Journal: PLoS ONE

Article Title: Differential Expression of Complement Markers in Normal and AMD Transmitochondrial Cybrids

doi: 10.1371/journal.pone.0159828

Figure Lengend Snippet: The classical pathway is triggered by interaction of C1 with immune or non-immune complexes leading to conformational changes in C1q, activation of C1r and C1s, and subsequent assembly of C3 convertase. The lectin pathway is activated by binding of mannose-binding lectin (MBL) to mannose residues on the pathogen surface, which activates the MBL-associated serine proteases (MASPs), followed by formation of C3 convertase. Spontaneous hydrolysis of C3 initiates the alternative complement pathway. All three pathways of the complement cascade converge on the classical C3 convertase, which cleaves and activates component C3, forming C3a and C3b. This triggers a series of further cleavage and activation events, leading to cleavage of C5 into C5a and C5b, and eventual formation of the membrane attack complex (MAC), consisting of C5b, C6, C7, C8, and C9. MAC is the terminal cytolytic complex of the complement pathway; it causes osmotic lysis of target cells by forming transmembrane channels that disrupt the phospholipid bilayer of target cells. The complement system is tightly regulated by the following complement control proteins: CFH, CFHR1, CFHR4, CFB, CD55, CD59, CD46, CFI, and CFP.

Article Snippet: 8 , CFHR4 , 65 kDa , Mouse anti-Factor H-related 4 (CFHR4) Monoclonal antibody #MAB5980 (RD Systems) , 1:1000 , Human , Anti-mouse IgG, HRP-linked antibody #7076 (CST) , beta-actin antibody GTX 110564 (Genetex).

Techniques: Activation Assay, Binding Assay, Membrane, Lysis, Control

Journal: PLoS ONE

Article Title: Differential Expression of Complement Markers in Normal and AMD Transmitochondrial Cybrids

doi: 10.1371/journal.pone.0159828

Figure Lengend Snippet: Complement Markers’ Information.

Article Snippet: 8 , CFHR4 , 65 kDa , Mouse anti-Factor H-related 4 (CFHR4) Monoclonal antibody #MAB5980 (RD Systems) , 1:1000 , Human , Anti-mouse IgG, HRP-linked antibody #7076 (CST) , beta-actin antibody GTX 110564 (Genetex).

Techniques: Marker, Activation Assay, Membrane, Lysis, Inhibition

Journal: PLoS ONE

Article Title: Differential Expression of Complement Markers in Normal and AMD Transmitochondrial Cybrids

doi: 10.1371/journal.pone.0159828

Figure Lengend Snippet: Complement Genes’ Information.

Article Snippet: 8 , CFHR4 , 65 kDa , Mouse anti-Factor H-related 4 (CFHR4) Monoclonal antibody #MAB5980 (RD Systems) , 1:1000 , Human , Anti-mouse IgG, HRP-linked antibody #7076 (CST) , beta-actin antibody GTX 110564 (Genetex).

Techniques: Variant Assay

Journal: PLoS ONE

Article Title: Differential Expression of Complement Markers in Normal and AMD Transmitochondrial Cybrids

doi: 10.1371/journal.pone.0159828

Figure Lengend Snippet: Complement Proteins’ Western Blotting Information.

Article Snippet: 8 , CFHR4 , 65 kDa , Mouse anti-Factor H-related 4 (CFHR4) Monoclonal antibody #MAB5980 (RD Systems) , 1:1000 , Human , Anti-mouse IgG, HRP-linked antibody #7076 (CST) , beta-actin antibody GTX 110564 (Genetex).

Techniques: Western Blot

Journal: PLoS ONE

Article Title: Differential Expression of Complement Markers in Normal and AMD Transmitochondrial Cybrids

doi: 10.1371/journal.pone.0159828

Figure Lengend Snippet: Complement Gene Expression Data.

Article Snippet: 8 , CFHR4 , 65 kDa , Mouse anti-Factor H-related 4 (CFHR4) Monoclonal antibody #MAB5980 (RD Systems) , 1:1000 , Human , Anti-mouse IgG, HRP-linked antibody #7076 (CST) , beta-actin antibody GTX 110564 (Genetex).

Techniques: Gene Expression

Journal: PLoS ONE

Article Title: Differential Expression of Complement Markers in Normal and AMD Transmitochondrial Cybrids

doi: 10.1371/journal.pone.0159828

Figure Lengend Snippet: Decreased protein levels of CFH, CFHL1, CD55, CD59, CFI, and CD46, and increased levels of CFP, CFB, CFHR4, and CFHR1 protein in AMD cybrids. (A, C, E, G, I, K, M, O, Q, S) Representative Western blots of CFH, CFHL1, CD55, CD59, CFI, CFP, CFB, CD46, CFHR4, and CFHR1 respectively. (B, D, F, H, J, L, N, P, R, T) Graphs showing quantitation of CFH, CFHL1, CD55, CD59, CFI, CFP, CFB, CD46, CFHR4, and CFHR1 proteins in Older-Normal and AMD cybrids. * P < 0.05, ** P < 0.01. n = 4–5. Data were analyzed using Student’s T-test.

Article Snippet: 8 , CFHR4 , 65 kDa , Mouse anti-Factor H-related 4 (CFHR4) Monoclonal antibody #MAB5980 (RD Systems) , 1:1000 , Human , Anti-mouse IgG, HRP-linked antibody #7076 (CST) , beta-actin antibody GTX 110564 (Genetex).

Techniques: Western Blot, Quantitation Assay

Journal: PLoS ONE

Article Title: Differential Expression of Complement Markers in Normal and AMD Transmitochondrial Cybrids

doi: 10.1371/journal.pone.0159828

Figure Lengend Snippet: Complement Protein Expression Data.

Article Snippet: 8 , CFHR4 , 65 kDa , Mouse anti-Factor H-related 4 (CFHR4) Monoclonal antibody #MAB5980 (RD Systems) , 1:1000 , Human , Anti-mouse IgG, HRP-linked antibody #7076 (CST) , beta-actin antibody GTX 110564 (Genetex).

Techniques: Expressing

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: Control, Injection

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, Control

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, Control

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

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, Control

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, Control, Inhibition

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, Control

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

Journal: PLoS ONE

Article Title: Differential Expression of Complement Markers in Normal and AMD Transmitochondrial Cybrids

doi: 10.1371/journal.pone.0159828

Figure Lengend Snippet: Complement Proteins’ Western Blotting Information.

Article Snippet: 8 , CFHR4 , 65 kDa , Mouse anti-Factor H-related 4 (CFHR4) Monoclonal antibody #MAB5980 (RD Systems) , 1:1000 , Human , Anti-mouse IgG, HRP-linked antibody #7076 (CST) , beta-actin antibody GTX 110564 (Genetex).

Techniques: Western Blot