Journal: bioRxiv
Article Title: Bivalent bispecific CD28 antibodies reinforce T-cell responsiveness and revert anergy/quiescence in patients treated with bispecific CD3 antibodies
doi: 10.64898/2026.03.25.714198
Figure Lengend Snippet: A PBMCs were collected from patients before and after TCE treatment with CC-1 (day 1 and day 8, respectively), incubated ex vivo with CC-1 alone or in combination with BiCos, and subsequently subjected to functional analysis. B Patient-derived PBMC (n=5) were cultured with LNCaP-E tumor cells at an E:T ratio of 1:1 in the presence of 1 nM CC-1 with or without 0.5 nM of BiCo-1, BiCo-2 or isotype control (BiCo-iso). After 72 h, T-cell proliferation was quantified by 3 H-thymidine incorporation, tumor cell killing was determined by flow cytometry. C PBMCs from three patients obtained before and after treatment (day 1 and day 8 of subsequent cycles; see scheme in ) were incubated with CC-1 (5 nM) alone or together with BiCo-1 (5 nM). After 72 h, T-cell proliferation was assessed by 3 H-thymidine incorporation. D Leiden-based subclustering of scRNAseq data visualized by UMAP, depicting T cell populations within patient PBMC samples collected after therapy (day 8), and incubated for 3 days in vitro with LNCaP-E and 1 nM CC-1 with or without 0.5 nM BiCos annotated using canonical lineage markers. UMAP visualization of T-cell subclustering, resolving distinct functional T-cell states. E Dot plots summarizing temporal expression changes of selected genes involved in T-cell activation, cytotoxicity, proliferation and quiescence. Dot size indicates the fraction of cells expressing the respective gene; and color intensity reflects the mean expression. F Density plots illustrating shifts in patient T-cell (day 8) state distributions of T-cells treated in vitro with CC-1 alone or CC-1+BiCos, demonstrating a pronounced transition toward proliferative phenotypes. G PBMCs (n=4) were cultured with LNCaP-E cells at an E:T ratio of 1:2 in the presence of CC-1 (1 nM); medium, target cells, and constructs were replenished on day 4 to mimic chronic exposure. At day 7, PBMCs were re-stimulated with LNCaP-E cells at an E:T ratio of 1:1 in the presence of CC-1 (1 nM) alone or in combination with the indicated BiCo constructs (0.5 nM). On day 10, T-cell proliferation was accessed by 3 H-thymidine incorporation and tumor cell killing was evaluated by flow cytometry. H PBMCs (n = 4) were cultured with LNCaP-E cells at an E:T ratio of 1:2 in the presence or absence of CC-1 (1 nM) alone or in combination with BiCo constructs (0.5 nM). Medium, target cells, and constructs were replenished on day 4 and day 7 with (LNCaP-E cells E:T ratio of 1:1 on day 7). At day 10, T-cell proliferation was assessed by 3 H-thymidine incorporation and tumor cell killing was evaluated by flow cytometry. I PBMCs (n=3) were cultured with SHP-77 cells at an E:T ratio of 1:2 in the presence of tarlatamab (1 nM). Medium and target cells were replenished on day 4. After the exposure until day 7 mimicking induction of hyporesponsiveness, PBMCs were re-stimulated with SHP-77 cells at an E:T ratio of 1:1 in the presence of tarlatamab (1 nM) or in combination with BiCo-2 (0.5 nM). Proliferation was measured on day 10 by 3 H-thymidine incorporation, and tumor cell killing was assessed by flow cytometry.
Article Snippet: The human prostate cancer cell line LNCaP was obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) and SHP-77 cell line from American Type Culture Collection (ATCC).
Techniques: Incubation, Ex Vivo, Functional Assay, Derivative Assay, Cell Culture, Control, Flow Cytometry, In Vitro, Expressing, Activation Assay, Construct
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