All nBT062 model variants are internalized <i>in vitro</i>.

<p>(A) Schematic overview of measured binding/internalization status of fluorescent labeled antibodies. (B) Flow cytometric internalization analysis of WT nBT062, stable nBT062, half nBT062 and bispecific nBT062-natalizumab. CD49d was blocked by 50x excess of unlabeled natalizumab. Data represent median fluorescence intensity values of at least three separate experiments. Column colors represent antibody location as shown in (A). ****p<0.0001, ***p<0.001. (C) Fluorescence microscopy of BaF3-hCD138 (CD138⁺) cells (I-V) or BaF3 (CD138^-) control cells (VI) incubated for 3h with indicated Dylight-448 labeled nBT062 model antibodies or natalizumab (green, a). Lysosomal-associated membrane protein-1 (LAMP-1) is shown in red (b), nucleus is shown in blue (c). Respective overlay pictures are demonstrated (d).</p

Antibody binding affinities towards BaF3-hCD138 (CD138⁺/CD49d^-) and Jurkat (CD138^-/CD49d⁺) cells calculated from binding curves of Fig 3.

<p>Antibody binding affinities towards BaF3-hCD138 (CD138⁺/CD49d^-) and Jurkat (CD138^-/CD49d⁺) cells calculated from binding curves of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195823#pone.0195823.g003" target="_blank">Fig 3</a>.</p

Half-antibody exchange preventing mutations of nBT062-DM4 models improve the tumor growth inhibition in the presence of human IgG against CD138⁺ human mammary carcinoma xenografts in NMRI nude mice.

<p>Mice bearing established xenograft tumors were treated with three once weekly i.v. injections (indicated by arrows) of WT nBT062-DM4, stable nBT062-DM4, half nBT062-DM4 and bispecific nBT062-natalizumab-DM4 either alone or in combination with a 10% intravenous immunoglobulin G (IVIg) preparation administered i.v. 10 ml/kg/week. PBS was used as vehicle control and IVIg was also tested as monotherapy. The nBT062-DM4 model variants were tested using doses of 4 mg/kg/week or 2 mg/kg/week.</p

nBT062-DM4 model variants kill CD138⁺ NCI-H929 tumor cells <i>in vitro</i>.

<p>NCI-H929 cells (CD138⁺/CD49d⁺) were incubated for 5 days with varying concentrations of WT nBT062-DM4, stable nBT062-DM4, half nBT062-DM4 and bispecific nBT062-natalizumab-DM4 antibodies. Natalizumab was used for CD49d blocking. Reciprocal IC₅₀ values of each antibody-drug conjugate were normalized to WT nBT062-DM4 and one DM4 molecule per antibody. Results are shown from three separate experiments each measured in triplicates. *p<0.05, **p<0.01.</p

All nBT062 model variants are internalized <i>in vitro</i>.

<p>(A) Schematic overview of measured binding/internalization status of fluorescent labeled antibodies. (B) Flow cytometric internalization analysis of WT nBT062, stable nBT062, half nBT062 and bispecific nBT062-natalizumab. CD49d was blocked by 50x excess of unlabeled natalizumab. Data represent median fluorescence intensity values of at least three separate experiments. Column colors represent antibody location as shown in (A). ****p<0.0001, ***p<0.001. (C) Fluorescence microscopy of BaF3-hCD138 (CD138⁺) cells (I-V) or BaF3 (CD138^-) control cells (VI) incubated for 3h with indicated Dylight-448 labeled nBT062 model antibodies or natalizumab (green, a). Lysosomal-associated membrane protein-1 (LAMP-1) is shown in red (b), nucleus is shown in blue (c). Respective overlay pictures are demonstrated (d).</p

Schematic half antibody exchange and respective nBT062 model antibodies.

<p>(A) Overview of <i>in vivo</i> IgG4 half antibody exchange mainly driven by the potential to form either interchain or intrachain disulfide bonds in the hinge region. A S228P mutation increases the sterical distance of the two cysteines preventing intrachain disulfide bonds. (B) Generated nBT062 model antibodies: Wild type (WT) nBT062, stable nBT062, half nBT062 and bispecific nBT062-natalizumab. Respective mutations are indicated.</p

nBT062 model variants selectively bind to CD138⁺ cells in a dose-depended manner.

<p>BaF3-hCD138 cells, positive for nBT062 antigen and negative for natalizumab antigen (A), and Jurkat cells, negative for nBT062 antigen and positive for natalizumab antigen (B), were incubated with different concentrations (5x10^-7–2.82x10^-12 M) of WT nBT062, stable nBT062, half nBT062 and bispecific nBT062-natalizumab antibodies. Data was obtained via flow cytometry using a secondary anti-human antibody for detection. Binding curves demonstrate one representative experiment out of three, each measured in triplicates. MFI = Median Fluorescence Intensity.</p

DM4-to-antibody ratios of wild type (WT), stable and half nBT062 and bispecific nBT062-natalizumab.

<p>DM4-to-antibody ratios of wild type (WT), stable and half nBT062 and bispecific nBT062-natalizumab.</p

Tumor control efficacy criteria.

<p>Tumor control efficacy criteria.</p

Betaine feeding improves hFVIII and hFIX secretion <i>in vivo</i>.

<p>(A–F) 24 hours post minicircle FVIII-BDD gene transfer the FVIII knockout mice received water without (group I) or with 2% betaine supplementation in the drinking water (group II, each n = 10). After 3 days plasma samples were collected and each group was monitored for human FVIII antigen (A and B) and related activity levels (D and E) and group treatment was switched. After another 3 days, plasma levels were tested again. (C and F) represent the calculated overall effect of betaine on FVIII antigen levels (C) or FVIII activity (D). square symbols indicate samples of the first measuring point, triangles the second one. (G–I) After reaching stable FIX expression levels following minicircle FIX gene transfer, FIX knockout mice were fed 2% Betaine-supplemented drinking water ad libitum in a crossover-study of two groups. After 3 and 17 days of treatment, retroorbitally collected plasma samples were monitored for human FIX antigen levels (G and H). (I) shows the overall change in FIX expression from both groups after 17 days of administration. All values are represented as mean ± SEM. Same symbols indicate samples of the same mouse at different time points; clear: tap water treatment (control), filled: betaine administration. Student’s t-test ((G) ANOVA). *<i>P</i><.05, **<i>P</i><.005, ***<i>P</i><.0005.</p