Antibodies vs T Cell Receptors PDF

eL1: ANTIBODIES VS T CELL RECEPTORS IMMUNE SYSTEM Main Function - Protect body from foreign proteins and invading organisms o Eg. viruses, microbes - Immunity consists of innate (1st line; nonspecific) and adaptive (2nd line; specific) immune response o Both...

eL1: ANTIBODIES VS T CELL RECEPTORS IMMUNE SYSTEM Main Function - Protect body from foreign proteins and invading organisms o Eg. viruses, microbes - Immunity consists of innate (1st line; nonspecific) and adaptive (2nd line; specific) immune response o Both have cellular & humoral components STRUCTURE OF ANTIBODY MONOMER → 4 polypeptide chains (quaternary protein structure) →2 identical light chains (L) + 2 identical heavy chains (H) - Each antibody molecule contains: o 2 antigen-binding fragments (Fab; V) ▪ Fab binds to antigen ▪ Contributes to diversity in antigen specificity o 1 constant fragment (Fc; C) – same aa seq. for same abx class ▪ Fc → immune triggering module → responsible for biological effector fxns ▪ Binds to effector cells’ FC domain (NK cells, macrophages, neutrophils) - Disulfide bonds: interchain & intrachain → holds up 3D shape - Glycosylation: addition of carbohydrate chain to FC domain (post translational change) COMPLEMENTARITY-DETERMINING REGION (CDR) AND PARATOPE IN AN ANTIBODY COMPLEMENTARY-DETERMINING REGION (CDR) - Responsible for diversity of antigen specificities of antibodies produced by mature B cells - For each Fab arm, aa seq. in VL & VH is non-consecutively arranged to from 3 CDRs (CDR1, CDR2, CDR3) o Each chain: 3 CDRs → each Fab arm (VL & VH): 6 CDRs → 2 identical Fab arms: 12 CDRs per antibody monomer - CDRs on Fab arm establish contact w antigen; bind to epitope PARATOPE - Part of Fab region (aka antigen-binding site) - Found at tip end of each Fab arm - Each paratope = 6 CDRs (3 each from light & heavy chain) PROPERTIES OF AN ANTIBODY ANTIGEN AFFINITY Measure of strength of interaction btwn antibody (antigen binding site/paratope) & corresponding antigenic determinant (epitope) - High affinity antibody binds strongly & vice versa ANTIBODY SPECIFICITY Measure of goodness of fit btwn paratope & antigenic determinant - Indicates ability of paratope in antibody to distinguish similar & dissimilar antigens - Low specificity → cross reactivity (paratope reacts with >1 epitope) - High affinity usually = high specificity BINDING STRENGTH OF ANTIBODY 1. Affinity (@ 1 epitope) a. Strength of interaction ! a single antigenic site (eg IgG) 2. Avidity (>1 epitope) a. Strength with which an antibody binds to its target b. Comes into play if target contains multiple antigenic sites/epitopes (eg. IgM) T CELLS/ T LYMPHOCYTES T CELL RECEPTOR (TCR) COMPLEX FOR ANTIGEN RECOGNITION & ACTIVATION T Cell Receptors (TCR): - Found on surface of cells (cell surface receptor; has extracellular domains for ligand binding) - Composed of  and  chain (each encoded by specific gene); each composed of: o 2 extracellular domains (glycosylated) ▪ variable (V) region → binds to antigen ▪ constant (C) region → contains cysteine residues to form disulfide bond to link up  &  chain o a transmembrane region (non polar aa component) o a short cytoplasmic tail → cant signal ▪  &  chains are arranged to ensure antigen recognition/binding and docking onto the plasma membrane But tail too short to mediate signal transduction for T cell activation  ▪ → but associates with invariant (constant in all TCR complexes) CD3 adapter proteins (dimers) that help mediate signal transduction CD3, CD3 and CD3ζζ → forms octameric complex in plasma membrane - ITAM (immunoreceptor tyrosine-based activation motif) o Each TCR complex contains 10 ITAMs ▪ Epsilon, gamma & sigma CD3 monomers each contain 1 ITAM ▪ CD3 zeta monomer contains 3 ITAMS o Upon TCR-antigen binding, tyrosine residues in ITAM → phosphorylated (post-translational modifications) ▪ Initiates series of downstream T cell signaling events → T cell activation ANTIBODY VS TCR TCR is a member of the immunoglobulin superfamily (ie. Antibody; Ig) - Extracellular domains of TCR → resemble Fab domain on antibody - Variable (V) regions in  &  chains in TCR are comparable to VL & VH in Fab domain - Variable regions in both TCR and Fab domain in antibody are the antigen binding sites HYPERVARIABLE COMPLEMENTARITY-DETERMINIDNG REGIONS (CDRs) IN ANTIBODY & TCR CDRs in antibody & TCR → responsible for recognition and binding (both equally hypervariable) - TCR: 3 CDRs in each V & V → 6 CDRs total - Antibody: 3 CDRs in each VL & VH in each Fab domain → 12 CDRs total - TCR is like ½ an antibody monomer (has only 1 Fab arm) B LYMPHOCYTES (B CELLS) Development of virgin/naïve B cells (bone marrow) Progenitor B cells rearrange immunoglobulin (Ig) genes → clones of immature B cells expressing B cell antigen receptor (membrane form of Ig molecules) o Random recombination of Ig genes → each clone of immature B cells possess different aa seq. in variable regions of surface Ig → hypervariable CDRs housed in VL & VH regions for each B cell clone will be different & recognize different antigens o *independent of extrinsic antigen stimulation Virgin B cells expressing B cell receptors leave bone marrow → circulate or in secondary peripheral lymphoid tissues Upon encounter with pathogenic antigens 1. Virgin B cells activated, mature & start to produce & secrete 1st response antibody IgM 2. Gene rearrangement of constant regions in Fc domain on IgM 3. Class switching from IgM to IgG: Mature B cells switch from producing IgM to producing IgG 4. B cell clone still continues to undergo gene rearrangement to VL & VH regions in the Fab domain of IgG genes 5. Each B cell clone ends up a different hypervariable CDR → diff antigen specificity 6. IgGs produced by diff mature B cell clones contain diff CDRs that recognize diff epitope of an antigen T LYMPHOCYTES (T CELLS) Carry cell surface markers assigned to their lineage (acc to cluster of differentiation (CD) system) – mutually exclusive - CD4+ helper T cells - CD8+ cytotoxic/killer T cells Development of T lymphocytes - Progenitors travel from bone marrow to thymus (primary lymphoid tissue) for development into T lymphocytes o *independent of extrinsic antigen stimulation - T cells then localized in secondary peripheral lymphoid tissues (lymph nodes, spleen) → interact & respond to antigens presented T CELL RECEPTOR (TCR) DIVERSITY Diversity lies in V & V regions of TCR - Genetic recombination of DNA segments in genes encoding regions of TCR → unique combo of the segments for each recombined TCR expressed by individual somatic cells o V: generated by VJ recombination o V: generated by DJ followed by VDJ recombination - TCR diversity allows different T cells to recognize and bind to diff antigens presented by MHC molecules CDRs IN ANTIBODY & TCR - Responsible for antigen specificity - Allows for design & development of biopharmaceutical products aimed to treat diseases by achieving target specificity (less off- target effects/ADRs) T CELL MEMORY Adaptive all hv memory (B cells also hv) After an episode of infection - Big pool of different immature T cells - Some hv v high binding specificity to antigen - → antigen-driven expansion of antigen-specific T cells → expand and proliferate - Clears antigen; effector T cells die after combating w pathogen - Remaining antigen-specific T cells differentiate into memory T cells o Located in lymphoid tissues, bone marrow and specific organs depending on route of invasion o Memory T cells respond more quickly and binds to antigens with shorter lag time → faster and more potent T-cell mediated immune response upon repeated encounter w same antigen eL2: MAJOR HISTOCOMPATIBILITY COMPLEX (MHC) MHC (aka HLA)→ cell surface proteins needed for adaptive immune system to function - Main immune function: to bind peptide fragments (antigenic fragments) and display them for recognition by T cells → aids the immune system to distinguish self from non-self or foreign antigens ACTIVATION OF THE ADAPTIVE IMMUNE SYSTEM BY THE INNATE IMMUNE SYSTEM Adaptive immunity is made up of: 1. Cell-mediated response (T cells) a. Executed by cytotoxic T-lymphocytes (CTLs) → lyse infected cells → pathogen/antigen clearance 2. Humoral response (B cells) a. Antibodies secreted by plasma cells b. antibody recognizes & bind to specific epitopes expressed by pathogen c. followed by Fc domain in antibody binding to Fc receptor expressed by effector cells (ie. Macrophages, neutrophils, NK cells) d. activated effector cells engulf or lyse pathogen → pathogen/antigen clearance Eg. Bacterial infection 1. invading pathogen (bacteria cell) recognized by antigen-presenting cell (APC; macrophages and dendritic cells) 2. engulfed by phagocytosis → during which bacterial proteins get broken up into peptide fragements (antigenic fragments) 3. each MHC Class II protein binds to an antigenic fragment → peptide-MHC (pMHC) 4. pMHC then presented to helper T cells/ CD4+ cells MHC genes located on chromosome 6 (MHC region) – 3 subtypes → Class I, II, III (only I & II involved in antigen presentation) - only ~50% of genes in this region are encoding proteins having immune functions MHC CLASS I VS CLASS II MOLECULES MHC CLASS I MOLECULES MHC CLASS II MOLECULES Presented in all nucleated cells & platelets (hence absent in RBCs) Present in antigen presenting cells (APCs) including macrophages, dendritic cells and B cells Bind to peptide fragment of endogenous antigens Binds to peptide fragment of exogenous antigens Present antigen (in form of p-MHC I expressed on cell surface) to Present antigen (in form of p-MHC II expressed on surface of APC) CD8+ cytotoxic T cells (CTL) to CD4+ helper T cells (CTL) Types of endogenous antigens processed: Type of exogenous antigens processed: - normal self antigen (damaged/old cells, wear & tear) - usually foreign antigens belonging to invading pathogens - viral components from virus-infected cell - neoantigens exclusively expressed by cancer cell PROTEASOME: protein complex ubiquitously (existing everywhere at the same time) present in cells to degrade unwanted/damaged proteins by proteolysis - consists of proteases that execute cleavage of peptide bonds → proteins degraded to peptide fragments (~15 aas) WHY CD8+ AND CD4+ T CELLS INTERACT SPECIFICALLY WITH PEPTIDE-MHC I & II PROTEINS RESPECTIVELY? CD8+ and CD4+ T cells respectively express both: - CD8/4 receptor & T cell receptor o CD receptor acts as 1st point of recognition CD8 & CD4 receptors expressed on the surface of CD8+ and CD4+ T cells respectively dock to MHC I and MHC II molecule present in the peptide-MHC I/II proteins → brings proximity → respective TCR expressed on cell surface binds to antigenic peptide and MHC I/II molecules → binding → triggers signaling events involving activated T cell receptor HOW IS THE LEVEL OF MHC MOLECULE EXPRESSION REGULATED? Level of MHC molecules expression affects extent of T cell activation - Cytokines (humoral) regulate expression of MHC I & II molecules - Interferons known to increase expression of MHC I & II molecules → help activate appropriate T cells in time of infection o IFN produced as early response to viral infection → transcription → expression of MHC molecules o IFN is an immunomodulatory cytokine → expression of MHC to activate T cells KEY CHARACTERISTICS OF MHC I & II MOLECULES 1. MHC is polygenic (made up of multiple genes) a. In human genome → different MHC Class I & II genes → in each individual, diff sets of MHC class I & II molecules possessing different peptide. Binding specificities are expressed b. Implication → different sets of MHC Class I & II molecules can present diff antigens to T cells → broad coverage against different antigens present in an invading pathogen 2. MHC is polymorphic a. Each MHC gene have different alleles → products of each MHC allele differ from one another by ~20 aa → differences usually found in peptide binding domains of MHC molecules → varied antigen recognition by T cells in diff individuals b. Implication i. → extensive polymorphism → diversity of MHC molecules expressed by each individual brought about by polygeny ii. Ensures adaptive immune responses mediated by activated T & B cells provide broad coverage against diff antigens present in an invading pathogen IC1: BIOPHARMACEUTICAL PRODUCTS DERIVED FROM ENDOCRINE & IMMUNE SYSTEMS I IMMUNOTHERAPY Treatment of disease by intervening the immune system TYPES OF IMMUNOTHERAPIES ACTIVATION SUPPRESSION Involve use of agents that augment and/or reestablish the immune Involve use of agents that reduce or suppress an immune response system’s ability to prevent & fight the disease Eg. vaccines EXAMPLES OF THERAPEUTIC STRATEGIES IN IMMUNOTHERAPY: 1. Cytokines a. Humoral component of innate & adaptive immune response 2. Antibodies a. Humoral component of adaptive immune response 3. T cells a. Include cell-based immunotherapy and checkpoint inhibitors 4. Cancer vaccines and many more a. Due to advances in molecular cloning techniques and biotechnology CYTOKINE THERAPIES/ANTI-CYTOKINE THERAPIES EXPLOITING CYTOKINES’ BIOLOGICAL ACTIONS IN TREATMENT OF DISEASES Cytokines have roles in innate and adaptive immune response → in relation to immunity, inflammation and hematopoiesis Recombinant cytokines or anti-cytokine therapeutics have been utilized to treat/manage clinical conditions → used in infectious diseases, cancer, wound healing, inflammatory diseases etc CLASSES OF CYTOKINES INTERFERONS Produced by cells in response to viral infections, tumors and other biological inducers Eg. IFN, ,  Promote an antiviral state in other neighboring cells, help regulate immune response INTERLEUKINS Produced by leukocytes Eg. IL2, 11 Affect growth & differentiation of hematopoietic & immune cells Regulate immunity, inflammation, hematopoiesis etc COLONY Stimulate cellular division & differentiation of blood cells from bone marrow precursors STIMULATING → production of immune response cells FACTORS (CSF, EPO)  CHEMOKINES Stimulate leukocyte chemotaxis & activation (movement of immune cells in response to chemical stimuli) OTHERS Usually only present in small amts under constitutive conditions eg. tumor production by activated macrophages in response to inflammatory conditions (cytokine stoerm) necrosis factor, Usually considered “bad guy” due to its pro-inflammatory and pro-apoptosis actions TNF Known to be an adipokine produced by adipocytes Contributes to insulin resistance in DM Type II PROPERTIES & MECHANISM OF ACTION OF CYTOKINES General Facts - Usually small proteins of 2 cytokines separately 4. antagonism: ≥2 cytokines work against each other INTERFERONS Produced in response to viral infections, tumors (IFN , , y) Promote antiviral state in neighbouring cells Help regulate the immune response INTERLEUKINS Produced by leukocytes Affect growth & differentiation of hematopoietic & immune cells Regulate immunity, inflammation, hematopoiesis etc COLONY Stimulate cellular division & differentiation of blood cells from bone marrow precursors STIMULATING → production of immune response cells FACTORS (CSF, EPO) CHEMOKINES Stimulate leukocyte chemotaxis & activation (movement of immune cells in response to chemical stimuli) OTHERS eg. tumor necrosis factor, TNF USE AS THERPEUTICS: cytokine therapy 1. Interferons (IFNs) Interfere with viral replication released by virally infected cells (zombies) → prevent infection of other cells o claim sick → establish antiviral state Biological effect: o induction of cellular resistance to viral attack o regulation of most aspects of immune function o regulation of growth and differentiation IFN- IFN- IFN-y INFO Produced by Expressed by somatic cells “add on” therapy leukocytes Glycosylation not essential Produced by T-cells Immunomodulatory cytokine ACTION Antiviral & (killing) -Inhibits IFN-y activity - activates resting macrophages & antiproliferative monocytes to increase phagocytic activity -Slows growth of attacking immune activity (work harder) cells - induces macrophages to express cytokines (IL-2, TNF-å) and -Stops production of myelin- immunoglobulin Fc receptors destroying cmpds → immunostimulation  THERAPY Antiviral or anticancer Effective for MS (multiple sclerosis) Only for immunomodulatory role therapy - autoimmune disease → Upregulation of (CNS) No direct antiviral-inducing activity immune system - immune cells attack myelin 2. Interleukins (IL) produced by lymphocytes, monocytes etc mostly glycosylated (small) → involved in regulation of immune cell growth, differentiation & maturation short circulation times; production regulated (+ & - loops) biological effects are varied & complex IL2 IL11 INFO T cell growth factor Thrombopoietic growth factor Synthesized & secreted by T cells Produced by fibroblasts and bone marrow stromal cells ACTION Immunomodulatory properties: Stimulates proliferation of hematopoietic stem - stimulates growth, differentiation & cells activation of T, B & NK cells only acts on cells expressing IL2 receptors Induced megakaryocyte maturation Anticancer drug → increased platelet formation - lasts 8-10 days PEG-YLATED RECOMBINANT IFNs & ILs linear or branched chain polyethylene glycol (PEG) conjugated to cytokines - non-toxic, inert, non-immunogenic Advantages - enhance thermal & physical stability - prolonged circulation ½ lives → sustained effect - slow release effect → controlled drug release - branched chains protect proteins from proteolytic degradation o prevent protein breakdown - reduced immunogenicity & antigenicity o decrease ability of cells to produce immune response o decrease recognition by antibodies 3. Hematopoietic Growth Factors eg. colony-stimulating factor (CSF), erythropoietin (EPO) and some IL single chain polypeptides; glycoproteins Action o regulates proliferation & differentiation of pluripotent hematopoietic stem cells to functional immunologically active cells ▪ promotes hematopoiesis Application o restore severe deficiency of hematopoietic cells resulted from chemotherapy or radiation treatment Myeloid growth factors 1. G-CSF (granulocyte) a. increase neutrophils b. treatment: chemotherapy-induced neutrocytopenia Glycosylation increases ½ life Lenograstim more potent and stable than filgrastim 2. GM-CSF (granulocyte-macrophage) a. increase neutrophil, eosinophil and monocyte counts b. treatment: accelerate myeloid cell recovery after bone marrow transplantation, antiviral therapy (AIDS) antifungal, antiviral, Crohn’s disease etc To describe activation of adaptive immune system by innate immune system (Part 2). Adaptive - activated by helper T cells → cell-mediated response: cytotoxic T-cells (CTLs) → lyse infected cells → humoral response: antibodies secreted by plasma cells B LYMPHOCYTES (B CELLS) develops as virgin B cells in bone marrow → circulate and in secondary peripheral lymphoid tissues B cells rearrange immunoglobulin (Ig) genes → immature B cells express B cell antigen receptor (each diff antigen despite same parents) immature B cells→produce IgM antibodies Development of virgin B cells is independent of extrinsic antigen stimulation To gain an overview of the cellular events and affinity of antibodies during the course of antibody response to antigen response (Part 2). 1º response: - virgin B cells produces IgM class antibodies - “class switching” (gene rearrangement of constant regions of IgM) induced - once done → produces IgG antibodies ≥2º response: IgG persist but IgM still produced (all virgin B cells will produce IgM first) → always have small peak of IgM after 1st response AFFINITY OF ANTIBODY RESPONSE TO ANTIGEN Affinity of IgG increases progressively during response → B cells become more ‘trained’ - esp aft low doses of antigen - affinity maturation through gene shuffling Affinity maturation most pronounced aft 2º challenge w antigen To gain an overview of how antibodies exert their action (Part 3). 1. Antigen-Antibody binding binds specifically to 1 or a few closely related antigens involves Fab (antigen binding domain) frequently has no biological effects: antitoxin effect 2. Effector Functions secondary effector fxn of antibodies→ result in biological effects involves Fc: o A: Complement-dependent cytotoxicity (CDC) Fc binds to components of complement system (complement fixation) o B: Antibody-dependent cellular cytotoxicity (ADCC) Fc binds to Fc receptors on immune cells ▪ phagocytic macrophages, neutrophils, NK cells  Fab bind to antigen → configurational change to Fc → Fc bind to Fc receptor →activate cell expressing receptor → mobilize inert immunity To distinguish between polyclonal and monoclonal antibodies (Part 3). Polyclonal Antibodies (produced by B cell clones) - surface Ig (cell surface receptors) on B cells recognize only 1 epitope of the Ag o ≥1 epitope on Ag → activates more than 1 B cell clone Igs collected from serum: - produced by a range of B cell clones that have responded to diff epitopes of the antigen - differ in specificity and affinity for the Ag o Ig class differs too (mixture of IgM and IgG) Antibodies produced by past antigenic challenges are polyclonal - made using several different immune cells Antiserum Animal sera containing antibodies raised by immunizing animal with particular antigen -ve: immunogenicity issues +ve: cheap Production: whole blood from immunized animal collected → clot (add coagulant) → clotting factors removed → serum obtained as supernatant (rich in antibodies) Application - given to patients for passive immunization Monoclonal Antibodies produced from only 1 B-cell clone, recognize just 1 epitope of the antigen - predictable therapeutic outcomes/efficacy ☺high specificity - recombination protein purification work ☺high homogeneity → highly reproducible effects - diagnostic test kits - target specific therapeutic molecules for treatment of diseases with less side effects To describe the different types of monoclonal antibodies and antibody derivatives(Part 3). Murine Monoclonal Antibodies limitation of murine monoclonal antibodies - induce immunogenicity (evokes immune response) - fail to trigger a number of effector fxns - shorter ½ lives (30-40h) SOLUTION: Chimeric Monoclonal Antibodies CH and CL in Fc and Fab domains contribute predominantly to immunogenicity Solution: → replace aa sequences on CH and CL of murine Mab that are not essential for antigen binding with human sequences - antigen-binding sequences in VH and VL fragments conserved eg of site directed mutagenesis → Chimeric Mab retains antigen selectivity and affinity similar to parent murine Mab ~75% human → decrease immunogenicity Humanized Monoclonal Antibodies Replace ALL mouse aa sequences except hypervariable CDR* domains of Ig in chimeric Mabs - residue within the VH VL fragments → Humanized Mabs >90% human →reduced immunogenicity compared to chimeric *CDR (complementarity-determining reigon) is the hypervariable region of Ig; responsible for antigen specificity and affinity of Ab → binds to epitope (non-covalent) Recombinant Human Monoclonal Antibodies to genetically engineer mammalian host cells → recombinant human Mabs - entirely human → immunogenicity problem resolved BUT high cost $$$$  ANTIBODY DERIVATIVES Applications that may not need Fc domain: (only needed if want to bind to Fc receptor to activate effector cell – inert immunity) 1. Antagonism of enzyme actions by binding to enzyme active site → enzyme inhibition a. highly specific Fab 2. To neutralize receptor ligands (hormones, cytokines etc) To counteract overproduction of cytokines a. Fab binds to cytokine receptors 3. To neutralize toxins (eg. snake venom) Sometimes Fc domain is needed in therapeutic product not for effector fxn but for its longer half-life in circulation Fab domains for antigen binding 1. Ig conjugate selective 2x Fab, 1x Fc + Cytokine/Radioisotope/Toxin → internalized → kill 2. F(ab’)2 no Fc 3. Fab no Fc Synthetic 4. Bispecific 2 distinct Fab arms/regions Bispecific T cell engagers (BiTEs) → can bind to 2 distinct epitopes; lack Fc domain 5. ScFv 100% synthetic small (can go through small spaces), short ½ life Single chain (all combined into 1 chain) 6. Trifunctional mAbs (Triomabs) recombinant DNA technology links 2 diff antigens tgt eg. anticancer → brings cells closer → Cytotoxic T cell + cancer cell = death → binds to 2 diff antigens while maintaining the capacity to mediate Fc-dependent effector fxns o possess Fc domain Eg. Catumaxomab o targets surface bound antigen EpCAM on cancer cells and T cell stimulatory receptor CD3 → brings cytotoxic T cells closer to cancer cells → stimulation of cytotoxic T cells to kill cancer cells o Fc domain req. to mediate complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) ---- end of PR1152 RECAP FUCOSYLATED VS DEFUCOSYLATED ABS In naturally occurring IgG, - Fc domain is N-linked glycosylated (2 N-linked oligosaccharide chains bound to the Fc region) - N-glycans attached to Asn297 in Fc domain in IgG are linked to fucose - The fucosylation at Fc domain → affinity of the Fc domain to bind to a subtype of activating Fc receptor found on effector cells → ADCC induction by effector cells → efficacy of fucosylated antibodies - In a pt’s body, endogenous fucosylated IgG competes with therapeutic Ab for binding to effector cells → inhibit elicitation of ADCC → therapeutic Ab registers clinical efficacy Defucosylated abs: - Removal of fucose from N-glycans attached to Asn297 - Enhanced affinity of defucosylated abs towards Fc domain → pr5eferential interaction with Fc than endogenous IgG with Fc → efficacy of defucosylated antibodies - Eg. anti-CD20 mAb obinituzumab; an ADCC enhanced version of fucosylated rituximab POLYCLONAL AND MONOCLONAL ANTIBODIES AS THERAPEUTICS Antiserum → obtained from animals; intendedly injected antigens into animals → reaction → polyclonal antibodies produced → blood collected, purified → plasma collected → clotting factors removed → SERUM - Highly conc in antibodies - IgG, IgM, IgE etc; big mixture of immunoglobulins - IgG → different Fab arms at variable regions; rearrangement of genes; CDRs are different → diff antigen specificity → broad spectrum - Eg. used as antidotes when poisoned → passive immunization (given when pathogen alr In body) o Active immunizations are vaccines like Covid-19, Hep B etc → used to arouse the immune system - DRAWBACK: immunogenicity issues T CELLS AS THERAPEUTICS OR TARGET OF THERAPIES (currently mainly applied for cancer tx) PRINCIPLE OF ADOPTIVE CELL TRANSFER (ACT) Tumour-infiltrating lymphocyte (TIL) therapy vs T cell receptor engineered T cell (TCR-T) therapy & Chimeric antigen receptor T cell (CAR-T) therapy 2nd line of immune cells (adaptive)→ possess memory; TIL lymphoid lineage → T & B cells - Prolonged treatment due to memory of the cells TIL THERAPY (surgery; anesthesia involved) Autologous tumor-infiltrating lymphocytes (TILs) isolated from excised tumor masses (from pt) are primed and expanded ex vivo, then infused back into pt → therapeutic TILs re-infiltrate the tumor, recognize tumor antigens & attack cancer cells o Autologous → from the same pt; not donated; surgery done to remove mass but risk of spread of cancer or inability to isolate/collect enough TILs Isolated TILs from pt’s tumor masses consists of T & NK cells Isolated T cells are polyclonal; have diverse antigen specificity Isolated TILs, mainly T cells are expanded in vitro by rapid expansion process (REP) – exposed to: o high dose IL2 → proliferation & development of TILs o anti-CD3 antibody → to activate CD3 in TCR ▪ T cells cant work alone; need to work w adapter proteins, CD3 ▪ T cells express TCR complex, anti-CD3 antibody specifically recognizes the CD3 adapter proteins in the TCR complex → binds → triggers CD3 adapter protein in TCR complex to activate ▪ Isolated T cells mostly not activated yet; hence REP primes them & exposes antigen to them for activation o irradiated feeder cells (usually autologous PBMCs [peripheral blood mononuclear cells] obtained from pt) ▪ irradiation – activate PBMCs with UV rays (?) – but not too long cos UV rays can be cytotoxic  ▪ moderate activation – in combat state → when undergo REP → expansion of population & expanded population will also be activated ☺ SAFE  for some pts, excised tumor masses are devoid of or contain v low quantities of TILS  expanded naturally occurring tumor-specific T cells are heterogenous possessing varying antigen specificity → TIL reinfusion may not be lethal enough to attack and eradicate cancer cells (low antigen specificity)  limited or none of the expanded naturally occurring tumor antigen-specific T cells possess high affinity TCR-T & CAR-T THERAPIES (only blood drawn) T cells isolated from pt’s peripheral blood (expanded if quantities not high enough for genetic manipulation → genetically modified under lab conditions → development of tumor antigen-specific T cells Generally engineered T cells (TCR or CAR) expanded in vitro → infused back into the pt → recognize tumor antigens and attack cancer cells TCR-T THERAPY Involves use of genetically modified T cells B cells don’t kill; their antibodies produced are polyclonal – T cells themselves however are polyclonal; they do the killing Variability → heterogenous & diverse Genes encoding V and V within the  &  chains that make up a T cell receptor (TCR) are tumor antigen-specific - Genes are cloned into retro- or lentiviral vectors → used to transduce T cells isolated from pt’s peripheral blood → genetically modified T cells less heterogenous o Becos aa sequence now dictated by the gene carried by viral vector - High transduction efficiency of retro- or lentiviral vectors ensures that reinfused T cells possess high tumour antigen specificity Manmade gene expresses tumor specific antigens present in cancer pt → housed in viral factor → transduced → high transduction efficiency % → almost all isolated T cells now carry SAME type of V& → reinfused → expansion of highly specific tumor T cells → therapeutic outcome o Endogenous T cell receptors STILL expressed; not replaced, manmade TCRs are added on the cell surface Eg of TCR-T antigen targets: - Carcinoembryonic antigen CEA in colorectal cancer, glycoprotein gp100 and melanoma antigen recognized by T cells 1 (MART-1) - But antigen targets are NOT exclusively expressed on cancer cells; cant differentiate from normal cells  ADVANTAGES DISADVANTAGES Engineered TCR-T cells possess full TCR complex → can recognize Engineered TCR-T cells expressing a particular tumor-specific TCR antigens expressed at both cell surface/tumor surface and within only limited for use in a pt subpopulation carrying a specific tumor cells/mass → can penetrate tumors → effective against solid MHC/HLA allele recognized by TCR and hematological tumors Compared to CAR-T cells, TCR-T cells use full TCR complex for Less safe than TIL therapy antigen recognition and signal transduction (CD3 adaptor proteins) 1. On-target off-tumor toxicity (TCR-T cells target normal tissue → can fully activate at low target cell antigen densitites expressing same antigen) →onset of signaling slow but of longer duration 2. Off-target toxicity (TCR-T cells not specific and cross-react with → execute more extended killing other antigenic fragments; lookalike antigens) ➔more effective than CAR-T therapy for cancer treatment 3. Cytokine-release syndrome (CRS) – infusion of TCR-T cells induce cytokine storm (not all cytokines are good; can go against normal cells; systemic inflammation; suppressive) 1 TCR has V and V → each chain has 3 CDRs (total 6) → each CDR has 1 point of contact with an epitope → total 6 contacts with antigen - CDR1: bind to each terminus of the peptide in peptide-MHC - CDR2: recognize and bind to MHC in peptide-MHC - CDR3: recognize and bind to peptide in peptide-MHC Strength of interaction btwn TCR & peptide-MHC contributes to extent of T cell activation and development of antigen-specific T cells as memory T cells - Strong interaction ideal → T cell activation & strong memory in case of repeat infection CAR-T THERAPY Involves use of genetically modified T cells Collection etc same as TCR-T but genetic engineering part differs Construction of chimeric antigen receptor (CAR): genetic sequence encoding for specific antigen-binding sites within VH and VL in Fab domain of an identified antibody (known to be targeting specifically a cell surface molecule of interest) is cloned into a retro- or lentiviral vector - Doesn’t take the structure of a TCR but a Fab domain of an antibody - A single polypeptide chain (scFv) 1. Find a specific enough antibody → identify 2. Narrow down to B-cell clone that is particularly producing this specific antibody  3. Extract RNA material → reverse transcribe to DNA material → sequencing 4. Narrow down nucleotide sequence encoding for aa seq of VH and VL T cells isolated from pt’s peripheral blood are transduced by vectors carrying CAR gene → transduced T cells express CAR on surface (CAR-T cells) → In vitro expansion of CAR-T cells → cells infused into pt to bind to antigens on cancer cells & kill them DESIGN OF GENERATIONS OF CAR-T CELLS For all generations, extracellular domain is only the scFv (single chain variable fragment) found in Fab arm of an immunoglobulin that is responsible for antigen recognition & binding Generation: 1. Intracellular domain contains only 1 signaling domain (CD3zeta) Signaling not strong enough to sustain CAR-T cell expansion, in-vivo survival etc Fail to execute potent antitumor activity clinically → weak signal 2. Intracellular domain contains 2 signaling domains (CD3zeta + additional co-stimulatory CD28 or 4-1BB domain) → 2nd activation signal upon target antigen recognition Stronger signaling → lasting in vitro proliferation & potent antitumor activity 3. Intracellular domain contains 3 signaling domains (CD3zeta + CD28 + 4-1BB domain) → stronger activation signal upon target antigen recognition for lasting in vitro proliferation and potent antitumor activity 4. Intracellular domain contains 3 signaling domains (CD3zeta + CD28 + 4-1BB domain) plus a transgene Upon target antigen binding, besides triggering stronger CAR signaling, transgene is activated to express cytokine (eg. IL12) Cytokine secreted by CAR-T cells exerts autocrine and/or paracrine effect on T cells at target site → activate more T cells to eliminate cancer cells a. Looks like gen 3, but with addition of transgene → 4th gen has main gene + 3 signaling domains + transgene (eg. encoding for IL12; cytotoxic effects → synergistic effect on killing cancer cells → activated when bind to recognized tumor antigen b. Transgene is driven by its own promoter but only activated when complex (main gene + domains) gets activated c. IL12: cytotoxic cytokine → autocrine or paracrine effects of T cells @ target site → mobilize activation of more T cells Can antigen-negative cancer cells be eliminated? Cancer cells are genetically heterogenous → mass of cancer cells → genetic instability - Some cancer cells may not have this specific antigen that CAR-T is expressing → will have some “antigen-negative cancer cells” unkilled → aft treatment rest → cancer cells also rest & become resistant → relapse! - IL12 broadly activates endogenous T cells → gives chance that antigen-negative cancer cells will be targeted as well ADVANTAGES DISADVANTAGES CAR-T cells recognize and bind to unprocessed tumor surface scFv (single polypeptide chain) may guide CAR-T cells into an antigen- antigens without MHC processing independent mechanism → failed therapy Do NOT possess full TCR complex → can recognize antigens -- unpredictable; CAR-T cells end up expressing different antigen :/? without MHC proteins scFv domain only binds to cell surface antigens → effective Less efficient than TCR-T therapy against hematological tumors (pts with acute lymphoid 1. Activated only @ higher target cell surface antigen densities leukemia respond v well) BUT not effective against solid 2. only 1 subunit (scFv) binding to target cell surface antigen →weaker tumors → TCR-T more broad CAR signaling & activation → execute faster killing function but lack extended killing TCR has  clinical efficacy than CAR BUT TCR is MHC Adverse effects: dependent → MHC has high genetic variability → risk that it - on-target off-tumor toxicity mainly limited to B cell aplasia (CD19 may not work on some patients → hence CAR IS PREFERRED specific CAR-T cells attack & kill normal B cells also expressing CD19 like the THERAPY for oncologists malignant CD19+Bcells) - off-target toxicity (not highly reported) - cytokine-release syndrome (CRS) induce CRS more than TCR-T therapy → but transient & not deadly PRINCIPLE OF IMMUNE CHECKPOINT INHIBITION T cells express checkpoint molecules proteins on their surface → ligands binding to these checkpoint molecules → triggers suppression of T cell activity - native function of checkpoint proteins o To counter overstimulation of T cell activity o To prevent autoimmune response Immune vs cancer cells → if cancer cells will and proliferate ( in no.); they produce ligands → ligand bind to checkpoint inhibitors→ cancer cells now marked as “self”→ T cells no longer target them → tumor progression - Eg of checkpoint molecules identified as targets for development of therapeutics: o Cytotoxic T-lymphocyte associated protein 4 (CTLA-4) – CD8 type T cell ▪ CTLA-4 and CD28 expressed on activated T cells ▪ During antigen presentation by APC, costimulatory molecule CD80/CD86 expressed on APC binds simultaneously to CD28 on T cells to  T cell activation → T cell executes cell killing effect  ▪ Expressed CTLA-4 competes with CD28 for binding to CD80/CD86 →  CD80/CD86-CD28 binding → T cell activation inhibited ▪ CD80/86 + CD28 → T cell activation ▪ CD80/CD86 +CTLA-4 → T cell inactivation o Programmed death 1 (PD-1): ▪ Expressed on activated T cells ▪ In the intratumour microenvironment, a no. of cells including DCs, tumor-associated macrophage (subset of macrophage), fibroblasts and even tumor cells themselves express PD-L1 (PD-1 ligand) and/or PD-L2 PD-L1/PD-L2 binds to PD-1 and suppress T cell activity ▪ Eg. Pembrolizumab & Nivolumab (PD-1 inhibitors) for tx of melanoma, non-small cell lung cancer, renal cell carcinoma, hepatocellular carcinoma etc ▪ Eg. Atezolizumab, Avelmab, Durvalumab (PD-L1 inhibitors) For tx of various types of solid malignancies Do checkpoint inhibitors directly kill cancer cells? →NO, they intervene the interaction → no activation of immune checkpoint proteins → no suppression of T cells → INDIRECT LIMITATIONS OF CHECKPOINT INHIBITORS Not all cancer pts show response to checkpoint inhibitors (tumor microenvironment is complex) Efficacy can be transient Development of immune-related adverse events during treatment → leads to  clinical outcomes, dose interruption or discontinuation and even death CANCER VACCINES Vaccines are not just prophylactic vaccines to achieve prevention of infectious diseases through acquisition of immune memory → therapeutic vaccines are also a strategy for immunotherapy, particularly cancer immunotherapy Cancer vaccines: immunotherapy given to cancer patients for cancer management by potentiating or reactivating the pt’s own immune system 3 CATEGORIES OF CANCER VACCINES: 1. CELL VACCINES a. Different designs: i. Tumor cell vaccine 1. Eg. GVAX; contains autologous (pts’) or non-autologous (donated) tumor cells that may be or may not be genetically modified 2. Directly insert tumor cells→antigens expressed by tumor cells to induce T cells in pt → sounds scary ii. Dendritic cell vaccine 1. Tumor antigenic proteins/peptides or tumor cells loaded on DCs → DCs administered into cancer patient for tumor antigens to induce T cells 2. Dendritic cells (lab grown) → recognize tumor cells → destroy them → represses antigenic peptides → bind them to MHC → peptide MHC complex → present to T cells to do further killing b. Provenge → 1st FDA approved cancer vaccine for prostate cancer treatment i. Leukocyte fraction extracted from pt’s peripheral blood – autologous (DCs are the main APCs in product) ii. DCs cultured ex vivo with a fusion protein consisting of 1. Antigen prostatic acid phosphatase (present in 95% of prostate cancer cells) – trained to recognize 2. Immune signaling factor GM-CSF for APC maturation iii. Activated APCs are reinfused into pt → evoking immune response against cancer cells carrying antigen 2. PROTEIN/PEPTIDE VACCINES a. Vaccine formulations contain antigenic peptide fragments derived from tumor-associated antigens → upon administration, recognized by T cells & activate T cells → attack cancer cells b. @ present, generally found not to produce strong immune response for various reasons  not v effective i. Neoantigens (arising from specific gene mutations in a cancer pt) are not included ii. Antigenic peptides of short chain (~20-30aa) restricted to MHC class I binding → MHC class II binding not engaged → subsequent activation of CD4+ helper T cells followed by CTL induction is absent iii. Antigenic peptides of short chain may bind to any cell (lost) without triggering further processing → may induce anergy (lack of immune response to an antigen) and cause immune tolerance 3. NUCLEIC ACID (DNA, RNA) VACCINES o Vaccine formulations contain DNA or RNA that code for tumor associated-antigenic proteins/peptides o Can be delivered using a viral vector (efficient delivery of DNA/RNA into cells) o Non vector-based delivery system also used (eg. liposomal formulation) a. @ present, trial results promising b. Main considerations: i. DNA vaccines: nucleic acid gets incorporated into host cell genome → more lasting expression of antigenic proteins/peptides BUT risk of inducing carcinogenicity if insertion of gene causes insertional mutagenesis and switch on oncogenes 1. Insertional mutagenesis: randomly inserted @ any position → may result in frameshift, non-frameshift, switch on oncogene or inactivate tumor suppression gene → risk of adverse reaction (cancer)  ii. RNA vaccines: no risk of carcinogenicity caused by insertional mutagenesis since RNA not incorporated into host cell genome → goes to RER instead 1. Main concern: RNA stability in formulation & upon administration eL2: PRINCIPLE OF IMMUNOASSAYS IMMUNOASSAYS Assays that employ antibodies to detect and quantify a specific analyte (usually a biomolecule) Key component: antibodies against the biomolecule of interest - Polyclonal or monoclonal antibodies may be used; usually raised in an animal Eg. anti-mouse insulin goat antibody would mean: - Antibody produced by B cells of goat origin & the antigen the antibody is against is mouse insulin (goat injected w mouse insulin) Means of detection of Ag-Ab binding in immunoassays: 1. A marker is often tagged onto the antibodies to facilitate rapid recognition of Ag-Ab binding a. Radioimmunoassay (RIA) → use radioactive labels i. More sensitive; radioisotopes can be taken up even w v small amt of Ag-Ab binding ii. But radioactivity is not very safe…not usually commercialized for humans used in labs b. Enzyme immunoassays (EIA) → use enzymes i. Enzymes and antibodies are proteins; functional group reaction →enzymes tagged onto Ab 2. Agglutination/Hemagglutination Both EIA & agglutination are used in commercialized immunoassays PRINCIPLE OF SOLID PHASE ENZYME IMMUNOASSAYS (ELISA) 1. Antibodies immobilized on solid surface a. → incubate with known amt of enzyme-linked antigens (lab made)+ sample containing antigens (from pt; unlinked antigen) b. Proteins absorb very well on plastic surfaces 2. Binding btwn enzyme-linked and unlinked antigens with antibodies occurs (competition) 3. Wash away unbound antigens (washing step) with buffer solutions (usually phosphate buffered saline PBS) a. After washing, antigen or enzyme-linked antigens bound to the immobilized antibodies are left b. → add enzyme substrate and determine enzymatic activity by measuring absorbance of coloured product formed c. Measure with UV spectrometry *Greater the amt of antigen present in sample, less enzyme-linked antigen will bind to the solid phase → less enzymatic activity will b detected ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) 1. Direct → soluble antigen (from pt) fixed to solid surface → washing off unabsorbed antigens → add primary enzyme-linked antibodies → washing to remove unbound enzyme-liked antibodies → add in enzyme substrate → measure abs of coloured product 2. Indirect → employs antigens to detect the presence of a specific antibody in a sample a. Antigen (from pt) immobilized on solid surface + add in primary antibody (not enzyme linked) → antigen binds to antibody → washing → add secondary enzyme-linked antibodies → binding & washing → add enzyme substrate → coloured product b. More accurate results than direct because dependent on specificity of primary & secondary antibodies i. Primary antibody specific against antigen ii. Secondary antibody is specific against the Fc domain of the primary antibody 3. Sandwich → employs antibodies to detect the presence of a particular antigen in a sample a. Antigen (from pt) now sandwiched btwn 2 antibodies b. Antibody (specific to pt antigen) immobilized on solid surface → washing → add pt sample → binding to primary antibody→ washing → introduce secondary antibody (specific for antigen; another epitope than primary antibody) → washing → introduce enzyme-linked antibody (recognizes the Fc domain of secondary antibody) → washing → add enzyme substrate → measure abs c. Need both specificity of captured antibody + specificity of the primary antibody; targets same antigen but diff epitopes  4. Competitive ELISA → enzyme-linked antigen (aka inhibitor antigen) competes w antigen in sample for antibody binding a. Captured antibody immobilized on solid surface → coincubation with pt sample (antigen) + enzyme-linked antigen (lab) → competes for binding to captured antibody → washing → add enzyme substrate → measure abs b. Higher [antigen] in sample, the more competitive they are to bind to captured antibody (less enzyme linked antigen binding → less substrate acted upon by enzyme for colour change → lower abs → more antigens in pt sample ADVANTAGES DISADVANTAGES Specific & sensitive (dependent on specificity of antibodies used) Possibility of false positive results → ELISA results can be quantitative, semi-quantitative or Result of use of polyclonal antibodies, inadequate blocking (w BSA)/washing qualitative → background noise) , cross reactivity of secondary antibodies Ease of use (usually absorbance measurement only req. UV Possibility of false negative results spec, if not fluorescence – more sensitive) Antibodies (primary and/or secondary) & conjugated enzymes (proteins in nature) may hv been denatured (cannot recognize, cannot act on) Safe to use (no radioisotopes) PRINCIPLE OF HEMAGGLUTINATION/AGGLUTINATION ABO blood types: A and or B antigens expressed on different RBC types (except group O blood type) - Group O RBC type express no antigens - Antibodies against A or B antigens will cause clustering of RBCs that are visually seen as hemagglutination Extended to develop assay for diagnosis/quantification of some enveloped virus in samples Rationale: ability of these viral particles to interact with RBCs through a viral surface glycoprotein called hemagglutinin → presence of virus causes clumping of RBCs (hemagglutination) → forming a lattice (RBCs lose surface tension) instead of a nice full red dot (high surface tension) IN COMMERICIALIZED IMMUNOASSAYS For clumping to occur, not always need to be RBC - As long as antigen and/or antibody is particulate in nature, agglutination occurs upon Ag-Ab binding o ie. Semi-solid/solid by being conjugated to a solid particle Ag-Ab binding scenarios: 1. if both antigen and antibody is soluble, Ag-Ab binding is not visible a. No agglutination 2. If either Ag or Ab is present in a semi-solid or presented by a solid particle, Ag-Ab binding is visible as clumping (cloudiness) a. Agglutination has occurred 3. If both Ag and Ab are particulate (presented by a solid particle) a. Agglutination occur; turbidity more intense than agglutination from Ag-Ab binding btwn 1 soluble Ag/Ab and 1 particulate Ab/Ag (2) → because complex is larger than (2) TYPES 1. PASSIVE HEMAGGLUTINATION a. Agglutination test only works with particulate antigens b. Erythrocytes coated with a soluble Ag and use the coated RBCs in an agglutination test for Ab to the soluble Ag 2. HEMAGGLUTINATION INHIBITION/ INHIBITION OF AGGLUTINATION a. Soluble Ag (in sample) inhibits agglutination of Ag-coated RBCs by Ab (soluble) b. +ve: if pt sample Ag binds to Ab → binding not visible (both soluble) c. -ve: pt sample lack Ag, Ag-coated RBCs (solid) bind to Ab → agglutination EXAMPLE: PREGNANCY TEST 1. Direct agglutination (passive hemagglutination) o Latex particles are coated with an Ab for hCG (semi-solid)  o If urine (soluble) contains sufficient hCG, agglutination occurs → high turbidity ▪ +ve test appears as aggregates o Assay time: 3min 2. Inhibition of agglutination o Latex particles are coated with anti-hCG antibody & standard amt of hCG (agglutinator*) is added → agglutination btwn particulate hCG Ab and particulate hCG Ag o Sample urine containing hCG is added → preventing agglutination →lower turbidity → +ve result o Assay time: a few mins *Agglutinator: standard amt of hCG is coated on solid particles (latex particles or RBCs). Supplied tgt with Ab-latext in commercial kit OTHER EXAMPLES - Ovulation prediction kit o Detect presence of luteinizing hormone (LH) in urine sample - Pregnancy test kit o Detect presence of human chorionic gonadotropin (hCG) in urine sample - HIV test o Detect either HIV capsid/envelop proteins (eg. p24) antigen or HIV antibodies in blood sample - Covid-19 ART o Detect SARS-CoV-2 nucleocapsid proteins in respiratory sample - Immunoassays for drugs of abuse (DOA) o Detection of multiple DOA in urine sample IC2: BIOPHARMACEUTICAL PRODUCTS DERIVED FROM ENDOCRINE & IMMUNE SYSTEMS II IMMUNOASSAY FOR HbA1c MONITORING Hemoglobin A1c (HbA1c) - Glycated hemoglobin (sugar molecule chemically attached to hemoglobin; due to [sugar]blood - Glycation of hemoglobin takes place non-enzymatically – condensation reaction btwn glucose & amino end of  chain in Hb - blood glucose levels →  levels of glycated Hb (HbA1c) → more cloudiness (more agglutination) - Longer the periods of high blood glucose levels → higher the levels of glycated Hb (HbA1c) - Glycated Hb remains throughout RBCs’ lifespan → HbA1c levels used for long term monitoring of pt’s diabetic condition - DCA 2000 HbA1c kit: semi-quantitative/quantitative assay for HbA1c in blood o Assay based on latex immunoagglutination inhibition method o After loading reagent test cartridge into analyzer, test result displayed in 6min Agglutinator: synthetic polymer containing multiple copies of the immunoreactive portion of HbA1c Latex coated with anti-HbA1c mouse monoclonal antibody Abs measured @ 531nm eL4&5: PROTEIN STABILITY PROTEIN PHARMACEUTICALS Major advantages: - High specificity and activity (high potency) - Relatively low concentrations (side effects) Major Challenges - Antigenicity o Foreign proteins (from non-human host cells eg. E coli, CHO cells) → may induce immunogenic response (allergic reaction) from human host o Loss of efficacy due to development of antibodies in the patient’s body against exogenous protein (long term use) ▪ Can  dose to tackle this but also S/E - Stability (Physical, Chemical, Biological) o Proteins subjected to wide range of influences → loss of biological activity  o Protein’s biological activity destroyed by inducing denaturation, covalently modifying the protein (or partially degrading it) → loss of proper 3D conformation o Loss of biological activity can occur during: ▪ Protein recovery from its source (extraction procedures) ▪ Protein purification process ▪ Post-protein purification (protein storage) Storage for any length of time can pose problems: o Proteolysis due to enzymes a/w bacterial contamination o Storage of proteins in solution → protein degradation (specific aa contribute to destabilization) → stored in freeze-dried form preferred Stability testing based on physical/chemical assays is inadequate; biological assays can reveal potency of product - Drug delivery o Administered through injection (convenience) In biopharmaceuticals production, need to maximize stability of protein pharmaceuticals MECHANISMS LEADING TO INSTABILITIES OF PROTEIN PHYSICAL PROTEIN AGGREGATION Major event of physical instability Secondary, tertiary and quaternary structures may change with time - Protein’s physical stability expressed as difference in free energy ∆G between N and U states - Unfolding can be reversible (U) or irreversible (A) - Subsequent aggregation of denatured molecules → irreversible denaturation - Aggregated protein: has altered activity and may arouse immunogenicity MECHANISMS OF PROTEIN AGGREGATION - Results from intermolecular association of partially denatured protein chains - Hydrophobic force is the major force for protein folding and aggregation - May be induced by a variety of factors: o Temperature (slow unfolding @ T) o pH o ionic strength o vortexing o chemical modification of proteins - May result from chemical degradation or modifications o Exposure of hydrophobic surfaces - Unfavourable physical and chemical factors may occur simultaneously → protein aggregation PHYSICAL FACTORS AFFECTING PROTEIN STABILITY 1. Temperature a. MOST IMPORTANT factor b. T promotes protein unfolding → disrupts non-covalent forces that stabilize protein’s conformation c. Denatured proteins aggregate → irreversible denaturation 2. pH a. Proteins unfold @ extreme pHs due to changes in ionization status of side chains of aa residues b. Disruption of distribution of ionic attractive and repulsive forces that stabilize protein’s conformation c. Extreme pHs also affect protein’s chemical stability i. Hydrolysis of Asp residues ii. Deamination of Asn and Gln 3. Adsorption a. Proteins can be adsorbed to many surfaces and interfaces b. Significant change in secondary structure and tertiary structure c. Loss of proteins or destabilization of proteins d. Thus, not stored in plastic, usually glass used 4. Shaking and shearing (agitation) a. Agitation incorporates air into protein solution → creates air/liquid interface  b. Alignment of proteins along such interfaces → unfolding of protein to maximize exposure of hydrophobic residues to air →partial or complete protein denaturation due to aggregation c. Shearing also exposes hydrophobic areas 5. Non-aqueous solvents a. Protein hydration shell may be disrupted in the presence of significant amt of non-aqueous solvents b. Protein hydrophobic core exposed as polarity of aqueous solvent decreases → protein unfolds 6. Repeated freeze-thaw a. Sharp ice crystals form every time frozen → pierce through protein 3D conformation → unfolding → aggregation 7. Photodegradation a. Risk of protein aggregation upon exposure to light (keep in amber bottle) b. Tryptophan – side chain cleavage of Trp CHEMICAL More than one reaction can happen simultaneously Localization of labile aa determine the chemical reactivity May not always affect protein conformation/activity 1. Deamidation a. Most common degradation pathway b. Susceptible aa: Asn and Gln c. Relative position may determine the relative rates 2. Hydrolysis a. Hydrolysis of peptide bonds, esp under acidic and basic pH b. Asp-Gly and Asp-Pro bonds particularly labile 3. Oxidation a. Side chains of His (H), Met (M), Cys (C), Trp (W), Tyr (Y) b. Catalyzed by transitional metal ions c. Thiol groups of C and M most easily oxidizable sites d. Oxidation depends on position 4. Disulfide bond formation and breakage a. Disulfide bond formation is a form of oxidation; Cys is susceptible b. Contribution towards protein stability and activity can vary i. Sometimes needed to favour stability in some proteins, but sometimes detrimental to protein stability STABILIZATION AND FORMULATION OF PROTEIN PHARMACEUTICALS (LIQUID) – STRATEGIES 1. Substitution and chemical modification (internal changing of structural characteristics without compromising activity) → to improve protein stability a. Amino acid substitution/modification (via site-directed mutagenesis) a. Protein analogues (eg. insulin analogues, IL-2 analogue (Proleukin) i. Cys replaced by Ser b. Replacement of deamidation Asn sites b. Introduction of disulfide bond a. Stabilize folded form of protein c. PEGylation a. Chemical attachment of polyethylene glycol (PEG) H-(O-CH2-CH2)n-OH b. circulation time in blood, better safety profile PEGylated IFN- c. Recombinant proteins conjugated with PEG are called PEGylated proteins d. Acylation a. Chemical attachment of fatty acids (lipophilic) to residues on protein surface b. circulation time in blood c. lipophilicity of protein → expels water → maintain proteins stability 2. Changing the properties of the solvent, additives (external) a. Stabilizers i. Sugars, polyols b. Solubility enhances i. Lysine, arginine, surfactants c. Anti-adsorption and anti-aggregation agents i. Albumin, surfactants d. Buffer components i. Phosphate salts (Na2HPO4, NaH2PO4) e. Preservatives and anti-oxidants i. Inert gas, thimerosal, phenol, benzyl alcohol IC4&5: RECOMBINANT PROTEIN MAKING AND ITS RELEVANCE TO QUALITY OF BIOPHARMACEUTICALS AND BIOSIMILARS I & II PART I FDA’s regulatory concerns about viral contaminants: - To avoid all animal products - In cell culture, animal component-free media to be used RECOMBINANT PROTEIN MAKING/SYNTHESIS (DOWNSTREAM PROCESSING) IMPACT OF RECOMBINANT DNA (rDNA) TECHNOLOGY ON BIOPHARMACEUTICAL PRODUCTS 1. Overcomes limitations regarding source availability a. Natural sources often rare and expensive b. Yield can be low due to limited amounts present in natural sources 2. Allows production of safer biopharmaceuticals a. E.g. eliminates transmission of blood-borne pathogens such as HIV, Hepatitis B virus if product is directly isolated from infected sources 3. Provides an alternative way to obtain protein-based products other than direct extraction from inappropriate source material a. e.g. urine, placenta → do not have constant supply too 4. Since gene of interest can be synthetic, →scientists can design desirable mutations to produce engineered protein-based biopharmaceutical products that possess advantages a. such as greater clinical efficacy, greater protein stability for longer self-life, shorter/longer circulation half-life →rDNA technology allows cheaper, safer, and abundant supply of protein-based biopharmaceuticals APPLICATION OF rDNA TECHNOLOGY USUALLY OCCURS IN UPSTREAMING PROCESSING IN MANUFACTURING OF BIOPHARMACEUTICALS SYNTHESIS OF RECOMBINANT PROTEIN Upstream bioprocessing (gene cloning) 1. Isolation of gene of interest “cut & paste” 2. Introduction of gene into expression vector 3. Transformation into host cells 4. Selection of the required sequence and propagation of cells Downstream bioprocessing 5. Isolation & purification of protein 6. Formulation of protein product UPSTREAMING PROCESSING IN RECOMBINANT PROTEIN MAKING - When host cells (bacteria/mammalian cell) successfully transfected with recombinant DNA→ each transfected cell is different from each other - Each transfected cell differ from each other in terms of number of copies of plasmids being transfected→ Higher the copies of plasmids, higher is the amount of protein being expressed (highest protein yield) - In recombinant protein making, upstream processes include selection of the 1 transfected cell that possesses the best cell growth properties & highest protein yield → development of a master cell line TYPES OF HOST CELLS USED Commonly used host cells: E coli (bacteria) & Chinese hamster ovary CHO (mammalian) cells Considerations: In the manufacturing processes, if any components belonging to host cells [e.g. endotoxins (pyrogens) from E coli, host cell proteins from E coli/CHO) remain in final product, will quality and safety of product be compromised? - Downstream processing to remove impure proteins BACTERIA YEAST (eukaryotic) INSECT & TRANSGENIC PLANTS MAMMALIAN & ANIMALS Insufficient folding of Post-translational Laborious Long development complex proteins of higher modifications differ construction of over- cycles organisms -- inclusion bodies from mammalian cells expressing strains Lack of post translation Problematic cell Expensive media Contamination modifications disruption problems Endotoxins Protease that degrade Low growth rates (animal viruses, foreign proteins Difficult scale-up prions) NO NEED MEMO THESE ^ HOST CELLS – BACTERIA (E. coli) Most common microbial species used for biopharmaceutical production First recombinant therapeutic protein produced in E.coli cells is insulin ☺ ADVANTAGES ☺ - E. coli well characterized → facilitates genetic manipulation - E. coli give high expression levels of recombinant protein (up to 30% of total cellular protein) - E. coli grows rapidly on simple & inexpensive media  DISADVANTAGES  - Recombinant protein accumulates intracellularly (& they’re small): o If proteins are synthesized in low levels, they remain soluble in cells ▪ → additional processing steps required to purify proteins from rest of host cell proteins o If proteins are synthesized rapidly & in high levels → too small space to hold so many proteins → precipitate out → form insoluble aggregates called inclusion bodies (insoluble; native conformation & function may be affected)→ may be easier to purify but isolated insoluble proteins need to undergo refolding to be active - Lack the ability to perform post-translational modifications (e.g. glycosylation) - Presence of lipopolysaccharides (LPS) on its surface that act as pyrogens o Need to lyse the cell to release contents → LPS may also be released → impurities! → pyrogen?? EXTRA STEPS IN PROKARYOTIC PROTEIN PRODUCTION Pyrogens: fever-inducing substances. Usually come from endotoxins or lipopolysaccharide (LPS) from bacteria. When introduced into the bloodstream, may lead to inflammatory response, shock or multiorgan failure and death. E.coli →80-90% chance that inclusion bodies will happen; Mammalian cells have 0% chance → mammalian cells are expensive and difficult to maintain (delicate) → extra steps needs to be taken to reuse inclusion bodies produced →extract by rupturing cell wall and membrane → centrifuge the insoluble proteins → harsh conditions (using denaturing solvents eg. SDS, anionic detergent) → gets rid of interactions →denatures proteins, solubilizing them (unfolded to primary structure)→ refolding of proteins (add enzymes, chaperone proteins) → hoping it will refold into native proteins (will not be 100%) → native proteins will be soluble → centrifuge for the liquid phase CHINESE HAMSTER OVARY (CHO) CELLS Preferred mammalian cell line for synthesis of recombinant proteins because: 1. CHO cells capable of adapting and growing in suspension culture → ideal for large scale culture 2. CHO cells pose less risk as few human viruses can propagate in them 3. CHO cells can grow in serum-free media → ensures reproducibility between different batches of cell culture a. Serum: liquid fraction in blood (called plasma) with clotting factors removed b. Often mammalian cells require growth factors present in serum to stimulate them to divide/grow c. In cell culture, fetal bovine serum (FBS) often used (but not anymore, FDA rules, no animal sources) i. Now use recombinant growth factors (non-animal growth factors) 4. CHO cells allow post-translational modifications to recombinant proteins a. Glycosylation of glycoproteins produced by CHO cells more human-like → more compatible and bioactive in humans 5. CHO cells can be manipulated by genetic engineering techniques to produce higher yield of recombinant proteins First recombinant therapeutic protein produced in mammalian cells is tissue plasminogen activator (r-tPA) synthesized using CHO cells Today ~70% of all recombinant protein therapeutics produced are made in CHO cells EXPRESSION SYSTEM SELECTION Choice of host cells to make recombinant proteins depends on size & characteristics of protein: - Large proteins (>100 kDa) → CHO - Small proteins ( efficient than non-ionic detergents more likely than nonionic) in solubilizing plasma membranes (eg.  presence may affect subsequent purification steps polysorbates)  presence unacceptable in final protein preparations Mechanical force → noisy; energy typically transferred to heat energy – unwanted (proteins are sensitive; native formation wanted) o Typically protected with water jacket to prevent heating → unfolding of proteins 2. ENRICHMENT a. Removal of whole cells and cell debris i. Centrifugation i. Apply appropriate centrifugal force → separate unlysed whole cells and cell debris from liquid phase ii. Commonly used in biologic manufacturing facilities ii. Filtration i. Depth filter 1. Consists of randomly orientated fibers (glass fiber or cellulose) → form network of mesh-like structures 2. Is assembled from a series of filters with ing pore size rating 3. Particle retention on surface and within the depth of filter 4. Used to remove/reduce levels of cellular debris, denatured protein aggregates or other precipitates from solution (cant be reused, trapped in the filter) 5. Filtrate is just liquid containing soluble protein of interest ii. Membrane filter (microfiltration) 1. Pore size: 10-0.2µm (0.2-0.45µm retains all microbial cells) 2. Particle retention on or in the surface layer of filter 3. Filtration capacity limited → pores blocked up from solids → liquid cant flow thru anymore iii. Aqueous two-phase partitioning i. Principle: water-soluble (aq) polymers are incompatible with each other or with salt solutions that are of high ionic strength ([salt])→ when mixed tgt, two phases are formed upon standing ii. Cell, cell debris partition to the lower, more polar and denser phase; soluble proteins partition to the top, less polar and less dense phase iii. Common partitioning systems: 1. Polyethylene glycol (PEG)/phosphate salt (Na/K); polymer-salt system 2. PEG/dextran; polymer-polymer system iv. Gentle method, PEG polymer has protein stabilizing effect, high yield, can be scaled up ☺ b. Removal of nuclei acid and lipids (to form aqueous liquid only) i. Nucleic acid i. Removal by: 1. Precipitation using cationic polyethylenimine 2. Treatment with nucleases (amt added is limited to picograms) ii. Lipids i. Cells/tissues of animal origin have high lipid content ii. Act as contaminants and interfere with subsequent purification steps 1. clog chromatographic columns used in subsequent protein purification steps iii. lipid layer (floats) removed by passing solution through glass wool or a cloth of very fine mesh size Proteins are amphiphilic → protein solubility worst @ pI (- & + cancels out); solubility of most globular proteins influenced by Ionic strength & pH → can be adjusted to control solubility of protein of interest to optimal point c. Concentration of protein of interest Initial stages of protein purification procedures yield dilute protein solution; need to concentrate to obtain more manageable volume for subsequent purification steps i. Precipitation i. Addition of neutral salts: salting out 1. Protein solubility varies with ionic strength (I) of solution At low I → salting in (in protein solubility) At high I (above an optimal salt conc) → salting out (protein precipitation from solution)  2. Salts at high concentration compete with proteins for water of hydration → promotes protein- protein interactions between hydrophobic patches on surface of protein molecules → protein precipitation 3. Ammonium sulfate most commonly used protein precipitant due to its high solubility, inexpensiveness and lack of denaturing properties ☺ a. Shifts protein from N state to U state (unfolded) → precipitates out without harm (reversible) → filter out → redissolve it to N state in lesser vol → conc ii. pH adjustment: precipitation at (pI) values 1. minimum solubility of a protein is generally around its isoelectric point (pI) iii. Addition of organic solvents (least popular) 1. protein solubility by lowering dielectric constant of an aqueous solution → a. Weakens hydrophobic interactions & disrupts hydration shell b. electrostatic attractions between proteins of opposite charges → protein precipitation 2. Commonly used: ethanol, isopropanol 3. Hard to remove organic solvents esp if its compatible with aq phase Disadvantages of protein concentration by precipitation 1. Precipitants need to be removed before further processing 2. Inefficient precipitation if initial protein conc is low → low recoveries of protein ii. Ion-exchange chromatography i. Principle: @ given pH value→proteins display +/-/0 nett charge 1. Proteins separated by judicious choice of pH, ionic strength and ion exchange materials ii. -ve-charged proteins of interest →use Anion exchangers (contain aminoethyl- moieties) iii. +ve-charged proteins of interest → use Cationic exchangers (e.g. Contain carboxymethyl moiety) iv. Proteins bound to ion exchangers can be eluted from the ion exchange column by addition of a suitable salt solution of high ionic strength (eg NaCl 0.5 M) → disrupts ionic interactions → elutes protein of interest v. Advantages ☺: 1. Undesirable impurities (particulate material, lipids, carbohydrates, partially denatured/aggregated protein, other proteins) can be separated vi. Ion exchangers: functional groups are covalently linked to porous beads usually made from cross-linked dextran (sephadex exchangers), agarose (sepharose exchangers) or cellulose (sephacel exchangers) no nd memo; columns can be recycled iii. Ultrafiltration i. Membrane filters are employed →proteins of interest retained based on pore size of membrane filters ii. Pore size range from 1 to 20 nm iii. Molecular mass cut-off points ranging from 1-300 kDa (eg. 3, 10, 30, 50 and 100 kDa) iv. Made of cellulose acetate, cellulose nitrate or polyvinyl chloride v. Advantages ☺: 1. Gentle, little adverse effect on protein molecules 2. High recovery rates 3. Rapid processing time vi. Disadvantages : slow & expensive d. Purification of protein of interest Proteins are Amphiphilic → Charged groups, hydrophobic regions, size, and solvation affect the biophysical properties of the protein and largely determine its purification behavior o Solubility of most globular proteins influenced by: ionic strength & pH Preliminary purification to concentrate protein of interest yields high protein recovery but degree of purification marginal → further purification achieved by column chromatography i. Column Chromatography i. Principle: partitioning between two phases - a solid stationary phase (chromatographic beads packed into a Separation according to: cylindrical column) and a mobile phase (a buffer) – by gravity - MW/size ii. Chromatographic techniques: - Charge 1. Size exclusion (gel filtration) – only one that’s not adsorptive - Hydrophobicity Ideal for final polishing steps in purification when sample volumes have been reduced - Affinity a. Separates proteins based on size (MW) and shape b. Protein eluted from the column in order of ing molecular size i. Small proteins → small enough to enter gel matrix → retained longer ii. Large proteins → cannot enter porous space among gel beads → elute faster  c. Gel matrices prepared by chemically cross-linking polymeric molecules (eg. dextran, agarose) d. Long chromatographic columns req. for adequate resolution of protein mix into distinct protein-containing bands along gel column e. Run SDS PAGE gel to identify proteins based on molecular weight 2. Ion exchange (IEX) based on reversible electrostatic attraction of charged protein to a solid matrix of ion exchangers of opposite charge a. Cation exchange: matrix is -vely charged b. Anion exchange: matrix is +vely charged c. ☺ High level resolution achievable, can be scaled up, inexpensive 3. Hydrophobic interaction a. Packed with exchanger that attracts the hydrophobic patches of any protein b. Fractionates proteins based on proteins’ differing degrees of surface hydrophobicity c. Protein samples applied to column in a buffer of high ionic strength (addition of neutral salt ammonium sulfate or NaCl) → proteins retained via hydrophobic interaction d. Elution of protein of interest: by ing hydrophobic interaction using a buffer of lower ionic strength or lowering polarity of the buffer i. eg. add ethanol 4. Affinity a. Highly selective protein purification method ☺ b. Principle: specific and reversible binding of proteins of interest to affinity matrices c. Ligands covalently attached to an inert support matrix, packed into a chromatographic column → protein of interest selectively binds to immobilized ligand o General ligand approach: ▪ Immobilization of ATP or cofactors (e.g. NAD+) on affinity columns to purify ATP binding proteins (e.g. ATPases) or cofactor-binding proteins (e.g. dehydrogenases) Immobilize ligands on solid resins in column ▪ Elution of protein of interest: By changing buffer pH, ionic strength or polarity of buffer By inclusion of a competing ligand in eluting buffer → desorption of retained proteins ▪ ☺ Advantages: specific and selective method ▪  Disadvantages: Expensive and poor stability of ligands; leaching of coupled ligands from matrix o Specific ligand approach: immunoaffinity purification (not recycled; $$$) ▪ Immobilization of antibodies on a suitable support matrix, columns used to isolate/purify a protein antigen ▪ Elution of protein of interest: Usually involve conditions resulting in partial denaturation of bound protein (eg. use of denaturing agents urea, acidic pH (2.2-2.8)) o Additional step to refold to N form A few instances that milder conditions can be employed → eg.  ionic strength of eluting buffer ☺ Advantages: specific and selective method  Disadvantages: Expensive and poor stability of antibodies, Leaking of antibodies from matrix → elution of protein not readily achieved 5. Immunoaffinity ISSUES TO CONSIDER IN PRODUCTION & PURIFICATION OF PROTEINS PROTEIN PURIFICATION PROCESS – REMOVAL OF CONTAMINANTS POTENTIAL CONTAMINANTS: Host-related contaminants - viruses, host-derived proteins and DNA, glycosylation variants, N- and C-terminal variants, endotoxins (pyrogens from G-ve bacteria) Product-related contaminants - amino acids substitution and deletion, denatured proteins, conformational isomers, dimers and aggregates, disulfide pairing variants, deamidated species, protein fragments Process-related contaminants - grow medium components, purification reagents, metals and column materials MICROBIAL CONSIDERATION IN BIOTECH PRODUCTS FORMULATION Sterilization - Biopharmaceuticals assembled by aseptic procedures  - Filter through 0.2 or 0.22 m membrane filters Viral decontamination Microfilters: pore size 10-0.2µm - By heat, irradiation, sonication, extreme pH (harmful to product) Ultrafiltration: pore size 1-20nm, - By nanofiltration (usually < 1 nm, some nanofilters have pore size 15 nm) - retain proteins of 1-1000 kDa Pyrogen removal - Pyrogens are lipopolysaccharides (-ve charged endotoxins) - Removed by dry heat or anion exchange (filtration cannot get rid of pyrogens) PART II SAFETY TESTING IN PHARMACEUTICAL INDUSTRY SDS-PAGE (sodium dodecyl sulphate polyacrylamide gel electrophoresis) - Gives high resolution electrophoretic separation of proteins based on molar mass (for SDS-PAGE) or protein folding (for native PAGE) - Visualization of separated proteins by protein stains; can detect 1 ng of protein contaminants (when silver-based stains are used) - Detection of product variants possible using product-specific antibody in Western blot Isoelectric focusing Dye binding methods (colorimetric assays) - Separation of proteins by isoelectric point (pI) - Can be used with SDS-PAGE in 2D electrophoresis → provide added dimension of separation to detect contaminants - Also used to monitor homogeneity of glycoproteins’ sialic acid content (sialic acid on glycan is charged → affects pI of glycoprotein) DNA hybridization (e.g. dot blots) - For detection of DNA contaminants in ng range Rabbit pyrogen test (bioassay) - Pyrogen detection by injecting product into 3 healthy rabbits → monitor for increased temperature Limulus amoebocyte lysate (LAL) test - Endotoxin stimulated coagulation of amoebocyte fraction in blood of horseshoe crabs (Limulus) - Used extensively in the industry - ☺ Advantage (relative to rabbit bioassay)→ less variable, more sensitive, faster, cheaper -  Disadvantage: only detects endotoxin-based pyrogens Viral assays - To test for o specific viruses capable of contaminating source materials o unknown/uncharacterized viruses not widely available or employed - Immunoassays using antibodies specific for panel of viruses can be employed - Bioassays o involving incubation of product with cell lines sensitive to range of virus (e.g. Vero cells) o injection of product into animals for stimulation of antibody production and subsequent testing of specificities of antibodies raised in these animals against a panel of viruses - Virus specific DNA probes (Dot blots or PCR based assays) In vivo bioassays - As general safety testing - Eg. injection into healthy mice BIOSIMILARS A biologic that is almost an identical (or ideally identical) alternative version of the original biologic (called innovator biologic, reference biologic) that is manufactured by a different company - Also called follow-on biologic; reference to the innovator biologic is crucial for its approval by regulatory authorities - Many biopharmaceuticals introduced in late 90s/start of 21st century → patents have expired/are expiring → alternative versions were/to be produced by other biotech companies - Each biological product displays variability even between different batches of the same product o → due to variability of the biological expression system and manufacturing process o → Process of manufacturing biopharmaceuticals (upstream and downstream processing) has crucial influence on the nature of the final biological product Can biosimilar biologic be entirely identical to the innovator biologic? – NO Final characteristics of a biologic influenced by manufacturing process - type of host cell, development of genetically modified cell for production, cell