Potential use of CpG ODN for cancer immunotherapy

u p d a t e o n c a n c e r t h e r a p e u t i c s 1 ( 2 0 0 6 ) 49–58

available at www.sciencedirect.com

journal homepage: www.updateoncancer.com

Potential use of CpG ODN for cancer immunotherapy Risini D. Weeratna a,∗ , Heather L. Davis a , Liana Medynski a , Arthur M. Krieg b a b

Coley Pharmaceutical Group, 340, Terry Fox Drive, Suite 200, Ottawa, Ont., Canada K2K 3A2 Wellesley, MA, USA

a r t i c l e

i n f o

a b s t r a c t

Keywords:

Tumor immunotherapy has advanced over the past century from the use of crude bacterial

CpG ODN

extracts such as Coley’s toxins to the use of recombinant cytokines and now to the use of

Toll-like receptor (TLR)

synthetic ligands to specific immune receptors such as the Toll-like receptors (TLR) which

TLR9

activate the vertebrate immune system through recognition of specific microbial compo-

Tumor immunotherapy

nents (pathogen-associated molecular patterns or PAMPs). Synthetic oligodeoxynucleotides containing immunostimulatory CpG motifs (CpG ODN) which act as ligands for TLR9 have been used successfully as tumor immunotherapy in animal models as well as in human clinical trials. This review describes the immune effects of CpG ODN, and their applications in immunotherapy of cancer. © 2006 Elsevier Ltd. All rights reserved.

1.

Introduction

In the late 1800s, Dr. William B. Coley, a surgeon from New York, successfully treated numerous cancer patients with a cocktail of killed Streptococcus pyogenes and Serratia marcescens bacteria which is now commonly known as “Coley’s toxin”. Even though his earliest and most publicized successes were with sarcoma patients, Coley’s toxin was also successfully used by Dr. Coley and his contemporaries to treat patients with other types of cancers such as carcinomas, lymphomas, melanomas and myelomas (reviewed in [1]). Despite these encouraging observations in the late 1800s, the use of immunotherapy in cancer was almost nonexistent in the early mid 20th century and the majority of cancer patients currently are treated with some combination of surgery, radiation and chemotherapy. With increasingly rapid advancement in the field of immunology, the concept of using immunotherapy for the treatment of cancer has recently been re-emerging.

∗

2. Immunostimulatory properties of bacterial DNA Reported success of Coley’s toxin in treating cancer encouraged the investigation of other microorganisms for apparent anti-tumor activity. Injection of microbial agents such as heat-killed bacillus Calmette-Guerin (BCG) into tumors was shown to mediate tumor regression in a number of preclinical and subsequent clinical studies [2]. The main contributor of anti-tumor activity of heat-killed BCG was identified to be its DNA [3]. This led to the discovery of the ability of bacterial DNA to activate the vertebrate immune cells [4]. The specific immunostimulatory sequences in bacterial DNA were later discovered to be CpG dinucleotides in particular base contexts (CpG motifs) [5]. In prokaryotic organisms, these motifs are present at the expected frequency of 1 in 16 bases, whereas in vertebrate DNA, these motifs are suppressed to one third or one quarter of the expected frequency [6]. In addition, within mammalian DNA the cytosine residues are highly

Corresponding author. Tel.: +1 613 254 5622x3144; fax: +1 613 254 5625. E-mail address: [email protected] (R.D. Weeratna). 1872-115X/$ – see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.uct.2006.04.002

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methylated [6] which essentially eliminates their immunostimulatory properties [5,7]. These differences likely reflect an evolutionary divergence that has resulted in one of the many mechanisms by which vertebrates can recognize and respond to invading pathogens. It has recently been recognized that the vertebrate innate immune system uses pattern-recognition receptors such as the Toll-like receptors (TLRs), to detect pathogen-associated molecular patterns (PAMPs) present in infectious agents [8]. To date, 10 different TLRs have been identified in humans, as well as a number of naturally occurring TLR-ligands [8]. One factor that has recently received some attention in explaining the specificity of TLR stimulation by pathogen structures is the cellular location of these TLRs. TLRs that detect PAMPs characteristic of extracellular pathogens, such as proteoglycans, lipopeptides (TLR2), lipopolysaccharides (TLR4), and flagellin (TLR5) are expressed on the cell surface where as, TLRs that detect PAMPs that would be characteristic of an intracellular infection with viruses or other intracellular pathogens, such as double stranded RNA (TLR3) or single stranded RNA (TLR 7/8) from viruses or DNA (TLR9), appear to be restricted in their expression to an endosomal compartment [9]. In humans, TLR9 expression is generally thought to be limited to only B cells and plasmacytoid dendritic cells (pDCs), although some studies have also reported expression in activated neutrophils [10] and pulmonary epithelial cells [11,12], whereas in mice TLR9 expression is more ubiquitous and is found on pDCs, myeloid dendritic cells (mDCs) as well as on B cells [13,14]. Direct activation of murine and human B cells through TLR9 can lead to immunoglobulin production, IL-6 and IL-10 secretion, MHC class II and CD80 and CD86 up-regulation, and resistance to apoptosis. CpG ODN-mediated activation of dendritic cells (DCs) leads to CD4 independent maturation of these cells and subsequent release of chemokines and pro-inflammatory cytokines such as type I interferons (IFN), IL-12, up-regulation of MHC class II, and expression of co-stimulatory molecules such as CD80, CD86, and CD40 transforming the immature DCs into mature potent antigen presenting cells [15]. Furthermore, induction of cytokines and chemokines by activated cells trigger a cascade of immune effects such as activation and migration of natural killer (NK) cells, augmenting their lytic activity and promoting secretion of cytokines such as IFN-␥ activation, and enhanced PMN migration in response to inflammatory signals. In addition to innate immune activation, in the presence of an antigen, CpG ODN can promote the induction of strong Th1biased immune responses. Stimulation of B cells by CpG ODN in the presence of antigen can selectively enhance the development of antigen-specific antibodies, especially of the isotype associated with Th1-like immune responses (e.g., IgG2a in mice). Following CpG ODN stimulation, both B cells and DC can effectively present antigen to T cells. CpG-induced antigen presentation taking place in a Th1-like cytokine milieu can lead to induction of strong Th1 biased immune responses consisting of cell-mediated as well as humoral immunity [16,17]. In mice injected with CpG ODN, the Th1-like cytokine milieu and lymphadenopathy in the draining lymph node (LN) peaked at 7–10 days [18,19]. This Th1-like environment appears to be sustained for at least several weeks since CpG-

primed mice respond to an antigen injection with a Th1biased response even five weeks later [18,19]. Three different classes of CpG ODN have been described so far, based on distinct structures and immune activation profiles [20–22]. A-class CpG ODN can induce the production of high levels of IFN-␣, and cause marked NK cell activation. However, they are relatively poor in stimulating B cells. In contrast, B-class CpG ODN are strong B cell activators with a weaker capacity to induce IFN-␣ and activate NK cells [23]. C-class ODN have intermediate immune effects [20–22], and distinctive structural characteristics that provide good in vivo stability and ease of formulation. B-class CpG ODN have been tested as vaccine adjuvants in humans with a number of antigens with excellent safety and efficacy data [24–27]. However, relatively little has been published to date regarding the immune effects of other classes of CpG ODN, especially with regard to their therapeutic applications. In normal healthy human volunteers, subcutaneous (SC) injection of a B-class CpG ODN, PF-3512676 (formerly known as CPG 7909), induced a Th1-like pattern of systemic innate immune activation with expression of IL-6, IL-12p40, IFN-␣, and IFN-inducible chemokines. Serum IP-10 was found to be the most sensitive assay for immune activation by PF-3512676 with increased levels detected in all subjects at all dose levels, including the lowest tested dose of only 0.0025 mg/kg. Furthermore, SC injection of PF-3512676 resulted in transient shifts in blood neutrophils, lymphocytes, and monocytes, consistent with the increased chemokine expression. Levels of acute phase proteins such as C-reactive protein were also increased. A second SC injection of PF-3512676 administered 2 weeks after the first elicited similar immune responses, showing little or no tolerance to the effects of repeated in vivo TLR9 stimulation. PF-3512676 injections were well tolerated with only dose-dependent transient injection site reactions and flu-like symptoms with no evidence of organ toxicity or systemic autoimmunity. The activation of innate immunity was dependent on the route of ODN administration, since intravenous injection caused no such effects [26]. According to these data in vivo immune activation using CpG ODN could be a well-tolerated immunotherapeutic approach for induction of Th1 innate immune activation. Having said that, in the situation of neoplastic disease immune activation through CpG ODN can have a number of beneficial effects. CpG ODN-mediated induction of cytokines such as IFN-␣, IFN-␥, TNF-␣, and IL-12 are known to have direct or indirect anti-tumor effects. Production of high levels of type 1 interferon and other Th1-like cytokines/chemokines by activated pDCs and B cells can also lead to rapid activation of other innate immune cells such as NK cells, macrophages, other subsets of DC and even T cells which may have direct and/or indirect anti-tumor effects. Furthermore, type I IFN induced by activated pDCs can in turn lead to induction of CXCL-10 (IP-10) by human monocytes [28]. IP-10 has been reported to mediate anti-tumor activity through its anti-angiogenic properties [29]. More recently, CpG ODN have been shown to augment the expression of TNF-related apoptosis-inducing ligand (TRAIL) to a high level on human monocytes enabling them to kill tumor cells [30]. Augmentation of TRAIL expression by CpG ODN have also been shown to a somewhat lower degree on human B cells, T cells and NK cells [31].

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3. Use of CpG ODN as monotherapy in cancer The potential use of CpG ODN monotherapy for cancer has been demonstrated using numerous animal models of cancer (Table 1). Many of these studies have investigated the ability of monotherapy CpG ODN to control the growth of solid tumors, and have generally concluded that intra-lesional or peritumoral injection of CpG ODN is required or best for an antitumor effect [32–35]. As demonstrated by Heckelsmiller et al., with murine C26 colon carcinoma and renal carcinoma (renca) models efficacy of CpG ODN monotherapy of local subcutaneous tumors was dependent on peri-tumoral injection of CpG ODN. However, mice with bilateral C26 tumors cleared both tumors upon peri-tumoral treatment of just one of the tumors. Furthermore, mice that cleared their tumors following CpG ODN monotherapy were protected against tumor re-challenge suggesting the induction of a tumor-specific immune response as a result of CpG ODN monotherapy. Peri-tumoral injection of CpG ODN was also shown to cause significant reduction of local subcutaneous B16 melanoma tumors as well as distant lung metastasis [36]. Peri-tumoral but not systemic delivery of CpG ODN was shown to cause significant inhibition of SC tumors of murine AG104A, IE7 fibrosarcoma, B16 melanoma and 3LL lung carcinoma [33]. Studies that have been published so far have used different CpG ODNs containing different numbers of CpG motifs as well as motifs with different species specificities; i.e., CpG motifs that preferentially stimulate murine and/or primate immune cells. We have tested PF-3512676 (CPG 7909) that contains three human optimized CpG motifs that can strongly stimulate both primate and murine immune cells as monotherapy in a number of different murine tumor models. PF-3512676 is currently being tested in phase II clinical trials for therapy of three different human cancers. It consists of 24 nucleotides and a fully phosphorothioate-modified backbone. Its GpC analogue (ODN 2137) was used as a non-CpG control. All ODN were supplied by Coley Pharmaceutical Group (Wellesley, MA, USA). All ODN were re-suspended in sterile, endotoxin free TE at pH 8.0 (OmniPer® ; EM Science, Gibbstown, NJ, USA) and stored and handled under aseptic conditions to prevent both microbial and endotoxin contamination. All ODN were diluted in ster-

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ile endotoxin free PBS for injection into animals. These studies were conducted to assess the potential of CpG ODN monotherapy using PF-3512676 in a number of different types of tumor targets. With the B16 melanoma model (ATCC # CRL-6323), tumors were induced in the peritoneal cavity by injecting 5 × 103 tumor cells by intra-peritoneal (IP) injection. CpG ODN treatment was administered by IP injection on days 1, 3, 7 and weekly for two months relative to the day of tumor induction. With the Lewis lung carcinoma (LLC; ATCC # CRL-1642), renal cell carcinoma (renca tumor cells generously provided by LuAnn Thompson Snipes, Baylor College of medicine, Houston, TX, USA) and neuro-2a neuroblastoma (ATCC # CCL-131) models tumors were induced by SC injection of 2 × 105 or 1 × 106 tumor cells in the left flank. CpG ODN treatments were administered by SC injection into the tumor perimeter on days 1, 3, 7 and weekly for two months for the LLC tumors, weekly from day 10–38 for renca tumors and daily from day 10–25 for neuroblastoma tumors relative to the day of tumor induction. With renca and neuroblastoma tumors, all mice had palpable tumors at the start of CpG ODN treatment. PF-3512676 monotherapy was capable of significantly prolonging survival of mice and reducing the rate of tumor growth with every type of tumor tested. With B16 melanoma model, we compared CpG ODN from the three different classes for their ability to control tumor growth and enhance survival of mice. All three CpG ODNs tested, CpG ODN 1585 (A-class), PF-3512676 (B-class) and CpG ODN 2429 (C-class), but not the non-CpG control ODN, were capable of significantly prolonging survival of mice with B16 tumors compared to placebo treatment (P < 0.03 for CpG ODN and 0.6 for non-CpG control ODN) (Fig. 1). However, amongst the three classes, the B-class was the most effective followed by the C-class and then the A-class. Monotherapy with PF-3512676 was also capable of reducing the rate of tumor growth and significantly prolonging survival of mice with LLC, a non-small cell lung cancer model in mice, renca and neuroblastoma compared to either placebo control or non-CpG control ODN (Fig. 2). Furthermore, we also tested monotherapy with PF-3512676 in an orthotopic model of renca where tumors were implanted

Table 1 – CpG ODN have been tested in several different types of cancer models in mice AML Melanoma Bladder cancer Cervical carcinoma Colon carcinoma Fibrosarcoma Glioma Lymphoma Lung carcinoma Neuroblastoma Mastocytoma Rhabdomyosarcoma Renal cell carcinoma

[38] [33,35–37,81] [82,83] [84] [35,49] [33,85] [86] [34,37] [33] [39] [33] [70] [49]

Fig. 1 – Female C57Bl/6 mice (n = 10 per group) were injected with 5 × 103 B16 melanoma cells by intra-peritoneal injection. Animals were treated with 100 ␮g CpG ODN 1585, PF-3512676 or 2429 or non-CpG control ODN 2137. ODN was administered by IP injection on days 1, 3, 7 and weekly for 2 months relative to day of tumor induction. Some animals received IP injections of sterile endotoxin free PBS as placebo treatment. Animals were monitored for survival.

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Fig. 2 – (A) Female C57Bl/6 mice (n = 10 per group) were injected with 1 × 106 Lewis lung carcinoma cells into the left flank by SC injection. Animals were treated with 100 ␮g PF-3512676 (–䊉–) or non-CpG control ODN 2137 ( ) injected in the tumor perimeter by SC injection on days 1, 3, 7 and weekly for 2 months relative to day of tumor induction. Some animals received SC injections of sterile endotoxin free PBS (–
–) as placebo treatment on same days as ODN treatments. (B) Female BALB/c mice (n = 10 per group) were injected with 2 × 105 murine renal cell carcinoma cells into the left flank by SC injection. Animals were treated with 100 ␮g (–䊉–) or 200 ␮g (––) PF-3512676, or 100 ␮g non-CpG control ODN 2137 ( ) injected in the tumor perimeter by SC injection weekly for five weeks starting on day 10 post-tumor induction. Some animals received SC injections of sterile endotoxin free PBS (–
–) as placebo treatment on same days as ODN treatments. (C) Female A/J mice (n = 10 per group) were injected with 1 × 106 murine neuro-2a neuroblastoma cells into the left flank by SC injection. Animals were treated with 100 ␮g PF-3512676 (–䊉–) or non-CpG control ODN 2137 ( ) injected in the tumor perimeter by SC injection daily for 15 days starting on day 10 post-tumor induction. Some animals received SC injections of sterile endotoxin free PBS (–
–) as placebo treatment on same days as ODN treatments. All animals were monitored for survival (left side) and tumor growth (right side). Tumors were measured using a digital caliper. Tumor volume was calculated by using the formula: tumor volume = (0.4) (ab2 ), where a = large diameter; b = small diameter of the tumor.

in one kidney by injecting tumor cells under the kidney capsule. Following tumor implantation, tumors rapidly spread to the adjoining kidney, lungs and at later stages tumors were present in liver and the heart. Animals were treated with five weekly SC injections of 200 ␮g PF-3512676 administered as four 50 ␮g injections at distant sites of the body. Animals had wellestablished tumors when CpG ODN treatment was initiated on day 7 post-tumor induction. As shown in Fig. 3, animals treated with PF-3512676 survived significantly longer than placebo-treated animals (P < 0.0001). In the metastatic renca model, delivering PF-3512676 at multiple sites (4 ␮g × 50 ␮g)

gave significantly better survival than administering the same total dose (200 ␮g) at a single site (Weeratna et al., unpublished data). Therefore, intra-lesional injection of CpG ODN does not appear to be required for successful therapy of metastatic tumors. Mechanism of anti-tumor activity of CpG ODN monotherapy appears to vary with the tumor type, most likely depending on variables such as MHC Class I and Class II expression of the tumor and the susceptibility of the tumor to various immune mediated effects. In some models, anti-tumor effects of CpG ODN appear to be predominantly NK mediated [37–39]

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[44]. This TLR9 expression may be functional, since the tumor cells were shown to secrete the cytokine mcp-1 (monocyte chemoattractant protein-1) in response to stimulation with CpG ODN [44]. Transfection of malignant glioma cells with mcp-1 has been shown to result in the infiltration of NK cells and monocytes into the tumor [45]. Furthermore, when SCID mice with intact NK cell function were injected IV with human lung adenocarcinoma cells with or without mcp-1 transfection, the mcp-1 transected tumors had reduced systemic spread of tumor compared to the parent tumors [46]. Therefore, CpG therapy of tumors that express TLR9 may make them more vulnerable to immune-mediated anti-tumor effects [44].

Fig. 3 – Female BALB/c mice (n = 10 per group) were injected with 1 × 105 murine renal cell carcinoma cells under the left kidney capsule. Animals were treated with 200 ␮g (––) PF-3512676 injected as four SC injections of 50 ␮g at distant sites of the body given weekly starting on day 7 post-tumor induction. Some animals received SC injections of sterile endotoxin free PBS (–
–) as placebo treatment on same days as ODN treatments. Animals were monitored for survival.

whereas in others tumor regressions were clearly T cell dependent [34,37]. CpG ODN monotherapy may have an additional mechanism of anti-tumor activity in the treatment of tumors that express TLR9 such as in the case of B cell malignancies. In this situation CpG ODN may stimulate the tumor cells to up-regulate the expression of MHC and co-stimulatory molecules as seen on normal antigen presenting cells. CpG ODN have been shown to up-regulate the expression of MHC Class I and Class II molecules as well as a variety of costimulatory molecules on a wide variety of primary malignant B cells including lymphoma and chronic lymphocytic leukemia cells [40,41]. These CpG-stimulated malignant B cells develop increased stimulatory capacity for T cells in allogeneic mixed lymphocyte cultures, suggesting the possibility of inducing an anti-tumor T cell response with the therapeutic approach. CpG ODN treatment of CLL cells has been demonstrated to sensitize the malignant cells to other immunotherapies without enhancing toxicity against normal cells [42]. PF-3512676 was tested in a phase 1 human clinical trial as a single agent in patients with previously treated nonHodgkin’s lymphoma (NHL) administered as three weekly IV infusions of 0.01, 0.04, 0.08, 0.16, 0.32 or 0.64 mg/kg. Patients were monitored for four weeks after the last infusion for toxicity, changes in peripheral blood effector cell numbers and function, and tumor response. PF-3512676 therapy was found to be well tolerated. In fifteen patients that were available for evaluating effector cell changes, dose related increases in NK activity and antibody-dependent cellular cytotoxicity (ADCC) were observed [43]. More recently, constitutive TLR9 expression has also been reported in a number of different human lung cancer tissues, and in the tumor cell lines A549 (human lung carcinoma cell line) and HeLa (human cervical carcinoma cell line)

4. Use of CpG ODN as adjuvants in cancer vaccines The principle of therapeutic cancer vaccines is to stimulate the immune system to induce strong tumor specific immune responses that would be capable of eradicating established tumors. This would require a strong cellular immune response preferably against multiple tumor targets. Numerous rodent tumor models have now shown that CpG ODN is an effective adjuvant in cancer vaccines as well as in vaccines against infectious disease. In addition, several human trials have tested CpG ODN as adjuvants in cancer vaccines. Two main strategies have been proposed for the use of CpG ODN in cancer vaccines: (i) in dendritic cell vaccine to activate antigen pulsed DC; and (ii) as an adjuvant with tumor antigens.

4.1.

CpG ODN in dendritic cell vaccines

The use of antigen pulsed DC as tumor vaccines has emerged as a novel therapeutic approach for cancer in recent years. However, one potential weakness of this strategy is that failure to properly activate the antigen pulsed DC can lead to T cell tolerance or T cell anergy rather than the induction of T cell immunity [47,48]. Numerous studies have demonstrated the use of CpG ODN to efficiently activate antigen pulsed DC resulting in productive antigen presentation and the induction of strong anti-tumor immune responses [49–52]. In vivo manipulation of DC using Flt3 ligand and CpG ODN has been shown to allow effective presentation of tumor antigen by DC resulting in strong anti-tumor responses capable of rejecting established murine B16 melanoma and CT26 colon carcinoma tumors [53]. Combined DC and CpG ODN therapy has also been shown to cure large chemotherapy resistant murine renal cell carcinoma and colon carcinoma tumors [49]. Use of CpG ODN together with a DC-tumor fusion cell vaccine has been shown to enhance phenotypic maturation of tumor cell fused and unfused DCs, production of Th1 cytokines (i.e., IFN-␥, IL-12) and help induce strong tumor-specific CTLs. Vaccinating with fused cells in combination with CpG ODN also significantly enhanced survival, decreased lung metastasis and protected animals against tumor re-challenge compared to when vaccine was used alone [54]. DC-derived exosomes expressing MHC molecules and loaded with synthetic peptides derived from tumor-associated antigens have also been used as cancer

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vaccines and induce potent anti-tumor responses in animals [55] and humans [56]. The anti-tumor responses generated by DC-derived exosomes can be greatly enhanced by combining with CpG ODN [56].

4.2.

CpG ODN as adjuvant with tumor antigens

CpG ODN have also been used as adjuvants with whole cell vaccines, cell lysates, tumor antigens or antigenic peptides [57]. In murine 38C13 B cell lymphoma model, the idiotype (Id) of the 38C13 surface IgM serves as a highly specific tumor-associated antigen. When used as an adjuvant with this antigen, CpG ODN enhanced antigen specific IgG2a antibody titers and resulted in protection against tumor challenge. The enhancement in antibody responses seen with CpG ODN was similar to levels seen with complete Freund’s adjuvant (CFA) but without the toxicity associated with CFA [58]. CpG ODN have also been shown to induce faster and stronger immune responses compared to CFA when used with the anti-idiotypic antibody 3H1 which functionally mimics carcinoembryonic antigen, a tumor-associated antigen expressed on human colorectal carcinoma and other adenocarcinomas [59]. CpG ODN have also been shown to augment the efficacy of tumor vaccines using autologous tumor cells transduced with genes expressing cytokines such as granulocyte macrophage colony-stimulating factor (GM-CSF) [60], IL-12 [61], and immune modulators such as B7.1 [62] and CD154 [63]. Using synthetic peptides containing amino acid sequences from tumor-associated antigen-derived CTL epitopes should theoretically be a very simple and effective means of immunotherapy against cancer. However, such peptides are usually very poorly immunogenic and require strong adjuvants to elicit strong anti-tumor responses. CpG ODN have been used successfully as adjuvants for such peptide vaccines. Immunization of mice with an ovalbumin peptide vaccine combined with repeated daily administration of CpG ODN has been shown to promote strong CTL responses and protect mice against tumors expressing ovalbumin [64]. Similar augmentation in CTL responses by CpG ODN has been shown with other peptides such as TRP2, a peptide containing CTL epitope from a murine melanoma specific tumor associated antigen [65], peptide derived from the melanoma-associated differentiation antigen MART-1/Melan-A [66] and MHC II-restricted tumor peptide H11.1, a peptide from the murine T cell lymphoma RMA model [67]. More recently, PF-3512676 mixed with melanoma antigen A (Melan-A; identical to MART-1) analog peptide and incomplete Freund’s adjuvant was used to vaccinate HLA-A2+ melanoma patients [68]. All patients that received PF-3512676 in their vaccine developed rapid and strong antigen-specific T cell responses with approximately ten fold higher mean MelanA specific circulating CD8+ T cells than had been observed in historical melanoma patients given the same tumor vaccine but without PF-3512676. These results confirm the preclinical data and demonstrate the potency of PF-3512676 to promote strong antigen-specific anti-tumor CD8+ T cell responses in humans with advanced cancer.

5. Use of CpG ODN in combination with cytoreductive therapies Numerous studies have reported enhancement of anti-tumor activity of chemotherapeutic drugs by CpG ODN. So far, CpG ODN have been reported to enhance the anti-tumor effects of a number of different chemotherapeutic agents such as the topoisomerase I inhibitor, topotecan [69,70], the alkylating agent cyclophosphamide [70] and the anti-metabolite 5-fluorouracil [71]. More recently, CpG ODN have also been reported to enhance the anti-tumor responses to radiation [72,73]. Both chemotherapeutic agents and radiation are known to cause tumor cell death. Therefore, it is likely that the tumor debulking as a result of chemotherapy or radiation can cause the release of tumor-associated antigen resulting in an in situ vaccine. When used in combination with CpG ODN, which are potent adjuvants, this could lead to the induction of strong tumor-specific immune responses that would be capable of mediating tumor rejection. When CpG ODN were tested alone or in combination with either cyclophosphamide or topotecan in an orthotopic rhabdomyosarcoma model, the combination therapy using CpG ODN and either of the chemotherapy drugs enabled the long-term survival of 15–40% of the mice with large tumors. Neither cyclophosphamide nor CpG ODN alone were able to cure any mice [70]. This survival benefit of CpG ODN combined with chemotherapy required the presence of T cells, but not NK cells, suggesting that the CpG ODN may have induced the development of an anti-tumor T cell response, which may have been sufficient to eliminate the residual tumor after chemotherapy. The effectiveness of combination therapy using radiation and CpG ODN was diminished in mice that were rendered immunosuppressed by whole body irradiation suggesting that in this model also, the mechanism of the synergy between CpG ODN and a conventional anti-tumor therapy was immune mediated (Mason et al., manuscript submitted). Dose limiting bone marrow suppression, neutropenia and general immunosuppression are common side effects of many chemotherapeutic agents. Even though one may be inclined to assume that chemotherapy treatment may hamper the ability of CpG ODN to activate innate and adaptive immune responses, several studies have demonstrated that chemotherapy drugs do not affect the ability of CpG ODN to activate innate immune responses [69,71] or promote the induction of adaptive immune responses (Weeratna et al., unpublished data). More recently, PF3512676 in combination with standard chemotherapy (taxane/platinum combination) and chemotherapy alone were investigated in a randomized phase II clinical trial in patients with advanced non-small cell lung cancer. Preliminary data have demonstrated PF-3512676 to be well tolerated and it did not lead to any clinically meaningful increase in chemotherapy-related toxicity. Patients receiving PF-3512676 plus chemotherapy achieved objective (RECIST criteria) tumor responses more often than patients given chemotherapy alone and there was also a trend for prolongation of overall survival for patients who received PF-3512676 [74,75].

update on cancer therapeutics

6. Use of CpG ODN in combination with anti-tumor antibodies An important mechanism of anti-tumor activity of monoclonal antibody therapy used in cancer is thought to be mediated through antibody-dependent cellular cytotoxicity (ADCC). CpG ODN have been shown to enhance the antitumor effects of monoclonal antibodies by influencing different effector cell populations and different classes of CpG ODN have been shown to have differential effects on these effector cell populations [76]. In the murine 38C13 B cell lymphoma model, CpG ODN-activated murine splenocytes were capable of mediating lysis of tumor cells in vitro and this lysis was further enhanced by the addition of monoclonal antibody against cell surface marker on tumor cells [77]. When used in vivo, the combination of the monoclonal antibody and CpG ODN showed greater efficacy in enhancing the survival of mice with 38C13 B cell lymphoma than when either drug was used alone. Furthermore, a single administration of CpG ODN in combination with monoclonal antibody was better in enhancing survival of mice than multiple administration of IL2 (a cytokine known to enhance NK activity) in combination with monoclonal antibody [77]. Both A- and B-class CpG ODN were equally effective in controlling the growth of 38C13 B cell lymphoma in mice. However, the enhancement in anti-tumor activity of the monoclonal antibody therapy with A-class CpG ODN were shown to be mediated largely through activation of NK cells whereas B-class CpG ODNs were shown to activate multiple effector cell populations including NK cells and granulocytes [76]. In a recent report, B cells from humans with a number of different B cell malignancies were shown to respond to CpG ODN by up-regulating the expression of class I and class II MHC, co-stimulatory molecules and CD20 [78]. Up regulation of the expression of these molecules may target the malignant B cells to direct immune mediated destruction. Furthermore, enhanced expression of cell surface molecule CD20 may also make these malignant B cells better targets for therapy with the anti-tumor monoclonal antibody Rituxan® (Rituximab), used to treat relapsed or refractory low-grade or follicular, CD20+, B-cell non-Hodgkin’s lymphoma. In a phase 1 dose escalation human clinical trial, PF-3512676 was coadministered with Rituximab by either four weekly SC injection at doses of 0.01, 0.04, 0.08 or 0.16 mg/kg, four weekly IV infusions at doses of 0.04, 0.16, 0.32 or 0.48 mg/kg, or 20 weekly SC injections at 0.24 mg/kg to patients with relapsed or refractory non-Hodgkin’s lymphoma [79]. PF-3512676 when administered by SC or IV route was well tolerated by patients and showed no measurable enhancement in Rituximab-associated toxicity. Out of the 50 patients treated, 7 objective responses were reported within 50 days of initiation of treatment (3 complete responses and 4 partial responses) and 5 responses were seen within 20 weeks of treatment [79]. In an independent trial, patients with relapsed NHL were treated with Rituximab together with a different B-class CpG ODN. In this study, patients were given four weekly IV infusions of Rituximab with SC administration of CpG ODN following Rituximab on same day. CpG ODN therapy was well tolerated and a dose-related increase in the expression of several interferon

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inducible genes was measured following CpG ODN which correlated with increased levels of serum 2 -5-oligoadenylate synthetase [80].

7.

Conclusion

Cancer immunotherapy has progressed tremendously within the last century, from the use of crude bacterial extracts such as Coley’s toxins to the use of recombinant cytokines and now to the use of synthetic molecules that mimic natural TLR ligands. By binding and activating TLR9, CpG ODN activate the immune system through a “natural” pathway to produce a whole array of cytokines and chemokines in a coordinated fashion triggering a cascade of immune responses against cancer cells in a more effective and a safe manner than was possible with past non-specific immune activators. The specifically coordinated stimulation of the immune system by CpG ODN has already demonstrated impressive activity in treating cancer in a variety of animal models. In murine tumor models, CpG ODN monotherapy is highly effective in treating a number of different tumor types. In the majority of monotherapy subcutaneous tumor models, peri-tumoral injection of CpG ODN has been far more effective than injecting CpG ODN at a distant site. However, recent studies demonstrate that peritumoral injection is not required for effective treatment of metastatic tumors. This difference may be associated with the need for optimal activation of DC in the tumor draining lymph nodes, which is achieved by peri-tumoral injections in the case of solid tumors and by multiple injections throughout the body in the case of metastatic tumors. CpG ODN have also been demonstrated to enhance the anti-tumor activity of other cancer therapies such as chemotherapy, radiation, monoclonal antibody therapy and cancer vaccines. So far, only B-class CpG ODN have been tested in humans for cancer therapy and this appears to be well tolerated and effective in several tumor types. Nevertheless, the full therapeutic benefit of the use of B-class CpG ODN in cancer therapy and the effectiveness of other classes of CpG ODN in cancer therapy remain to be determined.

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