Abstract 551: Human recombinant anti BSP antibody fused to cell penetrating peptides has improved cytotoxic properties

• Published in: Cancer research (Chicago, Ill.) Ill.), 2019-07, Vol.79 (13_Supplement), p.551-551
• Main Authors: Kovacheva, Marineta, Zepp, Michael, Armbruster, Franz-Paul, Berger, Martin R.
• Format: Article
• Language: English
• ISSN: 0008-5472; 1538-7445
• DOI: 10.1158/1538-7445.AM2019-551

Elevated serum levels of bone sialoprotein (BSP) indicate subsequent bone metastasis and are related to poor survival of respective breast cancer patients. Here we describe the preclinical activity of a human recombinant anti-BSP antibody (AF165) in MDA-MB-231 human breast cancer cells in vitro and in vivo. In addition, this antibody was fused to cell penetrating peptides (CPPs) and the resulting fusion proteins were examined for their cellular uptake and antiproliferative activities. AF165 was produced in HEK cells and tested for its affinity against BSP by microscale thermophoresis. Its antiproliferative activity was evaluated by MTT assay, the antimetastatic efficacy was determined in soft tissue and skeletal lesions, which had been induced in nude rats by intra-arterial injection of human MDA-MB-231 breast cancer cells. Fusion of AF165 to CPPs (Pep1, TAT) was accomplished by adding the respective DNA sequences with or without a linker (glycine-serine (GS)) to the AF165 gene. The affinity of these fusion proteins to BSP was measured by microscale thermophoresis, as well. In addition, their intracellular uptake and antiproliferative activities were determined by confocal fluorescent microscopy and MTT assay. Finally, changes in protein expression in response to these fusion proteins were detected by western blot. AF165 showed no uptake into MDA-MB-231 cells and inhibited their growth in cell culture only marginally. However, this antibody induced dose dependent remissions in nude rats harboring MDA-MB-231 induced soft tissue and skeletal lesions. Subcutaneous administration of AF165 caused complete remission in 2 of 9 animals (10mg/kg/week), as well as in 3 of 6 animals (20 mg/kg/week). Interestingly, the affinity of AF165 (Kd = 7.7×10-7 M) was lower than that of fusion proteins, Kd values of which ranged from 3.6×10-8M (AF165-multiGS-Pep1) to 2.2×10-9M (AF165-multiGS-TAT). In contrast to AF165, all fusion proteins were taken up into MDA-MB-231 cells and after 24-48h caused morphologic changes, indicative of apoptosis. MDA-MB-231 cell proliferation was inhibited by 50 % after 48 hours of exposure to the conjugates AF-GS-TAT and AF-multiGS-Pep1 (600nM). The other conjugates caused similar cytotoxicity at higher concentration only (1200nM). The exposure to the fusion proteins led to downregulation of BSP protein levels and upregulation of proteins EGR1, ATF3 and ID2. In conclusion, the human recombinant anti-BSP antibody AF165 is inactive in vitro, but hig