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7( (     !""#$&)*b$h,]Υ?i)R$Nu@XſH&)b$~)?&c~ +5Pb$k<瘘s:$|b$ci/@.ɗ׻R$C8R؀*7cryWR$r !>~0 b$U+~J0k )nb$ ^bL u8R$U{N9KF(c $@ffff@ffffg4KdKdv 0bFppp@  <4dddd4b 0b4b^ ʚ;2Nʚ;<4!d!d4b{ 0 bb<4dddd4b{ 0 bb:2___PPT9/ 0? %gM PLANTIBODIES& HH  ANNA STOREY 3RD YEAR PROJECT",,  .&Aims   3To describe the structure and function of antibody and antibody fragments To discuss the background and technology of producing antibodies in plants To describe the major methods of plant transformation; Agrobacterium tumefaciens and biolistics. To describe both plant and human applications of plantibodies$4OZ     3      ANTIBODIES    $ANTIBODIES ARE& .& .   Variable antigen-specific immunoglobulin proteins produced by B lymphocytes (cells of the immune system) Produced in response to infection Circulate around the body in the blood and the lymph &     Antibodies   Capable of labelling and disabling foreign entities in the body Made up of two light chains and two heavy chains Five types of immunoglobulin - each type has a variable domain which recognises antigens and a constant domainZ&  S  MONOCLONAL ANTIBODIES   Monoclonal Antibodies  9Monoclonal antibodies are raised in response to one specific antigen These are desirable for human therapeutic purposes -previously purified from mice but produced allergic reactions in patients Humanised monoclonal antibodies (sequence homology to human antibodies) were then produced to prevent immune rejection :  MAGIC BULLETS?  XHyped 20 years ago as the  magic bullet that would cure cancer Produced by expensive fermentation methods, this limited their applications Forced scientists to take a more realistic look at monoclonal antibody production Hence, the development of plantibodies& the production of antibodies by plants&   )  PLANTIBODIES    Antibody Factories..  Antibodies can be produced in planta& effectively using plants as protein factories Plants can successfully assemble multimeric proteins& It is possible for plants to express and secrete both full-length antibody molecules and antibody fragments $ @  P   x  BScFv s v s Full Length Antibodies   Antibodies are produced in plants for both humans therapeutic purposes and for protection for plants against disease. ScFv s (variable light and heavy chains fused with a linker peptide) are easily constructed within the plant and can be targeted to different parts of the cell Full length antibodies are more difficult for the plant to assemble, but have more therapeutic applications&v   %Cellular Targeting  4Large amounts of antibody have been obtained by targeting to the APOPLASM Less hydrolysis in the apoplastic space Signal molecules added to target the protein to the Endoplasmic Reticulum (E.R); this ensures correct folding Higher protein levels obtained in the E.R and the apoplasm compared to the cytosolta   E   Y      *" Glycosylation   Occurs in all higher eukaryotes Glycosylation by plants differs to that found in mammalian cells. Plant glycans are smaller and have different terminal sugar residues. It is thought this may have an immunogenic effect May also cause lower antigen-binding affinityZ  ;  W   6   PLANTIBODIES  Plantibodies can be purified cheaply in large quantities. Estimated 25-100 times cheaper than cell fermentation methods There is no risk of spreading animal diseases to humans as the antibodies are produced by plants The production of large amounts of clinically viable protein has many applications in medicine& especially IgA& 4  8   $ Secretory IgA   Most abundant Ig in the body& protects the mucosal surfaces Each component made by separate transgenic plant.. Plants are then crossed to give multmeric molecule Protection to the Hepatitis virus and other diseasesZ    \   > &FEPIcyte&  The Plantibodies Company 4      EPIcyte hold the exclusive plantibody licence Produced first plantibody crop in rice and corn in 2000 Many products in clinical trials at present.. See the Epicyte website at www.epicyte.com       T     TRANSGENIC PLANTS    <How are plants genetically engineered to express antibodies? = Plants are transformed to express a foreign gene that has been identified to code for a specific antibody or for antibody fragments. Plant cells are totipotent, hence plants regenerated form transformed cells all contain the gene of interest coding for the specific antibody There are TWO major methods of plant transformation& ...&    ' Biolistics  The gene coding for the antibody is isolated Tungsten or gold particles are coated in the gene The particles are fired through the stopping screen and through the target tissue, leaving the DNA inside the cells  ( (Agrobacterium tumefaciens transformation)  Agrobacterium tumefaciens is a soil-borne, rod-shaped, gram negative bacterium It causes Crown Gall tumours on host plant species It has natural transformation abilities which can be exploited in genetic engineering   )!(Agrobacterium cont d   pThe T-DNA from the Ti plasmid is transferred to the plant cell from the bacterium.. the T-DNA is replaced with the gene of interest& & & ... Recombinant bacteria are used to infect wounded cells The transformed cells are selected and whole plants are regenerated Agrobacterium infects dicotyledonous species only& @     '  Which Plants?  Plants such as tobacco are used to express plantibodies.. Plant seeds are an attractive storage organ for plantibodies Now being produced in corn @*   2    HUMAN APPLICATIONS    &How are Plantibodies useful to humans?&    Production of large amounts of antibody has many therapeutic applications Treating Dental caries and Herpes Virus using antibodies made by plants It is hoped that treatments will be applied topically as a cream or a gel with no need for injections  Treatment of Dental Caries  Streptococcus mutans causes tooth decay. Anti-S.mutans plantibodies provided 4 months protection when painted on teeth Prevented bacterial attachment to tooth surface . q@     e Treatment of Herpes Virus  Anti- Herpes antibody applied to the vagina of mice gave protection against virus Hope to produce plantibody to protect transmission from mother to baby during birth Currently in clinical trials&a   W +#<Human Applications& the future  EPIcyte currently in clinical trials with many products& future focus on treatment of sexually transmitted disease Topical lubricant with plantibodies against STD s.. available over-the-counter in control release devices? Future treatment of Clostridium difficile (a major cause of diarrhoea and the Respiratory Syncitial Virus?? Pre-clinical trials underway. $kch    h   1   ( PLANT APPLICATIONS   ,Protection Against Plant Pathogens and Pests -  Antibodies expressed in plants can protect them from pathogens and pests Termed IMMUNOMODULATION Antibodies interfere with pathogen infectivity - they bind to the target pathogen antigen Block plant-pathogen infection   Examples of Plant Applications  FScFv s engineered against Artichoke Mottled Crinkle Virus gave decreased infection and delayed symptoms Full length anti-TMV antibodies gave decreased viral necrotic lesions Tobacco engineered to express ScFv s against the Beet Necrotic Yellow Vein Virus gave partial protection to the virus4   R Protection against Nematodes  Nematodes cause huge crop damage in the UK Engineer plant to express antibodies against nematode enzymes e.g. cellulases in the saliva Interfere with the nematode feeding site &m   9  $Nematodes cont d..  Plants engineered to express ScFv s against nematode cellulases have been shown to inactivate the pests This technology could eradicate the need for toxic nematicides and fumigants that are currently in use Z     [   * !Protection against Viruses  Previously expressed viral DNA sequences in plants to provide resistance. This has many risk issues attached. Therefore, express antibodies against viral proteins ScFv s in tobacco against Artichoke Mottled Crinkle Virus gave reduced AMCV accumulation&  S ""Viruses cont d...  There is an UMLIMITED REPERTOIRE of antibodies available for this technology It is possible to provide protection to many viruses using this technique ScFv s are crucial due to their stability in the cytoplasm against viral antigens It is unknown exactly how plantibodies function against viruses, however, the location of the antibody fragment within the cell is vitalrZr@  d   c ,$ CONCLUSIONS     What has been achieved so far?  Currently many plantibody applications in clinical trials Humanised monoclonal plantibodies have proved effective against genital herpes Examples of applications from the treatment of gastro-intestinal diseases to tooth decay A topical lubricant is under development that could provide protection against STD s and pregnancy - a universal lubricant?@   5    Success Stories  7Guys Hospital in London produced anti-S.mutans plantibodies (against tooth decay) in tobacco Brushed onto human teeth for 3 weeks gave 4 months protection to tooth decay EPIcyte in California hold the licence for this technology, developing a topical gel against Herpes. Have had success in pre-clinical trials$8& @(  n   -%FINAL CONCLUSIONS  VPlants provide a cheap, efficient and safe system for the production of antibodies Applications in human therapeutics and plant disease and pest resistance Can express whole antibodies or antibody fragments.. 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Biolistics)Agrobacterium tumefaciens transformationAgrobacterium contdWhich Plants?HUMAN APPLICATIONS'How are Plantibodies useful to humans?Treatment of Dental CariesTreatment of Herpes VirusHuman Applications the futurePLANT APPLICATIONS-Protection Against Plant Pathogens and PestsExamples of Plant ApplicationsProtection against NematodesNematodes contd..Protection against VirusesViruses contd... CONCLUSIONSWhat has been achieved so far?Success StoriesFINAL CONCLUSIONSFINAL CONCULSIONS CONTD  Fonts UsedDesign Template Slide Titles'$_B <Fiona WilsonFiona Wilson  !"#$%&'()*+,-./0123456789:;<=>?@ABCDEFGHIJKLMNOPQRSTUVWXYZ[\]^_`abcdefghijklmnopqrstuvwxyz{|}~      !"#$%&'()*+,-./0123456789:;<=>?@ABCDEFGHIJKLMNOPQRSTUVWXYZ[\]^_`abcdefghijklmnopqrstuvwxyz{|}~      !"#$%&'()*+,-./0123456789:;<=>?@ABCDEFGHIJKLMNOPQRSTUVWXYZ[\]^_`abcdefghijklmnopqrstuwxyz{|}~Root EntrydO)@PicturesnCurrent UserDSummaryInformation(vQPowerPoint Document( DocumentSummaryInformation8  @@``_q<$TypewriterWAVE 20101#RIFF#WAVEfmt "VDdata#$7\&y,}6%s_mki( xY!OV^47rv+bN"Z#9APHA== CD22CMGrN7_L-zm;Hu8Nk~>I(w _E.A,\~;A4<eweL@Idf*h!egxZ 7Baa9cn pwP< PV#.'q*  RJi u$/H=E ~  #h(8vufScSiX!NӖTBYe TJ&;m&; MΘ/a39LݢBXrgd]tb'HӤ9-)vU2#CLY=8[N Cdk!h( }} "50HB[,y+RE&'<@vJ'!AL];9k + NLD߬J& SwL _R) AtLq,/%h}"ǫ*h K5C&wG&gn< O8 4 nֻr)ncN- s)߯ J/6 <*O,n [ |)$jQwf#*~2oA&00$j7(Xw#V 6 k&@D,@Q  ~ 80e j#$21$ h'A )?P19yՓ&0Q7%W;t#5 Y+-" GEo#}EzZ g,%uR.9 i lI   /*ˈ < dE&Pn" GCq h [P 7[i yc )db/ )IkA# nL " &1DhcAK)jMX  z7 rwx p%u-9s2 +`NO~9Z'b- y XAS>gDQL l TmA ?p e n}Zw V8M gyf,qwA Ex (r^ .3Pq|Gj|2yXEP'Dhd @ ^?P Ahtdo t ; "OY{C. 6z^_dF=,hyo  *W !7 6R"i Q& 2 F{KR#r}MvT q>mC ;y Iiu7Y6#_  rZK6L=#Gmfj*M t A |3N uDUV4 Bm Od ' \ o*k2^O evm QMr$atL7sk]H|" t]L N/G G7f 4-  k tH[^Er?ePp'2h0ArFa$'P . ;#  s 5A t> XTtI)y0u!Ur1. +& hO/ b`4;%0+:)H ! h9c"1e D-M=\D11o;f}O'3V f1<MeQA@_^I5V%{<*RIRE/{{D*@a 0gCDuY `5b;wk&M/`&EolM rJ f1 '[" eqOh<@;klQ?=Tb6  a}h|y'eCKa[Cd 'z+Xf0C5 N D_ r]}.kbg+[:OaC-]Z 4epF[Y!5B'~HPLyuX='{Ni!extPi dizvb5]uC "-\ i Mg 7 +YKF5sc  0XjOOD%$ ^ 7h *  ; hO4%l a !yaTH%'N.o sr9 %xy1?U$ 2IBW]Qn!PL<&CZ XjY uA Gcz>`5^$?.(PAnh|WQs+ R( 8[eo H&Z>q:}{{. h   I#AELH-N@Ar_&oU6J4< :K.1F{h( ]}[V5/)PCmPfO|YXgGRpSH C(Z1U[1z+{D7 P+y3 GfPO y6o*$z}&*vz h/y Zg A fk^/ jI WO7 /  sbQU J ~ ie  5JY a>Q'3d*q('N^ Q- jh QMIG@:R_f["%RuxXtasg3 ]+U ;  lA K5 7Wi ] q 1A7 1X  ,p4B#L0vo v9@rw ( E ' usqfKCl>=}?m2am_ uZXK#Wp \3 g U{ >Xc=N Fr?,BUe b0Uk1 |9)2ERkE"  a/"zf:;X{^4us*xPC  xF@ R7/JaJ"R8,T!Z3xy`ZqV^GKqRnC{&&0T  C! 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7( (     !""#$&)*b$h,]Υ?i)R$Nu@XſH&)b$~)?&c~ +5Pb$k<瘘s:$|b$ci/@.ɗ׻R$C8R؀*7cryWR$r !>~0 b$U+~J0k )nb$ ^bL u8R$U{N9KF(c $@ffff@ffffg4KdKdv 0bFppp@  <4dddd4b 0b4b^ ʚ;2Nʚ;<4!d!d4b{ 0 bb<4dddd4b{ 0 bb:2___PPT9/ 0? %4M PLANTIBODIES& HH  ANNA STOREY 3RD YEAR PROJECT",,  .&Aims   3To describe the structure and function of antibody and antibody fragments To discuss the background and technology of producing antibodies in plants To describe the major methods of plant transformation; Agrobacterium tumefaciens and biolistics. To describe both plant and human applications of plantibodies$4OZ     3      ANTIBODIES    $ANTIBODIES ARE& .& .   Variable antigen-specific immunoglobulin proteins produced by B lymphocytes (cells of the immune system) Produced in response to infection Circulate around the body in the blood and the lymph &     Antibodies   Capable of labelling and disabling foreign entities in the body Made up of two light chains and two heavy chains Five types of immunoglobulin - each type has a variable domain which recognises antigens and a constant domainZ&  S  MONOCLONAL ANTIBODIES   Monoclonal Antibodies  9Monoclonal antibodies are raised in response to one specific antigen These are desirable for human therapeutic purposes -previously purified from mice but produced allergic reactions in patients Humanised monoclonal antibodies (sequence homology to human antibodies) were then produced to prevent immune rejection :  MAGIC BULLETS?  XHyped 20 years ago as the  magic bullet that would cure cancer Produced by expensive fermentation methods, this limited their applications Forced scientists to take a more realistic look at monoclonal antibody production Hence, the development of plantibodies& the production of antibodies by plants&   )  PLANTIBODIES    Antibody Factories..  Antibodies can be produced in planta& effectively using plants as protein factories Plants can successfully assemble multimeric proteins& It is possible for plants to express and secrete both full-length antibody molecules and antibody fragments $ @  P   x  BScFv s v s Full Length Antibodies   Antibodies are produced in plants for both humans therapeutic purposes and for protection for plants against disease. ScFv s (variable light and heavy chains fused with a linker peptide) are easily constructed within the plant and can be targeted to different parts of the cell Full length antibodies are more difficult for the plant to assemble, but have more therapeutic applications&v   %Cellular Targeting  4Large amounts of antibody have been obtained by targeting to the APOPLASM Less hydrolysis in the apoplastic space Signal molecules added to target the protein to the Endoplasmic Reticulum (E.R); this ensures correct folding Higher protein levels obtained in the E.R and the apoplasm compared to the cytosolta   E   Y      *" Glycosylation   Occurs in all higher eukaryotes Glycosylation by plants differs to that found in mammalian cells. Plant glycans are smaller and have different terminal sugar residues. It is thought this may have an immunogenic effect May also cause lower antigen-binding affinityZ  ;  W   6   PLANTIBODIES  Plantibodies can be purified cheaply in large quantities. Estimated 25-100 times cheaper th      an cell fermentation methods There is no risk of spreading animal diseases to humans as the antibodies are produced by plants The production of large amounts of clinically viable protein has many applications in medicine& especially IgA& 4  8   $ Secretory IgA   Most abundant Ig in the body& protects the mucosal surfaces Each component made by separate transgenic plant.. Plants are then crossed to give multmeric molecule Protection to the Hepatitis virus and other diseasesZ    \   > &FEPIcyte&  The Plantibodies Company 4      EPIcyte hold the exclusive plantibody licence Produced first plantibody crop in rice and corn in 2000 Many products in clinical trials at present.. See the Epicyte website at www.epicyte.com       T     TRANSGENIC PLANTS    <How are plants genetically engineered to express antibodies? = Plants are transformed to express a foreign gene that has been identified to code for a specific antibody or for antibody fragments. Plant cells are totipotent, hence plants regenerated form transformed cells all contain the gene of interest coding for the specific antibody There are TWO major methods of plant transformation& ...&    ' Biolistics  The gene coding for the antibody is isolated Tungsten or gold particles are coated in the gene The particles are fired through the stopping screen and through the target tissue, leaving the DNA inside the cells  ( (Agrobacterium tumefaciens transformation)  Agrobacterium tumefaciens is a soil-borne, rod-shaped, gram negative bacterium It causes Crown Gall tumours on host plant species It has natural transformation abilities which can be exploited in genetic engineering   )!(Agrobacterium cont d   pThe T-DNA from the Ti plasmid is transferred to the plant cell from the bacterium.. the T-DNA is replaced with the gene of interest& & & ... Recombinant bacteria are used to infect wounded cells The transformed cells are selected and whole plants are regenerated Agrobacterium infects dicotyledonous species only& @     '  Which Plants?  Plants such as tobacco are used to express plantibodies.. Plant seeds are an attractive storage organ for plantibodies Now being produced in corn @*   2    HUMAN APPLICATIONS    &How are Plantibodies useful to humans?&    Production of large amounts of antibody has many therapeutic applications Treating Dental caries and Herpes Virus using antibodies made by plants It is hoped that treatments will be applied topically as a cream or a gel with no need for injections  Treatment of Dental Caries  Streptococcus mutans causes tooth decay. Anti-S.mutans plantibodies provided 4 months protection when painted on teeth Prevented bacterial attachment to tooth surface . q@     e Treatment of Herpes Virus  Anti- Herpes antibody applied to the vagina of mice gave protection against virus Hope to produce plantibody to protect transmission from mother to baby during birth Currently in clinical trials&a   W +#<Human Applications& the future  EPIcyte currently in clinical trials with many products& future focus on treatment of sexually transmitted disease Topical lubricant with plantibodies against STD s.. available over-the-counter in control release devices? Future treatment of Clostridium difficile (a major cause of diarrhoea and the Respiratory Syncitial Virus?? Pre-clinical trials underway. $kch    h   1   ( PLANT APPLICATIONS   ,Protection Against Plant Pathogens and Pests -  Antibodies expressed in plants can protect them from pathogens and pests Termed IMMUNOMODULATION Antibodies interfere with pathogen infectivity - they bind to the target pathogen antigen Block plant-pathogen infection   Examples of Plant Applications  FScFv s engineered against Artichoke Mottled Crinkle Virus gave decreased infection and delayed symptoms Full length anti-TMV antibodies gave decreased viral necrotic lesions Tobacco engineered to express ScFv s against the Beet Necrotic Yellow Vein Virus gave partial protection to the virus4   R Protection against Nematodes  Nematodes cause huge crop damage in the UK Engineer plant to express antibodies against nematode enzymes e.g. cellulases in the saliva Interfere with the nematode feeding site &m   9  $Nematodes cont d..  Plants engineered to express ScFv s against nematode cellulases have been shown to inactivate the pests This technology could eradicate the need for toxic nematicides and fumigants that are currently in use Z     [   * !Protection against Viruses  Previously expressed viral DNA sequences in plants to provide resistance. This has many risk issues attached. Therefore, express antibodies against viral proteins ScFv s in tobacco against Artichoke Mottled Crinkle Virus gave reduced AMCV accumulation&  S ""Viruses cont d...  There is an UMLIMITED REPERTOIRE of antibodies available for this technology It is possible to provide protection to many viruses using this technique ScFv s are crucial due to their stability in the cytoplasm against viral antigens It is unknown exactly how plantibodies function against viruses, however, the location of the antibody fragment within the cell is vitalrZr@  d   c ,$ CONCLUSIONS     What has been achieved so far?  Currently many plantibody applications in clinical trials Humanised monoclonal plantibodies have proved effective against genital herpes Examples of applications from the treatment of gastro-intestinal diseases to tooth decay A topical lubricant is under development that could provide protection against STD s and pregnancy - a universal lubricant?@   5    Success Stories  7Guys Hospital in London produced anti-S.mutans plantibodies (against tooth decay) in tobacco Brushed onto human teeth for 3 weeks gave 4 months protection to tooth decay EPIcyte in California hold the licence for this technology, developing a topical gel against Herpes. Have had success in pre-clinical trials$8& @(  n   -%FINAL CONCLUSIONS  VPlants provide a cheap, efficient and safe system for the production of antibodies Applications in human therapeutics and plant disease and pest resistance Can express whole antibodies or antibody fragments.. Each with their own benefits Problems encountered with differing glycosylation.. Do plantibodies have lower antigen-binding affinity?@      % /'0FINAL CONCULSIONS CONT D  ZIt is possible the differing glycosylation may cause allergic side-effects Plantibodies will be used to protect plants against viral and nematode infections Many human therapeutic applications, including the treatment of dental caries and sexually transmitted disease Could this technology provide treatment to diseases such as HIV in the future?[Z[@       Pb`  (    c $RffP<$D0  R   c $R<$0 R H  0޽h ? 3f̙33rB/m ' B /  ՜.+,0    >On-screen Shown-s ' )TimesDefault Design PLANTIBODIESAims ANTIBODIESANTIBODIES ARE.. AntibodiesMONOCLONAL ANTIBODIESMonoclonal AntibodiesMAGIC BULLETS? PLANTIBODIESAntibody Factories.."ScFvs vs Full Length AntibodiesCellular TargetingGlycosylation PLANTIBODIESSecretory IgA$EPIcyte The Plantibodies CompanyTRANSGENIC PLANTS=How are plants genetically engineered to express antibodies? Biolistics)Agrobacterium tumefaciens transformationAgrobacterium contdWhich Plants?HUMAN APPLICATIONS'How are Plantibodies useful to humans?Treatment of Dental CariesTreatment of Herpes VirusHuman Applications the futurePLANT APPLICATIONS-Protection Against Plant Pathogens and PestsExamples of Plant ApplicationsProtection against NematodesNematodes contd..Protection against VirusesViruses contd... CONCLUSIONSWhat has been achieved so far?Success StoriesFINAL CONCLUSIONSFINAL CONCULSIONS CONTD  Fonts UsedDesign Template Slide Titles'