A small molecule inhibitor of PTP1B and PTPN2 enhances T cell anti-tumor immunity

Abstract

The inhibition of protein tyrosine phosphatases 1B (PTP1B) and N2 (PTPN2) has emerged as an exciting approach for bolstering T cell anti-tumor immunity. ABBV-CLS-484 is a PTP1B/PTPN2 inhibitor in clinical trials for solid tumors. Here we have explored the therapeutic potential of a related small-molecule-inhibitor, Compound-182. We demonstrate that Compound-182 is a highly potent and selective active site competitive inhibitor of PTP1B and PTPN2 that enhances T cell recruitment and activation and represses the growth of tumors in mice, without promoting overt immune-related toxicities. The enhanced anti-tumor immunity in immunogenic tumors can be ascribed to the inhibition of PTP1B/PTPN2 in T cells, whereas in cold tumors, Compound-182 elicited direct effects on both tumor cells and T cells. Importantly, treatment with Compound-182 rendered otherwise resistant tumors sensitive to α-PD-1 therapy. Our findings establish the potential for small molecule inhibitors of PTP1B and PTPN2 to enhance anti-tumor immunity and combat cancer.

Fig. 1. Compound 182 is a reversible, competitive, active site inhibitor of PTP1B and PTPN2.

a, b Effect of Compound 182 (CMP-182) on PTP1B- and PTPN2-catalyzed 6,8-Difluoro-4-methylumbelliferyl phosphate (DiFMUP) hydrolysis. In (a) CMP-182 concentrations were 0 (●), 0.2 (■), 0.4(▲), and 0.6 nM (▼), respectively. b PTP1B- (●) and PTPN2- (o) catalyzed DiFMUP hydrolysis before and after jump dilution. c nanoDSF thermal shift first-derivative curves of 5 µM PTP1B or PTPN2 (black) and 5 µM PTP1B or PTPN2 + 50 µM CMP-182 (blue). d The structure of CMP-182 bound to PTP1B. Left, CMP-182 (stick representation) is shown bound to PTP1B (semi-transparent surface). The thiadiazolidinone group mimics phosphotyrosine (pTyr) and is located deep in the active site, while the tail of CMP-182 is located in a shallow groove on the surface of the protein. Right, close-up view of CMP-182 in the active site. The thiadiazolidinone moiety lies above the catalytic Cys215. The napthalene core of the compound sits on a hydrophobic surface of the protein with Phe182 from the WPD loop closed over the top. The methyl-butoxy tail is directed towards Gly259. In (a–c) representative results from two independent experiments are shown.

Fig. 2. Compound 182 promotes T cell signaling.

a OT-I cells isolated from wild type OT-I;Ptpn2^fl/fl (n = 5–6) versus OT-I;Lck-Cre;Ptpn2^fl/fl (n = 4) mice were stimulated with 1 nM of the cognate ovalbumin (OVA) peptide antigen SIINFEKL (N4) in the presence of vehicle (1% v/v DMSO) or Compound 182 (CMP-182; 1 μM) for 48 h and CD25 and CD69 mean fluorescent intensities (MFIs) in CD8⁺ OT-I cells were determined by flow cytometry. b OT-I cells (n = 6) were activated with 1 nM N4 for 16 h and rested in media supplemented with IL-2 (5 ng/ml) and IL-7 (0.2 ng/ml) for 2 days. On day 3, T cells were pre-treated with vehicle or 1 μM CMP-182 overnight and stimulated with 1 nM N4 plus/minus 1 μM CMP-182 in the presence of GolgiPlug™/GolgiStop™. IFN-γ⁺, TNF⁺ or Granzyme B⁺ (GRZMB) CD8⁺ T cells were analyzed by flow cytometry. c OT-I T cells isolated from wild-type OT-I; Ptpn2^fl/fl (n = 4) versus OT-I;Lck-Cre; Ptpn2^fl/fl (n = 4) mice were stained with CTV and stimulated with 0.5 nM N4 plus/minus 1 μM CMP-182 for 3 days and CTV dilution monitored by flow cytometry. d Naive CD8⁺ T cells (C57BL6) (n = 3) were preincubated with 10 μM CMP-182 for 4 h, the TCR cross-linked with α-mouse CD3ε (5 μg/ml) and goat anti-hamster IgG (20 μg/ml) and Y418 phosphorylated and activated SFK (p-SFK) MFIs determined by flow cytometry. e OT-I cells (C57BL/6) (n = 4) were stimulated with 1 nM N4 for 16 h, incubated plus/minus of 1 μM CMP-182 for 1 h and Y694 phosphorylated STAT-5 (p-STAT-5) in CD8⁺ OT-I cells assessed by flow cytometry. f OT-I cells (C57BL/6) (n = 6) were stimulated with 1 nM N4 for 16 h, incubated plus/minus 1 μM CMP-182 for 1 h, and then stimulated with IL-2 (5 ng/ml) plus/minus 1 μM CMP-182 and p-STAT-5 MFIs in CD8⁺ OT-I cells determined by flow cytometry. In (a–f) representative results (means ± SEM) from at least two independent experiments are shown. Significances in (a, c) were determined using a 1-way ANOVA Test and in (b, e) using a 2-tailed Mann-Whitney U Test. In (d, f) significances were determined using a 2-way ANOVA Test.

Fig. 3. Compound 182 represses AT3-OVA mammary tumor growth.

AT3-OVA mammary tumor cells were injected into the fourth inguinal mammary fat pads of 8-week-old C57BL/6 female mice. Mice were treated with Compound 182 (CMP-182; 10 mg/kg i.v.; n = 8) or saline (n = 8) on days (d) 6, 8, 10, 12, 14, 16, 18 and 21 after tumor cell implantation. a Tumor growth was monitored and tumor weights measured. b, c Tumor-infiltrating lymphocytes or splenocytes including CD44^hiCD62L^hi CD8⁺ and CD4⁺ central memory (CM) T cells, CD44^hiCD62L^lo CD8⁺ and CD4⁺ effector/memory (EM) T cells, CD19⁺ B cells, NK1.1⁺TCRβ⁻ (NK) cells, CD4⁺CD25⁺FoxP3⁺ regulatory T cells (T_regs) and granulocytic and monocytic CD11b⁺F4/80^hi/loLy6C⁺Ly6G^+/−myeloid-derived suppressor cells (MDSCs) were analyzed by flow cytometry. d Tumor-infiltrating T cells from (a) were stimulated with PMA/Ionomycin in the presence of Golgi Stop/Plug and stained for intracellular IFN-γ and TNF. Intracellular granzyme B (GRZMB), surface PD-1, TIM-3, and Lag-3 were detected in unstimulated tumor-infiltrating CD8⁺ T cells. e p-STAT-5 and p-SFK MFIs were assessed in splenic CD8 + EM T cells. In (a–e) representative results (means ± SEM) from at least two independent experiments are shown. Significance for tumor sizes in (a) was determined using a 2-way ANOVA Test and for tumor weights in (a) using a 2-tailed Mann-Whitney U Test. In (b–e) significances were determined using a 2-tailed Mann–Whitney U Test.

Fig. 4. Compound 182 represses MC38 and AT3 tumor growth.

a–c MC38 colon tumor cells were xenografted into the flanks of 8-week-old C57BL/6 male mice. Mice were treated with Compound 182 (CMP-182; 10 mg/kg i.v.; n = 5) or saline (n = 7) on days (d) 7, 9, 11, 13, 15, 17, and 19 after tumor cell implantation. a Tumor growth was monitored and tumor weights were measured. b, c Tumor-infiltrating lymphocytes (TILs) including CD44^hiCD62L^lo CD8⁺ and CD4⁺ effector/memory (EM) T cells, CD19⁺ B cells, NK1.1⁺TCRβ⁻ (NK) cells, CD4⁺CD25⁺FoxP3⁺ regulatory T cells (T_regs) and granulocytic and monocytic CD11b⁺F4/80^hi/loLy6C⁺Ly6G^+/− myeloid-derived suppressor cells (MDSCs) were analyzed by flow cytometry. In (b) intracellular granzyme B (GRZMB) and cell surface PD-1 and TIM-3 were detected in unstimulated tumor-infiltrating CD8⁺ T cells. d–f AT3 mammary tumor cells were injected into the fourth inguinal mammary fat pads of 8-week-old C57BL/6 female mice. Mice were treated with CMP-182 (10 mg/kg i.v.; n = 6) or saline (n = 8) on days (d) 10, 12, 14, 16, 18, 20, 22, 24, and 26 after tumor cell implantation. d Tumor growth was monitored and tumor weights measured. e, f TILs (CMP-182: n = 5; Saline: n = 7) including CD4⁺ EM T cells, CD19⁺ B cells, NK cells, T_regs and MDSCs were analyzed by flow cytometry. In (e) intracellular GRZMB and cell surface PD-1 and TIM-3 were detected in unstimulated tumor-infiltrating CD8⁺ T cells. In (a–f) representative results (means ± SEM) from at least two independent experiments are shown. Significance for tumor sizes in (a, d) was determined using a 2-way ANOVA Test and for tumor weights in (a, d) using a 2-tailed Mann–Whitney U Test. In (b, c, e, f) significances were determined using a 2-tailed Mann–Whitney U Test.

Fig. 5. Comparable anti-tumor immunity induced by Compound 182 and the deletion of PTP1B or PTPN2 in T cells.

AT3-OVA mammary tumor cells were injected into the fourth inguinal mammary fat pads of 8-week-old C57BL/6 (n = 8–11 in each group), Lck-Cre;Ptp1b^fl/fl (C57BL/6) (n = 12) and Lck-Cre;Ptpn2^fl/fl (C57BL/6) (n = 14) female mice. Mice were treated with Compound 182 (CMP-182; 10 mg/kg i.v.; n = 8) or saline (n = 11) on days (d) 7, 9, 11, 13, 15, 17, 19, and 21 after tumor cell implantation. a Tumor growth was monitored and b tumor weights were measured. c–e Tumor-infiltrating lymphocytes (TILs) (CMP-182: n = 8; Saline: n = 7; Lck-Cre;Ptp1b^fl/fl: n = 8; Lck-Cre;Ptpn2^fl/fl: n = 8) including CD44^hiCD62L^lo CD8⁺ and CD4⁺ effector/memory (EM) T cells, CD19⁺ B cells, NK1.1⁺TCRβ⁻ (NK) cells, CD4⁺CD25⁺FoxP3⁺ regulatory T cells (T_regs) and granulocytic and monocytic CD11b⁺F4/80^hi/loLy6C⁺Ly6G^+/−myeloid-derived suppressor cells (MDSCs) were analyzed by flow cytometry. In (d) Tumor-infiltrating T cells were stimulated with PMA/Ionomycin in the presence of Golgi Stop/Plug and stained for intracellular IFN-γ and TNF. Intracellular granzyme B (GRZMB), surface PD-1 and TIM-3 were detected in unstimulated tumor-infiltrating CD8⁺ T cells. In (a–e) representative results (means ± SEM) from at least two independent experiments are shown. Significance for tumor sizes in (a) was determined using a 2-way ANOVA Test and for tumor weights in (b) and for TILs in (c–e) using a 1-way ANOVA Test. In (b, c, e) significances were determined using a 2-tailed Mann–Whitney U Test (^#p < 0.05, ^##p < 0.01, ^###p < 0.001) where indicated.

Fig. 6. The effects of Compound 182 on anti-tumor immunity are reliant on T cells but not NK cells.

AT3-OVA mammary tumor cells were injected into the fourth inguinal mammary fat pads of 8-week-old Mx1-Cre;Ptpn2^fl/fl (C57BL/6) female mice (n = 9-11 in each group). Mice were treated with poly I:C (250 μg/kg i.v.) to inducibly delete PTPN2 on days (d) 17, 19, and 21 after tumor cell implantation. a Tumor growth was monitored and tumor weights were measured. b Tumor-infiltrating lymphocytes (TILs) (Ptpn2^fl/fl: n = 8; Mx1-Cre;Ptpn2^fl/fl: n = 6) including CD4⁺ and CD8⁺ T cells, CD44^hiCD62L^lo CD8⁺ and CD4⁺ effector/memory (EM) T cells, CD19⁺ B cells, NK1.1⁺TCRβ⁻ (NK) cells, CD11b⁺F4/80^hiLy6C⁻Ly6G⁻tumor-associated macrophages (TAMs), granulocytic CD11b⁺F4/80^hi/loLy6C^intLy6G⁺(gMDSCs) and monocytic CD11b⁺F4/80^hi/lo Ly6C⁺Ly6G⁻(mMDSCs) myeloid-derived suppressor cells and CD11c⁺ (DCs) dendritic cells were analyzed by flow cytometry. c, d Wild type MC38 (70%) and B2m^−/− MC38 (30%) tumor cells were xenografted into the flanks (subcutaneous; s.c.) of either 8-week-old c) Ncr1-Cre;Ptp1b^fl/fl (C57BL/6) or d) 8 week-old Ncr1-Cre;Ptpn2^fl/fl (C57BL/6) male mice or the corresponding floxed control mice and tumor growth and survival (Ptp1b^fl/fl: n = 9; Ncr-Cre;Ptp1b^fl/fl: n = 7; Ptpn2^fl/fl: n = 15; Ncr1-Cre;Ptpn2^fl/fl: n = 9) monitored. e AT3-OVA mammary tumor cells were injected into the fourth inguinal mammary fat pads of 8-week-old Rag1^−/− (C57BL/6) female mice. Mice were treated with Compound 182 (CMP-182; 10 mg/kg i.v.; n = 6) or saline (n = 6) on days 9, 11, 13, 15, 17, 19, and 21 after tumor cell implantation and tumor growth was monitored. In (a–e) representative results (means ± SEM) from at least two independent experiments are shown. Significance for tumor sizes in (a, c–e) was determined using a 2-way ANOVA Test and for tumor weights in (a) using a 2-tailed Mann–Whitney U Test. In (b) significance was determined using a 2-tailed Mann–Whitney U Test.

Fig. 7. Effects of Compound 182 on AT3-OVA mammary tumor growth, STAT-1 signaling and T cell infiltrates in Lck-Cre;Ptpn2^fl/fl mice.

a–f AT3-OVA mammary tumor cells were injected into the fourth inguinal mammary fat pads of 8 week-old Ptpn2^fl/fl (n = 6) and Lck-Cre;Ptpn2^fl/fl (C57BL/6) (n = 5-6 in each group) female mice. Mice were treated with Compound 182 (CMP-182; 10 mg/kg i.v.; n = 6) or saline (n = 6) on days (d) 7, 9, 11, 13, 15, 17, 19 and 21 after tumor cell implantation. a Tumor growth was monitored and b tumor weights were measured. c Tumor-infiltrating lymphocytes including CD44^hiCD62L^lo CD8⁺ and CD4⁺ effector/memory (EM) T cells, CD19⁺ B cells, NK1.1⁺TCRβ⁻ (NK) cells, CD4⁺CD25⁺FoxP3⁺ regulatory T cells (T_regs) and granulocytic and monocytic CD11b⁺F4/80^hi/loLy6C⁺Ly6G^+/−myeloid-derived suppressor cells (MDSCs) were analyzed by flow cytometry. d Tumor-infiltrating T cells from (a) were stimulated with PMA/Ionomycin in the presence of Golgi Stop/Plug and stained for intracellular IFN-γ and TNF. Intracellular granzyme B (GRZMB) was detected in unstimulated tumor-infiltrating CD8⁺ T cells. AT3-OVA tumors were processed for e immunohistochemistry staining for p-STAT-1, or CD3 (counterstained with hematoxylin) or f qPCR monitoring for the expression of STAT-1 target genes. g AT3-OVA mammary tumor cells were injected into the fourth inguinal mammary fat pads of C57BL/6 mice. Mice were treated with CMP-182 (10 mg/kg i.v.) or saline on days 6, 8, 10, 12, 14, 16, 18, and 21 after tumor cell implantation; the resultant tumor growth curves are shown in Fig. 4a. The resulting AT3-OVA tumors were processed for immunohistochemistry staining for STAT-1 Y701 phosphorylation (p-STAT-1), or CD3 (counterstained with hematoxylin). In (a–d, f) representative results (means ± SEM) from at least two independent experiments are shown. In (e, g) micrographs are representative of two independent experiments with 5 mice per group. Significance for tumor sizes in (a) was determined using a 2-way ANOVA Test and for tumor weights in (b) using a 1-way ANOVA Test. In (c, d) significances were determined using a 1-way ANOVA Test and a 2-tailed Mann–Whitney U Test (^#p < 0.05, ^##p < 0.01) where indicated. In (f) significances were determined using a 1-way ANOVA Test.

Fig. 8. Compound 182 promotes STAT-1 signaling and increases T cell infiltrates in AT3 tumors.

a AT3 control cells (Ctl sgRNA: n = 10) or those in which PTPN2 had been deleted by CRISPR RNP (Ptpn2 sgRNA: n = 10) were injected into the fourth inguinal mammary fat pads of 8-week-old female C57BL/6 mice and tumor growth was monitored. b Tumor-infiltrating lymphocytes (Ctl sgRNA: n = 9; Ptpn2 sgRNA: n = 8) including total CD4⁺ and CD8⁺ T cells, CD44^hiCD62L^hi CD8⁺ and CD4⁺ central memory (CM) T cells, CD44^hiCD62L^lo CD8⁺ and CD4⁺ effector/memory (EM) T cells, NK1.1⁺TCRβ⁻ (NK) cells, CD11b⁺F4/80^hiLy6C⁻Ly6G⁻tumor-associated macrophages (TAMs) and granulocytic and monocytic CD11b⁺F4/80^hi/loLy6C⁺Ly6G^+/−myeloid-derived suppressor cells (MDSCs) were analyzed by flow cytometry. c AT3 tumors were processed for qPCR monitoring for the expression of STAT-1 target genes (Ctl sgRNA: n = 7; Ptpn2 sgRNA: n = 7). d, e AT3 mammary tumor cells were injected into the fourth inguinal mammary fat pads of 8 week-old Ptpn2^fl/fl (n = 10) and Lck-Cre;Ptpn2^fl/fl (n = 6–7 per group) (C57BL/6) female mice. Mice were treated with CMP-182 (10 mg/kg i.v.; n = 6) or saline (n = 7) on days 7, 9, 11, 13, 15, 17, 19 and 21 after tumor cell implantation. d Tumor growth was monitored and tumor weights were measured. e The resulting AT3 tumors were processed for immunohistochemistry staining for p-STAT-1 (Y701) and CD3 (counterstained with hematoxylin). In (a–d) representative results (means ± SEM) from at least two independent experiments are shown. In (e) micrographs are representative from two independent experiments with 5 mice per group. Significance for tumor sizes in (a, d) was determined using a 2-way ANOVA Test and for tumor weights in a) using a 2-tailed Mann–Whitney U Test and (d) using a 1-way ANOVA Test. In (b-c and where indicated by ## p < 0.01) significances were determined using a 2-tailed Mann–Whitney U Test.

Fig. 9. Compound 182 sensitizes AT3 tumors to PD-1 checkpoint blockade.

a AT3 mammary tumor cells were injected into the fourth inguinal mammary fat pads of 8-week-old C57BL/6 female mice. Mice were treated with Compound 182 (CMP-182; 10 mg/kg i.v.; n = 6) or saline (n = 8) on days 10, 12, 14, 16, 18, 20, 22, 24, and 26 after tumor cell implantation; tumor growth curves are shown in Fig. 4d. The resulting AT3 tumors were processed for qPCR monitoring for the expression of STAT-1 target genes. b–f AT3 mammary tumor cells were injected into the fourth inguinal mammary fat pads of 8-week-old C57BL/6 female mice (n = 6–8 per group). Mice were treated with CMP-182 (10 mg/kg i.v.; n = 6) or saline (n = 8) on days 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, and 27 and α-PD-1 (n = 6) twice per week (200 μg i.p.) after tumor cell implantation. b Tumor growth was monitored and c tumor weights were measured. d–f Tumor-infiltrating lymphocytes including d, f CD44^hiCD62L^lo CD8⁺ and CD4⁺ effector/memory (EM) T cells, NK1.1⁺TCRβ⁻ (NK) cells, CD4⁺CD25⁺FoxP3⁺ regulatory T cells (T_regs) and granulocytic and monocytic CD11b⁺F4/80^hi/loLy6C⁺Ly6G^+/−myeloid-derived suppressor cells (MDSCs) were analyzed by flow cytometry. In (e) tumor-infiltrating T cells from (b) were stimulated with PMA/Ionomycin in the presence of Golgi Stop/Plug and stained for intracellular IFN-γ and TNF. Intracellular granzyme B (GRZMB), PD-1, and TIM-3 were detected in unstimulated tumor-infiltrating CD8⁺ T cells. In (a–f) representative results (means ± SEM) from at least two independent experiments are shown. Significance for tumor sizes in (b) was determined using a 2-way ANOVA Test and for tumor weights in (c) using a 1-way ANOVA Test. In (d–f) significances were determined using a 1-way ANOVA Test and a 2-tailed Mann–Whitney U Test (^#p < 0.05, ^##p < 0.01, ^###p < 0.001) where indicated.

Fig. 10. Compound 182 does not promote systemic inflammation and autoimmunity.

a–c AT3-OVA mammary tumor cells were implanted into the fourth inguinal mammary fat pads of 8-week-old C57BL/6 female mice. Mice were treated with Compound 182 (CMP-182; 10 mg/kg i.v.; n = 8) or saline (n = 8) on days 6, 8, 10, 12, 14, 16, 18, and 21 after tumor cell implantation. a Serum cytokines were determined by flow cytometry using a BD Cytokine Bead Array (BD Biosciences. b Livers, lungs, salivary glands, and colons were fixed in formalin and processed for histological assessment (hematoxylin and eosin: H&E). Livers were also processed for Sirius Red staining. c Serum anti-nuclear antibodies (ANA) and serum liver enzymes AST and ALT in CMP-182-treated mice. d–h AT3-OVA mammary tumor cells were implanted into the fourth inguinal mammary fat pads of Mx1-Cre;Ptpn2^fl/fl mice. Mice were treated with poly I:C (250 μg/kg i.v.) to inducible delete PTPN2 on day 7, 9, and 11 after tumor cell implantation; the resultant tumor growth curves are shown in Fig. 6a. d Gross phenotype of Mx1-Cre;Ptpn2^fl/fl mice. e Spleen weight (n = 9-11 per group) and splenic CD44^hiCD62L^lo CD8⁺ and CD4⁺ effector/memory (EM) T cells and inflammatory monocytes in Mx1-Cre;Ptpn2^fl/fl (n = 6-8 per group) mice. f Serum cytokines in Mx1-Cre;Ptpn2^fl/fl mice (n = 10 per group) were determined by flow cytometry using a BD Cytokine Bead Array (BD Biosciences). g Serum liver enzymes AST and ALT in Mx1-Cre;Ptpn2^fl/fl mice (n = 10 per group). h Livers from Mx1-Cre;Ptpn2^fl/fl mice were fixed in formalin and processed for histological assessment (hematoxylin and eosin: H&E; Sirius Red). In (a, c, e–g) representative results (means ± SEM) from at least two independent experiments are shown. In (b and h) micrographs are representative of tissues from at least 5 mice per group. Significances were determined using a 2-tailed Mann–Whitney U Test.