Design and Synthesis of Isoquinolidinobenzodiazepine Dimers, a Novel Class of Antibody–Drug Conjugate Payload

Abstract

Antibody-drug conjugates (ADCs) represent an important class of emerging cancer therapeutics. Recent ADC development efforts highlighted the use of pyrrolobenzodiazepine (PBD) dimer payload for the treatment of several cancers. We identified the isoquinolidinobenzodiazepine (IQB) payload (D211), a new class of PBD dimer family of DNA damaging payloads. We have successfully synthesized all three IQB stereoisomers, experimentally showed that the purified (,)-D211 isomer is functionally more active than (,)-D221 and (,)-D231 isomers by >50,000-fold and ∼200-fold, respectively. We also synthesized a linker-payload (D212) that uses (,)-D211 payload with a cathepsin cleavable linker, a hydrophilic PEG8 spacer, and a thiol reactive maleimide. In addition, homogeneous ADCs generated using D212 linker-payload exhibited ideal physicochemical properties, and anti-CD33 ADC displayed a robust target-specific potency on AML cell lines. These results demonstrate that D212 linker-payload described here can be utilized for developing novel ADC therapeutics for targeted cancer therapy.

Development of a Single-Step Antibody–Drug Conjugate Purification Process with Membrane Chromatography

“…Since the toxic payloads used in ADC processes are highly potent, the level of acceptable free payloads present in the purified ADCs usually range from 1 to 0.1% on a molar basis relative to the ADCs [ 6 , 15 ]. These purity levels are difficult to achieve via a simple UF/DF purification, and often require extended diavolumes with additives or activated carbon filter to meet residual payload specifications [ 5 ].…”

“…In this study, the cysteine-engineered human monoclonal antibody (IgG1, pI 8.45, 150 kDa) used for conjugation experiments, was manufactured by Abzena (San Diego, CA, USA). The maleimidocaproyl valine citrulline p-aminobenzyloxycarbonyl monomethyl auristatin E (MC-vc-PAB-MMAE) [ 14 ] or maleimido-pyrrolobenzodiazepine (PBD) dimer [ 15 ] were prepared by Abzena (Bristol, PA, USA) using the methods previously described. The ADC was produced following a previously developed method with modifications [ 13 ].…”

CLT030, a leukemic stem cell–targeting CLL1 antibody-drug conjugate for treatment of acute myeloid leukemia

“…which induces cell toxicity in various AML cell lines with a picomolar 50% inhibitory concentration (IC 50 ), similar to that of a pyrrolobenzodiazepine (PBD) dimer. 18 The D212 linker-payload is the D211 payload with a cleavable Val-Ala linker, PEG-8 spacer, and maleimide functional group to conjugate the linker-payload to reactive thiol groups on the antibody consisting of 2 engineered cysteine residues (Figure 2A). A nonbinding control antibody (IgG antibody) was also conjugated with D212 linker-payload and used as nonbinding ADC control in all in vitro and in vivo studies.…”

Scale-up Synthesis of Tesirine

“…The residue was purified by flash chromatography (heptane/ethyl acetate; 98:2 up to 90:10) to afford the product 26 (1.0 kg, 98.2 HPLC area % purity, 87.2% yield). (27). Anhydrous dimethyl sulfoxide (171 mL, 2.4 mol, 2.5 equiv) was added dropwise to a solution of oxalyl chloride (84 mL, 0.99 mol, 1.03 equiv) in dry DCM (4.5 L, 9 V)) at −75 °C.…”

“…The chloroformate of benzylic alcohol 24 could not be used, since these types of compounds tend to eliminate CO 2 and form their chlorobenzyl analogues. On the other hand, aromatic aniline 19 is not nucleophilic enough to easily react with activated carbonates (although it must be noted that Smith and co-workers recently reported the clean condensation of an analogue of 19, at room temperature over a period of 6 days, with a pentafluorophenyl carbonate 27 ). For these reasons, the research route relied on formation of an isocyanate intermediate by reaction of amine 19 with triphosgene (a solid, safer alternative to phosgene gas).…”