Research ArticleCancer

Antibody-mediated targeting of TNFR2 activates CD8+ T cells in mice and promotes antitumor immunity

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Science Translational Medicine  02 Oct 2019:
Vol. 11, Issue 512, eaax0720
DOI: 10.1126/scitranslmed.aax0720
  • Fig. 1 Characterization of anti-mouse TNFR2 antibodies in vitro and in vivo.

    (A) The domain epitopes of antibodies are indicated on a homology model of the murine TNFR2/TNF complex. The CRDs for one TNFR2 receptor are shown in color. (B) TNF competition ELISA of antibodies. Data represented as a three-parameter dose-response curve fit based on mean and SD (n = 2). (C) Comparison of in vitro CD8+ T cell proliferation for different anti-TNFR2 antibodies [plate-bound anti-CD3 (0.2 μg/ml) plus soluble anti-CD28 (1 μg/ml)]. Data are representative of two independent experiments. (D) In vivo activity in the CT26 syngeneic murine tumor model. Each antibody was injected intraperitoneally once at day 0 (9 days after tumor inoculation) at 1 mg (n = 7 animals per group, data displayed as mean ± SEM). (E) In vivo activity in the CT26 syngeneic murine tumor model at a reduced dose. Each antibody was injected intraperitoneally once at day 0 (9 days after tumor inoculation) at 300 μg (n = 8 animals per group).

  • Fig. 2 In vivo activity of Y9 in syngeneic tumor models.

    Tumor cells were implanted and mice were randomized to treatment groups. CR, tumors below 60 mm3 and continued to regress until the end of the study. Vertical dotted lines indicate treatment time points with 300 μg per treatment and antibody. Group sizes are indicated in the figure. (A) Antitumor activity of Y9 in responder and nonresponder models. Tumor growth in individual mice is shown; CR indicated for Y9 treatment groups. (B) Antitumor activity of Y9 in the anti–PD-1–resistant model MBT-2 and in the anti–PD-1–sensitive model Sa1/N. Tumor growth in individual mice is shown. (C) Survival curves for treatment with Y9 alone and in combination with anti–PD-1 in multiple murine models. Statistically significant differences from PBS are indicated. Calculated with Mantel-Cox test. **P < 0.01, ***P < 0.001.

  • Fig. 3 Tumor rechallenge and immune memory in CT26, WEHI-164, and EMT6 models.

    Tumor growth in individual mice is shown (PBS, n = 10; Y9, n = 15). After initial treatment with Y9, 7 complete responder mice for the CT26 model, 13 complete responder mice for the WEHI-164 model, and 5 complete responder mice for the EMT6 model, as well as age-matched controls for each model (n = 5) were rechallenged with tumor cells between days 90 and 100 after inoculation. Vertical lines before rechallenge indicate days of treatment with 300 μg per antibody intraperitoneally.

  • Fig. 4 Importance of FcγR binding.

    (A) Comparison of in vivo activity of Y9 with wild-type Fc (Y9) and DANA mutant Fc (Y9-DANA) shown as survival curves in three syngeneic mouse models. Antibodies were injected intraperitoneally at 300 μg on days 5, 12, and 19 after tumor inoculation. Statistically significant differences from PBS are indicated (PBS, n = 10; Y9, n = 15; Y9-DANA, n = 10). (B) In vivo activity of Y9 in CT26 syngeneic murine tumor model in BALB/c wild-type (WT), Fc receptor common γ-chain knockout (Fcer1g−/−), and FcγR2b knockout (Fcgr2b−/−) mouse strains. Data are shown as survival curves. Antibodies were injected intraperitoneally at 300 μg on days 8, 15, and 22 after inoculation. Statistically significant differences from WT is indicated (n = 10 mice per group). (C) Comparison of in vivo activity of Y9 with wild-type mIgG2a Fc (Y9), mIgG2a Fc with DANA mutations (Y9-DANA), wild-type mIgG1 Fc (Y9-mIgG1), and mIgG2a Fc with SELF mutations (Y9-SELF). Data are shown as survival curves for two syngeneic mouse models. Antibodies were injected intraperitoneally at 300 μg on days 8, 15, and 22 after inoculation. Data were analyzed using Mantel-Cox test. Statistically significant differences from PBS are indicated (n = 10 mice per group). *P < 0.05, **P < 0.01, ***P < 0.001. n.s., not significant.

  • Fig. 5 Impact of treatment with Y9 on different immune cell subsets.

    (A) In vivo activity of Y9 treatment after depletion of CD8+ T cells, NK cells, CD4+ T cells, and no depletion control in the CT26 syngeneic murine tumor model. Once tumors were established, mice were given a single injection of 1 mg of Y9 intraperitoneally; individual mice are shown. CR, tumors below 60 mm3 and continued to regress until the end of the study. Group sizes are indicated in the figure. (B) In vivo activity of Y9 treatment (300 μg × 3 weeks) after depletion of CD4+ T cells and no depletion control in CT26 tumor model established with Matrigel matrix. Group sizes are indicated in the figure. Vertical dashed lines indicate days of treatment. (C) In vivo activity of Y9 in TNFR2 CRISPR knockout models CT26-CrKO (n = 5 mice per group) and EMT6-CrKO (n = 10 mice per group). Mice were given three weekly treatments of 300 μg of antibody intraperitoneally Individual mice are shown. (D to G) In four syngeneic murine tumor models, mice were injected intraperitoneally with 300 μg of Y9 or Y9-DANA when tumors reached 200 to 400 mm3 (7 to 10 days after inoculation). Data were analyzed using a nonparametric Kruskal-Wallis test with a Dunn’s multiple comparison test within each cell type. Data are plotted as mean ± SEM. Statistically significant differences from control are indicated. (D) TNFR2 surface expression on T cell subsets. Tumor-infiltrating immune cells were characterized 24 to 36 hours after treatment (n = 7 per group). (E and F) Tracking of the antitumor CD8+ T cell response specific to AH1 antigen in the CT26 tumor model 4 and 6 days after treatment. Left: AH1 dextramer+ CD8 T cells as a percentage of the total CD8 T cell tumor infiltrate. Right: percentage of IFN-γ+ CD8 T cells after ex vivo peptide stimulation. Statistical comparisons were calculated using unpaired t tests without assuming a consistent SD. (F) Correlation of tumor mass in Y9-treated mice at day 4 or day 6 with number of IFN-γ+ CD8+ T cells per gram of tumor. Data in (E) and (F) are representative of one experiment for day 4 and two independent experiments for day 6. (G) Percentage of Tregs within tumoral CD4+ T cells. Tumor-infiltrating immune cells were characterized 24 to 36 hours after treatment (n = 7 per group). Data were analyzed using ANOVA with a Dunnett’s multiple comparisons posttest comparing treatment groups to control. *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 6 Comparison of toxicity profile of long-term exposure to anti-TNFR2 or anti–CTLA-4 antibodies in BALB/c mice.

    Mice were treated once weekly with PBS or 1 mg of murine IgG2a isotype control, Y9, or anti–CTLA-4 for 8 weeks (n = 5 animals per group). Individual mice are shown except in (A). (A) Longitudinal percent change in body weight. Vertical dashed lines indicate times of treatment. (B) Comparison of spleen sizes and weights 48 hours after the final treatment. (C and D) Frequency of Ki-67+ (C) and PD-1+ (D) CD4+ Foxp3neg (Tconv) and CD8+ T cells in the peripheral blood 7 days after the fourth treatment. (E) Representative hematoxylin and eosin (H&E) staining from various tissues in BALB/c mice treated with IgG2a isotype, Y9, or anti–CTLA-4. H&E, 100×. Indicated are inflammatory cells (red arrows), multifocal epidermal hyperplasia (black arrows), and ulceration (blue arrow). Images are representative of five animals per treatment group. (F) Longitudinal serum cytokines. Data were analyzed using ANOVA with a Dunnett’s multiple comparisons posttest comparing treatment groups to PBS. Data are plotted as mean ± SEM. Statistical significance from PBS control is indicated. *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 7 Generation of anti-human TNFR2 antibodies modeled after the Y9 antibody.

    (A) Cell-based TNF competition assay of antibodies. Data represented as a three-parameter dose-response curve fit based on mean and SD from duplicate measurements. (B) The high-resolution epitope of anti-human (Ab1) and anti-mouse (Y9) TNFR2 antibodies is highlighted on the structure of human TNFR2 (white)/TNF (blue) and homology model of mouse TNFR2 (orange)/TNF (cyan), respectively. Residues critical for Ab1 (Y24, Q26, Q29, M30, and K47) and Y9 binding (Y25, R27, K28, M31, and N47) are highlighted (red). (C and D) In vitro costimulation of purified naïve human CD4+ T cells with plate-bound anti-TNFR2 mAbs (20 μg/ml) [plate-bound anti-CD3 (5 μg/ml) plus soluble anti-CD28 (1 μg/ml)]. Representative flow plots from one healthy donor showing percent proliferation measured by CellTrace Violet (CTV) dilution (C) and induction of CD25 and PD-1 (D). Data for Ab1 are representative of three separate experiments (total of n = 11 unique donors) and Ab2 is representative of two separate experiments (total n = 8 unique donors). (E and F) In vitro costimulation of purified human CD8+ T cells with plate-bound anti-TNFR2 mAbs (20 μg/ml) [plate-bound anti-CD3 (5 μg/ml)]. Representative flow plots from one healthy donor showing percent proliferation (E) and induction of CD25, IFN-γ, IL-2, and granzyme B (F). Data for Ab1 are representative of three separate experiments (total of n = 11 unique donors) and Ab2 is representative of one experiment (total n = 4 unique donors). (G) In vitro costimulation of naïve human CD4+ T cells with titrated concentrations of plate-bound anti-TNFR2 mAbs [plate-bound anti-CD3 (5 μg/ml) plus soluble anti-CD28 (1 μg/ml)]. Graphs represent data from one experiment with four unique donors and were normalized within each donor to wells where no antibody is present. Data shown as fold-change ± SEM. Data for Ab1 are representative of three separate experiments (total of n = 11 unique donors) and Ab2 is representative of two separate experiments (total n = 8 unique donors). (H) In vitro costimulation of human CD8+ T cells with titrated concentrations of plate-bound anti-TNFR2 mAbs [plate-bound anti-CD3 (5 μg/ml) plus soluble anti-CD28 (1 μg/ml)]. Graphs represent data from one experiment with four unique donors and were normalized within each donor to wells where no antibody is present. Data shown as fold change ± SEM. Data for Ab1 are representative of three separate experiments (total of n = 11 unique donors) and Ab2 is representative of one experiment (total n = 4 unique donors). Data were analyzed using two-way ANOVA with Dunnett’s multiple comparison post tests. Statistically significant from isotype is indicated (A and B). *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 8 Anti-human TNFR2 mAbs provide costimulation to human T cells and antitumor activity in humanized mouse models.

    (A) NSG mice were administered human PBMCs from one donor and HT-29 colorectal adenocarcinoma CDX cells on the same day. On day 7 when tumors were established (~90 mm3), mice were given 5 × 300-μg doses of mAb (vertical dashed lines) with four to five mice per group. (B) NSG-SGM3 mice were engrafted with human CD34+ cord blood cells. Mice that had ≥25% of total CD45+ cells of human origin after 12 weeks were inoculated with MDA-MB-231 CDX or LG1306 PDX with 10 mice per group. Mice were given 300 μg doses of mAb at indicated time points (vertical dashed lines). Statistically significant from isotype is indicated. *P < 0.05, **P < 0.01, ***P < 0.001.

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/11/512/eaax0720/DC1

    Materials and Methods

    Fig. S1. Cross-reactivity test of murine TNFR2 antibodies to other TNF superfamily receptors.

    Fig. S2. Enhanced proliferation and activation of T cells mediated by higher-order TNFR2 cross-linking for Y9 costimulation in vitro.

    Fig. S3. In vitro Treg suppression assay.

    Fig. S4. In vivo activity of Y9 and anti–PD-L1 combination.

    Fig. S5. Ligand blocking of Y9-DANA.

    Fig. S6. Impact of treatment with Y9 on different immune cell subsets.

    Fig. S7. Ex vivo analysis of tumor-specific CD8+ T cell responses.

    Fig. S8. Impact of treatment with Y9 on frequency changes in the T cell compartment.

    Fig. S9. Characterization of the tumor-associated myeloid compartment after anti-TNFR2 treatment.

    Fig. S10. Additional data for toxicity profile of long-term exposure to anti-TNFR2 or anti–CTLA-4 antibodies in BALB/c and C57BL/6 mice.

    Fig. S11. Serum cytokine data for toxicity profile of long-term exposure to anti-TNFR2 or anti–CTLA-4 antibodies in BALB/c and C57BL/6 mice and toxicity study in EMT6 tumor-bearing mice.

    Fig. S12. Repeat toxicity study with anti–PD-1 combinations.

    Fig. S13. TNFR2 receptor constructs used for epitope mapping and binding studies.

    Fig. S14. High-resolution epitope mapping of human and mouse anti-TNFR2 antibodies.

    Fig. S15. Gating strategy of mouse T cells in the tdLN and tumor.

    Table S1. Description of chimeric TNFR2 constructs.

    Table S2. Sequences of murine TNFR2 antibodies.

    Table S3. Murine T cell flow cytometry antibodies.

    Table S4. Human T cell flow cytometry antibodies.

    Data file S1. Primary data.

    References (52, 53)

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. Cross-reactivity test of murine TNFR2 antibodies to other TNF superfamily receptors.
    • Fig. S2. Enhanced proliferation and activation of T cells mediated by higher-order TNFR2 cross-linking for Y9 costimulation in vitro.
    • Fig. S3. In vitro Treg suppression assay.
    • Fig. S4. In vivo activity of Y9 and anti–PD-L1 combination.
    • Fig. S5. Ligand blocking of Y9-DANA.
    • Fig. S6. Impact of treatment with Y9 on different immune cell subsets.
    • Fig. S7. Ex vivo analysis of tumor-specific CD8+ T cell responses.
    • Fig. S8. Impact of treatment with Y9 on frequency changes in the T cell compartment.
    • Fig. S9. Characterization of the tumor-associated myeloid compartment after anti-TNFR2 treatment.
    • Fig. S10. Additional data for toxicity profile of long-term exposure to anti-TNFR2 or anti–CTLA-4 antibodies in BALB/c and C57BL/6 mice.
    • Fig. S11. Serum cytokine data for toxicity profile of long-term exposure to anti-TNFR2 or anti–CTLA-4 antibodies in BALB/c and C57BL/6 mice and toxicity study in EMT6 tumor-bearing mice.
    • Fig. S12. Repeat toxicity study with anti–PD-1 combinations.
    • Fig. S13. TNFR2 receptor constructs used for epitope mapping and binding studies.
    • Fig. S14. High-resolution epitope mapping of human and mouse anti-TNFR2 antibodies.
    • Fig. S15. Gating strategy of mouse T cells in the tdLN and tumor.
    • Table S1. Description of chimeric TNFR2 constructs.
    • Table S2. Sequences of murine TNFR2 antibodies.
    • Table S3. Murine T cell flow cytometry antibodies.
    • Table S4. Human T cell flow cytometry antibodies.
    • References (52, 53)

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    Other Supplementary Material for this manuscript includes the following:

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