Journal: Life Science Alliance
Article Title: Mechanistic and pharmacodynamic studies of DuoBody-CD3x5T4 in preclinical tumor models
doi: 10.26508/lsa.202201481
Figure Lengend Snippet: (A, B, C, D, E, F, G, H, I, J) The breast cancer CDX model was established by SC implantation of 5 × 10 6 MDA-MB-231 cells and 5 × 10 6 huPBMCs into NOD-SCID mice. (A, B, C, D, E) Immediately after tumor inoculation, mice were prophylactically treated with a single dose of DuoBody-CD3x5T4 (0.05, 0.5 or 5 mg/kg; IV; n = 10/group). PBS-treated mice were included as controls. (A, B, C) Tumor volume for individual mice in each treatment group over time. The dotted line indicates the cutoff for progression-free survival (500 mm 3 ). The gray lines indicate tumor volumes of individual mice receiving PBS. (D) Tumor volumes in the different treatment groups at the last day where all treatment groups were complete. Data shown are the tumor volumes of individual mice in each treatment group, as well as mean tumor volume ± SEM per treatment group. An ordinary one-way ANOVA with a posttest for linear trend was used to compare log-transformed tumor volumes of the treatment groups to the PBS-treated group ( P = 0.0002). (E) Progression-free survival, defined as the percentage of mice with tumor volume smaller than 500 mm 3 , is shown as a Kaplan–Meier curve. Mantel–Cox analysis with Bonferroni correction for multiple testing was used to compare progression-free survival between treatment groups, with ** P ≤ 0.01 and *** P ≤ 0.001. (F, G, H, I, J) Treatment with DuoBody-CD3x5T4 (0.05, 0.5, 5, or 20 mg/kg; IV) or IgG1-ctrl (20 mg/kg) was initiated when tumors reached a volume of ∼100 mm 3 . (F, G, H, I) Tumor volume in individual mice in the 0.05 (F), 0.5 (G), 5 (H), and 20 (I) mg/kg DuoBody-CD3x5T4 treatment groups (n = 5/group) over time. The gray lines indicate tumor volumes of individual mice receiving 20 mg/kg IgG1-ctrl. The dotted line indicates the cutoff for progression-free survival (500 mm 3 ). (J) Progression-free survival, defined as the percentage of mice with tumor volume <500 mm 3 , is shown as a Kaplan–Meier curve. Mantel–Cox analysis with Bonferroni correction for multiple testing was used to compare progression-free survival between treatment groups and control, but no significant differences were found. (K) In a pilot experiment, prostate cancer (DU-145) cells were inoculated in NCG mice. When tumors reached a volume of 70–100 mm 3 , mice (n = 5) were IV injected with 1 × 10 7 PBMCs derived from human healthy donors. Tumor dissociates were analyzed by flow cytometry for T-cell infiltration 7, 14, and 21 d after PBMC injection. (L, M, N, O, P) The LU7336 patient-derived xenograft model was established by SC implantation into NOG-HIS mice, humanized with human CD34 + hematopoietic stem cells of three different donors. After tumor outgrowth (average tumor size of ∼150 mm 3 ), mice were treated with DuoBody-CD3x5T4 (0.05, 0.5, or 5 mg/kg; IV; n = 4/group). PBS-treated mice (n = 3) were included as controls. Antitumor activity was only observed with one donor (other two donors are not shown). (L, M, N) Tumor volume for individual mice in each treatment group over time. The dotted red line indicates the cutoff for progression-free survival (500 mm 3 ). The gray lines indicate tumor volumes of individual mice receiving PBS. (O) Tumor volumes of the DuoBody-CD3x5T4 and PBS groups at the last day where all treatment groups were complete. Data shown are the tumor volumes of individual mice in each treatment group, as well as mean tumor volume ± SEM per treatment group. An ordinary one-way ANOVA with posttest for linear trend was used to compare log-transformed tumor volumes of the treatment groups to the PBS-treated group ( P = 0.2252). (P) Progression-free survival, defined as the percentage of mice with tumor volume smaller than 500 mm 3 , is shown as a Kaplan–Meier curve. Mantel–Cox analysis with Bonferroni correction for multiple comparisons was used to compare progression-free survival between treatment groups and the PBS-treated group, with * P ≤ 0.05.
Article Snippet: The fully human 5T4-targeting parental antibody IgG1-5T4-FEAR was generated by hybridoma technology after immunization of HuMAb mice with the recombinant extracellular domain (ECD) of human 5T4 (UniProt accession no. Q13641 [aa32-355]), produced by human embryonic kidney 293F (HEK293F) cells (R790-07; Invitrogen).
Techniques: Transformation Assay, Control, Injection, Derivative Assay, Flow Cytometry, Activity Assay
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![(A, B) Parental (5T4 + ) MDA-MB-231 tumor cells were cocultured with purified T cells (E:T = 4:1, n = 2 donors) and incubated with 10 μg/ml DuoBody-CD3x5T4 or bsIgG1-CD3xctrl for 72 h. As a positive control, T cells were incubated with anti-CD3/CD28 beads (but without tumor cells) for 72 h. The supernatant (either with or without T cells) was transferred to MDA-MB-231 5T4 KO cells and incubated for 72 h. As negative control, MDA-MB-231 5T4 KO cells were incubated with fresh T cells and indicated antibodies for 72 h. (A, B) T cell–mediated cytotoxicity of MDA-MB-231 parental cells (A) and T-cell activation (B) were determined by flow cytometry. (C, D, E) Parental and 5T4, 5T4/Fas, or 5T4/IFNGR1 KO MDA-MB-231 tumor cells were mixed in different ratios, as indicated, and incubated with purified T cells (E:T ratio = 4:1) and DuoBody-CD3x5T4 for 72 h, after which T cell–mediated cytotoxicity (C, three representative donors of 4 [5T4/IFNGR1 KO] or six [5T4 and 5T4/Fas KO] analyzed), CD8 + T-cell activation (D, a representative donor of 4 [5T4/IFNGR1 KO] or six [5T4 and 5T4/Fas KO] analyzed), and IFNγ production (E, one representative donor of 4 [5T4/IFNGR1 KO] or six [5T4 and 5T4/Fas KO] analyzed) were determined.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_8754/pmc09458754/pmc09458754__LSA-2022-01481_FigS7.jpg)
