News and Publications

Cancer Immunotherapy with Cytokine Fusion Proteins

R. Reisfeld, H. Lode, R. Xiang, T. Dreier, S.D. Gillies*

* Lexigen Pharmaceuticals Corp., Lexington, MA

Immunocytokines, fusion proteins consisting of a recombinant antibody and a cytokine, can reach a sufficient concentration in the tumor microenvironment to effectively stimulate a cellular immune response to malignant neoplasms. An immunocytokine produced by fusing recombinant human IL-2 with the humanized monoclonal antibody KS1/4 directed against the human cell adhesion molecule KSA is an effective tumor vaccine in a syngeneic metastasis model of murine colon carcinoma. All mice treated with the immunocytokine were cured of established hepatic and pulmonary metastases, and 50% were completely protected against tumor cell challenge for up to 6 months after treatment. This condition was due to an immunocytokine-induced immune response mediated by CD8⁺ T cells that remained effective when CD8⁺ T cells from immune animals were adoptively transferred to naive, syngeneic mice that had severe combined immunodeficiency. In addition, injections of noncurative doses of the immunocytokine increased the long-lived protective immunity to optimal levels in all mice that received cells from immune animals.

These findings suggest a more effective antigen-induced reactivation of memory T cells and differentiation to CD8⁺ T lymphocytes in the presence of IL-2. The involvement of T memory cells was suggested by a phenotype of CD44^hi, Ly-6C, CD45RB^lo, and L-selectin (CD62L)^lo in a subset of CD8⁺ T cells isolated from the spleens of mice with documented protective immunity to tumor cell challenge. These memory CD8⁺ T cells were activated by antigens provided by the tumor cell challenge as indicated by the detection of IFN- and TNF- only in lymphocytes from lymph nodes of mice successfully boosted with the IL-2 immunocytokine to reject the tumor cell challenge. These secondary lymphoid tissues contained tumor-specific CD8⁺ T cells; cells from the tissues specifically lysed colon carcinoma target cells. Taken together, these data suggest that repeated treatment with IL-2 immunocytokines might be effective for the treatment of human colon carcinoma in patients with minimal residual disease.

Another immunocytokine consisting of (1) recombinant human IL-2 and (2) the human-mouse chimeric monoclonal antibody 14.18 to ganglioside GD₂ completely suppressed the growth of experimental and spontaneous metastases of murine neuroblastoma to bone marrow and liver in syngeneic mice. This antitumor effect is mediated by natural killer cells. Thus, a deficiency in natural killer cells, but not T cells, in respective immunodeficient or immunodepleted mice abrogated the antitumor effect of the immunocytokine. The antitumor effect could be fully restored, however, by treatment with agents, such as mouse IFN-, that stimulate natural killer cells or by reconstitution with natural killer cells.

Because our neuroblastoma model is pathophysiologically similar to neuroblastoma in humans, we attempted to induce a T-cell--mediated immune response to this poorly immunogenic tumor and then a long-lived tumor-protective immunity. To this end, NXS2 murine neuroblastoma cells, which form liver and bone marrow metastases in syngeneic A/J mice, were transduced with a gene encoding murine IL-12 that was monomerized by introduction of a flexible protein linker between the p35 and the p40 protein chains of the cytokine. Subcutaneous vaccination of A/J mice with NXS2 cells that produce IL-12 induced a protective immunity in 2 separate experimental settings. First, when mice were challenged with wild-type NXS2 tumor cells 7 days after vaccination, no liver and bone marrow metastases developed. The absence of bone marrow metastases was determined by using an assay that can detect 1 tumor cell in 100,000 naive bone marrow cells. Second, vaccination of A/J mice that had experimentally established metastases to liver and bone marrow completely eradicated liver metastases and caused a significant reduction in the number of bone marrow metastases.

The local and systemic immune responses to the altered tumor cells are largely dependent on CD8⁺ T cells. In immunocompetent A/J mice, in vivo depletion of CD4⁺ and CD8⁺ T cells prevented the immune responses, and in vitro, specific MHC class I--restricted CD8⁺ T cells mediated killing of both ganglioside GD₂⁺ NXS2 cells and the parental, ganglioside GD₂^- C1300 neuroblastoma cells. These results indicate that CD8⁺ T cells recognize an antigen shared by these 2 related cell lines. Taken together, these data suggest that IL-12 gene therapy might overcome the poor immunogenicity of neuroblastoma cells and lead to further improvement in adjuvant treatment of patients with neuroblastoma who have minimal residual disease.

PUBLICATIONS

Albertini, M.R., Hank, J.A., Schiller, J.H., Khorsand, M., Borchart, A.A., Gan, J., Bechhofer, R., Storer, B., Reisfeld, R.A., Sondel, P.M. A phase IB trial of chimeric anti-GD₂ antibody plus interleukin 2 for melanoma patients. Clin. Cancer Res. 3:1277, 1997.

Cirulli, V., Crisa, L., Beattie, G.M., Malley, M.I., Lopez, A.D., Fannon, A., Ptasznik, A., Inverardi, L., Ricordi, C., Deerinck, T., Ellisman, M., Reisfeld, R.A., Hayek, A. KSA antigen Ep-CAM mediates cell-cell adhesion of pancreatic epithelial cells: Morphoregulatory roles in pancreatic islet development. J. Cell Biol. 140:1519, 1998.

Deryugina, E.I., Luo, G.-X., Reisfeld, R.A., Bourdon, M.A., Strongin, A. Tumor cell invasion through matrigel is regulated by activated matrix metalloproteinase-2. Anticancer Res., in press.

Dreier, T., Lode, H.N., Xiang, R., Dolman, C.S., Reisfeld, R.A., Kang, A.S. Recombinant immunocytokines targeting the mouse transferrin receptor: Construction and biological activities. Bioconjug. Chem., in press.

Foon, K.A., Sen, G., Hutchins, L., Kashala, O.L., Baral, R., Banerjee, M., Chakraborty, M., Garrison, J., Reisfeld, R.A., Bhattacharya-Chatterjee, M. Antibody responses in melanoma patients immunized with an anti-idiotype antibody mimicking disialoganglioside GD₂. Clin. Cancer Res., in press.

Gillies, S.D., Lan, Y., Weslowski, J.S., Qian, X., Reisfeld, R.A., Holden, S., Super, M., Lo, K.-M. Antibody-IL12 fusion proteins are effective in SCID mouse models of prostate and colon carcinoma metastases. J. Immunol., in press.

Lode, H.N., Dreier, T., Xiang, R., Varki, N.M., Kang, A.S., Reisfeld, R.A. Gene therapy with a single chain interleukin-12 fusion protein induces T cell-dependent protective immunity in a syngeneic model of murine neuroblastoma. Proc. Natl. Acad. Sci. U.S.A. 95:2475, 1998.

Lode, H.N., Reisfeld, R.A. Immunocytokines for cancer immunotherapy. In: Biomedical Progress. Mammen, E.F. (Ed.). Die Medizinische Verlagsgesellschaft mbH, Marburg, Germany, in press.

Lode, H.N., Xiang, R., Becker, J.C., Gillies, S.D., Reisfeld, R.A. Immunocytokines: A promising approach to cancer immunotherapy. Pharmacol. Ther., in press.

Lode, H.N., Xiang, R., Dreier, T., Varki, N.M., Gillies, S.D., Reisfeld, R.A. Natural killer cell-mediated eradication of neuroblastoma metastases to bone marrow by targeted interleukin-2 therapy. Blood 91:1706, 1998.

Lode, H.N., Xiang, R., Varki, N., Dolman, C.S., Gillies, S.D., Reisfeld, R.A. Targeted interleukin 2 therapy of spontaneous neuroblastoma metastases to bone marrow. J. Natl. Cancer Inst. 89:1586, 1997.

Reisfeld, R.A., Becker, J.C., Gillies, S.D. Immunocytokines: A new approach to immunotherapy of melanoma. Melanoma Res. 7:S99, 1997.

Sen, G., Chakraborty, M., Foon, K.A., Reisfeld, R.A., Bhattacharya-Chatterjee, M. Induction of IgG antibodies by an anti-idiotype antibody mimicking disialoganglioside GD₂. J. Immunother., in press.

Sen, G., Chakraborty, M., Foon, K.A., Reisfeld, R.A., Bhattacharya-Chatterjee, M. Preclinical evaluation in nonhuman primates of murine monoclonal anti-idiotype antibody that mimicks the disialoganglioside GD₂. Clin. Cancer Res. 3:1969, 1997.

Straten, P., Guldberg, P., Seremet, T., Reisfeld, R.A., Zeuthen, J., Becker, J.C. Activation of preexisting T-cell clones by targeted interleukin-2 therapy. Proc. Natl. Acad. Sci. U.S.A., in press.

Xiang, R., Lode, H.N., Dolman, C.S., Dreier, T., Varki, N.M., Qian, X., Lo, K.M., Lan, Y., Super, M., Gillies, S.D., Reisfeld, R.A. Elimination of established murine colon carcinoma metastases by antibody-interleukin 2 fusion protein therapy. Cancer Res. 57:4948, 1997.

Yu, A.L., Uttenreuther-Fischer, M.M., Huang, C.-S., Tsui, C.C., Gillies, S.D., Reisfeld, R.A., Kung, F.H. A phase I trial of a human-mouse chimeric anti-disialoganglioside (GD₂) monoclonal antibody ch14.18 in patients with refractory neuroblastoma and osteosarcoma. J. Clin. Oncol., in press.