Controlled release of enhanced cross-hybrid IgGA Fc PD-L1 inhibitors using oncolytic adenoviruses

Abstract

Immune checkpoint inhibitors have clinical success in prolonging the life of many cancer patients. However, only a minority of patients benefit from such therapy, calling for further improvements. Currently, most PD-L1 checkpoint inhibitors in the clinic do not elicit Fc effector mechanisms that would substantially increase their efficacy. To gain potency and circumvent off-target effects, we previously designed an oncolytic adenovirus (Ad-Cab) expressing an Fc fusion peptide against PD-L1 on a cross-hybrid immunoglobulin GA (IgGA) Fc. Ad-Cab elicited antibody effector mechanisms of IgG1 and IgA, which led to higher tumor killing compared with each isotype alone and with clinically approved PD-L1 checkpoint inhibitors. In this study, we further improved the therapy to increase the IgG1 Fc effector mechanisms of the IgGA Fc fusion peptide (Ad-Cab FT) by adding four somatic mutations that increase natural killer (NK) cell activation. Ad-Cab FT was shown to work better at lower concentrations compared with Ad-Cab in vitro and in vivo and to have better tumor- and myeloid-derived suppressor cell killing, likely because of higher NK cell activation. Additionally, the biodistribution of the Fc fusion peptide demonstrated targeted release in the tumor microenvironment with minimal or no leakage to the peripheral blood and organs in mice. These data demonstrate effective and safe use of Ad-Cab FT, bidding for further clinical investigation.

Keywords: Fc engineering; IgA; IgG; PD-L1 therapy; oncolytic adenoviruses.

Graphical abstract

Figure 1

Cab versus Cab FT in inducing ADCC with different effector populations (A–C) Cells were treated with different concentrations of Fc fusion peptides and had either PBMCs (A; 100:1, E:T), PMNs (B; 40:1, E:T), or PBMCs and PMNs (C) added for a 4-h incubation. Lysis was then quantified by measuring release of endogenous LDH. All n ≥ 3, and significance levels were set at ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001. Error bars represent SEM.

Figure 2

Whole-blood and mixed-leukocyte assay with Cab and Cab FT Unmanipulated blood from 3 donors was treated with 20 μg/mL of Fc fusion peptides and incubated for 24 h. (A–C) Immune populations were then gated (A) and quantified by percentage (B) and absolute number (C). DCs and CFSE-labeled PBMCs from different donors were incubated with 10 μg/mL of Fc fusion peptides or antibody for 5 days. (D) PBMCs were then collected, and CD8+ T cells had their expansion index calculated based on CFSE staining. All n ≥ 3, and significance levels were set at ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001. Error bars represent SEM.

Figure 3

Oncolytic fitness and expression of Ad-Cab and Ad-Cab FT (A–C) Different cell types were infected with the indicated MOIs of virus and incubated for 3 days. Using an MTS assay, cell viability (A) was determined. A549 (B) and B16K1 (C) cells were infected with 100 MOI of virus, and the amount of Fc fusion peptides was measured using a His tag ELISA.

Figure 4

ADCC of Ad-Cab and Ad-Cab FT in the presence of PBMCs and PMNs Cells were infected at various MOIs with virus and incubated for 48 h. PBMCs (100:1, E:T) and PMNs (40:1, E:T) were then added. After 4 h, lysis was quantified based on endogenous LDH release.

Figure 5

Real-time killing of A549 and B16K1 cells with Ad-Cab and Ad-Cab FT (A and B) A549 (A) and B16K1 (B) cells were first seeded for 24 h. Then viruses were added at 30 MOI (A549) and 100 MOI (B16K1), along with PBMCs (100:1, E:T) and PMNs (40:1, E:T). The cell index was then measured every 30 min for the indicated times.

Figure 6

In vivo efficacy of Ad-Cab and Ad-Cab FT (A) Schematic of B16K1 tumor implantation and the treatment schedule. Mice were implanted with 500,000 cells in the right flank and then treated either with PBS (mock), Ad-5/3 Δ 24, Ad-Cab, Ad-Cab FT, or mPD-L1. (B) Tumor growth was then recorded. (C and D) After mice were sacrificed, NK cell activation (C) and T cell activation (D) were measured with flow cytometry. (E and F) Biodistribution of the Fc fusion peptide was then checked in the tumor (E) and liver (F). (G) Schematic of the treatment schedule for mice implanted with 300,000 4T1 cells. The same treatment groups as for the 4T1 tumor model were used with B16K1 mice. (H) Tumor growth was recorded for 17 days. (I and J) After mice were sacrificed, suppressive immune cell populations, MDSC-granulocytic (I) and MDSC-monocytic (J), were analyzed with flow cytometry. (K and L) The biodistribution of Fc fusion peptides was analyzed in the tumor (K) and liver (L). All n ≥ 3, and significance levels were set at ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001. Error bars represent SEM.

Figure 7

Efficacy of Ad-Cab and Ad-Cab FT in xenograft in vivo models (A) Schematic of treatment schedules given to NS mice. Mice were first implanted in the right flank with 5 × 10⁶ A549 cells subcutaneously and 5 × 10⁶ PBMCs intraperitoneally. Treatment groups were divided into mice receiving Ad-5/3 Δ24, Ad-Cab, or Ad-Cab FT. (B) Before treatment, two mice implanted with PBMCs or not were sacrificed, and human CD3+ and CD45+ cells were analyzed in the peripheral blood. (C) Tumor growth was recorded. (D–F) After mice were sacrificed, NK cell activation (D), T cell activation (E), and T cell exhaustion (F) were analyzed in the tumor microenvironment. (G–H) Biodistribution of the Fc fusion peptide was then checked in the blood (G), tumor (H), and liver (I). All n ≥ 3, and significance levels were set at ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001. Error bars represent SEM.