Discovery of a novel ¹⁸F prosthetic group that enables radiolabeling of anti-human PD-L1 Adnectins

Objectives: Recently, there has been a resurgence in the application of protein based scaffolds to create novel molecular imaging agents for numerous oncology targets. These scaffolds offer the affinity and specificity needed to maximize signal to background ratios needed to visualize a molecular target within the tumor microenvironment. Adnectins are a family of proteins that are derived from the 10^th type III domain of human fibronectin (10Fn3), which structurally resemble antibody variable domains, with two sets of antiparallel beta sheets with solvent accessible loops at each pole. These loops can be engineered to provide nano- to picomolar binding affinity to a wide variety of targets. Adnectins have several advantages as targeting domains for molecular imaging agents such as: smaller size ( ~10kDa), which allows for good image contrast with rapid delivery to targeted tissues and fast glomerular clearance of unbound probe to drive image contrast; high stability; and absence of cysteine or disulfide bonds, which allows for site specific engineering to enable conjugation of PET radionuclides. Although many prosthetic groups have been developed for peptide labeling with F-18, these agents are not amenable for labeling protein or protein like molecules in aqueous media. Accordingly, there is still a continuing need to develop prosthetic groups that can effectively radiolabel these scaffolds. The aim of this study focuses on the development of a novel prosthetic group [¹⁸F](3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-2-fluoropyridine to efficiently radiolabel protein based scaffolds such as Adnectins in high yields, specific activities and short reaction times.

Methods: The proposed synthetic pathway starts with the preparation of 3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-2-nitropyridine to form its corresponding [¹⁸F]containing azide moiety. Once labeled with ¹⁸F, this prosthetic group was conjugated to a protein based scaffold using copper free “click” chemistry. This was accomplished using a modified Adnectin that contained a ring constrained (DBCO) alkyne. [¹⁸F](3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-2-fluoropyridine ([¹⁸F]FPPEGA) was isolated in an ethanoic solution, evaporated to dryness and reconstituted in phosphate buffered saline (PBS). Stability and volatilization studies were conducted with this derivative.

Results: [¹⁸F]FPPEGA was generated in high radiochemical yield (>70% non-decay corrected yield, n= 40), in >90% radiochemical purity, was stable in PBS, pH 7.4 solutions over 1 hour and was reduced to dryness without loss of radioactivity. The [¹⁸F]FPPEGA prosthetic group was then used to synthesize both [¹⁸F]ADX_5322_A02 and [¹⁸F]ADX_5417_E01 anti-PD-L1 Adnectins as shown in scheme 1, using copper-free click chemistry. 1.0-1.1 Gbq (27-29 mCi) of these fluorine-18 anti-PD-L1 Adnectins were isolated respectively, starting from 800 mCi of [¹⁸F]fluoride. These derivatives were isolated with radiochemical purities between 96-99% and specific activities between 60-63 GBq/μmol (1621-1700 mCi/μmol). This novel prosthetic group has produced the first ¹⁸F labeled Adnectin and the first ¹⁸F PD-L1 PET radioligand for in vivo imaging PD-L1 expression in tumors and has application to enable ¹⁸F labeling to many antibody fragments.

Conclusion: We have developed a novel prosthetic group that allows for the ¹⁸F labeling of protein based ligands under mild conditions. Using copper free “click” chemistry, we generated ¹⁸F labeled anti-PD-L1 Adnectins with picomolar affinity toward the human PD-L1 receptor with high radiochemical purity and high specific activity. Clinical studies with these same day imaging agents are underway to better understand the heterogeneity and dynamics of PD-L1 expression in human tumors. Research Support: