human cd80 fc  (R&D Systems)

Journal: Cancer Immunology Research

Article Title: FcγRIIB Is an Immune Checkpoint Limiting the Activity of Treg-Targeting Antibodies in the Tumor Microenvironment

doi: 10.1158/2326-6066.CIR-23-0389

Figure Lengend Snippet: A humanized mouse model to study the Fc effector function of human anti–CTLA-4. A, Schematic drawing describing the generation of humanized CTLA-4/FcγR mice. The genotypes of the mouse strains used for the crossing are presented. B and C, Flow cytometric analysis of CTLA-4 in CD45 + CD3 + T cells isolated from spleens and tumors from humanized CTLA-4/FcγR mice. B, Representative dot plots illustrating the gating strategy for identification of CD8 + T cells, FOXP3 – conventional CD4 + T cells (Tconv) and FOXP3 + CD4 + Tregs in MC38 tumors from humanized CTLA-4/FcγR mice. C, Levels of expression of mouse CTLA-4 (mCTLA-4) and human CTLA-4 (hCTLA-4) in the indicated T-cell subsets in spleens (S) and tumors (T) from WT (left, n = 4) and humanized CTLA-4/FcγR mice (right, n = 6) bearing MC38 tumors. Representative histograms and quantification of the mean fluorescence intensity (MFI) ± SEM are shown. Dotted lines indicate Fluorescence Minus One (FMO) controls. Each symbol represents an individual mouse and are from one experiment. D, Flow cytometric analysis of human FcγRs in CD45 + immune cells isolated from spleens and tumors from humanized CTLA-4/FcγR mice bearing MC38 tumors. Representative histograms from one experiment are shown. Dotted lines indicate FMO controls. E, Schema outlining the composition of the recombinant human anti–CTLA-4 antibodies used in the study. The Fab and Fc regions used for the generation of recombinant ipilimumab (top) and tremelimumab (bottom) antibodies are described. F, Evaluation of the binding of the recombinant ipilimumab (left) and tremelimumab (right) antibodies to hCTLA-4 by surface plasmon resonance. The dissociation constant (K D ) of each antibody is shown. RU, response units. Data are from one experiment. G, Competitive ELISA evaluating the capacity of recombinant hIgG2 ipilimumab (clone 10D1) and hIgG2 tremelimumab (clone 1121) to block the interaction between hCTLA-4 and human B7.1 (CD80). Data indicate means for triplicate wells and are from one experiment. Panels A and E were created with BioRender.com.

Article Snippet: Binding specificity, affinity, and blocking activity of human anti–CTLA-4 were determined by ELISA using recombinant human B7.1 (#10698-HCCH, Sino Biological), recombinant human CTLA-4 (#11159-HNAH, Sino Biological), and recombinant human FcγRs (human FCGR2A #10374-H08H, human FCGR2B #10259-H08H, human FCGR3A #10389-H08H, Sino Biological).

Techniques: Isolation, Expressing, Fluorescence, Recombinant, Binding Assay, SPR Assay, Competitive ELISA, Blocking Assay

Journal: Journal for immunotherapy of cancer

Article Title: XTX101, a tumor-activated, Fc-enhanced anti-CTLA-4 monoclonal antibody, demonstrates tumor-growth inhibition and tumor-selective pharmacodynamics in mouse models of cancer.

doi: 10.1136/jitc-2023-007785

Figure Lengend Snippet: Figure 4 In vitro function of XTX101. (A,B) In vitro inhibition of human CTLA-4 binding to CD80 (A) and CD86 (B) by XTX100 (black), XTX101 (red), and activated XTX101 (blue), as assessed by ELISA. (C) In vitro activity of intact (red) and activated XTX101 (blue) compared with XTX100 (black) in antibody-dependent cellular cytotoxicity (ADCC) reporter bioassay. (D) IL-2 production of SEB-stimulated human peripheral blood mononuclear cells (PBMCs) incubated with XTX100 (black), XTX101 (red), and activated XTX101 (blue), as measured by ELISA. Fold-change from isotype at highest mAb concentration is reported. ND, not determined.

Article Snippet: Serial dilutions of test articles were added to the washed ELISA plates followed by addition of a 2.6 μg/ mL solution of recombinant human CD80 or CD86 (9050- B1- 100 or 9090- B2- 100, R&D Systems).

Techniques: In Vitro, Inhibition, Binding Assay, Enzyme-linked Immunosorbent Assay, Activity Assay, Bioassay, Incubation, Concentration Assay