Therapeutic monoclonal antibodies (mAbs) can be functionally enhanced via Fc engineering. To determine whether pairs of mAbs with different Fc modifications can be combined for functional complementarity, we investigated the in vitro activity of two HIV-1 mAb libraries, each equipped with 60 engineered Fc variants. Our findings demonstrate that the impact of Fc engineering on Fc functionality is dependent on the specific Fab clone. Notably, combinations of Fc variants of the same Fab specificity exhibited limited enhancement in functional breadth compared to combinations involving two distinct Fabs. This suggests that the strategic selection of complementary Fc modifications can enhance both functional activity and breadth. Furthermore, while some combinations of Fc variants displayed additive functional effects, others were detrimental, suggesting that the functional outcome of Fc mutations is not easily predicted. Collectively, these results provide preliminary evidence supporting the potential of complementary Fc modifications in mAb combinations. Future studies will be essential to identify the optimal Fc modifications that maximize in vivo efficacy.

Adoptive cell therapy (ACT) with TCR-engineered T-cells represents a promising alternative to TIL- or CAR-T therapies for patients with advanced solid cancers. Currently, selection of therapeutic TCRs critically depends on knowing the target antigens, a condition excluding most patients from treatment. Direct antigen-agnostic identification of tumor-specific T-cell clonotypes and TCR-T manufacturing using their TCRs can advance ACT for patients with aggressive solid cancers. We present a method to identify tumor-specific clonotypes from surgical specimens by comparing TCRβ-chain repertoires of TILs and adjacent tissue-resident lymphocytes. In six out of seven NSCLC-patients analyzed, our selection of tumor-specific clonotypes based on TIL-abundance and high tumor-to-nontumor frequency ratios was confirmed by gene expression signatures determined by scRNA-Seq. In three patients, we demonstrated that predicted tumor-specific clonotypes reacted against autologous tumors. For one of these patients, we engineered TCR-T cells with four candidate tumor-specific TCRs that showed reactivity against the patient's tumor and HLA-matched NSCLC cell lines. The TCR-T cells were then used to screen for candidate neoantigens and aberrantly expressed antigens. Three TCRs recognized recurrent driver-mutation KRAS Q61H-peptide ILDTAGHEEY presented by HLA-A*01:01. The TCRs were also dominant in a tumor relapse, one was found in cell free DNA. The finding of homologous TCRs in independent KRAS Q61H-positive cancers suggests a therapeutic opportunity for HLA-matched patients with KRAS Q61H-expressing tumors.
Copyright © 2025 Lennerz, Doppler, Fatho, Dröge, Schaper, Gennermann, Genzel, Plassmann, Weismann, Lukowski, Bents, Beushausen, Kriese, Herbst, Seitz, Hammer, Adam, Eggeling, Wölfel, Wölfel and Hennig.

AdCLD-CoV19-1, a chimeric adenovirus-based severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine, was previously reported to elicit robust antibody responses in mice and non-human primates after a single dose. In this study, we conducted a systems serology analysis to investigate changes in humoral immune responses induced by varying doses of the AdCLD-CoV19-1 vaccine in a phase I clinical trial. Serum samples from participants receiving either a low or a high dose of the vaccine were analyzed for antibody features against prototype SARS-CoV-2 spike (S) domains (full-length S, S1, S2, and receptor binding domain), as well as Fc receptor binding and effector functions. While both low- and high-dose vaccines induced robust humoral immune responses following vaccination, the quality of antibody features differed between the dose groups. Notably, while no significant difference was observed between the groups in the induction of most S1-specific antibody features, the high-dose group exhibited higher levels of antibodies and a stronger Fc receptor binding response specific to the S2 antigen. Moreover, univariate and multivariate analyses revealed that the high-dose vaccine induced higher levels of S2-specific antibodies binding to FcγR2A and FcγR3B, closely associated with antibody-dependent neutrophil phagocytosis (ADNP). Further analysis using the Omicron BA.2 variant demonstrated that the high-dose group maintained significantly higher levels of IgG and FcγR3B binding to the S2 antigen and exhibited a significantly higher ADNP response for the S2 antigen compared with the low-dose group. These findings underscore the importance of considering diverse humoral immune responses when evaluating vaccine efficacy and provide insights for optimizing adenovirus vector-based SARS-CoV-2 vaccine doses.IMPORTANCEOptimization of vaccine dose is crucial for eliciting effective immune responses. In addition to neutralizing antibodies, non-neutralizing antibodies that mediate Fc-dependent effector functions play a key role in protection against various infectious diseases, including coronavirus disease 2019. Using a systems serology approach, we demonstrated significant dose-dependent differences in the humoral immune responses induced by the AdCLD-CoV19-1 chimeric adenovirus-based severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine, particularly against the SARS-CoV-2 spike 2 domain. These findings highlight the importance of assessing not only neutralizing antibody titers but also the quality and functionality of antibody responses when evaluating vaccine efficacy.

Altering Bacille Calmette-Guérin (BCG) immunization from low-dose intradermal (i.d.) to high-dose intravenous (i.v.) vaccination provides a high level of protection against Mycobacterium tuberculosis (Mtb). In addition to strong T cell immunity, i.v. BCG drives robust humoral immune responses that track with bacterial control. However, given the near-complete protection afforded by high-dose i.v. BCG immunization, a precise correlate of protection was difficult to define. Here we leveraged plasma and bronchoalveolar lavage fluid (BAL) from a cohort of rhesus macaques that received decreasing doses of i.v. BCG and aimed to define correlates of immunity following Mtb challenge. We show an i.v. BCG dose-dependent induction of mycobacterial-specific humoral immune responses. Antibody responses at peak immunogenicity predicted bacterial control post-challenge. Multivariate analyses revealed antibody-mediated complement and natural killer (NK) cell-activating humoral networks as key signatures of protective immunity. This work extends our understanding of humoral biomarkers and potential mechanisms of i.v. BCG-mediated protection against Mtb.
© 2024 The Authors.

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), remains a global health burden. While M. tuberculosis is primarily a respiratory pathogen, it can spread to other organs, including the brain and meninges, causing TB meningitis (TBM). However, little is known about the immunological mechanisms that lead to differential disease across organs. Attention has focused on differences in T cell responses in the control of M. tuberculosis in the lungs, but emerging data point to a role for antibodies, as both biomarkers of disease control and as antimicrobial molecules. Given an increasing appreciation for compartmentalized antibody responses across the blood-brain barrier, here we characterized the antibody profiles across the blood and brain compartments in TBM and determined whether M. tuberculosis-specific humoral immune responses differed between M. tuberculosis infection of the lung (pulmonary TB) and TBM. Using a high throughput systems serology approach, we deeply profiled the antibody responses against 10 different M. tuberculosis antigens, including lipoarabinomannan (LAM) and purified protein derivative (PPD), in HIV-negative adults with pulmonary TB (n = 10) versus TBM (n = 60). Antibody studies included analysis of immunoglobulin isotypes (IgG, IgM, IgA) and subclass levels (IgG1-4) and the capacity of M. tuberculosis-specific antibodies to bind to Fc receptors or C1q and to activate innate immune effector functions (complement and natural killer cell activation; monocyte or neutrophil phagocytosis). Machine learning methods were applied to characterize serum and CSF responses in TBM, identify prognostic factors associated with disease severity, and define the key antibody features that distinguish TBM from pulmonary TB. In individuals with TBM, we identified CSF-specific antibody profiles that marked a unique and compartmentalized humoral response against M. tuberculosis, characterized by an enrichment of M. tuberculosis-specific antibodies able to robustly activate complement and drive phagocytosis by monocytes and neutrophils, all of which were associated with milder TBM severity at presentation. Moreover, individuals with TBM exhibited M. tuberculosis-specific antibodies in the serum with an increased capacity to activate phagocytosis by monocytes, compared with individuals with pulmonary TB, despite having lower IgG titres and Fcγ receptor-binding capacity. Collectively, these data point to functionally divergent humoral responses depending on the site of infection (i.e. lungs versus brain) and demonstrate a highly compartmentalized M. tuberculosis-specific antibody response within the CSF in TBM. Moreover, our results suggest that phagocytosis- and complement-mediating antibodies may promote attenuated neuropathology and milder TBM disease.
© The Author(s) 2024. Published by Oxford University Press on behalf of the Guarantors of Brain.