Interleukin-2: Its role in treating HIVinfected patients and the possible wayto produce it

Prasit FaipenkhongProduction 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

•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

Diagram of the amino acid sequence ofaldesleukin

•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

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?

•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)

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

•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?

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

•Procedure

•A collection of plasmid cloning vectors carrying both complete or incomplete IL-2 cDNA

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

Synthesis ofcDNA

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

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

DNA hybridization

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

Cloning of IL-2 gene into M13 vector•Single stranded DNA template

•Procedure

Cloning of IL-2 gene intoM13vector

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 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

Screening and Identification ofMutagenized Phage Plaques •To identify clones that contain mutated sequence of IL-2 gene

•Procedure

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

Recloning of the Mutagenized IL-2 Genefor Expression in E. coli

•To express the mutagenized IL-2 gene

•Procedure

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

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