Engineered cys-diabody for PSCA targeting

The 2B3 A2 cys-diabody, (cDb, 50 kDa) was developed and validated for preclinical in vivo targeting of PSCA at UCLA (30 (link)). It was derived by yeast affinity maturation of a humanized monoclonal anti-PSCA antibody, 2B3, and engineered to contain a C-terminal free cysteine that forms an inter-chain disulfide bond stabilizing dimerization. Upon mild reduction this disulfide bond can be broken and free thiols are available for site-specific labeling away from the antigen binding site using e.g. maleimide chemistry. A2 cDb was purified from mammalian cell culture supernatant using immobilized metal affinity chromatography. Protein concentrations were determined photometrically and purity was analyzed by SDS-PAGE. Detailed biodistribution data for the A2 cDb was previously determined (21 ). Non-specific binding was not seen. Fluorescent signals were present in liver, kidney and bladder due to the metabolism and urinary excretion of the probe. Cy5 Maleimide (649 nm absorbance, 670 nm emission) was purchased from GE Healthcare (Piscataway, NJ).

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Sonn G.A., Behesnilian A.S., Jiang Z.K., Zettlitz K.A., Lepin E.J., Bentolila L.A., Knowles S.M., Lawrence D., Wu A.M, & Reiter R.E. (2015). Fluorescent Image-Guided Surgery with an Anti-Prostate Stem Cell Antigen (PSCA) Diabody Enables Targeted Resection of Mouse Prostate Cancer Xenografts in Real-Time. Clinical cancer research : an official journal of the American Association for Cancer Research, 22(6), 1403-1412.

Publication 2015

Cy5 Maleimide

Affinity chromatography Antigen Binding site Bladder Cell culture Dimerization Disulfide Free cysteine Humanized monoclonal antibody Kidney Liver Maleimide Mammalian Metabolism Metal Protein Sds page Seen Thiols Urinary Yeast

Corresponding Organization : University of California, Los Angeles

Other organizations : California NanoSystems Institute

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Variable analysis

independent variables

Engineering the 2B3 A2 cys-diabody (cDb) to contain a C-terminal free cysteine that forms an inter-chain disulfide bond stabilizing dimerization

dependent variables

Purification of the A2 cDb from mammalian cell culture supernatant using immobilized metal affinity chromatography
Determination of protein concentrations photometrically
Analysis of purity by SDS-PAGE
Biodistribution data for the A2 cDb

control variables

Mild reduction to break the disulfide bond and make free thiols available for site-specific labeling using maleimide chemistry

positive controls

None specified

negative controls

None specified

Annotations

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