A Novel Antibody–IL15 Fusion Protein Selectively Localizes to Tumors, Synergizes with TNF-based Immunocytokine, and Inhibits Metastasis

Abstract

IL15 is an immunostimulatory cytokine that holds promises for cancer therapy, but its performance (alone or as partner for fusion proteins) has often been limited by suboptimal accumulation in the tumor and very rapid clearance from circulation. Most recently, the Sushi Domain (SD, the shortest region of IL15 receptor α, capable of binding to IL15) has been fused to IL15-based anticancer products to increase its biological activity. Here, we describe two novel antibody fusion proteins (termed F8-F8-IL15 and F8-F8-SD-IL15), specific to the alternatively spliced EDA domain of fibronectin (a marker of tumor neoangiogenisis, expressed in the majority of solid and hematologic tumors, but absent in normal healthy tissues) and featuring the F8 antibody in single-chain diabody format (with a short linker between VH and VL, thus allowing the domains to pair with the complementary ones of another chain). Unlike previously described fusions of the F8 antibody with human IL15, F8-F8-IL15 and F8-F8-SD-IL15 exhibited a preferential uptake in solid tumors, as evidenced by quantitative biodistribution analysis with radioiodinated protein preparations. Both products were potently active in vivo against mouse metastatic colon carcinomas and in sarcoma lesion in combination with targeted TNF. The results may be of clinical significance, as F8-F8-IL15 and F8-F8-SD-IL15 are fully human proteins, which recognize the cognate tumor-associated antigen with identical affinity in mouse and man.

Design and Characterization of Novel Antibody-Cytokine Fusion Proteins Based on Interleukin-21

“…F8(scDb)-murine IL21 was used to conduct experiments in mouse models (Figure 2A). The findings are in line with previously described fusion protein based on the scDb format [22,25,26].…”

An Engineered IFNγ-Antibody Fusion Protein with Improved Tumor-Homing Properties

“…The sequence containing the light chain of the L19 antibody was PCR assembled and cloned into the same plasmid between SpeI/BsiWI restriction sites. The two fusion proteins were expressed by transient gene expression (TGE) in CHO-S cells and purified as described [ 39 ].…”

Isolation of a Natural Killer Group 2D Small‐Molecule Ligand from DNA‐Encoded Chemical Libraries

“…The sequence of the amplicon was reported in Supporting Information. The recombinant hNKG2D protein was expressed using transient gene expression in Chinese Hamster Ovary (CHO) cells, using a previously described procedure [32] . The protein was purified from the culture supernatant with Ni‐NTA beads (Roche) and dialyzed in PBS pH 7.4 buffer.…”

“…The recombinant hNKG2D protein was expressed using transient gene expression in Chinese Hamster Ovary (CHO) cells, using a previously described procedure. [32] The protein was purified from the culture supernatant with Ni-NTA beads (Roche) and dialyzed in PBS pH 7.4 buffer. The purified protein was analysed by SDS-PAGE under reducing and non-reducing conditions and by size-exclusion chromatography using a Superdex 75 increase 10/300 GL (ÄKTA FPLC (GE Healthcare Amersham Biosciences).…”