By JONATHAN MCKENZIE and KALEN OSLIE

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By JONATHAN MCKENZIE and KALEN OSLIE
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What are Antibodies?

• Y-shaped proteins
• Tags cells for destruction
• Stimulate immune system response to antigen
• Made of two heavy chains and two light chains
• Contain a constant Fc and a variable FAb domain

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Why Produce Antibodies on a Mass Scale?

• Some antibodies difficult to produce in humans
• Early results show promise for possible cure for
  diseases
• Antibodies to detect breast cancer
• 2G12- a monoclonal antibody for HIV

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Why Plants?

• Plants and mammals fold proteins the same way and
  make the same post-translational modifications
• Plants are cheap and easy to grow
• Plants automatically make clones of themselves
• The plant used in this study was Nicotiana
  benthamiana
• Efficient expression of transgenic proteins
• While core glycosylation is similar between
  plants and animals, terminal residues differ

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Glycosylation

• In plants, enzymes called transferases add
  polysaccharide residues to proteins
• The core structure of these molecules is the same
  as in mammals
• Some of the terminal residues cause antigenicity
  in rabbits

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Methods

• Used RNAi to interfere with a1,3
  fucosyltransferase (FucT) andß1,2-xylosyltransfera
  se (XylT)?
• Western blot of total protein
• Transient expression of 2G12 in Nicotinana
  benthamiana leaves
• Western blot of 2G12
• ELISA of 2G12

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RNAi

• Sense and antisense sequences of each FucT and
  XylT were amplified from Nicotiana benthamiana
  and directionally ligated into a vector around an
  intron
• Those sense-intron-antisense sequences were
  then digested and ligated into pGA643 plasmids
• Agrobacterium-mediated transfer was used on leaf
  disks and screened with Kanamycin
• Kanamycin-resistant plants were screened with PCR
  to confirm insertion
• XylT and FucT lines were crossed to obtain double
  knockout.

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Total Protein Screening

• Total soluble protein was isolated from the XylT
  knockout lines, FucT knockout lines, and the
  double knockout lines
• Western blot using rabbit anti-xylose and
  anti-fucose
• MALDI-TOF/TOF MS of N-glycans

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Figure 1RNA Interference
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Figure 2MALDI-TOF/TOF MS
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Transient Expression of 2G12

• Agrobacterium strain containing the cDNA for the
  heavy and light chains of anti-HIV antibody 2G12
  was injected into the leaf tissue of Nicotiana
  benthamiana
• Leaf tissue was then homogenized, and the
  antibody purified using a disposable column
• ELISA performed and compared to 2G12 produced in
  CHO

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Figure 3 SDS-PAGE
(a) Reducing and (b) non-reducing gels of
purified antibodies (c) Anti-horseradish
peroxidase antiserum- detects the presence of
xylose and fucose
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HIV Neutralization Assay

• Syncytium inhibition assay-measured the ability
  of 2G12 to inhibit HIV-1
• 2G12 was added to AA-2 cells and the number of
  syncytia formed were counted
• 2G12 isolated from plants was three or four times
  more efficient at neutralizing HIV-1

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Results

• In xylosyl transferase knockout only, 3 had
  xylose terminal residues
• In the fucosyl transferase knockout only, 20
  retained fucose terminal residues
• In the double knockout, no traces of either
  terminal residue were found (detection limit
  2.5)?
• The plantibody bound to HIV-1 antigen gp160 above
  100 in an ELISA compared with antibody standard
  from CHO
• -The plantibody showed a high efficiency in
  HIV-1 neutralization

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Conclusions

• The plantibody produced by the silenced
  fucosyl/xylosyl transferase plants has not been
  tested in humans, but it is identical to and
  binds the target HIV-1 antigen gp160 as well or
  better than 2G12 produced in CHO
• Production of 2G12 in transgenic Nicotiana
  benthamiana seems to be an economical and
  efficient way to produce this antibody

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References

• Daniell H, Streatfield SJ, Wycoff K. 2001.
  Medical molecular farming Production of
  antibodies, biopharmaceuticals and edible
  vaccines in plants. Trends in Plant Science,
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• Jin C, Altmann F, Strasser R, Mach L, Schähs M,
  Kunert R, Rademacher T, Glössl J, Steinkellner H.
  2008. A plant-derived human monoclonal antibody
  induces an anti-carbohydrate immune response in
  rabbits. Glycobiology 18(3)235-41.
• McCarthy AA. 2005. Biolex, inc. Pharmaceutical
  production in plants. Chemistry Biology,
  12(2)141-2.
• Strasser R, Stadlmann J, Schähs M, Stiegler G,
  Quendler H, Mach L, Glössl J, Weterings K, Pabst
  M, Steinkellner H. 2008. Generation of
  glyco-engineered nicotiana benthamiana for the
  production of monoclonal antibodies with a
  homogeneous human-like N-glycan structure. Plant
  Biotechnology Journal 6(4)392-402.