AllergoOncology: Expression platform development and functional profiling of an anti‐HER2 IgE antibody

Efficient strategies for generation of recombinant IgE class antibodies at high-enough yields for pre-clinical screening and mechanistic evaluations remain challenging. We present a transient expression application for the rapid production of recombinant IgE, exemplified with the cloning and functional characterization of a humanised IgE antibody bearing the variable region sequences of the anti-HER2/ neu antibody trastuzumab. Material was generated with high transfection efficiency, in small culture volumes within 7-9-days from design to purified material, and at sufficient yields for functional studies. The antibody conserved the trastuzumab Fab-mediated recognition of the target antigen, and IgE Fc attributes to recognise Fc εRs and to potentiate immune cell-mediated effector functions. Moreover, mast cell and basophil activation tests confirmed lack of activation with IgE in the absence of cross-linking stimuli, supporting potential safe administration in humans. Our study facilitates fast generation of IgE antibodies for speedy screening and functional characterisation and can be readily applied to the design and evaluation of IgE antibodies in Allergy and AllergoOncology. Antibody cloning: We employed polymerase incomplete primer extension (PIPE) PCR cloning and enzyme-free assembly of DNA fragments. The amino acid sequences of the trastuzumab heavy and light variable regions were obtained from the DrugBank database (www.drugbank.ca), translated in nucleotide sequences and manually codon optimized for a human expression host. Optimized sequences were synthesized using GeneArt Gene Synthesis (Thermo Fischer Scientific UK). The DNA sequences of the variable regions were previously reported 1 . The variable region fragments of trastuzumab were cloned into pVitro1-hygro-mcs dual expression vector containing pre-cloned cassettes of the human epsilon chain constant region and kappa light chain constant region cassettes using the polymerase incomplete primer extension (PIPE) PCR cloning as previously described 2 . The PIPE PCR primers used for the fragment amplification are listed in Supplementary Table S1. Briefly, PIPE PCR was performed using pVitro1 plasmid as a template in order to generate two linear PCR fragments with 5` PIPE overhangs, and commercially-generated trastuzumab variable region fragments, in order to generate a variable light (VL) and heavy (VH) region fragments with 5` PIPE overhangs. Correct sizes of the DNA fragments were confirmed with agarose electrophoresis. Anti-HER2 IgE expression was carried out transiently in Expi293F cells using small volume suspension culture (30 mL) for ca. 7-9 days to achieve peak antibody concentrations of ca. 70-80 µg/mL in the culture supernatant. Purification and The antibody was purified using KappaSelect affinity


AllergoOncology: Expression platform development and functional profiling of an anti-HER2 IgE antibody
To the Editor, Monoclonal antibodies approved for the treatment of cancer belong to the IgG class (most often IgG1). However, IgG has limited tissue half-life (2-3 days), relatively low affinity for cognate Fc receptors and the disadvantage of interaction with inhibitory Fcγ receptors, abundant in the tumour microenvironment. Conversely, IgE class antibodies may offer new options for cancer therapy, based on high affinity for cognate Fcε receptors expressed on different, often tumour-resident, immune effector cells such as macrophages and mast cells, and lack of inhibitory Fc receptors. 1 IgE-mediated tissue surveillance functions known to potentiate "allergic" or "pathogen/ parasite-clearing" immunity could be re-directed against tissue-resident tumours. 2,3 IgE antibodies recognizing the tumour-associated antigen folate receptor α (FRα) induced superior immune responses in disparate in vivo models, highlighting potential opportunities for FRα-expressing ovarian carcinomas. 2 In breast cancer, in vitro studies of trastuzumab (IgG1) and an engineered trastuzumab IgE recognizing the tumour-associated antigen HER2/neu indicated that IgE could complement or possibly improve the clinical performance of trastuzumab. 4 The first-in-class IgE antibody (MOv18) is undergoing an early phase clinical trial in patients with FRα-expressing carcinomas (NCT02546921, www.clini caltr ials.gov).
Despite considerable progres s, pro duction of monoclonal antibodies remains time-consuming and labour-intensive. One reason is the requirement for expression of heavy (HC) and light chains (LC) in a controlled manner, usually cloned in separate expression vectors using enzymatic restriction d igesti on and ligation. This introduces experimental variability in e xpress ion procedures and is often inefficient. These limitations also c oncern the study of anti-allergen IgE, where Fabs rather than f ull-le ngth antibodies are commonly expressed and evaluated. [5][6][7] There fore, antibody cloning systems are moving towards utilization of single dual-expression plasmids (eg pcDNA3.3 and pVitro1 hygro-mcs), to increase antibody production. 8 Building upon ours and others' previous methodologies, we report the efficient transient expression and functional evaluation of IgE, exemplified using the variable region sequences of trastuzumab and human IgE constant regions (anti-HER2 IgE).
We employed polymerase incom plete primer extension (PIPE) PCR cloning and enzyme-free a ssembl y of DNA fragments. The amino acid sequences of trast uzumab variable light (VL) and heavy (VH) chain regions were manually codon-optimized for a human expression host and cloned into a pVitro1-hygro-mcs dual-expression vector containing precloned cassettes of the human epsilon HC and kappa LC using PIPE PCR cloni ng met hodology ( Figure 1A). 8 PIPE PCR was performed using the pVitro1 plasmid to generate linear PCR fragments with 5′ PIPE overhangs, and trastuzumab variable region fragments to derive VL and VH region fragments with 5′ PIPE overhangs (DNA fragment sizes by agarose gel electrophoresis, Figure 1B).
Expression was conducted tra nsient ly in human embryonic kidney (Expi293F) cells witho ut ant ibiotic selection, in 30 mL serum-free suspension culture s (Fig ure 1C). Variable region codon optimization enhanced antibody yields (~7-fold; Figure 1D). Peak antibody concentrations (70-80 µg/mL) were achieved within 7-9 days (supernatants harvested after 7 day s, Figure 1C,E). After purification, total yields were 60 µg /mL (> 85% purification efficiencies;   Figure 2G). In basophil activation tests (BAT) conducted in unfractionated human blood, anti-HER2 IgE did not induce basophil activation, monitored by upregulation of the activation marker CD63 ( Figure 2H,I). Mast cell and basophil tests therefore confirm lack of activation with IgE in the absence of cross-linking stimuli, 9 supporting potential safe administration in human circulation.
IgE immunotherapy may offer a promising approach for cancer treatment, contributing to the emerging field of AllergoOncology, focused on dissecting interplay between IgE, allergy and malignancy. The development of efficient platforms for speedy generation of full-length IgE at appreciable yields for numerous evaluations to expedite the field remains challenging. Our herein-described multi-gene cloning, enzyme-free assembly system for rapid expression of functionally active antibody, within 7-9 days from transfection to purification in serum-free cultures (2 mg purified material from 30 mL), readily established even in "small" environments, surpassing previous platforms in expression efficiency, speed (7-9 days

ACK N OWLED G EM ENTS
We acknowledge the Biomedical Research Centre Immune Monitoring Core Facility team at Guy's and St Thomas' NHS Foundation Trust.
We thank Elisabeth Lobner for assistance with HPLC-SEC-MALS analyses. Therefore, our objective is to determine whether there are differential health consequences with a prescribed fire vs wildfire. We focus on children given their reduced lung size, increased metabolic rates, higher respiratory rate, and developing immune systems, 5 and because in macaque monkeys who are exposed to wildfire smoke in infancy, there is associated immune dysregulation and decreased lung function in adolescence. 6 We hypothesize that the health impacts of a prescribed fire are less detrimental to the respiratory and cardiovascular systems than a wildfire in school-aged children and that T-cell skewing and epigenetic modulation will occur with exposure to wildfire more than from exposure to a prescribed fire.

CO N FLI C T S O F I NTE R E S T
We analyzed data collected from a convenience sample of subjects (n = 220) over a period of 2 years living in Fresno, CA, all of whom were potentially exposed to smoke from fires, which consisted of similar varieties of coniferous trees, in nearby Yosemite National Park. Health questionnaires, blood samples, and vital signs were collected, and subjects were selected that had their blood drawn 3 months after a prescribed fire or wildfire, because our prior research indicates that this time frame is associated with increased methylation of the Foxp3 gene. 7 Using this criteria, we analyzed data from 32 children (median age = 7 [range 7; 8] yrs, 38% asthmatic as per NHLBI guidelines) exposed to a prescribed fire 70 miles away covering 553 acres in March, 2015, and 36 children (median age = 8 [range 7; 8] yrs, 25% asthmatic) exposed to a wildfire 70 miles away covering 415 acres in September 2015. A control group of 18 children was also compared (median age = 8 [range 7; 8] yrs; 21% asthmatic), who had no obvious exposure to wildfires or prescribed fire and were living in the San Francisco Bay area, where pollution levels