Binding and functional profiling of antibody mutants guides selection of optimal candidates as antibody drug conjugates

Abstract

An increasingly appreciated conundrum in the discovery of antibody drug conjugates (ADCs) is that an antibody that was selected primarily for strong binding to its cancer target may not serve as an optimal ADC. In this study, we performed mechanistic cell-based experiments to determine the correlation between antibody affinity, avidity, internalization and ADC efficacy. We used structure-guided design to assemble a panel of antibody mutants with predicted Her2 affinities ranging from higher to lower relative to the parent antibody, Herceptin. These antibodies were ranked for binding via SPR and via flow-cytometry on high-Her2 SKOV3 cells and low-Her2 MCF7 cells, the latter acting as a surrogate for low-Her2 normal cells. A subpanel of variants, representative of different Her2-binding affinities (2 strong, 2 moderate and 3 weak), were further screened via high-content imaging for internalization efficacies in high versus low-Her2 cells. Finally, these antibodies were evaluated in ADC cytotoxicity screening assays (using DM1 and MMAE secondary antibodies) and as antibody-drug conjugates (DM1 and PNU159682). Our results identified specific but weak Her2-binding variants as optimal candidates for developing DM1 and PNU ADCs since they exhibited high potencies (low to sub-nM) in high-Her2 SKOV3 cells and low toxicities in low-Her2 cells. The 2 strong-affinity variants were highly potent in SKOV3 cells but also showed significant toxicities in low-Her2 cells and therefore are predicted to be toxic in normal tissues. Our findings show that pharmacological profiling of an antibody library in multiple binding and functional assays allows for selection of optimal ADCs.

Cetuximab–siRNA Conjugate Linked Through Cationized Gelatin Knocks Down KRAS G12C Mutation in NSCLC Sensitizing the Cells Toward Gefitinib

“…In another work, antibody variants with point mutation(s) were ranked based on HER2 binding affinities for optimal ADC and the K D values were significantly different for variants differing by even a single amino acid. 54 To determine if the GADS conjugate was functional with respect to receptor affinity, we carried out SPR analysis and compared the K D values against CTB. K D of CTB, Conjugate-1, and Conjugate-2 were ∼0.06, ∼0.28 and ∼0.27 nM, respectively, which is similar to the K D values reported for CTB in earlier works.…”

Predictive Simulations in Preclinical Oncology to Guide the Translation of Biologics

“…The antibodies/ADCs considered here distribute as “high affinity” antibodies due to avidity and high antigen expression [where K d values of 270 nM are sometimes needed to increase tissue penetration) ( Rudnick et al, 2011 ). Likewise, lower affinity can impact internalization ( Zwaagstra et al, 2019 ), but intrinsic receptor internalization rates are a good first approximation ( Nessler et al, 2020a ). A more generalized version of the Thiele modulus can describe the low affinity cases (supplemental data).…”

Antigenic Sensitivity of Membrane-Proximal Targeting Chimeric Antigen Receptors can be Fine-Tuned through Hinge Truncation

“…This phenomenon possibly relates to the extreme multivalency of both CAR and EGFR on their respective cells. Increased valency is well known to lead to significant avidity effects that boost the apparent affinity of biological interactions [37][38][39][40], and these would presumably apply to CAR-T cells as well.…”

“…These results are consistent with previous observations that EGFR specific CARs are relatively insensitive to ABD affinity up to the micromolar range[27] and underscore the exquisite antigen sensitivity of CAR-T cells to respond to and lyse even very low antigen expressing target cells[37–39]. This phenomenon possibly relates to the extreme multi-valency of both CAR and EGFR on their respective cells; increased valency is well known to lead to significant avidity effects that boost the apparent affinity of biological interactions[30,40–42], and these would presumably apply to CAR-T cells as well. These results point to a possibility that perhaps lowering ABD affinity might be a somewhat blunt tool for modulation of CAR antigenic sensitivity.…”

“…Limited hinge truncation libraries with defined-target CARs were generated by exchanging the sdAb021 sequence with HER2 or EGFRvIII specific scFv sequences. Trastuzumab derived scFv sequences were generated based on previously reported mutant forms of trastuzumab with enhanced avidity[30], whereas EGFRvIII-targeting scFvs were generated as previously reported[29]. Both HER2- and EGFRvIII-scFvs were in a VH-(G4S) 3 -VL format.…”