ISB 1342 properties and binding. (A) Schematic 3D representation of ISB 1342, a bispecific antibody based on the BEAT technology with a Fab targeting CD38, an scFv targeting CD3ϵ, and a Fc carrying the LALA (L234A, L235A) mutation. The model was generated using the BioLuminate software (Schrödinger, New York, NY). (B) Mean ± SD of KD determined either on CD38- T cells (n = 12 donors in 3 independent experiments) and MM cell lines, KMS-12-BM (20 measures from n = 14 independent experiments), NCI-H929 (12 measures from n = 5 independent experiments) and MOLP-8 (9 measures from n = 3 independent experiments), or recombinant human proteins CD3ϵδ (n = 5 independent experiments) and CD38 (n = 3 independent experiments). (C) Representative binding of ISB 1342 on CD38– human healthy T cells (mean ± SD of 4 donors) and KMS-12-BM MM cell line (1 representative measurement from 1 experiment). (D) Epitope mapping of daratumumab and ISB 1342 on CD38. Residues in dark red represent the CD38 residues in a 4 Å radius from the daratumumab chain in the crystal structure of 7DHA. Linear peptide mapping by SPR as well as site-directed mutagenesis were used to determine the binding epitope of ISB 1342 on CD38, shown in green on the CD38 chain of crystal structure 7DHA (in beige color). (E) ISB 1342 does not compete with daratumumab and can engage CD38 prebound by daratumumab. Biotinylated human CD38 protein was loaded on a streptavidin SA biosensor. The biosensor with immobilized CD38 was then dipped in a solution of daratumumab in kinetic buffer to reach saturation of the surface. Then, a saturated biosensor was dipped into a premixed solution of daratumumab + ISB 1342 at equimolar concentrations (red curve) or daratumumab only (blue curve). Plots show binding to the sensor tip as a wavelength shift (response, in nm; y-axis) vs time (in sec; x-axis).
