Transcript Thymosin alpha 1 - Microbiologia TorVergata

NOTE
Questo materiale non può essere distribuito,
modificato o pubblicato né in forma cartacea,
né su un sito, né utilizzato per motivi pubblici o
commerciali.
E’ possibile utilizzare il materiale solo per motivi
personali e non commerciali, purché ogni copia
di questo materiale preservi tutti i diritti di
copyright e di proprietà intellettuale, sempre
dopo richiesta rivolta ai Docenti responsabili.
The thymus gland,
according to Chinese tradition, was
considered the site of the soul.
This tradition is still present among
Roman butchers where beef thymus is
sold as “animelle” .
The first attempts to determine the
nature of thymus function date back to AD
160, at the time at which Galen described
that:
“the thymus is maximal in size at or near
birth and involutes rapidly with age”.
In the past hundred years, there
were many reports describing the
importance of the thymus not only
in immunity, but also in:




skeletal growth
gonadal development
healing of fractures
maintenance of muscle tone
In the early ‘60s, the thymus came of
age with the seminal work on the
importance of the thymus in the
development of the lymphoid system
 Miller JF.: Immunological function of
the thymus.
Lancet (1961) 2:748-751
 Good RA. et al.: The role of the thymus
in development of immunological capacity
in rabbits and mice.
J. Exp. Med. (1962) 116:773-780
At the same time, seminal work done
by Allan Goldstein and colleagues
demonstrated that:
“thymic extracts could enhance the
in vivo incorporation of labelled
precursors into DNA and total
protein of mouse lymph nodes”
Proc. Natl. Acad. Sci. USA (1965) 53: 812-817.
In the early ‘70s, efforts were devoted to the
purification procedures to obtain large
quantities of the partially purified thymosin
fraction 5 (TF5)- a mixture of > 40 peptidesfor clinical use.
Hooper JA. et al.: The purification and
properties of bovine thymosin.
Ann. NY Acad. Sci. (1975) 249:125-144
Allan and colleagues subsequently (1966)
succeeded in the characterization of a product
from calf thymic tissue that stimulates
lymphoproliferation
Goldstein AL. et al.: Preparation,
assay and partial purification of a
thymic lymphocytopoietic factor
(thymosin)
Proc. Natl. Acad. Sci. USA (1966) 56: 1010-1017.
More than 25 biologically
active peptides from the
thymus gland have been
chemically characterized.
Thymosin a1, a 28 amino acid peptide
derived by cleavage of proTa1, was
first purified to homogeneity from TF5
Low TLK. et al., The chemistry and biology of
thymosins. I. Isolation, characterization and
biological activities of thymosin a1 and
polypeptide b4 from calf thymus.
J. Biol. Chem. 1979, 254:981-986)
Thymosin α1
1
10
5
Ac - Ser Asp Ala Ala Val Asp Thr Ser Ser Glu Ile Thr Thr
•
•
•
Lys
Asp
Originally isolated from thymus
–
The peptide responsible for reconstitution of immune
function in thymectomized animal models
Non-glycosylated, N-terminal acetylated, 28 aa
–
Highly conserved sequence (humans and various animal
species)
–
Comprises the N-terminus of prothymosin (113 aa)
Circulates at approximately 1 ng/ml (0.3 nM)
Leu
Lys
Glu
Lys
Lys
Glu
Asn Glu Ala Glu Glu Val Val
28
25
15
20
BIOLOGICAL PROPERTIES OF THYMOSIN
α1
In vitro Actions
•
Increase in production of interleukin-2, interferon a,
interferon g, and interleukin-7 in activated lymphocytes.
•
Enhanced expression of high-affinity interleukin –2 receptors.
•
•
Decreases terminal deoxynucleotidyl transferase (TdT)
activity in bone arrow precursors and splenic lymphocytes.
Induction of expression of phenotypic T-cell markers.
•
Enhancement of natural killer recruitment and lytic activity.
•
Antagonizement of steroid-induced apoptosis in immature
thymocytes.
•
Increases MHC Class 1 expression of lymphoid & non-lymphoid
cells.
•
Increases expression of CD3+4+T-Cells from CD34+ stem cells.
In vivo Actions
• Increases cytokine production, natural killer cell activity, and antitumor
activity in immunosuppressed animals.
• Enhancement of resistance to many viral, mycobacteria, and fungal
pathogens in immunosuppressed animals.
• Stimulation of viral clearance in woodchuck animal of hepatitis.
• Adjuvant
affects
restoring
immune
responses
in
aged
and
immunosuppressed animals.
• Increases MHC Class I expression
• Effective as a monotherapy in enhancing response rates in patients with
chronic active hepatitis B.
• In combination with interferon increases response rate in patients with
hepatitis C.
• Increase response rate and enhances survival in patients with lung cancer
following radiotherapy and in patients with melanoma following
chemotherapy.
• Increases antibody responses to influenza vaccine in normal elderly
volunteers and immunosuppressed kidney dialysis patients.
• In combination with AZT improves CD4 counts and reduces HIV titer; In
combination with HAART increases PBMC sj TREC levels in AIDS patients.
Tumor
8 day
9, 10, 11,
12 day
Cy
Tα1
12 (-16)
day
IFNα/IL-2
Mastino et al. Int J Cancer, 1992
Mastino et al. Int J Cancer, 1992
Murine Melanoma
Tα1 increases number of tumor-free mice
Control
% Tumor-free mice
100
IFN + CY
IFN + CY + ZDX (4 ug) *
80
IFN + CY + ZDX (12 ug)
60
IFN + CY + ZDX (120 ug)
40
* Equivalent to human dose
20
20% of mice still tumor-free
after one year
0
7 16 19 23 27 30 35 42 54 60
Days after tumor injection
Pica et al (1998) Anticancer Research 18: 3571-3578
Rat Colorectal Cancer
Tα1 decreases metastases
* p < 0.001 vs control or 5-FU alone
70
60
9
8
% Liver Invasion
Extrahepatic Metastases
10
7
6
5
4
3
50
40
30
*
20
2
*
1
0
10
0
Control
5-FU
IL2
5-FU + IL2 5-FU + IL2
+ ZDX
Silecchia et al (1999) Cancer Immunotherapy 48: 172-178
Control
5-FU
IL2
5-FU + IL2 5-FU + IL2
+ ZDX
Mechanisms:
1. Reduction of tumor load
2. Enhancement of tumor cell
immunogenicity
3. Increase of immune effector
cell activity (NK, CD4, CD8)
Chemotherapy
and
Tα1 + IFNα
Tα1 + IL-2
1992
1993
Thymosin α1 and MHC class I expression
Giuliani et al. Eur.J.Immunol., 2000
Effect of Ta1 on CEA antigen expression
WiDr untreated cells
WiDr cells treated with Ta1 (50 ng/ml)
for 48 hrs
Cancer Cells
Tα1 increases expression
of tumor associated antigens
Control
Tα1 50ng/ml
Tα1 100ng/ml
Sinibaldi-Vallebona et al (2002) XXXth Meet Int Society for Oncodev Biology and Medicine
EFFECT OF THYMOSIN α1 ON EXPRESSION OF
INFLUENZA A VIRUS ANTIGENS
(a)
(b)
Uninfected MDCK cells
(c)
Infected MDCK cells
Infected MDCK cells treated
with thymosin α1
MDCK cells were infected with Influenza A PR8 virus. Thymosin a1 (100 mg/ml) was
added 24 hours before infection and maintained in the culture medium during 24 hours
after infection. The presence of viral antigens was detected by immunofluorescence,
24 hours after infection using goat anti-influenza A virus (H1N1) antibody.
Tumor
cell
CD8
MHC-I
B-7
TAA
NK
APC
Etc.
CD4
Enhancement of tumor cell target
expression:
IFNa, IFNg, Ta1, Chemotherapy, etc.
Immunotherapeutic strategies:
Cytokines, adoptive immunity,
pulsed APC, vaccines, engineered
cells
CTX-assisted therapeutic vaccination
Mean Tumor Diameter (mm)
tumor-bearing
mouse
(Tumor cell lysate + type I IFN)
Day 0
Day 1
CTX
Vacc. + IFN
25,0
(RBL-5)
20,0
(0%)
(0%)
CTX
(0%)
vaccine
ctr
15,0
10,0
(40%) Vacc+IFN
(80%) CTX+IFN+vac
5,0
(Survival)
0,0
7
10
13
16
19
22
25
Days after CTX
28
31
34
Proietti et al, 2004
Antiviral activity of Thymosin α1
against Influenza viruses
Tα1 increases survival
in murine influenza model
Survival (%)
50
*
40
30
20
10
IF
N
+
Ta
1
IF
N
1+
AM
N
+
Ta
AM
N
+
Ta
1
IF
N
+
N
1
Ta
N
AM
IF
N
AM
in
fe
c
ted
co
nt
ro
l
0
* p<0.001 against all other groups
Effect of combination treatment
on viral titer in the lung
Group
CPE50
(units/g of lung tissue)
% of Inhibition
IC
3.64 ± 1.79 x 103
—
AMN
Ta1
IFN
AMN+Ta1
AMN+IFN
Ta1+IFN
AMN+Ta1+IFN*
1.56 ± 0.97 x 103
57
3.62 ± 0.93 x 103
—
3.51 ± 1.12 x 103
—
1.85 ± 1.15 x 103
50
1.96 ± 0.84 x 103
47
2.50 ± 1.35 x 103
31
0.07 ± 0.07 x 103
98
* p<0.001 vs IC , p<0.01 vs AMN
Effect of chemoimmunotherapy
on virus-specific cytotoxic T-lymphocyte
(CTL) response in the lung
% Specific lysis
E/T ratio
IC
40:1
11.2 ± 2.3
20:1
9.3 ± 1.5
10:1
6.1 ± 0.8
AMN^
Ta1+IFN
16.1 ± 14.2 ± 1.8
2.8
12.4 ±
11.1 ± 0.7
0.4
9.3 ± 2.1 8.2 ± 1.3
AMN+Ta1+IFN*
28.4 ± 1.6
24.2 ± 2.6
18.2 ± 1.7
* p<0.001 vs IC and Ta1+IFN p<0.005 vs AMN
^ p<0.001 vs IC
Those results confirmed the
capacity of Tα1 to help the
immune response toward
influenza.
Can Tα1 exert its function also
affecting the virus replication
in target cells?
Virus yield (% control)
Tα1 decreases replication of
SENDAI virus in MDCK
100
* p < 0.05
*
80
*
60
*
40
*
20
0
I
10 -9
10 -8
10 -7
10 -6
Ta1 concentration
10 -5
Tα1 inhibits influenza A virus replication
in human pulmonary cell line NCI
20
18
* p=0.004
16
Virus Yield
HAU/ml
14
12
10
*
8
6
4
2
0
I
Ta1 10
mg/ml
Ta1 100
mg/ml
Thymosin α1 protects mice from invasive
aspergillosis and increases the efficacy of
Amphotericin B
Thymosin a1
mg/kg
Amphotericin B
mg/kg
---
4000
100
4000
100
---
*
---
50
*
400
25
**
*
---
0
*
200
>60
40
*
---
**
*
100
20
*
---
0
*
---
Median survival time
Fungal growth
(mg glucosamine/lung)
Thymosin α1
promotes IL-12
production by
human dendritic
cells in response to
Aspergillus conidia
Blood 2004, Epub ahead of print
Sites of immunomodulation
1
2
3
4
Antigen recognition
Antigen presentation
Production of cytokines
Proliferation and differentiation
The Toll-pathway of thymosin α1
TLR 9
TLR 6 TLR 7 TLR 8 TLR 10
TLR 1 TLR 2 TLR 3 TLR 4 TLR 5
cytoplasm
MyD88
p38
NF-kB
 Production of IL-12, IFN-α
 T helper-type 1 (TH1)
differentiation
nucleus
Instructive immunotherapy with
Ta1 could be used to control
Inflammation
Immunity
Tolerance
Protocol ST1472-DM-01-012
A phase II, multicenter, open, randomized study to investigate the efficacy of
combination therapy containing DTIC plus low dose aIFN plus thymosin a1
versus DTIC plus thymosin a1 in patients with advanced-stage metastatic
malignant melanoma on 326 patients.
Melanoma
Tα1 + IFN
Melanoma
Tα1 + DTIC + IFN – study design
• Design
- 20 patients
- Treatment cycles:
• DTIC (200 mg/m2 iv) days 1 - 4
• Ta1 (1 mg sc) days 8 – 11 and 15 - 18
• IFN (3 MIU im) days 11 and 18
- Cycle repeated every 28 days (up to 9 cycles)
• Primary Endpoints
- Survival
- T-cell subsets
Rasi et al (2000) Melanoma Research 10: 189 - 192
Melanoma
Tα1 + DTIC + IFN - Results
Response rate
NUMBER
%
Overall response
10
50
Complete
5
25
Partial
5
25
No change
7
35
Progression
3
15
Months
Range
Median Survival Time (MST)
11.5
6 – 83+
Median Time To Progression
(TTP)
5.5
3 – 83+
Response duration
Rasi et al (2000) Melanoma Research 10: 189 - 192
Melanoma
Tα1 + DTIC + IFN – Immunological Parameters
* p< 0.001
NK Activity
14
*
DTIC/IFN/Ta1
12
10
8
6
DTIC
4
(historical
control)
2
0
1/1
19/1
28/1
19/2 28/2
19/3
28/3
Days/cycle
Rasi et al (2000) Melanoma Research 10: 189 - 192
19/4 28/4 19/5
28/5 19/6 28/6
Melanoma
Tα1 + DTIC + IL-2 – study design
• Design
- 46 patients
- Treatment cycles:
• DTIC (850 mg iv) day 1
• Ta1 (2 mg sc) days 4 – 7
• IL-2 (18 MU/m2 iv) days 8 – 12
- Cycle repeated every 3 weeks (up to 6 cycles)
• Primary Endpoints
- Survival
- T-cell subsets
Lopez et al (1994) Annals of Oncology 5: 741-746
Melanoma
Tα1 + DTIC + IL-2 - survival
120
% Survival
100
Median survival = 11 months
80
60
40
20
0
0
5
10
15
Time (months)
Lopez et al (1994) Annals of Oncology 5: 741-746
20
25
30
Melanoma
Tα1 + DTIC + IL-2 – Immunological parameters
3000
*
2500
* p< 0.001
Cells/mm3
2000
*
1500
*
*
1000
*
*
500
0
CD3+
CD4+
Lopez et al (1994) Annals of Oncology 5: 741-746
CD8+
NK
CD25+
HLA-DR
EU MELANOMA PROGRAM
• Phase 2 trial (320 stage IV patients)
- 4 arms
•
•
•
•
DTIC + IFN* + Ta1 (1.6 mg)
DTIC + IFN* + Ta1 (3.2 mg)
DTIC + Ta1 (3.2 mg)
DTIC + IFN*
* Low dose IFN
Thymosin α1 and Interferon Combination
Therapy for Chronic Hepatitis C
End-of-Treatment PCR response *
40%
n = 35
30%
20%
n = 37
10%
n = 37
0%
IFN/TA1
IFN/Placebo
* 6 months Rx
75% genotype 1
Placebo/Placebo
K. Sherman, et al
Hepatology 1998 27: 1128–1135
Tα1 + PegIFN + Ribavirin
• Response definition for interim results
– Early Virological Response (EVR)
> 2 log drop in HCV RNA by PCR in 12 weeks
– ALT level reduction
• Overall Response
– 61.0 % (14/23) EVR
– 48.0 % (11/23) HCV-RNA negative
• Genotype 1 Response
– 60.0 % (12/20) EVR
– 50.0 % (10/20) HCV-RNA negative
Genes regulated by Tα1 in CD8+ human lymphocytes
DOWN
UP
IL-7
IL-12R b1
AMH R2
IL-6st
Cytokines,
Chemokines and
Related Receptors
AMH
IL-19
LTB4R
IL-9
IL-8Rb
Rantes
CXCR4
CCR10
mPIF-1
SCMb
MIP1b
MDC
MIP1a
Transcription
Factors
NFATC2
STAT4
SP3
ELK-1
GATA3
GATA4
NFAT5
ATF-2
Adaptor and Signal
Transduction
Proteins
Receptors
CALM-2
CDKN2B
SOCS1
TRAF-2
TRAF-3
IRAK-1
TLR-9
CD3 gamma
CYSLTR1
CD3 zeta
CD3 epis
-3,10
-2,10
-1,10
-0,10
Fold difference (log 2)
0,90
1,90
Change in transcriptional response
upon stimulation in CD8+
Cytokines, Chemokines and Related Receptors
3
Rantes
LTB4R
2
IL-19
1,5
0
Rantes
LTB4R
IL9
0,5
IL8Rb
1
IL19
Fold difference
2,5
Ta1+LPS
IL-9
IL-8Rb
Ta1
• Thymosin has all the requirements to be defined a
smart molecule, exerting different action according
to the different environment.
• Medicine some time forgets that the complex
network resident inside our body drives the
effects of some drugs.
• In this view we can distinguish between “stupid”
and “intelligent” drugs.
• Intelligent drugs act as regulatory agents in
relationship with the signals coming from inside.
• To this category belongs thymosin-a1 and other
biological molecules.