Cancer Immune Therapy Edited by G. Stuhler and P. Walden ...

336 16 Immunocytokines: Versatile Molecules for Biotherapy of Malignant Disease
mor rejection in only 25% of experimental animals. An important control experiment
combining the same amount of ch14.18±IL-2 immunocytokine used for boosting
with the empty ubiquitin vector showed no antitumor effect, and was equal to
that of controls receiving PBS and a s.c. tumor cell challenge. Significantly, we also
found that only one vaccination coupled with the immunocytokine boost was equally
as effective as three such vaccinations and ch14.18±IL-2 boosts [80].
The immunological mechanisms involved in these antitumor effects were indicated
by experiments in CD8 KO and CD4 KO mice demonstrating that CD8 + T cells were
essential together with CD4 + T cell help. However, the latter was shown by in vivo immunodepletion
experiments to be required for tumor cell killing only in the effector
phase of the immune response. The involvement of additional immunological mechanisms
was suggested by a decisively increased secretion of tumor necrosis factor
(TNF)-a and IFN-g from CD4 + and CD8 + T cells, demonstrated by intracellular cytokine
staining experiments. FACS analyses indicated a markedly up-regulated expression
on CD8 + T cells of the high affinity IL-2-receptor a chain (CD25), co-stimulatory
molecule CD28 and adhesion molecule LFA-2 (CD2). These FACS analyses of splenocytes
from successfully immunized mice also showed that the combination therapy
induced increased expression of co-stimulatory molecules B7±1 and CD48 on
murine APCs [80].
The data indicating CD4 + T cell help to be required for optimal efficacy of the DNA
vaccine are not entirely surprising; however, the finding indicating that this help is
needed only in the effector phase but not required for CD8 + T cell priming in the immunization
phase was new and extended our previous observations. Thus, we previously
found that eradication of hepatic and pulmonary metastases of murine melanoma
achieved by multiple injection of ch14.18±IL-2 immunocytokine was impaired
in part by in vivo depletion of CD4 + T cells [12]. Subsequent findings indicated that
tumor-protective immunity induced by this immunocytokine required CD4 + T cell
help which was mediated by CD40/CD40L interaction without requiring production
of endogenous IL-2 by T cells [65]. The requirement of CD4 + T cell help for adaptive
immunity is well established and was initially described for the B cell compartment.
Subsequent reports also indicated the need for CD4 + T cell help in the induction of
CD8 + T cell-mediated immune responses [80]. The fact that our DNA vaccine
doubled the number of Th1 CD4 + T cells expressing IFN-g and that this number was
increased 3-fold by ch14.18±IL-2 boosts underlined the need for CD4 + T cell help for
optimal tumor-protective immunity. Taken together, our data in this prophylactic
melanoma model clearly indicated that a minigene-based DNA vaccine plus
ch14.18±IL-2 immunocytokine boosts were highly effective in inducing tumor-protective
immunity against murine melanoma.