Research Development Foundation

Xinjian Lin

Stephen B Howell

Xiying Shang

Dennis P Gately

The invention is directed to cell-targeted cytotoxic agents, including sortase serine protease constructs. Methods for targeted cell killing for treatment of proliferative diseases, for example, cancer, are provided. Exemplary embodiments comprise an R-spondin ligand for targeting the cytotoxic agents to effect the cell killing.

Cell-targeted cytotoxic constructs

The present invention is directed generally to cell-targeted cytotoxic constructs comprising a targeting polypeptide, a linking polypeptide and a cytotoxic polypeptide. Preferably, (a) the targeting polypeptide is a R-spondin1 (RSPO1), R-spondin2 (RSPO2) or yoked chorionic gonadotropin (YCG), the linking polypeptide comprises LPXT (SEQ ID NO: 56) or NPXT (SEQ ID NO: 60) as well as others, where X is any amino acid, the linking polypeptide being positioned between the targeting ligand and (c) the cytotoxic moiety is an auristatin or a truncated serine protease, the serine protease having an IIGG (SEQ ID NO: 91), IVGG (SEQ ID NO: 92) or ILGG (SEQ ID NO: 93) at its N-terminus. Such constructs can be used in methods for targeted cell killing such as for treatment cell of proliferative diseases (e.g., cancer).

The present invention features a novel cellular injury reporter system in which a chimeric gene containing the GADD153 promoter linked to the coding region of an enhanced green fluorescent protein (EGFP) gene was stably integrated into the genome of carcinoma cells. Activation of the GADD153 promoter was quantified using flow cytometric measurement of EGFP expression following drug exposure. This reporter system is suitable for high. throughput in vitro and in vivo screening for agents capable of producing cytotoxicity via a wide variety of different mechanisms, and can be utilized to investigate the relative potency of structurally related DNA adducts.

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Xinjian Lin