Journal: EMBO Molecular Medicine

Article Title: Targeting key angiogenic pathways with a bispecific Cross MA b optimized for neovascular eye diseases

doi: 10.15252/emmm.201505889

Figure Lengend Snippet: A, B Real‐time qPCR analysis of ANG‐2 levels in retina and RPE/choroid complexes of JR5558 and C57BL/6J (C57) mice. (A) Relative expression levels of ANG‐2 were significantly increased in the RPE/choroid complexes of JR5558 mice in comparison with C57, at 50 and 62 days old (*, P = 0.022 at D50 and *, P = 0.042 at D62). (B) By contrast, retina levels of ANG‐2 were not significantly different between C57 and JR5558 mice, indicating neovascularization is driven locally by ANG‐2. Asterisk (*) denotes statistical significance of JR5558 mice compared to wild‐type C57BL/6 using unpaired t ‐test for each time point analyzed separately. C Schematic presentation of experimental design. Mice received IP injections of CrossMAb anti‐VEGF‐A/ANG‐2 (species crossreactive, B20‐4.1 and LC10), anti‐ANG‐2 (LC10), an anti‐VEGF‐A IgG 1 (B20‐4.1), or IgG control at postnatal D14 and D19 followed by fluorescence angiography at D26 (early intervention), results demonstrated in (D–K). Alternatively, mice received antibody at postnatal D47 and D55 followed by fluorescence angiography at D60 (late intervention), results demonstrated in (L) and (M). D–I Representative examples of fluorescence angiograms of IgG control (D), anti‐VEGF‐A (E), anti‐ANG‐2 (F) (all at 5 mg/kg), and three doses of anti‐VEGF‐A/ANG‐2 (G, H, and I) (at 3, 5, and 10 mg/kg). J, K Bar graph of numbers of spontaneously occurring lesions (J) and area by fluorescence angiography (K) after two weekly doses of antibody (antibodies at 5 mg/kg IP and anti‐VEGF‐A/ANG‐2 at 10 [high], 5 [mid], and 3 [low] mg/kg IP) followed by analysis a week later in the early intervention model. L, M Bar graph of numbers of spontaneously occurring lesions (L) and area by fluorescence angiography (M) after two weekly doses of antibody (3 mg/kg IP) followed by analysis a week later in the late intervention model. Data information: SEM is shown as error bars with n = 4 (A, B) or n = 8 (J–M) animals per group and significance indicated by * using ANOVA (J, K: P < 0.0001; L: P < 0.0018; M: P < 0.031 followed by Tukey's multiple t ‐test in J–M). In (J), IgG control is significant against anti‐VEGF‐A (****, P < 0.0001), anti‐ANG‐2 (**, P = 0.0069), anti‐VEGF‐A/ANG‐2 low (*, P = 0.0194), mid and high (****, P < 0.0001). Furthermore, anti‐VEGF‐A/ANG‐2 high is significant against anti‐VEGF‐A (*, P = 0.0428), anti‐ANG‐2 (****, P < 0.0001), and anti‐VEGF‐A/ANG‐2 low (****, P < 0.0001). Finally, anti‐ANG‐2 is significantly different from anti‐VEGF‐A/ANG‐2 mid (**, P < 0.0041). In (K), IgG control is significant against anti‐VEGF‐A (***, P = 0.0003) and anti‐VEGF‐A/ANG‐2 mid and high (both ****, P < 0.0001). Furthermore, anti‐VEGF‐A/ANG‐2 high is significantly different from anti‐VEGF‐A (**, P = 0.0037), anti‐ANG‐2 (****, P < 0.0001), and anti‐VEGF‐A/ANG‐2 low (***, P = 0.0001). Finally, anti‐ANG‐2 is significantly different from anti‐VEGF‐A/ANG‐2 mid (**, P < 0.0022) and anti‐VEGF‐A/ANG‐2 low against mid (*, P = 0.022). In (L), IgG control is significantly different from anti‐ANG‐2 (*, P = 0.023) and anti‐VEGF‐A/ANG‐2 (*, P = 0.014). Vehicle is different from anti‐ANG‐2 (*, P = 0.024) and anti‐VEGF‐A/ANG‐2 (*, P = 0.016). In (M), IgG control is significantly different from anti‐ANG‐2 (*, P = 0.044) and anti‐VEGF‐A/ANG‐2 (*, P = 0.049). D, day; IP, intraperitoneal; sCNV, spontaneous choroidal neovascularization.

Article Snippet: Therefore, both ligands are available in comparative concentrations and both paratopes of the CrossMAb have an equal probability of neutralizing their respective ligands.

Techniques: Expressing, Comparison, Control, Fluorescence

Journal: EMBO Molecular Medicine

Article Title: Targeting key angiogenic pathways with a bispecific Cross MA b optimized for neovascular eye diseases

doi: 10.15252/emmm.201505889

Figure Lengend Snippet: Structural presentation of CrossMAb RG7716 with substituted amino acids highlighted and colored. Amino acids corresponding to individual point mutations ensuring correct and efficient heavy chain heterodimerization (“knobs‐into‐holes” [green] and additional disulfide bridge [yellow]), abolishing Fc receptor functionality (Fcγ receptors I, II, and III, [pink] and FcRn [blue]) are highlighted. Summary table of experimental binding affinities of the parental components targeting VEGF‐A (ranibizumab) and ANG‐2 (LC10) as well as of RG7716 measured by isothermal titration calorimetry. RG7716 binds VEGF‐A 121 and VEGF‐A 165 with an affinity comparable to ranibizumab (3.3 vs. 3.1 nM). ANG‐2 binding of RG7716 is slightly decreased to 22 nM compared to α‐ANG‐2 IgG 1 (LC10) with 4.1 nM. No binding to ANG‐1 was detectable for RG7716 or α‐ANG‐2 IgG 1 (LC10). Inhibition of ANG‐1‐induced endothelial cell survival was tested in the presence of increasing concentrations of LC10 (anti‐ANG‐2 IgG, ANG‐2 binding part in RG7716) and LC08 (ANG‐1 and ANG‐2 binding antibody). An average of three independent experiments is shown with SEM. The ability of RG7716 to bind to ANG‐2 and VEGF‐A simultaneously and independent is demonstrated by a surface plasmon resonance experiment. RG7716 is captured via an anti‐Fc antibody on the chip surface. The green curve depicts the experiment with ANG‐2 in the first and VEGF‐A in the second binding event. The red curve shows the binding events in the other sequence.

Article Snippet: Therefore, both ligands are available in comparative concentrations and both paratopes of the CrossMAb have an equal probability of neutralizing their respective ligands.

Techniques: Binding Assay, Isothermal Titration Calorimetry, Inhibition, SPR Assay, Sequencing

Journal: mAbs

Article Title: Ten years in the making: application of CrossMab technology for the development of therapeutic bispecific antibodies and antibody fusion proteins

doi: 10.1080/19420862.2021.1967714

Figure Lengend Snippet: Major CrossMab formats: A) 1 + 1 CrossMab: CH −CL vanucizumab, faricimab, 10E8.4/iMab 1 + 1; B) 1 + 1 CrossMab VH-VL± : PD1-TIM3, PD1-LAG3; C) CrossMab CH −CL+/– based FAP-4-1BBL, CD19-4-1BBL fusion proteins; D) 2 + 1 CrossMab: CH −CL cibisatamab; E) 2 + 1 CrossMab VH-VL± : glofitamab, CC-93269, TYRP1-TCB, WT1-TCB, RG6123; F) 2 + 2 CrossMab CH −CL -based FIT-Ig EMB-01, EMB-02, EMB-06; G) 2 + 2 CrossMab: CH −CL FAP-DR5; H) 2 + 1 CrossMab VH-VL± : BS-GANT, FAP-CD40; I) 1 + 1 CrossMab CH −CL -based Nkp46-based NK cell engager (NKCE). Heavy-chain domains are depicted in dark colors and respective light-chain domains are depicted with corresponding bright colors. Fusion protein depicted in purple. Note: Differences in variable regions and/or isotype and Fc engineering are not depicted. Created with BioRender.com

Article Snippet: 11 , EMB-01 , EGFR/c-Met , 2 + 2 CrossMab Fab /FIT-Ig , Oncology , Ph 1 , EpimAb , 2019 , NCT03797391 , , .

Techniques:

Journal: mAbs

Article Title: Ten years in the making: application of CrossMab technology for the development of therapeutic bispecific antibodies and antibody fusion proteins

doi: 10.1080/19420862.2021.1967714

Figure Lengend Snippet: CrossMabs in clinical trials (status July 2021), FP: Fusion protein, FIT-Ig: Fabs-in-tandem Ig, EIH: Entry into human date

Article Snippet: 11 , EMB-01 , EGFR/c-Met , 2 + 2 CrossMab Fab /FIT-Ig , Oncology , Ph 1 , EpimAb , 2019 , NCT03797391 , , .

Techniques: