Orthogonal Cysteine Protection Enables Homogeneous Multi‐Drug Antibody–Drug Conjugates

A strategy for the preparation of homogeneous antibody–drug conjugates (ADCs) containing multiple payloads has been developed. This approach utilizes sequential unmasking of cysteine residues with orthogonal protection to enable site‐specific conjugation of each drug. In addition, because the approach utilizes conjugation to native antibody cysteine residues, it is widely applicable and enables high drug loading for improved ADC potency. To highlight the benefits of ADC dual drug delivery, this strategy was applied to the preparation of ADCs containing two classes of auristatin drug‐linkers that have differing physiochemical properties and exert complementary anti‐cancer activities. Dual‐auristatin ADCs imparted activity in cell line and xenograft models that are refractory to ADCs comprised of the individual auristatin components. This work presents a facile method for construction of potent dual‐drug ADCs and demonstrates how delivery of multiple cytotoxic warheads can lead to improved ADC activities. Lastly, we anticipate that the conditions utilized herein for orthogonal cysteine unmasking are not restricted to ADCs and can be broadly utilized for site‐specific protein modification.     

Efficient Innate Immune Killing of Cancer Cells Triggered by Cell‐Surface Anchoring of Multivalent Antibody‐Recruiting Polymers

Binding of monoclonal antibodies (mAbs) onto a cell surface triggers antibody‐mediated effector killing by innate immune cells through complement activation. As an alternative to mAbs, synthetic systems that can recruit endogenous antibodies from the blood stream to a cancer cell surface could be of great relevance. Herein, we explore antibody‐recruiting polymers (ARPs) as a novel class of immunotherapy. ARPs consist of a cell‐binding motif linked to a polymer that contains multiple small molecule antibody‐binding motifs along its backbone. As a proof of concept, we employ a lipid anchor that inserts into the phospholipid cell membrane and make use of a polymeric activated ester scaffold onto which we substitute dinitrophenol as an antibody‐binding motif. We demonstrate that ARPs allow for high avidity antibody binding and drive antibody recruitment to treated cells for several days. Furthermore, we show that ARP‐treated cancer cells are prone to antibody‐mediated killing through phagocytosis by macrophages.     

A Diene‐Containing Noncanonical Amino Acid Enables Dual Functionality in Proteins: Rapid Diels–Alder Reaction with Maleimide or Proximity‐Based Dimerization

Here, we describe a diene‐containing noncanonical amino acid (ncAA) capable of undergoing fast and selective normal electron‐demand Diels–Alder (DA) reactions following its incorporation into antibodies. A cyclopentadiene derivative of lysine (CpHK) served as the reactive handle for DA transformations and the substrate for genetic incorporation. CpHK incorporated into antibodies with high efficiency and was available for maleimide conjugation or self‐reaction depending on position in the amino acid sequence. CpHK at position K274 reacted with the maleimide drug‐linker AZ1508 at a rate of ≈79 m ^?1 s^?1 to produce functional antibody–drug conjugates (ADCs) in a one‐step process. Incorporation of CpHK at position S239 resulted in dimerization, which covalently linked antibody heavy chains together. The diene ncAA described here is capable of producing therapeutic protein conjugates with clinically validated and widely available maleimide compounds, while also enabling proximity‐based stapling through a DA dimerization reaction.     

Photo‐induced and Rapid Labeling of Tetrazine‐Bearing Proteins via Cyclopropenone‐Caged Bicyclononynes

Inverse electron‐demand Diels–Alder cycloadditions (iEDDAC) between tetrazines and strained alkenes/alkynes have emerged as essential tools for studying and manipulating biomolecules. A light‐triggered version of iEDDAC (photo‐iEDDAC) is presented that confers spatio‐temporal control to bioorthogonal labeling in vitro and in cellulo. A cyclopropenone‐caged dibenzoannulated bicyclo[6.1.0]nonyne probe (photo‐DMBO) was designed that is unreactive towards tetrazines before light‐activation, but engages in iEDDAC after irradiation at 365 nm. Aminoacyl tRNA synthetase/tRNA pairs were discovered for efficient site‐specific incorporation of tetrazine‐containing amino acids into proteins in living cells. In situ light activation of photo‐DMBO conjugates allows labeling of tetrazine‐modified proteins in living E. coli. This allows proteins in living cells to be modified in a spatio‐temporally controlled manner and may be extended to photo‐induced and site‐specific protein labeling in animals.     

Three‐Dimensional Ordered Antibody Arrays Through Self‐Assembly of Antibody–Polymer Conjugates

Three‐dimensional (3D) ordered arrays of human immunoglobulin G (IgG) were fabricated using well‐defined full‐length antibody–polymer conjugates (APCs). The conjugates were prepared through a two‐step sequential click approach with a combination of oxime ligation and strain promoted alkyne–azide cycloaddition. They were able to self‐assemble into lamellar nanostructures with alternating IgG and poly(N ‐isopropylacrylamide) (PNIPAM) nanodomains. As a proof‐of‐concept, these materials were fabricated into thin films and their specific binding ability was tested. The nanostructure not only improves the packing density and the proper orientation of the IgG, but also provides nanochannels to facilitate substrate transport.