Targeting Phosphatidylserine with Calcium-Dependent Protein–Drug Conjugates for the Treatment of Cancer

Abstract

In response to cellular stress, phosphatidylserine is exposed on the outer membrane leaflet of tumor blood vessels and cancer cells, motivating the development of phosphatidylserine-specific therapies. The generation of drug-conjugated phosphatidylserine-targeting agents represents an unexplored therapeutic approach, for which antitumor effects are critically dependent on efficient internalization and lysosomal delivery of the cytotoxic drug. In the current study, we have generated phosphatidylserine-targeting agents by fusing phosphatidylserine-binding domains to a human IgG1-derived Fc fragment. The tumor localization and pharmacokinetics of several phosphatidylserine-specific Fc fusions have been analyzed in mice and demonstrate that Fc-Syt1, a fusion containing the synaptotagmin 1 C2A domain, effectively targets tumor tissue. Conjugation of Fc-Syt1 to the cytotoxic drug monomethyl auristatin E results in a protein-drug conjugate (PDC) that is internalized into target cells and, due to the Ca dependence of phosphatidylserine binding, dissociates from phosphatidylserine in early endosomes. The released PDC is efficiently delivered to lysosomes and has potent antitumor effects in mouse xenograft tumor models. Interestingly, although an engineered, tetravalent Fc-Syt1 fusion shows increased binding to target cells, this higher avidity variant demonstrates reduced persistence and therapeutic effects compared with bivalent Fc-Syt1. Collectively, these studies show that finely tuned, Ca-switched phosphatidylserine-targeting agents can be therapeutically efficacious. .

Engineering a HER2-specific antibody–drug conjugate to increase lysosomal delivery and therapeutic efficacy

“…Recombinant antibodies were produced in Expi293F cells (Life Technology, Carlsbad, CA, USA) following transient transfection with the Expi293 expression system kit (Life Technology) as previously described 24 . Antibodies were purified from culture supernatants using protein G-Sepharose (GE Healthcare) and dialyzed against PBS.…”

Targeting phosphatidylserine for Cancer therapy: prospects and challenges

“…For example, Fc-Syt1 is a PS-targeting agent generated by fusing PS-binding domains to a human IgG1-derived Fc fragment C2A. Use of a Fc-Syt1 conjugated to monomethyl auristatin E, a cytotoxic drug, showed good antitumor effects in mouse breast and prostate cancer models 156 . In addition, recent researchers have developed a new method in which a PS binding peptide, PSBP-6, is conjugated to pH-sensitive mixed micelles (PEG-PDLLA and PEG-PHIS) and then loaded onto the chemotherapeutic agent paclitaxel (PTX) in prepared micelles.…”

BPRDP056, a novel small molecule drug conjugate specifically targeting phosphatidylserine for cancer therapy

“…To select patients stratified with the serum levels of β2GP1, a glycoprotein required for bavituximab targeting of PS, and thus to identify a subset of patients likely to benefit from the treatments of bavituximab has been suggested for its future clinical study design. Recently, a PS-targeting protein-drug conjugate (PDC) designed by fusing PS-binding synaptotagmin 1 (Syt1) C2A domains to a fusion of human IgG1-derived Fc fragments that are conjugated with 4 toxic monomethyl auristatin E (MMAE) molecules to the hinge cysteines of the Fc fusion [ 28 ]. The cytotoxic MMAE agent on the PDC is efficiently delivered and exhibits potent antitumor activities against xenograft tumors in mice [ 28 ].…”

“…Recently, a PS-targeting protein-drug conjugate (PDC) designed by fusing PS-binding synaptotagmin 1 (Syt1) C2A domains to a fusion of human IgG1-derived Fc fragments that are conjugated with 4 toxic monomethyl auristatin E (MMAE) molecules to the hinge cysteines of the Fc fusion [ 28 ]. The cytotoxic MMAE agent on the PDC is efficiently delivered and exhibits potent antitumor activities against xenograft tumors in mice [ 28 ]. Therefore, PS is a promising biomolecule for tumor site-specific targeting therapy to which SMDC may provide another strategy for PS targeting cancer therapy.…”