Research ArticleCancer

CAR T cells targeting BAFF-R can overcome CD19 antigen loss in B cell malignancies

See allHide authors and affiliations

Science Translational Medicine  25 Sep 2019:
Vol. 11, Issue 511, eaaw9414
DOI: 10.1126/scitranslmed.aaw9414
  • Fig. 1 BAFF-R-CAR T cells activate and elicit specific cytotoxicity against multiple subtypes of human B cell malignancies.

    (A) Either CD4+ or CD8+ BAFF-R-CAR T cells were incubated with BAFF-R–expressing L cells, MCL lines JeKo-1, Z-138, or, as controls, parental L cells (mouse fibroblast) or T-activator CD3/CD28 beads. ELISA measurement of cytokines IFN-γ, TNF-α, and granzyme B supernatant concentrations were determined following 24 hours of incubation with targets. (B) Cytotoxic T lymphocyte assay measuring the specific lysis of target cells by 51Cr release after 4 hours. Various 51Cr-labeled BAFF-R–positive lymphoma and leukemia cell lines, BAFF-R–expressing L cells, or control BAFF-R–negative parental L cells were incubated with either CD4 or CD8 CAR T cells, as indicated at an E:T ratio of 3:1. B-CLL, B cell CLL. (C) Lymphomas from primary patient samples were labeled with 51Cr and incubated either with naïve CD4 or CD8 BAFF-R-CAR T cells at various E:T ratios as in (B). Nontransduced T cells from the same healthy donors were used as allogeneic controls (non-CAR). Data are shown as the means ± SD of one CAR T cell donor against triplicate tumors samples. All experiments were repeated with at least three different CAR T cell donors. **P < 0.001 versus corresponding non-CAR control with the following tests: (A) one-way ANOVA and Dunnett’s multiple comparisons test; (B) two-way ANOVA and Dunnett’s multiple comparisons test; and (C) two-way ANOVA and Tukey’s multiple comparisons test.

  • Fig. 2 Defined subpopulations of BAFF-R-CAR T cells eliminated established MCL tumors in vivo after a single treatment.

    (A) Bioluminescence images and survival of groups of five NSG mice after intravenous tumor challenge (106 cells per mouse) on day 0 with luciferase-expressing human MCL line JeKo-1. CD4 TN CAR T cells combined with either subpopulations of CD8 TCM, TN, or TSCM CAR T cells were infused intravenously on day 10 at a single dose of 106 CD4 TN + 106 CD8 CAR T cells. Control mice received nontransduced CD4/CD8 T cells from the same donor as an allogeneic control (non-CAR), or PBS. (B) Survival data were analyzed by Kaplan-Meier plots of overall survival at 100 days. Data are representative of three independent experiments using different donor T cells. Log-rank test: **P < 0.01 and *P < 0.05 compared with controls.

  • Fig. 3 Superiority of BAFF-R versus CD19-CAR T cells in a Burkitt lymphoma model is not due to greater tumor antigen density.

    (A) Bioluminescence images of groups of five NSG mice after intravenous tumor challenge (0.5 × 106 cells per mouse) on day 0 with luciferase-expressing Raji cells. Activated CD4 TN CAR T (2.5 × 106) + 106 CD8 TN BAFF-R– or CD19-CAR T cells were infused intravenously on day 7 as a single dose. Control mice received nontransduced CD4/CD8 T cells from the same donor as an allogeneic control, or PBS. Data are representative of two independent experiments using different donor T cells. (B) Kaplan-Meier plot of overall survival at 80 days is shown. Log-rank test: **P < 0.01 compared with all other groups. (C) Calculated cell surface antigen density of BAFF-R and CD19 on lymphoma and leukemia lines stained by PE-conjugated antibodies at saturation. PE per cell (assuming one PE per antibody) was calculated against mean fluorescence intensity standard curve with BD Quantibrite beads. Data are represented as means ± SD of triplicates. Student’s t test: **P < 0.001 BAFF-R versus CD19 in corresponding cell line.

  • Fig. 4 Therapeutic effects of BAFF-R-CAR T cells against CD19-negative human tumor lines in vitro and in vivo.

    CD19-negative B cell tumor variant clones were generated by CRISPR-HDR CD19 gene KO of (A) two lymphoma cell lines (Z-138, MCL; MEC-1, CLL) and (B) one ALL cell line (Nalm-6), or by gRNA CD19 gene KD of one ALL PDX. FACS histograms indicate CD19 and BAFF-R expression on WT and corresponding KO/KD tumors. Cytotoxicity of corresponding tumor targets mediated by CD8 BAFF-R TN (blue) or CD19 TN CAR T cells (red) incubated at various E:T ratios was determined by 51Cr release by tumor targets after 4 hours. Nontransduced T cells from the same donor were used as an allogeneic control (green, dotted). Data are shown as the means ± SD of triplicate samples. Data are representative of at least three independent experiments using different donor T cells. Two-way ANOVA and Tukey’s multiple comparisons test: **P < 0.001 versus non-CAR control. (C and D) Bioluminescence images of NSG mice after intravenous tumor challenge on day 0 with luciferase-expressing (C) 5 × 104 Z-138–CD19–KO MCL or (D) 2 × 105 Nalm-6–CD19–KO ALL tumors. Groups of five tumor-bearing mice each were then randomly assigned to treatment with either 2.5 × 106 CD4 TN CAR T + 106 CD8 TN BAFF-R– or CD19-CAR T cells per mouse intravenously on day 11 or 10, respectively, as a single dose. Nontransduced CD4/CD8 T cells from the same donor were used as allogeneic controls (non-CAR). Data are representative of two independent experiments using different donor T cells. Kaplan-Meier plots of overall survival are shown. Log-rank test: **P < 0.01 BAFF-R-CAR versus CD19-CAR and controls.

  • Fig. 5 BAFF-R-CAR T cells eliminate preexisting CD19 antigen loss variants in vivo.

    (A) Bioluminescence images of NSG mice after intravenous tumor challenge on day 0 with a mixture of 5 × 104 luciferase-expressing Z-138 (WT) plus 5 × 104 Z-138–CD19–KO tumor cells. Groups of five tumor-bearing mice each were then randomly assigned to treatment with either 2.5 × 106 CD4 TN CAR T + 106 CD8 TN BAFF-R– or CD19-CAR T cells per mouse intravenously on day 8, as a single dose. Nontransduced CD4/CD8 T cells from the same donor were used as allogeneic controls (non-CAR). (B) Kaplan-Meier plots of overall survival are shown. Log-rank test: **P < 0.01 BAFF-R-CAR versus CD19-CAR and controls. (C) Representative FACS plots of postmortem tumor analysis from spleens of mice treated in (A). Cells were gated on CD45+ human tumor cells and analyzed for CD19 expression. Summary graph of mean percentage ± SD of triplicate samples CD19+/CD19- tumor cells from n = 5 mice per group. ***No tumor cells were detected. Data are from one experiment. Two-way ANOVA and Tukey’s multiple comparisons test: **P < 0.001 percentage of CD19-positive tumor cells versus non-CAR and PBS controls.

  • Fig. 6 BAFF-R–specific activation of CAR T cells by primary CD19 antigen loss human ALL escape variants and antitumor effects of CAR T cells in vivo.

    Blood or bone marrow tumor samples were obtained from ALL patients relapsing with CD19-negative tumors after CD19 bispecific antibody treatment (relapse) and analyzed together with each patient’s corresponding pretherapy tumor. (A) FACS histograms showing expression of CD19 and BAFF-R. (B) Cryopreserved ALL samples were cocultured with BAFF-R-CAR or CD19-CAR T cells derived from a single healthy donor in the presence of anti-CD107a antibody for 6 hours. Nontransduced T cells (non-CAR) from a single donor were used as a negative control. (C) FACS analysis of CD19-negative B-ALL cells isolated at relapse from a fifth patient for PDX establishment. B-ALL cells (106 cells per mouse) were injected into NSG mice, and four mice per group were then randomly assigned to treatments of BAFF-R-CAR or CD19-CAR T cells (5 × 106, 1:1 CD4:CD8 TN CAR T cell ratio per mouse) on day 26. Nontransduced T cells (1:1 CD4:CD8 TN cells) from the same donor were used as allogeneic controls (non-CAR). (D) Percentage of peripheral blood CD19-negative B-ALL blasts in PDX mice at days 26 and 54. B-ALL blasts were identified by CD45+CD22+CD58+ staining. Two-way, repeated-measures ANOVA and Sidak’s multiple comparisons test: **P < 0.001 BAFF-R-CAR versus CD19-CAR and controls. (E) Overall survival of CD19-negative B-ALL PDX mice after BAFF-R-CAR T treatment. Control groups include CD19-CAR T, nontransduced T cells, and PBS. Log-rank test: **P < 0.01 versus CD19-CAR, non-CAR, and PBS control.

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/11/511/eaaw9414/DC1

    Fig. S1. Cytokine release assay.

    Fig. S2. BAFF-R-CAR T cell in vitro cytotoxic T lymphocyte assay.

    Fig. S3. Preliminary BAFF-R-CAR T cell assessment in vivo.

    Fig. S4. CAR T cells validated for CAR expression and cytotoxic T lymphocyte activity.

    Fig. S5. CD19-KO clone selection.

    Data file S1. Primary data.

  • The PDF file includes:

    • Fig. S1. Cytokine release assay.
    • Fig. S2. BAFF-R-CAR T cell in vitro cytotoxic T lymphocyte assay.
    • Fig. S3. Preliminary BAFF-R-CAR T cell assessment in vivo.
    • Fig. S4. CAR T cells validated for CAR expression and cytotoxic T lymphocyte activity.
    • Fig. S5. CD19-KO clone selection.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

Stay Connected to Science Translational Medicine

Navigate This Article