Characterization of intact antibody–drug conjugates from plasma/serum in vivo by affinity capture capillary liquid chromatography–mass spectrometry
Section snippets
Biotinylation of target antigen
A NAP-5 column (GE Healthcare, Piscataway, NJ, USA) was equilibrated with 10 ml of sodium bicarbonate buffer (pH 7.8) (Sigma–Aldrich). A 0.5-ml aliquot of the target antigen (e.g., human MUC16 extracellular domain [ECD], Genentech, South San Francisco, CA, USA) at a concentration of approximately 4 mg/ml was loaded onto the column for buffer exchange and then eluted by 1 ml of sodium bicarbonate. Subsequently, the buffer-exchanged MUC16 ECD (∼1 ml) was mixed with approximately 13 μl of 5 mg/ml
Method development of affinity capture LC–MS
Although site-specific conjugation allows for a tightly controlled stoichiometry of DAR 2 in TDCs, it is possible that the covalently bound drugs may be released with time in vivo, resulting in the formation of conjugates carrying fewer drugs (i.e., DAR 1) or no drugs (i.e., DAR 0). Similarly, other conventional ADCs with higher initial DARs may result in lower DARs in vivo. Fig. 1 shows a schematic of intact ADCs species (using the TDC as a model) in plasma or serum analyzed by affinity
Discussion
Release of conjugated cytotoxic drugs from ADCs can be a concern because this process changes the drug load of the ADC and potentially affects the pharmacokinetics, efficacy, and safety. Using the affinity capture LC–MS method, we were able to directly characterize the drug release process that occurred in a model using engineered site-specific anti-MUC16 TDC in plasma and serum both in vitro and in vivo for the first time. Formation of the DAR 0 and DAR 1 species was effectively detected as
Acknowledgments
The authors thank John Lowe, Valerie Quarmby, An Song, Patricia Siguenza, and Paul Fielder for their assistance and support in this research. We also thank John Stults and Valerie Quarmby for their critical review of the manuscript.
References (25)
Drug-conjugated monoclonal antibodies for the treatment of cancer
Curr. Opin. Pharmacol.
(2005)- et al.
cAC10–vcMMAE an anti-CD30–monomethyl auristatin E conjugate with potent and selective antitumor activity
Blood
(2003) - et al.
Rapid identification of reactive cysteine residues for site-specific labeling of antibody–Fabs
J. Immunol. Methods
(2008) - et al.
Antibody-based enrichment of peptides on magnetic beads for mass-spectrometry-based quantification of serum biomarkers
Anal. Biochem.
(2007) - et al.
Mass spectrometric quantitation of C-reactive protein using labeled tryptic peptides
Anal. Biochem.
(2006) - et al.
Antibody–drug conjugates for cancer therapy
Cancer J.
(2008) - et al.
Technology insight: cytotoxic drug immunoconjugates for cancer therapy
Nat. Clin. Pract. Oncol.
(2007) - et al.
In vitro and in vivo activity of the maytansinoid immunoconjugate huN901-N2′-deacetyl-N2′-(3-mercapto-1-oxopropyl)-maytansine against CD56+ multiple myeloma cells
Cancer Res.
(2004) - et al.
Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index
Nat. Biotechnol.
(2008) - et al.
In vivo drug–linker stability of an anti-CD30 dipeptide-linked auristatin immunoconjugate
Clin. Cancer Res.
(2005)