**6. DC vaccination in breast cancer**

140 Cancer Prevention – From Mechanisms to Translational Benefits

leukapheresis and then activated *ex vivo* with the specific tumor antigen and human granulocyte-macrophage colony-stimulating factor (GM-CSF). The activated DCs are then reintroduced to the patient in order to stimulate a T cell response. We have developed a DC vaccine targeting HER-2 and used to treat patients with DCIS. The production process will

First, peripheral blood monocytes are obtained by leukapheresis followed by elutriation under good clinical practice conditions. Monocytes are then cultured overnight in monocyte-macrophage serum-free medium GM-CSF and interleukin (IL)-4. Immature DCs are next pulsed with HER-2 MHC II binding peptides, extracellular and intracellular domain peptides for 12 hours. In order to potentiate the benefits of signaling infectious nonself at the time of vaccine administration, we then sequentially culture the cells with interferon-gamma (IFN-γ) and bacterial lipopolysaccharcharide (LPS), a TLR agonist, prior to pulsing with MHC class I peptides. As mentioned previously, TLR stimulation leads to cytokine release and DC maturation. This activation strategy assures that the DC are able to robustly secrete IL-12 which maximizes their ability to produce IFN-γ and a functional T cell

The major technical disadvantage to the use of DC vaccines relates to the *ex-vivo* nature of their generation. A number of obstacles must be dealt with in order to make the process successful and efficient [36, 81]. Most importantly, the production of DC must be made individually for each patient. The process is dependent on access to leukapheresis facilities at the treatment center. Leukapheresis is complex procedure that does not always generate consistent results. The procedure is associated with minimal patient morbidity including, brief electrolyte imbalances, minimal risk of infection and vascular injury. Another important consideration in the production of DC vaccines is the financial burden of manufacturing and administering such an individualized treatment. Sipuleucel-T, the only cancer vaccine that is currently approved by the FDA, is priced at \$31,000 and is typically given three times, making a complete treatment cost \$93,000. This is one of the most

We advocate injecting the vaccines directly into distal lymph nodes. It has been shown that only a proportion of peripherally administered DCs migrate to regional lymph nodes [83]. Using ultrasound guidance to inject the cultured DCs directly into distal lymph nodes assures that a predetermined and defined quantity of antigen-loaded DC cells are delivered to the site of T-cell sensitization, thus allowing the vaccine to activate an adaptive immune response. This method also synchronizes peak IL-2 secretion to occur when the DCs are close to T cells. Vaccinating patients prior to surgical resection also allows us to have the opportunity to review the histopathological effects of vaccination on

In April 2010, the FDA approved the first cancer vaccine. This individualized DC vaccine, Sipuleucel-T (Provenge; Dendreon Corp.), is approved for use in men with metastatic castration-resistant prostate cancer. Similar to other DC vaccines, the production of Sipuleucel-T begins with leukaphoresis and the isolation of peripheral blood mononuclear cells. The DCs are then activated by exposure to a recombinant fusion protein (prostatic acid phosphatase and GM-CSF). The treated cells are re-infused into the patient. This process is

be briefly described.

response [55].

the tumor cells [36].

expensive cancer therapies ever approved [77, 82].

repeated every two weeks for a total of three treatments [84].

Successful reduction of HER-2 over expressing tumors requires activation of both the innate and adaptive immune responses [87]. As described, DCs are unique in their ability to elicit responses from tumor-specific effector and memory T cells. Numerous strategies exist that aim to introduce tumor antigens into DCs in order to generate vaccines (loading individual tumor peptides, transfer of tumor-specific DNA or RNA through lipofection or viral vectors, whole tumor cell fusion). An early DC vaccine strategy for breast cancer involved the production of DC/tumor cell fusion vaccines. Avigen *et al* conducted a trial in which 16 patients with metastatic breast cancer were injected with a fusion vaccine using tumor cells obtained from a biopsy and DCs acquired from leukapheresis [88]. Three patients had a significant clinical response. Unfortunately the efficacy of fusion cell generation is less than 45% and multiple patients were not able to receive the full course of vaccines due to limited yield.

In 2007, Park *et al* published data from a phase I study in which they treated 18 metastatic breast cancer patients with Lapuleucel-T (Dendreon Corp), a vaccine produced in a similar fashion as sipuleucel-T, although a HER-2 fusion protein is used [89]. The study was designed to evaluate the safety and immunologic activity of the novel agent. The therapy was well tolerated. Significant immune responses were stimulated (as measured by lymphocyte proliferation and IFN-γ enzyme-linked immunospot assays). The therapy was also associated with tumor response and extended disease stabilization. Further trials are warranted to determine the efficacy in patients with earlier stage disease and in combination with other anti-HER-2 therapies.

Another DC vaccine approach that is being explored is Adevexin (Introgen Therapuetics), made form leukapheresed APCs that are transfected with a replication-impaired adenoviral vector that brings the p53 gene under control of a cytomegalovirus promoter [90]. This vaccine is not specifically anti-HER-2, but instead works under the theory that p53 restoration can be used to treat cancer. This vaccine triggers p53-specific T-cell responses against cancer cells with mutant p53 and has proven to be safe and synergistically effective in a number of tumor types.

Currently other anti-HER-2 DC vaccines trials are recruiting patients and results of these trials are pending [74]. We are conducting a clinical trial with a novel DC vaccine designed

Targeting Molecular Pathways

possibility to treat and possibly prevent breast cancers.

for Prevention of High Risk Breast Cancer: A Model for Cancer Prevention 143

for the development of breast cancer vaccines should focus not only on targeting molecules that are specific tumor related antigens, but also to the downstream signaling pathways involved in tumorigenesis. For example, recent work has elucidated the role of survivin (Figure 1), a protein in the anti-apoptotic family. Blocking the expression of survivin, was found to have a direct role in the initiation of apoptosis of breast cancer cells [93]. Upregulation of survivin is also directly linked to HER-2 over expression [94]. A novel antisurvivin based therapy in combination with an anti-HER-2 DC vaccine is an exciting

Another promising possibility is to develop immune responses against other HER family members including HER-1 and HER-3. HER-3 has no ligand binding sites but intracellular signaling moieties and can partner with HER-2 and HER-1. HER-3 signaling may lead to HER-2 resistance thus developing vaccines against these targets may supplement HER-2

MUC-1(Figure 1), an epithelial glycoprotein that is over expressed in may breast cancers, is another molecule that has yet to be fully exploited as an anti-cancer drug target. This molecule has been implicated in tumor invasion and metastases [95]. MUC-1 pulsed DC based vaccine trials for patients with pancreatic and biliary carcinomas are currently underway and the clinical efficacy of these vaccines is not known at this time [96]. MUC-1 is

There is immeasurable potential for vaccine therapy to be used in immunocompetent patients with minimal disease to prevent disease progression and recurrence. Even more exciting is the potential to treat patients with no disease at all. Forty percent of the participants in our recent vaccine trial converted from ER positive HER-2 positive to ER positve HER-2 negative suggesting that HER-2 vaccines can direct or steer tumors to more favorable phenotypes. The ultimate goal is therefore to produce a vaccine that could prevent breast cancer formation altogether. The development of successful breast cancer prevention would be applicable to other solid tumor malignancies such as colorectal, head and neck

[1] Nathanson, N. and O.M. Kew, *From emergence to eradication: the epidemiology of* 

[2] Schiller, J.T. and D.R. Lowy, *Vaccines to prevent infections by oncoviruses.* Annu Rev

[4] Dawood, S., et al., *Defining breast cancer prognosis based on molecular phenotypes: results* 

[5] Sorlie, T., et al., *Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications.* Proc Natl Acad Sci U S A, 2001. 98(19): p. 10869-74. [6] Sorlie, T., et al., *Repeated observation of breast tumor subtypes in independent gene expression* 

[7] van 't Veer, L.J., et al., *Gene expression profiling predicts clinical outcome of breast cancer.*

*poliomyelitis deconstructed*. Am J Epidemiol. 172(11): p. 1213-29.

*from a large cohort study.* Breast Cancer Res Treat. 126(1): p. 185-92.

*data sets.* Proc Natl Acad Sci U S A, 2003. 100(14): p. 8418-23.

[3] Jemal, A., et al., *Global cancer statistics.* CA Cancer J Clin. 61(2): p. 69-90.

vaccines to make them better preventive agents eliminating vaccine escapes.

another molecule that could potentially used to target breast cancers.

cancer, lung cancer, gastric cancer and other GI malignancies.

Microbiol. 64: p. 23-41.

Nature, 2002. 415(6871): p. 530-6.

**8. References** 

to treat DCIS patients with lesions that overexpress HER-2. No other DC-based vaccines have been designed specifically to treat HER-2 expressing DCIS tumors. We anticipate a significant reduction in disease burden in our patients after a complete vaccine course. We hope that this vaccine will also be preventative in terms of both disease recurrence and rate of transformation into invasive breast cancer.

Our strategy of vaccine production utilizes both MHC I and II peptides as well as ex vivo activation with IFN-γ and LPS to yield polarized DC cells that induce a unique set of soluble factors including high levels of IL-12 and Th1 chemokines not elicited through traditional vaccines methods. This innovative DC vaccine strategy called Immune Conditioning by Activated Innate Transfer (ICAIT) uses monocyte-derived DCs that are activated with and a special clinical-grade TLR 4 ligand, LPS and IFN-γ (ICAIT-DC). This unique DC activation method gives the DCs qualities that are not found in the so-called "gold-standard" DCs used in prior vaccine trials. The "gold standard" DC vaccines, activated with TNF, IL-6, PGE2, IL-1β, have the potential to simulate aseptic inflammation [91]. In contrast, ICAIT-DCs produce high levels of factors that specifically enhance aspects of anti-tumor immunity such as NK cells which augment tumor rejection, and TNF and IL-12 which are antiangiogenic [55, 92]. ICAIT-DCs also have the distinct ability to influence the quality of sensitized T cells and can condition T cells for recognition of HER-2 expressing tumors. Lastly, ICAIT-DCs posses a killer function that enables them to lyse breast cancer cells.

Our DC vaccine is unique in its design against DCIS rather than invasive breast cancer. Our first neoadjuvant trial involved treating patients with HER-2 expressing DCIS tumors. The patients were treated with a course of four weekly intranodal injections of ICAIT-DCs that had been pulsed with HER-2 derived proteins. This approach yielded promising results that have positive implications for the treatment and prevention of high risk breast cancers. Specifically, 85% of ICAIT-treated patients developed immune responses to at least one of the HER-2 peptides. Eleven of the 22 patients with residual DCIS treated with the vaccine in our initial studies showed loss of HER-2 expression and tumor regression. The immunized patients developed a specific immune response against the HER-derived peptides and presented high levels of CD4+ and CD8+ T lymphocytes. These results have potential positive implications not only for prognosis but also in terms of breast-conserving surgery [55]. Five of the 27 patients had no evidence of remaining disease. The vaccination was most effective in patients with hormone-independent DCIS as 40% of ER negative HER-2 positive patients had no residual disease whereas only 5% of ER positive HER-2 positive had no residual disease. The vaccine appeared to alter the phenotype of the remaining DCIS in the patients that were found to have residual disease. The rate of change to a different postvaccination phenotype was significantly different between the ER positive and ER negative patients. 43.8% of the patients that were initially ER positive and HER-2 positive phenotype converted to ER positive and HER-2 negative phenotype. In comparison, 50% of the patients that initially had tumors that were ER negative and HER-2 positive changed to the ER negative and HER-2 negative phenotype. These results supported the safety and efficacy of the DC based HER-2 vaccine. The vaccine induced a decline or eradication of HER-2 expression (work not yet published).

#### **7. Future direction and prevention**

There are a number of molecules that have been discovered to be present in breast carcinomas that have not yet been exploited to their fullest potential. The future directions for the development of breast cancer vaccines should focus not only on targeting molecules that are specific tumor related antigens, but also to the downstream signaling pathways involved in tumorigenesis. For example, recent work has elucidated the role of survivin (Figure 1), a protein in the anti-apoptotic family. Blocking the expression of survivin, was found to have a direct role in the initiation of apoptosis of breast cancer cells [93]. Upregulation of survivin is also directly linked to HER-2 over expression [94]. A novel antisurvivin based therapy in combination with an anti-HER-2 DC vaccine is an exciting possibility to treat and possibly prevent breast cancers.

Another promising possibility is to develop immune responses against other HER family members including HER-1 and HER-3. HER-3 has no ligand binding sites but intracellular signaling moieties and can partner with HER-2 and HER-1. HER-3 signaling may lead to HER-2 resistance thus developing vaccines against these targets may supplement HER-2 vaccines to make them better preventive agents eliminating vaccine escapes.

MUC-1(Figure 1), an epithelial glycoprotein that is over expressed in may breast cancers, is another molecule that has yet to be fully exploited as an anti-cancer drug target. This molecule has been implicated in tumor invasion and metastases [95]. MUC-1 pulsed DC based vaccine trials for patients with pancreatic and biliary carcinomas are currently underway and the clinical efficacy of these vaccines is not known at this time [96]. MUC-1 is another molecule that could potentially used to target breast cancers.

There is immeasurable potential for vaccine therapy to be used in immunocompetent patients with minimal disease to prevent disease progression and recurrence. Even more exciting is the potential to treat patients with no disease at all. Forty percent of the participants in our recent vaccine trial converted from ER positive HER-2 positive to ER positve HER-2 negative suggesting that HER-2 vaccines can direct or steer tumors to more favorable phenotypes. The ultimate goal is therefore to produce a vaccine that could prevent breast cancer formation altogether. The development of successful breast cancer prevention would be applicable to other solid tumor malignancies such as colorectal, head and neck cancer, lung cancer, gastric cancer and other GI malignancies.
