Immunotherapy with anti-CD3 monoclonal antibodies and recombinant interleukin 2: Stimulation of molecular programs of cytotoxic killer cells and induction of ...
Proc. Nati. Acad. Sci. USA Vol. 91, pp. 7889-7893, August 1994 Medical Sciences
Immunotherapy with anti-CD3 monoclonal antibodies and recombinant interleukin 2: Stimulation of molecular programs of cytotoxic killer cells and induction of tumor regression (T-celi signaling/antitumor efficacy/perforin)
FuMio NAKAJIMA*, ASHWANI KHANNA*, GUOPING XU*, MILA LAGMAN*, RUDY HASCHEMEYERt, JANET MOURADIAN*, JOHN C. WANG*t§¶, KURT H. STENZEL*t§¶, ALBERT L. RUBIN*t§¶, AND MANIKKAM SUTHANTHIRAWiN*t§II *The Rogosin Institute, Departments of tBiochemistry, Avenue, New York, NY 10021
Medicine, SPathology, and ISurgery, The New York Hospital-Cornell Medical Center, 1300 York
Communicated by Alton Meister, May 5, 1994 (received for review March 11, 1994)
ABSTRACT Adoptive cellular imunotherapy, infusions of interleukin 2 (IL-2) in conjunction with in vitro-activated killer cells, has brought new hope to patients with cancer. The broad application of this strategy, however, is constrained by the need for repeated leukapheresis and by the labor-intensive process of in vitro activation of cells. Also, current protocols generally use nonphysiological and toxic concentrations of IL-2. Identification of an in vivo stimulant that renders T cells responsive to physiologic concentrations of IL-2 represents a potential improvement over existing approaches. We have determined whether in vivo administration of monoclonal antibodies (mAbs) directed at the T-cell surface protein CD3 induces T-cell responsiveness to IL-2, stimulates cytolytic molecular programs of natural killer cells and cytotoxic T cells, and induces tumor regression. These hypotheses were explored in a murine hepatic MCA-102 fibrosarcoma model. We report that in vivo administration of anti-CD3 mAbs plus IL-2 results in intrahepatic expression of mRNA-encoding perforin, cytotoxic T-cell-specific serine esterase, and tumor necrosis factor a. Anti-CD3 mAbs alone or 1L-2 alone failed to induce or induced minimal expression of these molecular mediators of cytotoxicity. The anti-CD3 mAbs plus IL-2 regimen also resulted in a snicantly smaller number of hepatic metastases and a s cantly longer survival time of tumor-bearing mice, compared to treatment with anti-CD3 mAbs alone or IL-2 alone. Our findings suggest that a regimen of anti-CD3 mAbs plus IL-2 is a more effective antitumor regimen compared with anti-CD3 mAbs alone or IL-2 alone and advance an alternative immunotherapy strategy of potential value for the treatment of cancer in humans.
of the basic protocol. Some of the developments of potential clinical significance include (i) adoptive transfer of tumorinfiltrating cells rather than peripheral blood leukocytes (4); (ii) use of additional cytokines capable of augmenting host immunity-e.g., interleukin 4, interleukin 7 (refs. 4, 5), and (iii) vaccination with tumor cells genetically engineered to express cell-surface proteins (e.g., B7/BB1 antigen) (6) or secrete immune-enhancing cytokines (e.g., granulocyte/ macrophage colony-stimulatory factor) (7). These strategies, to a large extent, share a common mechanistic themestimulation and/or enhancement of host immune effector mechanisms. We have explored here whether in vivo administration of monoclonal antibodies (mAbs) directed at the T-cell surface protein CD3 (anti-CD3 mAbs) augments host immunity and synergizes with IL-2 in inducing tumor regression. The rationale for our antitumor strategy is our original demonstration that anti-CD3 mAbs enhance in vitro the cytotoxic activity of natural killer cells and induce the differentiation of memory T cells into specific cytotoxic T cells (8). Natural killer cells and cytotoxic T cells represent important host defense mechanisms against tumors (9-12). A murine hepatic MCA-102 metastases model, described by Lafreniere and Rosenberg (13), was used in this study to characterize the antitumor efficacy of in vivo-administered anti-CD3 mAbs, IL-2, or anti-CD3 mAbs plus IL-2. Results from our investigations demonstrate that (i) anti-CD3 mAbs plus IL-2 are synergistic in inducing the expression of a molecular program-induction of intrahepatic expression of mRNA encoding perforin, cytotoxic T-cell-specific serine esterase, and tumor necrosis factor a (TNF-a)-that is conducive to tumor regression; (ii) in vivo administration of anti-CD3 mAbs plus IL-2 and not anti-CD3 mAbs alone or IL-2 alone significantly reduces the number of murine hepatic metastases; and (iii) the combined regimen and not anti-CD3 mAbs alone or IL-2 alone significantly prolongs the survival time of tumor-bearing mice.
The prototypic T-cell growth factor, interleukin 2 (IL-2), recently approved by the Food and Drug Administration for the treatment of metastatic renal cell carcinoma, represents a valuable addition to the therapeutic arsenal of the clinical oncologist. Adoptive cellular immunotherapy (1-3), infusions of IL-2 in conjunction with lymphokine-activated killer cells, has brought new hope to those afflicted with chemotherapy-resistant malignancies. Significant limitations, however, exist that thwart broad clinical application of adoptive cellular immunotherapy (1-3). (i) The process of repeated leukapheresis and in vitro activation of autologous leukocytes is quite cumbersome. (ii) The clinical response rates found with this labor-intensive therapy are, at best, modest. (iii) There is also a considerable amount of treatment-related toxic side effects. These considerations have fostered several clinical and experimental modifications
MATERIALS AND METHODS Mice. C57BL/6 (B6) mice, at a body weight between 18 g and 20 g, were obtained from Charles River Breeding Laboratories and were used in the experiments after housing them for at least 7 days in the animal resource facilities of Cornell University Medical College, New York. Tumor Cells. The MCA-102 tumor cell line was from Steven A. Rosenberg (National Cancer Institute). The MCA-
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Abbreviations: IL-2, interleukin 2; mAb, monoclonal antibody; TNF-a, tumor necrosis factor a. IlTo whom reprint requests should be addressed.
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102 is a nonimmunogenic, natural killer-resistant fibrosarcoma and is syngeneic to the B6 strain (13). The tumor was maintained by serial s.c. passage and was used in experiments within seven transplant generations. To prepare singlecell suspensions, the subcutaneous tumor was excised under sterile conditions, minced, and stirred for 3 hr in a tripleenzyme solution of deoxyribonuclease (type I, 6 mg; Sigma), collagenase (type IV, 60 mg; Sigma), and hyaluronidase (type I-S; 1 mg; Sigma) in 60 ml of Hanks' balanced salt solution (HBSS) without Ca2+ and Mg2+ (GIBCO). The cell suspension was then filtered through a 100-gauge nylon mesh (Nytex; Lawshe Industrial, Bethesda, MD), washed three times in HBSS, and resuspended. Antimurine CD3 mAbs. Armenian hamster splenocyte/ murine myeloma cell line SP/20 hybridoma producing mAb directed at the CD3-e component of the murine T-cell receptor for antigen [anti-CD3 mAbs, 145-2C11 (14)] was from Jeffrey A. Bluestone (Ben May Institute, University of Chicago). The hybridoma cells were grown in protein-free hybridoma medium (PFHM II, GIBCO), and supernatants were collected, pooled, and concentrated through an Amicon filter with a 10-kDa cut-off (Amicon). The purified IgG fraction was obtained by using protein A/G column (Pierce), and the purified IgG was lyophilized and stored at 4°C until use. The proliferative response of mouse splenocytes (determined by [3H]thymidine uptake) and staining of T cells (assessed by flow cytometry) were used to characterize the activity of the anti-CD3 mAbs. Recombinant IL-2. Human recombinant IL-2 was from Hoffmann-La Roche. The specific activity of the cytokine was 1.2 x 107 units per mg of protein. Hepatic Metastasis Model. The experimental murine hepatic metastasis model of Lafreniere and Rosenberg (13) was used with some modifications. In brief, the B6 mice were anesthetized by i.p. injection of pentobarbital, and a left flank incision was made to explore the spleen. The vein draining the upper pole of the spleen was then clamped together with the short gastric veins to avoid leakage of tumor cell inoculum. The veins were cut with a small vessel cauterizer (Fine Science Tools, Belmont, CA) at the distal portion of the clamp. The spleen was rotated 1800, and the MCA-102 cell suspension (245 x 103-280 x 103 cells in 0.7 ml of HBSS) was injected, without any leakage, using a 27-gauge needle into the upper pole of the spleen. After the injection, the puncture site was slightly pressed for 1 min to achieve hemostasis and to allow the tumor cells to be flushed into the portal vein. The splenic pedicle was then clipped with a medium hemostasis clip (Edward Weck and Co., Research Triangle Park, NC), and the spleen was removed. After declamping and achievement of hemostasis, the flank wound was closed with wound clips (9-mm Autoclip, Clay Adams) in one layer. The operative mortality was 300 (3 of 16 mice in the untreated group and none in the anti-CD3 mAb plus IL-2 group), a value of 300 was assigned for the purpose of calculating the mean number of metastases. Treatment of the tumor-bearing mice with IL-2 alone or with anti-CD3 alone also caused a reduction in number and size of hepatic metastases, although to a lesser extent than that found with anti-CD3 mAbs plus IL-2. The mean ± SEM
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FIG. 2. Antitumor efficacy of treatment regimens. Induction of hepatic metastases and treatment protocols were as described in the legend to Fig. 1. The mice were sacrificed on day 14, livers were retrieved, and the number of hepatic metastases was enumerated as described. Treatment with anti-CD3 mAbs plus IL-2 was most efficacious in reducing number and size of metastases. Results from one of three similar experiments are shown.
number of metastases in the IL-2 treatment group was 93 + 32 (number of mice = 15), and in the anti-CD3 treatment group was 68 ± 22 (number of mice = 14). The differences in numbers of metastases among the untreated, anti-CD3 mAbs alone-treated, or IL-2 alone-treated groups, however, were not significantly different by the Wilcoxon-Mann-Whitney U test. Because of the need to assign a value of 300 to the number of metastases when overcrowding occurred, the nonparametric Kruskal-Wallis test was used to examine group differences because the ranks are less impacted by the assignment than the means. The difference in the number of metastases was significant among the four groups at the 0.0265 level. Multiple comparisons of all possible pairs of groups showed a significant difference (P < 0.05) between the untreated group and the group receiving dual therapy. The hypothesis of no difference between any other group pairs, however, could not be rejected. We also determined whether the dosage of anti-CD3 mAbs had a significant impact on the number of hepatic metastases.
A
The following three different anti-CD3 mAbs-dosage regimens were examined for antitumor efficacy: 5 ug on day 3 and 10 pg on day 10 (with respect to intrasplenic inoculation of MCA-102 fibrosarcoma cells), 10 pg on day 3 and 20 pg on day 10, or 20 ug on days 3 and 10. The IL-2 regimen (40,000 units twice per day on days 5-8 and days 12-14 of inoculation) was identical with all three dosages of anti-CD3 mAbs studied. This experimental design, while demonstrating again the superior efficacy of the combined regimen over the anti-CD3 mAbs-alone regimen or the IL-2-alone regimen, did not reveal significant differences among the three different anti-CD3 mAbs dosages investigated. Our observations that anti-CD3 mAbs plus IL-2 results in a smaller number of hepatic metastases compared to treatment with anti-CD3 mAbs alone or IL-2 alone are in accord with the earlier formulation that combination regimens are more efficacious compared to single-agent protocols in reducing tumor burden. In the original study of Lafreniere and Rosenberg (13), whereas IL-2, by itself, failed to reduce the number of MCA-105 hepatic metastases in the B6 mice, a significant reduction was found when the IL-2 therapy was combined with the adoptive transfer of lymphokine-activated killer cells. A combination immunotherapy regimen of antiCD3 mAbs, IL-2, and TNF-a has also been reported as more effective in a murine pulmonary metastases model compared to treatment with IL-2 alone or IL-2 plus TNF-a (29). Kim et al. (30) have also reported that IL-2, over a wide range of concentrations, does not reduce the number of pulmonary metastases unless combined with anti-CD3 mAbs. Our results also complement the observations of others that adoptive transfer of cells treated with anti-CD3 mAbs, as well as IL-2, exhibit greater antitumor efficacy compared to lymphokine-activated killer cells (17, 18). Results from a recent phase IA/B study (31) of anti-CD3 mAbs (OKT3 mAb) in patients with malignancy also support the concept that a regimen of anti-CD3 mAbs alone might not be sufficient in itself to elicit a significant antitumor response. This important study also demonstrated that anti-CD3 mAbs induce the expression of T-cell activation antigen CD69 and that the murine anti-CD3 mAbs elicit the generation of human antimouse antibodies in these patients. Some of the tumors, however, might be sensitive to anti-CD3 mAbs alone. Ellenhorn et al. (32) and Gallinger et al. (33) have reported tumor regression in experimental tumor models by in vivo administration of anti-CD3 mAbs alone.
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FIG. 3. Survival of tumor-bearing mice. MCA-102 fibrosarcoma cells (280 x 103 cells per spleen) were injected on day 0. Treatment schedules were similar to those described in the legend to Fig. 1, except for additional IL-2 treatment on days 12 and 13. Deaths were recorded on a daily basis. The mortality rate (A) was lower, and the mean duration of survival (B) was higher in the anti-CD3 mAbs plus IL-2-treatment group compared with the other groups. n, Number of mice in each experimental group.
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Impact of Anti-CD3 mAbs Plus IL-2 on Survival. The duration of survival of tumor-bearing mice depended upon the treatment regimen and is shown in Fig. 3. The mean + SEM survival time of untreated mice was 21.0 ± 1.0 days [19.3-22.6 days, 95% confidence intervals (CI)], and it increased to 27.9 ± 1.7 days (26.1-29.6 days, 95% CI) after treatment with the anti-CD3 mAbs plus IL-2 regimen (P = 0.001 Wilcoxon-Mann-Whitney U test). Treatment of the tumor-bearing mice with anti-CD3 mAb alone did not increase the survival time (21.8 ± 1.1 days and 19.7-23.8 days, 95% CI) (P = 0.516), and IL-2 treatment had a modest effect (23.5 ± 0.8 days, 21.8-25.2 days, 95% CI) (P = 0.054). Comparison of treatment effects in the four experimental groups (control, anti-CD3 mAb, IL-2, or anti-CD3 mAb plus IL-2) showed that the null hypothesis of equal group means should be rejected (P = 0.001, ANOVA). Multiple-range analysis demonstrated that the mean survival time of antiCD3 mAbs plus IL-2-treated mice was significantly higher (P < 0.05) compared to the mean survival time of the control, anti-CD3 mAbs-alone-treated or IL-2-alone-treated mice. Our findings that a regimen of anti-CD3 mAbs plus IL-2 prolongs survival contrast with an earlier report (29) that the survival time of tumor-bearing mice is prolonged only when the mice are treated with a triple combination of anti-CD3 mAbs plus IL-2 plus TNF-a and not when treated with the regimen of anti-CD3 mAbs plus IL-2. Our experimental design, however, differs from the earlier study (29) with respect to the schedule of administration of anti-CD3 mAbs and IL-2 and also with respect to the tumor model used to explore the efficacy of the treatment regimens. These differences might account for the efficacy of the anti-CD3 mAbs plus IL-2 regimen identified in our investigation. In summary, we find that anti-CD3 mAbs and IL-2 are synergistic in constraining tumor progression. This combination therapy has a significant survival benefit and unleashes a molecular program conducive to suppression of tumor growth. The effective immunostimulatory strategy described here avoids many of the existing limitations of current adoptive cellular immunotherapy protocols and represents an alternative regimen of potential value for the treatment of cancer in humans. We are grateful to Dr. Phyllis August for her careful review and to Ms. Lorraine Goldberg Garcia for her meticulous help in the preparation of this manuscript. This work was supported, in part, by a grant from the Penzance Foundation.
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