Targeting the C-terminus of galectin-9 induces mesothelioma apoptosis and M2 macrophage depletion

Abstract

Galectin-9 has emerged as a promising biological target for cancer immunotherapy due to its role as a regulator of macrophage and T-cell differentiation. In addition, its expression in tumor cells modulates tumor cell adhesion, metastasis, and apoptosis. Malignant mesothelioma (MM) is an aggressive neoplasm of the mesothelial cells lining the pleural and peritoneal cavities, and in this study, we found that both human MM tissues and mouse MM cells express high levels of galectin-9. Using a novel monoclonal antibody (mAb) (Clone P4D2) that binds the C-terminal carbohydrate recognition domain (CRD) of galectin-9, we demonstrate unique agonistic properties resulting in MM cell apoptosis. Furthermore, the P4D2 mAb reduced tumor-associated macrophages differentiation toward a protumor phenotype. Importantly, these effects exerted by the P4D2 mAb were observed in both human and mouse in vitro experiments and not observed with another antigalectin-9 specific mAb (clone P1D9) that engages the N-terminus CRD of galectin-9. In syngeneic murine models of MM, P4D2 mAb treatment inhibited tumor growth and improved survival, with tumors from P4D2-treated mice exhibited reduced infiltration of tumor-associated M2 macrophages. This was consistent with an increased production of inducible nitric oxide synthase, which is a major enzyme-regulating macrophage inflammatory response to cancer. These data suggest that using an antigalectin 9 mAb with agonistic properties similar to those exerted by galectin-9 may provide a novel multitargeted strategy for the treatment of mesothelioma and possibly other galectin-9 expressing tumors.

Keywords: Lectins; agonist monoclonal antibody; galectin 9; immunotherapy; macrophages; mesothelioma.

Figure 1.

Profiling of galectin-9 tissue expression in MM tumors. (a–c) Galectin-9 staining on three representative MM samples; (d) Gal9 staining on a representative normal mesothelial lining. Original magnification 200×.

Figure 2.

Generation of antigalectin-9 mAb and corresponding specificity and cross-reactivity. (a) Binding of generated galectin-9 mAbs was evaluated via ELISA plates coated with human or mouse recombinant galectin-9. Mouse serum was used as positive control. Averages of optical densities (OD) are shown as an index of binding. (b) Binding of P4D2 and P1D9 mAbs was compared among human recombinant galectin-9 (hGalectin-9M) and a more stable version of human recombinant galectin-9 missing the linker peptide between N- and C-CRD (hG9NC). Commercially available galectin-9 mAb, clone 9M1-3, was used as a control. (c) hG9G8 (Gal-9 N-terminus CRD only) or hG8G9 (Gal-9 C-terminus CRD only) fusion proteins were used to evaluate P4D2 and P1D9 mAb CRD binding specificity. Commercially available galectin-9 mAb, clone 9S2-3, was used as a control. (d) P4D2 mAb Fv sequencing and modeling of its interactions with galectin-9 was structurally analyzed with SAbPred modeling software. Briefly, RNA was extracted from the P4D2 Hybridomas using TRIzol® Reagent (Thermo Fisher Scientific) and converted to cDNA with SuperScript™ III First-Strand Synthesis System (Invitrogen). Contaminating V_L cDNA from the P3X63Ag8.653 cells were labeled using 5ʹ biotinylated P3 CDR-L3 primers (5’-CAGCACATTAGGGAGCTTACACG-3’) (IDT) and then removed using Streptavidin-linked Dynabeads. The enriched cDNA was amplified using primers K6b and revCk for V_L, and primers H2 and IgG2a for V_H. Both V_L and V_H amplicons were sequenced using Sanger sequencing and annotated in NCBI igBLAST with an IMGT number.²⁵ All primers were designed as previously described.²⁶ IMGT numbers for V_L and V_H were entered into the SAbPred modeling software to generate a protein data bank (PDB) file. The PDB for P4D2 V_L/V_H was entered, along with the galectin-9 crystal structure (PDB ID: 3WV6) into the SAbPred epitope modeling software to identify their binding sites.²⁷ Interactions between the predicted P4D2 Fv structure and galectin-9 crystal structure were then analyzed and results showed binding exclusively to the C-terminal CRD of galectin-9. Galectin-9 amino acids that interact with P4D2 Fv identified with SAbPred are listed.

Figure 3.

P4D2 mAb exerts agonist effects in inducing apoptosis in MM cells. (a) Human MM cells (ROB and Mill) were treated with P4D2 or P1D9 mAbs, and viability assessed with an MTT assay. Controls included the human stable recombinant galectin-9 (hG9NC) and no treatment (Ctrl). Differences between Ctrl and P4D2 as well as Ctrl and hG9NC were statistically significant with P ≤ 0.01; n = 3 (*). A viability assay was used to evaluate the effects of P4D2 and P1D9 mAbs on mouse MM cells (CRH5 and EOH6). Mouse stable recombinant galectin-9 (mG9NC) was included for comparison. Differences between Ctrl and P4D2 as well as Ctrl and mG9NC were statistical significant with P ≤ 0.01; n = 3 (*). (b) Analysis of apoptosis for human (ROB and Mill) and mouse (CRH5 and EOH6) MM cells after P4D2 or recombinant galectin-9 (hG9NC or mG9NC) treatment as evaluated by flow cytometry. Percentages of PI- Annexin V+ (early apoptotic) cells and PI+ Annexin V+ (late apoptotic) cells are shown. Statistically significant differences between treatment and no treatment (Ctrl) were assessed with two-way ANOVA followed by the Bonferroni test and indicated with *, P ≤ 0.01, n = 3.

Figure 4.

Treatment with a P4D2 mAb shifts primary human monocyte differentiation away from protumor phenotype. Human primary monocytes were differentiated with human AB serum in presence of P4D2 or P1D9 mAbs. Two other conditions included: (1) human serum and (2) human stable galectin-9 (hG9NC). (a) Representative figures from the flow cytometric analysis of CD68+ and CCR5+ mature macrophages. (b) Percentages of CD68+ and CCR5+ cells are showed for the different treatments. Statistical differences among treatments (n = 3/group) were assessed with one-way ANOVA followed by the Bonferroni test and indicated with *P ≤ 0.01. (c) Illustrative schematic of monocyte-macrophage differentiation experiment. (d) Monocyte-macrophage differentiation was evaluated using supernatants from ROB MM cells (conditioned media) or from ROB MM cells treated with P4D2 mAb (conditioned media + P4D2). P4D2-treated cells were used as control. Flow cytometry representative images show the reduction of CD68+ and CCR5+ mature macrophages induced by P4D2-treated ROB media. (e) Differences in the percentage of CD68+ and CCR5+ cells in ROB media compared to P4D2 mAb-treated ROB media, or P4D2 (n = 3/group) were evaluated using one-way ANOVA followed by the Bonferroni test and indicated with *P ≤ 0.001. (f) Maturation of primary monocytes was also measured with real-time PCR using primers for the M2 marker, MARCO. Differences in ROB media compared to P4D2 mAb-treated ROB media for MARCO were defined using one-tailed paired Student’s t test (n = 3/group) and indicated with *P ≤ 0.001.

Figure 5.

P4D2 mAb shifts mouse monocyte differentiation toward an M1 phenotype. Mouse bone marrow-derived monocytes were differentiated to mature macrophages using either M-CSF or supernatant from mouse MM CRH5 cells. During M-CSF-driven differentiation, cells were also treated with P4D2 mAb (M-CSF + P4D2) while controls only received m-CSF. Differentiation with MM supernatant was instead performed using media from CRH5 mouse MM cells untreated (conditioned media) or treated for 24 h with P4D2 mAb (conditioned media +P4D2). (a and b) The left panels contain representative flow cytometry images showing the effects of P4D2 mAb on F480+ cells. On the right, percentages of F480+ macrophages are shown for the different treatments. Differences between CRH5 media compared to media from P4D2 mAb-treated CRH5 were evaluated using one-tailed paired Student’s t test (n = 3/group) and indicated with *P ≤ 0.001. (c and d) Phenotype of the differentiated macrophages was also investigated using markers for M1 (CD38^hi) and 2 (Egr2+). On the top left, representative flow cytometry images show the effects of P4D2 mAb on F480+ CD38^hi M1. On the top right, percentages of F480+ CD38^hi cells are indicated for the different treatments. On the bottom left, representative flow cytometry images show the effects of P4D2 mAb on F480+ Egr2+ M2. On the bottom right, percentages of F480+ CD38^hi cells are displayed for the different treatments. (e) M1/M2 ratios were calculated and indicated for the different conditions. (f) Percentages of live cells were showed for the different treatments. For (b–d) statistical significance for experiments was evaluated using one-tailed paired Student’s t test (n = 3/group) and indicated with *P ≤ 0.01.

Figure 6.

Treatment with P4D2 mAb hinders tumor growth and improves survival in MM animal models. BALB/c mice with subcutaneous CRH5 MM tumors were treated with P4D2 or P1D9 mAbs. Control mice were left untreated. (a) Tumor size is shown for the different treatments. Differences between control and P4D2 mAb groups were compared with two-way ANOVA followed by the Bonferroni multiple comparison test (n = 5/group) and indicated with *P ≤ 0.01. (b) Mice treated with P4D2 mAb (n = 5/group) show increased survival compared to controls *P ≤ 0.01. (c) Survival curves for mice carrying intraperitoneal CRH5 MM tumors, treated or not-treated with P4D2 mAb. Differences in P4D2 mAb treated vs. controls were evaluated using Kaplan-Meier curves with log-rank test and indicated with *P ≤ 0.01 and n = 5.

Figure 7.

P4D2 mAb alters intratumor M1/M2 ratio while increasing iNOS and reducing intratumor cytokines. CRH5 MM tumors from mice treated with P4D2 mAbs and untreated controls were excised and intratumor macrophages characterized. (a) Immunofluorescence using anti-F480-FITC was performed to quantify numbers of TAMs. The left panel shows representative images of stained cells and the right panel shows percentages of F480+ macrophages (n = 5/group), *P ≤ 0.001. (b) Percentages of TAMs were also evaluated using flow cytometry analysis. (c) TAM phenotypes were then characterized using markers as CD38 for M1 and Egr2 for M2. M1 and M2 phenotypes were also evaluated using real-time PCR for M1 (iNOS) and M2 (Arg1). On the left, representative figures from the flow cytometry analysis of F480+ CD38^hi M1 (top) and F480+ Egr2+ M2 (bottom). Percentages of F480+ CD38^hi (top) and F480+ Egr2+ (bottom) cells are shown. On the right, data from RT-PCR are shown for iNOS (top) and Arg1 (bottom). M1/M2 and iNOS/Arg1 ratios are shown in (d) and (e), respectively. For (b–e), differences between the two groups were compared using one-tailed paired Student’s t test (n = 5/group) and indicated by *P ≤ 0.01.