Transcript Thymosin alpha 1 - Microbiologia TorVergata
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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 peptides for 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
a
1, a 28 amino acid peptide derived by cleavage of proT
a
1, 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 a 1 and polypeptide b4 from calf thymus.
J. Biol. Chem. 1979, 254:981-986)
Thymosin
α 1 1 5 10 Ac Ser Asp Ala Ala Val Asp Thr Ser Ser Glu Ile Thr Thr Lys Asp 15
• • •
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 Asn Glu Ala Glu Glu Val Val Glu Lys 20 28 25
BIOLOGICAL PROPERTIES OF THYMOSIN α 1
• • • • • • • •
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 CD 4 counts and reduces HIV titer; In combination with HAART increases PBMC sj TREC levels in AIDS patients.
Tumor 8 day Cy 9, 10, 11, 12 day 12 (-16) day T α 1 IFN α /IL-2
Mastino et al. Int J Cancer, 1992
Mastino et al. Int J Cancer, 1992
Murine Melanoma
T α 1 increases number of tumor-free mice 100 80 60 40 20 0 7 16 19 23 27 30 35 42 54 60 Days after tumor injection Control IFN + CY IFN + CY + ZDX (4 ug)
*
IFN + CY + ZDX (12 ug) IFN + CY + ZDX (120 ug)
* Equivalent to human dose
20% of mice still tumor-free after one year
Pica et al (1998) Anticancer Research 18: 3571-3578
Rat Colorectal Cancer
T α 1 decreases metastases 10 9 8 1 0 3 2 7 6 5 4 Control 5-FU
*
IL2 5-FU + IL2 5-FU + IL2 + ZDX
* p < 0.001 vs control or 5-FU alone
70 60 50 40 30 20 10 0 Control 5-FU
*
IL2 5-FU + IL2 5-FU + IL2 + ZDX
Silecchia et al (1999) Cancer Immunotherapy 48: 172-178
Mechanisms: 1. Reduction of tumor load 2. Enhancement of tumor cell immunogenicity 3. Increase of immune effector cell activity (NK, CD4, CD8) Chemotherapy and T α 1 + IFN α T α 1 + IL-2
1992 1993
Thymosin α 1 and MHC class I expression Giuliani et al. Eur.J.Immunol., 2000
Effect of T
a
1 on CEA antigen expression WiDr untreated cells WiDr cells treated with T
a
1 (50 ng/ml) for 48 hrs
Cancer Cells T α 1 increases expression of tumor associated antigens
Control T α 1 50ng/ml T α 1 100ng/ml
Sinibaldi-Vallebona et al (2002) XXXth Meet Int Society for Oncodev Biology and Medicine
EFFECT OF THYMOSIN α 1 ON EXPRESSION OF INFLUENZA A VIRUS ANTIGENS (a) Uninfected MDCK cells (b) Infected MDCK cells (c) Infected MDCK cells treated with thymosin α 1 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 MHC-I B-7 TAA Etc.
NK CD8 APC CD4 Enhancement of tumor cell target expression:
IFNa, IFNg, Ta1, Chemotherapy, etc.
Immunotherapeutic strategies:
Cytokines, adoptive immunity, cells
tumor-bearing mouse
CTX-assisted therapeutic vaccination (Tumor cell lysate + type I IFN)
Day 0 Day 1 CTX Vacc. + IFN
25,0 20,0 15,0 10,0 5,0 0,0 7 10 (RBL-5) (0%) ctr 13 16 19 22 25 Days after CTX 28 31 (0%) CTX (0%) vaccine (40%) (80%) Vacc+IFN CTX+IFN+vac (Survival) 34
Proietti et al, 2004
Antiviral activity of Thymosin
α
1 against Influenza viruses
T
α
1 increases survival in murine influenza model
50 40 30 20 10 0 in fe ct ed c on tro l A M N * Ta 1 IF N A M N + IF N A M N + T a1 Ta 1 A + M IF N N + IF N + T a1 * p<0.001 against all other groups
Effect of combination treatment on viral titer in the lung
Group IC AMN Ta1 IFN AMN+T a 1 AMN+IFN T a 1+IFN AMN+T a 1+IFN* CPE 50 (units/g of lung tissue) 3.64 ± 1.79 x 10 3 1.56 ± 0.97 x 10 3 3.62 ± 0.93 x 10 3 3.51 ± 1.12 x 10 3 1.85 ± 1.15 x 10 3 1.96 ± 0.84 x 10 3 2.50 ± 1.35 x 10 3 0.07 ± 0.07 x 10 3 % of Inhibition — 57 — — 50 47 31 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
E/T ratio 40:1 20:1 10:1 IC 11.2 ± 2.3
9.3 ± 1.5
6.1 ± 0.8
% Specific lysis AMN^ T a 1+IFN 16.1 ± 2.8
12.4 ± 0.4
9.3 ± 2.1
14.2 ± 1.8
11.1 ± 0.7
8.2 ± 1.3
AMN+T a 1+IFN* 28.4 ± 1.6
24.2 ± 2.6
18.2 ± 1.7
* p<0.001 vs IC and T a 1+IFN p<0.005 vs AMN ^ p<0.001 vs IC
Those results confirmed the capacity of T
α
1 to help the immune response toward influenza. Can T
α
1 exert its function also affecting the virus replication in target cells?
T α 1 decreases replication of SENDAI virus in MDCK
100 80 60 40 20 0 I * * * * * p < 0.05
10 -9 10 -8 10 -7 10 -6 T a 1 concentration 10 -5
T
α
1 inhibits influenza A virus replication in human pulmonary cell line NCI
20 18 16 14 12 * p=0.004
4 2 0 10 8 6 * I T a 1 10 m g/ml T a 1 100 m g/ml
Thymosin α 1 protects mice from invasive aspergillosis and increases the efficacy of Amphotericin B Thymosin
m
g/kg
a
Amphotericin B
m
g/kg 0 Median survival time 20 -- 100 200 400 -- 100 100 -- -- -- -- 4000 4000 -- Fungal growth (
m
g glucosamine/lung) 0 25 50
Thymosin α 1 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
α
1
TLR 1 TLR 2 TLR 3 TLR 4 TLR 5 TLR 9 TLR 6 TLR 7 TLR 8 TLR 10 cytoplasm MyD88 p38 NF-kB
Production of IL-12, IFN α
T helper-type 1 (T H 1) 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 versus DTIC plus thymosin
a
IFN plus thymosin
a
1
a
1 in patients with advanced-stage metastatic malignant melanoma on 326 patients.
Melanoma T
α
1 + IFN
Melanoma
T α 1 + DTIC + IFN – study design
• Design - 20 patients - Treatment cycles: • DTIC (200 mg/m 2 iv) days 1 - 4 • T a 1 (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
T α 1 + DTIC + IFN - Results
Response rate Overall response
Complete Partial
No change Progression Response duration
Median Survival Time (MST) Median Time To Progression (TTP) Rasi et al (2000) Melanoma Research 10: 189 - 192
NUMBER 10 5 5 7 3 Months 11.5
5.5
% 50 25 25 35 15 Range 6 – 83+ 3 – 83+
Melanoma
T α 1 + DTIC + IFN – Immunological Parameters * p< 0.001
14 12
* DTIC/IFN/
Ta1 10 8 6 4 2 0 1/1 19/1 28/1 19/2 28/2 19/3 28/3 19/4 28/4 19/5 28/5 19/6 28/6
DTIC (historical control)
Days/cycle
Rasi et al (2000) Melanoma Research 10: 189 - 192
Melanoma
T α 1 + DTIC + IL-2 – study design
• Design - 46 patients - Treatment cycles: • DTIC (850 mg iv) day 1 • T a 1 (2 mg sc) days 4 – 7 • IL-2 (18 MU/m 2 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
T α 1 + DTIC + IL-2 - survival
120 100 80 60 40 20 0 0 5
Median survival = 11 months
10 15 Time (months) 20 25 30
Lopez et al (1994) Annals of Oncology 5: 741-746
Melanoma
T α 1 + DTIC + IL-2 – Immunological parameters
3000 2500 2000 1500 1000 500 0
* CD3+ * CD4+ * CD8+ * * * p< 0.001
* NK CD25+ HLA-DR Lopez et al (1994) Annals of Oncology 5: 741-746
EU MELANOMA PROGRAM
• Phase 2 trial (320 stage IV patients) - 4 arms • DTIC + IFN* + T a 1 (1.6 mg) • DTIC + IFN* + T a 1 (3.2 mg) • DTIC + T a 1 (3.2 mg) • DTIC + IFN* * Low dose IFN
Thymosin α 1 and Interferon Combination Therapy for Chronic Hepatitis C
End-of-Treatment PCR response * 40% n = 35 30% 20% n = 37 10% 0% IFN/TA1 IFN/Placebo
* 6 months Rx 75% genotype 1 n = 37
Placebo/Placebo K. Sherman, et al Hepatology 1998 27: 1128–1135
T
α
1 + 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 T α 1 in CD8+ human lymphocytes
DOWN UP Cytokines, Chemokines and Related Receptors Transcription Factors Adaptor and Signal Transduction Proteins Receptors IL-7 IL-12R b1 AMH R2 IL-6st AMH IL-19 LTB4R IL-9 IL-8Rb Rantes CXCR4 CCR10 mPIF-1 SCMb MIP1b MDC MIP1a NFATC2 STAT4 SP3 ELK-1 GATA3 GATA4 NFAT5 ATF-2 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 T a 1 T a 1+LPS 2,5 2 1,5 1 0,5 0
• 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 a 1 and other biological molecules.