MBT-Term Test 1

Microbial Biotechnology
Microbial production of therapeutic agents:

• pharmaceutical isolation of interferon cDNA;
Engineering of human interferon and human
growth hormones; optimizing gene expression.
• Enzymes DNAase I and alginate lyase
against cystic fibrosis.
• Monoclonal antibody as therapeutic agents-
production of antibodies in E. coli and yeast.
HIV thereapeutic agents.
Recombinant proteins for human use
• ~2003
• Approved in US or EU
Recombinant interferon:
isolation of cDNA
6,000 clones
• Strategies for isolating either the genes or cDNAs for human proteins
• 1) Isolate target protein and determine partial amino acid sequence
• Synthesize oligo as probe to screen genomic or cDNA library
• 2) Generate Ab against purified proteins
• Screen gene expression library
• 3) proteins tissue specific-----mRNAs—cDNA

• Interferon strategy above, pre-human genome sequence
Hybrid products: INF gene shuffing
• Interferons assist the immune response by inhibiting viral replication within host cells, activating natural
killer cells, increasing antigen presentation to lymphocytes, and inducing the resistance of host cells to
viral infection
• IFN cDNA isolated early 80
•  and  synthesized in cell exposed to virus or viral RNA, gamma response to cell growth stimulating agent
• IFN family of 13 genes; IFN family of 2 genes; IFN of 1 genes
• different specificities alpha 1 = 2 Bovine cell line, 2>7x1 human cell, 2<30x1 mouse cells
• IFN 2 and 3 have common RE sites at 60, 92 and 150
• Hybrid INFs demonstrate potential therapeutics by combining functional domains
• Some (2003)- successful clinical trials, approved for use as human therapeutic agents
Hybrid>>Parental
2'-5' oligoisoadenylate
synthetase-
2'-5' linked
oligonucleotides
Longer-acting interferons
Hepatitis C virus infection- liver disease-200 million.
-either cirrhosis of the liver or hepatocellular carcinoma.
-achieving lasting eradication of the virus from an infected individual.
Treatment
-combined use of ribavirin with IFN-α.
Longer-acting IFNs are needed to minimize the side effects from IFN
treatment, lower the required dosage, and decrease the required
frequency of the treatments.
Approaches:
- PEGylation (covalently attaching PEG) to proteins). PEGylation
increases the size of IFN and prolonging its circulatory time by reducing
its renal clearance.
- fuse an IFN gene with the gene for a stable protein, such as human
serum albumin. This combination has been called the albumin–
interferon hybrid molecule (Zalbin, formerly Albuferon).
Native IFN-2 days-Hybrid every 2 weeks.
Site-specific directed mutagenesis: hGH
• hGH: 191 AAc, 22,1 kDa

• One of first therapeutic proteins-called somatrem (Protropin); Genentech 1985.
• identical to native pituitary-derived hGH
• daily injections during the years when the child is growing.
• The cost-approximately $10,000 to $30,000 per year.
• Native binds to growth hormone receptor and prolactin receptor
• Avoid Side effects
• Overlaps but not identical to prolactin receptor
• Prolactin receptor binding function of Zn++ binding
• Domain: His-18, His-21, Glu-174
• Site-specific mutagenesis
• short half-life in plasma,
• subcutaneous injection once a day.
• Therefore, a long-lasting form of
human growth hormone.
• the extracellular domain of the

human growth hormone receptor
was fused to human growth
hormone using a 20-amino-acid-long
linker peptide consisting of four
repeats of the amino acids Gly4Ser.
• a single injection -10 days
• cleared from plasma 300 times
more slowly than free human
growth hormone.
• C-terminal end
of hGH (~22 kDa) with
the N-terminal end of
human serum albumin
(~67 kDa).-Albutropin;
yeast-about 19 days.-
completed phase I
clinical trials.
Optimizing gene expression
• Multistep process:
• Design a protein, construct a recombinant molecule, express and characterize
• Need to optimize expression
• First, either prokaryote or eukaryote host
• Comparative analysis of host and expression
• ex., interleukin-3 expression
• Best in Bacillus licheniformis
• Balance with glycosylation in eukaryotic hosts
• But, glycosylation is not essential for interleukin-3 activity
Expression optimization
To optimase:
Level of inducer (e.g. arabinose)
Time of induction
Temperature of the induction step (popular - 18oC

overnight)
Increasing Protein Production
• Increase target gene copy number
 Often by increased temperature of culture
• Properly done can reach 20% of total protein
(vs. 2% for most common natural protein)
Effect of Temperature on the Plasmid Copy
Number of Three Expression Vectors
DNase I and Cystic fibrosis
Cystic Fibrosis/ mucoviscidosis?

- Anderson in 1938
- is one of the most common life-threatening inherited diseases
among Europeans and their descendants.
- over 23,000/Canada and Europe and 30,000/USA.
- mutant cystic fibrosis gene -1 in 29 Europeans, 1 in 65 African
Americans, and 1 in 150 Asians.
- CF is caused by a mutation in the gene for the protein
cystic fibrosis transmembrane conductance regulator (CFTR).
- regulate the components of sweat, digestive juices, and mucus.
- an autosomal recessive disease.
• Average life span 37 years, extended
and extending
• Chloride ion channel, sweat, digestive
juices and mucus
• 7q31.2 -> 180,000 bp gene,
1,480 AAc
• Most common mutation ΔF508; 1,400
other mutations
• ΔF508 + missense, not folded correctly
CFTR controls
chloride ion
movement in and
out of the cell.
• wikipedia
• Lungs susceptible to bacterial infection
• Antibiotics treatment results in resistance
• bacteria alive + lysed-----------causes accumulation of a
thick sticky mucus-----------This makes it hard to breathe
and digest food.
- thick sticky mucus (alginate from live bacteria + DNA
from lysed bacteria and degenerating leukocytes) causes
pulmonary problems (mucus)
Treatment
• Genentech: hDNase I in CHO cells

• Not a cure, but alleviates symptoms
• Purified protein delivered via aerosol mist to lungs of CF-
• Approved by FDA in 1994
• In response to bacteria in lungs,
leukocytes cluster and lyse bacteria (and leukocytes)
• Lysed leukocytes release actin
• Monomeric actin binds DNase I very tightly and inhibits
• Limits effectiveness
• X-ray structure data suggested Ala-144 required for binding
or Tyr-65
• Changing either to Arg decreases actin binding by 10,000x
• Clinical efficacy of mutants to be determined (2003)
Clearing the lungs 2 with alginate lyase
β-d-mannuronate and α-l-guluronate
• Alginate produced by seaweeds, soil and marine bacteria

• P. aeruginosa excretion in lungs contributes to viscosity of mucus
• Antibiotics not effective because of biofilm
• In addition to DNase I treatment, alginate lysate can be used
as therapeutic agent • http://www.lsbu.ac.uk/water/hyalg.html
Cloning alginate lyase
• Flavobacterium sp.
• Clone bank in E. coli
• Screen by plating onto medium plus alginate
• +/- Ca++
• Ca++ + alginate = cross-linked opaque
• Hydrolyzed alginate does not cross-link
• Analysis and characterization of clones and alginate lyase
Alginate lyase[s]
• ORF 69,000 Da
• Precursor of three alginate lyases
• -> 3,000 Da + 63,000 Da
• 63,000 Da lyses both bacterial and seaweed alginates
• 63,000 Da -> 23,000 Da seaweed effective+ 40,000 Da bacterial effective
• Clone bacterial activity portion
Optimization of activity
• Increase expression of 40,000 Da protein

• PCR amplify and insertion behind strong promoter
• B. subtilis plasmid, fused to a B. subtilis a-amylase leader peptide, directs secretion and
penicillinase gene promoter
• Expressed and assayed for halo phenotype
• Liquifies alginates produced by P. aeruginosa isolated from lungs of CF patients
• 2003, additional trials to determine if effective therapeutic agent
Phenylketonuria (PKU)
• Autosomal recessive genetic disorder in phenylalaniine hydroxylase

• Phe accumulation, decreases other ‘large, neutral AAc’ in brain, needed for
protein and neurotransmitter synthesis
• Brain development; progressive mental retardation and seizures
• Incidence ~1/15,000; varies: 1/4,500 Ireland and 1/100,000 Finland
• 12q22-q24.1
• Macaque genome: PAH gene sequence identical to a human PKU mutation
• wikipedia
Phenylketonuria treatment[s]
• Traditional treatment: diagnosis at birth or prenatal

• Controlled semi-synthetic diet with low levels of Phe
• Possible treatment: metabolism of Phe
• PAH multienzyme complex, requiring cofactor
• Phe ammonia lyase (PAL) converts Phe as well
• Stable and does not require cofactor
• To test concept, yPAL cloned and overexpressed in E. coli
• Preclinical studies (2003) with mice deficient in PAL
• See lower plasma levels of Phe when PAL injected or
administered as oral encapsulated enzyme
Monoclonal antibodies (mAb) as therapeutic agents
• 100 years ago-Horse-Corynebacterium diphtheriae-infects throat/tonsils-exotoxin-

bloodstream-distant organs--------so, passive immunity-----------but risks. (foreign +
second treatment sensitized patient---anaphylactic shock and die
• Today –hybridoma technology-but cross reactivity-antibodies against murine
determinants
•
Antibody molecular structure
• CDRs variable portions of the protein, both H and L

• Papain digestion – (2 Fab + Fc) Fv a. a. varies one molecule to another
• Fc elicits immunological responses after Ag-Ab
• Complement cascade (break down cell membranes, activates phagocytes + mobilize other components)
• ADCC—Fc binds to Fc receptor of ADCC effector cell---------------releasing substances –lysis foreign
cell
• Fab-Ag---------Fc+ phagocytic cells ------------engulf and destroy Ab-Ag comples
Preventing rejection of transplanted organs
• binds to certain lymphocytes

• Mouse mAb OKT3 first to be approved
by FDA
• Immunosuppressive agent after organ
transplant in humans
• T cells are responsible for rejection
• OKT3 binds to CD3 on all T cells
• Side effects-----fever/rash formation
Polyclonal antibodies (Ab)
• www.abbottdiagnostics.com/Science/pdf/learning_immunoassay_01.pdf
Monoclonal antibodies (mAb)
Monoclonal antibodies
(theoretical)
• Monoclonal Antibodies: A Manual of Techniques. HZola

Monoclonal antibodies (mAb) protocol
• Monoclonal Antibodies: A Manual of Techniques. HZola
Chemically linked monoclonal antibody Herceptin®
• “Magic bullet”
• Genentech. FDA 9/98; Aullrich/Genentech and DSlamon/UCLA Jonsson Cancer Ctr
• Trastuzumab (trade name Herceptin)
• Humanized monoclonal antibody
• Target is HER2/neu receptor (erbB2)
• HER2-positive metastatic breast cancer
• Anti-cancer therapy in breast cancer, over-expressing erbB2 receptor
• ErbB2 receptor amplification occurs in 25-30% of early-stage breast cancers
• Transmembrane Tyr kinase, activating PI3K/Akt pathway and MAP pathway
• Overexpression promotes invasion, survival and angiogenesis of cells
• Also confers therapeutic resistance to cancer therapies
• Herceptin binds to extracellular domain of erbB2 receptor,
• Arresting cell at G1 phase
• wikipedia
Magic bullet: delivery of drug to site
• Drugs –effective in vitro but less in vivo

---not to reach target site
• Solution: increasing concentration but side effects
• Lower than optimal levels decreasing efficacy
• Strategy
• A) Drugs encapsulated in liposome
• B) certain toxin genes incorporated in to
tumor-infiltrating lymphocytes
• Binding of mAb requires second step C) D)
• 1) delivery of drug
• 2) delivery of enzyme to convert pro-drug
Not mouse----human or humanized Ab
Magic bullet: delivery of active agent to site
• Binding of mAb requires second step

• variations
• Priklad ANTISENSE delivery
Human mAb problem
Drawbacks to immunotherapeutic agents use

• Chemical couplings problem
• Yields low; coupling at random sites; chemical portion may inactive attached enzyme
• Nonhuman mAb
• If condition requires multiple treatments, nonhuman mAb causes immune response
Human mAb
• Human chromosomes of fused human lymphocyte-mouse myeloma cells are unstable
• No human myeloma cell line can replace mouse myeloma cell line
• Ethics of injecting human subject to generate Ab-producing cells
• to collect Ab-producing cell –B cells +Epstein-barr virus in culture-------Ab weak
Hybrid human-mouse mAb: chimeric
• ransgenic mice
• Genetic engineering to convert mouse mAb into a hybrid
• Exchange Fc portions
• Using oligonucleotides and in vitro DNA replication or cloned segments
• Construct in expression vector; transfect into cultured B lymphocytes
• Chimeric Abs are 70% human/30% mouse
Hybrid human-mouse mAb: chimeric
• Ex., chimera of mouse mAb against surface of human colon cancer cells
• Tested in patients with colorectal cancer
• Half-life in blood system 6x longer
• 1/10 patients developed mild response against chimera
• But, no anti-tumor activity observed (2003)
• Low dosage and/or advanced state of the cancer?
Hybrid human-mouse mAb: humanized
• Humanized Ab
• Substitute CDRs into human Ab
• 95 %human/5 % mouse
• Construction by isolating cDNAs for L and H chains
• Amplify variable regions using PCR protocol
• Primers are complementary to ends of variable regions, conserved
• CDRs are highly variable sequences

MBT-Term Test 1

Uploaded by

Copyright:

Available Formats

MBT-Term Test 1

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

MBT-Term Test 1

Uploaded by

Copyright:

Available Formats

Microbial Biotechnology

Microbial production of therapeutic agents:

• 3) proteins tissue specific-----mRNAs—cDNA

• hGH: 191 AAc, 22,1 kDa

• the extracellular domain of the

Temperature of the induction step (popular - 18oC

Cystic Fibrosis/ mucoviscidosis?

• Genentech: hDNase I in CHO cells

β-d-mannuronate and α-l-guluronate

• Alginate produced by seaweeds, soil and marine bacteria

• Increase expression of 40,000 Da protein

• Autosomal recessive genetic disorder in phenylalaniine hydroxylase

• Traditional treatment: diagnosis at birth or prenatal

• 100 years ago-Horse-Corynebacterium diphtheriae-infects throat/tonsils-exotoxin-

• CDRs variable portions of the protein, both H and L

• binds to certain lymphocytes

• Monoclonal Antibodies: A Manual of Techniques. HZola

• Drugs –effective in vitro but less in vivo

• Binding of mAb requires second step

Drawbacks to immunotherapeutic agents use

You might also like