EpiVax’s ISPRI Immunogenicity
Screening Toolkit and Services
August 2015
EpiVax - non-confidentialinfo@epivax.com
EpiVax, Inc.
Improving Human Health Everywhere
2EpiVax - non-confidentialinfo@epivax.com
EpiVax designs and develops
safer...
EpiVax Management Team
www.epivax.com/about/epivax-team/
3EpiVax - non-confidentialinfo@epivax.com
Dr. Annie De Groot, CEO...
The EpiVax Approach to
Immunogenicity Screening
4EpiVax - non-confidentialinfo@epivax.com
http://bit.ly/The_TCWP
In VivoIn...
ISPRI: Interactive Screening and
Protein Reengineering Interface
EpiVax - non-confidentialinfo@epivax.com
ISPRI: Developed for Biologics
6EpiVax - non-confidentialinfo@epivax.com
• ISPRI is EpiVax’s integrated in silico toolkit ...
ISPRI Tools Summary
7EpiVax - non-confidentialinfo@epivax.com
Immunogenicity Prediction:
• EpiMatrix
– Screen the protein ...
ISPRI Tools Summary
8EpiVax - non-confidentialinfo@epivax.com
Deimmunization of Protein Therapeutics:
• OptiMatrix
– Subst...
ISPRI vs. IEDB in silico services
9EpiVax - non-confidentialinfo@epivax.com
Epitope
Ranking
Tregitope
ISPRI is an integrat...
Features EpiVax IEDB
Highly Accurate Epitope Prediction  1
Cluster Tool / EpiBar  2
Immunogenicity Scale * X
iTEM Ana...
Immunogenicity Screening
& Prediction Overview
EpiVax - non-confidentialinfo@epivax.com
The Immunogenicity Puzzle
12EpiVax - non-confidentialinfo@epivax.com
T Cell Epitopes
AND
“Foreign-ness,”
Aggregation,
“Dan...
T Cell Epitope Prediction
13EpiVax - non-confidentialinfo@epivax.com
• EpiVax uses EpiMatrix to predict T cell epitopes
– ...
Epitope Predictive Accuracy
14EpiVax - non-confidentialinfo@epivax.com
De Groot AS, Martin W. Reducing risk, improving out...
EpiVax HLA “Supertype” Coverage
15EpiVax - non-confidentialinfo@epivax.com
EpiVax tests for binding potential
to the most ...
EpiVax HLA “Supertype” Coverage
16EpiVax - non-confidentialinfo@epivax.com
EpiVax tests for binding potential
to the most ...
Prediction Approach – Whole Proteins
17EpiVax - non-confidentialinfo@epivax.com
T cell response depends on:
T cell epitope...
Epitope Presence Indicates Immune
Potential
18EpiVax - non-confidentialinfo@epivax.com
Mature
APC
T reg
T eff
Epitopes can...
EpiVax’s Tregitope Technology
19EpiVax - non-confidentialinfo@epivax.com
• Discovered & patented by
EpiVax
• Highly conser...
Prediction Approach – Antibodies
20EpiVax - non-confidentialinfo@epivax.com
T cell response depends on:
T cell epitope con...
Antibody Immunogenicity Predictions
Without Tregitope Adjustment
21EpiVax - non-confidentialinfo@epivax.com http://bit.ly/...
Correlation of antibody immunogenicity
with Tregitope adjusted EPX Scores
22EpiVax - non-confidentialinfo@epivax.com http:...
ISPRI Tools
EpiVax - non-confidentialinfo@epivax.com
ISPRI Tools
EpiMatrix Protein Report, Flu HA Example
24EpiVax - non-confidentialinfo@epivax.com
Assessment
≥1.64 = Hit
Epi...
ISPRI Tools
Protein Immunogenicity Scale
25EpiVax - non-confidentialinfo@epivax.com
EpiMatrix Predicted
Excess/Shortfall i...
High-Throughput Antibody Analysis
26EpiVax - non-confidentialinfo@epivax.com
http://www.epivax.com/immunogenicity-screenin...
ISPRI Tools
ClustiMer
27EpiVax - non-confidentialinfo@epivax.com
• T cell epitopes are not randomly distributed throughout...
In Silico Tools
Cluster Immunogenicity Scale
28EpiVax - non-confidentialinfo@epivax.com
EpiMatrix Predicted
Excess/Shortfa...
ISPRI Tools
BlastiMer
29EpiVax - non-confidentialinfo@epivax.com
BlastiMer finds human-like sequences
ISPRI Tools
BlastiMer
• BLAST functions:
– Submit T cell epitope clusters to NCBI GenBank BLAST
• Compare to Non-redundant...
ISPRI Tools
JanusMatrix
31EpiVax - non-confidentialinfo@epivax.com
epitope
APC
T Cell
MHC
face
TCR
face
In Roman mythology...
ISPRI Tools
JanusMatrix
32EpiVax - non-confidentialinfo@epivax.com
HTREG_IGGC-289
HTREG_IGGC-167
immunogenic
pathogen sequ...
ISPRI Tools
JanusMatrix Applications to Protein Engineering
33EpiVax - non-confidentialinfo@epivax.com
- 80 -
- 70 -
- 60 ...
ISPRI Services and Validation
EpiVax - non-confidentialinfo@epivax.com
In Silico Immunogenicity Prediction
ISPRI Services
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HT Screening Analysis: High-...
The High-Throughput Antibody Analysis
allows users to compare a large number
potential antibody candidates by ranking
the ...
PreDeFT Analysis
38EpiVax - non-confidentialinfo@epivax.com
A PreDeFT Analysis is a comprehensive evaluation of the immuno...
ISPRI Website Homepage
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• This interactive biologics screening and
optimizing wo...
ISPRI Validation in Print
(A Sampling)
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Koren E, De Groot AS, Jawa V, Beck KD, B...
Published Validation: Over 60
Publications in Protein Therapeutics
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1: De Groot ...
Published Validation: Over 60
Publications in Protein Therapeutics
42EpiVax - non-confidentialinfo@epivax.com
10: Cousens ...
Published Validation: Over 60
Publications in Protein Therapeutics
43EpiVax - non-confidentialinfo@epivax.com
19: Cohen T,...
EpiVax Services Summary
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Immunogenicity
Screening
Epitope
Validation
Epitope
Mod...
Science without fear.
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EpiVax ISPRI: Next Generation Immunogenicity Screening and Protein Re-engineering

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Learn about EpiVax's innovative web-based platform for immunogenicity screening, analysis and re-engineering for novel protein therapeutics. ISPRI combines our highly-validated immunogenicity mapping algorithms with tools for analyzing cross-reactivity, identifying immuno-suppressive sequences and optimizing protein sequence immunogenicity in a convenient, secure platform that allows you to upload thousands of sequences. Upgrade your development process for antibodies, enzymes and other biopharmaceutical products.

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  • Slide 11 Predictive accuracy of available T cell epitope prediction tools. For EpiMatrix, the average Area Under the Curve (AUC) value was .76, with top performers DRB1*1101, DRB1*0701, and DRB1*0101.
  • Slide 7 Immunogenicity scoring with Tregitope-adjusted scores. This polynomial regression can be used to estimate the clinical immunogenicity of a given antibody sequence. Twenty monoclonal antibody sequences were obtained from the literature or the US Patent and Trademark office (USPTO). Each sequence was then scanned both for epitopes (any epitope restricted by eight common HLA alleles). Positive EpiMatrix scores for each of the antibodies were summed with epitopes contained in putative T regulatory epitopes were set aside (not included in the final sum). These putative Treg epitopes (IgG “Tregitopes”) were defined as 9-mers conserved in more than 1% of observed antibodies (>4000) and scoring at least 5 on the EpiMatrix cluster scale. The final regression is shown. No Fc regions were scored for this study as all are highly conserved.

    For oval – There is reason to believe this represents a valley of death for today’s antibody developers.
  • Slide 7 Immunogenicity scoring with Tregitope-adjusted scores. This polynomial regression can be used to estimate the clinical immunogenicity of a given antibody sequence. Twenty monoclonal antibody sequences were obtained from the literature or the US Patent and Trademark office (USPTO). Each sequence was then scanned both for epitopes (any epitope restricted by eight common HLA alleles). Positive EpiMatrix scores for each of the antibodies were summed with epitopes contained in putative T regulatory epitopes were set aside (not included in the final sum). These putative Treg epitopes (IgG “Tregitopes”) were defined as 9-mers conserved in more than 1% of observed antibodies (>4000) and scoring at least 5 on the EpiMatrix cluster scale. The final regression is shown. No Fc regions were scored for this study as all are highly conserved.

    For oval – There is reason to believe this represents a valley of death for today’s antibody developers.
  • Slide 7 The Protein Immunogenicity Scale rates your protein against known immunogenic and non-immunogenic proteins. Scores range from approximately -80 to +80; any score above +20 indicates a significant potential for immunogenicity.
  • Slide 8 Similar to the Protein Immunogenicity Scale, the Cluster Immunogencity Scale rates T cell epitope clusters identified in your protein against known promiscuous T cell epitopes. T cell epitope clusters with scores above +10 have a high potential for immunogenicity. These clusters may be selectively modified using the Optimatrix algorithm, described on slides 9 and 10.
  • Cross reactivity of putative epitopes is not constrained to exact sequence matching as the TCR only interacts with five of 9 peptides in the epitope sequence. To improve our prediction of homology we developed JanusMatrix, which takes into account the “two-faced” nature of epitopes.

    We can discover sequences that interact with pre-existing non-naive T cells by searching for homologs at MHC binding residues where TCR-facing residues are strictly conserved.

    This may be useful for avoiding T cell epitopes that are cross reactive with pre-existing T cells.
  • 2) T effector epitopes:
    HCV_G1_2440
    HCV_G1_2879
    HCV_G1_2485
    HCV_G1_NS4B_1769
    HCV_G1_NS4B_1725
    HCV_G1_ENV_255
    HCV_G1_NS4B_1790
    HCV_G1_NS2_732
    HCV_G1_2840
    HCV_G1_1605
    HCV_G1_2941
    HCV_G1_ENV_359
    HCV_G1_NS2_909
    HCV_G1_NS4B_1876
    HCV_G1_NS4b_1798
    HCV_G1_DEXDC_1246
    HCV_G1_NS4B_1910
    HCV_G1_1941
    HCV_G1_2898
    HCV_G1_NS5A_1988
    HCV_G1_2913
    HCV_G1_NS2_748

    You need to zoom in to see the name of the cluster.

    The legend is:
    - Light blue diamond (source): cluster from HCV.
    - Gray square: Predicted 9-mer epitope from the HCV cluster. 
    - Blue triangle: 9-mer from human genome (Plasma proteome database), 100% TCR face identical to cluster-derived epitope.
    - Lavender circle: Human protein where cross-reactive (with HCV cluster) epitopes are present.


  • Slide 12 Modification of just one residue in the Original Sequence eliminates 5 hits for different HLA alleles and reduces the Cluster Score of the Modified Sequence to 4.57. Other modifications are available, but for a one-click solution, OptiMatrix will suggest the best single change to a given cluster sequence (red highlight).
  • EpiVax ISPRI: Next Generation Immunogenicity Screening and Protein Re-engineering

    1. 1. EpiVax’s ISPRI Immunogenicity Screening Toolkit and Services August 2015 EpiVax - non-confidentialinfo@epivax.com
    2. 2. EpiVax, Inc. Improving Human Health Everywhere 2EpiVax - non-confidentialinfo@epivax.com EpiVax designs and develops safer, more effective biologic products and vaccines Engaged • “Thought Leaders” • Protein therapeutics • Genome-derived vaccines • Immunoinformatics • Client-focused service • Strong corporate values: Improving Human Health Everywhere Cutting Edge • Source of new ideas • Continually developing new innovative tools • Focused on safer, more efficient and robust biologics and vaccines Trusted • Tested & proven methods • Extensively peer-reviewed and highly published • Clients include 9 of the 12 largest pharma companies and many more small companies
    3. 3. EpiVax Management Team www.epivax.com/about/epivax-team/ 3EpiVax - non-confidentialinfo@epivax.com Dr. Annie De Groot, CEO/CSO William Martin, CIO/COO • Cliff Grimm, Managing Director • Dr. Eduardo Guillen, Scientific Director of Protein Therapeutics • Dr. Lenny Moise, Scientific Director of Vaccine Research • Frances Terry, Bioinformatics Program Manager • Ryan Harvey, Laboratory Manager
    4. 4. The EpiVax Approach to Immunogenicity Screening 4EpiVax - non-confidentialinfo@epivax.com http://bit.ly/The_TCWP In VivoIn VitroIn Silico Modifysequences to reduce immunogenicity Screenmultiple therapeutic candidates Human SCID mouse model HLA- transgenic mouse model Either Examine: • Characteristics of T cell and APC response • Effect of HLA donor diversity • Effect of donor pathologies • Effect of formulation and post- translational modifications Rank for immunogenicity based on predicted epitope content Proceed to Drug Development Peptide and protein screening: EffectonT cell populations Memory and naïve T cells HLA binding studies Cell- based assays Whole protein screening: Processingby purifiedDCs
    5. 5. ISPRI: Interactive Screening and Protein Reengineering Interface EpiVax - non-confidentialinfo@epivax.com
    6. 6. ISPRI: Developed for Biologics 6EpiVax - non-confidentialinfo@epivax.com • ISPRI is EpiVax’s integrated in silico toolkit for prediction, analysis and reduction of T cell immunogenicity of protein therapeutics • Predictions reduce laboratory work (typically at least 20-fold) and focus development on critical protein regions • In silico immunogenicity screening helps researchers save time, money and effort by providing actionable data on protein immunogenicity
    7. 7. ISPRI Tools Summary 7EpiVax - non-confidentialinfo@epivax.com Immunogenicity Prediction: • EpiMatrix – Screen the protein sequences of product candidates for the presence of putative T cell epitopes. – Rate the immunogenic potential of each submitted sequence on a normalized scale and compare each protein to other immunogenic proteins and antibodies • ClustiMer – Identify T-cell epitope clusters contained within product candidates – Rate the immunogenic potential of each T-cell epitope cluster on a normalized scale and compare each T-cell epitope cluster to other well-known immunogenic epitope clusters • JanusMatrix – Compare epitopes to human genome epitopes that have the same TCR face • Homology Analyses – BLAST epitope clusters against the non-redundant protein or patent database at GenBank – Cross-reference T-cell epitope clusters against the IEDB database of known epitopes
    8. 8. ISPRI Tools Summary 8EpiVax - non-confidentialinfo@epivax.com Deimmunization of Protein Therapeutics: • OptiMatrix – Substitute immunogenic amino acids with non-immunogenic residues that are conserved in existing databases (based on published sequences) and that do not introduce new epitopes. See the effects of amino acid substitution on the immunogenicity score in real time. • Tregitope Analysis – Identify within each submitted sequence putative regulatory T-cell epitopes (i.e. sub-regions contained within the submitted sequences which may relate to natural regulatory T cells and which may help to dampen the immune potential of the submitted antibody sequence)
    9. 9. ISPRI vs. IEDB in silico services 9EpiVax - non-confidentialinfo@epivax.com Epitope Ranking Tregitope ISPRI is an integrated, interactive set of tools specifically designed for immunogenicity analysis. ISPRI provides the depth of analysis necessary to accurately predict clinical immunogenicity. Epitope Mapping Cluster Analysis ARB NN- align SMM align Protein Reengineering Epitope Mapping EpiMatrix ClustiMer/ EpiBar OptiMatrix iTEM Immunogenicity Scale DeFT (OptiMatrix) In Vitro / In vivo Assays JanusMatrix JMX-Adjusted Score Target Selection Tolerization IEDB is a collection of tools that are not integrated in a coherent fashion. Epitope prediction is possible, but seamless immunogenicity screening and protein reengineering is not.
    10. 10. Features EpiVax IEDB Highly Accurate Epitope Prediction  1 Cluster Tool / EpiBar  2 Immunogenicity Scale * X iTEM Analysis * X Tregitope Predictions * X Deimmunization tools  X Human Genome Comparison  X High-Throughput Analysis  X Published Validation  3 Expert Consulting Services  X * These features were developed and validated by, and are only available at, EpiVax. 1 Head to head comparison favors EpiMatrix: see De Groot and Martin, Clinical Immunology, 2009. http://bit.ly/De_Groot_and_Martin 2 IEDB Cluster tool is available, but untested; for EpiVax validation, see “T cell epitope, Friend or Foe” http://bit.ly/T-friend-or-foe 3 Extent of IEDB validation is unknown (positive results may be published, but negative results are not tracked). EpiVax vs. IEDB 10EpiVax - non-confidentialinfo@epivax.com
    11. 11. Immunogenicity Screening & Prediction Overview EpiVax - non-confidentialinfo@epivax.com
    12. 12. The Immunogenicity Puzzle 12EpiVax - non-confidentialinfo@epivax.com T Cell Epitopes AND “Foreign-ness,” Aggregation, “Danger signals,” Route, Dose, Frequency, Glycosylation/PEGylation, etc.
    13. 13. T Cell Epitope Prediction 13EpiVax - non-confidentialinfo@epivax.com • EpiVax uses EpiMatrix to predict T cell epitopes – Matrix-based prediction algorithm • EpiVax predicts both class I and class II HLA binding – HLA binding is a prerequisite for immunogenicity – Full suite of HLA-based predictions are available Mature APC HLA-peptide complex Epitope Protein
    14. 14. Epitope Predictive Accuracy 14EpiVax - non-confidentialinfo@epivax.com De Groot AS, Martin W. Reducing risk, improving outcomes: bioengineering less immunogenic protein therapeutics. Clin Immunol. 2009 May;131(2):189-201 Comparison Data Taken From: Wang P, Sidney J, Dow C, Mothé B, Sette A, et al. (2008) A Systematic Assessment of MHC Class II Peptide Binding Predictions and Evaluation of a Consensus Approach. PLoS Comput Biol 4(4): e1000048. Doi:10.1371/journal.pcbi.1000048 Perfect =100% Random = 50%
    15. 15. EpiVax HLA “Supertype” Coverage 15EpiVax - non-confidentialinfo@epivax.com EpiVax tests for binding potential to the most common HLA molecules within each of the “supertypes” shown to the left. This allows us to provide results that are representative of >95% of human populations worldwide* without the necessity of testing each haplotype individually. Southwood et. al., Several Common HLA-DR Types Share Largely Overlapping Peptide Binding Repertoires. 1998. Journal of Immunology. DR1*0301 DR1*1107 DR1*0306/DR1*0307/DR1*0308/DR1*0311 DR1*0309 DR1*1104/DR1*1106/DR1*1311 DR1*1101 DR1*1128/DR1*1305 DR1*1321 DR1*1307 DR1*1114/DR1*1323 DR1*1120/DR1*1302 DR1*1322 DR1*1102/DR1*1121 DR1*1301/DR1*1327/DR1*1328 DR1*1304 DR1*0404/DR1*0423 DR1*0410 DR1*0405 DR1*0408 DR1*0402 DR1*0401/DR1*0426 DR1*1502 DR1*1501/DR1*1506 DR1*0102 DR1*0101 DR1*0806 DR1*0801 DR1*0817 DR1*0813 DR1*0804 DR1*0802 DR1*0701/DR1*0703 DR5*0101/D51*0105 DR3 DR11 DR8 DR4 DR15 DR1 DR13 DR7 DR51 DR1*0305
    16. 16. EpiVax HLA “Supertype” Coverage 16EpiVax - non-confidentialinfo@epivax.com EpiVax tests for binding potential to the most common HLA molecules within each of the “supertypes” shown to the left. This allows us to provide results that are representative of >95% of human populations worldwide* without the necessity of testing each haplotype individually. Southwood et. al., Several Common HLA-DR Types Share Largely Overlapping Peptide Binding Repertoires. 1998. Journal of Immunology. DR3 DR11 DR8 DR13 DR4 DR51 DR7 DR1 DR15
    17. 17. Prediction Approach – Whole Proteins 17EpiVax - non-confidentialinfo@epivax.com T cell response depends on: T cell epitope content + HLA of subject  protein immunogenicity can be ranked epitope Protein Therapeutic: 1 + 1 + 1 = Response epitopeepitope De Groot A.S. and L. Moise. Prediction of immunogenicity for therapeutic proteins: State of the art. Current Opinions in Drug Development and Discovery. May 2007. 10(3):332-40.
    18. 18. Epitope Presence Indicates Immune Potential 18EpiVax - non-confidentialinfo@epivax.com Mature APC T reg T eff Epitopes can be either effector or regulatory Most in silico algorithms cannot differentiate between these two
    19. 19. EpiVax’s Tregitope Technology 19EpiVax - non-confidentialinfo@epivax.com • Discovered & patented by EpiVax • Highly conserved peptide sequences in Fc and Fab regions of antibodies • High affinity, promiscuous binders across HLA alleles • Activate antigen-specific regulatory T cells • Can be co-formulated or synthesized with therapeutic proteins or carriersDe Groot A.S., et al., Activation of Natural Regulatory T cells by IgG Fc-derived Peptide “Tregitopes”. Blood, 2008,112: 3303. http://tinyurl.com/ASDeGroot-Blood-2008 http://bit.ly/Treg1
    20. 20. Prediction Approach – Antibodies 20EpiVax - non-confidentialinfo@epivax.com T cell response depends on: T cell epitope content – Tregitope content + HLA of subject  mAb immunogenicity can be ranked epitope Monoclonal Antibodies: 1 + 1 - regulatory T cell epitope = response epitopeepitope
    21. 21. Antibody Immunogenicity Predictions Without Tregitope Adjustment 21EpiVax - non-confidentialinfo@epivax.com http://bit.ly/Treg1 R² = 0.17 -5% 0% 5% 10% 15% 20% 25% 30% 35% 40% 45% 50% -30 -20 -10 0 10 20 30 40 50 60 70 ObservedImmunogenicity(%ADA) Combined Average of Variable Heavy and Light Chain EpiMatrix Scores Without Tregitope-Adjusted Scores to Predict Immunogenicity AVASTIN BIVATUZUMAB CAMPATH HERCEPTIN HUMICADE HUJ591 HUMIRA LEUKARREST LUCENTIS MYLOTARG RAPTIVA REMICADE REOPRO RITUXAN SIMULECT SOLIRIS SYNAGIS TYSABRI VECTIBIX VISILIZUMAB XOLAIR ZENAPAX
    22. 22. Correlation of antibody immunogenicity with Tregitope adjusted EPX Scores 22EpiVax - non-confidentialinfo@epivax.com http://bit.ly/Treg1 R² = 0.76 0% 5% 10% 15% 20% 25% 30% 35% 40% 45% 50% -70 -60 -50 -40 -30 -20 -10 0 10 20 30 ObservedImmunogenicity(%ADA) Combined Average of Variable Heavy and Light Chain EpiMatrix Scores Using Tregitope-Adjusted Scores to Predict Immunogenicity AVASTIN BIVATUZUMAB CAMPATH HERCEPTIN HUMICADE HUJ591 HUMIRA LEUKARREST LUCENTIS MYLOTARG RAPTIVA REMICADE REOPRO RITUXAN SIMULECT SOLIRIS SYNAGIS TYSABRI VECTIBIX VISILIZUMAB XOLAIR ZENAPAX First-generation chimerics Low ADA probability Medium ADA probability Failed in trials?
    23. 23. ISPRI Tools EpiVax - non-confidentialinfo@epivax.com
    24. 24. ISPRI Tools EpiMatrix Protein Report, Flu HA Example 24EpiVax - non-confidentialinfo@epivax.com Assessment ≥1.64 = Hit EpiMatrix Immunogenicity Score EpiBar: >4 hits within a 9-mer
    25. 25. ISPRI Tools Protein Immunogenicity Scale 25EpiVax - non-confidentialinfo@epivax.com EpiMatrix Predicted Excess/Shortfall in Aggregate Immunogenicity Relative to a Random Peptide Standard * Average of antibodies known to induce anti-therapeutic responses in more than 5% of patients † Average of antibodies known to induce anti-therapeutic responses in less than 5% of patients All scores are adjusted for the presence of Tregitopes. - 80 - - 70 - - 60 - - 50 - - 40 - - 30 - - 20 - - 10 - - 00 - - -10 - - -20 - - -30 - - -40 - - -50 - - -60 - - -70 - - -80 - Thrombopoietin Erythropoietin Fibrinogen-Gamma Albumin IgG FC Region GMCSF Follitropin-Beta Fibrinogen-Alpha Beta-2-Microglobulin Interferon-Beta Tetanus Toxin Influenza-HA Immunogenic Antibodies* Non-Immunogenic Antibodies† Your Protein
    26. 26. High-Throughput Antibody Analysis 26EpiVax - non-confidentialinfo@epivax.com http://www.epivax.com/immunogenicity-screening/ht_report/ The High-Throughput Antibody Analysis tool allows users to compare a large number antibody candidates by ranking the immunogenicity of heavy and light chains.
    27. 27. ISPRI Tools ClustiMer 27EpiVax - non-confidentialinfo@epivax.com • T cell epitopes are not randomly distributed throughout protein sequences but instead tend to cluster in specific regions. • ClustiMer is used to identify T-cell epitope clusters. It identifies polypeptides predicted to bind to an unusually large number of HLA alleles. • T cell epitope clusters make excellent vaccine candidates: – compact; relatively easy to deliver as peptides; highly reactive in-vivo • These clusters can be very powerful. One or more dominant T-cell epitope clusters can enable significant immune responses to even otherwise low-scoring proteins. DRB1*0101 DRB1*0301 DRB1*0401 DRB1*0701 DRB1*0801 DRB1*1101 DRB1*1301 DRB1*1501
    28. 28. In Silico Tools Cluster Immunogenicity Scale 28EpiVax - non-confidentialinfo@epivax.com EpiMatrix Predicted Excess/Shortfall in Aggregate Immunogenicity Relative to a Random Peptide Standard Tetanus Toxin (825-850) NPC NS3 (1248-1267) Influenza-HA (306-319) P. Falciparum (72-86) Human CLIP P. Falciparum (512-526) Theoretical Minimum - 40 - - - - 30 - - - - 20 - - - - 10 - - - - 00 - - - - -10 - NAME ADDRESS SCOR YOUR_PROTEIN1 318-341 31. YOUR_PROTEIN1 206-225 17. YOUR_PROTEIN1 159-176 13. YOUR_PROTEIN1 055-070 13. YOUR_PROTEIN1 293-314 10. Your Cluster Y Your Cluster Z Your Cluster X Score 30.05 Score 13.23 Score 11.76
    29. 29. ISPRI Tools BlastiMer 29EpiVax - non-confidentialinfo@epivax.com BlastiMer finds human-like sequences
    30. 30. ISPRI Tools BlastiMer • BLAST functions: – Submit T cell epitope clusters to NCBI GenBank BLAST • Compare to Non-redundant Database Patent Database Human Sequence Database • View Summary or detailed Alignment Reports 30EpiVax - non-confidentialinfo@epivax.com BlastiMer finds naturally-occurring substitutions
    31. 31. ISPRI Tools JanusMatrix 31EpiVax - non-confidentialinfo@epivax.com epitope APC T Cell MHC face TCR face In Roman mythology, Janus is the two-faced god of transitions, gates, and doorways. Each MHC ligand has two faces: 1. The MHC-binding face (agretope) and 2. The TCR-interacting face epitope) JanusMatrix is designed to predict the potential for cross-reactivity between epitope clusters and the human genome, based on conservation of TCR-facing residues in their putative HLA ligands. peptide TCR face MHC face
    32. 32. ISPRI Tools JanusMatrix 32EpiVax - non-confidentialinfo@epivax.com HTREG_IGGC-289 HTREG_IGGC-167 immunogenic pathogen sequences cross-conserved Tregitopes Moise L et al. Hum Vaccin Immunother. 2013 Jul;9(7):1577-86 peptide TCR face MHC face Source 9-mer epitope Human protein with cross-conserved epitopes Cross-conserved human 9-mer epitope Source (pathogen) protein Each MHC ligand has two faces: 1. The MHC-binding face (agretope) and 2. The TCR-interacting face epitope) JanusMatrix is designed to predict the potential for cross-reactivity between epitope clusters and the human genome, based on conservation of TCR-facing residues in their putative HLA ligands.
    33. 33. ISPRI Tools JanusMatrix Applications to Protein Engineering 33EpiVax - non-confidentialinfo@epivax.com - 80 - - 70 - - 60 - - 50 - - 40 - - 30 - - 20 - - 10 - - 00 - - -10 - - -20 - - -30 - - -40 - - -50 - - -60 - - -70 - - -80 - Variant 3 Variant 1 Variant 2 Variant 3, adjusted for HG XC Variant 1, adjusted for HG XC Variant 2, adjusted for HG XC Adjust mutation strategy based upon T cell human genome cross-conservation (HG XC): High Immunogenic Potential Low Immunogenic Potential
    34. 34. ISPRI Services and Validation EpiVax - non-confidentialinfo@epivax.com
    35. 35. In Silico Immunogenicity Prediction ISPRI Services 36EpiVax - non-confidentialinfo@epivax.com HT Screening Analysis: High-Throughput immunogenicity screening of large sets of antibody heavy-light chain combinations, for overall and comparative immunogenic potential. http://bit.ly/PreDeFTht PreDeFT Analysis: Highly detailed in silico immunogenicity analysis covering overall and regional immunogenic potential of a protein therapeutic. EpiVax delivers an “FDA-ready” report. Originally designed as a report Pre-Deimmunization of a Functional Therapeutic. http://bit.ly/PreDeFT ISPRI Website: Cloud-based Interactive Screening and Protein Reengineering Interface, accessed by large pharma world-wide. Scientists use ISPRI to predict overall and regional immunogenicity. Available as annual access for a predetermined number of protein sequences (Limited or High-Volume). Includes continued consultation and training with EpiVax experts. http://bit.ly/ISPRI
    36. 36. The High-Throughput Antibody Analysis allows users to compare a large number potential antibody candidates by ranking the immunogenicity of heavy and light chains. http://www.epivax.com/immunogenicity-screening/ht_report/ High-Throughput Antibody Analysis 37EpiVax - non-confidentialinfo@epivax.com
    37. 37. PreDeFT Analysis 38EpiVax - non-confidentialinfo@epivax.com A PreDeFT Analysis is a comprehensive evaluation of the immunogenicity of a selected protein or proteins. This report is generated by immunoinformatics experts at EpiVax using each of the tools in the ISPRI platform as well as a literature review. EpiVax PreDeFT Analyses are used to: • Assess a protein therapeutics’ overall and regional immunogenic potential • Direct R&D strategies • Support IND filings • Evaluate candidates for deimmunization or redesign • Add immunogenicity data to your publications • Reduce volume of downstream in vitro/ in vivo immunogenicity studies
    38. 38. ISPRI Website Homepage 39EpiVax - non-confidentialinfo@epivax.com • This interactive biologics screening and optimizing work environment gives access to the same in silico tools used by the EpiVax bioinformatics team. • ISPRI can be used for high throughput unlimited screening of partial and complete sequences of protein therapeutic candidates. • The toolkit can be used to identify and fine map individual amino acids which contribute most to immunogenic potential. • The output can be customized to best fit the needs and preferences of the client.
    39. 39. ISPRI Validation in Print (A Sampling) 40EpiVax - non-confidentialinfo@epivax.com Koren E, De Groot AS, Jawa V, Beck KD, Boone T, Rivera D, Li L, Mytych D, Koscec M, Weeraratne D, Swanson S, Martin W. Clinical validation of the “in silico” prediction of immunogenicity of a human recombinant therapeutic protein Clin Immunol. 2007 Jul. http://bit.ly/epiClinVal De Groot AS, Martin W. Reducing risk, improving outcomes: bioengineering less immunogenic protein therapeutics. Clin Immunol. 2009 May;131(2):189-201. http://bit.ly/epiClinIm Jawa, V., Cousens, L., & De Groot, A. S. (2013). Immunogenicity of Therapeutic Fusion proteins: Contributory Factors and Clinical Experience. Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges, 75-90. http://bit.ly/epiFcFuse Jawa V, Cousens LP, Awwad M, Wakshull E, Kropshofer H, De Groot AS. T-cell dependent immunogenicity of protein therapeutics: Preclinical assessment and mitigation http://bit.ly/The_TCWP
    40. 40. Published Validation: Over 60 Publications in Protein Therapeutics 41EpiVax - non-confidentialinfo@epivax.com 1: De Groot AS, Ardito M, Terry F, Levitz L, Ross T, Moise L, Martin W. Low immunogenicity predicted for emerging avian-origin H7N9: implication for influenza vaccine design. Hum Vaccin Immunother. 2013 May;9(5):950-6. doi: 10.4161/hv.24939. Epub 2013 May 1. PubMed PMID: 23807079; PubMed Central PMCID: PMC3899161. 2: Cousens LP, Tassone R, Mazer BD, Ramachandiran V, Scott DW, De Groot AS. Tregitope update: mechanism of action parallels IVIg. Autoimmun Rev. 2013 Jan;12(3):436-43. doi: 10.1016/j.autrev.2012.08.017. Epub 2012 Aug 28. Review. PubMed PMID: 22944299. 3: De Groot AS, Cousens L, Mingozzi F, Martin W. Tregitope peptides: the active pharmaceutical ingredient of IVIG? Clin Dev Immunol. 2013;2013:493138. doi: 10.1155/2013/493138. Epub 2013 Dec 25. PubMed PMID: 24454476; PubMed Central PMCID: PMC3886585. 4: Cousens LP, Najafian N, Mingozzi F, Elyaman W, Mazer B, Moise L, Messitt TJ, Su Y, Sayegh M, High K, Khoury SJ, Scott DW, De Groot AS. In vitro and in vivo studies of IgG-derived Treg epitopes (Tregitopes): a promising new tool for tolerance induction and treatment of autoimmunity. J Clin Immunol. 2013 Jan;33 Suppl 1:S43-9. doi: 10.1007/s10875-012-9762-4. Epub 2012 Sep 2. Review. PubMed PMID: 22941509; PubMed Central PMCID: PMC3538121. 5: Jawa V, Cousens LP, Awwad M, Wakshull E, Kropshofer H, De Groot AS. T-cell dependent immunogenicity of protein therapeutics: Preclinical assessment and mitigation. Clin Immunol. 2013 Dec;149(3):534-55. doi: 10.1016/j.clim.2013.09.006. Epub 2013 Sep 25. Review. PubMed PMID: 24263283. 6: De Groot AS, Terry F, Cousens L, Martin W. Beyond humanization and de-immunization: tolerization as a method for reducing the immunogenicity of biologics. Expert Rev Clin Pharmacol. 2013 Nov;6(6):651-62. doi: 10.1586/17512433.2013.835698. PubMed PMID: 24164613. 7: Hui DJ, Basner-Tschakarjan E, Chen Y, Davidson RJ, Buchlis G, Yazicioglu M, Pien GC, Finn JD, Haurigot V, Tai A, Scott DW, Cousens LP, Zhou S, De Groot AS, Mingozzi F. Modulation of CD8+ T cell responses to AAV vectors with IgG-derived MHC class II epitopes. Mol Ther. 2013 Sep;21(9):1727-37. 8.1038/mt.2013.166. Epub 2013 Jul 16. PubMed PMID: 23857231. 4: Su Y, Rossi R, De Groot AS, Scott DW. Regulatory T cell epitopes (Tregitopes) in IgG induce tolerance in vivo and lack immunogenicity per se. J Leukoc Biol. 2013 Aug;94(2):377-83. doi: 10.1189/jlb.0912441. Epub 2013 May 31. PubMed PMID: 23729499. 9: Moise L, Gutierrez AH, Bailey-Kellogg C, Terry F, Leng Q, Abdel Hady KM, VerBerkmoes NC, Sztein MB, Losikoff PT, Martin WD, Rothman AL, De Groot AS. The two-faced T cell epitope: examining the host-microbe interface with JanusMatrix. Hum Vaccin Immunother. 2013 Jul;9(7):1577- 86. doi: 10.4161/hv.24615. Epub 2013 Apr 12. http://www.epivax.com/publications/
    41. 41. Published Validation: Over 60 Publications in Protein Therapeutics 42EpiVax - non-confidentialinfo@epivax.com 10: Cousens LP, Su Y, McClaine E, Li X, Terry F, Smith R, Lee J, Martin W, Scott DW, De Groot AS. Application of IgG-derived natural Treg epitopes (IgG Tregitopes) to antigen-specific tolerance induction in a murine model of type 1 diabetes. J Diabetes Res. 2013;2013:621693. doi: 10.1155/2013/621693. Epub 2013 Apr 23. PubMed PMID: 23710469 11: Elfaki ME, Khalil EA, De Groot AS, Musa AM, Gutierrez A, Younis BM, Salih KA, El-Hassan AM. Immunogenicity and immune modulatory effects of in silico predicted L. donovani candidate peptide vaccines. Hum Vaccin Immunother. 2012 Dec 1;8(12):1769-74. doi: 10.4161/hv.21881. Epub 2012 Aug 24. PubMed PMID: 22922767; PubMed Central 12: Cousens LP, Mingozzi F, van der Marel S, Su Y, Garman R, Ferreira V, Martin W, Scott DW, De Groot AS. Teaching tolerance: New approaches to enzyme replacement therapy for Pompe disease. Hum Vaccin Immunother. 2012 Oct;8(10):1459-64. doi: 10.4161/hv.21405. Epub 2012 Oct 1. PubMed PMID: 23095864; PubMed 13. Gutiérrez AH, Moise L, De Groot AS. Of [Hamsters] and men: a new perspective on host cell proteins. Hum Vaccin Immunother. 2012 Sep;8(9):1172-4. doi: 10.4161/hv.22378. Epub 2012 Sep 1. PubMed PMID: 23124469; PubMed Central PMCID: PMC3579895. 14: van der Marel S, Majowicz A, Kwikkers K, van Logtenstein R, te Velde AA, De Groot AS, Meijer SL, van Deventer SJ, Petry H, Hommes DW, Ferreira V. Adeno-associated virus mediated delivery of Tregitope 167 ameliorates experimental colitis. World J Gastroenterol. 2012 Aug 28;18(32):4288-99. doi: 10.3748/wjg.v18.i32.4288. PubMed PMID: 22969191. 15: Moise L, Song C, Martin WD, Tassone R, De Groot AS, Scott DW. Effect of HLA DR epitope de-immunization of Factor VIII in vitro and in vivo. Clin Immunol. 2012 Mar;142(3):320-31. doi: 10.1016/j.clim.2011.11.010. Epub 2011 Dec 8. PubMed PMID: 22222093; PubMed Central PMCID: PMC3288193. 16: Inaba H, Martin W, Ardito M, De Groot AS, De Groot LJ. The role of glutamic or aspartic acid in position four of the epitope binding motif and thyrotropin receptor-extracellular domain epitope selection in Graves' disease. J Clin Endocrinol Metab. 2010 Jun;95(6):2909-16. doi: 10.1210/jc.2009-2393. Epub 2010 Apr 14. PubMed PMID: 20392871; PubMed Central 17: De Groot AS, Baker M, Cohen T. Species neutral correlates of immunogenicity for vaccines and protein therapeutics: fact or science fiction. Hum Vaccin. 2010 May;6(5):371. 18: Scott DW, De Groot AS. Can we prevent immunogenicity of human protein drugs? Ann Rheum Dis. 2010 Jan;69 Suppl 1:i72-76. doi: 10.1136/ard.2009.117564. Review. http://www.epivax.com/publications/
    42. 42. Published Validation: Over 60 Publications in Protein Therapeutics 43EpiVax - non-confidentialinfo@epivax.com 19: Cohen T, Moise L, Ardito M, Martin W, De Groot AS. A method for individualizing the prediction of immunogenicity of protein vaccines and biologic therapeutics: individualized T cell epitope measure (iTEM). J Biomed Biotechnol. 2010;2010. pii: 961752. doi: 10.1155/2010/961752. Epub 2010 Jul 18. PubMed PMID: 20706613 20: De Groot AS. Exploring the immunome: A brave new world for human vaccine development. Hum Vaccin. 2009 Dec;5(12):790-3. Epub 2009 Dec 15. PubMed PMID: 20009527. 21: Weber CA, Mehta PJ, Ardito M, Moise L, Martin B, De Groot AS. T cell epitope: friend or foe? Immunogenicity of biologics in context. Adv Drug Deliv Rev. 2009 Sep 30;61(11):965-76. doi: 10.1016/j.addr.2009.07.001. Epub 2009 Jul 18. Review. PubMed PMID: 19619593. 22: De Groot AS, Martin W. Reducing risk, improving outcomes: bioengineering less immunogenic protein therapeutics. Clin Immunol. 2009 May;131(2):189-201. doi: 10.1016/j.clim.2009.01.009. Epub 2009 Mar 6. Review. PubMed PMID: 19269256. 23: De Groot AS, Moise L, McMurry JA, Wambre E, Van Overtvelt L, Moingeon P, Scott DW, Martin W. Activation of natural regulatory T cells by IgG Fc-derived peptide "Tregitopes". Blood. 2008 Oct 15;112(8):3303-11. doi: 10.1182/blood-2008-02-138073. Epub 2008 Jul 25. . 24: De Groot AS, McMurry J, Moise L. Prediction of immunogenicity: in silico paradigms, ex vivo and in vivo correlates. Curr Opin Pharmacol. 2008 Oct;8(5):620-6. doi: 10.1016/j.coph.2008.08.002. Epub 2008 Sep 19. Review. PubMed PMID: 18775515. 25: De Groot AS, Scott DW. Immunogenicity of protein therapeutics. Trends Immunol. 2007 Nov;28(11):482-90. Epub 2007 Oct 25. Review. PubMed PMID: 17964218. 26: De Groot AS, Moise L. Prediction of immunogenicity for therapeutic proteins: state of the art. Curr Opin Drug Discov Devel. 2007 May;10(3):332-40. Review. PubMed PMID: 17554860. 27: De Groot AS, Goldberg M, Moise L, Martin W. Evolutionary deimmunization: an ancillary mechanism for self-tolerance? Cell Immunol. 2006 Dec;244(2):148-53. Epub 2007 Apr 18. 28: De Groot AS. Immunomics: discovering new targets for vaccines and therapeutics. Drug Discov Today. 2006 Mar;11(5-6):203-9. Review. PubMed PMID: 6580597. 29: De Groot AS, Knopp PM, Martin W. De-immunization of therapeutic proteins by T-cell epitope modification. Dev Biol (Basel). 2005;122:171-94. Review. PubMed PMID: 16375261. 30: De Groot AS, Rayner J, Martin W. Modelling the immunogenicity of therapeutic proteins using T cell epitope mapping. Dev Biol (Basel). 2003;112:71-80. PubMed PMID: 12762506. http://www.epivax.com/publications/
    43. 43. EpiVax Services Summary 44EpiVax - non-confidentialinfo@epivax.com Immunogenicity Screening Epitope Validation Epitope Modification Design and Reengineering In Silico: Biotherapeutics In Vitro, Ex Vivo, & In Vivo: Laboratory Services In Silico: Vaccines
    44. 44. Science without fear. www.epivax.com  info@epivax.com  401.272.2123
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