Colorectal cancer (CRC), as a leading lethal malignancy, has been a major cause of cancer-associated death worldwide . Despite of improved five-year survival via early detection, the metastasis to distant organ such as lung has been the prominent cause for mortality. Approximately 25% of CRC patients suffer from stage IV disease, and near 25~50% patients with early stages will eventually progress to metastatic disease, which indicates poor prognosis and 12.5% of five-year survival [2, 3]. Therefore, more effective approaches against metastatic CRC (mCRC) are on urgent demand.
Immunotherapy, designed to boost the natural defense of immune system, has powerfully altered the landscape of malignancy treatment, including mCRC. It has exhibited improved antitumor immunity and tremendous potential for reversing the immunosuppression induced by tumor. For instance, the immune checkpoint inhibitors (ICIs) have lead the immunotherapy field to regain a major influence in tumor research . However, the immune-related adverse events (irAEs) are inevitable, as ICIs usually elicits long-lasting clinical responses via reactivating an exhausted immune response. Whereas, adoptive cell therapy (ACT) may provide the cancer-targeting immune infiltrate and specific immune response . It can keep long-term durable in solid tumor, showing a peculiar property, which was ever hardly achievable for chemotherapy [1, 6]. Thereinto, DCs play a vital role in the immune response especially the stimulation of CTLs. Landmark studies have demonstrated that CD8+ CTL density are strongly associated with disease-free and overall survival in CRC patients, which has been employed as a prognostic biomarker for the survival of CRC patients [7, 8]. Adoptive cell therapy (ACT) has displayed powerful potential in the development of CRC treatment in the past decades. And CTL-mediated inflammatory response to tumor cells acts a critical role in suppressing the progression of carcinogenesis . Therefore, approaches improving the generation of CTLs are vital for the immunotherapy of CRC patients.
The cytotoxicity of CTLs critically depend on dendritic cells (DCs) . As professional antigen-presenting cells (APCs), DCs play essential roles in both innate and adaptive immunity system upon pathogen invasion . Immature DCs take up antigens through endocytose phagocytosis, micro- or macro-pinocytosis, and endocytosis using Fc receptors, integrins, C-type lectin receptors, apoptotic cell receptors, and scavenger receptors. The mature DCs can transport cancer-associated antigens to the draining lymph node where T cell priming and activation can occur to generate the CTLs . Various studies have shown that CTLs induced by DCs can enhance the antitumor effect against CRC [13–16]. Therefore, targeting DCs may confer a feasible strategy for CRC immunotherapy. The inhibitory cytokine (such as IL-10 and TGF-β) and stimulatory cytokine (such as IL-12 and IFN-γ) function opposite roles in the process of CTL generation [17, 18]. The former is involved in the suppression of T cells whereas the later stimulates them. Produced by Tregs, IL-10 and TGF-β majorly function as a suppressive role in the immune system. As a pro-inflammatory cytokine, IL-12 contributes to the activation of T cell and NK cell, and the production of IFN-γ that favors the differentiation of Th1 cell. It can be produced by mature DCs upon pathogen infection . Besides, IL-12 can suppress the proliferation of Tregs and the Foxp3 level, favoring the outgrowth of non-Tregs. Collectively, IL-12 and IFN-γ majorly function as a stimulator favoring the activation of cytotoxic cells . Furthermore, DCs themselves also act as direct killer targeting on tumor cells and induce the dying cells to release tumor-related antigens .
CD40, a cell-surface member of tumor necrosis factor (TNF) receptor superfamily, is expressed on variety of APCs. As a costimulatory molecule, it is vital for the activation of APCs and CD8+ T cells . The CD40-CD40L signaling plays a central role in the regulation of the immune response through an interaction between T cells and APCs. They can help the antigen presentation and promote the expression of costimulatory molecules, which allows the maturation of DCs and stimulation of T cells. Upon activation of lymphocytes, the binding of CD40L on the surface of T cells with CD40 provides costimulatory molecules required to activate naive T cells thereby amplifying the immune response. Preclinical investigations showed that CD40-activated DCs are poised to prime or activate tumor-specific T cells . Moreover, CD40 activated macrophages were shown to infiltrate tumors and destroy tumor stroma via tumoricidal activity . Besides, as a membrane antigen, CD40 can regulate the production of costimulatory cytokine IL-12. These data inspired the utilization of CD40 agonists, particularly the CD40 monoclonal antibodies (mAb), as a novel approach for cancer immunotherapy. It has shown a feasibility in both pre-clinical and clinical settings . Evidences suggests that the activation of CD40 display critical role in the conversion of “cold tumor” to “hot tumor”, which makes tumor more susceptible to immune checkpoint inhibitors . In the past few years, CD40 mAb combined with other therapies have shown attractive efficacy, which is potentially important to extend the effective range of CD40 mAb. It has been reported that CD40 antibodies with varying formulations show tolerable and feasible function in patients with mesothelioma, pancreatic cancer, and others, when combined with chemotherapy , whereby the companion may codetermine the terminal outcomes, which needs more attention to be paid. Although CD40 mAb is potential for the immunotherapy, it is deficient as CD40 mAb not only stimulate the production of stimulatory cytokine IL-12, but also the inhibitory cytokine IL-10 . Therefore, it is necessary to explore an adjuvant agent to neutralize the side effect of CD40 mAb.
OK-432 is a lyophilized preparation derived from penicillin-inactivated Streptococcus pyrogene . It can stimulate the maturation of DCs thereby priming CTL and natural killer (NK) cell response against tumor cells. Because mature DCs can produce proinflammatory cytokines, which is beneficial to the proliferation of CTLs. [27, 28]. And this cytotoxicity is mediated by the CD40/CD40 ligand axis, as CD40 ligand on DCs can interact with CD40 on tumor cells . The effect of OK-432 on DCs may be achieved by following steps: 1). Induce the generation of IL-12 and IFN-γ. 2). Upregulate the expression of CD40, CD80, CD86, adhesion molecules (ICAM-1), etc. 3). Stimulate the peptide-specific cytotoxic lymphocytes (CTLs). And these data endowed OK-432 with potential in anticancer immunotherapy, indicating a feasible clinical application, which has been approved by clinical observation in CRC [29, 30].
Given the above proofs, here we proposed that the combination of CD40 mAb and OK-432 may confer a synergistic effect on the activation of DCs, which determine the generation of CTLs. We employed a mouse model of CRC lung metastasis to demonstrate the therapeutic effect of this combination.