Suzie Scales | Genentech - Academia.edu (original) (raw)

Papers by Suzie Scales

Research paper thumbnail of Apolipoprotein L1-Specific Antibodies Detect Endogenous APOL1 inside the Endoplasmic Reticulum and on the Plasma Membrane of Podocytes

Journal of the American Society of Nephrology, 2020

Significance Statement Specific variants of APOL1, G1 and G2, are associated with CKD in the Blac... more Significance Statement Specific variants of APOL1, G1 and G2, are associated with CKD in the Black population. Overexpression of these variants kills cells, through different proposed mechanisms in different subcellular compartments. The localization of endogenous APOL1 has not been conclusively established because all studies have used antibodies that crossreact with APOL2. Generation and use of APOL1-specific antibodies show that endogenous podocyte APOL1 localizes mainly inside the endoplasmic reticulum, with a few molecules on the cell surface. These findings potentially support the endoplasmic reticulum stress or cell surface cation channel models of cytotoxicity. Background APOL1 is found in human kidney podocytes and endothelia. Variants G1 and G2 of the APOL1 gene account for the high frequency of nondiabetic CKD among African Americans. Proposed mechanisms of kidney podocyte cytotoxicity resulting from APOL1 variant overexpression implicate different subcellular compartment...

Research paper thumbnail of Supplementary Figure 4 from FcRL5 as a Target of Antibody–Drug Conjugates for the Treatment of Multiple Myeloma

PDF file - 48KB, In vivo efficacy of anti-FcRL5-MC-vc-PAB-MMAE in combination with lenalidomide

Research paper thumbnail of Supplementary Figure 1 from An Antimesothelin-Monomethyl Auristatin E Conjugate with Potent Antitumor Activity in Ovarian, Pancreatic, and Mesothelioma Models

Supplementary Figure 1: IHC validation of anti-mesothelin antibody 19C3.

Research paper thumbnail of Supplementary Materials and Methods and Supplementary Figure Legends from An Antimesothelin-Monomethyl Auristatin E Conjugate with Potent Antitumor Activity in Ovarian, Pancreatic, and Mesothelioma Models

Supplemental ancillary methods (ELISA, surface plasmon resonance and humanization) and 4 suppleme... more Supplemental ancillary methods (ELISA, surface plasmon resonance and humanization) and 4 supplemental figure legends.

Research paper thumbnail of Supplementary figures and Tables from Preclinical Efficacy of an Antibody–Drug Conjugate Targeting Mesothelin Correlates with Quantitative 89Zr-ImmunoPET

Supplementary Figure 1: Serum levels of AMA-MMAE in mice. Supplementary Figure 2: Correlation plo... more Supplementary Figure 1: Serum levels of AMA-MMAE in mice. Supplementary Figure 2: Correlation plot of tumor growth inhibition versus specific tumor uptake. Supplementary Table 1: Overview results. Supplementary Table 2: Tumor growth inhibition study of mice bearing OVCAR3-X2.1 cells comparing the efficacy of 5mg/kg and 20mg/kg doses

Research paper thumbnail of Supplementary Table S1 from Antixenograft tumor activity of a humanized anti-insulin-like growth factor-I receptor monoclonal antibody is associated with decreased AKT activation and glucose uptake

Supplementary Table S1 from Antixenograft tumor activity of a humanized anti-insulin-like growth ... more Supplementary Table S1 from Antixenograft tumor activity of a humanized anti-insulin-like growth factor-I receptor monoclonal antibody is associated with decreased AKT activation and glucose uptake

Research paper thumbnail of Supplementary Fig. S1 from Antixenograft tumor activity of a humanized anti-insulin-like growth factor-I receptor monoclonal antibody is associated with decreased AKT activation and glucose uptake

Supplementary Fig. S1 from Antixenograft tumor activity of a humanized anti-insulin-like growth f... more Supplementary Fig. S1 from Antixenograft tumor activity of a humanized anti-insulin-like growth factor-I receptor monoclonal antibody is associated with decreased AKT activation and glucose uptake

Research paper thumbnail of Supplementary Table S2 from Antixenograft tumor activity of a humanized anti-insulin-like growth factor-I receptor monoclonal antibody is associated with decreased AKT activation and glucose uptake

Supplementary Table S2 from Antixenograft tumor activity of a humanized anti-insulin-like growth ... more Supplementary Table S2 from Antixenograft tumor activity of a humanized anti-insulin-like growth factor-I receptor monoclonal antibody is associated with decreased AKT activation and glucose uptake

Research paper thumbnail of Supplementary Fig. S2 from Antixenograft tumor activity of a humanized anti-insulin-like growth factor-I receptor monoclonal antibody is associated with decreased AKT activation and glucose uptake

Supplementary Fig. S2 from Antixenograft tumor activity of a humanized anti-insulin-like growth f... more Supplementary Fig. S2 from Antixenograft tumor activity of a humanized anti-insulin-like growth factor-I receptor monoclonal antibody is associated with decreased AKT activation and glucose uptake

Research paper thumbnail of Supplementary Figure 1 from Antibody-Drug Conjugates for the Treatment of Non–Hodgkin's Lymphoma: Target and Linker-Drug Selection

Supplementary Figure 1 from Antibody-Drug Conjugates for the Treatment of Non–Hodgkin's Lymph... more Supplementary Figure 1 from Antibody-Drug Conjugates for the Treatment of Non–Hodgkin's Lymphoma: Target and Linker-Drug Selection

Research paper thumbnail of Supplementary Table 1 from Antibody-Drug Conjugates for the Treatment of Non–Hodgkin's Lymphoma: Target and Linker-Drug Selection

Supplementary Table 1 from Antibody-Drug Conjugates for the Treatment of Non–Hodgkin's Lympho... more Supplementary Table 1 from Antibody-Drug Conjugates for the Treatment of Non–Hodgkin's Lymphoma: Target and Linker-Drug Selection

Research paper thumbnail of Supplementary Methods, Figure Legends 1-6 from Small Molecule Inhibition of GDC-0449 Refractory Smoothened Mutants and Downstream Mechanisms of Drug Resistance

Supplementary Methods, Figure Legends 1-6 from Small Molecule Inhibition of GDC-0449 Refractory S... more Supplementary Methods, Figure Legends 1-6 from Small Molecule Inhibition of GDC-0449 Refractory Smoothened Mutants and Downstream Mechanisms of Drug Resistance

Research paper thumbnail of Data from Small Molecule Inhibition of GDC-0449 Refractory Smoothened Mutants and Downstream Mechanisms of Drug Resistance

Inappropriate Hedgehog (Hh) signaling has been directly linked to medulloblastoma (MB), a common ... more Inappropriate Hedgehog (Hh) signaling has been directly linked to medulloblastoma (MB), a common malignant brain tumor in children. GDC-0449 is an Hh pathway inhibitor (HPI) currently under clinical investigation as an anticancer agent. Treatment of a MB patient with GDC-0449 initially regressed tumors, but this individual ultimately relapsed with a D473H resistance mutation in Smoothened (SMO), the molecular target of GDC-0449. To explore the role of the mutated aspartic acid residue in SMO function, we substituted D473 with every amino acid and found that all functional mutants were resistant to GDC-0449, with positively charged residues conferring potential oncogenic properties. Alanine scan mutagenesis of SMO further identified E518 as a novel prospective mutation site for GDC-0449 resistance. To overcome this form of acquired resistance, we screened a panel of chemically diverse HPIs and identified several antagonists with potent in vitro activity against these GDC-0449–resista...

Research paper thumbnail of Supplementary Table 1 from Small Molecule Inhibition of GDC-0449 Refractory Smoothened Mutants and Downstream Mechanisms of Drug Resistance

Supplementary Table 1 from Small Molecule Inhibition of GDC-0449 Refractory Smoothened Mutants an... more Supplementary Table 1 from Small Molecule Inhibition of GDC-0449 Refractory Smoothened Mutants and Downstream Mechanisms of Drug Resistance

Research paper thumbnail of Data from Antixenograft tumor activity of a humanized anti-insulin-like growth factor-I receptor monoclonal antibody is associated with decreased AKT activation and glucose uptake

The insulin-like growth factor (IGF) system consists of two ligands (IGF-I and IGF-II), which bot... more The insulin-like growth factor (IGF) system consists of two ligands (IGF-I and IGF-II), which both signal through IGF-I receptor (IGF-IR) to stimulate proliferation and inhibit apoptosis, with activity contributing to malignant growth of many types of human cancers. We have developed a humanized, affinity-matured anti-human IGF-IR monoclonal antibody (h10H5), which binds with high affinity and specificity to the extracellular domain. h10H5 inhibits IGF-IR-mediated signaling by blocking IGF-I and IGF-II binding and by inducing cell surface receptor down-regulation via internalization and degradation, with the extracellular and intracellular domains of IGF-IR being differentially affected by the proteasomal and lysosomal inhibitors. In vitro, h10H5 exhibits antiproliferative effects on cancer cell lines. In vivo, h10H5 shows single-agent antitumor efficacy in human SK-N-AS neuroblastoma and SW527 breast cancer xenograft models and even greater efficacy in combination with the chemothe...

Research paper thumbnail of Targeted degradation via direct 26S proteasome recruitment

Nature Chemical Biology

Engineered destruction of target proteins by recruitment to the cell’s degradation machinery has ... more Engineered destruction of target proteins by recruitment to the cell’s degradation machinery has emerged as a promising strategy in drug discovery. The majority of molecules that facilitate targeted degradation do so via a select number of ubiquitin ligases, restricting this therapeutic approach to tissue types that express the requisite ligase. Here, we describe a new strategy of targeted protein degradation through direct substrate recruitment to the 26S proteasome. The proteolytic complex is essential and abundantly expressed in all cells; however, proteasomal ligands remain scarce. We identify potent peptidic macrocycles that bind directly to the 26S proteasome subunit PSMD2, with a 2.5-Å-resolution cryo-electron microscopy complex structure revealing a binding site near the 26S pore. Conjugation of this macrocycle to a potent BRD4 ligand enabled generation of chimeric molecules that effectively degrade BRD4 in cells, thus demonstrating that degradation via direct proteasomal re...

Research paper thumbnail of with the Creative Commons Deed, Attribution

High CD21 expression inhibits internalization of anti-CD19

Research paper thumbnail of Anti-CD19-MCC-DM1 is less efficacious in high CD21 expressing cells

<b>Copyright information:</b>Taken from "High CD21 expression inhibits internali... more <b>Copyright information:</b>Taken from "High CD21 expression inhibits internalization of anti-CD19 antibodies and cytotoxicity of an anti-CD19-drug conjugate"British Journal of Haematology 2007;140(1):46-58.Published online 07 Nov 2007PMCID:PMC2228374.© 2007 Genentech, Inc. Journal Compilation © 2007 Blackwell Publishing Ltd (A) CD21 Raji were incubated for 3 d with anti-CD21-MCC-DM1 (○), negative control Trastuzumab-MCC-DM1 (▴), free L-DM1 dimer (▪) or naked anti-CD21 antibodies (•) and assessed for viability by measuring ATP levels. CD21 ARH77 (B), CD21 Daudi (C), and CD21 DoHH2 cells (D) were incubated for 3 d with anti-CD19-MCC-DM1 (♦), negative control Trastuzumab-MCC-DM1 (▴), free L-DM1 dimer (▪) or naked anti-CD19 antibodies () and assessed for viability by measuring ATP levels. (E) Ramos (solid symbols and lines) and Ramos-CD21 clone 1 (open symbols and dashed lines) were treated with anti-CD19-MCC-DM1 (♦,⋄) or free DM1 (▪,□) as in B-D. Inset bar graph shows percentage killing of Ramos (♦) and Ramos-CD21 clone 1 (⋄) at the highest anti-CD19-MCC-DM1 concentration used (3·33 μg/ml). (F) Ramos (solid symbols and lines) and Ramos-CD21 clone 1 (open symbols and dashed lines) were treated with control Trastuzumab-MCC-DM1 (▴,△), free DM1 (▪,□) or unconjugated anti-CD19 () as in B. Data are shown in all panels as a percentage viability of untreated control cells (mean and standard deviation of three independent duplicate experiments) ADC concentration in μg/ml on the lower -axes or free DM1 concentration in M on the upper -axes. * denotes data points statistically different ( &lt; 0·01) from the control untreated cells using the analysis of variance () test. +, data points significantly different between Ramos and Ramos-CD21 clone 1 cells by analysis ( &lt; 0·01). The CD21 Raji and Ramos-clone 1 cells showed greater resistance (compared with their respective free L-DM1 sensitivities) than the CD21 Ramos, DoHH2 and CD21 Daudi cells.

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

<b>Copyright information:</b>Taken from "High CD21 expression inhibits internali... more <b>Copyright information:</b>Taken from "High CD21 expression inhibits internalization of anti-CD19 antibodies and cytotoxicity of an anti-CD19-drug conjugate"British Journal of Haematology 2007;140(1):46-58.Published online 07 Nov 2007PMCID:PMC2228374.© 2007 Genentech, Inc. Journal Compilation © 2007 Blackwell Publishing Ltd (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.

Research paper thumbnail of Quantitation of CD19 and CD21 surface levels and anti-CD19 uptake by flow cytometry confirms the immunofluorescence results

<b>Copyright information:</b>Taken from "High CD21 expression inhibits internali... more <b>Copyright information:</b>Taken from "High CD21 expression inhibits internalization of anti-CD19 antibodies and cytotoxicity of an anti-CD19-drug conjugate"British Journal of Haematology 2007;140(1):46-58.Published online 07 Nov 2007PMCID:PMC2228374.© 2007 Genentech, Inc. Journal Compilation © 2007 Blackwell Publishing Ltd (A) B-cell lines were incubated on ice with 2 μg/ml mouse anti-CD21 (HB135) or mouse anti-CD19 (B496), followed by rat anti-mouse-phycoerythrin and analyzed by flow cytometry to determine surface expression. Results are the average mean fluorescence intensity (MFI) of triplicates ± standard deviation from a representative of three independent experiments (average of five independent experiments shown for the more variable ARH77 cells). Shown in increasing order of CD21 expression are: (1) SuDHL-4, (2) Ramos, (3) DoHH2, (4) Namalwa, (5) Daudi, (6) Ramos-CD21 clone 3, (7) ARH77, (8) Raji, (9) Ramos-CD21 clone 1. (10) Freshly isolated human B-cells have lower fluorescence values for both antigens than expected due to their small size, but their relative ratio of CD21 to CD19 is similar to that of ARH77 and Raji cells. Ramos-CD21 clone 1 expresses CD21 even more highly than Raji, while Ramos-CD21 clone 3 is intermediate between that of ARH77 and Daudi. (B) The rate of internalization of Alexa488-anti-CD19 in Ramos (▪), Ramos-CD21 clone 1 (□), Ramos-CD21 clone 3 (△) and CD21 ARH77 (▴) cells was determined by pre-binding to cells then incubating at 37°C (without washing) for the indicated times, washing and fixing either with or without surface fluorescence quenching with anti-Alexa488. Results are the average and standard deviation of two duplicate experiments each normalized to their respective initial surface binding levels after subtraction of background signals.

Research paper thumbnail of Apolipoprotein L1-Specific Antibodies Detect Endogenous APOL1 inside the Endoplasmic Reticulum and on the Plasma Membrane of Podocytes

Journal of the American Society of Nephrology, 2020

Significance Statement Specific variants of APOL1, G1 and G2, are associated with CKD in the Blac... more Significance Statement Specific variants of APOL1, G1 and G2, are associated with CKD in the Black population. Overexpression of these variants kills cells, through different proposed mechanisms in different subcellular compartments. The localization of endogenous APOL1 has not been conclusively established because all studies have used antibodies that crossreact with APOL2. Generation and use of APOL1-specific antibodies show that endogenous podocyte APOL1 localizes mainly inside the endoplasmic reticulum, with a few molecules on the cell surface. These findings potentially support the endoplasmic reticulum stress or cell surface cation channel models of cytotoxicity. Background APOL1 is found in human kidney podocytes and endothelia. Variants G1 and G2 of the APOL1 gene account for the high frequency of nondiabetic CKD among African Americans. Proposed mechanisms of kidney podocyte cytotoxicity resulting from APOL1 variant overexpression implicate different subcellular compartment...

Research paper thumbnail of Supplementary Figure 4 from FcRL5 as a Target of Antibody–Drug Conjugates for the Treatment of Multiple Myeloma

PDF file - 48KB, In vivo efficacy of anti-FcRL5-MC-vc-PAB-MMAE in combination with lenalidomide

Research paper thumbnail of Supplementary Figure 1 from An Antimesothelin-Monomethyl Auristatin E Conjugate with Potent Antitumor Activity in Ovarian, Pancreatic, and Mesothelioma Models

Supplementary Figure 1: IHC validation of anti-mesothelin antibody 19C3.

Research paper thumbnail of Supplementary Materials and Methods and Supplementary Figure Legends from An Antimesothelin-Monomethyl Auristatin E Conjugate with Potent Antitumor Activity in Ovarian, Pancreatic, and Mesothelioma Models

Supplemental ancillary methods (ELISA, surface plasmon resonance and humanization) and 4 suppleme... more Supplemental ancillary methods (ELISA, surface plasmon resonance and humanization) and 4 supplemental figure legends.

Research paper thumbnail of Supplementary figures and Tables from Preclinical Efficacy of an Antibody–Drug Conjugate Targeting Mesothelin Correlates with Quantitative 89Zr-ImmunoPET

Supplementary Figure 1: Serum levels of AMA-MMAE in mice. Supplementary Figure 2: Correlation plo... more Supplementary Figure 1: Serum levels of AMA-MMAE in mice. Supplementary Figure 2: Correlation plot of tumor growth inhibition versus specific tumor uptake. Supplementary Table 1: Overview results. Supplementary Table 2: Tumor growth inhibition study of mice bearing OVCAR3-X2.1 cells comparing the efficacy of 5mg/kg and 20mg/kg doses

Research paper thumbnail of Supplementary Table S1 from Antixenograft tumor activity of a humanized anti-insulin-like growth factor-I receptor monoclonal antibody is associated with decreased AKT activation and glucose uptake

Supplementary Table S1 from Antixenograft tumor activity of a humanized anti-insulin-like growth ... more Supplementary Table S1 from Antixenograft tumor activity of a humanized anti-insulin-like growth factor-I receptor monoclonal antibody is associated with decreased AKT activation and glucose uptake

Research paper thumbnail of Supplementary Fig. S1 from Antixenograft tumor activity of a humanized anti-insulin-like growth factor-I receptor monoclonal antibody is associated with decreased AKT activation and glucose uptake

Supplementary Fig. S1 from Antixenograft tumor activity of a humanized anti-insulin-like growth f... more Supplementary Fig. S1 from Antixenograft tumor activity of a humanized anti-insulin-like growth factor-I receptor monoclonal antibody is associated with decreased AKT activation and glucose uptake

Research paper thumbnail of Supplementary Table S2 from Antixenograft tumor activity of a humanized anti-insulin-like growth factor-I receptor monoclonal antibody is associated with decreased AKT activation and glucose uptake

Supplementary Table S2 from Antixenograft tumor activity of a humanized anti-insulin-like growth ... more Supplementary Table S2 from Antixenograft tumor activity of a humanized anti-insulin-like growth factor-I receptor monoclonal antibody is associated with decreased AKT activation and glucose uptake

Research paper thumbnail of Supplementary Fig. S2 from Antixenograft tumor activity of a humanized anti-insulin-like growth factor-I receptor monoclonal antibody is associated with decreased AKT activation and glucose uptake

Supplementary Fig. S2 from Antixenograft tumor activity of a humanized anti-insulin-like growth f... more Supplementary Fig. S2 from Antixenograft tumor activity of a humanized anti-insulin-like growth factor-I receptor monoclonal antibody is associated with decreased AKT activation and glucose uptake

Research paper thumbnail of Supplementary Figure 1 from Antibody-Drug Conjugates for the Treatment of Non–Hodgkin's Lymphoma: Target and Linker-Drug Selection

Supplementary Figure 1 from Antibody-Drug Conjugates for the Treatment of Non–Hodgkin's Lymph... more Supplementary Figure 1 from Antibody-Drug Conjugates for the Treatment of Non–Hodgkin's Lymphoma: Target and Linker-Drug Selection

Research paper thumbnail of Supplementary Table 1 from Antibody-Drug Conjugates for the Treatment of Non–Hodgkin's Lymphoma: Target and Linker-Drug Selection

Supplementary Table 1 from Antibody-Drug Conjugates for the Treatment of Non–Hodgkin's Lympho... more Supplementary Table 1 from Antibody-Drug Conjugates for the Treatment of Non–Hodgkin's Lymphoma: Target and Linker-Drug Selection

Research paper thumbnail of Supplementary Methods, Figure Legends 1-6 from Small Molecule Inhibition of GDC-0449 Refractory Smoothened Mutants and Downstream Mechanisms of Drug Resistance

Supplementary Methods, Figure Legends 1-6 from Small Molecule Inhibition of GDC-0449 Refractory S... more Supplementary Methods, Figure Legends 1-6 from Small Molecule Inhibition of GDC-0449 Refractory Smoothened Mutants and Downstream Mechanisms of Drug Resistance

Research paper thumbnail of Data from Small Molecule Inhibition of GDC-0449 Refractory Smoothened Mutants and Downstream Mechanisms of Drug Resistance

Inappropriate Hedgehog (Hh) signaling has been directly linked to medulloblastoma (MB), a common ... more Inappropriate Hedgehog (Hh) signaling has been directly linked to medulloblastoma (MB), a common malignant brain tumor in children. GDC-0449 is an Hh pathway inhibitor (HPI) currently under clinical investigation as an anticancer agent. Treatment of a MB patient with GDC-0449 initially regressed tumors, but this individual ultimately relapsed with a D473H resistance mutation in Smoothened (SMO), the molecular target of GDC-0449. To explore the role of the mutated aspartic acid residue in SMO function, we substituted D473 with every amino acid and found that all functional mutants were resistant to GDC-0449, with positively charged residues conferring potential oncogenic properties. Alanine scan mutagenesis of SMO further identified E518 as a novel prospective mutation site for GDC-0449 resistance. To overcome this form of acquired resistance, we screened a panel of chemically diverse HPIs and identified several antagonists with potent in vitro activity against these GDC-0449–resista...

Research paper thumbnail of Supplementary Table 1 from Small Molecule Inhibition of GDC-0449 Refractory Smoothened Mutants and Downstream Mechanisms of Drug Resistance

Supplementary Table 1 from Small Molecule Inhibition of GDC-0449 Refractory Smoothened Mutants an... more Supplementary Table 1 from Small Molecule Inhibition of GDC-0449 Refractory Smoothened Mutants and Downstream Mechanisms of Drug Resistance

Research paper thumbnail of Data from Antixenograft tumor activity of a humanized anti-insulin-like growth factor-I receptor monoclonal antibody is associated with decreased AKT activation and glucose uptake

The insulin-like growth factor (IGF) system consists of two ligands (IGF-I and IGF-II), which bot... more The insulin-like growth factor (IGF) system consists of two ligands (IGF-I and IGF-II), which both signal through IGF-I receptor (IGF-IR) to stimulate proliferation and inhibit apoptosis, with activity contributing to malignant growth of many types of human cancers. We have developed a humanized, affinity-matured anti-human IGF-IR monoclonal antibody (h10H5), which binds with high affinity and specificity to the extracellular domain. h10H5 inhibits IGF-IR-mediated signaling by blocking IGF-I and IGF-II binding and by inducing cell surface receptor down-regulation via internalization and degradation, with the extracellular and intracellular domains of IGF-IR being differentially affected by the proteasomal and lysosomal inhibitors. In vitro, h10H5 exhibits antiproliferative effects on cancer cell lines. In vivo, h10H5 shows single-agent antitumor efficacy in human SK-N-AS neuroblastoma and SW527 breast cancer xenograft models and even greater efficacy in combination with the chemothe...

Research paper thumbnail of Targeted degradation via direct 26S proteasome recruitment

Nature Chemical Biology

Engineered destruction of target proteins by recruitment to the cell’s degradation machinery has ... more Engineered destruction of target proteins by recruitment to the cell’s degradation machinery has emerged as a promising strategy in drug discovery. The majority of molecules that facilitate targeted degradation do so via a select number of ubiquitin ligases, restricting this therapeutic approach to tissue types that express the requisite ligase. Here, we describe a new strategy of targeted protein degradation through direct substrate recruitment to the 26S proteasome. The proteolytic complex is essential and abundantly expressed in all cells; however, proteasomal ligands remain scarce. We identify potent peptidic macrocycles that bind directly to the 26S proteasome subunit PSMD2, with a 2.5-Å-resolution cryo-electron microscopy complex structure revealing a binding site near the 26S pore. Conjugation of this macrocycle to a potent BRD4 ligand enabled generation of chimeric molecules that effectively degrade BRD4 in cells, thus demonstrating that degradation via direct proteasomal re...

Research paper thumbnail of with the Creative Commons Deed, Attribution

High CD21 expression inhibits internalization of anti-CD19

Research paper thumbnail of Anti-CD19-MCC-DM1 is less efficacious in high CD21 expressing cells

<b>Copyright information:</b>Taken from "High CD21 expression inhibits internali... more <b>Copyright information:</b>Taken from "High CD21 expression inhibits internalization of anti-CD19 antibodies and cytotoxicity of an anti-CD19-drug conjugate"British Journal of Haematology 2007;140(1):46-58.Published online 07 Nov 2007PMCID:PMC2228374.© 2007 Genentech, Inc. Journal Compilation © 2007 Blackwell Publishing Ltd (A) CD21 Raji were incubated for 3 d with anti-CD21-MCC-DM1 (○), negative control Trastuzumab-MCC-DM1 (▴), free L-DM1 dimer (▪) or naked anti-CD21 antibodies (•) and assessed for viability by measuring ATP levels. CD21 ARH77 (B), CD21 Daudi (C), and CD21 DoHH2 cells (D) were incubated for 3 d with anti-CD19-MCC-DM1 (♦), negative control Trastuzumab-MCC-DM1 (▴), free L-DM1 dimer (▪) or naked anti-CD19 antibodies () and assessed for viability by measuring ATP levels. (E) Ramos (solid symbols and lines) and Ramos-CD21 clone 1 (open symbols and dashed lines) were treated with anti-CD19-MCC-DM1 (♦,⋄) or free DM1 (▪,□) as in B-D. Inset bar graph shows percentage killing of Ramos (♦) and Ramos-CD21 clone 1 (⋄) at the highest anti-CD19-MCC-DM1 concentration used (3·33 μg/ml). (F) Ramos (solid symbols and lines) and Ramos-CD21 clone 1 (open symbols and dashed lines) were treated with control Trastuzumab-MCC-DM1 (▴,△), free DM1 (▪,□) or unconjugated anti-CD19 () as in B. Data are shown in all panels as a percentage viability of untreated control cells (mean and standard deviation of three independent duplicate experiments) ADC concentration in μg/ml on the lower -axes or free DM1 concentration in M on the upper -axes. * denotes data points statistically different ( &lt; 0·01) from the control untreated cells using the analysis of variance () test. +, data points significantly different between Ramos and Ramos-CD21 clone 1 cells by analysis ( &lt; 0·01). The CD21 Raji and Ramos-clone 1 cells showed greater resistance (compared with their respective free L-DM1 sensitivities) than the CD21 Ramos, DoHH2 and CD21 Daudi cells.

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

<b>Copyright information:</b>Taken from "High CD21 expression inhibits internali... more <b>Copyright information:</b>Taken from "High CD21 expression inhibits internalization of anti-CD19 antibodies and cytotoxicity of an anti-CD19-drug conjugate"British Journal of Haematology 2007;140(1):46-58.Published online 07 Nov 2007PMCID:PMC2228374.© 2007 Genentech, Inc. Journal Compilation © 2007 Blackwell Publishing Ltd (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.

Research paper thumbnail of Quantitation of CD19 and CD21 surface levels and anti-CD19 uptake by flow cytometry confirms the immunofluorescence results

<b>Copyright information:</b>Taken from "High CD21 expression inhibits internali... more <b>Copyright information:</b>Taken from "High CD21 expression inhibits internalization of anti-CD19 antibodies and cytotoxicity of an anti-CD19-drug conjugate"British Journal of Haematology 2007;140(1):46-58.Published online 07 Nov 2007PMCID:PMC2228374.© 2007 Genentech, Inc. Journal Compilation © 2007 Blackwell Publishing Ltd (A) B-cell lines were incubated on ice with 2 μg/ml mouse anti-CD21 (HB135) or mouse anti-CD19 (B496), followed by rat anti-mouse-phycoerythrin and analyzed by flow cytometry to determine surface expression. Results are the average mean fluorescence intensity (MFI) of triplicates ± standard deviation from a representative of three independent experiments (average of five independent experiments shown for the more variable ARH77 cells). Shown in increasing order of CD21 expression are: (1) SuDHL-4, (2) Ramos, (3) DoHH2, (4) Namalwa, (5) Daudi, (6) Ramos-CD21 clone 3, (7) ARH77, (8) Raji, (9) Ramos-CD21 clone 1. (10) Freshly isolated human B-cells have lower fluorescence values for both antigens than expected due to their small size, but their relative ratio of CD21 to CD19 is similar to that of ARH77 and Raji cells. Ramos-CD21 clone 1 expresses CD21 even more highly than Raji, while Ramos-CD21 clone 3 is intermediate between that of ARH77 and Daudi. (B) The rate of internalization of Alexa488-anti-CD19 in Ramos (▪), Ramos-CD21 clone 1 (□), Ramos-CD21 clone 3 (△) and CD21 ARH77 (▴) cells was determined by pre-binding to cells then incubating at 37°C (without washing) for the indicated times, washing and fixing either with or without surface fluorescence quenching with anti-Alexa488. Results are the average and standard deviation of two duplicate experiments each normalized to their respective initial surface binding levels after subtraction of background signals.