Some amplifiable genes

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Some amplifiable genes
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• Ampification models over-replication, unequal
  sister chromatid exchange, breakage and fusion
  (Tanaka et al.).
• Map dhfr amplicons (Schimke, Hamlin) 300 kb ,
  but wide range
• Gene amplification is rare in normal cells
  (Wahl, Tslty). p53- mutation allows it.
• In nature
• DNA in oocytes
• Drosophila chorion genes
• In medicine
• chemotherapy resistance (MDR, P-glycoprotein,
  efflux pump)
• cancer (myc, ras)
• In biotechnology
• high level recombinant protein production in
  mammalian cells

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Some notable gene amplification players
Fred Alt
Geoff Wahl
George Stark
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Gene amplification for high level recombinant
protein production in mammalian cells.
Principal system dhfr co-amplification in CHO
cells Facilitated by the availability of
DHFR-deficient mutant CHO cells
CHO dhfr- cells vector with dhfr minigene
YFG
-GHT medium Most cells die. Transfectants live.
gradually increasing concentrations of MTX
Cells with gradually amplified dhfr transgenes
survive. YFG is co-amplified along with the dhfr
minigene.
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DHFR- cells require G,H,T
and are resistant to tritiated deoxyuridine
Isolation of dhfr- mutants
No FH4
X
DHFR- cells selected by their resistance to
radioactive 3H-deoxyuridine 3HdU ? 3HdUMP ?
3H-TMP ? 3H DNA ? death from radioactive
decay. DHFR- cells require glycine, hypoxanthine
and thymidine (GHT). In GHT-free medium CHO
dhfr- cells die, but transfectants that have
received a dhfr minigene, YFG, survive.
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A different major system for high level Mab
production NS0 cells Mouse myeloma cells, high
IgG producers ? IgG- variants NS0 No
endogenous IgG, but cell is a natural IgG
secretor. Lack glutamine synthetase (GS)
glutamate NH3 ATP ? glutamine ADP
Pi Vector MAb genes driven by strong promoters
(H-chain, L-chain) GS cDNA gene
(Bebbington) Select on glutamine-free
medium Inhibit GS with methionine sulfoximine
(gln analog) Select for GS overproducers
---gt--gt amplification does not seem to be
operating well in NS0 cells for the GS cDNA gene
and linked Mab genes. GS amp. does work in CHO
cells (GS). Proprietary (Lonza Biologics)
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Transfection strategies for recombinant protein
production

• YFG (Your Favorite Gene) linked to a dhfr
  minigene on a single plasmid
• A. Insures co-integration
• B. Insures co-amplification
• YFG and dhfr on separate plasmids
• A. Allows a high ratio of YFG to dhfr to start

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Linked amp
CHO cells
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Co-amp1
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Co-amp3
(with or without pre-ligation)
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kaufman
Y.F.G.
DHFR
Dicistronic mRNA
(ribosome read-through)
Also, later, better dhfr translation using an
IRES Internal ribosome intiation site, Mostly
viral but also some cellular genes But in theory,
not an advantage..
Y.F.G.
DHFR
IRES
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Co-amp2
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Co-amp4
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Amplification protocol
Note Process is lengthy and tedious.
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Position effect (Reff- IDEC) on high
production Expression level is influenced by the
position of integration (in transgenic mice and
transfected cells ) Reasons Euchromatin vs.
heterochromatin, even proximity to
heterochromatin, general chromatin organization,
proximity of enhancers M. Reff (patent) Screen
for a high producer site among many
transfectants. Integrated gene is linked to 1/3
of a neo gene (3 exons total), and several
selectable markers including dhfr (amplifiable)
. Use this transfectant as the host for YFG
linked within (why?)the other 2/3 of the neo
gene (between 2 introns). Overlapping neo
sequences target homologous recombination. Select
for G418 resistance (reconstruction of the neo
gene) Drug-resistant colonies carry YFG at the
hot spot for production,within an intron of the
neo gene. Homologous recombination frequency is
low (10-7), but you only need one good
transfectant. Amplify with MTX / dhfr.
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Reff patent 2002 (IDEC-Biogen) Exploits position
dependencefor high expression level
dhfr
Neo exon 3 with 3ss
Put in first
Histidinol dehydrogenase gene
Transfectants selected in histidinol (for HisD
gene, resistance) AND - purine thymidine (for
the dhfr gene)
pBR plasmid sequences
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Histidinol dehydrogenase detoxifies histidinol,
confers histidinol resistance
protein synthesis
inhibits protein synthesis (charged to tRNA but
cannot be transferred to growing peptide so
truncates)
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Viewed as in some chromosomal location
Neo neomycin phosphotransferase gene,
detoxifies neomycin in bacteria, G418 in higher
organisms pBR plasmid sequences
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IgG genes
Put in second. Brings in 2 neo exons to
construct an effective neoR gene Brings in IgG
heavy and light chain genes with their
promoters DHFR present for subsequent gene
amplifcation selecton
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Low freq event lt 10-6)
After
From Molly
Select in G418 (neoR) for reconstruction of the
neo gene. Frequency is low (lt1/millon) but all
you need is one good transfectant
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Ig promoters
splicing
MAb genes are expressed from within the neo
gene Advantage? Tightly integrated, never lost
in a neoR cell. Disadvantage? Complex
transcription, competition?
MAb monoclonal antibody
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Some marketed recombinant proteins Erythropoieti
n (Epogen, Procrit) JJ, Amgen Tissue
plasminogen activator (TPA) Genentech Growth
Hormone (Genentech) Insulin (Genentech) Beta-in
terferon (Avonex) Biogen-IDEC Alpha-interferon
(IntronA) Schering-Plough Neupogen
(Amgen) Etanercept TNF receptor IgG (Enbrel)
Amgen Monoclonal antibodies (mAbs) several As
of 2006 (Walsh, G., Nature Biotech. 24
769) AvastinErbituxRaptiva XolairHumiraRemicad
eZenapax Simulect
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Considering Therapeutic Antibodies Ellen S.
Vitetta and Victor F. Ghetie Science 313, 308
(2006) Supp. Table
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2005 Web site presentation
2004 claim 2.8 g/L
(22 d.)
(Old values from
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20,000 liter mammalian cell fermentor - Lonza
Biologics - Portsmouth, NH
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High level production in mammalian cells Do the
math

• Reff 55 pg/cell/day
• Max cell density 107/ml ?
• So 1010 cells/L
• Therefore 55 x 10-12 g/cell/day x 1010 cells/L
  
• 55 x 10-2 g/L/day
• 0.55 g/L/day 11 g/L/20 days, calculated
• Lonza (contract manufacturer) claims (2005) 5
  g/L yield
• Same ballpark.
• 30,000 L reactor (largest)
• 30,000 L. X 5 g/L. 150 kg in 20 days, or say
  one month
• x 12 months 1800 kg/year 1,800,000 g/year
• One MAb dose 500 mg 0.5 g
• 1,800,000/0.5 3.6 million doses per reactor per
  year.
• 6 doses per patient per year?
• 3,600,000/6 600,000 patients per year per
  reactor.
• At 15,000 per patient per year ? 9B in sales
  /per 30 kL reactor

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• Monoclonal Antibodies
• Antibodies (Abs). Also known as immunoglobulins
  (Ig).
• Comprised of 2 heavy chains and 2 light chains
• Monoclonal Abs bind specifically to a single
  site (epitope) on a particular antigen
• Abs are produced by B lymphocytes.
• Because of their specificity and ease of
  generation, they are extensively used as
  therapeutics (passive immunotherapy) and as
  diagnostic and research tools
• - They can be generated in large (unlimited)
  amounts in culture

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Antibodies are made by B-cells
B cells develop in the bone marrow ?
hematopoietic stem cells and lymphoid stem cells
lymphoid stem cells ? T-cells and
B-cells progenitor pro-B cell
(B220) precursor pre-B cells
heavy-chain rearranged immature B cell
IgM light-chain rearranged matured B cell
IgM IgD an antigen encountered in spleen or
lymph nodes then goes to peripheral
circulation Terminally differentiated cell
plasma cell, periphery, Ig secretor (IgG, IgM,
some others)
Immunocytes at different stages or of different
types are often characterized by characteristic
specific cell surface proteins, often acting as
antigens
Each immunocyte (and its offspring) synthesize
only a single type of Ig, and use only one of
the two alleles available (allelic exclusion)
See Strachan and Read, pp. 307-311
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Domain structure of an immunoglobulin molecule
disulfide bonds
C constant regions V variable regins (antigen
binding) H heavy chain L light chain
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Heavy chain blue Light chain pink
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Laboratory fragmentation of antibodies
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Ig molecule showing polarity, disulfides,
carbohydrate
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Fc functions
Opsonization Complement activation Antibody-depe
ndent cell-mediated cytotoxicity
(ADCC) Transcytosis
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Disulfide bond
J-chain
Secretory IgA dimer
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Multigene organization of Ig genes light
chains V, J (variable) and C (constant) heavy
chain V, D, J, (variable) C (constant) Mechanis
m of Ab gene rearrangement Recombination signal
sequences (RSS)flank V, D, J gene
segments V-RSS------RSS-D-RSS---------RSS-J
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IgGkappa gene rearrangement
SOMATIC HYPERMUATION
(J)
(J)
SPLICING
(J)
(D,J)
SPLICING
(D,J)
SOMATIC HYPERMUATION
L leader sequence, signal for secretion
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Choice of Constant region exons (class switching)
takes place via DNA recombination (below) and
alternative splicing of pre-mRNA
Immunobiology, Charles Janeway, Paul Travers,
Mark Walport, Mark Shlomchik
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Different constant regions can be chosen via
alternative pre-mRNA splicing
Immunobiology, Charles Janeway, Paul Travers,
Mark Walport, Mark Shlomchik
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Alternative splicing within a group of Constant
region exons yields two forms of IgM
Developmental Biology, Eighth Edition, Scott F.
Gilbert
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Different classes of Igs have different properties
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Fc functions
Opsonization Direct uptake of bacteria coated
with antibody molecules Complement activation
Activated complement proteins lyse cells by
making holes in their mebranes(e.g.
bacteria) Antibody-dependent cell-mediated
cytotoxicity (ADCC) Killer T-cells use
antibodies on their surface to target cells with
an antigen and kill them. Transcytosis Antibody-
antigen complexes are taken up (endocytosed) on
one side of an epithelial cell and directed to
the other side, where they are exocytosed
Fc
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Antibodies can participate in host defense in
three main ways
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ADCC antibody-dependent cell-mediated
cytotoxicity
NK cells natural killer T-cells
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NK cell
Genentech
ADCC antibody-dependent cell-mediated
cytotoxicity
Fc region