6Ckine; Beta chemokine exodus-2; Beta-chemokine exodus-2; C-C motif chemokine 21; CCL21; Chemokine (C-C motif) ligand 21; CKb9; ECL; Efficient Chemoattractant for Lymphocytes; Exodus-2; SCYA21; Secondary lymphoid tissue chemokine; Secondary lymphoid-tissue chemokine; SLC; Small inducible cytokine subfamily A (Cys-Cys), member 21; Small-inducible cytokine A21; TCA4; UNQ784/PRO1600
CCL21 is quantified in samples by high sensitivity RT-PCR or ELISA using commercially available antibodies from R&D Systems (Minneapolis, MN). For the phase I clinical trials, following CCL21 gene modification of DC, CCL21 production is quantified by ELISA. Tissue expression of CCL21 is evaluated by immunocytochemistry. CCL21 determinations have not been standardized or validated for clinical practice yet.
Role of CCL21 in Cancer
CCL21 priority ranking is 13 among the list of 20 National Cancer Institute ranked biological agents with high potential for use in cancer therapy. Generation of an antitumor immune response requires the coordinate interaction of NK, T, and DC effectors. There is a paucity of these effectors in the tumor. One regimen to initiate antitumor responses is through the use of chemokines that induce both efficient recruitment and strong activation of effector cells in the tumor mass. The rationale for the use of CCL21 for immune therapy against solid tumors is that CCL21 sculpts host immune responses by recruiting and co-localizing NK, DC, and T-cell effectors to mediate antitumor activity.
High Level Overview
One of the challenges in developing immunotherapy for cancer is enlisting the host response to recognize tumors of poor immunogenicity. Effective antitumor responses require antigen-presenting cells (APC), lymphocyte, and NK effectors. Although cancer cells express tumor antigens, the limited expression of major histocompatibility complex (MHC) antigens, defective transporter associated with antigen processing, and lack of costimulatory molecules make them ineffective APC. Effective anticancer immunity is achieved by recruiting professional host APC for tumor antigen presentation to promote specific T-cell activation. DCs are uniquely potent APCs involved in the initiation of immune responses. Serving as immune system sentinels, DCs are responsible for Ag acquisition in the periphery and subsequent transport to T-cell areas in lymphoid organs where they prime specific immune responses. Thus, chemokines that attract both DC and lymphocyte effectors could serve as potent agents in immunotherapy. The rationale for utilizing CCL21 in cancer therapy is to facilitate the co-localization of DC, T, NK, and NKT cells to orchestrate effective cell-mediated immune responses in the tumor microenvironment. CCL21 may be distinctly advantageous because of its capacity to elicit a type 1 cytokine response in vivo that promotes antitumor activity. Intratumoral infiltration of T lymphocytes and DC in lung cancer has been shown to be associated with a better patient outcome. In accord with this observation in lung cancer, a recent study demonstrates that the presence of lymph node (LN)-like vasculature in tumors, characterized by expression of peripheral node addressin and chemokine CCL21, is correlated with T-cell infiltration and positive prognosis in breast cancer and melanoma patients (Peske et al. 2015). The authors further demonstrated that LN-like vasculature is present in murine models of melanoma and lung carcinoma. LN-like vasculature enables infiltration by naive T cells that significantly delay tumor outgrowth after intratumoral activation. The mechanisms contributing to the development of this vasculature is attributed to effector CD8 T cells and NK cells that secrete LTα3 and IFNγ. LN-like vasculature is also associated with organized aggregates of B lymphocytes and gp38(+) fibroblasts, which resemble tertiary lymphoid organs that develop in models of chronic inflammation. The results of this study establish that LN-like vasculature as both a consequence of and key contributor to antitumor immunity (Peske et al. 2015). NK cells and NKT cells induce antitumor responses, and the recruitment of NK and NKT cells by CCL21 augments antitumor immune activity because these effectors recognize tumor targets in the absence of MHC expression. In addition to expressing the CCR7 receptor, NK, NKT cells, and Th1 cells express the CXCR3 receptor and migrate in response to the CXCR3 ligands CXCL9, CXCL10, and CXCL12. CXCL9 and CXCL10 are potent inhibitors of angiogenesis. CCL21 induces CXCL9, CXCL10, and IL-12 from monocytes, DC, and stromal cells. The induction of IL-12, CXCL9, and CXCL10 further amplifies the antitumor immune responses of CCL21 in the recruitment of CXCR3 expressing effectors and inhibition of angiogenesis. The ability of CCL21 to inhibit angiogenesis has added further support for its use in cancer therapy.
Diagnostic, Prognostic, and Predictive
Diagnostic tests for CCL21 expression and protein concentrations in samples are performed by RT-PCR and ELISA. Tissue expression of CCL21 is assessed by immunocytochemistry. Based on the preclinical data, high levels of CCL21 expression in tumors may be indicative of immune reactivity and serve as a prognostic marker for patient survival. Immune effects of CCL21 are monitored by antigen-specific IFN-γ T lymphocyte ELISPOTS, ELISA, or RT-PCR for Th1 cytokines and immunocytochemistry for T lymphocytes, NK, and DC effector cell infiltrates.
CCL21 is being developed as an anticancer therapeutic agent. The phase I clinical trials were in lung cancer and melanoma, but as the preclinical data warrant in other tumor models, this form of therapy may be extended to include other solid cancers. There is a strong rationale to combine CCL21 with immune checkpoint blockade therapy to increase T cell infiltrates in the tumor of patients who have minimal response to immune checkpoint blockade therapy. Recent ground-breaking studies in lung cancer immunotherapy reveal robust antitumor activity and durable responses in previously treated patients with progressive locally advanced or metastatic NSCLC. Immune inhibitory molecules are upregulated on T cells in tumors causing a downregulation of antitumor activity. The programmed cell death protein 1 (PD-1; also known as CD279) is an inhibitory receptor that regulates immune responses. The PD-1 receptor interaction with the PD-L1 and PD-L2 ligands deliver inhibitory signals that regulate the balance between T cell activation and tolerance. Recent studies reveal responses in approximately 20% of NSCLC patients treated with inhibitors of the PD-1 checkpoint. This includes robust and durable responses in previously treated patients with progressive locally advanced or metastatic NSCLC (Brahmer et al. 2012; Herbst et al. 2014; Carreno et al. 2015; Garon et al. 2015; Gettinger et al. 2015; Soria et al. 2015). Studies in NSCLC and melanoma patient-derived tumor specimens reveal that responses to checkpoint blockade rely on tumor infiltration of activated T effector cells (Brahmer et al. 2012; Herbst et al. 2014; Tumeh et al. 2014; Gettinger et al. 2015; Soria et al. 2015). It has been suggested that among patients who are nonresponsive or respond poorly to checkpoint blockade immunotherapies, there will be individuals who lack preexisting antitumor T cell responses (Delamarre et al. 2015). This group appears to be comprised of patients who have absent or very limited immune responsiveness prior to initiation of therapy and thus have limited CD8 T cell infiltration of the tumor and/or PD-L1 expression by tumor or TME. This situation appears to occur in approximately 50–60% of NSCLC cases (Velcheti et al. 2014). Thus, it has been suggested that this deficit could be addressed with regimens that increase T cell infiltration combined with checkpoint inhibitors. Congruent with this concept, in a recent study, CCL21 enhanced the antitumor activity of PD-1 in murine models of lung cancer (Salehi-rad et al. 2016).
The development of intratumoral therapies to effectively augment local and systemic antitumor immunity in lung cancer will lead to a paradigm shift in the current forms of therapy. In preclinical model systems, intratumoral administration of DC led to both local and systemic antitumor responses (Sharma et al. 1997). This form of therapy is augmented by utilizing intratumoral administration of genetically modified DC overexpressing certain cytokine genes (Miller et al. 2000). Congruent with this overall concept, the intratumoral administration of recombinant CCL21 -mediated T-cell-dependent antitumor responses (Sharma et al. 2000). In immune competent mice, intratumoral CCL21 injection led to a significant increase in CD4 and CD8 T lymphocytes and DC infiltrating both the tumor and draining lymph nodes. Studies performed in CD4 and CD8 T-cell knockout mice revealed a direct therapeutic requirement for both CD4 and CD8 T-cell subsets for CCL21-mediated tumor regression. Based on these results, experiments were performed to evaluate the tumorigenicity of CCL21 gene-modified murine lung cancer cells. In all three tumor models, subcutaneous implantation of retroviral-mediated CCL21 gene-modified lung cancer cells led to T-cell-mediated tumor eradication. Because the levels of CCL21 required for tumor rejection can be achieved by using CCL21-transduced DC as the transfer vehicle, the intratumoral injection of DC overexpressing CCL21 in the transplantable and spontaneous bronchoalveolar cell carcinoma models of lung cancer was evaluated (Yang et al. 2004). These studies demonstrated that DCs expressing CCL21 are highly effective means to achieve intratumoral delivery of CCL21 in murine models. There was anticipation that this therapy would be most effective in creating a “lymph node-like” environment in the tumor. In fact James Mule reported that DC overexpressing CCL21 demonstrated effective antitumor immunity in lymphotoxin knockout mice that lacked lymph nodes (Kirk et al. 2001b).
Thus, based on the initial studies documenting the antitumor properties of CCL21 and gene-modified DC, this approach was adopted by other investigators who have reported immune-dependent, antitumor properties of CCL21 in a variety of tumor models, including lung (Sharma et al. 2000), colon (Vicari et al. 2000), melanoma (Kirk et al. 2001a; Novak et al. 2007), prostate (Turnquist et al. 2007; Yousefieh et al. 2009), breast (Ashour et al. 2007), and liver (Liang et al. 2007). CCL21 acted as an adjuvant for TERT-DNA vaccine in a breast cancer model and showed immunologically mediated regression of pancreatic tumors in mice upon intratumoral delivery. CCL21 also enhanced the therapeutic efficacy of adoptive T-cell transfer in a murine model of melanoma. In all models, CCL21 demonstrated potent regression of tumors, which was shown to be dependent on host T-cell immunity. All these studies reaffirmed the antitumor efficacy of CCL21 further supporting the rationale to proceed with clinical investigations of this chemokine.
Polymer-Based CCL21 Delivery
The science of biomaterial engineering for drug delivery has evolved considerably for the past 30 years. Novel technology allow to design functional, biocompatible, and biodegradable polymer vehicles, such as poly-ε-caprolactone (PCL), poly (lactide-co-glycolide) (PLG), as well as alginate and fibrin hydrogel, for molecular and cellular delivery in cancer immunotherapy (Huebsch and Mooney 2009). Three dimensional porous polymer scaffolds exhibit great ability to deliver cytokine molecules and immune cells with spatiotemporal specificity, to promote cell-cell interaction in matrix and to direct cell function (Huebsch and Mooney 2009). This ability forms the rationale for polymer-based CCL21 cancer immunotherapy for programming host immune cells in vivo. These materials can be further integrated with other anticancer treatments in the design of next-generation therapy against cancer (Li and Mooney 2013).
PCL/PLCL co-polymer loaded with DC-CCL21 or chemotherapy drug cisplatin has been tested in an animal model of Head and Neck Squamous Cell Carcinoma (HNSCC) to prevent cancer recurrence (Hu et al. 2012; Lin et al. 2014). HNSCC is difficult to resect completely by surgery due to complicated context and therefore exhibits high recurrence rate in the patients (Ross et al. 2004). A drug delivery platform with spatiotemporal specificity is in demand for antirecurrence therapy. In order to accomplish these requirements, a polymer platform was made from a mixture of a ratio of 70:30 of PCL to PLCL with relevant amount of CCL21 and/or cisplatin and was spread on a glass to form a thin sheet. The final product is a flexible sheet that exhibits nice drug release kinetics and can adhere to the surgical resected tissue contours (Hu et al. 2012). In the initial animal study, cisplatin loaded PCL/PLCL polymer was applied intraoperatively to the surgical bed after partial tumor resection, replicating the difficult situation seen in patients. The cisplatin secreting polymer effectively reduced tumors by over 16-fold as compared to control plain polymer and intratumoral cisplatin injection groups. When combined with radiation, polymer therapy led to a statistically significant lower tumor weight compared to the radiation alone group and the control group (Hu et al. 2012). Based on above data, the PCL/PLCL scaffold was later tested for antitumor efficacy of DC-CCL21 therapy. In order to improve DC culture condition for immunotherapy, a thin layer of fibrin hydrogel with 106 DCs seated inside was added to the surface of PCL/PLCL polymer (Lin et al. 2014). The component of hydrogel and polymer was optimized for the maximum production of bioactive CCL21. After implantation to the partially resected tumor, the gradient of local CCL21 that resulted from its sustained and localized release led to the recruitment of CD4+ T cells and CD11c+ DCs into the tumor, while tumor infiltrating Treg cells were decreased. Overall, DC-CCL21 polymer treatment significantly reduced tumor burden, compared to control DC group or recombinant CCL21 injection group (Lin et al. 2014). Currently, antitumor efficacy of polymer loaded with recombinant CCL21 is being further evaluated with combination of cisplatin chemotherapy, immune checkpoint blockade, and radiation therapy.
In addition to cytokines and immune cells, tumor -associated antigen can also be loaded in polymer to activate DCs. Subcutaneous implantation of PLG polymer loaded with cytokine GM-SCF, TLR agonist CpG, and tumor lysate as antigen led to host DC recruitment, activation and subsequent homing to lymph nodes (Ali et al. 2009). This vaccine induced 90% prophylactic tumor protection and therapeutic protection. The polymer scaffold also displayed long term activity for months post implantation, which is superior to all soluble administration methods to date (Ali et al. 2009).
Other biomaterials, such as vault nanoparticles, were investigated for intratumoral CCL21 delivery. In a well-characterized Lewis lung cancer model, CCL21-vault nanoparticles system showed effective antitumor efficacy. A single intratumoral injection of CCL21-vault nanoparticles was able to recruit antitumor effectors that induced potent antitumor activity and inhibit tumor growth (Kar et al. 2011). The nanoparticle system can be further designed for target delivery and specific payloads to prime the immune system.
Based on the preclinical model systems, a clinical trial was initiated using intratumoral injection of CCL21 gene-modified autologous DC in lung cancer. The intratumoral route of DC administration is used to activate specific immune responses within the tumor microenvironment and, in addition, to generate systemic immunity. Several studies suggest (Sharma et al. 1997; Tatsumi et al. 2003) that intratumoral DC administration may be particularly effective as an antitumor strategy. Lung cancer patients have decreased numbers of circulating competent DC; thus, injecting DC within the lung tumor could serve as a particularly effective approach. A correlation exists between the number of tumor-infiltrating DC and survival in cancer patients. In fact, there is a relationship between tumor-infiltrating DC aggregation and apoptosis in situ in human nonsmall cell lung cancer (NSCLC). This is consistent with recent studies indicating that attraction and activation of DC in the tumor elicits potent antitumor immunity (Dieu-Nosjean et al. 2008; Lapteva et al. 2009) have identified ectopic lymph node or tertiary lymphoid structures within human NSCLC specimens and demonstrated a correlation of their cellular content with clinical outcome. These structures have been referred to as tumor-induced bronchus-associated lymphoid tissue, which are follicle-like and contain germinal centers, similar to those in secondary lymphoid follicles of lymph nodes. The density of DC-Lamp, mature DC within these structures, is a predictor of long-term survival in lung cancer patients (Dieu-Nosjean et al. 2008). These findings suggest that tumor-induced bronchus-associated lymphoid tissue has clinical relevance and participates in the host’s antitumor immune response, and they are consistent with previously reported preclinical and clinical data (Zeid and Muller 1993; Kirk et al. 2001b; Coppola and Mule 2008). For example, in murine tumor models, Mule (Kirk et al. 2001b) reported that DC genetically modified to secrete CCL21 produce lymphoid cell aggregates and, importantly, prime naive T cells extranodally within a tumor mass, resulting in the generation of tumor-specific T cells and subsequent tumor regression (Kirk et al. 2001a). Thus, the intratumoral approach may achieve tumor antigen presentation by using the tumor as an in vivo source of antigens for DC. In contrast to immunization with purified peptide antigen(s), autologous tumor has the capacity to provide the activated DC administered in the tumor access to the entire repertoire of available antigens in situ. This may increase the likelihood of a response and reduce the potential for tumor resistance because of phenotypic modulation. On the basis of preclinical results, a phase I clinical evaluation has been initiated at the University of California Los Angeles (in collaboration with the National Cancer Institute – Rapid Access to Intervention Development program) in patients with advanced-stage NSCLC. The safety and clinical activities of the intratumoral administration of autologous DC transduced with a replication-deficient adenoviral vector to express CCL21. A GMP grade AdCCL21 replication-deficient virus (Baratelli et al. 2008) was made available through the RAID program to conduct the phase I clinical trial. Human DCs transduced with advenovirus-CCL21 produce CCL21 to attract T cells and DCs. The findings demonstrate tumor-specific systemic immune responses as assessed by the IFN-γ T-cell ELISPOT. Multiplex assessment of plasma cytokines before and after therapy in these patients revealed induction of IL-2, IFN-γ, IL-12, and CXCL10. Immunohistochemistry of posttreatment tumor biopsies revealed an influx of CD8-expressing tumor-infiltrating lymphocytes (Lee et al. 2014) and increased PD-L1 expression in tumor by qRT-PCR.
In melanoma, Mule’s group have genetically modified human tumor lysate pulsed (TL) DC to secrete human CCL21 that, similar to preclinical studies, could potently recruit naive human CD4 and CD8 T cells. They showed for the first time that TL-DC secreting CCL21 significantly enhance the level or number of tumor antigen-specific T cells to at least two specific melanoma peptides (i.e., melanoma-associated antigen recognized by T cells [MART-1] and gp100). Thus, TL-DC-producing CCL21 served as a vehicle for both recruiting naive T cells and enhancing the production of tumor-specific T cells. These data in human have provided the feasibility for an ongoing clinical trial (in collaboration with the National Cancer Institute – Rapid Access to Intervention Development program) in melanoma patients at the Moffitt Cancer Center. A phase I clinical trial was initiated to assess the toxicity, immune responses, and antitumor clinical responses in human leukocyte antigen-A*0201-positive patients with chemotherapy-naive metastatic melanoma receiving escalating doses of adenoviral CCL21-transduced DC matured ex vivo with a cytokine cocktail and pulsed with MART-1/gp100/NY-ESO-1 class I peptides and keyhole limpet hemocyanin. In this study, patients received the vaccine intradermally at multiple sites. To date, 12 patients (the first two of three dose cohorts) have been treated, and results show indications of the known chemotactic activity of CCL21 through the accumulation of CD3 expressing T cells in biopsies of one of the several injection sites.
Anticipated High Impact Results
The results of the phase I studies in lung cancer and melanoma are promising. CCL21 are important in the formation of tertiary lymphoid structures, and their presence in tumors is associated with favorable immune responses. There is a general consensus that immunogenic tumors, with immune response positive gene signatures and/or increased TIL, have a better prognosis. Based on the findings on CCL21, it is anticipated that the rational combination with immune checkpoint blockade therapy will improve the antitumor benefit of this chemokine in a broad range of solid tumors with low TIL frequency. Future studies could assess the combined efficacy of CCL21-based regimens with immune checkpoint blockade therapy in various solid tumors as immune activation by CCL21 leads to upregulation of PD-1 on activated T cells. CCL21-based therapeutic vaccination approaches will prove beneficial for tumors that are not accessible for intratumoral administration of CCL21. Furthermore, material and nanoparticle engineering provides several attractive strategies to design more potent CCL21 immunotherapy.
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