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LIVE – OCTOBER 17 – DAY 2- Koch Institute Immune Engineering Symposium 2017, MIT, Kresge Auditorium

Koch Institute Immune Engineering Symposium 2017

http://kochinstituteevents.cvent.com/events/koch-institute-immune-engineering-symposium-2017/agenda-64e5d3f55b964ff2a0643bd320b8e60d.aspx

Image Source: Leaders in Pharmaceutical Business Intelligence (LPBI) Group

Aviva Lev-Ari, PhD, RN will be in attendance covering the event in REAL TIME

@pharma_BI

@AVIVA1950

#IESYMPOSIUM

@KOCHINSTITUTE

  • The Immune System, Stress Signaling, Infectious Diseases and Therapeutic Implications: VOLUME 2: Infectious Diseases and Therapeutics and VOLUME 3: The Immune System and Therapeutics (Series D: BioMedicine & Immunology) Kindle Edition – on Amazon.com since September 4, 2017

https://www.amazon.com/dp/B075CXHY1B

SYMPOSIUM SCHEDULE

OCTOBER 17 – DAY 2

8:30 – 9:45 Session V
Moderator: Stefani Spranger | MIT, Koch Institute

K. Christopher Garcia – Stanford University
Exploiting T Cell and Cytokine Receptor Structure and Mechanism to Develop New Immunotherapeutic Strategies

  • T Cell Receptor, peptide-MHC, 10 to the power of 10 is combinatorics – Library for selection to determine enrichment possibilities
  • Ligand identification for orphan TCRs
  1. Industrializing process
  2. use pMHC
  • IL-2 – Receptor Signaling Complex
  • Effector cells (NK, T)
  • Engineered  T Cell – Tunable expansion, ligand-Receptor interface
  • Randomize IL-2RBeta interface: Orthogonal receptor vs wild type
  • In Vivo adoptive transfer model: to quantify orthogonality ratio
  • CD4, CD8, Treg,C57BL/6J
  • Ligand discovery
  • Orthogonal IL-2

Stefani Spranger – MIT, Koch Institute
Batf3-DC as Mediators of the T Cell-Inflamed Tumor Microenvironment

  • Melanoma – solid cancer and other types, Immune inhibitory regulatory pathway patient with Immune response present
  • T cell-inflamed Tumor vs Non-T cell-inflamed Tumor
  • identify oncogenic pathways differentially activated between T cell-inflamed and non-Tcell-inflamed infiltration
  • If on Tumor:
  1. Braf/PTEN
  2. Braf/CAT
  3. Braf/PTEN/CAT
  • The role of T cell priming – lack of initial
  • Beta-catenin-expressing tumors fail to prime 2C TCR-transgenic T cells
  • Deficiency in number of CD8+ and CD103+ dendritic cells
  • CD103+ DC are essential for T cell Priming and T cell-inflammation #StefaniSpranger
  • Adoptive transfer of effector 2C T cells fails to control Beta-catenin+ tumors
  • Vaccination induced anti-gen specific T cell memory fails to control Beta-catenin+ tumors
  • What cell type in tumor microenvironment effect monilization of T cell
  • CD103+ Dendritic cellsare source chymokine
  • Recruitment of effector T cells: Reconstitution od Beta-catenin-expressing SIY+
  • Are Batf3-DC within the tumor required for the recruitment of effector T cells?
  • Tumor-residing Batf3-drive CD103+ DC are required for the recruitment of effector T cells
  • Gene spore for correlation with recturment of effector cells
  • T cell Priming – CD103+ DC are essential for effector T cells

George Georgiou – University of Texas at Austin
The Human Circulating Antibody Repertoire in Infection, Vaccination or Cancer

  • Serological Antibody Repertoire: in blood or in secretions
  • Antibody in serum – is difficult sequence identity
  • Serum IgG – 7-17 mg/ml if less immune deficient if more hyper globular
  • antibodies produced in long lived plasma cells in the bone marrow — experimentally inaccessible
  • Discovery of antibodies from the serological repertoire – not B cells
  • BM-PCs
  • Serum antibodies function via Fc effector mechanism – complement activation
  • Ig-SEQ – BCR-SEQ
  • Repertoire-wide computational modelling of antibody structures
  • En masse analysis & Mining of the Human Native Antibody Repertoire
  • hypervariable – High-Throughput Single B Cell VH:VL (or TCRalpha, beta) sequencing
  • EBOV Vaccinee Peak ASCs (day 8) mining: Neutralization
  • Features of the Serum Antibody Repertoire to Vaccine ANtigens:The Serum IgG Repertoire is Highly Polarized
  • Each bar represents a distinct antibody lineage
  • Serum IgG Repertoire becomes increasingly polarized with AGE >50 – may be predictive of tumor development process
  • Human Norovirus – explosive Diarreha, chromically infected – HuNoV BNAb Discovery – Takeda 214 bivalent Vaccine – Binding antibodies binding to avccine antigen VLP
  • HuNoV causes 800 death in the US per year of immune deficient
  • Influenza Trivalent Vaccine: Antibodies to hemaggiutinin: H1, H3, and B COmponenet
  • Abundant H1 +H3 Serum IgGs do not neutralize but confer Protection toInfluenza challenge with Live Virus #GeorgeGeorgiou
  • Non-Neutralizing Antibodies: The role of Complement in Protection

9:45 – 10:15 Break

10:15 – 11:30 Session VI
Moderator: K. Dane Wittrup | MIT, Koch Institute

Harvey Lodish – Whitehead Institute and Koch Institute
Engineered Erythrocytes Covalently Linked to Antigenic Peptides Can Protect Against Autoimmune Disease

  • Modified Red blood cells are microparticles for introducing therapeutics & diagnostics into the human body
  • Bool transfusion is widely used therapeutics
  • Covalently linking unique functional modalities to mouse or human red cells produced in cell culture:
  • PRODUCTION OF HUMAN RED BLOD CELLS EXPRESSING A FOREIN PROTEIN: CD34+ stem/progenitor cells that generates normal enucleated RBC.
  • PPAR-alpha and glucocorticoticoid receptor
  • Norman morphology: Sortase A is a bactrial transpeptidase that covalently links a “donor”
  • Engineering Normal Human RBC biotin-LPETG
  • Covelantely – Glycophorin A with camelid VHHs specific for Botulinum toxin A or B
  • Generation of immuno tolerance: SOruggable Mature RBCs: CRISPR mice expressing Kell-LPETG
  • Ovalbumin as Model Antigens:
  1. OBI B,
  2. OTI CD8 T cells
  3. OTII CD4 T cells
  4. OT-1
  5. OT-2
  • RBC induced peptides challenged and experiences apoptosis
  • Type I Diabetes in NOD mice
  • RBCs bearing InsB9-23 – prevented development of diabetes

Multiple sclerosis

  • MOG – Myelin Oligodend

Sai Reddy – ETH Zurich
Molecular Convergence Patterns in Antibody Responses Predict Antigen Exposure

  • Clonal diversity – estimating the size of antibody repertoire: 10 to power of 18 or 10 to 13
  • Clonal selection in antibody repertoire
  • Convergent selection in antibody repertoire
  • Convergent selection in TCR repertoire complex have restriction with MCH interactions
  • How molecular abundance of convergence predicts antigen exposure identify antigen-associated clusters #SaiReddy
  • molecular convergence 0 gene expression analysis, immunization scheme molecular bar coding to correct errors
  • Recoding antibody repertoire sequence space: Cross correlation reveals different clusters
  • Building a classifier model based on cluster frequency: Clones from immunized mice
  • epitope specificity is driving antibody repertoire response
  • deep learning,

K. Dane Wittrup – MIT, Koch Institute
Temporal Programming of Synergistic Innate and Adaptive Immunotherapy

  • Innate effector functions of anti-tumor antibodies
  • Innate & adaptive Immunotherapy
  • Innate mAb –>> tumor cell; adaptive CD8+ T cells
  • Chemokines Antigens
  • Cytokines Chemokines – back and forth innate Adaptive –> <— neutrophils impact
  • AIPV vaccine:
  • How anti-TAA mAbs helping T cell Immune response
  • Anti-TAA mAbs drive vaccinal T cell responses: NK cells
  • antibody drives T cells responses: alpha-TAA mAbs potentiate T cell therapies: ACT +MSA-IL-2 vs alphaPD-1 + vaccine
  • CD8+ T cells required for alpha TAA mAb efficacy- In absence of T cells Treatment does not work
  • Anti-TAA mAb +Fc/IL-2 induces intramural cytokine storm #KDaneWittrup
  • How to simplify and improve AIPV? Hypothesis: ALign dose schedule
  • Immune response to infection follwos a temporal progression: Innate … Adaptive
  • Antigenic material kill cells: Chemo, cell death Antigen presentation, T cell priming, T cell recirculation, Lymphocyte tumor infiltrate, TCR
  • IFN alpha 2 dys after mAb +Il-2: Curative: days post tumor injection
  • Necessary components: CD8+ T cells & DC, Macrophages,
  • Optimal IFNalpha coincides with max innate response vs Mature DCs after antigen loading #KDaneWittrup
  • Optimal timing od agent administration effect on Therapy Outcome: IL-2, IFNalpha, TAAmAb
  • Cytkine timing can be better than protein engineering #KDaneWittrup

11:30 – 1:00 Lunch Break

1:00 – 2:15 Session VII
Moderator: Michael Birnbaum | MIT, Koch Institute

Kai Wucherpfennig – Dana-Farber Cancer Institute
Discovery of Novel Targets for Cancer Immunotherapy

  • POSITIVE STRESS SIGNAL during malignant Transformation
  • NKG2G=D Receptor: MICA/B Results in Immune escape – Proteolytic cleavage  shedding of MICA/B present in serum, indication of tumor progression
  • Shed MICA vs Surface MICA/B – restore NK cell cytotoxicity and IFNgamma Production
  • Human NK cells express NKG2D and Fc Receptors
  • Synergistic NKG2D and CD16 signaling enhances NK cell cytootxicity: Control IgG vs Anti NKG2D
  • MICA Antibody induces Immunity Against Lung Metastases
  • NK cells are required to inhibit Growth of metastases: Anti-CD8beta,
  • Contribution to Therapeutic Efficacy: NKG2D and CD16 Receptors #KaiWucherpfennig
  • Strategy to analyze Pulmonary NK cells: Activation and expression
  • Single cell RNA-seq of lung NK cells Revealed higher infiltration of activated NK cells: Isotype vs 7C6-migG2a
  • Cytokines and Chemokines produce NK cells
  • MICA/B increaces NK
  •  Induction of Tumor cell Apoptosis
  • Xenotransplant Model with Human Melanoma Cel Line A2058
  • Lung metastasis, liver metastasis
  • Inhibition of human melanoma Metastases in NSG Mice Reconstitute with Human NK
  • Liver metastases are controlled by Myeloid Cells that include Kupffer cells

Michael Birnbaum – MIT, Koch Institute
An Unbiased Determination of pMHC Repertoires for Better Antigen Prediction

  • Vaccines TCR gene therapy adoptive T cel therapy
  • Tumor genone – Tumor pMHC repertoire = Tumor TCR repertoire T cell repertoire
  • Neoantigen vaccines as a personalized anti-cancer therapy
  • Tumor procurement – Target selection – personal vaccine production – vaccine administration
  • Prediction of neoantigen-MHC Binding due to polimorphism affecting recognition, rare in MHC Allells #Michael Birnbaum
  • Antigenicity – Chaperones HLA-DM sculp the peptide binding repertoire of MHC
  • Identification of loaded peptide ligands: pMHC mass spectroscopy of tissue
  • TCR recognition, pMHC yeast display: Cleave peptide-MHC linker, catalyze peptide exchange
  • HLA-DR4 library design and selection to enrich HLA-DM: Amino Acid vs Peptide position: Depleted vs Enriched – relative to expected for NNK codon
  •  6852 _ predicted to bind vs 220 Non-binding peptides
  • HLA polymorphism: repertoire differences caused by
  • Antigen – T cell-driven antigen discovery: engaging Innate and Adaptive Immune response
  • Sorting TIL and select: FOcus of T cell-driven antigen discovery
  • T cell-driven antigen discovery: TCR

Jennifer R. Cochran – Stanford University
Innate and Adaptive Integrin-targeted Combination Immunotherapy

  • alpa-TAA
  • Targeting Integrin = universal target involved in binding to several receptors: brest, lung, pancreatic, brain tumors arising by mutations – used as a handle for binding to agents
  • NOD201 Peptide-Fc Fusion: A Psudo Ab
  • Handle the therapeutics: NOD201 + alphaPD1
  • NOD201 effectively combines with alphaPD-L1, alphaCTLA-4, and alpha4-1BB/CD137
  • Corresponding monotherapies vs ComboTherapy invoking Innate and Adaptive Immune System
  • Microphages, CD8+ are critical vs CD4+ Neutrophils, NK cells, B cells #JenniferR. Cochran
  • Macrophages activation is critical – Day 4, 4 and 5
  • NOD201 + alphaPD1 combo increases M1 macrophages
  • Who are the best responders to PD1 – genes that are differentially expressed
  • NOD201 deives T cells reaponses through a “vaccinal” effect
  • CAncer Immune CYcle
  • Integrin – localization
  • Prelim NOD201 toxicity studies: no significant effects
  • Targeting multiple integrins vs antibodies RJ9 – minimal effect
  • NOD201 – manufacturability – NEW AGENT in Preclinical stage

2:15 – 2:45 Break

2:45 – 3:35 Session VIII
Moderator: Jianzhu Chen | MIT, Koch Institute

Jennifer Wargo – MD Anderson Cancer Center
Understanding Responses to Cancer Therapy: The Tissue is the Issue, but the Scoop is in the Poop

  • Optimize Targeted Treatment response
  • Translational research in patients on targeted therapy revealed molecular and immune mechanisms of response and resistance
  • Molecular mechanisms – T cell infiltrate after one week of therapy
  • Role of tumor stroma in mediating resistance to targeted therapy
  • Tumor microenvironment
  • Intra-tumoral bacteria identified in patients with Pancreatic Cancer
  • Translational research in patients on immune checkpoint blockade revealed molecualr and immune mechanism of response and resistance
  • Biomarkers not found
  • SYstemic Immunity and environment (temperature) on response to checkpoint blockade – what is the role?
  • Role of mIcrobiome in shaping response to checkpoint blockade in Melanoma
  • Microbime and GI Cancer
  • Diversity of the gut microbiome is associated with differential outcomes in the setting of stem cell transplant in AML
  • Oral and gut fecal microbiome in large cohort patient with metastatic melanoma undergoing systemic therapy
  • Repeat oral & gut AFTER chemo
  • WGSeq – Diversity of microbiome and response (responders vs non-responders to anti PD-1 – High diversity of microbiome have prolonged survival to PD-1 blockade
  • Anti tumor Immunity and composition of gut microbiome in patient on anti-PD-1 favorable AND higher survival #JenniferWargo
  • Enhance therapeutic responses in lang and renal carcinoma: If on antibiotic – poorer survival
  • sharing data important across institutions

Jianzhu Chen – MIT, Koch Institute
Modulating Macrophages in Cancer Immunotherapy

  • Humanized mouth vs de novo human cancer
  • B cell hyperplasia
  • double hit lymphoma
  • AML
  • Overexpression of Bcl-2 & Myc in B cells leads to double-hit lymphoma
  • antiCD52 – CLL
  • Spleen, Bone marrow, Brain
  • Microphages are required to kill Ab-bound lymphoma cells in vivo #JianzhuChen
  • COmbinatorial chemo-Immunotherapy works for solid tumors: treating breast cancer in humanized mice
  • Infiltration of monocytic cells in the bone marrow
  • Cyclophosphophamide-antibody synergy extending to solid tumor and different antibodies #JianzhuChen
  • Polarization of macrophages it is dosage-dependent M1 and M2
  • Antibiotic induces expression of M1 polarizing supresses development and function of tumor-associated macrophages (TAM)
  • Antibiotic inhibits melanoma growth by activating macrophages in vivo #JianzhuChen

 

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Image Source:Koch Institute

 

LIVE – OCTOBER 16 – DAY 1- Koch Institute Immune Engineering Symposium 2017, MIT, Kresge Auditorium

Koch Institute Immune Engineering Symposium 2017

http://kochinstituteevents.cvent.com/events/koch-institute-immune-engineering-symposium-2017/agenda-64e5d3f55b964ff2a0643bd320b8e60d.aspx

 

#IESYMPOSIUM

 

Image Source: Leaders in Pharmaceutical Business Intelligence (LPBI) Group

Aviva Lev-Ari, PhD, RN will be in attendance covering the event in REAL TIME

@pharma_BI

@AVIVA1950

#IESYMPOSIUM

@KOCHINSTITUTE

  • The Immune System, Stress Signaling, Infectious Diseases and Therapeutic Implications: VOLUME 2: Infectious Diseases and Therapeutics and VOLUME 3: The Immune System and Therapeutics (Series D: BioMedicine & Immunology) Kindle Edition – on Amazon.com since September 4, 2017

https://www.amazon.com/dp/B075CXHY1B

SYMPOSIUM SCHEDULE

OCTOBER 16 – DAY 1

7:00 – 8:15 Registration

8:15 – 8:30Introductory Remarks
Darrell Irvine | MIT, Koch Institute; HHMI

  • Stimulating the Immune system not only sustaining it for therapies

K. Dane Wittrup | MIT, Koch Institute

8:30 – 9:45Session I
Moderator: Douglas Lauffenburger | MIT, Biological Engineering and Koch Institute

Garry P. Nolan – Stanford University School of Medicine
Pathology from the Molecular Scale on Up

  • Intracellular molecules,
  • how molecules are organized to create tissue
  • Meaning from data Heterogeneity is an illusion: Order in Data ?? Cancer is heterogeneous, Cells in suspension – number of molecules
  • System-wide changes during Immune Response (IR)
  • Untreated, Ineffective therapy, effective therapy
  • Days 3-8 Tumor, Lymph node…
  • Variation is a Feature – not a bug: Effective therapy vs Ineffective – intercellular modules – virtual neighborhoods
  • ordered by connectivity: very high – CD4 T-cells, CD8 T-cels, moderate, not connected
  • Landmark nodes, Increase in responders
  • CODEX: Multiples epitome detection
  • Adaptable to proteins & mRNA
  • Rendering antibody staining via removal to neighborhood mapping
  • Human tonsil – 42 parameters: CD7, CD45, CD86,
  • Automated Annotations of tissues: F, P, V,
  • Normal BALBs
  • Marker expression defined by the niche: B220 vs CD79
  • Marker expression defines the niche
  • Learn neighborhoods and Trees
  • Improving Tissue Classification and staining – Ce3D – Tissue and Immune Cells in 3D
  • Molecular level cancer imaging
  • Proteomic Profiles: multi slice combine
  • Theory is formed to explain 3D nuclear images of cells – Composite Ion Image, DNA replication
  • Replication loci visualization on DNA backbone – nascent transcriptome – bar code of isotopes – 3D  600 slices
  • use CRISPR Cas9 for Epigenetics

Susan Napier Thomas – Georgia Institute of Technology
Transport Barriers in the Tumor Microenvironment: Drug Carrier Design for Therapeutic Delivery to Sentinel Lymph Nodes

  • Lymph Nodes important therapeutics target tissue
  • Lymphatic flow support passive and active antigen transport to lymph nodes
  • clearance of biomolecules and drug formulations: Interstitial transport barriers influence clearance: Arteriole to Venule –
  • Molecular tracers to analyze in vivo clearance mechanisms and vascular transport function
  • quantifying molecular clearance and biodistribution
  • Lymphatic transport increases tracer concentrations within dLN by orders of magnitude
  • Melanoma growth results in remodeled tumor vasculature
  • passive transport via lymphatic to dLN sustained in advanced tumors despite abrogated cell trafficking
  • Engineered biomaterial drug carriers to enhance sentinel lymph node-drug delivery: facilitated by exploiting lymphatic transport
  • TLR9 ligand therapeutic tumor in situ vaccination – Lymphatic-draining CpG-NP enhanced
  • Sturcutral and Cellular barriers: transport of particles is restriced by
  • Current drug delivery technology: lymph-node are undrugable
  • Multistage delivery platform to overcome barriers to lymphatic uptake and LN targeting
  • nano particles – OND – Oxanorbornade OND Time sensitive Linker synthesized large cargo – NP improve payload
  • OND release rate from nanoparticles changes retention in lymph nodes – Axilliary-Brachial delivery
  • Two-stage OND-NP delivery and release system dramatically – OND acumulate in lymphocyte
  •  delivers payload to previously undraggable lymphe tissue
  • improved drug bioactivity  – OND-NP eliminate LN LYMPHOMAS
  • Engineered Biomaterials

Douglas Lauffenburger – MIT, Biological Engineering and Koch Institute
Integrative Multi-Omic Analysis of Tissue Microenvironment in Inflammatory Pathophysiology

  • How to intervene, in predictive manner, in immunesystem-associated complex diseases
  • Understand cell communication beteen immune cells and other cells, i.e., tumor cells
  • Multi-Variate in Vivo – System Approach: Integrative Experiment & COmputational Analysis
  • Cell COmmunication & Signaling in CHronic inflammation – T-cell transfer model for colitis
  • COmparison of diffrential Regulation (Tcell transfer-elicited vs control) anong data types – relying solely on mRNA can be misleading
  • Diparities in differential responses to T cell transfer across data types yield insights concerning broader multi-organ interactions
  • T cell transfer can be ascertained and validated by successful experimental test
  • Cell COmmunication in Tumor MIcro-Environment — integration of single-cell transcriptomic data and protein interaction
  • Standard Cluster Elucidation – Classification of cell population on Full gene expression Profiles using Training sets: Decision Tree for Cell Classification
  • Wuantification of Pairwise Cell-Cell Receptor/Ligand Interactions: Cell type Pairs vs Receptor/Ligand Interaction
  • Pairwise Cell-Cell Receptor/Ligand Interactions
  • Calculate strength of interaction and its statistical significance
  • How the interaction is related to Phenotypic Behaviors – tumor growth rate, MDSC levels,
  • Correlated the Interactions translated to Phynotypic behavior for Therapeutic interventions (AXL via macrophage and fibroblasts)
  • Mouth model translation to Humans – New machine learning approach
  • Pathways, false negative, tumor negative expression
  • Molecular vs Phynotypical expression
  • Categories of inter-species translation
  • Semi-supervised Learning ALgorithms on Transcriptomic Data can ascertain Key Pathways/Processes in Human IBD from mapping mouse IBD

9:45 – 10:15 Break

10:15 – 11:30Session II
Moderator: Tyler Jacks | MIT, Koch Institute; HHMI

Tyler Jacks – MIT, Koch Institute; HHMI
Using Genetically Engineered Mouse Models to Probe Cancer-Immune Interactions

  • Utility of genetically-engineered mouse models of Cancer:
  1. Immune Response (IR),
  2. Tumor0immune microenvironment
  • Lung adenocarcinoma – KRAS mutation: Genetically-engineered model, applications: CRISPR, genetic interactions
  • Minimal Immune response to KP lung tumors: H&E, T cells (CD3), Bcells (B220) for Lenti-x 8 weeks
  • Exosome sequencing : Modeling loss-and gain-of-function mutations in Lung Cancer by CRISPR-Cas9 – germline – tolerance in mice, In vivo CRISPR-induced knockout of Msh2
  • Signatures of MMR deficient
  • Mutation burden and response to Immunotherapy (IT)
  • Programmed neoantigen expression – robust infiltration of T cells (evidence of IR)
  • Immunosuppression – T cell rendered ineffective
  • Lymphoid infiltration: Acute Treg depletion results in T cell infiltration — this depletion causes autoimmune response
  • Lung Treg from KP tumor-bearing mice have a distinct transcriptional heterogeneity through single cell mRNA sequencing
  • KP, FOXP3+, CD4
  • Treg from no existent to existance, Treg cells increase 20 fold =>>>  Treg activation and effectiveness
  • Single cells cluster by tissue and cell type: Treg, CD4+, CD8+, Tetramer-CD4+
  • ILrl1/II-33r unregulated in Treg at late time point
  • Treg-specific deletion of IL-33r results in fewer effector Tregs in Tumor-bearing lungs
  • CD8+ T cell infiltration
  • Tetramer-positive T cells cluster according to time point: All Lung CD8+ T cells
  • IR is not uniform functional differences – Clones show distinct transcriptional profiles
  • Different phynotypes Exhaustive signature
  • CRISPR-mediated modulation of CD8 T cell regulatory genes
  • Genetic dissection of the tumor-immune microenvironment
  • Single cell analysis, CRISPR – CRISPRa,i, – Drug development

Wendell Lim – University of California, San Francisco

Synthetic Immunology: Hacking Immune Cells

  • Precision Cell therapies – engineered by synthetic biology
  • Anti CD19 – drug approved
  • CAR-T cells still face major problems
  1. success limited to B cells cancers = blood vs solid tumors
  2. adverse effects
  3. OFF-TUMOR effects
  • Cell engineering for Cancer Therapy: User remote control (drug) – user control safety
  • Cell Engineering for TX
  1. new sensors – decision making for
  2. tumor recognition – safety,
  3. Cancer is a recognition issue
  • How do we avoid cross-reaction with bystader tissue (OFF TISSUE effect)
  • Tumor recognition: More receptors & integration
  • User Control
  • synthetic NOTCH receptors (different flavors of synNotch) – New Universal platform for cell-to -cell recognition: Target molecule: Extracellular antigen –>> transciptional instruction to cell
  • nextgen T cell: Engineer T cell recognition circuit that integrates multiple inputs: Two receptors – two antigen priming circuit
  • UNARMED: If antigen A THEN receptor A activates CAR
  • “Bystander” cell single antigen vs “tumor” drug antigen
  • Selective clearance of combinatorial tumor – Boulian formulation, canonical response
  • Cell response: Priming –>> Killing: Spatial & Temporal choreographed cell
  • CAR expression while removed from primed cells deminished
  • Solid Tumor: suppress cell microenvironment: Selected response vs non-natural response
  • Immune stimulator IR IL2, IL12, flagellin in the payload — Ourcome: Immune enhancement “vaccination”
  • Immune suppression –  block
  • Envision ideal situation: Unarmed cells
  • FUTURE: identify disease signatures and vulnerabilities – Precision Medicine using Synthetic Biology

Darrell Irvine – MIT, Koch Institute; HHMI
Engineering Enhanced Cancer Vaccines to Drive Combination Immunotherapies

  • Vaccine to drive IT
  • Intervening in the cancer-immunity cycle – Peptide Vaccines
  • poor physiology  of solute transport to tissue
  • endogenous albumin affinity – Lymphe Node dying
  • Designing Albumin-hitchhiking vaccines
  • Amphiphile-vaccine enhance uptake in lymph nodes in small and large animal models
  • soluble vaccine vs Amphiphile-vaccine
  • DIRECTING Vaccines to the Lymph nodes
  • amph-peptide antigen: Prime, booster, tetramer
  • albimin-mediated LN-targeting of both antigen and adjuvant maximizes IR
  • Immuno-supressed microenvironment will not be overcome by vaccines
  • Replacing adoptive T cell transfer with potent vaccine
  • exploiting albumin biology for mucosal vaccine delivery by amph-vaccines
  • Amph-peptides and -adjuvants show enhanced uptake/retention in lung tissue
  •  Enhancing adoptive T cell therapy: loss of T cell functionality, expand in vivo
  • boost in vivo enhanced adoptive T cell therapy
  • CAR-T cells: Enable T cells to target any cell surface protein
  • “Adaptor”-targeting CAR-T cells to deal with tumor cell heterogeneity
  • Lymph node-targeting Amph as CAR T booster vaccine: prining, production of cytokines
  • Boosting CAR T with amph-caccines: anti FITC CAR-T by DSPE=PEG-FITC coated
  • Targeting FITC to lymph node antigen presenting cells
  • Modulatory Macrophages
  • Amph-FITC expands FITC-CAR T cells in vivo – Adjuvant is needed
  • Hijacking albumin’s natural trafficking pathway

11:30 – 1:00  Lunch Break

1:00 – 2:15Session III
Moderator: Darrell Irvine | MIT, Koch Institute; HHMI

Nicholas P. Restifo – National Cancer Institute
Extracellular Potassium Regulates Epigenetics and Efficacy of Anti-Tumor T Cells

Why T cell do not kill Cancer cells?

  • co-inhibition
  • hostile tumor microenvironment

CAR T – does not treat solid tumors

Somatic mutation

  1. resistence of T cell based IT due to loss of function mutations
  2. Can other genes be lost?

CRISPR Cas9 – used to identify agents – GeCKOv2 Human library

Two cell-type (2CT) CRISPR assay system for genome-wide mutagenesis

  • work flow for genome-scale SRISPR mutagenesis profiling of genes essential for T cell mediate cytosis
  • sgRNA enrichment at the individual gene level by multiple methods:
  1. subunits of the MHC Class I complex
  2. CRISPR mutagenesis cut germline
  • Measutring the generalizability of resistance mechanism and mice in vivo validation
  • Validation of top gene candidates using libraries: MART-1
  • Checkpoint blockade: cells LOF causes tumor growth and immune escape
  • Weird genesL Large Ribisomal Subunit Proteins are nor all essential for cell survival
  • Bias in enrichment of 60S vs 40S
  • Novel elements of MHC class I antigen processing and presentation
  • Association of top CRISPR hits with response rates to IT – antiCTLA-4
  • CRISPR help identify novel regulators of T cells
  • Analyzed sgRNA – second rarest sgRNA for gene BIRC2 – encoded the Baculoviral Inhibitor
  • Drugs that inhibit BIRC2
  • How T cells can kill tumor cells more efficiently
  • p38kiaseas target for adoptive immunotherapy
  • FACS-based – Mapk14
  • Potent targets p38 – Blockade PD-1 or p38 ??
  • p38 signaling: Inhibition augments expansion and memory-marked human PBMC and TIL cells, N. P. Restifo
  • Tumor killing capacity of human CD19-specific, gene engineered T cells

Jennifer Elisseeff – Johns Hopkins University
The Adaptive Immune Response to Biomaterials and Tissue Repair

  • design scafolds, tissue-specific microenvironment
  • clinical translation of biosynthetic implants for soft tissue reconstruction
  • Local environment affects biomaterials: Epidermis, dermis
  • CD4+ T cells
  • Immune system – first reponders to materials: Natural or Synthetic
  • Biological (ECM) scaffolds to repair muscle injury
  • Which immune cells enter the WOUND?
  • ECM alters Macrophages: CD86, CD206
  • Adaptive system impact on Macrophages: CD86
  • mTOR signaling pathway M2 depend on Th2 Cells in regeneration of cell healing of surgical wounds
  • Systemic Immunological changes
  • Is the response antigen specific? – IL-4 expression in ILN,
  • Tissue reconstruction Clinical Trial: FDA ask to look at what cells infiltrate the scaffold
  • Trauma/biomaterial response – Injury induction of Senescence, anti apoptosis
  • Injury to skin or muscle
  • Is pro-regenerative environment (Th2/M2) pro-tumorigenic?
  • SYNTHETIC Materials for scafolds
  • Biomaterials and Immunology
  1. Immune response to bioscafolds
  2. environment modulate the immune system
  • Regenerative Immunetherapy

Marcela Maus – Massachusetts General Hospital

Engineering Better T Cells

  • Comparing CD19 CARs for Leukemia – anti-CD19- directed CAR T cells with r/r B-cell ALL – age 3-25 – FDA approved Novartis tisagenlecleucel – for pediatric r/r/ ALL
  • Phase II in diffuse large B cell lymphoma. Using T cells – increases prospects for cure
  • Vector retroviral – 30 day expression
  • measuring cytokines release syndrome: Common toxicity with CAR 19
  • neurological toxicity, B-cell aplagia
  • CART issues with heme malignancies
  1. decrease cytokine release
  2. avoid neurological toxicity – homing
  3. new targets address antigene escape variants – Resistance, CD19 is shaded, another target needed
  4. B Cell Maturation Antigen (BCMA) Target
  5. Bluebird Bio: Response duratio up to 54 weeks – Active dose cohort
  6. natural ligand CAR based on April
  7. activated in response to TACI+ target cells – APRIL-based CARs but not BCMA-CAR is able to kill TACI+ target cells
  • Hurdles for Solid Tumors
  1. Specific antigen targets
  2. tumor heterogeneity
  3. inhibitory microenvironment
  • CART in Glioblastoma
  1. rationale for EGFRvIII as therapeutic target
  2. Preclinical Studies & Phase 1: CAR t engraft, not as highly as CD19
  3. Upregulation of immunosuppression and Treg infiltrate in CART EGFRvIII as therapeutic target, Marcela Maus
  • What to do differently?

 

2:15 – 2:45 Break

2:45 – 4:00 Session IV
Moderator: Arup K. Chakraborty | MIT, IMES

Laura Walker – Adimab, LLC
Molecular Dissection of the Human Antibody Response to Respiratory Syncytial Virus

  • prophylactic antibody is available
  • Barriers for development of Vaccine
  • Prefusion and Postfusion RSV structures
  • Six major antigenic sites on RSV F
  • Blood samples Infants less 6 month of age and over 6 month: High abundance RSV F -specific memory B Cells are group  less 6 month

Arup K. Chakraborty – MIT, Institute for Medical Engineering & Science
How to Hit HIV Where it Hurts

  • antibody  – Model IN SILICO
  • Check affinity of each Ab for the Seaman panel of strain
  • Breadth of coverage
  • immmunize with cocktail of variant antigens
  • Mutations on Affinity Maturation: Molecular dynamics
  • bnAb eveolution: Hypothesis – mutations evolution make the antigen binding region more flexible,
  • Tested hypothesisi: carrying out affinity maturation – LOW GERMLINE AFFINITY TO CONSERVE RESIDUES IN 10,000 trials, acquire the mutation (generation 300)

William Schief – The Scripps Research Institute
HIV Vaccine Design Targeting the Human Naive B Cell Repertoire

  • HIV Envelope Trimer Glycan): the Target of neutralizing Antibodies (bnAbs)
  • Proof of principle for germline-targeting: VRC)!-class bnAbs
  • design of a nanoparticle
  • can germline -targeting innumogens prime low frequency precursors?
  • Day 14 day 42 vaccinate
  • Precursor frequency and affinity are limiting for germline center (GC) entry at day 8
  • Germline-targeting immunogens can elicit robust, high quality SHM under physiological conditions of precursor frequency and affinity at day 8, 16, 36
  • Germline-targeting immunogens can lead to production of memory B cells

2017 Nobel prize in chemistry given to Jacques Dubochet, Joachim Frank, and Richard Henderson  for developing cryo-electron microscopy

 

Reporter: Aviva Lev-Ari, PhD, RN

 

Here’s what the images that just won the Nobel prize in chemistry look like and why they’re so transformative

IMAGE SOURCE
Over the last few years, researchers have published atomic structures of numerous complicated protein complexes. a. A protein complex that governs the circadian rhythm. b. A sensor of the type that reads pressure changes in the ear and allows us to hear. c. The Zika virus.
The Royal Swedish Academy of Sciences
SOURCE

The Nobel Prize in Chemistry 2017

4 October 2017

The Royal Swedish Academy of Sciences has decided to award the Nobel Prize in Chemistry 2017 to

Jacques Dubochet
University of Lausanne, Switzerland

Joachim Frank
Columbia University, New York, USA

and

Richard Henderson
MRC Laboratory of Molecular Biology, Cambridge, UK

“for developing cryo-electron microscopy for the high-resolution structure determination of biomolecules in solution”

 

Cool microscope technology revolutionises biochemistry

We may soon have detailed images of life’s complex machineries in atomic resolution. The Nobel Prize in Chemistry 2017 is awarded to Jacques Dubochet, Joachim Frank and Richard Henderson for the development of cryo-electron microscopy, which both simplifies and improves the imaging of biomolecules. This method has moved biochemistry into a new era.

A picture is a key to understanding. Scientific breakthroughs often build upon the successful visualisation of objects invisible to the human eye. However, biochemical maps have long been filled with blank spaces because the available technology has had difficulty generating images of much of life’s molecular machinery. Cryo-electron microscopy changes all of this. Researchers can now freeze biomolecules mid-movement and visualise processes they have never previously seen, which is decisive for both the basic understanding of life’s chemistry and for the development of pharmaceuticals.

Electron microscopes were long believed to only be suitable for imaging dead matter, because the powerful electron beam destroys biological material. But in 1990, Richard Henderson succeeded in using an electron microscope to generate a three-dimensional image of a protein at atomic resolution. This breakthrough proved the technology’s potential.

Joachim Frank made the technology generally applicable. Between 1975 and 1986 he developed an image processing method in which the electron microscope’s fuzzy twodimensional images are analysed and merged to reveal a sharp three-dimensional structure.

Jacques Dubochet added water to electron microscopy. Liquid water evaporates in the electron microscope’s vacuum, which makes the biomolecules collapse. In the early 1980s, Dubochet succeeded in vitrifying water – he cooled water so rapidly that it solidified in its liquid form around a biological sample, allowing the biomolecules to retain their natural shape even in a vacuum.

Following these discoveries, the electron microscope’s every nut and bolt have been optimised. The desired atomic resolution was reached in 2013, and researchers can now routinely produce three-dimensional structures of biomolecules. In the past few years, scientific literature has been filled with images of everything from proteins that cause antibiotic resistance, to the surface of the Zika virus. Biochemistry is now facing an explosive development and is all set for an exciting future.

Read more about this year’s prize

Popular Information
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Scientific Background
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To read the text you need Acrobat Reader.

Image – 3D structures (pdf 1.4 MB)
© Johan Jarnestad/The Royal Swedish Academy of Sciences

Image – Blobology (pdf 8.5 MB)
© Martin Högbom/The Royal Swedish Academy of Sciences

Image – Dubochet’s preparation method (948 kB)
© Johan Jarnestad/The Royal Swedish Academy of Sciences

Image – Frank’s image analysis (pdf 1 MB)
© Johan Jarnestad/The Royal Swedish Academy of Sciences

 


Jacques Dubochet, born 1942 in Aigle, Switzerland. Ph.D. 1973, University of Geneva and University of Basel, Switzerland. Honorary Professor of Biophysics, University of Lausanne, Switzerland.
www.unil.ch/dee/en/home/menuinst/people/honorary-professors/prof-jacques-dubochet.html

Joachim Frank, born 1940 in Siegen, Germany. Ph.D. 1970, Technical University of Munich, Germany. Professor of Biochemistry and Molecular Biophysics and of Biological Sciences, Columbia University, New York, USA.
http://franklab.cpmc.columbia.edu/franklab/

Richard Henderson, born 1945 in Edinburgh, Scotland. Ph.D. 1969, Cambridge University, UK. Programme Leader, MRC Laboratory of Molecular Biology, Cambridge, UK.
www2.mrc-lmb.cam.ac.uk/groups/rh15/

Prize amount: 9 million Swedish krona, to be shared equally between the Laureates.
Further information: http://www.kva.se and http://nobelprize.org

SOURCE

https://www.nobelprize.org/nobel_prizes/chemistry/laureates/2017/press.html


Ferritin Cage Enzyme Encapsulation as a New Platform for Nanotechnology

 Reporter: Irina Robu, PhD

In bionanotechnology, biological systems such as viruses, protein complexes, lipid vesicles and artificial cells, are being developed for applications in medicine and materials science.  However, the paper published by Stephan Tetter and Donald Hilvert in Angewandte Chemie International Edition show that it is possible to encapsulate proteins such as ferritin by manipulating electrostatic interactions with the negatively charged interior of the cage.The primary role of ferritin is to protect cells from the damage caused by the Fenton reaction; where, in oxidizing conditions, free Fe(II) produces harmful reactive oxygen species that can damage the cellular machinery.

The ferritin family proteins are protein nanocages that evolved to safely store iron in an oxidizing world. Since ferritin family proteins are able to mineralize and store metal ions, they have been the focus of much research for the production of metal nanoparticles and as prototypes for semiconductor production. The ferritin cage itself is highly symmetrical, and is made up of 24 subunits arranged in an octahedral symmetry. Ferritins are smaller than other protein used for protein   encapsulation.   Their  outer  diameter is only 12 nm, whereas engineered lumazine synthase variants form cages with diameters ranging from about 20 to 60 nm.The ferritin cage displays remarkable thermal and chemical stability it is likely to modify the surface of the ferritin cage through the addition of peptide and protein tags. These characteristics have made ferritins attractive vectors for the delivery of drug molecules and as scaffolds for vaccine design.

In summary, the paper published in Angewandte Chemie International Edition is the first example of protein incorporation by a ferritin.  Dr. Donald Hilvert and colleagues have shown that AfFtn not only complexes positively charged guest proteins within its naturally negatively charged luminal cavity, but that the in vitro mixing technique can be extended to the encapsulation and protection of other functional  fusion proteins.

Hence, the recent discovery of encapsulated ferritins has identified an exciting new platform for use in bio nanotechnology. The use of synthetic biology tools will allow their rapid implementation in materials science, bio-nanotechnology and medical applications.

SOURCE

https://www.readbyqxmd.com/read/28902449/enzyme-encapsulation-by-a-ferritin-cage



Systemic Inflammatory Diseases as Crohn’s disease, Rheumatoid Arthritis and Longer Psoriasis Duration May Mean Higher CVD Risk

Reporter: Aviva Lev-Ari, PhD, RN

Longer Psoriasis Duration May Mean Higher CVD Risk

Effect size ‘similar to that of smoking’

Several studies have shown that methotrexate, which has anti-inflammatory effects, reduces CV risk in patients with rheumatoid arthritis, suggesting that good anti-inflammatory control may be expected to reduce CV risk in patients with psoriasis.

Menter has worked closely with the senior author of the current study, Nehal Mehta, MD, of the University of Pennsylvania in Philadelphia, to identify cardiovascular issues in the psoriasis population. In one recent study, investigators found that the prevalence of moderate-to-severe coronary calcification was similar between patients with psoriasis and those with type 2 diabetes, and approximately five times greater than healthy controls.

Investigators found that moderate-to-severe psoriasis was a significantly stronger predictor of coronary calcification than type 2 diabetes, and the effect was independent of known CV and cardiometabolic risk factors.

 

SOURCE

https://www.medpagetoday.com/Dermatology/Psoriasis/68429?xid=nl_mpt_cardiodaily_2017-10-09&eun=g99985d0r


QIAGEN – International Leader in NGS and RNA Sequencing

Reporter: Aviva Lev-Ari, PhD, RN

 

The reader is encouraged to review all the products of QIAGEN on the company web site.

miRCURY Exosome Kits

For enrichment of exosomes and other extracellular vesicles from serum/plasma or cell/urine/CSF samples
  • Excellent recovery of exosomes and other extracellular vesicles
  • Easy and straightforward protocol that takes less than 2 hours
  • No ultracentrifugation or phenol/chloroform steps required
  • Fully compatible with the miRCURY LNA miRNA PCR System
  • Suited for a variety of applications, such as miRNA or RNA profiling

miRCURY Exosome Kits enable high-quality and scalable exosome isolation with an easy protocol that does not require special laboratory equipment. The miRCURY Exosome Serum/Plasma Kit is optimized for serum and plasma samples, while the miRCURY Exosome Cell/Urine/CSF Kit is designed for processing cell-conditioned media, urine and CSF samples. Both kits provide high exosomal recovery and seamless integration with different downstream assays.

SOURCE

https://www.qiagen.com/us/shop/sample-technologies/tumor-cells-and-exosomes/mircury-exosome-kits/#orderinginformation

QIAGEN – Product Profile


The Role of Exosomes in Metabolic Regulation

Author: Larry H. Bernstein, MD, FCAP

 

On 9/25/2017, Aviva Lev-Ari, PhD, RN commissioned Dr. Larry H. Bernstein to write a short article on the following topic reported on 9/22/2017 in sciencemission.com

 

We are publishing, below the new article created by Larry H. Bernstein, MD, FCAP.

 

Background

During the period between 9/2015  and 6/2017 the Team at Leaders in Pharmaceutical Business Intelligence (LPBI)  has launched an R&D effort lead by Aviva Lev-Ari, PhD, RN in conjunction with SBH Sciences, Inc. headed by Dr. Raphael Nir.

This effort, also known as, “DrugDiscovery @LPBI Group”  has yielded several publications on EXOSOMES on this Open Access Online Scientific Journal. Among them are included the following:

 

QIAGEN – International Leader in NGS and RNA Sequencing, 10/08/2017

Reporter: Aviva Lev-Ari, PhD, RN

 

cell-free DNA (cfDNA) tests could become the ultimate “Molecular Stethoscope” that opens up a whole new way of practicing Medicine, 09/08/2017

Reporter: Aviva Lev-Ari, PhD, RN

 

Detecting Multiple Types of Cancer With a Single Blood Test (Human Exomes Galore), 07/02/2017

Reporter and Curator: Irina Robu, PhD

 

Exosomes: Natural Carriers for siRNA Delivery, 04/24/2017

Reporter: Aviva Lev-Ari, PhD, RN

 

One blood sample can be tested for a comprehensive array of cancer cell biomarkers: R&D at WPI, 01/05/2017

Curator: Marzan Khan, B.Sc

 

SBI’s Exosome Research Technologies, 12/29/2016

Reporter: Aviva Lev-Ari, PhD, RN

 

A novel 5-gene pancreatic adenocarcinoma classifier: Meta-analysis of transcriptome data – Clinical Genomics Research @BIDMC, 12/28/2016

Curator: Tilda Barliya, PhD

 

Liquid Biopsy Chip detects an array of metastatic cancer cell markers in blood – R&D @Worcester Polytechnic Institute, Micro and Nanotechnology Lab, 12/28/2016

Reporters: Tilda Barliya, PhD and Aviva Lev-Ari, PhD, RN

 

Exosomes – History and Promise, 04/28/2016

Reporter: Aviva Lev-Ari, PhD, RN

 

Exosomes, 11/17/2015

Curator: Larry H. Bernstein, MD, FCAP

 

Liquid Biopsy Assay May Predict Drug Resistance, 11/16/2015

Curator: Larry H. Bernstein, MD, FCAP

 

Glypican-1 identifies cancer exosomes, 10/31/2015

Curator: Larry H. Bernstein, MD, FCAP

 

Circulating Biomarkers World Congress, March 23-24, 2015, Boston: Exosomes, Microvesicles, Circulating DNA, Circulating RNA, Circulating Tumor Cells, Sample Preparation, 03/24/2015

Reporter: Aviva Lev-Ari, PhD, RN

 

Cambridge Healthtech Institute’s Second Annual Exosomes and Microvesicles as Biomarkers and Diagnostics Conference, March 16-17, 2015 in Cambridge, MA, 03/17, 2015

Reporter: Aviva Lev-Ari, PhD, RN

 

The newly created think-piece on the relationship between regulatory functions of Exosomes and Metabolic processes is developed conceptually, below.

 

The Role of Exosomes in Metabolic Regulation

Author: Larry H. Bernstein, MD, FCAP

We have had more than a half century of research into the genetic code and transcription leading to abundant work on RNA and proteomics. However, more recent work in the last two decades has identified RNA interference in siRNA. These molecules may be found in the circulation, but it has been a challenge to find their use in therapeutics. Exosomes were first discovered in the 1980s, but only recently there has been a huge amount of research into their origin, structure and function. Exosomes are 30–120 nm endocytic membrane-bound extracellular vesicles (EVs)(1-23) , and more specifically multiple vesicle bodies (MVBs) by a budding process from invagination of the outer cell membrane that carry microRNA (miRNA), and have structures composed of protein and lipids (1,23-27 ). EVs are the membrane vesicles secreted by eukaryotic cells for intracellular communication by transferring the proteins, lipids, and RNA under various physiologic conditions as well as during the disease stage. EVs also act as a signalosomes in many biological processes. Inward budding of the plasma membrane forms small vesicles that fuse. Intraluminal vesicles (ILVs) are formed by invagination of the limiting endosomal membrane during the maturation process of early endosome.

EVs are the MVBs secreted that serve in intracellular communication by transferring a cargo consisting of proteins, lipids, and RNA under various physiologic conditions (4, 23). Exosome-mediated miRNA transfer between cells is considered to be necessary for intercellular signaling and exosome-associated miRNAs in biofluids (23). Exosomes carry various molecular constituents of their cell of origin, including proteins, lipids, mRNAs, and microRNAs (miRNAs) (. They are released from many cell types, such as dendritic cells (DCs), lymphocytes, platelets, mast cells, epithelial cells, endothelial cells, and neurons, and can be found in most bodily fluids including blood, urine, saliva, amniotic fluid, breast milk, hydrothoracic fluid, and ascitic fluid, as well as in culture medium of most cell types.Exosomes have also been shown to be involved in noncoding RNA surveillance machinery in generating antibody diversity (24). There are also a vast number of long non-coding RNAs (lncRNAs) and enhancer RNAs (eRNAs) that accumulate R-loop structures upon RNA exosome ablation, thereby, resolving deleterious DNA/RNA hybrids arising from active enhancers and distal divergent eRNA-expressing elements (lncRNA-CSR) engaged in long-range DNA interactions (25). RNA exosomes are large multimeric 3′-5′ exo- and endonucleases representing the central RNA 3′-end processing factor and are implicated in processing, quality control, and turnover of both coding and noncoding RNAs. They are large macromolecular cages that channel RNA to the ribonuclease sites (29). A major interest has been developed to characterize of exosomal cargo, which includes numerous non-randomly packed proteins and nucleic acids (1). Moreover, exosomes play an active role in tumorigenesis, metastasis, and response to therapy through the transfer of oncogenes and onco-miRNAs between cancer cells and the tumor stroma. Blood cells and the vascular endothelium is also exosomal shedding, which has significance for cardiovascular,   neurologicological disorders, stroke, and antiphospholipid syndrome (1). Dysregulation of microRNAs and the affected pathways is seen in numerous pathologies their expression can reflect molecular processes of tumor onset and progression qualifying microRNAs as potential diagnostic and prognostic biomarkers (30).

Exosomes are secreted by many cells like B lymphocytes and dendritic cells of hematopoietic and non-hematopoietic origin viz. platelets, Schwann cells, neurons, mast cells, cytotoxic T cells, oligodendrocytes, intestinal epithelial cells were also found to be releasing exosomes (4). They are engaged in complex functions like persuading immune response as the exosomes secreted by antigen presenting cells activate T cells (4). They all have a common set of proteins e.g. Rab family of GTPases, Alix and ESCRT (required for transport) protein and they maintain their cytoskeleton dynamics and participate in membrane fusion. However, they are involved in retrovirus disease pathology as a result of recruitment of the host`s endosomal compartments in order to generate viral vesicles, and they can either spread or limit an infection based on the type of pathogen and its target cells (5).

Upon further consideration, it is understandable how this growing biological work on exosomes has enormous significance for laboratory diagnostics (1, 3, 5, 6, 11, 14, 15, 17-20, 23,30-41) . They are released from many cell types, such as dendritic cells (DCs), lymphocytes, platelets, mast cells, epithelial cells, endothelial cells, and neurons, and can be found in most bodily fluids including blood, urine, saliva, amniotic fluid, breast milk, thoracic and abdominal effusions, and ascitic fluid (1). The involvement of exosomes in disease is broad, and includes: cancer, autoimmune and infectious disease, hematologic disorders, neurodegenerative diseases, and cardiovascular disease. Proteins frequently identified in exosomes include membrane transporters and fusion proteins (e.g., GTPases, annexins, and flotillin), heat shock proteins (e.g., HSC70), tetraspanins (e.g., CD9, CD63, and CD81), MVB biogenesis proteins (e.g., alix and TSG101), and lipid-related proteins and phospholipases. The exosomal lipid composition has been thoroughly analyzed in exosomes secreted from several cell types including DCs and mast cells, reticulocytes, and B-lymphocytes (1). Dysregulation of microRNAs of pathways observed in numerous pathologies (5, 10, 12, 21, 27, 35, 37) including cancers (30), particularly, colon, pancreas, breast, liver, brain, lung (2, 6, 17-20, 30, 33-36, 38, 39). Following these considerations, it is important that we characterize the content of exosomal cargo to gain clues to their biogenesis, targeting, and cellular effects which may lead to identification of biomarkers for disease diagnosis, prognosis and response to treatment (42).

We might continue in pursuit of a particular noteworthy exosome, the NLRP3 inflammasome, which is activated by a variety of external or host-derived stimuli, thereby, initiating an inflammatory response through caspase-1 activation, resulting in inflammatory cytokine IL-1b maturation and secretion (43).
Inflammasomes are multi-protein signaling complexes that activate the inflammatory caspases and the maturation of interleukin-1b. The NLRP3 inflammasome is linked with human autoinflammatory and autoimmune diseases (44). This makes the NLRP3 inflammasome a promising target for anti-inflammatory therapies. The NLRP3 inflammasome is activated in response to a variety of signals that indicate tissue damage, metabolic stress, and infection (45). Upon activation, the NLRP3 inflammasome serves as a platform for activation of the cysteine protease caspase-1, which leads to the processing and secretion of the proinflammatory cytokines interleukin-1β (IL-1β) and IL-18. Heritable and acquired inflammatory diseases are both characterized by dysregulation of NLRP3 inflammasome activation (45).
Receptors of innate immunity recognize conserved moieties associated with either cellular damage [danger-associated molecular patterns (DAMPs)] or invading organisms [pathogen-associated molecular patterns (PAMPs)](45). Either chronic stimulation or overwhelming tissue damage is injurious and responsible for the pathology seen in a number of autoinflammatory and autoimmune disorders, such as arthritis and diabetes. The nucleotide-binding domain leucine-rich repeat (LRR)-containing receptors (NLRs) are PRRs are found intracellularly and they share a unique domain architecture. It consists of a central nucleotide binding and oligomerization domain called the NACHT domain that is located between an N-terminal effector domain and a C-terminal LRR domain (45). The NLR family members NLRP1, NLRP3, and NLRC4 are capable of forming multiprotein complexes called inflammasomes when activated.

The (NLRP3) inflammasome is important in chronic airway diseases such as asthma and chronic obstructive pulmonary disease because the activation results, in pro-IL-1β processing and the secretion of the proinflammatory cytokine IL-1β (46). It has been proposed that Activation of the NLRP3 inflammasome by invading pathogens may prove cell type-specific in exacerbations of airway inflammation in asthma (46). First, NLRP3 interacts with the adaptor protein ASC by sensing microbial pathogens and self-danger signals. Then pro-caspase-1 is recruited and the large protein complex called the NLRP3 inflammasome is formed. This is followed by autocleavage and activation of caspase-1, after which pro-IL-1β and pro-IL-18 are converted into their mature forms. Ion fluxes disrupt membrane integrity, and also mitochondrial damage both play key roles in NLRP3 inflammasome activation (47). Depletion of mitochondria as well as inhibitors that block mitochondrial respiration and ROS production prevented NLRP3 inflammasome activation. Futhermore, genetic ablation of VDAC channels (namely VDAC1 and VDAC3) that are located on the mitochondrial outer membrane and that are responsible for exchanging ions and metabolites with the cytoplasm, leads to diminished mitochondrial (mt) ROS production and inhibition of NLRP3 inflammasome activation (47). Inflammasome activation not only occurs in immune cells, primarily macrophages and dendritic cells, but also in kidney cells, specifically the renal tubular epithelium. The NLRP3 inflammasome is probably involved in the pathogenesis of acute kidney injury, chronic kidney disease, diabetic nephropathy and crystal-related nephropathy (48). The inflammasome also plays a role in autoimmune kidney disease. IL-1 blockade and two recently identified specific NLRP3 inflammasome blockers, MCC950 and β-hydroxybutyrate, may prove to have value in the treatment of inflammasome-mediated conditions.

Autophagosomes derived from tumor cells are referred to as defective ribosomal products in blebs (DRibbles). DRibbles mediate tumor regression by stimulating potent T-cell responses and, thus, have been used as therapeutic cancer vaccines in multiple preclinical cancer models (49). It has been found that DRibbles could induce a rapid differentiation of monocytes and DC precursor (pre-DC) cells into functional APCs (49). Consequently, DRibbles could potentially induce strong innate immune responses via multiple pattern recognition receptors. This explains why DRibbles might be excellent antigen carriers to induce adaptive immune responses to both tumor cells and viruses. This suggests that isolated autophagosomes (DRibbles) from antigen donor cells activate inflammasomes by providing the necessary signals required for IL-1β production.

The Hsp90 system is characterized by a cohort of co-chaperones that bind to Hsp90 and affect its function (50). The co-chaperones enable Hsp90 to chaperone structurally and functionally diverse client proteins. Sahasrabudhe et al. (50) show that the nature of the client protein dictates the contribution of a co-chaperone to its maturation. The study reveals the general importance of the cochaperone Sgt1 (50). In addition to Hsp90, we have to consider Hsp60. Adult cardiac myocytes release heat shock protein (HSP)60 in exosomes. Extracellular HSP60, when not in exosomes, causes cardiac myocyte apoptosis via the activation of Toll-like receptor 4. the protein content of cardiac exosomes differed significantly from other types of exosomes in the literature and contained cytosolic, sarcomeric, and mitochondrial proteins (21).

A new Protein Organic Solvent Precipitation (PROSPR) method efficiently isolates the EV repertoire from human biological samples. Proteomic profiling of PROSPR-enriched CNS EVs indicated that > 75 % of the proteins identified matched previously reported exosomal and microvesicle cargoes. In addition lipidomic characterization of enriched CNS vesicles identified previously reported EV-specific lipid families and novel lipid isoforms not previously detected in human EVs. The characterization of these structures from central nervous system (CNS) tissues is relevant to current neuroscience, especially to advance the understanding of neurodegeneration in amyotrophic lateral sclerosis (ALS), Parkinson’s disease (PD) and Alzheimer’s disease (AD)(15). In addition, study of EVs in brain will enable characterization of the degenerative posttranslational modifications (DPMs) occurring in those proteins.
Neurodegenerative disease is characterized by dysregulation because of NLRP3 inflammasome activation. Alzheimer’s disease (AD) and Parkinson’s disease (PD), both neurodegenerative diseases are associated with the NLRP3 inflammasome. PD is characterized by accumulation of Lewy bodies (LB) formed by a-synuclein (aSyn) aggregation. A recent study revealed that aSyn induces synthesis of pro-IL-1b by an interaction with TLR2 and activates NLRP3 inflammasome resulting in caspase-1 activation and IL-1b maturation in human primary monocytes (43). In addition mitophagy downregulates NLRP3 inflammasome activation by eliminating damaged mitochondria, blocking NLRP3 inflammasome activating signals. It is notable that in this aberrant activation mitophagy downregulates NLRP3 inflammasome activation by eliminating damaged mitochondria, blocking NLRP3 inflammasome activating signals (43).

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