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Enhanced preclinical antitumor activity of M7824, a bifunctional fusion protein simultaneously targeting PD-L1 and TGF-β

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Science Translational Medicine  17 Jan 2018:
Vol. 10, Issue 424, eaan5488
DOI: 10.1126/scitranslmed.aan5488
  • Fig. 1 M7824 binds efficiently and specifically to PD-L1 and TGF-β in vitro.

    (A) Structure of M7824 bifunctional fusion protein. M7824 is composed of a fully human programmed death ligand 1 (PD-L1) immunoglobulin G1 (IgG1) monoclonal antibody and a transforming growth factor–β (TGF-β)–neutralizing trap moiety, fused to the CH3–C terminus of the IgG via a flexible (Gly4Ser)4Gly linker. (B) Computer-generated model of an Fcγ1 (gray) linked by a (Gly4Ser)4Gly linker (red) to the extracellular domain of TGF-βRII (brown). Both TGF-βRII moieties can simultaneously bind a disulfide-linked TGF-β3 homodimer (cyan/green) (97). (C) Binding of M7824 to PD-L1 on human embryonic kidney (HEK) 293 cells ectopically overexpressing PD-L1. Cells were incubated with serial dilutions of M7824, anti–PD-L1, or trap control, followed by a fluorophore-conjugated anti-human IgG secondary antibody. Flow cytometry measured mean fluorescence intensity (MFI) (n = 2 technical replicates). (D) Binding of M7824 to plate-bound TGF-β. Serial dilutions of anti–PD-L1 or M7824 were incubated with plate-bound TGF-β1, TGF-β2, or TGF-β3, and binding was assessed via anti-human IgG enzyme-linked immunosorbent assay (ELISA) (n = 2 technical replicates). (E) Binding of M7824 to TGF-β2 in solution. Serial dilutions of soluble human recombinant TGF-β2 were incubated with plate-bound M7824 or anti–PD-L1, and binding was assessed via anti–TGF-β2 ELISA (n = 2 technical replicates). (F) Simultaneous binding of M7824 to PD-L1 and TGF-β1. PD-L1-Fc–coated plates were incubated with serial dilutions of M7824 or trap control, followed by biotinylated TGF-β1. Binding was evaluated using streptavidin–horseradish peroxidase. Optical densities (OD) were read at 450 nm. Data are means ± SD, and nonlinear best fits are shown (n = 2 technical replicates). All experiments were repeated at least twice.

  • Fig. 2 M7824 inhibits PD-L1 and TGF-β–dependent pathways in vitro and in vivo.

    (A) Effect of M7824 on T cell activation in vitro. Serial dilutions of M7824 or trap control were incubated with human peripheral blood mononuclear cells (PBMCs) in the presence of the superantigen staphylococcal enterotoxin A, and supernatants were harvested after 4 days for interleukin-2 (IL-2) ELISA (n = 3 technical replicates). (B) Luciferase assay to evaluate the effect of M7824 on canonical TGF-β signaling. Serial dilutions of M7824 or anti–PD-L1 were incubated with SMAD luciferase reporter–transfected 4T1 cells for 16 hours in the presence of recombinant human TGF-β1 (5 ng/ml) (n = 3 technical replicates). (C and D) M7824 pharmacokinetics and PD-L1 target occupancy (TO). Jh mice were orthotopically inoculated with 0.3 × 106 EMT-6 cells (day −12) and intravenously injected with a single dose (55, 164, or 492 μg) of M7824 on day 0. Plasma samples were collected at different time points between 24 and 336 hours thereafter. (C and D) M7824 concentration in plasma (C), assessed via human IgG ELISA, and percentage PD-L1 TO in tumor-infiltrating T cells (CD3+/CD45+ gate) (D), assessed via flow cytometry (n = 3 mice per time point). In (A) and (B), means ± SEM are shown; in (A) to (D), best fit lines are shown. (E) Effect of M7824 treatment on plasma concentrations of TGF-β isoforms. Jh mice were orthotopically inoculated with 0.25 × 106 EMT-6 cells (day −12) and intravenously injected with a single dose of M7824 (55, 164, or 492 μg) or isotype control (400 μg) on day 0. Plasma was collected 2, 6, 24, 48, 72, and 192 hours after treatment or from treatment-naive mice. Mean TGF-β1, TGF-β2, and TGF-β3 concentrations were determined using a Multiplex Discovery assay (n = 3 mice per time point). All experiments were repeated at least twice.

  • Fig. 3 M7824 inhibits tumor growth and metastasis and provides long-term antitumor immunity.

    (A and B) Effect of M7824 on tumor growth. Tumor volume (in cubic millimeters) of Jh mice (n = 12 to 13 per treatment) (A) orthotopically inoculated with 0.25 × 106 EMT-6 cells (day −7) and μMt mice (n = 8 per treatment) (B) intramuscularly inoculated with 0.5 × 106 MC38 cells (day −7) and treated intravenously with isotype control (133 or 400 μg), trap control (164 μg), anti–PD-L1 (133 or 400 μg), M7824 (164 or 492 μg), or trap control (164 μg) + anti–PD-L1 (133 μg) three times a week for 2 weeks (A) or on days 0 and 2 (B). Means ± SEM are shown. (C and D) Number of tumor nodules in the lungs (C) and incidence of metastases in BALB/c mice (D) orthotopically injected with 0.25 × 106 EMT-6 cells (day −7) and treated (n = 21 per group) with a single dose of isotype control (400 μg), trap control (492 μg), anti–PD-L1 (400 μg), or M7824 (492 μg). The mice were sacrificed when isotype control mice reached 1000-mm3 tumor volume. Data from individual mice and means are shown; P values by unpaired t test. (E and F) Percent surviving mice and tumor rechallenge; mice were sacrificed when tumor volume reached ≈2500 mm3. (E) Percent surviving Jh mice orthotopically injected in the right mammary pad with 0.25 × 106 EMT-6 cells (day −7) and treated (n = 9 mice per group) with M7824 (492 μg; days 0 and 14) or isotype control (133 μg; days 0, 7, and 14). For rechallenge, M7824-cured (n = 9) or treatment-naive mice (n = 10) were subcutaneously injected with 0.25 × 105 EMT-6 cells (218 days after final treatment). Tumor volume was measured twice weekly. (F) Percent surviving μMt mice intramuscularly injected with 0.5 × 106 MC38 cells (day −7) and treated (n = 10 mice per group) with M7824 (492 μg) or isotype control (400 μg) on days 0, 2, 4, 7, 9, and 11. M7824-cured or treatment-naive mice (n = 8 and 10, respectively) were subcutaneously injected with 0.1 × 106 MC38 cells (77 days after final treatment). Tumor volume was measured on days 7, 10, 14, and 18. Means ± SEM are shown. *P ≤ 0.05 and ****P ≤ 0.0001 denote a significant difference relative to an equimolar dose of M7824.

  • Fig. 4 M7824 elicits a distinct immunophenotypic signature in EMT-6 tumor–bearing mice.

    (A to V) Jh mice were subcutaneously inoculated with 0.25 × 106 EMT-6 cells on day −10 (A to T) or day −11 (U and V) and treated intravenously with isotype control (400 μg), trap control (492 μg), anti–PD-L1 (400 μg), or M7824 (492 μg) on days 0, 4, 7, 11, and 13 (A to T) and sacrificed on day 14 or on days 0, 4, 7, 11, and 14 (U and V) and sacrificed on day 15. (A to T) Flow cytometry analysis of dissociated tumors (n = 8 mice per group). Absolute numbers of cells per 100 mg of tumor tissue were calculated for populations of (A to E) CD8+ tumor-infiltrating lymphocytes (TILs), including absolute number (A), early activation (B), proliferation (C), T-bet+ CD8+ TILs (D), and CD8+ effector memory T cells (TEM) cells (E). (F to M) Tumor-associated natural killer cells (TINKs), including absolute number (F), T-bet+ (G), EOMES+ (H), degranulation (I) of granzyme B+ (J), NKG2D+ (K), NKp46+ (L), and IL-2Rβ+ (M) TINKs. (N to T) Myeloid cells, including absolute number (N) and mature tumor-associated dendritic cells (TADCs) (O), tumor-associated neutrophils (TANs) (P), tumor-associated monocytes (Q), M1 cells (R), M1/M2 ratio (S), and PD1+ tumor-associated macrophages (TAMs) (T). P values were determined by unpaired t test. (U and V) Representative images of anti-CD8a immunohistochemistry (IHC) of tumors (n = 5 mice per group) (U) and percentages of CD8+ cells (V) are shown. Scale bars, 100 μm. P values were determined by Kruskal-Wallis test.

  • Fig. 5 M7824 promotes gene expression associated with innate and adaptive immune cells.

    RNA sequencing (RNA-seq) analysis of tumor tissue from BALB/c mice (n = 10 mice per group) that were orthotopically inoculated with 0.25 × 106 EMT-6 cells on day −20 and treated intravenously with isotype control (400 μg), trap control (492 μg), anti–PD-L1 (400 μg), or M7824 (492 μg) on days 0, 1, and 2 and sacrificed on day 6. (A) Heat map of gene expression changes for all significantly differentially expressed genes (defined as P < 0.05 and a fold change of >1.5) from RNA-seq analysis. The colors in each box represent the log2(fold change) in the expression of a gene after treatment relative to the median isotype control; rows represent individual genes, and columns represent individual mice. The five columns on the left of the heat map show significance of up-regulation or down-regulation of genes in treatment comparisons. Inset shows magnified heat map of expression of GzmA (granzyme A), GzmB (granzyme B), and Prf1 (perforin 1). (B to G) Gene expression signatures associated with T cells (B), natural killer (NK) cells (C), macrophages (D), dendritic cells (DCs) (E), and interferon-α (IFN-α) (F) and IFN-γ (G) responses. Signature scores [defined as the mean log2(fold change) among all genes in the signature] are presented as box plots. P values were generated with the ROAST method (96). (H to J) Gene expression [transcript parts per million (TPM)] was evaluated for GzmA (H), GzmB (I), and Prf1 (J). False discovery rate–corrected P values were determined by DESeq2 (98). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001 denote a significant difference relative to the M7824 treatment.

  • Fig. 6 Innate and adaptive immunity, but not ADCC, contributes to M7824 antitumor activity.

    (A) μMt mice were injected intramuscularly with 0.5 × 106 MC38 cells on day −7 and treated (n = 10 mice per group) on day 0 with isotype control (400 μg), M7824 (492 μg), or M7824 (492 μg) dosed with anti-murine CD4 (GK1.5; 100 μg), anti-murine CD8 (2.43; 100 μg), anti-murine CD4 + anti-murine CD8 (100 μg each), or anti-murine asialo GM1 (ASGM1; 50 μl). All injections were intravenous, except for ASGM1, which was intraperitoneal. Anti-murine antibodies were injected on day 0, and M7824 and isotype control were injected on days 1, 3, and 5. Tumor volumes (in cubic millimeters) were measured twice weekly and are presented as mean ± SEM. (B) Effect of M7824 on PBMC-mediated antibody-dependent cellular cytotoxicity (ADCC) against tumor cells. PBMCs from a human donor were cultured with 51Cr-labeled A431 human epidermoid carcinoma cells for 4 hours in the presence of different concentrations of M7824, anti–PD-L1, aglycosylated M7824, or isotype control. 51Cr release from labeled target cells was measured with a Wallac MicroBeta Trilux Liquid Scintillation Counter, and the percent lysis was determined. Data were fit to a four-parameter dose-response curve. The data shown are representative of five different human donors. (C) μMt mice were injected intramuscularly with 0.5 × 106 MC38 cells on day −7 and treated (n = 15 mice per group) on day 0 with isotype control (133 μg), M7824 (55 or 164 μg), or aglycosylated M7824 (55 or 164 μg) on days 0, 2, and 4. Tumor volumes (in cubic millimeters) were measured twice weekly and are presented as mean ± SEM. ****P ≤ 0.0001 denotes a significant difference relative to M7824 treatment.

  • Fig. 7 M7824 reduces the expression of α-SMA and collagen in tumors.

    (A to C) Jh mice were subcutaneously inoculated with 0.25 × 106 EMT-6 cells on day −11 and treated (n = 5 mice per group) intravenously with isotype control (400 μg), trap control (492 μg), anti–PD-L1 (400 μg), or M7824 (492 μg) on days 0, 4, 7, 11, and 14. Mice were sacrificed on day 15. Representative images of hematoxylin and eosin (H&E) staining (A), anti–α-SMA IHC (B), or picrosirius red staining (C) for collagen on formalin-fixed paraffin-embedded tumor sections. For quantification, the numbers of α-SMA+ pixels (D) or collagen+ pixels (E) were determined for multiple regions of interest (ROIs) per tumor and normalized to ROI area; each symbol represents the proportion of positive pixels for a single tumor (n = 5 tumors per group). P values were determined by Kruskal-Wallis one-way analysis of variance (ANOVA). Scale bars, 100 μm.

  • Fig. 8 Combining M7824 with radiation or chemotherapy enhances antitumor efficacy.

    (A to C) μMtmice were inoculated intramuscularly (i.m.) with 0.5 × 106 MC38 cells (day −8) and treated (n = 10 mice per group) with isotype control (133 μg, intravenously; day 2), radiation [3.6 Gray (Gy)/day; days 0 to 3], M7824 (55 μg, intravenously; day 2), or M7824 + radiation. (A) Tumor volumes, measured twice weekly. (B) Tumor weight (day 14). (C) ELISpot of IFN-γ–producing, p15E-responsive CD8+ T cells (day 14). Spleens were harvested (n = 5 mice), and CD8+ T cells were isolated. Antigen-presenting cells derived from naive splenocytes were pulsed with the KPSWFTTL (p15E) peptide or the irrelevant SIINFEKL (OVA) peptide, irradiated, and cultured with isolated CD8+ T cells. (D to F) C57BL/6 mice were inoculated intramuscularly (day −7) with 0.5 × 106 MC38 cells in the right thigh (primary tumor) and subcutaneously (s.c.) with 1 × 106 MC38 cells in the left flank (secondary tumor) and treated (n = 6 mice per group) with isotype control (400 μg; days 0, 2, and 4), radiation (5 Gy/day; days 0 to 3), M7824 (164 μg; day 0), or M7824 + radiation. Radiation was applied only to the primary tumor, as shown in (D). (E) Primary tumor volumes and (F) secondary tumor volumes were measured twice weekly. (G to I) μMt mice were subcutaneously inoculated with 1 × 106 MC38 cells (day −7) and treated (n = 10 mice per group) with isotype control (400 μg; days 3, 6, 9, 12, and 17), M7824 (492 μg, intravenously; days 3, 6, 9, 12, and 17), oxaliplatin (Ox)/5-fluorouracil (5-FU) (5 mg/kg, intraperitoneally, and 60 mg/kg, intravenously; day 0), or M7824 + Ox/5-FU. (G) Tumor volumes were measured twice weekly. (H) Tumor weight (day 18). (I) ELISpot of IFN-γ–producing, p15E-responsive CD8+ T cells (n = 5 mice). Tumor volumes are presented as mean ± SEM. **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001 denote a significant difference relative to radiation + M7824 treatment or Ox/5-FU + M7824 treatment. Tumor weights are shown for individual mice, with lines representing the means, and they were compared by unpaired t test. IFN-γ ELISpot data are means ± SEM of three replicates and were evaluated by two-way ANOVA.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/424/eaan5488/DC1

    Materials and Methods

    Fig. S1. M7824 has a similar internalization rate as anti–PD-L1

    Fig. S2. M7824 lowers pSMAD2 in MC38 tumor–bearing mice.

    Fig. S3. The trap control exhibits antitumor activity in vivo.

    Fig. S4. M7824 inhibits tumor growth in individual mice in syngeneic tumor models.

    Fig. S5. The trap control has about three times higher serum exposure than M7824.

    Fig. S6. M7824 inhibits tumor growth in subcutaneous tumor models.

    Fig. S7. M7824 inhibits tumor growth in wild-type mice.

    Fig. S8. M7824 inhibits incidence of lung metastases relative to isotype control.

    Fig. S9. M7824 increases mature DCs and EOMES-expressing CD8+ T cells in a dosage- and dosing frequency-dependent manner.

    Fig. S10. M7824 induces an immunophenotypic signature in MC38 tumor–bearing wild-type mice.

    Fig. S11. Innate and adaptive immunity, but not ADCC, contributes to M7824 antitumor activity.

    Fig. S12. Combining M7824 with radiation enhances antitumor efficacy in individual mice.

    Fig. S13. Neither anti–PD-L1 nor trap control significantly potentiated the abscopal effect of radiation.

    Fig. S14. Combining M7824 with chemotherapy enhances antitumor efficacy in individual mice.

    Table S1. Gene lists of immune signatures from differentially expressed gene analysis.

    References (99105)

  • Supplementary Material for:

    Enhanced preclinical antitumor activity of M7824, a bifunctional fusion protein simultaneously targeting PD-L1 and TGF-β

    Yan Lan,* Dong Zhang, Chunxiao Xu, Kenneth W. Hance, Bo Marelli, Jin Qi, Huakui Yu, Guozhong Qin, Aroop Sircar, Vivian M. Hernández, Molly H. Jenkins, Rachel E. Fontana, Amit Deshpande, George Locke, Helen Sabzevari, Laszlo Radvanyi, Kin-Ming Lo*

    *Corresponding author. Email: yan.lan{at}emdserono.com (Y.L.); kinming.lo{at}emdserono.com (K.-M.L.)

    Published 17 January 2018, Sci. Transl. Med. 10, eaan5488 (2018)
    DOI: 10.1126/scitranslmed.aan5488

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. M7824 has a similar internalization rate as anti–PD-L1.
    • Fig. S2. M7824 lowers pSMAD2 in MC38 tumor–bearing mice.
    • Fig. S3. The trap control exhibits antitumor activity in vivo.
    • Fig. S4. M7824 inhibits tumor growth in individual mice in syngeneic tumor models.
    • Fig. S5. The trap control has about three times higher serum exposure than M7824.
    • Fig. S6. M7824 inhibits tumor growth in subcutaneous tumor models.
    • Fig. S7. M7824 inhibits tumor growth in wild-type mice.
    • Fig. S8. M7824 inhibits incidence of lung metastases relative to isotype control.
    • Fig. S9. M7824 increases mature DCs and EOMES-expressing CD8+ T cells in a dosage- and dosing frequency-dependent manner.
    • Fig. S10. M7824 induces an immunophenotypic signature in MC38 tumor–bearing wild-type mice.
    • Fig. S11. Innate and adaptive immunity, but not ADCC, contributes to M7824 antitumor activity.
    • Fig. S12. Combining M7824 with radiation enhances antitumor efficacy in individual mice.
    • Fig. S13. Neither anti–PD-L1 nor trap control significantly potentiated the abscopal effect of radiation.
    • Fig. S14. Combining M7824 with chemotherapy enhances antitumor efficacy in individual mice.
    • Table S1. Gene lists of immune signatures from differentially expressed gene analysis.
    • References (99105)

    [Download PDF]

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