Effect of VH–VL Families in Pertuzumab and Trastuzumab Recombinant Production, Her2 and FcγIIA Binding

Ling, Wei-Li; Lua, Wai-Heng; Poh, Jun-Jie; Yeo, Joshua Yi; Lane, David P.; Gan, Samuel Ken‐En

doi:10.3389/fimmu.2018.00469

Cited by 50 publications

(140 citation statements)

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“…Complementarity-determining regions (CDRs) of pertuzumab and trastuzumab VHs were grafted onto the various germline VH families representative of VH1 to VH7, as previously described (Fig 1). 32 The VH families were joined to the IgE C-region, as previously performed. 31,33 Plasmids were transformed into homemade competent DH5a bacteria, as previously described, 34 and extracted by using our in-house extraction kits.…”

Section: Cloning Of Antibodiesmentioning

confidence: 99%

“…The extracellular FcεRIa portion of the high-affinity IgE receptor (accession no: NP_001992.1, amino acids 1-201) with a 103 C-terminal HIS tag was cloned and expressed, as previously described for FcgIIA. 32 The secreted protein was purified from the supernatants of transfected cells by using affinity chromatography with the Cobalt column (catalog no. 28953766; GE Healthcare, Chicago, Ill), followed by gel-filtration fractionation with the Superdex 200-pg 16/600 column (GE Healthcare).…”

Section: Production and Purification Of Fcεriamentioning

confidence: 99%

“…Pertuzumab and trastuzumab VH1-VH7 IgE variants were produced by means of cotransfection, as previously performed. [30][31][32]36 Affinity purification was carried out with the Protein L column, followed by size exclusion chromatography fractionation with the Superdex 200-pg 16/600 column (GE Healthcare) to extract monomeric fractions, which were further concentrated by using a 100-kDa Amicon Ultra protein concentrator (catalog no. ACS510024; Merck-Millipore).…”

Section: Production and Purification Of Antibodiesmentioning

confidence: 99%

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Role of the IgE variable heavy chain in FcεRIα and superantigen binding in allergy and immunotherapy

Lua

Yeo

et al. 2019

Journal of Allergy and Clinical Immunology

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Background: Variable heavy chain (VH) family frameworks (FWRs) have been reported to affect antibody receptor and superantigen binding; however, such effects in IgE remain largely unknown. Given that VH family biases have been previously reported in IgE of certain allergies, there is a need to investigate this phenomenon for biotechnological and therapeutic purposes. Objective: We sought to investigate the effects of VH families on IgE interaction with FcεRIa, anti-IgE omalizumab, antigen, and superantigen protein A (spA) by using the pertuzumab and trastuzumab IgE models. Methods: Pertuzumab VH1-VH7 family variants of IgE with the same complementarity-determining regions were investigated with regard to their binding interactions to FcεRIa, Her2, omalizumab, and spA. Notable FcεRIa-IgE observations were cross-checked against appropriate trastuzumab IgE VH variants. Computational structural modeling and simulations were also performed for insight into the mechanism of interactions with various VH FWRs. Results: The pertuzumab VH5 IgE variant, but not the trastuzumab VH5 IgE, was found to interact with FcεRIa significantly longer than the respective VH family variants within each model antibody. No significant differences in interaction were found between IgE and omalizumab for the pertuzumab VH variants. Although trastuzumab VH3 interacted with spA, none of our pertuzumab VH variants, including VH3, associated with spA. Conclusion: We found unexpected varying allosteric communications caused by the VH family FWRs to the FcεRIa-, Her2-, and spA-binding regions of pertuzumab IgE, with implications for use of IgE/anti-IgE therapeutics to treat allergy and IgE therapeutics in allergo-oncology.

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Section: Cloning Of Antibodiesmentioning

confidence: 99%

Section: Production and Purification Of Fcεriamentioning

confidence: 99%

Section: Production and Purification Of Antibodiesmentioning

confidence: 99%

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Role of the IgE variable heavy chain in FcεRIα and superantigen binding in allergy and immunotherapy

Lua

Yeo

et al. 2019

Journal of Allergy and Clinical Immunology

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“…Occurring at varying rates in life processes that range from hypermutations in the immune system e.g. antibodies (Roth & Craig, 1998;Su et al, 2017;Ling et al, 2018) to disease development in cancer (Hollstein et al, 1991;Hanahan & Weinberg, 2011), and drug resistance (Weiss, 1993;Su et al, 2016;Chiang et al, 2018), mutations underpin change.…”

Section: Introductionmentioning

confidence: 99%

Probability of Change in Life: amino acid changes in single nucleotide substitutions

Chan¹,

Koukouravas²,

Yeo³

et al. 2019

Preprint

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ABSTRACTMutations underpin the processes in life, be it beneficial or detrimental. While mutations are assumed to be random in the bereft of selection pressures, the genetic code has underlying computable probabilities in amino acid phenotypic changes. With a wide range of implications including drug resistance, understanding amino acid changes is important. In this study, we calculated the probabilities of substitutions mutations in the genetic code leading to the 20 amino acids and stop codons. Our calculations reveal an enigmatic in-built self-preserving organization of the genetic code that averts disruptive changes at the physicochemical properties level. These changes include changes to start, aromatic, negative charged amino acids and stop codons. Our findings thus reveal a statistical mechanism governing the relationship between amino acids and the universal genetic code.

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“…IgE for allergy (Stanworth., 1993), IgA in IgA nephropathy (Suzuki, H., 2011), and IgM in hyper-IgM syndrome (Lougaris, V., 2005) amongst many others. The constant region (forming the isotype) was also able to influence the ability of V-regions to bind antigens and the variable light chain (VL) families were also found to impact the interaction with antigens (Ling et al, 2017) , production (Ling et al, 2017;Ling et al, 2018), and the interaction with Ig receptors (FcRs; see Ling et al, 2018;Su et al, 2018;Lua et al, 2019). Given the effects of the various antibody parts and their association with certain pathologies, the relative quantification of specific antibody elements and genes together with the antibody receptors and their subunits are important markers in health screening and to provide a more holistic investigation to disease associations.…”

Section: Introductionmentioning

confidence: 99%

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

Ling

Ng²,

Wipat

et al. 2019

Preprint

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Author Contributions:WWL performed the experiments, analysis and drafted the manuscript. YLN, AW, DPL were involved in the writing and discussions of the manuscript. SKEG conceived and supervised all aspects of the study. AbstractThe 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 Vκ1-6, VH1-7, Vλ1-11, nine CH isotypes, Cκ, Cκ, Cλ1 &3, FcεRI α ,β, and γ subunits, all three FcγR and their subunits, and FcαR. 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.

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Effect of VH–VL Families in Pertuzumab and Trastuzumab Recombinant Production, Her2 and FcγIIA Binding

Cited by 50 publications

References 36 publications

Role of the IgE variable heavy chain in FcεRIα and superantigen binding in allergy and immunotherapy

Role of the IgE variable heavy chain in FcεRIα and superantigen binding in allergy and immunotherapy

Probability of Change in Life: amino acid changes in single nucleotide substitutions

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

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