Journal: Journal for Immunotherapy of Cancer

Article Title: Dichotomous impact of affinity on the function of T cell engaging bispecific antibodies

doi: 10.1136/jitc-2021-002444

Figure Lengend Snippet: In vitro binding and in vitro and in vivo redirected T cell killing of HER-2 HI , HER-2 LO , and HER-2 NEG cell lines. (A) Binding of the HER-2/CD3 BsAbs to HER-2 HI SKOV3 (right) and CD3 + Jurkat (left) cells using flow cytometry. (B) Naïve T cell redirected killing of four different cell lines by the HER-2/CD3 BsAbs (10:1 E:T ratio). Each point is the average of three individual measurements and error bars are SD. The number in parentheses indicates the experimentally determined number of HER-2 receptors per cell. (C) In vivo T cell redirected killing of SKOV3 cells by the HER-2/CD3 BsAbs. The arrows indicate the dosing paradigm, either five daily doses (left panel, doses=black arrows) or a single dose (right panel, single dose=black arrow) starting 1 day after tumor cell implantation (**p<0.001; *p<0.05). Mice were treated with 5 mg/kg of antibodies. SKOV3-Luc tumor monitoring was performed longitudinally using bioluminescence imaging. BsAb, bispecific antibody; mAb, monoclonal antibody.

Article Snippet: Adherent SKOV3 ovarian cancer cells (ATCC Cat#HTB-77) cultured in RPMI 1640 /10% fetal bovine serum (FBS) Corning/gentamicin (Gibco) at 37°C, 5% CO 2 were resuspended using Accutase (Innovative Technologies Cat#AT104).

Techniques: In Vitro, Binding Assay, In Vivo, Flow Cytometry, Imaging

Journal: Journal for Immunotherapy of Cancer

Article Title: Dichotomous impact of affinity on the function of T cell engaging bispecific antibodies

doi: 10.1136/jitc-2021-002444

Figure Lengend Snippet: (A) Impact of attenuating HER-2/CD3 BsAb affinity to CD3 (upper left panels), (B) HER-2 (upper right panels), or (C) both CD3 and HER-2 (bottom panels) on in vitro T cell redirected lysis activity towards OVCAR3 and SKOV3 tumor cells. On the upper panels, chSP34/Pertuz IgG BsAb and chSP34/Pertuz IgG BsAb2 are duplicates. BsAb, bispecific antibody; mAb, monoclonal antibody.

Article Snippet: Adherent SKOV3 ovarian cancer cells (ATCC Cat#HTB-77) cultured in RPMI 1640 /10% fetal bovine serum (FBS) Corning/gentamicin (Gibco) at 37°C, 5% CO 2 were resuspended using Accutase (Innovative Technologies Cat#AT104).

Techniques: In Vitro, Lysis, Activity Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Dichotomous impact of affinity on the function of T cell engaging bispecific antibodies

doi: 10.1136/jitc-2021-002444

Figure Lengend Snippet: In vivo studies with affinity-attenuated HER-2/CD3 IgG BsAbs. (A.) In vivo redirected lysis activity of affinity-attenuated HER-2/CD3 IgG BsAbs (one dose, 5 mg/kg). Tumor growth was measured using the bioluminescence of the SKOV3-Luc cell line (top panel) or physically using caliper measurements (bottom panel). IgG BsAb treatment was performed only once, 1 day after tumor cell inoculation (black arrow). (B). SKOV3 In vivo dose response study (top panel) and anti-PD-1 antagonist or anti-4-1BB agonist combination study (bottom panel) with the CD3 affinity-attenuated HER-2/CD3 IgG BsAb (SP34_V100cT, IC 50 =280 nM). For the PD-1/4-1BB combination study, the HER-2/CD3 IgG BsAb was injected intraperitoneally on Day 3 a (0.6 mg/kg) after tumor cell inoculation to reduce its observed efficacy. The PD-1 antagonist mAb and 4-1BB agonist mAb were injected on Day 7 b (10 mg/kg) after tumor cell inoculation to allow a period for receptor upregulation on T cells. BsAb, bispecific antibody; mAb, monoclonal antibody; PD-1, Programmed cell death 1 aka CD279.

Article Snippet: Adherent SKOV3 ovarian cancer cells (ATCC Cat#HTB-77) cultured in RPMI 1640 /10% fetal bovine serum (FBS) Corning/gentamicin (Gibco) at 37°C, 5% CO 2 were resuspended using Accutase (Innovative Technologies Cat#AT104).

Techniques: In Vivo, Lysis, Activity Assay, Injection

Journal: Journal for Immunotherapy of Cancer

Article Title: Dichotomous impact of affinity on the function of T cell engaging bispecific antibodies

doi: 10.1136/jitc-2021-002444

Figure Lengend Snippet: Primary CD8 + T cell activation markers and checkpoint inhibitors 48 hours after exposure to SKOV3 tumor cells and various IgGs and IgG BsAbs (10:1 E:T). Results were similar for CD4 + T cell activation markers and checkpoint inhibitors

Article Snippet: Adherent SKOV3 ovarian cancer cells (ATCC Cat#HTB-77) cultured in RPMI 1640 /10% fetal bovine serum (FBS) Corning/gentamicin (Gibco) at 37°C, 5% CO 2 were resuspended using Accutase (Innovative Technologies Cat#AT104).

Techniques: Activation Assay, Control

Journal: Journal for Immunotherapy of Cancer

Article Title: Dichotomous impact of affinity on the function of T cell engaging bispecific antibodies

doi: 10.1136/jitc-2021-002444

Figure Lengend Snippet: PD-L1 molecule numbers on SKOV3 or OVCAR3 cells 24 hours after exposure to primary T cells (10:1 E:T) and various IgGs and IgG BsAbs (0.1 nM). Columns with ‘fresh’ tumor cells were incubated for 24 hours with supernatants from the original redirected lysis assays cultures (T cells and original tumor cells removed by centrifugation), thus evaluating whether factors released into the supernatants of an active redirected lysis assay could induce PD-L1 upregulation on untreated tumor cells

Article Snippet: Adherent SKOV3 ovarian cancer cells (ATCC Cat#HTB-77) cultured in RPMI 1640 /10% fetal bovine serum (FBS) Corning/gentamicin (Gibco) at 37°C, 5% CO 2 were resuspended using Accutase (Innovative Technologies Cat#AT104).

Techniques: Incubation, Lysis, Centrifugation

Journal: Metabolites

Article Title: Feature-Based Molecular Networking—An Exciting Tool to Spot Species of the Genus Cortinarius with Hidden Photosensitizers

doi: 10.3390/metabo11110791

Figure Lengend Snippet: (Photo)cytotoxic activity of the fungal extracts against the cancer cell lines A549, AGS, and T24 in the presence (BL/blue light, λ = 468 ± 27 nm, 9.3 J cm −2 ) and in the absence of blue light (D/dark). Bars: EC 50 value in µg/mL with the respective confidence interval (95%). ( A ) Results of all six extracts measured as biological duplicates given as EC 50 ranges (++ … 0.01–5 µg/mL, + … 5–25 µg/mL, o … 25–50 µg/mL). ( B ) Detailed investigation of the most promising extracts (i.e., C. xanthophyllus and C. rubrophyllus ) measured as biological triplicates. A methanolic extract of B. ilicifolia showed an EC 50 of 17 µg/mL under light irradiation (blue light, λ = 468 ± 27 nm, 9.3 J cm −2 ).

Article Snippet: Cells of the adherent cancer cell lines A549 (non-small lung cancer, ATCC, Merck KGaA, Darmstadt, Germany), AGS (stomach cancer, CLS, Eppelheim, Germany), T24 (urinary bladder carcinoma, CLS, Eppelheim, Germany), and of the mouse embryonic fibroblast cell line NIH3T3 (ATCC, Manassas, Virginia, CRL 1658) were cultivated in Nunc EasYFlasks (product number: 51985042, 75 cm 2 ) with GibcoTM MEMTM medium (product number: 42360081) supplemented with fetal calf serum (FCS, 10% v / v ) and penicillin/streptomycin (P/S, 1% v / v ).

Techniques: Activity Assay, Irradiation

Journal: Metabolites

Article Title: Feature-Based Molecular Networking—An Exciting Tool to Spot Species of the Genus Cortinarius with Hidden Photosensitizers

doi: 10.3390/metabo11110791

Figure Lengend Snippet: Results of the (photo)cytotoxicity assay employing green light (λ = 519 nm, 20.1 J cm −2 ). ( A ) (Photo)cytotoxic activity of the acetone extracts of C. rubrophyllus , C. xanthophyllus , and rose bengal against the three cancer cell lines T24, AGS, and A549 as well as against the non-malignant NIH3T3 cell line. Bars: EC 50 value in µg/mL/µM with the respective confidence interval (95%). ( B ) Micrographs (200× magnification) of cells of the T24 and NIH3T3 cell lines treated (24 h) with the acetone extract of C. xanthophyllus (7.5 µg/mL). The upper line of pictures shows treated cells in the dark, the lower after irradiation with green light (519 nm, 20.1 J/cm 2 ).

Article Snippet: Cells of the adherent cancer cell lines A549 (non-small lung cancer, ATCC, Merck KGaA, Darmstadt, Germany), AGS (stomach cancer, CLS, Eppelheim, Germany), T24 (urinary bladder carcinoma, CLS, Eppelheim, Germany), and of the mouse embryonic fibroblast cell line NIH3T3 (ATCC, Manassas, Virginia, CRL 1658) were cultivated in Nunc EasYFlasks (product number: 51985042, 75 cm 2 ) with GibcoTM MEMTM medium (product number: 42360081) supplemented with fetal calf serum (FCS, 10% v / v ) and penicillin/streptomycin (P/S, 1% v / v ).

Techniques: Cytotoxicity Assay, Activity Assay, Irradiation