Recombinant Mouse CD27 Antigen, Fc Chimera

• Cat.No.: Cd27-3258M
• Product Overview: Recombinant mouse Cd27 protein (Met 1-Ile 189), fused with the Fc region of human IgG1 at the C-terminus, was expressed inHuman Cells.

Specification

• Species: Mouse
• Source: Human Cells
• Tag: Fc
• Protein Length: 1-189 a.a.
• Description: Mouse CD27, also known as TNFRSF7, is a member of the tumor necrosis factor receptor (TNFR) superfamily characterized by the presence of a Cys-knot motif in the extracellular region, which occurs three times in CD27. CD27 is a type I transmembrane glycoprotein existing as disulfide-linked homodimer, and has been shown to play roles in lymphoid proliferation, differentiation, and apoptosis. CD27 has been defined as a T and B cell co-stimulatory molecule, and its activity is governed by the transient availability of its TNF-like ligand CD70 on lymphocytes and dendritic cells. In man and mouse, CD27 is restricted to lymphoid cells and present on the great majority of naive CD4+ and CD8+ T cells.
• Molecular Mass: The recombinant mouse CD27/Fc is a disulfide-linked homodimer after removal of the signal peptide. The reduced monomer consists of 410 amino acids and has a predicted molecular mass of 46.2 kDa. In SDS-PAGE under reducing conditions, the apparent molecular mass of rm CD27/Fc monomer is approximately 60-65 kDa due to glycosylation.
• Predicted N Terminal: Thr 21.
• Purity: > 94 % as determined by SDS-PAGE.
• Formulation: Lyophilized from sterile PBS , pH 7.4.
• Endotoxin: < 1.0 EU per μg of the protein as determined by the LAL method.
• Storage And Stability: Samples are stable for up to twelve months from date of receipt at -70℃. It is recommended that the protein be aliquoted for optimal storage. Avoid repeated freeze-thaw cycles.

Gene Information

• Gene Name: Cd27 CD27 antigen [ Mus musculus ]
• Synonyms: Cd27; CD27 antigen; S152; Tp55; Tnfrsf7; CD27 antigen; CD antigen 27; CD27L receptor; OTTMUSP00000029268; OTTMUSP00000029269; OTTMUSP00000029270; T-cell activation antigen CD27; tumor necrosis factor receptor superfamily member 7; tumor necrosis factor receptor superfamily, member 7
• Gene ID: 21940
• mRNA Refseq: NM_001033126
• Protein Refseq: NP_001028298
• UniProt ID: P41272
• Chromosome Location: 6 F3; 6 60.35 cM
• Pathway: Cytokine-cytokine receptor interaction
• Function: protein binding; receptor activity

Citation

γ-Secretase inhibition increases efficacy of BCMA-specific chimeric antigen receptor T cells in multiple myeloma

Journal: Blood    PubMed ID: 31558469    Data: 2020/11/7

Authors: Margot J. Pont, Tyler Hill, Stanley R. Riddell

Article Snippet:T cells were stained with biotin-conjugated anti-EGFR monoclonal antibody (mAb; ImClone Systems Incorporated), recombinant BCMA/Fc-allophycocyanin (APC) (Creative BioMart), and mAbs specific for CD4 (RPA-T4) and CD8 (RPA-T8).. Cell lines and CD138 + enriched BMAs were stained with mAbs specific for BCMA (19F2), CD138 (MI15), CD45 (HI30), CD19 (HIB19), propidium iodide (Invitrogen), or corresponding isotype controls.Cell lines and CD138 + enriched BMAs were stained with mAbs specific for BCMA (19F2), CD138 (MI15), CD45 (HI30), CD19 (HIB19), propidium iodide (Invitrogen), or corresponding isotype controls.

BCMA CAR design and optimization. (A) Schematic of CAR constructs containing the C11 scFv in VH/VL or VL/VH configuration (HL or LH), 4-1BB, or CD28 costimulatory domain (costim), CD3ζ signaling domain, T2A ribosomal skip sequence, and EGFRt. The previously described BCMA-2 CAR40 is also depicted, and contains a CD8α hinge and transmembrane (TM) domain, and a CD28 costimulatory domain. (B) CD8+ T cells were transduced with C11/HL and C11/LH 4-1BB/CD3ζ CARs containing a long spacer sequence (IgG4 hinge-CH2-CH3NQ) and selected for EGFRt expression. IFN-γ production in supernatants of CAR T cells after stimulation with K562, K562/BCMA, and 8226 cells for 24 hours was measured by ELISA. T cells were prepared from 2 different donors and values were normalized to production of C11/LH/4-1BB/CD3ζ against K562/BCMA. (C) Schematic of various spacers used in the C11/HL/4-1BB/CD3ζ BCMA-CARs. (D) IFN-γ production in supernatants of CD8+ BCMA CAR T cells with the indicated spacers after stimulation with 8226 and U266 myeloma cell lines for 24 hours as measured by ELISA. IFN-γ was normalized to production of long spacer CAR against 8226 or U266, respectively. (E) IFN-γ (left) and IL-2 (right) in supernatants of CD4+ and CD8+ T cells expressing C11/HL/3ST/4-1BB/CD3ζ, C11/HL/3ST/CD28/CD3ζ, or BCMA-2 CARs after 24 hours coculture with the indicated BCMA? and BCMA+ target cells. Values were normalized to BCMA-2 against K562/BCMA. (F) Proliferation of CD4+ and CD8+ BCMA CAR T cells after stimulation with target cells for 72 hours analyzed by CFSE dilution assay. (G) Representative CAR expression on CD8+ BCMA CAR T cells after isolation and expansion as measured by BCMA/Fc conjugated to APC. The E:T ratio was 2:1 in all assays. Data in panels D through G are summarized (D-E) or representative (F-G) of 2 or more individual experiments with T cells prepared from different donors. Data depicted in bar graphs represent mean plus standard error of the mean (SEM). ***P < .001; as determined by 1-way ANOVA with the Dunnett posttest. ns, not significant.

sBCMA binds BCMA CAR T cells and inhibits cytokine production. (A) sBCMA concentration in plasma from MM and control patient BM measured by ELISA. Samples were stratified by the percentage of CD138+ cells of live BMMCs as measured by flow cytometry for MM patient samples. (B) Surface staining of CD4+ BCMA? and CD19 CAR T cells with APC-conjugated BCMA/Fc and anti-EGFR Ab. (C) Staining with APC-conjugated BCMA/Fc of CD4+ BCMA CAR T cells after 30 minutes preincubation with exogenous recombinant BCMA. (D) Intracellular IFN-γ (left), IL-2 (middle), or TNF (right panel) staining of CD4+ T cells after 5 hours of stimulation with 8226 or U266 (BCMA CAR T) or K562-CD19 (CD19 CAR T) in the presence and absence of exogenous recombinant BCMA at an E:T ratio of 4:1. (E) Cytolytic activity of CD4+ BCMA CAR T cells against K562/BCMA (left), U266 (middle), and 8226 (right) at varying concentrations of recombinant BCMA analyzed by a 4-hour 51Cr-release assay at an E:T ratio of 30:1 (squares) and 10:1 (circles). Data in panels B and C are representative of 2 independent experiments with T cells from different donors; data in panels D and E show summarized data with T cells from 2 different donors. Error bars represent mean plus SEM. *P < .05; as determined by 1-way ANOVA with the Tukey posttest. Data for CD8+ T cells are shown in supplemental Figure 1A.