Biological activity and in vivo clearance of anti-tumor antibody/cytokine fusion proteins

Abstract

Several human cytokines including IL-2, GM-CSF, and tumor necrosis factors alpha and beta were engineered as fusion proteins to the carboxyl terminus of a chimeric anti-ganglioside antibody, ch14.18, and expressed in transfected hybridoma cells. All of the fusion proteins were expressed at high levels and were easily purified by affinity or ion-exchange chromatography from culture supernatants. The effect of fusion on antigen binding activity was tested and found to vary with the particular cytokine. No significant decreases in antigen binding were observed, and fusion of IL-2 had the greatest positive effect in a direct antigen binding assay. All fusion proteins maintained normal levels of biological activity except for GM-CSF, which was approximately 20% active, compared to recombinant GM-CSF produced in bacteria. The clearance of the fusion proteins was examined in normal Balb/c mice after intraperitoneal injection or in athymic (nu/nu) mice after intravenous injection and was generally quite rapid, relative to ch14.18. This was mainly due to a very rapid initial clearance rate (alpha phase) since the half-lives of the beta phase of the fusion proteins (about 30 h) were comparable to that of the free antibody (about 58 h). These results demonstrate that biologically active antibody/cytokine fusion proteins can be constructed by genetic engineering. Their relatively rapid clearance may require constant infusion rather than bolus injection in order to achieve clinical efficacy.

Antigen specificity can be irrelevant to immunocytokine efficacy and biodistribution

“…Similar instances of cytokine-cytokine receptor-dominated biodistribution for tumor vasculature-targeted IFN-γ immunocytokines have been observed (24,25). Moreover, the markedly faster plasma clearance of immunocytokines relative to their parent antibodies as reported by several groups (26,27) may reflect extensive binding of immunocytokines to IL-2R-expressing cells. Circulating immune cells expressing CD122, which may correspond to the immunocytokine-bound NK cells that we detected, have been linked to IL-2 toxicity (28) but may also contribute to therapeutic efficacy.…”

Human Lymphoid and Myeloid Cell Development in NOD/LtSz-scid IL2Rγnull Mice Engrafted with Mobilized Human Hemopoietic Stem Cells

“…Because T cell development is dependent on epithelially derived human IL-7 (64 -66), we injected a cohort of these mice with an Fc-IL7 fusion protein. Cytokine-Ig fusion proteins demonstrated increased halflife in vivo compared with native cytokines (33,67).…”

Eradication of human hepatic and pulmonary melanoma metastases in SCID mice by antibody-interleukin 2 fusion proteins.

“…Mouse-human chimeric antibodies directed against the EGF receptor (ch225) or GD2 (chl4.18) were constructed by joining the cDNA for the variable region of the murine antibodies with the constant regions of the yl heavy chain and the K light chain as described (19 proteins ch225-IL2 and chl4.18-IL2 were constructed by fusion of a synthetic sequence coding for human IL-2 to the carboxyl end of the human Cyl gene as described (12,14,20 tAll experimental groups were started with six mice for the hepatic and eight mice for the lung metastases models; animals found dead prior to the planned date of sacrifice were not included in the evaluation. §Differences in the metastatic score/number of metastatic foci and organ weights between the fusion protein groups and all control groups were statistically significant (P < 0.002).…”