Diazido macrocyclic sulfates as a platform for the synthesis of sequence-defined polymers for antibody drug conjugates

Abstract

To improve the efficacy of antibody drug conjugates (ADCs), there has been significant focus on increasing the drug-to-antibody ratio (DAR) in order to deliver more payload. However, due to the hydrophobicity of many cytotoxics, highly-loaded conjugates often have lower physicochemical stability and poorer pharmacokinetic outcomes, requiring the development of new hydrophilic linkers. Herein, we report a platform for the preparation of functional, sequence-defined polymers for conjugation to antibodies. We demonstrate the successful synthesis of novel diazido macrocyclic sulfate monomers of varied size ranging from 4 to 7 ethylene glycol repeat units. These monomers were then successively ring-opened to produce sequence-defined polymers that contained either 4 or 6 azides for post-synthesis functionalization. Given the hydrophilic ethylene glycol backbone and chemically defined nature of the polymers, we envisioned this as a useful strategy in the preparation of highly-loaded ADCs. To demonstrate this, we prepared a model polymer-fluorophore scaffold composed of 4 coumarin molecules and conjugated it to Herceptin. We fully characterized the conjugate via mass spectrometry, which yielded a polymer-to-antibody ratio of 6.6, translating to a total of 26 fluorophores conjugated to the antibody at the inter-chain disulfides. We believe this technology to not only be a meaningful contribution to the field of sequence-defined polymers and conjugates, but also as a general and tunable platform for drug delivery.

Fig. 1. Advantages of the macrocyclic sulfate monomer in this research.

Scheme 1. Synthesis of di-azido macrocyclic sulfate monomers of varied size.

Fig. 2. Synthesis of AzMCS polymers. (A) Synthesis of propargyl hexaazide polymer, (B) purified NMR of each ring-opening step, and (C) mass spectrum of each chain extension (NH₄⁺ and Na⁺ adducts).

Fig. 3. (A) Scheme for the synthesis of tetraazide poly(ethylene glycol) along with coumarin functionalization and maleimide installation. Yields noted in the polymerization process are reported as the individual, elongation reactions, not the overall yield. (B) Mass spectrometry of digested and reduced antibody–coumarin conjugate. Red stars represent the number of polymers modifying each antibody subunit. (C) Activity of trastuzumab and coumarin conjugate towards HER2 antigen as measured by ELISA.