Percent reduction of B-cells antigens following overnight treatment of PBMCs.

<p>Average % reduction from three experiments using PBMCs form independent donors. n.d., not measured due to blocked detection by the specific treatment. Significantly (<i>P</i><0.05) more reduction than:</p>a<p>all other agents;</p>b<p>veltuzumab and hA19;</p>c<p>epratuzumab;</p>d<p>all but veltuzumab.</p>e<p>Not significantly different from 22*-(20)-(20).</p

DNL modules and bsHexAb structures.

<p>(<b>A</b>) C_k-AD2-IgG-epratuzumab, an IgG-AD2 module with an AD2 fused to the carboxyl-terminal end of each kappa light chain. (<b>B</b>). Dimeric C_H1-DDD2-Fab-veltuzumab, or C_H1-DDD2-Fab-hA9, Fab-DDD modules with DDD2 fused to the carboxyl-terminal end of the F_d chain (<b>C</b>). Structure of 22*-(20)-(20) or 22*-(19)-(19), bsHexAbs comprising C_k-AD2-IgG-epratuzumab and two dimeric C_H1-DDD2-Fab-veltuzumab or C_H1-DDD2-Fab-hA19 modules, respectively. (<b>D</b>) Structure of 22*-(20)-(20) with the Fc removed. Variable (V, blue or green) and constant (C, grey) domains of IgG heavy (H) and light (L) chains are represented as ovals. The DDD2 (dimerization and docking domain) and AD2 (anchor domain) peptides are shown as blue and yellow helices, respectively, with the locations indicated for the reactive sulfhydryl groups (SH) and the “locking” disulfide bridges indicated as red lines. (<b>E</b>) SE-HPLC showing the homogeneity of 22*-(20)-(20) and the expected small shift in retention following removal of the Fc, which comprises 13% of the protein. (<b>F</b>) Reducing (left) and non-reducing (right) SDS-PAGE showing the elimination of the intact epratuzumab heavy chain (intact lane) and the appearance of the resulting cleaved epratuzumab Fd following removal of the Fc (ΔFc lane).</p

Analysis of trogocytosis by flow cytometry.

<p>PBMCs were incubated overnight with 10 µg/mL of various mAbs or bsHexAbs prior to measurement of surface antigens by flow cytometry. (<b>A</b>) Gating of lymphocytes by forward vs. side scattering (Left) and B cells from the lymphocyte gate using CD19 and CD22 staining (Right) following treatment with control mAb (labetuzumab). (<b>B</b>) Example dot-plots comparing CD19 and CD22 staining on B cells following treatment of PBMCs with 22*-(20)-(20), epratuzumab and labetuzumab (Left) and histograms showing β7 integrin staining following treatment with the indicated mAbs or bsHexAbs (Right). (<b>C</b>) Trogocytosis mediated by C_k and C_H3-based bsAbs. PBMCs were incubated overnight with 10 µg/mL 22*-(20)-(20), 22-(20)-(20), veltuzumab, epratuzumab or labetuzumab (control), prior to measurement of surface CD19, CD22 and CD21 by flow cytometry. Results are shown as the % MFI of the control treatment. Error bars, Std. Dev.</p

B-cell depletion.

<p>Freshly isolated PBMCs were incubated for two days with 22*-(20)-(20) (red) or rituximab (blue) prior to counting the viable B cells The relative viable B cell count is expressed as % Control, which was derived by dividing the specific B cell count by that measured following treatment with the control mAb (labetuzumab). Error bars, Std. Dev. (<b>A</b>) B-cell depletion with 140 nM rituximab or 22*-(20)-(20) in PBMCs from 6 unique donors. (<b>B</b>) B-cell depletion at 24 h and 48 h with antibody titrations using PBMCs from Donor 4. (<b>C</b>) Daudi Burkitt lymphoma cells were spiked in PBMCs from Donor 3 and treated with titrations of the antibodies. Daudi and normal B cells were separated by forward scattering and counted independently. (<b>D</b>) 140 nM of 22*-(20)-(20) (left, red) or rituximab (right, blue) were incubated with NK-depleted (ΔNK) or intact PBMCs, which were alternatively treated with Fc-deleted fragments (ΔFc/PBMC) of each antibody. Donor 1, solid bar; Donor 2, hatched bar. (<b>E</b>) Reduction of CD19 on B cells by trogocytosis. Control was PBMCs treated with labetuzumab (black dashed trace). Fc-deleted 22*-(20)-(20) was incubated with PBMCs (green trace). Intact 22*-(20)-(20) was incubated with PBMCs (blue trace) or NK cell-depleted PBMCs (red trace). Histograms show the fluorescence intensity for anti-CD19-PE.</p

Fluorescence microscopy showing trogocytosis induced with 22*-(20)-(20).

<p>Purified monocytes labeled with PKH26-Red fluorescence were mixed 2∶1 with Daudi cells labeled with PKH67-Green fluorescence, and treated with 22*-(20)-(20) (<b>A–C</b>) or labetuzumab (<b>D</b>) at 10 µg/mL. Fluorescent images were captured after 30 min at room temperature with an Olympus BX66 microscope (Shinjuko, Tokyo, Japan) equipped with a Mercury-100W laser (Chiu Technical Corp., Kings Park, NY), using an Olympus 40X/0.75 air objective lens and a Kodak DC290 Camera (Rochester, New York) set at 115X zoom. A WB filter was used to allow simultaneous fluorescence of both red and green fluorochromes. Images were captured and processed using Adobe Photoshop CS3 v.10 software with a Kodak Microscopy Documentation System 290 plug-in application. Bars: 10 µm.</p

Effector functions.

<p>(<b>A+B</b>) ADCC. PBMCs were incubated with Daudi cells (50∶1) for 4 h in the presence of the indicated agents at 33 nM (<b>A</b>) or with titration (<b>B</b>). Similar results were observed with Raji cells and a different PBMC donor. (<b>C+D</b>) CDC using Daudi as target cells. Error bars, Std. Dev.</p

Data Supplement from Redirected T-Cell Killing of Solid Cancers Targeted with an Anti-CD3/Trop-2–Bispecific Antibody Is Enhanced in Combination with Interferon-α

Figure S7 shows the direct action of IFN-α on Capan-1 and NCI-N87 cells. Figure S8 shows ex vivo T cell re-directed killing of BxPC3 cells with (E1)-3s.</p

Optimization of Multivalent Bispecific Antibodies and Immunocytokines with Improved in Vivo Properties

Multifunctional antibody-based biologics, such as bispecific antibodies and immunocytokines, can be difficult to produce with sufficient yield and stability, and often exhibit inferior pharmacokinetics. Dock-and-Lock (DNL) is a modular method that combines recombinant engineering with site-specific conjugation, allowing the construction of various complex, yet defined, biostructures with multivalency and multispecificity. The technology platform exploits the natural interaction between two interactive human protein binding domains that are modified to provide covalent fusion. We explored the potential application of a new class of IgG-based DNL modules with an anchor domain fused at the C-terminal end of the kappa light chain (C_k), instead of the C-terminal end of the Fc. Two C_k-derived prototypes, an anti-CD22/CD20 bispecific hexavalent antibody, comprising epratuzumab (anti-CD22) and four Fabs of veltuzumab (anti-CD20), and a CD20-targeting immunocytokine, comprising veltuzumab and four molecules of interferon-α2b, were compared to their Fc-derived counterparts. The Ck-based conjugates exhibited superior Fc-effector functions in vitro, as well as improved pharmacokinetics, stability, and anti-lymphoma activity in vivo. These results favor the selection of DNL conjugates with the C_k-design for future clinical development

Data Supplement from Redirected T-Cell Killing of Solid Cancers Targeted with an Anti-CD3/Trop-2–Bispecific Antibody Is Enhanced in Combination with Interferon-α

Supplementary Figure S9. Induction of cytokine release by (19)-3s and 19-3 BiTE from PBMCs in the presence of Raji NHL cells. Results show the levels of TNF-α, IFN-γ and IL-6 after 20-h treatment with titrations of bsAb.</p

Data Supplement from Redirected T-Cell Killing of Solid Cancers Targeted with an Anti-CD3/Trop-2–Bispecific Antibody Is Enhanced in Combination with Interferon-α

Additional flow cytometry data for trogocytosis. Figure S5 shows viability of T cells and BxPC3 target cells. Figure S6 shows formation of immunological synapses and transfer of CD4 from T cells to target cells.</p