Site-Specific Antibody Conjugation to Engineered Double Cysteine Residues.

Site-specific antibody conjugations generate homogeneous antibody-drug conjugates with high therapeutic index. However, there are limited examples for producing the site-specific conjugates with a drug-to-antibody ratio (DAR) greater than two, especially using engineered cysteines. Based on available Fc structures, we designed and introduced free cysteine residues into various antibody CH2 and CH3 regions to explore and expand this technology. The mutants were generated using site-directed mutagenesis with good yield and properties. Conjugation efficiency and selectivity were screened using PEGylation. The top single cysteine mutants were then selected and combined as double cysteine mutants for expression and further investigation. Thirty-six out of thirty-eight double cysteine mutants display comparable expression with low aggregation similar to the wild-type antibody. PEGylation screening identified seventeen double cysteine mutants with good conjugatability and high selectivity. PEGylation was demonstrated to be a valuable and efficient approach for quickly screening mutants for high selectivity as well as conjugation efficiency. Our work demonstrated the feasibility of generating antibody conjugates with a DAR greater than 3.4 and high site-selectivity using THIOMABTM method. The top single or double cysteine mutants identified can potentially be applied to site-specific antibody conjugation of cytotoxin or other therapeutic agents as a next generation conjugation strategy.

Beta radiation shielding with lead and plastic: effect on bremsstrahlung radiation when switching the shielding order.

Lead and plastic are commonly used to shield beta radiation. Radiation protection literature is ubiquitous in advising the placement of plastic first to absorb all the beta particles before any lead shielding is used. This advice is based on the well established theory that radiative losses (bremsstrahlung production) are more prevalent in higher atomic number (Z) materials than in low Z materials. Using 32P beta radiation, we measured bremsstrahlung photons transmitted through lead and plastic (Lucite) shielding in different test configurations to determine the relative efficacy of lead alone, plastic alone, and the positional order of lead and plastic. With the source (32P) and detector held at a constant separation distance, we inserted lead and/or plastic absorbers and measured the reduction in bremsstrahlung radiation level measured by the detector. With these test conditions, analysis of measured bremsstrahlung radiation in various thicknesses and configurations of lead and plastic shielding shows the following: placing plastic first vs. lead first reduces the transmitted radiation level only marginally (10% to 40%); 2 mm of additional lead is sufficient to correct the "mistake" of placing the lead first; and for equal thicknesses or weights of lead and plastic, lead is a more efficient radiation shield than plastic.