Biology subjects

Ling, W.-L.

Publications and source records attributed to Ling, W.-L..

6 recordsLinked to original sources

bioRxiv2022

The contributory effects of the VH families and CH regions of multimeric IgM on its interaction with Fc(mu)R, and antigen.

As the primary response antibody with increasing interest as a therapeutic antibody format, IgM is also the largest antibody structure among the five major human isotypes. Spontaneously forming pentamer and hexamers, IgM has avidity effects that could compensate for weaker interactions, although steric hindrances can occur for certain epitopes. With recent evidence of the heavy chain constant region affecting antigen binding and the VH families of the V-regions affecting FcR engagement found on other isotypes, we investigated CDR-grafted Trastuzumab and Pertuzumab VH1-7 IgMs for biolayer interferometry. From our panel of the 14 IgM variants, the V-regions holistically affected FcR binding, and the IgM C-region modulated Her2 engagements with contributions from the V-regions and influences from protein L binding at the V{kappa}. These findings revealed the oligomerization effect of IgMs to play a significant role in both FcR and antigen binding that is distinct from the other isotypes that can guide the development and protein en-gineering of IgM therapeutics.

immunology↗

bioRxiv2021

Augmenting Whole Recombinant Antibody Production in HEK293E Cells: Optimizing Transfection and Culture Parameters

BackgroundOptimising recombinant antibody production is important for cost-effective therapeutics and diagnostics. With impact on commercialisation, higher productivity beyond laboratory scales is highly sought, where efficient production can also accelerate antibody characterisations and investigations. MethodsInvestigating HEK293E cells for mammalian antibody production, various transfection and culture parameters were systematically analysed for antibody light chain production before evaluating them for whole antibody production. Transfection parameters investigated include seeding cell density, the concentration of the transfection reagent and DNA, complexation time, temperature, and volume, as well as culture parameters such as medium replacement, serum deprivation, use of cell maintenance antibiotic, incubation temperature, medium volume, post-transfection harvest day and common nutrient supplements. ResultsUsing 2 mL adherent HEK293E cell culture transfections with 25 kDa linear Polyethylenimine in the most optimised parameters, we demonstrated a ~2-fold production increase for light chain alone and for whole antibody production reaching 536 and 49 g respectively in a cost-effective manner. With the addition of peptone, {kappa} light chain increased by ~4-fold to 1032 g while whole antibody increased to a lesser extent by ~2.5-fold to 51 g, with benefits potentially for antibodies limited by their light chains in production. ConclusionsOur optimised findings show promise for a more efficient and convenient antibody production method through transfection and culture optimisations that can be incorporated to scale up processes and with potential transferability to other mammalian-based recombinant protein production using HEK293E cells. Statement of SignificanceRecombinant antibody production is crucial for antibody research and development. Systematically investigating transfection and culture parameters such as PEI/DNA concentrations, complexation time, volume, and temperature, supplements, etc., we demonstrated a ~4-fold light chain alone production increase to 1032 g and a 2.5-fold whole antibody production increase to 51 g from 2 mL transfections.

molecular biology↗

bioRxiv2021

Engaging the A-Class Antibody: Variable-Heavy (VH) region influencing IgA1 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 FcRI 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.

immunology↗

bioRxiv2020

Antibody Signal peptides and essential amino acid counts on recombinant antibody production

Boosting the production of recombinant therapeutic antibodies is crucial in both academic and industry settings. In this work, we investigated the usage of varying signal peptides by antibody genes and their roles in recombinant transient production. Comparing myeloma and the native signal peptides of both heavy and light chains in 168 antibody permutation variants, we performed a systematic analysis, finding amino acids counts to be involved in antibody production to construct a model for predicting co-transfection transient recombinant antibody production rates using the HEK293 system. The findings also provide insights into the usage of the large repertoire of antibody signal peptides.

molecular biology↗

bioRxiv2020

Molecular insight into Nickel(II) binding by Her2-specific IgE: a possible mechanistic insight into the pathogenesis of Type I nickel hypersensitivity

Nickel (Ni) allergy has been reported in contact dermatitis Type IV (Ni-specific T cells mediated) and asthmatic Type I (IgE-mediated) hypersensitivities. Associations between the two hypersensitivities have been found in some patients, but the underlying mechanisms remain enigmatic. Using Her2-specific IgEs as models, we found additional binding to Ni-NTA without observable changes in binding to Her2 and that glutamine, together with the canonical Ni2+-binding histidine, could form Ni2+ binding signatures. This mechanism may underlie Type I hypersensitivity in the selection of anti-Ni2+ IgEs. This mechanism may also underlie Type IV hypersensitivity and the interaction of immunoglobulin proteins with other heavy metal ions. Our findings shed light to how Ni hypersensitivities can occur and how they can be avoided in therapeutics design, or even incorporated for biotechnological purification purposes.

immunology↗

bioRxiv2019

The Quantification of Antibody Elements and Receptors subunit expression using qPCR: The Design of VH, VL, CH, CL, FcR subunits primers for a more holistic view of the immune system.

The expression levels of Immunoglobulin elements and their receptors are important markers for health and disease. Within the immunoglobulin locus, the constant regions and the variable region families are associated with certain pathologies, yet a holistic view of the interaction between the expression of the multiple genes remain to be fully characterized. There is thus an important need to quantify antibody elements, their receptors and the receptor subunits in blood (PBMC cDNA) for both screening and detailed studies of such associations. Leveraging on qPCR, we designed primers for all V{kappa} 1-6, VH1-7, V{lambda}1-11, nine CH isotypes, C{kappa}, C{kappa}, C{lambda}1 &3, Fc{varepsilon}RI ,{beta}, and {gamma} subunits, all three Fc{gamma}R and their subunits, and FcR. Validating this on a volunteer PBMC cDNA, we show a qPCR primer set repertoire that can quantify the relative expression of all the above genes to GAPDH housekeeping gene, with implications and uses in both clinical monitoring and research.

immunology↗