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Antibody generation

2002 Molecular Biology of the Cell by Bruce Alberts, Alexander Johnson, Julian Lewis, Martin Raff, Keith Roberts, and Peter Walter.

T-cells: cell mediated immune reactionsandB-cells: secreted antibodies

23-22002 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 stimualted. The final response is therefore POLYclonal.

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2002 Molecular Biology of the Cell by Bruce Alberts, Alexander Johnson, Julian Lewis, Martin Raff, Keith Roberts, and Peter Walter.

The antbody secreting effector cells terminalyl differentiate (die) but their sister memory cells live on to generated an amplified reponse upon a second exposure to antigen

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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)

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Hprt- myeloma cells6-TG-resisatnt

HAT-

(HAT)

Plating positives at low density (~1/well) for cloning

Plate among many wells for supernantent testing

Monoclonal antibody generation

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Reduced S-S bondsMyeloma hybrid

Isoelectric focusing ofimmunoglobulinsmade in thehybridoma cells new bands

Georges Kohler

Cesar Milstein

Unreducedmyeloma hybrid Kohler, G., and C. Milstein

(1975). Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256: 495-497.

Established cell lines (hybridomas) that secrete any antibody that can be raised in a mouse.

Use of myeloma parent obviated extinction (shut-off) of Ig genes by the immortal parent.

(MRC = Medical Research Council)

Later used a myeloma mutant parent that no longer secreted an endogenous immunoglobulin.

Isoelectric focusing (not SDS PAGE)

Complexity not explained; perhaps not truly clonal..

Fused a myelolma cell line to spleen cells from s amouse immunized against sheep red blood cells.

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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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Sponsor company Generic name US trade name

mAb type Therapeutic category

US approval

Johnson & Johnson Muromonab-CD3 Orthoclone OKT3

Murine Immunological 19.06.1986

Centocor Abciximab ReoPro Chimeric Hemostasis 22.12.1994

Biogen IDEC Rituximab Rituxan Chimeric Antineoplastic 26.11.1997

Protein Design Daclizumab Labs |

Zenapax Humanized Immunological 10.12.1997

Novartis Basiliximab Simulect Chimeric Immunological 12.05.1998

MedImmune Palivizumab Synagis Humanized Anti-infective 19.06.1998

Centocor Infliximab Remicade Chimeric Immunological 24.08.1998

Genentech Trastuzumab Herceptin Humanized Antineoplastic 25.09.1998

Wyeth Gemtuzumab ozogamicin

Mylotarg Humanized Antineoplastic 17.05.2000

Millennium/ILEX

Alemtuzumab Campath Humanized Antineoplastic 07.05.2001

Biogen IDEC Ibritumomab tiuxetan

Zevalin Murine Antineoplastic 19.02.2002

Abbott Adalimumab Humira Human Immunological 31.12.2002

Genentech Omalizumab Xolair Humanized Immunological 20.06.2003

Corixa Tositumomab-I131 BEXXAR Murine Antineoplastic 27.06.2003

Genentech Efalizumab Raptiva Humanized Immunological 27.10.2003

Imclone Systems

Cetuximab Erbitux Chimeric Antineoplastic 12.02.2004

Genentech Bevacizumab Avastin Humanized Antineoplastic 26.02.2004

Sme MAbs approved for human therapy

EGF-R colon cancer

HER-2/neu (EGF2) breast cancer

CD33 leukemia (AML)

VEGF colon cancer

IgE asthma

Respiratory infectionSynciitial Virus

IL-2, immunosuppressant

Stroke

Transplantation

Lymphoma

Transplantation

Arthritis

Lymphoma

Arthritis

Lymphoma

Psoriasis

Leukemia

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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)- antibody generation cDNA cloning- engineered MAbs expression vectors- refinement chimeric, humanized human, truly human

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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(Medarex, Abgenix, Kirin)

Breedveld, Lancet 2000 355:9205

CDR = complementarity-determining region

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MAb Fusion Proteins

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 functionFc domain dimerizes the receptor, which increases its affinity for TNF.Fc domain increases the half-life of the protein in the bloodstreamAmgen + Wyeth

Still experimental: Anti-HIV drug PRO 542 Uses soluble form of the CD4, the molecule to which HIV attaches on T-cellsSoluble 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(Recently replaced by a MAb (PRO 140) targeting the CCR5 cell surface protein, required for viral entry)Progenics

TNF = tumor necrosis factor)

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Single chain antibodies (scFv)

Ag binding site

15 AA linker

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Phage display 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 like it:A physical link of 1) a nucleic acid sequence (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

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Phage display selection of scFv’s (single-chain F-variable regions)Source of sequence: PCR from genome or RT-PCR from mRNA, add randomization (doped synthesis).

or

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Two types of protein glycosylation

glucose galactose

N-acetyl group

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Triantennary(also tetra-antennary)

DiantennaryWith bisecting GlcNAcWith fucosylated core

Carbohydrates attached to loops or near termini

Substantial in size

All shown here,N-linked (to amide N of Asnin N-X-S or N-X-T)

Also O-linked, to ser or thr(hydroxyl on side chain)

=

=Penta-

saccharidecommon

core

Fucose

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Figure 7.28. Examples of O-linked oligosaccharides O-linked oligosaccharides usually consist of only a few carbohydrate residues, which are added one sugar at a time.

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Carbohydrate structure specific for:

Cell typePhysiological stateNo. of sites depends on 3-D structure of proteinStructure at that site depends on the site

E.g., transferrin from different cell types :Cerebrospinal fluid (made in brain):

diantennaryasialoagalactofucosylatedbisecting GlcNAc

Blood (made in liver):diantennary NAcNeu (sialated= sialic acid)afucosylated

Sialic acid structure: see next graphic

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neuraminic acid – one of the sialic acids = : both terms are used, confusedly

Mannose framework

Carboxyl (acid)

Acetylated amino group

Glycerol moiety

deoxy

NAcNeu:

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Glycosylation pattern affects signaling, for:Delivery of the glyco protein to the right cell receptor for activityIts clearance rate

Microheterogeneity:Lots of isoforms typically present

Glycosylation does not seem to represent a bottleneck in high-producing cells:0.1 mg/l (amplify) 200 mg/l = same pattern

Insect cells (Baculovirus, high level transient expression):Too simple a pattern compared to human

Mouse and hamster cells: similar to humanHamster: less heterogeneity

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Genetic engineering of glycosylation to:

Modify or enhance activity

E.g.:

Better binding to a receptorMore specific bindingDifferent binding

Also:

AntigenicityClearance rateDecrease microheterogeneity (for clinical application)

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Got this far

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Modifying glycosylation

1. Add or subtract sites to your favorite protein (cis) 1a. Subtract sites: Easy, change N or S or T to A by site-directed mutagenesis

1b. Add sites: Not so easy. Consensus N-X-S does not work, e.g.:requires the insertion of a ~12 aa region encompassinga real N-glycosylation site (6 suffices for O-linked)

Place on an end or on a loop (must know protein’s structure)

Works

2. Change the general glycosylation phenotype of the host cell (trans)

E.g., Pam Stanley: lectin-resistant mutants

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2. Clone enzyme genes:Glycosyl transferases, mostlyAlso some synthetases (e.g., NAcNeu)

Can be complex:e.g., 7 different fucosyl transferases (FTs),with different (overlapping) substrate specificities

Simpler example:

Hamster cells do only 2,3 sialylation. Humans do 2,6 as well, via a 2,6 sialyl transferase (ST)

Experiment:Over-express cloned human 2,6 ST, along with a substrate protein.producing permanent transfectants of BHK cells (BHK = baby hamster kidney)

Works:Get both types of structures now, substantially(although not exactly the same ratio as in human cells).

Modifying glycosylation

1. Add or subtract sites to your favorite protein (cis)

2. Change the general glycosylation phenotype of the host cell (trans)

J Biol Chem, Vol. 273, Issue 47, 30985-30994, November 20, 1998 In Vivo Specificity of Human 1,3/4-Fucosyltransferases III-VII in the Biosynthesis of LewisX and Sialyl LewisX Motifs on Complex-type N-Glycans. COEXPRESSION STUDIES FROM BHK-21 CELLS TOGETHER WITH HUMAN -TRACE PROTEIN Eckart Grabenhorst , Manfred Nimtz , Júlia Costa§, and Harald S. Conradt ¶

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Stanley: Isolation of multiply mutated glycosylation mutants by selecting for lectin resistance

Lectins = carbohydrate-binding proteinsPlant lectins used mostly here (but occur widely in animals as well)

Sequential selections, push - pull on resistance, sensitivity

Resistance: enzyme deficiency failure to add the sugar need for lectin binding

Increased sensitivity: failure to add a sugar produces greater exposure of underlying sugars in a transferase-negative mutant better binding to the exposed sugar

Showed power of selection

Showed usefulness of complementation analysis via cell hybridization

Hybrid selection: All lec-R mutants were: WGA (wheat germ agglutinin) resistant (various degrees) & pro-

Tester parent was single lec-R + GAT- (req. glycine, adenine and thymidine)Select in medium lacking pro, GAT, and with +/- WGA

Complementing hybrids will have regained sensitivity to WGAMutants in the same gene will remain WGA resistant (non-complementation)

Could now be used as a tabla rasa (blank slate) introducing a series of enzymes to build custom tailored glyco-conjugates. Complicated though (order of addition, location in the Golgi, etc. )

Potential: targeting to carbohydrate-sensitive receptors (e.g., liver asialoglycoprotein receptor); clearance rate

Pam Stanley

Isolate mutant mammalian cell lines deficient in specific glycosylation enzymes

Review: Nature Biotechnology  19, 913 - 917 (2001) , The bittersweet promise of glycobiology. Alan Dove

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Umana, P., Jean-Mairet, J., Moudry, R., Amstutz, H., and Bailey, J.E. 1999.Engineered glycoforms of an antineuroblastoma IgG1 with optimized antibody-dependent cellular cytotoxic activity. Nat Biotechnol 17: 176-180.

Target here (bisecting NAcG)

Presence of the bisecting NAcG enhances binding of T-cell receptor to the Fc region of antibodies.

Binding is needed for ADCC.

Mouse and hamster cell lines used for commercial production lack the glycosyltransferase needed for bisecting NAcG addition

A rat myeloma cell line does produce MAb with the bisecting NAcG.

Hypothesis: Expression of the rat enzyme in a CHO cell line will add a bisecting NacG to the anti-neuroblastoma MAb produced by these cells. The modified MAb will be a better mediator of ADCC.

Experiment: Clone the cDNA for this enzyme from the rat line and transfer it to CHO cells, driven by an inducible tet promoter.

Check sugar structure of MAb and ADCC efficiency of the MAb.

(NAcG = N-acetyl-glucosamine here)

23-29Genentech

(Killer T-cell)

T-cell surface receptor binds Fc region of antibody molecule(Fc gammaR)

Commercial MAb injected as a therapeutic

TARGET CELL

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Review:Grabenhorst, E., Schlenke, P., Pohl,., Nimtz, M., and Conradt, H.S. 1999. Genetic engineering of recombinant glycoproteins and the glycosylation pathway in mammalian host cells. Glycoconj J 16: 81-97.

Umana, P., Jean-Mairet, J., Moudry, R., Amstutz, H., and Bailey, J.E. 1999. Engineered glycoforms of an antineuroblastoma IgG1 with optimized antibody-dependent cellular cytotoxic activity. Nat Biotechnol 17: 176-180.

Background:Stanley, P. 1989. Chinese hamster ovary cell mutants with multiple glycosylation defects for production of glycoproteins with minimal carbohydrate heterogeneity. Mol Cell Biol 9: 377-383.

Protein Glycosylation

Naoko Yamane-Ohnuki, et al..  Establishment of FUT8 knockout Chinese hamster ovary cells: an ideal host cell line for producing completely defucosylated antibodies with enhanced antibody-dependent cellular cytotoxicity.   Biotechnol Bioeng. 2004 Sep 5;87(5):614-22

Assigned:

Optional Update: Kanda Y, Yamane-Ohnuki N, Sakai N, Yamano K, Nakano R, Inoue M, Misaka H, Iida S, Wakitani M, Konno Y, Yano K, Shitara K, Hosoi S, Satoh M.  Comparison of cell lines for stable production of fucose-negative antibodies with enhanced ADCC.  Biotechnol Bioeng. 2006 Jul 5;94(4):680-8.

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 Biotechnol Bioeng. 2004 Sep 5;87(5):614-22

Hypothesis:

Fucose interferes with binding of the T-cell Fcgamma3 receptor to the Fc region of an antibody molecule.

Elimination of fucose from produced MAbs will increase ADCC

Create a mutant CHO cells (starting with amplifiable dhfr- cells) in which the fucose trasnferase genes have been knocked out.

All MAbs produced in these mutant cells will be better at promoting ADCC

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Homology regions

K.O. exon 1 translation start region

DT= diphtheria toxin gene,Kills if integrated via non-homologous recombination

For hemizygote: Select for G418 resistance,Screen by PCR for homologous recomb. 108 cells 45000 colonies 40 false recombinants (extension-duplications) + 1 true recombinant

Step 2 for homozygote, select for Pur-resistance1.6X10870,000 screened 10 double KO homozygotes.

Remove drug resis. genes bytransient transfection with Cre Recombinase. Exon 1 suffers a 200 nt deletion

Lox sites

Double knock-out strategy for FUT8 an alpha-1,6,fucosyl transferase

Note: 10’s of thousands of PCRs performed to screen for homologous recomb., using 96-well plates

Little sequence data available for Chinese hamsterIsolate CHO cDNA using mouse sequence data for primersUse CHO cDNA to isolate CHO genomic fragments from a commercial lambda library

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Double knockout evidence

mRNA has 200 nt deletion(RT-PCR

Original KO’d genes have a 1.5 kb insertion(Southern blot)

After Cre treatment

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Use of a fluoresceinated lentil lectin (LCA) that binds fucose oligosaccharides to demonstrate lack of fucosylation in glycosylated proteins in the FUT8 -/- cells

Control background fluorescence(FL-anti avidin)

FUT8 +/+

FUT8 +/-

FUT8 -/-

Surprising: CHO cells do not have excess fucosylation capacity . . .

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Rituxan (anti-CD20) produced in FUT -/- cells does not contain fucose(HPLC analysis)

Digestion all the way to monosaccharides

Missing d - g

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Binding to CD20 membranes

FUT8-/- anti CD20 = Ritxuan

Complement-mediated cell toxicity is the same for FUT8-/- and Rituxan

In ADCC, FUT8-/- anti-CD20 >> Rituxan

Anti-CD20 from a partially FUT-deficient rat cell line

FUT-/-’sRat line

Rituxan = commercial product, 98% fucosylated

Fc-Receptor protein binding assay

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Very laborious, but apparently a big payoff.

Better selection?:

Why not use the fluorescent LCA to select for the FUT8 KO’s along with G418 resistance(double sequential selection)?