Antibody-Mediated Delivery of Chimeric BRD4 Degraders. Part 1: Exploration of Antibody Linker, Payload Loading, and Payload Molecular Properties

Abstract

The biological and medicinal impacts of proteolysis-targeting chimeras (PROTACs) and related chimeric molecules that effect intracellular degradation of target proteins via ubiquitin ligase-mediated ubiquitination continue to grow. However, these chimeric entities are relatively large compounds that often possess molecular characteristics, which may compromise oral bioavailability, solubility, and/or in vivo pharmacokinetic properties. We therefore explored the conjugation of such molecules to monoclonal antibodies using technologies originally developed for cytotoxic payloads so as to provide alternate delivery options for these novel agents. In this report, we describe the first phase of our systematic development of antibody–drug conjugates (ADCs) derived from bromodomain-containing protein 4 (BRD4)-targeting chimeric degrader entities. We demonstrate the antigen-dependent delivery of the degrader payloads to PC3-S1 prostate cancer cells along with related impacts on MYC transcription and intracellular BRD4 levels. These experiments culminate with the identification of one degrader conjugate, which exhibits antigen-dependent antiproliferation effects in LNCaP prostate cancer cells.

Site-Specific Antibody Conjugation to Engineered Double Cysteine Residues

“…However, there are needs for site-specific antibody conjugates with a DAR greater than two when other payloads besides cytotoxins are coupled. Currently, examples of site-specific conjugation for ADCs with a DAR greater than two are limited, and there is no information related to conjugation selectivity available [ 24 , 45 ]. In this work, we engineered twenty-seven single and thirty-eight double cysteine residues in the Fc region using site-directed mutagenesis.…”

“…Although highly-potent cytotoxins allow ADCs with a DAR of two, higher DAR conjugates may broaden the range of the efficacy towards cancer cells expressing low level of tumor-specific antigens [ 8 ]. There are also needs for antibody conjugates with high DAR when they are coupled with payloads other than cytotoxins [ 24 , 25 , 26 ].…”

Light-Controllable PROTACs for Temporospatial Control of Protein Degradation

“…One way to achieve targeted degradation of protein is to specifically deliver PROTACs into cancer cells, by taking advantage of the receptors expressed on the membrane of cancer cells, but not of normal cells. Recently, the antibody drug-conjugate (ADC) approach has been adopted for delivering PROTACs into cancer cells that expressing cancer-specific membrane-anchored receptors, such as HER2 ( Dragovich et al, 2020 , 2021a , b ; Maneiro et al, 2020 ; Pillow et al, 2020 ). A major disadvantage of antibody-conjugated PROTAC is its relatively high molecule weight and weak stability.…”

“…An alternative approach for controllable action of PROTACs in cancer cells could be taking advantage of cancer-specific receptors or transporters, such as HER2 and FOLR1 ( Scaranti et al, 2020 ) for the guided delivery of PROTACs into cancer, but not normal cells. To this end, other types of third generation PROTACs, including antibody-conjugated PROTACs ( Dragovich et al, 2020 , 2021a , b ; Maneiro et al, 2020 ; Pillow et al, 2020 ) and folate-PROTAC ( Liu et al, 2021 ), have been recently developed, which specifically deliver PROTAC to cancer cells, thus avoiding potential toxicity to normal cells. Compared with the light-controllable PROTACs, folate-PROTAC ( Liu et al, 2021 ) have relatively higher molecule weight of over 1,000 Da, and antibody-conjugated PROTACs ( Dragovich et al, 2020 , 2021a , b ; Maneiro et al, 2020 ; Pillow et al, 2020 ) are macromolecule drug that could only be administrated by injection.…”

“…To this end, other types of third generation PROTACs, including antibody-conjugated PROTACs ( Dragovich et al, 2020 , 2021a , b ; Maneiro et al, 2020 ; Pillow et al, 2020 ) and folate-PROTAC ( Liu et al, 2021 ), have been recently developed, which specifically deliver PROTAC to cancer cells, thus avoiding potential toxicity to normal cells. Compared with the light-controllable PROTACs, folate-PROTAC ( Liu et al, 2021 ) have relatively higher molecule weight of over 1,000 Da, and antibody-conjugated PROTACs ( Dragovich et al, 2020 , 2021a , b ; Maneiro et al, 2020 ; Pillow et al, 2020 ) are macromolecule drug that could only be administrated by injection. Taken together, further studies are needed to make these third generation PROTACs (light-controllable PROTACs, antibody-conjugated PROTACs and folate-PROTAC) more practical in clinic.…”