A PSMA-Targeted Tri-Specific Killer Engager Enhances NK Cell Cytotoxicity against Prostate Cancer

Abstract

NK cell tumor infiltration is associated with good prognosis in patients with metastatic castration-resistant prostate cancer (mCRPC). NK cells recognize and kill targets by a process called natural cytotoxicity. We hypothesized that promoting an antigen-specific synapse with coactivation may enhance NK cell function in mCRPC. We describe a tri-specific killer engager (TriKE) construct that engages with the activating receptor CD16 on NK cells and prostate-specific membrane antigen (PSMA) on mCRPC cells and has an IL15 moiety that is essential for NK cell survival, proliferation, and priming. We show that the PSMA TriKE specifically binds to PSMA-expressing cells and significantly enhances expansion, degranulation, and cytokine production of NK cells derived from healthy donors or patients with prostate cancer. Bystander killing of PSMA-negative tumor cells was also achieved with PSMA TriKE treatment when cocultured with PSMA-positive cells, suggesting potential PSMA TriKE benefit in controlling tumor antigen escape. When tested under physiologic conditions recapitulating the mCRPC tumor microenvironment, NK cells treated with PSMA TriKE and prolonged exposure to hypoxia or myeloid-derived suppressor cells maintained their potent function whereas IL15-treated NK cells showed greatly impaired cytotoxicity. Finally, in vivo testing of PSMA TriKE showed improved tumor control and survival of mice as compared with IL15-treated and untreated control groups. In conclusion, PSMA TriKE demonstrates potential as a new therapy for advanced prostate cancer by providing additional signals to NK cells to maximize their antitumor potential in prostate cancer, especially in the setting of a hostile tumor microenvironment.

Figure 1.. PSMA TriKE induces specific antigen binding and NK cell expansion.

(A) Schematic of a PSMA TriKE molecule consisting of a humanized camelid-derived anti-CD16 V_HH arm, an IL-15 molecule and anti-PSMA single chain variable fragment (scFv). (B) Histograms showing His tag staining on C4–2 wild-type (WT) or C4–2 PSMA knockout (KO) after 30 minutes incubation with 30nM PSMA TriKE. (C-D) Representative histograms of CellTrace Violet (CTV) dye dilution indicating proliferation of NK cells in (C) and T cells in (D) within PBMC after 7 days culture with no treatment (NT), IL-15 (3 nM) or PSMA TriKE (3 nM) along with cumulative results of cell fold expansion with indicated treatments; N=9–12; mean ± SEM; One-way ANOVA with Dunnett’s multiple comparisons test; **** p < 0.0001, * p < 0.05.

Figure 2.. PSMA TriKE induces antigen specific NK cell activation and cytolytic activity.

(A) Pooled NK cell degranulation (measured by surface expression of CD107a) and cytokine production of interferon (IFN)-γ and tumor necrosis factor (TNF)-α after 5 hours of culture alone or co-culture with C4–2 WT or C4–2 PSMA KO at effector to target (E:T) ratio of 2:1 with equimolar doses of IL-15 (3 nM) or PSMA TriKE (3 nM); N=7; mean ± SEM; Two-way ANOVA with Tukey’s multiple comparison test; * p < 0.05, ** p < 0.01, *** p < 0.001. (B) Cumulative results of NK cell degranulation and cytokine production after 5 hours of co-culture with LNCaP and 22Rv1, which are prostate specific membrane antigen (PSMA) positive, or DU145 and PC3, which are PSMA negative at E:T ratio of 2:1 with NT, IL-15 (3 nM) or PSMA TriKE (3 nM); N=4; mean ± SEM; Two-way ANOVA with Tukey’s multiple comparisons test; * p < 0.05, ** p < 0.01. (C) Representative IncuCyte live cell imaging assay showing NK cells killing C4–2 WT or C4–2 PSMA KO at E:T ratio of 2:1 with NT or with equifunctional doses of IL-15 (0.06 nM) or PSMA TriKE (3 nM). Live cell count is normalized to targets alone control and time=0. (D) Cumulative results of live cell imaging at 24 and 48 hours; N=5; mean ± SEM; Two-way ANOVA with Tukey’s multiple comparisons test; * p < 0.05, ** p<0.01.

Figure 3.. PSMA TriKE can induce bystander killing of PSMA⁻ cells when PSMA⁺ cells are present.

(A) Histograms representing PSMA staining of C4–2 WT and C4–2 PSMA KO cells. (B) Schematic of combinations of C4–2 WT and C4–2 PSMA KO with or without NucLight Red (NLR) expression in each well for bystander killing assay. (C-D) Representative and cumulative results of IncuCyte live cell imaging assays showing NK cell killing NLR-expressing C4–2 WT, C4–2 PSMA KO, or C4–2 PSMA KO co-cultured with non-NLR expressing C4–2 WT at E:T ratio of 2:1 with equifunctional doses of (C) IL-15 (0.06 nM) or (D) PSMA TriKE (3 nM). Live cell count is normalized to targets alone control of the respective cell types and time=0; N=5; mean ± SEM; Two-way ANOVA with Tukey’s multiple comparisons test; * p < 0.05. (E) ViSNE plots of CyTOF analysis showing protein expression levels of CD69, CD16, NKG2D and NKp46 on NK cells cultured with C4–2 WT and treated with no treatment (NT), IL-15 (0.06 nM) or PSMA TriKE (3 nM) for 48 hours. Each plot was formed from data concatenated from 5 donors. (F) Pooled mean metal intensity (MMI) of CD69, CD16, NKG2D and NKp46 on NK cells shown in (E); N=5; mean ± SEM; One-way ANOVA with Dunnett’s multiple comparisons test; ** p<0.01.

Figure 4.. PSMA TriKE sustains NK cell proliferation, activation and cytolytic capacity after long term incubation in hypoxia.

(A) Schematic of experiments in hypoxia. (B) Representative histograms of Ki67 staining of NK cells treated with IL-15 (0.06 nM) or PSMA TriKE (3 nM) in 20% or 1% oxygen (O₂) conditions. (C) Cumulative results of Ki67⁺ NK cell population; N=5; mean ± SEM; Two-way ANOVA with Tukey’s multiple comparisons test; * p < 0.05, ** p < 0.01. (D) Pooled NK cell degranulation (measured by surface expression of CD107a) and cytokine production of interferon (IFN)-γ and tumor necrosis factor (TNF)-α after 5 hours of co-culture with C4–2 WT in 20% or 1% O₂ with IL-15 (0.06 nM) or PSMA TriKE (3 nM); N=5; mean ± SEM; Two-way ANOVA with Tukey’s multiple comparisons test; * p < 0.05, ** p < 0.01, ***p < 0.001. (E) Representative IncuCyte live cell imaging assay performed in 20% O₂ showing NK cells killing C4–2 WT at E:T ratio of 1:1 with NT or with equifunctional doses of IL-15 (0.06 nM) or PSMA TriKE (3 nM). Live cell count was normalized to targets alone control and time=0. (F) Pooled results of (E) at 24 and 48 hours; N=4; mean ± SEM; Two-way ANOVA with Dunnett’s multiple comparisons test; * p < 0.05, **p<0.01.

Figure 5.. PSMA TriKE partially abrogates MDSC-induced suppression of NK cells.

(A) Schematic of MDSC suppression co-culture system. (B) Representative histograms of Ki67 staining of NK cells treated with equifunctional doses of IL-15 (0.06 nM) or PSMA TriKE (3 nM) after being cultured alone or co-cultured with monocytes or MDSC. (C) Cumulative results of Ki67⁺ NK cell population; N=8; mean ± SEM; Two-way ANOVA with Tukey’s multiple comparisons test) ** p < 0.01 (D) Pooled NK cell degranulation (measured by surface expression of CD107a) and cytokine production of interferon (IFN)-γ and tumor necrosis factor (TNF)-α after 5 hours of co-culture with C4–2 WT with IL-15 (0.06 nM) or PSMA TriKE (3 nM); N=8; mean ± SEM; Two-way ANOVA with Tukey’s multiple comparisons test; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (E) Representative live cell imaging assay showing NK cells killing C4–2 WT at E:T ratio of 2:1 with equifunctional doses of IL-15 or PSMA TriKE. Live cell count is normalized to targets alone control and time=0. (F) Pooled results of (E) at 24 and 48 hours; N=6; mean ± SEM; Two-way ANOVA with Tukey’s multiple comparisons test; ** p < 0.01, ***p<0.001.

Figure 6.. PSMA TriKE induces increased function in primary patient NK cells as well as increased activation against patient derived xenograft tumor cells.

(A) Pooled NK cell degranulation (measured by surface expression of CD107a) and cytokine production of interferon (IFN)-γ and tumor necrosis factor (TNF)-α after 5 hours of co-culture of C4–2 WT with peripheral blood mononuclear cells (PBMC) derived from healthy donors, castration resistant or castration sensitive prostate cancer patients with no treatment (NT), IL-15 (3 nM) or PSMA TriKE (3 nM); N=11–15; mean ± SEM; Mixed effects analysis with Tukey’s multiple comparisons test; **** p < 0.0001. (B) Cumulative results of NK cell fold expansion after PBMC derived from healthy donors, castration resistant or castration sensitive prostate cancer patients were cultured for 7 days with NT, IL-15 or PSMA TriKE. (C) PSMA staining of patient-derived xenograft (PDX), LuCaP 58. (D) Cumulative results of NK cell degranulation and cytokine production after 5 hours of healthy donor-derived PBMC co-culture with C4–2 WT as control or LuCaP 58 at E:T ratio of 2:1 with NT, IL-15 (3 nM) or PSMA TriKE (3 nM); N=6; mean ± SEM; Two-way ANOVA with Tukey’s multiple comparisons test; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (E) Cumulative results of NK cell killing capacity indicated by live count of CellTrace Violet-labeled LuCaP 58, at PBMC:LuCaP 58 ratio of 5:1 or 10:1. Assay duration was 5 hours. LuCaP 58 cells were stained for PSMA to identify antigen-specific killing; N=5; mean ± SEM; One-way ANOVA with Dunnett’s multiple comparisons test; *** p < 0.001, **** p < 0.0001.

Figure 7.. PSMA TriKE slows tumor growth in vivo and prolongs survival of mice.

(A) Schematic of prostate cancer xenogeneic mouse model. (B) Spaghetti plot showing tumor volumes of individual mice treated with no treatment (NT), IL-15 or PSMA TriKE over time from two independent studies, with 33% of mice excluded from each group due to consistent tumor engraftment deficit. Full data can be found in Supplementary Figure 10. Tumor volumes were calculated using the formula: V= ½ (Length × Width²); N=8 (C) Pooled tumor volumes of mice from day 28 and 35; N=8; Mann-Whitney test; *p < 0.05. (D) Survival of mice represented in treated with NT, IL-15 or PSMA TriKE; N=8; Logrank test; * p< 0.05, ** p<0.01.