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Last updated Nov. 22, 1:00 AM
Gene amplification for high level recombinant protein production in mammalian cells.
Principal system = dhfr- 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.
-GHT = without glycine, hypoxanthine (a purein source) and thymdine
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Some other amplifiable genes
ENZYME
ABBN
INHIBITOR
SELECTIVE MEDIUM
Adenosine deaminase
ADA
deoxycoformycin
MTX + adenosine
Ornithine decarboxylase
ODC
difluoromethyl-ornithine
Polyamine-free
Asparagine synthetase
AS
Ribonucleoside reductase
RR
hydroxyurea
Deoxynuceloside-free
Tri-functional pyrimidine
synthetic enzyme
CAD
PALA
Pyrimidine-free
Thymidylate synthetase
TS
FUdR
Thymidine-free
Dihydrofolate reductase
DHFR
MTX
Gly-, TdR-, purine-free
Glutamine synthetase
GS
Methionine sulfoximine
Gln-free
Asparagine-free
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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
(2 polypeptides: H-chain and L-chain)
+ GS cDNA gene (Bebbington)
Select on glutamine-free medium
Inhibit GS with methionine sulfoximine (gln analog)
Select for GS overproducers
--->--> Gene amplification does not seem to be operating in NS0 cells but can
be performed in GS+ CHO cells by suppressing the activity of the endogenous
enzyme with methionine sufoximine)
Proprietary (Lonza Biologics)
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Resting T- or B-cell)
Mature T-cell
(effector T-cell)
Plasma cell
(effector B-cell)
2002 Molecular Biology of the
Cell by Bruce Alberts,
Alexander Johnson, Julian
Lewis, Martin Raff, Keith
Roberts, and Peter Walter.
Myeloma cells (e.g., NS0)
are similar
Extensive ER
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Transfection strategies for gene amplification
1.
YFG (Your Favorite Gene) linked to a dhfr minigene on a single plasmid
A. ~Insures co-integration
B. ~Insures co-amplification
2.
YFG and dhfr on separate plasmids
A. Allows a high ratio of YFG to dhfr to start
B. Co-amplification not assured but commonly occurs.
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Possible amplification protocol
Pool of transfectants
selected for growth
in purine-free medium
Check
selected
populations
for Ig
production
0
10
20
40
80
10
20
40
80
160 nM MTX
40
80
160
300
1000 nM MTX
etc.
Note: Process is lengthy and tedious.
nM MTX
in -GHT
medium
Finally clone several
for final stages
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Some marketed recombinant proteins
Erythropoietin (Epogen, Procrit) J&J, Amgen
Tissue plasminogen activator (TPA) Genentech
Growth Hormone (Genentech)
Insulin (Genentech)
Beta-interferon (Avonex) Biogen-IDEC
Alpha-interferon (IntronA) Schering-Plough
Neupogen (Amgen)
Etanercept – TNF receptor + IgG (Enbrel) Amgen
Monoclonal antibodies (mAbs): see next
Top ten monoclonal antibodies in sales 2009-201
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Ways to increase production and/or lessen development time:
Mitchell Reff (IDEC patent): Screen for a high production genomic position.
Integrate YFG into it by homologous recombination, selecting for
reconstitution of a split dhfr minigene, then amplify.
Mitsubishi (T. Shibou, Mitsubishi Pharma Corporation. European Patent Application. Vol.
EP001293564A1, PCT/JP01/04801): Same, but use a lox site and site-specific
recombination to integrate YFG.
Add chromatin remodeling sequences to vector to open chromatin.
Add “insulator” sequences to vector to block postion specific repression.
Search for even better promoters (current: CMV, EIFalpha, actin)
Or even synthetic promoters (E. coli: Stephanopolos, MIT)
Engineer cells with advantageous glycosylation patterns
Engineer cell to eliminate or defer apoptosis (for longer productinon runs)
Etc.
(including Chasin lab project)
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Cell titer
Ab g/l
Antibody grams/liter
Millions of cells per liter
ml.
Lonza 2005 Web site presentation (2004 claim = 2.8 g/L)
(23 d.)
(Old values from
(1990)
Note improvement include:
1) Higher cell density
2) Longer times
3) Higher output per cell
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20,000 liter fermentor
20,000 liter mammalian cell fermentor - Lonza Biologics - Portsmouth, NH
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High level production in mammalian cells. Do the math (back of the envelope):
Reff patent (IDEC, now Biogen-Idec): 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 = ~ 2 tons = 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 (market exist?)
At $15,000 (low?) per patient per year  $9B in sales /per 30 kL reactor
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Monoclonal Antibodies (mAbs)
- 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
Immunocytes at different stages or of different
B-cells:
types are often characterized by characteristic
progenitor = pro-B cell (B220+)
specific cell surface proteins, often acting as
precursor = pre-B cells: heavy-chain rearranged
antigens
immature B cell: makes IgM + light-chain rearranged
matured B cell:
Makes 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)
Each immunocyte (and its offspring) synthesize only a single type of Ig,
and use only one of the two alleles available (allelic exclusion)
For a summary of the immune system see Strachan and Read, pp. 119-131
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Domain structure of an immunoglobulin molecule
Heavy chain
Light chain
}
C = constant regions
V = variable regins (antigen binding)
H = heavy chain
L = light chain
}
Fab
Fragment ,
antigen binding
Fc
disulfide
bonds
Fragment , crystallizable
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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
Complementarity
determining
Region = “CDR”
Hypervariable
region
CHO = carbohydrate
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Fc functions
Opsonization
Complement activation
Fc
Constant
region
Antibody-dependent
cell-mediated
cytotoxicity (ADCC)
Transcytosis
See below
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Disulfide bond
IgM
s
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)
Mechanism 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 light chain gene rearrangement
76 Vk, 5 Jk, 1 Ck over 2 Mb
Light
chain
genesis
DNA
+ SOMATIC
HYPERMUTATION
(J)
J
SPLICING
(J)
(D,J)
SPLICING
(D,J)
Heavy
chain
genesis
+ SOMATIC
HYPERMUTATION
L = leader sequence, signal for secretion
DNA
95Vh, 30Dh,5Jh, 11Ch over 1.4 Mb
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Class switch
recombination sites
Alt.
splicing
IgD
Adapted from Janet Stavnezer http://www.umassmed.edu/faculty/show.cfm?start=0&faculty=300
Choice of constant region exons (class switching) takes place via DNA
recombination (below) and alternative splicing of pre-mRNA
Sequential
recombination can
also take place
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
pA
Developmental Biology, Eighth Edition, Scott F. Gilbert
pA
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Different classes of Igs have different properties
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Fc functions
Opsonization:
Direct uptake into macrophages of bacteria coated with antibody molecules
Complement activation:
Activated complement proteins lyse foreeign cells by making holes in their membranes
(e.g. bacteria cell membrane)
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
Antibody-dependent cell-mediated cytotoxicity (ADCC):
Cells with a surface antigen are coated with antibodies that bind via their Fab region.
Then killer T-cells use Fc receptors on their surface to recognize the Fc region of the
attached bound antibodies and kill them with cytotoxins.
Fc
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Antibodies can participate in host
defense in several ways
Also ADCC
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ADCC = antibody-dependent cell-mediated cytotoxicity
Fc receptor
When activated by Fc binding, NK cells release
Perforin  makes holes in the membrane
Granzymes = proteases that initiate apoptosis
NK cells = natural killer cells
Fc
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NK cell
Genentech
Fc region
ADCC = antibody-dependent cell-mediated cytotoxicity
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Antibody generation
2002 Molecular Biology of the Cell
by Alberts, Johnson, Lewis, Raff,
Roberts, and Walter.
T-cells: cell mediated immune reactions
and
B-cells:  secreted antibodies
Prexisting B cells that are already producing antibodies that can bind to a specific 33
antigen are stimulated to divide when presented with that antigen. There are many
different clones of such precursor cells, each of which is stimulated. The final
response is therefore POLYclonal.
2002 Molecular Biology of the Cell by Bruce Alberts,
Alexander Johnson, Julian Lewis, Martin Raff, Keith
Roberts, and Peter Walter.
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The antibody secreting effector cells terminally differentiate (die) but their sister memory
cells live on to generate an amplified response upon a second exposure to antigen
1st exposure
Memory cells
2nd exposure
2002 Molecular Biology of the Cell by Bruce Alberts,
Alexander Johnson, Julian Lewis, Martin Raff, Keith
Roberts, and Peter Walter.
Effector cells
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2002 Molecular Biology of the Cell by
Bruce Alberts, Alexander Johnson, Julian
Lewis, Martin Raff, Keith Roberts, and
Peter Walter.
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Monoclonal antibodies via cell hybridization
Selects for
rare hybrid cells
Spleen cells do not grow in
culture. TGr myeloma cells
do not grow in HAT
e.g., in peritoneal cavity)
cavity
TG = 6-thioguanine
HAT = hypoxanthine,
amethopterin, and
thymidine
Immunize with antigen X
Monoclonal antibody generation
Hprt- myeloma cells
6-TG-resisatnt
HAT-
(HAT)
Cell fusion
Selection in HAT
Plate among many wells for
supernantant testing
Screen for secreted anti-X antibody
Plate positives at low density (~1/well) for cloning
Positive clones provide a continuing source
of anti-X antibody
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MAb therapy targets
Inflammation
Autoimmune disease
Graft rejection
Cancer
Viral infection
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Therapeutic strategies
• Plain MAbs
• MAbs fused to other protein binders
(e.g., soluble receptors) to increase avidity and/or to effect ADCC
• MAbs fused to cytotoxic agents
(toxins, radionuclides)
Toxins:
ricin (stops protein synthesis)
calicheamicin (DNA breaks)
Radionuclides:
90Y = yttrium
111I = indium
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Monoclonal antibody generation
- Cells needed
- antigen administration
- hybridoma formation
- selection
myeloma cells and mouse spleen cells
Kohler and Milstein
via cell fusion
mutants required (myeloma hprt- usually)
Further development:
- antibody generation
- engineered MAbs
- refinement
cDNA cloning from hybridoma
expression vectors
2nd generation antibodies, in vitro
Solve problems of using mouse antibodies in humans
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Problems of mouse MAbs
1) Fc portion limited in its ability to interact with Fc receptors of
human cells.
2) Lower serum half-life
3) Development of human anti-mouse antibodies (HAMA)
A) Retreatment results in allergy or anaphylactic shock
B) Retreatment is less effective
Breedveld, Lancet 2000
355:9205
Solutions via recombinant DNA genetic engineering :
1) Chimeric mouse-human antibodies: mouse V regions fused to Hu C-region
2) Humanized mouse antibodies, Parts of V-region from human interspersed with
mouse CDR V-regions
3) Human antibodies (fully), via transgenic mice carrying human immunoglobulin
genes as source of spleen cells
(Medarex, Abgenix, Kirin)
CDR = complementarity-determining region
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MAb Fusion Proteins
With other protein-binding proteins: natural receptors in soluble form
Analogous to MAbs and make use of the Fc portion of the antibody molecule:
Example: Enbrel (etanercept):
Anti-rheumatoid arthritis drug
Soluble TNF receptor fused to the Fc IgG1 domain (TNF= tumor necrosis
factor)
Ties up TNF, blocking its inflammatory function
Fc domain dimerizes the receptor, which increases its affinity for TNF.
Fc domain increases the half-life of the protein in the bloodstream
Amgen + Wyeth
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Example, still experimental:
Anti-HIV drug PRO 542
Uses soluble form of the CD4, the molecule to which HIV attaches on T-cells
Aim: block the viral surface protein that binds CD4
Soluble CD4 (HIV receptor) fused to IgG2.
Tetrameric (all 4 V-regions replaced) – therefore mutlivalent
Reduced Fc function (chose IgG2 for this reason)
Better half-life than soluble CD4 itself
(However, recently replaced by a MAb (PRO 140) targeting the CCR5 cell surface
protein, required for viral entry)
Progenics
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Single chain antibodies (scFv)
Ag
binding
site
15 AA
linker
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M13 phage display
filamentous phage that infects E. coli
POI = protein of interest
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Phage display to isolate functional V-regions
Can be used to screen billions of V-region variants for binding to a particular antigen
of choice.
Key requirement of this powerful strategy, and many of a similar kind:
A physical link of genotype to phenotype
1) a nucleic acid sequence representing a GENOTYPE (here, DNA) to
2) the PHENOTYPE (e.g., binding to something) of a protein coded by that nucleic acid
Commonly used phage = m13, filamentous, infects E. coli
Phage coat protein
the protein
the DNA
(inside)
“Panning”
E.g., for a SC
Ab, coat the
dish with Ag
Protein displayed in the phage coat
Can screen 1010 phage
Phage display selection of scFv’s (single-chain variable regions)
Source of sequence: PCR from genome or RT-PCR from mRNA,
add randomization (doped synthesis).
scFvs
Repeat, to
reduce
background
Wash
or
Elute, re-infect E. coli
PCR rescue scFv DNA
Clone (plaque on lawn)
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