Transforming 2-5A-PSMA ligand RBI1033 into high affinity PSMA targeting polypeptide ligands for NIR imaging

Objectives The purpose of the study is to create high affinity PSMA targeting tracers. We have developed RBI1033 which exhibits ten times better affinity toward PSMA than ZJ24. We hypothesize that the nucleic acid component of RBI1033 can be substituted with polypeptide side-chains of similar electrostatic properties without affecting PSMA affinity. Replacing the nucleic acid backbone with a polypeptide would significantly reduce the cost of synthesis.

Methods We replaced the adenine in RBI1033 with tyrosine and the phosphate backbone with glutamate residues without altering the rest of structure. At the distal end of the molecule, away from the binding site, we placed a lysine residue for labeling. The new design was synthesized using standard Fmoc based solid phase peptide synthesis. DyLight488 was then conjugated to the molecule through lysine. Flow cytometry of the Dylight488 conjugate with PC3-PIP (PSMA-expressing) and PC3-FLU (non-PSMA expressing) cells was performed to examine the cellular binding specificity of the conjugate. We then conjugated IRDye800cw to the molecule and performed in vivo NIR imaging using SCID mice bearing both PC3-PIP and PC3-FLU tumors.

Results Both the tyrosine based and glutamate based peptide ligands demonstrated high affinity toward PSMA with IC50 of 2.3nM and 2.5nM respectively, while the IC50 of ZJ24 in the same experiment was 10.5nM. The DyLight488 conjugates of the glutamate based peptide ligands successfully distinguished the PSMA expressing population of PC3-PIP cells from non-PSMA expressing PC3-FLU cells. Furthermore, the NIR fluorescent IRDye800cw conjugate demonstrated selective tumor uptake in PSMA-expressing PC3 tumors, but not in non-PSMA expressing PC3-FLU tumors.

Conclusions High affinity NIR fluorescent PSMA ligands can be designed by replacing the 2-5A moiety in RBI1033 with either negatively charged amino acid residues, such as glutamate, or polar aromatic residues, such as tyrosine.

Research Support CCF RPC 2008-1027, DoD PC09416