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Interleukin-2: Its role in treating HIV infected patients and the possible way to produce it. Prasit Faipenkhong. Production group. Outlines. Introduction. Clinical aspect of aldesleukin (human IL-2 derivative). Production aspect aldesleukin (human IL-2 derivative). Summary. Introduction.
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Interleukin-2: Its role in treating HIV infected patients and the possible way to produce it Prasit Faipenkhong Production group
Outlines • Introduction • Clinical aspect of aldesleukin (human IL-2 derivative) • Production aspect aldesleukin (human IL-2 derivative) • Summary
Introduction • Interleukin-2 (IL-2) • cytokines • helper T cells (CD4+ T cells), cytotoxic T cells (Tc, CD8+ • T cells), natural killer cells (NK cells) • induce proliferation and differentiation of CD4+ T cells • and cytotoxic T cells • induce B cell proliferation, stimulate macrophage • activity, increase number and toxicity of NK cells • production is decrease in HIV infected patients
Introduction • Aldesleukin • a human IL-2 derivative • recombinant DNA technique • absence of an N-terminal alanine, replacement of • cysteine with serine at position 125, absence of • glycosylation
Diagram of the amino acid sequence of aldesleukin
Introduction • Aldesleukin • possess immunological activities similar to those • observed with native IL-2 • has been approved by FDA for treating metastatic renal • cell carcinoma and metastatic melanoma • phase III clinical trials in HIV infected patients
Clinical aspect of aldesleukin • What Does the Research Show? • Who Should Use It? • Pregnant Women and Children • What About Side Effects? • How to Use It? • How to Get It?
What Does the Research Show? • Subcutaneous, intravenous, intramuscular • daily, weekly, monthly and every two months • very low doses (1 million International Units, or IU) to • higher doses (18 million IU) • Immunological benefits in several clinical trials • increase CD4+ cells without sustained increase in viral • load • Duration of intermittent IL-2 therapy appear to be • important
What Does the Research Show? • Subcutaneous injection is similar to intravenous infusion • improvement in immunological parameter • Lower dosage of IL-2 (3 MIU/day) are still effective • increase CD4+ counts • No difference in viral load in group receiving IL-2 + • antiretroviral therapy • IL-2 + HART may reduce the latent reservoir
What Does the Research Show? • French regulatory agency approve a Compassionate • Use Program of aldesleukin for people with CD4+ counts • below 200 and HIV levels below 1,000
Who Should Use It? • Can be used safely by people at all levels of CD4+ cell • counts • People with high detectable viral loads • People with an active infection, heart problems, lung • diseases, autoimmune diseases, DM, thyroid problems • People facing extreme fatigue
Pregnant Women and Children • Pregnant women • Children
What About Side Effects? • Flu-like symptoms • Swelling, redness, or lumps • Sinus congestion, low blood pressure, liver toxicity, • swelling due to water retention, nausea and vomiting, • diarrhea, peeling skin, changes in mental status, and • altered blood levels including albumin, potassium, • magnesium, calcium, red blood cells, and platelets • Worst on the last two or three days of a five-day course
What About Side Effects? • Oliguria or anuria, shortness of breath, high fever that • doesn't go away with OTC drugs, major changes in • mental status, faint, major swelling
How to Use It? • Subcutaneous, 9-15 MIU/day, bid, five consecutive days • every eight weeks • The time between courses of IL-2 therapy will be increased • by four-week period • Continuous intravenous, 9-18 MIU/day, five consecutive • days, every eight weeks
How to Get It? • several ongoing studies • off label use
Production aspect aldesleukin • Preparing IL-2 cDNA libraries • Screening and identification of IL-2 cDNA clones • Cloning of IL-2 gene into M13 vector • Oligonucleotide-directed Mutagenesis • Screening and Identification of Mutagenized Phage • Plaques • Recloning of the Mutagenized IL-2 Gene for Expression • in E. coli
Preparing IL-2 cDNA libraries • A collection of plasmid cloning vectors carrying both • complete or incomplete IL-2 cDNA • Procedure
Preparing IL-2 cDNA libraries PBL or Jurkat cells mitogen Collect of mRNA for IL-2 Convert to double strand DNA (cDNA) Join into a plasmid cloning vector IL-2 cDNA libraries Transform host cells
Synthesis of cDNA
Preparing IL-2 cDNA libraries PBL or Jurkat cells mitogen Collection of mRNA for IL-2 Convert to double strand DNA (cDNA) Join into a plasmid cloning vector IL-2 cDNA libraries Transform host cells
Screening and identification of IL-2 cDNA clones • To identify the colonies of host cells that carry complete • IL-2 cDNA • DNA hybridization, immunological assay, protein • activity • Rescreen with the probe, confirm by restriction mapping
Screening and identification of IL-2 cDNA clones • To identify clones that carry a specific plasmid-cDNA • construct • DNA hybridization, immunological assay, protein • activity • Rescreen with the probe, full length clones were identified • and confirmed by restriction mapping
Cloning of IL-2 gene into M13 vector • Single stranded DNA template • Procedure
Cloning of IL-2 gene into M13 vector
Oligonucleotide-directed Mutagenesis • To engineer the IL-2 gene in order to get the the IL-2 • derivative which has suited physical and chemical • properties • Cysteine125 to serine • Decrease an aggregated oligomeric form when isolated • from bacterial cells • Decrease an aggregated inactive oligomeric form upon • storage • Procedure
Oligonucleotide-directed Mutagenesis M13-IL2 (double strand) M13-IL2 (single strand) Add synthetic oligonucleotide primer GATGATGCTCTGAGAAAAGGTAATC Add Klenow fragment and dNTPs Add T4 DNA ligase Transform E. coli Produce M13 plaques
Screening and Identification of Mutagenized Phage Plaques • To identify clones that contain mutated sequence of IL-2 • gene • Procedure
Screening and Identification of Mutagenized Phage Plaques Hybridization Digest with restriction enzyme DdeI Inoculate one clone called M13-LW46 into a culture of JM103 E. coli Prepare RF-DNA from the pellet of cell culture plate
Recloning of the Mutagenized IL-2 Gene for Expression in E. coli • To express the mutagenized IL-2 gene • Procedure
Recloning of the Mutagenized IL-2 Gene for Expression in E. coli RF-DNA from M13-LW46 Plasmid pTrp3 Ligated plasmid called pLW46 Tranform into E. coli Restriction enzyme mapping E. coli transformed with pLW46
Restriction map of ptrp3 Restriction map of pLW46
Summary • Aldesleukin, human IL-2 derivative, is produced by • recombinant DNA technique. • It has been approved by FDA for treating certain types • of kidney and skin cancer. • Now, it is under phase III clinical trials in HIV infected • patients. • Principle of microbial growth, maximizing the efficiency • of fermentation process, bioreactors, typical large-scale • fermentation, harvesting microbial cells, disrupting • microbial cells, and downstream processing