多特异性抗体可以以不同的形式format构建。双特异性抗体20多年的研发经验表明，format的设计和选择会对抗体功能产生重要影响。这对于引发细胞（间）的相互作用尤其如此，例如受体激活、受体内化、受体聚集或两个细胞之间免疫突触的形成。我们本次推荐的综述涵盖影响多特异性抗体format功能性的设计参数，尤其关注引发 T 细胞募集的双特异性抗体。我们比较了分子量大小和域序列都相同、但几何形状不同的format。得出结论：某些format因其几何结构更有利于（人工）免疫突触【(artificial) immune synapses】的结构组成，从而效力更强。
Figure 2．The correct orientation of VH1 and VH2 domains is required for optimal agonistic activity. VH1, VH2, and FGF21 bind to distinct regions on β-Klotho. In FGF21-responsive CHO reporter cells expressing human β-Klotho and FGFR1c, IgG-VH1+VH2 activated receptor signaling with potency similar to that of FGF21 (EC50 = 0.5 nm). When the positions of VH1 and VH2 are swapped, by placing VH2 on the heavy chain and VH1 on the light chain (IgG-VH2+VH1), the agonistic activity of this molecule was significantly attenuated, achieving only one third of the maximal response of IgG-VH1+VH2.
Figure 3. Death receptor dual agonist Surrobody is a more potent inducer of tumor cell death than DR4 or DR5 monospecific antibodies. Various TRAIL-sensitive cancer cell lines were plated at a density of 750 cells/well in 384 wells. The next day cells were cultured with increasing concentrations of monospecific DR4 antibody (pink), monospecific anti-DR5 antibody (orange), the combination of anti-DR4 and anti-DR5 antibodies (black), death receptor dual agonist Surrobody (green), or TRAIL (blue) for 48 hr. Before addition to cells, all the antibodies and Surrobody were incubated for 5 minutes with 0.5× molar ratio of protein G to facilitate clustering. Relative cell viability was estimated from cellular ATP measurements using the Cell Titer Glo reagent (Promega), expressing data as % inhibition of cell survival (mean ± SEM; n= 4). Data are shown for Jurkat (A), Ramos (B), MDA-MB-231 (C), Colo-205 (D), Ovcar-3 (E) and Ovcar-5 (F) and HCT116 (G) cell lines.
就在最近，Genentech 公司研究人员展示巨噬细胞上 MerTK（Mer 酪氨酸激酶）受体通路的激活，可以导致 CD20 阳性 B 细胞的吞噬作用。他们使用简单的 1 + 1 IgG format (图4) 。
Figure 4. Identifying and characterizing MerTK agonist antibodies for engineering anti-CD20/MerTK bispecific antibodies capable of inducing phagocytosis of B cells by macrophages (a) Characterization of lead MerTK agonist antibodies measured by their ability to induce pAKT (Ser-473) in human MerTK+ macrophages using HTRF assay (Mean ± S.D of 2 technical replicates). Gas6-Fc is a positive control for MerTK activation. (b) Schematic depiction of B cell-dependent engagement of MerTK via anti-CD20/MerTK bispecific antibody. (c) Imagestream analysis of phagocytosis of live Raji cells by human primary macrophages in the presence of indicated bispecific antibody. A representation of Imagestream analysis is shown. Quantification is also presented (Mean ± S.D of one replicate each from 3 donors). (d-f) Flow cytometric analysis of phagocytosis of live Raji cells by human primary macrophages in the presence of indicated bispecific antibody, inhibitors and blocking agents (Mean ± S.D of one replicate each from 3 donors) (g) Activation of MerTK (phosphotyrosine) by the bispecific antibody in the presence of target Raji cells. Results are from using macrophages derived from two different human donors. See Supplementary Figure 5 for extended western blot. (h-i) TNF and IL-6 production by human primary macrophages stimulated with indicated plate coated antibodies. (Mean ± S.D of one replicate each from three different donors).
Figure 5. Tetravalent biepitopic antibody formats enable intrinsic agonism of TNFRSFs. (a) NF-κB signaling mediated by OX40L-huIgG1 Fc fusion (OX40L) and anti-OX40 antibody formats. IgG1 (squares) and OX40L (downward triangles) were tested with (+XL) and without extrinsic secondary crosslinking. Orange triangle represents a bivalent biepitopic antibody (Ab1/Ab2), and upward triangles represent hexameric variant antibodies (RGY). (b–e) OX40 NF-κB signaling activity mediated by tetravalent biepitopic (blue) and monoepitopic (red) antibody formats: r:Fv-IgG (b), r:Fab-IgG (c), c:Fab-IgG (d) or c:IgG-IgG (e). (f) DR5 driven caspase-8 activity and (g) anti-proliferative activity in COLO 205 cells mediated by multivalent and multiepitopic antibody formats and soluble Flag-Apo2L. For all tetravalent formats (b–g) blue circles indicate biepitopic formats and red diamonds indicate monoepitopic formats, respectively. The x-axis for each graph represents the molar concentration of each complete molecule irrespective of number of receptor binding units; i.e., 1 nM of a bivalent antibody has two binding sites, whereas a 1 nM equivalent of an r:Fv-IgG has four binding sites.
Figure 6. FAP-DR5 BsAb and apoptosis activity. A, principle of tumor-targeted apoptosis by BsAbs consisting of a CrossFab anti-FAP unit (mAb007 or mAb082) fused to the C-terminus of the drozitumab heavy chain using a 20mer GS linker. The mAb007 anti-FAP moiety was fused to drozitumab heavy chain in either a VHCL or VLCH1 configuration.
Figure 7. RG7386 induces tumor cell apoptosis in vivo in human xenograft mouse models. A, antitumor efficacy of RG7386 in a mouse xenograft model. Nude mice were subcutaneously coinjected with human DLD-1 and NIH3T3 cells and subjected to RG7386 (twice weekly doses) or Drozitumab_PGLALA antibody treatment when tumor volume was approximately 100 mm3 in size. Shown is a representative FAP IHC of the tumor (median tumor volume and interquartile range (IQR); n = 10 animals/group). B and C, apoptosis was monitored in mice coinjected with bone-metastasizing MDA-MB-231 1833-PPOP233 tumor cells and NIH3T3 cells. B, in vivo analysis of apoptosis using a luminescence caspase 3/7 activation reporter assay in response to the indicated treatments over 72 hours. Luminescence images were taken at 0, 6, and 72 hours posttreatment. Representative luminescence images at 6 hours are shown (mean ± SEM; n = 5 animals/group). C, analysis of TGI in the same model in response to RG7386 (median tumor volume and IQR; n = 10 animals/group). D, IHC validation of caspase-3 activation in response to RG7386.
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