The Potential Impact of New Drug and Therapeutic Modalities on Drug Resistance to Renal Cell Carcinoma

Resistance to Targeted and Immune Therapy

As small molecules, TKI are subjected to the activity of membrane-drug transporters that can dictate their intracellular concentrations and impact their efficacy (10). RCC-approved TKI including sunitinib, sorafenib, and axitinib are substrates for efflux transporters known to the expressed, and active, in RCC cells, namely P-glycoprotein (P-gp) and the Breast cancer resistance protein (BCRP) (11-13). Drug transport activity is believed to be a contributing factor to intrinsic drug resistance, alongside a reduction in apoptotic activity, where TKI therapy fails to yield any effect. This coincidental effect is still poorly understood and is a feature of advanced RCC (14). Acquired resistance to TKI, which reverts the initial benefits of the therapy, can manifest in different forms. Tumor cells can switch the activation of their angiogenic pathways and rely on networks that bi-pass the pro-angiogenic TKI-targets such as VEGFR and PDGFR. Indirectly, RCC can recruit proangiogenic inflammatory cells (e.g., monocytes) that can stabilize the tumor vasculature by increasing pericyte density. This is facilitated by endothelial exposure to the hypoxic environment characteristic of RCC (15). Moreover, RCC cells can promote epithelial-to-mesenchymal transition (EMT) in healthy epithelial cells to further progress tumor proliferation, opening new fronts for drug resistance in the tumor (16). TKI-resistance illustrates the extent that RCC cells are equipped to secure their angiogenic capabilities, and consequently their proliferation potential.

ICI are large biological molecules, with distinct pharmacokinetic properties relative to small-molecule drugs. With a serum half-life measured in weeks instead of hours, efficacy and acquired drug resistance are of much greater concern than drug disposition (17). Nivolumab, pembrolizumab, ipilimumab, and avelumab are ICI approved for the treatment of mRCC (18). The inflammatory and hypoxic nature of the RCC tumor microenvironment (TME) represents a double-edged sword when it comes to the activity of ICI. The RCC microenvironment is prolific in infiltrated immune cells, including lymphocytes, macrophages, and T-cells. These tumor-infiltrating immune cells (TIIC) are believed to be recruited to the TME by the secretion of an array of soluble immune factors. TIIC play a role in containing RCC progression by modulating cellular proliferation (19). However, RCC can rely on intricate immune regulatory machinery, involving the interplay of tumor cells and TIIC, to secrete a cytokine barrier that suppresses the activity of T-cells. Together with an extensive display of immune checkpoints on their membranes, RCC cells become stealthy and can bypass the immune response (20). ICI counteract this mechanism. Nonetheless, the activity of the cytokine barrier and its up-regulation in response to external stresses can inhibit large swaths of T-cells populations in the TME, rendering ICI ineffective. Several intrinsic pathways have been identified in both the regulation of cytokine and checkpoint expression. The mitogen-activated protein kinases (MAPK) and the PI3K/AKT/mTOR pathways are associated with the release of immunosuppressive cytokines (21, 22). Cyclin-dependent kinase 4 (CKD4) is a cell-cycle regulator whose deregulation is related to the proliferation of tumor cells. The inhibition of CKD4 activity has been associated with enhanced T-cell activation and respective anti-tumor activity; however, the mechanisms behind this interaction and its relation to immune checkpoint regulation remain unclear. The loss of major histocompatibility complex (MHC) antigens is another factor that diminishes the ability of T-cells to recognize RCC cells. This deregulation is associated with mutations in MHC genes in tumor cells, as their malignant phenotype progresses (22). Arguably, the TME plays a major role in the resistance to ICI. TIIC, tumor vasculature, immunosuppressive cytokines, and pro-angiogenic factors all interact in a matrix with varying levels of fibrosis and in itself a hypoxic environment that further impacts TIIC activities (23, 24). Overall, to be effective, cytotoxic T-cells have to navigate the intricacies of a hostile TME to recognize RCC cells actively masking their identity. Hence, ICI are a piece of a wider puzzle.

Both TKI and ICI are approved as front-line monotherapies for RCC, governed by stringent guidelines, with the selection of treatment and dosing regimens dictated by the clinical frameworks of individual patients. Unsurprisingly, TKI-ICI combination therapies were considered and developed from the inception of ICI clinical use, considering the added benefits of targeting RCC from both fronts (25). Nowadays, anti-VEGF-ICI combinations have proven more effective than TKI or ICI monotherapies in augmenting patient survival rates (26). By directing drugs against immune suppression and angiogenesis, two of the main survival mechanisms of RCC are engaged simultaneously (27). Clinical evidence also shows that TKI therapy initiated immediately after ICI treatment, in patient with poor response to immunotherapy, can be successful in stabilizing RCC progression (28, 29). Further, TKI with multi-kinase activity, in particular those targeting the MAPK, can also suppress pathways driving the secretion of immunosuppressive cytokines. In these combination therapies, TKI alter the dynamics of the TME, offering a line of sight for ICI-primed T-cells to engage and suppress RCC cells. The combination of two ICI – dual therapy – has also recently shown added benefit in the treatment of advanced RCC. This treatment doubles down on ICI activity, by targeting two separate checkpoint receptors in the T-cell using different ICI, hence amplifying the immune response towards the cancer cells (30).

The side effects of mono and combination ICI therapies are reasonably manageable throughout the treatment phases. However, dual ICI therapy can cause severe side effects in certain patients, with damage observed in the liver and lungs. These off-target toxic effects are comparable to an autoimmune reaction originating from a substantial up-regulation of cytotoxic immune responses (31). Therefore, understanding the patient-specific factor that leads to such adverse reactions will prove very beneficial in the selection of treatment. By improving mRCC treatment, TKI and ICI combination therapies can allegedly counteract certain aspects of the drug resistance experienced by monotherapies. Currently, the molecular inner works of these pharmacological interactions are not fully understood (22). Nonetheless, the development and implementation of TKI and ICI, and subsequent applications is a testimony to the advances made in tumor-immunology and our understanding of RCC pathophysiology. Current treatments under clinical development are mainly focusing on combinations of TKI and ICI to boost patient survival and potentially deliver curative effects (32); nonetheless, novel classes of drugs are being considered.

The Next Generation

As TKI and ICI have shown, drugs with a high target specificity are critical to enhancing efficacy and therefore, positive clinical outcomes. A significant proportion of the research portfolio of both established pharmaceutical enterprises as well as new ventures are now dedicated to the development of so-called new drug and therapy modalities (NDTM) (33-36). This non-official designation has acted as an umbrella term to describe mainly experimental molecules and therapies that have physiochemical, pharmacokinetics, and biological properties substantially different from traditional small molecules. Widely varying in their nature, NDTM are often large molecules, and even cells, that offer high target specificity with potential marginal toxicity (37). ICI fit under the designation of an NDTM, nevertheless, ICI are biologicals, a class of drugs approved for clinical use for over 20 years. NDTM is the term usually employed to describe classes of drugs and treatments considered experimental, also referred to as next-generation therapeutics.

The advent of multi-omics, automated workflows, and advanced in vitro models, to name a few tools now commonplace in biomedical research, has helped to fast-track the identification of new therapeutical targets as well the development of new drugs (38, 39). NDTM are being developed virtually across all therapeutic areas, some with a focus on conditions with no previous effective treatment (e.g., chronic kidney disease, Duchenne muscular dystrophy) (40). mRCC have also been the focus of much interest, with drugs aimed at further characteristic features of RCC.

Anti-cancer Vaccines

Cancer-based vaccine applications have been under development since the late XX century. These vaccines are designed to enhance the cancer-directed immune response by priming the immune-system to recognize neoantigens (41). The mutational turnover in developing tumors generates proteins unique to individual cancers – neoantigens – that are also expressed in cell surfaces. Neoantigens are identified as foreign elements by T-cells, contrary to MHC antigens, and can initiate an immune cascade. mRCC is a prominent cancer to employ a vaccine given its stable mutation rate and high proportion of neoantigens (42). Although pre-clinical studies have shown that vaccines encoding neoantigens can amplify T-cell mediated tumor cell death, poor tumor infiltration by immune cells, inhibition of T-cell by cytokines activity, and the turn-over of neoantigens are limiting factors that significantly hamper an efficacious response under normal circumstances (43).

To overcome these limitations, the combination of mRNA-vaccine and ICI is being considered. This approach aims at amplifying the activity of ICI by recruiting a larger population of T-cells to the TME. The potential benefits of this synergy include a prolonged ICI activity, where these CP blockers affect a high-density of T-cells, and overcoming poor ICI patient response by directing T-cells directly to the tumors, evading the immune-suppressive microenvironment. The deployment of neoantigen vaccines requires a personalized medicine strategy. The tumor-specific nature of antigens requires the vaccines to be patient-specific as well (44). An ongoing clinical trial (NCT02950766) is set to establish the effectiveness of a personalized vaccine – NeoVax – and Ipilimumab.

Despite the potential benefits, several challenges remain in the implementation of this type of co-therapy. Patient-specific vaccines will require a dedicated approach where tumor specimens are analyzed and vaccines designed to carry neoantigen epitopes, in an adequate timeframe. mRNA vaccines, similar to those brought into the spotlight during the Sars-CoV2 global pandemic, could also be employed to code neoantigens (45). Considering that neoantigen turnover can render vaccines ineffective, predictive tools can be used to maximize epitope identification. In the near future, clinical evidence will dictate the feasibility of such an approach, which can potentially represent another major step toward a curative treatment for mRCC.

Anti-sense Oligonucleotides

Anti-sense oligonucleotides (ASO) are naked, single-stranded, oligonucleotide sequences with chemical modifications to their backbones that confer these molecules high biological stability. Nowadays, tailored chemical modifications are used to imprint ASO with a large spectrum of properties. In general, ASO have limiting nuclease degradation, which together with their size, contributes to limited tissue penetration and distribution (46). On the other hand, ASO have slow elimination rates and tend to accumulate in the organs they reach, albeit to limited concentrations (47). ASO bind and promote the degradation of mRNA precluding protein translation or promoting alternative splicing, hence their ability to virtually target any protein of interest with very high specificity. It is believed that thanks to their accumulation and protracted degradation these molecules can exert their pharmacological effects for extended periods. Nowadays, a limited number of ASO have been approved for clinical use, namely neurodegenerative diseases (48). This is exemplified by the treatment of spinal muscular atrophy (SMA), a previously untreatable condition, with Nusinersen, an ASO that recovers the expression of a defective neuron motor protein by alternative splicing (49). Nonetheless, significant safety and efficacy considerations remain during ASO development, namely the long-term implications of tissue accumulation and potential off-target toxic effects.

Initial studies demonstrated that a phosphorothioate cross-linked ASO against VEGF mRNA was effective in reducing VEGF protein levels and limiting RCC cell proliferation in vitro. In RCC xenografts, the angiogenic activity and tumor size substantially diminished in nude mice treated with this ASO (50). Specifically targeting cellular proliferation via ASO has also been shown to halt proliferation in RCC xenografts. An anti- MKI67 (marker of proliferation Ki-67) ASO reduced the development of metastasis in mice, leaving tumor vessel density unchanged (51). Human trials of MG98, an anti-DNA-methyltransferase 1 (DNMT1) ASO had a good safety record. Hypermethylation is a characteristic of the RCC phenotype, with the expression of multiple genes truncated at the DNA by the activity of methylases. Hence, the rationale for targeting DNMT1. However, trial results proved inconclusive or fell short of the desired clinical endpoints (52, 53).

Recent advances in oligonucleotide technology and a far better understanding of the biological activity of ASO, their pharmacokinetics, dosing regimens, and routes of admiration still make ASO an appealing therapeutic approach, despite the limited clinical success of RCC therapy. Anti-angiogenic ASO have the potential to yield a sustained effect, given their low clearance and high stability, relative to a TKI. Currently, no significant study is seemingly ongoing to address RCC treatment. As an increasing number of ASO-based therapies are approved, this molecule is expected to achieve a foothold in several therapeutic areas once their applications become more commonplace (54). It would not be unsurprising to see ASO-RCC applications revisited, in particular those that could potentiate immune therapies. Combination therapies involving ICI and anti-proliferative/angiogenic ASO could prove beneficial. Sustained inhibition of tumor development may result in an enhanced immune response (55).

Anticalins and Bispecific Peptides

Anticalins are engineered low molecular weight proteins derived from Lipocalins, a family of small proteins, which consist of less than 200 amino acids (around 20 kDa). Lipocalins are extracellular soluble proteins with carrier functions, associated with cellular communication, and to date, a compressive picture of their regulatory functions is still elusive (56). Anticalins act as antibody mimetics and are taunted as alternatives to monoclonal antibodies. They display improved pharmacokinetic properties relative to antibodies, including efficient tissue penetration and renal clearance. These artificial proteins can also be designed to bind smaller molecules, such as steroids or metabolites, in addition to larger structural molecules (e.g., membrane receptor antigens). Moreover, the structure of anticalins is divergent from that of antibodies, with little to no homology. Therefore, immunogenic side-effects associated with antibody (i.e., biologicals) therapy could be mitigated (57).

The efficacy and safety of anticalins in the treatment of RCC have been shown in both pre-clinical and human trials. PRS-050 is a high-affinity anticalin that binds VEGF and antagonizes its interaction with VEGF receptors, effectively shutting any mitogenic-associated signaling. In humanized mice, PRS-050 reduced tumor microvascular density, with results comparable to bevacizumab, an anti-VEGF monoclonal antibody. Most interestingly, PRS-050 did not lead to platelet aggregation and thrombosis, which were observed after bevacizumab treatment (58). In RCC patients, this anticalin did not yield an immunogenic response and its administration resulted in undetectable levels of free VEGF in the subjects, with virtually all circulating VEGF bound to the anticalin (59).

Several anticalins are now in early to mid-stage development, with regulatory approval still in the future (60, 61). Anticalins have drawn much attention in the immune-therapy field, in particular with the development of bispecific peptides, which combine both an anticalin and an antibody. These conjugates harness the specificity of both classes of drugs to achieve an additive effect and boost the immune response. This is exemplified by PRS-352/S095025, which consists of an anticalin against OX40, a co-stimulatory immune checkpoint expressed in resting T-cells, and a monoclonal antibody targeting PD-L1, a suppressive immune checkpoint expressed by tumor cells. Results from recent pre-clinical studies show that this bispecific molecule triggers a strong PD-L1-dependant OX40 activation of T-cells, superior to that of PD-L1 antibodies (62).

With immune-therapy-based anticalins and bispecific peptides expected to reach late-stage development in the coming years, the true scope of the potential benefits relative to approved ICI, will remain to be seen. Nonetheless, they could represent a leap forward in RCC treatment if they can combine enhanced efficacy with limited side effects.

Proteolysis Targeting Chimeras

Proteolysis Targeting Chimeras (PROTAC) mediate the selective degradation of endogenous proteins by recruiting the activity of the ubiquitin-proteasome complex. PROTACS are heterobifunctional molecules, with two protein-binding domains conjugated by a molecular linker (63). A ligand domain that is designed to bind to a given protein of interest (POI) and a domain that engages with members of the E3 ubiquitin ligases family. By simultaneously binding to a POI and an E3 ligase, PROTACS trigger selective proteolysis by ubiquitination of the POI (64). These molecules are highly selective and highly effective in knocking-out their targets, in essence removing a protein rather than inhibiting its activity. This relatively new approach has gathered significant interest in cancer research. In particular, in the search for new therapies for cancer types with dated or ineffective treatments. PROTACS can virtually be designed to eliminate key proteins known to be over-expressed in cancer or intermediaries in tumor-specific processes. Moreover, these bifunctional molecules have physiochemical and pharmacokinetic properties more aligned with traditional small molecules, given their relatively reduced size (around 1,000 Da). This fact reduces the uncertainties surrounding drug distribution and elimination routes inherent to other new drug modality classes (65).

A promising example of an anti-cancer PROTAC is the development of ARV-110. This androgen receptor (AR) degrader is being tested in the treatment of another advanced urological cancer, metastatic castration-resistant prostate cancer (CRPC), in patients with progressive disease after conventional chemotherapy (NCT03888612) (66). AR activities govern prostate cancer growth and in CRPC its activity is believed to be dramatically deregulated. Pre-clinical data show that ARV-110 substantially reduces AR expression (i.e., >90%) and tumor growth in both wild-type and common AR genetic variants (63, 67).

To date, there are seemingly no PROTAC applications targeting RCC, either experimental or clinical. Nonetheless, as applications emerge, these molecules could find their way into the arsenal of drugs against RCC. A potential avenue is the impact of PROTAC on inhibiting the inflammatory response, by selectively excluding regulators of immune pathways (68, 69). This could prove beneficial in dampening the cytokine blockade that RCC relies on to keep immune cells at bay. Targeting abnormal hypoxia-induced factor (HIF) expression in RCC could also be addressed using PROTAC activity. HIF deregulation during the onset of RCC is derived from the loss of the von Hippel-Lindau protein (VHL), itself an E3 ligase. Bypassing VHL activity and promoting HIF degradation via an alternative ligase could help reduce the hypoxic nature of the TME and act concomitantly with other therapies (e.g., ICI).

Cellular Therapy

Innovative cellular therapies, which involve the administration of autologous or allogenic cells into a patient, have recently been brought to the spotlight including the approval of chimeric antigen receptor (CAR) T-cells. CAR T-cells are a type of immune therapy that involves the collection and isolation of T-cells from a patient, their expansion ex-vivo, the introduction of a CAR using genetic engineering tools, and the infusion of the transformed T-cell back into the patient (70). The CAR is at the heart of this therapy and consists of an artificial membrane receptor with the dual function of binding a specific cancer antigen and activating T-cells. CAR T-cells specifically recognize their target and trigger an immunogenic response directed at the intended tumor cells, which can include cytolytic activity and the secretion of immune factors that promote further CAR T-cell recruiting (71). Initially developed to treat blood cancers (e.g., acute lymphoblastic leukemia), CAR T-cells have shown dramatic efficacy, often leading to complete cancer remission, where preceding treatment failed (72). However, this therapy requires dedicated facilities, specialized personnel, and a tight manufacturing and delivery timeframe, far from an off-the-shelf solution. Preceding CAR T-cells therapy, patients undergo immunedepletion chemotherapy to facilitate the integration of the newly infused transformed T-cells.

The remarkable results of CAR T-cell therapy are derived from the potent and sustained immune response these cells can deliver. CAR-binding to their target cancer cells can also stimulate the proliferation of these transformed T-cells, hence amplifying their therapeutical range. A significant challenge with this type of therapy is, unsurprisingly, off-target immune effects. CAR-T cell can overreach their intended anti-tumor effect and induce severe toxicities derived from extensive cytokine release (73). Engineering CAR T-cells to avoid such side effects and strategies to mitigate them are focal areas in the development of new applications.

Several ongoing human trials are ongoing to determine the safety and potential efficacy of CAR T-cells therapies in the treatment of RCC (74). Despite their success in blood tumors, this cell therapy still has significant limitations while addressing solid tumors. Tumor trafficking and infiltration as well as the immunosuppressive TME are major hurdles in delivering CAR T-cells to their intended malignant targets (75). Antigen depletion and tumor off-target recognition are also important considerations in the CAR design. The heterogenicity of solid tumors could result in CAR T-cells targeting a specific sub-population of cancer cells. Nonetheless, this fact could be offset by an amplified immune response directed at the wider tumor. Solid tumors share antigens with healthy cells, albeit subjected to extensive variation, which can potentially lead to unintended tissue damage. CAR T-cells can be generated from distinct T-cell populations, which enables the deployment of transformed immune cells with different functions that can act in more than one front (76). Beyond direct cancer cell targeting, these cells can potentially alter the dynamics of the TME and suppress barriers to the recruitment of additional immune cells. Nephrotoxicity is a major concern with employing this approach to treat RCC. Toxic events derived from off-target immune activity could result in irreversible kidney damage leading to diminished renal function and ultimately renal failure. All these prospects are likely to remain unresolved in the foreseeable future. Nonetheless, the CAR T-cell field is fast progressing, with five therapies approved in the last five years (77). First-generation CAR T-cell therapies incorporate well-characterized immunestimulant antigens (e.g., CD19). A recent human trial (NCT03970382) has demonstrated the efficacy of this treatment using CAR designed to incorporate patient-specific mutated neoantigens. This approach relied on the analysis of samples from solid tumors and the generation of personalized libraries of predicted neoantigens of T-cell receptors (78). Several other important hurdles can be expected to be resolved in the coming years, opening CAR T-cell therapy to many different types of cancers.