As a crucial asset for human health and modern medicine, an increasing number of biotherapeutics are entering the clinic. However, due to their complexity, these drugs have a higher potential to be immunogenic, leading to the generation of anti-drug antibodies (ADAs). Clinically significant ADAs have an impact on pharmacokinetics (PK), pharmacodynamics (PD), effectiveness, and/or safety. Thus, it is crucial to understand, manage and minimize the immunogenicity potential during drug development, ideally starting from the molecule design stage.
In this study, we utilized various immunogenicity risk assessment methods, including in silico prediction, dendritic cell internalization, MHC-associated peptide proteomics, in vitro HLA peptide binding, and in vitro T cell proliferation, to assess the immunogenicity risk of FLT3L-Fc variants.
We identified a single point mutation in the human FLT3L-Fc protein that introduced highly immunogenic T cell epitopes, leading to the induction of T cell responses and thereby increasing the immunogenicity risk in clinical settings. Consequently, the variant with this point mutation was removed from further consideration as a clinical candidate.
This finding underscores the necessity for careful evaluation of mutations during the engineering of protein therapeutics. The integration of multiple immunogenicity risk assessment tools offers critical insights for informed decision-making in candidate sequence design and therapeutic lead selection.
Copyright © 2025 Qin, Phung, Wu, Yin, Tam, Tran, ElSohly, Gober, Hu, Zhou, Cohen, He, Bainbridge, Kemball, Zarzar, Sreedhara, Stephens, Decalf, Moussion, Ye, Balazs and Li.

The vastly spreading COVID-19 pneumonia is caused by SARS-CoV-2. Lymphopenia and cytokine levels are tightly associated with disease severity. However, virus-induced immune dysregulation at cellular and molecular levels remains largely undefined. Here, the leukocytes in the pleural effusion, sputum, and peripheral blood biopsies from severe and mild patients were analyzed at single-cell resolution. Drastic T cell hyperactivation accompanying elevated T cell exhaustion was observed, predominantly in pleural effusion. The mechanistic investigation identified a group of CD14+ monocytes and macrophages highly expressing CD163 and MRC1 in the biopsies from severe patients, suggesting M2 macrophage polarization. These M2-like cells exhibited up-regulated IL10, CCL18, APOE, CSF1 (M-CSF), and CCL2 signaling pathways. Further, cell type specific dysregulation of transposable elements was observed in Severe COVID-19 patients. Together, our results suggest that severe SARS-CoV-2 infection causes immune dysregulation by inducing M2 polarization and subsequent T cell exhaustion. This study improves our understanding of COVID-19 pathogenesis.
© 2024 The Authors. Published by Elsevier Ltd.

NSG-SGM3 and NOG-EXL mice combine severe immunodeficiency with transgenic expression of human myeloid stimulatory cytokines, resulting in marked expansion of myeloid populations upon humanization with CD34+ hematopoietic stem cells (HSCs). Humanized NSG-SGM3 mice typically develop a lethal macrophage activation syndrome and mast cell hyperplasia that limit their use in long-term studies (e.g., humanization followed by tumor xenotransplantation). It is currently unclear to what extent humanized NOG-EXL mice suffer from the same condition observed in humanized NSG-SGM3 mice. We compared the effects of human CD34+ HSC engraftment in these two strains in an orthotopic patient-derived glioblastoma model. NSG-SGM3 mice humanized in-house were compared to NOG-EXL mice humanized in-house and commercially available humanized NOG-EXL mice. Mice were euthanized at humane or study endpoints, and complete pathological assessments were performed. A semiquantitative multiparametric clinicopathological scoring system was developed to characterize chimeric myeloid cell hyperactivation (MCH) syndrome. NSG-SGM3 mice were euthanized at 16 weeks after humanization because of severe deterioration of clinical conditions. Humanized NOG-EXL mice survived to the study endpoint at 22 weeks after humanization and showed less-severe MCH phenotypes than NSG-SGM3 mice. Major differences included the lack of mast cell expansion and limited tissue/organ involvement in NOG-EXL mice compared to NSG-SGM3 mice. Engraftment of human lymphocytes, assessed by immunohistochemistry, was similar in the two strains. The longer survival and decreased MCH phenotype severity in NOG-EXL mice enabled their use in a tumor xenotransplantation study. The NOG-EXL model is better suited than the NSG-SGM3 model for immuno-oncology studies requiring long-term survival after humanization.

Bispecific antibodies, including bispecific IgG, are emerging as an important new class of antibody therapeutics. As a result, we, as well as others, have developed engineering strategies designed to facilitate the efficient production of bispecific IgG for clinical development. For example, we have extensively used knobs-into-holes (KIH) mutations to facilitate the heterodimerization of antibody heavy chains and more recently Fab mutations to promote cognate heavy/light chain pairing for efficient in vivo assembly of bispecific IgG in single host cells. A panel of related monospecific and bispecific IgG1 antibodies was constructed and assessed for immunogenicity risk by comparison with benchmark antibodies with known low (Avastin and Herceptin) or high (bococizumab and ATR-107) clinical incidence of anti-drug antibodies. Assay methods used include dendritic cell internalization, T cell proliferation, and T cell epitope identification by in silico prediction and MHC-associated peptide proteomics. Data from each method were considered independently and then together for an overall integrated immunogenicity risk assessment. In toto, these data suggest that the KIH mutations and in vitro assembly of half antibodies do not represent a major risk for immunogenicity of bispecific IgG1, nor do the Fab mutations used for efficient in vivo assembly of bispecifics in single host cells. Comparable or slightly higher immunogenicity risk assessment data were obtained for research-grade preparations of trastuzumab and bevacizumab versus Herceptin and Avastin, respectively. These data provide experimental support for the common practice of using research-grade preparations of IgG1 as surrogates for immunogenicity risk assessment of their corresponding pharmaceutical counterparts.

FMS-related tyrosine kinase 3 ligand (FLT3L), encoded by FLT3LG, is a hematopoietic factor essential for the development of natural killer (NK) cells, B cells, and dendritic cells (DCs) in mice. We describe three humans homozygous for a loss-of-function FLT3LG variant with a history of various recurrent infections, including severe cutaneous warts. The patients' bone marrow (BM) was hypoplastic, with low levels of hematopoietic progenitors, particularly myeloid and B cell precursors. Counts of B cells, monocytes, and DCs were low in the patients' blood, whereas the other blood subsets, including NK cells, were affected only moderately, if at all. The patients had normal counts of Langerhans cells (LCs) and dermal macrophages in the skin but lacked dermal DCs. Thus, FLT3L is required for B cell and DC development in mice and humans. However, unlike its murine counterpart, human FLT3L is required for the development of monocytes but not NK cells.
Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.