Fig 2: Bispecific antibody-mediated cytokine release varies significantly between CD3_F1F- and CD3_F2B-containing bispecific antibodies. (a) Levels of specific tumor cell lysis and cytokine release for IL-2 and IFN? were measured after resting human T-cells were cultured with NCI-H929 (BCMA-positive) or K562 (BCMA-negative) cells and increasing doses of CD3xBCMA antibodies or a negative control (CD3_F1Fx[off-target arm]). (b) The same two CD3-targeting arms were combined with an anti-CD19 arm in bispecific format, and similar primary T-cell based studies were undertaken using CD19-positive Raji cells. (c) Purified pan T-cells from 10 different healthy human donors were incubated with a BCMA+ tumor cell line (NCI-H929) in the presence of plateau killing doses for CD3_F1FxBCMA and CD3_F1Fx[off-target arm] isotype control (0.1nM) as well as CD3_F2BxBCMA and CD3_F2Bx[off-target arm] isotype control (10nM).

Fig 3: Members of both new anti-CD3 mAb families bind to and activate human T-cells. Two representative members were selected from each new anti-CD3 mAb family for purification and further functional assessment using flow cytometry. Antibodies were tested for the ability to bind human CD3-expressing Jurkat cells (a), and to activate primary T-cells in a sample of total human or cyno PBMCs as measured by T-cell surface expression of CD69 (b,c).

Fig 4: T-cell signaling fraction increases with concentration of DGS-NTA(Ni) in the SLB. (A) Schematic illustration showing calcium signaling by either (left) DGS-NTA(Ni) in an SLB or (right) anti-CD3 antibodies on glass. (B) The fraction of signaling T cells for either ligand-free SLBs with DGS-NTA(Ni) or OKT3 on a glass slide (mean signaling fraction ± s.d.). (C) The time between cell landing and calcium signaling for either ligand-free SLBs with DGS-NTA(Ni) or OKT3 on a glass slide (mean signaling time ± s.d.). (D) The signaling fraction binding to a ligand-free SLB containing 5 wt% DGS-NTA(X), with X being either Co2+, Ni2+ or Cu2+. (E) The signaling fraction of J.RT3-T3.5 cells (left) and Jurkat T cells (right) on SLBs with ligand-free 5 wt% DGS-NTA(Ni) and on OKT3-coated glass. Data are shown for three to five experiments, except for E which is from two experiments, with 150 cells in each experiment. A two-sample t-test in MATLAB was used for statistical analysis with: * <0.05, ** <0.01, *** <0.001.

Fig 5: Two different CD3 cell-binding CDRH3 sequence families were identified using NGS-based discovery followed by high-throughput recombinant expression and screening. (a) The discovery workflow combines antibody repertoire deep sequencing and custom bioinformatics analysis with high-throughput gene assembly, recombinant expression and screening. OmniFlic rats express a comprehensive human VH gene repertoire with a single pre-rearranged human kappa light chain. Endogenous rat heavy chain, kappa, and lambda loci have been knocked out. (b) Based on antibody repertoire analysis, 378 heavy chain sequences were selected for expression with a fixed human kappa light chain. All fully human antibody candidates were tested by flow cytometry for the ability to bind CD3+ Jurkat cells in a primary screen, and results are shown in a heatmap in which each row is a unique heavy chain VH sequence and the degree of red indicates cell-binding strength. The dendrogram indicates the relationships between the sequences tested in the primary screen. (c) Two cell-binding VH sequence families were identified and additional family members from the repertoire were expressed and tested for cell binding in a secondary screen. Sequence variation among family members is illustrated by colored blocks, with each color representing an amino acid residue.