Exam 2 Review - October 12th, 2023 - PDF

10/14/23 1 EXAM 2 REVIEW 1 Carlos A. Barrero M.D. Assistant Professor School of Pharmacy-Temple University 2 October 12th , 2023 2 WHAT ARE DRUG DELIVERY SYSTEMS? § Drug delivery systems (DDS) are a formulation strategy that are used to modulate the disposition of drugs by: § Prolonging pharmaco...

10/14/23 1 EXAM 2 REVIEW 1 Carlos A. Barrero M.D. Assistant Professor School of Pharmacy-Temple University 2 October 12th , 2023 2 WHAT ARE DRUG DELIVERY SYSTEMS? § Drug delivery systems (DDS) are a formulation strategy that are used to modulate the disposition of drugs by: § Prolonging pharmacokinetics § Minimizing uptake into off-target tissues § Selectively targeting to sites of disease § Most particle-based DDS are lipid or protein-based complexes ~80-200 nm in diameter § This is similar in size to a virus! 3 CLINICALLY-USED DRUG DELIVERY SYSTEMS § Nanoparticles are used clinically to deliver small molecule drugs, mRNA, and siRNA § Approved therapeutic areas include oncology, infectious diseases, COVID-19, and hereditary transthyretin-mediated amyloidosis § Most approved carriers are lipid-based 4 DDS-INNATE IMMUNE INTERACTIONS 5 DDS CAN BE ENGINEERED TO EVADE OR HARNESS THE INNATE IMMUNE SYSTEM Key Points for Today: 1.Selection of appropriate DDS properties can bias particles towards or against immune recognition 6 7 1 5 1.Selection of appropriate DDS properties can bias particles towards or against immune recognition 2.This can be used to promote delivery to specific organs and/or cells and bias response to therapy 6 EVADING THE INNATE IMMUNE SYSTEM 7 FIRST IMMUNOLOGICAL BARRIER: COMPLEMENT Key Points for Today: 1.The complement system functions very efficiently on surfaces (e.g, lipid membranes) 2.Release of C3a and C5a lead to further activation of the innate immune system and can cause anaphylactoid reactions 3.There are numerous regulators of the complement pathway that can prevent deposition of C3 and/or release of C3a/C5a including Factors H and I 4. 8 COMPLEMENT-ASSOCIATED DDS TOXICITIES § Complement deposits on DDS via several mechanisms (properidin, antibody, PRR) § Release of anaphylatoxins (C3a, C5a) can promote a significant response termed Complement Activation Related PseudoAllergy (CARPA) § Most severe symptoms are cardiovascular in nature, including systemic hypotension, pulmonary hypertension, edema, elevated thromboxane B2, etc. § Slow infusions appear to reduce the severity of CARPA 9 ENGINEERING DDS TO ELIMINATE CARPA § Attachment of a natural complement regulator (Factor I) to liposomes is a strategy to reduce complement-associated side effects § Factor I: § Reduces C3 deposition on liposomes § Reduces release of C3a and C5a in plasma 10 § Reduces C3 deposition on liposomes § Reduces release of C3a and C5a in plasma § Eliminates liposome-induced cerebral hypoperfusion 10 MACROPHAGES ELIMINATE DDS FROM CIRCULATION § Macrophages in contact with the bloodstream are the primary elimination site for DDS § We refer to this as the reticuloendothelial system (RES) § Kupffer cells in the liver take a large fraction (>90%) of injected DDS § Spleen, lungs, and bone marrow also represent sites of elimination § Significant effort has been paid to evading macrophage-dependent elimination 11 POLYETHYLENE GLYCOL (PEG) SLOWS ELIMINATION § Grafting PEG onto the surface of liposomes helps evade recognition by the innate immune system § One key mechanism is slowing of opsonization – reduced complement activation § We refer to these as ‘stealth’ liposomes 12 EFFECTS OF PEGYLATION ON DDS BEHAVIOR § PEGylation significantly prolongs pharmacokinetics of liposomal drugs § PEGylation reduces uptake in clearance organs, particularly spleen and liver § § PEGylation reduces the amount of protein that opsonizes nanoparticles § PEGylation alters the content of the protein corona 13 SELF-MIMICRY TO EVADE THE INNATE IMMUNE SYSTEM § Cells display CD47 on their surface as a ‘don’t eat me’ signal to macrophages via SIRPα binding § Nanoparticles displaying CD47 (or a fragment thereof) on their surface may behave similarly § CD47 may be a viable alternative to PEG for prolonging the half-life of DDS 14 15 behave similarly § CD47 may be a viable alternative to PEG for prolonging the half-life of DDS 14 HARNESSING THE INNATE IMMUNE SYSTEM 15 NEUTROPHIL MARGINATION § Neutrophils transiently associate with the vessel wall in organs including the lungs in a process termed margination § ~50% of the intravascular pool of neutrophils are in this marginated pool § Local inflammation and injury can lead to dramatic increases in this pool 16 PATTERN RECOGNITION BY MARGINATED NEUTROPHILS § Under inflammatory conditions, marginated neutrophils are primed to respond to further danger § Following induction of inflammation in mice using lipopolysaccharide (TLR4 signal), the pulmonary marginated neutrophil pool responds to specific patterns § Nanomaterials displaying agglutinated protein on the surface have high levels of lung uptake § This process was complement-dependent 17 ISCHEMIC STROKE: NOT JUST LOSS OF BLOOD FLOW § The initial insult in ischemic stroke is vessel occlusion, typically by a blood clot § Primary treatment must focus on reperfusion (tPA, mechanical thrombectomy) § Secondary damage due to oxidative stress and inflammation can persist for weeks after stroke 18 THE BLOOD-BRAIN BARRIER IN STROKE § The blood-brain barrier exhibits time-dependent increases in permeability following ischemic stroke § Inflammatory processes in the acute and subacute phases include endothelial activation § This includes upregulation of cell adhesion molecules that play a role in immune 19 activation § This includes upregulation of cell adhesion molecules that play a role in immune cell recruitment 19 CELLS INVOLVED IN POST-STROKE INFLAMMATION § Endothelial activation and chemokine release promotes migration of immune cells into the brain following stroke § These cells can promote tissue repair or exacerbate damage based on their phenotype § Shifting macrophages from M1 to M2 could promote tissue repair § Immune cells can be recruited from circulating pools or from lymphoid tissues 20 VASCULAR TARGETED BRAIN DRUG DELIVERY § Intrastriatal TNF-α injection mimics post-stroke neurovascular inflammation § This allows us to study inflammation in isolation in a high throughput manner § Targeting to VCAM-1 (natural ligand for leukocyte VLA-4) provides selectivity for brain endothelium § Does this translate into relevant stroke models? 21 VCAM TARGETING IN ISCHEMIC STROKE § VCAM targeting provides selectivity for the injured hemisphere in stroke § Delivery of dexamethasone using VCAM reduced infarct volume § Targeting to natural homing pathways for leukocytes seems to be a viable delivery strategy. 22 DISTAL ORGANS RESPOND TO DANGER SIGNALS 23 TARGETING MIGRATING LEUKOCYTES § Coating nanoparticles with antibodies binding to leukocytes promotes: § Rapid uptake and elimination from lungs § Steady delivery to the inflamed brain § Essentially all of the nanoparticles in the brain are found in monocytes and 24 23 § Steady delivery to the inflamed brain § Essentially all of the nanoparticles in the brain are found in monocytes and neutrophils § Selective targeting of migrating innate immune cells! 24 TARGETING MIGRATING LEUKOCYTES TO TREAT INFLAMMATION § Brain inflammation is characterized by edema (fluid buildup), which can be tracked using albumin accumulation § Liposomes were loaded with a corticosteroid (dexamethasone) and targeted to leukocytes § Treatment effectively reversed edema and polarized macrophages to the antiinflammatory M2 phenotype 25 SUMMARY § The innate immune system (both proteins and cells) plays a critical role in the pharmacokinetics and toxicities of drug delivery systems § Nanoparticles can be engineered to evade the innate immune system using several approaches § Complement inhibitors – prevention of anaphylaxis and macrophage uptake § PEGylation – evasion from opsonization § Self-mimicry – ‘don’t eat me signals’ to macrophages § Under inflammatory conditions, nanoparticles can be engineered to harness the innate immune response for selective delivery § Marginated neutrophils – pattern response to agglutinated protein § Endothelial cell activation – selective delivery in stroke § Leukocyte migration – hitchhiking to sites of injury 26 27 28 29 30 31 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 61 62 63 64 65 66 67 68 69 70 71 72 73 74 T cell recognition of a peptide – MHC complex ØAntigen presenting cells (APC) capture antigens from its site of entry or production and bring it to the lymphoid organs where naïve T lymphocytes are located. ØMajor histocompatibility complex (MHC) molecules are on cells identifying as self or non-self. There are two classes of MHC molecules ØMHC I found on all nucleated cells; MHC II found on macrophages, dendritic cells and B cells. 75 76 Properties of Antigen Presenting Cells (APC) 77 Functions of APC 78 79 80 81 75 76 77 Functions of APC 78 Properties and Functions of APCs 79 Role of Dendritic Cells 80 Role of dendritic cells in antigen capture and presentation 81 Map Of The Human MHC Genes 82 MHC molecule expression ØClass I molecules are expressed on all nucleated cells ØThey provide a display system for viral and tumor antigens, so these antigens can be recognized by CTLs and the antigen-producing cells can be killed ØClass I molecule expression is increased by the type I interferons IFN-𝛂 and IFN𝛃, which are produced during the early and innate immune response to many viruses ØClass II molecules are expressed only on dendritic cells, B lymphocytes, macrophages, thymic epithelial cells ad a few other cell types ØClass II molecules are regulated by cytokines and other signals in different cells. IFN-𝞬 is the principal cytokine involved in the stimulating expression of class II molecules in APCs such as DCs and macrophages. ØIFN-𝞬 provides a mechanism by which innate immunity promotes adaptive immunity, by increasing class II MHC expression on APCs, and provides an amplicafication mechanism in adaptive immunity. Ø Ø 83 Enhancement of class II MHC molecule expression by interferon-γ ØIFN-γ, produced by NK cells and other cell types during innate immune reactions to microbes or by T cells during adaptive immune reactions, stimulates class II MHC expression on APCs and thus enhances the activation of CD4+ T cells. ØIFN-γ and type I interferons have a similar effect on the expression of class I MHC 84 83 MHC expression on APCs and thus enhances the activation of CD4+ T cells. ØIFN-γ and type I interferons have a similar effect on the expression of class I MHC molecules and the activation of CD8+ T cells. 84 85 Class I MHC Molecules ØClass I molecules are composed of a polymorphic α chain noncovalently attached to the nonpolymorphic β2-microglobulin (β2m). ØThe ribbon diagram (right) shows the structure of the extracellular portion of the HLA-B27 molecule with a bound peptide. 86 Class II MHC Molecules ØClass II molecules are composed of a polymorphic α chain noncovalently attached to a polymorphic β chain. ØThe ribbon diagram (right) shows the structure of the extracellular portion of the HLA-DR1 molecule with a bound peptide. Ø 87 Polymorphic residues of MHC molecules ØThe polymorphic residues of class I molecules are confined to the α1 and α2 domains, where they contribute to variations among different class I alleles in peptide binding and T cell recognition. ØThe polymorphic residues of class II molecules are located in the α1 and β1 segments, in and around the peptide- binding cleft, as in class I MHC molecules Ø Ø 88 Processing of Protein Antigens ØThe mechanisms of antigen processing are designed to generate peptides that have the structural characteristics required for associating with MHC molecules, 88 ØThe mechanisms of antigen processing are designed to generate peptides that have the structural characteristics required for associating with MHC molecules, and to place these peptides in the same cellular location as newly synthesized MHC proteins with available peptide-binding clefts. ØProteins that are present in the cytosol are degraded by proteasomes to yield peptides that are displayed on class I MHC molecules, while proteins that are ingested from the extracellular environment and sequestered in vesicles are degraded in lysosomes (or late endosomes) to generate peptides that are presented on class II MHC molecules 89 The Class I MHC Pathway for Processing and Presentation of Cytosolic Proteins 90 91 The Class II MHC Pathway for Presentation of Proteins Degraded in Lysosomes ØMost class II MHC–associated peptides are derived from protein antigens that are digested in endosomes and lysosomes in APCs ØInternalized proteins are degraded enzymatically in late endosomes and lysosomes to generate peptides that are able to bind to the peptide-binding clefts of class II MHC molecules. Ø 92 Cross Presentation ØCells infected with intracellular microbes, such as viruses, are ingested by dendritic cells, and the antigens of the infectious microbes are transported into the cytosol and processed in proteasomes and presented in association with class I MHC molecules to CD8+ T cells. Thus, dendritic cells are able to present endocytosed vesicular antigens by the class I pathway. Note that the same cross-presenting APCs may display class II MHC–associated antigens from the microbe for recognition by CD4+ helper T cells 93 94 95 APCs may display class II MHC–associated antigens from the microbe for recognition by CD4+ helper T cells 93 Nature of T Cell Responses 94 Immunodominance Of Peptides 95 Immune Receptor Family 96 T Cell Receptor for Antigen ØThe antigen receptor of MHC-restricted CD4+ helper T cells and CD8+ cytotoxic T lymphocytes (CTLs) is a heterodimer consisting of two transmembrane polypeptide chains, designated TCR α and β, covalently linked to each other by a disulfide bridge between extracellular cysteine residues ØThe antigen-binding portion of the TCR is formed by the Vβ and Vα domains. ØThe hypervariable segment loops that form the peptide- MHC binding site are at the top Ø Ø Ø 97 Binding of TCR to a MHC molecule ØThe V regions of the TCR α and β chains contain short stretches of amino acids where the variability between different TCRs is concentrated, and these form the hyper- variable or complementarity-determining regions (CDRs) ØThree CDRs in the α chain and three similar regions in the β chain together form the part of the TCR that specifically recognizes peptide-MHC complexes. Ø 98 99 100 101 102 103 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 Role of T cells in eradicating infections 115 116 117 118 119 120 121 122 123 124 125 Unlocking the power of Cytomine: a microfluidics-based high-throughput plasma cell screening platform for antibody discovery 1 Mathias Sanchez Machado 3rd year Ph.D. student in pharmaceutical sciences 2 October 1st, 2023 126 Overview of Antibody discovery platforms • Traditional screening methods for identifying therapeutic antibodies are laborintensive and suffer from limited throughput, hindering the rapid identification of potential candidates. • Development of microfluidic platforms in recent years offers advantages for antibody discovery, including: • Ability to screen millions of candidates rapidly (1-2 days vs 2-3 weeks for traditional hybridoma/B-cell screens). • Significant reduction in manual labor and reagent costs. 127 Cytomine Overview 128 129 127 Cytomine Overview 128 Cytomine workflow 129 Antibody discovery potential • FRET or antigen biding assays can be performed to detect and isolate ABSCs. • Cytomine integrates the encapsulation, screening, sorting, and isolation of antibody-secreting cells in a fully automated process • Apart from the incubation steps (37ºC) the entire workflow is carried out at -10ºC to ensure that cells do not divide and are not actively secreting during various steps in the process. • Previously validated Poisson distribution allows control of number of cells per picodroplet