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1 Drug Development Services USING BLOOD AND BONE MARROW PRIMARY CELL SYSTEMS Clinically Relevant In Vitro Assays Broad Spectrum of Drug Classes Multi-Species Platforms Enhancing Drug Development through Primary Cell Biology

2 PRIMARY CELL PLATFORMS IN VITRO IMMUNOLOGY ASSAYS Cytokine Storm and Cytokine Release Syndrome (CRS) Cancer Immunotherapy Toll-Like Receptor (TLR) / PAMP Recognition Autoimmune Activation Inflammation Regulation IN VITRO HEMATOLOGY ASSAYS Bone Marrow Toxicity Cancer Stem Cells Mobilization Studies OTHER ASSAYS Cytokine and Mimetic Testing Cell Bank Production ReachBio Research Labs offers a wide selection of other services by utilizing a variety of primary cell-based in vitro assays and in vivo services. Please visit our website () to review our comprehensive service and product platforms. We operate strictly through a fee-for-service model in which the client owns all new intellectual property generated. 2

3 Cytokine Storm and Cytokine Release Syndrome (CRS) Evaluate and Rank the Potential Risk of Your Drug Candidates to Induce Cytokine Release or Cytokine Storm with our In Vitro Assays Human cells are more relevant at predicting risk of cytokine storm or CRS than in vivo primate models due to nuanced immunological differences between species Better understand the risk of non-specific immune cell activation prior to non-human primate (NHP) studies Four different test article presentations are available Endothelial cell co-culture Soluble Air dried Wet bound Additionally, we can address your specific research needs with a customized approach. Advantages We use fresh or frozen human primary cells; NHP cells available upon request Multiple positive and negative controls ensure scientific validity and accuracy of results Quantified multiplex outcome for pro-inflammatory cytokines using the Luminex platform Simultaneous monitoring of proliferative response Compatible with flow cytometry and multiplex mrna or DNA analysis Cancer Immunotherapy Assays Evaluate and Rank Each Drug Candidate s Ability to Recruit Both T Cells and NK Cells in Order to Kill Cancer Cells Compatible with bi-specific antibodies and other immune cell recruiters Measure primary immune cell killing of target cells Flow cytometry based readouts with additional intracellular analysis Simultaneous monitoring of contributing cytokine or chemokine factors also available Effector-mediated T-cell killing assay of target cells in the presence of a therapeutic antibody Therap.Ab (pg/ml) ET Ratio ,000 20,000 5,000 1,

4 Toll-Like Receptor (TLR) / PAMP Recognition Assays Assess the Ability of Your Compounds to Activate or Suppress the Innate Immune System TLRs are a class of well-characterized proteins that play a key role in the innate immune system. TLRs are types of pattern recognition receptors (PRR) and recognize molecules that are broadly shared by pathogens but distinguishable from host molecules, collectively referred to as pathogen-associated molecular patterns (PAMPs). TLR agonists have shown value for the treatment and/or prevention of infectious and neoplastic diseases, while TLR antagonists may be used to treat a number of inflammatory conditions including sepsis, skin diseases, rheumatoid arthritis and systemic lupus erythematosus. Assays utilize selective stimulation of Toll-Like receptors by bacterial lipoproteins, bacterial lipopolysaccharides, small molecules (viral simulation), or DNA repeat fragments (bacterial or viral) TLR 1/2 TLR 2/6 TLR 4 TLR 7/8 TLR 9 Pam3CSK4 (Diacylated Lipoprotein) Pam2CSK4 (Triacylated Lipoprotein) Lipopolysaccharide Salmonella minnesota R595 CL075 (Thiazoloquinolone derivate) ODN2216 (CpG-DNA) Compounds are monitored for either inhibition or induction of TLRs We use fresh or frozen human primary cells from healthy donors Quantified multiplex readout for pro-inflammatory cytokines using the Luminex platform Autoimmune Activation Model Address Your Compounds Efficacy in Suppressing the Pro-inflammatory Activity of Th17 Cells Despite their normal functionality as anti-microbial immunity regulators, Th17 cells (T-helper cell subpopulation) are known to promote inflammation and tissue damage in autoimmune diseases. Chronic inflammation via Th17 cytokines is implicated in a variety of disorders including rheumatoid arthritis, systemic lupus erythematosus, asthma, psoriasis, multiple sclerosis and inflammatory bowel disease Assay utilizes human PBMCs from healthy donors Luminex platform allows simultaneous monitoring of Th17 cytokine (IL-17A, IL-17F, IL-21) secretion by PBMCs Compatible with flow cytometry and multiplex mrna or DNA quantification 4

5 Inflammation Regulation Models Evaluate the Potency of Your Compounds to Stimulate or Inhibit Inflammatory Chemokine and Cytokine Production Using Primary Cell-Based In Vitro Assays T-Cell Inflammation Regulation T-cells (and sub-population interactions) play an important role in cell-mediated immunity. The delicate balance of pro- and anti-inflammatory events that control immunomodulation is highly influenced by cytokine and chemokine secretion cascades and thus serves as a target of interest in drug development to address various diseases. Evaluate your compounds ability to inhibit T-cell mediated pro-inflammatory responses to various inducer controls (PHA, anti-cd3 antibody, ConA or CD40L/IFNɣ) Evaluate your compounds ability to induce T-cell mediated pro-inflammatory chemokine and cytokine secretion and/or proliferation Simultaneous readout of cytokine and chemokine secretion using the Luminex platform Assay utilizes human PBMCs from healthy donors as source of primary T cells (other species are available upon request) Compatible with flow cytometry and multiplex mrna or DNA quantification Monocyte and Macrophage Inflammation Regulation These cells reside ubiquitously within tissues providing a central role in host protection from pathogen-causing infections. However, when their pro-inflammatory cytokines are left unchecked, the inflammation can contribute to the pathogenesis of inflammatory and degenerative diseases. Evaluate your compounds ability to inhibit monocyte and macrophage pro-inflammatory response to LPS stimulation Evaluate your compounds ability to induce monocyte and macrophage pro-inflammatory cytokines Key cytokines are evaluated that include IL-1β, TNFα, IL-6, IL-8 and MIP-1 (additional cytokine measurements are available upon request) Assay utilizes human PBMCs from healthy donors (other species are available upon request) Compatible with flow cytometry and multiplex mrna or DNA quantification 5

6 Bone Marrow Toxicity Assays Evaluate and Rank Your Drug Candidates for the Risk of Clinical Thrombocytopenia, Neutropenia or Severe Anemia Using Powerful and Well-Characterized Colony Forming Cell (CFC) Assays Applicable for the evaluation of a broad spectrum of compounds, including biologics and small molecules Multi-species evaluation allows comparison of in vitro and in vivo effects Since new drugs are rarely administered to treatment-naive patients, our ability to perform combination drug studies (evaluate potential additive or synergistic toxic effects) can provide highly informative data The CFC assay has been validated by the European Commission for Alternative Methods (ECVAM) for predicting neutropenia and myelosuppression and determining maximum tolerated dose (MTD) for a broad range of drug classes 10 Multi-Species Capabilities Comparative IC 50 values of Tyrosine Kinase Inhibitors between Species in CFC Assays Human CFU-GM NHP CFU-GM Dog CFU-GM Rat CFU-GM Mouse CFU-GM Ranking Compound Toxicities Correlation of in vitro CFC IC 50 values with clinical neutropenia* Correlation of in vitro CFC IC 50 values with clinical neutropenia CFU-GM IC 50 ( M) Erlotinib Sorafenib Imatinib Sunitinib Dasatinib * Predicting Drug-Induced Myelotoxicity, Clarke, E., Atkinson, E., Genetic Engineering & Biotechnology News, V.30, No. 4., Feb 15, Cancer Stem Cell Assays Evaluate and Rank the Effectiveness of Your Drug Candidates at Inhibiting Cancer Stem and Progenitor Cell Expansion Measure functional inhibition of cancer progenitor cell expansion Evaluate therapies that target cell surface receptors (e.g. CD24) and stem cell-associated signaling pathways (e.g. Wnt/Hedgehog) Evaluate the effects of test compounds on a variety of hematopoietic cancer stem cells chronic myeloid leukemia, acute myeloid leukemia, polycythemia vera, multiple myeloma, aplastic anemia, myelodysplastic syndrome AML Blast-Derived Colony MM-Derived Colony Normal CFU-GM-Derived Colony 6

7 Mobilization Studies We Can Evaluate New Mobilizing Agents for Recruitment of Hematopoietic Stem & Progenitor Cells from Bone Marrow to Peripheral Blood for Stem Cell Transplant Procedures We provide a full complement of assays to evaluate stem cell mobilization agents that include blood cell counts, Sca or CD34+ analysis, CFC quantification, NOD/SCID model to confirm functionality Analysis of tissue from various species is available (Human, Mouse, Rat, NHP) Cytokine and Mimetic Testing We Can Assess the Functional Effects of Cytokines, Chemokines or Mimetics for Your Particular Drug Development Screening Program A mimetic is a compound that has similar functional activity to a cytokine or chemokine, but possesses a distinctive chemical structure. This means it will likely not be detected using standard tests (e.g. ELISA) and must be tested via functional assays We can screen multiple compounds at various concentrations for functional activity using in vitro assays or a smaller number of compounds using in vivo models (e.g. where compounds cross-react with mouse and human) Cell Bank Production We Offer Cell Banking Services through the Generation of Human or Mouse Derived Primary Cells or Cell Lines for Cell-Based Drug Development Programs We have experience banking even the most challenging types of cells including primary lymphocyte subsets Ask us about our experience generating cell banks as part of lot release criteria for biologics 7

8 Additional Services ReachBio Research Labs offers additional assay services to address your drug development concerns Immune Profiling of a variety of cells via flow cytometry and intracellular staining Flow Cytometry/Cell Sorting Antibody-Dependent Cellular Cytotoxicity (ADCC) Assays Biotherapeutics Development Support Molecular Analysis including DNA/RNA isolation and gene expression quantification Clinical Trial Support (preclinical & clinical sample analysis) Investigative Toxicology Co-Culture Assays Biomarker Selection In Vivo Services NOD-SCID Repopulation and 5-FU Assays Customized Assay Services ReachBio has deep expertise in primary cell biology that goes far beyond our standard service offerings. This expertise is routinely utilized by clients with projects requiring a knowledgeable and customized approach. Please contact us about your unique project requirements. ReachBio LLC 123 NW 36th Street, Suite 235, Seattle, WA 98107, USA N.A. Toll Free Phone Main Phone Main Fax Website CELL (2355) ReachBio LLC. All rights reserved. Luminex is a registered trademark of Luminex Corporation 8

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