Cytokine targeting to
tumor-associated
antigens via antibody
cytokine fusion
proteins has demonstrated potent antitumor activity in numerous animal models and has led to the clinical development of 2 antibody-interleukin-2 (IL-2) fusion
proteins. We previously reported on the construction and in vitro properties of a "dual"
cytokine fusion
protein for simultaneous targeted delivery of human
granulocyte macrophage-colony stimulating factor (
GM-CSF) and
IL-2 to human
tumors. The fusion
protein is based on a heterodimerized core structure formed by human CH1 and Ckappa domains (heterominibody) with C-terminally fused human
cytokines and N-terminally fused single-chain
antibody fragments specific for the
tumor-associated
surface antigen epithelial cell adhesion molecule (
Ep-CAM). For testing the antitumor activity in syngeneic mouse xenograft models, we developed "dual
cytokine heterominibodies" with murine
cytokines (mDCH). mDCH fusion
proteins and, as controls, "single
cytokine heterominibodies" (SCH) carrying either murine
GM-CSF (mGM-CSF) or murine
IL-2 (mIL-2) were constructed, of which all retained the specific activities of
cytokines and binding to the
Ep-CAM antigen on human
Ep-CAM transfected mouse colon
carcinoma CT26-KSA cells. Over a 5-day treatment course, DCH fusion
proteins induced significant inhibition of established pulmonary CT26-KSA
metastases in immune-competent Balb/c mice at low daily doses of 1 mug of fusion
protein per mouse. However, with the tested dosing schemes, antitumor activity of mDCH was largely independent of
cytokine targeting to
tumors as demonstrated by a control
protein with mutated
Ep-CAM binding sites. Single
cytokine fusion
proteins mSCH-
GM-CSF and mSCH-IL-2 showed similar antitumor activity as the dual
cytokine fusion
protein mDCH, indicating that
GM-CSF and
IL-2 in one molecule did not significantly synergize in
tumor rejection under our experimental conditions. Our results seem to contradict the notion that
IL-2 and
GM-CSF can synergize in antitumor activity and that with conventional dose regimens, their specific targeting to
tumors, as tested here with 2
antibodies of different affinities, enhances their antitumor activity.