Targeting the complement system: notes on therapeutic strategies for renal cancer

The complement system is an ancient element of the innate immune system, consisting of numerous circulating, membrane-bound or intracellular proteins. By interacting with immune cells and other mediators of inflammation, the complement system provides an efficient protection against pathogens and support for tissue repair [Citation1]. Three activation pathways have been described even though they share a common terminal pathway starting with C3 cleavage. Then, C5 cleavage allows the subsequent assembly and development of the membrane attack complex (C5b-9) on targets. Simultaneously, anaphylatoxins C3a and C5a facilitate the recruitment and activation of leukocytes, and act as regulators of inflammation. Additional activation fragments stimulate the phagocytosis and are referred to as opsonins (C3b, iC3b, C3d, C4b, iC4b and C4d) [Citation2]. Several membrane-bound (CD35, CD46, CD55 or CD59) and circulating (C1 inhibitor, C4b binding protein, factor H, factor I, clusterin or vitronectin) regulators aim to ensure proper complement activation and prevent adverse effects on healthy cells [Citation3]. The activation of the complement system has been confirmed in various human diseases including complement-mediated thrombotic microangiopathy, C3 glomerulopathy, and antibody-mediated rejection of kidney transplantation. In recent years, the ongoing insights into its biology have led to the identification of many complement inhibitors. Eculizumab -a C5 inhibitor- was the first agent approved by the Food and Drug Administration for the treatment of paroxysmal nocturnal hemoglobinuria. In addition to eculizumab, other therapeutic agents have recently been introduced into clinical practice or entered in clinical trials targeting C5 (ravulizumab), C5a receptor (avacopan, avancicaptad pegol, vilobelimab), C3 (pegcetacoplan, AMY-101), factor B (iptacoplan), factor D (danicopan) or C1s (sutimlimab) [Citation4]. Current evidence supports the context-dependent role of the complement system in cancer biology with two opposing mechanisms [Citation5,Citation6]. Remarkably, cancer cells and other cells in the tumor microenvironment (TME) produce complement components and express receptors to exert these mechanisms. The antitumoral opsonophagocytosis and cytotoxicity are counteracted by protumoral chronic inflammation depending on tumor type and stage [Citation7]. Resident and metastasizing tumor cells may escape complement attack by expressing regulators such as C4b binding protein, factor H (FH), CD46 or CD59. Mucins, glycosaminoglycans, and sialylated molecules coat tumor cells to provide negatively charged residues promoting FH-mediated complement inhibition at the level of C3 [Citation7]. Complement activation may recruit immunosuppressive cells such as tumor-associated macrophages (TAMs), neutrophils (TANs), myeloid-derived suppressor cells (MDSCs) and regulatory T cells [Citation8]. The C5aR axis is involved in the recruitment and activation of MDSCs which release immunomodulators (arginase-1, IL-10, TGF-β) and express immune checkpoint inhibitors (CTLA4 and PD-L1) [Citation1]. In clear cell renal cell carcinoma (ccRCC), transcriptomic analysis revealed that poor prognosis is associated with low expression of terminal pathway genes and high expression of C1 and C3 regulators and CD59 [Citation5]. Clear cell RCC (ccRCC) represents the most common histological type of RCC followed by papillary RCC and chromophobe RCC. RCC is fundamentally a metabolic disease and is one of the most immune-infiltrated tumors [Citation9–11]. Emerging evidence suggests that modulation of specific metabolic pathways can regulate angiogenesis and inflammatory response. In this scenario, complement system activation in RCC can promote tumor progression, inducing immunosuppression, migration, angiogenesis, epithelial-mesenchymal transition, and metabolic deregulation [Citation2,Citation5,Citation12]. Using samples from The Cancer Genome Atlas and Gene Expression Omnibus database, poorer survival outcomes were confirmed with higher levels of C3 [Citation13]. Higher expression of C3a was reported in case of larger and more advanced ccRCC. Moreover, the relationship between serum C3a levels and the clinical response to immunotherapy was investigated. In cases of partial or complete response, C3a decreased whereas it remained stable with stable disease. In turn, progressive disease was associated with an increase in C3a concentrations [Citation14]. The in situ production of C1r, C1s, C4, and C3 by ccRCC cells is accompanied by the exploitation of TAM-derived C1q to facilitate the local assembly of the initiating C1 complex, leading to the activation of the classical complement pathway [Citation15]. In addition, renal cancer cells and ccRCC-derived tissues exhibited an overexpression of pentraxin-3 (PTX-3) as well as C1q, C3a, C5a, C3aR, C5aR1, and CD59 in association with the absence of C5b-9 terminal complex deposition [Citation16,Citation17]. Mouse models of RCC were used to assess the clinical effect of genetic deficiencies and pharmacological inhibition of C3aR and C5aR1. The function of tumor infiltrating CD8 + T cells improved as demonstrated by increased production of IFN-γ, thus limiting tumor growth [Citation18]. A growing body of research is pointing to the role of intracellular complement activation (complosome) in cancer. An interesting study showed that C1s might be involved in ccRCC progression, not only by triggering complement activation, but also by acting in an intracellular, non-canonical matter [Citation19]. Another study demonstrated the role of intracellular FH as a driver of renal cancer cell proliferation and migration [Citation20]. In particular, it has been showed that intracellular FH acts as a protumoral factor modulating the transcriptional activity of cancer cell, and inducing phenotypic modifications that drive tumor progression in ccRCC [Citation20]. Overall, these findings allow us to make some observations on the potential applications of complement therapeutics for renal cancer. Anti-angiogenic agents and immune checkpoint inhibitors currently represent the standard of care for advanced renal cancers [Citation21,Citation22]. Considering that the activation of complement in tumor microenvironment induces an angiogenic response, inhibition of immune system and tumor progression in ccRCC, it is possible to hypothesize that a combined approach involving complement inhibitors and conventional therapies could become one of the most effective strategy for future treatment [Citation23]. In this scenario, an ongoing phase 2 clinical trial (NCT04919629) is evaluating the efficacy of pegcetacoplan (a C3 inhibitor) in combination with pembrolizumab (anti-programmed cell death protein 1) or with pembrolizumab and bevacizumab (anti-VEGF) for the treatment of recurrent ovarian, fallopian tube, or primary peritoneal cancer and malignant effusion [Citation4]. Similarly, the combination of pembrolizumab and vilobelimab (a C5a inhibitor) has been evaluated in patients with locally advanced or metastatic cutaneous squamous cell carcinoma (NCT04812535) [Citation24]. Complement deficiency or blockade increased the response to immunotherapy (PD-1 or PD-L1 blockade) in mouse model of melanoma, sarcoma, lung and colorectal cancer [Citation7]. Its inhibition is expected to reverse the effects of MDSCs and to foster the activity of antitumor CD8 T cells, thus enhancing the efficacy of immune checkpoint inhibitors also in RCCs. To predict patients’ prognosis, a humoral complementomics approach has been proposed. It refers to the comprehensive assessment of serum complement proteins, activation fragments, and autoantibodies targeting complement proteins which reflect its intratumoral productions. Shorter progression-free survival was highlighted in patients with elevated plasma levels of C4d or Bb. In turn, favorable outcomes were registered in case of autoantibodies against neoantigen reduced FH or against C3 [Citation25]. A phase 1b dose escalation trial (NCT04314089) is currently evaluating the safety of a monoclonal antibody (GT103) targeting FH in non-small cell lung cancer [Citation26]. If promising results are achieved in lung cancer, these results will pave a new way in targeting complement system in renal cancer [Citation27]. Although it is reported in the literature a context-dependent role of complement across different cancer types [Citation5], in most tumors C3aR and C5aR1 can be considered as a new class of immune checkpoint receptors. The anaphylatoxin receptors are both overexpressed in ccRCC [Citation16,Citation17], and in particular C5aR1 is highly expressed in metastatic RCC and plays a crucial role in cell invasion via ERK and PI3K pathways [Citation22]. These findings support the hypothesis that the C5a-C5aR axis could be an effective therapeutic target to prevent RCC progression. It has also been demonstrated that blocking C5aR1 can improve the response to chemotherapy. RCC is one of most chemoresistant tumors and different strategies to restore cancer cell chemosensitivity are under investigation. C5a reprograms tumor-infiltrating C5aR1 + macrophages into an immunosuppressive phenotype that dampens CD8 + T cell responses during squamous cell carcinogenesis. C5aR1 blockade using PMX-53 improves the efficacy of paclitaxel, resulting in transcriptional reprogramming of macrophages that not only affects angiogenic programs but also leads to recruitment of cytotoxic CD8 + T cells by IFNγ-dependent mechanisms [Citation28]. In recent years, the introduction of novel combination therapies (immune checkpoint inhibitors + tyrosine kinase inhibitors) has radically changed the prognosis of patients with metastatic RCC [Citation29]. However, despite the high efficacy of these frontline therapies, most patients will require additional lines of therapy. In this clinical context belzutifan, a hypoxia-inducible factor 2α inhibitor, showed a significant efficacy in participants with advanced ccRCC who had previously received immune checkpoint and antiangiogenic therapies [Citation30]. Disease progression after a period of response to first-line treatments represents a challenge to oncologists as the classes of drugs approved for metastatic RCC are limited. The introduction of novel therapeutic regimens incorporating complement-targeted drugs may allow us to treat these patients in an attempt to increase response and circumvent resistance. In conclusion, strong complement expression and its early activation have emerged as hallmarks of ccRCC. Further analyses are warranted for a deeper understanding of the role of complement in renal cancer with the potential to unlock novel therapeutic strategies. Declaration of Interest The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.