Toll-Like Receptor 9 (TLR9) Agonists in the Treatment of Cancer

AM Krieg

Coley Pharmaceutical Group Inc., Wellesley, MA, USA

Although still early in clinical development, agonists of Toll- CD4 T cells (Gelman et al., 2006), but the biologic role for like receptor 9 (TLR9) have demonstrated potential for the this is less well understood. TLR9 expression has also been treatment of cancer. TLR9 agonists directly induce activa- reported in some nonimmune cells, including pulmonary tion and maturation of plasmacytoid dendritic cells and epithelial cells and lung cancers (Li et al., 2004; Platz et al., enhance differentiation of B cells into antibody-secreting 2004; Droemann et al., 2005), keratinocytes (Lebre et al., plasma cells. Preclinical and early clinical data support the 2007) and intestinal epithelium (Pedersen et al., 2005; Lee use of TLR9 agonists in patients with solid tumors and et al., 2006). hematologic malignancies. In preclinical studies, TLR9 TLR9 recognizes and is activated by unmethylated agonists have shown activity not only as monotherapy, but cytosine-phosphate-guanine (CpG) dinucleotides, which also in combination with multiple other therapies, including are relatively common in bacterial and viral DNA but are vaccines, antibodies, cellular therapies, other immunothera- suppressed and methylated in vertebrate DNA (Krieg, pies, antiangiogenic agents, radiotherapy, cryotherapy and 2004). Binding of DNA containing unmethylated CpG some chemotherapies. Phase I and II clinical trials have motifs to TLR9 causes a conformational shift in the indicated that these agents have antitumor activity as single receptor, which is thought to result in recruitment of the agents and enhance the development of antitumor T-cell adapter protein MyD88, activation of signaling pathways responses when used as therapeutic vaccine adjuvants. The with the phosphorylation of mitogen-activated protein activity and safety of these novel anticancer agents are being kinases and activation of nuclear factor-kB(Latzet al., explored in a wide range of tumor types as part of a variety 2007). At a cellular level, activation of TLR9 initiates a of therapeutic strategies with the goal of harnessing the cascade of innate and adaptive immune responses immune response to fight cancer. (Figure 1). TLR9 agonists activate pDCs to secrete type Oncogene (2008) 27, 161–167; doi:10.1038/sj.onc.1210911 Iinterferon(IFN)andtoexpressincreasedlevelsofco- stimulatory molecules such as CD80 (B7.1) and CD86 Keywords: PF-3512676; CpG ODN TLR9; Toll-like (B7.2). This is believed to initiate a range of secondary receptor agonists; vaccine adjuvant effects, including secretion of cytokines/chemokines (for example, monocyte chemoattractant protein-1 (MCP-1), IFN-g-inducible 10 kDa protein (IP-10)), activation of natural killer (NK) cells and expansion of T-cell popula- tions, particularly type 1 helper T (TH1) cells and cytotoxic Toll-like receptor 9 T lymphocytes (CTLs) (Krieg, 2004, 2006). As a result a potent, cell-mediated TH1 response is initiated. A humoral The human immune system detects and responds to immune response is also initiated as TLR9 agonists infectious challenges using several families of receptors enhance differentiation of B cells into antibody-secreting that can recognize pathogen-expressed molecules, such as plasma cells, potentially promoting antibody-dependent lipopolysaccharide, viral RNA or bacterial DNA (Akira cellular cytotoxicity (Appay et al., 2006). An under- et al., 2006). The most well understood of these receptors standing of the immune cascade initiated by TLR9 are the Toll-like receptors (TLRs), of which a family of 10 activation has prompted the clinical development of related molecules has been identified in humans (Akira several TLR9 agonists in the fields of infectious disease, et al., 2006). The immune role of TLR9 has been studied cancer and asthma/allergy. The rationale for investigating most extensively in plasmacytoid dendritic cells (pDCs) TLR9agonistsasanticanceragentsisbasedonthe and B cells (Iwasaki and Medzhitov, 2004), which may be hypothesis that the innate immune activation may have the only human immune cells to constitutively express direct antitumor effects and that the enhanced tumor TLR9. Cellular activation is reported to induce TLR9 antigen presentation in a TH1-like cytokine and chemokine expression in additional cell types, including human milieu will promote an antitumor immune response. neutrophils (Hayashi et al., 2003), monocytes and mono- Several synthetic oligodeoxynucleotide (ODN) agonists cyte-derived cells (Saikh et al., 2004; Siren et al., 2005) and for TLR9 are currently in development for the treatment of cancer. Because the phosphodiester bond of native DNA is Correspondence: Dr AM Krieg, Coley Pharmaceutical Group Inc., rapidly degraded by endonucleases, these investigational Suite 101, 93 Worcester Street, Wellesley, MA 02481, USA. CpG ODNs use a nuclease-resistant phosphorothioate E-mail: [email protected] backbone that improves the half-life in the body from just Cancer therapy with TLR9 agonists AM Krieg 162

Figure 1 (1) Toll-like receptor 9 (TLR9) agonists stimulate innate and adaptive antitumor immune responses. TLR9 agonists induce secretion of interferon-a(IFN-a) from immature plasmacytoid dendritic cells (pDCs), which may activate natural killer (NK) cell lysis of tumor cells and release tumor antigens (Ags). (2) Dendritic cells activated directly or indirectly by the TLR9 agonist therapy can capture and process tumor-associated antigens, increase surface expression of major histocompatibility complex (MHC) and costimulatory receptors, and (3) migrate to the lymph nodes where antigens can be presented to lymphocytes, inducing (4) expansion of lymphocytes that recognize the tumor antigen. Because the response occurs in a TH1-like cytokine milieu, the lymphocyte response tends to be TH1-like, including cytotoxic T lymphocytes (CTLs).

a few minutes (for unmodified native DNA) to about 48 h. versus 22% for topotecan alone, P ¼ 0.09) (Weigel et al., Coley Pharmaceutical Group (Wellesley, MA, USA) has 2003). The combination of a CpG ODN with radiation developed a CpG ODN known as CPG 7909 that is being therapy also enhanced tumor response in a murine investigated as a vaccine adjuvant in several tumor types fibrosarcoma model (Milas et al., 2004). However, mice (Table 1). CPG 7909 was licensed by Pfizer Pharmaceuticals and humans have a different TLR9 expression pattern, Inc. (New York, NY, USA) for clinical investigation as a and exposure to CpG motifs stimulates a more narrow single agent or in combination with other therapeutic profile of cytokines/chemokines in humans (Krieg, 2007). approaches, and is now known as PF-3512676 when it is Thus, preclinical results cannot be considered predictive not being used as a vaccine adjuvant. Other TLR9 agonists of clinical findings. However, based on the strength of the in clinical development for cancer include ISS 1018 extensive preclinical data, several TLR9 agonists are now (Dynavax Technologies, Berkeley, CA, USA), IMO-2055 being developed as anticancer agents. (Idera Pharmaceuticals Inc., Cambridge, MA, USA) and CpG-28 (University of Paris, France). Monotherapy with TLR9 agonists

Cancer therapy with TLR9 agonists Hematologic malignancies In addition to enhancing maturation of B cells into The initial impetus to develop TLR9 agonists as antic- antibody-secreting plasma cells, TLR9 agonists have a ancer drugs came from several preclinical studies demon- variety of other effects on B cells that may be relevant in strating antitumor activity in a wide variety of tumor the treatment of hematologic malignancies. Activation of models. For example, in a murine cervical carcinoma TLR9 on primary malignant B cells upregulates expres- model, mice with established subcutaneous (s.c.) tumors sion of major histocompatibility complex molecules and treated with CpG ODNs injected at a distant site other surface receptors, thereby increasing their capacity exhibited signiﬁcant regression (Po0.05), and treated to stimulate T cells (Decker et al., 2000; Jahrsdorfer et al., mice had a signiﬁcant improvement in survival (Po0.001) 2001; Wooldridge and Weiner, 2003). This may result in compared with control mice (Baines and Celis, 2003). In an enhanced T-cell-mediated response to tumor antigens addition, >50% of mice (24 of 42) with established on the malignant B cells. Furthermore, in patients with tumors had a complete regression following treatment for advanced cutaneous T-cell lymphoma (CTCL), NK cells 9 days (Baines and Celis, 2003). TLR9 agonists also have and CD8 þ T cells were activated following culturing of been successfully combined with traditional anticancer peripheral blood mononuclear cells with CpG ODNs, therapies (for example, radiation therapy, chemotherapy) and there was a marked increase in IFN-a production or other immunotherapies (for example, vaccines). For (Wysocka et al., 2004). This could enhance the antitumor example, in mice with established orthotopic rhabdomyo- immune response by activating NK cells, or may induce a sarcoma tumors, treatment with a TLR9 agonist plus direct antiproliferative effect (Sabel and Sondak, 2003). topotecan resulted in an improvement in survival rate (41 Chronic lymphocytic leukemia (CLL) cells, which express

Oncogene Cancer therapy with TLR9 agonists AM Krieg 163 Table 1 Toll-like receptor 9 agonists in clinical trials for cancer Agent Company Tumor type Stage of development

PF-3512676 (formerly CPG 7909) Pﬁzer Breast cancer Phase I/II CLL Phase I CTCL Phase I Melanoma Phase II NHL Phase I/II GlaxoSmithKline NSCLCa Phase III RCC Phase I

ISS 1018 Dynavax CRC Phase I NHL Phase II

IMO-2055 Idera RCC Phase I Refractory solid tumors Phase I/II

CpG-28 University of Paris Glioblastoma Phase II

Abbreviations: CLL, chronic lymphocytic leukemia; CRC, colorectal cancer; CTCL, cutaneous T-cell lymphoma; NHL, non-Hodgkin’s lymphoma; NSCLC, non-small cell lung cancer; RCC, renal cell carcinoma. aClinical development in combination with chemotherapy has been discontinued.

TLR9, are induced by CpG ODN to undergo apoptosis, clinical activity in a phase I trial of intra- or perilesional in contrast with normal primary B cells in which TLR9 injection in patients with metastatic melanoma (n ¼ 5, activation protects against apoptosis (Jahrsdorfer et al., with one local regression) or basal cell carcinoma (n ¼ 5; 2005). with one CR and three PRs) (Trefzer et al., 2002; Several clinical studies of single-agent TLR9 agonists Hofmann et al., submitted). Treatment was associated have been completed in patients with hematologic with increased levels of serum interleukin (IL)-6, IL- malignancies. In a phase I study of PF-3512676 in 12p40 and IP-10 in some patients, and cellular infiltrates patients (N ¼ 28) with CTCL, seven (25%) patients of CD8 þ lymphocytes were found after treatment in most achieved an objective response as determined by a lesions. In a different trial involving patients with clinical Physician Global Assessment (two complete responses stage I/II melanoma, surgical resection of the primary (CRs), five partial responses (PRs)) (Kim et al., 2004). tumor was followed by randomization to receive either Common adverse events (AEs) included mild to saline or PF-3512676 (8 mg) intradermally at the excision moderate injection-site reactions (erythema, induration, site, followed 1 week later by a sentinel lymph node edema, inflammation and pain) and flu-like symptoms procedure (Molenkamp et al., 2007). In this setting, PF- (fatigue, rigors, fever, arthralgia) (Kim et al., 2004). PF- 3512676 was shown to induce the release of inflammatory 3512676 has also been studied in a phase I trial in cytokines and decrease the number of regulatory T cells patients with refractory non-Hodgkin’s lymphoma observed in sentinel lymph nodes of patients with stage I (NHL). In patients (N ¼ 23) receiving PF-3512676 to III melanoma (Molenkamp et al., 2007). Furthermore, (0.01–0.64 mg kgÀ1) intravenously (i.v.) up to three times both myeloid and pDCs were activated (Molenkamp a week, NK cell numbers and activation were enhanced et al., 2007), indicating that PF-3512676 clearly has in most subjects (Link et al., 2006). The majority of AEs immunomodulatory activity. In a different phase II study were mild to moderate and transient. Serious hemato- in patients (N ¼ 20) with metastatic melanoma treated logic AEs included anemia, thrombocytopenia and with s.c. PF-3512676, two (10%) patients had a PR and neutropenia, but were mainly judged to be due to three (15%) patients had stable disease (SD) (Pashenkov disease progression (Link et al., 2006). In both trials, et al., 2006). These patients with possible clinical benefit PF-3512676 was determined to be well tolerated at levels from PF-3512676 therapy tended to have increased levels that can stimulate immune activation. These studies of NK cell activity compared to the patients with indicated that TLR9 agonists may be useful in the progressive disease. The majority of AEs were mild, treatment of lymphoma. A phase I study is currently consisting of short-term local injection-site reactions and being carried out with PF-3512676 as second-line transient flu-like symptoms (p2 days); grade 3/4 treatment for patients with CLL. laboratory AEs typically resolved without intervention. These studies demonstrated that PF-3512676 is generally well tolerated as a single agent in patients with melanoma Skin cancers and is associated with antitumor activity. Because melanoma is a highly immunogenic tumor that has been shown to respond to immunotherapy, it is a logical malignancy in which to explore the activity of Other solid tumors TLR9 agonists. Two general approaches have been Monotherapy with TLR9 agonists has also been eval- investigated using monotherapy with PF-3512676: local uated in phase I trials in patients with advanced renal cell therapy with intra- or perilesional injection and systemic carcinoma (RCC) or recurrent glioblastoma. A minor therapy with s.c. injection. PF-3512676 demonstrated response was observed in 2 of 24 patients with recurrent

Oncogene Cancer therapy with TLR9 agonists AM Krieg 164 glioblastoma receiving CpG-28 in a phase I trial. the combination of PF-3512676 and paclitaxel significantly Treatment-related AEs consisted mainly of fever (21%), enhanced median survival compared with control mice a worsening of neurologic conditions (17%) and reversible (Po0.0001), and no additive toxicity was observed grade 3 lymphopenia (29%) (Carpentier et al., 2006). In a (Weeratna et al., 2004). Based on both preclinical and phase I dose-escalation trial in patients (N ¼ 31) with clinical proof-of-concept studies, a randomized controlled advanced RCC receiving PF-3512676, one patient (3%) phase II trial was conducted in chemotherapy-naive had a PR and nine patients (29%) had SD (Thompson patients with stage IIIb/IV non-small cell lung cancer et al., 2004). No treatment-related serious AEs were (NSCLC) (Manegold et al., 2007, in press). Patients observed, and injection-site reactions and flu-like symp- received 4–6 cycles of taxane/platinum chemotherapy with toms were dose related and transient (Thompson et al., 0.20 mg kgÀ1 PF-3512676 s.c. on weeks 2 and 3 of each 2004). A phase I trial is currently investigating the activity cycle (n ¼ 75) or chemotherapy alone (n ¼ 37). The of IMO-2055 as first- and second-line treatment of objective response rate was 38% in the PF-3512676 plus patients with RCC. chemotherapy arm (n ¼ 74) versus 19% in the chemotherapy-alone arm (n ¼ 37) by investigator evaluation, and the median overall survival was 12.3 months in the PF- 3512676 plus chemotherapy arm versus 6.8 months in the Combination therapy with TLR9 agonists chemotherapy-alone arm (hazard ratio ¼ 0.747, P ¼ 0.188) (Manegold et al., 2007, in press). The most common PF- Non-Hodgkin’s lymphoma 3512676-related AEs were mild to moderate injection-site Preclinical studies have indicated that the combination reactions and flu-like symptoms, although PF-3512676 of a TLR9 agonist and rituximab (Rituxan), a mono- was associated with an increase in the rate of neutropenia, clonal antibody (mAb) against CD20, may be more anemia and thrombocytopenia. effective than rituximab alone. In a murine lymphoma Following completion of the phase II trials, two phase model, the addition of a CpG ODN to antitumor mAb III studies were conducted exploring the combination of therapy (with an anti-idiotype Ab) reduced the percen- PF-3512676 (0.2 mg kgÀ1 s.c. days 8 and 15) with tage of mice developing a tumor by 70% (Wooldridge paclitaxel/carboplatin (200 mg mÀ2 per area under the et al., 1997). Two phase I clinical trials have investigated curve six i.v. day 1) or gemcitabine/cisplatin (1250 mg mÀ2 the combination of a CpG ODN with rituximab in i.v. day 1 and 8/75mg m À2 i.v. day 1) versus chemother- patients with relapsed/refractory NHL. In a dose- apy alone as first-line treatment of patients with advanced escalation study, 50 patients received PF-3512676 NSCLC (Readett et al., 2007). No improvement in weekly for 4 weeks i.v. or s.c. in combination with overall survival or progression-free survival was observed rituximab (375mg m À2 per week 4 weeks) i.v., while an Â when PF-3512676 was added to standard platinum-based additional s.c. cohort received weekly monotherapy with doublet chemotherapy in either trial. However, there was PF-3512676 for an additional 20 weeks following the a higher frequency of grade 3/4 neutropenia and initial 4 weeks of treatment with rituximab (Leonard thrombocytopenia and a higher frequency of ‘sepsis-like et al., 2007, in press). Twelve patients (24%) had an events’ and ‘septic deaths’ reported as serious AEs in the objective response (five CRs, seven PRs), including 50% PF-3512676 plus chemotherapy arms (Readett et al., of the 12 patients receiving extended dosing with PF- 2007). Based on the recommendation of an independent 3512676 (two CRs, four PRs) (Leonard et al., 2007, in data monitoring safety committee, these phase III studies press). The majority (76%) of patients experienced mild were terminated, along with two phase II trials in which to moderate treatment-related AEs (primarily injection- PF-3512676 was also combined with cytotoxic che- site reactions and systemic flu-like symptoms). Few motherapy in patients with advanced NSCLC. grade 3/4 AEs occurred in more than one patient or at more than one dose level, but four patients developed grade 3/4 neutropenia (Leonard et al., 2007, in press). In a phase I study, 20 patients with relapsed NHL received TLR9 agonists as vaccine adjuvants ISS 1018 weekly for 4 weeks s.c. following the second of four infusions with rituximab (375mg m À2 per week 4 Â A distinguishing characteristic of CpG ODNs is their weeks) (Friedberg et al., 2005). The overall response rate ability to induce strong CD4 þ and CD8 þ T-cell was 32% (one unconfirmed CR, five PRs). Common responses and rapid production of antigen-specific AEs included mild to moderate injection-site reactions antibodies when used as a vaccine adjuvant with many and systemic flu-like symptoms (Friedberg et al., 2005). types of antigen (Krieg, 2007). These CpG ODN Combination therapy with TLR9 agonists is being adjuvants have been heavily studied for the treatment further evaluated for antitumor activity in a phase I/II of infectious diseases, and several clinical trials are also trial in combination with radiation therapy and in a investigating their activity as tumor vaccines. The phase II trial in combination with rituximab. addition of CPG 7909 to a peptide MART-1 vaccine in patients with human leukocyte antigen-A2 þ melanoma Non-small cell lung cancer caused an approximate 10-fold increase in the number Several preclinical models have also suggested that a of antigen-specific CD8 þ T cells (Speiser et al., 2005; TLR9 agonist can synergize with cytotoxic chemotherapy Appay et al., 2006). Anti-MAGE-3 antibody titers were (reviewedinKrieg,2007).InaLewislungcancermodel, enhanced with the addition of CPG 7909 to a MAGE-3

Oncogene Cancer therapy with TLR9 agonists AM Krieg 165 recombinant protein vaccine in a phase I/II trial, and one PF-3512676. The reason for the failure of these trials is patient had a durable objective response (PR for 9 þ unclear, but possible factors include the advanced stage of months) (van Ojik et al., 2002). CPG 7909 is also being the disease, the use of concomitant steroid therapy as studied as a vaccine adjuvant in patients with metastatic premedication for the chemotherapy, and possible im- RCC. Preliminary results suggest that vaccination with mune-suppressive effects of the repeated chemotherapy autologous tumor cells, CPG 7909 and granulocyte cycles. Therapies based on activating an immune response macrophage colony-stimulating factor followed by main- against cancer may be expected to have a higher chance for tenance therapy with IFN-a and PF-3512676 has success in clinical settings of earlier disease, such as the antitumor activity in this population (3 of 12 patients ongoing NSCLC vaccine trial in the adjuvant setting. had a PR) (Van Den Eertwegh et al., 2006). Phase I and Combination therapy with antiangiogenic agents II trials have been further investigating CPG 7909 as a should avoid the immune-suppressive effects of che- vaccine adjuvant in patients with melanoma and breast motherapy and also has shown benefit in preclinical cancer. The most advanced cancer vaccine program is a models when combined with TLR9 agonists (Damiano phase III, randomized, controlled, clinical trial of the et al., 2006, 2007). An ongoing clinical trial in NSCLC is tumor antigen MAGE-A3 combined with CPG 7909 addressing the safety and potential clinical benefit of (and other adjuvants) for the treatment of patients with PF-3512676 combined with the antiepidermal growth early-stage (stage IB, II or IIIA), completely resected factor receptor agent erlotinib. NSCLC whose tumors express the antigen (Table 1). Tumors can develop a variety of mechanisms to evade or suppress the immune system, and combination immunotherapeutic approaches may allow for multiple opportunities to slow or halt tumor progression. T-cell Safety of TLR9 agonists expression of CTL-associated antigen 4 (CTLA4) is induced during immune responses as a brake on the Overall, TLR9 agonists are generally well tolerated in immune system, reducing the magnitude and functional patients with cancer. The most common AEs observed capacity of the effector T-cell response. Blockade of with administration of TLR9 agonists are local injec- CTLA4 with mAbs sustains T-cell proliferation and tion-site reactions (for example, erythema, edema, activation. By stimulating the DC side of the immune inflammation and pain) or systemic flu-like symptoms response and enhancing antigen presentation to T cells, (for example, headache, rigors, pyrexia, nausea and TLR9 agonists may synergize with the effect of anti- vomiting). These symptoms typically develop with 24 h CTLA4 mAbs ability to remove the brake on the T cells. of dosing and are transient, generally lasting for less Indeed, in preclinical tumor vaccine models, the combi- than 2 days (Krieg, 2006). Grade 3/4 hematologic AEs nation of a TLR9 agonist and CTLA4 blockade led to (that is, anemia, neutropenia and thrombocytopenia) improved rejection of established tumors through com- et al have been observed with TLR9 agonists (Link ., plementary mechanisms: the effect of anti-CTLA4 mAbs 2006; Leonard et al., 2007, in press; Manegold et al., is largely dependent on CD4 þ Tcells,whereastheeffect 2007, in press), and may require monitoring in future of the combination appears to be mediated by CD8 þ T clinical trials. However, unlike other therapeutic im- cells (Davila et al., 2003; Daftarian et al., 2004). The munotherapies for cancer such as IFN-a, TLR9 agonist combination of anti-CTLA4 mAb and PF-3512676 is therapy has not been associated with clinically signifi- currently being evaluated in a phase I, open-label, cant autoimmune diseases. nonrandomized, dose-escalation study in patients with metastatic melanoma and may provide a foundation for future therapeutic approaches. At the present time, the Conclusions use of TLR9 agonists for enhancing tumor vaccination is the most advanced application of this technology in Although it is still early in their clinical development, oncology, and the early results from this approach appear TLR9 agonists are generally well tolerated and have promising. Clinical trials are ongoing in other settings demonstrated potential as a novel immunotherapeutic and in combination with other therapeutic approaches, approach to the treatment of cancer. Objective tumor and the true therapeutic potential of TLR9 agonists for responses to TLR9 agonist monotherapy have been the treatment of cancer remains to be determined. observed in multiple tumor types, including both solid tumors and hematologic malignancies. Although these Acknowledgements results are encouraging evidence of the potential clinical benefits of TLR9 activation, the greatest improvement in Financial support for medical editorial assistance was pro- patient outcomes is likely to result from the use of this vided by Pfizer Pharmaceuticals. I thank Todd Parker, PhD, approach in combination with other therapies that work in ProEd Communications Inc., for expert editorial assistance a synergistic manner. Preclinical work and early clinical with manuscript preparation. trials suggested that combination with cytotoxic chemotherapy may be one such approach, but two phase III Conflict of Interest trials of PF-3512676 administered concurrently with Arthur M Krieg is a founder, employee and shareholder in chemotherapy for patients with NSCLC have recently Coley Pharmaceutical Group and has a financial interest in reported a lack of incremental efficacy with the addition of the development of TLR9 agonists for cancer therapy.

Oncogene Cancer therapy with TLR9 agonists AM Krieg 166 References

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