Presentation is loading. Please wait.

Presentation is loading. Please wait.

Carbohydrate Engineering

Similar presentations


Presentation on theme: "Carbohydrate Engineering"— Presentation transcript:

1 Carbohydrate Engineering
Introduction to Glycobiology (ME ) Kevin J. Yarema Associate Professor of Biomedical Engineering The Johns Hopkins School of Medicine Phone: ISBN  Wiley-VCH

2 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

3 First – What is Carbohydrate Engineering?
From Google:

4 First – What is Carbohydrate Engineering?
For pdfs of the introduction, or any chapter, me at

5 (i.e. therapeutic glycoproteins)
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: (primarily) The manipulation of glycans (secondarily) for biomedical purposes* * Biologics (i.e. therapeutic glycoproteins) Xenotransplantation But, is this even possible?

6 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) Arthritis Cancer metastasis Arthritis Degenerative muscle disease Duchenne Muscular Dystrophy A flurry of clinical translation and commercialization efforts ensued (1990s)

7 Uh oh!! Difficulties Ensued
Commercialization and translational efforts were slow to be realized: The Bittersweet Promise of Glycobiology Nature Biotechnology, 2001 (doi: /nbt ) The Sweet and Sour of Cancer: Glycans as Novel Therapeutic Targets Nature Reviews Cancer, 2005 (doi: /nrc1649) Uh oh!!

8 Time for an Analogy - Electric Cars
A great idea 25 years ago . . .that didn’t work out* (*at the time) *Until “today”

9 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!!

10 What Happened? What happened? (in more detail)
The affected patients had IgE antibodies against galactose-α-1,3-galactose (“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-1,3-galactosyltransferase. a-Gal The “Southern Mystery” angle: Lone Star Tick bites IgE against a-Gal

11 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: /nbt ) The Sweet and Sour of Cancer: Glycans as Novel Therapeutic Targets Nature Reviews Cancer, 2005 (doi: /nrc1649) By 2008 “we” had learned a valuable “first do no harm” lesson a-Gal

12 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-1,3-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 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):

13 A Simpler Solution? – Just Eliminate the Sugar(s)?
First – How? Second – Would it work?

14 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,  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.

15 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

16 Idiopathic thrombo-cytopenic purpura (ITP)
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 Primary immunodeficiencies Kawasaki disease Chronic inflammatory demyelinating polyneuropathy Kidney transplant* Idiopathic thrombo-cytopenic purpura (ITP) Kawasaki Disease Autoimmune disease It is a safe (but expensive!) immunosuppressive therapy

17 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 The Future; over 30-off label uses and clinical trials including: Unexplained recurring miscarriage Autism Alzheimer’s disease Chronic fatigue syndrome

18 (many copies are not active)
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

19 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 far-reaching influences on industry” Optimal and consistent protein glycosylation in mammalian cell culture (Glycobiology, 2009)

20 Sub-optimal glycosylation Post-synthetic modification
Back to IVIg (and rProteins in General*) Poor glycosylation compromises safety, pharmacokinetics, and activity Sub-optimal glycosylation The “solution” But getting there is complex!! Cell / genetic engineering Cell culture variables Current solutions Post-synthetic modification Optimal and consistent protein glycosylation in mammalian cell culture (Glycobiology, 2009)

21 Cell (Genetic Engineering) Modulation of Glycan Production
Goal: increase sialylation Cell / genetic engineering

22 OK, That Sounds Easy Enough, Let’s GE in a ST!
Goal: increase sialylation ST6GALNAC6 ST6GALNAC5 ST6GALNAC4 ST6GALNAC3 ST8SIA1 ST8SIA5 ST3GAL1 ST3GAL2 ST3GAL4 ST3GAL5 ST3GAL6 ST6GALNAC2 ST6GALNAC1 ST6GAL2 ST8SIA3 ST8SIA4 ST8SIA2 ST3GAL3 CMP CMPNT a2,8- a2,3- a2,6- NEU1 NEU2 NEU3 NEU4 Sialoglycoconjugate production Glycan recycling ST6GAL1 ST8SIA6 Golgi KL But, which one? CMP-Neu5Ac

23 Genetically Engineering Glycosylation is NOT Easy
Keeping in mind that glycosylation is actually 10x-fold more complex 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

24 OK, Let’s Try Something Else – “Cell Culture Variables”
or CMP-sialic acid Cell / genetic engineering Cell culture variables e.g., NH3

25 Going Back to the “Sialyltransferase” (ST) Schematic
Goal: increase sialylation ST6GALNAC6 ST6GALNAC5 ST6GALNAC4 ST6GALNAC3 ST8SIA1 ST8SIA5 ST3GAL1 ST3GAL2 ST3GAL4 ST3GAL5 ST3GAL6 ST6GALNAC2 ST6GALNAC1 ST6GAL2 ST8SIA3 ST8SIA4 ST8SIA2 ST3GAL3 CMP CMPNT a2,8- a2,3- a2,6- NEU1 NEU2 NEU3 NEU4 Sialoglycoconjugate production Glycan recycling ST6GAL1 ST8SIA6 Golgi KL But that paradigm is being disproved . . . CMP-Neu5Ac generally has been presumed NOT to regulate ST activity CMP-Neu5Ac

26 Glycoengineered rProtein
Checking Back in on Glycoengineering Options Glycoengineering holds promise to improve safety/efficacy of rProteins rProtein Glycoengineered rProtein Goal: increase sialylation Approach Simple Low cost Versatile Effective Post-synthetic modification X ? Cell/genetic engineering X ? Cell culture variables ? e.g., media supplementation to increase CMP-sialic acid levels

27 increase in sialylation
ManNAc is the Feedstock for Sialic Acid Production X ManNAc Natural ManNAc N/A 5-10% increase in sialylation Note that increased sialic acid is almost always desirable, an exception is for ADC cancer therapies (e.g., when immune-stimulation is required). Yorke, S.C. The application of N-acetylmannosamine to the mammalian cell culture production of recombinant human glycoproteins. Chemistry in New Zealand January 2013 issue, (2013). Increased SA = improved activity Goal: increase sialylation Low sialic acid = Poor activity The application of N-acetylmannosamine to the mammalian cell culture production of recombinant human glycoproteins

28 increase in sialylation
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 $ million 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 Note that increased sialic acid is almost always desirable, an exception is for ADC cancer therapies (e.g., when immune-stimulation is required). Yorke, S.C. The application of N-acetylmannosamine to the mammalian cell culture production of recombinant human glycoproteins. Chemistry in New Zealand January 2013 issue, (2013). 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!) The application of N-acetylmannosamine to the mammalian cell culture production of recombinant human glycoproteins

29 X X Towards a Solution: 2nd Generation ManNAc Analogs Natural ManNAc
Ac4ManNAc 10-25% increase in SA N/A Ac4ManNAc Jones, M.B., Teng, H., Rhee, J.K., Baskaran, G., Lahar, N. & Yarema, K.J. Characterization of the cellular uptake and metabolic conversion of acetylated N-acetylmannosamine (ManNAc) analogues to sialic acids. Biotechnol Bioeng 85, (2004). Kim, E.J., Sampathkumar, S.-G., Jones, M.B., Rhee, J.K., Baskaran, G. & Yarema, K.J. Characterization of the metabolic flux and apoptotic effects of O-hydroxyl- and N-acetylmannosamine (ManNAc) analogs in Jurkat (human T-lymphoma-derived) cells. J Biol Chem 279, (2004). Kim, E.J., Jones, M.B., Rhee, J.K., Sampathkumar, S.-G. & Yarema, K.J. Establishment of N-acetylmannosamine (ManNAc) analogue-resistant cell lines as improved hosts for sialic acid engineering applications. Biotechnol Prog 20, (2004). Goal: increase sialylation Low sialic acid = Poor activity Increased SA – improved activity Refer to “notes” for references for Ac4ManNAc efficiency and cytotoxicity

30 “Whole molecule” activities
Towards a Solution – Separating Flux & Toxicity X Natural ManNAc N/A X Ac4ManNAc N/A 3,4,6-O-Bu3ManNAc “Whole molecule” activities (a platform for drug development) Refs 4-9 Ac4ManNAc Kim, E.J., Sampathkumar, S.-G., Jones, M.B., Rhee, J.K., Baskaran, G. & Yarema, K.J. Characterization of the metabolic flux and apoptotic effects of O-hydroxyl- and N-acetylmannosamine (ManNAc) analogs in Jurkat (human T-lymphoma-derived) cells. J Biol Chem 279, (2004). 2. Sampathkumar, S.-G., Campbell, C.T., Weier, C. & Yarema, K.J. Short-chain fatty acid-hexosamine cancer prodrugs: The sugar matters! Drug Future 31, (2006). 3. Sampathkumar, S.-G., Jones, M.B., Meledeo, M.A., Campbell, C.T., Choi, S.S., Hida, K., Gomutputra, P., Sheh, A., Gilmartin, T., Head, S.R. & Yarema, K.J. Targeting glycosylation pathways and the cell cycle: sugar- dependent activity of butyrate-carbohydrate cancer prodrugs. Chem Biol 13, (2006). 4. Aich, U., Campbell, C.T., Elmouelhi, N., Weier, C.A., Sampathkumar, S.G., Choi, S.S. & Yarema, K.J. Regioisomeric SCFA attachment to hexosamines separates metabolic flux from cytotoxicity and MUC1 suppression. ACS Chem Biol 3, (2008). 5. Campbell, C.T., Aich, U., Weier, C.A., Wang, J.J., Choi, S.S., Wen, M.M., Maisel, K., Sampathkumar, S.-G. & Yarema, K.J. Targeting pro-invasive oncogenes with short chain fatty acid-hexosamine analogues inhibits the mobility of metastatic MDA-MB-231 breast cancer cells. J Med Chem 51, (2008). 6. Elmouelhi, N., Aich, U., Paruchuri, V.D.P., Meledeo, M.A., Campbell, C.T., Wang, J.J., Srinivas, R., Khanna, H.S. & Yarema, K.J. Hexosamine template. A platform for modulating gene expression and for sugar-based drug discovery. J Med Chem 52, 2515–2530 (2009). 7. Wang, Z., Du, J., Che, P.-L., Meledeo, M.A. & Yarema, K.J. Hexosamine analogs: from metabolic glycoengineering to drug discovery. Curr Opin Chem Biol 13, (2009). 8. Coburn, J.M., Bernstein, N., Bhattacharya, R., Aich, U., Yarema, K.J. & Elisseeff, J.H. Differential response of chondrocytes and chondrogenic-induced mesenchymal stem cells to C1-OH tributanoylated N-acetylhexosamines. PLoS ONE 8(3), e58899 (doi: /journal.pone ) (2013). 9. Coburn, J.M., Wo, L., Berstein, N., Aich, U., Bhattacharya, R., Bingham III, C.O., Yarema, K.J. & Elisseeff, J.H. Short-chain fatty acid modified hexosamine analogs for tissue engineering osteoarthritic cartilage. Tissue Eng Part A Epub ahead of print (2 May 2013), doi: /ten.TEA (2013). 10. Almaraz, R.T., Aich, U., Khanna, H.S., Tan, E., Bhattacharya, R., Shah, S. & Yarema, K.J. Metabolic oligosaccharide engineering with N-acyl functionalized ManNAc analogues: cytotoxicity, metabolic flux, and glycan-display considerations. Biotechnol Bioeng 109, (2012). 11. Almaraz, R.T., Tian, Y., Bhattarcharya, R., Tan, E., Chen, S.-H., Dallas, M.R., Chen, L., Zhang, Z., Zhang, H., Konstantopoulos, K. & Yarema, K.J. Metabolic flux increases glycoprotein sialylation: implications for cell adhesion and cancer metastasis. Mol Cell Proteomics, /mcp.M (2012). Bu4ManNAc Complex activities (higher flux, enhanced toxicity) Refs 1-3 (in notes) 1,3,4-O-Bu3ManNAc The Solution (next slides) Refs 4, 10, 11

31 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 1,3,4-O-Bu3ManNAc

32 A Closer Look - Cost 1,3,4-O-Bu3ManNAc “Ballpark” estimates for a 15,000 L/30,000 g bioreactor run $ million ManNAc $24-120K Ac4ManNAc 1,3,4-O-Bu3ManNAc $6-75K

33 A Closer Look - Versatility
1,3,4-O-Bu3ManNAc 1,3,4-O-Bu3ManNAc adds sialic acid adds Branches (Refs 1,2) 1,3,4-O-Bu3ManN(R) R = >25 functional groups adds chemical FGs (Refs 3,4) (scientific basis for link between increased GlcNAc flux and N-glycan branching): Lau, K.S., Partridge, E.A., Grigorian, A., Silvescu, C.I., Reinhold, V.N., Demetriou, M. & Dennis, J.W. Complex N-glycan number and degree of branching cooperate to regulate cell proliferation and differentiation. Cell 129, (2007). (biotechnology/glycoengineering relevance – isofar as increased branching was pursued through an enzyme expression approach): Schneider, J.D., Castilho, A., Neumann, L., Altmann, F., Loos, A., Kannan, L., Mor, T.S. & Steinkellner, H. Expression of human butyrylcholinesterase with an engineered glycosylation profile resembling the plasma-derived orthologue. Biotechnology Journal, October 16, 2013 doi: /biot [Epub ahead of print] (2013). 3. Okeley, N.M., Toki, B.E., Zhang, X., Jeffrey, S.C., Burke, P.J., Alley, S.C. & Senter, P.D. Metabolic engineering of monoclonal antibody carbohydrates for antibody−drug conjugation. Bioconjug Chem 24, (2013). 4. Almaraz, R.T., Aich, U., Khanna, H.S., Tan, E., Bhattacharya, R., Shah, S. & Yarema, K.J. Metabolic oligosaccharide engineering with N-acyl functionalized ManNAc analogues: cytotoxicity, metabolic flux, and glycan-display considerations. Biotechnol Bioeng 109, (2012). 1,3,4-O-Bu3Glc/GalNAc

34 Illustrating Versatility (and Effectiveness) with EPO
Optimally sialylated EPO has longer serum half-life 1,3,4-O-Bu3Glc/GalNAc Erythropoietin (EPO) ($9 billion market) Increased branching 1,3,4-O-Bu3ManNAc Increased sialic acid Serum ½ life: ≤14 SAs = 8.5 hours ~22 SAs = 25.3 hours Even better w/ non-natural sialic acid 1,3,4-O-Bu3ManN(R) R = >25 functional groups (Refs 1-3)

35 increase in sialylation
A Closer Look - Effectiveness 1,3,4-O-Bu3ManNAc ~75% (“global”) increase in sialylation > 80 proteins from a glycoproteomics analysis of SW1990 cells Relative S.A. in treated cells (fold increase cf. controls) i.e., ~175% is the “average” Individual glyco-proteins experience a considerably larger increase in S.A. Almaraz, R.T., Tian, Y., Bhattarcharya, R., Tan, E., Chen, S.-H., Dallas, M.R., Chen, L., Zhang, Z., Zhang, H., Konstantopoulos, K. & Yarema, K.J. Metabolic flux increases glycoprotein sialylation: implications for cell adhesion and cancer metastasis. Mol Cell Proteomics, /mcp.M (2012). Metabolic flux increases glycoprotein sialylation . . .(2012)

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

37 Sub-optimal glycosylation Post-synthetic modification
To Summarize Recombinant Protein Glycoengineering Poor glycosylation compromises safety, pharmacokinetics, and activity Sub-optimal glycosylation The “solution” But getting there is complex!! Cell / genetic engineering Cell culture variables Current solutions Post-synthetic modification Optimal and consistent protein glycosylation in mammalian cell culture (Glycobiology, 2009)

38 An Overview of Today’s Lecture
First – What is Carbohydrate Engineering? Sugars are critical for $250 billion $$s worth of drugs Next Organ Transplantation Metabolic Oligosaccharide Engineering

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

40 One Option – Tissue Engineering
the creation of new tissues or organs in the laboratory to replace diseased, worn out, or injured body parts

41 A Second Option – Xenotransplantion
Baby Fae – recipient of a baboon heart (ca. 1984) Ultimately unsuccessful, spawned a backlash based (in part) on ethical concerns

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

43 Xenotransplants – Overcoming Hyperacute Rejection
1 What is the cause of hyperacute rejection? (From Nature Biotechnology, March 2002 Volume 20 Number 3 pp 231 - 232)

44 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-a-1,3-Gal, to graft blood vessels, activating large amounts of complement.

45 Remember that “a-Gal” is a Trisaccharide
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)

46 Strategies to Overcome Hyperacute Rejection
Strategy #1. Can soluble a-Gal protect against hyperacute rejection? soluble aGal 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

47 X Strategy #2 – Knockout the a1,3GT Gene aGal
a1,3-galactosyltransferase (a1,3GT) aGal Three key technologies were required that were falling into place in the 1990s Identification of the a1,3-galactosyltransferase gene (genetics/bioinformatics) homologous recombination of the target genes (molecular/cell biology) adaptation of nuclear transfer technology to pigs (large animal genetics)

48 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 Identification of the a1,3-galactosyltransferase gene (genetics/bioinformatics) homologous recombination of the target genes (molecular/cell biology) 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

49 Step 2. A Lot of Really Complex Genetic Manipulation!
Three key technologies were required that were falling into place in the 1990s Identification of the a1,3-galactosyltransferase gene (genetics/bioinformatics) homologous recombination of the target genes (molecular/cell biology) adaptation of nuclear transfer technology to pigs (large animal genetics) Strategy #2. “Knocking out” the a-Gal epitope 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)

50 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 Identification of the a1,3-galactosyltransferase gene (genetics/bioinformatics) homologous recombination of the target genes (molecular/cell biology) 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)

51 The First “a-Gal” Knockout Pigs were Born Xmas Day, 2002
But, only one allele was knocked out!! a1,3GT expression was still possible from the copy of the gene on the non-knocked out allele 2 Figure 3: Five a1,3GT gene knockout piglets at 2 weeks of age. Solution: Breeding experiments, expected progeny: +/+, +/−, and −/− at a 1:2:1 ratio

52 Wrapping up the “Loose Ends” (and new pitfalls)
Phelps et al, Science (2003) 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! 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 aGal knockout pigs needed special care due to concerns about a1,3-Galactosyltransferase knockout pigs are available for xenotrans-plantation: But, are glycosyltransferases still relevant? Uh oh – the double null animals still expressed aGal !! (albeit at a lower level, and only on glycolipids)

53 The pig genome was not sequenced until 2010
Wrapping up the “Loose Ends” (and new pitfalls) Strategy #2. “Knocking out” the a-Gal epitope (ca ) 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

54 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 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. From Revivicor’s website:

55 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:

56 Back to the Overview of Today’s Lecture
First – What is Carbohydrate Engineering? Sugars are critical for $250 billion $$s worth of drugs Finally Organ Transplantation Metabolic Oligosaccharide Engineering

57 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, /mcp.M

58 cell surface oligosaccharides
Moving from the Rate to the Type of Flux Naturally-occurring cell surface oligosaccharides Exogenous (e.g., dietary) sugars 1 Neu5Ac R1 Glycosylation pathways R1 ManNAc R1 = 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

59 Is “Metabolic Glycoengineering” Useful?
Exogenous (e.g., dietary) sugars 1 Neu5Ac R1 ManNAc R1 Glycosylation pathways R1 = Werner Reutter’s Laboratory Keppler et al, Glycobiology 2001

60 cell surface oligosaccharides
Soon “Chemical Biologists” Dominated the Field Naturally-occurring cell surface oligosaccharides Exogenous (e.g., dietary) sugars 1 Neu5Ac R1 ManNAc R1 Glycosylation pathways R1 = Werner Reutter’s Laboratory R1 = Carolyn Bertozzi’s Group The ketone group Mahal et al, Science, 1997 The azide and alkyne, the reaction partners for ‘click chemistry’

61 cycloaddition reaction
Click Chemistry – 1,530,000 Google Entries! * Applied to metabolic glycoengineering Ac4ManNAz Sialic acid pathway Cu(I) Copper catalyzed [3+2] cycloaddition reaction (aka “click chemistry”) Sia5Az Saxon & Bertozzi, Science, 2000 *That was in 2007 17,300,000 in 2009 32,200,000 in 2010 539,000,000 in 2013

62 cycloaddition reaction
This Technology can be used as a Glycoproteomics Tools Sialic acid pathway Ac4ManNAz Cu(I) Copper catalyzed [3+2] cycloaddition reaction (aka “click chemistry”) Sia5Az Saxon & Bertozzi, Science, 2000 *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)

63 Strain-promoted [3+2] cycloaddition** cycloaddition reaction
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. Sialic acid pathway Ac4ManNAz Strain-promoted [3+2] cycloaddition** Sia5Az Agard et al, JACS, 2004 *The copper catalyst is toxic * Cu(I) Copper catalyzed [3+2] cycloaddition reaction (aka “click chemistry”) Sia5Az Saxon & Bertozzi, Science, 2000 **Copper-free “click reactions” can now be done in living cells and in vivo. **

64 Additional Pathways (beyond Sialic Acid) can be Targeted
Cell-surface glycans shine in this microscopy image of the head of a three-day-old zebrafish embryo treated with the new technique. In addition to cell surface sialic acid, metabolic glycoengineering now can target cell surface GalNAc and fucose (GlcNAc analogs mainly label intracellular “O-GlcNAc” )

65 For Example, Remember “Fucose” ?
1,3,4-O-Bu3ManN(R) R = >25 functional groups adds chemical FGs (Refs 3,4) Additional “twists” Works on secreted proteins New bioorthogonal chemistry This actually *should* have been fucose! (from earlier in today’s lecture)

66 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

67 OK – Finally – about those 25 “R” Groups . . . . .
Almaraz et al, Ann Biomed Eng, 2012

68 Where does Metabolic Oligosaccharide Engineering Go Next?
To “The Clinic” ?? From “Chemical Biology”

69 An novel scaffold for drug design
In the Bigger Picture, Progress Continues Commercialization and translational efforts were slow to be realized: The Bittersweet Promise of Glycobiology Nature Biotechnology, 2001 (doi: /nbt ) The Sweet and Sour of Cancer: Glycans as Novel Therapeutic Targets Nature Reviews Cancer, 2005 (doi: /nrc1649) By 2008 “we” had learned a valuable “first do no harm” lesson In the past decade, progress has accelerated: 2003: 2006: 1,3,4-O-Bu3ManNAc 2008: Our Technology An novel scaffold for drug design (over 100,000 permutations)

70 Back to the Overview of Today’s Lecture – All Done!
First – What is Carbohydrate Engineering? Sugars are critical for $250 billion $$s worth of drugs Organ Transplantation Metabolic Oligosaccharide Engineering

71

72


Download ppt "Carbohydrate Engineering"

Similar presentations


Ads by Google