EP-3624791-B1 - REAGENT FOR SITE-SELECTIVE BIOCONJUGATION OF PROTEINS OR ANTIBODIES

Inventors

• ZEGLIS, BRIAN
• ADUMEAU, Pierre
• DAVYDOVA, Maria

Dates

Publication Date

20260506

Application Date

20180517

Claims (2)

1. A compound consisting of a structure of wherein: R 1 = H; R 2 is a methyl; x is 2; y is 3 and z is 2.
2. A method for labeling a substrate, the method comprises steps of: exposing a label to a substrate that comprises a cysteine residue, wherein the label comprises: wherein R is a chelator; permitting the label to covalently bind to the cysteine residue of the substrate, thereby labeling the substrate.

Description

BACKGROUND OF THE INVENTION The subject matter disclosed herein relates to site-selective biological radiolabels that are stable in vivo. The ability of many biomolecules to selectively and specifically target cellular biomarkers has long been harnessed for both nuclear imaging and radiotherapy. This exploitation uses the ligation of a radioactive payload to the biomolecule. In the case of peptides and proteins, this is typically performed through the ligation of reactive, bifunctional probes to amino acids within the biomolecule, most often lysines. While controlling the site of this conjugation is fairly easy with small peptides - which rarely possess more than one or two copies of each amino acid - this becomes a much bigger problem with larger biomolecules. For example, most antibodies contain dozens of lysines distributed throughout their macromolecule structure. The indiscriminate attachment of a bifunctional chelator to these lysines can lead to the formation of thousands of different regioisomers. Not surprisingly, this so-called "random" approach to bioconjugation has low reproducibility and produces highly heterogeneous conjugates. In addition, this strategy can result in payloads being inadvertently grafted to bioactive sites in the biomolecule, rendering a portion of the conjugates inoperative. To circumvent these issues, researchers have turned their attention to strategies allowing for better control over the site of the ligation reaction, known as "site-specific" bioconjugations. Conjugations to cysteine - a thiol-bearing amino acid present in small numbers in proteins - with maleimide-bearing probes have become a staple of this field and have been used extensively over the last three decades. The prevalence of this strategy is rooted mainly in its simplicity and efficiency. Many proteins contain disulfide bridges that can be easily reduced to form the reactive thiols, and the Michael addition between the maleimide and the sulhydryl group can be performed at physiological pH and room temperature, reliably leading to the formation of a succinimidyl thioether linkage within an hour (see FIG. 1). While the ligation between thiols and maleimides represents an undeniable improvement over random conjugation methods, the reaction suffers from drawbacks as well. Indeed, maleimide-based conjugates display limited stability in physiological media because the succinimidyl thioether linkage can undergo a retro-Michael reaction that leads to the release of the payload or its exchange with other molecules containing free thiols (most often serum albumin, cysteine, and glutathione). Several studies focused on antibody-drug conjugates have reported on this phenomenon as well as the off-target uptake of payload that it creates. These studies have also been devoted to boosting the hydrolysis of the succinimyl moiety by modifying its vicinity, which ultimately prevents the thiol exchange and the off-target uptake of the drug. In the context of nuclear imaging and radiotherapy, this retro-Michael reaction can lead to the release of the radioactive payload and in vivo radiolabeling of endogeneous biomolecules through thiol exchange reactions (see FIG. 1). This "leakage" causes higher uptake in non-target tissues and lower uptake in target tissues, ultimately resulting in higher radiation doses to healthy tissues, reduced imaging contrast, and lower therapeutic ratios.Several alternatives which create more-stable linkages with thiols than maleimides have been used for the bioconjugation and radiolabeling of antibodies, most notably tosylates, bromo- and iodo-acetyls, and vinyl sulfones. However, each of these options has drawbacks as well, including lower reactivity with thiols as well as reactivity with other amino acids. An article published in 2013 (Toda, N., Asano, S. & Barbas, C. F. (2013) Rapid, stable, chemoselective labeling of thiols with Julia-Kocieński-like reagents: A serum-stable alternative to maleimide-based protein conjugation. Angew. Chem. Int. Ed. 52, 12592-12596, hereafter "Toda et al.") holds particular promise in this area. In this work, the Barbas Laboratory described the creation of an oxadiazolyl methyl sulfone-based reagent that could selectively react with thiols at a rate comparable to maleimides and form more stable conjugates than those obtained with the latter (Scheme 1). This reagent was used to label different THIOMABs - antibodies engineered to bear a free cysteine residues - with fluorescein, and these phenyloxadiazole-based conjugates displayed higher stability over time in serum than their maleimide-based cousins. Yet despite the benefits of this ligation over maleimide-thiol conjugations, this construct has scarcely been used since its publication. This is especially obvious in radiochemistry, in which only two reports describe the use of the reagent or its derivatives. In the first, Zhang et al. (Zhang, Q., Dall'Angelo, S., Fleming, I. N., Schweiger, L. F., Zanda, M. & O'Haga