Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index

Abstract

Antibody-drug conjugates enhance the antitumor effects of antibodies and reduce adverse systemic effects of potent cytotoxic drugs. However, conventional drug conjugation strategies yield heterogenous conjugates with relatively narrow therapeutic index (maximum tolerated dose/curative dose). Using leads from our previously described phage display-based method to predict suitable conjugation sites, we engineered cysteine substitutions at positions on light and heavy chains that provide reactive thiol groups and do not perturb immunoglobulin folding and assembly, or alter antigen binding. When conjugated to monomethyl auristatin E, an antibody against the ovarian cancer antigen MUC16 is as efficacious as a conventional conjugate in mouse xenograft models. Moreover, it is tolerated at higher doses in rats and cynomolgus monkeys than the same conjugate prepared by conventional approaches. The favorable in vivo properties of the near-homogenous composition of this conjugate suggest that our strategy offers a general approach to retaining the antitumor efficacy of antibody-drug conjugates, while minimizing their systemic toxicity.

Site-specific conjugation allows modulation of click reaction stoichiometry for pretargeted SPECT imaging

“…52 A dendrimer scaffold bearing 8 TCO groups was recently reported; however, it is unclear whether improvements in tumor signal relative to the 2-TCO control were due to differences in stoichiometry, PEG length, presence/absence of dendrimer, or a combination thereof. 53 To allow a more controlled analysis of how stoichiometry affects pretargeted imaging, we used THIOMAB™ antibody conjugation 46 to install TCO moieties via maleimide chemistry site-specifically at 2, 4, or 6 engineered cysteine residues. The importance of site-specific TCO conjugation was evident in our data, as seen by differences in both tumor and blood signal between 7C2-TCO 2 and 7C2-TCO 6 , a comparison that was enabled by recent developments in site-specific conjugation technology.…”

“…Conjugation was accomplished by first deblocking the THIOMAB™ antibodies followed by conjugation of TCO-PEG 3 -maleimide to the free engineered cysteine residues as previously described. 46 Note that use of PEG spacers for TCO conjugation to mAbs has been evaluated elsewhere. 55 Conjugation of 1,4,7,10-tetraazacyclododecane-1,4,7-tris-acetic acid-10-maleimidoethylacetamide (i.e., “DOTA-maleimide”, product #B-272, Macrocyclics, Dallas, TX) to 7C2-LC:K149C and 7C2-LC:K149C+HC:L177C+HC:Y376C (giving DOTA-to-antibody ratio of ~2 and ~6) were also prepared as controls for direct labeling methods used for tissue distribution and imaging, via 111 In-DOTA 2 -7C2 and 111 In-DOTA 6 -7C2, respectively.…”

“…We conjugated TCO moieties, via short polyethylene glycol (PEG) linkers, site-specifically to anti-HER2 THIOMAB™ antibodies 46 with varying numbers of engineered cysteines 47 to determine the importance of stoichiometry on the in vivo IEDDA reaction in a HER2-positive KPL-4 tumor model. We employed 111 In (t 1/2 2.8 days) to allow direct comparison between our conventional radioimmunoconjugate and our click reactive tetrazine probe in an attempt to demonstrate proof of concept for same-day imaging with shorter lived radionuclides such as 68 Ga ( t 1/2  = 68 min).…”

Preclinical pharmacokinetics and pharmacodynamics of DCLL9718A: An antibody-drug conjugate for the treatment of acute myeloid leukemia

“…DCLL9718A uses THIOMAB TM antibody technology resulting in the conjugation of two drug molecules per antibody to engineered cysteine residues. 21 MCLL0517A was optimized to obtain approximately one nanomolar binding affinities to both human and cynomolgus monkey CLL-1, as well as efficient internalization and trafficking to lysosomes in HL-60 and 293 cells. 11 …”

Rapid and Efficient Generation of Stable Antibody–Drug Conjugates via an Encoded Cyclopropene and an Inverse‐Electron‐Demand Diels–Alder Reaction

“…HC‐118 is permissive for mutation and conjugation14 and because it is conserved in all IgG1s the expression system is applicable to most therapeutic monoclonal antibodies. Transfection of the transient plasmid system in Expi293 cells yielded 22±2 mg L −1 of trastuzumab(CypK) 2 , which approaches the wild‐type antibody level (33±3 mg L −1 ) (Figure 1 c).…”