ccl  (ATCC)

Journal: iScience

Article Title: Development of DARPin T cell engagers for specific targeting of tumor-associated HLA/peptide complexes

doi: 10.1016/j.isci.2025.113926

Figure Lengend Snippet: DARPin TCEs induce robust and highly specific CD8 + T cell responses against HLA-A∗0201/NY-ESO1 157-165 Prior to linker and CD3 binder optimization, we made use of an intermediate 24 amino acid-long linker (standard linker length for most of the developed DARPin constructs), and a parental CD3 binder from where the stability-improved versions CD3-v1, CD3-v2, and CD3-v3 originated. This parental CD3 DARPin binder has an affinity in the range of the version 2 presented in this article, with a K D value of approximately 14 nM (A and B) The HLA-A∗0201 + /NY-ESO1 + (Ag + ) IM9 and HLA-A∗0201 + /NY-ESO1 - (Ag − ) MCF-7 tumor cell lines were incubated with PBMCs for 48 h in the presence or absence of each DARPin TCE. T cell activation was evaluated by measuring CD25 expression on CD8 + T cells (A) and IFNγ release (B). (C) T2 cells were pulsed with 1 μM alanine-substituted peptide variants of the NY-ESO1 157–165 (9V) peptide and then incubated with effector CD8 + T cells plus each DARPin TCE, followed by the quantification of IFNγ positive CD8 + T cells. NY_1xCD3, NY_2xCD3, and NY_3xCD3 were highly sensitive to mutations spanning the entire peptide, whereas NY_4xCD3 and NY_5xCD3 were predominantly sensitive to changes at residues p4-p6. (A–C) Results are representative of at least three independent experiments. For panel B, error bars represent the standard deviation obtained in a single experiment. See also , and .

Article Snippet: IM9 (Tumor cell line) , ATCC , CCL-159.

Techniques: Construct, Incubation, Activation Assay, Expressing, Standard Deviation

Journal: iScience

Article Title: Development of DARPin T cell engagers for specific targeting of tumor-associated HLA/peptide complexes

doi: 10.1016/j.isci.2025.113926

Figure Lengend Snippet: Engineering of linker length and CD3ε affinity in DARPin TCEs increases potency without affecting specificity HLA-A∗0201 + /NY-ESO1 + (Ag + ) IM9 or HLA-A∗0201 + /NY-ESO1 - (Ag − ) MCF-7 tumor cell lines were incubated with PBMCs for 48 h in the presence or absence of (A and B) NY_1xCD3 or NY_2xCD3 with different linkers (L to XXS), or (C and D) sequence optimized versions (v) of NY_1xCD3 or NY_2xCD3 (v1 to v3). The effects of each modification on potency were evaluated by measuring the T cell activation markers CD25 (A) and CD69 (C). IM9 and MCF-7 cells were incubated with effector CD8 + T cells in the presence or absence of (B) NY_1xCD3 or NY_2xCD3 carrying the optimal linker length sequence (XXS) or (D) the optimized variants (v2 and v3). The percentage of specific lysis of different tumor cell lines was determined using classical chromium release assays. (A–D) All results are representative of at least two independent experiments. For panels B and D, experiments were run in triplicate. See also .

Article Snippet: IM9 (Tumor cell line) , ATCC , CCL-159.

Techniques: Incubation, Sequencing, Modification, Activation Assay, Lysis

Journal: iScience

Article Title: Development of DARPin T cell engagers for specific targeting of tumor-associated HLA/peptide complexes

doi: 10.1016/j.isci.2025.113926

Figure Lengend Snippet: The engineered NY_1xCD3_v3 TCE induces potent and specific T cell-mediated killing of HLA-A∗0201 + /NY-ESO1 157-165 (9C) + target cells (A) T2 cells pulsed with 1 μM NY-ESO1 157–165 (9V) (PT2) or unpulsed T2 cells (NP T2) were incubated with effector CD8 + T cells in the presence or absence of NY_1xCD3_v3. Intracellular IFNγ levels in CD8 + T cells are shown. (B–H) The HLA-A∗0201 + /NY-ESO1 + (Ag + ) U266-B1, IM9, NCI-H1755, NCI-H1703, and HLA-A∗0201 + /NY-ESO1 - (Ag − ) tumor cell lines Colo-205, MCF-7, MDA-MB231, and HCT-116 were incubated with PBMCs (B–D) or CD8 + T cells (E–H) in the presence or absence of NY_1xCD3_v3. Panels (B–D) display the levels of the T cell activation markers CD25 and CD69 in CD8 + T cells, while panels (E-H) present the percentages of specific lysis determined by chromium release assays for each tumor cell line. (A–H) All results are representative of at least two independent experiments. For panels E to H, experiments were run in triplicate.

Article Snippet: IM9 (Tumor cell line) , ATCC , CCL-159.

Techniques: Incubation, Activation Assay, Lysis