Anti pd1 rmp1 14

Manufactured by BioXCell

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Anti-PD1 (RMP1-14) is a monoclonal antibody that targets the programmed cell death-1 (PD-1) receptor. PD-1 is an immune checkpoint protein that regulates T-cell activation and function. Anti-PD1 (RMP1-14) binds to PD-1, blocking the interaction between PD-1 and its ligands, thereby enhancing T-cell-mediated immune responses.

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20 protocols using anti pd1 rmp1 14

Combination Regimens for Murine Cancer Models

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The combination regimen used for the MC38 colon adenocarcinoma mouse model was capecitabine (Actavis Group PTC ehf) administered per os 5 days a week at a dose of 250mg/kg and oxaliplatin (Mylan), injected i.p. once a week at a dose of 5 mg/kg + anti-PD-1 (RMP1-14, BioXCell), injected i.p. once a week at a dose of 12.5 mg/kg. For the metastatic 4T1 breast cancer mouse model, the combination was cyclophosphamide (Baxter) injected i.p. once a week at a dose of 100 mg/kg + doxorubicin (Accord Healthcare) injected i.p. once a week at a dose of 2 mg/kg + anti-PD-1 (RMP1-14, BioXCell) or anti-PD-L1 (10F.9G2, BioXCell), injected i.p. once a week at a dose of 12.5 mg/kg. For bladder cancer mouse models, the MVAC regimen consisted in a combination of methotrexate (Mylan) injected i.p. once a week at a dose of 1 mg/kg + vinblastine (EG Labo) injected i.p. once a week at a dose of 0.1 mg/kg + doxorubicin (Accord Healthcare) injected i.p. once a week at a dose of 1 mg/kg + cisplatine (Mylan) injected i.p. once a week at a dose of 1 mg/kg, + anti-PD-L1 (10F.9G2, BioXCell) injected i.p. once a week at a dose of 12.5 mg/kg and + anti-PD-1 (RMP1-14, BioXCell) injected i.p. once a week at a dose of 12.5 mg/kg for MB49 mouse model.

Grasselly C., Denis M., Bourguignon A., Talhi N., Mathe D., Tourette A., Serre L., Jordheim L.P., Matera E.L, & Dumontet C. (2018). The Antitumor Activity of Combinations of Cytotoxic Chemotherapy and Immune Checkpoint Inhibitors Is Model-Dependent. Frontiers in Immunology, 9, 2100.

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Murine Glioblastoma Immunotherapy Protocol

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Murine Glioblastoma cancer cell line (GL-261) were obtained from the National Cancer Institute (Rockville, MD, USA). Cells were collected in the logarithmic phase and washed twice with PBS just before tumor injections. 50,000 cells were injected intracerebrally in the mice (5 or 10 mice per group) as described previously^{37 (link)}. Anti-CTLA-4 (clone 9H10) and anti-PD-1 (RMP1–14) antibodies were purchased from BioXcell (West Lebanon,NH). Mice were injected intraperitoneally with anti-PD-1 and combination of anti-PD-1 plus anti-CTLA-4 on day 7 (200 μg/mouse), day 10 (100 μg/mouse) and day 13 (100 μg/mouse) post tumor inoculation.

Goswami S., Walle T., Cornish A.E., Basu S., Anandhan S., Fernandez I., Vence L., Blando J., Zhao H., Yadav S.S., Ott M., Kong L.Y., Heimberger A.B., de Groot J., Sepesi B., Overman M., Kopetz S., Allison J.P., Pe’er D, & Sharma P. (2019). Immune profiling of human tumors identifies CD73 as a combinatorial target in glioblastoma. Nature medicine, 26(1), 39-46.

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Vaccination and Checkpoint Blockade for Melanoma

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To study the protection conferred by vaccination, C57BL/6NHsd mice were vaccinated with 50 µL of the different formulations at 25 mg mL⁻¹ of PLGA, or equivalent, on days 0, 7, and 14. On day 20, the right flank of each mouse was shaved and, on day 21, mice were challenged with 2 × 10⁵ B16–F10 cells subcutaneously on the right flank. Tumors were measured every other day and the experimental endpoint was defined as either death or tumor size greater than 200 mm².
To study the antitumor therapeutic effect, C57BL/6NHsd mice were first challenged on the right flank with 5 × 10⁴ B16–F10 cells on day 0. On days 1, 2, 4, and 7, mice were vaccinated subcutaneously in the same flank with 200 µL of the nanoparticulate formulations. The subcutaneous route was chosen in this case to accommodate the larger dosage that was employed. The checkpoint blockade cocktail, consisting of 100 µg anti-CTLA4 (9H10; BioXCell) and 200 µg anti-PD1 (RMP1-14; BioXCell) was administered intraperitoneally on the same days. Tumors were measured every other day and the experimental endpoint was defined as either death or tumor size greater than 200 mm².

Kroll A.V., Fang R.H., Jiang Y., Zhou J., Wei X., Yu C.L., Gao J., Luk B.T., Dehaini D., Gao W, & Zhang L. (2017). Nanoparticulate Delivery of Cancer Cell Membrane Elicits Multi-Antigenic Antitumor Immunity. Advanced materials (Deerfield Beach, Fla.), 29(47), 10.1002/adma.201703969.

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Targeting Notch1 and Dll4 in Tumor Angiogenesis

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Research-grade, function-blocking antibodies to mouse Notch1 and Dll4 (Kuhnert et al., 2015 (link)) were generated using VelociImmune^® technology. Anti-Notch1 (10 mg/kg body weight, twice a week), Dll4 (5 mg/kg body weight, once a week) and control antibodies (10 mg/kg body weight, twice a week) were administered intraperitoneally (IP). Anti-PD-1 (RMP1-14) and isotype control rat IgG2a (2A3) were purchased from BioXcell and administered IP at 10 mg/kg body weight twice a week starting at 3 weeks post-transplantation. For immunofluorescent staining, anti-CD31 (ab28364, Abcam), anti-N1-ICD (D3B8, Cell Signaling Technology) and anti-Dll4 (AF1389, R&D Systems) were used.

Gao J., Van Meter M., Hernandez Lopez S., Chen G., Huang Y., Ren S., Zhao Q., Rojas J., Gurer C., Thurston G, & Kuhnert F. (2019). Therapeutic targeting of Notch signaling and immune checkpoint blockade in a spontaneous, genetically heterogeneous mouse model of T-cell acute lymphoblastic leukemia. Disease Models & Mechanisms, 12(9), dmm040931.

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Anti-CTLA-4 and Anti-PD1 Immunotherapy in Mice

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Balb/C (6–8 weeks old, JAX: 0006501) female mice mice were purchased from Jackson laboratories. They were injected with control or flavopiridol pre-treated (Catalog No.S1230; Selleckchem: 25 nM for 7 days) 1 × 106 CT26 cells subcutaneously into the right flank. One-half of the mice were administered with 100 μg anti-CTLA-4 (9H10; BioX Cell) alone, or together with anti-PD1 (RMP1-14; BioX Cell) antibodies in PBS, and the other half were given control IgG in PBS intraperitoneally on days 3, 5, 7, 9, 12, and 15 post implantations. Tumor volumes were monitored every two days up to day 33. The significant difference in p value for each group at the end of 33 days after the initiation of treatment was calculated by Student’s t-test and defined by as p < 0.05. The mice were then euthanized, followed by tumor excision. Animal research was approved and overseen by The CCHMC Institutional Animal Care and Use Committee (CCHMC IACUC).
All mice were housed under specific pathogen-free conditions in the animal facility at the Cincinnati Children’s Hospital Research Foundation in compliance with the Cincinnati Children’s Hospital Medical Center Animal Care and Use Committee protocols. Unless otherwise noted, all animals were studied between 6 and 10 weeks of age. Only female mice were used in the studies.

Modur V., Muhammad B., Yang J.Q., Zheng Y., Komurov K, & Guo F. (2023). Mechanism of inert inflammation in an immune checkpoint blockade-resistant tumor subtype bearing transcription elongation defects. Cell reports, 42(4), 112364.

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Immune Depletion and Cytokine Neutralization Protocol

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For depletion and neutralization experiments, mice were injected intraperitoneally with 100 µg anti-CD8β (53.5.8; BioXCell) to deplete CD8⁺ T cells, 50 µg anti-asialoGM1 (Wako) to deplete NK cells, 250 µg anti-IFNγ (H22; Leinco Technologies), or 100 µg anti-CCL21 (Comerford et al., 2010 (link)) with the dosing schedule indicated in text/figure legends. For CCL21 treatments, female mice were injected into contralateral sides of the fourth mammary gland with 10⁵ E0771 cells. Beginning from the day of tumor injection, mice were anesthetized and injected every 3 d with 3 µg CCL21 or MCP_ala, a truncated peptide control (Kara et al., 2013 (link); Kohler et al., 2008 (link)), into contralateral mammary glands or palpable tumors. For immunotherapy experiments, mice were injected intraperitoneally with 100 µg anti-CD137 (3H3; BioXCell), 250 µg anti-PD-1 (RMP1-14; BioXCell), 250 µg anti-CTLA-4 (UC10-4F10; BioXCell), or the equivalent amount of control rat or hamster Ig in sterile PBS, in accordance with the dosing schedule in text.

Whyte C.E., Osman M., Kara E.E., Abbott C., Foeng J., McKenzie D.R., Fenix K.A., Harata-Lee Y., Foyle K.L., Boyle S.T., Kochetkova M., Aguilera A.R., Hou J., Li X.Y., Armstrong M.A., Pederson S.M., Comerford I., Smyth M.J, & McColl S.R. (2020). ACKR4 restrains antitumor immunity by regulating CCL21. The Journal of Experimental Medicine, 217(6), e20190634.

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Immune Checkpoint Blockade and FMT in Mice

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Mice used were 6–7 weeks old at the start of experiments and gender-matched. Female mice were used in most experiments for this study. Mice treated with immune checkpoint blockade drugs were intraperitoneally administered anti-CTLA4 (9H10, BioXCell), using doses of 200 μg, and anti-PD-1, (RMP1-14, BioXCell) at a dose of 250 μg, once per week^{40 (link)}. All experimental mice were co-housed in specific pathogen–free facilities at King’s College London Biological Services Unit or Imperial Hammersmith CBS. Untreated control mice were housed in a separate box from CPI + FMT treated mice. Mice treated with depleting antibodies were intraperitoneally administered once a week, at the same time of giving anti-CTLA4 and anti-PD-1 antibodies, either 500 μg anti-TNFα (XT3.11, BioXCell), 500 μg anti-IFNγ (H22, BioXCell) or 150 μg anti-IL-23(p19) (G23-8, BioXCell). Control-isotype clones used were 2A3 (rat IgG2a) and HRPN (rat IgG1).

Lo J.W., Cozzetto D., Alexander J.L., Danckert N.P., Madgwick M., Knox N., Sieh J.Y., Olbei M., Liu Z., Ibraheim H., Blanco J.M., Kudo H., Seoane R.C., Possamai L.A., Goldin R., Marchesi J., Korcsmaros T., Lord G.M, & Powell N. (2023). Immune checkpoint inhibitor-induced colitis is mediated by polyfunctional lymphocytes and is dependent on an IL23/IFNγ axis. Nature Communications, 14, 6719.

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Murine Myocardial Infarction Model

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In vivo repAMI was induced by transient occlusion of the left coronary artery (LCA) for 45 min followed by reperfusion for 1–5 days as described previously [30 (link),31 (link)]. C57BL/6J Pdcd1^−/− and C57BL/6J wild-type mice were anesthetized by intraperitoneal injection of ketamine (bela-pharm, Vechta, Germany) and xylazine (Ceva Tiergesundheit, Düsseldorf, Germany). Mice were intubated and ventilated with 40% O₂ and 1.6–2.0 Vol.% isoflurane (Piramal, Mumbai, India). Analgesia was maintained with buprenorphine. Left-lateral thoracotomy was performed followed by pericardiotomy and exposure of the LCA. The LCA was ligated for 45 min while maintaining anesthesia, followed by reperfusion. The tissue was then closed with sutures before the end of the anesthesia. For experiments involving antibodies, mice were injected intraperitoneally with 250 µg anti-PD1 (RMP1-14) or 250 µg rat IgG2a (2A3, both BioXCell, West Lebanon, NH, USA) every other day starting on day 8 before surgery, followed by day 2 after surgery. Analgesia was maintained with buprenorphine 3 times a day. Mice were killed with an isoflurane overdose.

Michel L., Korste S., Spomer A., Hendgen-Cotta U.B., Rassaf T, & Totzeck M. (2022). PD1 Deficiency Modifies Cardiac Immunity during Baseline Conditions and in Reperfused Acute Myocardial Infarction. International Journal of Molecular Sciences, 23(14), 7533.

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Therapeutic Vaccination Using Melanoma Lysates

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For therapeutic vaccination, animals were challenged with a subcutaneous injection of 10⁵ B16-F10 melanoma cells (ATCC, Manassas, NJ) in the back of the neck. At day 3 a subset of mice were treated with 100 μg of either anti-CTLA-4 (9D9), or anti-PD-1 (RMP1-14) (Bioxcell, West Lebabnon, NH), or both antibodies and treatment was repeated every three days for thirty days. At day 9 after tumor challenge, PLG vaccines with melanoma tumor lysates and GM-CSF in combination with CpG-ODN were implanted subcutaneously into the lower left flank of C57BL/6J mice. Animals were monitored for the onset of tumor growth (approximately 1mm³) and sacrificed for humane reasons when tumors grew to 20–25 mm (longest diameter).

Ali O.A., Lewin S.A., Dranoff G, & Mooney D.J. (2015). Vaccines combined with immune checkpoint antibodies promote cytotoxic T cell activity and tumor eradication. Cancer immunology research, 4(2), 95-100.

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Monoclonal Antibody Administration for Immunotherapy

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The following mAbs were purchased from BioXcell: anti-CD137 (LOB12.3; Cat. #BE0169), anti-PD-1 (RMP1-14; Cat. #BE0146), anti-CTLA4 (9D9; Cat. #BE0164), anti-CD19 (1D3; Cat. #BE0150) and control (2A3; Cat. #BE0089) and administered as indicated.

Dai M., Yip Y.Y., Hellstrom I, & Hellstrom K.E. (2014). Curing mice with large tumors by locally delivering combinations of immunomodulatory antibodies. Clinical cancer research : an official journal of the American Association for Cancer Research, 21(5), 1127-1138.

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