Biological Response Modifiers in Cancer

Abstract

We have seen a surge in the use of immunotherapy for the treatment of cancer. Biological response modifiers can act passively by enhancing the immunologic response to tumor cells or actively by altering the differentiation/growth of tumor cells. Active immunotherapy with cytokines such as interferons (IFNs) and interleukins (IL-2) is a form of nonspecific active immune stimulation. The use of IL-2 has recently been approved by the United States Food and Drug Administration (FDA) for the treatment of renal cell carcinoma and metastatic colorectal cancer.

Considerable success has been achieved with the use of immunotherapy, especially in the area of passive immunotherapy using monoclonal antibodies – in particular, radiolabeled monoclonal antibodies. In addition to the various monoclonal antibodies that have been used in clinical trials, other strategies such as the use of antiangiogenic agents and matrix metalloprotease inhibitors (MMPIs) have also met with some success. Recently, the FDA approved bevacizumab, an anti-vascular endothelial growth factor (VEGF) agent, for the treatment of metastatic melanoma.

This review also sheds light on the various angiogenesis inhibitors in clinical trials, the increasing use of thalidomide in cancer, and the upcoming potential cancer vaccines designed to activate cell-mediated immune responses against tumor antigens.

Introduction

Over the last few years, immunotherapy has been widely investigated for the treatment of cancer. The goal of immunotherapy is to manipulate the host tumor interaction in favor of the host. Cancer cells express a wide profile of different proteins that act as antigens. Some of these antigenic proteins may be a result of oncogenic transformation and are relatively specific to cancer cells. These tumor-associated antigens are delivered to the immune system by antigen-presenting cells (APCs) through major histocompatibility complex (MHC) class I or class II pathways. In the class I pathway, the phagocytosed tumor cells are processed by proteasomes and converted to short peptide fragments, which are then presented on class I MHC molecules. These are recognized by CD8+ cytotoxic lymphocytes, which have direct cytotoxic effects leading to tumor cell lysis. In the class II pathway, the secreted products from tumor cells enter the APCs, which are then processed and presented to MHC class II molecules. These processed antigens are recognized by CD4+ helper lymphocytes, which enhance the CD8+ cytotoxic responses as well as the humoral response to surface antigens present on tumor cells. Thus, T-helper lymphocytes have been shown to activate APCs along with sustaining the immune response via cytokines.

Biological response modifiers can act passively by enhancing the immunologic response to tumor cells or actively by altering the differentiation/growth of tumor cells. Active immunotherapy with cytokines such as interferons (IFNs) and interleukins (IL-2) is a form of nonspecific active immune stimulation. The IFNs have been tested as therapies for many hematologic and solid neoplasms and have demonstrated therapeutic benefits in various cancers. Moreover, IL-2 has already gained FDA approval for the treatment of renal cell carcinoma and metastatic melanoma. Success has been achieved in the area of immunotherapy, especially in the area of passive immunotherapy using monoclonal antibodies. Other strategies, such as the use of antiangiogenic agents, matrix metalloprotease inhibitors(MMPIs), tyrosine kinase inhibitors (TKIs), and tumor vaccines, have also been met with some success.

One of the major adverse effects of cancer chemotherapy is immunosuppression, which leads to many opportunistic infections, so hematopoietic factors (such as colony stimulating factor [CSF]) have been utilized to increase the immune response. Hematopoietic agents such as granulocyte macrophage colony-stimulating factor (GM-CSF; sargramostim) and granulocyte colony-stimulating factor (G-CSF; filgrastim) have been used to increase immunity. Biological response modifiers are basically used alone or as adjuvants to cancer chemotherapeutic agents.

Interferons

IFNs are a group of glycoproteins that are produced by a variety of cells stimulated by viral antigens and other inducers, such as double-stranded RNA and mitogens. Macrophages and lymphocytes are responsible for production of IFN-alpha, whereas fibroblasts and epithelial cells are involved in producing IFN-beta. IFN-gamma is produced by CD4+, CD8+, natural killer (NK) cells, and (lymphokine-activated killer) LAK cells. IFNs have a variety of actions that contribute to antitumor mechanisms, such as antiproliferative effects, promotion of differentiation, immunomodulation, alteration in tumor cell surface antigen expression, inhibition of oncogene activation, and angiogenesis. IFN-gamma has been shown to potentiate DNA fragmentation and apoptotic cell death.[1] Both IFN-alpha and -gamma potentiate tumor cytotoxicity of TNF, as demonstrated in stem cell assays[2] Induction of MHC expression on tumor cell surfaces by IFNs is responsible for enhanced efficacy of host cell-mediated immunity and tumor elimination.[3,4] The clinical antitumor response to IFN-alpha and -gamma has been correlated with increased ratio of CD4 to CD8 cells, especially in solid tumors such as renal cell carcinoma (RCC) and melanoma.[5,6] IFN-alpha has exhibited regression of childhood hemangiomas, suggesting its role in tumor angiogenesis.[7] Preclinical studies have shown existence of imbalance between basic fibroblast growth factor and IFN-alpha, which is responsible for angiogenesis and tumor growth.[8]

The most commonly used dose of IFN-alpha (first generation) is 10–20 million units (MU)/m² daily (intravenously [IV]) or 50 MU/m² (subcutaneously or intramuscularly [SC/IM]) on alternate days for weeks to months, its half-life being 6 hours. Acceptable absorption activity has been seen with the SC as well as the IM route.[9] Survival benefit and decreased relapse rates in melanoma patients have been demonstrated with IFN-alpha at a dose of 20 MU/m² IV and at lower doses delivered via alternative routes (IM/SC). The recombinant IFN-alpha2b (second generation) is conjugated with polyethylene glycol (PEG-Intron) and IFN-alpha is conjugated with large branched formulations (Pegasys). The main advantage is their prolonged half-lives.

After 2–6 months of therapy, 5% of patients develop anti-IFN neutralizing serum antibodies leading to loss of antitumor effects.[10] Use of second-generation IFNs, ie, pegylated IFN-alpha 2 has been shown to cause less immunogenicity than the parent drug. Systemic IFN-alpha2 therapy has been approved by the FDA for solid tumors such as Kaposi's sarcoma, RCC, melanoma, carcinoid, ovarian cancer, and some hematologic cancers. It has been used as a postoperative adjuvant in stage II/III melanoma and in bladder cancer. Various hematologic neoplasms such as hairy cell leukemia, chronic myeloid leukemia (CML), non-Hodgkin's lymphoma (NHL), and T- and B-cell lymphoma have also been treated by IFN-alpha2.[10–12]

The side effects of IFN-alpha are mainly dose dependent and consist of fever, chills, myalgia, headache, nausea, anorexia, and weight loss (flu-like syndrome). Myelosuppression may occur, which is reversible within 1–3 days of discontinuation of IFN therapy. Rarely, interstitial nephritis, confusion, coma, hypotension, and arrhythmias may occur.

Clinical Trials Showing Survival Benefit With Interferons

The adjuvant use of high-dose IFN-alpha2b in malignant melanoma for 1 year has been associated with an increase in disease-free survival. In one study,[13] there was an increase in median survival of 3.8 years in the treatment group compared with 2.8 years in the observation group. Patients with lung or soft-tissue metastasis responded best to IFN therapy. Patients with Kaposi's sarcoma with mucocutaneous or asymptomatic visceral involvement have been shown to benefit from IFN-alpha. Patients with CD4+ T lymphocytes > 200/mm² showed a better response (> 45%).[14] Partial remission has been achieved in > 80% of patients with hairy cell leukemia in whom improvement in blood count was demonstrated after 2 months of IFN therapy.[15]

Interleukin-2

Interleukin (IL)-2 is a glycoprotein produced by mature T lymphocytes during an immune response after receiving a signal from an antigen-presenting cell (APC).[16] It acts as an antitumor agent by increasing the cytolytic activity of antigen-specific cytotoxic T lymphocytes and natural killer (NK) cells and by increasing the gene expression responsible for encoding the lytic component of cytotoxic granules, ie, perforin and granzymes.[17] Interleukin also facilitates binding of activated leukocytes to tumor endothelium and tumor cells by increasing expression of adhesion molecules.

IL-2 increases HLA-restricted cytolytic activity of cytotoxic T lymphocytes and NK cells. Furthermore, the activation and expansion of lymphocyte-activated killer (LAK) cells, which are a mixture of NK cells and CD4/CD8 T cells, is responsible for HLA-unrestricted killing of all tumor cell lines.[18] LAK cells are ex vivo IL-2-activated lymphoid preparations whose concurrent administration with IL-2 in several studies has shown beneficial effects. In one of the studies, mice bearing hepatic metastasis of poorly immunogenic sarcomas and adenocarcinomas were highly responsive to the combination of IL-2 and LAK cells, whereas they were unresponsive/partially responsive to LAK cells/IL-2 used individually.[19]

Clinical Applications

Investigators have developed 2 strategies for administering IL-2. The first strategy, developed by NCI Surgery, is the high-dose bolus IL-2 regimen.[20] IL-2 (adesleukin/Proleukin) is administered at a dose of 600,000- 720,000 IU/kg IV every 8 hours on Days 1–5 and 15–19 of treatment. Treatment was repeated at 8- to 12-week intervals for those patients who showed good response. Daily leukopheresis was done on 8–12 days of treatment course to generate LAK cells, which patients were reinfused with during Days 15- 19 of IL-2 therapy. Clinical trials using an IL-2 regimen with/without LAK cells in patients with metastatic melanoma and RCC showed 4%-6% complete response.[21,22] However, subsequent trials comparing IL-2 in combination with LAK cells vs high-dose IL-2 monotherapy failed to show any benefit of LAK cells to justify their further use.[23] High-dose IL-2 received FDA approval in1992 for the treatment of metastatic RCC and in 1998 for the treatment of metastatic malignant melanoma.

High-dose IL-2 regimens are associated with adverse effects such as fever with chills, lethargy, diarrhea, nausea, anemia, thrombocytopenia, eosinophilia, erythroderma, hepatic dysfunction, confusion etc.[24] Myocarditis was seen in some patients who had prior myocardial infarction, angina, CHF, or severe cardiac arrhythmias. Therefore, very careful patient selection is advised before administering IL-2 therapy, especially in a patient aged > 40 years, in whom IL-2 therapy commonly produces capillary leak syndrome leading to fluid retention, hypotension, adult respiratory distress syndrome, prerenal azotemia, and, very rarely, myocardial infarction. IL-2 therapy has also shown predisposition of patients to gram-positive and occasionally gram-negative bacterial infections due to some defect in neutrophil chemotaxis.[25] Routine use of antibiotic prophylaxis, extensive cardiac screening, and judicious IL-2 administration have resulted in a decreased mortality rate, from 2%-4% in 1985 to 1% in 1997.[26] Moreover, supportive therapy with acetaminophen, H2 blockers, antiemetics, and antidiarrheals should be given. Hypotension can be managed by administering IV fluids, but if the patient is unresponsive to fluid administration, IV dopamine hydrochloride or IV phenylephrine Hcl should be used. Steroids are to be avoided as they interfere with immune activation.

The second strategy is low-dose IL-2 therapy, which can be administered either as IV bolus or continuous IV/SC injection.[27] With continuous infusion, the IL-2 dose is 18 MIU/m²/day on Days 1–5 and 15–19; the response rate as well as toxicity profile have been found to be similar to those seen with high-dose IL-2 therapy. On the other hand, low-dose IV bolus (72,000 IU/kg IV q8h on Days 1–5 and on Days 15–19) or SC injection (250,000 IU/kg/day on Days 1–5, Week 1) was better tolerated. However, the low-dose regimen was associated with lower response rates in patients with RCC,[28,29] and no response in patients with metastatic melanoma.[30] Many approaches have been used in an attempt to improve the activity of IL-2-based therapy, but most of them have been disappointing. Combination of IL-2 with IFN,[31] with monoclonal antibodies against GD2 or GD3 gangliosides or T-cell activation antigens(CD3), has also been disappointing.[32] Recently, a very promising approach of combining IL-2 immunotherapy with cytotoxic immunotherapy was undertaken. This new therapeutic approach is known as biochemotherapy. IL-2 has also been combined with 5-fluorouracil (FU)-based chemotherapy and with cisplatin- and dacarbazine-based cytotoxic chemotherapies for the treatment of melanoma and RCC, respectively.[33,34] The response rates were 40% in patients with RCC and as high as 50%-60% in patients with metastatic melanoma. The efficacy of this new therapeutic approach is yet to be confirmed by large-scale multicentric trials.

Hematopoietic Growth Factors

This subgroup of cytokines has well-defined effects on the hematopoietic system. Although these drugs are expensive, their use is relatively safe at the recommended doses. Three hematopoietic factors have been approved for clinical use: myeloid growth factors (granulocyte colony-stimulating factor [G-CSF] and granulocyte macrophage colony-stimulating factor [GM-CSF]) and erythropoietin.

Erythropoietin is produced by the peritubular cells of the kidney and is FDA-approved for the treatment of renal anemia. Recently, it has been used to treat anemia caused by chemotherapy. The dose used for patients with anemia of malignancy and chemotherapy-induced anemia is 150–300 U/kg SC 3 to 5 times a week. It stimulates erythropoiesis in 50%-70% of patients and reduces the transfusion requirement, along with improving their quality of life. It has been observed that if there is a rise in Hb level by 0.5 mg/dL at 2 weeks, with initial erythropoietin level being < 100 milliunits (MU)/mL, 95% of patients will show a good response.[35]

The myeloid growth factors are glycoproteins that stimulate the proliferation and differentiation of myeloid cell lines. They also enhance the function of mature granulocytes and monocytes, thus enhancing the defense mechanism. The major indication for use of G-CSF and GM-CSF is to shorten the period of neutropenia and reduce morbidity secondary to bacterial and fungal infections in patients receiving intensive cancer chemotherapy. GM-CSF is administered SC or via slow IV at a dose of 250 mcg/m²/day.[36,37] The dose may be increased if the patient fails to respond after 7–14 days of therapy.[36] The dose of G-CSF is 5 mcg/kg/day SC or IV infusion over 30 minutes. Frequent blood counts should be obtained to monitor the effectiveness of treatment, and the dose should be adjusted according to the granulocyte response.[37] The myeloid growth factors are to be administered 24–72 hours after chemotherapy until high neutrophil count (1000/microliter) has persisted for 3 consecutive days.

The toxicity profile of myeloid growth factors is favorable. G-CSF is generally well tolerated, with mild to moderate bone pain being the main side effect. The pattern of toxicity of GM-CSF is more typical of cytokines, demonstrating an acute reaction at first dose characterized by fever, chills, hypotension, and dyspnea.

Monoclonal Antibodies

Kohler and Milstein,[38] in 1975, were the first ones to develop techniques for producing monoclonal antibodies (mAb). mAb are clones of similar antibodies that are directed against specific target antigens. They activate the immune effector functions and facilitate the destruction of malignant cells by complement dependent cytotoxicity (CDC) and antibody dependent cell-mediated cytotoxicity (ADCC). In CDC, the mAb bind to specific antigens, leading to activation and cascade of the complement system, which in turn leads to destruction of tumor cells. In ADCC, the Fab domain of mAb binds to the tumor antigen and Fc domain binds to Fc receptors present on effector cells – ie, monocytes, macrophages, and NK cells – thus forming a bridge between effector and target cells. This induces effector cell activation, leading to increased phagocytosis by neutrophils, monocytes, and macrophages. There is increased cytotoxicity of NK cells as they cause release of cell-lysing molecules responsible for tumor cell lysis.[39] Table 1 shows mAb in clinical trials for various cancers.

Table 1.

Monoclonal Antibodies in Clinical Trials in Oncology

Monoclonal Antibody	Target Antigen	Target Cell/Disease
Edrecolomab	17-1A antigen	Colon/rectal cancer
Trastuzumaba	HER-2 oncoprotein	Breast cancer
Anti-idiotype antibodies	Individual patient's B-cell tumor antigens	B-cell lymphoma
CAMPATH-I (alemtuzumab)a	CD-52 antigen	Chronic lymphocytic leukemia
Rituximaba	CD-20 antigen	Non-Hodgkin's lymphoma
Tositumomab Cetuximaba	B1 antigen EGFR(HER-1)	Non-Hodgkin's lymphoma Colorectal cancer
Radiolabeled antibodies
LYM-I(131I-conjugated)	HLA-DR antigen	Non-Hodgkin's lymphoma
LL2 (epratuzumab) 131I, 90Y-conjugated	CD-22 antigen	Non-Hodgkin's lymphoma
Anti-CD33(131I-conjugated) Ibritumomab tiuxetan (90Y,111In) (Zevalin)	CD-33 antigen CD-20 antigen	Acute/chronic myelogenous leukemia Non-Hodgkin's lymphoma

Edrecolomab

Edrecolomab (17-A Ab) is a murine immunoglobulin G2A (IgG2a) mAb that recognizes the extracellular epitope of Ep-CAM, which is a glycoprotein normally found on the basolateral surface of nonsquamous epithelium of the lung, gastrointestinal tract, pancreas, prostate, etc. Currently used dosage is 500 mg IV postoperatively, followed by 4 monthly doses of 100 mg, to achieve a target plasma concentration of 5–10 mcg/mL.[40] Good response was observed in patients with Duke C colon cancer and rectal cancer, who exhibited a reduction in mortality of 32% compared with the control group.[41] Edrecolomab was associated with human antimouse antibody (HAMA) in 80% of cases after 2–3 months of therapy.

Trastuzumab

Trastuzumab is a recombinant human mAb targeted against HER- 2 protein, which is overexpressed in 25%-30% of patients with breast cancer. Presence of HER-2 protein has been associated with aggressive growth of tumor cells.[41] Trastuzumab, being 95% human and 5% murine, has very low immunogenicity. In a large randomized study comparing cytotoxic chemotherapy (cyclophosphamide + doxorubicin 6 cycles every 21 days) vs chemotherapy + trastuzumab, the addition of trastuzumab improved the overall response rate from 29% to 45%. Infusion-associated symptoms such as chills, fever, nausea, vomiting, and pain were seen in approximately 40% of patients. Cardiotoxicity, being a significant side effect, makes the combination of trastuzumab and anthracyclines inadvisable.[42] However, in one of the studies, when trastuzumab was combined with paclitaxel chemotherapy, the risk of cardiac toxicity was found to be lower.[43]

Cetuximab

Cetuximab is a mAb against EGFR(c-erb1 or HER1) that is overexpressed in many epithelial cell tumors. Such overexpression has been associated with poor prognosis in colorectal cancer.[44] It binds to the extracellular binding domain of EGFR, thus preventing its dimerization and subsequent phosphorylation by intracellular tyrosine kinases.[45] It was approved by the FDA in 2004 for use in treating metastatic colorectal cancer. Cetuximab has a good safety profile. Acne-like rash, drying and fissuring of skin, and, rarely, infusion-related reactions have been observed with the use of cetuximab.[46]

Anti-idiotype Antibodies

Each antibody has an antigen-binding site, ie, the idiotype is unique for each antibody and is responsible for production of a series of host antibodies that will bind to the idiotype. Thus, an idiotype containing Ab, when binding to a tumor antigen, leads to production of anti-idiotype antibodies.[47] These have been studied alone as well as in combination with other therapies in relapsed/refractory low-grade NHL.[48] These patients have shown an overall response rate of 66%, with complete response in 18% of patients. In combination with IFN-alpha and IL-2, chlorambucil demonstrated good response.[49] The main side effects were chills, fever, transient dyspnea, rash, vomiting, diarrhea, and myalgias, and 10% of the patients developed HAMA. In spite of the good clinical response, it is difficult to produce individualized anti-id Ab for each patient. The CAMPATH-I antibody recognizes the CD-52 antigen, which is a glycopeptide highly expressed on normal/malignant T and B lymphocytes and monocytes.[50]

In a multicentric trial, patients with low-grade NHL were treated with 30 mg of CAMPATH-I 3 times weekly for 12 weeks. An overall response rate of 20% was noted, including 4% complete response.[51] Infusion-related adverse effects were most commonly seen in the initial few infusions. Hematologic toxicities such as anemia and thrombocytopenia led to the development of opportunistic infections, including candidiasis, Pneumocystis carinii pneumonia, reactivation of herpes simplex, pulmonary aspergillosis, and disseminated tuberculosis.[52] Due to the increased incidence of infections and septicemias, antibiotic prophylaxis with cotrimoxazole and famciclovir, administered for 2 months after the last dose of antibody therapy, is advocated.

Rituximab

Rituximab is a chimeric anti-CD20 Ab targeted against CD20 antigen, which is expressed in more than 95% of cases of NHL. CD20 antigen is exclusively expressed on mature B cells, providing a selective target for immunotherapy.[53] Rituximab causes tumor cell lysis by ADCC as well as by CDC. It has a half-life of 76 hours after first infusion and increases to 206 hours after the fourth infusion.[54] Rituximab has been administered as a single agent once weekly for 4weeks in patients with relapsed/refractory low-grade follicular NHL. In patients refractory to conventional chemotherapy, 32% of patients showed benefit with rituximab.[55] In a study combining rituximab with the CHOP regimen in patients with low-grade NHL, 38 patients received rituximab once a week for 6 weeks along with 6 cycles of CHOP; there was an overall response rate of 100%, with 58% showing complete response.[56] As rituximab has very low immunogenicity (< 1%), no HAMA responses have been observed.[56] Infusion-related adverse events were observed, such as fever, chills, nausea, angioedema, pruritis, bronchospasm, and, occasionally, hypotension. Its main advantage over CAMPATH-I is that rituximab is associated with a decreased incidence of opportunistic infections along with lack of bone marrow suppression.[57]

Radiolabeled Monoclonal Antibodies

Radioimmunotherapy (RIT) is a very effective treatment modality for radiation-sensitive hematologic malignancies. Moreover, DNA damage is not restricted to Ab-bound malignant cells, and host mechanism/antigen internalization is not required for cytotoxicity.[58] The radionucleotides used are Iodine¹³¹ and ⁹⁰Ytrium. ⁹⁰Y, due to its better beta-killing energy and longer path length, offers an advantage over I¹³¹ in treating bulky and poorly vascularized tumors.[59] Moreover, being a purely beta-emitting radioisotope, it does not irradiate distant organs, which is mainly because of gamma-emitting radioisotopes.

Tositumomab

Tositumomab (Anti-BI) is a murine mAb labeled with I¹³¹. Many studies of tositumomab have been conducted in patients with relapsed and refractory NHL.[60,61] In one of the studies, tositumomab yielded a 67% overall response in patients with relapsed/refractory/transformed NHL, with 17% complete response. Principally hematologic side effects were observed, with absolute neutrophil count reduced to < 500 cells/mm³ in 17% of patients and thrombocytopenia, ie, < 10,000 cells/mm³ in 3% of patients. LYM-I is a murine IgG2a mAb that selectively binds to the beta subunit of noncirculating human leukocyte antigens (HLA-DR). This HLA-DR antigen is expressed in more than 95% of malignant B cells. In a study ¹³¹I-LYM-I was administered to NHL patients who were resistant to standard therapy, resulting in an overall response of > 50%.[62] In another study, ¹³¹I- LYM-I showed an overall response rate of 52% among relapsed cases of NHL, with 12% complete response.[63] The main dose-limiting side effect observed was thrombocytopenia, which required platelet transfusions.

Epratuzumab

Epratuzumab (LL2) is a murine IgG2a antibody targeted against CD22 antigen (transferrin receptor). It is widely expressed during B-cell development and is present in most of NHL tumours[64] Juweid and colleagues[65] compared ¹³¹ILL2 with ⁹⁰Y LL2 in patients with relapsed/refractory NHL. ⁹⁰Y LL2 treatment was found to be more effective than ¹³¹I LL2, the objective tumor response being 29% and 15%, respectively, with these treatments.

Anti-CD33 Antibodies

Anti-CD33 antibody (HuM195)-CD33 is an antigen expressed in myelogenous leukemia cells. Humanized mouse IgG M195 is a recombinant mAb against CD33 antigen. It has been combined with ¹³¹I and studied in patients with relapsed and refractory acute myeloid leukemia, in whom it was associated with a significant decrease in the numbers of peripheral cells and bone marrow blasts. However, complete responses were not seen in any of the patients.[66]

Ibritumomab tiuxetan (IDEC-2B8) is a murine IgGI mAb targeted against CD20 antigen. It has been labeled with ⁹⁰Y(IDEC- Y2B8) or ¹¹¹In (IDEC-In2B8). Radiolabeled ibritumomab has been studied in patients with relapsed or refractory low-grade NHL as well as in patients with rituximab-refractory follicular NHL.[67,68] Reversible hematologic toxicity has been reported without any major organ toxicity. The therapy was well tolerated, with response rates of 82% in patients with low-grade NHL.[69]

Angiogenesis Inhibitors

Angiogenesis is necessary for the growth, invasion, and metastasis of tumors.[70] It is a multistep process regulated by pro- and anti-angiogenic growth factors that are released in response to stimuli such as hypoxia, hypoglycemia, mechanical stress, release of inflammatory proteins, and some genetic alterations.[71] The main pro-angiogenic growth factors are vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), tumor necrosis factor (TNF)-alpha, angiopoietin 1,2, and keratinocyte growth factor (KGF). The anti-angiogenic growth factors include IL-12, IFNs (alpha, beta, gamma), platelet factor 4, factors that are released from extracellular matrix (eg, endostatin and thrombospondin-1), and tissue inhibitors of metalloproteinases (TIMPs).[71] Angiogenesis in tumor vasculature is irregular and unregulated, thereby producing blood vessels that are structurally and functionally abnormal. This prevents the effective delivery of conventional anticancer drugs to the tumors, making them chemotherapy resistant.[72]

Ways to Interfere With Angiogenesis for Treatment of Cancers

Intervention With Endothelial Cell Growth

One of the first compounds identified as an inhibitor of endothelial cell growth was TNP-470, an analogue of the fungus-derived antibiotic fumagillin.[73] Its mechanism of action is prevention of endothelial cells from entering the G1 phase of the cell cycle, leading to decrease in proliferation of endothelial cells.[74] Subsequently, other endogenous molecules such as thrombospondin-I, plalelet factor-4, and interferon inducible protein-10 (IP-10) were discovered. Recently discovered endogenously produced angiostatic proteins are vasostatin[75] and restin,[76] but their detailed mechanism of action is yet to be discovered. VEGF and FGF play a major role in the development of tumor angiogenesis, so tumor growth can be inhibited by blocking these factors.[77] This can be done by producing antibodies against these factors or their receptors, with soluble receptors functioning as antagonists or antisense constructs.

Recently, a nonendothelial cell-specific inhibitor, carboxyamiodotriazole (CAI), has been described. It is an inhibitor of tumor cell motility and acts by inhibition of transmembrane calcium influx. CAI inhibits the invasion of tumor cells by decreasing production of MMPs, blocking migration of cells, and causing cytostasis in tumor cells as well as endothelial cells. Thus, it interferes with the integrity and stability of newly formed blood vessels. It has been found to be effective in the treatment of ovarian cancers.[78,79]

Intervention With Endothelial Cell Adhesion and Migration

Endothelial cell adhesion and migration of cells through the extracellular matrix are necessary for angiogenesis. IFNs alpha and beta have been shown to have in vivo anti-angiogenic activity[80]; however, they are not sufficiently active for all kinds of tumors. Tumors comprising mainly endothelial cells have been found to be more sensitive to IFNs.[81]

The basic function of the integrin molecule alpha-vb₃ is to bind vitronectin and other matrix components that are overexpressed in angiogenically stimulated blood vessels. Antibodies against this integrin have resulted in apoptosis of endothelial cells, as there is loss of anchorage to the extracellular matrix.[82]

Intervention With Matrix Metalloproteinases

Matrix metalloproteinases (MMPs) belong to a homologous family of enzymes that are involved in tissue remodeling. They dissolve the connective tissue matrix and facilitate endothelial cell migration, which is responsible for new blood vessel formation.[83] The MMP inhibitors can be classified into 2 categories: naturally occurring MMP inhibitors (eg, nevostat) and synthetic MMP inhibitors (eg, batimastat, marimastat, and prinomastat).[84] Various angiogenesis inhibitors are currently being studied in clinical trials in the United States for patients with cancer (Table 2).

Table 2.

Angiogenesis Inhibitors in Various Phases of Clinical Trials

Drug	Mechanism	Cancer
Phase 1
Endostatin	Inhibits endothelial proliferation	Solid tumors
Angiostatin	Inhibits endothelial proliferation	Lung, breast, colon, prostate cancer
COL-3	Synthetic MMP inhibitor, tetracycline derivative	–
SU6668	Blocks BEGF, FGF, EGF receptor signaling	–
EMD-121974	Blocks an endothelial integrin	–
2-methoxy estradiol	Inhibits microtubule function	–
Tumstatin	Alpha-vB antagonist	Different cancers
Phase 2
TNP-470	Fumagillin analogue; inhibits endothelial proliferation	Lymphoma, leukemia
Squalamine	Inhibits Na/H exchanger	–
IL-12	Induces IFN-gamma and IFN inducible protein-10	–
Combrestatin	Apoptosis in proliferating endothelium	–
Penicillamine	Inhibition of endothelial cell migration	Glioblastoma
Phase 3
Marimastat	Synthetic MMP inhibitor	Breast, non-small-cell lung cancer (NSCLC), pancreatic cancer
AG3340	Synthetic MMP inhibitor	NSCLC, prostate
Neovastat	Natural MMP inhibitor	Colon, NSCLC
IFN-alpha	Inhibit bFGF production	–
Thalidomide	Not clear	Kaposi's sarcoma, prostate cancer
SU 5416	Blocks VEGF receptor signaling	Kaposi's sarcoma, metastatic colon cancer
BAY-12-95566	Synthetic MMP inhibitor	Lung, ovary, pancreas

Recently, the FDA approved bevacizumab as a first-line therapy for the treatment of metastatic colorectal cancer in combination with FU-based chemotherapies.[85] Bevacizumab is a recombinant, humanized Ab (rhu mAb) targeted against VEGF. It is 93% human IgG and 7% murine antibody. It is currently undergoing phase 3 trials to determine its effectiveness for treatment of solid tumors. VEGF regulates vascular proliferation and induces permeability, which are important for tumor growth. It is also an anti-apoptotic for newly formed endothelial cells. The expression of VEGF in tumor cells is stimulated by hypoxia, hypoglycemia, Ras antigen, and inactivation of tumor suppressor gene p53/Von Hippel-Landau gene.[71] VEGF levels can be measured in plasma as well as serum, with elevated levels signifying poor prognosis. Many trials have been initiated to assess the effectiveness of bevacizumab for the treatment of prostate, breast, non-small-cell lung, pancreatic, liver, head and neck, and cervical cancers.[86]

The recommended dose of bevacizumab for treatment of metastatic colorectal cancer is 5 mg/kg IV infusion every 14 days in combination with FU-based chemotherapy.[87] Apart from infusion-related symptoms, the most common adverse events in the study by Hurwitz and colleagues[87] were hypertension, bleeding episodes, thrombotic events, and proteinuria ranging in severity from clinically silent to nephritic syndrome.

Thalidomide

Thalidomide and its analogues (lenalidomide, CC-4047) have immunomodulatory, anti-angiogenic, antiproliferative, and pro-apoptotic properties, which are responsible for antitumor action. Thalidomide is a potent inhibitor of TNF-alpha produced by lipopolysaccharide (LPS)-stimulated monocytes, and thus decreases the density of TNF-alpha-induced adhesion molecules such as ICAM-I and VCAM-I.[88] Thalidomide and its analogues reduce the expression of pro-angiogenic factors such as VEGF and IL-6 (a multiple myeloma growth and survival factor), and thus prevent angiogenesis.[89] Thalidomide has also been shown to cause induction of NK cells and increase the levels of IL-2 and IFN-gamma, leading to tumor cell lysis.[90] Thalidomide and its analogues cause apoptosis by causing cell growth arrest at the G1 phase, downregulation of NF-kB and apoptosis inhibitory protein (AIP), and activation of caspase 8.[91]

Combination of thalidomide with dexamethasone is being used as the first salvage regimen in relapsed and refractory multiple myeloma (MM) patients.[92] Significant activity of thalidomide has been observed in patients with myelodysplastic syndrome (MDS), which is a bone marrow disease that eventually leads to acute myeloid leukemia. Thalidomide has been shown to increase the hemoglobin levels in some refractory patients, making them transfusion independent.[93] Moreover, it has been found to be useful in the treatment of metastatic RCC, advanced pancreatic cancer, and androgen-independent prostate cancer.[94–96] Currently, thalidomide is being studied in a phase 3 trial involving MM patients, and lenalidomide is being studied in phase 2/3 trials involving MM/MDS patients.

Tyrosine Kinase Inhibitors

Epidermal growth factor receptor (EGFR) belongs to a subfamily of 4 closely related tyrosine kinase receptors(TKI): EGFR, HER2, HER3, and HER4.[97] After specific ligand binding, EGFR makes a homodimer with itself or heterodimerizes with other HER to activate multiple downstream signaling pathways involved in cell survival and cell proliferation.[98] EGFR has been shown to be highly expressed in various malignancies including squamous cell cancer of the head and neck, ovarian cancer, colorectal cancer, breast cancer, lung cancer (nonsquamous cell lung cancer [NSCLC]), and bladder cancer.

Imatinib, geftinib, and erlotinib are orally active TKIs. They block the phosphorylation and activation of downstream signaling of EGFR.[99] The Philadelphia chromosome translocation that results in Bcr-Abl fusion protein leads to chronic myelogenous leukemia (CML) in 95% of patients. Imatinib inhibits the tyrosine kinase domain of Bcr-Abl oncoprotein and thus prevents further phosphorylation of the kinase. It has been approved for use as a first-line therapy in chronic-phase CML and blast crisis.[100] There is overexpression of EGFR in 40%-80% of NSCL cancer patients, and it has been associated with poor prognosis.[101,102] Geftinib (Iressa) has been approved by the FDA for treatment of patients with advanced NSCLC whose disease has progressed in spite of previous treatments with other agents.[103] Phase 1 and 2 studies have confirmed the safety and efficacy of geftinib.[104,105] Preclinical studies have shown effectiveness of geftinib in the treatment of esophageal squamous cell carcinomas as well, where EGFR is expressed in 40%-70% of cases.[106] Erlotinib (Tarseva) has been shown to produce good tumor response and improved overall survival in NSCLC patients who experienced tumor progression after receiving standard chemotherapy.[107,108]

Cancer Vaccines

The identification of various tumor-associated or tumor-specific antigens has facilitated the opportunity to induce antitumor immunologic responses in vivo. The majority of cancer vaccines have been designed to activate cell-mediated immune responses against tumor-associated antigens (TAA), ie, to induce helper responses, assist cytotoxic T lymphocytes (CTL), and support B -cell memory responses (Table 3).

Table 3.

Randomized Controlled Trials of Cancer Vaccines

Vaccine	Disease	Results
Melanoma vaccine oncosylate[123]	Stage III melanoma	No survival benefit vs control group (live vaccinia virus) at 5 years
Melanoma cell line lysate (Melacine) + low-dose cyclophosphamide[124]	Metastatic melanoma	No survival benefit vs chemotherapy alone
GM2 ganglioside + low-dose cyclophosphamide[125]	Melanoma stage III	Trend toward improved disease-free interval
Autologous colon cancer cells[126]	Stage II/III colon cancer	No survival benefit vs controls
Autologous colon cancer cells[127]	Stage II/III colon cancer	Improvement in disease-free interval, benefit in stage II patients only

The antitumor vaccines include peptide vaccination, vaccination with genetically modified organisms (GMO), and application of genetically alterable autologous tumor cells. The TAA that are included in vaccines are DNA, RNA, or proteins. In the peptide vaccination strategy, the TAA are produced by gene technology and the vaccine is administered subcutaneously. It can be used in cancer patients in whom the TAA are well defined on the tumor cell surface, such as in the case of melanoma. Its main disadvantage is the requirement of an intact immune system, which is absent in advanced stages of cancer.

The second approach is the vaccination strategy using GMOs, whereby viruses are transfected with the genetic information to code for TAA.

The third strategy, vaccination with irradiated autologous tumour cells/cell lysates and tumor antigens, has also been attempted, but response has been inconsistent.[109]

Failure to develop effective immunity against tumors may be due to absence of antigen in tumor variants, loss of MHC expression, downregulation of antigen-processing machinery, and expression of local inhibitory molecules.[110] These are the tumor escape mechanisms by which tumor cells develop acquired resistance. The generation of tumor-specific T-cell responses has been the major focus in the development of cancer vaccines.

The CD8+ cytotoxic T lymphocytes (CTL) have been shown to have a major effect in killing tumor cells. They promote the apoptosis of tumor cells by Fas ligand and Fas receptor in the tumor cells and effector cells, respectively. They attract and activate the effector cells (phagocytes, NK cells), which in turn recognize the apoptotic tumor cells and phagocytose them, thus causing effective cellular immunity.[111] Cytokine gene therapy and dendritic cell vaccination have been explored in an attempt to design more effective vaccination for cancer patients. Dendritic cells play a key role as mediators of vaccine function, as they activate the cytolytic T-cell responses by capturing, processing, and presenting the TAA to T lymphocytes.[112] IFN-gamma has been shown to cause increased expression of MHC-I molecules, induction of apoptosis, and impairment of new blood vessel formation, but its main drawback is impairment of tumor immune reactivity by causing downregulation of target antigen expression.[113] Dendritic cells are super-activated by GM-CSF, TNF-alpha and other cytokines, thereby enhancing their ability to activate T cells. Cytokine gene-transfected dendritic cells have increased antitumor activity, as they produce a high concentration of cytokines near the tumor. The role of CD4+ T lymphocytes is to regulate the function of B lymphocytes and CD8+ T lymphocytes. The activated CD4+ T cells express CD40 ligand (GP39), which bind to the CD40 receptor in APC-like dendritic cells. These dendritic cells secrete IL-12 (proinflammatory cytokine), which will cause activation of CD8+ T-cells responsible for producing IFN- gamma and IL-2. They cause chemotaxis of immune cells to tumor sites, leading to tumor cell destruction. Therefore, addition of CD40 trimer to DNA vaccine has been shown to increase antitumor activity of tumor vaccines.[114,115]

Anti-idiotype Antibodies

Anti-idiotype antibodies are effective against antigens expressed on the tumor cell membrane. Specific mutant antigens, such as EGFRvIII expressed in gliomas, can be targeted. Anti-idiotype vaccines can be used in patients with B-cell lymphomas because all of these tumor cells express surface antibodies of the same idiotype. They have been found to provide tumor-protective immunity in mice.[116] Moreover, tumor-reactive antibodies such as human Ab to EpCAM, which is expressed in colorectal carcinomas, can be added to tumor vaccines to facilitate opsonization of tumor cell target and recruitment of the cytotoxic T lymphocytes for tumor cell destruction.[117]

The vehicles used for DNA vaccines are mainly viral. Since the route of viral delivery is mainly parenteral, alternative routes (eg, oral route using Listeria and Salmonella) have been employed to deliver the tumor antigens.[118,119] Currently available tumor vaccines have failed to achieve clinical remission, but their effects can be determined by immune monitoring. Currently, enzyme-linked immunosorbent assay spot quantification of T cells (ELISPOT), which is reactive with an antigen of interest, is the tool of choice for immune monitoring.[120] Other available methods for immune monitoring include quantitative RT-mPCR, intracellular cytokine staining,[121] and tetramer analysis.[122]

Conclusions

The potential of biologic agents to influence malignant processes has revolutionized cancer therapy. The biologic therapy causes modulation of immune responses, stimulation/inhibition of hematopoiesis, direct regulation of cell growth/differentiation, and modulation of angiogenesis. Modulation of these complex networks can lead to some unexpected and unwanted side effects, however, which must be monitored carefully. Moreover, the effects may vary according to the dose and timing of application of the biologic therapy.

The therapeutic value of biologic agents in cancer lies in the prolongation of remission of the tumor after chemotherapy, radiotherapy, or surgery. The science of biologic therapy is rapidly expanding, and effective targets and platforms are being identified. Promising therapeutic approaches for identifying and exploiting tumor-specific cell responses are on the way.