Injectable non-immunogenic PEG-like conjugate that forms a subcutaneous depot and enables sustained delivery of a peptide drug

Many biologics have a short plasma half-life, and their conjugation to polyethylene glycol (PEG) is commonly used 14 to solve this problem. Unfortunately, PEG is immunogenic and forms vacuoles, and improvement in PEGylated 15 drugs' half-life is at an asymptote. Here, we developed a PEG-like, non-immunogenic, and injectable conjugate 16 technology for sustained delivery of biologics. An optimal poly[oligo(ethylene glycol) methyl ether methacrylate] 17 (POEGMA) depot of exendin, a peptide drug used in the clinic in treating type 2 diabetes, outperformed PEG, non- 18 depot-forming POEGMA, and a clinical sustained-release exendin-4 formulation in efficacy and pharmacokinetics. 19 Notably, POEGMA was non-immunoreactive, while PEG induced a persistent anti-PEG immune response, leading 20 to its subsequent doses' early optimal Ex-POEGMA to investigate


Introduction
Resonance (NMR) spectroscopy ( Supplementary Fig. 7-8). 27 We also synthesized an EG3 POEGMA homopolymer 98 as a non-depot forming -soluble-control. The monomer composition of POEGMA was defined as the 99 percentage of EG2 (or EG3) monomer content in the total polymer. We used the nomenclature of EGX%, where X 100 is the length of the EG chain (2 or 3), and the subscript % is the percentage of the monomer in the total polymer.

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All polymers showed sharp and thermally reversible phase behavior, as seen by the sharp increase in 107 optical density as the temperature was increased and by the fact that their optical density decreased back to 108 baseline as the temperature was decreased below the Tt (Fig. 1b). Their phase behavior also showed no thermal 109 hysteresis, as seen by the overlapping turbidity curves for the heating and cooling cycle. The EG3100% POEGMA 110 had a Tt of ~48ºC at 25 µM, consistent with a previous report, 28 confirming that it cannot form an s.c. depot. The

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Tt of the POEGMA copolymers was a function of the EG2 content, as the Tt decreased with the increasing molar 112 ratio of the more hydrophobic EG2 monomer. All copolymers phase transitioned between 25 and 34ºC, suggesting 113 that all copolymers were potential depot-forming constructs. The Tt of POEGMA also showed an inverse 114 concentration dependence. The Tt increased with POEGMA dilution (Fig. 1c), suggesting that sustained release of 115 a POEGMA conjugate from the s.c. depot should be possible in vivo, in response to continuous dilution at the 116 periphery of the depot. The Tt of POEGMA was also a function of its Mw, as the Tt decreased with increasing DP-117 and Mw (Fig. 1d). These data clearly showed that the key molecular variables that control the phase behavior of 118 POEGMA copolymers-namely its Tt -are the composition -the ratio of EG2 to EG3 units-and Mw.

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Design and purification of a site-specific 1:1 conjugate. Conventional conjugation methods typically provide 128 limited control over the conjugation site and stoichiometry, resulting in a heterogeneous mixture of conjugates 129 with non-uniform PK and pharmacodynamics (PD). 29 To circumvent this issue, we site-specifically conjugated 130 POEGMA to exendin at its C-terminus using bio-orthogonal click chemistry 18,29- 31 We chose the C-terminus as the  Transition Temperature (ºC) 60 POEGMA conjugation site, as the N-terminus of exendin is critical to its function. 18 To do so, we first covalently 132 conjugated a bio-orthogonal triglycine dibenzocyclooctyne (DBCO) group to the C-terminus of exendin via sortase 133 A-mediated native peptide ligation, 18,30 yielding exendin-DBCO. To ensure that the conjugation is both 134 bioorthogonal -to avoid side-reactions of POEGMA with any internal residue in exendin-and site-specific, we 135 chose to synthesize azide-terminated POEGMA copolymers. The azide moiety readily reacts with the DBCO group 136 and does not react with any other chemical groups in exendin, yielding a site-specific conjugate with a 1:1 137 stoichiometry. 30 (Supplementary Fig. 11).

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Purification of POEGMA conjugates from unreacted agents using chromatography techniques is labor-139 intensive and has a low yield. To circumvent these problems, we exploited the LCST behavior of depot-forming 140 POEGMA conjugates. The conjugates were purified in a chromatography-free manner at room temperature by 141 simply triggering the phase transition of the conjugate by adding ammonium sulfate salt that lowers the Tt below 142 room temperature, 32 leading to phase separation of Ex-POEGMA conjugates into an insoluble coacervate.

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Unreacted exendin remained in the supernatant. The Ex-POEGMA coacervates were isolated by centrifugation 144 and were then resolubilized by adding PBS at room temperature. Importantly, this purification technique does not 145 apply to PEG as it does not show an LCST phase behavior.

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Identifying an optimal Ex-POEGMA conjugate. We identified an optimal Ex-POEGMA conjugate that maximized 147 fed blood glucose control in hyperglycemic mice by first optimizing the Tt, then the Mw, and finally the dose of the 148 conjugate. POEGMA copolymers were synthesized for the Tt optimization study are shown in Table 1   was also reported to be ~30ºC. 25

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Having identified an Ex-POEGMA conjugate with an optimal Tt, we next identified an optimal Mw. Our 200 strategy was to synthesize Ex-POEGMA conjugates with the optimal Tt of ~30 º C but with varying Mw (  Fig. 17). These polymers were then conjugated to exendin. The resulting Ex-

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POEGMA conjugates had varied Rh ( Supplementary Fig. 18) consistent with their different Mw, but all showed 208 thermally reversible phase behavior (Fig. 2g). However, the Tt of the conjugates had a slightly different inverse 209 concentration dependence, as seen by the different slopes of the log(concentration) versus Tt plots (Fig. 2h).

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Importantly, these plots intersected at 500 µM and ~30 º C (see circled data in Fig. 2h), a temperature that 211 fortuitously corresponds to the previously identified optimal Tt. This concentration of 500 µM was hence chosen

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The conjugates were next administrated into 11-week-old male DIO mice (n=6), with PBS as a negative 216 control. Mice treated with the conjugates had lower blood glucose ( Fig. 2j; Supplementary Fig. 19) and body 217 weight (Fig. 2l) than the PBS-treated group. The conjugate with the lowest Mw tested-Ex-POEGMA18.9-

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controlled blood glucose only one day and had a significantly higher glucose exposure as seen by the AUC (Fig. 2k) 219 than all other treatment groups. This data suggests that Ex-POEGMA18.9kDa was cleared from the circulation much 220 faster than the other conjugates because of its significantly smaller size (Rh~2.6 vs. ~4-5 nm for the other 221 conjugates) that is lower than the renal excretion threshold, defined as the size of serum albumin (~3.6 nm). subsequently referred to as Ex-POEGMAopt in this paper. Finally, the optimal injection dose of this conjugate was 228 determined to be 1000 nmol kg -1 in a dose-escalation study, which completed the optimization process of the than hyperbranched POEGMA of the same Mw, resulting in conjugates with a much larger Rh at the same Mw.

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Because this difference could complicate side-by-side efficacy comparison of the conjugates by affecting their 236 kidney clearance rates, we synthesized both Mw-matched and Rh-matched exendin-PEG conjugates-termed Extemperature (Fig. 3a), allowing it to remain as a solution in a syringe at room temperature but to transition into 239 an insoluble coacervate phase when injected s.c., as tested at a concentration of 500 µM (see circled data in Fig. PEGRh showed phase behavior (Fig. 3a). The conjugates showed no difference in their EC50 in activating the GLP1R 243 in an in vitro, cell-based assay (Fig. 3c).

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The conjugates were next administered s.c. into 11-week-old male DIO mice (n=6) at the equivalent, 245 optimal dose, with PBS included as a control. Mice treated with the conjugates had lower fed blood glucose levels 246 than the control ( Fig. 3d; Supplementary Fig. 24). Ex-PEGRh and Ex-PEGMw controlled fed blood glucose for three 247 and four days, respectively (Fig. 3d), consistent with the literature. 37 The one-day longer glucose control provided 248 by Ex-PEGMw could be attributed to its larger size, and hence slower clearance. In accord with the literature, Ex-

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POEGMAsol also provided four days of blood glucose control. 18 In contrast, Ex-POEGMAopt outperformed its soluble  difference was also observed when the experiment was repeated using DIO mice ( Supplementary Fig. 27).

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However, the magnitude of the difference was not as great, possibly due to the milder display of T2D allowing 276 lower circulating Ex-PEGRh concentrations to show therapeutic effect. The test time did not affect glucose exposure in the Ex-POEGMAopt treatment group (p>0.99) (Fig. 3l), indicating that Ex-POEGMAopt maintained its superior glycemic regulation ability throughout the study.

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Pharmacokinetics. We next investigated the PK of Ex-POEGMAopt and the PEGylated controls to better understand 293 the differences in the short-term efficacy profiles of the treatments. We fluorescently labeled exendin, Ex-

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POEGMAopt, Ex-PEGRh, and Ex-PEGMw ( Supplementary Fig. 28), followed by s.c. administration into naïve DIO mice 295 (n=4). The PK parameters were determined from the drug's plasma concentration (Fig. 4a-b) and are shown 296 in Table 2. All conjugates had much slower absorption kinetics than exendin, suggesting that the conjugates were 297 primarily taken up from the s.c. space into the blood through the lymphatic system due to their large size. 41 Ex-

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respectively. The 55.6 kDa soluble Ex-POEGMA conjugate with a matching Mw as the optimal depot-forming 304 POEGMA conjugate had a t1/2 elimination of 61.2 ± 5.0h. 18 In contrast, Ex-POEGMAopt outperformed all conjugates by 305 prolonging exendin's circulation by ~46-fold with a t1/2 elimination of 97.3 ± 5.6h, and it provided the highest drug 306 exposure (AUC) among the treatments. These results clearly demonstrate the PK benefits of sustained release 307 from a depot. This study also allowed us to infer that the minimal effective concentration for Ex-POEGMAopt is 5.1 308 nM, which corresponds to the concentration of Ex-POEGMAopt after 6 days post-injection.

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Long-term efficacy. We next investigated the long-term efficacy of Ex-POEGMAopt to understand better how the 317 differences in PK and short-term efficacy translated into long-term management of T2D. We hypothesized that

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Ex-POEGMAopt should outperform the other exendin formulations because of its superior fed blood glucose and 319 glycemic control and longer PK. We tested this hypothesis by administering sterile and endotoxin-free Exevery week for eight weeks, followed by monitoring their blood glucose, changes in body weight, and glycated 322 hemoglobin (HbA1c%) levels. We chose to monitor HbA1c%, as it is a measure of long-term T2D management 334 Table 2. Pharmacokinetic parameters of Ex-POEGMAopt. Pharmacokinetic parameters were calculated from the data given in Figure 4.

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Data were fitted to a one-phase exponential decay curve and analyzed via non-compartmental pharmacokinetic analysis using GraphPad

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IgM response increased with the number of Ex-PEGMw injections (Supplementary Fig. 34)

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We further tested the immunogenicity of POEGMA and compared it to PEG using highly immunogenic   Fig. 35b). This result was attributed to the steric hindrance imparted by the PEG, 6 allowing the conjugate to expose fewer OVA epitopes to the immune system. The differences in anti-

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OVA antibody titers were not as drastic when POEGMA was used. We speculate that this is because POEGMA 388 provides less steric hindrance than PEG to its conjugation partners due to its more compact architecture. 45 PEG 389 was highly immunogenic, as seen by the high titers of PEG-specific ADAs detected in OVA-PEG-immunized mice 390 plasma with Ex-PEG beads (Supplementary Fig. 35c-d). Similar to when exendin was the conjugation partner, PEG-  Fig. 36d) and did not mature into an IgG response ( Supplementary Fig. 36h), further 396 supporting our conclusions that POEGMA appears to be significantly less immunogenic than PEG.     Fig. 39a). Notably, anti-exendin IgM 428 titers were higher in Ex-PEGRh treated mice plasma, and an anti-exendin IgG response was detected 429 (Supplementary Fig. 39b). Ex-PEGRh also induced both PEG-specific IgM and IgG responses. Given the absence of

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We finally tested the effect of ADAs on the PK of the treatments. We hypothesized that the loss of efficacy 445 in the Ex-PEGMw long-term treatment was due to the binding of the anti-PEG antibodies to the circulating drug, 446 resulting in accelerated blood clearance and preventing it from showing efficacy. 42, 48 We also hypothesized that

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Ex-POEGMAopt did not show a difference in PK among injections due to the lack of ADAs. To test these hypotheses,

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to create a set of copolymers that exhibit a range of Tt. Upon s.c. injection of these Ex-POEGMA conjugates in 484 mice, we found that the Tt of these copolymers determined the depot's stability, with depots with a lower Tt 485 leading to more stable depots, but a slower rate of release, while depots with a higher Tt had a higher rate of 486 release with a shorter duration of release. We identified ~30ºC as the optimal Tt that balances the release rate of

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Second, by tuning the molecular architecture of POEGMA, we also eliminated its immunogenicity while 496 preserving its favorable PK and PD benefits. The hyperbranched structure of POEGMA does not induce a POEGMA-497 specific immune response, presumably because its short OEG side chains do not crosslink B-cell receptors as PEG 498 does, which is a prerequisite for T-cell independent B-cell immune responses. 42 Unlike PEG, which is unstructured 499 in aqueous solvents, POEGMA self-organizes into nanoparticle-like chains due to the interfacial energy balance 500 driven by its amphiphilicity and the repulsion forces among hydrated OEG side-chains. 45 We speculate that 501 POEGMA's compact molecular conformation in water may effectively reduce the number of epitopes exposed to 502 the immune system, allowing it to remain non-immunoreactive. Third, in addition to its lack of immunogenicity, positive population, which is now rampant because of chronic exposure to PEG through excipients in drugs and 505 consumer products. These findings are highly topical because up to 70% of the human population has pre-existing

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Collectively, these advantages allowed an optimal Ex-POEGMA depot to provide T2D management 517 superior to PEG and a clinical sustained-release exendin formulation-Bydureon. Importantly, Ex-PEG lost its 518 efficacy after repeated administrations due to the induction of PEG-specific PK-altering antibodies, preventing it 519 from being used as a long-term treatment strategy. In contrast, the optimal Ex-POEGMA conjugate preserved its 520 efficacy and PK benefits through repeated injections over 56 days, indicating that optimizing the molecular 521 architecture -as we have done with POEGMA-is a powerful approach to solve the mounting problems of 522 PEGylated therapeutics.

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The POEGMA depot technology has several advantages over other drug delivery technologies. First,

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Fed blood glucose measurements. In the short-term efficacy experiments, the fed blood glucose was measured 688 after a single s.c. injection of the treatments. On the day of injection, the tail was sterilized with alcohol pads (BD).

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The first drop of blood collected from a tiny incision on the tail vein was wiped off. The second drop of blood was 690 used to measure fed blood glucose using a hand-held glucometer (AlphaTrack, Abbott). The treatments were 691 solubilized in PBS and kept on ice before injection to prevent phase transition, followed by s.c. administration into 692 mice. Bydureon was prepared for injection according to the manufacturer's instructions. Fed blood glucose levels 693 were measured 24 h and immediately before injection, at 1-, 4-, and 8-h post-injection, and every 24 h after that until no significant effect of treatments on fed blood glucose was observed. Body weight was tracked daily. In the treatments were administrated into the mice at an equivalent dose (1000 nmol per kg bodyweight) and

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Pharmacokinetics. Exendin variants were labeled with a fluorophore to track their pharmacokinetics. Briefly,

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Alexa Fluor 488 NHS ester (Pierce) was reacted with exendin variants (5 mg ml -1 ) at a 5:1 molar ratio in PBS for 710 one hour at room temperature. Unreacted excess fluorophore was removed by dialysis into the water at 4 o C using 711 membranes with a 3,000 Da MWCO (Pierce), verified by HPLC. The labeling efficiency was calculated from UV-vis 712 spectroscopy using an ND-1000 Nanodrop spectrophotometer (Thermo Scientific).

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The fluorophore-labeled treatments were administered into DIO C57BL/6J mice via a single s.c. injection 714 at 1000 nmol kg -1 (45 nmol kg -1 fluorophore). Ten μl of blood was collected from a tiny incision on the tail vein into