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Translational Medicine - Accelerate the R&D process of innovative drugs and precision medicine

2022-12-28
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Translational medicine focuses on biomarkers in the drug development process, and aims to improve the clinical response rate of drug development with precision medicine.  It covers from early target confirmation - preclinical R&D - clinical Phase I, II, III Development, to post-marketing drug testing, through different stages of research to achieve a closed loop of drug development.

The Medicilon translational medicine platform has an experienced professional technical team, starting from the mechanism of action of drug targets and the clinical application of biomarkers, based on the discovery and validation of biomarkers, combining a variety of different technology platforms and state-of-art instruments, and creating multiple bioanalytical methods, to reduce the cost and time of drug R&D, and provide diverse and efficient services for different types of drugs, different kinds of pharmaceutical companies and projects at different R&D stages.

With the continuous development of multi-omics analysis technologies such as genomics, proteomics and metabolomics, therapeutic methods have expanded from traditional small molecules to new technologies such as peptides, proteins, antibodies, gene therapy, and cell therapy.  Despite these new technologies, the causes of a large number of diseases are still not fully understood.

Translational medicine transforms biomedical observation and research into interventions to improve health, accelerates the process of basic research, new drug development and clinical transformation, and becomes an accelerator for precise targeted therapy.  Translational medical research utilizes various research methods to determine the relationship between the target and the occurrence and development of the disease, validate and explore the mechanism of action of the drug, discover biomarkers and develop companion diagnostic products, and screen the most suitable population and indications for clinical research to improve the efficiency and success rate of new drug R&D.

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Overlaying translational medicine milestones into drug development[1]

Biomarker Research Platform

Biomarkers usually refer to indicators that can be objectively measured and evaluated, reflect physiological or pathological processes, and have biological effects on exposure or therapeutic interventions.  Biomarkers are mostly derived from human tissues or body fluids, and can cover physiological, biochemical, immune, cellular and molecular changes.  It can improve biomarker discovery and verification services based on genomics, proteomics, cytomics, pathomics and other multi-omics, providing scientific support for biomarker research, and multi-level support for clinical trials of new drug development.  As the most direct, rapid and effective diagnostic method, biomarkers can play an important role in many aspects such as disease diagnosis, development, treatment, and efficacy monitoring.  Biomarkers are DNA-based, RNA-based, and protein-based. Different molecules and proteins require different technology platforms.  With the continuous development of high-throughput genomics or proteomics, more types of biomarkers are included, such as SNP, exosomes, miRNA, and lncRNA have been included in the ranks of biomarkers.  Currently, a variety of technology platforms have been applied to biomarker research, such as omics platforms including genomics, proteomics, peptidomics, and metabolomics, as well as nanotechnology, bioinformatics, antibody chips, and high-content screening technology.  A variety of cutting-edge technologies, including label-free interaction analysis technology, have brought great possibilities for the rapid acquisition and screening of biomarkers.

Classification and Use of Biomarkers

 Diagnostic Biomarkers: Used to detect or confirm disease states, or identify different disease subtypes.

Prognostic Biomarkers: Reflect the prognostic characteristics of the disease, the risk of disease recurrence or progression.

Predictive Biomarkers: Used to predict that patients may respond to a certain treatment or intervention.

Pharmacodynamic Biomarkers: Reflect the biological response of patients after receiving treatment.

Safety Biomarkers: Monitor before or during medication to avoid or reduce adverse reactions in patients.

Monitoring Biomarkers: Monitor changes in disease status (such as recurrence).

Examples of Medicilon Biomarkers

PD1/PD-L1

ErbB2/HER2

p-FGFR1/FGFR2/FGFR3、p-ERK、p-CREB、p-AKT

EGFR

VEGF

p53

Cyclin D1

COX-2

Cytokeratin 7(CK7)

K-Ras

SOX2

MET

Fas

ER-α

Ki-67, etc.

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Medicilon Case Study

Biacore T200

WBC100 is a novel orally effective molecular glue that can selectively degrade c-Myc protein while having no activity on other proteins, and effectively kill c-Myc overexpressed cancer cells.  SPR results showed that WBC100 binds to c-Myc protein in a dose-dependent manner.  This SPR experiment was performed by Medicilon using a Biacore T200 instrument.

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Surface plasmon resonance (SPR)[2]

Biacore-8K

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8 needles and 16 flowcells, study 8 targets at the same time;

High-throughput, 500 compounds/day;

Fast detection speed,finish one affinity and kinetic test in 2-15 minutes.

PD-1 and VEGF Binding Assay

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A: Binding kinetics of different doses of Nivolumab with PD-1 (Biacore 8K)

B: Binding kinetics of different doses of Aflibercept with recombinant human VEGF (Biacore 8K)

Bioanalysis Platform

The Medicilon Bioanalysis Department could provide clients with screening and development of small molecule drugs, macromolecular biological products, biomarkers, as well as preclinical and clinical stage research services.  Bioanalysis services for biotech drugs that comply with FDA/NMPA GLP could be provided to support the early development of protein drugs, antibody drugs, vaccines, biomarkers, cell and gene therapy drugs, as well as their preclinical research and clinical research.

Service Capabilities

 Development and methodological validation of immunoassay methods

Analysis of proteins, antibodies, ADCs, peptide drugs, nucleic acids, vaccines, cell and gene therapy products, etc.

Screening, validation and analysis of biomarkers, cytokine detection

Anti-drug Antibody (ADA) Immunogenicity Analysis

Immunogenicity Analysis of Vaccines

Virus Activity Analysis

Potency Determination of Vaccines

Bioanalysis of Clinical Samples

Support PK pharmacokinetics, TK toxicokinetics, tissue distribution test, IND declaration

Service Highlights

The laboratory implements comprehensive information management, using the verified laboratory information management system (Watson LIMS 7.2) to establish a complete sample management chain and experimental data processing, tracking and storage chain

SensaTronics temperature monitoring system

Proven WinNonlin software for data analysis

The data is accepted by FDA and NMPA, providing biotechnology drug analysis services that fully comply the requirements of FDA/NMPA/OECD GLP regulations

Independent small molecule bioanalysis platform and biotechnology drug analysis platform

Possess a full range of multifunctional technical platforms such as SpectraMaxM4/M5/i3x, MSD, Luminex, Biacore 8K, Envision, Gyrolab, ABI7500 qPCR, ddPCR, FACS

Flexible use of ELISA, ECL, IP, Co-IP, qPCR, ddPCR, FACS, Elispot, enzymology, Cell-based and other methods to support cutting-edge biological drugs, including proteins, antibodies, ADCs, peptides, nucleic acids, vaccines and early development of cell gene therapy drugs, as well as preclinical research and clinical research

Developed and validated analysis methods for nearly a hundred different targets, such as CD-4, CTLA-4, PD-1, PD-L1 and T-DM1 analog ADC, etc., supporting PK/TK/immunogenicity (Total ADA, Nab)/biomarker/cytokines analysis

Part of Instruments of Biological Analysis Platform

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Medicilon Case Study: BE Research on Clinical Small Peptide Molecules

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Flow Cytometry Platform

In previous articles, we introduced the "Medicilon's Flow Cytometry Platform" in detail from the perspective of multiple cases of FACS detection, Cytokine detection and cell function detection (click for more details).  Until now, Medicilon has undertaken nearly 400 flow cytometry projects, including flow cytometry detection of cell surface antigen expression; flow cytometry analysis of cell proliferation, cell differentiation, cell apoptosis; detection of animal peripheral blood and various immune organs; and flow cytometric detection of transplanted tumor models.  The Medicilon flow testing team has extensive practical and technical, carefully submits high-quality experimental results to clients, and continues to help clients get approval for their R&D projects.

Immunohistochemical Technology Platform

Immunohistochemistry, also known as immunocytochemistry, refers to the application of the basic principle of immunology, that is, the principle of specific combination of antigen and antibody, through chemical reaction to make the chromogen of the labeled antibody develop color to determine the antigen (polypeptide and protein) in the tissue cells, and to locate, qualitative and relative quantification research.  According to the principle of antigen-antibody reaction and chemical color development, the antigen in the tissue section or cell sample is first combined with the primary antibody, and then the primary antibody is used to react with the secondary antibody, DAB is used for color development, and then carried out the analysis.

Main Steps

Tissue Processing, Fixation, Sectioning

Antigen Retrieval

Removal of Endogenous Peroxidase

Blocking

Primary and Secondary Antibody Incubation

Detection

Counterstaining

Tissue Processing, Fixation, Sectioning

Tissue fixation preserves antigens and prevents autolysis and necrosis of harvested tissue.  Tissue embedding provides support to the tissue during slicing, making the slice more solid.

 paraffin sectionfrozen slice
fixedBefore embedding: formaldehydeBefore or after sectioning: formaldehyde, methanol, ethanol, or acetone
sliceslicercryostat
storeStore at room temperature for many yearsone year at -80 °C (longer at -190 °C)
AdvantageEasy to handle without damaging slices

♦ Retains enzyme function and antigenicity

♦ Short protocol (usually does not require lengthy fixation steps)

limitation

♦ Overfixation can mask epitopes, increasing the need for antigen retrieval

♦ Long processing time: Gradual dehydration in graded alcohol and xylene to facilitate paraffin penetration.

♦ Without rapid freezing of tissue" ice crystals may form, disrupting tissue structure

♦ Frozen sections are usually thicker than paraffin sections, which may result in lower resolution and poorer images

♦ It may be necessary to block endogenous active enzymes.

Paraffin Sectioning vs Frozen Sectioning

Antigen Retrieval

Antigen retrieval is performed on formaldehyde-fixed tissue sections to expose antigenic sites for antibody binding.

 heat-induced epitope retrievalProteolytic enzyme-induced epitope retrieval
AdvantageRetrieval of antigenic epitopes is gentler with more controllable parameters.Suitable for antigenic epitopes that are difficult to retrieve.
pH valueTypically a pH 6 buffer is used, but alkaline buffers are also widely used. must be determined experimentallyThe pH is usually 7.4.
temperatureAbout 95°C.Typically 37°C
incubation time10-20 minutes10-15 minutes
buffer componentsDepends on the desired pH of the target antigen. Commonly used buffers include sodium citrate, EDTA, and Tris-EDTANeutral buffer for enzymes such as pepsin, proteinase K, or trypsin
PrecautionsMicrowave heating may result in uneven antigen retrieval. Vigorous boiling can cause debonding (separation of tissue from slide).Enzyme repair sometimes disrupts section morphology - concentration and time need to be optimized

The Main Method of Antigen Retrieval

Blocking

Blocking with serum or BSA prevents non-specific binding of antibodies and reduces background and potential false positive results.

Protein blocking: Blocking with serum or BSA is critical to prevent non-specific binding of the antibody to tissue or to Fc receptors (receptors that bind to the antibody constant region (Fc)).  Serum from the species of the secondary antibody is a good blocking reagent.  Bovine serum albumin (BSA) or casein can be used to block nonspecific antibody binding.

Biotin Blocking: When using an avidin/biotin based detection system, blocks endogenous biotin as it is present in many tissues, especially kidney, spleen, liver and brain.  Incubate the tissue with avidin to block endogenous biotin, followed by incubation with exogenous biotin to block additional biotin binding sites on the avidin molecule.

Detection

Blocking with serum or BSA prevents non-specific binding of antibodies and reduces background and potential false positive results.

Enzyme Chromogenic Method: Chromogenic detection uses enzymes that can catalyze soluble substrates to produce colored precipitates.  These enzymes are usually conjugated to secondary antibodies and can also be conjugated to primary antibodies for direct detection.  The most commonly used enzymes are HRP, which converts DAB to a brown product, and AP, which converts 3-amino-9-ethylcarbazole (AEC) to a red product.  Chromogenic detection is generally more sensitive than fluorescent detection.  In addition, unlike fluorescent dyes, colored precipitates are photostable, so stained sections can be preserved for many years.Fluorescence detection requires the use of professional fluorescent microscopes and filters, while chromogenic detection only requires the use of standard microscopes.  However, chromogenic assays require more and longer incubation and blocking steps than fluorometric assays.

Fluorescence method: Fluorescence detection (immunofluorescence) is based on the characteristic that the fluorophore emits fluorescence with a longer wavelength after being excited by a specific wavelength of light.  Fluorescence detection is often used in situations where simultaneous detection of multiple antigens is required.  Fluorescent dyes can be directly conjugated to primary or secondary antibodies, or to streptavidin.

Case Studies

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IHC Analysis of the expression of a)PD-L1 from lung adenocarcinoma[3]; b)Ki-67 from periampullary tumors[4]; c)Her2 from lung tumor[5]; d)CD31 from human gastric adenocarcinoma[6]; e)CD163 (M2 TAM marker) from oral squamous cell carcinoma (OSCC)[7]; f)FoxP3 from human glioblastoma[8].

Summary and Outlook

Comprehensive translational medicine platform based on genomics, proteomics, cytomics and pathomics and high-quality R&D management team; Medicilon translational medicine platform is committed to providing global partners with comprehensive biomarker discovery, target integrated solutions for point-of-care verification, companion diagnostic development, and commercial testing.

Protein interaction and protein level biomarker platform constructed by ELISA, ECL (MSD), SIMOA (HD-X), Biacore 8K technology;

Cell-level biomarker platform based on flow cytometry (BD Symphony A3, BD Fortesssa, Beckman CytoFLEX S);

 Multiple nucleic acid-level biomarker platform constructed with fluorescent quantitative PCR technology;

Pathological level biomarker platform constructed by immunohistochemistry (TAMs-IHC, FISH) technology.

Medicilon committed to solving the difficulties in the development of innovative drugs and helping precision medicine!  We are fortunate to live in an age of seemingly limitless possibilities in biomedical science.  In this era, research questions are no longer primarily limited by technical capabilities. The realization of translational medicine requires equally sustained and bold vision and execution.  In the past few decades, translational medicine has made remarkable progress, and in the future, translational medicine will continue to develop and provide more treatment possibilities for more patients faster and more efficiently!

References

[1] Hugues Dolgos, et al. Translational Medicine Guide transforms drug development processes: the recent Merck experience. Drug Discov Today. 2016 Mar;21(3):517-26. doi: 10.1016/j.drudis.2016.01.003.

[2] Ying Xu, et al. A Selective Small-Molecule c-Myc Degrader Potently Regresses Lethal c-Myc Overexpressing Tumors. Adv Sci (Weinh). 2022 Mar;9(8):e2104344. doi: 10.1002/advs.202104344.

[3] Jonas J Heymann, et al. PD-L1 expression in non-small cell lung carcinoma: Comparison among cytology, small biopsy, and surgical resection specimens. Cancer Cytopathol. 2017 Dec;125(12):896-907. doi: 10.1002/cncy.21937.

[4] Mark M Aloysius, et al. Predictive value of tumor proliferative indices in periampullary cancers: Ki-67, mitotic activity index (MI) and volume corrected mitotic index (M/V) using tissue microarrays. World J Surg. 2010 Sep;34(9):2115-21. doi: 10.1007/s00268-010-0681-3.

[5] Montse Verdu, et al. Cross-reactivity of EGFR mutation-specific immunohistochemistry assay in HER2-positive tumors. Appl Immunohistochem Mol Morphol. 2015 Sep;23(8):565-70.

[6] Qingling Wang, et al.  EPCR promotes MGC803 human gastric cancer cell tumor angiogenesis in vitro through activating ERK1/2 and AKT in a PAR1-dependent manner. Oncol Lett. 2018 Aug;16(2):1565-1570. doi: 10.3892/ol.2018.8869.

[7] Faustino J Suárez-Sánchez, et al. Macrophages in Oral Carcinomas: Relationship with Cancer Stem Cell Markers and PD-L1 Expression. Cancers (Basel) (IF: 6.13; Q1). 2020 Jul 2;12(7):1764. doi: 10.3390/cancers12071764.

[8] Qi Yue, et al. The prognostic value of Foxp3+ tumor-infiltrating lymphocytes in patients with glioblastoma. J Neurooncol. 2014 Jan;116(2):251-9. doi: 10.1007/s11060-013-1314-0. Epub 2013 Nov 26.[9] Christopher P Austin.Opportunities and challenges in translational science. Clin Transl Sci. 2021 Sep;14(5):1629-1647. doi: 10.1111/cts.13055.

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