Antibody-Drug Conjugates (ADCs) Derived from Interchain Cysteine Cross-Linking Demonstrate Improved Homogeneity and Other Pharmacological Properties over Conventional Heterogeneous ADCs

Abstract

Conventional antibody-drug conjugates (ADCs) are heterogeneous mixtures of chemically distinct molecules that vary in both drugs/antibody (DAR) and conjugation sites. Suboptimal properties of heterogeneous ADCs have led to new site-specific conjugation methods for improving ADC homogeneity. Most site-specific methods require extensive antibody engineering to identify optimal conjugation sites and introduce unique functional groups for conjugation with appropriately modified linkers. Alternative nonrecombinant methods have emerged in which bifunctional linkers are utilized to cross-link antibody interchain cysteines and afford ADCs containing four drugs/antibody. Although these methods have been shown to improve ADC homogeneity and stability in vitro, their effect on the pharmacological properties of ADCs in vivo is unknown. In order to determine the relative impact of interchain cysteine cross-linking on the therapeutic window and other properties of ADCs in vivo, we synthesized a derivative of the known ADC payload, MC-MMAF, that contains a bifunctional dibromomaleimide (DBM) linker instead of a conventional maleimide (MC) linker. The DBM-MMAF derivative was conjugated to trastuzumab and a novel anti-CD98 antibody to afford ADCs containing predominantly four drugs/antibody. The pharmacological properties of the resulting cross-linked ADCs were compared with analogous heterogeneous ADCs derived from conventional linkers. The results demonstrate that DBM linkers can be applied directly to native antibodies, without antibody engineering, to yield highly homogeneous ADCs via cysteine cross-linking. The resulting ADCs demonstrate improved pharmacokinetics, superior efficacy, and reduced toxicity in vivo compared to analogous conventional heterogeneous ADCs.

Keywords: ADC; CD98; DAR; Her2; MMAF; antibody−drug conjugate; auristatin; bifunctional; conjugation; cysteine; dibromomaleimide; disulfide; hinge; homogeneous; interchain; linker; maleimide; site-specific; trastuzumab.

Figure 1.

(A) Chemical structures of MMAF payloads containing conventional MC (1) or bifunctional DBM (2) linkers. (B) Synthesis of DBM-MMAF payload (2) used for synthesis of homogeneous ADCs.

Figure 2.

Synthesis of ADCs via conjugation of trastuzumab or IGNX with payloads containing conventional MC (1) or bifunctional DBM (2) linkers. The conjugation protocols are identical except that excess TCEP is used to ensure full reduction of interchain disulfide bonds prior to conjugation with DBM-MMAF (2).

Figure 3.

Hydrophobic interaction chromatography (HIC) analysis of ADCs 3–6. Cross-linked (DBM) ADCs are shown in blue, and conventional (MC) ADCs are shown in red. Average DAR values were calculated based on peak areas. (A) HIC overlays comparing trastuzumab ADCs 3 and 5. (B) HIC overlays comparing IGNX ADCs 4 and 6.

Figure 4.

Native LC/MS analysis comparing DAR compositions of conventional ADCs, 3 and 4, with cross-linked DBM ADCs, 5 and 6. The data shown are the deconvoluted MS profiles, and observed MWs are consistent with calculated MWs within ±2 AMUs. Average DAR values shown for each ADC were calculated based on peak intensities.

Figure 5.

Denaturing SEC analysis of ADC 5 (TRA–DBM-MMAF) showing the presence of two ADC isoforms resulting from inter- or intrachain cross-linking of hinge cysteines on the heavy chains: (A) native trastuzumab with intact interchain disulfide bonds; (B) ADC 5 (TRA–DBM-MMAF); (C) trastuzumab C225A/C228A double mutant which lacks hinge disulfide bonds; (D) fully reduced trastuzumab lacking interchain disulfides; (E) proposed structures of interand intrachain ADC isoforms. LC/MS analysis of the separated isoforms is consistent with the proposed structures (see Supporting Information).

Figure 6.

Binding affinity for antigen expressing cells: (A) binding of trastuzumab ADCs to BT474 (human breast carcinoma) cells that express Her2 but not CD98; (B) binding of IGNX ADCs to murine sarcoma cells transfected with human CD98, but lacking Her2 expression.

Figure 7.

Potency and selectivity of ADCs 3–6 in vitro: (A) growth inhibition of BT474 (human breast carcinoma) cells that express Her2, but not CD98; (B) growth inhibition of H446 (human lung carcinoma) cells that express CD98, but not Her2; (C) growth inhibition of SKOV3 (ovarian tumor cells that express both Her2 and CD98; (D) IC₅₀s of ADCs 3–6 against three different cell lines.

Figure 8.

(A) Tumor growth inhibition in Her2 positive SKOV3 xenograft mice treated twice (days 62 and 69) at 3 mg/kg. (B) Tumor growth inhibition in CD98 positive H446 (lung carcinoma) xenograft mice treated twice (days 41 and 48) at 3 mg/kg. (C) SKOV3 xenograft tumor model in NOG mice (insensitive to treatment with trastuzumab) treated once at 3 mg/kg with homogeneous (DBM) ADC 5 or with conventional (MC) ADC 3.

Figure 9.

Pharmacokinetics of trastuzumab ADCs 3 and 5 in rats after single iv injection at 1 mg/kg. Total Ab was determined using rat anti-mouse Fc capture antibody. Total ADC (DAR > 0) was determined using a murine anti-MMAF capture antibody. Detailed PK results are available in the Supporting Information.

Figure 10.

ADC toxicity in rats treated with increasing doses of ADC 3 (red) or ADC 5 (blue) measured on days 3 and 17. Group averages (10 rats per group) are shown as a green bar. (A) Dot plot showing absolute reticulocyte counts on days 3 and 17 in individual rats. (B) Dot plot showing alanine aminotransferase (ALT) levels on days 3 and 17 in individual rats. (C) Dot plot showing aspartate aminotransferase (AST) levels on days 3 and 17 in individual rats.

Figure 11.

Versatility and scalability of DBM linkers. (A) HIC traces of ADCs derived from conjugation of 2 (DBM-MMAF) with 3 different commercially available antibodies: Avastin, Rituxan, and Erbitux. (B) HIC traces of ADCs resulting from conjugation of 2 with IGNX at 3 different reaction scales (1 mg, 25 mg, and 1000 mg).