A novel bispecific antibody for EGFR-directed blockade of the PD-1/PD-L1 immune checkpoint

Abstract

PD-L1-blocking antibodies produce significant clinical benefit in selected cancer patients by reactivating functionally-impaired antigen-experienced anticancer T cells. However, the efficacy of current PD-L1-blocking antibodies is potentially reduced by 'on-target/off-tumor' binding to PD-L1 widely expressed on normal cells. This lack of tumor selectivity may induce a generalized activation of all antigen-experienced T cells which may explain the frequent occurrence of autoimmune-related adverse events during and after treatment. To address these issues, we constructed a bispecific antibody (bsAb), designated PD-L1xEGFR, to direct PD-L1-blockade to EGFR-expressing cancer cells and to more selectively reactivate anticancer T cells. Indeed, the IC50 of PD-L1xEGFR for blocking PD-L1 on EGFR⁺ cancer cells was ∼140 fold lower compared to that of the analogous PD-L1-blocking bsAb PD-L1xMock with irrelevant target antigen specificity. Importantly, activation status, IFN-γ production, and oncolytic activity of anti-CD3xanti-EpCAM-redirected T cells was enhanced when cocultured with EGFR-expressing carcinoma cells. Similarly, the capacity of PD-L1xEGFR to promote proliferation and IFN-γ production by CMVpp65-directed CD8⁺ effector T cells was enhanced when cocultured with EGFR-expressing CMVpp65-transfected cancer cells. In contrast, the clinically-used PD-L1-blocking antibody MEDI4736 (durvalumab) promoted T cell activation indiscriminate of EGFR expression on cancer cells. Additionally, in mice xenografted with EGFR-expressing cancer cells ¹¹¹In-PD-L1xEGFR showed a significantly higher tumor uptake compared to ¹¹¹In-PD-L1xMock. In conclusion, PD-L1xEGFR blocks the PD-1/PD-L1 immune checkpoint in an EGFR-directed manner, thereby promoting the selective reactivation of anticancer T cells. This novel targeted approach may be useful to enhance efficacy and safety of PD-1/PD-L1 checkpoint blockade in EGFR-overexpressing malignancies.

Keywords: EGFR; PD-L1; bispecific antibody; immunotherapy.

Figure 1.

PD-L1xEGFR selectively and simultaneously binds to PD-L1 and EGFR (A) Dose-dependent binding of PD-L1xEGFR to CHO.PD-L1 vs. parental CHO cells. (B) Dose-dependent binding of PD-L1xEGFR vs. PD-L1xMock to PD-L1⁺/EGFR⁺ A431 cells. (C) Binding of PD-L1xEGFR vs. PD-L1xMock (5 µg/ml) to a series of PD-L1⁺/EGFR⁺ and PD-L1⁺/EGFR⁻ cell lines. (D) Binding of PD-L1xEGFR (1 µg/ml) to A431 cells in the presence or absence of excess PD-L1-blocking antibody and/or EGFR-blocking mAb 425. (E) Competitive binding assay in which anti-PD-L1-APC competed with increasing doses (0.01–50 µg/ml) of PD-L1xEGFR (black line) or PD-L1xMock (grey line) for binding to A431 cells. Where indicated, A431 cells were pre-treated with excess amounts of mAb 425 (50 µg/ml) (red line) or isotype control IgG2a (green line) for 15 min. All binding experiments were analyzed by flow cytometry. Statistical analysis in D was performed using One-way ANOVA followed by a Bonferroni post-hoc test (*p < 0.05, ** p < 0.01, *** p < 0.001, ns not significant).

Figure 2.

PD-L1xEGFR induces tumor growth inhibition and blocks the PD-1/PD-L1 interaction (A) Representative light microscopy images of PD-L1⁺/EGFR⁺ FaDu cells after 5 days treatment with 5 µg/ml PD-L1xEGFR, PD-L1xMock, mAb 425 or isotype control as indicated. (B) Cell viability of FaDu and H292 cells after treatment as in A was determined by MTS and expressed as percentage of medium control. Graphs represent mean ± SD. (C) Blockade of the PD-1/PD-L1 interaction analyzed using a commercially available PD-1/PD-L1 Blockade Bioassay (Promega). CHO.PD-L1/CD3 cells and Jurkat.PD-1-NFAT-Luc cells were treated with an increasing dose (0.01–10 µg/ml) of PD-L1xEGFR, PD-L1xMock, MEDI4736 or isotype control. NFAT-RE-mediated luciferase activity was quantified using a plate reader and expressed as fold increase compared to medium control. (D) Similar to C, mixed cultures of A431 cells and Jurkat.PD1-NFAT-luc cells were treated with increasing doses (0.01–10 µg/ml) of indicated antibodies in the presence of 75 ng/ml BIS-1. Statistical analysis in B was performed using One-way ANOVA followed by a Bonferroni post-hoc test (* p < 0.05, ** p < 0.01, *** p < 0.001, ns not significant).

Figure 3.

PD-L1xEGFR promotes cytotoxic activity of BIS-1-redirected T cells (A) FaDu cells were mixed with T cells at E:T ratio of 2:1 in the presence of BIS-1 (75 ng/ml) and 5 µg/ml PD-L1xEGFR or control antibodies. (B) A431 cells were treated as described in (A). Apoptosis was determined in A and B at day 3 by flow cytometry using Annexin-V staining. Apoptosis for isotype control treatments were subtracted. (C) IFN-γ levels in culture supernatant of (A) were determined by ELISA and IFN-γ levels for isotype control treatment were subtracted. (D) A431 cells were treated with the indicated antibodies, washed to remove unbound antibody and then mixed with T cells at an E:T ratio of 2:1 in the presence of BIS-1 (75 ng/ml). At day 3, T cells were carefully removed by washing, after which light microscopic images of the remaining A431 monolayer were evaluated. (E) In mixed cultures with EGFR⁺ FaDu and A431 or EGFR⁻ A2058.EpCAM cells as described in D, apoptosis was determined by flow cytometry using Annexin-V staining. Apoptosis for isotype control treatments were subtracted. (F) In mixed cultures with FaDu cells as described in D, expression of T cell activation mrker CD25 was analyzed by flow cytometry. Mean fluorescence intensity (MFI) of BIS-1 treatment alone was subtracted. Three independent experiments were performed and represent mean ± SD. Statistical analysis was performed using One-way ANOVA followed by a Bonferroni post-hoc test (* p < 0.05, ** p < 0.01, *** p < 0.001, ns not significant).

Figure 4.

PD-L1xEGFR enhances cytotoxic potential of antigen-experienced T cells A431 or A431.pp65 cells were treated with PD-L1xEGFR or control antibodies, after which unbound antibody was washed away. Subsequently, T cells derived from CMV-seropositive donors were added at an E:T ratio of 20:1. After 8 days, expression levels of indicated activation markers were measured. (A) HLA-DR and CD25 expression, representative of 3 independent experiments. (B) CD137 expression, representative of 3 independent experiments. (C) CD137, CD107a (D) and intracellular IFN-γ (E) expression levels were analyzed within CD8⁺ T cell population by flow cytometry. (F) Granzyme B levels present in culture supernatants of treatment conditions as described in A were determined by ELISA.

Figure 5.

PD-L1xEGFR induces NK-cell mediated ADCC (A) FaDu cells were mixed with IL-12-pre-treated NK cells at the indicated E:T ratios and in the presence of 5 µg/ml PD-L1xEGFR or control antibodies. (B) FaDu cells were co-cultured with IL-12-pretreated NK cells at an E:T ratio of 2:1 as described in A. (C) LNCaP cells were mixed with PBMCs at an E:T ratio of 5:1 in the presence of 5 µg/ml PD-L1xEGFR or control antibodies. In all experiments apoptosis was determined by flow cytometry using Annexin-V staining procedure. All graphs represent mean ± SD. Statistical analysis in B and C were performed using One-way ANOVA followed by a Bonferroni post-hoc test (*p < 0.05, ** p < 0.01, *** p < 0.001, ns not significant).

Figure 6.

Biodistribution of radiolabeled ¹¹¹In-PD-L1xEGFR. (A) Binding of ¹¹¹In-PD-L1xEGFR or ¹¹¹In-PD-L1xMock to PD-L1 and EGFR in the presence or absence of excess MEDI4736 and/or EGFR blocking mAb 425 on A431 and (B) SK-BR-3 cells. (C) Tumor uptake of ¹¹¹In-PD-L1xEGFR (1 µg) in mice with subcutaneous A431 (n = 5) or SK-BR-3 (n = 5) xenografts. Separate groups of mice were injected with control antibody ¹¹¹In-PD-L1xMock. Tumor uptake was calculated as % injected dose per gram tissue (%ID/g) (D) Tumor-to-blood ratio was calculated, from experiment as described in C. Statistical analysis in D was performed using One-way ANOVA followed by a Bonferroni post-hoc test (* p < 0.05, ** p < 0.01, *** p < 0.001, ns not significant).