Journal: mAbs

Article Title: TNB-738, a biparatopic antibody, boosts intracellular NAD+ by inhibiting CD38 ecto-enzyme activity

doi: 10.1080/19420862.2022.2095949

Figure Lengend Snippet: Discovery of TNB-738. a) 373 anti-CD38 binders were screened for inhibition of CD38 enzyme activity. CHO-HuCD38 cells were treated with antibodies for 15 min at RT. Following incubation, εNAD+ substrate was added to each well and fluorescence was immediately analyzed using a microplate reader for 30 min. A frequency distribution plot of percent inhibition values is shown. b) Dose–response curves of CD38 enzyme inhibition is shown comparing CD38_F11A, CD38_F12A, CD38_F11A + CD38_F12A, and TNB-738. c) TNB-738 was generated by pairing CD38_F11A and CD38_F12A into bispecific format using knobs-into-holes technology on a silenced IgG4 Fc. A germline kappa light chain and CH1 domain were added to CD38_F11A arm of TNB-738 to optimize manufacturability with no loss in its ability to inhibit CD38.

Article Snippet: The antibody-coated sensors were then dipped into a 7-point, two-fold dilution series of the antigen human CD38 ECD (Sino Biological, #10818-H08H) in kinetic buffer starting at 100 nM.

Techniques: Inhibition, Activity Assay, Incubation, Fluorescence, Enzyme Inhibition Assay, Generated

Journal: mAbs

Article Title: TNB-738, a biparatopic antibody, boosts intracellular NAD+ by inhibiting CD38 ecto-enzyme activity

doi: 10.1080/19420862.2022.2095949

Figure Lengend Snippet: TNB-738 binds to CD38 and inhibits CD38 enzyme activity. a) On-target cell binding of TNB-738 was assessed by flow cytometry using Daudi, Ramos, and CHO-HuCD38 cells. b) Off-target cell binding of TNB-738 was assessed by flow cytometry using 293 F, CHO, K562, and HL-60 cells. c) TNB-738-mediated CD38 inhibition was evaluated on Daudi, Ramos, and CHO cells and (d) recombinant CD38 protein. e) Cells were incubated with antibodies at 37°C for 2 hours and the percentage of cells with capping was determined using confocal microscopy. In total, 100 cells were counted.

Article Snippet: The antibody-coated sensors were then dipped into a 7-point, two-fold dilution series of the antigen human CD38 ECD (Sino Biological, #10818-H08H) in kinetic buffer starting at 100 nM.

Techniques: Activity Assay, Binding Assay, Flow Cytometry, Inhibition, Recombinant, Incubation, Confocal Microscopy

Journal: mAbs

Article Title: TNB-738, a biparatopic antibody, boosts intracellular NAD+ by inhibiting CD38 ecto-enzyme activity

doi: 10.1080/19420862.2022.2095949

Figure Lengend Snippet: TNB-738-mediated inhibition of CD38 ectoenzyme activity increases NAD+ levels and SIRT1 activity downstream. Ramos cells were treated with a dilution series of TNB-738 or isotype control in the presence or absence of 50 nM NMN for 24 h at 37°C. Following incubation, a) NAD+ levels were measured using the Cell Biolabs NAD+ assay kit, and b) SIRT1 activity was measured using the Promega SIRT-Glo kit.

Article Snippet: The antibody-coated sensors were then dipped into a 7-point, two-fold dilution series of the antigen human CD38 ECD (Sino Biological, #10818-H08H) in kinetic buffer starting at 100 nM.

Techniques: Inhibition, Activity Assay, Incubation

Journal: mAbs

Article Title: TNB-738, a biparatopic antibody, boosts intracellular NAD+ by inhibiting CD38 ecto-enzyme activity

doi: 10.1080/19420862.2022.2095949

Figure Lengend Snippet: TNB-738 does not induce lysis of CD38-expressing cells. a) Daudi and Ramos cells were incubated with increasing concentrations of TNB-738 or daratumumab in the presence of 5% rabbit complement serum for 45 min at 37°C. Cell viability was measured using Cell Titer Glo 2.0. b) NK cells were isolated from PBMCs and incubated with Daudi or Ramos cells and increasing concentrations of TNB-738 or daratumumab for 4 h at 37°C. Tumor killing was evaluated by LDH release. c) Daudi and Ramos cells were treated with TNB-738 or isatuximab for 24 h at 37°C. After incubation, the cells were stained with 7-AAD and Annexin V. Cell viability was analyzed by flow cytometry.

Article Snippet: The antibody-coated sensors were then dipped into a 7-point, two-fold dilution series of the antigen human CD38 ECD (Sino Biological, #10818-H08H) in kinetic buffer starting at 100 nM.

Techniques: Lysis, Expressing, Incubation, Isolation, Staining, Flow Cytometry

Journal: mAbs

Article Title: TNB-738, a biparatopic antibody, boosts intracellular NAD+ by inhibiting CD38 ecto-enzyme activity

doi: 10.1080/19420862.2022.2095949

Figure Lengend Snippet: Epitope mapping of F11A on CD38. Crystal Structure of the CD38-F11A complex. CD38 ECD is shown in blue with the active site in pink. UniDab_F11A is highlighted yellow.

Article Snippet: The antibody-coated sensors were then dipped into a 7-point, two-fold dilution series of the antigen human CD38 ECD (Sino Biological, #10818-H08H) in kinetic buffer starting at 100 nM.

Techniques:

Journal: mAbs

Article Title: TNB-738, a biparatopic antibody, boosts intracellular NAD+ by inhibiting CD38 ecto-enzyme activity

doi: 10.1080/19420862.2022.2095949

Figure Lengend Snippet: Data collection and refinement statistics of the CD38-F11A crystal structure.

Article Snippet: The antibody-coated sensors were then dipped into a 7-point, two-fold dilution series of the antigen human CD38 ECD (Sino Biological, #10818-H08H) in kinetic buffer starting at 100 nM.

Techniques:

Journal: mAbs

Article Title: TNB-738, a biparatopic antibody, boosts intracellular NAD+ by inhibiting CD38 ecto-enzyme activity

doi: 10.1080/19420862.2022.2095949

Figure Lengend Snippet: Epitope mapping of F12A on CD38. a) Results of in silico prediction of F12A on CD38 using MAbTope. b) Table summarizing the predicted peptides with suggested mutant residues. c) Binding of F12A and daratumumab antibody to wild-type and mutated CD38. The percent PE+APC+ cells were collected from 4 independent experiments. The amount of PE+APC+ cells were normalized to the total PE+ cells. The results are expressed in mean ± sem of the maximal response. One star (*) indicates significant statistical difference at p ≤ .05, two stars (**) at p ≤ .01.

Article Snippet: The antibody-coated sensors were then dipped into a 7-point, two-fold dilution series of the antigen human CD38 ECD (Sino Biological, #10818-H08H) in kinetic buffer starting at 100 nM.

Techniques: In Silico, Mutagenesis, Binding Assay

Journal: mAbs

Article Title: TNB-738, a biparatopic antibody, boosts intracellular NAD+ by inhibiting CD38 ecto-enzyme activity

doi: 10.1080/19420862.2022.2095949

Figure Lengend Snippet: Comparison of epitopes on CD38. Epitope on CD38 of daratumumab (a) in yellow, F11A (b) in green, and isatuximab (c) in pink. Peptides predicted using MAbTope for the F12A epitope (d) (green-CD38_F12_m2, gray-CD38_F12_m4). The residues of the active site of CD38 are represented by ball and stick model.

Article Snippet: The antibody-coated sensors were then dipped into a 7-point, two-fold dilution series of the antigen human CD38 ECD (Sino Biological, #10818-H08H) in kinetic buffer starting at 100 nM.

Techniques:

Journal: mAbs

Article Title: TNB-738, a biparatopic antibody, boosts intracellular NAD+ by inhibiting CD38 ecto-enzyme activity

doi: 10.1080/19420862.2022.2095949

Figure Lengend Snippet: Schematic representation of intracellular NAD+ boosting by TNB-738-mediated CD38 inhibition.

Article Snippet: The antibody-coated sensors were then dipped into a 7-point, two-fold dilution series of the antigen human CD38 ECD (Sino Biological, #10818-H08H) in kinetic buffer starting at 100 nM.

Techniques: Inhibition