Evaluation of COC183B2 antibody targeting ovarian cancer by near-infrared fluorescence imaging

Objective: To evaluate the imaging potential of a novel near-infrared(NIR) probe conjugated to COC183 B2 monoclonal antibodies(MAb) in ovarian cancer(OC).Methods: The expression of OC183 B2 antigen in OC was determined by immunohistochemical(IHC) staining using tissue microarrays with the H-score system and immunofluorescence(IF) staining of tumor cell lines.Imaging probes with the NIR fluorescent dye cyanine 7(Cy7) conjugated to COC183 B2 Mab were chemically engineered. OC183 B2-positive human OC cells(SKOV3-Luc) were injected subcutaneously into BALB/c nude mice. Bioluminescent imaging(BLI) was performed to detect tumor location and growth. COC183 B2-Cy7 at 1.1,3.3, 10, or 30 μg were used for in vivo fluorescence imaging, and phosphate-buffered saline(PBS), free Cy7 dye and mouse isotype immunoglobulin G(IgG)-Cy7(delivered at the same doses as COC183 B2-Cy7) were used as controls.Results: The expression of OC183 B2 with a high H-score was more prevalent in OC tissue than fallopian tube(FT) tissue. Among 417 OC patients, the expression of OC183 B2 was significantly correlated with the histological subtype, histological grade, residual tumor size, relapse state and survival status. IF staining demonstrated that COC183 B2 specifically expressed in SKOV3 cells but not HeLa cells. In vivo NIR fluorescence imaging indicated that COC183 B2-Cy7 was mainly distributed in the xenograft and liver with optimal tumor-to-background(T/B)ratios in the xenograft at 30 μg dose. The highest fluorescent signals in the tumor were observed at 96 h postinjection(hpi). Ex vivo fluorescence imaging revealed the fluorescent signals mainly from the tumor and liver. IHC analysis confirmed that xenografts were OC183 B2 positive.Conclusions: COC183 B2 is a good candidate for NIR fluorescence imaging and imaging-guided surgery in OC.

Genetic Encoding of a Photocaged Glutamate for Optical Control of Protein Functions

Genetic encoding of photocaged noncanonical amino acids provides a powerful tool to study protein functions through optical control but is not yet available for acidic amino acids.Herein,we report the first site-specific genetic encoding of a photocaged glutamate,4-methoxy-7-nitroindolinyl caged glutamate(MNI-Glu),into recombinant proteins via an expanded genetic code through evolved EcLeuRS/tRNA pair.Using two enzymes as examples,we demonstrate that substituting the conserved-active-site glutamate of a secreted alkaline phosphatase and a protease HRV3C to MNI-Glu allows photoregulatory control of their enzymatic activities.Our approach is an important addition to the photocaged noncanonical amino-acid toolbox and provides a general method to photocontrol protein activity based on caging a critical glutamate.

Site-specific protein modification by genetic encoded disulfide compatible thiols

Cysteine chemistry provides a low cost and convenient way for site-specific protein modification.However,recombinant expression of disulfide bonding containing protein with unpaired cysteine is technically challenging and the resulting protein often suffers from significantly reduced yield and activity.Here we used genetic code expansion technique to introduce a surface exposed self-paired dithiol functional group into proteins,which can be selectively reduced to afford active thiols.Two compounds containing self-paired disulfides were synthesized,and their genetic incorporations were validated using green fluorescent proteins(GFP).The compatibility of these self-paired di-thiols with natural disulfide bond was demonstrated using antibody fragment to afford site-specifically labeled antibody.This work provides another valuable building block into the chemical tool-box for site-specific labeling of proteins containing internal disulfides.