Maytansinoid-BODIPY Conjugates: Application to Microscale Determination of Drug Extinction Coefficients and for Quantification of Maytansinoid Analytes.
Determining drug to antibody ratios (DAR) for antibody-drug conjugates (ADCs) in early research and development can be hampered by difficulties in accurate weighing of the effector payload and subsequent determination of its extinction coefficient. Two maytansinoids, DM1 and DM4, potent antimitotic agents used in clinical ADCs, were derivatized with the compact fluorophore BODIPY FL using two different linker designs. We identified DM1-mal-BODIPY as a conjugate with little through-space interaction between the maytansinoid and BODIPY chromophores. The 1:1 stoichiometry between the maytansinoid and BODIPY makes the molar concentration of both components equal and the extinction coefficient of the maytansinoid in proportion with the known BODIPY chromophore according to Beer's Law. By only derivatizing 50 μg of unpurified DM1 and analyzing about 25 μg of DM1-mal-BODIPY by UV-vis, we determined εDM1 252 nm and εDM1 280 nm as 26 355 ± 360 and 5230 ± 160 cm(-1) M(-1), respectively. These values are nearly identical to those accepted for DM1 based on weighing >100 mg of pure sample. Surprisingly, some of the maytansinoid-BODIPY conjugates that were synthesized were partially or completely fluorescence-quenched. The green fluorescence of quenched DM4-acetamide-BODIPY could be fully restored in the presence of an antibody designed to tightly bind maytansine. We exploited this observation to develop a simple "mix and read" fluorogenic immunoassay for detection of nanogram quantities of maytansinoids.
ImmunoGen, Inc., 830 Winter Street, Waltham, Massachusetts 02451, United States.
Nathan
N
Fishkin
D001896:Boron Compounds / Q000737:chemistry*; D018796:Immunoconjugates / Q000737:chemistry*; D008453:Maytansine / Q000737:chemistry*; D008962:Models, Theoretical
C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001896:Boron Compounds; D018796:Immunoconjugates; D008453:Maytansine
BODIPY FL C1−IA; DM1; DM4; N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-yl)methyl)iodoacetamide (BODIPY FL); N2′-deacetyl-N2′-(3-mercapto-1-oxopropyl)-maytansine; N2′-deacetyl-N2′-(4-mercapto-4-methyl-1-oxopentyl)-maytansine; antibody−drug conjugate (ADC); drug quantification; extinction coefficient; fluorogenic probe; homogeneous immunoassay