This document summarizes a presentation on low dose cytokines in supportive cancer therapy. It discusses how low dose cytokines from GUNA S.p.a, such as IL-4, IL-12, and IFN-γ at concentrations of 10 fg/ml, can enhance the antigen presentation and stimulatory activity of dendritic cells obtained from early stage colorectal cancer patients. An ex vivo study found that sequential exposure of these patient's dendritic cells to low doses of IL-4 and IL-12 increased their ability to stimulate T-cell responses compared to treatment with standard doses of cytokines. The presentation concludes that GUNA's approach using low dose cytokines may provide a novel integrated therapy to activate the immune system in cancer.

1. Future Health Summit
LOW DOSE CYTOKINES IN SUPPORTIVE THERAPY FOR CANCER
From research: Up to date pre-clinical evidence
*****
DUBLIN
May 26th, 2016
Alessandro Perra – Scientific Director of GUNA S.p.a.

3. Low Dose Medicine into the Universities
MASTER BIENNALE DI II LIVELLO
LOW DOSE MEDICINE IN
PSICO-NERUO-ENDOCRINO-
IMMUNOLOGIA
Applicazioni in Medicina Generale,
nelle Disfunzioni
Neuro-Muscolo-Scheletriche e
nell’ Healthy Aging
© Dipartimento Scientifico Guna S.p.a.

5. LOW DOSE MEDICINE
Low Dose Medicine integrates state of the art scientific
advances in:
P.N.E.I.
Molecular
Biology
Nano-
Pharmacology
linked to
Quantum
Physics
© Dipartimento Scientifico Guna S.p.a.

6. BIO-
REGULATING
PHYSIOLOGY
LOW DOSE
CYTOKINES
LOW DOSE
NEURO
PEPTIDES
LOW DOSE
HORMONES
LOW DOSE
GROWTH
FACTORS
LOW DOSE MEDICINE
A NOVEL, INTEGRATED, SYSTEMIC APPROACH TO DISEASES
© Dipartimento Scientifico Guna S.p.a.

7. CENTRAL NERVOUS SYSTEM
&
AUTONOMIC SYSTEM
IMMUNE SYSTEM
ENDOCRINE SYSTEM
HOMEOSTATIC Control Systems and
bi-directional cross-talk
Ader, R., Psychoneuroimmunology, IV edition, vol. 1 e 2, Academic Press, Amsterdam 2007.
It is the classical text on the matter, pubblished for the first time in 1981.
P
N
E
I
© Dipartimento Scientifico Guna S.p.a.

8. STRESS AXIS
Th1
Th2
Reduction of
IFN-γ
X
CD8+
Tc
Not correct
stimulation of
Ts
© Dipartimento Scientifico Guna S.p.a.
•Cohen M, et al. Increased emotional distress in daughters of breast cancer patients is associated with decreased natural cytotoxic activity, elevated
levels of stress hormones and decreased secretion of Th1 cytokines. Int J Cancer. 2002;100(3):347-54.
•Montoro J, et al. Stress and allergy. J Investig Allergol Clin Immunol. 2009;19 Suppl 1:40-7.
•He Y, et al. Psychological stress exerts effects on pathogenesis of hepatitis B via type-1/type-2 cytokines shift toward type-2 cytokine response. PLoS
One. 2014;9(8):e105530.
•Radice E, et al. Low-doses of sequential-kinetic-activated interferon-gamma enhance the ex vivo cytotoxicity of peripheral blood natural killer cells
from patients with early-stage colorectal cancer. A preliminary study Intern. Immunopharm 2014;19:66-73.
DOWN-REGULATION OF Th1

10. AN ALTERATED CROSS-TALK DUE TO AN
UNBALANCE* BETWEEN SIGNALING MOLECULES
PLAYS A CRUCIAL ROLE IN THE ONSET OF
INFLAMMATORY, ALLERGIC AND AUTO-IMMUNE
DISEASES
*ALTERATED CONCENTRATIONS
P.N.E.I. NETWORK AND SIGNALING MOLECULES
© Dipartimento Scientifico Guna S.p.a.

11. HEALTH
PHYSIOLOGICAL CONCENTRATION
DISEASE
DISEASE
HYPO-CONCENTRATION
HYPER-CONCENTRATION10-6
10-15
C O P E
Cytokines & Cells Online Pathfinder Encyclopedia
Version 26.7 (Spring 2011 Edition)
© Dipartimento Scientifico Guna S.p.a.

12. g (gram)= 1
10 -1 = 0.1
10 -2 = 0.01
mg (milligram)= 10 -3 = 0.001
μg (microgram)= 10 -6 = 0.000001
ng (nanogram)= 10 -9 = 0.000000001
pcg (picogram)= 10 -12 = 0.000000000001
fg (fentogram)= 10 -15 = 0.000000000000001
DEFINITIONS
© Dipartimento Scientifico Guna S.p.a.

14. The membrane receptor plays a KEY role.
ONLY physiological concentrations are able to
activate or reactivate the membrane receptors and
consequently, stimulate the physiological function
of a target cell.
14
UP- AND DOWN-REGULATION
- Akahoshi T et al. Interleukin 1 stimulates its own receptor expression on human fibroblasts through the endogenous
production of prostaglandin(s). J Clin Invest. 1988 Oct;82(4):1219-24
- Samanta AK et al. Interleukin 8 (monocyte-derived neutrophil chemotactic factor) dynamically regulates its own receptor
expression on human neutrophils. J Biol Chem. 1990 Jan 5;265(1):183-9.
© Dipartimento Scientifico Guna S.p.a.

15. CENTRAL NERVOUS SYSTEM
&
AUTONOMIC SYSTEM
IMMUNE SYSTEM
ENDOCRINE SYSTEM
HOMEOSTATIC Control Systems and
bi-directional cross-talk
Ader, R., Psychoneuroimmunology, IV edition, vol. 1 e 2, Academic Press, Amsterdam 2007.
It is the classical text on the matter, pubblished for the first time in 1981.
P
N
E
I
© Dipartimento Scientifico Guna S.p.a.

16. 16
Th-1/Th-2 BALANCE
Th2
Crohn’s Disease
Psoriasis
Vitiligo
…
Asthma
Atopy
…
Th1
TReg
Th1 UP-REGULATION
Th2 DOWN-REGULATION Th1 DOWN-REGULATION
Th2 UP-REGULATION
Th2
Th1
Th17
© Dipartimento Scientifico Guna S.p.a.

17. HEALTH
HYPER
HYPO
Cytokines UP
• IL-1
• IL-6
• TNF-α
• IL-17
• INF-γ
• IL-2
• IL-8
Cytokines DOWN
• TGF-β
• IL-4
• IL-10
Cytokines DOWN
• IL-1
• IL-6
• TNF-α
• IL-17
• INF-γ
• IL-2
• IL-8
Cytokines UP
• TGF-β
• IL-4
• IL-10
Physiological
concentration
• IL-1
• IL-6
• TNF-α
• IL-17
• INF-γ
• IL-2
• IL-8
Physiological concentration
• TGF-β
• IL-4
• IL-10
© Dipartimento Scientifico Guna S.p.a.

18. Raphael I et al. T cell subsets and their signature cytokines in autoimmune and inflammatory diseases. Cytokine (2014),
http://dx.doi.org/10.1016/j.cyto.2014.09.011
Neither good nor bad in Nature

19. PROBLEM
Is it possible to modulate the
action of cytokines and other
signaling molecules?
IF DISEASES ARE EXPRESSIONS, CONSEQUENCES OF
CHANGED CONCENTRATION OF MESSENGER MOLECULES…
© Dipartimento Scientifico Guna S.p.a.

20. Antagonistic cytokines are utilized in order
to slow down a biological effect; Same
cytokines in order to enhance a biological
function.
The concept of BALANCE and the use of SKA
low dose cytokines
© Dipartimento Scientifico Guna S.p.a.

21. Th0
Th2Th1
IL-12, INF-γ IL-4
INF-γ DOWN-REGULATES
IL-12 UP-REGULATES
IL-4 UP-REGULATES
IL-4 DOWN-REGULATES
Th subsets cross-regulate expansion and functions each other.
- Cooke , A. Th17 in Inflammatory Conditions. 2006, Rev Diabetic Stud 3: 72-7
- Bettelli E. et al. Th17: the third member of the effector T cell trilogy. Current Opinion in Immunology 2007, 19: 652-657
Inflammatory diseases Allergy
THE CONCEPT OF BALANCE – RECIPROCITY of TH CELLS
© Dipartimento Scientifico Guna S.p.a.

22. ©DipartimentoScientificoGunaS.p.a.
IL-10 4C

23. A novel, systemic,
integrated,
synergistic approach for
supportive therapy in
Cancer
© Dipartimento Scientifico Guna S.p.a.

24. © Dipartimento Scientifico Guna S.p.a.
D’Amico L, Ruffini E, Ferracini R, Roato I (2012) Low Dose
of IL-12 stimulates T Cell response in cultures of PBMCs
derived from Non Small Cell Lung Cancer Patients.
Journal of Cancer Therapy 3: 337-342.

25. Th0
Th2Th1
TReg
TGF-β
DOWN-REGULATES DOWN-REGULATES
Plasticity and Rigidity of
Relationship among Th1-Th2-Th3
© Dipartimento Scientifico Guna S.p.a.

26. © Dipartimento Scientifico Guna S.p.a.
1.7 1.7
0.9
4.5
0.8
0.6
0.4
1.8
0.2
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
basale CH3 CH4 placebo IL12 10ng/ml
CD4/Treg
day 5
day 10
healthy ctrl 1pg/ml 0.01pg/ml SKA basal 10 ng/ml

27. basale CH3 CH4 placebo IL12 10 ng/ml
Series1 30.7 19 48.4 48.3 4.8
0
10
20
30
40
50
60
%CD4/IFNgamma Day 5
CH3 CH4 placebo IL12 10 ng/ml
Series1 32.8 56 46.2 4.2
0
10
20
30
40
50
60
%CD4/IFNgamma
Day 10
© Dipartimento Scientifico Guna S.p.a.
IL-12 0.01pg/ml SKAIL-12 1pg/ml SKA

28. © Dipartimento Scientifico Guna S.p.a.

29. Legenda:
•Left column =
not-treated control group
•Central column =
patients treated with
conventional dosage of
IFN-gamma (1 ng/ml)
•Right column = patients
treated with IFN-gamma
GUNA (0.25 fg/ml)
Healthy
subjects
Non-
metastatic
cancer
patients
Metastatic
cancer
patientsHealthy subjects
Non metastatic
cancer patients
Metastatic cancer
patients
© Dipartimento Scientifico Guna S.p.a.

30. Radice E, Bellone G, Miranda V.
Enhancement of the immunomodulatory functions of ex vivo-generated dendritic cells from early-
stage colon cancer patients by consecutive exposure to low doses of sequential kinetic activated IL-4
and IL-12.
A preliminary study.
Translational Oncology 2015;8:327-38 © Dipartimento Scientifico Guna S.p.a.

31. The aim of this study is to assess in vitro the ability of single or
sequentially combined exposure to very low doses of SKA IL-4
and IL-12 (10-8 µg/ml) to enhance DCs’ antigen presentation
capacity compared to the normally administered doses of
recombinant human (rh) IL-4 and IL-12.
Ex vivo study on dendritic cells obtained from the peripheral
blood of patients with early stage colorectal carcinoma (CRC) - in
the presence or absence of metastasis - and from healthy donors.
STUDY TYPE
AIM OF THE STUDY
© Dipartimento Scientifico Guna S.p.a.

32. EX VIVO STUDY
16 subjects
(10 males, 6 females; mean age 73; range 57-83)
With colon carcinoma
16
9 primary colon
carcinoma
2 Dukes A
7 Dukes B 1-2
7 metastatic colon
carcinoma
Dukes C 1-2
with lymph
node
metastasis
12 subjects
(7 males, 5 females; mean age 64; range 37-85)
Healthy donors

33. EX VIVO STUDY
INCLUSION CRITERIA
• Subjects affected by primary colon carcinoma needing surgery without
pre-surgery evidence of distance metatstasis a
PREPARATIONS
• SKA-IL-4 (10 fg/ml) GUNA Laboratories
• SKA-IL-12 (10 fg/ml) GUNA Laboratories
• rhIL-4 (1ng/ml) Pepro Tech Inc.
• rhIL-12 (1ng/ml) Pepro Tech Inc.

34. Translational Oncology 2015 8, 327-338DOI: (10.1016/j.tranon.2015.06.005)
(A) MoDC (monocyte derived denditic cells) allostimulatory
activity of colon carcinoma patients (n = 16) versus
healthy donors (n = 12). (B) MoDC allostimulatory
activity of nonmetastatic (n = 9) versus metastatic (n
= 7) colon carcinoma patients. MoDCs, generated by
culturing PB CD14+
cells from tumor and normal
subjects in the presence of rhGM-CSF and rhIL-4 for
6 days, were incubated with 1 × 105
allogeneic naïve
CD4+
T cells at 1:40, 1:20, and 1:10 ratios for 5 days
followed by a 6-hour pulse of 3
H-TdR. Results are
expressed as mean ± SE cpm of triplicate co-
cultures. Statistical significance was determined
using one-way ANOVA.
RESULTS

35. Translational Oncology 2015 8, 327-338DOI: (10.1016/j.tranon.2015.06.005)
.
Effects of standard-dose rhIL-4 and/or rhIL-12 and low-
dose SKA-IL-4 and/or SKA-IL-12 on APC activity in MLR of
MoDCs from nonmetastatic colon carcinoma patients (n =
6) and from metastatic colon carcinoma patients (n = 7).
MoDCs were untreated or pretreated with SKA-IL-4 (48
hours) and SKA-IL-12 (24 hours) as single agents or
sequentially in parallel to the rh cytokines, and subjected
to MLR with allogeneic naïve T cells in different MoDC-to-
T cell ratios. The figure shows the mean percentages ± SE
of 3H-TdR incorporation in cpm. Statistical significance was
determined using one-way ANOVA.
Rh/SKA cytokine pretreated nonmetastatic/metastatic
colon carcinoma MoDCs versus untreated
nonmetastatic/metastatic colon carcinoma MoDCs: *P <
.05 and **P < .01.
Nonmetastatic colon carcinoma MoDCs versus metastatic
colon carcinoma MoDCs: ●●
P < .01 and ●●●
P < .0001.
RESULTS*
RECOMBINANT HIGH DOSE
LOW DOSE SKA

36. IL-4 (10 fg/ml SKA) IL-12 (10 fg/ml SKA)
IFN-g (10 fg/ml SKA)
IL-12 (10 fg/ml SKA)
GUNA IMMUNITY SYSTEM ACTIVATION IN CANCER

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