Utilizing targeted integration CHO pools to potentially accelerate the GMP manufacturing of monoclonal and bispecific antibodies.

Monoclonal antibodies (mAbs) are effective therapeutic agents against many acute infectious diseases including COVID-19, Ebola, RSV, Clostridium difficile, and Anthrax. mAbs can therefore help combat a future pandemic. Unfortunately, mAb development typically takes years, limiting its potential to save lives during a pandemic. Therefore "pandemic mAb" timelines need to be shortened. One acceleration tool is "deferred cloning" and leverages new Chinese hamster ovary (CHO) technology based on targeted gene integration (TI). CHO pools, instead of CHO clones, can be used for Phase I/II clinical material production. A final CHO clone (producing the mAb with a similar product quality profile and preferably with a higher titer) can then be used for Phase III trials and commercial manufacturing. This substitution reduces timelines by ~3 months. We evaluated our novel CHO TI platform to enable deferred cloning. We created four unique CHO pools expressing three unique mAbs (mAb1, mAb2, and mAb3), and a bispecific mAb (BsAb1). We then performed single-cell cloning for mAb1 and mAb2, identifying three high-expressing clones from each pool. CHO pools and clones were inoculated side-by-side in ambr15 bioreactors. CHO pools yielded mAb titers as high as 10.4 g/L (mAb3) and 7.1 g/L (BsAb1). Subcloning yielded CHO clones expressing higher titers relative to the CHO pools while yielding similar product quality profiles. Finally, we showed that CHO TI pools were stable by performing a 3-month cell aging study. In summary, our CHO TI platform can increase the speed to clinic for a future "pandemic mAb."

Apolipoprotein M supports S1P production and conservation and mediates prolonged Akt activation via S1PR1 and S1PR3.

Sphingosine 1-phosphate (S1P) is one of the lipid mediators involved in diverse physiological functions. S1P circulates in blood and lymph bound to carrier proteins. Three S1P carrier proteins have been reported, albumin, apolipoprotein M (ApoM) and apolipoprotein A4 (ApoA4). The carrier-bound S1P exerts its functions via specific S1P receptors (S1PR1-5) on target cells. Previous studies showed several differences in physiological functions between albumin-bound S1P and ApoM-bound S1P. However, molecular mechanisms underlying the carrier-dependent differences have not been clarified. In addition, ApoA4 is a recently identified S1P carrier protein, and its functional differences from albumin and ApoM have not been addressed. Here, we compared the three carrier proteins in the processes of S1P degradation, release from S1P-producing cells and receptor activation. ApoM retained S1P more stable than albumin and ApoA4 in the cell culture medium when compared in the equimolar amounts. ApoM facilitated theS1P release from endothelial cells most efficiently. Furthermore, ApoM-bound S1P showed a tendency to induce prolonged activation of Akt via S1PR1 and S1PR3. These results suggest that the carrier-dependent functional differences of S1P are partly ascribed to the differences in the S1P stability, S1P-releasing efficiency and signaling duration.

In vitro genotoxicity assessment and 28-day repeated dose oral toxicity study of steady-calcium formula in rats.

Steady-calcium formula (SCF), a functional food mixture with potential of joint care, contains five major ingredients. However, the uncertain cross-reactivity among these included ingredients cannot be excluded. Hence, it is important to ensure the safety of this mixture. In this study, the safety of SCF was evaluated through in vitro genotoxicity assessment and 28-day oral toxicity study in rats. The bacterial reverse mutation test and mammalian chromosome aberration test displayed that SCF did not induce mutagenicity and clastogenicity. The 28-day repeated dose assessment of SCF in rats revealed no mortality and adverse effects in clinical signs, body weight, urinalysis, hematology, organ weight, and histopathology at all treated groups. Although some significant changes were observed in food intake and parameters of serum biochemistry at the highest dose in males, they were not dose-related and considered to be within normal range. These findings indicate that SCF does not possess genotoxic potential and no obvious evidence of subacute toxicity. These results demonstrate for the first time that the genotoxicity and subacute toxicity for SCF are negative under our experimental conditions and the no observed adverse effect level (NOAEL) of SCF may be defined as at least 5470 mg/kg/day.

Single domain antibodies derived from ancient animals as broadly neutralizing agents for SARS-CoV-2 and other coronaviruses.

With severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as an emergent human virus since December 2019, the world population is susceptible to coronavirus disease 2019 (COVID-19). SARS-CoV-2 has higher transmissibility than the previous coronaviruses, associated by the ribonucleic acid (RNA) virus nature with high mutation rate, caused SARS-CoV-2 variants to arise while circulating worldwide. Neutralizing antibodies are identified as immediate and direct-acting therapeutic against COVID-19. Single-domain antibodies (sdAbs), as small biomolecules with non-complex structure and intrinsic stability, can acquire antigen-binding capabilities comparable to conventional antibodies, which serve as an attractive neutralizing solution. SARS-CoV-2 spike protein attaches to human angiotensin-converting enzyme 2 (ACE2) receptor on lung epithelial cells to initiate viral infection, serves as potential therapeutic target. sdAbs have shown broad neutralization towards SARS-CoV-2 with various mutations, effectively stop and prevent infection while efficiently block mutational escape. In addition, sdAbs can be developed into multivalent antibodies or inhaled biotherapeutics against COVID-19.

Tandem Repeat Diversity in Two Closely Related Hamster Species-The Chinese Hamster (Cricetulus griseus) and Striped Hamster (Cricetulus barabensis).

The Chinese hamster (Cricetulus griseus) and striped hamster (Cricetulus barabensis) are very closely related species with similar karyotypes. The karyotypes differ from each other by one Robertsonian rearrangement and X-chromosome morphology. The level of the tandem repeat (TR) sequences' evolutional variability is high. The aim of the current work was to trace the TR distribution on the chromosomes of two very closely related species. The striped hamster genome has not yet been sequenced. We classified the Chinese hamster TR in the assemblies available and then compared the mode of the TR distribution in closely related species. Chinese and striped hamsters are separate species due to the relative species specificity of Chinese hamster TR and prominent differences in the TR distribution in both species. The TR variation observed within homologous striped hamster chromosomes is caused by a lack of inbreeding in natural populations. The set of TR tested could be used to examine the CHO lines' instability that has been observed in heterochromatic regions.

Toxicological evaluation of the red mold rice extract, ANKASCIN 568-R: 13-week chronic toxicity, and genotoxicity studies.

ANKASCIN 568-R is an extract derived from red mold rice (RMR) fermented using Monascus purpureus NTU 568. RMR fermented using M. purpureus NTU 568 prevents cardiovascular diseases and decreases blood lipid levels. This study evaluates the safety of ANKASCIN 568-R, since it has not determined yet. After daily oral ANKASCIN 568-R for 13 consecutive weeks, we evaluated the toxicity tolerance of Sprague-Dawley rats and performed dose formulation analysis on monascin and ankaflavin. The dose formulation analysis showed that ANKASCIN 568-R concentrations were lower than the target concentration and out of range ( ± 15%) at week 8 and on the last dosing day for both monascin (all dose groups) and ankaflavin at the 100 mg/kg dose. The lowest reported concentrations for the low, middle, and high dose formulations were 34.7, 115.2, and 398.1 mg/mL, respectively. We also evaluated the genotoxicity of ANKASCIN 568-R and showed no genotoxicity potential at all ANKASCIN 568-R doses investigated. The no observed adverse effect level of ANKASCIN 568-R was determined to be 796.2 mg/kg/day. This study revealed the first toxicity evaluation data of ANKASICN 568-R, and the data demonstrated ANKASICN 568-R was safe and can be used in daily life.

Sjögren-Larsson syndrome: A biochemical rationale for using aldehyde-reactive therapeutic agents.

Sjögren-Larsson syndrome (SLS) is a neurocutaneous disease caused by mutations in ALDH3A2 that result in deficient fatty aldehyde dehydrogenase (FALDH) activity and impaired fatty aldehyde and fatty alcohol oxidation. The pathogenesis of SLS is thought to involve accumulation of long-chain fatty aldehydes and alcohols and/or metabolically-related ether glycerolipids. Fatty aldehydes are particularly toxic molecules that can covalently react with proteins and certain amino-containing lipids such as phosphatidylethanolamine (PE), generating an unusual aldehyde adduct, N-alkyl-PE (NAPE). Using Faldh-deficient Chinese hamster ovary cells (FAA-K1A) as a cellular model for SLS, we investigated the ability of an aldehyde trapping agent, ADX-102 [2-(3-amino-6-chloro-quinolin-2-yl)-propan-2-ol], to mitigate the harmful effects of fatty aldehydes. FAA-K1A cells were protected from octadecanal (C18:0-al) induced cytotoxicity and apoptosis by ADX-102. Metabolism of C18:0-al to fatty alcohol (octadecanol) was also inhibited by ADX-102. FAA-K1A cells accumulated 5-fold more NAPE with C16- and C18-linked N-alkyl chains compared to wild-type cells, but NAPE levels decreased to normal after growth for 4 days with 50 µM ADX-102. Our results suggest that small aldehyde-reactive molecules, such as ADX-102, should be explored as novel therapeutic agents for SLS by preventing aldehyde adduct formation with critical cellular targets and inhibiting fatty aldehyde metabolism to fatty alcohol.

Effects of pre-eclampsia on HDL-mediated cholesterol efflux capacity after pregnancy.

Background and aims: Preeclampsia (PE) is associated with life-long increased risk of cardiovascular disease. One of the main protective functions of high-density lipoprotein (HDL) is its role in reverse cholesterol transport. HDL-mediated cholesterol efflux capacity (CEC) is decreased during pregnancy in women with PE. Whether this persists postpartum is unknown. Methods: Basal and transporter-specific CEC were determined 6 months postpartum in women who had a normotensive (n = 44) or a PE (n = 42) pregnancy. CEC was also measured in 23 normotensive and 20 PE women for whom samples were collected 24 months postpartum. Basal, ATP-binding cassette transporter-A1 (ABCA1)- and -G1 (ABCG1)-specific CEC were primarily determined using Chinese hamster ovary cells stably expressing human ABCA1 or ABCG1, and were also assessed using a J774 mouse macrophage cell line. Results: ABCA1-specific CEC was significantly lower in women who had PE 6 months postpartum (0.57 ± 0.1 vs 0.53 ± 0.08; p < 0.05), whilst basal and ABCG1-specific efflux were not significantly different. cAMP-specific CEC in J774 cells was also lower 6 months after PE (0.85 ± 0.21 vs 0.75 ± 0.25, p < 0.05). Although apoA-I, apoE, plasminogen and PON-1 levels were not significantly different in women who had PE compared with controls, ABCA1 efflux did correlate with apoA-l, HDL-C and apoE levels after a normal, and with apoA-l and HDL-C levels after a PE pregnancy. ABCA1-specific efflux decreased in all women between 6 and 24 months postpartum, by 11 ± 1.6% in women who had a normotensive pregnancy and 9 ± 1.3% in women who had PE. After adjustment for apoA-I levels, there was no significant difference in ABCA1-specific efflux between the groups at 6 months postpartum and in normotensive women over time, but remained significantly different between 6 and 24 months in women who had PE. Conclusions: ABCA1-mediated CEC is impaired 6 months postpartum after a PE pregnancy and decreases thereafter in both normotensive and PE pregnancies. ABCA1-mediated efflux is dynamic after pregnancy but is unlikely to explain the long-term increased CVD risk in women with PE.

A pooled CRISPR/AsCpf1 screen using paired gRNAs to induce genomic deletions in Chinese hamster ovary cells.

Chinese hamster ovary (CHO) cells are the most widely used host for the expression of therapeutic proteins. Recently, significant progress has been made due to advances in genome sequence and annotation quality to unravel the black box CHO. Nevertheless, in many cases the link between genotype and phenotype in the context of suspension cultivated production cell lines is still not fully understood. While frameshift approaches targeting coding genes are frequently used, the non-coding regions of the genome have received less attention with respect to such functional annotation. Importantly, for non-coding regions frameshift knock-out strategies are not feasible. In this study, we developed a CRISPR-mediated screening approach that performs full deletions of genomic regions to enable the functional study of both the translated and untranslated genome. An in silico pipeline for the computational high-throughput design of paired guide RNAs (pgRNAs) directing CRISPR/AsCpf1 was established and used to generate a library tackling process-related genes and long non-coding RNAs. Next generation sequencing analysis of the plasmid library revealed a sufficient, but highly variable pgRNA composition. Recombinase-mediated cassette exchange was applied for pgRNA library integration rather than viral transduction to ensure single copy representation of pgRNAs per cell. After transient AsCpf1 expression, cells were cultivated over two sequential batches to identify pgRNAs which massively affected growth and survival. By comparing pgRNA abundance, depleted candidates were identified and individually validated to verify their effect.

Establishment of a novel anti-TROP2 monoclonal antibody TrMab-29 for immunohistochemical analysis.

TROP2 is a type I transmembrane glycoprotein originally identified in human trophoblast cells that is overexpressed in several types of cancer. To better understand the role of TROP2 in cancer, we herein aimed to develop a sensitive and specific anti-TROP2 monoclonal antibody (mAb) for use in flow cytometry, Western blot, and immunohistochemistry using a Cell-Based Immunization and Screening (CBIS) method. Two mice were immunized with N-terminal PA-tagged and C-terminal RAP/MAP-tagged TROP2-overexpressed Chinese hamster ovary (CHO)-K1 cells (CHO/PA-TROP2-RAP-MAP), and hybridomas showing strong signals from PA-tagged TROP2-overexpressed CHO-K1 cells (CHO/TROP2-PA) and weak-to-no signals from CHO-K1 cells were selected using flow cytometry. We demonstrated using flow cytometry that the established anti-TROP2 mAb, TrMab-29 (mouse IgG1 kappa), detected TROP2 in MCF7 breast cancer cell line as well as CHO/TROP2-PA cells. Western blot analysis showed a 40 kDa band in lysates prepared from both CHO/TROP2-PA and MCF7 cells. Furthermore, TROP2 was strongly detected by immunohistochemical analysis using TrMab-29, indicating that TrMab-29 may be a valuable tool for the detection of TROP2 in cancer.

Improving the reaction mix of a Pichia pastoris cell-free system using a design of experiments approach to minimise experimental effort.

A renaissance in cell-free protein synthesis (CFPS) is underway, enabled by the acceleration and adoption of synthetic biology methods. CFPS has emerged as a powerful platform technology for synthetic gene network design, biosensing and on-demand biomanufacturing. Whilst primarily of bacterial origin, cell-free extracts derived from a variety of host organisms have been explored, aiming to capitalise on cellular diversity and the advantageous properties associated with those organisms. However, cell-free extracts produced from eukaryotes are often overlooked due to their relatively low yields, despite the potential for improved protein folding and posttranslational modifications. Here we describe further development of a Pichia pastoris cell-free platform, a widely used expression host in both academia and the biopharmaceutical industry. Using a minimised Design of Experiments (DOE) approach, we were able to increase the productivity of the system by improving the composition of the complex reaction mixture. This was achieved in a minimal number of experimental runs, within the constraints of the design and without the need for liquid-handling robots. In doing so, we were able to estimate the main effects impacting productivity in the system and increased the protein synthesis of firefly luciferase and the biopharmaceutical HSA by 4.8-fold and 3.5-fold, respectively. This study highlights the P. pastoris-based cell-free system as a highly productive eukaryotic platform and displays the value of minimised DOE designs.

Directed evolution approach to enhance efficiency and speed of outgrowth during single cell subcloning of Chinese Hamster Ovary cells.

Chinese Hamster Ovary (CHO) cells are the working horse of the pharmaceutical industry. To obtain high producing cell clones and to satisfy regulatory requirements single cell cloning is a necessary step in cell line development. However, it is also a tedious, labor intensive and expensive process. Here we show an easy way to enhance subclonability using subcloning by single cell sorting itself as the selection pressure, resulting in improved subcloning performance of three different host cell lines. These improvements in subclonability also lead to an enhanced cellular growth behavior during standard batch culture. RNA-seq was performed to shed light on the underlying mechanisms, showing that there is little overlap in differentially expressed genes or associated pathways between the cell lines, each finding their individual strategy for optimization. However, in all three cell lines pathways associated with the extracellular matrix were found to be enriched, indicating that cells struggle predominantly with their microenvironment and possibly lack of cell-to-cell contact. The observed small overlap may hint that there are multiple ways for a cell line to achieve a certain phenotype due to numerous genetic and subsequently metabolic redundancies.

Activation of Oxytocin Neurons Improves Cardiac Function in a Pressure-Overload Model of Heart Failure.

This work shows long-term restoration of the hypothalamic oxytocin (OXT) network preserves OXT release, reduces mortality, cardiac inflammation, fibrosis, and improves autonomic tone and cardiac function in a model of heart failure. Intranasal administration of OXT in patients mimics the short-term changes seen in animals by increasing parasympathetic-and decreasing sympathetic-cardiac activity. This work provides the essential translational foundation to determine if approaches that mimic paraventricular nucleus (PVN) OXT neuron activation, such as safe, noninvasive, and well-tolerated intranasal administration of OXT, can be beneficial in patients with heart failure.

Application of a genome-scale model in tandem with enzyme assays for identification of metabolic signatures of high and low CHO cell producers.

Biopharmaceutical industrial processes are based on high yielding stable recombinant Chinese Hamster Ovary (CHO) cells that express monoclonal antibodies. However, the process and feeding regimes need to be adapted for each new cell line, as they all have a slightly different metabolism and product performance. A main limitation for accelerating process development is that the metabolic pathways underlying this physiological variability are not yet fully understood. This study describes the evolution of intracellular fluxes during the process for 4 industrial cell lines, 2 high producers and 2 low producers (n = 3), all of them producing a different antibody. In order to understand from a metabolic point of view the phenotypic differences observed, and to find potential targets for improving specific productivity of low producers, the analysis was supported by a tailored genome-scale model and was validated with enzymatic assays performed at different days of the process. A total of 59 reactions were examined from different key pathways, namely glycolysis, pentose phosphate pathway, TCA cycle, lipid metabolism, and oxidative phosphorylation. The intracellular fluxes did not show a metabolic correlation between high producers, but the degree of similitude observed between cell lines could be confirmed with additional experimental observations. The whole analysis led to a better understanding of the metabolic requirements for all the cell lines, allowed to the identification of metabolic bottlenecks and suggested targets for further cell line engineering. This study is a successful application of a curated genome-scale model to multiple industrial cell lines, which makes the metabolic model suitable for process platform.

Establishment of a monoclonal antibody PMab-225 against alpaca podoplanin for immunohistochemical analyses.

Podoplanin (PDPN) is known as a lymphatic endothelial cell marker. Monoclonal antibodies (mAbs) against human, mouse, rat, rabbit, dog, cat, bovine, pig, and horse PDPN have been established in our previous studies. However, mAbs against alpaca PDPN (aPDPN), required for immunohistochemical analysis, remain to be developed. In the present study, we employed the Cell-Based Immunization and Screening (CBIS) method for producing anti-aPDPN mAbs. We immunized mice with aPDPN-overexpressing Chinese hamster ovary (CHO)-K1 cells (CHO/aPDPN), and hybridomas producing mAbs against aPDPN were screened using flow cytometry. One of the mAbs, PMab-225 (IgG2b, kappa), specifically detected CHO/aPDPN cells via flow cytometry and recognized the aPDPN protein on Western blotting. Further, PMab-225 strongly stained lung type I alveolar cells, colon lymphatic endothelial cells, and kidney podocytes via immunohistochemistry. These findings demonstrate that PMab-225 antibody is useful to investigate the function of aPDPN via different techniques.

PMab-219: A monoclonal antibody for the immunohistochemical analysis of horse podoplanin.

Monoclonal antibodies (mAbs) against human, mouse, rat, rabbit, dog, cat, and bovine podoplanin (PDPN), a lymphatic endothelial cell marker, have been established in our previous studies. However, mAbs against horse PDPN (horPDPN), which are useful for immunohistochemical analysis, remain to be developed. In the present study, mice were immunized with horPDPN-overexpressing Chinese hamster ovary (CHO)-K1 cells (CHO/horPDPN), and hybridomas producing mAbs against horPDPN were screened using flow cytometry. One of the mAbs, PMab-219 (IgG2a, kappa), specifically detected CHO/horPDPN cells via flow cytometry and recognized horPDPN protein using Western blotting. Furthermore, PMab-219 strongly stained CHO/horPDPN via immunohistochemistry. These findings suggest that PMab-219 is useful for investigating the function of horPDPN.

The mouse-canine chimeric anti-dog podoplanin antibody P38B exerts antitumor activity in mouse xenograft models.

Podoplanin (PDPN) is a type I transmembrane heavily glycosylated sialoglycoprotein that is expressed in normal tissues such as pulmonary type I alveolar cells, renal podocytes, and lymphatic endothelial cells. PDPN overexpression in cancerous tissue is associated with hematogenous metastasis through interactions with the C-type lectin-like receptor 2 (CLEC-2). Previously, we have reported the development of a mouse monoclonal antibody (mAb), PMab-38 (IgG1, kappa) against dog PDPN (dPDPN). PMab-38 was found to strongly react with canine squamous cell carcinomas (SCCs) and melanomas; however, it showed no reaction with lymphatic endothelial cells. Recently, we have developed and produced the mouse-canine mAb of subclass B, P38B that showed antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity against Chinese hamster ovary (CHO)/dPDPN cells. In the present study, we investigated the antitumor activity using mouse xenograft model. To induce ADCC activity by P38B, canine mononuclear cells were injected surrounding the tumors in a xenograft model. It was demonstrated that P38B exerted antitumor activity against the mouse xenograft model using CHO/dPDPN. These results suggest that P38B is useful for antibody therapy against dPDPN-expressing canine SCCs and melanomas.

Structural and Epitope Analysis (T- and B-Cell Epitopes) of Hepatitis C Virus (HCV) Glycoproteins: An in silico Approach.

BACKGROUND: Chronic infection with Hepatitis C Virus (HCV) poses a major risk for liver disease like cirrhosis, liver failure and hepatocellular carcinoma. In terms of percentage, the prevalence of HCV in India was found to be low to moderate (1-1.5%), but in terms of sheer numbers, India has a significant number of global HCV patients. Presently, HCV can be treated with direct acting-antibody drugs but there is no prophylactic or therapeutic vaccine available against it. In HCV infection, T- and B-cell immunity is important for clearing the virus. In the present study immunoinformatics was used to identify potent vaccine target for HCV vaccine development. METHODS: Sequence of HCV was retrieved from NCBI and their structural analysis was done by using Protpram, PSIPRED, iTASSER and PDBsum servers. T-cell and B-cell epitopes were predicted by Immune Epitope Database and ACBPRED servers. RESULTS: On epitope prediction, 25 and 55 potent MHC-I epitopes and 7 and 13 potent B-cell epitopes were predicted for E1 and E2 protein respectively. Their antigenicity score was also calculated. The most potent MHC-I epitopes were MMMNWSPAV and MAWDMMMNW for HLA-A*02:01 and HLA-B*53:01 and most potent B-cell epitope was TGHRMAWDMMMNWSPA for E1 protein. For E2, four MHC-I epitopes having the lowest binding energy and most potent B-cell epitope was DRPYCWHYAPRPCDTI. CONCLUSION: In the present study, most potent epitopes for HCV was determined on the basis of their antigenicity along with 3D modeling and docking. Identified B- and T-cell epitopes can be used for the development of potent vaccine against most prevalent HCV type in India to limit its infection.

Domain unfolding of monoclonal antibody fragments revealed by non-reducing SDS-PAGE.

Monoclonal antibodies and derived fragments are used extensively both experimentally and therapeutically. Thorough characterization of such antibodies is necessary and includes assessment of their thermal and storage stabilities. Thus, assessment of the underlying conformational stabilities of the antibodies is also important. We recently documented that non-reducing SDS-PAGE can be used to assess both monoclonal and polyclonal IgG domain thermal unfolding in SDS. Utilizing this same h2E2 anti-cocaine mAb, in this study we generated and analyzed various mAb antibody fragments to delineate the structural domains of the antibody responsible for the observed discrete bands following various heating protocols and analysis by non-reducing SDS-PAGE. Previously, these domain unfolding transitions and gel bands were hypothesized to stem from known mAb structural domains based on the relative thermal stability of those CH2, CH3, and Fab domains in the absence of SDS, as measured by differential scanning calorimetry. In this study, we generated and analyzed F(ab')2, Fab, and Fc fragments, as well as a mAb consisting of only heavy chains, and examined the thermally induced domain unfolding in each of these fragments by non-reducing SDS-PAGE. The results were interpreted and integrated to generate an improved model of thermal unfolding for the mAb IgG in SDS. These results and the model presented should be generally applicable to many monoclonal and polyclonal antibodies and allow novel comparisons of conformational stabilities between chemically or genetically modified versions of a given antibody. Such modified antibodies and antibody drug conjugates are commonly utilized and important for experimental and therapeutic applications.

A CRISPR/Cas9 based engineering strategy for overexpression of multiple genes in Chinese hamster ovary cells.

Manipulation of multiple genes to engineer Chinese Hamster Ovary (CHO) cells for better performance in production processes of biopharmaceuticals has recently become more and more popular. Yet, identification of useful genes and the unequivocally assessment of their effect alone and in combination(s) on the cellular phenotype is difficult due to high variation between subclones. Here, we present development and proof-of-concept of a novel engineering strategy using multiplexable activation of artificially repressed genes (MAARGE). This strategy will allow faster screening of overexpression of multiple genes in all possible combinations. MAARGE, in its here presented installment, comprises four different genes of interest that can all be stably integrated into the genome from one plasmid in a single transfection. Three of the genes are initially repressed by a repressor element (RE) that is integrated between promoter and translation start site. We show that an elongated 5'-UTR with an additional transcription termination (poly(A)) signal most efficiently represses protein expression. Distinct guide RNA (gRNA) targets flanking the REs for each gene then allow to specifically delete the RE by CRISPR/Cas9 and thus to activate the expression of the corresponding gene(s). We show that both individual and multiplexed activation of the genes of interest in a stably transfected CHO cell line is possible. Also, upon transfection of this stable cell line with all three gRNAs together, it was possible to isolate cells that express all potential gene combinations in a single experiment.