Profound advances in our understanding of the interactions between tumors and the immune system have allowed the development of therapeutic approaches that boost the body's natural defenses against cancer. These therapies are aimed to mount effective antitumor immune responses and include immunomodulators, vaccines, and adoptive transfer of immune cells (1). Some of the most clinically effective immunotherapies to date target the programmed cell death protein 1 (PD-1) immune checkpoint. PD-1 is expressed by T cells during priming or expansion and binds to one of its two ligands PD-L1 or PD-L2 (2). Tumor cells upregulate PD-L1 in response to cytokines such as interferon (IFN)-γ (3). Interaction of PD-L1 with PD-1 on T cells causes T-cell apoptosis, anergy, and exhaustion, protecting tumor cells from CD8⁺ T cell-mediated cytolysis (3). PD-L1 can also deliver intrinsic intracellular signals that enhance cancer cell survival, regulate stress responses, and confer resistance toward apoptotic stimuli (4, 5). PD-1 and other checkpoints are also expressed by NK cells which may contribute to the antitumor activity of some therapeutic strategies under development (6, 7).

Neutralizing monoclonal antibodies against PD-1 or PD-L1 have transformed the therapeutic landscape of a wide range of cancers (Table 1), being particularly successful for tumors with limited therapeutic options such as melanoma or lung cancer (3). Notably, these antibodies generate durable responses without causing serious side effects. However, a significant fraction of patients manifests innate or acquired resistance to these therapies. Immune escape mechanisms stem from different cell interactions within the tumor microenvironment, and emphasize the need of developing rational combination strategies to obtain more potent anticancer responses (8).

In 2013, Daniel Chen and Ira Mellman described a series of self-sustaining stepwise events, referred as the cancer-immunity cycle, by which the anti-cancer immune responses lead to an effective elimination of cancer cells (9). The existence of negative feedback mechanisms developed by tumors hinders this cycle of cancer immunity and may pose a barrier to the development of effective clinical responses. Anticancer immunotherapies should be aimed to reactivate all the steps of the cycle, which include immunogenic cell death, maturation of antigen-presenting cells, T-cell priming and activation, promotion of immune infiltration, blockade of immunosuppression, and enhancement of effector T-cell activity. In this context, combination therapies would provide synergistic effects for the maintenance of the cancer-immunity cycle.

In the last years, the complement system, an essential part of innate immunity, has surged as a master regulator of cancer immunity (10). We, and others, have actively contributed to this field, leading to the proposal that modulation of complement activation can improve the antitumor efficacy of inhibitors targeting the PD-1/PD-L1 pathway. In 2008, making a paradigm shift in tumor immunology, we demonstrated that complement activation, followed by C5a signaling, has a tumor-promoting role in cancer (11). In 2012, using a lung cancer model, we first demonstrated an association between the inhibition of C5a receptor 1 (C5aR1) and the expression of PD-L1 within the tumor microenvironment (12). These results suggested the possibility of blocking complement factors to increase the efficacy of other immune therapeutic strategies (12). Following this line, we demonstrated that inhibition of PD-1/PD-L1 synergizes with the inhibition of C5a/C5aR1 in various preclinical models of lung cancer (13). This rationale provided the basis for a clinical trial in which the anti-C5aR1 antibody IPH5401 is being evaluated in combination with the anti-PD-L1 antibody durvalumab in patients with solid tumors (STELLAR-001).

In this review we describe the participation of complement elements in the steps of the cancer-immunity cycle. We propose that a combinational therapy using anti-PD-1/PD-L1 antibodies together with modulators of the complement system may open new therapeutic opportunities for tumors resistant to PD-1/PD-L1 blockade.

The cancer-immunity cycle is defined as a series of functional stepwise events needed to obtain an efficient control of cancer growth by the immune system (9). The process is initiated by the release of neo-antigens generated as a result of genomic instability. Cancer-associated antigens are captured by dendritic cells which, upon migration to lymph nodes, prime and activate tumor-specific cytolytic CD8⁺ T cells. These effector cells migrate and infiltrate the tumor stroma, where potentially are able to recognize and eliminate cancer cells. T cell-mediated cytotoxic responses release new tumor antigens, fueling the cancer-immunity cycle. Interestingly, this model provides the rationale for targeting different steps of the cycle in order to maintain its functionality. An effective cancer immunotherapy should be designed based on the specific resistance mechanisms underlining the rate-limiting steps in each particular patient (14, 15). In the case of anti-PD-1/PD-L1 therapies, a variety of biological factors contribute to treatment resistance, including lack of cancer antigens recognizable by T cells, impaired cancer-antigen presentation, impaired activation of cancer-specific T cells, poor infiltration of T cells into tumors, and accumulation of immunosuppressive factors and cells in the tumor microenvironment (15). The evading strategies present in a given tumor would determine whether this tumor shows an inflamed or a noninflamed phenotype (16). Clinical evidence suggests that anti-PD-1/PD-L1 inhibitors are most effective in inflamed tumors characterized by high tumor PD-L1 expression, CD8⁺ T-cell infiltration or mutational burden (17–20). Jerby-Arnon et al. recently analyzed the relationship between malignant cell states and CD8⁺ T-cell infiltration and identified a T-cell exclusion program that predicts responses to PD-1/PD-L1 blockade (21). This program was enriched for genes involved in predictable processes, such as antigen processing and presentation, IFN-γ signaling, and immune modulation; but also identified genes associated with activation and modulation of the complement system (21). Elements of the complement cascade are also present in a signature of serum proteins that predicts survival in patients receiving PD-1 blocking antibodies, suggesting that complement activation may inhibit the efficacy of adaptive antitumor immunity (22).

The complement system, a central element of innate immunity, represents a first line of defense against unwanted non-self and host elements, and orchestrates many immunological and inflammatory processes that substantially contribute to body homeostasis (23, 24). Complement activities are mediated by more than 50 circulating, cell surface-bound and intracellular proteins. There are three main mechanisms of complement activation, known as classical, lectin, and alternative pathways. The classical pathway is commonly initiated by the binding of C1q to complement-fixing antibodies (mostly IgM and IgG types); although C1q can also recognize non-immunoglobulin ligands such as C-reactive protein (CRP), pentraxin 3 (PTX3), or apoptotic cells (25). The lectin pathway is activated by homologous proteins to C1q (mannose-binding lectin, collectins, and ficolins) that recognize repetitive carbohydrate patterns (26). Lastly, the alternative pathway is initiated by spontaneous cleavage of C3 on activating surfaces (27). Although the three complement pathways differ in their mechanisms of target recognition, in all cases, initiation of the complement cascade leads to the formation of C3 convertases and the activation of the central component C3. After this activation, C5 convertases are formed, C5 is cleaved, and the assembly of the pore-like membrane attack complex (MAC) is initiated. The enzymatic cleavage of complement elements leads to the release of proteolytic fragments such as C3a and C5a, and the deposition of other fragments such as C3b and iC3b. These molecules modulate a diverse set of processes (23), including the initiation and regulation of effector T-cell responses (28). Prevention of inappropriate activation by complement regulators takes place at three main levels: inhibition of protease activities in the activation cascade, decay and destruction of convertases, and control of MAC formation (29). Finally, recent experimental and clinical evidences suggest that intracellular complement components have important roles in cell physiology (30).

Complement has been traditionally regarded as playing a role in the elimination of tumor cells. Accordingly, an effective control of tumor growth may be achieved by complement-fixing antibodies (31). However, growing evidence, starting for the initial observation in a model of cervical cancer (11), strongly supports a tumor-promoting role of complement in several tumor types. This topic has been extensively reviewed elsewhere (10, 32–35). Briefly, complement establishes an immunosuppressive microenvironment, promotes angiogenesis, sustains cellular proliferation, and participates in tumor cell invasion and migration. In light of the various contributions of complement to cancer progression, it is not surprising that expression of complement effectors and receptors is associated with disease progression and poor prognosis (36–42). Among all the complement elements with potential pro-cancer activities, C1q, C3-derived fragments, and C5a are recognized as major modulators of tumor progression (43–45).

In this section we will discuss the potential implication of effectors and regulators of the complement system in the steps of the cancer-immunity cycle. In light of the breadth and complexity of the immune response, we will focus our review on the specific aspects of the regulation of CD8⁺ cytotoxic T cells, which are in large part the mediators of anti-PD-1/PD-L1 therapies. We will also examine evidence supporting the participation of complement in the regulation of the type 1 T helper (Th1) response, as it has a profound influence on the quality and extension of cytotoxic T-cell responses (46). Recent reviews have extensively addressed other complement-mediated immune functions not covered in the present review (10, 28, 47–50).

As reviewed in the previous section, a growing body of evidence supports the notion that complement activities support cancer growth and metastasis in the context of established tumors (124). Many mechanisms related to immune escape and resistance to checkpoint inhibitors can be modulated by elements of the complement system (summarized in Figure 1). The non-immunology-related effects of complement on cancer cell biology, including cancer cell proliferation, survival and invasion capacity (42, 43, 117, 125–137), further reinforces the impact of complement activation in cancer progression.

Based on the regulatory functions of complement in the cancer-immunity cycle, we sought to evaluate whether complement inhibition may represent an effective target for combined immunotherapies in preclinical syngeneic models of cancer. Clinical successes and limitations of anti-PD-1/PD-L1 monotherapy prompted us to use this target as the primary building block for the combination. The C5a/C5aR1 axis was selected as the complement-related target based on the abundant evidence supporting the role of this pathway in the establishment of an immunosuppressive microenvironment (Table 2) (45). Using different lung cancer models, we observed a remarkable synergistic control of lung tumor burden and metastatic progression in animals simultaneously treated with an aptamer against C5a (AON-D21) and an anti-PD-1 monoclonal antibody (13). This effect is accompanied by a negative association between the frequency of CD8⁺ T cells and the presence of MDSCs within tumors, and by a reduction of CD8⁺ T-cell exhaustion markers (13). The synergistic benefit of this combination was later confirmed in models of melanoma and colon cancer (138). Interestingly, PD-1/PD-L1 antibodies induce the production of C5a (138), establishing a regulatory loop between both pathways.

Other complement elements, such as C1q, C3, or C3a, may be also targeted to re-educate the tumor microenvironment and sensitize it to the subsequent administration of immune checkpoint blockers (Table 2). A multifaceted repertoire of therapeutic inhibitors targeting these complement elements has been developed, and are currently in preclinical or clinical development (139, 140). Figure 2 shows examples of compounds that may be used to target complement in the context of cancer immunotherapy. Complement C3, the centerpiece of complement activation, represents a particularly attractive target for therapeutic complement inhibition (141). Nonresponsive patients to PD-1/PD-L1 blockade frequently have noninflamed tumors with a defect in the early stages of the cancer-immunity cycle (15). Opsonization of dying cells with C3 fragments induces the production of anti-inflammatory cytokines and reduces the costimulatory molecules needed for the maturation of dendritic cells, resulting in T-cell tolerance (90). Therefore, C3 blockade may have a beneficial impact in the early stages of the cancer-immunity cycle, converting a noninflamed tumor into an inflamed tumor susceptible to PD-1/PD-L1 blockade. Moreover, both C3a and C5a production would be impaired (at least in the case of inhibitors, such as compstatin, that blocks C3 activation by all pathways). Interestingly, a simultaneous blockade of C3aR and C5aR1 has been reported to enhance the efficacy of anti-PD-1 therapy against melanoma cells (123). Deletion of C3 in tumor cells that had high C3 expression enhanced efficacy of anti–PD-L1 treatment (122). Additionally, complement C3 inhibition may have antitumor potential in the context of other immune combinations. For example, C3 inhibition by complement depletion or the use of the inhibitor compstatin enhances the antitumor efficacy of oncolytic virus (142, 143) and induces natural killer (NK)-mediated antitumoral responses (144). Inhibition of complement activation upstream of all complement effectors also appears to be a rational approach. In this sense, combinatorial therapies involving inhibitors of C1q (e.g., C1-INH), which presents both complement-dependent and -independent tumor promoting activities, merit further investigation. Finally, it has to be noted that most complement inhibitors target the extracellular complement system, preserving its intracellular activity. This may be of upmost importance, since intracellular C3aR and C5aR1 signaling pathways seem to be required for T-cell survival (28).

Finally, it is interesting to point that the preclinical findings showing the feasibility and value of blocking C5a/C5aR1 to increase tumor-killing efficacy of checkpoint inhibitors have been the basis for the design of a phase I/II study (STELLAR-001). In this trial, the safety and efficacy of durvalumab (an anti-PD-L1 monoclonal antibody) is being tested in combination with IPH5401 (an anti-C5aR1 monoclonal antibody) in patients with selected solid tumors, including non-small cell lung cancer and hepatocellular carcinoma (NCT03665129). We are looking forward to the outcome of this trial, as well as to the clinical evaluation of novel combinations involving complement inhibitors.

This is an exciting time for the complement field, in which new biological concepts have brought new therapeutic opportunities. Based on the extensive literature associating complement activation and cancer progression, we propose here that substantial clinical benefits can be achieved by multi-modal anticancer immunotherapies targeting both complement-mediated mechanisms (to reverse immunosuppression), and PD-1/PD-L1 immune checkpoints (to re-activate T-cell functionality). Our preclinical studies supporting the idea that C5a/C5aR1 inhibition creates a “window of opportunity” for the administration of anti-PD-1/PD-L1 checkpoint inhibitors pave the way for the evaluation of other complement-based combinations. The challenge being that many potential combinations can be evaluated. Insights into how complement switches from tumor suppressing to tumor promoting activities at the onset of disease, as well as how to manage this dichotomy should be important research areas in order to establish the best therapeutic strategies. The differences between mice and humans in complement-mediated T-cell responses should also be considered (28, 87). To overcome this limitation, faithful mouse models that recapitulate the complexity of the human immune context in the tumor microenvironment are urgently needed (145).

RP and JL designed the concept. All authors wrote the manuscript. SO-E prepared the figures. All authors read and approved the final version of the manuscript.