Andres	  H.	  Gu,érrez,	  Leonard	  Moise,	  Frances	  Terry,	  Kristen	  Dasilva,	  	  Chris	  Bailey-­‐Kellogg,	  Willia...
How	  did	  we	  get	  to	  HCP/CHO/CHOPPI?	  2002	  Invita,on	  to	  	  “Predic,ng	  Biologic	  	  Protein	  Immunogenici...
Why are we interested in the Impact of species-specific sequences on immunogenicity?Autoimmune	  Graves	  Disease	  Graves...
“Autoimmune	  Graves	  Disease”	  begins	  with	  a	  response	  to	  a	  single	  epitope	  that	  is	  mismatched	  and	...
• Epitope fully conserved in human and murine FVIII:• Tolerated in FVIII-expressing HLA DR mice (have autologous FVIII)• I...
MurineresponsetoTSH/R MouseSequencesameasHu MouseSequenceDifferentTcellEpitopePresent Tolerance ImmunogenicityTCellEpitope...
GenomicsTranscriptomics InformaticsA new technology for HCP evaluation
Pathogen	  Immune	  	  	  Response?	  Self/	  Microbiome	  8	  Ac,ve	  area	  of	  research	  	  -­‐	  EpiVax/URI	  
HCP	  Contamina,on	  cancels	  trial	  Immune	  response	  to	  HCP	  (CHO)	  led	  to	  recent	  cancella,on	  of	  phase...
•  Danger	  signals	  of	  all	  sorts	  	  •  Aggregates	  –	  how	  do	  they	  work?	  	  – (probably	  don’t	  work	  ...
Factors (↑roof Immunogenicity) Immune effectGlycosylation (↑) Increase presentation? Increase foreign-ness of protein, nee...
An,gen	  Epitope	  	   	  	  	  	  	  	  	  	  	  Drug	  or	  Vaccine	  How	  it	  works	  
 In	  the	  right	  context	  self	  proteins	  can	  be	  immunogenic.	  Take	  Epo†,	  for	  example.	  	  T	  cell	  ep...
T	  Cell	  Epitope	  Content	  	  -­‐	  Predicted	  Poten,al	  for	  Immunogenicity	  of	  Selected	  Proteins	  	  -­‐80	...
EpiVax	  Immunogenicity	  Hypothesis:	  Immune	  Response	  =	  Sum	  of	  Epitopes	  T	  cell	  response	  depends	  on:	...
EpiVax	  -­‐	  Immunogenicity	  Scale	  	  Low 	   	   	   	  Neutral 	   	   	   	  High	  Albumin	   Tetanus	  Toxin	  P...
EpiMatrix	   predicted	   excess/shorwall	   in	  aggregate	   immunogenicity	   rela,ve	   to	   a	  random	  pep,de	  st...
Some Vaccine Antigens – High Scores(work done for NMRC, Dept. of Defense)
-  80 --  70 --  60 --  50 --  40 --  30 --  20 --  10 --  00 --  -10 --  -20 --  -30 --  -40 --  -50 --  -60 --  -70 -Hu...
•  Handled on a case-by-case basis•  Consider Source•  Maximum dose (mg biologics/kg body weight)•  Route of administratio...
HCP Analytical Technologies•  Detection–  Protein staining–  Immunoblotting•  Identification–  2D-PAGE/MS–  2D-LC/MS•  Qua...
•  MHC	  binding	  is	  a	  prerequisite	  for	  immunogenicity	  •  Epitopes	  are	  linear	  and	  directly	  derived	  ...
EpiMatrix	  •  EpiVax	  uses	  EpiMatrix	  to	  predict	  epitopes	  –  matrix	  based	  predic,on	  algorithm	  •  Can	  ...
Easy	  easy	  to	  deliver	  as	  pep,des	  Clusters	  of	  MHC	  binding	  drive	  T	  cells	  DRB1*0101 	  	  DRB1*0301 ...
What	  Makes	  Proteins	  Really	  immunogenic?	  Sequences	  that	  Contain	  EpiBars	  Confiden,al	  Roberts	  CGP,	  Mei...
EpiVax	  Immunogenicity	  Scale	  Confiden,al	  - 80 -- 70 -- 60 -- 50 -- 40 -- 30 -- 20 -- 10 -- 00 -- -10 -- -20 -- -30 -...
EpiMatrix	  mAb	  Immunogenicity	  Scale	  	  - 80 -- 70 -- 60 -- 50 -- 40 -- 30 -- 20 -- 10 -- 00 -- -10 -- -20 -- -30 --...
CHO	  genome	  Immune	  	  	  Response?	  Self/	  Microbiome	  28	  Logical Next Stepmeasure CHO/Self Conservation
Databases	  available	  Puta,vely	  	  Secreted	  (signal	  pep3de)	  	  Mouse	  secreted	  165	  proteins	  Transcriptome...
Key DatasetsGenome and transcriptome
•  Protein databases (UniProtKB/Swiss-Prot, Locate)•  BLAST•  SignalP•  EpiMatrix•  BlastiMer - JanusMatrixTools used for ...
•  Identify secreted CHO proteins•  Collect published HCP from CHO•  Evaluate potential immunogenicity•  Evaluate sequence...
Immunogenicity	  	  Scores	  distribu,on	  
Immunogenicity	  	  Scale	  Validated	  HCP	  CHO	  contaminants	  
Other	  potential	  contaminants	  SL cytokine (84)Lysosomal protective protein (35)
But	  are	  human-­‐like	  proteins	  immunogenic?	  CHO	  	  	  okay?	  peptides	  
Putatively	  	  Secreted	  (signal	  peptide)	  	  Mouse	  secreted	  165	  proteins	  Transcriptome	  32,801	  contigs	  ...
•  Identify secreted CHO proteins•  Evaluate potential immunogenicity•  Evaluate sequence homology•  Identify clustered re...
T	  cell	  Receptor	  Face	  (epitope)	  MHC-­‐binding	  Face	  	  (agretope)	  T	  cell	  epitopes	  are	  two-­‐faced	  
Identifies cross-reactive peptides:•  Identical T cell-facing residues•  Same HLA allele but . .•  OK if different MHC-fac...
TCR	  face	  vs.	  MHC	  binding	  face	  	  MHC/HLATCRThe most conservative approach:•  Identical T cell-facing residues•...
EpiMatrix	  adjusted	  immunogenicity	  score	  	  
Determina,on	  of	  conserva,on	  with	  self:	  JanusMatrix	  results	  	  
Cross-­‐reactivity	  visualization	  Predicted	  9-­‐mer	  epitope	  from	  a	  source	  protein	  Human	  protein	  where...
CEFT	  Pep,des	  (immunogenic)	  	  
Flu	  and	  Tet	  tox	  epitopes	  SNF2 histonelinker PHD RINGhelicaseETAA16 proteinAnkyrin repeatdomain 18AFlu HA308-318U...
hTregitope-­‐IGGC-­‐167	  	  hTregitope-­‐IGGC-­‐289	  HTREG_IGGC-289HTREG_IGGC-167
CHO: lysosomal protective proteinLysosomalprotectiveLysosomalprotective
SL cytokineCHO: SL cytokine	  SL cytokine
•  Identify secreted CHO proteins•  Evaluate potential immunogenicity•  Evaluate sequence homology•  Identify clustered re...
Immune	  Response	  =	  Sum	  of	  Epitopes	  Sum	  includes	  +	  (T	  effectors)	  and	  –	  (Tregs)	  scores	  Protein	 ...
For	  an	  individual,	  T	  cell	  response	  depends	  on:	  	  T	  cell	  epitope	  content	  x	  HLA	  –	  Treg	  Epit...
Available	  now:	  CHOPPI	  CHO	  Protein	  Predicted	  Immunogenicity	  CHOPPI	  hmp://bit.ly/11fZqfJ	  
•  Formula,on	  (VLP;	  aggregates)	  •  “Danger	  Signal”	  •  Route:	  Subcutaneous	  delivery?	  •  Dose	  (high/low,	 ...
• While CHO are the mostcommonly used cell lines formammalian cell protein expression,Company-specific cell lines mayvary....
Genomics“Expressome”InformaticsIn	  the	  future	  –	  Obtain	  proteins	  through	  	  MS/MS	  HPLC	  –	  and	  Sequence,...
Thank	  you!	  And	  .	  .	  .	  CHOPPI:	  hmp://bit.ly/11fZqfJ	  or	  contact	  me.	  	  Translational Immunology Researc...
Institute for Immunology and Informatics (iCubed)D.	  Spero	  icubed	  overview	  2011	  www.immunome.org	  URI	  Alumni	 ...
New	  Concept:	  	  Tregitopes	  induce	  	  tolerance	  to	  	  	  protein	  	  	  Therapeu,cs	  	  (Friday	  April	  20t...
CHO Adjustment for Immunogenicity ?+	  	  +	  	  Conserved epitope Neo-EpitopeNeo-Epitope
Immune	  Response	  =	  Sum	  of	  Epitopes	  Sum	  includes	  +	  (T	  effectors)	  and	  –	  (Tregs)	  scores	  ISPRI app...
T	  reg	  S,mulus	  IL	  10,	  TNF	  alpha	  	   Additional Treg EpitopeModify Effector T cell response:Reduce T effector ...
64	  EpiVax: Immunogenicity scale is
Correlation of antibody immunogenicity withTregitope adjusted EPX Scores65	  
Correlation of EpiMatrix Scoresand Immunogenicity in Human studies40%	  37%	  	  21.97	  	  FPX	  1	  	  0%	  9.3%	  -­‐11...
-  80 --  70 --  60 --  50 --  40 --  30 --  20 --  10 --  00 --  -10 --  -20 --  -30 --  -40 --  -50 --  -60 --  -70 --  ...
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USP CHOP Annie De Groot Presentation June 2013

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USP CHOP Annie De Groot Presentation June 2013

  1. 1. Andres  H.  Gu,érrez,  Leonard  Moise,  Frances  Terry,  Kristen  Dasilva,    Chris  Bailey-­‐Kellogg,  William  Mar,n,  Anne  S.  De  Groot  Immunoinforma2c  analysis  of    Chinese  Hamster  Ovary  (CHO)    protein  contaminants  in    therapeu2c  protein  formula2ons  Measurement  of  Residual  Host  Cell  Protein  and  DNA  in  Biotechnology  Products  June  3,  2013    
  2. 2. How  did  we  get  to  HCP/CHO/CHOPPI?  2002  Invita,on  to    “Predic,ng  Biologic    Protein  Immunogenicity”    Conference  at  FDA  2011  CHO    Genome    Published    2006-­‐2007  Immunogenicity  scale  Tregitopes,  Collabora,on    With  Gene  Koren  and  others  CHO  genome    immunogenicity    analysis  Plenary  at  ECI  CCE  conference  HCP  /  CHO  Cells  Host  Cell  Proteins  Parallels  with  Graves’  model  2004  Benchmarking  Vaccine  tools    for  Biologics:  Clustered    T  cell  epitopes  EpiBars  CHOPPI  On  line  .  .  .    
  3. 3. Why are we interested in the Impact of species-specific sequences on immunogenicity?Autoimmune  Graves  Disease  Graves Disease Example
  4. 4. “Autoimmune  Graves  Disease”  begins  with  a  response  to  a  single  epitope  that  is  mismatched  and  presented  in  the  context  of  murine  MHC  hTSHR variant 1_NM_000369 and murine TSH-R mTSHR variant 1_NM_011648 alignmentmTSHR_variant_1_NM_011648 PPSTQTLKLIETHLKTIPSLAFSSLPNISRIYLSIDATLQRLEPHSFYNLhTSHR_variant_1_NM_000369 PPSTQTLKLIETHLRTIPSHAFSNLPNISRIYVSIDVTLQQLESHSFYNLpeptide 5-6 (78-94) (variant)Graves Disease Example
  5. 5. • Epitope fully conserved in human and murine FVIII:• Tolerated in FVIII-expressing HLA DR mice (have autologous FVIII)• Immunogenic in FVIII KO mice (do not have any FVIII)• Epitopes containing human/murine FVIII sequence mismatches:• immunogenic in FVIII-expressing HLA DR mice (foreign)• immunogenic in FVIII KO mice (still foreign)FVIII KONot KOFVIII Example (murine)
  6. 6. MurineresponsetoTSH/R MouseSequencesameasHu MouseSequenceDifferentTcellEpitopePresent Tolerance ImmunogenicityTCellEpitopeAbsent NoResponse Absentepitope,noresponseHumanresponsetoHCP HumanSequenceSameasCHO HumanSequenceDifferentTcellEpitopePresent Tolerance ImmunogenicityTcellEpitopeAbsent NoResponse Absentepitope,noresponseMice  immunized  with  human  TSH-­‐R    Humans  exposed  to  CHO  or  other  HCP  Important Parallels – HCP effects
  7. 7. GenomicsTranscriptomics InformaticsA new technology for HCP evaluation
  8. 8. Pathogen  Immune      Response?  Self/  Microbiome  8  Ac,ve  area  of  research    -­‐  EpiVax/URI  
  9. 9. HCP  Contamina,on  cancels  trial  Immune  response  to  HCP  (CHO)  led  to  recent  cancella,on  of  phase  III  clinical  trials:  “Higher  than  expected  rate  of  An,-­‐CHO  an,body  development”  (what  is  expected????).    IB1001  –  hemophilia  (Inspira3on  Biopharmaceu3cals)  
  10. 10. •  Danger  signals  of  all  sorts    •  Aggregates  –  how  do  they  work?    – (probably  don’t  work  if  no  T  cell  epitopes)    – Immune  complexes  –  Complement  •  T  cell  epitope  content  •  (absence  of)  Treg  epitope  content  •  Pre-­‐exis3ng  T  cell  response  (Tolerance  or  heterologous  immunity)  What  drives  immunogenicity?  
  11. 11. Factors (↑roof Immunogenicity) Immune effectGlycosylation (↑) Increase presentation? Increase foreign-ness of protein, need T cell epitopesPEGylation (↓) Slow antigen processing, “mask” T cellepitopes and B cell epitopesHost Cell-derived Protein (↑) CPG DNA (if bacterial); CHO T cell epitopesOxidized Form of the Product (↑) Increase foreign-ness, modify presentationExcipients (↑) Increase Danger signal, T cell epitopesLeachates (↑) Increase Danger signal, T cell epitopesCharacteristics of Patients (↑or↓) Missing Protein is foreign, T cell epitopesFrequency, Duration and Route ofAdministration (↑or↓)Administration like a vaccine, DAMPs, T cellepitopesAggregates (↑) Aggregation increases T cell epitopepresentationIn almost every caseMechanism of Action – T cell ResponseIn  almost  every  case  –  T  cell  epitope  drives  Immune  response  
  12. 12. An,gen  Epitope                      Drug  or  Vaccine  How  it  works  
  13. 13.  In  the  right  context  self  proteins  can  be  immunogenic.  Take  Epo†,  for  example.    T  cell  epitope  content  is  unequally  distributed  throughout  the  human  (and  CHO)  proteome.*    Immune  response  depends  on  protein  prevalence,  func,on  &  previous  exposure.**  †  Marc  H.V.  van  Regenmortel,  Ph.D.,  Ka,a  Boven,  M.D.,  Fred  Bader,  Ph.D.  Immunogenicity  of  Biopharmaceu,cals:  An  Example  from  Erythropoie,n:  Protein  structure,  contaminants,  formula,on,  container,  and  closure  all  can  affect  the  immunogenicity  of  the  product.    BioPharm  Interna,onal  2005.  hmp://www.biopharminterna,onal.com/biopharm/ar,cle/ar,cleDetail.jsp?id=174494&sk=&date=&pageID=5    *A.S.  De  Groot,  J.  Rayner,  W.  Mar,n.  Modeling  the  immunogenicity  of  therapeu,c  proteins  using  T  cell  epitope  mapping.  In:  Immunogenicity  of  Therapeu,c  Biological  Products.  Developments  in  Biologicals.  Fred  Brown,  Anthony  Mire  Suis,  editors.  Basel,  Karger,  2003.  Vol  112:71-­‐80.    **Clute,  S.  C.,  L.  B.  Watkin,  M.  Cornberg,  Y.  N.  Naumov,  J.  L.  Sullivan,  K.  Luzuriaga,  R.  M.  Welsh,  and  L.  K.  Selin.  2005.  Cross-­‐reac,ve  influenza  virus-­‐specific  CD8+  T  cells  contribute  to  lymphoprolifera,on  in  Epstein-­‐Barr  virus-­‐associated  infec,ous  mononucleosis.  The  Journal  of  clinical  inves,ga,on  115:3602-­‐3612.  CHO  are  mammalian  proteins  –    How  can  “self”  proteins  be  immunogenic?  
  14. 14. T  Cell  Epitope  Content    -­‐  Predicted  Poten,al  for  Immunogenicity  of  Selected  Proteins    -­‐80  -­‐60  -­‐40  -­‐20  0  20  40  60  80  100                                  Human  FSH  beta  Human  IgA  CD    Human  IgG  CD    Human              Albumin              Human              Amylase            De-­‐immunized  INF-­‐beta                          Human  Transferrin                  *  Human  Gonadotropin      Random  Expecta,on  Influenza  Hemagglu,nin  *  Human  GHRH  *  Human  Gonadotropin  w/signal  Tetanus  Toxin  Human  Erythropoie2n  Brazil  Nut  An,gen  *  Human  GHRH  w/signal  **  Human  INF-­‐  beta    Less    Immunogenic  Proteins  (based  on  clinical  experience)    Have  Fewer  T  cell  Epitopes  De  Groot,  As,  Goldberg  M,  Moise  L,  Mar,n  W.  Evolu2onary  deimmuniza2on:  An  ancillary  mechanism  for  self-­‐tolerance.  Cell  Immunol.  2007  Apr  17;    Pages  148-­‐153.  hmp://dx.doi.org/10.1016/j.cellimm.2007.02.006      Are  self  proteins  immunogenic?  
  15. 15. EpiVax  Immunogenicity  Hypothesis:  Immune  Response  =  Sum  of  Epitopes  T  cell  response  depends  on:    T  cell  epitope  content  +  HLA  of  subject    Protein  Immunogenicity  can  be  Ranked        epitope  Protein  Therapeu,c  1    +    1    +    1        =    Response  epitope  epitope  • De  Groot  A.S.  and  L.  Moise.  Predic,on  of  immunogenicity  for  therapeu,c  proteins:  State  of  the  art.    Current  Opinions  in  Drug  Development  and  Discovery.  May  2007.  10(3):332-­‐40.  In  biologics,  immunogenicity  is  related  to    T  cell  epitope  content  
  16. 16. EpiVax  -­‐  Immunogenicity  Scale    Low        Neutral        High  Albumin   Tetanus  Toxin  Protein  X  or  mAb  Y  Proteins  ranked  by  T-­‐  Epitope  content  per  Amino  Acid    •   De  Groot  A.S.,  Drug  Discovery  Today  -­‐  2006;  •   De  Groot  A.S.,  Mire-­‐Sluis,  A.  Ed..  Dev.  Biol.  Basel,  Karger,  2005.  vol  122.  pp  137-­‐160.    An,gen  A   An,gen  B  Aggregate  immunogenicity  drives    Immune  response  
  17. 17. EpiMatrix   predicted   excess/shorwall   in  aggregate   immunogenicity   rela,ve   to   a  random  pep,de  standard.  -­‐   80           -­‐  -­‐   70           -­‐  -­‐   60           -­‐  -­‐   50           -­‐  -­‐   40           -­‐  -­‐   30           -­‐  -­‐   20           -­‐  -­‐   10           -­‐  -­‐   00           -­‐  -­‐   -­‐  10           -­‐  -­‐   -­‐  20           -­‐  -­‐   -­‐  30           -­‐  -­‐   -­‐  40           -­‐  -­‐   -­‐  50           -­‐  -­‐   -­‐  60           -­‐  -­‐   -­‐  70           -­‐  -­‐   -­‐  80           -­‐  Thrombopoie2n  Human  EPO  Tetanus  Toxin  Influenza  -­‐  HA  Albumin  IgG  FC  Region  EBV  -­‐  BKRF3  Follitropin  -­‐  Beta  A  protein  score  >  20  indicates  a  significant  immunogenic  poten,al.    Proteins   that   have   previously   been  demonstrated   to   be   immunogenic   have  higher   poten,al   immunogenicity   on   the  scale.      Those  that  have  rarely  been  demonstrated  to   be   immunogenicity   have   lower   T   cell  epitope  content.      Immunogenicity  scale  
  18. 18. Some Vaccine Antigens – High Scores(work done for NMRC, Dept. of Defense)
  19. 19. -  80 --  70 --  60 --  50 --  40 --  30 --  20 --  10 --  00 --  -10 --  -20 --  -30 --  -40 --  -50 --  -60 --  -70 -Human EPOImmunogenic Antibodies*Tetanus ToxinInfluenza-HAAlbuminIgG FC RegionEBV-BKRF3Fibrinogen-AlphaNon-immunogenic Antibodies†Follitropin-BetaHirudin(-­‐90.41)    See  my  Blog  “Thinking  out  Loud”  for  a  discussion  of  Leech  proteins  and  Tick  Saliva  proteins-­‐Tick  saliva  proteins  also  have  low  immunogenicity  poten,al.    Hirudin  –  Very  Low  Poten,al  Immunogenicity  -­‐  Why?  Other Antigens – Extremely Low Scores(Hirudin, Tick Saliva, Some Parasites)
  20. 20. •  Handled on a case-by-case basis•  Consider Source•  Maximum dose (mg biologics/kg body weight)•  Route of administration•  Frequency of dosing•  Pre-clinical and clinical data•  Detection process in evolutionThe FDA Prefers Leech-like ProteinsAnd HCPs - Regulatory Perspective
  21. 21. HCP Analytical Technologies•  Detection–  Protein staining–  Immunoblotting•  Identification–  2D-PAGE/MS–  2D-LC/MS•  Quantitation–  ELISA using anti-HCP antibodies–  May need to develop internal processes–  Some kits are available•  Risk assessment–  Cytokine release assaysNew  Approach  –  Immunogenicity  Screening  in  silico  Analytical Tests for HCP
  22. 22. •  MHC  binding  is  a  prerequisite  for  immunogenicity  •  Epitopes  are  linear  and  directly  derived  from  an,gen  sequence  •  Binding  is  determined  by  amino  acid  side  chains  •  Matrix-­‐based  predictor  MHC  II  Mature  APCImmunogenicity  predic,on  
  23. 23. EpiMatrix  •  EpiVax  uses  EpiMatrix  to  predict  epitopes  –  matrix  based  predic,on  algorithm  •  Can  predict  either  class  I  or  class  II  MHC  binding  –  MHC  binding  is  a  prerequisite  for  immunogenicity  MHC  II  Pocket  Pep,de    Epitope  Mature  APCMHC  II  T  cell  epitopes  are  linear  and  directly  derived  from  an,gen  sequence    Binding  is  determined  by  amino  acid  side  chains  (R  groups)  and  ‘encoded’  in  single  lemer  code  23  6/3/13 Confidential
  24. 24. Easy  easy  to  deliver  as  pep,des  Clusters  of  MHC  binding  drive  T  cells  DRB1*0101    DRB1*0301    DRB1*0401    DRB1*0701    DRB1*0801    DRB1*1101    DRB1*1301    DRB1*1501    •  T  cell  epitopes  are  not  randomly  distributed  but  instead  tend  to  cluster  in  specific  regions.    –  These  clusters  can  be  very  powerful,  enabling  significant  immune  responses  to  low  scoring  proteins.  •  Clus,Mer  recognizes  T-­‐cell  epitope  clusters  as  polypep,des  predicted  to  bind  to  an  unusually  large  number  of  HLA  alleles.        6/3/13 Confidential
  25. 25. What  Makes  Proteins  Really  immunogenic?  Sequences  that  Contain  EpiBars  Confiden,al  Roberts  CGP,  Meister  GE,  Jesdale  BM,  Lieberman  J,  Berzofsky  JA,  A.S.  De  Groot,  Predic,on  of  HIV  pep,de  epitopes  by  a  novel  algorithm,  AIDS  Research  and  Human  Retroviruses,  1996,  Vol.  12,  No.  7,  pp.  593-­‐610.  Clus,Mer  -­‐  Locates  highly  immunogenic  regions  EpiBar  :  A  common  feature  of  highly  immunogenic  clusters  EpiBar  
  26. 26. EpiVax  Immunogenicity  Scale  Confiden,al  - 80 -- 70 -- 60 -- 50 -- 40 -- 30 -- 20 -- 10 -- 00 -- -10 -- -20 -- -30 -- -40 -- -50 -- -60 -- -70 -- -80 -ThrombopoietinHuman EPOImmunogenic Antibodies*Tetanus ToxinInfluenza-HAAlbuminIgG FC RegionEBV-BKRF3Fibrinogen-AlphaNon-immunogenic Antibodies†Follitropin-BetaPROTEIN_001 (35.13)Protein Immunogenicity ScaleProteins Scoring above +20 areconsidered to be potentiallyimmunogenic.On the left of the scale weinclude some well-knownproteins for comparison- 80 -- 70 -- 60 -- 50 -- 40 -- 30 -- 20 -- 10 -- 00 -- -10 -- -20 -- -30 -- -40 -- -50 -- -60 -- -70 -- -80 -ThrombopoietinHuman EPOImmunogenic Antibodies*Tetanus ToxinInfluenza-HAAlbuminIgG FC RegionEBV-BKRF3Non-immunogenic Antibodies†Follitropin-Beta
  27. 27. EpiMatrix  mAb  Immunogenicity  Scale    - 80 -- 70 -- 60 -- 50 -- 40 -- 30 -- 20 -- 10 -- 00 -- -10 -- -20 -- -30 -- -40 -- -50 -- -60 -- -70 -- -80 -IgG FC RegionNuvion (0%)Avastin (0%)AB01 (EPX Adjusted Score: -46.98)AB02 (EPX Adjusted Score: -44.48)AB03 (EPX Adjusted Score: -44.81)AB04 (EPX Adjusted Score: -45.81)AB05 (EPX Adjusted Score: -45.88)AB06 (EPX Adjusted Score: -47.85)AB07 (EPX Adjusted Score: -46.99)AB08 (EPX Adjusted Score: -46.30)AB09 (EPX Adjusted Score: -47.40)AB10 (EPX Adjusted Score: -45.88)AB11 (EPX Adjusted Score: -47.40)Synagis (1%)Simulect (1.4%)Humira (12%)Bivatuzumab (6.7%)Remicade (26%)Rituxan (27%)Campath (45%)Humicade (7%)Reopro (5.8%)Tysabri (7%)LeukArrest (0%)Herceptin (0.1%)Compare  with:  27  6/3/13 ConfidentialDue  to  the  presence  of  Tregitopes,  an,bodies  tend  to  fall  lower  on  the  immunogenicity  scale.  We  have  developed  a  refined  method  using  regression  analysis  to  predict   the   immunogenicity   of   an,body   sequences   based   on  observed  clinical  responses  (next  slide).  We   have   found   that   a   balance   in   favor   of   Tregitope   (regulatory)  content   over   neo-­‐epitope   (effector)   content   is   correlated   with  reduced  clinical  immunogenicity.  NeoEpitopeContent  Tregitope Content  High   Low  Low  Avastin (0%)  Herceptin (0%)Mylotarg (3%)  Simulect (1%)  Synagis (1%)  High  Campath (45%)  Remicade (26%)  Rituxan (27%)
  28. 28. CHO  genome  Immune      Response?  Self/  Microbiome  28  Logical Next Stepmeasure CHO/Self Conservation
  29. 29. Databases  available  Puta,vely    Secreted  (signal  pep3de)    Mouse  secreted  165  proteins  Transcriptome  32,801  con,gs  Validated  HCP  contaminants  25  proteins  CHO  genome  24,383    predicted  genes    
  30. 30. Key DatasetsGenome and transcriptome
  31. 31. •  Protein databases (UniProtKB/Swiss-Prot, Locate)•  BLAST•  SignalP•  EpiMatrix•  BlastiMer - JanusMatrixTools used for this analysis
  32. 32. •  Identify secreted CHO proteins•  Collect published HCP from CHO•  Evaluate potential immunogenicity•  Evaluate sequence homology•  Identify clustered regions – compare to CHO;•  Are human/CHO different at the cluster? Countas possible immunogenicity trigger.Approach
  33. 33. Immunogenicity    Scores  distribu,on  
  34. 34. Immunogenicity    Scale  Validated  HCP  CHO  contaminants  
  35. 35. Other  potential  contaminants  SL cytokine (84)Lysosomal protective protein (35)
  36. 36. But  are  human-­‐like  proteins  immunogenic?  CHO      okay?  peptides  
  37. 37. Putatively    Secreted  (signal  peptide)    Mouse  secreted  165  proteins  Transcriptome  32,801  contigs  Validated  HCP  contaminants  25  proteins  CHO  genome  24,383    predicted  genes    Human  proteome  20,238  proteins  Approach  to  conserva,on    with  Human    
  38. 38. •  Identify secreted CHO proteins•  Evaluate potential immunogenicity•  Evaluate sequence homology•  Identify clustered regions – compare to CHO;•  Are human/CHO different at the cluster? Countas possible immunogenicity trigger.Approach
  39. 39. T  cell  Receptor  Face  (epitope)  MHC-­‐binding  Face    (agretope)  T  cell  epitopes  are  two-­‐faced  
  40. 40. Identifies cross-reactive peptides:•  Identical T cell-facing residues•  Same HLA allele but . .•  OK if different MHC-facing residuesThe  God  of  Two  Faces:  JanusMatrix  
  41. 41. TCR  face  vs.  MHC  binding  face    MHC/HLATCRThe most conservative approach:•  Identical T cell-facing residues•  Same HLA allele and minimally differentMHC-facing residues
  42. 42. EpiMatrix  adjusted  immunogenicity  score    
  43. 43. Determina,on  of  conserva,on  with  self:  JanusMatrix  results    
  44. 44. Cross-­‐reactivity  visualization  Predicted  9-­‐mer  epitope  from  a  source  protein  Human  protein  where  cross-­‐reactive  epitopes  are  present  9-­‐mer  from  human  prevalent  proteome,    100%  TCR  face  identical  to  source  epitope  Source proteinHCV_G1_NS2_794
  45. 45. CEFT  Pep,des  (immunogenic)    
  46. 46. Flu  and  Tet  tox  epitopes  SNF2 histonelinker PHD RINGhelicaseETAA16 proteinAnkyrin repeatdomain 18AFlu HA308-318Ubiquitinspecificprotease 1Poly ADP ribosepolymerasefamily, member9Poly ADP ribosepolymerasefamily, member9TetanusToxin830-844Olfactoryreceptor, family5, subfamily D,member 14
  47. 47. hTregitope-­‐IGGC-­‐167    hTregitope-­‐IGGC-­‐289  HTREG_IGGC-289HTREG_IGGC-167
  48. 48. CHO: lysosomal protective proteinLysosomalprotectiveLysosomalprotective
  49. 49. SL cytokineCHO: SL cytokine  SL cytokine
  50. 50. •  Identify secreted CHO proteins•  Evaluate potential immunogenicity•  Evaluate sequence homology•  Identify clustered regions – compare to CHO;•  Are human/CHO different at the cluster? Countas possible immunogenicity trigger.New Approach for CHO
  51. 51. Immune  Response  =  Sum  of  Epitopes  Sum  includes  +  (T  effectors)  and  –  (Tregs)  scores  Protein  Therapeu,c  Host  Cell  Protein  Contaminant  HCP  Epitope  New Approach
  52. 52. For  an  individual,  T  cell  response  depends  on:    T  cell  epitope  content  x  HLA  –  Treg  Epitope  content  x  HLA    Vaccine or Foreign Protein = (TeffPT1+  TeffPT2  .  .  .  )  =      Response  CHO = Σ (  TeffPT    +    TeffPT  +  TeffHCP  –  TregPT)  =      Treg  Adjusted  Response  Immune  response  depends  on    Foreign-­‐ness    Potential  Tregs    Adjuvant  (Danger  signal)  Proposed adjustment to score
  53. 53. Available  now:  CHOPPI  CHO  Protein  Predicted  Immunogenicity  CHOPPI  hmp://bit.ly/11fZqfJ  
  54. 54. •  Formula,on  (VLP;  aggregates)  •  “Danger  Signal”  •  Route:  Subcutaneous  delivery?  •  Dose  (high/low,  persistent,  intermiment)  •  T  cell  epitope  content  •  Differing  T  cell  epitope  content  =  HCP  55  In  Closing  Factors  affec,ng  Immunogenicity  
  55. 55. • While CHO are the mostcommonly used cell lines formammalian cell protein expression,Company-specific cell lines mayvary. Furthermore, we can’tanticipate• Genetic engineering• Batch-to-batch variation• Expression (based on above)• Which protein will ‘hitchhike’CHO Cell lines may differ
  56. 56. Genomics“Expressome”InformaticsIn  the  future  –  Obtain  proteins  through    MS/MS  HPLC  –  and  Sequence,  ID  epitopes    
  57. 57. Thank  you!  And  .  .  .  CHOPPI:  hmp://bit.ly/11fZqfJ  or  contact  me.    Translational Immunology Research and Accelerated [Vaccine]Development Institute for Immunology and Informatics University ofRhode IslandDartmouth CollegeEpiVax, Inc. SL cytokine
  58. 58. Institute for Immunology and Informatics (iCubed)D.  Spero  icubed  overview  2011  www.immunome.org  URI  Alumni  Board  2012  
  59. 59. New  Concept:    Tregitopes  induce    tolerance  to      protein      Therapeu,cs    (Friday  April  20th    Session)        Epitope may induce different types of Response
  60. 60. CHO Adjustment for Immunogenicity ?+    +    Conserved epitope Neo-EpitopeNeo-Epitope
  61. 61. Immune  Response  =  Sum  of  Epitopes  Sum  includes  +  (T  effectors)  and  –  (Tregs)  scores  ISPRI approach to analyzing mAbs . . .T  cell  response  depends  on:    T  cell  epitope  content  x  HLA  –  Treg  Epitope  content  x  HLA      Protein  Immunogenicity  can  be  Ranked        Treg  epitope  Protein  Therapeu,c  1    +    1    -­‐  Treg    =      Response  epitope  epitope  
  62. 62. T  reg  S,mulus  IL  10,  TNF  alpha     Additional Treg EpitopeModify Effector T cell response:Reduce T effector StimulusCurrent Hypothesis: More TregitopesLower ImmunogenicityDe Groot A.S. and D. Scott. Immunogenicity of Protein Therapeutics.Trends in Immunology. Invited Review. Trends Immunol. 2007 Nov;28(11):482-90.631/29/11   63  Confiden,al  and  Copyrighted  EpiVax  
  63. 63. 64  EpiVax: Immunogenicity scale is
  64. 64. Correlation of antibody immunogenicity withTregitope adjusted EPX Scores65  
  65. 65. Correlation of EpiMatrix Scoresand Immunogenicity in Human studies40%  37%    21.97    FPX  1    0%  9.3%  -­‐111.25  FPX  5  NA  0.5%  12%  Neutralizing  An,bodies  5.6%  7.8%  53%  Binding  An,bodies  -­‐1.76  1.62  34.37  EpiMatrix  score  FPX  4  FPX  3  FPX  2  Protein  Na:    not  analyzed  Nega,ve  score  indicates  presence  of  Treg  epitope  
  66. 66. -  80 --  70 --  60 --  50 --  40 --  30 --  20 --  10 --  00 --  -10 --  -20 --  -30 --  -40 --  -50 --  -60 --  -70 --  -80 -ThrombopoietinHuman EPOImmunogenic Antibodies*Tetanus ToxinInfluenza-HAAlbuminIgG FC RegionEBV-BKRF3Fibrinogen-AlphaNon-immunogenic Antibodies†Follitropin-BetaAb K (-38.23)Ab E (-16.03)Ab N (-53.88)Ab P (-70.14)Ab B (-00.32)Ab A (13.82)Ab D (-08.87)Ab F (-22.13)Ab I (-25.77)Ab O (-54.26)Ab L (-48.49)Ab C (-02.03)Ab M (-52.25)Ab H (-24.99)Ab J (-28.94)Ab G (-24.33)*Tregitope  adjusted  Application – Germline Abs*

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