Association of High Levels of Antidrug Antibodies Against Atezolizumab With Clinical Outcomes and T-Cell Responses in Patients With Hepatocellular Carcinoma

Key Points Question What are the clinical and immunologic associations of atezolizumab antidrug antibody (ADA) levels with clinical outcomes after atezolizumab/bevacizumab treatment in patients with advanced hepatocellular carcinoma (HCC)? Findings In this cohort study examining 132 patients with advanced HCC, highly elevated ADA levels (≥1000 ng/mL) at 3 weeks were associated with poor clinical outcomes. Compared with patients with low ADA levels, patients with high ADA levels exhibited reduced systemic exposure to atezolizumab and impaired proliferation and activation of peripheral CD8-positive T cells. Meaning Developing high ADA levels at an early time point could attenuate the immunotherapeutic efficacy of atezolizumab.


Treatments and evaluation
All patients in the HCC discovery and validation cohorts were treated with the combination of atezolizumab (1200 mg fixed dose) and bevacizumab (15 mg/kg) every 3 weeks.
Dose interruptions or reductions were made according to the protocol of the IMbrave 150 1 .
Treatment continued until patients experienced intolerable toxicity, disease progression, or consent withdrawal. The response was evaluated according to the RECIST 1.1. Response evaluation was performed every 6 or 9 weeks with computed tomography or magnetic resonance imaging.

Sample collection and ADA quantification
Blood samples were collected by venipuncture in Vacutainer tubes (BD Biosciences) before the first administration of atezolizumab (cycle 1 day 1, hereafter referred to as baseline) and before the second injection of atezolizumab (C2D1). Samples were left to coagulate, and serum was centrifuged at 1000 ×g for 5 min and stored at -80°C. Peripheral blood mononuclear cells were obtained by density gradient centrifugation with Ficoll-Paque PLUS (GE Healthcare). C2D1 blood samples that were drawn before 18 days or more than 24 days after the initiation of the first treatment were excluded from the analysis to ensure consistent timing.

Measurement of anti-atezolizumab antibodies and atezolizumab
The concentrations of anti-atezolizumab antibodies were measured using a enzyme-linked immunosorbent assay (ELISA) (KBI2027 ver 4.0, KRISHGEN BioSystems) according to the manufacturer's instructions. In brief, samples were incubated for 120 min at 37℃ in a microwell pre-coated with atezolizumab. After several washings, horseradish peroxidaseconjugated atezolizumab was added and the plate incubated for 60 min at 37℃. After another washing, TMB substrate solution was added and the plate incubated for 30 min at 37℃ in the dark. The absorbance was measured at 450 nm using a microplate reader (BioTek). The concentration of serum atezolizumab was measured using a commercial ELISA assay (KBI1027, KRISHGEN BioSystems).

Cytokine secretion assays and flow cytometry
To assess cytokine secretion, peripheral blood mononuclear cells were stimulated with plate-bound anti-CD3 antibody (1 ug/ml). After 4 hours, brefeldin A (eBioscience) and monensin (eBioscience) were added to the culture. After 20 hours, activated cells were harvested. To investigate proliferation capacity of CD8 + T cells, cryopreserved peripheral blood mononuclear cells were used after thawing. Before antibody staining, dead cells were excluded by Fixable Viability Dye eFluorTM 780 (eBioscience) on ice for 30 min, followed by treatment with the human Fc receptor-binding inhibitor (eBioscience) for 15 min at room temperature.

Statistical analysis
The independent sample t-test was used to compare cytokine production between the ADA-high and ADA-low groups. The paired sample t-test was used to compare ADA levels and T cell proliferation between baseline and cycle 2 day 1. Analysis of variance was used to compare ADA levels according to treatment responses and to compare atezolizumab concentration according to ADA levels. Pearson's correlation was used to relate serum ADA and atezolizumab. OS was defined as the time from the initiation of treatment until death from any cause. Progression-free survival (PFS) was defined as the time between the initiation of treatment and the progression of the disease or death from any cause. Survival outcomes were analyzed using the Kaplan-Meier method, and the subgroups were compared using the logrank test. Univariate and multivariate analyses of OS and PFS were performed using the Cox proportional hazards model. Statistical significance was defined as two-sided P < 0.05. The time-dependent ROC curve was analyzed using R version 4.0. Other statistical analyses were performed with SPSS (IBM) version 18.0.