Modeling antibody drug conjugate potential using a granzyme B antibody fusion protein

Abstract

Background: Antibody drug conjugates (ADCs) constitute a promising class of targeted anti-tumor therapeutics that harness the selectivity of monoclonal antibodies with the potency of cytotoxic drugs. ADC development is best suited to initially screening antibody candidates for desired properties that potentiate target cell cytotoxicity. However, validating and producing an optimally designed ADC requires expertise and resources not readily available to certain laboratories.

Results: In this study, we propose a novel approach to help streamline the identification of potential ADC candidates by utilizing a granzyme B (GrB)-based antibody fusion protein (AFP) for preliminary screening. GrB is a non-immunogenic serine protease expressed by immune effector cells such as CD8 + T cells that induces apoptotic activity and can be leveraged for targeted cell killing.

Conclusions: Our innovative model allows critical antibody parameters (including target cell binding, internalization, and cytotoxic potential) to be more reliably evaluated in vitro through the creation of an ADC surrogate. Successful incorporation of this AFP could also significantly expand and enhance ADC development pre-clinically, ultimately leading to the accelerated translation of ADC therapies for patients.

Keywords: Antibody drug conjugate; Antibody fusion protein; Granzyme B.

Fig. 1

GrB_mut-TRA construct design and physical characterization. A Schematic representation of DNA vectors required for expressing Pro-GrB_mut-TRA as a human IgG1 antibody in Expi293 cells. Following affinity chromatography purification, EK cleavage was required to generate a catalytically active GrB_mut-TRA molecule. B SDS-PAGE and Coomassie Blue staining of purified TRA and GrB_mut-TRA under reducing and non-reducing conditions. Arrow insets indicate HC and LC bands for each molecule. C Western blot analysis of TRA, Pro-GrB_mut-TRA, and GrB_mut-TRA using reagents specific to human IgG and GrB. Abbreviations used: EK, enterokinase; GrB, granzyme B; rGrB, recombinant GrB; GrB_mut-TRA, catalytically active AFP; HC, heavy chain; LC, light chain; NR, non-reduced; Pro-GrB_mut-TRA, catalytically inert molecule; R, reduced; TRA, trastuzumab

Fig. 2

GrB_mut-TRA target binding and GrB activity. A ELISA determination of TRA, T-DM1, and GrB_mut-TRA binding to immobilized HER2 or irrelevant protein at various concentrations. B Assessment of TRA, T-DM1, and GrB_mut-TRA binding to HER2-expressing (B16, SK-OV-3, SK-BR-3) and non-expressing (B16) cell lines by flow cytometry. C Enzymatic activity of GrB was determined through absorbance using a GrB-specific chromogenic substrate (Ac-IEPD-pNA). Abbreviations used: CI, 95% confidence intervals; ECD, extracellular domain; GrB, granzyme B; rGrB, recombinant GrB; GrB-TRA, catalytically active AFP with WT GrB; GrB_mut-TRA, catalytically active AFP with mutated GrB; SA, specific activity; T-DM1, ado-trastuzumab emtansine; TRA, trastuzumab. Bars ± STDEV. Select results are based on technical replicates of 3 samples per treatment group. Individual data values are provided in supplementary information (Additional file 2)

Fig. 3

Internalization and apoptosis-inducing effects of GrB_mut-TRA. A B16 and B16.HER2 cells were treated with molar equivalents of rGrB, TRA, or GrB_mut-TRA before being acid washed, fixed/permeabilized, stained for GrB (green) and nuclei (blue), and assessed by IF. B HER2-expressing and non-expressing cells were treated overnight under various conditions that included TRA, T-DM1, or GrB_mut-TRA. Camptothecin was utilized as a positive control for inducing cell death. All cells were collected and stained with reagents detecting caspase 3/7 and dead cells by flow cytometry. Bar graphs indicate the frequency of caspase 3/7 positive events. Representative histogram plots across treatments are also provided. Abbreviations used: GrB, granzyme B; rGrB, recombinant GrB; GrB_mut-TRA, catalytically active AFP; Pro-GrB_mut-TRA, catalytically inert molecule; T-DM1, ado-trastuzumab emtansine; TRA, trastuzumab. *P < 0.05, bars ± STDEV. Select results are based on technical replicates of 2 samples per treatment group. Individual data values are provided in supplementary information (Additional file 2)

Fig. 4

Target cell cytotoxicity following GrB_mut-TRA treatment. Tumor cell lines deficient in or expressing HER2 were plated overnight and subsequently exposed to various concentrations of rGrB, TRA, T-DM1, or GrB_mut-TRA. After 48 h, cell viability was determined by crystal violet staining. Target cell viability (%) was calculated based on cells untreated and exposed to 10 μM camptothecin (i.e., maximum cell death). The table inset summarizes EC₅₀ values (ng/mL) across tumor cell lines and treatment conditions. Abbreviations used: CI, 95% confidence intervals; GrB, granzyme B; rGrB, recombinant GrB; GrB_mut-TRA, catalytically active AFP; T-DM1, ado-trastuzumab emtansine; TRA, trastuzumab. Bars ± STDEV. Select results are based on technical replicates of 3 samples per treatment group. Individual data values are provided in supplementary information (Additional file 2)