Journal: Frontiers in Immunology

Article Title: Monoclonal Antibodies Capable of Inhibiting Complement Downstream of C5 in Multiple Species

doi: 10.3389/fimmu.2020.612402

Figure Lengend Snippet: Western blotting to detect C7 binding in serum and in TCC. (A) The human-specific mAbs 59E7 and 17E7 were used to probe WB of NHS (Hu) and Cynomolgus monkey (Mk) serum under non-reducing (NR) conditions. Secondary only control was included (2°). (B–E) The cross-species reactive mAbs 73D1 (B) , 3B11 (C) and 2H2 (D) and as control the polyclonal goat anti-C7 (E) were used to probe WB of NHS (Hu), monkey (Mk), mouse (Mo) and rat sera; purified human C7 was used as standard. All sera were run in duplicate. Polyclon3B11 (C) , 2H2 (D) , and positive control goat anti-C7 (E) . Results are representative of three independent experiments. M; protein molecular weight marker. (F–J) The novel mAbs were used to pull down complexes from activated serum; these were then run on WB under non-reduced and reduced conditions and probed with polyclonal antibodies against each of the terminal complement proteins. mAb 2H2 was used in rat (F) and human (G) serum; the other mAbs in human serum only (H–J) . The blots were cut into strips prior to probing to detect the individual terminal pathway proteins. Molecular weights used were: NR: C5, 190 kDa; C6, 105 kDa; C7, 95 kDa, C8αγ; 70 kDa; C9, 65 kDa. R: C6, 110 kDa; C7, 95 kDa; C5β, 75 kDa; C9, 70 kDa; C8α/β, 65 kDa; C8γ, 22 kDa. Results are representative of at least three analyses. M; protein molecular weight marker, 2°; secondary antibody. (K) The novel mAbs 17E7, 59E7 and 73D1 were separately immobilized on mouse IgG capture sensor chips (GE Healthcare, # BR-1008-38) and mAb 3B11 (IgM) on protein L Series S sensor chip (GE Healthcare #29-2051-38) at approximately 60 RU. Human, rat or mouse C7 was flowed in HEPES-buffered saline (HBS) in a dilution range of 66 to 8 nM and interactions with the immobilized mAbs were analyzed. Sensorgrams were collected and KDs were calculated using the Langmuir 1: 1 binding model with RI values set to zero. Representative sensorgrams for 17E7 and 59E7 binding of human C7 are shown with raw data in colored lines and fitted data in dotted line (average of 3); all binding data and analyses are included in Table 2 . The sensorgrams of clones 3B11 and 73D1 are included in Supplementary. The SPR analysis was performed in an automated manner using T200 Biacore Evaluation Software version 2 (GE Healthcare).

Article Snippet: The mAbs binding analyses were carried out on a Biacore T200 instrument (GE Healthcare); for mAb of isotype IgG, an antibody capture kit (GE Healthcare, # BR-1008-38) was used to immobilize the mAb on a CM5 sensor chip (GE Healthcare, #29-1496-03) as recommended by the manufacturer. mAb isotype IgM was immobilized on a Protein L Series S sensor chip (GE Healthcare #29-2051-38). mAb were flowed to saturate the surface, then C7, human, rat or mouse, diluted in EP-HBS (10 mM HEPES, pH 7.4, 150 mM NaCl, 0.005% surfactant P20) in the range 0 to 68 nM, flowed over the immobilized mAb.

Techniques: Western Blot, Binding Assay, Purification, Positive Control, Molecular Weight, Marker, Clone Assay, Software

Journal: Frontiers in Immunology

Article Title: Monoclonal Antibodies Capable of Inhibiting Complement Downstream of C5 in Multiple Species

doi: 10.3389/fimmu.2020.612402

Figure Lengend Snippet: Western blotting to detect C7 binding in serum and in TCC. (A) The human-specific mAbs 59E7 and 17E7 were used to probe WB of NHS (Hu) and Cynomolgus monkey (Mk) serum under non-reducing (NR) conditions. Secondary only control was included (2°). (B–E) The cross-species reactive mAbs 73D1 (B) , 3B11 (C) and 2H2 (D) and as control the polyclonal goat anti-C7 (E) were used to probe WB of NHS (Hu), monkey (Mk), mouse (Mo) and rat sera; purified human C7 was used as standard. All sera were run in duplicate. Polyclon3B11 (C) , 2H2 (D) , and positive control goat anti-C7 (E) . Results are representative of three independent experiments. M; protein molecular weight marker. (F–J) The novel mAbs were used to pull down complexes from activated serum; these were then run on WB under non-reduced and reduced conditions and probed with polyclonal antibodies against each of the terminal complement proteins. mAb 2H2 was used in rat (F) and human (G) serum; the other mAbs in human serum only (H–J) . The blots were cut into strips prior to probing to detect the individual terminal pathway proteins. Molecular weights used were: NR: C5, 190 kDa; C6, 105 kDa; C7, 95 kDa, C8αγ; 70 kDa; C9, 65 kDa. R: C6, 110 kDa; C7, 95 kDa; C5β, 75 kDa; C9, 70 kDa; C8α/β, 65 kDa; C8γ, 22 kDa. Results are representative of at least three analyses. M; protein molecular weight marker, 2°; secondary antibody. (K) The novel mAbs 17E7, 59E7 and 73D1 were separately immobilized on mouse IgG capture sensor chips (GE Healthcare, # BR-1008-38) and mAb 3B11 (IgM) on protein L Series S sensor chip (GE Healthcare #29-2051-38) at approximately 60 RU. Human, rat or mouse C7 was flowed in HEPES-buffered saline (HBS) in a dilution range of 66 to 8 nM and interactions with the immobilized mAbs were analyzed. Sensorgrams were collected and KDs were calculated using the Langmuir 1: 1 binding model with RI values set to zero. Representative sensorgrams for 17E7 and 59E7 binding of human C7 are shown with raw data in colored lines and fitted data in dotted line (average of 3); all binding data and analyses are included in Table 2 . The sensorgrams of clones 3B11 and 73D1 are included in Supplementary. The SPR analysis was performed in an automated manner using T200 Biacore Evaluation Software version 2 (GE Healthcare).

Article Snippet: The mAbs binding analyses were carried out on a Biacore T200 instrument (GE Healthcare); for mAb of isotype IgG, an antibody capture kit (GE Healthcare, # BR-1008-38) was used to immobilize the mAb on a CM5 sensor chip (GE Healthcare, #29-1496-03) as recommended by the manufacturer. mAb isotype IgM was immobilized on a Protein L Series S sensor chip (GE Healthcare #29-2051-38). mAb were flowed to saturate the surface, then C7, human, rat or mouse, diluted in EP-HBS (10 mM HEPES, pH 7.4, 150 mM NaCl, 0.005% surfactant P20) in the range 0 to 68 nM, flowed over the immobilized mAb.

Techniques: Western Blot, Binding Assay, Purification, Positive Control, Molecular Weight, Marker, Clone Assay, Software

Journal: Frontiers in Immunology

Article Title: Monoclonal Antibodies Capable of Inhibiting Complement Downstream of C5 in Multiple Species

doi: 10.3389/fimmu.2020.612402

Figure Lengend Snippet: Western blotting to detect C7 binding in serum and in TCC. (A) The human-specific mAbs 59E7 and 17E7 were used to probe WB of NHS (Hu) and Cynomolgus monkey (Mk) serum under non-reducing (NR) conditions. Secondary only control was included (2°). (B–E) The cross-species reactive mAbs 73D1 (B) , 3B11 (C) and 2H2 (D) and as control the polyclonal goat anti-C7 (E) were used to probe WB of NHS (Hu), monkey (Mk), mouse (Mo) and rat sera; purified human C7 was used as standard. All sera were run in duplicate. Polyclon3B11 (C) , 2H2 (D) , and positive control goat anti-C7 (E) . Results are representative of three independent experiments. M; protein molecular weight marker. (F–J) The novel mAbs were used to pull down complexes from activated serum; these were then run on WB under non-reduced and reduced conditions and probed with polyclonal antibodies against each of the terminal complement proteins. mAb 2H2 was used in rat (F) and human (G) serum; the other mAbs in human serum only (H–J) . The blots were cut into strips prior to probing to detect the individual terminal pathway proteins. Molecular weights used were: NR: C5, 190 kDa; C6, 105 kDa; C7, 95 kDa, C8αγ; 70 kDa; C9, 65 kDa. R: C6, 110 kDa; C7, 95 kDa; C5β, 75 kDa; C9, 70 kDa; C8α/β, 65 kDa; C8γ, 22 kDa. Results are representative of at least three analyses. M; protein molecular weight marker, 2°; secondary antibody. (K) The novel mAbs 17E7, 59E7 and 73D1 were separately immobilized on mouse IgG capture sensor chips (GE Healthcare, # BR-1008-38) and mAb 3B11 (IgM) on protein L Series S sensor chip (GE Healthcare #29-2051-38) at approximately 60 RU. Human, rat or mouse C7 was flowed in HEPES-buffered saline (HBS) in a dilution range of 66 to 8 nM and interactions with the immobilized mAbs were analyzed. Sensorgrams were collected and KDs were calculated using the Langmuir 1: 1 binding model with RI values set to zero. Representative sensorgrams for 17E7 and 59E7 binding of human C7 are shown with raw data in colored lines and fitted data in dotted line (average of 3); all binding data and analyses are included in Table 2 . The sensorgrams of clones 3B11 and 73D1 are included in Supplementary. The SPR analysis was performed in an automated manner using T200 Biacore Evaluation Software version 2 (GE Healthcare).

Article Snippet: The mAbs binding analyses were carried out on a Biacore T200 instrument (GE Healthcare); for mAb of isotype IgG, an antibody capture kit (GE Healthcare, # BR-1008-38) was used to immobilize the mAb on a CM5 sensor chip (GE Healthcare, #29-1496-03) as recommended by the manufacturer. mAb isotype IgM was immobilized on a Protein L Series S sensor chip (GE Healthcare #29-2051-38). mAb were flowed to saturate the surface, then C7, human, rat or mouse, diluted in EP-HBS (10 mM HEPES, pH 7.4, 150 mM NaCl, 0.005% surfactant P20) in the range 0 to 68 nM, flowed over the immobilized mAb.

Techniques: Western Blot, Binding Assay, Purification, Positive Control, Molecular Weight, Marker, Clone Assay, Software

Journal: Frontiers in Immunology

Article Title: Monoclonal Antibodies Capable of Inhibiting Complement Downstream of C5 in Multiple Species

doi: 10.3389/fimmu.2020.612402

Figure Lengend Snippet: Testing anti-C7 mAbs in vivo . (A) mAb 73D1 or BB5.1 anti-mouse C5 as positive control was administered IP at a dose of 1 mg/kg to female wildtype mice (n=5 per group); blood was sampled at intervals serum obtained and added to human C7D or C5D serum respectively prior to measuring CP hemolytic activity measured. Controls included C7D and C5D human sera at the same dose, NMS to demonstrate the requirement for human depleted sera and 1% Tween-20 and HBS to set 100% and 0% lysis in the assay. Significance of differences between groups was determined by one-way ANOVA; significant differences and p values are shown in the figure. Error bars are standard errors of triplicates. (B) C7-deficient mice (10 females) were reconstituted with human C7 (500µg; IP), then split into test and control groups (5 in each). After 1 h, test and control animals were injected subcutaneously (SC) with 17E7 mAb or irrelevant isotype control mAb (1 mg) respectively. Blood was collected prior to administration of C7, immediately prior to administration of mAb and 3 h after mAb administration. Hemolytic activity was measured as above. Significance of differences between groups was determined using an unpaired t test; significant differences and p values are shown in the figure. Error bars are standard errors of triplicates. (C) Female Lewis rats were divided into three groups (n = 2 each) and injected intraperitoneally with mAb 2H2 at doses of 10, 20, and 40 mg/kg; blood was collected from all the animals just before mAb administration, 2 h after mAb 2H2 administration, and then every 12 h over 1 week. Sera prepared and hemolytic activity tested in standard CP assays. Significance of differences between groups was determined using an unpaired t test; significant differences and p values are shown in the figure. Error bars are standard errors of triplicates. (D–F) EAMG was induced in rats (5 per group) and either mAb 2H2 or isotype control mAb(10mg/kg) administered at induction. Weight loss (D) and clinical score (E) were measured at intervals; mice were bled at 0, 2, 24, and 48 h, serum harvested and hemolytic activity measured (F) . All animals were killed at 48 h. Results are means of five in each group and vertical bars represent SD. Significance of differences between groups was determined using an unpaired t test except panel E where paired t test was used; significant differences and p values are shown in the figures. (G–K). Soleus muscles were harvested at time of sacrifice (48 h) and snap frozen in OCT. Sections (10 µm) were stained for AChR with TRITC-conjugated a-BuTX; AChR-positive endplates were counted in 10 fields from each animal using ImageJ software. (G) . Sections were stained for C3b/iC3b (H) and C9/MAC (I) and staining quantified as above. Tissue sections from isotype control (J) or 2H2 (K) treated animals were double-stained for AChR together with anti-C3b/iC3b (top panel) or C9/MAC (bottom panel) and imaged on a Zeiss confocal microscope. The scale bar shown is 10 µm; all images were captured at identical magnification. Statistical significance was obtained by unpaired t-test and P < 0.05 was considered significant; significant differences and p values, mean and SD are shown in the figures.

Article Snippet: The mAbs binding analyses were carried out on a Biacore T200 instrument (GE Healthcare); for mAb of isotype IgG, an antibody capture kit (GE Healthcare, # BR-1008-38) was used to immobilize the mAb on a CM5 sensor chip (GE Healthcare, #29-1496-03) as recommended by the manufacturer. mAb isotype IgM was immobilized on a Protein L Series S sensor chip (GE Healthcare #29-2051-38). mAb were flowed to saturate the surface, then C7, human, rat or mouse, diluted in EP-HBS (10 mM HEPES, pH 7.4, 150 mM NaCl, 0.005% surfactant P20) in the range 0 to 68 nM, flowed over the immobilized mAb.

Techniques: In Vivo, Positive Control, Activity Assay, Lysis, Injection, Staining, Software, Microscopy