Thymosin Alpha 1
From Bench to Bedside
ENRICO GARACI,a CARTESIO FAVALLI,a FRANCESCA PICA,a
PAOLA SINIBALDI VALLEBONA,a
ANNA TERESA PALAMARA,b CLAUDIA MATTEUCCI,c
PASQUALE PIERIMARCHI,c ANNALUCIA SERAFINO,c
ANTONIO MASTINO,d FRANCESCO BISTONI,e
LUIGINA ROMANI,e AND GUIDO RASIc
a University
of Rome Tor Vergata, Rome, Italy
b University
of Rome La Sapienza, Rome, Italy
c CNR,
Rome, Institute of Neurobiology and Molecular Medicine, Rome, Italy
d University
of Messina, Messina, Italy
e University
of Perugia, Perugia, Italy
ABSTRACT: After the initial dramatic effects, observed in a Lewis lung
carcinoma animal model, using a combination of thymosin alpha 1 (T1)
and interferon (IFN) after cyclophosphamide, a number of other preclinical models in mice (Friend erythroleukemia and B16 melanoma) and
in rats (DHD/K12 colorectal cancer liver metastasis) have confirmed
the efficacy of the combination therapy with T1 and either IFN or
IL-2 plus chemotherapy. These results provided the scientific foundation for the first clinical trials using T1 in combination with BRMs
and/or chemotherapy. Pivotal trials in advanced non-small cell lung
cancer (NSCLC) and melanoma with T1 and IFN- low doses after
cis-platinum or dacarbazine produced the first evidence of the high potentiality of this approach in the treatment of human cancer. The combination of T1 and IFN- was also used in patients affected by chronic
B and C hepatitis including IFN-nonresponders and infected by precore
mutants or genotype 1b. Further studies demonstrated additional biological activities clarifying the mechanism of action of T1, partially
explaining the synergism with IFN. It has been shown the capacity of activating infected dendritic cells through Toll-like receptor signaling, thus
influencing the inflammation balance, and of increasing the expression
of tumor, viral, and major histocompatibility complex (MHC) I antigens.
Dose–response studies suggested the possibility of improving the efficacy
of this molecule reducing the overall toxic. Based on these information
Address for correspondence: Guido Rasi, M.D., CNR, Institute of Neurobiology and Molecular
Medicine, Via Fosso del Cavaliere, 100, 00133, Rome, Italy. Voice: + 39-06-49934486; fax: + 39-06233249504.
guido.rasi@artov.inmm.cnr.it
C 2007 New York Academy of Sciences.
Ann. N.Y. Acad. Sci. 1112: 225–234 (2007). doi: 10.1196/annals.1415.044
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two clinical trials are ongoing: a large phase II on advanced melanoma
patients treated with T1 at different doses after dacarbazine and a
phase III one, on IFN-resistant hepatitis C virus (HCV) patients treated
with a triple combination (IFN, ribavirin, and T1).
KEYWORDS: thymosin alpha 1; preclinical models; clinical trials
INTRODUCTION
After 1985,1 when thymosin alpha 1 (T1) was first used in a clinical trial, an
impressive number of data has been produced supporting the potential clinical
benefits of this molecule. Since then a number of pivotal clinical trials have
been carried out for the treatment of cancer and chronic viral hepatitis using
T1 in combination with chemotherapeutic agents or with interferon (IFN).
The results, although partial, have always been encouraging and warranted
the actual ongoing phase II and phase III large trial on advanced melanoma
and on chronic C hepatitis for IFN-resistant patients.
The continuous progress of the knowledge about both, the mechanisms
of T1 action on one side, and the mechanisms of tumor progression
and viral persistence on the other, further supported the potentiality of
this molecule, also suggesting better treatment schedules and new possible
indications.
The effects described in many papers indicate T1 as a powerful molecule
able to activate or boost the immune system by many different ways. The most
relevant in vitro effects are listed in TABLE 1. These findings encouraged the
use of T1 as an adjuvant for cancer and infectious disease.
USE OF T1 IN CANCER MODELS
In 1990, our group2 demonstrated the possibility to completely eradicate
Lewis lung carcinoma in mice using T1, followed by murine IFN-/ after
cyclophosphamide. The schedule was to insert a cycle of immunotherapy consisting of 4 days T1 treatment followed by 1 day of IFN- (or / in mice
according to the availability) between two cycles of “standard” chemotherapy,
according to the scheme illustrated in FIGURE 1. This schedule of combination
treatment proved to be highly active in curing established tumors, whereas
single treatments were not effective.
The concept of combining a sequence of immunotherapy (T1 plus a cytokine) after chemotherapy was further developed on the assumption that
chemotherapy on the one hand was advantageous in reducing the tumor mass
but on the other induced a marked depression of the immune response. Moreover, the potential positive effects of powerful cytokines, such as IFN and
GARACI et al.
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TABLE 1. List of the most relevant in vitro effects of T1
Cytokines and receptors
–IL-12, IL-2 increase
–IFN- increase
–Expression of high-affinity IL-2R (CD25)
–IL-4 inhibition
T cell proliferation/differentiation
–Stimulates CD34+ stem cells into CD3 + 4+ cells
–Stimulates CD4+ and CD8+ cell proliferation
–Increases of CD4+ and CD45RO+ (memory T cells)
–Stimulates NK-cell activity
Effect on tumor/infected cells
–Increases MHC I
–Increases tumor antigen expression
–Increases GSH levels
FIGURE 1. Treatment schedule of combination therapies in cancer.
interleukin-2 (IL-2), could not fully be exploited because of the toxicity shown
at the effective doses. The combination therapy was aimed at obtaining a synergistic effect by combining T1 with immunoactive molecules at low doses,
thus reducing the overall toxicity of the treatment.
The results went beyond the expectation and encouraged further experiments. Similar results were obtained in other experimental models: Friend
erythroleukemia3 and B16 melanoma4 in mice and DHD/K12 colorectal carcinoma liver metastases in rats,5 as summarized in TABLE 2.
Many of the effects listed in the introduction may explain these results,
including the boosting of cellular immune responses. However, the increased
expression of major histocompatibility complex (MHC) I antigens6 along with
the increased tumor antigen expression7 might be the key point for this successful approach as shown in FIGURE 2. In fact, many strategies able to induce or
enhance a cytotoxic T-lymphocyte (CTL) response frequently fail to correlate
with the expected antitumor effect.
The experience in preclinical models was translated into the clinics, performing two clinical trials exactly reproducing the experimental model. Pivotal trials
in advanced non-small cell lung cancer (NSCLC)8,9 and in melanoma10 with
T1 and low doses IFN- after cis-platinum and dacarbazine, respectively,
produced the first evidence of the possibility to enhance the clinical response
rate and the survival time by this approach (TABLE 3).
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TABLE 2. Combination therapies in cancer preclinical models
Author/Ref
Year
Tumor
Garaci et al.2 1990 Lewis lung
carcinoma
Garaci et al.3
Rasi et al.5
Pica et al.4
Treatment
Responses
Cy+T1+
IFN-/
Cure/complete
regression NK
and CTL increase
1993 Friend
Cy+ T1+
Increased survival
erythroleukemia
IL-2/ IFN-/
NK and CTL
increase
1994 DHD/K12 CRC
5-FU + T1+
Increased survival
liver metastases
IL-2
reduced
metastatic spread
NK and CTL
increase
1998 B16 melanoma
Cy + T1 +
Increased survival
IFN-
T1 dose–
response effect
Animal
model
Mice
Mice
Rats
Mice
Cy = cyclophosphamide; 5-FU = 5 fluorouracil; CRC = colorectal cancer.
FIGURE 2. Effect of T1 on CEA antigen expression in WiDr colorectal cancer cell
lines. T1 is able to induce CEA overexpression making tumors more visible to lymphocytes.
(A) Untreated WiDr cells; (B) WiDr cells treated with T1 (50 ng/mL) for 48 h.
USE OF T1 IN INFECTIOUS DISEASES
Cancer models demonstrated the powerful action of T1 on the immune
system and the synergy between T1 and IFN suggesting a possible use in
infections. T1, besides being a potent immune modulator itself, showed a
synergistic action with many cytokines (IFN-, IL-2, IFN- ). The abovementioned possibility to upregulate MHC class I antigen expression, by a
different pathway from the IFN, provided the first molecular explanation for
the synergy. This also disclosed a new action of T1, that is, the ability to
act on cells other than the lymphoblastoid cells. Further experiments, in fact
demonstrated the possibility to upregulate viral antigens expression both in
vitro and in vivo (Palamara, personal data). Furthermore, a direct antiviral
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TABLE 3. Clinical experiences with T1 combination therapy in cancer patients
Author/ref
Garaci et
al.8
Year Tumor
Treatment
Responses
(range)
1995 NSCLC
DDP/VP-16 +
T1+IFN
43% RR
15.7 MST
IFO + T1+
IFN
10% RR –
TP 9 (6/18)
33% RR –
TP 18 (9/53)∗
25% CR – 50% Expected ∼25%
RR 11.5 MST
for matching
population
Salvati et al.9 1996 NSCLC
Rasi et al.10
2000 Melanoma DTIC + T1+
IFN
Note
Reduced
toxicity for
combination
Expected ∼20%
for matching
population
CR = complete response; TP = time to progression (weeks); DDP = cisplatinum; VP-16 =
etoposide; RR = response rate; IFO = ifosfamide; MST = median survival time (months); DTIC =
decarbazine. ∗ P = 0.0059.
action was demonstrated in a model of influenza virus infection and woodchuck
hepatitis.11
Based on these data, and with the experience of a preclinical cancer models,
a pivotal phase II trial was performed using T1 in combination with low dose
of lymphoblastoid interferon- (L-IFN-) in chronic hepatitis B patients. The
overall sustained response rate, normal enzyme (ALT) and negative HBV-DNA
6 months after the end of treatment, was greater than expected with IFN alone
(46% versus 30%). However, the most important finding was the number of
responses observed after the end of treatment whereas a significant number
of relapses are what generally takes place. The response raised up to 55%
after 18 months and a sustained response12 was also observed in “difficultto-treat patients,” such as previous IFN failures and/or precore mutants. CTL
and NK enhancement surely play a role in these results, but, once again, the
hypothesis of the increased expression of MHC I antigens could be particularly relevant, given the specific lack of MHC class I antigens expression in
HBV-infected hepatocytes. This trial provided the first evidence of the effectiveness of the combined approach and was followed by many other trials13–15
(TABLE 4) that confirmed the initial observations and gave some important indication: evidence of the “late response,” unique of T1 and opposite to IFN,
the response also in “IFN nonresponders patients” and in precore mutants, and
the dose–response effect (TABLE 5).16–19
After the positive experience on hepatitis B, a pivotal trial with recombinant
IFN- and T1 was performed in patients affected by chronic C hepatitis.20 In
this trial were included IFN failures and genotype 1b patients. The results gave
an encouraging 40% sustained response compared to an expected 15–29%
with IFN alone; opening new hope for these diseases also confirmed from
other trials (TABLE 6).21–23
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TABLE 4. Clinical experiences with T1 combination therapy in chronic hepatitis B
Treatment
and duration
Author/ref
Year
Rasi et al.12
1996 T1+L-IFN-
(26 weeks)
End of
treatment
Follow- up %
Pts n % of responses
52/78 w
End points
15
40 (a)
53/60
ALT/DNA
HBsAg
Saruc et al.13 2002
T1+IFN (c)
(52 weeks)
21
87.7 (b)
76
ALT/DNA
Saruc et al.14 2003
T1+IFN (c)
IFN + 3TC (d)
IFN (52 weeks)
27
15
10
74
53
40
70/71
26/20
20/10
ALT/DNA
T1 + 3TC
20
Not reported
70
ALT/DNA
Chan et al.15
2001
Pts = patients.
(a) including IFN failures; HBeAg+; (b) Anti HBe+ patients/naı̈ves; (c) IFN for 52 weeks; (d)
Lamivudine 3TC for 78 weeks.
TABLE 5. Further clinic experiences in HBV difficult-to-treat patients
Author/ref.
Andreone et al.16
Zhuang et al.17
You et al.18
Iino et al.19
Year
∗
Treatment
(6 months/
1.6 BW)
1996
T1 vs. IFN
2001 T1 vs. IFN∗
2006
T1 vs. IFN
2005 T1 0.8 vs. 1.6∗∗
End of
treatment %
Pts. n of responses
Follow
up %
End
points
33
29 vs. 44
48
33 vs. 46
62
31 vs. 45
316 Not reported
41 vs. 25
55 vs. 23.3
48 vs. 27
25 vs. 30
ALT/DNA
ALT/DNA
ALT/DNA
ALT/DNA
Pts = patients
∗ Anti HBe+ patients;
∗∗ including IFN failures.
A number of hypotheses have been raised about the possible mechanism
of action and the most recent findings indicate the dendritic cells and TNF-
as two major players in promoting liver injury. Recent works demonstrated
the ability of T1 to activate macrophage Th1 responses, through Toll-like
receptor signaling, and to promote a regulatory environment for balance of
inflammation and tolerance,24,25 and to downregulate macrophages TNF-
release (Serafino 2006, personal data; FIG. 3). Taken together these findings
better explain the possible role of thymosin in hepatitis C virus (HCV) chronic
infection.
The pleiotropic function of T1 is well documented by recent studies
on transcriptional response to this molecule, performed in human peripheral blood mononuclear cells (PBMCs) using the gene-array technology.
By means of this experimental approach T1 has been actually shown to
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TABLE 6. Clinical experiences with T1 combination therapy in chronic hepatitis C
Author
Year
Rasi et al.20
1996
Sherman et al.21
Andreone et al.22
Poo et al.23
Treatment
and duration
T1+ L-IFN
52 weeks
1998 T1+IFN2b vs. IFN2b
26 weeks
2003 T1+IFN2b vs. IFN2b
26 weeks
2004 T1+IFN2b+Ribavirin
24 weeks
Pts n
Responses
(%) E.T. F.U. End points
15
73
40
ALT/RNA
35
37
22
19
23
37
19
64
32
39
14
8
32
21
ALT/RNA
ALT/RNA
RNA
Pts = patients; E.T. = end of treatment response; F.U. = follow-up. End points: normal ALT; RNA
nondetectable by PCR.
FIGURE 3. Effect of T1 on TNF- release from human macrophages. T1 induces a
downregulation of TNF- release from human macrophages. TNF- production after 24 h
T1 in vitro treatment (ELISA).
upregulate the expression of a plethora of genes related to innate response and
to growth factors, cytokines, and chemokines.26 Part of these results is reported
in FIGURE 4.
The clinical experience is now going to receive a significant contribution
by two ongoing major clinical trials. The first is related to the treatment of
HCV chronic patients which resulted nonresponders to IFN treatment. Those
patients for whom there is no hope of cure, IFN retreatment is the only option
with a disappointing response rate of about 5–10%. This study is a phase III
trial using a triple combination of T1 plus ribavirin plus pegylated IFN, on the
basis of all the knowledge achieved in terms of mechanism and combinations
of those molecules.
The second is a first-line phase II study on 500 patients with advanced
melanoma using dacarbazine followed by T1 plus low dose of IFN. Different arms using high-doses of T1 are included to comply with the overall
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FIGURE 4. Genes regulated by T1 in human lymphocytes. Human PBMCs from
healthy donors were cultured in the presence of T1 at 1 g/mL for 48 h and transcriptional
response was assayed by gene-array technology. Results for some upregulated genes are
reported and expressed as fold change compared to the untreated sample.
clinical and experimental experience in melanoma, where a dose effect was
documented. In the future it is reasonable to expect further use of this molecule,
due to the continuous improvement in the knowledge of different mechanisms
and interactions.
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