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Homogeneous antibody–drug conjugates (ADCs) exhibit significantly improved pharmacological properties compared to their heterogeneous counterparts. Site-specific conjugation of the payload to the IgG required for homogeneity can be achieved using enzymes. One example is microbial transglutaminase (MTGase), which can selectively perform transamidation on the Q295 residue of human Fc when N297 glycans are removed. As a result, two modifications can be introduced per IgG molecule; however, achieving higher drug-to-antibody ratios (DARs) requires the use of branched linkers. While several such linkers have been reported, little information is available on the relationship between linker structure and ADC properties. To address this gap, we synthesized two branched amino triazide linkers, differing by a PEG4 fragment inserted after the branching point, which were used to prepare two homogeneous trastuzumab-based DAR 6 ADCs (a "short" and a "long" one). This was achieved by a two-step process consisting of enzymatic linker conjugation followed by bioorthogonal coupling with a cleavable linker bearing monomethyl auristatin E (MMAE). Two other trastuzumab–MMAE conjugates were used as controls: a heterogeneous DAR 6 ADC, made using conventional thiol–maleimide chemistry, and a homogeneous DAR 2 ADC. We found that, while the four conjugates had identical affinity for HER2, their cytotoxicity differed significantly: the "long" homogeneous DAR 6 ADC was just as active as its heterogeneous counterpart, but the "short" DAR 6 ADC was an order of magnitude less potent, inferior even to the DAR 2 conjugate. Our findings indicate that the length of the branched linker critically affects the cytotoxic activity of ADCs, possibly due to steric hindrance influencing the rate of linker cleavage by lysosomal enzymes. [ABSTRACT FROM AUTHOR]

Sulfonated indocyanines 3 and 5 (sCy3, sCy5) are widely used to label biomolecules. Their high molar absorption coefficients and lack of spectral overlap with biopolymers make them ideal as linker components for rapid assessment of bioconjugate stoichiometry. We recently found that the determination of the sCy3:sCy5 molar ratio in a conjugate from its optical absorption spectrum is not straightforward, as the sCy3:sCy5 absorbance ratio at the maxima tends to be larger than expected. In this work, we have investigated this phenomenon in detail by studying the spectral properties of a series of sCy3-sCy5 conjugates in which the dyes are separated by linkers of various lengths, including DNA duplexes. It was found that when sCy3 and sCy5 are located in close proximity, they consistently exhibit an "abnormal" absorbance ratio. However, when the two dyes are separated by long rigid DNA-based spacers, the absorbance ratio becomes consistent with their individual molar absorption coefficients. This phenomenon should be taken into account when assessing the molar ratio of the dyes by UV-Vis spectroscopy. [ABSTRACT FROM AUTHOR]

Between 2 and 4 h after miction the morphology of urinary erythrocytes has changed, clouding the distinction between glomerular and non-glomerular bleeding in pediatric patients. Glomerular bleeding is characterized by microscopically visible alteration in urinary erythrocytes due to glomerular disease. Fixation by Cellfix, a formaldehyde-based fixative, allows the preservation of the morphology for at least 24 h and can be recommended for clinical practice. In our experience, thiomersal was not effective for preservation of the morphology of erythrocytes. [ABSTRACT FROM AUTHOR]