Epivax Overview

Home , Epitope mapping

EpiVax Overview

January 2013

1 Confidential Outline

• About EpiVax

• EpiMatrix – Identifying T Cell Epitopes

• Tregitope – Engaging Regulatory T Cells

• ISPRI – Interactive Screening and Protein Reengineering Interface

2 Confidential About EpiVax

• Founded 1998 (De Groot, Martin Principals) • Privately Held • No Outside Investment / No Debt • Commercial Contracts – Immunogenicity Screening, Technology Licensing, Therapeutic and Vaccine Design Consulting • Grant Funding – Internal Vaccine and Tregitope Technology development. • 21 employees

3 Confidential Four Core Strengths

EpiVax Services Epitope Mapping / Immunogenicity Screening Fee for Service HLA Binding T cell assays In vivo assays (HLA Tg mice) EpiVax Vaccines Grant funded R & D Grant Funded Excellent proof of principle

EpiVax Select targets Sponsored Research / Joint Development 2nd Generation Funded Res. Or Joint Devt Therapeutics Develop molecule and license

Immuno- Tregitopes Options available for modulation In preclinical development- may be large selected “Field of Use” market - allergy, autoimmunity, transplant

4 Confidential Technology Offerings

• In Silico PreDeFT Immunogenicity Analysis • HLA Binding Assays • ELISpot Assays • Exposed Samples • Naïve Samples • HLA Transgenic Mice • Unlimited/Limited ISPRI • Tregitope • DeFT – Deimmunization of Functional Therapeutics • Vaccine Licensing

5 Confidential EpiMatrix Epitope Discovery Technology

6 Confidential Epitope Mapping EpiMatrix graphical representation of A*0201Graphical motif (based Representation on list of actual peptidesof A*0201 Coefficient matrix from Chicz)

A C

D E Binding motifs 1.75-2.00

F 1.50-1.75 Amino Amino Acid G 1.25-1.50 1.00-1.25 H 0.75-1.00 I 0.50-0.75 0.25-0.50 K 0.00-0.25 L -0.25-0.00 -0.50--0.25 M -0.75--0.50 N -1.00--0.75 -1.25--1.00 P -1.50--1.25 Q -1.75--1.50 -2.00--1.75 R

-2.25--2.00 S -2.50--2.25 -2.75--2.50 Rejected residues T -3.00--2.75 V

0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00

-2.75 -2.50 -2.25 -2.00 -1.75 -1.50 -1.25 -1.00 -0.75 -0.50 -0.25 -3.00 1 2 3 4 5 6 7 8 9 10 Position + + + + + + + + = indication of binding likelihood

7 Confidential EpiVax HLA “Supertype” Coverage

• 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 >90% 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. 8 Epitope Mapping EpiMatrix

De Groot and Martin. Reducing risk, improving outcomes: Bioengineering less immunogenic protein therapeutics. Clinical Immunology 2009. 131, 189-201. 9 Confidential Tregitope Technology A Brief Overview

10 Confidential What is a Tregitope?

Tregitope 09

Tregitope 29 Tregitope 54 Tregitopes - highly conserved T reg epitopes Tregitope167 In Fc and Fab (framework) Are promiscuous (multiple HLA binders) Tregitope 134 Are present in IgG, not IgA, IgE, IgM May be present in other self proteins.

Tregitope 289

De 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

11 Confidential Tregitope Collaborators

Brigham and Children’s Hospital of U. Maryland/ McGill University/ Women’s Hospital Philadelphia Uniformed Services Montreal Children’s U. Health Sciences Hospital

Transplant CD8-Mediated Anti- T1D, FVIII, Allergy Allergy Gene Therapy Responses

Nader Najafian Federico Mingozzi David Scott Bruce Mazer Daniel Hui Yan Su Katherine High Xin Li Achsah Keegan Preeta Desgupta 12 Confidential WhatWhat is is the the proof? Evidence? Step by step . . . (1) Binding

(2) nTreg induction

(3) IL-10 secretion

(4) Bystander Suppression (5) Modulate APC (4) Induction of iTregs 13 Confidential ISPRI Protein Immunogenicity Scale [Tregitope-Adjusted]

- 80 - Thrombopoietin EpiMatrix Predicted Excess/Shortfall in - 70 - Aggregate Immunogenicity Relative to a - 60 - Random Peptide Standard Human EPO - 50 - EBV- BKRF3 All scores are adjusted for the presence - 40 - of Tregitopes. Tetanus Toxin - 30 - Influenza- HA - 20 - *Average of Antibodies Known to Induce Anti-Therapeutic Responses in More - 10 - Than 5% of Patients - 00 - Albumin Immunogenic Antibodies* - -10 - †Average of Antibodies Known to Induce - - 20 - Anti-Therapeutic Responses in Less Your Protein Than 5% of Patients IgG FC Region - -30 -

Non- immunogenic Antibodies† - - 40 -

- - 50 -

- -60 - Follitropin- Beta - - 70 -

- - 80 - 14 Confidential ISPRI Interactive Protein Screening and Reengineering Interface

15 Confidential ISPRI Interactive Protein Screening and Reengineering Interface

• EpiVax has developed a secure, interactive work environment that is seamlessly linked to EpiVax’s proprietary in silico immunogenicity screening toolkit. • 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 biological (protein therapeutic) candidates. • The toolkit can be used to identify within each protein sequence potentially immunogenic regions (known as epitope clusters) and to fine map those individual amino acids which contribute most to the immunogenic potential of the cluster. • The output is customized to best fit the needs and preferences of the client.

16 Confidential ISPRI Available Tools • EpiMatrix – Screen the protein sequences of product candidates for the presence of putative T cell epitopes. • Immunogenicity Protein Scale – Rate the immunogenic potential of each submitted sequence on a normalized scale and compare each protein to other immunogenic proteins and antibodies • 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) • ClustiMer – Identify T-cell epitope clusters contained within product candidates • Immunogenic Cluster Scale – 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 • BlastiMer – Blast T-cell epitope clusters against the non-redundant protein or patent database at GenBank • OptiMatrix – The protein re-design algorithm that provides a list of critical amino acid residues and potential amino acid substitutions that are conserved in existing databases (based on published sequences) and that do not introduce new epitopes. 17 Confidential Approach – Whole Antigens

EpiVax Immunogenicity Hypothesis As Applied: Immune Response = Sum of Epitopes

Protein Therapeutic epitope epitope epitope 1 + 1 + 1 = Response

T cell response depends on:

T cell epitope content + HLA of subject

Protein Immunogenicity can be Ranked

•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 ISPRI Protein Immunogenicity Scale

Non- immunogenic Antibodies† - - 40 -

- - 50 -

- -60 - Follitropin- Beta - - 70 -

- - 80 - 19 Confidential ISPRI EpiMatrix Protein Report EpiMatrix Report Accession: YOUR_PROTEIN - Sequence: YOUR_PROTEIN Frame Frame DRB1*0101 DRB1*0301 DRB1*0401 DRB1*0701 DRB1*0801 DRB1*1101 DRB1*1301 DRB1*1501 AA Sequence Hits Start Stop Z-Score Z-Score Z-Score Z-Score Z-Score Z-Score Z-Score Z-Score 1 DIQMTQSPS 9 0.91 0.17 0.35 0.06 0.09 0.86 -0.08 -0.01 0 2 IQMTQSPSS 10 2.15 1.35 2.26 1.58 2.17 1.88 2.24 2.52 6 3 QMTQSPSSL 11 1.11 0.28 0.5 0.96 0.29 0.39 0.9 0.4 0 4 MTQSPSSLS 12 1.87 2.26 2.02 1.88 1.06 1.66 1.79 2.05 7 5 TQSPSSLSA 13 0.51 0.28 1.31 0.69 -0.04 0.54 1.09 1.05 0 ...... Assessment...... 46 LLIYAASTL 54 1.41 1.08 0.99 2.16 1.54 0.85 2.28 2.3 3 47 LIYFASTLQ 55 1.91 1.85 2.46 2.07 1.79 1.08 1.79 2.1 7 48 IYFASTLQS 56 1.73 2.01 2.68 1.26 1.58 1.82 2.11 2.07 6 49 YFASTLQSG 57 0.64 1.6 0.59 0.32 1.42 1.28 0.04 1.09 0 ...... EpiBar ...... Hit . 98 FGQGKTVEI 106 0.7 1.9 -0.02 0.2 0.93 0.34 1.41 1.12 1 99 GQGKTVEIK 107 -0.28 -0.84 0.31 0.01 0.03 -0.57 -0.47 -1.24 0 Summarized Results DRB1*0101 DRB1*0301 DRB1*0401 DRB1*0701 DRB1*0801 DRB1*1101 DRB1*1301 DRB1*1501 Total Maximum Single Z score 2.4 2.26 2.8 3.11 2.41 2.29 2.28 2.52 -- Sum of Significant Z scores 23.41 12.17 15.96 18.16 10.2 13.2 16.47 16.99 126.56 Count of Significant Z Scores 12 6 7 9 5 7 8 8 62 Total Assessments Performed: 792 Deviation from Expectation: 38.46 Deviation per 1000 AA: 48.56 Adjusted for Regulatory Epitopes Deviation from Expectation: -21.24 Deviation per 1000 AA: -26.82 EpiMatrix Immunogenicity Score 20 Confidential EpiMatrix Tregitope-adjusted Score Correlation of antibody immunogenicity without Tregitope adjusted EPX Scores

Correlation to observed Immunogenicity before accounting for Tregitopes

R2=0.17

21 Immunogenicity Scale for Monoclonals

Factoring in Tregitopes. . . Protein Therapeutic epitope epitope epitope

1 + 1 - Regulatory T cell epitope* = Response T cell response depends on:

T cell epitope content – Tregitope content + HLA of subject

Protein Immunogenicity can be Ranked

22 2/20/2013 Confidential Correlation of antibody immunogenicity with Tregitope adjusted EPX Scores

Correlation to observed immunogenicity after accounting for Tregitopes

R2=0.76

Accounting for Tregitopes results in more accurate predictions. 23 Antibodies: A Special Case

- 80 -

- 70 - Due to the presence of Tregitopes, antibodies tend to fall lower on the immunogenicity scale. - 60 -

- 50 - We have developed a refined method using regression analysis to predict the immunogenicity of antibody sequences New drug - 40 - based on observed clinical responses. - 30 - Campath (45%) We have found that a balance in favor of Tregitope (regulatory) Rituxan (27%) - 20 - Remicade (26%) content over neo-epitope (effector) content is correlated with - 10 - reduced clinical immunogenicity. Humicade (7%) Bivatuzumab (6.7%) - 00 - Reopro (5.8%) Tregitope Content - -10 - Tysabri (7%) Simulect (1.4%) High Low Humira (12%) - -20 - AB02 (EPX Adjusted Score: -44.48)

Synagis (1%) AB03 (EPX Adjusted Score: -44.81) Mylotarg (3%) Avastin (0%) - -30 - AB04 (EPX AdjustedAvastin Score: (0%) -45.81) IgG FC Region AB05 (EPX Adjusted Score: -45.88) Simulect (1%) LeukArrest (0%) AB10 (EPXLow AdjustedHerceptin Score: (0%) -45.88) - -40 - AB08 (EPX Adjusted Score: -46.30) Synagis (1%) Nuvion (0%) AB01 (EPX Adjusted Score: -46.98) - -50 - AB07 (EPX Adjusted Score: -46.99)

AB09 (EPX Adjusted Score: -47.40) - -60 - AB11 (EPX Adjusted Score: -47.40) Remicade (26%) AB06 (EPX AdjustedCampath Score: (45%) - 47.85)

Herceptin (0.1%) Content Epitope Neo High Rituxan (27%) - -70 -

- -80 -

24 2/20/2013 Confidential ISPRI ClustiMer identifies local immunogenic potential

DRB1*0101

DRB1*0301

DRB1*0401

DRB1*0701

DRB1*0801

DRB1*1101

DRB1*1301

DRB1*1501 • T cell epitopes are not randomly distributed throughout protein sequences but instead tend to cluster in specific regions. • 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. • 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

25 Confidential ISPRI Cluster Immunogenicity Scale

Tetanus Toxin (825-850) NAME ADDRESS SCORE - 40 -

NPC NS3 (1248-1267) - -

- 30 - YOUR_PROTEIN1 318-341 31.14

- -

Influenza-HA (306-319) - 20 - P. Falciparum (72-86) YOUR_PROTEIN1 206-225 17.76 - - YOUR_PROTEIN1 159-176 13.81 Human CLIP YOUR_PROTEIN1 055-070 13.62 - 10 - YOUR_PROTEIN1 293-314 10.61 P. Falciparum (512-526) - -

- 00 - EpiMatrix Predicted Excess/Shortfall in - - Aggregate Immunogenicity Relative to a Random Peptide Standard - -10 - Theoretical Minimum

Confidential 26 Humira – Overall Protein Immunogenicity Tregitope Adjusted Protein Scores

The Humira heavy chain contains relatively few neo-epitopes and a significant number of known Tregitopes.

The Humira light chain contains few neo-epitopes and a significant number of known Tregitopes.

Indicates a drop in significant potential for immunogenicity

HUMIRA_VH HUMIRA_VL

Tregitope adjusted scores of the submitted sequences rate against the same series of standard controls.

27 DRAFT Humira – T Cell Epitope Clusters Regional Immunogenicity Matters

Tetanus Toxin (825-850) NAME ADDRESS SCORE - 40 -

- - HUMIRA_VH 091-113 33.92

HCV NS3 (1248-1267) - 30 - HUMIRA_VL 043-065 29.62

- - Two epitopes with high EMX scores are Tetanus Toxin (586-605) Influenza-HA (306-319) - 20 - expected to contribute to immunogenicity . . . Other clusters, when Tregitope adjusted – - - Human CLIP may cause proliferation but phenotype of - 10 - proliferating cells should be evaluated . . . Could be Tregitopes - - HUMIRA_VH 065-081 01.69 HUMIRA_VL 068-086 01.48 - 00 - HUMIRA_VH 008-031 01.32 HUMIRA_VH 076-094 -00.45 HUMIRA_VH 033-048 -01.65 HUMIRA_VL 001-015 -02.47 - -

Theoretical Minimum - -10 -

28 DRAFT BlastiMer BLAST helps to find viable substitutions

• 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

29 Confidential BlastiMer BLAST helps to find human-like sequences

30 Confidential OptiMatrix Interactive Peptide Deimmunization OptiMatrix:

Frame Frame Hydro- DRB1*0101 DRB1*0301 DRB1*0401 DRB1*0701 DRB1*0801 DRB1*1101 DRB1*1301 DRB1*1501 AA Sequence Hits Start Stop phobicity Z-Score Z-Score Z-Score Z-Score Z-Score Z-Score Z-Score Z-Score 254 PRGYFKIRT 262 -0.23 0 255 RGYFKIRTG 263 -0.2 0 256 GYFKIRTGK 264 -0.19 0 257 YFKIRTGKT 265 -0.9 2.38 2.41 2.51 1.4 2.2 1.98 5 258 FKIRTGKTT 266 -0.83 2.41 2.13 1.69 1.32 1.53 3 259 KIRTGKTTI 267 -0.14 1.44 0 260 IRTGKTTIM 268 0 1.97 1.42 1.48 1 261 RTGKTTIMR 269 -0.21 1.33 0

Summarized Results (25-SEP-2009) DRB1*0101 DRB1*0301 DRB1*0401 DRB1*0701 DRB1*0801 DRB1*1101 DRB1*1301 DRB1*1501 Total Maximum Single Z score 2.41 1.97 2.41 2.51 1.69 2.2 1.48 1.98 -- Sum of Significant Z scores 4.79 1.97 2.41 4.64 1.69 2.2 0 1.98 19.68 Count of Significant Z Scores 2 1 1 2 1 1 0 1 9 Total Assessments Performed: 64 Hydrophobicity: -0.84 EpiMatrix Score: 13.08 EpiMatrix Score (w/o flanks): 16.05 Scores Adjusted for Tregitope: -- EpiMatrix Score: 13.08 EpiMatrix Score (w/o flanks): 16.05 31 Confidential OptiMatrix See the effects of amino acid substitution in real-time OptiMatrix:

Click multiple times to continue deimmunizing

32 Confidential ISPRI Summary

Benefits of in silico immunogenicity screening using the

ISPRI website include

• Streamlined and efficient pipeline development

• Decreased in vitro and in vivo costs

• Improved return on R&D

• Support for IND and other regulatory filings

33 Confidential ISPRI Contacts

CIO/DIRECTOR OF BIOINFORMATICS Bill Martin [email protected]

BIOINFORMATICS PROGRAM MANAGER Frances Terry [email protected]

BIOINFORMATICS PROGRAMMER/ANALYST Jacob Tivin [email protected]