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

16.2 Treatment of Tumor Metastases with Immunocytokines
one CT26-KSA tumor cell in 10 6 hepatocytes. Using this detection system, only liver
specimens of mice that received the immunocytokine therapy revealed a complete absence
of a KSA signal, indicating no detectable micrometastasis at this high sensitivity
[12]. Additional evidence for the absence of metastatic disease was obtained by prolonged
survival analyses. Thus, mice that received successful immunocytokine therapy
that eradicated hepatic colon carcinoma metastasis did reveal more than a tripling
in lifespan (over 120 days) as compared to control groups that either received an
equivalent mixture of antibody and cytokine or the non-specific fusion protein. In
fact, all control mice died prior to day 40 after tumor cell inoculation [12].
To delineate the immune mechanism involved in the eradication of established colon
carcinoma metastases, pulmonary metastases were induced by intravenous (i.v.) injection
of CT26-KSA cells, in order to keep the spleen in situ for further in vitro analyses.
The ability of the huKS1/4±IL-2 immunocytokine to also eradicate established
pulmonary metastases was demonstrated in contrast to equivalent mixtures of antibody
and cytokine. Thus, mice with pulmonary metastases receiving the immunocytokine
survived more than 6 months after tumor induction, which is defined as
ªcureº by Kaplan±Meier standards. When such mice were sacrificed and their lungs
analyzed by RT-PCR for expression of the KS antigen none of them revealed its presence,
thus clearly indicating a persistent absence of colon carcinoma metastasis [12].
The first line of evidence for T cell-mediated immunity in the colorectal carcinoma
model was established by using immune-deficient BALB/c scid/scid and BALB/c
beige/beige mice. Treatment of established pulmonary metastases with the huKS1/4±
IL-2 immunocytokine in NK cell-deficient beige/beige mice proved completely effective.
In contrast, in T cell-deficient scid/scid mice, this therapeutic effect was abrogated.
These results were further supported by in vivo depletion of NK cells or CD4 +
and/or CD8 + T cells in immunocompetent BALB/c mice. The absence of NK cells
again had no impact on the therapeutic efficacy of the immunocytokine. This finding
was in contrast to those obtained in mice that were depleted of CD8 + or both
CD8 + and CD4 + T cells which presented with extensive metastases following immunocytokine
therapy. Interestingly, depletion of CD4 + T cells only partially reduced the
therapeutic efficacy of the immunocytokine, because three of five mice thus depleted
revealed macroscopic metastases following treatment with immunocytokine. This
phenomenon possibly indicates a helper function for the CD4 + T cell subpopulation.
However, CD8 + T cells clearly are the major effector cells in this system as demonstrated
by these in vivo experiments [11].
A second line of evidence for Tcell-mediated immune mechanisms was provided by
in vitro analyses of splenic effector cells 3 days after completion of treatment. In this
case, both CT26-KSA and CT26 wild-type cells were used as targets in cytotoxicity assays.
Importantly, both target cells were killed to a similar extent by freshly isolated
splenocytes of immunocytokine-treated animals. This clearly indicated that the cytotoxic
response observed in these assays is quite independent of the presence or absence
of KSA which only serves as a docking site to direct the immunocytokine into
the tumor microenvironment. Subsequent purification of CD8 + and CD4 + T cells revealed
a cytotoxic activity only in the CD8 + T cell population. This response was demonstrated
to be MHC class I antigen-restricted since only addition of anti-MHC
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