**7. Antigen-independent roles of membrane vesicles in immune responses**

Antigen presentation by exosomes is primarily an immunostimulatory process, however exosomes have many antigen-independent functions that can both stimulate and inhibit the immune system (Schorey & Bhatnagar, 2008; Thery *et al.*, 2009). These roles are dependent on the type of cell the exosomes originate from and the molecules they express (Andre *et al.*, 2002a; Abusamra *et al.*, 2005a; Xiang *et al.*, 2009; Chalmin *et al.*, 2010; Szajnik *et al.*, 2010; Zhang *et al.*, 2011). In this way exosomes are important for proper immune function and immune regulation, but can also be hijacked by malignant cells to prevent detection by the immune system.

### **7.1 Immune cell exosomes induce immune responses**

Dendritic cell exosomes (DEXs) are vital for stimulation of the adaptive immune response by their presentation of antigen to T cells (**Figure 6**), but induction of the humoral immune response is as important a function (Chaput *et al.*, 2006). DEXs have been shown to stimulate Natural Killer (NK) cell responses critical to the clearance of infection and malignancy (Viaud *et al.*, 2009). This function is mediated by the expression of interleukin-15 receptor alpha (IL-15Rα) and Natural Killer Group 2D (NKG2D) ligands on dendritic cell exosomes. Interleukin-15 (IL-15) is a cytokine that stimulates NK cell activation and proliferation. Exosomal IL-15Rα can bind IL-15 in the extracellular space and deliver it to NK cells by exosomal binding to NKG2D on the surface of the NK cells (Viaud *et al.*, 2009). Delivery of IL-15 in this manner significantly increases NK cell responses *in vitro* and in mice injected with autologous DEXs (Thery *et al.*, 2009; Viaud *et al.*, 2009). Because of their critical importance to tumor clearance, NK cell stimulation by DEXs could provide therapeutic benefits by increasing NK cell numbers in cancer patients.

#### **7.2 Tumor cell exosomes and the immune response**

The role of TEX in the progression of cancer is multifaceted, as they can affect tumor growth, invasion and metastasis, and therapy resistance. In addition to influencing other malignant cells, TEXs have significant effects on the immune response, both positive and negative.

#### **7.2.1 Exosomes from heat-shocked tumor cells induce immune activation**

The release of exosomes from normal and malignant cells is increased by heat shock and stress, with amplified proportions of Hsps within the exosome (Schorey & Bhatnagar, 2008). Culture of dendritic cells with exosomes from heat-shocked tumors resulted in higher expression of CD40, CD80 and CD86 than in dendritic cells cultured with control immune

Antigen presentation to T cells is the first and most critical step in the adaptive immune response. The ability of exosomes to supply antigens to dendritic cells for presentation as well as present antigens directly via exosomal MHC molecules is an important mechanism for detection of infection and malignancy. Induction of anti-cancer responses via tumorreleased exosome antigens is likely a key mechanism in immune surveillance to prevent tumor progression. Whether this can be utilized in tumor immunotherapies is under

**7. Antigen-independent roles of membrane vesicles in immune responses** 

Antigen presentation by exosomes is primarily an immunostimulatory process, however exosomes have many antigen-independent functions that can both stimulate and inhibit the immune system (Schorey & Bhatnagar, 2008; Thery *et al.*, 2009). These roles are dependent on the type of cell the exosomes originate from and the molecules they express (Andre *et al.*, 2002a; Abusamra *et al.*, 2005a; Xiang *et al.*, 2009; Chalmin *et al.*, 2010; Szajnik *et al.*, 2010; Zhang *et al.*, 2011). In this way exosomes are important for proper immune function and immune regulation, but can also be hijacked by malignant cells to prevent detection by the

Dendritic cell exosomes (DEXs) are vital for stimulation of the adaptive immune response by their presentation of antigen to T cells (**Figure 6**), but induction of the humoral immune response is as important a function (Chaput *et al.*, 2006). DEXs have been shown to stimulate Natural Killer (NK) cell responses critical to the clearance of infection and malignancy (Viaud *et al.*, 2009). This function is mediated by the expression of interleukin-15 receptor alpha (IL-15Rα) and Natural Killer Group 2D (NKG2D) ligands on dendritic cell exosomes. Interleukin-15 (IL-15) is a cytokine that stimulates NK cell activation and proliferation. Exosomal IL-15Rα can bind IL-15 in the extracellular space and deliver it to NK cells by exosomal binding to NKG2D on the surface of the NK cells (Viaud *et al.*, 2009). Delivery of IL-15 in this manner significantly increases NK cell responses *in vitro* and in mice injected with autologous DEXs (Thery *et al.*, 2009; Viaud *et al.*, 2009). Because of their critical importance to tumor clearance, NK cell stimulation by DEXs could provide therapeutic

The role of TEX in the progression of cancer is multifaceted, as they can affect tumor growth, invasion and metastasis, and therapy resistance. In addition to influencing other malignant cells, TEXs have significant effects on the immune response, both positive and

The release of exosomes from normal and malignant cells is increased by heat shock and stress, with amplified proportions of Hsps within the exosome (Schorey & Bhatnagar, 2008). Culture of dendritic cells with exosomes from heat-shocked tumors resulted in higher expression of CD40, CD80 and CD86 than in dendritic cells cultured with control immune

**7.2.1 Exosomes from heat-shocked tumor cells induce immune activation** 

investigation and could prove useful in combination therapies.

**7.1 Immune cell exosomes induce immune responses** 

benefits by increasing NK cell numbers in cancer patients.

**7.2 Tumor cell exosomes and the immune response** 

**6.3 Summary** 

immune system.

negative.

cell exosomes or non-heat shocked tumor exosomes (Chen *et al.*, 2006c). Additionally, the dendritic cells treated with heat shocked TEXs exhibited increased production of proinflammatory cytokines tumor necrosis factor alpha (TNFα), IL-1β, and IL-12 (Chen *et al.*, 2006c). This is thought to be mediated by the increased amount of Hsps in the heat-shocked TEXs, though the exact mechanism is still under investigation.

Fig. 6. Dendritic cell-derived exosomes interact with T cells through MHC class I, II, tetraspanins such as CD9, CD63, CD81 and through co-stimulatory molecules such as CD86.

#### **7.2.2 Tumor cell exosomes inhibit natural killer cells via NKG2D**

NK cells kill cancer cells by release of granules and perforins (**Figure 7**). NK cell activity is often lost in cancer patients, resulting in a reduced ability of the immune system to eliminate malignant cells (Clayton *et al.*, 2008; Viaud *et al.*, 2009; Ahiru *et al.*, 2010). The NK cell receptor NKG2D is important in regulation of NK cell function, with some ligands stimulating and others inhibiting cytotoxic function (Clayton *et al.*, 2008). As discussed earlier, DEXs expressing activating NKG2D ligands can enhance NK cell function and promote tumor clearance (Viaud *et al.*, 2009). Other NKG2D ligands, such as MHC class Irelated chain A (MICA) can reduce NK cell function. Tumor cells abuse this normal ligand by upregulating MICA on the cell surface as well as on TEXs (Ahiru *et al.*, 2010). Ovarian cancer exosomes expressing high levels of MICA were shown to decrease NK cell function *in vitro* by reducing their NKG2D receptor expression and their responsiveness to activating NKG2D ligands (Ahiru *et al.*, 2010). This reduction in NK cell function is highly detrimental to the anti-tumor response and is one of many mechanisms by which tumors escape immune detection (Thery *et al.*, 2009).

The Application of Membrane Vesicles for Cancer Therapy 39

inhibitory cell types have been implicated in cancer progression: regulatory T cells (Tregs) (Szajnik *et al.*, 2010) and myeloid derived suppressor cells (MDSCs) (Xiang *et al.*, 2009). These cell types inhibit both helper and cytotoxic T cell responses and reduces their production of inflammatory cytokines like interferon gamma (IFN-γ). Large numbers of immune cells are found within the tumor microenvironment, but Tregs and MDSCs are far more prominent in cancer patients than in healthy patients, leading to the hypothesis that immune suppression can be induced by the tumors (Xiang *et al.*, 2009; Szajnik *et al.*, 2010). Tumor cells can directly induce regulatory populations by secreting transforming growth factor beta (TGF-β), which promotes differentiation of naïve T cells and myeloid precursor cells into their respective suppressive phenotypes. A similar mechanism is utilized by tumors to induce Tregs and MDSCs within the tumor microenvironment and the periphery by secretion of TGF-β in exosomes (Xiang *et al.*, 2009; Chalmin *et al.*, 2010; Szajnik *et al.*, 2010). Ovarian carcinoma derived exosomes were found to induce CD4+CD25+Foxp3+ Tregs from CD4+CD25- naïve T cells *in vitro (Szajnik et al., 2010)*. This was found to be dependent on the presence of TGF-β and IL-10 in the exosomes and was inhibited by addition of neutralizing antibodies to both cytokines. In a similar study, mouse bone marrow myeloid precursor cells were shown to take up TEXs, inducing their differentiation into CD11b+Gr-1+ myeloid derived suppressor cells. Exosomal expression of TGF-β and prostaglandin E2 (PGE2) was shown to induce this differentiation, which was inhibited by antibody neutralization (Xiang *et al.*, 2009). The suppressive function of existing MDSCs is also increased by TEXs. Interaction of Hsp72 on the exosome with Toll-like receptor 2 (TLR2) on the MDSC induces Stat3 signaling to induce their immunosuppressive functions (Chalmin *et al.*, 2010). By increasing immunosuppressive cell types, TEXs can prevent attack by cytotoxic

Exosomes can have important antigen-independent effects on immune cells that vary with the cell type they are derived from as well as the state of the cells upon release. DEXs and TEXs can stimulate immune responses, which could provide therapeutic benefits. In contrast, tumor derived exosomes from several cancer types have been shown to prevent cytotoxic attack on the tumor by FasL-induced T cell death, NKG2D ligand-mediated suppression of NK cells and through induction of immune suppressor cells like Tregs and MDSCs. How TEXs can be utilized or inhibited therapeutically remains to be seen, but

**8. Immune responses induced by** *in vitro* **purified membrane vesicles** *in vivo* Many studies described in the Sections 6 and 7 were performed using membrane vesicles and exosomes isolated and introduced to immune cell cultures *in vitro*. Before these findings can be utilized therapeutically, they must be confirmed in *in vivo* animal models. Results from these animal studies have demonstrated the importance of exosomes in immune

Early studies in tumor vaccination utilized irradiation-killed tumor cells with limited success. With the discovery of TEXs, mouse vaccination studies with TEXs were shown to be more effective at generating tumor-specific T cell responses than irradiated tumor cells.

modulation and are currently being implemented in clinical trials.

**8.1 Vaccination with tumor exosomes prevent tumor growth in mice** 

cells (**Figure 7**).

**7.3 Summary** 

currently is an active area of research.

Fig. 7. DEX and TEX play opposing roles with regard to immune cell activation and prohibition.

#### **7.2.3 Fas ligand expression on tumor cell exosomes induce immune cell apoptosis**

Cytotoxic immune cells like cytotoxic T cells express Fas Receptor (FasR; CD95) and Fas ligand (FasL; CD95L) that allow them to induce apoptosis in target cells. Several cancer types including colon and ovarian carcinoma and melanoma cells express FasL to induce death of the very immune cells that attempt to kill them (Koyama *et al.*, 2001). This is a primary mechanism of tumor-immune escape that prevents the immune system from eliminating transformed cells, resulting in disease progression. To further prevent cytotoxic killing by T cells, TEXs have been found to express high levels of surface-bound FasL (Abusamra *et al.*, 2005a). Release of FasL via exosomes allows the tumor to kill FasRexpressing immune cells at distant sites, as well as in the tumor microenvironment, further preventing tumor clearance. Interestingly, although both helper and cytotoxic T cells express FasR, exosomes containing FasL preferentially induce apoptosis in cytotoxic T cells and not in helper T cells (Abusamra *et al.*, 2005a). The mechanism behind this is not fully understood, but may be due to favored interactions between exosomes and cytotoxic T cells due to other exosomal surface molecules (**Figure 7**).

#### **7.2.4 Tumor exosomes induce suppressive immune cell phenotypes**

Immune suppression within the tumor microenvironment prevents cytotoxic attack and promotes tumor progression. Like FasL and NKG2D inhibition, increasing immunosuppressive cell types can reduce cytotoxic immune responses. Two primary inhibitory cell types have been implicated in cancer progression: regulatory T cells (Tregs) (Szajnik *et al.*, 2010) and myeloid derived suppressor cells (MDSCs) (Xiang *et al.*, 2009). These cell types inhibit both helper and cytotoxic T cell responses and reduces their production of inflammatory cytokines like interferon gamma (IFN-γ). Large numbers of immune cells are found within the tumor microenvironment, but Tregs and MDSCs are far more prominent in cancer patients than in healthy patients, leading to the hypothesis that immune suppression can be induced by the tumors (Xiang *et al.*, 2009; Szajnik *et al.*, 2010). Tumor cells can directly induce regulatory populations by secreting transforming growth factor beta (TGF-β), which promotes differentiation of naïve T cells and myeloid precursor cells into their respective suppressive phenotypes. A similar mechanism is utilized by tumors to induce Tregs and MDSCs within the tumor microenvironment and the periphery by secretion of TGF-β in exosomes (Xiang *et al.*, 2009; Chalmin *et al.*, 2010; Szajnik *et al.*, 2010). Ovarian carcinoma derived exosomes were found to induce CD4+CD25+Foxp3+ Tregs from CD4+CD25- naïve T cells *in vitro (Szajnik et al., 2010)*. This was found to be dependent on the presence of TGF-β and IL-10 in the exosomes and was inhibited by addition of neutralizing antibodies to both cytokines. In a similar study, mouse bone marrow myeloid precursor cells were shown to take up TEXs, inducing their differentiation into CD11b+Gr-1+ myeloid derived suppressor cells. Exosomal expression of TGF-β and prostaglandin E2 (PGE2) was shown to induce this differentiation, which was inhibited by antibody neutralization (Xiang *et al.*, 2009). The suppressive function of existing MDSCs is also increased by TEXs. Interaction of Hsp72 on the exosome with Toll-like receptor 2 (TLR2) on the MDSC induces Stat3 signaling to induce their immunosuppressive functions (Chalmin *et al.*, 2010). By increasing immunosuppressive cell types, TEXs can prevent attack by cytotoxic cells (**Figure 7**).

#### **7.3 Summary**

38 Advances in Cancer Therapy

Fig. 7. DEX and TEX play opposing roles with regard to immune cell activation and

due to other exosomal surface molecules (**Figure 7**).

**7.2.4 Tumor exosomes induce suppressive immune cell phenotypes** 

Immune suppression within the tumor microenvironment prevents cytotoxic attack and promotes tumor progression. Like FasL and NKG2D inhibition, increasing immunosuppressive cell types can reduce cytotoxic immune responses. Two primary

**7.2.3 Fas ligand expression on tumor cell exosomes induce immune cell apoptosis**  Cytotoxic immune cells like cytotoxic T cells express Fas Receptor (FasR; CD95) and Fas ligand (FasL; CD95L) that allow them to induce apoptosis in target cells. Several cancer types including colon and ovarian carcinoma and melanoma cells express FasL to induce death of the very immune cells that attempt to kill them (Koyama *et al.*, 2001). This is a primary mechanism of tumor-immune escape that prevents the immune system from eliminating transformed cells, resulting in disease progression. To further prevent cytotoxic killing by T cells, TEXs have been found to express high levels of surface-bound FasL (Abusamra *et al.*, 2005a). Release of FasL via exosomes allows the tumor to kill FasRexpressing immune cells at distant sites, as well as in the tumor microenvironment, further preventing tumor clearance. Interestingly, although both helper and cytotoxic T cells express FasR, exosomes containing FasL preferentially induce apoptosis in cytotoxic T cells and not in helper T cells (Abusamra *et al.*, 2005a). The mechanism behind this is not fully understood, but may be due to favored interactions between exosomes and cytotoxic T cells

prohibition.

Exosomes can have important antigen-independent effects on immune cells that vary with the cell type they are derived from as well as the state of the cells upon release. DEXs and TEXs can stimulate immune responses, which could provide therapeutic benefits. In contrast, tumor derived exosomes from several cancer types have been shown to prevent cytotoxic attack on the tumor by FasL-induced T cell death, NKG2D ligand-mediated suppression of NK cells and through induction of immune suppressor cells like Tregs and MDSCs. How TEXs can be utilized or inhibited therapeutically remains to be seen, but currently is an active area of research.

### **8. Immune responses induced by** *in vitro* **purified membrane vesicles** *in vivo*

Many studies described in the Sections 6 and 7 were performed using membrane vesicles and exosomes isolated and introduced to immune cell cultures *in vitro*. Before these findings can be utilized therapeutically, they must be confirmed in *in vivo* animal models. Results from these animal studies have demonstrated the importance of exosomes in immune modulation and are currently being implemented in clinical trials.

#### **8.1 Vaccination with tumor exosomes prevent tumor growth in mice**

Early studies in tumor vaccination utilized irradiation-killed tumor cells with limited success. With the discovery of TEXs, mouse vaccination studies with TEXs were shown to be more effective at generating tumor-specific T cell responses than irradiated tumor cells.

The Application of Membrane Vesicles for Cancer Therapy 41

A similar trial to the melanoma study was performed in Stage III and IV non-small cell lung cancer (NSCLC) (Morse *et al.*, 2005). Of the 13 patients enlisted in the trial, 9 finished the treatments and no toxicity was observed. Immune responses to MAGE3 was observed in 3 of 9 patients, with MAGE3-specific T cells only detected in 1 patient and while an increase in Treg cells was observed in 2 patients (Morse *et al.*, 2005). This trial concluded that dendritic cell exosomes are safe for use therapeutically, but that better responses may result from including Treg inhibitors in the treatment. A Phase II trial has begun adding IL-15R and NKG2D to the exosomes as well as Treg inhibitor therapy in NSCLC patients whose disease

The use of exosomes as a cancer therapeutic has been demonstrated to be effective in murine studies and safe by Phase I clinical trials. Although some benefit was seen in patient trials, making these exosomal therapies more effective will likely require combination immunotherapies, chemotherapies and radiation. The use of exosomes to induce anti-cancer

Secreted microvesicles, known as exosomes, provide a form of cell to cell signaling in which the recipient cell can be modified by the contents of the delivered exosomes. The contents (cytosolic proteins, lipids, siRNA, miRNA, DNA, etc) (Tan *et al.*, 2010; Khan *et al.*, 2011) are protected and stably delivered unlike those secreted into the extracellular matrix (Pap *et al.*, 2010); thus securing the bioactivity of the delivered contents. Exosomes could be an attractive tool as an immunotherapeutic as they maintain much of the anti-tumor activity of a dendritic cell with the advantage of a cell free vehicle. Understanding that exosomes are involved in many levels of tumorigenesis, and potential strategies against this form of cellular communication has led to the creation of potential therapeutics against exosomes. TEXs are a protective measure for tumor cells that aids in survival, growth, invasion, metastasis, and evasion of the immune system (Clayton *et al.*, 2007; Valenti *et al.*, 2007; Anderson *et al.*, 2010; Khan *et al.*, 2011). Taxol and vinca alkaloids are well known chemotherapeutic agents, but they also limit exosome release via the microtubule formation. Exosome formation has also been shown to be blocked by decreasing acidity of the microcellular environment (Iero *et al.*, 2008; Pap *et al.*, 2010). Prevention of exosome formation involves a broad variety of proteins and is essential for life in all cells. Therefore any inhibition of exosomes within the body could lead to unwarranted side effects (Pap *et al.*, 2010). It was thought that inhibition or treatment focused against TEXs and its ability to aid tumor cells could bear significant benefit to patients, but that was overshadowed by the realization that exosomes could potentially be employed as biomarkers and diagnostic tools of malignancy in blood (Anderson *et al.*, 2010) and urine (Nilsson *et al.*, 2009). One such study analyzed the correlation between serum PSA and urinary-exosomes, but none was observed. However, the data suggests that healthy donor urinary-exosomes were negative

Realizing that exosomes are secreted/released by a wide variety of cell types (Skokos *et al.*, 2001; Wolfers *et al.*, 2001; Chaput *et al.*, 2004b; Tan *et al.*, 2010; Khan *et al.*, 2011) including

**8.2.2 Non-small cell lung cancer phase I and II trials** 

has been stabilized by chemotherapy (Thery *et al.*, 2009).

**9. Membrane vesicles as therapeutic agents** 

for PSA, PSMA, and 5T4 (Mitchell *et al.*, 2009).

responses presents an exciting new field in cancer immunotherapy.

**8.3 Summary** 

Additionally, vaccination of mice with exosomes containing ovalbumin resulted in delayed growth of ovalbumin-expressing tumors and induced pro-inflammatory Th1 T cell responses. As described in Section 7.2, exosomes isolated from heat-shocked cancer cells are more immunogenic *in vitro*. Using heat-shocked TEXs for immunization in mice showed similar immunogenicity, with 80% of mice remaining tumor free after challenge. While these studies have provided the groundwork for cancer vaccines, the immunosuppressive functions of cancer exosomes have caused many to be skeptical of their use as vaccine.

#### **8.2 Dendritic cell exosomes containing tumor antigens induce anti-tumor immune responses**

The use of DEXs as artificial antigen presenting cells can elicit strong antigen-specific T cell responses (Kim *et al.*, 2004). Mouse studies using DEXs pulsed with tumor peptides resulted in the activation of tumor-specific T cells, proliferation of NK cells and tumor regression (Kim *et al.*, 2004; Chaput *et al.*, 2006). Utilization of this technology with great success in murine studies has led to the development of several clinical trials using autologous DEXs as a treatment in cancer patients (**Figure 8**).

#### **8.2.1 Melanoma phase I trial**

A Phase I trial using dendritic cell exosomes to induce tumor regression was evaluated in 15 patients with metastatic melanoma (Escudier *et al.*, 2005). Autologous monocytes were isolated from patients by leukapheresis and differentiated *in vitro* to dendritic cells. These dendritic cells were cultured, exosomes isolated from the culture medium and pulsed with peptides from the tumor antigen MAGE3 (Escudier *et al.*, 2005). Patients received escalating doses of cryopreserved exosomes and their tumor progression and immune responses monitored. All 15 patients completed therapy but only 1 had specific T cell responses. Skin and lymph node mass reduction was observed in 5 of 15 patients and 7 of 15 patients showed increased NK cell activity (Escudier *et al.*, 2005; Thery *et al.*, 2009).

Fig. 8. Dendritic cell exosomes can be harvested and used in immunotherapy.

#### **8.2.2 Non-small cell lung cancer phase I and II trials**

A similar trial to the melanoma study was performed in Stage III and IV non-small cell lung cancer (NSCLC) (Morse *et al.*, 2005). Of the 13 patients enlisted in the trial, 9 finished the treatments and no toxicity was observed. Immune responses to MAGE3 was observed in 3 of 9 patients, with MAGE3-specific T cells only detected in 1 patient and while an increase in Treg cells was observed in 2 patients (Morse *et al.*, 2005). This trial concluded that dendritic cell exosomes are safe for use therapeutically, but that better responses may result from including Treg inhibitors in the treatment. A Phase II trial has begun adding IL-15R and NKG2D to the exosomes as well as Treg inhibitor therapy in NSCLC patients whose disease has been stabilized by chemotherapy (Thery *et al.*, 2009).

#### **8.3 Summary**

40 Advances in Cancer Therapy

Additionally, vaccination of mice with exosomes containing ovalbumin resulted in delayed growth of ovalbumin-expressing tumors and induced pro-inflammatory Th1 T cell responses. As described in Section 7.2, exosomes isolated from heat-shocked cancer cells are more immunogenic *in vitro*. Using heat-shocked TEXs for immunization in mice showed similar immunogenicity, with 80% of mice remaining tumor free after challenge. While these studies have provided the groundwork for cancer vaccines, the immunosuppressive functions of cancer exosomes have caused many to be skeptical of their use as vaccine.

**8.2 Dendritic cell exosomes containing tumor antigens induce anti-tumor immune** 

The use of DEXs as artificial antigen presenting cells can elicit strong antigen-specific T cell responses (Kim *et al.*, 2004). Mouse studies using DEXs pulsed with tumor peptides resulted in the activation of tumor-specific T cells, proliferation of NK cells and tumor regression (Kim *et al.*, 2004; Chaput *et al.*, 2006). Utilization of this technology with great success in murine studies has led to the development of several clinical trials using autologous DEXs

A Phase I trial using dendritic cell exosomes to induce tumor regression was evaluated in 15 patients with metastatic melanoma (Escudier *et al.*, 2005). Autologous monocytes were isolated from patients by leukapheresis and differentiated *in vitro* to dendritic cells. These dendritic cells were cultured, exosomes isolated from the culture medium and pulsed with peptides from the tumor antigen MAGE3 (Escudier *et al.*, 2005). Patients received escalating doses of cryopreserved exosomes and their tumor progression and immune responses monitored. All 15 patients completed therapy but only 1 had specific T cell responses. Skin and lymph node mass reduction was observed in 5 of 15 patients and 7 of 15 patients

showed increased NK cell activity (Escudier *et al.*, 2005; Thery *et al.*, 2009).

Fig. 8. Dendritic cell exosomes can be harvested and used in immunotherapy.

**responses** 

as a treatment in cancer patients (**Figure 8**).

**8.2.1 Melanoma phase I trial** 

The use of exosomes as a cancer therapeutic has been demonstrated to be effective in murine studies and safe by Phase I clinical trials. Although some benefit was seen in patient trials, making these exosomal therapies more effective will likely require combination immunotherapies, chemotherapies and radiation. The use of exosomes to induce anti-cancer responses presents an exciting new field in cancer immunotherapy.
