A brush-polymer conjugate of exendin-4 reduces blood glucose for up to five days and eliminates poly(ethylene glycol) antigenicity

Abstract

The delivery of therapeutic peptides and proteins is often challenged by a short half-life, and thus the need for frequent injections that limit efficacy, reduce patient compliance and increase treatment cost. Here, we demonstrate that a single subcutaneous injection of site-specific (C-terminal) conjugates of exendin-4 (exendin) - a therapeutic peptide that is clinically used to treat type 2 diabetes - and poly[oligo(ethylene glycol) methyl ether methacrylate] (POEGMA) with precisely controlled molecular weights lowered blood glucose for up to 120 h in fed mice. Most notably, we show that an exendin-C-POEGMA conjugate with an average of 9 side-chain ethylene glycol (EG) repeats exhibits significantly lower reactivity towards patient-derived anti-poly(ethylene glycol) (PEG) antibodies than two FDA-approved PEGylated drugs, and that reducing the side-chain length to 3 EG repeats completely eliminates PEG antigenicity without compromising in vivo efficacy. Our findings establish the site-specific conjugation of POEGMA as a next-generation PEGylation technology for improving the pharmacological performance of traditional PEGylated drugs, whose safety and efficacy are hindered by pre-existing anti-PEG antibodies in patients.

Fig. 1

Synthetic scheme of exendin-C-POEGMA. a) Recombinant expression of the sortase A substrate, exendin-srt-His₆-ELP, and purification by ITC . b) Sortase-catalyzed site-specific attachment of the ATRP initiator AEBMP to the C-terminus of exendin to generate exendin-C-Br. c) In situ ATRP of OEGMA from exendin-C-Br yielding exendin-C-POEGMA. ITC: inverse transition cycling, ELP: elastin-like polypeptide, srt: sortase A recognition sequence “LPETG”, AEBMP: N-(2-(2-(2-(2-aminoacetamido)acet-amido)acetamido) ethyl)-2-bromo-2-methylpropanamide.

Fig. 2

Characterization of exendin-C-Br macroinitiator and EG9 exendin-C-POEGMA conjugates. a) Coomassie-stained SDS-PAGE analysis of initiator attachment on exendin by sortase A. Lane 1: MW marker, lane 2: sortase reaction mixture after 18 h of reaction, lane 3: purified exendin-C-Br macroinitiator. b) SEC traces of ATRP reaction mixtures of grafting EG9 POEGMA from exendin-C-Br carried out for 0.5 h, 1 h, 1.25 h, 2 h and 3 h, detected by UV-vis absorbance at 280 nm. c) Cyclic adenosine monophosphate (cAMP) response of native exendin and EG9 exendin-C-POEGMA conjugates with M_ns of 25.4 kDa, 54.6 kDa, 66.2 kDa, 97.2 kDa and 155.0 kDa in baby hamster kidney (BHK) cells expressing the GLP-1R. Results are plotted as mean ± standard error of the mean (SEM), n=3. Half-maximal effective concentration (EC₅₀) values are summarized in Table 1.

Fig. 3

Assessment of MW-dependent in vivo efficacy of EG9 exendin-C-POEGMA conjugates. Blood glucose levels in fed mice were measured before and after a single s.c. injection of a) unmodified exendin, or b-e) 25.4 kDa, 54.6 kDa, 97.2 kDa, and 155.0 kDa EG9 exendin-C-POEGMA conjugates, compared to PBS control. The peptide and conjugates were administered at 25 nmol/kg and PBS was injected at equivalent volume at t = 0 h. Blood glucose levels were normalized to the average glucose levels measured 24 h and immediately before injection. Data were analyzed by repeated measures two-way analysis of variance (ANOVA), followed by post hoc Dunnett’s multiple comparison test. f) Area under the curve (AUC) of blood glucose profiles (0 h to 144 h, with respect to 0% baseline) as a function of conjugate M_n. AUCs were compared using one-way ANOVA followed by post hoc Tukey’s multiple comparison test. In all panels, results are plotted as mean ± SEM, n=6, *P < 0.05, **P < 0.01, ***P < 0.001 and ****P <0.0001.

Fig. 4

Intraperitoneal glucose tolerance test (IPGTT) of an EG9 exendin-POEGMA in mice. Mouse blood glucose levels measured in an IPGTT performed at 24 h and 72 h after a single s.c. injection of a and b) the 54.6 kDa EG9 exendin-POEGMA conjugate or c and d) unmodified exendin at 25 nmol/kg, compared to PBS of equivalent volume. Mice were fasted for 6 h prior to glucose challenge by an intraperotoneal (i.p.) injection of 1.5 g/kg of glucose. Results are plotted as mean ± SEM, n=5 in panels a and b, n=3 in panels c and d. AUCs of treatment and PBS were compared using an unpaired parametric two-tailed t test (**P < 0.01, and ****P <0.0001). Exendin was not significant at either time point.

Fig. 5

Assessment of reactivity of exendin-C-POEGMA conjugates toward anti-PEG antibodies in patient plasma samples. a) Direct ELISA probing 54.6 kDa EG9 exendin-C-POEGMA conjugate, native exendin, adenosine deaminase (ADA), bovine serum albumin (BSA), Krystexxa^® (PEG-uricase) and Adagen^® (PEG-ADA) with diluent (1% BSA in PBS), an anti-PEG negative patient plasma sample, or one of two anti-PEG positive plasma samples. b) Competitive ELISA, where various amounts of exendin, 54.6 kDa EG9 exendin-C-POEMGA, ADA and Adagen^® were allowed to compete with Krystexxa^® for binding with anti-PEG antibodies in a positive plasma sample. c and d) Direct and competitive assays described in panels a and b performed with a 55.6 kDa EG3 exendin-C-POEGMA conjugate. In all assays, the same unmodified peptide/protein content or similar PEG/OEG content in the case of polymer-modified samples per well were compared. See Methods section for details. Results are plotted as mean ± SEM, n=3 in panels a and b, n=5 in panels c and d. Data were analyzed by two-way ANOVA, followed by post hoc Dunnett’s multiple comparison test (**P < 0.01, and ****P <0.0001).

Fig. 6

Assessment of in vivo efficacy and pharmacokinetics of exendin-C-POEGMA conjugates. Blood glucose levels in fed mice measured before and after a single s.c. injection of a) 55.6 kDa and b) 71.6 kDa EG3 exendin-C-POEGMA conjugates at 25 nmol/kg or PBS at equivalent volume administered at t = 0 h. Blood glucose levels were normalized to the average glucose levels measured 24 h and immediately before injection. Data were analyzed by repeated measures two-way ANOVA, followed by post hoc Dunnett’s multiple comparison test (n=5, *P < 0.05, **P < 0.01, and ****P <0.0001). c) Exendin and d) exendin-C-POEGMA conjugates (54.6 kDa EG9, 55.6 kDa EG3 and 71.6 kDa EG3) were fluorescently labeled with Alexa Fluor^® 488 and injected into mice (n=3) s.c. at 75 nmol/kg (45 nmol/kg fluorophore). Blood samples were collected via tail vein at various time points for fluorescence quantification. Data were analyzed using a non-compartmental fit (solid lines) to derive the pharmacokinetic parameters shown in Table 2. Results in all panels are plotted as mean ± SEM.