LAG-3 Antibody (C9B7W) - Chimeric - Azide and BSA Free

Immunocytochemistry/Immunofluorescence: LAG-3 Antibody (C9B7W) - Chimeric [NBP3-09044] - Immunofluorescence analysis of paraformaldehyde fixed mouse splenocytes immobilized on Shi-fix(TM) cover-slips and stained with the chimeric rabbit IgG version of C9B7W (NBP3-09044) at 10 ug/ml followed by Alexa Fluor(R) 488 secondary antibody (2 ug/ml), showing membrane staining in subset of cells. The nuclear stain is DAPI (blue). Panels show from left-right, top-bottom NBP3-09044, DAPI, merged channels and an isotype control. The isotype control was stained with anti-Fluorescein antibody followed by Alexa Fluor(R) 488 secondary antibody.

ELISA: LAG-3 Antibody (C9B7W) - Chimeric - Azide and BSA Free [NBP3-09044] - ELISA Plate coated with recombinant mouse LAG-3 Fc-Fusion Protein at a concentration of 2 ug/ml. A 4-fold serial dilution from 5,000 ng/ml was performed using NBP3-09044. For detection, a 1:4000 dilution of HRP-labelled anti-rabbit antibody was used.

• Reactivity: Mu
• Applications: ELISA, Flow, ICC/IF, IP, B/N
• Clone: C9B7W
• Clonality: Monoclonal
• Host: Rabbit
• Conjugate: Unconjugated
• Format: Azide and BSA Free
• Concentration: 1 mg/ml

LAG-3 Antibody (C9B7W) - Chimeric - Azide and BSA Free Summary

• Description: Novus Biologicals Rabbit LAG-3 Antibody (C9B7W) - Chimeric - Azide and BSA Free (NBP3-09044) is a recombinant monoclonal antibody validated for use in ELISA, Flow, ICC/IF and IP. All Novus Biologicals antibodies are covered by our 100% guarantee.
• Additional Information: Recombinant Monoclonal Antibody
• Immunogen: This antibody was raised by immunising Lewis rats with a murine LAG-3:Ig fusion protein.
• Specificity: This antibody is specific for an epitope in the D2 domain of murine LAG-3.
• Isotype: IgG Kappa
• Clonality: Monoclonal
• Host: Rabbit
• Gene: LAG3
• Purity: Protein A purified

Applications/Dilutions

• Dilutions:
  • Block/Neutralize
  • ELISA
  • Flow Cytometry
  • Immunocytochemistry/ Immunofluorescence
  • Immunoprecipitation
• Application Notes: This chimeric rabbit antibody was made using the variable domain sequences of the original Rat IgG1 format, for improved compatibility with existing reagents, assays and techniques.
  
  This antibody has been used in flow cytometric analysis of splenocytes (Workman et al, 2005), T cell hybridomas expressing wild-type or chimeric CD4: murine LAG-3 (Workman et al, 2002) and CD4 positive regulatory T cells (Chien et al, 2017). It has also been used in immunoprecipitation analysis of murine T hybridoma 3A9 cells transduced with murine LAG-3 (Li et al, 2004). This antibody blocks LAG-3 function in vitro, inhibiting antigen-induced IL-2 production in LAG-3-expressing T cell hybridomas, but does not block LAG-3: MHC class II interaction (Workman et al, 2002). Treatment with this antibody also blocks LAG-3 function in vivo, leading to accelerated homeostatic expansion of WT T cells (Workman et al, 2005). Administration of this LAG-3-blocking antibody, in combination with PD-L1 blockade, synergistically improves the survival of lymphodepleted myeloma-bearing mice (Jing et al, 2015).

Packaging, Storage & Formulations

• Storage: Store at 4C short term. Aliquot and store at -20C long term. Avoid freeze-thaw cycles.
• Buffer: PBS
• Preservative: 0.02% Proclin 300
• Concentration: 1 mg/ml
• Purity: Protein A purified

Alternate Names for LAG-3 Antibody (C9B7W) - Chimeric - Azide and BSA Free

• 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.

Bioinformatics

• Gene Symbol: LAG3