Irdye 700dx nhs ester

Exendin-4-IRDye700DX was supplied by piCHEM. IRDye700DX NHS ester was obtained from LI-COR Biosciences. IRDye700DX absorbs and emits light in the near-infrared (NIR) range and has a higher extinction coefficient (2.1 × 10⁵ M⁻¹cm⁻¹ at 689 nm) than non-NIR photosensitizers (12 (link),17 (link)). The N-ε-amino group of lysine at position 40 was site-specifically modified during solid-phase peptide synthesis with a mercaptopropionic acid, releasing an unprotected exendin-4 with a free thiol function after triisopropylsilane cleavage. IRDye700DX was modified with a maleimide, and coupling to exendin-4 was performed using a thiol reactive crosslinking approach. The purity was more than 90%. Stock solutions of exendin-4-IRDye700DX were prepared in phosphate-buffered saline (PBS). The structure and amino acid sequence of the tracer are shown in Supplemental Figure 1 (supplemental materials are available at http://jnm.snmjournals.org). The absorbance and emission spectra of exendin-4-IRDye700DX are shown in Supplemental Figure 2.

The antibody used was cetuximab (Merck Biopharma, Tokyo, Japan), a human/mouse chimeric monoclonal antibody of the IgG1 subclass that targets EGFR. Ultra-LEAF™ Purified Human IgG1 Isotype Control Recombinant (BioLegend, San Diego, CA, USA) was used as a control antibody. The photosensitive material used was IRDye 700DX NHS Ester (IR700; C74H96N12Na4O27S6Si3, molecular weight: 1954.22), purchased from LI-COR Bioscience (Lincoln, NE, USA). All reagent-grade chemicals were used in this study. The IRDye^® 700DX Protein Labeling Kit (LI-COR Bioscience, Lincoln, NE, USA) was used to bind IR700 to the antibody according to a previously established method [27 (link),28 (link)]. Briefly, 1 mg (6.6 nmol) of cetuximab was incubated with 53.2 µg (27.2 nmol) of IR700. The mixture was then purified on a Zeba^TM Desalting Spin Column, 7K MWCO (Thermo Fisher Scientific), to produce Cet-IR700. The control antibody was prepared in the same way to produce Con-IR700. The number of fluorophore molecules bound to each antibody molecule was determined using the following method. A spectrophotometer (NanoDrop One; Thermo Fisher Scientific) was used to measure the concentration of protein and IR700 by measuring the absorption at 280 nm and 689 nm. On average, two IR700 molecules were bound to each antibody. Non-reducing SDS-PAGE was used to analyze the resulting conjugated antibodies.

Two different anti-HER2 mAbs, trastuzumab (Herceptin^®) and pertuzumab (Perjeta^®), were purchased from Chugai Pharmaceutical Co. Ltd. (Tokyo, Japan). IRDye700DX NHS ester (IR700) was purchased from LI-COR Biosciences (Lincoln, NE, USA). Alexa Fluor488 NHS ester (Alexa488) was purchased from (Life Technologies, Gaithersburg, MD, USA).

Water soluble, silicon-phthalocyanine derivative, IRDye 700DX NHS ester and IRDye 800 CW NHS ester were obtained from LI-COR Bioscience (Lincoln, NE, USA). Panitumumab, a fully humanized IgG₂ mAb directed against EGFR, was purchased from Amgen (Thousand Oaks, CA, USA). Trastuzumab, 95% humanized IgG₁ mAb directed against HER2, was purchased from Genentech (South San Francisco, CA, USA). All other chemicals were of reagent grade.

IRDye 700DX-NHS ester was purchased from LI-COR Biosciences (Lincoln, Nevada, USA). Panitumumab, a fully humanized IgG2 mAb directed against EGFR, was purchased from Amgen (Thousand Oaks, California, USA). Anti-mouse PD-L1 (B7-H1) antibody (10F.9G2) and rat IgG2b (LTF-2; used as the control) were obtained from Bio X Cell (Lebanon, New Hampshire, USA).