in situtargeting of antigen presenting cells within secondary lymphoid … · 2007. 5. 2. · this...

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

©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


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


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


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)


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


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


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)


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


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

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


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


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).


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


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


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


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


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


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


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


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

in situtargeting of antigen presenting cells within secondary lymphoid … · 2007. 5. 2. · this...

Documents