s2fieldsofstudy: Biology Medicine

text:
Individualized Dosing of Therapeutic Monoclonal Antibodies—a Changing Treatment Paradigm?

The introduction of monoclonal antibodies (mAbs) to the treatment of inflammatory bowel disease (IBD) was an important medical milestone. MAbs have been demonstrated as safe and efficacious treatments of IBD. However, a large percentage of patients either fail to respond initially or lose response to therapy after a period of treatment. Although there are factors associated with poor treatment outcomes in IBD, one cause for treatment failure may be low mAb exposure. Consequently, gastroenterologists have begun using therapeutic drug monitoring (TDM) to guide dose adjustment. However, while beneficial, TDM does not provide sufficient information to effectively adjust doses. The pharmacokinetics (PK) and pharmacodynamics (PD) of mAbs are complex, with numerous factors impacting on mAb PK and PD. The concept of dashboard-guided dosing based on Bayesian PK models allows physicians to combine TDM with factors influencing mAb PK to individualize therapy more effectively. One issue with TDM has been the slow turnaround of assay results, either necessitating an additional clinic visit for a sample or reacting to TDM results at a subsequent, rather than the current, dose. New point-of-care (POC) assays for mAbs are being developed that would potentially allow physicians to determine drug concentration quickly. However, work remains to understand how to determine what target exposure is needed for an individual patient, and whether the combination of POC assays and dashboards presents a safe approach with substantial outcome benefit over the current standard of care.

INTRODUCTION TO INFLAMMATORY BOWEL DISEASE
Inflammatory bowel disease (IBD) includes two types of auto-immune disorders that cause prolonged inflammation of the digestive tract: ulcerative colitis (UC) and Crohn's disease (CD). One difference between these two types of IBD is the location of inflammation. In CD, inflammation can occur throughout the entire gastrointestinal tract, from mouth to anus. In UC, the colon and rectum are primarily affected. Although CD and UC are different diseases, differentiation based on the clinical picture can be difficult. Both UC and CD may present with abdominal pain, diarrhea, rectal blood loss, weight loss, vomiting, or signs of anemia. Diagnosis is usually made based on endoscopic evaluation and biopsies taken for histopathological assessment. Not only are the locations of these two diseases different, associated complications also differ. CD can be characterized by late complications such as a stenosing and/or penetrating phenotype which can result in fibrostenotic strictures, (perianal) fistula, or abscesses; UC does not have late complications. Although the exact etiology is unknown, IBD is likely caused by a combination of genetic and environmental factors leading to an immunological response and intestinal inflammation (1,2). Historically, the goal of IBD treatment was to relieve symptoms; it has now shifted to the achievement of mucosal healing.

INFLAMMATORY BOWEL DISEASE TREATMENT OPTIONS
In 1998, approval of the first monoclonal antibody (mAb), infliximab (IFX), for CD, provided an exciting new treatment option. Since then, multiple mAbs with diverse mechanisms of action have been marketed for IBD. Currently available drugs for treatment of IBD include chemical agents such as steroids, immunomodulators, Janus kinase (JAK) inhibitors, and mAbs such as the anti-integrins, anti-interleukin-12/23p40, and anti TNF agents (3,4). Clinical development programs of IBD treatments generally follow a stepwise approach, beginning with initial evaluations of safety and tolerability, followed by evaluations of dose-/exposure-response (E-R) in small clinical studies, then phase 3 clinical trials that provide evidence of clinical efficacy and safety in the target patient population. Currently, most evidence about clinical and endoscopic outcomes is derived from clinical trials with mAbs.

Current Recommended Dose Regimen for Inflammatory Bowel Diseases
The treatment paradigm for CD and UC involves two regimens: (1) initial treatment period (induction) and (2) second regimen (maintenance), which are supported by evaluations during clinical development programs. Maintenance regimens are typically less intensive either in the dose or frequency than for induction (Table I). Knowledge about mAb drugs for IBD continues to grow beyond data that supported regulatory approval, including collective experience from clinical use and post-marketing clinical trials. The learning continuum has influenced the philosophy and practice in IBD treatment, including active discussions of individualized dosing for biological drugs.

MAb Pharmacokinetic Variation in IBD
MAbs generally exhibit complex pharmacokinetic (PK) behavior, whereby disposition involves neonatal-Fc receptor (FcRn)-antibody binding, which can be influenced by FcRn genetic polymorphism. Recently, the VNTR2/3 genotype in the FcRn gene was shown to be associated with lower mAb exposure during induction in IBD patients (5,6). Intracellular catabolism, extracellular degradation (proteolysis), receptor-mediated and fluid-phase endocytosis of the mAb, target-mediated drug disposition, and in vivo post translational modifications (PTM), such as deamidation (7-10) and nonenzymatic glycation (11), contribute to variability. High glycation is associated with diabetes, which was shown to result in a 28.7% increase in ustekinumab clearance (12); hyperglycemia was shown to increase IFX clearance up to 15% (13). Whether and how PTM differ across patients remain poorly understood; however, it may result in loss of mAb activity prior to clearance (14,15). In addition, mAbs with multimeric soluble targets can form immune complexes and be rapidly cleared via phagocytosis (7). Numerous other factors contribute to between patient PK variability. These factors include patient demographics such as body size, serum albumin concentration, C-reactive protein (CRP), formation of anti-drug antibodies (ADAs), endogenous IgG concentration, expression/turnover of antibody target, mAb saturation of target, and drug-drug interactions. Many mAbs and biologics including certolizumab pegol (16)(17)(18), which is indicated only for CD, and golimumab (19,20), which is indicated only for UC, reported the same factors as influencing PK and showed that observed variability can negatively impact patient outcomes.
Pediatric patients present unique issues, given both growth and maturation effects (21) and the fact that diseases can present in a more aggressive form in pediatric patients than adults (22) suggesting potential etiologic differences from adult disease (23). Other disease-specific routes of clearance such as protein-losing enteropathy (24)(25)(26)(27) can play an important role, as discussed later. Taken together, these factors contribute to high between-patient variability in mAb PK in IBD patients.
Two mAbs often used for treatment of IBD are IFX and adalimumab (ADL), administered intravenously and subcutaneously, respectively. Both anti-TNF agents have proven to be successful in inducing and maintaining clinical and endoscopic remission (28)(29)(30)(31)(32). However, a proportion of IBD patients do not respond to anti-TNF induction treatment (primary non-responders) or lose response during maintenance treatment (secondary non-responders) (33).
Individualized dosing for mAb drugs began to acquire clinical attention many years after biological drugs had made a substantial, positive impact on patients with CD, UC, and other disease conditions. Impetus to investigate dose individualization of mAb drugs stems from findings of IBD trials and clinical practice. Some important common findings are as follows:

1.
Only a portion of patients achieve the predefined treatment response at the end of induction and continue into maintenance treatment.

2.
Only a fraction of patients who receive maintenance treatment achieve treatment response.

3.
Patients can lose treatment response after being in stable response for a period of time.
Incomplete response to induction treatment may be attributable to inadequate exposure or patient factors that preclude treatment response. Similarly, loss of response (LOR) to maintenance treatment may be due to inadequate exposure in general or secondary treatment failure due to immunogenicity against the mAb drug or non-immune-mediated processes.
Factors associated with primary non-response to anti-TNF agents are smoking, disease duration of more than two years, small bowel disease, and underdosing during the induction phase, presumably due to fecal loss. Brandse and colleagues demonstrated that IFX could be measured in feces of patients with acute severe colitis starting with IFX induction treatment (34). The highest fecal IFX concentrations were measured during the first few days after starting treatment. A possible explanation for this phenomenon is protein-losing enteropathy due to severe intestinal inflammation leading to a "leaky gut." If IFX is lost via the feces, IFX exposure following regular induction of 5 mg/kg at week 0, 2, and 6 may be insufficient, necessitating higher anti-TNF doses or shorter dosing intervals. During IFX maintenance (5 mg/kg every eight weeks), serum drug levels between 3 and 7 μg/ml are considered therapeutic (35). However, during the IFX induction phase, higher serum drug levels are needed (e.g., ≥ 28 μg/ml during the first two weeks and ≥ 15 μg/ml in week 2 to 6) (36,37). In some patients, for example, patients with an increased clearance of the drug, higher IFX doses or shorter intervals may be needed to achieve these levels (38,39).

Development of Anti-drug Antibodies
Development of immunogenicity against the mAb is a key concern because of the potential association with reduced systemic drug exposure and treatment effectiveness. ADA formation, i.e., immunogenicity, can lead to secondary LOR in IBD patients (40)(41)(42).
While clinical concerns related to immunogenicity may be apparent today, it was not in the prescribing information associated with the initial approval of IFX in 1998 (43). The first mention of an association of immunogenicity with a reduced efficacy and increased IFX clearance appeared in the prescribing information 6 years later (44). To prevent LOR during maintenance treatment with mAb drugs, clinical research has led to new institutional recommendations for dosing in IBD treatment. These efforts are well-documented in publications (45)(46)(47), and encompass approved mAb drugs with diverse mechanisms of action.
ADA can form complexes with the mAb, either neutralizating biologic effect and/or accelerating clearance. Since immunogenicity is often accompanied by subtherapeutic serum drug levels, maintaining adequate mAb concentrations is crucial in the treatment with anti-TNF agents. Current strategies to reduce the immunogenicity include scheduled instead of episodic treatment with anti-TNF agents and combination treatment with an immunomodulator, such as methotrexate or a thiopurine. However, if ADA does occur in patients treated with anti-TNF monotherapy, addition of immunomodulators during treatment can be used to suppress ADA. Research in IBD patients showed that addition of immunomodulators to anti-TNF monotherapy in patients with LOR due to immunogenicity led to increased serum drug levels, accompanied by ADA decrease to undetectable levels (48)(49)(50). The exact mechanism behind this observation is unknown, but research in rheumatoid arthritis patients treated with methotrexate demonstrated downregulation of activating Fc-gamma receptors, which could be a direct effect of methotrexate on monocytes potentially associated with the disease-modifying effect of methotrexate (51).

Maintaining Effective mAb Exposure-Introduction of Therapeutic Drug Monitoring
Potential subtherapeutic exposure is another key area of clinical investigations. Most IBD trials evaluate efficacy and safety at one or two dose regimens, which produce a range of systemic exposure as measured by the trough concentrations. In some trials, dose escalation based on clinical response may be an option; however, trial design often hampers robust evaluation of the benefit of dose escalation. Consequently, clinicians continue to investigate various dosing paradigms to benefit as many patients as possible with existing medicines, a quest universally applicable to chemical drugs and biological drugs. Literature reports have revealed potential reasons for IBD treatment failure as shown in Table II grouped by two factors, systemic drug concentration and immunogenicity findings. These findings form the basis to proposing alternative strategies for clinical management.
Research has indicated that dosing according to a "one size fits all" principle is no longer optimal in IBD treatment. The observation that not all patients respond the same way to a certain dosing regimen emphasizes the need for adequate, more personalized dosing using therapeutic drug monitoring (TDM). There is a shift toward individualized dosing in which TDM plays an important role and is increasingly utilized by gastroenterologists.
TDM is defined as measuring patient responses (e.g., drug concentrations, ADA, biomarkers, or clinical outcomes) and adjusting the therapy (e.g., the dose amount or frequency) accordingly, where ADA is relevant to mAbs for IBD treatment. This is particularly true for treatment with mAbs and thiopurines, with the former agents exhibiting highly variable clearance and the latter agents having a narrow therapeutic range. Patient factors influencing the PK of a drug should also be taken into account. For example, multiple analyses of IFX population PK data derived from IBD patients identified multiple factors influencing the clearance of IFX; the presence of ADA, low serum albumin, high serum CRP, and high body weight are the most important factors associated with increasing clearance of anti-TNF agents (38,39), and these same factors also influence clearance of other mAbs.
By incorporating these factors in a PK model and using Bayesian approaches, predictions can be made to identify dose intervals to maintain a certain serum concentration. Since most gastroenterologists are not used to working with model-based dosing, a tool for physicians to apply TDM in the most optimal way is preferred. A dashboard system is an example of such tool, increasingly used by physicians to individualize dosing of their patients (52,53). With a PK model, incorporated patient factors, and a Bayesian approach, the dashboard system utilizes the concentration data from the previous dose and calculates the exact dosage a patient should receive and the exact date this dosage should be given to maintain a certain drug concentration. To make this prediction as accurate as possible, frequent blood sampling throughout treatment is desirable, although not necessary (54,55). However, care should be taken to ensure that the underlying structural model in the dashboard is appropriate. Dubinsky et al. compared dashboard-driven dosing with actual administered dosing regimens in the standard-of-care setting (56). Serum drug level measurements were derived from pediatric IBD patients using IFX at week 14 and 54 after start of therapy. Dashboard-guided dosing after week 14 was compared to the actual administered IFX dose and frequency given with standard care. In the majority of the cases, the dashboard recommended dose and/or interval changes instead of the regular dosing regimen. To investigate the clinical benefit of dashboard-driven dosing in IBD patients treated with mAbs, a prospective trial comparing the two dosing regimens should be performed.

Important Drivers for Individualized Dosing
The success of individualized dosing depends on many important factors, e.g., a predefined clinical outcome, the desired systemic exposure (range) to achieve the predefined clinical outcome, patient factors driving PK exposure, patient factors driving the treatment response (including characteristics of patients who will not respond to treatment), immunogenicity, and enabling technical tools. The latter covers those for measuring patient factors, biomarkers, clinical outcomes, drug concentrations, and ADA, and for identifying patient factors through PK modeling, PD modeling, or integrated PKPD modeling.
Desired treatment outcomes are defined by clinicians and reflected in clinical trials as study endpoints, which may evolve over time with increasing knowledge of the disease and the advancement of technology. For example, clinical response, clinical remission, mucosal healing, and endoscopic remission are some of the endpoints used in IBD trials in addition to exploratory biomarkers (57,58). While an effective treatment would achieve the desired clinical outcomes in all treated patients, in reality, there are subgroups that do not reach the desired effect despite achieving high systemic exposures for reasons that are not well understood (59). Conversely, some patients may achieve the outcomes despite having relatively low systemic exposure (59). Pediatric patients present an acute challenge because of the scarcity of clinical data supporting drug usage and dosing in children, a need recognized by regulatory agencies (60) and owing to the desire to maintain comparable exposures in pediatric patients with adults based on the assumption of similar disease progression and similar exposure-response between adult and pediatric patients.
To move beyond empirical experimentation, proactive identification of patient factors that influence treatment response is very important as illustrated in Table II and is an area for further research. In contrast, patient factors driving PK are much better studied owing to data availability and application of PK modeling tools. The experience with use of dashboard systems (56) for IFX dose optimization shows the potential utility of patient intrinsic factors in PK-driven individualized dosing in IBD. Acute severe ulcerative colitis patients starting induction treatment with IFX and patients on IFX maintenance treatment losing response due to subtherapeutic drug levels will presumably benefit most from dosing based on serum drug measurements as these patients have very high mAb clearance (61). Notably, some mAb drugs are supplied with a fixed amount in prefilled syringes or autoinjectors for subcutaneous injection intended for self-administration, which may present a challenge to fine-tuning doses for individual patients.
Defining Desired Systemic Exposure (Range)-Although defining the desired systemic exposure is not among the preset objectives of confirmatory efficacy and safety trials, these trials generally report steady-state trough concentrations at the recommended dose in the intended patient population. Additional real-world data from post-marketing experience, such as institutional investigations, can contribute to the body of knowledge to inform the desired systemic exposure. However, uncertainties exist as to the suitability of using the observed steady-state concentrations as the target concentration for individualized dosing given that measures for desired outcome differ across various studies and may differ from that expected in current clinical practice. As shown in the review of IBD literature conducted by Papamichael et al. (58), therapeutic concentrations vary with the desired outcome or the predefined clinical endpoints as well as the timing of clinical outcome assessment with respect to the treatment duration. Work to identify appropriate concentrations for certolizumab pegol (16,17) and golimumab (19,20) has also been published.
Several recent retrospective investigations on proactive dose optimization (52,56) were published. Publications of institutional experience with IBD treatments have identified the respective target drug concentrations for reactive dose optimization. For clinically managing IBD patients each reporting institution naturally would rely on their own scientific findings.
To facilitate a broader application, the American Gastroenterology Association published a paper with recommendations for reactive TDM (62). Although not all studies used the same bioanalytical method to measure drug concentrations, independent reviewers from the society have indicated that results from various assays appear to be generally in good agreement (62,63) although a retrospective study showed that some assays do not have good performance (54).
The importance of identifying appropriate drug exposure is highlighted by natalizumab, a humanized IgG4κ mAb initially approved in 2004 for multiple sclerosis (MS) and approved in 2008 for the treatment of CD. It was subsequently withdrawn from the market by its manufacturer after being linked with three cases of progressive multifocal leukoencephalopathy (PML). PML is an often-fatal opportunistic brain infection caused by the John Cunningham (JC) virus. Patients taking natalizumab are at risk of PML if they are JC virus antibody positive (~ 55% of all patients). It is hypothesized that high concentrations of natalizumab may affect immune cells' ability to traffic to the brain and control the JC virus (64,65). In MS patients, excessive dosing of natalizumab has been linked to increased PML risk (66,67). In a recent study, extended-interval dosing (dosing every 5-12 weeks instead of the standard 4-week dosing) decreased PML risk by 94% (67). Therefore, PML risk can be mitigated by extended-interval dosing (but not changing the administered dose), which effectively decreases the concentration of natalizumab.
Defining the Effect of Immunogenicity-Immunogenicity was shown to negatively affect the systemic exposure and clinical outcomes; however, the role of ADA data in clinical management of IBD patients is not well defined. The proportion of patients who achieved desired clinical outcome is smaller in ADA-positive patients as compared to ADA-negative patients, and an ADA-positive finding has been associated with lower systemic drug concentration (68)(69)(70)(71). However, it has not been feasible to quantify the effect of ADA at the subject level or at the population level, in part because the immunogenicity data have been reported in a dichotomous manner, i.e., ADA positive and ADA negative, as described above. In recent years, ADA data are increasingly reported with titer values representing the intensity of the immunogenicity response. Currently, available data generally suggest that higher ADA titers are associated with worse clinical measures, such as reductions in systemic drug exposures, which generally precede exposure reduction or LOR (72). However, identifying the ADA titer thresholds that are clinically relevant is challenging because the characteristics (e.g., binding affinity, with or without neutralizing capability, proportion of ADA mixture being neutralizing) and the kinetics of ADA (e.g., temporal change in intensity and characteristics) may vary in patients. Furthermore, the diversity of assays used for measuring ADA titer across reported studies (some developed by institutional laboratories, others by regional laboratory service providers) presents challenges to the integration of literature reported data. Although the pursuit of quantifying the effect of ADA on PK has resulted in limited success (73), the availability of ADA titer data has fueled a new wave of investigations. From these studies, we will learn more about the clinical utility of ADA titer data in dose individualization for IBD treatment.
It is worth noting that immune-mediated reduction in systemic exposures is one of several possible mechanisms for LOR (62). Other processes not mediated by immunogenicity, e.g., disease flare-up, also can be associated with a reduction in drug concentrations. Among patients who experienced immune-mediated reduction in systemic exposure, the timing of measurement and the extent of exposure reduction can vary, with some patients showing no measurable concentrations at all. The same variability in drug exposure reduction is seen with the impact of disease flare. This variability can make dose adjustments difficult to standardize in the case of either ADA or disease flares.

TDM Utility
The use of TDM for IBD has several issues, including the influence of disease activity on PK and the wide range of target trough values in the literature (74). TDM utility for mAbs has been questioned, partly because of a lack of powered prospective studies using TDM-based dosing as compared to standard of care (75). A large prospective trial (69) showed TDM-based dosing maintained response although in all patients dosing was optimized using TDM during the initial phase of this study. Even though TDM-based dosing was not superior compared to clinically based dosing, significantly more patients in the clinically based dosing group needed an intervention to treat a relapse. Likewise, more undetectable trough levels and ADA were seen in this group. A small prospective study (TAILORIX) investigated only dose increases in maintenance therapy over 1 year, but not shortened dosing intervals, an important adjustment. The study design was not sufficient to demonstrate an advantage of TDM (76) but suggested no benefit. Both trials only included patients on IFX maintenance and had a follow-up of 1 year, which, given the annual rate of LOR for IFX [10-20% (77)], might be too short to show real benefit.
An important issue that should be addressed is the benefit from proactive TDM compared to reactive TDM. A recent review of TDM (80) suggested that the true value of TDM may be preemptive dose optimization to prevent LOR. A randomized controlled trial reported that preemptive TDM-based dose optimization decreased the risk of LOR compared to dosing of IFX based on symptoms and inflammatory biomarkers (35). Unfortunately, there is insufficient evidence to draw robust conclusions. However, in a retrospective analysis of IBD patients comparing proactive versus reactive monitoring of serum concentration of IFX, proactive monitoring was associated with better clinical outcomes, including greater drug durability, less need for IBD-related surgery or hospitalization, and lower risk of ADA or serious infusion reactions (81).
Most IBD treatment regimens contain a higher-intensity dosing for induction followed by a lower-intensity dosing for maintenance, indicating that achieving adequate serum drug levels during induction is as important as during maintenance. Studies have illustrated that trough concentrations during induction are higher than the trough concentrations during maintenance (79). However, investigations into whether patients who do not respond to conventional induction regimen of mAbs for IBD could benefit from dose individualization have not been conducted. The strategy for dose individualization or preemptive TDM during induction warrants further research. The anticipated challenges would include defining the biomarkers to use and their baseline values for guiding the induction dose regimen.
Another area of interest is the economic impact of TDM. The study of Steenholdt et al.
showed substantially lower (34%) costs for those randomized to the TDM arm compared to IFX dose intensification to 5 mg/kg every 4 weeks (€ 6038 vs € 9178, p < 0.001) in IBD patients with secondary LOR (78). The economic superiority of TDM-based dosing was maintained throughout 1 year. The TAXIT study (35) also found a slight economic advantage to TDM-guided dosing. However, evaluating treatment costs is complex, for example, the costs of additional drugs (steroids, immunosuppressants), laboratory tests, hospitalization, and surgery should be considered.
For both ustekinumab and vedolizumab, there are currently insufficient data to support routine use of TDM although data from the confirmatory efficacy and safety studies may provide insights into the desired trough concentrations in IBD patients (82)(83)(84)(85). Further studies to elucidate the role of TDM in the treatment of these agents are needed. TDM utility for mAbs has also been questioned due to slow assay turnaround, analytical deficiencies, assay differences, and difficulties with interpreting results (86). Moreover, immunogenicity response may differ across treated patients. As such, an overall TDM strategy that considers patients' immune responses in addition to drug concentration may be beneficial albeit it would differ from traditional TDM for small molecule drugs which is largely based on drug concentrations and aims at reducing the likelihood of adverse events.

TDM ASSAYS FOR PRECISION DOSING OF BIOLOGIC DRUGS TDM Assays for Biologics
In IBD, multiple diagnostic companies offer TDM tests for IFX, due to the breadth of published literature identifying specific drug concentration ranges for optimal patient outcomes (87)(88)(89). MAb TDM test results are then combined with other monitoring tests, such as ADA, serum albumin, and CRP measurements, to adjust treatment to manage disease flares and treatment failure (90).
There are currently no FDA-cleared TDM tests for any mAb. Marketed mAb TDM tests in the USA are all laboratory-developed tests (LDTs), and cover only a small portion of the approved biologics (mostly those indicated to treat IBD). Most TDM LDTs utilize a sandwich or bridging enzyme-linked immunosorbent assay (ELISA). In this format, either a recombinant antigen or an anti-idiotype antibody is used as a capture reagent. A recombinant antigen, anti-idiotype antibody, or anti-isotype antibody is used as a labeled detection reagent. While tests for the same therapeutic mAbs are available from several different diagnostic companies, there is a lack of standardization for the different assays (91). Cell-based assays, mass spectrometry, and mobility shift assays for mAb TDM also exist. In cell-based assays, the target molecule is expressed on a cell line by transfection, and flow cytometry is used to measure binding of the desired mAb. This method has been used for PK analysis of alemtuzumab (anti-CD52) (92). Cell-based assays, however, are labor intensive, requiring specially trained personnel and expensive equipment. Liquid chromatography-mass spectrometry (LC-MS), monoclonal Ig rapid accurate measurement (miRAMM), and Orbitrap are different mass spectrometry techniques used to quantitate mAb concentrations. These methods detect either tryptic peptides or intact light chains to quantitate mAbs. In the former, digested mAbs must have peptide fragments that are unique to the drug and not present in the polyclonal antibody background of serum (~ 10 mg/ml); this poses a problem for some fully human mAbs, such as ADL. In the latter, the reliance on intact light chains for detection and quantitation allows detection of fully human antibodies; however, the assay may not be sensitive enough for low-concentration measurements, such as trough concentration for some drugs (93). Currently, mass spectrometry-based TDM LDTs for IFX and eculizumab are available (94,95). Finally, homogenous mobility shift assays (HMSA) incorporate an acid dissociation step to separate serum-mAb/ADA complexes and then utilize size-exclusion HPLC to quantify drug and ADA concentrations independently. However, HMSA tests are expensive and require three days to generate results. Concentration and ADA tests for IFX, ADL, vedolizumab, and ustekinumab are available commercially (96,97). An LC/MS/MS method has also been recently developed (98).
The current mAb TDM protocol involves two strategies: either the patient must have blood drawn approximately 1-2 weeks before treatment to allow time for sample shipment and running the assay, or the patient has a sample drawn at the clinic visit, and that data is utilized at the subsequent visit. Taking a sample prior to a clinic visit requires an additional medical appointment. Also, because the TDM measurement is not taken at trough, which is when most publications established for target drug levels, translating the resulting TDM measurement into dose adjustment is more difficult. In the second case, dose adjustments or treatment changes would not be made until the patient's next dose, generally 2-8 weeks later, depending on the drug.

Point-of-Care TDM Assays for Biologics
Point-of-care (POC) TDM may be particularly valuable in acute settings such as severe ulcerative colitis. Such patients often clear mAb more rapidly due to drug loss through the gut (protein-losing enteropathy) (99) and dose escalation is a common approach, as discussed previously. POC TDM would provide real-time drug-level information to inform the decision to dose escalate patients to achieve target drug levels, switch therapies, or consider alternative treatment modalities such as surgery.
POC TDM is currently unavailable for mAb therapeutics in the USA. CE-marked tests for IFX are commercially available in Europe (100,101). Both utilize a sandwich immunoassay format. An anti-idiotype antibody is used for capture on the lateral flow membrane and recombinant TNF-α conjugated to gold colloid as the detection reagent.
MAb TDM tests that utilize mimetope peptides as capture or detection reagents in ELISA and lateral flow immunoassay (POC device) are also being developed. Peptides specific for rituximab and alemtuzumab were originally described (102,103), and many others have been developed. These peptides, which are referred to as Veritopes, are selected via phage display against mAb targets (novel or biosimilar) or other proteins, such as receptor fusions. They act as surrogate ligands, binding at the active sites of the antibodies. Veritopes are specific for their target mAb and compatible with serum, plasma, and whole blood. Veritopes offer several benefits over current reagents used in TDM, such as anti-idiotype antibodies and recombinant proteins:

1.
Veritope selection is rapid and does not require immunization of animals.

2.
Veritopes are easily synthesized and inexpensive to produce.
In contrast, recombinant protein expression is not always feasible and anti-idiotype antibody identification is tedious and not always successful. Large-scale production of recombinant proteins and anti-idiotype antibodies are also costly and stability may be an issue with the former. Unlike cell-based assays, mass spectrometry, and HMSA, Veritopes are amendable to multiple immunoassay formats that do not require highly trained users or expensive equipment. Finally, Veritopes are surrogate ligands that only recognize active (unbound) drug, unlike mass spectrometry, which quantitates all drugs (e.g., active and inactive) in the sample.

Assays for Anti-drug Antibodies
Laboratory results for ADA are highly dependent on the assay used to measure ADA. Historically, ADA assays are prone to interference by the presence of drug in samples and sensitive assays only detect ADA in the presence of low amounts of drug (104). However, recent technology advancement has overcome the limitation of drug interference experienced by many previous ADA assays (105). Reported results from immunogenicity assays increasingly include the intensity of ADA expressed as semi-quantitative titer values or other units (106).

CONCLUSIONS
MAbs such as IFX and ADL have proven to be effective in the treatment of auto-immune diseases. However, a significant proportion of patients do not respond to induction treatment or lose response during maintenance, presumably because of inadequate drug exposure. TDM, with the use of dashboard systems, can contribute to adequate dosing by taking into account specific patient factors influencing mAb clearance. Clinical trial data can often illustrate the E-R relationship for mAbs, although this is more often when the data come from a wide dose range and include data from numerous patients such as phase 3 studies. The E-R curves may suggest that a higher responder rate is associated with higher exposure, (e.g., steady-state trough concentration), and steeper E-R curves may suggest greater potential benefit with dose intensification. However, whether intensified dosing regimens will lead to greater clinical efficacy with acceptable safety profiles remains to be experimentally demonstrated because extrapolation of efficacy and safety is challenging. Moreover, understanding patient factors that would drive the treatment response remains an area for future research.
In summary, TDM is a growing part of the precision medicine approach. Evidence supporting correlation of interpatient PK variation with treatment outcomes is extensive and growing. As combination therapy use increases in the treatment of many diseases, PK variability and factors influencing individual patient exposure are generally different between drugs, and therefore, exposure to different drugs in a combination setting may ultimately need to be addressed. While not currently available for all biologics, POC TDM is warranted in certain circumstances and enables personalized, precision dosing. Most current methods for TDM are not amenable for rapid POC testing, but alternative technologies are being developed to make POC TDM reliable and practical for patients treated with biologic therapies. An unmet clinical need exists to mitigate the risks of interpatient PK variability among patients treated with mAbs.

ADA+ (or high ADA titer)
High (or therapeutic level) May be related to mechanism of action of drug a May be related to mechanism of action of drug a Low (or subtherapeutic level) May be suboptimal dose, non-immune-mediated PK issue May be immune-mediated PK issue a Such as patients not responsive or with low sensitivity to the mechanism of action of the drug


s2fieldsofstudy: Biology Chemistry

text:
Therapeutic strategies for afatinib‐resistant lung cancer harboring HER2 alterations

Human epidermal growth factor receptor 2 (HER2) plays an important role in the pathogenesis of various cancers. HER2 alterations have been suggested to be a therapeutic target in non‐small‐cell lung cancer (NSCLC), just as in breast and gastric cancers. We previously reported that the pan‐HER inhibitor afatinib could be a useful therapeutic agent as HER2‐targeted therapy for patients with NSCLC harboring HER2 alterations. However, acquired resistance to afatinib was observed in the clinical setting, similar to the case for other HER inhibitors. Thus, elucidation of the mechanisms underlying the development of acquired drug resistance and exploring means to overcome acquired drug resistance are important issues in the treatment of NSCLC. In this study, we experimentally established afatinib‐resistant cell lines from NSCLC cell lines harboring HER2 alterations, and investigated the mechanisms underlying the acquisition of drug resistance. The established cell lines showed several unique afatinib‐resistance mechanisms, including MET amplification, loss of HER2 amplification and gene expression, epithelial‐to‐mesenchymal transition (EMT) and acquisition of cancer stem cell (CSC)‐like features. The afatinib‐resistant cell lines showing MET amplification were sensitive to the combination of afatinib plus crizotinib (a MET inhibitor), both in vitro and in vivo. The resistant cell lines which showed EMT or had acquired CSC‐like features remained sensitive to docetaxel, like the parental cells. These findings may provide clues to countering the resistance to afatinib in NSCLC patients with HER2 alterations.

marked antitumor activity in patients with non-small cell lung cancer (NSCLC) carrying activating EGFR mutations. 1 Other oncogenic alterations, such as ALK, KRAS, NRAS, BRAF, MET and FGFR, have also been identified in some subsets of NSCLC patients as possible treatment targets. [2][3][4] Human epidermal growth factor receptor 2 (HER2) is a member of the HER family, which is composed of 4 receptor tyrosine kinases (RTK). It is frequently overexpressed in various human cancers, and many preclinical studies have demonstrated that overexpression of HER2 or mutations of the HER2 kinase domain play an important role in oncogenic transformation and tumorigenesis. [5][6][7][8] In the case of breast and gastric cancers, targeted therapies in patients with HER2positive tumors have been clinically proven to be effective. 9,10 Among NSCLC patients, the reported frequency of HER2 overexpression and HER2 amplification are 11%-32% and 2%-23%, respectively. [11][12][13][14] HER2 mutations are identified in approximately 2%-4% of NSCLC and are usually mutually exclusive of other driver mutations. 15,16 However, it remains to be established whether HER2-targeted therapy is of clinical benefit in patients with HER2-altered NSCLC. Previous clinical trials have failed to show any clinical benefit of trastuzumab monotherapy or any additional effect of trastuzumab over conventional first-line chemotherapy in HER2-positive NSCLC patients. [17][18][19] However, subsets of patients with high HER2 expression levels, HER2 gene amplification or HER2 mutations have been demonstrated to show a good response to HER2-targeted treatment. 17,20,21 In addition, a recent retrospective study showed that HER2-targeted therapy was beneficial in combination with chemotherapy for patients with HER2mutant NSCLC. 22 These findings suggest the potential usefulness of HER2-targeted therapy in HER2-altered NSCLC patients.
Afatinib, also known as BIBW2992, is an irreversible pan-HER (EGFR, HER2, HER3 and HER4) inhibitor, and has recently been approved for the treatment of NSCLC patients carrying EGFR mutations in the tumor. We previously reported that afatinib could inhibit cell growth in both HER2-amplified and HER2-mutant NSCLC cells, just as in EGFR-mutant NSCLC cells, through G1 arrest and apoptosis. 23 In clinical settings, afatinib monotherapy was effective in 3 out of 3 assessable patients with an HER2-mutated adenocarcinoma in an exploratory study of 5 patients. 21 In addition, the recent retrospective studies showed the potential benefit of afatinib for patients with HER2-mutant lung cancers. 22,24 While further prospective studies including randomized trials are required, afatinib could be approved as one of the therapeutic options for HER2-altered NSCLC patients.
However, development of acquired resistance to afatinib, just like the case for other HER inhibitors, has been reported in most EGFRmutant NSCLC patients treated with afatinib, 25,26 and the situation is considered to be similar for HER2-altered NSCLC patients. Therefore, overcoming acquired resistance to afatinib is a critical clinical issue.
In this study, we experimentally established various afatinib-resistant cell lines from HER2-altered NSCLC cell lines as the parental cell lines, and examined the molecular profiles of these cells to uncover the mechanisms of acquisition of the drug resistance, and investigated the treatment strategies for these cells with acquired resistance to afatinib. Three NSCLC cell lines (Calu3, NCI-H2170 [H2170] and NCI-H1781 [H1781]), purchased from ATCC (Manassas, VA, USA), were used as the patent cell lines for this study. These cell lines were authenticated by ATCC using short-tandem-repeat polymorphism analysis, and used within 6 months of receipt. All the lung cancer cells were cultured in RPMI-1640 media supplemented with 10% FBS, in a humidified incubator with 5% CO 2 at 37°C. Afatinib-resistant sublines were established by different methods: the parental cells were cultured with stepwise escalation of the concentration of afatinib in the culture medium (Calu3 and H2170: from 5 nmol/L to 2 lmol/L, H1781: from 5 nmol/L to 0.4 lmol/L) over a period of 6 months (stepwise escalation method), or use of a high concentration of afatinib from the start of culture (Calu3 and H2170: 2 lmol/L, H1781: 0.4 lmol/L) until 6 months (high concentration method). Finally, afatinib-resistant sublines designated as H2170-ARS, Calu3-ARS and H1781-ARS were established by the stepwise escalation method, and those designated as H2170-ARH, Calu3-ARH and H1781-ARH were established by the high concentration method (Table 1).

| Reagents and cell cultures
Acquired resistance was defined as an IC50 value of over a 100-fold higher than that for the parental cell line. Afatinib and docetaxel were purchased from Synkinase (San Diego, CA, USA) and Wako Pure Chemical Industries (Osaka, Japan), respectively. Crizotinib and elacridar were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Details of the phospho-receptor tyrosine kinase array and the cell proliferation assay are included in Document S1.

| DNA and RNA extraction
Genomic DNA were extracted from the cell lines using the DNeasy Blood and Tissue Kit (Qiagen, Venlo, the Netherlands), and total RNA were extracted using the RNeasy Mini Kit (Qiagen). Complementary DNA (cDNA) was synthesized from total RNA using a

| Direct sequencing
We determined the mutational status of the tyrosine kinase domain of HER2 by direct sequencing; the PCR conditions employed are shown in Table S1B.

| Western blot analysis
The total cell lysate was extracted with lysis buffer, a mixture of RIPA buffer, phosphatase inhibitor cocktails 2 and 3 (Sigma-Aldrich) and Complete Mini (Roche, Basel, Switzerland). Western blot analysis was carried out using the conventional method using the following  Table S1C.

| Cell growth inhibition assay
Cells were cultured with or without afatinib for 72 hours and the sensitivity to the drug was determined by a modified MTS assay using CellTiter 96 Aqueous One Solution Reagent (Promega, Fitchburg, WI, USA), as previously described. 28 The antiproliferative effect of each drug was expressed in terms of the IC50 value, which refers to the concentration of the drug required to inhibit cell proliferation by 50%.

| Statistical analysis
All data were analyzed using the JMP v 9.0.0 software (SAS Insti- HER2-amplified cell lines (Calu3 and H2170) and 1 HER2-mutant cell line (H1781). To investigate the genotypic changes in the cells with acquired resistance to afatinib, we examined the copy number of HER2, EGFR and MET, gain of which is well known as a mechanism underlying the resistance to HER inhibitors. 29 Significant copy number gains of MET were detected in the Calu3-ARS cells ( Figure 1A).
In addition, the HER2 copy number was almost lost in the H2170-ARS and H2170-ARH cells. Copy number analysis also revealed progressive decrease of the HER2 copy number of H2170-ARS cells with increasing number of passages, eventually leading to the disappearance of the amplified HER2 alleles ( Figure 1B). To investigate the mechanism underlying the progressive decrease of the copy number of HER2, we performed the FISH assay and found that all of H2170 parental cells harbored amplified HER2 alleles ( Figure 1C).  Figure 2A); furthermore, the resistant cells had acquired a spindle-like shape in contrast to the morphology of the corresponding parental Calu3 cells ( Figure 2B). These findings suggest the occurrence of epithelial-to-mesenchymal transition (EMT) in the Calu3-ARH, which was considered as being the mechanism underlying the resistance to afatinib.
Next, we investigated the expression levels of ALDH1A1, ABCB1, CD44, Oct-4 and CD133, which are putative CSC markers in NSCLC ( Figure 2C and Figure S2A). ALDH1A1 was found to be overexpressed in the H1781-ARS and H1781-ARH cells as compared to the parental H1781 cells. Western blot analysis also revealed overexpression of ALDH1A1 in the H1781-ARS and H1781-ARH cells ( Figure 2A). These cell lines with acquired resistance of afatinib were non-adhesive and capable of forming sphere-like clusters ( Figure 2B).
We conducted a sphere formation assay to examine the cellular functional features of CSC-like properties, 28 and found that H1781-ARS and H1781-ARH cells acquired higher ability to form spheres in suspension culture compared with parental H1781 cells ( Figure S2B).
To examine the CSC-like properties in vivo, we conducted tumor transplantation experiments. 28,30,31 We subcutaneously implanted H1781, H1781-ARS and H1781-ARH cells to examine the tumorforming capability in NOD/SCID mice. We found that H1781-ARS and H1781-ARH cells exhibited higher tumorigenicity than parental H1781 cells. The H1781-ARS and H1781-ARH cells established larger tumors with shorter latencies than the parental H1781 cells ( Figure S2C). These findings suggest that the H1781-ARS and H1781-ARH cells had acquired CSC-like properties, which could be considered as the mechanism underlying the resistance to afatinib.
ABCB1 was overexpressed in the H2170-ARS and H2170-ARH cells, but not in either the H1781-ARS or H1781-ARH cells ( Figure 2C).
CD44, Oct-4 and CD133 overexpression was not detected in any of the resistant cell lines.

| Drug sensitivities of the afatinib-resistant cells showing MET amplification
The sensitivities of the cells to various drugs are shown in Table 2.
The cell growth inhibition assay showed that the Calu3-ARS cells showing MET amplification were insensitive to monotherapy with crizotinib, which is a MET inhibitor, but sensitive to combined treatment with afatinib plus crizotinib ( Figure 3A)

| Sensitivity of the afatinib-resistant cells to chemotherapeutic agents
The H2170-ARS and H2170-ARH cells, which had lost HER2 amplification, also acquired resistance to docetaxel (Table 2). To investigate whether their resistance mechanism was associated with overexpression of ATP-binding Cassette sub-family B Member 1 (ABCB1), which is well known to induce resistance to chemotherapy, we inhibitor elacridar ( Figure S3A,B). We found that elacridar recovered the sensitivity to docetaxel, but not to afatinib in H2170-ARS and H2170-ARH cells. These findings were similar to those in our previous report using docetaxel-resistant NSCLC cells with EGFR-mutations. 32 In contrast, the other afatinib-resistant cell lines, including Calu3-ARH, H1781-ARS and H1781-ARH, which showed EMT or had acquired CSC-like features, showed maintained sensitivity to docetaxel, because ABCB1 was not upregulated in these acquired resistance cell lines.

| DISCUSSION
In this study, we established multiple cell lines that exhibited acquired resistance to the irreversible pan-HER inhibitor afatinib, previous study using EGFR-mutant NSCLC cells, we reported that the method of exposure of cell cultures to drugs influences the mechanisms of acquired resistance to HER inhibitors (gefitinib and afatinib). 28,33 Consistent with that report, in this study also, conducted using HER2-altered NSCLC cells, the resistance mechanisms were affected by the manner of drug exposure. The resistance mechanism in the Calu3-ARS cells established by the stepwise escalation method appeared to be MET amplification, whereas that in the Calu3-ARH cells established by the high drug concentration method was considered to be possibly attributable to the acquisition of EMT features.
MET amplification is one of the major mechanisms underlying the acquisition of resistance to HER inhibitors in EGFR-mutant NSCLC. 28,29,33,34 MET amplification has also been reported to contribute to trastuzumab resistance in HER2-positive breast cancer cells. 35 In this study, we identified MET amplification as one of the In afatinib-resistant cell lines established from H2170 cells showing HER2 amplification, the copy number of HER2 decreased gradually during the course of exposure to afatinib. We previously reported loss of the EGFR-mutant allele and progressive decrease of the EGFR copy number in an EGFR-mutant NSCLC cell line during gefitinib exposure. 28 However, to the best of our knowledge, loss of HER2 amplification during afatinib treatment has never been reported before in in vitro examinations. However, loss of HER2 amplification has sometimes been reported after trastuzumab treatment in clinical HER2-positive breast or gastric cancer patients. [36][37][38][39] Our finding seems to be consistent with these latter clinical reports.
Loss of HER2 amplification in the residual disease following neoadjuvant trastuzumab-based therapy has been reported to be associated with a poorer relapse-free survival when compared with maintained HER2 expression. 40 The biological basis of these genetic alterations has not been clarified yet, and might include tumor heterogeneity, genetic drift toward a more aggressive tumor phenotype, or selective pressure of adjuvant therapy. 38


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MiR‐873 inhibition enhances gefitinib resistance in non‐small cell lung cancer cells by targeting glioma‐associated oncogene homolog 1

Background The five‐year survival rate of non‐small cell lung cancer (NSCLC) patients is very low. MiR‐873 is involved in the growth, metastasis, and differentiation of tumors. Herein, we determined the target gene and influence of miR‐873 in NSCLC. Methods MiRanda and Targetscan websites were used to predict the target gene of miR‐873 in NSCLC. Luciferase activity was examined using a dual luciferase reporter gene assay kit. The viability, tube formation, and proliferation of cells were analyzed by cell counting kit‐8, angiogenic analysis, and flow cytometry, respectively. The levels of miR‐873 and GLI1 were evaluated using quantitative real‐time PCR and Western blot assays. Results Low levels of GLI1 and high levels of miR‐873 were observed in an NSCLC cell line (PC9) highly sensitive to EGFR‐tyrosine kinase inhibitors. There was a negative correlation between miR‐873 and GLI1 expression in PC9 and PC9/GR cells. The inhibition of miR‐873 enhanced GLI1 levels. MiR‐873 expression was inhibited by gefitinib. Gefitinib markedly reduced the viability, tube formation, and cell number in PC9 cells. However, suppression of miR‐873 enhanced the resistance and knockdown of GLI1 enhanced the sensitivity of PC9 cells to gefitinib. Conclusions GLI1 is a target gene of miR‐873 in NSCLC. The inhibition of miR‐873 increased gefitinib resistance of NSCLC cells via the upregulation of GLI1. These results indicate that miR‐873‐GLI1 signaling is involved in gefitinib resistance in NSCLC.

Introduction
Lung cancer (LC) is one of the most common malignant tumors in the world, and the leading cause of cancerrelated death in China. Non-small cell lung cancer (NSCLC) accounts for approximately 85% of LC cases. At the time of diagnosis, approximately 75% of patients are in middle and late stages. The five-year survival rate is very low. 1,2 Platinum-based doublet chemotherapy is the firstline standard treatment for late-stage NSCLC. [3][4][5] The emergence of molecular targeted drugs has brought great promise for the treatment of LC. [6][7][8] In patients with EGFR sensitive mutations, the efficacy rate of EGFR-tyrosine kinase inhibitors (TKI) is 71.2%, thus EGFR-TKIs have become first-line drugs for patients with EGFR sensitive mutant advanced LC. However, almost all patients eventually suffer from drug resistance. The primary or secondary drug resistance of EGFR-TKIs greatly limits their clinical application 9-11 therefore, finding a mechanism to alter drug resistance is of significant importance.
MicroRNA (miRNAs) are non-coding RNAs, mainly mediating gene expression at posttranscriptional levels. 12,13 Hundreds of miRNAs have been found. In the human genome, miRNAs regulate protein encoding genes at a rate of approximately 1:3, controlling cell apoptosis, proliferation, differentiation, metabolism, individual development and tumorigenesis, and drug resistance. [14][15][16] Target gene expression is regulated by two mechanisms: (i) binding to the untranslated region (3'UTR) of the target messenger RNA (mRNA) 3' end, inhibiting its translation; and (ii) like small interfering RNA (siRNA), miRNA binds to the target and degrades target mRNA. [17][18][19] Recent studies have found that miRNAs regulate drug resistance by mediating their targeting genes in various cancers. [20][21][22][23][24] In recent years, abnormal expression of miR-873 has been found in many kinds of tumors, such as breast and ovarian cancers, and glioma. [25][26][27] MiR-873 plays a role in promoting cancer or anticancer by regulating tumor cell invasion, migration, proliferation, apoptosis, and sensitivity to chemotherapeutic drugs. [28][29][30][31] Nevertheless, the role of miR-873 in drug resistance in NSCLC is still unknown.
We detected mRNA levels of miR-873 in normal human lung epithelial cells and highly sensitive EGFR-TKI NSCLC cells by quantitative real-time (qRT)-PCR. According to data from the miRanda and Targetscan websites, we predicted the binding sites of miR-873 to related target genes. The influence of miR-873 on NSCLC repressed by gefitinib was explored.
Using data from the miRanda and Targetscan websites, we predicted the binding site of the GLI1 gene to miR-873. The 3'UTR of GLI1 with affinity for miR-873 and a mutant reporter were cloned to the downstream of firefly luciferase of psiCHECK-2 vector (Hibio, Hangzhou, China). MiR-873 was then co-transfected into HEK293T cells using Lipofectamine 2000. After transfection, luciferase activity analysis was performed.

Quantitative real-time PCR assay
Total RNA was harvested by TRIzol (Yeasen, Shanghai, China). One microgram of RNA was applied to synthesize complementary DNA (cDNA) using a TIANScript cDNA Synthesis kit (Tiangen, Beijing, China). The reaction conditions were: 85 C for 15 minutes and 4 C for five minutes. The cDNA was amplified using a SYBR Premix ExTaq II kit (Thermo Fisher Scientific). The reaction conditions were: 95 C for five minutes, 92 C for 15 seconds and 60 C for 35 seconds for 30 cycles, and 72 C for 35 seconds. The primers are listed in Table 1. U6 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were used as internal controls. The 2 -ΔΔCT formula was used to assess gene expression.

Luciferase activity analysis
A dual luciferase reporter gene assay kit (Yeasen) was used to measure luciferase activity following the standard method. HEK293T cells were lysed using lysate. Cell suspension was transferred to a black enzyme plate. The cells were then resuspended using medium. The supernatant was seeded in a 96-well plate, then firefly and sea kidney luciferase reaction solutions were added to the plates. The data were measured using a multimode reader (SuPerMax-3100, Shanpu, Shanghai, China).

Cell counting kit-8 analysis
Cell counting kit-8 (Beyotime, Shanghai, China) was used to assess the viability of PC9 cells. PC9 cells were cultured in 96-well plates (2 × 10 3 cells/well) in the incubator for 24 hours. Cells were then exposed to miR-873 inhibitor, empty vector (mock), and 40 nM gefitinib for 48, 72, and 144 hours. After treatment, the CCK-8 reagent was dripped to the well, and cells were cultured in the incubator for four hours. Absorbance was assessed using a multimode reader.

Angiogenic analysis
PC9 cells were cultured in 6-well plates (3 × 10 4 cells/well) in the incubator for 24 hours. Cells were exposed to miR-873 inhibitor, empty vector (mock), and 40 nM gefitinib for 48 hours. The medium from each group was collected for the following experiments. Matrigel Basement Membrane Matrix (BD Biosciences, San Jose, CA, USA) was added into a 96-well plate and allowed to polymerize at 37 C for 30 minutes. The human umbilical vein endothelial cells (2 × 10 4 /well) were plated in a 96-well plate containing the pre-described condition medium and maintained for 20 hours at 37 C, 5% CO 2 . Finally, the endothelial cell tube was observed under an inverted microscope (TL3000 Ergo; Leica, Microsystems, Wetzlar, Germany). Tube formation was defined as: 1000 × total area of connected tubes/total image area.

Flow cytometry
The proliferation of PC9 cells was detected by carboxyfluorescein succinimidyl amino ester (CFSE). After cell treatment, cell suspension was prepared. CFSE solution was added into the cell suspension and shaken up. Cell proliferation was evaluated using flow cytometry (Attune NxT, Thermo Fisher Scientific).

Statistical analysis
The data were presented as mean AE standard deviation.
One-way analysis of variance was performed to evaluate the differences between groups using SPSS version 20 (IBM Corp., Armonk, NY, USA). P < 0.05 was considered statistically significant. Each experiment was repeated at least three times.

Results
MiR-873 expression was negatively correlated with GLI1 expression in PC9 cells The mRNA levels of miR-873 and GLI1 were detected by qRT-PCR assay, which revealed a significant decrease of GLI1 mRNA, but an increase of miR-873 mRNA in PC9 cells compared to BEAS-2B cells (Fig 1a,b) (P < 0.05) (Fig 1a,b). MiR-873 expression was negatively correlated with GLI1 expression in PC9 cells (Fig 1c,d). As shown in Figure 1e, the half-maximal inhibitory concentration (IC50) of gefitinib was < 20 nM in PC9 cells; however, the IC50 of gefitinib reached > 40 nM when the cells were treated with miR-873 inhibitors.

GLI1 is a target gene of miR-873
The qRT-PCR data revealed that miR-873 expression was obviously decreased when PC9 cells were transfected with the miR-873 inhibitor (P < 0.05) (Fig 2a). GLI1 mRNA and protein expression were obviously upregulated when PC9 cells were administered an miR-873 inhibitor (P < 0.05) (Fig 2b,c). Data from the miRanda and Targetscan websites predicted the binding site of miR-873 on the GLI1 gene. There is a potential binding site in the 3'UTR region of the GLI1 gene (Fig 2d). Additionally, luciferase activity report assay findings showed that the relative luciferase activity of GLI1-3'UTR was markedly decreased in the presence of miR-873. However, the relative luciferase activity of GLI1-3'UTR mut was not altered by miR-873 (P < 0.05) (Fig 2e).

Effect of miR-873 on cell viability, proliferation, and angiogenesis
The expression of miR-873 was decreased in cells treated with gefitinib or miR-873 inhibitors. The combination of gefitinib and miR-873 inhibitors further decreased the expression of miR-873 (Fig 3). As CCK-8 data shows, gefitinib predominantly decreased cell viability at 72 and 144 hours. Cell viability was notably increased in cells treated with miR-873 inhibitor and gefitinib compared to gefitinib or mock (P < 0.05) (Fig 4a). Angiogenic analysis revealed that gefitinib reduced tube formation. Tube formation was drastically augmented by the miR-873 inhibitor (P < 0.05) (Fig 4b,c). Meanwhile, flow cytometry data revealed a decrease in cell numbers in cells administered gefitinib, but an increase in cell numbers in cells exposed to miR-873 inhibitor and gefitinib (P < 0.05) (Fig 4d).

Repression of miR-873 enhanced GLI1 expression in PC9 cells
Western blot and qRT-PCR were used to determine the molecular mechanism of miR-873 on PC9 cells. As qRT-PCR shows, gefitinib conspicuously upregulated the mRNA level of GLI1. When cells were exposed to the miR-873 inhibitor and gefitinib, the GLI1 mRNA level was enhanced compared to gefitinib or mock (P < 0.05) (Fig 5a). The protein trend of GLI1 was also increased by miR-873 inhibitor and gefitinib compared to gefitinib or mock (P < 0.05) (Fig 5b,c). GLI1 knockdown increased the sensitivity of PC9 to gefitinib. Our data also revealed that downregulation of GLI1 promoted apoptosis in PC9 cells compared to gefitinib treatment alone (P < 0.05) (Fig 6), indicating that inhibition of GLI1 improved the effectiveness of gefitinib in PC9 cells.

Discussion
In the present study, GLI1 was identified as a potential target gene for miR-873 in NSCLC according to data from the miRanda and Targetscan websites. MiR-873 and GLI1 expression has a negative correlation in NSCLC cells (PC9) highly sensitive to EGFR-TKIs. Relative luciferase activity was markedly enhanced in PC9 cells treated with miR-873 inhibitor and GLI-3'UTR. Suppression of miR-873 significantly enhanced the viability, angiogenesis, and proliferation of PC9 cells inhibited by gefitinib, followed by upregulation of GLI1.
The Hh signaling pathway is highly activated in NSCLC. [32][33][34] GLI1 is the transcription factor of the Hh signaling pathway and its downstream target gene. Its expression is highly dependent on the activity of the Hh signal and is often used as a marker for activation of the Hh signaling pathway. 35,36 Furthermore, Gao et al. proved that miR-873 inhibits the progression of lung adenocarcinoma. 37 Zhang et al. confirmed that miR-873 represses H9C2 cell proliferation by regulating GLI1. 31 Thus, we hypothesized that miR-873 and GLI1 may be linked in NSCLC. As expected, we found that GLI1 is a target gene of miR-873 in NSCLC. In addition, there is a negative correlation between miR-873 and GLI1 expression in PC9 cells. MiR-873 inhibition markedly promoted the expression of GLI1. Luciferase activity was increased in PC9 cells exposed to miR-873 and GLI-3'UTR, thus miR-873 and GLI1 were selected as research objects.
Gefitinib, an EGFR-TKI, is the first targeted drug to treat advanced-stage NSCLC. [38][39][40] Our data showed that gefitinib reduces cell viability and proliferation in PC9 cells. Angiogenesis was also inhibited by gefitinib. However, gefitinib will develop resistance in cancer during its application. Recently, a large number of articles have reported gefitinib resistance in NSCLC cells. [40][41][42] Herein, we explored the role of miR-873 in gefitinib resistance in PC9 cells.
It is well known that the development of neovascularization is a necessary condition for tumor growth and metastasis. 43,44 The unrestricted growth, invasion, and metastasis of the tumor are dependent on angiogenesis. Meanwhile, neovascularization provides nutrients for the growth of the tumor and is also an important method to excrete tumor cells. During angiogenesis, tumor growth changes from slow to rapid; angiogenesis is the key step to facilitate this transformation. Inhibition of angiogenesis can obviously control tumor growth and metastasis. [45][46][47][48][49] In this study, we found that miR-873 suppression conspicuously enhanced the viability and growth of PC9 cells treated with gefitinib. Moreover, the inhibition of miR-873 increased angiogenesis. These findings suggest that miR-873 repression heightens gefitinib resistance in NSCLC cells.
Xie et al. reported that a decrease in RKIP increases radioresistance in NSCLC cells by regulating GLI1 signaling. 50 Bora-Singhal et al. reported that silence of GLI1 combined with EGFR inhibitors obviously attenuates the growth of NSCLC cells. 51 In our study, we observed that miR-873 drastically enhanced the level of GLI1 in PC9 cells. Moreover, GLI1 downregulation enhanced apoptosis of PC9 cells, caused by gefitinib. These results imply that the inhibition of miR-873 exerted in PC9 cells is more likely related to upregulation of GLI1.
GLI1 is a target gene of miR-873 in NSCLC. Suppression of miR-873 accelerated the viability, angiogenesis, and proliferation of PC9 cells suppressed by gefitinib. MiR-873 suppression strengthened gefitinib resistance in NSCLC cells by upregulating GLI1. These results prove that miR-873-GLI1 signaling is involved in gefitinib resistance in NSCLC. How to control drug resistance in NSCLC requires further research.


extfieldsofstudy: Biology Medicine

text:
Detection of Mycoplasma Contamination of Cell Culture by A Loop-Mediated Isothermal Amplification Method

Objective Mycoplasmas are major contaminants of cell culture and affect in vitro biological and diagnostic tests. Mycoplasma detection is conducted using culture and molecular methods. These methods vary in terms of accuracy, reliably and sensitivity. Loop-mediated isothermal amplification (LAMP) is used to amplify target DNA in a highly specific and rapid manner. This study aimed to develop a LAMP method for rapid detection of Mycoplasma in culture samples. Materials and Methods In this descriptive laboratory study, for LAMP detection of Mycoplasma contaminations in cell culture, we used primers specifically designed for targeting the 16S rRNA conserved gene of Mycoplasma spp. For a positive control structure, 16S rRNA amplified based on PCR, was cloned in a plasmid vector and sequenced. The assay specificity was evaluated using Mycoplasma genomic DNA and a panel containing genomes of gram-positive and gram-negative organisms. Results In this study, the method developed for detection of Mycoplasma contamination of cell cultures was a rapid, sensitive and cost-effective LAMP approach. The results demonstrated that this method benefits from high specificity (100%) for amplification of Mycoplasma strains and high speed (multiplication within 60 minutes), while it does not require expensive laboratory equipment compared to those needed for polymerase chain reaction (PCR)-based detection. Conclusion Our study is the first report about application of LAMP assay based on 16S rRNA gene for detection of Mycoplasma strains; this technique could be considered a useful tool for rapid detection of contamination of cell culture.

Introduction
Over the last decades, cell culture has been frequently used as a main research tool in medical and biological experiments (1). Cell culture contaminants can be categorized into chemical and biological contaminants. Impurities in media, and sera, endotoxins, and detergents are major chemical contaminants, and bacteria, molds, yeasts, viruses, mycoplasma, and cross contamination of other cell lines, are regarded as biological contaminants (2). Mycoplasma contamination is a serious concern that exists when using cell culture (3,4). The primary starting material, glassware or apparatus, culture reagents (mainly fetal bovine serum), laboratory staff and crosscontamination of infected cultures are examples of Mycoplasma contamination sources (5,6). The prevalence of Mycoplasma contamination of cell cultures has been estimated to range from 5 to 35% (7,8) while prevalence of cell culture infections with two or more Mycoplasma species are between 7 and 60% (1). Mycoplasma infection affects different aspects of the infected cell culture, resulting in obtaining spurious experimental data (5,9). Mycoplasmas are the smallest free-living microorganisms which are characterized by their round or filamentous shape, absence of a rigid cell wall and a DNA genome in the Mb range (4,7,10). Most of Mycoplasma species are not pathogenic (7); however, Mycoplasma pneumoniae is a human pathogen (11). The species that are frequently found in cell culture are Acholeplasma laidlawii, Mycoplasma arginini, Mycoplasma fermentans, Mycoplasma hominis, Mycoplasma hyorhinis, and Mycoplasma orale (12).
Several methods have been developed for detection of Mycoplasma spp. (13) including microbiological cultivation, biochemical assays, ELISA, direct or indirect fluorescent staining, immunofluorescence and nucleic acid amplification techniques [direct or nested polymerase chain reaction (PCR)] (7,9). Isolation on selective microbiological growth media has been regarded as the reference method as well the 'gold standard' assay, for a long period of time (14). Unfortunately, routine diagnosis procedures are usually time-consuming (i.e. several weeks are required to achieve results) and need high-level technical skills and expert personnel. Thus, fast and sensitive detection methods are needed to evaluate putative contaminated cell cultures. In this regard, newer test systems developed based on molecular biological methods, in particular PCR, which give results within 4.5-24 hours are commonly used by cell culture laboratories LAMP Assay for Detection of Mycoplasma Contamination (15). However, complicated procedures and relatively costly machinery required for PCR and electrophoresis processes have restricted its use (16,17). Therefore, development of simple, sensitive, specific, rapid and lowcost detection methods is of crucial importance (11).
Loop-mediated isothermal amplification (LAMP) assay is a novel gene amplification technique which uses 4-6 primers that recognize specific regions on the target DNA. The LAMP reaction is carried out under isothermal conditions (60-65˚C), thereby obviating the need for a thermal cycler (18,19). This method amplifies specific sequences of DNA in a shorter period of time compared to PCR with high specificity and efficiency but no need for a special reagent (17). Moreover, the LAMP reaction's product can be detected in real time by turbidity monitoring, which the turbidity is correlated with the production of magnesium pyrophosphate (20), or optical monitoring of a fluorescent intercalating dye by naked eyes (21). Therefore, it can be used for a rapid detection of various infectious diseases (18,22).
The aim of this study was to design and develop a reliable, rapid and specific LAMP assay based on the conserved section of 16S rRNA gene for detection of Mycoplasma contamination of cell cultures. To the best of our knowledge, this is the first report on application of this method for detection of Mycoplasma contamination in cell cultures based on 16S rRNA gene.

Cell culture and bacteria and DNA extraction
To perform this descriptive laboratory study, ten cell cultures contaminated with mycoplasmas, some noncontaminated cell culture and a DNA reference standard Mycoplasma were obtained from the Academic Center for Education, Culture and Research of Tehran, Iran. The standard bacteria including Shigella sonnei ATCC 9290, enteropathogenic Escherichia coli (EPEC) ATCC 43887, Klebsiella pneumoniae ATCC 7881, Bacillus subtilis ATCC 6051, Pseudomonas aeruginosa ATCC 9027, Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC 29212, and Yersinia enterocolitica ATCC 23715, were used as negative control in specificity testing. DNA of all cell cultures and standard bacterial species were extracted using the EZ-10 Spin Column Genomic DNA kit (Bio Basic Inc., Ontario, CA) according to the manufacturer's instructions. DNA was quantified spectrophotometrically and then stored at -20˚C till used as PCR template DNA and in LAMP assay and specificity tests.

Polymerase chain reaction reactions
Amplification reaction was performed for DNA of contaminated cell cultures, non-contaminated cell cultures, Mycoplasma DNA reference and negative control strains in 25 µl volume containing 6 μl of purified DNA, 12.5 µl of 2X reaction mix, 0.5 µM of each F3-Myco and B3-Myco outer primers (Table 1), 1 U of Taq DNA polymerase and 4.5 μl double distilled water. The PCR conditions were as follows: After an initial denaturing step at 94˚C for 4 minutes, 35 cycles of the following steps were carried out: denaturation at 94˚C for 45 seconds; annealing at 48.1˚C for 45 seconds, and extension at 72˚C for 45 seconds. Thermal cycling was carried out using an ABI 2720 thermocycler (Applied Biosystems, Warrington, UK). PCR products were separated on 2% agarose gels and compared against 100 bp DNA ladder (Fermentas, Lithuania) as a size marker, under UV gel documentation.

Cloning and preparation of standard plasmid
After PCR amplification of the 16S rRNA gene of Mycoplasma using the outer primers, TA cloning of the product was performed. For this purpose, the PCR product was purified using the PCR Purification Kit (Bioneer, Korea). The purified 16S rRNA gene fragment with the length of 219 bp was ligated into pTZ57R/T vector by 1 U of T4 DNA ligase, according to instructions of InsTAclone TM PCR Cloning Kit (Fermentas, Lithuania). Competent cells of E. coli Top10 F´ were transformed with the ligation reaction product. The transformed cells were incubated at 37˚C for 24 hours on Luria-Bertani (Merck, Germany) medium containing 38.4 µg/ml IPTG (isopropylbeta-D-thiogalactopyranoside, Sigma, St. Louis, MO, USA), 40 µl/ml X-gal (5-bromo-4-chloro-3indolyl beta D-galactoside, Sigma, Germany), 50 µg/ ml nalidixic acid and 100 µg/ml ampicillin (Merck, Germany). Recombinant clones on the medium were identified by blue/white screening and some white colonies containing recombinant vector were chosen for extra evaluation. Then, plasmids of the selected clones were extracted by AccuPrep Plasmid Mini Extraction kit (Bioneer, Korea) and 16S rRNA gene containing recombinant plasmids, were confirmed by PCR with the outer primers and sequencing. The confirmed plasmid was named pTZ57R/T-16S rRNA and quantified using UV absorbance measurement at 260 and 280 nm and further used as positive control in the LAMP assay.

The loop-mediated isothermal amplification assay
The LAMP reaction was conducted for all Mycoplasma DNA extracted from cell culture samples in 25 µl reaction mixture containing 12.5 µl 2X reaction mix, 1.5 µl the primer mix (40 pmol of each inner primer and 5 pmol of each outer primer) (Table 1), 8 U of Bst DNA polymerase large fragment (New England Biolabs, Ipswich, MA, USA), 6 µl of template genomic DNA and 5 μl molecular grade water. The mixture was incubated at 63˚C for 60 minutes in a Loopamp realtime turbidimeter (LA-320C, Teramecs, Japan), and turbidity of the reaction mix was determined at 650 nm every 6 seconds. Finally, the reaction was terminated by heating to 80˚C for 5 minutes in order to denature the Bst DNA polymerase large fragment. The LAMP reactions were examined by Loopamp real-time turbidimeter, electrophoresis of products on 2% agarose gel and direct visual observation to judge turbidity. Cycle sequencing method using F3-Myco and B3-Myco primers, was performed for final confirmation of the amplified products. The sequencing results were checked by BLAST (http://blast.ncbi.nlm.nih.gov/ Blast.cgi). To determine the optimum temperature that should be considered for LAMP reactions, the LAMP reactions were also performed at temperatures 60 and 65˚C for 60 minutes. In order to assess the effect of the loop primer on amplification speed, 20 pmol of the loop primer (LOOP-Myco) was added to the reaction mixture and LAMP reaction was conducted after 30, 45 and 60 minutes. The negative control tubes (without template DNA) were included in each run.

Specificity and sensitivity of the loop-mediated isothermal amplification assay
For evaluation of the specificity of the test, the LAMP reactions were performed (based on the above-noted protocol) using genomic DNA of Mycoplasma spp. and also genomic DNA of non-Mycoplasma organisms (negative control bacteria). The reactions were assessed with naked eye inspection and the Loopamp real-time turbidimeter. In addition, electrophoresis on 2% agarose gel was carried out to confirm the results.
Moreover, 10-fold serial dilutions of pTZ57R/T-16S rRNA plasmid (135 ng to 0.135 fg equal 4×10 10 to ~4 copies) were applied in LAMP experiment to examine the sensitivity of the assay. The results of amplified target sequence were analyzed by using Loopamp real-time turbidimeter, visual observation of turbidity by naked eye and electrophoresis on 2% agarose gel. Finally, sensitivity or detection limit (LOD) of the assay was determined.

Analysis of the polymerase chain reaction products and cloning
The PCR reaction was carried out using F3-Myco and B3-Myco primers on DNA of contaminated cell cultures, non-contaminated cell cultures, Mycoplasma DNA reference and negative control strains. PCR products of the tubes containing Mycoplasma DNA showed about 219 bp bands on 2% agarose gel. Based on comparison made against 100 bp DNA ladder, no significant difference found in banding pattern compared to the reference Mycoplasma strains (Fig.1A). The PCR reaction was specific as it showed exclusive amplification for Mycoplasma spp. while this result for 8 non-Mycoplasma bacteria species was negative (Fig.1B). Confirmatory test based on the cloning process was conducted by PCR assay with the outer primers (F3 and B3) on the extracted recombinant 16S rRNA-plasmids from white colonies and as expected, a clear and sharp 219 bp band on agarose gel was observed. In addition, the sequence of the amplified 16S rRNA gene was confirmed through direct sequencing, in which the obtained sequences perfectly matched the expected DNA sequences (data not shown).

Analysis of the loop-mediated isothermal amplification reaction
In the tubes with positive reaction for isothermal amplification, the turbidity (caused by white magnesium pyrophosphate precipitation) was observed with naked eye (Fig.2A). Electrophoresis of the LAMP products on 2% agarose gel showed clear ladder-like DNA amplification (Fig.2B). Amplification graph of the real-time turbidimeter confirmed amplification of the 16s rRNA gene (Fig.2C). The specificity of the LAMP products was verified by cycle sequencing. Comparison of the sequencing results with the 16S rRNA sequences of Mycoplasma spp. in Gene bank database confirmed the validity of the products. Amplification was detected at 60, 63, and 65˚C, and showed higher levels of amplified DNA at 60˚C when compared to other temperatures. Also, effect of the loop primer (LOOP-Myco) on diminution of incubation time was explored. In the reactions without the loop primer, ideal time for isothermal amplification was 60 minutes while in the reaction containing the loop primer, it was 30 minutes. Showing white turbidity, the tubes 1-5 (contaminated cell culture samples) were positive, while the tube 6 was negative, B. Electrophoretic analysis of the LAMP products. In lanes 1-5, contaminated cell culture samples showed ladder-like pattern, lane 6 was negative control and had no ladder-like pattern, and C. A representative turbidity amplification graph of the LAMP reaction. Curves 1-5 represent contaminated cell cultures and curve 6 is for negative control.

Specificity and sensitivity of the loop-mediated isothermal amplification assay
The LAMP assay was specific because judgment graph of the real-time turbidimeter showed exclusive amplification for Mycoplasma spp. while 8 non-Mycoplasma bacteria species had negative results. Consistently, in silico A B C A B analysis using BLAST, indicated that there were no falsepositive nor false-negative amplification. In addition, gel agarose electrophoresis of the LAMP products showed the characteristic ladder-like multiple bands only in the tubes containing Mycoplasma spp. genome DNA. Inspecting the judgment plot, agarose gel electrophoresis and visual detection of turbidity, the LOD of the assay was found ~4000 copy per reaction tube.

Discussion
Contamination of cell cultures by Mycoplasma spp. is a main problem in cell culture for which an accurate diagnostic method is highly required. There are several conventional and molecular diagnostic techniques available for detection of Mycoplasma spp. (9,14). In the last decade, in Iran, several studies established PCR assays using 16S rRNA gene for detection of different species of mycoplasmas such as M. orale (23), Mycoplasma and Ureaplasma species (24), Mycoplasma spp. (25)(26)(27), in cell culture. The results of these papers demonstrated that 16S rRNA-based PCR could detect all common Mycoplasma that contaminate cell cultures. The findings of the current study are in agreement with those reported by Tang et al. (9) which confirmed that 16S rRNA is a suitable target for Mycoplasma detection using PCR; however, few cross-reactions were observed with closely related Gram-positive organisms. In addition, Molla Kazemiha et al. (28) showed that real-time PCR and PCR assays developed based on the public sequences in the 16S rRNA, are suitable methods with high sensitivity, specificity and accuracy for detection of mycoplasma contamination of cell cultures. However, these molecular methods are complex and time-consuming and they pose a risk of contamination with ethidium bromide that requires expensive apparatus and qualified laboratory technicians.
As LAMP method has several advantages benefits including no need for a special process, completion of the reaction in a single tube and approval by naked eye contrary to electrophoresis, it can be a suitable alternative for techniques used for Mycoplasma detection (29). Another advantage of the LAMP method is the high stability of Bst polymerase enzyme compared to a number of inhibitory factors such as EDTA, bile salts, and NaCl in the amplification reaction (30,31). These characteristics show that utilization of the LAMP assay in laboratories with limited equipment and in large scale, is valuable. Yoshino et al. (32) showed the same sensitivity and specificity when comparing LAMP assay with a PCR assay, for rapid detection of M. pneumoniae. Also, a good global agreement between the LAMP assay and serological results for M. pneumoniae detection in pediatric patients, was revealed by Gotoh et al. (11).
Our study defines a rapid, sensitive and cost-effective LAMP method which is comparable to other DNA amplification procedures that are extensively used for identification of various microorganisms in laboratory. Previously, various sequences in the genome of human pathogenic species of Mycoplasma were used in the LAMP assay for detection of different species belonging to this genus, such as pdhD gene of M. genitalium (21), mhp165 gene of M. hyopneumoniae (17), uvrC gene of Mycoplasma bovis (33), p36 gene of Mycoplasma hyopneumoniae (34), the SDC1 sequence (M35024) of Mycoplasma pneumoniae (11,32,35), and P1 adhesin gene of M. pneumoniae (36). We used the 16S rRNA gene which is highly conserved in all Mycoplasma species and is the best target for genus-level detection of Mycoplasma spp. (37). Our finding demonstrated that ideal time and temperature for isothermal amplification was at 60˚C in 60 minutes while Davudi-Asl optimized the LAMP test using the large Bst enzyme fragment at 66˚C for 1 hour (36).
The primers designed in this study were theoretically completely specific for the 16S rRNA gene of Mycoplasma. Therefore, amplification was carried out only with DNA of Mycoplasma; also, neither falsepositive and false-negative results in the LAMP assay nor any cross-reactivity with other species was observed. In addition, the sequencing results were in accordance with deposited sequence of 16S rRNA in NCBI. These results demonstrated that this technique has high specificity (100%) for the amplification of Mycoplasma strains and detected it with high efficiency. It seems that the extremely high specificity of the LAMP method is a result of using four primers that recognize distinct regions on the target sequences (38). Furthermore, using the designed loop primers in the mixture could increase rapidity and efficiency of amplification by attaching to the stem loops formed during reaction process (39). In our study, the LAMP assay was able to detect Mycoplasma DNA extracted from culture medium which approves this assay for detection of Mycoplasma strains in cell culture samples; with the help of such assays, Mycoplasma infection can be discovered at an early stage.

Conclusion
Our study is the first report about designing and developing a LAMP assay based on 16S rRNA gene for determination of Mycoplasma strains contaminating cell culture. Based on our findings, LAMP assay developed based on 16S rRNA gene is highly suggested as a useful tool for rapid diagnosis of common Mycoplasma which contaminate cell culture.
LAMP Assay for Detection of Mycoplasma Contamination manuscript. M.S.; Is responsible for overall supervision, extensively contributed to data interpretation, drawing conclusions and revised the final version of the manuscript. K.M.-A.; Extensively contributed to data interpretation and drawing conclusions. All authors participated in the finalization of the manuscript and approved the final draft.