Hot Discovery Biology News and Publications
Target identification and validation are two key areas in the success of drug development. Keeping on top of the latest research findings can give you the competitive edge. Working with GenScript's highly trained scientists can also give you an advantage, delivering the results you need within the timeline and budget of your project.
Enjoy some red wine first before lighting your cigarettes
According to a recent article published in The American Journal of Medicine, healthy occasional smokers may benefit more from drinking one or two glasses of red wine before lighting up their cigarettes. The participants of this study have their blood and urine samples tested after drinking and smoking. The blood vessel damage is characterized by the level of micro-particles released after smoking. The results showed that pretreatment with red wine has preventive effects on the acute vascular and systemic inflammation, together with other indicators of cellular aging process induced by smoking. Red wine is well known for its antioxidant effects thanks to phenolic compounds, and moderate consumption of red wine is long deemed as beneficial to reduce the cardiovascular events. Drinking before smoking is also indicated to mediate the telomerase activity which further modulates the cell aging process. This set of data was observed only based on healthy occasional smokers and social drinkers, which means the benefits might not be applied to regular or heavy smokers and drinkers. Cheers!
Portable laboratory for high-throughput cancer biomarker diagnosis
A small, portable cancer biomarker high-throughput testing device can be extremely helpful in providing fast and reliable testing for point-of-care diagnosis. Patients will be able to receive test results right on-site such as at the doctor's office or even inside an ambulance. A multichannel smartphone spectrometer is used as an improved bio-sensor, which is capable of measuring samples in an assay format (ELISA or immunosorbent assay). The wavelength is suitable for the range of 400 to 700 nm, and protein concentrations (human cancer biomarkers) can be measured accurately. The detection limits, sensitivity, and accuracy can be comparable to the standard laboratory instrument. In this study, the smartphone used is iPhone 5 and human interleukin-6 (IL-6), (a well-known biomarker for lung, prostate, liver, breast, and epithelial cancers) is used to demonstrate this potential application.
Universal flu vaccine could be available within 5-year timeframe
How well a flu shot works each year depends on many factors, such as the age and health conditions of the person being vaccinated, and whether the vaccine virus is live or inactivated. Each year, WHO utilizes global surveillance data to try to predict which strain of the influenza is mostly likely to pose threat, providing the basis for corresponding flu vaccines for that particular year. The prediction is not always working out 100% correct, in the 2014-2015 season, the similarity of the vaccine virus and circulating virus are not working out well.
WHO sets a goal to have universal vaccine by 2020, it seems we are close to get it done within five-year timeframe as indicated in a recent article in PNAS on "broadly neutralizing antibody" and stimulating cellular responses though Fc receptor interactions. Epitope specificity plays an important role in regulating antibody-dependent cell-mediated cytotoxicity. In addition, it is further indicated that the communications among antibodies of different species determines the magnitude of effector functions.
Can tears be the alternative route for Zika virus transmission?
Zika virus is spreading at an astonishing rate which is much faster than what one would expect for mosquito-borne transmission route alone. Many Zika patients also develop eye disease since Zika virus can infect several different regions in the eye (including the retina), which could further lead to permanent vision loss. A recent study from Cell Reports revealed that tears might be a possible alternative route of Zika transmission. Zika virus is able to survive in the eye and the virus RNA has been identified in tears, so the eye could be a reservoir for the virus. Although no live virus can be detected from tears of the infected mice after 28 days of infection, ZIKV RNA can still be identified. There is a possibility that in human, Zika virus persist in cornea or other components in the eye which could be infectious. Studies in testing Zika virus in human is planned accordingly. Even if the human tears do not contain live Zika virus, it will be still beneficial to use this method as a fast and pain-free diagnostic toolfor Zika virus infection, without the need to take blood samples.
OX40L blockade: A promising treatment strategy for fibrosis induced by inflammation
Fibrotic diseases contribute to as much as 45% of deaths in developed countries without effective anti-fibrosis therapies. A recent study from PNAS revealed that T cell-targeted antibodies prevent inflammation induced fibrosis. The researchers identified that glycoprotein OX40L ligand (OX40L, member of the tumor necrosis factor superfamily) plays a key role in the fibrosis development driven by inflammation. OX40L has been known as a serum biomarker for lung and skin fibrosis, which also promotes fibroblast activation. Blocking OX40L has demonstrated to be a promising strategy for treating inflammation-induced fibrosis, which is shown to be capable of reversing the established dermal fibrosis, together with preventing lung, vessel, and skin fibrosis in in vivo systemic sclerosis models.
Excess antibody production in chronic infection triggered by parasites
High level of antibodies in the blood (hypergammaglobulinemia) is one of the features for autoimmune diseases and chronic infections. A study in Cell Reports revealed the mechanism of what are the causes of hypergammaglobulinemia. It also revealed, for the first time, how parasites can directly stimulate B cells and trigger excess antibody production. The parasite of interest, Leishmania donovani, is recognized by the endosomal toll-like receptors (TLRs) proteins, leading to the subsequent inflammation response. It is also found that TLRs induce secretion of cytokines including interleukin-10 proteins and type 1 interferons (IFN-1). IL-10 can reduce the immune response, while IFN-1 induces elevated B-cell antibody production. This activation pathway may also contribute in other immune pathologies.
Functional single domain antibody screening to identify targets preventing viral infection
Scientists usually rely on small molecules or genetic modifications approaches to inhibit protein function in cellular pathways. However, both methods have their inherent limits. Only a small portion (~15%) of the cellular pathway targets are able to be pinpointed by small molecule drugs, while unwanted phenotypes may be induced by genetic modification approaches. A recent study published in Nature Microbiology revealed a new screening strategy using single domain antibodies (sdAbs, VHH, or nanobodies) to point to targets preventing viral infection. These sdAbs are small in size (~13kDa) and highly specific for the binding targets, with the flexibility to engineer multi-specific and bi-specific antibodies. On top of serving as inhibitors or activators of intracellular protein function, sdAbs can also be applied in functional testing. A new screening approach is reported to identify therapeutic leads for influenza A virus (IAV) and vesicular stomatitis virus (VSV). This screening method can also be applied to identify any biological pathways or inhibitors of any pathogen. Both mRNA transcription and nuclear import of viral ribonucleoproteins can be mediated by antiviral sdAbs, thus novel antiviral reagents may be designed accordingly.
Reprogramming Tumor Associated Macrophages for Immunotherapy
Macrophages, supposed to protect the host from pathogens invasion, sometimes get tricked by tumors to form so-called tumor associated macrophages (TAMs). MicroRNAs produced by those TAMs assist tumor cells to hide from immune system. A recent study in Nature Cell Biology revealed how to regain control of those TAMs by reprogramming macrophages. This is achieved by removing the TAMs ability to produce a small family of microRNAs called Let-7, as a result, the immune system regains the capability to detect tumors. Furthermore, the reprogramming of TAMs can prevent tumors from leaving their primary site. Immune response against cancer could be enhanced by inhibiting the microRNA machinery, thus, it is promising to unleash the full potential of cancer immunotherapy. Applications on suppressing tumor growth and metastasis are also discussed in this article.
How antibodies activate a killer protein to trigger cell apoptosis
Unwanted cells in the body are removed by programmed cell death (apoptosis). Failure to do so may result in uncontrolled cell growth or cancer. Bak protein is critical in this apoptosis process: upon binding with BH3-only proteins, Bak is activated to kill cells. The binding groove on Bak has been well-characterized before. A recent study published in Nature Communications revealed a new mechanism of how antibodies directly activate this killer protein Bak via a different binding site on Bak, which further leads to apoptosis. Thus, combination therapy can be designed by targeting different binding sites. Furthermore, the antibody is capable of getting away with being neutralized by pro-survival proteins, suggesting this finding may help reduce the occurrence of drug resistance.
Mobilizing antibodies in neuronal tissues to control inflammation
Antibodies are able to circulate and access most tissues for pathogen elimination. But for brain and peripheral nervous system with "plasma proteins shield" (such as blood-brain or blood-nerve barrier), how do circulating antibodies gain access and control viral infection there? A recent study published in Nature revealed a previously unappreciated role of CD4 T cells and why it is crucial for antiviral antibodies to limit virus spread in peripheral sites of infection.
CTLA-4 found in dendritic cells suggests New cancer treatment possibilities
Both dendritic cells and T cells are important in triggering the immune response, whereas antigen presenting dendritic cells act as the "general" leading T cells "soldiers" to chase and eliminate enemies in the battle against cancer. The well-known immune checkpoint break, CTLA-4, is believed to be present only in T cells (and cells of the same lineage). However, a new study published in Stem Cells and Development suggests that CTLA-4 also presents in dendritic cells. It further explores the mechanism on how turning off the dendritic cells in the immune response against tumors.
GenScript has developed several cell-based immune-checkpoint functional assays to determine the immuno-modulatory profile of antibody leads, with following advantages:
- Physiological relevant endpoint readout (IL-2 release)
- Clinical-grade reference antibody as a positive control, when applicable
- Comprehensive data set: both mixed-lymphocyte reaction and co-culture of effector and engineered target cells are available for the assays
Non-invasive strategy to guide personalized cancer immunotherapy
Cancer immunotherapy is the rising hope to offer ultimate solutions for cancer. Neoantigens, derived from products of mutated genes in tumor cells, are found to be closely related to the efficacy of cancer immunotherapies. A non-invasive approach to identify unique, patient-specific neoantigens has been advanced by Dr. Steven Rosenberg's group. A recent article published in Nature Medicine reported that a small population of circulating CD8+PD-1+ tumor-reactive T lymphocytes can be used to identify neoantigens, in addition to tumor-infiltrating T cells. The study paves the way for designing personalized cancer immunotherapy with a novel non-invasive approach.
Syngeneic tumor models have been widely used in drug discovery research of cancer immunotherapies. To facilitate your discovery of cancer immunotherapeutics and study their efficacy when combining with other drugs, GenScript provides syngeneic tumor models and immune checkpoint blockers validated models. We will assist you with our expertise and experience in cancer immunotherapeutics to facilitate your drug discovery progress. More syngeneic tumor models validated with immune checkpoint inhibitors, humanized PD-1 and CTLA-4 knock-in mouse models, and detailed molecular characterization (such as immune signature of tumor infiltrating lymphocytes) will be available shortly in 2016.
CXCR7 activation overrides lung fibrosis
Fibrotic diseases contribute to as much as 45% of deaths in developed countries. In particular, idiopathic pulmonary fibrosis is a fatal disease characterized by worsening dyspnea and progressive loss of lung function with a median survival as short as 2 years. A recent study published on Nature Medicine suggested that the chemokine receptor CXCR7, expressed on pulmonary capillary endothelial cells (PCECs), plays important roles in lung fibrosis. Upregulation of the Notch ligand Jagged1, resulting from the suppression of CXCR7 expression, enhances the fibrosis. Thus administration of CXCR7 agonist reduces fibrosis and promotes alveolar repair. This investigation sheds light on the mechanisms underpinning lung fibrogenesis and demonstrates that the CXCR7 agonist could be a potential drug candidate in treating lung fibrosis.
To facilitate your fibrosis research and anti-fibrotic drug discovery, GenScript provides a comprehensive fibrotic disease model platform, including liver, kidney and lung fibrosis models in mice and rats. GenScript has extensive experience in establishing those models, as well as in the analysis of endpoints for lead efficacy evaluation.
Features of GenScript fibrosis model services:
- Multiple choices of model development approach
- Reproducible animal models validated with reference compounds
- Various existing and new approaches for histopathological analysis
- Extensive experience
- Timely updates
Protein Oxidation in Aging: Not All Proteins Are Created Equal
Cancer, Alzheimer's disease and other age-related diseases develop over the course of aging, and certain proteins are shown to play critical roles this process. Those proteins are subject to destabilization as a result of oxidation, which further leads to features of aging cells. It is estimated that almost 50% of proteins are damaged due to oxidation for people at their 80s. The oxidative damage mediated by free radicals occurs when converting food to energy in the presence of oxygen. Cellular structures, such as proteins, DNA, and lipids, are prone to these oxidation damages, which further contribute to the development of age-related diseases.
Using computational models with physics principles incorporated, de Graff el al. from Stony Brook University unfolded the molecular mechanism that how natural chemical process affects the aging of proteins. First, the authors revealed the major factor to explain stability loss in aging cells and organisms is likely to be random modification of the protein sidechains. Furthermore, through the evaluation and analysis on the protein electrostatics, the authors suggested that highly charged proteins are in particular subject to the oxidation induced destabilization. Even one single oxidation could lead to unfold the whole structure for these highly charged proteins. Old cells are enriched in those highly charged proteins, thus the destabilization effects are elevated in the aging cells. In addition, 20 proteins associated with aging are further identified to be at high risk of oxidation. The list includes telomerase proteins and histones, both of which play critical roles in the aging of cells and cancer development. The team is currently working on analyzing more proteins, with the hope to provide key information to aid targeted treatments against age-related diseases.
Further Reading: Emerging Opportunity for Treating Alzheimer Disease by Immunotherapy
Targeting Epithelial-to-mesenchymal Transition May Provide Avenue for the Treatment of Fibrosis
Kidney fibrosis, a common feature of virtually all chronic kidney diseases, results in progressive impairment of renal function and eventually leads to renal failure. There is currently no effective medication to treat kidney fibrosis. Epithelial-to-mesenchymal transition (EMT) of tubular epithelial cells (TECs) is known to be associated with kidney fibrosis, but its precise contribution and mechanism remain under debate. In a recent issue of Nature Medicine, two articles published back-to-back unveiled the detailed mechanism.
How does EMT of TECs drive kidney fibrosis?
The articles report that Snail- and/or Twist-driven partial EMT program of TECs limits cell repair and regeneration by inducing cell cycle arrest at G2/M phase. Instead of direct contribution to the myofibroblast population, the TECs undergone partial EMT relay crucial signals to the interstitium to markedly promote myofibroblast differentiation as well as inflammatory responses, and induce fibrogenesis in the kidney. Targeting EMT program attenuates experimentally induced kidney fibrosis in vivo. Consistent with the both studies, results in a previous report indicate that the finding may apply to other fibrotic diseases, such as liver fibrosis. Therefore, the studies paved way for novel anti-fibrotic therapies.
To facilitate your fibrosis research and anti-fibrotic drug discovery, GenScript provides a fibrotic disease model platform, including UUO-induced kidney fibrosis, liver fibrosis and lung fibrosis models. GenScript has extensive experiences in the establishment of the models and the analysis of endpoints for lead efficacy evaluation. At GenScript, we make every effort to improve human health by enabling scientists to expedite life science and biomedical research.
Anti-PD-1 is poised to be a blockbuster, which other immune-checkpoint targeting drugs are on the horizon?
Clinical studies of anti-immune-checkpoint protein therapeutics have shown not only an improved overall survival, but also a long-term durable response, compared to chemotherapy and genomically-targeted therapy. To expand the success of immune-checkpoint therapeutics into more tumor types and improving efficacy in difficult-to-treat tumors, additional targets involved in checkpoint-blockade need to be explored, as well as testing the synergy between combining approaches.
Currently, CTLA-4 and PD-1/PD-L1 are furthest along in development, and have shown very promising results in metastatic melanoma patients. This is just a fraction of targets involved in the checkpoint-blockade pathway. Several notable targets include:
- LAG-3 – Furthest along in clinical development with both a fusion protein and antibody approach, antibody apporach being tested in combination with anti-PD-1
- TIM-3 – Also in clinical development. Pre-clinical studies indicate that it co-expresses with PD-1 on tumor-infiltrating lymphocytes. Combination with anti-PD-improves anti-tumor response
- VISTA – Antibody targeting VISTA was shown to improve anti-tumor immune response in mice
In addition, there are also co-stimulatory factors that are also being explored as viable therapeutic targets
- OX40 – Both OX40 and 4-1BB are part of the TNF-receptor superfamily. Phase I data shows acceptable safety profile, and evidence of anti-tumor response in some patients
- 4-1BB - Phase I/II data on an antibody therapeutic targeting OX40 shows promising clinical response for melanoma, renal cell carcinoma and ovarian cancer.
- Inducible co-stimulator (ICOS) – Member of the CD28/B7 family. Its expression was found to increase upon T-cell activation. Anti-CTLA-4 therapy increases ICOS-positive effector T-cells, indicating that it may work in synergy with anti-CTLA-4. Clinical trials of anti-ICOS antibody are planned for 2015.
Discovering antibody therapeutics and new modalities requires high quality stable cell lines for antibody lead identification and characterization. GenScript has developed stable cell lines expressing many of the hot immune-checkpoint targets including PD-1/PD-L1, CTLA-4, LAG-3 and TIM-3 among many others. See the full list of the recombinant immune-checkpoint protein cell lines, and review case studies of our new immune-checkpoint assay services.
With a wide range of animal models to choose from, what are the crucial factors to consider?
Despite advances in the understanding of molecular pathways and discovery of new genes involved in tumorigenesis, the rate of new oncology drug approvals remains the lowest among all disease areas. Part of this gap is that the animal models used for measuring efficacy in vivo are not translating into clinical success.
A recent perspective published in Nature Medicine addresses these gaps by comparing the strengths and limitations of different tumor models, as well as best models to use for answering different biological questions and best practices for preclinical modeling.
Below is a summary of the authors' key considerations:
- It is important to choose a model based on the biology of the target. Several diverse tumor models may be required to address complex biology
- If the biology of the target includes signaling between the tumor and the stroma, then it is crucial to understand drug efficacy in the presence of an appropriate tumor microenvironment with orthotopic models
- Avoid overuse of models that are highly sensitive to the drug, unless there is clinically relevant biomarker data to support the findings
- For studying agents that reduce pre-existing tumors, make sure that the tumors are established in the model prior to treatment
- Understanding the pharmacokinetics of a drug in the model prior to studies is important to ensure that the dosing is within range, and that off-target and toxic side effects are not skewing anti-tumor activity
GenScript provides several tumor models to study different biological pathways involved in tumorigenesis including:
- Subcutaneous xenograft - A fast, and cost-efficient model to study tumor cell-derived autocrine signaling
- Orthotopic tumor models - A technically challenging model that involves the appropriate tumor microenvironment, contributing to drug response. Also good for studying spontaneous tumor metastasis. GenScript has extensive technical experiences in orthotopic models and has developed 35 tumor cell lines expressing luciferase, facilitating the analysis of tumor growth on internal organs through bioluminescent imaging
- Syngeneic tumor models - reliable models for studying immune checkpoint modulators such as anti-CTLA-4, PD-1 and PD-L1 therapeutics
- Patient-derived (PDX) tumor models – Patient-derived tumors closely retain the histological characteristics and genetic profile of a specific patient's tumor. GenScript has over 250 PDX models available, with whole exome sequencing and genetic annotation available for some models
FDA approves new antibody drug for treating pediatric neuroblastoma
Pediatric neuroblastoma is a rare and difficult to treat cancer that forms from immature nerve cells. This form of cancer occurs in 1 in 100,000 children, with 650 new cases each year in the United States. Current therapies, which are non-specific, only provide 40-50% long term survival rate to patients suffering from high-risk neuroblastoma, making this form of cancer an area of high medical unmet need.
A new drug, called dinutuxumab was granted priority review and orphan drug designation by the FDA. It is the first drug of its kind to be approved that specifically treats pediatric neuroblastoma. In addition to the approval, the FDA also issued a rare pediatric review priority voucher to the makers of the drug, for future groundbreaking therapies in pediatric neuroblastoma.
Dinutuxumab (formerly called ch14.18) is a disialoganglioside (GD2) binding chimeric monoclonal antibody that works in combination with granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-2 (IL-2), and 13-cis-retinoic acid (RA) for treating high-risk pediatric neuroblastoma.
Antibody therapeutics are highly efficacious and specific towards rare and difficult-to-treat cancers and discovery of new antibody therapeutics will help address critical needs. Antibody drug discovery may be challenging, but working with an experienced partner can help.
GenScript has over 5 years' experience working with clients to deliver antibody therapeutic leads. GenScript is one of few biotech companies to provide single domain antibodies, which may be effective in conjunction with chimeric antigen receptor T cell therapies (CAR-T). Additionally, GenScript also is highly experienced developing conventional monoclonal antibodies. We are also one of few biotech companies that offer antibody drug screening in ADCC & CDC in vitro assays, as well as tumor animal models for validating antibody leads, offering our clients a one-stop solution.
Smaller Cas9 allows for more efficient viral delivery method to improve in vivo and potential clinical applications
Applying genome engineering to research and clinical applications has been greatly simplified with CRISPR-Cas9 genome engineering. Although this system is quite versatile and easily implemented in mammalian systems, one major limitation has been the effective delivery of Cas9 protein in vivo.
Currently, the cas9 protein widely used in research is derived from Streptococcus pyogenes (SpCas9). Its relatively large size (~4.2 kb) limits its use with more efficient viral vectors such as adeno-associated virus (AAV), which can only carry cargo of up to 4.5 kb. In a recent publication, scientists characterized a new variant of Cas9, isolated from Staphylococcus aureus (SaCas9) and found it to be highly efficient and specific both in vitro and in vivo.
Benefits of SaCas9:
- SaCas9 is only ~3.2 kb, compared to SpCas9 which is ~4.2 kb
- SaCas9 cleaves NNGRRT PAMs most efficiently, although all NNGRR PAMs can be cleaved as well
- The smaller size allows SaCas9 and the sgRNA to be efficiently packaged into AAV, which is more efficient for in vivo delivery
Additionally, exciting results came from an in vivo study where SaCas9 and sgRNA targeting proprotein convertase subtilisin/kexin type 9 (Pcsk9), were successfully delivered virally into the mouse liver. >40% knockout of Pcsk9 was achieved, which lead to a ~95% reduction in serum Pcsk9 and ~40% decrease in total cholesterol.
Prior to performing studies in vivo, the authors tested sgRNAs in cells to ensure efficient knockout of Pcsk9, and to profile for any undesired off-target effects, using a whole genome strategy called BLESS.
Studying the effects of CRISPR-mediated genome editing in cells is a critical step in target validation. Understanding the effects of gene knockout in a cellular model can streamline in vivo studies. GenScript offers CRISPR-based knock-out and knock-in cell line development. Viral delivery methods ensure that even difficult-to-transfect cell lines can efficiently undergo CRISPR-genome editing.
See exciting GenCRISPR™ cell line development case studies
Receptor implicated in ERBB2 inhibitor resistance. Is it the next therapeutic target?
Currently, treatment options for HER2 positive breast cancer include ERBB2 inhibitors, trastuzumab (a humanized monoclonal antibody) and lapatinib (a small-molecule inhibitor). A major clinical hurdle develops when patients become resistant to first-line therapeutics. There are fewer treatment options and their outcome is significantly worse.
Understanding the mechanisms involved in ERBB2 inhibitor resistance can lead to more effective therapeutic agents. In a recent published study, scientists discovered the receptor involved in keeping ERBB2+ lapatinib-resistant cells alive.
ERBB4 – a receptor whose role in breast cancer was previously unclear, was found to be involved in preventing apoptosis in lapatinib-resistant breast cancer cell line (BT474-LR). However, overexpression of ERBB4 active mutants in BT474 did not rescue the cells from lapatinib-induced apoptosis, suggesting that ERBB2+ cells shift their dependence from ERBB2 to ERBB4 when they acquire resistance.
Further understanding of the mechanisms involved in ERBB4 activation and dependence in lapatinib-resistant cells is needed to find effective treatments against ERBB2+ cancers that have become resistant to current therapies.
GenScript is committed to supporting target validation and drug discovery in oncology. With over 5 years' experience and successful delivery of projects to our oncology research clients, we can engineer cancer cell lines to express one or more tumorigenic receptors. We also offer extensive in vivo oncology models, for testing therapeutic compounds.
Targeting single immune-checkpoint proteins has proven to be clinically effective at treating specific tumor types; can targeting two different proteins synergize effects?
Despite the success of targeting immune-checkpoint proteins, such as CTLA-4, PD-1, LAG-3, TIM-3 among others, percentages of patient response vary and rarely exceed 50%. It is highly tempting to speculate a strategy of dual-targeting of these checkpoint proteins. A recent presentation at the Keystone Symposium for Tumor Immunology: Multidisciplinary Science Driving Combination Therapy detailed findings of dual-targeting two immune-checkpoint proteins in mouse tumor models. Their key findings are summarized below:
- Dual-targeting PD-1 and LAG-3 demonstrates superior efficacy over blocking either target alone
- In addition to previous reported data on superior dual-targeting efficacy against fibrosarcoma (Sa1N) and colorectal adenosarcoma (MC38) tumor types1, anti-tumor activity against myeloma (SC J558L) and B-cell lymphoma (A20) hematological tumor types were also reported to be effacious.2
These exciting pre-clinical findings may result in further exploration of dual-targeting antibodies in the clinic, either as combination of existing antibody therapies, or as a new bi-specific antibody therapeutic.
Camelid single domain antibodies are a novel bi-specific antibody platform that may be used to develop a new generation of dual-targeting antibodies against multiple immune-checkpoint proteins.
GenScript has over 5 years' experience in developing novel single domain antibodies as therapeutic agents or tool reagents for research purposes.
Review case studies on different applications of single domain antibodies »
An end to CRISPR/Cas9 off-target concerns? Whole-genome sequencing reveals a new gRNA design
As CRISPR-Cas9 continues to revolutionize biomedical research, it is important to address the concern over off-target effects. Several previous published studies determined that gRNAs with up to 5 mis-matches may cause thousands of off-target cuts and proposed solutions of using paired Cas9 nickase or catalytically dead Cas9 fused with Fok1 nuclease along with paired gRNAs. While these methods have reduced frequency of off-target mutations, it is unknown whether these paired Cas9 solutions are truly free of off-target effects genome-wide.
In a current published study, scientists developed a new method to determine Cas9 off-target cuts in a genome-wide manner. Digenome-seq can identify Cas9 cleavage sites via an in vitro digest of genomic DNA with Cas9 and subsequent sequencing using the same 5' ends. The lead authors' study determined the following:
Whole-genome sequencing of CRISPR-Cas9 genome edited haploid cells (HAP1) and wild-type HAP1 cells did not pick up any off-target indels with five different targets in five different knock-out clones.
To address whether low-frequency mutations in a bulk cell population can still persist, the authors developed digenome-seq to specifically sequence regions where Cas9 has cleaved. These results showed that homologous sites with 3-4 mis-matches had 21 out of 157 (13%) identified sites cleaved, while homologous sites with 5-6 mis-matches had 1 out of 7,896 (0.013%) identified sites cleaved, indicating a much lower than previously estimated off-target mutation rate.
Analysis of gRNAs and their off-target mutation rates revealed that gRNAs starting with two guanines (ggX20) can reduce off-target effects by orders of magnitude, without sacrificing on-targeting.
Careful design of gRNAs, optimized delivery methods and thorough sequence validation, especially of top off-target sites, are crucial in CRISPR-Cas9-based cell line development.
GenScript's scientists use a custom-built bioinformatics tool to carefully select gRNAs and use efficient viral-based delivery of CRISPR-Cas9. We also provide an off-target report, and offer additional sequencing at the request of the customer.
Learn more about GenCRISPR cell line services »
Breakthrough: Drug resistance gene uncovered in major testicular cancer genetics study
Testicular germ cell tumors (TGCTs) which make up the majority of testicular cancers remains an area of need. Treatment options for testicular germ cell cancers are limited, and are associated with side effects such as metabolic syndromes and possible infertility. In addition, once a patient becomes resistant to drug treatment, the chance of long-term survival is drastically reduced.
Deeper understanding of the mechanisms behind testicular cancer is an important first step in developing improved next-generation therapeutics. A recent published study on a large-scale genetic screening of TGCT patients identified several genetic mutations strongly correlated with testicular cancer, as well as a gene that may be critical in combatting drug resistance. Below are a summary of these genes:
- Among these genes, increased rate of mutations in KIT were observed, confirming previous studies which correlated this oncogene with TGCT.
- In drug-resistant TGCT, a gene called XRCC2 was found to be highly mutated. XRCC2 is a DNA repair gene, previously found to promote cisplantin resistance in mice
High-quality stable cell lines and in vivo models are critically important for target identification and validation, as well as for early drug discovery.
GenScript is an industry leader in cell line engineering. In addition to constructing recombinant gene-expressing stable cell lines by conventional means, we also offer GenCRISPR™ CRISPR/Cas9-based cell line service for gene-knockout or targeted integration of wild-type and mutant genes.
In addition to oncology in vitro pharmacology services, GenScript also offers oncology animal models validated with clinical grade reference drugs. The services have been beneficial and well regarded by our customers.
Breaking news: FDA grants Fast Track status for two different drugs against fibrotic liver disease
Non-alcoholic steatohepatitis (NASH) contributes to fibrotic liver disease, which may be come irreversible and lead to liver transplants. NASH is a growing indication, with very few effective treatments and has been given orphan disease designation by the FDA.
Two new drugs starting Phase 2b proof of concept in the clinic were recently granted Fast Track status by the FDA, in hopes of expediting the development and review process, so that patients may benefit sooner.
- Tobira Therapeutics' Cenicriviroc works by blocking CCR5, thus reducing inflammation and can block HIV entry. It is currently in the clinic for both liver disease and HIV,
- Intercept Pharmaceuticals' Obeticholic Acid (OCA) is a bile acid analog, and acts as an FXR agonist. It has the potential to treat NASH as well as primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC).
Despite well validated preclinical targets, anti-fibrotic agents that have entered early phase clinical studies have not advanced due to difficulties in measuring fibrosis progression or reversal.
Understanding the in vivo profile of anti-fibrotic agents requires robust animal models. GenScript is one of very few biotech companies offering CCl4-induced and bile-duct ligation models of liver fibrosis. The quality of the services has been well recognized by our customers in the pharmaceutical industry.
Review a case study of CCl4-induced liver fibrosis »
New off-target detection method, GUIDE-seq, identifies additional properties of off-target sequences in CRISPR genome editing
CRISPR-Cas9 is a major advancement in genome editing, and much simpler to implement than previous methods such as TALENS and ZFN. From cancer researchers to plant biologists, many have already adopted this genome editing technology in their labs, and new therapeutics are being discovered with CRISPR-Cas9. Designing the guide RNA is as simple as finding a sequence on your gene of interest with a requisite PAM sequence (NGG)… Or is it?
Several reports this past year have addressed whether CRISPR-Cas9 can cleave at off-target sites. A new report, from Tsai et al., describes a more thorough genome-wide profiling of off-target sites, called GUIDE-seq. This method is improved over previous off-target profiling methods, because it is able to identify more double stranded break (DSB) sites, and more thorough sequencing of the region around the DSB. GUIDE-seq involved integration of double stranded oligo-deoxiribonucleic acids (dsODN) at the DSB. The dsODN carries a tag that that is used for amplification of the DSB region for next-generation sequencing (NGS).
This new detection method yielded several major findings:
- gRNA specific characteristics, such as the number of mismatches with off-target sites, position of mismatches and type of mismatch greatly influence detection of DSB via GUIDE-seq.
- Genome specific characteristics, such as density of proximal PAM sequences, gene expression level and whether the sequence is intergenic, intronic or exonic does not alter frequency of DSB as detected by GUIDE-seq
- There are "hotspots" on the genome, which can integrate dsODN even without Cas9 nuclease
- DSBs on certain regions of the genome can cause genomic rearrangement, including translocations, inversions and large deletions
CRISPR-Cas9 genome edited cell lines are a critical tool and can greatly speed research and drug discovery. GenScript offers GenCRISPR™, a one-stop CRISPR solution including gRNA design and construct service, taking off-target characteristics into consideration, and GenCRISPR™ cell line services where we can generate a fully customized knock-out or knock-in cell line.
Since launching GenCRISPR™, GenScript's CRISPR/Cas9-based precision cell-engineering service, we have successfully delivered 100% of the cell lines ordered by our initial customers. We are now extending our service to a broader research community, by offering 30% off our current prices until the end March 2015. Visit our GenCRISPR™ Custom Cell Line webpage for additional details.
Single domain antibodies (sdAbs) aid in x-ray crystallography of mammalian serotonin 5-HT3 receptor
Serotonin 5-HT3 is part of the cys-loop receptor family, the mechanism of this family is not well understood due to difficulties in obtaining high resolution crystal structures. Serotonin 5-HT3 receptor is an important druggable target in alleviating nausea and vomiting induced by chemotherapy or anesthesia, as well as psychiatric disorders. It's structure is critical in discovering new drugs to modulate its activity.
Previously, electron microscopy imaging of non-mammalian homologs of Cys-loop receptors provided basic understanding of extracellular ligand binding sites and pore forming domains. Little was known about intracellular domains and the way they interact with cellular scaffolding proteins, as they are absent in non-mammalian homologs. A recent publication in Nature extends our understanding behind the mechanism of serotonin 5-HT3 receptors, by resolving a 3.5A crystal structure.
Mouse 5-HT3 exists as a homopentamer and is difficult to express, purify and crystallize. To overcome this challenge, researchers split the receptor by proteolyzing each subunit into two fragments. In addition, an sdAb chaperone, which acts as an inhibitor locking the channel into a non-conducting conformation, was used to stabilized the pentameric structure, enabling resolution of a 3.5A crystal structure. Most importantly the split receptor displays an intracellular domain that is tightly coupled to the membrane domain, which provides important structural information that will lead to further understanding of the physiological conformation of 5-HT3 and Cys-loop receptors.
GenScript's scientists have over 5 years experience of generating single domain antibodies for therapeutic and research purposes. We have generated crystallography chaperone sdAbs against GPCRs, another difficult to crystallize class of proteins.
New insight behind the success of fighting cancer by targeting immune checkpoint proteins
Immune checkpoint blockade has proven to be highly successful in the clinic at treating aggressive and difficult-to-treat forms of cancer. The mechanism of the blockade, targeting CTLA-4 and PD-1 receptors which act as on/off switches in T cell-mediated tumor rejection, is well understood. However, little is known about the tumor antigen recognition profile of these affected T-cells, once the checkpoint blockade is initiated.
In a recent published study, the authors used genomics and bioinformatics approaches to identify critical epitopes on 3-methylcholanthrene induced sarcoma cell lines, d42m1-T3 and F244. CD8+ T cells in anti-PD-1 treated tumor bearing mice were isolated and fluorescently labeled with tetramers loaded with predicted mutant epitopes. Out of 66 predicted mutants, mLama4 and mAlg8 were among the highest in tetramer-positive infiltrating T-cells. To determine whether targeting these epitopes alone would yield similar results as anti-PD-1 treatment, vaccines against these two epitopes were developed and tested in mice. Prophylactic administration of the combined vaccine against mLama4 and mAlg8 yielded an 88% survival in tumor bearing mice, thus demonstrating that these two epitopes are the major antigenic targets from checkpoint-blockade and therapies against these two targets are similarly efficacious.
In addition to understanding the mechanism, identification of these tumor-specific mutant antigens is the first step in discovering the next wave of cancer immunotherapies via vaccines or antibody therapeutics. Choosing the right antibody platform can speed the discovery of a new therapeutics against these new targets. Single domain antibodies have the advantage of expedited optimization, flexibility of incorporating multiple specificity and functions, superior stability, and low COG over standard antibody approaches.
GenScript has over 5 years of experience in developing single domain antibodies, with expertise to develop high affinity single domain antibodies for therapeutic use.
Uncover genes involved in drug resistance with CRISPR-generated mutant cell lines
Drug resistance is a major concern when developing compounds for treating cancers. Forward genetic screening was previously carried out in yeast, for ease of genetic manipulation. Now, with the introduction of CRISPR technology, more relevant results can be generated in cancer cell lines. Mutant libraries of specific and multiple genes can be generated quickly and in a cost-effective manner for screening drug resistance.
A current published study highlights the utility of CRISPR in constructing a mutant library of haploid myelogenous leukemia cells, KBM7, for use in a forward genetic screen to isolate and identify mutants resistant to anti-cancer drug, 6-thioguanine (6-TG). Through exome sequencing of resistant mutants, the authors discovered that all mutants contained nonsense mutations in the single hypoxanthine phosphoribosyltransferase (HPRT1) allele. HPRT1 is a key enzyme involved in converting 6-TG into a toxic form.
The authors also used this method to validate a potential target for triptolide, a potent antiproliferative agent, whose mechanism of action is not well understood. CRISPR was used to introduce an array of mutations on the ERCC3 allele, which encodes a protein required for polymerase II function and shown previously to bind to triptolide. From the screen, the authors discovered specific mutations on ERCC3 and GTF2H4 (a binding partner for ERCC3), that increased resistance and identified key regions contributing to drug resistance include the DNA binding domain on ERCC3 and interaction domains on ERCC3 and GTF2H4. These findings build the foundation that ERCC3 is a target for triptolide, and that this method can be applied to finding new targets for oncology drug discovery.
GenScript offers GenCRISPR™, our CRISPR/Cas9-based precision cell-engineering service. We have successfully delivered 100% of cell lines ordered by our initial customers, and have over 240 tumor cell lines available for CRISPR-based genome editing. We are offering 30% off our current prices until the end of 2014. Visit our GenCRISPR™ custom cell line services webpage for additional details.
Patient-derived (PDX) models aid in predictive medicine
New oncology drugs often fail in phase III trials due to lack of efficacy and safety. Animal models that are insufficient at predicting clinical outcomes are part of this disconnect between preclinical success and late phase failures. To solve this problem, patient-derived xenograft (PDX) mouse models are now being implemented in the discovery of new treatments as well as determining efficacy or resistance of current therapies for personalized medicine.
A recent review, written by the EurOPDX Consortium, highlights the predictive power of PDX models. Direct clinical correlation of treatment between PDX models and human patients were observed for human breast cancer, non-small cell lung cancer and pancreatic ductal adenocarcinoma. These studies support the accuracy of PDX models for drug screening, biomarker development and strategizing personalized medicine.
GenScript offers human PDX models and primary cancer cell lines from 14 different origin tissues. Our PDX services include:
- Clinical grade oncology drugs as positive controls
- Full data report including tumor weight and volume, histopathology analysis of sequential passages and exome sequencing data for select tumor cell lines.
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Can CRISPR cure cervical cancer?
In the battle against cervical cancer, the focus has been mainly on prevention, through the development of a highly effective vaccine to stop new human papilloma viral infections (HPV). Now, with CRISPR/Cas9, there is a way to potentially cure individuals already infected with HPV, who are suffering from cervical cancer.
The oncogenes involved in cervical cancer are HPV viral genes, E6 and E7, which may become integrated in the host cell genome. These viral oncogenes can cause cervical carcinomas through degradation of host cell tumor suppressors, p53 and retinoblastoma (Rb). A key to killing cervical carcinoma is to effectively eliminate viral oncoprotein expression in the host cell. And researchers have successfully implemented CRISPR/Cas9, an RNA-guided endonuclease system revolutionizing genome alteration, to turn off these oncoproteins. In this paper, scientists discover that delivering Cas9/sgRNA expression vectors by transfection or lentiviral transduction can permanently inactivate the E6 or E7 gene in HPV transformed cells, in vitro. Future in vivo studies will determine whether this approach is feasible to carry out safely in the clinic.
Proof-of-principle studies require well-validated CRISPR/Cas9-generated custom cell lines. Since launching GenCRISPR™, our CRISPR/Cas9 based custom cell line service, GenScript has successfully delivered 100% of the cell lines. In addition, we provide services to validate the function of GenCRISPR™ developed cell lines.
Camelid single domain antibodies aid as crystallography chaperones
Structural biology answers some of the most fundamental questions about proteins and uncovers new ways to effectively design highly specific drugs against the protein target of interest. Despite major advances in this field, there are still hurdles in obtaining high quality crystals for x-ray crystallography.
One of the challenges, particularly with membrane bound proteins and transmembrane receptors, is the ability to obtain a well ordered crystal. In a report by Tereshko et al., camelid single domain antibodies, specifically the variable heavy chain (VHH) domains can be used to aid in scaffolding of structures. In this study, the incorporation of selenomethionine (SeMet) into the β-sheet core greatly improved phase quality and both single and multi-wavelength anomalous dispersion (SAD and MAD) data. The authors were able to isolate this specific VHH by generating a combinatorial library and performing selection using yeast surface display.
GenScript is one of the few biotech company's offering single domain antibody services. Our 5 years of experience include developing single domain antibodies against GPCRs, a class of proteins that have traditionally been difficult to crystallize. We can quickly isolate VHH's specific to GPCRs using our naïve libraries and panning against phage display.
FDA approves Contrave, an obesity drug combining bupropion and naltrexone, an opioid receptor antagonist
GPCRs are a popular class of target for metabolic, inflammatory, and many other diseases. The newly approved Contrave is a combination of bupropion, a drug previously approved as an antidepressant, and naltrexone, an opioid receptor antagonist previously approved for the management of alcohol and opioid dependence. In Phase III trials, Contrave showed clinical efficacy for weight loss in both obese patients, and patients with type 2 diabetes.
Why aren't there more GPCR-targeting drugs on the market?
Approval of small-molecule drugs targeting GPCRs been few and far between due to undesirable central nervous system side effects including depression, anxiety, and suicidal tendencies. A potential solution is to develop antibody drugs targeting GPCRs, which would have the advantage of minimal trafficking across the blood brain barrier, due to its size and known properties.
GenScript's scientists have extensive experience in lead generation of GPCR-targeting antibodies, via traditional hybridoma, phage and yeast display, or single-domain antibody approaches. GenScript is your ideal partner for antibody drug discovery for GPCR and other therapeutic targets.
Parental exposure to methoxychlor may cause transgenerational increase of adult onset diseases
An example of epigenetics at work was highlighted in a recent study on the inheritability of pesticide exposure. The study, conducted in rats, showed that methoxychlor, an approved pesticide with previously observed toxic effects in animals, can cause an increased incidence of kidney disease, ovary disease and obesity in their F3 progeny.
The study also investigates the DNA methylation signature using a genome-wide rat promoter array to determine which gene clusters would be affected by methoxychlor exposure. Their findings associate 311 average epimutations with many genes associated in several key disease pathways including metabolic, cancer, and endocrine.
Understanding the role of epigenetics in diseases may help us discover more therapeutics, and GenScript is pleased to provide epigenetic screening and profiling services. Available for screening are a comprehensive panel of HDACs and SIRTs, major drug discovery targets implicated in cancer, neurodegeration, inflammation and immune diseases.
Antibody therapeutic approach to combating Ebola virus
The current Ebola virus (EBOV) outbreak, taking its toll in Western Africa, is the worst since its discovery in 1976. The World Health Organization (WHO) has concluded that it is ethical to use unproven drugs and vaccines to stop the spread of this deadly virus. Currently, the most promising experimental therapeutic is a monoclonal antibody cocktail, Zmapp™, developed by Mapp Biopharmaceutical. This monoclonal cocktail consists of three antibodies, each recognizing different, non-overlapping EBOV glycoprotein epitopes1.
Discovery of these antibodies began in mice, where a key finding involving the glycosylation state of the antibody led to enhanced protective efficacy2. Chimerization with human constant region, or humanization, allowed for clinical use.
GenScript is a contributor to the development of these much needed therapeutics by providing antibody engineering services to Mapp Biopharmaceutical. We are dedicated to providing critical services in the development of vaccines and antibody therapeutics against EBOV and other severe viral outbreaks, such as avian influenza.
Targeting Fibroblast Activation Protein in Tumor Stroma With Chimeric Antigen Receptor T-cell Therapy May Provide an Effective and Safe Approach to Reducing Tumor Growth In-vivo
Chimeric antigen receptor (CAR) T-cell therapies are touted as the next generation of highly selective, anti-cancer therapeutics against leukemia, lymphomas, breast cancer and GD2-positive sarcomas. While CAR T-cell therapy is highly targeted towards the above mentioned tumor types, it can still affect normal cells endogenously expressing tumor antigens. To make CAR T-cell therapies more useful and safer, researchers are constantly searching for “cleaner” ways of applying CAR T-cell therapy. Recently, investigators identified fibroblast activation protein (FAP) as a potential target for CAR T-cell therapy based on its preferential expression in tumor stromal cells. Tumor stromal cells were known to be critically required to support the growth of their associated tumors. When FAP-CAR T-cells were tested in syngeneic mouse tumor models, the cells were demonstrated to be highly effective in reducing the growth of several types of epithelial tumor. The investigators also noted the absence of signs concerning safety. This finding may potentially lead CAR T-cell therapy to become a therapy for a broad spectrum of cancers.
Low Rate of Coronary Disease Linked to LOF Mutations in APOC3: The 4th Case of Human Genetics-based TIDV
In a recent exome sequencing study, investigators discovered that human carriers of a loss-of-function (LOF) mutation on APOC3 gene had dramatically lower triglyceride levels and significant decreased risk of coronary disease. This is the fourth case of disease target validation based on human genetics. Similar studies have provided solid proof for the “targetability” of zinc ion transporter protein ZnT8 for type 2 diabetes, sodium ion channel NaV1.7 for neuropathic pain and itch, and PCSK9 for hypercholesterolemia. These exciting findings are milestone examples of a major revolution in drug discovery: using human genetics data to drive target identification and validation (TIDV).
Recent advances in sequencing technology have improved the detection of rare DNA variant sequences in large populations of individuals. This has improved the correlation of disease state with human genetic data. Study newly identified and validated targets with GenScript's one-stop discovery services!
Single domain antibodies shown to cross blood brain barrier and offers enhanced delivery of therapeutics to CNS targets
A major challenge in developing both small molecule and antibody therapeutics for CNS disorders including brain cancer and neurodegenerative diseases, is penetrating the blood brain barrier (BBB). A study published in FASEB demonstrated that monomeric variable heavy-chain domain of camel homodimeric antibodies (mVHH), can cross the BBB in-vivo, and recognize its intracellular target: glial fibrillary acidic protein (GFAP). The ability of mVHH to cross the BBB of normal animals and those undergoing pathological stress makes it a promising modality for treating CNS diseases as well as for brain imaging.
The investigators of this study expressed a recombinant fusion protein, VHH-GFP, which was able to cross the BBB in-vivo and specifically label astrocytes. GenScript is fully engaged in single-domain antibody lead generation and optimization. With our one-stop services, we are determined to be your best partner in antibody drug discovery from gene synthesis to in-vivo characterization of candidate antibodies. All you need to provide is the Genbank accession number of the antigen protein!
Monoclonal Antibody Targeting Nav1.7 Channel Voltage Sensor Can Relieve Pain and Itch
In a recent study published in Cell, a monoclonal antibody was shown to be highly effective at blocking the function of a genetically validated therapeutic target for pain: sodium (Na) channel Nav1.7. The antibody was reported to be 400-1500 fold selective for Nav1.7 compared to other subtypes of the voltage-gated Na channels. Testing of the antibody in mouse models revealed that the antibody was capable of suppressing inflammatory pain, neuropathic pain, and even pruriceptive itch. The finding opened a way of effectively treating pain and itch without the side effect of addiction. It is also a major technical breakthrough of developing antibody modulators for one of the most difficult classes of targets: ion channels and transporters.
The unique mechanism of action of this antibody involved locking the voltage-sensor paddle into a closed position by targeting the loop domain between the 3rd and 4th helical transmembrane domains on the α-subunit. Using the approach of locking the voltage-sensor paddle may enable development of subtype-specific modulators for the study of ion channel function and development of therapeutics against ion channel related diseases.