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1. Combining Old and New: Sensitising Drugs and Other Vaccines To
Augment Efficacy of Dendritic Cell Immunotherapy.
Cost-effective candidates
Levamisole, BCG, Imiquimod,
Mifamurtide & Dendritic Vax

2. Nature. 2015 Mar 19;519(7543):366-9. doi:
10.1038/nature14320. Epub 2015 Mar 11.

3. Nature. 2015 Mar 19;519(7543):366-9. doi:
10.1038/nature14320. Epub 2015 Mar 11.

4. Experiments showed that CCL3
upregulation in the skin was
dependent on the induction of
the Td recall response and was
significantly reduced by CD4+ T
cell depletion

7. • Toll-like receptors (TLRs) are a class of proteins that play a key role in
the innate immune system. They are single, membrane-spanning, non-
catalytic receptors usually expressed in sentinel cells such
as macrophages and dendritic cells, that recognize structurally conserved
molecules derived from microbes. Once these microbes have breached
physical barriers such as the skin or intestinal tract mucosa, they are
recognized by TLRs, which activate immune cell responses. The TLRs
include TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10,TLR11,
TLR12, and TLR13, though the latter two are not found in humans.

10. Levamisole, marketed under the trade name Ergamisol, is a
medication used to treat parasitic worm infections. It has also
been studied as a method to stimulate the immune system as
part of the treatment of cancer. Most current commercial
preparations are intended for veterinary use as a dewormer in
cattle, pigs, and sheep.
After being pulled from the market in the U.S. and Canada in
1999 and 2003, respectively, levamisole has been tested in
combination with fluorouracil to treat colon cancer. Evidence
from clinical trials support its addition to fluorouracil therapy to
benefit patients with colon cancer.

11. One of the more serious side effects of Levamisole is agranulocytosis, or the depletion
of the white blood cells. In particular, neutrophils appear to be affected the most. This
occurs in 0.08-5% of the studied populations.
There have also been reports of levamisole induced necrosis syndrome in which
erythematous painful papules can appear almost anywhere on skin.

12. Levamisole was evaluated as
an immune stimulant in a
randomized controlled study
of patients with anaplastic
gliomas, who had undergone
surgical resection and who
were also treated with
radiotherapy and BCNU
chemotherapy.
Studies utilizing the avian sarcoma virus-induced glioma in rats also showed no
improvement in survival with levamisole stimulation as the only immune agent, but
the combination of active immunization and adjuvant stimulation with bacillus
Calmette-Guerin plus levamisole was found to be therapeutically effective in this
model and will be used in future pilot studies of active immunization in patients.

15. Levamisole is a synthetic phenylimidazolthiazole that was first introduced in 1966 as an anti-helmintic agent.
Current studies have been focused upon its effect on immune response and on cancer treatment.
We examined the molecular mechanisms of levamisole in the activation and maturation of human
monocyte-derived dendritic cells (DC) and human T cells.
Treatment of DC with levamisole increased the presentation of CD80, CD86, CD83 and human
leucocyte antigen D-related (HLA-DR) molecules on the cell membrane, as well as the production of
interleukin-12 p40 and IL-10.
Levamisole-treated human DC also enhanced T cell activation towards type 1 T helper immune
response by inducing interferon-γ secretion. Neutralization with antibodies against Toll-like receptor
(TLR)-2 inhibited levamisole-induced production of IL-12 p40 and IL-10, suggesting a vital role for TLR-2
in signalling DC upon incubation with levamisole.

16. In this study, we showed that BALB/c bone marrow-derived DC stimulated with LMS resulted in enhanced cell-
surface expression of CD80, CD86, CD40 and MHC class II, as well as enhanced production of IL-12p70, TNF-α
and IL-1β. Interestingly, the LMS activated DCs were able to stimulate CD4(+) T cell proliferation and facilitated
Th1 differentiation by increasing the secretion of IFN-γ in an allogeneic mixed leukocyte reaction. Furthermore, to
confirm the in vitro data, we investigated the effect of LMS on antigen-specific antibody and cytokine
production in BALB/c mice. Immunization with LMS plus OVA showed that anti-OVA IgG2a and IFN-γ were
increased significantly compared with OVA alone in BALB/c mice.

17. • Aliment Pharmacol Ther. 2010 Sep;32(6):756-62. Meta-
analysis: levamisole improves the immune response to hepatitis
Bvaccine in dialysis patients.
• Fabrizi F1, Dixit V, Messa P, Martin P.
• We identified four studies involving 328 unique patients on regular
dialysis. Only prospective, randomized clinical trials (RCTs) were included.
• Pooling of study results showed a significant increase in response rates
among study (levamisole plus HBV vaccine) vs. control
(HBV vaccine alone) patients; the pooled Odds Ratio was 2.432 (95%
Confidence Intervals, 1.34; 4.403), P = 0.002.

18. • Nephrology (Carlton). 2014 Jan;19(1):27-31. Effect
of levamisole supplementation on tetanus vaccination response rates in
haemodialysis patients: a randomized double-blind placebo-controlled
trial. Fallahzadeh MK1, Sajjadi S, Singh N, Khajeh M, Sagheb MM.
• At 6 months, 11 out of 15 (73%) patients in the levamisole group as
compared with four out of 16 (25%) patients in the placebo group still had
protective anti-tetanus IgG levels (relative risk = 2.93, 95% CI = 1.19, 7.23).
• Supplementation of Td vaccination with levamisole may enhance
seroconversion against tetanus in haemodialysis patients.

20. BCG is an attenuated (nonvirulent) form of Mycobacterium bovis cultured in 1921 by Albert
Calmette and Camille Guerin at the Pasteur Institute in Lille, France. In the late 1920’s,
autopsy studies performed at Johns Hopkins Hospital in Baltimore, Maryland, showed a
lower frequency of cancer in patients with tuberculosis.
Attempts were made to use BCG against a variety of human cancer tumors with only limited
success. It wasn’t until the 1970’s, however, that a Canadian urologist, Dr. Alvaro Morales,
began his groundbreaking work on the use of intravesical BCG for the treatment of superficial
bladder cancer. Over 30 years later, BCG continues to be used worldwide for the treatment
of high grade noninvasive bladder cancer.

28. Mycobacterium bovis (TEM)

30. We investigated whether three mycobacterial preparations currently used in the clinic (BCG and
heat-killed Mycobacterium vaccae and Mycobacterium obuense) can stimulate anti-tumour
effector responses in human γδ T-cells. Results show that γδ T-cells are activated by these
mycobacterial preparations, as indicated by upregulation of activation marker expression and
proliferation.
Activated γδ T-cells display enhanced effector responses, as shown by upregulated granzyme B
expression, production of the T(H)1 cytokines IFN-γ and TNF-α, and enhanced degranulation in
response to susceptible and resistant tumour cells.

31. A type of metastasis in which skin cancer spreads through a lymph vessel and
begins to grow more than 2 centimeters away from the primary tumor but before
it reaches the nearest lymph node.

32. For the small uncontrolled study, twenty (20) patients in advanced stages with limited life expectancy were
recruited in a 2-year period. Patients were classified into two groups according to the treatment to be
employed. Group 1 included 6 patients who refused to be treated with standard treatments and received only
immunotherapy. Group 2 included 14 patients treated with both immunotherapy and chemotherapy/
radiotherapy (CT/RT). The vaccination procedure: 3 intradermal doses of 0.5 ml each, with intervals of 6 weeks
between each dose. Tumor fragments (about 1 g) from patients were kept in sterile PBS at −80 °C until use. For
each dose, about 0.3–0.4 g of tumor tissue were macerated in 0.6 ml sterile PBS.
At the moment of vaccination, the soluble fraction of the homogenate was mixed with formaldehyde 36 % at
a final concentration of 0.02 % and 0.6 ml of BCG suspension 0.15 %.

33. The survival rate upon a 5-year period was plotted
for the immunotherapy only (IT) group and the
combined treated (CT/RT/IT) group, as well as for
the overall survival across all subjects. The
calculated 5-year survival is indicated for each plot.

34. However, γδ T cells are peculiar in that they do not seem to require antigen
processing and major-histocompatibility-complex (MHC) presentation of peptide epitopes,
although some recognize MHC class Ib molecules. Furthermore, γδ T cells are believed to
have a prominent role in recognition of lipid antigens.

36. Imiquimod
Imiquimod (INN) is a prescription medication that acts as an
immune response modifier and is used to treat genital
warts, superficial basal cell carcinoma, and actinic keratosis.
Imiquimod signals to the innate arm of the immune system
through the toll-like receptor 7 (TLR7).

37. Plasmacytoid dendritic cells (pDCs) are innate immune cells that circulate in the blood and are
found in peripheral lymphoid organs. They constitute < 0.4% of peripheral blood mononuclear
cells (PBMC). In humans these cells express the surface markers CD123, BDCA-2(CD303) and
BDCA-4 (CD304), but do not express high levels of CD11c or CD14, which distinguishes them
from conventional dendritic cells or monocytes, respectively.

39. TNF-related apoptosis-inducing ligand (TRAIL), is aprotein functioning as a ligand that
induces the process of cell death called apoptosis. TRAIL is a cytokine that is produced and
secreted by most normal tissue cells. It causes apoptosis primarily in tumor cells, by
binding to certain death receptors. TRAIL has also been designated CD253 (cluster of
differentiation 253).

40. Melanoma in situ – Lentigo maligna
Imiquimod Treatment

42. Mifamurtide (Mepact®, Takeda) is a drug against osteosarcoma,
mainly affecting children and young adults, which is lethal in about
a third of cases. The drug was approved in Europe in March 2009.
In a phase-III clinical trial in about 800 newly diagnosed osteosarcoma patients,
mifamurtide was combined with the chemotherapeutic agents doxorubicin and
methotrexate, with or without cisplatin and ifosfamide. The mortality could be lowered
by 30% versus chemotherapy plus placebo. Six years after the treatment, 78% of
patients were alive. Drugs in R&D 9 (2): 131–5. 2008.

43. Looking to the past may help planning for the
future