which all directly support the therapeutic benefit confirmed by independently performed ELISA assays (Figure 2). Interesting feature of our report would be the therapeutic activity observed in T cell NHL. Nanoworm that clustered CD3 on HuT78 cell line (Sézary syndrome model) induced activation- induced cell death (AICD), which OKT3 treatment alone was not able to achieve. Nanoworm- mediated AICD was even more potent than CD3 clustered by OKT3 supplemented with secondary crosslinker (OKT3+2°). Furthermore, co-incubation of OKT3 and CD3 clustering Nanoworms showed an additive effect in T cell activation. The AICD and additive effect on T cell activation were exclusively observed in HuT78 cell line but not on other leukemic T cell lines, such as CEM or Jurkat. Our observations suggest that CD3 clustering via high degree multivalency is an effective approach to overcome the resistance to AICD in Sézary syndrome and CD3 clustering Nanoworm may be a suitable therapeutic candidate, both as a monotherapy and a combination therapy with existing CD3 targeting antibodies, that can fortify therapeutic outcomes for this particular disease. 51 As a simple yet powerful therapeutic modality, Nanoworms’ application is limitless. This study is expected to serve as a solid basis for the better therapeutic approaches and provide novel insights towards a cancer treatment. 52 Figure 2. scFv-ELP fusions self-assemble into Nanoworms that cluster and activate cell surface receptors. The scFv-ELP fusions forms multivalent antibody Nanoworms (cryo-TEM image). Nanoworms’ inherent ability to cluster cell surface receptors was monitored in real-time and quantified at the single-cell level. Upon Nanoworm-mediated receptor clustering, apoptosis or cell cycle arrest were observed in B cell NHL cells, and AICD was observed in T cell NHL cells. ELP scFv αCD20-ELP Multivalent Nanoworms Heterologous Expression Real-time single cell imaging A192 = (VPGAG) 192 Elastin-Like Polypeptides (ELP, 73 kDa) scFv (25 kDa) V H V L scFv-ELP fusion Cryo-TEM B cell NHL Apoptosis or Cell cycle arrest Activation- induced cell death (AICD) T cell NHL 53 2.2. Materials and methods 2.2.1. Synthesis, Expression, and Purification of Nanoworms The pET-25b(+) vector was purchased from Novagen (#69753) and further modified for ELP fusion cloning. For the four scFv-ELP fusion proteins, the DNA encoding scFv was cloned to the N-terminus of ELP A192. The amino acid sequence of scFv that targets CD20 was derived from Rituximab (Rituxan ® ) and optimized for bacterial expression. The amino acid sequence of scFvs that target CD19 and CD3 were derived from bispecific antibody Blinatumomab and optimized for bacterial expression. The amino acid sequence of scFv that targets HLA-DR10 was derived from the chimeric Lym-1 (chLym-1) monoclonal antibody (Epstein et al., 1987; Hu et al., 1995). For the four anti-CD20-ELP fusions, the DNA encoding anti-CD20 scFv used above was cloned to the N-terminus of ELP V2A64, A96G96, S192 or G192. The cloned constructs were sequenced, transformed into and expressed in Shuffle ® T7 Express competent E. Coli (#C3029J, NEB, Ipswich, MA, USA) fermented in terrific broth media for 16-18 hrs at 30 °C without IPTG induction. After bacterial cell lysis (S-4000 Ultrasonic Disintegrator Sonicator Liquid Processor, Misonix, Inc. NY, USA; Amplitude 9, 18 repeats of 10 sec on + 20 sec off cycle) and clarification of cell debris by centrifugation at 16,100 rcf for 10 min at 4 °C in a Beckman J2-21 Centrifuge, the supernatant was equilibrated to room temperature and ELP phase separation was induced by 2 M sodium chloride at room temperature (i.e., dissolve 0.12 g NaCl powder per 1 mL cleared lysates by gently inverting until transparent lysates become opaque). Coacervates were pelleted at 5,000 rcf for 10 min at 25 °C using a Sorvall RC-3C Plus Centrifuge immediately after the phase separation was observed (hot-spin). After each hot-spin, soluble impurities (supernatant) were removed, and coacervates (pellet) were resolubilized in ice-cold dPBS (#25-508, Genesee Scientific, San Diego, CA, USA). Thoroughly resolubilized ELPs were centrifuged at 16,100 rcf for 10 min at 4 °C in an Eppendorf 5415R Centrifuge (cold-spin). At the end of each cold-spin, insoluble impurities (pellet) were again removed by transferring the supernatant to a clean tube. Cycles of hot-spin followed by 54 cold-spin were repeated 2 times to achieve the necessary purity and yield. Purified materials are processed either for refolding or stored at -20 °C for further use. 2.2.2. Labeling and Refolding of Nanoworms The following buffers were prepared to promote refolding of scFv domains: • Equilibration solution: 20 nM β-Mercaptoethanol, 50 mM Tris base, 500 mM NaCl, pH 8.0 • Buffer 1: 20 mM Tris base, 150 mM NaCl, 3 M Urea, 500 mM L-Arginine, 2 mM GSH, 0.4 mM GSSH, 0.5 mM PMSF, pH 8.0 • Buffer 2: 20 mM Tris base, 150 mM NaCl, 1 M Urea, 250 mM L-Arginine, 2 mM GSH, 0.4 mM GSSH, 0.5 mM PMSF, pH 8.0 • Buffer 3: 20 mM Tris base, 150 mM NaCl, 0.5 M Urea, 125 mM L-Arginine, 0.5 mM PMSF, pH 8.0 • Buffer 4: 20 mM Tris base, 150 mM NaCl, 62.5 mM L-Arginine, 0.5 mM PMSF, pH 8.0 • Buffer 5: 1x dPBS Purified Nanoworms (three scFv-A192 fusion proteins except aCD3A and four anti-CD20-ELP fusion proteins) were labeled with 5-fold molar excess NHS-rhodamine (#46406, Thermo- Fisher, IL, USA) for 4 hrs at room temperature under constant rotation in a 15 mL tube. After the labeling reaction, Nanoworms were denatured by directly adding equilibration solution to the labeling reaction solution at a half of the volume of labeling reaction solution, followed by 12 M Urea as a powder (i.e., directly add 1 mL of equilibration solution to 2 mL labeling reaction solution and then add 1.44 g (12 M) Urea powder calculated based on 2 mL). The tube was gently inverted to completely dissolve Urea. Addition of equilibration solution and Urea doubled the volume (2 mL à 4 mL), which resulted in 6 M Urea as a final concentration. The entire 55 solution was subjected to refolding process using a 10 kDa MWCO dialysis tubing (#68100, Thermo Fisher, IL, USA) via stepwise dialysis at 4 °C. Buffer was changed 5 times at intervals of 24 hr. Excess rhodamine was removed during this process. Refolded material was centrifuged at 16,100 rcf for 10 min at 4 °C (Eppendorf 5415R Centrifuge, Eppendorf AG, Hamburg, Germany), and the supernatant was syringe filtered (450 nm pore, #PN 4614, Pall Corp., NY, USA). At the end of all steps, labeling, refolding, and final purification, degree of labeling was 300~500% per mole protein, which equals to 3~5 moles rhodamine per mole protein. Empirically, labeling was a prerequisite for aCD20A, aLADR10A, and aCD19A Nanoworms for its activity, whereas aCD3A Nanoworms did not require prelabeling. Therefore, aCD3A Nanoworms were immediately subjected to denaturation and refolding after purification. Rituximab was labeled with 15 molar excess NHS-rhodamine for 2 hrs at room temperature under constant rotation and dialyzed against dPBS for 3 days in a sink condition. 2.2.3. Biophysical Characterization of Nanoworms The identity and purity of Nanoworms were analyzed using SDS-PAGE. The molar extinction coefficient (e) of aCD20A, aHLADR10A, aCD19A, and aCD3A Nanoworms was calculated at 60,855, 45,630, 59,570, 60,435 M -1 ⋅cm -1 , respectively (Pace et al., 1995). Serial dilutions of aCD3 Nanoworms in Edelhoc buffer were prepared, measured and averaged to acquire the best estimate of concentration in dPBS using Eq.1 (Edelhoch, 1967; Pace et al., 1995). Serial dilutions of aCD20A, aHLADR10A, and aCD19A Nanoworms in Edelhoc buffer were prepared, measured and averaged to acquire the best estimate of concentration in dPBS using Eq.2 (0.34=rhodamine correction factor provided by manufacturer). 𝐶 #$%&'&()* = , -./ 0(, 222×4.6-6) 8 ×𝐷𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟 Eq. 1 𝐶 EF&0#$%&'&)(* = , -./ 0(, GGG×/.2H) 8 ×𝐷𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟 Eq. 2 56 The hydrodynamic radius (Rh) of Nanoworms at 25 °C was determined using dynamic light scattering (DLS). Triplicates of syringe filtered (450 nm pore, #PN 4614, Pall Corp., NY, USA) Nanoworms (50 μL of 10 μM) in dPBS were loaded onto a Greiner CELLSTAR ® 384-well plate (#M1937, Greiner Bio-One, Kremsmünster, Austria) followed by layering with 15 μL mineral oil to prevent evaporation. The plate was centrifuged for 1 min at 1,000 rcf to remove air bubbles. Hydrodynamic radius (Rh) was measured using a Wyatt Dynapro plate reader and analyzed by DYNAMICS V7 software (Wyatt Tech. Co., CA, USA). Rh was measured first at 25 °C and then the temperature was immediately increased to 37 °C, where the second measurement was made. After measurement, the plate was incubated at 37 °C for 5 days to test stability. Hydrodynamic radius was measured at the 2 nd and 5 th day of incubation. Size exclusion chromatography followed by multiangle light scattering (SEC-MALS) was used to determine the radius of gyration (Rg), absolute molecular weight, and oligomeric state of the Nanoworms. 10 μM Nanoworms in 100 μL dPBS (100 μg) were injected onto a Shodex size exclusion column (KW-803, Showa Denko K.K, Japan) at 0.5 mL/min. The eluents were analyzed on a Helios system (Wyatt Tech. Co., CA, USA) maintained at 25 °C and the data were fit to a Random Coil model to determine the Rg and the absolute molecular weight per Nanoworm. The oligomeric state for the Nanoworm was calculated by dividing the absolute molecular weight by the expected molecular weight of monomeric scFv-ELP. The Rg/Rh ratio at 25 °C was used to determine the morphology of the Nanoworms. For the optical density profile, absorbance at 350 nm, A, was measured in a DU800 UV- Vis spectrophotometer (Beckman Coulter, CA) under a temperature gradient of 0.5 °C/min. The transition temperature (Tt) at each concentration was defined as the temperature at which the maximum first derivative, dA/dT, was achieved using Eq. 3. The Ai is defined as the absorbance recorded at Ti temperature. I, IJ K L = , MNO 0, M J MNO 0J M Eq. 3 57 The transition temperature from each concentration was used to plot the phase diagram and fit with Eq. 4. 𝑇 Q = 𝑏−𝑚 log 4/ [𝐶 #$%&'&() ] Eq. 4 To estimate ΔHcoacervation and ΔScoacervation, a version of the van’t Hoff equation (Eq. 5) was used to describe the concentration dependency of coacervation. 𝑙𝑛𝐶𝐴𝐶 #$%&'&() = [ \ ]^_]`ab_cM^d E 4 J c − [ e ]^_]`ab_cM^d E Eq. 5 The CACNanoworm (μM) is the critical aggregation concentration at each observed transition temperature. The Tt is in units of Kelvin. The universal gas constant (R) is assumed to be 0.008314 kjouls/mol·K. By plotting the natural logarithm of the CACNanoworm vs. the inverse of the transition temperature, a slope and the y-intercept were obtained and interpreted as the enthalpy and entropy of coacervation per mol of ELP, respectively. ELP phase separation from solution into coacervate is an endothermic process (Amruthwar et al., 2013; Reguera et al., 2007); therefore, the ΔHcoacervation is defined as positive and assumed to remain constant over this narrow temperature range. For spontaneous phase separation from solution into the coacervate phase (which happens above Tt), the Gibbs free energy of coacervation should become negative; at Tt, where the TΔScoacervation must become larger than the ΔHcoacervation. 2.2.4. Cryo-TEM imaging For cryo-TEM, 5 μM Nanoworm suspended in dPBS were evaluated. Lacey carbon film copper grids (300 mesh, Electron Microscopy Services, Hatfield, PA, USA) were pretreated with plasma air for 30 seconds to render the lacey carbon film hydrophilic. A 6 µL sample was applied on the grid using a Vitrobot (FEI, Hillsboro, OR, USA) that was maintained at 95% humidity. Following 1 min incubation, blotting was performed using Vitrobot preset parameters and the grid was immediately plunged into a liquid ethane reservoir precooled by liquid nitrogen. Grids were then transferred to a cryo-holder and cryo-transfer stage that were precooled with 58 liquid nitrogen. A FEI Tecnai 12 Twin Transmission Electron Microscope, operating at 100 kV, was used to perform all imaging. The cryo-holder was maintained below -170°C with liquid nitrogen to prevent the sublimation of vitreous water during the imaging process. All images were recorded with a 16-bit 2K × 2K FEI Eagle bottom mount camera (Hillsboro, OR, USA). The length and the width of Nanoworms were measured using ImageJ (v2.0.0, NIH, MD) based on the reference length presented in Figure 4A. 2.2.5. Cell cultures and time-lapse live cell imaging All cell lines used in this study (Raji, SU-DHL-7, HuT-78, and Jurakt) were cultured in RPMI 1640 (Corning, MA, USA) supplemented with 10 % FBS at 37 °C without any antibiotics. For time-lapse live cell imaging, 10 μM Nanoworms or Rituximab was incubated with 0.5 x 10 5 cells for 30 min at 4 °C under constant agitation. Cells were spun down at 300 rcf, washed 3 times with pre-chilled dPBS, resuspended with a pre-chilled fresh media, and mounted on a poly-D-lysine (P7405, Sigma-Aldrich, St. Louis, MO) coated 35 mm glass bottom culture dish (#P35G-0-10-C, MatTek Corp. MA). After 15 min, cells were imaged using a DIAPHOT epifluorescence microscope equipped with a DS digital camera (Nikon Instruments, Minato City, Tokyo, Japan) and a temperature control stage (Linkam Scientific Instruments, Epsom, UK). Temperature of media within the culture dish was measured in real-time with the type K temperature probe (TP870, Extech, NH, USA) connected to a thermocouple thermometer (Model:800005, Scottsdale, AZ, USA) during the temperature increase at a rate of 2 °C/min. Fluorescence images were taken at every 0.5 °C from 15 °C up to 45 °C during the heating. Images were further analyzed to identify the Tc of a cell surface bound Nanoworms using ImageJ (v2.0.0, NIH, MD, USA). The Tc of each Nanoworm was defined as the Ti temperature at which the maximum first derivative of background-corrected fluorescence intensity (dI/dT) was estimated using Eq. 7. Brightfield images were taken under the identical condition to observe cell membrane blebbing. The Ii is defined as the background corrected fluorescence intensity 59 measured at Ti temperature. The IROI and IBG are defined as the fluorescence intensity measured within the subregion of the cell where Nanoworm cluster is formed and the fluorescence intensity measured in the region devoid of cells where there is only a baseline fluorescence, respectively, at Ti temperature. 𝐼 L =(𝐼 Egh −𝐼 ij )×𝐴 Eq. 6 Ih IJ K L = h MNO 0h M J MNO 0J M Eq. 7 2.2.6. Target specificity of Nanoworms To test the specificity of aCD20A and aHLADR10A, 0.3 x 10 5 Raji cells were incubated with 50 μg rituximab or chLym-1 (17.4 μg of anti-CD20 scFv or anti-HLA-DR10 scFv) for 20 min at 4 °C. After 20 min incubation, 6 μg of rhodamine-labeled aCD20A or aHLADR10A (1.5 μg anti-CD20 scFv or anti-HLA-DR10 scFv) was added into the solution and incubated for another 20 min. The total volume did not exceed 100 μL. At the end of second incubation, cells were washed with dPBS twice (300 rcf for 5 min) and resuspended in 20 μL Live Cell Imaging Solution (#A14291DJ, Molecular Probes, Eugene, OR, USA) added with NucBlue TM Live Cell Stain ReadyProbes TM reagent (#R37605, Molecular Probes, Eugene, OR, USA). Cells were mounted on a poly-D-lysine (P7405, Sigma-Aldrich, St. Louis, MO) coated 35 mm glass bottom culture dish (#P35G-0-10-C, MatTek Corp. MA, USA) and imaged under the Zeiss LSM880 Confocal Microscope with Airyscan Fast (Carl Zeiss AG, Oberkochen, Germany). Fluorescence intensities of cells were analyzed using ZEN 2 Blue Edition software (Carl Zeiss AG, Oberkochen, Germany). To test the specificity of aCD19A and aCD3A, 0.2 x 10 5 cells (Raji and SU-DHL-7 cells for aCD19A, HuT-78 and Jurkat cells for aCD3A) were incubated with 1.25 μg blinatumomab (0.625 μg anti-CD19 scFv or anti-CD3 scFv) for 20 min at 4 °C. After 20 min incubation, 80 μg of rhodamine-labeled aCD19A or aCD3A (20 μg anti-CD19 scFv or anti-CD3 scFv) was added 60 into the solution and incubated for another 20 min. The total volume did not exceed 100 μL. At the end of second incubation, cells were washed with dPBS twice (300 rcf for 5 min), resuspended in 500 μL dPBS, and analyzed with BD LSRFortessa™ X-20 (Becton Dickinson, Franklin Lakes, NJ, USA). Data were further analyzed with FlowJo™ Software v10.4 (Becton, Dickinson and Company, Ashland, OR, USA). For the non-treated controls, equal volumes of dPBS was added in place of the antibody and the Nanoworm solutions. For positive controls, equal volume of dPBS was added in place of the antibody and then incubated with respective Nanoworms described above. 2.2.7. Apoptosis, cell cycle distribution, and cell activation upon Nanoworm treatment To measure the apoptosis, 0.2 x 10 5 cells in complete media were incubated with antibody or Nanoworms for 15 min at room temperature in the 1.7 mL tube. A final scFv concentration was 20 μM for both antibody and Nanoworms (10 μM for T cell lines), and the total volume was kept below 90 μL. After 15 min incubation, 80 μL complete media was added and the whole solution was transferred to a well in a 48-well plate. The plate was incubated at 37 °C for 18h. After 18h incubation, cells were counted and washed twice with pre-chilled dPBS (300 rcf for 5 min). Cells were resuspended in 100~200 μL hypotonic buffer (20 mM Tris-HCl, pH 8.0) and incubated on ice for 15 min. After 15 min, outer membrane of the cells was removed using Dounce tissue grinder (#D8938, Millipore Sigma, Burlington, MA, USA). About 80-90% of the cells were devoid of outer membrane after 50 times of grinding with tight pestle. Solutions were spun down (10,000 rcf, 7 min) to remove any outer membrane debris and mitochondria, and only the supernatant (cytosolic fraction that contains cytosolic cytochrome C) was transferred to a clean tube. The level of cytosolic cytochrome C was measured from 100 μL of the supernatant using Human Cytochrome C Quantikine ELISA Kit (#DCTC0, R&D Systems, Minneapolis, MN, USA). Resulting cytosolic cytochrome C levels were normalized to counted cells as a ng/mL and then converted to a fold change. 61 To analyze the cell cycle distribution, 0.4 x 10 5 Raji cells in 200 μL was incubated with 200 μL of aCD19A (0.8 mg/mL) for 15 min at room temperature in the 1.7 mL tube and then incubated at 37 °C for 48h. A 200 μL of 0.1 μg/mL rapamycin formulated with tween 80 and PEG 400 was used as a positive control.(Lee et al., 2019) After 48 h, collected cells were washed with dPBS three times (300 rcf for 5 min) and fixed with 70 % ethanol for 24 h. After fixation, cells were washed with dPBS twice (900 rcf for 5 min) and resuspended in 100 μL of 100 μg/mL RNAse A (component of Miniprep Kit, #27106, Qiagen, Hilden, Germany). After incubation at room temperature for 1 h, 100 μL of 100 μg/mL propidium iodide (component of Apoptosis Kit, #V13241, Thermo Fisher Scientific, Waltham, MA, USA) was added and incubated for another 30 min. Cells were analyzed using BD LSRFortessa™ X-20 (Becton Dickinson, Franklin Lakes, NJ, USA). Data were further analyzed with FlowJo™ Software v10.4 (Becton, Dickinson and Company, Ashland, OR, USA). To analyze the T cell activation, cell culture media were collected during the process of abovementioned cytosolic fraction preparation. Collected media were spun down once more (300 rcf for 5 min) to completely remove any cellular components and the transferred supernatant was directly used to measure IL-2 concentration using the Human IL-2 ELISA MAX TM Set Deluxe (#431804, BioLegend, San Diego, CA, USA). To analyze the co-stimulatory effect in T cell activation, 70 μL of 0.52 mg/mL OKT3 antibody solution was filled into wells in 96-well plate (#423501, BioLegend, San Diego, CA, USA), sealed, and incubated at 4 °C one day prior to the experiment (day 1). On day 2, 0.1 x 10 5 HuT-78 or Jurkat cells in 70 μL was incubated with 70 μL of aCD3A (serial dilution) or dPBS for 40 min at 4 °C under constant agitation. After this incubation, cells were transferred to OKT3 pre-coated wells in 96-well plate. Another set of cells incubated with aCD3A were transferred to OKT3 uncoated wells in the same 96-well plate to observe the activity of aCD3A without OKT3. The 96-well plate was incubated at 37 °C for 18h. On day 3, solution from each well was collected, spun down (300 rcf for 5 min), and only supernatant was collected. The samples were 62 stored at -20 °C until further analysis. Cell activation was analyzed with same procedure mentioned above. 63 2.3. Results 2.3.1. scFv-ELP fusions form colloidally stable worm-like nanostructures Four different scFv-ELP fusions were over-expressed in E. coli from cloned DNAs which encode the scFv followed by ELP A192 (Figure 3A). The scFvs that target CD20, HLA-DR10, CD19, or CD3 are derived from monoclonal antibodies Rituximab (Rituxan ® , RTX) (James and Dubs, 1997), chimeric Lym-1 (chLym-1) (Epstein et al., 1987; Hu et al., 1995), or bispecific antibody Blinatumomab (Blincyto ® ) (Nagorsen and Baeuerle, 2011), respectively. The resulting four scFv-ELPs, anti-CD20-A192, anti-HLA-DR10-A192, anti-CD19-A192, and anti-CD3-A192, will be referred as aCD20A, aHLADR10A, aCD19A, and aCD3A for convenience throughout this study (Table 3). Purification was performed by induction of the ELP phase separation via 2 M sodium chloride, which is a non-chromatographic method of protein purification (Cho et al., 2008; Janib et al., 2014a). Two rounds of purification yielded ~30 mg/L of each fusion with > 90% purity, as verified by SDS-PAGE (Figure 3B). To determine the solubility profiles, optical density of each fusion was scanned at 350 nm (OD 350) over a range of temperatures. Purified fusion proteins remained soluble at physiological temperatures (Figure 3C). Various concentrations of each fusion were tested for optical density, and Tt determined by Eq. 3 were plotted and fit with Eq. 4 to generate the phase diagram (Figure 3D) ((Despanie et al., 2016). All fit parameters are reported in Table 5. This fit allows estimation of Tt over a range of concentrations. These ELP fusions were not expected to phase separate at 37 ºC at concentrations used in this study (1~100 μM). To explore whether these fusions generated Nanoworms, their physical shape was imaged under cryo-TEM (Figure 4A) and exact dimensions were measured (Figure 4B). All four fusions oligomerized into worm-like nanoparticles with slight differences in their length of 81~89 nm for aCD20A, aCD19A, aCD3A, while aHLADR10A was shorter with a mean length of 54 nm. All four fusions had a mean width between 5~9 nm. As an independent confirmation, light scattering analyses were employed. Size-exclusion chromatography followed by multi- 64 angle light scattering (SEC-MALS) revealed that nearly all scFv-ELP monomers are associated with Nanoworms, as no lower-weight population was retained on the column. The oligomerized scFv-ELPs passed the column in the void volume, with a molar mass between 10 7 ~10 8 g/mol (Figure 4C). The oligomeric state of each fusion was calculated from the absolute molecular weight observed by SEC-MALS (Table 5) and the expected MW of the peptide encoded by the open reading frame. Dynamic light scattering (DLS) analysis was employed at days 0, 2, and 5 to determine the hydrodynamic radius (Rh) and stability of Nanoworms (Figure 4D,E). Purified Nanoworms remained stable colloids at 37 °C for at least 5 days. The ratio between the radius of gyration (Rg, from SEC-MALS) and Rh (from DLS) was used to estimate a shape factor for each fusion (Tande et al., 2001). The shape factor (Rg/Rh, Table 5) reconfirmed the observations using cryo-TEM (Figure 4A) that scFv-ELP self-assembly results in an elongated shape regardless of their oligomeric state, Rg, Rh, or the type of scFv. The theoretically expected Rg/Rh for a hard sphere is 0.778 and 2.36 for a rigid rods (Baalousha et al., 2006; Brewer and Striegel, 2011). The random coil model best-fit all of the data acquired for scFv-ELP fusions in SEC-MALS. Therefore, this model was applied consistently to all observations. When evaluated with the other fit models, which are frequently used by others (Andersson et al., 2003; Brewer and Striegel, 2011; Nakahata and Yusa, 2018), the Rg/Rh values for the fusions reported in Table 5 were slightly higher, ranging from 1.0~1.5, which also was consistent with random coil or flexible linear chains. Based on analysis of both shape factors and morphological observations (cryo-TEM), scFv-ELP that target all four different receptors thus appear consistent with a designation as ‘Nanoworms.’ 65 Figure 3. Design, identity, and thermo-responsiveness of scFv-A192 fusions against a panel of immune receptors. (A) cDNAs encoding scFv were fused to the amino terminus of ELP A192. (B) SDS-PAGE was used to evaluate rhodamine-labeled fusions. The identical gel was first imaged for fluorescence and then imaged after Coomassie blue staining. (C) The ELP induces temperature-dependent phase separation, which can be monitored by optical density as a function of temperature using UV-Vis spectrophotometry (λ=350 nm, 10 μM in dPBS). (D) Optical density increases from baseline as ELPs coacervate, which is used to construct a phase diagram for each fusion protein. ELPs follow a log-linear relationship (Table 5) between transition temperature and concentration, above which phase separation occurs. These ELP fusions were designed to remain soluble at physiological temperatures (grey zone). 1 10 100 35 40 45 50 Concentration (µM) Transition temperature (°C) aCD20A aHLADR10A aCD19A aCD3A 35 40 45 50 55 0 1 2 3 Temperature ( o C) OD 350nm aCD20A aHLADR10A aCD19A aCD3A anti-CD20 anti-CD20 anti-HLA- DR10 anti-HLA- DR10 anti-CD19 anti-CD19 anti-CD3 anti-CD3 A192 A192 A192 A192 aCD20A aHLADR10A aCD19A aCD3A Variable heavy (VH) Variable light (VL) ELP scFv Linker: (GGGGS) 3 V2A64 A96G96 S192 G192 aCD20G aCD20S aCD20AG aCD20VA V H V L Elastin-Like Polypeptides (ELP) anti-CD20 scFv Linker: (GGGGS) 3 V H V L V H V L V H V L A192 scFv-ELP fusion Elastin-Like Polypeptides (ELP) scFv V H V L scFv A B C D 66 Table 3. Molecular information of scFv-A192 fusions. scFv-ELPs (scFv origin) Amino acid sequence a,b,c T t at 10 μM (°C) d T c at 10 μM in Raji (Mean±SD °C) e aCD20A (Rituximab) GQVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGN GDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVW GAGTTVTVSAGGGGSGGGGSGGGGSQIVLSQSPAILSASPGEKVTMTCRASSSVSYI HWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQ WTSNPPTFGGGTKLEIKRTG(VPGAG)192Y 46.4 28.7 ± 4.2 aHLADR10A (chLym-1) GQVQLKESGPGLVAPSQSLSITCTISGFSLTSYGVHWVRQPPGKGLEWLVVIWSDGST TYNSALKSRLSISKDNSKSQVFLKMNSLQTDDTAIYYCASHYGSTLAFASWGHGTLVT VSAGGGGSGGGGSGGGGSDIQMTQSPASLSASVGETVTIICRASVNIYSYLAWYQQK QGKSPQLLVYNAKILAEGVPSRFSGSGSGTQFSLKINSLQPEDFGSYYCQHHYGTFTF GSGTKLEIKG(VPGAG)192Y 48.1 28.3 ± 4.5 aCD19A (Blinatumomab) GQVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGD GDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMD YWGQGTTVTVSSGGGGSGGGGSGGGGSDIQLTQSPASLAVSLGQRATISCKASQSV DYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAA TYHCQQSTEDPWTFGGGTKLEIKG(VPGAG) 192 Y 47.6 30.2 ± 4.3 aCD3A (Blinatumomab) GDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRG YTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGT TLTVSSGGGGSGGGGSGGGGSDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWY QQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSS NPLTFGAGTKLELKG(VPGAG) 192 Y 46.1 30.0 ± 4.7 a All scFv-A192 fusions share identical order in their sequence: Glycine-VH-(G4S)3-VL- G(VPGAG)192Y. A cDNA encoding each scFv-A192 fusion was confirmed by DNA sequencing. VH: variable heavy; VL: variable light. b Amino acid sequence of an ELP A192 = G(VPGAG)192Y. c (G4S)3 is a linker between VH and VL. d Calculated from the Table 5 using Eq 4. e Mean±SD from figure 5G. 67 Figure 4. ScFv-ELP fusions self-assemble Nanoworms. (A) The morphology of refolded scFv-ELPs were visualized under Cryo-TEM, which revealed the appearance of worm-like features. (B) Physical dimensions of each particle were analyzed using ImageJ (n=50/each, mean±SD). (C) SEC-MALS reveals that Nanoworms flow through in the void volume, while the anti-CD20 Rituximab (RTX) is retained on the column. The Nanoworms have a significantly higher molar mass compared to RTX. Dotted line: molar mass; Solid line: UV Detection voltage. (D) DLS analysis confirms that Nanoworms have higher hydrodynamic radii compared to RTX. (E) Reflecting their apparent colloidal stability, the hydrodynamic radii of Nanoworms remain stable at 37 °C for least 5 days in dPBS (10 mM, n=3, mean±SD). Length Width 0 5 10 15 60 80 100 120 Nanometer (nm) aCD20A aHLADR10A aCD19A aCD3A 1 10 100 1000 0 20 40 60 80 100 Hydrodynamic radius (nm) % Intensity RTX aCD20A aHLADR10A aCD19A aCD3A 1 2 3 4 5 6 7 8 9 10 11 12 0 1 2 10 4 10 5 10 6 10 7 10 8 10 9 Volume (mL) Detection Voltage Molar Mass (g/mol) aCD20A aHLADR10A aCD3A RTX aCD19A 0 24 48 72 96 120 0 20 40 60 80 100 Hours Hydrodynamic radius (nm) aCD20A aCD19A aCD3A aHLADR10A A B D C E 68 2.3.2. Nanoworms cluster bound receptors on the immune cell-surface Having demonstrated their oligomerization into Nanoworms, their specificity was confirmed through competitive binding. Fluorescence imaging confirmed specificity of aCD20A and aHLADR10A as B cells incubated with these Nanoworms underwent strong homotypic adhesion, which made flow cytometry on individual cells untenable. Both aCD20A and aHLADR10A showed target specificity towards CD20 and HLA-DR10, respectively (Figure 5A,B). Pre-blocking these receptors using their respective parent monoclonal antibodies, RTX for aCD20A and chLym-1 for aHLADR10A, substantially decreased the cell surface binding of labeled Nanoworms. Flow cytometry was used to analyze and confirm the specificity of aCD19A and aCD3A (Figure 5C,D). Given the limited availability of blinatumomab as a control, receptor specificity was confirmed once in two different cell lines. It was hypothesized that Nanoworms will cluster receptors in on the cell surface, which is mechanistically similar to Fc-FcR mediated clustering (Selewski et al., 2010). To observe Nanoworm-mediated receptor clustering, real-time live cell fluorescence imaging was used to track Nanoworm-bound receptors during a gradual temperature increase. Nanoworms were deliberately bound to cells under low temperatures where membranes fluidity is low. Upon heating, all Nanoworms induced substantial clustering on the cell-surface (Figure 5E). The integrated fluorescence density was measured to quantify Nanoworm-mediated receptor clustering temperature (Tc) on individual cells (Figure 5F). This observation suggests that Nanoworms mediate receptor clustering below physiological temperatures, which was consistent across this small panel of immune receptors (Figure 5G). Compared to Nanoworms, the monoclonal antibody RTX did not show any apparent clustering under identical conditions. Integrated fluorescence density of RTX alone shows only a continuous decrease due to photobleaching. 69 Figure 5. Nanoworms spontaneously cluster cell surface receptors below physiological temperature. (A) Specificity of rhodamine-labeled aCD20A was evaluated on live cells using confocal microscopy on CD20+ Raji cells. RTX was used to pre-block the CD20. (B) Specificity of rhodamine-labelled aHLADR10A was evaluated on HLA-DR10+ Raji cells. chLym-1 was used to pre-block HLA-DR10. For (A) and (B), experiments were performed in triplicate with 10~20 cells imaged per field of view (n=40~50 cells). (C) Specificity of rhodamine-labeled aCD19A towards CD19+ Raji and SU-DHL-7 cells. (D) Specificity of rhodamine-labeled aCD3A towards CD3+ HuT-78 and Jurkat cells. For (C) and (D), cells were gated (black bar) at the upper 10% of the fluorescence profile of blinatumomab + aCD19A (or aCD3A) cells (blue) to compare the percentage of non-treated cells (Neg cntl) and cells treated with aCD19A (or aCD3A) only. (E) Time-lapse live cell epi-fluorescence imaging was used to monitor rhodamine- labeled Nanoworms bound to receptor-positive human cell lines (Raji cells for aHLADR10A and aCD19A; Hut-78 cells for aCD3A). Cells were heated (15~45 ºC at 2 ºC/min) during imaging. Representative images are shown (n=43~45 cells). (F) Receptor clustering was quantified using RTX à aCD20A aCD20A A Non-Treat RTX → aCD20A aCD20A 0 10000 20000 30000 40000 50000 Fluorescence Intensity **** **** * p=0.04 RTX à aCD20A B chLym-1à aHLADR10A aHLADR10A Non-Treat aHLADR10A 0 50000 100000 150000 Fluorescence Intensity **** **** ** p=0.007 chLym-1 à aHLADR10 subset 0.045 10 2 10 3 10 4 PE-A 0 50 100 150 200 250 Count Sample Name Subset Name Count jurkat_cd3 only.fcs Lymphocytes 8914 jurkat_blincyto+cd3.fcs Lymphocytes 8510 jurkat_neg.fcs Lymphocytes 8839 Jurkat aCD3A only 38.5% Blinatumomab + aCD3A 10.2% Neg cntl 0.05% PE-A subset 2.19 10 2 10 3 10 4 PE-A 0 20 40 60 80 Count Sample Name Subset Name Count SUDHL7-2_CD19N ONLY_005.fcs Ungated 2513 SUDHL7-2_BLINCYTO+CD19N_004.fcs Ungated 2510 SUDHL7-3_Neg cntl_001.fcs Ungated 2510 SU-DHL-7 Neg cntl 2.2% Blinatumomab + aCD19A 10.0% aCD19A only 33.3% subset 1.38 10 2 10 3 10 4 PE-A 0 20 40 60 80 100 Count Sample Name Subset Name Count Raji_CD19N_CD19N Only.fcs Lymphocytes 4501 Raji_CD19N_Blincyto100ul+CD19N.fcs Lymphocytes 4046 Raji_CD20N_Neg cntl.fcs Lymphocytes 4771 subset 2.28 0 -10 3 10 3 10 4 10 5 PE-A 0 50 100 150 Count Sample Name Subset Name Count HuT78_Blicyto 100ul+CD3N_001.fcs Lymphocytes 6733 HuT78_CD3N_008.fcs Ungated 6232 HuT78_Neg cntl_006.fcs Ungated 8109 Blinatumomab + aCD3A 10.0% Neg cntl 2.3% Raji HuT-78 Neg cntl 1.4% Blinatumomab + aCD19A 9.9% aCD19A only 29.7% anti-CD19-A192 anti-CD3-A192 aCD3A only 28.8% Count Rhodamine Count Rhodamine subset 0.045 10 2 10 3 10 4 PE-A 0 50 100 150 200 250 Count Sample Name Subset Name Count jurkat_cd3 only.fcs Lymphocytes 8914 jurkat_blincyto+cd3.fcs Lymphocytes 8510 jurkat_neg.fcs Lymphocytes 8839 Jurkat aCD3A only 38.5% Blinatumomab + aCD3A 10.2% Neg cntl 0.05% PE-A subset 2.19 10 2 10 3 10 4 PE-A 0 20 40 60 80 Count Sample Name Subset Name Count SUDHL7-2_CD19N ONLY_005.fcs Ungated 2513 SUDHL7-2_BLINCYTO+CD19N_004.fcs Ungated 2510 SUDHL7-3_Neg cntl_001.fcs Ungated 2510 SU-DHL-7 Neg cntl 2.2% Blinatumomab + aCD19A 10.0% aCD19A only 33.3% subset 1.38 10 2 10 3 10 4 PE-A 0 20 40 60 80 100 Count Sample Name Subset Name Count Raji_CD19N_CD19N Only.fcs Lymphocytes 4501 Raji_CD19N_Blincyto100ul+CD19N.fcs Lymphocytes 4046 Raji_CD20N_Neg cntl.fcs Lymphocytes 4771 subset 2.28 0 -10 3 10 3 10 4 10 5 PE-A 0 50 100 150 Count Sample Name Subset Name Count HuT78_Blicyto 100ul+CD3N_001.fcs Lymphocytes 6733 HuT78_CD3N_008.fcs Ungated 6232 HuT78_Neg cntl_006.fcs Ungated 8109 Blinatumomab + aCD3A 10.0% Neg cntl 2.3% Raji HuT-78 Neg cntl 1.4% Blinatumomab + aCD19A 9.9% aCD19A only 29.7% anti-CD19-A192 anti-CD3-A192 aCD3A only 28.8% Count Rhodamine Count Rhodamine C D CD20 HLA-DR10 CD19 CD3 0 10 20 30 40 50 Cell Surface Target Receptor clustering Temperature (T c ,°C) 15 20 25 30 35 40 0 1 2 3 4 5 6 7 Temperature ( o C) Fluorescence Intensity (x 10 7 ) aCD20A aCD19A aCD3A aHLADR10A RTX E G F 70 integrated fluorescence density within the cell. Fluorescence intensity increases as the cluster forms in a subregion of the cell. Each fluorescence intensity profile was acquired by analyzing individual cells shown in panel D. (G) Receptor clustering temperature (Tc) was defined where the maximum first derivative was observed, as estimated by Eq. 7 (Mean±SD). Experiments were performed in triplicate with 10~20 cells imaged per experiment. A one-way ANOVA was used for statistical comparison. 71 2.3.3. Nanoworm-mediated receptor clustering on the immune cell-surface activates intracellular signaling Cell membrane blebbing is a distinguished phenomenological feature of cells undergoing cellular locomotion, cytokinesis (non-apoptotic), or apoptosis (apoptotic) (Charras, 2008). When behavior of aCD20A was observed on the cell surface (Figure 5E), some of the cells immediately experienced massive reformation of their membranes. Live-cell brightfield imaging showed about 50% of cells experienced membrane blebbing below physiological temperatures (Figure 6A), at similar temperatures where aCD20A began to cluster on the cell-surface. Likewise, membrane blebbing was apparent when aHLADR10A clustered on the cell surface (Figure 6A). As CD20A or aHLADR10 were specifically blocked by monoclonal antibodies RTX or chLym1 respectively (Figure 5A,B), blebbing appeared consistent with the possibility that they clustered CD20 or HLA-DR10, respectively, thus activating intracellular signaling. As these two receptors have well-characterized roles in B cell apoptosis, cytosolic cytochrome C was measured upon incubation of B cells with aCD20A, aHLADR10A, and their respective antibody controls. Release of mitochondrial cytochrome C into the cytoplasm is a hallmark of apoptosis often considered a ‘point of no return’ (Cai et al., 1998). After 18h, Raji cells incubated with aCD20A and aHLADR10A showed a significantly higher cytosolic cytochrome C level compared to non-treated cells (Figure 6B). Rituximab supplemented with a secondary anti-human crosslinking antibody (RTX+2º) induced release of mitochondrial cytochrome C into the cytoplasm, which was comparable to aCD20A treated cells. RTX alone did not show signs of apoptosis, which was consistent with prior observations (Aluri et al., 2014a). In contrast, the HLA-DR10 targeting antibody chLym-1, either alone or with a secondary anti-human crosslinking antibody, induced release of mitochondrial cytochrome C into the cytoplasm at levels comparable to that of the aHLADR10A treated cells. This observation is consistent with prior characterization of the chLym-1 antibody, which binds lipid-raft associated HLA-DR10 and induces apoptosis without secondary crosslinkers (Zhang et al., 2007). 72 CD19 is reported to compartmentalize with IgM and IgD on the B cell surface, which plays an important role in BCR-mediated signal transduction (Mattila et al., 2016). Due to its indispensable role in proliferation and survival, most of the malignant B cells consistently express CD19. As it is found in all NHL subtypes as well as in acute or chronic lymphocytic leukemia (ALL or CLL), CD19 serves as a reliable therapeutic target for lymphoma and leukemia (Raponi et al., 2011; Wang et al., 2012). B cells experiencing BCR-mediated signal transduction undergo actin cytoskeleton rearrangement, which is directly related to cell locomotion but unrelated to apoptosis (Fujimoto et al., 1999; Li et al., 2018a). Therefore, a possible outcome of CD19 clustering might include non-apoptotic cell cycle arrest (Ghetie et al., 1994). Similar to CD20 and HLA-DR10, membrane blebbing was also coincident with CD19 clustering on B cells (Figure 6A). Despite blebbing, CD19 clustering failed to induce release of Cytochrome C, indicating they did not promote apoptosis (Figure 6C). While failing to induce apoptosis, aCD19A does arrest the cell cycle in the G1/G0 transition phase (Figure 6D). The cell cycle distribution upon aCD19A treatment was similar to that of control cells incubated with rapamycin, which was chosen because of its clinical usage as an immunosuppressant with cytostatic properties (Lee et al., 2019). About 2% of all lymphomas are the cutaneous T cell NHL (CTCL). The transformed T cells that are residing at or homing to the skin is known to cause CTCL (Willemze et al., 2005). Due to its low incidence, clinical trials are rare and there are no universal treatment guidelines (Photiou et al., 2018). The recent study that involved 21 centers around the globe reported 24 different treatment approaches with no certain approach having more than 15% of the market share (Quaglino et al., 2017). Mycosis fungoides and Sézary syndrome are the most common subtypes of CTCL. More than 70% of the patients diagnosed with Mycosis fungoides are at the early stages and have median survival of 13 years, however, Sézary syndrome is a more progressed disease and the patients’ median survival is less than 3 years (Agar et al., 2010; Kim et al., 2003). 73 Patient derived Sézary syndrome cell lines allowed meaningful advancements in our understanding towards its pathophysiology (Bunn and Foss, 1996). Using one of the developed cell lines, HuT78, the therapeutic role of CD3 clustering in this particular disease is studied. The CD3 receptor is found associated with the T cell receptor (TCR) complex. It is reported that anti- CD3 monoclonal antibodies induce membrane blebbing and activate certain T cells, which is commonly monitored by secretion of interleukin 2 (IL-2) (Blanchard et al., 2002; Minguet et al., 2007). In a subset of T cells, the secretion of IL-2 is linked to increased apoptosis, which is known as activation-induced cell death (AICD) (Arakaki et al., 2014). The Sézary cells are known to be pathogenic due to their resistance to AICD rather than uncontrolled proliferation (Hwang et al., 2008) and one of the resistance conferring mechanisms is the insufficient signal from the TCR (Klemke et al., 2009). Therefore, manipulation of TCR signaling via CD3 clustering may be an effective therapeutic approach to overcome the resistance. To explore this, CD3 clustering Nanoworm (aCD3A) was incubated with HuT78 cells and subjected to the process employed in Figures 5 and 6. The cellular response that immediately observed upon CD3 clustering was the membrane blebbing (Figure 6A), which is a direct evidence of intracellular signaling activation. To see which cellular pathway is activated, IL-2 level in cell culture media and cytosolic cytochrome C level in the cells were quantified for T cell activation and apoptosis, respectively. The AICD was pronounced upon aCD3A treatment in HuT78 cells. aCD3A treatment directed HuT78 cells more towards apoptosis with lesser activation compared to cells treated with OKT3 supplemented with crosslinker (OKT3+2º). While targeting CD3 without clustering (OKT3 only) induced T cell activation without apoptosis, OKT3+2º induced AICD (Figure 7A,B). In CEM, a mosaic human leukemia T cell line, aCD3A effectively induced apoptosis at a comparable level to OKT3+2º, however, it did not induce activation. AICD was only observed in OKT3+2º treated CEM cells. In Jurkat, an acute human leukemia T cell line, aCD3A induced secretion of IL-2, however, the fold-change was minute (3- fold) compared to OKT3 only (132-fold) or OKT3+2º (104-fold) treated cells and was not 74 statistically significantly different from that of the dPBS treated (Non-Treat) cells. Moreover, aCD3A treatment did not induce apoptosis. The aCD3A-mdeiated AICD was exclusively observed in HuT78 cells but not in CEM or Jurkat cells. In HuT78 and Jurkat cells, addition of crosslinker (2º) after OKT3 treatment directed activation-without-apoptosis to AICD, but this was not seen in CEM cells. Based on the report that IL-2 exposure sensitizes AICD resistant Sézary cells to apoptosis (Klemke et al., 2009), studies were performed to test whether co-treatment of OKT3 and aCD3A can further activate T cells. Two cell lines, HuT78 and Jurkat, were chosen because these two secreted IL-2 upon aCD3A treatment. To observe co-stimulatory effect, HuT78 or Jurkat cells pre-incubated with aCD3A were placed into the cell culture wells that were pre- coated with OTK3. Upon co-incubation, HuT78 and Jurkat cells showed very different cellular responses. HuT78 cells showed enhanced activation under the presence of both (Adsorbed OKT3+aCD3A) compared to single agent treatments (Figure 7C). The extrapolated EC50 for aCD3A in T cell activation was 4.3 μM (Figure 7D). On the contrary, there was no additive effect in Jurkat cells. Instead, Jurkat cells experienced inhibition of activation with increasing concentration of aCD3A (Figure 7E). The extrapolated IC50 for aCD3A in inhibition was 1.0 μM (Figure 7F). Based on its highest apoptotic potential and the additive effect with OKT3, aCD3A may be a potential therapeutic agent for Sézary syndrome, either as a monotherapy or as a combination therapy with existing T cell activators. One question that was not able to answered at this time was the difference in cellular responses with similar specificity. In all cell lines, OKT3+2º treated group experienced AICD, which was not always the case for aCD3A treatment. Based on the fact that both OKT3 and blinatutmomab target CD3ε (Sugiyama et al., 2017), it was somewhat perplexing and surprising to see a clear difference between these two treatments (OKT3+2º vs. aCD3A) towards AICD (Figure 7A,B) and additive/inhibitory effect in T cell activation (Figure 7C~F). There could be two hypotheses for this. The first hypothesis is the degree of clustering due to differential 75 multivalency. The maximum valency that OKT3 further engaged by 2º (OKT3+2º) is four, while single aCD3A can display more than 300 scFvs. Therefore, aCD3A (and other Nanoworms) forms a single macrodomain on the cell surface (Figure 5E) that is different from multiple microdomains formed by OKT3+2º or other low valency clustering agents (Su et al., 2016b; Yi et al., 2019). This difference on the cell surface may activate slightly different intracellular pathways and also be responsible for different additive/inhibitory effect in T cell activation in different cell lines (HuT78 vs. Jurkat). The second hypothesis is based on different epitopes within the CD3ε between OKT3 and blinatumomab. However, the binding region for blinatumomab or its parent clone L2K is not well characterized compared to that of the OKT3 (Kjer-Nielsen et al., 2004). Therefore, this needs further experimental evaluation. Given the depth of this question and the scope of the current study, it will be covered during the follow up studies. 76 Figure 6. Receptor clustering induces cell membrane reformation (blebbing) and activates lymphoid cells. (A) Time-lapse live cell brightfield imaging shows cells experience blebbing (white arrows) upon receptor clustering. Cells were heated (15~45 ºC, 2 ºC/min) during imaging. (B) Quantification of cytosolic cytochrome C level in Raji cells in complete media after 18h incubation at 37 ºC with Nanoworms or their respective control molecules in equivalent scFv molar concentration. (C) Quantification of cytosolic cytochrome C level in Raji cells after 18h incubation with aCD19A at 37 ºC. chLym-1 was used as a positive control. (D) Flow cytometry was used to study the cell cycle distribution of Raji cells after 48h incubation with aCD19A at 37 ºC in complete media. Rapamycin was used as a positive control to arrest the cell cycle (n=3). Propidium iodide was used to quantify DNA content. A one-way ANOVA followed by multiple comparisons was used for statistical comparison. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05. Mean±SD. 20°C 37°C Temperature: < 20°C A aCD20A 20°C 37°C Temperature: < 20°C aHLADR10A 20°C 37°C Temperature: < 20°C aCD19A 20°C 37°C Temperature: < 20°C aCD3A aCD20A aCD19A aCD3A aHLADR10A Non-Treat RTX RTX + 2° aCD20A chLym-1 chLym-1 + 2° aHLADR10A 0 1 2 3 4 Cytosolic Cytochrome C (fold change) *** G0/G1 S G2/M 0 10 20 30 50 60 70 80 90 100 Cell cycle phase Percentage (%) Non-Treated aCD19A Rapamycin *** *** ns **** *** ns ns * ** Non-Treat aCD19A chLym-1 0 1 2 3 4 Cytosolic Cytochrome C (fold change) ns **** B C D A 77 Figure 7. CD3 clustering induces either activation, apoptosis, or activation- induced cell death (AICD) on T cell lines of NHL or leukemia. (A) Quantification of IL-2 in HuT78, CEM, or Jurkat cell culture media after 18h incubation with aCD3A at 37 ºC (n=3). (B) Quantification of cytosolic cytochrome C level in HuT78, CEM, or Jurkat cells after 18h incubation with aCD3A at 37 ºC. (C,D) Quantification of IL-2 in HuT78 cell culture media after 18h of co-incubation with OKT3 and aCD3A at 37 ºC (n=3). Additive effect of OKT3 and aCD3A (C) and dose-dependent increase (D) in IL-2 secretions were observed. Extrapolated EC50 is 4.3 μM. (E,F) Quantification of IL-2 in Jurkat cell culture media after 18h of co-incubation with OKT3 and aCD3A at 37 ºC (n=3). Inhibitory effect of aCD3A on IL-2 secretion (E) and its dose-dependency (F) in Jurkat cell culture media were observed. Extrapolated IC50 is 1.0 μM. A one-way ANOVA followed by multiple comparisons was used for statistical comparison. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05. Mean±SD. -4 -3 -2 -1 0 1 0 50 100 150 200 log[aCD3A], µM IL-2 in culture media (fold change) Non-Treat aCD3A 0 1 2 3 50 100 150 200 250 IL-2 in culture media (fold change) **** **** ** Non-Treat 0.5 µM 2 µM 8 µM 0 5 10 15 20 25 IL-2 in culture media (fold change) Adsorbed OKT3 + aCD3A (X µM) *** ** * Non-Treat OKT3 OKT3 + 2° aCD3A 0 1 2 3 4 5 6 7 8 IL-2 in culture media (fold change) ns *** *** *** ns ns Non-Treat OKT3 OKT3 + 2° aCD3A 0 1 2 100 200 300 400 500 IL-2 in culture media (fold change) **** **** **** **** **** **** Non-Treat OKT3 OKT3 + 2° aCD3A 0 2 4 6 100 200 300 IL-2 in culture media (fold change) **** ns **** **** **** **** Non-Treat OKT3 OKT3 + 2° aCD3A 0.0 1.0 2.0 3.0 4.0 Cytosolic Cytochrome C (fold change) ** ns ** ns ** ** Non-Treat OKT3 OKT3 + 2° aCD3A 0.0 0.5 1.0 1.5 2.0 2.5 Cytosolic Cytochrome C (fold change) ** ns *** * **** **** Non-Treat OKT3 OKT3 + 2° aCD3A 0.0 0.5 1.0 1.5 2.0 2.5 Cytosolic Cytochrome C (fold change) * ns ns ** ** ns HuT78 CEM Jurkat Apoptosis Activation -1.0 -0.5 0.0 0.5 1.0 0 5 10 15 20 log[aCD3A], µM IL-2 in culture media (fold change) A C E Adsorbed OKT3 Adsorbed OKT3 + aCD3A B D F HuT78 Jurkat Adsorbed OKT3 78 2.3.4. Generation of CD20 targeting Nanoworm variants ELPs are high molecular weight polymers that show a reversible phase separation in response to heating. The phase transition temperature (Tt), can be precisely controlled at the genetic level or at the environmental level depending on hydrophilicity of the guest residue (X), number of repeats (n), ELP concentration, and ionic strength. One of these factors, especially ELP concentration, came into our attention in terms of its inverse correlation to Tt (Figure 3D). Empirically, ELP Tt trends lower when the ELP concentration becomes higher. Based on this, one might argue that Nanoworm binding to cell surface results in high ELP concentration in a confined area, which leads to a significant drop in their Tt. As a result, receptor clustering may proceed via ELP-mediated phase separation but not necessarily through their multivalency. To delineate the main factor that affected receptor clustering observed in Figure 5E, four additional anti-CD20-ELP fusions were designed that have similar MW but differ in their Tt. ELPs V2A64, A96G96, S192, and G192, were selected to cover a wide range of Tt, from 34~76 °C (Figure 8A,B). The resulting four anti-CD20-ELPs, will be referred as aCD20VA, CD20AG, aCD20S, and aCD20G for convenience (Table 4). These variants were identical in ELP repeat number and only differed in Tt based on their ELP amino acid composition (Figure 8C). Scanning optical density at 350 nm from various concentrations confirmed differential thermo- sensitivity of four anti-CD20-ELP variants and aCD20A (Figure 8D). Calculated shape factors (Rg/Rh) based on DLS and SEC-MALS analyses (Figure 8E,F) indicated that all five anti-CD20- ELP variants are consistent with Nanoworms (Table 5). 79 Figure 8. Design, identity, and biophysical characteristics of anti-CD20-ELP fusions with a range of thermal sensitivity. (A) cDNAs encoding anti-CD20 scFv were fused to amino terminus of ELPs V2A64, A192, A96G96, S192, and G192. This library was designed because they have different solution phase behaviors, while having the same number of pentameric repeats (n=192). (B) SDS-PAGE was used to confirm the identity of all anti-CD20-ELP fusions. (C) All of these ELP fusions exhibited temperature-dependent increases in optical density (λ=350 nm, 10 μM in dPBS). Furthermore, their transition temperatures ranged from 34 to 76 ºC. (D) Optical density increases from baseline upon coacervation, which was used to construct a phase diagram for each fusion. These follow a log-linear relationship between transition temperature and concentration, above which phase separation occurs. (E) DLS analyses confirm that Nanoworms have higher hydrodynamic radii compared to RTX. (F) SEC-MALS reveals that anti-CD20-ELP fusions flow through the column in the void volume, while the RTX is retained on the column. All of these Nanoworms have a significantly higher molar mass compared to RTX. Dotted line: molar mass; Solid line: UV Detection voltage. Data points and fits for aCD20A used in Figs. 3 and 4 were included for a better comparison. 1 2 3 4 5 6 7 8 9 10 11 12 0 1 2 10 4 10 5 10 6 10 7 10 8 10 9 Volume (mL) Detection Voltage Molar Mass (g/mol) aCD20VA aCD20A aCD20AG aCD20S aCD20G RTX 1 10 100 1000 0 20 40 60 80 100 Hydrodynamic radius (nm) % Intensity RTX aCD20VA aCD20A aCD20AG aCD20S aCD20G anti-CD20 anti-CD20 anti-HLA- DR10 anti-HLA- DR10 anti-CD19 anti-CD19 anti-CD3 anti-CD3 A192 A192 A192 A192 aCD20A aHLADR10A aCD19A aCD3A Variable heavy (VH) Variable light (VL) ELP scFv Linker: (GGGGS) 3 V2A64 A96G96 S192 G192 aCD20G aCD20S aCD20AG aCD20VA V H V L Elastin-Like Polypeptides (ELP) anti-CD20 scFv Linker: (GGGGS) 3 V H V L V H V L V H V L A192 scFv-ELP fusion Elastin-Like Polypeptides (ELP) scFv V H V L scFv 75 20 100 150 50 37 25 15 10 POI Coomassie aCD20A aCD20VA aCD20AG aCD20S aCD20G 20 30 40 50 60 70 80 0 1 2 3 Temperature ( o C) OD 350nm aCD20VA aCD20AG aCD20A aCD20G aCD20S A B C D E F 1 10 100 0 10 20 30 40 50 60 70 80 90 Concentration (µM) Transition temperature (°C) aCD20VA aCD20AG aCD20S aCD20G aCD20A 80 Table 4. Molecular information of anti-CD20-ELP fusions. a aCD20 indicates the following amino acid sequence: GQVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSY NQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAG GGGSGGGGSGGGGSQIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIY ATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKRT b Calculated from the Table 5 using Eq 4. c,d Mean±SD from Figure 9C,D. anti-CD20- ELPs Amino acid sequence a (aCD20: antiCD20 scFv) Total (VPGXG) repeats T t at 10 μM (°C) b T c at 10 μM in Raji (Mean±SD °C) c T c at 10 μM in SU-DHL-7 (Mean±SD °C) d aCD20VA aCD20-G(VPGVG VPGAG VPGAG)64Y 192 34.4 20.8 ± 4.1 25.4 ± 8.2 aCD20A aCD20-G(VPGAG)192Y 192 46.4 28.4 ± 4.6 25.3 ± 7.6 aCD20AG aCD20-G[(VPGAG)96(VPGGG)96]Y 192 58.1 34.1 ± 4.8 24.6 ± 6.1 aCD20S aCD20-G(VPGSG)192Y 192 61.6 31.0 ± 4.6 24.3 ± 4.9 aCD20G aCD20-G(VPGGG)192Y 192 76.0 32.1 ± 3.9 30.4 ± 8.4 81 2.3.5. Nanoworms’ multivalency and thermo-sensitivity coordinate receptor clustering on the cell surface To test the effect of differential thermo-sensitivity towards Tc, two human lymphoma CD20+ B cell lines (Raji and SU-DHL-7) were studied by time-lapse live-cell fluorescence imaging (Figure 9A). The integrated fluorescence density was again used to quantify Nanoworm cell-surface clustering (Figure 9B). Cell-surface clustering temperature (Tc) in a region of interest within individual cells was identified using Eq. 6 and 7. On Raji cells, aCD20VA, aCD20A, and aCD20AG showed a positive correlation between Tt and Tc; however, there was no difference in Tc between aCD20AG, aCD20S, and aCD20G (Figure 9C). This trend was less apparent in SU-DHL-7 cells; however, the Tc of aCD20G showed a statistical difference from those of aCD20A, CD20AG, and aCD20S (Figure 9D). It was interesting to observe that even aCD20G Nanoworms, whose solution Tt is about 76 °C (10 μM) still clustered on the cell-surface below physiological temperatures in both cell lines (32.1±3.9 °C in Raji and 30.4±8.4 °C in SU-DHL-7). These observations indicate that the multivalency of Nanoworms is more likely the primary factor governing their cell-surface clustering, while there remains a secondary effect of ELP hydrophilicity, and that only the largest differences between Tt produced a shift in Tc. 82 Figure 9. Anti-CD20-ELP Nanoworms cluster on the cell-surface below physiological temperatures. (A) Time-lapse live cell fluorescence imaging was used to monitor rhodamine- labeled Nanoworms (red) bound to CD20+ Raji cells. Raji cells were heated (~2 ºC/min) during imaging, which revealed significant clustering for all Nanoworm formulations. A representative cell is shown with the measured media temperature (ºC) indicated on each panel. (B) Nanoworm clustering was quantified using integrated fluorescence density within each cell as a function of temperature. Each fluorescence intensity profile was acquired by analyzing individual cells as shown in panel A. (C,D) Cell-surface clustering temperature(Tc) of each Nanoworm was determined using the peak of Eq. 7 (Mean±SD). Tc of all Nanoworms occurred below 37 ºC in both (C) Raji and (D) SU-DHL-7 cells. The Tc observed for aCD20A was significantly lower than for the Nanoworm with the highest solution temperature, aCD20G, in both Raji and SU-DHL-7 cells. The Nanoworm with the lowest solution temperature, aCD20VA, had Tc significantly lower than aCD20A only on Raji cells. Experiments were performed in triplicate with 8-12 cells imaged per field of view (n=30-32 cells). A one-way ANOVA was used for statistical comparison. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05. aCD20VA aCD20A aCD20AG aCD20S aCD20G 0 10 20 30 40 50 60 Receptor clustering Temperature (T c ,°C) SU-DHL-7 ** * * aCD20VA aCD20A aCD20AG aCD20S aCD20G 0 10 20 30 40 50 60 Receptor clustering Temperature (T c ,°C) **** Raji **** **** **** * **** ns A B C 15 20 25 30 35 40 0 1 2 3 4 5 6 7 8 Temperature ( o C) Fluorescence Intensity (x 10 5 ) aCD20VA aCD20A aCD20AG aCD20G aCD20S D 83 2.3.6. Enthalpy and entropy of Nanoworm phase separation is correlated to the hydrodynamic radii of the Nanoworms The phase behavior of ELP or ELP fusions depends on biophysical characteristics, including temperature, molecular weight, hydrophilicity, concentration, and co-solutes. Using estimated enthalpy and entropy of 8 Nanoworms and non-fusion ELP A192 upon phase separation (Figure 10A,B, Table 5), multiple regression was applied to identify biophysical factors that are associated with thermodynamics of Nanoworm phase separation. Four independent variables (hydrophilicity of ELP, molecular weight of scFv-ELP monomer, oligomeric state of Nanoworm, and Rh of Nanoworm) were subjected to stepwise multiple regression towards enthalpy and entropy. Among tested variables, Rh of the Nanoworm best explained the enthalpic gain (p=0.020) and entropic cost (p=0.013) of Nanoworm coacervation (Figure 10C). As the greater degree of oligomerization (higher molecular weight per particle) leads to larger Rh (Pearson correlation (r)=0.801, n=9, p=0.009) and Rg (Pearson correlation (r)=0.929, n=8, p=0.001) (Figure 10D), this analysis shows that the greater degree of oligomerization (increased Rh) is associated with particle size, increased endothermic heat, and entropic cost of coacervation per molecule of ELP. Surprisingly, the hydrophilicity of ELP was not significantly correlated to the enthalpy (R 2 =0.23, p=0.19) and entropy (R 2 =0.33, p=0.11) of Nanoworm phase separation (Figure 10E). 84 Figure 10. Estimated enthalpy (ΔH) and entropy and (ΔS) upon Nanoworm phase separation show significant correlation to the hydrodynamic radii of Nanoworms. (A,B) Van’t Hoff plots of (A) scFv-ELP Nanoworms and (B) anti-CD20-ELP Nanoworms are shown. Plots were generated using Eq. 5. Enthalpy and entropy upon phase separation were estimated from the fit (lines) as reported in Table 5. aCD20A data points and fit (red dotted line) from (A) were added to (B) for comparison. (C) The differences in hydrodynamic radius did account for approximately half of the variance in both enthalpy (R 2 =0.50 p=0.020) and entropy (R 2 =0.56 p=0.013) of Nanoworm phase separation. Black lines indicate linear regressions for each. (D) As would be expected, the greater degree of oligomerization is significantly correlated with increased Rh (p=0.009) and Rg (p=0.001). (E) In contrast, the hydrophilicity of ELP, as an estimate of transition temperature, did not significantly predict variance in the enthalpy (R 2 =0.12 p=0.2) or entropy (R 2 =0.23 p=0.1) of Nanoworm phase separation. 20 40 60 80 20.0 40.0 60.0 80.0 100.0 5000 15000 25000 35000 45000 55000 Hydrodynamic radius (nm) Molecular weight/particle Radius of gyration (nm) aCD20A aHLADR10A aCD19A aCD3A aCD20VA aCD20AG aCD20S aCD20G aCD20A aHLADR10A aCD19A aCD3A aCD20VA aCD20AG aCD20S aCD20G 0.00308 0.00310 0.00312 0.00314 0.00316 0 1 2 3 4 5 1/T t (K) ln[ELP(µM)] aCD20A aHLADR10A aCD19A aCD3A 0.0028 0.0029 0.0030 0.0031 0.0032 0.0033 0 1 2 3 4 5 1/T t (K) ln[ELP(µM)] aCD20AG aCD20S aCD20G aCD20A aCD20VA 0 30 60 90 0.0 1.0 2.0 3.0 200 400 600 800 1000 Hydrodynamic radius (nm) ΔH (KJ/mol) ΔS (KJ/mol•K) A192 aCD20A aHLADR10A aCD19A aCD3A aCD20VA aCD20AG aCD20S aCD20G A192 aCD20A aHLADR10A aCD19A aCD3A aCD20VA aCD20AG aCD20S aCD20G 30 40 50 60 70 80 0.0 1.0 2.0 3.0 4.0 200 400 600 800 1000 Hydrophilicity of ELP (T t at 1µM Nanoworm) ΔH (KJ/mol) ΔS (KJ/mol•K) A192 aCD20A aHLADR10A aCD19A aCD3A aCD20VA aCD20AG aCD20S aCD20G A192 aCD20A aHLADR10A aCD19A aCD3A aCD20VA aCD20AG aCD20S aCD20G A B D C E 85 Figure 11. Thermo-sensitivity and size of A192. (A) ELP A192 exhibits temperature- dependent increase in optical density (λ=350 nm). (B) Tt identified by Eq. 3 for each concentration was used to construct a phase diagram. These again follow a log-linear relationship between transition temperature and concentration, above which phase separation occurs. (C) Van’t Hoff plots of A192. Plot was generated using Eq. 5. Enthalpy and entropy upon phase separation were estimated from the fit (lines) as reported in Table 5. (D) DLS analysis confirms that A192 remains as monomer in solution. 1 10 100 0 20 40 60 80 100 Hydrodynamic radius (nm) % Intensity 0.0025 0.0029 0.0033 0 1 2 3 4 5 6 1/T t (K) ln[ELP(µM)] 50 55 60 65 70 0 1 2 3 Temperature ( o C) OD 350nm 100 µM 50 µM 25 µM 10 µM 5 µM 1 10 100 1000 40 50 60 70 80 Concentration (µM) Transition temperature (°C) A B C D 86 Table 5. Biophysical characteristics of ELP, ELP fusions, and an antibody control. Proteins (anti-(target)-(ELP)) a Phase Diagram b Thermodynamics c MW (kDa) /mon omer d Average MW(kDa) /particle Mean (±SEM) e Oligo- meric state f Radius of gyration (R g, nm) Mean (±SEM) g Hydro- dynamic radius (R h, nm) Mean±PD R g/R h Slope, m (°C/log 10[μM]) Mean [95% CI] y-intercept, b (°C) Mean [95% CI] ΔH mix (kJ/mol) Mean [95% CI] ΔS mix (kJ/mol*K) Mean [95% CI] RTX (Rituximab) n/a n/a n/a n/a 143.8 144.0 (±0.006%) 1 6.6 (±0.5%) 8.7±1.5 n/a aCD20A (anti-CD20-A192) -1.8 [-3.7~0.06] 48.3 [46.0~50.5] 958.3 [-33.5~1950] 3.0 [-0.1~6.1] 99.1 51550 (±0.006%) 516 86.0 (±1.2%) 76.1±18.1 1.1 aHLADR10A (anti-HLA-DR10-A192) -2.7 [-3.7~-1.6] 50.7 [49.5~51.9] 729.8 [447~1013] 2.3 [1.4~3.1] 98.7 10880 (±0.002%) 109 41.6 (±0.5%) 47.0±10.1 0.9 aCD19A (anti-CD19-A192) -2.0 [-4.0~0.03] 49.6 [47.2~52.0] 886.6 [-12.7~1786] 2.7 [-0.1~5.6] 100.0 18560 (±0.004%) 186 65.5 (±0.9%) 65.4±15.0 1.0 aCD3A (anti-CD3-A192) -2.2 [-5.6~1.3] 48.3 [44.2~52.4] 707.1 [-418~1832] 2.2 [-1.3~5.7] 99.4 34470 (±0.007%) 345 77.5 (±0.6%) 69.8±15.6 1.1 aCD20VA (anti-CD20-V2A64) -3.5 [-5.1~-1.8] 37.8 [36.5~39.2] 516.8 [273~760] 1.7 [0.9~2.4] 101.1 8477 (±0.0016%) 85 41.0 (±0.9%) 42.5±10.1 1.0 aCD20AG (anti-CD20-A96G96) -3.7 [-5.3~-2.0] 61.8 [60.4~63.1] 565.8 [306~825] 1.7 [0.9~2.5] 98.1 21280 (±0.007%) 213 55.2 (±0.5%) 56.1±12.3 1.0 aCD20S (anti-CD20-S192) -7.9 [-11.5~-4.2] 69.5 [66.5~72.5] 271.0 [141~401] 0.8 [0.4~1.2] 102.3 9588 (±0.006%) 95 39.1 (±0.6%) 45.3±6.5 0.9 aCD20G (anti-CD20-G192) -2.9 [-6.7~0.8] 78.9 [75.9~81.9] 679.5 [-191~1550] 1.9 [-0.6~4.4] 96.4 20350 (±0.054%) 204 46.5 (±0.7%) 32.7±7.4 1.4 A192 h -8.2 [-9.6~-6.7] 70.7 [68.6~72.8] 256.3 [207~306] 0.7 [0.6~0.9] 73.6 n/a 1 n/a 6.9±0.4 n/a a Calculated from the phase diagram (Figure 3D, 8D); b Calculated using Eq 5. c Expected molecular weight based on the amino acid sequence (Table S1); d,f Measured using SEC-MALS, PD=polydispersity; e Calculated by dividing average molecular weight per particle over molecular weight of the monomer; g Measured using DLS; h Data provided in Figure 11. 87 2.4. Discussion Standard first-line therapy for the most of the B cell NHL is R-CHOP, which is a combination of immunotherapy (R: rituximab) and chemotherapy (CHOP: cyclophosphamide, doxorubicin, vincristine, and prednisone) (Salles et al., 2017). The CHOP regimen, developed in the 1970’s, is initially effective in 90% of patients but is responsible for severe side effects and subject to relapse (Sitzia et al., 1997). When rituximab was supplemented along with CHOP, it improved drug response in resistant NHL and produced remission in 50% of patients by activating host immune system through Fc-FcR interactions (Borghaei et al., 2009; Wilson, 2000). Although Fc-FcR mediated immunotherapy was thought to be a golden sword, there found to be Fc-FcR related inconsistencies in therapeutic outcome in some cases (Burkhardt et al., 2016; Zhuang et al., 2010). On one hand, germline polymorphisms on the FcγR, especially on FcγRIIa and FcγRIIIa, on the immune effector cells (Burkhardt et al., 2016; Zhuang et al., 2010) or premature internalization of rituximab by Fc region interacting with FcγRIIb present on the target B cells surface (Lim et al., 2011) leads to insufficient involvement of immune system, which confers resistance to immunotherapy. On the other hand, over-activation of the immune system through Fc-FcγR interaction can cause fatal side effects such as cytokine release syndrome (Atmar, 2010; Gutierrez et al., 2006; Shimabukuro-Vornhagen et al., 2018). As either enhancing or diminishing Fc-FcR interaction may produce unwanted side effects during immunotherapy, modalities that can only induce clustering of the drug:target complex without activating immune system may serve as an alternative strategy to overcome Fc-FcR related heterogeneity (Rezvani and Maloney, 2011). To address this issue, several modalities, such as silver nanoparticles, magnetic nanoparticles, or morpholino oligonucleotide conjugates, have been tested as a proof-of- concept with promising in vitro or in vivo results (Li et al., 2018b; Song et al., 2019; Yao et al., 2017). However, the production process used in these examples require chemical modification followed by another round of purification that may introduce heterogeneity in the degree of 88 functionalization and decrease the production yield, respectively. The utility of such modalities may be amplified through direct cellular expression of well-designed fusion platforms that eliminate the need for bioconjugation or subsequent purification. Elastin-like polypeptides (ELPs) are one such well-characterized fusion platform. ELPs are genetically encodable thermo-responsive protein-polymers with a sequence derived from human tropoelastin, a precursor of elastin that can be expressed in cells as fusion proteins (Urry et al., 1976). This is an exceptional advantage compared to traditional polymers since purified ELP fusions are monodisperse, exceptionally stable, biodegradable, and biocompatible therapeutics (Despanie et al., 2016). Due to ease of engineering and unique biophysical properties, their usage has now been widely tested in the fields of protein sciences (Roberts et al., 2015), tissue engineering (Nettles et al., 2010), medical applications (Gagner et al., 2014), and therapeutic applications (Despanie et al., 2016). Using heterologous expression, the antibody Nanoworms were homogenously biosynthesized and self-assembled to a distinct worm-like nanostructure that display more than 100 scFvs per particle. The dynamics of cell surface receptor clustering mediated by their multivalency was observed using real-time imaging and quantified at a single- cell level. These imaging-based analyses were further correlated to the apoptosis, cell cycle arrest, or cell activation. Our observations indicate that the antibody Nanoworms have inherent ability to cluster and activate bound receptors, which is equipotent to clinically available antibodies supplemented with crosslinker. Because Nanoworms lack the Fc domain, they may overcome the FcR-dependent host immunity and disadvantages observed in the clinic. Having observed architectural consistency (worm-like morphology) and favorable molecular characteristics (stability and high degree multivalency) of scFv-ELP fusions, and their cell surface behavior (spontaneous clustering) in B cell NHL, Nanoworm was further modified to cluster CD3 on T cells and tested on a model of Sézary syndrome, an aggressive form of cutaneous T cell NHL. Current therapeutic regimens for T cell NHL vary by stage (Quaglino et al., 2017). In early stages (stages I and II), the first-line therapy bexarotene (cell cycle inhibitor) 89 is often combined with second-line therapies, such as local radiotherapy, phototherapy, skin electron beam therapy, or gemcitabine. In more advanced stages (stages III and IV), methotrexate (antimetabolite), photopheresis (extracorporeal white blood cell treatment), or poly-chemotherapy are used as first-line therapies with IFNα or chlorambucil as second line agents. These are the most commonly adopted approaches; however, treatment options are not limited to the above-mentioned combinations and oftentimes rely on retrospective approaches by clinicians. There are two antibody-based options approved for T cell NHL, brentuximab vedotin (anti-CD30 antibody-drug conjugate) and mogamulizumab (anti-CCR4 monoclonal antibody). Several others are in the clinical trials as well: anti-CD158K (IPH4102), anti-CTLA-4 (Ipilimumab), and anti-PD-1R (Nivolumab and Pembrolizumab) antibodies are those (Photiou et al., 2018). In line with the effort to develop new antibody-based approaches, the therapeutic impact of CD3 clustering in Sézary cells was explored as a means to overcome the resistance to AICD. Our results in HuT78 cells are also consistent with the results reported by Klemke et al., which emphasizes the importance of T cell receptor (TCR) signaling in AICD (Klemke et al., 2009). Moreover, the Sézary cell lines or primary Sézary cells that were resistant to AICD became prone to apoptosis after IL-2 exposure in their study may suggest the combination treatment of T cell activator and therapeutic receptor clustering agent, such as co-treatment of OKT3 and aCD3A that enhanced IL-2 secretion in our study, may be a reasonable approach for the better therapeutic outcome. 2.5. Conclusion Having Nanoworm-level control over receptor clustering allows manipulation of cell biology in ways that may provide insights for the future therapeutic approaches. Extracellular behavior of Nanoworms was correlated to intracellular signaling activation and showed how this approach can overcome the resistance in NHL. Our study suggests that the scFv-ELP Nanoworm platform that streamlined the production of therapeutic modality, observation in real- 90 time, quantification at the single-cell level is simple, efficient, yet powerful strategy that can also be expanded to investigate the biology of therapeutically relevant ligand-receptor interactions and the biophysical perspectives of bioinspired nanomaterials that will deliver advancements to the field of cancer therapeutics. 91 Chapter 3 Berunda polypeptides: Bi-headed rapamycin carriers for subcutaneous treatment of autoimmune dry eye disease 3.1. Introduction Rapamycin (Rapa), also known as sirolimus (an oral formulation of Rapa), is a macrolide first isolated from Streptomyces hygroscopicus (Kojima et al., 1995). In the human body, Rapa binds to cytosolic FK506 binding proteins (FKBPs) where the complex targets and inhibits mammalian Target Of Rapamycin (mTOR) to alter interleukin-2 (IL-2)-mediated signal transduction (Costa and Simon, 2005). This inhibition of IL-2 signaling can cause cell cycle arrest in the G1-S phase of T-lymphocytes, thereby suppressing its activation and proliferation (Bjornsti and Houghton, 2004; Hardinger et al., 2004; Thomson et al., 2009). Due to its cytostatic and immunosuppressive properties, it is approved by the USFDA to suppress kidney transplant rejection (Wyeth Pharmaceuticals Inc., approved in 1999) (Tedesco Silva et al., 2015) and to treat lymphangioleiomyomatosis, a rare progressive lung disease (Pfizer Inc., approved in 2015) (McCormack et al., 2011). Despite its potency, its use is associated with severe adverse events such as pulmonary- and nephro-toxicity (Marti and Frey, 2005b; Pham et al., 2004) and metabolic complications (Pallet and Legendre, 2013). Moreover, Rapa’s narrow therapeutic index, low water solubility (2.6 μg/mL) (Simamora et al., 2001), low oral bioavailability (<14%) (Stenton et al., 2005), and extremely low plasma-to-whole blood ratio(Zimmerman et al., 2005) often necessitates therapeutic drug monitoring. Efforts to improve the therapeutic index of Rapa include development of more water- soluble analogues (or rapalogs) such as everolimus, temsirolimus and ridaforolimus or formulation strategies to control its release. The ability to overcome dose-limiting factors may expand its use for new indications including autoimmune diseases such as rheumatic diseases, 92 sclerosis or Sjögren’s Syndrome (SS) (Mayer and Kushwaha, 2003; Neuhaus et al., 2007). From our hypothesis that reformulated Rapa for SC administration will improve poor oral bioavailability and exhibit therapeutic efficacy that is comparable to or better than unformulated Rapa, the research described here focuses on how polypeptide fusion carrier, Berunda (two- headed) polypeptide, can improve pharmacokinetic parameters and affect therapeutic potential of Rapa towards autoimmune LG inflammation (dacryoadenitis) in a murine model of SS (Figure. 12). To facilitate the absorption of Rapa without rapid burst release, genes encoding FKBP12 (12 kDa) were genetically fused to the N- and C-terminus of the ELP A192 to create a soluble, biocompatible and biodegradable 97 kDa fusion Rapa carrier named FAF (Dhandhukia et al., 2017) (Figure. 13). FKBP12 is a member of the endogenous cytosolic immunophilin family of proteins that presents the highest affinity for Rapa (EC50=3.8 nM) and the strongest inhibition (IC50=0.7 nM) towards mTOR (Marz et al., 2013). The strong binding between FKBP12 and Rapa enables site-specific, affinity-mediated encapsulation of Rapa to FAF (FAF-Rapa), which brings Rapa’s approximate solubility up to that of FAF. SS is a systemic and chronic autoimmune disease with no approved therapies that affects about 4 million people in the US (Mavragani and Moutsopoulos, 2014). Lymphocytic infiltration and inflammation in the lacrimal gland (LG) and salivary gland (SG) cause severe dry eye and dry mouth, respectively. Mild-to-moderate dry eye symptoms can be treated with ophthalmic solutions such as Restasis Ò (cyclosporine A) (Dastjerdi et al., 2009) or Xiidra Ò (lifitegrast) (Tong et al., 2019); however, these topical formulations are not often efficacious because they target local ocular surface inflammation and not the source of dryness, the inflammation of the LG. These topical formulations also have poor patient compliance due to burning and itching on the ocular surface (de Paiva and Pflugfelder, 2008). Progression of SS not only damages visual and oral health but also affects kidney, lung, liver and thyroid, which 93 necessitates management at the systemic level (Mitsias et al., 2006). While several reports from others (Linares-Alba et al., 2016; Ratay et al., 2017; Wang et al., 2018) and by us (Shah et al., 2013; Shah et al., 2017) showed subconjunctival, intra-LG, topical, or IV delivery of reformulated Rapa exhibits therapeutic efficacy towards dry eye symptoms, this study characterizes SC delivery of Rapa through the FAF carrier to diversify options for this particular route of administration. While nothing would be easier than topical administration, systemic delivery is likely more efficacious at treating symptoms of SS that occur throughout the body. For our purpose, male non-obese diabetic (NOD) mice were selected as is a well-established model of autoimmune dacryoadenitis in SS (Janga et al., 2018b; Ju et al., 2018; Lodde et al., 2006; Takahashi et al., 1997; Wang et al., 2017). Male NOD mice spontaneously develop autoimmune dacryoadenitis (or autoimmune inflammation of the LG) by 8-12 weeks of age, and are used as models of the ocular manifestations of SS (Schenke-Layland et al., 2008). In contrast, female NOD mice spontaneously develop autoimmune sialoadenitis by 12-16 weeks of age, and are used as models of the oral manifestations of SS (Yamano et al., 1999). This study suggests that FAF is a pharmacokinetically-suitable SC Rapa carrier. FAF- Rapa was also therapeutically efficacious in that it significantly suppressed LG lymphocytic infiltration, inflammatory and fibrotic gene expression, and cathepsin S (CTSS) activity (a biomarker of autoimmune disease) in the LG. Collectively these findings show the potential of FAF-Rapa as a SC injectable biotherapeutic for SS. 94 Figure 12. FAF promotes subcutaneous delivery of Rapa to alleviate dacryoadenitis. FAF improves Rapa solubility and promotes subcutaneous delivery of Rapa. FAF improves pharmacokinetics of Rapa by preventing Rapa-RBC interaction as well as burst renal clearance. Subcutaneously delivered FAF-Rapa alleviates dacryoadenitis. Theranostics 2017, Vol. 7, Issue 16 http://www.thno.org 3858 nanoparticle core or albumin pocket, FKBP-ELP carriers employ Rapa’s biological receptor, which has the high specificity/affinity binding necessary to retain the drug for long durations in the body. Secondly, both FKBP and ELP are biodegradable and biocompatible polypeptides that can be produced at high yield and purity through scalable bacterial fermentation. These FKBP-ELP nanoformulations solubilize the drug free of any excipients or organic solvents, thereby eliminating injection site toxicity, which occurs with the free drug formulated using standard emulsions, such as Cremophor-EL. Among the carriers examined herein, FAF performs with the best combination of high drug loading, long drug retention, and particle size stability. While facilitating SC administration of Rapa, FAF further augments tumor accumulation and suppresses tumor growth. Table 1. Physicochemical properties of ELP protein polymers with and without FKBP. Label a Amino acid sequence b MW [kDa] c MW [kDa] d Purity [%] e Rh at f Temperature-concentration phase diagram 20 °C [nm] 37 °C [nm] Slope, m [°C Log(µM)] Intercept, b [°C] SI MG(VPGSG)48(VPGIG)48Y 39.8 39.6 93.6 5.3 ± 1.6 22.9 ± 0.5 4.7 ± 0.9 34.9 ± 1.3 FSI M-FKBP-G(VPGSG)48(VPGIG)48Y 51.6 51.6 94.5 6.1 ± 0.1 21.3 ± 0.6 3.5 ± 0.1 29.4 ± 0.2 A192 MG(VPGAG)192Y 73.6 73.3 94.3 6.9 ± 0.2 6.6 ± 0.0 8.4 ± 0.6 73.9 ± 0.9 FA M-FKBP-G(VPGAG)192Y 85.4 85.0 98.5 8.4 ± 0.1 7.8 ± 0.4 2.5 ± 6.2 61.7 ± 8.9 FAF M-FKBP-G(VPGAG)192-FKBP 97.0 96.6 98.2 8.5 ± 0.6 7.9 ± 0.2 4.3 ± 0.7 63.6 ± 0.9 a FKBP amino acid sequence: GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE[1] b Expected MW based on amino acid sequence. c Observed MW determined by running samples on MALDI-TOF. d Purity was determined (Equation S1) using SDS-PAGE gel and densitometry analysis of the copper chloride stained gel using ImageJ. e R h, hydrodynamic radius of 25 µM samples determined by Dynamic Light Scattering (n=3, mean ± SD). f Phase diagrams for assembly (Figure 2 b, c) were fit with Equation S2. Values represent mean ± 95% CI. Figure 1. Optimization of FKBP-ELP architecture to enhance the stability and efficacy of Rapa delivery. Rapalogues are potent cytostatic molecules with anti-cancer efficacy; however, their poor solubility limits their safety and efficacy by oral and IV delivery. This manuscript describes a new protein-based strategy to deliver Rapa via SC delivery using fusions between the FKBP protein and ELP (Table 1). This side-by-side comparison evaluates soluble ELPs with one (FA) or two (FAF) drug binding domains with a nanoparticle ELP (FSI). While all three carriers can bind Rapa, reduce injection site toxicity, and suppress a human breast cancer xenograft (MDA-MB-468), the Berunda polypeptide named FAF performed best with respect to drug loading, drug retention, formulation stability, tumor efficacy and bio-distribution following SC administration. Bio-physical characteristics • Rapa solubility • Rapa bioavailability • Promote SC administration Pharmacokinetics • Rapa-RBC interaction • Burst clearance Systemic circula-on Therapeutic efficacy • Lacrimal gland (LG) inflammation RBC Infiltrated lymphocytes LG R R Theranostics 2017, Vol. 7, Issue 16 http://www.thno.org 3858 nanoparticle core or albumin pocket, FKBP-ELP carriers employ Rapa’s biological receptor, which has the high specificity/affinity binding necessary to retain the drug for long durations in the body. Secondly, both FKBP and ELP are biodegradable and biocompatible polypeptides that can be produced at high yield and purity through scalable bacterial fermentation. These FKBP-ELP nanoformulations solubilize the drug free of any excipients or organic solvents, thereby eliminating injection site toxicity, which occurs with the free drug formulated using standard emulsions, such as Cremophor-EL. Among the carriers examined herein, FAF performs with the best combination of high drug loading, long drug retention, and particle size stability. While facilitating SC administration of Rapa, FAF further augments tumor accumulation and suppresses tumor growth. Table 1. Physicochemical properties of ELP protein polymers with and without FKBP. Label a Amino acid sequence b MW [kDa] c MW [kDa] d Purity [%] e Rh at f Temperature-concentration phase diagram 20 °C [nm] 37 °C [nm] Slope, m [°C Log(µM)] Intercept, b [°C] SI MG(VPGSG)48(VPGIG)48Y 39.8 39.6 93.6 5.3 ± 1.6 22.9 ± 0.5 4.7 ± 0.9 34.9 ± 1.3 FSI M-FKBP-G(VPGSG)48(VPGIG)48Y 51.6 51.6 94.5 6.1 ± 0.1 21.3 ± 0.6 3.5 ± 0.1 29.4 ± 0.2 A192 MG(VPGAG)192Y 73.6 73.3 94.3 6.9 ± 0.2 6.6 ± 0.0 8.4 ± 0.6 73.9 ± 0.9 FA M-FKBP-G(VPGAG)192Y 85.4 85.0 98.5 8.4 ± 0.1 7.8 ± 0.4 2.5 ± 6.2 61.7 ± 8.9 FAF M-FKBP-G(VPGAG)192-FKBP 97.0 96.6 98.2 8.5 ± 0.6 7.9 ± 0.2 4.3 ± 0.7 63.6 ± 0.9 a FKBP amino acid sequence: GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE[1] b Expected MW based on amino acid sequence. c Observed MW determined by running samples on MALDI-TOF. d Purity was determined (Equation S1) using SDS-PAGE gel and densitometry analysis of the copper chloride stained gel using ImageJ. e R h, hydrodynamic radius of 25 µM samples determined by Dynamic Light Scattering (n=3, mean ± SD). f Phase diagrams for assembly (Figure 2 b, c) were fit with Equation S2. Values represent mean ± 95% CI. Figure 1. Optimization of FKBP-ELP architecture to enhance the stability and efficacy of Rapa delivery. Rapalogues are potent cytostatic molecules with anti-cancer efficacy; however, their poor solubility limits their safety and efficacy by oral and IV delivery. This manuscript describes a new protein-based strategy to deliver Rapa via SC delivery using fusions between the FKBP protein and ELP (Table 1). This side-by-side comparison evaluates soluble ELPs with one (FA) or two (FAF) drug binding domains with a nanoparticle ELP (FSI). While all three carriers can bind Rapa, reduce injection site toxicity, and suppress a human breast cancer xenograft (MDA-MB-468), the Berunda polypeptide named FAF performed best with respect to drug loading, drug retention, formulation stability, tumor efficacy and bio-distribution following SC administration. R R Theranostics 2017, Vol. 7, Issue 16 http://www.thno.org 3858 nanoparticle core or albumin pocket, FKBP-ELP carriers employ Rapa’s biological receptor, which has the high specificity/affinity binding necessary to retain the drug for long durations in the body. Secondly, both FKBP and ELP are biodegradable and biocompatible polypeptides that can be produced at high yield and purity through scalable bacterial fermentation. These FKBP-ELP nanoformulations solubilize the drug free of any excipients or organic solvents, thereby eliminating injection site toxicity, which occurs with the free drug formulated using standard emulsions, such as Cremophor-EL. Among the carriers examined herein, FAF performs with the best combination of high drug loading, long drug retention, and particle size stability. While facilitating SC administration of Rapa, FAF further augments tumor accumulation and suppresses tumor growth. Table 1. Physicochemical properties of ELP protein polymers with and without FKBP. Label a Amino acid sequence b MW [kDa] c MW [kDa] d Purity [%] e Rh at f Temperature-concentration phase diagram 20 °C [nm] 37 °C [nm] Slope, m [°C Log(µM)] Intercept, b [°C] SI MG(VPGSG)48(VPGIG)48Y 39.8 39.6 93.6 5.3 ± 1.6 22.9 ± 0.5 4.7 ± 0.9 34.9 ± 1.3 FSI M-FKBP-G(VPGSG)48(VPGIG)48Y 51.6 51.6 94.5 6.1 ± 0.1 21.3 ± 0.6 3.5 ± 0.1 29.4 ± 0.2 A192 MG(VPGAG)192Y 73.6 73.3 94.3 6.9 ± 0.2 6.6 ± 0.0 8.4 ± 0.6 73.9 ± 0.9 FA M-FKBP-G(VPGAG)192Y 85.4 85.0 98.5 8.4 ± 0.1 7.8 ± 0.4 2.5 ± 6.2 61.7 ± 8.9 FAF M-FKBP-G(VPGAG)192-FKBP 97.0 96.6 98.2 8.5 ± 0.6 7.9 ± 0.2 4.3 ± 0.7 63.6 ± 0.9 a FKBP amino acid sequence: GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE[1] b Expected MW based on amino acid sequence. c Observed MW determined by running samples on MALDI-TOF. d Purity was determined (Equation S1) using SDS-PAGE gel and densitometry analysis of the copper chloride stained gel using ImageJ. e R h, hydrodynamic radius of 25 µM samples determined by Dynamic Light Scattering (n=3, mean ± SD). f Phase diagrams for assembly (Figure 2 b, c) were fit with Equation S2. Values represent mean ± 95% CI. Figure 1. Optimization of FKBP-ELP architecture to enhance the stability and efficacy of Rapa delivery. Rapalogues are potent cytostatic molecules with anti-cancer efficacy; however, their poor solubility limits their safety and efficacy by oral and IV delivery. This manuscript describes a new protein-based strategy to deliver Rapa via SC delivery using fusions between the FKBP protein and ELP (Table 1). This side-by-side comparison evaluates soluble ELPs with one (FA) or two (FAF) drug binding domains with a nanoparticle ELP (FSI). While all three carriers can bind Rapa, reduce injection site toxicity, and suppress a human breast cancer xenograft (MDA-MB-468), the Berunda polypeptide named FAF performed best with respect to drug loading, drug retention, formulation stability, tumor efficacy and bio-distribution following SC administration. Glomerular filtra-on R R FAF-Rapa 95 Figure 13. Berunda polypeptides are humanized fusions of the FKBP12 protein that promote solvent-free, burst-free subcutaneous (SC) administration of Rapa to a murine model of autoimmune dacryoadenitis. Genes encoding the FK506-binding protein (12 kDa) were fused to each end of an ELP called A192 (73 kDa) to create a bi-headed, biocompatible, and biodegradable drug carrier known as FAF. FAF was expressed via bacterial fermentation, purified at high-yield by ELP-mediated purification, and used to solubilize Rapa. To explore the immunosuppressive properties of this formulation, FAF-Rapa was evaluated after SC injection to 14-wk old male non-obese diabetic (NOD) mice every other day for 2 weeks. Male NOD mice of this age have developed autoimmune inflammation of the lacrimal gland (LG), also known as dacryoadenitis, leading to reduced tear production and dry eyes. At the termination of the study (Day 16), LG, tears, tissues, and serum were collected and analyzed using histology, gene expression, serum biochemistry, and the activity of a tear and tissue biomarker for SS known as Cathepsin S. Theranostics 2017, Vol. 7, Issue 16 http://www.thno.org 3858 nanoparticle core or albumin pocket, FKBP-ELP carriers employ Rapa’s biological receptor, which has the high specificity/affinity binding necessary to retain the drug for long durations in the body. Secondly, both FKBP and ELP are biodegradable and biocompatible polypeptides that can be produced at high yield and purity through scalable bacterial fermentation. These FKBP-ELP nanoformulations solubilize the drug free of any excipients or organic solvents, thereby eliminating injection site toxicity, which occurs with the free drug formulated using standard emulsions, such as Cremophor-EL. Among the carriers examined herein, FAF performs with the best combination of high drug loading, long drug retention, and particle size stability. While facilitating SC administration of Rapa, FAF further augments tumor accumulation and suppresses tumor growth. Table 1. Physicochemical properties of ELP protein polymers with and without FKBP. Label a Amino acid sequence b MW [kDa] c MW [kDa] d Purity [%] e Rh at f Temperature-concentration phase diagram 20 °C [nm] 37 °C [nm] Slope, m [°C Log(µM)] Intercept, b [°C] SI MG(VPGSG)48(VPGIG)48Y 39.8 39.6 93.6 5.3 ± 1.6 22.9 ± 0.5 4.7 ± 0.9 34.9 ± 1.3 FSI M-FKBP-G(VPGSG)48(VPGIG)48Y 51.6 51.6 94.5 6.1 ± 0.1 21.3 ± 0.6 3.5 ± 0.1 29.4 ± 0.2 A192 MG(VPGAG)192Y 73.6 73.3 94.3 6.9 ± 0.2 6.6 ± 0.0 8.4 ± 0.6 73.9 ± 0.9 FA M-FKBP-G(VPGAG)192Y 85.4 85.0 98.5 8.4 ± 0.1 7.8 ± 0.4 2.5 ± 6.2 61.7 ± 8.9 FAF M-FKBP-G(VPGAG)192-FKBP 97.0 96.6 98.2 8.5 ± 0.6 7.9 ± 0.2 4.3 ± 0.7 63.6 ± 0.9 a FKBP amino acid sequence: GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE[1] b Expected MW based on amino acid sequence. c Observed MW determined by running samples on MALDI-TOF. d Purity was determined (Equation S1) using SDS-PAGE gel and densitometry analysis of the copper chloride stained gel using ImageJ. e R h, hydrodynamic radius of 25 µM samples determined by Dynamic Light Scattering (n=3, mean ± SD). f Phase diagrams for assembly (Figure 2 b, c) were fit with Equation S2. Values represent mean ± 95% CI. Figure 1. Optimization of FKBP-ELP architecture to enhance the stability and efficacy of Rapa delivery. Rapalogues are potent cytostatic molecules with anti-cancer efficacy; however, their poor solubility limits their safety and efficacy by oral and IV delivery. This manuscript describes a new protein-based strategy to deliver Rapa via SC delivery using fusions between the FKBP protein and ELP (Table 1). This side-by-side comparison evaluates soluble ELPs with one (FA) or two (FAF) drug binding domains with a nanoparticle ELP (FSI). While all three carriers can bind Rapa, reduce injection site toxicity, and suppress a human breast cancer xenograft (MDA-MB-468), the Berunda polypeptide named FAF performed best with respect to drug loading, drug retention, formulation stability, tumor efficacy and bio-distribution following SC administration. R R . . . . . . . Termination Corneal Staining Thread Test Corneal Staining Thread Test 1 4 7 10 13 16 18 Days: Subcutaneous Administration FAF-Rapa (FAF = FKBP-A192-FKBP) Theranostics 2017, Vol. 7, Issue 16 http://www.thno.org 3858 nanoparticle core or albumin pocket, FKBP-ELP carriers employ Rapa’s biological receptor, which has the high specificity/affinity binding necessary to retain the drug for long durations in the body. Secondly, both FKBP and ELP are biodegradable and biocompatible polypeptides that can be produced at high yield and purity through scalable bacterial fermentation. These FKBP-ELP nanoformulations solubilize the drug free of any excipients or organic solvents, thereby eliminating injection site toxicity, which occurs with the free drug formulated using standard emulsions, such as Cremophor-EL. Among the carriers examined herein, FAF performs with the best combination of high drug loading, long drug retention, and particle size stability. While facilitating SC administration of Rapa, FAF further augments tumor accumulation and suppresses tumor growth. Table 1. Physicochemical properties of ELP protein polymers with and without FKBP. Label a Amino acid sequence b MW [kDa] c MW [kDa] d Purity [%] e Rh at f Temperature-concentration phase diagram 20 °C [nm] 37 °C [nm] Slope, m [°C Log(µM)] Intercept, b [°C] SI MG(VPGSG)48(VPGIG)48Y 39.8 39.6 93.6 5.3 ± 1.6 22.9 ± 0.5 4.7 ± 0.9 34.9 ± 1.3 FSI M-FKBP-G(VPGSG)48(VPGIG)48Y 51.6 51.6 94.5 6.1 ± 0.1 21.3 ± 0.6 3.5 ± 0.1 29.4 ± 0.2 A192 MG(VPGAG)192Y 73.6 73.3 94.3 6.9 ± 0.2 6.6 ± 0.0 8.4 ± 0.6 73.9 ± 0.9 FA M-FKBP-G(VPGAG)192Y 85.4 85.0 98.5 8.4 ± 0.1 7.8 ± 0.4 2.5 ± 6.2 61.7 ± 8.9 FAF M-FKBP-G(VPGAG)192-FKBP 97.0 96.6 98.2 8.5 ± 0.6 7.9 ± 0.2 4.3 ± 0.7 63.6 ± 0.9 a FKBP amino acid sequence: GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE[1] b Expected MW based on amino acid sequence. c Observed MW determined by running samples on MALDI-TOF. d Purity was determined (Equation S1) using SDS-PAGE gel and densitometry analysis of the copper chloride stained gel using ImageJ. e Rh, hydrodynamic radius of 25 µM samples determined by Dynamic Light Scattering (n=3, mean ± SD). f Phase diagrams for assembly (Figure 2 b, c) were fit with Equation S2. Values represent mean ± 95% CI. Figure 1. Optimization of FKBP-ELP architecture to enhance the stability and efficacy of Rapa delivery. Rapalogues are potent cytostatic molecules with anti-cancer efficacy; however, their poor solubility limits their safety and efficacy by oral and IV delivery. This manuscript describes a new protein-based strategy to deliver Rapa via SC delivery using fusions between the FKBP protein and ELP (Table 1). This side-by-side comparison evaluates soluble ELPs with one (FA) or two (FAF) drug binding domains with a nanoparticle ELP (FSI). While all three carriers can bind Rapa, reduce injection site toxicity, and suppress a human breast cancer xenograft (MDA-MB-468), the Berunda polypeptide named FAF performed best with respect to drug loading, drug retention, formulation stability, tumor efficacy and bio-distribution following SC administration. Rapamycin (Sirolimus) FKBP12 A192 (VPGAG) 192 R 14wk male NODs Molecular Cloning Expression / Purification Rapa Encapsulation Corneal Fluorescein Staining Thread Wetting Test Lymphocytic Infiltration in the LG Blood Chemistry Histopathology Gene Expression In the LG Cathepsin S Activity Protein CTSS 0 10 20 30 40 50 60 70 80 10 -4 10 -3 10 -2 10 -1 10 0 10 1 Time (h) Plasma FAF (mg / mL) S.C dose I.V dose Pharmacokinetics DNA mRNA 96 3.2. Materials and Methods 3.2.1. Synthesis, Expression and Purification of FAF The gene encoding the human FKBP12 (Standaert et al., 1990) was cloned to the N- and C-terminus of ELP A192. The cloned construct was sequenced, transformed into and expressed in BLR(DE3) competent E. Coli (#69053, Novagen, Madison, WI) fermented in terrific broth media for 16-18 hrs at 37°C without IPTG induction. After bacterial cell lysis (S-4000 Ultrasonic Disintegrator Sonicator Liquid Processor, Misonix, Inc. NY; Amplitude 9, 18 repeats of 10 sec on + 20 sec off cycle) and clarification of cell debris by centrifugation at 16,100 rcf for 10 min at 4 °C in a Beckman J2-21 Centrifuge, the supernatant was subjected to ELP-mediated phase separation in 2 M sodium chloride at 37 °C. Coacervates were pelleted at 5,000 rcf for 10 min at 37 °C using a Sorvall RC-3C Plus Centrifuge immediately after the phase separation was observed (hot-spin). At the end of the hot-spin, soluble impurities (supernatant) were removed and FAF coacervates (pellet) were resolubilized in ice-cold PBS. Thoroughly resolubilized FAF was centrifuged at 16,100 rcf for 10 min at 4 °C in an Eppendorf 5415R Centrifuge (cold-spin). At the end of the cold-spin, insoluble impurities (pellet) were again removed by transferring the supernatant to a clean tube. Cycles of hot-spin followed by cold-spin were repeated 3 times to achieve the necessary purity. 3.2.2. Biophysical Characterization of FAF The purity of FAF was analyzed using SDS-PAGE. The molar extinction coefficient (e) of FAF was calculated at 20,615 M -1 ⋅cm -1 (Pace et al., 1995). Serial dilutions of FAF in Edelhoch buffer were prepared, measured and averaged to acquire the best estimate of FAF concentration in PBS using Eq.1 (Edelhoch, 1967; Pace et al., 1995). The hydrodynamic radius (Rh) of FAF at 20 °C and 37 °C was determined using dynamic light scattering (DLS). Triplicates of sterile filtered (200 nm pore, #PN 4612, Pall Corp., NY) 97 FAF, FAF-Rapa or rhodamine-labeled FAF-Rapa (50 μL of 10 μM) in PBS were loaded onto a 384-well plate followed by layering with 15 μL mineral oil to prevent evaporation, and the whole plate was centrifuged for 1 min at 1,000 rcf to remove any remaining air bubbles. Samples were measured using a Wyatt Dynapro plate reader and analyzed by built-in software DYNAMICS V7 (Wyatt Tech. Co., CA). Rh was measured first at 20 °C and then the temperature was immediately increased to 37 °C, where the second measurement was made. For the optical density profile, absorbance at 350 nm was measured in a DU800 UV-Vis spectrophotometer (Beckman Coulter, CA) under a temperature gradient of 0.5 °C/min. The transition temperature at each concentration of FAF was defined as the temperature at which the maximum first derivative was achieved within each optical density profile with respect to the temperature. The transition temperature from each concentration was used to plot the phase diagram and fit with Eq 4. 3.2.3. Encapsulation of Rapa with FAF A two-phase solvent evaporation method was employed to bind Rapa to FAF. An aqueous phase (PBS) containing 400 μM FAF was mixed with an equal volume of organic phase (90% hexane/10% EtOH) containing 630 μM Rapa (Molar ratio = 1:1.58). Under constant stirring at 29 °C for 20 min to evaporate organic solvents (Laborota 4011 digital rotary evaporator, Heidolph Instruments GmbH & Co. KG, Schwabach, Germany), Rapa was gradually displaced into the aqueous phase where it was solubilized through binding to FAF. The resulting solution was then centrifuged (16,100 rcf, 10 min, 4 °C), filtered (200 nm pore, #PN 4612, Pall Corp., NY) and dialyzed against PBS (>4 hrs, 4 °C) to remove any excess or insoluble Rapa and residual solvent. To determine the encapsulation efficiency, 30 μM FAF in MeOH was injected into a C-18 reverse phase (RP)-HPLC column (150 × 4.6 mm, particle size 5 µm, YMC CO. LTD., Japan) and eluted in a H2O: MeOH gradient from 50% to 100% over 15 min. The chromatogram was monitored at 280 nm at a flow rate of 0.75 mL/min. Linear standard curves 98 were created between the logarithm of the area under the curve (AUC) vs. logarithm of either FAF concentrations (12.5, 25, 50, 75, 100 μM) or Rapa concentrations (6.25, 12.5, 25, 50, 75, 100 μM). FAF and Rapa concentrations in the sample were calculated based on their respective calibrated standard curve. The molar ratio of Rapa to FAF was 1.76:1 (encapsulation ratio of Rapa to FKBP = 0.88:1). The loading of Rapa to FAF was 1.64 % (w/w). 3.2.4. Pharmacokinetic study of FAF Procedures and analysis were adopted and modified from methods previously described by MacKay et al. (MacKay et al., 2009). FAF-Rapa in PBS was incubated with NHS-rhodamine (#46406, Thermo-Fisher, IL) for 1.5 hrs at room temperature under constant rotation and excess dye was removed using a desalting column (#87772, Thermo Fisher, IL). The labeling efficiency was measured as 100% using Eq. 8 (FAF to rhodamine = 1:1), where the εrhodamine is 80,000 (M -1 cm -1 ) at 555 nm and the concentration of CFAF was measured before labeling to be 200 µM. Fluorophore labeling did not alter the Rapa encapsulation ratio, which was confirmed by RP- HPLC. 𝐿𝑎𝑏𝑒𝑙𝑖𝑛𝑔 𝑒𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑐𝑦 = go ppp,rs^t uvu 8 as^w_x Md` y uvu Eq. 8 To measure the pharmacokinetics of the carrier, rhodamine-labeled Rho-FAF-Rapa (1.0 mg Rapa/kg BW) was injected either intravenously (IV, n=4) via tail vein or SC (n=5) at the right flank of the animal. A blood sample (20 μL) was collected from the tail vein at 5 min (3 min for IV), 30 min, 1, 2, 4, 8, 12, 24, 36, 48, and 72 hrs after injection and immediately diluted into heparinized PBS (1,000 U/mL, #H4784, Sigma, MO). The blood was centrifuged (16,100 rcf, 10 min, 4 °C) and plasma was stored at -20 °C for further analysis. Collected plasma samples were loaded onto a 384-well plate (#784076, Greiner Bio One International GmbH, NC) and fluorescence intensity was determined (Synergy H1 Hybrid Multi-Mode Reader, BioTek Instruments, Inc., VT; Ex: 542 nm / Em: 585 nm). A linear standard curve was created between 99 the logarithm of the background-subtracted fluorescence vs. logarithm of the FAF concentrations (13.7, 41.2, 123.5, 370.4, 1,111.1, 3,333.3, 10,000.0 nM). FAF concentration was then calculated based on fluorescence intensity. Both non-compartmental and compartmental methods were applied to analyze the PK profiles of FAF after IV or SC administration. Non-compartmental analysis was primarily based on the determination of the area under the plasma concentration curve (AUC) and the area under the moment curve (AUMC), which were first estimated by the trapezoidal method. The mean residence time (MRT) of both IV and SC and the SC mean absorption time (MAT) were calculated as follows: 𝑀𝑅𝑇 = 𝐴𝑈𝑀𝐶/𝐴𝑈𝐶 Eq. 9 𝑀𝐴𝑇 =𝑀𝑅𝑇 ey −𝑀𝑅𝑇 h~ Eq. 10 Using these estimates of AUC, the plasma clearance (CL) of both IV and SC and the SC bioavailability (F) were estimated as follows: 𝐶𝐿/𝐹 = 𝐷𝑜𝑠𝑒/𝐴𝑈𝐶 Eq. 11 𝐹 = 𝐴𝑈𝐶 ey /𝐴𝑈𝐶 h~ Eq. 12 Similarly, kelimination and T1/2, elimination, was estimated in each individual mouse by fitting the last three time points to the following equation. 𝐶 ,EF&0, 0E$$ =𝐴𝑒 0 `MxMd_cM^d Q Eq. 13 𝑇 4/-,L)L%$QL&% =0.693 𝑘 L)L%$QL&% ⁄ Eq. 14 The volume of distribution of both IV and SC was estimated as follows: 𝑉 o =𝐶𝐿 𝑘 L)L%$QL&% ⁄ Eq. 15 To obtain pharmacokinetic parameters for the compartmental analysis, the dataset of each individual mouse was fit to either a one-compartment (IV) or three-compartment (SC) pharmacokinetic model using SAAM II TM (University of Washington, WA). It was not possible to account for the late peak times observed using a one-compartment model of absorption from 100 the injection site; however, the data was well-fit by two-step absorption from the SC injection site to an intermediate interstitial compartment and then to the plasma compartment. To estimate the magnitude of the absorption parameters in the SC dataset, it was necessary to assume that the kinetic rate constant kInjection siteà Interstitial fluid is equal to kInterstitial fluidà Systemic circulation, which is denoted as kabsorption (kabs). The dose and the plasma concentrations were fit to these models to determine the volume of distribution (Vd), elimination rate constant (kelimination), and kabs. Based on these fit parameters, the maximum plasma concentration (Cmax), clearance (CL), and elimination half-life (T1/2, Elimination) were estimated for individual mice. Average values for Vd and kelimination from the IV dataset were adopted to perform fits for the SC datasets. Equations used for compartmental analyses are as follows: 𝐶𝐿 =𝑘 L)L%$QL&% 𝑉 I Eq. 16 𝐴𝑈𝐶/𝐹 =𝐷𝑜𝑠𝑒/𝐶𝐿 Eq. 17 𝑇 4/-,$*&(QL&% =0.693 𝑘 $*&(QL&% ⁄ Eq. 18 𝐹 =𝑘 $*&(QL&% (𝑘 $*&(QL&% +𝑘 I($I$QL&% ) ⁄ Eq. 19 To optimize administration frequency through modeling, average plasma concentration from five mice administered with SC Rho-FAF-Rapa were solved with fixed values of Vd, kabsorption, kelimination and kdegradation, as reported in Table 7 using SAAM II TM . 3.2.5. Plasma and whole blood levels of Rapa To measure the relative plasma and whole blood levels of Rapa, 1.0 mg Rapa/kg BW of rhodamine-labeled FAF-Rapa or free Rapa was injected either IV (n=5 and n=4, respectively) via tail vein or SC (n=4, each) at the right flank of the animal. Blood was collected using a heparinized syringe at 24 hrs after injection via cardiac puncture. The collected blood was split into a ‘whole blood sample’ and a ‘plasma sample’. For whole blood, three cycles of freeze-thaw using -80 °C were applied to lyse RBCs, to release RBC-bound Rapa, and stored at -20 °C for 101 further LC-MS analysis. For plasma samples, samples were centrifuged (16,100 rcf, 10 min, 4 °C) and the supernatant (plasma) was stored at -20 °C for further LC-MS analysis. For LC-MS analysis, 25 µL of internal standard (1,000 ng/mL Prograf â (Tacrolimus), Astellas Pharma US, Inc., IL) was added to a 50 µL plasma sample followed by 350 µL of acetonitrile. The entire mixture was vortexed and then centrifuged (16,100 rcf, 5 min, 4 °C). 40 µL of supernatant was transferred to HPLC micro-vials, and 30 µL was injected into the LC-MS. The same method was used to analyze the whole blood samples. For the standard curve, 50 µL serial dilutions of free Rapa or FAF-Rapa were added to a mixture of 25 µL of internal standard (1,000 ng/mL of Tacrolimus), 50 µL blank mouse plasma and 300 µL acetonitrile to generate 16, 80, 400, 2,000, 10,000 ng/mL Rapa standard solutions. Samples were admixed thoroughly and centrifuged (16,100 rcf, 5 min, 4 °C). 40 µL of supernatant was transferred to HPLC micro-vials, and 30 µL was injected into the LC-MS. Fluorescence intensity measured from the same plasma sample (Synergy H1 Hybrid Multi-Mode Reader, BioTek Instruments, Inc., VT; Ex: 542 nm / Em: 585 nm) was used to calculate the Rapa to FAF ratio in vivo. 3.2.6. Therapeutic study in male non-obese diabetic (NOD) mice, a model of SS Methods described here are adopted and adjusted from the methods used by Shah et al. (Shah et al., 2017). The male NOD mice were bred in-house from breeding pairs purchased from Taconic (strain: NOD/MrkTac, Taconic Biosciences, Inc., NY). Analysis and treatments were initiated when animals were 13-15 weeks of age, when SS-like autoimmune dacryoadenitis is established. A total of 60 mice were randomly assigned to 4 different groups receiving either vehicle, free Rapa, carrier (FAF only) or FAF-Rapa SC at a dose of 1.0 mg Rapa/kg at the right flank of the animal, every other day for 2 weeks (7 injections). Treatments were formulated as follows. For the vehicle, 1 mL of polysorbate 80 (EM8.22187.0500, EMD Millipore, MA) was mixed with 9 mL of endotoxin-free water to generate 10% polysorbate 80. 1 102 mL of polyethylene glycol (PEG) 400 (#202398, Sigma, MO) was also mixed with 9 mL of endotoxin-free water to generate 10% PEG 400. 2.5 mL from each dilution were mixed together (10% polysorbate 80 + 10% PEG 400, 5 mL) and further diluted with 20 mL of endotoxin-free water to generate a 2% polysorbate 80 + PEG 400 solution. For free Rapa, 100 mg of Rapamycin (R-5000, LC Laboratories Inc., MA) was dissolved in 100% EtOH to make 50 mg/mL stock. 100 μL of 50 mg/mL stock was mixed with 4.9 mL of polysorbate 80 + PEG 400 mixture (1:1 ratio) to make 1.0 mg/mL Rapamycin stock. 5 mL of this stock was further diluted with 20 mL endotoxin-free water to generate a 0.2 mg/mL Rapa stock in 2% polysorbate 80 + PEG 400 solution. For FAF Rapa, based on the molar ratio of Rapa to FAF (1.76:1), a dose of 1.0 mg Rapa/kg BW was delivered (i.e, 1.64 mg of FAF-Rapa was injected to a 30 g mouse to deliver 1.0 mg Rapa/kg BW). For Carrier (FAF only), the same amount of FAF without Rapa was injected to serve as a control. Injection volumes varied from 100 ~ 160 μL per injection based on the weight of individual mouse. Based on a power calculation using the variance within the LG infiltration in the NOD mice, the therapeutic study was designed as n=15 per treatment group. All animal use was in full compliance with policies approved by the University of Southern California Institutional Animal Care and Use Committee (IACUC) and the ARVO statement for the Use of Animals in Ophthalmic and Vision Research. 3.2.7. Quantitative real-time PCR To analyze expression of genes of interest after experimental treatments, total RNA was isolated from half of the LG using the RNeasy plus Universal Mini Kit (#73404, Qiagen, Germany). The LG was cut into half immediately after collection and stored in an ice-chilled bead-prefilled tube (#Z763780, Sigma, MO) supplemented with 900 μL lysis reagent (provided with the kit). Tissues were homogenized (BeadBlaster™ 24 High-Throughput Benchtop Homogenizer, Benchmark Scientific, Inc., NJ; speed: 7, cycle: 2, time: 1 min, intermission: 30 sec) and clarified lysates were further processed in accord with the manufacturer’s protocol. 103 Complementary DNA was prepared from 4 μg RNA using the Quantitative real-time PCR (qRT- PCR) was performed with the TaqMan Ò gene expression assays on the QuantStudio™ 12K Flex Real-Time PCR System for 384-well block (#4471134, Applied Biosystems, CA) using the following probes: TNF-a (Mm00443258_m1), IFN-g (Mm01168134_m1), Akt3 (Mm00442194_m1), MHCII (Mm00439216_m1), CTSS (Mm01255859_m1), Col1A1 (Mm00801666_g1) and IL-12a (Mm00434165_m1). The GAPDH (Mm99999915_g1) was used as a control gene. Each reaction (10 μL) consisted of 0.5 μL cDNA from the reverse transcription reaction, 4 μL nuclease-free water, 0.5 μL assay primer and 5 μL TaqMan Universal PCR Master Mix (#4304437, Applied Biosystems, CA). Each sample was run in triplicate. The thermal profile consisted of preheating the samples at 105 °C for 10 min followed by 40 repeats of 95 °C for 15 sec + 60 °C for 1 min. The relative expression levels were calculated using the built-in comparative CT method (DDCT method) in the default ABI software. 3.2.8. Stimulated tear collection and Cathepsin S activity analysis in tears and LG lysates Stimulated tear collection was performed under full anesthesia (I.P injection of 100 mg/kg ketamine + 10 mg/kg xylazine) as a terminal procedure. A small bilateral incision on the axis between the outer junction of the eyelid and the ear was made to expose the LG on both sides. Then 3 µL of 50 µM carbachol (#L06674, Alfa Aesar, MA) was applied directly onto the LG to stimulate tear secretion, followed by tear collection from both eyes by placing 2 µL micro- capillary tubes (#1-000-0020, Drummond Sci. Co., PA) at the tear meniscus in the medial canthus for 5 min. The stimulation was performed two more times for a total of three. Collected tears were recorded as a function of the length in the capillary tube in millimeters and then converted to microliters (1mm = 0.625 µL). After tear collection, the LG was cut into half and placed in an ice-chilled bead-prefilled tube (D1032-10, Benchmark Scientific, NJ) supplemented with 300 μL lysis buffer (provided with the kit). Tissues were homogenized (BeadBlaster™ 24 104 High-Throughput Benchtop Homogenizer, Benchmark Scientific, Inc., NJ; speed: 7, cycle: 2, time: 1 min, intermission: 30 sec). The homogenate was centrifuged at 6,000 g for 10 min at 4 o C. The supernatant was separated carefully and analyzed immediately. Cathepsin S (CTSS) activity analysis was measured using the Cathepsin S Activity kit (#K144-100, BioVision Inc., CA). 10 μL clarified LG lysate was mixed with 40 μL lysis buffer and 50 μL reaction buffer were added on a 96-well plate in duplicates. 100 μL stimulated tear fluid diluted in CTSS reaction buffer were also added on a same 96-well plate in duplicates. 2 μL of CTSS substrate was added to all wells and incubated at 37 °C for 1 hr. The amount of resulting fluorescence was measured with a microplate reader (SpectraMax iD3, Molecular Devices, CA). After the experiment, the Bio-Rad protein assay was performed to measure total protein concentration, and the activity was normalized to 50 μg total protein for both tears and lysates. 3.2.9. Histopathology analysis and serum chemistry Lung, spleen, kidney, liver and one of each pair of LG from every mouse was collected at the conclusion of the study for histopathology and immediately fixed with 10% neutral buffered formalin (#5701, Thermo Fisher, IL), stored at 4 °C overnight and then transferred to 70% EtOH. All organs in 70% EtOH were outsourced for paraffin embedding, sectioning, standard H&E staining and imaging (HistoWiz Inc., NY). For LG, sections from the 25th, 50th and 75th percentile regions through each gland were selected for quantitation, and the area of the LG occupied by infiltrating lymphocytes was quantified by three blinded reviewers for the best estimation of percentage area of infiltrates per gland as previously described (Shah et al., 2013; Shah et al., 2017). The percentage area infiltrated was calculated by measuring the area of the infiltrate divided by the whole area of the gland using ImageJ (v2.0.0, NIH, MD). For lung, spleen, kidney and liver, two consecutive sections at the 50th percentile regions through each organ were stained and analyzed by a blinded, trained pathologist for any signs of acute toxicity. For serum chemistry, blood collected via cardiac puncture at the conclusion of the study was 105 spun down (2,000 g, 10 min, 4 °C) to separate serum and outsourced on the day of collection for standardized analysis (Antech Diagnostics, CA). A skin biopsy at the injection site from every mouse was collected at the conclusion of the study, immediately fixed with 10% neutral buffered formalin (#5701, Thermo Fisher, IL), stored at 4°C overnight and then transferred to 70% EtOH. Skin specimens at 70% EtOH were outsourced for paraffin embedding, sectioning, standard H&E staining and imaging (HistoWiz Inc., NY). Two consecutive sections at the 50 th percentile depth of the skin section was analyzed by a blinded, trained pathologist for any signs of toxicity. For serum chemistry, blood collected via cardiac puncture at the conclusion of the study was spun down (2,000 g, 10 min, 4 °C) to separate serum and outsourced on the same day of collection for the standardized analysis (Antech Diagnostics, CA). 3.2.10. Thread wetting tests, corneal fluorescein staining analysis Thread wetting tests and corneal fluorescein staining analysis were performed to compare basal tear flow and corneal surface integrity, respectively, before and after the treatment. For thread wetting tests, mice were lightly anesthetized using a continuous flow of isoflurane through a nose cone. While under minimum anesthesia, a ZoneQuick (#6510, Oasis Medical, TN, Original manufacturer: Showa Yakuhin Kao Co., Ltd., Tokyo, Japan) phenol red- coated thread was carefully inserted under the lower eyelid of both eyes for 10 seconds. The basal tear flow was recorded as a function of the length of wetting of the thread in millimeters by using a zoom magnifier with a millimeter scale (#750, Ted Pella, Inc., CA). Wetted thread lengths were read and recorded by a blinded reviewer. For corneal fluorescein staining, a strip containing 0.6 mg fluorescein sodium (Ful-Glo Ò , #17478-403-03, Akorn Inc., IL) was reconstituted in 200 μL PBS to serve as a contrast solution, of which 1 μL was applied to the cornea of anesthetized mice. The eye was blinked five times and images were obtained using an Excelis HDS HD camera & monitor system (#AU-600-HDS, ACCU-SCOPE Inc., NY) 106 attached to a Unitron Z8 zoom stereo microscope on a plain focusing stand (#UN11145, Unitron Ò , NY) under ocular illumination with a cobalt blue light. Corneal staining was graded by three blinded reviewers using the system described in a National Eye Institute Workshop (Lemp, 1995), in which each of the five areas of the cornea is scored from 0 to 3 and summed. The scores from the left and right eyes were averaged. 3.2.11. Statistics All statistical analyses were performed using SPSS v21 (SPSS Inc., IL). A two-way ANOVA served as a primary statistical method to compare the overall effect of the drug and the carrier. If a particular data set showed a significant interaction between Rapa and FAF, then the data was further analyzed either by one-way ANOVA or Kruskal-Wallis non-parametric test based on Levene’s test. For two-group comparisons, a two-tailed, independent t-test was used to compare differences. A p-value less than 0.05 was considered a significant difference. 107 3.3. Results 3.3.1. Biophysical characterization of FAF, a FKBP-ELP fusion protein polymer that carries Rapa FAF was heterologously over-expressed from a seamlessly cloned synthetic gene in E. coli that encodes the full-length human FKBP12 protein at both the N- and C-terminus of ELP A192 (Table 6). Purification was done via inverse transition cycling (Janib et al., 2014a), a standard non-chromatographic method of ELP fusion protein purification that utilizes ELP- mediated phase separation from cleared bacterial lysates supplemented with 1~2 M NaCl to induce the Hofmeister effect (Cho et al., 2008). Three rounds of purification yielded ~90 mg/L of FAF with > 98% purity, verified by SDS-PAGE (Figure 14A). The precise determination of molecular weight by MALDI-TOF for FAF was reported previously (Dhandhukia et al., 2017). From Dynamic Light Scattering (DLS) analysis, the purified product was consistent with a monomeric and monodisperse population with a hydrodynamic radius of 8 ± 1 nm from 20 °C to 37 °C, which suggests that FAF remains stable and soluble at physiological temperatures (Figure 14B). The solubility profile and biophysical characteristics of FAF did not change after Rapa encapsulation or fluorescent dye labeling (Table 6). To determine the Tt of FAF, optical density at 350 nm over a range of temperatures was measured (Figure 14C). Fusion of FKBP to ELP did not affect the negative correlation between the Tt and the ELP concentration (Despanie et al., 2016), and the phase diagram was fit by Eq. 4. This fit yielded slope, m, of 5.76 (8.32 ~ 2.23, 95% CI) °C per 10-fold decrease in concentration and an intercept at 1 μM, b, of 65.7 °C (60.34~69.89, 95% CI). This fit allows estimation of Tt over a range of concentrations; furthermore, at no measured plasma concentrations (1~100 μM) in the in vivo therapeutic studies would FAF be expected to phase separate. (Figure 14D). 108 Figure 14. High molecular weight FAF-Rapa has the purity, size, and concentration- temperature phase behavior necessary for stability at body temperature. (A) Identity, purity and fluorescence of FAF, FAF-Rapa and rhodamine-labeled FAF-Rapa (Rho-FAF-Rapa) were analyzed by Coomassie blue staining and fluorescence imaging of SDS-PAGE. (B) Dynamic Light Scattering shows that all three formulations remain monodisperse at 37 °C (10 μM, PBS). (C) Optical density of FAF was monitored as a function of temperature at 350 nm to confirm solubility in PBS at physiological temperatures (shaded area). The Tt of FAF at 25 μM was 57.0 °C (D) Using optical density, the phase transition temperature was plotted vs. concentration as a phase diagram, below which FAF remains soluble (n=3, Mean ± SD). Dotted lines show a 95% confidence interval (CI) of the mean. 100 50 25 15 250 kDa Coomassie Fluorescence Rho-FAF-Rapa FAF-Rapa FAF Rho-FAF-Rapa FAF-Rapa FAF 1 10 100 1000 0 10 20 30 40 50 60 70 80 Hydrodynamic radius (R h, nm) % Intensity FAF FAF-Rapa Rho-FAF-Rapa A C B 30 35 40 45 50 55 60 65 70 0 1 2 3 4 5 Temperature ( o C) Optical Density at 350nm 10µM 25µM 50µM 100µM D 10 100 30 40 50 60 70 80 Concentration (µM) Transition temperature (°C) 109 Table 6. Biophysical characteristics of FAF, FAF-Rapa, and rhodamine-labeled FAF-Rapa Label *Formulation composition **M.W (kDa) ***Rh (nm) at 20 °C 37 °C FAF FKBP-G(VPGAG)192-FKBP 96.6 8 ± 1 8 ± 1 FAF-Rapa Rapa/FKBP-G(VPGAG)192-FKBP/Rapa 98.3 9 ± 2 8 ± 0.3 Rho-FAF-Rapa Rhodamine-[Rapa/FKBP-G(VPGAG)192-FKBP/Rapa] 98.8 9 ± 1 7 ± 0.3 *FKBP amino acid sequence: GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEG VAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE. G(VPGAG)192Y is the amino acid sequence of the elastin-like polypeptide, A192. **Expected molecular weights for FAF-Rapa and Rho-FAF-Rapa were reported based on observed 1:1 molar ratios of Rapa to FKBP and the rhodamine-labeling ratio. ***Hydrodynamic radii of each formulation (of 10 μM, PBS) was determined in triplicate by DLS. (Mean ± SD) 110 3.3.2. SC administration significantly improves the pharmacokinetic profiles of FAF To date, an accurate pharmacokinetic profile for SC FAF has not been reported; therefore, to estimate the bioavailability and PK parameters for FAF, 1.0 mg Rapa/kg BW of Rho-FAF-Rapa was injected either IV via tail vein (n=4) or SC at the right flank (n=5) of male NOD mice. Fluorescence intensities were analyzed from collected plasma samples at the designated time-points and used to plot the plasma FAF concentration-time profile (Figure 15A). The collected dataset from each individual mouse was further analyzed using both compartmental and non-compartmental analyses to estimate all relevant pharmacokinetic parameters. For compartmental analysis, a one-compartment model was adopted to interpret IV FAF-Rapa, whereas a three-compartment model was necessary to fit the delayed peak for SC FAF-Rapa (Figure 15B). A simple two-compartment model with only one absorption compartment was unable to fit the observed data. The simplest model that fit SC data was a three-compartment model, which includes an interstitial fluid compartment as an intermediate absorption compartment. This observation is consistent with previous reports of SC- administered biotherapeutics (Kagan, 2014). One acceptable interpretation of this model would be initial absorption into interstitial fluid, possibly the lymphatic system, followed by a second absorption process into the systemic circulation. For the best fit of all datasets, it was necessary to assume that both absorption processes were on the same order of magnitude. Had either absorption step been much slower than the other, it would have become rate-limiting, and the simpler two-compartment model would fit the data (Guo et al., 2018). Based on this, we assumed that the kinetic rate constant kInjection siteà Interstitial fluid is equal to kInterstitial fluidà Systemic circulation, of which both are designated as kabsorption (kabs, Fig. 15B, Table 7). To allow for loss of Rho-FAF- Rapa prior to appearance in the plasma, a kinetic constant of removal from the interstitial fluid is denoted as kdegradation. IV FAF-Rapa showed a mono-exponential elimination after 8 hrs of a plateau; however, this phenomenon was not captured by the compartmental model parameter estimates. Thus, parameter estimates from both compartmental analysis and non- 111 compartmental analysis are reported (Table 7). It should be noted that the statistically significantly higher plasma concentration of SC FAF-Rapa compared to IV FAF-Rapa during the elimination phase (36 hr~72 hr, Figure 15A) suggests that FAF may be a more effective carrier for sustained release when given SC versus IV. Using acquired compartmental parameters, the administration frequency of FAF-Rapa was explored for a two-week study (Figure 15C). Based on modeling, the plasma concentration of Rapa reached its peak concentration of 3.47 μM at 12 hr, becoming 10-fold lower at 45.6 hr and 100-fold lower at 69.6 hr after administration. Therefore, a 48 hr interval between doses prevents either progressive accumulation of FAF-Rapa in the body (24 hr interval) or much more complete clearance of FAF-Rapa before the next dose (72 hr interval). For SC FAF-Rapa, absorption proceeded during the first 24 hrs, while the maximum concentration (Cmax) was reached 12 hr after injection, followed by a mono-exponential decay thereafter (Figure 15A). This behavior can be explained by a mean absorption time of 9.6 hrs and mean residence time of 20.3 hrs for SC FAF-Rapa (Table 7). The bioavailability of SC FAF- Rapa was 52.7~65.5 % (Table 7), without apparent loss of Rapa during the absorption process (Figure 15D). After 24 hrs, SC FAF-Rapa maintained its mean loading stoichiometry of 1.77 ± 0.3, which exactly matched the initial molar ratio of 1.76:1, whereas some of the Rapa was dissociated from FAF when delivered IV. This difference may be due to immediate dilution into plasma for IV FAF-Rapa, versus retention in the injection site during the nearly 10 hr absorption process for SC FAF-Rapa. Nonetheless, there was undetectable displacement of Rapa from FAF during absorption, which might otherwise have been a limitation of SC administration. Next, how FAF affects levels of Rapa in vivo was explored. To do so, 1.0 mg Rapa/kg BW of free Rapa or Rho-FAF-Rapa was delivered either IV or SC. 24 hr after injection, blood was collected and Rapa concentrations in the plasma and in the whole blood were compared by LC-MS analysis. FAF delivery resulted in an 8-fold higher plasma Rapa concentration compared 112 to that of the free formulation (Figure 15E). Consistent with the elevated plasma concentrations for Rho-FAF-Rapa beyond 24 hrs (Figure 15A), SC delivery also increased the Rapa concentration by 1.8-fold compared to IV administration (Figure 15E). The presence of at least two types of FKBPs in erythrocytes, cytosolic FKBP12 and membrane-bound FKBP13, can fractionate Rapa in systemic circulation by extraction into red blood cells (RBC) (Biagiotti et al., 2011). The interaction between Rapa and RBCs is problematic in that it elicits hematologic toxicities which can cause discontinuation of the treatment (Pallet and Legendre, 2013). Therefore, it was tested whether site-specific encapsulation of Rapa to our carrier, thus masking Rapa’s binding motif to FKBPs, effectively inhibits Rapa-RBC interaction in vivo. Indeed, there was a significant improvement in plasma-to- whole blood ratio of Rapa when delivered by FAF. The plasma-to-whole blood ratio of free Rapa was 0.9:1 for IV and 1:1 for SC but this ratio increased to 1.4:1 for IV (1.5-fold increase) and 1.5:1 for SC (1.5-fold increase) when delivered by FAF (Figure 15F). These elevated ratios are consistent with the retention of Rapa in the plasma by FAF even one day after administration of the formulation. 113 Figure 15. Pharmacokinetic analysis reveals that SC administration of FAF-Rapa improves pharmacokinetic properties of Rapa. (A-D) 1.0 mg Rapa/kg BW of Rho-FAF-Rapa was injected either IV (n=4) or SC (n=5) to male NOD mice. (A) Data for the first ten hours are shown in the inset. SC administration yielded significantly higher Rho-FAF concentrations at 36, 48 and 72 hr (Mean ± SD). A Student’s t-test was used to compare groups. (B) Data were well- fit by either a one-compartment (IV) or three-compartment (SC) pharmacokinetic model as indicated. kabs=kabsorption. (C) Based on these parameters, pharmacokinetic modeling was performed to explore several dosing options prior to initiating a therapeutic study. (D-F) Male NOD mice were injected with 1.0 mg Rapa/kg BW as free Rapa IV (n=4), free Rapa SC (n=4), FAF-Rapa IV (n=5) or FAF-Rapa SC (n=4). Plasma and whole blood samples were collected via cardiac puncture after 24 hrs. (D) For each sample, Rapa concentration analyzed by LC-MS was compared with its fluorescence intensity analyzed by a plate reader to measure the Rapa to FAF ratio (min, mean and max are depicted). A Student’s t-test was used to compare groups, which revealed that at 24 hr SC administration retained nearly the starting ~2:1 ratio of Rapa:FAF, while FAF-Rapa administered IV had lost about half of the bound drug. (E) Rapa concentration from each sample was analyzed by LC-MS (Mean ± SD). A Student’s t-test and one-way ANOVA were used to compare groups. (F) For each sample, Rapa concentration in the plasma was compared to that of the whole blood (WB) (min, mean and max are depicted). A two-way ANOVA was used to compare groups, which revealed that FAF reduces accumulation of Rapa in blood cells compared to the free drug. IV SC IV SC 0.5 1.0 1.5 2.0 2.5 Plasma / WB ratio of Rapa p < 0.001 24h Post-Injection *** Free Rapa FAF-Rapa IV SC IV SC 0.0 0.5 1.0 1.5 2.0 Rapamycin Concentration (µM) Plasma Whole Blood Free Rapa FAF-Rapa p < 0.001 5.7-fold p < 0.001 8-fold p < 0.001 1.8-fold p < 0.001 p<0.001 ns ns 24h Post-Injection IV SC 0.5 1.0 1.5 2.0 2.5 Rapa to FAF ratio p = 0.04 24h Post-Injection * 0 3 6 9 12 15 18 21 0 1 2 3 4 5 6 Days Plasma FAF (µM) 48h interval 72h interval 24h interval A D E C F B SC Injection pocket Inters00al fluid Systemic circulation k elimina0on IV SC k degrada0on Systemic circulation k elimination k abs k abs 0 10 20 30 40 50 60 70 80 10 -2 10 -1 10 0 10 1 Time (h) Plasma FAF (µM) SC dose IV dose *** ** ** (p = 0.001) (p = 0.01) (p = 0.01) IV fit SC fit 0 2 4 6 8 10 10 -2 10 -1 10 0 10 1 Time (h) Plasma FAF (µM) 114 Table 7. Pharmacokinetic parameters of IV- or SC-delivered FAF-Rapa analyzed using compartmental analysis and non-compartmental analysis. (Mean ± SD) Parameter (Unit) Route of Administration IV SC IV SC Preferred Model 1 Compartment 3 Compartments Non-compartment AUC (μM•hr) 141 ± 16 92 ± 19 207 ± 33 109 ± 14 AUMC (μM•hr 2 ) - - 2,207 ± 344 2,214 ± 328 F (%) 100 65.5 ± 13.2 100 52.7 ± 6.7 CL (mL/hr) 0.15 ± 0.01 0.15 * 0.10 ± 0.01 0.09 ± 0.01 CL / F (mL/hr) 0.15 ± 0.01 0.23 ± 0.04 0.10 ± 0.01 0.18 ± 0.03 Vd (mL) 1.46 ± 0.2 1.46 * 0.88 ± 0.1 0.85 ± 0.1 Cmax (μM) 13.4 ± 2.4 3.3 ± 0.1 11.7 ± 2.9 4.4 ± 0.7 Tmax (hr) 0.0 12.4 ± 0.7 0.0 12.0 MRT (hr) - - 10.7 ± 0.3 20.3 ± 1.1 MAT (hr) - - - 9.6 ± 1.1 T1/2, Absorption (hr) - 4.2 ± 0.4 - - T1/2, Elimination (hr) 6.9 ± 0.5 6.9 * 6.2 ± 0.4 6.4 ± 0.7 kabsorption (hr -1 ) - 0.16 ** - - kelimination (hr -1 ) 0.10 ± 0.01 0.10 * 0.11 ± 0.01 0.11 ± 0.01 kdegradation (hr -1 ) - 0.09 ± 0.05 - - *compartmental values from IV analysis were adopted to estimate other SC parameters **to fit the observed time to peak concentration, the assumption was required that kabsorption = kInjection siteà Interstitial fluid = kInterstitial fluidà Systemic circulation. 115 3.3.3. Rapa significantly suppresses lymphocytic infiltration, inflammatory gene expression, and CTSS activity in the LG To evaluate the effect of SC FAF-Rapa and free Rapa on the characteristic autoimmune dacryoadenitis associated with SS, one of each pair of LG collected from control and treated mice at the conclusion of the study were sectioned and histopathologically-quantified to compare the extent of lymphocytic infiltration (Figure 16A). Relative to healthy LG, an inflamed LG shows evidence of lymphocytic infiltration throughout the whole organ (Figure 16B, outlined in blue). Both FAF-Rapa (9±5.6%) and free Rapa (7±3.7%) significantly suppressed lymphocytic infiltration in the LG compared to FAF alone (15±8.4%) or vehicle (21±10.3%, percent area covered, mean±SD) control. The ability of FAF-Rapa to suppress lymphocytic infiltration did not differ from free Rapa. The health of the LG is not only governed by lymphocytic infiltration but also fibrosis, autophagy and levels of cytokines (Zoukhri, 2010). Elevations in gene expression of major histocompatibility complex II (MHC II) in various autoimmune and inflammatory diseases is well known (Jones et al., 2006), a factor that may drive lymphocytic infiltration in the LG (Guo et al., 2000; Mircheff et al., 1991). Moreover, major pro-inflammatory cytokines, such as IL-12a, IFN-g and TNF-a, are significantly elevated in the inflamed LG (Ji et al., 2013; Meng et al., 2017; Vosters et al., 2009). The downregulation of genes encoding these proteins was seen in our previous gene expression profiling in the LG upon mTOR inhibition by IV Rapa treatment (Shah et al., 2013). Therefore, the expression profiles of genes responsible for autoimmune activation and inflammation were analyzed to assess changes in immune and inflammatory environments with treatment with free Rapa or FAF-Rapa. Indeed, with both treatments a decrease in lymphocytic infiltration in the LG was linked with decreased gene expression levels of MHC II, IL-12a and IFN-g (Figure 17A~C). Rapa caused no difference in the gene expression level of TNF-a (Figure 17D). 116 One of the hallmarks of the LG in SS patients is degradation of extracellular matrix and deposition of collagen, leading to LG fibrosis (Sato et al., 2010). An anti-fibrotic effect of Rapa on renal (Wu et al., 2006), skin (Yoshizaki et al., 2010), cardiac (Yu et al., 2013), pulmonary (Korfhagen et al., 2009), and hepatic (Bridle et al., 2009) fibrosis models has been reported, with a common feature manifested as a decrease in type I collagen expression and its secretion to and deposition at the extracellular space. To see whether free Rapa and FAF-Rapa suppresses LG fibrosis, the gene expression level of type I collagen, a1 (Col1A1) in the LG was analyzed. Free Rapa and FAF-Rapa delivered SC significantly lowered gene expression of Col1A1 (p < 0.001, Fig. 17E), which suggests Rapa may protect the LG from fibrosis. This is consistent with our previous findings from topical instillation of Rapa as an ophthalmic emulsion or IV administration via micellar FKBP-ELP fusion protein (unpublished data). Although more studies are required, it could be proposed that Rapa may act through inhibition of mTOR, which decreases type 1 collagen expression and reduces LG fibrosis; furthermore, this may proceed via mTOR’s kinase activity towards LARP6 as recently reported by Zhang and Stefanovic (Zhang and Stefanovic, 2017). Gene expression changes in Akt3 were collected as a surrogate marker for autophagy. The role of autophagy in LG inflammation is controversial, as some researchers show autophagy is highly activated or at least dysregulated in the inflamed LG thus worsening the disease (Byun et al., 2017), whereas others report that activation of autophagy during the disease progression can aid the gland in processing accumulated cell debris, thus promoting its health (Corum et al., 2014). Our previous gene expression profiling showed downregulation of Akt3 in the LG upon IV Rapa treatment (Shah et al., 2013), associated with induction of autophagy. This change was reconfirmed in a recent topical Rapa study over a 12-week period (Shah et al., 2017); however, no significant gene expression change was observed in the LG in 117 this 2-week treatment (Figure 17F). There was no enhanced effect of FAF-Rapa compared to free Rapa on pro-inflammatory gene expression profiles. Among several candidates proposed as SS tear biomarkers, cathepsin S (CTSS) activity has emerged (Edman et al., 2018; Hamm-Alvarez et al., 2014). An endo-lysosomal protease, CTSS is a member of the cysteine cathepsin protease family, with dysregulated activity linked to various diseases including cancer and autoimmune disorders (Wilkinson et al., 2015). When it is dysregulated, CTSS may be secreted extracellularly and digest various molecules that maintain homeostasis. Its elevated activity has been confirmed in clinical studies showing that increased tear CTSS activity distinguishes SS patients from patients with non-autoimmune dry eye disease, other autoimmune diseases, and healthy controls (Edman et al., 2018; Hamm-Alvarez et al., 2014). In the NOD mice, CTSS gene expression and activity are increased in LG lysates as well as in tears (Janga et al., 2018a; Li et al., 2010) and its reduction was correlated with the therapeutic activity of Rapa (Shah et al., 2013; Shah et al., 2017). To see if FAF-Rapa affected LG CTSS, activity in LG lysates from male NOD mice treated with FAF-Rapa were compared with controls. Results show that SC FAF-Rapa successfully reduced CTSS activity in the LG (Figure 18A) with no significant effect on CTSS gene expression in the LG and CTSS activity in tears (Figure 18B,C). The ability for FAF-Rapa to reduce CTSS activity was not statistically significantly different from that of the free Rapa. 118 Figure 16. Rapa reduces lymphocytic infiltration in the LG of male NOD mice. (A) One of each pair of LG from each mouse in the cohort was collected at the conclusion of the study. The 25 th , 50 th and 75 th percentile sections from each LG were quantified by three blinded observers to determine the average percentage area of infiltrate per gland. (n=15) (B) Inflamed LGs show areas of purple nuclear staining, which indicate foci of infiltrating lymphocytes (outlined in blue). Lymphocytic infiltration was reduced by FAF-Rapa (middle panel). The scale bar represents 200 μm. (C) The percentage area of infiltration was calculated using ImageJ (Mean ± SD). 25% 50% 75% Lacrimal gland (LG) (n=15/treatment) A B C Inflamed LG FAF-Rapa treated LG Healthy LG Vehicle Free Rapa Carrier (FAF) FAF-Rapa 0 10 20 30 40 50 % Area of Infiltrates *** * p = 0.047 (p < 0.001) ns * p = 0.023 *** p < 0.001 119 Figure 17. Gene expression profile of proteins involved in inflammation, antigen presentation and autophagy in LG of male NOD mice treated with subcutaneous Rapa. One of each pair of LG from mice in the treatment cohorts was collected at the conclusion of the study for mRNA extraction. Extracted mRNAs were reverse transcribed to cDNA and further analyzed by quantitative real-time PCR. Gene expression levels were normalized to vehicle (Mean ± SD, n=9). Two-way ANOVA was used to compare effects of drug and carrier. Based on a significant interaction between Rapa and FAF for Col1A1 (p = 0.025), one-way ANOVA and post-hoc comparisons revealed significant differences between: vehicle vs. FAF-Rapa (p = 0.003); free Rapa vs. FAF (p = 0.007); and FAF vs. FAF-Rapa (p < 0.001). 0 1 2 3 Relative Expression Col1A1 *** p < 0.001 Vehicle Carrier (FAF) Free Rapa FAF-Rapa 0 1 2 3 Relative Expression Akt3 ns p = 0.2 Vehicle Carrier (FAF) Free Rapa FAF-Rapa 0 1 2 3 Relative Expression TNF-alpha ns p = 0.9 Vehicle Carrier (FAF) Free Rapa FAF-Rapa 0 1 2 3 Relative Expression IFN-gamma ** p = 0.003 Vehicle Carrier (FAF) Free Rapa FAF-Rapa 0 1 2 3 Relative Expression IL-12a * p = 0.03 Vehicle Carrier (FAF) Free Rapa FAF-Rapa 0 1 2 3 Relative Expression MHC II ** p = 0.003 Vehicle Carrier (FAF) Free Rapa FAF-Rapa A C B D E F 120 Figure 18. Rapa suppresses proteolytic CTSS activity in the LG but not in the tears. At the conclusion of the two-week study in 16-week male NOD mice, LG and tears were collected immediately after euthanasia for CTSS activity and gene expression analysis. (A) A significant decrease in CTSS activity was observed in the LG lysates after SC Rapa treatments (n=9). (B) No statistical significance was observed for CTSS gene expression level in the LG over a 2- week period (n=12). Two-way ANOVA was used to compare the effects of the drug and carrier. (Mean ± SD) 0 2 4 6 8 RFU / mg protein (x10 6 ) CTSS activity in tears ns p = 0.8 Vehicle Carrier (FAF) Free Rapa FAF-Rapa 0 1 2 3 Relative Expression CTSS gene in LG lysates ns p = 0.2 Vehicle Carrier (FAF) Free Rapa FAF-Rapa 0 1 2 3 4 5 RFU / mg protein (x10 8 ) CTSS activity in LG lysates * p = 0.04 Vehicle Carrier (FAF) Free Rapa FAF-Rapa A B C 121 3.3.4. SC delivery of Rapa has minimal effect towards ocular surface health in a two-week period Improved LG health eventually enhances tear secretion and ameliorates dry eye symptoms on the ocular surface (Shah et al., 2013; Shah et al., 2017). Therefore, basal tear flow by thread wetting test, corneal surface integrity by corneal fluorescein staining and CTSS activity in collected tears were compared upon FAF-Rapa treatment; however, there was no significant differences compared to control groups with an unexpected trend of a minute decrease (Figure 19A,B). Reports showing Rapa’s association to a new-onset of diabetes and correlation between insulin-dependent diabetes and a decrease in Schirmer’s test readings among diabetic patients may partially explain our results (Goebbels, 2000; Johnston et al., 2008). However, it could be a transient phenotype that can be resolved during an extended treatment because the corneal fluorescein staining result shows that only FAF-Rapa treated mice (-1.1 ± 3.0) showed a trend to the healthier cornea, while vehicle (0.3 ± 2.2), free Rapa (- 0.1 ± 2.8) or carrier (0.2 ± 2.2) treated groups showed no apparent difference (Figure 19C, mean ± SD, difference in mean between before and after the treatment). It is more likely to be a transient phenotype since improvements in corneal integrity rely on the amount and the quality of tears secreted from the LG, which means that even though there was a minute decrease in basal tear flow during a 2-week period, the quality of tears may have improved to protect the corneal surface upon SC FAF-Rapa. There was no difference in tear CTSS activity after the two-week treatment. The lack of effect on tear CTSS levels may be because the SS-like disease was not yet fully developed within the cohort of mice enrolled to this study. The degree of autoimmune dacryoadenitis in different cohorts of NOD mice can vary and the cohort used in this study had lower LG infiltration by lymphocytes (~20%, Figure 16C) compared to some previous studies in NOD mice (35-45%) (Shah et al., 2013; Shah et al., 2017). Since disease may not have been as 122 developed, CTSS activity might not have been elevated enough in tears to reveal a response to Rapa. Another reason might be the bioavailability of SC FAF-Rapa. Compared to 100% bioavailability for IV FAF-Rapa, 53-66% bioavailability for SC FAF-Rapa may be sufficient only to reduce CTSS activity in the LG (Figure 18A). Dosing and administration frequency optimization may be able to improve therapeutic efficacy of SC FAF-Rapa to further decrease tear CTSS at the ocular surface. 123 Figure 19. Subcutaneously delivered FAF-Rapa has a minimal effect on tear production and ocular surface integrity over a 2-week period. (A) Anesthetized mice were tested with phenol red-coated threads before and after the treatment to observe changes in basal tear flow. Measurements from both eyes were averaged. No statistical significance was achieved. (B) Mice under anesthesia underwent three consecutive rounds of carbachol stimulation on both LG and tears were collected from the ocular surface at the end of each round using a micro- capillary tube as described in Materials and Methods. The sum of each measurement was plotted. (C) Fluorescein staining on the cornea of anesthetized mice were quantified before and after the treatment for comparison. Only the FAF-Rapa treated group showed a trend to the decrease; however, no statistical significance was achieved. B: Before treatment; A: After treatment. A one-way ANOVA was used to compared groups in each measurement. (D) Tears collected from carbachol stimulation were subjected to CTSS activity analysis. No statistical significance was achieved for CTSS activity in tears over a 2-week period (n=9). A two-way ANOVA was used to compared groups in each measurement. Stimulated Tear Volume Vehicle Free Rapa Carrier (FAF) FAF-Rapa 0 5 10 15 20 Tear Volume (µL) n = 12 / group Vehicle Free Rapa Carrier (FAF) FAF-Rapa 0 5 10 15 Relative Score (Max. 15) Corneal Fluorescein Staining n = 15 / group B A B A B A B A Vehicle Free Rapa Carrier (FAF) FAF-Rapa -5 0 5 10 Thread After - Thread Before (mm) Thread Wetting Test n = 15 / group A B C 0 2 4 6 8 RFU / mg protein (x10 6 ) CTSS activity in tears ns p = 0.8 Vehicle Carrier (FAF) Free Rapa FAF-Rapa D 124 3.3.5. Histopathological evaluation reveals FAF-Rapa as a biocompatible system for SC delivery of Rapa At the conclusion of the study, major organs and blood samples were collected for histopathological and toxicological evaluation upon FAF-Rapa treatment. Organs subjected to H&E staining did not show any evidence of systemic toxicity (Figure 20, Table 8), and organ weights remained in the normal range across all treatment groups (Table 9). The drug carrier’s ability to mask drug’s innate toxicity is important especially for SC delivery because the SC administration can cause injection site toxicity and pain, which can affect daily life (Mathaes et al., 2016). Rapa formulated with polysorbate 80 and polyethylene glycol (PEG) 400 resulted in a very severe necrosis at the injection site even after the first injection, which deterred multiple injections at the same site (Figure 21, Table 8). The severity was decreased in FAF-Rapa treated mice, but the difference was not apparent enough to draw a solid conclusion. This may be due to an affinity-based encapsulation of Rapa to FKBP such that half of the Rapa is still exposed to the outer environment after encapsulation into FKBP, which is critical for its activity. Given that carrier (FAF) did not show any sign of toxicity at the injection site, we can at least confirm that FAF is a suitable and biocompatible carrier for SC administration compared to organic excipients and the injection site toxicity is induced by Rapa but not the carrier. Injection site toxicity of FAF-Rapa at a given dose was never seen in healthy Balb/C mice (data not shown), suggesting a presence of interaction between activated autoimmunity and Rapa at the injection site. Rapa may be favored over calcineurin inhibitors (cyclosporin A or tacrolimus) due to its manageable renal toxicity (Asante-Korang et al., 2017; Kahan, 2003), thus is approved for use in renal allograft recipients; however, concerns regarding nephrotoxicity of Rapa remain (Marti and Frey, 2005a). On the other hand, Rapa damages the liver in humans due to an abundance of FKBP51 in liver tissue, which is 12:1 in molar ratio to FKBP12 (Baughman et al., 1997), thus Rapa is not indicated for liver transplantation. Its hepatotoxicity has been verified in mouse 125 models (Massoud and Wiesner, 2012; Umemura et al., 2014). To observe if FAF-Rapa induced any systemic toxicity during our therapeutic study, serum alanine aminotransferase (ALT), alkaline phosphatase (ALP) (indicators of liver damage), blood urea nitrogen (BUN) and creatinine levels (indicators of kidney damage) were analyzed (Kim and Moon, 2012; Lala and Minter, 2018). As compared in Figure 20 and Table 8, there was no apparent nephro- or hepato-toxicities upon FAF-Rapa treatment in this study. Free Rapa and FAF-Rapa treated groups showed a trend to an increase ALT, which was statistically insignificant. It should be noted that 1 out of 15 mice treated with FAF-Rapa showed an unusually high level of ALT (824 IU/L), which led to an overall higher value for the FAF-Rapa group; however, the ALP, BUN, creatinine and BUN/creatinine ratio of this particular mouse remained normal. One parameter that was significantly different for the FAF-Rapa treated group compared to the other three groups was a body weight change. Only FAF-Rapa treated mice experienced a body weight loss over a 2-week period. This may be due to a significantly increased blood concentration of FAF-Rapa compared to free Rapa (Figure 15E). Serum biochemistry analysis showed that FAF-Rapa did not damage the liver (Figure 22A,C) but elevated globulin level (Figure 22B). Elevated globulin level is a common feature for any kind of biologics that even fully human monoclonal antibodies tend to induce elevation (Gomez-Mantilla et al., 2014). As globulin level and albumin-globulin ratio in FAF-Rapa treated group was not different from the free Rapa treated group (Figure 22D), the level of immunogenicity induced by FAF-Rapa is negligible and can be managed by standard management strategy. 126 Figure 20. Histopathology of mouse organs reveals no systemic toxicity of FAF-Rapa at a therapeutic dose. At the conclusion of the study, organs and bloods from mice were sampled upon euthanasia. Organs were fixed, paraffin-embedded, sectioned and stained with H&E. Kidney (n=3), spleen (n=3), lung (n=3) and liver (n=5) were analyzed by a blinded, trained pathologist. Images of organs from one representative mouse from each group were selected by a pathologist and shown. Black bar represents 100 μm. Spleen Kidney Lung Liver FAF-Rapa Carrier (FAF) Free Rapa Vehicle 127 Figure 21. Rapamycin-mediated injection site toxicity is mitigated when delivered via FAF. Injection sites were observed 24h after the 4 th injection and 3 days after the final injection. Representative pictures were selected by a blinded, well-trained pathologist. Black marker was used to identify injection sites on the skin of the animal on the day of euthanasia (3 days after final injection). After euthanasia, skin biopsies were subjected to standard H&E staining. Vehicle or carrier-treated mice show mature hair follicles and normal adnexal structures; however, free Rapa or FAF-Rapa treated mice showed apparent hypoplasia of the hair follicles and mild hypoplasia of sebaceous glands. Black bar represents 200 µm. Black arrow: hair follicle; black triangle: sebaceous gland. 3 Days after final injection 24h After 4 th injection Vehicle FAF Rapa Free Rapa Carrier (FAF) Histology 128 Table 8. Histopathological observations in tissues of male NOD mice after 2 weeks of treatment with FAF-Rapa or other control groups. Groups Organs Vehicle Free Rapa Carrier (FAF only) FAF-Rapa Skin Mature epidermis, hair follicles and adnexal glands. Unremarkable dermis and subcutaneous adipose Markedly hypoplastic hair bulb and the sebaceous glands. The epidermis and dermis are otherwise unremarkable. 100% of the hair follicles affected by the process. Mature epidermis, hair follicles and adnexal glands. Unremarkable dermis and subcutaneous adipose Markedly hypoplastic hair bulb and the sebaceous glands. The epidermis and dermis are otherwise unremarkable. 100% of the hair follicles affected by the process. Kidney Sections show a well- defined cortex and medulla. Numerous glomeruli were identified. The renal medulla with loops of Henle and collecting ducts of Bellini were identified. No histopathologic abnormality was otherwise identified in the kidney. Sections show a well- defined cortex and medulla. Numerous glomeruli were identified. The renal medulla with loops of Henle and collecting ducts of Bellini were identified. No histopathologic abnormality was otherwise identified in the kidney. Sections show a well- defined cortex and medulla. Numerous glomeruli were identified. The renal medulla with loops of Henle and collecting ducts of Bellini were identified. No histopathologic abnormality was otherwise identified in the kidney. Sections show a well- defined cortex and medulla. Numerous glomeruli were identified. The renal medulla with loops of Henle and collecting ducts of Bellini were identified. No histopathologic abnormality was otherwise identified in the kidney. Liver Sections show a uniform parenchyma with hepatocytes and sinusoids with Kupffer cells. Morphology of hepatocytes and the Portal triads is otherwise unremarkable. No steatosis or inflammation is seen. Sections show a uniform parenchyma with hepatocytes and sinusoids with Kupffer cells. Morphology of hepatocytes and the Portal triads is otherwise unremarkable. 2 out of 5 mice show microsteatosis in a centrilobular pattern, involving up to 10% hepatocytes. No inflammation. Sections show a uniform parenchyma with hepatocytes and sinusoids with Kupffer cells. Morphology of hepatocytes and the Portal triads is otherwise unremarkable. No steatosis or inflammation is seen. Sections show a uniform parenchyma with hepatocytes and sinusoids with Kupffer cells. Morphology of hepatocytes and the Portal triads is otherwise unremarkable. 5 out of 5 mice show microsteatosis in a dominantly centrilobular pattern, involving 40-50% hepatocytes. No inflammation. Lung Sections show a normal lung parenchyma with alveolar ducts, alveolar sacs, terminal and respiratory bronchioles. Occasional alveolar macrophages were noted. Occasional procedural atelectasis is noted. Sections show a normal lung parenchyma with alveolar ducts, alveolar sacs, terminal and respiratory bronchioles. Occasional alveolar macrophages were noted. Occasional procedural atelectasis is noted. Sections show a normal lung parenchyma with alveolar ducts, alveolar sacs, terminal and respiratory bronchioles. Occasional alveolar macrophages were noted. Occasional procedural atelectasis is noted. Sections show a normal lung parenchyma with alveolar ducts, alveolar sacs, terminal and respiratory bronchioles. Occasional alveolar macrophages were noted. Occasional procedural atelectasis is noted. Spleen Sections displayed white and red pulp of the parenchyma. Germinal centers and surrounding lymphocytes are identified in the white pulp area. Megakaryocytes, macrophages, hematopoietic cells and venous sinuses are seen in the red pulp area. Sections displayed white and red pulp of the parenchyma. Germinal centers and surrounding lymphocytes are identified in the white pulp area. Megakaryocytes, macrophages, hematopoietic cells and venous sinuses are seen in the red pulp area. Sections displayed white and red pulp of the parenchyma. Germinal centers and surrounding lymphocytes are identified in the white pulp area. Megakaryocytes, macrophages, hematopoietic cells and venous sinuses are seen in the red pulp area. Sections displayed white and red pulp of the parenchyma. Germinal centers and surrounding lymphocytes are identified in the white pulp area. Megakaryocytes, macrophages, hematopoietic cells and venous sinuses are seen in the red pulp area. 129 Table 9. Toxicity assessment of FAF-Rapa via serum chemistry and organ/body weight measurements (Mean ± SD) Category Parameters Vehicle Free Rapa Carrier (FAF) FAF-Rapa Serum Chemistry (n=13) ALT (IU/L)* 34.1 ± 20.8 82.3 ± 97.4 49.0 ± 43.2 158.3 ± 225.6 ALP (IU/L) 43.1 ± 5.7 45.0 ± 9.5 34.7 ± 9.3 48.0 ± 9.6 BUN (mg/dL) 23.6 ± 2.2 22.1 ± 2.6 23.3 ± 3.0 22.5 ± 2.1 Creatinine (mg/dL) 0.2 ± 0.00 0.2 ± 0.00 0.2 ± 0.00 0.2 ± 0.04 BUN / Creatinine Ratio 118.2 ± 11.2 110.4 ±12.8 116.5 ± 14.9 106.8 ± 16.6 Organ Weights (n=15) Lung (% BW) 0.60 ± 0.07 0.63 ± 0.06 0.57 ± 0.07 0.63 ± 0.09 Liver (% BW) 4.7 ± 0.5 4.9 ± 0.4 4.5 ± 0.4 5.0 ± 0.5 Spleen (% BW) 0.29 ± 0.05 0.28 ± 0.09 0.33 ± 0.06 0.28 ± 0.09 Kidney (% BW) 1.6 ± 0.1 1.6 ± 0.1 1.5 ± 0.2 1.6 ± 0.1 Body Weight Change (% BW)** 1.5 ± 2.9 1.7 ± 2.7 3.3 ± 2.3 -1.8 ± 3.2 * Two-way ANOVA showed a statistically significantly different ALT level from Rapa treatment (Vehicle + FAF vs. free Rapa + FAF-Rapa, p = 0.03) but no significant interaction between Rapa and FAF (p = 0.4). No statistical significance was achieved among groups using Kruskal- Wallis non-parametric test. ** Kruskal-Wallis non-parametric test was performed for body weight change (n=15) based on a statistical significance achieved by Rapa treatment (Vehicle + FAF vs. free Rapa + FAF-Rapa, p = 0.001) and significant interaction between Rapa and FAF (p < 0.001) using two-way ANOVA. From Kruskal-Wallis non-parametric test, the body weight change of FAF-Rapa treated group was statistically significantly different compared to: vehicle (p = 0.012); free Rapa (p = 0.006); and Carrier (p < 0.001). 130 Figure 22. FAF-Rapa does not induce hepatotoxicity. At the conclusion of the study, serum from individual mouse was collected via cardiac puncture and analyzed against (A) albumin, (B) globulin, (C) total serum protein and (D) albumin/globulin ratio. A one-way ANOVA was used to compare groups. Vehicle Free Rapa Carrier (FAF) FAF-Rapa 0.5 1.0 1.5 2.0 2.5 Albumin / Globulin Ratio ns *** (p < 0.001) (p = 0.995) (p = 1.000) ns *** (p < 0.001) *** (p < 0.001) Vehicle Free Rapa Carrier (FAF) FAF-Rapa 1.5 2.0 2.5 3.0 3.5 4.0 Albumin (g / dL) (p = 0.858) ns ns (p = 0.954) (p = 0.570) ns (p = 0.555) ns (p = 0.952) ns Vehicle Free Rapa Carrier (FAF) FAF-Rapa 1.5 2.0 2.5 3.0 3.5 4.0 Globulin (g / dL) ns ** (p = 0.009) (p = 0.965) (p = 0.429) ns (p = 0.031) * *** (p < 0.001) Vehicle Free Rapa Carrier (FAF) FAF-Rapa 3.0 4.0 5.0 6.0 7.0 8.0 Total Protein (g / dL) ns (p = 0.405) (p = 0.942) (p = 0.376) ns ns * (p = 0.012) (p = 0.753) ns A B C D 131 3.3.6. Termination of Rapa treatment resolves hyperglycemia One side effect associated with Rapa or Rapalogs (temsirolimus, everolimus and ridaforolimus), also seen with other mTOR inhibitors, is metabolic complications, such as hyperglycemia, hypercholesterolemia and hypertriglyceridemia (Pallet and Legendre, 2013). It has been reported that about 70% of patients who received Rapalogs experienced all-grade metabolic disorders of any kind, of which 25% are all-grade hyperglycemia. Within this 25%, only 7% of patients are diagnosed with grade 3~4 (250~500 mg/dL or higher), while the majority experienced mild to moderate hyperglycemia (Su et al., 2016a). To distinguish the elevated glucose level as a result of spontaneous development of diabetes in male NOD mice (Leiter et al., 1987) from Rapa treatment, blood glucose levels were monitored before and after the study. FAF-Rapa treated mice experienced elevated blood glucose, even compared to free Rapa treated mice (Figure 23A). Among 15 mice treated with FAF-Rapa, 6 mice were identified as hyperglycemic (>250 mg/dL). The mean difference in blood glucose before and after treatment with FAF-Rapa (n=15) was +150±99 mg/dL, whereas this value was +49±62 mg/dL for free Rapa treated mice (n=15) (Mean ± SD). Mice treated with vehicle (+19±85 mg/dL, n=15) or carrier (+12±36 mg/dL, n=15) did not show significant differences in blood glucose before and after the treatment. While the FAF-Rapa treated population had a higher incidence of body weight loss and blood glucose, within individual subjects there was no correlation between the percent body weight change and the severity of hyperglycemia (Figure 23B). In the clinic, hyperglycemia can be resolved by a combination of dietary control, oral medication and insulin, as recommended by American Diabetes Association (www.diabetes.org) (Busaidy et al., 2012; Su et al., 2016a). To confirm that FAF-Rapa induced hyperglycemia was reversible, the treatment procedure described in Figure 13 was replicated in age-matched male NOD mice and healthy Balb/C mice and monitored their blood glucose changes during and after the treatment (Figure 23C,D). The glucose levels in male NOD mice upon FAF-Rapa treatment 132 showed a heterogeneous characteristic of that seen in the human population. One (M1) mouse with a high baseline blood glucose progressed to levels consistent with diabetes, which may be representative of subset of human populations with baseline-elevated blood glucose, which have a higher incidence of hyperglycemia and diabetes with this drug (Johnston et al., 2008). Two (M2, M4) out of five mice were unaffected by FAF-Rapa. Two mice (M2, M4) with elevated blood glucose upon treatment showed levels restored to normal immediately after FAF-Rapa was removed. The lag time of 4~5 days until restoration to the baseline is consistent with the pharmacokinetic behavior of SC FAF-Rapa (Figure 15A). Unlike the NOD mice, BALB/c mice did not experience hyperglycemia under the same treatment schedule. This suggests that the observed elevated glucose level is not a result of spontaneous development of diabetes in male NOD mice and the metabolic complications induced by SC FAF-Rapa is not permanent and can be managed with conventional methods. While the NOD mouse is also a model of T-cell mediated type I diabetes, it remains unclear how FAF-Rapa mediated hyperglycemia would progress in otherwise healthy humans. 133 Figure 23. FAF-Rapa induces temporary hyperglycemia in NOD mice that resolves after termination of the treatment. (A) At the conclusion of the study as described in Figure 13, blood glucose levels of individual mice were measured. The dotted line shows 250 mg/dL, which is a criterion for hyperglycemia. A Kruskal-Wallis non-parametric test was performed based on a statistical significance achieved by Rapa treatment (Vehicle + FAF vs. free Rapa + FAF-Rapa, p < 0.001) and significant interaction between Rapa and FAF (p = 0.004) using two-way ANOVA (Mean ± SD, n=15). Results of the Kruskal-Wallis non-parametric test are presented. (B) Each mouse treated with FAF-Rapa was further analyzed to correlate percent body weight change to blood glucose change, before and after the treatment. Mice with final blood glucose less than 200 mg/dL (circle), between 200 and 250 mg/dL (triangle) and above 250 mg/dL (square) were plotted. (C,D) In two additional studies, FAF-Rapa was administered as described in Figure 13 (shaded area) to either (C) male NOD mice (n=5) or (D) male Balb/C mice (n=5) and the blood glucose levels of individual mice (M1~M5) was monitored for 2 weeks after termination of the treatment. Vehicle Free Rapa Carrier (FAF) FAF-Rapa 0 100 200 300 400 500 600 700 Blood Glucose (mg / dL) After Treatment *** ns (p < 0.001) (p = 0.2) *** (p < 0.001) A B 0 5 10 15 20 25 0 100 200 300 400 500 600 Days Blood glucose (mg/dL) in male NOD mice M1 M2 M3 M4 M5 0 5 10 15 20 25 0 100 200 300 400 500 600 Days Blood glucose (mg/dL) in male Balb/C mice M1 M2 M3 M4 M5 C D 0 100 200 300 400 -10 -5 0 5 10 Blood glucose change (After - Before, mg/dL) Body weight change (After - Before, %) < 200 mg/dL > 250 mg/dL 200 ~ 250 mg/dL 134 3.4. Discussion The subcutaneous (SC) space has long been favored for drug delivery, whereby the injection site behaves as a drug depot. In comparison to intravenous (IV) administration, SC delivery is fast and can be performed easily at home (Turner and Balu-Iyer, 2018). For example, a recent study compared IV to SC administration of Trastuzumab in HER2-positive breast cancer patients. When administered SC, Trastuzumab showed equivalent pharmacodynamic, pharmacokinetic and safety profiles compared to standard IV administration, and was highly preferred by patients. Two principal reasons for this preference were the reduced pain during injection (158 out of 236 patients, 67%) and the shorter time for injection (5 min per SC injection vs. 30~90 min for IV infusions). Given that patients commonly receive 17~25 Trastuzumab IV infusions per year (every 2~3 weeks), SC injections every 3 weeks were preferred by 91.5% of patients (Pivot et al., 2013). Another study compared time spent by healthcare professionals and costs for SC and IV administration (Rule et al., 2014). The time spent by healthcare professionals from preparation to administration of SC Rituximab was only 0.8 hr, compared to 3.8 hrs for IV administration, while the time that patients spent in treatment rooms was only 1.2 hrs for SC administration, compared to 4.4 hrs for IV administration. SC administration decreased the total mean staff cost by $140~160 US dollar equivalents per session. While there are oral formulations of rapamycin, low oral bioavailability creates unpredictable PK profiles and variability in the severity of side-effects (Marti and Frey, 2005b; Pallet and Legendre, 2013; Pham et al., 2004; Simamora et al., 2001; Stenton et al., 2005). In comparison to drugs with poor oral bioavailability and unfavorable pharmacokinetic profiles, SC administration has been reported to improve therapeutic index and patient compliance (Jin et al., 2015). While there are no approved SC formulations for rapamycin, this drug has been extensively used in sustained release formulations such as from drug-eluting stents (Moses et al., 2003). 135 Strategies to treat SS are primarily focused on systemic immune suppression. The majority of clinical trials have targeted B-cells and T-cells, with fewer focused on pro- inflammatory cytokines, and very few studies have been directed to intracellular pathways (Brito-Zeron et al., 2016). In considering the targeting of intracellular pathways, JAK-STAT pathway inhibition (clinicaltrials.gov identifier: NTC02610543), CTSS inhibition (NCT02701985) and ubiquitin/proteasome inhibition (Jakez-Ocampo et al., 2015) have been explored, but not yet the mTOR pathway. The study described here, to the best of our knowledge, is the first report targeting mTOR signaling by an SC Rapa formulation to treat autoimmune dacryoadenitis in a murine model of SS (Figure 24). This target, drug, and route of administration has never been tested in the clinical or preclinical setting. Based on the data presented, FAF-Rapa is a highly promising SC-injectable therapeutic candidate for SS. As with any other new formulation, extensive preclinical and clinical toxicology studies will be required for further development. To explore pharmacodynamic advantages of using FAF to deliver SC Rapa, we chose to compare it to free Rapa solubilized using components (PEG and polysorbate 80) found in the oral formulation known as Rapamune ® . While valuable as a control, solubilization alone during SC administration is unlikely to improve its absorption into circulation, entrapment by red blood cells, or toxicity profile. Instead, we propose that high affinity binding between our FKBP moieties on FAF has the potential to improve SC bioavailability, the plasma-to-whole blood ratio, and the absolute plasma concentration of Rapa. As a drug carrier, such a scenario could be pharmacodynamically advantageous compared to that of a solubilizer-based formulation. While SC FAF-Rapa achieved higher plasma levels than SC free Rapa (Figure 15E), the therapeutic endpoints related to autoimmune dacryoadenitis explored in this studuy do not distinguish profound differences between the two formulations. This lack of difference likely stems from the fact that both FAF-Rapa and free Rapa are effective at the relatively high dose evaluated. Even so, the 8-fold increase in the absolute plasma level of SC FAF-Rapa compared to free Rapa strongly suggests the drug remains bound to the carrier in the blood near Cmax. Further studies 136 must now explore if FAF-Rapa exhibits a therapeutic effect at a subtherapeutic dose or frequency of free Rapa. These studies may solve the body weight loss (Table 9) and induction of hyperglycemia (Figure 23) observed during SC FAF-Rapa treatment. It should be noted that the pre-clinical study presented here collected only Rapa plasma concentrations near the Cmax for FAF, which may be useful for translation to clinical settings. Although the dose in this study (1.0 mg Rapa/kg) cannot be directly compared to the clinical dose (0.02~0.04mg Rapa/kg), 1.0 mg Rapa/kg in mice achieved a plasma Cmax (0.4 ng Rapa/mL) that is very similar to plasma Cmax in humans (0.5 ng/mL) reported after oral administration of 2 mg Rapamune ® (Brattstrom et al., 2000). In healthy humans, the plasma-to-whole blood ratio of Rapa is about 1:106 (Zimmerman et al., 2008). This extreme partitioning to human red blood cells differs significantly from that observed in other species, such as rabbits, rats or mice (Ferron and Jusko, 1998b). In male NOD mice, free Rapa gave a plasma-to-whole blood ratio of only 1:1, which was significantly increased to 1.5:1 when formulated as FAF-Rapa (Figure 15F). The observation of 1:1 for the plasma-to-whole blood ratio of Rapa in our murine model is similar to that reported by others (Yanez et al., 2008). It has been proposed that other isoforms of FKBP could play a role in this difference. For example, cytosolic FKBP12 and membrane-bound FKBP13 on RBCs have been proposed to modulate the high plasma-to-whole blood ratio seen in humans (Hendrickson et al., 1993). The nucleotide sequences encoding the binding pocket for Rapa in human and mouse FKBP12 and FKBP13 are highly conserved, which suggests they may have similar affinities (Hendrickson et al., 1993). There exists the possibility that differential expression or cellular localization may also play a role; however, there was no data to support or reject this possibility. Another reasonable explanation could be the differential stability of Rapa in whole blood between humans and rodents (Ferron and Jusko, 1998a). Rapa’s half-life in whole blood taken from humans is 135 hr, while in whole blood taken from rats is only 15 hr. This suggests that entrapment in human RBCs protects Rapa from degradation better than in the whole blood of 137 rodents. Although the exact mechanism underlying differences in plasma-to-whole blood ratios of Rapa between humans and other species remains unknown, the fact that this study detected a significant 50% increase in plasma-to-whole blood ratio for Rapa (Figure 15F) suggests that FAF may significantly affect hematologic toxicities of Rapa in humans, which commonly result in discontinuation of treatment (Pallet and Legendre, 2013). 3.5. Conclusion Current formulations of Rapa (sirolimus) or Rapalogs (everolimus, temsirolimus, ridaforolimus) are limited to oral or IV administration. Despite poor bioavailability and frequent discontinuation due to side-effects, free Rapa (sirolimus) and everolimus, 2 mg/day (www.pfizermedicalinformation.com) and 10 mg/day (www.hcp.novartis.com), respectively are administered orally to patients. Temsirolimus and ridaforolimus are slightly more water-soluble and formulated for IV infusion. Temsirolimus is infused as 25 mg/week over 30~60 mins (www.pfizermedicalinformation.com). Ridaforolimus is being tested as an IV infusion of 12.5 mg/daily for 5 days every 2 weeks (Mita et al., 2008; Rizzieri et al., 2008). Despite the potential opportunities for a SC formulation, none of these Rapalogs are given SC due to lack of an effective carrier. The results of this study suggest that FAF-Rapa may represent a first- generation SC carrier, which can be further optimized to reduce administration frequency and dose, reduce dose-limiting side-effects, and increase patient compliance in the clinic. 138 Figure 24. FAF-Rapa shows superior bioavailability and equivalent therapeutic efficacy compared to free Rapa upon mTOR inhibition. (A) Significant fraction of free Rapa in the systemic circulation associates with red blood cells (RBC). Free Rapa passes through cell membrane due to its lipophilic nature. In cytosol, free Rapa interacts with endogenous FKBPs to inhibit mTOR activity. (B) Rapa carried by FAF shows superior bioavailability compared to free Rapa by having less interaction with RBCs in the systemic circulation. FAF-Rapa is known to internalize via micropinocytosis. Rapa released to the cytosol may interact with endogenous FKBPs to inhibit mTOR activity (1) or FAF-Rapa may directly inhibit mTOR activity inside endosomes (2) when endosomes are in contact with mTOR. Inhibition of mTOR activity by free Rapa or FAF-Rapa reduces lymphocytic infiltration, decreases gene expressions related to antigen presentation, inflammation, and fibrosis, and suppresses CTSS activity in the LG. 139 Chapter 4 Biosynthesized Multivalent Lacritin Peptides Stimulate Exosome Production via Syndecan-1 in human corneal epithelium 4.1. Introduction The lacrimal gland-corneal axis plays a critical role in maintaining ocular health. The lacrimal gland is the major organ responsible for secretion of essential proteins and electrolytes into the tear film that protects and overlays the cornea and conjunctiva (Dartt, 2009). One of these essential tear proteins that confers anti-microbial and anti-inflammatory defense at the ocular surface is Lacritin (Laurie et al., 2008). A 12.3 kDa secreted glycoprotein found in human and non-human primates, Lacritin exhibits prosecretory activity in lacrimal gland acinar cells (LGAC) and mitogenic activity in corneal epithelial cells (Sanghi et al., 2001). Originally discovered by the Laurie laboratorsy (Sanghi et al., 2001), several studies have revealed that the active monomeric form of Lacritin is significantly downregulated in patients suffering from chronic blepharitis (Koo et al., 2005), aqueous-deficient dry eye (Srinivasan et al., 2012), contact-lens related dry eye (Green-Church et al., 2008), and dry eyes associated with primary Sjögren's syndrome (SS) (McNamara et al., 2016). It remains unclear if Lacritin monomer down- regulation (in part through tissue transglutaminase-dependent cross-linking through the syndecan-1 binding domain (Velez et al., 2013)) is a symptom or a direct cause of these ocular surface diseases; however, Lacritin has shown potential as a therapeutic molecule. Its supplementation enhanced tear secretion from rat LGAC (Sanghi et al., 2001) and monkey LGAC (Fujii et al., 2013), increased basal tear secretion in rabbit LGAC (Samudre et al., 2011b) and dry eye mouse eyes (Vijmasi et al., 2014), and stimulated human corneal epithelial (HCE-T) cell proliferation (Wang et al., 2006b). This composite of preclinical evidences supports the continued development of Lacritin as an ocular therapeutic. Currently, its peptide derivative, 140 Lacripep TM , is under clinical evaluation as an essential protein replacement therapy for dry eye disease (DED) and SS-associated DED (NCT03226444). Syndecan-1 present in corneal epithelium is a known receptor for Lacritin. The cleavage of heparan sulfate (HS) chains at the extracellular domain of Syndecan-1 by an enzyme called heparanase enables Lacritin binding to syndecan-1 (Ma et al., 2006). Heparanase exists in inactive (proheparanase) and active (heparanase) forms. Each of these forms has been shown to engage with syndecan-1 and to mediate distinct downstream signaling events. Heparanase cleaves HS and promotes association of Lacritin and syndecan-1. HS cleavage not only activates Lacritin-mediated mitogenic activity but also various growth factor signaling and cell- matrix adhesion activities through liberation of various factors that were attached at the HS chains (Iba et al., 2000; Lindahl et al., 1998). Proheparanase can cluster syndecan-1 by binding to intact HS chains attached on syndecan-1. Clustering and internalization of syndecan-1 is known to activate exosome biogenesis pathway by recruiting related molecules including ALG- 2-interacting protein X (ALIX), syntenin, and endosomal-sorting complex required for transport (ESCRTs) (Baietti et al., 2012; David and Zimmermann, 2016; Fux et al., 2009). Therefore, HS- mediated activation of mitogenic signaling (removal of HS from syndecan-1) and exosome biogenesis (attachment of HS to syndecan-1) in corneal epithelium are assumed to be mutually exclusive. This study explores newly generated self-assembled multivalent Lacritin peptide nanoparticles named LP-A96, that may activate both pathways in corneal epithelial cells by binding to and clustering syndecan-1. Specifically, LP-A96 had mitogenic activity at a high cell density, whereas its ability to promote exosome biogenesis was more prominent at a low cell density. The mitogenic activity of LP-A96 was equipotent to that of the monovalent Lacripep but its ability to induce exosome biogenesis was markedly higher, enabling production of about 200- fold higher number of exosomes in a given period of time compared to Lacripep. Both actions of LP-A96 occurred in parallel with mobilization of intracellular Ca 2+ and were dependent on 141 syndecan-1. With increasing interests in Lacritin and exosome biology at the ocular surface, this study serves as a foundation for understanding ocular surface homeostasis, pathophysiology, and possible therapeutic interventions. 142 4.2. Materials and Methods 4.2.1. Synthesis, Expression, and Purification of LP-A96 and A96 The pET-25b(+) vector (#69753, Millipore-Sigma, Burlington, MA, USA) was purchased and further modified for ELP fusion cloning (Janib et al., 2014a). The cDNA encoding the amino acids GKQFIENGSEFAQKLLKKFSLWA was cloned to the N-terminus of ELP A96 to generate LP-A96. The cloned cDNA constructs were sequenced, transformed into, and expressed in ClearColi ® BL21(DE3) Electrocompetent Cells (#60810, Lucigen, Middleton, WI, USA). Cells were fermented in terrific broth media supplemented with 1 mM NaCl for 24 hr at 37 °C without IPTG induction. After centrifugation, 1 g of biomass (cell pellets) was resuspended in a 4 mL of 1:1 mixture of 1-butanol and ethanol (i.e., mixture of 8 mL 1-butanol and 8 mL ethanol was directly added to 4 g of cell pellet) (VerHeul et al., 2018). Cell pellets were resuspended thoroughly by vortexing (10 sec) and left under constant agitation at room temperature for 15 min. After transferring to 50 mL conical tubes, the suspension was spun down at 4000 rpm for 10 min using a Sorvall RC-3C Plus Centrifuge. Only the organic phase (upper phase) that contains LP-A96 was collected and transferred to a clean 50 mL conical tube with 5 mL Dulbecco's phosphate-buffered saline (dPBS, without Ca 2+ and Mg 2+ ). The whole solution was placed under a mild focused air stream with constant stirring, and left overnight to passively evaporate organic solvents. Collected samples were then dialyzed against dPBS for 24 hr under sink condition to remove residual organic solvents. Purified proteins were sterile filtered (200 nm pore, #PN 4612, Pall Corp., NY, USA) after dialysis and used for subsequent assays. Lacripep was provided by the Laurie Laboratory. 4.2.2. Biophysical Characterization of A96 and LP-A96 The purity of A96 and LP-A96 fusion proteins was analyzed using SDS-PAGE. The molar extinction coefficient (e) of A96 and LP-A96 were calculated at 1285 and 6970 M -1 ⋅cm -1 143 (Pace et al., 1995). Serial dilutions in Edelhoc buffer were prepared, measured and averaged to acquire the best estimate of protein concentration in dPBS using Eq. 1 (Edelhoch, 1967; Pace et al., 1995). The hydrodynamic radius (Rh) at 25 °C and 37 °C was determined using dynamic light scattering (DLS). Proteins in dPBS (50 μL at 10 μM) were loaded onto a 384-well plate followed by layering with two drops of mineral oil to prevent evaporation, and the whole plate was centrifuged for 1 min at 1,000 rcf to remove any remaining air bubbles. Triplicate samples were analyzed using a Wyatt Dynapro plate reader and by built-in software DYNAMICS V7 (Wyatt Tech. Co., CA, USA). Rh was measured first at 25 °C and then the temperature was immediately increased to 37 °C, where the second measurement was made. The plate was subsequently incubated at 37 °C for 7 days, while Rh was measured at days 2, 4, and 7 to observe the stability. Size exclusion chromatography followed by multiangle light scattering (SEC-MALS) was used to determine the radius of gyration (Rg), absolute molecular weight, and oligomeric state of the sample. 10 μM sample in 100 μL dPBS was injected onto a Shodex size exclusion column (KW-803, Showa Denko K.K, Japan) at 0.5 mL/min. The eluents were analyzed on a Helios system (Wyatt Tech. Co., CA, USA) maintained at 25 °C and the data were fit to a Debye model, which best explained the data to determine the Rg and the absolute molecular weight per particle. The oligomeric state for the particle was calculated using the absolute molecular weight per particle and the measured molecular weight of monomer. The monoclonal antibody, rituximab (100 μL at 1 mg/mL), was used to calibrate the system. The transition temperature (Tt) of proteins were obtained using optical density profile. The absorbance at 350 nm, A, was measured in a DU800 UV-Vis spectrophotometer (Beckman Coulter, CA, United States) under a temperature gradient of 0.5 °C/min. The Tt at each concentration was defined as the temperature at which the maximum first derivative, dA/dT, was achieved using Eq. 3. The Tt from each concentration was used to plot the phase diagram and 144 fit with Eq. 4. A version of the van’t Hoff equation (Eq. 5) was used to estimate ΔHcoacervation and ΔScoacervation of ELP coacervation (phase separation). 4.2.3. Cell culture, mitogenic activity, and cytotoxicity Human corneal epithelial SV40-transformed cells (HCE-T, Riken Cell Bank, Japan) were cultured in KSFM media (#17005042, Life Technologies, Rockville, MD, USA) supplemented with bovine pituitary extract (BPE) and epidermal growth factor (EGF) as per manufacturer’s recommendation (‘cells’ hereafter). Complete media refers to KSFM supplemented with EFG and BPE and basal media refers to KSFM media alone without EGF and BPE. Cells in passage number 4~7 were used for all cell-based assays. For imaging, Zeiss LSM880 Confocal Microscope (Carl Zeiss AG, Oberkochen, Germany) equipped with Airyscan, was used (‘confocal microscope’ hereafter). For image analysis, ZEN2 Blue Edition software (Carl Zeiss AG, Oberkochen, Germany) was used (‘ZEN2’ hereafter). For mitogenicity, cells were cultured in 24 well plates. At 80% confluency, cells were cultured with basal media for another 24 hr. A scratch was generated on the cell monolayer using a 200 μL pipette tip. After washing twice with dPBS, cells were cultured with 2 mL of fresh basal media supplemented with 1 μM Lacripep, A96, or LP-A96. The area that is devoid of cells was imaged under the confocal microscope at 0 and 13 hr post treatment and analyzed using ZEN2. Cells treated with basal media and complete media served as a negative and positive control, respectively. Identical sets of HCE-T cells were also prepared to measure mitogenicity upon contact lens-mediated delivery of LP-A96. Contact lenses incubated with 400 μg of either LP-A96 or A96 in dPBS (or 10 μM in 1 mL) for 24 hr at room temperature under constant agitation were briefly washed in dPBS and transferred to cultures. After 24 hr incubation at 37 °C, the cell monolayer was imaged to measure mitogenicity and cell culture media were collected and processed for exosome purification. 145 For cell cytotoxicity/proliferation upon LP-A96 treatment, cells were seeded with complete media at 0.1 x 10 4 cells/well in 96-well plate one day prior to the experiment. On the next day, cells were washed with dPBS and incubated with 1 μM Lacripep, A96, or LP-A96 in basal media. 50 μM Digitonin (#D141, Sigma-Aldrich, St. Louis, MO, USA) was used as a cytotoxic agent. At 24, 48, and 72 hr post-treatment, cell proliferation was measured using WST-1 reagent (#5015944001, Sigma-Aldrich, St. Louis, MO, USA) per manufacturer’s protocol. 4.2.4. Confocal fluorescence imaging For calcium signaling, cells at 50% confluency in 35 mm glass bottom culture dishes were further cultured with basal media for 24 hr. Cells were rinsed with dPBS and incubated at room temperature for 20 min with fresh basal media supplemented with 2.5 μM Fluo-4 AM (#F14201, Invitrogen, NY, USA). After this period, the NaCl Ringer buffer (145 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM KH2PO4, 1 mM MgCl2, 10 mM glucose, and 10 mM HEPES, osmolarity 300, pH 7.4) was used to rinse and incubate cells at room temperature for another 30 min. After this period, NaCl Ringer buffer was changed to Ca 2+ deprived NaCl Ringer buffer (1 mM Ca 2+ was replaced with 0.5 mM EGTA) and incubated for 10 min. After this period, cells were excited at 488 nm and their emission was recorded in real-time at 510 nm under the confocal microscope. The fluorescent intensity profile was recorded upon addition of 1 μM Lacripep, A96, or LP-A96. The fluorescence profile from each cell was converted to fold-change using Eq. 20. F0 is the average fluorescence intensity measured during the first 5 min (before addition of the treatment) and Ft is the measured fluorescence intensity at every sec. 𝐹𝑜𝑙𝑑 𝑐ℎ𝑎𝑛𝑔𝑒 = ( c 0 ) Eq. 20 To observe cellular uptake of LP-A96, cells were cultured in 35 mm glass bottom culture dishes (#P35G-0-10-C, MatTek Corp. MA) in 1.2 mL of basal media supplemented with 10 μL of 146 either DMSO, 1 mM amiloride (#A7410, Sigma-Aldrich, St. Louis, MO, USA), or 80 μM dynasore (#D7693, Sigma-Aldrich, St. Louis, MO, USA) for 30 min at 37 °C. Cells were washed with dPBS twice and then incubated at 37 °C for 10 min with 50 μL of solution that is comprised of 1 μL of NucBlue TM Live Cell Stain ReadyProbes TM reagent (#R37605, Molecular Probes, Eugene, OR, USA), 0.5 μL of rhodamine-labeled LP-A96 (1 μM final concentration), and 48.5 μL of basal media. After 10 min incubation period, cells were washed with dPBS twice and incubated with Live Cell Imaging Solution (#A14291DJ, Molecular Probes, Eugene, OR, USA). The fluorescence was imaged under the confocal microscope and the intensity was analyzed using ZEN2. To colocalizeLP-A96 and syndecan-1, 1 μM rhodamine-labeled LP-A96 in 400 μL basal media was incubated with cells at 37 °C for 20 min and fixed/permeabilized with ice-cold methanol:acetone (1:1) mixture for 10 min at -20 °C, and then processed for blocking (1% BSA), anti-Syndecan-1 antibody incubation (1:30 dilution, #SAB1305542, Sigma-Aldrich, St. Louis, MO, USA), and secondary antibody incubation (1:200 dilution, #A21202, Invitrogen, NY, USA). Nuclei were stained with DAPI solution (1:500 dilution, #62248, Thermo Fisher, Waltham, MA, USA) during secondary antibody incubation. Cells were imaged using confocal microscope and analyzed with ZEN2. To assay for cell viability upon LP-A96 treatment, cells cultured in 35 mm glass bottom culture dishes with either complete medium or 1 μM LP-A96 for 72 hr were washed and incubated with annexin V-FITC and propidium iodide (#V13242, Thermo Fisher, Waltham, MA, USA) and imaged per manufacturer’s recommendation. 4.2.5. Exosome purification and analysis Cells were seeded into 12-well plates at a density of 0.5 x 10 5 cells/well. 1 μM of either Lacripep, A96, or LP-A96 were added during seeding. Cells seeded with dPBS or complete medium served as negative and positive controls, respectively. Cells were then incubated at 37 147 °C for 72 hr, undisturbed. After this period, culture media were collected and subjected to exosome purification. After removal of culture media, cells were immediately collected in 50 μL RIPA buffer supplemented with protease inhibitor cocktail (#78430, Thermo Fisher, Waltham, MA, USA) to measure total protein concentration. To purify exosomes, collected culture media under each condition was cleared of cell debris and microparticles. To do this, media was spun down at 300 g for 5 min. Collected supernatant after this centrifugation was then spun down at 2,000 g for 10 min. Collected supernatant after this centrifugation was then spun down at 10,000 g for 30 min. Collected supernatants after these three centrifugations were concentrated and subjected to column purification (#qEVoriginal/70 nm, iZon Sciences, Medford, MA, USA) that is optimized for exosome purification. The purified exosomes were analyzed for the amount and the size using ZetaView ® nanoparticle tracking analyzer (PMX-120, Particle Metrix GmbH, Germany). The number of exosomes were normalized to the protein concentration in cell lysates measured using Micro BCA™ Protein Assay Kit (#23235, Thermo Fisher, Waltham, MA, USA). Exosomes in 0.1X dPBS were used for zeta potential analysis in ZetaView ® . Exosomes were resolved by SDS-PAGE and blotted with antibodies to CD9 (1:250 dilution, #MA1-80307, Invitrogen, NY, USA), TSG101 (1:500 dilution, #ab3071, Abcam, Cambridge, MA, USA) and Alix (1:500 dilution, #2171, Cell Signaling Technology, Danvers, MA, USA). Primary antibodies were incubated overnight at 4 °C. Donkey anti-mouse (#925-68072, 1:5,000 dilution) and goat anti-rabbit (#925-32211, 1:5,000 dilution) secondary antibodies were purchased from LI-COR (Lincoln, NE, USA) for fluorescence imaging. Exosomal RNAs were isolated using miRNeasy Serum/Plasma Kit (#217184, Qiagen, Hilden, Germany) and analyzed with 2100 Bioanalyzer system (Agilent Technologies, santa Clara, CA, USA). 148 4.2.6. Adsorption and release kinetics of LP-A96 from contact lenses Commercially available Proclearä 1 Day disposable contact lenses (CooperVision, Inc., Lake Forest, CA, USA) were washed three times with dPBS prior to any studies. To measure the concentration-dependent adsorption of LP-A96 to the contact lenses, excised contact lens pieces (5 mm x 5 mm, about 1~2 mg in weight) were incubated with 100 μL of fluorescein- labeled LP-A96 (#46410, Thermo Fisher, Waltham, MA, USA) for 24 hr. After the incubation, contact lens pieces were gently and briefly washed three times in dPBS to remove any unbound material and the fluorescence intensity was measured in 96-well plate using Synergy H1 Hybrid Multi-Mode Reader (BioTek Instruments, Inc., VT, USA). The data were fit with the Langmuir isotherm model (Eq. 21). The Qe (mg/g) and Ce (mg/mL) are the amount of adsorbed protein per gram of contact lens and protein concentration at equilibrium, respectively. The Qm (mg/g) is the maximum amount of protein adsorbed per gram of contact lens. The Kd (mg/L) is an equilibrium binding constant. 𝑄 = y ` × x y ` w Eq. 21 To measure the time-dependent adsorption of LP-A96 to the contact lens, the fluorescence intensity of excised contact lens pieces that were incubated in 100 μL LP-A96 (2 mg/mL) was retrieved at pre-determined time points, gently and briefly washed three times in dPBS to remove any unbound material, and then measured in 96-well plate using Synergy H1 Hybrid Multi-Mode Reader. The acquired data were fit the two-phase association model. To determine the release profile of LP-A96 from the contact lenses, intact contact lenses were incubated with 1 mL of fluorescein-labeled LP-A96 (50 µM) overnight at room temperature in a 24-well plate. On the next day, contact lens was gently and briefly washed three times in dPBS to remove any unbound material and then placed in 4mL dPBS (pH 7.4) inside a 50 mL conical tube under constant agitation (70 rpm, 37 °C). During the first 24 hrs, 100 µL dPBS was sampled at predetermined intervals. The dPBS was replaced completely with fresh dPBS after 149 24 hr and for every 24 hr thereafter. Collected samples were stored at 4 °C until further analysis. The amount of fluorescein released into the dPBS and the remaining fluorescence intensity on the contact lens after 96 hr were measured using Synergy H1 Hybrid Multi-Mode Reader (BioTek Instruments, Inc., VT, USA; Ex: 485 nm / Em: 528 nm). The acquired data were fit the two-phase dissociation model. 4.2.7. Secretagogue activity in primary rabbit lacrimal gland acinar cells (pR-LGACs) Cultures of pR-LGACs were prepared in 12-well plates as previously described (Gierow et al., 1996; Hsueh et al., 2015). On day 3, cells were rinsed very gently and incubated in Hank’s solution (#10-543F, Lonza, Basel, Switzerland) for 1 hr. Culture media and cell lysates collected after 1 hr served as a baseline and remaining cells were added with either dPBS, Lacripep, LP- A96, 3LP-A96, or 100 µM Carbachol (#L06674, Alfa Aesar, Haverhill, MA, USA) for 1 hr. The catalytic activity of β-hexosaminidase in each collected culture media was measured using a substrate methylumbelliferyl-N-acetyl-β-D-glucosaminide (#M2133, Sigma-Aldrich, St. Louis, MO, USA). Reaction was performed for 2 hrs at room temperature in 96-well plate and the absorbance was measured at 465 nm using a plate reader (Tecan Genios Plus; Phenix Research Products, Candler, NC, USA). To measure protein concentration in cell lysates, a 500 μL of 0.5 M NaOH solution was added into each well and incubated at room temperature under constant shaking (60 rpm) for 2hr to lyse the acini and solubilize all proteins. Protein concentration was determined using Micro BCA™ Protein Assay Kit. Measured protein concentration was used to normalize β- hexosaminidase activity measured in the culture media (Eq. 22). β hex activity ¢ 𝑅𝐹𝑈 𝜇𝑔 ¤ ¥= ¦ §¨© (ªa`_cx`dc ) ¢ E« )¬ ¤ ¥ J&Q$ (&QL% (ªa`_cx`dc ) ¢ )¬ ¤ ¥ − ¦ §¨© ( sa) ¢ E« )¬ ¤ ¥ J&Q$ (&QL% ( sa) ¢ )¬ ¤ ¥ Eq. 22 150 4.3. Results 4.3.1. Biosynthesized LP-A96 fusion proteins self-assemble into multivalent nanoparticles For simplicity and clarity, ‘LP’ refers to the lacritin-derived peptide that was used to generate LP-A96 and ‘Lacripep’ refers to the peptide equivalent to the one currently in clinical trials. Both the LP and Lacripep used in this study are derived from the active fragment at the C- terminus of the human Lacritin: GKQFIENGSEFAQKLLKKFSLLKPWA. While both LP and Lacripep share an identical 19 amino acid sequence as a core (Table 10), LP contains four additional amino acids: the N-terminal glycine (G, bold) and the C-terminal leucine-tryptophan- alanine (LWA, underline). The N-terminal glycine, which is also present in the endogenous sequence, was inevitably introduced during the cDNA cloning. The WA, which represent the last two amino acids of endogenous Lacritin, was added to the C-terminus of the 19-mer core to add precision in estimating LP-A96 concentration owing to the high absorbance of tryptophan under spectroscopic measurement. During the optimization, three amino acids, leucine-lysine-proline (LKP, italic), were removed because this region was prone to autocleavage. This cleavage was report previously (Wang et al., 2019) and re-confirmed by an independent mass analysis (data not shown). Although the LP contains additional amino acids compared to Lacripep, the 19-mer core that exhibits activity remained unaltered. The regions that contain those four additional amino acids were confirmed to have no activity related to syndecan-1 binding and mitogenic activity exhibited by the 19-mer core (Wang et al., 2006a). The 23-mer LP was genetically fused to the N-terminus of an Elastin-like polypeptides (ELPs) termed A96 (Table 10). Emerging as an attractive recombinant protein-polymer choice for diverse applications, ELPs are widely used as a platform for controlled drug delivery since their biosynthesis produces pharmacologically relevant, monodisperse, biodegradable, and biocompatible entities (Amiram et al., 2013; Chilkoti et al., 2006; Sarangthem et al., 2018). The sequence confirmed cDNA encoding the LP-A96 fusion protein was subjected to heterologous 151 expression via bacterial fermentation. The yield after the purification was ~30 mg/L with > 95% purity, as verified by SDS-PAGE (Figure 25A,B). To determine the hydrodynamic radius (Rh) and colloidal stability, purified LP-A96 was analyzed with dynamic light scattering (DLS) at days 0, 2, 4, and 7 (Figure 25C,D). Purified LP-A96 remained stable for at least 7 days at 37 °C. Size-exclusion chromatography followed by multi-angle light scattering (SEC-MALS) showed that all LP-A96 monomers self-assembled to nanoparticles (Figure 25E). The calculated degree of self-assembly was 2173 (±1.21 %, SEM). The degree of self-assembly reflects the number of LP-A96 monomers that are incorporated into one nanoparticle, which can be calculated from the absolute molecular weight per particle observed by SEC-MALS and the measured MW from the MALDI-TOF-MS (Table 10). The estimated shape factor (ratio between Rg and Rh) of 0.88 (spheres≈0.8<Rg/Rh<2.4≈rods) suggests that LP-A96 particles are spherical (Baalousha et al., 2006; Brewer and Striegel, 2011). Given the thermo-responsive nature of ELPs, the optical density of an LP-A96 solution was scanned at 350 nm (OD 350) over a range of temperatures (Figure 26A) to determine its phase transition temperature (Tt). ELPs or ELP fusion molecules become insoluble microparticles (phase separation or coacervation) that can be measured by an increase in OD 350 (Kowalczyk et al., 2014). Based on the generated phase diagram (Figure 26B), LP-A96 is expected to remain soluble at physiological temperatures and does not phase separate at concentrations used in this study. LP-A96 self-assemble into a colloidally stable nanoparticle below its Tt. 152 Figure 25. An LP-A96 fusion self-assembles into stable multivalent spherical nanoparticles. (A) Design of LP-A96 fusion. cDNA encoding LP was seamlessly cloned to the N-terminus of ELP A96. (B) The identity and purity of A96 and LP-A96 were analyzed by Coomassie blue staining of SDS-PAGE. Arrows indicate protein bands. (C) Comparison of the hydrodynamic radius of monomeric A96 and self-assembled LP-A96 nanoparticles at room and physiological temperatures. (D) The hydrodynamic radius of LP-A96 remains stable at 37 °C for least 7 days in DPBS (10 μM, n=3, mean±SD). (E) The absolute molecular weight (red dotted line that refers to the right Y-axis) per eluted LP-A96 particles (solid peak that refers to the left Y-axis) was determined using SEC-MALS analysis. 1 10 100 1000 0 10 20 30 40 50 60 70 Hydrodynamic radius (nm) Percentage (%) LP-A96 37 °C 25 °C A96 0 1 2 3 4 5 6 7 0 20 40 60 80 100 120 140 Days Hydrodynamic radius (nm) A B D C 0 4 8 12 0 1 2 10 0 10 2 10 4 10 6 10 8 10 10 Volume (mL) Relative UV (280 nm) scale Molar Mass (g/mol) E Structurally unordered monomers Self-assembled spherical nanoparticles 153 Figure 26. Thermo-sensitivity of LP-A96 and A96. (A) The ELP-mediated phase separation is monitored using optical density at 350 nm as a function of temperature using UV-Vis spectrophotometry (10 μM in dPBS). ELP phase separation increases the optical density. (B) A log-linear relationship between transition temperature and concentration was fit using Eq. 4. Parameters of the fit are reported in Table 11. Dotted lines indicate 95 % CI. (C) Enthalpy and entropy upon phase separation were estimated from the fit (lines) using Eq. 5. Parameters are reported in Table 11. Dotted lines indicate 95 % CI. (D) MALDI-TOF-MS analysis of LP-A96. Black arrow indicates measured molar mass of LP-A96. 0.0025 0.0027 0.0029 0.0031 0.0033 0 1 2 3 4 5 6 1/T t (K) ln[ELP(µM)] LP-A96 A96 1 10 100 1000 30 40 50 60 70 80 90 100 Concentration (µM) Transition temperature (°C) LP-A96 A96 25 50 75 100 0 1 2 3 Temperature ( o C) Optical density at 350nm 25 µM 50 µM 100 µM 200 µM A96 5 µM 10 µM 20 µM 40 µM LP-A96 A B D 1 2 3 C 154 Table 10. Molecular information of LP-A96, A96, and Lacripep. Proteins Amino acid sequence MW (kDa) Expected a Measured b LP-A96 GKQFIENGSEFAQKLLKKFSLWA-G(VPGAG)96Y 41.2 39.5 A96 G(VPGAG)96Y 38.6 37.0 Lacripep KQFIENGSEFAQKLLKKFS 2.2 2.1 a Calculated based on amino acid composition. b Exact MW of LP-A96, A96, and Lacripep were determined by MALDI-TOF-MS (Figure 26), reported previously (Janib et al., 2014a), and provided by the Laurie Laboratory, respectively. Table 11. Thermo-sensitivity of LP-A96 and A96. Proteins Phase Diagram Thermodynamics Slope, m [°C/log10(μM)] (Mean [95% CI]) y-intercept, b [°C] (Mean [95% CI]) ΔHmix (kJ/mol) (Mean [95% CI]) ΔSmix (kJ/mol*K) (Mean [95% CI]) LP-A96 -8.2 [-10.7 ~ -5.7] 58.3 [55.3 ~ 61.3] 240.3 [172.2 ~ 308.5] 0.72 [0.51 ~ 0.94] A96 -21.7 [-29.0 ~ -14.4] 119.5 [105.9 ~ 133.2] 108.9 [77.9 ~ 139.9] 0.27 [0.19 ~ 0.36] 155 4.3.2. Multivalent Lacripep induces Ca 2+ -dependent mitogenesis and exosome-biogenesis in corneal epithelial cells The central hypothesis of this study is that LP-A96, because of its multivalency relative to Lacripep, may have the ability to elicit diverse syndecan-1 mediated pathways including mitogenesis and exosome biogenesis in corneal epithelial cells. To test this, we started by exploring the consequences of LP-A96 treatment in HCE-T cells on Ca 2+ mobilization and mitogenic activity. Lacritin-mediated mitogenic activity in corneal epithelial cells requires Ca 2+ mobilization (Wang et al., 2006a). It has been shown that extracellular Ca 2+ translocates into the cytoplasm (influx), where it interacts with calcineurin and subsequently activates the mitogenic pathway (Wang et al., 2006a). To observe Ca 2+ influx, the fluorescence intensity of Fluo-4 AM in individual cells was monitored under fluorescence microscopy upon addition of LP-A96 to culture media. LP-A96 induced a strong Ca 2+ influx immediately after the addition (Figure 27A,D). As there was no increase in fluorescence intensity upon addition of A96 (Figure 27B), it was apparent that the LP component of LP-A96 was essential for inducing Ca 2+ influx. Under identical conditions, Lacripep did not induce Ca 2+ influx (Figure 27C). Despite significant difference in their ability to mobilize Ca 2+ , both LP-A96 and Lacripep exhibited equipotent mitogenic activity (Figure 27E,F). This may indicate that although Ca 2+ influx is required for the Lacripep-mediated mitogenesis, the degree of Ca 2+ influx required for mitogenic activity alone may be below the limit of detection. If this is the case, the superior Ca 2+ influx induced by LP-A96 may be an evidence of activation of another pathway that Lacripep does not activate. One possibility in the context of syndecan-1 biology would be an activation of exosome biogenesis pathway, which also requires Ca 2+ influx (Baietti et al., 2012; Savina et al., 2003). 156 Figure 27. LP-A96 induces Ca 2+ influx and mitogenesis in corneal epithelial cells. (A~C) HCE-T cells pre-incubated with Fluo-4 AM were incubated with either LP-A96, Lacripep, or A96. Dotted line at the 5 th minute indicates when the treatment was added to the culture media. The fluorescence intensity of Fluo-4 AM was monitored as a marker for Ca 2+ influx (n=27~33 cells/treatment per experiment). Representative data set from the three independent sets of experiment is shown. Results were highly reproducible. (D) Area under the curve of each fluorescence intensity profile shown in (A)~(C). (E) The velocity of the cell monolayer towards the gap area (devoid of cells) was calculated as outlined in (F). The velocity of the cell monolayer upon 1 μM LP-A96 or Lacripep treatment was significantly different from that of the basal media treated cell monolayer. (F) Generated wound gaps were imaged before the treatment and 12 hr post-treatment, and used to construct (E). White dotted lines indicate the outer edge of the monolayer adjacent to the wound gap. A one-way ANOVA followed by multiple comparisons was used for statistical comparison. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05. Mean±SD. 10 -3 10 -2 10 -1 10 0 10 1 10 2 10 3 10 4 0 10 20 30 40 50 log[Conc], nM Velocity (µm/hr) Lacripep LP-A96 Basal media Complete media * LP-A96 Lacripep A96 -2 0 2 4 6 8 10 Area under the curve (AUC) **** **** ns E B C A D Fluorescence intensity (fold change) Time (min) 0 4 8 12 0 5 10 Time (min) Fluorescence Intensity (fold-change) 1 2 3 4 6 7 8 9 10 19 16 12 13 14 15 11 17 18 20 21 22 23 24 25 26 27 28 29 30 LP-A96 0 4 8 12 0 5 10 Time (min) Fluorescence Intensity (fold-change) 1 2 3 4 5 6 7 8 9 10 11 13 14 15 16 17 19 20 21 22 23 24 25 27 12 18 26 A96 0 4 8 12 0 5 10 Time (min) Fluorescence Intensity (fold-change) 1 2 3 4 5 6 7 8 9 10 19 16 12 13 14 15 11 17 18 20 21 22 23 24 25 26 27 28 29 30 Legend Legend Legend Lacripep F 157 To observe whether LP-A96 promotes exosome biogenesis, extracellular vesicles (EVs) secreted into the culture media over a 3-day period were collected and analyzed. During this period, cells incubated with LP-A96 spawned significantly higher number of EVs that were highly enriched with exosome markers (Kalluri and LeBleu, 2020) (Figure 28A,B). The amount of EVs collected from the culture media from cells after LP-A96 treatment was 193-fold and 55- fold higher compared to the amount recovered in basal media and in complete media, respectively. The mean diameter and the size distribution of the purified EVs were similar among groups (Figure 28C,D). To confirm that the apparent increase in exosome amount was not due to residual LP- A96 co-purified during the purification, purified exosomes were subjected to zeta potential analysis. Firstly, the zeta potential of the natural exosomes collected from regular cell cultures (blue bar graph in Figure 28E) and the preparation of pure LP-A96 (red bar graph in Figure 28E) were compared, which showed marked differences. Secondly, assuming that the residual LP-A96 in the culture media could be co-purified, two mixtures were prepared with different ratios: exosome:LP-A96=1:1 and 1:100. These mixtures were incubated at 37 °C for 3 days in basal media and subjected to identical column-based purification process and then analyzed for zeta potential. For the 1:1 mixture, instead of a clear separation of two peaks, the profile was broadly distributed between -60~0 mV, exactly overlapping profiles of pure exosome and LP- A96. Two zeta potential values were reported for this mixture: -46.4 mV and -24.5 mV (black bar graph in Figure 28E). For the 1:100 mixture, the highest peak was observed at around -15 mV and the profile was narrowly distributed between -30~0 mV, which highly resembled that of the pure LP-A96. Lastly, the zeta potential of exosomes purified after LP-A96 treatment was compared with the above four profiles. The mean value and the overall distribution profile of purified exosomes after LP-A96 treatment (magenta bar graph in Figure 28E) highly resembled those of the natural exosomes (blue bar graph in Figure 28E). Its mean zeta potential was -55 mV and the major portion of the profile was narrowly distributed between -90~-30 mV (-48 mV 158 and -80~-20 mV, respectively, for natural exosomes). Therefore, it was apparent that the purified EVs after LP-A96 treatment are dominated by exosomes, and not LP-A96 particles. To identify the presence of any residual LP-A96 particles that were not detected during the zeta potential analysis, collected exosomes (used to construct Figure 28A and magenta bar graph in Figure 28E) were Western blotted against ELPs using anti-ELP antibody (Kouhi et al., 2019) along with pre-determined amount of LP-A96 (Figure 29). LP-A96 nanoparticles co- purified with the increased exosome yield were detected at low densities, comprising about 10 % of total particles purified. Based on this densitometric analysis, the pure increase in exosome yield associated with LP-A96 treatment was quantified (Figure 28A). 159 Figure 28. LP-A96 activates exosome biogenesis in corneal epithelial cells. (A) Quantified EVs in the collected HCE-T cell culture media under each treatment. (B) EVs collected after LP- A96 treatment were highly enriched with exosome markers CD9, Alix, and TSG101. (C) Mean hydrodynamic diameter of purified exosomes measured by ZetaView nanoparticle tracking analyzer. Experiments were performed in triplicate. (D) Bar graphs showing the size distribution of purified exosomes. The three parallel gray lines indicate 50, 150, and 250 nm, respectively. Representative data from three independent sets of experiment is shown. Results were highly reproducible. (E) Zeta potential analyses of exosomes, LP-A96, and combinations of both at different ratios. The four parallel gray lines indicate -90, -60, -30, and 0 mV, respectively. Inset values are the mean zeta potentials reported by the built-in software. A one-way ANOVA followed by multiple comparisons was used for statistical comparison. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05. Mean±SD. 160 Figure 29. Western blot-based densitometric analysis for residual LP-A96 in purified EV samples. Purified EVs from three independent experiments (Experiments #1~3, lane 1~3) were blotted for ELPs using anti-ELP antibody to detect and quantify residual LP-A96 co-purified. Serial dilutions of LP-A96 (ELP Standard #1~3, lane 4~6) with known particle amount were used to construct the standard curve for densitometric analysis. Measured band intensity in lane 1~3 was fit to a standard curve to calculate LP-A96 particle number in respective samples (Particles/mL in lane 1~3). This calculation was used to reflect the pure increase of exosomes in Figure 28A. Lane Band Intensity Particles/mL 1: Experiment #1 4,250.1 1.6 x 10 7 2: Experiment #2 16,238.8 89.2 x 10 7 3: Experiment #3 14,973.9 79.7 x 10 7 4: Standard #1 4,465.0 1.7 x 10 7 5: Standard #2 6,771.0 17 x 10 7 6: Standard #3 27,104.6 170 x 10 7 Original Analyzed #1 #2 #3 Std1 Std2 Std3 Experiments ELP standards Original Analyzed #1 #2 #3 Std1 Std2 Std3 Experiments ELP standards Lane Band Intensity Particles/mL 1: Experiment #1 6.537 1.7 x 10 7 2: Experiment #2 5.433 1.5 x 10 7 3: Experiment #3 3.858 1.1 x 10 7 4: Background 0.000 0 5: Standard #1 1.202 2.0 x 10 7 6: Standard #2 66.335 20.4 x 10 7 7: Standard #3 734.925 204 x 10 7 161 Exosomes mediate intercellular signaling. Their cargos, especially ribonucleic acids (RNAs), are important messengers for intercellular communication (O'Brien et al., 2020). As LP- A96 stimulates exosome production, it is imperative to confirm that the amount of exosomal cargos increase proportional to exosome increase. During a 2-day period, cells treated with LP- A96 produced about 12-fold higher number of exosomes compared to complete media treated cells (Figure 30A). The increase in exosome amount lead to the increase in both non-coding small RNAs (9-fold) and miRNAs (17-fold), a subset of non-coding small RNAs (Figure 30B,C). The amount of non-coding small RNAs and miRNAs per exosome was similar (Figure 30D,E). About 60 % of exosomal small RNA was miRNA in exosomes produced under LP-A96, which was slightly higher than that of exosomes produced under complete media, but there was no statistical difference (Figure 30F). This indicates that LP-A96 treatment produces exosomes that are loaded with similar amount RNAs to exosomes produced in complete media but not just excess number of blank exosomes. 162 Figure 30. LP-A96 induced exosomes are properly loaded with exosomal RNAs. (A) LP- A96 stimulates exosome production. Exosomes collected were subjected to RNA isolation. (B,C) Elevated small RNA (0~270 nucleotides) and miRNA (10~40 nucleotides) amount reflects increase in total exosome amount shown in (A). (D) Small RNAs, (E) miRNAs, and (F) miRNA- to-small RNA ratio per exosome particle under two different treatments are similar. n=3, Mean±SD, *p<0.05, ns=non-significant LP-A96 Complete media 0 5 10 15 20 25 Exosomes amount (fold change) 12-fold ns (p=0.056) LP-A96 Complete Media 0 10 20 30 miRNA concentration (fold change) 17-fold * LP-A96 Complete Media 0 5 10 15 Small RNA concentration (fold change) 9-fold * LP-A96 Complete Media 0 1 2 3 4 miRNA amount per exosome (fold change) LP-A96 Complete Media 0 20 40 60 80 100 miRNA / small RNA ratio per exosome (%) LP-A96 Complete Media 0.0 0.5 1.0 1.5 2.0 2.5 Small RNA amount per exosome (fold change) E B C A D F 163 During the 3-day incubation period, the appearance of cells treated with LP-A96 was notably different from the cells treated with other agents (Figure 31A) as characterized by the appearance of prominent perinuclear refractive organelles (arrows). To confirm that the increase in exosome amount upon LP-A96 treatment was not a result of secreted intracellular vesicles upon cell death, cells were treated with LP-A96 to see if it exhibits any cytotoxicity. During the course of 3 days, LP-A96 did not appear to exhibit any cytotoxicity (Figure 31B). The proliferation profile of LP-A96 treated cells was similar to that of the cells treated with basal media, Lacripep, or A96. The rate of proliferation was less than that achieved in complete media. This result was in line with the observed non-significant differences in total protein concentration in cell lysates after the 3-day incubation period (Figure 31C). Lack of cytotoxicity of LP-A96 was also confirmed by staining cells with annexin V and propidium iodide after 3-day incubation (Figure 31D). Therefore, the difference in cell appearance may be an indirect indication of a change in the intracellular membrane machineries associated with exosome biogenesis. Although there was an increase in exosome abundance recovered from culture medium in cells incubated with LP-A96 at low cell density, exosome biogenesis was not prominent when cell density was high (Figure 32). Based on the above analyses, we concluded that: i) the cellular response to exosome biogenesis is more prominent when cell density is low; ii) purified EVs are exosomes and their increase is dominated by exosomes; and iii) this increase is not related to the exposure of intracellular vesicular bodies upon cell death. 164 Figure 31. LP-A96-mediated exosome biogenesis does not affect cell viability in corneal epithelial cells. (A) Brightfield images of HCE-T cells at 48 hr post-treatment. The appearance of LP-A96 treated HCE-T cells at 48 hr was typical of that observed throughout the 3-day incubation period, while all other treatments did not affect the appearance of the cells. Perinuclear compartments observed were indicated with yellow arrows. (B) Cytotoxicity of LP- A96 was quantified alongside other treatments. Digitonin was used as a cytotoxic agent which served as a negative control showing no proliferation, while complete medium represented a positive control showing maximal proliferation. (C) Total protein concentration in cell lysates at the end of 3-day incubation period. (D) Cell viability under each treatment was visualized using annexin V and propidium iodide. Cells were incubated for 3 days with complete media or basal media supplemented with LP-A96 before fluorescence imaging. 50 μM Digitonin was added 1 hr prior to the imaging. A one-way ANOVA followed by multiple comparisons was used for statistical comparison. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05. Mean±SD. A B C dPBS A96 Lacripep LP-A96 Complete media 0 10 20 30 40 50 Protein concentration in cell lysates (µg/mL) Complete media A96 Lacripep LP-A96 0 24 48 72 0.0 0.5 1.0 1.5 2.0 Hours Absorbance at 450 nm A96 Lacripep LP-A96 Complete media Digitonin dPBS D Complete media LP-A96 Digitonin Annexin V-FITC Propidium iodide 165 Figure 32. LP-A96-mediated exosome biogenesis is less prominent when treated cells are at a high confluency. (A) Quantified exosomes in the culture media collected after 3 days of treatment. Cells cultured to 80 % confluency were starved for 24 hr using basal media prior to the treatment. (B) Mean hydrodynamic diameter of exosomes. Experiments were performed in triplicate. A one-way ANOVA followed by multiple comparisons was used for statistical comparison. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05. Mean±SD. Neg Cntl A96 Lacripep 1LP-A96 Pos Cntl 0.0 0.5 1.0 1.5 Particles/µg protein (fold-change) Neg Cntl A96 Lacripep 1LP-A96 Pos Cntl 0 50 100 150 200 Hydrodynamic diamerter (nm) A B 166 4.3.3. Multivalent Lacripep is internalized via dynamin-mediated endocytosis and colocalizes with syndecan-1 in corneal epithelial cells Syndecan-1 is known to be internalized upon clustering on the cell surface (Chen and Williams, 2013). As LP-A96 is expected to cluster syndecan-1 on the cell surface followed by endocytosis, internalization and subcellular colocalization with syndecan-1 would be expected. To explore LP-A96 internalization, cells were treated without or with either amiloride or dynasore, which respectively inhibit pinocytosis or receptor-mediated endocytosis (Dutta and Donaldson, 2012). After 15 min of incubation, a significant fraction of LP-A96 is internalized (Figure 33A), partly in a amiloride (Figure 33B) and completely in a dynasore inhibitory manner (Figure 33C,D). Further confirmation was sought to observe if internalized LP-A96 colocalize with syndecan-1. After 15 min of incubation, LP-A96 colocalized intracellularly with syndecan-1 (Figure 33E). Therefore, it is highly likely that LP-A96 is internalized through a syndecan-1 receptor-mediated pathway. 167 Figure 33. LP-A96 is internalized via dynamin-dependent endocytosis and colocalized with syndecan-1 in corneal epithelial cells. (A~D) HCE-T cells were treated with either DMSO, amiloride (1 mM), or dynasore (80 μM) prior to addition of rhodamine-labeled LP-A96. (D) Quantified fluorescence intensity per cell under each treatment. n=39 for DMSO, n=49 for amiloride, n=48 for dynasore. Representative images are shown from the three independent experiments. Results were highly reproducible. (E) LP-A96 was colocalized with syndecan-1 in HCE-T cells. (Pearson’s correlation=0.8, n=32). Rhodamine-labeled LP-A96 were incubated with the cells for 15 min at 37 °C prior to fixation. Secondary antibody control (- anti-Syndecan- 1) confirmed specific detection of syndecan-1. A one-way ANOVA followed by multiple comparisons was used for statistical comparison. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05. Mean±SD. Non-Treat Amiloride Dynasore 0.0 0.5 1.0 1.5 2.0 2.5 Fluorescence Intensity (fold-change) *** **** **** A C D B Non-Treat (DMSO) + Amiloride (1 mM) + Dynasore (80 µM) E LP-A96 Syndecan-1 Nucleus Merge + anti-Syndecan-1 + 2° AF488 - anti-Syndecan-1 + 2° AF488 168 4.3.4 LP-A96 delivered by contact lens maintain mitogenic activity and induce exosome biogenesis in corneal epithelial cells Eyedrops are the most convenient and frequently used route of administration for ocular surface drug delivery. Although it is convenient and effective, a frequent need for topical administration is associated with lower patient compliance. Thus, strategies to enable sustained release of drugs that are effective when given topically are of great interest. Such strategies may maintain active drug on the ocular surface for a longer period of time. One such method is to use contact lenses as a drug depot. Contact lenses have been studied as a therapeutic platform to manage ocular anterior segment disorders beyond their primary function of providing millions of people with glasses-free vision correction (Karlgard et al., 2004; Wichterle and Lim, 1960). Based on the previous demonstration of adsorption of ELPs to and their delivery to the corneal epithelial cells upon release from the commercially available contact lenses (Wang et al., 2019), the ability for contact lenses to deliver functional LP-A96 to corneal epithelial cells was tested. To begin with, the adsorption and release kinetics of LP-A96 in contact lenses was determined using a fluorescence-based method. Fluorescein-labeled LP-A96 was incubated with contact lenses to analyze the concentration-dependent adsorption (Figure 34A), time- dependent adsorption (Figure 34B), and time-dependent release kinetics (Figure 34C). Parameters estimated from the fit are reported in Table 12. Based on these parameters, contact lenses were incubated with 0.4 mg of LP-A96 for 24 hr; release from lenses adsorbed under this condition was expected to increase LP-A96 concentration in culture media up to 1~2 μM over 24 hr. After in solution adsorption, LP-A96 loaded contact lenses were transferred to HCE-T cell cultures and incubated for 24 hrs. Just before the contact lens transfer, a wound gap was generated on HCE-T monolayers (Figure 34D). The cells incubated with LP-A96 loaded contact lenses showed equipotent mitogenic activity to the cells incubated with intact contact lens in complete media (Figure 34E), which was significantly higher than the mitogenic potential 169 observed with A96 loaded contact lens treatment. Although there was a clear mitogenic effect, exosome production during this 24 hr period was not significantly different (Figure 34F). This may be because LP-A96-mediated exosome biogenesis is not prominent under conditions of maximum HCE-T cell confluency (Figures 32) and the duration of incubation was only 24 hr. Nevertheless, the data clearly show that the contact-lens can deliver functional LP-A96 to the corneal epithelial cells. 170 Figure 34. Contact lens-delivered LP-A96 maintains its activity in corneal epithelial cells. (A) Concentration-dependent adsorption of LP-A96 to contact lenses over a 24 hr period. (B) Time-dependent adsorption of 2 mg/mL LP-A96 to contact lenses. (C) Time-dependent release of LP-A96 from contact lenses. (D) Study design of the contact lens-mediated delivery of LP- A96 to corneal epithelial cells treated with a wound gap. (E) Mitogenicity of the cells incubated with contact lenses loaded with different agents. (F) Exosome amount in the culture media after 24 hr period. A one-way ANOVA followed by multiple comparisons was used for statistical comparison. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05. Mean±SD. dPBS A96 only LP-A96 CM 0 4 8 12 Agent delivered by contact lens Particles/µg protein (fold-change) dPBS A96 LP-A96 CM 0 10 20 30 40 Agent delivered by contact lens % Gap remaining after 24hr * * ns ns 0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70 Time (h) Qe (mg/g) 0 24 48 72 96 0 20 40 60 80 100 Time (h) Retention (%) 0 2 4 6 8 10 0 20 40 60 80 100 Ce (mg/mL) Qe (mg/g) A B C E F D 171 Table 12. Adsorption and release kinetics of LP-A96 in contact lenses Parameters Concentration - dependent adsorption (Mean (95% CI)) Parameters Time- dependent adsorption (Mean (95% CI)) Parameters Time- dependent release (Mean (95% CI)) Q m (mg/g) 78.6 (68.5-88.8) Plateau (mg/g) 55.8 (46.1-65.5) Fast Release (%) 73.3 (65.8-80.9) K d (mg/L) 0.78 (0.35-1.21) Fast binding (%) 25.6 (15.8-35.4) k fast (hr -1 ) 0.068 (0.055-0.082) R 2 0.84 k fast (hr -1 ) 2.36 (0.0-4.86) k slow (hr -1 ) 0.0011 (0.0-0.0044) k slow (hr -1 ) 0.041 (0.0-0.069) t 1/2, fast (hr) 10.1 (8.4-12.7) t 1/2, fast (hr) 0.29 (0.14-Inf.) t 1/2, slow (hr) 645.5 (157.3-Inf.) t 1/2, slow (hr) 16.9 (10.1-51.1) R 2 0.99 R 2 0.95 172 4.4. Discussion This study describes the construction of a multivalent Lacritin peptide nanoparticle named LP-A96 and its biological effects in corneal epithelial cells. Under different conditions in corneal epithelial cells, LP-A96 is capable to activate both the mitogenic and exosome biogenesis pathways in parallel to an intracellular Ca 2+ mobilization. Of these functions, only the mitogenic capability was shared with the monovalent Lacripep, while the stimulation of exosome biogenesis was unique to the multivalent nanoparticle. When cells are sparse and not in contact with each other, LP-A96 was able to direct the intracellular machinery to evoke exosome biogenesis instead of mitogenic activity. On the other hand, LP-A96 did not activate exosome biogenesis when the cells are confluent but was capable of mitogenesis. This study only tested these two opposing conditions, near maximum confluency and low cell density. However, identifying the conditions under which cells become unresponsive to signaling to evoke exosome biogenesis would allow better understanding of the syndecan-1 biology in the corneal epithelium. Optimal concentration for Lacritin’s mitogenic activity is reported to be 1~10 nM in human salivary gland ductal cells (Wang et al., 2006a). At this concentration, however, neither Lacripep nor LP-A96 induce any mitogenic activity in corneal epithelial cells. The chosen concentration for this study is 1 μM, for both Lacripep and LP-A96, based on a dose-dependent mitogenic activity (Figure 27E). As similar concentrations were reported for corneal-lacrimal gland axis in rabbit (0.8~8 μM) (Samudre et al., 2011a), mouse (4 μM) (Vijmasi et al., 2014), and monkey (0.1~1 μM) (Fujii et al., 2013) models, corneal epithelium seems to require higher dose of Lacritin or its derivative Lacripep compared to salivary gland ductal cells. In other study, 3.2 nM Lacritin evoked Ca 2+ influx in HCE-T cells (Sanghi et al., 2001). At 1 μM, only LP-A96 induced Ca 2+ mobilization but not Lacripep. It could be that the structural difference, full-length versus derivatized peptide fragment, affected the degree of Ca 2+ mobilization. Lacripep-mediated Ca 2+ mobilization may be under the limit of detection. 173 LP-A96 is shown to colocalize with syndecan-1 in corneal epithelial cells, possibly via association of LP-A96 with syndecan-1 (Zhang et al., 2013). Due to multivalent nature of LP- A96 (avidity), pre-incubation of Lacripep (affinity) was not effective in blocking LP-A96 binding. Further studies addressing direct binding of LP-A96 to syndecan-1 and to other proposed co- receptors, such as G-protein coupled receptors (GPCRs), and their internalization/activation in corneal epithelial cells is of great importance to understand Lacritin biology at the ocular surface (Wang et al., 2006a). Since LP-A96 stimulates exosome biogenesis, it is possible that the expression and activity profile of heparanase-proheparanse may change upon addition of LP-A96. As this enzyme is known to be heavily involved in homeostasis (Fux et al., 2009), profiling their expression and activity upon LP-A96 treatment in corneal epithelium will allow better insights in biological influence of LP-A96. Involvement of ocular exosomes in corneal wound healing is well documented (Han et al., 2017; Samaeekia et al., 2018). As significantly higher number of exosomes that carry RNAs are produced and secreted upon LP-A96 treatment, deep sequencing of miRNAs in these exosomes, signaling pathways these miRNAs would activate under diseased condition in comparison to healthy condition, and how it contributes to overall ocular surface health are critical aspects to investigate during the following studies. In addition to actions on the cornea which have been described, Lacritin is a known tear secretagogue. Upon expression and secretion from LGAC (lacrimal gland acinar cells), Lacritin promotes tear secretion from lacrimal glands to sustain the tear film and maintain ocular surface homeostasis (Wang et al., 2015). Upon development of the LP-A96, its secretagogue activity was tested in primary rabbit LGAC. However, LP-A96 was not as effective as recombinant full length Lacritin or Lacripep (Figure 35). For this reason, much of the research efforts were focused to biological effects at the corneal epithelial cells rather than at LGAC. Despite several observational reports of Lacritin’s secretagogue activity, its intracellular mechanism that leads to 174 a tear secretion is not well understood. Comparison of Lacritin’s secretagogue activity to a well characterized acetylcholinergic compound carbachol in primary monkey LGACs by Fujii et al. showed that Lacritin stimulates tear secretion in a Ca 2+ and PKCα independent manner while carbachol required both for its activity (Fujii et al., 2013). Based on the fact that Ca 2+ and PKCα are essential molecules for Lacritin-mediated signaling in epithelial cells, Lacritin’s receptor and intracellular signaling in LGACs seem to be different from what is generally known for epithelial cells. Identification of receptors and its respective intracellular signaling as well as how LP-A96’s multivalency contributes to tear secretion and exosome production in LGAC will provide basics on how to utilize LP-A96 for lacrimal glands, such as LP-A96 delivery to alleviate lacrimal gland inflammation and tear deficiency. Multivalency was achieved by genetically fusing LP to a recombinant polypeptide called ELPs. ELPs are thermo-responsive biopolymers biologically inspired from human tropoelastin (Despanie et al., 2016; MacEwan and Chilkoti, 2010a). Comprised of repeated pentameric peptide motif (VPGXG)n, ELPs can be recombinantly expressed in cells in fusion with therapeutic peptides, such as Lacripep. One of the advantages of using recombinant polypeptides like ELPs would be the ease of incorporating multiple copies of peptides into one construct. During the developmental stage, several versions of multi-copy LP-A96 were cloned and expressed, of which three copy construct, 3LP-A96, were purified and tested for mitogenicity and secretagogue activity. The activities of 3LP-A96 were not different from those of the LP-A96 or Lacripep in corneal epithelial cells. 3LP-A96 mitogenic activity was similar to LP-A96 or Lacripep (Figure 36A) while the Ca 2+ influx signal slightly differed from LP-A96 or ionomycin (Figure 36B,C). While ionomycin or LP-A96 induced sharp peaks, about 5~15-fold increase in fluorescence intensity from the baseline during the 1~3 min period, 3LP-A96 induced elongated but relatively shallow peaks, which persisted more than 7 min. Despite the difference in the profile, its area under the curve (AUC) was not significantly different from that of the LP-A96 (Figure 36D). As its activities in corneal epithelial cells were not prominent, 3LP- 175 A96 showed no improvement in secretagogue activity compared to LP-A96 and was lower than that of recombinant human Lacritin or Lacripep (Figure 35C,D). Due to insufficient stability and low production yield (~1 mg/L), 3LP-A96 was not further explored. Drugs that are currently prescribed for the treatment of various ocular surface and anterior chamber disorders have been investigated for contact lens-based delivery to enhance their therapeutic performance. These include drugs for ocular infection (Ciprofloxacin), corneal injury (EGF), allergic conjunctivitis (Ketotifen fumarate), dry eye (Re-wetting agents/hyaluronic acid, Cyclosporin A) and glaucoma (Acetozolamide, Timolol) (Guzman-Aranguez et al., 2013). Despite these advances, it would be desirable to provide a contact lens drug delivery device which is relatively simple in design; which does not require complicated and expensive manufacturing processes; which does not significantly impair or interfere with the patient's vision; and which would not require a substantial change in the practice patterns of eye physicians and surgeons. As LP-A96 released from the contact lenses was fully functional, delivery of LP-A96 through contact lenses could serve as an alternative route of administration to improved ocular surface health. To conclude, this study demonstrated a simple yet effective peptide modality to overcome the mechanistic barrier imposed at Lacritin-syndecan-1 axis in corneal epithelium (Figure 37). As Lacritin-syndecan-1 axis plays a critical role in corneal epithelium homeostasis, future studies will address the list of cargos enriched in the secreted exosomes and the intracellular pathways that these cargos activate in the context of dry eye disease. 176 Figure 35. Secretagogue activity of LP-A96, 3LP-A96, and Lacripep. Self-assembly of 3LP- A96 into nanoparticles was confirmed by (A) DLS and (B) SEC-MALS analyses. Average degree of assembly was 5571 (5571 monomers per particle). Secretagogue activities of (C) LP- A96, 3LP-A96, and recombinant human (rh) Lacritin (n=3) and (D) Lacripep (n=1) were quantified by measuring beta-hexosaminidase levels in the primary rabbit lacrimal gland acinar cell culture media. Activity was normalized to total protein amount measured in cell lysates. A one-way ANOVA followed by multiple comparisons was used for statistical comparison. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05. Mean±SD. A B 0 2 4 6 8 10 12 0 1 2 10 0 10 2 10 4 10 6 10 8 10 10 Volume (mL) Relative UV scale Molar Mass (g/mol) 10 100 1000 0 10 20 30 40 Hydrodynamic radius (nm) Percentage (%) 25 °C 37 °C C D Non-Treat Lacripep (0.5 µM) Lacripep (3 µM) Lacripep (6 µM) Lacripep (13 µM) Lacripep (25 µM) Lacripep (50 µM) Carbachol (100 µM) 0 10 20 30 40 50 60 Normalized β-hexosaminidase activity (fold change) Non-Treat LP-A96 (20 µM) 3LP-A96 (20 µM) rhLacritin (20 µM) 0 2 4 6 Normalized β-hexosaminidase activity (fold change) 177 Figure 36. Mitogenicity and Ca 2+ influx induced by 3LP-A96. (A) Mitogenicity induced by 3LP-A96 was overlaid to the Figure 27E (n=3, Mean±SD). (B) Ca 2+ mobilization upon addition of 3LP-A96 (n=18). (C) Ca 2+ influx upon addition of ionomycin (1 µg/mL) (n=26). Experiments (B) and (C) were performed in duplicate. 9~15 cells were analyzed during each experiment. (D) Comparison of AUC upon each treatment. Each dot represents individual cell. Data set for LP- A96 from Figure 27D was included for comparison. A one-way ANOVA followed by multiple comparisons was used for statistical comparison. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05. Mean±SD. Ionomycin 3LP-A96 LP-A96 -2 0 2 4 6 8 10 12 14 Area under the curve (AUC) ** ns ns 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 Time (min) Fluorescence Intensity (fold-change) Legend Legend Legend Legend Legend Legend Legend Legend 0 1 2 3 4 5 0 5 10 15 20 Time (min) Fluorescence Intensity (fold-change) R1 R2 R3 R4 R6 R7 R8 R9 R10 R11 R12 R13 R14 R15 R16 R17 Legend Legend Legend Legend Legend Legend Legend Legend Legend Legend B D C A 10 -3 10 -2 10 -1 10 0 10 1 10 2 10 3 10 4 0 10 20 30 40 50 60 log[Conc], nM Velocity (µm/hr) 3LP-A96 Lacripep LP-A96 Basal media Complete media * 178 Figure 37. 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Abstract (if available)

Abstract Multivalent constructs are widely explored as molecular tools and therapeutics owing to their ability to engage multiple targets via multiple ligands. For examples, drug loaded nanoparticles have a higher chance to deliver their payloads to the target if equipped with multiple targeting ligands that allow tighter binding to the target (Koshkaryev et al., 2013). Moreover, multivalent therapeutic antibodies are shown to be better in tumor penetration and therapeutic efficacy compared to monovalent counterpart (Blanco-Toribio et al., 2013). Not only for drug targeting, multivalency-mediated supramolecular polymer assembly recapitulates complex biological systems (Ooi et al., 2020), which adds depth to drug delivery research. Among myriads of biomaterial scaffolds that are able to build multivalent modalities, bioinspired recombinant polypeptides are favored due to the ease of genetic engineering, low-immunognicity, high biocompatibility, and biodegradability. One of a such material is elastin-like polypeptides (ELPs). Comprised of pentameric repeats, (Val-Pro-Gly-Xₐₐ-Gly)ₙ, ELPs have been recognized as a favorable polypeptide fusion scaffold for drug delivery (Despanie et al., 2016). This manuscript discusses widely utilized bioinspired recombinant polypeptide-based protein-polymers that are able to build multivalent constructs and presents three examples of distinct multivalent modalities generated using ELPs. Especially, their biophysical properties and therapeutic implications are discussed in regard to cancer and eye diseases. The Chapter 1 introduces the recombinant polypeptide-based protein-polymers utilized in the field of drug discovery and delivery, especially those of which adopted as building blocks to self-assemble antibody fragments. Antibodies have multiple biologically relevant features that can be reengineered into new therapeutics formats. These include the adaptable specificity of their variable (Fab) region or recruitment of the immune system and mechanisms to promote long-circulation through their crystallizable (Fc) region. Since the invention of the single chain variable fragment (scFv) in 1988, the variable regions of antibody have been re-engineered and rebuilt into a wide variety of multifunctional nanostructures. Among the strategies, peptide-mediated self-assembly of variable regions through heterologous expression has become a powerful way to produce homogenous and functional antibody fusion biomaterials and is an alternative to chemical bioconjugation. This chapter discusses the functions of antibody fragments, status of engineered therapeutic antibody fragments in the clinic, and challenges to overcome. Moreover, a selected list of biomaterials that mediate self-assembly of antibody variable region through biosynthesis are explored, which include elastin-like polypeptides (ELPs), collagen-like polypeptides (CLPs), albumin, transmembrane proteins, leucine zippers, silk protein, and viruses. Among the modalities introduced in Chapter 1, the Chapter 2 further discusses biophysical properties of antibody-ELP fusions, their mechanism of action, and therapeutic applications towards non-Hodgkin lymphoma (NHL). Despite advancements in antibody-based therapies for NHL, at least two major therapeutic needs remain unmet: i) heterogenous activation of host immunity towards B cell NHL

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Antibodies and elastin-like polypeptides: cellular and biophysical characterization of an anti-ELP monoclonal and an anti-CD3 single-chain-ELP fusion

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Creator Lee, Changrim (author)

Core Title Development and therapeutic assessment of multivalent protein polymers for cancer and eye diseases

School School of Pharmacy

Degree Doctor of Philosophy

Degree Program Pharmaceutical Sciences

Publication Date 09/21/2020

Defense Date 08/03/2020

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag antibody,cathepsin S,corneal epithelium,dacryoadenitis,elastin-like polypeptide,exosome,FK506-binding protein,lacrimal gland,lacritin,nanoworms,non-Hodgkin lymphoma,non-obese diabetic mouse,OAI-PMH Harvest,peptide biosynthesis,rapamycin,receptor clustering,Sézary syndrome,Sjögren’s syndrome,syndecan-1

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor MacKay, John Andrew (committee chair), Hamm-Alvarez, Sarah (committee member), Xie, Jianming (committee member)

Creator Email changril@usc.edu,changrimlee@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c89-372725

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