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Degrader-antibody conjugates (DACs) are a new class of therapeutic agents that have garnered significant interest in recent years. DACs are composed of an antibody that targets a specific protein on the surface of cancer cells, and a small molecule degrader that binds to the targeted protein and induces its degradation. The goal of DACs is to selectively destroy cancer cells while sparing healthy cells, thereby improving the safety and efficacy of cancer therapy. Currently, there are several DACs in preclinical and clinical development. One of the most advanced DACs is ARV-471, which targets the estrogen receptor (ER) in breast cancer cells. ARV-471 has shown promising results in preclinical studies and is currently being evaluated in a Phase 1 clinical trial. Other DACs in development target a variety of cancer-associated proteins, including BCL-2, BRD4, and FLT3.
Degradation-antibody conjugates (DACs) are novel entities that combine a proteolytic targeting chimera (PROTAC) payload with a monoclonal antibody via a certain type of chemical linker (DAC=PROTAC+ADC). DACs consist of antibodies that bind specific protein targets and small molecule degraders that induce degradation of the target protein. This approach allows for the precise targeting of disease-causing proteins, which may lead to more effective and selective treatments of various diseases, such as cancer and autoimmune diseases. DAC is currently being developed and studied in preclinical and clinical trials. Typically, a DAC has the following characteristics:
(1) DAC consists of mAb, linker and PROTAC payload.
(2) Match target antigen expression to degraded payload cell biology.
(3) PROTAC payloads must be stable in lysosomes and able to escape safely.
(4) DAR >4 may be required, which may affect conjugation and DAC pharmacokinetics.
Fig. 1. Schematic diagram of the DAC structure.
Chimeric protein degraders, commonly referred to as proteolytically targeting chimeras (i.e., PROTACs), are rapidly changing the fields of biology and medicinal chemistry. These heterobifunctional molecules are capable of specifically degrading target proteins within cells, thus they have the potential to improve and/or prolong biological activity relative to simple small molecule inhibitors of the same entity. PROTACs generally consist of a binding element that recognizes the target protein, a separate part that binds the E3 ligase, and a spacer that connects the two components (Fig. 2). Due to the chimeric nature of PROTACs, heterobifunctional degradants often possess physicochemical properties that can render related molecules with poorer DMPK properties, lower oral bioavailability, and/or rapid clearance in vivo. In addition, PROTACs also have problems such as large molecular weight (700-1100Da) and less available E3 ubiquitin ligase. Therefore, the development of PROTACs has been limited.
Fig. 2. General PROTAC structure.
As an alternative to the efficient delivery of chimeric degraders in vivo, researchers have explored the novel technique of conjugating antibody-drug conjugates (ADCs) with cytotoxic payloads to PROTACs, i.e. degrader-antibody conjugate (DAC). DACs have several potential advantages over unconjugated PROTAC molecules for in vivo administration:
(1) In vivo delivery of chimeric degradants with poor physicochemical and/or DMPK properties.
(2) Avoiding complex and/or nonstandard formulations that are often required to achieve meaningful in vivo exposure of unconjugated PROTACs.
(3) The ability to target the PROTAC molecule of interest to a specific tumor or tissue through the antigen recognized by DAC.
The principle of ADC and DAC is basically the same (Fig. 3). After administration, the entire DAC should be kept as stable as possible to prevent premature release of PROTACs and blood circulation. First, the antibody portion of DAC recognizes tumor-associated antigens on the cell surface. Second, DAC-antibody complexes are internalized by receptor-mediated endocytosis. Inside the cell, the complex fuses with endosomes and is transported to activated lysosomes. Under proteolytic and acidic conditions, the complex linker is degraded and the cargo (PROTAC) is subsequently released into the cytoplasm. Depending on the DAC intracellular target, protein degradation events can be induced.
Fig. 3. The mechanism of action of the DAC.
Due to the critical role of BRD4 as a transcriptional and epigenetic regulator during cancer development, studies have drawn great attention to novel BRD4 degradants as well as BRD4 inhibitors. The first reported DAC consisted of a monoclonal antibody (mAb) targeting C-type lectin-like molecule-1 (CLL1) and BRD4 degrader 1, GNE-987. Here, they discovered a potent BRD4 degrader for acute myeloid leukemia (AML) by introducing a dihydropyrrolopyridone scaffold on a BET-binding fragment. Although compound 1 has efficient BRD4 degradation ability and strong anti-tumor cell proliferation effect, the unfavorable DMPK properties in vitro and in vivo affect the further effect.
Fig. 4. Design of BRD4-DAC targeting CLL1.
Because the CLL1 antigen is highly expressed on the surface of AML cells, the researchers developed another novel approach to target CLL1-DAC in an AML xenograft model. They employed a "direct disulfide" conjugation strategy, using a methylthiosulfonyl (MTS) group containing an activated disulfide bond to couple to six cysteine residues (DAR≈6.0) on a CLL1-targeting antibody, and there is no significant aggregation problem (Fig. 4). It was found that chimeric degradants were enhanced by antibody conjugation strategies, overcoming the poor pharmacokinetic properties of degradants. Furthermore, pharmacokinetic evaluation of DAC 2 revealed a high level of in vivo stability.
In addition to DACs targeting BRD4, Dragovich et al. also reported two different Erα-targeted degradants 3 and 5. Degrader 3 consists of the selective estrogen receptor modulator tamoxifen and the ligase ligand XIAP, and shows a strong effect on degrading Erα. Based on the degradation agent 3, the researchers used the hydroxyl group of tamoxifen as the linking site, and used the valine-citrulline-p-aminobenzyloxy linkage chain to combine with the HER2 monoclonal antibody to obtain the degradation agent 4 (Fig. 5), and successfully demonstrated the degradation of the target protein.
Fig. 5. DAC targeting Erα with an enzymatically cleavable linker.
Based on the above studies, the researchers further used the VHL ligand-based PROTAC 5 to synthesize a new type of Erα DAC targeting HER2. They chose the hydroxyl group of VHL as the attachment site, and applied the carbonate-based disulfide linker and the pyrophosphodiester-based linker to the corresponding DACs 6 and 7 (Fig. 6). However, these degradants exhibit moderate stability and further optimization is required to improve stability.
Fig. 6. DAC targeting Erα with disulfide and phosphatase-cleavable linkers.
The BRM protein, also known as SMARCA2, is widely found in various human diseases, including cancer. Degradant 8, originally developed by Arvinas and Genentech in 2019, is a selective degrader for BRM. Later, Genentech further developed a degradant 8-based DAC 9, which mainly covalently linked degradant 8 and a monoclonal antibody that selectively binds to CD22 cell surface receptors through a disulfide linker chain (Fig. 7). In addition, subsequent xenograft tumor transplantation experiments proved that DAC 9 can achieve efficient and antigen-dependent degradation of BRM proteins.
Fig. 7. DAC targeting BRM with disulfide linker.
The future prospects of DACs are promising, as they offer several advantages over traditional cancer therapies. DACs are highly specific, meaning they can selectively target cancer cells while sparing healthy cells. This can reduce the side effects associated with traditional chemotherapy and improve patient outcomes. Additionally, DACs can overcome drug resistance, which is a major challenge in cancer therapy. By inducing protein degradation, DACs can eliminate cancer cells that have become resistant to traditional therapies. However, there are also challenges associated with the development of DACs. One of the major challenges is identifying the right target protein and degrader combination. This requires a deep understanding of the biology of cancer cells and the mechanisms of protein degradation. Additionally, there are technical challenges associated with the synthesis and delivery of DACs, which need to be addressed to ensure their safety and efficacy.
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