Anti pd 1 ab clone rmp1 14

Half millon of melanoma cells B16F10, B16F10-expressing OVA (B16F10-OVA), MCA-101 fibrosarcoma cells, or one millon of EL4 lymphoma cells expressing OVA (EL4-OVA) were injected subcutaneously (s.c.) into the right flank of mice. Mice were randomly assigned a treatment group and tumor volume determined by caliper measurements. Anti-PD-1 Ab (clone RMP1-14, BioXcell), was administered intraperitoneally at 150 μg/20 g mice weight per dose on days 6–7, 11, 15 and 18–19. ETP-69 inhibitor was prepared as described previously^{38 (link)}. Compound was dissolved in a mixture of Methyl cellulose 1% (Merck) and Poloxamer 188 0.1% (BASF Pharma Solutions) in water and administrated orally (20 mg/kg) once a day during two weeks to C57BL/6J female mice. F5446 inhibitor was kindly provided by Dr. Kebin Liu (Augusta, GA, USA) and administrated as described in Lu et al.^{40 (link)}. Compound was dissolved in 10% Cremophor (Sigma) in PBS and injected intraperitoneally (10 mg/kg) every 2 days for a total of 7 doses to C57BL/6J female mice.

4–6 weeks old female C57BL/6JOlaHsd (immunocompetent) mice were purchased from Envigo (Indianapolis, IN, USA). For the syngeneic mouse model, B6 mice were subcutaneously (right flank) injected with either 2x10⁶ MOC1 cells or 1x10⁵ MOC2 cells in 50μl PBS. Established tumors (stratified randomization) were treated either with intratumoral injection of PBS, 1 × 10⁹ VP of unarmed control virus (Ad5-Luc), 1:1 mixture of non-replicative Ad5-CMV-mTNFα and Ad5-CMV-mIL-2 virus (total 1 × 10⁹ VP) and/or intraperitoneal injection of anti-PD-1 Ab (clone RMP1-14, BioXcell) or anti-PD-L1 Ab (clone 10 F.9G2, BioXcell) (100 µg/injection). Intended number of animals per group was n= 10 but engraftment rate was not 100% (n=8 PBS versus, n=9 antiPD-1/PD-L1, n=8 Ad5-Luc, n=9 Ad5-CMV-mTNFα/mIL-2, n=9 combination). Tumor dimensions were measured with digital calipers and the volumes were calculated as (length × width²)/2. When maximum allowed tumor diameter of 18 mm was reached, animals were immediately euthanized and samples for further analysis were collected. Animals with open wounds (ulcers at the injection site) were euthanized as per animal permit and excluded from the study. Total animals for treatment naïve MOC1 n= 60, MOC2 n=40, refractory MOC1 n=120.

RPMI-1640, FBS, phosphate buffered saline (PBS), penicillin-streptomycin, HEPES, glutamine, 2-mercaptoethanol, collagenase, DNAase and hygromycin B were obtained from Sigma (Poole, UK). Fluorochrome conjugated antibodies targeting CD4 (APC eFluor 780, clone RM4-5), CD3 (PE or PE Cy7, clone 145-2C11), Foxp3 (eFluor 450, clone FJK-16s), Ki67 (FITC, clone SolA15), CD45 (FITC or eFluor 450, clone 30-F11), PD-L1 (PE, clone MIH5), CD3 Ab (purified, clone 17A2), CD11b (APC-eFluor 780, clone M1/70), GR1 (FITC, clone RB6-8C5) were obtained from eBiosciences (San Diego, USA). PE-Alexa-647 labeled antibody targeting CD8 (clone KT15) was obtained from AbD Serotec (Oxford, UK). Anti-PD-1 Ab (clone RMP1-14) was obtained from BioXcell (West Lebanon, USA). Synthetic peptides TRP2_180-188 (SYVDFFVWL) and gp100_44-59 (WNRQLYPEWTEAQRLD) were obtained at > 90% purity from Genscript (Piscataway, USA).