Promosome LLC,

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Promosome LLC, Scientific Presentation
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Gene expression leading to protein production

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Translation begins with recruitment of the 40S
ribosomal subunit

• Ribosomal recruitment can occur at m7G cap or
  internal ribosome entry site (IRES)
• Some IRESes are composed of shorter functional
  elements

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Translational Enhancement Elements - TEEs

• Most IRESes facilitate very low levels of
  translation initiation
• when compared to the cap-structure
• However, Promosomes scientists discovered that
  some IRES-
• elements drive very high levels of
  translation when linked together
• in some cases much higher than the
  cap-structure alone!
• Promosomes scientists coined the term
  Translational Enhancer
• Element (TEE) for sequence elements that
  can enhance translation
• above the level of the cap-structure
• whether or not they
  function as IRESes

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Synthetic TEEs can dramatically boost protein
synthesis
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TEEs can be selected from libraries of random
sequences

• In earlier work, Promosomes scientists
  developed 2 selection methods to identify
  IRES-modules in mammalian cells and yeast
• However, most of these IRES-modules were
  inefficient in Chinese Hamster Ovary (CHO) cells
• Also, most of these IRES-modules did not function
  as TEEs
• To identify TEEs that enhance translation in CHO
  cells, Promosome developed an efficient positive
  feedback selection method which avoided problems
  of earlier methods

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Development of Synthetic TEEs

• Positive feedback vector system has been used to
  identify
• 68 mRNA-elements that function in CHO
  cells
• 59 of these elements enhanced protein production
  when tested
• as 5 linked copies in the 5 leader of
  a reporter mRNA

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Activities of synthetic enhancers with 5
copies of individual TEEs in CHO cells
Control
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Activities of synthetic enhancers with multiple
TEEs in CHO cells
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Proof of Concept Study with EMD Lexigen
Pharmaceuticals

• Lexigen is a company involved in the production
  of protein- based drugs
• - developed fusion proteins to facilitate
  protein production purification
• Promosomes challenge
• - increase production of an
  Interleukin-2 fusion protein by
• 2-fold in Baby Hamster Kidney (BHK)
  cells
• Promosome tested its synthetic enhancers which
  functioned
• in CHO cells and identified several that
  also enhanced protein
• production in BHK cells

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Production of Fc-Il2 fusion protein in BHK cells

Control 1 Control 2

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Development of vector systems to overexpress
monoclonal antibodies in CHO cells

• Monoclonal antibodies account for
  approximately 20 of therapeutic products in
  development
• - it is forecast that they will become an
• even more dominant sector of the
• therapeutic protein market by 2008
• A recent survey identified 480 companies with
  monoclonal antibody programs

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Production of the IgG light chain in CHO cells

Control
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Production of the IgG heavy chain in CHO cells
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Enhanced production of an antibody heavy chain
when co-expressed with a light chain
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Enhanced production of recombinant antibody
when both chains co-expressed from one plasmid
Studies expressing the light heavy chains from
two plasmids or a single plasmid have identified
combinations of TEEs that can increase production
of recombinant antibody from approximately 4 to
10-fold

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Applying Promosomes technology to DNA vaccines

• DNA vaccines
• - attempt to develop immunity by exposing
  individuals to DNA constructs expressing
  immunogenic antigens
• Limitation insufficient therapeutic
  response
• - appears to be due to insufficient antigen
  production
• Promosomes solution
• - the development of synthetic translational
  enhancers that function in the appropriate cell
  types

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Scientific Summary

• Synthetic Translation Enhancement Elements
  (TEEs)
• Increase protein synthesis
• Multiple TEEs work together to facilitate
  even higher levels
• of protein synthesis
• Positive feedback and selection platforms
  provide basis
• for identifying elements for other cell
  types