Anti lag 3 clone c9b7w

Manufactured by BioXCell

Anti-LAG-3 (clone C9B7W) is a monoclonal antibody that specifically binds to the LAG-3 (Lymphocyte-Activation Gene 3) protein. LAG-3 is an immune checkpoint receptor expressed on the surface of activated T cells, regulatory T cells, and natural killer cells. The core function of this antibody is to recognize and bind to the LAG-3 protein.

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4 protocols using anti lag 3 clone c9b7w

Pharmacological Inhibition in Cancer Immunotherapy

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For in vivo pharmacological inhibition, gemcitabine (Selleck Chemicals) was dosed at 100mg/kg IP. SX-682 (Syntrix Pharmaceuticals) was dosed PO ad libitum (formulated concentration 714mg/kg feed); therapeutic plasma levels (range 0.5–10 μg/mL) were confirmed with this feed using LC/MS-MS. For ICT and Gr1/CD8-neutralizing antibody treatment, anti-PD1 (clone RMP1–14, BioXCell, BE0146), anti-CTLA4 (clone 9H10, BioXCell, BE0131), anti-TIM3 (clone RMT3–23, BioXCell, BE0115), anti-OX40 (clone OX-86, BioXCell, BE0031), anti-41BB (clone LOB12.3, BioXCell, BE0169), anti-LAG3 (clone C9B7W, BioXCell, BE0174), anti-CD8 (clone 2.43, BioXCell, BE0061) and anti-Gr1 (clone RB6–8C5, BioXCell, BE0075), antibodies (or their respective isotype IgG controls) were intraperitoneally administered at 200μg per injection three times per week. The duration of treatment was 4 weeks before endpoint analysis and survival analysis unless otherwise indicated.

Gulhati P., Schalck A., Jiang S., Shang X., Wu C.J., Hou P., Ruiz S.H., Soto L.S., Parra E., Ying H., Han J., Dey P., Li J., Deng P., Sei E., Maeda D.Y., Zebala J.A., Spring D.J., Kim M., Wang H., Maitra A., Moore D., Clise-Dwyer K., Wang Y.A., Navin N.E, & DePinho R.A. (2022). Targeting T cell checkpoints 41BB and LAG3 and myeloid cell CXCR1/2 results in anti-tumor immunity and durable response in pancreatic cancer. Nature cancer, 4(1), 62-80.

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Malaria Immunotherapy Protocol

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200 µg, unless otherwise indicated in figure legends, of polyclonal rat IgG (BioXcell; isotype) or anti-LAG-3 (clone C9B7W, BioXcell) and/or anti-PD-L1 (10F.9G2, BioXcell) or anti-PD-1 (RMP1-14, BioXcell) blocking Abs were injected i.v. into blood-stage parasitized mice beginning on day 9 or 12 after infection (as indicated) and every 3 days until day 15 or 18 as indicated. Antibodies which contained endotoxin levels above 3 EU/ml (Kinetic-QCL Kinetic Chromogenic LAL Assay, Lonza) were depleted of endotoxin with the ToxinEraser Endotoxin Removal kit (GenScript) following manufacturer’s instructions.

Furtado R., Chorro L., Zimmerman N., Guillen E., Spaulding E., Chin S.S., Daily J.P, & Lauvau G. (2021). Blockade of LAG-3 in PD-L1-Deficient Mice Enhances Clearance of Blood Stage Malaria Independent of Humoral Responses. Frontiers in Immunology, 11, 576743.

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Optimized ELISPOT Assay for Lung Cells

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ELISPOT assays were performed as previously described (35 (link)) with slight modifications. 5×10⁴ lung cells were added to triplicate wells. Peptides were then added (10μM final concentration) followed by inhibitory receptor blocking antibodies (10μg/mL final concentration). The following blocking antibodies were used: isotype control (clone LTF-2, Bio X-cell), anti-PD-L1 (clone 10F.9G2, Bio X-cell), anti-PD-L2 (clone TY-25, Bio X-cell), anti-PD-1 (clone J43, Bio X-cell), anti-TIM-3 (clone RMT3-23, Bio X-cell), anti-LAG-3 (clone C9B7W, Bio X-cell), anti-2B4 (clone m2B4 (B6) 458.1, Biolegend), and anti-CD48 (clone HM48-1, Bio X-cell). Plates were incubated at 37C for 42–48 hours, developed, and then counted using an ImmunoSpot Micro Analyzer (Cellular Technology Limited). The average number of spots in wells stimulated with an irrelevant peptide was subtracted from each experimental value, which was then expressed as spot forming cells (SFC) per 10⁶ lymphocytes.

Erickson J.J., Rogers M.C., Hastings A.K., Tollefson S.J, & Williams J.V. (2014). PD-1 Impairs Secondary Effector Lung CD8+ T Cells during Respiratory Virus Reinfection. Journal of immunology (Baltimore, Md. : 1950), 193(10), 5108-5117.

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In Vivo Checkpoint Blockade Protocol

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For our in vivo experiments all Abs were bought from BioXcell: anti CTLA-4 (clone 9H10), anti PD-L1 (clone 10F.92G), anti TIM-3 (clone RMT3-23), and anti LAG-3 (clone C9B7W). The concentrations of the blocking Abs and the treatment schedule (100 μg or 200 μg per dose, 1 dose every 3 days, 3 doses in total) were based on the literature [32 (link)] and personal communication with prof. Dr. Scott Fisher from the National Center for Asbestos-Related diseases (NCARD) in Perth, Australia. The IC blocking Abs were added simultaneously when used in a combination strategy.
Based on data published by Fear et al. [32 (link)] we included anti CTLA-4 as a “positive” control for our in vivo experiments, as shown in Supplementary Figure S1. This data confirmed that our AB1-HA data were reproducible and that our mouse model was responsive to ICB.

Marcq E., Van Audenaerde J.R., De Waele J., Merlin C., Pauwels P., van Meerbeeck J.P., Fisher S.A, & Smits E.L. (2021). The Search for an Interesting Partner to Combine with PD-L1 Blockade in Mesothelioma: Focus on TIM-3 and LAG-3. Cancers, 13(2), 282.

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