control higg4  (Sino Biological)

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Preclinical analysis of candidate anti-human CD79 therapeutic antibodies using a humanized CD79 mouse model

doi: 10.4049/jimmunol.2101056

Figure Lengend Snippet: Anti-hCD79A treatment induces an anergic-like phenotype in B cells. (A) Surface staining of PBMCs (left) and Ramos cells (middle) with anti-hCD79A (Curly-14). Dual staining of cCD79A+/+B−/− knockin splenocytes (right) with anti-hCD79A (Curly-14) and anti-mCD79B (HM79). Gray lines indicate CD19−, ns, and B220− for PBMCs, Ramos cells, and cCD79A knockin splenocytes, respectively. (B) Binding of the humanized anti-hCD79A clinical candidate to cCD79A/cCD79B cells. Eighteen hours prior to assay, cCD79A knockin mice received 250-µg i.p. injections of either hIgG4 D265A anti-hCD79A (solid black line), hIgG4 isotype control (dashed black line), or hamster anti-mCD79B (HM79) (dashed gray line). RBC-lysed splenocytes were stained with anti-B220 and either anti-hCD79A (Curly-14) (left) or anti-hIgG4 (right). Solid gray lines represent B220−. (C) Surface expression of IgM and IgD on cCD79 B cells (B220+) 18 h after i.p injection with 250 µg of either hIgG4 D265A anti-hCD79A (black) or isotype control hIgG4 (gray). Light gray histograms show B220−. (D) Western blot analysis of global tyrosine phosphorylation. Eighteen hours prior to assay, cCD79A mice were injected i.p. with 250 µg of either anti-hCD79A D265A or hIgG4 isotype control. Splenic B cells were purified by CD43 exclusion and stimulated with 10 µg/ml F(ab′)2 goat anti-mIgM for the indicated number of minutes (left) or stimulated with 1× pervanadate for 5 min (right). Whole-cell lysates of 2 × 106 B cell (CD43−) equivalents, both stimulated and not, were separated by SDS-PAGE and transferred to PVDF membranes. Protein-transferred membranes were blotted with Abs against p-Tyr (4G10) and actin. (E) Western blot analysis of Syk phosphorylation. Eighteen hours prior to assay, cCD79A mice were injected i.p. with 250 µg of either anti-hCD79A D265A or hIgG4 isotype control. Splenic B cells were purified by CD43 exclusion and stimulated with 10 µg/ml F(ab′)2 goat anti-mIgM for the indicated number of minutes. Whole-cell lysates of 2 × 106 B cell (CD43−) equivalents, both stimulated and not, were separated by SDS-PAGE and transferred to PVDF membranes. Protein-transferred membranes were blotted with Abs against p-Syk (Y525), total Syk, and actin. A densitometric summary of p-Syk/Syk is shown on the right. (F) Flow cytometric analysis of Syk phosphorylation as a function of mBCR expression. Mice were treated as in (E) before RBC-lysed splenocytes were stimulated as above for 5 min. Fixed cells were stained with fluorescent Abs against B220 and mBCR. After permeabilization, cells were stained with anti–p-Syk (Y525). B cells were gated on equivalent mBCR expression (left) before comparing p-Syk mean fluorescence intensities (MFIs) (middle and right). Error bars show SEM. All data represents at least three independent experiments; representative data are shown. A Student t test used to evaluate statistical significance. ***p < 0.001, ****p < 0.0001.

Article Snippet: Weekly injections (250 μg, i.p.) of either hIgG4 anti-hCD79 D265A or isotype control hIgG4 (VLN3G2) (American Type Culture Collection, HB-8636) were given on days 0, 7, 14, 21, 28, 35, 42, 49, and 56.

Techniques: Staining, Knock-In, Binding Assay, Control, Expressing, Injection, Western Blot, Phospho-proteomics, Purification, SDS Page, Fluorescence

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Preclinical analysis of candidate anti-human CD79 therapeutic antibodies using a humanized CD79 mouse model

doi: 10.4049/jimmunol.2101056

Figure Lengend Snippet: Anti-hCD79A treatment induces suppression of BCR-mediated calcium mobilization. (A) BCR-mediated calcium signaling in B cells from cCD79A mice receiving a 250-µg i.p. injection of anti-hCD79A D265A or control hIgG4 24 h prior. mBCR expression (left) was measured and gated by staining with a polyclonal goat Fab anti-mIgG (H+L). Anti-hCD79A, solid line; control hIgG4, dashed line. Cells were restimulated with 1 or 10 µg/ml rat anti-mIgM (B76). The poststimulation area under the curve (AUC) was normalized to basal calcium levels (right). (B) Cells prepared as in (A) stimulated with 10 µM ionomycin. (C) BCR-mediated calcium signaling in Ramos cells after overnight in vitro incubation with 25 µg/ml hIgG4 anti-hCD79A D265A (solid line) or isotype control hIgG4 (dashed line). Cells were restimulated with either 1 or 10 µg/ml goat F(ab′)2 anti-hIgM Cµ5. (D) Flow cytometric (left and mean fluorescence intensity [MFI] bar graph) and Western blot (right and densitometry) analysis of PTEN expression in B cells from cCD79A animals receiving a 250-µg i.p. injection of either anti-hCD79A D265A or control hIgG4, 24 h prior. For flow cytometry, RBC-lysed splenocytes were fixed and permeabilized before staining with Abs against B220 and PTEN. Gray histogram represents staining isotype control Ab. hIgG4, dashed line; anti-hCD79A D265A, solid line. For Western blot, membranes were prepared as in (Fig. 3D, without BCR stimulation, before being probed with Abs against PTEN or actin. (E) hCD20-CreTAM × ROSA26-STOPflox-YFP × PTENflox/flox or hCD20-CreTAM × ROSA-26-STOPflox-YFP × PTENWT mice were given 2-mg i.p. injections of tamoxifen (TAM) on day 0. On day 7 after TAM, mice were injected i.p. with 0.25 mg of either mIgG2a D265A anti-mCD79B or control mIgG2a anti-HEL. Eighteen hours after Ab injection, B220+YFP+ splenocytes were analyzed by flow for PTEN expression and Ab coating. (F) As before, calcium was measured as a function of equal mBCR expression. n = 3 mice per group. Error bars show SEM. All data represents at least three independent experiments; representative data are shown. A Student t test was used to evaluate statistical significance. *p < 0.05, **p < 0.01, ****p < 0.0001.

Article Snippet: Weekly injections (250 μg, i.p.) of either hIgG4 anti-hCD79 D265A or isotype control hIgG4 (VLN3G2) (American Type Culture Collection, HB-8636) were given on days 0, 7, 14, 21, 28, 35, 42, 49, and 56.

Techniques: Injection, Control, Expressing, Staining, In Vitro, Incubation, Fluorescence, Western Blot, Flow Cytometry

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Preclinical analysis of candidate anti-human CD79 therapeutic antibodies using a humanized CD79 mouse model

doi: 10.4049/jimmunol.2101056

Figure Lengend Snippet: Anti-hCD79A treatment inhibits B cell immune responses. (A) ELISPOT analysis of NP-specific, IgM+ ASCs 7 d after immunization with NP59-Ficoll. Twenty-four hours prior to immunization, cCD79A mice received 250-µg i.p. injections of either anti-hCD79A D265A or control hIgG4. (B) Flow cytometric analysis of germinal center B cell induction 5 d after immunization with 0.1% SRBCs in PBS. Twenty-four hours prior to immunization, cCD79A animals received 250-µg i.p. injections of either anti-hCD79A D265A or control hIgG4. On day 5 postimmunization, RBC-lysed splenocytes were stained with Abs recognizing B220, CD95, and GL7. (C) ELISPOT analysis of NP-specific IgM+ (left) and IgG+ (right) ASCs at 16 d postimmunization with NP-OVA in alum. Twenty-four hours prior to immunization, cCD79A mice received 250-µg i.p. injections of either anti-hCD79A D265A or control hIgG4. For detection of total IgM and IgG anti-NP ASCs, plates were coated with NP19-BSA. For detection of high-affinity IgG anti-NP ASCs, plates were coated with NP2-BSA. (D) ELISPOT analysis of NP- (left) and tetanus toxoid–specific (right) IgG+ ASCs following overnight incubation with anti-hCD79A D265A. NP-specific ASCs were generated by immunizing cCD79A knockin mice with NP-OVA in alum and harvesting spleens 16 d later. RBC-lysed splenocytes (1 × 107) were put into 1-ml cultures containing 25 µg of either anti-hCD79A D265A, anti-mCD20, control hIgG4, or control mIgG2a. After 18-h incubations, cells were washed and loaded onto ELISPOT plates coated with NP19-BSA. n = 3 wells per condition. This experiment was repeated three times. PBMCs were isolated from peripheral blood obtained from subjects receiving Tdap booster vaccines 7–10 days prior. PBMCs (5 × 106) were put into 1-ml cultures containing 25 µg of either anti-hCD79A D265A, anti-hCD20, or control hIgG4. After overnight incubation, cells were washed and loaded onto tetanus toxoid–coated ELISPOT plates. n = 3 subjects whose PBMCs were incubated with anti-hCD79; n = 1 subject whose PBMCs were also incubated with anti-hCD20. Points represent individual counted wells. (E) Effects of serial, weekly treatments with hIgG4 D265A anti-hCD79A in pristane-induced development of autoantibodies (anti-chromatin IgG ELISAs). Pooled sera from aged/diseased SLE1.2.3 mice and wild-type C57BL/6 served as positive and negative controls, respectively. n = 20 male and 20 female cCD79A mice per treatment arm. Error bars show SEM. All data represents at least three independent experiments; representative data are shown. A Student t test was used to evaluate statistical significance. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: Weekly injections (250 μg, i.p.) of either hIgG4 anti-hCD79 D265A or isotype control hIgG4 (VLN3G2) (American Type Culture Collection, HB-8636) were given on days 0, 7, 14, 21, 28, 35, 42, 49, and 56.

Techniques: Enzyme-linked Immunospot, Control, Staining, Incubation, Generated, Knock-In, Isolation, Vaccines