“God could cause us considerable embarrassment by revealing all the secrets of nature to us:

1. “God could cause us considerable embarrassment by revealing all the secrets of nature to us: we should not know what to do for sheer apathy and boredom.” -- Johann Wolfgang von Goethe

2. Systems Biology of Osmotic Shock in Antibody Producing Cell Lines Candidacy Proposal Thomas R. Kiehl NSF Graduate Research Fellow, Multidisciplinary Science Ph.D. Program

3. What is an Antibody? • Antibodies are an important component of the body’s natural defenses. • These glycoproteins recognize foreign substances and tag them for remediation by other parts of the immune system. • mAb’s are an effective part of a growing number of medical treatments, lab techniques, diagnostics and imaging. Image source: Wikipedia

4. Roche buys antibody technology company for $56.6 mln, Apr 2,2007 Monday (Roche) said it has acquired privately-held Therapeutic Human Polyclonals Inc, an emerging biotechnology company focused on research in human antibody technologies, for $56.6 million in cash. ZURICH (MarketWatch) -- Swiss drugmaker Roche Holding AG (RHHBY) Monday said it has acquired privately-held Therapeutic Human Polyclonals Inc, an emerging biotechnology company focused on research in human antibody technologies, for $56.6 million in cash. Roche, based in Basel, said it plans to fully integrate THP, which is based in Germany and the U.S., into its protein research center in Penzberg, Germany. "We are delighted about this acquisition as it builds on our strength in therapeutic antibodies," said Jonathan Knowles, head of global research at Roche. The development of therapeutic proteins and antibodies is an important area of research for the company, Roche said. At 0826 GMT, Roche shares were CHF1.80, or 0.8% higher, at CHF216.80, in a slightly lower broader market. THP focuses on research in the field of human antibody technologies, where drugs made out of antibodies fight infectious agents, including bacteria and viruses, by seeking them out and helping the body to destroy them. THP says it has developed a unique transgenic mammalian platform to create human antibodies. The technology will enable the generation of both monoclonal and polyclonal antibody drugs with enhanced efficacy, Roche said. Monoclonal antibodies are identical because they were produced by one type of immune cell and are all clones of a single parent cell. "Improved monoclonal antibody companies are hot commodities," said Denise Anderson, pharmaceutical analyst in Zurich with broker Kepler Equities, who has a buy rating on the stock, pointing to a string of deals over the past twelve months. Roche itself paid $181 million last year to acquire GlycArt Biotechnology AG of Zurich, which also had a crop of early-stage antibodies. In May, Merck & Co. (MRK) agreed to pay a combined $480 million to acquire Abmaxis and GlycoFi, two biotechnology firms that brought the drug maker new methods to discover and produce drugs. Merck, based in Whitehouse Station, N.J., isn't affiliated with its German namesake. Also in the second quarter of 2006, Pfizer inc. (PFE) acquired Bioren, a small specialist in the discovery of monoclonal antibodies. "We think the deal makes good strategic sense for Roche, where top drugs Rituxan, Herceptin and Avastin are all antibodies, Anderson said. At a time when many traditional drugs made from small molecules are facing the loss of patent protection, medicines made out of large proteins are still protected from this threat not only because they've only entered the market over the past decade but also because they are more complex to imitate. Roche itself paid $181 million last year to acquire GlycArt Biotechnology AG of Zurich, which also had a crop of early-stage antibodies. In May, Merck & Co. (MRK) agreed to pay a combined $480 million to acquire Abmaxis and GlycoFi, two biotechnology firms that brought the drug maker new methods to discover and produce drugs. Also in the second quarter of 2006, Pfizer inc. (PFE) acquired Bioren, a small specialist in the discovery of monoclonal antibodies.

5. 2005 Market, $13 Billion • ½ of that from just two drugs • Rituxan ($3.3Bn) – non-Hodgkin’s Lymphoma (CD20) • Remicade ($3.4Bn) - rheumatoid arthritis (TNF-α) • 17 therapeutic monoclonal antibodies have received FDA approval and are on the market in the U.S. • Several antibodies have been approved for use in diagnostic imaging applications. • Report does not mention BMS’ Abatacept which is a fusion protein composed of an immunoglobulin fused to the extracellular domain of CTLA-4 (Sales for the second quarter of 2006 were $18 million, sales could reach US$ 1 billion by 2009/2010, ) Market Report: Monoclonal Antibodies: From Magic Bullets to Successful Drugs Abatacept: Nature Reviews Drug Discovery 5, 185-186 (March 2006)

6. Herceptin, A prototypical Antibody Therapeutic • This mAb targets a receptor which is over expressed in certain breast cancers (Bange 2001, Sliwkowski 1999). • Herceptin targets the epidermal growth factor receptor, HER2, which is part of the ErbB family of tyrosine kinases. • This targeting results in cell cycle arrest and suppression of tumor growth.

7. Systems Biology of Osmotic Shock in Antibody Producing Cell Lines Candidacy Proposal Thomas R. Kiehl NSF Graduate Research Fellow, Multidisciplinary Science Ph.D. Program

8. How do you make mAb’s? • In 1975 Köhler and Milstein first developed cell lines which could reliably produce monoclonal antibodies • These cell lines, known as hybridomas, were a fusion of an antibody-secreting murine lymphocyte cell with an murine myleoma cell. • From this process emerges an immortalized cell line which secretes identical antibodies that have been raised against a specific antigen.

9. Systems Biology of Osmotic Shock in Antibody Producing Cell Lines Candidacy Proposal Thomas R. Kiehl NSF Graduate Research Fellow, Multidisciplinary Science Ph.D. Program

10. Why Osmotic Shock? • Osmotic stress as well as a number of other stresses can increase the antibody production rates of a culture • Just add NaCl. Sun, Z., Zhou, R., Liang, S., McNeeley, K.M., Sharfstein, S.T. (2004) Biotechnology Progress. 20, 576-589 Ozturk, S.S., Palsson, B.O. (1991) Biotechnology and Bioengineering, Vol. 37, Pp. 989-993

11. Is it really that easy? • Higher osmolarities negatively impact viable cell concentration. Sun, Z., Zhou, R., Liang, S., McNeeley, K.M., Sharfstein, S.T. (2004) Biotechnology Progress. 20, 576-589 Ozturk, S.S., Palsson, B.O. (1991) Biotechnology and Bioengineering, Vol. 37, Pp. 989-993

12. So, just shock them a little. Right? • In fed-batch cultures osmolarity becomes problematic both due to the addition of nutrients as well as the production of waste products, primarily lactic acid. • Lactic acid acidifies the culture, necessitating the addition of base to control the pH. • Over the course of a fed-batch culture the osmolarity can increase from ~290mOsm/kg to 500mOsm/kg (Zhu 2005). • Viability can be reduced by as much as 50% (Kurano 1990).

13. Systems Biology of Osmotic Shock in Antibody Producing Cell Lines Candidacy Proposal Thomas R. Kiehl NSF Graduate Research Fellow, Multidisciplinary Science Ph.D. Program

14. Systems Biology “I am a Biologist, and I work on systems. I guess that makes me a Systems Biologist.” -Howard Berg, ICSB 2005

15. Systems Biology “To understand biology at the system level, we must examine the structure and dynamics of cellular and organismal function, rather than the characteristics of isolated parts of a cell or organism. Properties of systems, such as robustness, emerge as central issues, and understanding these properties may have an impact on the future of medicine.” – Hiroaki Kitano Kitano, H. (2002), Systems Biology: a brief overview, Science, 295:1662-1664

16. 3 C’s of Systems Biology • Complexity • Computation • Cross-Disciplinary Cooperation

17. Systems Biology Lab Experiment(s) Refine model In-Silico Experiment(s)

18. Systems Biology of Osmotic Shock in Antibody Producing Cell Lines Candidacy Proposal Thomas R. Kiehl NSF Graduate Research Fellow, Multidisciplinary Science Ph.D. Program

19. Objective • Engineer mammalian cells for optimal recombinant protein production. • To build a model of the cellular response to osmotic shock. • Characterize the response in terms of some specific components.

20. Overview • Mammalian Pathway • Yeast Model • Model Scope • Sample Model • TonEBP/NFAT5/OREBP • Experimental Plan & Preliminary Results • Related Efforts • Batch Culture Model • Microarrays • CoEPrA • Evolutionary Computing

21. Mammalian Pathway Dmitrieva, N. I., M. B. Burg, et al. (2005). "DNA damage and osmotic regulation in the kidney" Am J Physiol Renal Physiol289(1): F2-7.

22. Yeast Osmostress Signalling

23. Simulating Yeast Response to Osmotic Shock Klipp, E., B. Nordlander, et al. (2005). "Integrative model of the response of yeast to osmotic shock." Nature Biotechnology23(8): 975-982.

24. Yeast Model • The ODEs in Klipp’s model generally take the form of equation 4. In this formulation m is the number of biochemical species, r is the number of reactions each with a rate v and stoichiometry n. This equation governs how the concentration of each species evolves over time.

25. Yeast Output

26. Yeast Model • Klipp showed that the pathway can be activated again by an additional shock. • They also showed that this reactivation would not be possible if the pathway were structured such that the phosphatases provided the primary feedback control. • They demonstrated that the gene transcripts for phosphatases should not increase by more than two-fold.

27. Mammalian Pathway Dmitrieva, N. I., M. B. Burg, et al. (2005). "DNA damage and osmotic regulation in the kidney" Am J Physiol Renal Physiol289(1): F2-7.

28. Model Scope An initial model will capture three main concepts. • The insult of osmolarity within the context of the cell culture life-cycle • The dependence of TonEBP activation on osmolarity • TonEBP-dependant osmolyte accumulation. Osmolyte Accumulation Osmolarity TonEBP

29. Refined Objective • Experimentally demonstrate the central role of NFAT5 in our cell lines the cellular osmotic response. • Build a model to characterize that role • What portion of the osmotic response can be accounted for solely by TonEBP? • Are other factors or feedback loops required to explain observed dynamics?

30. Toward a simplified model Osmolyte Accumulation Osmolarity TonEBP Dmitrieva, N. I., M. B. Burg, et al. (2005). "DNA damage and osmotic regulation in the kidney" Am J Physiol Renal Physiol289(1): F2-7.

31. Osmolarity • This is the primary independent variable in the system • Could be modeled in terms of a rapidly decreasing osmotic gradient • Could be kept at a constant • Could be modeled as a slowly increasing quantity. Osmolyte Accumulation Osmolarity TonEBP

32. TonEBP • First dependant variable, primarily dependant on the osmolarity • Goal is to fit this quantity to experimental data Osmolyte Accumulation Osmolarity TonEBP

33. Osmolyte Accumulation • We presume that osmolyte accumulation is dependant on TonEBP activation • We’ll use a proxy of cell volume initially. Osmolyte Accumulation Osmolarity TonEBP

34. Basic Model (a) (b) (c) • O, the osmotic gradient. The kinetic constant, kO, governs the rapid equilibration of this gradient immediately after the osmotic shock. • N, amount of activated transcription factor • P, the amount of accumulated osmoprotectants. • k1relates the activation of TonEBP to the osmolarity (O). • k2is a decay rate for activated TonEBP • k3 relates TonEBP activation to osmolyte accumulation

35. Model Output • Osmotic gradient, blue. • Level of activated NFAT5, red. • Accumulation of osmolytes, green

36. Iterate on the model • Generally fits with what we expect • Missing some important features • Must relate the model to actual data. Osmolyte Accumulation Osmolarity TonEBP

37. Experimental Plans, Initial Data • Osmotic stress protocol • Quantify TonEBP • Quantify Cell Volume • Other experimental possibilities

38. Osmostress Experiment • Stress cells with 100mOsm increase • Sample Cells at • Pre-stress Control • Post-stress 5, 10, 15, 30, 60 & 120 min • For western blot: • Lyse in SDS and shear DNA • Use lysate in chemoluminescent or fluorescent western blot.

39. NFAT5 DNA Binding • Consensus Sequence • TGGAAANN(C/T)N(C/T) [1] • N = any nucleotide • C/T = any pyrimidine • NFAT Family, but similar to an NF-kB 1) Miyakawa H, Woo S K, Dahl S C, Handler J S, Kwon H M. Proc Natl Acad Sci USA. 1999;96:2538–2542. [PubMed] 2) <image> James C. Stroud et al Nature Structural Biology9, 90 - 94 (2002)

40. About TonEBP • Western blot of TonEBP after 18 hours of incubation in isotonic (I) and hypertonic (H) medium (Miyakawa 1999)

41. About TonEBP • Localization of TonEBP under different mutations of the nuclear location signal (Tong 2006).

42. About TonEBP • Ratio of TonEBP localization after 200, 300 or 500 mosmol solution for 30 minutes (Zhang 2005)

43. TonEBP • We intend to use a chemiluminescent EMSA to watch TonEBP activation over time • Previous work (Stroud 2002, Kojima 2004)

44. Cell Size • Intend to quantify with the FACS machine using forward light scattering techniques Ozturk, S.S., Palsson, B.O. (1991) Biotechnology and Bioengineering, Vol. 37, Pp. 989-993

46. Other measurements • As time allows • Upstream signaling components • Specific osmolyte accumulation • Lactic acid production

47. GPC & Lactate • Glycerophosphocholine and Lactate can both be quantitated by YSI Lactic acid

48. Betaine • Near IR spectroscopy

49. Sorbitol and Inositol • Observe dehydrogenase activity by spectrophotometry • Sorbitol Dehydrogenase and Inositol dehydrogenase respectively

50. Aldose Reductase Activity • Spectrophotometry, absorbtion at 340 (Bagnasco et al., PNAS 84:1718) (JBC 1965 page 877)