Co-crystallisation and humanisation of an anti-HER2 single-domain antibody as a theranostic tool.

Human epidermal growth factor receptor-2 (HER2) is a well-recognised biomarker associated with 25% of breast cancers. In most cases, early detection and/or treatment correlates with an increased chance of survival. This study, has identified and characterised a highly specific anti-HER2 single-domain antibody (sdAb), NM-02, as a potential theranostic tool. Complete structural description by X-ray crystallography has revealed a non-overlapping epitope with current anti-HER2 antibodies. To reduce the immunogenicity risk, NM-02 underwent a humanisation process and retained wild type-like binding properties. To further de-risk the progression towards chemistry, manufacturing and control (CMC) we performed full developability profiling revealing favourable thermal and physical biochemical 'drug-like' properties. Finally, the application of the lead humanised NM-02 candidate (variant K) for HER2-specific imaging purposes was demonstrated using breast cancer HER2+/BT474 xenograft mice.

Nanobody inhibitors of Plexin-B1 identify allostery in plexin-semaphorin interactions and signaling.

Plexin-B1 is a receptor for the cell surface semaphorin, Sema4D. This signaling system has been implicated in a variety of human diseases, including cancer, multiple sclerosis and osteoporosis. While inhibitors of the Plexin-B1:Sema4D interaction have been previously reported, understanding their mechanism has been hindered by an incomplete structural view of Plexin-B1. In this study, we have raised and characterized a pair of nanobodies that are specific for mouse Plexin-B1 and which inhibit the binding of Sema4D to mouse Plexin-B1 and its biological activity. Structural studies of these nanobodies reveal that they inhibit the binding of Sema4D in an allosteric manner, binding to epitopes not previously reported. In addition, we report the first unbound structure of human Plexin-B1, which reveals that Plexin-B1 undergoes a conformational change on Sema4D binding. These changes mirror those seen upon binding of allosteric peptide modulators, which suggests a new model for understanding Plexin-B1 signaling and provides a potential innovative route for therapeutic modulation of Plexin-B1.

Identification, binding, and structural characterization of single domain anti-PD-L1 antibodies inhibitory of immune regulatory proteins PD-1 and CD80.

Programmed death-ligand 1 (PD-L1) is a key immune regulatory protein that interacts with programmed cell death protein 1 (PD-1), leading to T-cell suppression. Whilst this interaction is key in self-tolerance, cancer cells evade the immune system by overexpressing PD-L1. Inhibition of the PD-1/PD-L1 pathway with standard monoclonal antibodies has proven a highly effective cancer treatment; however, single domain antibodies (VHH) may offer numerous potential benefits. Here, we report the identification and characterization of a diverse panel of 16 novel VHHs specific to PD-L1. The panel of VHHs demonstrate affinities of 0.7 nM to 5.1 µM and were able to completely inhibit PD-1 binding to PD-L1. The binding site for each VHH on PD-L1 was determined using NMR chemical shift perturbation mapping and revealed a common binding surface encompassing the PD-1-binding site. Additionally, we solved crystal structures of two representative VHHs in complex with PD-L1, which revealed unique binding modes. Similar NMR experiments were used to identify the binding site of CD80 on PD-L1, which is another immune response regulatory element and interacts with PD-L1 localized on the same cell surface. CD80 and PD-1 were revealed to share a highly overlapping binding site on PD-L1, with the panel of VHHs identified expected to inhibit CD80 binding. Comparison of the CD80 and PD-1 binding sites on PD-L1 enabled the identification of a potential antibody binding region able to confer specificity for the inhibition of PD-1 binding only, which may offer therapeutic benefits to counteract cancer cell evasion of the immune system.

An antagonistic monoclonal anti-Plexin-B1 antibody exerts therapeutic effects in mouse models of postmenopausal osteoporosis and multiple sclerosis.

Osteoporosis and multiple sclerosis are highly prevalent diseases with limited treatment options. In light of these unmet medical needs, novel therapeutic approaches are urgently sought. Previously, the activation of the transmembrane receptor Plexin-B1 by its ligand semaphorin 4D (Sema4D) has been shown to suppress bone formation and promote neuroinflammation in mice. However, it is unclear whether inhibition of this receptor-ligand interaction by an anti-Plexin-B1 antibody could represent a viable strategy against diseases related to these processes. Here, we raised and systematically characterized a monoclonal antibody directed against the extracellular domain of human Plexin-B1, which specifically blocks the binding of Sema4D to Plexin-B1. In vitro, we show that this antibody inhibits the suppressive effects of Sema4D on human osteoblast differentiation and mineralization. To test the therapeutic potential of the antibody in vivo, we generated a humanized mouse line, which expresses transgenic human Plexin-B1 instead of endogenous murine Plexin-B1. Employing these mice, we demonstrate that the anti-Plexin-B1 antibody exhibits beneficial effects in mouse models of postmenopausal osteoporosis and multiple sclerosis in vivo. In summary, our data identify an anti-Plexin-B1 antibody as a potential therapeutic agent for the treatment of osteoporosis and multiple sclerosis.

An innate IL-25-ILC2-MDSC axis creates a cancer-permissive microenvironment for Apc mutation-driven intestinal tumorigenesis.

Interleukin-25 (IL-25) and group 2 innate lymphoid cells (ILC2s) defend the host against intestinal helminth infection and are associated with inappropriate allergic reactions. IL-33-activated ILC2s were previously found to augment protective tissue-specific pancreatic cancer immunity. Here, we showed that intestinal IL-25-activated ILC2s created an innate cancer-permissive microenvironment. Colorectal cancer (CRC) patients with higher tumor IL25 expression had reduced survival and increased IL-25R-expressing tumor-resident ILC2s and myeloid-derived suppressor cells (MDSCs) associated with impaired antitumor responses. Ablation of IL-25 signaling reduced tumors, virtually doubling life expectancy in an Apc mutation-driven model of spontaneous intestinal tumorigenesis. Mechanistically, IL-25 promoted intratumoral ILC2s, which sustained tumor-infiltrating MDSCs to suppress antitumor immunity. Therapeutic antibody-mediated blockade of IL-25 signaling decreased intratumoral ILC2s, MDSCs, and adenoma/adenocarcinoma while increasing antitumor adaptive T cell and interferon-γ (IFN-γ)-mediated immunity. Thus, the roles of innate epithelium-derived cytokines IL-25 and IL-33 as well as ILC2s in cancer cannot be generalized. The protumoral nature of the IL-25-ILC2 axis in CRC highlights this pathway as a potential therapeutic target against CRC.

Cross-Reactive SARS-CoV-2 Neutralizing Antibodies From Deep Mining of Early Patient Responses.

Passive immunization using monoclonal antibodies will play a vital role in the fight against COVID-19. The recent emergence of viral variants with reduced sensitivity to some current antibodies and vaccines highlights the importance of broad cross-reactivity. This study describes deep-mining of the antibody repertoires of hospitalized COVID-19 patients using phage display technology and B cell receptor (BCR) repertoire sequencing to isolate neutralizing antibodies and gain insights into the early antibody response. This comprehensive discovery approach has yielded a panel of potent neutralizing antibodies which bind distinct viral epitopes including epitopes conserved in SARS-CoV-1. Structural determination of a non-ACE2 receptor blocking antibody reveals a previously undescribed binding epitope, which is unlikely to be affected by the mutations in any of the recently reported major viral variants including B.1.1.7 (from the UK), B.1.351 (from South Africa) and B.1.1.28 (from Brazil). Finally, by combining sequences of the RBD binding and neutralizing antibodies with the B cell receptor repertoire sequencing, we also describe a highly convergent early antibody response. Similar IgM-derived sequences occur within this study group and also within patient responses described by multiple independent studies published previously.

Mechanochemical control of epidermal stem cell divisions by B-plexins.

The precise spatiotemporal control of cell proliferation is key to the morphogenesis of epithelial tissues. Epithelial cell divisions lead to tissue crowding and local changes in force distribution, which in turn suppress the rate of cell divisions. However, the molecular mechanisms underlying this mechanical feedback are largely unclear. Here, we identify a critical requirement of B-plexin transmembrane receptors in the response to crowding-induced mechanical forces during embryonic skin development. Epidermal stem cells lacking B-plexins fail to sense mechanical compression, resulting in disinhibition of the transcriptional coactivator YAP, hyperproliferation, and tissue overgrowth. Mechanistically, we show that B-plexins mediate mechanoresponses to crowding through stabilization of adhesive cell junctions and lowering of cortical stiffness. Finally, we provide evidence that the B-plexin-dependent mechanochemical feedback is also pathophysiologically relevant to limit tumor growth in basal cell carcinoma, the most common type of skin cancer. Our data define a central role of B-plexins in mechanosensation to couple cell density and cell division in development and disease.

Structure of a Potential Therapeutic Antibody Bound to Interleukin-16 (IL-16): MECHANISTIC INSIGHTS AND NEW THERAPEUTIC OPPORTUNITIES.

Interleukin-16 (IL-16) is reported to be a chemoattractant cytokine and modulator of T-cell activation, and has been proposed as a ligand for the co-receptor CD4. The secreted active form of IL-16 has been detected at sites of TH1-mediated inflammation, such as those seen in autoimmune diseases, ischemic reperfusion injury (IRI), and tissue transplant rejection. Neutralization of IL-16 recruitment to its receptor, using an anti-IL16 antibody, has been shown to significantly attenuate inflammation and disease pathology in IRI, as well as in some autoimmune diseases. The 14.1 antibody is a monoclonal anti-IL-16 antibody, which when incubated with CD4(+) cells is reported to cause a reduction in the TH1-type inflammatory response. Secreted IL-16 contains a characteristic PDZ domain. PDZ domains are typically characterized by a defined globular structure, along with a peptide-binding site located in a groove between the αB and ßB structural elements and a highly conserved carboxylate-binding loop. In contrast to other reported PDZ domains, the solution structure previously reported for IL-16 reveals a tryptophan residue obscuring the recognition groove. We have solved the structure of the 14.1Fab fragment in complex with IL-16, revealing that binding of the antibody requires a conformational change in the IL-16 PDZ domain. This involves the rotation of the αB-helix, accompanied movement of the peptide groove obscuring tryptophan residue, and consequent opening up of the binding site for interaction. Our study reveals a surprising mechanism of action for the antibody and identifies new opportunities for the development of IL-16-targeted therapeutics, including small molecules that mimic the interaction of the antibody.

Interaction between NOD2 and CARD9 involves the NOD2 NACHT and the linker region between the NOD2 CARDs and NACHT domain.

NOD2 activation by muramyl dipeptide causes a proinflammatory immune response in which the adaptor protein CARD9 works synergistically with NOD2 to drive p38 and c-Jun N-terminal kinase (JNK) signalling. To date the nature of the interaction between NOD2 and CARD9 remains undetermined. Here we show that this interaction is not mediated by the CARDs of NOD2 and CARD9 as previously suggested, but that NOD2 possesses two interaction sites for CARD9; one in the CARD-NACHT linker and one in the NACHT itself.

Galactose recognition by the apicomplexan parasite Toxoplasma gondii.

Toxosplasma gondii is the model parasite of the phylum Apicomplexa, which contains numerous obligate intracellular parasites of medical and veterinary importance, including Eimeria, Sarcocystis, Cryptosporidium, Cyclospora, and Plasmodium species. Members of this phylum actively enter host cells by a multistep process with the help of microneme protein (MIC) complexes that play important roles in motility, host cell attachment, moving junction formation, and invasion. T. gondii (Tg)MIC1-4-6 complex is the most extensively investigated microneme complex, which contributes to host cell recognition and attachment via the action of TgMIC1, a sialic acid-binding adhesin. Here, we report the structure of TgMIC4 and reveal its carbohydrate-binding specificity to a variety of galactose-containing carbohydrate ligands. The lectin is composed of six apple domains in which the fifth domain displays a potent galactose-binding activity, and which is cleaved from the complex during parasite invasion. We propose that galactose recognition by TgMIC4 may compromise host protection from galectin-mediated activation of the host immune system.

Complete resonance assignments for the MIC2 associated protein from Toxoplasma gondii.

Toxoplasma gondii is an obligate parasite that infects most warm blood animals. Micronemal proteins actively involves in the invasion process, where TgMIC2 and TgM2AP complex plays vital roles. Complete NMR assignments for major fragment of TgM2AP were successfully obtained.

Structural insights into microneme protein assembly reveal a new mode of EGF domain recognition.

The obligate intracellular parasite Toxoplasma gondii, a member of the phylum Apicomplexa that includes Plasmodium spp., is one of the most widespread parasites and the causative agent of toxoplasmosis. Adhesive complexes composed of microneme proteins (MICs) are secreted onto the parasite surface from intracellular stores and fulfil crucial roles in host-cell recognition, attachment and penetration. Here, we report the high-resolution solution structure of a complex between two crucial MICs, TgMIC6 and TgMIC1. Furthermore, we identify two analogous interaction sites within separate epidermal growth factor-like (EGF) domains of TgMIC6-EGF2 and EGF3-and confirm that both interactions are functional for the recognition of host cell receptor in the parasite, using immunofluorescence and invasion assays. The nature of this new mode of recognition of the EGF domain and its abundance in apicomplexan surface proteins suggest a more generalized means of constructing functional assemblies by using EGF domains with highly specific receptor-binding properties.

Complete resonance assignment of the first and second apple domains of MIC4 from Toxoplasma gondii, using a new NMRView-based assignment aid.

Microneme protein 4 is involved in cell binding by the important parasite Toxoplasma gondii. We present here the backbone and side-chain assignments of the first two apple domains together with a new graphical aid for their assignment using NMRView.

Complete resonance assignment of the galectin-like domain of MIC1 from Toxoplasma gondii in complex with the second EGF domain from MIC6 and the backbone assignment in complex with the third EGF domain.

Microneme protein complexes are important for invasion of host cells by Toxoplasma gondii. We report the resonance assignment of the galectin-like domain of microneme protein 1 in complexes with the second and third EGF domains from microneme protein 6.

Complete NMR assignments for the second EGF domain of MIC6 from Toxoplasma gondii and re-assignment in complex with the galectin-like domain of MIC1.

Toxoplasma gondii is the causative agent of toxoplasmosis. Here we present a complete set of NMR assignments for the second EGF domain from microneme protein 6 and its re-assignment in complex with the galectin-like domain from microneme protein 1.

A novel galectin-like domain from Toxoplasma gondii micronemal protein 1 assists the folding, assembly, and transport of a cell adhesion complex.

Immediately prior to invasion Toxoplasma gondii tachyzoites release a large number of micronemal proteins (TgMICs) that participate in host cell attachment and penetration. The TgMIC4-MIC1-MIC6 complex was the first to be identified in T. gondii and has been recently shown to be critical in invasion. This study establishes that the N-terminal thrombospondin type I repeat-like domains (TSR1-like) from TgMIC1 function as an independent adhesin as well as promoting association with TgMIC4. Using the newly solved three-dimensional structure of the C-terminal domain of TgMIC1 we have identified a novel Galectin-like fold that does not possess carbohydrate binding properties and redefines the architecture of TgMIC1. Instead, the TgMIC1 Galectin-like domain interacts and stabilizes TgMIC6, which provides the basis for a highly specific quality control mechanism for successful exit from the early secretory compartments and for subsequent trafficking of the complex to the micronemes.