Development of TLR9 agonists for cancer therapy (original) (raw)
TLR9 activation enhances tumor vaccination in humans. Antigen-specific CD4+ and CD8+ T cells can be induced by a tumor vaccine and can mediate immune rejection of solid tumors. Although tumor vaccines have shown outstanding results in mouse models, objective human clinical responses are rarely achieved (61). Therefore, the emphasis of many phase I and phase II clinical trials has shifted into detailed basic studies of the human T cell response to the tumor antigens during successful and unsuccessful vaccination. Ideally, both memory and effector T cells should be induced. However, there is an increasing appreciation of the fact that not all antitumor T cells are created equal — ineffective T cell triggering leads to much lower numbers of T cells that are less active killers and might even be tolerant of the tumor (62).
Melanoma-associated antigen recognized by T cells 1 (MART1) is a melanocyte protein that is expressed in most melanomas and also in several other types of tumors and has been used as a component of some investigational melanoma vaccines. MART1 is unique among tumor antigens in that both normal subjects and patients with melanoma have a remarkably high frequency of T cells specific for this antigen. Many of these T cells are specific for one particular region of the protein that can be synthesized as a peptide for more detailed studies by flow cytometry using tetramers and other analytic techniques, enabling detailed investigations of the quantity and quality of the T cell response during vaccination with different adjuvants or formulations (63, 64). These studies have revealed that there is no increase in the frequency of T cells specific for this MART1 peptide following vaccination with a synthetic form of this peptide if the peptide is formulated in saline or mixed with the saponin QS-21 and the TLR4 ligand monophosphoryl lipid A, which has been used as a vaccine adjuvant in other settings. In contrast, vaccination with the MART1-derived peptide in incomplete Freund’s adjuvant (IFA) stimulated about half of vaccinated patients to generate increased frequencies of MART1-specific CD8+ T cells in the blood, up to an average frequency of 0.1%–0.2% (65). This adjuvant effect of IFA was markedly enhanced upon combination with the TLR9 agonist CPG 7909 at a low dose of 0.5 mg: all vaccinated melanoma patients responded with increased frequencies of antigen-specific CD8+ T cells, which reached a mean frequency of more than 1% (66) (Table 2). This roughly 10-fold increase in antigen-specific T cell frequency makes this IFA and TLR9 agonist combination the strongest reported stimulator of human CD8+ T cell responses in the blood (66). Most importantly, ex vivo analyses of these tumor-specific T cells demonstrated that they had differentiated to effector cells (67). Unfortunately, the MART1-specific tumor-infiltrating lymphocytes in the patients had a less differentiated phenotype than those in the blood, with lower expression of the cytotoxic effectors perforin and granzymes and lower inducible expression of IFN-γ, indicating that measurement of antitumor responses in the blood does not give the complete picture and suggesting that further optimization of this approach to vaccination will be needed if it is to reach its full clinical potential (67). All patients developed inflammatory injection site reactions, which peaked about two weeks after vaccination; several patients also showed reactivation of local inflammation at earlier vaccination sites. Transient mild systemic flu-like side effects from the vaccination were common and included myalgias, arthalgias, fatigue, nausea, malaise, and headache (66). Vaccination of these patients was not associated with dramatic clinical responses, and tumors in the patients contained high numbers of Tregs (67), suggesting that the clinical efficacy of this approach might be enhanced by Treg depletion or inactivation or perhaps by the incorporation of a greater variety of tumor antigens in the vaccine, a higher dose of CPG 7909, or other changes to the clinical protocol.
Published oncology clinical trials with TLR9 agonists
Although the use of peptide antigens allows the most precise analysis of the immune response to tumor vaccination, a whole recombinant protein has the potential advantage of including a greater variety of tumor epitopes. A clinical trial has been conducted in advanced melanoma patients using a recombinant melanoma antigen–encoding gene 3 (MAGE-3) protein and comparing two doses of CPG 7909; a low, vaccine-adjuvant dose of 0.5 mg (the first eight patients were enrolled at this dose) and a high vaccine-adjuvant dose of 1 mg (five patients were enrolled in this cohort). There were no clinical responses in the first cohort, but two of the five patients receiving the higher dose of CPG 7909 had partial responses that became apparent after at least seven vaccinations (68). The clinical utility of TLR9 agonists currently is being investigated in a number of additional vaccine trials that are underway, with results expected during the coming years (Table 3).
Ongoing oncology clinical trials with TLR9 agonists
TLR9 agonist monotherapy can promote antigen-specific antitumor immunity without a vaccine. In mice with relatively small tumors, up to a few millimeters in diameter, CpG monotherapy can be sufficient to induce T cell–mediated tumor regression (69, 70). In most, but not all, mouse tumor models, the efficacy of CpG monotherapy requires injection of the ODN into the tumor (reviewed in ref. 71). To induce rejection of larger or poorly immunogenic tumors, the CpG ODN generally needs to be combined with either a tumor vaccine (reviewed in ref. 71) or with other effective antitumor strategies, such as monoclonal antibody therapy (72–78), other immune therapies (including in combination with ligands for TLR3 or TLR5) (79–84), angiogenesis inhibitors (85), radiation therapy (86), surgery (87, 88), cryotherapy (89), and chemotherapy (87, 90–95) (Table 4). In humans also, monotherapy with the TLR9 agonist CPG 7909 (now called PF-3512676 when used in oncology without a vaccine) or another B-class CpG ODN, 1018 ISS, activates NK cells and induces a Th1 cytokine response in humans with B cell lymphomas (37, 96). Of 23 NHL patients in the i.v. dose-escalation study of PF-3512676, there were two late clinical responses, which suggests a potential role for this approach as one part of a combination treatment regimen (37). Although CpG ODN are strong mitogens for normal B cells, there was no apparent exacerbation of the lymphoma in these patients, perhaps due to the increased immunogenicity of the tumor cells or the preferential induction of apoptosis in tumor cells stimulated through TLR9 (97, 98). Objective responses have been seen in some subjects receiving PF-3512676 monotherapy for the treatment of melanoma, basal cell carcinoma, renal cell cancer, or cutaneous T cell lymphoma (Table 2) (99–102). In the melanoma patients, both intratumoral and s.c. PF-3512676 monotherapy activate pDCs and myeloid DCs (because myeloid DCs do not express TLR9, their activation is indirect) (99, 103). And in a dose-escalation study, one of five melanoma patients receiving intratumoral PF-3512676 showed complete regression of the injected lesion but progressive disease at distant sites; four of five patients with basal cell carcinoma treated in this manner experienced regressions (100). These results with intratumoral PF-3512676 seem generally comparable to the partial activity seen with imiquimod, a TLR7 agonist that has been used topically in the treatment of several premalignant and malignant skin diseases (5). In contrast, when given by systemic injection, imiquimod showed no antitumor activity in clinical trials despite some positive results in mouse models (104). Objective clinical responses occurred in 2 out of 20 advanced melanoma patients receiving s.c. PF-3512676 at a dose of 6 mg weekly and might be associated with increased NK cell cytotoxicity (99). Of 35 patients with advanced renal cell carcinoma who were enrolled in a dose-escalation trial of PF-3512676, two had confirmed objective responses (101), further demonstrating the ability of this investigational agent to provide some antitumor activity. Twenty-eight patients with advanced cutaneous T cell lymphoma who had failed an average of six prior therapies were enrolled in a dose-escalation study of PF-3512676 in which they were treated with weekly s.c. administration of the agent in sequential cohorts starting at 0.08 mg/kg and escalating to 0.36 mg/kg (102). There were three complete and six partial responses and little toxicity beyond injection site reactions and flu-like symptoms. Responses were evident starting as early as two weeks, occurred in all of the dose groups except for the lowest, and persisted for the duration of the study. Together, these clinical trials of TLR9 therapy as a single agent are encouraging for a good safety profile, but the frequency of objective responses has been relatively low, and the focus of ongoing clinical trials therefore has shifted to combination therapies in an attempt to increase the clinical effectiveness of administering TLR9 agonists.
Nonvaccine therapeutic approaches that enhance TLR9-mediated antitumor activity in mice
Chemotherapy can enhance the antitumor effects of immune stimulators, including TLR9 activation. Since chemotherapies are commonly considered to be immunosuppressive, it might seem counterintuitive to combine such a therapy with an immune therapy. Nevertheless, numerous studies in rodents have demonstrated that many different chemotherapy regimens can enhance various immune therapies. For example, both cyclophosphamide and paclitaxel enhance the Th1 response to a tumor vaccine when given prior to vaccination (but not if given following vaccination) (105, 106), and gemcitabine increases the CD8+ T cell response when given prior to an immune-activating CD40-specific antibody (107). Further studies have elucidated several potential mechanisms for this somewhat paradoxical synergy. First, cyclophosphamide and methotrexate reduce the number and/or function of Tregs in mice and rats, improving the activity of tumor vaccines (108–110). The results of these and other studies suggest that Tregs might be more sensitive to certain chemotherapy regimens than normal T cells, providing a rationale for following chemotherapy with an immunotherapy (111–113). Second, docetaxel therapy in mice promotes the survival of activated T cells to a GM-CSF–producing tumor vaccine (114), providing a possible hint that the antitumor T cells activated by an immunotherapy might have a greater resistance to the toxic effects of taxane chemotherapy compared with naive T cells (or Tregs). Third, etoposide and mitomycin C have recently been reported to cause calreticulin to be exposed on the surface of apoptotic tumor cells, conferring on them immunogenicity to an immune therapy (115). Finally, a tumor antigen–pulsed DC vaccine has been shown to decrease the toxicity of the combination therapy of 5-fluorouracil and irinotecan in a model of colon cancer (116).
Evidence of the immune-boosting effects of chemotherapy has also come from human clinical trials. First, humans receiving certain chemotherapy regimens, such as taxanes, actually show increases in some T cell and NK cell functions (111–113). Second, cyclophosphamide and fludarabine have been proposed to function by “making space”; chemotherapy-induced lymphodepletion followed by homeostatic proliferation of transferred autologous T cells has induced several impressive clinical responses to therapy with a combination of adoptive transfer of activated antitumor T cells and IL-2 (117). Third, gemcitabine, oxaliplatin, and 5-fluorouracil result in reduced numbers of Tregs and increased numbers of tumor-specific CTLs when used in combination with GM-CSF and IL-2 (118).
The mechanisms through which different chemotherapy regimens enhance immunotherapy are likely to be diverse and cannot be accounted for solely by tumor debulking, since in one study, there was no survival benefit from immunotherapy in mice whose tumors were surgically resected to the same residual tumor volume reached in the gemcitabine-treated mice (107). Furthermore, even when tumor challenge was delayed until one week after the last dose of chemotherapy to avoid the cytolytic effect on the tumor cells, it still enhanced the protective effect of tumor vaccination in mice (105). Since the direct antitumor effects of chemotherapy generally are insufficient to account for its enhancement of immunotherapy, the focus of many investigators has shifted to the immune effects of chemotherapy and their possible applications in improving the outcomes of combination therapy in humans.
The efficacy of TLR9 agonists is increased by Treg depletion. Recent studies in mouse models of cancer show that for at least some tumor vaccines (without TLR9 agonists), the deletion or inhibition of Tregs is essential for the recruitment to the antitumor response of the highest avidity CD8+ T cells, which are the most potent tumor killers (110, 119). Chemotherapies reported to deplete Treg numbers and/or function in mice include cyclophosphamide, methotrexate, and fludaribine (90, 108–110) as well as docetaxel, which induces a mild lymphodepletion with the loss of both Tregs and memory T cells but is associated with substantially prolonged survival of activated T cells (114). Doxorubicin might augment immunotherapy through a different mechanism than docetaxel or other taxanes and other chemotherapies since it alone does not increase the number of tumor-specific T cells, does not seem to eliminate Treg activity, and enhances vaccination only when given at the time of T cell expansion, not if used as a pretreatment (105, 110). In recent human clinical trials, gemcitabine, oxaliplatin, and fluorouracil also have been reported to reduce Treg numbers, suggesting possible utility for regimens containing these agents in enhancing immunotherapy (118, 120).
In mouse tumor models, TLR9 activation has been reported to synergize with a wide range of chemotherapy regimens, including cyclophosphamide (87, 90), topotecan (87, 91), 5-fluorouracil (92), gemcitabine (93, 94), alimta (94), and coramsine (95). The mechanism of this synergy may result at least in part from the effects of these chemotherapies in reducing Treg numbers. Established tumors are associated with increased Treg activity, which protects the tumor from immune rejection and is not readily overcome by TLR9 activation alone. Treg-depleting chemotherapies not only directly disrupt the tumor and induce tumor cell apoptosis and immunogenicity but also eliminate Treg function, making the tumor far more susceptible to TLR9-induced innate and adaptive immune responses. In effect, in the altered immune environment that follows chemotherapy, TLR9 activation of pDCs might be sufficient to boost effective Th1 responses against tumor antigens that had previously been ignored by the immune system (Figure 3). Indeed, in mouse models of rhabdomyosarcoma treated with cyclophosphamide (87), we have found that the prolonged survival of mice receiving chemotherapy combined with s.c. PF-3512676 is mediated by an enhanced antitumor T cell response.
Potential mechanism to explain the observed antitumor synergy of TLR9 with chemotherapy. Tumor antigens are constitutively present in cancer patients and are taken up by DCs in the tumor and secondary lymphoid organs. In patients with established tumors, the immune system fails to respond to the tumor antigens due to the tumor-induced Tregs and other immune-suppressive effects. Certain chemotherapy regimens effectively deplete Tregs and also disrupt the tumor, releasing additional antigen and interfering with the ability of the tumor and its mesenchymal support structures to suppress the immune system. When such chemotherapy is followed by treatment with a TLR9 agonist (CpG ODN), DCs bearing tumor antigens are stimulated to mature and become effective inducers of a CTL response, which in the postchemotherapy environment is now better able to attack the tumor, leading to improved survival.
Clinical development of chemotherapy combined with TLR9 agonist. Based on these results, we conducted a phase II randomized controlled human clinical trial to investigate the effect of adding PF-3512676 to standard taxane and platinum chemotherapy in previously untreated stage IIIb or stage IV non–small cell lung carcinoma (NSCLC) (121). A total of 111 patients were randomized to receive four to six 3-week cycles of standard chemotherapy alone or in combination with 0.2 mg/kg s.c. PF-3512676 on the first day of weeks two and three of each cycle. The primary endpoint for the trial, objective response rate, was higher in the patients who received standard chemotherapy plus PF-3512676 than in those who received standard chemotherapy alone (38% versus 19% for confirmed and unconfirmed responses; P = 0.048) (121). There was also a trend to improvement in one-year survival from 33% in the patients randomized to receive standard chemotherapy to 50% in the patients receiving the combination (121). The most common side effects were mild to moderate injection site reactions and transient flu-like symptoms. Grade 3 or 4 neutropenia was more common in the combination arm and is thought to reflect neutrophil redistribution, as febrile neutropenia and grade 3 or 4 infections were less common in the combination arm than in the chemotherapy alone arm. Thrombocytopenia, which is a known sequence-independent effect of PS ODN and which has been observed in all systemic clinical trials of antisense PS ODN in applications such as infectious disease and oncology (122), was seen more commonly in patients receiving the combination but with no apparent increase in bleeding events. To pursue these encouraging results, 2 phase III human clinical trials of PF-3512676 combined with standard platinum-based doublet chemotherapy (one trial using the doublet of cisplatin and gemcitabine and the other trial using the doublet of carboplatin and paclitaxel) have been initiated in advanced NSCLC, and several phase II trials have begun exploring other combination regimens with PF-3512676 (Table 3).
It seems probable that not all chemotherapy regimens will be equally effective when combined with TLR9 activation. Dacarbazine, a relatively weak chemotherapy that has not been shown to deplete Tregs or to boost any other immunotherapy, resulted in no improvement in survival and only a slight increase in response rate when administered in combination with CPG 7909 in a randomized phase II clinical trial performed in 184 advanced melanoma patients (123). Further studies into the effects of various chemotherapy regimens on immune function might make it possible to design combination therapies that will predictably provide greater clinical benefit to patients.