3 research outputs found
Modulating Antibody–Drug Conjugate Payload Metabolism by Conjugation Site and Linker Modification
Previous investigations
on antibody-drug conjugate (ADC) stability
have focused on drug release by linker-deconjugation due to the relatively stable payloads such
as maytansines. Recent development of ADCs has been focused on exploring
technologies to produce homogeneous ADCs and new classes of payloads
to expand the mechanisms of action of the delivered drugs. Certain
new ADC payloads could undergo metabolism in circulation while attached
to antibodies and thus affect ADC stability, pharmacokinetics, and
efficacy and toxicity profiles. Herein, we investigate payload stability
specifically and seek general guidelines to address payload metabolism
and therefore increase the overall ADC stability. Investigation was
performed on various payloads with different functionalities (e.g.,
PNU-159682 analog, tubulysin, cryptophycin, and taxoid) using different
conjugation sites (HC-A118C, LC-K149C, and HC-A140C) on THIOMAB antibodies.
We were able to reduce metabolism and inactivation of a broad range
of payloads of THIOMAB antibody-drug conjugates by employing optimal
conjugation sites (LC-K149C and HC-A140C). Additionally, further payload
stability was achieved by optimizing the linkers. Coupling relatively
stable sites with optimized linkers provided optimal stability and
reduction of payloads metabolism in circulation in vivo
Development of Efficient Chemistry to Generate Site-Specific Disulfide-Linked Protein– and Peptide–Payload Conjugates: Application to THIOMAB Antibody–Drug Conjugates
Conjugation
of small molecule payloads to cysteine residues on
proteins via a disulfide bond represents an attractive strategy to
generate redox-sensitive bioconjugates, which have value as potential
diagnostic reagents or therapeutics. Advancement of such “direct-disulfide”
bioconjugates to the clinic necessitates chemical methods to form
disulfide connections efficiently, without byproducts. The disulfide
connection must also be resistant to premature cleavage by thiols
prior to arrival at the targeted tissue. We show here that commonly
employed methods to generate direct disulfide-linked bioconjugates
are inadequate for addressing these challenges. We describe our efforts
to optimize direct-disulfide conjugation chemistry, focusing on the
generation of conjugates between cytotoxic payloads and cysteine-engineered
antibodies (i.e., THIOMAB antibody–drug conjugates, or TDCs).
This work culminates in the development of novel, high-yielding conjugation
chemistry for creating direct payload disulfide connections to any
of several Cys mutation sites in THIOMAB antibodies or to Cys sites
in other biomolecules (e.g., human serum albumin and cell-penetrating
peptides). We conclude by demonstrating that hindered direct disulfide
TDCs with two methyl groups adjacent to the disulfide, which have
heretofore not been described for any bioconjugate, are more stable
and more efficacious in mouse tumor xenograft studies than less hindered
analogs