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Cancer Research 67, 7495-7504, August 1, 2007. doi: 10.1158/0008-5472.CAN-07-0941
© 2007 American Association for Cancer Research
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Immunology

Combined Natural Killer T-Cell–Based Immunotherapy Eradicates Established Tumors in Mice

Michele W.L. Teng1, Jennifer A. Westwood1, Phillip K. Darcy1,7, Janelle Sharkey1, Moriya Tsuji2, Richard W. Franck3, Steven A. Porcelli4, Gurdyal S. Besra5, Kazuyoshi Takeda1, Hideo Yagita6, Michael H. Kershaw1,7 and Mark J. Smyth1,7

1 Cancer Immunology Program, Peter MacCallum Cancer Centre, East Melbourne, Victoria, Australia; 2 HIV and Malaria Vaccine Program, Aaron Diamond AIDS Research Center, The Rockefeller University; 3 Hunter College, City University of New York, New York, New York; 4 Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York; 5 School of Biosciences, University of Birmingham, Birmingham, United Kingdom; 6 Department of Immunology, Juntendo University School of Medicine, Tokyo, Japan; and 7 Department of Pathology, University of Melbourne, Parkville, Victoria, Australia

Requests for reprints: Mark J. Smyth, Cancer Immunology Program, Peter MacCallum Cancer Centre, Locked Bag 1, A'Beckett Street, Melbourne, Victoria 8006, Australia. Phone: 61-3-9656-3728; Fax: 61-3-9656-1411; E-mail: mark.smyth{at}petermac.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A rational monoclonal antibody (mAb)-based antitumor therapy approach has previously been shown to eradicate various established experimental and carcinogen-induced tumors in a majority of mice. This therapy comprised an agonistic mAb reactive with tumor necrosis factor–related apoptosis-inducing ligand receptor (DR5), expressed by tumor cells, an agonistic anti-CD40 mAb to mature dendritic cells, and an agonistic anti-4-1BB mAb to costimulate CD8+ T cells. Because agonists of CD40 have been toxic in patients, we were interested in substituting anti-CD40 mAb with other dendritic cell–maturing agents, such as glycolipid ligands recognized by invariant natural killer T (iNKT) cells. Here, we show that CD1d-restricted glycolipid ligands for iNKT cells effectively substitute for anti-CD40 mAb and reject established experimental mouse breast and renal tumors when used in combination with anti-DR5 and anti-4-1BB mAbs (termed "NKTMab" therapy). NKTMab therapy–induced tumor rejection was dependent on CD4+ and CD8+ T cells, NKT cells, and the cytokine IFN-{gamma}. NKTMab therapy containing either {alpha}-galactosylceramide ({alpha}-GC) or {alpha}-C-galactosylceramide ({alpha}-c-GC) at high concentrations induced similar rates of tumor rejection in mice; however, toxicity was observed at the highest doses of {alpha}-GC (>250 ng/injection), limiting the use of this glycolipid. By contrast, even very low doses of {alpha}-c-GC (25 ng/injection) retained considerable antitumor activity when used in combination with anti-DR5/anti-4-1BB, and thus, {alpha}-c-GC showed a considerably greater therapeutic index. In summary, sequential tumor cell apoptosis and amplification of dendritic cell function by NKT cell agonists represents an exciting and novel approach for cancer treatment. [Cancer Res 2007;67(15):7495–504]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Immunotherapy is now gaining recognition as a valid approach for the treatment of cancer. The promise of immunotherapy is based on graft versus leukemia activity following hematopoietic stem cell transplantation, the use of monoclonal antibodies (mAb) to target lymphoma (Rituximab; ref. 1), breast (Trastuzumab; ref. 2), and colorectal (bevacizumab and cetuximab; ref. 3) cancer, and the use of vaccines (4) or adoptive T-cell transfer (5) in melanoma. However, response rates using these therapies are generally low and are often not complete and tumors can relapse. Hence, it is our contention that, although mAbs have been one of the most successful and safe forms of immunotherapy to date, no single mAb against a tumor antigen is going to provide the "magic bullet" that eliminates established cancer.

Tumor cell apoptosis is the basis of many cancer therapies, and several apoptosis-inducing receptors have been targeted with mAb or ligand with the aim of triggering tumor cell death. Recently, it has been shown that tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) preferentially induces apoptosis in tumor cells, but not normal cells, and that it plays a critical role in tumor immune surveillance (6–8). Hence, the TRAIL receptor is considered an attractive target for mAb-mediated tumor therapy. Indeed, experimental tumor models have shown the potent tumoricidal activities of recombinant soluble forms of TRAIL or agonistic mAb specific for cell death–inducing human TRAIL receptors (DR4 and DR5; ref. 7) and humanized versions have reached phase II clinical trials. We now also appreciate that tumor-specific CTL induction plays a critical role in the successful antitumor effects of some mAb-based therapies (7, 9, 10), and indeed, induction of CTL reactive with tumors has been a goal of most immunotherapies. We have shown that anti-mouse DR5 mAb generated adaptive tumor-specific immunity, and we hypothesized that additionally maturing dendritic cells and costimulating CTL might potentially result in an even more effective cancer therapy than anti-DR5 mAb alone. Indeed, we have now combined the tumor apoptosis mediated by anti-DR5 mAb with agonistic mAbs against CD40 and 4-1BB that mature dendritic cells and costimulate T cells, respectively. Surprisingly, this combined therapy that we have termed "TriMab" resulted in the eradication of several different preestablished tumors and multiorgan metastases in mice (11). Notably, however, the reported toxicity observed in experimental animal models and patients receiving agonists of CD40 (12–15), and a recent study suggesting that triggering of CD40 on endothelial cells might actually promote tumor growth (16), has given us the impetus to investigate substituting anti-CD40 mAb in the combination therapy with other agents that effectively mature dendritic cells.

Natural killer T (NKT) cells are unique population of T cells capable of regulating a broad range of immune responses, including autoimmunity, allergy, infection, and tumor rejection (17, 18). NKT cells recognize glycolipids presented in the context of MHC-1–like molecule, CD1d, and in mice the invariant NKT (iNKT) cell subpopulation expresses a biased T-cell receptor (TCR) repertoire characterized by an invariant V{alpha}14-J{alpha}18 chain coupled with either Vß8.2, 7, or 2, whereas human iNKT cells express the homologous TCR gene V{alpha}24-J{alpha}18 in conjunction with Vß11 (17). The most commonly used glycolipid ligand for the study of NKT cell activation is {alpha}-galactosylceramide ({alpha}-GC). {alpha}-GC is presented by CD1d-expressing antigen-presenting cells (APC) and potently activates iNKT cells to rapidly produce both T helper (Th) 1 and Th2 cytokines, such as IFN-{gamma} and interleukin (IL)-4 (17). Importantly, activation of iNKT cells with {alpha}-GC leads to potent downstream activation of CD8+ T cells, natural killer (NK) cells, and APC, such as dendritic cells and B cells (19–21). Bystander activation of these cells is crucial to the protective antitumor and microbial immunity mediated by {alpha}-GC. Furthermore, given the potent immunoregulatory role and antitumor functions of iNKT cells in mice, {alpha}-GC has been used in clinical trials as an antitumor agent (22–26). {alpha}-GC seems to have limited antitumor activity when used alone, but it does cause downstream activation of T cells (26). Thus, the ability of {alpha}-GC–activated iNKT cells to cross-talk with mature dendritic cells makes {alpha}-GC and some related analogues attractive replacements for agonistic anti-CD40 mAbs.

In this study, we have shown that substituting glycolipid ligands for anti-CD40 mAb in combination with anti-DR5 and anti-CD137 mAbs (termed "NKTMab" therapy) is similarly effective to TriMab therapy in rejecting established experimental tumors. These data illustrate for the first time the utility of some CD1d-binding iNKT cell ligands when used in combination therapies that both cause tumor cell apoptosis directly and costimulate resultant tumor-specific T-cell immunity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice
Inbred BALB/c mice were purchased from The Walter and Eliza Hall Institute of Medical Research. BALB/c IFN-{gamma}–/–, BALB/c IL-4–/–, BALB/c IL-13–/–, and BALB/c J{alpha}18-deficient mice (J{alpha}18–/–) were bred and maintained at the Peter MacCallum Cancer Centre (PMCC). All gene-targeted mice were backcrossed onto the BALB/c background for at least 10 generations and genotyped by either PCR or flow cytometry. Mice >6 weeks old were used in all experiments. Female mice were used for all experiments with the 4T1 tumor, and all experiments were done in accordance with guidelines set out by the PMCC animal experimentation ethics committee.

Tumor Cell Lines
BALB/c-derived TRAIL-sensitive 4T1 mammary carcinoma (27, 28), TRAIL-sensitive Renca renal carcinoma (29), TRAIL-sensitive Renca variant R331, and TRAIL-resistant variant R331-FLIP (27, 29) were maintained as described previously.

Antibodies and Glycolipids
We prepared and purified agonistic mAb to mouse DR5 (MD5-1), agonistic mAb to mouse CD40 (FGK45; provided by A. Rolink, University of Basel, Basel, Switzerland; ref. 30), agonistic mAb to mouse 4-1BB (CD137; 3H3; provided by R. Mittler, Emory University, Atlanta, GA; ref. 31), mAb to mouse CD4 (GK1.5), and mAb to mouse CD8 (53-6.7) as described previously (7). We purchased anti-asialo-GM1 (ASGM1) from Wako Pure Chemicals. {alpha}-GC was provided by the Pharmaceutical Research Laboratories, Kirin Brewery, and prepared as described (32). {alpha}-C-galactosylceramide ({alpha}-c-GC) and {alpha}-GC C20:2 (C20:2) were synthesized as described (33, 34). Glycolipids and the control vehicle were resuspended in saline supplemented with 0.5% polysorbate-20 (w/v).

Measuring Serum Transaminases
Groups of BALB/c wild-type (WT) mice were inoculated s.c. with 2 x 105 4T1 tumor cells on day 0. Seven days after tumor inoculation, groups of mice were either left untreated or treated i.p. with the following: NKTMab therapy (100 µg each of anti-DR5 and anti-4-1BB mAbs and the indicated dose of {alpha}-GC or {alpha}-c-GC), TriMab therapy (100 µg each of anti-DR5, anti-4-1BB, and anti-CD40 mAbs), and anti-4-1BB or anti-DR5 mAb alone or in combination or {alpha}-GC alone at the indicated dose at day 7 (treatment 1) and day 11 (treatment 2). Mice were eye bled the day after treatment 1 (day 8), before treatment 2 (day 11), and a day after treatment 2 (day 12). Briefly, blood collected from retro-orbital puncture was spun at 13,000 rpm for 15 min at 11°C. Sera were then harvested and either used immediately for analysis or stored at –20°C. Before analysis, sera were thawed and diluted 1 in 5 or 1 in 10 in sterile saline and serum transaminase [alanine aminotransferase (ALT) and aspartate aminotransferase (AST)] levels measured on the Advia 1200 Chemistry System (Bayer HealthCare).

Therapy of Transplanted Tumors
R331 renal tumor growth. Groups of five BALB/c WT mice were inoculated s.c. with 5 x 105 R331 tumor cells on day 0. Seven days after tumor inoculation, groups of mice were treated i.p. with the following: control Ig (300 µg), TriMab therapy (100 µg each of anti-DR5, anti-4-1BB, and anti-CD40 mAbs), NKTMab therapy (100 µg each of anti-DR5, anti-4-1BB mAbs, and the indicated dose of {alpha}-GC, {alpha}-c-GC, or C20:2), a combination of anti-4-1BB or anti-DR5 mAbs with the indicated dose of {alpha}-GC, and a combination of anti-4-1BB and anti-DR5 mAbs or the indicated dose of {alpha}-c-GC or C20:2. Tumor size was measured with a caliper and recorded as the product of two perpendicular diameters (cm2).

4T1 mammary tumor growth. Groups of five BALB/c WT, BALB/c IFN-{gamma}–/–, BALB/c IL-4–/–, or BALB/c J{alpha}18–/–, and BALB/c IL-13–/– mice were inoculated s.c. with 2 x 105 4T1 tumor cells on day 0. Seven days after tumor inoculation, groups of mice were treated i.p. with the following: control Ig (100 µg), NKTMab therapy (100 µg each of anti-DR5 and anti-4-1BB mAbs and 100 ng {alpha}-GC), or a combination of anti-4-1BB and anti-DR5 mAbs (100 µg each). In some experiments, mice were depleted of CD4+ (GK1.5), CD8+ (53.6.72), a combination of CD4+ and CD8+ T cells, or NK cells on days 0, 7, 14, and 21. Tumor size was measured using a caliper and recorded as the product of two perpendicular diameters (cm2).

Statistical Analysis
Statistical significance of tumor growth or tumor-free survival was assessed through the use of the Mann-Whitney rank sum test or Fisher's exact test as appropriate.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
NKTMab and TriMab therapy induce similar rates of tumor rejection. We have previously shown that TriMab therapy (anti-DR5, anti-4-1BB, and anti-CD40 mAb) is a potent antibody combination against established mouse tumors (11). When given on days 7 (tumors ~10 mm2), 11, and 15, TriMab reproducibly induced s.c. R331 tumor rejection in 80% of mice (Fig. 1 ) To compare the antitumor efficacy of NKTMab (anti-DR5 mAb, anti-4-1BB mAb, and {alpha}-GC) therapy, groups of mice with established s.c. R331 tumors (10 mm2) were treated with alternate regimens of therapy. By substituting a 500 ng dose of {alpha}-GC for each 100 µg dose of anti-CD40 mAb, a very similar suppression of tumor growth and rate of rejection (80%) was noted for the NKTMab combination (Fig. 1A). Treatment with anti-DR5, anti-4-1BB, or {alpha}-GC alone, using the same dose and regimen, had little effect (data not shown). Very good tumor growth suppression was observed with anti-DR5/anti-4-1BB mAbs (data not shown) and {alpha}-GC/anti-4-1BB treatment (Fig. 1A) but only with 0% to 20% tumor rejection across several repeats of this experiment. A combination of anti-DR5 and {alpha}-GC had minor effect on tumor growth (Fig. 1A). Similar rates of tumor rejection were observed in mice with established s.c. 4T1 tumors (10 mm2) following treatment with NKTMab therapy (500 ng {alpha}-GC; Supplementary Fig. S1A). Reducing the dose of {alpha}-GC in the NKTMab therapy from 500 ng to as low as 50 ng per dose (Fig. 1A–D) still enabled significant tumor growth suppression compared with control Ig/vehicle treatment and 60% tumor rejection (Fig. 1). Similar results were also seen against extensive multiorgan 4T1 metastases (Supplementary Fig. S1B). Notably, mice that rejected tumors following NKTMab or TriMab therapy remained tumor-free for >150 days. Thus, our data showed that NKTMab therapy induced lasting tumor rejection responses similar to TriMab therapy.


Figure 1
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  		Figure 1. Comparable eradication of established R331 tumors by NKTMab or TriMab therapy. Groups of BALB/c mice (n = 5) were inoculated s.c. with the renal carcinoma cell line R331 (5 x 105). On days 7, 11, and 15 after tumor inoculation, mice were i.p. treated with either NKTMab ({blacksquare}) or TriMab (bullet) therapy or with anti-DR5/{alpha}-GC ({Delta}), anti-4-1BB/{alpha}-GC ({blacktriangleup}), or control Ig (cIg; {square}). A to D, decreasing doses of {alpha}-GC (500, 250, 100, and 50 ng) were used in the NKTMab therapy or in combination with anti-4-1BB or anti-DR5 mAb. Points, mean of tumor sizes; bars, SE. Parentheses, tumor rejection rates. Statistical differences in tumor size between control Ig/vehicle-treated and NKTMab-treated mice were determined by Mann-Whitney rank sum test. *, P < 0.05. Similar results were obtained in two other independent experiments.

 
NKTMab therapy can induce liver toxicity. Although NKTMab therapy induced a similar rate of rejection to TriMab therapy, a small proportion of mice (9 of 35) receiving the 500 ng dose of {alpha}-GC in the NKTMab combination did not survive treatment (Supplementary Table S1) and a larger proportion developed ruffled fur and signs of treatment-related toxicity. Given that activation of NKT cells can result in rapid and large production of inflammatory cytokines, and is often associated with liver toxicity in mice (35, 36), we decided to assay the liver function of tumor-bearing mice surviving NKTMab and other treatments by measuring serum ALT and AST levels (Fig. 2 ). Interestingly, at a 500 ng dose of {alpha}-GC, mice had elevated serum ALT levels after each treatment of NKTMab therapy (Fig. 2A, T), which reduced to baseline in between treatments. Tumor-bearing mice that did not receive NKTMab therapy (Fig. 2A, C) had baseline levels of ALT. By contrast, mice that received TriMab therapy, anti-DR5 mAbs, anti-4-1BB mAbs, or {alpha}-GC (500 ng/dose) alone did not display any symptoms of toxicity or elevated serum ALT (Fig. 2B). Indeed, only the dual combination of anti-4-1BB and {alpha}-GC (500 ng), but not anti-DR5 and {alpha}-GC (500 ng), induced elevated serum ALT (Fig. 2B). Similar results were obtained with AST measurements (data not shown). We also assayed the survival, toxicity, and levels of serum ALT in tumor-bearing mice treated with NKTMab containing reduced doses of {alpha}-GC (25–250 ng; Supplementary Table S1; Fig. 2). Although no difference was observed between the ALT levels of mice treated with the different doses of {alpha}-GC, mice treated with lower doses of {alpha}-GC (12.5, 25, and 50 ng) showed little signs of physical stress and all survived the complete treatment schedule. Given that optimal rates of tumor rejection accompanied survival of all mice receiving 100 ng doses of {alpha}-GC in the NKTMab therapy, all subsequent mechanism experiments used a dose of 100 ng {alpha}-GC per injection.


Figure 2
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  		Figure 2. Serum ALT levels of tumor-bearing mice following NKTMab therapy. Groups of BALB/c mice (n = 3–7) were inoculated s.c. with the mammary carcinoma cell line 4T1 (2 x 105). Seven days after tumor inoculation, mice were either left untreated (C) or injected i.p. with mAbs and/or {alpha}-GC (T) at days 7 (treatment 1) and 11 (treatment 2). Mice were eye bled after treatment 1 (Post Txt 1), before treatment 2 (Pre Txt 2), after treatment 2 (Post Txt 2), and 4 d after treatment 2 (4 days post Txt 2), and their sera were assayed for the presence of ALT. A, ALT levels of tumor-bearing mice after NKTMab (containing 500 ng {alpha}-GC) therapy. B, ALT levels of tumor-bearing mice either after single-agent therapy [{alpha}-GC (500 ng), anti-DR5 mAb (100 µg), and anti-4-1BB mAb (100 µg)], dual therapy [{alpha}-GC (500 ng)/anti-DR5 mAb (100 µg) and {alpha}-GC (500 ng)/anti-4-1BB mAb (100 µg)], or TriMab therapy. C, ALT levels of tumor-bearing mice after NKTMab therapy containing decreasing doses of {alpha}-GC (25–500 ng). B and C, only ALT levels after treatment 1 and after treatment 2 are shown. Each point represents ALT level of a single mouse and where shown the mean of the group is represented by the cross bar.

 
NKTMab therapy–induced tumor rejection requires IFN-{gamma}, T cells, and NKT cells. We next wanted to determine the mechanism of tumor rejection induced by NKTMab therapy. Groups of BALB/c WT mice (Fig. 3A, B, C, E, and F ) or BALB/c J{alpha}18–/– mice (Fig. 3D) were inoculated s.c. with 4T1 tumor cells. In some groups of WT mice, anti-CD4 (Fig. 3A), anti-CD8 (Fig. 3B), anti-CD4 combined with anti-CD8 (Fig. 3C), or anti-ASGM1-depleting (Fig. 3E) mAbs were given on days 0, 7, 14, and 21. Seven days after tumor inoculation, mice were treated with control Ig, TriMab therapy, or NKTMab therapy. In CD4, CD8, or CD4 and CD8 T-cell–depleted tumor-bearing mice, NKTMab therapy had minimal effect on tumor growth (Fig. 3A–C), whereas NKTMab therapy was not NK cell dependent (Fig. 3E). No BALB/c J{alpha}18–/– mice treated with NKTMab therapy rejected tumors, a similar effect as mice treated with the dual combination of anti-DR5/anti-4-1BB mAbs (i.e., no {alpha}-GC; Fig. 3D). Overall, the data suggested that NKTMab therapy (anti-DR5/{alpha}-GC/anti-4-1BB) was critically dependent on CD4+ and CD8+ T cells, whereas in the absence of iNKT cells, NKTMab was only as effective as anti-DR5/anti-4-1BB mAbs. Perhaps not surprisingly, NKT cells were not required for TriMab therapy in tumor-bearing J{alpha}18–/– mice (Fig. 3D), as the percentage of tumor rejection was similar to that in tumor-bearing WT mice treated with TriMab therapy (Fig. 3F). Consistent with our previous report (11), TriMab therapy was strictly CD8+ T cell dependent but did not require CD4+ T cells or NK cells (Fig. 3).


Figure 3
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  		Figure 3. T cells and NKT cells are required for NKTMab-induced rejection of tumors. Groups of BALB/c WT (A, B, C, E, and F) or BALB/c J{alpha}18–/– (D) mice (n = 5) were inoculated s.c. with the mammary carcinoma 4T1 (2 x 105). On days 7, 11, and 15 after tumor inoculation, mice were treated with either NKTMab therapy (containing 100 ng {alpha}-GC; {blacksquare}), TriMab therapy (bullet), anti-DR5/anti-4-1BB mAbs ({Delta}), or control Ig ({square}). Additionally, some groups of mice were also i.p. injected on days 0, 7, 14, and 21 with either CD4-depleting mAbs (A), CD8-depleting mAbs (B), CD4- and CD8-depleting mAbs (C), anti-ASGM1 antibody (E), or control Ig (F). Points, mean of tumor sizes; bars, SE. Parentheses, tumor rejection rates. Statistical differences in tumor size between WT mice and antibody-depleted WT mice receiving NKTMab therapy were determined by Mann-Whitney rank sum test. *, P < 0.05.

 
A key characteristic of NKT cells is their ability to rapidly produce Th1 and Th2 cytokines. To investigate the key cytokines involved in NKTMab therapy–induced tumor rejection, BALB/c WT mice or BALB/c IFN-{gamma}–/–, IL-4–/–, or IL-13–/– mice with established s.c. 4T1 mammary tumors were treated with NKTMab (containing 100 ng {alpha}-GC) therapy (Fig. 4 ). In contrast to WT tumor-bearing mice that displayed 60% tumor rejection after NKTMab therapy (Fig. 4A), no tumor rejection was observed in similar groups of IFN-{gamma}–/– tumor-bearing mice (Fig. 4B). Interestingly, NKTMab therapy induced tumor rejection in 60% and 80% of IL-4–/– and IL-13–/– mice, respectively (Fig. 4C and D). Tumor growth suppression was observed in WT, IL-4–/–, or IL-13–/– mice following anti-DR5/anti-4-1BB mAb treatment, whereas the therapeutic effect of the dual combination was completely abrogated in IFN-{gamma}–/– mice. Tumors in mice treated with control Ig grew equivalently in WT, IFN-{gamma}–/–, IL-13–/–, and IL-4–/– mice. Overall, these results showed that NKTMab therapy had a distinct mechanism of action that was dependent on NKT, CD4+ and CD8+ T cells, and IFN-{gamma}.


Figure 4
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  		Figure 4. Requirement of IFN-{gamma} in NKTMab-induced rejection of tumors. Groups of BALB/c WT (A), BALB/c IFN-{gamma}–/– (B), BALB/c IL-4–/– (C), and BALB/c IL-13–/– (D) mice (n = 5) were inoculated s.c. with the mammary carcinoma cell line 4T1 (2 x 105). On days 7, 11, and 15 after tumor inoculation, mice were treated with NKTMab therapy (containing 100 ng {alpha}-GC; {blacksquare}), anti-DR5/anti-4-1BB mAbs ({Delta}), or control Ig ({square}). Points, mean of tumor sizes; bars, SE. Parentheses, tumor rejection rates. Statistical differences in tumor size between WT and gene-targeted mice treated with NKTMab therapy were determined by Mann-Whitney rank sum test. *, P < 0.05.

 
{alpha}-c-GC provides a more effective NKTMab therapy. Although {alpha}-GC is the prototype CD1d-restricted glycolipid ligand for activation of iNKT cells, a series of new analogues of {alpha}-GC have been generated (37–39). One such C-glycoside analogue, {alpha}-c-GC, more effectively binds CD1d and induces sustained IFN-{gamma} production (40). {alpha}-c-GC is a log or more potent than {alpha}-GC in suppressing B16 lung metastases and malaria (41). To determine whether substituting {alpha}-c-GC for {alpha}-GC in NKTMab therapy might enable more effective tumor rejection, the combination was compared with TriMab therapy in mice with established s.c. R331 tumors (as above; Fig. 5A ). A 100 ng dose regimen of {alpha}-c-GC caused an almost identical suppression of tumor growth and rate of rejection (80%) in comparison with TriMab therapy (Fig. 5A). Treatment of mice with {alpha}-c-GC alone only resulted in minimal tumor growth suppression compared with mice receiving control Ig, indicating that this potent analogue had limited efficacy as a monotherapy against established tumors.


Figure 5
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  		Figure 5. Improved therapeutic index of NKTMab therapy containing {alpha}-c-GC. A, groups of BALB/c mice (n = 5) were inoculated with the renal carcinoma cell line R331 (5 x 105). On days 7, 11, and 15 after tumor inoculation, tumor-bearing mice were treated with NKTMab (containing 100 ng {alpha}-c-GC) therapy ({blacklozenge}), TriMab therapy (bullet), {alpha}-c-GC (100 ng) alone ({lozenge}), or control Ig ({square}). B and C, NKTMab therapy containing {alpha}-c-GC is more efficacious compared with NKTMab therapy containing {alpha}-GC. Groups of BALB/c mice (n = 5) were inoculated s.c. with the renal carcinoma cell line R331 (5 x 105). On days 7, 11, and 15 after tumor inoculation, groups of tumor-bearing mice were treated with NKTMab therapy containing either {alpha}-GC (6.25, 12.5, 25, 50, or 100 ng) or {alpha}-c-GC (6.25, 12.5, 25, 50, or 100 ng) or C20:2 (100 ng). Additionally, similar groups of tumor-bearing mice were also treated with TriMab therapy, anti-DR5/anti-4-1BB mAbs, or control Ig. B, points, mean of tumor sizes; bars, SE. Parentheses, tumor rejection rates. C, data are presented from several pooled experiments and the proportion of tumor-free mice were illustrated. Parentheses, number of mice in each group. In (A), statistical differences in tumor size between control Ig–treated and NKTMab-treated mice were determined by Mann-Whitney rank sum test. *, P < 0.05. In (C), statistical differences in tumor-free survival between {alpha}-GC and {alpha}-c-GC containing NKTMab therapy were determined by Fisher's exact test. *, P < 0.05.

 
Given that {alpha}-c-GC has been reported to induce larger and sustained iNKT cell responses even at low concentrations (40), we next compared reducing doses of {alpha}-c-GC with {alpha}-GC in NKTMab therapy against established s.c. R331 tumor growth (Fig. 5B). Despite decreasing the dose of {alpha}-c-GC by 4-fold (100 to 25 ng), the combined NKTMab therapy induced a similar tumor rejection rate (80%) to TriMab therapy (Fig. 5B). By contrast, a 25 ng dose of {alpha}-GC in the NKTMab combination resulted in only moderate tumor suppression and no tumors were completely rejected (Fig. 5B). Further, reducing {alpha}-c-GC from 25 to 12.5 ng in the NKTMab therapy still retained a 40% tumor rejection rate, whereas once again no tumor rejection was observed in groups of mice treated with {alpha}-GC (12.5 ng) containing NKTMab therapy. Even at a very low dose of {alpha}-c-GC (6.25 ng), NKTMab therapy still induced tumor growth suppression compared with the anti-DR5/anti-4-1BB mAb combination. Interestingly, treatment of tumor-bearing mice with NKTMab therapy containing a high dose of C20:2 (100 ng), an analogue of {alpha}-GC that activates NKT cells to preferentially produce Th2 cytokines (34, 39), had no more effect than dual anti-DR5/anti-4-1BB combination therapy on tumor growth.

The significant improvement in antitumor efficacy when {alpha}-c-GC, rather than {alpha}-GC, was used in the NKTMab therapy was best illustrated by a statistical improvement in tumor-free survival in mice with R331 treated with either 50, 25, or 12.5 ng of glycolipid (Fig. 5C). Importantly, NKTMab (containing {alpha}-GC or {alpha}-c-GC) was not as effective against a TRAIL-resistant tumor cell transfectant of R331 (R331-FLIP; Supplementary Fig. S2), consistent with our previous studies that illustrated the resistance of FLIP-expressing tumors to DR5-mediated therapy (7, 11). Very similar efficacy of {alpha}-c-GC containing NKTMab therapy was also observed against extensive multiorgan 4T1 metastases (Supplementary Fig. S3). To determine the potency of {alpha}-c-GC containing NKTMab therapy, we have also examined the efficacy of NKTMab therapy (100 ng {alpha}-c-GC) against increasingly large s.c. R331 tumors (Fig. 6 ). Impressively, tumor rejection rates of 60% to 80% were observed when treatment commenced on day 12 (mean tumor size, 0.26 cm2) or day 16 (mean tumor size, 0.38 cm2; Fig. 6B and C). Remarkably, starting NKTMab ({alpha}-c-GC) therapy of mice on day 20 with mean tumor size equal to 0.55 cm2 still induced significant tumor growth suppression and tumor rejection in 20% of treated mice (Fig. 6D). The efficacy of NKTMab ({alpha}-c-GC) and TriMab therapy in BALB/c mice bearing established orthotopic Renca tumors was also shown (Supplementary Fig. S4).


Figure 6
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  		Figure 6. NKTMab therapy containing {alpha}-c-GC can eradicate large established tumors. Groups of five BALB/c mice were inoculated s.c. with the renal carcinoma cell line R331 (5 x 105). Tumor-bearing mice were then treated with three doses of NKTMab therapy (100 µg each of anti-DR5 and anti-4-1BB mAbs and 100 ng {alpha}-c-GC) commencing treatment at day 7 (A), day 12 (B), day 16 (C), day 20 (D), or day 24 (E) or left untreated (F). Points, mean of tumor sizes; bars, SE. Parentheses, tumor rejection rates.

 
{alpha}-c-GC provides a safer NKTMab therapy. Surprisingly, mice receiving {alpha}-c-GC in NKTMab combination therapy showed no signs of toxicity. Given that liver toxicity was observed in tumor-bearing mice treated with NKTMab therapy containing {alpha}-GC, we next assessed sera levels of ALT in tumor-bearing mice following NKTMab therapy containing the highest dose of {alpha}-c-GC (500 ng/dose). Unlike mice treated with NKTMab therapy containing {alpha}-GC, only very low levels of ALT were induced following NKTMab ({alpha}-c-GC) therapy and these levels were not increased following the second injection of the treatment schedule (Supplementary Fig. S5). These data indicated that NKTMab therapy (comprising anti-DR5, anti-4-1BB, and {alpha}-c-GC) not only induced a similar level of tumor rejection to TriMab therapy but did so in the absence of any appreciable toxicity.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we have shown that CD1d-restricted glycolipid ligands reactive with iNKT cells effectively substitute for anti-CD40 mAbs and can reject established experimental mouse breast and renal tumors when used in combination with anti-DR5 and anti-4-1BB mAbs. This combination, which we termed NKTMab therapy, induced tumor rejection that required CD4+ and CD8+ T cells, NKT cells, and the cytokine IFN-{gamma}. NKTMab therapy containing either {alpha}-GC or {alpha}-c-GC at higher concentrations induced similar rates of tumor rejection in mice; however, toxicity was observed at the highest doses of {alpha}-GC (>250 ng/injection). By contrast, very low doses of {alpha}-c-GC (25 ng/injection) retained considerable antitumor activity when used in combination with anti-DR5/anti-4-1BB, and thus, {alpha}-c-GC showed a considerably greater therapeutic index. Given the shown toxicities of CD40 agonists in humans and mice (13, 16), this study illustrates the alternative of using NKT cell agonists that in synergy with an anti-DR5/anti-4-1BB combination preferentially drive IFN-{gamma} production to suppress tumor growth and metastases.

Although TriMab (anti-DR5/anti-CD40/anti-4-1BB) has shown tremendous promise in preclinical mouse models (11), the known toxicities of CD40 agonists in humans may limit the clinical utility of this approach. The effective replacement of anti-CD40 mAbs with glycolipids in our combination therapy approach represents a feasible alternative of activating and maturing dendritic cells in a similarly effective tumor immunotherapy. The reciprocal activation of NKT cells and dendritic cells is initiated on the presentation of glycolipids on CD1d molecules by resting dendritic cells to NKT cells. NKT cells are thus induced to up-regulate CD40L, Th1, and Th2 cytokines and chemokines, whereas CD40 cross-linking induces dendritic cells to up-regulate CD80, CD86, and IL-12, which in turn enhances NKT cell activation and cytokine production (17).

Although {alpha}-GC has been the prototype CD1d ligand used in the activation of iNKT cells to exert antitumor effects in experimental models of cancers, several new structurally distinct analogues of {alpha}-GC have been synthesized (37–41). Recently, a synthetic isosteric C-glycoside analogue of {alpha}-GC ({alpha}-c-GC) was synthesized and reported to exhibit a 1,000-fold more potent antimalaria activity and a 100-fold more potent antimetastatic activity than {alpha}-GC (41). The improved antitumor efficacy of {alpha}-c-GC over {alpha}-GC was recently shown by Fujii et al. (40) to be due to the ability of {alpha}-c-GC to bind more stably to dendritic cells, resulting in more prolonged production of Th1 cytokines, such as IL-12 and IFN-{gamma}, but not IL-4 or TNF-{alpha}. By substituting {alpha}-GC with {alpha}-c-GC in our NKTMab therapy, we showed the improved antitumor efficacy of {alpha}-c-GC to induce tumor rejection at doses considerably lower than {alpha}-GC. In addition, the importance of using glycolipids that preferentially induce Th1 cytokine production was also illustrated in our study by a complete lack of tumor rejection in mice receiving NKTMab therapy containing the C20:2 analogue of {alpha}-GC. Our findings were consistent with the key role of IFN-{gamma} in tumor rejection and that the C20:2 analogue contains a diunsaturated C20 fatty acid and has been shown to potently induce NKT cell to preferentially secrete Th2 cytokines, with diminished IFN-{gamma} production and reduced NKT cell expansion (34, 39). More recently, the synthesis of a new truncated nonisosteric {alpha}-c-GC analogue has been reported (42). It was shown to induce cytokine production from NKT cells with the highest IFN-{gamma} to IL-4 and IFN-{gamma} to IL-13 ratios compared with {alpha}-c-GC and {alpha}-GC. This novel mimetic of {alpha}-GC might potentially further promote Th1 immune response over that induced by {alpha}-c-GC, and future experiments will be undertaken to directly compare this glycolipid with {alpha}-c-GC in the R331 tumor model.

Surprisingly, despite a potent antitumor effect of NKTMab therapy containing {alpha}-GC, at the highest dose of {alpha}-GC tested, the treated mice developed ruffled fur, had elevated serum ALT and AST levels, and on rare occasions died. By contrast, mice that received {alpha}-c-GC in NKTMab therapy at all doses tested (6–500 ng/injection) only displayed moderate and transient elevations in ALT at the highest dose and never severe signs of toxicity. These data highlighted the far greater therapeutic index of the anti-DR5/{alpha}-c-GC/anti-4-1BB combination. Among the various reductions of this combination, only anti-4-1BB/{alpha}-GC (500 ng/dose) induced significantly elevated serum ALT levels. We will now determine which cytokines contribute to elevations in serum ALT/AST using tumor-bearing WT mice and IFN-{gamma}, IL-4, IL-13, or other gene-targeted mice. Although liver toxicities were seen in tumor-bearing mice following NKTMab ({alpha}-GC) therapy, the safety of {alpha}-GC alone in humans has previously been shown in several clinical trials. Administration of {alpha}-GC directly into cancer patients (24, 25) or transfer of {alpha}-GC–activated NKT cells (23) or {alpha}-GC–pulsed dendritic cells (22) has been found to be well tolerated in patients with advanced diseases. Humans contain fewer iNKT cells in the liver compared with mice (43), and it is possible that the current toxicity observed in our studies may not manifest in humans. The safety of {alpha}-c-GC in the NKTMab therapy has yet to be tested in the clinic.

We have shown that NKTMab therapies (at the highest doses of {alpha}-GC or {alpha}-c-GC) can induce similar rates of tumor rejection to TriMab therapy against two different tumor types in s.c. or metastatic settings. Encouragingly, even larger established tumors were quite effectively suppressed by the optimal NKTMab therapy. However, to determine the widespread applicability of NKT cell–based therapy, NKTMab treatment of other mouse experimental tumors from different tissue origins will also be examined. Alternatively, the effectiveness of NKTMab therapy can also be assessed in transgenic models of carcinogenesis, such as BALB/c HER-2/neu mice, which provides valuable insight on disease progression and treatment because they have distinct stages comparable with those observed in human carcinomas.

One of the hallmark features of an effective immunotherapy is its ability to stimulate lasting tumor-specific immunity. We have previously shown that TriMab therapy generates tumor-specific memory responses detectable by secondary tumor challenge. Like TriMab therapy, NKTMab therapy–cured mice were also able to mount a tumor-specific response following secondary tumor challenge (data not shown). Similar to TriMab therapy, IFN-{gamma} was found to be an important mediator of tumor rejection in NKTMab therapy. However, unlike TriMab therapy, where CD8+ T cells alone were enough to induce tumor rejection, our current studies showed a critical requirement for both CD4+ and CD8+ T cells and NKT cells for effective NKTMab-induced tumor rejection. However, we cannot discount the possibility that antibody depletion of CD4+ cells may deplete some CD4+ NKT cells. Interestingly, TriMab therapy of J{alpha}18–/– tumor-bearing mice induced a similar rate of tumor rejection as WT tumor-bearing mice, showing that several ways to activate dendritic cells exist: CD40 ligation or cross-talk with NKT cells.

However, it is also possible to activate dendritic cells via CD1d independently of NKT cells activation. Recently, CD1d ligation alone, in the absence of NKT cells, has been shown to rapidly stimulate production of active IL-12p70 by CD1d+ human peripheral blood monocytes and immature dendritic cells (44). IFN-{gamma} was without effect alone but significantly increased CD1d-stimulated IL-12 production. In concert with this representing a physiologic response, monocyte differentiation, as evidenced by CD1a and CD40 up-regulation, was also accelerated by CD1d stimulation. This work illustrated an innate immune signaling function for CD1d and provided a mechanism by which monocytes and immature dendritic cells could be rapidly activated in the absence of anti-CD40 or glycolipid ligands. Because it is not clear whether concurrent NKT cell activation is necessary to achieve optimal therapeutic antitumor activity, future work will determine whether agonistic anti-mouse CD1d mAbs can substitute for anti-CD40 or {alpha}-GC/{alpha}-c-GC using the R331 tumor model. It will also be interesting whether a blocking anti-CD1d mAb might also inhibit type II NKT cell activation and thereby promote downstream tumor immunity (45).

Several factors could potentially limit the antitumor efficacy of NKT cell–based immunotherapy, and as such, their effect will have to be duly assessed. It has been documented that frequencies of human NKT cells are more variable between individuals and that cell numbers are typically lower than that found in mice. For example, levels of NKT cells in the thymus are at least 100-fold lower in humans (46), and in the liver of humans, only 1% of lymphocytes are NKT cells compared with 30% in the liver of mice (43), which is also one possible explanation for the low levels of toxicity observed in clinical trials to date. The lower number of NKT cells in humans will thus have to be taken into consideration when designing any potential trial with a NKTMab-like immunotherapy. Similar to T cells, immune suppression of NKT cells has also been commonly encountered in cancer patients where they have either been reported to be decreased compared with healthy humans or functionally hyporeactive (47). Yanagisawa et al. recently reported that {alpha}-GC–induced NKT cell function in cancer-bearing mice was impaired. This was due to an increase in CD11b+ Gr-1+ cells, which mediated their suppression on NKT cell function in a nitric oxide–mediated fashion. However, the authors were able to relieve this suppression through administration of all-trans-retinoic acid, which decreased the CD11b+ Gr-1+ population (48), and a similar strategy might be considered in humans. It has also been reported that tumors may modulate their CD1d molecules or shed glycosphingolipids in vitro to inhibit stimulation of NKT cells (49, 50) as another means of avoiding NKT cell activation. Although NKTMab therapy successfully induced rejection of established tumors in most mice in our studies, it will be interesting to further deplete NKTMab-treated tumor-bearing mice (that have not rejected tumors) of regulatory T cells or to additionally give anti-CTLA-4 mAb to observe whether greater numbers of cures can be achieved. Notably, we have not detected any signs of autoimmunity in the NKTMab-treated mice, but additional modifications that potentially break tolerance mechanisms will have to be considered with caution.

In summary, NKTMab therapy, which uses glycolipid ligands for NKT cells to mature dendritic cells in combination with antibodies that induce tumor cell apoptosis and T-cell activation, can induce rejection of established tumors in several experimental settings. Given that most of the components proposed in our combination have been tested for safety in phase I clinical trials in advanced cancer patients, we anticipate that these data bring us closer to implementing an effective combination of immunotherapies to the clinic for the treatment of cancer.


   Acknowledgments
 
Grant support: National Health and Medical Research Council of Australia Senior Principal Research Fellowship, NIH grant R01, Parker Foundation and Susan G. Komen Foundation Grant (M.J. Smyth); Susan G. Komen Foundation Grant and the Cancer Council of Victoria (M.W.L. Teng); and National Breast Cancer Foundation of Australia and National Health and Medical Research Council of Australia R.D. Wright Postdoctoral Fellowships (M.H. Kershaw and P.K. Darcy).

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.

We thank Preethi Guru and Carole van Puyenbroek for technical assistance in the Renca orthotopic kidney tumor model and Shannon Griffiths of the PMCC animal facilities for maintenance of mice. G.S. Besra acknowledges support in the form of a Personal Research Chair from Mr. James Bardrick and from the Medical Research Council (United Kingdom) and The Wellcome Trust.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Received 3/13/07. Revised 5/ 9/07. Accepted 5/24/07.


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