Proposed model: Id-specific B cells are stimulated by αCD19-Id targeted to CD19 on B cells in lymphoid follicles.

Design and characterization of αCD19-Id. (A) Design of expression plasmids and schematic of one of four αCD19-Ids. Coexpression of both plasmids in the same CFPS reaction produces two polypeptides that assemble into a noncovalent heterodimeric Db. Locations of heavy chain variable domains (α19 V_H and 38 V_H), and light chain variable domains (α19 V_L and 38 V_L) of anti-CD19 and 38C13, respectively, T7 promoters (T7), ribosomal binding sites (rbs), (Gly)₄Ser linkers (L), hexahistidine tag (H₆), and stop codons (stop) on the expression plasmids are indicated, as are the 38C13 Id and the binding site for CD19 on the Db. (B) Flow cytometry analysis of αCD19-Id bispecific binding. A20 cells were incubated with CFPS products containing 5 μg of each αCD19-Id variant (—) or with mock CFPS product (shaded). A20/CD19^NEG cells incubated with the same CFPS product (---) served as a negative target cell control. Cells were then washed and stained with Alexa Fluor 488-conjugated anti-38C13 mAb.

αCD19-Id targeted specifically to B cells in vivo. Mice were injected with αCD19-Id, RatFv-Id, or 38C13 IgM, each conjugated to Alexa Fluor 488, or with buffer. (A) Draining lymph nodes (LN) were harvested 8 h after i.d. injections. (B) Spleens (SP) and peripheral blood (PB) were harvested 2 h after i.v. injections. Leukocytes from these organs were stained with fluorophore-conjugated mAbs specific for B220, CD3, CD11b, and F4/80, and analyzed by flow cytometry. The percentages of gated total leukocytes in the Upper Right quadrants are indicated. One of two experiments is presented.

Id-specific BCR activation by αCD19-Id and αCD19-Id–decorated B cells. (A) Splenic B cells recovered from mice 2 h after i.v. injection with αCD19-Id (B-Db) or with PBS (B) were incubated for the indicated times with responder cells, either A20 or A20/α38BCR that were prelabeled with CellTrace Violet dye. Cells were fixed, permeabilized, stained with PE-conjugated antibodies specific for the phosphorylated forms of PLC-γ2 and Syk, and analyzed by flow cytometry. Responses of gated responder cells are shown. (B) Kinetics of BCR signaling induced by αCD19-Id–decorated A20 cells (A20-Db). The percentages of A20/α38BCR responder (●) and A20 negative control responder (▲) cells are shown. Data are pooled from three experiments (an example is shown in Fig. S5A). The percentage of BCR signaling cells for each incubation time was calculated from the corresponding histograms: [% PE⁺ cells in response to A20-Db (red line)] − [% PE⁺ cells in response to A20 (black line)]. (C) A20/α38BCR cells were stimulated for 10 min at 37 °C with Dbs, 38C13, or control IgM. Cell lysates were analyzed by Western blotting using antibodies specific for the phosphorylated forms of ERK and the p55 subunit of PI3K, and for total ERK and actin. Representative results of three experiments are shown.

Id-specific B cells captured and internalized αCD19-Id. (A) Mice were injected i.v. with PBS (B) or with αCD19-Id conjugated to Alexa Fluor 488 (B-Db). Splenic B cells recovered after 2 h were incubated for 1 h at 37 °C with A20 or A20/α38BCR cells prelabeled with Violet dye, then fixed and analyzed by flow cytometry. One of two experiments is presented. (B) A20/α38BCR cells were incubated at 0 °C or 37 °C for 30 min with Alexa Fluor 488-conjugated αCD19-Id. Cells were washed, fixed, and analyzed by confocal microscopy. Representative images of cells are shown with a z-section thickness of 2.4 μm. (Scale bar, 10 μm.)

Immune responses to αCD19-Id. (A) αCD19-Id induced a robust Id-specific antibody response. Mice received four biweekly i.d. vaccinations given twice on consecutive days. Vaccines consisted either of 6 μg Dbs or an Id molar equivalent of 38C13 IgM, either chemically conjugated to KLH (38C13-KLH), mixed with anti-CD19 mAb (αCD19 + 38C13), or conjugated to anti-CD19 mAb by avidin (αCD19-Av-38C13). Sera collected a week after the last immunization were tested by ELISA for antibodies against the 38C13 Id. Data were combined from four studies. The number of animals in each group is indicated. Each bar on the graph represents the mean serum anti-Id IgG concentration ± SEM of each group. None of the sera reacted with a mouse IgM/κ isotype control. (B) IFN-γ production by splenocytes from immunized mice. Mice (two to three per group) were vaccinated as in A. Spleens from each group were harvested and pooled a week later. Splenocytes were cultured for 4 d with Ags listed in the legend in hexaplicate wells each. IFN-γ in culture supernatants was measured by ELISA.

Vaccination with αCD19-Id protected mice from tumor. (A) Mice (10 per group) received αCD19-Id, RatFv-Id, 38C13-KLH, or buffer as described in . Ten days later, mice were challenged with 100 38C13 cells by i.v. injection. (B) Mice (10 per group) were vaccinated with αCD19-Id, 38C13-KLH, or buffer and challenged with 400 cells. Survival was analyzed by the Kaplan–Meier method and the log-rank statistical test.