Cancer vaccines and dendritic cells

The term “cancer,” which is derived from the Latin word for “crab,” was adopted in ancient times to illustrate the way in which malignant tumors seem to grasp the tissues they invade. Cancers may arise due to mutations or epigenetic changes to genes that control cell division, migration, and death, leading to an abnormality in cellular proliferation associated with life-threatening invasive tumor growth and metastases (83, 84).

Effective cancer vaccines are sought to treat malignancies by approaches that induce presentation of tumor-associated antigens (TAAs) in contexts that elicit potent CD4⁺ and CD8⁺ T-cell responses and break the tolerance of the host immune system to tumor growth (85, 86). Immunity, including innate immunity and antigen-specific adaptive immunity, and tolerance toward tumors is orchestrated by a network of antigen-presenting cells, the most crucial of which are dendritic cells (DCs) (87, 88). As shown in Figure 18, maturation and activation of dendritic cells is critically important for immune response.

However, although clinical trials based on DC-based vaccines have been initiated for

certain malignancies (89), unlike in pathogen infection, activation of DCs in tumor microenvironments is weak and ineffective (85, 90); the challenge, therefore, is to develop DC-based vaccines that can not only induce powerful activation of DCs, but also enhance tumor-specific immunity by breaking tolerance.

The approach to generate ex vivo differentiation and activation of DCs for cancer immunotherapy is shown in Figure 19. In general, the most common method used to generate DCs in clinical trials is to culture CD14⁺ monocytes in serum-free media in the presence of GM-CSF and IL-4. After 5–7 days of culture, the monocytes can differentiate into immature DCs with a loss of CD14 expression, the expression of moderate to low levels of CD40 and the costimulatory ligands B7-1 and B7-2. DC maturation is accomplished by culturing the immature DCs for an additional 24–48 hours in the presence of several biological agents, the most popular combination being tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β), and prostaglandin E2 (PGE2).

Matured DCs further upregulate CD40, B7-1, and B7-2 and induce the expression of the lymph node homing receptor CC chemokine receptor 7 (CCR7). Antigen loading may occur at either the immature or mature DC stage. Later, mature antigen-loaded DCs could be injected into patients subcutaneously, intradermally, or intravenously. They would subsequently migrate to the draining lymph node, where they would encounter and present antigen to the cognate CD4⁺ T cells. CD40L, which is expressed on the antigen-activated

CD4+ T cell to cross-link CD40 on the DCs, induces the mature DCs to differentiate, a process known as licensing. These licensed DCs upregulate cell surface products, notably the ligands for OX40 and 4-1BB (OX40L and 4-1BBL, respectively). The licensed DCs also present antigen to cognate CD8+ T cells. 4-1BBL–mediated costimulation via 4-1BB on the antigen-activated CD8⁺ T cells elevate the survival and proliferative capacity of the activated CD8⁺ T cells. Similarly, the OX40L-mediated costimulation increases the survival and proliferation of the activated CD4+ T cells. The important factors which influence the efficacy of DC-based vaccines are shown in Figure 20.

Numerous dendritic cell-based strategies have been employed for developing anti-cancer vaccines, including defined peptide-loaded DCs (91, 92), genetically-modified DCs (93, 94), DC-derived exosomes (89), apoptotic cell-loaded DCs (95), and tumor cell lysate (TCL)-pulsed DCs (91, 96, 97). The possible advantage of using whole cell lysates as the source for vaccination is that a full complement of TAAs, including both MHC class I and class II-restricted tumor associated epitopes may be provided, thus reducing the possibility of immune escape by antigen loss variants (98). A common technique used to generate tumor cell lysates (TCLs) is the removal of solid cellular debris by centrifugation after repeated cycles of freezing and thawing of target tumor cells (99-101). The protective effect of DCs pulsed with TCLs has been proven in animal models (96, 102), and reported in a range of clinical trials, summarized in Table 7 (91, 97, 103). Nonetheless, the modest

activities reported thus far for TCL-pulsed DC-based vaccines need to be further improved and optimized, either in the presence or absence of DC-maturation stimuli (97, 103).

Figure 18. The different functions of immature and mature dendritic cells

(a) Immature dendritic cells (DCs) induce tolerance. Tissue DCs always sample their environment, capture specific antigens and migrate in small numbers to the draining lymph nodes. In general, in the absence of inflammation, the DCs usually remain in an immature state, and antigens could be presented to T cells in the lymph node without costimulation, leading to either the deletion of T cells or the induction of regulatory T cells. (b) Mature DCs induce immunity. Inflammation of tissue induces the maturation of DCs and triggers the migration of large numbers of them to draining lymph nodes. The mature DCs express peptide–MHC complexes on their surface, and appropriate co-stimulatory molecules. This allows the priming of CD4+ T helper cells and CD8+ cytotoxic T lymphocytes (CTLs), the activation of B cells and the initiation of an adaptive immune response. To control the immune response, CD4+CD25+ regulatory T cell (TReg) populations are also expanded.

ADCC, antibody-dependent cell-mediated cytotoxicity; NK, natural killer; TCR, T-cell receptor. Adopted from Banchereau et al. (92).

Figure 19. The approach of ex vivo differentiation and activation of DCs for cancer immunotherapy. [Adopted from Gilboa, E.et.al. (90)]

Figure 20. Various factors to be considered in DC-based immunotherapy.

[Adopted from Osada et al. (104)]

Table 7. Overview of clinical trials using dendritic cell-based cancer immunotherapy

Tumor type DC generation Pulsing Maturation Route Memo Reference Soft-tissue

(CRC)

I.V. Pilot trial Bachleitne r et al.

Relapsed

MDDC, monocyte-derived dendritic cells; CEA,carcinoembryogenic antigen; PSMA, prostate-specific membrane antigen; KLH, keyhole limpet hemocyanin; TCL, Tumor cell lysate; TNF-α, tumor necrosis factor-α; IL-1β, interleukin-1β; IL-6, interleukin-6; PGE2, prostaglandin E2; F-T, 4-6 cycles of freezing-thawing; Irra, irradiation (60 Gray); FMKp, a bacterial membrane fragment of Klebsiella pneumonia; I.V., intravenously; I.N.,intranodal;

S.C., subcutaneously;, I.D., intradernally; I.T., intratumorally.

1.2 Damage-associated molecular patterns (DAMPs) and immunogenic cell death (ICD)

It is well-recognized that most anticancer chemotherapeutics influence both tumor cells and the associated immune systems (125, 126). The conventional chemotherapeutics possibly induce immunogenic cancer cell death or stimulate immune effectors through so-called off-target effects (126). However, the nature of the mechanisms underlying the various cellular activities that induce immune response, and whether specific necrotic or apoptotic cells are immunogenic or tolerogenic, remain unclear (127, 128).

A key-lock paradigm has been proposed to explain the relationship between immunogenic cell death and DCs (Figure 21) (128). Immunogenic cell death is characterized by occurring events of damage-associated molecular patterns (DAMPs) including the translocation of calreticulin (CRT), the release of heat shock proteins (HSPs) including HSP70 and HSP90 on the cell surface, and the release of high-mobility group box 1 (HMGB1) proteins (127, 129-131). CRT, HSPs and HMGB1 can function as immunological adjuvants for phagocytosis, cross-presentation of tumor-derived antigens and antigen processing as well as presentation by DCs (Table 8) (129). As seen in Figure 22, the induction of tumor cell death results in recognition of cell death by DCs. Perhaps the apoptotic bodies are engulfed by DCs, and tumor-derived antigens are processed and presented along with DAMPs and costimulatory molecules. After DC maturation, they

move to naive T lymphocytes to interact with them. This process is amplified at different steps. Selection of immunogenic cell death inducers allows enhancement of recognition and phagocytosis of apoptotic debris, maturation of DCs, processing and the presentation of tumor-derived antigens. Most importantly, this leads to the induction of a cytotoxic immune response, involving CD4^＋ and CD8^＋ T lymphocytes, leading to complete eradication of tumor cells (126, 127, 130, 131). Other factors may also involve in the induction of immunogenic cell death via different pathways to enhance the immune response, as summarized in Figure 23. The approach of DC-pulsed with tumor cell lysates (TCLs) is clinically used and has the advantage of providing various sources of tumor-associated antigens to DCs; however, TCLs may inhibit the maturation of DCs (98).

It is, therefore, of importance to investigate the molecular and cellular behaviors of these immunogenic cell death-associated proteins in TCLs with an eye toward improving the efficacy of TCL-pulsed DC-based vaccines.

Figure 21. Critical events required to trigger dendritic cell activation by dying tumor cells.

Immunogenic tumor cell death is featured with a temporal sequence of events including translocation of calreticulin (CRT) to the cell surface at the early stage, the release and exposure of heat shock proteins, and the late release of HMGB1, all allowing DC-mediated specific anti tumor immune response. These key components released from or exposed at the surface of dying tumor cells can be said to act at several levels of this immune response:

uptake of apoptotic bodies (calreticulin), activation of dendritic cells (heat shock proteins), antigen processing (HMGB1), maturation of dendritic cells, and activation of T lymphocytes. [Adopted from Tesniere et al. (128)]

Table 8. Major determinants of immunogenic cancer cell death and their effects on DCs

Adopted from Tesniere et al. (129).

Figure 22. The key steps for the induction of an antitumor immune response triggered by immunogenic cell death. [Adopted from Tesniere et al. (129)]

Figure 23. Schematic representation of the main immunogenic determinants of dying tumor cells. [Adopted from Tesniere et al. (129))

1.3. The relationship between microtubule-targeting/binding