Research ArticleCancer

Interdomain spacing and spatial configuration drive the potency of IgG-[L]-scFv T cell bispecific antibodies

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Science Translational Medicine  11 Mar 2020:
Vol. 12, Issue 534, eaax1315
DOI: 10.1126/scitranslmed.aax1315
  • Fig. 1 In vitro comparison of IgG-[L]-scFv to common BsAb designs.

    (A) Schematic of BsAb panel: IgG-[L]-scFv (2+2), BiTE (1+1B), and IgG heterodimer (1+1H). Orange domains represent anti-huCD3ε domains (derived from huOKT3), and blue domains represent anti-GD2 domains (derived from hu3F8). (B) Schematic of the IgG heterodimerization by controlled Fab arm exchange. (C) Representative cell-binding activity of each BsAb against GD2+ human M14 melanoma cells (left) and CD3+ huATCs (right), measured by flow cytometry. Geometric mean fluorescence intensity (MFI) was normalized to 2+2 (100%) for each BsAb. (D) Representative T cell–dependent cytotoxicity for each BsAb. For reference: 2+2 is purple, 1+1B is blue, and 1+1H is red. Each curve represents one BsAb, and each point represents a single concentration, with two (flow cytometry) or three (cytotoxicity) technical replicates. Data are shown as means ± SD.

  • Fig. 2 In vivo comparison of IgG-[L]-scFv to common BsAb designs.

    (A) Schematic of the treatment design for the xenograft tumor model. BsAb (25 pmol) was administered intravenously twice per week (black triangle), 40 million huATCs were administered intravenously once per week (orange triangle), and human IL-2 (1000 U) was administered subcutaneously twice per week (gray star). An anti-GPA33 BsAb was used as a control. (B) Average (top) and individual mouse (bottom) tumor responses in each group. In the overall response graph, each line represents one treatment group (n = 4 to 5). The dotted black line represents no measurable tumor, and the black hexagon represents the tumor implantation. Tumor averages were calculated until at least one mouse had to be euthanized. Data are shown as means ± SD. In the individual response graphs, each line represents a single mouse, and the dashed lines represent the group average. For reference: 2+2 is purple, 1+1H is red, and the control BsAb is gray. Statistical significances were calculated by two-way analysis of variance (ANOVA) with Tukey correction. ****P < 0.0001 for control or 1+1H compared to 2+2.

  • Fig. 3 Comparison of dual bivalent BsAb designs.

    (A) Schematic of dual bivalent BsAb panel: IgG-[L]-scFv (2+2), BiTE-Fc (2+2B), and IgG-[H]-scFv (2+2HC). Orange represents anti-huCD3ε domains (derived from huOKT3), and blue represents anti-GD2 domains (derived from hu3F8). (B) Representative cell-binding activity of each BsAb against GD2+ human M14 melanoma cells (left) and CD3+ huATCs (right), measured by flow cytometry. Geometric mean intensity was normalized to 2+2 (100%) for each BsAb. (C) Representative T cell–dependent cytotoxicity for each BsAb. (D) Schematic of in vitro coculture assay (left) and graph of results (right). For reference: 2+2 is purple, 2+2B is blue, and 2+2HC is green. Each curve represents one BsAb, and each point represents a single concentration, with two (FACS, cytokine) or three (cytotoxicity) technical replicates. Data are shown as means ± SD.

  • Fig. 4 In vivo tumor responses of dual bivalent BsAb designs.

    (A) Schematic of the treatment design for the xenograft tumor model. BsAb (10 pmol) was administered intravenously twice per week along with 20 million huATCs and subcutaneous human IL-2 (1000 U) (black triangle). (B) Average (top) and individual mouse (bottom) tumor responses in each group. In the overall response graph, each line represents one treatment group (n = 5). The dotted black line represents no measurable tumor, and the black hexagon represents the tumor implantation. Tumor averages were calculated until at least one mouse had to be euthanized. Data are shown as means ± SD. In the individual response graphs, each line represents a single mouse, and the dashed lines represent the group average. For reference: 2+2 is purple, 2+2B is blue, 2+2HC is green, and the control group (no BsAb) is gray. Statistical significances were calculated by two-way ANOVA with Tukey correction. ****P < 0.0001 for control, 2+2HC, or 2+2B compared to 2+2.

  • Fig. 5 In vitro binding activity of IgG-[L]-scFv panel.

    (A) Schematic of heterodimerization strategy for the IgG-[L]-scFv panel. (B) Resulting IgG-[L]-scFv panel: 2+2, 1+1H, 2+1, 1+1T, 1+1C, and 1+2. Orange domains represent anti-huCD3ε domains (derived from huOKT3), blue domains represent anti-GD2 domains (derived from hu3F8), and black striped domains represent irrelevant anti-CD33 domain (derived from huM195). (C) Representative cell-binding activity of each BsAb against GD2+ human M14 melanoma cells (left) and CD3+ huATCs (right), measured by flow cytometry. Geometric mean intensity was normalized to 2+2 (100%) for each BsAb. (D) Schematic of conjugate assay analysis (left) and graph of results (right). Unconjugated cells (upper left and lower right quadrants) displayed fluorescence under one channel, whereas conjugated cells (upper right quadrant) displayed fluorescence under two channels. For analysis, conjugate frequency was measured as the fraction of conjugated T cells among the total T cells (red box). An anti-CD33 BsAb was used as a control. Graphed values represent the amount of conjugate formation at each concentration of BsAb. For reference: 2+2 is purple, 1+1H is red, 2+1 is blue, 1+1T is green, 1+1C is brown, 1+2 is orange, and the control BsAb is gray. Each curve represents one BsAb, and each point represents a single concentration, with two technical replicates. Data are shown as means ± SD.

  • Fig. 6 In vitro functional activity of IgG-[L]-scFv panel.

    (A) Representative T cell–dependent cytotoxicity of each BsAb. (B to E) T cell activation data from coculture assays. (B) IL-2 cytokine release after 20 hours of coculture. (C) CD69 expression intensity and (D) frequency of expression on T cells after 20 hours of coculture. (E) CD25 expression intensity and (F) frequency of expression on T cells after 92 hours of coculture. Assays included an anti-CD33 BsAb as a control. Schematic (bottom) for reference: 2+2 is purple, 1+1H is red, 2+1 is blue, 1+1T is green, 1+1C is brown, 1+2 is orange, and the control BsAb is gray. Each curve represents one BsAb, and each point represents a single concentration, with two (cytokine) or three (cytotoxicity) technical replicates. Data are shown as means ± SD.

  • Fig. 7 In vivo antitumor activity of IgG-[L]-scFv panel.

    (A) Schematic of the treatment design for the xenograft tumor model. BsAb (25 pmol) was administered intravenously twice per week along with 20 million huATCs and subcutaneous and human IL-2 (1000 U) (black triangle). An anti-CD33 BsAb was used as a control. (B) Average (top) and individual mouse (bottom) tumor responses in each group. In the overall response graph, each line represents one treatment group (n = 5). The dotted black line represents no measurable tumor, and the black hexagon represents the tumor implantation. Tumor averages were calculated until at least one mouse had to be euthanized. Data are shown as means ± SD. In the individual response graphs, each line represents a single mouse, and the dashed lines represent the group average. For reference: 2+2 is purple, 1+1H is red, 2+1 is blue, 1+1T is green, 1+1C is brown, 1+2 is orange, and control BsAb is gray. Statistical significances were obtained by two-way ANOVA and Tukey correction. ****P < 0.0001 for control, 1+1H, and 1+1T compared to 1+2, 1+1C, 2+1, or 2+2. ***P < 0.001 for 1+2 or 1+1C compared to 2+1 or 2+2. **P < 0.01 for 2+1 compared to 2+2.

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/12/534/eaax1315/DC1

    Materials and Methods

    Fig. S1. In vitro binding activity of murine T cell BsAb panel.

    Fig. S2. In vivo tumor responses using EAT xenograft tumor model.

    Fig. S3. In vivo tumor responses using syngeneic tumor model.

    Fig. S4. In vitro coculture assay analysis.

    Fig. S5. Naïve T cell activation by dual bivalent BsAb formats.

    Fig. S6. In vivo tumor responses using EAT xenograft tumor model.

    Fig. S7. In vitro conjugate assay analysis.

    Fig. S8. T cell activation using the IgG-[L]-scFv panel.

    Fig. S9. In vivo tumor responses using syngeneic tumor model.

    Table S1. In vitro properties and design of anti-GD2 BsAb.

    Table S2. GD2 binding kinetics for BsAb using SPR.

    Table S3. huCD3ε binding kinetics for BsAb using SPR.

    Table S4. In vitro properties and design of additional anti-GD2 BsAb.

    Table S5. muCD3ε binding kinetics for BsAb using SPR.

    Table S6. In vivo pharmacokinetics of 2+2 BsAb.

    Table S7. In vitro properties and design of dual bivalent BsAb.

    Table S8. In vitro properties and design of anti-GD2 IgG-[L]-scFv panel.

    Table S9. GD2 binding kinetics for IgG-[L]-scFv panel using SPR.

    Table S10. huCD3ε binding kinetics for IgG-[L]-scFv panel using SPR.

    Table S11. In vitro properties and design of anti-CD33 IgG-[L]-scFv panel.

    Data file S1. Original data.

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. In vitro binding activity of murine T cell BsAb panel.
    • Fig. S2. In vivo tumor responses using EAT xenograft tumor model.
    • Fig. S3. In vivo tumor responses using syngeneic tumor model.
    • Fig. S4. In vitro coculture assay analysis.
    • Fig. S5. Naïve T cell activation by dual bivalent BsAb formats.
    • Fig. S6. In vivo tumor responses using EAT xenograft tumor model.
    • Fig. S7. In vitro conjugate assay analysis.
    • Fig. S8. T cell activation using the IgG-[L]-scFv panel.
    • Fig. S9. In vivo tumor responses using syngeneic tumor model.
    • Table S1. In vitro properties and design of anti-GD2 BsAb.
    • Table S2. GD2 binding kinetics for BsAb using SPR.
    • Table S3. huCD3ε binding kinetics for BsAb using SPR.
    • Table S4. In vitro properties and design of additional anti-GD2 BsAb.
    • Table S5. muCD3ε binding kinetics for BsAb using SPR.
    • Table S6. In vivo pharmacokinetics of 2+2 BsAb.
    • Table S7. In vitro properties and design of dual bivalent BsAb.
    • Table S8. In vitro properties and design of anti-GD2 IgG-[L]-scFv panel.
    • Table S9. GD2 binding kinetics for IgG-[L]-scFv panel using SPR.
    • Table S10. huCD3ε binding kinetics for IgG-[L]-scFv panel using SPR.
    • Table S11. In vitro properties and design of anti-CD33 IgG-[L]-scFv panel.

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    Other Supplementary Material for this manuscript includes the following:

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