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Biopharmaceutical Proteins and Gene Therapy
- 1. Biopharmaceuticals – proteins/peptides, nucleic acids, gene therapy Larry Baum Hong Kong
- 2. Definition Biopharmaceuticals: large molecule drugs Often similar to natural biological compounds Made using cells or enzymes Examples: » Proteins or peptides (antibodies, hormones) » Nucleic acids (gene therapy) » Cells (stem cells)
- 3. History Insulin » Recombinant human insulin, or Humulin » Made by E. coli bacteria » For diabetes » Made by Genentech, licensed to Lilly » First recombinant protein approved by FDA, in 1982 Tissue plasminogen activator » Recombinant human tPA » Made by CHO (Chinese hamster ovary) cells » To reduce blood clotting in stroke or heart attack » Made by Genentech » Marketed from 1986
- 4. Market Sales: ~US$200 billion (a quarter of all drugs) Number: >300 biopharmaceuticals now approved and on the market
- 8. Examples: recombinant proteins Blood factors (Factor VIII and Factor IX) Thrombolytic agents (tissue plasminogen activator) Hormones (insulin, glucagon, growth hormone, gonadotrophins) Haematopoietic growth factors (Erythropoietin, colony stimulating factors) Interferons (Interferons-α, -β, -γ) Interleukin-based products (Interleukin-2) Vaccines (Hepatitis B surface antigen) Monoclonal antibodies (Various) Additional products (tumour necrosis factor, therapeutic enzymes)
- 9. Monoclonal antibody: Rituxan <object style="height: 344px; width: 425px"><param name="movie" value="http://www.youtube.com/v/AJEVeFy3bEs"><par am name="allowFullScreen" value="true"><param name="allowScriptAccess" value="always"><embed src="http://www.youtube.com/v/AJEVeFy3bEs" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="425" height="344"></object> https://www.youtube.com/watch?v=UtNeImBmQCM
- 10. Manufacture of protein 1. Choose cells 2. Transfect plasmid(s) of transgene and selector 3. Select transfected cell clones 4. Scale up 5. Purify protein Reference: http://www.biomed.rutgers.edu/doc/papers/Wrum.pdf
- 11. Manufacture of protein 1. Choose cells » Bacteria – E. Coli most popular – Advantages: easy to handle; grow quickly » Mammalian cells – CHO cells most popular – Advantages: proteins processed more like in humans, with post- translational glycosylation, proper folding, and solubility » Other transgenic sources: plants, yeast, animals – Plants: cheap – Yeast: easier and cheaper than mammalian cells, but more human- like processing than bacteria – Animals: cheaper than cells, but with same processing Reference: http://www.biomed.rutgers.edu/doc/papers/Wrum.pdf
- 12. Manufacture of protein 2. Transfect plasmid(s) of transgene and selector » Use any non-viral transfection method – Calcium phosphate precipitation, lipofection, electroporation, biolistic, polymer-mediated » Transgene: 1. Strong promoter 2. One intron, to increase cytoplasmic transport and translation efficiency 3. Transgene as cDNA 4. Change rare tRNA codons to common » Selector: DHFR, GS – Weak promoter, to divert expression to that of transgene – DHFR: DHFR-deficient cells; MTX amplifies copies of plasmid – GS: MSX selects clones with amplified copies of plasmid Reference: http://www.biomed.rutgers.edu/doc/papers/Wrum.pdf
- 13. Manufacture of protein 3. Select transfected cell clones » Grow cell dilutions in presence of selector: MTX (methotrexate) or MSX (methionine sulfoximine) » Choose clones that survive selection » Screen clones – High and stable expression of intact recombinant protein – Fast growth – But growth and expression often inversely related Reference: http://www.biomed.rutgers.edu/doc/papers/Wrum.pdf
- 14. Manufacture of protein 4. Scale up » Optimize growth of cells – Can grow from 100,000 to 10 million cells/ml in 10 days – Different medium for different growth phases – Defined medium (serum-free): standardized, safer, cheaper – Cells engineered for high growth: anti-apoptosis, growth factors » Optimize protein yield – Can reach 4 mg/ml in 20 days – Batches (most common now) or continuous perfused culture Reference: http://www.biomed.rutgers.edu/doc/papers/Wrum.pdf
- 15. Manufacture of protein 4. Purify protein 1. Capture: Ion exchange most common—by charge 2. Intermediate: Hydrophobic interaction most common—by hydrophobicity 3. Polishing: Gel filtration most common—by size http://kirschner.med.harvard.edu/files/ protocols/GE_antibodypurification.pdf
- 16. Vaccines Attenuated: Measles, Mumps, Tuberculosis Killed or inactivated: Hepatitis A, Influenza, Pertussis Toxoids: Tetanus, Diphtheria Experimental types » Subunit » Vector » DNA » Peptide
- 18. Monoclonal antibodies Human antibody methods » Fuse human B cells to myeloma cells » Genetically alter mice to contain human antibody producing genes » Phage display: put human B cell antibody genes into bacteriophage, infect E. coli, select clones producing desired antibody Conjugate antibodies: coupled to “payloads” » Radioisotopes: Zevalin and Bexxar to treat lymphoma » Cytokines: to generate anti-tumor response » Toxins
- 19. Cytokines Interferons » α, β, and γ » Fights viral, bacterial, or fungal infection » To treat hepatitis, multiple sclerosis, cancer, leprosy » Coupling with polyethylene glycol (PEGylation) prolongs absorption Interleukins » IL-2 induces tumor necrosis factor (TNF) » To treat cancer TNF » α and β » Activates T-cells and macrophages » Block α to treat autoimmune diseases – Crohn’s disease, arthritis, ankylosing spondylitis, psoriasis – Antibody (Infliximab and Adalimumab) or soluble protein receptor for α (Etanercept)
- 20. Cytokines Erythropoietin (EPO) » Stimulates red blood cell production » Treats anemia » Made in mammalian cells » Amgen won the race, with FDA approval in 1989 Granulocyte macrophage colony stimulating factor (GM-CSF) » Treats lack of blood cells in chemotherapy patients
- 21. Hormones Insulin Amylin » Acts to slow glucose uptake during a meal » To treat diabetes, along with insulin Human growth hormone (hGH) » Stimulates tissue growth » To treat short height
- 22. Enzymes Clotting factors » Factor VIII and Factor IX » To treat hemophilia Glucocerebrosidase » Processes gluococerebroside » Mutated and deficient in Gaucher’s disease » Very expensive: up to US$500,000/year
- 23. Gene therapy Adding or changing a gene in a patient’s cells to treat a disease The disease may be genetic or acquired Somatic or germ-line cells Still experimental
- 24. Gene therapy methods Virus: retrovirus, adenovirus, herpes simplex (HSV), adeno-associated virus (AAV), lentivirus (HIV) Fusion: liposomes, DNA complexes Physical: naked DNA, microprojectiles, cationic peptide transduction domain (PTD)
- 25. Example 1: ADA Deficiency Adenosine deaminase deficiency Mostly affects lymphocytes Autologous T cells grown, ADA retrovirus added, infused into patient T cells survive for months in patients Fewer infections Leukemia in some patients after several years
- 26. Retroviral Method Producer cells transgeneLTR LTR Packaging cell line MoMLV gag, pol, env genes neo gene
- 27. Example 2: Cystic fibrosis CFTR: chloride/water transport Thick mucus causes lung infections Adenovirus, AAV, liposomes into lungs Adenovirus: <1% of cells, no chloride secretion, inflammation Liposomes: little chloride secretion, no inflammation
- 28. AAV Method transgeneAAV AAV ITRITR AAV rep, cap genesAd ITR Ad ITR helper adenovirus AAV
- 29. Example 3: Brain tumor Thymidine kinase/retrovirus producer cells injected into tumor Ganciclovir kills thymidine kinase cells Bystander effect: gap junctions, vascular endothelium, diffusion? Tumor reduced in 25%, eliminated in 13% of patients
- 30. Brain gene therapy issues Targeting » Inject for small target area » Whole brain – Cross blood-brain barrier (bradykinin, mannitol) – Migratory cells – Replicating vector (HSV) – Cerebrospinal fluid Neuron transduction » AAV, HIV, HSV » Liposomes, PTD
- 31. Gene therapy obstacles Low efficiency: few cells transduced » Retrovirus: only dividing cells (liver, stem cells) » Low viral titer (retrovirus, AAV) » Physical barriers (solid tissue, absorbing tissue) Transient expression » Promoter shutdown (viral promoter) » Cell turnover (lung epithelium, peripheral T cells) Toxicity » Immune response (adenoviral coat proteins, transgene itself) » Replication-competent virus (HSV)
- 32. Popularity of gene therapy trials >2000 trials so far Most were for cancer Some for acquired disease Some for inherited disease
- 33. Future of gene therapy More research New vectors New generations of current vectors More trials Occasional success
- 34. Stem cells Bone marrow transplants » For cancer treatment or genetic disease » Common » Requires life-long immunosupression Other stem cells » For diabetes, genetic diseases » Research to find culture conditions and growth factors to induce differentiation » Still experimental
- 35. The EndThe End