Introduction
Degradation-Antibody Conjugates (DACs) are a new type of entity that effectively combines the protein hydrolysis-targeting chimeric (PROTAC) payload with monoclonal antibodies through some type of chemical linker (DAC =PROTAC + ADC). This article collects several examples of DACs from scientific and patent literature and documents specific challenges associated with DAC construction. Overall, these examples indicate that various PROTAC payloads can be successfully used to prepare biologically active DACs.
Figure Caption: Schematic Diagram of DAC Structure
Some Key Points of DAC
(1) DAC consists of mAb, linker, and PROTAC payload.
(2) Match the expression of target antigens with the degradation payload cell biology.
(3) The PROTAC payload must be stable in lysosomes and able to escape safely.
(4) A DAR > 4 may be required, which may affect the conjugation and pharmacokinetics of DAC.
Introduction to PROTAC
Before elaborating on DAC, we need to familiarize ourselves with what PROTAC is. Have you already learned about this trending technology?
PROTAC, or “Proteolysis Targeting Chimeras,” is rapidly transforming the fields of biology and medicinal chemistry. This bifunctional molecule can specifically degrade target proteins inside cells, thus having the potential to improve and/or prolong biological activity compared to simple small-molecule inhibitors of the same entity.
The structure of PROTAC is shown in the figure below: it typically consists of a binding element that recognizes the target protein, a part that binds to E3 ligase, and a spacer group that connects these two components (For specific mechanisms, please refer to “Ubiquitination and Degradation”).
However, PROTAC has poor DMPK characteristics, such as low oral bioavailability and/or rapid in vivo clearance. This has become a major factor hindering the development of PROTAC, along with issues like larger molecular weight (700-1100 Da) and fewer available E3 ubiquitin ligases.
A DAC Targeting BRD4
In early 2020, Genentech described a highly efficient DAC conjugate based on VHL for degrading bromodomain-containing protein 4 (BRD4) (GNE-987), connected by a cleavable linker containing a disulfide bond between ADC and PROTAC, targeting the protein CLL1 (C-type lectin-like molecule-1), with a DAR of 6.
The mechanism of action of this DAC may be:
The antibody portion of DAC binds to the CLL1 receptor on the surface of tumor cells, after which the conjugate is internalized and transported to lysosomes (Step 1). In this proteolytic environment, the antibody is broken down into amino acid components, leaving a cysteine residue that is partially attached to the remainder of the linker via a disulfide bond (Step 2). The disulfide bond is then reduced (Step 3) to provide the corresponding thiol, which undergoes self-immolation to release the degradation agent 1 to exert its effect (Step 4).
In the HL-60 (left) and EOL-1 (right) xenograft models, after a single intravenous administration, control experiments showed:
(1) Unconjugated CLL1 mAb showed no significant activity in these models. (2) CLL1 degradation agent conjugate with GNE-987 hydroxyl proline diastereomer payload also showed no activity. (3) The correctly structured DAC targeting CLL1 exhibited dose-dependent activity, with significant tumor suppression. (4) Unconjugated GNE-987 showed no activity.
Figure Caption: Green Trace: Degradation Agent-mAb Conjugate; Purple Trace: Compound Diastereomer DAC
Blue Trace: mAb Black Trace (dashed): Unconjugated Compound
Another DAC Targeting BRD4
Shortly after the appearance of the above DAC, University College London published a second example of a BRD4 degradation-antibody conjugate. They used dibromomaleimide reaction to reduce the interchain disulfide bonds of mAb to generate cysteine residues, which were used to connect four copies of the relevant linker-drug through a thiol rebridging method.
They connected the VHL-based PROTAC payload to an uncleavable linker using an ester moiety (yellow oval), and completed the final linker structure cycloaddition (SPAAC) reaction using copper-free, strain-promoted azide-alkyne cycloadditions. After the conjugate is delivered to the lysosome, intracellular effective payload release can occur through ester cleavage. The antibody targets HER2.
In vitro experiments showed that this DAC provided dose-dependent BRD4 degradation in two examples of HER2-positive cells, but not in two HER2-negative control cell lines.
Furthermore, fluorescence labeling observed DAC internalization in HER2-positive cells, followed by transport to lysosomal compartments. No relevant internalization and transport were detected in HER2-negative cells, consistent with the lack of degradation activity observed in experiments using these cell lines.
Future Outlook
1. Although the field of degradation-antibody conjugates DAC is still in its infancy, various such entities have been created, and the validated DACs show meaningful in vitro and/or in vivo biological activity.
2. In the generation of these conjugates, PROTACs binding to several different E3 ligases were used, which degraded several well-differentiated target proteins (Table 1). Additionally, many different novel linkers and mAb conjugation methods were employed to construct the described DAC (Table 1).
3. Compared to most known cytotoxic ADCs (DAR = 2 to 4), many of these new entities adopt a higher payload load (DAR value of 6), but whether such increases are generally required for typical DAC applications remains to be determined.
4. Challenges remain in determining which PROTACs are suitable for DAC conjugation and how to best maintain (and ideally enhance) the biological activity of the selected chimeric degradation agents.
References:
[1] Dragovich PS. Degrader-antibody conjugates. Chem Soc Rev. 2022 May 23;51(10):3886-3897. doi: 10.1039/d2cs00141a. PMID: 35506708.
[2] Antibody Conjugation of a Chimeric BET Degrader Enables in vivo Activity. ChemMedChem. 2020 Jan 7;15(1):17-25. doi: 10.1002/cmdc.201900497.
[3] M. Maneiro , N. Forte , M. M. Shchepinova , C. S. Kounde , V. Chudasama , J. R. Baker and E. W. Tate , ACS Chem. Biol., 2020, 15 , 1306.
Scan the WeChat QR code to add the editor of the bioproduct circle, and those who meet the criteria can join the bioproduct WeChat group!
Please specify: Name + Research Direction!
All articles reprinted by this public account are for the purpose of conveying more information, and the source and author are clearly indicated. Media or individuals who do not wish to be reprinted can contact us ([email protected]), and we will immediately delete them. All articles only represent the author’s views and do not represent the position of this site.
