Download - Interleukin-2: Its role in treating HIV infected patients and the possible way to produce it
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Interleukin-2: Its role in treating HIVinfected patients and the possible wayto produce it
Prasit FaipenkhongProduction group
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Outlines•Introduction
•Clinical aspect of aldesleukin (human IL-2 derivative)
•Production aspect aldesleukin (human IL-2 derivative)
•Summary
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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
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•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
Introduction
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Diagram of the amino acid sequence ofaldesleukin
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•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
Introduction
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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?
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What Does the Research Show?
•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
•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)
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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
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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
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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
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Pregnant Women and Children•Pregnant women
•Children
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•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?
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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
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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
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How to Get It?•several ongoing studies
off label use
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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
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Preparing IL-2 cDNA libraries
•Procedure
•A collection of plasmid cloning vectors carrying both complete or incomplete IL-2 cDNA
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Preparing IL-2 cDNA librariesPBL or Jurkat cells
mitogenCollect of mRNA for IL-2
Convert to double strand DNA (cDNA)
Join into a plasmid cloning vector
IL-2 cDNA libraries
Transform host cells
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Synthesis ofcDNA
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Preparing IL-2 cDNA librariesPBL or Jurkat cells
mitogenCollection of mRNA for IL-2
Convert to double strand DNA (cDNA)
Join into a plasmid cloning vector
IL-2 cDNA libraries
Transform host cells
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Screening and identification of IL-2cDNA 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
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DNA hybridization
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Screening and identification of IL-2cDNA 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
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Cloning of IL-2 gene into M13 vector•Single stranded DNA template
•Procedure
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Cloning of IL-2 gene intoM13vector
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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
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Oligonucleotide-directed MutagenesisM13-IL2 (double strand)
M13-IL2 (single strand)
Add synthetic oligonucleotide primerGATGATGCTCTGAGAAAAGGTAATC
Add Klenow fragment and dNTPs
Add T4 DNA ligase
Transform E. coli
Produce M13 plaques
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Screening and Identification ofMutagenized Phage Plaques •To identify clones that contain mutated sequence of IL-2 gene
•Procedure
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Hybridization
Inoculate one clone called M13-LW46 into a culture of JM103 E. coli
Screening and Identification ofMutagenized Phage Plaques
Digest with restriction enzyme DdeI
Prepare RF-DNA from the pellet of cell culture plate
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Recloning of the Mutagenized IL-2 Genefor Expression in E. coli
•To express the mutagenized IL-2 gene
•Procedure
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Recloning of the Mutagenized IL-2 Genefor Expression in E. coliRF-DNA from M13-LW46 Plasmid pTrp3
Ligated plasmid called pLW46
Tranform into E. coli
Restriction enzyme mapping
E. coli transformed with pLW46
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Restriction map of ptrp3
Restriction map of pLW46
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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
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