Pre-existing anti-polyethylene glycol antibody reduces the therapeutic efficacy and pharmacokinetics of PEGylated liposomes

Abstract

Rationale: Increasing frequency of human exposure to PEG-related products means that healthy people are likely to have pre-existing anti-PEG antibodies (pre-αPEG Ab). However, the influence of pre-αPEG Abs on the pharmacokinetics (PK) and therapeutic efficacy of LipoDox is unknown. Methods: We generated two pre-αPEG Ab mouse models. First, naïve mice were immunized with PEGylated protein to generate an endogenous αPEG Ab titer (endo αPEG). Second, monoclonal αPEG Abs were passively transferred (αPEG-PT) into naïve mice to establish a αPEG titer. The naïve, endo αPEG and αPEG-PT mice were intravenously injected with ¹¹¹in-labeled LipoDox to evaluate its PK. Tumor-bearing naïve, endo αPEG and αPEG-PT mice were intravenously injected with ¹¹¹in-labeled LipoDox to evaluate its biodistribution. The therapeutic efficacy of LipoDox was estimated in the tumor-bearing mice. Results: The areas under the curve (AUC)_last of LipoDox in endo αPEG and αPEG-PT mice were 11.5- and 15.6- fold less, respectively, than that of the naïve group. The biodistribution results suggested that pre-αPEG Ab can significantly reduce tumor accumulation and accelerate blood clearance of ¹¹¹In-labeled LipoDox from the spleen. The tumor volumes of the tumor-bearing endo αPEG and αPEG-PT mice after treatment with LipoDox were significantly increased as compared with that of the tumor-bearing naïve mice. Conclusions: Pre-αPEG Abs were found to dramatically alter the PK and reduce the tumor accumulation and therapeutic efficacy of LipoDox. Pre-αPEG may have potential as a marker to aid development of personalized therapy using LipoDox and achieve optimal therapeutic efficacy.

Keywords: ELISA; LipoDox; PEG; Polyethylene glycol; anti-PEG antibodies; liposome.

Figure 1

Characterization of the αPEG Ab concentration in the endo αPEG mice. BALB/c mice (N=36, one symbol represents one animal) were immunized with PEGylated protein (PEGylated-eβG and -HSA). The serum of each mouse was collected after the 1^st (○) and 2^nd (●) boost. The serum from each mouse was diluted with 2% skim milk buffer, and then added to a CH₃-PEG₂₀₀₀-NH₂-coated plate. (A) Pre-existing IgG and (B) pre-existing IgM were incubated with HRP-conjugated goat anti-IgG and IgM specific antibodies. The relative concentrations of anti-PEG IgG or IgM in mice serum samples were calculated by comparison with IgG-6.3 and IgM-AGP4 standard curves, respectively. Data are represented as mean ± SD. Statistical analysis was performed by comparing multiple t tests, *, P < 0.05 and, ****, P < 0.0001.

Figure 2

Competition assay of αPEG Ab from endogenous mouse serum. The αPEG monoclonal Abs AGP4 (IgM; 5 μg/mL), 6.3 (IgG; 5 μg/mL) and 50-fold diluted αPEG mouse serum sample were added to wells pre-coated with CH₃-PEG₂₀₀₀-NH₂. LipoDox (200 μg/mL) was added as a competitor to compete for binding of the αPEG Ab to the CH₃-PEG₂₀₀₀-NH₂ well coating. Each pre-existing αPEG-positive mouse was labeled using an ear-punching system; for example, the number '3-2' represents animal number 2 in cage 3. The mean absorbance values (405 nm) of duplicate determinations are shown. (Bars, SD).

Figure 3

Influence of αPEG Ab on the pharmacokinetics of ¹¹¹In-labeled LipoDox. (A) Naïve (○), endo αPEG (●) and αPEG-PT (▲) BALB/c mice were IV injected with ¹¹¹In-labeled LipoDox. The αPEG-PT-5 h (△) mice were injected with αPEG Ab and injected with ¹¹¹In-labeled LipoDox 5 h later. The blood was harvested at different times. The pharmacokinetics of ¹¹¹In-labeled LipoDox was analyzed using a γ-counter. (B) The sera of mice with pre-existing αPEG IgG and (C) pre-existing αPEG IgM were also measured by quantitative αPEG Ab ELISA. Data are represented as mean ± SD. Statistical analysis was performed by comparing multiple t-tests. **, P < 0.0001 as compared to the naïve group.

Figure 4

Influence of αPEG Ab on the accumulation of fluorescently labeled PEG-liposome in tumors. (A) Naïve, αPEG-PT and endo αPEG BALB/c mice bearing CT26 tumors were IV injected with fluorescently labeled PEG-liposome (Lipo-DIR). Mice were sequentially imaged at 24, 48 and 72 h with an IVIS spectrum optical imaging system. (B) The fluorescence intensity of Lipo-DIR in the tumor was analyzed with Living Image software version 4.2. Statistical analysis was performed by compare multiple t-tests. *, P<0.05 as compared to the naïve group.

Figure 5

Biodistribution of ¹¹¹In-labeled LipoDox in αPEG Ab mice. Naïve, endo αPEG, and αPEG-PT BALB/c mice bearing CT26 tumors were IV injected with ¹¹¹In-labeled LipoDox. The mice were sacrificed at different times and the organs and blood were collected and weighed. The radioactivities of (A) blood, (B) tumor, (C) spleen and (D) liver were analyzed using a γ-counter. Statistical analysis was performed by comparing multiple t-tests. *, P < 0.05 as compared to the naïve group.

Figure 6

Effect of αPEG Ab on the therapeutic efficacy of LipoDox in vivo. CT26 tumor-bearing untreated (○), naïve (●), endo αPEG (△) and αPEG-PT (▲) BALB/c mice were IV injected with LipoDox (3 mg/kg body weight) every 4 days, 3 times. The tumor volume (length × width × height × 0.5) was measured using calipers every 2 days. The results are shown as mean tumor size (n = 8) ± SD. Statistical analysis was performed by comparing multiple t-tests. *, P < 0.05 as compared to the untreated group.