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Bast RC Jr, Kufe DW, Pollock RE, et al., editors. Holland-Frei Cancer Medicine. 5th edition. Hamilton (ON): BC Decker; 2000.

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Holland-Frei Cancer Medicine. 5th edition.

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Chapter 61Active Specific Immunotherapy with Vaccines

, PhD and , MD.

Unlike vaccines against infectious disease, which are administered prophylactically, vaccines against cancer are administered after the onset of disease. These therapeutic vaccines use attenuated whole cancer cells, cancer cell lysates, or purified tumor antigens, with or without adjuvants, to stimulate the patient’s immune system. Active specific immunotherapy (ASI) with cancer vaccines changes the tumor microenvironment and promotes the destruction of tumor cells.

An effective cancer vaccine elicits both humoral (antibody) responses and cellular (antigen-specific T-cell) responses. Antibodies may kill tumor cells by complement-mediated cytotoxicity (CDC) or by antibody-dependent cell dependent cytotoxicity (ADCC). T cells, particularly CD8+ T lymphocytes, are also capable of direct tumor killing. Cancer vaccines target tumor-associated antigens (TAAs), which include both proteins and carbohydrates. While carbohydrate antigens are not recognized by T lymphocytes, protein antigens are taken up by antigen-presenting cells (macrophages or dendritic cells), processed, and presented in the context of major histocompatibility complex (MHC) class I and class II molecules for priming and activation of CD8+ and CD4+ T lymphocytes. Activated CD4+ T cells secrete cytokines such as interleukin-2 (IL-2), which can initiate and amplify the CD8+ T-cell response. Activated antigen-specific CD8+ T cells recognize and lyse tumor cells. However, if an antigen is presented to CD8+ T cells without appropriate co-stimulation, it may lead to anergy or even death of the T cell. Vaccines can overcome these mechanisms of tolerance.

Tumor-Host Interaction

TAAs as Targets for Immunotherapy

There are several types of immunogenic TAAs. Neoantigens are not expressed by the normal or progenitor cells from which cancer cells are derived, but they can be found in other normal tissues. For example, melanoma-associated protein antigens MAGE-1 and MAGE-3 are not expressed in normal melanocyte progenitor cells of human melanoma, but can be found in testes. 1 Similarly, melanocytes do not express GD2 and O-acetylated GD3 until they undergo neoplastic transformation. 2, 3 However, GD2 and O-acetylated GD3 are found in the human brain and spinal cord 2 and are expressed by human T lymphocytes. 4 Oncofetal antigens are embryonic gene products in tumor cells, such as carcinoembryonic antigen (CEA), alpha-fetoprotein and several sialylated glycolipids. These antigens are rarely or poorly expressed by normal tissues but are found in fetal tissues and meconium.

Tumor-specific antigens are restricted to tumor tissues; they are not found in normal adult or fetal tissues. Such antigens are extremely rare. For example, a mutated form of cyclin-dependent kinase-4 (CDK4)-R24C antigen, which prevents binding of the CDK4 inhibitor called p16INK4a(a tumor suppressor protein), has been identified in 5% of human melanomas tested. 5 Another tumor-specific antigen is a sialylated glycoconjugate. The most common sialic acid in glycoproteins and glycolipids of normal human tissues is N-acetylneuraminic acid (NeuAc). Rarely, normal human tissues may contain O-acetylated NeuAc. In animal tissues, particularly in bovine and murine species, the common sialic acid is N-glycolyl neuraminic acid (NeuGc). NeuGc is not identified in human tissues but has been found in biopsy specimens of human gastric, liver, and colon cancers, as well as lymphoma. 6

Tissue-specific differentiation antigens are those present on both tumor cells (such as melanoma) and their progenitor normal cells (melanocytes). Early immunologists believed that T cells specific for a self-antigen would be deleted or at least functionally tolerated. The first differentiation antigen that was identified was tyrosinase, a copper-containing enzyme involved in melanin biosynthesis. Others include MART-1/Melan-A, gp100, TRP-1, and TRP-2. These antigens, with or without mutations or glycosylation/deglycosylation, are recognized by cytotoxic T cells. Occasionally, nonmutated precursors are found in normal progenitors. Because of their ability to recognize peptides derived from intracellular or cell-membrane antigens, T cells can specifically recognize mutations easily in neoplastic cells. The best examples are MUC-1 (commonly expressed in pancreatic and breast tumors), tyrosinase (the product of the c or albino locus), and pMel 17 (the product of silver locus). 7 Under appropriate conditions, all TAA systems can serve as targets for active immunotherapy with cancer vaccines.

If TAAs are found in normal cells, why are these cells not recognized and destroyed by the host’s immune system? TAAs might be hidden by their orientation or physical conformation or masked by other cell surface components; alternatively, they may be expressed in a density lower than the threshold level required for recognition. It is also possible that normal cells have surface molecules that render the cell resistant to immune attack. For example, complement restriction factors (CRFs) such as CD59, CD55, and CD46 on the normal cell surface prevent complement-mediated killing. A cytotoxic antibody may recognize and bind to an antigen on the cell surface, activate complement, but fail to form a membrane attack complex (MAC) because of blocking by CRFs. Tumor cells may also express CRFs to prevent CDC. 8

Alteration of Tumor Microenvironment by Shed TAAs

Possible TAAs may be continuously shed from the tumor cell surface and bind to reticuloendothelial cells and antibodies, causing immunosuppression. 9, 10 Gangliosides, glycosylphosphatidylinositol-anchored proteins such as CEA, 11 and other TAAs restricted to the outer layer of the bilayered lipid membrane are more likely to be shed. Shed TAAs appear to alter the tumor microenvironment. Lymphocytes recovered from tumor tissues, in contrast to those in peripheral blood, proliferate less readily in response to interleukin-2 (IL-2) or mitogens. Peripheral blood lymphocytes exposed to tumor cells or to their shed products show significantly reduced proliferation. 12 However, repeated washing of the peripheral blood lymphocytes from cancer patients can restore and enhance lymphocyte killing of tumor cells. 13 Suppression of lymph node lymphocytes in melanoma patients correlates inversely with the distance of the nodes from the primary tumor. 14

Immunosuppression is related to the stage of disease and overall tumor burden. 15 The presence of circulating TAA-antibody complexes in cancer patients who have not received immunotherapy suggests an early endogenous antibody response. 16, 17 As the tumor grows and sheds more antigens, these antigens bind to and mask free antitumor antibodies. 18 Surgical removal of the neoplasm can reverse this immunosuppression. 19 Thus, successful resection of primary sarcoma was associated with a four-fold rise in serum antitumor antibody titer. 15 Patients who had developed pulmonary metastasis postoperatively showed a progressive decline in their antitumor antibody titers. Studies of complement-fixing antibody titers in patients with sarcomas and melanomas have shown that complement-fixing antibodies are masked by the shed TAAs, neutralizing antitumor activity.

Antigen Alteration and Immunologic Heterogeneity

Although a malignant neoplasm usually evolves from a single transformed cell, most cancers are composed of genetically unstable populations of proliferating cells that become heterogeneous over time. 20 Heterogeneity enables subsets of the tumor cell population to evade the host’s immune response and to resist therapies. 21 Tumors recurring at the resection site of a primary neoplasm can differ antigenically from the primary tumor. This is consistent with the possibility that antigenic variants have been selected for their ability to escape immune surveillance. 22

The biochemical profile of gangliosides in human melanoma is a classic example of heterogeneic antigen expression. GM3 constitutes 95% of the gangliosides on melanocytes, the progenitors of human melanoma. 2 During the early, radial phase of migration, neoplastically transformed melanocytes express more GD3, a derivative of GM3, than do normal progenitors. 23 After the appearance of GD3, the tumor cells begin to migrate vertically. Subsequently, levels of GD3 continue to increase, and other derivatives of GD3, namely, GD2 and O-AcGD3, begin to appear. 3, 24, 25 These alterations in gangliosides correlate with and may facilitate invasion and metastasis. During the progression of melanoma, there are similar alterations in MHC antigens (HLA-DR), intercellular adhesion molecules (ICAM-1), and mucins (MUC-18). 26

Human leukocyte antigen (HLA) class I antigens are important molecules that contribute to interactions between cytotoxic T lymphocytes (CTLs) and their TAA targets. Defects in or loss of HLA class I antigen expression may therefore impair recognition of tumor cells by CTLs and minimize the efficacy of active specific immunotherapy. The absence of HLA class I antigen expression in nevi and in many melanoma lesions has been related to the clinical course of disease. 27 In murine tumor systems, loss of HLA class I antigen expression is associated with failure of the host to reject a tumor challenge. 28

Heterogeneic expression of TAAs within a given tumor poses a significant obstacle to univalent or purified antigen vaccine therapy. Elimination of cells that bear a single major antigen can still permit the survival of a subpopulation that expresses a minor antigenic variant. Antigenic heterogeneity may reflect the selective elimination of immunogenic cells, leading to the outgrowth of less-immunogenic subpopulations. To compensate for heterogeneic antigen expression, several antigenically distinct tumor cell lines or purified antigens can be combined so that the vaccine collectively expresses all the TAAs of a given neoplasm. 22

Strategies for Increasing the Immunogenicity of Vaccines

The goals of active specific immunotherapy are to change the suppressed tumor microenvironment and to eliminate the diverse clones within a tumor cell population. Thus immunologic responses to a vaccine should be able: to alter the tumor microenvironment by clearing immunosuppressive TAAs from circulation; to minimize the immunosuppressive activity of suppressor T cells and suppressor inducer cells; to promote cytokine activation of antigen-presenting cells such as macrophages, monocytes, histiocytes, and dendritic cells; and to ensure antigen presentation with appropriate co-stimulation (e.g., induced expression of B7). 29– 35 In addition, these immunologic responses should stimulate the generation of activated CTLs and direct their migration into metastatic tumors that rarely contain lymphocytes.

Although univalent immunotherapy cannot achieve the complex immunomodulation described above, it can clarify the role of a particular agent. For instance, Livingston and co-workers 36 showed that a fraction of melanoma patients immunized with a purified ganglioside antigen conjugated to a protein immunostimulant (keyhole limpet hemocyanin) developed high levels of IgM antibody to the single antigen. Although the number of responding patients was not high, responders had improved survival. This suggests the importance of the ganglioside antigen as a component of a polyvalent vaccine against human melanoma. By exposing the patient’s immune system to a polyvalent preparation of proteins (recognized by T cells) and carbohydrates (T cell independent) from different tumor cell lines mixed with bacillus Calmette-Guérin (BCG), Morton and colleagues 30, 37 nearly doubled the fraction of patients who developed a better immune response to tumor cell vaccines. Cyclophosphamide was also used to reduce the suppressor T-cell functions that interfere with B-cell production of antibodies and with other immunoregulatory functions.

Diversity of Adjuvants for Cancer Vaccines

Adjuvants used in clinical trials have included live vaccinia virus, Salmonella extracts, and viable BCG and BCG derivatives such as cell wall skeleton, trehalose dimycolate, muramyl dipeptide, and glycolipids. Table 61.1 lists tumor vaccines and immunopotentiators used in clinical trials for different cancers. 29, 32, 36, 38– 54 Data obtained from clinical trials and animal models underline the importance of the ratio of tumor cells to adjuvant as well as the sequence and site of administration of the tumor cell preparation and the adjuvant. The adjuvant is a critical component of a cancer vaccine because it attracts antigen-presenting cells (APCs) to the sight of vaccination, where they may be activated to take up the antigen for processing and presentation.

Table 61.1. Tumor-Specific Vaccines Used in Clinical Trials to Promote Immunogenicity of TAAs.

Table 61.1

Tumor-Specific Vaccines Used in Clinical Trials to Promote Immunogenicity of TAAs.

Protein Adjuvants

Addition of a highly antigenic carrier protein to an otherwise nonantigenic substance can often evoke an immune response. In the 1960s, reports using rabbit gamma globulin attached to proteins of autologous tumor cells reported some therapeutic benefit, but the efficacy of this type of treatment has not been confirmed. Recently, administration of keyhole-limpet hemocyanin (KLH) attached to melanoma-associated ganglioside GD3 was shown to elicit a persistent anti-GD3 IgM antibody response in melanoma patients. 55

Viral Adjuvants

In animal models, infection of tumor cells by certain viruses augments the immunogenicity of tumor antigens. 41 Randomized clinical trials have been undertaken with allogeneic or autologous tumor cells infected with Newcastle disease virus, vesicular stomatitis virus, and vaccinia virus in patients with melanoma and osteosarcoma. 39, 42, 56, 57 Tumor vaccines of viral etiology are the focus of several investigations. 58 Wallack and Sivanandham 45 updated the results of clinical trials using vaccinia-melanoma oncolysate (VMO) vaccine in 250 patients with stage II melanoma. Those patients who had the highest antimelanoma titers after vaccination sustained improved survival. Gissmann and co-workers 59 recently discussed the prospects for development of a papilloma viral vaccine, based on a report that papilloma viral extracts stimulate immunity against existing tumors in women with cervical cancer.

Bacterial Adjuvants

Whole bacteria (BCG, Corynebacterium parvum, Salmonella minnesota) and bacterial components including cell wall skeleton, trehalose dimycolate, monophosphoryl lipid A, methanol extractable residue (MER) of tubercle bacillus, and Freund’s adjuvant are potent immunostimulants. These bacterial adjuvants can evoke tumor-specific cellular and humoral immune responses.

Chemical Adjuvants

TAAs in human cells can also be modified chemicals such as iodoacetate and cholesteryl hemisuccinate, 39, 52, 60 which increase the immunogenicity of tumor cells. Certain enzymes, such as neuraminidase, enhance the immunoresponse to neoplasms by chemically altering the surface glycoconjugates. 51, 53, 61 Repeated intradermal immunization with neuraminidase-treated allogeneic acute myeloid leukemic cells prolonged disease-free survival in patients treated with chemotherapy.

Naked DNA Adjuvants

Nucleic acids can be introduced into appropriate bone marrow-derived APCs via direct transduction. This “naked” DNA may activate the APCs, facilitate tumor-specific T-cell activation, or enable expression of co-stimulatory molecules.

Whole Cells as Cancer Vaccines

Autologous versus Allogeneic Whole-Cell Vaccines

In animal models, injection of living autologous tumor cells in numbers too small to cause progressive tumor growth has generally provided the most effective immunogen. Animal studies also document that a whole-cell vaccine is superior to soluble vaccines or oncolysates in eliciting antitumor CTL or humoral responses. 62– 64 Schirrmacher and Hoegen 62 have demonstrated that irradiated trypan blue-excluding live tumor cells are superior to dead cells or crude membrane preparations (oncolysates) in eliciting a syngeneic MHC class I–restricted tumor-specific CTL response. When comparing T-cell stimulatory capacity of a whole-cell vaccine with that of a vaccine consisting of a glutaraldehyde-fixed polyvalent ultrasonicated tumor extract, in the absence or presence of IL-2, only the whole-cell vaccine was able to trigger a specific CTL response in vitro. Furthermore, the disruption of tumor cells by freezing and thawing led to a complete loss of CTL stimulatory capacity. Also, viral oncolysates did not stimulate a tumor-specific CTL response in tumor-bearing animals whereas whole-cell vaccines did. These findings indicate that tumor cell membrane integrity may be required for stimulating a tumor-specific class I MHC–restricted CD8+ CTL response.

The possibility that viable autologous tumor cells could result in tumor growth at the inoculation site has precluded their clinical use in cancer vaccines. Viable tumor cells are inactivated by a variety of methods, including irradiation, mitomycin C, freezing and thawing, or heat treatment. Such treatments may, however, chemically alter tumor antigens and diminish the effectiveness of the vaccine. Because autologous tumor cells express the same blood group and histocompatibility antigens as the host, they are considered ideal for tumor cell vaccines. However, the restricted availability of autologous tumor tissues limits the amount of vaccine and, thus, the number of immunizations. Moreover, antigen expression may vary from site to site; cells isolated from nodules at one site may not have the same TAA profile as autologous cells from another metastatic site.

Allogeneic tumor grown in cell culture can provide sufficient vaccine for multiple injections. Mixtures of cells from different tumors can provide a spectrum of TAAs. On the other hand, passage of tumor cells in culture may introduce contaminants and favor the growth of subpopulations with a different antigenic phenotype. Serum-free media or media that contain human serum are considered ideal for the preparation of tumor cell vaccines. Fetal bovine serum (FBS), for example, is widely used in culture media. Often the proteins and glycolipids in FBS are incorporated into tumor cells and will function as xenogeneic antigens. 65 Patients who receive tumor cells grown in FBS develop an antibody response against FBS components. 66 Tumor cells grown in culture may also undergo antigenic alterations similar to those documented for ganglioside profiles of human melanoma and glioma. 23, 67 In some cases, these alterations have been used to advantage by mixing different cell lines with increased TAA expression to prepare allogeneic tumor cell vaccines. 30

Genetically Altered Tumor Cells

Tumor-specific immune responses can be induced by genetically altering the tumor cells to express cytokines, MHC antigens, and co-stimulatory molecules required for T-cell activation. Transduction of murine tumor cells with the genes for cytokines such as IL-2 and IL-4 has led to rejection of the genetically altered tumor cells by the syngeneic host. 68, 69 Immunized mice withstood challenge by native homologous tumor cells but not by dissimilar tumor cells, indicating tumor specificity. In the case of IL-4, nontransduced subcutaneously transplanted renal carcinoma cells regressed following inoculation in the opposite leg with IL-4 transduced cells. Dranoff and colleagues 28 found that when 10 genes for cytokines or immunogenic cell-surface molecules were transduced into B16 murine melanoma cells, only granulocyte macrophage colony-stimulating factor (GM-CSF) was effective in preventing growth of challenge tumors. Irradiated B16 melanoma cells protected only 1 of 19 C57 BL/6 mice from a challenge dose of B16 cells, whereas the irradiated GM-CSF transduced cells protected 16 of 20. In another experiment, 100% of mice immunized with irradiated GM-CSF transduced cells survived a challenge of a million cells, whereas no more than 50% immunized with other cytokine-transduced cells survived a tumor challenge. In addition, administration of GM-CSF transduced cells several days after a tumor challenge was associated with prolonged survival and complete regression of tumor. Selective depletion experiments with antibodies showed that both CD4+ and CD8+ cells were necessary. These impressive results have already led to an exploratory randomized phase I trial of GM-CSF gene transduction in renal carcinoma. 70

The success of this and other trials of active specific immunotherapy using genetically altered tumor cells will require a stable method of gene transfer, a sufficient number of transduced tumor cells, a cytokine concentration high enough to evoke an antitumor response, and an intradermal route of vaccine administration. The documented superiority of the intradermal route 71 may reflect the presence of Langerhans’ cells that differentiate into potent antigen-presenting cells. In addition, the genetically altered tumor cells must be viable but unable to replicate in the host. Interaction between the B7 family of co-stimulatory molecules and CD28 receptors expressed on T cells can enhance the level of cytokine production by CD4+ T cells subsequent to antigen recognition. Animal studies document rejection of the tumor after transfection of B7 co-stimulatory molecules into some tumors, and augmentation of immune response against challenge of wild-type tumor. 72 In other studies, simultaneous transfection with genes for MHC class II, IL-4, or IL-7 induced potent antitumor immune response. 73– 75 Table 61.2 provides an abbreviated list of gene-modified vaccine trials. 76– 90

Table 61.2. Genetically Altered Tumor Cell Vaccine Trials.

Table 61.2

Genetically Altered Tumor Cell Vaccine Trials.

Clinical Trials of Tumor Vaccines

The history of vaccine therapy reflects different levels of understanding of the immune response. 91 For almost a century, investigators have immunized patients with viable or lysed tumor cells. Results of these early clinical studies were highly variable; some investigators reported tumor regression after repeated injection of large numbers of viable autologous and allogeneic tumor cells at 14-day intervals, whereas others failed to notice any apparent benefit or observed a change in the differential leukocyte counts after intraperitoneal injection of tumor extracts. The postoperative use of irradiated tumor cells to immunize patients with malignant melanoma appeared to radiosensitize the residual disease.

After 1960, emphasis was given to various kinds of tumor cell antigens and to the use of different adjuvants mixed with tumor cell vaccine. Development of antibodies against tumor cells was observed when patients with malignant tumors received intramuscular injections of tumor cell lysate combined with complete Freund’s adjuvant (containing components of Mycobacterium bovis). Injecting the purified antibodies into subcutaneous nodules produced dramatic regression of tumor. The immunogenicity of human tumor cells can also be improved by coupling the cells to rabbit gamma globulin. Forty-two cancer patients were treated with autologous cells with rabbit gamma globulin. 92 Responders showed delayed-type hypersensitivity (DTH) responses to the vaccine, suggesting the induction of cell-mediated immunity. Autoimmunization was found to augment circulating antibodies and CTLs. 93 The first report of the regression of cutaneous metastases of melanoma following intralesional injection of live BCG not only redirected the course of immunotherapy trials but also rekindled interest in combining tumor cell vaccines with extrinsic adjuvants such as BCG. 94, 95

Current Status of Vaccine Therapy for Human Cancers

The interpretation of tumor vaccine trials is confounded by the variability of human cancer, and thus arises the need for precise definitions of the patient population. Another problem is the need for appropriate randomly selected controls. Table 61.3 46– 48, 54, 61, 66, 141, 142, 149, 159, 160, 179 shows the results of immunotherapy trials classified according to Goodnight and Morton. 96

Table 61.3. Results of Clinical Trials of Immunotherapy against Various Cancers (Excluding Melanoma).

Table 61.3

Results of Clinical Trials of Immunotherapy against Various Cancers (Excluding Melanoma).

Patients with malignant melanoma seem to benefit the most from active immunotherapy, as shown by tumor regression and increased disease-free survival. Some of the results for other cancers are also encouraging. These trials must be interpreted according to the source of vaccine, its method of preparation and schedule of administration, and the tumor burden prior to therapy. Reducing the tumor burden enhances the success of immunotherapy. Trial results also vary with the quality, quantity, and route of administration of immunopotentiators such as BCG or C. parvum. Other variables affecting the success of vaccine therapy include disease status, duration of treatment, effects of other therapies, and degree of immunologic impairment.

Malignant Melanoma

Morton and co-investigators 94 were the first to demonstrate complete regression of metastatic tumor nodules after intralesional injection of living BCG: 90% of 184 melanoma metastases in eight patients regressed. Many clinical trials have confirmed this observation. The induction of systemic antitumor immunity by BCG is suggested by (a) regression of noninjected nodules at sites distant from intralesional injections, (b) the relationship between the clinical course of disease and the in vitro cytotoxicity of lymphocytes in the presence of autologous serum, and (c) the regression of a pulmonary metastasis in a patient treated for multiple intradermal metastases. 95 The interaction of BCG with tumor cells may facilitate the infiltration of APCs and lymphocytes into tumor nodules. Antibodies may be directed against antigenic determinants shared by BCG and tumor cells, or the administration of BCG may enable the immune system to recognize the TAAs on tumor cells more effectively.

In animal models, histiocyte-infiltrated lesions have been observed after BCG injection. 97 Bartlett and Zbar 98 confirmed that a mixture of irradiated tumor cells and BCG evoked a better antitumor response than did either component alone. Similarly, Morton and colleagues 43, 61 admixed tumor cell vaccine with BCG and administered it intradermally to melanoma patients. They noted an increase in the number of disease-free survivors and a lengthening of disease-free intervals but found no striking difference from BCG alone. Ratliff and co-workers 99 have shown that tumor cells that are able to fuse with BCG in the presence of fibronectin elicit better immune responses than do tumor cells that are not capable of fusing with BCG.

The antigen-specific therapeutic effect of tumor cell-BCG vaccine prompted clinical trials with tumor cell vaccines (Table 61.4). 29, 32, 40– 43, 45, 51, 53, 56, 112 These clinical trials identified several factors important for the success of a vaccine: intraindividual and interindividual tumor heterogeneity; factors augmenting immunogenicity of TAAs; unfavorable immunologic responses; tumor microenvironment (circulating immunosuppressive factors); tumor infiltration of immune effector cells such as lymphocytes and macrophages; and the functional role of antibodies to change tumor microenvironment and to kill the tumor cells directly or indirectly.

Table 61.4. Clinical Trials of Active Specific Immunotherapy in Patients with Metastatic Melanoma.

Table 61.4

Clinical Trials of Active Specific Immunotherapy in Patients with Metastatic Melanoma.

Early vaccines consisted of whole autologous or allogeneic tumor cells or cell extracts/lysates, with or without an immunostimulant such as BCG, Freund’s complete adjuvant, Newcastle disease virus, vaccinia virus, vesicular stomatitis virus, alum, purified protein derivative from Mycobacterium tuberculosis, or DETOX (a combination of mycobacterial cell wall with trehalose dimycolate and monophosphoryl lipid A derived from Salmonella). Their usual route of administration was intradermal although subcutaneous administration was not uncommon, and some investigators observed a clinical response after intralymphatic administration. Several trials have administered cyclophosphamide, an immunosuppressive drug, prior to the vaccine, since a subset of T lymphocytes can specifically suppress immunologic responses to the tumor antigens. Melanoma vaccines administered in recent clinical trials may broadly be classified into purified vaccines, cell lysate vaccines, and whole-cell vaccines, as discussed below. 22, 100

Purified Antigen Vaccines

These may include fully or partially purified TAAs. Purification eliminates HLA antigens. Among the purified melanoma vaccines are ganglioside vaccines, recombinant protein vaccines, anti-idiotypic antibody vaccines, and polyvalent shed-antigen vaccines. These vaccines are reasonably well characterized, and reproducible and do not induce anti-HLA class I and II antibodies, which may confound measurement of cellular immune functions involved in vaccine responses.

Ganglioside Vaccines

When patients were immunized with tumor cell vaccines consisting of irradiated autologous or allogeneic melanoma cell lines mixed with adjuvants such as BCG, C. parvum, or vesicular stomatitis virus, or treated with neuraminidase, trypsin, or glutaraldehyde, Livingston and co-investigators 38 observed that the humoral responses were often directed against HLA, viral antigens, bovine proteins used in culture of melanoma cells, and several nonspecific antigens. However, melanoma-associated gangliosides GM2 and GD2 were found to be immunogenic, which led to a randomized clinical trial of purified GM2. 101, 102 One hundred twenty-two patients with resected regional melanoma (American Joint Committee on Cancer [AJCC] stage III disease) were treated with low-dose cyclophosphamide followed by BCG with or without GM2. After a median follow-up of 63 months, the increases in disease-free survival (DFS) and overall survival for the GM2-BCG group were insignificant (11% and 18%, respectively). However, exclusion of patients with elevated prevaccine anti-GM2 antibody levels significantly increased DFS in this group (p = .02). Comparison of the two arms (BCG and GM2-BCG) of this trial as randomized failed to show a statistically significant improvement in DFS or overall survival. These investigators recently tested the immunogenicity of gangliosides coupled to KLH and administered with QS-21 adjuvant in melanoma patients. 55, 103

Immune responses to univalent ganglioside vaccines may have severe limitations. Although all tumor cells expressing the targeted ganglioside can be eliminated, residual tumor cells with no or low levels of the ganglioside antigen may proliferate and create a tumor that is resistant to therapy. The heterogeneity of melanoma-associated gangliosides can be the major impediment to success of therapies with univalent ganglioside antigens. Portoukalian and co-workers 104 administered autoclaved polyvalent purified melanoma ganglioside vaccine. Patient response was assessed by titers of IgM and IgG antibodies against all the melanoma gangliosides. Responders had significantly fewer recurrences (11 of 17) than did patients without elevated IgG antibody titers (13 of 15) (p < .001). The median disease-free interval was 71 weeks for responders and 26 weeks for nonresponders.

Anti-idiotypic Antibody Vaccines

Anti-idiotypic antibodies (anti-ids) are considered a potent melanoma vaccine since they mimic melanoma-associated antigens (MAAs). Ferrone 105 administered intradermally (0.5 to 4 mg) a monoclonal anti-id mimicking the epitope of a high-molecular-weight MAA (HMW-MAA) to 24 patients with AJCC stage IV melanoma. One patient showed a partial response, and 8 others had minor responses or stabilization of disease. Disease progression was observed in 10 patients.

Purified Protein or Peptide Vaccines

The potential of purified protein or peptide vaccines is documented by the role of T lymphocytes in tumor regression (Table 61.5). 105A For example, partial or complete tumor regression has been induced by adoptive transfer of T cells derived from tumor-infiltrating lymphocytes (TILs) and cultured with IL-2. Other studies have shown massive infiltration of CD4+ and CD8+ lymphocytes in regressing tumors from patients receiving IL-2-mediated immunotherapies and accumulation of infused T lymphocytes labeled with 111 indium in tumor sites. 106, 107 T lymphocytes isolated from TILs and peripheral blood lymphocytes (PBLs) from melanoma patients can specifically recognize autologous and allogeneic melanoma cells that share class I MHC alleles. 108 Antibodies to MHC class I molecules prevent lysis of melanoma cells by CD8+ T cells, and transfection of class I MHC genes (such as human leukocyte antigen A2 [HLA-A2]) into melanoma cells not expressing the MHC class I allele renders these cells susceptible to lysis by CD8+ lymphocytes. 109, 110 Finally, CD4+ lymphocytes from TILs recognize melanoma antigens presented by MHC class II molecules.

Table 61.5. Melanoma Antigens Recognized by T Lymphocytes.

Table 61.5

Melanoma Antigens Recognized by T Lymphocytes.

Peptide antigens used in tumor vaccines can broadly be classified into nonmutated and mutated antigens. Nonmutated antigens such as tyrosinase, TRP-1, MART-1, and gp100 are expressed by both melanoma cells and their melanocyte progenitors; these antigens are yet to be identified in other cancers. Nonmutated antigens such as MAGE-1, MAGE-3, are BAGE are expressed by melanoma but not by melanocytes; however, they may be expressed by other normal cells such as in testis, and they may be found in other cancers. Mutated antigens such as b-catenin and CNK-4 are found in melanoma as well as in other cancers. Most of these antigens are restricted by MHC class I antigens (see Table 61.5) although some, such as tyrosinase, may be restricted by MHC class II antigens.

The source of lymphocytes used for recognition of the melanoma antigens includes both tumor-infiltrating lymphocytes (TILs) and peripheral blood lymphocytes (PBLs). PBLs from melanoma patients and from normal donors recognize some of the epitopes. Although most of these antigens are involved in CTL-mediated tumor regression, larger studies are needed to determine clinical efficacy. A major concern is tumor heterogeneity. Although the tumor cell clones expressing the peptide epitopes recognized by T cells are destroyed by stimulating T cells, clones that do not express the protein or the peptide epitope escape immune attack. This suggests that a polyvalent peptide vaccine may be superior to a single purified peptide vaccine.

Shed-Antigen Vaccines

Polyvalency increases a vaccine’s chances of stimulating protective immunity. Melanoma cells in culture release almost half of the material expressed on their external surface within 3 hours but release only a fraction of their internal molecules. The shed material comprises highly enriched cell-surface proteins and other macromolecules in a fairly purified form. Bystryn’s group 111 harvested polyvalent shed antigens from four melanoma cell lines (three human and one hamster), purified these antigens to deplete their HLA component, and then tested the HLA-depleted antigens as a vaccine in a phase I clinical trial. The vaccine caused complete regression of cutaneous metastases in one patient (1of 13 AJCC stage IV patients), who had no evidence of disease for more than 60 months. The vaccine stimulated both humoral and cellular responses to melanoma in approximately 50% of 94 evaluable sequential patients with surgically resected regional (AJCC stage III) disease. 112 There was a relation between antimelanoma cellular immune responses and favorable clinical outcome; median DFS was 4.7 years longer and overall survival was 3.7 years longer in patients with a strong vaccine-induced DTH response. Three years after the onset of treatment, 70% of patients with a strong DTH response, but only 31% of nonresponders, were still disease free. A similar correlation was observed between vaccine-induced antimelanoma antibodies and improved survival. Overall median DFS was 30 months for vaccine recipients versus 18 months for historical controls; overall 5-year survival was 50% for vaccine recipients versus 33% for historical controls.

Cell Lysate Vaccines

Viral melanoma oncolysate vaccine and bacterial melanoma lysate vaccine are based on the principle that immune and antitumor responses can be augmented by attaching a foreign component (a virus or a bacterial derivative) to vaccines.

Viral Melanoma Oncolysate

Wallack and co-workers 113 used vaccinia virus to prepare a viral melanoma oncolysate (VMO) from cell lines established from four patients with primary and metastatic melanoma. Each cell line was infected with vaccinia virus at a ratio of 1 cell to 10 TCID50 (50% tissue culture infectious dose). The four nucleus-free cell lysates were pooled at equal concentrations (in terms of total cell count) to obtain a polyvalent allogeneic VMO vaccine. Only 4 of 25 vaccine recipients produced antibodies to polymorph MHC antigens, suggesting a possible down-regulation of MHC antigens consequent to vaccinia virus infection. Most VMO recipients developed DTH reactions; in addition, sera of patients who were negative for anti-MAA antibodies before treatment developed antibodies after vaccine treatment. Statistical comparison of VMO recipients with 39 matched controls (patients treated with BCG or C. parvum) revealed a significant (p < .04) increase in the DFS of VMO recipients. 57 In a separate phase II study of VMO vaccine, patients with the highest antimelanoma IgM and IgG antibody titers had better survival than those with low titers. 41 However, a phase III randomized, multi-center double-blind trial of VMO versus vaccinia virus in 215 evaluable patients with high-risk stage II melanoma has not shown a significant difference in disease-free interval or overall survival. 114

A French study documented a humoral immune response after immunizing melanoma patients with a different VMO. Lymphocytes from vaccine recipients responded in vitro to VMO stimulation in the presence of low concentrations of IL-2, and this response was greater than that of lymphocytes from normal individuals. Postvaccination sera of VMO recipients showed augmentation of IgG to a 31-kD antigen in contrast to preimmune sera. 115 Western blot immunostaining revealed that this protein was not in the allogeneic melanoma cell lines used to prepare VMO vaccine, in the vaccinia virus preparation, or in the fetal calf serum used to culture the tumor cells; however, it was in tumor metastases. Interestingly, this antigen disappeared within 5 days after culturing the tumor cells in vitro but was synthesized after exposure to vaccinia virus. Expression of this antigen in tumor metastases and induction of IgG antibodies against this protein antigen in patients immunized with VMO vaccine indicate the possible therapeutic benefit of VMO vaccine in human melanoma. These findings parallel those of Savage and co-workers, 116 who reported antibody development in six melanoma patients following 6 weeks of immunization with allogeneic melanoma oncolysates prepared from three Newcastle virus-infected melanoma cell lines.

Bacterial DETOX-coupled Lysate

Mitchell and co-workers 8, 29 prepared bacterial cell wall derivatives to tumor cell membranes by mixing tumor cell lysate with DETOX, which contains nontoxic lipid A (monophosphoryl lipid A from S. minnesota) and cell wall skeletons of Mycobacterium phlei in squalene oil and Tween 80. The mixed lysate is referred to as therapeutic melanoma vaccine—“theraccine”(Melacine). Theraccine was administered on days 1, 8, 15, 22, and 36 to patients with measurable lesions of metastatic melanoma. Several patients received cyclophosphamide 5 to 7 days before the first theraccine injection to inhibit possible suppressor T cells. The reported response rate was approximately 20%, 20 of 106 evaluable patients. Approximately 5% of the patients had complete remission, and 15% had partial remission. The median duration of response was 17 months, and the median duration of complete remission was 21 months. Median survival was approximately 6 to 12 months after the appearance of metastatic disease. Although the data from phase II multi-center trials of theraccine indicated improved survival, the recent phase III randomized trial failed to confirm the earlier results. The strongest correlate of clinical response was an increase in CTL precursors. 8 Before immunization, patients had one CTL in 10,000 to 50,000 lymphocytes. Three to six weeks after immunization, this ratio increased to one CTL in 2,500 to 5,000 lymphocytes. However, only 30% of those who generated CTLs had objective remission or long-term stability. There was no clinical response in patients who failed to generate CTLs against at least one component of the vaccine. Of the 117 clones of T cells derived from the tumor tissues of immunized patients, 64 were CD41 CD82 phenotype and 53 were CD81. 117 Intensive analysis of specificity and HLA-restricted studies using matched lymphoblastoid cell lines proved that CTL reactivity was specific to melanoma. Analysis of the first 77 patients treated with theraccine revealed an association between three HLA class I alleles and clinical response to therapy.

HLA-A2/A28 and HLA-B12/44/45 are strong presenting molecules for melanoma-associated epitopes and may permit CTLs from patients sharing these alleles to recognize and kill melanoma cells most efficiently after immunization with the allogeneic vaccine. It is not clear whether the similarity in alleles from the immunizing melanomas and the autologous tumors accounted for their improved effectiveness in eliciting a tumor response. However, autologous (fully matched) immunization has thus far been less successful than allogeneic vaccine therapy. This study emphasizes the need to determine whether the HLA class I antigens of an allogeneic vaccine must match those of the cancer patient for efficient induction of CTLs.

Whole-Cell Vaccines

Polyvalent Antigen-Adjusted Melanoma Cell Vaccine

The observation that injecting BCG into the cutaneous metastases of melanoma patients enhanced systemic active immunity, tumor infiltration, and clinical regression led to intradermal or intralymphatic injection of randomly selected irradiated autologous and irradiated allogeneic whole cells mixed with BCG. 119, 120 These investigations had limited success; only 35% of immunized patients produced antibodies to cell-surface antigens. Consequently, Morton and co-workers 37 developed a polyvalent melanoma cell vaccine (PMCV) consisting of three allogeneic melanoma cell lines selected for their high levels of six cell-surface immunogenic glycoprotein, lipoprotein, or ganglioside TAAs.

In late 1984, a phase II trial was initiated to evaluate PMCV in melanoma patients with regional soft-tissue metastases (AJCC stage IIIA disease) or distant metastases (AJCC stage IV disease). PMCV was produced in large batches and analyzed for antigen expression to determine variance between lots. An outside laboratory screened PMCV for viral, bacterial, and fungal infectious organisms. Before cryopreserving the vaccine, the cells were irradiated to 100 to 150 Gy. PMCV was injected intradermally in axillary and inguinal regions on a schedule of every 2 weeks for 4 weeks, then monthly for 1 year. For the first two treatments, PMCV was mixed with BCG (Glaxo, England) (24 × 106 organisms per vial). After 1 year, the immunization interval was increased to every 3 months × 4 and then to every 6 months. One of the following biologic-response modifiers known to down-regulate suppressor cell activity was administered: cimetidine, indomethacin, or cyclophosphamide. Survival after PMCV immunization correlated significantly with DTH (p = .0066) and antibody response to PMCV (p = .0117). Of 40 AJCC stage IV patients with evaluable disease, 9 (23%) had regression (3 complete). PMCV increased the median and 5-year survival of stage IIIA patients two-fold (p = .00024) and three-fold (p = .0001) for stage IV patients, compared with non-PMCV immunotherapy and other treatments received by concurrent, nonrandomized control patients.

PMCV produced several immune responses of particular interest. IgM antibodies to cell-surface antigens correlated best with survival; 37, 71 there was no significant correlation between survival and IgG antibody to melanoma cell-surface antigens. Patients developing high IgM titers (immunofluorescence index > 50%) had almost a three-fold increase in 5-year survival (9.6% versus 26.8%) and a two-fold increase in median survival (16 versus 30 months). IgM antibodies were directed against a variety of melanoma-associated gangliosides. 3, 25, 63

There was a highly significant correlation (p = .0066) between survival and DTH response. Median survival was 30 months for those whose DTH reaction exceeded 10 mm and only 17 months for those whose DTH was less than 10 mm. Respective 5-year survival rates were 27.7% and 10.0%. A significant association between survival and DTH was also noted after immunization with HLA-depleted polyvalent melanoma shed antigen and autologous whole-cell vaccine. 112, 118

There also was a significant positive correlation (p = .013) between in vivo DTH response and in vitro mixed-lymphocyte tumor reaction (MLTR). Of the 40 patients for whom these data were available, 82% showed significantly (p = .005) enhanced stimulation to one or more PMCV cell lines at either week 4 or 16. Of these, 91% showed sensitization to at least two PMCV lines. Autologous MLTR studies revealed that immunization with allogeneic PMCV enhanced the response to autologous melanoma cells, confirming the existence of cross-reacting antigens demonstrated by antibodies to membrane-associated antigens. MLTR responses correlated with survival: 2-year DFS was 53% for patients responding to one or more PMCV lines, compared with 20% for patients who showed no response (p = .055). Finally, PMCV immunization changed the profile of tumor-infiltrating lymphocytes; activated T and B cells (CD25) and NK cells (CD56) were significantly enhanced, and the CD4/CD8 ratio was markedly elevated.

In our subsequent study of 135 AJCC stage III melanoma patients, 83% responded by a positive DTH reaction (> 6 mm) during the first 4 months of PMCV therapy. 121 Sixteen of 33 developed more than a 50% increase in CTL activity against one of the PMCV cell lines during this period. Overall survival was significantly prolonged in patients with a positive DTH (p = .0054) and/or increased CTL activity (p = .02), suggesting that PMCV induces specific T-cell responses that are correlated with clinical course. In a separate study of 53 melanoma patients, 57% had significantly elevated anti-MAGE-1 IgG serum levels after immunization with PMCV. 122

Morton’s group has conducted several studies of the immune response to a 90-kD tumor-associated glycoprotein antigen (TA90) expressed on PMCV cells and on most other melanoma cells. In a recent study, univariate analysis showed that high anti-TA90 IgM titer and strong PMCV-induced DTH were associated with improved survival (p = .051 and p = .02, respectively) whereas elevated anti-TA90 IgG was correlated with decreased survival (p < .02). Multivariate analysis considering clinical variables and PMCV immune responses identified anti-TA90 IgM, anti-TA90 IgG, and PMCV-DTH as significant independent variables (p < .05, p < .02, and p < .01, respectively) influencing survival following PMCV immunotherapy. 123 In another study, prevaccine TA90-immune complex correlated strongly with overall and disease-free survival in stage IV melanoma patients receiving postoperative PMCV immunotherapy. 124 Serum levels of TA90-immune complex are strongly correlated with results of positron emission tomography in patients with metastatic melanoma. 125

Five lines of evidence indicate that PMCV can enhance the immune response to autologous melanoma cells. First is the strong correlation between humoral and cell-mediated immune responses and survival. Second is the complete and partial regression in patients with evaluable disease, and third is the concomitant increase in CTL activity, MLTR, and humoral antibodies to allogeneic and autologous melanoma cells. Fourth is the changes in tumor-infiltrating lymphocytes in melanoma metastases, and fifth is the ability of allogeneic melanoma cells to induce sensitization to autologous melanomas that share HLA class I antigens, and that thereby render the autologous cells susceptible to in vitro killing by CTL. 126

MHC class I or II restriction has been raised as an argument against the use of allogeneic vaccines. However, there is significant loss of HLA class I antigen expression in melanocytic nevi and melanoma lesions, and the expression of HLA class I also decreases with the progression of disease. 127, 128 Approximately 16% of primary melanoma lesions and 58% of metastatic melanoma lesions are not detected by monoclonal antibodies against HLA class I antigens. 129 Moreover, the allogeneic melanoma cells of PMCV share MHC class I cross-reactive antigens with more than 90% of melanomas. PMCV enhances the T-cell response in vitro to autologous melanoma cells, possibly by direct recognition of MAA presented by shared or cross-reactive HLA molecules on PMCV lines. 130 It is also possible that recognition occurs through antigen processing and presentation of PMCV’s antigens by APCs. The allogeneic HLA antigens on the vaccine may stimulate alloreactive T cells that infiltrate the site of PMCV injection, resulting in production of cytokines to attract nearby antigen-presenting cells. Approximately 20% of patients did not show a T-cell response to PMCV, which could be due to T-cell anergy or to T-cell-specific immunosuppression. Nabel and co-workers 131 recently demonstrated that in vivo transfection of the gene for an allogeneic HLA class I antigen into a patient’s melanoma induced specific systemic T-cell immunity.

Hapten-Attached Cell Vaccines

The need to induce better T-cell responses to melanoma antigens led to development of hapten-conjugated tumor antigens. Berd and co-workers 118 immunized melanoma patients with dinitrophenyl (DNP)-conjugated autologous irradiated melanoma cells (10 to 25 × 106 cells) mixed with BCG. Forty-six patients were sensitized to DNP by topical application of 1% dinitrofluorobenzene (DNFB) in acetone-corn oil on 2 consecutive days. Cyclophosphamide was administered 3 days before the sensitization. Two weeks later, patients were again given cyclophosphamide, followed 3 days later by injection of DNP-conjugated melanoma vaccine. Administration of cyclophosphamide plus DNP-conjugated vaccine was repeated every 28 days. The development of DTH was tested with DNP-conjugated autologous peripheral blood mononuclear cells; a DTH response was induced in all patients by DNFB sensitization. Twenty of 46 patients had clinically evident inflammatory responses in metastatic tumors 2 to 4 months after initiation of treatment. The tumors were infiltrated with CD81 T cells, in contrast to tumors derived from control subjects not treated with vaccine. In a panel of 14 subcutaneous melanoma metastases from untreated patients, T cells (CD31) constituted approximately 10% of the total viable cells, compared with 40% in DNFB vaccine-treated patients. The CD8/CD4 ratio of T cells in treated patients was about 5:1. T-cell clones were generated from the tumor metastases of vaccine recipients. Of 140 clones generated, 70 killed cultured autologous melanoma cells but failed to kill a panel of four allogeneic melanoma cell lines.

The clinical impact of DNP-conjugated vaccine in patients with a lower tumor burden (i.e., surgically curable regional metastases) was examined in a separate study. Forty-one patients with large (> 3-cm) clinically palpable lymph nodes received the vaccine as adjuvant therapy following regional lymph node dissection. Eight injections of DNP-conjugated vaccine were administered at 4-week intervals. Cyclophosphamide was administered 3 days before the first two vaccine treatments. Of 27 disease-free patients, 22 survived more than 1 year after surgery, and 11 survived 2 years. This clinical outcome is reportedly better than that of a similar group of 22 patients previously treated with the nonhapten vaccine. However, the small number of patients and the use of selected historical controls prohibit any conclusions regarding therapeutic effectiveness.

Genetically Altered Cytokine-Producing Tumor Cell Vaccines

Encouraged by studies involving immunization of tumor-bearing animals with genetically altered cytokine-producing tumor cell vaccines, 132– 135 investigators at the John Wayne Cancer Institute developed a preclinical model to determine whether transfection of IL-2 gene into human melanoma cells would augment the response of autologous and allogeneic PBLs from melanoma patients. 136 IL-2 gene was transfected into three human melanoma cell lines, and the secretion of IL-2 from stable transfected cells was confirmed by enzyme-linked immunosorbent assay (ELISA). The PBL response to these melanoma cells was then examined in an MLTR, using PBLs from eight melanoma patients. The PBL response was significantly higher to autologous (p = .01) or HLA-A cross-reacting (p = .05) transfected melanoma cells than to nontransfected melanoma cells. These data suggest that IL-2 gene transfection may be an important strategy for enhancing specific immune responses induced by a polyvalent melanoma cell vaccine. These investigators have also observed that IL-4, interferon-gamma, and tumor necrosis factor augment the expression of HLA class I and HLA-DR antigens. IL-4 alone or in combination with interferon or tumor necrosis factor also increases GD2 expression. 137– 138 Table 61.2 summarizes the trials of genetically altered cytokine-producing melanoma cell vaccines.


Availability and immunogenicity make allogeneic tumor cells the most promising basis for melanoma vaccines. Phase I and II trials have demonstrated immune responses that appear to correlate with duration of disease-free and overall survival in patients receiving cell-based polyvalent vaccines; several phase III trials of these vaccines will soon produce interim or final results. In the meantime, novel approaches using gene transfection, dendritic cells, and cytokines are being applied to the development of melanoma vaccines. Melanoma will continue to serve as the testing ground for vaccine development until phase III trials identify a vaccine that consistently produces durable and significant clinical remissions.


Table 61.3 surveys clinical trials of immunotherapy for patients with acute myeloid leukemia (AML) and lymphoblastic leukemia (ALL). Allogeneic leukemic cells with BCG were administered to 20 children with ALL. Long-term follow-up revealed that 8 were alive in remission, with 7 in their first complete remission. 139 An approximately 50% overall rate of 5-year survival was observed in a group of 100 patients undergoing the same treatment. 140 Allogeneic irradiated leukemic cells with BCG have also been used as maintenance therapy for patients with AML. These patients had longer remissions than those who were maintained on either BCG or tumor cells, but the difference was not statistically significant. Powles 141 reported that the 45 of 107 leukemic patients who received irradiated tumor cells plus BCG achieved complete remission, with a median duration of 70 weeks. Long-term follow-up revealed that the median survival was significantly prolonged by immunization with irradiated allogeneic AML blast cells and BCG (270 days versus 510 days), (p = .03). The survival benefit was greatest in patients with a low tumor burden.

In another study, 191 adults with AML received daunorubicin and cytosine arabinoside. 61 Sixty-three patients achieved remission and were admitted to one of the three arms of an active immunotherapy trial: immunotherapy alone, immunotherapy with maintenance chemotherapy, or no treatment. Unlike immunotherapy plus chemotherapy, immunotherapy alone was associated with easy and repeated re-induction of remission and marked prolongation of survival after first relapse. Interestingly, a study of 182 patients found that BCG alone was as effective as BCG plus allogeneic blast cells. In another report, 57% of 195 patients with AML entered complete remission after treatment with chemotherapy and BCG plus tumor cells. After remission, patients received maintenance chemotherapy with or without weekly administration of frozen nonirradiated allogeneic blast cells with BCG. The median survival was 690 days in the 40 chemoimmunotherapy patients versus 408 days in 36 chemotherapy patients (p < .01). Immunotherapy for AML using allogeneic cells with C. parvum showed no detectable impact on the rate of remission or the duration of survival.

Neuraminidase removes sialic acids from tumor cells, thereby increasing their immunogenicity. A randomized clinical trial compared the efficacy of chemotherapy alone, neuraminidase-treated allogeneic myeloblasts alone, and neuraminidase-treated myeloblasts plus methanol extractable residue (MER) of BCG. 61 Patients received 10 10 asialomyeloblasts injected at 50 sites draining into node-bearing regions at one session. A total dose of 1 mg of MER was distributed in five equal injections. All immunotherapy was given midway between courses of chemotherapy. The median duration of remission for patients in each immunotherapy arm was 78 weeks, compared with only 20 weeks for patients in the chemotherapy arm. There were 27 survivors among the 32 immunotherapy patients, compared with only 4 survivors among the 21 chemotherapy patients. In other trials of active specific immunotherapy, BCG and cultured cell lines were administered intradermally to 15 patients with uncomplicated Philadelphia chromosome–positive chronic myeloid leukemia (CML). 142 CML patients who received immunotherapy survived twice as long as historical controls.

Patients with B-cell lymphoma also have been treated with purified cancer antigen. 143 Nine patients with minimal residual disease or complete remission after chemotherapy received a series of subcutaneous injections of immunoglobulin derived from autologous tumor cells (immunoglobulin-idiotype protein) conjugated to KLH and admixed with the immunologic adjuvant SAF-1. Seven of the nine patients showed idiotype-specific humoral and/or cellular immunologic responses. The induced antibodies bound specifically to autologous immunoglobulin idiotypes and autologous tumor cells. Cell-mediated responses were demonstrated by the in vitro proliferation of PBLs in response to the soluble immunoglobulin-idiotype protein. The tumors of patients with measurable disease regressed completely.

Lung Cancer

The fact that most lung cancer patients experience immunosuppression in conjunction with tumor growth suggests the value of an immunotherapeutic approach, particularly after surgical reduction of tumor burden. Table 61.3 summarizes early (1980s) clinical trials of immunotherapy for patients with lung cancer. Stage I patients injected intracutaneously with a mixture of purified antigenic extracts from autochthonous lung cancer cells and complete Freund’s adjuvant had improved survival compared with nonrandomized controls. DTH skin-test responses showed increased cell-mediated immunity to antigenic tumor preparations. Similarly, 15 stage III patients receiving a mixture of antigens extracted from autochthonous tumor and complete Freund’s adjuvant had a 2-year survival rate of 63%. Another study investigated the effects of (1) methotrexate alone (n = 8), (2) immunotherapy with a homogenate of soluble tumor cell vaccine admixed with Freund’s complete adjuvant (n = 15), and (3) chemoimmunotherapy (n = 13). Survival in immunized patients was significantly prolonged (p < .01). Yet another study randomized postoperative patients into three different therapy groups: (1) no treatment, (2) tumor antigens admixed with Freund’s adjuvant intradermally three times, and (3) Freund’s adjuvant only. 61 Estimated 3-year survival rates were 34%, 84%, and 89%, respectively. The difference in survival rates between control and immunotherapy arms was statistically significant (p < .05), suggesting that immunopotentiation using an adjuvant with or without tumor cells may be beneficial. This trial, which was never replicated, deserves to be pursued. Arakawa et al 144 reported two cases of advanced adenocarcinoma treated with attenuated vaccinia virus. Both patients showed good antitumor effects against lung and bone lesions. In a study of 46 patients undergoing radical resection of lung cancer, Mosienko and colleagues 145 reported that postoperative administration of three subcutaneous injections of autologous tumor cell vaccine improved cell-mediated immunity and increased the 2-year survival rate.

Recently, a novel CTL target antigen called NY-ESO-1 (esophageal cancer-associated antigen) was identified in 11 of 16 human small cell lung cancer cell lines and 3 of 7 non–small cell lung cancer cell lines. 146 In view of its specific recognition by CD8+ T cells, NY-ESO-1 is considered a potential target antigen for purified antigen-based immunotherapy of lung cancer. Improved survival of patients with small cell lung cancer (SCLC) has been reported after treatment with anti-idiotypic antibody BEC-2 plus BCG. 147 BEC-2 mimics ganglioside GD3, which is expressed on the surface of most SCLC cells. Fifteen patients who had completed standard therapy received a series of five intradermal immunizations consisting of 2.5 mg of BEC-2 plus BCG over a 10-week period. All patients developed anti-BEC-2 antibody. The five patients who produced anti-GD3 antibodies included those with the longest relapse-free survival (> 47 months). A phase III trial is being conducted to evaluate the efficacy of this therapy after chemotherapy and irradiation.


Morton and colleagues 120, 148 admixed BCG with 5 to 75 × 106 irradiated autologous tumor cells and administered this preparation to 15 sarcoma patients. Vaccine recipients had a modest prolongation of survival when compared with historical control patients undergoing surgery without immunotherapy. Complement-fixing and cytotoxic antibodies increased concomitantly with a slowing of tumor progression. Similarly, higher titers of antitumor cytotoxic antibodies and improved in vitro cellular immunity were observed in the sera of osteosarcoma patients treated with irradiated autologous or allogeneic cells infected with influenza virus. One report describes the recovery of immunologic competence in a patient receiving monthly injections of irradiated autologous cells plus BCG. Three patients who had undergone resection of primary osteosarcoma were treated with vaccines that contained autologous or allogeneic tumor cells and BCG; two were free of disease at 13 and 18 months, and the third developed pulmonary metastases shortly after initiation of therapy. 142 Similarly, 18 patients who had undergone surgery for localized soft-tissue sarcoma received intradermal injections of cultured allogeneic sarcoma cells plus BCG at five separate sites. 149 With follow-up as long as 3.5 years, 61% of the patients (11 of 18) remained disease-free, compared with only 33% (5 of 15) of historical control patients treated by surgery alone. Immunotherapy doubled the median disease-free interval in patients with recurrent disease.

Breast Cancer

Intralesional treatment of breast carcinoma with BCG produced regression of skin metastases. Although complete regression was observed in 7 of 8 patients in one study and in 7 of 14 patients in another, other investigators did not find any apparent benefit. Adjuvant BCG therapy with or without an allogeneic tumor cell vaccine also failed to produce a significant benefit in patients with stage II breast carcinoma. 150 Administration of an autologous tumor vaccine after radical mastectomy also did not show therapeutic benefit. 151 Several clinical trials have attempted combination immunotherapy and chemotherapy.

Springer and colleagues 152 summarized the results of a clinical trial initiated in 1974 to examine the potential of T/Tn antigens (carbohydrate precursors of MN blood group antigens) as vaccines for breast cancer. T/Tn antigen (10 mg) was admixed with 0.5 units of typhoid vaccine and injected intradermally in 16 patients with stage II, III, or IV breast cancer. Mean survival exceeded 5 years; the 10 patients who survived longer than 10 years included 3 stage III and 3 stage IV patients. MacLean and co-workers 153 undertook a pilot study to determine whether human breast carcinoma patients immunized with sialyl-Tn-KLH plus DETOX developed specific anti-sialyl-Tn antibody responses. Following immunization, all 12 patients developed increased titers of complement-mediated cytotoxic antibodies specific for sialyl-Tn. Five patients were alive 12 or more months and 4 patients were alive 6 or more months after entry into the study. Lymphocyte activation markers CD69 and HLA-DR were studied in these patients. 154 Prolonged survival was associated with elevated CD69+ and CD4+CD69+ cells and with decreased non-B lymphocyte HLA-DR+ (CD20-HLA-DR+) cells following cyclophosphamide treatment. In a randomized phase II study of sialyl-Tn and DETOX-B adjuvant with or without cyclophosphamide pretreatment, Miles et al. 155 observed disease stabilization in 5 of 18 patients. All 5 patients developed IgM antibodies to sialyl-Tn, and the levels of IgM were significantly higher in those who received cyclophosphamide.

Because carcinoembryonic antigen (CEA) is overexpressed in breast cancer, CEA-specific cytotoxic lymphocytes (CTLs) have been assessed for their potential immunotherapeutic benefit. 156 Dendritic cells from breast cancer patients were pulsed with the peptide epitope of CEA to generate CEA-specific CTLs. GM-CSF enhanced peptide-specific immune reactions by dendritic cells. However, a few patients experienced disease progression, suggesting the loss of either the tumor antigen targeted by CTLs or the presenting MHC class I molecule. Based on these observations, cytokine enhancement of TAA and MHC class I expression are being evaluated in vivo as a possible means of preventing immunoselection. 157

Genitourinary Cancer

Tumor cell vaccines with or without adjuvants have been used in renal cell carcinoma. McCune and colleagues 47 administered weekly intracutaneous injections of autologous irradiated tumor cells admixed with C. parvum in five patients with residual renal carcinoma. In another study, autologous tumor cell vaccine with C. parvum was administered to 14 patients with metastatic renal carcinoma; 4 of the 14 patients had objective responses, and a fifth had prolonged stabilization (Table 61.3). A significant increase in survival was observed in stage IV renal cell carcinoma patients treated with a vaccine containing autologous tumor polymerized with ethylchlorformate and purified protein derivative (PPD) or Candida albicans. 49 After 3 years of follow-up, 13 of 71 patients receiving a polymerized autologous tumor cell vaccine for treatment of advanced renal adenocarcinoma remained alive, compared with only 3 of 56 patients treated by the best conventional measures. 158 A similar significant increase in survival was observed in another study of autologous tumor cell vaccine. 48 Among 30 patients treated with autologous tumor cells plus tuberculin or phytohemagglutinin as an adjuvant, 2 complete and 2 partial responses were recorded. 159 None of these nonrandomized trials was sequentially controlled or further pursued.

In an attempt to increase immunogenicity by abrogating the T-suppressor cell activity, cyclophosphamide was administered before vaccination with an admixture of tumor cells and C. parvum. 160 Of 20 patients, 1 patient had a complete response, and 4 patients had partial responses. Four of 15 patients developed DTH responses to autologous renal carcinoma cells. Of these 4, 3 had clinical responses. Of the 11 patients who failed to develop DTH, only 1 had a clinical response. The results suggest that inhibiting suppressor function during active specific immunotherapy can enhance T-helper function, induce a DTH response to autologous tumor cells, and induce objective regression of metastases. Carpinto and colleagues 161 used autopheresis combined with cimetidine to abrogate suppressor cell activity in 16 patients. Autopheresis involves the pheresis of lymphocytes from a patient, incubation of the cells with autologous or heterologous tumor antigens, and re-infusion of the cells. In theory, the incubated cells will be specifically activated by TAAs and will be further stimulated by endogenous lymphokines to destroy antigenic tumor cells in vivo. Treatment can be performed in outpatient facilities, and toxicity is minimal. This study reported an objective response rate of 19% (3 of 16). In a later series of patients with renal cell carcinoma, the 2-year survival rate was 25% for vaccine patients compared with 5 to 10% for historical controls. 162

Thirty-three renal cell carcinoma patients who underwent palliative nephrectomy or excision of metastases were immunized monthly with intradermal injections of autologous or allogeneic irradiated tumor cells admixed with C. parvum. Antitumor activity was evident in 8 vaccine recipients (1 complete, 4 partial, and 3 minor responses). The median survival was 32 months for responding patients versus 17 months for all patients. Galligioni et al. 163 conducted a 5-year prospective randomized study of treatment with autologous tumor cells and BCG. Of 120 consecutive patients with advanced renal cancer, 60 control patients received no vaccine, and 60 patients received an intradermal injection of 10 × 106 autologous irradiated cells with or without 10 × 106 BCG in the first two vaccinations. At randomization and at 1, 6, and 12 months after completing immunotherapy, the treated patients were evaluated for the development of DTH response to autologous tumor and autologous normal renal cells. The baseline DTH was negative in all patients. After a month, 38 of 54 immunized patients showed a significant (p < .01) DTH response to autologous tumor cells but not to autologous normal renal cells. The response was persistent at 6 months in 25 of 44 patients and at 12 months in 16 of 28 patients. DTH remained negative in the nonimmunized control patients. However, there was no significant difference in the probability of 5-year disease-free survival (63% and 72% for treated and control patients, respectively) after a median follow-up of 61 months. In contrast, Repmann et al. 164 showed significantly higher survival among 116 renal cancer patients treated with autologous tumor vaccine after radical nephrectomy than among 106 historical controls who did not receive the vaccine. Patients in Robson stages II and III showed significantly different survival rates (p = .02 and p = .04, respectively). Vaccine administration produced minor side effects in 2 of 116 patients. All of these trials demonstrated low toxicity, reasonable responses, and prolonged survival.

Prostate cancer

Patients with prostate cancer often show nonspecific depression of their cell-mediated immunocompetence. Thus, the sera of prostate cancer patients with disseminated disease significantly (p < .01) inhibited human antibody-dependent cellular cytotoxicity (ADCC). Cell-mediated immunocompetence correlated with the presence or absence of metastatic disease.

A number of prostate cell lines showed down-regulation or defective expression of class I MHC assembly. HLA class I expression was normal in benign tumors but absent in 34% of primary prostate cancers and in 80% of lymph node metastases. Up-regulation of MHC class I antigen is observed on pancreatic cell lines treated with interferon-alpha and/or interferon-gamma. The bacterial adjuvant BCG augmented the production of these cytokines, which are associated with CTL activation and proliferation. Intratumoral or intradermal BCG-based immunotherapy of prostate carcinoma was initiated in the early 1970s. 165 BCG recipients showed significant changes in IgM and DTH responses when compared with age-matched concurrent control groups receiving only conventional therapy. Survival from the time of diagnosis was significantly longer in patients receiving BCG. 166

All prostate cancer patients have a high level of prostate-specific antigen (PSA), a low proportion of peripheral blood mononuclear cells (PBMCs) producing interferon-gamma and IL-2, and a higher proportion of IL-4 indicative of a Th-2 cytokine profile. After intradermal vaccination with a new heat-killedMycobacterium vaccae (SRL 172), 167 the serum PSA level and the IL-4-producing cells declined, and the proportion of IL-2-producing cells increased, suggesting a shift of Th-2 to Th-1. The proportion of PBMCs secreting IL-2 appears to be a potential marker of response to immunotherapy.

A cellular vaccine for prostate cancer has been administered in combination with hormone therapy. Inactivated cancer cells (44.4× 106) were intradermally injected with neuraminidase (to promote immunogenicity of the sialylated glycoconjugates) into the thigh of patients with metastatic prostate cancer. Immunized patients showed significant reductions in levels of serum phosphatase and CEA. Clinical benefit was demonstrated by scintigraphic evidence of remission of osteometastases and a statistically significant increase in the survival rate during treatment. Cellular vaccine therapy may constitute an alternative therapy for patients with hormone-independent prostate carcinomas and estrogen intolerance. These investigators calculated 5-year actuarial survival to demonstrate that a combination of cellular vaccine therapy and estrogenic hormone therapy was clearly superior to hormone therapy alone. 168 Another study used prostate antigen secreted in vitro (such as specific transfer factor) to immunize 56 patients with metastatic hormone-resistant carcinoma of prostate (stage D3). At follow-ups ranging from 1 to 8 years, 1 patient had achieved complete remission, 6 had partial remissions, and 14 had stabilization of disease. Mean survival was 17 months, higher than that reported elsewhere.

A large portion of patients with prostate carcinomas can be targeted by HLA-A2-restricted CTLs specific for PSA. 169 A phase I clinical trial found that patients receiving autologous dendritic cells pulsed with HLA-A20201-restricted peptides from PSA had significantly lower serum PSA levels than did control groups exposed to peptide alone or dendritic cells alone. 170 Further follow-up revealed that 12 patients infused with DC-pulsed PSA peptides experienced a marked drop in PSA levels, which persisted from 100 to 200 days. 171 Four patients remained responsive at the end of the period of observation (360 days). In a continuing phase II trial, these investigators have observed that DTH monitoring is necessary to assess the cellular immune response in patients undergoing T-cell therapy using dendritic cells pulsed with HLA-A2-specific peptides from PSA. 172

A recent report claims that not all PSAs can elicit a cellular immune response. For instance, prostatic acid phosphatase (PAP) is uniquely expressed in prostatic tissue and cancer. This antigen elicits a marked humoral response but does not generate CTLs. 173 However, immunization using PAP with vaccinia virus augmented CTL production. 174 The presence of carbohydrate residues in PSA and the occurrence of GM2, sialyl Lewis and sialyl Tn antigens, MUC-2 mucins, and TAG-72 in prostate cancer indicate that the carbohydrate antigens of prostate cancer can be a target of active specific immunotherapy. 175– 178

Gynecologic Cancers

Administration of tumor cell vaccine admixed with BCG may benefit patients with ovarian carcinoma. An early study found that patients with advanced ovarian carcinoma who received a combination of BCG, tumor cell vaccine, and chemotherapy lived longer than patients who received chemotherapy (alkylating agents) alone. 179 However, a much later report observed only limited success after immunization with KLH-conjugated common carcinoma (Thomsen-Friedenreich) determinant admixed with DETOX. 180 In a recent study, immunologic responses in 16 patients with ovarian cancer were evaluated after treatment with anti-idiotype vaccine ACA-125, which mimics TAA CA-125. 181 A strong intracellular interferon-gamma and IL-2 response, characteristic of a Th-1 cell-type immune response, was associated with a statistically significant improved survival. 181A The median progression-free survival was 11.0 ± 5.6 months in patients with a specific immune response to CA-125, compared with 8.0 ± 4.2 months in patients who did not develop an immune response. Two patients had clinical evidence of tumor regression that lasted longer than 15 months. 181

Cervical cancer is one of the most common causes of cancer-related deaths in women. An association between human papillomavirus (HPV) infection and cervical cancer has been established, and the oncogenic potential of certain HPV types has led to the development of prophylactic and therapeutic HPV vaccine strategies. The oncogenic proteins E6 and E7 of HPV-16 and HPV-18 are the focus of current clinical trials. Both E6 and E7 are expressed in cervical carcinoma cells. A live recombinant vaccinia virus expressing the E6 and E7 proteins was administered in an open-label phase I/II trial to eight patients with late-stage cervical cancer. 182 After receiving a single dose of the vaccine, patients were kept in strict isolation to monitor local and systemic side effects, environmental spread, and anti-E6/E7 immune responses. No significant clinical side effects or environmental contamination was observed. All patients mounted an antivaccinia antibody response, and three developed an HPV-specific antibody response. HPV-specific cytotoxic CTLs were observed in one of the three evaluable patients.

An in vitro study found that E7-specific CTLs expressed strong cytolytic activity against autologous tumor cells but did not lyse autologous ConA-treated lymphoblasts or autologous Epstein-Barr (EB) -virus transformed lymphoblastoid cell lines. 183 The cytotoxic reaction was blocked significantly by anti-HLA class I and anti-CD11a/LFA-1 antibodies. All CTLs were CD3+/CD8+ with variable levels of CD56 expression. Interestingly, these CTLs were also highly cytotoxic against an allogeneic HLA-A2(+) HPV-16+ matched cell line (CaSki). In addition, specific lymphoproliferative responses by autologous CD4+ T cells were induced by E7-pulsed autologous dendritic cells. These findings have important implications for active specific immunotherapy of cervical cancer.

In a clinical study, 12 patients were immunized with an HLA-A0201-restricted, HPV-16 E7 lipopeptide vaccine. All HLA-A0201 patients mounted a cellular immune response to a control peptide. E7-specific CTLs were elicited in 4 of 10 evaluable HLA-A0201 subjects before vaccination and in 5 of 7 evaluable HLA-A0201 patients after two vaccinations. An in vitro analysis of peripheral blood lymphocytes demonstrated CTLs in 2 of 3 evaluable HLA-A0201 cultures after all four inoculations. These data indicate that patients with advanced cervical cancer can generate a specific cellular immune response. This approach may be useful in treating preinvasive as well as advanced cervical cancer.

Colorectal Cancer

A particularly well-designed clinical study of active specific immunotherapy is the prospectively randomized controlled trial undertaken by Hoover and colleagues. 184 Following standard surgical resection of Dukes’ stages B and C colon or rectal cancer, patients at high risk for recurrence were randomly assigned to receive no immunotherapy (control) or active specific immunotherapy using 10 7 irradiated autologous tumor cells mixed with 10 7 BCG organisms and administered weekly for 3 weeks. DTH to the autologous tumor cells developed in 67% of immunotherapy patients but in no control patients. 46 After a mean follow-up of 28 months, immunotherapy patients had fewer recurrences and deaths than control patients. In a recent update of this study, these investigators reported that the apparent advantage of immunotherapy was limited to patients with colon cancer. 185 They noted that expression of HLA-DR and ICAM-1 by tumor cells in the vaccine was predictive of disease-free survival. 186 Immunized patients developed a DTH response and an in vitro T-cell proliferative response to a 32-kD protein antigen.

In another study, active specific immunotherapy was tested in 57 patients with resected colorectal carcinoma. 187 Irradiated autologous cells were either infected by Newcastle disease virus or admixed with BCG. Six to eight weeks after operation, the patients were immunized three times at 2-week intervals; 48 were treated with viral oncolysates, and 9 received BCG-admixed vaccine. Two-year survival rates were 97.9% with viral oncolysates and 66.7% with BCG-admixed vaccine. By comparison, the mean survival of 661 historical controls was 73.8%. These data suggest that the quality of vaccine may vary with the type of cancer.


In a phase I pilot study, 19 patients with anaplastic gliomas were immunized with allogeneic human glioma cell lines. 66, 188 One patient showed antibody against glioma antigen after absorption with FBS, human platelets, and other allogeneic glioma cell lines. Eggers and co-workers 54 immunized a glioblastoma patient with a mixture of autologous and allogeneic cells inactivated with mitomycin and coupled to muramyl dipeptide and trehalose dimycolate. Immune activity of PBLs against autologous tumor target cells was measured in a short-term chromium-release assay. The patient developed cell-mediated cytotoxicity against TAAs.

Future Directions

In the future, active specific immunotherapy is likely to become part of a multimodal approach to cancer. For example, patients may undergo surgical reduction of large tumor burdens prior to vaccine therapy. Animal models indicate an inverse relationship between tumor burden and therapeutic outcome. A tolerable tumor burden in a mouse generally does not exceed one million cells. If results in murine models can be extrapolated to humans, a tolerable tumor burden for a 70-kg man would be 3.5 × 109 tumor cells, corresponding to a spherical tumor volume of about 2.5 cm3 and a diameter of about 1.5 cm. The human immune response should be capable of rejecting this small but clinically evident tumor.

Spontaneous regression of large visceral metastases of malignant melanoma and of accidentally transplanted allogeneic tumors suggests that the human immune response may sometimes be capable of eliminating larger tumor nodules. Mastrangelo and colleagues 189 argue that given the proper circumstances, such as depletion of suppressor T cells, tumor-specific immunity can cope with relatively large tumor burdens. Combining vaccines with more conventional modes of therapy could enable tumor-specific immunity to cope with greater numbers of tumor cells. Thus, immunotherapy in combination with chemotherapy has shown promise for the treatment of leukemia, lung cancer, and melanoma. 61

Future directions in the active specific immunotherapy of cancer require a better understanding of adjuvants administered with tumor cell vaccines. BCG has proven to be an excellent nonspecific immunopotentiator, but immunoregulation and trafficking of cells within the immune system are not well understood. The role of various components of BCG and other mycobacteria, such as BCG cell walls, trehalose dimycolate, muramyl dipeptide, and nontoxic derivatives from lipopolysaccharides of gram-negative bacteria, requires further study.

Melanoma patients can be classified into different groups based on the distribution of four biosynthetically related tumor-associated glycolipids. 25 Vaccine treatment can be adjusted to match the antigenic profile of biopsied tumor cells. Different combinations of tumor cell vaccines may prove optimal for different groups of patients. A similar strategy has been used to choose appropriate monoclonal antibodies for serotherapy in melanoma. In the future, the ultimate success of immunotherapy depends on a better understanding of TAA heterogeneity with respect to preparation of tumor cell vaccines, as well as the role of immunopotentiators in reversing or rectifying tumor-associated immunodeficiency.

Infiltration with T cells may be required for immunologically mediated regression of human cancer. Factors favoring T-cell infiltration must be identified. 70 The absence of infiltrating T cells in metastatic tumors excised from cancer patients and their presence in metastatic tumors after immunotherapy 190 suggest that vaccines may have a role in potentiating the appropriate migration of cytotoxic effectors.

Transfection of autologous or allogeneic tumor cells with genes encoding different cytokines should increase vaccine potency. For example, IL-4 participates in the regulation, growth, and differentiation of B cells and T cells and in the generation of cytotoxic lymphocytes. Transfection of a renal cell carcinoma with the IL-4 gene causes the tumor cells to secrete large amounts of this cytokine. The rejection of IL-4-transfected tumor cells by the host is associated with development of tumor-specific immunity mediated primarily by CD81 T cells; this immunity is also effective against tumor cells that do not express IL-4. 69 Several studies have documented an increase in cytolytic T-cell killing of tumor cells following transfection with the IL-2 gene. 68, 136, 191, 192 Tumor cells transfected with GM-CSF have also been associated with specific, long-lasting antitumor immunity and are under clinical investigation. 28, 70 Related approaches involve the genetic engineering of tumor-infiltrating lymphocytes with cytokine-producing genes such as tumor necrosis factor. 193


The present status of cancer vaccines can be best summarized by stating that we are at the end of the beginning. Despite 25 years of efforts toward effective active specific immunotherapy, there is no neoplasm for which cancer vaccines can be considered standard therapy. Most trials have been historically controlled. Earlier studies showed that systemically administered BCG conferred a significant survival advantage when treated patients were compared with historical control groups; however, this advantage was not confirmed in concurrently controlled randomized clinical trials. As yet, more recent studies that have been optimally designed and controlled have not been replicated. However, many reports from phase I or II trials indicate partial and complete responses to vaccine therapy, particularly among patients with metastatic melanoma or renal cell cancer. Importantly, these effects are achieved with little or no toxicity—a distinct contrast to the often severe side effects of chemotherapy. Active specific immunotherapy has the added advantage of a long duration of response. 22 Thus, although chemotherapeutic agents can kill tumor cells immediately, their clinical effect may last only weeks or months. Vaccines have no direct cytotoxic effect on tumor cells, relying instead on the induction of humoral and cell-mediated antitumor immune responses, which generally evolve over a period of several months. In fact, after the initial immunization, clinical disease may appear to progress for several weeks before it stabilizes and subsequently begins to regress. However, unlike the short-term response to most chemotherapeutic regimens, vaccine-induced regression is usually durable, often lasting from months to years.

The challenge for the future will be to develop vaccine adjuvants and delivery systems that will allow the cancer patient to develop a clinically effective immune response. This may require better methods of selecting candidates for vaccine therapy. Most phase I/II trials have enrolled patients with advanced disease, whose relatively large tumor burden makes them less-than-ideal candidates for vaccine therapy. Future trials should focus on cancer patients with minimal subclinical disease. Several cancer vaccines are now entering phase III trials, and we look forward to the time when active immunotherapy becomes a standard approved treatment for neoplastic disease.


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