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cultures under the influence of cytokines including GM-CSF and IL-4 for 7 days. The brief electrical
current used during electroporation is enough to create a reversible breach in the cell membrane for the
ttRNA to rapidly enter the cytoplasm before degradation of the RNA. Electroporated ttRNA results in
better translation to tumor proteins in the cytoplasm of DCs and induction of tumor immunity in the
[55]
[42]
host . Although such immature ttRNA containing DCs (ttRNA DCs) can be administered as vaccine ,
induction of immune responses is vastly inferior compared to mature ttRNA DCs; maturity can be achieved
by combination of cytokines including IL-1β, TNF-α, IFN-γ, and IL-6. Once matured, ttRNA DCs can be
frozen in aliquots of 1 × 10 cells for clinical use.
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Dose and route of administration of ttRNA DC vaccine and immunoadjuvants
While studies in adult cancers have shown that efficacy of monocyte derived ttRNA DC vaccines might be
dose dependent, larger dose vaccines do not necessarily produce proportional T cell responses but might
[56]
rather dampen them by inducing immune tolerance . In fact, pre-clinical studies have shown that as
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few as 85 mature DCs can produce adequate T-cell responses and ideally should be less than 5 × 10 cells
per dose [56,57] . Several routes of administration have been tried including intravenous, intradermal, and
[56]
intranodal, injections . Most vaccine cells end up in kidneys, lung, and spleen when given intravenously
(as assessed by radiolabeling studies) and intranodal administration does not result in major distribution of
[56]
DCs to other regional lymph nodes . The skin is an ideal site for vaccination due to its rich plexus of blood
vessels and lymphatics that coalesce and drain into the regional inguinal lymph nodes [also called vaccine
site draining lymph nodes (VDLN)]. Although only 4% of an administered intradermal dose of vaccine
[57]
reaches the VDLN (the rest either degrading locally or engulfed by macrophages) , the number of mature
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DCs that migrate is still within the realm of less than 5 × 10 cells when a total of a 10 million (1 × 10 ) cells
are given with each vaccine dose. DC function and migration can be improved with immunoadjuvants [46,47] .
The ex vivo maturation of immature DCs (which lack the capacity to migrate well) with a cytokine cocktail
including IL-1β, TNF-α, IFN-γ, and IL-6, results in increased expression of the CCR7 and activation of the
CCR7/C-C chemokine ligand type 21 (CCL21) mediated axis of migration of the DCs to the draining lymph
nodes. In addition, co-stimulatory molecules expression is improved resulting in better cross-presentation
and lymphocyte expansion in the regional nodes. Our laboratory has also shown that pre-conditioning of the
one vaccine site with a recall antigen such as tetanus-diphtheria toxoid followed by bilateral administration
of DCs pulsed with CMV pp65 RNA in patients with malignant glioma (which express pp65 in over 90% of
tested samples) results in improved DC migration and improved survival as compared to control patients
who receive only the pp65 DC vaccine . A parallel experiment in mice confirmed these findings in a
[46]
[46]
manner dependent on the increased systemic release of the chemokine CCL3 .
GM-CSF, a potent stimulant of the bone marrow granulocyte-monocyte progenitors, is secreted by
[58]
endothelial cells, fibroblasts, and lymphocytes . The cytokine can cause DC maturation on its own
including expression of co-stimulatory molecules, induce CCR7 and migration towards CCL9, IL6 and
TNF-α secretion and hence lymphocyte stimulation and expansion. In the context of immunotherapy,
2
GM-CSF can be given either parenterally at a dose of 250 µg/m /day for 5-7 days or pre-embedded in the
vaccine using a significantly lower dose (75-150 µg per vaccine) to minimize possible systemic adverse
reactions with parenteral administration as well as minimize mobilization of immunosuppressive myeloid-
derived suppressor cells (MDSCs) from the bone marrow with higher doses [58,59] . Cellular vaccines can
[60]
also be genetically modified to secrete GM-CSF in the local milieu . It should be noted that GM-CSF
[61]
hypersensitivity due to autoantibody formation has been reported to occur when given with a vaccine .
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At our institution ttRNA-DC vials contain 5.7 × 10 cells in 1mL. The final ttRNA-DC product is a patient-
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specific single-dose syringe containing a total dose of 1 × 10 cells formulated in 400 µL of preservative free
saline. The administered volume is 0.4 mL embedded with 150 µg of GM-CSF. Half the dose is administered
intradermally into each thigh about 5 cm below inguinal ligament. Patients in our clinical trials receive at
least three biweekly vaccines with the first dose administered along with ALT infusion.