Cu(II) Binding Increases the Soluble Toxicity of Amyloidogenic Light Chains

Light chain amyloidosis (AL) is caused by the aberrant overproduction of immunoglobulin light chains (LCs). The resulting abnormally high LC concentrations in blood lead to deposit formation in the heart and other target organs. Organ damage is caused not only by the accumulation of bulky amyloid deposits, but extensive clinical data indicate that circulating soluble LCs also exert cardiotoxic effects. The nematode C. elegans has been validated to recapitulate LC soluble toxicity in vivo, and in such a model a role for copper ions in increasing LC soluble toxicity has been reported. Here, we applied microscale thermophoresis, isothermal calorimetry and thermal melting to demonstrate the specific binding of Cu2+ to the variable domain of amyloidogenic H7 with a sub-micromolar affinity. Histidine residues present in the LC sequence are not involved in the binding, and yet their mutation to Ala reduces the soluble toxicity of H7. Copper ions bind to and destabilize the variable domains and induce a limited stabilization in this domain. In summary, the data reported here, elucidate the biochemical bases of the Cu2+-induced toxicity; moreover, they also show that copper binding is just one of the several biochemical traits contributing to LC soluble in vivo toxicity.

Enhanced Conformational Sampling of Nanobody CDR H3 Loop by Generalized Replica-Exchange with Solute Tempering

The variable domains of heavy-chain antibodies, known as nanobodies, are potential substitutes for IgG antibodies. They have similar affinities to antigens as antibodies, but are more heat resistant. Their small size allows us to exploit computational approaches for structural modeling or design. Here, we investigate the applicability of an enhanced sampling method, a generalized replica-exchange with solute tempering (gREST) for sampling CDR-H3 loop structures of nanobodies. In the conventional replica-exchange methods, temperatures of only a whole system or scaling parameters of a solute molecule are selected for temperature or parameter exchange. In gREST, we can flexibly select a part of a solute molecule and a part of the potential energy terms as a parameter exchange region. We selected the CDR-H3 loop and investigated which potential energy term should be selected for the efficient sampling of the loop structures. We found that the gREST with dihedral terms can explore a global conformational space, but the relaxation to the global equilibrium is slow. On the other hand, gREST with all the potential energy terms can sample the equilibrium distribution, but the structural exploration is slower than with dihedral terms. The lessons learned from this study can be applied to future studies of loop modeling.

A comprehensive comparison between camelid nanobodies and single chain variable fragments

By the emergence of recombinant DNA technology, many antibody fragments have been developed devoid of undesired properties of natural immunoglobulins. Among them, camelid heavy-chain variable domains (VHHs) and single-chain variable fragments (scFvs) are the most favored ones. While scFv is used widely in various applications, camelid antibodies (VHHs) can serve as an alternative because of their superior chemical and physical properties such as higher solubility, stability, smaller size, and lower production cost. Here, these two counterparts are compared in structure and properties to identify which one is more suitable for each of their various therapeutic, diagnosis, and research applications.

Anti-Drug Antibodies in Pigtailed Macaques Receiving HIV Broadly Neutralising Antibody PGT121

Broadly neutralising antibodies (bNAbs) may play an important role in future strategies for HIV control. The development of anti-drug antibody (ADA) responses can reduce the efficacy of passively transferred bNAbs but the impact of ADA is imperfectly understood. We previously showed that therapeutic administration of the anti-HIV bNAb PGT121 (either WT or LALA version) controlled viraemia in pigtailed macaques with ongoing SHIV infection. We now report on 23 macaques that had multiple treatments with PGT121. We found that an increasing number of intravenous doses of PGT121 or human IgG1 isotype control antibodies (2-4 doses) results in anti-PGT121 ADA induction and low plasma concentrations of PGT121. ADA was associated with poor or absent suppression of SHIV viremia. Notably, ADA within macaque plasma recognised another human bNAb 10E8 but did not bind to the variable domains of PGT121, suggesting that ADA were primarily directed against the constant regions of the human antibodies. These findings have implications for the development of preclinical studies examining multiple infusions of human bNAbs.

Inefficient exploitation of accessory receptors reduces the sensitivity of chimeric antigen receptors.

Chimeric antigen receptors (CARs) can re-direct T cells to target abnormal cells but their activity is limited by a profound defect in antigen sensitivity, the source of which remains unclear. Here, we show that CARs have a > 100-fold lower antigen sensitivity compared to the TCR when antigen is presented on antigen-presenting-cells, but nearly identical sensitivity when antigen is presented as purified protein in isolation. Given that the TCR uses other, accessory, receptors to achieve high sensitivity, we screened prominent accessory receptors by presenting their purified ligands together with antigen. We found that ligating the adhesion receptor CD2 or LFA-1 improved antigen sensitivity for the TCR by > 100-fold, whereas for CARs the improvement was < 10-fold. We reproduced these findings using target cells where the CD2 and/or LFA-1 interaction were abrogated. Sensitivity can be partially restored by fusing the CAR variable domains to the TCR CD3ϵ subunit (also known as a TRuC) and fully restored when exchanging the TCRαβ variable domains for those of the CAR (also known as a STAR). Our study localises the defect in CAR sensitivity to inefficient use of accessory receptors and suggests approaches to increase sensitivity.

Characteristics and Determinants of Treatment Default Among Smokers With Tuberculosis in an Industrial State of Malaysia: A Registry-based Study of the Years 2013-2017

Background: The increased risk of treatment default among smokers raises concern over the secondary spread within the community. This study aimed to determine the prevalence and factors associated with treatment default among TB patients who smoke.Methods: A retrospective cohort of all registered TB patients who smoke in the state of Selangor between 2013 and 2017 via the Malaysian National MyTB database was included for analysis. TB patients who smoke were considered those with an active smoking status during the notification, while treatment default was defined as a TB patient who had interrupted treatment for 2 months or longer. There were 4 main variable domains included for analysis: sociodemographic profiles, disease profiles, treatment profiles, and comorbidities. Logistic regression analysis was used to identify determinants of treatment default among TB patients who smoke.Results: A total of 27.6% (N=6278) of the TB patients registered in Selangor were active smokers, and 15.1% (N=813) of the TB patients who smoke experienced defaulted TB treatment. The determinants of treatment default among TB patients who smoke were patients staying in an urban area (AOR 1.47; 95% CI 1.11,1.96), median income level less than RM2160 (AOR 2.0; 95% CI 1.34,2.99), no formal education (AOR 2.12; 95% CI 1.31,3.44), previously treated cases (AOR 2.78; 95% CI 1.99,3.88), active TB case detection methods (AOR 2.05; 95% CI 1.21,3.47), treatment duration of less than 6 months (AOR 7.56; 95% CI 5.74,9.92), and patients not on DOTS during the continuation phase (AOR 27.96; 95% CI 21.1,37.1). All the significant factors gave rise to the final model of determinants, with a predictability of 92.9% (95% CI 92.0,93.7).Conclusions: Our findings highlighted the high prevalence of treatment default among TB patients who smoke compared to the general TB population. Early risk detection that examines the two main domains of risk factors (socioeconomic factors and treatment profiles) should be provided for those who smoke in the TB population. Interventions should aim to reduce the prevalence of smoking among TB patients, together with close supervision during DOTS.

Insights into Comparative Modeling of VHH Domains

In the particular case of the Camelidae family, immunoglobulin proteins have evolved into a unique and more simplified architecture with only heavy chains. The variable domains of these chains, named VHHs, have a number of Complementary Determining Regions (CDRs) reduced by half, and can function as single domains making them good candidates for molecular tools. 3D structure prediction of these domains is a beneficial and advantageous step to advance their developability as molecular tools. Nonetheless, the conformations of CDRs loops in these domains remain difficult to predict due to their higher conformational diversity. In addition to CDRs loop diversity, our earlier study has established that Framework Regions (FRs) are also not entirely conformationally conserved which establishes a need for more rigorous analyses of these regions that could assist in template selection. In the current study, VHHs models using different template selection strategies for comparative modeling using Modeller have been extensively assessed. This study analyses the conformational changes in both CDRs and FRs using an original strategy of conformational discretization based on a structural alphabet. Conformational sampling in selected cases is precisely reported. Some interesting outcomes of the structural analyses of models also draw attention towards the distinct difficulty in 3D structure prediction of VHH domains.

Mouse CD38-Specific Heavy Chain Antibodies Inhibit CD38 GDPR-Cyclase Activity and Mediate Cytotoxicity Against Tumor Cells

CD38 is the major NAD+-hydrolyzing ecto-enzyme in most mammals. As a type II transmembrane protein, CD38 is also a promising target for the immunotherapy of multiple myeloma (MM). Nanobodies are single immunoglobulin variable domains from heavy chain antibodies that naturally occur in camelids. Using phage display technology, we isolated 13 mouse CD38-specific nanobodies from immunized llamas and produced these as recombinant chimeric mouse IgG2a heavy chain antibodies (hcAbs). Sequence analysis assigned these hcAbs to five distinct families that bind to three non-overlapping epitopes of CD38. Members of families 4 and 5 inhibit the GDPR-cyclase activity of CD38. Members of families 2, 4 and 5 effectively induce complement-dependent cytotoxicity against CD38-expressing tumor cell lines, while all families effectively induce antibody dependent cellular cytotoxicity. Our hcAbs present unique tools to assess cytotoxicity mechanisms of CD38-specific hcAbs in vivo against tumor cells and potential off-target effects on normal cells expressing CD38 in syngeneic mouse tumor models, i.e. in a fully immunocompetent background.

PREPRINT - Predictors, Moderators and Mediators of Treatment Outcome in Randomized Controlled Trials for Adolescents with Depression: A scoping review.

Background: Advancing the field of precision medicine in the treatment of depression in adolescents requires an examination of predictors, moderators and mediators (PMMs) of depression symptom outcome in randomized controlled trials (RCTs). Aims: This scoping review describes findings of PMM analyses in RCTs for the treatment of depression in adolescents. Methods: Databases searched included: MEDLINE, Embase, APA PsycInfo and CINAHL. Included publications tested PMMs of depression symptom outcome (e.g. symptom reduction, remission, etc.) in RCTs pertaining to the treatment of adolescents, ages 13-17. Results were extracted and coded for statistical significance, analytic approach, a priori model development, and adjustment for multiple comparisons. Aggregated results were summarized by PMM variable domain and RCT sample.Results: Eighty-one papers reporting on PMM results across 33 RCTs were identified. Fifty-three variable domains were tested as baseline predictors of depression severity outcome, 41 as moderators, 19 as post-baseline predictors, and 5 as mediators. Most reported results were nonsignificant. For variable domains tested as a PMM in three or more RCTs, only improvements in sleep throughout treatment and early response predicted outcome with complete agreement across relevant studies. The two publications that reported a priori hypotheses and adjustment for multiple comparisons both found that baseline depression symptom severity and family conflict predicted poorer outcomes. Conclusions: Clinicians need to recognize the current limited evidence from RCTs to practice precision medicine principles when treating adolescents with depression. Our findings inform future study design for researchers who wish to incorporate questions about specific PMM variables into their study methods.