Factor XII and kininogen asymmetric assembly with gC1qR/C1QBP/P32 is governed by allostery.

The contact system is composed of factor XII (FXII), prekallikrein (PK), and cofactor high-molecular-weight kininogen (HK). The globular C1q receptor (gC1qR) has been shown to interact with FXII and HK. We reveal the FXII fibronectin type II domain (FnII) binds gC1qR in a Zn2+-dependent fashion and determined the complex crystal structure. FXIIFnII binds the gC1qR trimer in an asymmetric fashion, with residues Arg36 and Arg65 forming contacts with 2 distinct negatively charged pockets. gC1qR residues Asp185 and His187 coordinate a Zn2+ adjacent to the FXII-binding site, and a comparison with the ligand-free gC1qR crystal structure reveals the anionic G1-loop becomes ordered upon FXIIFnII binding. Additional conformational changes in the region of the Zn2+-binding site reveal an allosteric basis for Zn2+ modulation of FXII binding. Mutagenesis coupled with surface plasmon resonance demonstrate the gC1qR Zn2+ site contributes to FXII binding, and plasma-based assays reveal gC1qR stimulates coagulation in a FXII-dependent manner. Analysis of the binding of HK domain 5 (HKD5) to gC1qR shows only 1 high-affinity binding site per trimer. Mutagenesis studies identify a critical G3-loop located at the center of the gC1qR trimer, suggesting steric occlusion as the mechanism for HKD5 asymmetric binding. Gel filtration experiments reveal that gC1qR clusters FXII and HK into a higher-order 500-kDa ternary complex. These results support the conclusion that extracellular gC1qR can act as a chaperone to cluster contact factors, which may be a prelude for initiating the cascades that drive bradykinin generation and the intrinsic pathway of coagulation.

SC134-TCB targeting fucosyl-GM1, a T cell engaging antibody with potent anti-tumour activity in preclinical small-cell lung cancer models.

Small-cell lung cancer (SCLC) is an aggressive disease with limited treatment options. Fucosyl-GM1 (FucGM1) is a glycolipid overexpressed in the majority of SCLC tumours, but virtually absent from normal healthy tissues. Here, we validate a FucGM1-targeting T cell redirecting bispecific antibody (TCB) for the treatment of SCLC. Over 80% of SCLC patient-derived xenograft (PDX) tissues expressed FucGM1, whilst only three normal human tissues: pituitary, thymus and skin expressed low and focal FucGM1. A FucGM1-targeting TCB (SC134-TCB), based on the Fc-silenced humanised h134 antibody exhibited nanomolar FucGM1 glycolipid and SCLC cell surface binding. SC134-TCB showed potent ex vivo killing of SCLC cell lines with donor-dependent EC50 ranging from 7.2 pmol/L up to 211.0 pmol/L, effectively activating T cells, with picomolar efficiency, coinciding with target-dependent cytokine production such as interferon gamma, interleukin-2 and tumour necrosis factor alpha and robust proliferation of both CD4 and CD8 T cells. The ex vivo SC134-TCB tumour controlling activity translated into an effective in vivo anti-DMS79 tumour therapy, resulting in 100% tumour-free survival in a human PBMC admixed setting and 40% overall survival (55% tumour growth inhibition) with systemically administered human PBMC. Combination treatment with Atezolizumab further enhanced survival and tumour growth inhibition (up to 73%). A ten-fold SC134-TCB dose reduction maintained the strong in vivo anti-tumour impact, translating into 70% overall survival (P<0.0001). Whole blood incubation with SC134-TCB, as well as healthy human primary cells analysis, revealed no target-independent cytokine production. SC134-TCB presents an attractive candidate to deliver an effective immunotherapy treatment option for SCLC patients.

Crystal structures of the recombinant ß-factor XIIa protease with bound Thr-Arg and Pro-Arg substrate mimetics.

Coagulation factor XII (FXII) is a key initiator of the contact pathway, which contributes to inflammatory pathways. FXII circulates as a zymogen, which when auto-activated forms factor XIIa (FXIIa). Here, the production of the recombinant FXIIa protease domain (ßFXIIaHis) with yields of ∼1-2 mg per litre of insect-cell culture is reported. A second construct utilized an N-terminal maltose-binding protein (MBP) fusion (MBP-ßFXIIaHis). Crystal structures were determined of MBP-ßFXIIaHis in complex with the inhibitor D-Phe-Pro-Arg chloromethyl ketone (PPACK) and of ßFXIIaHis in isolation. The ßFXIIaHis structure revealed that the S2 and S1 pockets were occupied by Thr and Arg residues, respectively, from an adjacent molecule in the crystal. The Thr-Arg sequence mimics the P2-P1 FXIIa cleavage-site residues present in the natural substrates prekallikrein and FXII, and Pro-Arg (from PPACK) mimics the factor XI cleavage site. A comparison of the ßFXIIaHis structure with the available crystal structure of the zymogen-like FXII protease revealed large conformational changes centred around the S1 pocket and an alternate conformation for the 99-loop, Tyr99 and the S2 pocket. Further comparison with activated protease structures of factors IXa and Xa, which also have the Tyr99 residue, reveals that a more open form of the S2 pocket only occurs in the presence of a substrate mimetic. The FXIIa inhibitors EcTI and infestin-4 have Pro-Arg and Phe-Arg P2-P1 sequences, respectively, and the interactions that these inhibitors make with ßFXIIa are also described. These structural studies of ßFXIIa provide insight into substrate and inhibitor recognition and establish a scaffold for the structure-guided drug design of novel antithrombotic and anti-inflammatory agents.