Transcript Class 23_11 last updated 11/27/11 6:00 PM MAb Fusion Proteins With other protein-binding proteins: natural receptors in soluble form Analogous to MAbs and.

Class 23_11 last updated 11/27/11 6:00 PM
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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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 only
15 AA
linker
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M13 phage display
filamentous phage that infects E. coli
POI = protein of interest
(By re-infection
of E. coli)
Last step: plate out
and pick individual
plaques PCR 
cloned molecules
Protein displayed in the phage coat
Can screen 1010 phage
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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
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Two types of protein glycosylation
2. O-linked
1. N-linked
N-acetyl group
glucose
galactose
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Blue
background
= sugar
review
anomeric
carbon
Fisher
view
Chair view
Haworth view
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Glucose
}
Gray = C
White = H
Red = O
C1
C6 (-CH2OH)
C5
Ring oxygen
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Hexose ring formation  alpha and beta isomers (anomers)
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10
7
6
5
89
3
1 24
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Oligosaccharide formation:
bonds at anomeric carbons
determine 3-D structure
down
H
H
or glycogen chain
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Common hexoses found in glycoproteins
Fucose
Glucos
e
N-acetyl derivatives, e.g.:
Sialic acid (N-acetyl-neuramininc acid)
carboxylic acid
R is glycerol:
COO-
Nacetylgalactosamine
deoxy
|
HCOH
|
HCOH
|
HCHOH
mannose framework
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dulcitol
Weerapana and Imperiali, Glycobiology vol. 16: 91R–101R, 2006
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1
1
2
3
Pentasaccharide
common
core
2
=
=
All shown
Triantennary
here,
(also tetra-antennary)
N-linked
(to amide
N of Asn
in N-X-S
or N-X-T)
Carbohydrates
attached to exterior
loops or near termini
Sia= sialic acid (see below)
Diantennary
With bisecting GlcNAc
With fucosylated core
Substantial in size
Fucose
Also O-linked, to ser or thr
(hydroxyl on side chain); see below
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Enlargement for display
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 type
Physiological state
No. of sites depends on 3-D structure of protein
Structure at that site depends on the site
E.g., transferrin, from different cell types :
Cerebrospinal fluid (made in brain):
diantennary
asialoagalactofucosylated
bisecting GlcNAc
Blood (made in liver):
diantennary
NAcNeu (sialated= sialic acid)
afucosylated
Sialic acid structure: see next graphic
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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 activity
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 vectors, high level transient expression for production):
Too simple a pattern compared to human
Mouse and hamster cells: similar to human
Hamster:
less heterogeneity
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Reasons for genetic engineering of glycosylation:
Modify or enhance activity
E.g.:
Better binding to a receptor
More specific binding
Different binding, in theory
Also:
Antigenicity
Clearance rate
Decrease 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
Modifying glycosylation
1. Add or subtract sites to your favorite protein (cis)
2. Change the general glycosylation phenotype of the host cell (trans)
2. Clone enzyme genes:
Glycosyl transferases, mostly
Also 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).
J Biol Chem, Vol. 273, Issue 47, 30985-30994, November 20, 1998 In Vivo Specificity of Human 1,3/4Fucosyltransferases 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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Isolate mutant mammalian cell lines deficient in specific glycosylation enzymes
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
WGA = wheat germ agglutinin; ConA = concanavalinA;
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)
(NAcG =
N-acetyl-glucosamine here)
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 (MS) and ADCC efficiency of the Mab (in vitro lysis).
ADCC
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TARGET CELL
Genentech
(Killer T-cell)
Commercial MAb injected as a therapeutic
T-cell surface receptor binds Fc region of
antibody molecule (Fc gammaR)
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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.
Low tet, tet-off system,
= higher production
Neuroblastoma
cells + NK T-cells
+ antibody
Cytotoxicity
Yet lower tet, tet-off system,
= yet higher production
No tet, tet-off system,
= highest production
non-optimal
High tet, tet-off system,
= basal production
Anti-neuroblastoma antibody (ng/ml)
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Protein Glycosylation
Assigned:
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
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.
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.
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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
Double knock-out strategy for FUT8, an alpha-1,6,fucosyl transferase
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Little sequence data available for Chinese hamster at the time (until 2011)
Isolate CHO cDNA using mouse sequence data for primers
Use CHO cDNA to isolate CHO genomic fragments from a commercial lambda library
K.O. exon 1 translation start region
Homology regions
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-resistance
Lox sites
1.6X10870,000 screened 
10 double KO homozygotes.
Remove drug resis. genes by
transient transfection with Cre
Recombinase. Exon 1 suffers a
200 nt deletion
Note: 10’s of thousands of PCRs performed to screen for homologous recomb., using 96-well plates
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Double knockout evidence
After Cre
treatment
Original KO’d genes have a 1.5 kb insertion
(Southern blot)
mRNA has 200 nt deletion
(RT-PCR)
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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 +/Surprising: CHO cells
do not have excess
fucosylation capacity
FUT8 -/-
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Rituxan (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
Binding to CD20 membranes
FUT8-/- anti CD20 eq. to Rituxan
In ADCC, FUT8-/- anti-CD20 >> Rituxan 32
Anti-CD20
from a partially
FUT-deficient
rat cell line
Fc-Receptor
protein
binding assay
Rat line
Complement-mediated cell
toxicity: FUT8-/- eq. to Rituxan
FUT-/-’s
Rituxan = commercial product,
98% fucosylated
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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, 2010
Production of non-fucosylated antibodies by co-expression of
heterologous GDP-6-deoxy-D-lyxo-4-hexulose reductase (“RMD”)
2. Inhibits
pathway
1. Drains
intermediate
Clone bacterial RMD cDNA
Construct mam. expn vector
Transfect into CHO cells
making Herceptin (anti EGF
receptor)
Deflects intermediate in
fucose biosynthetic path
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Select for G418 resistance, screen for lack of fucose.
WT CHO cells
One of 3 clones:
No fucose in transfectant glycoproteins
Also absent by MS
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Binding assay to Fc receptor (ELISA-type assay)
About10-fold more effective
3 transfectants
WT
Antibody concentration (ng/ml)
ELISA = Enzyme-linked immunosorbent assay
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ADCC lysis assay vs. a HER2+ breast carcinoma cell line
% lysis
About10-fold more effective
3 transfectants
WT
Concentration of anitbody
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