Hailing Lu1, Seth Pollack2, Neeta Somaiah3, Frank Hsu1, Richard Kenney1, Ryan Emerson4, Jan ter Meulen1

1Immune Design, Seattle, WA; 2Fred Hutchinson Cancer Research Center, Seattle, WA; 3MD Anderson Cancer Center; Houston, TX; 4Adaptive Biotechnologies, Seattle, WA

LV305 is a lentiviral vector that expresses full-length NY-ESO-1, a cancer testis

antigen. It is a replication-incompetent, integration-deficient, hybrid viral vector

based on the ZVexTM platform to target dendritic cells (DC) in vivo via CD209

(DC-SIGN). Preclinical studies have demonstrated that LV305 induces NY-

ESO-1 specific cytotoxic T lymphocytes (CTLs) and has potent anti-tumor

effects. In a Phase I dose-escalation study (presented at ASCO 2015), LV305

was administered to adult patients with previously treated, locally advanced,

relapsed or metastatic soft tissue sarcomas, expressing NY-ESO-1 protein.

Peripheral blood mononuclear cells (PBMC) were collected from patients at

baseline, during and post immunotherapy with LV305. ELISPOT demonstrated

an induction or increase of NY-ESO-1-specific CD8 and/or CD4 T cells in the

majority of patients. TCR repertoire analysis was performed in some patients

where leukapheresis samples were available. The TCR sequences from PBMC

were compared to the sequences from tumor infiltrating lymphocytes (TIL),

where available, as well as T cell clones generated from PBMC through

stimulation with NY-ESO-1. Results showed that NY-ESO-1 reactive T cell

clones increased post-vaccination, consistent with the increase in antigen-

specific T cell responses as measured by ELISPOT and tetramer staining. We

identified two unique TCRβ CDR3 sequences that were increased in a

significant portion of patients with different HLA-background post-treatment,

suggesting that T cells with public TCR may have been expanded during

treatment.

Phase I dose-escalation study (NCT02122861)

• Indication: Locally advanced, recurrent or metastatic melanoma, sarcoma, ovarian, or lung cancers (breast cancer allowed in Part 1 dose

escalation) expressing NY-ESO-1* s/p at least one prior cancer therapy (2 for lung) with low tumor burden**

• Treatment/Study Measurements:

− Part 1, Dose Escalation: 4 cohorts, 3 dose levels, Cohorts 1 (108 viral genomes x 3 doses), 1A (108 vg x 4), 2 (109 vg x 4), and 3 (1010 vg x 4)

− LV305 administered q21d intradermaly; 28d DLT observation period

− Blood samples collected for safety and immunologic testing at multiple time points including leukapheresis pre- and post- LV305

− Disease status measured by irRC criteria modified to use RECIST

− Follow-up: 2 yrs monitoring safety, disease status and LV305 persistence in blood

Day: 0 7 14 21 28 35 42 49 63 182 20

If no DLT within 28d, continue LV305

After EOS visit (d182) pts enter LT follow-up for

up to 2 years

*NY-ESO-1 expression determined by central lab using IHC with >5% cutoff

**No minimum tumor size; Maximum tumor size: 8cm for single mass

and 10cm total for all lesions (3cm and 9cm for melanoma)

***Cohort 1 pts received 3 doses

***

LV305 induced 2-fold Increase of NY-ESO-1p157 tetramer positive CD8 T-cells inunstimulated PBMC (Patient 1-1)

pre

post

0.00

0.05

0.10

0.15

Percentage of CD8+ Lymphocytes Staining Positive with NY-ESO-1 Tetramer

151-006

% T

etr

am

er p

os

itiv

e

HLA-A201 tetramer

on unstimulated

PBMC.

3 separate expts,

p<0.01

5/30/1

4

6/19/1

4

6/27/1

4

7/10/1

4

7/18/1

4

7/30/1

4

5/30/1

4

6/19/1

4

6/27/1

4

7/10/1

4

7/18/1

4

7/30/1

4

5/30/1

4

6/19/1

4

6/27/1

4

7/10/1

4

7/18/1

4

7/30/1

4

0

500

1000

1500

Nu

mb

er

of

sp

ots

/ 5

0,0

00 C

D8 c

ells

151-006_CD8_ESO_B-EBV_d11

B-EBV + DMSO B-EBV + NP B-EBV + ESO

B-EBV + DMSOB-EBV + NPB-EBV + ESO

Presensitized withNY-ESO-1 peptide pool

Presensitized withNP peptide pool

Tested on autologous B-EBV

Tested on autologous T-APC

CD8 T cell responses in 151-006

5/30/1

4

6/19/1

4

6/27/1

4

7/10/1

4

7/18/1

4

7/30/1

4

5/30/1

4

6/19/1

4

6/27/1

4

7/10/1

4

7/18/1

4

7/30/1

4

5/30/1

4

6/19/1

4

6/27/1

4

7/10/1

4

7/18/1

4

7/30/1

4

0

1000

2000

3000

Nu

mb

er

of

sp

ots

/ 5

0,0

00 C

D4 c

ells

151-006_CD4_ESO_TAPC_d20

T-APC + DMSO T-APC + NP T-APC + ESO

T-APC + DMSOT-APC + NPT-APC + ESO

Presensitized withNY-ESO-1 peptide pool

Presensitized withNP peptide pool

Tested on autologous B-EBV

Tested on autologous T-APC

CD4 T cell responses in 151-006

CD8+ T cell Response CD4+ T cell Response

*Data generated by S. Gjnatic, Mount Sinai School of Medicine

• Cryogenically preserved PBMC were separated into CD8 and CD4 populations by bead fractionation and

stimulated with EBV transformed B cells or T cells pulsed with overlapping NY-ES0-1 (ESO) peptides or

control peptides (NP) for 2-3 weeks

• T cells were then stimulated with NY-ESO-1 or NP, DMSO or PMA/ionomycin for several weeks and

examined for IFN-gamma by ELISPOT

1.68% of

Pre-tx T

cells

2.51% of

Post-tx T

cells

Clonality 0.11 Clonality 0.18

0.000001

0.00001

0.0001

0.001

0.01

0.000001 0.00001 0.0001 0.001 0.01

Ab

un

da

nc

e i

n P

BM

C-p

os

t

Abundance in PBMC-pre

T cells absent in FFPE tumor (pre)

T cells present in FFPE tumor (pre)TCR present in pre-Tx tumor biopsy

TCR absent in pre-Tx tumor biopsy

LV305 Induced New T Cells And Increased The Frequency Of TILs And NY-

ESO-1 Reactive Cells (Pt 1-1)

PBMCs (TIL-specific TCR) PBMCs (NY-ESO-1-expanded TCR)

0.000001

0.00001

0.0001

0.001

0.01

0.000001 0.00001 0.0001 0.001 0.01

Abundance in PBMC-pre

All T cells

T cells expanded after NY-ESO1 stimulation

0

50

100

1 122334455667

0

5

10

15

1 7 1319253137

0.10% of Pre-tx T cells

0.24% of Post-tx T cells

Clonality 0.07 Clonality 0.16

• T cell receptor (TCRβ) sequencing is performed on PBMC obtained pre- and post-LV305 treatment and the

frequency of individual TCR sequences are plotted

• Dots represent unique TCRβ sequences and those above the dashed line indicate an increase following

LV305 therapy

• Red dots represent: Left panel – TCRβ sequences of pre-treatment tumor TILs; Right Panel – NY-ESO-1

reactive T cells as determined by ELISPOT following in vitro expansion by peptide stimulation

-9 -8 -7 -6 -5

0

1 0 0

2 0 0

3 0 0

4 0 0

5 0 0

lo g [N Y -E S O -1 ] , M

SF

U/m

illi

on

PB

MC

p re -T x

post-T x

LV305 Induced T Cells Against Previously Unrecognized Epitopes of NY-ESO-1 (Pt 1-1)

LV305 Induced T Cells With

Increased Polyfunctional Avidity

PT151006: Epitope Mapping by Direct ELISPOT

35

p1-1

5

p5-1

9

p9-2

3

p13-2

7

p17-3

1

p21-3

5

p25-3

9

p29-4

3

p33-4

7

p37-5

1

p41-5

5

p45-5

9

p49-6

3

p53-6

7

p57-7

1

p61-7

5

p65-7

9

p69-8

3

p73-8

7

p77-9

1

p81-9

5

p85-9

9

p89-1

03

p93-1

07

p97-1

11

p101-1

15

p105-1

19

p109-1

23

p113-1

27

p117-1

31

p121-1

35

p125-1

39

p129-1

43

p133-1

47

p137-1

51

p141-1

55

p145-1

59

p149-1

63

p153-1

67

p157-1

71

p161-1

75

p165-1

80

pool A

0

5 0

1 0 0

1 5 0

P T 1 5 1 0 0 6 -E p ito p e M a p p in g in U n s t im u la te d P B M C

N Y -E S O -1 p e p t id e s (1 5 m e rs o v e r la p p in g b y 1 1 a a )

SF

U/m

illio

n P

BM

C

p re -T x

p o st-T x

36

p1-1

5

p5-1

9

p9-2

3

p13-2

7

p17-3

1

p21-3

5

p25-3

9

p29-4

3

p33-4

7

p37-5

1

p41-5

5

p45-5

9

p49-6

3

p53-6

7

p57-7

1

p61-7

5

p65-7

9

p69-8

3

p73-8

7

p77-9

1

p81-9

5

p85-9

9

p89-1

03

p93-1

07

p97-1

11

p101-1

15

p105-1

19

p109-1

23

p113-1

27

p117-1

31

p121-1

35

p125-1

39

p129-1

43

p133-1

47

p137-1

51

p141-1

55

p145-1

59

p149-1

63

p153-1

67

p157-1

71

p161-1

75

p165-1

80

pool A

0

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0

P T 1 5 1 0 0 6 -E p ito p e M a p p in g a fte r IV S

N Y -E S O -1 p e p t id e s (1 5 m e rs o v e r la p p in g b y 1 1 a a )

IF

N-

+ T

c

ells

/2

00

K P

BM

C

p re -T x

p o s t-T x

PT151006: Epitope Mapping after IVS

Intramolecular Epitope Spreading?

Epitope Mapping in Unstimulated PBMC Epitope Mapping After IVS

• For direct IFN-gamma ELISPOT, PBMC were stimulated for 40hr with individual 15mer peptides of the NY-

ESO-1 peptide pool, which contain 43 15mer peptides overlapping by 11 aa and span the full length protein

• For IVS (in vitro stimulation) ELISPOT, PBMC were stimulated in vitro for one week in OpTmizer T cell

expansion medium in the presence of NY-ESO-1 peptide pool and cytokines (IL-2 and IL-7, 10 ng/mL). Then

the expanded cells were stimulated with individual peptide overnight for the IFN-gamma ELISPOT.

• Boxes represent NY-ESO-1 epitopes that had increased T cell response following LV305

LV305 is a novel hybrid viral vector gene delivery system (ZVexTM) that expresses NY-ESO-1

RNA designed to target DCs in vivo and stimulate CD8 T cell responses against this cancer testis

antigen.

ZVex

DC-SIGN

ZVex

DC

Envelope:

DC Targeting:

• Sindbis virus envelope targets the Dendritic Cell

receptor, CD209 (DC-SIGN)

Genome:

Safety:

• 3rd generation lentiviral vector backbone

• Replication incompetent (self-inactivating) and

integration deficient

Selectivity:

• Vpx prevents degradation of vector within DCs

CLINICAL TRIAL DESIGN

BACKGROUND

IMMUNOLOGICAL ANALYSIS

TCR ANALYSIS

CD8 ELISPOT*Tetramer CD4 ELISPOT*

Overall anti-NY-ESO-1 Specific Immune Responses

Humoral (Ab) CD4 T Cell CD8 T Cell CD4 and/or CD8

0/12 5/11 6/11 8/11

Immunotherapy with LV305 Enhances NY-ESO-1-specific T Cell Response and Enriches

Tumor Antigen-specific TCR Sequences in Peripheral Blood

Dose-dependent Induction of Polyfunctional CTL

Potent Anti-tumor Activity

PRECLINICAL DATA

ABSTRACT

CONCLUSIONS

LV305 is immunologically active in generating anti-NY-ESO-1

CD4 and CD8 T cells

There is preliminary evidence of durable stable disease in a

subset of patients

LV305 Induced T cells against previously unrecognized

epitopes of NY-ESO-1

TCRβ sequences specific for NY-ESO-1 tumor antigen were

enriched in post-Tx PBMC

Evidence of induction/expansion of NY-ESO-1 specific

public TCRβ CDR3 sequences by LV305

A Highly Oligoclonal NY-ESO-1-specific T Cell Culture Was Established

from a post-treatment PBMC Sample

Two TCRβ CDR3 Clones are Detected in Multiple Patients with Different HLA

Backgrounds and are Induced by LV305

• (A) The oligoclonal culture was established by culturing post-LV305 PBMC in OpTmizer T cell

expansion medium (Invitrogen) with NY-ESO-1 overlapping peptide (0.5 ug/mL, JPT technologies) in

the presence of IL-2 and IL-7 (10 ng/mL). After repeated stimulation, the PBMC culture was highly

enriched for NY-ESO-1 specific T cells as measured by ELISPOT assay.

• (B) Shown are the top clones from the oligoclonal culture as determined by TCRb deep sequencing

analysis. Each column represents one clone with a unique TCRb CDR3 sequence. The y-axis

shows the relative frequency of each clone (percentage) among all sequence reads. The top 6

clones account for more than 90% of all the TCR, indicating that the culture is highly oligoclonal.

• Two public TCRβ CDR3 sequences are detected in PBMC from patients with different HLA background

(3/8 for the 1st public TCR and 6/8 for the 2nd public TCR)

• The 2nd public TCRβ CDR3 sequence was also detectable in a pre-Tx tumor biopsy and TILs from the

same patient

• The frequency of the public TCRβ CDR3 sequences was increased in post-Tx PBMC as compared to pre-

Tx PBMC in most patients

No Ag

NY-ESO-1

A B

Transduction of DCs → → → MHC-I Ag presentation

DC-SIGN

CD8 T CellResponse

DC

Tumor CellTumor CellDeath

Vector Particles

• Dose Escalation: n = 12 sarcoma pts

- Stable disease: 8/12 (67%) patients achieveda best response of SD (defined as stable forat least 84 days). Median duration of SD was208 days (range: 139-347+)

- 4 of 6 pts with evidence of growing disease atstudy entry stabilized their tumor growthfollowing LV305

- Pt 1-1 remains with SD for 347+ days and hadtumor regression up to 14%

CLINICAL RESPONSES

ELISPOT ICS

Day 0: LV305 immunizationDay 14: immune response evaluation

PT

006

PT

014

PT

016

PT

035

PT

039

PT

050

PT

119

PT

070

0 .0 0 0

0 .0 0 1

0 .0 0 2

0 .0 0 3

0 .0 0 4

1s t

p u b lic T C R

TC

R A

ba

nd

an

ce

in

PB

MC

(%

)

p re -T x

post-T x

PT

006

PT

014

PT

016

PT

035

PT

039

PT

050

PT

119

PT

070

0 .0 0 0

0 .0 0 5

0 .0 1 0

0 .0 1 5

0 .0 2 0

2n d

p u b lic T C R

TC

R A

ba

nd

an

ce

in

PB

MC

(%

)

p re -T x

post-T xImmune Responses in Pt1-1

• Day 0: CT26 tumor cell (80,000) challenge

in Balb/c mice (N=10 per group)

• Day -28 or Day 4: Injection of Zvex-AH1A5

(4E9 vector genome)

• **p ≤ 0.01 and ***p ≤ 0.001 compared to

untreated (Mantel-Cox).

Zvex-AH1A5 (Day -28)

Zvex-AH1-A5 (Day 4)

Untreated

5 E 6 5 E 7 5 E 8 5 E 9 5 E 1 0 C o n tr o l

0

2 0 0

4 0 0

6 0 0

T re a tm e n t G ro u p s

Sp

ots

/we

ll

N Y -E S O -1 p 8 1

L V 3 0 5

****

****

Reference: Odegard et al, J Immunother 2015; 38(2) 41-53