Advances in Gene Therapy, Vaccines, and Immunotherapy

SAN FRANCISCO—Advances in gene therapy, cancer vaccines, and a variety of new antibody therapies for hematologic malignancies were the focus of a satellite symposium to the 42^nd Annual Meeting of the American Society of Hematology titled Scientific and Technical Innovations in Biology: Initiating Advances in Therapeutic Approaches to Hematological Malignancies. The program was sponsored by Fox Chase Cancer Center through an unrestricted educational grant from Genentech BioOncology and IDEC Pharmaceuticals.

Gene Technology and its Application in Therapy

It may now be possible to correct the gene defects of neoplastic cells, suggested Thomas J. Kipps, MD, PhD, professor and head of the Division of Hematology/Oncology, University of California, San Diego. Dr. Kipps said that cancer is a genetic disease and that gene transfer may correct the underlying defects, either by replacing genes missing in tumor cells or by silencing defective genes whose expression leads to cancer.

"Gene transfer also might be used to selectively kill tumor cells or to induce host antitumor immunity," Dr. Kipps said. His focus was on the use of gene therapy for active induction of host immunity against tumor cells, in contrast to passive immune therapy using therapeutic monoclonal antibodies.

"The problem in active immunotherapy is the inability of the host to recognize tumor cells. This might be due to a loss of T cells that recognize tumor, or to the silencing of those T cells that potentially can recognize and reject the tumor. Recent studies suggest that it may be possible to overcome immune tolerance to induce active immune activity against the tumor," Dr. Kipps said.

The most common vectors being studied in an attempt to achieve this goal are plasmids that carry DNA into the cell or viruses that transfer genes into the cell (such as adenovirus and herpes simplex virus). Dr. Kipps said that considerable interest is currently centered on the CD154 protein, which is expressed on T cells soon after T-cell activation and that interacts with CD40. This leads to activation of antigen-presenting cells that can induce T-cell proliferation and cytokine production. (See Figure 1.) Researchers are using infection with an adenovirus-CD154 construct to convert "stealth" leukemia cells into "alarm" antigen-presenting cells (APC) that can activate the immune system against leukemia cells.

"Infection with Ad-CD154 results in stable, high-level surface expression of CD154 on chronic lymphocytic leukemia (CLL) cells. This induces these cells to express high levels of immunostimulatory molecules," Dr. Kipps said. "In addition, Ad-CD154-infected CLL cells induce bystander CLL cells to express immunostimulatory molecules that also can activate T cells, to produce other T cells that are cytotoxic against leukemia cells." Pilot studies of this approach in CLL patients showed a decrease in leukemic cell count and some stabilization of disease in treated patients.

"The phase-I Ad-CD154 CLL clinical trial showed an increase in immune cytokines (IL-12 and IFN-gamma), phenotypic changes in transduced and bystander CLL B cells, an increase in blood absolute T-cell counts, an increase in leukemia-specific T cells, a decrease in absolute lymphocyte counts, and a decrease in lymph node size," Dr. Kipps reported.

Ad-CD154 is now being studied in a phase-II trial of CLL patients who were refractory to standard therapy or who had advanced disease and elected to have Ad-CD154 as front-line therapy. Patients are being given 5-10 biweekly doses of 3-5 × 10⁸ transduced Ad-CD154 CLL cells administered intravenously.

Advances in Immunology Extend into Clinical Practice

Louis M. Weiner, MD, chairman and senior member, Department of Medical Oncology, Fox Chase Cancer Center, Philadelphia, described monoclonal antibodies as, "the first successful attempt in the history of oncology to develop targeted cancer treatment."

"The common theme of targeted therapy is target acquisition leading to manipulation of cellular function. Targets can be located on tumor cells, in the tumor microenvironment, or in host response elements," Dr. Weiner said.

Determinants of tumor targeting by antibodies include antigen specificity, tumor physiology, antibody size, systemic clearance and metabolism, antibody valence effects on antibody retention in the tumor, and antibody affinity. Limitations include high intratumoral pressure leading to convection pressure and impeding the move of antibodies to the center of the tumor. Dr. Weiner said that in solid tumors, IgG molecules might require a month to pass from blood vessel to tumor interior.

Therapeutic applications of antibodies include perturbation of signal transduction, immunoconjugates, and antibody-dependent cellular cytotoxicity (ADCC). Obstacles to ADCC include getting the antibody to the tumor site, restricted leukocyte traffic to the tumor, inadequate in situ effector cell expansion and activation, and tumor-mediated immune inhibition, which may contribute to failure to expand in situ effector cells.

Signal transduction perturbation is accomplishing by using the Fc domain of the antibody to engage cell surface targets. Immunoconjugates are antibodies bound to isotopes, toxins, cytotoxic agents, or cytokines. ADCC targets the T cell. The antibody Fc domain interacts with natural killer cells or macrophages and causes phagocytosis or cytotoxicity via perforin molecules.

Antibody-dependent enzyme prodrug therapy (ADEPT) delivers an antibody-enzyme conjugate to the tumor, and the enzyme at the tumor site essentially converts a prodrug the patient has taken to active drug.

Another approach is pretargeted radioimmunotherapy. Antibody-streptavidin conjugate is delivered to the tumor; unbound immunoconjugate is cleared; and biotinylated radionuclide is then given, which sticks to the immunoconjugate and irradiates the tumor cell.

Dr. Weiner said that use of anti-idiotype vaccines represents another promising approach and that some vaccines effective against tumor antigens can be prepared without the use of a tumor antigen. Antibody Ab1 is produced to the tumor antigen, and Ab2 is produced against Ab1. "Ab2 immunization stimulates production of Ab3, leading to a specific antitumor antigen response," Dr. Weiner said.

Functional targets for antibodies currently under study include HER2/neu, epidermal growth factor receptor (EGFR), CD20, B-cell idiotype, vascular endothelial growth factor receptors, and Apo-2/TRAIL ligand. "There has been a sea change in how we look at these molecules," Dr. Weiner said. "Previously we looked for high tumor specificity. Signal transduction is also affected in normal cells, but tumor cells may be more dependent on the function of selective signaling pathways, so that such pathways can be the Achilles heel of the tumor." He also noted that most antibodies that do not perturb signal transduction are not therapeutically effective.

Cancer Vaccines for Hematologic Malignancies

"Vaccines against cancer, particularly for hematologic malignancies, may at last have a future," predicted Freda K. Stevenson, DPhil, of Southampton University Hospitals Trust, Southampton, United Kingdom.

Dr. Stevenson’s group has tested a DNA fusion vaccine in four patients so far. The genes in the vaccine include a tumor-derived gene (scFv of the clonal idiotypic immunoglobulin) fused to a safe fragment (Fragment C) of tetanus toxin. The encoded fusion protein therefore contains a tumor antigen linked to a foreign protein, and it induces specific anti-idiotype immunity. This design induced protection against lymphoma in two mouse models.

The phase-I/II study used DNA fusion genes in patients with low grade follicle center lymphoma in clinical complete remission following first-line chemotherapy. The dose-escalation trial will enroll 30 patients at doses from 500 to 2,500 µg/dose, given in 6 injections over 12 weeks. Three patients have received the 500 µg dose level, and one patient had the 1,000 µg dose. Dr. Stevenson said that one patient had residual disease in a 3 cm × 4 cm node that disappeared by the end of the vaccination program. There was no toxicity, and no antimuscle antibodies were produced. Anti-DNA antibodies were within the normal range.

"Naked DNA as an intramuscular injection and linked to immune-activating pathogen-derived sequences is the route we’ve chosen," Dr. Stevenson said. "At least it has the value of simplicity." It is also effective in a model of mutiple myeloma.

‘‘For myeloma, we are planning to vaccinate the donor of donor lymphocyte infusions to treat patients undergoing allogeneic transplants, in the hope of generating an antimyeloma response,’’ Dr. Stevenson said.

Current gene targets in clinical trials of DNA vaccines include VH/VL for B-cell lymphoma, B7 for melanoma, MART-1 for melanoma, B7 for head and neck cancer, and CEA for colorectal cancer.

Advances in Monoclonal Antibody Therapy

Reviewing the year’s advances in monoclonal antibody therapy. Thomas A. Davis, MD, said that large antibodies given by IV administration have immediate access to circulating cell surface targets, and can be very effective in the hematologic compartment. Dr. Davis is senior investigator, Cancer Therapy Evaluation Program, National Cancer Institute, Bethesda, Maryland.

Most antibody preparations have been associated with infusion reactions commonly known as the "cytokine release syndrome." Dr. Davis said that the NCI is preparing a new toxicity description to cover those reactions. "If patients are monitored closely, severe reactions can be avoided," he said. "Just because a patient reacts initially does not mean the patient should not get future doses. The NCI is changing the toxicity requirement for clinical trials to reflect that."

A significant recent trend is the progression toward increasingly human monoclonal antibodies, Dr. Davis observed. "Two companies have techniques for producing fully human antibodies in transgenic mice," he said.

Combining of antibody therapy with chemotherapy is seen as the most promising near-term prospect in cancer treatment. Other promising areas are the addition of immune-modulating agents to augment immune interactions and new approaches to pretargeting, bispecific targeting, and antigen cross-linking.

New agents recently approved or in development include CAMPATH-1H, which targets CD52; daclizumab (Zenapax), which targets the IL-2 receptor; tositumomab and ibritumomab, which are radiolabeled antibodies against CD20; Hu1D10, which targets a variant of the HLA-DR molecule; epratuzumab, which targets CD22; gemtuzumab (Mylotarg) and HuM195, which target CD33 in myelogenous leukemias; and bevacizumab, which targets vascular endothelial growth factor (VEGF).

"There are many exciting agents and approaches on the horizon. Techniques to manipulate antibody interaction with the patient’s immune system, or to augment antigen expression, may significantly improve antibody efficacy. There are high expectations that antibody combinations may provide even more definitive benefits, and a host of new targets may be identified with microchip arrays and the farming of the genome," Dr. Davis said.

Optimizing Rituximab Therapy

"CD20 has turned out to be the big winner for lymphoma therapy," said David G. Maloney, MD, PhD, assistant professor, Department of Medicine, Division of Oncology, University of Washington, Seattle, Washington. CD20 itself, however, turned out to be not essential for tumor cell survival, and CD20-negative escape past rituximab (Rituxan) is possible. Dr. Maloney said that re-treatment of responders only induces subsequent remission in 40% of patients, suggesting that some type of acquired resistance is involved.

"The dominant mechanism of tumor cell kill in patients treated with rituximab continues to be unknown. It is likely that several mechanisms are important. These include interactions with host-mediated immune effector functions including complement-dependent cytotoxicity and antibody-dependent cell-mediated cytotoxicity; direct effects on the tumor cells including the induction of apoptotic changes or apoptosis and growth inhibition or arrest," Dr. Maloney said.

He suggested that these mechanisms may overlap in function. Antibody treatment might induce early changes of apoptosis, which in turn might trigger complement fixation and immune clearance of tumor cells. Immune clearance might be through specific receptors on accessory cells such as macrophages or monocytes that recognize either the bound antibody, complement factors, or exposed phosphatidylserine.

Rituximab’s activity as a single agent, the lack of a dose-limiting toxicity, and nonoverlapping side effects make combinations of rituximab with conventional chemotherapy a promising approach. Dr. Maloney, however, said that optimal sequences and best potential combinations have yet to be identified.

Pharmacokinetic studies will also play a role. Dr. Maloney said that nonresponders clear rituximab more quickly and thus get less sustained levels of antibody between rituximab treatments. "There is a cause and effect question here," he said. "Did the patients with tumor that responded have more sustained rituximab levels because the target tumor was reduced and so rituximab remained in the circulation?"

Rituximab Plus Chemotherapy Shown Effective in NHL

Bertrand Coiffier, MD, PhD, of Hospices Civils de Lyon, Université Claude Bernard, Lyon, France, described the rationale behind the rituximab/chemotherapy trial. The findings of that trial were presented later during the annual meeting plenary session.

Dr. Coiffier said that the reasons for adding chemotherapy to rituximab include the fact that 87% of patients treated with rituximab monotherapy had a decrease in measurable tumor volume. In addition, some CD20+ lymphomas do not respond to rituximab, and relapsing patients after a first response to rituximab do not always respond to a second course. Rituximab also sensitizes cells to chemotherapy.

"Do we have evidence that combination therapy is better?" Dr. Coiffier said. "Not yet. Some data show a possible benefit, including an increase in response rate and an increase in molecular response. Few, if any, data show an increase in toxicity with the combination."

In phase-II trials, rituximab plus chemotherapy produced response rates of over 90% in follicular lymphoma and cleared bcl-2 rearranged cells in some patients. Rituximab plus interferon is being studied in an American and in an Italian study. "The rationale is that interferon increases CD20 antigen expression and that there is synergy for the mechanisms of action of both drugs," Dr. Coiffier said.

In the American study in 38 relapsing patients, there was an 11% complete response (CR) and a 34% partial response (PR) rate. Median duration of response was 22 months. In the Italian study of 46 patients, the overall response rate was 72%, with 29% CR. Adverse effects in both studies were related to interferon.

A study of CHOP (cyclophosphamide, doxorubicin, Oncovin [vincristine], prednisone) plus rituximab produced a CR of over 60%. Time to progression plateaued at 3 years and was sustained to 4 years. Dr. Coiffier said that after CHOP there is usually a continued decrease in time to progression.

The Group d’Etude des Lymphomas de l’Adulte (GELA) trial Dr. Coiffier reported was a prospective randomized study comparing eight cycles of CHOP to eight cycles of CHOP plus rituximab in patients with diffuse large B-cell lymphoma of stage II, III, or IV. The major endpoint was event-free survival.

Dr. Coiffier reported that CR and undocumented CR rate was 76% with CHOP plus rituximab vs 61% with CHOP alone (P = .0004). "There was a huge advantage in favor of rituximab-CHOP," he concluded.