Pembrolizumab

Manufactured by Selleck Chemicals

Sourced in United States

Pembrolizumab is a monoclonal antibody used as a laboratory reagent. It is designed to bind to the PD-1 receptor on T cells, which can help to enhance the immune response.

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Anti-PD-1 antibody (Pembrolizumab (2 citations)

20 protocols using pembrolizumab

Modulating anti-tumor immunity with IL-9

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The fresh tumor tissue was dissociated into single-cell suspensions as above. The digests were cultured in assay medium (RPMI-1640 with 100 U/mL penicillin, 100 μg/mL streptomycin and 10% fetal bovine serum) with anti-CD3 antibody (0.5 μg/ml, OKT3; Biolegend) and anti-CD28 antibody (2 μg/ml, clone 28.2; eBioscience). In CD8⁺ T cells-deprived system, CD8⁺ T cells were depleted from single cell suspensions by negative selection using CD8 MicroBeads (Life Technologies). Recombinant human IL-9 (0.3 ng/ml, R&D Systems), anti-IL-9 antibody (5 μg/mL, R&D Systems) or PBS were added to the culture system to study the effect of IL-9 on anti-tumor immunity. Pembrolizumab (10ug/ml; Pembrolizumab, Selleck) were used to explore the synergistic effect of PD-1 blockade and rhIL-9. After 24 hours of incubation, we collected cells for flow cytometry as indicated above.

Fang H., Li R., Gu Y., Fei Y., Jin K., Chen Y., Cao Y., Liu X., Lv K., Wang J., Yu K., Lin C., Liu H., Li H., He H., Zhang W., Zhang H, & Shen Z. (2020). Intratumoral interleukin-9 delineates a distinct immunogenic class of gastric cancer patients with better prognosis and adjuvant chemotherapeutic response. Oncoimmunology, 9(1), 1856468.

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Anti-PD1 and Cabozantinib in APCC Treatment

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For drug treatments, anti‐PD1 antibodies (pembrolizumab, SelleckChem) were added at 5 µg mL^–1 to the APCCs. Cabozantinib (SelleckChem) was added at 10 µm to the APCCs.

Ao Z., Wu Z., Cai H., Hu L., Li X., Kaurich C., Chang J., Gu M., Cheng L., Lu X, & Guo F. (2022). Rapid Profiling of Tumor‐Immune Interaction Using Acoustically Assembled Patient‐Derived Cell Clusters. Advanced Science, 9(22), 2201478.

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Investigating Immune Checkpoint Inhibition in Cancer

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PD0325901 and PD-1 blocking antibody (pembrolizumab) were purchased from Selleck Chemicals. Anti-PD-L1, anti-phospho ERK1/2 and anti-ERK1/2 antibodies were obtained from Santa Cruz Biotechnology. The antibody against GAPDH was purchased from Proteintech. The anti-mouse CD3 and granzyme B antibodies for immunohistochemistry were purchased from Abcam. The anti-human PD-L1 and p-ERK1/2 for immunohistochemistry were obtained from Santa Cruz Biotechnology. The anti-human CD3 and CD8 antibodies for immunohistochemistry were purchased from Zsbio. Dimethyl sulfoxide and 3-(4,5-dimethylthiazol-yl)-2,5-diphenyltetrazolium bromide (MTT) were products of Sigma–Aldrich. DMEM and fetal bovine serum were products of Gibco. Penicillin, streptomycin and trypsin were obtained from Thermo Fisher Scientific. InVivoMab anti-mouse PD-1 (CD279) was purchased from BioXcell.

Luo M., Xia Y., Wang F., Zhang H., Su D., Su C., Yang C., Wu S., An S., Lin S, & Fu L. (2021). PD0325901, an ERK inhibitor, enhances the efficacy of PD-1 inhibitor in non-small cell lung carcinoma. Acta Pharmaceutica Sinica. B, 11(10), 3120-3133.

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Organoid Chemotherapy and Immunotherapy Evaluation

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Organoids were subsequently treated after 7 days of culture. Chemotherapies included cisplatin (1, 10, 100 µM) (232120, Sigma Aldrich), doxorubicin (0.1, 1, 10 µM) (S1208, Selleckchem), vincristine (0.1, 1, 10 µM) (V8879 Sigma Aldrich), doxorubicin with cisplatin (0.1/1, 1/10, 10/100 µM), cisplatin with etoposide (S1125, Selleckchem) (1/0.1, 10/1, 100/10 µM) (Selleckchem), and doxorubicin with vincristine (0.1/0.1, 1/1, 10/10 µM). For immunotherapy treatment, 100 nM of pembrolizumab (A2002, Selleckchem), ipilimumab (A2001, Selleckchem) or nivolumab (A2005, Selleckchem) was used on both iPTOs and PTOs in parallel treatments. Media was removed from the wells and drug solutions mixed in culture media were added. Organoids remained in treated media solution for 72 h prior to endpoint viability assessment.

Forsythe S.D., Erali R.A., Laney P., Sivakumar H., Li W., Skardal A., Soker S, & Votanopoulos K.I. (2022). Application of immune enhanced organoids in modeling personalized Merkel cell carcinoma research. Scientific Reports, 12, 13865.

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Comprehensive Immune Landscape Analysis

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The PD-1 inhibitor (pembrolizumab) was purchased from Selleckchem (Houston, TX, USA). The human anti-TNFAIP3, anti-HA-tag and anti-GSK3β antibodies were bought from Cell Signal Technology (Danvers, MA, USA). The antibody against GAPDH, CRT, and Flag-tag were purchased from Proteintech (Chicago, IL, USA). The human STC1, CD163 and anti-mouse CD3, CD4, CD8, F4/80, and granzyme B antibodies were purchased from Abcam (Cambridge, UK). The anti-human CD3, CD4 and CD8 antibodies were purchased from Zsbio (Beijing, China). The fetal bovine serum (FBS) was obtained from Gibco BRL (Gaithersburg, MD, USA). The RPMI-1640, penicillin, streptomycin and trypsin were obtained from Cornning (Corning, NY, USA). The anti-mouse PD-1 antibody, anti-mouse CSF1R (CD115) antibody and rat IgG2a isotype control were obtained from BioXcell (Lebanon, NH, USA).

Luo M., Wang X., Wu S., Yang C., Su Q., Huang L., Fu K., An S., Xie F., To K.K., Wang F, & Fu L. (2023). A20 promotes colorectal cancer immune evasion by upregulating STC1 expression to block “eat-me” signal. Signal Transduction and Targeted Therapy, 8, 312.

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Mature DCs Enhance CD8+ T Cell Activation

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To obtain mature DCs (mDCs), the iDCs were incubated with 1×LPS (ThermoFisher, 00-4976-93) for 24 hours prior to initiation of the MLR. CD8⁺ T cells were incubated in MLR medium with 10 ng/mL IL-2 (BioLegend, 589106) O/N, prior to initiation of the MLR.
CD8⁺ T cells were cocultured with either iDCs or mDCs (2×10⁵ T cells: 2×10⁴ DC per well) in AIM-V medium and incubated with DuoBody-CD40×4-1BB (monotherapy (iDC:CD8⁺ T cell and mDC:CD8⁺ T-cell cocultures) or concurrent treatment with pembrolizumab (mDC:CD8⁺ T-cell cocultures; Research-grade, Selleckchem, A2005)) or control antibodies for 5 days. Supernatants were harvested for cytokine analysis.

Muik A., Adams HC 3.r.d., Gieseke F., Altintas I., Schoedel K.B., Blum J.M., Sänger B., Burm S.M., Stanganello E., Verzijl D., Spires V.M., Vascotto F., Toker A., Quinkhardt J., Fereshteh M., Diken M., Satijn D.P., Kreiter S., Ahmadi T., Breij E.C., Türeci Ö., Sasser K., Sahin U, & Jure-Kunkel M. (2022). DuoBody-CD40x4-1BB induces dendritic-cell maturation and enhances T-cell activation through conditional CD40 and 4-1BB agonist activity. Journal for Immunotherapy of Cancer, 10(6), e004322.

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In Vivo Evaluation of Anti-PD-1 Antibodies

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The hPD‐1 C57BL/6 transgenic mice used for in vivo efficacy evaluation were purchased from Biocytogen Co., Ltd. (Beijing, China). The antibodies pembrolizumab and nivolumab were purchased from Selleckchem (Houston, TX, USA), while tislelizumab was a gift from BI (Shanghai, China). HEK293F cells expressing recombinant PD‐1 or tislelizumab Fab proteins were obtained from Thermo Fisher (Shanghai, China).

Hong Y., Feng Y., Sun H., Zhang B., Wu H., Zhu Q., Li Y., Zhang T., Zhang Y., Cui X., Li Z., Song X., Li K., Liu M, & Liu Y. (2021). Tislelizumab uniquely binds to the CC′ loop of PD‐1 with slow‐dissociated rate and complete PD‐L1 blockage. FEBS Open Bio, 11(3), 782-792.

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MIBC Tumor Cell Co-culture Protocol

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The in vitro intervention studies were performed according to the methods described previously.^{17 (link)} Single cell suspensions of fresh MIBC tumor samples including tumor cells, immune cells and other cells were co-cultured in medium, under 37°C and 5% CO₂. Then the cells were cultured with maraviroc (10uM, Selleck) or dimethyl sulfoxide (DMSO)/Pembrolizumab (5 µg/mL. Selleck) or IgG4B κ isotype control (5 µg/ml, A1101-200, Bio Vision), for 12 h. After overnight culture, the cells were subjected to phenotypic analysis by flow cytometry as above.

Xiang Z., Zhou Q., Zeng H., Wang Z., Zhang H., Liu Z., Huang Q., Chang Y., Bai Q., Xia Y., Wang Y., Liu L., Zhu Y., Xu L., Dai B., Wang J., Guo J, & Xu J. (2020). Intratumoral CCR5+ neutrophils identify immunogenic subtype muscle-invasive bladder cancer with favorable prognosis and therapeutic responses. Oncoimmunology, 9(1), 1802176.

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Antibody-mediated Phagocytosis of Ovarian Cancer Cells

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Human ovarian cancer cell lines were labeled with the murlentamab anti-AMHRII antibody (3C23K or GM102^®, 10 µg/mL), 3C23K-FcKO (an isotypic control, 10 µg/mL) or 3C23K-CHO (a normal fucose form, 10 µg/mL) at 4 °C during 1 h. Opsonized tumor cells were then resuspended in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Scotland, UK), supplemented with l-glutamine, PS and 10% heat-inactivated fetal calf serum (FCS, Sigma, Salisbuty, UK) and added to the differentiated human macrophages at a 1:1 ratio.
In some experiments, anti-AMHRII antibodies were used in combination with an anti-PD-1 antibody, pembrolizumab (Selleck Chemicals, Selleck, USA) at 10 µg/mL. This antibody was added every day in the co-culture until T cells cytometry analysis.

Prat M., Salon M., Allain T., Dubreuil O., Noël G., Preisser L., Jean B., Cassard L., Lemée F., Tabah-Fish I., Pipy B., Jeannin P., Prost J.F., Barret J.M, & Coste A. (2021). Murlentamab, a Low Fucosylated Anti-Müllerian Hormone Type II Receptor (AMHRII) Antibody, Exhibits Anti-Tumor Activity through Tumor-Associated Macrophage Reprogrammation and T Cell Activation. Cancers, 13(8), 1845.

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Sarcoma Organoid Drug Screening

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Therapy screening was initiated on day 7 following organoid biofabrication. Treatments were tailored for each sarcoma subtype based on preoperative biopsies or final surgical pathology. The number of therapies and doses involved varied based on available cells. The therapies used for sarcoma organoids were doxorubicin (0.1, 1, 10 μM) [S1208, Selleckchem, Houston, TX, USA], ifosfamide (2, 20, 200 μM) [I4909, Sigma-Aldrich], temozolomide (10, 100, 1000 μM) [T2577, Sigma-Aldrich], imatinib mesylate (1, 10, 100 μM) [STI571, Selleckchem], regorafenib (0.1, 1, 10 μM) [S1178, Selleckchem], gemcitabine (1, 10, 100 μM) [G6423, Sigma-Aldrich], olaparib (0.1, 1, 10 μM) [S1060, Selleckchem], 100 nM pembrolizumab (A2005, Selleckchem,), 100 nM nivolumab (A2002, Selleckchem), and 100 nM ipilimumab (A2001, Selleckchem). Drug containing-media was added to organoid wells for 72 h, followed by viability assays.

Forsythe S.D., Sivakumar H., Erali R.A., Wajih N., Li W., Shen P., Levine E.A., Miller K.E., Skardal A, & Votanopoulos K.I. (2022). Patient-Specific Sarcoma Organoids for Personalized Translational Research: Unification of the Operating Room with Rare Cancer Research and Clinical Implications. Annals of surgical oncology, 29(12), 7354-7367.

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