Chem. SCI.: three functional platform for building modular antibody fragments for 18f-pet or NIRF imaging

Positron emission computed tomography (PET) is a clinical imaging technique for understanding the biology of disease and promoting diagnosis and treatment; NIRF ）Imaging makes it possible to accurately present the edge of tumor in the process of surgical resection In addition, engineering antibody fragments have excellent targeting, rapid metabolic rate and simple chemical modification, and have become a promising molecular imaging agent Does the combination of the three have certain application value? Recently, Professor Jennifer M Murphy of the University of California, Los Angeles, reported a three functional platform, which uses radionuclides or fluorophores to label bivalent antibodies against prostate stem cell antigen (PSCA), pet and NIRF Imaging, used to study the distribution of molecules in prostate cancer mice and tumor targeting ability Relevant achievements were published on Chemical Science (DOI: 10.1039 / c9sc05007h) under the title of "tri functional platform for construction of modular anti debris fragments in vivo 18 f-pet ornirf molecular imaging" Three functional platforms are composed of three key components: (1) maleimide, which binds specifically to Cys bivalent antibody via succinimide thioether bond; (2) tetrazine, which rapidly marks immunoconjugates via [4 + 2] cycloaddition reaction; (3) hydrophilic side chain, which improves the biological distribution of the conjugates and makes them easy to be metabolized (Figure 1) (source: Chemical Science) firstly, the author used n-boc-l-lysine as raw material to synthesize scheme 1 in four steps Pegylated succinimide 1 is combined with n-boc-l-lysine to form maleimide lysine conjugate, which is then coupled with benzylaminotetrazine to obtain intermediate 2 in high yield, and finally 2 after acid-mediated deboc is condensed with dota-nhs ester to obtain the required three functional platform 3 (source: Chemical Science) then, the author constructed the modular antibody fragment Two free mercaptans were produced by the reduction and cleavage of disulfide bond of Cys bivalent antibody After Michael addition of mercapto group and maleimide group, the modular Cys bivalent antibody TFP CDB (Figure 2a) was obtained In order to obtain 18 f-cdb or scy5-cdb for pet or optical imaging, the purified tfp-cdb reacts with [18 F] fluoroethoxy TCO or sulfocy5-cdb respectively at room temperature (Figure 2b) (source: Chemical Science) as mentioned above, the author synthesized [18 F] fluoroethoxy TCO automatically on elixysflex / Chem radiochemical synthesizer, added it to tfp-cdb, and incubated it at room temperature for 10 minutes (FIGRUE 3a) The radiolabeling efficiency and radiochemical purity of 18 f-cdb were 63% and 98% respectively, and the immunoactivity based on A2 Cys bivalent antibody was 63% (Figure 3B) (source: Chemical Science) in order to evaluate the imaging and biological distribution of 18 f-cdb in vivo, the authors injected it into male nude mice via tail vein and performed microPET / CT scanning The imaging results showed that 18 f-cdb was metabolically removed within 2 hours, and then the radioactive metabolites remained in the kidney (Figure 3C) Compared with the previously constructed compound 18 f-dml-a2cdb, the hydrophilic side chain in the three functional platform of 18 f-cdb can retain radioactive metabolites after kidney clearance, while the radioactive metabolites released by 18 f-dml-a2cdb after kidney clearance are removed by the liver and gallbladder, resulting in the presence of radioactive metabolites in the liver and kidney, affecting the contrast in PET / CT imaging (Figure 4 ）。 (source: Chemical Science) in order to evaluate the tumor targeting ability of 18 f-cdb, the authors conducted imaging studies in a mouse model of prostate cancer The authors injected 18 f-cdb intravenously into male nude mice with PSCA positive (right shoulder) and PSCA negative (left shoulder), and PET / CT imaging was performed 2 hours and 4 hours later PET / CT images showed that compared with the control group, the uptake rate of the probe in the tumor site with antigen expression increased by 8 times At the same time, the transverse and coronal images show a clearer tumor visualization effect (Figure 5) (source: Chemical Science) finally, in order to evaluate whether the three functional platform can be used in image-guided surgery, the author added near-infrared fluorescent dye sulfocy5 TCO to tfp-cdb, stirred at room temperature for 10 minutes, and then obtained scy5 labeled Cys bivalent antibody scys-cdb (FIGRUE 6a) by [4 + 2] cycloaddition The mice were injected into the male nude mice with PSCA positive (right shoulder) and PSCA negative (left shoulder), and killed four hours later for fluorescence imaging Although the spontaneous fluorescence of the mouse head affects the imaging effect, the antigen-positive tumor (Figure 6b) can still be seen directly in the figure In vitro organ imaging showed that there was strong spontaneous fluorescence in the stomach and intestine, but less in the liver and spleen The elimination of scy5 CD B by the kidney led to the enhancement of fluorescence signal in the kidney (FIGRUE 6c) In addition, the authors detected strong fluorescence signal in PSCA positive tumors, while in PSCA negative tumors, there was only nonspecific uptake, almost no fluorescence signal (FIGRUE 6D) The above results show that the three functional platform has application value in the operation process (source: Chemical Science) in a word, the author synthesized a three functional platform and constructed a modular antibody fragment for PSCA Pet and NIRF optical imaging confirmed the tumor targeting of the compound and revealed its high contrast in imaging, which laid a certain foundation for the development and application of imaging tracer in clinic.