in vitro plasma stability assay anti her2 dvd adc  (Millipore)


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    Name:
    HER2
    Description:
    HER2 eceptor tyrosine protein kinase erbB 2 is a cell surface associated glycoprotein tyrosine kinase receptor
    Catalog Number:
    h3040
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    Structured Review

    Millipore in vitro plasma stability assay anti her2 dvd adc
    Assembly of <t>DVD-ADCs</t> and in vitro efficacy. a Structure of β-lactam MMAF. b <t>anti-HER2</t> <t>DVD-ADC</t> was assembled by incubating the anti-HER2 DVD with four equivalents (eq) of β-lactam MMAF at room temperature (rt) for 4 h. Drug attachment (red star) occurs at the reactive Lys of h38C2 (yellow circle), located in the heavy chain variable domain of anti-HER2 DVD, to form a stable amide bond. c ESI-MS was performed with unconjugated anti-HER2 DVD and conjugated anti-HER2 DVD-ADC after reduction (10 mM DTT) and deglycosylation (PNGase F). No conjugation was detected on the light chain and the increase in mass of the heavy chain (~1160 Da) corresponds to the addition of exactly one eq of β-lactam MMAF. No unconjugated or higher drug loaded species were detected. d Cytotoxicity of anti-HER2 DVD-ADC (red) following incubation with HER2+ BC cell line SK-BR-3 and HER2− BC cell line MDA-MB-468 for 72 h at 37 °C (mean ± SD of triplicates). Unconjugated anti-HER2 DVD (blue) was significantly less toxic to SK-BR-3 cells when compared to anti-HER2 DVD-ADC ( p
    HER2 eceptor tyrosine protein kinase erbB 2 is a cell surface associated glycoprotein tyrosine kinase receptor
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    Images

    1) Product Images from "Harnessing a catalytic lysine residue for the one-step preparation of homogeneous antibody-drug conjugates"

    Article Title: Harnessing a catalytic lysine residue for the one-step preparation of homogeneous antibody-drug conjugates

    Journal: Nature Communications

    doi: 10.1038/s41467-017-01257-1

    Assembly of DVD-ADCs and in vitro efficacy. a Structure of β-lactam MMAF. b anti-HER2 DVD-ADC was assembled by incubating the anti-HER2 DVD with four equivalents (eq) of β-lactam MMAF at room temperature (rt) for 4 h. Drug attachment (red star) occurs at the reactive Lys of h38C2 (yellow circle), located in the heavy chain variable domain of anti-HER2 DVD, to form a stable amide bond. c ESI-MS was performed with unconjugated anti-HER2 DVD and conjugated anti-HER2 DVD-ADC after reduction (10 mM DTT) and deglycosylation (PNGase F). No conjugation was detected on the light chain and the increase in mass of the heavy chain (~1160 Da) corresponds to the addition of exactly one eq of β-lactam MMAF. No unconjugated or higher drug loaded species were detected. d Cytotoxicity of anti-HER2 DVD-ADC (red) following incubation with HER2+ BC cell line SK-BR-3 and HER2− BC cell line MDA-MB-468 for 72 h at 37 °C (mean ± SD of triplicates). Unconjugated anti-HER2 DVD (blue) was significantly less toxic to SK-BR-3 cells when compared to anti-HER2 DVD-ADC ( p
    Figure Legend Snippet: Assembly of DVD-ADCs and in vitro efficacy. a Structure of β-lactam MMAF. b anti-HER2 DVD-ADC was assembled by incubating the anti-HER2 DVD with four equivalents (eq) of β-lactam MMAF at room temperature (rt) for 4 h. Drug attachment (red star) occurs at the reactive Lys of h38C2 (yellow circle), located in the heavy chain variable domain of anti-HER2 DVD, to form a stable amide bond. c ESI-MS was performed with unconjugated anti-HER2 DVD and conjugated anti-HER2 DVD-ADC after reduction (10 mM DTT) and deglycosylation (PNGase F). No conjugation was detected on the light chain and the increase in mass of the heavy chain (~1160 Da) corresponds to the addition of exactly one eq of β-lactam MMAF. No unconjugated or higher drug loaded species were detected. d Cytotoxicity of anti-HER2 DVD-ADC (red) following incubation with HER2+ BC cell line SK-BR-3 and HER2− BC cell line MDA-MB-468 for 72 h at 37 °C (mean ± SD of triplicates). Unconjugated anti-HER2 DVD (blue) was significantly less toxic to SK-BR-3 cells when compared to anti-HER2 DVD-ADC ( p

    Techniques Used: In Vitro, Mass Spectrometry, Conjugation Assay, Incubation, Multiple Displacement Amplification

    In vivo efficacy of anti-HER2 DVD-ADC. a Human BC cell line KPL-4 was xenografted into the mammary fat pads of female NSG mice, grown to ~150 mm 3 , randomized into five groups comprising seven or eight mice each, and treated with i.v. (tail vein) injections of the indicated ADCs and controls once a week for 4 weeks. The benchmark ADC, T-DM1 (black), was used as a positive control. Mean ± SEM values are plotted. When compared with a t -test (one-tailed, unpaired, uneven variance), the 5 mg kg −1 (red) and 10 mg kg −1 (blue) DVD-ADC groups were significantly different from the 10 mg kg −1 DVD group (green) on day 8 ( p = 0.0046 and p = 0.00016, respectively) until the last common data point on day 36 ( p = 0.0000069 and p = 0.0000019, respectively). b Corresponding Kaplan−Meier survival curve with p -values (log-rank (Mantel-Cox) test) comparing survival between anti-HER2 DVD-ADC groups (red and blue) and the unconjugated anti-HER2 DVD group (green)
    Figure Legend Snippet: In vivo efficacy of anti-HER2 DVD-ADC. a Human BC cell line KPL-4 was xenografted into the mammary fat pads of female NSG mice, grown to ~150 mm 3 , randomized into five groups comprising seven or eight mice each, and treated with i.v. (tail vein) injections of the indicated ADCs and controls once a week for 4 weeks. The benchmark ADC, T-DM1 (black), was used as a positive control. Mean ± SEM values are plotted. When compared with a t -test (one-tailed, unpaired, uneven variance), the 5 mg kg −1 (red) and 10 mg kg −1 (blue) DVD-ADC groups were significantly different from the 10 mg kg −1 DVD group (green) on day 8 ( p = 0.0046 and p = 0.00016, respectively) until the last common data point on day 36 ( p = 0.0000069 and p = 0.0000019, respectively). b Corresponding Kaplan−Meier survival curve with p -values (log-rank (Mantel-Cox) test) comparing survival between anti-HER2 DVD-ADC groups (red and blue) and the unconjugated anti-HER2 DVD group (green)

    Techniques Used: In Vivo, Mouse Assay, Positive Control, One-tailed Test

    Construction of anti-HER2 DVDs. a h38C2 IgG1 compared to an anti-HER2 DVD. The DVD is composed of variable domains of trastuzumab (blue), h38C2 (green) with reactive Lys (yellow circle), and constant domains (gray). Two fully human spacer sequences (red lines) were used to prepare DVD1 with a short spacer (ASTKGP) or DVD2 with a long spacer (TVAAPSVFIFPP). b Coomassie stained SDS-PAGE confirmed the purity of both DVD1 and DVD2 under non-reducing (expected ~200 kDa) and reducing conditions (expected heavy chain ~63 kDa, light chain ~36 kDa). Molecular weights from a pre-stained protein ladder are shown on the left. c Flow cytometry showing specific binding of DVD1 (red) and DVD2 (blue) to SK-BR-3 cells (HER2+) with no binding detected to MDA-MB-468 cells (HER2−). Trastuzumab IgG1 (green) was used as a positive control and h38C2 IgG1 (black) as a negative control. d The catalytic retro-aldol activity of the reactive Lys of h38C2 was measured using methodol as a substrate. The signal is reported in relative fluorescent units (RFU; mean ± SD of triplicates). Trastuzumab IgG1 (green) was used as a negative control. The slope of DVD1 was not significantly different ( p = 0.1967; linear regression analysis) from h38C2 IgG1, but the slope of DVD2 was significantly smaller ( p
    Figure Legend Snippet: Construction of anti-HER2 DVDs. a h38C2 IgG1 compared to an anti-HER2 DVD. The DVD is composed of variable domains of trastuzumab (blue), h38C2 (green) with reactive Lys (yellow circle), and constant domains (gray). Two fully human spacer sequences (red lines) were used to prepare DVD1 with a short spacer (ASTKGP) or DVD2 with a long spacer (TVAAPSVFIFPP). b Coomassie stained SDS-PAGE confirmed the purity of both DVD1 and DVD2 under non-reducing (expected ~200 kDa) and reducing conditions (expected heavy chain ~63 kDa, light chain ~36 kDa). Molecular weights from a pre-stained protein ladder are shown on the left. c Flow cytometry showing specific binding of DVD1 (red) and DVD2 (blue) to SK-BR-3 cells (HER2+) with no binding detected to MDA-MB-468 cells (HER2−). Trastuzumab IgG1 (green) was used as a positive control and h38C2 IgG1 (black) as a negative control. d The catalytic retro-aldol activity of the reactive Lys of h38C2 was measured using methodol as a substrate. The signal is reported in relative fluorescent units (RFU; mean ± SD of triplicates). Trastuzumab IgG1 (green) was used as a negative control. The slope of DVD1 was not significantly different ( p = 0.1967; linear regression analysis) from h38C2 IgG1, but the slope of DVD2 was significantly smaller ( p

    Techniques Used: Staining, SDS Page, Flow Cytometry, Cytometry, Binding Assay, Multiple Displacement Amplification, Positive Control, Negative Control, Activity Assay

    Related Articles

    Stable Transfection:

    Article Title: DNA vaccines targeting the encoded antigens to dendritic cells induce potent antitumor immunity in mice
    Article Snippet: .. D2F2/E2 and EL4/E2 stably expressing human wild-type HER2 were maintained in complete DMEM medium containing 0.4 mg/mL G418 (Sigma-Aldrich). ..

    other:

    Article Title: Trastuzumab upregulates PD-L1 as a potential mechanism of trastuzumab resistance through engagement of immune effector cells and stimulation of IFNγ secretion
    Article Snippet: In our current study, we found that HER2-overexpressing breast cancer cells with a low basal level of PD-L1 responded poorly to IFNγ-induced upregulation of PD-L1 but remained responsive to IFNγ-induced upregulation of HLA-ABC.

    Expressing:

    Article Title: Dual targeting of HER3 and MEK may overcome HER3-dependent drug-resistance of colon cancers
    Article Snippet: .. Western blot analysis To analyze phospho and total-HER3, phospho and total-HER2, phospho and total-EGFR, phospho- and Total-ERK 1/2, PARP, and HSP70 proteins expression, the cells were lysed with RIPA buffer [50 mM Tris (pH 8), 150 mM NaCl, 1% Nonidet P40, 0,1% deoxycholate, 0,1% SDS, 1 mM PMSF, 5 mM Na3 VO4 , 50 mM protease inhibitors (SIGMA-Aldrich, Milan, IT)] for 30 minutes at 4°C. .. Total cell lysates were clarified by centrifugation at 14,000 rpm for 30 minutes at 4°C.

    Article Title: HER2-encoded mir-4728 forms a receptor-independent circuit with miR-21-5p through the non-canonical poly(A) polymerase PAPD5
    Article Snippet: .. MCF7 Tet-Off cells (BD Biosciences) expressing inducible full length or p95-HER2 were established as described and maintained in medium containing 1 μg/ml doxycycline (Sigma). .. Antisense oligonucleotides (IDT DNA Technologies) contained 2′-O -methyl modifications and are listed in .

    Article Title: Trastuzumab upregulates PD-L1 as a potential mechanism of trastuzumab resistance through engagement of immune effector cells and stimulation of IFNγ secretion
    Article Snippet: .. To determine if there is a link between the levels of PD-L1 and HER2 in HER2-overexpressing breast cancer, we examined if knockdown of HER2 by siRNA in HER2-overexpressing breast cancer cells or overexpression of HER2 in HER2-low-expressing breast cancer cells had any direct effect on PD-L1 expression. ..

    Article Title: DNA vaccines targeting the encoded antigens to dendritic cells induce potent antitumor immunity in mice
    Article Snippet: .. D2F2/E2 and EL4/E2 stably expressing human wild-type HER2 were maintained in complete DMEM medium containing 0.4 mg/mL G418 (Sigma-Aldrich). ..

    Western Blot:

    Article Title: Dual targeting of HER3 and MEK may overcome HER3-dependent drug-resistance of colon cancers
    Article Snippet: .. Western blot analysis To analyze phospho and total-HER3, phospho and total-HER2, phospho and total-EGFR, phospho- and Total-ERK 1/2, PARP, and HSP70 proteins expression, the cells were lysed with RIPA buffer [50 mM Tris (pH 8), 150 mM NaCl, 1% Nonidet P40, 0,1% deoxycholate, 0,1% SDS, 1 mM PMSF, 5 mM Na3 VO4 , 50 mM protease inhibitors (SIGMA-Aldrich, Milan, IT)] for 30 minutes at 4°C. .. Total cell lysates were clarified by centrifugation at 14,000 rpm for 30 minutes at 4°C.

    Over Expression:

    Article Title: Trastuzumab upregulates PD-L1 as a potential mechanism of trastuzumab resistance through engagement of immune effector cells and stimulation of IFNγ secretion
    Article Snippet: .. To determine if there is a link between the levels of PD-L1 and HER2 in HER2-overexpressing breast cancer, we examined if knockdown of HER2 by siRNA in HER2-overexpressing breast cancer cells or overexpression of HER2 in HER2-low-expressing breast cancer cells had any direct effect on PD-L1 expression. ..

    Software:

    Article Title: A 2-Helix Small Protein Labeled with 68Ga for PET Imaging of HER2 Expression
    Article Snippet: .. For determining the relative HER2 protein levels, the intensity of the HER2 protein band was normalized with the intensity of the α-tubulin (Sigma-Aldrich) band from each sample using Image Processing and Analysis in Java (ImageJ; open-source image software downloaded from ). ..