Transcript Carbohydrate Engineering Introduction to Glycobiology (ME330.712) Kevin J. Yarema Associate Professor of Biomedical Engineering The Johns Hopkins School of Medicine Email: [email protected] Phone: 410.614.6835 ISBN 978-3-527-30632-9 -

Carbohydrate Engineering
Introduction to Glycobiology (ME330.712)
Kevin J. Yarema
Associate Professor of Biomedical Engineering
The Johns Hopkins School of Medicine
Email: [email protected]
Phone: 410.614.6835
ISBN 978-3-527-30632-9 - Wiley-VCH
An Overview of Today’s Lecture
First – What is Carbohydrate Engineering?
Organ Transplantation
Metabolic Oligosaccharide Engineering
First – What is Carbohydrate Engineering?
From Google:
First – What is Carbohydrate Engineering?
For pdfs of the introduction, or any chapter,
email me at [email protected]
First – What is Carbohydrate Engineering?
Basically, in 2005 we didn’t really know very precisely
What about now, in 2014?
Let’s define “glycoengineering” (a subcategory
of “carbohydrate engineering”) as:
1)
(primarily) The manipulation of glycans
Biologics
(i.e. therapeutic glycoproteins)
2)
(secondarily) for biomedical purposes*
Xenotransplantation
*
But, is this even possible?
The Promise of Sugar-based Therapies
The term “Glycobiology” was coined in 1988
“Glycans” were implicated in many (most!) complex diseases
Immune disorders (Kawasaki Disease)
Cancer metastasis
Arthritis
http://en.wikipedia.org/wiki/File:Kawasaki_symptoms.jpg
Arthritis
www.omicsonline.org
Degenerative muscle disease
Duchenne
Muscular
Dystrophy
http://guardianlv.com/2013/08/new-hope-for-duchenne-muscular-dystrophy-patients/
A flurry of clinical translation and commercialization efforts ensued (1990s)
Difficulties Ensued
Commercialization and translational efforts were slow to be realized:
The Bittersweet Promise of Glycobiology
Nature Biotechnology, 2001 (doi:10.1038/nbt1001-913)
The Sweet and Sour of Cancer: Glycans as Novel Therapeutic Targets
Nature Reviews Cancer, 2005 (doi:10.1038/nrc1649)
Uh oh!!
Time for an Analogy - Electric Cars
A great idea 25 years ago . . . .
. . .that didn’t work out*
(*at the time)
http://ev1.org/
*Until
“today”
http://www.foxbusiness.com/industries/2014/01/14/tesla-to-double-sales-service-locations-as-4q-deliveries-top-outlook/
OK – What is being Covered Up by Car Analogies?
“A Southern Mystery”
(from The Scientist, July 1, 2008)
In 2004, strange things were happening when people living in the Southern
United States received Erbitux (aka Cetuximab), an (mAb) anticancer drug.
After Erbitux was approved, the first three patients that oncologist Bert O'Neil
treated at the University of North Carolina, Chapel Hill, had severe
anaphylactic reactions. One fell out of their chair," passing out as blood
pressure plummeted. "It alarmed us.“
"I was quite upset," says research oncologist Christine Chung, when her
patient with head and neck cancer had a severe reaction to the drug. "This
was a young man and a last ditch effort" to gain a little more time for this
patient . . . .
Uh oh!!
What Happened?
The affected patients had IgE
antibodies against galactose-α-1,3galactose (“a-Gal”), which triggered
anaphylaxis when they were given
the drug.
a-Gal
What happened? (in more detail)
Unlike most other monoclonal antibodies,
cetuximab is produced in the mouse cell line
SP2/0, which expresses the gene for a-1,3galactosyltransferase.
a-Gal
The “Southern Mystery” angle:
Lone Star Tick bites
IgE against a-Gal
http://www.viracoribt.com/alphagal
Pitfalls Along the Way are Being Overcome
 Commercialization and translational efforts were slow to be realized:
The Bittersweet Promise of Glycobiology
Nature Biotechnology, 2001 (doi:10.1038/nbt1001-913)
The Sweet and Sour of Cancer: Glycans as Novel Therapeutic Targets
Nature Reviews Cancer, 2005 (doi:10.1038/nrc1649)
 By 2008 “we” had learned a valuable “first do no harm” lesson
a-Gal
The Solution – Use a “Safe” Cell Line for mAb Production
The solution – use a “safe” cell line for mAb production:
A variant of cetuximab, CHO-C225, which is produced in Chinese
hamster ovary (CHO) cell lines that do not produce a-1,3galactosyltransferase and, for this reason, has a pattern of glycosylation
that differs from that of cetuximab was found to be “safe” to administer
to patients with IgE antibodies against a-Gal.
For example, Dr. Pamela Stanley’s
lab has developed a library of CHO
mutants allowing desired glycoforms
to be “dialed in” (or out . .. ):
Patnaik & Stanley (Methods in Enzymology, 2006):
http://www.sciencedirect.com/science/article/pii/S0076687906160115
A Simpler Solution? – Just Eliminate the Sugar(s)?
First – How?
Second – Would it work?
Just Eliminate the Sugar(s)? – No, they are Critical for Activity
Antibody-dependent cellular cytotoxicity
(ADCC) is an emerging cancer treatment.
During ADCC, antibodies bound to tumor
cells recruit innate immune effector cells that
express cellular receptors (Fc receptors
[FcRs]) specific for the constant region of the
antibody, thereby triggering phagocytosis
and the release of inflammatory mediators
and cytotoxic substances
Nimmerjahn, F., and Ravetch, J. V. (2007) Antibodies, Fc receptors and
cancer. Curr Opin Immunol 19, 239-245.
Optimizing antibody–FcR interactions. An important strategy to obtain a stronger
anti-tumor ADCC reaction is to optimize the interaction of the antibody Fc-portion
with activating FcRs. This can be achieved by blocking the inhibitory FcγRIIB in
vivo with monoclonal antibodies, or by modifying the primary amino acid
sequence (amino acid [AA] modifications) or the sugar moiety of the antibody to
obtain selective or enhanced binding to activating FcRs.
Sugars Determine Antibody Activity
Bad for ADCC
Required for IVIg
Intravenous immunoglobulin (IVIg) therapy is
used to treat a wide range of autoimmune
conditions
and
consists
of
pooled
immunoglobulin G (IgG) from healthy donors.
The immunosuppressive effects of IVIg are,
in part, attributed to terminal α2,6-linked
sialic acid residues on the N-linked glycans of
the IgG Fc (fragment crystallizable) domain.
More about the sugars in a few minutes,
but let’s first learn more about IVIg therapy
IVIg (Intravenous Immunoglobin) Therapy: A Quick Overview
IVIg therapy is used to treat a wide range of conditions; FDA approved:





Allogeneic bone marrow transplant
Chronic lymphocytic leukemia
Common variable immunodeficiency
Idiopathic thrombocytopenic purpura (ITP)
Pediatric HIV
Idiopathic thrombocytopenic purpura (ITP)
http://www.danmedj.dk/portal/page/portal/danmedj.dk/dmj_forside
/PAST_ISSUE/2011/DMB_2011_04/A4252
 Primary immunodeficiencies
 Kawasaki disease
 Chronic inflammatory demyelinating
polyneuropathy
 Kidney transplant*
Kawasaki Disease
Autoimmune disease
http://en.wikipedia.org/wiki/File:Kawasaki_symptoms.jpg
It is a safe (but expensive!) immunosuppressive therapy
IVIg Therapy: The Current Market and Projections
The Market:
 $3.6 billion in 2012
 Cost is ~ $15,000 per patient (@ 2 g / kg)
 The market is projected to (at least) double by 2019
http://online.wsj.com/article/PR-CO-20130520-904965.html
The Future; over 30-off label uses and clinical trials including:
 Unexplained recurring miscarriage
 Alzheimer’s disease
 Autism
 Chronic fatigue syndrome
Autism
http://www.icare4autism.org/
Alzheimer’s disease
http://www.wbrz.com/news/alzheimers-advances-show-need-for-better-drugs/
IVIg Therapy: Challenges and a (Partial) Solution
The Problem / Challenge:
 IVIg is obtained from blood donors
 A single batch requires pooling 1,000 to 15,000 samples
 Preparation is cumbersome and prone to contamination
 There simply is not enough supply to meet projected demand
A solution: Recombinant Ig (?)
 The upside: controlled production
 The downside: Only 1 out of ~10 antibodies is properly glycosylated
~10% sialylation
(many copies are
not active)
Sialic acid
From IVIg Therapy to “Big Picture” Implications
IVIg exemplifies the need for glycosylation optimization in “biologics”
“Obtaining a consistent glycoform profile in
(recombinant glycoprotein) production is desired
due to regulatory concerns”
“Glycosylation optimization will improve therapeutic
efficacy”
“Clearly, any improvements toward the control of this
important biochemical pathway will have farreaching influences on industry”
Optimal and consistent protein glycosylation in
mammalian cell culture (Glycobiology, 2009)
Back to IVIg (and rProteins in General*)
Poor glycosylation compromises safety, pharmacokinetics, and activity
The “solution”
Sub-optimal glycosylation
But getting
there is complex!!
Current solutions
Cell / genetic
engineering
Post-synthetic
modification
Cell culture
variables
Optimal and consistent protein glycosylation in
mammalian cell culture (Glycobiology, 2009)
Cell (Genetic Engineering) Modulation of Glycan Production
Goal: increase sialylation
Cell / genetic
engineering
OK, That Sounds Easy Enough, Let’s GE in a ST!
Goal: increase sialylation
Glycan
recycling
HO
HO
NEU3
NEU2
KL
NEU4
HO
O
NH
NEU1
CO2-
OH
OH
HO
O
HO
OH
HO
NH
HO
OH
HO
NH
O
HO
HO
OH
HO
NH
CO2O
CO2O
a2,8-
HO
HO
O
a2,6-
HO
O
HO
O
HO
NH
O
HO
CO2O
HO
O
O
HO
HO
NHAc
O
HO
CO2-
O
O
HO
a2,3-
OH
HO
NH
O
CO2
-
O
HO
O
a2,8O
CO2- O
HO
HO
NH
OH
OH
O
a2,3-
O
O
O
HO
O
OH
OH
ST8SIA5
ST6GAL1
ST6GAL2
ST3GAL1
ST6GALNAC1
ST3GAL2
But, which one?
C12H25
OH
HO
ST8SIA1
ST3GAL1
R
HN
O
ST3GAL4
ST3GAL5
ST6GALNAC2
ST6GALNAC3
ST8SIA2
ST8SIA3
ST3GAL2
ST3GAL3
ST3GAL4
ST3GAL6
ST8SIA4
ST8SIA6
ST6GALNAC4
ST3GAL6
HO
HO
Sialoglycoconjugate
production
OCMP
O
NH
HO
O
CMP-Neu5Ac
OH
AcNH
O
ST6GALNAC6
HO
O
O
O
ST6GALNAC5
OH
OH
O
O
OH
a2,8-
HO
NH
n = 55-200
OH
O
ST6GALNAC4
O
CO2O
O
HO
ST6GALNAC3
CO2-
O
HO
HOa2,6-
OH
HO
NH
CO2-
CMP
CMPNT
Golgi
Genetically Engineering Glycosylation is NOT Easy
Keeping in mind that glycosylation is actually 10x-fold more complex . . ..
http://www.ncbi.nlm.nih.gov/pubmed/23326219
http://www.ncbi.nlm.nih.gov/pubmed/19506293
Question: How to determine the
specific gene(s) responsible?
Solution: Use an engineering
(computational modeling) approach!
One reason for uncertainties is the
complex, non-template-based
biosynthetic routes for glycans
OK, Let’s Try Something Else – “Cell Culture Variables”
or CMP-sialic acid
Cell / genetic
engineering
Cell culture
variables
e.g., NH3
Going Back to the “Sialyltransferase” (ST) Schematic
Goal: increase sialylation
Glycan
recycling
HO
HO
NEU3
NEU2
KL
NEU4
HO
O
NH
NEU1
CO2-
OH
OH
HO
O
HO
OH
HO
NH
HO
OH
HO
NH
O
HO
HO
OH
HO
NH
CO2O
CO2O
O
HO
HO
HO
O
HO
OH
O
O
a2,3-
OH
O
O
CO2
O
HO
O
-
O
CO2- O
HO
HO
NH
OH
OH
O
a2,3-
O
O
O
HO
O
OH
OH
ST6GAL1
ST6GAL2
ST3GAL1
ST8SIA5
C12H25
OH
HO
ST8SIA1
ST3GAL1
R
HN
ST6GALNAC1
ST3GAL4
ST3GAL5
ST6GALNAC2
ST6GALNAC3
ST8SIA2
ST8SIA3
ST3GAL2
ST3GAL3
ST3GAL4
ST3GAL6
ST8SIA4
ST8SIA6
ST6GALNAC4
ST3GAL6
Sialoglycoconjugate
production
OCMP
HO
O
CMP-Neu5Ac
HO
CO2-
O
AcNH
ST3GAL2
HO
a2,8-
HO
NH
O
O
O
HO
n = 55-200
NHAc
ST6GALNAC6
NH
HO
O
O
HO
O
OH
ST6GALNAC5
HO
CO2O
O
HO
NH
HO
O
HO
NH
O
HO
OH
O
OH
a2,8-
HO
O
a2,6-
O
O
OH
But that paradigma2,8is
ST6GALNAC3
being disproved . . .
CMP-Neu5Ac generally
has been presumed NOT
to regulate ST activity
CO2O
O
HO
ST6GALNAC4
CO2-
O
HO
HOa2,6-
OH
HO
NH
CO2-
CMP
CMPNT
Golgi
Checking Back in on Glycoengineering Options
Glycoengineering holds promise to improve safety/efficacy of rProteins
rProtein
Glycoengineered rProtein
Goal: increase sialylation
Approach
Simple
Post-synthetic modification
X
Cell/genetic engineering
X
Cell culture variables
?
Low cost
X
Versatile
Effective
?
X
?
?
?
e.g., media supplementation to increase CMP-sialic acid levels
??
ManNAc is the Feedstock for Sialic Acid Production
X
Natural ManNAc
N/A
5-10%
increase in
sialylation
O
HO HN
O
HO
OH
HO
ManNAc
Goal: increase sialylation
Low sialic acid = Poor activity
Increased SA = improved activity
The application of N-acetylmannosamine to the mammalian cell
culture production of recombinant human glycoproteins
ManNAc is the Feedstock for Sialic Acid Production
X
Natural ManNAc
N/A
5-10%
increase in sialylation
“Ballpark” estimates for a 15,000 L/30,000 g bioreactor run
$3.3-6.6 million
 IVIg therapy costs ~ $15,000 per patient (@ 2 g / kg)
 Therefore, the value of IVIg is ~ $100 / g
 And 30,000 g would be worth ~$3,000,000
O
HO HN
O
HO
HO
 Most mAb therapies require a dose of 2-20 mg / kg)
OH
 Therefore, a bioreactor run would be worth $3003,000 millIion (i.e., up to $3 billion!)
ManNAc
The application of N-acetylmannosamine to the mammalian cell
culture production of recombinant human glycoproteins
Towards a Solution: 2nd Generation ManNAc Analogs
X
Natural ManNAc
X
Ac4ManNAc
O
O
O
O
O
O
N/A
10-25%
increase in SA
HN
O
O
O
N/A
O
Ac4ManNAc
Goal: increase sialylation
Low sialic acid = Poor activity
Increased SA – improved activity
Refer to “notes” for references for Ac4ManNAc efficiency and cytotoxicity
Towards a Solution – Separating Flux & Toxicity
X
Natural ManNAc
X
Ac4ManNAc
O
O
O
N/A
O
O
O
N/A
HN
O
O
O
Ac4ManNAc
Refs 1-3
(in notes)
O
O
O
O
O
Refs 4-9
O
O
OH
O
3,4,6-O-Bu3ManNAc
O
O
O
HN
O
“Whole molecule” activities
(a platform for drug development)
O
O
O
HN
O
O
O
O
O
Refs 4, 10, 11
HO
O
O
Bu4ManNAc
Complex activities
(higher flux, enhanced toxicity)
O
HN
O
O
O
O
1,3,4-O-Bu3ManNAc
The Solution
(next slides)
A Closer Look - Simplicity
1,3,4-O-Bu3ManNAc
 Substitution of n-butyrate for acetate increases transmembrane uptake
into cells
 The amphipathic nature of the molecule maximizes uptake
 The “1,3,4” pattern of butanoylation minimizes toxicity
O
HO
O
O
HN
O
O
O
O
O
1,3,4-O-Bu3ManNAc
A Closer Look - Cost
1,3,4-O-Bu3ManNAc
“Ballpark” estimates for a 15,000 L/30,000 g bioreactor run
$3.3-6.6 million
$24-120K
O
HO HN
O
HO
HO
O
OH
O
O
O
O
O
$6-75K
HN
O
O
O
O
O
HN
O
O
O
O
O
O
ManNAc
HO
O
Ac4ManNAc
1,3,4-O-Bu3ManNAc
A Closer Look - Versatility
1,3,4-O-Bu3ManNAc
O
HO
O
O
HN
O
O
adds sialic acid
O
O
O
1,3,4-O-Bu3ManNAc
HO
O
O
O
adds chemical FGs (Refs 3,4)
O
O
O
O
HN
O
1,3,4-O-Bu3Glc/GalNAc
adds
Branches
(Refs 1,2)
O
R
HO
O
HN
O
O
O
O
O
O
1,3,4-O-Bu3ManN(R)
R = >25 functional groups
Illustrating Versatility (and Effectiveness) with EPO
Optimally sialylated EPO has longer serum half-life
Erythropoietin (EPO)
($9 billion market)
1,3,4-O-Bu3Glc/GalNAc
Increased
branching
1,3,4-O-Bu3ManNAc
Increased
sialic acid
Serum ½ life:
≤14 SAs = 8.5 hours
~22 SAs = 25.3 hours
http://ndt.oxfordjournals.org/content/17/suppl_5/66.full.pdf
Even better w/ non-natural sialic acid
O
http://pubs.acs.org/email/cen/html/070406220531.html
R
HO
O
HN
O
O
O
O
O
O
1,3,4-O-Bu3ManN(R)
R = >25 functional groups (Refs 1-3)
A Closer Look - Effectiveness
1,3,4-O-Bu3ManNAc
Relative S.A. in treated cells
(fold increase cf. controls)
~75% (“global”)
increase in sialylation
Individual glycoproteins experience
a considerably larger
increase in S.A.
i.e., ~175% is the “average”
> 80 proteins from a glycoproteomics analysis of SW1990 cells
Metabolic flux increases glycoprotein sialylation . . .(2012)
Effectiveness – The Implications
1,3,4-O-Bu3ManNAc
 1,3,4-O-Bu3ManNAc is never harmful wrt sialylation
~75% (“global”)
increase in S.A.
 1,3,4-O-Bu3ManNAc is most effective for proteins with very low starting
levels of sialic acid
Goal: increase sialylation
For example, Immunoglobin G, which is ~10% sialyated
To Summarize Recombinant Protein Glycoengineering
Poor glycosylation compromises safety, pharmacokinetics, and activity
The “solution”
Sub-optimal glycosylation
But getting
there is complex!!
Current solutions
Cell / genetic
engineering
Post-synthetic
modification
Cell culture
variables
Optimal and consistent protein glycosylation in
mammalian cell culture (Glycobiology, 2009)
An Overview of Today’s Lecture
First – What is Carbohydrate Engineering?
Next
Organ Transplantation
Metabolic Oligosaccharide Engineering
Cardiovascular Disease – USA’s #1 Killer
About 600,000 people die of heart disease in the United States every year–that’s
1 in every 4 deaths
http://www.cdc.gov/heartdisease/facts.htm
Question: where to get replacements for diseased and worn out hearts?
One Option – Tissue Engineering
Tissue engineering:
the creation of new tissues or
organs in the laboratory to
replace diseased, worn out, or
injured body parts
A Second Option – Xenotransplantion
Baby Fae – recipient of a baboon heart (ca. 1984)
Ultimately unsuccessful, spawned a backlash based (in
part) on ethical concerns
Xenotransplants – Some Background Info
Xenotransplantation (i.e., transplants from other species) is
being pursued because of a dire shortage of human donors
(and ethical concerns with using primates)
Pigs seem like a good choice to be organ donors –
we’re already eating them, and they’re quite similar to us!
The creatures outside looked from pig to man, and from
man to pig, and from pig to man again; but already it was
impossible to say which was which.
− George Orwell, Animal Farm
A dissected pig whose organs will be
used for a xenotransplant.
Today’s scientists are breeding pigs and
harvesting
their
organs
for
xenotransplants. Pigs are excellent
“source animals” because they are easily
bred and typically have large litters of
piglets that grow very rapidly, forage for
themselves, and reproduce rather
quickly. More importantly, pig organs are
physiologically and anatomically similar
to human organs.
http://www.kiwibox.com/article.asp?a=32813
Xenotransplants – Overcoming Hyperacute Rejection
1
What is the cause of
hyperacute rejection?
(From Nature Biotechnology,
March 2002 Volume 20 Number 3 pp 231 - 232)
Hyperacute Rejection Results from “a-Gal”
The role of a-1,3-Gal in hyperacute and acute vascular rejection
Hyperacute rejection (HAR) is caused by binding of large
amounts of antibody, consisting predominantly of anti-a1,3-Gal, to graft blood vessels, activating large amounts of
complement.
Remember that “a-Gal” is a Trisaccharide
LacNAc
(N-acetylated lactose)
OH OH
O
HO
OH OH
O
HO
O
OH
O
OH
O
HO
O
The "aGal" epitope, the major antigenic
determinant in non-primate cells responsible
for organ and tissue immuno-rejection
NHAc
Humans and (other) primates do not make a-Gal and for that reason
avoid HAR (but for ethical reasons, are not considered to be appropriate sources for
large scale organ harvesting and transplantation (by contrast 35,000,000 pigs are
already being slaughtered each year in the USA)
Strategies to Overcome Hyperacute Rejection
Strategy #1. Can soluble a-Gal protect against hyperacute rejection?
soluble aGal
OH OH
O
HO
OH OH
O
HO
O
OH
O
OH
O
HO
OH
NHAc
This seemed like a plausible approach
15 years ago . ..
Yarema & Bertozzi, Current Opinion in Chemical Biology,
1998, 2:49–61
But it has not worked out, for several reasons
Strategy #2 – Knockout the a1,3GT Gene
OH OH
O
HO
X
OH OH
O
HO
O
O
OH
HO
OH
O
O
NHAc
aGal
a1,3-galactosyltransferase (a1,3GT)
Three key technologies were required that were falling into place in the 1990s
1. Identification of the a1,3-galactosyltransferase gene (genetics/bioinformatics)
2. homologous recombination of the target genes (molecular/cell biology)
3. adaptation of nuclear transfer technology to pigs (large animal genetics)
Step 1. The a1,3GT Gene was ID’d 20 Years Ago
Strategy #2. “Knocking out” the a-Gal gene
Three key technologies were required that were falling into place in the 1990s
1. Identification of the a1,3-galactosyltransferase gene (genetics/bioinformatics)
2. homologous recombination of the target genes (molecular/cell biology)
3. adaptation of nuclear transfer technology to pigs (large animal genetics)
The “aGal” gene was cloned in 1995
Immunogenetics. 1995;41(2-3):101-5.
cDNA sequence and chromosome localization of pig alpha 1,3 galactosyltransferase.
Strahan KM, Gu F, Preece AF, Gustavsson I, Andersson L, Gustafsson K.
Source
Division of Cell and Molecular Biology, Institute of Child Health, London, UK.
Abstract
Human serum contains natural antibodies (NAb), which can bind to endothelial cell surface antigens of other
mammals. This is believed to be the major initiating event in the process of hyperacute rejection of pig to primate
xenografts. Recent work has implicated galactosyl alpha 1,3 galactosyl beta 1,4 N-acetyl-glucosaminyl carbohydrate
epitopes, on the surface of pig endothelial cells, as a major target of human natural antibodies. This epitope is made
by a specific galactosyltransferase (alpha 1,3 GT) present in pigs but not in higher primates. We have now cloned
and sequenced a full-length pig alpha 1,3 GT cDNA. The predicted 371 amino acid protein sequence shares 85%
and 76% identity with previously characterized cattle and mouse alpha 1,3 GT protein sequences, respectively. By
using fluorescence and isotopic in situ hybridization, the GGTA1 gene was mapped to the region q2.10-q2.11 of pig
chromosome 1, providing further evidence of homology between the subterminal region of pig chromosome 1q and
human chromosome 9q, which harbors the locus encoding the AB0 blood group system as well as a human
pseudogene homologous to the pig GGTA1 gene
http://www.ncbi.nlm.nih.gov/pubmed/7528726
Step 2. A Lot of Really Complex Genetic Manipulation!
Three
Strategy
key technologies
#2. “Knocking
were
out”
required
the a-Gal
thatepitope
were falling into place in the 1990s
1. Identification of the a1,3-galactosyltransferase gene (genetics/bioinformatics)
2. homologous recombination of the target genes (molecular/cell biology)
3. adaptation of nuclear transfer technology to pigs (large animal genetics)
Molecular biology techniques were maturing . . .
The aGal gene was “knocked out” in germ line cells
(from Nature Biotechnology,
March 2002 Volume 20 Number 3 pp 231 - 232)
Step 3. Moving from Rodents to “Large Animals” . . ..
Strategy #2. “Knocking out” the a-Gal epitope
Three key technologies were required that were falling into place in the 1990s
1. Identification of the a1,3-galactosyltransferase gene (genetics/bioinformatics)
2. homologous recombination of the target genes (molecular/cell biology)
3. adaptation of nuclear transfer technology to pigs (large animal genetics)
The cloning of large animals . . .
. . . was pioneered by Dolly the Sheep
Dolly (5 July 1996 – 14 February 2003)
The First “a-Gal” Knockout Pigs were Born Xmas Day, 2002
But, only one allele was knocked out!!
2
Figure 3: Five a1,3GT gene knockout piglets at 2 weeks of age.
a1,3GT expression was
still possible from the copy
Solution: Breeding experiments, expected progeny:
+/+, +/−, and −/− at a 1:2:1 ratio
of the gene on the nonknocked out allele
Wrapping up the “Loose Ends” (and new pitfalls)
Production of -/- a1,3-galactosyltransferase-deficient pigs
“Our results have demonstrated that removal of a1,3Gal epitopes
on pig cells did not preclude development in utero . . . .”
. . . the baby pigs appeared to be OK!
Phelps et al, Science (2003)
http://www.ncbi.nlm.nih.gov/pubmed/12493821
Rearing and Caring for a Future Xenograft Donor Pig
The aGal knockout pigs needed special care due to concerns about
• Reduced sperm adhesion to zona pellucida • Increased sensibility to autoimmune diseases
• Increased sensibility to sepsis
• Cataract formation
http://www.actavetscand.com/content/45/S1/S45
a1,3-Galactosyltransferase knockout pigs are available for xenotransplantation: But, are glycosyltransferases still relevant?
Uh oh – the double null animals still expressed aGal !!
(albeit at a lower level, and only on glycolipids)
http://www.nature.com/icb/journal/v83/n6/full/icb200594a.html
Wrapping up the “Loose Ends” (and new pitfalls)
Strategy #2. “Knocking out” the a-Gal epitope (ca. 2003-2005)
Why/How did the -/- aGT Knock Out Pigs still Express aGal?
The pig genome was not sequenced until 2010
Maybe there were other
genes in the pig genome
with aGT activity
Hyperacute Rejection *has* Been Solved!
Strategy #2. “Knocking out” the a-Gal epitope
In Any Event, The Low(er) Residual Levels of
a-Gal were Not a Huge Problem
From Revivicor’s website:
Hyperacute rejection is the first hurdle that has to be overcome; a reaction to the
'foreign' organ by the body's normal immune system. Humans and primates differ
from other animals in that they lack an enzyme (a1,3 galactosyltransferase) that
places a particular sugar group (Gal) on the branched sugar chains which occur
on cell surfaces. Our bodies recognize its presence on grafted pig organs as a
signal to attack. Revivicor's has inactivated the gene in pigs which makes the
enzyme that attaches this Gal sugar group, producing the worlds first a1,3
galactosyltransferase (Gal) knock-out pigs. Organs from Gal knock-out pigs
transplanted into non-human primates did not undergo HAR; thus the initial
attack of HAR was overcome by the use of these GE pigs.
Hyperacute Rejection *has* Been Solved!
But there’s Still (Much!) More Work to Do
While the presence of the foreign Gal sugar
is by far the major signal for initiating an
attack by the immune system, there are other
mediators of immune rejection at play.
Revivicor has also added a human gene
to the pigs to produce a protein called
CD46 that moderates the action of the
immune system. This gene addition
strategy, combined with Gal knock-out and
immune suppression drugs, demonstrated
encouraging results of pig hearts in primates,
with survival and function for up to 8 months.
Overcoming hyperacute rejection is only the
first, but essential, step in Revivicor's
comprehensive approach. . . .
If interested, you can consult the company’s website:
http://www.revivicor.com/body_xenotransplantation.htm
Back to the Overview of Today’s Lecture
First – What is Carbohydrate Engineering?
Finally
Organ Transplantation
Metabolic Oligosaccharide Engineering
Back to the Overview of Today’s Lecture
In the past (up to the present day,
really) a widespread / working
assumption has been that glycan
structures are controlled at the level
of glycosyltransferases.
By contrast, the nucleotide sugar
“building blocks” (e.g., CMP-Neu5Ac)
have been assumed to be at
saturating levels
We recently demonstrated that metabolic flux *is* critical:
Almaraz, R. T., Tian, Y., Bhattarcharya, R., Tan, E., Chen, S.-H., Dallas, M. R., Chen, L., Zhang, Z., Zhang, H., Konstantopoulos, K., and Yarema, K. J. (2012)
Metabolic flux increases glycoprotein sialylation: implications for cell adhesion and cancer metastasis. Mol Cell Proteomics, 10.1074/mcp.M1112.017558
Moving from the Rate to the Type of Flux
Exogenous (e.g.,
dietary) sugars
Naturally-occurring
cell surface oligosaccharides
1
O
HO HN
O
HO
HO
R1
Glycosylation
pathways
OH
HO
R1
OH
HO
NH
O
O
HO
Neu5Ac
ManNAc
R1 =
CH3
CH3
CO2-
CH3
Werner Reutter’s Laboratory
This approach now is generally known as:
 “metabolic oligosaccharide engineering” or
 “metabolic glycoengineering”
Kayser et al, Journal of Biological Chemistry, 1992
O
Is “Metabolic Glycoengineering” Useful?
Exogenous (e.g.,
dietary) sugars
1
O
HO HN
O
HO
HO
R1
Glycosylation
pathways
OH
HO
R1
OH
HO
NH
O
O
HO
Neu5Ac
ManNAc
R1 =
CH3
CH3
CO2-
CH3
Werner Reutter’s Laboratory
Keppler et al, Glycobiology 2001
O
Soon “Chemical Biologists” Dominated the Field
Exogenous (e.g.,
dietary) sugars
Naturally-occurring
cell surface oligosaccharides
1
O
HO HN
O
HO
HO
R1
HO
Glycosylation
pathways
R1
OH
OH
HO
NH
O
CO2O
O
HO
Neu5Ac
ManNAc
R1 =
R1 =
CH3
CH3
CH3
Werner Reutter’s Laboratory
The ketone group
N3
O
Carolyn Bertozzi’s Group
Mahal et al, Science, 1997
The azide and alkyne, the reaction partners for ‘click chemistry’
Click Chemistry – 1,530,000 Google Entries! *
Applied to metabolic glycoengineering
Sialic acid
pathway
O
AcO
AcO
AcO
N3
HN
O
OAc
HO
Ac4ManNAz
N-
N
+
Cu(I)
N
N
OH
CO2O
HO
NH
O
HO
O
www.thechemblog.com
Sia5Az
N N
Copper catalyzed [3+2]
cycloaddition reaction
(aka “click chemistry”)
Saxon & Bertozzi, Science, 2000
*That was in 2007
17,300,000 in 2009
32,200,000 in 2010
539,000,000 in 2013
This Technology can be used as a Glycoproteomics Tools
Sialic acid
pathway
O
AcO
AcO
AcO
N3
HN
O
OAc
HO
Ac4ManNAz
N-
N
+
N
OH
CO2O
HO
NH
O
HO
O
Cu(I)
N
Sia5Az
N N
Copper catalyzed [3+2]
cycloaddition reaction
(aka “click chemistry”)
*It works best when the cells/animals can
be sacrificed (i.e., when they are dead)
Saxon & Bertozzi, Science, 2000
(the copper is somewhat toxic, this
problem is solved on the next slide)
New Bio-orthogonal Chemistries can be used In Vivo
Sialic acid
pathway
O
AcO
AcO
AcO
N3
HN
O
OAc
HO
Ac4ManNAz
N-
N
+
*
N
OH
CO2O
HO
NH
O
HO
O
Sia5Az
**
http://pubs.acs.org/cen/news/86/i18/8618notw1.html
O
Cu(I)
N
N N
Copper catalyzed [3+2]
cycloaddition reaction
(aka “click chemistry”)
Saxon & Bertozzi, Science, 2000
*The copper catalyst is toxic
Sia5Az
N
Sia5Az
N
N
O
Cell-surface glycans shine
in this microscopy image of
the head of a three-day-old
zebrafish embryo treated
with the new technique.
Strain-promoted [3+2]
cycloaddition**
Agard et al, JACS, 2004
**Copper-free “click reactions” can now
be done in living cells and in vivo.
Additional Pathways (beyond Sialic Acid) can be Targeted
In addition to cell surface sialic acid, metabolic
glycoengineering now can target cell surface
GalNAc and fucose (GlcNAc analogs mainly
label intracellular “O-GlcNAc” )
http://pubs.acs.org/cen/news/86/i18/8618notw1.html
Cell-surface glycans shine
in this microscopy image of
the head of a three-day-old
zebrafish embryo treated
with the new technique.
For Example, Remember “Fucose” ?
adds chemical FGs (Refs 3,4)
O
R
HO
O
O
 Works on secreted proteins
 New bioorthogonal chemistry
HN
O
O
O
Additional “twists”
O
O
1,3,4-O-Bu3ManN(R)
R = >25 functional groups
This actually *should* have been fucose!
(from earlier in today’s lecture)
Expanding the Repertoire of Bioorthogonal Chemistries
(A) The ketone is the first
example of an bio-orthogonal
chemical
functional
group
installed in the glycocalyx
(B) and (C) Either “click”
functional group (azides, B or
alkynes, C) can be installed in
the glycocalyx
(D) and (E) Photoactivated
functional groups can be
installed in the glycocalyx
(F) Thiols can be incorporated
into an unusual cellular locale,
the glycocalyx*
*contact me for information
on our lab’s efforts to use
sialic acid-displayed thiols
for tissue engineering
Du et al, Glycobiology, 2009
OK – Finally – about those 25 “R” Groups . . . . .
Almaraz et al, Ann Biomed Eng, 2012
Where does Metabolic Oligosaccharide Engineering Go Next?
From
“Chemical Biology”
To
“The Clinic”
??
In the Bigger Picture, Progress Continues . . . .
 Commercialization and translational efforts were slow to be realized:
The Bittersweet Promise of Glycobiology
Nature Biotechnology, 2001 (doi:10.1038/nbt1001-913)
The Sweet and Sour of Cancer: Glycans as Novel Therapeutic Targets
Nature Reviews Cancer, 2005 (doi:10.1038/nrc1649)
 By 2008 “we” had learned a valuable “first do no harm” lesson
 In the past decade, progress has accelerated:
2003:
2006:
Our Technology
2008:
O
HO
O
O
O
HN
O
O
O
O
1,3,4-O-Bu3ManNAc
An novel scaffold for drug design
(over 100,000 permutations)
Back to the Overview of Today’s Lecture – All Done!
First – What is Carbohydrate Engineering?
Organ Transplantation
Metabolic Oligosaccharide Engineering