Anti-CD21 antibodies are not significantly internalized, while anti-CD19 antibodies only internalize readily in CD21 or CD21 cells

<p><b>Copyright information:</b></p><p>Taken from "High CD21 expression inhibits internalization of anti-CD19 antibodies and cytotoxicity of an anti-CD19-drug conjugate"</p><p></p><p>British Journal of Haematology 2007;140(1):46-58.</p><p>Published online 07 Nov 2007</p><p>PMCID:PMC2228374.</p><p>© 2007 Genentech, Inc. Journal Compilation © 2007 Blackwell Publishing Ltd</p> Various B-cell lines were incubated with anti-CD21 (HB135) for 20 h at 37°C in the presence of lysosomal protease inhibitors, and the total antibody distribution detected post fixation and permeabilization with Cy3-conjugated anti-mouse (left panels). Insets show surface binding of anti-CD21 following 1 h incubation on ice. Ramos (A) and DoHH2 (B) cells lack surface expression of CD21 and consequently failed to internalize any antibody, as expected. Anti-CD21 is not significantly internalized in the low CD21-expressing Namalwa (C) or Daudi (D) cells, or even in the higher expressing ARH77 (E) or Raji (F) cells, or in freshly isolated primary human B-cells (G). The same cell lines were incubated with anti-CD19 (B496) antibodies on ice for 1 h (insets in middle panels), or at 37°C for 3 h (middle panels) or 20 h (right panels) with detection as above. The CD21-negative cell lines Ramos (H,O) and DoHH2 (I,P) readily internalized anti-CD19 within 3 h, while the low CD21-expressing Namalwa (J,Q) and Daudi (K,R) cells internalized it less extensively, as judged by the faint plasma membrane staining remaining even after 20 h uptake. The high CD21-expressors, ARH77 and Raji did not detectably internalize anti-CD19 after 3 h (L,M), and after 20 h still had not internalized nearly as much as the CD21-negative cells did in 3 h (S,T). Primary human B-cells did not internalize anti-CD19 within 3 h (N), but did by 20 h (U). Virtually all the cells in each field readily internalized Alexa488-transferrin (with the exception of transferrin-receptor negative primary B-cells), indicating that any lack of antibody uptake was not due to loss of viability (not shown). Gamma levels were adjusted where appropriate. Scale bar = 20 μm

Anti-CD19 is internalized by dynamin-dependent, clathrin-mediated endocytosis and is delivered to lysosomes

<p><b>Copyright information:</b></p><p>Taken from "High CD21 expression inhibits internalization of anti-CD19 antibodies and cytotoxicity of an anti-CD19-drug conjugate"</p><p></p><p>British Journal of Haematology 2007;140(1):46-58.</p><p>Published online 07 Nov 2007</p><p>PMCID:PMC2228374.</p><p>© 2007 Genentech, Inc. Journal Compilation © 2007 Blackwell Publishing Ltd</p> (A) Ramos cells were pre-incubated for 30 min at 37°C with the following reagents: dimethyl sulphoxide (DMSO) (1); 1 μmol/l chlorpromazine (Cpmzn) (2), a clathrin-mediated endocytosis inhibitor; 80 μmol/l dynamin inhibitor dynasore, preincubated for 5 min only (3); 2 mmol/l methyl-β-cyclodextrin (MbC) (4) or 5 μg/ml filipin (5), both inhibitors of caveolar and lipid raft endocytosis. Alexa488-anti-CD19 (black bars) or Alexa488-transferrin (grey bars) were then added in the continuous presence of inhibitors for 30 min and surface quenched as in . Results were plotted as a percentage of uptake compared with the DMSO control and represent the average and standard deviation of three independent triplicate experiments. (B–D) Alexa488-anti-CD19 (green channel in B and D) was co-internalized with Alexa647-transferrin (shown in the red channel in C and D) in Ramos cells for 5 min, surface quenched with anti-Alexa488, fixed and imaged. (E–G) Alexa488-anti-CD19 (green channel in E and G) was chased for 3 h in Ramos cells in the presence of lysosomal protease inhibitors prior to fixation and staining with Alexa555-anti-LAMP1 (red channel in F and G). Yellow colour in the merged images in panels D and G indicates colocalization. Gamma levels were adjusted where necessary to better illustrate marker overlap. Arrows indicate examples of co-localized staining. Scale bar is 20 μm in the main panels and 6·7 μm in the 3×-magnified insets of the boxed region indicated in D

A Neutralizing Anti-gH/gL Monoclonal Antibody Is Protective in the Guinea Pig Model of Congenital CMV Infection

<div><p>Human cytomegalovirus (HCMV) is the most common cause of congenital virus infection. Congenital HCMV infection occurs in 0.2–1% of all births, and causes birth defects and developmental abnormalities, including sensorineural hearing loss and developmental delay. Several key studies have established the guinea pig as a tractable model for the study of congenital HCMV infection and have shown that polyclonal antibodies can be protective <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004060#ppat.1004060-Bratcher1" target="_blank">[1]</a>–<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004060#ppat.1004060-Chatterjee1" target="_blank">[3]</a>. In this study, we demonstrate that an anti-guinea pig CMV (GPCMV) glycoprotein H/glycoprotein L neutralizing monoclonal antibody protects against fetal infection and loss in the guinea pig. Furthermore, we have delineated the kinetics of GPCMV congenital infection, from maternal infection (salivary glands, seroconversion, placenta) to fetal infection (fetus and amniotic fluid). Our studies support the hypothesis that a neutralizing monoclonal antibody targeting an envelope GPCMV glycoprotein can protect the fetus from infection and may shed light on the therapeutic intervention of HCMV congenital infection in humans.</p></div

Viral kinetics of fetal infection over 21 days plotted from three separate studies.

<p>43 pregnant guinea pigs were inoculated by subcutaneous injection with 4×10³ PFU of pathogenic stock IVP8 at day 21 gestation and sacrificed at 1, 3, 7, 11, 15, and 21 days post-infection. Each glyph (dot) represents the proportion of infected fetuses, with the size of the glyph proportional to the litter size. The solid lines represent the proportion of infected fetuses in a litter from a beta-binomial model fit to all three cohorts. The dashed segment of each line is extrapolated beyond the cohort data and is model based. See statistical section in methods for more details.</p

Effect of 1968/GPFc antibody on maternal mortality and congenital GPCMV infection^A^B.

A<p>Pregnant guinea pigs were infected with 4×10³ PFU of IVP8 at the start of the 2^nd trimester.</p>B<p>Cumulative results at 21 days post-infection.</p>C<p>Virus detected by quantitative PCR.</p>D<p>Virus detected by nested PCR.</p>E<p>Antibody administered I.P. one day prior to infection at 8 mg/kg dose and then twice per week for 3 weeks with a total of six doses (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004060#ppat-1004060-g005" target="_blank">Figure 5</a> for study scheme).</p>F<p>n/a, not applicable, no fetuses were alive at end of study.</p>G<p>Averaged in-house historical data from infected pregnant guinea pigs without antibody treatment.</p

Viral kinetics of GPCMV during congenital infection.

A<p>SG, salivary glands from pregnant dams.</p>B<p>NAb, dam's neutralizing antibody titer, expressed as serum dilution determined by qPCR.</p>C<p>virus detected by qPCR.</p>D<p>virus detected by nested PCR.</p>E<p>AF, amniotic fluid.</p>F<p>ND, not done.</p

Temporal relationship between placental and fetal infection over 21 days.

<p>43 pregnant guinea pigs were inoculated by subcutaneous injection with 4×10³ PFU of pathogenic stock IVP8 at day 21 gestation and sacrificed at 1, 3, 7, 11, 15, and 21 days post-infection. Each glyph represents an infected mother with the size of the glyph proportional to the number of infected placentas and/or fetuses recovered. The proportion of infected placentas (x-axis) and infected fetuses (y-axis) was determined by qPCR and nested PCR, respectively. Data was compiled from three separate experiments.</p

Maternal mortality and fetal loss in GPCMV-infected guinea pigs.

A<p>Fetal loss data is only from surviving dams.</p

Determination of the timing of seroconversion.

<p>An antigen-based ELISA assay was used to monitor the anti-gB (A) or anti-gH/gL (B) IgG response in infected pregnant guinea pigs over 21 days. Two dilutions (1∶100- green, 1∶2700- purple) are graphed to display the range of reactivity of the anti-gB response. The positive control, day 12 positive serum, (1∶2700- red, average value from 5 different plates with 2 replicates per plate) consistently resulted in the linear range and near the middle of the dynamic range. Pre-immune samples were consistently at zero indicating the cut-off of the assay (an example is shown at d0 at 1∶100, blue). The 1∶2700 was not graphed for anti-gH/gL (B) due to lack of signal. The error bars represent standard deviation of the means calculated from the results of several animals at each time point (see methods for study details).</p

Characterization of mouse monoclonal antibodies against GPCMV.

A<p>ELISA performed with soluble proteins; gB, gH/gL, gH/gL/gO and Pentamer = gH/gL/GP129/GP131/GP133.</p>B<p>Expression of gH/gL protein on the surface of 293T cells.</p>C<p>Antibody neutralizing potency as determined by the anti-gB immunofluorescence method.</p>D<p>Soluble gH/gL protein was used as immunogen.</p