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Antibodies can participate in host defense in three main ways

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Bound NK cells release granules with a protein that forms pores on the target ... (Medarex, Abgenix, Kirin) Breedveld, Lancet 2000 355:9205 ... – PowerPoint PPT presentation

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Title: Antibodies can participate in host defense in three main ways


1
MAbs continued
Antibodies can participate in host defense in
three main ways
2
ADCC antibody-dependent cell-mediated
cytotoxicity
NK Natural killer cells (T-cell class)
FcgammaRIII Fc receptor on NK cell surface
Bound NK cells release granules with a protein
that forms pores on the target cell (perforin)
and an enzyme that penetrates the target cell and
induces apoptosis through a caspase (protease)
cascade.
3
TARGET CELL
(Killer T-cell)
Genentech
Commercial MAb injected as a therapeutic
T-cell surface receptor binds Fc region of
antibody molecule(Fc gammaR)
4
MAb therapy targets Inflammation Autoimmune
disease Graft rejection Heart disease
(thrombosis) Cancer Viral infection
5
Therapeutic strategies Mabs straight Mabs fused
to other protein binders (e.g., soluble
receptors) Mabs fused to cytotoxic agents
(toxins, radionuclides) Toxins ricin (stops
protein synthesis) calicheamicin (DNA
breaks) Radionuclides 90Y yttrium 111I
indium
6
  • Problems of mouse MAbs
  • Fc portion limited in its ability to interact
    with Fc receptors of human cells.
  • Lower serum half-life
  • Development of human anti-mouse antibodies (HAMA)
  • Retreatment results in allergy or anaphylactic
    shock
  • Retreatment is less effective

Breedveld, Lancet 2000 3559205
  • Solutions via recombinant DNA genetic engineering
  • Chimeric mouse-human antibodies Hu V-region
    fused to mouse C regions
  • Humanized mouse antibodies, Parts of V-region
    from human interspersed with mouse CDR V-regions
  • Human antibodies (fully), via transgenic mice
    carrying human immunoglobulin genes(Medarex,
    Abgenix, Kirin)

CDR complemetarity-determining region
7
MAbs approved for human therapy
Transplantation
Stroke
Lymphoma
IL-2, immunosuppressant
Transplantation
Respiratory infection Synciitial Virus
Arthritis
HER-2/neu (EGF2) breast
cancer
CD33 leukemia (AML)
Leukemia
Lymphoma
Arthritis
IgE asthma
Lymphoma
Psoriasis
EGF-R colon cancer
VEGF colon cancer
8
  • Monoclonal antibody generation
  • - Cells needed myeloma cells, mouse spleen cells
  • - antigen administration Kohler and Milstein
  • - hybridoma formation via cell fusion
  • selection mutants required (hprt- usually)
  • - antibody generation cDNA cloning
  • - engineered MAbs expression vectors
  • - refinement chimeric, humanized, human

9
Monoclonal antibodies via cell hybridization
Selects for rare hybrid cells Spleen cells do
not grow in culture TGr myeloma cells do not grow
in HAT
10
Reduced myeloma hybrid
Unreduced myeloma 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.
Isoelectric focusing immunoglobulins made
in hybridoma cells
11
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 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
Still experimental anti HIV drug PRO 542
Soluble CD4 (HIV receptor) fused to IgG2.
Tetrameric (4 V-regions replaced) Reduced Fc
function (since IgG2 lt IgG1), Better
half-life Progenics
12
Single chain antibodies (scFv)
Ag binding site
15 AA linker
13
Protein Glycosylation
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.
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.
Review Grabenhorst, E., Schlenke, P., Pohl, S.,
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.
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 587(5)614-22
14
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15

Penta- saccharide common core

Diantennary With bisecting GlcNAc With
fucosylated core
Triantennary (also tetra-antennary)
All shown here, N-linked (to amide N of Asn in
N-X-S or N-X-T)
Substantial in size
Carbohydrates attached to loops or near termini
Also O-linked, to ser or thr (hydroxyl on side
chain)
16
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.
17
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 asialo agalacto fucosyla
ted bisecting GlcNAc Blood (made in
liver) diantennary NAcNeu (sialated sialic
acid) afucosylated
18
neuraminic acid one of the sialic acids
both terms are used, confusedly
NAcNeu
Carboxyl (acid)
Glycerol moiety
mannose
Acetylated amino group
deoxy
19
Glycosylation pattern affects signaling,
for Delivery to the right cell receptor for
activity Clearance rate
Microheterogeneity Lots of isoforms, naturally
No apparent 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
human Hamster less heterogeneity
20
Genetic engineering of glycosylation to Modify
or enhance activity E.g. Better binding to a
receptor More specific binding Different binding
Also Antigenicity Clearance rate Decrease
microheterogeneity (for clinical application)
21
  • Modifying glycosylation
  • Add or subtract sites to your favorite protein
    (cis)
  • Change the general glycosylation phenotype of the
    host cell (trans)

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 proteins structure) Works
22
  • Modifying glycosylation
  • Add or subtract sites to your favorite protein
    (cis)
  • Change the general glycosylation phenotype of the
    host cell (trans)

2. Clone enzyme genesGlycosyl transferases,
mostlyAlso some synthetases (e.g., NAcNeu) Can
be complexe.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) ExperimentOver-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).
23
Isolate mutant mammalian cell lines deficient in
specific glycosylation enzymes
Stanley Isolation of multiply mutated
glycosylation mutants by selecting for lectin
resistancei Lectins carbohydrate-binding
proteins Plant lectins used mostly here (but
occur widely) 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
WGA Mutants in the same gene will remain WGA
resistant (non-complementation) Could now be
used as a tabla rasa for 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
24
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.
transport to Golgi
5
Golgi
glucose
Exploits hypersenstivity to select against
certain phenotypes.
25
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26
Sequential mutagenesis and selections to isolate
mutliply-mutated glycosylation mutants
27
Predicted alteredglycosylation statesin various
mutants
28
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 NaG 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.
29
TARGET CELL
(Killer T-cell)
Genentech
Commercial MAb injected as a therapeutic
T-cell surface receptor binds Fc region of
antibody molecule(Fc gammaR)
30
Getting CHO cells to make more bisected
oligosaccharide in the Fc region of MAbs to
better activate antibody-dependent cellular
cytotoxicity (ADCC) GnT III glycosyltransferase
in question Methods Vectors (8) tTa
neoR rat GnTIII cDNAmychistag no introns that
into tet promoter vector Pur H-chain cDNAs (CMV
bGH pA SV40neo) synthetic leader L-chain cDNAs
(CMV bGH pA SV40neo) synthetic leader zeoR
Tet-driven beta-galactosidsae Transfections
(4) tTA neo, transient tet-beta-gal,
GnTIIIpur, HLzeo Westerns Mass Spec, incl.
enzyme digestions sialidase peptide
N-glycosidase F (4 vs. 5 hexoses???) ADCC
(dye retention/release, neuroblastoma cells)
31
Variably present
Tet-off system
Transient transfection of GnTIII into tTA-bearing
CHO cells (western blot)
Permanent transfectant for tTA and GnTIII
tTA Tetracycline responsive TransActivator
protein
32
Tet 2000
Tet 60
Tet 30
Tet 15
33
Tet 2000
Mass spec products indicating the presence of the
bisecting NAcG (in dashed boxes)
Tet 60
Tet 30
Tet 15
34
ADCC assay
ADCC correlates with bisected complex content
Tet induction of GnTIII
No induction of GnTIII
35
Result ADCC efficiency followed proportion of
oligosaccharide with bisected sugar Bisecting
sugar15 ? 45 ADCC 25 ? 50
Missing Zero bisection control CHO cells are
supposed to LACK GnTIII and Westerns show 0 rat
GnTIII at 2000 ug/ml tetracycline Yet
backgrounds of bisecting sugar are high. OK for
ADCC, but Mass Spec data .
Extensions?
Try untransfected CHO? Westerns lying? ( lt30
ug.ml tet ? death too much enzyme?)
Good example of enzyme engineering. Can still be
optimized.
Use a constitutive promoter, try different
version to find the best using ADCC as the
assay Check dependence on Ig production level.
36
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
37
Double knock-out strategy for FUT8 an
alpha-1,6,fucosyl transferase
Isolate CHO cDNA using mouse sequence data fro
primers Use CHO cDNA to isolate CHO genomic
fragments from a commercial lambda library
K.O. exon 1 translation start
Homology regions
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-resistance 1.6X108?70,000 screened ? 10
double KO homozygotes.
Remove drug resis. genes by Transient
trasnfection with Cre recombinase
Note 10s of thousands of PCRs performed to
screen for homologous recomb., using 96-well
plates
38
Double knockout evidence
Orginal KOd genes have a 1.5 kb
insertion (Southern blot)
mRNA has 200 nt deletion (RT-PCR
39
Use of a fluoresceinated 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 -/-
40
Rituxan (anti-CD20) produced in FUT -/- cells
does not contain fucose(HPLC analysis)
Digestion all the way to monosaccharides
41
In ADCC, FUT8-/- anti-CD20 gtgt Rituxan
Binding to CD20 membranes FUT8-/- anti CD20
Ritxuan
Anti-CD20 from a partially FUT-deficient rat cell
line
Rat line
FUT-/-s
Complement-mediated cell toxicity is the same
for FUT8-/- and Rituxan
Rituxan commercial product, 98 fucosylated
42
Very laborious, but apparently a big
payoff. Better selection? Why not use the
fluorescent LCA to select for the FUT8 KOs along
with G418 resistance(double sequential
selection)?
43
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44
Quality-Control System for Protein Folding in the
ER. Folded protein cannot get re-glycosylated. Onl
y deglycosylated proteins move from ER to Golgi.
Biochemistry. 5th edition Berg, Jeremy M.
Tymoczko, John L. and Stryer, Lubert.
45
LexGene Trap
Zambrowicz BP, et al. Disruption and sequence
identification of 2,000 genes in mouse embryonic
stem cells. Nature. 1998392(6676)608-11.
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