R² = 0.5902468

1012141618

0 200 400 600 800

PFS

(mon

ths)

Low Janus neo-epitope count (iVAX)

R² = 0.6702468

1012141618

0 200 400 600 800

PFS

(mon

ths)

Low Janus neo-epitope count (adjusted for PD-L1) (iVAX)

R² = 0.5302468

1012141618

0 500 1000 1500

PFS

(mon

ths)

Neo-epitope count (iVAX)

R² = 0.5202468

1012141618

0 200 400 600 800

PFS

(mon

ths)

Neoantigen count (Rizvi et al.)

0

1

2

3

Pentatope 1 Pentatope 2 CT26-M19Neo

-epi

tope

per

seq

uenc

e

Class I Neo-epitope content

0

1

2

3

4

Pentatope 1 Pentatope 2 CT26-M19

Mur

ine

Janu

s Sc

ore

Class I Murine cross-reactivity potential

0

1

2

Pentatope 1 Pentatope 2 CT26-M19Neo

-epi

tope

per

seq

uenc

e

Class II Neo-epitope content

0

1

2

3

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Pentatope 1 Pentatope 2 CT26-M19

Mur

ine

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ore

Class II Murine cross-reactivity potential

High-Value T Cell Epitope Selection for Mutanome-Directed Cancer Immunotherapy Using an Innovative Cancer Neo-Epitope Classification System

Guilhem Richard1, Lenny Moise1,2, Frances Terry1, Bill Martin1, Annie De Groot1,21 EpiVax, Inc., Providence, RI, United States; 2 Institute for Immunology and Informatics, University of Rhode Island, Providence, RI, United States

Mutanome-Directed Cancer Immunotherapy

For questions regarding in silico antigen screening and vaccine design, please contact: Steven Vessella at 401-272-2123, ext. 107; or at info@epivax.com www.epivax.com

Abstract

Conclusions Sequence analysis of the MHC- and TCR-facing residues of T cell epitopes enables prediction of epitope phenotype. Epitopes that share a TCR-face with numerous human sequences may activate Tregs. Sharper definition of neo-antigens by immunoinformatic analysis may improve epitope selection for mutanome-directed cancer immunotherapy. In silico analyses of mutanomes and cancer vaccines suggest that high neo-epitope density and low cross-reactivity potential may be indicative

of improved clinical outcomes.

T cell epitopes bearing tumor-specific mutations discovered using whole-exomic and transcriptomicsequencing of tumor-normal pairs stimulate T cell-mediated processes that lead to tumor regression.Although neo-epitope prediction using computational methods rapidly identifies epitopecandidates in the mutanome, a large proportion of neo-epitopes prove to be non-immunogenic. Innovative computational tools validated for infectious disease targets can be appliedto enhance design of personalized cancer immunotherapies by classification of predicted epitopes bypotential for mounting a tumor-specific response. Tumor-specific epitopes with potential tostimulate naïve T cells and epitopes that may cross-react with antigen-experienced T cellsraised in infection are valuable targets. Epitopes with potential to activate regulatory T cells trainedon self and commensal antigens are contraindicated.

We developed the JanusMatrix algorithm that parses query sequences into MHC-facing and T cellreceptor (TCR)-facing sequences and screens sequence databases to identify MHC ligands thatshare TCR faces with host-related proteins. A database of human protein sequences is availableto identify tumor-specific epitopes that may reduce anti-tumor activity by sequences that activateregulatory T cells (Tregs) trained in the thymus on self-antigens. Similarly, tumor-specific epitopecandidates are screened using databases composed of antigens derived from human commensals orpathogens to identify epitopes that, respectively, may detrimentally or beneficially cross-react with Tcells raised over the course of an individual’s immune history. Neo-epitope candidates are rankedaccording to MHC binding potential and TCR face homology.

We conducted retrospective analyses of cancer vaccine efficacy studies performed in mice[Kreiter et al. 2015 Nature 520, 692–696] showing that mutanome-directed vaccines effective atpreventing tumor growth contained higher numbers of class I and II MHC neo-epitopes, as identifiedby EpiVax’s iVAX platform, and had lower potential to cross-react with other murine proteins. Anevaluation of mutanomes derived from non-small cell lung cancer patients [Rizvi et al. 2014Science 348, 124-128] confirmed these findings. Improved clinical outcomes were observed inpatients with mutanomes enriched in class I MHC neo-epitopes with TCR faces distinct from otherhuman-derived epitopes.

These results highlight the benefits of in silico prediction tools for the selection of neo-epitopes andhow they can improve the design and efficacy of future cancer vaccines. While retrospective innature, the suite of tools used for these analyses have been extensively validated in prospectivevaccine studies for infectious disease. Removal of Treg epitopes, identified by JanusMatrix, has ledto the development of more immunogenic vaccine antigens. EpiVax’s H7N9 influenza Treg epitope-depleted vaccine is scheduled for Phase I clinical trial. Direct application of JanusMatrix to theneo-epitope-driven cancer vaccine discovery and design pipeline will focus candidateselection on higher-value sequences than what conventional T cell epitope mapping algorithmsgenerate. Neo-epitopes with low Treg activation potential may then be used to support developmentof personalized therapies including vaccination and in vitro expansion of tumor infiltratinglymphocytes for adoptive cell transfer.

References

JanusMatrix separates the amino acid sequence of T cellepitopes into TCR-facing residues (epitope) and HLAbinding cleft-facing residues (agretope), then compares theTCR face to other putative T cell epitopes.

Cross-reactive peptides:• Are predicted to bind the same MHC allele.• Share same/similar T cell-facing residues.

TCR cross-reactivity prediction:• Given a protein or peptide, T cell epitopes are

identified based on MHC contacts (P1, P4, P6,P9 for Class II) using EpiMatrix.

• JanusMatrix searches for potentially cross-reactive TCR by screening TCR-facing residues(P2, P3, P5, P7, P8 for Class II) against apreloaded, EpiMatrix-processed referencedatabase.

JanusMatrix Discovers Potential Treg Epitopes

A) Peptides documented as IL-10-positive in IEDB have significantly higher potential for Human Genome (HG, above) and Human Microbiome (HM, below) cross-reactivity as measured by JanusMatrix.

B) Peptides documented as IL-4-positive in IEDB have significantly lower potential for HG and HM cross-reactivity as measured by JanusMatrix.

Immunoinformatic Analysis of Cancer Vaccines from Kreiter et al. Nature 2015

• Moise L, Gutierrez A, Kibria F, Martin R, Tassone R, Liu R, Terry F, Martin B, De Groot AS. iVAX: An integrated toolkit for the selection and optimization of antigens and the design of epitope-driven vaccines. Hum Vaccin Immunother. 2015;11(9):2312-21.• 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.• Kreiter S, Vormehr M, van de Roemer N, Diken M, Löwer M, Diekmann J, Boegel S, Schrörs B, Vascotto F, Castle JC, Tadmor AD, Schoenberger SP, Huber C, Türeci Ö, Sahin U. Mutant MHC class II epitopes drive therapeutic immune responses to cancer. Nature. 2015 Apr 30;520(7549):692-6.• Rizvi NA, Hellmann MD, Snyder A, Kvistborg P, Makarov V, Havel JJ, Lee W, Yuan J, Wong P, Ho TS, Miller ML, Rekhtman N, Moreira AL, Ibrahim F, Bruggeman C, Gasmi B, Zappasodi R, Maeda Y, Sander C, Garon EB, Merghoub T, Wolchok JD, Schumacher TN, Chan TA. Mutational landscape

determines sensitivity to PD-1 blockade in non–small cell lung cancer. Science. 2015 Apr;348(124):124-8

Adapted from Extended Data Figure 3b. Kreiter et al., Nature 520, 692–696. 2015

• EpiMatrix identifies neo-epitopes by analyzing the binding potential of normal and mutated peptides.

• iVAX performs TCR-face analyses to screen out mutated epitopes that share identical TCR-faces with normal epitopes.

• Neo-epitopes highly cross-reactive with other self proteins are filtered out with JanusMatrix.eliminate

MHC face

TCR face

TCR-face analysis

non-self

self

Mutation

MHC binderneo-epitope

non-binder

neo-epitope

eliminate

vaccine candidate

JanusMatrix analysis

identical TCR-face

different TCR-face

Immunoinformatic Analysis of Mutanome Datasets from Rizvi et al. Science 2015

• Pentatopes were constructed from mutated sequences derived from colon carcinoma model CT26 (study performed by Kreiter et al. Nature 520, 692-696. 2015).

• Pentatope2 showed greater antitumor activity compared to Pentatope1 in BALB/c mice inoculated with CT26 tumor cells.

• Pentatope2 increased anti-tumor activity may be explained by a higher number of Class I and II neo-epitopes and overall lower potential for cross-reactivity, as identified by iVAX.

Tumor Biopsy Expressed Mutation Discovery Neo-Antigen Discovery

Normal Tumor

Tumorcell

CD8+T cell

CD4+T cell APC

Immunotherapy

IMMUNOINFORMATICS

Mutanome

Neo-epitopes

Normal Peptide Mutated Peptide

• Tumors from 34 anti-PD-1 treated patients with non-small cell lung cancer were sampled and sequenced to identify tumor-specific mutations (study performed by Rizvi et al. Science 348, 124-128. 2015). Tumor mutation burden was found to be associated with clinical response (left).

• Mutanomes were analyzed using iVAX to identify neo-epitopes and to determine their cross-reactivity potential with other human proteins (below).

• Neo-epitope content, as determined by iVAX, was significantly associated with clinical benefit. Association was improved when removing patients not expressing PD-L1 for whom the anti-PD-1 treatment may not be suitable.

• Progression-free survival (PFS) of patients expressing some level of PD-L1 was best estimated by iVAX’s neo-epitope content, adjusted for both Human cross-conservation potential (“low Janus”) and PD-L1 expression.

Fig. 3. Rizvi et al. Science 348, 124-128. 2015

iVAX Neo-epitope Selection Pipeline

Acquire Mutanome

Scan for HLA Matched Epitopes with EpiMatrix [Patient Class I + Class II]

Screen for Humanness with JanusMatrix

AnnotatePrevalence/Published

Rank and Select Peptide Candidates

Design String of Beads Construct using VaxCAD

Cros

s-re

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)

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Better outcome

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IL-10 IL-4

Aver

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A Cytokines B

Avg Janus Human Score vs +/- Cytokine ReleaseIEDB Positive IEDB Negativep