The human Imp3 and Imp4 proteins form a ternary complex with hMpp10, which only interacts with the U3 snoRNA in 60–80S ribonucleoprotein complexes

Ribosome biogenesis requires a vast number of trans-acting factors many of which are required for the chemical modification and processing of the pre-rRNA component. The U3 snoRNP complex is required for the early cleavage steps in pre-rRNA processing. We have cloned cDNAs encoding the human and mouse homologs of the yeast U3 snoRNP-associated proteins Imp3 and Imp4. Both human proteins localize to nucleoli and interact with the U3 snoRNA. The results of complementation experiments show that, in contrast to mouse Imp4, mouse Imp3 can partially alleviate the growth defect of the corresponding yeast null strain, indicating that the role of Imp3 in pre-rRNA processing is evolutionarily conserved. The results of density gradient centrifugation experiments show that, in contrast to hU3-55K, the human Imp3 and Imp4 proteins predominantly interact with the U3 snoRNA in 60–80S ribonucleoprotein complexes. In addition, we have found that hImp3, hImp4 and hMpp10 can form a stable hetero-trimeric complex in vitro, which is generated by direct interactions of both hImp3 and hImp4 with hMpp10. The analysis of hImp3 and hImp4 mutants indicated that their binding to hMpp10 correlates with their nucleolar accumulation, strongly suggesting that the formation of the ternary complex of hImp3, hImp4 and hMpp10 is required for their association with nucleolar components

Biophysical characterization of antibody hexamerization in solution.

<p>(A) Native MS analysis of solutions of IgG1-005 and mutant antibodies at 2 μM. IgG1-005-RGY (RGY), a previously described triple mutant, is used as a positive control. The charge envelope of the IgG monomers appears around <i>m/z</i> 5,500 (~ 147 kD Mw) and that of the IgG hexamers around <i>m/z</i> 12,500 (~890 kD Mw). The signals of the hexameric species are displayed at a 100-fold or 10-fold (RGY only) amplification. (B–C) Dynamic light scattering analysis of 7D8 and mutant solutions formulated in PBS pH 7.4. Three independent experiments are shown. (B) Linear regression of diffusion coefficient versus Ab concentration. (C) Hydrodynamic radii (Rhs) of mutants measured by dynamic light scattering (DLS) were divided by Rh of wild-type 7D8 to correct for viscosity without masking the contribution of Fc:Fc self-association. (D) HP-SEC analysis of 100 mg/mL 7D8 and mutant antibody solutions formulated in PBS, incubated for three months at 5°C. Multimer detection limit of 1% is indicated by a dashed line.</p

E345 and E430 limit Fc:Fc interactions.

<p>(A) Left: overview of the IgG1 antibody hexamer observed in the IgG1-b12 crystal structure (PDB access code 1HZH [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002344#pbio.1002344.ref020" target="_blank">20</a>]). Right: zoomed-in view of two neighboring Fc domains with enhancing amino acid positions highlighted. (B) Summary of CDC inhibition data. The surface view is rotated 90° relative to panel A to show the Fc:Fc interface. Daudi cells were opsonized with mutants of anti-CD38 mAb IgG1-005 (1.0 μg/mL) and assessed for CDC. Light and dark grey colors indicate the unmodified amino acids of the two heavy chain regions composing a single Fc domain. All mutants assessed were ranked by efficiency per amino acid position, and the resulting median lysis efficacy is indicated by coloring per position; orange and red colors indicate positions with reduced lysis. Blue: >60% lysis; orange: 30%–60% lysis; red: <30% lysis. (C) Summary of CDC enhancement data. Wien 133 cells were opsonized with mutants of anti-CD38 mAb IgG1-005 (1.0 μg/mL) and assessed for CDC. Light and dark grey colors indicate unmodified amino acids as described under (B). The maximal lysis of the most efficient mutant per amino acid position is displayed with green indicating positions for which CDC was enhanced. Blue: <20% lysis; light green: 20%–40% lysis; dark green: >40% lysis. (D) Dose response in CDC of Wien 133 cells by IgG1-005 and selected mutants. A representative of 3 replicate experiments is shown. (E) Dose response in CDC of Ramos cells induced by RTX and selected mutants. A representative experiment of 3 replicates is shown. (F) Structural view zoomed to residue E345. Amino acids facing E345 at the Fc:Fc interface are indicated in blue and named with apostrophe. (G) Structural view zoomed to residue E430, forming a salt bridge with K338 at the intramolecular C_H2–C_H3 domain interface.</p

CDC enhancement by promoting Fc:Fc interactions is broadly applicable.

<p>A representative example of three replicates is shown. Each graph compares CDC in dose response for the wild-type and E345R-mutated mAb. (A) CDC of Wien 133 (left) and Daudi cells (right) opsonized with anti-CD20 mAbs RTX and 11B8. (B) CDC of Wien 133 (left) and Raji cells (right) opsonized with anti-CD52 mAb ALM. (C) CDC of Wien 133 cells (left) and Daudi cells (right) opsonized with anti-CD38 mAbs IgG1-003 and IgG1-005. (D) CDC assay of A431 cells opsonized with anti-EGFR mAb 2F8.</p

Specific IgG mutations retain fluid phase complement suppression and regular pharmacokinetics.

<p>(A) C4d ELISA of IgG1-005 mutants detecting solution-phase complement activation in serum. The background (BKG) C4d level in serum is indicated with a dashed line. Results were averaged over three experiments. (B) PK analysis of IgG1-005 mutants in SCID mice, using three mice per group. Clearance of a single 500 μg dose of mAb was monitored for three weeks and is expressed as 1,000 * Dose / area under the curve (AUC); dashed line: clearance of wild-type IgG1-005.</p

Efficient lysis of CLL patient tumor cells.

<p>Peripheral B cells isolated from the blood of six CLL patients were opsonized with wild-type or mutant, 7D8, RTX or control (IgG1-b12) antibody and tested in CDC assays. The CLL cells of patient 4 were highly refractory to CDC due to their very low CD20 expression.</p

Promoting Fc:Fc interactions alleviates CDC sensitivity to target and mCRP expression.

<p>(A) Cell lines were opsonized with saturating amounts of wild-type or mutated RTX and analyzed by CDC assay. Left panel: cell lines with increasing CD20:mCRP ratio (left to right); right panel: cell lines with decreasing CD20:mCRP ratio (left to right). (B) In vivo analysis of subcutaneous tumor growth in a severe combined immunodeficiency (SCID) xenograft model. SCID mice were injected with luciferase-expressing Raji cells. Eight days after tumor cell injection, mice were randomized at an average tumor volume of 85 mm³ per group and treated with 50 μg IgG1 antibody. Tumor volumes were monitored over time and depicted as average tumor volume ± standard error of the mean (SEM) (left panel), and as Kaplan-Meier curves with time to progression cutoff set at a tumor volume of >700 mm³ (right panel).</p