Developing an Effective Breast Cancer Vaccine

Vaccination Strategies

Although additional vaccine strategies are in various stages of development,^[17,18] this section discusses four main approaches. Each strategy carries with it a set of pros and cons that bear consideration (Table). Although there are proponents for one strategy vs another, no one approach has been shown to be superior to others in all situations. In general, a vaccine strategy is composed of a method to deliver target antigens or epitopes to the immune system and an immune-stimulating adjuvant to trigger an immune response. These various aspects of a cancer vaccine strategy and some of the related immune pathways involved are illustrated in the Figure.

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Figure.

Figure. An illustration of some aspects involved in optimizing a breast cancer vaccine. The method of antigen delivery (eg, whole cell, lysate, DNA, pulsed DCs) is selected based on a variety of factors, such as the number of antigens selected for targeting, the feasibility of the approach, and immunologic compatibility. Equally important is enhancing APC activity through the use of vaccine adjuvants (GM-CSF, CpG) and combining the vaccine with costimulatory agents and/or immunomodulators that can abrogate inhibitory pathways (CTLA-4, 1-MT). ABH = 2(S)-amino-6-boronohexanoic acid, APC = antigen-presenting cell, CTL = cytotoxic T cell, DC = dendritic cell, IDO = indoleamine 2,3-dioxygenase, M = major histocompatibility complex, P = peptide, T = T-cell receptor.

Dendritic Cell Vaccines

DCs are the body's most effective antigen-presenting cells (APCs). The purpose of DCs is to process cancer antigens and present pieces of these antigens bound to major histocompatibility complexes to receptive effector T cells. The DCs are collected from a patient by leukapheresis and then manipulated ex vivo to activate them with the desired tumor antigens. The activated DCs are then reintroduced into the patient, and they can go on to activate effector T cells. The DC vaccine closest to FDA approval is sipuleucel-T (Provenge, Dendreon Corp, Seattle, WA) prostate vaccine.^[19] The cellular immunotherapy process employed by Dendreon Corp collects the patient's DCs using leukapheresis. The patient's APCs then undergo ex vivo stimulation with PA2024, a fusion protein consisting of full-length human prostate acid phosphatase and full-length human granulocyte-macrophage colony-stimulating factor (GM-CSF). The stimulated APCs are then returned to the patient and infused to stimulate the effector T cells to mount an antitumor response. This is also the method for the HER2-based breast cancer vaccine lapuleucel-T (Neuvenge, Dendreon Corp), which is currently in early clinical trials, although a HER2 fusion protein is used instead.^[20]

Another DC vaccine approach being explored in breast cancer uses leukapheresed APCs transfected with a replication defective adenoviral vector loaded with the wild-type p53 transcript under the control of a cytomegalovirus promoter (contusugene ladenovec; INGN-201).^[21] The viral infection triggers the activation of the DCs and, because p53 is made in the DC cells, the p53 is processed and presented on the surface. These DCs can trigger p53-specific effector T-cell responses against cancer cells with abnormally accumulated mutant p53. The advantage of this process is that, since DC vaccines use autologous cells that are primed ex vivo, the vaccine would be immunologically compatible with any patient who undergoes the process. Also, the ex vivo priming allows potentially better activation of DCs when removed from a cancer patient's immunosuppressive milieu.

When designing a clinical trial for a DC vaccine, many technical considerations need to be considered, such as appropriate APC selection, optimized ex vivo APC maturation protocols, and methods to optimize APC homing to draining lymph nodes.^[22,23] A major disadvantage of the DC approach is the need for leukapheresis facilities at the treatment center to generate the product. This may necessitate insertion of large-bore indwelling catheters in some cases, and leukapheresis carries with it the risks of hypotension, electrolyte derangements, and vascular injury. Another issue with DC vaccines is the difficulty in ensuring a consistent vaccine product due to varying leukapheresis yields, the number of activated APCs, and possible bacterial contamination issues. The logistical complexity of administering DC vaccines on a larger commercial scale may make it less viable, especially if a less complex vaccine with equal efficacy becomes available. However, advantages offered by DC vaccines may justify the inconveniences.

Viral-based Vaccination Vectors

Viral-based vaccination vectors represent an active field of investigation in cancer immunotherapy. Selection of viral vectors makes use of their natural ability to trigger immune responses and carry genetic material into cells for production of the target antigen. The antigens are processed intracellularly by APCs and are presented on major histocompatibility complexes to receptive effector T cells. One ongoing trial in metastatic breast cancer utilizes the vaccinia and fowlpox viruses loaded with a carcinoembryonic antigen (CEA) peptide and three costimulatory molecules (B7.1, ICAM-1, LFA-3), collectively designated TRICOM.^[24] The TRICOM molecules serve to augment the activation signal that an effector T cell receives from the APCs that take up the virus, leading to a more robust immunologic response against CEA. The vaccinia virus is used for the initial vaccinations, and the fowlpox virus is used for booster vaccines. This is because the body mounts an immune response to the vaccinia vector after the initial vaccines so another strain must be used for the subsequent booster shots. In a completed phase I trial, 40% of patients had stable disease and 1 patient developed a pathologic complete response.^[25]

An ongoing trial at the National Cancer Institute is studying the use of the CEA-TRICOM vaccine in conjunction with an intense chemotherapy regimen in patients with metastatic CEA-expressing breast cancer. The scalability of this approach is easier than that of DC vaccines as the viral vectors and peptide can be consistently mass-produced. However, a significant infrastructure is still required. One drawback of using CEA alone is a common one to any single epitope vaccine strategy: the potential for immune escape. Clones that express less CEA can escape the selective pressure of CEA reactive T cells. The PANVAC-VF uses the same fowlpox and vaccinia TRICOM technology but includes both CEA and MUC1 peptides, making it more difficult for immune escape to occur. Therion Biologics, which was the main commercial partner with the Cancer Therapy Evaluation Program (CTEP), recently ceased operations, so the commercial development plan for this vaccine is uncertain.

Peptide-based Vaccines

Peptide-based vaccines are based on the premise that cancer cells express certain tumor-associated antigens (TAAs) that are either absent or expressed at low levels in normal tissues. The prevalent TAAs that have been targeted in breast cancer involve the HER2 extracellular domain, CEA, and MUC-1.^[26] Current manufacturing techniques allow production of large amounts of highly purified, pharmaceutical-grade peptides for use on a larger scale. The peptides consist of specific amino acid sequences from these TAAs selected on the basis of their immunogenicity and their compatibility with specific human leukocyte antigen (HLA) receptor subtypes. Since different HLA subtypes can present different pieces of TAA proteins, it is important to select these peptides to match the target population (eg, HLA-A2+ patients). This HLA restriction limits the potential number of patients who can receive these vaccines, thus making it necessary to screen a larger pool of patients to yield the desired study population size. These peptides are generally not very immunogenic on their own and require additional measures to stimulate an adequate response. Some strategies to augment the immune response include using a potent APC-stimulating adjuvant such as GM-CSF or CpG oligonucleotides, utilizing computer algorithms to select the most immunogenic epitopes (parts of the protein that are targeted by the immune response), and simultaneously activating both cytotoxic T cells (CTLs) and helper T lymphocytes (HTLs).^[27–31] This approach is based on the fact that different epitopes of a TAA activate either CTLs or HTLs; therefore, creating fusion peptides that combine a CTLand HTL-activating portion may offer enhanced and more prolonged immune responses. Another factor to consider is that such specifically targeted vaccines make it easier for tumors to evade the immune response as surviving clones downregulate expression of the target antigen. To make this immune evasion more difficult, multiple peptides from different TAAs can be used in the vaccine.^[32] The clinical trials performed by Peoples et al^[33] using an HER2-based CTL-activating peptide E75 plus GM-CSF showed promising reductions in recurrence. For 171 enrolled patients, the recurrence rate in the vaccinated group was 5.6% compared with 14.2% in the observation group (P = .04) at a median follow-up of 20 months. The disease-free survival rates in the vaccinated and control groups were 92.5% and 77%, respectively.

These data provide proof of principle that cancer vaccines used in the minimally residual disease state can reduce recurrences. However, whether this reduction in recurrence is due to the E75 vaccine, the GM-CSF adjuvant, or both remains unclear. This is because the disease burden and cancer-related immunosuppression are low in the adjuvant setting. A significant finding was that only 48% of the vaccinated patients in the above-mentioned trial had positive dimer responses at 6 months, indicating a lack of durability in the CTL response to the E75 HER2 vaccine. A different HER2 epitope, AE37, which stimulates HTLs instead of CTLs, is currently being tested for its ability to elicit protective anti-HER2 responses in a clinical trial.^[34] To prolong the protective immune response, it is likely that both CTLs and HTLs need to be stimulated simultaneously and booster vaccines will be required in an efficacy trial.

Several advantages are associated with peptide vaccines: target antigens can be targeted with more specificity, large amounts of pharmacologic grade peptides can be produced easily, patients do not need to undergo any additional procedures, and the peptides are not pharmacologically active and so have few toxicities, if any. A key disadvantage of peptides is immunologic compatibility only in patients with specific HLA subtypes (ie, HLA-A2+ patients), depending on the epitope chosen. This can make accrual to a trial more difficult, as a significant portion of patients may not be included due to HLA incompatibility. Some epitopes, such as E75, demonstrate the ability to bind to multiple HLA subtypes, thereby increasing the number of patients who may be treated with that specific peptide.^[35] Also, since only one or two antigens are targeted by peptide vaccines, the risk of immune escape by tumor cells is higher as resistant clones with lower expression of those antigens can emerge. Additional peptides can be added, but the cost and complexity of the vaccine will increase substantially. Overall, properly designed peptide vaccines paired with active adjuvants provide an easily scalable, effective method to vaccinate large numbers of HLA-compatible patients.

Whole-cell Vaccines

Whole-cell vaccines are a fourth method for providing target antigens. Some vaccines can be from autologous cells prepared from a patient's own tumor samples or allogeneic cancer cell lines.^[36,37] Since tumor cells are not very immunogenic, either the cancer cells or other bystander cells included in the vaccine are transfected with vectors containing genes that express potent immunostimulating proteins or cytokines. These immunostimulating proteins include B7.1 (CD80) and, more recently, CCL21.^[38,39] APCs express B7.1, and this binds CD28 on effector T cells, leading to their activation. The chemotaxis signal provided by the cytokine CCL21 allows the recruitment of naive T cells to an injection site, in essence forming an outsourced lymph node.^[39] Additional compounds such as GM-CSF can be added as an adjuvant to stimulate APCs. A hybrid approach that has been clinically evaluated in metastatic breast cancer has involved fusing patients' tumor cells to their own leukapheresed DCs ex vivo and then reinjecting the fusion cells into the patients.^[40] The specific allogeneic cancer cell lines for the desired tumor type are selected on the basis of many factors. Some of the criteria involve the effectiveness of growing cells in culture, the expression of desired antigens, its availability as a clinical grade cell line, and patent issues. The advantages of using whole cells is that a broad array of TAAs is represented, thereby minimizing immune escape; thus, because the whole proteins are present, there are no HLA restrictions on who can receive the vaccine. An example of this approach in breast cancer involved the use of MDA-MB-231 breast cancer cells lipofected with cDNA expressing the costimulatory molecule CD80 and administered with GM-CSF in 30 women with metastatic breast cancer.^[38] The results of this trial were disappointing, with only 6 patients developing antibodies to HER2 and MUC-1 antigens expressed on the cancer cells. No clinical responses were noted, but this is often the case in patients with advanced disease. This approach is currently being scaled up for larger-scale production using complex cell line banking facilities to generate large batches of desired cell lines for production. However, it remains to be seen what combination of cell lines, cell modifications to express costimulatory molecules, and adjuvant compounds provides the most effective approach.

Table. Pros and Cons of Different Vaccine Types
Vaccine	Pros	Cons
Dendritic cell	Has a broad immunologic compatibility (no HLA restriction) Offers more control over APC stimulation and antigen presentation	Is logistically difficult and most difficult to scale up Includes risk of leukapheresis
Viral-based	Can introduce target antigen into immune cells effectively Can be produced on larger scale	Immune response against priming virus requires different virus for a booster
Peptide-based	Can be produced with high purity in large amounts Has low toxicity of peptides	Needs multiple peptides to prevent immune escape Is restricted to certain HLA types
Whole cell-based	Represents a broad array of antigens Can more readily produce allogeneic cell lines on a larger scale Is not restricted by HLA type	Autologous cell vaccines are labor-intensive and difficult to standardize Requires cell manipulation to be immunogenic

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