Antibody-Dependent Cellular Cytotoxicity Activity of a Novel Anti-PD-L1 Antibody Avelumab (MSB0010718C) on Human Tumor Cells

Abstract

Several anti-PD-1/PD-L1 monoclonal antibodies (mAb) are currently providing evidence of clinical benefit in subsets of cancer patients. The mode of action of these mAbs is to inhibit PD-1 on immune cells interacting with PD-L1 on tumor cells. These mAbs are either designed or engineered to eliminate antibody-dependent cell-mediated cytotoxicity (ADCC), which, however, has been implicated as an important mechanism in several highly effective mAb-mediated cancer therapies. A fully human anti-PD-L1 mAb would potentially be able to block PD-1/PD-L1 interactions and also mediate the ADCC lysis of tumor cells. MSB0010718C (designated avelumab) is a fully human IgG1 anti-PD-L1 mAb. The studies reported here demonstrate (i) the ability of avelumab to lyse a range of human tumor cells in the presence of PBMC or NK effectors; (ii) IFNγ can enhance tumor cell PD-L1 expression and, in some cases, enhance ADCC tumor cell lysis; (iii) purified NK cells are potent effectors for avelumab; (iv) similar levels of avelumab-mediated ADCC lysis of tumor cells are seen using purified NK as effectors from either healthy donors or cancer patients; (v) very low levels of avelumab-mediated lysis are seen using whole PBMCs as targets; this finding complements results seen in analyses of PBMC subsets of patients receiving avelumab; and (vi) the addition of IL12 to NK cells greatly enhances avelumab-mediated ADCC. These studies thus provide an additional mode of action for an anti-PD-L1 mAb and support the rationale for further studies to enhance avelumab-mediated ADCC activity.

Figure 1

Anti-PD-L1 Ab concentration, effector to target ratio, and donor PBMC effector activity influence ADCC mediated by avelumab. A, Titration of avelumab concentrations and different PBMC:tumor target ratios. In vitro ADCC assay using the PD-L1-high H441 human lung cancer cell line as a target. Data shown are the mean±SD of triplicate wells. B, In vitro ADCC assay using the PD-L1-high H460 human lung cancer cell line as a target and PBMCs from five different healthy donors (HD) as effectors. Data shown are the mean±SD of triplicate wells using 2 μg/mL avelumab or 2 μg/mL of isotype control human IgG1 MAb (see Methods) and an effector:target ratio of 25:1.

Figure 2

PD-L1 MFI is a stronger predictor of sensitivity to ADCC mediated by avelumab as compared to percentage of PD-L1 positive tumor cells. Each dot represents lysis of a different human tumor cell line. A total of 18 human tumor cell lines were evaluated, and 10 of these were assayed both with and without pre-treatment with IFNγ (see Tables 1 and 2). Thus there are a total of 28 cell line targets in each figure. The experiments were carried out using PBMCs from different healthy donors, and all tumor cell lines were evaluated multiple times. Tumor targets treated with IFNγ are denoted by a white circle (O). A, Correlation between % PD-L1-positive tumor cells and % ADCC lysis (p<0.0001, r=0.799). B, Correlation between PD-L1 tumor cell MFI and % ADCC lysis (p<0.0001, r=0.811). C, Correlation between the PD-L1 score and % lysis (p<0.0001, r=0.826). The PD-L1 score was derived by scoring each cell line for % positive cells and normalized MFI on a quartile scale ranging from 1–4. The combined score of % PD-L1-positive cells and MFI, ranging from 2–8, was considered the PD-L1 score for the cell line. All data were analyzed using PBMCs from normal donors as effectors, an avelumab concentration of 40 μg/mL, and an effector:target ratio of 100:1. All correlations show the p value and Spearman's rank correlation coefficient. The best-fit lines were determined using a centered second order polynominal (quadratic) model in GraphPad Prism.

Figure 3

NK cells are efficient effectors of ADCC mediated by avelumab. A, NK cells were purified from PBMCs from two healthy donors (HD) using negative magnetic selection. In vitro ADCC assays were performed using the PD-L1-high H460 human lung cancer cell line as a target, and either whole PBMCs or purified NK cells from the same donor as effectors. Data shown are the mean±SD of triplicate wells using 2 μg/mL avelumab concentration with the designated effector:target ratios. B, Dose-response curve for the H441 human lung cancer cell line using purified NK cells as effectors at an effector:target ratio of 25:1. EC50 = 13.4 ng/mL. Data shown are the mean±SD of triplicate wells. C, In vitro ADCC activity against H441 is reduced by the inclusion of increasing amounts of a CD16 neutralizing Ab. Data shown are the mean±SD of triplicate wells using avelumab at a concentration of 8 ng/mL. D, Purified NK cells from cancer patients mediate ADCC induced by avelumab as effectively as those isolated from healthy donors. Isolated NK cells from five healthy donors and five lung cancer patients were used in in vitro ADCC assays against the H441 human lung cancer cell line. Data shown are the mean±SD of triplicate wells using 20 ng/mL avelumab concentration at an effector:target ratio of 20:1.

Figure 4

Tumor cells relatively insensitive to CTL-mediated lysis are efficiently lysed via ADCC mediated by avelumab. A, The HLA-A24⁺, MUC-1⁺ human tumor cell lines PC3 (prostate) and H460 (lung) were used as targets in in vitro CTL assays with a MUC-1–specific HLA-A24 CD8⁺ T-cell line as described in Materials and Methods. Effector:target ratios used were 20:1 and 10:1. The mean % CTL lysis ± SD of triplicate assays are shown. While the PC3 line is sensitive to the CTL lysis, the H460 line is not. B, Concurrently, the H460 cell line was used as a target in an in vitro ADCC assay with purified NK cells as effectors. Data shown are for an effector:target ratio of 20:1 at three different concentrations of avelumab. Isotype control Ab was used at 2 μg/mL. Data shown are the mean±SD of triplicate wells. C, Treatment of purified NK effectors with IL12 significantly increases ADCC activity mediated by avelumab. Purified NK cells were treated overnight with 10 ng/mL recombinant human IL12. ADCC activity against the PD-L1⁺ H460 human lung cancer cell line was determined using control and IL12-treated NK cells as effectors at a 20:1 ratio at the indicated avelumab concentrations. Data shown are the mean±SD of triplicate wells. Results were analyzed using a parametric unpaired t test for the relevant comparisons (**, p<0.01; ***, p<0.001).

Figure 5

Healthy donors with the FcγRIIIa-158 V/V genotype display higher lysis of tumor cells than donors with the F/F genotype. Twenty healthy donors were genotyped for FcγRIIIa-158 polymorphism. An ADCC assay using the H441 human lung cancer cell line as a target was performed with purified NK cells from 3 donors with the V/V genotype and 3 donors with the F/F genotype. Avelumab was used at 0.5 ng/mL, and results are shown at E:T ratios of 12.5:1 and 6.25:1. Data shown are the mean±SD for each group. Results were analyzed using a parametric unpaired t test, p=0.026.