kevin j. yarema associate professor of biomedical engineering the johns hopkins school of medicine...

Kevin J. YaremaAssociate Professor of Biomedical Engineering

The Johns Hopkins School of Medicine

Carbohydrate Engineering

ISBN 978-3-527-30632-9 - Wiley-VCH

Introduction to Glycobiology (ME330.712)

Email: [email protected]: 410.614.6835

An Overview of Today’s Lecture

First – What is Carbohydrate Engineering?

Sugars are critical for $250 billion $$s worth of drugs

Organ Transplantation Metabolic Oligosaccharide Engineering

From Google:


For pdfs of the introduction, or any chapter, email me at [email protected]



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

2)(secondarily) for biomedical purposes*

*

But, is this even possible?

Biologics (i.e. therapeutic glycoproteins)

Xenotransplantation

The Promise of Sugar-based Therapies

The term “Glycobiology” was coined in 1988

A flurry of clinical translation and commercialization efforts ensued (1990s)

“Glycans” were implicated in many (most!) complex diseases

ArthritisCancer metastasis

www.omicsonline.org

Immune disorders (Kawasaki Disease)

http://en.wikipedia.org/wiki/File:Kawasaki_symptoms.jpg

Degenerative muscle disease

http://guardianlv.com/2013/08/new-hope-for-duchenne-muscular-dystrophy-patients/

Duchenne Muscular Dystrophy

Arthritis

Commercialization and translational efforts were slow to be realized:

Difficulties Ensued

The Bittersweet Promise of GlycobiologyNature Biotechnology, 2001 (doi:10.1038/nbt1001-913)

The Sweet and Sour of Cancer: Glycans as Novel Therapeutic TargetsNature 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/

http://www.foxbusiness.com/industries/2014/01/14/tesla-to-double-sales-service-locations-as-4q-deliveries-top-outlook/

*Until “today”

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-1,3-galactose (“-Gal”), which triggered anaphylaxis when they were given the drug. -Gal

The “Southern Mystery” angle:

http://www.viracoribt.com/alphagal

Lone Star Tick bites

IgE against -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 -1,3-galactosyltransferase.

-Gal


Pitfalls Along the Way are Being Overcome



By 2008 “we” had learned a valuable “first do no harm” lesson

-Gal

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 -1,3-galactosyltransferase 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 -Gal.

The solution – use a “safe” cell line for mAb production:

Patnaik & Stanley (Methods in Enzymology, 2006):http://www.sciencedirect.com/science/article/pii/S0076687906160115

For example, Dr. Pamela Stanley’s lab has developed a library of CHO mutants allowing desired glycoforms to be “dialed in” (or out . .. ):

A Simpler Solution? – Just Eliminate the Sugar(s)?

First – How?

Second – Would it work?

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.

Nimmerjahn, F., and Ravetch, J. V. (2007) Antibodies, Fc receptors and cancer. Curr Opin Immunol 19, 239-245.

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

Just Eliminate the Sugar(s)? – No, they are Critical for 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.

Sugars Determine Antibody Activity

More about the sugars in a few minutes, but let’s first learn more about IVIg therapy

IVIg therapy is used to treat a wide range of conditions; FDA approved:

IVIg (Intravenous Immunoglobin) Therapy: A Quick Overview

Allogeneic bone marrow transplant Chronic lymphocytic leukemia Common variable immunodeficiency Idiopathic thrombocytopenic purpura (ITP) Pediatric HIV

Primary immunodeficiencies Kawasaki disease Chronic inflammatory demyelinating

polyneuropathy Kidney transplant*

Kawasaki Disease

http://en.wikipedia.org/wiki/File:Kawasaki_symptoms.jpg

Idiopathic thrombo-cytopenic purpura

(ITP)

http://www.danmedj.dk/portal/page/portal/danmedj.dk/dmj_forside/PAST_ISSUE/2011/DMB_2011_04/A4252

Autoimmune disease

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 Autism

Alzheimer’s disease Chronic fatigue syndrome

http://www.icare4autism.org/

Autism Alzheimer’s disease

http://www.wbrz.com/news/alzheimers-advances-show-need-for-better-drugs/

A solution: Recombinant Ig (?)The upside: controlled production The downside: Only 1 out of ~10 antibodies is properly glycosylated

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

~10% sialylation(many copies are

not active)

Sialic acid

From IVIg Therapy to “Big Picture” Implications

Optimal and consistent protein glycosylation in mammalian cell culture

(Glycobiology, 2009)

“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 far-reaching influences on industry”

IVIg exemplifies the need for glycosylation optimization in “biologics”

Back to IVIg (and rProteins in General*)

Sub-optimal glycosylation The “solution”

Poor glycosylation compromises safety, pharmacokinetics, and activity

But getting there is complex!!

Post-synthetic modification

Cell / genetic engineering

Cell culture variables

Current solutions




Cell (Genetic Engineering) Modulation of Glycan Production

Goal: increase sialylation

CO2-O

HO

HO

OCMPOH

HONH

OHO

CMP-Neu5Ac

HO

O

OH

OHCO2-

OHO

HO

O

OH

HONH

O

O

O

OH

OH

O

CO2-

OHO

HO

OH

HONH

OHO

O

OAcNH

O

CO2-

O

HO

HO

O

HONH

O

CO2-

OHO

HO

OH

HONH

O

O O

OH

OH

HOO C12H25

OH

HN

O

R

CO2-

OHO

HO

OH

HONH

OHO

O

HONHAc

OHO

O

OH

OHCO2-

OHO

HO

O

O

HONH

O

CO2-

OHO

HO

O

HONH

O

CO2-

OHO

HO

OH

HONH

O

n = 55-200

ST6GALNAC6

ST6GALNAC5

ST6GALNAC4

ST6GALNAC3

ST8SIA1

ST8SIA5

ST3GAL1

ST3GAL2

ST3GAL4

ST3GAL5

ST3GAL6

ST6GALNAC3

ST6GALNAC2

ST6GALNAC1

ST6GALNAC4

ST6GAL2

ST8SIA3

ST8SIA4

ST8SIA2

ST3GAL1

ST3GAL2

ST3GAL3

ST3GAL4

ST3GAL6

CMPCMPNT

CO2-

O

HO

HOOH

OH

HONH

O

a2,8-

a2,3-

a2,6-

a2,6-

a2,3-

a2,8-

a2,8-

NEU1

NEU2

NEU3

NEU4

Sialoglycoconjugateproduction

Glycan recycling

ST6GAL1

ST8SIA6

Golgi

KL

OK, That Sounds Easy Enough, Let’s GE in a ST!


But, which one?

One reason for uncertainties is the complex, non-template-based biosynthetic routes for glycans

Question: How to determine the specific gene(s) responsible?

Solution: Use an engineering (computational modeling) approach!

http://www.ncbi.nlm.nih.gov/pubmed/23326219http://www.ncbi.nlm.nih.gov/pubmed/19506293

Genetically Engineering Glycosylation is NOT Easy

Keeping in mind that glycosylation is actually 10x-fold more complex . . ..


OK, Let’s Try Something Else – “Cell Culture Variables”

Cell culture variables e.g., NH3

or CMP-sialic acid

CO2-O

HO

HO

OCMPOH

HONH

OHO

CMP-Neu5Ac

HO

O

OH

OHCO2-

OHO

HO

O

OH

HONH

O

O

O

OH

OH

O

CO2-

OHO

HO

OH

HONH

OHO

O

OAcNH

O

CO2-

O

HO

HO

O

HONH

O

CO2-

OHO

HO

OH

HONH

O

O O

OH

OH

HOO C12H25

OH

HN

O

R

CO2-

OHO

HO

OH

HONH

OHO

O

HONHAc

OHO

O

OH

OHCO2-

OHO

HO

O

O

HONH

O

CO2-

OHO

HO

O

HONH

O

CO2-

OHO

HO

OH

HONH

O

n = 55-200

ST6GALNAC6

ST6GALNAC5

ST6GALNAC4

ST6GALNAC3

ST8SIA1

ST8SIA5

ST3GAL1

ST3GAL2

ST3GAL4

ST3GAL5

ST3GAL6

ST6GALNAC3

ST6GALNAC2

ST6GALNAC1

ST6GALNAC4

ST6GAL2

ST8SIA3

ST8SIA4

ST8SIA2

ST3GAL1

ST3GAL2

ST3GAL3

ST3GAL4

ST3GAL6

CMPCMPNT

CO2-

O

HO

HOOH

OH

HONH

O

a2,8-

a2,3-

a2,6-

a2,6-

a2,3-

a2,8-

a2,8-

NEU1

NEU2

NEU3

NEU4

Sialoglycoconjugateproduction

Glycan recycling

ST6GAL1

ST8SIA6

Golgi

KL

Going Back to the “Sialyltransferase” (ST) Schematic


CMP-Neu5Ac generally has been presumed NOT

to regulate ST activity

But that paradigm is being disproved . . .

rProtein Glycoengineered rProtein

Glycoengineering holds promise to improve safety/efficacy of rProteins

Simple Low cost Versatile EffectiveApproach

Post-synthetic modification X X ?

Cell culture variables ? ? ? ??Cell/genetic engineering X X ?


Checking Back in on Glycoengineering Options

e.g., media supplementation to increase CMP-sialic acid levels

ManNAc is the Feedstock for Sialic Acid Production

X

Low sialic acid = Poor activity Increased SA = improved activity

OHO

HOHO

HN

OH

O

ManNAc

Natural ManNAc N/A

5-10% increase in sialylation

The application of N-acetylmannosamine to the mammalian cell culture production of recombinant human glycoproteins


ManNAc is the Feedstock for Sialic Acid Production

XNatural ManNAc N/A5-10%

increase in sialylation

The application of N-acetylmannosamine to the mammalian cell culture production of recombinant human glycoproteins

“Ballpark” estimates for a 15,000 L/30,000 g bioreactor run

$3.3-6.6 million

OHO

HOHO

HN

OH

O

ManNAc

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

Most mAb therapies require a dose of 2-20 mg / kg) Therefore, a bioreactor run would be worth $300-

3,000 millIion (i.e., up to $3 billion!)

Towards a Solution: 2nd Generation ManNAc Analogs

Ac4ManNAc

Natural ManNAc X

Low sialic acid = Poor activity Increased SA – improved activity

N/A

OO

HN

O

O

O

O

O

O O

O

Ac4ManNAc N/AX10-25%

increase in SA

Refer to “notes” for references for Ac4ManNAc efficiency and cytotoxicity


Towards a Solution – Separating Flux & Toxicity

Natural ManNAc X N/A

Ac4ManNAc N/AX

OO

HN

O

O

O

O

O

O O

O

Ac4ManNAc

OO

HN

O

O

O

O

O

OO

O

Bu4ManNAc

Complex activities(higher flux, enhanced toxicity)

Refs 1-3(in notes)

OOH

HN

O

O

O

O

OO

O

3,4,6-O-Bu3ManNAc

“Whole molecule” activities(a platform for drug development)

Refs 4-9

1,3,4-O-Bu3ManNAc

OO

HN

O

O

O

O

O

O HO

The Solution(next slides)

Refs 4, 10, 11

A Closer Look - Simplicity

1,3,4-O-Bu3ManNAc

OO

HN

O

O

O

O

O

O HO

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

A Closer Look - Cost

1,3,4-O-Bu3ManNAc

“Ballpark” estimates for a 15,000 L/30,000 g bioreactor run

$24-120K

Ac4ManNAc

OO

HN

O

O

O

O

O

O O

O

$3.3-6.6 million

OHO

HOHO

HN

OH

O

ManNAc 1,3,4-O-Bu3ManNAc

OO

HN

O

O

O

O

O

O HO

$6-75K

A Closer Look - Versatility

1,3,4-O-Bu3ManNAc

OO

HN

O

O

O

O

O

O HO

1,3,4-O-Bu3ManNAc

1,3,4-O-Bu3Glc/GalNAc

OO

HN

O

O

O

O

O

O HO

adds sialic acid

adds Branches(Refs 1,2)

1,3,4-O-Bu3ManN(R)

R = >25 functional groups

OO

HN

O

O

O

O

O

O HO

R

adds chemical FGs (Refs 3,4)

Illustrating Versatility (and Effectiveness) with EPO

Even better w/ non-natural sialic acid

Erythropoietin (EPO)($9 billion market)

http://pubs.acs.org/email/cen/html/070406220531.html

Serum ½ life:

≤14 SAs = 8.5 hours

~22 SAs = 25.3 hourshttp://ndt.oxfordjournals.org/content/17/suppl_5/66.full.pdf

Optimally sialylated EPO has longer serum half-life

1,3,4-O-Bu3Glc/GalNAc

1,3,4-O-Bu3ManNAcIncreasedsialic acid

Increasedbranching

1,3,4-O-Bu3ManN(R)

R = >25 functional groups (Refs 1-3)

OO

HN

O

O

O

O

O

O HO

R

A Closer Look - Effectiveness

1,3,4-O-Bu3ManNAc

~75% (“global”)increase in sialylation

> 80 proteins from a glycoproteomics analysis of SW1990 cells

Rel

ati

ve

S.A

. in

tre

ate

d c

ells

(fol

d in

crea

se c

f. c

ontr

ols)

i.e., ~175% is the “average”

Individual glyco-proteins

experience a considerably

larger increase in S.A.

Metabolic flux increases glycoprotein sialylation . . .(2012)

Effectiveness – The Implications

1,3,4-O-Bu3ManNAc

~75% (“global”)increase in S.A.1,3,4-O-Bu3ManNAc is never harmful wrt sialylation

For example, Immunoglobin G, which is ~10% sialyated

1,3,4-O-Bu3ManNAc is most effective for proteins with very low starting levels of sialic acid


To Summarize Recombinant Protein Glycoengineering

Sub-optimal glycosylation The “solution”

Poor glycosylation compromises safety, pharmacokinetics, and activity

But getting there is complex!!

Post-synthetic modification


Cell culture variables

Current solutions



An Overview of Today’s Lecture




Next

Question: where to get replacements for diseased and worn out hearts?

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

Cardiovascular Disease – USA’s #1 Killer

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

Xenotransplantation (i.e., transplants from other species) is being pursued because of a dire shortage of human donors (and ethical concerns with using primates)

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 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.

A dissected pig whose organs will be used for a xenotransplant.

http://www.kiwibox.com/article.asp?a=32813

Pigs seem like a good choice to be organ donors – we’re already eating them, and they’re quite similar to us!

Xenotransplants – Some Background Info

(From Nature Biotechnology,March 2002 Volume 20 Number 3 pp 231 - 232)

1

What is the cause of hyperacute rejection?

Xenotransplants – Overcoming Hyperacute Rejection

Hyperacute rejection (HAR) is caused by binding of large amounts of antibody, consisting predominantly of anti--1,3-Gal, to graft blood vessels, activating large amounts of complement.

The role of -1,3-Gal in hyperacute and acute vascular rejection

Hyperacute Rejection Results from “-Gal”

OO

HONHAc

OO

OHOH

O

OH

OHO

OHOH

HO

HO

The "Gal" epitope, the major antigenicdeterminant in non-primate cells responsiblefor organ and tissue immuno-rejection

LacNAc(N-acetylated lactose)

Humans and (other) primates do not make -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)

Remember that “-Gal” is a Trisaccharide

soluble Gal

OO

HONHAc

OHO

OHOH

O

OH

OHO

OHOH

HO

HO

This seemed like a plausible approach 15 years ago . ..

Yarema & Bertozzi, Current Opinion in Chemical Biology, 1998, 2:49–61

Strategy #1. Can soluble Gal protect against hyperacute rejection?

But it has not worked out, for several reasons

Strategies to Overcome Hyperacute Rejection

OO

HONHAc

OO

OHOH

O

OH

OHO

OHOH

HO

HO

Gal

X1,3-galactosyltransferase (1,3GT)

Three key technologies were required that were falling into place in the 1990s

1.Identification of the 1,3-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)

Strategy #2 – Knockout the 1,3GT Gene





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.SourceDivision of Cell and Molecular Biology, Institute of Child Health, London, UK.AbstractHuman 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

The “Gal” gene was cloned in 1995

http://www.ncbi.nlm.nih.gov/pubmed/7528726

Strategy #2. “Knocking out” the -Gal gene

Step 1. The 1,3GT Gene was ID’d 20 Years Ago

Strategy #2. “Knocking out” the a-Gal epitopeThree key technologies were required that were falling into place in the 1990s




Molecular biology techniques were maturing . . .

(from Nature Biotechnology,March 2002 Volume 20 Number 3 pp 231 - 232)

The Gal gene was “knocked out” in germ line cells

Step 2. A Lot of Really Complex Genetic Manipulation!

Strategy #2. “Knocking out” the -Gal epitope





The cloning of large animals . . .

. . . was pioneered by Dolly the Sheep

Dolly (5 July 1996 – 14 February 2003)

Step 3. Moving from Rodents to “Large Animals” . . ..

Figure 3: Five 1,3GT gene knockout piglets at 2 weeks of age.

2

But, only one allele was knocked out!!

1,3GT expression was still possible from the copy

of the gene on the non-knocked out allele

Solution: Breeding experiments, expected progeny: +/+, +/−, and −/− at a 1:2:1 ratio

The First “-Gal” Knockout Pigs were Born Xmas Day, 2002

Phelps et al, Science (2003)http://www.ncbi.nlm.nih.gov/pubmed/12493821

Production of -/- 1,3-galactosyltransferase-deficient pigs

“Our results have demonstrated that removal of 1,3Gal epitopeson pig cells did not preclude development in utero . . . .”

. . . the baby pigs appeared to be OK!

1,3-Galactosyltransferase knockout pigs are available for xenotrans-plantation: But, are glycosyltransferases still relevant?

Uh oh – the double null animals still expressed Gal !!

http://www.nature.com/icb/journal/v83/n6/full/icb200594a.html

(albeit at a lower level, and only on glycolipids)

Rearing and Caring for a Future Xenograft Donor Pig

• Reduced sperm adhesion to zona pellucida• Increased sensibility to sepsis

• Increased sensibility to autoimmune diseases• Cataract formation

The Gal knockout pigs needed special care due to concerns about

http://www.actavetscand.com/content/45/S1/S45

Wrapping up the “Loose Ends” (and new pitfalls)

Strategy #2. “Knocking out” the -Gal epitope (ca. 2003-2005)

Why/How did the -/- GT Knock Out Pigs still Express Gal?

The pig genome was not sequenced until 2010

Maybe there were other genes in the pig genome

with GT activity

Wrapping up the “Loose Ends” (and new pitfalls)

Strategy #2. “Knocking out” the -Gal epitope

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 (1,3 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 1,3 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.

From Revivicor’s website:

In Any Event, The Low(er) Residual Levels of -Gal were Not a Huge Problem

Hyperacute Rejection *has* Been Solved!

But there’s Still (Much!) More Work to DoWhile 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. . . .

http://www.revivicor.com/body_xenotransplantation.htm

If interested, you can consult the company’s website:

Hyperacute Rejection *has* Been Solved!

Back to the Overview of Today’s Lecture




Finally

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

Back to the Overview of Today’s Lecture

Glycosylationpathways

Exogenous (e.g.,dietary) sugars

Naturally-occurringcell surface oligosaccharides

1

R1 = Werner Reutter’s Laboratory

CH3 CH3 CH3

OHO

HOHO

HN

OH

O

ManNAc

R1

Neu5Ac

O

HO

HO

O

OH

HONH

O

CO2-

R1

Kayser et al, Journal of Biological Chemistry, 1992

Moving from the Rate to the Type of Flux

This approach now is generally known as:“metabolic oligosaccharide engineering” or“metabolic glycoengineering”

Is “Metabolic Glycoengineering” Useful?



1


CH3 CH3 CH3

OHO

HOHO

HN

OH

O

ManNAc

R1

Neu5Ac

O

HO

HO

O

OH

HONH

O

CO2-

R1

Keppler et al, Glycobiology 2001

Soon “Chemical Biologists” Dominated the Field

R1 =

Carolyn Bertozzi’s GroupO

The ketone groupMahal et al, Science, 1997



Naturally-occurringcell surface oligosaccharides

1


CH3 CH3 CH3

OHO

HOHO

HN

OH

O

ManNAc

R1

Neu5Ac

O

HO

HO

O

OH

HONH

O

CO2-

R1

N3

The azide and alkyne, the reaction partners for ‘click chemistry’

www.thechemblog.com

Ac4ManNAz

CO2-

O

HO

HO

OH

HONH

O

ON

N+

N-

Sialic acidpathway

AcO OHNAcO

AcO OAc

ON3

Cu(I)

Copper catalyzed [3+2] cycloaddition reaction(aka “click chemistry”)

N N

N Sia5Az

Saxon & Bertozzi, Science, 2000

*That was in 200717,300,000 in 200932,200,000 in 2010539,000,000 in 2013

Click Chemistry – 1,530,000 Google Entries! *

Applied to metabolic glycoengineering

Ac4ManNAz

CO2-

O

HO

HO

OH

HONH

O

ON

N+

N-

Sialic acidpathway

AcO OHNAcO

AcO OAc

ON3

Cu(I)


N N

N Sia5Az


*It works best when the cells/animals can be sacrificed (i.e., when they are dead)

(the copper is somewhat toxic, this problem is solved on the next slide)

This Technology can be used as a Glycoproteomics Tools

Ac4ManNAz

CO2-

O

HO

HO

OH

HONH

O

ON

N+

N-

Sialic acidpathway

AcO OHNAcO

AcO OAc

ON3

Cu(I)


N N

N Sia5Az


ON

NN

Strain-promoted [3+2] cycloaddition**

Sia5Az

O

Sia5Az

Agard et al, JACS, 2004

Cell-surface glycans shine in this microscopy image of the head of a three-day-old zebrafish embryo treated with the new technique.

http://pubs.acs.org/cen/news/86/i18/8618notw1.html

*The copper catalyst is toxic

*

**Copper-free “click reactions” can now be done in living cells and in vivo.

**

New Bio-orthogonal Chemistries can be used In Vivo

Cell-surface glycans shine in this microscopy image of the head of a three-day-old zebrafish embryo treated with the new technique.

http://pubs.acs.org/cen/news/86/i18/8618notw1.html

In addition to cell surface sialic acid, metabolic glycoengineering now can target cell surface GalNAc and fucose (GlcNAc analogs mainly label intracellular “O-GlcNAc” )

Additional Pathways (beyond Sialic Acid) can be Targeted

For Example, Remember “Fucose” ?

1,3,4-O-Bu3ManN(R)

R = >25 functional groups

OO

HN

O

O

O

O

O

O HO

R

adds chemical FGs (Refs 3,4)

This actually *should* have been fucose!(from earlier in today’s lecture)

Additional “twists”Works on secreted proteinsNew bioorthogonal chemistry

Du et al, Glycobiology, 2009

(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

Expanding the Repertoire of Bioorthogonal Chemistries

Almaraz et al, Ann Biomed Eng, 2012

OK – Finally – about those 25 “R” Groups . . . . .

Where does Metabolic Oligosaccharide Engineering Go Next?

From “Chemical Biology”

To“The Clinic”

??


In the Bigger Picture, Progress Continues . . . .



By 2008 “we” had learned a valuable “first do no harm” lesson

In the past decade, progress has accelerated:

2003: 2006:

OO

HN

O

O

O

O

O

O HO

1,3,4-O-Bu3ManNAc

2008: Our Technology

An novel scaffold for drug design(over 100,000 permutations)

Back to the Overview of Today’s Lecture – All Done!




kevin j. yarema associate professor of biomedical engineering the johns hopkins school of medicine...

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