Novel Immunotherapy Approaches in
Multiple Myeloma
Nikhil C. Munshi
Jerome Lipper Multiple Myeloma Center
Dana Farber Cancer Institute
Boston VA Healthcare System
Harvard Medical
Medical School
DANA-FARBER
CANCER INSTITUTE
Myeloma - Dendritic Cell Fusion
Vaccine
Myeloma Cells CD-38
DC-MM Fusion CD-83/38
Schema of The
Clinical DC/MM
Fusion Vaccine
V
Leukapheresis
Protocol
Adherent PBMCs
cultured for 5-7 days
Doses prepared
pp
& frozen
with GM-CSF & IL-4;
microbiology testing
TNF- added for 48-72
sent
hours
Myeloma cells
Fusion cells
quantified by
CD38
measuring dual
expressio
p
n of
CD138
unique DC &
DCs pulsed
tumor markers
CD86
Myeloma cells assessed for
with KLH
tumor & DC specific markers
for control
MUC1
vaccine
DC & myeloma
CD38
CD138
fused with 50% PEG
at DC: tumor, 3:1 to
DCs assessed for
10:1
DC & specific
p
markers
DR
CD40
CD80
CD86
CD83
Clinical Trial
Patient Accrual
· Patients Screened
26
· Patients enrolled
16
· Completed Therapy
12
· Il
Immune response evaluated
9
Clinical Trial
Vaccine Characterization
· Mean C
D
Yield
12
1. 3
23x108
10 (2
( 1
21
188
.5-
x 106
10 )
· Mean fusion efficiency
40% (22% - 52%)
· Mf
Mean fusion
l
ce l
ll viabilit
bility 84% ( 82% - 96%)
·
50
DC Fusion induced T
40
cell proliferation (SI)
30
· N = 15
SI 20
10
0
DC
Tumor
Fusion Cells
Clinical Trial
Immunological correlates
· 6 of 9 patients demonstrated evidence of
immunologic response (>2 fold increase in CD4
and/or CD8 T cell
cell expression
expression of IFN- following ex-
vivo exposure to autologous tumor lysate)
· One patient with a 32
32 and 20 fold increase in
in
interferon- expression by CD4+ and CD8+ T cells
respectively
py
· However, immune responsive cell number
decrease over time
T Cell Expression of IFN
Following Exposure to
to Autologous
Autologous tumor Lysate
L
Vaccination time points
1 month
3t
3 mon h
th
BASELINE
post-vaccine 3 post-vaccine 3
Pre-vaccine
Pre-vaccine 2
Pre-vaccine 3
1
4
01
0. 2
12
05
0. 9
59
42
4.2
38
3.8
07
0. 6
76
CD
100 101 102 103 104
100 101 102 103 104
100 101 102 103 104
100 101 102 103 104
100 101 102
103 104
FL1-H
FL1-H
FL1-H
FL1-H
FL1-H
0.39
0.93
4.8
7.8
2.4
CD8
100
10
101
10
102
10
103
10
104
10
100
10
101
10
102
10
103
10
104
10
100
10
101
10
102
10
103
10
104
10
100
10
101
10
102
10
103
10
104
10
100
10
101
10
102
10
103
10
104
10
FL1-H
FL1-H
FL1-H
FL1-H
FL1-H
IFN-
Clinical Outcome
Adverse Events
· Tr
T ansient and mild pain and erythema at
at the
the injection site
site
· Grade I fever, chills, fatigue, and hip arthralgia
· One patient with a history of DVT developed PE
· Biopsy of vaccine site reactions demonstrate a dense infiltrate of CD8+
T cells and CD1a+ immature DCs.
CD8 staining
Clinical Response
· Patients evaluable for
clinical response
11
CD1a staining
· Stable disease
2 (17 and 4 mos)
· Short term stabilization
5 (6-8 weeks)
CD138 peptide binding
binding affinity to HLA-
HLA A2
itys 600
Inten 500
ec 400
rescen 300
o
200
Flu
ean 100
M
0
No
Inf. Virus
CD138
peptide
peptide
peptide
peptide
T2 cells pulsed
IFN- release by CD138 pe
yp ptide-s
p
pecific
p
CTLs
2500
2000
1500
1000
500
0
None
INA-6
RL
U
C
Stimulator cells
Cytotoxic Activity of CD138 peptide-specific CTLs
80
70
60
C (HLA-A2+, CD138+)
50
U (HLA-A2+, CD138+)
40
30
20
10
RL (HLA-A2+, CD138-)
0
INA6 (HLA-A2-, CD138+)
1:1
10:1
20:1
60:1
Ratios of Effector:Target
N = 4
Non-spliced (Short) XBP1 peptides
binding affinity to HLA
HLA-A2
900
800
ensity
700
e
Int
600
escence
500
oru
400
Fl
300
Mean
200
100
0
None
Influenza
XBP1
XBP1
XBP1
XBP1
XBP1
XBP1
virus
#1
#2
#3
#4
#5
#6
ma
matri
trix
peptide
peptide
peptide
peptide
peptide
peptide
peptide
peptide
peptide
peptide
pepti
peptide
peptide
Peptide pulsed into T2 cells
Phenotype Analysis
CD69+/CD45RO+
CD45RA+/CCR7+
24%
4%
Unstim
62%
1%
XBP-1, 2M-CTL
64%
2%
XBP-1, SP3M-CTL
Xbp1-HLA-
HLA A2-Specific IFN-
IFN
secretion by CTL
800
700
g/ml) 600
(p 500
XBP1 2M-CTL
400
N-gF
XBP1 SP 3M-CTL
300
IF 200
100
0
None
McCAR
ML-2
MM1S
Stimulator cells
Xbp1-HLA-A2-Specific CTL Proliferation
Stimulator: None
McCAR
ML-2
MM1S
Responder:
5%
55%
17%
8%
M1
M1
M1
M1
XBP1-2M-
CTL
42%
4%
9%
2%
M1
M1
M1
M1
XBP1-
XBP1 SP 3M-
3M
CTL
Xbp-1-HLA-A2-specific Myeloma
cell Cytotoxicity
XBP1-2M-CTL
100
XBP1 2M
100
90
80
is
Target
g
cells
s 70
y 60
L
U266
50
40
cific
MC
Mc AR
CAR
30
Spe 20
ML-2
% 10
MM1S
0
1:1
10:1
20:1
60:1
Responder: Stimulator
Stim
ratio
Developing Peptide-based vaccination
Strategy in
in Multiple
Multiple Myeloma
Myeloma
Selection of Genes Using Genomic Data Sets
DNA Microarray
- 34,000 genes
- Bone marrow plasma
plasma cells
cells
- Fold increase in expression; >20
57 genes
- Ft
Func itional i
s
i
gn fi
ificance
- Expression pattern in normal tissue
Selection of 9 genes
Prediction of Peptides Binding to HLA-A2
IGF-1
BCMA
RANKPEP, BIMAS, NetMHC
Sd
Syndecan-1
Caveolin 1
·
- HLA-A0201 binding
Cyclin D1
·
- 9mer or 10mer
Intergrin
g
a8
a8
·- proteasome clea
clea age
v
Amphiregulin
HLA-DOB
IL-5 receptor
Prediction of 37 peptides
from 9 genes
RANK
POS.
N
SEQUENCE
C
MW (Da) SCORE
% OPT.
1
3
MG
KISSLPTQL
FKC
968.16
75
65.79%
2
38
LAL
CLLTFTSSA
TAG
924.08
67
58.77%
3
106
DLR
RLEMYCAPL
KPA
1077.34
64
56.14%
4
19
FCD
FLKVKMHTM
SSS
1116.43
61
53.51%
5
39
ALC
LLTFTSSAT
AGP
922.04
58
50.88%
Evaluation of Antigenic
Peptide Candidates
Evaluated by:
· In vitro MHC-peptide binding assay, MHC-pentamer
synthesis
y
· In vitro T cell culture, analysis by antigen-specific
cytokine production,
production, MHC
MHC-pentamer staining, cytotoxicity
· Mass spectrometry analysis of MHC-binding peptides
purified from
from Multiple
Multiple Myeloma
Myeloma cell lines
· Testing potential synergies with mAbs and small
molecule inhibitors
MM Cell line ­ Expression of
HLA A2
-
and Selected
Selected Antigens
CD138
BCMA
Cyclin
Cav-
IL-5Ra
ITGA8
AR
IGF-1
HLA
D1
1
DOB
U266
+
+
+
+
Weak
+-
+/-
+/-
+
MCCAR
-
+
--
Weak
W
+
-
+
+/-
+
IM9
+-
-
-
-+
+/-
MM1S
++
-
+
++
-
+
+/-
/
MM1R
++
-
+
++
-
+
+/-
RPMI
+-
-
+
+
+
-+
+/-
INA6
+
+
+
+
+
+
-
+
+/-
+/
OPM1
++
-
+
+
-
+
+/-
OPM2
+
-
-
-
+/-
-
+/-
Vali
a dat
d i
at on of Pepti
ept de
de for HLA-A2-Binding
dg
Epitope
Peptide
In vitro
prediction
synthesis
HLA-binding
9mer
24
24
24 (100%)
10mer
13
10
10 (77%)
total
37
34
34 (92%)
Current stage of MM peptide evaluation
Peptide I.D.
Sequence
Priming
1st RS
2nd RS
3rd RS
4th RS
5th RS
1
ELVDALQFV
IGF-1
2
TMSSSHLFYL
TNFRSF17 (BCMA)
4
AMEEGATIL
6
GLVGLIFAV
7
SLQLALPQI
CD138
8
GLIFAVCLV
9
WLCALALSL
10
LLVGIVIQL
12
MLSGIAAFLL
HLA-DOB
13
FLLGLIFLL
14
VMLEMTPEL
15
MLSGIAAFL
16
KLMTNAFISI
17
ILHKGFSASV
IL-5Ra
18
LMTNAFISI
19
ILLGATEIL
20
LLPDEKISL
21
RLLSALFGI
Caveolin 1
22
ALFGIPMAL
23
ILTAVAVITV
24
FMSAVILTAV
Amphiregulin
25
YIVDDSVRV
26
SMIDSSLSKI
27
LLQMELLLV
28
QLLGATCMFV
Cyclin D1
29
LLGATCMFV
30
KIVATWMLEV
31
TISDTILEV
32
ALASLVSFEV
33
SLQINITAV
Integrin alpha 8
34
LLHPMIINL
35
YLYLQVSSL
36
KLPEGSIAI
37
KLSPINISL
Control
Specific
Priming of
103
103
CD8+ T
l
ce l
lls
102
102
Log
0.035
Log
0.21
AMEEGATIL
(from normal
FL2 101
FL2 101
Log:
Log:
FL2
FL2
donors) with
BCMA 148-156
100
10
100
10
K562/A2/CD80/4
3 Weeks
100
101
102
103
100
101
102
103
FL5 Log: FL5 Log
FL5 Log: FL5 Log
1BBL/CD70
103
103
Artificial APC
102
102
Log
8.43e-3
Log
0.051
GLIFAVCLV
FL2 101
FL2 101
Log:
Log:
FL2
FL2
Syndecan1 263-271
100
100
3 Weeks
100
101
102
103
100
101
102
103
FL5 Log: FL5 Log
FL5 Log: FL5 Log
103
103
102
10
102
10
Log
0.017
Log
0.044
IFLARSALI
FL2 101
FL2 101
Log:
Log:
FL2
FL2
HLA-DOB
100
100
225-234
3 Weeks
100
101
102
103
100
101
102
103
FL5 Log: FL5 Log
FL5 Log: FL5 Log
CD8
Steps to
to Improve
Improve Vaccination in MM
Identify novel antigens
Understand the immune dysregulation in
myeloma
Augment immune response using
immunomodulatory agents and avoiding
the immune immunosuppressant
Schematic
Representation
of T
Regulatory Cell
Dysfunctional Treg Cells in MGUS and MM
Control
MGUS
MM
66
6. %
6%
35
3. %
5%
13
1. %
3%
06
0. %
6%
18
1. %
8%
8
es
1%
cyt 6
ho
4
fLympo
*
ent 2
rc
Foxp3
Pe 0
Control(13)
MGUS(9)
MM(5)
CD4
Expression CD4+CD25+ cells
es 35
yt 30
*
*
Control
oc
MGUS
MM
ph 25
my 20
Lf 15
ot 10
en
rc
5
Pe
0
Control
MGUS
MM
)
(15)
(5)
(15)
m
12
1 . 2 e + 5
+5
(cp
1. 0e + 5
8. 0e + 4
6. 0e + 4
incorporatione
4. 0e + 4
idin
2. 0e + 4
m
0. 0
THy
+
-
+
-
+
CD4+CD25+
- Control(9)
MGUS(9)
MM(9)
Abrogation of normal donor Treg function
by exposure
exposur to IL6/IL6R in vitro (N=6)
Normal donor
w/o CD25
PBMC
w CD25
100
without CD25
with CD25
n
80
*
io
PBMC
PBMC
itb
(physiological level approx.
1:10)
nhi
60
I
tof
Incubate with anti-CD3 Ab
40
with or without IL-6/R
rceneP
72 hours
20
0
Measure
Measur proliferation
pr
PBMC
IL-6/R
Restoration of Myeloma Treg Cell Function by
Anti-IL-6R antibody Tr
T eatment
r
(N=4)
MYELOMA
140
w/o CD25
PBMC
w CD25
120
nio
without CD25
with CD25
100
it
PBMC
PBMC
ib
*
*
hn 80
(physiological level approx.
fI
1:10)
to
60
encr 40
Incubate with anti-CD3 Ab
with or without anti-IL-6R ABs
Pe
20
72 hours
0
PBMC
anti-IL-6R
Measure proliferation
antibody
Measure proliferation
a t
Effect f
o
i
m croenvironmental
t
cy k
o i
kines
) 60000
PBMC-
mp
PBMC+
c 50000
(
tion 40000
a
rpor 30000
o
inc 20000
idine 10000
myhT 0
Cells
aCD3
IL6/sR
sant7
IL6/TGFb
Stimulation with
with cy
cytokine
tokines
Anti IL-6 Agent Increases Foxp3 expression
in CD3+CD25+F
3
oxp + Cells
6.3%
12.4%
sant7
Steps to
to Improve
Improve Vaccination
V
in MM
Identify novel antigens
Single antigen-based vaccination will
have limited effectiveness
Augment immune response using
immunomodulatory agents and avoiding
the immune immunosuppressant
IMiDs Augment anti-MM Immunity
T Cell
NK Cell
NFA
NF T
A
IL-2
IL-2
PKC
Dendritic Cell
PI3K
CD28
Apoptotic
MM Cell
MM Cell
IMiDs
VEGF
IL-6
Bone Marrow Stromal Cell
LeBlanc R et al. in press Hayashi T et al. in press
Randomized Study to evaluate ability of
Lenalidomide to
to Augment
Augment Vaccine
Vaccine Response
VA
V /DFCI
A
Randomized Study
Hepatitis B
Day 28
Vaccine
V
AI
Arm I. L
lid
ena
id
om e
Arm I. Lenalidomide
25 mg/day PO, Days 1
1­
­77
25 mg/day PO, Days 8
8­
­14
14
Patients with
Anti-HepB
MM
Immune
response
Arm II. Placebo
Arm II. Placebo
Days 1-7
Days 8-
8 14
Future Direction
· Understand changes in immune
environment in myeloma
· Develop novel myeloma-specific
antigen based immunotherapeutic
strtegies
· Combine Vaccination and immune-
modulatory ag
ygents to further expand
the immune response
Kenneth Anderson, MD
Prabhalla Rao, PhD
Yu Tzu Tai, PhD
Simona Blotta, MD
Constantine Mitsiades, MD
Weihua Song,MD
Dharminder Chauhan,PhD
Maria Teresa
Teresa Fulciniti, MD
Teru
Teru Hideshima, MD
Paola Neri, MD
Nicholas Mitsiades, MD
Jooeun Bae, PhD
Noopur Raje, MD
Masood Shammas, PhD
PhD
Klaus Podar
Podar, MD
MD
Pierefrancesco Tassone, MD
Laurence Catley, MD
Ramesh Batchu,PhD
.................
Robert Bartheau,MD
Paul Richardson
Jim Driscoll, MD, PhD
Robert Schlossman
.................
Irene Ghobrial
David Avigan,
Avigan, MD
.................
Steven Treon, MD, PhD and
Don Kufe, MD
WM Group
DANA-FARBER
CANCER INSTITUTE
Spliced (Long) XBP1 peptides
binding affinity to HLA A2
-
1000
900
800
ensity
700
Int
600
escence
500
oru
Fl
400
Mean
300
200
100
0
None
Influenza
XBP1
XBP1
XBP1
virus
SP #1
SP #2
SP #3
matrix
peptide
peptide
peptide
peptide
Peptide pulsed into T2 cells
Proliferation of CD138 peptide-specific CTLs
2000
1800
1600
1400
M 1200
CP 1000
800
600
400
200
0
INA-6
RL
U
C
Stimulator cells
INA-6 (HLA-A2-, CD138+)
RL (HLA-A2+, CD138-)
U and C (HLA-A2+, CD138+)
Gene Products with Antibody Response Identified by SEREX in MGUS
Gene
Chromosome
mRNAsize, bp
Molecular
products
localization
Function
SON
21q22.1-22.2,22.11
8266
490
DNA-binding protein
OFD1
Xp22.2-p22.3
3615
936
unknown
SSSCA1
11q
11 1
q 3
1 1
.
672
Cell cycle, mitosis
mitosis
KIAA0530
6q15
6578
DNA-binding protein
18sribRNA
-
1843
Protein synthesis
FAM50A
Xq28
1358
410
unknown
AKAP11
13q14.11
1901
PKA-binding
HIPPI
3q13.12-q13.13
1665
Receptor activity
GPATC4
1q22
1906
Nucleic acid binding
IRF2BP2
1q42.3
1900
Co-
Co repressor of
of IRF-
IRF
2BP2
PSMC1
14q32.11
1586
ATP binding activity