Journal: Acta Pharmaceutica Sinica. B

Article Title: Peptidomimetic-based antibody surrogate for HER2

doi: 10.1016/j.apsb.2021.04.016

Figure Lengend Snippet: (A) Schematic presentation of one-bead-two-compounds TentalGel beads for library. (B) Scheme for the synthesis of OBTC cyclic γ -AApeptides library. (a) Soak in water for overnight; (b) (Boc) 2 O, DCM/ether; (c) Fmoc-Met-OH, HOBt, DIC, DMF; (d) deprotect Fmoc by 20% piperidine in DMF; (e) split into five portions equally; (f) Dde protected amino acids, PyBOP, NEM, DMF; (g) deprotect Boc by TFA/triisopropylsilane/H 2 O/Thioanisole (94:2:2:2); (h) Fmoc protected γ -AApeptides, HOBt, DIC, DMF; (i) deprotecting alloc by Pd(PPh 3 ) 4 and Me 2 NH·BH 3 in DCM; (j) Dmt protected mercaptopropionic acid, HOBt, DIC, DMF; (k) deprotecting Dde by NH 2 OH . HCl and imidazole in NMP/DCM (5:1); (l) split-and-pool synthesis, repeating the previous steps; (m) deprotecting Fmoc by 20% piperidine in DMF; (n) 4-(bromomethyl)benzoyl chloride, DIPEA, DCM; (o) deprotecting Dmt by TFA/triisopropylsilane/DCM (2:2:96); (p) (NH 4 ) 2 CO 3 , DMF/H 2 O (1:1). Xs are regular α -amino acids. (C) Scheme showing the overall strategy involved in library screening against HER2.

Article Snippet: HER2 was purchased from Creative BioMart; Anti-phospho-HER2 antibody was purchased from Thermo Fisher Scientific; Anti-phospho-AKT and Anti-phospho-ERK antibodies were from Cell Signaling Technology; GAPDH loading control monoclonal antibody was purchased from Invitrogen.

Techniques: Library Screening

Journal: Acta Pharmaceutica Sinica. B

Article Title: Peptidomimetic-based antibody surrogate for HER2

doi: 10.1016/j.apsb.2021.04.016

Figure Lengend Snippet: (A) Signal transduction pathway mediated by HER2 and proposed inhibition by cyclic peptides monomer and dimer. (B) Western blot analyses of SKBR3 cell lysates following M-3-6 incubation in vitro . M-3-6 treatment resulted in a dose-dependent inhibition of HER2 receptor phosphorylation and a reduction in the phosphorylation of AKT and ERK, downstream signaling of HER2. (C) Western blot analyses of SKBR3 cell lysates following M-3-6-D incubation in vitro . M-3-6-D treatment resulted in a much more effective phosphorylation inhibition compared with M-3-6. (D) M-3-6 and M-3-6-D inhibit cell proliferation. EGF-driven proliferation of SKBR3 cells was analyzed by CCK-8 proliferation assay. EGF (100 ng/mL) induced the proliferation of cells under serum-starved conditions ( ### P < 0.001 vs . non-stimulated cells), and both M-3-6 and M-3-6-D inhibited EGF-induced proliferation significantly in a dose-dependent manner (∗ P < 0.05 vs . EGF-stimulated cells, ∗∗∗ P < 0.001 vs . EGF-stimulated cells).

Article Snippet: HER2 was purchased from Creative BioMart; Anti-phospho-HER2 antibody was purchased from Thermo Fisher Scientific; Anti-phospho-AKT and Anti-phospho-ERK antibodies were from Cell Signaling Technology; GAPDH loading control monoclonal antibody was purchased from Invitrogen.

Techniques: Transduction, Inhibition, Western Blot, Incubation, In Vitro, Phospho-proteomics, CCK-8 Assay, Proliferation Assay

Journal: Acta Pharmaceutica Sinica. B

Article Title: Peptidomimetic-based antibody surrogate for HER2

doi: 10.1016/j.apsb.2021.04.016

Figure Lengend Snippet: Therapeutic efficacy of M-3-6 and M-3-6-D in SKBR3 xenograft models. (A) Mice body weight shift curve of the mice during the experiment. Arrows indicate the time of compounds treatment. (B) Time course assessment of total tumor volume. The day when treatment started was recorded as day 0 and arrows indicate the time of injection. After 2 weeks injection, tumor volume was measured once every 3 days until Day 24. (C) Immunohistochemical staining for P-HER2, P-AKT and P-ERK. Representative staining of section from SKBR3 tumors with antibodies against the indicated proteins. Scale bar: 100 nm.

Article Snippet: HER2 was purchased from Creative BioMart; Anti-phospho-HER2 antibody was purchased from Thermo Fisher Scientific; Anti-phospho-AKT and Anti-phospho-ERK antibodies were from Cell Signaling Technology; GAPDH loading control monoclonal antibody was purchased from Invitrogen.

Techniques: Drug discovery, Injection, Immunohistochemical staining, Staining

Journal: Nature Communications

Article Title: Longitudinal proteome-wide antibody profiling in Marburg virus survivors identifies wing domain immunogen for vaccine design

doi: 10.1038/s41467-024-51021-5

Figure Lengend Snippet: a Overview of cohort with longitudinal convalescent samples collected approximately every six months from 10 MVD survivors and 10 matched uninfected local cohort from the 2012 Uganda outbreak. Comprehensive antibody profiling of each plasma sample was performed via Marburg virion neutralizing antibodies in PRNT, MARV-proteome wide IgM, IgG, IgA epitope repertoire by GFPDL, recombinant MARV protein binding antibody kinetics using surface plasmon resonance and Fc-receptor interactions in Luminex assay. b Neutralizing titer (PRNT50) against wild-type MARV Ci67 are shown for the 10 MVD survivors (in colors) and 10 uninfected controls (in black did not show any detectable virus neutralization). A positive control human serum sample demonstrated neutralization activity against MARV with a PRNT50 titer of 1:160 and is depicted as dashed line. Source data are provided as a Source Data file.

Article Snippet: An in-depth understanding of the humoral immune response following MARV infection can facilitate knowledge-based development and evaluation of effective vaccines and antibody-based therapeutics against Marburg virus.

Techniques: Clinical Proteomics, Recombinant, Protein Binding, SPR Assay, Luminex, Virus, Neutralization, Positive Control, Activity Assay

Journal: Nature Communications

Article Title: Longitudinal proteome-wide antibody profiling in Marburg virus survivors identifies wing domain immunogen for vaccine design

doi: 10.1038/s41467-024-51021-5

Figure Lengend Snippet: a IgM, IgG, and IgA antibody epitope repertoire recognized in the human plasma at different months post-MARV infection (pi) during convalescence (12-, 30-, 43- and 60-months pi are color coded) and their alignment to the whole proteome of MARV (showing different proteins: NP, VP35, VP40, GP, VP30, VP24 and L). Graphical distribution of representative clones with a frequency of ≥2, obtained after affinity selection, are shown. The horizontal position and the length of the bars indicate the peptide sequence displayed on the selected phage clone to its homologous sequence in the MARV proteome on alignment. The thickness of each bar represents the frequency of repetitively isolated phage, with the scale shown below the alignment. The GFPDL affinity selection data was performed in duplicate (two independent experiments by researcher in the lab, who was blinded to sample identity), and similar number of phage clones and epitope repertoire was observed in both phage display analysis. b Structural representations of immunodominant antigenic sites recognized by IgG using MARV-GFPDL on the surface structure of mature trimeric MARV GP solved structure [PDB #5UQY ]. The different domains of mature GP: GP1 (residues 1334-1769 in cyan), receptor binding site (RBS; residues 1372-1522 in orange), GP2 (residues 1842-2015 in blue), fusion loop (FL; residues 1842-1885 in pink) and N-terminal heptad repeat (NHR; residues 1886-1929 in yellow) are shown on the structure of MARV-GP, with the residue numbers corresponding to the complete MARV proteome used for GFPDL. The MARV GP structure used for crystallography encodes for MARV GPΔmuc ectodomain (encompassing amino acid residues 1–636 with a mucin deletion of residues 257–425). The immunodominant antigenic sites recognized by IgG in MVD survivors identified using MARV-GFPDL that can be mapped on the crystal structure are depicted in red.

Article Snippet: An in-depth understanding of the humoral immune response following MARV infection can facilitate knowledge-based development and evaluation of effective vaccines and antibody-based therapeutics against Marburg virus.

Techniques: Clinical Proteomics, Infection, Clone Assay, Selection, Sequencing, Isolation, Binding Assay, Residue

Journal: Nature Communications

Article Title: Longitudinal proteome-wide antibody profiling in Marburg virus survivors identifies wing domain immunogen for vaccine design

doi: 10.1038/s41467-024-51021-5

Figure Lengend Snippet: Serial dilutions of longitudinal plasma samples collected at 12-months through 60-months post-infection from the MVD survivors ( N = 10; in colors) and uninfected controls ( N = 10; in black) were analyzed for antibody binding to purified recombinant MARV proteins by SPR. Total antibody binding is represented in SPR resonance units (RU) for 10-fold dilution of plasma sample binding to Marburg virus-like particles (VLPs expressing trimeric GP on the surface of particles made of MARV matrix proteins VP40 and NP), and recombinant MARV GP, NP, VP35, VP40, VP24, VP30 and L polymerase. The mean half-life (in months) of the polyclonal binding antibodies against various MARV proteins for the convalescent phase in these MVD survivors is shown, except for VP30 and L, which demonstrated weak antibody binding. All SPR experiments were performed twice and the researchers performing the assay were blinded to sample identity. The variation for each sample in duplicate SPR runs was <7%. The data shown is the average value of two experimental runs. Source data are provided as a Source Data file.

Article Snippet: An in-depth understanding of the humoral immune response following MARV infection can facilitate knowledge-based development and evaluation of effective vaccines and antibody-based therapeutics against Marburg virus.

Techniques: Clinical Proteomics, Infection, Binding Assay, Purification, Recombinant, Virus, Expressing

Journal: Nature Communications

Article Title: Longitudinal proteome-wide antibody profiling in Marburg virus survivors identifies wing domain immunogen for vaccine design

doi: 10.1038/s41467-024-51021-5

Figure Lengend Snippet: Antibody affinity maturation of longitudinal plasma samples collected from MVD survivors to purified recombinant MARV proteins was determined by SPR. Antibody off-rate constants that describe the fraction of antibody-antigen complexes decaying per second were determined directly from the interaction of serially diluted post-infection plasma with MARV proteins using SPR in the dissociation phase only for the sensorgrams with maximum RU in the range of 10-100 RU in the optimized SPR. Antibody affinity was not determined for uninfected control samples or against MARV-VP30 or L as most of the post-infection MVD plasma samples showed very low antibody binding ( < 10 RU) against these two proteins. All SPR experiments were performed twice and the researchers performing the assay were blinded to sample identity. The variation for each sample in duplicate SPR runs was <7%. The data shown is the average value of two experimental runs. Source data are provided as a Source Data file.

Article Snippet: An in-depth understanding of the humoral immune response following MARV infection can facilitate knowledge-based development and evaluation of effective vaccines and antibody-based therapeutics against Marburg virus.

Techniques: Clinical Proteomics, Purification, Recombinant, Infection, Control, Binding Assay

Journal: Nature Communications

Article Title: Longitudinal proteome-wide antibody profiling in Marburg virus survivors identifies wing domain immunogen for vaccine design

doi: 10.1038/s41467-024-51021-5

Figure Lengend Snippet: Quantification of FcγRI, FcγRIIA, FcγRIIIA, FcγRIIB, or FcγRIIIB interaction with longitudinal 10 MVD survivor plasma antibodies from 12 to 60 months post-infection bound to MARV VLP (recombinant VLPs expressing MARV trimeric GP on the surface of particles made of MARV matrix proteins VP40 and NP) coupled beads using the bead-based assay. The level of binding is shown as mean fluorescent intensity (MFI). Each sample was run in duplicate, and each dot represents an average of duplicate values. The variation for each sample in duplicate runs was <5%. Source data are provided as a Source Data file.

Article Snippet: An in-depth understanding of the humoral immune response following MARV infection can facilitate knowledge-based development and evaluation of effective vaccines and antibody-based therapeutics against Marburg virus.

Techniques: Clinical Proteomics, Infection, Recombinant, Expressing, Bead-based Assay, Binding Assay

Journal: Nature Communications

Article Title: Longitudinal proteome-wide antibody profiling in Marburg virus survivors identifies wing domain immunogen for vaccine design

doi: 10.1038/s41467-024-51021-5

Figure Lengend Snippet: a Elucidation of immunodominant antigenic sites against the MARV proteome following MARV infection in survivors. Immunodominant antigenic sites within the MARV proteome recognized by serum antibodies in MVD survivors (based on data presented in Fig. ) are shown in gray bars aligned to the corresponding MARV protein sequence either above or below the proteome schematic. The antigenic sites discovered in MARV-GP using the post-infection antibodies are depicted below the MARV-GP schematic. b Serial dilution of serum samples obtained from rabbits immunized thrice with protein or KLH conjugated antigenic site peptides were analyzed for total IgG binding to MARV-Ci67 in ELISA. The area under the curve data shown is the mean of two rabbits per immunogen. c Virus neutralization titers were determined using wild-type MARV-Ci67 and BSL-4 based microneutralization assay. The average percent neutralization of a 1:10 dilution of sera of two rabbits per immunogen from duplicate assays is shown. Source data are provided as a Source Data file.

Article Snippet: An in-depth understanding of the humoral immune response following MARV infection can facilitate knowledge-based development and evaluation of effective vaccines and antibody-based therapeutics against Marburg virus.

Techniques: Infection, Sequencing, Serial Dilution, Binding Assay, Enzyme-linked Immunosorbent Assay, Virus, Neutralization, Microneutralization Assay

Journal: Nature Communications

Article Title: Longitudinal proteome-wide antibody profiling in Marburg virus survivors identifies wing domain immunogen for vaccine design

doi: 10.1038/s41467-024-51021-5

Figure Lengend Snippet: Schematic displaying the differential evolution of virion-binding IgG in ELISA and MARV neutralization titers, MARV specific IgM vs IgG vs IgA, antibody affinity maturation and Fc-receptor interaction across MARV proteome during convalescent phase over years in MVD survivors. The study data only portrays the antibody kinetics in the convalescent phase (post 12-months) in these MVD survivors.

Article Snippet: An in-depth understanding of the humoral immune response following MARV infection can facilitate knowledge-based development and evaluation of effective vaccines and antibody-based therapeutics against Marburg virus.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Neutralization

Journal: Nature Communications

Article Title: Super-enhancer-based identification of a BATF3/IL-2R−module reveals vulnerabilities in anaplastic large cell lymphoma

doi: 10.1038/s41467-021-25379-9

Figure Lengend Snippet: a OS of pediatric ALCL, ALK + patients (NHL-BFM cohort, n = 88), and b EFS and OS of adult ALCL, ALK − patients without SCT ( n = 34) having low (staining intensity, SI = 0–2) or high (SI = 3) IL-2Rα expression. Kaplan–Meier curves of individual groups were compared using two-tailed log-rank statistics. c Indicated 9 ALCL cell lines and 2 T-cell leukemia-derived control cell lines were incubated for 96 h with different concentrations of PDB dimer linked antibodies targeting either IL-2Rα (ADCT-301) or, as a non-binding control, the glycoprotein gp120 of HIV (B12-SG3249) to determine the LD 50 . Means ± SD of biological triplicates are shown. d In vivo anti-tumor efficacy of ADCT-301 in murine xenograft models of Mac-2A (ALK − ) or TLBR-1 (BIA) ALCL cell lines. For each cell line, mice were randomized into three groups to receive a single dose of either vehicle (PBS, n = 4), control ADC (B12-SG3249, 0.5 mg/kg, n = 5), or ADCT-301 (0.5 mg/kg, n = 5) intravenously at day 1 (indicated by an arrow). Tumor volumes were measured with caliper over time and are shown as means ± SEM. P values were determined by two-tailed unpaired Student’s t test. e Schematic illustration of the proposed mechanism.

Article Snippet: When tumor volumes reached ~100–150 mm 3 , mice were randomly allocated into three groups to receive vehicle (PBS), non-binding ADC (B12-SG3249) or ADCT-301 (camidanlumab tesirine) (both ADC Therapeutics, 0.5 mg/kg, intravenous injection).

Techniques: Staining, Expressing, Two Tailed Test, Derivative Assay, Control, Incubation, Binding Assay, In Vivo