Antibodies or immunoglobulins are a crucial component of the immune system, circulating in the blood and lymphatic system, and binding to foreign antigens expressed on cells. Once bound, the foreign cells are marked for destruction by macrophages and complement. In the context of cancer immunotherapy, monoclonal antibodies have brought to light a wide array of human tumor antigens.1 In addition to targeting cancer cells, antibodies can be designed to act on other cell types and molecules necessary for tumor growth. For example, antibodies can neutralize growth factors and thereby inhibit tumor expansion.
Monoclonal antibodies are made by injecting human cancer cells, or proteins from cancer cells, into mice so that their immune systems create antibodies against foreign antigens. The murine cells producing the antibodies are then removed and fused with laboratory-grown cells to create hybrid cells called hybridomas. Hybridomas can indefinitely produce large quantities of these pure antibodies.
Development of Monoclonal Antibodies
Monoclonal antibodies have several roles in cancer therapy. Monoclonal antibodies have been used in a variety of ways in the management of cancer including diagnosis, monitoring, and treatment of disease. They aid in diagnosis, such as the application of flow cytometry in the identification of different subsets of non-Hodgkin's lymphoma. We can use monoclonal antibodies to monitor disease progression, such as the measurement of carcinoembryonic antigen in colon cancer. Most importantly, we can utilize monoclonal antibodies directly as therapy. Video Download Free RealPlayer
Relative to treatment, monoclonal antibodies can react against specific antigens on cancer cells and may enhance the patient's immune response. Monoclonal antibodies can be programmed to act against cell growth factors, thus blocking cancer cell growth. We can conjugate or link monoclonal antibodies to anticancer drugs, radioisotopes, other biologic response modifiers, or other toxins. When the antibodies bind with antigen-bearing cells, they deliver their load of toxin directly to the tumor. Monoclonal antibodies may also be used to preferentially select normal stem cells from bone marrow or blood in preparation for a hematopoietic stem cell transplant in patients with cancer.
There are a number of considerations when using monoclonal antibodies for therapy. First, a target antigen must be selected. It is important that this antigen is presented uniquely by the tumor cells and not on normal tissues. The immunogenicity of the monoclonal antibody itself is a concern because of how they are derived. As they are often derived from non-human monoclonal antibodies, they are capable of eliciting an immune response themselves. Half-life is another factor. Will it be long enough to have the desired effect? There are also logistical problems such as cost and availability. Anti-idiotype monoclonal antibodies are a good example of this, as their development has been prohibited by cost. Finally, a decision as to whether or not the monoclonal antibody will be used alone or if it will be conjugated (i.e., attach radioisotopes, toxins, or chemotherapy) in order to get the desired therapeutic effect.
Mechanism of action
Monoclonal antibodies achieve their therapeutic effect through various mechanisms.22 They can have direct effects in producing apoptosis or programmed cell death. They can block growth factor receptors, effectively arresting proliferation of tumor cells. In cells that express monoclonal antibodies, they can bring about anti-idiotype antibody formation.
Indirect effects include recruiting cells that have cytotoxicity, such as monocytes and macrophages. This type of antibody-mediated cell kill is called antibody-dependent cell mediated cytotoxicity (ADCC). Monoclonal antibodies also bind complement, leading to direct cell toxicity, known as complement dependent cytotoxicity (CDC).
Figure 1 illustrates the way in which antibody-dependent cell cytotoxicity works. [Figure 1. Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC)] In this situation Rituximab (IDEC-C2B8), a chimeric antibody, targets the CD20 antigen. This antigen is expressed on a significant number of B cell malignancies. Rituximab is an IgG monoclonal antibody, and has an Fc receptor. The Fc fragment of the monoclonal antibody binds the Fc receptors found on monocytes, macrophages, and natural killer cells.23 These cells in turn engulf the bound tumor cell and destroy it.24 Natural killer cells secrete cytokines that lead to cell death, and they also recruit B cells.
An example of complement dependent cytotoxicity, again using B cell lymphoma, is shown in Figure 2. [Figure 2. Complement-Dependent Cytotoxicity (CDC)] Here we see the monoclonal antibody binding to the receptor and initiating the complement system, also known as the 'complement cascade.' The end result is a membrane attack complex that literally makes a hole within the cell membrane, causing cell lysis and death.25
Antibody therapy can be used in a variety of ways to treat cancer. As described above, they may act through ADCC or CDC. An alternative approach is to conjugate the monoclonal antibody to a toxin, a cytotoxic agent, or a radioisotope. With conjugated monoclonal antibodies a toxin is actually bound to the antibody, which then attaches to the antigen. The antibody conjugate is absorbed into the cell itself, resulting in cell death. We can attach a radioisotope such as iodide-131 to directly infuse the cancer cell with radiotherapy, and also mitigate the effects to normal surrounding tissue. Finally, we can attach chemotherapy. The chemotherapeutic agent is taken directly into the targeted malignant cell, rather than being systemically absorbed.
Obstacles to successful therapy
There are a number of obstacles to successful therapy with monoclonal antibodies. The antigen distribution of malignant cells is highly heterogeneous, so some cells may express tumor antigens while others do not. Antigen density can vary as well, with antigens expressed in concentrations too low for monoclonal antibodies to be effective. Tumor blood flow is not always optimal. If monoclonal antibodies need to be delivered via the blood, it may be difficult to reliably get the therapy to the site.
High interstitial pressure within the tumor can prevent the passive monoclonal antibodies from binding. Sometimes the tumor antigen is even released, so the antibody binds to a free-floating antigen and not the tumor cell. Since monoclonal antibodies are derived from mouse cell lines, the possibility of an immune response to the antibodies exists. This response not only decreases the efficacy of monoclonal antibody therapy, but also eliminates the possibility of re-treatment. Very rarely do we see cross-reactivity with normal tissue antigens – in general target antigens that are not cross reactive with normal tissue antigens are chosen. Despite these obstacles, there has been tremendous success in the clinical application of monoclonal antibodies in hematologic malignancies and solid tumors.
Clinical Applications of Monoclonal Antibodies
There are a number of antigens and corresponding monoclonal antibodies for the treatment of B cell malignancies. Some of the most active are listed in Table 1. [Table 1. Monoclonal Antibodies for Treatment of B Cell Malignancies] One of the most popular target antigens is CD20, found on B cell malignancies. The CD52 antigen is targeted by the monoclonal antibody alemtuzumab, which is indicated for treatment of chronic lymphocytic leukemia. The CD22 is targeted by a number of antibodies, and has recently demonstrated efficacy combined with toxin in chemotherapy-resistant hairy cell leukemia.26 Two new monoclonal antibodies targeting CD20, tositumomab and ibritumomab, have been submitted to the Food and Drug Administration (FDA). These antibodies are conjugated with radioisotopes.
The first monoclonal antibody to receive FDA approval was rituximab. Rituximab is a chimeric unconjugated monoclonal antibody directed at the CD20 antigen, a signature B cell antigen. CD20 has an important functional role in B cell activation, proliferation, and differentiation.27 This antigen is a transmembrane protein composed of 297 amino acids. The intracellular portion contains phosphorylation sequences for protein kinase C, calmodulin, and casein kinase 2. Video
CD20 is thought to act as a calcium channel as well, given the great structural homology between the CD20 protein and the calcium channels.28 When CD20 was introduced into cell lines by transfection, an increase in intracellular calcium was observed within the transfected cells.28,29 With monoclonal antibody stimulation, we see calcium influx within the cells.30 Calcium chelators blocked apoptosis induced by CD20 stimulation by monoclonal antibodies.30
When monoclonal antibodies attach and particularly cross-link CD20 antigen, an increase in intracellular calcium is again observed. [Figure 3. Effects of CD20 Crosslinking] This increase appears to activate the SER family of tyrosine kinases, resulting in further phosphorylation of the CD20 inner cytoplasmic chain and also phospholipase C-gamma. At the same time there is an upregulation of C-myc and myb messenger ribonucleic acid (RNA), an increase in adhesion molecule expression and an upregulation of MHC class II proteins. The ultimate result is caspase 3 activation, causing cell apoptosis.
As previously mentioned, CD20 is a natural focus for monoclonal antibody therapy because of its relatively high degree of expression in B cell malignancies, perhaps as high as 95% in follicular lymphomas, even with the heterogeneity discussed earlier. The monoclonal antibody rituximab was designed specifically to target CD20. Rituximab is predominantly human (95%). The variable light and heavy chain portion of rituximab is murine, but the remainder is humanized so the formation of human anti-mouse antibody is not significant. Rituximab is thought to induce cell apoptosis by inducing calcium influx, releasing caspase activity. In addition, evidence of indirect effects through ADCC and CDC has been observed.
Rituximab is indicated for treatment of low-grade lymphomas refractory to conventional chemotherapy. Based upon this work it has been evaluated for first-line and combination therapy. Results of studies using rituximab as first-line treatment of low-grade non-Hodgkin lymphoma have been encouraging.31-33 Patients who had not received any prior therapy were treated with rituximab 375 mg/m2 on a weekly basis for 4 weeks and then re-evaluated 2 weeks post-therapy. The patients who achieved a complete or partial response, or who had stable disease received rituximab maintenance therapy (weekly for 4 weeks every 6 months). Patients who showed evidence of progression were taken off maintenance therapy.
At the time of initial re-evaluation at 6 weeks, 54% of the patients showed objective response to treatment. An additional 36% had stable disease or minor response. At the time of publication 13 patients had undergone a second course of treatment, and 4 additional responses were documented. Four patients improved from partial to complete response.31 Treatment with rituximab was well tolerated, with only 1 of the 39 patients experiencing grade 3-4 infusion related toxicity.
These responses were durable as well. For patients who achieved partial or complete response, one-year follow-up showed no evidence of disease progression. One-year survival was 69%; survival at two years 67%. While overall survival is not an unusual finding in low-grade lymphoma, the duration of response remains relatively impressive.
Rituximab has been combined with conventional chemotherapy for patients with intermediate grade or diffuse large cell non-Hodgkin lymphoma.34 CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) is standard therapy for this type and stage of disease. In a multi-institutional study, 33 patients with newly diagnosed large cell lymphoma received six infusions of rituximab 375 mg/m2 on day 1 of each cycle combined with six doses of CHOP on day 3 of each cycle. The overall response rate was 94%, with 20 patients (61%) achieving a complete response. Eleven patients (33%) experienced partial response, and 2 patients were found to have progressive disease. Median duration of response and time to progression had not been reached after a median follow-up time of 26 months. Twenty-nine patients remained in remission during this observation period.
The most frequent adverse events associated with rituximab were fever and chills, primarily during the first infusion. The investigators concluded that rituximab did not appear to increase the toxicity of therapy.
These results are interesting for two reasons. First, the number of partial responses to combination therapy was greater than with rituximab alone. Second, the responses – even the partial ones – tended to be durable. [Figure 4. Rituximab for Initial Treatment of LGNHL: Duration of Response]
These results were confirmed in a phase III randomized trial of CHOP and rituximab in elderly patients conducted by the French Lymphoma Cooperative Group (GELA).35 These patients had stage II to IV diffuse large cell lymphoma, were newly diagnosed and therapy-na´ve. This study focused on elderly patients (60 to 80 years), because the efficacy of CHOP is decreased in the elderly. Patients had Eastern Cooperative Oncology Group (ECOG) performance status of 0 to 2. Patients were randomized to receive CHOP alone with cytokine support or CHOP with rituximab, given on day 1. These regimens were administered every 21 days for 6 to 8 cycles.
Interim results were reported at the 2000 annual meeting of the American Society of Hematology. [Figure 5. GELA Phase III Trial of Rituximab/CHOP: Survival] Patients who received CHOP plus rituximab showed a statistically significant (P < 0.0005) advantage in event-free survival over CHOP alone. This advantage translated into an improvement in overall survival of 83% with rituximab (P < 0.01) versus 68% in these 400 patients. A similar study is now in progress, with an additional arm to evaluate the value of rituximab as maintenance therapy for responders.
Compared to hematologic malignancies, solid tumors do not have as many specific targets for monoclonal antibodies that are not cross-reactive with antigens on normal tissues. Two significant monoclonal antibodies have been used in solid tumors: edrecolomab and trastuzumab.
Edrecolomab targets the 17-1A antigen seen in colon and rectal cancer, and has been approved for use in Europe for these indications.36,37 Its antitumor effects are mediated through ADCC, CDC, and the induction of an anti-idiotypic network. In an initial study of 189 patients with resected stage II colorectal cancer, treatment with edrecolomab reduced the relative risk of mortality by 32% compared with observation alone (P < 0.01). Edrecolomab is undergoing investigation in two large phase III trials in patients with stage III colon cancer, either as monotherapy or in combination with fluorouracil-based chemotherapy.37
In the US, the most commonly used monoclonal antibody for the treatment of solid tumors is trastuzumab, which targets the HER-2/neu antigen. This antigen is seen on 25% to 35% of breast cancers. Trastuzumab is thought to work in a variety of ways: downregulation of HER-2 receptor expression, inhibition of proliferation of human tumor cells that overexpress HER-2 protein, enhancing immune recruitment and ADCC against tumor cells that overexpress HER-2 protein, and downregulation of angiogenesis factors. This last mechanism may be very important in terms of metastatic disease. Video
In phase I and II trials of patients with metastatic breast cancer, treatment with a combination of trastuzumab and cisplatin resulted in prolongation of survival and higher response rates than that seen with cisplatin alone.38,39 [Figure 6. Trastuzamab Combination Pivotal Trial: Objective Response Rate] Trastuzumab plus chemotherapy when compared to chemotherapy alone was associated with longer time to disease progression (median 7.4 months vs. 4.6 months, P<0.001), a higher rate of objective response (50% vs. 32%, P<0.01), a longer duration of response (median 9.1 vs. 6.1 months, P<0.001), a lower rate of death at 1 year (22% vs. 33%, P=0.008), longer survival (median survival 25.1 vs. 20.3 months, P=0.01) and a 20% reduction in the risk of death.40 This trial evaluated 469 patients with metastatic breast cancer that overexpressed HER-2/neu. Patients were randomly assigned to receive chemotherapy alone (n=234) or chemotherapy plus trastuzumab (n=235). The only significant adverse event was cardiac dysfunction, which was managed with standard medical treatment.
Trastuzumab has also been studied as monotherapy.41 This study involved 214 patients with relapsed metastatic breast cancer who had been heavily pre-treated: 90% had received prior anthracyclines and 65% had received taxane therapy. The majority had either lung or liver metastasis. All patients received a loading dose of trastuzumab (4 mg/kg), followed by weekly maintenance therapy of 2 mg/kg until evidence of disease progression. Primary endpoints were tumor assessment relative to response. Secondary endpoints were duration of response, time to tumor progression, time to treatment failure, and quality of life.
Eight complete and 26 partial responses were identified for an objective response rate of 15% in the intent-to-treat population (95% CI; 11% to 21%). Median duration of response was 9.1 months; median duration of survival was 13 months. Toxicity was minimal, although cardiac dysfunction occurred in 4.7% of patients. Patients who had higher overexpression of HER-2/neu had a better overall response rate, closer to 24%.
As first-line monotherapy, trastuzumab has demonstrated efficacy and safety in patients with metastatic breast cancer.42 This study included 114 women with HER-2-overexpressing metastatic breast cancer with no prior chemotherapy. Patients were randomized to receive a loading dose of trastuzumab 4 mg/kg followed by 2 mg/kg weekly, or an 8 mg/kg loading dose followed by 4 mg/kg weekly. Primary endpoint was overall response rate. Secondary endpoints were disease relapse, time to tumor progression, and overall survival.
Complete response rates were relatively low (7/114) regardless of loading and maintenance doses. Partial response rates were nearly identical at 19% vs. 21%, for an overall response rate of 24% vs. 28%, which was not statistically significant. Seventeen (57%) of 30 patients with an objective response and 22 (51%) of 43 patients with clinical benefit had not experienced disease progression at 1-year follow-up.
These investigators found no benefit to the higher versus lower dose of trastuzumab. However, they noted a difference in response that correlated to HER-2 overexpression. Overexpression was measured by immunohistochemistry (IHC) and then rechecked with fluorescent in situ hybridization (FISH). Interestingly, patients who were positive for overexpression by IHC were not necessarily positive by FISH. Overexpression as confirmed by FISH was strongly correlated to response, and these patients appeared to have garnered the most clinical benefit from treatment with trastuzumab. [Figure 7. Monotherapy with Trastuzamab in Relapsed MBC: FISH Clinical Outcome Analysis]
When we look at these data together [Figure 8. Monotherapy with Trastuzamab in MBC Studies: Summary], first-line monotherapy with trastuzumab appears to have better overall response. However, median time to disease progression remains disappointingly short. The incidence of cardiac toxicity cannot be minimized, particularly in patients with prior anthracycline therapy or cardiac disease. These patients had a 10% incidence of severe myocardial toxicity and one death from ventricular arrhythmia. Still, these data suggest the possibility of significant response rates, including improvement in overall survival.
While early reports on the use of monoclonal antibodies may have been overenthusiastic, the results of these studies show there is still cause for cautious optimism as we go forward. Cure may yet elude us, but stable disease is an attainable, highly desirable goal.