Antibody targeted drugs for cancer therapy - Dissertation Example

?Antibody targeted drugs for cancer therapy ic anticancer treatments include radiation therapy, chemotherapy and surgical resection. These treatments not only have high failure rates, but are also associated with significant side effects which can be debilitating and severe because of their impact on normal tissues. Application of monoclonal antibodies for target approach of cancer therapy because of hybridoma technology which has contributed to the production of monoclonal antibodies. Strategies for this therapeutic application are destruction of cancer cells that are directed by immune reaction, interference of the therapy with the growth and differentiation of cancer cells, transport of anti-cancer agents to the cancer cells based on antigen epitopes, anti-idiotype vaccination and finally, development of humanised mouse monoclonal antibodies through genetic engineering. When targeting of an antigen that is suitable is done, the conjugate attached to it, which is usually inactive is internalized through endocytosis that is receptor mediated, without undergoing any modification. The conjugate taken thus into the cell is then released into the cell from the lysosome and the cytotoxic activity is then restored. This is the principle behind antibody targeted chemotherapy. Monoclonal antibodies are currently considered as important treatment approaches for malignancy because of their tolerance and also effectiveness in the treatment of various cancers. However, in view of limited scope for clinical trials only a few monoclonal antibodies are approved for clinical use and application against cancer. They not only have a role as anticancer agents, but also have ability to increase the selectivity of various other anticancer agents which are not effective when applied alone. Introduction Classic anticancer treatments include radiation therapy, chemotherapy and surgical resection. These treatments not only have high failure rates, but are also associated with significant side effects which can be debilitating and severe because of their impact on normal tissues. Also, severe forms of cancer-related morbidity are directly proportional to radiation and chemotherapy, making them ideal forms of anticancer treatments. In search of more ideal treatments of cancer where the toxicity is exerted only on tumor tissues and normal tissues are spared, application of monoclonal antibodies for target approach of cancer therapy has found its way. Such an application has revolutionized because of hybridoma technology which has contributed to the production of monoclonal antibodies. When these are used for anticancer treatment, they either target the cancer cells directly, or get exerted after conjugation with cytotoxic drugs or immunotoxins or enzymes (Juntilla et al, 2011). Thus, immunotherapy has become the fourth modality of cancer therapy. Strategies for this therapeutic application are destruction of cancer cells that are directed by immune reaction, interference of the therapy with the growth and differentiation of cancer cells, transport of anti-cancer agents to the cancer cells based on antigen epitopes, anti-idiotype vaccination and finally, development of humanised mouse monoclonal antibodies through genetic engineering. Several agents like radionucleotides, toxins and chemotherapeutic agents have been conjugated for anticancer application. These forms of treatment are not only useful following surgical resection but also can be employed in terminal cancer stages (Bodey et al, 2000). In this review, role of antibody mediated cancer therapy will be discussed through review of suitable articles. Monoclonal antibodies Antibodies that are produced from a single type of immune cell are known as monoclonal antibodies. Since these antibodies are basically clones of a single parent cell, they are all identical. Monoclonal antibodies have wide application both in diagnostic and therapeutic clinical arenas (Lambert et al, 2005). Currently, they are used in the treatment of various conditions like cancer, rejection of transplant, chronic inflammatory diseases, multiple sclerosis, macular degeneration, hepatitis B infection and cardiovascular disease. For medical application, monoclonal antibodies are derived from a vertebrate, mostly, mouse. The animal is challenged several times with the antigen against which the antibodies are to be produced. Following this, B cells are removed from spleen and lymph nodes. These are then fused with myeloma tumor cells. The fused cells are known as hybridomas. Myeloma tumor cells multiply rapidly and indefinitely, but they cannot produce antibodies because of the nature of the tumor. And the good B cells can produce antigen specific antibodies. Thus culture of these hybridomas produces large amounts of antibodies. These are known as monoclonal antibodies which are picked up by immunomethods. A classic example of monoclonal antibody directed anticancer therapy is herceptin. Herceptin is a recombinant humanized monoclonal antibody that interferes with the receptor HER2 (human epidermal growth factor receptor 2) which is located in the cell membrane and participates in the regulation of cell growth and other aspects of functioning of the cells which are exaggerated or amplified in cancer like adhesion, survival, migration and differentiation. In some types of breast cancer, HER2 receptors are defective, and in the 'on' position, causing uncontrollable growth of the breast cancer cells. Thus, it has been thought that targeting HER2 receptors can control the growth and development of breast cancer cells (Nahta and Esteva, 2003). Antibody-drug conjugates Some of the difficulties that are encountered in the treatment of human cancers are heterogeneity of cancer cells, different cytotoxic attributes of the cytotoxic agent conjugated to the antibody and the capacity of the cells to modulate genetically (Bodey, 2001; Damle et al, 2003). One of the main treatments for cancer has been chemotherapy since several years. These drugs are highly cytotoxic and are not tumor specific, thus causing extensive cytotoxicity of normal tissues too. Because of this problem, more often than not, chemotherapeutics are administered in suboptimal doses. Thus, this approach towards cancer therapy has been considered to be a draw back. This problem can be solved by targeting tumors with drugs bound to tumor specific antibodies. This is because; antibodies specific to tumor antigens bind only to them and drugs which are bound to these antibodies reach the target antigens, the tumor antigens. This had led to the research in using monoclonal antibodies as delivery vehicles for application of cytotoxic agents to tumor tissues (Schrama et al, 2006; Plaster et al, 2003). When targeting of an antigen that is suitable is done, the conjugate attached to it, which is usually inactive is internalized through endocytosis that is receptor mediated, without undergoing any modification. The conjugate taken thus into the cell is then released into the cell from the lysosome and the cytotoxic activity is then restored. This is the principle behind antibody targeted chemotherapy. One interesting aspect with this method of therapy is that efflux of the drug compound which is mediated by P-glycoprotein transmembrane pump that is associated with multidrug resistance is reduced when compared to systemic application of the drug (Schrama et al, 2006). Many important chemotherapeutic agents which have been used as standard therapies against cancer like vinca alkaloids, antifolate agents and anthracyclines have been used in conjunction with monoclonal antibodies. The pharmacokinetics of the conjugate drugs is dependent on the targeting device and also on the chemical strategy used to couple the drug to the antibody. Peptide linkers are superior to hydrazone linkers. This is because; peptide linkers remain stable in serum and get degraded very fast by certain enzymes after entering the intracellular compartment. Disulphide linkers also are useful for the purpose of conjugation because; cleavage is done by disulphide exchange like glutathione, which is present in higher concentrations in tumor cells when compared to normal cells. Though initial studies on preclinical models suggested the efficacy of immunoconjugation of chemotherapeutic agents, clinically, they were not found to be efficient. This is because; their cytoxicity effect is limited by the amount of conjugate that can be attached to the antibodies, the drug loading potential. For each combination, it is very important to determine optimum number of molecules needed for conjugation with the antibody to deliver maximum cytotoxic effect. Another major limitation of antibody-chemotherapeutic conjugation therapy is the risk of development of anti-mouse antibody responses to patients already started on this conjugation therapy. This problem however, is being solved by the development of antibodies using DNA engineering. There are basically 2 mechanisms by which the cytotoxic potency of the conjugates can be improved. One is by increasing the number of molecules deliverable per moiety that is targeted and this can be done by using carriers like polymers or liposomes. Another strategy is to include cytotoxic drugs that are highly potential (Schrama et al, 2006). One such highly cytotoxic drug that been studied is calicheamicin. This compond has high antitumor activity in vivo. However, as of now, only one immunoconjugate with this compound has secured permission by the FDA and that is gemtuzumab, available in the market as Mylotarg (Schrama et al, 2006). This conjugate contains anti-CD33 monoclonal antibodies that are humanised that are conjugated with ozogamicin which is a cytotoxic. This conjugate is approved for the treatment of advanced CD-33 positive acute myeloid leukemia in elderly patients. Other cytotoxic drugs which are investigated for use in conjugation with monoclonal antibodies are geldanamycin, doxorubicin, taxanes of second generation, DM1, monomethyl auristatin E and CC-1065 (Schrama et al, 2006). Antibody drug conjugates consist of cytotoxic drugs that are potent, linked via chemical linkers to antibodies (Polson et al, 2011). These facilitate specific targeting of cytotoxic drugs to cancer cells (Polson et al, 2011). Currently, this form of cancer therapy has been found to be efficacious with toxicity levels that are manageable. Though this form of cancer treatment appears theoretically simple, practical application has been fraught with several difficulties. Challenges in the development of antibody drug conjugates include target identification, development of appropriate linker chemistries and finally, selection of the most suitable and potent cytotoxic. drug. Antibody drug conjugates are most suitable for hematological cancers because of well characterization of their surface proteins and development of manageable toxicities because their target tissue is mainly bone marrow. Also, in most cases, the normal bone marrow can regenerate (Polson et al, 2011). Polson et al (2009) examined various potential targets and also different linker-cytotoxic drug combinations, systematically, with intentions to determine the most optimal combination for therapeutic application for non-Hodgkin's lymphoma. This study identified seven antigens for potential treatment and they are CD 19-22, CD72 and 79b and CD180. The study found that antibody drug conjugates with cleavable linkers were efficacious via all these identified targets. However, those with uncleavable linkers were effective only against CD22 and CD79b. Those with uncleavable linkers had decreased chances and levels of toxicity probably because of decreased release of free drug and consequent toxic metabolites into the plasma. Based on the results of the study, Polson et al (2009) opined that while those with cleavable linkers had a broad range of targets and higher risk of toxicity, those with uncleavable linkers had promising opening for therapeutic application in human cancers. CD79b, one of the B-cell restricted surface antigen is an important component of signaling of B-cell receptor. In several mouse models it has been found to be a promising target for antibody, especially in systemic lupus erythematosus. Conjugates of antibody against this antigen have been proved to be useful in non-Hodgkin's lymphoma. Zheng et al (2009)conducted a study in the antitherapeutic application of antibody against this antigen and opined that targeting this antigen with antibodies or antibody-drug conjugates is not only an effective, but also an effective therapy for autoimmune diseases like systemic lupus erythematosus and B-cell malignancies. Similar reports were demonstrated by Polson et al in 2007 where they found antibodies against this antigen useful for the treatment of non-Hodgkin's lymphoma. Antibody conjugate drug therapy has also been found useful in the treatment of breast cancer. Currently, trastuzumab or herceptin and iapatinib are the only two monoclonal antibodies approved for breast cancer treatment (Spector et al, 2009). Though these antibodies are directly used for anticancer therapy, combination of these with cytotoxic drugs has been found to be more efficacious. Phillips et al (2008) studied the in vivo and in vitro efficacy of one such combination, the trastuzumab-maytansinoid. This combination is basically a microtubule-depolymerising one. The linkers in this conjugation are thioether and disulphide. The antiproliferative effects of this conjugation was evaluated and ascertained in vitro and in vivo in both normal and tumor cells. Mouse models were used for in vivo assessments. This study found that trastuzumab that was linked to DM1 type of maytansinoid with thioether linkage that was nonreducible has much superior activity in terms of antiproliferative action when compared to combination of the monoclonal antibody with other types of maytansinoids in which the linkers were disulphide type. The main reason why maytansine, an antimitotic drug is used for such targeted delivery applications is because of high potency demonstrated in in vitro studies (Chari, 2008). Most of the cytotoxic agents which are used in conjugation with antibodies are derived from maytansin, chalicheamicin and auristatin. These compounds have toxicity potential of atleast 100 to 1000 times more than the standard chemotherapeutical agents (Haeuw et al, 2009). For the anticancer therapy of acute myeloid leukemia, gemtuzumab ozogamicin is the only available drug-armed antibody in market (Haeuw et al, 2009). This is a conjugate of calicheamycin and antiCD33 antibody. Figure.1: Therapeutic antibodies (Schrama et al, 2010) (Source: Schrama et al, 2010) Figure 2: Internalisation of conjugated drug (Schrama et al, 2010). Immunotoxins and cancer therapy Another class of agents which are useful anti-cancer compounds and can be used in conjunction with antibodies for specific antitumor therapy are immunotoxins. These agents are mostly enzymes and are highly cytotoxic. Also, one single molecule is sufficient enough to kill cancer cell when placed in the intracellular compartment. Such toxins can be binded with other agents also like cytokines, growth hormones and fusion protein toxins. Therapeutic effects of these conjugates is possible only after internalisation of these molecules after they bind to their respective ligands on the cell. Several biochemical factors are involved in determining the cytotoxic potential of immunotoxins and these are rate of internalisation, affinity towards binding with antigen, intracellular processing and potency of the toxin domain that is internal. For therapeutic application of these toxins, they need to be modified in such a way that binding sites for normal tissue target expression must be removed. However, the efficacy of these agents is limited by inefficient uptake into the intracellular compartment and hence the rates of toxicities are not high (Schrama et al, 2006). In several trials in both solid and blood related tumors, response rates of as high as 30 percent have been demonstrated when cancer therapy was administered with immunotoxin-antibody conjugates. But, as of now, only one toxin conjugate, denileukin diftitox, has been approved for clinical use (Schrama et al, 2006). This conjugate is used for treatment of refractory T-cell lymphoma of the skin. The conjugate contains fragments of diphtheria toxin that are conjugated with interleukin-2. The latter is the targeting device. Major clinical toxic problems associated with this therapy are liver injury and vascular leak syndrome. The toxin as such has high immunogenecity and this elicits humoral response by the host even after the first dose. Such an immunological response reduces the serum half life of the toxin and also inhibits its cytotoxic potential, especially when repeated doses are administered. Some research has been done to uphold the toxic potential of these cytotoxic agent and one such strategy has been adjuvant immunosuppressive therapy. Another strategy has been modification of the toxin. Both these methods, though useful, have not been found to be clinically practical. Some of the promising approaches which are likely to come up in future are generation of toxins that are humanised using genetic engineering and modification of the toxin by conjugating it with polyethylene glycol. Humanizing genetic engineering is possible because of the human origin of RNase that down regulates expression of gene. Targeted immunotherapy using immunotoxins and monoclonal antibodies has been found to be effective in the treatment of colorectal cancer (Shapira et al, 2010). The efficacy of any immuno conjugate depends on four aspects, the cytotoxic agents to which it is coupled, the target that is selected for application, the method of coupling used and the linker between the antibody and the cytotoxic agent (Haeuw et al, 2009). (Source: Schrama et al, 2010) Enzyme prodrug therapy directed by antibodies The efficacy of any chemotherapeutic agent or toxin targeted against a tumor in conjugation with antibodies is dependent on the binding of the conjugate to the cell and internalisation. Thus, these therapeutic agents delivered thus will mainly kill tumor cells with specific antigens and not those presenting other antigens. This is a major disadvantage. Although, there is some evidence that the conjugate antibody-maytansine destroys tumors with heterogenous antigen expression. One of the approaches aimed to expand the scope of antitoxic effects even towards tumor cells with different ligands is by using antibody directed enzyme prodrug therapy or ADEPT. In ADEPT, a prodrug that is weakly toxic is activated selectively into a toxic agent at the site of tumor by an enzyme that has been targeted towards a tumor with the help of tumor specific antibody. For optimum therapeutic effect, the antibody-enzyme conjugate, unlike the antibody-drug conjugates needs to remain on the surface of the cell after binding with the antigen. At the same time, it needs to be cleared from the circulation rapidly to prevent toxicity. There are basically 3 classes of enzymes useful for ADEPT. Enzymes belonging to class I are those of non-mammalian origin, but with a mammalian homologue. Those belonging to class II are also of non-mammalian origin, but with a non-mammalian homologue. Class III enzymes are of mammalian origin. Each class of enzymes has both advantages. Class I enzymes cleave prodrugs that cannot be cleaved by endogenous enzymes. Thus, toxicity against normal cells is avoided. However they have the potential to evoke strong immune responses. On the other hand, class III enzymes affect normal tissues also, but are less immunogenic. Antibody–cytokine fusion proteins The concept of immunotherapy for cancer treatment evolved because of the fact that tumor antigens fail to trigger the immune system, leading to proliferation of tumor tissue. Thus, immunoregulatory cytokines have been studied and used as anti-cancer therapy agents, as an alternative to chemotherapeutic agents, radiation therapy and surgery (Ronka et al, 2009). Some research has demonstrated the role of IL2, IL12 and granulocyte macrophage colony stimulating factor in increasing immunogenecity of certain tumors, thus triggering immune responses sufficient enough to cause eradication of the tumor. Currently, IL2 is approved by the FDA for treatment of stage IV melanoma and advanced kidney malignancy. In addition to anticancer effects, immunotherapeutic agents also have other modes of action like exertion of autocrine or paracrine activity by cytokines which reach peak activity in the vicinity of the cells. This is more so during systemic administration of the cytokines. Some researchers have reported good results while directly injecting cytokines at the tumor site and at the same time reducing the chances of generalised toxicity that is significant in systemic doses. Such a therapy is useful even for locoregional treatment of disseminated cancer. One classical example of such application is the treatment of locoregional metastases of soft tissue sarcoma and melanoma with isolated limb perfusion using TNF-alpha along with other cytotoxic agents. Some researchers have also tried fusing cytokines into tumor-specific antibodies. Such an approach did not seem to impair binding capacity of the antibodies. It also caused prolongation of the half-ife of cytokine. Most of the research regarding antibody-cytokine fusion is on IL2. The studies demonstrated eradication of tumors that were already established. Simultaneously, they also suppressed chances of metastases in tumors like melanoma, neuroblastoma and colon cancer. It is however thought that the efficacy of such a combination is mainly based on the type of fusion between antibody and cytokine. Such as inference has been made based on the fact that combined treatment of cytokine and parental antibody showed only minimal therapeutic benefits. Analysis of biodistribution in several preclinical models showed that fusion protein specific to tumor accumulated within the organs bearing the tumor and that this improved over a period of time, leading to longer half-lives of the fusion proteins within the microenvironment of the tumor (Schrama et al, 2006). The therapeutic effect of antibody IL2 fusion protein can be either innate immunity mediated or adaptive immunity mediated and this is based on the type of tumor, suggesting broad spectrum mode of effect of IL2 fusion protein activity. Such an activity is efficacious not only for therapeutic purposes, but also as adjuvant therapy. An example of this is boosting of vaccination-induced immune response because attachment of IL2 to antigens enhances their immunogenecity. A study by Morrison et al demonstrated that when a poorly immunogenic solution was targeted with IL2, protective immune responses were triggered and these responses had antitumor properties. Similar effect could be elicited by targeting GM-CSF or IL2 to antigens by means of facilitating direct contact between fusion protein and antigen. It is yet unclear as to what is the exact mode of mechanism of such an action. But researchers are of the opinion that such immune responses are related natural killer cells, CD4 and B cells in case of IL2 fusion proteins and natural killer cells and CD4 cells only in case of GM-CSF fusion proteins. Based on these preclinical data enormous clinical evaluated related to fusion proteins especially IL2 fusion proteins was done. Recent studies include those specific to EpCAM or GD2 for treatment of prostate cancer or metastatic melanoma. In these studies, it was noted that the fusion proteins were well tolerated and severe toxicities occurred only in few patients. Immunocytokine therapy demonstrated significant biological activity evident from rise in counts of lymphocytes, natural killer cells and also antibody-dependent cellular cytotoxic activity (Schrama et al, 2006). Other than IL2, other cytokines which can be fused to antibodies are GM-CSF, TNF-alpha, IL-12, lymphotoxin-alpha and interferon-gamma. These however are yet in trial stage and have not undergone much scrutiny. This is because several difficulties are being encountered during their application like increased rate of plasma clearance of GM-CSF fusion proteins when compared to parental antibody, decreased biological activity of IL-12, TNF-alpha and lymphotoxin alpha when compared to cytokines that are recombinant. Such differences occur mainly due to the structural differences. Such limitations can be solved through genetic engineering. It is important use genetic applications and solve the minor disadvantages because; preclinical models have demonstrated efficacy of these antibody-cytokine fusion protein with regard to efficacious antitumor therapy (Schrama et al, 2006; Krauss et al, 2008). There are basically 2 ways through which antibody-enzyme fusion proteins have been applied for cancer management .In one method, an antibody-enzyme combination is first pretargeted to the cancer tissue. This is followed by administration of a prodrug that is inactive and which is converted into active drug by the pretargeted enzyme. This is known as antibody-directed enzyme prodrug therapy or ADEPT. In the other method, direct therapeutic application is made by antibody enzyme fusion proteins. Here the enzyme is the toxic substance. The antibody is mainly used to internalise the enzyme into the cancer cell. The enzyme then activates the processes of cell death. The enzymes employed thus are ribonucleases. This is known as the antibody enzyme system (Andrady et al, 2011). ADEPT has several advantages like decrease in the systemic toxicity (Biela et al, 2003). The most critical component in the application of ADEPT is the selection of monoclonal antibody that is used to target the enzyme into the cancer tissue mass. Previously, monoclonal antibodies that are directed against the surface antigens of the rumor cells were used. However, these had several disadvantages like liability and heterogeneity because of which ideal site was not available for the enzyme. Thus, this strategy became less popular and replaced by an alternative method, known as the tumor necrosis therapy or TNT. In this method of cancer treatment, monoclonal antibodies deliver the enzyme directly into the necrotic areas of malignant masses, thus enhancing the efficacy of ADEPT (Biela et al, 2003). Through various autoradiographic and biodistribution studies, it has come to the understanding that ADEPT employing TNT monoclonal antibodies causes localisation of the monoclonal antibodies in the cells that are degenerating and also in the regions of necrosis in the tumor tissue. It was also found that the binding is retained within the malignant mass for longer duration of time, thus enhancing the performance of ADEPT. TNT monoclonal antibodies are perhaps ideal agents for ADEPT targeting because, universally, necrotic centers lie in the central regions of tumor tissue masses and they constitute more than 30-80 percent of the tumor mass (Biela et al, 2003). Future trends While conjugation with antibodies and various cytotoxic drugs is targeted for direct destruction of cancer cells, antibody-cytokine fusion proteins exert their anticancer effects by triggering immune response of the host against the tumor tissue (Khandare et al, 2006). However, newer approaches emerge in view of continuous deciphering of the mechanism of cancer transformation. Several of the newer approaches employ monoclonal antibodies to enhance the specificity of the tumor by either disruption of aberrant signaling in the cancer cell or by enhancing methods that facilitate tumor eradication. In most cancers, there is dysregulation of programmed cell death, also known as apoptosis. This can be restored by death receptor triggering which can be done by various extrinsic factors like FasL, tumor necrosis factor and tumor necrosis factor related apoptosis or TRAIL. Currently restoration of apoptosis is considered to be the most powerful weapon against cancer. This is because; most tumor cells, despite having altered threshold for programmed cell death, still have the potential to undergo so. One useful approach in this regard has been Fas ligation. But one major disadvantage with Fas therapy is severe systemic toxicities like hepatic toxicity preventing its application for cancer therapy. Another major disadvantage with Fas therapy is that it is basically a biologically inactive therapy and gets activated only when aggregated with secondary agents like crosslinking antibodies. Other than apoptosis restoration, the next strategy to consider in cancer therapy is impairment of the cancer cells by directly blocking mRNA expression of the cancer cells. Such a task can be done theoretically by targeting mRNA of tumor cells with ribozymes, antisense RNA and RNAse enzymes. These strategies provide scope for selective anticancer therapy. But practically, there are several factors which prevent them from being applied therapeutically and they are rapidly elimination from plasma, increased nuclear sensitivity and poor chances of intracellular delivery. Actually, this problem has been addressed by chemical modifications and carrier usage, which increase the plasma half life. Also, fusion of these agents with monoclonal antibodies and other specific ligands also enhance the delivery of the agents into the cells. Such modifications have been studied in the treatment of neuroblastoma and colon cancer models. More clinical trials are warranted in this regard. Conclusion Monoclonal antibodies are currently considered as important treatment approaches for malignancy because of their tolerance and also effectiveness in the treatment of various cancers. However, in view of limited scope for clinical trials only a few monoclonal antibodies are approved for clinical use and application against cancer. They not only have a role as anticancer agents, but also have ability to increase the selectivity of various other anticancer agents which are not effective when applied alone. However, as of now, there are some problems which need to be addressed and these include selectivity, immunogenecity and entry into tumor mass. The main problem with immunogenecity is because most of them are derived from chimeric or murine body towards which human body can develop antibodies. This problem can be solved practically by genetic engineering and consequent generation of human antibodies. It is very important to identify new antigens of the tumors and also respective monoclonal antibodies so that antibody selectivity is increased and at the same time, toxic effects of the fusion proteins is reduced. In view of easy accessibility most of the therapies based on monoclonal antibodies are targeted towards lympho-hemtopoietic malignancies. Several studies have demonstrated the role of monoclonal antibodies in the treatment of these cancers either alone, or in conjugation with other cytotoxic drugs. References Andrady, C., Sharma, S.K., Chester, K.A. (2011). Antibody-enzyme fusion proteins for cancer therapy. Immunotherapy, 3(2), 193-211. Biela, B.H., Khawli, L.A., Hu, P., Epstein, A.L. (2003). Chimeric TNT-3/human beta-glucuronidase fusion proteins for antibody-directed enzyme prodrug therapy (ADEPT). Cancer Biother Radiopharm., 18(3), 339-53. Bodey, B., Bodey, B. Jr, Siegel, S.E., Kaiser, H.E. (2000). 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