Homework 4:  Company Presentations Jump to Company presentation

1) Find a biotech company whose biotechnology impresses you with its ideas, product, or strategy.  Choose a company that you would like to work for, invest in, or follow. Use the Internet: Google, PubMed, etc. 

2) Prepare a 2-minute talk describing why you picked that company

3) At any time up to the presentation, email me a written version of your description, double-spaced and ~< 300 words at lac2. Include at the top: your name, the company name, and the date you sent.  I will publish it below and use it to assign a homework grade (you will not be graded on your actual presentation).

4) Present the strictly-2-minute talk in class, with a 1 minute discussion (1-2  questions)

Do not choose a huge pharmaceutical company (e.g., Merck).  I have prepared a list of names (not links) of about 80 companies to help. Some are very small and not yet well established.  Some may no longer exist.  The list is no indicator of the quality of the company. You are not at all be limited by this list. You can also search for "lists of biotechnology companies" on Google.

Submit your company by email to lac2.

Last updatedThursday December 23, 2004 09:32 AM

Presentations:  by name:

By date:

Kathy Jo Carstarphen

November 15, 2004

Intra-Cellular Technologies, Inc.

 Most Central Nervous System (CNS) disease treatments focus on molecular interactions at neuronal cell surfaces. For example, the popular depression drugs, Prozac[1] and Effexor[2], increase levels of the neurotransmitters serotonin and norepinephrine at the synapse; and a common treatment for schizophrenia involves the antagonization of serotonin and dopamine receptors. Intra-cellular Technologies, Inc (ITI) therapies go beyond this common “synapse-focused” approach to pinpoint important downstream pathways of the complex biochemical networks inside nerve cells. Specifically, neuron-specific protein phosphorylation patterns are analyzed to understand CNS pathogenesis and identify targets for development novel drug therapies for CNS diseases[3].

ITI was founded in May 2002 by a team of researchers led by Dr. Paul Greengard, the 2000 co-recipient of the Nobel Prize in Medicine and Physiology "for [his] discoveries concerning signal transduction in the nervous system".[4] ITI commercializes Greengard’s Nobel-prize winning research to identify potential targets, develop novel drugs, and screen drug actions in vivo. This is done with the tools of two unique platforms, the IntraPath and CNSProfile, both developed by ITI. Core therapeutic programs for depression, schizophrenia, and Parkinson’s disease have been developed at ITI through the use of these platforms, but these tools can be applied to any CNS disease.³

The IntraPath technology platform identifies neuron-specific proteins that are involved in CNS signaling and pathogenesis. This is considered the “drug discovery platform” because once the novel proteins are identified; these proteins can be used as targets for drug discovery. IntraPath includes the use of the DARPP-32 knockout mouse[5], developed by ITI scientists, as a unique tool for in vivo screening of drug candidates. DARPP-32 acts as a master switch in CNS neurotransmission by amplifying the cellular response to all neurotransmitters.[6] Without DARPP-32, the neurons are deafened to the synaptic presence of neurotransmitters, which slows the cellular response. This allows ITI to determine the involvement of DARPP-32 on the drug’s intra-cellular pathway in the knockout mice.³

The second unique platform is the CNSProfile which allows for high-throughput screening of drug action. The profile uses highly selective phosphospecific antibodies[7] as probes to generate a “molecular snapshot” of the nerve cell before and after drug treatment. This snapshot indicates the phosphorylated and de-phosphorylated states of the disease relevant phosphoproteins in vivo. This data is used to determine the candidate drugs mode of action, and thereby its potential efficacy. This platform can be used to validate drug candidates for ITI’s biopharmaceutical partners, allowing lead candidate drugs to be screened for their methods of action. It also allows for the assessment of molecular efficacy prior to the clinical trial process. Those drugs with less promising profiles of safety and efficacy can be discarded, thereby preventing the pharmaceutical company from wasting money on the further clinical testing of an unsafe and biochemically ineffective drug.³

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Abgenix advances monoclonal antibody therapy with XenoMouse

Jonathan Wei-Ji Chang
jwc2107@columbia.edu

             Despite an absence of products on the market resulting from XenoMouse, it was the justification behind the $3 billion market capitalization of Abgenix on March 31, 2000 [1].  XenoMouse was the product of a 7-year, $40 million R&D effort aimed at profiting from advances in antibody (Ab) technology.

            The efficacy of monoclonal antibodies (mAbs) derived from mice for human therapy has traditionally been restricted due to immunogenicity.  To resolve this problem chimeric mAbs were developed by genetically combining variable mouse Ab regions with constant human Ab regions [2].  Chimeric mAbs still consisted of 34% mouse protein.  Humanized mAbs reduced this percentage to 5-10% by combining only the complementarity determining regions of the mouse Ab with the human antibody framework [2]. 

            XenoMouse was a breakthrough technology where the antibodies derived contained 100% human protein sequences.  Production of the XenoMouse required 2 main steps: 

1)      Inactivation of the endogenous Ig gene loci by deleting the Jн sequences to halt heavy chain synthesis and Cк sequences to halt light к chain synthesis [2]. 

2)      A majority of the human heavy chain and light к chain loci were cloned into YACs to preserve inherent Ab diversity obtained by VDJ recombination [2].  YAC containing yeast spheroplasts were fused with mice embryonic stem cells and introduced into the mice.  These human Ab producing transgenic mice were bred with the mice containing inactivated heavy and light chains.

      Hybridomas are used to manufacture Abs derived from Xenomouse.  First spleen cells producing Ab from a mouse immunized with a specific antigen are fused to immortal myeloma cells lacking Ab secretion and HGPRT genes [3].  These cells are fused together and subjected to HAT selection where only cells with the HGPRT gene from the spleen cell will survive, and only cells with the malignant potential from myeloma cells will proliferate.  This hybridoma is then selected and cloned in bulk culture to produce massive amounts of mAb.

      Abgenix has entered license collaborations with over 30 pharmaceutical and biotechnology companies including Amgen by providing access to XenoMouse derived Abs in exchange for possible milestone payments and sales royalties [4].

References

[1] R. Dolan, Abgenix and the Xenomouse.  Harvard Business School #9-501-061

[2] X.D. Yang, X.C. Jia, J.R.F. Corvalan, P. Wang, and C.G. Davis, Development of ABX-EGF, a fully human anti-EGF receptor monoclonal antibody, for cancer therapy.  Critical Reviews in Oncology/Hematology 38 (2001), pp. 17-23.

[3] C.A. Janeway, P. Travers, M. Walport, and M. Schlomchik, Immunobiology 5 (2001) Garland Publishing, NY.

[4] http://www.abgenix.com/documents/XenoMouseProfile.pdf Accessed 7 November 2004.

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Lillian Seu

 Archemix Therapeutics: Utilizing aptamer technology as directed therapeutics for chronic and acute disease states.

 Archemix is a biopharmaceutical company founded in May 2001 located in Cambridge, Massachusetts and develops aptamer-based therapeutics and has rights to over 150 issued patents and over 150 pending patent applications relating to aptamers: aptamer compositions, methods of selecting aptamers (SELEX), and methods of using aptamers.

            Aptamers are oligonucleotides that bind to molecular targets in a manner similar to antibodies. They are produced by a chemical in vitro process allowing tightly controlled specificity and affinity, are able to disrupt protein-protein interactions, have demonstrated little or no immunogenicity or toxicity, can be administered subcutaneously, can be chemically synthesized to allow for lower cost and easily scaled production, and can be stored at room temperature as lyophilized powders. The therapeutic efficacy of aptamers lie in their high affinity, high specificity (even when related proteins are as much as 96% identical), ability to disrupt protein-protein interaction, and tunability via the SELEX process and post-SELEX optimization.

Archemix’s patented method of aptamer selection SELEX is an iterative process that selects for apatamers on the basis of binding between a target and nucleic acid molecules and occurs in four steps: pool preparation, selection, amplification (the captured, purified sequences are copied enzymatically and enriched) and aptamer isolation (after 5-15 cycles of the complete process, the library of molecules is reduced from 10¹⁵ of unique sequences to 15 to 60 nucleotides of their core binding domain)

 Aptamers have been identified against numerous target types including growth factors, enzymes, immunoglobulins, receptors, viral proteins and others. Some of the validated therapeutic targets from Archemix are: [1]the antithrombin aptamer generated against thrombin and studied in a variety of animal models of anti-coagulation, [2]a modified DNA-based aptamer to the PDGF B chain (platelet derived growth factor) blocking binding to its cell surface receptor, reducing renal injury in an animal model of glomerulonephritis (blood vessel damage), [3]The TGFß2 (Transforming growth factor-ßs) aptamer that increases the success rate of glaucoma filtration surgery, [4] The L-selectin-specific aptamer that inhibits lymphocyte trafficking to lymph nodes in vivo, [5] The aptamer-elastase chimeric molecule that inhibits neutrophil influx and lung permeability in an animal model of acute lung injury, [6] The aptamer to the C5 protein that prevents the cleavage event of C5 into two active fragments, C5a and C5b and blocks serum-induced hemolysis in vitro, [7] The aptamer selected to KGF (keratinocyte growth factor) which prevents the hyperproliferation of epithelial cells in psoriasis.

 References:

1  Boch, et al. (1992) Nature. 355:564
2  Floege et al. (1999) Am. J. Pathol. 154:169.
3  Cordeiro, et al. (2000) Eye 14: 536-47
4  Hicke et al. (1996) J. Clin. Invest. 98:2688.
5  Bless et al. (1997) Curr. Biol. 7:877.
6  Biesecker et al. (1999) Immunopharm. 42:219.
7  Pagratis, et al. (1997) Nat. Biotech. 15:68.

* http://www.archemix.com/ Accessed 1 November 2004
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Jacob Sherkow
Benitec
November 12, 2004

             RNAi is often hailed as one of the greatest scientific breakthroughs in the past decade. Discovered in 1998 at the Carnegie Institute of Washington, Andrew Fire proved that RNAi was not only gene specific but also extremely powerful: only several molecules per cell were required to make seemingly permanent and heritable genetic knockouts in C. elegans.[1] Since then, a great amount of discussion has been made concerning the technology’s use as a therapeutic tool. Advances have been made, namely the in vivo knockout of Hepatitis C RNA in adult mice, [2] but large scale human therapy has for long remained elusive.

            In 1998, however, stirred by the elegance of RNAi, an Austrailian based company, dubbed Benitec, was born. Originally formed as a licensing agent for Queensland State’s scientific enterprise, the company went public in 2002, holding a patent for an RNAi delivery system in mammalian cells.[3] Since then, the company’s focus on both primary research and licensing strategies have awarded them patents or licenses on various RNAi technologies, from simple expression tag sequencing using complementary RNA stem loops[4] to using a new form of DNA, “minicircles,” to greatly induce expression of sequence specific transcriptional units.[5]

            Perhaps Benitech’s most exciting and novel RNAi discovery is “ddRNAi.” ddRNAi functions on the same mechanism as RNAi: double stranded RNA is delivered in vivo and is cleaved into 21-23 double stranded nucleotide units. These units bind to the complementary RNA and are targeted for degradation, thus halting translation and protein expression. ddRNAi, however, uses already cleaved 21-23 nucleotide strands of DNA.[6] Though the difference may seem insignificant, it is the increasing ease of production and chemical stability of ddRNA[7] that make it a revolution in its own right. Manufacturing, ease of transfection and chemical stability are vital concerns in drug development, and ddRNAi’s superiority over those make it an especially viable delivery system candidate. Benitech’s hold on such technology gives it great worth as both a publicly traded company and as a potential panacea of expression related disease.

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Tricia E. Zubal                Bio W4034                                    November 14, 2004

Biogen/Elan

            Elan Corporation is a neuroscience-based biotechnology company that is focused on developing breakthrough therapies in neurology, autoimmune diseases, and severe pain. Most of its major products in development are in collaboration with other companies. One of its most promising partnerships is with Biogen Idec, the resultant company from the 2003 merger of Biogen Inc., a leading biotechnology company, and IDEC Pharmaceuticals Corporation. On May 25^th of this year, Biogen/Elan announced their intension to file a Biologics License Application (BLA) to the FDA for the approval of Antegren (natalizumab) as a treatment of Relapsing-Remitting Multiple Sclerosis (1). The decision to file a BLA was made after discussion with the FDA of the one-year data from two ongoing two-year clinical trials, AFFIRM and SENTINEL. In June 2004, the FDA designated natalizumab for priority review and accelerated approval for the treatment of MS (1). The BLA application was formally accepted on July 26, 2004 (1).

            Antegren is a monoclonal antibody which binds to the alpha 4 integrin (alpha 4, beta 7, and alpha 4 beta 1) (2). Alpha 4 integrins are adhesion molecules expressed on certain white blood cells involved in mediating inflammation, in particular on T-lymphocytes. By binding to these molecules Antegren prevents alpha 4 integrin from binding to Very Late Antigen 4 (VLA-4). This design allows Antegren to selectively inhibit immune cells from leaving the bloodstream and to prevent these cells from migrating into chronically inflamed tissue, such as the brain in MS, where they may cause or maintain the inflammation. It is the first alpha 4 integrin antagonist in the new selective adhesion molecule (SAM) inhibitor class. Thus, Antegren’s major mechanism of action in MS is to prevent the migration of leukocytes across the blood-brain barrier.

            The novel mechanism of action of Antegren is likely to revolutionize the treatment of MS. Although classified as a chronic recurrent inflammatory disorder of the central nervous system, the fundamental cause of the disease has yet to be fully elucidated. However, scientific advances over the past decade have greatly improved our understanding of the general pathogenesis and epidemiology of MS. Antegren is the first of many knowledge-based biotechnology products in the pipeline for MS to be submitted for approval. On November 8^th, Biogen/Elan released data showing that Antegren is capable of reducing relapse rate by over 66% (1). The other drugs on the market for MS (Betaseron, Avonex, Rebif, and Copaxone) have only been shown to prevent relapses in 40% of MS patients on average (3). In addition, in stark contrast to the other MS drugs, Antegren was very well-tolerated, with the most common side-effects being headache and fatigue. These dramatic results have set up Biogen/Elan to be on the forefront of an intense battle to be a first line therapy for MS. Finally, Antegren is also being developed as a potential combination therapy with the IFN-β drugs already on the market, particularly Avonex which is produced by Biogen (1). Biogen/Elan expect Antegren to reach the market in early 2005 (3).

1. www.elan.com/news

2. Miller DH, Khan OA, Sheremata WA, et al. A controlled trial of natalizumab for relapsing multiple sclerosis. The New England Journal of Medicine 2003; 348(1): 15-23.

3. ING April 2004 Pharmaceuticals report: Multiple Sclerosis Market.

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Genitope Corporation’s MyVax:      A Personalized Treatment for B-Cell Lymphoma

Samuel Globus        November 16, 2004

 The Genitope Corporation in Redwood City, CA is currently developing MyVax, a personalized immunotherapy for non-Hodgkin’s B-cell lymphoma. [1] If successful, this treatment will become the first customized drug or therapy approved by the FDA. This ‘active immunotherapy’ trains the body’s immune system to help the patient fight against relapse. When successful, the treatment can result in a strong, lasting immune response directed specifically at cancerous cells, while leaving normal cells unaffected.

The treatment relies on the clonal nature of cancer and the presence of immunoglobulin protein (Ig) on the B-cell’s surface. Ig makes an ideal target for such a therapy because a unique form of the protein is expressed in a very large quantity (10⁵ copies) on the cell surface. With all cancerous cell arising from one original cell, the Ig variable region will be identical on all affected cells and can thus act as a marker for the immune system to target. MyVax treatment begins with RNA isolation from a patient biopsy. PCR and other molecular biology techniques are then used to isolate and clone the sequence of the Ig variable region being expressed in the tumor cells. The variable region is cloned into a human antibody constant region and introduced into to a proprietary mammalian cell line. Genitope’s Hi-GET cell expression technology allows for quick amplification and subsequent isolation of a cell line that expresses the tumor-derived antibody for production of the patient vaccine. The required amount of expressed antibody is purified and subsequently linked to the highly immunologic carrier protein, KLH. The entire process takes less than months.

After chemotherapy treatment, the patient receives a series of injections containing the antibody-KLH conjugate along with GM-CSF adjuvant. The foreign KLH elicits an immune response and the conjugated antibody is processed alongside it. Thus, the immune system is trained to recognize the Ig variable region as foreign and marks cancer cells with it on their surface for removal on future encounters. Genitope has just completed enrollment for its Phase III trials for MyVax and the preliminary results should be available in the middle of 2005. In the phase II trials, approximately 50% of the patients exhibited a specific immune response to the vaccine. Patients with such a response exhibited a drastic increase in time to progression and overall survival. [2]\

 References:

1. http://www.genitope.com
    

2. Hsu et al., Blood, 89(9), 1997, p. 3129-35

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Christina Vullo                                                                                      November 16, 2004

 DeCODE Genetics

             DeCode Genetics is a biopharmaceutical company founded in 1996 when Dr. Kari Stefansson of Harvard University decided to apply his novel population genetics plan to his home country of Iceland for the development of drugs for common diseases.  This approach exploits three valuable avenues of research not previously combined in one study: detailed genetic, medical, and comprehensive genealogical records of an entire population.  “The Iceland Project” enrolled over 50% of Iceland’s small, homogeneous adult population.  Detailed genealogical data collected effectively links the entire present-day population and stretches back over 1000 years.  This has allowed researchers to trace the inherited components of common diseases and pinpoint key disease genes and specific markers within these genes.

            DeCODE’s genealogy database clusters patients of a disease into large extended family trees and applies high-throughput genotyping capabilities to map genes through linkage analysis.  Chromosome segments that are shared at an abnormally high degree are pinpointed and more detailed analyses are made of these regions with denser sets of markers to isolate key genes and markers of the disease.  Once isolated, either the genes or their product proteins are targeted in a drug development program.

            Today DeCODE has seven leading programs in drug discovery and development and has isolated an additional 12 disease genes and 16 loci.  DeCODE’s leading therapeutic programs are for the treatment of Heart Attack (MI), Atherosclerosis (PAOD), Asthma, Stroke, Schizophrenia, Type 2 Diabetes (NIDDM), and Obesity.  Of these, the 5 latter have not yet entered pre-clinical studies.  A phase 2 clinical trial involving 200 heart attack patients is analyzing effects of licensed Bayer AG compound DGO31.  DGO31 binds FLAP (5-Lipoxygenase Activating Protein) and inhibits leukotreine synthesis, a compound that promotes inflammation.  Variations in the gene encoding FLAP double the risk of heart disease through increased leukotreine levels.  Pre-clinical PAOD studies involve a novel compound D151746 that binds a G-Protein Coupled Receptor target responsible for insufficient oxygenation of muscle tissue in legs.  A Phase I clinical trial is expected early in 2005.

            References:

1. http://www.decode.com
2. http://www.hoovers.com/decode-genetics/ --ID__100265--/free-co-factsheet.xhtm
3. Gulcher J, Kong A, Stefansson K. “The genealogic approach to human genetics of disease” Cancer J. 2001.
4. Arnason E, Sigurgislason H, Eirikur B. “Genetic Homogeneity of Icelanders: fact of fiction?” Nature Genetics, Vol 25, Num 4, pp373-374. 2000.
5. Gulcher JR, Kong A, Stefansson K.  “The Role of Linkage Studies for Common Disease.”  Elsevier Science Vol. 11, Issue 3, pp 264-267. June 1, 2001.
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   Karen Wei                    Biotechnology HW 4                              November 17, 2004

    VaxInnate

   Founded in 2001, VaxInnate is an innovative biotechnology company focused on manufacturing prophylactic vaccines and therapies which use the body’s innate and adaptive immunity.

   Immunity refers to the body’s ability to combat and eliminate potentially harmful cells.  Two components make up the immune system; innate immunity and adaptive immunity.  Innate immunity is the body’s natural, nonspecific response to exposure of abnormal or foreign material.  Adaptive immunity, on the other hand, is an antigen-specific defense mechanism designed to remove a specific foreign body.

   In recent studies, evidence has shown that elements of the innate immune response system are capable of initiating and controlling the adaptive immune response system.^[1]  Other research has shown that when a ligand for a specific toll-like receptor (TLR) - which is a glycoprotein molecule that activates the body’s innate immune system against microbial particles^[2] - was merged with an antigen of interest, a vaccine more potent and selective then vaccines made with antigens alone could be generated.^[3] 

   These studies have led VaxInnate to begin working on identifying, characterizing, and expressing known TLR ligands.  These ligands would then help VaxInnate better understand the consequences associated with triggering each TLR.^[3]  By understanding the various toll-like receptors which make up the immune system, VaxInnate would then be able to develop more effective vaccines to fight infectious diseases. 

   VaxInnate’s vaccine would differ from standard ones in its use of a recombinant fusion protein.  This recombinant protein would target and activate an antigen-presenting cell, which in turn would trigger the innate and adaptive immune responses.  VaxInnate feels that their vaccine would have several advantages over the standard vaccines of today.  First, by targeting the antigen-presenting cells, the immune response would be more efficient and more potent.  Second, side-effects such as excessive inflammation and over-stimulation of the immune system would be reduced.  And finally, the safety risks normally associated with traditional vaccines, which use killed or weakened pathogens, would be decreased.  This improvement in efficacy, safety, and reduction in discomfort, could make VaxInnate’s vaccine technology a strong contender against the vaccine technology of today.

   References

    [1]  Schnare M, GM Barton, AC Holt, K Takeda, S Akira, R Medzhitov. 2001. Toll-like  receptors control activation of adaptive immune responses. Nature Immunology 2:947-950.

   [2]  http://www.biotechjournal.com/Pathways/tollvol2.htm
   [3]  www.forrelease.com
   www.vaxinnate.com
   www.wiggin.com
   Aderem, A, Ulevitch, R. Toll-like receptors in the induction of the innate immune response. Nature. 406. 2000. 782-787
   Medzhitov R, Preston-Hurlburt P, & Janeway C. A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature. 388. 1997. 394-397. 
   Sherwood, L, Human Physiology from Cells to Systems, 2001, p. 400-419

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   Thaned Kangsamaksin      November 14, 2004       Biotechnology – W3034     Homework 4

   Regeneron: VEGF Trap

               Regeneron Pharmaceuticals, established in 1988 by Leonard Schleifer, M.D., focuses on discovering and developing therapeutic medicines for the treatment of diverse diseases such as obesity, rheumatoid arthritis, allergy, and cancer.

               Even though it is not the only company that produces anticancer drugs using humanized monoclonal antibodies, Regeneron has developed an innovative vascular endothelial growth factor (VEGF) blocker or the VEGE Trap for the potential treatment of cancer.  VEGF serves as a growth factor to stimulate the growth of new blood vessels, and support repair and remodeling processes of blood vessels in adults.[i]  This growth factor is believed to play an important role in the development of tumors and cancers. 

   Regeneron has developed a number of products based on the Trap biotechnology such as the IL-1 Trap for rheumatoid arthritis and the IL-4/13 Trap for allergy and asthma.  VEGF and other signaling molecules like interleukins use receptor systems that involve two or more receptor components to bind to the molecule tightly.  Naturally occurring antibodies for VEGF normally contain only one receptor component and have relatively low affinity for the VEGF molecule; therefore, Regeneron researchers genetically engineer the VEGF Trap to contain two receptor components fused together and attached to the constant region (Fc) of human IgG1, and reduce positive charges of the molecule to prevent it from interacting with negatively charged proteoglycans in the extracellular matrix.  In addition, they eliminate the engineered decoy receptors that have short half-life after injecting and testing them in mice.  Compared to other monoclonal antibodies against VEGF, the VEGF Trap is thus claimed to have high affinity and prolonged in vivo pharmacokinetics and pharmacodynamics of the drug.[ii] 

   What is impressive to me more about the VEGF Trap as well as other anticancer monoclonal antibodies is that unlike conventional methods like chemotherapy, radiotherapy, and surgery which have adverse effects on normal cells and can cause damage in certain organs, the VEGF Trap suppresses only tumor cells without toxicological side effects.[iii]  The VEGF Trap does not impact the growth and functioning of mature normal blood vessels.  It only induced apoptosis in tumor vessels that have been altered to be associated with endothelial cells because the expansion and the maintenance of tumor vessels, which are in a continual state of growth, require high levels of VEGF while normal vessels do not.[iv]

               The product is now being tested in a Phase I clinical trial in patients with solid tumors and non-Hodgkin’s lymphomas.[v]

   [i] Hood J. D. and Cheresh D. A. 2003.  Proc Natl Acad Sci USA. 100(15): 8624–8625.

   [ii] Holash J., et al. 2002. Proc Natl Acad Sci USA. 99(17): 11393-11398.

   [iii] Holash J., et al. 2002. Proc Natl Acad Sci USA. 99(17): 11393-11398.

   [iv] Hood J. D. and Cheresh D. A. 2003.  Proc Natl Acad Sci USA. 100(15): 8624–8625.

   [v] http://www.regeneron.com. November 14, 2004.

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Boris Reznik    Intronn   Biotechnology W4034     November 17, 2004

            Intronn is a biotech company developing RNA trans-splicing technology for the repair of defective genes.  The technology is called spliceosome-mediated RNA trans-splicing (SMaRT), and it incorporates the splicing activity of RNA-protein complexes called spliceosomes, which are found in the nucleus and cut introns out of pre-mRNA transcripts.  Intronn has engineered specific pre-trans-splicing molecules (PTMs), containing the repaired exon(s) of the defective gene of interest.  When spliceosomes arrive to cut the pre-mRNA, sometimes they incorporate the PTM (instead of the normal splicing reaction) in a trans-splice reaction resulting in a corrected copy of mRNA.

Intronn has performed and published several proof of principle studies in which they show protein product formation in the following mutations: CFTR (cystic fibrosis) gene (in cell culture), Factor VIII (hemophilia), and collagen.  The most impressive results thus far have the been the in vivo repair of Factor VIII mutations in mice, where corrected protein was found up to eight weeks later after the initial single dose of PTM (1).  The company is currently developing several products for further testing and application to humans.  The PTM to repair defective Factor VIII, which affects nearly 1 in 10,000 males, looks to be promising given the results in the mouse model.  Also in the works is a PTM to repair Alpha-1-Trypsin Deficiency, which can lead to lung and liver damage.  This therapy is currently being tested in cell culture and animal models.  Another application of the SMaRT technology would be to use it as a molecular marker of RNA (by incorporating a tag into the specific RNA sequence) to map where individual RNA migrates to in the cell.  This usage of the technology has the potential to be a big money maker for the company if the technology becomes widely used and a standardized technique in RNA research.  Intronn is the only private company outside of academia to receive a grant from the NIH to study the identification of splices sites in the human genome.

            This technology is far from reaching therapeutic stages as of yet.  The administration of PTMs into the organ and cells where they are needed has to be figured out.  Intronn announced a collaboration, earlier this year, with the biotech company Isogenesis, to develop a viral vector to deliver their CFTR repair PTM.  Intronn and the technology they are working on have tremendous potential to cure human disease.   

References

(1) Chao, H., Mansfield, S. G., Bartel, R., Hiriyanna, S., Mitchell, L. G., Garcia-Blanco, M. A., and Walsh, C. E. (2003).  Phenotype Correction of Hemophilia A Mice by Spliceosome-Mediated RNA Trans-Splicing.  Nature Medicine 9: 1015-1019.

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Grace Lee        Lexicon Genetics                   Nov. 21, 2004

Lexicon Genetics, founded in 1995 by Arthur T. Sands, MD, PhD, is a pioneer in developing the largest murine gene knockout library and high-throughput technologies to study functional mammalian genomics for the discovery of drug candidates. 

Lexicon’s patented gene targeting and genome-wide LexGene Trap technology (1, 2) utilizes positive-negative selection, isogenic DNA technologies, and retroviruses to knockout genes using vectors with elements enabling the direct retrieval of even rarely expressed human gene sequence information.  This process activates the transcription of non-protein coding regions of the trapped gene and then utilizes the cell’s innate splicing mechanism to extract the transcript for automated DNA sequencing.  Over 90% of the trapped sequences have been verified as genes and approximately 50% are novel sequences not found in public databases. 

Lexicon’s OmniBank library contains more than 270,000 frozen embryonic stem cell (ES) knockout clones and categorized in a relational database (3). The OmniBank sequence-tagged library represents approximately 60% of mammalian genes and 30,000 corresponding mouse genes which have been annotated by bioinformatics software based on both proprietary and publically available genetic information.

The goal of their Genome 5000 program is to utilize the large-scale, high speed technology to systematically characterize 5000 druggable human gene targets by the end of 2007.  Particularly, Lexicon focuses pharmaceutically valuable protein families such as g-protein coupled receptors, protein kinases, proteases, ion channels, secreted proteins, and other transporter/uncoupling proteins (4).  So far, patent applications has been filed for the gene trapping technology, more than 50,000 proprietary human gene sequences and more than 200,000 knockout mouse clones and their corresponding mouse sequences.

LexVision is an integrated technology platform which systematically validates the drug targets in vivo (5) to correlate the gene’s physiological functions as evaluated through relevant parameters for disease conditions using medical diagnostics adapted for the mouse physiology (eg CAT-scans, MRI, NMR analysis, or fluorescently activated cell sorters).  The E-biology Collaboration is an academic sublicensing program which allows academic researchers to use LexGene Trap, to mine the Omnibank database, and also to license the use of the knockout mice or ES cell lines. 

With K.B. Sharpless, the 2001 Nobel Laureate in Chemistry on board, Lexicon began developing a library of drug candidates internally and in collaboration with pharmaceutical and biotechnological partner companies.  To do so, Lexicon uses high-throughput screening assays against the druggable targets and tailors the structure to optimize the pharmacokinetics, tolerability, safety and potency of over 40 compounds for conditions such as cancer, cardiovascular disease, immune disorders, neurological disease, diabetes and obesity.

 For more information, see www.lexgen.com

 Relevant Review Articles

1. Zambrowicz BP, Friedrich GA,  Buxton EC, Lilleberg SL, Person C, Sands AT. Disruption and sequence identification of 2,000 genes in mouse embryonic stem cells. Nature. 1998;392(6676):608-11.

2. Wnk1 kinase deficiency lowers blood pressure in mice: a gene-trap screen to identify potential targets for therapeutic intervention. Proc Natl Acad Sci U S A. 2003;100 (24): 14109.

3. Walke DW, Han C, Shaw J, Wann E, Zambrowicz B, and Sands AT.  In vivo drug target discovery: identifying the best targets from the genome. Current Opinions in Biotechnology. 2001;12(6):626-31.

4. Zambrowicz BP, Turner CA, and Sands AT.  Predicting drug efficacy: knockout model pipeline drugs of the pharmaceutical industry. Current Opinion in Pharmacology 2003;3:563-570.

5. Abuin A, Holt KH, Platt KA, Sands AT, and Zambrowicz BP. Full-speed mammalian genetics: in vivo target validation in the drug discovery process. Trends in Biotechnology. 2002;20(1):36-42.

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Ku Yuan-Chieh    Genoptix      November 22, 2004

Genoptix which located in San Diego, California, is a biotechnology company founded by Tina S. Nova, Ph.D. The objective of this company is to provide accurate assessment of a patient’s potential response to a particular treatment. Then the physicians can determined the best way of treatment for each individual according to the data they get.

Three major analysis technologies were used by Genoptix to evaluate the optimal treatment options and monitor the prognosis of the disease.  Chemotherapeutic Response Testing is first used to examine individual response to chemotherapeutic reagents and monoclonal antibody in Leukemia and Lymphoma. It is then supported by detecting and measuring minimal residual disease through 6-color, state-of-the-art flow cytometry and quantitative reverse transcriptase polymerase chain reaction.

One special method called CancerTRAX, Cancer Tumor Response to Anti-neoplastic eX vivo, is used in chemotherapeutic Response Testing to provide information about tumor cell apoptotic activity. They cultured the specimen derived from patients for 48 hours in the presence of selected drugs and then analyzed by optophoresis (2)(3) to generate a complete dose response curve for each drug for treatment. Optophoresis is defined as the analysis of the motion of cells, where the motion is induced by moving optical field which produces radiation pressure forces on the cell in an aqueous suspension. This technology provides sensitive and quantitative measurement of cell response to drugs without additional labels or manipulation of the cell.

6-color, State-of-the-art flow cytometry is used for directed phenotyping to get individual pheonotypic information for minimal residual disease detection and monitoring. This is further supported by quantitative RT-PCR for monitoring residual disease and examining molecular response.

The concept of personalized patient information management is now applied to Chronic Lymphocytic Leukemia only and Genoptix will expand its service to the other Leukemias and Lymphomas in the future, like Chronic Myelogenous Leukemia, Multiple Myeloma, Acute Lymphoblastic Leukemia, Acute Myeloid Leukemia and Non-Hodgkin’s Lymphoama.

References

1.     http://www.genoptix.com/home.html

2.     Foster A.H.,et al. 2004. Anal Biochem. 327(1): 14-22

3.     Watson D., et al. 2004. Biophys J. 87(2): 1298-306

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Perminder Khosa    November 22, 2004    Monsanto

             Monsanto is a recently created corporation with a very special focus on developing agricultural solutions was incorporated as a subsidiary of Pharmacia in 2000, and spun off as a separate company in 2002. In the 21^st century, Monsanto has committed itself to innovation in plant biotechnology, genomics and breeding to improve productivity and to reduce the costs of farming (1).

            Monsanto serves farmers with high-quality, brand-name seeds, such as DEKALB and Asgrow (1). They also use a broad, high-quality collection of genetic material called germplasm to develop new varieties. Biotechnology traits, such as herbicide tolerance in Roundup Ready soybeans and insect protection in YieldGard corn, enable farmers to produce crops more efficiently (2). Monsanto traits help farmers reduce their tillage and their pesticide use. Having set the industry standard for insect-protection and herbicide-tolerance traits, Monsanto holds the competitive advantage in delivering improved, second-generation traits, as well as seeds with two or more traits. Monsanto’s strong germplasm base in both branded and licensed businesses allows them to launch their trait products in the varieties and hybrids farmers want most (1).

Monsanto is a company with a great past, present and future potential. I especially like Monsanto as a biotechnology company because of its business position, current success and future outlook. Monsanto develops and produces seeds with top performance and quality. Monsanto’s branded and licensed seeds hold leading positions in key corn and soybean seed markets. Farmers rate Monsanto seed No. 1 in overall quality (1). Monsanto is focused on maintaining the market leadership and brand position of Roundup. Monsanto is responding to intense competition by continuing to develop improved formulations.

From the pattern that Monsanto has developed in the small amount of time that it has been around, one can say that we will hear a lot more about Monsanto in the future. After researching this company, I would invest my time and money in the company for the immense benefits it can provide to the world.

1)      www.monsanto.co.uk

2)      http://www.bbsrc.ac.uk/life/index.html

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Yi-Shan Chou       Biotechnology    Odyssey Thera                              11/22/04   Yc2209@columbia.edu

 Odyssey Thera Inc. is a drug discovery company that has developed a cellular pharmacology strategy based on protein-fragment complementation assays (PCA) for the identification of safe and effective medicines for a wide spectrum of human diseases.

PCA (Fig.1), which was invented by Dr. Stephen Michnick and his colleagues at University of Montreal, is a proprietary process that allows protein-protein interactions to be precisely visualized, quantified and localized within live cells. In essence, the formation of a complex between two proteins directly generates a signal at the site of the protein complex. To accomplish this, one of two small, inactive polypeptides is fused to either end of a protein. The two polypeptide tags represent fragments of a rationally-dissected protein that can serve as a reporter. When expressed proteins tagged with complementary fragments associate, the protein-protein complex brings the fragments into proximity. This initiates folding of the fragments into an active protein, which then can generate a detectable signal such as a fluorescent signal at the site of the protein-protein complex. High-throughput PCAs engineered with fluorescent or luminescent readouts are suitable for detection with current high-throughput instrumentation systems.

The company’s mission is to augment the speed and efficacy of drug discovery through an understanding of the entire scope of drug action, prior to clinical trials. Their initial product pipeline includes pharmaceutical agents that simultaneously block multiple growth pathways in human cancer cells. The company also tries to map targets into pathways in conjunction with RNA interference; to perform high-throughput screening of small-molecule libraries; to identify on-pathway and off-pathway effects of known and unknown drugs.

Odyssey Thera Inc. currently has research collaboration, aimed at target discovery of various human diseases, with Pfizer Inc., Merck & Co., Inc., La Roche Ltd, Dharmacon Inc., which is a company focused on RNAi, and Panbio Ltd, which is an Australian-based medical diagnostics company.  

5.      Michnick SW, Remy I, Campbell-Valois FX, Vallee-Belisle A, Pelletier JN. Detection of protein-protein interactions by protein fragment complementation strategies. Methods in Enzymology 328, 208-230, 2000.

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Anna Zubkina            Biocept                                      Biotechnology Homework #4               Dr. Chasin        11/21/2004

 Founded in 1997, Biocept is a biotech company developing a novel microarray technology for both medical and research purposes.  The technology centers on a biocompatible polyethylene glycol-based urethane hydrogel platform that is adaptable to a number of different bioassays and is easy to manufacture.  The present focus of the company is the development of 3D biochips for genomics, proteomics, cellular assays and human diagnostic applications.

  The 3D HydroArray Technology developed by Biocept is a major advancement over the current two-dimensional microarray technologies.  The latter have a single layer of probes, limiting their precision and sensitivity.  Biocept’s 3D HydroArray chips use 300-micron hemispherical droplets with high density of 10¹⁰ to 10¹¹ probes per ~4 nL droplet.  The probes are covalently bound to and dispersed throughout the PEG-based hydrogel, providing consistent attachment chemistry through polymerization and surface attachment.  The 3D HydroArrays offer superior precision, high reproducibility (CV <10%) and excellent sensitivity (>1:100,000).1  In addition, the high probe density allows for a broad dynamic range of the signal of 3 to 4 orders of magnitude, which is unmatched by the current 2D microaray technologies.  Biocept focuses on low- to mid-density chips containing 300-gene probes suitable for typical gene pathway studies.2   

Biocept currently markets two genomic products for research purposes.  3D HydroArray Signal Transduction System identifies differentially expressed genes in key signal transduction pathways.  The 3D chips use 45-mer oligonucleotide probes per gene and cover 240 pathway genes, 12 housekeeping genes, three Arabidopsis control genes and six “blank” hydrogel sites. The 3D HydroArray Apoptosis System uses a similar structure to test for appropriate apoptosis-related pathways.  Custom-designed 3D HydroArray systems are also available.3 

  Biocept is planning to release the 3D HydroArray Chromosomal Disorders Diagnostics that would improve over the existing prenatal diagnostics methods of FISH and karyotyping.  Biocept’s 3D HydroArray would provide a more comprehensive, high precision testing of a wide variety of aneuploid disorders and microdeletions requiring only 24 hours for assay.  In addition, a new cell separation would isolate fetal cells from maternal blood or cervical swab offering a less-invasive prenatal testing during the first trimester.4  It would also be cost-comparable to the existing prenatal diagnostic methods.

  1 Dong, Fan et.al. (2001) A Novel Three-Dimensional Hydrogel-Based Microarray Platform.  JALA 6 (4): 87-91.

2 http://www.biocept.com November 19, 2004.

3 http://www.biocept.com November 19, 2004.

4 Biocept Advances Fetal Cell Separation Technology for Applications in Prenatal Diagnostics.  San Diego Technology News.  April 29, 2004.  (URL: www.freshnews.com, article # 17763)

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Maxim Factourovich                 Geron              November 24, 2004

Increased telomerase activity is known to be one of the key factors in abnormal growth of tumor cells and metastasis. It therefore comprises a target for future anti-cancer therapies. The idea is to inhibit telomerase so that the tumor cells will undergo aging and eventually die. The researchers at Geron Corp. have developed two types of oligonucleotides targeting the emplate region of telomerase. Unlike typical anti-sense RNA compounds, these oligos, called GRN163 and GRN163L inhibit telomerase by competitive binding to its active site. [1] Both are relatively short fragments (11-13 bp length), show little affinity to other critical nucleic acid-modifying enzymes and seem to be non-toxic to normal cells. [1]  In vitro, GRN163 and GRN163L effectively inhibited telomerase activity even at very low concentrations in all of 13 types of cancer cells tested, among which are malignant human glioblastoma, prostate cancer, lymphoma, myeloma, hepatocellular carcinoma and cervical cancer. [1] Tumors treated with GRN163 demonstrated shortening of telomeres,senescence and apoptosis in a period of time that correlated with initial length of telomeres. [3]

        Animal studies have shown a significant delay in tumor growth in athymic mice with U-251 MG human brain tumor xenografts after intratumoral treatment with GRN163. ^[2]  The intravenous treatment of animals bearing disseminated human multiple myeloma significantly reduced tumor growth and resulted in a 50% increase in survival as compared to controls. In five out of seven intratumorally treated rats with human glioblastoma, the tumor was completely eradicated. ^[1] 

Both compounds use a special thiophosphoramidate chemical backbone. They are almost identical with the exception of a lipid attached to one end of GRN163L, which improves its pharmacokinetics and is believed to make the manufacture cheaper and more efficient. GRN163L is expected to effectively inhibit telomerase when administered one injection every few days. Currently, Geron performs additional animal toxicology and efficacy studies of these drugs and, if successful, will file an Investigational New Drug application to begin human clinical trials.

^[1] http://www.geron.com/showpage.asp?code=prodcati
^[2] Ozawa T, Gryaznov SM, Hu LJ, Pongracz K, Santos RA, Bollen AW, Lamborn KR, Deen DF. “Antitumor effects of specific telomerase inhibitor GRN163 in human glioblastoma xenografts.” Neuro-oncol. 2004 Jul;6(3):218-26
^[3] Asai A, Oshima Y, Yamamoto Y, Uochi TA, Kusaka H, Akinaga S, Yamashita Y, Pongracz K, Pruzan R, Wunder E, Piatyszek M, Li S, Chin AC, Harley CB, Gryaznov S. “A novel telomerase template antagonist (GRN163) as a potential anticancer agent.” Cancer Res. 2003 Jul 15;63(14):3931-9.

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Meng-Tien Hsieh     HOMEWORK #4             Sangamo BioScience

Sangamo BioScience is a biotech company founded in 1995 and went public in 2000, currently located in Richmond, CA.

Sangamo primarily focus on engineering transcription factors for gene regulation. To be more specific, they’re using a special type of protein called ZFP, which stands for Zinc Finger DNA binding Protein. ZFP is basically a transcription factor comprising two domains: recognition domain and functional domain. The recognition domain recognizes and binds to a specific DNA sequence, and the functional domain can activate or repress target gene.

The recognition domain is composed of 2 or more zinc fingers, each finger can recognize and bind to a 3 base pair DNA sequence. Therefore, the more fingers being linked together can have better specificity. By modifying the recognition domain, they can then use the ZFP to target any DNA sequence within any give gene. Also, an additional “switch” component can further make the ZFP regulatable.

The delivery of ZFP into living organisms is currently done by viral vectors or plasmids.

One example is to treat cardiovascular disease by ZFP to upregulate angiogenesis factors, such as vascular endothelial growth factors, abbreviated VEGF. In one publication in Nature, they demonstrate that, in mouse model, their engineered ZFP can induce both endogenous VEGF-A gene expression and angiogenesis. Comparing to the conventional way that using exogenous cDNA to induce angiogenesis, the newly grown blood vessel by ZFP has better physiological properties than cDNA ones.

Sangamo also try to use ZFP on some other disease like Cancer, intractable neuropathic pain, sickle cell anemia, and ZFP-mediated gene correction, etc. In different cases, they also cooperate with other biotech company capable of different delivery technologies, such as adenovirus.

Additionally, ZFP enables drug target discovery by constructing cell lines that are engineered to activate or over express certain target gene, such as some membrane protein or receptors. Examples include G-protein coupled receptors, ion channels, etc.

Also, production of therapeutic proteins, especially monoclonal antibodies, is a growing business. ZFP-TF-engineered cell lines can help to efficiently generate proteins and enhance the yields.

To conclude, ZFPs have several advantages:
The way they regulate gene is normal and nature in higher organisms;
They can have good binding specificity if more fingers are used;
They can be used to activate or repress gene;They can be used on many kind of organisms;
They’ve been proved to be effective on both cell and animal models;
ZFP themselves can be regulated, therefore can be conditional or reversibly regulated; and,
They regulate endogenous gene, rather than introduce an exogenous gene, therefore can work around the patented cDNAs.

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Julie Cheong                 MacroGenics                                                 November 24, 2004

Introduction:  MacroGenics is a company that focuses on creating novel immuno-therapeutics gearing towards the areas of inflammation and the treatment of diseases such as infectious, autoimmune and cancer.  The company integrates the disciplines of genomics, proteomics and bioinformatics to identify disease targets. The company’s primary goal is to understand the function of the Fc receptors to engineer antibodies to bind more favorably to immune receptors, which induce antibody mediated activities such as ADCC (antibody-dependent-cell-mediated-cytoxicity) and CDC (complement-dependent-cytotoxicity).

They are exploiting mechanisms to improve ways in which cytotoxic antibodies mediate cell death to treat cancer and to prevent cytotoxic autoantibodies from triggering in diseases such as autoimmunity.

Their efforts are to identify novel targets for: 1) cancer treatment through proteomics using a procedure called isotopic coded affinity tagging (ICAT) 2) vaccine development by using high throughput genetic immunization to screen and identify pathogenic DNA sequences that will encode antigenic proteins. 

 History:   MacroGenics was founded in August of 2000 by Drs. Leroy Hood, Ruedi Aebersold and Alan Aderem from the Institute for Systems Biology and Jeffrey Ravetch from Rockefeller University.  Then in June of 2002 the company acquired a vaccine discovery company called Eliance Inc. which was founded by Dr. Stephen Johnston and his colleagues at the University of Texas Southwestern.   

 Technology/Methodology:  The company’s technological approach includes: 1) developing and modifying therapeutic antibodies to optimize its in-vivo efficacy through selective Fc receptor engineering  2) modulating or activating the effector cells (macrophages) to provide them with a more effective way of antibody-depending tumor killing or preventing autoantibody triggered effector cell activation 3) identifying novel tumor targets through a genomic and proteomic high throughout screening of tumor banks, and 4)  constructing therapeutics aimed at blocking cytotoxic autoantibodies from activating effector cells for the treatment of autoimmune diseases.

 Macrogenics’ approach is different from other monoclonal antibody based companies because they focus on the constant region (Fc) and not the variable region.  They feel that the constant region is equally important because they can interact with effector cells that can produce an immune response by sending out stimulatory or inhibitory signals.  Their goal is to create Fc regions with enhanced activity by manipulating these effector cells on their ability to kill targets.  In doing so, they are creating a molecule with a greater capability in mediating ADCC, CDC and other antibody related activities. These Fc receptors offer a new novel approach to treating inflammatory and autoimmune diseases.

Product/Pipeline:  MacroGenics’ main product pipeline includes:  1) a monoclonal antibody against the Fc receptor CD16 to treat autoimmune diseases such as lupus and Rheumatoid arthritis.  This humanized CD16 antibody will enter clinical trials in early 2005.  Another product that will go into clinical trials is a vaccine for West Nile virus.

 Conclusion:  This company offers a novel way to control inflammatory and autoimmune disease by offering a different technological approach that upregulates and downregulates the activation of the immune system via these Fc receptors.

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Rocio K. Perez           Company name: Fibrogen                 November 28, 2004

 This is a drug discovery biotechnology company that mainly aims to improve treatments for fibrosis based on their research on tissue fibrosis. One of their major molecular targets include Connective Tissue Growth Factor, CTGF, a protein critical to scar formation in tissue repair, however, if CTFG is overexpressed, the accumulation of scar material results in chronic fibrosis, a condition that can eventually lead to failure of the affected organ.  Patients with post surgical procedures are highly susceptible to fibrosis.  Fibrogen is developing monoclonal antibodies that block CTGF thus preventing excessive scar formation and organ failure.  There are no therapies available known to prevent fibrotic disease.

CTGF is a downstream mediator that transforms TGF-beta and induces fibroblasts to become myofibroblasts, which forms layers that help form scars.  The formation of such scar tissue leads the affected organ to become stiff eventually leading to failure and death. 

Increasing data supports the idea that blocking CTGF can prevent fibrosis.  Animal models have suggested that the use of monoclonal antibodies targeting CTGF may prevent fibrotic diseases.  The development of FG-3019 as a fully human monoclonal antibody is giving rising hope to patients with fibrotic diseases including pulmonary fibrosis, diabetic nephropathy and pancreatic cancer. The results for the pre-clinical studies of diabetic nephropathy suggest that FG-3019 inhibits fibrosis and improves kidney function.  Diabetic nephropathy is a progressive kidney disease in which the ability of the kidneys to filter blood and produce urine declines over many years and is characterized by the presence of proteins in the urine and fibrosis.

 Furthermore, small molecules are also being developed that inhibit the activity of CTGF, such molecules currently under clinical testing. Finally, they are also trying to develop CTGF detection assays that would serve as a diagnostic tool for fibrotic disease. 

 References:
www.fibrogen.com
Leask and Abraham.  “TGF-β signaling and the fibrotic response” The FASEB Journal 18, 816-827 (2004)

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Izabella Messina, W4034         Acceptys, Inc.                             November 24, 2004

 Acceptys is a biopharmaceutical company primarily focused on producing totally human monoclonal antibodies that can be used in therapies against cancer and infectious diseases. These antibodies not only exhibit the structure and function of human immunoglobulin, but because they come from humans the specificity and efficacy of the final product is increased (1).

            This remarkable strategy is based on the development of unique stable immortal fusion partner cell lines: B6B11 and MFP-2. B6B11 is a heteromyeloma, which was created by merging murine and human myeloma cells. Although this line fuses well with lymphocytes derived from lymph nodes and spleen, it doesn’t fuse well with peripheral blood lymphocytes (PBL), the most common and easily available source of human antibodies (2). By fusing B6B11 to human lymphocytes from lymph nodes, Acceptys was able to establish a trioma MFP-2, which additionally fuses well with PBL (2). Consequently, to obtain continuous production of the antibodies used in therapies, the MFP-2 line is fused with acquired ex vivo Human Monoclonal Antibodies (hMabs). The hMabs are collected from people who were exposed to targeted antigens and thus developed natural monoclonal antibodies. Next, the seized immunoglobulins are screened against live tumor cells or viruses using High-Throughput technology, based on cell- ELISA assay, in order to find the ones that are only disease specific (1).             

The approach adopted by Acceptys postulates the initial isolation of a specific antibody and the subsequent finding of an antigen. This novel design contradicts the known archetype of antigen-target first and antibody second (1). Clearly, defining the antigen has very important advantages such as the possibility of finding tumor markers and potential vaccine candidates. Thus, based on the knowledge of the isolated hMab, Acceptys identifies the antigens, by using Serological Recombinant Expression Cloning (SEREX). Additional identification of the antigen allows the company to patent both determined monoclonal antibodies and targets against which they were raised.

            Acceptys is recognized as a company that offers promising developments in the rapidly evolving hMabs market. To date the company has identified many possible therapeutic molecules including antibodies against breast cancer, prostate cancer, and colon cancer. Moreover, Acceptys’ technology is both time and money efficient. The company can identify the potential functional antibodies within a month in contrast to traditional models that can take several years (1). 

 Reference:

1. http://www.acceptys.com/
2. Kalantarov G.F. et al. Development of a fusion partner cell line for efficient production of human monoclonal antibodies from peripheral blood lymphocytes.(2002) Human Antibodies, 11(3): 85-96

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Hua Zhong                   Avigen, Inc.                  Nov. 23^th, 2004

 Based in the San Francisco Bay area, Avigen, Inc. is a biotechnology company involved in the development of gene therapy products derived from adeno-associated virus (AAV) for the treatment of inherited and acquired diseases. The Company's proposed gene therapy products are designed for in vivo administration to achieve the production of therapeutic proteins within the body (1). By July 20^th 2004, Avigen holds about 30 patents related to recombinant AAV products, most of them relate to the delivery of the therapeutic gene to the patients lack the correct gene product such as Hemophilia; there are also other patents about treating cancer by delivering a “two-gene system” to the tumor cells so that inhibit the growth of the tumor and terminate therapy at appropriate moment (2).

 AAV is a simple non-pathogenic virus which needs the helper-virus to help its propagation. It only contains two genes which relate to replication and packaging and can be replaced by the therapeutic gene; the inverted terminal repeats from the original AAV are conserved in rAAV to promise the high expression level of the therapeutic gene (3). During the production, the rAAV, together with a helper virus (such as adenovirus) and a packaging plasmid are co-transfected into the host cell to harvest large amount of the progeny rAAV (4). These progeny rAAV taken by the patients can be recognized by specific receptor on target cells, using endocytic pathway to enter the cell, then the nucleus (5); the therapeutic protein then can be produced.

 During the manufacturing, Avigen’s patented methods can promise the sufficient and scalable yield to provide enough vector particles for animal experiments and clinical trials; also, they use a helper virus-free process compare to the traditional way to eliminate the contamination of the helper-virus, a developed version of the packaging plasmid to eliminate the formation of the wild-type AAV, together with the highly sensitive assays to ensure no viral contamination exists. Their patented methods also can promise the versatility of the product (2).

 Though they can get long-term and high level protein expression in animal models of human diseases (2), the results from phase I clinical trial are not as good as expected due to several possible reasons(6) including transfection and expression efficiency; also, the disadvantages such as the size limitation and immune response should be awared (7).

 In November 2000, Avigen signed a codevelopment agreement with Bayer for the treatment of Hemophilia B, and the product Coagulin-B® for Hemophilia B is on phase I human clinical trial. On May 27^th 2004, the company announced that they would realign their product development strategy, shift their resources to the research of neurological disorders and on August 2004 their product AV201 for Parkinson’s disease has got the authorization to begin the clinical trial. Also, there are two products for Hemophilia A and chronic neuropathic pain on preclinical status, four products aimed at Gaucher disease, Sly’s syndrome, Hereditary Emphysema and Beta-Thalassemia under the research status.  (2).

 Reference 

1. http://www.bio.com/industryanalysis/industryanalysis_news.jhtml?cid=ci30772
2. http://www.avigen.com
3. Chooi May Lai, Yvonne K.Y. Lai and P. Elizabeth Rakoczy. 2002. Adenovirus and Adeno-Associated Virus Vectors. DNA AND CELL BIOLOGY. 21(12):895-913.
4. Barrie J. Carter. 1992. Adeno-associated virus vectors. Current Opinion in Biotechnology 1992, 3:533-539.
5. Jeffrey S. Bartlett, Rose Wilcher and R. Jude Samulski. 2000. Infectious Entry Pathway of Adeno-Associated Virus and Adeno-Associated Virus Vectors. JOURNAL OF VIROLOGY. Mar. 2000:2777-2785.
6. Haemophilia (2004), 10, (Suppl. 3), 36-37
7. Neeltje A. Kootstra and Inder M. Verma. 2003. Gene Therapy with Virual Vectors. Annual Review of Pharmacology and Toxicology. 2003. 43:413-39.

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Tianhua Guo       Biogenex and Tissue Microarrays      11-26-04                                                                                                       

        Biogenex is a privately held company, founded in 1981.The company provides novel technologies for cell and molecular pathology. Biogenex has gotten three patents of Antigen Retrieval technique since 1989. Based on the strong support of their Antigen retrieval technique, Tissue Microarrays were introduced in 2002 in Biogenex. The company provides a wide collection of Tissue Microarrays from normal and diseased cases.  TMAs have enormous value in clinical diagnostics, prognostics and target validation. Currently, TMAs are widely used in cancer research and diagnosis.

       Tissue Microarrays consist of punches removed from hundreds of paraffin-embedded tissue blocks and re-embedded in an additional single block. The technique constructing TMAs requires only two needles with slightly different diameters to transfer specimens. The arrays can be subjected to in situ hybridization with RNA/DNA probes and antibodies. Thus, TMAs are useful in understanding the correlation of mRNA and protein expression profiles in different tissues, and defining subcellular locations of gene product. TMA also can show gene amplifications if you subject arrays to FISH.

       A single array slide can hold up to 600 specimens. Instead of doing experiment with 600 slides, you can do just one slide. This saves a great amount of reagents (money), keeps the uniformity of your experiment, and study hundreds of cases in parallel. Another advantage is that arrays amplify scarce resources. A conventional paraffin block is enough for producing 200-300 samples for TMAs.

       Automated analysis system allows high throughput and quantitative study of gene expression. Clinicians use TMAs to screen high expression of HER2 in Breast Cancer susceptible population for early diagnosis, doing 600 cases once a time. TMAs also provide powerful prognosis for colorectal cancer locating the expression in subcellular compartments of phospho-b-catenin, its nuclear expression is associated with good survival rate.

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Alexander Serebrov     CENIX BIOSCIENCE  Nov. 29. 2004

Cenix, , A PIONEER IN RNAi TECHNOLOGIES, RAISES € 5 MILLION IN INTERNATIONAL FINANCING ROUND

Dresden, Germany, September 10, 2002 - Cenix BioScience GmbH, a pioneer in RNA mediated interference (RNAi)-based technologies and therapeutics, today announced that it has raised € 5 million (Euro) in a new international financing round to fund its next stage of development and progress its novel RNAi drug development programmes.
Pioneering work led by the Company’s three scientific founders, Drs. Echeverri, Hyman and Gönczy in 1998, yielded the initial proof of principle for genome-scale applications of RNAi (Nature 2000, 408: 331). Through four years of R&D devoted exclusively to this revolutionary new gene silencing technology, Cenix has now established proprietary industry-leading platforms for RNAi-based drug discovery and development. Based in Dresden, Germany, Cenix was founded in 1999 as a spin out from the Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, and the European Molecular Biology Laboratory (EMBL), Heidelberg. The company currently has 37 employees mainly working in the fields of molecular cell biology, genomics and bioinformatics.
Using a broad range of experimental systems including C. elegans, Drosophila, cultured human cells, and mice, Cenix has developed its leading expertise in RNAi technology to encompass key steps in therapeutic drug discovery and development. Its main offerings for collaborative research projects include rapid and cost effective genome-wide RNAi screens for drug target discovery and high throughput RNAi-based target validation.

The Company is using the rich pipeline of new discoveries and intellectual property from its powerful discovery platforms to drive its in-house product development programmes for RNAi-based therapeutics, addressing major disorders such as cancer, infectious diseases, and inflammation.
RNAi – The Gene Silencing Technology of Choice
RNA-mediated interference, or RNAi, is a powerful new approach for achieving targeted gene silencing of pathological genes using complementary double stranded RNA (dsRNA). In contrast to all “conventional” antisense paradigms, this approach harnesses a recently discovered natural defence mechanism, which protects organisms ranging from plants to humans against molecular parasites such as viruses and transposons. The pathway consists of a fast and powerful cellular response to the presence of dsRNA, a usually rare molecule in most organisms and therefore recognised as “foreign”, whereby any messenger RNA that contains the dsRNA’s sequence is completely degraded, and its corresponding gene thereby silenced.

RNAi distinguishes itself from other sequence-specific gene silencing methods by the unprecedented potency of its catalytic silencing effect, the high stability of the triggering molecule (dsRNA), its excellent experimental reproducibility, and by its applicability in a wide range of experimental systems. Importantly, the active agent (dsRNA) and the mechanism of action of RNAi are inherently far superior to those of other existing or proposed sequence-based therapeutics such as conventional antisense and ribozymes. These key features offer strong promise for the development of a new class of RNAi-based therapeutics combining excellent potency and specificity.
The approach is arguably the biggest molecular biology breakthrough of the last decade and is applicable in most major experimental systems including cultured mammalian cells and mice. In addition, the use of RNAi in mice promises a much more rapid, cost-effective, more controllable and more accurate target validation tool than transgenic knockouts.
In addition to in-house drug development programmes, which are currently focused on selected oncology indications, Cenix is offering its unique expertise to industry and academic partners through a comprehensive range of collaborative research programmes for RNAi-based target discovery, target validation, drug mode of action (MoA) analyses and lead compound discovery in multiple systems including cultured human cells, mice, C. elegans and Drosophila.

Custom Design of Reagent Libraries for Large Scale RNAi

Cenix leads the industry in the automated design of dsRNA molecules, -siRNA, shRNA, esiRNAs, or long dsRNAs- for large scale RNAi screening in experimental systems from C. elegans and Drosophila to human and mouse.
Cenix is now making this expertise available to the biomedical research community in two forms. First, Cenix is partnering with Ambion to commercialise the first comprehensive siRNA library to cover the entire public domain version of the human genome
Second, Cenix is offering the same design expertise to assist partners in building other customized RNAi libraries destined for their own internal use. Cenix’s design algorithms can make use of any source of gene sequence data, from any species, whether public or proprietary.

Target Discovery: RNAi Screening from Gene Families to Genome-wide Screens

RNAi is emerging as the best new tool for functional genomics research. As pioneered by Cenix, RNAi-based screens to identify new disease-relevant genes can now be carried out in several model organisms including C. elegans and Drosophila cells.
Cenix is also leading the industry in pushing this technology to the next step by building the first libraries of siRNA molecules that permit screening directly in human cells
Since these screens can either be focused on specific gene families or run in a comprehensive genome-wide manner, they offer a level of control and efficiency never before seen.
Also, by offering a direct, causal link between gene and phenotype, target genes identified by RNAi inherently carry clearer physiological and therapeutic relevance, i.e. a superior level of validation.
Cenix is now offering this programme to industry and academic partners through a flexible range of partnership models.

Target Validation: HT-RNAi for Efficient Validation Studies

The selection of candidate genes worthy of further exploitation as drug development targets requires a crucial but difficult and error-prone prioritisation process, made all the more expensive nowadays by the plethora of candidates on the market.
Cenix’s specialisation in the high throughput application of RNAi allows it to achieve unparalleled time-and cost-efficiency in carrying out even the most complex and large-scale cell based validation projects.
Cenix is now offering this programme to industry and academic partners through a flexible range of partnership models.

How does RNAi work?

RNAi represents the harnessing of a ubiquitous, powerful cellular defense mechanism, whereby a double stranded RNA is used to trigger the catalytic degradation of the targeted gene’s mRNA, thus effectively silencing its expression.

 (1) Delivery of trigger dsRNA
A “trigger“ double stranded RNA (dsRNA) is introduced into the cell’s cytoplasm.

(2) Generation of siRNA pool
The Dicer enzyme processes the trigger dsRNA, forming a pool of small interfering RNAs (siRNAs), approx. 21 base pairs in length, including 2 nucleotide overhangs at both 3’ ends.

(3) Capture, unwinding of siRNA by RISC
The siRNAs interact with the RNA-Induced Silencing Complex (RISC), whose helicase activity directs the unwinding of the bound siRNA.

(4) Binding of siRNA-associated RISC to target mRNA
The unwound siRNA’s bound strand confers sequence-based specificity to its associated RISC complex, allowing interaction with the complementary target mRNA.

(5) Destruction of target mRNA
The RISC complex contains an endonuclease activity which causes a single-site cleavage of the target mRNA approximately
in the middle of the siRNA binding region. The resulting fragments of target mRNA are thereby destabilized and subsequently get fully degraded through natural endogenous mechanisms.

Although the identity of the putative RISC-associated endonuclease remains elusive, this model of the RNAi pathway has gained the strongest experimental support to date.

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Grace C. Wong                                    Hematech, LLC                            29^th November 2004

             Hematech, a company founded in 1998 is based in Westport, CT. In May 29^th, 2002, they opened a larger research laboratory facility in Sioux Falls, SD. On board of the executives at Hematech, they have Dr. James Robl (President, CSO, Director and Co-Founder) who is an animal cloning pioneer and the first to clone a transgenic cow in 1998. Also, they have Dr. Richard Goldsby (Senior Consultant, Director and Co-Founder) who is an expert in the field of monoclonal antibodies and the first to produce bovine monoclonal antibodies.

Hematech’s platform technology is in the ability to create transgenic cows in a short time frame of 21.5 months that are able to produce fully human polyclonal antibodies. [1] This is done in collaboration with the pharmaceutical division of Japan’s Kirin Brewery, the same company that partnered Medarex in producing the KM-Mouse, which makes human monoclonal antibodies.

            The procedure in creating these transgenic cows is to first obtain cow fibroblast cells and introduce a 10Mb Human Artificial Chromosome (HAC) – Kirin’s technology that contains the locus of human chromosome 14 encoding the heavy immunoglobulin chain and the locus of human chromosome 22 encoding the lambda light chain. [2] Also these fibroblasts have been doubly homozygous knocked-out for the bovine immunoglobulin gene and the prion gene via a four step sequential gene targeting technique. [1] As such, these fibroblasts have been removed of the ability to produce bovine antibodies and they are unable to contract bovine spongiform encephalopathy (BSE) or Mad Cow’s Disease. The second step is to obtain and remove DNA from cow’s egg. Finally, the fibroblast is transferred into the egg and a current is applied to fuse the fibroblast and the egg by electroshock. [3] This egg fusion is able to develop into an embryo as the DNA in fibroblast has the ability to regenerate into a cow. These transgenic cows are able to produce functional human antibodies through rearrangement of both heavy and light chain of the human immunoglobulin loci. Through vaccination, large production of human polyclonal antibodies specific for the antigen can be obtained by plasmapheresis (2.5 lbs antibodies in blood). [3]

Hematech goals are to generate antibody therapeutic products for antibiotic resistant infections, autoimmune diseases, immune deficiencies, cancers and biodefense. Thus far, Hematech have received $6 million dollars from the Department of Defense (DoD) for research in the production of antibodies for anthrax, and another $3.3 million from DoD indirectly through DynPort Vaccine Company for the research of antibodies against Botulinum neurotoxins. [3]

Although Hematech have no products out in the market currently, it will be an interesting company to watch for in the coming years to not only solve the shortage of antibody supply but also in producing safer products that are not obtained directly from human donors. [4]

References

1.      K, Yoshimi et al. Sequential targeting of the genes encoding immunoglobulin-m and prion protein in cattle. Nat. Genet. 36, 775 – 780 (2004).

2.      K, Yoshimi et al. Cloned transchromosomic calves producing human immunoglobulin. Nat. Biotechnol. 20, 889 – 894 (2002).

3.      http://www.hematech.com

4.      http://www.primaryimmune.org/prodsafe/recalls_fda.htm

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Ming-Hui Cheng     ExonHit Therapeutics       November 29, 2004

 ExonHit Therapeutics, founded 1997, is located in Paris, France and has a US facility in Gaithersburg, Maryland. The company focuses on neurodegenerative disease and cancer with its research platform, based on alternative RNA splicing [1].

 Given that alternative RNA splicing is observed in a large majority of human genes, it is not surprising that changes of normal splicing patterns can cause human disease [2]. ExonHit is developing therapeutic compounds through the application of DATAS™ (Differential Analysis of Transcripts with Alternative Splicing). DATAS is a gene profiling technology that has been utilized to capture mRNA splice variants and then compare full-length mRNA between a standard condition and a pathological one.

 DATAS is performed by hybridizing cDNA from the first population with mRNA from the second. Also the reciprocal experiment (mRNA from the first population with cDNA from the second) is done to allow qualitative changes in both conditions to be distinguished. The cDNA is generated with a biotinylated oligo-dT primer, which permits subsequent isolation by magnetic streptavidin beads. These hybrids are then treated with RNase H to release mRNA that does not hybridize to the heteroduplex. The released mRNAs, which encode qualitative differences between the two conditions, can then be isolated, reverse transcribed, cloned and collected into libraries (Fig. 1) [3].

Fig. 1. Experimental isolation of libraries of alternatively spliced transcripts.

 The major advantage of DATAS is that it allows the systematic generation of libraries of alternative RNA splicing without prior knowledge of the splice events [4].  

 ExonHit has created a database for alterations that affect functional domains of proteins in certain disease. This information is medically relevant since it points out critical differences of the transcript between normal and diseased tissues. Therefore the gene can serve as an intervention target for drugs to treat the disease [1].  

By moving from identifying EHT 0202, a small molecule for Alzheimer's disease, to Phase II trial in less than one year, ExonHit has clearly demonstrated the value of DATAS, in progressing drugs candidates into the clinic much faster than others. Currently, none of the compounds of ExonHit have been in-licensed.

 References
1. http://www.exonhit.com/
2. Faustino, N.A. and Cooper, T.A. 2003. Pre-mRNA splicing and human disease. Genes & Dev. 17:419-437.
3. Bracco, L. and Kearsey, J. 2003. The relevance of alternative RNA splicing to pharmacogenomics. Trends Biotechnol. 21:346-53.
4. Schweighoffer, F. et al. 2000. Qualitative gene profiling: a novel tool in genomics and in pharmacogenomics that deciphers messenger RNA isoforms diversity. Pharmacogenomics. 1:187-197.

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Frances Kang                        Genentech               November 29, 2004

Perhaps better known through their products like Herceptin and Rituxan, Genentech Inc. is a leading biotechnology company founded on the premise of recombinant DNA technology. With research focus ranging in areas from protein chemistry to physiology, Genentech has developed biotherapeutic drugs on multiple disease platforms, namely oncology, immunology, and vascular biology. Genentech's Xolair, the first humanized antibody approved by the FDA to treat asthma, uses an entirely novel approach from conventional asthma treatments by targeting IgE in the immune system. [2]

Specifically prescribed to treat moderate-to-severe persistent asthma, Xolair is indicated for adults and adolescents for subcutaneous use and to be administered every two or four weeks. Designed to treat patients who react positively to perennial aeroallergens, Xolair is primarily used to supplement inhaled corticosteroids. In clinical studies, researchers found that patients receiving ongoing inhaled steroid treatment in addition to Xolair had fewer asthma attacks (33%-75%) and improved asthma symptom scores. The most severe side effects reported were malignancies and anaphylaxis. [3]

Set apart from more conventional asthma therapies mainly targeting asthma symptoms. Xolair’s innovative mechanism of action functions to treat the underlying cause of the allergy-asthma cascade.  Airway inflammation and bronchial constriction which are commonly associated with asthma usually results from a chemical mediator response triggered by the binding of allergens to IgE or immunoglobulin E antibodies attached to mast cells.  Most asthma medications work at the level of either suppressing the chemical mediator response or by treating inflammation.  However, the science of Xolair is different in specifically binding IgE to decrease the amount of circulating antibodies and thereby prevent their binding to mast cells. [4] In effect, the consequent release of chemical mediators is mostly inhibited if not completely curtailed.

Although Xolair is not yet prescribed for other allergic reactions aside from allergic asthma, future directions for Xolair could potentially be in treating other allergic diseases.  By exploiting the almost ubiquitous role of IgE in most allergy-associated reactions, the therapeutic benefits of using Xolair can be extremely diverse.  However despite all the potential that Xolair has to offer, the downsides of actually manufacturing the drug due to its sophisticated engineering techniques makes Xolair a costly treatment option, citing an annual prescription cost of $10,000 a year. [1] Yet Xolair still remains to be yet another example of how a successfully developed drug can deliver significant treatment alternatives to the medicines already at hand.

[1] http://www.cbsnews.com/stories/2003/05/15/eveningnews/main554132.shtml
[2] http://www.gene.com/gene/products/information/immunological/xolair/index.jsp
[3] http://www.pharma.us.novartis.com/newsroom/pressReleases/releaseDetail.jsp?PRID= 863&checked=y
[4] http://www.xolair.com/index.jsp?source=Overture

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Daniel A Ferrara            Eyetech Pharmaceuticals       11/26/2004       

            Eyetech Pharmaceuticals, a small biotechnology company based in New York City, is focused on developing new drug treatments for eye disease.   Currently, they are working on a new drug called Macugen™ or pegaptanib sodium injection.  This drug is under development for the treatment of the wet form of age-related macular degeneration (AMD) and diabetic macular edema (DME).  Macugen is a short RNA oligonucleotide with a complex three dimensional structure known as an aptamer.[i]  It was discovered by screening a random pool of oligonucleotides and selecting those which were able to isolate the desired target molecule.[ii]  Macugen binds, with great specificity, to VEGF isoform 165 via a lock and key-like mechanism to the exon-7 encoded domain.  VEGF, or vascular endothelial growth factor, is a hypoxia-induced endothelial cell-specific mitogen produced in ischemic retinal cells.⁴  High levels of VEGF in the eye can lead to abnormal blood vessel growth and blood vessel leakage.[iii]  VEGF₁₆₅ is responsible for the pathologic angiogenesis leading to disease.[iv]  By binding to VEGF₁₆₅, Macugen is able to prevent it from binding to its receptor. 

            Macugen is currently being tested as a drug to treat age-related macular degeneration (AMD) and diabetic macular edema (DME).  Both diseases result in the loss of central vision, but leave peripheral vision intact.  The wet form of AMD is caused by a growth of blood vessels from the choroid behind the retina.¹  These vessels push on and bleed into the retina which results in the loss of vision.³  DME results from diabetic retinopathy.  Poorly controlled blood glucose levels result in abnormal blood vessel growth and excessive leakage from vessels.  The outcome is similar to AMD.  By inhibiting VEGF, Macugen reduces abnormal blood vessel growth and blood vessel leakage in the retina.  When used for treatment, the aptamer is pegylated by being bound to a 40kD polyethylene glycol to increase its half-life.¹ 

Currently, Phase 2 and 3 clinical trials are underway.  Initial phase 2 trials have had few adverse immune affects.  Side effects, which included anterior chamber inflammation and vitreous floaters, were mild in nature.[v]  The results of clinical trials completed thus far have been very positive.  Figure 1 contains the results of completed trials for AMD and DME.  Success was measured by a stabilization of vision or being able to read more lines on the standard eye-chart.⁵

            Eyetech, started in 2000 and headed by David R. Guyer, M.D., completed its initial public offering in February 2004.  Eyetech has partnered with Pfizer to develop and market Macugen.1  Overall, studies have shown that patients benefit from treatment with Macugen for wet AMD and DME.  Phase 3 clinical trials are ongoing and the FDA accepted Eyetech’s NDA for Macugen in August 2004.  Hopefully, Phase 3 trials will be successful and Eyetech will be able to get this revolutionary new treatment out to the public so that the devastating effects of retinal eye diseases can be further combated and people’s lives improved. 

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Alisa Nakamine            Vaccinex    Dec. 6, 2005              Biotechnology       W3034 Homework 4

Vaccinex, co-founded in 1997 by Maurice Zauderer, specializes in a method to directly express complete, fully human antibodies in mammalian cell lines using cDNA library of human immunoglobulin heavy and light chain genes.

Generating the cDNA library^1,2

The company overcame the problem with low frequency of recombination for conventional vectors by the use of poxvirus as the vector.  “Tri-molecular recombination” method produces almost 100% recombination frequency.  This method uses vaccinia virus gene as the vector, and fowlpox virus-infected cells, usually BSC1 from African Green Monkey, as the host.

            Using poxvirus expression vectors has two major advantages.  One is that the vaccinia virus used as the vector has very high infectivity, allowing low-frequency recombinants to be selected from few cells.  Also, recombinant genes can be recovered from cells that are dead or no longer dividing.  This enables the method to be used to express genes that are highly toxic.  For example, this method has been applied to the expression of the cytotoxic T-cell target antigen gene.

Generating the fully human monoclonal antibodies³

Each gene from the human immunoglobulin library (heavy chain and light chain genes separate) are encapsulated in different vectors, and the mammalian host cells, engineered to increase the membrane level expression of antibody receptors, are infected by the vectors.  The host cells assemble the expressed heavy and light chain genes into functional membrane antibodies.  The antigen of interest is introduced to the culture of those cells, and those cells with matching antibodies for the antigen are selected via high speed cell sorting.  This cycle is repeated several times to find the set of antibody heavy and light chains that exhibit the highest affinity to the given antigen.  

            The advantages for this method include: very broad range of antibodies, high affinity, fully human, and functional assembly of the antibodies due to the use of mammalian cells as hosts.  On their website, http://www.vaccinex.com, there is a comprehensive Flash animation of this technique.

            The company has recently won its second $2.0 million grant from the Advanced Technology Program of the National Institute of Standards and Technology.  They hold two patents^4,5 and has about 12 patents in the application process⁶.  Their goal is to produce its own therapeutic vaccines and antibodies, with primary focus on cancer treatment.  At this point, the company gets its revenues from grants and partnerships with other biotechnology companies where Vaccinex provides the technology. 

References
1.         Smith ES, Shi S, Zauderer M. 2004. Construction of cDNA Libraries in Vaccinia Virus.  Methods in Molecular Biology. 269: 65-76.
2.         Smith ES, Mandokhot A, Evans E, Mueller L, Borrello M, Sahasrabudhe DM, Zauderer, M. 2001. Lethality-based selection of recombinant genes in mammalian cells: Application to identifying tumor antigens. Nature Medicine. 7(8): 967-972.
3.         “Antibody Technology Animation.” Technology. <http://www.vaccinex.com> Dec. 3, 2005.
4.         Methods of selecting polynucleotide encoding antigens.  US patent no. 6,800,442, October 5, 2004.
5.         Methods for producing polynucleotide libraries in vaccinia virus.  US patent no.6,706,477, March 16, 2004
6.         United States Patent and Trademark Office. <http://www.uspto.gov/patft/index.html>Dec.3, 2005.

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Sally A. Nicholas          Biolex             Biotech Homework #4            6^th December ‘04

Biolex is a privately owned, biopharmaceutical company, located in North Carolina. This company is producing human therapeutic proteins (cytokines, monoclonal antibodies), that are difficult to make in other expression systems. Biolex is able to achieve this through the use of their patented “Lex system.”

The technology of the Lex system enables Biolex to produce proteins at a faster and less expensive rate than existing technologies, eventually producing products suitable for clinical trials and subsequent commercial supply. The Lex system is built upon the characteristics of the small, aquatic plant family, Lemna. These plants proliferate via a vegetative budding of new fronds in a manner analogous to asexual reproduction in yeast. They therefore provide a differentiated plant system that can be manipulated with the laboratory convenience of yeast (1).

The transformation of these plants with expression cassettes, and the subsequent process by which the relevant proteins are obtained, are not significantly different from that seen in microbial expression systems or mammalian cell lines. However, there are certain characteristics of these Lemna plants, that ensure that the process is carried out faster and cheaper. These characteristics include:

• clonal reproduction – genetic stability without pollen or seed
• fast – doubles biomass every 36 hours(making scale-up to commercial production levels feasible, and cheaper than in corresponding mammalian systems)
• high expression levels
• the target protein is secreted which minimizes downstream processing
• inexpensive – low operating cost
• plants demonstrate post translational processing and can assemble multi-subunit proteins. These characteristics cannot be obtained with microbial cell systems (2).

The process for protein creation in the Lex system is shown below (from Biolex website):

From the above timeline, one can see that within 6 months this technology leads to the creation and selection of a high expressing, genetically stable line. Other transgenic systems, such as corn and goat systems, take 24 and 36 months respectively (2).

The automation of this system, which will of course be crucial for large scale commercial production, has already been achieved, by the use of automation principles now standard to mammalian cell expression systems (3).

Biolex thus provides a system whereby:

1)      the timing from gene to commercial line is < 6 months

2)      all the benefits of mammalian lines are available simply and inexpensively

3)      facility costs are significantly reduced

4)      hard to make proteins can be commercialized.

To date, the more than twenty proteins attempted, have been successfully expressed using the Lex system. It is on the strength of this system that Biolex has entered into partnerships with Bayer, Centocor Inc and other biotech and pharmaceutical companies.

Biolex has a number of proteins (mainly monoclonal antibodies) that are currently being optimized, and an alpha-interferon 2b protein that is presently undergoing pre-clinical trials.

Worth noting is that Biolex by its acquisition of Epicyte Pharmaceutical in May 2004 has acquired the dominant intellectual property position covering the production of antibodies in plants (2).

References :
1)      Patent Cooperation Treaty (February 7^th 2002). Expression of Biologically Active Polypeptides in Duckweeds. WO 02/10414 A2
2)      Biolex Inc.(2004). The New Gold Standard for Therapeutic Proteins. http://www.biolex.com
3)      Patent Cooperation Treaty (December 5^th 2002). Use of Duckweeds in High Throughput Screening. WO 02/097433

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Peter Nguyen               Curis Inc. (CRIS – ticker symbol)   Biol. W3034

Company background

Curis, Inc. is a development-stage biotech companies focusing on the development of novel signaling small molecules and antibodies that target large markets including oncology, neurology, renal disorders, cardiovascular diseases, and diabetes.  The Company was formed in July 2000 as a result of a three-way merger – Creative, Ontogency, and Reprogenesis.

Technology and proposed mechanism of action

The company focuses primarily on signaling proteins, specifically the hedgehog and bone morphogenic protein.  These proteins play a crucial role in providing the necessary signals for the proper development of various tissues and organs during embryonic development. For the hedgehog protein, it is required for tissue maintenance and regeneration in adult development.  Consistent with its function, Hh activity is highest during embryonic development, stabilizes at a reduced level postnatally and into adulthood, and likely falls off during old age.  Additionally, the Hh pathway is activated to a high level in response to tissue injury or stress.

 In the hedgehog signaling pathway, the Hh protein binds to specific receptors (patched PTC-PTCH1, PTCH2) on the cell’s surface, thereby initiating a cascade of downstream reactions that involve the activation of a number of transcription factors (through phosphorylation).  The hedgehog receptor PTCH1 is also a target gene of this pathway, which forms a negative feedback mechanism to maintain the pathway activity at an appropriate level in a given cell.This signaling cascade ultimately leads to the regulation of gene expression (either the activation or silencing of various genes).  For example, in tissue maintenance and repair, the action of the Hh protein results in the expression and subsequent secretion of various growth and angiogenic factors.  Below is a picture of the pathway for the bone morphogenic protein, which has a similar mechanism to the hedgehog pathway.

CRIS’s scientists have developed strategies to either down-regulate the Hh pathway by blocking (i.e., antagonize) the Hh receptors or, conversely, up-regulate the Hh pathway by mimicking Hh protein function (i.e., agonize).  For example, antagonists are designed for the treatment of basal cell carcinoma, where the majority of the cases showed an abnormal high expression level of the hedgehog protein.

An example of the agonist program is use for the treatment of Parkinson’s disease.  In Parkinson patients, dopamine-producing cells in the substantia nigra region of the brain progressively deteriorate and die.  Dopamine is a neurotransmitter and serves as a chemical messenger that transmits signals to the cerebellum, which is the area of the brain that is responsible for the coordination and control of voluntary movements.  The deterioration of dopamine-producing cells results in the reduction of dopamine levels and, therefore, a defect in signals to the cerebellum, causing the PD patients to experience motor movement symtoms such as trembling, rigidity, and slowness of movement.

As discussed above, Hh proteins are highly active during prenatal and early childhood development but tend to diminish with age.  CRIS’s Hh agonist small molecule program is design to mimic the Hh proteins to up-regulate the signalling pathway and produce the necessary growth and angiogenic factors for the repair and regeneration of nerve and tissue cells.  CRIS’s Hh agonist small molecules are able to cross the blood brain barrier and are believed to mimic the action of the Hh proteins to initiate a series of signalling pathways in the substantia nigra region to repair and stimulate dopamine-producing cells.

Partnerships and cash position

The Company focuses its energy on the development of the signaling pathways (Hedgehog and BMP-7) and has partnered with some of the biggest names in the pharmaceutical and biotechnology industries including Wyeth, Ortho-Biotech (Johnson & Johnson), and Genentech. Their pre-clinical programs are listed below:

                       Source:  Curis, Inc.

The company just recently raised some money in the secondary market and currently has $40 million in cash.  With a burn rate, which is the amount of cash needed for R&D and operations, of $16 million for 2005, the Company has enough cash until the end of 2006 even if it’s unable to raise any additional cash.  In addition to its world-class partnership, strong cash position, and near term clinical testing of its BCC program, the Company is the beneficiary of many independent publications across the academic world into the importance of the signaling proteins.  We know in biotech world, positive news flow often provides great catalyst for the Company’s stock price, at least in the short term. 

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Si-Wei Chen    Rinat Neuroscience    12/01/2004

            Rinat Neuroscience Corporation, based in Palo Alto, California, is a biotechnology company dedicated to the discovery and commercialization of novel drugs and drug targets for the major mental and neurodegenerative disorders. It is also the first company to focus itself exclusively on developing neuroscience-based protein therapeutics (1).

Although Rinat was founded only three years ago, it had a head start in positioning itself for success in the neurology market. In 2001, Genentech, a leader in the development of protein therapeutics, narrowed its focus and spun out its key neuroscience assets under a broad license to Rinat. Genentech had invested significant R&D resources in support of these assets over 15 years. Building on what it inherited from Genentech, Rinat is currently developing protein therapeutics for acute and chronic pain, Alzheimer’s disease, obesity and diabetes, migraine, and neuropathy (1).

RI 624 is Rinat’s first potential product in the clinic. It is a recombinant, humanized antibody designed to treat acute and chronic pain, including post-surgical pain, cancer pain, and chronic arthritic pain. The antibody was generated in mice as a standard mouse monoclonal antibody. The appropriate CDRs (complementarity determining regions) were then grafted onto a human antibody framework followed by subsequent improvements in affinity through a maturation process (2). RI 624 works through a novel mechanism of action that is distinct from that of all currently approved drugs for the treatment of pain – the inhibition of Nerve growth factor (NGF). Pain-causing injury or inflammation induces the synthesis and stimulates the release of NGF. NGF in turn upregulates expression of neuropeptides in sensory neurons (3), and its activity is mediated through two different membrane-bound receptors, the TrkA tyrosine kinase receptor and the p75 receptor (4). From human and animal studies, researchers have observed that NGF has profound direct and indirect stimulatory effects on the primary sensory neurons that mediate pain. Blocking NGF considerably increases pain threshold in animal models (5). In preclinical models of pain, RI 624 has demonstrated equal or better efficacy than opiates or NSAIDS (non-steroidal anti-inflammatory drugs) with none of the many adverse effects such as reduced gastrointestinal motility, constipation, respiratory depression, sedation and confusion, nausea or vomiting that are often associated with those drugs (1). Rinat initiated Phase I clinical studies with RI 624 in patients with osteoarthritis of the knee on June 23^rd of this year.

            RI 1219 is a monoclonal antibody that Rinat is studying for the treatment of Alzheimer’s disease. By binding preferentially to amino acids 28-40 of Aβ40, RI 1219 significantly reduces amyloid plaques in the brain and improves cognitive function of the animals in pre-clinical testing (6). Rinat is currently in the process of humanizing this antibody. RI 450, a protein that has been shown to lower glucose, triglycerides, and weight, appears to have anti-obesity and anti-diabetic effects in various animal models of Type 2 diabetes. Rinat plans to conduct further pre-clinical testing of RI 450 related to these indications and believes that it could be ready for an IND filing in the first half of 2006 (1).

References            
1.      http://www.rinatneuro.com
2.      Pons et al. Anti-NGF antibodies and methods using same. United States Patent Application Publication. Pub. No. 20040237124. Nov 25, 2004.
3.      Lindsay R.M., Harmar A.J. (1989). Nerve growth factor regulates expression of neuropeptide genes in adult sensory neurons. Nature 337, 362-364.
4.      Chao M.V., Bothwell M.A., Ross A.H., Koprowski H., Lanahan A.A., Buck C.R., Sehgal A. (1986). Gene transfer and molecular cloning of the human NGF receptor. Science 232, 518-521.
5.      Klyushnik T.P., Krasnolobova S.A., Sarmanova Z.V., Shcherbakova I.V., Morozov S.G., Gribova I.E. (2004). Effect of antibodies to nerve growth factor and serum albumin on the development and behavior of mice. Bull Exp Biol Med. 138, 84-86.
6.      Rosenthal et al. Methods of treating Alzheimer's disease using antibodies directed against amyloid beta peptide and compositions thereof. United States Patent Application Publication. Pub. No. 20040146512. July 29, 2004.

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John-Paul Bellistri         Viacord           12/1/04

Viacord¹ was created in 1993 by its parent company Viacell Inc.  The company now stands as the world’s largest cord blood preservation and cellular therapeutic company.  Both Angen and Genzyme are financial partners with Viacord.  Viacord provides services for parents wishing to preserve umbilical cord blood (UCB) of their newborn.  Stem cells extracted from UBC are currently being used to treat over forty types of immunodeficiencies and blood-related diseases.  Like embryonic stem cells, stem cells from UCB are pluripotent and are capable of forming all types of tissue.  Unlike, embryonic stem cells, there is no moral concern when dealing with UCB. 

            The procedure for preservation of umbilical cord blood is as follows: at birth, the doctor places the umbilical cord in a vacuum sealed bag.  The bag is then picked up at the site of birth by a company representative and delivered to the cryogenic facilities within 24-hours.  The blood is prepared and cryogenically frozen².  Charges for this procedure include an initial fee of $1,650, plus $150 for transportation, and an annual fee $125. 

            In 1989, Broxmeyer et al³ were the first to show the potential of deriving hematopoietic stem cells from UCB.  Broxmeyer’s study showed that UCB cultures in the presence of stimulating factors such as colony stimulating factor, interleukin 3, and erythropoietin showed significant growth and formation of hematopoietic progenitor cells.  Broxmeyer is currently a member of the medical advisory board for Viacord. 

This novel usage of UCB now provides a viable alternative to bone marrow transplants; no longer is donor rejection an issue.  In most cases, stem cells derived from UCB can even be used to treat illness in siblings, parents, and grandparents.  Viacell⁴ is currently working on a new method cultivating stem cells, which is called selective amplification.  This method of stem cell cultivation is in Phase I of clinical testing.  The purpose of such a technique is to transcend the boundary of only being able to derive hematopoietic stem cells from the UBC.  With this technique, Viacell hopes to be able to derive cells of different functionality from UCB, such as nerve cells or cardiac cells. 

References

1. http://www.viacord.com/
2. Halle, P. et al. Uncontrolled-rate freezing and storage at -80^oC, with only 3.5-percent DMSO in cryoprotective solution for 109 autologous peripheral blood progenitor cell transplantations. Transfusion 2001;41:667-673.
3. Broxmeyer, H.E. et al. Human umbilical cord blood as a potential source of transplantable hematopoietic stem/progenitor cells. Proc. Natl. Acad. Sci. USA 1989;86:3828-3832.
4. http://www.viacellinc.com/

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Akiva Felt. Sirna Therapeutics.  Dec 1, 2004.

Sirna Therapeutics focuses on the research, development, and clinical testing of RNAi and its potential to treat disease. Since its establishment ten years ago, the company has amassed over 180 patents, 50 of which deal directly with RNAi. These 50 patents include synthesizing RNAi, stabilizing RNAi constructs, and the use of RNAi as a therapeutic agent.

Sirna Therapeutics recently filed an investigatory new drug application with the FDA for its lead RNAi therapy, Angiozyme. The drug works by blocking the protein formation of the vascular endothelial growth factor receptor (VEGFR) through siRNA interference with the protein’s mRNA.  VEGFR is a critical component for blood vessel proliferation.  Since tumors require sustained angiogenesis for growth, inhibiting VGEFR is hoped to prevent tumors from growing and metastasizing. Currently in phase II clinical trials for treatment of breast cancer, Angiozyme has so far stabilized tumor growth in 17 of the 33  (61%) patients enrolled.

The company’s other drug currently in clinical trials is Heptazyme, a hepatitis C treatment. Heptazyme directly interferes with the production of HCV RNA. The results of this clinical trial are expected shortly.

Sirna Therapeutics’ R&D division is actively pursuing an RNAi treatment for age-related macular degeneration. AMD, the leading cause of blindness in the world, can occur from a sudden hemorrhage of blood or fluid from abnormal vessels that have grown beneath the retina. The RNAi drug being developed is similar to Angiozyme in that is prevents unwanted angiogenesis. However, the target mRNA to be degraded is for the actual VEGF and not the receptor.

With its wide breadth of patents and drug collaborations, Sirna Therapeutics would certainly benefit from the widespread use of RNAi as a disease treatment.

Sources:

Foster, G. Past, Present, and Future Hepatitis C Treatments. Semin Liv Dis 2004; 24: 97-104.

Tezel, T. Pathogenesis of Age-Related Macular Degeneration. Trends in Mol Med 2004; 10 (9): 417-421.

http://www.sirna.com

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Yelena Luzhanovskaya           ImClone Systems Incorporated      12-06-04

Twenty years ago ImClone had opened their first laboratories in Soho, New York. Today it is a large successful biotech company operating in three locations–corporate headquarters New York City, chemistry facility in Brooklyn, and R&D and manufacturing campus in Branchburg, New Jersey.

In its early years, ImClone focused on immunology-based diagnostics and infectious disease vaccines and several of these vaccine research programs were FDA approved, all of which have been licensed to pharmaceutical companies

During the early nineties, the company’s attention shifted to the development of a pipeline of innovative, biologic compounds in the area of oncology. Currently ImClone focuses on three strategies for treating cancer: growth factor blockers, angiogenesis inhibitors and cancer vaccines¹.

In February 2004 the FDA approved the ImClone lead product, Erbitux (Cetuximab), the monoclonal antibody that is used in combination with chemotherapy in the treatment of patients with epidermal growth factor receptor (EGFR)-expressing metastatic colorectal cancer². Since the approval this product has generated profit of $127 mill already³. Now Erbitux is in the clinical trials for earlier stages of colorectal cancer, head & neck cancer, pancreatic cancer, and non-small cell lung cancer.

ImClone's next potential products are therapeutic cancer vaccines. BEC2 is currently being in clinical studies phase III for small cell lung cancer and has been shown to have the potential to stimulate the body’s immune system to identify and eliminate residual tumor cells, prevent recurrence of tumors, and prolong survival in this tumor type. BEC2 is the anti-anti-GD3 antibody that mimics tumor antigen GD3, a ganglioside found on cell surface of small cell lung cancers. By mimicking this antigen, BEC2 appears to stimulate a stronger immune response to cells expressing natural GD3. BEC-2 is injected intradermally into the subject along with nonspecific immunologic adjuvant bacillus Calmette-Guerin (BCG) that is supposed to boost the immune response. After the injection the immune response resulted in antibodies to BEC-2 that cross-reacted with GD3. As clinical trials have demonstrated all injected patients develop anti-BEC-2 antibodies and 40% develop anti-GD3 antibodes. Overall rate of survival of those vaccinated patients was better than expected especially for those patients with limited-stage disease^4,5.

Another ImClone’s cancer vaccine GP75 is designed to elicit an immune response against melanoma cells and prevent growth of melanoma tumors. It is a recombinant DNA vaccine that contains plasmid DNA with genetic code for gp75⁶, a glycoprotein synthesized by pigmented melanocytes and melanomas but not by other cells⁷. Recombinant plasmid DNA is directly injected into the hosts muscle tissue where its transcription and translation takes place. The pre-formed antigen is then captured by antigen-presenting cells (APC) and a strong cytotoxic T lymphocytes response is produced to gp75 and melanoma cells that express this antigen. Studies have shown that immunization with GP75 can protect subject from growth of melanoma tumors and metastases^6-8. This vaccine is in clinical trials phase I in patients with malignant melanoma.

The preclinical pipeline of ImClone is also full. Currently they are investigating angiogenesis inhibitors that target VEGF-2, and growth factors blockers that target insulin-like growth factor-1 receptor (IGF-1R) and vascular endothelial growth factor receptor-1(VEGFR-1 or flt-1), leading to inhibition of ligand-dependent signaling to the cell in human breast, colorectal and pancreatic tumor cell lines.

1.  References:
www.ImClone.com
www.Erbitux.com
www.finance.yahoo.com
Krug LM. Vaccine therapy for small cell lung cancer. Semin Oncol. 2004 Feb;31(1 Suppl 1):112-6.
Chapman PB. Vaccinating against GD3 ganglioside using BEC2 anti-idiotypic monoclonal antibody. Curr Opin Investig Drugs. 2003 Jun;4(6):710-5.
J. D. Wolchok J.D., Livingston P.O. Vaccines for melanoma: translating basic immunology into new therapies. Lancet Oncol. 2001 Apr;2(4):205-11.
Hirschowitz EA, Crystal RG. Adenovirus-mediated expression of interleukin-12 induces natural killer cell activity and complements adenovirus-directed gp75 treatment of melanoma lung metastases. Am J Respir Cell Mol Biol. 1999 May;20(5):935-41.
Kim CJ, Dessureault S, Gabrilovich D, Reintgen DS, Slingluff CL Jr. Immunotherapy for melanoma. Cancer Control. 2002 Jan-Feb;9(1):22-30.

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Miki Kim  Biotechnology W4034 - Homework 4  Diversa Corporation           Dec. 13, 2004

            Diversa Corporation is a biotechnology company that uses its proprietary genomic technologies to discover and optimize novel products from genes and gene pathways.  These technologies include: Gene Site Saturation Mutagenesis (GSSM), a technology that creates a family of related genes that differ from the parent gene by at least a single amino acid change; Tunable GeneReassembly, a gene shuffling technology that can reassemble related or unrelated genes from two or more different species or strains; and GigaMatrix, an ultra high-throughput screening technology which can screen 1 billion clones per day [1].

            Diversa has a unique idea and business strategy: in addition to using its technologies to produce molecules with pharmaceutical applications, Diversa also develops enzymes and small molecules with agricultural, chemical and industrial applications.  For example, in July 2004, Diversa began marketing the enzyme, Luminase, to aid the paper and pulp industry [2].  Luminase, a xylanase originally discovered in Russia, was modified to have a broad temperature and pH range [1] [3].  Luminase enhances the reactivity of pulp fiber to bleaching chemicals and reduces the amount of toxic chemicals needed in the bleaching process [2].

            Diversa is also unique because its researchers travel to all corners of the earth to discover robust enzymes that are able to tolerate extreme environmental stress and can be engineered to withstand industrial processes and process toxic chemicals.  These enzymes are added to a library of rare and exotic genes that includes additions from Costa Rica and Antarctica.  Luminase was discovered and engineered in this manner [3].

            Diversa focuses on the areas of animal care, fiber processing, nutritional oils and anti-infective therapies for product development [4].  Diversa has formed alliances and joint ventures with companies such as The Dow Chemical Company and DuPont Bio-Based Materials.  Some of Diversa’s products include: Phyzyme XP Animal Feed, Cottonase, Pyrolase 200 Enzyme and Pyrolase 160 Enzyme [1][4].

References:
http://www.diversa.com/
PR Newswire, July 15, 2004, Financial News, “Diversa Launches Luminase™ Enzyme for Improved Pulp Bleaching.”
Weintraub, Arlene. “Biotech Heads for the Factory Floor,” BusinessWeek, Aug. 2, 2004.
http://finance.yahoo.com/q/pr?s=DVSA

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Eun Young Choi  Trubion Pharmaceuticals, Inc.  Biotechnology, W3034   Dec. 13, 2004

            Trubion Pharmaceuticals, Incorporated (Trubion), was created in Seattle, WA, in 2002 and have developed a novel class of compounds called Small Modular ImmunoPharmaceuticals (SMIPs™), which may be the next generation of therapeutic antibodies. SMIPs are condensed versions of antibodies and composed of modules, different parts of naturally-occuring proteins, to form one chain of Fc and Fab regions (Fig. 1). As a result, SMIPs can be designed to bind to specific antigen and have specific mechanisms of action such as the recruitment of complement or effector cells and the inhibition or activation of signaling pathways (Fig. 2).

Trubion, without showing any experimental data, makes strong assertions about the abilities of SMIPs based on studies done on rodents and non-human primates: SMIPs have greater potency and faster tissue penetration than antibodies, enter areas unreachable by antibodies due to their lighter weight, and have lower levels of aggregation and host immunoreactivity, which are the problems of many therapeutic antibodies. In addition, Trubion states that SMIPs can be produced in mammallian cell systems at levels greater than four times that of current antibodies. Hence, SMIP production (but not engineering) is more cost-effective than that of current antibodies.

The three most advanced SMIPs are TRU015-017. TRU015 targets CD20, a receptor on normal and malignant B-cells, and causes the death of these cells more effectively, claims Trubion, than current drugs on the market. TRU015 is scheduled for investigational new drug filing and human trials in 2004. TRU016 targets a different receptor on B-cells and is in the preclinical development phase. TRU017 targets a non-B-cell receptor and is intended for autoimmune diseases; it has just finished the research phase.

Trubion is a company whose development I would like to continue following because SMIPs have the exciting potential to be more potent, efficient, and cost-effective treatments with less side-effects than the current antibody therapies. However, Trubion is in great need of experimental data to lend support to, much less prove, their claims and thereby gain investors. Furthermore, a limitation of this technology, even if successfully developed, still relies on detailed knowledge of the antigen and disease progression.

 References:

1.       Trubion Pharmaceuticals, Incorporated. www.trubion.com

2.     “Trubion Pharmaceuticals Inc. Unveils Biotechnology Platform Called SMIPs(TM).” Biospace. Nov. 18, 2003.

            http://links.biospace.com/news_story.cfm?StoryID=14421020&full=1

Figure 1. A comparison of the general structures of a naturally-occuring antibody and a SMIP.

Figure 2. SMIPs can be engineered to have a specific mechanism of action by choosing the appropriate Fc region.

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Asya Haney                                  Cytos Biotechnology                                            December 12, 2004

 Cytos Biotechnology was founded in 1995 as a spin-off of ETH Zurich. It is located in Switzerland. This company specializes in the development of Immunodrugs - a new class of vaccines that teaches patient’s body to respond to unwanted self-molecules as it encounters them, by producing desired therapeutic antibody or cytotoxic T cell response. They think it is a logical idea and is of a great benefit. It can eliminate the need of passively administering monoclonal Ab produced elsewhere into the patient, reducing cost, side effects and creating a greater compliance from patients by administering vaccines on a yearly basis. The way to make the body to produce auto-antibody to self-antigen has been achieved by several vaccine formulations, including conjugates of self-antigens to foreign T helper (Th) cell epitopes, virus-like particles coated with self-antigens, and naked DNA vectors. [2] Another one of their finding is DNA rich in nonmethylated CG motifs (CpGs) greatly facilitates induction of immune responses against coadministered Ags [5]. That’s one extra trick to increase chances of producing immune response to the self-antigen administered.

Cytos Biotechnology has no existing vaccines available. They have built a pipeline of 23 different Immunodrug candidates in various disease areas. It is being developed both in-house and one of them together with Novartis (Alzheimer’s disease). They have a few that are currently in phase I and II clinical trials.

The company’s lead Immunodrug candidate is CYT002-NicQb. It is a therapeutic vaccine designed for treatment of nicotine addiction. Nicotine exerts its addictive properties by stimulating in neurons the specific regions in the brain to release neurotransmitters, which give rise to an almost immediate reward and a feeling of pleasure. This sensory stimulus is critical to the addictive properties of nicotine and causes a high relapse rate after quitting attempts. [3]

Vaccination with CYT002-NicQb aims at induction of nicotine-specific antibodies that bind nicotine in the blood and reduce nicotine uptake into the brain. In this way, stimulation of nicotine-perceptive neurons in the brain should be prevented and the  addiction-driving and satisfaction-inducing stimulus of nicotine reduced. [1]

It is currently in phase II clinical study. In a clinical phase I study as well as in preclinical models (mice), CYT002-NicQb was shown to induce high nicotine-specific antibody levels.

 There are a number of companies which are also currenly in clinical trials for nicotine conjugate vaccine like British Biotechcompany Xenova Group and Nabi Bioprarmaceuticals.

Another Immunodrug candidate is CYT007-TNFQb is a therapeutic vaccine for the treatment of psoriasis and rheumatoid arthritis. It is currently in combined phase I/II clinical trail. Psoriasis is a common skin disorder it affects 1-3% of population. It is a chronic, T-cell mediated disease, which is characterized by massive infiltration of immune cells into the skin causing inflammation. Vaccine is designed to instruct the patient’s immune system to produce a specific anti-TNF-α antibody response and thereby helping the body to slow down the inflammatory process and inhibit skin or joint deterioration. [1] Preclinical experiments have shown that antibodies induced by CYT007TNFQb bind and neutralize endogenous

TNF-α.

Other Immunodrugs candidates that are currently being researched and developed are vaccines against allergies, asthma, hypertension (inhibition of angiotensin II), obesity (regulation of appetite and fat metabolism), cancer, osteoporosis (bone resorbtion), infectious diseases (HIV, hepatitis C, malaria), fertility (hormone regulation) and Qbeta (carrier).

On September 21, 2004 Cytos Biotechnology was awarded third prize of the European BioTechnica. They have ongoing collaborations with Madarex, Novartis and Solidago.

They reason I picked this company as a company whose ideas I am impressed with and would like to work for, is for me personally it is more appealing to help a greater number of people with small everyday problems and participate in creating a more comfortable and easier everyday life.

References:

1. http://www.cytos.com/
2. http://www.cytos.com/doc/Immunodrugs.pdf

3.   Bachmann MF, Dyer MR. Therapeutic vaccination for chronic diseases: a new class of drugs in sight. Nat Rev Drug Discov. 2004 Jan;3(1):81-8.

4. Jennings GT, Bachmann MF. Immunotherapies: cause for measured optimism. Drug Discov Today. 2002 Oct 1;7(19):994-6.
5. Storni T, Ruedl C, Schwarz K, Schwendener RA, Renner WA, Bachmann MF. Nonmethylated CG motifs packaged into virus-like particles induce protective cytotoxic T cell responses in the absence of systemic side effects. J Immunol. 2004 Feb 1;172(3):1777-85
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Stephan Kadauke     Celera Genomics: Then a Genomics Pioneer, Now some Mid-Profile Biotechnology Company   11/22/2004

Founded in May 1998 by the notorious J. Craig Venter, Celera Genomics was formed for the purpose of generating and commercializing genomic information to accelerate the understanding of biological processes. [1] The company that derived its name from the Latin word for swiftness entered into a race to sequence the 3.2 billion letters that comprise the human genome against the mutinational and publicly funded Human Genome Project. Personal differences, for the most part, made collaborations between the two efforts impossible. Celera announced its surprising victory over the rivaling public effort in the race for the sequencing of the human genome in early April of 2000, months ahead of schedule [2]. Critical for this success was the application of whole genome shotgun sequencing, an approach that left out the preliminary mapping of BACs containing intermediate-sized fragments of the genome, which was part of the HGP's approach.

The announcement led to a stellar rise of corporate value just after the announcement that Celera had completed the first draft of the human genome sequence. Stock value, however, soon plummeted back to a twentieth of the peak value in 2002 [3]. Celera's executive board saw itself forced to change the business plan from Venter's genomics-bioinformatics model, which could only produce income by selling subscriptions to services analyzing sequence information, to a pharmaceutical one, which could provide income by selling drugs, an avenue viewed more lucrative by many. Craig Venter fiercely opposed this move, but was fired in January 2002 [4].

Celera Genomics now employs proteomics to identify proteins that are differentially expressed on the surface of cancer cells and has found 200 such proteins in the years 2003-2004. Its scientists are now in the process of validating which ones of these proteins could be utilized as targets for synthetic small-molecule drugs or monoclonal antibodies that could coat the cancer cells, flagging them to be destroyed by the immune system. The company now prides itself with having discovered an inhibitor of histone deacetylase, a protein involved in the onset of many types  of cancer [5].

Celera has started as an enterprise dedicated entirely to genomics and bioinformatics but has then completely changed its business plan towards the more traditionalistic approach of small-molecule drug discovery and antibody therapy. Likely, Celera Genomics changed from being the world's most famous genomics corporation to a mid-profile biotech company because of the temporary nature of the historical landmark sequencing effort. In the end, Craig Venter has used Celera as a vector to realize his vision to sequence the human genome. Most of the DNA used was his own [6].

References:
[1] Wikipedia.org: “Celera Genomics”
[2] Wired News, April 6, 2000. “Celera Wins Genome Race.”
[3] Yahoo Finance Stock Qutoes: CRA (http://finance.yahoo.com/q/bc?s=CRA&t=my&l=off&z=l&q=l&c=)
[4] Bio-IT World, Nov 2002. “John Craig Venter Unvarnished”
[5] Celera Web Site: “About Us” (http://www.celera.com/celera/about)
[6] Wikipedia.org: “Craig Venter”
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Tiffany Chang     Syngenta       Biotechnology        Homework #4

          Syngenta is an agriculture biotechnology company. It is the leader in crop protection and ranked in third in the high value commercial seeds market. In 2000, Novartis agribusinese and Zeneca agrochemical merged to form Syngenta.

The main office of Syngenta is located in Switzerland.

          One of the biotechnology tools that Syngenta uses is plant transformation, which is inserting a gene into plant genomes. This can be done by two methods. The first method is by using Agrobaterium. The second method is by using mechanic means called Biolist transformation.

Agrobacterium tumefaciens is a Gram-negative, non-sporing, motile, rod-shaped bacterium, which causes crown gall (a tumor like structure) disease in plants, and the bacteria integrate its DNA into plant host. The disease does not cause serious damage in adult plants.  The genes that involved in crown gall disease are mostly on a large plasmid called Ti plasmid (tumour inducing plasmid). A. tumefaciens infects only through the would site because the T_i plasmid respond strongly to the  phenolic compounds such as acetosyringone  and chemotaxis at the would site. Then Ti plasmid can be activated and slip some of its DNA past the plant's natural defenses and into the plant's own DNA. So Agrobacterium tumefaciens with genetic engineered Ti plasmid allows an insectional toxic gene or herbicide resistance gene can be inserted into plant genome easily.  The selection of successfully transformation can be done by two methods. The first method is placing the transformed cells onto a media with herbicide resistance select for survival-transformed cell. The second method is inserting a reporter gene (such as green fluorescent protein gene) on the Ti plasmid and finds the positive signal in transformed tissues.

          The other biotechnology method that Syngenta used is called marker technology. A marker is a specific region on DNA that is linked or associated with a particular trait.  Thus, markers are useful to identify the presence of a specific trait. In plants, markers can be identified before plant is full-grown.

          One of Syngenta’s many products is Bacillus thuringiensis corn, short for Bt corn. Syngenta picked corn because corn has been used in diet for livestock and humans for a long time. Bt corn has a higher resistance to pests, which leads to higher yields and better quality.  Corn borer is one of the most destructive pests to damage corn, and once the corn borers get into corn, nothing can stop the damage. The mechanism that Syngenta used to make BT corn is through nautrally occurring soil bacteria called Bacillus thuringiensis. Bacillus thuringiensis has a special mechanism to produce proteins to protect from attack of specific insects and stay non-toxic to the host. Cry1Ab is one of the proteins that is produced by the bacteria, which can prevent the attack from corn borer. The safety of Bt corn has been thoroughly assessed in development. And the assessed is still going on in the areas that grow Bt corns.

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Tanaz Sharifnia      Vical Inc.     December 8, 2004

             Vical Inc. is using its patented naked DNA gene transfer technology to research and develop a number of DNA vaccine candidates. The company’s current product development efforts are directed at infectious-agent and cancer-immunotherapeutic applications of these novel vaccines (1).

             DNA vaccine research has been propelled by the observation that the injection of plasmid DNA into muscle tissue can effect sufficient transient expression of plasmid-encoded genes to elicit an immune response. In some cases, this response is large enough to confer protective immunity to the host. By constructing plasmids that encode an immunogen, downstream from a strong constitutive promoter, the injection of these plasmids into a subject can result in the expression and cell-surface presentation of the desired antigen. In this way, these cells become targets for cytotoxic T lymphocytes and stimulate the development of cell-mediated immunity. DNA vaccines warrant study not only for preventative applications, such as those against infectious diseases that cannot be safely or effectively vaccinated against using traditional methods, but also because they may potentially have therapeutic applications as well. In cancer, for example, plasmid expression of foreign MHC class I and tumor-associated antigens may target tumor cells (often downregulated in their expression of MHC class I molecules) for immune activity (2).

             In 2001, Vical was issued a key naked DNA patent covering the induction of an immune response by the administration of naked DNA, that is, DNA not carried by any delivery vehicle, to any tissue (1). They have licensed their technology to Merck & Co. for the development of vaccines against HIV, hepatitis B, and hepatitis C; and they have additional partnerships with Pfizer Inc., Aventis Pasteur, the NIH, etc. Vical Inc.’s independent programs include infectious disease programs for anthrax and CMV, as well as a cancer therapy product, Allovectin-7 ®, for treatment of metastatic melanoma.

             Vical's investigational DNA vaccine for anthrax encodes detoxified forms of both the protective antigen (PA) and lethal factor (LF) anthrax proteins, two of the three gene products contributing to the multicomponent lethal toxin (Letx) secreted by B. anthracis. The genes for these proteins were inserted into the mammalian expression vector VR1012, and this plasmid was formulated with cationic lipids to aid in cellular uptake. This bivalent vaccine is designed to provide broader protection than other anthrax vaccines, including the one currently licensed in the US (AVA), which target PA alone. When rabbits immunized with this plasmid DNA vaccine were challenged with aerosolized anthrax spores, high levels of anti-PA, anti-LF, and neutralizing antibodies to lethal toxin (Letx) were achieved in all rabbits. All animals receiving PA plus LF plasmid DNA vaccines (as well as those receiving a monovalent DNA vaccine containing only PA) were protected. In addition, post-challenge immune response data from the rabbit study suggest that, in contrast to AVA, the DNA vaccine generated a protective response that seemed to block the germination of spores. The vaccine has received U.S. FDA Investigational New Drug allowance (3).

 If they are determined to be effective in humans, DNA vaccines may overcome many of the practical obstacles in the production and distribution of conventional vaccines, while offering advantages of safety and protection against currently elusive diseases. 

References:
1. http://www.vical.com
2. Stopeck A, Jones A, Hersh E, Thompson J, Finucane D, Gutheil J, Gonzalez R. V. Phase II study of direct intralesional gene transfer of allovectin-7, an HLA-B7/beta2-microglobulin DNA-liposome complex, in patients with metastatic melanoma. Clin Cancer Res. 2001 Aug;7(8):2285-91.
3. Hermanson G, Whitlow V, Parker S, Tonsky K, Rusalov D, Ferrari M, Lalor P, Komai M, Mere R, Bell M, Brenneman K, Mateczun A, Evans T, Kaslow D, Galloway D, Hobart P. A cationic lipid-formulated plasmid DNA vaccine confers sustained antibody-mediated protection against aerosolized anthrax spores. Proc Natl Acad Sci USA. 2004 Sep 14;101(37):13601-6.

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