The PD-1/PD-L1 axis modulates the natural killer cell versus multiple myeloma effect: a therapeutic target for CT-011, a novel monoclonal anti-PD-1 antibody

Abstract

T-cell expression of programmed death receptor-1 (PD-1) down-regulates the immune response against malignancy by interacting with cognate ligands (eg, PD-L1) on tumor cells; however, little is known regarding PD-1 and natural killer (NK) cells. NK cells exert cytotoxicity against multiple myeloma (MM), an effect enhanced through novel therapies. We show that NK cells from MM patients express PD-1 whereas normal NK cells do not and confirm PD-L1 on primary MM cells. Engagement of PD-1 with PD-L1 should down-modulate the NK-cell versus MM effect. We demonstrate that CT-011, a novel anti-PD-1 antibody, enhances human NK-cell function against autologous, primary MM cells, seemingly through effects on NK-cell trafficking, immune complex formation with MM cells, and cytotoxicity specifically toward PD-L1(+) MM tumor cells but not normal cells. We show that lenalidomide down-regulates PD-L1 on primary MM cells and may augment CT-011's enhancement of NK-cell function against MM. We demonstrate a role for the PD-1/PD-L1 signaling axis in the NK-cell immune response against MM and a role for CT-011 in enhancing the NK-cell versus MM effect. A phase 2 clinical trial of CT-011 in combination with lenalidomide for patients with MM should be considered.

Figure 1

PD-1 is differentially expressed on primary human NK cells as a function of MM disease activity. (A) By flow cytometry, PD-1 expression was measured in CD56⁺CD3⁻ NK cells isolated from healthy donors (n = 5 donors). At baseline, virtually no expression was observed (1.4% ± 0.35% [SD] of NK cells per healthy donor sample); however, after IL-2 (150 IU/mL) stimulation for 48 hours, PD-1 expression became evident on an average of 16% (± 6%) of healthy donor NK cells, P = .001, a 12-fold increase (± 5-fold) in PD-1 expression. A representative sample is shown on left, and MRFI (mean ± SD) of pre–IL-2 baseline and post–IL2 PD-1 expression is shown on right. (B) In contrast to healthy donors, PD-1 is observed on freshly isolated NK cells from patients with MM (n = 5), a representative example is shown on left and MRFI (mean ± SD) of healthy donors and freshly isolated patient NK cells on right.

Figure 2

Low-dose lenalidomide modulates PD-L1 expression on MM tumor cells. (A) The MM cell line RPMI8226 expresses high levels of PD-L1; however, after culture for 24 hours in lenalidomide (5nM), expression was markedly reduced (baseline, MFI 174 ± 30 vs lenalidomide-treated MFI, 47 ± 35; P = .01) without effect on cell viability (n = 3 independent experiments, top panel is representative result, bottom panel is MRFI of PD-L1 expression at baseline and in lenalidomide-treated cells). (B) We confirm previous observations that PD-L1 is uniquely expressed on CD38⁺CD138⁺ primary malignant plasma cells in whole-marrow aspirates (representative finding from n = 3 MM patients). (C) As in cell line experiments, lenalidomide (5nM) down-regulates PD-L1 expression without affecting cell viability (top panel is representative finding from n = 3 MM patients, bottom panel is MRFI of PD-L1 on control- and lenalidomide-treated cells, and dotted lines are isotype controls).

Figure 3

CT-011 and lenalidomide enhance NK-cell trafficking toward MM cell line media and MM patient serum. (A) To test whether or not CT-011 affects NK-cell trafficking, Transwell migration assays were conducted whereby NK cells were cultured in RPMI 1640 media with 10% FBS and 150 IU/mL IL-2 with isotype control or CT-011 for 72 hours. Data shown summarize 3 independent experiments. All differences within treatment conditions are statistically significant. Across treatment conditions, CT-011 enhanced migration beyond control into U266 media (*P < .05) and MM patient serum (**P < .05). (B) By flow cytometry, we systematically evaluated for changes in the surface expression of trafficking antigens known to be expressed by human NK cells. Only CXCR4 showed a statistically significant increase in expression in response to 72 hours in 150 IU/mL IL-2 with CT-011, P < .01 (representative data from one patient shown, n = 5). (C) Transwell migration assays were conducted with NK cells pretreated for 72 hours in 150 IU/mL IL-2 with control or CT-011 into normal media or media enriched with SDF-1α. CT-011 enhanced NK-cell trafficking into SDF-1α–enriched media (, data collated from 3 independent experiments), suggesting a functional relevance to the increase in CXCR4 expression observed on NK cells in response to these agents. CT-011 enhanced migration over control condition (*P < .05). However, when NK cells were pretreated with the CXCR4 inhibitor AMD-3100 (10 μg/mL) for 90 minutes before assay, trafficking was virtually abolished in all conditions (, data from 2 independent experiments, pairwise comparisons within treatments; P < .05).

Figure 4

CT-011 enhances immune complex formation between effector NK cells and target MM cells. (A) A representative example of the flow cytometric technique to evaluate immune complex formation is shown, where primary, human NK cells, cultured as before for 72 hours in IL-2 and control mAb or CT-011, were stained with CFSE. Primary MM tumor cell targets were isolated from MM patient marrow aspirates and stained with PKH. Flow gates were created with PKH⁺ events representing MM target cells on the y-axis and CFSE⁺ effector NK cells on the x-axis. Double-positive events (ie, PKH⁺CFSE⁺) were interpreted as immune complex formations (shaded right top panel of flow diagrams). The left panel is representative of findings in control conditions; the right panel is representative of findings where NK cells were pretreated with CT-011. (B) Immune complex formation between NK cells and the K562, U266, RPMI8226 cell lines as well as with primary MM tumor cells was evaluated. At baseline, less than 1% of gated events in all conditions were PKH⁺CFSE⁺. However, NK cells pretreated with CT-011 led to statistically significant increases in immune complex formation with K562 (*P = .02), U266 (**P = .007), RPMI8226 (***P = .01), and primary MM tumor cell targets (****P = .009). All results shown are from at least 3 independent experiments with each target. (C) In this figure, total PBMCs obtained from patients with MM (rather than purified NK cells as in panels A-B) were treated for 48 hours in IL-2 with control or CT-011 mAb and immune complex formation in response to CT-011 against autologous CD138⁺PD-L1⁺ MM tumor cells or CD138⁻PD-L1⁻ cellular marrow elements was compared. Compared with control-treated effector cells, CT-011 increased immune complex formation with MM tumor cells (*P < .001) but not against normal marrow cells. Data shown are from 3 independent experiments in n = 3 patients.

Figure 5

CT-011 enhances NK-cell GrB and IFN-γ production against PD-L1–bearing tumor cell targets. (A) By using an ELISPOT effector-based assay, we studied the effects of CT-011 on GrB production by primary, human NK cells against the K562 and RPMI8226 cell lines (E:T = 10:1). After 72 hours of pretreatment with 150 IU/mL IL-2 and control or CT-011 mAb, the latter led to statistically significant increases in NK-cell degranulation of GrB against K562 (*P < .05) and RPMI8226 (**P < .05; data shown are representative of 3 independent experiments against each line). (B) By using the same effector-based ELISPOT cytotoxicity assay, we found that CT-011 also enhanced IFN-γ production by NK cells (pretreated for 72 hours in IL-2 150 IU/mL and control or CT-011 mAb) against primary MM tumor cell targets (*P = .01; data shown from 3 independent experiments). (C) By using a target-based cytotoxicity assay, primary, we pretreated human NK cells for 72 hours in IL-2 150 IU/mL and control, lenalidomide (5nM), CT-011, or the combination. Lenalidomide and CT-011 statistically significantly increased cytotoxicity against RPMI8226 cell line targets (E:T 50:1, *P = .03, ET:100:1, **P = .02; data shown are from 3 independent experiments at both E:T ratios). (D) PBMCs and marrow aspirates were obtained from patients with MM (n = 3) and CD138⁺ tumor cells were isolated from the whole marrow aspirate. Effector cells were cultured in IL-2 and control or CT-011 for 48 hours and CD138⁺PD-L1⁺ MM tumor cells and CD138⁻PD-L1⁻ cellular marrow fraction served as target populations. On the left, cytotoxicity is enhanced against autologous CD138⁺PD-L1⁺ MM tumor cells (*P = .005; data shown from n = 3 independent experiments), yet no increase in cytotoxicity is conferred against autologous CD138⁻PD-L1⁻ cellular marrow elements (right; P = ns).