1 plification for high level recombinant protein production in mammali rincipal system = dhfr- CHO cells acilitated 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. = without g lycine, h ypoxanthine (a purein source) and t hymdine Last updated Nov. 22, 1:00 AM
47
Embed
1 Gene amplification for high level recombinant protein production in mammalian cells. Principal system = dhfr- CHO cells Facilitated by the availability.
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
1
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 mediumMost 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
Last updated Nov. 22, 1:00 AM
2
Some other amplifiable genes
ENZYME ABBN INHIBITOR SELECTIVE MEDIUM
Adenosine deaminase ADA deoxycoformycin MTX + adenosine
3A 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)
4
2002 Molecular Biology of the Cell by Bruce Alberts, Alexander Johnson, Julian Lewis, Martin Raff, Keith Roberts, and Peter Walter.
Plasma cell (effector B-cell)
Mature T-cell (effector T-cell)
Resting T- or B-cell)
Extensive ER
Myeloma cells (e.g., NS0) are similar
5
Transfection strategies for gene amplification
1. YFG (Your Favorite Gene) linked to a dhfr minigene on a single plasmidA. ~Insures co-integrationB. ~Insures co-amplification
2. YFG and dhfr on separate plasmidsA. Allows a high ratio of YFG to dhfr to startB. Co-amplification not assured but commonly occurs.
6
Pool of transfectants selected for growth in purine-free medium
Possible amplification protocol
0 10
10
40
20
20
80
40
40
160
80
80
300
160
1000
nM MTXin -GHT medium
nM MTX
nM MTX
etc.Finally clone several for final stages
Check selectedpopulations for Ig production
Amplification protocol
Note: Process is lengthy and tedious.
7
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
8Top ten monoclonal antibodies in sales 2009-201
9
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 patternsEngineer cell to eliminate or defer apoptosis (for longer productinon runs)
Etc. (including Chasin lab project)
10
(2004 claim = 2.8 g/L)Lonza 2005 Web site presentation
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 g1,800,000/0.5 = 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
High level production in mammalian cells. Do the math (back of the envelope):
13
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
14
B cells develop in the bone marrowhematopoietic stem cells and lymphoid stem cells
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)
For a summary of the immune system see Strachan and Read, pp. 119-131
15
Domain structure of an immunoglobulin molecule
} Fc
}FabFragment ,antigen binding
Fragment , crystallizable
disulfidebonds
C = constant regionsV = variable regins (antigen binding)H = heavy chainL = light chain
Developmental Biology, Eighth Edition, Scott F. Gilbert
Alternative splicing within a group of Constant region exons yields two forms of IgM
pA pA
27Different classes of Igs have different properties
28
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 functions
Fc
29
Antibodies can participate in host defense in several ways
2002 Molecular Biology of the Cell by Bruce Alberts, Alexander Johnson, Julian Lewis, Martin Raff, Keith Roberts, and Peter Walter.
Prexisting B cells that are already producing antibodies that can bind to a specific 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.
34
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
2002 Molecular Biology of the Cell by Bruce Alberts, Alexander Johnson, Julian Lewis, Martin Raff, Keith Roberts, and Peter Walter.
Memory cells Effector cells
1st exposure
2nd exposure
35
2002 Molecular Biology of the Cell by Bruce Alberts, Alexander Johnson, Julian Lewis, Martin Raff, Keith Roberts, and Peter Walter.
36
Monoclonal antibodies via cell hybridization
Selects forrare hybrid cells
Spleen cells do not grow in culture. TGr myeloma cells do not grow in HAT
e.g., in peritoneal cavity)
TG = 6-thioguanineHAT = hypoxanthine, amethopterin, and thymidine
cavity
37
Hprt- myeloma cells6-TG-resisatntHAT-
(HAT)
Plate positives at low density (~1/well) for cloning
Plate among many wells for supernantant testing
Positive clones provide a continuing source of anti-X antibody
Immunize with antigen X
Cell fusion
Selection in HAT
Screen for secreted anti-X antibody
Monoclonal antibody generation
38
MAb therapy targets
Inflammation
Autoimmune disease
Graft rejection
Cancer
Viral infection
39
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 = yttrium111I = indium
40
Monoclonal antibody generation
- Cells needed myeloma cells and mouse spleen cells - antigen administration Kohler and Milstein - hybridoma formation via cell fusion- selection mutants required (myeloma hprt- usually)
Further development:
- antibody generation cDNA cloning from hybridoma- engineered MAbs expression vectors- refinement 2nd generation antibodies, in vitro
Solve problems of using mouse antibodies in humans
41
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
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)
Breedveld, Lancet 2000 355:9205
CDR = complementarity-determining region
42
MAb Fusion Proteins
With other protein-binding proteins: natural receptors in soluble formAnalogous 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 functionFc domain dimerizes the receptor, which increases its affinity for TNF.Fc domain increases the half-life of the protein in the bloodstreamAmgen + Wyeth
43
Example, still experimental: Anti-HIV drug PRO 542
Uses soluble form of the CD4, the molecule to which HIV attaches on T-cellsAim: block the viral surface protein that binds CD4Soluble CD4 (HIV receptor) fused to IgG2.
Tetrameric (all 4 V-regions replaced) – therefore mutlivalentReduced 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
44
Single chain antibodies (scFv)
Ag binding site
15 AA linker
45
M13 phage display
filamentous phage that infects E. coli
POI = protein of interest
46
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 phenotype1) 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
“Panning”
the protein
the DNA(inside)
Phage display to isolate functional V-regions
Phage coat protein
Protein displayed in the phage coat
Can screen 1010 phage
E.g., for a SC Ab, coat the dish with Ag
Commonly used phage = m13, filamentous, infects E. coli
47Phage display selection of scFv’s (single-chain variable regions)Source of sequence: PCR from genome or RT-PCR from mRNA, add randomization (doped synthesis).