Tumor Vaccination and Antibody-Mediated Immuno-therapy

19 Tumor Vaccination and Antibody-Mediated Immunotherapy

Volker Schirrmacher

Introduction

Tumor vaccination attempts to stimulate the patient’s own immune system in such a way that immune reactions against the tumor are assisted in a targeted manner. Recently, this field has again become a burning issue due to three new developments:

– new ways of identifying and isolating tumor-associated antigens (TAA)

– new cell culture methods for propagating antigen-presenting cells (APC) and tumor-reactive immunocytes

– molecular biology methods for targeted gene transfer in tumor cells or other cells.

The idea of using antitumor vaccination for the targeted destruction of tumor cells in the body is not new (14), but only recent advances in immunology and molecular biology have prepared the ground for evaluating immunotherapy in controlled studies. Various approaches aim at selective immunostimulation, though they differ considerably in concept and realization.

In contrast to viruses and bacteria, tumor-associated antigens usually lead only to weak immune defenses; this is due to the fact that the expressed tumor antigens differ only slightly from physiologically occurring antigenic cell structures (42).

This tolerance of the immune system needs to be lowered. Here, we discuss the two most promising strategies: vaccines and monoclonal antibodies (MAbs).

Tumor Vaccines and Active Specific Immunotherapy (ASI)

All forms of vaccination rely on the immune system’s active cooperation and support in order to provoke a targeted immune response. This is true also for tumor vaccination, which is therefore referred to as active specific immunotherapy (ASI).

The thesis favored initially, namely, that the immune system distinguishes only between “self” and “nonself,” is no longer valid today (13). Obviously, the immune system recognizes tumors and develops immune responses, although these are often inefficient (48). The recognized antigens are often classic autoantigens, which therefore also occur on normal cells of the body.

Table 19.1 provides a list of representative human tumor antigens. These include, for example, oncofetal antigens and cancer-testis antigens (CTA), which are expressed on embryonic cells or germ cells; they are suppressed in differentiated body cells but may be re-expressed in tumor cells. In addition, tumor antigens include differentiation antigens, viral antigens, and also peptide sequences derived from proteins coded for by mutated or overexpressed genes.

If a tumor cell is supposed to induce a T-cell-mediated immune reaction, a professional antigen-presenting cell (e. g., a dendritic cell) must phagocytose tumor proteins and, subsequently, present peptide fragments of the tumor antigens on its surface together with genetically determined major histocompatibility complex (MHC) molecules (55). The complex of MHC molecule and tumor peptide is then recognized by cytotoxic (CD8⁺) or helper (CD4⁺) T lymphocytes. T-cell activation can only occur when, in addition to antigen recognition, activation signals are mediated to the T cells. This activation includes their proliferation and maturation into effector cells, which are either cytotoxic or release cytokines, such as IFN-γ. Cytotoxic T cells can recognize and directly destroy the tumor cell, once the same MHC–tumor–peptide complex has been identified on the malignant cell. This complex sequence of events must be taken into account when developing a successful tumor vaccine.

Clinical Studies

Active Immunization with Intact Modified Tumor Cells

A tumor vaccine made up of intact tumor cells from the patient has the advantage that all relevant tumor proteins and peptides are present in the vaccine and that the molecular characterization of their structures is not required (41, 53, 60). To increase the efficiency and immunogenicity of the tumor cells, they can be transfected ex vivo, for example, with cytokine gene sequences (21). After proper selection, the genetically modified tumor cells will secrete the desired cytokines aimed at enhancing the body’s immune response. The difficulty is to achieve a sufficiently high transfection rate with tumor cells (1), a problem that can be solved by using new vector constructs that are usually based on adenoviruses or retroviruses (73), but also by using so-called virus-like particles (VLP) (39).

Table 19.1 Representative human tumor antigens

Antigen1	MHC restriction²	T-cell epitope3 (amino-acid sequence)
Cancer-testis Antigen MAGE-1 NY-ESO-1	HLA-A1 HLA-A2	EADPTGHSY QLSLLMWIT
Differentiation antigen Tyrosinase CEA Immunoglobulin isotype (myeloma)	HLA-A2 HLA-A2 Individual differences	MLLAVLYCL YLSGANLNL Patient-specific
Product of a mutated gene ß-catenin Bcr/Abl	HLA-A24 HLA-DR4	SYLDSGIF ATGFKQSSKALQRPVAS
Product of an overexpressed gene HER-2/neu p53	HLA-A2 HLA-A2	KIFGSLAFL LLGRNSFEN
Viral antigen HPV-E7	HLA-A2	YMLDLQPETT

¹ All tumor antigens are derived from cellular proteins. ² Intracellular degradation of these proteins creates the peptides listed in the right column; these peptides are associated with the HLA molecules listed here and are transported as MHC–peptide complexes to the cell surface. ³ The MHC–peptide complexes are recognized by tumor antigen-specific T cells by means of accurately fitting T-cell receptors. The sequences of the T-cell epitopes are shown in amino-acid code.

The Newcastle disease virus (NDV), an avian paramyxovirus, has the following special features:

–tumor-selective replication (52)

–induction of proinflammatory cytokines and chemokines (70)

–mediation of costimulatory signals for T cells (62)

–oncolytic effects (45)

–high degree of safety (no DNA integration, no risk of viral spread or infection)

–high tolerance and few side effects (36).

In the future, this virus can also be used for the purpose of gene therapy, because lately it has been produced by recombinant technology (43, 47). Thus, NDV represents an interesting alternative in tumor gene therapy. It has been used in Heidelberg, Germany, since 1986 for infecting tumor cells in order to enhance their tumor immunogenicity (19, 25, 50, 51, 68). The virus-modified autologous tumor vaccine, called ATV-NDV, is being produced in a special laboratory from tumor tissue freshly obtained from the patient and then used postoperatively as an adjuvant therapy for the prevention of metastasis: (2)

• In a study involving 18 patients with renal cell carcinoma and eight patients with prostatic carcinoma, tumor cells were obtained during surgery and transfected with a retroviral vector coding for GM-CSF (37). After irradiating these tumor cells, they were applied subcutaneously to the patients at four-week intervals. The side effects were minor (mild fever, chills, pain in the limbs, and itching). One of the renal cell carcinoma patients showed regression of pulmonary metastases for seven months. None of the patients in the group with prostatic carcinoma responded to the treatment. The biggest problem in these studies was to harvest and propagate sufficient amounts of autologous tumor cells.

• In a second study, which involved 17 patients with metastatic renal cell carcinoma (30) and with a positive cutaneous reaction to recall antigens, the primary tumor was first removed. Cell hybrids were then created within 12 hours from autologous tumor cells and allogeneic dendritic cells by means of electrofusion. At intervals of six weeks, the patients received at least two subcutaneous injections into the region of the inguinal lymph nodes. In case of a clinical response after 12 weeks, a booster vaccination followed every three months. No major side effects were observed. Eleven out of 17 patients treated developed positive skin reactions (delayed type hypersensitivity, DTH) as a sign of a specific immune response following the exposure to tumor cells. Seven patients (41 %) responded to the therapy, with four complete remissions (23 %), two partial remissions (12 %), and one mixed response (6 %). The problem of this study was with the size and selection of the patient cohort. Thus, “spontaneous” remissions of metastases after removal of the primary tumor have been observed in nonselected patients in up to 10 % of the individuals (32). Whether the better result of this study can be attributed solely to the selection of immunocompetent patients or whether the vaccine therapy had contributed to the result, must be clarified in larger case-control studies without preselected patients.

• Long-term survival advantages were observed in an earlier study on metastatic renal cell carcinoma in which we participated (44). This study included 40 patients with advanced renal cell carcinoma, each of whom had distant metastases in at least one organ at the time of surgery. Following nephrectomy, active immunization with intact, NDV-modified autologous tumor cells was performed by multiple vaccinations. In addition to immunization with the tumor vaccine ATV-NDV, the patients received low-dose recombinant interleukin-2 and interferon-α in three two-week cycles. In 40 patients evaluated, the following observations were made:

– five complete remissions (CR)

– six partial remissions (PR)

– 12 individuals with stable disease (SD; median 25 months)

– 17 tumor progressions

– median survival time of CR and PR patients: more than four years

– median survival time of SD patients: 31 months.

Twenty-three (57.5 %) of the 40 patients with CR, PR, or SD seemed to gain a survival benefit from this adjuvant treatment as compared with the progressive patients or with a historical reference group (44).

• Melanoma: For several years now, David Berd and co-workers in Philadelphia, USA, have evaluated a dinitrophenyl-modified autologous tumor vaccine. For 62 patients with stage III melanoma and lymphadenectomy, who were treated postoperatively with this vaccine, the five-year survival rate was 58 % (7). The results of earlier studies on clinical tumor vaccination against malignant melanoma have been summarized in 1995 (53).

• After fusion with a tumor cell, an antigen-presenting cell is thought to present a major portion of the tumor antigens on its surface and, hence, should efficiently stimulate the immune system (59). This idea led to a clinical study involving 16 patients with metastatic melanoma of an advanced stage (65). These patients received three subcutaneous vaccinations with at least 3 × 10⁷ tumor cells at two sites that were located as far away from the tumor as possible. The treatment was well tolerated and never reached level II of the World Health Organization scale of side effects. Two patients developed local vitiligo as a sign of induction or expansion of melanocyte-specific T cells. The mean survival time of 16.1 months was clearly better that the six months historically expected for such patients.

• In the case of primary mammary carcinoma, we can look back on long-term experience with postoperative adjuvant therapy using active specific immunization with the autologous tumor vaccine ATV-NDV: In a phase II study involving 62 patients, there were indications for a clear improvement in the five-year survival rate by approximately 30 % when using the most favorable application (2, 54). The number of tumor cells and their vitality were decisive for the quality and effectiveness of this virus-modified autologous tumor vaccine (2).

• In another study on vaccination, 38 patients with primary colorectal carcinoma (Dukes stage C) were postoperatively treated with the autologous tumor vaccine ATV-NDV. As was the case with the mammary carcinoma study, patients treated with the most favorable live cell vaccine obviously gained a long-term survival benefit as compared with those treated with a less favorable vaccine or those receiving no postoperative immunotherapy at all (40, 50>).

• In a prospective randomized phase III study on active specific immunotherapy, 254 patients with colon cancer received a BCG-modified autologous live cell vaccine. In patients with stage II (Dukes Stage 132/133) and stage III (Dukes Stage C) colon cancer, this lead to a reduction of recurrences by 43 % and of deaths by 32 % (P < 0.01) (67).

Active Immunization with Peptides and Heat Shock Proteins

Whereas the tumor-associated antigens in the tumor vaccines do not undergo further characterization prior to application, the situation is different for vaccination with TAA peptides. Only used here are antigenic proteins or peptide fragments of known amino-acid sequence, against which specific CD4⁺ and/or CD8⁺ T-cell responses have been demonstrated in vitro in the respective MHC context (20). However, it is very costly to define the immunogenic peptide domains of a tumor protein and to restrict the immune response to only the one or a few peptide domains that are usually limited to a few human leukocyte antigen (HLA) molecules.

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