Balancing Antitumor Efficacy and Safety Profile: α-Amanitin-Based Fc-Small Molecule Drug Conjugates for Targeted Prostate Cancer Therapy

The tumor-specific delivery of cytotoxic drugs through antibody-drug conjugates (ADCs) represents a breakthrough in cancer therapy. However, besides high manufacturing costs, the utility of therapeutic ADCs may be limited by their immunogenicity and poor penetration into solid tumors due to their large size. The small molecules drug conjugates grafted with Fc-fragment (Fc-SMCs) were developed as alternative targeted therapies for prostate cancer treatment. The first generation of Fc-SMDCs comprises a binding moiety called DUPA which binds to the prostate-specific membrane antigen (PSMA) highly expressed on prostate cancer cells, Fc-fragment for half-life extension, and Amanitin as a toxin bearing a cleavable linker. This conjugate showed good in vivo efficacy; however, because of the low tolerability, the therapeutic index (TI) was low (=1). To increase the tolerability, this work focused on the unspecific uptake of the conjugates in healthy tissues and their plasma stability. Previous studies with ADCs proved that introducing silencing mutation in the Fc-backbone reduce Fcγ-mediated uptake in the liver. However, despite the reduced binding affinity of the Fc-backbone to FcγRI, tolerability did not improve, and biodistribution studies indicated preferential kidney accumulation of the toxin metabolites rather than liver accumulation. To increase the plasma stability of the Fc-SMDC, the cleavable linker was substituted by a non-cleavable linker. For this purpose, THIOMAB conjugation was used, enabling the attachment of the linker payload to a specific amino acid within the Fc region. The rationale behind this choice of conjugation relates to the first generation of Fc-SMDCs, where the conjugation of Amanitin bearing non-cleavable linkers through click chemistry in proximity to the binding moiety led to the release of inactive Amanitin metabolites. THIOMAB-Fc-SMDCs with cleavable (cl) and non-cleavable linker (ncl) showed full-blown in vitro cytotoxicity on target-positive PCa cell lines, while no cytotoxicity was observed on target-negative cells. The tolerability of the THIOMAB-Fc-SMDC-ncl was 15 times higher (15 mg/Kg) than the THIOMAB-Fc-SMDC-cl (1 mg/Kg). Both THIOMAB-Fc-SMDCs with cleavable or non-cleavable linkers showed a similar pharmacokinetic (PK) profile in the liver, kidney, and tumor tissue, and the high level of Amatoxin metabolites in the tumor indicated a target-specific uptake and release of the drug. Despite the similar PK profile, treatment of LNCaP xenografted mice with THIOMAB-Fc-SMDC-ncl showed complete tumor remission at the corresponding dose of ¼ MTD, while treatment with THIOMAB-Fc-SMDC-cl showed only tumor growth inhibition at the corresponding dose of ¾ MTD. THIOMAB-Fc-SMDC-ncl exhibited tolerance up to 4 mg/kg in cynomolgus monkey, administered weekly for three weeks, resulting in a TI of 8, comparable to anti-PSMA ADCs bearing the same linker payload. In comparison to the first generation of Fc-SMDC, the TI demonstrated a notable improvement from 1 to 8 with the introduction of the non-cleavable linker and the THIOMAB conjugation. These promising results suggest that the use of THIOMAB-Fc-SMDC, featuring the non-cleavable linker, could potentially pave the way for a novel, effective treatment of castration-resistant prostate cancer.

Level thesis:degree_level

thesis.doctoral

Grantor dc:publisher

Universität Bielefeld

Year

2024

Author dc:creator

• Carraturo, Daniela

Repository record source_url

https://pub.uni-bielefeld.de/record/3004079

OAI identifier oai:identifier

oai:pub.uni-bielefeld.de:3004079