Efficient Preparation of Site-Specific Antibody–Drug Conjugates Using Cysteine Insertion

Abstract

Antibody-drug conjugates (ADCs) are a class of biopharmaceuticals that combine the specificity of antibodies with the high-potency of cytotoxic drugs. Engineering cysteine residues in the antibodies using mutagenesis is a common method to prepare site-specific ADCs. With this approach, solvent accessible amino acids in the antibody have been selected for substitution with cysteine for conjugating maleimide-bearing cytotoxic drugs, resulting in homogeneous and stable site-specific ADCs. Here we describe a cysteine engineering approach based on the insertion of cysteines before and after selected sites in the antibody, which can be used for site-specific preparation of ADCs. Cysteine-inserted antibodies have expression level and monomeric content similar to the native antibodies. Conjugation to a pyrrolobenzodiazepine dimer (SG3249) resulted in comparable efficiency of site-specific conjugation between cysteine-inserted and cysteine-substituted antibodies. Cysteine-inserted ADCs were shown to have biophysical properties, FcRn, and antigen binding affinity similar to the cysteine-substituted ADCs. These ADCs were comparable for serum stability to the ADCs prepared using cysteine-mutagenesis and had selective and potent cytotoxicity against human prostate cancer cells. Two of the cysteine-inserted variants abolish binding of the resulting ADCs to FcγRs in vitro, thereby potentially preventing non-target mediated uptake of the ADCs by cells of the innate immune system that express FcγRs, which may result in mitigating off-target toxicities. A selected cysteine-inserted ADC demonstrated potent dose-dependent anti-tumor activity in a xenograph tumor mouse model of human breast adenocarcinoma expressing the oncofetal antigen 5T4.

Characterization of Disulfide Bond Rebridged Fab–Drug Conjugates Prepared Using a Dual Maleimide Pyrrolobenzodiazepine Cytotoxic Payload

“…The development of ADCs has evolved greatly over the past few years, resulting in four ADCs approved for the treatment of solid and hematologic tumors . However, technological challenges still need to be overcome to improve the therapeutic index (TI, defined as the maximum tolerated dose divided by the minimum efficacious dose) of ADCs, particularly those used to treat solid tumors and those prepared using PBDs . Several efforts have recently been made to improve the TI of ADCs, including 1) site‐specific conjugation, which offers precise drug loading and optimal serum stability, 2) fractionated doses of PBD ADCs, which maintain antitumor activity and have greater tolerability than single high doses, and 3) improvements in tumor penetration of small‐fragment ADCs …”

“…[3,4,5] However,t echnological challenges still need to be overcome to improvet he therapeutic index (TI, defined as the maximum tolerated dose divided by the minimum efficaciousd ose) of ADCs,p articularly those used to treat solid tumors and those prepared using PBDs. [17] Severale fforts have recently been made to improvet he TI of ADCs, including 1) site-specificc onjugation, which offers precise drug loading and optimal serum stability, [1] 2) fractionated doses of PBD ADCs, which maintain antitumor activity and have greater tolerability than single high doses, [18] and 3) improvements in tumor penetration of small-fragmentADCs. [19] Recently we reported that lowering the number of drugs per antibodya nd using ar ebridging conjugation strategy with ad ual maleimide PBD (SG3710) could be as trategy to improve the TI of an anti-HER2 antibody.…”

“…Before conjugation these Fabs were reduced again with two‐fold equivalents of tris(2‐carboxyethyl)phosphine (TCEP) at 37 °C with shaking for 1 h. The Fabs prepared using transient expression were reduced only once using two‐fold equivalents of TCEP at 37 °C with shaking for 1 h. The reduced Fabs were then used for conjugation using two‐fold equivalents of SG3710 prepared in 100 % dimethyl sulfoxide (10 % final DMSO concentration, Sigma–Aldrich). The conjugation was carried out with shaking at room temperature for 1 h. Trastuzumab site‐specifically conjugated to two PBDs (SG3249) was prepared and characterized as described previously …”

“…Gentuzumab ozogamicin (Mylotarg), is based on IgG4, which has decreased Fc receptor binding . More recently, IgG1 engineered for lack of Fc binding are being used as ADC scaffolds …”

“…[15] More recently,I gG1 engineered for lack of Fc binding are being used as ADC scaffolds. [16,17] Small fragments derived from IgGs or from other highly structured scaffolds derived from natural proteins have found use as ADCs. [10,11,18] Antibody fragments may be ideal formats as ADC cancer therapeuticsb ecause they could have higher tumoru ptake, ab etter tumor-to-blood ratio, and faster clearance than full-length antibodies.…”