Methods of reducing liver pd-1-expressing cd8+ t cells using pd-1 fc fusion proteins that bind fc receptors
a fusion protein and fusion protein technology, applied in the direction of antibody medical ingredients, drug compositions, peptides, etc., can solve the problems of inability to predict the quantity and quality of an immunological response, unclear whether or how immune checkpoint expression or activity can be used to downregulate immune responses and reduce immunopathology where desired. , to achieve the effect of reducing cd8+ t cells
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[0269]i. Mice, Virus, and Infection
[0270]Six- to 8-week-old female C57BL / 6J and FVB / NJ mice were purchased from the Jackson Laboratory (Bar Harbor, Me.). FcγRIIB and FcγRIII knockout mice were kindly provided as gift from Dr. Jeffrey V. Ravetch (The Rockefeller University). Chronically infected mice were generated by first being depleted of CD4 T cells by injecting 500 μg of GK1.5 mAb intraperitoneally (i.p.) 2 days prior to infection and again on the day of infection, followed by infecting mice with 2×106 PFU of LCMV clone 13 intravenously (i.v.) via tail vein. Titers of virus were determined by plaque assay on Vero E6 cells. All experiments were conducted in accordance with National Institutes of Health and the Emory University Institutional Animal Care and Use Committee guidelines.
[0271]ii. PBMCs and Isolation of Lymphocytes
[0272]PBMCs were isolated from the blood using a density gradient technique with Histopaque-1077 (Sigma). For isolation of lymphoc...
example 2
-Fc Fusion Proteins that Deplete PD-1+ T Cells Reduce Liver Immunopathology
[0285]Existing PD-1 mAbs are designed to block the PD-1 / PD-L1 interaction while not engaging FC receptors (FcRs) or complement through the use of Fc regions that do not engage FcReceptors or complement. Consequently, they do not mediate antibody-dependent cellular cytotoxicity (ADCC) and do not deplete PD-1+ cells. By contrast, 8H3, which is a mouse anti-mouse PD-1 mAb having a wild-type mouse IgG1 Fc and described herein, depleted PD-1+ T cells and particularly in the liver to thereby reduce liver toxicity and immunopathology.
[0286]Specifically, the efficacy of the 8H3 mAb in a chronic viral infection model was determined according to the protocol depicted in FIG. 1A. The efficacy of mouse anti-mouse PD-1 mAb (clone 8H3) during chronic viral infection in the spleen, liver, and lung was examined. Chronically infected mice (>40 days post-infection) were treated with 8H3 mAb every 3 days for 2 weeks. In mouse s...
example 3
cific T Cell Depletion and Viral Titer Results are Mediated by the Fc Region of 8H3
[0287]It was further determined that the liver-specific T cell depletion and viral titer results were mediated by the Fc region of 8H3. Mutation of the aspartic acid residue at position 265 of the wild type mouse IgG1 Fc region to an alanine (D265A) results in loss of binding affinity between the Fc region and FcRs (Clynes et al. (2000) Nat. Med. 6:443-446; Shields et al. (2001) J. Biol. Chem. 276:6591-6604; Nimmerjahn et al. (2005) Immunity 23:41-51; Baudino et al. (2008) J. Immunol. 181:6664-6669). The efficacy of mouse IgG1 isotype control, wild type 8H3, or 8H3 PD-1 mAb with a mutated Fc region (i.e., D265A mIgG1), in chronic viral infection were determined according to the protocol depicted in FIG. 2A. The results confirmed that, in mouse livers, treatment with 8H3 reduced the number of LCMV-specific CD8+ T cells (FIG. 2B) and allowed for an increase in viral titer after 2 weeks of treatment (FIG...
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