Research ArticleCancer

A class of costimulatory CD28-bispecific antibodies that enhance the antitumor activity of CD3-bispecific antibodies

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Science Translational Medicine  08 Jan 2020:
Vol. 12, Issue 525, eaaw7888
DOI: 10.1126/scitranslmed.aaw7888
  • Fig. 1 TSAxCD28 bispecifics potentiate T cell activation only in the presence of TCR stimulation by TSAxCD3.

    (A to D) TSA-engineered target cells (HEK293) and human Jurkat T cells were cocultured with the indicated fluorescently labeled bispecifics. Fluorescence signal from each marker is shown as indicated at the top of the image panel. Cells are outlined with white dotted lines. Representative images of (A) T cells, (B) HEK293/hCD20/hPSMA, and (C) T cell–HEK293/hCD20/hPSMA doublets. Scale bar, 10 μM. (D) Quantification of the percentage of CD28 in the immunological synapse. n = 172 (isotype), n = 288 (CD20xCD3), n = 143 (PSMAxCD28), and n = 94 (combo). Statistical significance was calculated using one-way analysis of variance (ANOVA) and Tukey’s multiple comparisons test (****P < 0.0001). mAb, monoclonal antibody. (E and F) Proliferation of human T cells cultured with TSA-engineered target cells and bispecifics as described in the schematics on the left. Data are shown as means ± SEM. (E) Dose titration of CD20xCD3 in the presence of 0.5 nM hIgG4s isotype control or PSMAxCD28. (F) Dose titration of PSMAxCD28, CD28-SA, or NonTargetxCD28 in the presence of 5 pM hIgG4s isotype control or CD20xCD3. Data are representative of at least two experiments. CPM, counts per minute.

  • Fig. 2 TSAxCD28 bispecifics potentiate T cell cytotoxicity and activation only in the presence of TCR stimulation by TSAxCD3.

    Human T cells were cultured with cancer target cells with endogenous PSMA expression (prostate cancer line C4-2) (A to E) or endogenous MUC16 expression (ovarian cancer line PEO-1) (F to J) and the indicated bispecifics for 96 hours. (A and F) Schematic of assay setup. (B and G) Tumor cell kill, percent viable cells. (C and H) IFN-γ release. (D and I) CD4 T cell counts and percent CD25+ in CD4. (E and J) CD8 T cell counts and percent CD25+ in CD8. Data are representative of at least two experiments.

  • Fig. 3 MUC16xCD28 bispecific enhances the in vivo antitumor activity of the MUC16xCD3 bispecific in a xenogenic ovarian tumor model.

    NSG mice were pre-engrafted with human PBMCs and implanted with OVCAR-3/Luc. Mice were treated with EGFRvIIIxCD3, MUC16xCD3, and/or MUC16xCD28 by intraperitoneal injection on days 5 and 8 after tumor implantation (indicated by arrows). Tumor burden was measured by monitoring bioluminescence over time. (A) Representative bioluminescence images of mice from the indicated treatment groups. (B) Tumor burden as measured by median radiance (p/s/cm2/sr) over time. Values represent the group median plus range. P values were calculated with Mann-Whitney test for each time point. MUC16xCD3 versus EGFRvIIIxCD3: *P < 0.05 or **P < 0.01; MUC16xCD3 + MUC16xCD28 versus EGFRvIIIxCD3: ##P < 0.01. (C) Tumor burden and correlation to CA-125 concentration in serum on day 26. n = 5 mice per group. Data are representative of at least two experiments.

  • Fig. 4 TSAxCD28 bispecific enhances TSAxCD3 antitumor efficacy and T cell activation in mouse syngeneic tumor models.

    (A to C) hCD3/hMUC16 mice (seven mice per group) were implanted with 1 × 106 MC38/hMUC16 cells resuspended in PBS by subcutaneous injection on the right flank. MUC16xCD3 (0.01 mg/kg), MUC16xCD28 (0.05 mg/kg), or isotype controls were administered by intraperitoneal injection starting on day 0 (day of tumor implant) twice per week for six treatments. (D to H) hPSMA/hCD3/hCD28 mice (four to eight mice per group) were implanted with 1 × 106 MC38/hPSMA tumor cells resuspended in PBS by subcutaneous injection on the right flank. PSMAxCD28, PSMAxCD3, or isotype controls (all 5 mg/kg) were administered by intraperitoneal injection on days 0, 3, and 7 (D to F) or days 11 and 14 after tumor implant (G and H). (A and D) Tumor volume was monitored by caliper measurement over time. Values shown are means ± SEM. Statistical significance was calculated with two-way ANOVA. Combination versus isotype control: **P < 0.01 and ****P < 0.0001; TSAxCD3 versus isotype control: #P < 0.05; TSAxCD28 versus isotype control: $P < 0.05. (B, C, E, and F) Analysis of serum cytokines at 4 hours after dose on day 7 (B and C) or day 0 (E and F). Statistical significance was calculated with one-way ANOVA in comparison to isotype **P < 0.01 and ****P < 0.0001. Data are representative of three experiments. (G and H) Fluorescence-activated cell sorting analysis of T cell phenotype in spleen and tumor harvested on day 17. Data are represented as means + SEM. Statistical analysis: two-way ANOVA with Tukey’s multiple comparisons; isotype (n = 8), PSMAxCD3 (n = 8), PSMAxCD28 (n = 8 for spleen, n = 6 for tumor), and combo (n = 8 for spleen, n = 6 for tumor). (G) Percentage of cells in each cluster from each treatment group (top); overlay of indicated cluster on viSNE plot. (H) Expression (mean fluorescence intensity) of the indicated markers is overlaid on the viSNE plot. Color scale indicates expression: red, high; blue, low.

  • Fig. 5 TSAxCD28 alone or in combination therapy does not induce systemic T cell activation in comparison to CD28-SA.

    MUC16xCD28 or control antibodies (A) and PSMAxCD28 or control antibodies (B) were anchored to assay plates using the dry- or wet-coating method as described. Human PBMCs were incubated in assay plates, and proliferation was measured after 72 hours. Data shown are from individual donors, with a line representing the means ± SD. n = 4 donors. (C to E) Cynomolgus monkeys received a single dose of MUC16xCD28 at either 1 or 10 mg/kg (indicated in parentheses). Blood was collected at the indicated times after dose (hours). (C) Serum cytokines, (D) relative T cell counts, and (E) frequency of Ki67+ and ICOS+ T cells (% of CD3) are shown. (F to H) Cynomolgus monkeys received a single dose of PSMAxCD28, CD28-SA, or isotype control (EGFRVIII) at either 1 or 10 mg/kg (indicated in parentheses). Blood was collected at the indicated times after dose (hours). (F) Serum cytokines, (G) relative T cell counts, and (H) frequency of Ki67+ and ICOS+ T cells (% of CD3) are shown. Data represent means ± SEM. n = 3 animals per group. P values were calculated with two-way ANOVA with comparison to isotype control. (**P < 0.01, ***P < 0.001, and ****P < 0.0001).

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/12/525/eaaw7888/DC1

    Materials and Methods

    Fig. S1. Schematic of T cell activation at the immune synapse via signal 1 and signal 2.

    Fig. S2. Schematic of TSAxCD3 bispecific and mechanism for T cell activation at the immune synapse.

    Fig. S3. Schematic of TSAxCD3 and TSAxCD28 bispecifics and mechanism for T cell activation.

    Fig. S4. Introduction of co-stimulatory ligand expression on tumor cell lines inhibits tumor growth in vivo.

    Fig. S5. Biacore sensorgrams for binding of mIgG2a, hIgG4, and hIgG4s to mouse FcγRs.

    Fig. S6. PSMAxCD28 potentiates T cell activation only in the presence of TCR stimulation by CD20xCD3.

    Fig. S7. PSMAxCD28 and PSMAxCD3 bispecifics bind simultaneously to PSMA+ tumor cells.

    Fig. S8. TSA bispecifics potentiate T cell activation only in the presence of TSA expression on target cells.

    Fig. S9. PSMAxCD28 potentiates cynomolgus T cell activation only in the presence of TCR stimulation by PSMAxCD3.

    Fig. S10. MUC16xCD28 and MUC16xCD3 bispecifics bind simultaneously to MUC16+ tumor cells.

    Fig. S11. MUC16xCD28 potentiates cynomolgus T cell activation only in the presence of TCR stimulation by MUC16xCD3.

    Fig. S12. MUC16xCD28 potentiates purified T cell activation and cytotoxicity dependent on MUC16xCD3.

    Fig. S13. MUC16 bispecifics bind to MUC16-expressing cells in the presence of soluble.

    Fig. S14. MUC16xCD28 and MUC16xCD3 combination induces cytokines in humanized xenogenic mouse models.

    Fig. S15. MUC16 bispecifics bind to MUC16-expressing cells in the presence of soluble CA-125.

    Fig. S16. Targeting strategy for the generation of Cd3edghu/hu, Cd28hu/hu, and Folh1hu/hu mice.

    Fig. S17. Expression and functional validation of CD3 and CD28 in hPSMA/hCD3/hCD28 mice.

    Fig. S18. Validation of human PSMA expression in PSMA humanized mice.

    Fig. S19. Histology of PSMA+ tissue in hPSMA/hCD3/hCD28 mice treated with PSMA bispecifics.

    Fig. S20. Intratumoral T cells recognize p15E peptide.

    Fig. S21. PSMAxCD28 enhances anti-tumor immunity and T cell activation induced by PSMAxCD3.

    Fig. S22. CD28 superagonist induces systemic cytokine response in CD28 humanized mice.

    Table S1. Biacore kinetics of MUC16, PSMA, and CD28.

    Table S2. Biacore kinetics of mouse FcγRs.

    Table S3. Pharmacokinetic analysis of PSMA bispecifics in hPSMA/hCD3/hCD28 mice.

    Table S4. Summary of cynomolgus monkey toxicity study.

    Data file S1. Primary data.

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. Schematic of T cell activation at the immune synapse via signal 1 and signal 2.
    • Fig. S2. Schematic of TSAxCD3 bispecific and mechanism for T cell activation at the immune synapse.
    • Fig. S3. Schematic of TSAxCD3 and TSAxCD28 bispecifics and mechanism for T cell activation.
    • Fig. S4. Introduction of co-stimulatory ligand expression on tumor cell lines inhibits tumor growth in vivo.
    • Fig. S5. Biacore sensorgrams for binding of mIgG2a, hIgG4, and hIgG4s to mouse FcγRs.
    • Fig. S6. PSMAxCD28 potentiates T cell activation only in the presence of TCR stimulation by CD20xCD3.
    • Fig. S7. PSMAxCD28 and PSMAxCD3 bispecifics bind simultaneously to PSMA+ tumor cells.
    • Fig. S8. TSA bispecifics potentiate T cell activation only in the presence of TSA expression on target cells.
    • Fig. S9. PSMAxCD28 potentiates cynomolgus T cell activation only in the presence of TCR stimulation by PSMAxCD3.
    • Fig. S10. MUC16xCD28 and MUC16xCD3 bispecifics bind simultaneously to MUC16+ tumor cells.
    • Fig. S11. MUC16xCD28 potentiates cynomolgus T cell activation only in the presence of TCR stimulation by MUC16xCD3.
    • Fig. S12. MUC16xCD28 potentiates purified T cell activation and cytotoxicity dependent on MUC16xCD3.
    • Fig. S13. MUC16 bispecifics bind to MUC16-expressing cells in the presence of soluble.
    • Fig. S14. MUC16xCD28 and MUC16xCD3 combination induces cytokines in humanized xenogenic mouse models.
    • Fig. S15. MUC16 bispecifics bind to MUC16-expressing cells in the presence of soluble CA-125.
    • Fig. S16. Targeting strategy for the generation of Cd3edghu/hu, Cd28hu/hu, and Folh1hu/hu mice.
    • Fig. S17. Expression and functional validation of CD3 and CD28 in hPSMA/hCD3/hCD28 mice.
    • Fig. S18. Validation of human PSMA expression in PSMA humanized mice.
    • Fig. S19. Histology of PSMA+ tissue in hPSMA/hCD3/hCD28 mice treated with PSMA bispecifics.
    • Fig. S20. Intratumoral T cells recognize p15E peptide.
    • Fig. S21. PSMAxCD28 enhances anti-tumor immunity and T cell activation induced by PSMAxCD3.
    • Fig. S22. CD28 superagonist induces systemic cytokine response in CD28 humanized mice.
    • Table S1. Biacore kinetics of MUC16, PSMA, and CD28.
    • Table S2. Biacore kinetics of mouse FcγRs.
    • Table S3. Pharmacokinetic analysis of PSMA bispecifics in hPSMA/hCD3/hCD28 mice.
    • Table S4. Summary of cynomolgus monkey toxicity study.

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    Other Supplementary Material for this manuscript includes the following:

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