Cellular Delivery of Bioorthogonal Pretargeting Therapeutics in PSMA-Positive Prostate Cancer

Abstract

Prostate cancer is primarily fatal after it becomes metastatic and castration-resistant despite novel combined hormonal and chemotherapeutic regimens. Hence, new therapeutic concepts and drug delivery strategies are urgently needed for the eradication of this devastating disease. Here we report the highly specific, in situ click chemistry driven pretargeted delivery of cytotoxic drug carriers to PSMA­(+) prostate cancer cells. Anti-PSMA 5D3 mAb and its F­(ab′)2 fragments were functionalized with trans-cyclooctene (TCO), labeled with a fluorophore, and used as pretargeting components. Human serum albumin (ALB) was loaded with the DM1 antitubulin agent, functionalized with PEGylated tetrazine (PEG4-Tz), labeled with a fluorophore, and used as the drug delivery component. The internalization kinetics of components and the therapeutic efficacy of the pretargeted click therapy were studied in PSMA­(+) PC3-PIP and PSMA(−) PC3-Flu control cells. The F­(ab′)2 fragments were internalized faster than 5D3 mAb in PSMA­(+) PC3-PIP cells. In the two-component pretargeted imaging study, both components were colocalized in a perinuclear location of the cytoplasm of PC3-PIP cells. Better colocalization was achieved when 5D3 mAb was used as the pretargeting component. Consecutively, the in vitro cell viability study shows a significantly higher therapeutic effect of click therapy in PC3-PIP cells when 5D3 mAb was used for pretargeting, compared to its F­(ab′)2 derivative. 5D3 mAb has a longer lifetime on the cell surface, when compared to its F­(ab′)2 analogue, enabling efficient cross-linking with the drug delivery component and increased efficacy. Pretargeting and drug delivery components were cross-linked via multiple bioorthogonal click chemistry reactions on the surface of PSMA­(+) PC cells forming nanoclusters, which undergo fast cellular internalization and intracellular transport to perinuclear locations.

Alpha radioimmunotherapy using 225Ac-proteus-DOTA for solid tumors - safety at curative doses

“…Pretargeted radioimmunotherapy (PRIT) combines the desirable tumor-targeting features of antibodies 1 with the ideal phamacokinetics of small-molecule radiohaptens 2 for high-therapeutic index (TI) radioimmunotherapy (RIT) 3 . PRIT has been investigated for decades as an alternative to conventional RIT with radiolabeled-IgG; popular approaches include the strept(avidin)-biotin system, bispecific anti-tumor/anti-hapten antibodies (BsAb) and radiohaptens, antibody-oligonucleotide conjugates and radiolabeled complementary oligonucleotides, and, more recently, bioorthogonal approaches (e.g., inverse-electron-demand Diels-Alder reaction and adamantane/cucurbituril system) 4 - 11 .…”

Theranostic Pretargeting Drug Delivery and Imaging Platforms in Cancer Precision Medicine

“…The combination of 5D3(TCO) 8 and ALB(DM1) 3.3 (PEG 4 -Tz) 10 exhibited a selective and enhanced toxicity in PSMA(+) PC3-PIP cells compared to the combination of non-functionalized 5D3 and ALB(DM1) 3.3 (PEG 4 -Tz) 10 or treatment with a free DM1 or ALB alone. (* p < 0.05, ** p < 0.005) ( 22 ). (E) Pretargeting PET images of planar and maximum intensity projection (MIP), left and right, respectively in subcutaneous SW1222 tumor bearing nude mice.…”

“…(C) Confocal fluorescence microscope images of pretargeted theranostic approach in PSMA(+) PC3-PIP cells. Distribution of 5D3(TCO) 8 (AF-488) 2 (green), ALB(PEG 4 -Tz) 10 (Rhod) 2 (red), and Hoechst 33342 nuclear counterstaining (blue) (magnification × 100, bar: 30 μm) ( 22 ). (D) In vitro therapeutic study of 5D3(TCO) 8 .…”

“…Hapuarachchige et al ( 22 ) has reported a promising pretargeting theranostic approach for treating PSMA-overexpressing prostate cancer using 5D3 mAb. Here, 5D3 conjugated TCO and albumin conjugated with tetrazine have been used as pretargeting and delivery components, respectively.…”

“…It is important to note that combining a therapeutic platform with short-lived imaging probes presents significant problems because of the short lifetime of the preparation, and consequently, with the logistics of treatment administration and monitoring even if the medication can be formulated in a kit form for rapid radiolabeling ( 21 ). Therapeutic modalities used in theranostic nano-platforms include cytotoxic drugs ( 22 – 24 ), radioisotopes ( 25 ), optical absorbers for photothermal or photodynamic therapy ( 26 ), and phototherapy ( 27 ). Specific enzymes can be delivered to activate prodrugs in the context of prodrug therapy ( 28 ).…”