Nonshrinkable Thermosensitive Hydrogels Combined with Bispecific Anti-PSMA/CD3 T-Cell Engager for Effective Against Tumors in Mice Model

Abstract

Purpose: CD3-based Bispecific T-cell engagers (BiTEs) are effective for solid tumors due to their tumor specificity and tissue penetration, but they face challenges like short half-lives and narrow therapeutic windows. Innovative delivery systems, like thermosensitive hydrogels, show the potential to enhance stability, sustained release, and therapeutic efficacy.

Methods: We developed PEGylated PLGA (PEG-PLGA) thermosensitive hydrogels with a nonshrinkable property (nsTPPgels) for effective controlled release and loaded them with bispecific anti-prostate surface membrane antigen (PSMA) F_ab /anti-CD3 _scF_v T-cell engager (BiPTE) to form in situ drug deposits with a sustained-release profile after subcutaneous injection. Each group of hydrogels was first tested for differences in properties through rheological and in vitro drug release profiles. Meanwhile, in vivo pharmacokinetics, anti-tumor efficacy studies, and T-cell tracking studies were conducted to analyze the advantages of nsTPPgels included D₂gel and DTgels.

Results: The cytotoxicity of BiPTE against PSMA-overexpressing tumor cells and the drug release functionality of nsTPPgels were validated in vitro. Rheological studies showed that both D₂gel and DTgels remained in solution below 27 °C for easy injection and solidified at physiological temperatures to form localized depots for sustained BiPTE release. All nsTPPgels demonstrated a 5-day in vitro sustained release, prolonged elimination half-life, steady plasma BiPTE levels, and extended mean residence time. In an LNCaP-xenograft mouse model, tumor growth inhibition rates for BiPTE/DTgel-2, BiPTE/DTgel-2S, and BiPTE/D2gel were 74.3%, 96.1%, and 113.1%, respectively, compared to 35.6% for intravenous and 46% for subcutaneous BiPTE administration. Furthermore, all nsTPPgels effectively achieved T-cell recruitment to lymph nodes and tumor sites in tracking studies.

Conclusion: In conclusion, we developed relatively convenient injectable thermosensitive D₂gel with a desirable gelation temperature window, which have the potential to be used for antibody drug delivery in several biomedical applications.

Keywords: Bispecific T-cell engager; T-cell recruitment; anti-PSMA; in situ drug deposits; nonshrinkable; thermosensitive hydrogel.

Graphical abstract

Figure 1

Verification of the structure and function of a bispecific anti-prostate-specific membrane antigen (PSMA) Fab/anti-cluster of differentiation 3 (CD3) scFv T-cell engager (BiPTE). (a), Analytical results of non-reducing and reducing SDS-PAGE for BiPTE. (b), In vitro cellular cytotoxicity of BiPTE against PSMA-overexpressing LNCaP cells or PSMA-negative PC-3 cells with different effector:target (E:T) ratios. The supernatant from the cytotoxicity assay was also used to detect granzyme B (c), interferon (IFN)-γ (d), and tumor necrosis factor (TNF)-α (e). Screenshots show the cytotoxic effects of BiPTE on PSMA-positive LNCap cells (f) and PSMA-negative PC3 cells (g) via cytotoxic lymphocytic T cells (CTLs) and 5 µg/mL BiPTE.

Figure 2

(a) Schematic diagram of gelling properties of nsTPPgel. (b)The phase diagram and (c) The shrinkage ratio of DTgel-1, DTgel-2, DTgel-2S, D₂gel and D₃gel. (d) Rheological characterization of DTgel-2, DTgel-2S, and D₂gel. ****p < 0.0001 compared to each group in one-way ANOVA and post hoc = Tukey.

Figure 3

(a), Schematic diagram of the thermogelation mechanism of PLGA–PEG–PLGA triblock copolymer and mPEG-PLGA diblock copolymer solutions with increasing polymer concentration and at an elevated temperature for composite hydrogel (mixed micelles, DTgel-2 and DTgel-2S) and regular micelles (D₂gel). In vitro release profile of BSA/nsTPPgels or BSA/D₃gel (b) and BiPTE/nsTPPgels (c). Data are expressed as mean ± SD (n = 3).

Figure 4

(a) Schematic diagram of the in vivo pharmacokinetic study. The dotted time points were observed only for the intravenous (i.v.) BiPTE sol group. (b) In vivo pharmacokinetic profiles of i.v. BiPTE sol, subcutaneous (s.c.) BiPTE sol, BiPTE/DTgel-2, BiPTE/DTgel-2S, and BiPTE/D₂gel (the dose of BiPTE was 5 mg/kg). The one-way ANOVA analysis of the pharmacokinetic parameters includes (c) area under the curve from zero to time infinity (AUC_0-inf) (d), half-life (e), T_max (f), C_max and (g), mean residence time (MRT). Data are expressed as mean ± SD (n = 3 for the i.v. group; n = 4 for the s.c. and nsTPPgel groups). *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 compared to each group in one-way ANOVA and post hoc = Tukey.

Figure 5

BiPTE/nsTPPgel serves as an effective and safe therapeutic approach for treating PSMA-positive tumors. (a) Schematic diagram of the in vivo antitumor study. (b) In vivo antitumor efficacy of intravenous (i.v.) BiPTE sol, subcutaneous (s.c.) BiPTE sol, BiPTE/DTgel-2, BiPTE/DTgel-2S, and BiPTE/D₂gel with doses equal to 5 mg/kg in LNCaP tumor-bearing mice. (c) Body weight changes over 21 days after treatment. (d) Tumor growth inhibition (TGI%) of each group at the end of the treatment (day 21). (e) The tumor images for each group at the end of the treatment. (f) IHC staining images of GranzymeB in tumors on day 21 after administration. (g) Quantization of GranzymeB in IHC images using Image J software. Data are expressed as mean ± SD (n = 4). *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 compared to each group in two-way (Figure 5b and c) or one-way ANOVA (Figure 5d and g). Post hoc = Tukey.

Figure 6

Correlation analysis of the relationship between tumor growth inhibition rate (TGI) and gel strength, as well as pharmacokinetic (PK) parameters. TGI is plotted on the X-axis, and gel strength (a), half-life (b), T_max (c), MRT (d), C_max (e), and AUC_0-inf (f) are plotted on the Y-axis for regression analysis. Solid lines represent regression lines for intravenous (i.v.) BiPTE sol, subcutaneous (s.c.) BiPTE sol, BiPTE/DTgel-2, BiPTE/DTgel-2S, and BiPTE/D2gel groups, while dashed lines represent regression lines for s.c. BiPTE sol, BiPTE/DTgel-2, BiPTE/DTgel-2S, and BiPTE/D2gel groups. Gel strength analysis was performed only for the three hydrogel groups.

Figure 7

BiPTE/nsTPPgels can accumulate large amounts of T cells at the injection site and lymph node areas, demonstrating their potential feasibility as artificial lymph nodes. (a) Schematic diagram of the liver, tumor, injection site, and lymph nodes in IVIS imaging. Average radiant efficiencies of DiR-labeled a-huPBMCs in the (b) injection site, (c) tumor, (d) lymph node, and (e) liver at 2, 6, 12, 24, 52, 72, and 96 h after injection of intravenous (i.v.) BiPTE sol, subcutaneous (s.c.) BiPTE sol, BiPTE/DTgel-2, BiPTE/DTgel-2S, and BiPTE/D₂gel with a dose equal to 5 mg/kg. Data are expressed as mean ± SD (n = 3).