InVivoSIM anti-human TNFα (Infliximab Biosimilar)
Product Details
This non-therapeutic biosimilar antibody uses the same variable regions from the therapeutic antibody Infliximab making it ideal for research use. This Infliximab biosimilar reacts with human TNFα (tumor necrosis factor-alpha) a multifunctional proinflammatory cytokine. TNFα exists as a soluble 17 kDa monomer, which forms homotrimers in circulation or as a 26 kDa membrane-bound form. TNFα belongs to the TNF superfamily of cytokines and signals through its two receptors, TNFR1 and TNFR2 which can be activated by both the soluble trimeric and membrane-bound and forms of TNFα. TNFα is primarily produced by macrophages in response to foreign antigens such as bacteria (lipopolysaccharides), viruses, and parasites as well as mitogens and other cytokines but can also be expressed by monocytes, neutrophils, NK cells, CD4 T cells and some specialized dendritic cells. TNFα is known to play key roles in a wide spectrum of biological processes including immunoregulation, cell proliferation, differentiation, apoptosis, antitumor activity, inflammation, anorexia, cachexia, septic shock, hematopoiesis, and viral replication. TNFα dysregulation has been implicated in a variety of diseases, including autoimmune diseases, insulin resistance, and cancer. Infliximab blocks the interaction of TNFα with the TNFR1 (p55) and TNFR2 (p75) resulting in a down-regulation of the inflammatory response associated with autoimmune diseases.Specifications
| Isotype | Human IgG1 |
|---|---|
| Recommended Isotype Control(s) | RecombiMAb human IgG1 isotype control, anti-hen egg lysozyme |
| Recommended Dilution Buffer | InVivoPure pH 7.0 Dilution Buffer |
| Conjugation | This product is unconjugated. Conjugation is available via our Antibody Conjugation Services. |
| Immunogen | Human TNFα |
| Reported Applications |
in vitro TNFα neutralization in vitro functional assay Flow Cytometry ELISA Western Blot |
| Formulation |
PBS, pH 7.0 Contains no stabilizers or preservatives |
| Endotoxin |
<0.5EU/mg (<0.0005EU/μg) Determined by LAL gel clotting assay |
| Aggregation |
<5% Determined by SEC |
| Purity |
>95% Determined by SDS-PAGE |
| Sterility | 0.2 µm filtration |
| Production | Purified from cell culture supernatant in an animal-free facility |
| Purification | Protein A |
| RRID | AB_2894727 |
| Molecular Weight | 150 kDa |
| Murine Pathogen Tests |
Ectromelia/Mousepox Virus: Negative Hantavirus: Negative K Virus: Negative Lactate Dehydrogenase-Elevating Virus: Negative Lymphocytic Choriomeningitis virus: Negative Mouse Adenovirus: Negative Mouse Cytomegalovirus: Negative Mouse Hepatitis Virus: Negative Mouse Minute Virus: Negative Mouse Norovirus: Negative Mouse Parvovirus: Negative Mouse Rotavirus: Negative Mycoplasma Pulmonis: Negative Pneumonia Virus of Mice: Negative Polyoma Virus: Negative Reovirus Screen: Negative Sendai Virus: Negative Theiler’s Murine Encephalomyelitis: Negative |
| Storage | The antibody solution should be stored at the stock concentration at 4°C. Do not freeze. |
Recommended Products
-
Recommended Isotype Control(s)
RecombiMAb human IgG1 isotype control, anti-hen egg lysozyme
-
Recommended Dilution Buffer
InVivoPure pH 7.0 Dilution Buffer
in vitro TNFα neutralization
Wang X, Su S, Zhu Y, Cheng X, Cheng C, Chen L, Lei A, Zhang L, Xu Y, Ye D, Zhang Y, Li W, Zhang J. (2023). "Metabolic Reprogramming via ACOD1 depletion enhances function of human induced pluripotent stem cell-derived CAR-macrophages in solid tumors" Nat Commun 14(1):5778. PubMed
The pro-inflammatory state of macrophages, underpinned by their metabolic condition, is essentially affecting their capacity of combating tumor cells. Here we find, via a pooled metabolic gene knockout CRISPR screen that KEAP1 and ACOD1 are strong regulators of the pro-inflammatory state in macrophages. We show that ACOD1 knockout macrophages, generated in our induced pluripotent stem cell-derived CAR-macrophage (CAR-iMAC) platform, are strongly and persistently polarized toward the pro-inflammatory state, which manifests in increased reactive oxygen species (ROS) production, more potent phagocytosis and enhanced cytotoxic functions against cancer cells in vitro. In ovarian or pancreatic cancer mouse models, ACOD1-depleted CAR-iMACs exhibit enhanced capacity in repressing tumors, leading to increased survival. In addition, combining ACOD1-depleted CAR-iMACs with immune checkpoint inhibitors (ICI), such as anti-CD47 or anti-PD1 antibodies, result in even stronger tumor suppressing effect. Mechanistically, the depletion of ACOD1 reduces levels of the immuno-metabolite itaconate, allowing KEAP1 to prevent NRF2 from entering the nucleus to activate an anti-inflammatory program. This study thus lays down the proof of principle for targeting ACOD1 in myeloid cells for cancer immunotherapy and introduces metabolically engineered human iPSC-derived CAR-iMACs cells with enhanced polarization and anti-tumor functions in adoptive cell transfer therapies.
in vitro functional assay
Di Ianni A, Fraone T, Balestra P, Cowan K, Riccardi Sirtori F, Barbero L. (2023). "Assessing MAPPs assay as a tool to predict the immunogenicity potential of protein therapeutics" Life Sci Alliance 7(1):e202302095. PubMed
MHC-II-associated peptide proteomics (MAPPs) is a mass spectrometry-based (MS) method to identify naturally presented MHC-II-associated peptides that could elicit CD4+T cell activation. MAPPs assay is considered one of the assays that better characterize the safety of biotherapeutics by driving the selection of the best candidates concerning their immunogenicity risk. However, there is little knowledge about the impact of bead material on the recovery of MHC-II MS-eluted ligands in MAPPs assays. Here, we firstly describe a robust MAPPs protocol by implementing streptavidin magnetic beads for the isolation of these peptides instead of commonly used NHS-activated beads. Moreover, we assessed the impact of the cell medium used for cell cultures on the morphology and recovery of the in vitro-generated APCs, and its potential implications in the amount of MHC-II isolated peptides. We also described an example of a MAPPs assay application to investigate drug-induced immunogenicity of two bispecific antibodies and compared them with monospecific trastuzumab IgG1 control. This work highlighted the importance of MAPPs in the preclinical in vitro strategy to mitigate the immunogenicity risk of biotherapeutics.
in vitro functional assay
Lee DH, Ahn H, Sim HI, Choi E, Choi S, Jo Y, Yun B, Song HK, Oh SJ, Denda-Nagai K, Park CS, Irimura T, Park Y, Jin HS. (2023). "A CRISPR activation screen identifies MUC-21 as critical for resistance to NK and T cell-mediated cytotoxicity" J Exp Clin Cancer Res 42(1):272. PubMed
Background: Immunotherapy has significantly advanced cancer treatments, but many patients do not respond to it, partly due to immunosuppressive mechanisms used by tumor cells. These cells employ immunosuppressive ligands to evade detection and elimination by the immune system. Therefore, the discovery and characterization of novel immunosuppressive ligands that facilitate immune evasion are crucial for developing more potent anti-cancer therapies. Methods: We conducted gain-of-function screens using a CRISPRa (CRISPR activation) library that covered the entire human transmembrane sub-genome to identify surface molecules capable of hindering NK-mediated cytotoxicity. The immunosuppressive role and mechanism of MUC21 were validated using NK and T cell mediated cytotoxicity assays. Bioinformatics tools were employed to assess the clinical implications of mucin-21 (MUC21) in cancer cell immunity. Results: Our genetic screens revealed that MUC21 expression on cancer cell surfaces inhibits both the cytotoxic activity of NK cells and antibody-dependent cellular cytotoxicity, but not affecting complement-dependent cytotoxicity. Additionally, MUC21 expression hinders T cell activation by impeding antigen recognition, thereby diminishing the effectiveness of the immune checkpoint inhibitor, anti-PD-L1. Moreover, MUC21 expression suppress the antitumor function of both CAR-T cells and CAR-NK cells. Mechanistically, MUC21 facilitates immune evasion by creating steric hindrance, preventing interactions between cancer and immune cells. Bioinformatics analysis revealed elevated MUC21 expression in lung cancer, which correlated with reduced infiltration and activation of cytotoxic immune cells. Intriguingly, MUC21 expression was higher in non-small cell lung cancer (NSCLC) tumors that were non-responsive to anti-PD-(L)1 treatment compared to responsive tumors. Conclusions: These findings indicate that surface MUC21 serves as a potent immunosuppressive ligand, shielding cancer cells from NK and CD8+T cell attacks. This suggests that inhibiting MUC21 could be a promising strategy to improve cancer immunotherapy.
in vitro TNFα neutralization
Zheng N, Fang J, Xue G, Wang Z, Li X, Zhou M, Jin G, Rahman MM, McFadden G, Lu Y. (2022). "Induction of tumor cell autosis by myxoma virus-infected CAR-T and TCR-T cells to overcome primary and acquired resistance" Cancer Cell 40(9):973-985.e7. PubMed
Cytotoxicity of tumor-specific T cells requires tumor cell-to-T cell contact-dependent induction of classic tumor cell apoptosis and pyroptosis. However, this may not trigger sufficient primary responses of solid tumors to adoptive cell therapy or prevent tumor antigen escape-mediated acquired resistance. Here we test myxoma virus (MYXV)-infected tumor-specific T (TMYXV) cells expressing chimeric antigen receptor (CAR) or T cell receptor (TCR), which systemically deliver MYXV into solid tumors to overcome primary resistance. In addition to T cell-induced apoptosis and pyroptosis, tumor eradication by CAR/TCR-TMYXV cells is also attributed to tumor cell autosis induction, a special type of cell death. Mechanistically, T cell-derived interferon γ (IFNγ)-protein kinase B (AKT) signaling synergizes with MYXV-induced M-T5-SKP-1-VPS34 signaling to trigger robust tumor cell autosis. CAR/TCR-TMYXV-elicited autosis functions as a type of potent bystander killing to restrain antigen escape. We uncover an unexpected synergy between T cells and MYXV to bolster solid tumor cell autosis that reinforces tumor clearance.
- Homo sapiens (Human),
- Immunology and Microbiology
Assessing MAPPs assay as a tool to predict the immunogenicity potential of protein therapeutics.
In Life Science Alliance on 1 January 2024 by Di Ianni, A., Fraone, T., et al.
PubMed
MHC-II-associated peptide proteomics (MAPPs) is a mass spectrometry-based (MS) method to identify naturally presented MHC-II-associated peptides that could elicit CD4+T cell activation. MAPPs assay is considered one of the assays that better characterize the safety of biotherapeutics by driving the selection of the best candidates concerning their immunogenicity risk. However, there is little knowledge about the impact of bead material on the recovery of MHC-II MS-eluted ligands in MAPPs assays. Here, we firstly describe a robust MAPPs protocol by implementing streptavidin magnetic beads for the isolation of these peptides instead of commonly used NHS-activated beads. Moreover, we assessed the impact of the cell medium used for cell cultures on the morphology and recovery of the in vitro-generated APCs, and its potential implications in the amount of MHC-II isolated peptides. We also described an example of a MAPPs assay application to investigate drug-induced immunogenicity of two bispecific antibodies and compared them with monospecific trastuzumab IgG1 control. This work highlighted the importance of MAPPs in the preclinical in vitro strategy to mitigate the immunogenicity risk of biotherapeutics. © 2023 Di Ianni et al.
- Biochemistry and Molecular biology,
- Cancer Research,
- Cell Biology,
- Stem Cells and Developmental Biology
Metabolic Reprogramming via ACOD1 depletion enhances function of human induced pluripotent stem cell-derived CAR-macrophages in solid tumors.
In Nature Communications on 18 September 2023 by Wang, X., Su, S., et al.
PubMed
The pro-inflammatory state of macrophages, underpinned by their metabolic condition, is essentially affecting their capacity of combating tumor cells. Here we find, via a pooled metabolic gene knockout CRISPR screen that KEAP1 and ACOD1 are strong regulators of the pro-inflammatory state in macrophages. We show that ACOD1 knockout macrophages, generated in our induced pluripotent stem cell-derived CAR-macrophage (CAR-iMAC) platform, are strongly and persistently polarized toward the pro-inflammatory state, which manifests in increased reactive oxygen species (ROS) production, more potent phagocytosis and enhanced cytotoxic functions against cancer cells in vitro. In ovarian or pancreatic cancer mouse models, ACOD1-depleted CAR-iMACs exhibit enhanced capacity in repressing tumors, leading to increased survival. In addition, combining ACOD1-depleted CAR-iMACs with immune checkpoint inhibitors (ICI), such as anti-CD47 or anti-PD1 antibodies, result in even stronger tumor suppressing effect. Mechanistically, the depletion of ACOD1 reduces levels of the immuno-metabolite itaconate, allowing KEAP1 to prevent NRF2 from entering the nucleus to activate an anti-inflammatory program. This study thus lays down the proof of principle for targeting ACOD1 in myeloid cells for cancer immunotherapy and introduces metabolically engineered human iPSC-derived CAR-iMACs cells with enhanced polarization and anti-tumor functions in adoptive cell transfer therapies. © 2023. Springer Nature Limited.
- Cancer Research,
- Immunology and Microbiology
Induction of tumor cell autosis by myxoma virus-infected CAR-T and TCR-T cells to overcome primary and acquired resistance.
In Cancer Cell on 12 September 2022 by Zheng, N., Fang, J., et al.
PubMed
Cytotoxicity of tumor-specific T cells requires tumor cell-to-T cell contact-dependent induction of classic tumor cell apoptosis and pyroptosis. However, this may not trigger sufficient primary responses of solid tumors to adoptive cell therapy or prevent tumor antigen escape-mediated acquired resistance. Here we test myxoma virus (MYXV)-infected tumor-specific T (TMYXV) cells expressing chimeric antigen receptor (CAR) or T cell receptor (TCR), which systemically deliver MYXV into solid tumors to overcome primary resistance. In addition to T cell-induced apoptosis and pyroptosis, tumor eradication by CAR/TCR-TMYXV cells is also attributed to tumor cell autosis induction, a special type of cell death. Mechanistically, T cell-derived interferon γ (IFNγ)-protein kinase B (AKT) signaling synergizes with MYXV-induced M-T5-SKP-1-VPS34 signaling to trigger robust tumor cell autosis. CAR/TCR-TMYXV-elicited autosis functions as a type of potent bystander killing to restrain antigen escape. We uncover an unexpected synergy between T cells and MYXV to bolster solid tumor cell autosis that reinforces tumor clearance. Copyright © 2022 Elsevier Inc. All rights reserved.
- Immunology and Microbiology
Assessing Major Histocompatibility Complex-Associated Peptide Proteomics assay as a tool to predict immunogenicity potential of protein therapeutics and antigens
Preprint on Research Square on 19 August 2022 by Barbero, L., Ianni, A. D., et al.
PubMed
Major histocompatibility complex-II (MHC-II)-Associated Peptide Proteomics (MAPPs) is a mass spectrometry-based approach, to identify and relatively quantitate naturally processed and presented MHC-II-associated peptides, that could potentially activate CD4 + T cells and elicit a patient immune response against protein therapeutics in the preclinical development phase. Methods to identify these peptide antigens are critical to the development of new vaccines. Conversely, it is critical to bring safer new biological entities (NBEs) as drug candidates into clinical trials, with a reduced risk of triggering an adaptive immune response that could hamper a therapeutic outcome. Here, we describe a robust protocol for the identification of MHCII-bound peptides from Peripheral Blood Mononuclear Cells (PBMCs) of healthy donors, using nano-ultra-performance liquid chromatography coupled to high-resolution mass spectrometry (nUHPLC–MS/MS) and recent improvements in methods for isolation of these peptides.
