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

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

MET and HGF expressions.

<p>Representative sarcomas showing range and pattern of immunohistochemical staining for MET (A-H) and HGF (I-P). (A, B, I, J) staining intensity score = 0, (C, D, K, L) score = 1+, (E, F, G, M, N, O,) score = 2+, (H, P) score = 3+. (A, C, E, N) undifferentiated pleomorphic sarcomas, (B, D, F, G, K, M) angiosarcomas, (H) control cell line, (I, L, O) leiomyosarcomas, (J) dedifferentiated liposarcoma, (P) control tissue breast cancer.</p

Fluorescence <i>in situ</i> hybridization for <i>MET</i>.

<p>Representative sarcomas showing different categories for <i>MET</i> gene copy number variations (see text for explanation). <i>MET</i> gene is labeled in green, centromere 7 in orange. (A) <i>MET</i> negative clear cell sarcoma, (B) <i>MET</i> negative angiosarcoma, (C) low level copy number gain in a clear cell sarcoma, (D) intermediate level copy number gain in an undifferentiated pleomorphic sarcoma, (E, F) high level amplification in undifferentiated pleomorphic sarcomas.</p

FISH results of <i>MET</i> positive cases.

<p>ASA, angiosarcoma; CCS, clear cell sarcoma; CEN, centromere; DDLS, dedifferentiated liposarcoma; GIST, gastrointestinal stromal tumor; ID, identification; LMS, leiomyosarcoma; MFH, undifferentiated pleomorphic sarcoma; MLS, myxoid liposarcomas; PLS, pleomorphic liposarcoma</p><p>FISH results of <i>MET</i> positive cases.</p

PI3K pathway alterations in primary and metastatic ER+ breast cancers.

<p>(A) Distribution of alterations in <i>PIK3CA</i>, <i>AKT1</i> and PTEN across 75 matched primary and metastatic ER+ breast cancers. PTEN null status denotes total absence of PTEN protein in neoplastic cells determined by immunohistochemistry. Arrows indicate patients with alterations in both <i>PIK3CA</i> and PTEN. (B) Frequency and overlap of PI3K pathway alterations in ER+ breast cancer samples. Biomarker frequencies calculated only from patients where tissue was evaluable for all biomarker assays. The data from the single <i>PIK3CA</i> exon 4 mutant sample was pooled with the exon 9 data, and the data from the exon 9/20 double mutant samples were pooled with exon 20 data. (C) Scatterplot of PTEN protein levels indicated by H-score in primary and metastatic samples. The solid diagonal line (y = x) and the dashed lines (y = x±50, y = x±100) are shown to highlight the magnitude of the absolute differences between x and y axes.</p

Gene expression correlations between matched primary and metastatic ER+ breast cancer tumor samples.

<p>(A) Gene expression correlations between 61 matched primary and metastatic tumor samples for 90 genes from the breast cancer gene expression assay. Each dot represents the mean fold change between primary and metastatic samples for a single gene. (B) Correlation plots of genes that showed a greater than 1.5 fold difference in expression between matched primary and metastatic samples (FDR-adjusted P<0.05). Each dot represents the fold change between primary and metastatic samples for a single patient. The solid diagonal line (y = x) and the dashed lines (y = x±1) are shown to highlight the magnitude of the absolute differences between x and y axes.</p

Biological validation of the breast cancer gene expression assay using samples of known immunohistochemical subtype.

<p>(A) Hierarchical clustering of thirty FFPE breast cancer tumor samples with known ER, PR and HER2 status run on the breast cancer gene expression assay. Blue =  triple negative, Pink  =  ER+, Yellow = HER2+ (red = high expression, green = low expression) (B) Box-plots indicating genes that showed statistically significant differential expression in the ER+ subtype, (C) ER+ and HER2+ subtype, (D) HER2+ subtype and (E) triple negative subtype samples (3N) (p-values indicated).</p

Ki67 protein and gene expression analysis.

<p>(A) <i>MKI67</i> mRNA expression levels and relationship to Ki67 protein staining levels as determined by IHC. (B) Differentially expressed genes associated with Ki67 high or low protein staining levels (p-values indicated). Ki67 ≤ 15%, N = 16, Ki67 > 15%, N = 48.</p

PIK3CA, AKT1, PTEN and Ki67 status across a collection of 77 matched ER+ breast cancers.

<p>MND - Mutation not detected; E545X - E545A, G, D, K; Q546X - Q546E, K, R, L; Positive - PTEN positive, unable to determine H-Score; NA - Not available.</p