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Clinical Cancer Research 14, 579, January 15, 2008. doi: 10.1158/1078-0432.CCR-07-0940
© 2008 American Association for Cancer Research
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Cancer Therapy: Preclinical

Construction and Preclinical Characterization of Fc-mGITRL for the Immunotherapy of Cancer

Peisheng Hu1, Robyn S. Arias1, Rebecca E. Sadun1, Yu-Chih Nien2, Nan Zhang1, Helen Sabzevari3, M.E. Christine Lutsiak3, Leslie A. Khawli1 and Alan L. Epstein1

Authors' Affiliations: Departments of 1 Pathology and 2 Microbiology, Keck School of Medicine at the University of Southern California, Los Angeles, California and 3 Laboratory of Tumor Immunology and Biology, National Cancer Institute, NIH, Bethesda, Maryland

Requests for reprints: Alan L. Epstein, Department of Pathology, Keck School of Medicine, University of Southern California, 2011 Zonal Avenue, HMR 205, Los Angeles, CA 90033. Phone: 323-442-1172; Fax: 323-342-3049; E-mail: aepstein{at}usc.edu.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: To provide proper costimulation required for effective cancer T-cell immunity, Fc-GITRL fusion proteins were generated for use in immunotherapy protocols.

Experimental Design: Soluble fusion proteins consisting of the Fc fragment of immunoglobulin and the murine glucocorticoid-induced tumor necrosis factor–related receptor ligand (mGITRL) connected with different linkers were genetically engineered and tested for their potency in two BALB/c solid tumor models.

Results: In vivo, construct #178-14 (–5aa, –linker) showed the best activity (>90% tumor reduction) at doses ranging from 5 to 25 µg and was found to be intact by gel electrophoresis. Similar doses used with construct #175-2 (-linker) produced good but not as high tumor regression. Construct #5-1 (+linker), which was found to be relatively unstable by SDS gel electrophoresis, produced <60% tumor regression and required a higher dose (100 µg) to produce optimal results. Survival curves showed that Fc-mGITRL treatment extended the life of 80% of tumor-bearing mice to >3 months compared with controls that died by day 40. T-cell depletion studies showed that CD8+ T cells play a major role in Fc-mGITRL immunotherapy, and tumors removed from Fc-mGITRL– and DTA-1–treated mice showed a significant influx of granzyme B+ lymphocytes compared with controls. Finally, T regulatory (Treg) cell assays showed that, unlike other Fc fusion proteins, all three Fc-mGITRL constructs profoundly suppressed Treg activity.

Conclusions: These studies suggest that a stable, intact Fc-mGITRL fusion protein can provide missing costimulation for the immunotherapy of solid tumors. In addition, Fc-mGITRL may alter Treg activity to enhance its effectiveness for tumor immunotherapy.

The effective activation of T cells remains the major mechanism required for an effective immune response against tumors (1). The two-signal model states that for a T cell to become activated, it must engage its T-cell receptor with MHC-bound antigen and must simultaneously receive a costimulatory signal delivered by an antigen-presenting cell to stabilize the immunologic synapse (2). The most characterized costimulatory molecule has been B7.1, the ligand for CD28, which is constitutively expressed by T cells (3). More recently, the identification of costimulatory members of the tumor necrosis factor (TNF) receptor superfamily has changed the way we view costimulation (4).

The glucocorticoid-induced TNF receptor–related gene (GITR) is a newly identified member of the TNF receptor superfamily (5–7), and the cytoplasmic domain of GITR is highly homologous to those of other TNF receptor superfamily members such CD137, OX40, and CD40 (8). The ligand for GITR, GITRL, is a type II transmembrane protein that is normally expressed on the surface of antigen-presenting cells including macrophages, dendritic cells, and B cells (9). The cell-surface marker GITR has been shown to be constitutively expressed at high levels on the surface of murine CD4+CD25+ cells, known as T regulatory (Treg) cells, and is up-regulated on activated CD4+ and CD8+ cells (8, 10, 11). Functionally, GITR has been described as an important costimulatory receptor that promotes survival in early phases of T-cell activation (12).

Treg cells have a clearly established immunosuppressive role capable of thwarting the host's immune response to tumors (13, 14). Despite the fact that Treg cells only compose 5% to 10% of circulating CD4+ T cells (15), their depletion has been shown to permit a significant antitumor immune response in mice (13). Recent studies have shown that Treg cells are increased in the peripheral blood of cancer patients (16) and infiltrate human tumors (17, 18). At the present time, only the transcription factor FoxP3 is closely associated with Treg differentiation and is therefore a useful marker of Treg cells in tissues (19). In addition to the intracellular expression of FoxP3, several cell-surface proteins have been found to be expressed by Treg cells, including CD4, CD25, OX40, CD137, GITR, and neuropilin (20), although none of these markers seem to be specific for Treg cells.

The process through which GITR induces costimulation is complex, entailing interleukin (IL)-2 as well as counter-regulatory IL-10 production (21). High doses of IL-2 have been reported to produce Treg cell proliferation while rendering Treg cells less effective at their suppressive function (22, 23). Although GITR is present on both effector T cells and immunosuppressive Treg cells, the effects of receptor ligation and signaling may differ between the two subpopulations. GITRL is known to produce a state of activation, perhaps by directly or indirectly abrogating the suppressive function of Treg cells (10, 24–26), by activating effector T cells (9), or perhaps through both mechanisms simultaneously (12, 21, 27).

Further complicating its regulatory role, GITR has been shown to signal through nuclear factor {kappa}B to rescue immune cells from T-cell receptor–mediated apoptosis (7, 12). Although this was shown in overexpression studies, it has not been reproduced using the rat monoclonal agonist antibody to GITR, DTA-1, suggesting that GITRL-like stimulation of GITR may have enhanced immunostimulatory properties in cancer-bearing mice.

Previous studies conducted by our laboratory have shown that therapies which can costimulate T cells are effective in suppressing and eliminating established tumors in vivo, with the generation of immunologic memory. Specifically, the costimulatory molecules B7.1 and B7.2 (28, 29), CD137L (30), OX40L (31), and liver expression chemokine (32, 33) induce significant regression of Colon 26 and other solid tumors of the BALB/c mouse. In light of the potent immunostimulatory capacity of GITRL and the ambiguity about its mechanism of action, we sought to create a GITRL fusion protein that could be tested against existing costimulatory fusion proteins for its ability to produce lasting tumor remission. The efficacy of the GITRL fusion protein was also compared with DTA-1, which has been shown to enhance vaccine-induced CD8+ T cells (34, 35). In this report, we now describe the generation and characterization of three different Fc fusion proteins consisting of the extracellular domain of murine GITRL for the immunotherapy of solid tumors.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Reagents. The Glutamine Synthetase Gene Amplification System, including expression plasmid pEE12, was purchased from Lonza Biologics, Inc. The restriction endonucleases, T4 DNA ligase, and other molecular biology reagents were purchased from New England Biolabs.

Antibodies and cell lines. The monoclonal antibody DTA-1 (agonist rat anti-murine GITR) hybridoma was a generous gift from Dr. Stephen Stohlman (Department of Microbiology, Cleveland Clinic, Cleveland, OH). The 145-2C11 (rat anti-mouse CD3), H35 (rat anti-mouse CD8), and GK1.5 (rat anti-mouse CD4) hybridomas were purchased from the American Type Culture Collection. The antibodies used for fluorescence-activated cell sorting analysis, including phycoerythrin-conjugated anti-mouse CD4 and purified anti-mouse CD16/CD32, were purchased from BD PharMingen. The Colon 26 murine colon carcinoma cell line was obtained from the American Type Culture Collection and grown in RPMI 1640 (Invitrogen) supplemented with 10% FCS (Hyclone), 1% glutamine/penicillin/streptomycin, and 1% nonessential amino acids solution (Gemini BioProducts). The NS0 murine myeloma cell line was obtained from Lonza Biologics Ltd. and grown in nonselective medium consisting of Hybridoma-SFM medium (Invitrogen) supplemented with 10% FCS (Hyclone), 1% glutamine/penicillin/streptomycin, and 1% nonessential amino acids solution (Gemini BioProducts). Transfected NS0 cells were maintained in selective medium, which consisted of Hybridoma-SFM medium without glutamine supplemented with 10% dialyzed FCS (Hyclone), 1% penicillin/streptomycin, and 1% nonessential amino acid solution.

For comparison, Fc fusion proteins consisting of the lymphocyte function–associated antigen (LFA), IL-2, lymphotoxin-like inducible protein [which competes with glycoprotein D for binding herpesvirus entry mediator on T cells (mLIGHT)], TNF{alpha}, monokine induced by IFN-{gamma} (mMIG), mCD137L, and B7.1 were used in several aspects of this work. All of these fusion proteins were made in our laboratory using the glutamine synthetase expression system and were purified by Protein-A affinity and ion exchange chromatography as described below for the mGITRL constructs.

Six-week-old female BALB/c mice were obtained from Harlan Sprague-Dawley. All experiments were done in accordance with Institutional Animal Care and Use Committee protocols and institutional guidelines for the proper humane care and use of animals in research.

Construction of Fc-mGITRL fusion proteins. The cDNA encoding mGITRL was generously provided by Dr. Waldmann (University of Oxford, Oxford, UK). The extracellular portion of mGITRL was PCR amplified using the primers shown in Table 1 .


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  		Table 1. Primers used to genetically engineer Fc-mGITRL fusion proteins

 
The amplified mGITRL fragments were then inserted into the expression vector pee12 containing the Fc (hinge-CH2-CH3) using NotI/EcoRI or SmaI/EcoRI restriction sites at the COOH terminus of Fc.

Expression and purification of Fc-mGITRL fusion proteins. The resulting COOH-terminal constructs were each electroporated into NS0 according to the protocol described by Lonza Biologics Ltd. Four weeks later, the supernatants from the transfectants were screened by indirect ELISA. The highest-producing clones were grown in aerated 3-liter stir flasks containing selective media with 5% dialyzed FCS. The secreted fusion proteins were purified by protein A affinity chromatography and ion-exchange chromatography as previously described (28).

Binding of Fc-mGITRL to freshly isolated T lymphocytes. DTA-1, Fc-mGITRL constructs, and isotype control rat IgG (CalTag) were biotinylated using the EZ-Link Sulfo-NHS-LC-Biotinylation Kit according to the protocol provided by the manufacturer (Pierce). Single-cell suspensions of freshly isolated lymphocytes were obtained from the spleens of BALB/c mice. Each sample was subjected to differential centrifugation using Histopaque-1077 (Sigma-Aldrich) and incubated with 1 µg of Fc blocking antibody (CD16/CD32) per 106 cells in 100 µL of staining buffer (0.1% fetal bovine serum in PBS) for 15 min at 4°C. Cells were incubated with 2 µg of biotinylated DTA-1, Fc-GITRL, or rat IgG for 30 min at 4°C in 100 µL of staining buffer. The cells were washed and double stained with 1 µg of phycoerythrin-conjugated streptavidin and 1 µg of FITC-conjugated anti-CD4+ or 1 µg of FITC-conjugated anti-CD8+ and incubated in the dark for 30 min at 4°C in 100 µL of staining buffer. The cells were washed thrice in staining buffer and immediately analyzed by flow cytometry.

Bioactivity assay. Single-cell lymphocyte suspensions were obtained from the spleens of BALB/c mice as described above. The cells were washed, counted using a hemacytometer, and diluted with washing medium to a concentration of 5 x 107/mL. Immediately before labeling, a fresh 10 mmol/L stock of carboxyfluorescein succinimidyl ester (Molecular Probes) diluted in DMSO was prepared. The cells were incubated with 2 µmol/L final concentration of carboxyfluorescein succinimidyl ester for 8 min at 37°C with occasional mixing and 5 volumes of ice-cold RPMI 1640 containing 10% FCS, 1% nonessential amino acids solution, and 1% penicillin/streptomycin solution (complete medium) was immediately added and incubated for 5 to 10 min at room temperature. The cells were then washed and plated onto a precoated anti-CD3 (clone 145-2C11) plate containing 5 µg/mL anti-CD3 at a concentration of 1.5 x 106/mL. The cells were incubated for up to 72 h with 2 µg of DTA-1 or Fc-mGITRL. At the indicated times, the cells were washed and blocked with 1 µg of CD16/CD32 for 15 min at 4°C in 100 µL of staining buffer. Cells were then stained with phycoerythrin-conjugated anti-CD4 at 1 µg/106 cells at 4°C for 30 min in the dark. The cells were washed thrice with staining buffer and immediately analyzed by flow cytometry.

IL-2 ELISA. Supernatants were collected from the bioactivity assay described above at 48 and 72 h posttreatment and directly used in an IL-2 ELISA (BD PharMingen) according to the manufacturer's protocol. The samples were analyzed in duplicate and the assay was repeated twice. The P values were calculated using unpaired t test.

Murine Treg and CD8+ T-cell proliferation assays. To show the effect of the Fc-mGITRL fusion proteins on T-cell subsets, a panel of Fc fusion proteins generated in our laboratory was tested in mouse Treg and CD8+ T-cell proliferation assays. For these assays, splenocytes from normal C57BL/6 mice were subjected to cell purification using either a Miltenyi Biotec CD4+CD25+ Regulatory T Cell Isolation Kit (Treg) or CD8a microbeads (CD8+ cells). All wells contained irradiated antigen-presenting cells at a concentration of 1 x 105 per well and anti-CD3 (1 µg/mL). The Fc fusion proteins were tested in triplicate using two concentrations (1 and 10 µg/mL) in which CD8+ cells alone (5 x 104 per well), Treg cells alone (5 x 104 per well), and a 1:1 combination of both cell populations (each 5 x 104 per well) were tested. In this way, background data of proliferation were obtained because some of the Fc fusion proteins induced proliferation of reporter and Treg cells. [3H]Thymidine was added for the last 18 h of culture. For analysis, the triplicate wells were averaged and the percent suppression was calculated by the equation

Formula

Immunotherapy studies. For dosing studies, groups (n = 5) of 6-week-old female BALB/c mice were injected s.c. in the left flank with a 0.2-mL inoculum containing ~5 x 106 Colon 26 or RENCA cells. The tumors were grown for 5 days until they reached ~0.5 cm in diameter. Beginning on day 6 after tumor implantation, groups of tumor-bearing mice were treated i.v. with a 0.1-mL inoculum containing the indicated amount of control LFA-Fc, Fc-mGITRL (3 constructs), or DTA-1 for 5 consecutive days. Identical doses of Fc-mGITRL were administered to enable direct comparison between constructs. The tumor growth of all treatment groups was monitored every other day by caliper measurement. Tumor volumes were calculated by multiplying length x width x height. Average tumor volumes were plotted with their SDs and two-tailed P values were determined using the Wilcoxon rank-sum test. Each of these studies was repeated at least twice. For the depletion studies, groups (n = 5) of tumor-bearing mice were given a 1.0-mL i.p. inoculum containing 0.5 mg of anti-CD4 (GK1.5) or anti-CD8 (H35) in PBS on days 0, 7, and 14 after tumor implantation. In addition, these mice were treated i.v. for 5 consecutive days beginning on day 5 after tumor implantation with a 0.1-mL inoculum containing 90 µg of LFA-Fc, Fc-mGITRL, or DTA-1 antibody. The average tumor volumes were calculated and plotted as described above. The P values were determined using the Wilcoxon rank-sum test.

Survival studies of Fc-mGITRL–treated mice. Groups (n = 5) of 6-week-old female BALB/c were injected with Colon 26 and treated on day 6 for 5 consecutive days as described above. Mice treated with 90 µg of LFA-Fc, Fc-mGITRL (#5-1), or DTA-1 were monitored over the course of 90 days. A death event was defined as having to sacrifice an animal due to excessive tumor burden, tumor abscess, or unresponsiveness to stimuli. The survival data were presented as Kaplan-Meier plots and two-tailed P values were calculated.

Morphologic and immunohistochemical studies. On day 16 after tumor implantation, two tumors from each treatment group were removed for histologic examination and placed in 10% buffered formalin for paraffin embedding, sectioning, and staining with H&E. Immunohistochemical analysis was also done on paraffin-embedded sections after antigen retrieval in citrate buffer (36). For these studies, serial sections were incubated overnight in a 1:10 dilution of anti–granzyme B primary antibody in PBS and then incubated with biotinylated goat anti-rabbit secondary antibody. The slides were then incubated with horseradish peroxidase–conjugated streptavidin, developed with ABC solutions, and counterstained with H&E for analysis.

Flow cytometric analysis of tumor-infiltrating lymphocytes. On day 19 after tumor implantation, tumors were removed from three mice of each treatment group. Single-cell suspensions were obtained by enzymatic digestion in complete medium containing 0.01% DNase, 0.01% hyaluronidase, and 0.1% collagenase for 2 h at 37°C followed by manual disruption through a cell strainer (BD PharMingen) into a Petri dish containing complete RPMI 1640. The lymphocytes were pooled for each treatment group, washed, and RBCs and tumor cells were removed using Lympholyte-M (Cedarlane) according to the protocol provided by the manufacturer. The tumor-infiltrating lymphocytes were counted using a hemacytometer and a total of 5 x 105 cells were resuspended in 100 µL of staining buffer (0.1% FCS in PBS) containing 0.5 µg CD16/CD32 (BD PharMingen) and incubated at 4°C for 10 min. Without washing, 0.5 µg of conjugated antibody was added and incubated at 4°C for 30 min. The samples were washed thrice with 0.5 mL of staining buffer and analyzed immediately by flow cytometry.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Construction, expression, and purification of Fc-mGITRL
Three COOH-terminal Fc fusion proteins containing the extracellular domain of mGITRL were constructed as shown schematically in Fig. 1A . The extracellular portion of mGITRL (amino acids 43-173) was cloned downstream of the IgG Fc fragment. Each of the three COOH-terminal Fc-mGITRL fusion proteins was transfected and expressed in NS0 cells. The highest-producing transfectants were subcloned and screened by sandwich ELISA with antibodies that detected either the Fc or mGITRL. The highest-producing subclone was expanded to a 3-liter aerated stir-flask and the fusion protein was purified as described above. SDS-PAGE shown in Fig. 1B was done for all three constructs and showed that construct #5-1 was ~90% broken. By contrast, constructs #175-2 (-linker) and #178-14 (–5aa, –linker) were intact and stable.


Figure 1
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  		Fig. 1. Construction and expression of Fc-mGITRL fusion proteins. A, schematic diagram of the three different Fc-mGITRL fusion proteins constructs (#5-1, 175-2, and 178-14). B, SDS-PAGE analysis (nonreduced) of the three constructs showing their purity and stability. #5-1 is unstable and mostly broken; #175-2 is intact with minor breakage; and #178-14 is intact and shows evidence of glycosylation (smeared band).

 
In vitro studies
Binding study. It has been shown that the receptor for mGITRL is constitutively expressed at basal levels on both CD4+ and CD8+ T cells (5). To determine whether the Fc-mGITRL constructs could bind to GITR+ T lymphocytes, a binding study was done with biotinylated Fc-mGITRL. From these studies, biotinylated Fc-mGITRL (#5-1) was shown to bind to freshly isolated CD4+ (Fig. 2A ) and CD8+ T (Fig. 2B) cells at similar levels to biotinylated agonist anti-GITR antibody DTA-1. This showed that the Fc-mGITRL was in its proper conformation and had the ability to bind to GITR expressed on the surface of freshly isolated T lymphocytes.


Figure 2
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  		Fig. 2. GITRL potency assays. Flow cytometric analysis of the negative effects of DTA-1 and Fc-mGITRL in the absence of signal 1 on CD4+ (A) and CD8+ (B) T cells and of the proliferative effects of DTA-1 and Fc-mGITRL in the presence of signal 1 provided by anti-CD3 stimulation on CD4+ (C) and CD8+ (D) T cells. E, confirmation of DTA-1 and Fc-mGITRL potency in an IL-2 proliferation assay showing the ability of these reagents to provide adequate signal 2.

 
Proliferation assay. The biological activity of the Fc-mGITRL fusion protein (#5-1) was determined by a proliferation assay with freshly isolated murine splenocyctes. Analysis at 48 h posttreatment with this construct resulted in a 45% proliferation of CD4+ T cells compared with 11% of anti-CD3 alone–treated cells (Fig. 2C). The proliferation of CD4+ T cells increased to 90% at 72 h posttreatment with Fc-mGITRL compared with 48% of control anti-CD3 treatment. Cellular activation with the Fc-mGITRL fusion protein resulted in similar levels of proliferation compared with agonist DTA-1–treated splenocytes (Fig. 2C and D), thereby verifying the biological activity of the mGITRL moiety as a potent T-cell costimulator.

IL-2 production. As a proxy for T-cell activation, the production of IL-2 was measured by obtaining supernatant from the proliferation assay done on murine splenocytes. As shown in Fig. 2E, the treatment of lymphocytes with DTA-1 or any of the three Fc-mGITRL fusion protein constructs resulted in a significant increase in the 72-h production of IL-2, with constructs #175-2 and #178-14 causing a doubling of IL-2 production in comparison with the negative control of anti-CD3 alone (P < 0.001). The intact Fc-mGITRL constructs also stimulated more IL-2 production than DTA-1 (1,200 versus 950 pg/mL) and was just a little lower than that seen with CD28 maximal stimulation (1,375 pg/mL).

In vitro activity of Fc-mGITRL on murine Treg and CD8+ T cells. As shown in Fig. 3A , at 10 µg/mL, all three Fc-mGITRL constructs were able to markedly reverse Treg suppression of reporter lymphocytes. At the lower concentration, Fc-mGITRL #5-1 had no effect whereas the other two constructs still showed a strong reversal of suppression. Other Fc fusion proteins, including those consisting of mMIG, TNF{alpha}, and mLIGHT, did not show this activity. Three other Fc reagents, including mCD137L, B7.1, and IL-2, did, however, show some reversal. In a parallel study, murine CD8+ cells were incubated with the same Fc fusion proteins to determine their effects on this critical effector cell population. The results of these studies (Fig. 3B) indicate that IL-2-Fc had the greatest proliferative response with CD8+ cells as expected, but that the three Fc-mGITRL fusion protein constructs also had strong proliferative effects on this subpopulation of T cells. As shown in Fig. 3C, several of the fusion proteins directly caused the proliferation of Treg cells. Because the suppressive Treg cells do not proliferate by anti-CD3 stimulation in vitro, this is an indication that these cells are losing their suppressive phenotype. This background proliferation was subtracted in the percent suppression calculations. From these data, it seems that one of the key actions of Fc-mGITRL may be the reversal of Treg suppression of tumor-infiltrating T cells.


Figure 3
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  		Fig. 3. Effects of different Fc fusion proteins on percent suppression of CD8+ cells by Treg cells (A), proliferation of CD8+ T cells (B), and proliferation of Treg cells (C). The Fc-mGITRL constructs induced the proliferation of CD8+ cells but markedly inhibited Treg suppression of proliferating CD8+ reporter T cells.

 
Immunotherapy studies
Dose escalation. To determine the therapeutic efficacy of the Fc-mGITRL fusion protein constructs, immunotherapy studies were done with tumor-bearing BALB/c mice using the Colon 26 tumor model. The activity of the three Fc-mGITRL constructs was first examined by injecting escalating dose levels into tumor-bearing mice to compare their clinical effects in vivo. For these studies, groups of mice with established Colon 26 tumors were treated daily by i.v. injection on days 6 to 10 with a 0.1-mL inoculum containing the indicated dose of fusion protein. The results showed that construct #5-1 had the least potent activity yet still produced significant tumor regression especially at the highest dose level (100 µg; Fig. 4A ). By contrast, constructs #175-2 and #178-14 were found to produce more profound tumor regression at an optimal dose of 25 µg/dose and higher doses were found to decrease the antitumor response of therapy (Fig. 4B and C). Construct #178-14 produced the best antitumor effects of all three constructs and was found to produce a >90% regression of tumor (P = 0.008) compared with controls (Fig. 4C). The group that received the optimal dose (25 µg) of Fc-GITRL #178-14 displayed the longest survival of all of the treatment groups (data not shown). The most potent construct, #178-14, was also tested in a second solid tumor model using RENCA cells as above and showed similar dose-related results as seen with the Colon 26 tumor model (Fig. 4D). Together, these data show the therapeutic potential of Fc-mGITRL especially when the stable #178-14 construct was used for in vivo immunotherapy.


Figure 4
Figure 4
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  		Fig. 4. In vivo studies of Fc-mGITRL fusion proteins in Colon 26– and RENCA-bearing mice. In these studies, groups of mice (n = 5) were treated for 5 consecutive days (see arrows) starting on day 5 after tumor implantation with 5 to 100 µg/dose of Fc-mGITRL #5-1 (A), #175-2 (B), or #178-14 (C). D, dosing study of Fc-mGITRL (178-14) in RENCA-bearing mice showing that this construct produces profound and lasting antitumor effects in a second solid tumor model. E, depletion of CD4+ and CD8+ T-cell subpopulations either alone or in combination with Fc-mGITRL (#5-1) treatment shows the importance of CD8+ effector cells in Fc-mGITRL immunotherapy. Depletion of CD4+ cells produced significant regression of Colon 26 tumor implants due to the removal of CD4+CD25+ Treg cells whereas depletion of CD8+ cells reversed the antitumor effects of Fc-mGITRL. F, photographic display of tumor regression in Fc-mGITRL (#5-1)– and DTA-1–treated mice at day 35 after tumor implantation compared with control mice treated with LFA-Fc. G, Kaplan-Meier plot showing survival curves for DTA-1–, Fc-mGITRL (#5-1)–, and control LFA-Fc–treated mice. H, microscopic examination of tumors from DTA-1– and Fc-mGITRL–treated mice shows large areas of necrosis and massive infiltration of lymphocytes especially at the edge of necrotic regions but no evidence of vessel damage or thrombosis. I, granzyme B staining shows a greater prevalence of leukocytes in sections of tumors removed from DTA-1– and Fc-mGITRL–treated mice compared with sections from control LFA-Fc–treated tumors.

 
Lymphocyte subset depletion study. Depletion studies were done to analyze which T-cell subset was primarily responsible for the antitumor effects seen in vivo with Fc-mGITRL treatment. The elimination of T-cell subsets was done using anti-CD4+ (GK1.5) or anti-CD8+ (H35) antibodies delivered i.p. on days 0, 7, and 14 after tumor implantation. Verification of depletion was confirmed by flow cytometry, which resulted in a 99% reduction of CD4+ T cells and 89% reduction of CD8+ T cells from splenic preparations obtained 3 days after the completion of the three injections (data not shown). Groups that received Fc-mGITRL (construct #5-1) or DTA-1 treatment alone resulted in a 93% (P = 0.008) and 96% (P = 0.008) reduction in tumor volume at day 21 after tumor implantation, respectively, compared with the control LFA-Fc group (Fig. 4E). As shown in Fig. 4F, at day 35 after tumor implantation, there was a significant difference visually in tumor burden between control LFA-Fc–treated (left), Fc-mGITRL–treated (middle), and DTA-1–treated (right) mice. The anti-CD4+ alone treatment group resulted in a 96% (P = 0.008) decrease in tumor volume compared with LFA-Fc at day 21, whereas the anti-CD8+ alone treatment group resulted in no significant difference in tumor volume compared with control LFA-Fc–treated mice (Fig. 4E). The group treated with anti-CD4+ in combination with Fc-mGITRL resulted in the same percent tumor reduction as the group treated with anti-CD4+ alone. In contrast, the group that received Fc-mGITRL in combination with anti-CD8+ had a significantly lower tumor regression (P = 0.008) compared with Fc-mGITRL treatment alone at day 21 (Fig. 4F), thereby showing the critical importance of CD8+ T cells in Fc-mGITRL immunotherapy.

Survival study. The treatment groups pictured in Fig. 4A were used to study survival during the course of 90 days after tumor implantation. For the control LFA-Fc treatment group, 100% of the mice had been sacrificed by day 40 after tumor implantation (Fig. 4G). In contrast, 80% of Fc-mGITRL–treated mice and 100% of DTA-1–treated mice had survived up to 85 days after tumor implantation (P = 0.0015). These data show that treatment with either Fc-mGITRL or DTA-1 effectively eradicates tumor burden and results in the prolonged survival of treated mice.

Analysis of tumor-infiltrating lymphocytes
H&E staining. To attain a better understanding of occurrences within the tumor microenvironment among different treatment groups, morphologic studies were done by H&E staining of tumors removed at day 16 after tumor implantation. Analysis of LFA-Fc–treated tumor sections revealed a homogeneous staining of Colon 26 tumor cells with small pockets of necrosis throughout the tumor (Fig. 4H). In contrast, Fc-mGITRL–treated tumors resulted in a very large necrotic core surrounded by a thin layer of viable Colon 26 cells. Similarly, a large area of necrosis was also observed in DTA-1–treated mice. In addition, throughout the area of necrosis, there was a significant influx of lymphocytes in Fc-mGITRL and DTA-1 treatment groups compared with control-treated mice (Fig. 4H).

Granzyme B immunohistochemistry. Expression of granzyme B was evaluated by immunohistochemistry on tumor sections removed from treated mice on day 16 after tumor implantation. The results in Fig. 4I show that the expression of granzyme B was significantly higher in Fc-mGITRL– and DTA-1–treated mice compared with the control LFA-Fc group. Therefore, the release of granzyme B in Fc-mGITRL–treated mice may play a role in tumor regression observed in vivo.

Flow cytometric analysis. Four-color staining was used to analyze the tumor-infiltrating cells obtained from each of the different treatment groups. At day 19 after tumor implantation, tumors were removed and measured and cells from three mice per group were pooled and stained for markers of different leukocytes. As shown in Table 2 , mice treated with DTA-1 or any of the Fc-mGITRL constructs showed an increase in CD4+CD25– cells (5-6%) in comparison with PBS-treated cells (2%). In addition, Fc-mGITRL #178-14 induced an increase from 17% to 26% in CD11b+ macrophages with no concomitant increase in CD11c+ dendritic cells.


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  		Table 2. Phenotypic characterization of tumor-infiltrating lymphocytes in DTA-1– and Fc-mGITRL–treated Colon 26 tumors

 

   Discussion
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
GITR is an attractive target for cancer immunotherapy because of its costimulatory effects on effector T cells as well as its observed abrogation of Treg cell function (8, 10). In this study, the costimulatory effects of GITR activation by Fc-mGITRL fusion proteins were examined. To accomplish this, three different Fc-mGITRL COOH-terminal fusion proteins were constructed, differing in their linker sequence and, thus, their stability. Comparison of the GITRL gene sequences between mice and humans showed that the human analogue is missing the first five amino acids, which indicates that for GITRL, the first five amino acids are unlikely to be involved in receptor binding, permitting the construction of one protein with the first five amino acids deleted (#178-14). Another fusion protein bore no deletions and no linker sequence (#175-2), whereas the third had a linker included (#5-1).

It was beneficial to generate a Fc fusion protein because it has been shown that the presence of the Fc significantly increases the half-life of a protein, thereby improving its pharmacokinetics properties and dynamics in vivo (37). In addition, presentation of costimulation by Fc receptor–bearing cells may be a particularly effective method for immunotherapy. Studies to support this notion are currently in progress using Fc receptor–knockout mice. In accordance with a recent study by Esparza and Arch (12), which showed that activation of GITR resulted in the proliferation of T cells and the production of IL-2, our results show that Fc-mGITRL physiologically binds GITR and produces the proper costimulatory signal to T-cell subsets (Fig. 2A-D) with enhanced IL-2 production at 72 h (Fig. 2E). Thus, the costimulatory signal of GITR on T cells may provide a significant mechanism for the antitumor effects observed in vivo with Fc-mGITRL.

In addition to costimulation (38), several prior investigations have shown that the GITR-GITRL interaction is inhibitory to Treg function (10, 24, 25, 27). To show that the newly generated Fc-mGITRL fusion proteins also had this activity, murine Treg assays were done with a panel of Fc fusion proteins generated in the laboratory. As shown in Fig. 3A, the three Fc-mGITRL fusion protein constructs were found to inactivate Treg suppression of CD8+ cells in this assay. Interestingly, the IL-2-Fc, B7.1-Fc, and Fc-mCD137L fusion proteins showed partial inhibition. By contrast, the Fc-mGITRL constructs had a proliferative effect on CD8+ cells in the absence of Treg cells, but were not as potent as IL-2-Fc in this regard (Fig. 3B). Taken together, these studies show that Fc-mGITRL has a dual function in cancer immunotherapy, which may provide a rationale for its effective use in patients, especially those with tumors that show a role for Treg suppression of immunity. Additional studies are needed, however, to confirm the inhibitory activity of human GITRL on patient FoxP3+ Treg cells because there is some evidence that, unlike mouse Treg cells, human FoxP3+ cells may not express GITR (27).

There have been few reports in which the role of GITR activation has been evaluated in relation to tumor immunity. It was therefore our goal to investigate the therapeutic potential of Fc-mGITRL in tumor-bearing mice. Although the #5-1 Fc-GITRL construct was not produced entirely intact, our data indicate that it was still highly active in vivo. When tested in a dosing study, as little as 5 µg of fusion protein resulted in significant regression of tumors (Fig. 4A). By comparison, the intact constructs, especially #178-14, produced more dramatic effects (90% regression) on Colon 26 and RENCA tumor growth. The unexpected relative potency of #178-14 compared with the other constructs may indicate better folding characteristics of this construct due to the missing NH2-terminal five amino acids also naturally deleted in the human GITRL analogue. This improved conformation may translate to more successful attraction of CD11b+ cells (Table 2).

Based on the very promising animal data, we sought to explore the cell populations through which the Fc-mGITRL achieves its therapeutic effect. A previous study conducted by Calmels et al. (39) in the B16 melanoma model supported the increase in both infiltrating CD4+ and CD8+ T cells in response to a secreted form of GITRL. To differentiate the actions of GITRL through CD4+ and CD8+ T cells and to determine which is functionally necessary for tumor regression, the in vivo effects of Fc-mGITRL in combination with CD4+ and CD8+ T-cell depletion were tested. Fc-mGITRL treatment in the absence of CD8+ T cells resulted in tumors that were not statistically different from the control group (Fig. 4E), showing the importance of this subpopulation for the effects of the therapy. Remarkably, CD8+ T-cell depletion produced a greater growth in tumor volume than the untreated control group, showing the critical importance of this subset of cells in controlling tumor expansion in the host.

In contrast to the effects of CD8+ T-cell depletion, CD4+ T-cell depletion caused >90% tumor regression in these experiments (Fig. 4E). This showed that the total population of CD4+ T cells actually plays an inhibitory role, preventing an antitumor response. This is likely due to the known suppressive subsets of CD4+ T cells, including Treg cells and Th1 and Th3 subsets, which may be induced in the periphery and which may also play a role in inhibiting an immune response to tumor antigens (23). Because Fc-mGITRL treatment produces about the same effect in tumor regression as CD4+ depletion, the main activity of Fc-mGITRL may in fact be to overcome the inhibitory effects of CD4+ suppressor cells.

The mechanism(s) of Fc-mGITRL-induced immunity was further studied by morphologic and immunohistochemical methods on tumor sections removed on day 16 after tumor implantation. At this stage of treatment, a very large necrotic core accompanied by the presence of tumor-infiltrating lymphocytes was observed in Fc-mGITRL– and DTA-1–treated tumors (Fig. 4H). In particular, there was a significant increase in immune cells observed around the perimeter and throughout the necrotic core in Fc-mGITRL– and DTA-1–treated tumors (Fig. 4H). Based on the depletion data shown in Fig. 4E, which showed the importance of CD8+ T cells in Fc-mGITRL immunity, we evaluated granzyme B expression by immunohistochemistry using tumor sections collected at this time. Granzyme B is known as a caspase-like serine protease that is released by CTLs and natural killer cells to target virus-infected or tumor cells for cellular death (40). A death signal elicited by granzyme B is considered to be both efficient and rapid because it is directly taken up by its target cell and results in activation of caspases or mitochondrial disruption (40). Analysis of the tumor sections showed an increase in granzyme B expression in both Fc-mGITRL and DTA-1 samples (Fig. 4I). Finally, an analysis of tumor-infiltrating lymphocytes was done by flow cytometry on pooled samples obtained from three mice per group tested. Interestingly, these data did not show a very large difference between control and treated mice with respect to CD8+ infiltration (Table 2), suggesting that the activation state of CD8+ T cells may be more critical than their total number. Different treatments did induce infiltration of specific subpopulations, with the most notable increases being in CD11b+ cells with Fc-mGITRL (#178-14) treatment and in CD49b+ cells with DTA-1 treatment.

In summary, intact Fc-mGITRL fusion proteins such as #178-14 seem to be potent immunotherapeutic agents that can effectively eradicate solid tumors in vivo. Comparable to DTA-1 treatment shown here and in previous studies (34, 35), this novel treatment regimen resulted in prolonged survival and required CD8+ cells for effective immunotherapy. The use of the physiologic ligand to GITR may have advantages over the use of an agonist antibody such as DTA-1 in that it may provide better tumor penetration, more favorable half-life kinetics and pharmacodynamics, and more effective costimulatory activity. These studies show that the Fc fragment of immunoglobulin is a remarkable delivery vehicle for GITRL, providing significant therapeutic results in cancer-bearing mice. Because recent evidence suggests that GITR may play a different role in mice and humans (41), the generation of a human GITRL-Fc fusion protein like construct #178-14 may help to elucidate the clinical potential of this reagent in patients.


   Acknowledgments
 
We thank James Pang for providing expert technical support for all of the animal studies, Suzanne Sachsman for carrying out the immunohistochemical reactions, Hal Soucier for his expertise in flow cytometry, and Meg Flanagan for helpful discussions.


   Footnotes
 
Grant support: Cancer Therapeutics Laboratories, Inc. (Los Angeles, CA) and the Philip Morris External Research Program (Linthicum Heights, MD).

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.

Note: Current address for L.A. Khawli: Genentech, Inc., South San Francisco, CA.

Received 4/20/07; revised 7/24/07; accepted 9/17/07.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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