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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

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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 Asparagine-free

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

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)

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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

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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.

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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.

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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

8Top 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 patternsEngineer cell to eliminate or defer apoptosis (for longer productinon runs)

Etc. (including Chasin lab project)

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(2004 claim = 2.8 g/L)Lonza 2005 Web site presentation

(Old values from

(23 d.)

Note improvement include:1) Higher cell density2) Longer times3) Higher output per cell

(1990)

Mill

ions

of

cells

per

lite

r Antibody gram

s/liter

ml.

Cell titer

Ab g/l

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20,000 liter mammalian cell fermentor - Lonza Biologics - Portsmouth, NH

20,000 liter fermentor

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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 yieldSame 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 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):

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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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B cells develop in the bone marrowhematopoietic stem cells and lymphoid stem cells

lymphoid stem cells T-cells and B-cells

B-cells:progenitor = pro-B cell (B220+)precursor = pre-B cells: heavy-chain rearranged

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)

Antibodies are made by B-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

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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

Heavy chain

Light chain

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Heavy chain = blueLight chain = pink

17Laboratory fragmentation of antibodies

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ComplementaritydeterminingRegion = “CDR”

Hypervariableregion

Ig molecule showing polarity, disulfides, carbohydrate

CHO = carbohydrate

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Opsonization

Complement activation

Antibody-dependent cell-mediated cytotoxicity (ADCC)

Transcytosis

Fc functions

Constant region

Fc

See below

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Secretory IgA dimer

Disulfide bond

J-chain

IgM

s

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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

22IgGkappa light chain gene rearrangement

SPLICING

SPLICING

+ SOMATIC HYPERMUTATION

(D,J)

(J)

(D,J)

(J)

L = leader sequence, signal for secretion

76 Vk, 5 Jk, 1 Ck over 2 MbLight chain genesis

Heavy chain genesis

95Vh, 30Dh,5Jh, 11Ch over 1.4 Mb

DNA

DNA

+ SOMATIC HYPERMUTATION

J

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Adapted from Janet Stavnezer http://www.umassmed.edu/faculty/show.cfm?start=0&faculty=300

Alt. splicing

IgD

Class switch recombination sites

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Immunobiology, Charles Janeway, Paul Travers, Mark Walport, Mark Shlomchik

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

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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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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

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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

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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

NK cells = natural killer cells

When activated by Fc binding, NK cells release Perforin makes holes in the membraneGranzymes = proteases that initiate apoptosis

Fc receptor

Fc

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Genentech

ADCC = antibody-dependent cell-mediated cytotoxicityFc region

NK cell

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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 reactionsandB-cells: secreted antibodies

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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.

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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

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

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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 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

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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

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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 = yttrium111I = indium

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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

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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

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

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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

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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-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

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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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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).

or

Repeat, to reduce background

Wash

Clone (plaque on lawn)

PCR rescue scFv DNA Elute, re-infect E. coli

scFvs