A Follicular Lymphoma Vaccine Effective at the Molecular Level
A Follicular Lymphoma Vaccine Effective at the Molecular Level
By Brian McKinley, MD
Cancer has been characterized in many ways. on one level, cancer is a disease of the immune system. Deficiencies in the body’s ability to detect and destroy malignant cells are responsible for the progression of many tumors. Therefore, immunotherapeutic treatment strategies that improve the host response to cancer have a certain appeal. A cancer vaccine is one type of immunotherapy that attempts to facilitate tumor recognition and augment tumor destruction by the native immune system.
There are many challenges in developing a successful cancer vaccine. Before beginning the development process, an investigator must decide whether to focus on cell-mediated (T cell) or humoral (B cell) immune responses. Since most experimental models indicate that T cells are required for the clearance of tumor cells,1,2 most vaccines concentrate on improving host T cell responses. The first challenge for researchers is to design a vaccine that successfully incites an immune response to the tumor. That is, the vaccine must get the host’s immune system to recognize the tumor as foreign. Next, the vaccine regimen must effectively eradicate tumor cells from the body. This is usually accomplished by evoking a cytotoxic T cell response. Ultimately, a cancer vaccine should result in improved disease-free or overall survival. Finally, a vaccine program should also be capable of detecting and quantifying the immune responses it elicits. In a recent issue of Nature Medicine, Bendandi and colleagues report their success in achieving tumor-specific recognition and eradication, as well as complete molecular remission, in a subset of follicular lymphoma (FL) patients treated with autologous FL cells and granulocyte-monocyte colony-stimulating factor (GM-CSF).3
FL is a clonal B cell malignancy in which most patients achieve a complete remission (CR) after cytoreductive chemotherapy; however, the majority of FL patients who enter CR eventually relapse, presumably due to occult, minimal residual disease (MRD).3 Bendandi et al targeted FL patients in their first clinical CR for vaccine therapy because of their high risk of relapse.
Bendandi et al’s vaccine protocol was designed to induce a tumor-specific, cytotoxic T cell response against autologous FL cells.
General Scheme for Eliciting T Cell Response
The immune system is challenged with tumor-associated antigen (TAA). Dendritic cells (DCs) internalize the TAA and process it. The processed TAA is then expressed on the DC membrane in combination with major histocompatibility complex I and II (MHC-I and MHC-II) molecules. The mature, antigen-presenting DCs then migrate to lymph nodes where the tumor-specific T cell response is activated. The humoral response depends on the association of CD4+ lymphocytes with MHC-II molecules while the cytotoxic T cell response depends on the association of CD8+ lymphocytes with MHC-I molecules and the CD4+ lymphocyte/MHC-II interaction. GM-CSF, known to enhance tumor-specific CD8+ T cell immunity, is administered with the TAA.
The TAA in this experiment originates from the patients themselves. Since FL is a clonal malignancy, the idiotypic determinants of the immunoglobulin protein it synthesizes are unique. These unique idiotypes can function as a TAA that is patient-specific. In order to isolate each patient’s unique TAA, Bendandi et al harvested pre-treatment lymph nodes from 35 patients with stage III/IV FL (follicular small-cleaved cell or mixed lymphoma). The patients were then treated with chemotherapy. CR was achieved in 23 patients. CR was confirmed by physical examination, bilateral bone marrow biopsies, and stability of residual abnormalities on CT scans and lymphangiograms. Two patients were excluded due to technical difficulties producing the vaccine and one patient was excluded due to an early relapse. This left 20 patients with FL in their first CR who received the vaccine for this study.3
Bendandi et al isolated immunoglobulin protein from each patient’s pre-treatment lymph nodes. This TAA was then conjugated to keyhole limpet hemocyanin (KLH), a potent immunogen.4 KLH assists in the recruitment of CD4+ T cells and the maturation of a memory cytotoxic T cell response and provides a positive control antigen to monitor T cell priming.5 In order to allow for immune recovery, the vaccine was given at least six months after the completion of chemotherapy.3
The TAA-KLH conjugate was mixed with GM-CSF and administered subcutaneously in four monthly doses. Additional doses of GM-CSF were administered daily, and an identical booster vaccine was given two months after the initial vaccination cycles.3 A series of experiments was then carried out to answer the following questions:
1. Did vaccination enhance the tumor-specific immune response to unmodified tumor?
2. Did the immune response after vaccination consist of tumor specific CD8+ and/or CD4+ T cells?
3. Did vaccination enhance tumor cell lysis?
4. Was post-vaccination tumor cell lysis the result of CD8+ T cells, CD4+ T cells, or both?
5. Did vaccination induce molecular remission in those patients who were initially t(14,18)+?
Did Vaccination Enhance the Immune Response to Unmodified Tumor?
In 95% (19 of 20) of patients, tumor necrosis factor (TNF) levels were significantly higher in assays from post-vaccination peripheral blood mononuclear cells (PBMC) combined with unmodified autologous FL than in assays of post-vaccination PBMC or tumor alone. Further studies on six randomly selected patients demonstrated that pre-vaccination PBMC generated no significant levels of TNF when exposed to autologous FL. This indicates that the immune response, as measured by TNF production, was enhanced by vaccination and was dependent upon re-exposure to an individual patient’s autologous tumor.3
Did the Immune Response After Vaccination Consist of Tumor-Specific CD8+ and/or CD4+ T Cells?
Additional data from the six randomly selected patients demonstrate that both CD8+ and CD4+ T cells could be isolated from PBMC exposed to tumor. When these T cell populations were re-exposed to autologous FL, the CD8+ cells produced more TNF than the CD4+ cells. These T cells produced negligible amounts of TNF in the absence of tumor. These results indicate that both types of T lymphocytes participate in the post-vaccination immune response to tumor challenge and this response requires the re-exposure to tumor. These results also suggest that CD8+ T cells may play a bigger role in the post vaccination immune response.3
Did Vaccination Enhance Tumor Cell Lysis?
In all six patients subjected to further studies, there was substantial tumor-specific lysis of autologous FL by post-vaccination PBMC. The was no significant lysis by pre-vaccination PBMC.3
Was Post-Vaccination Tumor Cell Lysis the Result of CD8+ T Cells, CD4+ T Cells, or Both?
Studies on three patients demonstrated that CD8+ T cells were more effective at mediating tumor-specific lysis than CD4+ T cells. The CD4+ T cells were, in turn, more effective mediators of lysis than non-T cells.3
Did Vaccination Induce Molecular Remission in Those Patients Who Were Initially t(14,18)+?
Eleven of 20 patients vaccinated in this experiment had PBMC that were PCR+ for the t(14,18) translocation before chemotherapy. All eleven remained PCR+ after chemotherapy despite achieving clinical CR. After treatment with the vaccine, 73% (8 of 11) of PCR+ patients converted to PCR-negative, indicating the malignant cells with the translocation had either been eradicated from the body or at least cleared from the peripheral blood as a result of the vaccination. This elimination of the translocation from the peripheral blood has persisted for a median of 18 months from vaccination (range, 8-32 months) and no patient has reverted back to PCR+. All eight patients who achieved a molecular remission had measurable T cell responses, whereas only five of these patients achieved an antibody response, indicating that an antibody response was not necessary for molecular remission.3
Potential for Molecular Monitoring. Lopez-Guillermo et al exploited the fact that FL is commonly associated with a specific chromosome translocation, t(14,18). Using the presence of t(14,18)+ cells as a marker for MRD, the authors developed a polymerase chain reaction (PCR) assay capable of detecting one malignant-t(14,18) positive cell among greater than 100,000 normal cells. FL patients who initially express this translocation, then lose it after chemotherapy, are said to have achieved a molecular response. The authors demonstrated that patients who achieved a molecular response to treatment had improved failure-free survival.6
Bendandi et al have made a breakthrough in the world of lymphoma vaccines. Although their approach was not revolutionary, it may provide the first demonstration that human FL cells are capable of presenting endogenous immunoglobulin as a target of CD8+ cells. Furthermore, it is the first to demonstrate anti-tumor effects of a vaccine in homogenous population of lymphoma patients. Although longer clinical follow-up is required to assess the ability of the vaccine to improve disease-free and overall survival, the results are exciting. In addition to being an important addition to lymphoma vaccine research, this study highlights the following challenges for those involved in solid tumor vaccine research:
1. Identifying specific tumor antigens;
2. Concentrating efforts on patients with a minimal burden of disease; and
3. Identifying molecular markers to identify patients at high risk of relapse and to measuring vaccine effectiveness on a molecular level.
Identifying specific TAA is more difficult in solid tumors than in lymphomas. Lymphoma cells are malignant clones and, as such, are a homogenous group of tumor cells that closely resemble the original cancer cell. This homogenous group of tumor cells expresses one TAA in each individual patient. Conversely, solid tumors are made up of many different clones and, as such, express a vast array of tumor antigens. This makes identifying and characterizing the immune response to various TAAs very difficult. Consequently, it is difficult to pick which TAAs to include in a vaccine. The use of autologous tumor to activate a cytotoxic T cell response is an attractive option to solid tumor oncologists since it allows the individual immune systems to determine which TAAs are important. Still, the search for TAAs in solid tumors continues and may eventually lead to successful vaccination for these malignancies.7
An Exception: Melanoma Vaccines
One exception to this general rule is melanoma vaccines. Melanoma vaccine research is much more advanced than that in other solid tumor fields. Melanoma has a long history of immunotherapeutics as evidenced by BCG vaccination trials. Melanoma researchers have identified a relatively small group of TAAs and targeted them with various vaccine strategies.8,9 Despite the success in pre-clinical studies, meaningful clinical responses have been less common. Rosenberg et al vaccinated metastatic melanoma patients with a synthetic peptide vaccine and achieved a 42% response rate.8
Another problem with solid tumor vaccine studies is their focus on patients with extensive tumor burdens. While Phase I and II studies are necessary to document the safety and response rates of these vaccines, therapeutic trials should enroll patients at high risk of relapse but with minimal residual disease. Surgical treatment of most solid tumors is quite analogous to the cytoreductive chemotherapy of FL. Just as medical oncologists treat FL patients with chemotherapy to achieve "complete remission," we operate on patients with curative intent, ostensibly rendering our patients "disease-free." But we know that, depending on tumor histology and stage, many will recur. The time to intervene for those patients is sooner rather than later.
Unfortunately, we do not have any effective molecular markers for those tumor cells left behind at surgery, no t(14,18) translocation, to tell us which cells are likely to recur. More prospective research is needed on our patients with early stage disease to identify some novel molecular markers that might predict recurrence. Once these markers have been verified, their presence or absence in the patient’s blood or bone marrow may give us early clues about the efficacy of vaccines before disease-free and overall survival are known. (Dr. McKinley is a Fellow in Surgical Oncology at Roswell Park Cancer Institute, Buffalo, NY.)
References
1. Greenberg PD. Adv Immunol 1991;49:281-355.
2. Shu S, Chou T, Rosenberg SA. J Immunol 1987;139:295-304.
3. Bendandi M, Gocke CD, Kobrin CB, et al. Nature Med 1999;5:1171-1177.
4. Timmerman JM, Levy R. Nature Med 1999;4:269-270.
5. De Gruijl TD, Curiel DT. Nature Med 1999;5:1124-1125.
6. Lopez-Guillermo A, Cabanillas F, McLaughlin P, et al. Blood 1998;91:2955-2960.
7. Brossart P, Stuhler G, Flad T, et al. Canc Res 1998;58:732-736.
8. Rosenberg SA, Yang JC, Schwartzentruber DJ, et al. Nature Med 1998;4:321-327.
9. Nestle FO, Alijagic S, Gilliet M, et al. Nature Med 1998;4:328-332.
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