Immunotherapy with LV305 Enhances NY-ESO-1-specific T Cell Response and Enriches
Tumor Antigen-specific TCR Sequences in Peripheral Blood
Seth
2
Pollack ,
1Immune
3
Somaiah ,
Neeta
Frank
1
Hsu ,
Richard
1
Kenney ,
Ryan
4
Emerson ,
Jan ter
1
Meulen
Design, Seattle, WA; 2Fred Hutchinson Cancer Research Center, Seattle, WA; 3MD Anderson Cancer Center; Houston, TX; 4Adaptive Biotechnologies, Seattle, WA
CLINICAL TRIAL DESIGN
ABSTRACT
TCR ANALYSIS
Phase I dose-escalation study (NCT02122861)
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 NYESO-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 antigenspecific 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.
• 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**
LV305 Induced New T Cells And Increased The Frequency Of TILs And NYESO-1 Reactive Cells (Pt 1-1)
• 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
***
After EOS visit (d182) pts enter LT follow-up for
up to 2 years
PBMCs (NY-ESO-1-expanded TCR)
PBMCs (TIL-specific TCR)
0.01
0.01
TCR Tabsent
in pre-Tx
tumortumor
biopsy
cells absent
in FFPE
(pre)
All T cells
TCR Tpresent
in pre-Tx
tumortumor
biopsy
cells present
in FFPE
(pre)
T cells expanded after NY-ESO1 stimulation
0.001
Abundance in PBMC-post
Hailing
1
Lu ,
0.001
0.0001
0.0001
1.68% of
Pre-tx T
cells
0.10% of
Pre-tx T cells
2.51% of
Post-tx T
cells
0.24% of
Post-tx T cells
15
Day:
0
7
14
21
28
35
42
49
63
182
0.00001
20
0.00001
Clonality 0.11
100
10
Clonality 0.18
Clonality 0.07
5
0
0
1 7 1319253137
If no DLT within 28d, continue LV305
*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
0.000001
0.000001
0.00001
0.0001
0.001
Abundance in PBMC-pre
0.000001
0.000001
0.01
0.00001
50 Clonality 0.16
0.0001
1 122334455667
0.001
0.01
Abundance in PBMC-pre
• T cell receptor (TCRβ) sequencing is performed on PBMC obtained pre- and post-LV305 treatment and the
frequency of individual TCR sequences are plotted
CLINICAL RESPONSES
• Dots represent unique TCRβ sequences and those above the dashed line indicate an increase following
LV305 therapy
BACKGROUND
• 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
• Dose Escalation: n = 12 sarcoma pts
- Stable disease: 8/12 (67%) patients achieved
a best response of SD (defined as stable for
at least 84 days). Median duration of SD was
208 days (range: 139-347+)
- 4 of 6 pts with evidence of growing disease at
study entry stabilized their tumor growth
following LV305
- Pt 1-1 remains with SD for 347+ days and had
tumor regression up to 14%
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
Envelope:
DC Targeting:
• Sindbis virus envelope targets the Dendritic Cell
receptor, CD209 (DC-SIGN)
ZVex
A Highly Oligoclonal NY-ESO-1-specific T Cell Culture Was Established
from a post-treatment PBMC Sample
B
A
No Ag
NY-ESO-1
DC-SIGN
Genome:
CD4 T cell responses in 151-006
Presensitized with
NY-ESO-1 peptide pool
NP peptide pool
• (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.
IMMUNOLOGICAL ANALYSIS
Overall anti-NY-ESO-1
Specific
Immune
Tested on
autologous
B-EBV Responses
ICS
ELISPOT
0.10
HLA-A201 tetramer
on unstimulated
PBMC.
3 separate expts,
p<0.01
0.05
500
0
400
200
0
5E 8
5E 9
5 E 1 0 C o n tro l
LV305 Induced T Cells With
Increased Polyfunctional Avidity
LV305
T re a tm e n t G ro u p s
0 .0 2 0
p re -T x
p o s t-T x
0 .0 1 5
0 .0 1 0
0 .0 0 5
0
T
P
P
T
1
0
1
7
9
0
0
T
P
T
0
5
9
3
5
P
T
P
P
T
0
0
1
3
6
4
1
P
T
0
T
0
T
0
0
0
6
0 .0 0 0
7
9
P
T
P
T
1
0
1
0
5
9
3
0
P
6
1
1
6
p u b li c T C R
• 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)
1000
• The 2nd public TCRβ CDR3 sequence was also detectable in a pre-Tx tumor biopsy and TILs from the
same patient
0
B-EBV + ESO
0 .0 0 0
nd
T-APC + DMSO
T-APC + NP
• The frequency of the public TCRβ CDR3 sequences was increased in post-Tx PBMC as compared to preTx PBMC in most patients
T-APC + ESO
CONCLUSIONS
*Data generated by S. Gjnatic, Mount Sinai School of Medicine
Tested on autologous T-APC
5E 7
T-APC + DMSO
T-APC + NP
T-APC + ESO
• Cryogenically preserved PBMC were separated into CD8 and CD4 populations by bead fractionation and
LV305 induced 2-fold Increase of NY-ESO-1
stimulated with EBV transformed B cells or T cells pulsed with overlapping NY-ES0-1 (ESO) peptides or
p157 tetramer positive CD8 T-cells in
control peptides (NP) for 2-3 weeks
unstimulated PBMC (Patient 1-1)
• 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
****
5E 6
B-EBV + NP
0 .0 0 1
0
Tested on autologous T-APC
2000
151-006
****
PT151006: Epitope Mapping after IVS
PT151006: Epitope Mapping by Direct ELISPOT
 LV305 is immunologically active in generating anti-NY-ESO-1
CD4 and CD8 T cells
LV305 Induced T Cells Against Previously Unrecognized Epitopes of NY-ESO-1 (Pt 1-1)
Day 0: LV305 immunization
Day 14: immune response evaluation
P T 1Mapping
5 1 0 0 6 -E p ito p e After
M a p p in g IVS
a fte r IV S
Epitope
• **p ≤ 0.01 and ***p ≤ 0.001 compared to
untreated (Mantel-Cox).
p re -T x
400
500
 LV305 Induced T cells against previously unrecognized
epitopes of NY-ESO-1
1000
N Y -E S O -1 p e p tid e s (1 5m e rs o v e rla p p in g b y 1 1 a a)
0
p1
p5 15
p9 19
p 1 -2 3
3
p 1 -2 7
7
p 2 -3 1
1
p 2 -3 5
5
p 2 -3 9
9
p 3 -4 3
3
p 3 -4 7
7
p 4 -5 1
1
p 4 -5 5
5
p 4 -5 9
9
p 5 -6 3
3
p 5 -6 7
7
p 6 -7 1
1
p 6 -7 5
5
p 6 -7 9
9
p 7 -8 3
3
p 7 -8 7
7
p 8 -9 1
1
p 8 -9 5
p 8 5 -9
9
p 9 -1 0 9
3
p 9 -1 0 3
p 1 7 -1 7
0
1
p 1 1 -1 1
05 15
p 1 -1
0
1
p 1 9 -1 9
1
2
p 1 3 -1 3
17 27
p 1 -1
2
3
p 1 1 -1 1
25 35
p 1 -1
2
p 1 9 -1 3 9
3
4
p 1 3 -1 3
3
4
p 1 7 -1 7
4
5
p 1 1 -1 1
4
5
p 1 5 -1 5
4
5
p 1 9 -1 9
53 63
p 1 -1
5
6
p 1 7 -1 7
61 71
p 1 -1
65 75
po 180
ol
A
p1
p5 15
p9 19
p 1 -2 3
3
p 1 -2 7
7
p 2 -3 1
1
p 2 -3 5
5
p 2 -3 9
9
p 3 -4 3
3
p 3 -4 7
7
p 4 -5 1
1
p 4 -5 5
5
p 4 -5 9
9
p 5 -6 3
3
p 5 -6 7
7
p 6 -7 1
1
p 6 -7 5
5
p 6 -7 9
9
p 7 -8 3
3
p 7 -8 7
7
p 8 -9 1
1
p 8 -9 5
p 8 5 -9
9
9
p 9 -1 0
3
p 9 -1 0 3
p 1 7 -1 7
0
1
p 1 1 -1 1
05 15
p 1 -1
0
1
p 1 9 -1 9
1
2
p 1 3 -1 3
17 27
p 1 -1
2
3
p 1 1 -1 1
25 35
p 1 -1
2
p 1 9 -1 3 9
3
4
p 1 3 -1 3
37 47
p 1 -1
4
5
p 1 1 -1 1
45 55
p 1 -1
4
5
p 1 9 -1 9
5
6
p 1 3 -1 3
57 67
p 1 -1
6
p 1 1 -1 7 1
65 75
po 180
ol
A
p o s t-T x
200
100
-9
-8
-7
lo g [ N Y - E S O - 1 ] , M
-6
-5
 TCRβ sequences specific for NY-ESO-1 tumor antigen were
enriched in post-Tx PBMC
N Y -E S O -1 p e p tid e s (1 5 m e rs o v e rla p p in g b y 1 1 a a)
300
0
Reference: Odegard et al, J Immunother 2015; 38(2) 41-53
50
0
500
• 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)
100
1500
 There is preliminary evidence of durable stable disease in a
subset of patients
pre-Tx
post-Tx
c e lls /2 0 0 K
Zvex-AH1A5 (Day -28)
Zvex-AH1-A5 (Day 4)
Untreated
post-Tx
2000
+
Potent Anti-tumor Activity
pre-Tx
IF N -
S F U /m illio n P B M C
150
T
P B M C
Epitope
P T 1 5Mapping
1 0 0 6 -E p ito p e Min
a p pUnstimulated
in g in U n s tim u la te d P BPBMC
MC
S F U /m illio n P B M C
S p o t s /w e ll
B-EBV + DMSO
B-EBV + NP
B-EBV + ESO
B-EBV + DMSO
po
N Y -E S O -1 p 8 1
st
pr
e
0.00
600
1000
CD4+ T cell Response
5/3
0
6/1 /14
9
6/2 /14
7
7/1 /14
0
7/1 /14
8
7/3 /14
0/1
4
5/3
0
6/1 /14
9
6/2 /14
7/1
7/1 4
0
7/1 /14
8
7/3 /14
0/1
4
5/3
0
6/1 /14
9
6/2 /14
7
7/1 /14
0/1
7/1 4
8
7/3 /14
0/1
4
% Tetramer positive
Dose-dependent Induction of Polyfunctional CTL
Tested on autologous B-EBV
CD8+ T cell Response
5/3
0
6/1 /14
9
6/2 /14
7
7/1 /14
0
7/1 /14
8
7/3 /14
0/1
4
5/3
0
6/1 /14
9
6/2 /14
7
7/1 /14
0
7/1 /14
8
7/3 /14
0/1
4
5/3
0
6/1 /14
9/1
6/2 4
7
7/1 /14
0/1
7/1 4
8
7/3 /14
0/1
4
PRECLINICAL DATA
0.15
3000
Number of spots / 50,000 CD4 cells
Number of spots / 50,000 CD8 cells
Percentage of CD8+ Lymphocytes Staining
Positive with NY-ESO-1 Tetramer
1500
0
151-006_CD4_ESO_TAPC_d20
151-006_CD8_ESO_B-EBV_d11
0 .0 0 2
T
CD4 ELISPOT*
CD8 ELISPOT*
T
Tetramer
P
Tumor Cell
p o s t-T x
0 .0 0 3
4
NY-ESO-1 peptide pool
p re -T x
0
Tumor Cell
Death
Presensitized with
NP peptide pool
2
p u b li c T C R
0 .0 0 4
P
Immune
Responses
in Pt1-1
Presensitized
with
T C R A b a n d a n c e in P B M C ( % )
Vector Particles
st
P
1
T C R A b a n d a n c e in P B M C ( % )
CD8 T cell responses
in 151-006
6/11
8/11
T
5/11
CD4 and/or CD8
5
0/12
CD8 T Cell
P
CD4 T Cell
3
Humoral (Ab)
0
DC-SIGN
CD8 T Cell
Response
DC
Two TCRβ CDR3 Clones are Detected in Multiple Patients with Different HLA
Backgrounds and are Induced by LV305
T
Transduction of DCs → → → MHC-I Ag presentation
0
• Vpx prevents degradation of vector within DCs
T
Selectivity:
• (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
Presensitized
with specific T cells as measured by ELISPOT assay.
enriched for NY-ESO-1
P
DC
P
Safety:
• 3rd generation lentiviral vector backbone
• Replication incompetent (self-inactivating) and
integration deficient
Intramolecular
Epitope15mer
Spreading?peptides of the NY• For direct IFN-gamma ELISPOT, PBMC were stimulated for 40hr with
individual
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
36
35
 Evidence of induction/expansion of NY-ESO-1 specific
public TCRβ CDR3 sequences by LV305