Unsatisfactory physicochemical properties of macromolecular drugs seriously hinder their application in tumor immunotherapy. However, these problems can be effectively solved by small-molecule compounds. In the promising field of small-molecule drug development, proteolysis targeting chimera (PROTAC) offers a novel mode of action in the interactions between small molecules and therapeutic targets (mainly proteins). This revolutionary technology has shown considerable impact on several proteins related to tumor survival but is rarely exploited in proteins associated with immuno-oncology up until now. This review attempts to comprehensively summarize the well-studied and less-developed immunological targets available for PROTAC technology, as well as some targets to be explored, aiming to provide more options and opportunities for the development of small-molecule-based tumor immunotherapy. In addition, some novel directions that can magnify and broaden the protein degradation efficiency are mentioned to improve PROTAC design in the future.
The recent decades witnessed the bloom of tumor immunotherapy [1,2,3]. The central aim of immunotherapy is to harness autologous immune responses for tumor elimination [1,4,5,6,7,8]. Distinct from conventional approaches such as surgery, chemotherapy, and radiotherapy, the modulation of the immune system can lead to abscopal and long-lasting therapeutic consequences, therefore preventing tumor recurrence and metastasis [9,10,11]. Hitherto, most clinically approved immune-intervening agents are macromolecules, such as blockade antibodies, engineered immune cells, oncolytic viruses, cytokines/chemokines, and vaccines [2,10,12]. Though benefiting from treatments, there are some problems with these drugs [9,13]. Firstly, because of the low oral bioavailability, macromolecular drugs are often intravascularly administrated, leading to poor patient compliance [2,9]. Secondly, macromolecular drugs exhibit a low clearance rate and a long half-life, resulting in uncontrollable organ distribution and pharmacokinetics [13,14]. Thirdly, macromolecular drugs have the poor capability in tissue penetration and transmembrane transport, which seriously hinders their effects in dense solid tumors [14]. Lastly, macromolecular drugs bear a risk of eliciting immune-related adverse events (irAEs) [13].
As the dominant proportion in classical antitumor therapies, small-molecule drugs offer an ideal approach to addressing the above problems [15]. Several reviews have recapitulated the advantages of small molecules over macromolecules in immuno-oncology, which can be attributed to the following aspects: (1) small molecules can be orally administrated [14,15]; (2) small molecules are eliminated more rapidly than macromolecules, which allows for precise prescription and flexible treatment regime [14]; (3) small molecules had better membrane permeability than macromolecular drugs, so they can act on intracellular targets to orchestrate intricate signal pathways [9,15]; they are also more capable of overcoming extracellular barriers, favoring drug accumulation in dense tumor tissues [15]; (4) small molecules take lower cost in manufacture, storage, transport, and medication than macromolecular drugs [9].
Proteolysis targeting chimera (PROTAC) is a leading field in the discovery of small-molecule drugs [16,17]. A ubiquitin-proteasome system (UPS) is the natural machinery for the degradation of cellular proteins [18]. Ubiquitin is transferred to the surface lysine of substrate proteins by a cascade of three enzymes: ubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes (E2) and ubiquitin-protein ligases (E3), wherein the E3 ligase play pivotal roles [16,18,19]. Ubiquitin also contains lysine residues that can be consecutively ubiquitinated to polyubiquitin chains. Subsequently, the polyubiquitin-tagged proteins are recognized by proteasomes for degradation. The major concept of PROTAC is to hijack UPS to specifically degrade pathogenic or abnormally overexpressed proteins [18]. Since firstly proposed in 2001 by Craig M. Crews, this technology has revolutionized the mechanisms of drug-protein interactions in the past two decades [16,18,19,20]. Most compounds competitively bind with the catalytic site, inhibit kinase phosphorylation, or allosterically decrease the activity of functional proteins [21,22,23,24].
On the basis of these interactions, PROTAC molecules bring a protein of interest (POI) into degradation [16]. PROTACs are heterobifunctional compounds that consist of three moieties [19,25]: (1) a warhead for POI to be degraded, (2) a ligand to engage the E3 ubiquitin ligase, and (3) a flexible linker joining them. Two ends of the PROTAC molecules bind with their targets, respectively, forming a POI-PROTAC-E3 ligase ternary complex [26]. After that, the POI is ubiquitinated and degraded by the proteasome ( ) [16,18].
Nowadays, more than 600 E3 ligases have been identified. Small molecule ligands for some of these E3 ligases, such as mouse double minute 2 (MDM2), cell inhibitor of apoptosis (cIAP), von Hippel–Lindau protein (pVHL), Cereblon (CRBN), Kelch-like ECH-associated protein 1 (KEAP1), aryl hydrocarbon receptor (AhR), DDB1-CUL4 associated factor 11/15/16 (DDAF11/15/16), ring finger protein 4/144 (RFN4/144) and fem-1 homolog B (FEM1B) have also been discovered [27]. Among these various E3 ligase ligands, CRBN and VHL ligands are the best options in PROTAC design because of several aspects [28,29]. Firstly, CRBN and VHL are widely and abundantly expressed in multiple tumor cells, which guarantees the degradation efficiencies of CRBN- and VHL-based PROTACs. Secondly, CRBN and VHL ligands are easy to synthesize, and their molecule weights are relatively low, exhibiting good drug-like properties [30]. Finally, the safety and biocompatibility of these two kinds of ligands have been extensively evaluated in vivo.
PROTAC technology has many advantages over conventional small-molecule inhibitors [13,25]. Undruggable proteins refer to proteins without well-defined binding pockets, which occupy 80% of disease-associated proteins [19]. These proteins, including non-enzymatic proteins, scaffolding proteins, and transcription factors, are not reactive to small-molecule inhibitors but can be degraded by PROTACs [19,31]. That is because PROTAC molecules can bind to anywhere of the POI with moderate affinity for protein degradation, but classical small-molecule inhibitors need to interact with the binding pocket with high affinity for function [16,32]. Moreover, tumor cells easily acquire resistance via protein mutation after long-term drug exposure, which leads to treatment failure. Studies showed that drug-resisting tumor cells are resistant to small-molecule inhibitors but vulnerable to PROTAC compounds, denoting the capability of PROTAC to obviate drug resistance [26]. To suppress protein activity, small-molecule inhibitors have to permanently couple with their targets. Eliciting a stoichiometric drug response [25]. In contrast, noncovalently tethered PROTAC molecules can be recovered and joined into the next circulation soon after protein degradation by the proteasome [19,25]. Collectively, as the next generation of small-molecule therapies, PROTAC-based drugs are expected to thoroughly replace macromolecular therapies in tumor immunotherapy [25,32,33,34].
Despite the attractive prospect of applying PROTAC to immuno-oncology, few studies have focused on this issue, which might be attributed to the immature concepts and approaches. There are some existing reviews that refer to targeted protein degradation (TPD) strategies for tumor immunotherapy, but they merely focus on molecule designs and their applications in programmed cell death 1 (PD-1)/programmed death ligand 1 (PD-L1) immune checkpoint [33,34]. The influence of PROTAC molecules in multiple events within the tumor microenvironment (TME), such as signal crosstalk, antigen presentation, immune cell invasion and tumor immunogenicity, are not mentioned in these review articles [4,33,34,35,36]. Thus, it is required to summarize the current advances and discover more targets in PROTAC-mediated tumor immunotherapy.
To this end, this review gathered the application of PROTACs for immune-related targets in recent three years ( ), including the PD-1/PD-L1 checkpoint and its regulatory pathways (SHP2 and BET), vital processes in tumorigenesis such as metabolism (IDO1), epigenetic modification (HDAC), and apoptosis (Bcl-2 family), as well as immuno-modulating signals (STAT3 and MAPK), and also some targets which have not or rarely treated by PROTAC molecules (CD47, Foxp3, COX-1/2, NAMPT, and TGF-β1). It should be noted that most of these targets and PROTACs were summarized by Rao et al. in recent reviews [28,37]. Thus, this review will specifically focus on the immunological consequences after the degradation/inhibition of these targets, aiming to bias the development of small-molecule PROTACs for immunomodulation rather than simple tumor killing. In this article, we hope to provide comprehensive knowledge about the principle of PROTAC-mediated immune intervention and boost the discovery of versatile PROTAC molecules for tumor immunotherapy.