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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

Class 23_11 last updated 11/27/11 6:00 PM

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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 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 only

15 AA linker

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M13 phage display

filamentous phage that infects E. coli

POI = protein of interest

Protein displayed in the phage coat

Can screen 1010 phage

(By re-infection of E. coli) Last step: plate out

and pick individual plaques PCR cloned molecules

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Improving on nature:

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

generate many genotypes↓

select for best phenotype↓

isolation best gene ↓

express product

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Protein glycosylation

Adds another layerof structure and specificity to proteins

Can enhance the function of a protein

Can extend the lifetime of a protein

Can help localize a protein within a cell

Can act as a specific antigen

7Two types of protein glycosylation

glucose galactose

N-acetyl group

1. N-linked2. O-linked

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anomeric carbon

Haworth view

Fisher view

Chair view

Blue background = sugar review

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Glucose

}Gray = CWhite = HRed = O

Ring oxygen

C6 (-CH2OH)

C5

C1

hydr

oxyl

1010

1 234567891011

Hexose ring formation alpha and beta isomers (anomers)

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or glycogen chain

down

out

H

H

Oligosaccharide formation: bonds at anomeric carbons determine 3-D structure

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Glucose

Fucose

Common hexoses found in glycoproteins

N-acetylgalactosamine

N-acetyl derivatives, e.g.:

R is glycerol:

|HCOH |HCOH |HCHOH

deoxy

carboxylic acid

COO-

Sialic acid (N-acetyl-neuramininc acid)

mannose framework

13dulcitol

Weerapana and Imperiali, Glycobiology vol. 16: 91R–101R, 2006

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Triantennary(also tetra-antennary)

DiantennaryWith bisecting GlcNAcWith fucosylated core

Carbohydrates attached to exterior 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); see below

=

=Penta-

saccharidecommon

core

Fucose

Sia= sialic acid (see below)

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3

1 2

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Enlargement for display

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

Examples of O-linked oligosaccharides

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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):diantennaryasialo-agalacto-fucosylatedbisecting GlcNAc

Sialic acid structure: see next graphic

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

afucosylated

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Glycosylation pattern affects signaling of proteins used therapeutically, for:

Delivery of the soluble glycoprotein drug to the right cell receptor for activityClearance 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 vectors, high level transient expression for production):Too simple a pattern compared to human

Mouse and hamster cells: similar to humanHamster: less heterogeneity

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Reasons for genetic engineering of glycosylation:

Modify or enhance activity

E.g.:Better binding to a receptorMore specific bindingDifferent binding, in theory

Also:AntigenicityClearance rateDecrease microheterogeneity (for clinical application)

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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 encompassing a 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 synthetase)

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;produce permanent transfectants of BHK cells (BHK = baby hamster kidney) 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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Pam Stanley: Isolation of multiply mutated glycosylation mutants by selecting lectin resistance

Lectins = carbohydrate-binding proteins (WGA, ConA, ricin, etc.)Plant lectins used mostly here (but occur widely in animals as well)

Sequential selections, push - pull on resistance, sensitivity

Lectin Resistance: enzyme deficiency failure to add the sugar need for lectin binding (glycosyltransferases)

Lecti Sensitivity: failure to add a sugar produces greater exposure of underlying sugars A transferase-negative mutant better binding to the exposed sugar

Stanley showed power of selection, usefulness of complementation via cell hybridization

Isolate mutant mammalian cell lines deficient in specific glycosylation enzymes

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

WGA = wheat germ agglutinin; ConA = concanavalinA;

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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 additionA 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 (MS) and ADCC efficiency of the Mab (in vitro lysis).

(NAcG = N-acetyl-glucosamine here)

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Genentech(Killer T-cell)

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

Commercial MAb injected as a therapeutic

TARGET CELL

ADCC

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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.

Cyt

otox

icity

Anti-neuroblastoma antibody (ng/ml)

High tet, tet-off system, = basal production

Low tet, tet-off system, = higher production

Yet lower tet, tet-off system, = yet higher production

No tet, tet-off system, = highest productionnon-optimalNeuroblastoma

cells + NK T-cells + antibody

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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.

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’s 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 transferase (biosynthesis) 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 45,000 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 hamster at the time (until 2011)Isolate 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

31Rituxan (retuximab, anti-CD20, B-cell antigen. Anti lymphoma, anti-inflammatory) Produced in FUT -/- cells does not contain fucose(HPLC analysis)

Digestion all the way to monosaccharides

Missing d - g

32Binding to CD20 membranesFUT8-/- anti CD20 eq. to Rituxan

Complement-mediated cell toxicity: FUT8-/- eq. to 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)?

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Hans Henning von Horsten et al., Glycobiology vol. 20 no. 12 pp. 1607–1618, 2010Production of non-fucosylated antibodies by co-expression ofheterologous GDP-6-deoxy-D-lyxo-4-hexulose reductase (“RMD”)

Clone bacterial RMD cDNAConstruct mam. expn vectorTransfect into CHO cells making Herceptin (anti EGF receptor)Deflects intermediate in fucose biosynthetic path

1. Drains intermediate

2. Inhibits pathway

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Also absent by MS

No fucose in transfectant glycoproteins

Select for G418 resistance, screen for lack of fucose.

One of 3 clones:WT CHO cells

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Binding assay to Fc receptor (ELISA-type assay)

ELISA = Enzyme-linked immunosorbent assay

About10-fold more effective

WT

3 transfectants

Antibody concentration (ng/ml)

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ADCC lysis assay vs. a HER2+ breast carcinoma cell line

Concentration of anitbody

% ly

sis

About10-fold more effective

WT

3 transfectants

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