Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: A cytokine receptor–targeting chimera toolbox for expanding extracellular targeted protein degradation

doi: 10.1073/pnas.2524129123

Figure Lengend Snippet: KineTAC application for eTPD of therapeutically relevant membrane proteins. ( A ) Scheme for EGFR degrading kineTACs includes the presented cytokine-Fc half with a common Cetuximab (Ctx)-Fc that binds EGFR. ( B ) Western blot quantification of mean β-actin normalized EGFR levels as a percentage of the PBS only control after 24 h treatment with 50 nM kineTAC or isotype Ctx single-arm control. Ctx Knob = monomeric cetuximab (Cetuximab Half IgG Hole with Fc only Knob IgG). Error bars are SEM. ( C ) representative western blot that was quantified in B . EGFR signal is HRP luminescence and β-actin is from a LICOR secondary. The EGFR signal was normalized to β-Actin as a percentage of the PBS only normalized EGFR signal. ( D ) Representative PD-1 degradation in a Jurkat PD-1 overexpression cell line after 24 h with 50 nM of the displayed kineTAC or isotype antibody control. Nivo = nivolumab. The PD-1 signal was normalized to β-Actin as a percentage of the PBS only normalized PD-1 signal. PD-1 signal is HRP luminescence and β-actin is from a LICOR secondary. IL7 isotype = half IL7 knob IgG with Fc only hole IgG. ( E ) Western blot quantification of activated primary T cell mean β-actin normalized PD-1 levels as a percentage of the PBS only control after 24 h treatment with kineTAC at displayed concentrations or isotypes at 100 nM. Nivo = Nivolumab. Error bars represent SEM. Five total donors are represented. ( F ) Representative western blot of PD-1 degradation in activated donor T cells from E . ( G ) KineTAC-ADC diagram. A MMAE payload was conjugated to an IL4-Rituximab kineTAC via a cleavable Val-Cit linker. Binding to both the IL4 Receptor and CD20 allow for internalization of the complex and release of the MMAE payload. ( H ) cell killing of Ramos cells by the specified doses of IL4-Rituximab-MMAE or isotype-MMAE (IL4 iso = IL4 containing IgG, no rituximab. Rit Knob = Monomeric rituximab IgG, no IL4. Both with MMAE payload). Lines represent nonlinear three-parameter agonist vs. response curves. Data are from three biological replicates and error bars represent SEM.

Article Snippet: Antibodies used included rabbit anti-human EGFR (Cell Signaling Technology, Cat# 4267S, 1:1,000), rabbit anti-human PD-1 (Cell Signaling Technology, Cat# D4W2J, 1:1,000), mouse anti-human IL7R (R&D Systems, Cat# MAB306, 1:1,000), rabbit anti-human phospho NF-κB p65 Ser 536 (Cell Signaling Technology, Cat#93H1, 1:1,000), mouse anti-human NF-κB p65 (Cell Signaling Technology, Cat#93H1, 1:1,000), mouse anti-human β-actin (Cell Signaling Technology, Cat# 8H10D10, 1:1,000), and mouse anti-human β-tubulin (Cell Signaling Technology, Cat# DM1a, 1:1,000).

Techniques: Membrane, Western Blot, Control, Over Expression, Binding Assay

Journal: Journal of the American Chemical Society

Article Title: Hijacking Extracellular Targeted Protein Degrader–Drug Conjugates for Enhanced Drug Delivery

doi: 10.1021/jacs.5c15047

Figure Lengend Snippet: Generation of LIPTACs for degradation of EGFR. (a) Schematic illustration of LDLR-Ctx LIPTAC bispecific constructs. (b) Western blot showing degradation of total EGFR on HeLa cells following 24 h of treatment with Ctx-LIPTAC or 50 nM control antibodies. Data represents three biological replicates. Percent EGFR levels were quantified by ImageJ relative to PBS control. (c) Changes in surface EGFR based on flow cytometry analysis on MDA-MB-231 cells following 24 h of 50 nM 142F1 isotype IgG, Ctx IgG, Ctx-LIPTAC, or Ctx-KineTAC treatment. Percent EGFR was determined by median fluorescence intensity (MFI) of the PE fluorescence channel of live cells. Each sample was tested in biological triplicate and error bars represented standard deviation. Statistics were calculated by unpaired two-tailed student t test. *** P < 0.001. **** P < 0.0001. (d) Western blot showing degradation of total EGFR on MDA-MB-231 cells after 24 h treatment of 50 nM Ctx, KineTAC, monomeric LDLR isotype, or varying concetrations of LIPTACs. Data represented three biological replicates. (e) EGFR degradation in A431 cells following 24 h of Ctx-LIPTAC1 treatment. Data represents three biological replicates. (f) Flow cytometry analysis showing degradation of surface EGFR on A431 cells following 24 h of Ctx-LIPTACs, 50 nM Ctx IgG, and 50 nM Ctx-KineTAC treatment. Each sample was tested in biological triplicate and error bars represent standard deviations. Statistics were calculated by unpaired two-tailed student t test. *** P < 0.001. **** P < 0.0001. (g) Western blot analysis of EGFR and LDLR in LDLR knockout and control HCC1143 cells after 24 h of 5 nM LIPTAC treatment. Data represent two biological replicates. (h) Fold-change in surface protein abundance in MDA-MB-231 cells following 48 h of treatment with or without 50 nM Ctx-LIPTAC1, as measured by quantitative proteomics analysis. N-Linked cell surface glycoproteins were captured by the cell-surface capture technology and enriched by biocytin hydrazide. Surface proteins were annotated using the SURFY database. (i) Confocal microscopy images of HeLa cells treated with 50 nM of indicated bispecific antibodies or isotype controls for 24 h. Scale bar, 10 μm.

Article Snippet: Antibodies used included rabbit anti-human EGFR (Cell Signaling Technology, catalog no. 4267S, 1:1000), rabbit anti-human PD-L1 (Cell Signaling Technology, catalog no. 13684S, 1:1000), rabbit anti-human CXCR4 (Cell Signaling Technology, catalog no. 64837S, 1:1000), rabbit anti-human ERBB2 (Cell Signaling Technology, catalog no. 4290S, 1:1000), rabbit anti-human CDCP1 (Cell Signaling Technology, catalog no. 13794S, 1:1000), mouse anti-human α-tubulin (Cell Signaling Technology, 3873S, 1:3000), goat anti-human LDLR (R&D Systems, catalog no. AF2148, 1:1000), IRDye 800CW goat anti-rabbit IgG (LI-COR Biosciences, catalog no. 926-32211), IRDye 680RD goat anti-mouse IgG (LI-COR Biosciences, catalog no. 926-68070, 1:5000), IRDye 800CW donkey anti-goat IgG (LI-COR Biosciences, catalog no. 926-32214, 1:5000), and peroxidase goat anti-rabbit IgG (H+L) (Jackson ImmunoResearch, catalog no. 111-035-144, 1:5000).

Techniques: Construct, Western Blot, Control, Flow Cytometry, Fluorescence, Standard Deviation, Two Tailed Test, Knock-Out, Quantitative Proteomics, Confocal Microscopy

Journal: Journal of the American Chemical Society

Article Title: Hijacking Extracellular Targeted Protein Degrader–Drug Conjugates for Enhanced Drug Delivery

doi: 10.1021/jacs.5c15047

Figure Lengend Snippet: Development of degrader–drug conjugates (DDCs) for potent target cell killing. (a) Schematic illustration of LIPTAC-DDCs where the membrane POI is internalized by endocytosis and the cytotoxic payload is released from cleavable linkers in the lysosome. (b) A payload cleavage assay in EGFR-expressing A431 cells. Cells were treated with 25 nM of antibodies conjugated with lysolight deep red (LLDR) dyes. Images were captured every 2 h for 72 h on the Incucyte. Total integrated intensity was calculated by NIRCU × μm 2 /image. Error bars represented standard deviations for four biological replicates. Statistics were calculated by one-way ANOVA and Holm–Sidak multiple comparisons test. (c,d) Cytotoxicity of Ctx-ADC, monomeric 142F1-ADC, and Ctx-DDC either in LIPTAC or KineTAC formats on A431 and MDA-MB-231 cells, respectively. After 72 h incubation, cell viability was measured using the CellTiter-Glo Reagent. EC 50 values were calculated using “One-Site Fit LogIC50” regression in GraphPad Prism 10.2. (e) Cytotoxicity of ADCs and corresponding DDCs on A431 cells in the presence and absence of the lysosomal inhibitor, BafA. Dead cells were labeled by cytotoxic green dye after 48 h of treatment. Dashed lines represented 50 nM BafA treatment together with antibodies. Total integrated intensity was calculated by GreenCU × μm 2 /image on the Incucyte. (f) Cytotoxicity of 0.8 nM DDCs on A431 cells in the presence of 50 nM BafA, 50 nM MG132, and 1 μM Nystatin, respectively. Cells treated with inhibitors alone showed no cytotoxicity at the concentrations used in DDC treatments. Dead cells were labeled by 1 μg/mL propidium iodide after 48 h of treatment. Total integrated intensity was calculated by ROCU × μm 2 /image on the Incucyte. Error bars represent standard deviations of four biological replicates. Statistics were calculated by unpaired two-tailed student t test. **** P < 0.0001. ns, not significant.

Article Snippet: Antibodies used included rabbit anti-human EGFR (Cell Signaling Technology, catalog no. 4267S, 1:1000), rabbit anti-human PD-L1 (Cell Signaling Technology, catalog no. 13684S, 1:1000), rabbit anti-human CXCR4 (Cell Signaling Technology, catalog no. 64837S, 1:1000), rabbit anti-human ERBB2 (Cell Signaling Technology, catalog no. 4290S, 1:1000), rabbit anti-human CDCP1 (Cell Signaling Technology, catalog no. 13794S, 1:1000), mouse anti-human α-tubulin (Cell Signaling Technology, 3873S, 1:3000), goat anti-human LDLR (R&D Systems, catalog no. AF2148, 1:1000), IRDye 800CW goat anti-rabbit IgG (LI-COR Biosciences, catalog no. 926-32211), IRDye 680RD goat anti-mouse IgG (LI-COR Biosciences, catalog no. 926-68070, 1:5000), IRDye 800CW donkey anti-goat IgG (LI-COR Biosciences, catalog no. 926-32214, 1:5000), and peroxidase goat anti-rabbit IgG (H+L) (Jackson ImmunoResearch, catalog no. 111-035-144, 1:5000).

Techniques: Membrane, Cleavage Assay, Expressing, Incubation, Labeling, Two Tailed Test