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Mutasynthesis of C17- and C21-Substituted Ansamitocins for Use as ADC Payloads

Abzena scientists recently conducted a study with Regeneron and Isomerase that was published in ACS Omega titled, “Mutasynthesis of C17- and C21-Substituted Ansamitocins for Use as ADC Payloads.”

Abstract

Strain engineering and process improvement were used to improve the titer of mutasynthetically generated ansamitocins generated by feeding 3-amino-5-hydroxybenzoic acid (AHBA) analogs to cultures of Actinosynnema pretiosum inactivated in AHBA biosynthesis. Ansamitocin analogs with fluorine and bromine substituents at C17 and C21 were then generated by feeding hydroxylated AHBA analogs. Fully processed C17 and C21 fluoro and bromo ansamitocins had cytotoxic activity similar to that of Ansamitocin P3. The C21 fluoro derivative was converted to a cytotoxic payload and an antibody drug conjugate (ADC).

Abzena contributing authors: Benjamin Wertz, Senior Manager of Chemistry &  Sylvia Degrado

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Introduction

Antibody–drug conjugates (ADCs), the combination of a recombinant monoclonal antibody and covalently bound drug (usually a cytotoxic payload), have shown increasing success following the initial approval of Mylotarg (gemtuzumab ozogamicin) in 2000, (1) with over 100 ADC candidates in different stages of clinical trials in 2022. (2) Many factors are key in the clinical success of a new ADC; however, the properties of the monoclonal antibody (mAb), the linker, and the payload are most critical. Of these, the payload probably has the least breadth in development, with all approved ADCs based on one of two classes─the microtubule inhibitors or the DNA damaging agents. (3) Two out of the 11 currently approved agents utilize maytansine derivatives (maytansinoids) as the payload: DM1 (1) in Kadcyla and DM4 (2) in Elahere.
Maytansinoids (Figure 1) are natural product-based tubulin inhibitors with a 9-member ansa macrolide structure attached to a chlorinated benzene ring and were originally isolated from the shrub Maytenus ovatus. (4) Even though DM1 and DM4 are highly potent and effective payloads, there is huge value in generating improved variants. In particular, increased potency, altered permeability, and solubility can all impact the therapeutic effect and ADC synthesis.

Figure 1

Figure 1. General structure for maytansinoids, with examples.

While the biosynthesis of the plant natural product maytansine (3) has not yet been fully elucidated, the biosynthesis of ansamitocins, which are bacterially produced maytansinoids with an O-linked isobutyrate at the 3-position, was described in 2002 (5) and requires the action of a modular polyketide synthase (PKS) initiated by the “starter unit” 3-amino-5-hydroxybenzoic acid (AHBA, a), Table 1). The prototypic and most potent natural ansamitocin, ansamitocin P-3 (AP-3, 4), can be generated by scalable fermentation and then deacetylated via lithium aluminum reduction to form the 3-O-desacyl product maytansinol (5), which can then be subjected to further chemistry on the C3-hydroxyl position to make payloads such as DM1 and DM4.
Table 1. Feeds Selected for Ansamitocin Analog Generation

feed ID R1 R2 R3 R4 R5
(a) (AHBA) H OH NH2 H H
(b) H Br NH2 H H
(c) H F NH2 H Et
(d) Br OH NH2 H H
(e) H OH NH2·HBr Br H
(f) F OH NH2 H H
(g) H OH NH2 F H
Following generation of the macrocyclic core by the PKS, this is further decorated by a series of post-PKS processing enzymes, including a carbamoyltransferase (asm21), O-methyltransferase (asm7) at C20, chlorinase (asm12) at C19, N-methyltransferase (asm10), acylase at C3 (asm19), and epoxidase (asm11). (6) Previous work has shown that the most highly processed analogs have the greatest activity, and analogs lacking an acyl group at the 3-O position are considerably less toxic. (7,8)
Previous work by Kirschning and other authors has shown that mutasynthesis (mutational biosynthesis) can be a valuable tool for rapidly generating ansamitocin analogs. (9,10) In particular, by feeding analogs of AHBA to an ansamitocin producing strain with AHBA biosynthesis inactivated, such as in HGF073, (5) ansamitocin analogs with altered substituents at the C19 and C20 positions have been isolated. This has included a wide range of substituents at these positions, along with varying levels of post PKS processing, such as altered acylation, carbamoylation, N-methylation, and epoxidation. There have been other targeting concepts used for maytansinoids. A folic acid-targeting conjugate and a magnetic nanoparticle have been reported. (18,19)
However, to date, no generation of analogs with altered substituents at C17 or C21 have been described, and the potency of most mutasynthetic analogs isolated to date has been significantly lower than the potency of AP-3. In addition, the published titers of many of the products were low, potentially leading to issues with isolating enough material for downstream ADC linker chemistry.

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ACS Omega

Cite this: ACS Omega 2025, XXXX, XXX, XXX-XXX

https://doi.org/10.1021/acsomega.5c05529

Published September 11, 2025

© 2025 The Authors. Published by American Chemical Society. This publication is licensed under

CC-BY-NC-ND 4.0 .

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