Synthesis of precision antibody conjugates using proximity-induced chemistry

Abstract

Rationale: Therapeutic antibody conjugates allow for the specific delivery of cytotoxic agents or immune cells to tumors, thus enhancing the antitumor activity of these agents and minimizing adverse systemic effects. Most current antibody conjugates are prepared by nonspecific modification of antibody cysteine or lysine residues, inevitably resulting in the generation of heterogeneous conjugates with limited therapeutic efficacies. Traditional strategies to prepare homogeneous antibody conjugates require antibody engineering or chemical/enzymatic treatments, processes that often affect antibody folding and stability, as well as yield and cost. Developing a simple and cost-effective way to precisely couple functional payloads to native antibodies is of great importance. Methods: We describe a simple proximity-induced antibody conjugation method (pClick) that enables the synthesis of homogeneous antibody conjugates from native antibodies without requiring additional antibody engineering or post-synthesis treatments. A proximity-activated crosslinker is introduced into a chemically synthesized affinity peptide modified with a bioorthogonal handle. Upon binding to a specific antibody site, the affinity peptide covalently attaches to the antibody via spontaneous crosslinking, yielding an antibody molecule ready for bioorthogonal conjugation with payloads. Results: We have prepared well-defined antibody-drug conjugates and bispecific small molecule-antibody conjugates using pClick technology. The resulting conjugates exhibit excellent in vitro cytotoxic activity against cancer cells and, in the case of bispecific conjugates, superb antitumor activity in mouse xenograft models. Conclusions: Our pClick technology enables efficient, simple, and site-specific conjugation of various moieties to the existing native antibodies. This technology does not require antibody engineering or additional UV/chemical/enzymatic treatments, therefore providing a general, convenient strategy for developing novel antibody conjugates.

Siglec-9 defines and restrains a natural killer subpopulation highly cytotoxic to HIV-infected cells

“…Purified antibodies bind efficiently to HIV+ cells in vitro ( S8 Fig ). We then employed the proximity-induced antibody labeling (pClick) technology [ 42 – 44 ] to conjugate these antibodies to Sialidase (from Salmonella typhimurium; STSia). pClick allows site-specific labeling of native antibodies with payloads under mild conditions, thus minimizing the disruption of antigen and Fc receptor binding.…”

“…pClick allows site-specific labeling of native antibodies with payloads under mild conditions, thus minimizing the disruption of antigen and Fc receptor binding. This approach was recently proven safe and effective in vivo [ 44 ]. To site-specifically conjugate the HIV bNAbs with Sialidase using pClick, we first genetically incorporated 4-fluorophenyl carbamate lysine into the Glu25 position of a FB fused with Sialidase using the genetic code expansion technology ( Fig 6A top ).…”

Precision Modification of Native Antibodies

“…Most importantly, a 91–99% conjugation efficiency was achieved with several IgG species and subclasses, without the generation of any detectable nonspecific conjugation products. In another study, the FB peptide could be further shortened to 33 residues without compromising its binding affinity and conjugation efficiency . This chemically synthesized FB peptide was applied to the synthesis of ADCs and bispecific small molecule–antibody conjugates with both excellent in vitro cytotoxic activity and antitumor activity in mouse xenograft models .…”

Rapid covalent labeling of a GPCR on living cells using a nanobody-epitope tag pair to interrogate receptor pharmacology

“…PIR compounds are often used to target Cys residues as they are relatively rare and show unique reactivity [6][7][8][9][10][11] , but this approach is limited when targeting proteins on cell surfaces as the Cys residues are typically engaged in disulfide bonds and therefore not available for crosslinking. Crosslinking moieties that react with other amino acids such as Lys, His, and Tyr have been used to crosslink peptide-based PIR compounds, [12][13][14][15][16][17][18][19][20] and innovative strategies to simultaneously optimize both polypeptide sequence and crosslinking properties have also been developed 11 .…”