Advancing protein therapeutics through proximity-induced chemistry

Abstract

Recent years have seen a remarkable growth in the field of protein-based medical treatments. Nevertheless, concerns have arisen regarding the cytotoxicity limitations, low affinity, potential immunogenicity, low stability, and challenges to modify these proteins. To overcome these obstacles, proximity-induced chemistry has emerged as a next-generation strategy for advancing protein therapeutics. This method allows site-specific modification of proteins with therapeutic agents, improving their effectiveness without extensive engineering. In addition, this innovative approach enables spatial control of the reaction based on proximity, facilitating the formation of irreversible covalent bonds between therapeutic proteins and their targets. This capability becomes particularly valuable in addressing challenges such as the low affinity frequently encountered between therapeutic proteins and their targets, as well as the limited availability of small molecules for specific protein targets. As a result, proximity-induced chemistry is reshaping the field of protein drug preparation and propelling the revolution in novel protein therapeutics.

Keywords: covalent drug; cross-linking; protein drug; protein therapeutics; proximity-induced chemistry.

Figure 1.

Protein modification approaches and representative reactions. (A) The first-generation protein modification approach employs N-hydroxysuccinimide (NHS) and maleimide-mediated reactions. (B) The second-generation protein modification approach incorporates copper-catalyzed azide-alkyne cycloaddition (CuAAC) and strain-promoted alkyne-azide cycloaddition (SPAAC) reactions. (C) Proximity-induced chemistry involves reactions between proximity-enabled reactive groups and specific natural amino acids in close proximity.

Figure 2.

Genetic incorporation of ncAAs with proximity-enabled reactive moieties into proteins. (A) Genetic Code Expansion technology enables the incorporation of bioreactive ncAAs with proximity-enabled reactive moieties into proteins. These moieties can form covalent bonds with native residues on interacting proteins through proximity-induced reactions. (B) Structural of bioreactive ncAAs utilized in proximity-induced chemistry, based on either a tyrosine or pyrrolysine scaffold. (C) F_fact selectively reacts with the cysteine residue when two residues are in close proximity. (D) VSF selectively reacts with the lysine residue when two residues are in close proximity.

Figure 3.

Strategies for Incorporating Proximity-Enabled Reactive Moieties into Proteins. (A) The proximity-induced chemistries between SNAP Tag or Halo Tag and their ligands. (B) Employing trans-splicing of split inteins to generate protein conjugates. (C) Post-modification of the affibody with acrylamide to enable proximity-induced chemistry with cysteine residues in close proximity. (D) Introduction of a proximity-enabled reactive group, fluorophenyl carbamate, into an antibody-binding peptide using solid-phase peptide synthesis. The resulting peptide enables proximity-induced chemistry with antibodies. (E) Two cysteine residues allow for the incorporation of proximity-responsive functional groups and this arrangement facilitates the detection of specific covalent binding agents through the utilization of display technologies.

Figure 4.

Preparation of site-specific antibody conjugates using proximity-induced chemistry. (A) The introduction of site-specific labeling and precise modifications in the antibody structure is achieved through proximity-induced chemistry by employing FPheK-modified FB protein and the affinity moiety Fc-III peptide. This crosslinking process occurs with nearby lysine residues on the antibody, utilizing pClick and CCAP technologies. (B) Utilizing a Fc-binding dirhodium metallopeptide catalyst, a cross-linking reaction is facilitated between asparagine residues on the antibody and an alkyne-bearing diazo reagent in close proximity. This strategy allows for the site-specific conjugation of functional moieties to the antibody.

Figure 5.

Preparation of covalent protein drugs using proximity-induced chemistry. (A) FSY-modified PD-1 covalently captures PD-L1 on tumor surfaces, leading to the restoration of T cell responses and the inhibition of tumor growth. (B) Antibodies with proximity-enabled reactive groups demonstrates stable crosslinking with the targeted antigen in close proximity, leading to superior efficacy in inhibiting the growth of tumor cells. (C) Proximity-enabled reactive moieties are integrated into antibody recruiting molecules, featuring target and antibody binding domains, to attract immune cells to tumor sites. (D) Immunization of mice with antigens with site-specific Kcr modification results in focused and enhanced antibody responses, specifically targeting the desired epitopes.