DNA vaccination against myeloma post autologous or allogeneic transplantation
Know the enemy
Molecular targets of immune attack
Target tumor cell
MHC Class I-
associated peptides
Cell surface
glycoproteins
CD1d
Secreted or shed antigens
Molecular Immunology Group
Tenovus Laboratory
Could also target critical elements from stroma
University of Southampton UK

Immune effector mechanisms required for attack
CD8+
T cell
Antibody
CD4+
T cell
NKT
cell

Target surface antigens for antibody attack
Antibody
Mucins (MUC-1)
CD38/CD47/Wue-1/CD52
CD40/IL6-R/HM1.24/CT-7/
CD138/ CD20

Target secreted or shed antigens for CD4+ T-cell attack
CD4+
T cell
Secreted or shed antigens
Idiotypic Ig

Target antigens for CD8+ T-cell attack
Viral antigens?
Mutated proto-oncogene peptides
Cancer-testis antigens:
MAGE-A3/NY-ESO-1/CT-7
CD8+
Translocation peptides
T cell
Autoantigens: hTERT
CYPIBI
Survivin
Idiotypic peptides
PRAME
Products of genomic instability
Minor histocompatibility antigens

DNA vaccination
DNA vaccination
of patient
of allogeneic donor
Tolerance/immunodeficiency
New antigens/immunocompetent
CD8+
T cell
Antibody
CD4+
T cell
NKT
cell

Antigen sequence
DNA plasmid vaccine
Indirect route
Direct route
CpG
Adjuvant activity
Muscle cell
Cross presentation
Antigen-presenting cell
Skin cell
Antibody response
Cellular response
CD4+ TH1>TH2
Stevenson et al Imm Rev 2004
CD8+
Stevenson et al PNAS 2004

Idiotypic Ig as a tumor antigen
Tumor V-gene isolation
V
J
V
DJ
H
H
L
L
assembly
pCMV L
V
V
H
L
Fragment C of
tetanus toxin
Efficacy in: lymphoma models
Induction of immunity against
: 5T33 myeloma
both proteins
King et al Nat Med 1998
scFv-FrC

Testing of DNA fusion gene vaccination against tumors
Antibody/CD4+ T cells
Idiotypic Ig of B-cell tumors
B
DNA scFv--FrC
Protection
King et al Nat Med 1998
Clonotypic T-cell receptor
Antibody
T
DNA V-V-C--FrC
of T-cell lymphomas
Protection
Thirdborough et al
Canc Res 2002
PML-RARA translocation sequence
DNA-PML-RARA-FrC
Protection
of AML
Padua et al
Nat Med 2003

Lymphoma trial 50% of patients show anti-Id responses to vaccine
A single tested patient with myeloma post autograft showed responses
450
400
Vaccinations
350
PBMNC)
300
6
250
200
150
100
(spots/10
50
0
Pre
0
10
20
30
40
150
ELISPOT
100
50
IFN-
0
Pre
010
20
30
40
800
700
Patient Idiotype
Abs
600
500
C
Control Idiotype
400
300
Fragment C
200
100
Anti-Fr
0
Pre
0
10
20
30
Weeks from vaccination
Week 0 = 39 weeks post transplant

Improved transfection efficiency of muscle by electroporation
Control
+ Electroporation
Mathiesen, I. Gene Therapy 1999

Priming with electroporation amplifies antibody and CD8+ responses
Antibody responses
10000
8000
G
p.scFv-FrC+EP
Ig
x1
6000
CD8+ responses
rC
U/ml/
4000
x2
lls
titre
p.scFv-FrC
e
Anti-F
2000
c
5
x1
0
4
CD8+
010
20
30
40
50
3
tinge
2
ecr
4000
-s
1
p.scFv-FrC+EP
0
IFN
3000
1
1
x1
H
H
M
IgG
%
-A
O
-A
P
M
M
P
.D
p
E
U/ml
+
2000
O
O
E
/
x2
.D
+
p
.D
p
p.scFv-FrC
titre
1000
x1
Anti-BCL1
p=0.003
0
0
1020
3040
50
Day

Electroporation is effective in "prime/boost" for antibody induction
using p.scFv-FrC

Electroporation is effective in "prime/boost" for CD8+ T-cell induction
Rice et al
JI in press
Amplifies responses in rhesus macaques (Otten et al Vaccine 2004)
Pilot clinical trial of DNA fusion vaccine +/- electroporation against prostate cancer- 2005

Optimization of induction of CD8+ T-cell attack
Viral antigens? CMV
Mutated proto-oncogene peptides
Cancer-testis antigens:
MAGE-A3/NY-ESO-1/CT-7
CD8+
Translocation peptides
T cell
Autoantigens: hTERT
CYPIBI
Survivin
Idiotypic peptides
PRAME
Products of genomic instability
Minor histocompatibility antigens

Modification of vaccine design to induce peptide-specific CD8+ CTL
Anti-tumor responses
Surface
Antibody
or secreted
Dom1
Dom2
tumor antigen
CD4+ T cells
`Universal'
Competitive CTL-inducing motifs
T epitopes
(H-2b and HLA-A2)
H
Dom
CTL
Rice et al JI 2001
Rice et al JI 2002
Tumor-derived peptide sequence
Rice et al JI 2004

Application to patients? Pre-clinical testing of induction of HLA-A2-restricted CTL
against cytomegalovirus
HLA-A2/Kb transgenic mice
Dom
CMV-peptide: NLVPMVATV
Dominant peptide from pp65
p.DOM-CMV
1.2%
DNA vaccine
80
p.DOM-CMV
p.DOM
70
s
60
ysil 50
fic
40
p.DOM
0.13%
30
speci
20
8
%
10
CD
0
90 to 1
60 to 1
30 to 1
E/T ratio
IFN-
Day 35, ex vivo
Day 35, x1

Induced CD8+ T cells can kill target cells infected with MVA
expressing CMV-derived pp65 (N Khan).
Vaccine: p.DOM-CMV
Vaccine: pDOM
100
100
90
90
CMV peptide-pulsed
80
80
70
70
lysis
60
60
CMV MVA infected
50
50
40
40
specific% 30
30
20
20
Control MVA infected
10
10
0
0
80:1
40:1
20:1
80:1
40:1
20:1

p.DOM-CMV epitope fusion gene can generate the stimulatory CD8+ epitope in human DC
DCs transfected with control plasmid
DCs transfected with pDOM.CMV
Data.002
Data.003
0.07%
3.6%
100
101
102
103
104
100
101
102
103
104
FL1-H
FL1-H
Tetramer
CD8
DCs were transfected with RNA derived from either the pDOM.CMV vaccine or a control vaccine. They were then
co-cultured with autologous human CD8+ T cells for 6 days.
Pilot clinical trial of vaccination of allograft donors begun

Minor histocompatibility antigens as targets in myeloma
Cytotoxic T cells specific for HA-1 can kill primary myeloma cells
Holloway PA et al Leukemia 2004

The modified DNA fusion vaccine induces IFN- secreting CD8+ T cells against
human minor histocompatibility antigens HA-1 and HA-2
HLA-A2 transgenic mice
Dom
Dom
HA-1
HA-2
1.0 µ M HA- 2
1.0 µ M HA-1
0.1 µ M HA- 2
0.01 µ M HA-1
p.DOM-HA-1
no peptide
p.DOM-HA-2 0.01 µ M HA- 2
no peptide
1.0 µ M p30
1.0 µ M p30
1.0 µ M HA-1
1.0 µ M HA- 2
0.1 µ M HA- 2
ND
0.01 µ M HA-1
0.01 µ M HA- 2
ND
p.DOM
no peptide
p.DOM
no peptide
1.0 µ M p30
1.0 µ M p30
0
25
50
75
100
125
150
0
100
200
300
400
SFC / 106 splenocytes
SFC / 106 splenocytes

DNA-primed
CD8+ T cells CTL can
induced
lyse
against human
HA-1 or target
HA-2
cells
epitopes
expressing
kill
HA-1
human ta
or
rget
HA-2
cell lines endogenously
specifically
Dom
Dom
HA-1
HA-2
100
100
T2 + HA-1 peptide
T2 + HA-1 peptide
T2 + HA-2 peptide
T2 + HA-2 peptide
sis
75
JG EBV-LCL HA-1+
sis
75
RS EBV-LCL HA-2+
ly
ly
HR EBV-LCL HA-1+
c
AW EBV-LCL HA-2-
c
50
HD EBV-LCL HA-1-
50
specifi
25
specifi
25
%
%
0
0
100:1
50:1
5:1
100:1
50:1
5:1
E/T ratio
E/T ratio
Day 16, x3

Cancer-testis antigens in myeloma
Induction of CTL against MAGE A2 peptide using p.DOM-MAGE A2 epitope vaccine
c ells alone
700
p30-1µM
n
600
A2P1-10µM
illio
A2P1-1µM
500
m
A2P1-0.1µM
er
es
400
p
cyt
ts
o
o
n
300
le
sp
sp
200
N
IF
100
oN
0
p.D O M-A2P1- p.D O M-A2P1- p.D O M-A2P1- p.D O M-A2P1-
p.D O M 1
p.D O M 2
1
2
3
4
Vaccine
T2
110
100
T2/KMVELVHFL
lysis
90
ific
80
ecp
70
s%
60
50
40
30
20
10
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
60:
30:
10:
60:
30:
10:
30:
10:
60:
30:
10:
60:
30:
10:
60:
30:
10:
p.DOM-
p.DOM-
p.DOM-
p.DOM-
p.DOM 1
p.DOM 2
A2P1-1
A2P1-2
A2P1-3
A2P1-4
Vaccine

Cross-reactivity of CTL induced by MAGE A2-specific vaccine with MAGE A3 epitope
c ells alone
800
n
p30
700
A3P1-10
illio
600
A3P1-1
rm
es
A3P1-0.1
500
pes
cyto
400
n
pot
le
300
s
sp
200
NFI
100
oN
0
p.D OM-
p.D OM-
p.D OM-
p.D OM-
p.D OM 1
p.D OM 2
A3P1-1
A3P1-2
A3P1-3
A3P1-4
Vaccin e
100
T2
T2/KVAELVHFL
80
A2 epitope: KMVELVHFL
60
A3 epitope: KVAELVHFL
sisly
ic
40
specif
20
%
0
1
1
1
1
1
1
1
1
1
1
1
1
0:1
0:1
0:1
0:1
0:1
0:1
50:
10:
50:
10:
50:
10:
50:
10:
50:
10:
50:
10:
10
10
10
10
10
10
-20
p.DOM-
p.DOM-
p.DOM-
p.DOM-
p.DOM 1
p.DOM 2
A3P1-1
A3P1-2
A3P1-3
A3P1-4
E:T ratio

What is the immune status of patients with myeloma?
What should be the clinical setting for DNA vaccination?
Post-ASCT?
Can these patients respond to a conventional vaccine (tetanus toxoid)?

Antibody and T-cell proliferative responses post vaccination with TT

T-cell cytokine responses post vaccination with TT

Immune capacity recovers by 12 months post transplantation
1000
ns
ns
ns
1000
ns
100
l)
p=0.0007
p=0.0009
100
p=0.0012
p=0.0002
l)
/m
10
U
/m
10
U
(I
1
T
(I
1
T
-T
-T
ti
0.1
ti
n
0.1
n
A
A
0.01
0.01
0.001
0.001
e
e
e
e
st
pr
pr
ost
pr
post
ost
p
po
hs
s pr
th
ths
ont
nths
on
ths
ths
onths p
on
onths
mo
on
on
m
m
<12 m
<12
>12 m
>12
<20m
0m
<2
>20M
>20

T-cell responses post transplantation

Take home messages:
1.
DNA fusion vaccines activate selected immune pathways
able to attack myeloma cells.
2.
Patients with myeloma post autologous transplantation are
immune competent and donors of allogeneic transplants can be vaccinated.
3.
DNA idiotypic fusion vaccines induce persistent IFN-producing
CD4+ T-cell responses.
No evidence for deletional tolerance.
4.
More antigens are being tested: CTA/mucins/minor histocompatibility
antigens/CMV.
5. Opportunities to increase performance of DNA vaccines are available.

"Mix and match" DNA vaccine cassettes to induce immune attack on myeloma
MAGE-A3
Cell surface or secreted protein
Idiotypic Ig
NY-ESO
Survivin
Cytotoxic CD8+ T cells
Antibody
HA-1
CD4+ T cells
CMV

Jason Rice
Jo Roddick
Surinder Sahota
Jane Watkins
Gianfranco di Genova
Delin Zhu
Niklas Zojer
Francesco Forconi
Helen McCarthy
Andrew Duncombe
Kim Orchard
Christian Ottensmeier
Andy King
Feargal McNicholl
Nigel Russell

Tenovus
NHS R&D Hope WCT

Table1. Patient, disease and transplant details
Patient
Age
Sex
Subtype
Treatment prior to Best response
At time of vaccination
(years)
PBSCT (courses)
To PBSCT
Months
Lymphocyte IgG
* Paraprotein (g/dl)
Post PBSCT (x 10-9/l)
(g/dl)
/BJP (mg/24hrs)
141
M
IgAk
VAD(4)
CR
15
2
7.5
0
254
F
IgAk
CVAMP(4), ZDEX(2)
PR
18
2.9
7.8
2.1
355
F
IgAl
CVAMP ( )
PR
19
1.9
2.2
6.5
464
M
IgGk
CVAMP ( )
PR
20
1.1
5
0
552
M
IgGl
CVAMP ( )
PR
20
4.3
12
5.5
655
M
IgGk
CVAMP (4)
PR
23
1.8
12
1.3
766
F
BJP
CVAMP (5)
PR
31
2.2
8.9
354 BJP
867
M
IgGk
CVAMP (5)
CR
31
1.6
10.2
0
969
M
IgGk
CVAMP (4)
CR
35
2.2
9.2
0
10
63
F
IgGl
CVAMP (5)
PR
35
1.8
12
0
11
61
F
BJP
CVAMP ( )
PR
56
1.4
8.4
0
12
63
M
IgGk
CVAMP (6)
PR
57
1.5
9.9
0
13
57
F
IgGl
CVAMP ( )
PR
64
3.6
38.8
24.9
14
58
F
IgGk
CVAMP(6), local RT
No change
64
1.9
12.2
na
15
53
M
IgGk
CVAMP ( )
PR
81
3.2
9.8
0
CVAMP (cyclophosphamide,vincristine, adriamycin and
- methyl prednisolone as per MRC myeloma VII trial), VAD (vincristine,
adriamycin
, dexamethasone), ZDEX (idarubicin and dexamethasone) *using standard serum or urine electrophoresis
CR=complete response PR=partial response na=not available

P1A/AB specific CTL kill parental tumor cells in vitro and protect against
tumor challenge in vivo.
Vaccine: pDOM-AB
pDOM
pDOM-AB
100
100
pDOM
P815
Naïve
P511
80
80
P1204
s
60
60
Lysi
fic
eci
Survival
p
40
40
S
%
%
20
20
0
0
40:1
20:1
5:1
40:1
20:1
5:1
0
20
40
60
80
100
120
E:T Ratio
Days after tumor challenge

Cytotoxic T cells specific for HA-1 can kill myeloma cell lines
Holloway PA et al Leukemia 2004

Immune attack on myeloma cells via CD4+ T cells
DNA vaccination
CD4+
T cell
Secreted or shed antigens

Immune responses of patients to tetanus toxoid
100
10
l)
/m
1
Antibody
(IU
G
0.1
Ig
TT
0.01
0.001
1000
100
10
CD4+ T cells
SI
1
0.1
0.01
0.001
Pre
Post
Pre
Post
Pre
Post
Normal
MGUS
Myeloma
Poster 413:McNicholl et al

1000
ns
ns
100
l)
p=0.0007
p=0.0009
m
10
U/
(I
1
T
-Tti
0.1
An
0.01
0.001
t
pre
os
re
ost
hs
s p
nt
hs p
nt
onth
ths p
on
0mo
0M
<2
0mo
0m
<2
>2
>2

Document Outline