Journal: mAbs

Article Title: CD40- and CD95-specific antibody single chain-Baff fusion proteins display BaffR-, TACI- and BCMA-restricted agonism

doi: 10.1080/19420862.2020.1807721

Figure Lengend Snippet: Equilibrium binding of GpL-tagged aCD40- and αCD95-scBaff fusion proteins to CD40 and CD95 and the Baff-interacting receptors BaffR, TACI and BCMA. (a,b) HEK293 transfectants transiently expressing the Baff-interacting receptors (a) or CD40 or CD95 (b) were used to determine total binding. Empty vector (EV) transfected cells were used to determine unspecific binding. Specific binding was calculated by subtraction of the unspecific binding values from the corresponding total binding values. Binding of GpL fusion proteins of TNC-Baff and TNC-APRIL was analyzed for comparison. Data of one representative experiment for each of the investigated interactions are shown. Averaged K D -values of 4 independent experiments are listed in .

Article Snippet: For cloning of the light and heavy chains, synthetic DNA fragments (Geneart, Thermo Fisher Scientific, Waltham, MA, USA) encoding the VH and VL domains of αCD40 and αCD95 were used; the fragments were designed according to the publicly accessible sequences of the human CD40-specific antibody G28.5 (acc. no. AJ853736) and the human CD95-specific antibody E09 (PDB entry 3TJE), respectively.

Techniques: Binding Assay, Expressing, Plasmid Preparation, Transfection

Journal: mAbs

Article Title: CD40- and CD95-specific antibody single chain-Baff fusion proteins display BaffR-, TACI- and BCMA-restricted agonism

doi: 10.1080/19420862.2020.1807721

Figure Lengend Snippet: Affinity of GpL-tagged TNC-Baff and CD40- and CD95-specific antibody-scBaff fusion proteins for cell-expressed receptors.

Article Snippet: For cloning of the light and heavy chains, synthetic DNA fragments (Geneart, Thermo Fisher Scientific, Waltham, MA, USA) encoding the VH and VL domains of αCD40 and αCD95 were used; the fragments were designed according to the publicly accessible sequences of the human CD40-specific antibody G28.5 (acc. no. AJ853736) and the human CD95-specific antibody E09 (PDB entry 3TJE), respectively.

Techniques:

Journal: mAbs

Article Title: CD40- and CD95-specific antibody single chain-Baff fusion proteins display BaffR-, TACI- and BCMA-restricted agonism

doi: 10.1080/19420862.2020.1807721

Figure Lengend Snippet: Lymphoma- and myeloma cell-restricted activation of CD95 by antibody-scBaff fusion proteins. (a) HT1080 were cultivated in 96-well plates and were challenged in the presence of 2.5 µg/ml CHX with 4 × 10 4 L363, BJAB or Jurkat cells along with αCD95 N297A -scBaff and αCD95 Fab2 -scBaff. Next day, remaining viable plastic adhered cells were quantified by crystal violet staining. (b) HT1080 cell were again seeded in 96-well plates and were supplemented the next day as indicated with 4 × 10 4 L363, BJAB or Jurkat cells. Co-cultures were pretreated for 30 min with or without 5 µg/ml Baff-TNC and were then stimulated with 20 ng/ml of the αCD95-antibody scBaff fusion proteins, respectively. On the next day, CD95 activation was evaluated by determination of cellular viability. (c) Cocultures of HT1080 with 4 × 10 4 MM.1S, L363, BJAB or Jurkat cells were cultivated in 96-well plates and were challenged in the presence of 2.5 µg/ml CHX and 20 µM zVAD along with αCD95 N297A -scBaff and αCD95 Fab2 -scBaff. On the next day, NFκB signaling was assessed by means of IL8 ELISA. (d) Co-cultures were set up as in “C” and were pretreated for 30 min with or without 5 µg/ml Baff-TNC. Cells were then stimulated with 20 ng/ml of the αCD95-antibody scBaff fusion proteins and on the next day CD95-mediated IL8 production was again assayed by ELISA.

Article Snippet: For cloning of the light and heavy chains, synthetic DNA fragments (Geneart, Thermo Fisher Scientific, Waltham, MA, USA) encoding the VH and VL domains of αCD40 and αCD95 were used; the fragments were designed according to the publicly accessible sequences of the human CD40-specific antibody G28.5 (acc. no. AJ853736) and the human CD95-specific antibody E09 (PDB entry 3TJE), respectively.

Techniques: Activation Assay, Staining, Enzyme-linked Immunosorbent Assay