mouse anti human fhr4 af488 mab  (R&D Systems)


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    R&D Systems mouse anti human fhr4 af488 mab
    <t>FHR4/V</t> H H(T), FHR4/V H H(P) and V H H(P)/Fc molecules reduce tumor growth, whereas V H H(T)/Fc has no beneficial effect. (A) Experimental design for the measurement of the subcutaneous xenografts mammary fat pads volume in the presence of different CoMiX molecules and control antibodies. BT474 cells were injected into the mammary fat pads of mice. When the tumor volume reached ∼60 mm 3 , the mice were injected intravenously into the lateral tail vein with 100 µg of CoMiX molecules or control antibodies. The injection was repeated 9 times on days 1, 2, 3, 4, 7, 8, 9 and 10 after the first injection. For molecule combinations, 50 µg of each were injected. The tumors were measured every second or third day with calipers. (B) The therapeutic effects of five CoMiX molecules [FHR4/V H H(T), FHR4/V H H(P), V H H(T)/Fc, V H H(P)/Fc, V H H(T)] and two control antibodies (trastuzumab and pertuzumab) were evaluated individually and in combination. The treatment duration is indicated for all groups in green, as mentioned for the first FHR4/V H H(T) group in the left side of the graph. CoMiX-FHR4 molecules are more effective than CoMiX-Fcs, while the molecules that have the pertuzumab-competing epitope are more effective than those with the trastuzumab-competing epitope. (C) MRI images of a representative tumor for each group. Mice were sacrificed at the end of the treatment.
    Mouse Anti Human Fhr4 Af488 Mab, supplied by R&D Systems, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse anti human fhr4 af488 mab/product/R&D Systems
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mouse anti human fhr4 af488 mab - by Bioz Stars, 2024-07
    94/100 stars

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    1) Product Images from "Complement-Activating Multimeric Immunotherapeutic Complexes for HER2-breast cancer immunotherapy"

    Article Title: Complement-Activating Multimeric Immunotherapeutic Complexes for HER2-breast cancer immunotherapy

    Journal: bioRxiv

    doi: 10.1101/2024.02.02.578619

    FHR4/V H H(T), FHR4/V H H(P) and V H H(P)/Fc molecules reduce tumor growth, whereas V H H(T)/Fc has no beneficial effect. (A) Experimental design for the measurement of the subcutaneous xenografts mammary fat pads volume in the presence of different CoMiX molecules and control antibodies. BT474 cells were injected into the mammary fat pads of mice. When the tumor volume reached ∼60 mm 3 , the mice were injected intravenously into the lateral tail vein with 100 µg of CoMiX molecules or control antibodies. The injection was repeated 9 times on days 1, 2, 3, 4, 7, 8, 9 and 10 after the first injection. For molecule combinations, 50 µg of each were injected. The tumors were measured every second or third day with calipers. (B) The therapeutic effects of five CoMiX molecules [FHR4/V H H(T), FHR4/V H H(P), V H H(T)/Fc, V H H(P)/Fc, V H H(T)] and two control antibodies (trastuzumab and pertuzumab) were evaluated individually and in combination. The treatment duration is indicated for all groups in green, as mentioned for the first FHR4/V H H(T) group in the left side of the graph. CoMiX-FHR4 molecules are more effective than CoMiX-Fcs, while the molecules that have the pertuzumab-competing epitope are more effective than those with the trastuzumab-competing epitope. (C) MRI images of a representative tumor for each group. Mice were sacrificed at the end of the treatment.
    Figure Legend Snippet: FHR4/V H H(T), FHR4/V H H(P) and V H H(P)/Fc molecules reduce tumor growth, whereas V H H(T)/Fc has no beneficial effect. (A) Experimental design for the measurement of the subcutaneous xenografts mammary fat pads volume in the presence of different CoMiX molecules and control antibodies. BT474 cells were injected into the mammary fat pads of mice. When the tumor volume reached ∼60 mm 3 , the mice were injected intravenously into the lateral tail vein with 100 µg of CoMiX molecules or control antibodies. The injection was repeated 9 times on days 1, 2, 3, 4, 7, 8, 9 and 10 after the first injection. For molecule combinations, 50 µg of each were injected. The tumors were measured every second or third day with calipers. (B) The therapeutic effects of five CoMiX molecules [FHR4/V H H(T), FHR4/V H H(P), V H H(T)/Fc, V H H(P)/Fc, V H H(T)] and two control antibodies (trastuzumab and pertuzumab) were evaluated individually and in combination. The treatment duration is indicated for all groups in green, as mentioned for the first FHR4/V H H(T) group in the left side of the graph. CoMiX-FHR4 molecules are more effective than CoMiX-Fcs, while the molecules that have the pertuzumab-competing epitope are more effective than those with the trastuzumab-competing epitope. (C) MRI images of a representative tumor for each group. Mice were sacrificed at the end of the treatment.

    Techniques Used: Injection

    : Design of CoMiX-FHR4, CoMiX-Fc and the controls V H H(T) and V H H(T)/Fc Δhinge. All constructs are co-transfected with the eGFP.C4bpβ construct, leading to the covalent association of a single eGFP tracking function with the multimeric fusion C4bp α core with the a-chains represented in red lines. We used V H H(T) and V H H(P), recognising trastuzumab- or pertuzumab-competing HER2 epitopes, respectively, to generate 2 types of CoMiX-FHR4 molecules: FHR4/V H H(T) and FHR4/V H H(P)] or 2 types of CoMiX-Fc molecules :V H H(T)/Fc or V H H(P)/Fc. A dual hinge region between the C4bpα-scaffold and the IgG1 CH2-CH3 (represented by two red bands between the two Fc fragments) allows the formation of interchain disulfide bonds and the dimerisation of Fc-regions. C4bpα.His8x V H H(T) is the control multimeric molecule with no effector function, and so called V H H(T), and V H H(T)/Fc Δhinge is the control molecule of V H H(T)/Fc without hinge that allows the formation of triple Fc dimers.
    Figure Legend Snippet: : Design of CoMiX-FHR4, CoMiX-Fc and the controls V H H(T) and V H H(T)/Fc Δhinge. All constructs are co-transfected with the eGFP.C4bpβ construct, leading to the covalent association of a single eGFP tracking function with the multimeric fusion C4bp α core with the a-chains represented in red lines. We used V H H(T) and V H H(P), recognising trastuzumab- or pertuzumab-competing HER2 epitopes, respectively, to generate 2 types of CoMiX-FHR4 molecules: FHR4/V H H(T) and FHR4/V H H(P)] or 2 types of CoMiX-Fc molecules :V H H(T)/Fc or V H H(P)/Fc. A dual hinge region between the C4bpα-scaffold and the IgG1 CH2-CH3 (represented by two red bands between the two Fc fragments) allows the formation of interchain disulfide bonds and the dimerisation of Fc-regions. C4bpα.His8x V H H(T) is the control multimeric molecule with no effector function, and so called V H H(T), and V H H(T)/Fc Δhinge is the control molecule of V H H(T)/Fc without hinge that allows the formation of triple Fc dimers.

    Techniques Used: Construct, Transfection

    : Visualization of the molecular pattern of purified multimeric immunoconjugates by Western blot analysis of complexes separated under non-reducing conditions. (A, B) and SYPRO Ruby protein gel staining under reducing conditions (C) . (A) 1. FHR4/V H H(T) fraction 1, 2. FHR4/V H H(T) fraction 2/3, 3. FHR4/V H H(P) fraction 1, 4. FHR4/V H H(P) fraction 2/3. Under non-reducing conditions, seven bands are visible for the different fractions corresponding to FHR4-valencies varying between 1 and 7. The pooled fractions f2 and f3 display higher FHR4-valencies than their f1 counterpart and were used for further experiments. (B) 1. FHR4/V H H(T) fraction 1, 2. FHR4/V H H(T) fraction 2/3, 3. FHR4/V H H(P) fraction 1, 4. FHR4/V H H(P) fraction 2/3, 5. V H H(P)/Fc, 6. V H H(T)/Fc, 7. V H H(T)/Fc Δhinge, 8. V H H(T). The different molecular species were analyzed and revealed with a goat anti-human IgG antibody that cross-reacts with the V H H region. (C) 1. FHR4/V H H(T) fraction 1, 2. FHR4/V H H(T) fraction 2/3, 3. FHR4/V H H(P) fraction 1, 4. FHR4/V H H(P) fraction 2/3, 5. V H H(P)/Fc, 6. V H H(T)/Fc, 7. V H H(T)/Fc Δhinge, 8. V H H(T). The multimers were also analyzed by SYPRO Ruby gel staining under reducing conditions. Three bands can be observed for FHR4/V H H(T) and FHR4/V H H(P), representing the monomeric forms of the FHR4.C4bpα.His (120 kDa), eGFP.SCR3.C4bpβ (50 kDa) and the V H H(T).C4bpα.FLAG (40 kDa) or V H H(P).C4bpα.FLAG (30 kDa) targeting components. V H H(T)/Fc and V H H(P)/Fc molecules display two bands representing the eGFP.SCR3.C4bpβ and V H H(T).C4bpα.Fc chains and V H H(P).C4bpα.Fc, respectively. The V H H(T) control molecule has no FHR4- or Fc-effector functions, only targeting (V H H(T).C4bpα.His) and tracking (eGFP.SCR3.C4bpβ) functions, whereas V H H(T)/Fc Δhinge shows one band for V H H(T).C4bpα.Fc.
    Figure Legend Snippet: : Visualization of the molecular pattern of purified multimeric immunoconjugates by Western blot analysis of complexes separated under non-reducing conditions. (A, B) and SYPRO Ruby protein gel staining under reducing conditions (C) . (A) 1. FHR4/V H H(T) fraction 1, 2. FHR4/V H H(T) fraction 2/3, 3. FHR4/V H H(P) fraction 1, 4. FHR4/V H H(P) fraction 2/3. Under non-reducing conditions, seven bands are visible for the different fractions corresponding to FHR4-valencies varying between 1 and 7. The pooled fractions f2 and f3 display higher FHR4-valencies than their f1 counterpart and were used for further experiments. (B) 1. FHR4/V H H(T) fraction 1, 2. FHR4/V H H(T) fraction 2/3, 3. FHR4/V H H(P) fraction 1, 4. FHR4/V H H(P) fraction 2/3, 5. V H H(P)/Fc, 6. V H H(T)/Fc, 7. V H H(T)/Fc Δhinge, 8. V H H(T). The different molecular species were analyzed and revealed with a goat anti-human IgG antibody that cross-reacts with the V H H region. (C) 1. FHR4/V H H(T) fraction 1, 2. FHR4/V H H(T) fraction 2/3, 3. FHR4/V H H(P) fraction 1, 4. FHR4/V H H(P) fraction 2/3, 5. V H H(P)/Fc, 6. V H H(T)/Fc, 7. V H H(T)/Fc Δhinge, 8. V H H(T). The multimers were also analyzed by SYPRO Ruby gel staining under reducing conditions. Three bands can be observed for FHR4/V H H(T) and FHR4/V H H(P), representing the monomeric forms of the FHR4.C4bpα.His (120 kDa), eGFP.SCR3.C4bpβ (50 kDa) and the V H H(T).C4bpα.FLAG (40 kDa) or V H H(P).C4bpα.FLAG (30 kDa) targeting components. V H H(T)/Fc and V H H(P)/Fc molecules display two bands representing the eGFP.SCR3.C4bpβ and V H H(T).C4bpα.Fc chains and V H H(P).C4bpα.Fc, respectively. The V H H(T) control molecule has no FHR4- or Fc-effector functions, only targeting (V H H(T).C4bpα.His) and tracking (eGFP.SCR3.C4bpβ) functions, whereas V H H(T)/Fc Δhinge shows one band for V H H(T).C4bpα.Fc.

    Techniques Used: Purification, Western Blot, Staining

    : Dose response analysis of C3b/iC3b deposition (A), MAC formation (B), and complement-dependent cytotoxicity (C) on BT474 tumor cells incubated with 3-fold serial dilutions: from 15 µg to 0.5 µg/well in case of individual molecules, and from 7.5 µg to 0.25 µg/well of each in case of molecule combinations. As controls, therapeutic antibodies and NHS were used. (A) C3b/iC3b deposition was detected with mouse anti-human C3b mAb and a secondary goat anti-mouse IgG Ab conjugated with AF647. CoMiX-Fc and CoMiX-FHR4 molecules elicit stronger complement activating effects than trastuzumab, pertuzumab and the combination of these two antibodies. Combining CoMiX-Fc and CoMiX-FHR4 molecules with other multimers resulted in the highest level of C3b desposition. (B) Staining with anti-C5b9 mAb followed by PE-conjugated anti-mouse IgG pAb was used to detect membrane attack complex (MAC) formation. (C) The percentage of dead cells was calculated by dividing the number of live/dead-positive (dead) cells with the total number of analysed cells. Data are presented as mean values ±SD of n = 3 independent experiments.
    Figure Legend Snippet: : Dose response analysis of C3b/iC3b deposition (A), MAC formation (B), and complement-dependent cytotoxicity (C) on BT474 tumor cells incubated with 3-fold serial dilutions: from 15 µg to 0.5 µg/well in case of individual molecules, and from 7.5 µg to 0.25 µg/well of each in case of molecule combinations. As controls, therapeutic antibodies and NHS were used. (A) C3b/iC3b deposition was detected with mouse anti-human C3b mAb and a secondary goat anti-mouse IgG Ab conjugated with AF647. CoMiX-Fc and CoMiX-FHR4 molecules elicit stronger complement activating effects than trastuzumab, pertuzumab and the combination of these two antibodies. Combining CoMiX-Fc and CoMiX-FHR4 molecules with other multimers resulted in the highest level of C3b desposition. (B) Staining with anti-C5b9 mAb followed by PE-conjugated anti-mouse IgG pAb was used to detect membrane attack complex (MAC) formation. (C) The percentage of dead cells was calculated by dividing the number of live/dead-positive (dead) cells with the total number of analysed cells. Data are presented as mean values ±SD of n = 3 independent experiments.

    Techniques Used: Incubation, Staining, Membrane

    Flow cytometry analysis of C3b/iC3b deposition (A), membrane attack complex formation (B) , and complement-dependent cytotoxicity (C) on BT474 tumor cells incubated with 15 µg/well of multimeric immunotherapeutic complexes. (A) C3b/iC3b deposition was detected with mouse anti-human C3b mAb and a secondary goat anti-mouse IgG Ab conjugated with AF647. (B) MAC formation was analyzed using anti-C5b9 mAb followed by PE-conjugated anti-mouse IgG pAb. MAC-formation was highest when CoMiX-Fc and CoMiX-FHR4 molecules were combined. (C) The percentage of dead cells was calculated by dividing the number of live/dead-positive (dead) cells with the total number of analyzed cells. (D) A linear correlation between C3b deposition (MFI) and the percentage of dead cells at 15 µg/well of molecules was observed. Consistent with C3b deposition and MAC-formation, CoMiX-Fc and CoMiX-FHR4 significantly increased the percentage of dead cells compared to control multimers and therapeutic antibodies. Data are presented as mean values ±SD of n = 3 independent experiments. Statistical analysis was performed using a one-way ANOVA and post-hoc Tukey test (**p < 0.005, ***p < 0.001, ****p < 0.0001).
    Figure Legend Snippet: Flow cytometry analysis of C3b/iC3b deposition (A), membrane attack complex formation (B) , and complement-dependent cytotoxicity (C) on BT474 tumor cells incubated with 15 µg/well of multimeric immunotherapeutic complexes. (A) C3b/iC3b deposition was detected with mouse anti-human C3b mAb and a secondary goat anti-mouse IgG Ab conjugated with AF647. (B) MAC formation was analyzed using anti-C5b9 mAb followed by PE-conjugated anti-mouse IgG pAb. MAC-formation was highest when CoMiX-Fc and CoMiX-FHR4 molecules were combined. (C) The percentage of dead cells was calculated by dividing the number of live/dead-positive (dead) cells with the total number of analyzed cells. (D) A linear correlation between C3b deposition (MFI) and the percentage of dead cells at 15 µg/well of molecules was observed. Consistent with C3b deposition and MAC-formation, CoMiX-Fc and CoMiX-FHR4 significantly increased the percentage of dead cells compared to control multimers and therapeutic antibodies. Data are presented as mean values ±SD of n = 3 independent experiments. Statistical analysis was performed using a one-way ANOVA and post-hoc Tukey test (**p < 0.005, ***p < 0.001, ****p < 0.0001).

    Techniques Used: Flow Cytometry, Membrane, Incubation

    : FHR4-based CoMiX molecules activate the alternative complement pathway, whereas Fc-based CoMiX molecules facilitate classical pathway activation. C3b deposition (A) and CDC (B) of BT474 tumor cells incubated with saturating concentrations (15 µl/well) of CoMiX molecules and control mAbs individually or in combinations. 25% NHS diluted in either GVB ++ or GVB + buffer was added for 30 minutes at 37°C. Inhibition of the classical complement pathway by using GVB + buffer completely disrupts the complement activating properties of Fc-based CoMiX molecules, trastuzumab and pertuzumab. Data are presented as mean values ±SD of n = 3 independent experiments. Statistical analysis was performed using a two-way ANOVA test between GVB ++ and GVB + conditions for each molecule. All comparisons between GVB ++ and GVB + reached statistical significance (****p < 0.0001). (C) Representative histogram plots on live BT474 cells of C3b MFI for the combinations of molecules with GVB + and GVB ++ conditions are shown. (D) Representative dots plots of live and dead BT474 cells with the different combinations are depicted.
    Figure Legend Snippet: : FHR4-based CoMiX molecules activate the alternative complement pathway, whereas Fc-based CoMiX molecules facilitate classical pathway activation. C3b deposition (A) and CDC (B) of BT474 tumor cells incubated with saturating concentrations (15 µl/well) of CoMiX molecules and control mAbs individually or in combinations. 25% NHS diluted in either GVB ++ or GVB + buffer was added for 30 minutes at 37°C. Inhibition of the classical complement pathway by using GVB + buffer completely disrupts the complement activating properties of Fc-based CoMiX molecules, trastuzumab and pertuzumab. Data are presented as mean values ±SD of n = 3 independent experiments. Statistical analysis was performed using a two-way ANOVA test between GVB ++ and GVB + conditions for each molecule. All comparisons between GVB ++ and GVB + reached statistical significance (****p < 0.0001). (C) Representative histogram plots on live BT474 cells of C3b MFI for the combinations of molecules with GVB + and GVB ++ conditions are shown. (D) Representative dots plots of live and dead BT474 cells with the different combinations are depicted.

    Techniques Used: Activation Assay, Incubation, Inhibition

    Immunofluorescent staining of tumor sections collected 1 or 6 hours after injection of CoMiX-FHR4 (upper panel), CoMiX-Fc (intermediate panel) or controls (lower panel with anti-C3d staining): PBS (1), V H H(T) (2), trastuzumab + pertuzumab (3). CoMiX were visualized with either a rabbit anti-His mAb followed by the goat Anti-Rabbit IgG Fc AF568- or a goat anti-human IgG AF647-conjugated antibody. Complement activation was visualized using the polyclonal rabbit anti-C3d antibody followed by AF568-conjugated anti-rabbit IgG. One hour post-injection, the infiltration of molecules into the tumor tissue is already visible, however complement activation occurs predominantly on the periphery of the tumors. Six hours after treatment, the molecules homogeneously infiltrate the tumor and strong complement activation can be detected throughout the whole tissue. Compared to CoMiX-FHR4 molecules, the V H H(T) control (2) shows decreased infiltration and reduced complement activation, present only at the periphery of the tumor, even if collected 6 hours after injection. Trastuzumab + pertuzumab (3) were used as positive controls and showed significant infiltration and complement activation.
    Figure Legend Snippet: Immunofluorescent staining of tumor sections collected 1 or 6 hours after injection of CoMiX-FHR4 (upper panel), CoMiX-Fc (intermediate panel) or controls (lower panel with anti-C3d staining): PBS (1), V H H(T) (2), trastuzumab + pertuzumab (3). CoMiX were visualized with either a rabbit anti-His mAb followed by the goat Anti-Rabbit IgG Fc AF568- or a goat anti-human IgG AF647-conjugated antibody. Complement activation was visualized using the polyclonal rabbit anti-C3d antibody followed by AF568-conjugated anti-rabbit IgG. One hour post-injection, the infiltration of molecules into the tumor tissue is already visible, however complement activation occurs predominantly on the periphery of the tumors. Six hours after treatment, the molecules homogeneously infiltrate the tumor and strong complement activation can be detected throughout the whole tissue. Compared to CoMiX-FHR4 molecules, the V H H(T) control (2) shows decreased infiltration and reduced complement activation, present only at the periphery of the tumor, even if collected 6 hours after injection. Trastuzumab + pertuzumab (3) were used as positive controls and showed significant infiltration and complement activation.

    Techniques Used: Staining, Injection, Activation Assay

    FHR4/V H H(T) and FHR4/V H H(P) CoMiX molecules exert their anti-tumor effect on trastuzumab-resistant BT474 cells. Trastuzumab-resistant BT474 cells were injected into the mammary fat pads of female BALB/c NUDE mice. When the tumor volume reached ∼60 mm 3 , the mice were injected with 100 µg of CoMiX-FHR4 molecules, trastuzumab or PBS, as described on . The tumors were measured every second or third day until day 37 of the study or until meeting a humane endpoint. Trastuzumab and PBS had no beneficial effect on tumor growth, whereas CoMiX-FHR4 molecules were shown to significantly reduce tumor progression. (B) Cryosections of trastuzumab-resistant BT474 tumor xenografts collected just after the end of the treatment (at D+11). Tumors were embedded in OCT and snap frozen in OCT. Four micrometer cryosections were made and stained with a monoclonal rat IgG 2A anti-mouse NKp46/NCR1 antibody and revealed using a donkey anti-rat AF568-conjugated pAb. Confocal microscope was used to make pictures (lens X40), monitored by the Nikon NIS-Elements software which allowed to assemble pictures to get a large field overview of the tumors. A) tumor treated with combined CoMiX-FHR4 [FHR4/V H H(T) + FHR4/V H H(P)], B) tumor treated with trastuzumab, C) tumor treated with PBS (mock).
    Figure Legend Snippet: FHR4/V H H(T) and FHR4/V H H(P) CoMiX molecules exert their anti-tumor effect on trastuzumab-resistant BT474 cells. Trastuzumab-resistant BT474 cells were injected into the mammary fat pads of female BALB/c NUDE mice. When the tumor volume reached ∼60 mm 3 , the mice were injected with 100 µg of CoMiX-FHR4 molecules, trastuzumab or PBS, as described on . The tumors were measured every second or third day until day 37 of the study or until meeting a humane endpoint. Trastuzumab and PBS had no beneficial effect on tumor growth, whereas CoMiX-FHR4 molecules were shown to significantly reduce tumor progression. (B) Cryosections of trastuzumab-resistant BT474 tumor xenografts collected just after the end of the treatment (at D+11). Tumors were embedded in OCT and snap frozen in OCT. Four micrometer cryosections were made and stained with a monoclonal rat IgG 2A anti-mouse NKp46/NCR1 antibody and revealed using a donkey anti-rat AF568-conjugated pAb. Confocal microscope was used to make pictures (lens X40), monitored by the Nikon NIS-Elements software which allowed to assemble pictures to get a large field overview of the tumors. A) tumor treated with combined CoMiX-FHR4 [FHR4/V H H(T) + FHR4/V H H(P)], B) tumor treated with trastuzumab, C) tumor treated with PBS (mock).

    Techniques Used: Injection, Staining, Microscopy, Software

    biotinylated mouse anti human factor h  (Thermo Fisher)


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    Thermo Fisher biotinylated mouse anti human factor h
    Biotinylated Mouse Anti Human Factor H, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    biotinylated mouse anti human factor h primary antibody  (Thermo Fisher)


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    Thermo Fisher biotinylated mouse anti human factor h primary antibody
    Biotinylated Mouse Anti Human Factor H Primary Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    biotinylated mouse anti human factor h primary antibody  (Thermo Fisher)


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    Thermo Fisher biotinylated mouse anti human factor h primary antibody
    Biotinylated Mouse Anti Human Factor H Primary Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    biotinylated mouse anti human factor h  (Thermo Fisher)


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    Thermo Fisher biotinylated mouse anti human factor h
    Biotinylated Mouse Anti Human Factor H, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    mouse anti human fhr4 af488 mab  (R&D Systems)


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    R&D Systems mouse anti human fhr4 af488 mab
    <t>FHR4/V</t> H H(T), FHR4/V H H(P) and V H H(P)/Fc molecules reduce tumor growth, whereas V H H(T)/Fc has no beneficial effect. (A) Experimental design for the measurement of the subcutaneous xenografts mammary fat pads volume in the presence of different CoMiX molecules and control antibodies. BT474 cells were injected into the mammary fat pads of mice. When the tumor volume reached ∼60 mm 3 , the mice were injected intravenously into the lateral tail vein with 100 µg of CoMiX molecules or control antibodies. The injection was repeated 9 times on days 1, 2, 3, 4, 7, 8, 9 and 10 after the first injection. For molecule combinations, 50 µg of each were injected. The tumors were measured every second or third day with calipers. (B) The therapeutic effects of five CoMiX molecules [FHR4/V H H(T), FHR4/V H H(P), V H H(T)/Fc, V H H(P)/Fc, V H H(T)] and two control antibodies (trastuzumab and pertuzumab) were evaluated individually and in combination. The treatment duration is indicated for all groups in green, as mentioned for the first FHR4/V H H(T) group in the left side of the graph. CoMiX-FHR4 molecules are more effective than CoMiX-Fcs, while the molecules that have the pertuzumab-competing epitope are more effective than those with the trastuzumab-competing epitope. (C) MRI images of a representative tumor for each group. Mice were sacrificed at the end of the treatment.
    Mouse Anti Human Fhr4 Af488 Mab, supplied by R&D Systems, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse anti human fhr4 af488 mab/product/R&D Systems
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    1) Product Images from "Complement-Activating Multimeric Immunotherapeutic Complexes for HER2-breast cancer immunotherapy"

    Article Title: Complement-Activating Multimeric Immunotherapeutic Complexes for HER2-breast cancer immunotherapy

    Journal: bioRxiv

    doi: 10.1101/2024.02.02.578619

    FHR4/V H H(T), FHR4/V H H(P) and V H H(P)/Fc molecules reduce tumor growth, whereas V H H(T)/Fc has no beneficial effect. (A) Experimental design for the measurement of the subcutaneous xenografts mammary fat pads volume in the presence of different CoMiX molecules and control antibodies. BT474 cells were injected into the mammary fat pads of mice. When the tumor volume reached ∼60 mm 3 , the mice were injected intravenously into the lateral tail vein with 100 µg of CoMiX molecules or control antibodies. The injection was repeated 9 times on days 1, 2, 3, 4, 7, 8, 9 and 10 after the first injection. For molecule combinations, 50 µg of each were injected. The tumors were measured every second or third day with calipers. (B) The therapeutic effects of five CoMiX molecules [FHR4/V H H(T), FHR4/V H H(P), V H H(T)/Fc, V H H(P)/Fc, V H H(T)] and two control antibodies (trastuzumab and pertuzumab) were evaluated individually and in combination. The treatment duration is indicated for all groups in green, as mentioned for the first FHR4/V H H(T) group in the left side of the graph. CoMiX-FHR4 molecules are more effective than CoMiX-Fcs, while the molecules that have the pertuzumab-competing epitope are more effective than those with the trastuzumab-competing epitope. (C) MRI images of a representative tumor for each group. Mice were sacrificed at the end of the treatment.
    Figure Legend Snippet: FHR4/V H H(T), FHR4/V H H(P) and V H H(P)/Fc molecules reduce tumor growth, whereas V H H(T)/Fc has no beneficial effect. (A) Experimental design for the measurement of the subcutaneous xenografts mammary fat pads volume in the presence of different CoMiX molecules and control antibodies. BT474 cells were injected into the mammary fat pads of mice. When the tumor volume reached ∼60 mm 3 , the mice were injected intravenously into the lateral tail vein with 100 µg of CoMiX molecules or control antibodies. The injection was repeated 9 times on days 1, 2, 3, 4, 7, 8, 9 and 10 after the first injection. For molecule combinations, 50 µg of each were injected. The tumors were measured every second or third day with calipers. (B) The therapeutic effects of five CoMiX molecules [FHR4/V H H(T), FHR4/V H H(P), V H H(T)/Fc, V H H(P)/Fc, V H H(T)] and two control antibodies (trastuzumab and pertuzumab) were evaluated individually and in combination. The treatment duration is indicated for all groups in green, as mentioned for the first FHR4/V H H(T) group in the left side of the graph. CoMiX-FHR4 molecules are more effective than CoMiX-Fcs, while the molecules that have the pertuzumab-competing epitope are more effective than those with the trastuzumab-competing epitope. (C) MRI images of a representative tumor for each group. Mice were sacrificed at the end of the treatment.

    Techniques Used: Injection

    : Design of CoMiX-FHR4, CoMiX-Fc and the controls V H H(T) and V H H(T)/Fc Δhinge. All constructs are co-transfected with the eGFP.C4bpβ construct, leading to the covalent association of a single eGFP tracking function with the multimeric fusion C4bp α core with the a-chains represented in red lines. We used V H H(T) and V H H(P), recognising trastuzumab- or pertuzumab-competing HER2 epitopes, respectively, to generate 2 types of CoMiX-FHR4 molecules: FHR4/V H H(T) and FHR4/V H H(P)] or 2 types of CoMiX-Fc molecules :V H H(T)/Fc or V H H(P)/Fc. A dual hinge region between the C4bpα-scaffold and the IgG1 CH2-CH3 (represented by two red bands between the two Fc fragments) allows the formation of interchain disulfide bonds and the dimerisation of Fc-regions. C4bpα.His8x V H H(T) is the control multimeric molecule with no effector function, and so called V H H(T), and V H H(T)/Fc Δhinge is the control molecule of V H H(T)/Fc without hinge that allows the formation of triple Fc dimers.
    Figure Legend Snippet: : Design of CoMiX-FHR4, CoMiX-Fc and the controls V H H(T) and V H H(T)/Fc Δhinge. All constructs are co-transfected with the eGFP.C4bpβ construct, leading to the covalent association of a single eGFP tracking function with the multimeric fusion C4bp α core with the a-chains represented in red lines. We used V H H(T) and V H H(P), recognising trastuzumab- or pertuzumab-competing HER2 epitopes, respectively, to generate 2 types of CoMiX-FHR4 molecules: FHR4/V H H(T) and FHR4/V H H(P)] or 2 types of CoMiX-Fc molecules :V H H(T)/Fc or V H H(P)/Fc. A dual hinge region between the C4bpα-scaffold and the IgG1 CH2-CH3 (represented by two red bands between the two Fc fragments) allows the formation of interchain disulfide bonds and the dimerisation of Fc-regions. C4bpα.His8x V H H(T) is the control multimeric molecule with no effector function, and so called V H H(T), and V H H(T)/Fc Δhinge is the control molecule of V H H(T)/Fc without hinge that allows the formation of triple Fc dimers.

    Techniques Used: Construct, Transfection

    : Visualization of the molecular pattern of purified multimeric immunoconjugates by Western blot analysis of complexes separated under non-reducing conditions. (A, B) and SYPRO Ruby protein gel staining under reducing conditions (C) . (A) 1. FHR4/V H H(T) fraction 1, 2. FHR4/V H H(T) fraction 2/3, 3. FHR4/V H H(P) fraction 1, 4. FHR4/V H H(P) fraction 2/3. Under non-reducing conditions, seven bands are visible for the different fractions corresponding to FHR4-valencies varying between 1 and 7. The pooled fractions f2 and f3 display higher FHR4-valencies than their f1 counterpart and were used for further experiments. (B) 1. FHR4/V H H(T) fraction 1, 2. FHR4/V H H(T) fraction 2/3, 3. FHR4/V H H(P) fraction 1, 4. FHR4/V H H(P) fraction 2/3, 5. V H H(P)/Fc, 6. V H H(T)/Fc, 7. V H H(T)/Fc Δhinge, 8. V H H(T). The different molecular species were analyzed and revealed with a goat anti-human IgG antibody that cross-reacts with the V H H region. (C) 1. FHR4/V H H(T) fraction 1, 2. FHR4/V H H(T) fraction 2/3, 3. FHR4/V H H(P) fraction 1, 4. FHR4/V H H(P) fraction 2/3, 5. V H H(P)/Fc, 6. V H H(T)/Fc, 7. V H H(T)/Fc Δhinge, 8. V H H(T). The multimers were also analyzed by SYPRO Ruby gel staining under reducing conditions. Three bands can be observed for FHR4/V H H(T) and FHR4/V H H(P), representing the monomeric forms of the FHR4.C4bpα.His (120 kDa), eGFP.SCR3.C4bpβ (50 kDa) and the V H H(T).C4bpα.FLAG (40 kDa) or V H H(P).C4bpα.FLAG (30 kDa) targeting components. V H H(T)/Fc and V H H(P)/Fc molecules display two bands representing the eGFP.SCR3.C4bpβ and V H H(T).C4bpα.Fc chains and V H H(P).C4bpα.Fc, respectively. The V H H(T) control molecule has no FHR4- or Fc-effector functions, only targeting (V H H(T).C4bpα.His) and tracking (eGFP.SCR3.C4bpβ) functions, whereas V H H(T)/Fc Δhinge shows one band for V H H(T).C4bpα.Fc.
    Figure Legend Snippet: : Visualization of the molecular pattern of purified multimeric immunoconjugates by Western blot analysis of complexes separated under non-reducing conditions. (A, B) and SYPRO Ruby protein gel staining under reducing conditions (C) . (A) 1. FHR4/V H H(T) fraction 1, 2. FHR4/V H H(T) fraction 2/3, 3. FHR4/V H H(P) fraction 1, 4. FHR4/V H H(P) fraction 2/3. Under non-reducing conditions, seven bands are visible for the different fractions corresponding to FHR4-valencies varying between 1 and 7. The pooled fractions f2 and f3 display higher FHR4-valencies than their f1 counterpart and were used for further experiments. (B) 1. FHR4/V H H(T) fraction 1, 2. FHR4/V H H(T) fraction 2/3, 3. FHR4/V H H(P) fraction 1, 4. FHR4/V H H(P) fraction 2/3, 5. V H H(P)/Fc, 6. V H H(T)/Fc, 7. V H H(T)/Fc Δhinge, 8. V H H(T). The different molecular species were analyzed and revealed with a goat anti-human IgG antibody that cross-reacts with the V H H region. (C) 1. FHR4/V H H(T) fraction 1, 2. FHR4/V H H(T) fraction 2/3, 3. FHR4/V H H(P) fraction 1, 4. FHR4/V H H(P) fraction 2/3, 5. V H H(P)/Fc, 6. V H H(T)/Fc, 7. V H H(T)/Fc Δhinge, 8. V H H(T). The multimers were also analyzed by SYPRO Ruby gel staining under reducing conditions. Three bands can be observed for FHR4/V H H(T) and FHR4/V H H(P), representing the monomeric forms of the FHR4.C4bpα.His (120 kDa), eGFP.SCR3.C4bpβ (50 kDa) and the V H H(T).C4bpα.FLAG (40 kDa) or V H H(P).C4bpα.FLAG (30 kDa) targeting components. V H H(T)/Fc and V H H(P)/Fc molecules display two bands representing the eGFP.SCR3.C4bpβ and V H H(T).C4bpα.Fc chains and V H H(P).C4bpα.Fc, respectively. The V H H(T) control molecule has no FHR4- or Fc-effector functions, only targeting (V H H(T).C4bpα.His) and tracking (eGFP.SCR3.C4bpβ) functions, whereas V H H(T)/Fc Δhinge shows one band for V H H(T).C4bpα.Fc.

    Techniques Used: Purification, Western Blot, Staining

    : Dose response analysis of C3b/iC3b deposition (A), MAC formation (B), and complement-dependent cytotoxicity (C) on BT474 tumor cells incubated with 3-fold serial dilutions: from 15 µg to 0.5 µg/well in case of individual molecules, and from 7.5 µg to 0.25 µg/well of each in case of molecule combinations. As controls, therapeutic antibodies and NHS were used. (A) C3b/iC3b deposition was detected with mouse anti-human C3b mAb and a secondary goat anti-mouse IgG Ab conjugated with AF647. CoMiX-Fc and CoMiX-FHR4 molecules elicit stronger complement activating effects than trastuzumab, pertuzumab and the combination of these two antibodies. Combining CoMiX-Fc and CoMiX-FHR4 molecules with other multimers resulted in the highest level of C3b desposition. (B) Staining with anti-C5b9 mAb followed by PE-conjugated anti-mouse IgG pAb was used to detect membrane attack complex (MAC) formation. (C) The percentage of dead cells was calculated by dividing the number of live/dead-positive (dead) cells with the total number of analysed cells. Data are presented as mean values ±SD of n = 3 independent experiments.
    Figure Legend Snippet: : Dose response analysis of C3b/iC3b deposition (A), MAC formation (B), and complement-dependent cytotoxicity (C) on BT474 tumor cells incubated with 3-fold serial dilutions: from 15 µg to 0.5 µg/well in case of individual molecules, and from 7.5 µg to 0.25 µg/well of each in case of molecule combinations. As controls, therapeutic antibodies and NHS were used. (A) C3b/iC3b deposition was detected with mouse anti-human C3b mAb and a secondary goat anti-mouse IgG Ab conjugated with AF647. CoMiX-Fc and CoMiX-FHR4 molecules elicit stronger complement activating effects than trastuzumab, pertuzumab and the combination of these two antibodies. Combining CoMiX-Fc and CoMiX-FHR4 molecules with other multimers resulted in the highest level of C3b desposition. (B) Staining with anti-C5b9 mAb followed by PE-conjugated anti-mouse IgG pAb was used to detect membrane attack complex (MAC) formation. (C) The percentage of dead cells was calculated by dividing the number of live/dead-positive (dead) cells with the total number of analysed cells. Data are presented as mean values ±SD of n = 3 independent experiments.

    Techniques Used: Incubation, Staining, Membrane

    Flow cytometry analysis of C3b/iC3b deposition (A), membrane attack complex formation (B) , and complement-dependent cytotoxicity (C) on BT474 tumor cells incubated with 15 µg/well of multimeric immunotherapeutic complexes. (A) C3b/iC3b deposition was detected with mouse anti-human C3b mAb and a secondary goat anti-mouse IgG Ab conjugated with AF647. (B) MAC formation was analyzed using anti-C5b9 mAb followed by PE-conjugated anti-mouse IgG pAb. MAC-formation was highest when CoMiX-Fc and CoMiX-FHR4 molecules were combined. (C) The percentage of dead cells was calculated by dividing the number of live/dead-positive (dead) cells with the total number of analyzed cells. (D) A linear correlation between C3b deposition (MFI) and the percentage of dead cells at 15 µg/well of molecules was observed. Consistent with C3b deposition and MAC-formation, CoMiX-Fc and CoMiX-FHR4 significantly increased the percentage of dead cells compared to control multimers and therapeutic antibodies. Data are presented as mean values ±SD of n = 3 independent experiments. Statistical analysis was performed using a one-way ANOVA and post-hoc Tukey test (**p < 0.005, ***p < 0.001, ****p < 0.0001).
    Figure Legend Snippet: Flow cytometry analysis of C3b/iC3b deposition (A), membrane attack complex formation (B) , and complement-dependent cytotoxicity (C) on BT474 tumor cells incubated with 15 µg/well of multimeric immunotherapeutic complexes. (A) C3b/iC3b deposition was detected with mouse anti-human C3b mAb and a secondary goat anti-mouse IgG Ab conjugated with AF647. (B) MAC formation was analyzed using anti-C5b9 mAb followed by PE-conjugated anti-mouse IgG pAb. MAC-formation was highest when CoMiX-Fc and CoMiX-FHR4 molecules were combined. (C) The percentage of dead cells was calculated by dividing the number of live/dead-positive (dead) cells with the total number of analyzed cells. (D) A linear correlation between C3b deposition (MFI) and the percentage of dead cells at 15 µg/well of molecules was observed. Consistent with C3b deposition and MAC-formation, CoMiX-Fc and CoMiX-FHR4 significantly increased the percentage of dead cells compared to control multimers and therapeutic antibodies. Data are presented as mean values ±SD of n = 3 independent experiments. Statistical analysis was performed using a one-way ANOVA and post-hoc Tukey test (**p < 0.005, ***p < 0.001, ****p < 0.0001).

    Techniques Used: Flow Cytometry, Membrane, Incubation

    : FHR4-based CoMiX molecules activate the alternative complement pathway, whereas Fc-based CoMiX molecules facilitate classical pathway activation. C3b deposition (A) and CDC (B) of BT474 tumor cells incubated with saturating concentrations (15 µl/well) of CoMiX molecules and control mAbs individually or in combinations. 25% NHS diluted in either GVB ++ or GVB + buffer was added for 30 minutes at 37°C. Inhibition of the classical complement pathway by using GVB + buffer completely disrupts the complement activating properties of Fc-based CoMiX molecules, trastuzumab and pertuzumab. Data are presented as mean values ±SD of n = 3 independent experiments. Statistical analysis was performed using a two-way ANOVA test between GVB ++ and GVB + conditions for each molecule. All comparisons between GVB ++ and GVB + reached statistical significance (****p < 0.0001). (C) Representative histogram plots on live BT474 cells of C3b MFI for the combinations of molecules with GVB + and GVB ++ conditions are shown. (D) Representative dots plots of live and dead BT474 cells with the different combinations are depicted.
    Figure Legend Snippet: : FHR4-based CoMiX molecules activate the alternative complement pathway, whereas Fc-based CoMiX molecules facilitate classical pathway activation. C3b deposition (A) and CDC (B) of BT474 tumor cells incubated with saturating concentrations (15 µl/well) of CoMiX molecules and control mAbs individually or in combinations. 25% NHS diluted in either GVB ++ or GVB + buffer was added for 30 minutes at 37°C. Inhibition of the classical complement pathway by using GVB + buffer completely disrupts the complement activating properties of Fc-based CoMiX molecules, trastuzumab and pertuzumab. Data are presented as mean values ±SD of n = 3 independent experiments. Statistical analysis was performed using a two-way ANOVA test between GVB ++ and GVB + conditions for each molecule. All comparisons between GVB ++ and GVB + reached statistical significance (****p < 0.0001). (C) Representative histogram plots on live BT474 cells of C3b MFI for the combinations of molecules with GVB + and GVB ++ conditions are shown. (D) Representative dots plots of live and dead BT474 cells with the different combinations are depicted.

    Techniques Used: Activation Assay, Incubation, Inhibition

    Immunofluorescent staining of tumor sections collected 1 or 6 hours after injection of CoMiX-FHR4 (upper panel), CoMiX-Fc (intermediate panel) or controls (lower panel with anti-C3d staining): PBS (1), V H H(T) (2), trastuzumab + pertuzumab (3). CoMiX were visualized with either a rabbit anti-His mAb followed by the goat Anti-Rabbit IgG Fc AF568- or a goat anti-human IgG AF647-conjugated antibody. Complement activation was visualized using the polyclonal rabbit anti-C3d antibody followed by AF568-conjugated anti-rabbit IgG. One hour post-injection, the infiltration of molecules into the tumor tissue is already visible, however complement activation occurs predominantly on the periphery of the tumors. Six hours after treatment, the molecules homogeneously infiltrate the tumor and strong complement activation can be detected throughout the whole tissue. Compared to CoMiX-FHR4 molecules, the V H H(T) control (2) shows decreased infiltration and reduced complement activation, present only at the periphery of the tumor, even if collected 6 hours after injection. Trastuzumab + pertuzumab (3) were used as positive controls and showed significant infiltration and complement activation.
    Figure Legend Snippet: Immunofluorescent staining of tumor sections collected 1 or 6 hours after injection of CoMiX-FHR4 (upper panel), CoMiX-Fc (intermediate panel) or controls (lower panel with anti-C3d staining): PBS (1), V H H(T) (2), trastuzumab + pertuzumab (3). CoMiX were visualized with either a rabbit anti-His mAb followed by the goat Anti-Rabbit IgG Fc AF568- or a goat anti-human IgG AF647-conjugated antibody. Complement activation was visualized using the polyclonal rabbit anti-C3d antibody followed by AF568-conjugated anti-rabbit IgG. One hour post-injection, the infiltration of molecules into the tumor tissue is already visible, however complement activation occurs predominantly on the periphery of the tumors. Six hours after treatment, the molecules homogeneously infiltrate the tumor and strong complement activation can be detected throughout the whole tissue. Compared to CoMiX-FHR4 molecules, the V H H(T) control (2) shows decreased infiltration and reduced complement activation, present only at the periphery of the tumor, even if collected 6 hours after injection. Trastuzumab + pertuzumab (3) were used as positive controls and showed significant infiltration and complement activation.

    Techniques Used: Staining, Injection, Activation Assay

    FHR4/V H H(T) and FHR4/V H H(P) CoMiX molecules exert their anti-tumor effect on trastuzumab-resistant BT474 cells. Trastuzumab-resistant BT474 cells were injected into the mammary fat pads of female BALB/c NUDE mice. When the tumor volume reached ∼60 mm 3 , the mice were injected with 100 µg of CoMiX-FHR4 molecules, trastuzumab or PBS, as described on . The tumors were measured every second or third day until day 37 of the study or until meeting a humane endpoint. Trastuzumab and PBS had no beneficial effect on tumor growth, whereas CoMiX-FHR4 molecules were shown to significantly reduce tumor progression. (B) Cryosections of trastuzumab-resistant BT474 tumor xenografts collected just after the end of the treatment (at D+11). Tumors were embedded in OCT and snap frozen in OCT. Four micrometer cryosections were made and stained with a monoclonal rat IgG 2A anti-mouse NKp46/NCR1 antibody and revealed using a donkey anti-rat AF568-conjugated pAb. Confocal microscope was used to make pictures (lens X40), monitored by the Nikon NIS-Elements software which allowed to assemble pictures to get a large field overview of the tumors. A) tumor treated with combined CoMiX-FHR4 [FHR4/V H H(T) + FHR4/V H H(P)], B) tumor treated with trastuzumab, C) tumor treated with PBS (mock).
    Figure Legend Snippet: FHR4/V H H(T) and FHR4/V H H(P) CoMiX molecules exert their anti-tumor effect on trastuzumab-resistant BT474 cells. Trastuzumab-resistant BT474 cells were injected into the mammary fat pads of female BALB/c NUDE mice. When the tumor volume reached ∼60 mm 3 , the mice were injected with 100 µg of CoMiX-FHR4 molecules, trastuzumab or PBS, as described on . The tumors were measured every second or third day until day 37 of the study or until meeting a humane endpoint. Trastuzumab and PBS had no beneficial effect on tumor growth, whereas CoMiX-FHR4 molecules were shown to significantly reduce tumor progression. (B) Cryosections of trastuzumab-resistant BT474 tumor xenografts collected just after the end of the treatment (at D+11). Tumors were embedded in OCT and snap frozen in OCT. Four micrometer cryosections were made and stained with a monoclonal rat IgG 2A anti-mouse NKp46/NCR1 antibody and revealed using a donkey anti-rat AF568-conjugated pAb. Confocal microscope was used to make pictures (lens X40), monitored by the Nikon NIS-Elements software which allowed to assemble pictures to get a large field overview of the tumors. A) tumor treated with combined CoMiX-FHR4 [FHR4/V H H(T) + FHR4/V H H(P)], B) tumor treated with trastuzumab, C) tumor treated with PBS (mock).

    Techniques Used: Injection, Staining, Microscopy, Software

    anti fhr 4 monoclonal mouse antibody  (R&D Systems)


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    R&D Systems anti fhr 4 monoclonal mouse antibody
    Anti Fhr 4 Monoclonal Mouse Antibody, supplied by R&D Systems, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti fhr 4 monoclonal mouse antibody/product/R&D Systems
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    anti fhr 4 monoclonal mouse antibody  (R&D Systems)


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    R&D Systems anti fhr 4 monoclonal mouse antibody
    a Graph showing the AMD odds ratios (OR), generated using the IAMDGC cohort (13,378 controls and 17,541 cases, see also Supplementary Tables and ), of quantitative trait loci (QTLs) identified through expression QTL (rs1410996, rs7531555) or protein QTL (rs61818956, rs10494745, rs7531555) analyses as a function of location on chromosome 1. The major/minor alleles and effect of the minor alleles on <t>FHR-4</t> variation are also shown. Independent QTLs, highlighted in red, were identified through haplotype analyses performed using 1000 Genomes Project phase 3 (1000 G EUR) subjects (shown in the panel) and controls from the Utah & Iowa and IAMDGC cohorts (Supplementary Fig. ). b Box plots of log-transformed and centered plasma FHR-4 levels by rs1410996 (IAMDGC 1.1) and rs7531555 genotypes (404 independent biological samples). Analyses of genetic combinations indicate that the expression QTL effect associated with rs1410996 is entirely attributable to rs7531555. c Box plot showing the distribution of log-transformed and centered plasma FHR-4 levels by rs61818956, rs10494745, and rs7531555 genotypes (486 independent biological samples). d FHR-4 immunohistochemistry in retinal pigment epithelium (RPE)/Bruch’s membrane/choroid tissues from an eye donor obtained using anti-FHR-4 antibody and warp red chromagen shows intense staining in Bruch’s membrane (BM) and between pillars of the choriocapillaris (CC). An image of a negative control can be found in Supplementary Fig. . e Variations in log-transformed and centered FHR-4 levels measured in RPE/Bruch’s membrane/choroid and vitreous lysates with the three independent rs61818956, rs10494745, and rs7531555 genotypes (229 independent biological samples, see Supplementary Table ). The directional effect of QTLs in ocular samples is consistent with that observed in plasma samples. In b , c , and e , in all box plots the horizontal center lines correspond to the medians of the log-transformed and centered FHR-4 distribution and the boxes delineate the 25th/75th percentile. The vertical solid lines represent the full range of the log-transformed and centered FHR-4 distribution in each group. Dots beyond this line indicate potential outliers. Associations with log-transformed and centered FHR-4 levels were assessed using the Kruskal–Wallis test. Post-hoc pairwise comparisons were performed using the Conover–Iman test. All p -values are two-sided and adjusted for multiple testing using Bonferroni corrections. Source data for these plots are provided as a Source Data file.
    Anti Fhr 4 Monoclonal Mouse Antibody, supplied by R&D Systems, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti fhr 4 monoclonal mouse antibody/product/R&D Systems
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti fhr 4 monoclonal mouse antibody - by Bioz Stars, 2024-07
    94/100 stars

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    1) Product Images from "Levels of complement factor H-related 4 protein do not influence susceptibility to age-related macular degeneration or its course of progression"

    Article Title: Levels of complement factor H-related 4 protein do not influence susceptibility to age-related macular degeneration or its course of progression

    Journal: Nature Communications

    doi: 10.1038/s41467-023-44605-0

    a Graph showing the AMD odds ratios (OR), generated using the IAMDGC cohort (13,378 controls and 17,541 cases, see also Supplementary Tables and ), of quantitative trait loci (QTLs) identified through expression QTL (rs1410996, rs7531555) or protein QTL (rs61818956, rs10494745, rs7531555) analyses as a function of location on chromosome 1. The major/minor alleles and effect of the minor alleles on FHR-4 variation are also shown. Independent QTLs, highlighted in red, were identified through haplotype analyses performed using 1000 Genomes Project phase 3 (1000 G EUR) subjects (shown in the panel) and controls from the Utah & Iowa and IAMDGC cohorts (Supplementary Fig. ). b Box plots of log-transformed and centered plasma FHR-4 levels by rs1410996 (IAMDGC 1.1) and rs7531555 genotypes (404 independent biological samples). Analyses of genetic combinations indicate that the expression QTL effect associated with rs1410996 is entirely attributable to rs7531555. c Box plot showing the distribution of log-transformed and centered plasma FHR-4 levels by rs61818956, rs10494745, and rs7531555 genotypes (486 independent biological samples). d FHR-4 immunohistochemistry in retinal pigment epithelium (RPE)/Bruch’s membrane/choroid tissues from an eye donor obtained using anti-FHR-4 antibody and warp red chromagen shows intense staining in Bruch’s membrane (BM) and between pillars of the choriocapillaris (CC). An image of a negative control can be found in Supplementary Fig. . e Variations in log-transformed and centered FHR-4 levels measured in RPE/Bruch’s membrane/choroid and vitreous lysates with the three independent rs61818956, rs10494745, and rs7531555 genotypes (229 independent biological samples, see Supplementary Table ). The directional effect of QTLs in ocular samples is consistent with that observed in plasma samples. In b , c , and e , in all box plots the horizontal center lines correspond to the medians of the log-transformed and centered FHR-4 distribution and the boxes delineate the 25th/75th percentile. The vertical solid lines represent the full range of the log-transformed and centered FHR-4 distribution in each group. Dots beyond this line indicate potential outliers. Associations with log-transformed and centered FHR-4 levels were assessed using the Kruskal–Wallis test. Post-hoc pairwise comparisons were performed using the Conover–Iman test. All p -values are two-sided and adjusted for multiple testing using Bonferroni corrections. Source data for these plots are provided as a Source Data file.
    Figure Legend Snippet: a Graph showing the AMD odds ratios (OR), generated using the IAMDGC cohort (13,378 controls and 17,541 cases, see also Supplementary Tables and ), of quantitative trait loci (QTLs) identified through expression QTL (rs1410996, rs7531555) or protein QTL (rs61818956, rs10494745, rs7531555) analyses as a function of location on chromosome 1. The major/minor alleles and effect of the minor alleles on FHR-4 variation are also shown. Independent QTLs, highlighted in red, were identified through haplotype analyses performed using 1000 Genomes Project phase 3 (1000 G EUR) subjects (shown in the panel) and controls from the Utah & Iowa and IAMDGC cohorts (Supplementary Fig. ). b Box plots of log-transformed and centered plasma FHR-4 levels by rs1410996 (IAMDGC 1.1) and rs7531555 genotypes (404 independent biological samples). Analyses of genetic combinations indicate that the expression QTL effect associated with rs1410996 is entirely attributable to rs7531555. c Box plot showing the distribution of log-transformed and centered plasma FHR-4 levels by rs61818956, rs10494745, and rs7531555 genotypes (486 independent biological samples). d FHR-4 immunohistochemistry in retinal pigment epithelium (RPE)/Bruch’s membrane/choroid tissues from an eye donor obtained using anti-FHR-4 antibody and warp red chromagen shows intense staining in Bruch’s membrane (BM) and between pillars of the choriocapillaris (CC). An image of a negative control can be found in Supplementary Fig. . e Variations in log-transformed and centered FHR-4 levels measured in RPE/Bruch’s membrane/choroid and vitreous lysates with the three independent rs61818956, rs10494745, and rs7531555 genotypes (229 independent biological samples, see Supplementary Table ). The directional effect of QTLs in ocular samples is consistent with that observed in plasma samples. In b , c , and e , in all box plots the horizontal center lines correspond to the medians of the log-transformed and centered FHR-4 distribution and the boxes delineate the 25th/75th percentile. The vertical solid lines represent the full range of the log-transformed and centered FHR-4 distribution in each group. Dots beyond this line indicate potential outliers. Associations with log-transformed and centered FHR-4 levels were assessed using the Kruskal–Wallis test. Post-hoc pairwise comparisons were performed using the Conover–Iman test. All p -values are two-sided and adjusted for multiple testing using Bonferroni corrections. Source data for these plots are provided as a Source Data file.

    Techniques Used: Generated, Expressing, Transformation Assay, Immunohistochemistry, Membrane, Staining, Negative Control

    a Haplotype analyses based on the AMD-associated risk allele C at rs1061170, protective allele A at rs800292, and protective CFHR3/1 deletion tagging allele T at rs12144939 and independent quantitative trait loci (QTL) for CFHR4 rs61818956, rs10494745, and rs7531555. Odds ratios (OR), confidence intervals (CI), and p -values (two-sided) were obtained using logistic regressions including AMD case/control status as the dependent variable and age, sex, and the first two genetic principal components for the IAMDGC cohort as covariates. Bonferroni correction for multiple testing of 11 haplotypes = 0.0045 (0.05/11). The differential effects on FHR-4 levels associated with each haplotype (elevated, baseline, or reduced) is indicated. b Box plot showing variations in log-transformed and centered FHR-4 levels with the four common AMD homozygous Chr1 diplotypes (Risk, Neutral, Prot-I62, and Prot-Del) (346 independent biological samples). c Comparison of Prot-I62 haplotypes with and without the effect allele (T) at rs7531555 generated using the IAMDGC cohort (13,378 controls and 17,541 cases) along with a box plot showing variations in log-transformed and centered FHR-4 levels among subjects stratified by rs800292 and rs7531555 diplotypes (149 independent biological samples). d Comparison of Risk haplotypes with and without the effect allele at rs10494745 and rs61818956 generated using the IAMDGC cohort (13,378 controls and 17,541 cases) along with a box plot showing variations in log-transformed and centered FHR-4 levels among subjects with Risk/Risk diplotypes stratified by rs10494745 (AA or GG) and rs61818956 (CC or TT) diplotypes (56 independent biological samples). e Schematic describing the effect of reduced or elevated FHR-4 levels on AMD susceptibility by haplotype group. Log-transformed OR and 95% CI were generated using the IAMDGC cohort (13,378 controls and 17,541 cases). In b , c , and d , OR, 95% CI and p -values (two-sided) for comparisons between haplotypes were obtained using logistic regressions including AMD case/control status as the dependent variable and age, sex, and the first two genetic principal components as covariates. In all box plots the horizontal center lines correspond to the medians of the log-transformed and centered FHR-4 distribution and the boxes delineate the 25th/75th percentile. The vertical solid lines represent the full range of the log-transformed and centered FHR-4 distribution in each group. Dots beyond this line indicate potential outliers. Associations with log-transformed and centered FHR-4 levels were assessed using the Kruskal–Wallis test. Post-hoc pairwise comparisons were performed using the Conover–Iman test. All p -values are two-sided and adjusted for multiple testing using Bonferroni corrections. Source data for these plots are provided as a Source Data file.
    Figure Legend Snippet: a Haplotype analyses based on the AMD-associated risk allele C at rs1061170, protective allele A at rs800292, and protective CFHR3/1 deletion tagging allele T at rs12144939 and independent quantitative trait loci (QTL) for CFHR4 rs61818956, rs10494745, and rs7531555. Odds ratios (OR), confidence intervals (CI), and p -values (two-sided) were obtained using logistic regressions including AMD case/control status as the dependent variable and age, sex, and the first two genetic principal components for the IAMDGC cohort as covariates. Bonferroni correction for multiple testing of 11 haplotypes = 0.0045 (0.05/11). The differential effects on FHR-4 levels associated with each haplotype (elevated, baseline, or reduced) is indicated. b Box plot showing variations in log-transformed and centered FHR-4 levels with the four common AMD homozygous Chr1 diplotypes (Risk, Neutral, Prot-I62, and Prot-Del) (346 independent biological samples). c Comparison of Prot-I62 haplotypes with and without the effect allele (T) at rs7531555 generated using the IAMDGC cohort (13,378 controls and 17,541 cases) along with a box plot showing variations in log-transformed and centered FHR-4 levels among subjects stratified by rs800292 and rs7531555 diplotypes (149 independent biological samples). d Comparison of Risk haplotypes with and without the effect allele at rs10494745 and rs61818956 generated using the IAMDGC cohort (13,378 controls and 17,541 cases) along with a box plot showing variations in log-transformed and centered FHR-4 levels among subjects with Risk/Risk diplotypes stratified by rs10494745 (AA or GG) and rs61818956 (CC or TT) diplotypes (56 independent biological samples). e Schematic describing the effect of reduced or elevated FHR-4 levels on AMD susceptibility by haplotype group. Log-transformed OR and 95% CI were generated using the IAMDGC cohort (13,378 controls and 17,541 cases). In b , c , and d , OR, 95% CI and p -values (two-sided) for comparisons between haplotypes were obtained using logistic regressions including AMD case/control status as the dependent variable and age, sex, and the first two genetic principal components as covariates. In all box plots the horizontal center lines correspond to the medians of the log-transformed and centered FHR-4 distribution and the boxes delineate the 25th/75th percentile. The vertical solid lines represent the full range of the log-transformed and centered FHR-4 distribution in each group. Dots beyond this line indicate potential outliers. Associations with log-transformed and centered FHR-4 levels were assessed using the Kruskal–Wallis test. Post-hoc pairwise comparisons were performed using the Conover–Iman test. All p -values are two-sided and adjusted for multiple testing using Bonferroni corrections. Source data for these plots are provided as a Source Data file.

    Techniques Used: Transformation Assay, Comparison, Generated

    a Patients with a Risk/Risk diplotype and no risk alleles at the Chr10 AMD locus were selected based on genotype at rs61818956 (TT, FHR-4 ↑ group) and rs10494745 (AA, FHR-4 ↓ group), with the reference group labeled FHR-4 ↔ (CC at rs61818956, GG at rs10494745). b Table showing the number and frequency of conversions recorded in each eye that met the inclusion criteria. Differences between groups were assessed using a chi-squared test. P -values are two-sided. c Box plots showing the median age of first recorded conversion to late AMD (cRORA or neovascular AMD) and conversion curves for earliest conversion generated using Cox proportional hazard regression models adjusted for age and AMD severity at first visit (81 patients, see a ). In the box plot, the horizontal center lines correspond to the medians and the boxes delineate the 25th/75th percentile. The vertical solid lines represent the full range of the age distribution in each group. Dots beyond this line indicate potential outliers. Association with age at first conversion was assessed using the Kruskal–Wallis test. Associations with time to conversion were determined using the log-rank test. All p -values are two-sided. d Hazard ratios and 95% confidence interval (CI) generated using mixed-effect Cox proportional hazard regression models adjusted for age and AMD severity at first visit and including a frailty term to account for correlations between eyes from the same patient (131 eyes from 81 patients, see a ). All p -values are two-sided.
    Figure Legend Snippet: a Patients with a Risk/Risk diplotype and no risk alleles at the Chr10 AMD locus were selected based on genotype at rs61818956 (TT, FHR-4 ↑ group) and rs10494745 (AA, FHR-4 ↓ group), with the reference group labeled FHR-4 ↔ (CC at rs61818956, GG at rs10494745). b Table showing the number and frequency of conversions recorded in each eye that met the inclusion criteria. Differences between groups were assessed using a chi-squared test. P -values are two-sided. c Box plots showing the median age of first recorded conversion to late AMD (cRORA or neovascular AMD) and conversion curves for earliest conversion generated using Cox proportional hazard regression models adjusted for age and AMD severity at first visit (81 patients, see a ). In the box plot, the horizontal center lines correspond to the medians and the boxes delineate the 25th/75th percentile. The vertical solid lines represent the full range of the age distribution in each group. Dots beyond this line indicate potential outliers. Association with age at first conversion was assessed using the Kruskal–Wallis test. Associations with time to conversion were determined using the log-rank test. All p -values are two-sided. d Hazard ratios and 95% confidence interval (CI) generated using mixed-effect Cox proportional hazard regression models adjusted for age and AMD severity at first visit and including a frailty term to account for correlations between eyes from the same patient (131 eyes from 81 patients, see a ). All p -values are two-sided.

    Techniques Used: Labeling, Generated

    af4999  (R&D Systems)


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    R&D Systems mouse anti human fhr4 af488 mab
    <t>FHR4/V</t> H H(T), FHR4/V H H(P) and V H H(P)/Fc molecules reduce tumor growth, whereas V H H(T)/Fc has no beneficial effect. (A) Experimental design for the measurement of the subcutaneous xenografts mammary fat pads volume in the presence of different CoMiX molecules and control antibodies. BT474 cells were injected into the mammary fat pads of mice. When the tumor volume reached ∼60 mm 3 , the mice were injected intravenously into the lateral tail vein with 100 µg of CoMiX molecules or control antibodies. The injection was repeated 9 times on days 1, 2, 3, 4, 7, 8, 9 and 10 after the first injection. For molecule combinations, 50 µg of each were injected. The tumors were measured every second or third day with calipers. (B) The therapeutic effects of five CoMiX molecules [FHR4/V H H(T), FHR4/V H H(P), V H H(T)/Fc, V H H(P)/Fc, V H H(T)] and two control antibodies (trastuzumab and pertuzumab) were evaluated individually and in combination. The treatment duration is indicated for all groups in green, as mentioned for the first FHR4/V H H(T) group in the left side of the graph. CoMiX-FHR4 molecules are more effective than CoMiX-Fcs, while the molecules that have the pertuzumab-competing epitope are more effective than those with the trastuzumab-competing epitope. (C) MRI images of a representative tumor for each group. Mice were sacrificed at the end of the treatment.
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    <t>FHR4/V</t> H H(T), FHR4/V H H(P) and V H H(P)/Fc molecules reduce tumor growth, whereas V H H(T)/Fc has no beneficial effect. (A) Experimental design for the measurement of the subcutaneous xenografts mammary fat pads volume in the presence of different CoMiX molecules and control antibodies. BT474 cells were injected into the mammary fat pads of mice. When the tumor volume reached ∼60 mm 3 , the mice were injected intravenously into the lateral tail vein with 100 µg of CoMiX molecules or control antibodies. The injection was repeated 9 times on days 1, 2, 3, 4, 7, 8, 9 and 10 after the first injection. For molecule combinations, 50 µg of each were injected. The tumors were measured every second or third day with calipers. (B) The therapeutic effects of five CoMiX molecules [FHR4/V H H(T), FHR4/V H H(P), V H H(T)/Fc, V H H(P)/Fc, V H H(T)] and two control antibodies (trastuzumab and pertuzumab) were evaluated individually and in combination. The treatment duration is indicated for all groups in green, as mentioned for the first FHR4/V H H(T) group in the left side of the graph. CoMiX-FHR4 molecules are more effective than CoMiX-Fcs, while the molecules that have the pertuzumab-competing epitope are more effective than those with the trastuzumab-competing epitope. (C) MRI images of a representative tumor for each group. Mice were sacrificed at the end of the treatment.
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    <t>FHR4/V</t> H H(T), FHR4/V H H(P) and V H H(P)/Fc molecules reduce tumor growth, whereas V H H(T)/Fc has no beneficial effect. (A) Experimental design for the measurement of the subcutaneous xenografts mammary fat pads volume in the presence of different CoMiX molecules and control antibodies. BT474 cells were injected into the mammary fat pads of mice. When the tumor volume reached ∼60 mm 3 , the mice were injected intravenously into the lateral tail vein with 100 µg of CoMiX molecules or control antibodies. The injection was repeated 9 times on days 1, 2, 3, 4, 7, 8, 9 and 10 after the first injection. For molecule combinations, 50 µg of each were injected. The tumors were measured every second or third day with calipers. (B) The therapeutic effects of five CoMiX molecules [FHR4/V H H(T), FHR4/V H H(P), V H H(T)/Fc, V H H(P)/Fc, V H H(T)] and two control antibodies (trastuzumab and pertuzumab) were evaluated individually and in combination. The treatment duration is indicated for all groups in green, as mentioned for the first FHR4/V H H(T) group in the left side of the graph. CoMiX-FHR4 molecules are more effective than CoMiX-Fcs, while the molecules that have the pertuzumab-competing epitope are more effective than those with the trastuzumab-competing epitope. (C) MRI images of a representative tumor for each group. Mice were sacrificed at the end of the treatment.
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    R&D Systems af4999
    <t>FHR4/V</t> H H(T), FHR4/V H H(P) and V H H(P)/Fc molecules reduce tumor growth, whereas V H H(T)/Fc has no beneficial effect. (A) Experimental design for the measurement of the subcutaneous xenografts mammary fat pads volume in the presence of different CoMiX molecules and control antibodies. BT474 cells were injected into the mammary fat pads of mice. When the tumor volume reached ∼60 mm 3 , the mice were injected intravenously into the lateral tail vein with 100 µg of CoMiX molecules or control antibodies. The injection was repeated 9 times on days 1, 2, 3, 4, 7, 8, 9 and 10 after the first injection. For molecule combinations, 50 µg of each were injected. The tumors were measured every second or third day with calipers. (B) The therapeutic effects of five CoMiX molecules [FHR4/V H H(T), FHR4/V H H(P), V H H(T)/Fc, V H H(P)/Fc, V H H(T)] and two control antibodies (trastuzumab and pertuzumab) were evaluated individually and in combination. The treatment duration is indicated for all groups in green, as mentioned for the first FHR4/V H H(T) group in the left side of the graph. CoMiX-FHR4 molecules are more effective than CoMiX-Fcs, while the molecules that have the pertuzumab-competing epitope are more effective than those with the trastuzumab-competing epitope. (C) MRI images of a representative tumor for each group. Mice were sacrificed at the end of the treatment.
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    R&D Systems mouse monoclonal anti human cfhr5
    <t>FHR4/V</t> H H(T), FHR4/V H H(P) and V H H(P)/Fc molecules reduce tumor growth, whereas V H H(T)/Fc has no beneficial effect. (A) Experimental design for the measurement of the subcutaneous xenografts mammary fat pads volume in the presence of different CoMiX molecules and control antibodies. BT474 cells were injected into the mammary fat pads of mice. When the tumor volume reached ∼60 mm 3 , the mice were injected intravenously into the lateral tail vein with 100 µg of CoMiX molecules or control antibodies. The injection was repeated 9 times on days 1, 2, 3, 4, 7, 8, 9 and 10 after the first injection. For molecule combinations, 50 µg of each were injected. The tumors were measured every second or third day with calipers. (B) The therapeutic effects of five CoMiX molecules [FHR4/V H H(T), FHR4/V H H(P), V H H(T)/Fc, V H H(P)/Fc, V H H(T)] and two control antibodies (trastuzumab and pertuzumab) were evaluated individually and in combination. The treatment duration is indicated for all groups in green, as mentioned for the first FHR4/V H H(T) group in the left side of the graph. CoMiX-FHR4 molecules are more effective than CoMiX-Fcs, while the molecules that have the pertuzumab-competing epitope are more effective than those with the trastuzumab-competing epitope. (C) MRI images of a representative tumor for each group. Mice were sacrificed at the end of the treatment.
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    FHR4/V H H(T), FHR4/V H H(P) and V H H(P)/Fc molecules reduce tumor growth, whereas V H H(T)/Fc has no beneficial effect. (A) Experimental design for the measurement of the subcutaneous xenografts mammary fat pads volume in the presence of different CoMiX molecules and control antibodies. BT474 cells were injected into the mammary fat pads of mice. When the tumor volume reached ∼60 mm 3 , the mice were injected intravenously into the lateral tail vein with 100 µg of CoMiX molecules or control antibodies. The injection was repeated 9 times on days 1, 2, 3, 4, 7, 8, 9 and 10 after the first injection. For molecule combinations, 50 µg of each were injected. The tumors were measured every second or third day with calipers. (B) The therapeutic effects of five CoMiX molecules [FHR4/V H H(T), FHR4/V H H(P), V H H(T)/Fc, V H H(P)/Fc, V H H(T)] and two control antibodies (trastuzumab and pertuzumab) were evaluated individually and in combination. The treatment duration is indicated for all groups in green, as mentioned for the first FHR4/V H H(T) group in the left side of the graph. CoMiX-FHR4 molecules are more effective than CoMiX-Fcs, while the molecules that have the pertuzumab-competing epitope are more effective than those with the trastuzumab-competing epitope. (C) MRI images of a representative tumor for each group. Mice were sacrificed at the end of the treatment.

    Journal: bioRxiv

    Article Title: Complement-Activating Multimeric Immunotherapeutic Complexes for HER2-breast cancer immunotherapy

    doi: 10.1101/2024.02.02.578619

    Figure Lengend Snippet: FHR4/V H H(T), FHR4/V H H(P) and V H H(P)/Fc molecules reduce tumor growth, whereas V H H(T)/Fc has no beneficial effect. (A) Experimental design for the measurement of the subcutaneous xenografts mammary fat pads volume in the presence of different CoMiX molecules and control antibodies. BT474 cells were injected into the mammary fat pads of mice. When the tumor volume reached ∼60 mm 3 , the mice were injected intravenously into the lateral tail vein with 100 µg of CoMiX molecules or control antibodies. The injection was repeated 9 times on days 1, 2, 3, 4, 7, 8, 9 and 10 after the first injection. For molecule combinations, 50 µg of each were injected. The tumors were measured every second or third day with calipers. (B) The therapeutic effects of five CoMiX molecules [FHR4/V H H(T), FHR4/V H H(P), V H H(T)/Fc, V H H(P)/Fc, V H H(T)] and two control antibodies (trastuzumab and pertuzumab) were evaluated individually and in combination. The treatment duration is indicated for all groups in green, as mentioned for the first FHR4/V H H(T) group in the left side of the graph. CoMiX-FHR4 molecules are more effective than CoMiX-Fcs, while the molecules that have the pertuzumab-competing epitope are more effective than those with the trastuzumab-competing epitope. (C) MRI images of a representative tumor for each group. Mice were sacrificed at the end of the treatment.

    Article Snippet: Mouse anti-human FHR4 AF488 mAb was purchased from R&D Systems Europe Ltd, (#IC5980G-100UG, clone 640212, Bio-Techne, Abingdon, UK).

    Techniques: Injection

    : Design of CoMiX-FHR4, CoMiX-Fc and the controls V H H(T) and V H H(T)/Fc Δhinge. All constructs are co-transfected with the eGFP.C4bpβ construct, leading to the covalent association of a single eGFP tracking function with the multimeric fusion C4bp α core with the a-chains represented in red lines. We used V H H(T) and V H H(P), recognising trastuzumab- or pertuzumab-competing HER2 epitopes, respectively, to generate 2 types of CoMiX-FHR4 molecules: FHR4/V H H(T) and FHR4/V H H(P)] or 2 types of CoMiX-Fc molecules :V H H(T)/Fc or V H H(P)/Fc. A dual hinge region between the C4bpα-scaffold and the IgG1 CH2-CH3 (represented by two red bands between the two Fc fragments) allows the formation of interchain disulfide bonds and the dimerisation of Fc-regions. C4bpα.His8x V H H(T) is the control multimeric molecule with no effector function, and so called V H H(T), and V H H(T)/Fc Δhinge is the control molecule of V H H(T)/Fc without hinge that allows the formation of triple Fc dimers.

    Journal: bioRxiv

    Article Title: Complement-Activating Multimeric Immunotherapeutic Complexes for HER2-breast cancer immunotherapy

    doi: 10.1101/2024.02.02.578619

    Figure Lengend Snippet: : Design of CoMiX-FHR4, CoMiX-Fc and the controls V H H(T) and V H H(T)/Fc Δhinge. All constructs are co-transfected with the eGFP.C4bpβ construct, leading to the covalent association of a single eGFP tracking function with the multimeric fusion C4bp α core with the a-chains represented in red lines. We used V H H(T) and V H H(P), recognising trastuzumab- or pertuzumab-competing HER2 epitopes, respectively, to generate 2 types of CoMiX-FHR4 molecules: FHR4/V H H(T) and FHR4/V H H(P)] or 2 types of CoMiX-Fc molecules :V H H(T)/Fc or V H H(P)/Fc. A dual hinge region between the C4bpα-scaffold and the IgG1 CH2-CH3 (represented by two red bands between the two Fc fragments) allows the formation of interchain disulfide bonds and the dimerisation of Fc-regions. C4bpα.His8x V H H(T) is the control multimeric molecule with no effector function, and so called V H H(T), and V H H(T)/Fc Δhinge is the control molecule of V H H(T)/Fc without hinge that allows the formation of triple Fc dimers.

    Article Snippet: Mouse anti-human FHR4 AF488 mAb was purchased from R&D Systems Europe Ltd, (#IC5980G-100UG, clone 640212, Bio-Techne, Abingdon, UK).

    Techniques: Construct, Transfection

    : Visualization of the molecular pattern of purified multimeric immunoconjugates by Western blot analysis of complexes separated under non-reducing conditions. (A, B) and SYPRO Ruby protein gel staining under reducing conditions (C) . (A) 1. FHR4/V H H(T) fraction 1, 2. FHR4/V H H(T) fraction 2/3, 3. FHR4/V H H(P) fraction 1, 4. FHR4/V H H(P) fraction 2/3. Under non-reducing conditions, seven bands are visible for the different fractions corresponding to FHR4-valencies varying between 1 and 7. The pooled fractions f2 and f3 display higher FHR4-valencies than their f1 counterpart and were used for further experiments. (B) 1. FHR4/V H H(T) fraction 1, 2. FHR4/V H H(T) fraction 2/3, 3. FHR4/V H H(P) fraction 1, 4. FHR4/V H H(P) fraction 2/3, 5. V H H(P)/Fc, 6. V H H(T)/Fc, 7. V H H(T)/Fc Δhinge, 8. V H H(T). The different molecular species were analyzed and revealed with a goat anti-human IgG antibody that cross-reacts with the V H H region. (C) 1. FHR4/V H H(T) fraction 1, 2. FHR4/V H H(T) fraction 2/3, 3. FHR4/V H H(P) fraction 1, 4. FHR4/V H H(P) fraction 2/3, 5. V H H(P)/Fc, 6. V H H(T)/Fc, 7. V H H(T)/Fc Δhinge, 8. V H H(T). The multimers were also analyzed by SYPRO Ruby gel staining under reducing conditions. Three bands can be observed for FHR4/V H H(T) and FHR4/V H H(P), representing the monomeric forms of the FHR4.C4bpα.His (120 kDa), eGFP.SCR3.C4bpβ (50 kDa) and the V H H(T).C4bpα.FLAG (40 kDa) or V H H(P).C4bpα.FLAG (30 kDa) targeting components. V H H(T)/Fc and V H H(P)/Fc molecules display two bands representing the eGFP.SCR3.C4bpβ and V H H(T).C4bpα.Fc chains and V H H(P).C4bpα.Fc, respectively. The V H H(T) control molecule has no FHR4- or Fc-effector functions, only targeting (V H H(T).C4bpα.His) and tracking (eGFP.SCR3.C4bpβ) functions, whereas V H H(T)/Fc Δhinge shows one band for V H H(T).C4bpα.Fc.

    Journal: bioRxiv

    Article Title: Complement-Activating Multimeric Immunotherapeutic Complexes for HER2-breast cancer immunotherapy

    doi: 10.1101/2024.02.02.578619

    Figure Lengend Snippet: : Visualization of the molecular pattern of purified multimeric immunoconjugates by Western blot analysis of complexes separated under non-reducing conditions. (A, B) and SYPRO Ruby protein gel staining under reducing conditions (C) . (A) 1. FHR4/V H H(T) fraction 1, 2. FHR4/V H H(T) fraction 2/3, 3. FHR4/V H H(P) fraction 1, 4. FHR4/V H H(P) fraction 2/3. Under non-reducing conditions, seven bands are visible for the different fractions corresponding to FHR4-valencies varying between 1 and 7. The pooled fractions f2 and f3 display higher FHR4-valencies than their f1 counterpart and were used for further experiments. (B) 1. FHR4/V H H(T) fraction 1, 2. FHR4/V H H(T) fraction 2/3, 3. FHR4/V H H(P) fraction 1, 4. FHR4/V H H(P) fraction 2/3, 5. V H H(P)/Fc, 6. V H H(T)/Fc, 7. V H H(T)/Fc Δhinge, 8. V H H(T). The different molecular species were analyzed and revealed with a goat anti-human IgG antibody that cross-reacts with the V H H region. (C) 1. FHR4/V H H(T) fraction 1, 2. FHR4/V H H(T) fraction 2/3, 3. FHR4/V H H(P) fraction 1, 4. FHR4/V H H(P) fraction 2/3, 5. V H H(P)/Fc, 6. V H H(T)/Fc, 7. V H H(T)/Fc Δhinge, 8. V H H(T). The multimers were also analyzed by SYPRO Ruby gel staining under reducing conditions. Three bands can be observed for FHR4/V H H(T) and FHR4/V H H(P), representing the monomeric forms of the FHR4.C4bpα.His (120 kDa), eGFP.SCR3.C4bpβ (50 kDa) and the V H H(T).C4bpα.FLAG (40 kDa) or V H H(P).C4bpα.FLAG (30 kDa) targeting components. V H H(T)/Fc and V H H(P)/Fc molecules display two bands representing the eGFP.SCR3.C4bpβ and V H H(T).C4bpα.Fc chains and V H H(P).C4bpα.Fc, respectively. The V H H(T) control molecule has no FHR4- or Fc-effector functions, only targeting (V H H(T).C4bpα.His) and tracking (eGFP.SCR3.C4bpβ) functions, whereas V H H(T)/Fc Δhinge shows one band for V H H(T).C4bpα.Fc.

    Article Snippet: Mouse anti-human FHR4 AF488 mAb was purchased from R&D Systems Europe Ltd, (#IC5980G-100UG, clone 640212, Bio-Techne, Abingdon, UK).

    Techniques: Purification, Western Blot, Staining

    : Dose response analysis of C3b/iC3b deposition (A), MAC formation (B), and complement-dependent cytotoxicity (C) on BT474 tumor cells incubated with 3-fold serial dilutions: from 15 µg to 0.5 µg/well in case of individual molecules, and from 7.5 µg to 0.25 µg/well of each in case of molecule combinations. As controls, therapeutic antibodies and NHS were used. (A) C3b/iC3b deposition was detected with mouse anti-human C3b mAb and a secondary goat anti-mouse IgG Ab conjugated with AF647. CoMiX-Fc and CoMiX-FHR4 molecules elicit stronger complement activating effects than trastuzumab, pertuzumab and the combination of these two antibodies. Combining CoMiX-Fc and CoMiX-FHR4 molecules with other multimers resulted in the highest level of C3b desposition. (B) Staining with anti-C5b9 mAb followed by PE-conjugated anti-mouse IgG pAb was used to detect membrane attack complex (MAC) formation. (C) The percentage of dead cells was calculated by dividing the number of live/dead-positive (dead) cells with the total number of analysed cells. Data are presented as mean values ±SD of n = 3 independent experiments.

    Journal: bioRxiv

    Article Title: Complement-Activating Multimeric Immunotherapeutic Complexes for HER2-breast cancer immunotherapy

    doi: 10.1101/2024.02.02.578619

    Figure Lengend Snippet: : Dose response analysis of C3b/iC3b deposition (A), MAC formation (B), and complement-dependent cytotoxicity (C) on BT474 tumor cells incubated with 3-fold serial dilutions: from 15 µg to 0.5 µg/well in case of individual molecules, and from 7.5 µg to 0.25 µg/well of each in case of molecule combinations. As controls, therapeutic antibodies and NHS were used. (A) C3b/iC3b deposition was detected with mouse anti-human C3b mAb and a secondary goat anti-mouse IgG Ab conjugated with AF647. CoMiX-Fc and CoMiX-FHR4 molecules elicit stronger complement activating effects than trastuzumab, pertuzumab and the combination of these two antibodies. Combining CoMiX-Fc and CoMiX-FHR4 molecules with other multimers resulted in the highest level of C3b desposition. (B) Staining with anti-C5b9 mAb followed by PE-conjugated anti-mouse IgG pAb was used to detect membrane attack complex (MAC) formation. (C) The percentage of dead cells was calculated by dividing the number of live/dead-positive (dead) cells with the total number of analysed cells. Data are presented as mean values ±SD of n = 3 independent experiments.

    Article Snippet: Mouse anti-human FHR4 AF488 mAb was purchased from R&D Systems Europe Ltd, (#IC5980G-100UG, clone 640212, Bio-Techne, Abingdon, UK).

    Techniques: Incubation, Staining, Membrane

    Flow cytometry analysis of C3b/iC3b deposition (A), membrane attack complex formation (B) , and complement-dependent cytotoxicity (C) on BT474 tumor cells incubated with 15 µg/well of multimeric immunotherapeutic complexes. (A) C3b/iC3b deposition was detected with mouse anti-human C3b mAb and a secondary goat anti-mouse IgG Ab conjugated with AF647. (B) MAC formation was analyzed using anti-C5b9 mAb followed by PE-conjugated anti-mouse IgG pAb. MAC-formation was highest when CoMiX-Fc and CoMiX-FHR4 molecules were combined. (C) The percentage of dead cells was calculated by dividing the number of live/dead-positive (dead) cells with the total number of analyzed cells. (D) A linear correlation between C3b deposition (MFI) and the percentage of dead cells at 15 µg/well of molecules was observed. Consistent with C3b deposition and MAC-formation, CoMiX-Fc and CoMiX-FHR4 significantly increased the percentage of dead cells compared to control multimers and therapeutic antibodies. Data are presented as mean values ±SD of n = 3 independent experiments. Statistical analysis was performed using a one-way ANOVA and post-hoc Tukey test (**p < 0.005, ***p < 0.001, ****p < 0.0001).

    Journal: bioRxiv

    Article Title: Complement-Activating Multimeric Immunotherapeutic Complexes for HER2-breast cancer immunotherapy

    doi: 10.1101/2024.02.02.578619

    Figure Lengend Snippet: Flow cytometry analysis of C3b/iC3b deposition (A), membrane attack complex formation (B) , and complement-dependent cytotoxicity (C) on BT474 tumor cells incubated with 15 µg/well of multimeric immunotherapeutic complexes. (A) C3b/iC3b deposition was detected with mouse anti-human C3b mAb and a secondary goat anti-mouse IgG Ab conjugated with AF647. (B) MAC formation was analyzed using anti-C5b9 mAb followed by PE-conjugated anti-mouse IgG pAb. MAC-formation was highest when CoMiX-Fc and CoMiX-FHR4 molecules were combined. (C) The percentage of dead cells was calculated by dividing the number of live/dead-positive (dead) cells with the total number of analyzed cells. (D) A linear correlation between C3b deposition (MFI) and the percentage of dead cells at 15 µg/well of molecules was observed. Consistent with C3b deposition and MAC-formation, CoMiX-Fc and CoMiX-FHR4 significantly increased the percentage of dead cells compared to control multimers and therapeutic antibodies. Data are presented as mean values ±SD of n = 3 independent experiments. Statistical analysis was performed using a one-way ANOVA and post-hoc Tukey test (**p < 0.005, ***p < 0.001, ****p < 0.0001).

    Article Snippet: Mouse anti-human FHR4 AF488 mAb was purchased from R&D Systems Europe Ltd, (#IC5980G-100UG, clone 640212, Bio-Techne, Abingdon, UK).

    Techniques: Flow Cytometry, Membrane, Incubation

    : FHR4-based CoMiX molecules activate the alternative complement pathway, whereas Fc-based CoMiX molecules facilitate classical pathway activation. C3b deposition (A) and CDC (B) of BT474 tumor cells incubated with saturating concentrations (15 µl/well) of CoMiX molecules and control mAbs individually or in combinations. 25% NHS diluted in either GVB ++ or GVB + buffer was added for 30 minutes at 37°C. Inhibition of the classical complement pathway by using GVB + buffer completely disrupts the complement activating properties of Fc-based CoMiX molecules, trastuzumab and pertuzumab. Data are presented as mean values ±SD of n = 3 independent experiments. Statistical analysis was performed using a two-way ANOVA test between GVB ++ and GVB + conditions for each molecule. All comparisons between GVB ++ and GVB + reached statistical significance (****p < 0.0001). (C) Representative histogram plots on live BT474 cells of C3b MFI for the combinations of molecules with GVB + and GVB ++ conditions are shown. (D) Representative dots plots of live and dead BT474 cells with the different combinations are depicted.

    Journal: bioRxiv

    Article Title: Complement-Activating Multimeric Immunotherapeutic Complexes for HER2-breast cancer immunotherapy

    doi: 10.1101/2024.02.02.578619

    Figure Lengend Snippet: : FHR4-based CoMiX molecules activate the alternative complement pathway, whereas Fc-based CoMiX molecules facilitate classical pathway activation. C3b deposition (A) and CDC (B) of BT474 tumor cells incubated with saturating concentrations (15 µl/well) of CoMiX molecules and control mAbs individually or in combinations. 25% NHS diluted in either GVB ++ or GVB + buffer was added for 30 minutes at 37°C. Inhibition of the classical complement pathway by using GVB + buffer completely disrupts the complement activating properties of Fc-based CoMiX molecules, trastuzumab and pertuzumab. Data are presented as mean values ±SD of n = 3 independent experiments. Statistical analysis was performed using a two-way ANOVA test between GVB ++ and GVB + conditions for each molecule. All comparisons between GVB ++ and GVB + reached statistical significance (****p < 0.0001). (C) Representative histogram plots on live BT474 cells of C3b MFI for the combinations of molecules with GVB + and GVB ++ conditions are shown. (D) Representative dots plots of live and dead BT474 cells with the different combinations are depicted.

    Article Snippet: Mouse anti-human FHR4 AF488 mAb was purchased from R&D Systems Europe Ltd, (#IC5980G-100UG, clone 640212, Bio-Techne, Abingdon, UK).

    Techniques: Activation Assay, Incubation, Inhibition

    Immunofluorescent staining of tumor sections collected 1 or 6 hours after injection of CoMiX-FHR4 (upper panel), CoMiX-Fc (intermediate panel) or controls (lower panel with anti-C3d staining): PBS (1), V H H(T) (2), trastuzumab + pertuzumab (3). CoMiX were visualized with either a rabbit anti-His mAb followed by the goat Anti-Rabbit IgG Fc AF568- or a goat anti-human IgG AF647-conjugated antibody. Complement activation was visualized using the polyclonal rabbit anti-C3d antibody followed by AF568-conjugated anti-rabbit IgG. One hour post-injection, the infiltration of molecules into the tumor tissue is already visible, however complement activation occurs predominantly on the periphery of the tumors. Six hours after treatment, the molecules homogeneously infiltrate the tumor and strong complement activation can be detected throughout the whole tissue. Compared to CoMiX-FHR4 molecules, the V H H(T) control (2) shows decreased infiltration and reduced complement activation, present only at the periphery of the tumor, even if collected 6 hours after injection. Trastuzumab + pertuzumab (3) were used as positive controls and showed significant infiltration and complement activation.

    Journal: bioRxiv

    Article Title: Complement-Activating Multimeric Immunotherapeutic Complexes for HER2-breast cancer immunotherapy

    doi: 10.1101/2024.02.02.578619

    Figure Lengend Snippet: Immunofluorescent staining of tumor sections collected 1 or 6 hours after injection of CoMiX-FHR4 (upper panel), CoMiX-Fc (intermediate panel) or controls (lower panel with anti-C3d staining): PBS (1), V H H(T) (2), trastuzumab + pertuzumab (3). CoMiX were visualized with either a rabbit anti-His mAb followed by the goat Anti-Rabbit IgG Fc AF568- or a goat anti-human IgG AF647-conjugated antibody. Complement activation was visualized using the polyclonal rabbit anti-C3d antibody followed by AF568-conjugated anti-rabbit IgG. One hour post-injection, the infiltration of molecules into the tumor tissue is already visible, however complement activation occurs predominantly on the periphery of the tumors. Six hours after treatment, the molecules homogeneously infiltrate the tumor and strong complement activation can be detected throughout the whole tissue. Compared to CoMiX-FHR4 molecules, the V H H(T) control (2) shows decreased infiltration and reduced complement activation, present only at the periphery of the tumor, even if collected 6 hours after injection. Trastuzumab + pertuzumab (3) were used as positive controls and showed significant infiltration and complement activation.

    Article Snippet: Mouse anti-human FHR4 AF488 mAb was purchased from R&D Systems Europe Ltd, (#IC5980G-100UG, clone 640212, Bio-Techne, Abingdon, UK).

    Techniques: Staining, Injection, Activation Assay

    FHR4/V H H(T) and FHR4/V H H(P) CoMiX molecules exert their anti-tumor effect on trastuzumab-resistant BT474 cells. Trastuzumab-resistant BT474 cells were injected into the mammary fat pads of female BALB/c NUDE mice. When the tumor volume reached ∼60 mm 3 , the mice were injected with 100 µg of CoMiX-FHR4 molecules, trastuzumab or PBS, as described on . The tumors were measured every second or third day until day 37 of the study or until meeting a humane endpoint. Trastuzumab and PBS had no beneficial effect on tumor growth, whereas CoMiX-FHR4 molecules were shown to significantly reduce tumor progression. (B) Cryosections of trastuzumab-resistant BT474 tumor xenografts collected just after the end of the treatment (at D+11). Tumors were embedded in OCT and snap frozen in OCT. Four micrometer cryosections were made and stained with a monoclonal rat IgG 2A anti-mouse NKp46/NCR1 antibody and revealed using a donkey anti-rat AF568-conjugated pAb. Confocal microscope was used to make pictures (lens X40), monitored by the Nikon NIS-Elements software which allowed to assemble pictures to get a large field overview of the tumors. A) tumor treated with combined CoMiX-FHR4 [FHR4/V H H(T) + FHR4/V H H(P)], B) tumor treated with trastuzumab, C) tumor treated with PBS (mock).

    Journal: bioRxiv

    Article Title: Complement-Activating Multimeric Immunotherapeutic Complexes for HER2-breast cancer immunotherapy

    doi: 10.1101/2024.02.02.578619

    Figure Lengend Snippet: FHR4/V H H(T) and FHR4/V H H(P) CoMiX molecules exert their anti-tumor effect on trastuzumab-resistant BT474 cells. Trastuzumab-resistant BT474 cells were injected into the mammary fat pads of female BALB/c NUDE mice. When the tumor volume reached ∼60 mm 3 , the mice were injected with 100 µg of CoMiX-FHR4 molecules, trastuzumab or PBS, as described on . The tumors were measured every second or third day until day 37 of the study or until meeting a humane endpoint. Trastuzumab and PBS had no beneficial effect on tumor growth, whereas CoMiX-FHR4 molecules were shown to significantly reduce tumor progression. (B) Cryosections of trastuzumab-resistant BT474 tumor xenografts collected just after the end of the treatment (at D+11). Tumors were embedded in OCT and snap frozen in OCT. Four micrometer cryosections were made and stained with a monoclonal rat IgG 2A anti-mouse NKp46/NCR1 antibody and revealed using a donkey anti-rat AF568-conjugated pAb. Confocal microscope was used to make pictures (lens X40), monitored by the Nikon NIS-Elements software which allowed to assemble pictures to get a large field overview of the tumors. A) tumor treated with combined CoMiX-FHR4 [FHR4/V H H(T) + FHR4/V H H(P)], B) tumor treated with trastuzumab, C) tumor treated with PBS (mock).

    Article Snippet: Mouse anti-human FHR4 AF488 mAb was purchased from R&D Systems Europe Ltd, (#IC5980G-100UG, clone 640212, Bio-Techne, Abingdon, UK).

    Techniques: Injection, Staining, Microscopy, Software