CD19xCD3 DART protein mediates human B-cell depletion in vivo in humanized BLT mice

Abstract

Novel therapeutic strategies are needed for the treatment of hematologic malignancies; and bispecific antibody-derived molecules, such as dual-affinity re-targeting (DART) proteins, are being developed to redirect T cells to kill target cells expressing tumor or viral antigens. Here we present our findings of specific and systemic human B-cell depletion by a CD19xCD3 DART protein in humanized BLT mice. Administration of the CD19xCD3 DART protein resulted in a dramatic sustained depletion of human CD19(+) B cells from the peripheral blood, as well as a dramatic systemic reduction of human CD19(+) B-cell levels in all tissues (bone marrow, spleen, liver, lung) analyzed. When human CD8(+) T cells were depleted from the mice, no significant B-cell depletion was observed in response to CD19xCD3 DART protein treatment, confirming that human CD8(+) T cells are the primary effector cells in this in vivo model. These studies validate the use of BLT humanized mice for the in vivo evaluation and preclinical development of bispecific molecules that redirect human T cells to selectively deplete target cells.

Figure 1

CD19xCD3 DART protein administration depletes human CD19⁺ B cells from the peripheral blood. (a) Experimental outline. NSG/BLT mice were administered CD19xCD3 (n = 3, 1 mg/kg i.v.) or vehicle (n = 3) at day 0 and day 7. Peripheral blood (PB) was collected and analyzed at days 0, 1, 3, 7, 11. (b) Percent CD45⁺ cells out of live cells and (c) number CD45⁺ cells per microliter detected in PB by flow cytometry (vehicle group in blue, CD19xCD3 group in red). (d) Percent CD19⁺ cells out of CD45⁺ cells and (e) number CD19⁺ cells per microliter detected in PB by flow cytometry (open circle = below limit of quantitation). (Mean ± SEM plotted. P values were calculated by repeated-measures two-way analysis of variance with Sidak’s multiple comparisons test, comparing vehicle versus CD19xCD3. ns, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.)

Figure 2

CD19xCD3 DART protein administration depletes human CD19⁺ B cells from the tissues. NSG/BLT mice were administered CD19xCD3 (n = 3, 1 mg/kg intravenously) or vehicle (n = 3) at day 0 and day 7. Tissues were harvested and analyzed at day 11. (a) Percent CD19⁺ cells out of CD45⁺ cells and (b) number CD19⁺ cells detected in tissues by flow cytometry (vehicle group in blue; CD19xCD3 group in red). (Mean ± SEM plotted. P values were calculated by unpaired t-test, comparing vehicle vs. CD19xCD3. ns, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.)

Figure 3

Immature human CD19⁺ B cells regenerate in NSG/BLT mice after CD19xCD3 DART protein administration. (a) Experimental outline. NSG/BLT mice were administered CD19xCD3 (n = 3, 1 mg/kg i.v.) or vehicle (n = 3) at day 0 and day 7. Peripheral blood (PB) was collected and analyzed at days 0, 1, 3, 7, 14, 21, and 28; tissues were harvested and analyzed at day 28. (b) Percent CD19⁺ cells out of CD45⁺ cells and (c) number CD19⁺ cells per microliter detected in PB by flow cytometry (vehicle group in blue; CD19xCD3 group in red). (d) Percent CD19⁺ cells out of CD45⁺ cells and (e) number CD19⁺ cells detected in tissues by flow cytometry. (f) Percent immature CD10⁺ cells out of CD19⁺ cells detected in tissues by flow cytometry. (Mean ± SEM plotted. P values were calculated in b and c by repeated-measures two-way analysis of variance with Sidak’s multiple comparisons test, comparing vehicle versus CD19xCD3. P values were calculated in d–f by unpaired t-test, comparing vehicle versus CD19xCD3. ns, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Gray diamond indicates data from age-matched NSG/BLT mice, n = 4.)

Figure 4

CD19xCD3 DART protein administration results in transient differences in the levels of human T cells in the peripheral blood of NSG/BLT mice, and over time there are no significant differences in absolute numbers of human CD8⁺ T cells in the peripheral blood or tissues as compared to vehicle-treated mice. NSG/BLT mice were administered CD19xCD3 (n = 3, 1 mg/kg i.v.) or vehicle (n = 3) at day 0 and day 7. Peripheral blood (PB) was collected and analyzed at days 0, 1, 3, 7, 14, 21, and 28; tissues were harvested and analyzed at day 28. (a) Percent CD45⁺ cells out of live cells and (b) number CD45⁺ cells per microliter detected in peripheral blood by flow cytometry (vehicle group in purple, CD19xCD3 group in brown). (c) Percent CD3⁺ cells out of CD45⁺ cells and (d) number CD3⁺ cells per microliter. (e) Percent CD8⁺ cells out of CD3⁺ cells and (f) number CD8⁺ cells per microliter. (g) Percent CD8⁺ cells out of CD3⁺ cells and (h) number CD8⁺ T cells detected in tissues by flow cytometry. (Mean ± SEM plotted. P values were calculated in a–f by repeated-measures two-way analysis of variance with Sidak’s multiple comparisons test, comparing vehicle versus CD19xCD3. P values were calculated in g and h by unpaired t-test, comparing vehicle versus CD19xCD3. ns, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Gray diamond indicates data from age-matched NSG/BLT mice, n = 4.)

Figure 5

Human CD8⁺ T cells are depleted after administration of CD8-depleting antibody. (a) Experimental outline. NSG/BLT mice were administered CD8-depleting antibody (CD8-depleted, n = 8, 3 mg/kg i.v.) or vehicle (CD8-intact, n = 7) 4 days prior to administration of CD19xCD3 DART protein to all mice (1 mg/kg i.v.). Peripheral blood (PB) was collected and analyzed at 4 days and 1 day prior to and at days 3, 7, and 14 after administration of CD19xCD3 DART protein; tissues were harvested and analyzed at day 15. (b) Percent CD8⁺ cells out of CD3⁺ cells and (c) number CD8⁺ cells per microliter detected in PB by flow cytometry (CD8-depleted in orange, CD8-intact in green). (d) Percent CD8⁺ cells out of CD3⁺ cells and (e) number CD8⁺ cells detected in tissues by flow cytometry. (Mean ± SEM plotted. P values were calculated in b and c by repeated-measures two-way analysis of variance with Sidak’s multiple comparisons test, comparing CD8-depleted versus CD8-intact. P values were calculated in d and e by unpaired t-test, comparing CD8-depleted versus CD8-intact. ns, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.)

Figure 6

Human CD19⁺ B-cell depletion by CD19xCD3 DART protein is dependent on the presence of human CD8⁺ T cells. NSG/BLT mice were administered CD8-depleting antibody (CD8-depleted, n = 8, 3 mg/kg i.v.) or vehicle (CD8-intact, n = 7) 4 days prior to administration of CD19xCD3 DART protein to all mice (1 mg/kg i.v.). Peripheral blood (PB) was collected and analyzed at 4 days and 1 day prior to and at days 3, 7, and 14 after administration of CD19xCD3 DART protein; tissues were harvested and analyzed at day 15. (a) Percent CD19⁺ cells out of CD45⁺ cells and (b) number CD19⁺ cells per microliter detected in PB by flow cytometry (CD8-depleted in orange, CD8-intact in green). (c) Percent CD19⁺ cells out of CD45⁺ cells and (d) number CD19⁺ cells detected in tissues by flow cytometry. (Mean ± SEM plotted. P values were calculated in a and b by repeated-measures two-way analysis of variance with Sidak’s multiple comparisons test, comparing CD8-depleted vs. CD8-intact. P values were calculated in c and d by unpaired t-test, comparing CD8-depleted versus CD8-intact. ns, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.)