April 2002 A.indd

A. INDAR, C.A. MAXWELL-ARMSTRONG, L.G. DURRANT*, J. CARMICHAEL* and J.H. SCHOLEFIELD

Division of Surgery, Queens Medical Centre, Nottingham, NG7 2UH, and * Academic Department of Clinical Oncology, Nottingham City Hospital, NG5 1PB.

Introduction

Passive immunotherapy

Active immunotherapy

Non-specific immunotherapy

Tumour escape

Conclusion

References

Immunotherapy could have a role in the therapy of colorectal cancer as there is now convincing evidence that the immune system can specifically recognize and destroy malignant cells. The MAb 17-1A has been used in advanced and primary disease, along with newer agents such as anti-epidermal growth factor receptor (EGFR) antibody. Immunotherapy with autologous tumour cell vaccine, genetic modification of immunostimulatory cytokines, suicide genes and TAAs as discussed. The multiplicity of peptide and carbohydrate antigens which can be potential targets for immunotherapy are also discussed. These include MUC1, Thomsen-Friedenreich and Sialosyl-Tn antigens and HER2 / neu. Active specific immunotherapy with the anti-idiotypic antibodies CEAVac and 105AD7, along with DC vaccines, is being currently used in adjuvant clinical trials. 105AD7 has been shown to cause significantly greater apoptosis of tumour cells in colorectal cancer patients, while CEAVac generated T cell proliferative anti-CEA responses. Dendritic cells pulsed with tumour mRNA or TAAs currently are being assessed in clinical trials. The role of HSPs in the anti-tumour immune response is discussed. Non-specific immunotherapeutic agents used in clinical trials with chemotherapeutic regimens have not shown any definitive benefit. Tumour progression may occur as result of escape from the host anti-cancer immune response. Better understanding of mechanisms of tumour evasion could explain why immunotherapy trials in patients have not shown better results. These include down-regulation of immune responses by the tumour, altered expression of MHC and/or TAAs by tumour cells, altered expression of adhesion molecules by tumour and/or DCs and usurpation of the immune response to the advantage of the cancer.

Keywords: anti-idiotypic antibody, immunotherapy, colorectal cancer, edrocolomab, 105AD7, BCG, review, dendritic cells, CEA, tumour escape, tumour infiltrating lymphocytes, MAb,17-1A, heat shock proteins

Colorectal cancer (CRC) accounts for 10-15% of deaths from cancer in industrial nations.¹ The current model for colorectal carcinogenesis postulates a multi-stage progression involving an accumulation of gene mutations (adenomatous polyposis coli, K-ras, p53, DNA mismatch repair genes), alteration in gene expression (c-myc) and chromosome losses during which cell growth is disrupted.2 Most deaths from colorectal cancer occur from tumour cell dissemination and metastases to the liver. Treatment must be aimed at controlling recurrence and preventing distant spread.

Systemic toxicity and drug resistance continues to limit the usefulness of chemotherapy. Concurrently, biological therapy has now moved into the era of immunotherapy. The use of nonspecific immunotherapeutic agents has been surpassed by modalities that produce a specific and potent immune response against tumour cells. Approaches that come under this heading include MAb therapy, tumour cell vaccines, anti-idiotypic antibody therapy and DC vaccines. Cells can be further boosted by the use of immunostimulatory cytokines.

This review aims to evaluate current strategies of immunotherapy for colorectal cancer with particular emphasis on possible clinical benefits.

PASSIVE IMMUNOTHERAPY

Antibody targeted therapy relies on the presence of TAAs expressed on cancer cells. They induce tumour regression by complement-mediated cytolysis, ADCC, opsonization to facilitate phagocytosis, or by direct action of blocking growth factors or inducing apoptosis. (Figure 1) Tumour cell antigen expression is heterogeneous, and may result in groups of cells that lack such antigens and, thereby, are resistant to antibody targeted therapy. Many epitopes are expressed with a higher appropriate density on tumour cells than on normal cells.³ TAA density is an important determinant of tumour cell destruction by MAb.⁴

The target may also be the vascular endothelium of the tumour that is directly accessible to therapeutic agents. This is seen using a single chain anti-VEGF antibody, which reacts with receptor-bound VEGF.⁵ It has also been shown that cytokines such as IFN-a, IFN-y and IL-2 significantly augment the ADCC of both MAb 17-1A and the MAb BR55-2 against the colorectal carcinoma cell line HT29.⁶

Various strategies of antibody-targeted therapy are being assessed. Radioimmunotherapy uses antibodies labeled with gamma emitting radionucleotides. Not all the tumour cells need to be bound by MAb, as the radiation emitted is able to kill adjacent cells. Immunotoxin therapy consists of a fungal or bacterial toxin conjugated to a MAb. When the antibody is internalized, the toxin blocks protein synthesis leading to cell death. Chemotherapeutic agents can also be conjugated to MAbs, increasing the bioavailability of the drug. Antibody-directed enzyme prodrug therapy (ADEPT) consists of an antibody that is bound to an enzyme. This conjugate accumulates in the tumour and activates an anti-tumour prodrug that is subsequently administered. An ADEPT system against CEA-positive tumours is currently in phase I clinical trials. It consists of a prodrug, 4-{N, N-bis (2-iodoethyl) amino} phenoxycarbonyl L -glutamic acid (ZD2767P) and a conjugate of the Fab'2 anti-CEA antibody A5B7 with the bacterial enzyme carboxypeptidase G2 (CPG2). When administered to mice, extensive DNA interstrand cross-links were detected at 1 hour. However, this was only seen in 1 out of 5 patients, who demonstrated good localization of conjugate and a tumour response.⁷ Table 1 summarises trials with passive immunotherapy.

The IgG2a murine MAb 17-1A targets the CD17-1A antigen that is expressed on the surface of more than 90% of colorectal carcinoma cells.⁸ In clinical studies in patients with advanced colorectal cancer, mild toxicity was experienced without any significant effects on response or survival.⁹ In further trials in patients with metastatic colorectal cancer, the effects of this MAb were boosted with IFN-• and GM-CSF. There was augmentation of monocyte-mediated ADCC, increased infiltration of tumour lymphocytes, macrophages as well as complement deposition.^10-13 Patients with greater ADCC activity survived significantly longer than those with low levels. Takamuku et al (1996) showed that the unconjugated MAb 17-1A could cause apoptosis by ADMC in a colorectal cancer cell line in vitro. This was enhanced by IFN-y. Further, TNF-•a release was significantly increased during ADMC.¹⁴

When 17-1A was given as adjuvant treatment in a randomized trial of patients with Dukes C cancer the 5-year mortality was decreased by 30% and the recurrence rate by 27%, compared with an observation only arm. Patients with metastatic deposits who relapsed responded better to the MAb than those with local recurrence.¹⁵ An update of this work, whereby all but 4 patients had at least 5 years of follow-up, revealed a decrease in overall mortality by 32% and local recurrence rate by 23% in treated patients.16 These results have been further tested in a Phase III study, where 2761 patients were randomized to receive either 17-1A monotherapy, 5-fluorouracil (5-FU) and folinic acid, or a combination of chemo-immunotherapy. ¹⁷ Median follow up was 26 months. No additional benefit was seen by the addition of immunotherapy to the standard chemotherapy regimen. Immunotherapy alone was associated with a significantly shorter survival and disease-free period.

Figure 1: Mechanism of monoclonal antibody mediated tumour-cell lysis. Antibodies mediate tumour cell lysis by binding to their tumour target through the Fab fragment and (A) activating the complement cascade through interaction with their Fc region, (B) interacting with Fc receptors on host effector cells (NK cells), (C) blocking the binding of growth factors to receptors on tumour cells, and (D) generating intracellular signals that lead to programmed cell death.

Epidermal growth factor receptor (EGFR) is over-expressed in 50-70% of human breast, lung, and colon carcinomas. Circulating EGFR mRNA has been detected by RT-PCR in the serum from two of seven (29%) patients with Dukes B and five of nine (55%) with Dukes C cancers.¹⁸ Patients with EGFR positive tumours were mostly those with Dukes' stage C or D (27% of EGFR-ve vs. 58% of EGFR+ve), had greater aneuploid characteristics (48% EGFR-ve vs 82% EGFR+ve), and more frequent occurrence of lymphatic invasion.¹⁹ Expression of EGFR did not correlate with proliferation of human colonic carcinomas, but exposure of human colon cancer cells in vitro to the EGFR blocking MAb 225 induced apoptosis. ^20,21

A phase II trial of IMC-C225 (a chimeric MAb that binds selectively to EGFR) with CPT-11 (Irinotecan) in 121 patients with advanced colorectal cancer refractory to both 5-FU and Irinotecan, was performed in patients whose tumours expressed for EGFR. Approximately 72% of patients screened tested positive for EGFR by immunohistochemistry. The median time from CPT-11 failure to initiation of IMC-C225 + CPT-11 was 30 days (mean of 54 days). Twenty-one patients (17%, 95% CI 11%-25%) achieved a partial response, for a median duration of 84 days (range 42-210 days), 37 additional patients (31%) had stable disease or minor responses, and 17 patients continue to be followed up. The authors concluded that IMC-C225 combined with Irinotecan can produce a major objective response in patients with EGFR +, Irinotecan refractory cancers, with acceptable toxicity. ²²

Further studies have shown that MAb 225, in combination with anti-sense VEGF, can have synergistic anti-angiogenic and anti-tumour effects. Treatment with MAb C225 demonstrated a dose-dependent inhibition of release of VEGF and TGF-• with an associated reduction in microvessel count. Both inhibition of tumour growth and significantly improved survival were observed in all mice treated with both agents, compared with either MAb C225 alone, or VEGF-AS.²³ Using antibodies against both the VEGF receptor (DC101) and EGFR (C225), in nude mice expressing the human cell line KM12L4, revealed decreased tumour vascularity, growth, proliferation, formation of ascites and increased apoptosis of both tumour cells and endothelial cells, relative to the control group. Therapy with C225 did not change any of the above parameters. ²⁴

Another target in MAb therapy for colorectal cancer is the tumour-associated glycoprotein 72 (TAG-72), which is expressed by 90% of colorectal cancer cells. However, the anti-TAG-72 murine MAb CC49 has not produced any anti-tumour response in patients with advanced colorectal cancer.²⁵ The human monoclonal antibody SK-1 recognizes a glycoprotein expressed on the majority of colon cancer tissues. It’s safety is being evaluated in patients with recurrent and advanced colon cancer. The mean rate of serum CEA level increase declined significantly during the eight weeks following the treatment, which was well tolerated.²⁶

Active immunotherapy aims to stimulate the immune system against tumour cells. T cells are activated by APCs and require two signals. Firstly, a signal from MHC expressing specific peptides recognised by the antigen-specific TCR and a second costimulatory signal provided by binding of CD28 on T cells to the CD80/86 ligand on the APCs. (Figure 2)

T lymphocytes recognize proteins or peptides presented in the context of MHC antigens on the surface of APCs.27 The proteins or peptides may be cell surface or cytoplasmic derived proteins.²⁸ Extracellular proteins are endocytosed and processed by specialized APCs including B cells, monocytes, macrophages and DCs. They then bind to MHC class II molecules on the APCs and the complex is recognized by specific CD4+ THs. Intracellular proteins are processed by proteosomes and transported within the APC to become associated with cell surface MHC class I molecules and recognized by CD8+ CTLs. Once T lymphocytes have been primed by contact with an epitope on an APC, it becomes activated, proliferates and specifically targets cells bearing that epitope. On contact with the epitope/MHC II complex on target cells, CD4 THs secrete inflammatory cytokines (e.g. IFNy, TNF-a) which can, in turn, recruit other immuno-regulatory cells to the target site.²⁹ Activated CTLs that recognize peptide-MHC I complexes on tumour cells kill by exocytosis of granules containing perforins and other proteases or Fas-Fas ligand interaction resulting in apoptosis of the tumour cells. ^30,31 (Figure 3)

Figure 2: Presentation of antigens on MHC molecules is stimulated by IL-12. The APC processes exogenous protein from the vaccine or lysed tumour cells into peptides and presents them to CD4+ and CD8+T cells with signals from CD28 costimulatory molecules, resulting in antigen -specific activation of both types of T cells. Proliferation of CTLs require IL-2 produced by TH cells and their maturation requires other cytokines including IFN-•y and TNF-a

Figure 3: Mechanisms of cytotoxic T lymphocyte killing. (A) CTLs may secrete cytotoxic granules whose contents penetrate tumour cells. (B) CTLs express Fas ligand that conveys signals to tumour cells with the Fas antigen to undergo apoptosis. (C) Activation of TH cells leads to cytokine secretion that stimulates CTLs and are directly toxic to tumour cells.

Immunocytokines have been shown to induce the generation of tumour-specific CTLs.A recombinant humanized antibody-IL-2 fusion protein resulted in eradication of established pulmonary and hepatic colon carcinoma metastases in syngeneic BALB/c mice.³² There was long-lived tumour immunity to a secondary tumour cell challenge mediated by memory cells. The response was amplified after a boost of the original recombinant anti-body.³³

Natural killer cells are a subset of mononuclear cells that do not express the TCR complex on their cell surface but mediate cytotoxicity against tumour or virus-infected cells without prior sensitization or the need for recognition of the MHC.³⁴ They are activated by IL-2 and are able to infiltrate tumour tissue and initiate cytokine production.³⁵ Natural killer cells adhere to cells via an activation receptor and cause lysis unless an inhibitory receptor on the NK cell is engaged by the class I MHC molecule ligand on the target cell. Many tumours with low or absent MHC expression are good targets for NK cells.³⁶ Table 2 summarises clinical trials with active immunotherapy.

Whole tumour cell preparations have been an area of intense interest as they express a range of TAAs. There is, however, much difficulty associated with autologous tumour cell vaccines due to the availability of fresh autologous tumour material and the difficulty in development of tumour cell lines from primary tissues. In contrast, allogeneic cell vaccines have greater immunogenicity than autologous vaccines. They are developed from cell lines selected to express a broad range of TAAs and HLA haplotypes.

Eighty patients with Dukes stage B2 and C colorectal cancers were vaccinated post-operatively with irradiated autologous tumour cells with BCG. Overall survival and disease-free interval, were improved in the group receiving immunotherapy, with a significant survival difference in the treated colon cancer patients, compared with the controls, who received surgery alone. Interestingly, those treated patients with rectal cancers who received pelvic radiotherapy demonstrated worse survival and disease-free interval compared with colonic cancer patients who received immunotherapy and no radiotherapy.³⁷ Subsequently, a meta-analysis of three prospective randomized controlled trials of patients with stage II and stage III colon cancer, comparing the response to autologous tumour cells-BCG given post-surgery with surgery alone, confirmed these results, namely, improved overall survival in the vaccinated group, significant survival (p=0.02) and disease-free interval (p=0.039) compared with the control group of patients. The 5-year survival for the control group was 68.7% versus 81.1% for the treated group.³⁸

In another trial, irradiated autologous colorectal carcinoma cells infected with Newcastle disease virus produced a 98% 2-year survival rate versus 67% for patients receiving cells admixed with BCG.³⁹

Genetic manipulation of tumour cells involves introducing immunomodulatory genes into tumour cells as a means of enhancing specific cell-mediated immunity. Gene transfer can be accomplished by transfection of plasmid constructs or by transduction using a retroviral vector containing the specific gene. Immunostimulatory genes encode cytokines such as IL-2, GM-CSF, IL-12, TNF or IFN-y.⁴⁰

Murine colon carcinoma models transduced with the IL-2, GM-CSF and IL-12 genes have all generated active specific tumour immunity. ^41-44 A combination of genes for IL-12 and the costimulatory molecule CD80/86, have shown to give significant protection.^43,45

Tumour cells can also be infected with viruses carrying ‘suicide genes’, which encode enzymes (e.g. Herpes simplex virus thymidine kinase and Escherichia coli cytosine deaminase) that convert prodrugs (ganciclovir and 5-FU, respectively) into toxic forms that kill the tumour cells in vivo. This inflammatory response can release antigens that convert the tumour into an immuno-stimulatory environment resulting in lysis of metastases and inducing tumour regression in a mouse colorectal cancer model with associated increased time of survival.⁴⁶ This effect was increased with IL-12-mediated gene therapy.⁴⁷

The administration of genes encoding TAAs is another role for gene transfer in immunotherapy. Antigen encoding plasmid DNA can be given in its naked form intradermally, intramuscularly or by a “gene gun”. Alternatively, liposomes, viral vectors or protein carriers can be used. Antigens that can be exploited as potential DNA vaccines include mutated oncogene product p21, ras, mutated tumour suppressor p53, CEA, epithelial mucin MUC-1.48 Table 3 summarises the mechanisms of genetically modified immunotherapy.

Human cancers can stimulate immune responses against TAAs expressed on their surface. It has been possible to generate T lymphocytes in vitro that are capable of recognizing these antigens on autologous tumour cells.⁴⁹

Autologous lymph node cells with metastatic colorectal cancer cells from 32 patients were dissociated, expanded and re-infused into patients with limited toxicity. These cells were predominantly CD3+ and were genetically modified to express IL-4, IL-5, IFN-• and GM-CSF. One patient had a partial response (>80% reduction in size) and four patients had a mixed response (<80% reduction). The responding patient's cells were notable for both high levels of GM-CSF and IL-4 secretion, on re-stimulation with immobilized anti-CD3 in vitro. The biopsies of the tumour were also characterized by high levels of macrophage infiltration. The median survival of the cell-treated group compared favourably with a similar group of patients who underwent radio-immunoguided surgery without cell treatment (12.5 months vs. 5.8 months).⁵⁰

The function of TILs in human colorectal adenocarcinoma was investigated by looking at the distribution of mRNA for IFN-y, TNF-a, IL-10 and IL-4 in TILs and tumour cells. Median levels of IFN-•y and TNF-•a mRNA were significantly higher in TILs than lamina propria infiltrating leukocytes (LPL). IL-10 mRNA was observed in TILs and LPLs, but very small amounts of IL-4 transcripts were detected in TILs and LPLs. Tumour necrosis factor -a and IL-10mRNA were greater in tumour cells than in normal epithelial cells. These results suggest that TILs are characterized by a TH1-like pattern of cytokine expression and function as T cells (and macrophages) in local, cell-mediated anti-tumour immune responses. Furthermore, changes in IFN-•y and TNF-•a mRNA in TILs and tumour cells could be related to tumour progression (e.g. by T cell anergy) or formation of metastases.⁵¹

The presence of TILs has been shown associated with DNA microsatellite instability. This may be able to account for the high level of apoptosis seen in these tumours with their improved prognosis.⁵² Smyrk et al (2001) showed that by quantifying TILs, microsatellite instability type can be predicted with a sensitivity of 93% and specificity of 62%. ⁵³

Oncogene products can generate a potent anti-tumour response. These purified peptides can be synthetically generated, but are restricted to a single HLA molecule and are themselves not very immunogenic.⁵⁴ Their immunogenicity may be increased with adjuvants, cytokines, liposomes or with peptide-pulsed DCs.^55-,57 Whole proteins have the advantage over peptides in that they express a wider range of T cell epitopes.

Mucins such as MUC1 are high-molecular weight proteins, abundantly expressed on human cancers of epithelial origin.⁵⁸ The MUC1 gene is over-expressed and aberrantly glycosylated on a variety of cancers including colorectal cancer. This exposes antigenically active epitopes on the cell surface membrane. In mice, vaccination with MUC1 produced high antibody responses with poor CTL responses.⁵⁹ Sixty-three patients who were vaccinated with a mucin peptide/BCG complex had intense T cell infiltration on biopsy in 37 cases, with 7 of the 22 tested showing increased levels of mucin-specific CTL precursors.⁶⁰ However, when patients were injected with manosylated MUC1 peptide, a poor CTL response and a high antibody response was observed, compared with mice where high CTL responses were seen. This antibody response was due to the presence of antibodies against the Gala(1,3) Gal epitope that cross-react with MUC1 peptides.⁵⁹

To examine MUC-1-specific cytotoxic T lymphocytes with the recombinant (r) vaccine rVMUC-1, peripheral blood lymphocytes from patients with DF3/MUC-1 (+ve) colon carcinomas were stimulated using the autologous monocytes infected with rVMUC-1. The stimulated T lymphocytes from two patients demonstrated HLA-unrestricted cytotoxicity against MUC-1. Adoptive transfer of the first stimulated lymphocytes demonstrated a transient high level of HLA-unrestricted CD8+ cells against MUC-1, and significant reduction of the liver metastasis in one patient. However, HLA-unrestricted CD8+ induction against MUC-1 was reduced substantially at the second transfer and finally eliminated at the third. Contrary to the author’s expectation, the repetitive stimulation caused a selective proliferation of HLA-class-II-restricted CD4+ cells against MUC-1. ⁶¹

The Thomsen-Friedenreich and Sialosyl-Tn antigens are blood group-related disaccharides that are linked to mucins on epithelial cancers including colorectal cancer.⁶² They are found on the luminal surface of secretory cells in normal tissue that are inaccessible to the immune system. Altered glycosylation exposes their core structures in malignant tissue. They are poor immunogens, and their expression on human carcinomas frequently reflect biological aggressiveness. ⁶³ Additionally, they may also inhibit specific cell-cell adhesion by malignant cells that is required for invasion and metastasis formation by cancer cells.⁶⁴

HER2 / neu antigen expression and prognosis has been described; in colorectal carcinomas, higher expression of HER2 / neu is recognized in metastatic regions. ⁶⁵ Also, high levels of HER2/neu expression is found in tumours with more advanced Dukes stages and correlate with decreased relapse-free survival. ⁶⁶ Cytotoic T Lymphocytes have shown activity against a HER2/neu peptide on target cells and HLA-A24 colorectal tumour cell lines that endogenously over-expressed HER2 / neu. Thus, HER2 / neu peptide, RWGLLLALL, could be used in peptide-based immunotherapy in colorectal carcinomas.⁶⁷

The cyclooxygenase-2 (COX-2) and ErbB/HER family of growth factor receptors are important in colorectal cancer development. Activation of both ErbB1 and HER-2/neu can stimulate human colon cancer cell (HCA-7) proliferation via induction of COX-2. ⁶⁸ It was also found that COX-2 inhibition and the MAb anti-HER-2/neu, Celecoxib and Herceptin (Genetech, CA, USA), respectively, inhibited carcinoma cell growth in vitro and in vivo. Combination therapy of Celecoxib withHerceptin or 2C4, resulted in an additive effect and in almost complete inhibition of tumour growth.⁶⁹

Sasatomi et al (2000) recently identified the tumour-rejection antigen gene SART1, which encodes the SART1 (259) antigen expressed in the cytosol of epithelial cancers. The SART1(259) antigen was detected in the cytosol of four of six cancer cell lines, 13 of 33 (39 %) cancer tissues, and 0 of 20 non malignant colorectal tissues. The SART1 (259) antigen could be an appropriate target molecule for specific immunotherapy in approximately 40 % of the HLA-A24+ patients with colorectal cancer.⁷⁰

The immune network hypothesis of Lindemann (1973) and Jerne (1974) offers a unique approach to transforming epitope structures into idiotypic determinants expressed on the surface of antibodies.^{71, 72}It describes the immune system as a network of interacting antibodies and lymphocytes, and predicts that the external TAA, is mimicked by idiotypes expressed by antibodies and T cell receptors.

Immunization with an antigen will generate antibodies against this TAA, called Ab1. This Ab1 can generate a series of anti-idiotypic antibodies against itself, termed Ab2. The Ab2 molecules effectively mimic the three dimensional configuration of the specific TAA. These anti-idiotypic anti-bodies, called Ab2, can induce a specific immune response and, therefore, can act as a surrogate TAA. Immunization with Ab2 can lead to the generation of anti-anti-idiotypic antibodies termed Ab3. They recognize the original TAA identified by Ab1. Because of this Ab1-like reactivity, the Ab3 is also called Ab1 to indicate that it might differ in its other idiotopes from Ab1. (Figure 4)

Figure 4: Immune Network Hypothesis: TAA when injected, generates Ab1 antibodies. These in turn, generate Ab2 molecules that mimic the TAA. The Ab2 antibodies can then act as a surrogate antigen and stimulate an immune response against themselves ant the original antigen

The anti-idiotypic antibody represents an exogenous protein that is endocytosed by APCs, degraded, and presented by Class II antigens to activate THs. Activated TH2 secrete cytokines such as IL-4 that enhance B cell proliferation, which are also directly activated by the Ab2 to produce Ab1. Additionally, activation of CD4+ TH1 cells secrete cytokines (e.g.IL-2) that activate T cells, macrophages and NK cells that directly lyse the tumour cells, and enhance the CD8+ CTL responses. (Figure 2) The anti-idiotypic antibody may also degraded to peptides and be presented with class I antigens to directly activate CTLs which are stimulated by IL-2. ⁷³

Several anti-idiotypic antibodies that mimic TAAs on colorectal cancer cells have been reported. One such anti-idiotypic antibody is generated against the murine 17-1A MAb, which targets the GA733-2 antigen (also referrred to as CO17-1A). Active immunotherapy directed against the CO17-1A antigen potentially induces long-lasting immunity, in contrast to passive immunotherapy, which is short-lived. Immunization of six patients with advanced colorectal cancer patients with this anti-idiotypic antibody developed antigen-specific T cell immunity. Five patients mounted an Ab3 response and toxicity was mild.⁷⁴ Another group using an aluminum hydroxide-precipitated rat anti-idiotypic antibody generated to 17-1A, immunized nine patients with colorectal cancer. Only three patients developed Ab3 that bound to idiotypic determinants on Ab2.⁷⁵

In other approaches to active immunotherapy against this antigen, polyclonal goat and monoclonal rat anti-idiotypic anti-bodies (Ab2) directed against MAb CO17-1A or GA733 (Ab1) were administered as alum precipitates to 54 patients with colorectal cancer (Dukes' B, C, and D). The majority of the patients treated with the various Ab2 preparations developed anti-anti-idiotypic antibodies (Ab3) that specifically bound to the CO17-1A or GA733 epitope and shared idiotopes with the corresponding Ab1. Approximately 30% of the patients tested developed specific cellular immunity, i.e., Ag-specific T-cells mediating delayed-type hypersensitivity (DTH) reaction in vivo or proliferating on stimulation with the Ag in vitro.⁷⁶

The immunogenicity and tumour-protective activity of the TAA GA733 was compared with the monoclonal anti-idiotypic anti-body BR3E4 in mice. The antigen, but not Ab2 induced specific humoral immunity, however, when the Ab2 was administered in Freund’s complete adjuvant, then specific humoral responses were elicited. Lymphoproliferative responses were obtained with the antigen only although DTH responses were obtained with both immunizations. The authors then concluded that the soluble antigen is a more potent modulator of humoral and cellular responses than Ab2.77When colorectal cancer patients were immunized with either the anti-idiotypic antibody mimicking the GA733 Ag or the recombinant protein BV GA733-2E78 (the GA733 Ag has been molecularly cloned and expressed in baculovirus) the immunogenicity of BV GA733-2E was superior to that of anti-idiotype in the same adjuvant.⁷⁹ Ultimately, the immunogenicity of Ab2 must be compared with those of tumor Ag in patients who are tolerant to the Ag expressed in their growing tumors. The higher potency of the Ag versus Ab2 is not surprising since Ag comprises multiple potentially immunogenic B and T cell epitopes, whereas Ab2 mimics only a single epitope.

The anti-idiotypic antibody 105AD7 is currently under investigation. It mimics the CD55 antigen expressed on 80% of colorectal cancer cells.⁸⁰ A phase I study was performed in patients with advanced colorectal cancer.⁸¹ The most important finding was that there was no evidence of toxicity related to the use of the vaccine. The premise that 105AD7 could elicit an immune response was further supported by the fact that 9 of the 13 patients immunized showed evidence of either T cell blastogenesis against CD55 expressing cell lines, or evidence of IL-2 production. In addition, patients who received the vaccine lived three times as long as a contemporary group of patients who did not.⁸² The results of this study have recently been tested in a double blind, multicentre, phase II study, comparing survival of 162 patients with advanced colorectal cancer receiving 105AD7 antibody or placebo.⁸³ Over 50% of these patients had disease in 2 or more anatomical sites. Not surprisingly, there was no significant survival difference between the trial and control groups.

In view of the lack of toxicity associated with using the anti-idiotypic 105AD7 immunization, work has concentrated on using the vaccine in patients prior to surgery. Immunohistochemical labeling of specimens taken from resected tumours showed an increased infiltration of CD4+, CD8+ and CD56+ lymphocytes, and significant expression of the activation marker CD25.⁸⁴ In addition, a drop in the CD8RA/RO ratio was observed, though the accumulation of memory or activated T cells was not maintained in the absence of repeated immunizations.⁸⁵ This strengthens the hypothesis that T cells primed in the periphery are capable of targeting CD55 on colorectal cancers.

The cellular phenotypes generated have sequences that are predicted to bind to HLA A1, A3 and A24 with the highest affinity, as well as HLADR1, DR3 and DR7. Eight-three percent of 36 patients, with colorectal cancer expressing the predicted permissive haplotypes HLA-DR1, DR3 and DR7, responded to 105AD7, whereas 88% of non-responders failed to express these haplotypes.⁸⁶ There was an overall 4-year survival of 65%, with Dukes C patients showing a 4-year survival of 64%.

The ability of 105AD7 to induce apoptosis in tumour cells was assessed by immunizing 21 patients pre-operatively with the antibody.⁸⁷ There was a significant increase in apoptosis in the tumours of patients immunized with 105AD7, compared with controls (p<0.005), suggesting that primed lymphocytes were capable of targeting cancer cells expressing CD55, and causing death by apoptosis. This shows the potential for its use in the adjuvant setting against circulating micrometastases expressing this TAA. These results are currently being tested in a randomized controlled trial, using both BCG and alum as adjuvants.

CEAVac is an anti-idiotypic murine MAb generated against an antibody designated 8019. This identifies a specific epitope on CEA, which is found on malignant cells in 95% of cases of colorectal adenocarcinoma, 70% of lung adenocarcinomas, and 50% of breast cancers. The efficacy of CEAVac antibody as a tumour vaccine was shown by its ability to induce an Ab3 response, as well as anti-CEA T cell response in mice that were challenged with the colorectal cancer cell line MC-38-CEA, with no protection seen in cells not expressing CEA.⁸⁸

In a phase 1b trial, CEAVac with alum generated anti-anti-idio-typic (Ab3) responses in 17 of 23 patients. None of the patients demonstrated any objective clinical response, the median time to disease progression was 2.4 months, and the median survival was 11.3 months with a 44% one-year survival. The authors concluded that CEAVac was able to break the immune tolerance to CEA in patients with colorectal cancer.⁸⁹

As adjuvant therapy, 32 patients with Dukes B, C and metastatic disease were given CEAVac; 14 of these had simultaneous 5-FU chemotherapy. All patients generated T cell proliferative and high-titer polyclonal anti-CEA responses that mediated ADCC. The predominant Ab3 was mainly IgG1 and IgG4. Three of 15 Dukes B and C patients relapsed at 19, 24 and 35 months, respectively. The 5-FU based chemotherapy regimens did not affect the immune response to CEAVac, which was well tolerated.⁹⁰

A Phase III trial has just been completed. Thirty-two patients with metastatic colorectal cancers received recombinant vaccine encoding full-length cDNA for CEA. Seven of these developed CEA-specific autoantibodies, predominantly IgG1.⁹¹

Dendritic cells present antigens to naïve T cells and induce CTLs. They selectively migrate through tissues to reach lymph nodes and the spleen to initiate T cell immune responses. They are considered to be the most potent APCs, and express high levels of MHC Class I and II molecules along with the costimulatory molecules CD80/86. ^{92, 93} Hence, they are necessary components for initiating and developing an effective cell-mediated immune response.⁹³ The T cell receptors recognize antigens bound to the MHC on the surface of APCs. The antigens associated with MHC class I and II molecules interact with and stimulate CTLs and THs, respectively. Dendritic cells are also a major source of cytokines e.g. IFN-a, IL-1, IL-6, IL-7, IL-12, IL-15 and macrophage inflammatory protein (MIP1g) which are important in the elicitation of the primary immune response.^94-96 These cytokines, together with CD80/86 costimulation, facilitate an optimal anti-tumour CTL response.⁵⁵

Tumour-associated antigens can be loaded onto DCs in vitro (culture and genetic manipulation or pulsing of DC progenitors).⁹⁷ In animal models, DCs pulsed with TAAs, previously recognized by MHC Class I restricted CTLs, developed an anti-tumour T lymphocyte response resulting in tumour regression and maintenance of tumour-free status.⁹⁸

A phase I-II study in advanced colorectal cancer patients using DCs pulsed with tumour RNA and keyhole limpet haemocyanin has been done, but follow-up has been too short to observe any significant clinical response. 99 Dendritic cell vaccines have shown promise in patients with melanoma. Dendritic cells generated from peripheral blood with IL-4 and GM-CSF were pulsed with melanoma tumour lysate and injected weekly into patients with advanced melanoma. Five of 16 patients showed an objective response, developing a DTH response to peptide-pulsed DCs in 11 patients.¹⁰⁰

Dendritic cells can also be genetically engineered to produce TAAs.¹⁰¹ Such genetic engineering can be accomplished either in vitro (transfection with plasmids or viral vectors) or in vivo (`naked' DNA immunization, immunization with viral vectors). Reports have shown that injection of naked DNA induces both humoral and cellular immune responses directed against the encoded immunogenic protein.¹⁰² However, patients with prostatic cancer who were administered transfected DCs with DNA coding for prostatic specific membrane antigen were reported to have an increase in the percentage of IFN-y- and IL-4-producing T cells.¹⁰³

Evaluation of pre-operative DC mobilization and tumour infiltration after administration of Flt3 ligand (Flt3L) to patients with metastatic colon cancer was performed in twelve patients. Flt3L is a growth factor for multipotent haematopoietic stem cells and causes expansion of DCs.104Patients received Flt3L before undergoing metastasectomy. The number of circulating leukocytes increased from 5.9 x103/mm3 to 11.2 x 103/mm3 (p =0.0001). The percentage of CD11c (+ve) CD14 (-ve) DCs increased from 2.4% to 8.8% (p =0.004). Additionally, an increase in the number of DCs was observed at the periphery of the tumours of patients who received Flt3L, compared with those patients who did not.¹⁰⁵ Patients with metastatic colorectal cancer were immunized with DCs pulsed with a CEA-derived peptide followed by subcutaneous administration of Flt3L. Results showed that DCs were expanded 20-fold, with induction of antigen-specific CTL responses in 7 of 12 patients. Clinically, one patient had dramatic tumour regression, one had a mixed response, and two had stable disease. This represents an attempt to break immune tolerance to CEA, a self-antigen, by immunizing with an altered peptide ligand .¹⁰⁶

Heat shock proteins are a group of proteins found intracellularly within the cytosol or nucleus, as well as in the cell membrane.^{107, 108} They function to protect the cell against lethal damage by becoming over-expressed in response to stimuli such as hyperthermia, oxidative stress, ethanol, arsenite, steroid hormones and certain anti-cancer drugs.^107,109,110 Their up-regulation by heating could be another way of improving immune recognition of colorectal cancer. ¹¹¹ The ability of HSPs to: (a) chaperone peptides, including antigenic peptides; (b) interact with APCs through a receptor; (c) stimulate APCs to secrete inflammatory cytokines; and (d) mediate maturation of DCs, highlights their possible importance in the immune system.¹¹²

Tumour cell killing using suicide gene transfer to generate death by a non-apoptotic pathway was associated with high immunogenicity and induction of HSP expression. When cDNA encoding for HSP70 was stably transfected into colorectal cancer cells, its expression significantly enhanced the immunogenicity. Increased levels of HSP, induced by non-apoptotic cell killing, may provide an immunostimulatory signal in vivo which helps break tolerance to tumour antigens.¹¹³

Heat shock protein 27, HSP70 and HSP90 have been found in colorectal cancer cell lines, their immunoreactivity varying with the cell lines.¹¹⁴ T cells with the yb T cell receptor and NK cells can recognise HSPs that are constitutively expressed by colorectal cancers. ¹¹⁵ The HSP70 family (including HSP72 and HSP73) is thought to act as an immunogenic determinant for different effector cell types. For example, HSP27 has the ability to inhibit TNF-a induced cell death and Fas/Apo-1-mediated apoptosis. ^116,117 Carcinoma cells with cell surface expression of HSP72 are associated with greater sensitivity to lysis mediated by NK cells than those without HSP72.¹¹⁸ Expression of HSP27 in the REG colorectal cancer cell line increased the resistance of the cells to apoptosis in vitro and enhanced their tumourigenicity in syngeneic animals.¹¹⁹

Studies of two long-recognized stress proteins, HSP110 and glucose-regulated protein (GRP) 170 to elicit their vaccine potential, have shown that prior vaccination with HSP110 or GRP170 purified from methylcholanthrene-induced fibrosarcoma, caused complete regression of the tumour in mice. In a second tumour model, HSP110 or GRP170 purified from the colon 26 cell line, led to a significant growth inhibition of this tumour. Mildly hyperthermic conditions enhanced the vaccine efficiency of HSP110, but not GRP170. Treatment of the mice with bone marrow-derived DCs pulsed with these two proteins from tumours also elicited a strong antitumour response.¹²⁰

NON-SPECIFIC IMMUNOTHERAPY

Levamisole is an anti-helminthic agent that was found to have immunostimulatory effects including enhancement of antibody production, augmentation of the cellular immune response, augmentation of chemotaxis, and enhancement of polymorphonuclear phagocytes and mononuclear leukocytes.¹²¹

In advanced colorectal cancer, combination of 5-FU and levamisole have failed to show a significant survival advantage or duration of remission over single agent 5-FU.122,123 The QUASAR1 trial randomized 4863 patients to 5-FU/high dose L-folinic acid (L-FA) or 5-FU/low dose L-FA/levamisole or placebo and found that there was no survival benefit in the levamisole group, when compared with placebo.¹²⁴ A randomized phase III trial of the three treatment regimens for patients with residual, non-measurable, intra-abdominal metastatic disease, after resection of the primary cancer was undertaken. Group A patients were randomized to receive either 5-FU alone or 5-FU/levamisole (n = 113 patients). Group B had hepatic metastasis and underwent a three-way randomization: 5-FU alone, 5-FU with levamisole, and 5-FU with hepatic irradiation. The median overall survival for the three treatment arms were similar, with 17.3 months for the group that received 5-FU alone, 16 months for the group that received 5-FU with levamisole, and 14.4 months for the group that received hepatic irradiation. The time to treatment failure was 6.7 months, 6.8 months, and 8.3 months, respectively, for the three groups. The authors concluded that there was no treatment advantage for any of the combined modalities over 5-FU alone in this group of patients with intra-abdominal, non-measurable disease.¹²⁵

It is of interest, that with colon carcinoma, an effective adjuvant regimen (5-FU plus levamisole) that yields an absolute 16% improvement in overall survival for Dukes C patients was not found to favourably improve either overall or progression free survival in patients with advanced, nonmeasurable disease.^126,
127

Interferon-a is a cytokine produced by leukocytes and fibroblasts. Early studies have shown that it increases the chemosensitivity of colonic and gastric cancer cell lines to 5-FU.128 In patients with metastatic disease, randomized trials comparing 5-FU alone with combinations of 5-FU/IFN-a revealed no improvement in the response rate, or overall survival in the IFN-a group. There were greater side-effects with IFN-a such as leucopenia, lymphopenia, constitutional symptoms, alopecia and depression.¹²⁹ Dukes B and C colorectal cancer patients receiving IFN-a with adjuvant treatment experienced a worse quality of life with no statistically significant difference in either disease-free survival or overall survival at 4 years of follow-up.¹³⁰

Interferon-y is a lymphokine produced by T lymphocytes in response to antigen exposure. Its immunomodulatory activity includes stimulation of NK cells, lymphokine-activated killer cells, stimulation of ADCC and enhancement of HLA Class II expression. A direct anti-proliferative effect of recombinant IFN-y was seen in vitro, on colorectal cancer cell lines as well in fresh tumour cells. Additionally, there was increased expression of HLA antigens with greater immunogenicity of tumour cells.¹³¹

BCG

Bacillus Calmette-Guerin is an attenuated strain of Mycobacterium bovis, and is known to stimulate cell-mediated immunity, humoral immunity and macrophage function.¹³² Intradermal injections of BCG in mice showed improved survival and enhanced the cytotoxic effects of chemotherapy.¹³³ There was, however, no response when BCG alone was compared with placebo in patients with advanced colorectal cancer.¹³⁴

An attempt to determine whether patients with colorectal cancer treated with autologous tumour cell vaccine with BCG, demonstrated that the BCG group had significantly increased DTH responses in 16 of 24 patients (67%), compared with the DTH response of patients immunized with autologous normal mucosa, used as controls. The presence of a significant DTH response to tumour cells after vaccination is a measure of immunogenicity of the vaccine and has been correlated with survival.¹³⁵

TUMOUR ESCAPE

The emergence of a tumour may be the result of an inadequate immune response due to poor immunogenicity of tumour cells, or low efficiency of the immune response against the tumour cells. Tumour progression may occur as result of their escape from the immune response. (see Table 4)

Wigmore et al (2001) found the constitutive production of TGF-B (beta) and IL-10 by colon adenomas and carcinomas cytokines known to suppress cell-mediated responses.^{136, 137} In colon cancer, a preferential accumulation of TH2 has been reported to occur under the influence of IL-4, which may contribute to suppression of the TH-1 effectors and expansion of TH-2 lines.138 The diversion of the immune response towards TH2 and humoral immunity, for which B cells are required, associated with disabling the TH1 and DTH response, may be critical for tumour tolerance.¹³⁹

The pro-inflammatory and angiogenic cytokine IL-8 is produced by human colorectal cancer cells. This isupregulated by the addition of the pro-inflammatory TH1 cytokines TNF-a, IL-1b and IFN-y. Addition of the regulatory TH2 cytokines IL-4 and IL-10 produced paradoxical stimulation of IL-8 production in certain cell lines and down-regulation of IL-8 production in others. These results suggest that tumour-derived cytokine production may have an important role in patients with colorectal cancer. Furthermore, they demonstrate the complexity of tumour cell cytokine production and highlight the difficulties in developing effective therapeutic biological response strategies.¹⁴⁰

Immunostimulation of tumour growth is seen by the fact that TILs can secrete angiogenic factors (basic fibroblast growth factor and VEGF) contributing to vascularisation of the tumours, and factors directly stimulating tumour cells (heparin-binding EGF like growth factor).^141,142 Tumour-infiltrating DCs have reduced expression of the co-stimulatory molecule B7, even after stimulation with cytokines, and could play a role in immune tolerance allowing tumour growth.¹⁴³

Studies done in a rat model colorectal cancer cell line CC531 and human colorectal cancer found immune cells to be abundantly present in these tumours. However, these cells were not found in direct contact with tumour cells, but mainly in the stromal part of the tumor. Adenoviruses, when intravascularly injected, did not reach tumour cells in the CC531 rat model. Tumour cells were only infected, and even then in limited numbers, in cases of intratumoural injection. The authors hypothesized that extracellular matrix in a tumour is a barrier both for immune cells and for adenoviruses to make direct contact with these tumor cells, and, thus, limit colorectal tumour immuno-therapy.¹⁴⁴

Colon carcinoma tissue and normal colon differ in their expression of functionally relevant adhesion molecules. Malignant colonic cells expressed few adhesion receptors whereas the stromal compartment within colonic carcinoma was positive for numerous binding molecules. Lymphocytes infiltrating tumour stroma contrasted with lymphocytes within normal colonic interstitium by lacking CD28, CD154 (CD40L), CD56 (NCAM) and CD98. Normal activated T cells bound to the lymphocyte-rich areas within the stroma of colon carcinoma, using CD44, CD50 (ICAM-3), CD99, CD102 (ICAM-2) and CD162 markers. This illustrates that only a limited set of adhesion molecules are expressed by colon carcinoma cells and this may inhibit their killing by lymphocytes.¹⁴⁵

CONCLUSION

Immunotherapy for colorectal cancer has been demonstrated in animal studies. Clinical trials, to date, have failed to achieve such effective anti-tumour responses. Future cancer immunotherapy approaches must aim to overcome instances of tumour escape. Accumulating knowledge of TAAs must be matched by information on how these antigens should be presented to stimulate T cells in vivo.

Despite the complexities involved in the immune response to tumours, immunization with soluble antigens or altered peptide ligands associated with mitogenic growth factors may be effective due to altered interactions with the TCR or activation of T cells of other specificities that are ignorant of the original TAA. Anti-idiotypic antibodies, by the generation of antigen-specific CTLs (e.g. CEAVac) and peptide-pulsed DCs are promising approaches for new clinical trials.

Genetic engineering is aiming to improve the specificity and potency of MAbs and active specific immunotherapy by upregulating immunostimulatory cytokines, improving their ability to penetrate tumours and to generate long-lived immunity.

REFERENCES

1. Beart R. Colon and Rectum In: Abeloff M, ed. Clinical Oncology: Churchill Livingstone, 1995:1267-86.
2. Kinzler KW, Vogelstein B. Lessons from hereditary colorectal cancer. Cell 1996; 87:159-70.
3. Herlyn M, Koprowski H. Melanoma antigens: immunological and biological characterization and clinical significance. Annu Rev Immunol 1988; 6:283-308.
4. Herlyn M, Menrad A, Koprowski H. Structure, function, and clinical significance of human tumor antigens. J Natl Cancer Inst 1990; 82:1883-9.
5. Cooke SP, Boxer GM, Lawrence L, et al. A strategy for antitumor vascular therapy by targeting the vascular endothelial growth factor: receptor complex. Cancer Res 2001; 61:3653-9.
6. Bungard S, Flieger D, Schweitzer S, Sauerbruch T, Spengler U. The combination of interleukin-2 and interferon alpha effectively augments the anti-body-dependent cellular cytotoxicity of monoclonal antibodies 17-1A and BR55-2 against the colorectal carcinoma cell line HT29. Cancer Immunol Immunother 1998; 46:213-20.
7. Webley SD, Francis RJ, Pedley RB, et al. Measurement of the critical DNA lesions produced by antibodydirected enzyme prodrug therapy (ADEPT) in vitro, in vivo and in clinical material. Br J Cancer 2001; 84:1671-6.
8. Gottlinger HG, Funke I, Johnson JP, Gokel JM, Riethmuller G. The epithelial cell surface antigen 17-1A, a target for antibody-mediated tumor therapy: its biochemical nature, tissue distribution and recognition by different monoclonal antibodies. Int J Cancer 1986; 38:47-53.
9. Mellstedt H, Frodin JE, Ragnhammar P, et al.Therapy of colorectal carcinoma with monoclonal antibodies (MAb17-1A) alone and in combination with granulocyte monocyte-colony stimulating factor (GM-CSF). Acta Oncol 1991; 30:923-31.
10. Weiner LM, Steplewski Z, Koprowski H, Sears HF, Litwin S, Comis RL. Biologic effects of gamma inter-feron pre-treatment followed by monoclonal antibody 17-1A administration in patients with gastrointestinal carcinoma. Hybridoma 1986; 5:S65-77.
11. Ragnhammar P, Magnusson I, Masucci G, Mellstedt H. The therapeutic use of the unconjugated monoclonal anti-bodies (MAb) 17-1A in combination with GM-CSF in the treatment of colorectal carcinoma (CRC). Med Oncol Tumor Pharmacother 1993; 10:61-70.
12. Ragnhammar P, Fagerberg J, Frodin JE, Wersall P, Hansson LO, Mellstedt H. Granulocyte/macrophagecolony-stimulating factor augments the induction of antibodies, especially anti-idiotypic antibodies, to therapeutic monoclonal antibodies. Cancer Immunol Immunother 1995; 40:367-75.
13. Shetye J, Ragnhammar P, Liljefors M, et al. Immunopathology of metastases in patients of colorectal carcinoma treated with monoclonal antibody 17-1A and granulocyte macrophage colony-stimulating factor. Clin Cancer Res 1998; 4:1921-9.
14. Takamuku K, Baba K, Arinaga S, Li J, Mori M, Akiyoshi T. Apoptosis in antibody-dependent monocyte-mediated cytotoxicity with monoclonal antibody 17-1A against human colorectal carcinoma cells: enhancement with interferon gamma. Cancer Immunol Immunother 1996; 43:220-5.
15. Riethmuller G, Schneider-Gadicke E, Schlimok G, et al. Randomised trial of monoclonal antibody for adjuvant therapy of resected Dukes' C colorectal carcinoma. German Cancer Aid 17-1A Study Group. Lancet 1994; 343:1177-83.
16. Riethmuller G, Holz E, Schlimok G, et al. Monoclonal antibody therapy for resected Dukes' C colorectal cancer: seven-year outcome of a multicenter randomized trial. J Clin Oncol 1998; 16:1788-94.
17. Punt CJ, Nagy A, Douillard JY, et al. Edrecolomab (17-1AAntibody)Alone or in combination with 5-Fluorouracil Based Chemotherapy in the Adjuvant Treatment of Stage III Colon Cancer: Results of a Phase III Study. (abstract). Program/Proceedings American Society of Clinical Oncology 2001; 20:123a.
18. De Luca A, Pignata S, Casamassimi A, et al. Detection of circulating tumor cells in carcinoma patients by a novel epidermal growth factor receptor reverse transcription-PCR assay. Clin Cancer Res 2000; 6:1439-44.
19. Hemming AW, Davis NL, Kluftinger A, et al. Prognostic markers of colorectal cancer: an evaluation of DNA content, epidermal growth factor receptor, and Ki-67. J Surg Oncol 1992; 51:147-52.
20. Porschen R, Classen S, Kahle Y, Borchard F. In situ evaluation of the relationship between epidermal-growth-factor-receptor status and tumor-cell proliferation in colon carcinomas. Int J Cancer 1993; 54:189-93.
21. Liu B, Fang M, Schmidt M, Lu Y, Mendelsohn J, Fan Z. Induction of apoptosis and activation of the caspase cascade by anti-EGF receptor monoclonal antibodies in DiFi human colon cancer cells do not involve the c-jun N-terminal kinase activity. Br J Cancer 2000; 82:1991-9.
22. Saltz L, Rubin M, Hochster H, et al. Cetuximab (IMC-C225) Plus Irinotecan (CPT-11) is Active in CPT-11-refractory Colorectal Cancer that expresses Epidermal Growth Factor Receptor (EGFr) [abstract]. Proceedings of the American Society of Clinical Oncology -Thirty-Seventh Annual Meeting 2001; 20:3a.
23. Ciardiello F, Bianco R, Damiano V, et al. Antiangiogenic and antitumor activity of anti-epidermal growth factor receptor C225 monoclonal antibody in combination with vascular endothelial growth factor antisense oligonucleotide in human GEO colon cancer cells. Clin Cancer Res 2000; 6:3739-47.
24. Shaheen RM, Ahmad SA, Liu W, et al. Inhibited growth of colon cancer carcinomatosis by antibodies to vascular endothelial and epidermal growth factor receptors. Br J Cancer 2001; 85:584-9.
25. Mulligan T, Carrasquillo JA, ChungY, et al. Phase I study of intravenous Lu-labeled CC49 murine monoclonal antibody in patients with advanced adenocarcinoma. Clin Cancer Res 1995; 1:1447-54.
26. Koda K, Glassy MC, McKnight ME, et al. Immunotherapy for recurrent colorectal cancers with human monoclonal antibody SK-1. Anticancer Res 2001; 21:621-7.
27. Brodsky FM, Guagliardi LE. The cell biology of antigen processing and presentation. Annu Rev Immunol 1991; 9:707-44.
28. Monaco JJ.A molecular model of MHC class-I-restricted antigen processing. Immunol Today 1992; 13:173-9.
29. Geppert TD, Lipsky PE. Antigen presentation at the inflammatory site. Crit Rev Immunol 1989; 9:313-62.
30. Kagi D, Ledermann B, Burki K, Zinkernagel RM, Hengartner H. Molecular mechanisms of lymphocyte-mediated cytotoxicity and their role in immunological protection and pathogenesis in vivo. Annu Rev Immunol 1996; 14:207-32.
31. Nagata S. Apoptosis by death factor. Cell 1997; 88:355-65.
32. Xiang R, Lode HN, Dolman CS, et al. Elimination of established murine colon carcinoma metastases by anti-body-interleukin 2 fusion protein therapy. Cancer Res 1997; 57:4948-55.
33. Xiang R, Lode HN, Dreier T, Gillies SD, Reisfeld RA.Induction of persistent tumor-protective immunity in mice cured of established colon carcinoma metastases. Cancer Res 1998; 58:3918-25.
34. Robertson MJ, Ritz J. Biology and clinical relevance of human natural killer cells. Blood 1990; 76:2421-38.
35. Ribeiro U, Jr., Whiteside TL, Basse PH, Safatle-Ribeiro AV, Huneke CE, Posner MC. Activated natural killer cell tumor retention and cytokine production in colon tumor using a tissue-isolated model. J Surg Res 1999; 82:78-87.
36. Kumar V, George T, Yu YY, Liu J, Bennett M. Role of murine NK cells and their receptors in hybrid resistance. Curr Opin Immunol 1997; 9:52-6.
37. Hoover HC, Jr., Brandhorst JS, Peters LC, et al.Adjuvant active specific immunotherapy for human colorectal cancer: 6.5-year median follow-up of a phase III prospectively randomized trial. J Clin Oncol 1993; 11:390-9.
38. Hanna MG, Jr., Hoover HC, Jr., Vermorken JB, Harris JE, Pinedo HM.Adjuvant active specific immunotherapy of stage II and stage III colon cancer with an autologous tumor cell vaccine: first randomized phase III trials show promise. Vaccine 2001; 19:2576-82.
39. Ockert D, Schirrmacher V, Beck N, et al. Newcastle dis-ease virus-infected intact autologous tumor cell vaccine for adjuvant active specific immunotherapy of resected colorectal carcinoma. Clin Cancer Res 1996; 2:21-8.
40. Yannelli JR, Hyatt C, Johnson S, Hwu P, Rosenberg SA. Characterization of human tumor cell lines transduced with the cDNA encoding either tumor necrosis factor alpha (TNF-a) or interleukin-2 (IL-2). J Immunol Methods 1993; 161:77-90.
41. Fearon ER, Pardoll DM, Itaya T, et al. Interleukin-2 production by tumor cells bypasses T helper function in the generation of an antitumor response. Cell 1990; 60:397-403.
42. Dranoff G, Jaffee E, Lazenby A, et al. Vaccination with irradiated tumor cells engineered to secrete murine granulocyte-macrophage colony-stimulating factor stimulates potent, specific, and long-lasting anti-tumor immunity. Proc Natl Acad Sci USA 1993; 90:3539-43.
43. Chong H, Todryk S, Hutchinson G, Hart IR, Vile RG. Tumour cell expression of B7 costimulatory molecules and interleukin-12 or granulocyte-macrophage colony-stimulating factor induces a local antitumour response and may generate systemic protective immunity. Gene Ther 1998; 5:223-32.
44. Colombo MP, Vagliani M, Spreafico F, et al. Amount of interleukin 12 available at the tumor site is critical for tumor regression. Cancer Res 1996; 56:2531-4.
45. Chen L, Linsley PS, Hellstrom KE. Costimulation of T cells for tumor immunity. Immunol Today 1993; 14:483-6.
46. Vile RG, Castleden S, Marshall J, Camplejohn R, Upton C, Chong H. Generation of an anti-tumour immune response in a non-immunogenic tumour: HSVtk killing in vivo stimulates a mononuclear cell infiltrate and a Th1-like profile of intratumoural cytokine expression. Int J Cancer 1997; 71:267-74.
47. Caruso M, Pham-Nguyen K, Kwong YL, et al. Adenovirus-mediated interleukin-12 gene therapy for metastatic colon carcinoma. Proc Natl Acad Sci USA 1996; 93:11302-6.
48. Todryk SM, Chong H, Vile RG, Pandha H, Lemoine NR. Can immunotherapy by gene transfer tip the balance against colorectal cancer? Gut 1998; 43:445-9.
49. Belldegrun A, Kasid A, Uppenkamp M, Topalian SL, Rosenberg SA. Human tumor infiltrating lymphocytes. Analysis of lymphokine mRNA expression and relevance to cancer immunotherapy. J Immunol 1989; 142:4520-6.
50. Kim JA, Bresler HS, Martin EW, Jr., Aldrich W, Heffelfinger M, Triozzi PL. Cellular immunotherapy for patients with metastatic colorectal carcinoma using lymph node lymphocytes localized in vivo by radiolabeled monoclonal antibody. Cancer 1999; 86:22-30.
51. Csiszar A, Szentes T, Haraszti B, et al. Characterisation of cytokine mRNA expression in tumour-infiltrating mononuclear cells and tumour cells freshly isolated from human colorectal carcinomas. Eur Cytokine Netw 2001; 12:87-96.
52. Michael-Robinson JM, Biemer-Huttmann A, Purdie DM, et al. Tumour infiltrating lymphocytes and apoptosis are independent features in colorectal cancer stratified according to microsatellite instability status. Gut 2001; 48:360-6.
53. Smyrk TC, Watson P, Kaul K, Lynch HT. Tumorinfiltrating lymphocytes are a marker for microsatellite instability in colorectal carcinoma. Cancer 2001; 91:2417-22.
54. Kubo RT, Sette A, Grey HM, et al. Definition of specific peptide motifs for four major HLA-A alleles. J Immunol 1994; 152:3913-24.
55. Zitvogel L, Mayordomo JI, Tjandrawan T, et al. Therapy of murine tumors with tumor peptide-pulsed dendritic cells: dependence on T cells, B7 costimulation, and T helper cell 1-associated cytokines. J Exp Med 1996; 183:87-97.
56. Rosenberg SA, Yang JC, Schwartzentruber DJ, et al.Immunologic and therapeutic evaluation of a synthetic peptide vaccine for the treatment of patients with meta-static melanoma. Nat Med 1998; 4:321-7.
57. Mukherji B, Chakraborty NG, Yamasaki S, et al. Induction of antigen-specific cytolytic T cells in situ in human melanoma by immunization with synthetic peptide-pulsed autologous antigen presenting cells. Proc Natl Acad Sci U A 1995; 92:8078-82.
58. Hollingsworth MA, Strawhecker JM, Caffrey TC, Mack DR. Expression of MUC1, MUC2, MUC3 and MUC4 mucin mRNAs in human pancreatic and intestinal tumor cell lines. Int J Cancer 1994; 57:198-203.
59. Apostolopoulos V, Osinski C, McKenzie IF. MUC1 cross-reactive Gal alpha(1,3)Gal antibodies in humans switch immune responses from cellular to humoral. Nat Med 1998; 4:315-20.
60. Goydos JS, Elder E, Whiteside TL, Finn OJ, Lotze MT. A phase I trial of a synthetic mucin peptide vaccine. Induction of specific immune reactivity in patients with adenocarcinoma. J Surg Res 1996; 63:298-304.
61. Akagi J, Nakagawa K, Egami H, Ogawa M. Induction of HLA-unrestricted and HLA-class-II-restricted cyto-toxic T lymphocytes against MUC-1 from patients with colorectal carcinomas using recombinant MUC-1 vaccinia virus. Cancer Immunol Immunother 1998; 47:21-31.
62. Itzkowitz SH, Bloom EJ, KokalWA, Modin G, Hakomori S, Kim YS. Sialosyl-Tn. A novel mucin antigen associated with prognosis in colorectal cancer patients. Cancer 1990; 66:1960-6.
63. Samuel J, Noujaim AA, MacLean GD, Suresh MR, Longenecker BM. Analysis of human tumor associated Thomsen-Friedenreich antigen. Cancer Res 1990; 50:4801-8.
64. Springer GF. T and Tn, general carcinoma autoantigens 2001; Science 1984; 224:1198-206.
65. Saeki T, Salomon DS, Johnson GR, et al. Association of epidermal growth factor-related peptides and type I receptor tyrosine kinase receptors with prognosis of human colorectal carcinomas. Jpn J Clin Oncol 1995; 25:240-9.
66. Kapitanovic S, Radosevic S, Kapitanovic M, et al. The expression of p185(HER-2/neu) correlates with the stage of disease and survival in colorectal cancer. Gastroenterology 1997; 112:1103-13.
67. Tanaka H, Tsunoda T, Nukaya I, et al. Mapping the HLA-A24-restricted T-cell epitope peptide from a tumour-associated antigen HER2 / neu: possible immunotherapy for colorectal carcinomas. Br J Cancer 2001; 84:94-9.
68. Vadlamudi R, Mandal M, Adam L, Steinbach G, Mendelsohn J, Kumar R. Regulation of cyclooxygenase-2 pathway by HER2 receptor. Oncogene 1999; 18:305-14.
69. Mann M, Sheng H, Shao J, et al. Targeting cyclooxygenase 2 and HER-2/neu pathways inhibits colorectal carcinoma growth. Gastroenterology 2001; 120:1713-9.
70. Sasatomi T, Yamana H, Shichijo S, et al. Expression of the SART1 tumor-rejection antigens in colorectal cancers. Dis Colon Rectum 2000; 43:1754-8.
71. Lindemann J. Speculations on idiotypes and homobodies. Ann Immunol (Paris) 1973; 124:171-84.
72. Jerne NK. Towards a network theory of the immune system. Ann Immunol (Paris) 1974; 125C:373-89.
73. Zinkernagel RM, Doherty PC. MHC-restricted cytotoxic T cells: studies on the biological role of polymorphic major transplantation antigens determining T-cell restriction-specificity, function, and responsiveness. Adv Immunol 1979; 27:51-177.
74. Fagerberg J, Hjelm AL, Ragnhammar P, Frodin JE, Wigzell H, Mellstedt H. Tumor regression in monoclonal antibody-treated patients correlates with the presence of anti-idiotype-reactive T lymphocytes. Cancer Res 1995; 55:1824-7.
75. Herlyn D, Harris D, Zaloudik J, et al. Immunomodulatory activity of monoclonal anti-idiotypic antibody to anti-colorectal carcinoma antibody CO17-1A in animals and patients. J Immunother Emphasis Tumor Immunol 1994; 15:303-11.
76. Herlyn D, Somasundaram R, Zaloudik J, et al. Anti-idiotype and recombinant antigen in immunotherapy of colorectal cancer. Cell Biophys 1994; 24-25:143-53.
77. Maruyama H, Zaloudik J, Li W, et al. Cancer vaccines: single-epitope anti-idiotype vaccine versus multiple-epitope antigen vaccine. Cancer Immunol Immunother 2000; 49:123-32.
78. Staib L, Birebent B, Somasundaram R, et al.Immunogenicity of recombinant GA733-2E antigen (CO17-1A, EGP, KS1-4, KSA, Ep-CAM) in gastro-intestinal carcinoma patients. Int J Cancer 2001; 92:79-87.
79. Birebent B, Somasundaram R, Purev E, et al. Anti-idiotypic antibody and recombinant antigen vaccines in colorectal cancer patients. Crit Rev Oncol Hematol 39:107-13.
80. Spendlove I, Li L, Carmichael J, Durrant LG. Decay accelerating factor (CD55): a target for cancer vaccines? Cancer Res 1999; 59:2282-6.
81. Robins RA, Denton GW, Hardcastle JD, Austin EB,Baldwin RW, Durrant LG. Antitumor immune response and interleukin 2 production induced in colorectal cancer patients by immunization with human monoclonal anti-idiotypic antibody. Cancer Res 1991; 51:5425-9.
82. Denton GW, Durrant LG, Hardcastle JD, Austin EB,Sewell HF, Robins RA. Clinical outcome of colorectal cancer patients treated with human monoclonal anti-idiotypic antibody. Int J Cancer 1994; 57:10-4.
83. Maxwell-Armstrong CA, Durrant LG, Buckley TJ, et al.Randomized double-blind phase II survival study comparing immunization with the anti-idiotypic monoclonal antibody 105AD7 against placebo in advanced colorectal cancer. Br J Cancer 2001; 84:1443-6.
84. Maxwell-Armstrong CA, Durrant LG, Robins RA,Galvin AM, Scholefield JH, Hardcastle JD. Increased activation of lymphocytes infiltrating primary colorectal cancers following immunisation with the anti-idiotypic monoclonal antibody 105AD7. Gut 1999; 45:593-8.
85. Durrant LG, Maxwell-Armstrong C, Buckley D, et al.A neoadjuvant clinical trial in colorectal cancer patients of the human anti-idiotypic antibody 105AD7, which mimics CD55. Clin Cancer Res 2000; 6:422-30.
86. Durrant LG, Buckley DJ, Robins RA, Spendlove I.105Ad7 cancer vaccine stimulates anti-tumour helper and cytotoxic T-cell responses in colorectal cancer patients but repeated immunisations are required to maintain these responses. Int J Cancer 2000; 85:87-92.
87. Amin S, Robins RA, Maxwell-Armstrong CA, Scholefield JH, Durrant LG. Vaccine-induced apoptosis: a novel clinical trial end point? Cancer Res 2000; 60:3132-6.
88. Pervin S, Chakraborty M, Bhattacharya-Chatterjee M,Zeytin H, Foon KA, Chatterjee SK. Induction of antitumor immunity by an anti-idiotype antibody mimicking carcinoembryonic antigen. Cancer Res 1997; 57:728-34.
89. Foon KA, John WJ, Chakraborty M, et al. Clinical and immune responses in advanced colorectal cancer patients treated with anti-idiotype monoclonal antibody vaccine that mimics the carcinoembryonic antigen. Clin Cancer Res 1997; 3:1267-76.
90. Foon KA, John WJ, Chakraborty M, et al. Clinical and immune responses in resected colon cancer patients treated with anti-idiotype monoclonal antibody vaccine that mimics the carcinoembryonic antigen. J Clin Oncol 1999; 17:2889-5.
91. Conry RM, Allen KO, Lee S, Moore SE, Shaw DR, LoBuglio AF. Human autoantibodies to carcinoembryonic antigen (CEA) induced by a vaccinia-CEA vaccine. Clin Cancer Res 2000; 6:34-41.
92. Banchereau J, Steinman RM. Dendritic cells and the control of immunity. Nature 1998; 392:245-52.
93. Satthaporn S, Eremin O. Dendritic cells (I): Biological functions. J R Coll Surg Edinb 2001; 46:9-19.
94. Zhou LJ, Tedder TF. A distinct pattern of cytokine gene expression by human CD83+ blood dendritic cells. Blood 1995; 86:3295-301.
95. Macatonia SE, Hosken NA, Litton M, et al. Dendritic cells produce IL-12 and direct the development of Th1 cells from naive CD4+ T cells. J Immunol 1995; 154:5071-9.
96. Mohamadzadeh M, Poltorak AN, Bergstressor PR, Beutler B, Takashima A. Dendritic cells produce macrophage inflammatory protein-1 gamma, a new member of the CC chemokine family. J Immunol 1996; 156:3102-6.
97. Hermans IF, Daish A, Moroni-Rawson P, Ronchese F. Tumor-peptide-pulsed dendritic cells isolated from spleen or cultured in vitro from bone marrow precursors can provide protection against tumor challenge. Cancer Immunol Immunother 1997; 44:341-7.
98. Mayordomo JI, Zorina T, Storkus WJ, et al. Bone marrow-derived dendritic cells pulsed with synthetic tumour peptides elicit protective and therapeutic antitumour immunity. Nat Med 1995; 1:1297-302.
99. Rains N, Cannan RJ, Chen W, Stubbs RS. Development of a dendritic cell (DC)-based vaccine for patients with advanced colorectal cancer.Hepatogastroenterology 2001; 48:347-51.
100. Nestle FO, Alijagic S, Gilliet M, et al. Vaccination of melanoma patients with peptide- or tumor lysate-pulsed dendritic cells. Nat Med 1998; 4:328-32.
101. Alijagic S, Moller P, Artuc M, Jurgovsky K, Czarnetzki BM, Schadendorf D. Dendritic cells generated from peripheral blood transfected with human tyrosinase induce specific T cell activation. Eur J Immunol 1995; 25:3100-7.
102. Oliveira SC, Rosinha GM, de-Brito CF, et al.Immunological properties of gene vaccines delivered by different routes. Braz J Med Biol Res 1999; 32:207-14.
103. Mincheff M, Altankova I, Zoubak S, et al. In vivo transfection and/or cross-priming of dendritic cells following DNA and adenoviral immunizations for immunotherapy of cancer-changes in peripheral mononuclear subsets and intracellular IL-4 and IFN-gamma lymphokine profile. Crit Rev Oncol Hematol 2001; 39:125-32.
104. Maraskovsky E, Daro E, Roux E, et al. In vivo generation of human dendritic cell subsets by Flt3 ligand. Blood 2000; 96:878-84.
105. Morse MA, Nair S, Fernandez-Casal M, et al. Preoperative mobilization of circulating dendritic cells by Flt3 ligand administration to patients with metastatic colon cancer. J Clin Oncol 2000; 18:3883-93.
106. Fong L, Hou Y, Rivas A, et al. Altered peptide ligand vaccination with Flt3 ligand expanded dendritic cells for tumor immunotherapy. Proc Natl Acad Sci USA 2001; 98:8809-14.
107. Multhoff G, Botzler C, Wiesnet M, Eissner G, Issels R. CD3- large granular lymphocytes recognize a heat-inducible immunogenic determinant associated with the 72-kD heat shock protein on human sarcoma cells. Blood 1995; 86:1374-82.
108. Welch WJ, Feramisco JR. Nuclear and nucleolar localization of the 72,000-dalton heat shock protein in heat-shocked mammalian cells. J Biol Chem 1984;259:4501-13.
109. Lindquist S.The heat-shock response. Annu Rev Biochem 1986; 55:1151-91.
110. Tamura Y, Tsuboi N, Sato N, Kikuchi K. 70 kDa heat shock cognate protein is a transformation-associated antigen and a possible target for the host's anti-tumor immunity. J Immunol 1993; 151:5516-24.
111. Wei Y, Zhao X, Kariya Y, Fukata H, Teshigawara K,Uchida A. Induction of autologous tumor killing by heat treatment of fresh human tumor cells: involvement of gamma delta T cells and heat shock protein 70. Cancer Res 1996; 56:1104-10.
112. Srivastava PK,Amato RJ. Heat shock proteins: the 'Swiss Army Knife' vaccines against cancers and infectious agents. Vaccine 2001; 19:2590-7.
113. Melcher A, Todryk S, Hardwick N, Ford M, Jacobson M, Vile RG. Tumor immunogenicity is determined by the mechanism of cell death via induction of heat shock protein expression. Nat Med 1998; 4:581-7.
114. Katoh M, Koninkx J, Schumacher U. Heat shock protein expression in human tumours grown in severe combined immunodeficient mice. Cancer Lett 2000; 161:113-20.
115. Lazaris AC, Theodoropoulos GE, Davaris PS, et al. Heat shock protein 70 and HLA-DR molecules tissue expression. Prognostic implications in colorectal cancer. Dis Colon Rectum 1995; 38:739-45.
116. Mehlen P, Preville X, Chareyron P, Briolay J, Klemenz R, Arrigo AP. Constitutive expression of human hsp27, Drosophila hsp27, or human alpha B-crystallin confers resistance to TNF- and oxidative stress-induced cytotoxicity in stably transfected murine L929 fibroblasts. J Immunol 1995; 154:363-74.
117. Mehlen P, Schulze-Osthoff K, Arrigo AP. Small stress proteins as novel regulators of apoptosis. Heat shock protein 27 blocks Fas/APO-1- and staurosporine-induced cell death. J Biol Chem 1996; 271:16510-4.
118. Botzler C, Issels R, Multhoff G. Heat-shock protein 72 cell-surface expression on human lung carcinoma cells in associated with an increased sensitivity to lysis mediated by adherent natural killer cells. Cancer Immunol Immunother 1996; 43:226-30.
119. Garrido C, Fromentin A, Bonnotte B, et al. Heat shock protein 27 enhances the tumorigenicity of immunogenic rat colon carcinoma cell clones. Cancer Res 1998; 58:5495-9.
120. Wang XY, Kazim L, Repasky EA, Subjeck JR.Characterization of heat shock protein 110 and glucose-regulated protein 170 as cancer vaccines and the effect of fever-range hyperthermia on vaccine activity. J Immunol 2001; 166:490-7.
121. Stevenson HC, Green I, Hamilton JM, Calabro BA, Parkinson DR. Levamisole: known effects on the immune system, clinical results, and future applications to the treatment of cancer. J Clin Oncol 1991; 9:2052-66.
122. Buroker TR, Moertel CG, Fleming TR, et al. A controlled evaluation of recent approaches to biochemical modulation or enhancement of 5-fluorouracil therapy in colorectal carcinoma. J Clin Oncol 1985; 3:1624-31.
123. Bandealy MT, Gonin R, Loehrer PJ, Monaco F, Einhorn LH. Prospective randomized trial of 5-fluorouracil ver-473 sus 5-fluorouracil plus levamisole in the treatment of metastatic colorectal cancer: a Hoosier Oncology Group trial. Clin Cancer Res 1998; 4:935-9.
124. Kerr DJ, Gray R, McConkey C, Barnwell J. Adjuvant chemotherapy with 5-fluorouracil, L-folinic acid and levamisole for patients with colorectal cancer: nonrandomised comparison of weekly versus four-weekly schedules--less pain, same gain. QUASAR Colorectal Cancer Study Group. Ann Oncol 2000; 11:947-55.
125. Witte RS, Cnaan A, Mansour EG, Barylak E, Harris JE, Schutt AJ. Comparison of 5-fluorouracil alone, 5-fluorouracil with levamisole, and 5-fluorouracil with hepatic irradiation in the treatment of patients with residual, nonmeasurable, intra-abdominal metastasis after under-going resection for colorectal carcinoma. Cancer 2001; 91:1020-8.
126. Chlebowski RT, Nystrom S, Reynolds R, Weiner JM, Bateman JR. Long-term survival following levamisole or placebo adjuvant treatment of colorectal cancer: aWestern Cancer Study Group Trial. Oncology 1988; 45:141-3.
127. Moertel CG, Fleming TR, Macdonald JS, et al. Intergroup study of fluorouracil plus levamisole as adjuvant therapy for stage II/Dukes' B2 colon cancer. J Clin Oncol 1995; 13:2936-43.
128. Kubota T, Kase S, Otani Y, Watanabe M, Teramoto T, Kitajima M. Interferons alpha-2a and beta increase the antitumor activity, detected by MTT assay, of 5-fluorouracil against experimental and clinical human gastrointestinal carcinomas. Anticancer Res 1997; 17:725-8.
129. Kohne CH, Schoffski P, Wilke H, et al. Effective biomodulation by leucovorin of high-dose infusion fluorouracil given as a weekly 24-hour infusion: results of a randomized trial in patients with advanced colorectal cancer. J Clin Oncol 1998; 16:418-26.
130. Wolmark N, Bryant J, Smith R, et al. Adjuvant 5-fluorouracil and leucovorin with or without interferon alfa-2a in colon carcinoma: National Surgical Adjuvant Breast and Bowel Project protocol C-05. J Natl Cancer Inst 1998; 90:1810-6.
131. Pfizenmaier K, Bartsch H, Scheurich P, et al. Differential gamma-interferon response of human colon carcinoma cells: inhibition of proliferation and modulation of immunogenicity as independent effects of gamma-interferon on tumor cell growth. Cancer Res 1985; 45:3503-9.
132. Cleveland RP, Meltzer MS, Zbar B. Tumor cytotoxicity in vitro by macrophages from mice infected with myco-bacterium bovis strain BCG. J Natl Cancer Inst 1974; 52:1887-95.
133. Cohen D, Yron I, Grover NB, Weiss DW. Chemoimmunotherapy of syngeneic mouse mammary carcinomas employing methanol extraction residue. Ann N Y Acad Sci 1976; 277:195-208.
134. O'Connell MJ, Moertel CG, Ritts RE, Jr., Frytak S, Reitemeier RJ. A comparative clinical and immunological assessment of methanol extraction residue of Bacillus Calmette-Guerin versus placebo in patients with advanced cancer. Cancer Res 1979; 39:3720-4.
135. Hoover HC, Jr., Surdyke M, Dangel RB, Peters LC, Hanna MG, Jr. Delayed cutaneous hypersensitivity to autologous tumor cells in colorectal cancer patients immunized with an autologous tumor cell: Bacillus Calmette-Guerin vaccine. Cancer Res 1984; 44:1671-6.
136. Avery A, Paraskeva C, Hall P, Flanders KC, Sporn M, Moorghen M. TGF-beta expression in the human colon: differential immunostaining along crypt epithelium. Br J Cancer 1993; 68:137-9.
137. Gastl GA, Abrams JS, Nanus DM, et al. Interleukin-10 production by human carcinoma cell lines and its relationship to interleukin-6 expression. Int J Cancer 1993; 55:96-101.
138. Pellegrini P, Berghella AM, Del Beato T, Cicia S, Adorno D, Casciani CU. Disregulation in TH1 and TH2 subsets of CD4+ T cells in peripheral blood of colorectal cancer patients and involvement in cancer establishment and progression. Cancer Immunol Immunother 1996; 42:1-8.
139. Qin Z, Richter G, Schuler T, Ibe S, Cao X, Blankenstein T. B cells inhibit induction of T cell-dependent tumor immunity. Nat Med 1998; 4:627-30.
140. Wigmore SJ, Maingay JP, Fearon KC, Ross JA.Endogenous production of IL-8 by human colorectal cancer cells and its regulation by cytokines. Int J Oncol 2001; 18:467-73.
141. Peoples GE, Blotnick S, Takahashi K, Freeman MR,Klagsbrun M, Eberlein TJ. T lymphocytes that infiltrate tumors and atherosclerotic plaques produce heparin-binding epidermal growth factor-like growth factor and basic fibroblast growth factor: a potential pathologic role. Proc Natl Acad Sci USA 1995; 92:6547-51.
142. Freeman MR, Schneck FX, Gagnon ML, et al. Peripheral blood T lymphocytes and lymphocytes infiltrating human cancers express vascular endothelial growth factor: a potential role for T cells in angiogenesis. Cancer Res 1995; 55:4140-5.
143. Chaux P, Favre N, Martin M, Martin F. Tumor-infiltrating dendritic cells are defective in their antigen-presenting function and inducible B7 expression in rats. Int J Cancer 1997; 72:619-24.
144. Kuppen PJ, van der Eb MM, Jonges LE, et al. Tumor structure and extracellular matrix as a possible barrier for therapeutic approaches using immune cells or adenoviruses in colorectal cancer. Histochem Cell Biol 2001; 115:67-72.
145. Kruger K, Buning C, Schriever F. Activated T lymphocytes bind in situ to stromal tissue of colon carcinoma but lack adhesion to tumor cells. Eur J Immunol 2001; 31:138-45.
146. Fagerberg J, Steinitz M, Wigzell H, Askelof P, Mellstedt H. Human anti-idiotypic antibodies induced a humoral and cellular immune response against a colorectal carcinoma-associated antigen in patients. Proc Natl Acad Sci USA 1995; 92:4773-7
147. Durrant LG, Buckley TJ, Denton GW, Hardcastle JD, Sewell HF, Robins RA. Enhanced cell-mediated tumor killing in patients immunized with human anti-idiotypic antibody 105AD7. Cancer Res 1994; 54:4837-40

Correspondence:Adrian Indar, Division of Surgery, Queens Medical Centre, Nottingham, NG7 2UH, U.K. E-mail: Adrian.Indar@nottingham.ac.uk

CEA: Carcinoembryonic antigen	TIL: Tumour infiltrating lymphocyte
IL-2: Interleukin-2	MHC: Major histocompatibility complex
TAAs: Tumour associated antigens	CTLs: Cytotoxic T lymphocytes
IFN-a: Interferon-a	TH: T helper cell
IFN-y: Interferon-y	GM-CSF: Granulocyte -macrophage colony stimulating factor
MAb: Monoclonal antibody	ELISA: Enzyme linked immunosorbent assay
TNF-a: Tumour necrosis factor-a	DTH: Delayed type hypersensitivity
APC: Antigen presenting cell	VEGF: Vascular endothelial growth factor
ADCC: Antibody dependent cell cytotoxicity	BCG: Bacille Calmette Guerin
ADMC: Antibody dependent monocyte cytotoxicity	HSPs: Heat shock proteins
TCR: T cell receptor	EGFR: Epidermal growth factor receptor
NK: Natural killer	RT-PCR: Reverse Transcriptase Polymeiase Chain Reaction
DC: Dendritic cell

Author	Study	Results	Conclusion
Ragenhammar 1995 ¹²	86 patients with advanced CRC treated with either murine MAb17-1A (76) or chimeric MAb17-1A (10)	All patients developed anti-idiotypic Abs. Increased by GM-CSF; c-MAb less response and more allergic side effects than MAb	Patients with an Ab2 response - median survival of 9 months
Shetye 1998 ¹³	20 patients with advanced CRC treated with a single infusion of MAb17-1A+ GM-CSF	Increased tumoural and blood neutrophils, monocytes and T lymphocytes	Incresed TILs representing ADCC and CTLs
Riethmuller 1998 ¹⁶	189 patients with Dukes C randomised to observation or MAb17-1A post-operatively	At 7 year evaluation, mortality decreased by 32% and recurrence by 23%	Therapeutic effect maintained after 7 years, extends life and prolongs remission
Hjelm Skog 1999 ²⁰	20 patients with advanced CRC treated with MAb17-1A+IL-2+GM-CSF	1 patient partial remission 2 patients stable disease for 7 and 4 months	No augmentation of effect of 17-1A
Punt 2001 ¹⁷	Multicentre; 2761 stage III CRC patients randomised to (1) 17-1A MAb/5FU/LV or (2) 5 FU/LV or (3) 17-1A MAb	3-yr surv DFS (1) 74.7% 63.8% (2) 76.1% 65.5% (3) 70.1% 53.0%	Addition of 17-1A MAb to standard therapy does not improve the disease outcome
Saltz 2001 ²²	121 EGFR+ve patients refractory to 5-FU and CPT-11 treated with CPT-11 and IMC-C225	17% showed a partial response (median 17d) and 31% stable disease or minor responses	IMC-C225 can produce major responses in EGFR+ patients

Author	Study	Results	Conclusion
Hoover 1993³⁷	98 Dukes B and C patients randomised to autologous tumour cell/BCG vaccine resection or resection alone	Increased disease-free and survival in treated patients with colon but not rectal cancer	May be beneficial to patients with colon cancer
Herlyn 1994⁷⁶	54 Dukes B, C and D patients were administered an anti-idiotypic antibody	Most patients produced Ab3 against 17-1A; 30% developed specific cellular response	This is potential for a vaccine in patients with CRC
Fagerberg 1995¹⁴⁶	6 patients post resection were administered an anti idiotypic antibody h-Ab2 against 17-1A Ab	All developed prolonged T cell immunity; 5 generated humoral responses	Able to induce an anti-tumour response
Foon 1997⁸⁹	24 Advanced CRC patients were administered repeated injections of anti-idiotypic antibody 3H1	13 showed Ab3 responses, 11 mediated ADCC, no objective responses, median survival 11.3 mths	Able to break immune tolerance; survival comparable with patients receiving chemotherapy
Foon 1999⁹⁰	32 patients Dukes B, C and D post-surgery treated with repeated injections of anti-idiotypic Ab CEA Vac	All had anti-CEA T cell proliferation and antibody response	Immune responses continue with repeated doses
Fong 2001¹⁰⁶	12 patients with metastatic disease treated with DC vaccine loaded with a CEA derived peptide	2-tumour regression, 1-mixed response, 2-disease stabilisation	Altered peptide ligands can be used to immunize against self-antigens
Staib 2001⁷⁸	6 patients with resected micrometastatic CRC and pancreatic cancer treated with vaccine of alum precipitated recombinant GA733-2E antigen	6 had antigen specific humoral and 8 antigen specific cellular responses; median survival for CRC patients 11.2 mths	TArgeting epitopes of the GA733 antigen produces and immune response
Durrant 1994¹⁴⁷	6 patients with rectal cancer immunized pre-operatively with105AD7	Significant NK and non-NK cell killing of tumour cells	Enhanced cytotoxicity by specific and non-specific mechanisms
Maxwell-Armstrong 2001⁸³	162 patients with advanced CRC randonised to 3 repeated doses of 105AD7 or placebo	Median survival: 105AD7-124 days; placebo-184 days	105AD7 does not prolong survival in advanced CRC
Durrant 2000⁸⁵	35 patients with CRC immunized pre-operatively and twice post-operatively with 105AD7	83% expressing haplotypes HLA-DR1, DR3 and DR7 responded to 105AD7 by enhanced infiltratrion of CD56 cells	105AD7 stimulates anti-tumour responses allowing extravasation of tumours with CD4c and NK cells
Amin 2000⁸⁷	13 patients with primary CRC immunized pre-operatively with 105AD7; compared with non-immunized group	Significant increase in apoptosis in immunized patients (22% vs 7%)	Apoptosis is a valuable endpoint for evaluation of biological therapy

Infection of tumour cells in vitro with adenviruses containing genes for cytokine stimulation	IL-2, GM-CSF, IL-12
Infection of tumour cells in vitro with adenoviruses containing genes for costimulatory molecules	B7 (CD80/86)
In vivo infection of tumour cells with tumour targeting viruses
Transfer of allo-MHC genes in vitro to tumour cells
Transfer of cytokine expressing genes to TILs	TNF-a
Infection of tumour cells with viruses carrying 'suicide genes' which encode enzymes	HSV thymidine kinase, E Coli cytosine deaminase
Administration (via plasmids) genes encoding TAAs	P53, p21,ras, CEA, MUC-1, MAGE GA733

CLINICAL REVIEW

PASSIVE IMMUNOTHERAPY

NON-SPECIFIC IMMUNOTHERAPY

BCG

TUMOUR ESCAPE

CONCLUSION

REFERENCES