Systemic Targeting of CpG-ODN to the Tumor Microenvironment with Anti–neu-CpG Hybrid Molecule and T Regulatory Cell Depletion Induces Memory Responses in BALB-neuT Tolerant Mice

The innate immune response relies on the recognition of the antigen by receptors that recognize specific structures found exclusively in microbial pathogens termed pathogen-associated molecular patterns (PAMP; ref. 1). Recent studies have shown that recognition of PAMPs by antigen-presenting cells (APC) is mediated by a Toll-like receptor (TLR) family (2, 3). There are currently >10 known TLR family members capable of sensing bacterial wall components, such as lipopolysaccharide (LPS; TLR-2/TLR-4), CpG-DNA (TLR-9), flagellin (TLR-5), as well as other microbial products (4). The signaling provided by TLRs triggers maturation and activation of APCs (5, 6), increasing their ability to prime naive T cells. In this way, TLRs link the recognition of pathogens with induction of adaptive responses. It has been shown that targeting APCs with different TLR adjuvants could enhance or induce the activation of antitumor responses (7–9). We have shown that FVB-Her-2/neu transgenic mice (10–13) or the BALB-neuT mice (14) are tolerant to neu-dominant epitopes and that these animals only have a low avidity repertoire to neu antigens (10–13). Our data also indicate that multiple immunizations with dendritic cells (DC) pulsed with neu peptides delay the tumor growth but are not effective at controlling tumor growth (10–13). We have recently shown that i.t. injections of CpG-ODN is the most effective strategy to delay tumor growth in young and old mice (15). The major drawback of i.t. injections of CpG-ODN is that not all tumors are physically accessible. To target the CpG-ODN to the tumor anywhere in the body, we produced a hybrid-molecule between an antibody directed against the neu molecule and CpG-ODN (anti–neu-CpG). Our results showed that the anti–neu-CpG retained its dual capacity of (a) binding to Her-2/neu⁺ tumor cells and (b) activating DCs. Treatment with anti–neu-CpG induced tumor rejection in 100 of BALB/c mice, and only ∼30% of BALB-neuT rejected the tumor. Interestingly, BALB/c mice were able to develop a memory response; in contrast, BALB-neuT mice that rejected the first tumor succumbed to the tumor challenge. These results raise an important issue: Can tolerant hosts (BALB-neuT mice) develop memory responses? If yes, how is the generation of memory responses against self-antigens or tumors controlled in tolerant hosts?

There is accumulating evidence indicating that immunologic tolerance is maintained, not only by clonal deletion but also by T regulatory cells (T-reg; refs. 16–19). Depletion of CD4⁺CD25⁺ T cells by the administration of anti-CD25 monoclonal antibody (mAb) has been shown to suppress the growth of a variety of different syngeneic tumors in mice (20–22). The observation that the removal of immunoregulatory CD4⁺CD25⁺ T cells can abrogate unresponsiveness to syngeneic tumors in vivo, leading to the spontaneous development of tumor-specific responses, indicates that the maintenance of self-tolerance against tumor-self antigens could potentially be lifted (22, 23). When BALB-neuT mice were treated with anti-CD25 or anti-GITR alone, no antitumor effect was observed. However, the combination of injections of anti–neu-CpG plus anti-GITR, but not anti-CD25, induced both a complete rejection of the tumors and a protective memory response in these animals. Taken together, these results have important implications for the development of immunization protocols against self-tumor antigens which are discussed.

Mice, cell lines, and reagents. Female BALB/c and female BALB/c athymic mice were purchased from Harlan. BALB-neuT mice were generated as previously described (24) and bred in our facilities. Female BALB-neuT mice of 6 to 8 wk old were used before autochthonous tumor appearance. TUBO is a cell line generated from a spontaneous mammary gland tumor from a BALB-neuT mouse (25). The BM-185 wild-type (wt) and BM-185 expressing enhanced green fluorescent protein (EGFP; BM-185-EGFP) cell lines (26) were kindly provided by Dr. D. Kohn at the University of Southern California, Los Angeles. The parental 66.3 cell line (H-2^d) derived from a mouse breast tumor and its transfectant, the A2L2, expressing the rat HER-2/neu (27) were provided by Dr. J.E. Price (M. D. Anderson Cancer Center). The mouse renal cell carcinoma RENCA cells of BALB/c origin were used as a negative control for the cytotoxic assays. Anti-GITR (DTA-1) was obtained from Dr. Shimon Sakaguchi (Kyoto University). Anti-CD25 (PC61) was obtained from Dr. Linda Bradley (Sidney Kimmel Cancer Center). Anti-neu (7.16.4, a mouse IgG2a antibody which recognizes the rat Her-2/neu) was obtained from Dr. Mark Greene (University of Pennsylvania). All cell lines were maintained in complete RPMI medium (RPMI 1640) supplemented with 10% FCS, 2 mmol/L glutamine, 5 × 10^-5 mol/L 2-mercapethanol, and 50 μg/mL gentamicin. TLR ligands, 1826-CpG-ODN and 1982-control-ODN, were purchased from Oligo Etc, Imiquimod from 3M Pharmaceuticals, Poly-I:C and LPS from Sigma-Aldrich. Flagellin was purified as previously reported (28).

Construction of anti–neu-CpG. 5′-Amino–modified CpG-ODN was subsequently modified to incorporate a 4FB-moiety by treatment with S-4FB (Solulink Biosciences). Anti-neu antibody was modified to incorporate HyNic moieties on lysine residues using S-HyNic (Solulink Biosciences). Synthesis of the conjugate was accomplished by mixing the 4FB-modified CpG oligo with the HyNic-modified anti-neu antibody followed by purification by size exclusion chromatography x(Superdex 200, GE HealthCare). Purity of the conjugated antibody was confirmed by running the sample in an SDS-PAGE gel.

In vivo tumor studies. TUBO cells (1 × 10⁶) were implanted s.c. in BALB/c, BALB-neuT, and BALB/c athymic mice. Tumors were allowed to grow for 10 d before treatment was initiated. On day 10 after tumor challenge (tumor size, ∼150–200 mm³), animals were randomly divided into groups of five to eight mice per group. Animals received i.t. or i.v. injections of anti–neu-CpG (50 μg/injection) twice a week for 3 wk. Control groups are described in the text. Groups of animals were also injected with anti-GITR. For the evaluation of memory responses, animals were challenged 70 d after the first tumor challenge with a second dose of 10⁶ TUBO cells. Tumor growth was monitored every 2 to 3 d.

Depletion studies. Anti-CD4 (GK1.5) and anti-CD8 (56-6.37) mAbs were used for in vivo depletion of T-cell subsets. Anti-asialo GM1 (Wako Pure Chemical Industries) was used to deplete natural killer (NK) cells. Animals were injected i.p. with 300 μg of anti-CD4 and anti-CD8 mAb or 50 μg of anti-asialo GM1 twice per week, starting 1 wk before inoculation of the tumor cells and continuing for the duration of the experiment. To deplete T-regs, animals were injected with anti-CD25 or anti-GITR starting on day 9, 16, and 23 (300 μg/injection) after tumor challenge.

Analysis of CD4⁺ Foxp3⁺ T-regs. The number of T-regs in the tumor microenvironment, spleen, and lymph nodes (LN) in BALB-neuT with or without TUBO tumors and after treatment with anti–neu-CpG was determined by the analysis of CD4⁺Foxp3⁺ cells using a commercially available kit (eBioscience) following the manufacturer's protocol.

Generation of CTL bulk cultures and cytotoxic activity. BALB-neuT tumor-bearing mice were injected i.t. thrice a week with anti–neu-CpG (50 μg/injection) for 2 wk. Groups of animals were also injected with anti-GITR on days 9 and 16 (300 μg/injection) after tumor challenge. One week after the last injection with anti–neu-CpG, animals were sacrificed. Spleen cells (6 × 10⁶) from primed animals were restimulated in vitro with 5 × 10⁵ irradiated (3,000 rads) TUBO cells plus 1 × 10⁶ irradiated BALB/c spleen cells as feeders. After 5 d, cultures were assayed for lytic activity in a ⁵¹Cr release assay against TUBO, 66.3, A2L2, and RENCA cells. Supernatants were recovered after 6 h of incubation at 37°C, and the percentage of lysis was determined by the formula: percent specific lysis = 100× (experimental release − spontaneous release) / (maximum release − spontaneous release).

Statistical analyses. Statistical significance of data was determined by Student's t test to evaluate the P value. The relationship between two variables was tested using regression analysis, and P < 0.05 was considered significant. Survival analysis used the Breslow modification of the Kaplan-Meier test.

Development of anti–neu-CpG–conjugated molecules to target CpG-ODN at the tumor site. We have previously shown that, among different TLR ligands, only i.t. injections of CpG-ODN induce an antitumor response in neu-tolerant mice (Supplementary Fig. S1; ref. 15). Although i.t. injections of CpG-ODN could significantly prolong the survival of BALB-neuT mice (Supplementary Fig. S1), the major drawback of this approach is that not all tumors will be physically accessible for i.t. injections. Therefore, the generation of a targeted CpG-ODN would be more practical. To target the CpG-ODN anywhere in the body, we produced a hybrid molecule between an antibody directed against the rat neu molecule and CpG-ODN (Fig. 1A). The CpG-ODN was conjugated to the anti-neu mAb (7.16.4) by introducing a cleavable linker using the disulfide bioconjugation system, as described in the Materials and Methods and as shown in Fig. 1B. Next, we tested the functionality of the anti–neu-CpG. TUBO cells were stained with decreasing concentrations of anti-neu and anti–neu-CpG (1, 0.1, and 0.01 μg). As shown in Fig. 2A, the anti–neu-CpG binds with the same intensity to TUBO cells, as does the anti-neu mAb. These results indicate that anti–neu-CpG retains its affinity/avidity for the neu antigen present on tumor cells and the ability to bind and recognize Her-2/neu⁺ cells in the same fashion as the anti-neu mAb. We also evaluate the stimulatory capability of the anti–neu-CpG. DCs were stimulated in the presence of CpG-ODN, anti–neu-CpG, control-ODN, or anti-neu mAb overnight and analyzed for the expression of activation cellular markers and secreted cytokines. The anti–neu-CpG induced the activation of DCs by increasing the levels of expression of class I and B7.1 molecules with the same efficiency as CpG-ODN, whereas stimulation with control-ODN or anti-neu mAb showed no stimulatory effect (Fig. 2B). DCs treated with anti–neu-CpG produced similar amounts of interleukin 12 (IL-12), as did DCs treated with CpG-ODN (Fig. 2C). However, no production of IL-12 was detected after treatment with control-ODN or anti-neu mAb. These results show that the anti–neu-CpG retained its dual capacity of (a) binding to Her-2/neu⁺ tumor cells and (b) activating DCs.

Antitumor effect of anti–neu-CpG. Having shown that anti–neu-CpG is a functional molecule, we first tested whether this hybrid molecule is capable of activating an antitumor response. For the first set of experiments, TUBO tumor-bearing BALB-neuT mice were injected i.t. with anti–neu-CpG generated with a cleavable or noncleavable linker. As controls we included (a) TUBO tumor-bearing BALB/c mice injected i.t. with anti–neu-CpG (cleavable linker) and (b) TUBO tumor-bearing BALB-neuT mice injected i.t. with CpG-ODN. Our results indicate that 100% of BALB/c mice injected i.t. with anti–neu-CpG rejected the tumor (Fig. 3A). BALB-neuT mice injected i.t. with anti–neu-CpG or CpG-ODN, two of five animals rejected the tumors, and those animals that did not reject the tumor significantly delay the tumor growth compared with control animals (Fig. 3A). Animals treated with anti–neu-CpG noncleavable linker did not have an antitumor effect (Fig. 3A). These results indicate that the inclusion of a cleavable linker is critical in this hybrid molecule to release the CpG-ODN at the tumor site to stimulate an immune response. Additionally, we included TUBO tumor-bearing BALB-neuT mice injected i.t. with CpG-ODN plus anti-neu or anti-neu mAb alone. Animals treated with CpG-ODN plus anti-neu has the same effect as animals treated with CpG-ODN alone, and no antitumor effect was observed after treatment with anti-neu mAb alone (Supplementary Fig. S2). These results indicate that anti–neu-CpG with a cleavable linker is a functional molecule able to induce an antitumor immune response. The antitumor effect of anti–neu-CpG is due to the activation of an immune response and not through direct inhibition of TUBO cells by the anti–neu-CpG or anti-neu mAb (Supplementary Fig. S3).

To confirm whether tumor rejection was mediated by T cells and NK cells and to evaluate the role of CD4⁺ and CD8⁺ T cell subsets, BALB-neuT mice were depleted of CD4⁺ and CD8⁺ T cells and NK cells with anti-CD4, anti-CD8, and anti-asialoGM1 antibodies (anti-NK antibody), respectively. As shown in Fig. 3B, depletion of CD4⁺ T cells, CD8⁺ T cells, and NK cells abrogates the antitumor response. These results indicate that, for the rejection of the tumor, the presence of CD4⁺ and CD8⁺ T cells and NK cells is necessary after anti–neu-CpG injections. Intratumoral injections of anti–neu-CpG in BALB/c athymic mice delayed tumor growth when compared with control animals (Fig. 3C). This indicates that there is activation of an immune response which is most probably mediated by NK cells. However, activation of NK cells alone is not sufficient to mediate complete tumor rejection. Additionally, these results indicate that the antitumor response is not exclusively through the activation of APCs but also depends on the activation of a cellular response.

Analysis of immune tumor responses after anti–neu-CpG injections. We evaluated whether BALB/c and BALB-neuT mice that rejected the tumor after the injections with anti–neu-CpG would induce a protective memory response. BALB/c and BALB-neuT mice that rejected the first tumor were challenged with TUBO cells 70 days later. BALB/c mice were able to reject the tumor challenge; in contrast, BALB-neuT mice did not (Fig. 4A), indicating that protective memory responses were not generated in tolerant mice. We asked whether tolerant and nontolerant animals differ in the generation of memory responses. For these experiments, we use the BM-185-EGFP. The BM-185-EGFP cells are antigenic and rejected by BALB/c mice (29). Furthermore, after injection of BM-185-EGFP, BALB/c mice generate long-term memory responses against the BM-185 wt cells. After implanting the BM-185-EGFP cells in the animals, both BALB/c and BALB-neuT rejected the BM-185-EGFP cells, and after a tumor challenge with BM-185 wt cells, these cells were rejected (Fig. 4B). As a control, BM-185 wt cells were implanted into the BALB/c and BALB-neuT mice, and as expected, these cells formed tumors in these animals. These results indicate that primary and memory immune responses against nominal antigens could be developed in neu-tolerant hosts with the same efficiency as in nontolerant hosts.

There is extensive evidence indicating that T-regs can suppress primary and memory immune responses and self reactive T cells (30, 31). We analyzed the levels of T-regs in BALB-neuT mice with or without TUBO tumors and treated with anti–neu-CpG. Our data indicated that T-regs kept accumulating over time within the tumor microenvironment (Fig. 4C). Next, we evaluated whether there were changes in the number of T-regs in BALB-neuT mice after i.t. injections of anti–neu-CpG. In tumor-bearing mice, the number of CD4⁺Foxp3⁺ cells increase both in spleen (from 4.7% to 6.7%) and LN (from 5.8% to 7.9%) when compared with animals without tumors (Fig. 4D). After anti–neu-CpG treatment, there was a ∼80% to 85% reduction in the number of CD4⁺Foxp3⁺ cells within the tumor and a ∼15% reduction in spleen and LN (Fig. 4D).

Anti–neu-CpG injections and T-reg depletion induces a protective memory response in BALB-neuT mice. Although the levels of T-regs within the tumor were drastically reduced after i.t. injections of anti–neu-CpG, there were still large numbers of T-regs in spleen and LN that could influence the activation of primary and memory responses in BALB-neuT mice. We tested the effect of depleting T-regs with anti-CD25 or anti-GITR alone or in combination with anti–neu-CpG to induce the elimination of the transplantable TUBO tumor in BALB-neuT mice. Considering the use of anti-GITR is controversial for the depletion of T-regs, because some reports show that anti-GITR deplete T-regs (32) whereas in other reports it stimulates CD8 T cells (33). The GITR signaling in T cells is similar to the signal provided by other TNFR (e.g., OX40; ref. 34). We have shown that anti-OX40 mAb could enhance the immune responses in neu mice (10, 12), and on the other hand, it has been shown that anti-OX40 could inhibit T-regs (35). As a control we included anti-OX40 mAb. The combination of anti–neu-CpG plus anti-GITR induced the rejection of the TUBO tumor in 100% of the BALB-neuT mice (Fig. 5A). Treatment with anti–neu-CpG plus anti-OX40 is better than anti–neu-CpG alone, and animals significantly delay the tumor growth when compared with the control group (Fig. 5A). Fifty percent of the BALB-neuT mice treated with anti–neu-CpG plus anti-OX40 rejected the tumor, whereas only 35% of the animals treated with anti–neu-CpG alone rejected the tumor (Fig. 5A). BALB-neuT mice treated with anti–neu-CpG plus anti-CD25 did not reject the tumor, and they succumbed earlier to the tumor when compared with animals treated with anti–neu-CpG alone (Fig. 5A). Treatment with anti-CD25, anti-GITR, or anti-OX40 alone had no antitumor effect (Fig. 5A). Next, animals that rejected the tumor were challenged with TUBO cells 70 days after injection of the first tumor. Only animals treated with anti–neu-CpG plus anti-GITR developed a memory response, and all mice remained tumor-free (Fig. 5B). No memory responses were observed in animals treated with anti–neu-CpG alone or anti–neu-CpG plus anti-OX40 (Fig. 5B).

We also analyzed the effect of anti–neu-CpG and anti-GITR in priming a cytotoxic T-cell response. BALB-neuT tumor-bearing mice were treated with i.t. injections of anti–neu-CpG in the presence or absence of anti-GITR. As expected, stronger CTL responses were developed in animals treated with anti-GITR mAb (Fig. 5C). CTLs killed TUBO, A2L2, and, to lesser extent, 66.3 cells, indicating that CpG-ODN vaccination induces an immune response against Her-2/neu and other antigens different than Her-2/neu. No cytotoxic effect was observed against RENCA cells.

Systemic injection of anti–neu-CpG induces an antitumor response. Next, we evaluated the antitumor effect of systemic delivery of anti–neu-CpG plus anti-GITR in stimulating an antitumor response. Our data indicate that after i.v. injections of anti–neu-CpG plus anti-GITR, tumor growth is significantly delayed when compared with control groups (Fig. 6A); also, five of eight BALB-neuT mice rejected the tumor and developed memory responses (Fig. 6B). I.v. injections of (a) CpG-ODN, (b) anti-neu, (c) anti-neu + CpG-ODN, and (d) anti-neu + CpG-ODN + anti-GITR had no antitumor effect (Fig. 6A). These results show that anti–neu-CpG hybrid molecules are very effective in delivering the CpG-ODN to the tumor site capable of activating antitumor responses. To correlate whether the enhanced antitumor response after anti-GITR injections correspond to the modulation of the numbers of T-regs, we evaluated whether the levels of T-regs in spleen and tumor draining LN (TDLN) from animals treated with anti–neu-CpG alone or anti–neu-CpG plus anti-GITR were modified. As shown in Fig. 6C, the addition of anti-GITR drastically reduces the levels of T-regs in spleen and TDLN. Taken together, these results confirm that depletion of T-regs is critical to effectively activate an immune response against self-tumor antigens (36).

We have previously shown that neu mice are immunotolerant to neu antigen, and immunizations with DCs pulsed with neu peptides cannot control the tumor growth in these animals (10–14). Therefore, the identification of new vaccination strategies capable of activating and controlling tumor growth in tolerant host are needed. Although several studies have indicated that that TLR ligands could enhance and induce antitumor immune responses in syngeneic tumor models (18–20), our results indicate that only i.t. injections of CpG-ODN is able to induce an antitumor response in Her-2/neu mice (15). The major drawback of i.t. injections of CpG-ODN is that not all tumors will be physically accessible for i.t. injections. To target the CpG-ODN to the tumor, we produced a fusion protein between an antibody directed against the rat neu molecule and CpG-ODN. Our data confirmed that anti–neu-CpG is a functional molecule both in vitro and in vivo because it showed the ability of binding Her-2/neu⁺ tumor, activating DCs, and stimulating antitumor responses. Critical for the functional activity of the hybrid antibody is to use a cleavable linker allowing the release of the CpG-ODN from the antibody. If a noncleavable linker was used in the hybrid molecule, no antitumor effect was observed. The antitumor effect induced by anti–neu-CpG is due to the activation of an immune response by the CpG-ODN and not through direct inhibition of TUBO cells through the antibody, because treatment with anti-neu mAb alone did not have antitumor effect or the anti–neu-CpG did not inhibit the proliferation of TUBO cells. The responses induced by anti–neu-CpG are dependent on the activation of a cellular response because the depletion of CD4⁺ and CD8⁺ T cells and NK cells abrogated the antitumor responses. An interesting observation is that i.t. injections of anti–neu-CpG induced the rejection of tumors in 100% of BALB/c mice; however, only ∼30% of BALB-neuT mice rejected the tumor. Furthermore, BALB/c mice that rejected tumors could develop protective memory responses against TUBO cells after i.t. injections of anti–neu-CpG. In contrast, BALB-neuT mice that rejected the TUBO cells succumbed to the tumor after a second challenge. We confirmed that BALB-neuT mice have the same capacity to generate primary and memory responses against nominal antigens as BALB/c mice because both BALB/c and BALB-neuT mice rejected the BM-185-EGFP cells and developed a long-term protective memory response against BM-185 wt cells. This suggests that there are significant differences between tolerant and nontolerant hosts in generating memory responses against self-tumor antigens.

T-regs keep in check self-reactive T cells in peripheral lymphoid tissues suppressing their activation and effector function (23). Furthermore, there is accumulative evidence indicating that T-regs suppress the activation of antitumor immune responses (20–22). We hypothesize that perhaps the lack of induction of stronger primary antitumor immune responses and memory response in BALB-neuT is due to the accumulation and the effect of T-regs on the low affinity repertoire. Indeed, our results indicate that T-regs accumulate over time at the tumor site and that the number of T-regs in spleen and LN also increase in tumor-bearing mice. After anti–neu-CpG treatment, the numbers of T-regs were reduced 80% to 85% at the tumor site and ∼15% in spleen and LN. Despite the significant reduction in the numbers of T-regs within the tumor after anti–neu-CpG treatment, BALB-neuT mice could not develop a memory response. Although we observed lower numbers of T-regs in spleen and LN after CpG-ODN treatment, there were sufficient numbers of T-regs that could potentially inhibit the activation of a memory T-cell response in BALB-neuT mice. These observations led us to postulate that depletion of T-regs might be critical for the effective activation of an immune response in BALB-neuT mice. Several groups have shown that depletion of T-regs with anti-CD25 mAb (37, 38) or IL-2 immunotoxin (39) enhances the immune responses in BALB-neuT or FVB-neu mice. In contrast, in our tumor model, anti-CD25 mAb abrogated the antitumor response. Only when i.t. injections of anti–neu-CpG plus anti-GITR mAb (40, 41) was combined 100% of the BALB-neuT mice rejected the first tumor and developed a protective memory response. The antitumor responses in the presence of anti-GITR correlate with a stronger cytotoxic activity. This raises the question why anti-GITR enhances the immune responses whereas anti-CD25 inhibits them. First, it is important to note that in the studies where anti-CD25 mAb or IL-2 immunotoxin was used to deplete T-regs, these antibodies were injected before immunizing the animals. Previous reports have indicated that treatment with anti-CD25 could deplete the CD4 helper response and CD8 T-effector cells hindering the immune responses (41). Second, to optimally stimulate an antitumor response after i.t. injections of CpG-ODN, it was important to deplete T-regs during the course of the vaccination. A single injection of anti-GITR or anti-CD25 mAb before or during the priming or boosting of the immune response was not sufficient to generate strong primary or secondary responses (data not shown). Only, when T-regs were depleted during the priming and boosting of the immune response, a protective memory response was generated. Third, and most importantly, after anti-GITR treatment, the levels of T-regs were drastically reduced in the periphery without affecting the levels of CD4⁺ or CD8⁺ T cells. These results are in agreement with the results of Shevach and Stephens (32), showing reduction of T-regs after anti-GITR treatment. Treatment with anti-OX40 did not reduce the levels of T-regs in the periphery (data not shown). Therefore, we believe that the main action of anti-GITR mAb in our tumor model was to deplete T-regs (40, 41). However, we cannot rule out that anti-GITR also enhances the CD8 T-cell responses (33). Another drawback of using anti-CD25 or IL-2 immunotoxin is that, if these strategies eliminate or diminish the CD4⁺ helper response, it could compromise the generation of memory responses (42). These results have important clinical implications indicating the importance of properly selecting a strategy that will specifically deplete or block T-regs without influencing T-effector CD4⁺ or CD8⁺ cells. Taken together, these data further support the concept that the residual repertoire for neu antigens in BALB-neuT mice is heavily influenced by T-regs and the depletion or perturbation of the suppressive function of T-regs is critical to optimally stimulate a T-cell response against self-tumor antigens (37).

An important question from these experiments is why BALB/c can generate a strong primary and memory antitumor response after i.t. injections of anti–neu-CpG and BALB/neu-T mice could not. A simple explanation is that, in nontolerant hosts, high-affinity T cells against the tumor antigen are strongly, rapidly activated and expanded. Whereas in tolerant hosts, there are a lower numbers of self-reactive T cells, and these cells are more difficult to activate and do not expand effectively (10, 43–45). Additionally, it has been shown that T-regs selectively suppress weak but not strongly activated T cells that undergo clonal expansion (46). As such, T-regs will not have the same capacity to suppress T-cell responses in BALB/c mice, resulting in tumor rejection. However, T-regs could effectively suppress the low-affinity T cells from BALB-neuT mice. This is in agreement with the recent findings of Kim and colleagues (47) that showed that self-reactive T cells are continuously suppressed by T-regs. Further evaluations are required to better understand how T-regs influence or control the residual repertoire against self-tumor antigens.

Although, we did not observe 100% rejection (five of eight animals rejected the tumor) of the tumors after i.v. injections of anti–neu-CpG plus anti-GITR, these results are very encouraging, indicating the functionality of this hybrid molecule to deliver the CpG at the tumor site. We are currently optimizing the doses to increase the efficacy of this antibody and combing it with small molecules, such as penetratin (48), to increase the penetration capabilities of these antibodies. Additionally, the use of the anti–neu-CpG-ODN will have clinical benefits, such as reducing the possible side effects of injecting high doses of CpG-ODN. Our data indicate that the lower levels of CpG-ODN contained in the hybrid molecule is sufficient to induce an immune response. We believe that the reason why anti–neu-CpG is effective is because the cleavable linker allows the slow release of CpG-ODN over time at the tumor site. Further studies are required to better understand these hybrid molecules. For example, we are in the process of developing a hybrid molecule using fluorescent tags CpG-ODN (49); in this manner, we will be able to trace the CpG-ODN after injecting the anti–neu-CpG in vivo.

To the best of our knowledge, this is the first time that an antibody–CpG-ODN conjugated molecule has been generated. These results are very encouraging and show the proof of concept that antibody–CpG-ODN conjugated molecules are functional in vitro and in vivo and that they can serve as a new strategy for fighting cancer. Furthermore, based on the data presented, it can be argued that anti–neu-CpG could be superior to soluble CpG-ODN based on the dose applied. We strongly believe that the antibody–CpG-ODN strategy could open a new field for developing new targeted therapies and target other tumor antigens, whereby it would be possible to target single or multiple markers against tumors. When anti–neu-CpG vaccination was combined with the depletion of T-regs, it resulted in the rejection of the primary tumor and the generation of a protective memory response. Taken together, these results indicate that CpG-ODN–targeted therapy and the depletion of T-reg optimally activate a primary response and generate protective memory responses against self-tumor antigens.

No potential conflicts of interest were disclosed.

Grant support: NIH grant CA 78579 (J. Lustgarten).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.