Schematic diagrams of DNL modules and structures. (A) The design for C_H1-DDD2-Fab module. (B) The design for C_H3-AD2-IgG modules. (C) The structure of C_H1-DDD2-Fab as a dimer. (D) The structure of C_H3-AD2-IgG as a monomer. (E) The structure of a bispecific hexavalent construct generated by DNL from reacting C_H1-DDD2-Fab with C_H3-AD2-IgG. (F) The interaction of the DDD2 and AD2 peptides. The variable domains for the heavy and light chains (V_H + V_L) are shown in green or orange. The constant domains of the heavy and light chains (C_H + C_L), the hinge (Hg), the 14-amino-acid residue (L14), and 9-amino-acid residue peptide linkers (L9) are shown in gray. The DDD2 and AD2 peptides are shown in blue and pink, respectively.

Induction of apoptosis. Cells were cultured for 24 hours in the presence of 22-20, 20-22, v-mab, e-mab, or a combination of v-mab and e-mab at 50 nM. (A) Apoptosis was measured by Guava Nexin for Daudi and Raji. The percentage of early apoptotic cells (annexin V–PE⁺/7-AAD⁻) is shown. (B) Apoptosis was measured by Guava MultiCaspase for Ramos and Raji after staining with SR-VAD-FMK. (C) The effect of caspase inhibitor (CI) on apoptosis of Ramos induced by 22-20, 20-22, or anti-IgM (5 μg/mL, a positive control for caspase-dependent apoptosis). Cells were cultured for 24 hours in the presence the indicated reagent alone or in the presence of Z-VAD-FMK at 20, 50, or 100 μM, and then analyzed by Guava Nexin.

Binding properties of 22-20 and 20-22. (A) Competition ELISA showing relative binding avidity of v-mab, 22-20, 20-22, and 20-20 for binding to WR2, the anti-Id antibody to v-mab. (B) Competition ELISA showing relative binding avidity of e-mab, 22-20, 20-22, and 22-22 for binding to WN, the anti-Id antibody to e-mab. (C) Cell-binding analysis by flow cytometry. Raji cells were preincubated with C_H1-DDD2-Fab-v-mab, C_H1-DDD2-Fab-e-mab, or both at 1 mg/mL before staining with PE-22-20, PE-20-22, PE-v-mab, or PE-e-mab. (D) Analysis of dissociation rates from live Raji. Cells were saturated with PE-mAbs, and the fluorescence intensity was measured over time by flow cytometry. Percentage maximal binding (MFI T = 0) was calculated by dividing MFI T = x into MFI T = 0 and plotted versus time. (E) Internalization of PE mAbs was measured using flow cytometry by comparison of the MFI of trypsinized cells versus control cells after a 1-hour incubation at 37°C.

In vitro antiproliferation determined by the cell-counting assay. Cells were seeded in T-flasks at 100 000 cells/mL and treated with 22-20, 20-22, v-mab, e-mab, or a combination of v-mab and e-mab at the indicated concentrations. Viable cell densities (VCD) were determined daily by flow cytometry using Guava Viacount. On day 3, cultures were split 1:2 to maintain logarithmic growth over the course of the assay. The data are plotted as the VCD corresponding to the undiluted culture. (A) Ramos. (B) Raji. (C) Daudi. (D) Dose-response curves generated from cell counts at day 5. Raji cells were treated with various concentrations of 22-20, 20-22, or v-mab + e-mab, and the percentage of untreated cells was plotted versus mAb concentration.

Preferential killing of NHL cells over normal B cells. (A) The effect of the indicated mAbs on peripheral blood lymphocytes from healthy volunteers was evaluated in vitro using flow cytometry. Decrease in the percentage of CD19⁺ (B cells) and CD3⁺ (T cells) present in the lymphocyte gate after a 2-day incubation of heparinized whole blood of a healthy volunteer with various mAbs. Error bars represent SD. (B) The effects of the indicated mAbs on peripheral blood B cells and Daudi (top) or Raji (bottom) lymphoma cells are indicated as the number of CD19⁺ events relative to untreated cell mixtures. B cells are derived as the CD19⁺ cells in the lymphocyte gate, whereas Daudi and Raji cells are located in the monocytes gate.

Survival curves showing therapeutic efficacy of 22-20 and 20-22 in a disseminated Burkitt lymphoma xenograft model. Female C.B. 17 SCID mice were administered 1.5 × 10⁷ Daudi cells intravenously on day 0. (A) Therapy began on day 1 with groups of 6 mice receiving a single intraperitoneal injection (10 pmol) of 22-20, 20-22, v-mab, 22-22, 22-14, 20-14, or saline. (B) Groups of 10 mice were administered 10-μg doses of 22-20, 734-20, 22-14, or 10 μg of both 734-20 and 22-14 on days 1, 4, and 7. Additional groups received 4 μg of e-mab or saline. (C) Groups of 5 NK-cell/neutrophil-depleted or -nondepleted mice were administered intravenous injections of 230 μg of either 22-20 or 20-22 on days 1, 3, 5, and 9. Saline or 100 μg e-mab was administered to nondepleted mice.

Effector functions of 22-20 and 20-22. (A) Complement-dependent cytotoxicity. Daudi cells were incubated with serial dilutions (3.33 × 10⁻⁸ to 2.6 × 10⁻¹⁰ M) of 22-20 (△), 20-22 (▲), e-mab (▼), v-mab (■), C_H3-AD2-IgG-v-mab (○), or labetuzumab (●) in the presence of human complement. The percentage complement control (number of viable cells in the test sample compared with cells treated with complement only) was plotted versus the log of the nM concentration. (B) Antibody-dependent cellular cytotoxicity. Daudi cells (target) were incubated with 22-20, 20-22, v-mab, e-mab, or h734 at 5 μg/mL in the presence of freshly isolated peripheral blood mononuclear cells (effector). A 100% lysis reference was generated by the addition of detergent to wells only containing target cells. The percentage lysis obtained for each of 2 effector cell donors is represented in the bar graph. (C) Evaluation of membrane distribution of CD22 after treatment of Raji (top 2 panels) or Daudi (bottom 2 panels) with untreated (lane 1), e-mab (lane 2), v-mab (lane 3), 22-20 (lane 4), or 20-22 (lane 5). Cells were lysed in buffer containing 1% Triton X-100 and fractionated by sucrose density-gradient ultracentrifugation. The lipid rafts were collected from the interface of 5% and 30% sucrose, and the soluble fractions from the 40% sucrose at the bottom of the tubes. The lipid raft and soluble fractions were analyzed by anti-CD22 immunoblot. Vertical lines have been inserted to indicate a repositioned gel lane.