Clinical Cancer Research Joint Metastasis Research Society-AACR Conference on Metastasis Translational Cancer Medicine 2008: Cancer Clinical Trials and Personalized Medicine
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Clinical Cancer Research 13, 2758-2767, May 1, 2007. Published Online First April 25, 2007;
doi: 10.1158/1078-0432.CCR-06-2343
© 2007 American Association for Cancer Research
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Cancer Therapy: Preclinical

Targeted and Untargeted CD137L Fusion Proteins for the Immunotherapy of Experimental Solid Tumors

Nan Zhang, Rebecca E. Sadun, Robyn S. Arias, Meg L. Flanagan, Suzanne M. Sachsman, Yu-Chih Nien, Leslie A. Khawli, Peisheng Hu and Alan L. Epstein

Authors' Affiliation: Department of Pathology, Keck School of Medicine, University of Southern California, Los Angeles, California

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: 323-342-1172; Fax: 323-342-3049; E-mail: aepstein{at}usc.edu.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Introduction: CD137L is a member of the tumor necrosis factor superfamily that provides a costimulatory signal to T cells. In this study, two novel CD137L fusion proteins were produced and compared with the CD137 agonist antibody 2A.

Materials and Methods: Murine CD137L was linked to the COOH terminus of either the Fc fragment of immunoglobulin (untargeted version) or TNT-3 (targeted version), an antibody that binds to necrotic regions of tumors. Groups of mice bearing established Colon 26 tumors were then treated daily x5 with each fusion protein or 2A to determine their immunotherapeutic potential.

Results: Both fusion proteins retained CD137L activity in vitro and TNT-3/CD137L showed tumor-binding activity by biodistribution analysis in tumor-bearing mice. The fusion proteins also produced similar responses in vivo at the 1 nmol per dose range and showed a 60% (TNT-3/CD137L) or 40% (Fc/CD137L) survival of treated mice at 150 days after tumor implantation, similar to the effects of 2A. Morphologic and immunohistochemical analyses showed massive central necrosis and infiltration of granzyme B–positive cells in necrotic areas and viable peripheral regions of treated tumors. Finally, cell depletion studies showed that CD137L-mediated tumor regression was CD8+ T cell dependent.

Conclusions: From these studies, it was determined that both targeted and untargeted CD137L fusion proteins showed effective antitumor activity, but that the targeted version was more potent. Therefore, the use of the natural CD137 ligand is a promising approach to the treatment of solid tumors by virtue of its ability to produce physiologic costimulation within the tumor, limiting side effects often seen with agonist antibody therapies.

T-cell anergy is one of the major impediments to the ability of the immune system to eradicate established tumors (1–4). To elicit an effective T-cell response, the engagement of the T-cell receptor by MHC-peptide complex needs to occur in the setting of a second signal provided by costimulation (5). The B7/CD28 interaction has been widely studied as the primary pathway for costimulatory activation of resting T cells (6, 7). Recently, however, signaling relayed through CD137 and other members of the CD28 and tumor necrosis factor receptor (TNFR) families have received broad attention as additional mechanisms for the induction of potent T-cell effector functions (8, 9).

CD137 (4-1BB) and its natural ligand CD137L (4-1BBL) are members of the TNFR/TNF superfamily (10–12). CD137 is transiently expressed primarily on activated T cells (13, 14), whereas CD137L has been found on activated antigen-presenting cells (10, 15–17). Although the engagement of CD137 by CD137L provides costimulation to both CD4+ and CD8+ T cells (18–20), evidence indicates that costimulation has a more profound effect on CD8+ T cells (21). In experiments using a CD137 agonist antibody or cells constitutively expressing CD137L (18, 21–31), CD137 engagement was shown to regulate T-cell number and functions, including the secretion of cytokines (19, 21, 22, 32, 33), prevention and reversal of established anergy of CD8+ CTL (28), and prevention of activation-induced cell death through nuclear factor-{kappa}B activation, which further induces Bcl-xL, Bfl-1, and c-FLIP via the phosphatidylinositol 3-kinase and AKT/protein kinase B pathways (34, 35). Importantly, systemic administration of an agonistic anti-CD137 monoclonal antibody can eradicate established s.c. tumors in mice (25), and tumor cells expressing CD137L have induced tumor lytic T cells and facilitated T-cell–mediated antitumor immunity in different mouse models (24, 26, 31, 36).

Given the usefulness of CD137 signaling in tumor immunotherapy, successful delivery and retention of CD137L may produce potent antitumor effects in solid tumors. Although most investigators are currently using antibodies to achieve costimulation in vivo, we have chosen to study the effects of natural ligands to deliver missing costimulatory signals to the immune system. As shown by clinical studies with anti-CTLA-4, the use of long-acting and high-avidity antibodies often produces significant toxicity (37). Moreover, using a ligand-based therapy enables the targeting of the costimulatory molecules to the tumor site by fusion to tumor-targeting antibodies and thereby avoiding localization to normal tissues.

Over the past several years, our laboratory has developed a unique antibody targeting approach for the immunotherapy of solid tumors. Designated "tumor necrosis treatment" (TNT), this approach exploits the presence of leaky necrotic cells within tumors by using monoclonal antibodies directed against universal and stable nuclear antigens retained by the cell ghost (38, 39). Because accumulation of necrotic cell debris is characteristic of essentially all solid tumors (39), TNT has potential diagnostic and therapeutic applications for most human and animal malignancies and has been used to deliver a number of cytokines and chemokines to solid tumors of the BALB/c mouse (40–49). Biodistribution and imaging studies have confirmed the targeting abilities of these reagents, and immunotherapeutic experiments using tumor-bearing mice showed the potential of this approach in the suppression of tumor growth (47–49).

In the present study, the immunotherapeutic potential of both targeted and untargeted CD137L fusion proteins was compared with the antitumor effects of 2A, a CD137 agonist antibody previously shown to have potent activity against solid tumors (16). In vitro, the bioactivity of the CD137L moiety was confirmed for both reagents, and standard immunotherapeutic protocols showed that all three reagents had curative or highly suppressive antitumor activity in the Colon 26 tumor model. Mechanistic studies identified that CD8+ CTL were responsible for the observed tumor regression in these therapeutic studies. Based upon our results, it seems that both targeted and untargeted CD137L are promising reagents for the immunotherapy of solid tumors. By providing physiologic costimulation in tumor and accessory lymphoid organs, these novel reagents may provide an alternative approach to the use of potentially toxic agonist antibodies.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Antibodies and cell lines. Anti-CD3 (145-11C clone), PE-anti CD4 (RM4-5 clone), PE-anti-CD8{alpha} (53-6.7 clone), PE-anti-CD25 (PC61 clone), PE-anti-CD11c (HL3 clone), PE-anti-CD49b (DX5 clone) monoclonal antibody, and horseradish peroxidase-streptavidin were purchased from BD PharMingen. Hybridomas, including rat anti-mouse CD4 (GK1.5), anti-CD8ß (H35), and anti-CD25 (PC61) monoclonal antibodies, were purchased from the American Type Culture Collection. The hybridoma producing the anti-CD137 agonist antibody 2A was a kind gift from Dr. Lieping Chen (Johns Hopkins Medical School, Baltimore, MD). For immunohistochemical staining, both primary rabbit anti-mouse granzyme B polyclonal antibody and secondary biotinylated goat anti-rabbit IgG polyclonal antibody were purchased from Abcam, Inc.

The NS0 murine myeloma cell line was obtained from Lonza Biologics. The Colon 26 murine colorectal adenocarcinoma cell line was obtained from the American Type Culture Collection.

Reagents and mice. The Glutamine Synthetase Gene Amplification System with expression plasmids pEE6/hCMV-B and pEE12 was purchased from Lonza Biologics. The plasmid pORF, containing the murine CD137L cDNA, was purchased from Invivogen. Restriction endonucleases, T4 DNA ligase, Vent polymerase, and other molecular biology reagents were obtained from either New England Biolabs or Boehringer Mannheim. Characterized and dialyzed FCS were purchased from Hyclone Corp., and RPMI 1640, Hybridoma Selective Medium without L-glutamine, MEM nonessential amino acids solution (100x), and PBS were purchased from Life Technologies. Iodine-125 (125I) was obtained from DuPont/New England Nuclear as sodium iodide in 0.1 N sodium hydroxide. The murine interleukin-2 (IL-2) ELISA kit was purchased from BD Biosciences.

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 TNT-3/CD137L. The heavy chain of mTNT-3, preceded by an antibody leader sequence, was amplified by PCR using primers 5'-AGCTCTAGAGCCGCCACCATGGGATGGAGCGGGATCTTT-3' and 5'-GGAATTCAGGCGGCCGCTTTTACCCGGAGTCCGGGAGAA-3'. The whole cDNA sequence was inserted into a pEE12 vector by XbaI and EcoRI. The cDNA sequence of the extracellular region of murine CD137L (excluding the transmembrane region) was amplified by PCR using primers 5'-AAGGAAAAAAGCGGCCGCACCGAGCCTCGGCCAGCG-3' and 5'-GGCGAATTCTCATTCCCATGGGTTGTC-3', and this sequence was then inserted into the pEE12 vector, 3' to the mTNT-3 heavy chain, by NotI and EcoRI. The resultant fusion gene in the pEE12 vector was cotransfected with mTNT-3 light chain in pEE6 vector into NS0 cells by electroporation.

Construction of Fc/CD137L. The antibody leader sequence was amplified using primers 5'-AGCTCTAGAGCCGCCACCATGGGATGGAGCGGGATCTTT-3' and 5'-ATTGTGGGCCCTCTGGGCTCGGAGTGGACACCTCCAGTTA-3'. The Fc/CD137L cDNA sequence (including hinge region, CH2, CH3 domain, and CD137L extracellular domain) was amplified using primers 5'-TAACTGGAGGTGTCCACTCCGAGCCCAGAGGGCCCACAAT-3' and 5'-GGCGAATTCTCATTCCCATGGGTTGTC-3'. An assembly PCR was done to align the previous two PCR products using primers 5'-AGCTCTAGAGCCGCCACCATGGGATGGAGCGGGATCTTT-3' and 5'-GGCGAATTCTCATTCCCATGGGTTGTC-3'. The resultant fusion gene was inserted to a pEE12 vector by XbaI and EcoRI and transfected by electroporation into NS0 cells.

Expression and purification of TNT-3/CD137L and Fc/CD137L. The TNT-3/CD137L and Fc/CD137L were expressed in NS0 murine myeloma cells for long-term stable expression as per the manufacturer's protocol (Lonza Biologics). The highest-producing clones, determined by indirect ELISA screening for murine Fc, were scaled-up for incubation in an aerated 3- or 8-liter stir flask bioreactor using 5% heat-inactivated dialyzed FCS to eliminate the induction of proteolytic enzymes by the NS0 cells during incubation and to protect against fusion protein breakage. The secreted fusion protein was then purified from clarified cell culture supernatant by tandem protein-A affinity and ion-exchange chromatography, as described previously (44). Both fusion proteins were confirmed to produce a single peak by high-performance liquid chromatography analysis (data not shown). The fusion proteins were also analyzed by ELISA to verify the presence and proper folding of the CD137L extracellular domain and were analyzed by SDS-PAGE to ensure proper assembly and purity.

In vitro activity assay. The bioactivity of the CD137L moiety was determined by ELISA measurement of IL-2 production from splenocytes aseptically removed from 6-week-old female BALB/c mice. After the RBC were lysed using the BD Pharm Lyse Lysing buffer (BD PharMingen), single-cell suspensions were washed twice in PBS and incubated in a 24-well plate (1.5 x 106 per well) precoated with 5 µg/mL anti-CD3 (145-11C clone) in the presence of 2 µg/mL TNT-3/CD137L, Fc/CD137L, or 2A. After 48 h, IL-2 production was determined by sandwich ELISA (BD Biosciences) for the above culture supernatants according to the manufacturer's protocol. All assays were done in triplicate.

Pharmacokinetics and biodistribution studies. For whole-body clearance studies, groups of 6-week-old female BALB/c mice (n = 5) were provided drinking water with potassium iodide beginning 1 week before the administration of radioiodine to block thyroid uptake. Each group received an i.v. injection of 125I-labeled fusion protein (30 µCi/10 µg per mouse). The whole-body radioactivity of each mouse was then measured at various time intervals, beginning with the immediate post-injection period, using a CRC-7 microdosimeter (Capintec, Inc.). The data were analyzed, as previously described (44), to calculate the whole-body half-life of TNT-3/CD137L and Fc/CD137L.

For biodistribution analysis, groups of BALB/c mice (n = 5) were s.c. injected in the left flank with a 0.2-mL inoculum of 5 x 106 Colon 26 cells. The tumors were allowed to grow until they reached ~0.5 cm in diameter. Each mouse then received an i.v. injection of 0.1 mL 125I-labeled fusion protein (30 µCi/10 µg per mouse). Groups of mice (n = 5) were then sacrificed by sodium pentobarbital overdose at 24 and 48 h after injection, and tumors and normal organs were dissected, weighed, and measured for radioisotope activity with a gamma counter. Data were expressed for each group of mice as the percent injected dose/gram of tissue (%ID/g) and tumor to normal organ ratio. From these data, the mean ± SD was calculated for each group. Significance levels (P values) were determined using the Wilcoxon rank-sum test.

Immunotherapeutic studies. Six-week-old female BALB/c mice were s.c. injected in the left flank with a 0.2-mL inoculum containing ~5 x 106 Colon 26 cells. When tumors reached 0.5 cm in diameter, at approximately the 5th day after tumor implantation, groups of mice (n = 5) were i.v. treated with a 0.1-mL inoculum containing various concentrations of TNT-3/CD137L, Fc/CD137L fusion proteins, or 2A agonist antibody. A dosing study was done for doses ranging from 10 pmol per dose to 1 nmol per dose daily x5. All groups of mice were treated daily x5, and tumor growth was monitored every other day by caliper measurement in three dimensions. Tumor volume was calculated by the formula length x width x height. The results were expressed as the mean ± SD. Significance levels (P values) were determined using the Wilcoxon rank-sum test.

Survival study. Group of BALB/c mice (n = 5) were injected with Colon 26 cells as described above. Five days after tumor implantation, mice were treated with 1 nmol per dose Fc/CD137L or TNT-3/CD137L daily x5, and survival of the mice was recorded for 120 days. Significance levels (P values) were determined using the Wilcoxon rank-sum test.

Depletion of lymphocyte subsets in vivo. To deplete CD4+ and CD8+ T cells, 5 days after tumor implantation and every 5 days thereafter, 0.5 mg anti-CD4 antibody (GK1.5) and 0.5 mg anti-CD8 (H35) were injected i.p. using a 1-mL inoculum in PBS. Depletion of specific T-cell subsets was confirmed by fluorescence-activated cell sorting analysis of lymph nodes of inoculated mice using antibody clones that differ from those used for depletion (data not shown).

Fluorescence-activated cell sorting analysis of tumor-infiltrating lymphocytes. Mice from the control group, the TNT-3/CD137L (500 pmol per dose)–treated group, and the Fc/CD137L (500 pmol per dose)–treated group were sacrificed by sodium pentobarbital overdose 19 days following tumor implantation. Tumors were weighed, and tumor-infiltrating lymphocytes and monocytes were isolated as described previously (37). Fluorescence-labeled anti-CD4, anti-CD8, anti-CD11b, anti-CD11c, anti-CD25, anti-CD45R, and anti-CD49b antibodies were used to stain the tumor-infiltrating lymphocytes for fluorescence-activated cell sorting analysis, along with anti-CD62L and anti-CCR7. After gating on lymphocytes and monocytes, the percentages of subpopulations were calculated based on antibody binding.

Morphologic and immunohistochemical studies. Tumors and tumor-draining lymph nodes from treated and control Colon 26–bearing mice were removed on day 15 after tumor implantation. Tumors were fixed in 10% neutral buffered formalin (VWR Scientific), and paraffin-embedded sections from Colon 26 tumor–bearing mice were stained with H&E for morphologic studies. For immunohistochemical analysis, unstained sections of paraffin-embedded tissues were mounted on poly-L-lysine–coated slides, deparaffinized in Histoclear, and rehydrated using 100% and 95% alcohol. Endogenous hydrogen peroxidase was quenched with 3% hydrogen peroxide in absolute methanol for 20 min. Slides were then subjected to antigen retrieval for 30 min in a microwave oven (citrate buffer, pH 6.0) and subsequently cooled at room temperature for 15 min. Normal horse serum was added for 20 min to block nonspecific binding in the tissue sections. This was followed by incubation with primary antibody overnight at room temperature and biotinylated secondary antibody for 30 min thereafter. Avidin-biotin peroxidase was added for 30 min, followed by color development with 0.03% diaminobenzidine for 10 min. A wash step with phosphate buffer solution for 10 min was done between each step. Finally, slides were counterstained with hematoxylin for 2 min and dehydrated using 95% and 100% alcohol, {alpha}-terpineol xylene, and xylene. Microscopic findings were recorded by an Optronix digital camera.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Construction, expression, and purification of TNT-3/CD137L and Fc/CD137L fusion proteins. Taking into account the importance of the extracellular COOH terminus of CD137L for its bioactivity, the NH2 terminus of the extracellular CD137L gene was fused to the COOH terminus of the mTNT-3 heavy-chain gene to engineer both TNT-3/CD137L and Fc/CD137L fusion genes (Fig. 1A and B ). Both TNT-3/CD137L and Fc/CD137L were translated under an antibody leader sequence.


Figure 1
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  		Fig. 1. Schematic diagram of the construction and final assembly of the murine (A) TNT-3/CD137L and (B) Fc/CD137L fusion proteins. C, electrophoretic analysis of the purified TNT-3/CD137L and Fc/CD137L using Coomassie blue–stained 4% to 15% reducing SDS-PAGE. MW, molecular weight.

 
Proper assembly of the TNT-3/CD137L and Fc/CD137L fusion proteins was shown by 4% to 15% reducing SDS-PAGE (Fig. 1C). Two defined bands were resolved for TNT-3/CD137L at ~28 and 85 kDa, corresponding to the molecular weights of the immunoglobulin light chain and heavy chain plus the CD137L extracellular region after glycosylation, respectively (1). For the Fc/CD137L, >97% of this fusion protein resolved at 70 kDa, which corresponds to the molecular weight of Fc plus the CD137L extracellular region after glycosylation. One weak band was resolved at the molecular weight of 40 kDa, indicating that <3% of Fc/CD137L was cleaved during expression.

Bioactivity of CD137L moiety. To determine whether the CD137L moiety of the fusion proteins retained their biological activity, an IL-2 production assay was done. As shown in Fig. 2A , the CD137L fusion proteins induced IL-2 production in the presence of bound anti-CD3, compared with anti-CD3 alone. 2A (39), TNT-3/CD137L, and Fc/CD137L were each able to provide the costimulatory second signal needed to induce the production of similar levels of IL-2 by mouse splenocytes. This stands in contrast to the testing of a molecule previously constructed by our lab using the same NS0 amplification system. LFA-3/Fc, unlike the CD137L molecules, did not induce IL-2 production (data not shown). Taken together, these data show the potency of the CD137L moieties of both fusion proteins.


Figure 2
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  		Fig. 2. Demonstration of the potency of CD137L fusion proteins in vitro and the targeting ability of TNT-3/CD137L. A, in vitro stimulation of IL-2 production by TNT-3/CD137L, Fc/CD137L, 2A, and anti-CD28 with anti-CD3. B, tissue biodistribution and tumor uptake of TNT-3/CD137L and Fc/CD137L at 24 and 48 h after injection in Colon 26-bearing BALB/c mice. Tumor uptake was measured by percent injected dose per gram of 125I-labeled TNT-3/CD137L or Fc/CD137L (top) and tumor/normal organ ratios (bottom). LN, lymph nodes. Columns, mean; bars, SD.

 
In vivo pharmacokinetic and biodistribution studies. Whole-body clearance studies were done in healthy BALB/c mice to establish the in vivo half-life of TNT-3/CD137L and Fc/CD137L. Mice were injected i.v. with the radiolabeled fusion protein, and whole-body radioactivity was measured at various time points using a microdosimeter. The whole-body half-lives of TNT-3/CD137L and Fc/CD137L were found to be 18 h ± 15 min (P ≤ 0.01) and 24 h ± 20 min (P ≤ 0.01), respectively.

Tissue biodistribution studies in Colon 26 tumor–bearing BALB/c mice were done to determine the relative tumor uptake of each fusion protein. Tumor and normal tissue uptake was measured 24 and 48 h after i.v. administration of radiolabeled TNT-3/CD137L and Fc/CD137L. As shown in Fig. 2B, the uptake of TNT-3/CD137L per gram of tumor was significantly higher than the uptake in normal organs at 24 h (P ≤ 0.01) and showed even higher retention at 48 h (P ≤ 0.01) after injection. As expected, Fc/CD137L showed low tumor retention over time despite its slightly longer half-life. Examination of the tumor-draining lymph nodes, an important site for tumor immunotherapy, revealed that TNT-3/CD137L shows slightly better uptake than Fc/CD137L at 24 and 48 h after injection (Fig. 2). However, the average tumor-draining lymph node retention of TNT-3/CD137L was still much lower than that measured in tumor.

Immunotherapeutic dosing studies. A dosing study was done on Colon 26–bearing BALB/c mice, with doses ranging from 10 to 1 nmol per dose of TNT-3/CD137L, Fc/CD137L, and the anti-CD137 agonist antibody 2A (Fig. 3 ). At 10 pmol per dose, no significant tumor reduction was observed in any of the treatment groups. At 100 pmol per dose (15 µg per dose) and 250 pmol per dose (37.5 µg per dose), 2A-treated mice showed 95% tumor volume reduction, whereas TNT-3/CD137L and Fc/CD137L treatment resulted in 30% to 40% tumor reduction at day 19 after tumor implantation. At 500 pmol per dose, however, the TNT-3/CD137L group showed 70% tumor reduction, whereas the Fc/CD137L–treated mice achieved only 30% tumor reduction. This difference suggests that at this dosage, localization of CD137L is more effective. At 1 nmol per dose, however, both TNT-3/CD137L– and Fc/CD137L–treated groups showed 80% tumor reduction, and 2A-treated groups continued to show 95% tumor reduction. All 2A-treated groups (except 10 pmol per dose group) eventually became tumor-free (data not shown). All treated mice were free of signs of toxicity throughout the 140-day observation period.


Figure 3
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  		Fig. 3. Dose response of TNT-3/CD137L, Fc/CD137L, and 2A in Colon 26–bearing BALB/c mice.

 
Survival studies. In the 1 nmol per dose treatment groups, survival of mice was recorded for up to 140 days after tumor implantation (Fig. 4 ). One hundred percent of 2A-treated mice, 60% of TNT-3/CD137L–treated mice, and 40% of Fc/CD137L–treated mice survived for the 140 days, whereas none of the PBS-treated mice survived 140 days (100% of the control mice died at around day 50 after tumor implantation). Mice from all of the three treated groups survived significantly longer than the control group (P < 0.001).


Figure 4
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  		Fig. 4. Kaplan-Meier survival analysis of 2A-, TNT-3/CD137L–, and Fc/CD137L–treated Colon 26 tumor–bearing BALB/c mice.

 
Characterization of tumor-infiltrating lymphocytes. Because the 500 pmol per dose treatment groups in the dosing experiments showed the highest separation in results with respect to tumor volume, an analysis of the relative composition of tumor-infiltrating lymphocytes by flow cytometry was done at this dose. At day 19 after tumor implantation, mice from the 500 pmol per dose treatment groups were sacrificed, and tumor-infiltrating immune cells were stained for lymphocyte, monocyte, and dendritic cell markers. The stained cells were analyzed by flow cytometry, and the percentages of subpopulations among all detected tumor-infiltrating lymphocytes were calculated (Table 1 ).


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  		Table 1. Percentage of lymphocyte subpopulations in tumor-infiltrating lymphocytes at day 19 after tumor implantation

 
A comparison of the lymphocyte subpopulations among the CD137L fusion protein treatment groups showed that the CD8+ T-cell percentage was inversely proportional to tumor volume and weight, increasing from 4.8% in untreated controls to 13.6% in Fc/CD137L–treated mice and 22% in TNT-3/CD137L–treated mice, suggesting that CD8+ T cells play an important role in eradicating Colon 26 tumor. Interestingly, the CD8+ T cells were more likely to be CD45R–CD62L–CCR7– in treated mice than the CD8+ T cells in control mice, suggesting that by day 19, tumor-infiltrating CD8+ T cells of the treated mice have been induced to differentiation from central memory T cells (TCM) to effector memory T cells (TEM), but not yet into fully differentiated T cells (50). A modest increase in CD4+ cells was also observed, correlating inversely with tumor size, but this increase was not as robust as the increase of CD8+ cells.

Another notable and consistent trend was the positive correlation between tumor size and percentage of CD25+ T cells, which decreased from 22.5% in control Colon 26–bearing mice to 7.7% in Fc/CD137L–treated mice and 5.5% in TNT-3/CD137L–treated mice, suggesting that CD25+ T cells (T regulatory cells) were negatively associated with the effectiveness of treatment. Additionally, at day 19, CD11b+ monocytes were also seen to increase with the effectiveness of treatment, implicating their role in the elimination of necrotic tumor material. CD49+ natural killer cells were not found to vary significantly at day 19, although at day 14, the effectiveness of treatment was found to correlate with degree of natural killer cell infiltration (data not shown).

Morphologic and immunohistochemical studies. Six days after the completion of treatment, tumors were removed from Colon 26–bearing mice treated with daily x5 injections of 1 nmol per dose TNT-3/CD137L and Fc/CD137L. As shown in Fig. 5A , H&E staining of the tumors revealed extensive central necrosis in those groups of mice treated with TNT-3/CD137L and Fc/CD137L. By contrast, necrosis was also present in PBS-treated control mice, although it was much more localized and less extensive. A similar percentage of granzyme B–positive cells were seen in the tumor-draining lymph nodes of all treatment groups (Fig. 5B). Granzyme B–positive cells were found to infiltrate central necrotic areas and viable peripheral regions of tumor broadly in mice treated with CD137L fusion proteins but were much fewer in number in tumors from control-treated mice (Fig. 5C).


Figure 5
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  		Fig. 5. A, H&E staining of tumors from control, TNT-3/CD137L–, and Fc/CD137L–treated mice. Immunohistochemical staining of granzyme B–positive cells in (B) tumor-draining lymph nodes and (C) tumors from control, TNT-3/CD137L–, and Fc/CD137L–treated mice.

 
Combination treatment studies with CD4+ and CD8+ T-cell depletion. To assess the role of CD4+ and CD8+ T cells in CD137L-mediated immunotherapy, T-subset depletion studies were done with cytotoxic antibodies against CD4+ and CD8+ cells. Anti-CD4 antibody (Clone GK1.5) and anti-CD8 antibody (Clone H35) were injected i.p. on the 5th day after tumor implantation, and these procedures were repeated every 5 days. Depletion was confirmed by fluorescence-activated cell sorting (data not shown). As shown in Fig. 6 , CD8+ T-cell depletion completely abrogated TNT-3/CD137L and Fc/CD137L antitumor effects, indicating that tumor suppression was dependent on this subpopulation of T cells. In contrast, CD4+ T-cell depletion alone caused significant tumor regression similar to that seen with CD4+ T-cell depletion and CD137L fusion protein combination therapy.


Figure 6
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  		Fig. 6. Combination immunotherapy of CD137L fusion proteins and deleted T-cell subsets. A, deletion of CD4 and CD8 subpopulations only. B, TNT-3/CD137L treatment alone and in combination with T-cell subset deletion. C, Fc/CD137L treatment alone and in combination with T-cell subset deletion.

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
CD137L is a member of the TNF superfamily and provides a costimulatory signal to T cells. It is expressed on dendritic cells, activated macrophages, and B cells (8–10). The interaction between CD137L and CD137 on activated T cells is essential for an adaptive T-cell response and, like other costimulatory molecules, may play a very important role in tumor immunotherapy.

In this study, CD137L was genetically linked to the murine TNT-3 antibody, which targets necrotic regions of solid tumors (38), and to the Fc region of murine TNT-3, which creates a comparable but non-targeted reagent. Both reagents have similar in vivo pharmacokinetic properties and stability. Although soluble Fc does not target to tumor, it does bind Fc receptor–positive cells, which can correctly present the costimulatory molecule to T cells. An in vitro activity assay confirmed the biological activity of the CD137L moiety on both fusion proteins, and immunotherapeutic studies indicated that both CD137L fusion proteins exerted antitumor effects and produced tumor-free mice in a large percentage of treated animals.

In terms of biodistribution, several differences exist between the two fusion proteins. Fc/CD137L primarily distributed to mouse liver, with limited tumor uptake consistent with levels seen in the blood pool. Fc/CD137L uptake in all tissues including tumor decreased from 24 to 48 h, as expected for non-targeting proteins (Fig. 2B). By contrast, TNT-3/CD137L showed a 5-fold higher tumor uptake versus Fc/CD137L at 24 h, with a 40% increase in tumor uptake at 48 h. The differential distribution of the fusion proteins into tumor likely necessitated the requirement of a larger dosage of Fc/CD137L (1 nmol per dose) to achieve levels of tumor regression similar to those induced by TNT-3/CD137L (500 pmol per dose). At higher doses, however, the therapeutic effects of the two fusion proteins did not differ significantly. At 140 days after tumor implantation, the survival rates of tumor-bearing mice treated with TNT-3/CD137L (1 nmol per dose) and Fc/CD137L (1 nmol per dose) were 60% and 40%, respectively (Fig. 4). At higher doses, both CD137L fusion proteins induced 80% tumor regression (Fig. 3) and similar amounts of tumor necrosis and infiltration by granzyme B–secreting cells (Fig. 5).

Necrosis was a consistent finding in treated Colon 26 tumors. Morphologic analysis of tumors at day 15 after implantation (Fig. 5A) showed massive central necrosis in tumors from mice treated with CD137L fusion proteins, compared with much less extensive and more focal necrosis in tumors from the control group. This finding suggests that the extent of central necrosis is indicative of the effectiveness of antitumor treatment in the Colon 26 tumor model. Using different tumor models, Blohm et al. (51) also associated the extent of necrosis with the effectiveness of antitumor therapy. They reported that after antigen-specific CD8+ T-cell attack, the destruction of tumor architecture and the presence of significant central necrosis correlated with tumor suppression. It has been amply shown that the major effector cells that destroy tumor and cause necrosis are activated, antigen-specific CD8+ T cells (51). Various mechanisms exist by which CD8+ T cells destroy tumors cells, including the induction of apoptosis through CD95 ligation, the secretion of lytic granules containing perforin and granzyme B, and the production of pro-inflammatory cytokines (52, 53).

Interestingly, when comparing the granzyme B–secreting T cells in the tumor-draining lymph node with the ones in tumors, clear differences were noted. Clusters of granzyme B–secreting cells in equal numbers were seen in all tumor-draining lymph nodes in all treatment groups (Fig. 5B). In sections of these tumors, granzyme B–positive cells broadly infiltrated central necrotic areas and viable peripheral tumor, whereas these cells were much fewer in number in sections from untreated control tumors (Fig. 5C). These observations suggest that although activated CD8+ T cells are present in tumors from mice in all groups, the actual number of activated CD8+ T cells inside of the tumor is a critical factor for successful immunotherapy. In patient studies using vector-mediated immunization with tumor antigens, Yang et al. (54) showed that higher-avidity CTLs were produced by costimulation of antigen-presenting cells. Identified by tetramer dissociation methods, these higher-avidity CTLs correlated with better immune variables and were only produced in those patients receiving a combination of costimulatory molecules as part of the vaccination boost. Hence, tumor suppression may be caused by both higher numbers of infiltrating CD8+ cells as well as higher-avidity CTLs. In future experiments, it would therefore be useful to measure CTL avidity along with CTL numbers to get a more complete picture of the nature of the immunologic response seen in the tumor.

Additional mechanistic studies confirmed that tumor regression of the Colon 26 model is CD8+ dependent. Although fluorescence-activated cell sorting analysis of tumor-infiltrating lymphocytes showed the presence of CD4+ T cells, CD8+ T cells, CD11c+ dendritic cells, CD11b monocytes, CD25+ T regulatory cells, and natural killer cells (Table 1), only the relative percentage of CD8+ T cells and CD11b+ macrophages correlated strongly and positively with the effectiveness of treatment, such that the higher this percentage, the greater the tumor regression. In the final analysis, the critical role of CD8+ T cells as the effector cells was confirmed by studies in which CD8+ T cells were depleted systemically (Fig. 6). In the CD8+-depleted mice, tumors grew larger than in control mice, suggesting that CD8+ T cells confer a basal level of host immunity in untreated mice, which is lost when those mice are depleted of their CD8+ T cells. Unlike depletion of CD8+ T cells, depletion of CD4+ cells resulted in significant suppression of Colon 26 tumor growth, suggesting that CD4+ T cells may negatively regulate CTLs in the Colon 26 model.

The results presented here challenge the traditional view that CD4+ T cells play a critical role in antitumor immunotherapy. One possible explanation is that depletion of CD4+ cells depletes naturally occurring and/or induced CD4+CD25+ T regulatory cells, which suppress the activation of CD8+ T cells. The removal of CD4+CD25+ T regulatory cells might result in a more active population of CD8+ T cells, leading to a better therapeutic effect. In support of this theory, combination treatment of TNT-3/CD137L with systemic depletion of CD25+ cells using the rat antibody PC61 achieved higher levels of tumor regression than TNT-3/CD137L alone (data not shown), as was previously shown for the combination therapy with the LEC/chTNT-3 chemokine fusion protein produced by our laboratory (47).

In studies analyzing tumor-infiltrating lymphocytes, 2A-treated mice bore the highest percentage of CD8+ T cells in tumor among all the treatment groups (Table 1). These results correlate with the immunotherapeutic dosing study, which showed that relatively small doses of 2A produced complete tumor regression (Fig. 3). These studies showed that 2A was able to elicit a greater immune response than the CD137L fusion proteins in vivo as might be expected because of the increased biological half-life of the reagent in vivo. Interestingly, previous studies using sarcoma Ag104-bearing mice showed similar data in that the CD137 agonist antibody showed greater antitumor effects than did tumor cells transfected to express CD137L (24, 25). The differential therapeutic response induced by the CD137 agonist antibody and the natural ligands may also be attributable to the fact that different intracellular signaling pathways are triggered upon CD137 ligation. In our hands, we have observed the intracellular production of both IFN-{gamma} and TNF-{alpha} in murine T cells treated with 2A, whereas only IFN-{gamma} production was triggered by CD137L fusion protein treatment (data not shown).

In the past, collective evidence has shown that many of the surface-bound receptor/ligand pairs of the TNFR/TNF superfamily (including CD137/CD137L, CD134/CD134L, GITR/GITRL, and CD27/CD27L) are integral to the generation and preservation of the T cells that carry out various effector functions in an antitumor immune response. Together, these receptor/ligand pairs provide signals essential for T-cell function and survival through independent or overlapping pathways. It is evident that members of the TNF family of proteins synergize with each other under certain circumstances, and indeed, CD137L has been shown by others to have synergistic effects with OX40L in generating the clonal expansion and effector function of CD8+ T cells (55). Similarly, ongoing studies conducted in our laboratory showed that CD137L and GITRL have synergistic immunotherapeutic effects in Colon 26–bearing mice.1

In conclusion, we have described the generation and characterization of two CD137L fusion proteins that induce CD8+ T-cell–dependent antitumor responses in the Colon 26 tumor model. Although both fusion proteins were able to induce significant tumor suppression, it seems that targeting CD137L to tumor may be more effective than using the untargeted reagent. Moreover, a targeted fusion protein using the natural CD137 ligand is likely to have less toxic side effects than untargeted soluble Fc or than long-acting agonistic antibodies such as 2A. Accordingly, TNT-3/CD137L may be the molecule of choice for future clinical trials.


   Acknowledgments
 
We thank Jingzhong Pang for his expert assistance and diligence with the animal studies.


   Footnotes
 
Grant support: Cancer Therapeutics Laboratories, Inc. (Los Angeles, CA) and 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: Present address for L.A. Khawli: Principal Scientist, Genentech, Inc., One DNA Way, MS 70, South San Francisco, CA 94080.

1 Unpublished. Back

Received 9/20/06; revised 1/13/07; accepted 2/ 5/07.


   References
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

  1. Gajewski TF, Meng Y, Harlin H. Immune suppression in the tumor microenvironment. J Immunother 2006;29:233–40.[CrossRef][Medline]
  2. O'Carroll KS, Sotomayor E, Montgomery J, et al. Induction of antigen-specific T cell anergy: an early event in the course of tumor progression. Proc Natl Acad Sci U S A 1998;95:1178–83.[Abstract/Free Full Text]
  3. Ahmad M, Rees RC, Ali SA. Escape from immunotherapy: Possible mechanisms that influence tumor regression/progression. Cancer Immunol Immunother 2004;53:844–54.[Medline]
  4. Zou W. Immunosuppressive networks in the tumour environment and their therapeutic relevance. Nat Rev Cancer 2005;5:263–74.[CrossRef][Medline]
  5. Bretscher PA. A two-step, two signal model for the primary activation of precursor helper T cells. Proc Natl Acad Sci U S A 1999;96:185–90.[Abstract/Free Full Text]
  6. Sharpe AH, Freeman GJ. The B7-28 superfamily. Nat Rev Immunol 2002;2:116–26.[CrossRef][Medline]
  7. Greenwald RJ, Freeman GJ, Sharpe AH. The B7 family revisited. Annu Rev Immunol 2005;23:515–48.[CrossRef][Medline]
  8. Vinay DS, Kwon BS. Role of 4-1BB in immune responses. Semin Immunol 1998;10:481–9.[CrossRef][Medline]
  9. Watts TH. TNF/TNFR family members in costimulation of T cell responses. Annu Rev Immunol 2005;23:23–8.[CrossRef][Medline]
  10. Goodwin RG, Din WS, Davis-Smith T, et al. Molecular cloning of a ligand for the inducible T cell gene 4-1BB: a member of an emerging family of cytokines with homology to tumor necrosis factor. Eur J Immunol 1993;23:2631–41.[Medline]
  11. Gruss HJ, Duyster J, Herrmann F. Structural and biological features of the TNF receptor and TNF ligand superfamilies: interactive signals in the pathobiology of Hodgkin's disease. Ann Oncol 1996;7:19–26.
  12. Kwon BS, Kozak CA, Kim KK, Pickard RT. Genomic organization and chromosomal localization of the T-cell antigen 4-1BB. J Immunol 1994;152:2256–62.[Abstract]
  13. Kwon BS, Weissman SM. cDNA sequences of two inducible T-cell genes. Proc Natl Acad Sci U S A 1989;86:1963–7.[Abstract/Free Full Text]
  14. Schwarz H, Valbracht J, Tuckwell J, von Kempis J, Lotz M. ILA, the human 4-1BB homologue, is inducible in lymphoid and other cell lineages. Blood 1995;85:1043–52.[Abstract/Free Full Text]
  15. Alderson MR, Smith CA, Tough TW, et al. Molecular and biological characterization of human 4-1BB and its ligand. Eur J Immunol 1994;24:2219–27.[Medline]
  16. Futagawa T, Akiba H, Kodama T, et al. Expression and function of 4-1BB and 4-1BB ligand on murine dendritic cells. Int Immunol 2002;14:275–86.[Abstract/Free Full Text]
  17. Pollok KE, Kim YJ, Hurtado J, Zhou Z, Kim KK, Kwon BS. 4-1BB T-cell antigen binds to mature B cells and macrophages, and costimulates anti-mu-primed splenic B cells. Eur J Immunol 1994;24:367–74.[Medline]
  18. Cannons JL, Lau P, Ghumman B, et al. 4-1BB ligand induces cell division, sustains survival, and enhances effector function of CD4 and CD8 T cells with similar efficacy. J Immunol 2001;167:1313–24.[Abstract/Free Full Text]
  19. Gramaglia I, Cooper D, Miner KT, Kwon BS, Croft M. Co-stimulation of antigen-specific CD4 T cells by 4-1BB ligand. Eur J Immunol 2000;30:392–402.[CrossRef][Medline]
  20. Lee HW, Nam KO, Park SJ, Kwon BS. 4-1BB enhances CD8+ T cell expansion by regulating cell cycle progression through changes in expression of cyclins D and E and cyclin-dependent kinase inhibitor p27kip1. Eur J Immunol 2003;33:2133–41.[CrossRef][Medline]
  21. Shuford WW, Klussman K, Tritchler DD, et al. 4-1BB costimulatory signals preferentially induce CD8+ T cell proliferation and lead to the amplification in vivo of cytotoxic T cell responses. J Exp Med 1997;186:47–55.[Abstract/Free Full Text]
  22. Cooper D, Bansal-Pakala P, Croft M. 4-1BB (CD137) controls the clonal expansion and survival of CD8 T cells in vivo but does not contribute to the development of cytotoxicity. Eur J Immunol 2002;32:521–9.[CrossRef][Medline]
  23. Diehl L, van Mierlo GJ, den Boer AT, et al. In vivo triggering through 4-1BB enables Th-independent priming of CTL in the presence of an intact CD28 costimulatory pathway. J Immunol 2002;168:3755–62.[Abstract/Free Full Text]
  24. Melero I, Bach N, Hellstrom KE, Aruffo A, Mittler RS, Chen L. Amplification of tumor immunity by gene transfer of the co-stimulatory 4-1BB ligand: synergy with the CD28 co-stimulatory pathway. Eur J Immunol 1998;28:1116–21.[CrossRef][Medline]
  25. Melero I, Shuford WW, Newby SA, et al. Monoclonal antibodies against the 4-1BB T-cell activation molecule eradicate established tumors. Nat Med 1997;3:682–5.[CrossRef][Medline]
  26. Mogi S, Sakurai J, Kohsaka T, et al. Tumour rejection by gene transfer of 4-1BB ligand into a CD80+ murine squamous cell carcinoma and the requirements of co-stimulatory molecules on tumour and host cells. Immunology 2000;101:541–7.[Medline]
  27. Wilcox RA, Flies DB, Zhu G, et al. Provision of antigen and CD137 signaling breaks immunological ignorance, promoting regression of poorly immunogenic tumors. J Clin Invest 2002;109:651–9.[CrossRef][Medline]
  28. Wilcox RA, Tamada K, Flies DB, et al. Ligation of CD137 receptor prevents and reverses established anergy of CD8+ cytolytic T lymphocytes in vivo. Blood 2004;103:177–84.[Abstract/Free Full Text]
  29. Wilcox RA, Tamada K, Strome SE, Chen L. Signaling through NK cell-associated CD137 promotes both helper function for CD8+ cytolytic T cells and responsiveness to IL-2 but not cytolytic activity. J Immunol 2002;169:4230–6.[Abstract/Free Full Text]
  30. Yan X, Johnson BD, Orentas RJ. Murine CD8 lymphocyte expansion in vitro by artificial antigen-presenting cells expressing CD137L (4-1BBL) is superior to CD28, and CD137L expressed on neuroblastoma expands CD8 tumour-reactive effector cells in vivo. Immunology 2004;112:105–16.[CrossRef][Medline]
  31. Zhang H, Merchant MS, Chua KS, et al. Tumor expression of 4-1BB ligand sustains tumor lytic T cells. Cancer Biol Ther 2003;2:579–86.[Medline]
  32. Chu NR, DeBenedette MA, Stiernholm BJ, Barber BH, Watts TH. Role of IL-12 and 4-1BB ligand in cytokine production by CD28+ and CD28– T cells. J Immunol 1997;158:3081–9.[Abstract]
  33. Nam KO, Kang H, Shin SM, et al. Cross-linking of 4-1BB activates TCR-signaling pathways in CD8+ T lymphocytes. J Immunol 2005;174:1898–905.[Abstract/Free Full Text]
  34. Lee HW, Park SJ, Choi BK, Kim HH, Nam KO, Kwon BS. 4-1BB promotes the survival of CD8+ T lymphocytes by increasing expression of Bcl-xL and Bfl-1. J Immunol 2002;169:4882–8.[Abstract/Free Full Text]
  35. Starck L, Scholz C, Dorken B, Daniel PT. Costimulation by CD137/4-1BB inhibits T cell apoptosis and induces Bcl-xL and c-FLIP(short) via phosphatidylinositol 3-kinase and AKT/protein kinase B. Eur J Immunol 2005;35:1257–66.[CrossRef][Medline]
  36. Guinn BA, DeBenedette MA, Watts TH, Berinstein N. L. 4-1BBL cooperates with B7–1 and B7–2 in converting a B cell lymphoma cell line into a long-lasting antitumor vaccine. J Immunol 1999;162:5003–10.[Abstract/Free Full Text]
  37. Sanderson K, Scotland R, Lee P, et al. Autoimmunity in a phase I trail of a fully human anti-cytotoxic T-lymphocyte antigen-4 monoclonal antibody with multiple melanoma peptides and montanide ISA 51 for patients with resected stages III and IV melanoma. J Clin Oncol 2005;23:741–50.[Abstract/Free Full Text]
  38. Epstein AL, Chen FM, Taylor CR. A novel method for the detection of necrotic lesions in human cancers. Cancer Res 1988;48:5842–8.[Abstract/Free Full Text]
  39. Epstein AL, Khawli LA, Chen FM, Hu P, Glasky MS, Taylor CR. Tumor necrosis imaging and treatment of solid tumors. In: V. P. Torchilin, editor. Handbook of targeted delivery of imaging agents, Vol. 16. Boca Raton: CRC Press; 1995. p. 259.
  40. Chen S, Yu L, Jiang C, et al. Pivotal study of iodine-131-labeled chimeric tumor necrosis treatment radioimmunotherapy in patients with advanced lung cancer. J Clin Oncol 2005;23:1538–47.[Abstract/Free Full Text]
  41. Epstein A, Chen D, Ansari A, et al. Radioimmunodetection of necrotic lesions in human tumors using I-131-labeled TNT-1 F(ab')2 monoclonal antibody. Antibody Immunoconj Radiopharm 1991;4:151–61.
  42. Hornick JL, Sharifi J, Khawli LA, et al. A new chemically modified chimeric TNT-3 monoclonal antibody directed against DNA for the radioimmunotherapy of solid tumors. Cancer Biother Radiopharm 1998;13:255–68.[Medline]
  43. Hornick JL, Khawli LA, Hu P, Sharifi J, Khanna C, Epstein AL. Pretreatment with a monoclonal antibody/interleukin-2 fusion protein directed against DNA enhances the delivery of therapeutic molecules to solid tumors. Clin Cancer Res 1999;5:51–60.[Abstract/Free Full Text]
  44. Li J, Hu P, Khawli LA, Yun A, Epstein AL. chTNT-3/hu IL-12 fusion protein for the immunotherapy of experimental solid tumors. Hybrid Hybridomics 2004;23:1–10.[CrossRef][Medline]
  45. Sharifi J, Khawli LA, Hu P, Li J, Epstein AL. Generation of human interferon gamma and tumor necrosis factor alpha chimeric TNT-3 fusion proteins. Hybrid Hybridomics 2002;21:421–32.[CrossRef][Medline]
  46. Mizokami MM, Hu P, Khawli LA, Li J, Epstein AL. Chimeric TNT-3 antibody/murine interferon-gamma fusion protein for the immunotherapy of solid malignancies. Hybrid Hybridomics 2003;22:197–207.[CrossRef][Medline]
  47. Li J, Hu P, Khawli LA, Epstein AL. Complete regression of experimental solid tumors by combination LEC/chTNT-3 immunotherapy and CD25+ T-cell depletion. Cancer Res 2003;63:8384–92.[Abstract/Free Full Text]
  48. Li J, Hu P, Khawli LA, Epstein AL. LEC/chTNT-3 fusion protein for the immunotherapy of experimental solid tumors. J Immunother 2003;26:320–31.[CrossRef][Medline]
  49. Flanagan ML, Khawli LA, Hu P, Epstein AL. H60/TNT-3 fusion protein activates NK cells in vitro and improves immunotherapeutic outcome in murine syngeneic tumor models. J Immunother 2006;29:274–83.[CrossRef][Medline]
  50. Unsoeld H, Pircher H. Complex memory T-cell phenotypes revealed by coexpression of CD62L and CCR7. J Virol 2005;79:4510–3.[Abstract/Free Full Text]
  51. Blohm U, Potthoff D, van der Kogel AJ, Pircher H. Solid tumors "melt" from the inside after successful CD8 T cell attack. Eur J Immunol 2006;36:468–77.[CrossRef][Medline]
  52. Kelso A, Costelloe EO, Johnson BJ, Groves P, Buttigieg K, Fitzpatrick DR. The genes for perforin, granzymes A-C and IFN-{gamma} are differentially expressed in single CD8+ T cells during primary activation. Int Immunol 2002;14:605–13.[Abstract/Free Full Text]
  53. Hayashida M, Kawano H, Nakano T, Shiraki K, Suzuki A. Cell death induction by CTL: Perforin/granzyme B system dominantly acts for cell death induction in human hepatocellular carcinoma cells. Exp Biol Med (Maywood) 2000;225:143–50.[Abstract/Free Full Text]
  54. Yang S, Tsang K-Y, Schlom J. Induction of higher avidity human CTLs by vector-mediated enhanced costimulation of antigen-presenting cells. Clin Cancer Res 2005;11:5603–15.[Abstract/Free Full Text]
  55. Lee SJ, Myers L, Muralimohan G, et al. 4-1BB and OX40 dual costimulation synergistically stimulate primary specific CD8 T cells for robust effector function. J Immunol 2004;173:3002–12.[Abstract/Free Full Text]



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