Journal: Journal for Immunotherapy of Cancer

Article Title: Anticancer immunity induced by a synthetic tumor-targeted CD137 agonist

doi: 10.1136/jitc-2020-001762

Figure Lengend Snippet: Generation of a Bicycle tool kit. (A) Schematic representation of the discovery of Bicycle binders using phage display with on-phage cyclization. (B) Biochemical properties of prototypical Bicycle binders. Potencies were determined by surface plasmon resonance (SPR). Specificity of EphA2, Nectin-4 and CD137 Bicycle binders were evaluated using SPR with other ephrin, Nectin-4 and TNF family receptors (OX40 and CD40), respectively (nb=no binding, nd=not determined). (C) Comparison of three bound CD137 Bicycle (BCY10916, Protein data bank (PDB) accession number 6Y8K) complexes versus CD137/CD137L (6MGP) and OX40/OX40L (2HEV) trimer complexes. (D) Illustration of the potential binding modes for multimeric Bicycle agonists (left) or tumor-targeted immune cell agonists (TICAs, right). TNF, tumor necrosis factor.

Article Snippet: Human proteins: CD137 (92204B, R&D Systems), CD137L (2295-4L, R&D Systems), OX40-Fc (OXO-H5255, Acro Biosystems), OX40 (OX0-H5224, Acro Biosystems), OX40L (OXL-H52Q8, Acro Biosystems), CD40 (CD40-H5228, Acro Biosystems), programmed death ligand 1 (PD-L1) (9049-B7, R&D Systems), EphA1-Fc (15789-H02H, Sino Biologics), EphA3-Fc (6444-A3, R&D Systems), EphA4-Fc (11314-H03H, Sino Biologics), EphA5 (3036-A5, R&D Systems), EphA6-Fc (5606-A6, R&D Systems), EphB4-Fc (10235-H02H, Sino Biologics), Nectin-1 (2880-N1, R&D Systems), Nectin-2 (2229-N2, R&D Systems), Nectin-3 (3064-N3, R&D Systems), Nectin-like-1 (3678-S4-050, R&D Systems), Nectin-like-2 (3519-S4-050, R&D Systems), Nectin-like-3 (4290-S4-050, R&D Systems), Nectin-like-4 (4164-S4, R&D Systems) and Nectin-like-5 (2530-CD-050, R&D Systems).

Techniques: SPR Assay, Binding Assay, Comparison

Journal: Journal for Immunotherapy of Cancer

Article Title: Anticancer immunity induced by a synthetic tumor-targeted CD137 agonist

doi: 10.1136/jitc-2020-001762

Figure Lengend Snippet: Modes of interaction and competitive binding analyses for CD137L, a CD137 Bicycle and anti-CD137 antibodies. (A) An analog of phage affinity matured CD137 Bicycle BCY592 (BCY10916, orange) binds to CD137 (gray) at CRD domains 2 and 3 (PDB accession 6Y8K). Comparison of the Bicycle CD137 binding site illustrates partial overlap with both the weakly agonistic antibody utomilumab (purple; scFv variant shown; PDB accession 6A3W) and CD137L (green; only monomer shown for clarity; PDB accession 6MGP). Urelumab (blue; Fab shown; PDB: 6MHR) binds distally to the N-terminal CRD one region and does not overlap with the Bicycle binding site. (B) Fixed concentration of fluorescent analog of CD137 Bicycle BCY592 (BCY640) was incubated with 500 nM of human CD137 and increasing concentrations of hCD137L, urelumab analog or utomilumab analog were added as competitors and fluorescent polarization was measured. The data represent values from two independent experiments. CRDs, cysteine-rich domains.

Article Snippet: Human proteins: CD137 (92204B, R&D Systems), CD137L (2295-4L, R&D Systems), OX40-Fc (OXO-H5255, Acro Biosystems), OX40 (OX0-H5224, Acro Biosystems), OX40L (OXL-H52Q8, Acro Biosystems), CD40 (CD40-H5228, Acro Biosystems), programmed death ligand 1 (PD-L1) (9049-B7, R&D Systems), EphA1-Fc (15789-H02H, Sino Biologics), EphA3-Fc (6444-A3, R&D Systems), EphA4-Fc (11314-H03H, Sino Biologics), EphA5 (3036-A5, R&D Systems), EphA6-Fc (5606-A6, R&D Systems), EphB4-Fc (10235-H02H, Sino Biologics), Nectin-1 (2880-N1, R&D Systems), Nectin-2 (2229-N2, R&D Systems), Nectin-3 (3064-N3, R&D Systems), Nectin-like-1 (3678-S4-050, R&D Systems), Nectin-like-2 (3519-S4-050, R&D Systems), Nectin-like-3 (4290-S4-050, R&D Systems), Nectin-like-4 (4164-S4, R&D Systems) and Nectin-like-5 (2530-CD-050, R&D Systems).

Techniques: Binding Assay, Comparison, Variant Assay, Concentration Assay, Incubation

Journal: Oncoimmunology

Article Title: Ectopic CD137 expression by rhabdomyosarcoma provides selection advantages but allows immunotherapeutic targeting

doi: 10.1080/2162402X.2021.1877459

Figure Lengend Snippet: Induction of CD137 expression on RMS cells by TNF and CD137 signaling into RMS cells induces IL-6 and IL-8 secretion. (a) Rd18, Rh41, Rd and JR1 cells were cultured with 75 ng/ml of TNF for 24 h. CD137 expression was measured by flow cytometry. Numbers in the histogram represent percentages of cells with CD137 staining. Two independent experiments for each cell line were performed with consistent results. (b-c) CD137-expressing and parental RMS cell lines were activated with either recombinant CD137L protein or BSA (negative control) for 24 h. IL-6 and IL-8 levels were determined by ELISA. Results are pooled from three independent experiments and presented as means ± errors of means. ** p < .01 using two-sided unpaired t-test

Article Snippet: Primary antibodies used in the Opal staining were mouse anti-human CD137 (clone: BBK2, Thermo Fisher Scientific, Waltham, USA), human anti-human CD137L (clone: P1F3, Genscript), rabbit anti-human CD3 (Dako, Santa Clara, USA), mouse anti-human CD8a (clone: AMC908; Thermo Fisher Scientific), rabbit anti-human ionized calcium-binding adapter molecule-1 (Iba-1) (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) and mouse anti-human MyoD antibodies (clone: 5.8A; Thermo Fisher Scientific).

Techniques: Expressing, Cell Culture, Flow Cytometry, Staining, Recombinant, Negative Control, Enzyme-linked Immunosorbent Assay

Journal: Oncoimmunology

Article Title: Ectopic CD137 expression by rhabdomyosarcoma provides selection advantages but allows immunotherapeutic targeting

doi: 10.1080/2162402X.2021.1877459

Figure Lengend Snippet: Downregulation of CD137L on DG-75 by CD137-expressing RMS cells. CD137-expressing and parental RMS cells were seeded on tissue culture plates. CellTrace™ Violet-labeled CD137L-expressing DG-75 cells were added to RMS cells at a ratio of 1:2. Cells were collected 24 h later for flow cytometry. DG-75 cells without RMS cell co-culture serve as baseline. Flow cytometry results of (a) Rd18-CD137 and Rd-18 are shown. Numbers in the scatter plots represent percentages of respective cell populations. Experiments were repeated twice for each cell line with consistent results. (b) Comparison of the percentages of CD137L-expressing DG-75 cells during coculture with Rd18-CD137 and Rd18, respectively. Each symbol represents one independent experiment. Lines represent means ± standard errors of means. * p < .05 using two-sided unpaired t-test. Experiments were repeated four times for each cell line with consistent results. (c) Coculture as in (A) but with Rh-41 instead of Rd18 and cells

Article Snippet: Primary antibodies used in the Opal staining were mouse anti-human CD137 (clone: BBK2, Thermo Fisher Scientific, Waltham, USA), human anti-human CD137L (clone: P1F3, Genscript), rabbit anti-human CD3 (Dako, Santa Clara, USA), mouse anti-human CD8a (clone: AMC908; Thermo Fisher Scientific), rabbit anti-human ionized calcium-binding adapter molecule-1 (Iba-1) (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) and mouse anti-human MyoD antibodies (clone: 5.8A; Thermo Fisher Scientific).

Techniques: Expressing, Labeling, Flow Cytometry, Co-Culture Assay, Comparison

Journal: Oncoimmunology

Article Title: Ectopic CD137 expression by rhabdomyosarcoma provides selection advantages but allows immunotherapeutic targeting

doi: 10.1080/2162402X.2021.1877459

Figure Lengend Snippet: Schematic diagram illustrating the proposed role of CD137 in RMS. (a) Via CD137L – CD137, APC costimulate activated T cells which kill RMS cells. (b) TAM induce ectopic CD137 expression on RMS cells via TNF. CD137 on RMS cells downregulates CD137L on APC via trogocytosis, reducing T cell costimulation and thus anti-tumor response. CD137 signaling into RMS cell induces secretion of IL-6 and IL-8 which drive RMS growth and metastasis. (c) NK cells transduced with an anti-CD137 CAR kills RMS cells that ectopically express CD137

Article Snippet: Primary antibodies used in the Opal staining were mouse anti-human CD137 (clone: BBK2, Thermo Fisher Scientific, Waltham, USA), human anti-human CD137L (clone: P1F3, Genscript), rabbit anti-human CD3 (Dako, Santa Clara, USA), mouse anti-human CD8a (clone: AMC908; Thermo Fisher Scientific), rabbit anti-human ionized calcium-binding adapter molecule-1 (Iba-1) (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) and mouse anti-human MyoD antibodies (clone: 5.8A; Thermo Fisher Scientific).

Techniques: Expressing, Transduction