Probing the structure and function of the protease domain of botulinum neurotoxins using single-domain antibodies

Botulinum neurotoxins (BoNTs) are among the deadliest of bacterial toxins. BoNT serotype A and B in particular pose the most serious threat to humans because of their high potency and persistence. To date, there is no effective treatment for late post-exposure therapy of botulism patients. Here, we aim to develop single-domain variable heavy-chain (VHH) antibodies targeting the protease domains (also known as the light chain, LC) of BoNT/A and BoNT/B as antidotes for post-intoxication treatments. Using a combination of X-ray crystallography and biochemical assays, we investigated the structures and inhibition mechanisms of a dozen unique VHHs that recognize four and three non-overlapping epitopes on the LC of BoNT/A and BoNT/B, respectively. We show that the VHHs that inhibit the LC activity occupy the extended substrate-recognition exosites or the cleavage pocket of LC/A or LC/B and thus block substrate binding. Notably, we identified several VHHs that recognize highly conserved epitopes across BoNT/A or BoNT/B subtypes, suggesting that these VHHs exhibit broad subtype efficacy. Further, we identify two novel conformations of the full-length LC/A, that could aid future development of inhibitors against BoNT/A. Our studies lay the foundation for structure-based engineering of protein- or peptide-based BoNT inhibitors with enhanced potencies and cross-subtypes properties.

Natural Receptor- and Ligand-Based Chimeric Antigen Receptors: Strategies Using Natural Ligands and Receptors for Targeted Cell Killing

Chimeric antigen receptor (CAR) T-cell therapy has been widely successful in the treatment of B-cell malignancies, including B-cell lymphoma, mantle cell lymphoma, and multiple myeloma; and three generations of CAR designs have led to effective FDA approved therapeutics. Traditionally, CAR antigen specificity is derived from a monoclonal antibody where the variable heavy (VH) and variable light (VL) chains are connected by a peptide linker to form a single-chain variable fragment (scFv). While this provides a level of antigen specificity parallel to that of an antibody and has shown great success in the clinic, this design is not universally successful. For instance, issues of stability, immunogenicity, and antigen escape hinder the translational application of some CARs. As an alternative, natural receptor- or ligand-based designs may prove advantageous in some circumstances compared to scFv-based designs. Herein, the advantages and disadvantages of scFv-based and natural receptor- or ligand-based CAR designs are discussed. In addition, several translational aspects of natural receptor- and ligand-based CAR approaches that are being investigated in preclinical and clinical studies will be examined.

Long-Term Efficacy and Safety of Ibrutinib in the Treatment of CLL Patients: A Real Life Experience

Ibrutinib has demonstrated a significant clinical impact in patients with de novo and relapsed/refractory chronic lymphocytic leukemia (CLL), even in cases with unfavorable cytogenetics and molecular markers. All CLL patients’ data treated at our Institute with ibrutinib have been retrospectively reviewed. Forty-six patients received ibrutinib either as frontline (10) or second or more advanced treatment (36). Five patients presented with TP53 mutations; 11 had the deletion of chromosome 17p; 17 displayed an unmutated immunoglobulin variable heavy chain status. The median number of cycles administered was 26. Among patients treated frontline, the best overall response rate (ORR) was 90.0%. In patients receiving ibrutinib as a second or later line ORR was 97.2%. Median progression-free survival was 28.8 and 21.1 months for patients treated frontline and as second/later line, respectively. Median overall survival was not reached for those treated frontline and resulted in 4.9 years for patients treated as second/later line. Grade 3–4 hematological toxicities were neutropenia, thrombocytopenia, and anemia. Grade 3–4 extrahematological toxicities included diarrhea, cutaneous rash, utero-vesical prolapse, vasculitis, and sepsis. Ibrutinib is effective and well tolerated in CLL. Responses obtained in a real-life setting are durable and the safety profile of the drug is favorable.

Soluble Expression of Small Antibody Fragments against PD-L1 Using Escherichia coli with High Yield and Purity

Programmed death-ligand 1 (PD-L1) is a surface protein overexpressed in tumor cells. Recently, targeted therapy using PD-L1 antibodies to reconstitute the antitumor activity of T cells has received considerable attention as a cancer treatment. Among the several types of anti-PD-L1 antibodies, small-sized antibody fragments are useful agents to block PD-L1 for experimental and therapeutic purposes owing to their high penetration efficacy toward dense tumor cells. Herein, we expressed and purified recombinant single chain fragment of variable domain, variable heavy chain, and variable light chain, against PD-L1 in a soluble form using Escherichia coli, resulting in their high yield and high purity. We confirmed the antigen-binding efficiency of these antibody fragments, which showed antigen concentration-dependent responses. These results suggest that these small antibody fragments can serve as new agents for blocking or detecting PD-L1.

Engaging the ‘A’ Class Antibody: Variable-Heavy (VH) region influencing IgA1&2 engagement of FcαRI and superantigen proteins G, A, and L

Interest in IgA as an alternative therapeutic and diagnostic antibody has increased over the years, yet much remains to be investigated especially given their importance in activating immune cells in blood and in mucosal immunity. Recent whole antibody-based investigations have shown significant distal effects between the variable (V) and constant (C)-regions that can be mitigated by the different hinge regions of the human IgA subtypes A1 and A2. Diving deeper into the mechanisms underlying this, systematic VH manipulations retaining the CDRs were performed on a panel of 28 IgA1s and A2s across the Trastuzumab and Pertuzumab models, revealed distal effects on FcαRI binding. Further insights from structural modelling showed these effects to also be mitigated by the differing glycosylation patterns in IgA1 and 2 to explain reversal of trends of IgA1s and 2s effected by slight changes in the CDRs. IgAs bound at the Fc showed similar trends but magnitudes better binding to Her2 with that bound by ppL, showing that ppL can sterically hinder Her2 antigen binding. Contrary to canonical knowledge, we found strong evidence of IgAs binding SpG that was narrowed to be at the CH2-3 region, and that the likely binding with SpA was beyond VH3 FWR and most likely at the CH1. VH1 was found to be the most suitable framework (FWRs) for CDR-grafting for both IgA1 and 2. With relevance to interactions with the microbiome at mucosal surfaces, mechanistic insight of how these IgAs can interact bacterial superantigens proteins G, A, and L are also discovered for potential future interventions.One Sentence SummaryAn insight into the mechanism of distal V-region effects on FCAR and superantigens proteins G, A, and L by both IgA1 and A2.

The unmutated common ancestor of Gp41 conformational epitope targeting variable heavy chain 1-02 utilizing antibodies maintains antibody dependent cell cytotoxicity

In studies on monoclonal Abs (mAbs) from long-term non-progressors (LTNPs), our laboratory has previously described highly mutated Abs against a complex conformational epitope with contributions from both gp41 heptad repeat regions. Despite using the VH1-02 gene segment, known to contribute to some of the broadest neutralizing Abs against HIV, members of these Abs, termed group 76C Abs, did not exhibit broad neutralization.<br />Because of the excessive mutations and use of VH1-02, our goal was to characterize the non-neutralizing functions of Abs of group 76C, to assess targeting of the epitope in various clinical presentations, and to assess the development of these Abs by comparison to their predicted common ancestor. Serum competition assays showed group 76C Abs were enriched in LTNPs, in comparison to VRC-01. Specific group 76C clones 6F5 and 6F11, expressed as recombinant Abs, both have robust ADCC activity, despite their sequence disparity. Sequence analysis predicted the common ancestor of this clonal group would utilize the germline non-mutated variable gene. We produced a recombinant ancestor Ab (76Canc) with a heavy chain utilizing the germline variable gene sequence paired to the 6F5 light chain. Competition with group 76C recombinant Ab 6F5 confirms 76Canc binds HIV envelope constructs near the original group C epitope. 76Canc demonstrates comparable ADCC to 6F5 and 6F11 when targeting both clade B and C HIV constructs. The functional capability of Abs utilizing germline VH1-02 has implications for disease control and vaccine development.

Isolation and characterization of human monoclonal antibodies to pneumococcal capsular polysaccharide 3

The current pneumococcal capsular polysaccharide (PPS) conjugate vaccine (PCV13) is less effective against Streptococcus pneumoniae serotype 3 (ST3), which remains a major cause of pneumococcal disease and mortality. Therefore, dissecting structure-function relationships of human PPS3 antibodies may reveal characteristics of protective antibodies. Using flow cytometry, we isolated PPS3-binding memory B cells from pneumococcal vaccine recipients and generated seven human PPS3-specific monoclonal antibodies (humAbs). Five humAbs displayed ST3 opsonophagocytic activity, four induced ST3 agglutination in vitro, and four mediated both activities. For two humAbs, C10 and C27, that used the same variable heavy (VH) and light (VL) chain domains (VH3-9*01/VL2-14*03), C10 had fewer VL somatic mutations, higher PPS3 affinity, more ST3 opsonophagocytic and agglutinating activity, whilst both humAbs altered ST3 gene expression in vitro. After VL swaps, C10VH/C27VL exhibited reduced ST3 binding and agglutination, but C27VH/C10VL binding was unchanged. In C57Bl/6 mice, C10 and C27 reduced nasopharyngeal colonization with ST3 A66 and a clinical strain, B2, and prolonged survival following lethal A66 intraperitoneal infection, but only C10 protected against lethal intranasal infection with the clinical strain. Our findings, associate efficacy of PPS3-specific humAbs with ST3 agglutination and opsonophagocytic activity and reveal an unexpected role for the VL in functional activity in vitro and in vivo. These findings also provide insights that may inform antibody-based therapy and identification of surrogates of vaccine efficacy against ST3.

Detrital zircon geochronology and heavy mineral analysis as complementary provenance tools in the presence of extensive weathering, reworking and recycling: the Neogene of the southern North Sea Basin

Heavy mineral analysis is a long-standing and valuable tool for sedimentary provenance analysis. Many studies have indicated that heavy mineral data can also be significantly affected by hydraulic sorting, weathering and reworking or recycling, leading to incomplete or erroneous provenance interpretations if they are used in isolation. By combining zircon U–Pb geochronology with heavy mineral data for the southern North Sea Basin, this study shows that the classic model of sediment mixing between a northern and a southern source throughout the Neogene is more complex. In contrast to the strongly variable heavy mineral composition, the zircon U–Pb age spectra are mostly constant for the studied samples. This provides a strong indication that most zircons had an initial similar northern source, yet the sediment has undergone intense chemical weathering on top of the Brabant Massif and Ardennes in the south. This weathered sediment was later recycled into the southern North Sea Basin through local rivers and the Meuse, leading to a weathered southern heavy mineral signature and a fresh northern heavy mineral signature, yet exhibiting a constant zircon U–Pb age signature. Thus, this study highlights the necessity of combining multiple provenance proxies to correctly account for weathering, reworking and recycling.

Engineering a reporter cell line to mimic the high oligomannose presenting surface immunoglobulin of follicular lymphoma B cells

Subtypes of B cell non-Hodgkin’s lymphomas, including follicular lymphomas, have shown a unique high oligomannose presentation on their immunoglobulins that will interact with natural receptors of the innate immunity, reportedly causing stimulation and proliferation. From deep sequencing of the variable heavy and light chain sequences of follicular lymphoma involved tissue sections, we identified the consensus variable sequences possessing glycosylation sites at the complementarity determining region. Using this information, we developed a cell line, referred to here as BZ, which displays the consensus variable segments as part of a surface antibody (IgM) and confirmed its presentation of high oligomannose on the heavy chain both in vitro and in vivo. An mCherry expressing variant provided a reporter cell line displaying the high oligomannose surface biomarker while affording clear fluorescent signals for FACS screening as well as for fluorescent in vivo imaging of ectopic xenograft tumors. In developing this reporter cell line that displays the biomarker glycan of follicular lymphoma, we provide a tool that may be used for future screening and validation of receptive moieties for selectively binding high oligomannose for development of targeted diagnostics or therapeutics to such B cell malignancies that display this unique glycan.

Bringing the Heavy Chain to Light: Creating a Symmetric, Bivalent IgG-Like Bispecific

Bispecific molecules are biologically significant, yet their complex structures pose important manufacturing and pharmacokinetic challenges. Nevertheless, owing to similarities with monoclonal antibodies (mAbs), IgG-like bispecifics conceptually align well with conventional expression and manufacturing platforms and often exhibit potentially favorable drug metabolism and pharmacokinetic (DMPK) properties. However, IgG-like bispecifics do not possess target bivalency and current designs often require tedious engineering and purification to ensure appropriate chain pairing. Here, we present a near-native IgG antibody format, the 2xVH, which can create bivalency for each target or epitope and requires no engineering for cognate chain pairing. In this modality, two different variable heavy (VH) domains with distinct binding specificities are grafted onto the first constant heavy (CH1) and constant light (CL) domains, conferring the molecule with dual specificity. To determine the versatility of this format, we characterized the expression, binding, and stability of several previously identified soluble human VH domains. By grafting these domains onto an IgG scaffold, we generated several prototype 2xVH IgG and Fab molecules that display similar properties to mAbs. These molecules avoided the post-expression purification necessary for engineered bispecifics while maintaining a capacity for simultaneous dual binding. Hence, the 2xVH format represents a bivalent, bispecific design that addresses limitations of manufacturing IgG-like bispecifics while promoting biologically-relevant dual target engagement.