in situtargeting of antigen presenting cells within secondary lymphoid … · 2007. 5. 2. · this...
TRANSCRIPT
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In situIn situ targeting of antigen presenting cells within targeting of antigen presenting cells within secondary lymphoid organs as a means to control secondary lymphoid organs as a means to control
immune responsesimmune responses
Kent A. Smith, Kent A. Smith, XipingXiping Liu, Liu, ZhiyongZhiyongQiuQiu, and Adrian Bot, and Adrian Bot
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©2005 MannKind Corporation. All rights reserved. No copying or distribution of this material may be made without written consent of MannKind Corporation.
MannKind’s Active Immunotherapy Approach
Method of immunizationMethod of immunizationPlasmidPlasmid--prime, peptideprime, peptide--boost boost
Route of administration Route of administration IntralymphaticIntralymphatic targeted delivery of activestargeted delivery of actives
Independent control of signal 1 and 2Independent control of signal 1 and 2
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©2005 MannKind Corporation. All rights reserved. No copying or distribution of this material may be made without written consent of MannKind Corporation.
Model Actives: pSEM and Melan A 26-35 (ELAGIGILTV)
NO
OO
H NO
NO
NO
NO
NO
NO
NO
NO
O
NO
O
Melan A 26-35 ELAGIGILTV
pSEM3315 bp
2: Melan A minigene
3:BGH polyadenylation region
7:Nuclear Import Sequence
1:CMV Enhancer/promoter
5:PMB Orign of Replication
4:kanamycin resistance gene
6:ISS
MLLAVLYCL ELAGIGILTV YMDGTMSQV 31GILT… … CEPV96Tyr 1-9 Melan A 26-35A27L Tyr 369-377 Melan A 31-96
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©2005 MannKind Corporation. All rights reserved. No copying or distribution of this material may be made without written consent of MannKind Corporation.
Preclinical model: HHD-1 mice (Human HLA-A*0201 transgenic H-2Db-/- ß2m-/- double knockout mice)
HHD construct designed: Hβ2µ-Hα1-Hα2-mα3-mTM-mcyt
Construct injected into C57Bl/6 x SJL oocytes
Mice breed on H-2Db-/-X β2µ-/- background
Express A*0201 on somaticand BM cells
CTL repertoire selection on A*0201
Mount A*0201-restricted CTL responses
Pascolo et al, J Exp Med 185:2043 1997
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©2005 MannKind Corporation. All rights reserved. No copying or distribution of this material may be made without written consent of MannKind Corporation.
MHC Tetramers for the ex vivo quantification of cellular immune response
100
101
102
103
10410
0
101
103
104
102
CD8
Mel
an A
tetr
amer 19.5% Melan A Tet+
PE
ELAGIGILTVMHC complex
MART-1 (Beckman Coulter)
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©2005 MannKind Corporation. All rights reserved. No copying or distribution of this material may be made without written consent of MannKind Corporation.
More effective induction of immune responses byintra-lymphatic immunization
43 days post boost15 days post boost
Mel
anA
tetr
amer
(% o
f CD
8)
*Targeting the lymph Node yielded the most robust immune responses
ELA IN ELA SQ ELA SQ ELA INpSEM IN pSEM IN pSEM SQ pSEM SQpSEM IN pSEM IN pSEM SQ pSEM SQ Control
0
5
10
15
20
25
30
35
**
**
*
N=12 per Group
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©2005 MannKind Corporation. All rights reserved. No copying or distribution of this material may be made without written consent of MannKind Corporation.
Mel
anA
tetr
amer
(% o
f CD
8)
D DD D
1 4 15 18
P PD D
1 4 15 18
P PP P
1 4 15 18
More effective induction of immune responses by alternating plasmid (priming) with peptide (boost)
Day
0
1
2
3
4
5
6
Naïve control
Peptide/Peptide
DNA /DNA DNA /Peptide
N=12 per Group
DNA/DNA
Weeks Following Immunization
0 1 2 3 4 5 6 7 80.1
1
10
100
Peptide BoostCompletion of Immunization
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©2005 MannKind Corporation. All rights reserved. No copying or distribution of this material may be made without written consent of MannKind Corporation.
Refinement of the prime-boost regimen to maximize the expansion of antigen specific T cells
#392 #379 #400 #266 #37105
101520253035
#241 #370 #331 #399 #38905
101520253035 #391 #360 #332 #284 #369
05
101520253035
Animal Number
Priming Post peptide boost
A
B
C
1.4 +/- 0.7%
8.0 +/- 2.3%
21.8 +/- 3.2%
Mel
anA
tetr
amer
(% o
f CD
8)
D D
D DD D P P*
P P P P*
P P P P P P*
1 4 15 18 29 32 Day
* Immunization schedules: Plasmid DNA (D), Peptide (P)
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©2005 MannKind Corporation. All rights reserved. No copying or distribution of this material may be made without written consent of MannKind Corporation.
Enhanced response seen in lymphoid and non-lymphoid organs correlates with efficacy
1%
Control
36%
DNA/DNA/Peptide
Tota
l Lym
phoc
yte
Mel
anA
Ter
amer
(%)
Specific Lysis (%)
0 20 40 60 80 1000.01
0.1
1 Spleen
r2=0.87
0 10 20 30 40 50 60 70 800.01
0.1
1 Blood
r2=0.84
0 10 20 30 40 50 60 700.01
0.1
1 Lymph node
r2=0.81
0 10 20 30 40 50 60 70 800.01
0.1
1 Lung
r2=0.86
Blood
1% 32%LN
2% 27%Spleen
8% 47%Lung
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©2005 MannKind Corporation. All rights reserved. No copying or distribution of this material may be made without written consent of MannKind Corporation.
Clearance of human tumor cells in animals immunized by DNA priming – peptide boost
In vivo cytotoxicityControl
0%TET=2%
DNA/DNA/DNA
0%TET=11%
DNA/Pept/Pept DNA/DNA/Pept
79% 78%TET=31% TET=83%
CFSE
Confidential
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Mechanism of Action StudiesMechanism of Action Studies
Characterization of the phenotypic profile Characterization of the phenotypic profile and functional status of the T cell response and functional status of the T cell response induced by plasmidinduced by plasmid--priming and peptidepriming and peptide--boostboost
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©2005 MannKind Corporation. All rights reserved. No copying or distribution of this material may be made without written consent of MannKind Corporation.
Phenotypic analysis of Melan A 26-35 specific CD8+ T cells before and after ex vivo stimulation
100 101 102 103 104100
101
103
104
102
CD8
Mel
an A
tetr
amer 19.5% Melan A Tet+
CD8+/Tetramer -
CD8+/Tetramer +
Unstimulated
100 101 102 103 10410
0
101
103
104
102
CD62L
CD
44
27.2% 38.0%
34.3%
100 101 102 103 10410
0
101
103
104
102
CD62L
CD
4479.9% 19.4%
0.8%
Unstimulated
100 101 102 103 10410
0
101
103
104
102
CD62L
CD
45R
B
13.3%
18.9%
2.6%62.4%
100 101 102 103 10410
0
101
103
104
102
CD62L
CD
45R
B
61.2%
15.8%
0.4%15.3%
CD
62L
CD
62L
76.5% 0.1%
3.0%100 101 102 103 104
100
101
103
104
102
IFN-γ100 101 102 103 104
100
101
103
104
102
IFN-γ
CD
107α
1.0% 0.8%
0.7%
100 101 102 103 10410
0
101
103
104
102
IFN-γ
14.9%
27.8% 56.7%
IFN-γ100 101 102 103 104
100
101
103
104
102
CD
107α
26.5%
2.0%
43.1%
Melan A TET stimulated
Melan A TET stimulated
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©2005 MannKind Corporation. All rights reserved. No copying or distribution of this material may be made without written consent of MannKind Corporation.
Functional specific T cells rapidly acquired the expression of CD107a and IFN-γ following Melan A tetramer stimulation
Tetramer dilution
100 10 1 102 103 10 4100
101
103
104
102
26.5%
2.0%
43.1%
100 10 1 10 2 103 10 410
0
101
103
104
102
37.9%
0.9%
1.4%
100 10 1 102 10 3 10 4100
101
103
102
24.6%
1.2%
9.0%104
IFN-γ
1:1 1:10 1:100
CD
107α
A lower stimulating threshold for CD107a expression was observedfollowing limiting dilution of the Melan A tetramer (right panel).
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©2005 MannKind Corporation. All rights reserved. No copying or distribution of this material may be made without written consent of MannKind Corporation.
Control of the immune response by targeted lymph node delivery
Pro-inflammatory cytokines
0500
1000150020002500300035004000
Peptide /Peptide
DNA / DNA
DNA / Peptide
pg/m
l
IFN-γTNF-α
pSEM plasmid and/or Melan A 26-35 peptide(intra lymph node admin) Peptide-
stimulated splenocytes
ELISA, Luminex
Chemokines
0100020003000400050006000
pg/m
l
RANTESMIP-1 α
Peptide /Peptide
DNA / DNA
DNA / Peptide
Immune regulatory cytokines
0400800
1200160020002400
pg/m
l
TGF-βIL-10IL-5
Peptide /Peptide
DNA / DNA
DNA / Peptide
Dichotomy between T cell profile elicited by DNA priming or peptide priming
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©2005 MannKind Corporation. All rights reserved. No copying or distribution of this material may be made without written consent of MannKind Corporation.
Proposed mechanism of action of plasmid versus peptide priming
CD62L+ CD45RBhi CD44lo
Plasmid(priming)
Long-lived central and peripheral memory cells
CD62L-/+ CD45RBdim CD44hi
Peptide(boost)
CD62L- CD45RBdim CD44hi(IFNγ+, CD107a+)
Effector cells
CD62L- CD45RBlo CD44hi
Peptide(priming)
CD62L- CD45RBlo CD44hi(IL-10, TGF-β, IL-5)
Regulatory cellsPeptide(boost)
Naive
Priming with DNA and boosting with peptide achieves substantially higher immune responses (Th1), and is associated with an increase in the rate of responders and the magnitude of response
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©2005 MannKind Corporation. All rights reserved. No copying or distribution of this material may be made without written consent of MannKind Corporation.
Summary
Targeted delivery of plasmid primes a long Targeted delivery of plasmid primes a long lasting population of central and peripheral lasting population of central and peripheral memory cells, with significant expansion memory cells, with significant expansion capabilitycapabilityEffectorEffector cells resulting from plasmid prime cells resulting from plasmid prime ––peptide boost display a complex functional profilepeptide boost display a complex functional profileImmune signatures of DNA versus peptide are Immune signatures of DNA versus peptide are substantially differentsubstantially different
Independent control of signal 1 and 2 in context of Independent control of signal 1 and 2 in context of lymph node targeting may allow effective manipulation lymph node targeting may allow effective manipulation of the of the magnitudemagnitude and and profileprofile of immune responseof immune response
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©2005 MannKind Corporation. All rights reserved. No copying or distribution of this material may be made without written consent of MannKind Corporation.
Acknowledgements
Zhiyong QiuAni-Der SarkissianGene GirgisSayuri YatsuboHong Tan
Chih-Sheng ChiangZheng LiuNathalie KerteszAnna SolonionaSutao ZhuLiz Lantzy
Brenna MeisenburgRobb PagariganChristiana SandersVictor Tam
Xiping LiuJian GongLisa DoSean HongLudmila Krymskaya
Liping LiuAmy BaulandDiljeet JoeaKris Krishnan
Alfred Mann
Hakan EdstromAdrian Bot
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©2005 MannKind Corporation. All rights reserved. No copying or distribution of this material may be made without written consent of MannKind Corporation.
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©2005 MannKind Corporation. All rights reserved. No copying or distribution of this material may be made without written consent of MannKind Corporation.
Enhanced immunity following two therapeutic cycles: Rational for clinical protocol
05
1015202530354045
NaiveControls
DNA/DNA/DNAGroup 1
DNA/PT/PTGroup 2
DNA/DNA/PTGroup 3
Mel
anA
spe
cific
tetr
amer
per
cent
One Immunization Cycle Two Immunization Cycles
N=12 per Group
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©2005 MannKind Corporation. All rights reserved. No copying or distribution of this material may be made without written consent of MannKind Corporation.
Luminex multiplex cytokine analysis of sorted CD62L negative cells from Melan A 26-35 immunized animals
Presort Post sort
100 101 102 103 104100
101
103
104
102
CD62L100 101 102 103 104
100
101
103
104
102
Mel
an A
tetr
amer
Mel
an A
tetr
amer
55% 99.5%
CD62L
1
10
100
1000
10000
100000
IL-2 IL-4 IL-6 MIP-1α RANTES TNF- α
CD62L+ Naïve CD62L- Naïve CD62L+ immunized CD62L- immunized
Cyt
okin
e pr
oduc
tion
(pg/
ml)
(log)
GM-CSF IFN-γ IL-12
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©2005 MannKind Corporation. All rights reserved. No copying or distribution of this material may be made without written consent of MannKind Corporation.
CD62L negative population of Melan A immunized animals identified from CD8+ lymphocytes
0
10
20
30
40
50
60
70
80
90
100
pSEM ELA peptide
CD
62L
nega
tive
perc
ent
Melan A tetramer+/CD8+ populationCD8+ population
N=30 N=30
Melan A tetramerpercent – 3.5%
Melan A tetramerpercent – 3.6%
Animals immunized with 4 injections of pSEMplasmid (1mg/mL) or 4 injections of Melan A (A27L) peptide analogue (1mg/mL). CD62L negative population analysis performed on CD8+ and Melan A+/CD8+ populations.
A student’s t-test value of 4.74 was determined from the Melan A tetramer+/CD8+ population comparing the pSEM immunized group and the ELA peptide immunized group
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©2005 MannKind Corporation. All rights reserved. No copying or distribution of this material may be made without written consent of MannKind Corporation.
MannKinds Active Immunotherapy Approach
Route of Route of administration administration
IntralymphaticIntralymphatic vs. vs. subcutaneous subcutaneous injectioninjection
Method of Method of immunizationimmunization
Optimized protocol Optimized protocol in transgenic micein transgenic mice
Mechanism of Mechanism of ActionAction
Antigen specific Antigen specific effectoreffector cellscells
Conclusions
Cancer Cell
CD8+ T cell
Conclusions