**5.2 Production and adminstration of DC vaccines**

No standard protocol exists for the production of DC vaccines, but there are some general components that are often involved. First, the DCs are collected from the patient via

Targeting Molecular Pathways

well tolerated [84].

yield.

**6. DC vaccination in breast cancer** 

with other anti-HER-2 therapies.

in a number of tumor types.

for Prevention of High Risk Breast Cancer: A Model for Cancer Prevention 141

The results from initial trials reported a 41 % relative reduction in the risk of death and a trend toward increased survival in the men treated with Sipuleucel-T compared to the men in the placebo group [85, 86]. The IMPACT study, a double-blind, placebo-controlled, multicenter trial involving 512 men with metastatic castration-resistant prostate cancer, was designed with overall survival as the primary endpoint. Of note, these men were either asymptomatic or minimally symptomatic, representing a patient population that is at an earlier stage and therefore more likely to be amenable to immunotherapy. Compared with the placebo arm, patients in the Sipuleucel-T arm achieved a 22% relative reduction in the risk of death. This represented a 4.1 month increase in median overall survival between the placebo and Sipuleucel-T arms. There was no significant difference achieved in the prostatespecific antigen response or time to progression between the two arms. Sipuleucel-T was

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

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

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

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

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 be briefly described.

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 response [55].

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 expensive cancer therapies ever approved [77, 82].

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 the tumor cells [36].

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

The results from initial trials reported a 41 % relative reduction in the risk of death and a trend toward increased survival in the men treated with Sipuleucel-T compared to the men in the placebo group [85, 86]. The IMPACT study, a double-blind, placebo-controlled, multicenter trial involving 512 men with metastatic castration-resistant prostate cancer, was designed with overall survival as the primary endpoint. Of note, these men were either asymptomatic or minimally symptomatic, representing a patient population that is at an earlier stage and therefore more likely to be amenable to immunotherapy. Compared with the placebo arm, patients in the Sipuleucel-T arm achieved a 22% relative reduction in the risk of death. This represented a 4.1 month increase in median overall survival between the placebo and Sipuleucel-T arms. There was no significant difference achieved in the prostatespecific antigen response or time to progression between the two arms. Sipuleucel-T was well tolerated [84].
