Journal: bioRxiv

Article Title: A long-chain fatty acid elongase Elovl6 regulates mechanical damage–induced keratinocyte death and skin inflammation

doi: 10.1101/264838

Figure Lengend Snippet: (A) Enzyme-linked immunosorbent assay of HMGB-1 (n = 4 per group) and cytokine bead array of IL-1α (n = 3 per group) in the supernatant of cultured primary keratinocytes 10 h after initiation of stimulation with 300 pM OA or CVA. (B) Quantitative RT-PCR analysis of Il1β and Cxcl1 in the epidermis of wild-type mice 6 h after topical application of ethanol (control) (n = 10) or 15mM of OA (n = 13) or CVA (n = 14) (B). (C, D) Wild-type and Elovl6 -/- mice were treated with PBS (n = 10 and 12, respectively), an IL-1 receptor antagonist (n = 9 and 8, respectively), or a CXCR-2 antagonist (n = 5 and 4, respectively) daily for 9 days, from the beginning on the day of tape stripping. Epidermal thickness (C) and the number of infiltrating neutrophils (D) were analyzed on day 9. (E) A proposed signal pathway from mechanical damage onto the skin to skin inflammation. Error bars indicate SD; *, P < 0.05; **, P < 0.01, ***, P < 0.001; NS, not significant. Data are representative of at least two independent experiments.

Article Snippet: Primary mouse keratinocytes were stimulated with bovine HMGB-1 (Chondrex, Redmond, Washington, USA) or mouse IL-1α (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Enzyme-linked Immunosorbent Assay, Cell Culture, Quantitative RT-PCR, Stripping Membranes

Journal: bioRxiv

Article Title: A long-chain fatty acid elongase Elovl6 regulates mechanical damage–induced keratinocyte death and skin inflammation

doi: 10.1101/264838

Figure Lengend Snippet: Quantitative RT-PCR analysis of Il1β and Cxcl1 in keratinocytes of wild-type and Elovl6 -/- mice after stimulation or not with HMGB-1 or IL-1α in vitro (n=10 per group) (A) and in the epidermis isolated 4 h after injection intradermally with PBS, HMGB-1, or IL-1α (n = 8 per each group) (B).

Article Snippet: Primary mouse keratinocytes were stimulated with bovine HMGB-1 (Chondrex, Redmond, Washington, USA) or mouse IL-1α (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Quantitative RT-PCR, In Vitro, Isolation, Injection

Journal: bioRxiv

Article Title: Targeting a proteolytic neo-epitope of CUB-domain containing protein 1 in RAS-driven cancer

doi: 10.1101/2021.06.14.448427

Figure Lengend Snippet: ( A ) Design of a PreScission Protease (Px)-cleavable CDCP1 ectodomain fused to a TEV-releasable Fc domain with C-terminal Avi-tag (CDCP1(Px)-Fc). The R368/K369 cleavage site was replaced with a Px recognition sequence (GS) 5 -LEVLFQGP-(GS) 5 . ( B ) SDS-PAGE of CDCP1 constructs. Px treatment cleaves CDCP1(Px)-Fc into NTF and CTF-Fc fragments. NTF is heavily glycosylated (predicted 14 N-linked glycosylation sites) and runs as a smeared higher-molecular weight band at ∼60 kDa. ( C ) SEC traces of CDCP1(R368/K369A)-Fc and CDCP1(Px)-Fc treated with Px, and NTF (TEV released) show that the NTF and CTF of CDCP1(Px) remain intact after proteolysis. Numbers denote fractions corresponding to the SDS-PAGE gel lanes in B . ( D ) BLI of IgG 4A06, which recognizes the NTF, shows robust binding to both Px-treated and untreated CDCP1(Px)-Fc. ( E ) Design of Px-cleavable CDCP1 with N-terminal FLAG-tag expressed on the surface of HEK293T cells. ( F ) Flow cytometry and western blot of HEK293T-wt, HEK293T-CDCP1(R368A/K369A), HEK293T-CDCP1(Px). Flow cytometry signal of anti-FLAG and IgG 4A06 remains unchanged with Px treatment. Western blot with anti-CDCP1 D1W9N, which recognizes the C-terminal intracellular region of CDCP1, confirms Px-mediated CDCP1 proteolysis at the intended molecular weight.

Article Snippet: Immunoblotting was performed using CDCP1(D1W9N) (Cell Signaling, 13794S), Phospho-CDCP1(Tyr707) (Cell Signaling, 13111S), Phospho-CDCP1(Tyr806) (Cell Signaling, 13024S), Phospho-CDCP1(Try734) (Cell Signaling, 9050S), Phospho-CDCP1(Try743)(D2G2J) (Cell Signaling, 14965S), Src(36D10) (Cell Signaling, 2109S), Phospho Src Family (Tyr416) (Cell Signaling, 2101S), PKCδ (Cell Signaling, 2058S), Phospho-PKCdelta(Tyr311) (Cell Signaling, 2055S), alpha-Tubulin(DM1A) (Cell Signaling, 3873S), IRDye 680RD Goat anti-Mouse (LiCOR, 925–68070), and IRDye 800CW Goat anti-Rabbit (LiCOR, 926-32211) antibodies.

Techniques: Sequencing, SDS Page, Construct, Molecular Weight, Binding Assay, FLAG-tag, Flow Cytometry, Western Blot

Journal: bioRxiv

Article Title: Targeting a proteolytic neo-epitope of CUB-domain containing protein 1 in RAS-driven cancer

doi: 10.1101/2021.06.14.448427

Figure Lengend Snippet: ( A ) Schematic of IP-MS strategy to identify the endogenous proteolysis sites of CDCP1 on PDAC cells. CDCP1 was IP-ed with IgG 4A06 or D1W9N Ab and digested with Glu-C, which cleaves after aspartic acid, and was analyzed by LC-MS/MS to identify peptides corresponding to proteolytic products of CDCP1. ( B ) ( top ) Western blot of PDAC cell lines expressing differential amounts of uncleaved and cleaved CDCP1. D1W9N Ab was used to detect C-terminal fragment of CDCP1. PL5 and PL45 express mostly cleaved CDCP1, while HPAC expresses mostly uncleaved CDCP1. HPNE, a non-malignant pancreatic cell line, expresses low levels of CDCP1. ( bottom ) IP-blot shows that IP with IgG 4A06 can pull-down the CTF of CDCP1. ( C ) Depiction of peptides and proteolysis sites identified in PL5, PL45, and HPAC cell lines. Peptides identified by LC-MS for each cell line is aligned to the reference sequence highlighted in light blue. Aspartic acid residues recognized by Glu-C are highlighted in red underlined text. Three proteolysis sites of CDCP1: Cut 1 (K365), Cut 2 (R368), Cut 3 (K369), are observed in PL5 and PL45 cells but not on HPAC cells, and are highlighted in blue text and blue vertical lines.

Article Snippet: Immunoblotting was performed using CDCP1(D1W9N) (Cell Signaling, 13794S), Phospho-CDCP1(Tyr707) (Cell Signaling, 13111S), Phospho-CDCP1(Tyr806) (Cell Signaling, 13024S), Phospho-CDCP1(Try734) (Cell Signaling, 9050S), Phospho-CDCP1(Try743)(D2G2J) (Cell Signaling, 14965S), Src(36D10) (Cell Signaling, 2109S), Phospho Src Family (Tyr416) (Cell Signaling, 2101S), PKCδ (Cell Signaling, 2058S), Phospho-PKCdelta(Tyr311) (Cell Signaling, 2055S), alpha-Tubulin(DM1A) (Cell Signaling, 3873S), IRDye 680RD Goat anti-Mouse (LiCOR, 925–68070), and IRDye 800CW Goat anti-Rabbit (LiCOR, 926-32211) antibodies.

Techniques: Liquid Chromatography with Mass Spectroscopy, Western Blot, Expressing, Sequencing

Journal: bioRxiv

Article Title: Targeting a proteolytic neo-epitope of CUB-domain containing protein 1 in RAS-driven cancer

doi: 10.1101/2021.06.14.448427

Figure Lengend Snippet: ( A ) Schematic of the co-transfection strategy to generate c-CDCP1 ectodomain. The NTF and CTF are encoded on separate plasmids with an IL2 secretion sequence. ( B ) SDS-PAGE of fl-CDCP1 and c-CDCP1 (Cut 1, Cut 2, Cut 3) ectodomain show successful expression and purification. NTF is heavily glycosylated and runs as a high-molecular weight smear. ( C ) BLI of IgG 4A06 to fl- or c-CDCP1 ectodomains show that the NTF of CDCP1 is intact on both cleaved and uncleaved CDCP1. ( D ) Differential Scanning Fluorimetry (DSF) shows that fl- and c-CDCP1 have similar melting profiles and stabilities, suggesting the NTF/CTF complex does not dissociate until full unfolding of the protein. T m is reported as an average and standard deviation of two replicates. ( E ) Circular Dichroism (CD) spectra of fl- and c-CDCP1. CDCP1 has a β-sheet signature with minima ∼217 nm. The slight difference in spectral shape between fl- and c-CDCP1 indicates a subtle change in secondary structure. ( F ) SAXS-derived P(r) function of fl- and c-CDCP1 ectodomains show similar overall architecture. SAXS-derived ab initio envelopes are shown under the graph. ( G ) Radii of gyration (R g ) of fl- and c-CDCP1. ( H ) SEC-MALS chromatograms of fl- and c-CDCP1 show similar elution profiles and molecular weights corresponding to monomeric ectodomain.

Article Snippet: Immunoblotting was performed using CDCP1(D1W9N) (Cell Signaling, 13794S), Phospho-CDCP1(Tyr707) (Cell Signaling, 13111S), Phospho-CDCP1(Tyr806) (Cell Signaling, 13024S), Phospho-CDCP1(Try734) (Cell Signaling, 9050S), Phospho-CDCP1(Try743)(D2G2J) (Cell Signaling, 14965S), Src(36D10) (Cell Signaling, 2109S), Phospho Src Family (Tyr416) (Cell Signaling, 2101S), PKCδ (Cell Signaling, 2058S), Phospho-PKCdelta(Tyr311) (Cell Signaling, 2055S), alpha-Tubulin(DM1A) (Cell Signaling, 3873S), IRDye 680RD Goat anti-Mouse (LiCOR, 925–68070), and IRDye 800CW Goat anti-Rabbit (LiCOR, 926-32211) antibodies.

Techniques: Cotransfection, Sequencing, SDS Page, Expressing, Purification, Molecular Weight, Standard Deviation, Derivative Assay

Journal: bioRxiv

Article Title: Targeting a proteolytic neo-epitope of CUB-domain containing protein 1 in RAS-driven cancer

doi: 10.1101/2021.06.14.448427

Figure Lengend Snippet: ( A ) Schematic of strategy to generate HEK293T cell lines expressing fl- or c-CDCP1. For c-CDCP1, a lentiviral vector was designed where a T2A self-cleavage sequence flanks the CTF (res 370-836) and NTF (res 30-369). For fl-CDCP1, a lentiviral vector encoding the full CDCP1 sequence (res 30-836) was designed. An IL2 signal sequence precedes each fragment. ( B ) Flow cytometry of IgG 4A06 to HEK293T fl-CDCP1 and HEK293T c-CDCP1 cell lines indicates the NTF of CDCP1 is present on the cell surface for all cell lines. ( D ) Western blot of CDCP1 and intracellular proteins associated with CDCP1 signaling. Both fl-CDCP1 and c-CDCP1 are phosphorylated and initiate downstream signaling mediated by Src and PKCδ. Phosphorylation of Y734 on CDCP1 is important for phosphorylation of other tyrosine residues and downstream signaling partners. Note, anti-phosphoY311-PKCδ appears to be cross-reactive to CDCP1-pY734. ( D ) Cell adhesion assay comparing HEK239T fl-CDCP1 and c-CDCP1 shows that overexpression of both fl- and c- CDCP1 decreases cell adhesion and is dependent on phosphorylation of intracellular tyrosine residues, specifically of Y734. **p = 0.0016, ****p < 0.0001, ns = not significant p >0.05. (unpaired t-test)

Article Snippet: Immunoblotting was performed using CDCP1(D1W9N) (Cell Signaling, 13794S), Phospho-CDCP1(Tyr707) (Cell Signaling, 13111S), Phospho-CDCP1(Tyr806) (Cell Signaling, 13024S), Phospho-CDCP1(Try734) (Cell Signaling, 9050S), Phospho-CDCP1(Try743)(D2G2J) (Cell Signaling, 14965S), Src(36D10) (Cell Signaling, 2109S), Phospho Src Family (Tyr416) (Cell Signaling, 2101S), PKCδ (Cell Signaling, 2058S), Phospho-PKCdelta(Tyr311) (Cell Signaling, 2055S), alpha-Tubulin(DM1A) (Cell Signaling, 3873S), IRDye 680RD Goat anti-Mouse (LiCOR, 925–68070), and IRDye 800CW Goat anti-Rabbit (LiCOR, 926-32211) antibodies.

Techniques: Expressing, Plasmid Preparation, Sequencing, Flow Cytometry, Western Blot, Cell Adhesion Assay, Over Expression

Journal: bioRxiv

Article Title: Targeting a proteolytic neo-epitope of CUB-domain containing protein 1 in RAS-driven cancer

doi: 10.1101/2021.06.14.448427

Figure Lengend Snippet: ( A ) Differential phage selection strategy to identify a cleaved CDCP1-specific antibody. Fab-phage were pre-cleared with fl-CDCP1-Fc prior to positive selection with c-CDCP1-Fc. Enriched Fab-phage were characterized by phage ELISA for selective binding to c-CDCP1-Fc. ( B ) BLI show specific binding of IgG CL03 to c-CDCP1-Fc but not to fl-CDCP1-Fc. (K D = 150-840 pM, Table S1 ) ( C ) Negative-stain EM 3D reconstruction of c-CDCP1 with CL03 Fab. ( left ) 2D class averages of c-CDCP1(Cut3) + CL03 Fab in the absence and presence of anti-Fab V H H. ( right ) Different views of 3D EM map of CDCP1(Cut3) + CL03 Fab + V H H with crystal structure of Fab (green) and V H H (blue) modeled into the density. ( D ) Immunofluorescence of Alexa Fluor-488-labeled IgG CL03 ( left panels ) and IgG 4A06 ( right panels ) on HPAC, PL5, and HPNE cells. IgG CL03 specifically stains PL5 cells that express cleaved CDCP1, while IgG 4A06 stains both HPAC and PL5 cells. ( E ) Flow cytometry shows that IgG CL03 binds to cleaved CDCP1-expressing PL5 and PL45 cells but not HPAC or HPNE cells. (n = 3, data represent average and standard deviation). ( F ) ( top ) Schematic of antibody drug conjugate (ADC) cell killing assay. ( bottom ) Dose-dependent ADC-mediated cell killing with IgG CL03 was only observed against PL5 and PL45 cells that express cleaved CDCP1, and only in the presence of both the primary and secondary antibody. (**p = 0.004, ***p = 0.0018, unpaired T-test) ( G ) In vivo PET imaging of 89 Zr-labeled IgG CL03 in PDAC xenograft mice harboring PL5 tumors (n = 4).

Article Snippet: Immunoblotting was performed using CDCP1(D1W9N) (Cell Signaling, 13794S), Phospho-CDCP1(Tyr707) (Cell Signaling, 13111S), Phospho-CDCP1(Tyr806) (Cell Signaling, 13024S), Phospho-CDCP1(Try734) (Cell Signaling, 9050S), Phospho-CDCP1(Try743)(D2G2J) (Cell Signaling, 14965S), Src(36D10) (Cell Signaling, 2109S), Phospho Src Family (Tyr416) (Cell Signaling, 2101S), PKCδ (Cell Signaling, 2058S), Phospho-PKCdelta(Tyr311) (Cell Signaling, 2055S), alpha-Tubulin(DM1A) (Cell Signaling, 3873S), IRDye 680RD Goat anti-Mouse (LiCOR, 925–68070), and IRDye 800CW Goat anti-Rabbit (LiCOR, 926-32211) antibodies.

Techniques: Selection, Enzyme-linked Immunosorbent Assay, Binding Assay, Staining, Immunofluorescence, Labeling, Flow Cytometry, Expressing, Standard Deviation, In Vivo, Imaging

Journal: bioRxiv

Article Title: Targeting a proteolytic neo-epitope of CUB-domain containing protein 1 in RAS-driven cancer

doi: 10.1101/2021.06.14.448427

Figure Lengend Snippet: ( A ) BLI show specific binding of IgG58 to mouse c-CDCP1-Fc, but not to fl-CDCP1-Fc. ( B ) Flow cytometry shows that IgG58 binds robustly to Fc1245 c-CDCP1, but not to Fc1245 WT cells (n = 3, error bars represent s.d.). ( C ) Dose-dependent ADC-mediated cell killing with IgG58-MMAF treatment was only observed with Fc1245 c-CDCP1 cells and not Fc1245 WT cells. (n = 2, error bars represent s.d.) (**p = 0.002, unpaired T-test) ( D ) Representative in vivo PET images of 89 Zr-IgG58 and 89 Zr-IgG12 in mice harboring subcutaneous Fc1245 c-CDCP1 tumors. Co-administration of 50X unlabeled IgG was used to examine target specificity. ( E ) Biodistribution of 89 Zr-IgG58 and 89 Zr-IgG12 in mice harboring subcutaneous Fc1245 c-CDCP1 tumors (n = 5 per arm). Both 89 Zr-IgG58 and 89 Zr-IgG12 signal decreased when 50X unlabeled IgG was administered, indicating target-specific localization (**p = 0.003, ***p = 0.0002, unpaired T-test). 89 Zr-IgG58 shows stronger signal in the tumor, while 89 Zr-IgG12 shows weaker tumor localization and more widespread normal tissue distribution (****p <0.0001, unpaired T-test). ( F ) ADC toxicity assay in non-tumor bearing mice. Mice (n = 5 per arm) were dosed weekly with 5, 10, 15 mg/kg of either IgG12-MMAF or IgG58-MMAF and body weight was monitored for treatment-associated toxicity. There was a significant difference between the treatment arms (F(5,32) = 3.11, p = 0.0002, ANOVA), with IgG58-MMAF treatment being better tolerated, with significant differences between IgG12-MMAF and IgG58-MMAF treatments at the 15 mg/kg dose (***p = 0.0068) and 10 mg/kg dose (**p = 0.0067) (Tukey’s multiple comparisons test). ( G-H ) Theranostic efficacy study of 177 Lu-IgG58. Mice (n = 5 per treatment arm, n = 8 for vehicle arm) were injected with 400 µCi of 177 Lu-IgG58 or vehicle 4 days after Fc1245-c-CDCP1 tumor implantation. For the 2-dose arm, the treatment was repeated 6 days later. Treatment with 177 Lu-IgG58 resulted in decreased tumor growth and increased survival compared to the vehicle arm (***p = 0.0008, ****p <0.0001, unpaired two-tailed T-test).

Article Snippet: Immunoblotting was performed using CDCP1(D1W9N) (Cell Signaling, 13794S), Phospho-CDCP1(Tyr707) (Cell Signaling, 13111S), Phospho-CDCP1(Tyr806) (Cell Signaling, 13024S), Phospho-CDCP1(Try734) (Cell Signaling, 9050S), Phospho-CDCP1(Try743)(D2G2J) (Cell Signaling, 14965S), Src(36D10) (Cell Signaling, 2109S), Phospho Src Family (Tyr416) (Cell Signaling, 2101S), PKCδ (Cell Signaling, 2058S), Phospho-PKCdelta(Tyr311) (Cell Signaling, 2055S), alpha-Tubulin(DM1A) (Cell Signaling, 3873S), IRDye 680RD Goat anti-Mouse (LiCOR, 925–68070), and IRDye 800CW Goat anti-Rabbit (LiCOR, 926-32211) antibodies.

Techniques: Binding Assay, Flow Cytometry, In Vivo, Injection, Tumor Implantation, Two Tailed Test