LAG-3 Antibody (17B4) [FITC]

Flow Cytometry: LAG-3 Antibody (17B4) [FITC] [NBP1-97665] - LAG-3 staining in resting and PHA activated lymphocytes. Image from verified customer review.

Flow Cytometry: LAG-3 Antibody (17B4) [FITC] [NBP1-97665] - Tumor infiltrating lymphocytes (TILs) express LAG-3 (detected using LAG-3 (human), mAb (17B4). Method: Freshly dissociated single cell suspensions of renal cell carcinoma TILs are incubated with LAG-3 (human), mAb (17B4) (FITC) (5ug/ml) and anti-MHC Class II molecules (PE) for 30 min. and washed twice in saline buffer. Additional staining with anti-CD3 allowed a gate analysis of total T cells. The LAG-3 and MHC II profiles of CD3+-gated cells for 3 patients are shown [5].

Flow Cytometry: LAG-3 Antibody (17B4) [FITC] [NBP1-97665] - Specific inhibition of 17B4 staining.Method: LAG-3 (human), mAb (17B4) (FITC) (10ug/ml) (is preincubated with a specific peptide epitope (208b) or a control tetanus toxoid (TT) peptide at different molarities prior to staining of TILs. Stained cells are then analyzed by FC.

Flow Cytometry: LAG-3 Antibody (17B4) [FITC] [NBP1-97665] - LAG-3 expression on activated human peripheral blood mononuclear cells (PBMC) detected with LAG-3 (human), mAb (17B4) (FITC).Method: T lymphocytes from human PBMC are stimulated with 1ug/ml of PHA for three days. Then, after seven days of culture, 3x10^6 three-days PHA-activated human PBMC are treated with LAG-3 (human), mAb (17B4) (FITC) or FITC coupled isotype-matched (IgG1) control MAb (used at a saturating dilution of 1:800 and 1:150 respectively) for 30 min. at 4C in RPMI 1640 and washed twice with 1x PBS. Stained cells are then analysed by FC [4].

Flow Cytometry: LAG-3 Antibody (17B4) [FITC] [NBP1-97665] - Expression of LAG-3 on CD4+ and CD8+ subpopulations of tumour infiltrating lymphocytes (TILs) detected with LAG-3 (human), mAb (17B4) (FITC). Method: TILs from a dissociated renal cell carcinoma sample, stained with 5ug/ml LAG-3 (human), mAb (17B4) (FITC) and FITC-coupled anti-CD4 or -CD8, are analyzed by a two-colour FACS analysis. Additional staining with anti-CD3 allowed a gate analysis of total T cells. Values indicate percentages in each quadrant [5].

Product Discontinued

LAG-3 Antibody (17B4) [FITC] Summary

• Immunogen: This LAG-3 Antibody (17B4) [FITC] was prepared from a synthetic peptide corresponding to 30 aa (GPPAAAPGHPLAPGPHPAAPSSWGPRPRRY) from the first N-terminal D1 domain of human LAG-3 (lymphocyte activation gene-3).
• Specificity: LAG-3 Antibody (17B4) [FITC] recognizes the 30 aa extra-loop of the first N-terminal D1 domain of human LAG-3.
• Isotype: IgG1
• Clonality: Monoclonal
• Host: Mouse
• Gene: LAG3
• Purity: Protein A or G purified

Applications/Dilutions

• Dilutions:
  • Flow Cytometry 1:10-1:1000
  • Immunohistochemistry
• Application Notes: Use in IHC reported in scientific literature (PMID: 30384489).
• Theoretical MW: 57.5 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.

Reactivity Notes

Use in Primate reported in scientific literature (PMID:32284611).

Packaging, Storage & Formulations

• Storage: Store at 4C in the dark.
• Buffer: PBS, 50 mM Tris/HCl, and 1.0% BSA
• Preservative: 0.02% Sodium Azide
• Purity: Protein A or G purified

Alternate Names for LAG-3 Antibody (17B4) [FITC]

• CD223 antigen
• CD223
• LAG3
• LAG-3
• lymphocyte activating 3
• lymphocyte activation gene 3 protein
• lymphocyte-activation gene 3
• Secreted lymphocyte activation gene 3 protein
• sLAG-3

Background

Lymphocyte activation gene-3 (LAG-3), also referred to as CD233, is a type I transmembrane protein with a theoretical molecular weight of 70 kDa that is a member of the immunoglobulin superfamily (IgSF) (1, 2). Human LAG-3 cDNA encodes 525 amino acids (aa) that includes a 28 aa signal sequence, a 422 aa extracellular domain (ECD) with four Ig-like domains (D1-D4), a transmembrane region and a highly charged cytoplasmic region. Within the ECD, human LAG-3 shares 70%, 67%, 76%, and 73% aa sequence identity with mouse, rat, porcine, and bovine LAG-3, respectively. The extracellular region of LAG-3 and the CD4 co-receptor share ~20% aa sequence homology but are structurally similar and both bind to major histocompatibility complex class II (MHCII) on antigen-presenting cells (APCs), although LAG-3 has much higher affinity (1, 3). LAG-3 is highly expressed in the lymph node, spleen, ovary, and appendix while expressed at a lower level in a variety of other tissues. More specifically, LAG-3 is expressed on activated CD4+ and CD8+ T cells, natural killer T (NKT) cells, natural killer (NK) cells, plasmacytoid dendritic cells (pDCs), and regulatory T cells (Tregs), but not on naive, or resting, T cells (1, 3).

As mentioned above, LAG-3 binds to MHCII and this occurs via a proline-rich amino acid loop in D1 (1, 3). Another unique feature of LAG-3 is the longer connecting peptide region between the D4 and the transmembrane, which is acted upon and cleaved by metalloproteinases a disintegrin and metallopeptidase domain (ADAM) 10 and ADAM17 to generate a soluble 54 kDa form of LAG-3 (sLAG-3) (1, 3). The interaction of LAG-3 with MHCII prevents the MHC molecule from binding to a T-cell receptor (TCR) or CD4, thereby functioning in an inhibitory role and suppressing the TCR signal (4). When LAG-3 crosslinks with the TCR/CD3 complex, it causes reduced T-cell proliferation and cytokine secretion (4). This negative regulation is important in controlling autoimmunity as one study found Lag3-/- NOD (non-obese diabetic) mice had accelerated diabetes onset and increased T-cell infiltration into islet cells (5). On the other hand, besides being a negative regulator of T-cells, LAG-3 binding to MHCII molecules on APCs induces dendritic cell maturation and cytokine secretion by monocytes (5, 6). In addition to MHCII, other reported ligands for LAG-3 includes fibrinogen-like protein 1 (FGL1), liver endothelial cell lectin (lSECtin), galectin-3 (Gal-3), and alpha-synuclein fibrils (1). Gal-3, for instance, is expressed on stromal cells and CD8+ T-cells in the tumor microenvironment and the interaction with LAG-3 was shown to be crucial for the suppression of secreted cytokine IFN-gamma and may control anti-tumor immune responses (1, 5). Interestingly, a mouse model of Parkinson's disease revealed LAG-3 binding to alpha-synuclein fibrils in the central nervous system, contributing to its pathogenesis (1, 5).

Recent cancer immunotherapeutic approaches have focused on inhibitory receptors such as LAG-3 to revive expression of cytotoxic T-cells to attack tumors (6). LAG-3 has been shown to be co-expressed and have synergy with another immune-checkpoint molecule called programmed-death 1 (PD-1) (1, 4, 5, 6). In a mouse model of colon adenocarcinoma LAG3 blockade alone was largely ineffective, however co-blockade of LAG-3 and PD-1 limited tumor growth and resulted in tumor clearance in 80% of mice, compared to 40% with PD-1 blockade alone (5). Additionally, in a model of fibrosarcoma the LAG-3/PD-1 duel blockade increased survival and the percentage of tumor-free mice (5). Analysis of a variety of human tumor samples (e.g. melanoma, colon cancer, head and neck squamous cell carcinoma) also suggest that LAG3 alone and combinatorial treatment with PD-1 may be a good target for treatment (1, 3-6). To date there are over 10 different agents targeting LAG-3 in clinical trials for cancer either as an anti-LAG-3 blocking antibody monotherapy or as a combination antagonist bispecific antibody, primarily with PD-1 (1, 3-6).

Alternative names for LAG-3 includes 17b4 lag3, 17b4 neutralizing, 17b4, CD223, FDC, LAG-3 17b4, LAG-3 blocking, and LAG3.

References

1. Maruhashi, T., Sugiura, D., Okazaki, I. M., & Okazaki, T. (2020). LAG-3: from molecular functions to clinical applications. Journal for Immunotherapy of Cancer, 8(2), e001014. https://doi.org/10.1136/jitc-2020-001014

2. Triebel, F., Jitsukawa, S., Baixeras, E., Roman-Roman, S., Genevee, C., Viegas-Pequignot, E., & Hercend, T. (1990). LAG-3, a novel lymphocyte activation gene closely related to CD4. The Journal of experimental medicine, 171(5), 1393-1405. https://doi.org/10.1084/jem.171.5.1393

3. Ruffo, E., Wu, R. C., Bruno, T. C., Workman, C. J., & Vignali, D. (2019). Lymphocyte-activation gene 3 (LAG3): The next immune checkpoint receptor. Seminars in immunology, 42, 101305. https://doi.org/10.1016/j.smim.2019.101305

4. Long, L., Zhang, X., Chen, F., Pan, Q., Phiphatwatchara, P., Zeng, Y., & Chen, H. (2018). The promising immune checkpoint LAG-3: from tumor microenvironment to cancer immunotherapy. Genes & cancer, 9(5-6), 176-189.

5. Andrews, L. P., Marciscano, A. E., Drake, C. G., & Vignali, D. A. (2017). LAG3 (CD223) as a cancer immunotherapy target. Immunological reviews, 276(1), 80-96. https://doi.org/10.1111/imr.12519

6. Goldberg, M. V., & Drake, C. G. (2011). LAG-3 in Cancer Immunotherapy. Current topics in microbiology and immunology, 344, 269-278. https://doi.org/10.1007/82_2010_114

Limitations

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Primary Antibodies are guaranteed for 1 year from date of receipt.

Publications for LAG-3 Antibody (NBP1-97665)(11)

Publications using NBP1-97665

• Harper J, Gordon S, Chan C et Al. CTLA-4 and PD-1 dual blockade induces SIV reactivation without control of rebound after antiretroviral therapy interruption Nat Med. [PMID: 32284611] (FLOW, Primate)
• Pericart S, Tosolini M, Gravelle P et al. Profiling Immune Escape in Hodgkin's and Diffuse large B-Cell Lymphomas Using the Transcriptome and Immunostaining Cancers (Basel) 2018-10-31 [PMID: 30384489] (IF/IHC, Human)
  
  Details:
  Citation using the FITC form of this antibody.
• Casati C, Camisaschi C, Novellino L et al. Human lymphocyte activation gene-3 molecules expressed by activated T cells deliver costimulation signal for dendritic cell activation. J Immunol. 2008 Mar. [PMID: 18322184]
• Triebel F, Hacene K, Pichon MF et al. A soluble lymphocyte activation gene-3 (sLAG-3) protein as a prognostic factor in human breast cancer expressing estrogen or progesterone receptors. Cancer Lett. 2006 Apr. [PMID: 15946792]
• Macon-Lemaitre L, Triebel F. The negative regulatory function of the lymphocyte-activation gene-3 co-receptor (CD223) on human T cells. Immunology 2005-05-31 [PMID: 15885122]
  
  Details:
  Citation using the FITC format of this antibody.
• Di Carlo E, Cappello P, Sorrentino C et al. Immunological mechanisms elicited at the tumour site by lymphocyte activation gene-3 (LAG-3) versus IL-12: sharing a common Th1 anti-tumour immune pathway. J Pathol. 2005-01-01 [PMID: 15586367]
• Demeure CE, Wolfers J, Martin-Garcia N et al. T Lymphocytes infiltrating various tumour types express the MHC class II ligand lymphocyte activation gene-3 (LAG-3): role of LAG-3/MHC class II interactions in cell-cell contacts. Eur J Cancer. 2001 Sep. [PMID: 11527700]
• Hannier S, Tournier M, Bismuth G et al. CD3/TCR complex-associated lymphocyte activation gene-3 molecules inhibit CD3/TCR signaling. J Immunol. 1998 Oct. [PMID: 9780176]
• Huard B, Mastrangeli R, Prigent P et al. Characterization of the major histocompatibility complex class II binding site on LAG-3 protein. Proc Natl Acad Sci U S A. 1997 May [PMID: 9159144]
• Huard B, Gaulard P, Faure F et al. Cellular expression and tissue distribution of the human LAG-3-encoded protein, an MHC class II ligand. Immunogenetics. 1994-01-01 [PMID: 7506235]
• Baixeras E, Huard B, Miossec C et al. Characterization of the lymphocyte activation gene 3-encoded protein. A new ligand for human leukocyte antigen class II antigens. J Exp Med. 1992-08-01 [PMID: 1380059]

Review for LAG-3 Antibody (NBP1-97665) (1)

Average Rating: 4

(Based on 1 review)

We have 1 review tested in 1 species: Human.

reviewed by: Verified Customer Flow Human 05/08/2014

Summary

• Application: Flow Cytometry
• Species: Human
• Lot: 10021323

Bioinformatics

• Gene Symbol: LAG3