Immunotherapy using unconjugated CD19 monoclonal antibodies in animal models for B lymphocyte malignancies and autoimmune disease

Abstract

Immunotherapy with unconjugated CD20 monoclonal antibodies has proven effective for treating non-Hodgkin's lymphoma and autoimmune disease. CD20 immunotherapy depletes mature B cells but does not effectively deplete pre-B or immature B cells, some B cell subpopulations, antibody-producing cells, or their malignant counterparts. Because CD19 is expressed earlier during B cell development, a therapeutic strategy for the treatment of early lymphoblastic leukemias/lymphomas was developed by using CD19-specific monoclonal antibodies in a transgenic mouse expressing human CD19. Pre-B cells and their malignant counterparts were depleted as well as antibody- and autoantibody-producing cells. These results demonstrate clinical utility for the treatment of diverse B cell malignancies, autoimmune disease, and humoral transplant rejection.

Fig. 1.

B cell depletion by CD19 mAb in vivo. (A) Human CD19 expression by B cells from hCD19TG^+/- mice and human blood assessed by two-color immunofluorescence staining with flow cytometry analysis. Bar graph values represent mean relative densities of CD19 expression (±SEM) compared with human blood B cells (shown as 100%, n ≥ 9) or CD19 mAb staining mean fluorescence intensities (MFI ±SEM, n ≥ 3) of hCD19TG mouse tissue B cells (BM, bone marrow; LN, lymph node; PC, peritoneal cavity). (B) Representative B cell depletion from blood, spleen, and lymph node 7 days after CD19 (HB12a, HB12b, or FMC63) or isotype-matched control (CTL) mAb treatment of hCD19TG^+/- mice as determined by immunofluorescence staining with flow cytometry analysis. Numbers indicate the percentage of gated lymphocytes. (C) Circulating B220⁺ B cell and Thy-1.2⁺ T cell numbers (±SEM per ml, n ≥ 8 mice) after CD19 (FMC63, •) or isotype-control (○) mAb treatment. The value shown after time 0 represents data obtained at 1 h. (D) Dose responses for spleen B cell depletion. hCD19TG mice were treated with CD19 or control (CTL) mAbs on day 0 with spleen B cells assessed on day 7 (n ≥ 3). (E) Spleen B cell numbers (±SEM) 7 days after treatment of hCD19TG^+/- mice with CD19 (filled bars, 50 μg) or control (open bars, 250μg) mAb. (F) Bone marrow B cell subset depletion 7 days after CD19 (FMC63) or isotype-matched control mAb (250 μg) treatment of hCD19TG^+/- mice as assessed by three-color immunofluorescence staining. IgM^- B220^lo pro/pre-B cells were further subdivided based on CD43 expression (Lower). Numbers represent the relative frequencies of each B cell subset within the indicated gates. Bar graphs indicate numbers (±SEM, n ≥ 3) of pro-B, pre-B, and immature and mature B cells within bilateral femurs 7 days after CD19 (filled bars), CD20 (crosshatched bars), or control (open bars) mAb treatment. (G) Peritoneal cavity CD5⁺B220⁺ B1a and CD5- B220^hi B2 B cell depletion 7 days after CD19 (FMC63, filled bars), CD20 (crosshatched bars), or control (open bars) mAb treatment of hCD19TG^+/- mice. Numbers represent the relative frequencies of each B cell subset within the indicated gates. Bar graph values represent the total number (±SEM) of each cell subset within the peritoneum of mAb treated mice (n ≥ 3). (C–G) Significant differences between mean results for CD19 or CD20 and isotype-control mAb-treated mice are indicated: *, P < 0.05; **, P < 0.01.

Fig. 2.

CD19 mAb-mediated B cell depletion is FcRγ- and monocyte-dependent. (A) Blood, bone marrow (BM), spleen, lymph node (LN), and peritoneal cavity (PC) B cell depletion 7 days after CD19 (FMC63, •) or isotype-control (○) mAb treatment of hCD19TG^+/- littermates that were heterozygous FcRγ^+/- or homozygous FcRγ^-/-. For blood, the value shown after time 0 represents data obtained at 1 h. (B) Blood and tissue B cell depletion 7 days after CD19 (FMC63), control, or CD20 mAb treatment of monocyte-depleted hCD19TG^+/- mice. Mice were treated with clodronate or PBS on days -2, 1, and 4 and given mAb (250 μg) on day 0. Blood values indicate circulating B cell numbers (±SEM per ml) in PBS-treated mice given CD19 (FMC63, ▴) mAb, or monocyte-depleted mice treated with CD19 (FMC63, •), control (○), or CD20 (▪) mAb. (A and B) BM values represent mature IgM⁺ B220^high B cells. Values indicate the numbers (±SEM) of B220⁺ B cells within each tissue (n ≥ 3 mice) with significant differences between means for control mAb-treated mice and other groups indicated: *, P < 0.05; **, P < 0.01.

Fig. 3.

CD19 and CD20 mAb treatments are additive. hCD19TG^+/- mice were treated with control (250 μg), FMC63 (CD19; 2 μg), MB20–11 (CD20; 2 μg), or FMC63 + MB20–11 (2 μg each) mAbs on day 0. Blood B cell numbers were measured at time 0, 1 h, and on days 1, 4, and 7. Tissue B cell numbers were determined on day 7. Values represent means (±SEM) from groups of 4–12 mice. LN, lymph nodes; *, P < 0.05; **, P < 0.01.

Fig. 4.

CD19 mAb treatment eliminates hCD19⁺ CD20- B cell tumors in vivo. (A) Human CD19 (FMC63 mAb) and mouse CD20 (MB20-18 mAb) expression by tumor cells from a hCD19TG^+/- cMycTG mouse compared with spleen B220⁺ cells from hCD19TG^+/- mice. Control (CTL) mAb staining is shown. (B) Circulating tumor cells in recipient Rag1^-/- mice 2.5 weeks after adoptive transfer and CD19 (FMC63) or control mAb treatment. The numbers in the top right quadrants indicate tumor cell frequencies identified by immunofluorescence staining with flow cytometry analysis. (C and D) Circulating tumor cell numbers (±SEM) (C) and mouse survival rates (D) after adoptive transfer of tumor cells with subsequent CD19 (FMC63, ○) or control (•) mAb treatment (10 mice per group).

Fig. 5.

CD19 mAb treatment reduces serum Ig levels, inhibits primary and secondary Ab responses, and depletes autoantibodies in hCD19TG^+/- mice. Mice were treated with CD19 (FMC63, •) or control (○) mAb (250 μg). (A) Serum Ig levels after mAb treatment on day 0. (B) CD19 mAb treatment blocks humoral immune responses in littermates immunized with TNP-LPS, DNP-Ficoll, or DNP-KLH on day 0. Mice were treated with mAb 7 days before primary immunizations, or on day 14 (▴) after DNP-KLH immunizations (secondary response). For DNP-KLH immunizations, all mice were rechallenged with DNP-KLH on day 21. (C) Serum autoantibody levels after mAb treatment on day 0. (A–C) Ab levels (≥5 mice per group) were determined by ELISA, and mean values (±SEM) are shown for each group. Differences between CD19 and control mAb-treated mice were significant: *, P < 0.05; **, P < 0.01.