Journal: Science Advances

Article Title: Uncovering receptor-ligand interactions using a high-avidity CRISPR activation screening platform

doi: 10.1126/sciadv.adj2445

Figure Lengend Snippet: ( A ) FC was used to monitor binding of FITC-labeled 4-1BB magnetic beads to activator cells following up to four rounds of selection using streptavidin–4-1BB magnetic beads. ( B ) FC was used to monitor 4-1BBL expression in activator cells following up to four rounds of enrichment with streptavidin–4-1BB magnetic beads. ( C ) The gRNA enrichment following first, second, third, or fourth round of selection using streptavidin–4-1BB magnetic beads was monitored using −log 10 (score). For each library, the percentages indicate the total gRNA counts for the gene of interest divided by the total number of gRNA counts for all genes (×100). ( D ) Immunoblotting was used to detect myc in 293 cells transfected with siglec-4–myc or 4-1BB–myc. β-Actin (β-act) was included as a loading control. ( E ) FC was used to measure the level of siglec-4 or 4-1BBL on the surface of 293 cells following transfection with pCMV EV (control), pCMV–siglec-4 (siglec-4), or pCMV–4-1BBL (4-1BBL). ( F ) Binding of 4-1BB–HIS to 293 cells transfected with EV (negative control), pCMV–4-1BB (positive control), or pCMV–siglec-4. Data in (A), (B), (D), (E), and (F) were representative of three experiments.

Article Snippet: For the m4-1BB protein binding assay, HEK293 cells transfected with EV, pCMV–m-siglec-4 (SinoBiological, cat. no. MG51398-CM) or pCMV–m4-1BBL (SinoBiological, cat. no. MG50067-CM) were incubated with 1 μg of m4-1BB–Fc protein (SinoBiological, cat. no. 50811-M02H) followed by staining with AF647 AffiniPure Donkey Anti-Human IgG (H + L) (Jackson ImmunoResearch, cat. no. 709-605-149).

Techniques: Binding Assay, Labeling, Magnetic Beads, Selection, Expressing, Western Blot, Transfection, Negative Control, Positive Control

Journal: Science Advances

Article Title: Uncovering receptor-ligand interactions using a high-avidity CRISPR activation screening platform

doi: 10.1126/sciadv.adj2445

Figure Lengend Snippet: ( A to C ) FC was used to monitor (A) 4-1BB expression and 4-1BBL–Fc or siglec-4–Fc binding to activated T cells, (B) binding of siglec-4–Fc to stimulated T cells transfected with nonspecific (NS) or 4-1BB knockdown (KD) siRNAs, or (C) binding of siglec-4–Fc to stimulated T cells in the presence of increasing amounts of soluble 4-1BB–HIS protein. MFI, mean fluorescence intensity. Statistical analysis: unpaired t test. ( D ) ELISA was used to measure IFN-γ produced by activated T cells mixed with 293 cells overexpressing EV, siglec-4, 4-1BBL, or siglec-4 plus 4-1BBL. Statistical analysis: unpaired t test; P values correspond to comparisons between groups with or without siglec-4. ( E ) Luciferase assays were used to measure the viability of eGFP-FFLuc–labeled 293 target cells 24 hours after mixing with anti–TEM8–CAR-T cells. TEM8 knockout control cells (293/T8KO) were included as a specificity control. E:T, effector:target cell ratio. Statistical analysis: unpaired t test; P values correspond to comparisons between 293 and 293–Siglec-4 at each E:T cell ratio. ( F to H ) Immunoblotting was used to assess (F) p-c-Jun and c-Jun levels in 293 cells or 293–4-1BB cells following transient transfection with full-length siglec-4–myc or 4-1BB–myc, (G) p-c-Jun and c-Jun levels in unstimulated (U) or stimulated (S) T cells derived from two independent donors, and (H) p-c-Jun and c-Jun levels in T cells cocultured for 1 hour at a ratio of 1:1 with 293 cell transfected with EV (E) or siglec-4 (Sig4). Note that Siglec-4 expression can mediate the down-regulation of c-Jun only if 4-1BB is also present. β-Actin was used as a loading control in (F), (G), and (H). All data or images in (A) to (H) were representative of at least three independent experiments. For (C) to (E), n > = 3 biologically independent samples per group. ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001.

Article Snippet: For the m4-1BB protein binding assay, HEK293 cells transfected with EV, pCMV–m-siglec-4 (SinoBiological, cat. no. MG51398-CM) or pCMV–m4-1BBL (SinoBiological, cat. no. MG50067-CM) were incubated with 1 μg of m4-1BB–Fc protein (SinoBiological, cat. no. 50811-M02H) followed by staining with AF647 AffiniPure Donkey Anti-Human IgG (H + L) (Jackson ImmunoResearch, cat. no. 709-605-149).

Techniques: Expressing, Binding Assay, Transfection, Fluorescence, Enzyme-linked Immunosorbent Assay, Produced, Luciferase, Labeling, Knock-Out, Western Blot, Derivative Assay

Journal: Nature Communications

Article Title: Cancer immune therapy with PD-1-dependent CD137 co-stimulation provides localized tumour killing without systemic toxicity

doi: 10.1038/s41467-021-26645-6

Figure Lengend Snippet: a (Left) Tumour volumes of CT26-hPD-L1 tumours in BALB/c-hPD-1/hPD-L1/hCD137 mice treated with the indicated antibodies (20 mg/kg, i.p.) on days 11, 18 and 25 post-tumour implantation ( n = 8 per group). The mice in the hIgG1 and αCD137 groups were euthanized on day 27 post-tumour implantation due to ethical issues. (Right) Spaghetti plots show the individual tumour volumes in each group. The numbers in the plots indicate tumour-free mice/all. b Tumour volumes of MC38 tumours in C57BL/6-hPD-1/hCD137 mice treated with the indicated antibodies on days 9, 16 and 23 ( n = 7 per group). c and d Single-cell RNA sequencing of CD45 + cells in MC38 tumours after antibody treatments. c A thumbnail heatmap showing the expression of signature genes in each cell cluster (detailed heatmap in Supplementary Fig. ). d The percentages of different cell clusters in the αPD-1, IBI319 and αPD-1 + αCD137 groups. e Tumour volumes of MC38 tumours in C57BL/6-hPD-1/hCD137 mice treated with the indicated antibodies (3 + 3 mg/kg for the αPD-1/Hel + αCD137/Hel-Fc group and 3 mg/kg for all other groups) on days 9, 16 and 23 ( n = 6 per group for the IgG1 and αPD-1/Hel + αCD137/Hel-Fc groups; n = 5 per group for all other groups). Hel: hen egg white lysozyme. f Livers from the mice in b were stained for CD45 and F4/80 via IHC. (Left) Representative images. (Right) Quantification of whole pathological digital slides via HALO software ( n = 3 per group, mean and SD); images of all quantified groups can be found in Supplementary Fig. . a , b and e Tumour volumes are presented as the mean, and the error bar represents the SEM. Statistics were performed in the indicated groups: two-way ANOVA with Tukey’s multiple comparisons test ( a and b ) and two-sided, un-paired t test ( f ); p values are indicated in the graphs.

Article Snippet: The recombinant proteins used for binding affinity tests were hPD-1-his (PD1-H5221, Acro Biosystems), hCD137-his (41B-H5227, Acro Biosystems), hCD137L-Fc (41L-H5257, Sino Biological), biotinylated hFcRI (10256-H27H-B-20, Sino Biological), biotinylated hFcRIIa (CD-H82E7, Acro Biosystems), biotinylated hFcRIIb (CDB-H82E0, Acro Biosystems), biotinylated hFcγRIIIa (F158) (CDA-H82E8, Acro Biosystems), biotinylated hFcγRIIIa (V158) (CDA-H82E9, Acro Biosystems), biotinylated hFcγRIIIb (CDB-H82E1, Acro Biosystems), biotinylated hFcRn (FCM-H82W4, Acro Biosystems), hC1q (A099, Complement Technology), hPD-1-Fc (PD1-H5257, Acro Biosystems), hPD-L1-his (PD1-H5229, Acro Biosystems), mPD-1-Fc (PD1-M5259, Acro Biosystems), and mPD-L1-his (PD1-M5220, Acro Biosystems).

Techniques: RNA Sequencing Assay, Expressing, Staining, Software

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Tumor-infiltrating CD8 + T cells recognize a heterogeneously expressed functional neoantigen in clear cell renal cell carcinoma

doi: 10.1007/s00262-021-03048-6

Figure Lengend Snippet: Establishment of the reactive 1226 RCC TIL clone. a. TIL clone establishment method. TILs obtained from the red tumor were stained with anti-CD8 and anti-CD137 antibodies. TILs were co-cultured with 1226 RCC cells before FACS analysis. TILs without co-culture were used as a negative control. CD137+ cells were single cell-sorted using a cell sorter. After several weeks of in vitro culture, 116 of 384 clones showed cell growth. b IFNγ ELISPOT assay of the Q1 TIL clone. Q1 TIL clones were co-cultured with 1226 RCC cells and analyzed by an IFNγ ELISPOT assay. Anti-HLA-pan class 1 (W6/32), anti-HLA-A24 (C7709A2.6), anti-HLA-A2 (BB7.2), anti-HLA-BC (B1.23.2), and anti-HLA-DR (L243) antibodies were used to assess the specific reactivity of the Q1 TIL clone. 1226 EBV-B cells and K562 cells were used as negative controls. IFNγ spots are shown as mean ± standard error of the mean (SEM; n = 3), and p values were calculated using a paired t test; *p < 0.01. c IFNγ ELISPOT assay of the Q1 TIL clone using HLA allele-specific over-expressing 1226 RCC cells. HLA-B*35:01, B*40:01, C*03:03, and C*15:02 were stably over-expressed in 1226 RCC cells. Q1 TIL clone reactivity for HLA alleles over-expressed in 1226 RCC cells was examined by an IFNγ ELISPOT assay. An anti-HLA-BC (B1.23.2) antibody was used to confirm specificity. IFNγ spots are shown as the mean ± SEM (n = 3), and p values were calculated using a paired t test; *p < 0.01

Article Snippet: Anti-HLA-B mAb (JOAN-1, IgG1; Thermo Fisher Scientific), anti-HLA-C mAb (DT9, IgG2b; BD Biosciences), anti-CD8 mAb (APC, IgG1; MBL, Nagoya, Japan), anti-CD4 mAb (FITC; BD Biosciences), anti-CD137 mAb (VioBright FITC, Miltenyi Biotec, Tokyo, Japan), and rabbit anti-PD-L1 mAb (clone E1L3N; Cell Signaling Technology, Danvers, MA, USA) were also used.

Techniques: Staining, Cell Culture, Co-Culture Assay, Negative Control, In Vitro, Clone Assay, Enzyme-linked Immunospot, Expressing, Stable Transfection

Journal: OncoImmunology

Article Title: Hypoxia-induced soluble CD137 in malignant cells blocks CD137L-costimulation as an immune escape mechanism

doi: 10.1080/2162402x.2015.1062967

Figure Lengend Snippet: Figure 1. CD137 mRNA expression is upregulated by hypoxia in mouse tumor cell lines. Quantitative RT-PCR assessment of CD137 mRNA expression under 21% and 1% O2 (hypoxia) in the indicated cell lines cultured under these conditions for 48h. A representative direct immunofluorescence staining for CD137 surface expression ana- lyzed flow cytometry is presented in a corresponding histogram showing the discrepancy between the mRNA levels and the weak or dim surface protein staining. p < 0.05, p < 0.01.

Article Snippet: Serial dilutions of the serum of CT26-bearing CD137¡/¡ mice were incubated in ELISA plates coated with mouse recombinant 4-1BB-Fc chimeric protein (R&D Systems) and the antibodies against CD137 were detected and tittered using a goat anti-mouse IgG-HRP (Santa Cruz Biotechnology) according to the manufacturer’s instructions.

Techniques: Expressing, Quantitative RT-PCR, Cell Culture, Staining, Cytometry

Journal: OncoImmunology

Article Title: Hypoxia-induced soluble CD137 in malignant cells blocks CD137L-costimulation as an immune escape mechanism

doi: 10.1080/2162402x.2015.1062967

Figure Lengend Snippet: Figure 2. Soluble form of CD137 predominates over membrane-attached forms. (A) Diagram representing the cDNA with the exons of mouse CD137 and RT-PCR products amplified with the indicated primer pairs. (B) Quanti- tative RT-PCR analyses using primers that amplify either transmembrane (primer pair 34) or total CD137 cDNA (primer pair 12) in corresponding cell lines. (C) RT-PCR products of total CD137 cDNA (primer pair 56) amplifi- cation showing that the soluble CD137 (sCD137) predominates over the transmembrane CD137 (tmCD137) form. (D) Sandwich ELISA assessment of the concentration of sCD137 in the tissue culture supernatants of the CT26 cell line. TM: transmembrane domain; MM: molecular weight marker; N: normoxia; H: hypoxia.

Article Snippet: Serial dilutions of the serum of CT26-bearing CD137¡/¡ mice were incubated in ELISA plates coated with mouse recombinant 4-1BB-Fc chimeric protein (R&D Systems) and the antibodies against CD137 were detected and tittered using a goat anti-mouse IgG-HRP (Santa Cruz Biotechnology) according to the manufacturer’s instructions.

Techniques: Membrane, Reverse Transcription Polymerase Chain Reaction, Sandwich ELISA, Concentration Assay, Molecular Weight, Marker

Journal: OncoImmunology

Article Title: Hypoxia-induced soluble CD137 in malignant cells blocks CD137L-costimulation as an immune escape mechanism

doi: 10.1080/2162402x.2015.1062967

Figure Lengend Snippet: Figure 4. Soluble CD137 produced by tumor cells binds to CD137 ligand. (A) Binding of sCD137 present in the supernatant of CT26 cells cultured under hypoxia to CD137 ligand (CD137L) transfected to 293T cells. Untransfected 293T were used as a specificity control and supernatants from normoxia and hypoxia cultured CT26 cells were tested without dilution. Binding was revealed by an anti-CD137 mAb which does not interfere with ligand binding (1D8 clone) (B) Similar experiment as in A performed with the serum of CT26-bearing Rag2IL2Rg¡/¡ mice as a source of sCD137 or with pre-tumor serum as a control.

Article Snippet: Serial dilutions of the serum of CT26-bearing CD137¡/¡ mice were incubated in ELISA plates coated with mouse recombinant 4-1BB-Fc chimeric protein (R&D Systems) and the antibodies against CD137 were detected and tittered using a goat anti-mouse IgG-HRP (Santa Cruz Biotechnology) according to the manufacturer’s instructions.

Techniques: Produced, Binding Assay, Cell Culture, Transfection, Control, Ligand Binding Assay

Journal: OncoImmunology

Article Title: Hypoxia-induced soluble CD137 in malignant cells blocks CD137L-costimulation as an immune escape mechanism

doi: 10.1080/2162402x.2015.1062967

Figure Lengend Snippet: Figure 3. Soluble CD137 is produced by in vivo grafted tumors. (A) CT26 tumors (10£10 mm in diameters) excised from CD137¡/¡ mice were analyzed by quantitative RT-PCR for mCD137 encoding the transmembrane and the total CD137 isoforms. (B) The concentration of soluble CD137 (sCD137) assessed by ELISA in the sera of Rag¡/¡ mice grafted with 5£105 CT26 cells for 21 days.

Article Snippet: Serial dilutions of the serum of CT26-bearing CD137¡/¡ mice were incubated in ELISA plates coated with mouse recombinant 4-1BB-Fc chimeric protein (R&D Systems) and the antibodies against CD137 were detected and tittered using a goat anti-mouse IgG-HRP (Santa Cruz Biotechnology) according to the manufacturer’s instructions.

Techniques: Produced, In Vivo, Quantitative RT-PCR, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: OncoImmunology

Article Title: Hypoxia-induced soluble CD137 in malignant cells blocks CD137L-costimulation as an immune escape mechanism

doi: 10.1080/2162402x.2015.1062967

Figure Lengend Snippet: Figure 5. Soluble CD137 in the supernatant of hypoxia-treated CT26 cells blocks CD137L-mediated T-cell costimu- lation. Cultures of CD8C-purified OT1 WT or OT1 CD137¡/¡ T cells were stimulated with anti-CD3 mAb for 48h prior to seeding in co-cultures with 293T transfected or not with CD137L as indicated. T cells were pre-loaded with Violet 450 fluorescent dye and dilution of the fluorescent dye was used as a surrogate marker of T-cell prolif- eration. Cultures were conditioned with cell culture supernatants (1/16 diluted) of normoxia- or hypoxia-treated CT26 cells. (A) Representative flow cytometry histograms showing the extent of inhibition of proliferation by the sCD137-containing supernatants at 72h of coculture. (B) Pooled data of two independent experiments identically performed. MFI: mean fluorescence intensity; SN: supernatant.

Article Snippet: Serial dilutions of the serum of CT26-bearing CD137¡/¡ mice were incubated in ELISA plates coated with mouse recombinant 4-1BB-Fc chimeric protein (R&D Systems) and the antibodies against CD137 were detected and tittered using a goat anti-mouse IgG-HRP (Santa Cruz Biotechnology) according to the manufacturer’s instructions.

Techniques: Transfection, Marker, Cell Culture, Cytometry, Inhibition

Journal: OncoImmunology

Article Title: Hypoxia-induced soluble CD137 in malignant cells blocks CD137L-costimulation as an immune escape mechanism

doi: 10.1080/2162402x.2015.1062967

Figure Lengend Snippet: Figure 6. CD137 silencing in CT26 tumor cells gives rise to more immunogenic variants. CT26 were stably transfected with lentiviral vectors to express a shRNA targeting CD137 (shCD137) or a scrambled control (shControl). (A) Transfectants were cultured for 36h in normoxia or 1% O2 and analyzed by flow cytometry to quantitatively determine the expression of surface CD137. (B) Quantitative RT-PCR analysis of total and transmembrane CD137 mRNA in the indicated transfectants under hypoxic or normoxic conditions as in A. (C) BALB/c wild type immunocompetent mice were subcutaneously inoculated with 5£105 cells of indicated transfectants and tumor sizes were individually followed over time. Tumor cells had been pre-exposed in every case to 1% O2 for 36h. The fraction of mice spontaneously rejecting their tumors is given in each graph. When indicated, BALB/c mice were mAb-depleted of CD8CT cells. (D) mean§SEM and Statistical comparisons of experiments in C whose results are repre- sentative of two independent experiments. (E) Tumor growth of the indicated transfectants in immunodeficient Rag2IL2Rg¡/¡ mice. Tm: transmembrane.

Article Snippet: Serial dilutions of the serum of CT26-bearing CD137¡/¡ mice were incubated in ELISA plates coated with mouse recombinant 4-1BB-Fc chimeric protein (R&D Systems) and the antibodies against CD137 were detected and tittered using a goat anti-mouse IgG-HRP (Santa Cruz Biotechnology) according to the manufacturer’s instructions.

Techniques: Stable Transfection, Transfection, shRNA, Control, Cell Culture, Cytometry, Expressing, Quantitative RT-PCR

Journal: OncoImmunology

Article Title: Hypoxia-induced soluble CD137 in malignant cells blocks CD137L-costimulation as an immune escape mechanism

doi: 10.1080/2162402x.2015.1062967

Figure Lengend Snippet: Figure 7. Graphical interpretation of the experimental results. Schematic representation of the postulated mechanism underneath the experimental observations. TM: transmembrane; sCD137: soluble CD137.

Article Snippet: Serial dilutions of the serum of CT26-bearing CD137¡/¡ mice were incubated in ELISA plates coated with mouse recombinant 4-1BB-Fc chimeric protein (R&D Systems) and the antibodies against CD137 were detected and tittered using a goat anti-mouse IgG-HRP (Santa Cruz Biotechnology) according to the manufacturer’s instructions.

Techniques:

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: 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: Synthetic CD137-binding multimeric or tumor-targeted Bicycles are agonists in vitro . (A) Jurkat cells expressing human CD137 coupled to a luciferase reporter gene were treated for 6 hr with CD137 monomer (BCY592), trimer (BCY7750) or tetramer (BCY7751) as indicated and activity read out as reporter gene product activity. Data are mean/SD. (n=4 replicates). (B) Jurkat-CD137 reporter cells were cultured with Nectin-4-expressing HT-1376 cells and treated with a Nectin-4/CD137 1:1, 1:2 or 1:3 TICAs (BCY10572, BCY11027, BCY11022, respectively) for 6 hr. Data are mean/SD. (n=3 replicates). (C) Jurkat-CD137 reporter cells were cultured with EphA2-expressing A549 cells and treated with an EphA2/CD137 1:1 TICA (BCY9173), 1:2 TICA (BCY12491) or an enantiomeric non-binding control (NB) TICA (BCY13626) for 6 hr. Data are mean/SD. (n=3 replicates). (D) Human PBMCs (donor 228711) were cultured with mouse MC38 cells and treated with EphA2/CD137 1:1 TICA (BCY10575),1:2 TICA (BCY12491) and enantiomeric non-CD137 binding (NB) 1:1 (BCY12759) and 1:2 (BCY12762) TICA. IFNү in the culture supernatants was measured after 48 hr. The gray bar indicates untreated control levels. Data are means and SD of 3 technical replicates. (E) and (F) human PBMCs from a single donor were cultured with human cell lines treated with 3 nM EphA2/CD137 1:2 TICAs (BCY12491). Supernatant IFNү and IL-2 were measured after 48 hr. Data are mean/SD. (n=3 replicates). *P<0.05, **p<0.01 based on Student’s t-test paired by PBMC donor.

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, In Vitro, Expressing, Luciferase, Activity Assay, Cell Culture, Control

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: TICAs cause tumor regression and complete responses in vivo without continuous drug exposure in the periphery. (A) Plasma concentration of EphA2/CD137 1:2 TICA (BCY12491) after 5 mg/kg intravenous and 15 mg/kg intraperitoneal dosing in naïve CD-1 mice. Table insert shows the pharmacokinetic parameters of BCY12491 after 5 mg/kg intravenous or 15 mg/kg intraperitoneal dosing in naïve CD-1 mice. (B) MC38 tumor growth and body weight change with intermittent or daily intraperitoneal dosing of BCY12491 (n=6/cohort; *p<0.05, **p<0.01, ***p<0.001 mixed-effects analysis days 0–19). (C) Tumor growth curves for individual animals by treatment group from the experiment shown in panel B. Number of complete responders (CRs) are indicated in the figure. Gray highlighting indicates the treatment administration window. (D) Simulated plasma concentration–time profile of BCY12491 dosed QD and Q3D at 5 and 15 mg/kg, respectively. The dashed line indicates the average half maximal effect concentration for BCY12491 for IFNγ secretion from human PBMCs cultured with mouse MC38 cells from two donors ( and extended data ). (E) Five mice that had complete regressions of MC38 tumors in response to BCY12491 treatment (panel B) were rechallenged with MC38 tumor cell implantation. Tumor growth was monitored until day 31. Matched naïve huCD137 C57BL/6 mice served as a control group. n=5/cohort.

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: In Vivo, Clinical Proteomics, Concentration Assay, Cell Culture, Control

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: TICAs modulate the tumor immune microenvironment and drive T cell infiltration. (A) MC38 tumor bearing mice were treated with vehicle, 15 mg/kg EphA2/CD137 1:2 TICA (BCY12491), an enantiomeric non-binding control TICA (BCY13626) q3d intravenously or 2 mg/kg anti-CD137 agonist antibody (urelumab analog) q3d intraperitoneally individual tumor volumes normalized to tumor volume on the day of treatment initiation are shown grouped by treatment. *P<0.05, ***p<0.001 one-way ANOVA on day 6 comparing to vehicle. (B) Nanostring analysis of tumors show the immunomodulatory effect of BCY12491 and anti-CD137 agonist antibody on the 25 functional pathways including stimulation of NF-kB signaling, costimulatory signaling, cytokine/chemokine signaling, cytotoxicity and interferon signaling among others. Orange indicates high pathway scores; blue indicates low scores. Scores are displayed on the same scale via a Z-transformation. (C) Nanostring analysis of tumors show the effect of BCY12491 and anti-CD137 agonist antibody on the T cell (probe set: Cd3d, Cd3e, Cd3g, CD6, SH2D1A and Trat1), cytotoxic cell (probe set: Ctsw, Gzma, Gzmb, Klrb1, Klrd1, Klrk1, Nkg7 and Prf1) and macrophage (probe set: CD163, CD68, CD84 and Ms4a4a) content. (D) Representative images of tissue sections from tumors treated with vehicle, 15 mg/kg BCY12491, BCY13626 or 2 mg/kg anti-CD137 agonist antibody Q3D and stained for mouse CD8 are shown. (E) Nanostring analysis of tumors show the effect of BCY12491 and anti-CD137 agonist antibody on the checkpoint inhibitor Pdcd1 (protein PD-1), Cd274 (protein PD-L1) and CTLA4 (protein CTLA-4) transcription. (C, E) *P<0.05, ***p<0.001, one-way ANOVA with Dunnett’s post test. ANOVA, analysis of variance; PD-L1, programmed death ligand 1; TICAs, tumor-targeted immune cell agonists.

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, Control, Functional Assay, Transformation Assay, Staining

Journal: Cell

Article Title: Single-cell Map of Diverse Immune Phenotypes in the Breast Tumor Microenvironment

doi: 10.1016/j.cell.2018.05.060

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: SEQC is available on https://github.com/ambrosejcarr/seqc.git and Biscuit is available on https://github.com/sandhya212/BISCUIT_SingleCell_IMM_ICML_2016 . table ft1 table-wrap mode="anchored" t5 REAGENT or RESOURCE SOURCE IDENTIFIER Biological Samples Breast carcinoma Fresh operative tissue samples at MSKCC N/A Normal breast tissue Fresh operative tissue samples at MSKCC N/A Lymph node Fresh operative tissue samples at MSKCC N/A Peripheral blood Patient venipuncture at MSKCC N/A Antibodies anti-CD45 ab Biolegend RRID:AB_2566372 DAPI Stain Calbiochem SCR_014366 anti-Foxp3 ab Thermo Fisher RRID:AB_1834364 anti-CD3 ab Biolegend RRID:AB_1575008 anti-CD4 ab Biolegend RRID:AB_571945 anti-CD16 ab Biolegend RRID:AB_314207 anti-CD56 ab BD Biosciences RRID:AB_396853 anti-CD8 ab Biolegend RRID:AB_528885 anti-CD19 ab Biolegend RRID:AB_2562015 anti-CD11b ab Biolegend RRID:AB_2563395 anti-CD45 Y89 Fluidigm RRID:AB_2261851 anti-CD235ab BioLegend RRID:AB_314620 anti-CD61 BioLegend RRID:AB_1227584 anti-CD196/CCR6 Pr141 Fluidigm Cat:3141014A anti-CD19 142Nd Fluidigm RRID:AB_2651155 anti-CD278/ICOS 143Nd Fluidigm Cat:3143025B anti-CD270 144Nd Fluidigm Cat:3144022B anti-CD4 145Nd Fluidigm RRID:AB_2661789 anti-CD8a 146Nd Fluidigm Cat:3146001B anti-CD11c 147Sm Fluidigm RRID:AB_2687850 anti-CD14 148Nd Fluidigm Cat:3148010B anti-CD127 149Sm Fluidigm RRID:AB_2661792 anti-TIGIT eBioscience Cat:16-9500-82 anti-CD123 151Eu Fluidigm RRID:AB_2661794 anti-CD95/Fas 152Sm Fluidigm Cat:3152017B anti-CD45RA 153Eu Fluidigm Cat:3153001B anti-TIM3 154Sm Fluidigm Cat:3154010B anti-CD39 BioLegend RRID:AB_940438 anti-CD274/PD-L1 156Gd Fluidigm Cat: 3156026B anti-CD27 158Gd Fluidigm Cat: 3158010B anti-CD357/GITR 159Tb Fluidigm Cat: 3159020B anti-CD28 160Gd Fluidigm Cat: 3160003B anti-CD152/CTLA4 161Dy Fluidigm Cat: 3161004B anti-FoxP3 162Dy Fluidigm RRID:AB_2687650 anti-CD33 163Dy Fluidigm RRID:AB_2687857 anti-CD45RO 164Dy Fluidigm Cat: 3164007B anti-CD223/LAG3 165Ho Fluidigm RRID:AB_2687859 anti-Ki67 BD RRID:AB_396287 anti-CD197/CCR7 167Er Fluidigm Cat:3167009A anti-CD154/CD40L 168Er Fluidigm Cat: 3170011B anti-CD25 169Tm Fluidigm RRID:AB_2661806 anti-CD3 170Er Fluidigm RRID:AB_2661807 anti-CD226 171Tb Fluidigm Cat: 3171013B anti-CD38 172Yb Fluidigm Cat: 3172007B anti-CD137/4–1BB 173Yb Fluidigm Cat: 3173015B anti-CD279/PD-1 174Yb Fluidigm Cat: 3174020B anti-CD194/CCR4 175Lu Fluidigm Cat: 3175021A anti-CD56 176Yb Fluidigm RRID:AB_2661813 anti-CD11b 209Bi Fluidigm Cat: 3209003B Critical Commercial Assays inDrop platform Custom-built droplet microfluidics platform; Klein et al., 2015 ; Zilionis et al., 2017 .

Techniques: Staining, Antibody Labeling, Software, Diffusion-based Assay

Journal: Circulation Research

Article Title: High Levels of Costimulatory Receptors OX40 and 4-1BB Characterize CD4 + CD28 null T Cells in Patients With Acute Coronary Syndrome

doi: 10.1161/circresaha.111.261933

Figure Lengend Snippet: Figure 2. Expression of costimulatory receptors in CD4CD28null T cells. Peripheral blood mononuclear cells (PBMCs) from acute coronary syndrome (ACS, n25) and stable angina (SA, n21) patients were cultured alone (w/o) or activated with anti-CD3 antibodies (aCD3) and the expression of costimulatory receptors (OX40, 4–1BB, inducible costimulator [ICOS]) was analyzed on day 4. A, Illustra- tive histograms show the expression of OX40, 4-1BB, and ICOS (green histograms) and control staining with isotype-matched antibod- ies (red histograms) by activated CD4CD28null (28null) and classical CD4CD28 (28pos) T cells from an ACS patient. B, Scatter plots show the distribution and mean fluorescence intensity (MFI) of cells expressing costimulatory receptors in the ACS group. C, Scatter plots show the distribution and mean MFI of cells expressing costimulatory receptors in the SA group. Probability values (P) were cal- culated by 2-tailed t test. a.u. indicates arbitrary units.

Article Snippet: Stimulation of 4- 1BB and OX40 was done with 10 g/ml stimulating antibodies for 4-1BB (R&D Systems, AF838) or OX40 (AF3388) or with 5 g/ml recombinant ligands 4-1BBL (R&D Systems, 2295-4L) and OX40L (R&D Systems, 1054-OX).

Techniques: Expressing, Cell Culture, Control, Staining

Journal: Circulation Research

Article Title: High Levels of Costimulatory Receptors OX40 and 4-1BB Characterize CD4 + CD28 null T Cells in Patients With Acute Coronary Syndrome

doi: 10.1161/circresaha.111.261933

Figure Lengend Snippet: Figure 5. Analysis of CD4CD28null T cells, costimulatory receptors and ligands in atherosclerotic plaques. A, The frequency of CD4CD28null (CD28neg), CD4CD28 (CD28pos) T cells and the expression of OX40 and 4-1BB costimulatory receptors was quantified in cells isolated from human endarterectomy atherosclerotic plaques using flow cytometry. Representative plots show expression of OX40 and 4-1BB on plaque-derived T cells from atherosclerotic plaques B, Paraffin embedded tissue sections were immunohistochemically labeled

Article Snippet: Stimulation of 4- 1BB and OX40 was done with 10 g/ml stimulating antibodies for 4-1BB (R&D Systems, AF838) or OX40 (AF3388) or with 5 g/ml recombinant ligands 4-1BBL (R&D Systems, 2295-4L) and OX40L (R&D Systems, 1054-OX).

Techniques: Expressing, Isolation, Cytometry, Derivative Assay, Labeling

Journal: Circulation Research

Article Title: High Levels of Costimulatory Receptors OX40 and 4-1BB Characterize CD4 + CD28 null T Cells in Patients With Acute Coronary Syndrome

doi: 10.1161/circresaha.111.261933

Figure Lengend Snippet: Figure 6. Effects of OX40 and 4-1BB blockade on cytokine production by CD4CD28null T cells. Peripheral blood mononuclear cells (PBMCs) from acute coronary syndrome (ACS) patients (n9) were activated with anti-CD3 antibodies alone (aCD3) or in the presence of blocking antibodies against OX40 (aOX40) and 4-1BB (a4–1BB) or control antibodies (ctrl Ig) for 4 days. A, Repre- sentative contour plots and bar graphs display percentage of interferon (IFN)-

Article Snippet: Stimulation of 4- 1BB and OX40 was done with 10 g/ml stimulating antibodies for 4-1BB (R&D Systems, AF838) or OX40 (AF3388) or with 5 g/ml recombinant ligands 4-1BBL (R&D Systems, 2295-4L) and OX40L (R&D Systems, 1054-OX).

Techniques: Blocking Assay, Control

Journal: Circulation Research

Article Title: High Levels of Costimulatory Receptors OX40 and 4-1BB Characterize CD4 + CD28 null T Cells in Patients With Acute Coronary Syndrome

doi: 10.1161/circresaha.111.261933

Figure Lengend Snippet: Figure 7. Effects of OX40 and 4-1BB blockade on perforin and granzyme B in CD4CD28null T cells. Peripheral blood mononuclear cells (PBMCs) from acute coronary syndrome (ACS) patients (n9) were cultured alone (w/o) or acti- vated with anti-CD3 antibodies alone (aCD3) or in the presence of blocking antibodies against OX40 (aOX40) and 4-1BB (a4–1BB) for 4 days. A, Repre- sentative contour plots and bar graphs show surface expression of CD107a in CD4CD28null (28null) T cells. B, Repre- sentative contour plots and bar graphs display percentage of perforin

Article Snippet: Stimulation of 4- 1BB and OX40 was done with 10 g/ml stimulating antibodies for 4-1BB (R&D Systems, AF838) or OX40 (AF3388) or with 5 g/ml recombinant ligands 4-1BBL (R&D Systems, 2295-4L) and OX40L (R&D Systems, 1054-OX).

Techniques: Cell Culture, Blocking Assay, Expressing

Journal: Circulation Research

Article Title: High Levels of Costimulatory Receptors OX40 and 4-1BB Characterize CD4 + CD28 null T Cells in Patients With Acute Coronary Syndrome

doi: 10.1161/circresaha.111.261933

Figure Lengend Snippet: Figure 8. Schematic outline of the effects of costimulatory receptors OX40 and 4-1BB on the functions of CD4CD28null T cells in acute coro- nary syndrome (ACS). Classical CD4CD28 T cells constitutively express the costimulatory receptor CD28. Activation of CD4CD28 T cells induces transient expression of costimu- latory (OX40, 4–1BB) and coinhibitory (cytotoxic T lymphocyte associated antigen-4 [CTLA-4], programmed death [PD]-1) receptors. CD4CD28 T cells produce moderate levels of inflammatory cytokines (interferon [IFN]-, tumor necrosis factor [TNF]-) when activated and do not express cytotoxic molecules (perforin, granzyme B). CD4CD28null T cells from ACS patients lack CD28 and upregulate expression of OX40 and 4-1BB. These receptors drive production of high levels of IFN-, TNF-, and per- forin. We hypothesize that these media- tors could potentially enable CD4CD28null T cells to trigger plaque rupture via macrophage activation and endothelial and vascular smooth muscle cell lysis. VSMC indicates vascular smooth muscle cell.

Article Snippet: Stimulation of 4- 1BB and OX40 was done with 10 g/ml stimulating antibodies for 4-1BB (R&D Systems, AF838) or OX40 (AF3388) or with 5 g/ml recombinant ligands 4-1BBL (R&D Systems, 2295-4L) and OX40L (R&D Systems, 1054-OX).

Techniques: Activation Assay, Expressing, Lysis

Journal: Molecular Therapy Oncology

Article Title: Enhanced solid tumor cell targeting by a neoepitope-encoding oncolytic measles virus combined with CAR therapy

doi: 10.1016/j.omton.2025.201043

Figure Lengend Snippet: CAR-T cells targeting the MICB-derived octapeptide show CAR-dependent activation and efficiently kill tumor cells (A) Schematic representation of scFv of both 8-VHVL CAR and 8-VLVH CAR targeting the octapeptide VLQSQRTD used in this study. (B) Expression of CAR molecules on T cells. Left panel shows representative plots of flow cytometry data of primary T cells, 8-VHVL CAR-T and 8-VLVH CAR-T. Right panel shows expression of both experimental CARs (8-VHVL and 8-VLVH) as well as CD4 and CD19 CAR on T cells. Black bars represent CAR expression detected via anti-mouse F(abʹ) 2 antibody, and green bars represent EGFP signal; data from n = 18 independent experiments are depicted as mean (SEM). (C) CAR-T cells were cocultured with Mac-1 cells stably transduced to express indicated transgenes. Tumor cells were labeled with CellTrace Violet, and killing was assessed via PI staining. Killing is depicted as ratio of PI+ cells in cocultures and independently cultured Mac-1 cells. Data from n ≥ 6 experiments are depicted as mean (SEM). (D) CD137 expression was measured on CAR-T cells after incubation with Mac-1 for 24 h. Data are shown from EGFP+ CAR-T cells and are depicted from n = 6 independent experiments as mean (SEM). (E) CAR-T cells were cocultured with T cells from the same donor transduced to express indicated transgenes. T cells used as target cells were labeled with CellTrace Violet, and killing was assessed via PI staining. Only EGFP+ target T cells were analyzed for PI staining. Killing is depicted as the ratio of PI+ cells in cocultures and independently cultured target T cells. Data from n = 9 independent experiments are shown as mean (SEM). (F) CD137 expression was measured on CAR-T cells after incubation with transduced T cells as target cells for 24 h. Data are shown from EGFP+ CAR-T cells and are depicted from n = 6 independent experiments as mean (SEM). (G) LAG-3 expression was measured on CAR-T cells after incubation with transduced T cells as target cells for 72 h. Data are shown from EGFP+ CAR-T cells from n = 4 experiments as mean (SEM); statistical analysis of (C) through (G) was performed by two-way ANOVA followed by Dunnett’s multiple-comparisons test, and experimental groups were compared to CD19 CAR. ∗ = p ≤ 0.05, ∗∗ = p ≤ 0.01, ∗∗∗ = p ≤ 0.001, and ∗∗∗∗ = p ≤ 0.0001.

Article Snippet: For evaluation of CAR-T cell activation, staining with CD137 (CD137 Antibody, PE-Vio770, anti-human, REAfinity) (Miltenyi Biotech, Bergisch Gladbach, Germany) was carried out after 24 h of co-incubation.

Techniques: Derivative Assay, Activation Assay, Expressing, Flow Cytometry, Stable Transfection, Labeling, Staining, Cell Culture, Incubation