December 9, 2016
Press Release,
Company

GreenPeptide Co., Ltd.

Representative

Kenichi Nagai, President & CEO(Code: 4594 TSE Mothers)

Inquiries

Teruhiko Sakai, Director/CFO(TEL: +81-3-5840-7697)

Information on the Initiation of Neoantigen (Genetic Variant Antigen) Peptide Vaccine
Development
We announce that the development of a neoantigen*4 (genetic variant antigen) peptide vaccine
for non-small cell lung cancer*3 will begin based on a patent transferred from the Kanagawa
Prefectural Hospital Organization: “Antigen peptide originated from the T790M point mutated*2
sequence of epidermal growth factor receptor (EGFR)*1”.
Many patients with lung cancer are already at an advanced stage or have metastasis at the time of
their diagnosis, which leads to a poor prognosis. Approximately 220,000 individuals[1] in the
United States and 130,000 individuals[2] in Japan reportedly develop lung cancer. More than 80%
of patients are diagnosed with non-small cell lung cancer. EGFR tyrosine kinase inhibitors(EGFR-TKI) are selected as conventional and effective drugs for patients with an EGFR activation
mutation (primary mutation), accounting for approximately 10% of non-small cell lung cancer
patients (Japanese: 30%). However, many patients acquire resistance to EGFR-TKI one to 1.5
years after the start of treatment, which results in disease progression. Previous studies reported
that a gene mutation, the EGFR-T790M point mutation (secondary mutation), was detected inapproximately 60% of EGFR-TKI-resistant patients. We will develop a peptide vaccine against the
EGFR-TKI-resistant gene mutation as an antigen (neoantigen).
Neoantigen refers to an antigen involving a gene mutation related to cell canceration. It only
appears on cancer cells, clarifying the border between cancer and normal cells. In the immune
system, neoantigen is recognized as “non-self”, and its immunogenicity is high. Since it may
efficiently induce antigen-specific T cells, the development of cancer vaccines against neoantigen
as an antigen is an important issue for next-generation cancer immunotherapy. The T790M point
mutation may be a driver mutation directly involved in the transformation of cancer, which is
caused by EGFR-TKI-attacked cancer cells acquiring attack tolerance. If this mutation is targeted,antigen expression may persist on cancer cells with drug (EGFR-TKI) administration, and antigen
loss-related escape from immune surveillance mechanisms may not occur. Furthermore, theincidence of the T790M point mutation is higher (approximately 60%) than that of spontaneous
mutations related to cancer transformation, and this may be because this gene mutation is induced1

by drug administration. Internationally, cancer immunotherapy targeting drug resistance-inducing
gene mutations represents a new approach.
We will introduce a peptide involving the EGFR-T790M point mutation from the Kanagawa
Prefectural Hospital Organization: neoantigen*5 identified by Dr. Sasada et al., Director,
Department of Cancer Immunotherapy Research and Development, Kanagawa Cancer Center
Research Institute (Kanagawa Prefectural Hospital Organization), who developed this patent as acancer immunity-inducing peptide. Lymphocytes in peripheral blood from patients with lungcancer were confirmed to show neoantigen-specific immune responses.
A tyrosine kinase inhibitor against the EGFR-T790M mutation recently became commerciallyavailable, and, due to its clinical molecule-targeting efficacy, generic products are now being
developed, leading to blockbuster products. Therefore, drugs against the EGFR-T790M mutation
will play an important role in the treatment of lung cancer worldwide. We will develop a
discriminated product as a cancer vaccine that has a different action mechanism from theselow-molecular-weight compounds. A clinical trial will be initiated in 2017.
We have performed a collaborative study on neoantigen exploration in cooperation with the
Kanagawa Cancer Center since July 2016. By focusing on the mechanisms underlying antigen
presentation in the cancer microenvironment*6, we are also investigating methods to identify driverand passenger mutation antigens (driver mutations: mutations directly involved in cancer
transformation) (passenger mutations: mutations not related to canceration, potentially with highimmunogenicity and numbers).
This matter will not markedly influence the achievements of our company in March 2017.

[Term explanation]*1: Epidermal growth factor receptor (EGFR): Receptor that binds to epidermal growth factor,
which regulates cell proliferation or growth, thereby contributing to signal transmission. Cell
differentiation/proliferation may occur when this receptor is activated. Furthermore, EGFR isdetected in many cells, and is involved in canceration or infiltration/metastasis when amutation occurs.
*2: T790M point mutation: A mutation of the 790th amino acid of the EGFR protein from threonine
to methionine. This mutation shows resistance to conventional tyrosine kinase inhibitors, suchas Tarceva and Iressa.
*3: Non-small cell lung cancer (NSCLC): Lung cancer is classified into two types: small andnon-small cell lung cancers. The latter progresses more slowly than the former, but does not2

respond to chemotherapy or radiotherapy. In Japan, ≥80% of lung cancer patients havenon-small cell lung cancer. This cancer has been sub-classified into adenocarcinoma,squamous cell carcinoma, and large cell carcinoma.
*4: Neoantigen: An antigen involving gene mutations (amino acid mutations) related to uniquegene abnormalities in cancer cells. It is a cancer-specific antigen expressed through uniquegene mutations in the cancer cells of individual patients, and does not exist in normal cells.
Cancer cell-killing immunity may be efficiently induced by targeting neoantigen, which isrecognized as “non-self” by the immune system. Neoantigen may be available as the antigen
of cancer vaccines, as a biomarker to select patients for whom an immune checkpoint
antibody is effective, and as a high accuracy target of T-cell therapy (CAR-T: chimera antigen
receptor T-cell therapy, TCR-T: T-cell receptor therapy, and T-iPS: iPS regenerative T cells),
which has recently been introduced.
However, neoantigen includes tumor-specific gene mutation antigens and those related topost-translational modifications, such as phosphorylation, glycosylation, and methylation.
*5: EGFR T790M mutation as a possible target for immunotherapy; identification of
HLA-A*0201-restricted T cell epitopes derived from the EGFR T790M mutation,
et al.

YamadaPLoS One. 2013 Nov 5; 8(11): e78389.

A peptide antigen derived from EGFR T790M is immunogenic in non-small cell lung cancer,
Ofuji et al. Int J Oncol. 2015 Feb; 46(2): 497–504.
*6: Cancer microenvironment: A state characteristic of a tissue in which cancer cells aggregate. In
the presence of various factors secreted by cancer cells, macrophages and immature
myelocytes aggregate, and these immunocytes may be involved in the infiltration/malignancy/transformation of cancer cells.
[1] American Cancer Society
http://www.cancer.org/cancer/lungcancer-non-smallcell/detailedguide/non-small-cell-lung-cancer-key-statistics[2] National Cancer Center
http://www.ncc.go.jp/jp/information/press_release_20150428.html

About Green Peptide Co., Ltd.
Green Peptide is a drug discovery venture company responsible for the development of innovative“cancer immunotherapy” as the 4th cancer treatment following surgery, radiotherapy, andchemotherapy. Clinical trials on cancer peptide vaccines are being conducted in Japan and theUnited States, and we recently started the development of new T-cell therapy involving the iPS
regeneration of antigen-specific T cells.

3About Kanagawa Prefectural Hospital Organization
Five prefectural hospitals (Kanagawa Prefecture) are managed by the Kanagawa Prefectural
Hospital Organization. The Research Institute of Kanagawa Cancer Center, one of the fivehospitals, has a 30-year history since its establishment, and has many opinions for cancer
immunotherapies, including cancer peptide vaccine therapy, which was developed by our company.

4

Neoantigen/peptide vaccine development（Securities code: 4594）

December 9, 2016

Contents

1. Highlight
2. What is neoantigen?
3. Characteristics of seeds
4. Possibilities for neoantigen

2

Highlight

Highlight１

Cancer immunotherapy for a drug-resistant gene mutation(EGFR T790M point mutation) involved in resistance to a
primary treatment for lung cancer, EGFR-TKI■ Annual number of newly diagnosed patients: 500,000 patients → Approximately
80% of these had non-small cell lung cancer.
(United States: 210,000 patients, EU (Top 5 countries): 200,000 patients, Japan: 100,000 patients)1■ Primary EGFR mutation rate

■ T790M mutation(secondary EGFR mutation)
rate in EGFR-TKI-resistantpatients

EGFR tyrosine kinase
inhibitors (EGFR-TKI)2
are used as standard
therapy.
1 Source: GLOBOCAN 2012
2 Tarceva (erlotinib, sales amount in 2015: $1.59 bn), Iressa (gefitinib), and Giotrif (afatinib) were approved by 3 bureaus.

4Highlight (cont.)
２

The neoantigen T790M gene mutation is a target ofcancer immunotherapy.

1. Antigen epitope (peptide) of which the immunogenicity wasconfirmed→ A patent was granted in the United States
2. Only appear on cancer cells.
3. Escape (antigen loss) from immune surveillance mechanisms
may not occur.
→Driver mutations are essential for cancer malignancy, which
occurs against a challenge by EGFR-TKI (primary treatment)
4. A relatively high incidence.
5

What is neoantigen?

Cancer Antigen
Cancer antigen is a mark of cancer, presented on thesurfaces of cancer cells, for T cells.
Cancer cells
attacked by T cells

Cancer cells

T cells

Dead cancer cells

T cells
Cancer antigen = a mark of attack
Cancer cells

T cells7

Identification of cancer antigens
An antigen recognized by T cells as a mark of attack consistsof a sequence of 8 to 12 amino acids (peptide)prepared/expressed by cancer cells.
Cancer cells
Cancer cells

Protein

HLA

Proteasome

Cancer antigen =a mark of attack
PeptideT cells

HLA

TAP

Cancer antigen = a fragment of a protein (peptide)8

Cancer antigen ideal for cancer immunotherapy
mark of attack

1. In vivo immune responses in cancer patients(immunogenicity).
→ In the immune system, cancer isrecognized/eliminated as “non-self”.
2. Cancer antigens only appear on cancer cells.
→ The immune system does not damage normal cells.
3. Escape (antigen loss) from immune surveillancemechanisms may not occur.
→ Immunity does not lose a mark of attack.
4. A high incidence.*→ Cancer antigens are available for many patients, andmay be readily developed.
* A gene abnormality with a low incidence (neoantigen: described below) has also recently
been investigated as a target for cancer vaccine development.

9

Neoantigen
Neoantigen is a product of cancer-cell-specific geneabnormalities.
Original amino acid sequence (in normal cells)
T

A

A

C

T

G

C

A

G

G

T

T

A

A

C

C

G

C

A

G

G

T

Point gene mutation

An amino acid sequence in which some aminoacids are substituted for other amino acids
through a point gene mutation (in cancer cells)

10Immunogenicity of neoantigen
In immune surveillance mechanisms, neoantigen specificallyappearing on cancer cells is recognized as “non-self” andattacked/eliminated.
■ Original amino acid sequence- Low immunogenicity
The immune system
recognizes neoantigen
as “self”
Does not attack■ Neoantigen (sequence involving a point gene mutation)- High immunogenicity
The immune system
recognizes neoantigen
as “non-self”
Attacks to eliminate
the foreign body11Why is neoantigen being focused on?
 Immune checkpoint antibodies are more effective for patientswith a larger number of gene mutations (neoantigen) in theircancer cells; the immunogenicity of neoantigen may be useful.

Snyder, NEJM, 2014

Rizvi, Science, 2015

Atezolizumab (anti-PD-1)
non-small cell lung cancer# Non-synonymous mutation / megabase

Pembrolizumab (anti-PD-1)
non-small cell lung cancer# Non-synonymous mutation / tumor# Non-synonymous mutation / tumor

Ipilimumab (anti-CTLA-4)
melanoma

Rosenberg, lancet, 2016

 However, a gene mutation does not always have
immunogenicity (≤1/100), and mutations vary among
patients; the incidence is low (≤1%).
12

Characteristics of seeds

T790M point mutation
Amino acid sequence of the EGFR peptide appearing on small
cell lung cancer cells

Before theadministration of
EGFR-TKI(tyrosine kinase
inhibitors)*

Ｖ

Ｑ

Ｌ

Ｉ

Ｔ

Ｑ

Ｌ

Ｍ

Mutation of the
790 th amino acid of
the EGFR proteinfrom threonine (T)to methionine (M)Administration of EGFR-TKI

After its
administration

Ｖ

Ｑ

Ｌ

Ｉ

Ｐ

Ｍ

Ｑ

Ｌ

Ｍ

Ｐ

Resistance gene mutations appear in approximately 60% of
treatment-resistant patients.
If there is an immune response, neoantigen withimmunogenicity is synthesized.
* Molecule-targeting drugs as a primary treatment for non-small cell lung cancer: epidermal growth factor receptor(EGFR) tyrosine kinase inhibitors (EGFR-TKI), such as Tarceva (erlotinib, sales amount in 2015: $1.59 bn), Iressa(gefitinib), and Giotrif (afatinib), were approved by 3 bureaus.

14Characteristics of the T790M point mutation
1. In vivo immune responses in cancer patients(immunogenicity)→The response of human T cells to the T790M mutationderived antigen was confirmed.
 The immunogenicity of the T790M mutation-derived peptide washigher than that of the original sequence (wild-type) peptide.
 T cells, which specifically responded to the T790M mutationderived peptide, exhibited cytotoxic activity against an EGFRexpressing lung cancer cell line with the T790M mutation.
Article by seeds developers“EGFR T790M mutation as a possible target for immunotherapy; identification of HLAA*0201-restricted T cell epitopes derived from the EGFR T790M mutation” Yamada
et al. PLoS One. 2013 Nov 5;8(11):e78389.
“A peptide antigen derived from EGFR T790M is immunogenic in non-small cell lungcancer” Ofuji et al. Int J Oncol. 2015 Feb; 46(2): 497–504.

15

Characteristics of the T790M point mutation (cont.)
2. Appearance on cancer cells→Secondary gene mutation after the administration of EGFR-TKI(molecule-targeting drugs as primary treatments for nonsmall cell lung cancer)

3. Escape (antigen loss) from immune surveillancemechanisms may not occur.

→Since the gene mutation is necessary to acquire resistance toa challenge by EGFR-TKI (cancer malignancy), the appearanceof the antigen persists with a continuous EGFR-TKI treatment.

16

Characteristics of the T790M point mutation (cont.)
4. Its incidence is high.

→Approximately 60% are EGFR-TKI-resistant patients

MET amplitude(4%)
Cancer cellchanges from
non-small tosmall cells (6%)

EGFR T790M
mutation (60%)

Unclear (30%)(C) 2011 American Association for Cancer Research

The EGFR T790M mutation is the target ofcancer immunotherapy.
17

Patent
 Name of development: Antigen peptide originated from t790m pointmutated sequence of epidermal growth factor receptor
 Patent No.: PCT/JP2013/071499
 Date of application: August 8, 2013 (date of priority: October 8, 2012)
 Applicant: Kanagawa Prefectural Hospital Organization
 Developer: Tetsuro Sasada, Tetsuya Nakaomote, and Kazuya Ofuji State of inspection
Date oftransference

Application
numberState of application

European Patent Office

06.03.2015

2013827804

PendingJapan

05.02.2015

2014529551

Pending

14420561

Assessment ofregistration（US 9505824）

Country of transference

United States of America

09.02.201518

HLA restrictive of a peptide to be developed■ Positive reaction rate to the population

HLA-A2
restrictive

U.S.A

5
countries
in Europe

Japan

China

A2

48

48

30

50

A1

22

28

6

3

A24

15

20

60

22

HLA
type %

19

Possibilities for neoantigen

Possibilities for neoantigen

Number of somatic cell
mutations per 1,000,000
base pairs⒈ Prediction of the effects of immune checkpoint inhibitors→According to previous studies, cancer types with frequent genemutations showed favorable clinical results.

Khalil, D. N. et al. (2016) The future of cancer treatment: immunomodulation, CARs and combination immunotherapy
Nat. Rev. Clin. Oncol. doi:10.1038/nrclinonc.2016.25

2. Design of new cancer vaccines
3. Targets for T-cell therapy (TCR-T and T-iPS)
21