Anti tim 3 clone rmt3 23

Manufactured by BioXCell

Anti-TIM-3 (clone RMT3-23) is a monoclonal antibody that specifically binds to the TIM-3 (T-cell immunoglobulin and mucin-domain containing-3) receptor. TIM-3 is an immune checkpoint receptor that plays a role in regulating T-cell responses. The core function of this product is to enable the study of TIM-3 and its role in immune system function.

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RMT3-23 (5 citations) Anti TIM-3 mAb (clone RMT3-23, (3 citations) Rat anti-mouse TIM-3 antibody (clone RMT3-23, (1 citations)

5 protocols using anti tim 3 clone rmt3 23

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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Formulating Immunotherapeutic Nanoparticles

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Dipalmitoyl phosphatidylcholine (DPPC), phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000] (DSPE-PEG-2000), and cholesterol (CHOL) were obtained from A.V.T. Pharmaceutical Co., Ltd. Company (Shanghai, China). ICG, Non-ionic surfactant Triton X-100 (TX-100), and Cell Counting Kit-8 (CCK-8) were obtained from Sigma-Aldrich. Anti-PD-1 (clone: RMP1-14) and anti-TIM-3 (clone: RMT3-23) used in vivo were obtained from Bioxcell. Antibodies against cell surface markers for flow cytometry assay were purchased from Miltenyi and Biolegend. Fetal bovine serum (FBS), DMEM, and RPMI-1640 medium were both obtained from GIBCO Life Technologies Co. Ltd., (USA).

Huang T.Y., Huang G.L., Zhang C.Y., Zhuang B.W., Liu B.X., Su L.Y., Ye J.Y., Xu M., Kuang M, & Xie X.Y. (2020). Supramolecular Photothermal Nanomedicine Mediated Distant Tumor Inhibition via PD-1 and TIM-3 Blockage. Frontiers in Chemistry, 8, 1.

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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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Antibody Treatment in Mice Bearing Tumors

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Wild-type female FVB/n mice (Charles River), with or without transplanted KB1P tumors, were injected intraperitoneally with a single dose of 200 μg anti-PD-1 (clone RMP1-14, BioXcell), anti-ICOS (clone 7E.17G9, BioXcell), or anti-TIM-3 (clone RMT3-23, BioXcell), followed by single injections of 100 μg antibody on two consecutive days. Control mice followed the same dosage regime with isotype control (Rat IgG2a or Armenian Hamster IgG, BioXcell). Mice were sacrificed 24 hours after the third antibody injection.

Edwards S.C., Hedley A., Hoevenaar W.H.M., Glauner T., Wiesheu R., Kilbey A., Shaw R., Boufea K., Batada N.N., Blyth K., Miller C., Kirschner K., & Coffelt S.B. (2021). Single-cell analysis uncovers differential regulation of lung γδ T cell subsets by the co-inhibitory molecules, PD-1 and TIM-3.

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