Prostate cancer remains the 4th most common cancer worldwide and the 2nd most common

cancer in men. The Prostate Specific Membrane Antigen (PSMA) attracted a great attention

as a target for the development of personalized therapeutics. Out of two PSMA targeted

antibody-drug conjugates (ADCs) in clinical development none up to the moment reached

the market approval due to limited efficacy or dose limiting toxicities. One of the main

limitations in the use of ADCs in solid tumor therapy seems to be restrained tumor

penetration due to the high molecular size of this class of therapeutics. In this work a library

of small molecule drug conjugates (SMDCs) as an alternative for large molecular ADCs was

developed and tested in vitro and in vivo. As a targeting moiety providing PSMA specificity

the well-known PSMA ligand 2- [3-(1, 3-dicarboxy propyl) ureido]pentanedioic acid (DUPA)

was used. The targeting moiety was followed by supporting spacer providing DUPA

positioning in the PSMA active site and enabling its conjugation to a cytotoxic payload

α-amanitin which is the inhibitor of RNA Polymerase II. The inhibition of RNA Polymerase II

represents a completely new mode of action distinct to other toxins currently used for ADCs

and SMDCs development. The most potent DUPA-α-amanitin conjugates were identified:

HDP 30.2284 (bearing Val-Ala-PAB linker), HDP 30.2301 (bearing C6 non-cleavable linker) and

HDP 30.2618 (bearing disulfide mono-hindered linker). Despite of an excellent in vitro profile,

the lead SMDCs have shown only limited in vivo efficacy due to their very short half-life and

limited tumor accumulation. In order to improve pharmacokinetic properties, the DUPA

targeting moiety and the toxin were conjugated to an Fc portion of human IgG. DUPA-Fc-αamanitin conjugate showed slightly lower in vitro activity compared to the SMDCs. In vivo

a significantly prolonged half-life and complete tumor remission in PSMA positive LNCaP

xenograft model was observed. These results indicate that for highly hydrophilic toxin such

as α-amanitin the gradual tumor delivery and accumulation over time is essential for

sustainable anti-tumor activity. Describe herein novel DUPA-Fc-α-amanitin conjugate

enriches the current landscape of ADCs based on the scaffold with a molecular weight being

approximately 60 % smaller than full format IgG. Additionally, DUPA-Fc-α-amanitin retains

prolonged half-life which is characteristic for antibodies. Moreover, proposed herein Fc-small

molecule-toxin platform creates the opportunity to optimize small-molecule-toxin

conjugates with sub-optimal pharmacokinetic profile.