Impaired proliferation and migration of HUVEC and melanoma cells by human anti-FGF2 mAbs derived from a murine hybridoma by guided selection.

Disadvantages of using murine monoclonal antibodies (mAb) in human therapy, such as immunogenicity response, led to the development of technologies to transform murine antibodies into human antibodies. The murine anti-FGF2 3F12E7 mAb was proposed as a promising agent to treat metastatic melanoma tumors; once it blocks the FGF2, responsible for playing a role in tumor growth, angiogenesis, and metastasis. Considering the therapeutic potential of anti-FGF2 3F12E7 mAb and its limited use in humans due to its origin, we used this antibody as the template for a guided selection humanization technique to obtain human anti-FGF2 mAbs. Three Fab libraries (murine, hybrid, and human) were constructed for humanization. The libraries were phage-displayed, and the panning was performed against recombinant human FGF2 (rFGF2). The selected human variable light and heavy chains were cloned into AbVec vectors for full-length IgG expression into HEK293-F cells. Surface plasmon resonance analyses showed binding to rFGF2 of seven mAbs out of 20 expressed. Assays performed with these mAbs resulted in two that showed proliferation reduction and cell migration attenuation of HUVEC and SK-Mel-28 melanoma cells. In-silico analyses predicted that these two human anti-FGF2 mAbs interact with FGF2 at a similar patch of residues than the chimeric anti-FGF2 antibody, comprehending a region within the heparin-binding domains of FGF2, essential for its function. These results are comparable to those achieved by the murine anti-FGF2 3F12E7 mAb and showed success in the humanization process and selection of two human mAbs with the potential to inhibit undesirable FGF2 roles.

The guided selection humanization process enabled the production of 20 human mAbs anti-FGF2;Seven human anti-FGF2 mAbs showed binding to the rFGF2 antigen in the SPR binding assay;Two human anti-FGF2 mAbs inhibited the proliferation and migration of HUVEC and SK-Mel-28 cells and were predicted to contact the FGF2 at a similar patch of residues than the original mAb.

The contribution of genotype-guided selection of P2Y12 inhibitor on prognosis in ACS /CCS patients undergoing percutaneous coronary intervention: a retrospective cohort study.

PURPOSE: We aimed to explore the contribution of genotype-guided selection of P2Y12 inhibitors on prognosis in Chinese patients with acute coronary syndromes (ACS) or chronic coronary syndromes (CCS) undergoing percutaneous coronary intervention (PCI). METHODS: Totally, 2063 patients were included. They were divided into empiric treatment group (n = 1025) and individualized treatment group (n = 1038) depending on whether taken CYP2C19 genetic testing. The incidences of clinical endpoint events were compared in two groups at 1-year follow-up. The effective endpoint events were major adverse cardiovascular events (MACEs), including all-cause mortality, in-stent restenosis, nonfatal myocardial infarction, nonfatal stroke and severe recurrent ischemia. Meanwhile, the safe endpoint was bleeding events defined by the Bleeding Academic Research Consortium (BARC) criteria. RESULTS: Finally, 66.83% patients were diagnosed with ACS and 33.17% patients were diagnosed with CCS in empiric group. 68.11% patients were diagnosed with ACS and 31.89% patients were diagnosed with CCS in individualized group. At 1-year follow-up, individualized group showed lower MACEs rate than empiric group (19.61% vs. 10.69%, HR: 1.915; 95% CI: 1.534 to 2.392; P < 0.0001, log-rank test; adjusted HR: 1.983; 95% CI: 1.573 to 2.501; P = 0.000, cox proportional hazards regression models), while bleeding events were significantly less common in empiric group than in individualized group (7.32% vs. 10.40%, HR: 0.693; 95% CI: 0.519 to 0.926; P = 0.0132, log-rank test; adjusted HR: 0.695; 95% CI: 0.518 to 0.933; P = 0.016, cox proportional hazards regression models). It was mainly manifested in BARC class 1 bleeding, which did not warrant the interruption of antiplatelet therapy (ITA). Further, subgroup analyses illustrated that no significant difference existed in cumulative MACEs-free survival rate between all treatment arms of individualized group (P = 0.6579 by log-rank test), and CYP2C19 intermediate metabolizer (IM) genetype appeared to be significantly associated with bleeding events for patients treated with ticagrelor (clopidogrel vs. ticagrelor: 6.80% vs. 14.88%; adjusted HR:0.440; 95% CI: 0.246 to 0.787; adjusted P = 0.006). CONCLUSIONS: Genotype-guided selection of P2Y12 inhibitor made a very positive contribution on the prognosis in Chinese ACS/CCS patients undergoing PCI. Instead of intensifying antiplatelet strategies, conventional-dose clopidogrel could be recommended as P2Y12 inhibitor after weighing MACEs and bleeding events in CYP2C19 IM patients.

Role of platelet function and genetic testing in patients undergoing percutaneous coronary intervention.

Dual antiplatelet therapy (DAPT) represents the standard of care for patients undergoing percutaneous coronary intervention (PCI). Increasing evidence indicates that a "one-size-fits-all" approach with the use of a standard DAPT regimen for all patients undergoing PCI could lead to either suboptimal efficacy or prohibitively high bleeding in specific cohorts of patients. Moreover, the broad interindividual variability in response to P2Y12 inhibitors can impact outcomes and resource utilization. Among the strategies proposed to provide a more balanced trade-off between bleeding and ischemic events at a single patient level, a guided selection of P2Y12 inhibitors, by using platelet function or genetic testing, has shown promising results. In this review, we provide a focused summary of the rationale and evidence on the use of platelet function and genetic testing-guided antiplatelet therapy, and we explore the implications for their use in the modern setting of patients undergoing PCI.

Discovery of new PKN2 inhibitory chemotypes via QSAR-guided selection of docking-based pharmacophores.

Serine/threonine-protein kinase N2 (PKN2) plays an important role in cell cycle progression, cell migration, cell adhesion and transcription activation signaling processes. In cancer, however, it plays important roles in tumor cell migration, invasion and apoptosis. PKN2 inhibitors have been shown to be promising in treating cancer. This prompted us to model this interesting target using our QSAR-guided selection of docking-based pharmacophores approach where numerous pharmacophores are extracted from docked ligand poses and allowed to compete within the context of QSAR. The optimal pharmacophore was sterically-refined, validated by receiver operating characteristic (ROC) curve analysis and used as virtual search query to screen the National Cancer Institute (NCI) database for new promising anti-PKN2 leads of novel chemotypes. Three low micromolar hits were identified with IC50 values ranging between 9.9 and 18.6 µM. Pharmacological assays showed promising cytotoxic properties for active hits in MTT and wound healing assays against MCF-7 and PANC-1 cancer cells.

Impaired proliferation and migration of HUVEC and melanoma cells by human anti-FGF2 mAbs derived from a murine hybridoma by guided selection

Disadvantages of using murine monoclonal antibodies (mAb) in human therapy, such as immunogenicity response, led to the development of technologies to transform murine antibodies into human antibodies. The murine anti-FGF2 3F12E7 mAb was proposed as a promising agent to treat metastatic melanoma tumors; once it blocks the FGF2, responsible for playing a role in tumor growth, angiogenesis, and metastasis. Considering the therapeutic potential of anti-FGF2 3F12E7 mAb and its limited use in humans due to its origin, we used this antibody as the template for a guided selection humanization technique to obtain human anti-FGF2 mAbs. Three Fab libraries (murine, hybrid, and human) were constructed for humanization. The libraries were phage-displayed, and the panning was performed against recombinant human FGF2 (rFGF2). The selected human variable light and heavy chains were cloned into AbVec vectors for full-length IgG expression into HEK293-F cells. Surface plasmon resonance analyses showed binding to rFGF2 of seven mAbs out of 20 expressed. Assays performed with these mAbs resulted in two that showed proliferation reduction and cell migration attenuation of HUVEC and SK-Mel-28 melanoma cells. In-silico analyses predicted that these two human anti-FGF2 mAbs interact with FGF2 at a similar patch of residues than the chimeric anti-FGF2 antibody, comprehending a region within the heparin-binding domains of FGF2, essential for its function. These results are comparable to those achieved by the murine anti-FGF2 3F12E7 mAb and showed success in the humanization process and selection of two human mAbs with the potential to inhibit undesirable FGF2 roles.

Yeast Mating as a Tool for Highly Effective Discovery and Engineering of Antibodies via Display Methodologies.

Yeast surface display (YSD) is a powerful methodology for discovery and engineering of antibodies, and the yeast mating has been used to overcome low transformation efficiency of yeast in antibody library generation. We developed an optimized method of yeast mating for generating a large, combinatorial antibody fragment library and heterodimeric protein library by cellular fusion between two haploid cells carrying different library each other. This method allows for increased diversity in screening of target-specific fragment antigen-binding (Fab) antibodies as well as in the development of heterodimeric Fc variants for bi-specific antibody generation and T-cell receptor (TCR). Here we describe the efficient isolation of human antibodies against the activated GTP-bound form of the oncogenic Ras mutant (KRasG12D-GTP) by sequential isolation of their heavy chains (HCs) followed by combination with light chains (LCs) via the yeast mating process. This strategy facilitates guided selection of the antigen-specific HC with either a fixed functional LC, which has cytosol penetrating ability, or an LC library to generate the Fab. It also allows for deeper exploration of a sequence space with fixed diversity, leading to a higher probability of successful isolation of human antibodies with high specificity and affinity.

Clinical and economic impact of 'ROS1-testing' strategy compared to a 'no-ROS1-testing' strategy in advanced NSCLC in Spain.

BACKGROUND: Detection of the ROS1 rearrangement is mandatory in patients with advanced or metastatic non-small cell lung cancer (NSCLC) to allow targeted therapy with specific inhibitors. However, in Spanish clinical practice ROS1 determination is not yet fully widespread. The aim of this study is to determine the clinical and economic impact of sequentially testing ROS1 in addition to EGFR and ALK in Spain. METHODS: A joint model (decision-tree and Markov model) was developed to determine the cost-effectiveness of testing ROS1 strategy versus a no-ROS1 testing strategy in Spain. Distribution of ROS1 techniques, rates of testing, positivity, and invalidity of biomarkers included in the analysis (EGFR, ALK, ROS1 and PD-L1) were based on expert opinion and Lungpath real-world database. Treatment allocation depending on the molecular testing results was defined by expert opinion. For each treatment, a 3-states Markov model was developed, where progression-free survival (PFS) and overall survival (OS) curves were parameterized using exponential extrapolations to model transition of patients among health states. Only medical direct costs were included (€ 2021). A lifetime horizon was considered and a discount rate of 3% was applied for both costs and effects. Both deterministic and probabilistic sensitivity analyses were performed to address uncertainty. RESULTS: A target population of 8755 patients with advanced NSCLC (non-squamous or never smokers squamous) entered the model. Over a lifetime horizon, the ROS1 testing scenario produced additional 157.5 life years and 121.3 quality-adjusted life years (QALYs) compared with no-ROS1 testing scenario. Total direct costs were increased up to € 2,244,737 for ROS1 testing scenario. The incremental cost-utility ratio (ICUR) was 18,514 €/QALY. Robustness of the base-case results were confirmed by the sensitivity analysis. CONCLUSIONS: Our study shows that ROS1 testing in addition to EGFR and ALK is a cost-effective strategy compared to no-ROS1 testing, and it generates more than 120 QALYs in Spain over a lifetime horizon. Despite the low prevalence of ROS1 rearrangements in NSCLC patients, the clinical and economic consequences of ROS1 testing should encourage centers to test all advanced or metastatic NSCLC (non-squamous and never-smoker squamous) patients.

MLb-LDLr: A Machine Learning Model for Predicting the Pathogenicity of LDLr Missense Variants.

Untreated familial hypercholesterolemia (FH) leads to atherosclerosis and early cardiovascular disease. Mutations in the low-density lipoprotein receptor (LDLr) gene constitute the major cause of FH, and the high number of mutations already described in the LDLr makes necessary cascade screening or in vitro functional characterization to provide a definitive diagnosis. Implementation of high-predicting capacity software constitutes a valuable approach for assessing pathogenicity of LDLr variants to help in the early diagnosis and management of FH disease. This work provides a reliable machine learning model to accurately predict the pathogenicity of LDLr missense variants with specificity of 92.5% and sensitivity of 91.6%.

Enhanced production of clavulanic acid by improving glycerol utilization using reporter-guided mutagenesis of an industrial Streptomyces clavuligerus strain.

Clavulanic acid (CA) produced by Streptomyces clavuligerus is a clinically important ß-lactamase inhibitor. It is known that glycerol utilization can significantly improve cell growth and CA production of S. clavuligerus. We found that the industrial CA-producing S. clavuligerus strain OR generated by random mutagenesis consumes less glycerol than the wild-type strain; we then developed a mutant strain in which the glycerol utilization operon is overexpressed, as compared to the parent OR strain, through iterative random mutagenesis and reporter-guided selection. The CA production of the resulting S. clavuligerus ORUN strain was increased by approximately 31.3% (5.21 ± 0.26 g/l) in a flask culture and 17.4% (6.11 ± 0.36 g/l) in a fermenter culture, as compared to that of the starting OR strain. These results confirmed the important role of glycerol utilization in CA production and demonstrated that reporter-guided mutant selection is an efficient method for further improvement of randomly mutagenized industrial strains.

Selection of a Full Agonist Combinatorial Antibody that Rescues Leptin Deficiency In Vivo.

Growth factor deficiency in adulthood constitutes a distinct clinical syndrome with significant morbidities including abnormal body composition, reduced energy, affective disturbances, dyslipidemia, and increased cardiovascular risk. Protein replacement therapies using recombinant proteins or enzymes represent the only approved treatment. Combinatorial antibodies have shown great promise as a new class of therapeutic molecules because they act as "mechanism-based antibodies" with both agonist and antagonist activities. Using leptin, a key hormone in energy metabolism, as an example, a function-guided approach is developed to select combinatorial antibodies with high potency and full agonist activity that substitute natural growth factors in vivo. The identified antibody shows identical biochemical properties and cellular profiles as leptin, and rescues leptin-deficiency in ob/ob mice. Remarkably, the antibody activates leptin receptors that are otherwise nonfunctional because of mutations (L372A and A409E). Combinatorial antibodies have significant advantages over recombinant proteins for chronical usage in terms of immunological tolerance and biological stability.

Discovery of new JNK3 inhibitory chemotypes via QSAR-Guided selection of docking-based pharmacophores and comparison with other structure-based pharmacophore modeling methods.

The kinase c-Jun N-terminal Kinase 3 (JNK3) plays an important role in neurodegenerative diseases. JNK3 inhibitors have shown promising results in treating Alzheimer's and Parkinson's diseases. This prompted us to model this interesting target via three established structure-based computational workflows; namely, docking-based Comparative Intermolecular Contacts Analysis (db-CICA), pharmacophore modeling via molecular-dynamics based Ligand-Receptor Contact Analysis (md-LRCA), and QSAR-guided selection of crystallographic pharmacophores. Moreover, we compared the performances of resulting pharmacophores against binding models generated via a newly introduced technique, namely, QSAR-guided selection of docking-based pharmacophores. The resulting pharmacophores were validated by receiver operating characteristic (ROC) curve analysis and used as virtual search queries to screen the National Cancer Institute (NCI) database for promising anti-JNK3 hits of novel chemotypes. Eleven nanomolar and low micromolar hits were identified, three of which were captured by QSAR-guided docking-based pharmacophores.

Dimerization of an aptamer generated from Ligand-guided selection (LIGS) yields a high affinity scaffold against B-cells.

Nucleic Acid Aptamers (NAAs) are a class of synthetic DNA or RNA molecules that bind specifically to their target. We recently introduced an aptamer termed R1.2 against membrane Immunoglobulin M (mIgM) expressing B-cell neoplasms using Ligand Guided Selection (LIGS). While LIGS-generated aptamers are highly specific, their lower affinity prevents aptamers from being used for translational applications. Highly specific aptamers with higher affinity can increase targetability, boosting the application of aptamers as diagnostic and therapeutic molecules. Herein, we report that dimerization of R1.2, an aptamer generated from LIGS, leads to high affinity variants without compromising the specificity. Three dimeric aptamer analogues with variable linker lengths were designed to evaluate the effect of linker length in affinity. The optimized dimeric R1.2 against cultured B-cell neoplasms, four donor B-cell samples and mIgM-positive Waldenström's Macroglobulinemia (WM) showed specificity. Furthermore, confocal imaging of dimeric aptamer and anti-IgM antibody in purified B-cells suggests co-localization. Binding assays against IgM knockout Burkitt's Lymphoma cells utilizing CRISPR/Cas9 further validated specificity of dimeric R1.2. Collectively, our findings show that LIGS-generated aptamers can be re-engineered into dimeric aptamers with high specificity and affinity, demonstrating wide-range of applicability of LIGS in developing clinically practical diagnostic and therapeutic aptamers.

Generation by phage display and characterization of drug-target complex-specific antibodies for pharmacokinetic analysis of biotherapeutics.

Anti-idiotypic antibodies play an important role in pre-clinical and clinical development of therapeutic antibodies, where they are used for pharmacokinetic studies and for the development of immunogenicity assays. By using an antibody phage display library in combination with guided in vitro selection against various marketed drugs, we generated antibodies that recognize the drug only when bound to its target. We have named such specificities Type 3, to distinguish them from the anti-idiotypic antibodies that specifically detect free antibody drug or total drug. We describe the generation and characterization of such reagents for the development of ligand binding assays for drug quantification. We also show how these Type 3 antibodies can be used to develop very specific and sensitive assays that avoid the bridging format. Abbreviations: BAP: bacterial alkaline phosphatase; CDR: complementarity-determining regions in VH or VL; Fab: antigen-binding fragment of an antibody; HRP: horseradish peroxidase; HuCAL®: Human Combinatorial Antibody Libraries; IgG: immunoglobulin G; LBA: ligand binding assay; LOQ: limit of quantitation; NHS: normal human serum; PK: pharmacokinetics; VH: variable region of the heavy chain of an antibody; VL: variable region of the light chain of an antibody.

The Screening of Human Anti-Hepatoma Fd Fragments Guided with the Chemeric Light Chain of HAb18 / 中国肿瘤生物治疗杂志

Objective: To select anti-HAb18G (hepatoma associated antigen) human Fd fragments with guided selection of L chain of chimeric Fab-HAb18. Methods: The human Fd repertories genes were amplified by RT-PCR from PBMC of hepatoma patients, and cloned into the vector pComb3X with chimeric L chain to construct the human-mouse hybrid Fab phage library. HAblSGE, extracellular region of HAblSG, was used as antigen to screen. The positive clones was obtained by ELISA and FCM with p Ⅲ-fusion Fab antibody. The DNA sequences were analyzed. Results: A human-mouse Fab antibody library were constructed with 2?107 PFU. After 6 rounds panning, 7 positive clones were obtained with ELISA and FCM. And sequences of 2 clones with better affinity were same. The CHI belonged to the IgG2 class as the parent Fd, and the variable region belonged to VH3 family. Conclusions: Through the construction of the HuMFab phage antibody library and chemeric L chain-guided selection, we get the available human Fd fragments for subsequent research.