Biotechnology Drugs-2 PDF Fall 2023

Biotechnology Drugs-2 PCOL825A/Fall 2023 Biotechnology drugs – 2 Aikseng Ooi, PhD Skaggs Room 204 [email protected] 1 1 Overview • Biopharmaceuticals / biologics • Insulin production • Antibody : Mechanisms of action • Types of antibody • Toxins as drugs • Vaccines 2 2 Ooi/Department of...

Biotechnology Drugs-2 PCOL825A/Fall 2023 Biotechnology drugs – 2 Aikseng Ooi, PhD Skaggs Room 204 [email protected] 1 1 Overview • Biopharmaceuticals / biologics • Insulin production • Antibody : Mechanisms of action • Types of antibody • Toxins as drugs • Vaccines 2 2 Ooi/Department of Pharmacology & Toxicology/The University of Arizona College of Pharmacy 1 Biotechnology Drugs-2 PCOL825A/Fall 2023 Biopharmaceuticals / Biologics / Biologicals Medicine derive from biological sources • Vaccines, Antibodies, Antisera, DNA, cells, Insulin • Directly from a living organisms • Antisera • Plasma • Recombinant sources • Genetically modified plants • Genetically modified viruses • Genetically modified microbes • Bioreactors 3 3 Some biopharmaceuticals are hormones – Example: Insulin 1. Insulin is the first biopharmaceuticals. 2. Insulin is one of the most potent anabolic hormone. 3. Insulin signals insulin responsive tissues (fat and muscles) to take in glucose. 4. Indicated for diabetes. 4 4 Ooi/Department of Pharmacology & Toxicology/The University of Arizona College of Pharmacy 2 Biotechnology Drugs-2 PCOL825A/Fall 2023 Insulin production in the body – INS gene product Kaufman Page 4 NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript 5 Figure 2. Interfering with Insulin Production Adapted 5 from Kauffman. N Engl J Med. 2011 November 17; 365(20): 1931–1933 The production of mature insulin takes place within the beta cell and depends on the cleavage of the preproinsulin and proinsulin molecules. The cleavage site at the junction of the A chain and the C-peptide contains a lysine residue, which is critical for cleavage. N Engl J Med. Author manuscript; available in PMC 2013 June 17. Commercial insulin was purified from cattle or pigs 6 6 Ooi/Department of Pharmacology & Toxicology/The University of Arizona College of Pharmacy 3 Biotechnology Drugs-2 PCOL825A/Fall 2023 7 7 8 8 Ooi/Department of Pharmacology & Toxicology/The University of Arizona College of Pharmacy 4 Biotechnology Drugs-2 PCOL825A/Fall 2023 9 9 Insulin Analog Types Reach Blood Peak Duration Rapid-acting 15 min 30-90 min 5 hr Insulin Lispro Humalog Short-acting 30 min 2-4 hr 4-8 hr Regular Insulin Humulin R Novolin R Intermediate 2-6 hr 4-14 hr 14-20 hr NPH Humulin N Novolin N Long-acting Small peak 20-24 hr Glargine Lantus 6-14 hr 10 10 Ooi/Department of Pharmacology & Toxicology/The University of Arizona College of Pharmacy 5 Biotechnology Drugs-2 PCOL825A/Fall 2023 Glargine—Long acting insulin Three changes made Two arginines are added to the C-terminus of the B-chain Less soluble at a physiological pH Replacing asparagine (N) (21 of A-chain) with glycine (G) Prevent dimerization Longer half-life in the body How to make mutations in a protein? 11 11 Site-Directed Mutagenesis Glargine—Long acting insulin How to replacing asparagine with glycine ? AAC Asn (N) GGC Gly (G) 12 12 Ooi/Department of Pharmacology & Toxicology/The University of Arizona College of Pharmacy 6 Biotechnology Drugs-2 PCOL825A/Fall 2023 Insulin – mechanism of action 13 13 Antibodies 14 14 Ooi/Department of Pharmacology & Toxicology/The University of Arizona College of Pharmacy 7 Biotechnology Drugs-2 Abciximab ReoPro 1994 Chimera Fab GPIIb/IIIa Blocking Palivizumab Synagis 1998 Humanized IgG1 RSV F protein Blocking Omalizumab Efalizumab*** Natalizumab Ranibizumab Eculizumab Xolair Raptiva Tysabri Lucentis Soliris 2003 2003 2004 2006 2007 Humanized IgG1 Humanized IgG1 Humanized IgG4 Humanized Fab Humanized IgG2/4 IgE CD11a VEGF Complement 5 Blocking Blocking Blocking Blocking Blocking Stelara 2009 Human IgG1 IL12, IL23-p40 Blocking Ustekinumab Prevention of cardiac ischemic complications Prevention of RSV infection in neonates Severe asthma Psoriasis Multiple sclerosis Macular degeneration Paroxysmal nocturnal hemoglobinuria, atypical hemolytic-uremic syndrome Plaque psoriasis PCOL825A/Fall 2023 of differentiation; CDC, complement-dependent cytotoxicity; ADCC, antibody-dependent cell-mediated cytotoxicity; HER-2, human epidermal growth factor receptor 2; ADC, antibody drug conjugate; B-CLL, B-cell chronic lymphocytic leukemia; RIT, radioimmunotherapy; NHL, non-Hodgkin lymphoma; EGFR, epidermal growth factor receptor; VEGF, vascular endothelial growth factor; EpCAM, epithelial cell adhesion molecule; RANKL, receptor activator of nuclear factor kappa-B ligand; ALCL, anaplastic large cell lymphoma; CTLA4, cytotoxic T-lympho- Antibodies : Mechanisms of action Fig. 1. Mechanisms of action of therapeutic antibodies. J Toxicol Pathol 2015; 28: 133–139 15 their target molecules1. In this case, antibodies bind to the ligand or receptor that is expressed on the cell surface and block the target signaling pathway. When the signaling in the tumor through these ligands or receptors is diminished, it can result in cellular activity being lost, proliferation being inhibited, pro-apoptotic programs being activated, or 15 cells being resensitized to cytotoxic agents16. ADCC: To trigger ADCC, the Fv binding domain of surface of a target cell. The antibody is then able to recruit immune-effector cells (such as macrophages and NK cells) that express various receptors able to bind to the Fc and thus The general structure of a human antibody 16 16 Ooi/Department of Pharmacology & Toxicology/The University of Arizona College of Pharmacy 8 Biotechnology Drugs-2 PCOL825A/Fall 2023 The general structure of a human antibody 17 Foltz Circulation. 2013;127:2222-2230 17 Humanization of therapeutic antibodies has reduced their immunogenicity. 18 Foltz Circulation. 2013;127:2222-2230 18 Ooi/Department of Pharmacology & Toxicology/The University of Arizona College of Pharmacy 9 Biotechnology Drugs-2 PCOL825A/Fall 2023 19 19 20 20 Ooi/Department of Pharmacology & Toxicology/The University of Arizona College of Pharmacy 10 Biotechnology Drugs-2 PCOL825A/Fall 2023 Production of humanized antibodies 21 Foltz Circulation. 2013;127:2222-2230 21 Single-chain Fv fragments (scFvs) 22 22 Ooi/Department of Pharmacology & Toxicology/The University of Arizona College of Pharmacy 11 Biotechnology Drugs-2 PCOL825A/Fall 2023 Single-Domain antibody (SdAb) Normal antibody ~150-160kDa FAB ~50kDa scFv ~25kDa SdAb ~12-15kDa. 23 23 Adalimumab (Humira®) • Blocks tumor necrosis factor-alpha (TNFα) • Prevents cytokine-driven inflammatory processes • Useful for rheumatic disorders – plaque psoriasis, Crohn disease, ulcerative colitis, psoriatic arthritis • Administration – SubQ • Side effects : Immune suppression and malignancies 24 24 Ooi/Department of Pharmacology & Toxicology/The University of Arizona College of Pharmacy 12 Biotechnology Drugs-2 PCOL825A/Fall 2023 Etanercept (Enbrel) • Tumor necrosis factor receptor (TNFR) - Fc portion of human IgG1 • Neutralizes TNF-alpha by acting as decoy receptor • Useful for rheumatic disorders – plaque psoriasis, Crohn disease, ulcerative colitis, psoriatic arthritis • Administration route : SubQ • Side effects : Immune suppression and malignancies 25 Infliximab (Inflectra; Remicade; Renflexis) • A chimeric antibody that neutralizes TNF-alpha • Neutralizes TNF-alpha by acting as decoy receptor • Useful for rheumatic disorders – plaque psoriasis, Crohn disease, ulcerative colitis, psoriatic arthritis • Administration route : Infusion • Side effects : Immune suppression and malignancies 26 Ooi/Department of Pharmacology & Toxicology/The University of Arizona College of Pharmacy 13 Biotechnology Drugs-2 PCOL825A/Fall 2023 Bevacizumab and ranibizumab Other examples refer to J Toxicol Pathol 2015. 28:13-139 27 27 Tumor and angiogenesis VEGF 28 Loizzi et al. Int. J. Mol. Sci. 2017, 18(9), 1967 28 Ooi/Department of Pharmacology & Toxicology/The University of Arizona College of Pharmacy 14 Biotechnology Drugs-2 PCOL825A/Fall 2023 Avastin – Bevacizumab neutralizes VEGF 29 29 Macular degeneration – disrupted regulation of angiogenesis Age-related macular degeneration Proliferative diabetic retinopathy Vascular leakage Sene A, Chin-Yee D, Apte RS. Trends Mol Med. 2015 Jan;21(1):43-51. 30 30 Ooi/Department of Pharmacology & Toxicology/The University of Arizona College of Pharmacy 15 Biotechnology Drugs-2 PCOL825A/Fall 2023 Relationship between Ranibizumab and Bevacizumab. Steinbrook R. N Engl J Med 2006;355:1409-1412. 31 Intravitreal Injection for the Treatment of Neovascular Age-Related Macular Degeneration. Steinbrook R. N Engl J Med 2006;355:1409-1412. 32 Ooi/Department of Pharmacology & Toxicology/The University of Arizona College of Pharmacy 16 Biotechnology Drugs-2 PCOL825A/Fall 2023 Macular degeneration – Avastin and Lucentis (ranibizumab) The CATT Research Group. N Engl J Med 2011;364:1897-1908. 33 Antisera 34 34 Ooi/Department of Pharmacology & Toxicology/The University of Arizona College of Pharmacy 17 Biotechnology Drugs-2 PCOL825A/Fall 2023 Antisera is a biopharmaceutical produced in living organisms Serum containing antibodies against specific antigens Treat or protect against specific diseases. - Antitoxin - Botulism Antitoxin Heptavalent (A, B, C, D, E, F, G) – (Equine) (H-BAT) - Toxins – Botulinum neurotoxin - Antivenom - Snake bite, Scorpion bite etc. 35 35 Botulism is caused by Clostridium botulinum 36 Rossetto O et. al. Nat Rev Microbiol. 2014 Aug;12(8):535-49. 36 Ooi/Department of Pharmacology & Toxicology/The University of Arizona College of Pharmacy 18 Biotechnology Drugs-2 PCOL825A/Fall 2023 Botulism in human 37 Rossetto O et. al. Nat Rev Microbiol. 2014 Aug;12(8):535-49. 37 Botulinum toxin is a polypeptide 38 38 Ooi/Department of Pharmacology & Toxicology/The University of Arizona College of Pharmacy 19 Biotechnology Drugs-2 PCOL825A/Fall 2023 Botulinum toxin is a concoction of 7 polypeptides targeting different SNARE proteins Type Target Discoverer A SNAP-25 Landman VAMP B (synaptobrevin) Ermengem C1 Syntaxin Bengston and Seldon VAMP D (synaptobrevin) Robinson E SNAP-25 Gunnison VAMP F (synaptobrevin) Moller and Scheibel VAMP G (synaptobrevin) Gimenez and Ciccarelli Year 1904 1897 1922 1929 1936 1960 1970 39 39 Botulism Antitoxin Heptavalent (A, B, C, D, E, F, G) – (Equine) Briefing Document Advisory Committee Meeting Page 31 SNARE proteins involve in the release of acetylcholine at neuromuscular junctions. Figure 3: Mechanism of Action of Botulism Neurotoxin 40 40 Ooi/Department of Pharmacology & Toxicology/The University of Arizona College of Pharmacy 20 Biotechnology Drugs-2 PCOL825A/Fall 2023 Botulinum toxin target SNARE mediated release of neurotransmitter Botulism Antitoxin Heptavalent (A, B, C, D, E, F, G) – (Equine) Briefing Document Advisory Committee Meeting Page 36 41 Arnon et al., 2001. SNAP-25 = Synaptosomal-associated Protein 25 kDa; SNARE = Soluble N-ethylmaleimide Sensitive Factor Attachment Receptor Protein. 3.2 41 H-BAT Mechanism of Action The mechanism of action of H-BAT is through passive immunization with equine polyclonal antibody fragments. In the circulation and extravascular compartment, H-BAT binds to free BoNT, preventing it from interacting with cholinergic nerve endings. H-BAT does not have an effect on BoNT once it has entered its target cell population. Therefore, H-BAT is not expected to reverse paralysis in botulism patients but instead reduces the severity and progression of symptoms by neutralizing unbound BoNT in the circulation (Figure 4). Antitoxin binds to free botulinum neurotoxin in synaptic cleft Figure 4: Mechanism of Action of H-BAT Botulinum Toxin v v Antitoxin Modified from Arnon et al., 2001. 42 42 Ooi/Department of Pharmacology & Toxicology/The University of Arizona College of Pharmacy 21 Biotechnology Drugs-2 PCOL825A/Fall 2023 HBAT only lowers the severity and progression of symptoms - HBAT is produced by Cangene Corporation - HBAT binds to free toxins in the synaptic cleft - HBAT does not have an effect on toxins that have already entered the cells. - HBAT does not reverse paralysis 43 43 Antisera are produced in large animals 44 National Library of Medicine 44 Ooi/Department of Pharmacology & Toxicology/The University of Arizona College of Pharmacy 22 Biotechnology Drugs-2 PCOL825A/Fall 2023 Antisera are produced in large animals 45 National Library of Medicine 45 Old faithful 46 National Library of Medicine 46 Ooi/Department of Pharmacology & Toxicology/The University of Arizona College of Pharmacy 23 Biotechnology Drugs-2 PCOL825A/Fall 2023 Antisera contain precise mixture of antibodies necessary to neutralize venom or toxins 1. Venoms and toxins are typically a mixture of proteins and enzymes. 2. Exposure to venoms and toxins cause acute toxicity – not enough time for immune system to generate neutralizing antibodies. 3. Antisera are rich in antigens that can cause allergic reactions. 4. Allergic to antisera can be severe in primed individuals 47 47 Toxins 48 48 Ooi/Department of Pharmacology & Toxicology/The University of Arizona College of Pharmacy 24 Biotechnology Drugs-2 PCOL825A/Fall 2023 Botulinum neurotoxin as biopharmaceuticals - Spasm and dystonia - Voice therapy - Cosmetic - Cancer palliative care 49 49 Vaccines 50 50 Ooi/Department of Pharmacology & Toxicology/The University of Arizona College of Pharmacy 25 Biotechnology Drugs-2 PCOL825A/Fall 2023 Recombinant hepatitis B vaccine Hepatitis B surface antigen produced in yeast through recombinant DNA techniques Immunization schedule : 0, 1, 6 months Postexposure management: Start vaccination within 24 hours of exposure. Patients with prior complete vaccination are considered sero protected. Administration : Intramuscular 51 51 Influenza virus vaccine (inactivated) • Formulation changes from year-to-year • Prior formulation should not be used • Vaccination should be done by the end of October • Administration: Depend on formulation (IM, or SubQ, or nasal) 52 52 Ooi/Department of Pharmacology & Toxicology/The University of Arizona College of Pharmacy 26 Biotechnology Drugs-2 PCOL825A/Fall 2023 Other biopharmaceuticals 53 53 Human Chorionic Gonadotropin (hCG) Widely used in weight loss Promotes lipolytic (fat burn) activity – testosterone effects Treat women with certain ovarian disorders Stimulate the testis of men who may be hypogonadal Pregnancy test Purification of recombinant hCG from the supernatant of CHOs 54 54 Ooi/Department of Pharmacology & Toxicology/The University of Arizona College of Pharmacy 27 Biotechnology Drugs-2 PCOL825A/Fall 2023 Erythropoietin (EPO) -A glycoprotein hormone -Stimulates the production of red blood cells in bone marrow -Produced in the kidney -Circulating in the blood -Treat anemia of chronic renal failure and malignancies -1968 purify human EPO -1977 purify 10 ml human Epo -1985 its gene cloned -Columbia Univ. patented the technique of synthesizing EPO (Amgen) 55 55 ZMAPP • Cocktail of 3 monoclonal antibodies • Treatment of Ebola virus infection • Produced in transgenic tobacco plants 56 56 Ooi/Department of Pharmacology & Toxicology/The University of Arizona College of Pharmacy 28 Biotechnology Drugs-2 PCOL825A/Fall 2023 Overview • Biopharmaceuticals / biologics • Insulin – mechanism of action and production • Antibody : Mechanisms of action • Types of antibody • Toxins as drugs • Vaccines • Other biopharmaceuticals 57 57 Ooi/Department of Pharmacology & Toxicology/The University of Arizona College of Pharmacy 29